Gene THERAPY

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EUROPEAN COMMISSION Directorate F – Health Unit F5 – Health Biotechnology Contact: Charles Kessler Office CDMA 2/188 Tel (32-2) 29 56112 Fax (32-2) 29 94693 E-mail: [email protected] EUROPEAN COMMISSION

NEW THERAPIES

EU-supported research in Genomics and Biotechnology for Health Sixth Framework Programme (2002-2006)

Edited by Charles Kessler

Directorate-General for Research 2007 Life Sciences, Genomics and Biotechnology for Health EUR 22841

NEW THERAPIES TABLE OF CONTENTS

inTRODUCTION 11

Regenerative

Medicine 15

EuroStemCell THERAPEUSKIN European consortium for stem cell research 17 Ex vivo gene therapy for recessive dystrophic epi- dermlysis bullosa : preclinical and clinical studies GENOSTEM 44 Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to BetaCellTherapy the bed side 22 Beta cell programming for treatment of diabetes 47 OsteoCord Bone from blood: optimised isolation, characteri- EuroSTEC sation and osteogenic induction of mesenchy- Soft tissue engineering for congenital birth mal stem cells from umbilical cord blood 27 defects in children: new treatment modalities for spina bifida, urogenital and abdominal wall TherCord defects 52 Development and preclinical testing of cord blood-derived cell therapy products 30 SC&CR Application and process optimisation of human EPISTEM stem cells for myocardium reapair 57 Role of p63 and related pathways in epithelial stem cell proliferation and differentiation and in STEMSTROKE rare EEC-related syndromes. 32 Towards a stem cell therapy for stroke 61

Ulcer Therapy STEMS Gene transfer in skin equivalnts and stem cells: Preclinical evaluation of stem cell therapy in novel strategies for chronic ulcer repair and tis- stroke 64 sue regeneration 37 STROKEMAP Skintherapy Multipotent Adult Progenitor Cells to treat Stroke Gene therapy for Epidermolysis Bullosa: a model 67 system for treatment of inherited skin diseases. 41 RESCUE From stem cell technology to functional restoration after spinal cord injury 69

New Therapies – Table of contents 5 NEW THERAPIES TABLE OF CONTENTS

Gene Therapy 83

STEM-HD CLINIGENE Embryonic stem cells for therapy and exploration European Network for the Advancement of Clini- of mechanisms in Huntington Disease 72 cal Gene Transfer and Therapy 85

NEUROscreen CONSERT The discovery of future neuro-therapeutic Concerted Safety & Efficiency Evaluation of Ret- molecules 75 roviral Transgenesis in Gene Therapy of Inherited Diseases 92 myoamp Amplification of human myogenic stem cells in GIANT clinical conditions 77 Gene therapy: an Integrated Approach for Neo- plastic Treatment 98 CRYSTAL CRYo-banking of Stem cells for human Therapeu- BACULOGENES tic AppLicationp 80 Baculovirus vectors for gene therapy 102

THOVLEN Targeted Herpesvirus-derived Oncolytic Vectors for Liver cancer European Network 104

THERADPOX Optimised and novel oncolytic adenoviruses and pox viruses in the treatment of cancer: Virothera- py combined with molecular chemotherapy 107

RIGHT RNA Interference Technology as Human Thera- peutic Tool 110

ZNIP Therapeutic in vivo DNA repair by site-specific double-strand breaks 115

SNIPER Sequence Specific Oligomers for in vivo DNA Repair 118

Improved precision Improved precision of nucleic acid based therapy of cystic fibrosis 121

6 New Therapies – Table of contents NEW THERAPIES

INTHER InVivoVectorTrain Development and application of transposons European labcourse: towards clinical gene thera- and site-specific integration technologies as py: preclinical gene transfer assessment 147 non-viral gene delivery methods for ex vivo gene- based therapies 124 IndustryVectorTrain European labcourse: advanced methods for in- Epi-Vector dustrial production, purification and characteri- Episomal vectors as gene delivery systems for sation of gene vectors 150 therapeutic application 129

PolExGene Biocompatible non-viral polymeric gene delivery systems for the ex vivo treatment of ocular and cardiovascular diseases with high unmet medical need 133

Magselectofection Combined isolation and stable nonviral transfection of hematopoietic cells ‘ a novel platform technology for ex vivo hematopoietic stem cell gene therapy 135

SyntheGeneDelivery Ex vivo gene delivery for stem cells of clinical interests using synthetic processes of cellular and nuclear import and targeted chromosomal integration 138

MOLEDA Molecular optimisation of laser/electrotransfer DNA administration into muscle and skin for gene therapy 141

ANGIOSKIN DNA electrotransfer of plasmids coding for antiangiogenic factors as a proof of principle of non-viral gene therapy for the treatment of skin disease 144

New Therapies – Table of contents 7 NEW THERAPIES TABLE OF CONTENTS

Immunotherapy and Transplantation 153

Allostem MimoVax The development of immunotherapeutic strate- Alzheimer’s disease-treatment targeting trun- gies to treat haematological and neoplastic dis- cated Aß40/42 by active immunisation 193 eases on the basis of optimised allogeneic stem cell transplantation 155 Pharma-Planta Recombinant pharmaceuticals from plants for DC-THERA human health 196 Dendritic cells for novel immmunotherapies 159 SAGE SME-led antibody glyco-engineering 200 DC-VACC Dendritic cells as natural adjuvants for novel vac- BMC cine technologies 165 Bispecific monoclonal antibody technology concept 202 THERAVAC Optimised delivery systems for vaccines targeted AUTOCURE to dendritic cells 169 Curing autoimmune disease. A translational approach to autoimmune diseases in the post-ge- DENDRITOPHAGES nomic era using inflammatory arthritis and my- Therapeutic cancer vaccines 173 ositis as prototypes and learning examples 204

Genomes To Vaccines INNOCHEM Translating genome and proteome information Innovative chemokine-based therapeutic strate- into immune recognition 177 gies for autoimmunity and chronic inflammation 209 COMPUVAC Ration design and standardised evaluation of CELLAID novel genetic vaccines. 181 European symposia for the evaluation of potentials and perspectives of curative cell therapies HEPACIVAC for autoimmune diseases 214 New preventative and therapeutic Hepatits C vaccines: from preclinical to phase I 186 STEMDIAGNOSTICS The development of new diagnostic tests, new BacAbs tools and non-invasive methods for the preven- Assessment of structural requirements in com- tion, early diagnosis and monitoring for haemat- plement-mediated bactericidal events: towards a opoietic stem cell transplantation 217 global approach to the selection of new vaccine candidates 189 RISET Reprogramming the immune system for the establishment of tolerance 221

8 New Therapies – Table of contents NEW THERAPIES

XENOME Engineering of the porcine genome for xenotransplantation studies in primates: a step towards clinical application 224

CLINT Facilitating international prospective clinical trials in stem cell transplantation 228

TRIE Transplantation research integration across Europe 230

INDEX OF PROJECTS 233

INDEX OF

COORDINATORS 234

New Therapies – Table of contents 9 10 NEW THERAPIES Introduction

ew therapies” as used in the title here factors and transplantation science. Projects refers to treatments such as gene and described here focus on regeneration of tissues “Ncell therapy, immunotherapy, tissue such as bone and cartilage, skin, muscle, heart, engineering and regenerative medicine which pancreas and nervous tissue, through the use of offer hope for therapy of diseases which are cur- haematopoietic, mesenchymal, cord blood, hu- rently untreatable, where life is at stake, and for man embryonic and adult stem cells. In many cas- regenerating diseased, damaged or defective es projects take a comparative approach to iden- tissues and organs. Their main characteristic is tify the best sources of cells and to see how they that they employ large biomolecules, genes, perform in different tissues. Such comparisons cells and tissues as therapy rather than drugs or can also reveal insights into underlying knowl- pharmaceuticals. By addressing such conditions edge of the biological processes involved. High- as arthritis, diabetes, heart and neurodegenera- lights of the research include using stem cells to tive disease, new therapies address problems of reverse muscular dystrophy in a dog model of an ageing population, and as high-value, new the disease, and developing a technique to grow technologies, they represent an opportunity for pure brain stem cells. developing industrial competitiveness. Gene Therapy. Gene therapy is the insertion The purpose of this compilation of information of genes into an individual’s cells and tissues to on EU-sponsored research in new therapies is treat a disease; its success depends on isolation to demonstrate the range of activities under- of effective therapeutic genes and on finding taken in this field during the course of the EU’s suitable vectors to deliver them into the patient’s Sixth Framework Programme for Research (2002- cells. The main approaches for gene delivery are 2006), notably under the heading “Applications viral, which often displays a high rate of gene of knowledge and technologies in the field of transfer but which can be immunogenic and dif- genomics and biotechnology for health” in the ficult to produce on a large scale, and nonviral, “Life sciences, genomics and biotechnology for which results in less effective gene transfer and health” thematic priority. limited expression periods, but which has no insert size limitation, is relatively non-immuno- The projects described have been ordered as genic and easy to manufacture. Cell-based vec- much as possible on the basis of similarity and tors for gene delivery use a patient’s own cells, have been grouped into three chapters: which are transfected ex vivo and injected back into the same patient, where they are non-im- Regenerative Medicine. This refers to innova- munogenic and recognised as self. Reports here tive medical therapies enabling the body to re- focus on development of tools and technologies pair, replace, restore and regenerate damaged to overcome the existing limitations and allow or diseased cells, tissues and organs. The field in- clinical application of gene therapy. cludes research areas such as cell therapy, tissue engineering, biomaterials engineering, growth

New Therapies – Introduction 11 NEW THERAPIES Introduction

Immunotherapy and Transplantation. Immunotherapy aims to modulate the immune system to achieve a prophylactic and/or therapeutic goal by inducing, enhancing, or suppressing an immune response. Projects in this sector include cell therapy using stem cell transplantation, dendritic cells, vaccines and antibodies to specifically target cells, and research on autoimmune diseases. Transplan- tation projects are also grouped here since immune suppression and tolerance are major research goals. Shortage of organs for transplantation is addressed through optimisation strategies, development of xenotransplants and the regenerative medicine approach.

While these three areas do possess their own identity, they also contain parts that are interrelated; hence use of the term new therapies to cover them all. A summary of the main topics covered by the activities is shown in schematic version in the figure below:

EU New Therapies Research (FP6)

REGENERATIVE MEDICINE IMMUNOTHERAPY GENE THERAPY

hESC Oligomers siRNA Mesenchymal Dendritic Cells Episomal Adult SC Cancer Non viral vectors hematopoietic Infectious diseases Zn-finger Electrotransfer Cord Blood SC Transposon Minichr Rheuma Plasmid romosome Cytokines Banking Transplantation Autoimmune Oncolytic Retro Mechanisms AAV Viral/ Viral vectors Hep C Alzheimer islets brain spine Lenti Baculo Bacteria Vaccinology Tissue repair skin Engineered cells heart & regeneration Selection Design Clinic Safety Soft tissue muscle Ab Production Standardisation

Main topics of new therapies research projects described in this review Integrated projects (16 projects). These are the largest size projects and integrate a range of differ- The aim of the projects described here was to ent activities, such as multidisciplinary re- create technological platforms for the develop- search, demonstration and training. They ment of new therapeutic tools. To achieve this also permit projects to take a translational projects were built around five different funding approach linking underlying biology and schemes: therapy and enable scientists, clinicians and other stakeholders to work together to achieve their deliverables.

12 New Therapies – Introduction NEW THERAPIES

Specific Targeted Research Projects The number of project supported in each of the (29 projects). These are smaller projects three areas and the EC financial contribution to which focus on specific research issues. them is shown in the table below. It can be seen They may be translational but are less that overall 59 projects were supported with an multi-disciplinary and wide-ranging than EC contribution of almost €270 million; this was the Integrated Projects. distributed among about 700 research teams, almost 100 of which are SMEs. SME-Specific Targeted Research Projects (7 projects). Targeted Research Projects European research projects are encouraged designed to encourage research and in- to be open towards the general public and to novation efforts of Small and Medium- engage with stakeholders and interest groups. Sized Enterprises and where research-led Accordingly many projects organise public SMEs play a leading role. meetings and dialogue and set up websites on the consortium, the research and results. Specific Support Actions (5 actions) for training, Website addresses are given in the details of conferences or prospective studies in sup- each project and provide more and more up-to- port of the programme. date information than this publication.

Networks of Excellence (2 networks), whose objective is to reduce fragmentation in EU research, structure the way it is carried out and strengthen its excellence.

Area Number of projects EC financial contribution (million €) Regenerative Medicine 19 80.6 Gene therapy 19 77.4

Immunotherapy 21 107.8 and Transplantation

Total 59 265.8

Numbers of projects supported and EC financial contribution to new therapies research

It is clear from this compilation that new therapies is an extremely active area of research for European scientists both from the public and private sectors. Since the projects described here take a platform approach to tool and technology development applications are expected in a range of different practical settings. The recently launched Seventh Framework Programme and approval of the Regulation on advanced therapy medicinal products will act as a further stimulus for the area and open the way for much-needed clinical applications.

New Therapies – Introduction 13 14 New Therapies – Regenerative Medicine

16 New Therapies – Regenerative Medicine Regenerative Medicine EuroStemCell European Consortium for Stem Cell Research

Contract No LSHB-CT-2003-503005 Project type Integrated Project EC contribution e 11 900 000 Starting date 1 February 2004 Duration 48 months Website www.eurostemcell.org

Background and objectives:

The EuroStemCell consortium aims to harness the intrinsic potential of embryonic, foetal and adult stem cells for the continuous generation of specialised cell types. The ability to produce differentiated cell populations at scale will create new opportunities for the assignment of gene function, identification and validation of drug targets, pharmaceutical screening, toxicology testing, and, in the longer perspective, for the repair of diseased or damaged tissue by cell Cell examines the ethical and societal impacts of transplantation. stem cell research and exploitation. The consortium develops outreach resources to engage pa- EuroStemCell is an integrated trans-European tient groups, high school students and lay public effort to develop well-characterised cellular re- in the issues that surround stem cell research and sources of therapeutic potential, derived from medicine. embryonic, neural, myogenic, haematopoietic and epithelial stem cells. EuroStemCell applies Approach and methodology: genomic and transgenic technologies in conjunc- tion with state of the art in vitro and in vivo func- The fundamental research work of EuroStem- tional assays, to dissect the molecular machinery Cell is focused on stem cell identification, isola- that governs self-renewal versus differentiation. tion, and in vitro propagation and differentia- The consortium undertakes comparative mo- tion. This includes the generation and validation lecular and functional evaluations of stem cells of key tools, such as antibodies and genetically from embryonic, foetal and adult sources. Knowl- engineered cell lines and mice. Allied to this, the edge thus acquired is used to achieve scaleable consortium is collating microarray expression production of identified and characterised cell profiling data on different stem cells. This foun- lineages for in vitro gene and compound screen- dation work is accompanied by the evaluation of ing. These cell line resources are also subjected candidate stem cell resources for in vitro up-scal- to preclinical evaluation of tissue integration and ing and drug screening potential. Longer-term repair potential in animal models. studies will evaluate the survival and functional integration of transplanted cells in normal and Alongside basic and applied research, EuroStem- injured tissue.

New Therapies – Regenerative Medicine 17 EuroStemCell

es between partner laboratories funded, while 2 three-day meetings of the entire consortium (more than 100 researchers) were held, to promote interaction and collaboration between partner laboratories, and refine project planning.

Moreover, EuroStemCell arranged a major Eu- ropean stem cell conference series, ‘Advances in Stem Cell Research’. The first conference took place in Milan in 2005, with an attendance of over 300. The conference was successfully repeated in Lausanne in 2006, and will continue in Stockholm in 2007. The series attracts leading international speakers and participants from around the world, providing an important opportunity to highlight European stem cell research. The consortium also organised annual summer schools on “Stem Cells and Regenerative Medicine” to develop and The overall flow of the project can be illustrated train the next generation of European stem cell schematically, as shown below: scientists. Each school is attended by 54 pre-and A number of parallel activities have been de- post-doctoral students, including clinicians and signed to support, extend and maximise the im- regulators, and is open to non-members of Eu- pact of the project partners’ scientific research. In roStemCell. the first 3 years, EuroStemCell has carried out the following: A ‘Glossary for Stem Cell Biology’, was prepared: • it developed a prototype stem cell da- it was published in Nature and adopted by the tabase to encourage data exchange and EMBO Science & Society Programme in its report dissemination; on stem cell research. Web-based information • it encouraged junior group leaders to about stem cell research was made accessible to take a higher profile in the project by ac- a range of audiences at www.eurostemcell.org. cording Associate Principal Investigator EuroStemCell responded individually to hun- status to 8 individuals; dreds of requests for information from media, • it organised specialist workshops fo- patient groups and individuals, and it also issued cusing on disease areas, to bring stem 13 press releases, and attracted worldwide me- cell researchers and clinical practitioners dia coverage for key publications. together and develop a ‘roadmap to the clinic’; The project partners successfully produced an • it held a series of workshops on the award-winning animated film, A Stem Cell Story, ethical implications of stem cell research, about stem cell research. The film has been distrib- involving scientists, ethicists, legislators, uted online, via iTunes, through festival and public clinicians and patient representatives in screenings, and on DVD, and has been viewed by discussions, and making outcomes widely audiences totalling more than 10 000. Three ad- available via the group’s website. ditional short films to provide in-depth resources on ethics, cell culture and cloning have been There were 25 training and workgroup exchang- developed, in addition to a careers workshop for

18 New Therapies – Regenerative Medicine Regenerative Medicine

schoolchildren aged 13 and over, and a hands-on Main findings: activity programme for younger children. EuroStemCell initiated joint activities and re- The consortium has now published 56 scientific search projects with the BetaCellTherapy Inte- papers directly supported by EuroStemCell. Many grated project, supported by ‘Bridge’ funding of these studies have appeared in the leading from the Juvenile Diabetes Research Foundation peer-review journals including Nature, Science, International. Cell, PLoS Biology, Developmental Cell, Genes & Development, Development, Neuron and Jour- Expected outcome: nal of Neuroscience.

Over the four-year funding period, EuroStemCell In the area of basic stem cell biology, EuroStem- has been contributing to advances in techno- Cell investigators have published fundamental logical platforms, cellular resources and scientific observations, challenging dogmas about the knowledge in stem cell biology. This will provide origins and properties of myogenic, haematopoi- the basics for translational research. EuroStem- etic and hair follicle stem cells (Nature 431; Genes Cell is accordingly beginning to establish criteria Dev 19; Cell 121; Development 133; Nature Cell for preclinical validation and future clinical trials Biology 8). in the areas of neurological repair, muscle repair and epithelial repair. A major outcome of the Partners in Edinburgh, Milan and Bonn, working project will be a ‘Roadmap to the clinic’ for cell on stem cells for the nervous system have collab- therapy. orated, achieving for the first time expansion of pure populations of neural stem (NS) cells, with EuroStemCell research will also facilitate biop- the potential to generate the three cell types of harmaceutical exploitation of stem cells, in part the central nervous system (PLoS Biology 3; Cer- mediated via the SME partners. In this regard, Eu- ebral Cortex 16). roStemCell partners have, to date, filed 24 patent applications, several of which have been licensed Importantly, these neural stem cells can be ob- for exploitation by SME partners. EuroStemCell is tained both from embryonic stem (ES) cells and committed to developing ethical guidelines and from in vivo tissue sources. The consortium has promoting public engagement with stem cell re- also reported advances in directing both mouse search, with the aim of positively impacting pub- and human embryonic stem cells into the nerv- lic and political opinion. ous system lineage by manipulation of defined

New Therapies – Regenerative Medicine 19 EuroStemCell

signalling pathways (PLoS Biology 4). Other part- Tonlorenzi, R., Innocenzi, A., Mognol, P., Thibaud, ners are investigating the potential of resident J.L., Galvez, B.G., Barthélémy, I., Perani, L., Mantero, stem cells in the adult brain to be harnessed for S., Guttinger, M., Pansarasa, O., Rinaldi, C., Cusella repair (Neuron 52). De Angelis, M.G., Torrente, Y., Bordignon, C., Bot- tinelli R., Cossu, G., ‘Mesoangioblast stem cells In the muscle stem cell field, members of the con- ameliorate muscle function in dystrophic dogs’, sortium have demonstrated the high capacity of Nature 444, 574 – 579. freshly isolated muscle progenitor cells to repair damaged muscle (Science 309) and the potential Shinin, V., Gayraud-Morel, B., Gomès, D., Tajba- of expanded mesangioblast cells for tissue repair khsh, S., ‘Asymmetric division and cosegregation in a large animal model of muscular dystrophy of template DNA strands in adult muscle satellite (Nature 444). cells’, Nature Cell Biology 8, 677 – 682.

Partners in Lausanne and London are advancing Lowell, S., Benchoua, A., Heavey, B., Smith, A.G., in the development of a large animal model for ‘Notch Promotes Neural Lineage Entry By epidermal stem cell transplantation. Pluripotent Embryonic Stem Cells’, PLoS Biology 4(5): e121. SME partners are coordinating projects to improve culture conditions for the expansion of Gustafsson, M.V., Zheng, X., Pereira, T., Gradin, K., various stem cell types. Several refined media for- Jin S., Lundkvist, J., Ruas, J.L., Poellinger, L., Len- mulations have been commercialised, including dahl, U., Bondesson, M., ‘Hypoxia requires Notch ESGRO complete media for the growth of murine signaling to maintain the undifferentiated cell ES cells in serum free conditions, HEScGRO for the state’, Developmental Cell 9: 617-628. growth of human ES cells in serum and animal component free conditions, and N2B27 and RHB- Montarras, D., Morgan, J., Collins, C., Relaix, F., Zaf- A media for the differentiation of human and fran, S., Cumano, A., Partridge, T., Buckingham, M., mouse stem cells into cells of the neural lineage. ‘Direct Isolation of Satellite Cells for Skeletal Mus- The goal is to develop fully defined conditions cle Regeneration’, Science 309: 2064-2067 for stem cell propagation for future therapeutic grade production. Conti, L., Pollard, S.M., Gorba, T., Reitano, E., Toselli, M., Biella, G., Sun, Y., Sanzone, S., Ying, Q.L., Cat- The EuroStemCell ethics group has produced re- taneo, E., Smith, A., ‘Niche-independent symmet- ports on stem cell registers and stem cell bank- rical self-renewal of a mammalian tissue stem ing following a workshop arranged jointly with cell’, PLoS Biology 3(9): e283 the Commission, and on commercialisation of stem cell research. These reports can be viewed Adolfsson, J., Månsson, R., Buza-Vidas, N., Hultquist, at the following websites: A., Liuba, K., Jensen, C.T., Bryder, D., Yang, L., Borge, http://www.eurostemcell.org/Ethics/ethics_wrk- O.J., Thoren, L.A.M., Anderson, K., Sitnicka, E., Sa- shp2005.htm saki, Y., Sigvardsson, M., Jacobsen, S.E.W., ‘Identifi- http://www.eurostemcell.org/Ethics/ethics_wrk- cation of Flt3+ Lympho-Myeloid Stem Cells Lack- shp2006.htm ing Erythro-Megakaryocytic Potential: A Revised Road Map for Adult Blood Lineage Commitment’, Major publications Cell 121: 295-306.

Sampaolesi, M., Blot, S., D’Antona, G., Granger N.,

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Coordinator Elena Cattaneo and Luciano Conti Milano University Austin Smith Milan, Italy Wellcome Trust Centre for Stem Cell Research University of Cambridge Giulio Cossu Tennis Court Road Milano Hospital SCRI Cambridge CB2 1QR, UK Milan, Italy E-mail: [email protected] Anders Haegerstrand Partners NeuroNova AB Stockholm, Sweden Tim Allsopp Stem Cell Sciences UK Meng Li Cambridge, UK Imperial College London, UK Ernest Arenas, Jonas Frisen, Urban Lendhal Karolinska Institute John McCafferty Stockholm, Sweden Wellcome Trust Sanger Institute Cambridge, UK Yann Barrandon Swiss Federal Institute for Technology Claus Nerlov and Liliana Minichiello Lausanne, Switzerland EMBL-Monterotondo Rome, Italy Anders Bjorklund, Goran Hermeren , Sten Eirik Jacobsen, Olle Lindvall, Zaal Kokaia Lars Wahlberg Lund University NsGene A/S Lund, Sweden Ballerup, Denmark

Clare Blackburn Alexander Medvinsky, Simon Tomlinson, Fiona Watt Val Wilson, Brian Hendrich Cancer Research UK University of Edinburgh Cambridge, UK Institute for Stem Cell Research Edinburgh, UK Christian Dani CNRS Olivier Brustle and Frank Edenhofer Nice, France University of Bonn Medical Centre Bonn, Germany Tariq Enver Weatherall Institute of Molecular Medicine Margaret Buckingham, Ana Cumano, Jean-François Oxford, UK Nicolas, Shahragim Tajbakhsh, Benoît Robert Institut Pasteur Paris, France

New Therapies – Regenerative Medicine 21 GENOSTEM Adult mesenchymal stem cells engineering for connective tissue disorders: from the bench to the bedside

Contract No LSHB-CT-2003-53161 Project type Integrated Project EC contribution e 8 752 000 Starting date 1 January 2004 Duration 48 months Website www.genostem.org

Background and objectives: nective tissues, since they are the multipotential stem cells that give rise to skeletal cells (osteob- Regeneration takes place in the body through- lasts, chondrocytes and tenocytes), vascular cells out life. However, bone, cartilage, tendons, ves- (endothelial cells, pericytes and vascular smooth sels and cardiac muscle have a limited capacity muscle cells), sarcomeric muscle (skeletal and for self-repair, and after injury or disease the re- cardiac) and adipocytes. generative power of adult tissue is often not sufficient, leading to non-functional scaring. When Approach and methodology: organs or tissues are irreparably damaged, they may be replaced by an artificial device or donor GENOSTEM will purify and optimise cultures of organ. However, the number of available donor MSCs collected from bone marrow, skeletal mus- organs is critically limited. cles, fat pads, cord blood vasculature, placental and adult peripheral blood of humans, as well as Generation of tissue-engineered replacement rodents. By combining different cells sources, iso- organs by means of ex vivo culture of autolo- lating different cell subsets, and providing better gous organ-specific cells seeding a scaffold may accessibility of growth factors (using scaffolds, represent a suitable substitute for donor organ matrices, microcarriers coated with cytokines, implantation. It also prevents the risks of disease etc.), the final aim is to obtain cell populations transmission from donors, a common problem with different differentiation and proliferation in organ implantation. Tissue engineering using potentials: stem cells takes full advantage of the high pro- • undifferentiated MSCs where the full liferative capacity and the multi-potentiality of differentiation potential would be pre- these immature cells. The use of embryonic stem served; cells is limited because of ethical drawbacks, as • committed MSCs that would express, well as the allogeneic immune response, induc- in addition to mesenchymal factors, at ing rejection of the implants. The alternative is least one lineage-specific marker; the use of adult stem cells. • differentiated progeny blocked at a certain differentiation stage; The goal of GENOSTEM is to establish a European • fully differentiated progeny. international scientific leadership for stem cell regenerative medicine in the field of connective The consortium will also identify novel factors tissue disorders. Autologous adult Mesenchymal essential for MSC proliferation and differentia- Stem Cells (MSCs) are optimal candidates to serve tion using genomic and proteomic analysis, and as building blocks for the engineering of con- engineered cells in gain (transfer of genes) and

22 New Therapies – Regenerative Medicine Regenerative Medicine

loss (transfer of small interfering RNAs) of function studies.

To engineer MSCs (or differentiated progeny) with the appropriate scaffold to secure repair of the target tissue and to define optimal conditions for transplantation of MSCs in normal and pathological tissue, is another approach undertaken by GENOSTEM. Undifferentiated MSCs or MSC-differentiated progeny will be assessed in normal and in pathological animal models, to investigate the reparative potential of MSC strains. The project strategy will include the use of sophisticated cul- Figure 1: Stem cells injected in merinos sheep differenti- ture conditions, using biodegradable scaffolds, ating to cartilage, visualised in vivo with confocal celviso system (Photo Inserm) matrices and microcarriers binding soluble growth factors, inhibitors and adhesive glycoproteins and oped scaffolds with a DNA delivery system, and a vectors containing gene sequences adapted to specific microsphere able to deliver differentia- the pathological state under study. Gene transfer tion factors. will be carried out using original lentiviral vectors, non-viral DNA delivery complexes (polymersomes) Standard conditions for culturing adult stem cells or matrix-anchoring, DNA-binding peptides. from umbilical cords, adipose tissue and bone marrow have been developed. The cells were The project will define the best strategy for trans- cultured in vitro and infected with Adeno-Smad8 lating GENOSTEM research into phase I clinical tri- and BMP-2 at different ratios, or just with Adeno- als in bone defect, in partnership with SMEs and Smad8, and seeded on a Collagen sponge and regulatory bodies for the scaling-up of safe proce- implanted intramuscularly in NOD/SCID mice. dures. GENOSTEM will be taking advantage of the Tendon-like tissue was generated with the com- experience already acquired by one of the part- bination of Smad8 and BMP-2 expression. The ners, in clinical trials using cultured muscle cells. factors involved in chondrogenesis and the engineering of the cells were successfully identified. Main findings: Chitosan scaffolds combined with TGF-treated GENOSTEM partners have compared in vivo the ovine stem cells have been injected in merino different scaffolds with MSCs, including hydro- sheep and followed for 2 months (Fig 1). Results gel, chitosan, polymer, PAMs and ceramics. The obtained with this pertinent osteoarthritis model project partners developed procedures that have validated the protocol, and will allow clini- could combine cell attachment efficiency with cal development of the procedure. local growth factor-releasing capacity. Further- more, a large-scale in vivo model for cartilage Moreover, the consortium has focused on bone repair was developed: a chitosan-poly(butylene repair for the setting-up of clinical trials. For this, succinate) (PBS)(50/50%wt) blend was produced animal models of femoral bone defect have and processed into scaffolds, using melt spin- been initiated. The partners developed a cranial ning and fibre bonding. This scaffold was used in critical size defect assay in nude mice for in vivo the merino sheep model for cartilage repair with implantation. A femur critical size defect model a chondral defect. The partners have also devel- using a novel external fixation device is avail-

New Therapies – Regenerative Medicine 23 GENOSTEM

able in immunodeficient mice for studying bone stem cells in bone bioengineering repair. The analysis of the osteogenic potential • Hofmann A, Gross G. Tendon and liga- of various hMSCs implanted with HA/TCP (with ment engineering: From cell biology to in various %) and different materials (hydrogel/ce- vivo application ramic scaffold, chitosan) has been performed in • Mendez-Peruz M, Hughes C, Annenkov this heterotopic assay in vivo. For bone induction, A, Daly G, Chernajovsky Y. Engineering the partners identified IGF2 and IGF-BP2 through stem cells for therapy the microarray analyses. Treatment with IGF2 or/ • Vilquin JT, Rosset P. Mesenchymal stem and IGF-BP2 at the appropriate dosage, slightly cells in bone and cartilage repair: current increased ALP staining, suggesting osteoblastic status. differentiation. Snedeker, J.G., Pelled, G., Zilberman, Y., Gerhard, F., Large-scale GMP production of autologous MSC Muller, R., Gazit, D., ‘Endoscopic cellular microsco- for clinical purposes is available. py for in vivo biomechanical assessment of tendon function’, J Biomed Opt, 2006, 11(6):064010. Major publications Chernayovsky, Y., ‘Gene therapy for arthritis Hoffmann, A., Pelled, G., Turgeman, G., Eberle, P., - where do we stand?’ Arthritis Res Ther, 2005, Zilberman, Y., Shinar, H., Keinan Adamsky, K., Win- 7(6):227-9. kel, A., Shahab, S., Navon, G., Gross, G., Gazit, D., ‘Neotendon formation induced by manipulation Arnulf, B., Lecourt, S., Soulier, J., Ternaux, B., Lacas- of the Smad8 signalling pathway in mesenchy- sagne, M.N., Crinquette, A., Dessoly, J., Sciaini, mal stem cells’, J. Clin. Invest, 2006, 116(4):940-52. A.K., Benbunan, M., Chomienne, C., Fermand, J.P., Marolleau, J.P., Larghero, J., ‘Phenotypic and func- Review articles Special Focus: Mesenchymal Stem tional characterization of bone marrow mesen- Cells, Regenerative Medicine, 2006, 1 (4):517-604 chymal stem cells derived from patients with • Jorgensen C. Tissue engineering multiple myeloma’, Leukemia, 2007, 21(1):158-63. through mesenchymal stem cells: role of Epub 2006 Nov 9. the Genostem Consortium • Delorme B, Chateauvieux S, Charbord Kulbe, H., Thompson, R., Wilson, J.L., Robinson, S., P. The concept of mesenchymal stem cells. Hagemann, T., Fatah, R., Gould, D., Ayhan, A., Balk- • Roche S, Provansal M, Tiers L, Jorgensen will, F., ‘The inflammatory cytokine tumor necro- C, Lehmann S. Proteomics of primary mes- sis factor-alpha generates an autocrine tumor- enchymal stem cells promoting network in epithelial ovarian cancer • Srouji S, Kizhner T, Livne E. 3D scaffolds cells’, Cancer Res, 2007, 67(2):585-92. for bone marrow stem cell support in bone repair Bianco, P., Kuznetsov, S.A., Riminucci, M., Gehron • Djouad F, Mrugala D, Noël D, Jorgensen Robey, P., ‘Postnatal skeletal stem cells’, Methods C. Engineered mesenchymal stem cells for Enzymol, 2007, 419:117-48. cartilage repair • Marie PJ, Fromigué O. Osteogenic dif- Delorme, B., Charbord, P., ‘Culture and charac- ferentiation of human marrow-derived terisation of human bone marrow mesenchymal mesenchymal stem cells stem cells’, Methods in Molecular Medicine – Tissue • Kimelman N, Pelled G, Gazit Z, Gazit D. Engineering 2nd Edition. Edited by Hansjörg Haus- Applications of gene therapy and adult er and Martin Fussenegger. (in press)

24 New Therapies – Regenerative Medicine Regenerative Medicine

Coordinator Louis Casteilla CNRS- UMR 5018 Christian Jorgensen Toulouse, France INSERM Unit 475 Ralph Müller Hôpital Lapeyronie, Service Immuno-Rhumatologie ETH - Swiss Federal Institute of Technology 34000 Montpellier, France Institute for Biomedical Engineering E-mail : [email protected] Zürich, Switzerland

Partners Dan Gazit The Hebrew University of Jerusalem Pierre Marie Skeletal Biotechnology Lab INSERM - U349 Jerusalem, Israel Hôpital Lariboisiere Paris, France Gerhard Gross Helmholtz Zentrum für Infektionsforschung Jean-Thomas Vilquin Department of Gene Regulation and Differentiation INSERM – U582 Braunschweig, Germany Institut de MyologieGroupe Hospitalier Pitié-Salpêtrière Paris, France Paolo Bianco Università di Roma La Sapienza Claudia Montero-Menei Institute for Cell Biology and Tissue Engineering INSERM – U646 Rome, Italy Laboratoire de Vectorisation Particulaire Angers, France Virgil Paunescu University of Medicine and Pharmacy Victor Babes Pierre Charbord Department of Physiology and Immunology Université François Rabelais Timisoara, Romania Laboratoire d’Hématopoïèse, Faculté de Médecine Tours, France Robert Oostendorp Technische Universität München Yuti Chernajovsky Labor für Stammzellphysiologie University of London Munich, Germany Queen Mary and Westfield College Bone & Joint Research Unit, Barts and The London Ulrike Nuber London, UK Lund University Stem Cell Centre Antoinette Hatzfeld Lund, Sweden Centre National de la Recherche Scientifique (CNRS) UPR 9045 - Laboratoire de Biologie des Cellules Souches Erella Livne Humaines Technion - Israel Institute of Technology Villejuif, France Anatomy and Cell Biology Faculty of Medicine Technion Haifa, Israel

New Therapies – Regenerative Medicine 25 GENOSTEM

Thomas Häupl Christiane Dascher-Nadel Charité, Medical Faculty Inserm-Transfert SA Department of Rheumatology Department of European and International Affaires Berlin, Germany Marseille, France

Jerónimo Blanco Jeffrey Hubble Centro de Investigación Cardiovascular EPFL - Swiss Federal Institute of Technology Barcelona, Spain Institute for Biological Engineering and Biotechnology Lausanne, Switzerland Eleni Papadaki University of Crete Medical School Heraklion, Crete, Greece

Maria Kalmanti University of Crete Heraklion, Crete, Greece

Jean-Pierre Pujol Université de Caen Laboratoire de Biochimie du Tissu Conjonctif Caen, France

Nuno Neves University de Minho Department of Polymer Engineering Braga, Portugal

Jean-Pierre Mouscadet Abcys SA Paris, France

John Watson Kuros Biosurgery AG Zürich, Switzerland

Nathalie Rougier Biopredic International Rennes, France

Andres Crespo GENOPOIETIC Miribel, France

26 New Therapies – Regenerative Medicine Regenerative Medicine OsteoCord Bone from blood: Optimised isolation, characterisation and osteogenic induction of mesenchymal stem cells from umbilical cord blood

Contract No LSHB-CT-2005-018999 Project type Specific Targeted Research Project EC contribution e 2 500 000 Starting date 1 January 2006 Duration 36 months Website www.bonefromblood.org

Background and objectives: a manner similar to bone marrow-derived MSCs. The aim of this project is to optimise the isolation Stem cells are ‘blank’ cells that can replicate in- and expansion of MSCs from human UCB (CB- definitely, or given the right triggers, grow into MSCs). The differentiation capacity of CB-MSCs specialised cells for specific areas of the body. In will be examined, with a specific focus on osteo- adults, a few types of cell, such as blood, bone genesis, and we will tissue engineer 3-D bone re- marrow or nerves, are unable to replicate them- placement structures. selves by normal cell division. Within these parts of the body, a small quantity of stem cells is found, Approach and methodology: that is used to repair and replace damaged cells. The intrinsic ability of stem cells to self-repair OsteoCord is a nine-partner EC FP7 project with on demand has generated a significant level of representatives from academia and industry. The interest in determining how scientists might be CB-MSCs will be characterised using a range of able to exploit stem cells for the treatment of cer- techniques, including examination of the gene tain disease conditions. and protein expression profiles, before and after osteogenic differentiation, compared to MSCs There is an urgent clinical requirement for ap- isolated from human bone marrow, as well as propriate bone substitutes that are able to re- embryonic stem cells. The integrated datasets place current grafting procedures for the repair will allow the consortium to identify specific and/ of diseased or damaged bone. Mesenchymal or novel signalling pathways associated with CB- stem cells (MSCs) are found predominantly in MSCs, that will help them to understand CB-MSC the bone marrow and are able to differentiate biology and may help identify new targets for into osteogenic (bone), chondrogenic (cartilage), cell-based therapies. adipogenic (fat) and tenogenic (tendon) tissue types, thus offering considerable therapeutic Bioimpedance measurements of CB-MSCs in 2- potential for tissue engineering applications in D and 3-D growth configurations will be deter- orthopaedic surgery. mined, using purpose-built microchips for high- throughput characterisation. The immune status However, invasive extraction procedures and of CB-MSCs will also be determined. Allogeneic poor cell yields have demanded the identifica- transplantation of MSCs between different indi- tion of alternative tissue sources of MSCs. Grow- viduals may be possible, as MSCs appear to be ing evidence suggests that umbilical cord blood ’immune-privileged’, in that they are not neces- (UCB) contains a population of rare MSCs that are sarily rejected when implanted into unmatched able to give rise to many different cell types in recipients. It is important to point out that MSCs

New Therapies – Regenerative Medicine 27 OsteoCord

display immuno-suppressive characteristics, and Main findings: are able to reduce an immune response and promote the engraftment of different cell types, Since its launch, the OsteoCord project and all of such as skin cells and blood cells. Therefore, it is its work packages have gained ground. The ma- necessary to determine how CB-MSCs react un- jor achievements , are set out below: der different immune environments. • determined growth characteristics and CD antigen expression profile of CB-MSCs, To deliver sufficient cell numbers for viable ther- following recovery from cryopreservation; apies, key components of the project are focused • determined electrochemical imped- on expansion protocols. Comparative analyses ance measurements of BM-MSCs, using a of growth rates and ageing characteristics will planar electrode chip system; identify the lifespan of CB-MSCs in culture. Novel • developed BM-MSC spheroids for 3-D techniques will be combined with scale-up pro- electrode chip assays; cedures and the generation of CB-MSC lines for • optimisation of MSC alloreactivity as- banking, following optimised cryopreservation says in progress; protocols. Biocompatibility assays using a range • analysed dynamic microcarrier-assist- of bespoke, biomimetic scaffolds will be used to ed bioreactors for CB-MSC growth; develop tissue engineering applications. • established osteogenic differentiation protocols; An independent ethical evaluation of the work • identified candidate osteogenic scaf- will determine how the work has contributed folds and initiated biocompatibility studies; to the ethics of stem cell research and other is- • completed an extensive international sues such as cord blood banking. OsteoCord patents review, on the therapeutic appli- will also provide a social science assessment of cations of MSCs. the different expectations of MSCs held across the research community. The aim is to assist the research community in better understanding broader technical, regulatory, commercial and clinical factors in the future shaping of MSCs, and articulate, robust and empirically informed sce- narios for the exploitation of MSCs.

Expected outcome:

OsteoCord’s integrated approach using existing and complementary expertise will provide a timely and thorough evaluation of CB-MSCs, and define appropriate routes for their therapeutic implementation. The work ranges from fundamental biological understanding through to the delivery of CB-MSC-based therapies, under appropriate regulatory conditions.

28 New Therapies – Regenerative Medicine Regenerative Medicine

Coordinator Helder Cruz ECBio Paul Genever Cell biotechnologies University of York Oeiras, Portugal Biomedical Tissue Research Department of Biology (Area 9) Robin Quirk York, YO10 5YW, UK RegenTec Ltd E-mail: [email protected] Nottingham, UK

Partners

Moustapha Kassem University Hospital of Odense Department of Endocrinology Odense, Denmark

Jens Andersen University of Southern Denmark Department of Biochemistry and Molecular Biology Odense, Denmark

Hagen Thielecke Fraunhofer Institute for Biomedical Engineering Biohybrid Systems Department St Ingbert, Germany

Lee Buttery University of Nottingham Tissue Engineering Group School of Pharmacy Nottingham, UK

Karen Bieback Ruprecht-Karls-Universität Heidelberg Institute of Transfusion Medicine and Immunology Stem Cell Laboratory Mannheim, Germany

Trevor Walter Angel Biotechnology Ltd Northumberland, UK

New Therapies – Regenerative Medicine 29 Thercord Development and preclinical testing of cord blood-derived cell therapy products

Contract No LHSB-CT-2006-018817 Project type Specific Targeted Research Project EC contribution e 1 800 000 Starting date 1 May 2006 Duration 36 months Website www.thercord.eu

Background and objectives: MSCs, the greater the chances of a successful transplant and long-term engraftment. Testing of As scientists continue to discover new applica- the MSCs also confirmed certain characteristics tions for human stem cells, they are targeting that help induce tissue repair, like the develop- diseases with a significant need for more effica- ment of bone and cartilage. cious treatment options. Until recently, patients affected by acute renal failure (ARF) were treated CB MSCs were positive for CD44, CD105, CD90, by dialytic and pharmacological approaches HLA class I and negative for CD31, CD45, CD34, therapies, with very limited efficacy. Human cord HLA class II. Moreover, within the CB MSCs, the blood (CB) mesenchymal stem cell (MSCs) could consortium identified a high percentage of represent an interesting alternative source for CD146+/34-/45- cells, consistent with the perivas- therapeutic purposes in renal repair. cular/pericyte-like phenotype, a new stem cell subpopulation. In the migration assay, they were Approach and methodology: able to observe that these cells were capable of migrating and integrating into the damaged re- The Thercord consortium isolated MSCs from nal cell line. full-term umbilical CB to test their therapeutic potential on renal tissue in vitro and in vivo. These With the proteome assay, they analysed several cells were extensively characterised by flow cy- solubles, including Hepatocyte Growth Factor, tometry, and the differentiation assays were Vascular Endothelial Growth Factor, Fibroblast performed towards adipogenic, osteogenic and Growth Factor — factors well known to be in- chondrogenic lineages in order to confirm their volved in renal protection and differentiation. mesenchymal features. Moreover, it tested their They tested the CB MSCs in immunocompro- ability to migrate in the presence of in vitro dam- mised mice with acute renal failure, showing that age, and to produce soluble factors to promote these cells strongly protect the mice from renal renal repair. Their ability to promote renal repair function impairment. Kidneys were assessed when transplanted into NOD-SCID mice with functionally (blood urea nitrogen: BUN), as well acute renal failure was also assessed. as by histology, immunohistochemistry and Tunel assay, effectively showing lower levels of Main findings: BUN, reduced tubular changes and reduced apoptosis in animals treated with CB MSCs. The group successfully isolated MSCs from CB units at a rate of approximately 18%. What is clear Interestingly, inflammatory cytokines such as IL- is that the greater the percentage of extracted alpha, IL-beta and TNF beta were statistically re-

30 New Therapies – Regenerative Medicine Regenerative Medicine

duced in mice receiving CB MSCs, in comparison Coordinator to the control group. In conclusion, they demonstrated that CB MSCs produce soluble factors Lorenza Lazzari that play a critical role, providing a protective Fondazione Ospedale Maggiore Policlinico and reparative effect in acute renal failure. And Mangiagalli e Regina Elena in vivo CB MSCs exhibit reparative potential in Milan, Italy acute renal failure. E-mail: [email protected]

Partners

Maurizio Pesce Centro Cardiologico Monzino Milan, Italy

Dominique Bonnet Cancer Research UK London, UK

Anna Magdalene Wobus Insitut fur Pflanzengenetik und Kulturpflanzenforschung Gatersleben, Germany

Willem Fibbe Leiden University Medical Centre Leiden, Netherlands

Joan Garcia Barcelona Cord Blood Bank Barcelona, Spain

Marcela Contreras National Blood Service London, UK

Rita Maccario IRCCS Policlinico San Matteo di Pavia Pavia, Italy

New Therapies – Regenerative Medicine 31 EPISTEM Role of p63 and related pathways in epithelial stem cell proliferation and differentiation and in rare EEC-related syndromes

Contract No LSHB-CT-2005-019067 Project type Integrated Project EC contribution e 8 130 000 Starting date 1 January 2006 Duration 48 months Website www.epistem.eu

Background and objectives: Approach and methodology:

The focus of EPISTEM is on generating new EPISTEM is collecting and culturing keratinocytes knowledge and translating it into applications from EDS patients with p63 mutations, as well as that enhance human health. To this end, both carrying out an extensive analysis of phenotype- fundamental and applied research will be in- genotype correlation. These keratinocytes will volved. EPISTEM integrates multidisciplinary and be used to uncover the role of p63 proteins and coordinated efforts to understand the molecular pathways in normal and abnormal skin develop- basis of factors involved in epidermal stem cell ment. generation, maintenance and differentiation, as well as skin disease. Moreover, the core molecule The consortium is building relevant in vitro and in that will be studied in this Integrated Project (IP) vivo skin disease models for studying the role of is p63 (and related pathways), a molecule geneti- p63 in EDS disease, and the genetic assessment cally proven to be involved in the development of novel pathways discovered during this project. of rare skin diseases. These models will be used for drug assessment as well. It will also provide insight into the regu- Collectively, the prevalence of ectodermal dys- lation and involvement of p63 and related path- plasia syndromes (EDS) is estimated at 7 cases in ways in skin differentiation, the maintenance of 10 000 births. Currently, there is no cure for these the proliferative capacity of epithelial stem cells patients. By creating the EPISTEM consortium, and the transition of ectodermal cells to epider- the project partners will address from different mal stem cells. angles (genetics, gene profiling, molecular and cellular biology, structural biology, drug design, Screening for and design of novel therapeutic bioinformatics) the molecular pathways involved drugs, based on three dimensional p63 models, in epidermal dysplasia syndromes, making use of which will refold/reactivate or inhibit p63 mu- different technologies (mutation analysis, micro- tants and induce biological responses in relevant array, ChiP, transgenes, proteomics, in vitro skin disease models, is also key. cultures, crystallography, etc.). The consortium brings together leading European clinicians, ge- Expected outcome: neticists, molecular and cellular biologists, structural biologists, a drug designer and bioinfor- First of all, the EPISTEM consortium will gener- matics specialists in the field of p63 (and related ate thorough insight into the molecular biology molecules) research. of a rare disease such as EDS, for which no cure is currently available. Second, characterising and

32 New Therapies – Regenerative Medicine Regenerative Medicine

understanding how epidermal stem cell maintenance is regulated by p63 could be beneficial for the treatment of burn victims, since these stem cells could be used for tissue regeneration. Therefore, as currently estimated, the EPISTEM research proposal may have a far broader impact on clinical practice and for biomedical industry in the long run. Third, this integrated project will generate knowledge and technology that is applicable not only to p63 itself, but also to its family members p53 and p73; p53 being an important target that is mutated in most cancers and p73 being an important molecule for neurogenesis. Fourth, from a technological point of view, the EPISTEM proposal will invest in the development of chIP (chromatin immunoprecipitation) on chip Figure 1: Various combinations of ectodermal dysplasia, technology. orofacial clefting and limb malformations are the hallmark of p63-associated syndromes. EEC syndrome is the prototype of these syndromes and together with LMS shows all three Main findings: hallmarks. ADULT syndrome patients never show orofacial clefting, whereas AEC and RHS never show limb defects. Non-syndromic limb defect condition (SHFM4) and non-syn- The project partners have collected material dromic cleft lip/palate (NSCL) are also caused by mutations from 226 patients in 119 families, all with muta- in the p63 gene. tions in the p63 gene. Expression vectors suitable for transient expression in mammalian cells p63 functions as a negative regulator of p21 are available for 6 of the p63 isoforms (ΔN and TA expression in keratinocytes through an unex- combined with alpha, beta and gamma). Trans- pected and complex mechanism that involves a activation assays have been carried out for sev- reciprocal negative cross-talk between the p63 eral p63 mutations found in patients. EPISTEM and Notch signaling pathways. has generated two transgenic mice lines overexpressing TAp63α and two lines overexpress- In order to determine the role of caspase-14, the ing ΔNp63α under the K5 promoter p63 wild-type consortium generated caspase-14 deficient mice. background. The consortium recently developed They determined the NMR solution structure of a model of ectodermal/epidermal differentia- the SAM domain mutant (L518F) which causes tion from mouse embryonic stem (ES) cells, and the Hay-Wells syndrome. showed that BMP4 leads to the apoptosis of ES- derived neuroectodermal cells while inducting Initial experiments using Saos-2 cells expressing epidermal fate. exogenous temperature sensitive (ts) A167P mutant TAp63γ revealed that treatment with PRIMA- Target genes for specific p63 isoforms selectively 1 inhibited cell growth in a mutant p63-depend- expressed in proliferating and differentiating ent manner, as shown by the WST-1 proliferation keratinocytes have been identified. EPISTEM has assay. Two human p63 point mutants have been validated several p63 target genes identified by engineered, TAp63γ-R204W and TAp63γ-R304W. the gene array in vivo, using the transgenic mice Both have been cloned in the pcDNAHA vector and the TAp63 and/or DNp63 genetically com- and in the pTRE2pur vector, to allow regulation plemented mice. The partners have shown that by Tet (doxycyclin).

New Therapies – Regenerative Medicine 33 EPISTEM

Figure 2: Pathogenic p63 mutations cause at least five different syndromes and two non-syndromic conditions. Mutations causing different diseeases are illustrated in different colours. Only mutations that are discussed in the text are indicated, an overview of all mutations is given in Table 1. EEC hostpot mutations are clustered in DNA binding domain, and RHS and AEC syndrome mutations in SAM and TI domains. Several mutations, such as R280, R313, I510, S541 and 1850 Del A, can have a variable clinical outcome, probably due to genetic background effects. Tha black asterisks illustrate sites needed for upiquiti- nation (K193, K194 and PY) and the white asterisk represents a sumoylation site (fKXD/E).40,42,58

Several data sets involving p53, p63 and p73 have transcriptional up-regulation of the IGF-I gene’, J been collected and constructed. Furthermore, a Biol Chem, 2006, 281, 30463-30470. web tool was developed and is now available in the private section of the website http://tools. Ponassi, R., Terrinoni, A., Chikh, A., Rufini, A., Lena, epistem.eu. This tool makes it possible to remain A.M., Sayan, B.S., Melino, G., Candi, E., ‘p63 and p73, informed of all the datasets and the tools that are members of the p53 gene family, transactivate available. PKCdelta’, Biochem Pharmacol, ,2006, 72, 1417- 1422. Major publications Rinne, T., Hamel, B., van Bokhoven, H., Brunner, Candi, E., Rufini, A., Terrinoni, A., Dinsdale, D., H.G., ‘Pattern of p63 mutations and their phe- Ranalli, M., Paradisi, A., De Laurenzi, V., Spagnoli, notypes—update’, Am J Med Genet A, 2006, 140, L.G., Catani, M.V., Ramadan, S., Knight, R.A., Melino, 1396-1406. G., ‘Differential roles of p63 isoforms in epidermal development: selective genetic complementa- Testoni, B., Borrelli, S., Tenedini, E., Alotto, D., Cast- tion in p63 null mice’, Cell Death Differ, 2006, 13, agnoli, C., Piccolo, S., Tagliafico, E., Ferrari, S., Viga- 1037-1047. no, M.A., Mantovani, R., ‘Identification of new p63 targets in human keratinocytes’, Cell Cycle, 2006, Devgan, V., Nguyen, B.C., Oh, H., Dotto, G.P., 5, 2805-2811. ‘p21WAF1/Cip1 suppresses keratinocyte differentiation independently of the cell cycle through Vigano, M.A., Lamartine, J., Testoni, B., Merico,

34 New Therapies – Regenerative Medicine Regenerative Medicine

D., Alotto, D., Castagnoli, C., Robert, A., Candi, E., Coordinator Melino, G., Gidrol, X., Mantovani, R., ‘New p63 targets in keratinocytes identified by a genome- Peter Vandenabeele wide approach’, Embo J, 2006, 25, 5105-5116. Flanders Interuniversity Institute for Biotechnology VIB/UGent Ghent, Belgium E-mail: [email protected] or [email protected]

Partners

Hans van Bokhoven, Gijs Schaftenaar, Martijn Huynen Radboud University Nijmegen Nijmegen, Netherlands

Gerry Melino and Vincenzo De Laurenzi Università Degli Studi di Roma Tor Vergata Rome, Italy

Gian Paolo Dotto University of Lausanne Lausanne, Switzerland

John McGrath King’s College London London, UK

Klas Wiman Karolinska Institutet Stockholm, Sweden

Volker Doetsch J.W. Goethe-Universitat Frankfurt am main Frankfurt, Germany

Roberto Mantovani Università degli Studi di Milano Milan, Italy

Massimo Gulisano Istituto Oncologico del Mediterraneo Viagrande, Catania, Italy

New Therapies – Regenerative Medicine 35 EPISTEM

Daniel Aberdam INSERM Nice, France

GeneSpin SrL Milano, Italy

Piranit Kantaputra CMU Chiang Mai, Thailand

Jingde Zhu SJTU Shanghai, China

36 New Therapies – Regenerative Medicine Regenerative Medicine Ulcer Therapy Gene transfer in skin equivalents and stem cells: novel strategies for chronic ulcer repair and tissue regeneration

Contract No LSHB-CT-2005-512102 Project type Specific Targeted Research Project EC contribution e 2 391 953 Starting date 1 July 2005 Duration 36 months Website http://ulcertherapy.idi.it

Background and objectives: peutic efficacy in wound healing, of a combination of the two previous approaches, based on the Chronic skin ulceration is a frequent pathological generation and optimisation of skin equivalents, condition and represents a major health problem engineered ex vivo to over-express therapeutic for humans. In particular, cutaneous ulcers are a proteins. In parallel, the therapeutic potential in common complication of diabetes (due to mac- wound healing of endothelial progenitor cells rovascular and microvascular disease), and pe- (EPCs) and mesenchymal stem cells (MSCs), ge- ripheral neuropathy, associated with skin more netically modified ex vivo to express angiogenic vulnerable to traumatic injuries and growth fac- molecules or genes involved in stem cell survival tor expression reduction. Furthermore, chronic and proliferation, is under evaluation. ulcers are characterised by a tissue imbalance between proteolytic enzymes and their inhibi- Approach and methodology: tors, often leading to enhanced growth factor degradation. The project’s final goal will be achieved through the three main objectives set out below. Conventional therapeutic approaches (relief of pressure at the wound site, surgical debridement, 1. Understanding the role of growth factors, control of infection, and in selected cases, arterial proteases, and protease inhibitors in the patho- reconstruction) are often not enough to guaran- physiology of wound healing. To this end, a ba- tee adequate healing. Non-conventional tools for sic research approach is used to characterise the the treatment of chronic ulcers include topical role and mechanism of the action of potentially application of recombinant growth factors — di- therapeutic proteins. Transgenic and conditional rectly or mediated by viral vector gene delivery knockout mice are generated for studying the — and transplantation of skin equivalents. How- involvement of novel growth factors in wound ever, the short half-life of exogenous growth fac- healing. In parallel, the analysis of the proteo- tors, their degradation, or the inefficient delivery lytic degradation of growth factors, the charac- of genes to target cells, are major limitations for terisation of the involved proteolytic enzymes, the treatment with recombinant factors. Analo- and the examination of presence and activity of gously, the therapeutic effect of skin-equivalent tissue protease inhibitors is performed. The cD- grafting is limited, mostly due to infections and NA’s coding for the studied growth factors and an impaired production of granulation tissue by protease inhibitors are inserted into adenoviral the host. vectors that are used to deliver these proteins into wounds practised on mice. For this activity, The goal of Ulcer Therapy is to assess the thera- animal models characterised by impaired wound

New Therapies – Regenerative Medicine 37 Ulcer Therapy

Figure 1. Schematic representation of the in vivo model of “skin-humanised mice”. Human skin equivalents are generated from cultured primary keratinocytes and fibroblasts obtained from skin biopsies. Skin equivalents are then orthotopically transplanted onto the back of nude mice. Figure 2. EGFP-bioengineered “skin-humanised mice”. Human cultured primary keratinocytes transfected with viral vectors carrying EGFP are used to generate skin equivalents healing are used. Most of the study is performed that are grafted onto nude mice. Epifluorescence on diabetic animals. microphotographs show the EGFP-positive epithelium, corresponding to the human grafted skin, surrounded by mouse skin. 2. Evaluating the therapeutic potential of genetically modified stem cells in impaired wound healing. Circulating endothelial precursor cells gene transfer efficacy into keratinocytes and skin (EPCs) and mesenchymal stem cells (MSCs) se- equivalents of adeno and adeno-associated viral crete angiogenic growth factors, and can dif- vectors, the nerve growth factor (NGF) and the ferentiate into endothelial cells, thus improving best candidates from the genes analysed in the angiogenesis. The capacity of isolated EPCs and course of this project, are employed for further MSCs per se, or infected with adenoviral vectors in vitro and in vivo preclinical studies using skin carrying angiogenic molecules or genes involved grafts on immunodeficient mice. in stem cell survival and proliferation, injected at the wound margin in mouse model of impaired Expected outcome: wound healing (NOD/SCID or diabetic mice), is therefore investigated. In parallel, a broad analy- Ulcer Therapy anticipates the following outcome: sis is conducted to identify the most suitable ad- • characterisation of the role and mech- enoviral and adeno-associated viral vectors for anism of the action of potentially thera- gene transfer into these stem cell types. peutic proteins in wound healing; • characterisation of the proteolytic en- 3. Assessing the safety and efficacy of geneti- zyme and the tissue protease inhibitor cally modified skin equivalents as a delivery sys- function in the ulcer environment; tem for therapeutic proteins in wound healing. • identification of the most suitable ade- To this end, quality controls, safety and efficacy noviral and adeno-associated viral vectors preclinical studies in vitro and on animal models for gene transfer into keratinocytes and are performed. Fibrin is used as an optimal sub- stem cells (EPCs and MSCs); strate to reconstitute genetically modified com- • assessment of the quality, safety and posite skin grafts for the proper delivery of thera- potential therapeutic effects of geneti- peutic factors. After a comparative analysis of the cally-modified skin equivalents;

38 New Therapies – Regenerative Medicine Regenerative Medicine

• assessment of the safety and efficacy different lineages, were chosen for as- in ulcer therapy of stem cells per se or in sessing the efficacy in wound healing of genetically modified form; MSCs. Preliminary data indicated a role • development of standard operating in promoting wound healing of AT-MSCs procedures for the ex vivo genetic modi- topically administered into wounds of fication of skin equivalents and their pro- diabetic mice. duction and testing; • Of the different viral vectors tested, • design of a protocol for a phase I trial first generation Ad5 vectors proved to be of gene therapy in humans affected by the most efficient in transfecting keratino- chronic diabetic ulcers. cytes, while serotype-2 adeno-associated vectors were able to efficiently transduce Main findings: genes into EPCs. • Quality control, safety and efficacy The main results obtained during the first period studies on cultured keratinocytes and skin of the Ulcer Therapy project are set out below: equivalents transfected with an adenovi- • The analysis of transgenic and knock- ral vector containing the NGF cDNA (Ad5- out mice indicated that increased ex- NGF-EGF) indicated that high transgene pression of the angiogenic factor PlGF or expression is achieved without any rele- activin or the soluble IGF-I isoform at the vant negative effect. Moreover, the analy- wound site, exerts a beneficial effect in sis of wound healing in skin equivalents wound healing. PlGF activity has also been transduced with a viral vector contain- investigated in diabetic mice and proved ing the keratinocyte growth factor (KGF) to accelerate healing by promoting ang- cDNA and grafted onto nude mice, indi- iogenesis. Importantly, adeno-mediated cated that KGF overexpression was able PlGF gene transfer to diabetic wounds to enhance closure. This data indicates was able to significantly accelerate heal- that humanised mice are a suitable model ing, approximating the wound closure for testing the effect of selected factors in time of healthy controls. Different aspects vivo. of the repair process were improved following PlGF gene transfer. Major publications • The analysis of growth factor degradation, protease and protease inhibitors Cianfarani, L., Zambruno, G., Brogelli, L., Sera, F., in the ulcer environment revealed that in Lacal, P.M., Pesce, M., Capogrossi, M.C., Failla, C.M., chronic wounds PlGF and HGF, as already Napolitano, M., Odorisio, T., ‘Placenta growth fac- described for VEGF, are targets of proteo- tor in diabetic wound healing: altered expression lytic cleavage that attenuates their activi- and therapeutic potential’, Am J Pathol, 2006, 169: ties. Site-directed mutagenesis protected 1167-1182. VEGF from proteolytic degradation and enhanced its therapeutic activity in dia- Roth, D., Piekarek, M., Christ, H., Paulsson, M., Bloch, betic wound healing. W., Krieg, T., Davidson, J., Eming, S.A., ‘Plasmin • Adipose tissue-derived MSCs (AT- modulates VEGF-A mediated angiogenesis dur- MSCs), which are easier to obtain and ing wound repair’, Am J Pathol, 2006, 168:670-684. propagate in culture compared to bone marrow-derived ones (BM-MSCs), and Carretero, M., Escàmez, M.J., Prada, F., Mirones, I., show capacity to differentiate towards Garcìa, M., Holguìn, A., Duarte, B., Podhajcer, O.,

New Therapies – Regenerative Medicine 39 Ulcer Therapy

Jorcano, J.L., Larcher, F., Del Rìo, M., ‘Skin gene Coordinator therapy for acquired and inherited disorders’, His- tol Histopathol, 2006, 21: 1233-1247. Giovanna Zambruno Istituto Dermopatico dell’Immacolata, IDI-IRCCS Eming, S.A., Krieg, T., ‘Molecular mechanisms of Lab. Biologia Molecolare e Cellulare VEGF-A action during tissue repair’, J Investig Der- Via dei Monti di Creta, 104 matol Symp Proc, 2006, 11: 79-86. 00167 Rome, Italy E-mail: [email protected] Eming, S.A., Smola-Hess, S., Kurschat, P., Hirche, D., Krieg, T., Smola, H., ‘A novel property of povidon- Partners iodine: inhibition of excessive protease levels in chronic non-healing wounds’, J Invest Dermatol, Maurizio Pesce 2006, 126: 2731-2733. Centro Cardiologico Monzino Milan, Italy Perabo, L., Goldnau, D., White, K., Endell, J., Boucas, J., Humme, S., Work, L.M., Janicki, H., Hallek, M., Sabine Werner Baker, A.H., Buening, H., ‘Heparan sulfate prote- Swiss Federal Institute of Technology oglycan binding properties of adeno-associated Zurich, Switzerland virus retargeting mutants and consequences for their in vivo tropism’, J Virol, 2006, 80: 7265-7269. Fernando Larcher Centro Investigaciones Energeticas, Medioambientales y Eming, S.A., Werner, S., Bugnon, P., Wickenahuser, Tecnologicas C., Siewe, L., Utermöhlen, O., Davidson, J., Krieg, T., Madrid, Spain Roers, A., ‘Accelerated wound closure in mice deficient for interleukin-10’, Am J Pathol, 2007, 170: Michael Hallek 188-202. Clinic I of Internal Medicine University of Cologne Cologne, Germany

Sabine Eming University of Cologne Department of Dermatology Cologne, Germany

Nadia Rosenthal European Molecular Biology Laboratory Monterotondo, Rome, Italy

Elena Dellambra IDI Farmaceutici S.p.A Pomezia, Italy

40 New Therapies – Regenerative Medicine Regenerative Medicine Skintherapy Gene therapy for edidermolysis bullosa: a model system for treatment of inherited skin diseases

Contract No LHSB-CT-2005-512073 Project type Specific Targeted Research Project EC contribution e 2 079 000 Starting date 15 April 2005 Duration 36 months Website www.debra-international.org/researche1a.htm

Background and objectives: premature death. In addition to physical suffering, patients are faced with social difficulties: Epidermolysis Bullosa (EB) is a rare genetic skin they cannot perform physical activities, and they disease, affecting approximately 30 000 individu- need permanent assistance and regular medical als in Europe, and approximately 400 000 to 500 treatments. EB patients are also excluded from 000 people worldwide. The patients are predom- the workforce, not only owing to their physical inantly children, and there is no treatment avail- appearance, but also because the public is unin- able yet. formed and unaware of the condition.

EB is characterised by an extreme fragility of the Approach and methodology: skin, resulting in unremitting blisters and ero- sions with unceasing wound healing. The skin Skintherapy will recruit patients, and perform di- lesions stem from the poor adhesion of the epi- agnoses and genetic analyses of DEB patients. A dermis to the underlying mesenchyme, which total of 120 patients have already been recruited. makes the skin vulnerable to damage caused by The consortium will develop viral vectors (on- mild friction and trauma. The genetic bases of the coretroviral, lentiviral, and hybrid adeno/AAV different clinical forms of EB have been elucidat- vectors) to integrate and firmly express the col- ed, and a precise correlation has now been made lagen VII cDNA in DEB epidermal stem cells ex between genetic defects of the basement mem- vivo; work is in progress to define the gene trans- brane components, and the types of EB which fer efficiency into clonogenic keratinocyte stem have been classified within the Simplex (EBS), cells. Skin equivalents made with genetically cor- Junctional (JEB), and Dystrophic (DEB) forms. rected DEB cells to assess the full morphological and functional reversion of the DEB phenotype In DEB, generalised blisters heal with scarring. will be constructed. Skintherapy will graft the Extensive and progressive mutilating scarring re- skin equivalents expressing the recombinant sults in increased disability and deformity of the collagen VII on appropriate immune competent fingers, as well as in contractures of the joints. The animal models. The consortium is already graft- disease may also affect other areas of the body ing and carrying out an analysis of the fate of the (mouth, throat, eyes), causing discomfort, and in transplanted recombinant skin equivalents in most of the cases, difficulties in performing vital additional dogs. activities, like eating. The project partners will develop a technology The most severe cases are often complicated by for the production and downstream processing the development of aggressive skin cancer and of clinical-grade viral vectors preparations, under

New Therapies – Regenerative Medicine 41 Skintherapy

Good Manufacturing and Laboratory Practices Major publications (GMP/GLP) standards. Different cell lines are being evaluated to obtain the best transduction Mavilio, F., Pellegrini, G., Ferrari, S., Di Nunzio, F., Di rate and lentiviral production. Iorio, E., Recchia, A., Maruggi, G., Ferrari, G., Provasi, E., Bonini, C., Capurro, S., Conti, A., Magnoni, C., Gi- Expected outcome: annetti, A., De Luca, M., ‘Correction of junctional epidermolysis bullosa by transplantation of ge- Skintherapy’s immediate goal is to develop a netically modified epidermal stem cells’,Nat Med, gene therapy approach to DEB and design ap- 2006, Dec;12(12):1397-402. Epub 2006 Nov 19. propriate phase I/II trials. The expected result is a model system for DEB treatment using ex vivo Carretero, M., Escámez, M.J., Prada, F., Mirones, I., gene therapy. Specifically, the project aims at García, M., Holguín, A., Duarte, B., Podhajcer, O., developing a gene therapy technology model Jorcano, J.L., Larcher, F., Del Río, M., ‘Skin gene based on autologous transplantation of skin therapy for acquired and inherited disorders’, made in vitro, using genetically modified epider- Histol Histopathol, 2006, Nov;21(11):1233-47. Re- mal stem cells. view.

This model can be extended to other genetic skin diseases requiring local or systemic delivery of active molecules. The model system particularly targets the development of significantly improved, efficient and safe delivery systems, which are urgently needed.

Main findings:

The feasibility of a phase I/II gene therapy trial has been evaluated on a JEB patient. Epidermal stem cells isolated from a patient affected by JEB were transduced using a retroviral vector, to make engineered epithelial grafts that were transplanted back onto the patient. Synthesis, proper assembly and long-term expression of the recombinant laminin 5, together with the development of a firmly adherent epidermis with no blisters and/or infection episodes, were observed for the duration of the follow-up (i.e. 12 months). The results will be used to establish guidelines for efficient preclinical and clinical trials for gene therapy of epidermal cells, and also for the development of a similar trial for RDEB and DDEB patients.

42 New Therapies – Regenerative Medicine Regenerative Medicine

Coordinator

Guerrino Meneguzzi INSERM Unité 634 27 Avenue Valombrose 06107 Nice, Cedex 02, France E-mail: [email protected]

Partners

Michele De Luca Centro Regionale di Recerca Sulle Cellule Staminali Epiteliali Fondazione Banca Degli Occhi del Veneto Venice, Italy

Fulvio Mavilio Università di Modena e Reggio Emilia Department of Biomedical Sciences Modena, Italy

Leena Bruckner-Tuderman University Hospital Freiburg Department of Dermatology Freiburg, Germany

Marcela Del Rio Centro de Investigaciones Energeticas Medioambiantales y Technologicas Department of Epithelial Damage, Repair and Tissue Engineering Madrid, Spain

Anna Stornaiuolo Molmed S.p.A Discovery Division Milan, Italy

John Dart DEBRA Europe Crowthorne, UK

Karine Baudin INSERM-TRANSFERT SA Paris, France

New Therapies – Regenerative Medicine 43 THERAPEUSKIN Ex vivo gene therapy for recessive dystrophic epidermolysis bullosa: preclinical and clinical studies Contract No LHSB-CT-2005-511974 Project type Specific Targeted Research Project EC contribution e 1 530 000 Starting date 26 June 2005 Duration 36 months Website www.lyon.inserm.fr/therapeuskin /download.htm

Background and objectives:

Individuals with recessive dystrophic epidermolysis bullosa (RDEB) suffer from life-long severe skin and mucosal blistering followed by scarring. The disorder causes considerable morbidity, as well as premature mortality associated with an increased risk of squamous cell carcinomas. RDEB is caused by loss-of-function mutations in the type VII collagen gene (COL7A1) encoding the major structural component of anchoring fibrils at the junction between the epidermis and the dermis. No specific treatment is currently available. The objective of the THERAPEUSKIN project Figure 1. General overview of the ex vivo gene therapy ap- is to prepare an ex vivo gene therapy approach proach for RDEB using SIN-COL7A1 retroviral vectors. by transplantation of genetically modified skin equivalents. of the SIN-pEF1alpha-COL7A1 vector (prepared from the packaging cell line). The SINpCOL7A1- Approach and methodology: COL7A1 vector was subsequently used to genetically correct primary RDEB keratinocytes The THERAPEUSKIN project partners have devel- and fibroblasts ex vivo, which were then used to oped safe [Self INactivating (SIN)] amphotropic prepare skin equivalents (SE). The SE were made MFG-derived retroviral vectors in which COL7A1 of a human plasma-derived fibrin gel incorporat- expression is driven by the tissue specific COL7A1 ing genetically corrected RDEB fibroblasts, onto or the ubiquitous EF1alpha promoter. The MFG which genetically corrected epidermal cells were vector was modified due to the large size (8.9 kb) grown. of the COL7A1 cDNA. Viruses were produced by tri-transfection (pCOL7A1-COL7A1 and pEF1a- Three months after grafting the genetically cor- COL7A1), or by a packaging cell line (InsertAgene rected SE onto nude mice, skin biopsies showed pEF1alpha-COL7A1). a fully differentiated epithelium and no signs of blisters, as well as positive, linear type VII col- Good transduction efficiencies were obtained lagen immunostaining at the dermal-epidermal with one batch of the SINpCOL7A1-COL7A1 vec- junction, as well as ultrastructural evidence of tor (prepared by tri-transfection) and batches anchoring fibril formation. Assessment of the

44 New Therapies – Regenerative Medicine Regenerative Medicine

graft after six months demonstrated the trans- Main findings: duction of a proportion of epidermal stem cells and fibroblasts sufficient for the self-renewal of The COL7A1-SIN vectors the partners have de- the genetically corrected graft. These grafting veloped have a strong therapeutic potential for experiments are currently being repeated using clinical application: they have no selection mark- the SIN-pEF1alpha-COL7A1 vector produced by ers; they are self-inactivating (reducing the risk the packaging cell line. of oncogenic events); they provide a human promoter driving COL7A1 expression; and they can Expected outcome: be used in an autologous SE system. A cohort of individuals expressing some type VII collagen Reproduction of these results with the SIN-pE- protein will then be selected for the grafting of F1alpha-COL7A1 vector will lead to the produc- genetically corrected skin equivalents onto lim- tion of a master cell bank suitable for the prepa- ited skin areas. ration of clinical grade GMP vector production.

Figure 2/ A-F. In vivo restoration of type VII expression and epidermal adherence in RDEB reconstructed skin 12 weeks post-grafting, using COL7A1 retroviral vectors Histological analysis (A,C,E) and type VII collagen immunostaining (B,D,F) at three months post-grafting of skin equivalents (SE) made of normal keratinocytes (NK) and fibroblasts (NF) (A,B), Krdeb and Frdeb (C,D), and genetically corrected Krdeb and Frdeb (E,F) with COL7A1 vector. Type VII collagen accumulates along the dermal-epidermal junction in normal SE and in genetically corrected SEs, while it is completely absent in SE made of uncorrected RDEB cells. Note the dermo-epidermal cleft () in the SE made of uncorrected RDEB cells (C,D), while no blistering is seen in genetically corrected SE (E-F).

New Therapies – Regenerative Medicine 45 THERAPEUSKIN

Coordinator Patricia Joseph-Mathieu INSERM Transfert Alain Hovnanian Paris, France INSERM U563 Purpan Hospital Department of Genetics 31300 Toulouse, France E-mail: [email protected]

Partners

Maria Antonietta Zanta-Boussif Généthon Evry, France

Olivier Danos INSERM U781 Necker Hospital Paris, France

Yann Barrandon EPFL/CHUV Lausanne, Switzerland

Irene Leigh QMUL London, UK

John McGrath KCL St Thomas Hospital London, UK

Christina Bodemer Necker Hospital Department of Dermatology Paris, France

Luis Moroder Max Planck Institute of Biochemistry Martinsried, Germany

John Dart DebRA Europe Crowthorne, UK

46 New Therapies – Regenerative Medicine Regenerative Medicine BetaCellTherapy Beta Cell Programming for Treatment of Diabetes

Contract No LSHB-CT-2005-512145 Project type Integrated Project EC contribution e 12 000 000 Starting date 1 April 2005 Duration 60 months Website www.betacelltherapy.org

Background and objectives: and to regenerate them in vivo. The central unit, reference centres and bio-industry function as Diabetes is a prevalent chronic disease that re- translation components towards clinical imple- duces quality of life and increases the risks of life- mentation. A multicentre team of clinicians carry threatening complications. Its onset in younger out clinical trials on the prevention and treat- patients is caused by a massive loss of insulin- ment of diabetes. Two trials are currently being producing beta cells. Regenerating a functional conducted: beta cell mass is thus a major goal, both in bio- • antibody intervention in recent-onset medicine and for society. Beta cell grafts pre- type 1 diabetes; pared from human pancreases can cure the dis- • beta cell transplant trial in advanced ease, but this form of cell therapy is hindered by diabetes. a shortage of donor organs. The Centre develops new diagnostics and thera- The BetaCellTherapy consortium, with leading peutics for these trials. It defines quality control teams in molecular, developmental and func- and safety criteria as a guide to preclinical test- tional biology, is conducting an integrated pro- ing. It also provides training, and interacts with gramme to generate insulin-producing beta cells the scientific and medical community. The Cen- in therapeutic quantities. This FP6-supported tre informs patients and the public of progress programme is carried out with the R&D platform and perspectives in the field of its activities and of the JDRF (Juvenile Diabetes Research Founda- objectives. It has also established a collaborative tion) Centre for Beta Cell Therapy in Diabetes. The link to the EuroStemCell consortium, another 6th Centre organises transfer of knowledge to asso- FP integrated project ciated bio-industry and multicentre clinical trials, as well as to the general public. It is an interna- Nature’s biological programme for develop- tional consortium composed of an R&D platform, ing and preserving a functional beta cell mass a multicentre clinical trial team and associated throughout life, is taken as a platform for direct- facilities, bio-industry and reference centres; its ing strategies towards the laboratory produc- central unit consists of a coordination core and tion of a therapeutic beta cell mass (Fig 2). Beta a beta cell bank. cells will be derived from embryonic stem cells and from transdifferentiating liver, intestinal and The R&D platform explores the normal biologi- pancreatic exocrine cells. cal processes that preserve and maintain an adequate beta cell mass, and applies this knowledge Functional genomics will be used to compare to produce functional beta cells in the laboratory phenotypes of beta cells from new sources with

New Therapies – Regenerative Medicine 47 BetaCellTherapy

Embryonic Stem Cells • Project III: Programming a therapeutic beta cell mass.

Endodermal Stem Cells These projects are based on knowledge on the embryonic development of beta cells; findings demonstrating plasticity of somatic cells of endodermal origin, suggesting their potential H epatic Pancreatic Intestinal transdifferentiation to beta cells; and analysis of Progenitor C ells key mechanisms that generate and maintain a functional beta cell mass. Duct/Acinar C ells Expected outcome:

Beta Cells BetaCellTherapy expects the following outcomes: • to use the physiological programming Functional Beta Cell Mass of the pancreatic beta cell mass as template for generating beta cells with therapeutic potential in the laboratory; Therapeutic Beta C ell Mass • to collect comparative data on the efficacy and safety of beta cell generation by for C linical T rials of B eta C ell T herapy in Diabetes embryonic stem cell and transdifferentiation strategies; Fig 2 - Plan of integrated programme. The numerals refer to • to increase the availability of beta cell the three projects. grafts for transplantation in diabetic patients; those isolated from the pancreas. This analy- • to define and disseminate quality con- sis will direct further research, and determine trol criteria for a standardised composi- the start of preclinical testing. It will also gener- tion of therapeutic beta cell grafts in clini- ate new tools and quality control criteria that cal protocols; will allow the standardisation of ongoing trials • to transfer knowledge for bioindustrial and adjustments in graft biology, to increase its and clinical implementation; long-term survival and function in patients. This • to establish a scientific and technolog- project should help develop a cure for diabetes ic platform for adjusting graft biology so by (re)programming cells for beta cell therapy. as to optimise its long-term survival and function in patients; Approach and methodology: • to organise a training programme on the consortium’s state of the art. The overall objectives are addressed by three interactive projects with the following specific Main findings: objectives: • Project I: Programming stem cells to- Major achievements are presented below: wards beta cells; • Project II: Transdifferentiation of endo- Project I. Programming stem cells towards beta dermal cells to beta cells; cells:

48 New Therapies – Regenerative Medicine Regenerative Medicine

• identification of steps in the transcrip- Major publications: tion factor cascade during embryonic formation of pancreatic cells: HNF1ß > HNF 6 Duvillie, B., Attali, M., Bounacer, A., Ravassard, P., > Pdx 1 > Nkx 6.1; Basmaciogullari, A., Scharfmann, R., ‘The Mesen- • identification of Ngn 3 targets with en- chyme Controls the Timing of Pancreatic Beta- docrinogenic properties: MyT1 and IA1; Cell Differentiation’, Diabetes, 2006, 55(3):582-9. • the role of FGF4, FGF10 and retinoic acid in driving endoderm to pancreas; Pedersen, J.K., Nelson, S.B., Jørgensen, M.C., • development of an in vitro model that Henseleit, K.D., Fujitani, Y., Wright, C.V.E., Sander, recapitulates the steps of beta cell forma- M., Serup, P., ‘Endodermal expression of Nkx6 tion in rat embryonic pancreas; genes depends differentially on Pdx1’, Develop- • proof-of-concept that hES cells can be mental Biology, 2005, 288(2):487-501 induced into beta-like cells. Brolen, G.K., Heins, N., Edsbagge, J., Semb, H., ‘Sig- Project II. Transdifferentiating endodermal cells nals from the embryonic mouse pancreas induce towards beta cells: differentiation of human embryonic stem cells • development of culture media for de- into insulin-producing beta-cell-like cells’, Diabe- riving beta-like cells from exocrine pan- tes, 2005, 54(10):2867-74. creas; • transdifferentiation of beta-like cells Poll, A.V., Pierreux, C.E., Lokmane, L., Haumaitre, C., from human foetal liver cells; Achouri, Y., Jacquemin, P., Rousseau, G.G., Cereghi- • discovery of insulin-expressing cells in ni, S., Lemaigre, F.P., ‘A vHNF1/TCF2-HNF6 cascade the biliary duct system; regulates the transcription factor network that • transcriptome of pancreatic duct cells controls generation of pancreatic precursor cells’, driven to neuroendocrine phenotype fol- Diabetes, 2006, 55(1):61-9. lowing ngn3-transfection. Baeyens, L., De Breuck, S., Lardon, J., Mfopou, J.K., Project III. Programming therapeutic beta cell Rooman, I., Bouwens, L., ‘In vitro generation of in- mass: sulin-producing beta cells from adult exocrine • assay developed to measure beta cell pancreatic cells’, Diabetologia, 2005, 48(1): 49-57. death in vitro and in vivo; • transcriptome of adult rat and human Mellitzer, G., Bonné, S., Luco, R.F., Van De Casteele, beta cells, and use to detect two novel M., Lenne, N., Collombat, P., Mansouri, A., Lee, J., beta cell, as well as specific markers, and to Lan, M., Pipeleers, D., Nielsen, F.C., Ferrer, J., Grad- identify beta-cell specific genes in ngn3- wohl, G., Heimberg, H., ‘IA1 is Ngn3-dependent transdifferentiated human duct cells; and essential for differentiation of the endocrine • regulation of phenotype at transla- pancreas’, Embo J, 2006; 25(6):1344-52. tional level; • development and phenotyping mice Zalzman, M., Anker-Kitai, L., Efrat, S., ‘Differentia- with Irs2-deletion in pancreatic endocrine tion of human liver-derived insulin-producing or beta cells; cells towards the beta-cell phenotype’, Diabetes, • In vitro model for screening neofor- 2005, 54(9):2568-75. med cells for susceptibility to cytokines. Bogdani, M., Suenens, K., Bock, T., Pipeleers- Marichal, M., In’t Veld, P., Pipeleers, D., ‘Growth and

New Therapies – Regenerative Medicine 49 BetaCellTherapy

Functional Maturation of {beta}-Cells in Implants Coordinator of Endocrine Cells Purified From Prenatal Porcine Pancreas’, Diabetes, 2005, 54(12):3387-94. Daniel Pipeleers Vrije Universiteit Brussel Casellas, A., Salavert, A., Agudo, J, Ayuso, E., Jimen- Diabetes Research Centre ez, V., Moya, M., Munoz, S., Franckhauser, S., Bosch, Laarbeeklaan 103 F., ‘Expression of IGF-I in pancreatic islets prevents 1090 Brussels, Belgium lymphocytic infiltration and protects mice from E-mail: [email protected] type 1 diabetes’, Diabetes, 2006, 55(12):3246-55. Partners Jackerott, M., Moldrup, A., Thams, P., Galsgaard, E.D., Knudsen, J., Lee, Y.C., Nielsen, J.H., ‘STAT5 ac- Daniel Pipeleers, Harry Heimberg, Luc Bouwens tivity in pancreatic beta-cells influences the se- Vrije Universiteit Brussel verity of diabetes in animal models of type 1 and Brussels, Belgium 2 diabetes’, Diabetes, 2006, 55(10):2705-12. Frédéric Lemaigre Christian de Duve Institute of Cellular Pathology Brussels, Belgium

Bernard Peers Université de Liège Liege, Belgium

Jens H. Nielsen University of Copenhagen Copenhagen, Denmark

Shimon Efrat Tel Aviv University Tel Aviv, Israel

Fatima Bosch Universitat Autònoma de Barcelona Barcelona, Spain

Henrik Semb Lund University Lund, Sweden

Pedro Herrera University of Geneva Geneva, Switzerland

50 New Therapies – Regenerative Medicine Regenerative Medicine

Jonathan Slack Petter Björquist University of Bath Cellartis AB Bath, UK Göteborg, Sweden

Dominic Withers Finn C. Nielsen University College London University of Copenhagen Hospital Rigshospitalet London, UK Copenhagen, Denmark

Christel Hendrieckx Zhidong Ling JDRF Centre for Beta Cell Therapy in Diabetes Academic Hospital VUB Brussels, Belgium Brussels, Belgium

Philippe Ravassard Yuval Dor Centre National de la Recherche Scientifique The Hebrew University Paris, France Hadassah Medical School Jerusalem, Israel Raphael Scharfmann and Gérard Gradwohl INSERM Paris, France

Jorge Ferrrer Institut d’Investigacions Biomediques A. Pi I Sunyer Barcelona, Spain

Anne Grapin-Botton Swiss Institute for Experimental Cancer Research Lausanne, Switzerland

Sue Swift Beta-Cell NV Brussels, Belgium

Luc Schoonjans Thromb-X NV Louvain, Belgium

Ole Madsen and Palle Serup Novo Nordisk A/S Gentofte, Denmark

Thomas Mandrup-Poulsen Steno Diabetes Centre Gentofte, Denmark

New Therapies – Regenerative Medicine 51 EuroSTEC European programme on Soft Tissue engineering for Children

Contract No LSHC-CT-2006-037409 Project type Integrated Project EC contribution e 7 828 500 Starting date 1 January 2007 Duration 60 months Website www.eurostec.eu

Background and objectives: Ethical and regulatory issues will be fully addressed before final clinical application, and par- The aim of the EuroSTEC project is to use modern ents and children will have to be able to under- tissue engineering approaches to treat children stand these new treatment options. A dialogue with structural disorders present at birth, such with the general public, including patient’s asso- as spina bifida, urogenital defects, gastroschisis, ciations, will be sought. Demonstration activities diaphragmatic hernia and esophageal atresia. will be carried out to increase the awareness of The project strives to take a translational route new treatment modalities based on tissue engi- through in vitro and animal experiments to early neering. Finally, surgeons will be trained to use clinical trials. Tailor-made ‘smart’ biomatrices the new operation techniques. (scaffolds) will be prepared, using natural scaf- The project combines European leaders in the fold molecules (collagen, elastin) and/or man- field of biomatrices, cell culture, animal models, made polymers (polylactic/glycolic acid), and will surgery, and ethical and regulatory issues. be substituted with regulatory molecules such as growth factors and glycosaminoglycans. Approach and methodology:

A variety of cells, including stem cells, fibroblasts, The EuroSTEC project has 5 research areas that muscle cells and urothelial/epithelial cells will be cover different aspects of soft tissue engineer- cultured in vitro and seeded into biomatrices. For ing for congenital birth defects in children. The major congenital birth defects, biomatrices, with first research area focuses on ‘biomatrices for tis- or without cells, will be prepared and implanted sue engineering’. Each tissue has its own unique using novel animal models, and evaluated for set and content of scaffold biomolecules. By their capacity to regenerate the correct tissues. selectively incorporating biologically active Biomatrices will degrade in time, and then be re- molecules, such as morphogens into tissue-en- placed by the bodies’ own tissues, thus assuring gineering constructs, cellular behaviour may be compliance with growth which is especially im- fine-tuned. The synthesis of biodegradable sup- portant in young children. Prenatal and postnatal port polymers can be achieved using approved reconstructive procedures will improve the final monomers, following established procedures in outcome of reconstructive surgery. Clinical trials the laboratory. In this project, the aim is to de- for diaphragmatic hernias will form the start of velop polymers that can carry a mechanical load the patient registry and protocol development for periods spanning 3 to 6 months, and that for future clinical studies. are processed to form suturable compliant supports for the above bioactive extracellular matrix (ECM).

52 New Therapies – Regenerative Medicine Regenerative Medicine

Figure 1: Flowchart showing the interactions of the dif- In research area 2, ‘cell culture systems and bi- ferent research areas (RAs) and management overview of the EuroSTEC project omatrix interactions’, in vitro cell culture systems, cell lines and human cell culture systems are applied to study epithelial and skin tissues, muscle phragmatic Hernia (sheep, rabbit) and oesopha- tissues, urogenital, esophagus and amniotic cells gus atresia (sheep). In foetal models and clinical and tissues. Interactions between vascular sup- invasive procedures the disruption of amniotic ply and biomatrices have been investigated for membranes plays an important role in second- extracellular matrices. ary morbidity. Now, various cell lines with different characteristics are available for study. This allows compara- Currently, children with severe structural anoma- tive analyses of scaffolds of different composi- lies such as spina bifida, bladder extrophy, gastro- tion to construct cell-matrix implants. schisis, congenital diaphragmatic hernia (CDH), and oesophageal atresia need extensive surgical Several of the university research centres have procedures with long-term additional morbid- developed different animal models for severe ity. Most reconstructions consist of local skin flap congenital structural anomalies which can be coverage of the defects, local tissue growth of the used for research area 3, ‘models for severe con- organ after closure of the defects, or excision of genital anomalies’. These models can be used to dysplastic tissues and additional reconstructions. study new treatment strategies, including tis- Scar tissue, dysplastic and ischemia can prevent sue engineering techniques. Currently, models a good functional outcome and may result in for foetal or postnatal investigations are avail- long-term complications. In EuroSTEC, ‘Clinical able for spina bifida (sheep), skin and epithelial treatments, pilot studies and instruments’ are investigations (rat), urogenital reconstructions part of research area 4, which includes a study of (rabbit, sheep), Gastroschisis and Congenital dia- intervention for CDH.

New Therapies – Regenerative Medicine 53 EuroSTEC

Figure 2: Graphical presentation of the different research In the last few decades, many European countries areas and the partners involved in the work. have had an ongoing debate about the following question: to what extent is it desirable and tal structural anomalies needing life-long medi- morally acceptable to treat these children with cal care. The involvement of industrial partners all kinds of technological means? This issue will will improve the technical aspects, offer larger be further studied in research area 5, ‘ethical and production facilities and a distribution network, clinical registry and study management.’ as well as quality control systems. The new ‘smart’ biomaterials will be distributed by European Expected outcome: companies in the field of tissue engineering, with worldwide applications both for children EuroSTEC forms a strategic combination of basic with different congenital structural anomalies molecular, cellular and tissue structure scientific and also for markets in the field of biomedical knowledge with clinical paediatric patient care devices involving all children’s hospitals. and surgical experience, as well as the development of new treatment strategies. The project The project should lead to a database with dis- combines scientific information and cooperation ease-specific information concerning children between different individual scientific groups with severe structural anomalies that can be cor- working in the field of tissue engineering in Eu- related with environmental epidemiological and rope. Technically, this will lead to new products genomic databases for further elucidation of which can be evaluated in a preclinical setting underling disease courses. The development of for applications in children with severe congeni- this specific patient registration and study proto-

54 New Therapies – Regenerative Medicine Regenerative Medicine

cols will enable the start of phase II studies (and Coordinator phase II/III studies). Wouter Feitz EuroSTEC will also lead to socio-ethical discus- Radboud University Nijmegen Medical Centre sions and protocol development concerning Department of Urology (659) the use of new biomaterials in children and the PO Box 9101 ethical aspects involved. The inventory of the dif- 6500 HB Nijmegen, Netherlands ferent ethical aspects and diverse social implica- E-mail: [email protected] tions in various European countries, in addition to the evaluation and analysis of this information, Partners will lead to applicable protocols in all involved clinical settings, and solutions for regulatory Jöns Hilborn problems before the start of clinical trials. Uppsala University Polymer Chemistry, Dept. of Materials Chemistry Uppsala, Sweden

Peter Frey Centre Hospitalier Universitaire Vaudois Dept. of Paediatric Urology and Surgery Lausanne, Switzerland

Jan Deprest Katholieke Universiteit Leuven Department of Gynaecology and Obstetrics Leuven, Belgium

Gerard Barki Karl Storz GmbH & Co.KG Tuttlingen, Germany

Martin Meuli University Children’s Hospital Zurich Department of Surgery Zurich, Switzerland

Roland Zimmermann University Hospital Zurich Department of Obstetrics Zurich, Switzerland

Amulya Saxena Medizinische Universität Graz Department of Paediatric Surgery Graz, Austria

New Therapies – Regenerative Medicine 55 EuroSTEC

Wim Witjes CuraTrial SMO & Research BV Arnhem, Netherlands

Noes de Vries European Medical Contract Manufacturing (E.M.C.M.) B.V. Nijmegen, Netherlands

Benjamin Herbage Symatese Biomateriaux Chaponost, France

Paul van den Berg University Medical Centre Groningen Department of Obstetrics and Gynaecology Groningen, Netherlands

Eduard Gratacos Institute d’Investigacions Biomediques August Pi I Sunyer Foetal Medicine + Therapy Research Group Barcelona, Spain

Kypros Nicolaides Foetal Medicine Foundation / King’s College Hospital Harris Birthright, Foetal Medicine Unit London, UK

Ingo Heschel Matricel GmbH Herzogenrath, Germany

56 New Therapies – Regenerative Medicine Regenerative Medicine SC&CR Application and process optimisation of human stem cells for myocardium repair

Contract No LHSB-CT-2004-502988 Project type Specific Targeted Research Project EC contribution e 1 954 200 Starting date 1 February 2004 Duration 48 months Website www.SC-CR.eu

Background and objectives: peutic potential of these cells. In general, there are several prerequisites to the validation of Sudden untreated occlusion of a coronary artery, large-scale use of stem cells for therapeutics, following plaque rupture and/or vasospasm, may namely: result in extensive necrosis of the myocardium, • identifying the most suitable stem cell i.e. a transmural myocardial infarction. Because type(s) to be used, to repair/regenerate a remaining cardiomyocytes are not able to repli- given organ; cate, the loss of cardiomyocytes after infarction is • knowing the combination of stimuli irreversible, and the tissue defect heals by fibrotic that might drive differentiation of stem scarring after eight weeks. Moreover, post-infarc- cells toward a specific lineage; tion geometric disarrangement of the myocytes • identifying genes important for stem and extracellular matrix, dilation of ventricular cell conversion, as well as setting up safe cavity and compensatory hypertrophy of the re- and effective vectors and protocols to maining myocardium, a process defined through induce cardiac myocyte differentiation ventricular remodelling, may contribute to devel- pathway(s) by gene transfer; opment of ventricular dysfunction. • assessing the functional properties of Autologous transplantation of bone marrow stem cell-derived differentiated cells; stem cells into the infarcted myocardium is an in- • unravelling the action of factors that novative and promising strategy for the therapy might be involved in recruitment and of heart failure due to ischemic heart disease. The homing of endogenous stem cell in tis- SC&CR project proposes a strongly integrated ap- sues where regeneration is needed; proach to verify human stem cells’ safety and ef- • evaluating the mid- and long-term ficacy for the treatment of ischemic myocardium, effects of stem cell administration in pa- and to identify interventions that can be rapidly tients, after having performed exhaustive translated to clinical practice, in order to prevent preliminary analysis of stem cell use in or reduce damage to ischemic tissue. animal preclinical models.

Approach and methodology: Expected outcome:

Despite the number of observations report- It is expected that autologous HSC transplanta- ing “transdifferentiation” of tissue-derived stem tion into the infarcted or chronically ischemic cells, there is no conclusive evidence on the myocardium may be highly effective in wound mechanism(s) underlying changes in stem cell repair, in terms of heart muscle regeneration and fate, and insufficient information on the thera- improvement of coronary blood flow. The con-

New Therapies – Regenerative Medicine 57 SC&CR

sortium notes that while the approach described bryonic stem cells. The clinical use of HSCs in the in SC&CR — i.e. pharmacological mobilisation, treatment of severe forms of ischemic heart dis- collection by means of apheresis and intra-myo- ease may represent an additional or alternative cardial or intra-coronary injection of stem cells therapeutic option that may beneficially modify under direct vision — has not been used by oth- the unfavourable course of the disease. HSCs er groups, it offers several advantages compared transplantation may be combined with con- to other methods such as the collection of stem ventional therapies in order to add further ben- cells from the bone marrow by direct aspiration, efit. Repopulating areas of muscle loss, restoring and from the skeletal muscle by biopsy. ¬— at least partly ¬— the systolic and diastolic properties of the left ventricle, and stimulating Firstly, it is painless, it does not require general the growth of new blood vessels may reduce the anaesthesia and it does not imply any blood loss. high incidence of mortality and complications This is a major advantage because it reduces the associated with heart disease. Both a significant overall procedural risk in certain subsets of pa- improvement in the quality of life of the patients, tients, like those affected by heart failure and re- and a decrease in the social-economic burden of fractory angina pectoris. Thus far, no major com- ischemic heart disease are expected. plication has emerged following the clinical use of the granulocyte colony stimulating factor, in Main findings: patients affected by ischemic heart disease. During the first reporting period of this project, There is a great deal of clinical experience of HSC work was carried out in order to understand mobilisation and collection by apheresis in the molecular events associated with cardiac myo- haematological field. This technique is adopted cyte differentiation of stem cells using cell cul- even in healthy donors without significant com- ture, physiology, genetic transfer, preclinical and plications. Additionally, HSCs do not require any clinical approaches. This work has involved the ex vivo pharmacological manipulation or culture development of individual activities sustained in order to modulate their differentiation poten- by a high intra-network dissemination of results, tial. Moreover, the number of collected cells is and the sharing of tools and common platforms. significantly higher than after bone marrow aspi- Work has been carried out in four areas: ration (60-80 x106 vs. 10-30 x106 BMSC). Clinical Activities: In this part of the project, two The intra-myocardial injection under direct vi- independent phase I trials of autologous bone sion, during a CABG procedure, allows precise an- marrow-derived stem cells, in patients suffering atomic identification of the target area, and even chronic ischemic heart disease are being carried of the distribution of the injections. By contrast, out. the intravenous route is limited by the first-pass attenuation effect on the BMSC into the pulmo- Physiologic assessment of stem cell differentia- nary circulation, and by the fact that the myo- tion: Differentiation of stem cells in the myocar- cardium receives only 3-5% of the whole cardiac dium was assessed morphologically in several output. Consequently, the number of stem cells studies in different preclinical models. However, populating an infarcted or ischemic area may be the extent of functional integration into the my- significantly reduced. ocardium is still a matter of debate. In this part of the project, the consortium targeted clarifying Finally, the use of autologous adult BMSC does at a functional level the differentiation of several not imply any ethical issues, in contrast with em- stem cell types, using cell culture techniques.

58 New Therapies – Regenerative Medicine Regenerative Medicine

In vivo preclinical studies: The consortium’s goal Brugh, S.A., Zahanich, I., Rüschenschmidt, C., Bekc, was to assess the effect of factors and genes po- H., Blyszczuk, P., Czyz, J., Heubach, J.F., Ravens, U., tentially involved in the recruitment, homing, dif- Horstmann, O., St-Onge, L., Braun, T., Brustle, O., ferentiation and proliferation of stem cells in the Boheler, K.R., Wobus, A.M., ‘Signals from embry- infarcted heart. onic fibroblasts induce adult intestinal epithelial cells to form nestin-positive cells with prolifera- In vitro studies: A number of groups belonging tion and multineage differentiation capacity in to the SC&CR consortium sought to analyse the vitro’, Stem Cells, 24:2085-2097. differentiation of stem cells by cell culture, gene manipulation, genomic analysis and biopolymers De Boer, T.P., Van der Heyden, M.A.G., Rook, embedding. M.B., Wilders, R., Broekstra, R., Kok, B., Vos, M.A., De Bakker, J.M.T., Van Veen, T.A.B., ‘Pro-arrhyth- Major publications mogenic potential of immature cardiomyocytes is triggered by low coupling and cluster size’, Car- Minetti, G.C., Colussi, C., Adami, R., Serra, C., diovasc Res, 2006, 71:704-714. Mozzetta, C., Parente, V., Fortuni, S., Straino, S., Sampaolesi, M., Di Padova, M., Illi, B., Gallinari, P., De Boer, T.P., Van Veen, T.A.B., Bierhuizen, M.F.A., Steinkuhler, C., Capogrossi, M.C., Sartorelli, V., Bot- Kok, B., Rook, M.B., Boonen, K.J.M., Vos, M.A., Do- tinelli, R., Gaetano, C., Pure, P.L., ‘Functional and evendans, P.A., De Bakker, J.M.T., Van der Heyden, morphological recovery of dystrophic muscles M.A.G., ‘Connexin 43 repression following epithe- in mice treated with deacetylase inhibitors’, Nat lium-to-mesenchyme transition in embryonal Med, 2006, 12:1147-1150. carcinoma cells requires Snail 1 transcription factor’, Differentiation, 2007, Mar;75(3):208-18. D’Arcangelo, D., Ambrosino, V., Giannuzzo, M., Gaetano, C., Capogrossi, M.C., ‘Axl receptor activation mediates laminar shear stress anti-apoptotic effects in human endothelial cells’, Cardiovasc Res, 2006, 71:754-63.

Lagostena, L., Avitabile, D., De Falco, E., Orlandi, A., Grassi, F., Iachininoto, M.G., Ragone, G., Fucile, S., Pompilio, G., Eusebi, F., ‘Electrophysiological properties of mouse bone marrow c-kit cells co- cultured onto neonatal cardiac myocytes’, Cardio- vascr Res, 2005, 66:482-492.

Nikolova, T., Wu, M., Brumbarov, L., Alt, R., Opitz, H., Boheler, Cross M., Wobus, A.M., ‘WNT-condi- tioned media differentially affect the proliferation and differentiation of cord blood-derived CD133+cells in vitro’, Differentiation, 2007, 74 Feb;75(2):100-11.

Wiese, C., Rolletscheck, A., Kania, G., Navarrete- Santos, A., Anisimov, S.V., Steinfarz, B., Tarasov, K.V.,

New Therapies – Regenerative Medicine 59 SC&CR

Coordinator Michael Hallek University of Cologne Clinical Department Maurizio C. Capogrossi Cologne, Germany Istituto Dermopatico dell’Immacolata – IDI – IRCCS Laboratorio di Patologia Vascolare Via dei Monti di Creta 104 – 00167 Rome, Italy E-mail: [email protected]

Partners

Maurizio Pesce Centro Cardiologico Monzino Milan, Italy

Antonio Zaza Università degli Studi Milano-Bicocca Milan, Italy

Aldona Dembinska-Kiec The Jagiellonian University Krakow, Poland

Anna Magdalena Wobus Institute of Plant Genetics and Crop Plant Research Gatersleben, Germany

Carsten Werner Institut für Polymerforschung Dresden e.V. Dresden, Germany

Jacques De Bakker Interuniversity Cardiology Institute of the Netherlands Utrecht, Netherlands

Nadia Rosenthal European Molecular Biology Laboratory Monterotondo, Rome, Italy

Maria Luisa Nolli Areta International Gerenzano, Varese, Italy

60 New Therapies – Regenerative Medicine Regenerative Medicine StemStroke Towards a stem cell therapy for stroke

Contract No LHSB-CT-2006-037526 Project type STREP EC contribution e 2 475 508 Starting date 1 January 2007 Duration 36 months Website www.stemstroke.eu

Background and objectives: tial, and many aspects related to basic biological properties of NSCs, and their possible application Stroke is a major cause of long-term disability as therapeutic tools, still has to be established. in humans, and there is a lack of effective treat- The limited capacity of NSCs to be expanded in ments. Stroke affects about 3.5 million people vitro, and the enormous need to develop repro- in the EU, with 700 000 new cases every year. ducible technology to obtain a stable and suffi- Although most stroke survivors spontaneously cient number of NSCs as a source for cell therapy recover to some degree, more than half of stroke in neurodegenerative diseases, makes it impor- patients suffer significant residual impairments, tant to characterise NSCs obtained from different creating enormous economic and societal bur- sources, and to explore their capacity to survive dens. For example, Sweden reports an incidence and integrate into the damaged brain. of 213 first-ever strokes per 100 000 individuals, indicating that the total excess direct and indi- A novel mechanism for neuronal replacement rect cost of stroke would be €1.5 billion, with al- after stroke, i.e., the formation of new neurons most 45% of the direct costs attributed to social from the adult brain’s own NSCs, was demon- services. strated recently. New neurons are generated in the subventricular zone and migrate to the The extension of the average human lifespan has striatum, where they differentiate into mature resulted in a steady increase in the incidence of neurons with the characteristics of those which stroke among the expanding elderly population, have died. This potential self-repair mechanism creating a serious hazard to quality of life and has raised a lot of interest in both the scientific also increasing health care costs. From this per- and clinical communities. Moreover, recent data spective, all efforts to bring the latest advances of indicate that stroke-induced neurogenesis is science and technology a step closer to the de- long-lasting and that endogenous NSCs in the velopment of therapies against stroke, is of great adult brain produce new mature neurons over importance. several months following a stroke. The long-lasting neurogenesis occurs concomitantly with the The possibility to isolate and propagate neural spontaneous recovery of motor function, which stem cells (NSCs) with self-renewal and multipo- is observed over several months in both animals tential properties from the foetal and adult nerv- and humans affected with stroke. Whether there ous system, as well as embryonic stem (ES) cells is any causal relationship between striatal neuro- and their potential applications in cell therapy, genesis and behavioural recovery after stroke is have attracted a lot of research interest in recent as yet unknown. years. This field is just starting to reveal its poten-

New Therapies – Regenerative Medicine 61 StemStroke

cognitive deficits. Finally, the transplantation- and endogenous neurogenesis-based strategies will be optimised in animal models of stroke, and with close collaboration between basic scientists and clinicians, a preclinical protocol for stem cell application in stroke will be developed.

Expected outcome:

By providing a virtually unlimited source of different types of neurons and glia, this stem cell Strategies for stem cell therapy in stroke patients ex- technology may become the scientific break- plored by StemStroke through that will render cell replacement a useful treatment strategy for stroke patients. The main aim of StemStroke is to develop novel strategies to repair the brain and restore function Involving top European scientists with comple- after stroke based on the transplantation of NSCs mentary expertise and one SME, StemStroke, or their derivatives, or stimulation of the produc- using NSCs to repair the stroke-damaged brain, tion of new neurons from the adult brain’s own can make this research state-of-the-art through NSCs. unique and diverse means: • by demonstrating the capacity of stem Approach and methodology: cells of various sources to expand ex vivo and to differentiate into specific neuronal Two main strategies to generate neurons for re- phenotypes; placement in the stroke-damaged brain will be • by identifying tools and molecules that used in StemStroke. In the first strategy, NSCs iso- can stimulate endogenous neurogenesis lated from human ES cells, or from human foetal based on detailed knowledge about its or adult brain tissue, are expanded in culture, ge- regulatory mechanisms; netically modified (if needed) and subsequently • by determining whether neurons gen- transplanted into the recipient subjected to erated from NSCs can be anatomically and stroke. In the second strategy, neurogenesis from functionally integrated into the stroke- endogenous NSCs is stimulated using tools de- damaged brain; veloped in the project, leading to the increased • by developing MRI-based imaging for survival, migration or maturation of newly monitoring the survival, migration and dif- formed neurons. ferentiation of endogenous, and grafted NSCs and their progeny, as well as the al- The morphological and functional integration of teration of the lesion in response to stem grafted and endogenously generated NSCs and cell therapy; their progeny in the stroke-damaged brain, will • by showing the level of functional re- be assessed using immunocytochemistry, ana- covery after stroke that can be induced by tomical tracing techniques and patch-clamp elec- grafted and endogenous NSCs; trophysiology. A new in vivo MR-based imaging • by developing the first, scientifically- and behavioural test battery, developed within based preclinical protocol for the applica- the proposed project, will be used to assess stem tion of stem cells in stroke patients. cell function and recovery of sensory, motor and

62 New Therapies – Regenerative Medicine Regenerative Medicine

Coordinator

Zaal Kokaia University Hospital Lund Strategic Research Centre for Stem Cell Biology and Cell Therapy Laboratory of Neural Stem Cell Biology and Neuro- science Programme, BMC B10 SE-221 84 Lund Sweden E-mail: [email protected]

Partners

Austin Smith University of Cambridge Wellcome Trust Centre for Stem Cell Research Cambridge, UK

Mathias Hoehn Max-Planck-Institute for Neurological Research In-vivo-NMR Laboratory Cologne, Germany

Liliana Minichiello European Molecular Biology Laboratory Mouse Biology Unit Monterotondo, Rome, Italy

Stephen B. Dunnett Cardiff University Brain Repair Group at School of Biosciences Cardiff, UK

Lilian Wikström NeuroNova AB Stockholm, Sweden

New Therapies – Regenerative Medicine 63 STEMS Pre-clinical evaluation of stem cell therapy in stroke

Contract No LHSB-CT-2006-037328 Project type Specific Targeted Research Project EC contribution e 2 400 000 Starting date 1 December 2006 Duration 36 months Website http://www.stemsproject.eu

Background and objectives: ment at long-term post-stroke delays. STEMS aims specifically at determining the extent and The use of stem cells with multipotent properties limits of SC therapy in stroke, in order to pave the has become a challenging area of research for way for clinical therapeutic trials. most clinical fields. It is of particular importance in disciplines that desperately lack treatment op- Approach and methodology: tions, such as brain disorders and lesions. In this context, stroke or ischemic cerebrovascular dis- To define the therapeutic potential of SC in stroke, ease is an important target. Stroke accounts for a multidisciplinary study from human stem cell roughly half of the patients hospitalised for neu- culture to the analysis of functional recovery in rological diseases, and is associated with a large animal models of stroke will be implemented. proportion of the healthcare costs in Europe. The main objectives are to define the following: • the most efficient cell source, by a Until now, all neuroprotective approaches that comparison of embryonic and adult neu- have yielded positive results in animal models ral stem cells; of stroke have proved ineffective in clinical trials. • the ideal differentiation stage for Given their expected capacity to self-renew and transplantation; differentiate efficiently into the desired cell type, • the window of opportunity for trans- clonal populations of stem cells (SC) promise to plantation; produce beneficial effects in a number of diseas- • the set of behavioural tests and images. Several studies already indicate that SC trans- ing parameters to improve animal model plantation has therapeutic potential for stroke, predictiveness of functional recovery. using embryonic, foetal and adult SC sources, and lines derived from teratoma. Expected outcome:

However, a great deal of crucial information must By addressing a fundamental roadblock in the be found before SC transplantation becomes a development pathway for stroke therapy, STEMS clinical reality. For instance, the standardisation will make available a series of validated methods, of the conditions to regulate SC proliferation and models and analytical tools that will greatly en- differentiation so as to produce region-specific hance the capability of consortium members, as grafts needs to be better defined; and changes in well as stimulate the development of new ave- the properties of SC, induced by transplantation nues of research for stroke therapy. The validated into lesioned brain structures, are poorly under- methods include a culture of specific hESC and stood, as is the full extent of functional improve- hANSC cell lines, up-scaling and shipping con-

64 New Therapies – Regenerative Medicine Regenerative Medicine

ditions for both cell types, and transplantation protocols in the stroke-injured adult brain. The validated models will include development of a nonhuman primate model of stroke, allowing an evaluation of higher cognitive functions. The validated analytical tools will include homogenised guidelines for functional evaluation among the groups, and the development of new MRI sequences for studies in monkeys.

STEMS will provide the Community with a unique set of quantified data in stroke research, new standards, and the proof of concept of a new therapeutic approach for stroke. It is the consorti- Figure A Nestin-positive cells from hESC-derived progeni- um’s belief that the results will encourage groups tors transplanted into a rat model of stroke indicate differentiation into neural cell types (picture Inserm). of researchers to cooperate, and stimulate industrial interest for the field, so as to rapidly regenerate European forces on stroke experimental and clinical research.

Firgure B Co-localisation of Nestin (red) and human nuclear marker (green) identifies the cells as transplanted ones (yellow indicates the co-localisation (picture Inserm).

Figure C Magnetic resonance imaging of the brain lesion induced by 2 hours occlusion of the middle cerebral artery in rats (picture Inserm/CEA)

New Therapies – Regenerative Medicine 65 STEMS

Coordinator Eva Sykova Institute of Experimental Medicine Brigitte Onteniente Academy of Sciences of the Czech Republic INSERM Unit 549 Prague, Czech Republic Neurobiologie de la Croissance et de la Sénescence 2 Ter, rue d’Alésia Christiane Dascher-Nadel 75014 Paris, France Inserm Transfert SA E-mail: [email protected] Department of European and International Affairs Marseille, France Partners

Patrik Brundin Lunds Universitet Faculty of Medicine Department of experimental medical science Lund, Sweden

Bente Finsen University of Southern Denmark Medical Biotechnology Centre Odense, Denmark

Jonas Frisen Karolinska Institute Department of Cell and Molecular Biology Stockholm, Sweden

Philippe Hantraye Centre à l’Energie Atomique URAD2210 CEA/CNRS: Unité d’imagerie isotopique, biochimique et pharmacologique Orsay, France

Johan Hyllner Cellartis AB Goteborg, Sweden

Klaus Reymann Leibniz-Institut für Neurobiologie Neuropharmacology Group Magdeburg, Germany

66 New Therapies – Regenerative Medicine Regenerative Medicine STROKEMAP Multipotent adult progenitor cells to treat stroke

Contract No LSHB-CT-2006-037186 Project type Specific Targeted Research Project EC contribution e 2 400 000 Starting date 1 October 2006 Duration 36 months

Background and objectives: MAPCs endothelial cells and neural cells; • to compare the efficacy of MAPCs or Stroke is caused by the occlusion of a cerebral their differentiated progeny with gold- artery, resulting in irreversible tissue damage for standard cell populations (that is, bone which there is no curative treatment available as marrow cells currently used in animal yet. Each year, approximately one million peo- models) to restore circulation and neural ple suffer strokes in the EU. Approximately 25% circuitry damaged by stroke; of men and 20% of women experience a stroke • to develop innovative, non-invasive if they live to 85 years, and stroke is the second imaging techniques that will allow it to most common cause of death worldwide. follow cell survival, migration and engraftment following transplantation, as well STROKEMAP hypothesises that a universal popu- as to gain insight into the mechanism(s) lation of stem cells that can restore bloodflow through which stem cells repair the blood through the ischemic region, and that can differ- supply and neural circuitry in the brain, entiate in vivo into neurons and astrocytes that and also to provide quantitative assess- died due to cerebral ischemia will be the superior ment of such repair; cell population for the vascular and neural repair • to develop strategies allowing trans- needed for therapy of stroke. plantation of universal donor human stem cells, and strategies to circumvent The overall hypothesis is that allogeneic Multipo- possible immunological rejection; tent Adult Progenitor Cells or MAPCs, a novel • to develop clinical scale and grade bone marrow stem cell population first described stem cell populations that could be used by the Verfaillie lab in 2002, are an ideal candi- in clinical trials upon completion of the date stem cell population, and this will be tested translational and preclinical studies pro- in STROKEMAP. Indeed, MAPCs can generate ar- posed in this project; terial the endothelial cells and vascular smooth • to develop an ethical and legal frame- muscle cells needed to restore the vasculature, work in which to initiate clinical trials with as well as the neural cells and neuroprogenitors stem cells for stroke. that could be used to restore neural circuitry. Approach and methodology: The objectives of STROKEMAP are: • to carry out studies to further un- Two work packages will determine the mecha- derstanding in processes that underlie nisms that underlie differentiation of rodent the differentiation and specialisation of and human MAPC to arterial versus venous en-

New Therapies – Regenerative Medicine 67 STROKEMAP

dothelium and to neural stem cells. This involves Coordinator differentiation in vitro, gene array studies, and for the endothelium testing, certain principles in ze- Catherine Verfaillie brafish. Stamcelinstituut, K U Leuven Onderwijs & Navorsing 1 Another two work packages are aimed at the Herestraat 49 immunological consequences of grafting MAPC 3000 Leuven, Belgium in vivo: one evaluating the principles in mouse, E-mail: [email protected] and a second where tests will be carried out to establish whether mice can be given a human Partners immune system using MAPC. The consortium will then test the immunological consequences Aernout Luttun, Stefaan Van Gool, Luc Mortelmans, Paul of MAPC themselves, or MAPC-derived endothe- Schotsmans, Bart De Moor lium and neural cells, in the autologous and al- Katholieke Universiteit Leuven logeneic setting. Leuven, Belgium

A fifth work package will evaluate whether Peter Carmeliet and Mieke Dewerchin MAPC can restore blood flow in the brain and Vlaams Interuniversitair Instituut voor Biotechnology neural circuitry. This will be performed chiefly on Leuven, Belgium rodents. A sixth work package will evaluate different methods for labelling stem cells to allow Felipe Prosper and Ivan Penuelas for their visualisation via PET, MRI and BLI, while a Universidad de Navarra seventh work package will focus on cell popula- Pamplona, Spain tions that will be developed, and that conform to GMP guidelines. These populations could then be José Manuel Garcia-Verdugo used at the end of the granting period in phase I Universidad de Valencia or II trials, if the results obtained are encouraging. Valencia, Spain Finally, there is also an ethics work package. Markus Manz Expected outcome: Institute for Research in Biomedicine Bellinzona, Switzerland The anticipated outcome of the STROKEMAP project is to: Ernest Arenas • establish the possible beneficial ef- Karolinska Institutet fects of MAPC therapy in the setting of Stockholm, Sweden stroke; • develop tools to image the fate of Gil Van Bokkelen stem cells in the brain; ReGenesys • understand the immunological conse- Brussels, Belgium quences of MAPC transplantation; • develop GMP procedures to expand Jean-Marc Idee MAPC or their differentiated progeny; Guerbet • develop an ethical framework for Roissy, France MAPC storage and use.

68 New Therapies – Regenerative Medicine Regenerative Medicine RESCUE From stem cell technology to functional restoration after spinal cord injury

Contract No LHSB-CT-2005-518233 Project type Specific Targeted Research Project EC contribution e 2 500 000 Starting date 1 December 2005 Duration 36 months Website www.rescueproject.eu

Background and objectives: SCI has long been regarded as intractable, largely According to the International Campaign for due to the alleged inability of the central nervous Cures of spinal cord injury Paralysis (ICCP), more system (CNS) to regenerate. However, over the than 130 000 people worldwide survive a trau- last two decades, technological advances, com- matic spinal cord injury (SCI) each year, leading bined with the understanding of the pathophysi- to permanent paralysis and a lifetime of disabil- ology of SCI, have progressed to the point where ity. Thus, with an average age at injury of 33.4 it is now conceivable to develop therapeutic years and a nearly normal life expectancy due to intervention strategies aimed at reconstructing advances in healthcare, it is clear that the popu- the neuronal circuitry damaged by the lesion. lation of people with SCI is steadily increasing globally. One of the most powerful tools for this objective is based on stem cells, which can be used in the It is estimated that by 2005, over 2.5 million peo- following ways: ple worldwide were living with SCI-induced pa- • to introduce permissive molecules ralysis. In Europe, there are approximately 330 and/or trophic agents at the level of the 000 people suffering from SCI, with more than 10 lesion to improve regeneration of the sev- 000 new cases occurring each year. ered axons; • to replace damaged cells, grafted locally to stimulate specific circuits such as the central pattern generator; • to enhance the therapeutic potential through the activation of intrinsic stem cells.

Rescue focuses on the combination of the most efficient technologies to direct the fate of intrinsic, as well as extrinsic stem cells, and/or their transformation, in order to obtain appropriate cell types at the right time and in the right place, to promote repair of the injured spinal cord.

New Therapies – Regenerative Medicine 69 RESCUE

Approach and methodology: of RESCUE can therefore be used as templates for the elaboration of therapeutic strategies whose RESCUE will investigate a number of different ap- application should be broadened to other CNS proaches that are currently available, to try to re- traumatic damages, such as traumatic brain inju- store function of the injured spinal cord through ries and stroke. the use of human stem cells from bone marrow and the central nervous system. These approaches are set out below. • Use of stem cells during the acute phase (within one week of the injury in man) to reduce inflammation and secondary degeneration in the spinal cord. This also reduces the formation of scar tissue which constitutes a major barrier to ax- onal regrowth. • Enhanced regeneration of neurons and axons in the spinal cord through increased availability of axon growth-permissive molecules and/or trophic agents produced by stem cells or their progeny. Such molecules may be produced by un- modified cells, or by cells specifically engineered to do so. • Regeneration/restoration of function by supporting and monitoring the activation of intrinsic spinal cord stem cells. Murine and human adult spinal cord stem cells • Restoration of function after replacement of the damaged cells, following local grafting.

Expected outcome:

RESCUE’s final objective is to translate experimental studies using human stem cells in preclinical animal models into the clinic. This will be achieved through the elaboration of a series of therapeutic tools for stem cell therapy to be used in a wide variety of clinical paradigms of SCI.

Spinal cord lesions represent an ideal model for the development of regenerative therapy for traumatic lesions of the central nervous system (CNS), as they are more prone to precise functional characterisation and follow-up than brain injuries. Preclinical results obtained in the context

70 New Therapies – Regenerative Medicine Regenerative Medicine

Coordinator Jack Price Centre for the Cellular Basis of Behaviour Alain Privat King’s College London INSERM London, UK Unit 583 Physiopathology and treatment of sensorial and motor deficiencies Jean-Philippe Hugnot Institut de Neuroscience Université Montpellier 2 Hôpital Saint-Eloi Montpellier, France 80 rue Augustin Fliche 34295 Montpellier Cedex 05, France E-mail: [email protected]

Partners

Jean Schoenen University of Liège Department of Neurology Liège, Belgium

Eva Sykova Institute of Experimental Medicine Academy of Sciences of the Czech Republic Prague, Czech Republic

Jacques Mallet Centre national de la recherché scientifique - CNRS Laboratoire Génétique de la Neurotransmission Paris, France

Manuel Gaviria Neuréva Montpellier, France

Christiane Dascher-Nadel Inserm-Transfert SA Department of European and International Affaires Marseille, France

Gary Brook University Hospital Aachen Aachen, Germany

Minerva Gimenez y Ribotta Consejo Superior de Investigaciones Cientificas Alicante, Spain

New Therapies – Regenerative Medicine 71 STEM-HD Stem cells for therapeutics and exploration of mechanisms in Huntington’s disease

Contract No LHSB-CT-2006-037349 Project type Specific Targeted Research Project EC contribution e 2 500 000 Starting date 1 December 2006 Duration 36 months Website www.stemhd.eu

Background and objectives: tribute in a decisive manner to the understanding of the mechanisms of HD, as a necessary Huntington’s Disease (HD) is a rare monogenic step toward finding a cure. The consortium aims disorder. Age of onset of clinical symptoms of HD at reaching two main complementary goals: to is quite variable, although the disease starts, in decipher molecular mechanisms of HD, and to the majority of cases, at adulthood (average 35 identify compounds endowed with therapeutic years of age). Pathological lesion primarily affects potential for HD. the GABAergic output medium-spiny neurons of the striatum. Clinical symptoms are character- Approach and methodology: ised by a rapidly progressive alteration of motor abilities (chorea, bradykinesia), psychological dis- To realise its goals, STEM-HD will perform the fol- turbances (depression and irritability) and cogni- lowing activities: tive impairment (of most functions, by the later • use an available human embryonic stages), leading to dementia. On average, death stem (ES) cell line, based upon the hypoth- occurs 15 to 20 years later. esis that ES cells expressing a disease-related mutant gene may be used for mo- HD is an autosomal, dominant monogenic dis- lecular modelling of that monogenic ease. The mutation consists of an enlarged CAG disease; triplet repeat that encodes an elongated stretch • implement large-scale technological of polyglutamine in the N-terminal portion of the resources, and combine resource-driven large protein huntingtin. Neither the physiologi- and hypothesis-driven analyses at all stag- cal role of huntingtin, nor the molecular mecha- es of the project. nisms of the pathology are currently known, but since its discovery, a number of cellular pathways, Expected outcome: interacting proteins and nucleic acids have been identified. The identification of new biomarkers and of the molecular pathway of HD may lead to possible HD research has benefited from a direct genetic therapeutic applications, and the development of screen for more than 12 years, and preimplan- new diagnostic tools. These tools will allow spe- tation genetic diagnosis has been proposed to cialists to follow the development of the disease at-risk couples for more than 6 years. However, in patients known as carriers of the mutation, no validated treatment — neither curative nor helping them to determine the best time to start symptomatic — is available for HD today. the palliative treatments (psychological care, ex- The fundamental objective of STEM-HD is to con- ercise, etc), or the treatments, once developed. It

72 New Therapies – Regenerative Medicine Regenerative Medicine

Figure A Colony of undifferentiated human embryonic Figure B hES cells differentiated into neuronal progeni- stem cells (200X, passage 19) on a monolayer of feeder tor cells (day 45). Immature neural cells stained in red cells. hES marker proteins Oct4 and Nanog stained in red (Nestin), young neurons in green (Tuj1); nuclear staining and green, respectively; nuclear staining in blue (DAPI). in blue (DAPI). will also be of help to facilitate the identification of the disease in patients not known as carriers of the mutation. Finally, it will be a valuable tool to evaluate in vitro in patients, the effectiveness of any new treatment in clinical trials for HD.

Although the focus of the consortium will be dedicated fully to HD, the participants’ common goal is to extend the value of their achievements by making it a model disease for designing protocols and infrastructures applicable to many other monogenic diseases. Because each rare monogenic disease affects only a limited number of people (less than 5 in 10 000), it has been dif- Figure C Mature GABAergic neurons (day 63 of difficult until now to allocate to each affected indi- ferentiation). GABAergic striatal neurons stained in red (DARPP-32), neuronal cells in green (MAP2); nuclear stain- vidual, the investments required by large-scale ing in blue (DAPI). approaches, that are used for non-rare diseases © Photos Inserm both for the exploration of mechanisms and for high throughput/high content drug screening. — and by restricting its workload to a minimum through systematic automation, thus providing The fact that these monogenic diseases are di- the opportunity to search for therapeutics of sev- verse in terms of their phenotype, and that each eral rare monogenic diseases. one is relatively rare, have made them compara- tively unattractive targets for drug discovery. STEM-HD aims at directly addressing these issues, by reducing the specificity of the process for each disease — through the standardisation of common protocols and infrastructures for all

New Therapies – Regenerative Medicine 73 STEM-HD

Coordinator Christiane Dascher-Nadel Inserm-Transfert SA Marc Peschanski Department of European and International Affairs INSERM Unit 421, I-STEM Marseille, France BP 118, 1 rue de l’Internationale 91004 Evry cedex, France E-mail: [email protected]

Partners

Joseph Itskovitz-Eldor Technion - Israel Institute of Technology Stem Cell Centre, Faculty of Medicine Haifa, Israel

Dorotea Rigamonti Dialectica srl Nerviano, Italy

Jacques Haiech Université Louis Pasteur Strasbourg Faculté de Pharmacie LC1 UMR7175 - Groupe Chimiogénomique et Pharma- cogénomique Illkirch, France

Nicholas Allen Cardiff University School of Biosciences Cardiff, UK

Elena Cattaneo Universita degli Studi di Milano Department of Pharmacological Sciences and Centre of Excellence on Neurodegenerative Diseases Laboratory of Stem Cell Biology and Pharmacology of Neurodegenerative Disorders Milan, Italy

Karen Sermon Vrije Universiteit Brussel Research Group Reproduction and Genetics Brussels, Belgium

74 New Therapies – Regenerative Medicine Regenerative Medicine NEUROscreen The discovery of future neuro-therapeutic molecules

Contract No LHSB-CT-2006-503005 Project type SME-Specific Targeted Research Project EC contribution e 2 587 100 Starting date 1 March 2007 Duration 36 months

Background and objectives:

NEUROscreen is an industry-led project combining novel neural stem cell bioassays and post- genomic chemical genetics, so as to discover innovative therapies in the fields ofneurological diseases, regenerative medicine and cancer. A key objective is to join fledgling and well-established European commercial partners in a pre-competitive, collective drive to translate fundamental stem cell biology into a state-of-the-art discovery platform, and successfully demonstrate the potential exploitability of the concept while simultaneously strengthening European bio-industry.

Approach and methodology: Novel neural stem (NS) cells stained with antibodies The project work plan consists of 6 interlinked specific for a defining marker of the cells work packages (WPs). WP1 focuses on the pro- curement, derivation, banking, annotation and establishment of quality control standards for WP3 and WP4 provide essential confirmation of a range of rodent and human neural stem cell specification potential for cell lines in use, via in lines, including brain stem cell tumour lines. WP2 vitro (WP3) and in vivo (organotypic slice culture; is the bioassay design component covering the WP4) assessment. WP5 focuses on the provision development of key genetic modification tech- of stem cell lines of adequate quality and quan- nology (BAC) and the establishment of a reper- tity for screening, as well as the compilation of toire of lines containing reporter constructs. WP2 technology components and processes for the will provide a valuable chemical ‘hit’ validation automated handling of cells for the screening platform involving the verification of protein activity. Cell formatting will be the precursor to function using RNA interference. the screening activities in WP6. A two-site, two- platform approach will be used for the screens Using modified cell lines to design bioassays re- using a large number of chemicals pre-selected quires a number of key validatory steps to be per- by in silico-based design. Leads will be validated formed in order to assess the integrity of the cells. through WP2, WP3 and WP4 activities.

New Therapies – Regenerative Medicine 75 NEUROscreen

Expected outcome: Partners

NEUROscreen will present a detailed description Dorotea Rigamonti of the comparative properties of human neural Dialectica s.r.l. stem cells derived either from tumor or normal Milan, Italy biopsies, and will offer a comparative fidelity of functional properties of neural stem cell-derived Pasquale De Blasio neurons, versus those isolated acutely ex vivo. BioRep s.r.l. Procedures for the automated expansion of sta- Milan, Italy ble, proliferating human neural stem cells, as well a repertoire of bioassays to measure differentia- Stefanie Terstegge tion and subtype specification from neural stem Life & Brain GmbH and progenitor cells, will be set up. The NEURO- Biomedizinische & Neurowissenschaftliche screen project will establish a repository of anno- Technologie-Plattform tated stem cell lines, with product release criteria Bonn, Germany based on defined QA/QC characteristics, as well as a defined set of bioactive, optimal compounds Austin Smith to be validated as contenders for prospective re- University of Cambridge generative medicines. The Wellcome Trust Centre for Stem Cell Research Cambridge, UK

Luciano Conti Coordinator Università Degli Studi di Milano Dipartimento di Scienza Farmacologiche Tim Allsopp Milan, Italy Stem Cell Sciences Plc Minerva Building 250, Babraham Research Campus Oliver Brüstle Babraham, Cambridge, CB22 4AT, UK University of Bonn E-mail: [email protected] Institute of Reconstructive Neurobiology Bonn, Germany Scientific coordinator François Guillemot Lilian Hook National Institute Medical Research European Research Programme Manager Molecular Neurobiology Division Stem Cell Sciences UK Ltd London, UK Roger Land Building Kings Buildings Cesare Spadoni West Mains Rd Albany Medical Research Inc Edinburgh, EH9 3JQ, UK (formerly ComGenex) E-mail: [email protected] Budapest, Hungary

76 New Therapies – Regenerative Medicine Regenerative Medicine myoamp Amplification of human myogenic stem cells in clinical conditions

Contract No LSHB-CT-2006-037479 Project type SME-Specific Targeted Research Project EC contribution e 2 480 000 Starting date 1 December 2006 Duration 36 months

Background and objectives: It will ensure that these conditions and guidelines are transferred to SME and clinicians, defining ef- Many groups have used animal models to inves- ficient integration through dedicated partners tigate the possibilities of using autologous cell within the 3-years duration of this project. therapy for muscular dystrophies, but these data are dispersed, not always comparable and little Many clinical trials using muscle cells have been attention has been focused on the transfer of this developed in the past for Duchenne Muscular knowledge towards applications for therapeutic Dystrophy with very limited success. The recent trials. emergence of new therapeutic venues, based upon post-transcriptional genetic corrections Data exist on injecting murine cells into mouse called “exon-skipping”, have raised new hope for muscle, but information regarding human cells this disease. Using viral transfer approaches it has is sparse. The feasibility of autologous myoblast given very promising results but cannot reach transfer therapy has already been demonstrated every muscle of the body and trigger a immune for cardiac repair, even if in cardiac therapy, in- response to the vector. Autologous cell therapy jected cells were mainly used to counteract the may bypass this reaction and be used as a com- development of fibrosis in patients devoid of any plement or alternative if the cell type used ful- defect in skeletal muscle. filled both being an efficient vector and bringing a functional benefit to the diseased muscle. The fact that preclinical trials developed in mouse models of muscular dystrophies have been suc- Autologous muscle cells cannot be used since cessful as compared to clinical trials, which used these are already defective in dystrophic mus- mostly allogenic cells and resulted in very limited cle, while stem cells from other origins are ideal clinical benefit for the patients, illustrates the ur- candidates, as long as their myogenic and pro- gent need for preclinical studies using human liferative potentials are ensured. In this perspec- cells. tive mesoangioblasts, which have already been used in a mouse model of muscular dystrophy, Myoamp will aim at defining conditions and and AC133 cells have a therapeutic potential guidelines to produce transduced human stem as demonstrated in the mouse, but very little is cells as vectors for clinical trials. Myoamp will syn- known about the conditions required to amplify ergise expertises from European leaders in their in GMP conditions these stem cells isolated from respective field to set up conditions for autolo- humans, which is an essential step required be- gous transfer of human stem cells in GMP condi- fore any clinical trial. tions for the treatment of DMD by exon-skipping.

New Therapies – Regenerative Medicine 77 MYOAMP

Myoamp will address the question of the amplifi- In addition to basic knowledge on the amplifica- cation of these cells used as autologous cell ther- tion mechanisms, myoamp will bring guidelines apy vectors and their safety. The cells transduced and standard operative procedures to obtain with a lentiviral construct allowing exon-skipping these cells in a reproducible and safe manner, will be selected and further amplified. It should that can be directly transferred to SMEs and clini- be noted that transduced cells may be cloned so cians for clinical applications. that their integration site is determined), as will These guidelines will therefore address technical, be proposed in the part of myoamp that focus on ethical and safety issues in a GMP environment. safety and ethics. In vitro and in vivo approaches will bring understanding on the regulation of Potential applications: proliferation, while the telomere length (reflecting the mitotic clock status of the cell) will be • preclinical protocols, standard oper- monitored, as well as a combination of receptors ating procedures to characterise, amplify known to trigger proliferation, (FGF, IGF1, ). and assess myogenic human stem cells for autologous cell therapy treatments in The stability of the parameters initially examined muscle disease. in non-GMP conditions, will be checked through • ethical and safety procedures to cover amplification in various conditions, to allow the the protocols. definition of both guidelines for GMP production • specific culture medium with a defined and key-parameters to be followed during the set of growth factors. GMP amplification. The number of cells to be injected at each implantation, which is purely empirical in many clinical trials, will be tested and optimised in a model of implantation of human cells in immuno-deficient mice, in order to define the maximum number of cells to be finally amplified in GMP conditions.

Role of SMEs

1. 3H Biomedical cell provider and responsible for defining SOP for cell handling; 2. CELLGENIX development of adapted serum-free culture medium and definition of SOP; 3. GENOSAFE development of safety procedures and assays for the process.

Expected results:

The main expected result is to obtain the final product, i.e. protocols to obtain amplified human stem cells, in a state that will allow an optimised efficiency in injections in vivo.

78 New Therapies – Regenerative Medicine Regenerative Medicine

Scientific coordinator Anton Ottavi Inserm-Transfert Vincent Mouly Paris, France INSERM UMR S 787 Myologie Institut de Myologie 105 bd de l’Hôpital 75634 Paris Cedex 13, France E-mail: [email protected]

Partners

Luis Garcia INSERM Paris, France

Yvan Torrente University of Milan Milan, Italy

Giulio Cossu Fondazione Centro San Raffaele del Monte Tabor Milan, Italy

Jenny Morgan Imperial College, London London, UK

Otto Merten Généthon, Evry, France

Mallen Huang 3H Biomedical Uppsala, Sweden

Roland Bosse Cellgenix Gmbh, Freiburg, Germany

Vincent Giuliani Genosafe SA Evry, France

New Therapies – Regenerative Medicine 79 CRYSTAL Cryo-banking of stem cells for human therapeutic application

Contract No LSHB-CT-2006-037261 Project type Specific Targeted Research Project EC contribution e 2 400 000 Starting date 1 February 2007 Duration 36 months Website www.crystal-eu.org

Background and objectives: In addition, for many stem cell types cryopreservation itself is neither optimised nor validated for Stem cells are currently at the centre of biomedi- different cell types, and there are multiple cellu- cal research. Besides advancing the basic under- lar and biophysical challenges to be addressed, standing of the human development and the in order to define optimised cryopreservation cellular differentiation processes, stem cells hold protocols. Current methods represent a trade-off the unique potential for novel therapies of de- between preventing formation of damaging ice generative diseases such as ischemia of the heart, crystals and toxic effects of cryoprotectants. In Parkinson’s disease, diabetes, and certain types of particular, the amenability of different stem cell tumours. populations to freezing conditions is not well understood. The viability of human embryonic Future human stem cell therapy, however, will stem cells is low after freezing, and short- and have to build on a readily available, safe and reli- mid-term effects of freezing on cellular proper- able supply of high-quality human stem cells that ties remain to be investigated. must be assured by cell banking. Current banking approaches still rely on storing sources of stem The aim of CRYSTAL is to develop tools and pro- cells rather than on the banking of defined, well- cedures to enable the cryopreservation of differ- characterised stem cell populations (both adult ent stem cell types for the generation of sufficient and embryonic). It should be noted that there is numbers of high-quality cells suitable for safe hu- room for improvement of the conditions for reli- man stem cell therapy. To this end, CRYSTAL will able outgrowth, since they are still at a rudimen- conduct focused research on the methods, tools tary stage. The isolation, identification and culture and protocols required for optimal cryopreserva- of stem cells are not standardised among labora- tion and banking of stem cells. tories and reproducibility of protocols is limited. In order to achieve this objective, the following As the culture of human embryonic stem cells unresolved methodological and experimental routinely requires the use of animal products or aspects of stem cell banking will be addressed: cells, their therapeutic use is ruled out. In vitro cul- • scientific validation and optimisation ture and expansion of haematopoietic stem cells, of protocols for identification, charac- either from adult bone marrow or cord blood, is terisation, maintenance and expansion of far from optimal, necessitating further research in stem cells; order to overcome problems related to the insuf- • establishment and validation of the ficient numbers of obtained stem cells and the cryopreservation of stem cells; ageing of the stem cell population. • definition of validation methods of the

80 New Therapies – Regenerative Medicine Regenerative Medicine

properties of stem cells after defreezing, including maintenance and expansion, engraftment capability, etc.

Approach and methodology:

The CRYSTAL project pursues an integrated approach, using both somatic and embryonic stem cells to address existing shortcomings that limit the routine application of stem cell banking with a therapeutic perspective. The consortium will develop tools and optimised procedures to en- Figure (© IBMT): Cryopreservation issues and novel ap- able cryopreservation of different stem cell types proaches to cryopreservation. and allow for the safe production of sufficient Fig. 1 - combined block face scanning electron microscopy numbers of high-quality cells for future human (back scattered mode) with freeze substitution of cryopre- served adherent murine fibroblasts. The arrow indicates an therapy. This will comprise standardised proto- intracellular ice domain. cols and tools for stem cell isolation, identification and culture, novel approaches to their cryopreservation (e.g. novel cryoprotectants, freezing in different conformations) and an automated quality control system for stem cell preparations.

Five stem cell research laboratories providing four different sources of adult (from cord blood, bone marrow and placenta) and human embryonic stem cells have teamed up with two partners specialising in applied banking and fundamental cryobiological research. The scientific experimental work is supported by a unit that Fig. 2 - scanning electron micrograph (secondary electron will create a common knowledge base for inte- mode) of adherent cells (murine fibroblasts, gold) on carbon based nanostructured fibres (blue). The image is manually grating pre-existing know-how from both inside colored, horizontal image width about 120 microns. and outside the consortium, and for guiding partner laboratories in the implementation and refinement of standard operating procedures for Expected outcome: culturing techniques, cryopreservation and validation of protocols. CRYSTAL plans to deliver a set of optimised, validated protocols, covering the core aspect of stem The scientific work is to be supported by profes- cell banking, and achieving significant innovation sional project management and a team ensur- in the three areas of preparation and cultivation ing the effective dissemination and use of the methods, preservation methods and validation obtained results. CRYSTAL is thus in a position methods. These optimised, validated methods to solve existing problems using an integrated, and tools will be made available to the scientific systematic approach, and to provide standard- community to underpin initiatives on stem cell ised, reproducible methods and tools to advance banking, thus providing a solid foundation for therapeutic stem cell research in Europe. the future development of stem cell therapies.

New Therapies – Regenerative Medicine 81 CRYSTAL

Coordinator

Jürgen Hescheler University of Cologne / Clinical Centre of the University of Cologne Institute of Neurophysiology Robert-Koch-Str. 39 50931 Cologne, Germany E-mail: [email protected]

Partners

Guy Wouters Life-Sciences Group N.V. HB Zutphen, Netherlands

Heiko Zimmermann Fraunhofer Institute for Biomedical Engineering (IBMT) St. Ingbert, Germany

Andrea Kolbus Medical University of Vienna Department of Obstetrics and Gynaecology Vienna, Austria

Andreas Zisch University Hospital Zurich Department of Obstetrics Zurich, Switzerland

Catherine Verfaillie Katholieke Universiteit Leuven Interdepartementaal Stamcelinstituut Leuven, Belgium

Peter Ponsaerts University of Antwerp Laboratory of Experimental Hematology Antwerp, Belgium

Annette Ringwald ARTTIC Paris, France

82 New Therapies – Regenerative Medicine

84 New Therapies – Gene Therapy Gene Therapy CLINIGENE European network for the advancement of clinical gene transfer and therapy

Contract No LSHB-CT-2004-018933 Project type Network of Excellence EC contribution e 12 000 000 Starting date 1 April 2006 Duration 60 months Website www.clinigene.eu

Background and objectives: CLINIGENE aims to: • foster interaction between all stake- As the field of gene therapy has matured, oppor- holders: regulators, preclinical and clinical tunities have been created that are both excit- investigators, scientists, companies (oth- ing and promising, and some treatments have erwise competitors), as well as patients’ already been proven clinically effective. However, groups, in order to streamline integration precise quality and safety standards for clinical of multidisciplinary expertise; gene transfer have yet to be defined. • establish quality, safety, efficacy and In this context, establishing optimal methods for morally acceptable standards for clinical the production of both standard and innovative gene transfer products; vector systems would pave the way for acceler- • identify the ‘critical path’ to accelerate ated product development and improved safety. the transit phase from the preclinical to This would be of enormous value to patients, the clinical phase by integrating expertise individual investigators, industry and regulatory and generating new knowledge; authorities alike. • improve European competitiveness by spreading excellence and disseminating The move from the preclinical to the clinical knowledge; phase calls for clinical-quality grade products • gain a clinically significant improve- manufactured for human use. In addition, the ment in the treatment of human disease safety profile of these products must ensure that through gene therapy. the potential benefits outweigh potential side effects, thus providing patients with a safe and Approach and methodology: efficacious treatment option. The role of CLINIGENE is to efficiently mobilise all The network initially analysed the bottlenecks that interested parties, involving academic research exist. From this assessment, partners were able to and production centres in particular, alongside better understand current and future limitations, companies, patients’ groups and regulatory bod- and could then examine and offer potential solu- ies. Its goal is to integrate multidisciplinary re- tions and methods (Table 1). search so as to decipher the key elements that The CLINIGENE project will not establish stand- can lead to improved safety and clinical efficacy ards in retrospect, based on previous trials; it of gene transfer/therapy products. Control and will rather augment the development of entirely test methods will be established, and can then be novel protocols using cutting-edge technology. applied as platforms for particular gene transfer Practical examples of new knowledge applica- products. tions and, in the longer term, of clinical success

New Therapies – Gene Therapy 85 CLINIGENE

Solutions to propose Other bottlenecks Technological Bottlenecks Current to be tested & if (not addressed by platforms to be addressed limitations possible exemplified CLINIGENE) by the NoE • Reproducibility • Size of the insert • Titers • Stable cell-lines • Intracellular barriers • Scale up AAV • Serotype production for each selected • Route of administration • Non-specific serotype • Vector targeting encapsidation • Lack of • Establish SOPs and standardized high improved monitoring yield production methods to achieve • Gutless vector system improved production and production • Unspecific interaction of purification • Free access to • Efficiency of • Improved production cell Adenovirus vector with non- transcomple- oncolytic Ad-vectors target cellular lines mentation cell- and extracellular • Non specific uptake/ • Capsid modification/ lines compartments polymer coating/ specific • Contamination by Targeting targeting helper virus • Low degree of • Genome optimisation Tumour spread (Oncolytic)

• Characterized packaging cell-lines • Insertional • Packaging cells with mutageneis in target specific integration sites cells • Integration • Novel purification, formulation & storage • Vector titers Retrovirus • Non-characterized • Vector titers improving titers • Size of transgenic packaging cell-lines • Purification • Pseudotyping with a sequences • Targeting • Stability variety of virus envelopes • Number and • Locus insultators in target distribution of vector cells integration sites per • Specific integration cell

• Safety issues • Mode of • Partial recombination production (stable • Validation of RCL assays versus transient), • Chromatography • Stable packaging cell-lines purification and based purification • Number and Lentivirus and toxicity of viral proteins quality • Test methods & distribution of Besides • Lentivirus associated • Low-yield Retroviruses tumour development in liver • Plasmid reference material vector integration • Exclusion of regulatory viral contaminants for replication sites per cell genes (rev sequences) • Synthesis of viral competent • Quality of vector proteins by target recombinant preparation for in vivo use cells

• Homogeneity of • Testing methods for Cell Therapy • Differentiation • Cell-transformation source & phenotype homing & distribution (gene- • Long-term function • Heterogeneity of • Viability/Survival • Cell-marking manipulated • Immune attack to autologous primary cells • Distribution and • Optimised culture cells) the cells • Adventitious viruses Homing conditions

• Acute toxicity of delivery method Non-viral • Bacterial sequences • Mitotic stability • Potential genotoxicity technologies (CpG, selection • Replicating • SMAR/ORI of physicochemical General genes) episomes • Minicircles methods (e.g. DNA damage due to electric forces in electroporation)

No vectorisation • Limitation to a few • Efficacy Naked DNA specific situations • Safety • Specific electrodes Physical • Perception of the • Control algorithms vectorology • SOP preparation, Electroporation technology (fears of evaluation, diffusion the electricity) • Training Chemical • Purity of the products • Lack of standards • Standard procedures vectorology Present • Too large number of • Lack of • Reference for material preparation DNA formulations options comparisons materials

86 New Therapies – Gene Therapy Gene Therapy

are to be provided. The network will address orphan diseases, such as Fanconi’s anaemia or Duchenne’s, as well as more prevalent pathologies, including cancer, cardiovascular and neurodegenerative disorders. Common standards of reference must be shared in order to establish quality. Firstly, such standards must be tested, broad consensus must then be reached and subsequently agreed upon by the academic centres and the companies specialising in the manufacture of gene transfer vectors for the purpose of human gene therapy. Due to the diversity of the emerging needs, the standards will be worked out along distinct timelines from one vector system to the next, during the course of the programme (Fig 1a&b).

CLINIGENE is developing platform databases for particular vectors with respect to their safety, efficacy and quality standards in order to accelerate the transition from research to clinical trials (Fig 1a&b). Safety will be considered both in Figure 1a & 1b: The European Network Structure and terms of pharmaco-toxicology and outcomes viral safety. Whatever the technology, viral safety ognised by regulatory authorities. assessment includes a series of common check- Pharmaco-toxicology profiles can be drawn only points to consider and also standardise. Another in a case-by-case manner, combining the vector important safety issue relates to vector integra- system of choice and the specific gene address- tion with a concurrent risk of insertional muta- ing the disease in question. A challenge for the genesis. The EMEA/CHMP-GTEG (now GTWP) has network is to successfully mobilise partner ex- reported on serious adverse events in clinical trials pertise in order to obtain useful clinical trial in- involving X-linked severe combined immunodefi- formation, which will form the basis on an inter- ciency patients. More work is needed on a case- national effort to use the existing technology for by-case basis in order to evaluate the risks accord- the treatment of rare diseases in particular. Prac- ing to the nature/function of the gene of interest tical clinical protocols will be considered, dealing and the disease to be treated. The network is ad- mainly with the definition of the requirements dressing potential improvements, using a variety for patients’ clinical monitoring. These include: of experimental approaches. CLINIGENE recently • thorough definitions of biological sam- submitted its comments to the FDA-Long Term ples to be taken and of those to be stored Follow-Up guideline, the relevance of which is rec- where retrospective studies are required;

New Therapies – Gene Therapy 87 CLINIGENE

Fig. 2

• points to be considered and steps to programmes to streamline innovation and opti- be taken towards the long-term follow- mise dissemination (Fig 2). up of patients, in particular those patients treated with integrating vectors at risk for To successfully integrate its efforts — given the insertional mutagenesis. breadth of the technological approaches and applications anticipated within CLINIGENE — the The CLINIGENE network has adopted an inte- network coordinator has developed the innova- grated approach targeting high-level education, tive scientific and strategic management soft- training and human mobility of post-doctoral ware called Clinisoft, accessible online through scientists, doctors and pharmacists in order to a confidential and restricted-access website. It support a European dimension of highly spe- serves several purposes, and functions in the fol- cialised researchers, health professionals and lowing ways: managers. The clinical development process is • a virtual and highly hierarchic work- expanded with this approach in collaboration space supporting information exchange with the European Society of Cell & Gene Thera- and collaborative work on documents; py. Contacts have already been established with • an archive for all important network- other EC-funded gene therapy-related research produced information and documents;

88 New Therapies – Gene Therapy Gene Therapy

• an online coordination, planning and Coordinator network monitoring tool; and • a direct communication tool between Odile Cohen-Haguenauer defined working groups or technology École Normale Supérieure de Cachan & AP-HP platforms. Laboratoire de Biotechnologies et Pharmacologie géné- tique Appliquée (LBPA) Expected outcome: 61, avenue du President Wilson 94235, Cachan Cedex, France The partnership will combine its efforts and skills E-mail: [email protected] to initiate clinical approaches and decipher the key elements that can lead to clinical success. Partners Implementing benchmarking activities will effectively establish good practices as well. In or- Alberto Aurricchio der to promote safe and high quality clinical TIGEM - Telethon Institute of Genetics and Medicine gene transfer treatments, CLINIGENE will apply Naples, Italy a series of imaging technologies in collaboration with the DiMI FP6 NoE on Molecular Imaging and Robin Ali Industry partners. CLINIGENE aims to achieve the Institute of Ophthalmology - University College London following: Division of Molecular Therapy Bath St • conducting of phase I trials addressing London, UK previously-unasked questions; • prevention of failures at the clinical Segolene Ayme stage, as they would have negative con- Orphanet - INSERM SC11 sequences for patients entering the study Hôpital Broussais and would penalise the field in general; Paris, France • delivery of practical results that would open new opportunities for funding re- Fatima Bosch search and clinical development in this Universitat Autònoma de Barcelona field, as well as promoting the expansion Centre de Biotecnologia Animal i Teràpia (CBATEG) of the high-tech industrial sector. Bellaterra, Spain

In developing the actions initiated by the Eure- Jan Bubenik genethy2 network, CLINIGENE seeks to reinforce Academy of Sciences of the Czech Republic links to the international community. The net- Institute of Molecular Genetics work will address NIH/OBA-RAC and FDA-CBER, Prague, Czech Republic contribute to the International Liaison Group, and promote interaction through the interna- Manuel Carrondo - Pedro Cruz tional committee of the American Society of Instituto de Biologia e Tecnologia (IBET) Gene therapy. By encouraging and favouring Oeiras, Portugal contacts, cooperation and data sharing with members of the international community, such Christopher Baum as the USA and Japan, CLINIGENE will also ad- Hannover Medical School vance the strengthening and creation of jobs in Department of Experimental Haematology the biotechnology sector. Hannover, Germany

New Therapies – Gene Therapy 89 CLINIGENE

Klaus Cichutek Nicolas Mermod Paul-Ehrlich-Institut Université de Lausanne Langen, Germany Institut de Biotechnologie Lausanne, Switzerland Nicole Deglon CEA - MIRCen Lluis Mir Orsay, France CNRS - UMR 8121 Institut Gustave-Roussy George Dickson Villejuif, France Royal Holloway & Bedford New College School of Biological Science Philippe Moullier Egham (Surrey), UK CHU Hôtel Dieu Nantes Laboratoire Thérapie Génique Gösta Gahrton Nantes, France Karolinska Institutet Department of Medicine, Huddinge University Hospital Amos Panet Stockholm, Sweden The Hebrew University Hadassah Medical School Bernd Gänsbacher Jerusalem, Israel Klinikum rechts der Isar der Technischen Universität München Marc Peschanski Institut für Experimentelle Onkologie und Therapiefor- ISTEM (Unité INSERM) schung Evry Cedex, France Munich Germany Maria-Grazia Roncarolo Hansjörg Hauser FCSR-TIGET Helmoltz Centre for Infection Research San Raffaele Telethon Institute for Gene Therapy Braunschweig, Germany Milan, Italy

Andreas Jacobs Daniel Scherman Klinik für Neurologie der Universität zu Köln INSERM Labors für Gentherapie & Molekulares Imaging am MPI Paris, France für Neurologische Forschung Cologne, Germany Richard Vile Mayo Clinic - Gene Therapy Programme David Klatzmann Rochester (Minnesota), USA UMR 7087-UPMC/CNRS-CERVI Hôpital de la Pitié Salpêtrière Christof Von Kalle Paris, France German Cancer Research Centre (DKFZ) National Centre for Tumour Diseases (NCT) and Stefan Kochanek Department of Translational Oncology, University of Ulm Heidelberg, Germany Division of Gene Therapy Ulm, Germany

90 New Therapies – Gene Therapy Gene Therapy

Seppo Yla-Herttuala Martin Wisher University of Kuopio Bioreliance A.I.Virtanen Institute Scotland, UK Kuopio, Finland Vincent Zuliani Heinz Zwierzina Genosafe Innsbruck University Evry, France Department of Internal Medicine Innsbruck, Austria Luçay Sautron Science Pratique SA Anne-Marie Masquelier Cachan, France Généthon Evry, France

Gilles Avenard BioAlliance Pharma SA Paris, France

Charlotte Dalba Epixis Paris, France

Frederic Henry Clean Cells Bouffere, France

Charles Irving Theravir Jerusalem, Israel

Monica Lusky and Jean-Yves Bonnefoy Transgene Strasbourg, France

Kyri Mitrophanous Oxford Biomedica Oxford, UK

Felicia M. Rosenthal CellGenix GmbH Freiburg, Germany

Martin Schleef Plasmid Factory Bielefeld, Germany

New Therapies – Gene Therapy 91 CONSERT Concerted Safety and Efficiency Evaluation of Retroviral Transgenesis for Gene Therapy of Inherited Diseases

Contract No LSHB-CT-2004-005242 Project type Integrated Project EC contribution e 11 635 000 Starting date 1 November 2004 Duration 48 months Website www.gene-therapy.eu

Background and objectives: the mechanistic understanding of transgene- host interactions. The project generates the basis The CONSERT project integrates leading Europe- for technology development and promotes pa- an activities for a structured implementation of tient safety. novel therapies, using genetically modified postnatal stem cells, with a focus on the treatment of Translational dissemination of know-how from monogenic immunodeficiencies, haemoglob- academia to industry creates a network of cell inopathies, anaemias and storage disorders. processing manufacturers with large economic CONSERT develops and evaluates methods for potential, and prepares researchers for future genetic stem cell modification with wide impli- clinical studies with improved predictability. cations for many other disorders, including viral infection and cancer. Genetic enhancement of cellular therapies is expected to have a major impact on the treatment A central theme of the project is an unbiased of numerous inherited and acquired disorders. safety and efficiency evaluation of the key tech- Introducing defined genes into transplantable nology used in the genetic modification of rep- cells will ultimately result in a better control of licating somatic cells: retroviral vector-mediated their expansion, migration, differentiation and transgenesis. This is made possible only through elimination in vivo, according to the individual’s concerted multi-centre studies. Lenti-, spuma- unique medical condition. Also, novel effector- and gamma-retroviral vectors are tailor-made functions can be introduced, such as the resto- for target disorders, and tested for potency and ration of defective genes or protection against safety in preclinical disease models. Designed drug toxicity, side effects or virus infection. Les- with a translational aim, basic studies in stem cell sons learnt in the context of selected inherited biology and selectable marker technology com- disorders will have immediate intellectual and plement this research. practical consequences for the development of novel treatment options against acquired dis- A highly important aspect of CONSERT is the eases such as cancer or viral infection. molecular and clinical monitoring of currently active and successful clinical trials of stem cell European scientists and SMEs that hold key pat- gene therapy in inherited diseases. This creates ents in gene transfer technologies and diagnos- a paradigmatic data-mining activity to obtain tic assays for patient monitoring have pioneered insights into crucial issues of clonal kinetics of this field. All are members of CONSERT, underlin- gene-modified cells in vivo. Molecular studies in ing its high profile; they have also provided the precise cell systems and animal models provide first clinical evidence of a cure for inherited dis-

92 New Therapies – Gene Therapy Gene Therapy

orders of haematopoiesis by cor- Stem cell Biology and Transduction rective gene transfer into haemat- In vivo Selection approaches Safety Manufacturing opoietic stem/progenitor cells. Fundamental Knowledge Interface Given this background, CONSERT’s projects major objective is the concerted A Genomic Interface Program safety and efficiency evaluation of retroviral transgenesis. This is exemplified with selected inherited disorders as clinical targets, which Disease Specific Projects are in urgent need of the develop- Outreach & Red cell disorders Training ment of novel curative approaches. Wiskott-Aldrich Syndrome Ethics CONSERT’s work programme re- Leukodystrophies & Lysosomal storage disorders Regulatory, IPR and legal issues Chronic Granulomatous Disease A tailored Education and Exchange programme flects that the full potential of gene therapy can only be developed through an integrated approach that targets ex- seminations and ethics component, can be visu- isting biological, clinical, technological, commer- alised, in a nutshell, as demonstrated below: cial and ethical hurdles. Main findings: CONSERT’s scientific structure of integrating work packages (WPs) directed at fundamental Highlights of the work completed are presented knowledge to treat specific diseases with cura- below. tive intent, as well as with a strong genomics and manufacturing component, and a proactive dis- Schematic representation of gene therapy for inherited diseases. Areas shaded in blue, mark pharmaceutical involvement and potential.

New Therapies – Gene Therapy 93 CONSERT

Members of CONSERT have developed break- mouse models in which the impact of vector throughs in basic vector technology, molecular design on insertional mutagenesis can be evalu- analysis of gene insertion profiles, and upscaling ated directly. of cell manipulation procedures for clinical use. Ever-increasing data obtained by CONSERT teams As with any new therapeutic approach, gene suggest that the risk of insertional mutagenesis transfer may also induce novel kinds of side ef- can be strongly reduced by improving the vector fects. Somewhat reflecting the leading status of design (using lentiviral vectors and other types the project’s research and application, members of integrating vectors with redesigned transgene of CONSERT have also demonstrated the first cassettes), highly purified and limited numbers cases of serious adverse events of gene transfer of cells used, and the use of cell lineage specific into haematopoietic cells, and designed preclini- expression of the transgenes. The consortium cal as well as clinical approaches to address and has succeeded in developing a new generation prevent the underlying mechanisms. of self-inactivating gammaretroviral and lentiviral vectors, that are currently tested for efficacy The population of stem cells that allows for the and safety. complete restoration of lymphohaematopoiesis is difficult to enrich and maintainin vitro. Several In the safety evaluation and genomics work participants have made a breakthrough by dem- packages, the consortium has unravelled the re- onstrating that growth factor stimulated stem lationship between gammaretroviral integration cells ex vivo under serum free conditions do not and gene expression profiles of immature stem lose their repopulating capacity and actually can cells. The results have provided novel insights be amplified ex vivo. This is now further imple- into retroviral transgenesis and the effects of mented in preclinical evaluation protocols. State- retroviral integrations on stem cell behaviour, as of-the-art preclinical assay systems have been well as yielded novel insights into stem cell biol- made available to the project, which include pri- ogy. These results provide a solid basis for safety mary cell culture models, murine models of regu- evaluation of the new generation of vectors lar and genetically altered haematopoiesis, and constructed. immunodeficient mice for the preclinical study of human haematopoiesis, to be validated by stud- Elaborating from our paradigms of successful ies in non-human primates if ethically justified. clinical studies and side effect mana¬g¬¬e¬ment, These are needed to address how the quality of the project is addressing a number of inherited cell purification, novel culture conditions, vector disorders for which a sufficient conventional improvements and cell application protocols can therapy is not available besides allogeneic bone be improved, to achieve long-term polyclonal re- marrow transplantation (alloBMT). Several of constitution of haematopoiesis in vivo. the diseases chosen are paradigms for a selective advantage of gene-modified cells (SCID-X1, Leukaemia induction is still a rare complication ADA-SCID, Wiskott-Aldrich-Syndrome, Fanconi of stem cell gene therapy, which requires the anemia), while others (chronic granulomatous simultaneous existence of clonal dominance disease, thalassemia, Diamond-Blackfan anemia) (which might even contribute to the therapeu- require stronger preparation of the patient for tic effect of retroviral gene transfer) of otherwise cell transplantation or the co-expression of a lin- benign haematopoietic cells and additional eage-restricted therapeutic gene with a selecta- cancerogenic signal alterations. Importantly, the ble gene operative at the level of stem cells. partners have successfully established various

94 New Therapies – Gene Therapy Gene Therapy

Animal models are available for all disorders, al- be a major educational event for the post-docs lowing disease-specific preclinical potency and and PhD students working within the project. In safety evaluation. Among the clinical targets those meetings, the ethical reflection has been chosen, thalassemia ultimately has the greatest implemented as a plenary workshop for all partic- social and healthcare impact. The metabolic stor- ipants. Two ethics conferences, directed at estab- age disorder metachromatic leukodystrophy lishing a European consensus for the ethical con- and Wiskott-Aldrich-Syndrome, have reached siderations involved in the development of gene the stage of pharmaceutical development pre- therapy for inherited diseases, are scheduled for paring for clinical trial. Members of the consor- 2007. In addition, the consortium participates in tium actively conduct clinical trials for SCID-X1 European training courses in collaboration with and ADA-SCID. other European projects and patient organisations, and periodically approaches the lay press. In future commercialisation development, the The European umbrella patient organisation leading role of European science in gene therapy EGAN has been included as a partner in the sec- development for inherited diseases as represent- ond phase of the project, to lead the patient and ed in CONSERT, also translates into SME activities public information platform of the consortium. with significant potential for intellectual prop- The consortium is maintaining a website for ac- erty management and product development. cess to public and professional information. Three SMEs or non-profit organisations engaged with CONSERT have documented their expertise Major publications in retroviral technology upscaling, clinical application of advanced cell products and process Berglin-Enquist, I., Nilsson, E., Ooka, A., Månsson, standardisation. Moreover, insertion site invento- J.E., Olsson, K., Ehinger, M., Brady, R.O., Richter, J., ries arising from current preclinical and clinical Karlsson, S., ‘Effective cell and gene therapy in a data mining approaches of retroviral transgene- murine model of Gaucher disease’, Proc. Natl. Acad. sis, create the basis for further SME participations, Sci. USA, 2006, 103:13819-13824. E-publ Sept 5. in which key technologies of functional genomics are developed and implemented. The IPR po- Fuchs, M., ‘Gene therapy. An ethical profile of a sition of the consortium has been fully evaluated, new medical territory,’ Journal of Gene Medicine, and alternative models for exploitation of the re- 2006, 8(11): p. 1358-1362. sults are under development. Kustikova, O., Fehse, B., Modlich, U., Düllmann, J., Public interactions, ethical reflections and teach- Kamino, K., von Neuhoff, N., Schlegelberger, B., Li, ing activities are integral components of CON- Z., Baum, C., ‘Clonal dominance of haematopoi- SERT’s scientific and technological objectives. etic stem cells triggered by retroviral gene mark- Expanding the well-documented activities of in- ing’, Science, 2005, 308: 1171-1174 dividual members with national and EU regulatory agencies (including EMEA), patient interest Kustikova, O., Geiger, H., Li, Z., Brugman, M.H., groups, ethical committees and gene therapy so- Chambers, S.M., Shaw, C.A., Pike-Overzet, K., de cieties (including ESGT), CONSERT is implement- Ridder, D., Staal, F.J.T., Keudell, G., Cornils, K., Nat- ing a central ethics project to address potential tamai, K.J., Modlich, U., Wagemaker, G., Goodell, conflicts arising from cutting-edge technology M.A., Fehse, B., Baum, C., ‘Retroviral vector inser- developments in somatic transgenesis. tion sites associated with dominant haematopoietic clones mark “stemness” pathways’, Blood, The annual scientific meetings are designed to 2006, E-publ. Nov 21.

New Therapies – Gene Therapy 95 CONSERT

Montini, E., Cesana, D., Schmidt, M., Sanvito, F., Kinnon, C., Ali, R.R., Thrasher, A.J., ‘Effective gene Ponzoni, M., Bartholomae, C., Sergi Sergi, L., Ben- therapy with nonintegrating lentiviral vectors’, edicenti, F., Ambrosi, A., Di Serio, C., Doglioni, C., Nature Medicine, 2006, Mar;12(3):348-53. von Kalle, C., Naldini, L., ‘Haematopoietic stem cell gene transfer in a tumor-prone mouse model un- covers low genotoxicity of lentiviral vector inte- Coordinator gration’, Nature Biotechnol, 2006, (6):687-96. Gerard Wagemaker Ott, M.G., Schmidt, M., Schwarzwaelder, K., Stein, Erasmus University Medical Centre S., Siler, U., Koehl, U., Glimm, H., Kuhlcke, K., Schilz, Faculty Building, Department of Haematology, Ee 1314 A., Kunkel, H., Naundorf, S., Brinkmann, A., Deich- Dr. Molewaterplein 50 mann, A., Fischer, M., Ball, C., Pilz, I., Dunbar, C., ,Du 3015 GE Rotterdam, Netherlands Y., Jenkins, N.A., Copeland, N.G., Luthi, U., Hassan, PO Box 2040, 3000 CA Rotterdam, Netherlands M., Thrasher, A.J., Hoelzer, D., von Kalle, C., Seger, R., E-mail: [email protected] Grez, M., ‘Correction of X-linked chronic granulomatous disease by gene therapy, augmented by Partners insertional activation of MDS1-EVI1, PRDM16 or SETBP1’, Nature Medicine, 2006, Apr;12(4):401-9. Didier Trono École Polytechnique Fédérale de Lausanne Pike-Overzet, K., de Ridder, D., Weerkamp, F., Baert, Laboratory of Virology and Genetics M.R., Verstegen, M.M., Brugman, M.H., Howe, S.J., Lausanne, Switzerland Reinders, M.J., Thrasher, A.J., Wagemaker, G., van Dongen, J.J., Staal, F.J., ‘Gene therapy: is IL2RG on- Christopher Baum cogenic in T-cell development?’ Nature, 2006, Sep Hannover Medical School 21;443(7109):E5; discussion E6-7. Department of Haematology Haemostaseology and Oncology Thrasher, A.J., Gaspar, H.B., Baum, C., Modlich, U., Laboratory of Experimental Cell Therapy Schambach, A., Candotti, F., Otsu, M., Sorrentino, Hannover, Germany B., Scobie, L., Cameron, E., Blyth, K., Neil, J., Abina, S.H., Cavazzana-Calvo, M., Fischer, A., ‘Gene thera- Christof Von Kalle py: X-SCID transgene leukaemogenicity’, Nature, National Centre for Tumour Diseases (NCT) 2006, Sep 21;443(7109):E5-6; discussion E6-7. Heidelberg, Germany

Verhoeyen, E., Wiznerowicz, M., Olivier, D., Izac, B., Klaus Kuehlcke Trono, D., Dubart-Kupperschmitt, A., Cosset, F.L., European Institute for Research and Development of ‘Novel lentiviral vectors displaying “early act- Transplantation Studies AG ing cytokines” selectively promote survival and Oberstein, Germany transduction of NOD/SCID repopulating human haematopoietic stem cells’, Blood, 2005, Nov Michael Fuchs 15;106(10):3386-95. Institut für Wissenschaft und Ethik Section of Biomedical Ethics Yanez-Munoz, R.J., Balaggan, K.S., MacNeil, A., Bonn, Germany Howe, S.J., Schmidt, M., Smith, A.J., Buch, P., Ma- cLaren, R.E., Anderson, P.N., Barker, S.E., Duran, Y., Bartholomae, C., von Kalle, C., Heckenlively, J.R.,

96 New Therapies – Gene Therapy Gene Therapy

Thomas M. Pohl Luigi Naldini GATC Biotech AG Fondazione Centro San Raffaele del Monte Tabor Konstanz, Germany DIBIT – TIGET Milan, Italy Jordi Barquinero Centre de Transfució i Banc de Teixit Maria Grazia Roncarolo Unitat de Diagnostic i Terapia Molecular Università Vita-Salute San Raffaele Barcelona, Spain Faculty of Medicine Milan, Italy Juan A. Bueren Centro de Investigaciones Energeticas Mediambientales Louise van den Bos Y Technolgicas Science & Technology Transfer Haematopoiesis Programme Rotterdam, Netherlands Madrid, Spain Stefan Karlsson François-Loïc Cosset Lunds Universitet INSERM Unit 412 Laboratory of Medicine Laboratoire de Vectorologie Retrovirale et Medical Faculty Therapie Genique –ENS Moleculair Medicine and Gene Therapy Lyon, France Lund, Sweden

Alain Fischer and Marina Cavazzana-Calvo Adrian Thrasher INSERM Unit 429 University College London Hopital Necker Enfants Malades Molecular Immunology Unit, Institute of Child Health Paris, France London, UK

Anne Galy Mary Collins Généthon University College London Evry, France Immunology and Molecular Pathology London, UK William Saurin Genomining George Vassilopoulos Montrouge, France Foundation of Medical and Biological Research of the Academy of Athens Nicholas Anagnou Laboratory of Cell and Gene Therapy, Centre of Basic National and Kapodistrian University of Athens Research Basic Sciences, University of Athens School of Medicine, Athens, Greece Laboratory of Biology Athens, Greece Manuel Grez Georg-Speyer Haus Fulvio Mavilio Frankfurt, Germany Molecular Medicine S.p.A Discovery Cor Oosterwijk Milan, Italy European Genetic Alliances Network Soestdijk, Netherlands

New Therapies – Gene Therapy 97 GIANT Gene therapy: an integrated approach

Contract No LSHB-CT-2004-512087 Project type Integrated Project EC contribution e 9 700 000 Starting date 1 January 2005 Duration 60 months Website www.giant.eu.com

Background and objectives: the transgene, and duration and stability of its expression all influence final vector choice and Screening for prostate cancer is likely to have design. With ongoing research and discoveries in an impact on the spectrum of disease, changing the field of molecular genetics, it is expected that the distribution towards organ-confined disease new generations of genetic vectors will be essen- that is susceptible to radical surgery, radiothera- tial for the translation of molecular research into py, and in 80% of cases, anti-androgen therapy. clinical practice. However, even with such improvements and ad- vancements, the disease burden in Europe in an ageing male population will be substantial, con- sidering the expense involved in screening as well as in radical surgery (with impotence as the most common consequence and incontinence encountered in 3-5% of cases).

In the longer term, when the tumour has escaped the prostatic capsule, and anti-androgen therapy has failed, the tumour returns and is almost in- variably fatal within 18-24 months. At this stage, most conventional cytotoxic therapies are ineffective, although immunotherapeutic approaches and taxotere treatment have shown some promise. Even these improvements result in little more than a two-month extension of life.

A novel gene-based therapy, which can take ac- EGFP expressing virus Infected xenografts count of the heterogeneity of the disease, will of- Adenovirus CMV EGFP fer the possibility of extended life of good quality, even if all of the tumour cells are not destroyed. Approach and methodology: To be a successful vehicle for gene therapy, each vector type (both viral or non-viral) has its own The GIANT project will provide an international particular qualities and limitations, and its degree resource to apply innovative technologies for the of development also depends on the intended modification of existing gene therapy vectors, fo- medical application. The choice of the target, cusing on increased prostate targeting and de-

98 New Therapies – Gene Therapy Gene Therapy

creased vector immunogenicity by ‘stealthing’. gene therapy (human adenovirus vectors); The pre-existing and innate immune response • prevention of attachment of retarget- against viruses requires the inoculation of large ed vectors to cells and matrix elements amounts of virus to produce a therapeutic effect within blood vessels and other tissues and with a high cost per treatment. The GIANT modi- organs, to permit ultimate intravenous ap- fied vectors will be tested in uniform in vitro and plication of gene therapy, and minimise in vivo models of human prostate for improved treatment side effects; efficacy, prior to GIANT sponsored and organised • promotion of gene therapy as a valid Phase I trials. anti-cancer strategy by early design and conduction of a Phase I trial, using the first However, this translational action will also serve generation of viral vectors. as a template for the development of innovative treatments based on gene therapy, which can be Main findings: applied in generic terms to other tumour types, using appropriate, tumour-specific targeting. The project partners have significantly improved The project also seeks to harmonise the regula- the technology to incorporate polypeptide-lig- tions for clinical trials in Europe, to encourage ands in the HI loop of the adenovirus fibre. Fur- multicentre, multinational clinical trials of cancer thermore, adenovirus fibres have been entirely gene therapy using common stocks of GLP grade replaced by a single-chain TCR and the viral pro- viruses as a ‘gene medicine’ in the same way as tein IX molecules have been fused with scTCRs, small molecules can now be tested in the clinic. with affibodies, as well as with scFv fragments. This technology allows the modified viruses to Expected outcome: bind with enhanced affinity to new target cells (e.g.prostate cancers) and to target the delivery The GIANT consortium seeks to secure the of therapeutic genes. following: New molecules have been designed using ad- • generation of vectors which prefer- vanced polymer chemistry, to de- and re-target entially attach to and penetrate prostate adenoviruses by coating the viral particles. The tumour cells; effectiveness of these novel polymer coated • preferential expression of optimised vectors has been determined in vivo. A novel hu- therapeutic genes in prostate tumour man phage display library has been generated cells, relative to other cell and tumour to provide new anti-PSMA scFv, Affibodies and types; MHC-restricted Fab fragments directed against • combination of the above to eliminate the Prostate-Specific Membrane Antigen. Good the expression of the prototype therapeu- manufacturing practice grade polyethylene- tic genes (HSVtk), and growth of oncolytic imine has been generated to initiate clinical trials adenoviruses in adjacent tissues; of non-viral vectors. • optimisation of therapeutic gene/prodrug combinations to maximise thera- GIANT has also achieved the retargeting of non- peutic effects; viral vectors with cell-specific receptors and hy- • further development of advanced drolytic degradation/reduction. Efficacy of the non-viral vector systems, and assessment Ad[I/PPT-E1A] prostate-specific oncolytic adeno- of their activity preclinically in controlled virus has been determined in vitro and in vivo as comparisons with the currently accepted a prelude to the first GIANT clinical trial. Finally, gold standard vector system for cancer logistics for a three-country phase I trial of the

New Therapies – Gene Therapy 99 GIANT

Ad[I/PPT-E1A] prostate-specific oncolytic adenovirus are mostly complete, and clinical grade material has been ordered for the trial.

Major publications

Kreppel, F., Gackowski, J., Schmidt, E., Kochanek, S., ‘Combined Genetic and Chemical Capsid Modi- fications Enable Flexible and Efficient De- and Retargeting of Adenovirus Vectors’, Molecular therapy: The Journal of the American Society of Gene Therapy, 2005, July: 12(1): 107-17

Kraaij, R., van Rijswijk, P., Oomen, M.H.A., Haisma, H.J., Bangma, C.H., ‘Prostate Specific Membrane Antigen (PSMA) Is a Tissue-Specific Target for Ad- enoviral Transduction of Prostate Cancer In vitro’, The Prostate, 2005, February 15; 62(3): 253-9

Vellinga, J., De Vrij, J., Myhre, S., Uil, T., Martineau, P., Lindholm, L., Hoeben, R.C., ‘Efficient Incorpora- tion of a Functional Hyper-Stable Single-Chain Antibody Fragment Protein-IX Fusion in the Ad- enovirus Capsid’, Gene Ther, 2007, Apr;14(8):664- 70. Epub 2007 Feb 1.

Dissemination ‘New treatments for an old man’s disease’ The House, Maitland, N.J., 2007, 32: 36-37.

Patents

100 New Therapies – Gene Therapy Gene Therapy

Coordinator Leif Lindholm Got-a-Gene AB Norman J. Maitland Kullavik, Sweden University of York YCR Cancer Research Unit Wystke van Weerden Department of Biology Scuron York YO10 5YW, UK Rotterdam, Netherlands E-mail: [email protected] Norman Maitland Scientific coordinator Procure York, UK Ellen Schenk-Braat Erasmus MC University Medical Centre Ernst Wagner Department of Urology H1072 Ludwig-Maximilians-Universitat Dr Molewaterplein 40 Munich, Germany 3015 GD Rotterdam, Netherlands E-mail: [email protected] Jean-Paul Behr Université Louis Pasteur de Strasbourg Partners Illkirch, France

Chris Bangma Patrick Erbacher Erasmus MC University Medical Centre Polyplus Transfection Rotterdam, Netherlands. Illkirch, France

Magnus Essand Kerry Fisher Uppsala University Hybrid Systems Uppsala, Sweden Oxford, UK

Len Seymour University of Oxford Oxford, UK

Rob Hoeben Leiden University Medical Centre Leiden, Netherlands

Stefan Kochanek University of Ulm Ulm, Germany

Karel Ulbrich Academy of Sciences of the Czech Republic Prague, Czech Republic

New Therapies – Gene Therapy 101 BACULOGENES Use of baculovirus as a vector for gene therapy

Contract No LHSB-CT-2006-037541 Project type Specific Targeted Research Project EC contribution e 2 499 746 Starting date 1 January 2007 Duration 36 months

Background and objectives: BV technology has been used for years for pro- Gene therapy is a technique that delivers thera- ducing recombinant proteins, and thus large- peutic nucleic acids into somatic cells; it is one scale production technology is readily adaptable of the most promising therapeutic methods for the exploitation of gene therapy approaches. under development for treating a large scope In addition, BV vectors can be used efficiently for of pathologies, ranging from genetic disorders producing other gene therapy vectors such as (e.g. myopathies) to degeneration syndromes AAVs. The BV genome is well-known, and several or cancers. The potential of gene therapy has selective targeting approaches engineered into not yet been fully exploited, mainly because of the virus envelope and capsid have been devel- significant limitations related to the safety, gene oped. delivery capacities and several other properties of the currently used vectors. BACULOGENES aims to develop clinically suitable methods for the development, production, Baculoviruses (BVs) are insect pathogenic DNA vi- testing and validation of next-generation stabi- ruses and are not known to replicate in mamma- lised and selective BV vectors for gene therapy lian cells; this gives them an advantage in terms applications, as well as to optimise the produc- of safety over classical mammalian viruses cur- tion of new AAV serotype vectors. Target diseases rently used as vectors, like the adeno-associated for in vivo gene delivery with selectively targeted virus (AAV), the adenovirus, murine retroviruses BV include muscle disorders, age-related macular and lentiviruses. The most promising baculovirus degeneration and prostate cancer. for gene therapy is the well-known Autographa californica multiple nucleopolyhedrovirus (Ac- The BACULOGENES consortium consists of eight MNPV). It is inherently safe and can deliver large partners from six countries, including pioneers in pieces of DNA on its genome (≥50 Kbp). the use of BVs for mammalian gene transfer applications, and two major established gene therapy BV replication and virus production does not occur vector-producing companies in the EU. The con- in mammalian cells and BV is not known to be asso- sortium will devote its efforts not only to BV gene ciated with any human disease. However, by using therapy applications, but also to the development a vertebrate active expression cassette as a part of of large-scale production, downstream process- baculovirus genome, efficient gene expression can ing, purification and analysis methods. The quality also be directed in non-target cells. A large range of control and validation assays, and all issues relat- vertebrate cells has been shown to be permissive ed to regulatory aspects required for the clinical for AcMNPV transduction, both in vitro and in vivo. exploitation of BV technology, will be covered.

102 New Therapies – Gene Therapy Gene Therapy

Approach and methodology: Coordinator

The innovative strategy of BACULOGENES relies John Martin on the original engineering of BVs (AcMNPV) to Ark Therapeutics Ltd make them safe, specific and efficient vectors for 79 New Cavendish Street gene therapy. BACULOGENES’ approach consists London W1W 6XB, UK of stabilising the BV genome through deletion E-mail: [email protected] and insertion of specific sequences, in combination with transgenes relevant to the disease Scientific coordinator targeted. The ‘stabilised’ BVs will then be engineered as follows: (a) to enhance the capacity to Anssi Mähönen deliver the therapeutic gene(s) to the right cells, Ark Therapeutics Oy (b) to optimise the therapeutic gene expression Kuopio, Finland in the right cells, and (c) to make BVs less immu- E-mail: [email protected] nogenic and potentially invisible for the immune system (stealth virus). Such improvements will be Partners obtained through envelope protein, capsid and genome modifications. Nick Hunt Altonabiotec In parallel, the baculovirus expression system Hamburg, Germany will be optimised for the production of different serotypes of AAV by using stabilised constructs. Paula Alves In addition, the development of baculovirus con- Instituto de Biologia Experimental e Tecnologica (IBET) structs allowing the production of recombinant Oeiras, Portugal AAV without the concomitant production of baculovirus is planned. Monique M. van Oers Wageningen University Expected outcome: Laboratory of Virology Wageningen, Netherlands BACULOGENES will strengthen European leadership, knowledge and competitiveness in BV Kari Airenne technology, through the delivery of novel and University of Kuopio validated efficient vectors, as well as in platform Kuopio, Finland technology, for the exploitation of potential clinical applications of gene therapy. This project will Lindsay Georgopoulos thus pave the way for BVs to be used in clinical University of York applications, an area not yet explored with ex- YCR Cancer Research Unit periments, and will provide an optimised BV- York, UK based AAV platform for gene therapy purposes. The project will also offer the biotech communi- Otto-Wilhelm Merten ty a highly efficient manufacturing process, and Généthon associated QC methods for BVs processing. Evry Cedex, France

New Therapies – Gene Therapy 103 THOVLEN Targeted herpesvirus-derived oncolytic vectors for liver cancer European network

Contract No LSHB-CT-2005-018649 Project type Specific Targeted Research Project EC contribution e 2 494 460 Starting date 1 January 2006 Duration 36 months Website http://www.thovlen.eu

Background and objectives: Another novelty is related to the ability of the HSV-1 vectors to permit a sophisticated and flex- THOVLEN seeks to develop safe and efficient ible combined approach against HCC. That is, in herpes simplex virus type 1 (HSV-1)-derived on- addition to optimising the oncolytic properties colytic vectors, designed to strictly target and of HSV-1 vectors, THOVLEN will exploit the very eradicate human hepatocellular carcinomas large transgenic capability of HSV-1, to generate (HCCs), the most common liver cancer in adults. vectors that will simultaneously display multiple HSV-1 is certainly one of the most promising vi- and multimodal anti-tumour activities acting ral platforms for the development of improved either locally or systemically. These include com- oncolytic vectors, as anticipated by the unique bined expressions of anti-angiogenic, immune- biological properties of this virus and confirmed modulatory, and oncolytic proteins. by the encouraging results generated by clinical trials in gliomas. However, the first generations of Approach and methodology: oncolytic HSV-1 vectors have also shown limitations regarding efficacy and safety. THOVLEN will design HCC-targeted virus vectors that will simultaneously display multiple New generations of innovative HSV-1 vectors, targeting elements acting at different steps of with improved potency and safety, are required the virus life cycle, in order to ensure maximum before the oncolytic strategy using HSV-1 be- aggressiveness for HCC cells with minimum or comes a standard therapeutic reality in the fight no virulence for healthy tissues. Moreover, the against cancer. This is the goal of THOVLEN. One unique advantage of the HSV genome to carry of the most important innovative contributions about 40 kbs of foreign DNA will be exploited in of this project concerns the overall approach to- the context of designing a multimodal approach wards the advancement of HSV-1-based oncolytic for cancer therapy, required for improvement of viruses. Instead of focusing on the development the inherent anti-tumour activity of the virus. of vectors carrying deletions, and in particular virus genes, THOVLEN will engineer competent, The availability and expertise in the use of sev- but replication-restricted HSV-1 vectors, strictly eral well-defined animal models for liver cancer, targeted to HCC. These vectors will combine will allow the consortium to evaluate the safety multiple HCC-targeting approaches, both at the and efficacy of their vectors in relevant systems. level of entry and at the level of gene expression Through fundamental research, they will gener- and replication, and will be able to multiply and ate novel genomic and proteomic information spread only in HCC, while displaying no virulence on the interactions between the oncolytic vec- in normal healthy tissues. tors and the normal and cancer cells, which will

104 New Therapies – Gene Therapy Gene Therapy

guide the rational design of vectors targeted to The first of these recombinant vectors has al- HCC cells. The emerging results will be the im- ready been achieved and THOVLEN is currently provement of the vector oncolytic potency and studying their biological properties. At the same of safety. Different ways of producing HSV-1 vec- time, animal models and immune tools that will tors conceived to specifically penetrate, express allow assessments of the anti-oncogenic proper- genes, and replicate into HCC will be investigat- ties, the toxicity and the immunogenicity of the ed, thus killing these cells and allowing the virus vectors, have been created. to spread and infect other tumour cells. Lastly, THOVLEN is now conducting proteomic These vectors should be unable to replicate and and transcriptomal studies to compare infected destroy normal surrounding cells. In addition versus non-infected hepatomas, in order to iden- to their inherent targeted oncolytic potential, tify changes in the expression patterns of the these vectors will express enhancing transgenic infected cells, as well as a way to identify novel sequences, encoding immune-modulators, anti- activated promoters that could eventually allow angiogenic molecules, fusion proteins, or toxic improving the targeting of the oncolytic vectors. proteins, which are expected to have an additive With several collaborations forged with hospitals negative effect on tumour growth. in France and Greece, fresh human hepatocytes have been secured, helping to serve to evaluate Expected outcome: the impact of the oncolytic vectors in these cells, and to confirm that the vectors can replicate and Once the project ends, the consortium expects disseminate in cultured hepatomas, but not in to produce a number of HSV-1-based oncolytic cultured hepatocytes. vectors specifically targeted to treat HCC. These vectors will combine targeting and enhancing functions, which will be evaluated for efficacy and toxicity on different HCC animal models, including standard and transgenic mice, as well as woodchucks. The potential applications of the results and observations generated by THOVLEN concern the development of novel strategies for the treatment of primary liver cancers of humans.

Main findings and results:

In the first year of the project, the consortium succeeded in constructing the majority of transcription units expressing essential HSV-1 genes under the control of cancer-specific promoters. They also set up transcription units expressing HSV-1 glycoproteins that were modified to allow specific entry of the vectors in liver cells. These genes are currently being introduced into the HSV-1 genome so as to generate the recombinant viruses.

New Therapies – Gene Therapy 105 THOVLEN

Coordinator

Alberto Epstein Centre de Génétique Moléculaire et Cellulaire Université Claude Bernard Lyon 1 Lyon, France E-mail: [email protected]

Partners

Roberto Manservigi Dipartimento di Medicina Sperimentale e Diagnostica Sezione di Microbiologia Università degli Studi di Ferrara Ferrara, Italy

Thomas Brocker Ludwig-Maximilians-Universitaet Muenchen Institute for Immunology Munich, Germany

Penelope Mavromara Hellenic Pasteur Institute Athens, Greece

Ruben Hernandez Alcoceba Fundacion para la investigacion medica aplicada (FIMA) Pamplona, Spain

Fernando Corrales Fundacion para la investigacion medica aplicada (FIMA) Pamplona, Spain

Andres Crespo Genopoietic Miribel, France

Jean-Jacques Diaz Centre de Génétique Moléculaire et Cellulaire CNRS – UMR 5534 Université Claude Bernard Lyon I (UCBL) Villeurbanne, France

106 New Therapies – Gene Therapy Gene Therapy THERADPOX Optimised and novel oncolytic adenoviruses and pox viruses in the treatment of cancer: virotherapy combined with molecular chemotherapy

Contract No LSHB-CT-2005-018700 Project type Specific Targeted Research Project EC contribution e 2 411 006 Starting date 1 December 2005 Duration 36 months Website www.theradpox.org

Background and objectives: inherent strong oncolytic potencies and safety records, pox virus- and adenovirus-based vectors Today, cancer is the second-leading cause of dis- have been chosen as the vehicle for pursuing the ease-induced mortality worldwide. Among the goal of the project, which is to improve the safety innovative treatments for cancer, virotherapy and therapeutic efficacy of OVsin vivo. Novel and holds great promise. Oncolytic viruses (OVs) are improved OVs will be engineered, with the fol- replicating micro-organisms (viruses) that have lowing traits in vivo: been selected or engineered, so as to grow in- • to specifically target colorectal, pan- side and kill tumour cells. It is well known that as creatic and ovarian cancer cells; a tumour evolves, mutations in multiple genes • to replicate exclusively in cancer cells contribute to the malignant phenotype. OVs that are armed with therapeutic genes specifically target cancer cells, because they are rendering only tumour cells sensitive to able to exploit the very same cellular defects that chemotherapy; promote tumour growth. • to widely spread within the tumour to permit total tumour eradication. Although the number of different types of OVs that have been tested in preclinical trials is grow- Through the proposed multidisciplinary work ing, only a few have made the transition into the plan and the strong and complementary ex- clinic, limiting the available clinical experience. pertise of the 10 European partners involved, These new therapeutics are therefore still the THERADPOX will succeed in performing the fol- subject of research as the scientific community lowing objectives: develops means to optimise their efficacy. • generate advanced knowledge that could be translated in safer cancer treat- The THERADPOX project focuses on specific on- ments with an increased therapeutic in- colytic vectors such as pox virus and adenovirus- dex; based vectors, owing to their inherent strong • contribute to the improved quality of oncolytic potencies and safety records. THERAD- life of cancer patients by offering fewer POX aims to improve the safety and therapeutic treatments with no toxic side effects; efficacy of OVs in vivo. • propose new guidelines and standards for the development of OVs; Ultimately, THERADPOX targets the improve- • strengthen the competitiveness of Eu- ment of cancer treatment, for which there is a rope in the war against cancer. high unmet medical need and where available effective modalities are missing. Owing to their

New Therapies – Gene Therapy 107 THERADPOX

Approach and methodology: Adenovirus and Pox viruses Normal cells Cancer cells Common strategies and methodologies were defined and adapted for OVs in the De-targeting • Mutations viral surface proteins THERADPOX project. The establishment and harmonisation of common strategies Normal cells Cancer cells related specifically to the following: vector • Surface expression of ligands which bind engineering, production and purification of Targeting to cancer cells receptors oncolytic Pox and Ad; reporter genes; quality controls; standardisation of therapeutic Normal cells Cancer cells potency and tumour selectivity; methods • Promoter mutations for tumour cell infection in vitro; common Replication • Gene deletion in vitro evaluation of the functionality of Normal cells FCU1; tumour models; and standardised

analysis of data (all partners). Cancer cells • Mutations Expected outcome: Spreading • Gene insertion • Gene deletion

It is anticipated that THERADPOX will Tumour cell fusion achieve the development of oncolytic vectors with increased tumour selectivity Arming • Gene insertion: suicide FCU1 gene of infection, replication and viral spread Normal cells through the tumour tissue that will subse- Prodrug quently enter clinical development. Cancer cells Drug Testing Main findings:

The findings of the project are set out be- Tumour cell lines In vivo: Human tumours and and biopsies in vitro metastasis in mice low: • Enhanced tumour infectivity and tumour selective replication con- • Confirmed selective replication of the cerning oncolytic adenoviruses through myxoma virus in tumour cells and their liver de-targeting and tumour targeting. lack of replication in normal cells concern- • Engineering of various optimised on- ing oncolytic Pox viruses. This set the basis colytic adenoviruses. These chimeric viral for further molecular engineering of these capsids were constructed in such a way viruses, so as to enhance their safety and as to target transcriptional deregulated selectivity for selective tumour cell kill- pathways in tumours; specifically, the E2F ing. Oncolytic vaccinia viruses were engi- pathway was targeted in most tumour neered, harbouring gene deletions, with types and the Wnt pathway in colon can- increased selectivity for tumour cells. cer cells. These engineered adenoviruses • Increased spreading of an oncolytic also exhibit increased replication in tu- adenovirus carrying a fusogenic protein mour cells as well as a significantly im- through tumour cells in culture, aiming proved tumour-to-liver ratio in vitro and in for increased spreading of OVs through vivo. the tumour tissue. Oncolytic adenoviruses

108 New Therapies – Gene Therapy Gene Therapy

targeting stromal barriers in tumour tis- Coordinator sue have also been engineered. • “Armed” oncolytic adenoviruses with a Monika Lusky gene coding for a non-toxic prodrug con- TRANSGENE SA verting enzyme, FCUI, targeting improved 11 rue de Molsheim therapeutic efficacy of OVs. 67082 Strasbourg Cedex, France • Testing of various formulations to al- E-mail: [email protected] low for controlled release of OVs for enhanced safety of OVs. • Selection of various tumour models for Partners the in vivo testing of selected candidates. • Development of a software applica- Ramon Alemany tion, allowing for a fast, sharp and stand- Institut Català d’Oncologia ardised analysis of data. The website www. Barcelona, Spain theradpox.org will enable the dissemination of results to the scientific, clinical and Stéphane Bertagnoli citizen communities. Institut National de la Recherche Agronomique Toulouse, France Conclusion: Richard Iggo Various model oncolytic Pox and adenoviruses University of St Andrews have been generated as proof of concept to- Bute Medical School wards the development of oncolytic vectors with St Andrews, UK increased tumour selectivity of infection, replication and viral spread through the tumour tissue. Harry Jalonen The results obtained during the first year of the DelSiTech Ltd project have set the groundwork for the devel- Turku, Finland opment of viral vectors with improved therapeutic efficacy. Therefore, selected oncolytic pox and Akseli Hemminki adenoviruses will be used to screen their tumour University of Helsinki selectivity in a panel of colorectal, ovarian and Helsinki, Finland pancreatic tumour models during the period following the THERADPOX project. Mohammed Benbouchaib NewLab Villers Les-Nancy, France

Gerd Sutter Paul Ehrlich Institut Langen, Germany

Victoria Smith Oncotest GmbH Freiburg, Germany

New Therapies – Gene Therapy 109 RIGHT RNA interference technology as human therapeutic tool

Contract No LSHB-CT-2004-005276 Project type Integrated Project EC contribution e 11 202 230 Starting date 1 January 2005 Duration 48 months Website www.ip-right.org/

Background and objectives: There are two main possibilities to apply RNAi: small interfering RNAs (siRNAs) that are deliv- The research initiative RIGHT aims at exploiting ered directly into cells as naked siRNAs or with and further developing the vast potential of RNA the help of a transfection reagent are used, while interference (RNAi) to provide effective thera- the other possibility is to use small hairpinRNAs peutic tools for the treatment of severe diseases, (shRNA) that are encoded by a vector and de- based on an advanced understanding of their livered into the cell, and are thereby expressed underlying mechanisms.RNA interference is a using the machinery of the cell itself. As a new naturally occurring mechanism that regulates technology, RNAi has revolutionised basic re- gene expression and is naturally employed by the search by enabling gene function analysis and organism as defence against viruses. Short (21- by providing potential new therapeutic strate- 23 bp) double-stranded RNA is used to induce gies. Only recently, the Nobel Prize in Medicine the sequence-specific degradation of mRNA and was awarded to Craig C. Mello and Andrew Z. Fire thereby block the synthesis of the corresponding for ‘RNA interference – gene silencing by double- protein. stranded RNA’.

Approach and methodology:

As a technique to control gene expression, RNAi has the potential to specifically regulate the production of disease-associated genes. For application as a therapeutic tool in vivo, it is necessary to overcome technical challenges, such as insufficient uptake or low stability of the inhibitors, undesired interferon response or non-specific silencing of other genes. To address the multiple facets of therapy development, the RIGHT project is divided into 5 competence domains. Experts from various scientific fields are working together to exploit the vast potential of this interesting new technology and to make the application of RNAi as a therapeutic tool possible, in a multidisciplinary way.

110 New Therapies – Gene Therapy Gene Therapy

1) Molecular Mechanisms and Technologies: The understanding of the molecular processes associated with RNAi and the naturally occurring counterpart microRNA is improved. This knowledge serves as a basis for the development of novel molecular strategies enabling the successful application of RNAi technology for human therapy. Use is made of large-scale RNAi libraries and high-throughput screening technologies to identify new target genes for therapeutic approaches and to analyse identified inhibitors.

2) Chemical Tools: New chemically modified siRNAs are synthesised and extensively tested in cell culture and living Overview of the five competence domains in RIGHT organisms in order to increase sensitivity, specificity, delivery, stability and cost-effectiveness, and with pharmacokinetic methods. Successful can- to reduce side effects. didates are then assessed in animal models, and extensive phenotyping of treated animals will be 3) Genetic Tools: performed. Potent viral or non-viral RNAi delivery vectors are generated and their features evaluated in 5) Cell Biology and Disease Models: relation to their chemical counterparts. Special Selected disease models are used for the para- emphasis is given to the development of tissue- digmatic assessment of RNAi as a therapeutic specific and inducible systems. tool. This generates RNAi leads for clinical tests. In particular, RIGHT focuses on infectious diseases 4) Pharmacokinetics: and genetic defects causing degenerative dis- For the development of a drug, synthetic or ge- eases and cancer. netic RNAi, reagents are tested in cultured cells Expected outcome:

Within 4 years, the potential of RNAi to diagnose and successfully treat diseases will be demonstrated and proof of principle will be provided for the value of RNAi as a therapeutic tool in living organisms.

Main findings:

In the RIGHT project, new tools for the application and evaluation of RNAi, such as random libraries or high throughput techniques have been developed. New microRNAs could be identified and their function analysed in more detail. It could be shown that miR-181 participates in muscle

New Therapies – Gene Therapy 111 RIGHT

models, like influenza and HBV. For these disease models, specific inhibitors were identifiedin vitro. These inhibitors will now be optimised for application in vivo and their effects tested in animal models.

Major publications

Piva, R., Pellegrino, E., Mattioli, M., Agnelli, L., Lom- bardi, L., Boccalatte, F., Costa, G., Ruggeri, B.A., Cheng, M., Chiarle, R., Palestro, G., Neri, A., Ing- hirami, G., ‘Functional validation of the anaplastic Press conference at Max Planck Institute in Berlin, from left lymphoma kinase signature identifies CEBPB and to right: Prof. Dr. Joachim W. Engels, Prof. Dr. Thomas F. Meyer, BCL2A1 as critical target genes’, J Clin Invest, 2006, Prof. Dr. Arndt Borkhardt 116(12):3171-82. cell differentiation (Naguibneva et al. 2005) and that miR-223 is involved in human granulocyte Pal, A., Severin, F., Lommer, B., Shevchenko, A., Ze- differentiation (Fazi et al., 2005). rial, M., ‘Huntingtin-HAP40 complex is a novel Rab5 effector that regulates early endosome mo- In the field of chemically-modified siRNAs, the tility and is up-regulated in Huntington’s disease’, invention of a new design for these inhibitors J. Cell Biol, 2006, 13;172(4):605-18. shows promising results. This new design leads to higher stability of the inhibitors and allows ad- Brown, B.D., Venneri, M.A., Zingale, A., Sergi, L.S., ditional modifications that are under evaluation Naldini, L., ‘Endogenous microRNA regulation in the consortium. These modifications could suppresses transgene expression in haematopoi- help to increase targeting and reduce undesired etic lineages and enables stable gene transfer’, side effects of the compounds. This new design Nat Med, 2006, 12(5):585-91. has already been submitted for patenting. Naguibneva, I., Ameyar-Zazoua, M., Polesskaya, A., Besides the construction of synthetic inhibitors, Ait-Si-Ali, S., Groisman, R., Souidi, M., Cuvellier, S., efforts are undertaken to develop new vector Harel-Bellan, A., ‘The microRNA miR-181 targets systems for the expression of shRNAs. Here, some the homeobox protein Hox-A11 during mamma- first promising results show the possibility to lian myoblast differentiation’, Nat Cell Biol, 2006, use lentiviral vectors that include miRNA target 8(3):278-84. sequences to regulate gene expression in different organs (Brown et al., 2006). With these vec- Varghese, O.P., Barman, J., Pathmasiri, W., Plas- tors, the delivery and segregated expression of hkevych, O., Honcharenko, D., Chattopadhyaya, shRNAs among different tissues is possible and J., ‘Conformationally constrained 2’-N,4’-C-eth- should provide a new tool for more efficient and ylene-bridged thymidine (aza-ENA-T): synthe- safe expression of shRNAs. sis, structure, physical, and biochemical studies of aza-ENA-T-modified oligonucleotides’, J Am In the first two years of the project several disease Chem Soc, 2006, 128(47):15173-87. models were established to assess the potential of RNAi approaches in vivo. These models include Casares, N., Pequignot, M.O., Tesniere, A., Ghiring- different cancer models and infectious disease helli, F., Roux, S., Chaput, N., Schmitt, E., Hamai, A.,

112 New Therapies – Gene Therapy Gene Therapy

Coordinator

Thomas F. Meyer Max-Planck-Institute for Infection Biology Charitéplatz 1 D 10117 Berlin, Germany E-mail: [email protected]

Partners

Thomas Rudel Max Planck Institute for Infection Biology Berlin, Germany

Jørgen Kjems University of Aarhus Aarhus, Denmark

Olli Kallioniemi VTT Technical Research Centre of Finland Turku, Finland

Jesper Wengel Poster from a public symposium organised by RIGHT in University of Southern Denmark Paris, Institut Pasteur in October 2006 Odense, Denmark

Hervas-Stubbs, S., Obeid, M., Coutant, F., Métivier, Jyoti Chattopadhyaya D., Pichard, E., Aucouturier, P., Pierron, G., Garrido, University of Uppsala C., Zitvogel, L., Kroemer, G., ‘Caspase-dependent Uppsala, Sweden immunogenicity of doxorubicin-induced tumor cell death’, J Exp Med, 2005, 19;202(12):1691-701. Annick Harel-Bellan Institut André Lwoff, CNRS Fazi, F., Rosa, A., Fatica, A., Gelmetti, V., De Marchis, Villejuif, France M.L., Nervi, C., Bozzoni, I., ‘A minicircuitry comprised of microRNA-223 and transcription factors Carola Ponzetto and Giorgio Inghirami NFI-A and C/EBPalpha regulates human granulo- University of Torino poiesis’, Cell, 2005, 123(5):819-31. Turin, Italy

Patents Marino Zerial Max Planck Institute of Molecular Cell Biology and One patent application was submitted within the Genetics RIGHT project by Jesper Wengel (Southern Den- Dresden, Germany mark University) and Jørgen Kjems (University of Aarhus) on a new design for small interfering RNAs (Danish Patent Application 2006 00433).

New Therapies – Gene Therapy 113 RIGHT

Michael Roberts Patrick Brian Arbuthnot Regulon University of the Witwatersrand Athens, Greece Johannesburg, South Africa

Joachim Engels Guido Kroemer Johann Wolfgang Goethe-Universität INSERM Frankfurt, Germany Villejuif, France

Piet Herdewijn Arndt Borkhardt Katholieke Universiteit Leuven Heinrich-Heine- Universität Düsseldorf - Universitätsk- Leuven, Belgium linikum Düsseldorf, Germany Luigi Naldini Fondazione Centro San Raffaele del Monte Tabor Milano, Italy

Wlodzimierz Krzyzosiak Polish Academy of Sciences Poznan, Poland

Jörn Glökler RiNA GmbH Berlin, Germany

Irene Bozzoni University of Rome “La Sapienza” Rome Italy

Johan Auwerx Insitut Clinique de la Souris (ICS) Illkirch, France

George Mosialos and George Kollias Biomedical Sciences Research Centre “Al. Fleming” Vari (Attica), Greece

Patrick Erbacher PolyPlus-transfection SA Illkirch, France

Sara Skogsäter ARTTIC Paris, France

114 New Therapies – Gene Therapy Gene Therapy ZNIP Therapeutic in vivo DNA repair by site-specific double-strand breaks

Contract No LSHB-CT-2006-037783 Project type SME-Specific Targeted Research Project EC contribution e 2 349 996 Starting date 1 January 2007 Duration 36 months

Background and objectives: tional gene targeting by HR increases with the length of homology between the donor DNA To date, more that 6 000 human single gene dis- and the target sequence. Although conventional orders have been identified, affecting about 2% gene targeting is an invaluable tool for creating of the population. To cure or prevent disorders transgenic laboratory animals, the technique is caused by single gene defects, direct in situ re- hampered by major limitations, including low pair would be an attractive strategy, particularly targeting frequencies, generally with only one for diseases where the therapeutic approach targeted cell in every 105-107 treated cells. could be based on short sequence substitutions, deletions or insertions. Moreover, in most mammalian cells, the ratio between site-specific homologous recombination Currently, most gene therapy approaches are and non-homologous end joining (NHEJ) is un- based on the delivery of a therapeutic gene favourable. Consequently, until now, gene target- coupled to promoter sequences. Some of the ing has depended on sensitive selection systems therapeutic sequences may be replacements for in order to identify the few cells where gene tar- a mutated gene, while other approaches aim to geting has occurred. Such selection strategies kill or protect particular cells. However, the de- normally require the donor DNA to contain extra livery of genes that are taken from their natural non-homologous sequences coding for selec- context poses a number of problems and limitation markers (often derived from bacteria or vi- tions. Thus, a strategy that corrects, or disrupts, ruses). These can result in undesired side effects a gene in situ is a central biomedical challenge on the cells, as exemplified by down regulation that unlocks entirely new strategies for func- of expression due to de novo methylation. tional genomics, and ultimately, for gene therapy. Direct and site-specific modification of a gene at Designed endonucleases have recently provided its natural locus, without the introduction of ad- a revolutionary tool for achieving high rates of ditional sequences like selection cassettes, offers targeted genome alteration. By stimulating ho- an appealing novel strategy in gene therapy. mologous recombination through the creation of site-specific targeted double strand breaks, in Such gene targeting is based upon the cells’ ca- vivo sequence alteration rates can be obtained. pacity to carry out homologous recombination (HR), a process which results in an accurate and In ZNIP, a major effort is in progress to generate precise exchange of genetic material between tools, both improved zinc finger endonucleases the introduced donor DNA and the homologous and second generation TFOs (Triplex-forming genomic target DNA. The efficiency of conven- oligonucleotides) for targeted sequence altera-

New Therapies – Gene Therapy 115 ZNIP

encounter DNA damage that challenges their genomic integrity, they generally activate DNA repair pathways and cell cycle checkpoint signalling in order to counteract the damage. Moreo- ver, if the DNA damage can not be repaired, cells can activate apoptotic signalling pathways.

Finally, the technology will be tested in primary stem cells and transgenic reporter mice as an important step towards clinical trials.

tion in living cells. The goal of the ZNIP consor- Approach and methodology: tium is to take gene repair protocols to a level where high rates can be obtained with consist- ZNIP will proceed by the following steps: ency, high efficacy and specificity, as well as with • computer-assisted design of new en- strongly reduced levels of side effects. To achieve donuclease specificities and in vitro vali- this goal, the consortium will proceed as follows: dation; • improve and test sequence-specific • development of new bioassays for in DNA zinc finger endonucleases (ZFNs); vivo optimisation of zinc finger endonu- • develop triple helix-forming oligonu- cleases; cleotides (TFOs) that induce high rates; • analysis of pathways involved in tar- • elucidate and constructively improve geted sequence correction with DNA the cellular mechanisms involved in the double strand breaks; process; • application of these pathways for • test the targeted sequence alteration improvement and increase of targeting in primary cells (including stem cells) and rates; in living test animals. • analysis of the cellular responses to targeted sequence alteration: aberrant in- Only limited knowledge is available on the cel- tegration and toxicity; lular pathways and proteins that are involved in • analysis of targeted sequence correc- and/or regulate targeted gene correction after tion in animal models and natural stem site-specific DNA double strand breaks. A bal- cell populations; ance between HR and NHEJ may determine the • analysis and improvement of delivery efficiency of gene correction. However, other and dose control. pathways may also be involved. To improve targeting frequencies and targeting safety (i.e. to Expected outcome: reduce genotoxic side-effects by non-homologous end-joining, for instance), it is of paramount ZNIP anticipates further development of a novel importance to understand the role of cellular fac- technique for targeted sequence alteration in liv- tors in the correcting pathway(s); this work will ing cells as a tool for gene therapy. Designed en- be carried out extensively by the consortium. donucleases are a revolutionary new tool for inducing site-specific genome alterations. They will Furthermore, it is important to establish whether have a significant impact on future gene therapy cellular pathways that could impair the viability protocols, as well as on a broad spectrum of bio- of the targeted cells are activated. When cells technological applications.

116 New Therapies – Gene Therapy Gene Therapy

Coordinator Ralf Wagner GENEART GmbH Stefan Krauss D-93053 Regensburg, Germany Rikshospitalet Section for cellular and genetic therapy Forskningparken Gaustadalleen 21 0349 Oslo, Norway E-mail: [email protected]

Partners

Toni Cathomen Institute of Virology Charité Universitätsmedizin Berlin Campus Benjamin Franklin Berlin, Germany

Klaus Schwarz University Hospital Ulm Transfusion Medicine Ulm, Germany

Luis Serrano Centre de Regulació Genomica Barcelona, Spain

Tom Brown University of Southampton School of Chemistry Highfield, Southampton, UK

Roland Kanaar Erasmus Medical Centre Department of Cell Biology and Genetics Rotterdam, Netherlands

Bengt Nordén Chalmers University of Technology Physical Chemistry Gothenburg, Sweden

ATDBio Ltd Highfield, Southampton, UK

New Therapies – Gene Therapy 117 SNIPER Sequence specific oligomers for in vivo DNA repair

Contract No LSHB-CT-2004-005204 Project type Specific Targeted Research Project EC contribution e 2 035 000 Starting date 1 January 2005 Duration 36 months

Background and objectives: focusing on strategies based on singles stranded oligonucleotides (ssODN) and triple helix form- Gene targeting can be defined as a method for ing oligonucleotides (TFO). introducing site-specific sequence alterations in the genomes of living cells. Successful gene tar- TFOs have demonstrated genome altering activ- geting would have far reaching implications for ity in vitro and in vivo. Improving selective site- the construction of mutant cell lines and animals, specific DNA recognition by TFOs is a central both for studies of gene function as well as for requirement, because the lack of a general recog- the development of disease models. Further- nition code restricts TFO applicability to a limited more, direct modification of a target gene at its number of loci in the genome. The consortium genomic location, without the introduction of is working towards modified bases in the triple- additional redundant sequences, would offer an helix-forming oligonucleotides that tolerate appealing strategy for gene therapy. pyrimidine interruptions and that contribute to increased triplex stability and binding kinetics. Use of gene targeting in order to create transgenic cell lines does not necessarily require high A second class of molecules capable of se- targeting rates as long as powerful selection bio- quence-specific DNA recognition is the peptide essays can be applied to facilitate isolation of the nucleic acids (PNAs). PNAs can recognise DNA by targeted cells. However, for a clinical application three modes: triplex invasion; duplex invasion; or in gene therapy protocols, the introduction of double duplex invasion. Of these, the triplex and selective markers in the target genome is not de- double duplex invasion approaches are of par- sirable and, consequently, such strategies require ticular interest for targeted gene repair and are higher targeting rates. For example, it has been being analysed by the consortium. Using vari- estimated that in order to observe a beneficial ef- ous chemical modifications, the project partners fect from the correction of the mutation under- have improved DNA binding properties of TFOs lying the monogenetic disease haemophilia, at and PNAs. The consortium has also strongly im- least 1%-5% of the treated cells need to express proved the properties of the sequence template the corrected protein. for the targeted repair reaction by a combination of structural improvements and a novel strategy Since targeted sequence alteration in living cells based on an active group transfer. is increasingly turning into a promising tool for gene therapy applications, it is important to ana- By using different mechanisms, ssODN have the lyse the various strategies needed for site spe- potential to achieve site-specific alterations in cific DNA alterations. The SNIPER consortium is the sequence of a genomic DNA template in liv-

118 New Therapies – Gene Therapy Gene Therapy

in the observed targeted repair; • develop improved cellular delivery systems; • provide a thorough analysis of potential side effects.

Expected outcome:

The project will further develop strategies for ing cells. The consortium has shown that ssODN targeted sequence alteration in living cells as a induce significant rates of in vivo sequence cor- tool for biotechnology and gene therapy. rection rates. Main findings: A critical aspect for the further development of gene repair approaches is the elucidation of the High rate site-specific targeted sequence altera- cellular responses needed to achieve targeted tion is likely to prove a viable strategy. Strategies sequence alteration. For this purpose, siRNA- based on a combination of site specific endonu- based screens are carried out in order to identify cleases and selected DNA templates are offering factors involved in gene repair. The consortium substantial promise for future biotechnological also seeks to gain an undestanding of the cellu- and gene therapy applications. lar components of the process by studying those components that make up the cellular mismatch Major publications repair machinery, as well as those of the replication machinery and of the cell cycle checkpoints. Olsen, P.A., Randol, M., Luna, L., Brown, T., Krauss, S., The consortium successfully demonstrated the ‘Genomic sequence correction by single-strand- role of the cell cycle on in vivo targeted sequence ed DNA oligonucleotides: role of DNA synthesis alteration. and chemical modifications of the oligonucle- otide ends’, J Gene Med, 2005, Dec;7 (12):1534-44. Despite the substantial challenges that remain, targeted sequence alteration based on ssODN Olsen, P.A., Randol, M., Krauss, S., ‘Implications of promises to become a part of the standard toolkit cell cycle progression on functional sequence for functional gene alterations. correction by short single-stranded DNA oligonucleotides’, Gene Ther, 2005, 12:546-51. Approach and methodology: Fox, K.R., Brown, T., ‘An extra dimension in nucleic SNIPER aims to: acid sequence recognition’, Q Rev Biophys, 2005, Nov;38 (4):311-20. • improve platform technology for targeted sequence alteration in living viable Alzeer, J., Scharer, O.D., ‘A modified thymine for cells using single strand oligonucleotides; the synthesis of site-specific thymine-guanine • improve TFO modules by chemical DNA interstrand crosslinks’, Nucleic Acids Res, modifications; 2006, 34(16):4458-66. • design and implement new reactions to induce single base pair alterations; Frykholm, K., Morimatsu, K., Norden, B., ‘Conserved • define the cellular pathways involved conformation of RecA protein after executing the

New Therapies – Gene Therapy 119 SNIPER

DNA strand-exchange reaction. A site-specific University of Southampton linear dichroism structure study’, Highfield, Southampton, UK Biochemistry, 2006, Sep 19;45(37):11172-8. Keith R. Fox Hanada, K., Budzowska, M., Modesti, M., Maas, A., School of Biological Sciences Wyman, C., Essers, J., Kanaar, R., ‘The structure- University of Southampton specific endonuclease Mus81-Eme1 promotes Southampton, UK conversion of interstrand DNA crosslinks into double-strands breaks’, EMBO J, 2006, 25:4921-32. Roland Kanaar Erasmus Medical Centre Li, H., Broughton-Head, V.J., Peng, G., Powers, V.E., Rotterdam, Netherlands Ovens, M.J., Fox, K.R., Brown, T., ‘Triplex staples: DNA double-strand cross-linking Bengt Nordén at internal and terminal sites using psoralen-con- Chalmers University of Technology taining triplex-forming oligonucleotides’, Biocon- Gothenburg, Sweden jug Chem, 2006, Nov-Dec;17 (6):1561-7. Peter E. Nielsen Wyman, C., Kanaar, R., ‘DNA double-strand break University of Copenhagen repair: all’s well that ends well’, Annu Rev Genet, Copenhagen, Denmark 2006; 40:363-83. Orlando D. Schärer Kim, K.H., Fan, X.J., Nielsen, P.E., ‘Efficient Sequence- Institute of Molecular Cancer Research Directed Psoralen Targeting Using Pseudocom- University of Zurich plementary Peptide Nucleic Acids’, Bioconjug Zurich, Switzerland Chem, 2007, Jan 26. Dorcas Brown ATDBio Ltd Coordinator Highfield, Southampton, UK.

Stefan Krauss Ralf Wagner Rikshospitalet GENEART GmbH Section for cellular and genetic therapy Regensburg, Germany Forskningparken Gaustadalleen 21 0349 Oslo, Norway E-mail: [email protected]

Partners

Ulf Ellervik Lund Institute of Technology Lund University Lund, Sweden

Tom Brown

120 New Therapies – Gene Therapy Gene Therapy Improved precision Improved precision of nucleic acid based therapy of cystic fibrosis

Contract No LHSB-CT-2004-005213 Project type Specific Targeted Research Project EC contribution e 3 504 000 Starting date 1 December 2004 Duration 26 months Website www.improvedprecision.com

Background and objectives: use optimised compounds in established foetal and adult ΔF 508del mice and in a new (β-ENaC Cystic fibrosis (CF) is a recessive congenital dis- overexpressing transgenic mouse) animal model ease with a high incidence in Caucasian popula- of cystic fibrosis. tions (1 in 3 000 newborns is affected). It is caused by mutations in the gene coding for CFTR (cystic Approach and methodology: fibrosis transmembrane conductance regulator), a chloride channel, which results in decreased This comprehensive approach of interfering with chloride secretion and hyper absorption of so- ENaC expression by delivering siRNA to the air- dium across epithelia. These disturbances in ion ways, includes the following objectives: fluxes result in water hyper-absorption from the 1. Develop compounds able to modulate ENaC airway surface liquid into the cells, which pro- expression by RNA interference: duce a highly viscous and elastic mucus. These • novel formulations of synthetic siRNA changes result in chronic bacterial infection of and plasmid expression systems, the airways, leading to chronic lung disease. • minichromosomes for expressing siRNA, • lentiviral vectors for expressing siRNA; Despite recent developments in the treatment of 2. Establish aerosolisation of such compounds CF there is no definite cure for this disease, and without damaging the compounds. life expectancy is around 30 years. Several obser- 3. Formulate such compounds as magnetic vations suggest that a downregulation of ENaC vectors, in order to make them susceptible to restores the perciliary liquid layer, thereby rehy- magnetic field guidance (“Magnetofection” tech- drating the mucus and improving ciliary clear- nology) and combine the magnetic approach ance in the lung. Therefore, the members of the with aerosolisation. consortium propose to specifically downregu- 4. Develop instrumentation to generate magnet- late ENaC expression by RNA interference. ic gradient fields for magnetic lung targeting. 5. Evaluate and validate the novel formulations The intention is to develop compounds able to and delivery technologies using meaningful modulate ENaC expression, either transiently by models in vitro and in vivo. synthetic siRNA constructs or plasmids expressing siRNA constructs, or long-term by lentiviral Expected outcome: gene transfer for stable chromosomal integration, or minichromosomes expressing siRNA The expected outcome of this project is to dem- constructs. After testing these compounds in onstrate that the downregulation of ENaC sub- murine cell lines, the consortium members will units by RNA interference in airway epithelial

New Therapies – Gene Therapy 121 Improved precision

cells in vitro and in vivo is feasible. Evidence for Xenariou, S., Griesenbach, U., Ferrari, S., Dean, P., efficient downregulation should be shown on Scheule, R.K., Cheng, S.H., Geddes, D.M., Plank, C., the level of mRNA- and protein concentration of Alton, E.W., ‘Using magnetic forces to enhance the three ENaC subunits: α,-β,-, and γ-. In addi- non-viral gene transfer to airway epithelium in tion, a correction of the pathologic sodium hyper vivo’, Gene Ther, 2006. absorption which characterises cystic fibrosis airway epithelia is expected by downregulation of Babincova, M., Babinec, P., ‘Aerosolized VEGF in ENaC-subunits. For the in vivo experiments, a CF- combination with intravenous magnetically tar- mouse model, as well as β -ENaC-overexpressing geted delivery of DNA-nanoparticle complex transgenic mouse model, will be tested. At the may increase efficiency of cystic fibrosis gene end of the project, enough evidence should be therapy’ Med Hypotheses, 2006, 67(4):1002. available to initiate a clinical trial on RNA interference for cystic fibrosis lung disease by targeting Schillinger, U., Brill, T., Rudolph, C., Huth, S., Gerst- ENaC. ing, S., Krotz, F., Hirschberger, J., Bergemann, C., Plank, C., ‘Advances in magnetofection - magneti- Main findings: cally guided nucleic acid delivery’, Journal of Mag- netism and Magnetic Materials, 2005, 293(1):501- Downregulation of ENaC mRNA and sodium 508. transport function in cell culture systems resulting in significant electrophysiologic effects Rudolph, C., Ortiz, A., Schillinger, U., Jauernig, J., on ENaC-mediated sodium uptake, could be Plank, C., Rosenecker, J., ‘Methodological optimi- achieved by RNA interference. Currently, experi- zation of polyethylenimine (PEI)-based gene de- ments in animal models are ongoing to confirm livery to the lungs of mice via aerosol application’, these data. Journal of Gene Medicine, 2005, 7(1):59-66.

OZ Biosciences, a SME partner in this project, as Rudolph, C., Schillinger, U., Ortiz, A., Plank, C., Go- in this project, commercialised three novel mag- las, M.M., Sander, B., Stark, H., Rosenecker, J., ‘Aero- netic nanoparticle formulations based on Mag- solized nanogram quantities of plasmid DNA netofection (SilenceMag, ViroMag and ViroMag mediate highly efficient gene delivery to mouse R/L), and one lipid-based formulation specific to airway epithelium’, Molecular Therapy, 2005, siRNA applications (Lullaby® siRNA transfection 12(3):493-501. reagent).

Major publications

Mykhaylyk, O., Vlaskou, D., Tresilwised, N., Pithay- anukul, P., Moller, W., Plank, C., ‘Magnetic nanoparticle formulations for DNA and siRNA delivery’, Journal of Magnetism and Magnetic Materials, 2007, in press.

Dames, P., Ortiz, A., Schillinger, U., Lesina, E., Plank, C., Rosenecker, J., Rudolph, C., ‘Aerosol gene delivery to the murine lung is mouse strain dependent’, J Mol Med, 2006, Dec 8.

122 New Therapies – Gene Therapy Gene Therapy

Coordinator Holger Schankin and Hans Schreier MCS Micro Carrier Systems GmbH Joseph Rosenecker Neuss, Germany Klinikum der Universität München Kinderklinik und Kinderpoliklinik im Dr. von Haunerschen Olga Zegarra-Moran Kinderspital Laboratorio di Genetica Molecolare Lindwurmstraße 4 Istituto Giannina Gaslini D-80337 München, Germany Genoa, Italy E-mail: [email protected] Peter Babinec and Melania Babincova Partners Comenius University Department of Nuclear Physics and Biophysics Massimo Conese, Elena Copreni Bratislava, Slovakia San Raffaele University Institute for Experimental Treatment of Cystic Fibrosis Detlef Eric Hinz and Lena Grimm - DIBIT Fraunhofer Patent Centre Milan, Italy Munich, Germany

Fiorentina Ascenzioni University of Rome “La Sapienza” Dipartimento di Biologia Cellulare e dello Sviluppo Rome, Italy

Olivier Zelphati OZ Biosciences Parc Scientifique et Technologique de Marseille-Luminy Marseille, France

Charles Coutelle and Suzy Buckley Imperial College London Faculty of Medicine Division of Biomedical Sciences Gene Therapy Research Group London, UK

Christian Plank and Olga Mykhaylyk Institut für Experimentelle Onkologie der TUM Klinikum r. d. Isar Munich, Germany

Bob J. Scholte, Patricia Spijkers Erasmus Medical Centre Cell Biology Department Rotterdam, Netherlands

New Therapies – Gene Therapy 123 INTHER Development and application of transposons and site-specific integration technologies as non-viral gene delivery methods for ev vivo gene-based therapies

Contract No LSHB-CT-2005-018961 Project type Specific Targeted Research Project EC contribution e 2 800 000 Starting date 1 November 2005 Duration 36 months Website www.mdc-berlin.de/izsvak/eng

Background and objectives: Transposable elements are recombinases that can be considered as natural, non-viral delivery vehi- Considerable effort has been devoted to the cles, capable of efficient genomic insertion. The development of gene delivery strategies for the use of transposable elements can address one of treatment of inherited and acquired disorders in the main problems of non-viral technologies: sta- humans. Ex vivo gene therapies are based on re- ble genomic integration provides long-term ex- moving cells from a patient, introducing a thera- pression of therapeutic genes. Using transposon peutic gene construct into the cells, and implant- vectors, integration from plasmid DNA into chro- ing the engineered cells back into the patient. Ex mosomes provides the basis for long-term expres- vivo gene therapy is less cost-effective and more sion of therapeutic genes in treated cells. labour-intensive than in vivo gene therapy, but from a safety perspective, it is potentially more This project will evaluate the available recombi- attractive. nase systems in terms of their efficacy and safety features. INTHER proposes to establish optimal Currently, both viral and non-viral methods are delivery protocols for the therapeutic/recombi- used for gene delivery. Adapting viruses for nase-systems into a variety of cells. It will deter- gene transfer is a popular approach, but safety, mine what type of cells and diseases models are immunogenicity and production issues hamper best suited to the recombinase technology that clinical progress. The establishment of non-viral, can offer an alternative to existing viral and nonintegrating vectors generated considerable in- viral technologies. terest in developing efficient and safe vectors for human gene therapy. Approach and methodology:

INTHER aims to establish and apply methods Novel gene transfer technologies will be estab- and protocols for efficient nucleic acid/protein lished by developing transposon vectors that delivery into therapeutically relevant target cells, mediate efficient and targeted integration of in the context of recombinase technologies. The therapeutic genes into the genome. INTHER aims overall objective of INTHER is to develop non-vi- at developing recombinases tailored for gene ral, recombinase-based technologies for ex vivo therapeutic purposes, by using three different gene therapeutic applications as an alternative recombinase systems: SB, FP and PhiC31. The to current viral/non-viral gene delivery technolo- project will evaluate which recombinase system gies, with the aim of circumventing the toxicity holds the greatest promise for further develop- and immunogenicity problems raised by viral ment, by performing head-to-head comparisons delivery systems. among the three recombinase systems.

124 New Therapies – Gene Therapy Gene Therapy

Figure 1. Experimental strategies for targeting Sleeping Beauty transposition. The common components of the targeting systems include a transposable element that contains the IRs (arrowheads) and a gene of interest equipped with a suitable promoter. The transposase (purple circle) binds to the IRs and catalyzes transposition. A DNA-binding protein domain (red oval) recognizes a specific sequence (turquoise box) in the target DNA (parallel lines). (a) Targeting with transposase fusion proteins. Targeting is achieved by fusing a specific DNA-binding protein domain to the transposase. (b) Targeting with fusion proteins that bind the transposon DNA. Targeting is achieved by fusing a specific DNA-binding protein domain to another protein (white oval) that binds to a specific DNA sequence within the transposable element (yellow box). In this strategy, the transposase is not modified. (c) Targeting with fusion proteins that interact with the transposase. Targeting is achieved by fusing a specific DNA- binding protein domain to another protein (light green oval) that interacts with the transposase. In this strategy, neither the transposase nor the transposon is modified.

Figure 2. Transposon targeting using a strategy based on protein-protein interactions between a targeting fusion protein and the SB transposase. (a) The targeting fusion protein consists of the tetracycline repressor (TetR) that binds to the TRE, a nuclear localization signal (NLS), a glycine-bridge (G) and the N-terminal protein interaction domain of the SB transposase (N-57). (b) Cells were cotransfected with the components of the transposon targeting system, and genomic DNA of pooled transformant cells was subjected to PCR as described in Fig. 5c. The agarose gel shows PCR products obtained from cells transfected with a vector expressing TetR/NLS/N-57 or with TetR/NLS/LexA with primers amplifying the left or the right IR of the transposon. M: size marker. (c) Mapping of targeted SB insertions (arrows) with respect to the TRE-EGFP target isolated from five independent experiments is shown. Multiple arrows represent independent insertions into the same site. Positions of the insertions are indicated below; the numbers correspond to the base pair numbering of the pTRE-d2EGFP plasmid (Clon- tech). (d) Frequency of targeted transposition. The agarose gel shows PCR products obtained from individual, transgenic cell clones, using primer pairs amplifying the right IR of the transposon. A PCR product recovered from pooled (P) DNA samples serves as a reference. M: size marker.

Figure 3. Bioenginered muscle implants

Figure 4. Compared to Mo-MuLV-LTR, Sleeping Beauty IRs exhibit moderate promoter activity. Luciferase-reporter experiments.

Figure 5. The expression of the neighbouring genes is influenced primarily by the promoter activity of the transgene. The shielding effect of the SH4 insulator sequences influences. Luciferase-reporter experiments.

New Therapies – Gene Therapy 125 INTHER

INTHER proposes to improve the performance systems are well established, ‘single therapeutic of transposon vectors by enhancing their transgene’ models. Selective advantage of the treated positional activity and by strengthening their bi- cells either exists or can be provided, using the osafety profiles. The project intends to improve human ABCG2 multidrug transporter protein the performance of the SB and FP systems, to en- as a selectable marker for several models. The sure that the expressed therapeutic gene does treatment of the selected diseases has not been not influence the genes at the integration site. solved by conventional treatments in these mod- It also aims to enhance the activity of the trans- els, and they are currently established to use viral posase by in vitro evolution (DNA shuffling of hy- vectors. The animal disease models of INTHER in- peractive versions of the transposases). clude copper metabolism diseases, anaemia, hy- percholesterolemia, bleeding disorders, chronic The project will target SB/FP transposon integra- granulomatous disease (CGD) and neurological tion into specific locations in the human genome disorders. in order to improve the safety profile of transposon vectors for gene therapeutic applications. INTHER will address the interaction between the Molecular strategies of targeted transposition recombinase systems and the treated cell, in par- employed by naturally occurring transposable ticular. Safety issues of transposon vector admin- elements will be adapted to the SB/FP systems. istration and the characteristics of genomic integration will also be tackled. Extensive datasets INTHER seeks to establish the optimal condi- with viral vectors are available in these models tions to deliver the recombinase system/thera- in order to evaluate the potential of the transpo- peutic gene combinations for the different cell son-based, non-viral approach. types. Since the transposon is a plasmid based non-viral delivery vehicle, cutting edge non-viral Expected outcome: nucleic acid delivery methods will be utilised. In the project’s framework, Nucleofection technol- INTHER is expected to establish new therapeutic ogy would be optimised specifically for each of tools for somatic gene therapy as an alternative the recombinase systems in the context of the to existing viral and non-viral technologies. The relevant cell types targeted in this project. Si- project will evaluate the potential of recombi- multaneously, the capabilities of cell-penetrating nase-based (Sleeping Beauty, Frog Prince and peptides (CPPs), in combination with the trans- PhiC31-mediated) vectors for safe and efficient poson systems, will be evaluated. In addition, the transgene expression in ex vivo models. It will de- application of reversible implantation systems velop and optimise delivery techniques suiting (collagen implants, skin biopumps, and encapsu- the transposon technology. lated cells) will be explored. INTHER is investigating seven animal disease A disease model system will be identified, where models to establish the advantages and limita- the recombinase technology has a clear advan- tions of the recombinase-mediated therapeutic tage over viral approaches and current non-viral approach. To address the problems of efficacy us- approaches. To ascertain whether recombinase- ing non-viral technology, the experimental mod- based technologies might offer an alternative el systems are carefully selected. In most of the to treatments based on viral transduction, the disease models, the treated cell population has a recombinase systems will be probed in seven selective advantage. The use of selectable mark- different ex vivo disease models, previously treat- ers, particularly ABCG2, is a promising strategy ed by viral vectors. The selected ex vivo model that will enhance the selection of engineered

126 New Therapies – Gene Therapy Gene Therapy

cells. The experimental data will provide a strong Main findings: basis for further validation of transposon-based vectors in preclinical and clinical settings. Breakthroughs: In the first year of the project, the proof of principle of manipulating the target site INTHER expects to deliver the results set out of integration was established. below. • significantly more active recombinase systems compared to the prototype ver- Major publications sions, to ensure the feasibility of the recombinase-based gene therapeutic ap- Liu, J., Jeppesen, I., Nielsen, K., Jensen, T.G., ‘Phi proach; c31 integrase induces chromosomal aberrations • proof of principle experiments tar- in primary human fibroblasts’, Gene Ther, 2006, geting the integration sites of Sleeping Aug;13(15):1188-90. Beauty and Frog Prince to predetermined genomic loci in human cells; Thorrez, L., Vandenburgh, H., Callewaert, N., • better penetration of the transposon Mertens, N., Shansky, J., Wang, L., Arnout, J., Collen, into cells, through the use of cell-penetrat- D., Chuah, M., Vandendriessche, T., ‘Angiogenesis ing peptides (CPPs); enhances factor IX delivery and persistence from • development of optimal protocols for retrievable human bioengineered muscle im- the combination of the transposons and plants’, Mol Ther, 2006, Sep;14(3):442-51. Nucleofector® technology; • utilising transposon vectors and ex vivo therapeutic approaches, INTHER will test the potential of clinically feasible and potentially safer approaches for the following: somatic gene therapy of disorders in copper metabolism; Biopump laser beam gene transfer (BPLBGT) technology as a cell factory for the production of human secretory proteins; gene therapy of the genetic bleeding disorders haemophilia and hereditary thrombocytopenia; for gene therapy of CGD, to develop a method to provide a selective advantage for modified stem cells, using the human ABCG2 multidrug transporter protein as a selectable marker; the use of encapsu- lated human cell lines and human neural progenitor cells; and cell therapy in LDL receptor-deficient mice; • evaluation of safety and toxicity issues associated with the use of transposons as gene vectors in human cells.

New Therapies – Gene Therapy 127 INTHER

Coordinator Péter Krajcsi Zsuzsanna Izsvák Solvo Biotechnology, Inc. Max-Delbrück-Center for Molecular Medicine Szeged, Hungary Robert Rössle Strasse 10 13125 Berlin, Germany Lars Wahlberg E-mail: [email protected] NsGene A/S Ballerup, Denmark Partners

Ülo Langel University of Stockholm Department of Neurochemistry Stockholm, Sweden

Thomas G. Jensen The Kennedy Institute National Eye Clinic Glostrup, Denmark

Eithan Galun Goldyne Savad Institute of Gene Therapy Hadassah Medical Organisation Jerusalem, Israel

Seppo Ylä-Herttuala University of Kuopio A.I.Virtanen Institute Department of Biotechnology and Molecular Medicine Kuopio, Finland

Marinee Chuah Flanders Interuniversity Institute for Biotechnology vzw University of Leuven Department of Transgene Technology and Gene Therapy Leuven, Belgium

Balázs Sarkadi National Medical Centre Institute of Haematology and Immunology Budapest, Hungary

Herbert Müller-Hartmann AMAXA GmbH Cologne, Germany

128 New Therapies – Gene Therapy Gene Therapy EpiVector Episomal vectors as gene delivery systems for therapeutic application

Contract No LSHB-CT-2004-511965 Project type Specific Targeted Research Project EC contribution e 2 100 000 Starting date 1 January 2005 Duration 36 months Website www.ls.manchester.ac.uk/epivector

Background and objectives:

There are many chronic human diseases that cause great suffering as a result of inherited or acquired mutations in our genetic material. Once present, these changes to the structure of DNA are passed from one cell to the next as cells divide. Inherited and acquired mutations are responsible for many diseases, common examples being cystic fibrosis and muscular dystrophy. In these cases, the debilitating consequences of Figure 1: Distribution of pEPI-1 in CHO cells, during and mutations in DNA arise because of changes to immediately following mitosis. The localisation of the episome was studied by FISH on spreads of metaphase the structure of the protein that is expressed chromosomes (A), and the equal distribution of vector from the mutated gene. molecules was monitored in postmitotic nuclei of dividing cells (B). The episome (green) was visualised by pEPI FISH. To-Pro-3 was used for DNA counterstaining (red). A The concept of gene therapy is to overcome the maximum intensity projection was rendered from a set damage caused by genetic mutations in human of 5 mid serial sections in (B). Arrows in (A) indicate a pEPI cells by providing, to the appropriate cells, a nor- signal pair, each signal localised on a sister chromatid. Note the extremely high efficiency with which episomes mal copy of the damaged gene. In principle, if the are segregates to daughter nuclei - in (B) both daughters normal protein is then expressed in these target have 7 single plasmids defined by FISH signals. The FISH spots also show low grade mirror symmetry, similar to cells it will be possible to replace the malfunc- that seen for chromosome territories following mitosis. tioning protein with a fully functioning counterpart. However, this is technically very challenging, and systems that are currently being evaluated cells, it is by no means certain that the gene will in clinical trials suffer from potential deficiencies be expressed faithfully. In fact, all too often the that compromise safety. protein is only made for a short time. One way of providing long-term expression is to use vec- The most challenging aspects of gene therapy tors that integrate into the chromosome of the arise from two main sources. First, available pro- target cell; this ensures that the new gene is not tocols for delivering DNA to the target cells are lost as cells divide. However, a potential problem generally inefficient. The most efficient systems with this is that by interfering with the cell ge- use viral particles, but the best systems are also nome, it is possible to alter the normal patterns associated with immunological side effects. Sec- of gene expression, and in some cases this can ond, even when the DNA is delivered to target lead to cancer.

New Therapies – Gene Therapy 129 EpiVector

Figure 2: Reporter gene expression in transgenic pig foetuses generated by sperm mediated gene transfer of epi-vectors. Shows an analysis of enhanced-green fluorescent protein expression in embryonic pig tissues after transfer of epi-vectors to oocytes using sperm mediated gene transfer. The episomal status of the epivectors was determined by blotting and plasmid rescue. Confocal images show reporter gene expression in six tissues (from left to right: skeletal muscle, heart, liver, kidney, lung, and skin) from a representative positive foetus (Top) and the same tissues from a negative control foetus (Below).

This project is designed to evaluate the possi- with no selection pressure. The main limitation bility of developing extra-chromosomal gene is the (low) efficiency with which highly mitoti- delivery systems for gene therapy and evaluate cally stable vectors are maintained. This appears protocols for their safe use in pre-clinical model to be a general property of extra-chromosomal systems. The episomal systems under study do DNA vectors. Establishing a stable maintenance not interfere with host cell chromosomes and so phenotype does not involve genetic changes do not have any secondary genetic effects. The (shown by plasmid rescue and sequencing), so key features of EpiVector are to define the ge- it is clearly dependent on epigenetic program- netic elements that are required for persistent, ming. This problem has been studied in detail long-term gene expression and maintenance of and important observations are beginning to the extra-chromosomal DNA molecules in ap- pinpoint the key features: propriate cells. 1. The Lipps and Jackson collaboration has shown that when stably maintained, vec- Approach and methodology: tors are stably associated with the most active nuclear compartment and retain an Key approaches are to use vector construction association with some chromosomal land- and engineering to define the behaviour/prop- marks throughout mitosis. This provides erties of different vector constructs in different a means of ensuring efficient inclusion cellular environments. Models systems used in- into daughter nuclei but also – though clude: general cell culture models; model systems the molecular mechanism is unknown – a for gene therapy (e.g. muscular dystrophy); gene means of uniform segregation during cell expression in primitive haematopoietic stem division. cells; mouse embryonic stem cells; and sperm 2. The Lipps and Azorin collaboration has mediated gene transfer to explore development revealed that the efficiency of establish- behaviour. ing stable clones is increased 5-10fold if DNA is assembled into chromatin before The key feature of epi-vectors is their excep- delivery to cells. tional mitotic and expressional stability in cells 3. Bode and colleagues have shown that

130 New Therapies – Gene Therapy Gene Therapy

generation of minicircles that do not con- observations made using sperm mediate trans- tain bacterial DNA also improved estab- fer of epi-vectors to pig oocytes. lishment efficiencies by 5 to10 times. 4. Studies by Lipps and collaborators have The oocyte is a permissive environment for epi- shown that epi-vectors can be introduced genetic re-programming and epi-vectors have into pig oocytes by sperm-mediated gene been shown to pass to all tissues of an embryo transfer to give an extremely high efficien- following their introduction into oocytes by cy of episomal gene transfer to all tissues sperm mediated gene transfer. This implies that of pig embryos. mouse embryonic stem cells will likely provide a 5. Establishment of human stem cell clones route for making transgenic animals with a sta- and mouse embryonic stem cell clones is ble expression of genes from epi-vectors. ongoing. Major publications Expected outcome: Jenke, A.C.W., Eisenberger, T., Baiker, A., Stehle, The expected outcome of the project is to I.M., Wirth, S., Lipps, H.J., ‘The nonviral episomal deliver a detailed understanding of the genetic replicating vector pEPI-1 allows long-term inhi- and epigenetic features that support the stable bition of bcr-abl expression by shRNA’, Human mitotic behaviour of episomal DNA molecules in Gene Therapy, 2005, 16:533-539. proliferating human cells. The project has defined the genetic elements required for stable mitotic Bode, J., Winkelmann, S., Götze, S., Spiker, S., Tsut- inheritance and is now attempting to analyse sui, K., Bi, C., Benham, C., ‘Correlations between the epigenetic features. An important aspect Scaffold/Matrix Attachment Region (S/MAR) of this activity is to understand in molecular Binding Activity and DNA Duplex Destabilisation detail how epi-vectors interact with functional Energy’, J. Mol. Biol, 2006, 358:597-613. http:// nuclear compartments and define the dynamic dx.doi.org/10.1016/j.jmb.2005.11.073 behaviour of these interactions. Live cell imaging of tagged epi-vectors is being used to address Jackson, D.A., Juranek, S., Lipps, H.J., Design- vector dynamics. ing nonviral vectors for efficient gene transfer and long term gene expression’, Mol. Ther, 2006, Main findings: 14:613-626.

Significant findings have included a phenotypic Winkelmann, S., Klar, M., Benham, C., A.K., P., description of the molecular behaviour of epi- Goetze, S., Gluch, A., Bode, J., ‘The vectors in proliferating cells. This has provided positive aspects of stress: Strain Initiates Domain early insight into the molecular behaviour and Decondensation (SIDD)’, Briefings in Functional provides a system that should allow the mo- Genomics and Proteomics, 2006, 5, 24-31 http:// lecular mechanism to be defined. It is clear that dx.doi.org/10.1093/bfgp/ell003 epigenetic programming of the vector is key to the way it behaves in proliferating cells. Modify- Manzini, S., Vargiolu, A., Stehle, I., Bacci, M.L., Cer- ing the sequence or chromatin architecture of rito, M.G., Giovannoni, R., Forni, M., Donini, P., Papa, the vector prior to introducing into target cells M., Lipps, H.J., Lavitrano, M., ‘Genetically modified significantly influences the efficiency with which pigs produced with a non vital episomal vector’, mitotically stable clones are established. A par- Proc. Natl, 2006, Acad. Sci. USA 103:17672-17677. ticular breakthrough in this regard comes from

New Therapies – Gene Therapy 131 EpiVector

Nehlsen, K., Broll, S., Bode, J., ‘Replicating minicir- Coordinator cles: Generation of nonviral episomes for the efficient modification of dividing cells’, Gene Ther Dean A Jackson and Mol Biol, 2006, 10B: 233-243. Manchester Interdisciplinary Biocentre University of Manchester Faculty of Life Sciences 131 Princess St M1 7DN Manchester, UK. E-mail: [email protected].

Partners

Roel van Driel University of Amsterdam Science Faculty Swammerdam Institute for Life Sciences Amsterdam, Netherlands

Hans Lipps University of Witten Department of Cell Biology Witten, Germany

George Dickson Royal Holloway - University of London Centre for Biomolecular Sciences Egham, UK

Fernando Azorin CSIC Institute of Molecular Biology Barcelona, Spain

Jürgen Bode GBF German Research Centre for Biotechnology Braunschweig, Germany

Ariella Oppenheim Hebrew University-Hadassah Medical School Department of Haematology Jerusalem, Israel

Ben Davis genOway Germany Hamburg, Germany

132 New Therapies – Gene Therapy Gene Therapy PolExGene Biocompatible non-viral polymeric gene delivery systems for the ex vivo treatment of ocular and cardiovascular diseases with high unmet medical need

Contract No LSHB-CT-2006-019114 Project type Specific Targeted Research Project EC contribution e 2 132 607 Starting date 1 June 2006 Duration 36 months

Background and objectives: ing the polymer membrane with cell interacting peptides (CIP). PolExGene aims to develop a non-viral ex vivo gene therapy that can find potential applications Approach and methodology: in the field of ocular and cardiovascular diseases with high unmet medical need. One of the key Two different strategies will be developed and innovative aspects of PolExGene is the develop- compared. In a first strategy (A), cells will be trans- ment of novel gene vectors from non-toxic and fected using CPP containing polyplexes, after non-immunogenic, biodegradable polymeric which the cells will be seeded on a biodegrad- carrier materials based on multifunctional poly- able polymer membrane, functionalised with CIP. α-aminoacids. Polymer membranes will be prepared by solvent casting or electrospinning. Overall DNA delivery will be improved through the combination of polyplexes with cell pen- Alternatively (strategy B), the polymer membrane etrating peptides (CPP). The internalisation effi- will be surface coated with CPP-containing poly- ciency is expected to be enhanced through the plexes prior to cell seeding. In both cases, the cell- use of Penetratin-like CPP, while the membrane- cell interaction will be improved by functionalis- Figure 1. Application of an ex vivo gene therapy approach for the treatment of ocular and cardiovascular diseases.

New Therapies – Gene Therapy 133 PolExGene

seeded biodegradable polymer membrane will Coordinator be implanted into the retina of the eye or used as coating for cardiovascular prosthesis. The biode- Etienne Schacht gradable polymers selected will be biocompat- Polymer Chemistry & Biomaterials Research Group ible: non-toxic and non-immunogenic. Department Organic Chemistry Ghent University Krijgslaan 281 S-4 In order to obtain the technological objec- 9000 Ghent, Belgium tives, the following scientific objectives will be E-mail: [email protected] achieved by implementing seven Work Packages (WP): Partners • WP1: selection of CIP and CPP; • WP2: development of CPP-containing Arto Urtti polymers; University of Helsinki • WP3: development of CIP-containing Faculty of Pharmacy/Drug Discovery and Development polymer membranes; Technology Center • WP4: preparation of plasmids and CPP- Helsinki, Finland containing polyplexes; • WP5: characterisation of polyplex-cell Hagen Thielecke and polymer membrane-cell interactions; Fraunhofer Institute for Biomedical Engineering • WP6: study on immunological prop- St. Ingbert, Germany erties of polyplexes and polymer membranes; Prof. Blanca. Rihova • WP7: polymer membrane implanta- Institute of Microbiology tion in test animals. Prague, Czech Republic)

An eighth Work Package, WP8, will be devoted to Alain Joliot the project management to ensure that the over- Ecole Normale Supérieure all project objective is met. Paris, France

The technological objectives addressed for the Eberhart Zrenner development of CPP containing biocompatible University Eye Hospital of Tübingen non-viral polymeric gene delivery systems for ex Tübingen, Germany vivo treatment are very ambitious: • development of non-toxic and non- Alain Yvorra immunogenic biodegradable polymeric Epytop CPP-containing DNA-carriers; Nîmes, France • development of a biohybrid cell-based CIP-containing implantation system for David Eckland ocular and cardiovascular applications; Ark Therapeutics • development of a cell-based micro ar- London, UK ray technology for the quantification of the biological activity (transfection effi- Seppo Ylä-Herttuala ciency and toxicity) of the DNA-carriers. University of Kuopio A.I. Virtanen Institute Kuopio, Finland

134 New Therapies – Gene Therapy Gene Therapy Magselectofection Combined isolation and stable nonviral transfection of haematopoietic cells – a novel platform technology for ex vivo haematopoietic stem cell gene therapy

Contract No LSHB-CT-2006-019038 Project type Specific Targeted Research Project EC contribution e 2 800 000 Starting date 1 May 2006 Duration 36 months Website www.magselectofection.eu

Background and objectives: logical tools, as well as magnetic resonance imaging. The therapeutic potential will be examined The feasibility of ex vivo gene therapy in humans in a SCID-X mouse model in direct comparison has been demonstrated with retrovirally trans- with established retroviral technology. Besides duced haematopoietic stem cells. At the same contributing to the progress of health care, Mag- time, risks associated with the use of retroviral netoselectofection will foster the competitive- vectors became apparent. In order to circum- ness of Europe’s biotechnology industry. vent problems associated with viral vectors, we will develop a novel, nonviral, combined ex vivo Magnetoselectofection seeks to combine mag- cell separation/transfection platform for haemat- netic cell sorting and transfection based on opoietic cells, suitable for site-specific genomic Miltenyi’s clinically approved MACS Technology integration of transfected nucleic acids into non- and Magnetofection (magnetically guided nu- coding regions of the host genome. This concept cleic acid delivery), for manipulation of haemat- will be applied to haematopoietic stem cells and opoietic cells. It also aims to achieve a stable and will be validated with established preclinical regulatable transfected gene expression in hae- models of SCID-X1. matopoietic stem cells, by site-specific genomic integration of delivered nucleic acids upon This platform technology for integrated cell iso- Magnetoselectofection, with plasmid constructs lation and transfection will be based on a clinical- harbouring the phiC31 integrase system, and ally approved magnetic cell separation technique ternatively, a drug-inducible AAV-derived repli- (MACS Technology), combined with magneti- case/integrase system. cally enhanced transfection (Magnetofection). It will also be based on nucleic acid constructs Magnetoselectofection will characterise this tech- that provide site-specific genomic integration nology through an analysis of genomic integration — either the phage phiC31 integrase system or, sites, transcriptom profiling, and a characterisation alternatively, a drug-inducible AAV-derived repli- of stable and inducible transfected gene expres- case/integrase system. sion. The project will validate the technology in transgenic SCID-X mouse models by evaluating Technology validation includes the following: homing, engraftment and persistence in trans- analysis of genomic integration sites; transcrip- genic animal models, via molecular biological tom profiling; characterisation of stable and in- tools and magnetic resonance imaging. Magne- ducible trans-gene expression, and evaluation toselectofection will demonstrate the therapeutic of homing; and engraftment and persistence in efficacy, as well as assess the associated risks of the transgenic animal models using molecular biotechnology in transgenic mouse models.

New Therapies – Gene Therapy 135 Magselectofection

The dissemination and use of Magnetoselecto- Expected outcome: fection will be made possible from its transfer into research and clinics through the participat- Magselectofection will generate a novel technol- ing companies. ogy for nucleic acid delivery which combines the ease of application of magnetic nanoparticle- Approach and methodology: based cell isolation and sorting, with the advantages of magnetic nanoparticle-based nucleic The clinical success in curing patients suffering acid delivery technology. While this novel plat- from SCID-X has demonstrated the great poten- form technology is expected to be broadly ap- tial of gene therapy. In the same study, the short- plicable to a variety of clinical and research ap- comings and serious biological risks associated plications, this project focuses on its use in gene with the use of current viral gene vector technol- therapy of a rare hereditary disease, SCID-X1. ogy became evident. Therefore, Magselectofection comprises detailed analyses in the stable genetic modification and Of the 11 patients treated with retrovirally trans- transplantation of haematopoietic stem cells, duced haematopoietic stem cells, 2 developed a while avoiding the use of viral gene vectors. lympho-proliferative disease due to insertional mutagenesis caused by the uncontrolled inte- Expected results include novel nucleic acid con- gration properties of the vector. Hence, safe alter- structs for stable genetic modification of cells natives are required to realise gene therapeutic and novel technology for transfecting such con- concepts involving stable genetic modification structs. In addition, they include insights into the of cells. molecular biology of such constructs once transfected, into the cell biology of haematopoietic Members of this consortium have previously de- stem cells modified in this manner, and into the veloped independent technologies for the fol- biodistribution, engraftment and homing of such lowing: magnetic cell separation; magnetic field- cells once transplanted in animal models. assisted nucleic-acid delivery to cells; nucleic acid constructs suitable for stable integration into Apart from the primary intended application, i.e. eukaryotic genomes; disease-relevant transgenic gene therapy of SCID-X1, there are multiple nu- mouse models, to evaluate the biological charac- cleic acid therapy applications that can be envis- teristics and therapeutic potential of transgenic aged, as is indicated below. cells under pre-clinical settings; and tools for ex- • using the method to induce RNA inter- pression profiling. ference (RNAi) in hematopoietic stem cells to fight HIV infections; There are strong personal and scientific links to • using the method for the transfection the EU-funded CONSERT project. The fundamen- of lymphocytes for adoptive immuno- tal objective of Magselectofection is to combine therapy of cancer; the named independent technologies and skills • transfection of neuronal stem cells for so as to generate a novel, yet simple, efficient and ex vivo gene therapy of central nervous safe integrated platform technology for the ge- system disorders; netic modification of cells in clinical and research • ex vivo transfection of stem cells or applications. This innovative technology is the progenitor cells for tissue engineering first developed towards the application in SCID- (bone, cartilage reconstruction, tendon X1 gene therapy; clinical and research protocols and wound healing etc.); are well established. • cell tracking, molecular imaging. The

136 New Therapies – Gene Therapy Gene Therapy

magnetic nanoparticles used in Magse- Peter Babinec lectofection are suitable contrast agents Comenius University for magnetic resonance imaging. Thus, the Bratislava, Slovakia method can be used to introduce these particles in any cell of interest, optionally Olivier Zelphati along with other agents for molecular im- OZ Biosciences S.A.R.L. aging (e.g. fluorescent dyes). Marseille, France

Joseph Rosenecker Coordinator Ludwig-Maximilians-Universität Munich, Germany Christian Plank Klinikum rechts der Isar der TU München Ulf Johann Institute of Experimental Oncology Fraunhofer Gesellschaft zur Förderung der angewandten Ismaninger Str. 22 Wissenschaft e.V. D-81675 Munich, Germany Munich, Germany E-mail: [email protected] Michele Calos Partners Stanford University Palo Alto (CA), USA Gerard Wagemaker Erasmus Medical Centre Rotterdam, Netherlands

Fulvio Mavilio Fondazione Centro San Raffaele del Monte Tabor Milan, Italy

Tsvee Lapidot Weizmann Institute of Science Rehovot, Israel

Zygmunt Pojda M. Sklodowska-Curie Memorial Cancer Centre and Insti- tute of Oncology Warsaw, Poland

Michael Apel and Ian Johnston Miltenyi Biotec GmbH Bergisch-Gladbach, Germany

Peter Steinlein Research Institute of Molecular Pathology Vienna, Austria

New Therapies – Gene Therapy 137 SyntheGeneDelivery Ex vivo gene delivery for stem cells of clinical interest using synthetic processes of cellular and nuclear import and targeted chromosomal integration

Contract No LHSB-CT-2005-018716 Project type Specific Targeted Research Project EC contribution e 2 400 000 Starting date 1 December 2005 Duration 36 months Website http://lepg.univ-tours.fr

Background and objectives:

The project will develop SyntheGeneDelivery (SGD), a new ex vivo gene delivery (evGD) protocol to provide stable long-term expression of integrated transgenes. The clinical objective is to provide gene therapy solutions for some ge- Murine mesenchymal stem cells transfected with netic diseases of the neuromuscular and skeletal plasmid DNA encoding GFP complexed with BGTC/DOPE liposomes. systems, as well as for circulating polypeptide deficiencies that together afflict more than 35 million patients in Europe. • the access to “good” target sites to ensure healthy integrations in chromosomes; In gene therapy for inherited disorders, the main • avoidance of undesirable transgene area of application of an efficient evGD proc- integrations. ess lies in the modification of stem cells. Indeed, the advantage of such systems is that cells can To achieve these goals and fully develop the be transfected and verified in vitro, before being technology, SyntheGeneDelivery will use two grafted in a patient with a debilitating genetic populations of stem cells. The first is a mesen- deficiency. chymal stem cell (MSC), which can be simply recovered from bone marrow and manipulated for Approach and methodology: the correction of some genetic disorders, such as those involving haematopoietic and mesen- The main limitation in developing these therapeu- chymal derivative cells. The second will be mus- tic strategies is the availability of efficient and safe cle stem cells that have the potential to correct processes. The SGD protocol is designed to over- some muscle genetic disorders, but that can also come these problems and to ensure long-term be used, after the intramuscular graft of the mod- maintenance of transgene expression by chromo- ified stem cells, as a factory for the production of somal integration, bypassing several barriers: polypeptides that are deficient in genetic and • the extra-cellular matrix and the cell acquired disorders, and that can be secreted by membrane; the muscle to diffuse toward some target organs • the traffic from endosomes to lyso- of the patient. somes and subsequent degradation; • the dissociation of DNA from its carrier; For DNA vector internalisation in cells, the con- • the transfer into the nucleus; sortium will use the BGTC-DOPE lipoplex system

138 New Therapies – Gene Therapy Gene Therapy

in the first version, and the Poloxamer block copolymer in the two latest versions. Transgene integration will be achieved using vectors derived from the mariner Mos1 transposon. These DNA vectors will be improved for their key properties The mariner Mos1 transposon. using molecular engineering methods. the fully non-cytolytic product approved by the First, their integration efficiency will be improved, FDA — the Propoplex block copolymer — using not only by optimising the vector nuclear import, procedures adapted to ex vivo transfection. but also by obtaining hyperactive transposases (the enzyme assuming the transposon mobility), The consortium will use the SGD process on mus- and ITR (the end of the transposon). Second, Syn- cle or mesenchymal stem cells, in an attempt to theGeneDelivery will define the configuration of assay its therapeutic potential in several diseases: vectors for which integration efficiency will not Duchenne Muscular Dystrophy (DMD), type 1 Os- be dependent on the transgene size. Finally, site- teogenesis imperfecta, Hemophilia A and Diabe- specific integration vectors will be developed tes mellitus type 1. The properties of SGD and its by fusing mariner Tpases with polydactyl Zinc evolutions are adequate to the therapeutic strat- Finger Domains, thus targeting the DNA integra- egies targeted on these diseases. It can be used tion at close proximity of a predefined site. The with cassettes containing the therapeutic trans- efficiency and the fidelity of integration of the gene that are less than 5 kpb for the DMD (using chimeric Tpases will be modelled in vivo before highly engineered microdystrophin constructs), their use for clinical purposes. and less than 3-4 kbp for other diseases. Moreo- ver, if mariner vector is able to carry larger trans- At each step, the modifications to the Synthe- genes, the consortium should then use cassettes GenTransfer process will be evaluated in foetal containing therapeutic transgenes of 20-30 kpb. and adult MSC, and in muscle stem cells. Their effect on retaining the dual stem cell properties on Main findings: self renewal and multilineage differentiation will be evaluated, in comparison to standard lentivi- Foetal mesenchymal stem cells and a clone of ral integration systems. murine muscle stem cells from a condition- ally immortilised mouse have been selected for Expected outcome: evaluation of all the SGD process. Human muscle stem cells will be used to evaluate the SGD V3. EvGD strategies for therapy purposes are of high interest when they concern stem cells, due to DNA internalisation in stem cells is actually per- the potential of differentiating them ex vivo and formed using the nucleofection system from in vivo in a wide range of cell types. Indeed, this Amaxa. A more efficient transfection reagent, a opens key possibilities that meet challenges lipidic aminoglycoside derivative, is now avail- posed by many diseases. The SGD process is well able from In Cell Art (Partners 2 and 7), and will adapted to develop evGD on stem cells because be used to develop SGD V2. of the properties of the BGTC-DOPE, which is the only identified chemical product able to internal- MRNA encoding the transposase internalisation ise DNA molecules in these cells. Developing the in HeLa cells is actually efficiently performed us- project, the innocuity of the cellular import will ing PEI. The Kozak modified and codon optimised be improved by replacing the BGTC-DOPE with Mos1 transposase has little activity in transfected

New Therapies – Gene Therapy 139 SyntheGeneDelivery

stem cells. A second version, which has no self- Coordinator annealing properties was developed by Partner 1 and will be used in SGD V2. Hyper-active trans- Yves Bigot posases are still available and will be used in SGD Université François Rabelais de Tours V2. Similarly, more efficient ITR were designed LEPG FRE 2969 and will be used in SGD V2. UFR Sciences & techniques 37200 Tours, France In conclusion, it must be underlined that SGD V1 E-mail: [email protected] was made from few optimised components (cellular import, nuclear import, protein and DNA Partners counterparts). Results were encouraging but not yet at the level expected at the end of the pro- Cyrille Grandjean gramme. However, the necessary elements are in In Cell Art place, to proceed with evaluation of the SGD V2. Nantes, France

Major publications Oscar Simmonson Avaris Sinzelle, L., Jegot, G., Brillet, B., Rouleux-Bonnnin, Stockholm, Sweden F., Bigot, Y., Augé-Gouillou, C., ‘Factors controlling Mos1 transposition in vitro and in bacteria’, Sub- Karin Lundin mitted. Karolinska Institutet Stockholm, Sweden Svahn, M.G., Hasan, M., Sigot, V., Valle-Delgado, J.J., Rutland, M.W., Lundin, K.E., Smith, C.I., ‘Self-as- Ronald Chalmers sembling supra-molecular complexes by single- University of Oxford stranded extension from plasmid DNA’, Oligonu- Oxford, UK cleotides, 2007, Spring;17(1):80-94. Dominic Wells Ge, R., Heinonen, J.E., Svahn, M.G., Mohamed, A.J., Imperial College Lundin, K.E., Smith, C.I., ‘Zorro locked nucleic acid London, UK induces sequence-specific gene silencing’, FASEB J, 2007, Apr 10. Bruno Pitard INSERM Nantes, France

140 New Therapies – Gene Therapy Gene Therapy MOLEDA Molecular optimisation of laser/electrotransfer DNA administration into muscle and skin for gene therapy

Contract No LHSB-CT-2004-512034 Project type Specific Targeted Research Project EC contribution e 2 447 972 Starting date 1 January 2005 Duration 36 months Website www.moleda.org

Background and objectives:

For gene therapy, the use of non-viral DNA offers the advantage of lack of immunogenicity, absence of size limit for the therapeutic expression cassette, simpler GMP production, and improved safety/toxicity profiles. However, the efficient, precise and safe delivery of plasmids or other forms of non-viral DNA remains to be improved.

Two different in vivo local tissue plasmid delivery techniques were recently introduced, and are Moleda’s Team - Brussels currently the most efficient in terms of gene expression level: electrotransfer using mild electric More precisely, MOLEDA aims at identifying the pulses (ET) and laser beam gene transfer using optimal conditions for plasmid delivery into femtosecond infrared titanium sapphire laser en- muscle and skin. This optimisation will be ac- ergy (LBGT). companied by a head-to-head comparison, using the same DNA preparations, between ET and Approach and methodology: LBGT. This optimisation/comparison will be performed in two different tissues: muscle and skin. MOLEDA aims to determine the optimal condi- MOLEDA will, for each tissue and each model of tions for precise and selective plasmid transfer delivery (ET or LBGT), assess which is the best into skeletal muscle and skin, in the frame of a promoter: cytomegalovirus (CMB) or two differ- head-to-head comparison between the two ent tissue-specific promoters. For muscle, two most promising in vivo plasmid DNA delivery strong skeletal muscle promoters will be exam- technologies at present, and to introduce molec- ined. For skin vaccination, one strongly active ular strategies to improve these technologies. keratinocyte promoter, and one dendritic cell Optimised conditions will then be used on ani- specific promoter will be used. mals with four different therapeutic applications. The researchers will assess and select the prefer- The overall objective is to develop non-viral DNA ential form of non-viral DNA to be used, from the technology into a preclinical phase. Consequent- following options: ly, MOLEDA will address safety issues (tissue dam- • conventional plasmid; age, inflammation, etc.) to ensure the success of • prokaryotic-backbone deleted, ‘mini- its efforts. plasmid’ devoid of antibiotic resistance

New Therapies – Gene Therapy 141 MOLEDA

Moleda’s big team gene (an approach pioneered by Team 1); • linear PCR produced eukaryotic ex- These optimisation results will be applied to pression cassette (technology mastered three different therapeutic paradigms, for four by Team 2). different medical applications: • long-term intracellular expression of MOLEDA will assess, for the secreted transgenic the dystrophin gene in skeletal muscle for protein part of the programme, the usefulness of the therapy of Duchenne muscular dys- an optimised enhancing secretion discovered re- trophy (an inherited neuromuscular dis- cently (Tauler et al., 1999), as well as examine for ease); each tissue (muscle and skin), and for each mode • long-term blood secretion of a circu- of delivery (ET and LBGT), what the best DNA for- lating protein, with erythropoietin (EPO) mulation is. The options are ‘naked’ versus self- and secreted monoclonal antibodies as associated to cationic lipid, or to non cationic the selected applications; lipid (discovered by Team 1; Patent Herscovici et • short-term transgene expression in al. 2002). To date, cationic lipids have proved in- skin for raising humoral and cellular im- efficient for naked DNA intramuscular injection, mune response: antitumour vaccination but except for that particular case, little is known (i.m. ET has been shown to enhance DNA about the effect of cationic lipids, and even less vaccine response). of the effect that neutral DNA formulation has on It is likely that each specific therapeutic appli- ET and on LBGT in different tissues. cation will require an adapted protocol of DNA administration. Internal data among the consor- The researchers will study if pre- or post-ionto- tium’s participants confirm this assumption. For phoresis may be beneficial for ET or LBGT. Ionto- instance, electrotransfer might represent the phoreisis involves applying low intensity electri- best technology for skeletal muscle DNA delivery cal currents, <0,5mA/cm2, for several minutes. It when maximal production and blood secretion has been shown to enhance transdermal drug of the transgenic protein is needed. Converesely, delivery and promote intratissular migration of LBGT might represent a more efficient technol- charged molecules. ogy for skin administration or for the production

142 New Therapies – Gene Therapy Gene Therapy

by muscle of a very potent hormone. Also, a mus- Coordinator cle specific promoter might prove more adaptive (i.e. leading to high level sustained transgene Daniel Scherman expression), only to ET or LBGT. In addition, the U640 Inserm, UMR 8151 CNRS precision of gene delivery might depend on the Faculté de Sciences Pharmaceutiques et Biologiques, delivery technology used. 4 avenue de l’Observatoire 75270 Paris Cedex 06, France Expected outcome: E-mail: [email protected]

Optimised conditions and constructs for ET and Partners LBGT will be applied in the context of the three different gene therapy paradigms, and four dif- Eithan Galun ferent medical applications. The safety issues (tis- Hadassah Medical Organisation sue damage, inflammation, integration, etc.) will Jerusalem, Israel be specifically addressed as well. The overall objective is to develop non-viral DNA technology Véronique Préat into a preclinical phase. Université Catholique de Louvain Louvain, Belgium Main findings: George Dickson In the first part of the programme, ET and LBGT Royal Holloway & Bedford New College were optimised in parallel by local DNA delivery University of London into skeletal muscle and skin, by assessing the London, UK following: • what the best promoter (ubiquitous or Volker Schirrmacher tissue-specific) is for each model of deliv- Deutsches Krebsforschungszentrum ery (ET or LBGT); Heidelberg, Germany • the preferential form of non-viral DNA — either conventional plasmid or linear Iacob Mathiesen PCR produced expression cassette; INOVIO • the usefulness of an optimised secre- Oslo, Norway tion sequence for secreted transgenic proteins; Paul Parren • what the best DNA formulation is — GENMAB ‘naked’ versus associated to cationic or to Utrecht, Netherlands non-cationic lipid; • if pre- or post-iontophoresis is beneficial. Stéphane Blot Ecole Nationale Vétérinaire d’Alfort In addition, MOLEDA has made great progress by Maison-Alfort, France constructing a new generation of high efficiency plasmids devoid of antibiotic resistance genes Alain Cimino and their corresponding genetically modified Inserm Transfert bacterial host. Paris, France

New Therapies – Gene Therapy 143 ANGIOSKIN DNA electrotransfer of plasmids coding for antiangiogenic factors as a proof of principle of non-viral gene therapy for the treatment of skin disease.

Contract No LSHB-CT-2005-512127 Project type Specific Targeted Research Project EC contribution e 2 780 683 Starting date 1 May 2005 Duration 48 months Website http://dl.ltfe.org/login_angioskin.asp

Background and objectives: trodes for safety and efficacy on normal skin in animals, and analyse the effects of The ANGIOSKIN consortium wants to establish the electrotransfer of the antiangiogenet- the proof of concept that therapeutic genes can ic factor on models of skin disease related be safely delivered to skin by DNA electrotrans- to excessive angiogenesis, using non non- fer (electrogenetherapy), in order to prevent or vasive biophysical, as well as histological to treat acquired or inherited skin diseases. methods to follow changes in the vascu- larisation of the lesion. The ANGIOSKIN project is based on the results of • electrotransfer the therapeutic gene the earlier CLINIPORATOR (Analysis of the mech- to a benign lesion in humans, as a proof anisms of DNA electrotransfer, and elaboration of of concept of the use of non-viral gene a CE labelled pulse generator), and ESOPE (Prep- therapy to treat acquired or inherited skin aration of the Standard Operating Procedures diseases. of Electrogenetherapy, electrotransferring of a reporter gene into humans) projects and on a Final goal: proprietary gene coding for a potent human antiangiogenic factor. This factor is the disintegrin ANGIOSKIN will bring the proof of concept of fragment of the Metargidin, also referred to as therapeutic gene non-viral electrotransfer into AMD-15 (MDC 15 in mouse). skin, to treat inherited or acquired diseases.

Approach and methodology: Main findings:

ANGIOSKIN will do the following: The consortium has been actively working on • electrotransfer the proprietary thera- the gene therapy vector that will be used in the peutic antiangiogenesis gene to melano- clinical trials foreseen in the project. The issues ma cutaneous metastasis in humans, us- related to the GMP production of the DNA for the ing the procedures validated in the ESOPE clinical trials have also been cleared with sub- project, in order to show its clinical ef- contractors. The sequences required for gene ex- ficacy. Noninvasive biophysical methods pression (promoters) have also been worked out, (Doppler ultrasounds, Near InfraRed Spec- resulting in the latest data showing improved troscopy, etc.) will be used to monitor the expression and efficacy of the vector. antiangiogenic effects. • develop new specific electrodes for The preparation of the clinical trials has started. skin lesions treatment, validate the elec- The clinical partners involved in the first clinical

144 New Therapies – Gene Therapy Gene Therapy

trials (related to malignant tissues, i.e. melano- Coordinator mas) have defined the protocols. Despite the delay in the submission of the protocols to the Lluis M. Mir ethics committees and regulatory agencies, the UMR 8121 CNRS - Institut Gustave-Roussy preparation of these documents and contacts Laboratory of Vectorology and Gene Transfer has continued. 39, rue C. Desmoulins F- 94805 Villejuif Cédex, France The technological aspects have resulted in proto- E-mail: [email protected] types for new devices. The extent of their future use is still under evaluation by the consortium. Partners Moreover, other items are also under experimental evaluation for eventual inclusion in future Gonzalo Cabodevila patent applications. Portions of the work of the Franche Comté Electronique Mécanique Thermique et consortium will soon be presented in specialised Optique meetings. Besançon, France

Major publications Damien Grenier and Caroline Jullien CNRS UMR 8029 SATIE (Systèmes et Applications de Patents: Technologies de l’Information et de l’Energie) Team BIOMIS (biomicrosystèmes) Two patent applications have been prepared and ENS Cachan Campus de Kerlann submitted. They cover some of the microfluidics Bruz, France aspects of the project, as well as the pulses delivered by the new type of electrodes on which Ruggero Cadossi the consortium is working. However, their sum- IGEA S.r.l. maries have yet to be published and no details Carpi (MO), Italy can be provided at the moment. Caroline Robert Mir, L.M., Miklavcic, D., ‘Combinations of electric Institut Gustave-Roussy pulses for DNA electrotransfer’. Dermatology Department of Medicine Villejuif Cédex, France Jullien, M.C., Hoel, A., Mir, L.M., ‘Fluid dispensing system’. Véronique Préat Université catholique de Louvain Unité de Pharmacie galénique Brussels, Belgium

Julie Geh Herlev Hospital Deptarment of Oncology, 54B1 Herlev, Denmark

New Therapies – Gene Therapy 145 ANGIOSKIN

Michael P. Schön University of Würzburg Rudolf Virchow Zentrum für Experimentelle Biomedizin Würzburg, Germany

Dominique Costantini BioAlliance Pharma SA Paris, France

Damijan Miklavcic University of Ljubljana Faculty of Electrical Engineering Ljubljana, Slovenia Subcontractor SC2 to partner University of Ljubljana:

Gregor Sersa Institute of Oncology Laboratory of Radiation Biology Ljubljana, Slovenia

Lone Skov Afdelingslæge Dermatologisk afdeling KAS Gentofte Hellerup, Denmark

146 New Therapies – Gene Therapy GVPNConference EUROLABCOURSE CONFERENCE & COURSE CONFERENCE & COURSE 4-5 October 2001 14-27 April 2002 1-14 February 2004 14-26 June 2004 EVRY EVRY BELLATERRA EVRY TOTAL TOTAL Participants in the TOTAL Participants in the TOTAL Participants in the Country participants participants practical course participants practical course participants practical course nb % % nb nb % nb nb Gene% nb n b Therapy% nb % 1. Algeria 1 0,6 2. Austria 1 0,36 1 0,70 3 1,97 1 0,6 3. Belgium 13 4,63 10 7,10 1 0,66 2 1,2 4. Brazil 1 0,6 5. Bulgaria 1 0,66 1 3,13 2 1,2 1 3,13 I6.nVi Canada voVector1 0,70 2 1,3T2 rain1 3,13 2 1,2 7. China 1 0,36 2 1,32 1 3,13 1 0,6 1 3,13 European8. Czech Republic labcourse: towards clinical gene1 therapy:0,66 preclinical gene transfer 9. Denmark 3 2,10 1 0,66 assessment10. Finland 1 0,6 1 3,13 11. France 223 79,36 42 30,00 8 25,0 45 29,61 4 12,5 64 38,3 4 12,5 12. Germany 8 2,85 17 12,10 2 6,25 3 1,97 8 4,8 1 3,13 13. Greece Contract No LSSB-CT-2003-5032192 1,32 1 3,13 1 0,6 1 3,13 14. Hungary 2 1,40 2 6,25 5 3,29 3 9,38 6 3,6 1 3,13 15. Iceland Project type Specific1 0, 66Support1 3,Action13 16. Iran EC contribution e 1611 5000,66 1 0,6 1 3,13 17. Ireland 3 1,8 1 3,13 18. Israel 4 1 ,42 2 Starting1,40 date1 3 ,13 1 June 2003 3 1,8 2 6,25 19. Italy 4 1,42 6 4,30 2 6,25 3 1,97 2 6,25 14 8,4 5 15,63 20. Japan Duration 21 months2 1,32 1 0,6 1 3,13 21. Korea 1 0,70Website 1 0,66 22. Latvia 1 0,70 www.vecteurotrain.org 23. Lithuania 2 1,40 1 3,13 2 1,32 2 6,25 24. Luxembourg 1 0,70 25. Mexico 1 0,66 26. Netherlands 3 1,07 6 4,30 1 3,13 3 1,97 1 3,13 5 3,0 27. Poland 1 0,66 1 3,13 1 0,6 1 3,13 28. Portugal 1 0,70 1 3,13 4 2,63 2 6,25 2 1,2 1 3,13 29. Romania 1 0,70 1 3,13 30. Russia 1 0,66 1 3,13 1 0,6 Background31. Slovenia and objectives: Main findings: 1 0,6 32. Spain 5 1,78 7 5,00 7 21,88 46 30,26 6 18,75 10 6,0 4 12,5 33. Sweden 2 0,71 11 7,90 4 12,5 3 1,97 1 3,13 3 1,8 The34. S InVivoVectorTrainwitzerland 3 event,1,07 sponsored5 3,60 by1 the 3,13 About3 1501,97 researchers hailing6 3, 6from1 different3,13 in- 35. Turkey 1 0,70 2 1,32 3 9,38 7 4,2 3 9,38 EC,36 .was Taiw athen third in a series of training initiatives stitutions across the EU and1 abroad0,6 participated 37. United Kingdom 12 4,27 12 8,60 1 3,13 5 3,29 10 6,0 on38 .gene Urugua yand cell therapy that Généthon devel- in the1 last0,66 three1 events.3,13 The success of the confer- oped,39. USA with the participation2 0,71 of other7 centres5,00 ac- ence6 and3,9 5course in Bellaterra8 was4,8 also2 due6,25 to the tive in this fieldTotal in 2the81 EU. 1Launched00 140 in1 0020013 2 with1 00 new152 training100 3format,2 100 that167 combined100 32 the10 0typical the extensive GVPN1 3conference countries 2(Evry2 countr ieofs France,13 count ries research29 countrie s conference18 countries (including29 countries invited18 coun tr lectures,ies 45 % women 44 % women 75 % women 49.5 % women 50 % women 39 % women 65 % women 4-5 October), the InVivoVector131 institutions Train project87 institut iconons - short communications73 institutions and poster101 institu sessions)tions with tinued with three workshops:(105 academic, 22 the(53 Eurolabcourse academic, 29 industries, 5 o ther a round(58 academ ic,table 13 industries, discussion 2 other (74 andacadem ic,a 25 practical industries, 2 other course industries, 4 other organisations) organisations) organisations) on vectorology in Evry,organisations 14-26(1)) April 2002, and two (see the table in the annex). After the basic tech- in 2004 in Bellaterra (Spain,T Othis T A Lreport) n u m b eand r o f again i n s t i t u t i oniques n s : 2 9 3 of( 2 2 6vector a c a d e m i c production, a n d 6 7 i n d u s t r purificationi e s ) and in Evry, 14-26 June . This educational activity ad- characterisationS P O N S O R S were delivered with the first EC-MC-FP5, AFM, EC-MC-FP5, AFM, INSERM, EC-LSH-FP6, FEBS, INSERM, EC-LSH-FP6, GENOPOLE, CG- dresses a primary needIndus tofries the scientificGENOPOLE, EcommuMBO, Indus-tries EurolabcourseGENOPOLE, Indu striesin 2002 in Essonne,Evry, INSERM, the InVivoVector Industries - (1) nitypatient’s to update associations, e tscientifichical/regulatory agenc conceptsies, scientific press and technolo- Train consortium thought it necessary to provide gies on gene therapy, in order to implement its training on the basic techniques of gene transfer developmental process towards a new medical in vivo into animals, which is the second essen- practice. tial step in gene therapy development. Therefore, lectures, short communications and posters on the principles of gene delivery into different or- Scheme of the training events in 2002 and 2004 (see gans (including gene expression assessment in www.vecteurotrain.org for more information)

4 days 8 days Conference Practical course Good practices (1), Deliverables: Concepts Examples business opportunities Experience

Activities: Tutorials Research Data Public round table Practical training Lectures, discussions Posters, short Discussions with large public communications exhibits, video-shows Teachers: International keynote Researchers Industries, regulatory agencies, Experts/ teachers/tutors speakers patients’ associations, opinion groups Participants from all institutions(2) Participants: 32 selected young researchers(3) (1) GLP, GMP, GCP, ethics, regulations... (2) 200 max PhD students, post docs, research staff, industries, regulators, other professionals (3) PhD students, post docs, young researchers, selected on the basis of scientific excellence and motivation to participate

New Therapies – Gene Therapy 147 InVivoVectorTrain

GVPNConference EUROLABCOURSE CONFERENCE & COURSE CONFERENCE & COURSE 4-5 October 2001 14-27 April 2002 1-14 February 2004 14-26 June 2004 EVRY EVRY BELLATERRA EVRY TOTAL TOTAL Participants in the TOTAL Participants in the TOTAL Participants in the Country participants participants practical course participants practical course participants practical course nb % % nb nb % nb nb % nb nb % nb % 1. Algeria 1 0,6 2. Austria 1 0,36 1 0,70 3 1,97 1 0,6 3. Belgium 13 4,63 10 7,10 1 0,66 2 1,2 4. Brazil 1 0,6 5. Bulgaria 1 0,66 1 3,13 2 1,2 1 3,13 6. Canada 1 0,70 2 1,32 1 3,13 2 1,2 7. China 1 0,36 2 1,32 1 3,13 1 0,6 1 3,13 8. Czech Republic 1 0,66 9. Denmark 3 2,10 1 0,66 10. Finland 1 0,6 1 3,13 11. France 223 79,36 42 30,00 8 25,0 45 29,61 4 12,5 64 38,3 4 12,5 12. Germany 8 2,85 17 12,10 2 6,25 3 1,97 8 4,8 1 3,13 13. Greece 2 1,32 1 3,13 1 0,6 1 3,13 14. Hungary 2 1,40 2 6,25 5 3,29 3 9,38 6 3,6 1 3,13 15. Iceland 1 0,66 1 3,13 16. Iran 1 0,66 1 0,6 1 3,13 17. Ireland 3 1,8 1 3,13 18. Israel 4 1,42 2 1,40 1 3,13 3 1,8 2 6,25 19. Italy 4 1,42 6 4,30 2 6,25 3 1,97 2 6,25 14 8,4 5 15,63 20. Japan 2 1,32 1 0,6 1 3,13 21. Korea 1 0,70 1 0,66 22. Latvia 1 0,70 23. Lithuania 2 1,40 1 3,13 2 1,32 2 6,25 24. Luxembourg 1 0,70 25. Mexico 1 0,66 26. Netherlands 3 1,07 6 4,30 1 3,13 3 1,97 1 3,13 5 3,0 27. Poland 1 0,66 1 3,13 1 0,6 1 3,13 28. Portugal 1 0,70 1 3,13 4 2,63 2 6,25 2 1,2 1 3,13 29. Romania 1 0,70 1 3,13 30. Russia 1 0,66 1 3,13 1 0,6 31. Slovenia 1 0,6 32. Spain 5 1,78 7 5,00 7 21,88 46 30,26 6 18,75 10 6,0 4 12,5 33. Sweden 2 0,71 11 7,90 4 12,5 3 1,97 1 3,13 3 1,8 34. Switzerland 3 1,07 5 3,60 1 3,13 3 1,97 6 3,6 1 3,13 35. Turkey 1 0,70 2 1,32 3 9,38 7 4,2 3 9,38 36. Taiwan 1 0,6 37. United Kingdom 12 4,27 12 8,60 1 3,13 5 3,29 10 6,0 38. Uruguay 1 0,66 1 3,13 39. USA 2 0,71 7 5,00 6 3,95 8 4,8 2 6,25 Total 281 100 140 100 32 100 152 100 32 100 167 100 32 100

13 countries 22 countries 13 countries 29 countries 18 countries 29 countries 18 countries 45 % women 44 % women 75 % women 49.5 % women 50 % women 39 % women 65 % women 131 institutions 87 institutions 73 institutions 101 institutions (105 academic, 22 (53 academic, 29 industries, 5 other (58 academic, 13 industries, 2 other (74 academic, 25 industries, 2 other industries, 4 other organisations) organisations) organisations) organisations(1)) T O T A L n u m b e r o f i n s t i t u t i o n s : 2 9 3 ( 2 2 6 a c a d e m i c a n d 6 7 i n d u s t r i e s )

S P O N S O R S EC-MC-FP5, AFM, EC-MC-FP5, AFM, INSERM, EC-LSH-FP6, FEBS, INSERM, EC-LSH-FP6, GENOPOLE, CG- Industries GENOPOLE, EMBO, Industries GENOPOLE, Industries Essonne, INSERM, Industries (1) patient’s associations, ethical/regulatory agencies, scientific press

Statistics on the participants in the training events on gene therapy since 2001

148 New Therapies – Gene Therapy

4 days 8 days Conference Practical course Good practices (1), Deliverables: Concepts Examples business opportunities Experience

live animals and the immune response), were followed by a practical training on gene deliver- Coordinator ing in vivo, i.e. administration into liver, lung, brain and muscle. Mauro Mezzina Généthon-CNRS FRE 3018 In this course, preclinical gene transfer, and the 1bis Rue de l’International theoretical and practical issues of gene vectors 91002 Evry, France as potential new bio-pharmaceuticals were pre- E-mail: [email protected] sented. For the first time, 32 researchers from 18 countries were trained on the techniques of in vivo gene transfer, based on the criteria of good laboratory practices and the ethical principles of animal experimentation.

Major publications

Mezzina, M., ‘Towards clinical gene therapy: preclinical gene transfer assessment’, Gene Therapy, 2004, vol. 11, S1 pp 1-172

New Therapies – Gene Therapy 149 IndustryVectorTrain European labcourse: advanced methods for industrial production, purification and characterisation of gene vectors

Contract No LSSB-CT-2003-505516 Project type Specific Support Action EC contribution e 172 000 Starting date 1 January 2004 Duration 21 months Website www.vecteurotrain.org

Background and objectives: Approach and methodology:

The topics developed in IndustryVectorTrain During the practical course, 32 selected research- focused on the biotechnological issues of gene ers acquired hands-on experience in advanced therapy (GT) products (vectors, plasmids, cell methods for the following: lines, etc.), and when they must be prepared at • the production of AAV (i.e. the tran- pharmaceutical scale. It is a mandatory step in sient transfection of 293 cells or infection the development process of gene therapy. A to- of insect cells with recombinant baculo- tal of 114 scientific communications (36 full lec- viruses) and purification with simple and tures, 23 short communications and 55 posters) fast methods using new ion-exchange were presented at a conference, highlighting the chromatographic devices; biology, use, and industrial development of the • the preparation of large batches main gene vectors with general and expert infor- of AdV purified with chromatographic mation on the following: procedures; • RVV, LVV, AdV and parvovirus-based, • the production of LVV vectors at a non-viral and other viral (alphavirus and large scale with improved packaging cell herpes-based) vector systems; lines; • some applications in gene transfer • new strategies to purify RVV and LVV into liver, muscle, CNS and HSC; pseudotyped with VSV-G protein; • biotechnological issues of scaling up, • the characterisation of viral vectors for i.e. theory and praxis of purification, cell basic bio-safety parameters (i.e. physical cultures techniques and devices for in- and functional titration and assessment dustrial production of RTV, AdV and AAV of adventitious contaminants and RCPs). vectors, as well as quality control analysis; • the regulatory and ethical issues of Main findings: gene transfer and GT; • in a special session or round table , a Researchers and other GT professionals attend- number of vector production facilities ing this course were trained in the scientific, and services currently available to the technological and regulatory issues concerning European and US scientific communities the evolutionary process of gene therapy prod- were presented. ucts towards new biopharmaceuticals. This could be achieved by integrating the skills of different professions: the researchers, who devise the gene transfer prototypes and protocols, and per-

150 New Therapies – Gene Therapy Gene Therapy

form animal experimentation to validate proofs Coordinator of principle; the regulators, who define the legal frame where gene therapy products must be Mauro Mezzina used in clinical trials; the industrialists, who allow Généthon-CNRS FRE 3018 technology transfers from the research benches 1bis Rue de l’International to the biotech factories in order to transform the 91002 Evry, France prototypes into exploitable manufactures; and E-mail: [email protected] the physicians, who are deeply involved in the last steps which lead to the completion of a clinical trial.

Major publications

Bagnis, C., Merten, O.W., Mezzina, M., ‘Advanced methods for industrial production purification and characterisation of gene vectors’, Gene Ther- apy, 2005, vol. 12, S1 pp 1-176

New Therapies – Gene Therapy 151 152 New Therapies – Immunotherapy and Transplantation

154 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation AlloStem The development of immunotherapeutic strategies to treat haematological and neoplastic diseases on the basis of optimised allogenic stem cell transplantation

Contract No LHSB-CT-2004-503319 Project type Integrated Project EC contribution e 8 000 000 Starting date 1 April 2004 Duration 48 months Website www.allostem.org

Background and objectives: AlloStem is developing protocols for the treat- Diseases of the blood, be they malignant or not, ment of patients with haematological disease, are often terminal. Over the last 30 years, im- and for the effective delivery of immunotherapy provements in chemotherapy and the use of to those patients. external agents in cancer treatments have been progressing steadily. Despite these advances, Approach and methodology: however, haematopoietic stem cell transplantation remains the only therapy that can result in The AlloStem consortium has brought together long-term disease-free survival of many patients, 29 clinical, research and industrial partners from especially those who have had relapses follow- the EU and abroad. The 24 biomedical research ing initial sessions of chemotherapy. teams and 5 SMEs involved as full partners are devoted to the advance of translational research It is also now possible to approach the treatment and new technology for the development of im- of these diseases through the exploitation of the munotherapeutic strategies to treat haemato- genetic differences between individuals, to pro- logical and neoplastic diseases. In line with their duce targets through which the immune system objectives, the consortium’s research efforts are can eliminate the diseased cells. This treatment, organised in three main work packages (WPs), immunotherapy, involves the stem cell transplant wherein the different participating groups coop- delivering specific immune effector cells expand- erate and constitute integrated task forces (TFs) ed from the donor. The scientific and technologi- to address the following topics: cal objectives of AlloStem are to develop new • development of immunotherapeutic strategies for optimising the use of allogeneic strategies to mediate anti-tumour activity; haematopoietic stem cell transplantation, based • the application of immunotherapeutic on the prediction and modulation of the immune strategies to control infectious diseases response in order to achieve the following: which threaten the lives of transplant re- • reduce the risk of graft versus host dis- cipients; ease (GvHD); • the optimisation of the transplant pro- • generate selective anti-tumour im- cedure as a basis for subsequent immuno- mune responses; therapy. • provide protective immunity against opportunistic infections; • extend the applicability of transplantation to a larger population of patients.

New Therapies – Immunotherapy and Transplantation 155 AlloStem

Expected outcome: HLA class I and class II ligands of HCMV, EBV and other relevant infectious pathogens. These new AlloStem targets improved healthcare for EU citi- ligands have been incorporated into assays for zens through new treatment protocols and phar- the monitoring of virus-specific immune recon- maceuticals. As a result of the AlloStem training stitution following allogeneic HSCT, as well as programme, knowledge in this important field in protocols of T cell stimulation and priming, will be broadened and developed in those coun- to generate virus-specific T cell lines and clones tries involved. for adoptive immunotherapy. Expression of toll- like receptors on dendritic cells has been studied Main findings: after stimulation with various infectious pathogens. Protocols were developed for the selection The results for the first 33 months have been im- of pathogen-specific T cells using the cytokine- pressive across each work package. The tumour capture assay. Standard operating procedures immunotherapy work package (WP1) has char- were developed for the generation of clinical acterised several new potentially immunogenic grade CMV- and EBV-specific T cell lines. peptides. Algorithms have been developed to allow for the determination of new potential The transplant modalities work package (WP3) targets of immunotherapy. Several important has defined new conditioning regimens of re- signals from toll-like receptors were identified, duced toxicity and sufficient immuno-suppres- marking these pathways in dendritic cells as po- sive qualities that allows more therapeutically tential targets, thus allowing manipulation of the targeted transplants of haematopoietic progeni- induction of a cellular immune response. tors from HLA mismatched donors, using different strategies of cell manipulation that enhance Protocols have been developed for the purposes the contributions of regulatory T cells, veto cells, indicated below: mesenchymal stem cells or NK cells. Protocols • selecting and enriching antigen spe- have been developed for the following purposes: cific T cells; • transplants of umbilical cord blood • allowing the modification of malig- supported by third party cells; nant cell populations, which result in the • cell selection and cell purging and/or generation of malignant antigen present- enrichment, using methods based on the ing cells; immune recognition of specific cell sur- • selecting and enriching antigen specif- face or functional markers. ic T cells following the activation by these cell populations, using cytokine capture Standard operating procedures for post-trans- reagents; plant assessment of tolerance (both in terms of • conducting important studies to bet- rejection and of GvHD), as well as for the recov- ter understand the interaction of Natural ery of protective immune functions were also Killer (NK) cells with target tumour cells. defined. Clinical assays based on these protocols were initiated. It should be noted that a number of crucial steps have also been taken to ensure new immuno- Major publications therapeutic strategies in the near future. Ghiringhelli, F., Puig, P.E., Roux, S., Parcellier, A., Sch- The infectious disease work package (WP2) has mitt, E., Solary, E., Kroemer, G., Martin, F., Chauffert, achieved the prediction and verification of new B., Zitvogel, L., ‘Tumor cells convert immature my-

156 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

eloid dendritic cells into TGF-beta secretors that N., Hertenstein, B., Rohde, F., ‘Oral valganciclo- stimulate the proliferation of CD4+CD25+ regu- vir leads to higher exposure to ganciclovir than latory T cells’, Journal of Experimental Medicine, intravenous ganciclovir in patients following al- 2005, 202:919-929 (Epub Sept 26). logeneic stem cell transplantation’, Blood, 2006, 107: 3002-3008. Marcenaro, E., Della Chiesa, M., Bellora, F., Paro- lini, S., Millo, R., Moretta, L., Moretta, A., ‘IL12 or IL4 prime human Natural Killer cells to mediate functionally divergent interactions with dendrit- Coordinator ic cells or tumors’, Journal of Immunology, 2005; 174:3992-3 Alejandro Madrigal The Anthony Nolan Trust Orabona, C., Puccetti, P., Vacca, C., Bicciato, S., Lu- The Royal Free Hospital chini, A., Fallarino, F., Bianchi, R., Velardi, E., Per- Pond Street ruccio, K., Velardi, A., Bronte, V., Fioretti, M.C., Hampstead, London NW3 2QG, UK. Grohmann, U., ‘Towards the identification of a E-mail [email protected] tolerogenic signacells’, Blood, 2005; Dec 8 (Epub ahead of print). Partners

Taieb, J, Chaput, N, Ménard, C, Apetoh, L, Péquignot, AlloStem Ltd M, Casares, N, Terme, M, Flament, C, Maruyama, K, London, UK Opolon, P, Lecluse, Y, Métivier, D, Tomasello, E, Vivi- er, E, Ghiringhelli, F, Martin, F, Klatzmann, D, Poy- Leonid Alexeev nard, T, Yagita, H, Ryffel, B, Kroemer, G., Zitvogel, State Research Centre Institute of Immunology L., ‘A novel dendritic cell subset involved in tu- Moscow, Russia mor immunosurveillance’, Nature Medicine, 2006, 12:214-219 (Epub Jan 29). Jirina Bartunkova Univerzita Karlova V Praze Stewart, C.A., Laugier-Anfossi, F., Vely, F., Saul- Prague, Czech Republic quin, X., Riedmuller, J., Tisserant, A., Gauthier, L., Romagne, F., Ferracci, G., Arosa, F.A., Moretta, A., Javier Bordone Sun, P.D., Ugolini, S., Vivier, E., ‘Recognition of pep- ITMO Fundacion Mainetti tide-MHC class I complexes by activating killer Buenos Aires, Argentina immunoglobulin-like receptors’, Proceedings Na- tional Academy of Sciences, USA, 2005, 102:13224- Dominique Charron 13229. HLA et Médecine Paris, France Shaw, B., Marsh, S.G.E., Mayor, N.P., Russell, N.H., Madrigal, J.A., ‘HLA-DPB1 matching status has Hans-Georg Rammensee significant implications for recipients of unre- Eberhard Karls-Universitaet lated donor stem cell transplants’, Blood, 2006, Tuebingen, Germany 107:1220-1226. Fred Falkenburg Einsele, H., Reusser, P., Bornhäuser, M., Kalhs, P., University of Leiden Medical Centre Ehninger, G., Hebart, H., Chalandon, Y., Kröger, Leiden, Netherlands

New Therapies – Immunotherapy and Transplantation 157 AlloStem

Manuel Fernandez Nikolai Schwabe Universidad Autonoma de Madrid Proimmune Ltd Madrid, Spain Oxford, UK

Juan Garcia Robert Rees Centre de Transfusio I Banc de Teixits Nottingham Trent University Barcelona, Spain Nottingham, UK

Els Goulmy Yair Reisner University of Leiden Medical Centre Weizmann Institute of Science Leiden, Netherlands Rehovot, Israel

François Romagne Dolores Schendel Innate Pharma GSF Forschungszentrum fuer Umwelt und Gesundheit Marseille, France GmbH Munich, Germany Andrzej Lange Ludwik Hirszfeld Institute Andrea Velardi Wroclaw, Poland Università Degli Studi di Perugia Perugia, Italy Franco Locatelli IRCCS Policlinico San Matteo Eric Vivier Pavia, Italy Centre National de la Recherche Scientifique Marseille, France Mario Assenmacher Miltenyi Biotec GmbH Laurence Zitvogel Bergisch Gladbach, Germany Institut Gustave Roussy Villejuif Cedex, France Alessandro Moretta Università degli studi di Genova Hermann Einsele Genoa, Italy Julius-Maximilians-University Wuerzburg, Germany Lorenzo Moretta Istituto Giannina Gaslini Oystein Aamellem Genoa, Italy Dynal Biotech ASA Oslo, Norway Ricardo Pasquini Universidade Federal do Parana Parana, Brazil

Pavel Pisa Karolinska Institutet Stockholm, Sweden

158 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation DC-THERA Dendritic cells for novel immunotherapies

Contract No LHSB-CT-2004-512074 Project type Network of Excellence EC contribution e 7 600 000 Starting date 1 January 2005 Duration 60 months Website www.dc-thera.org

Background and objectives:

Dendritic cell (DC) immunobiology has enormous potential for the development of new immunotherapies for cancer and infectious disease. Europe is home to a critical mass of leaders in the field, who have pioneered many advances and provided initial proof of principle for the approach. DC-THERA is a Network of Excellence (NoE) whose goal is to promote the integration of the activities of 26 participant groups of scientists and clinicians, along with 6 expert SMEs across Europe, over its five-year duration.

DC-THERA will incorporate additional groups, particularly from new and candidate Member States, as Associated Members of the Network. Fig 1. Langerhans cells (a type of dendritic cell) and Their collective expertise and resources will be gamma-delta T cells in skin epidermis forged into an ambitious joint programme of activities, so as to restructure the field. The project diseases. The network will provide a centralised will translate genomic, proteomic and bioinfor- European resource of databases for the field. matic information, with knowledge from molecular cell biology and preclinical models, into DC-THERA will develop new research tools, inte- therapeutic endpoints: clinical trials of DC-based grate existing and new technological platforms, therapies for cancer and HIV. recruit additional support staff, and make these available as shared resources for all partners. It will Approach and methodology: implement an ambitious education and training programme, including new PhD studentships, a Four thematic S&T Clusters have been defined, visiting scholars scheme, and high-quality train- with a fifth for horizontal activities. The latter ing courses, with the possibility of a postgradu- includes development of synergistic links with ate degree in translational DC immunobiology. other networks, providing added value to EC DC-THERA will contribute to the European bio- programmes by underpinning all projects de- technology sector and have a major impact on veloping new vaccine strategies for major killer European policymaking for the future.

New Therapies – Immunotherapy and Transplantation 159 DC-THERA

and MRI, has been used to visualise intracellular signalling events, intercellular communication in real time, and cell migration in living tissues. In WP5, intracellular signalling pathways have been investigated in different subsets of DC, and during their responses to different stimuli. Small inhibitory RNA (siRNA) constructs have been prepared and delivered to DC to explore the effect of interfering with these pathways. In WP6, the responses of DC to ‘intrinsic’ stimuli (e.g. inflammatory cytokines, CD40 ligation) and ‘extrinsic’ stimuli (e.g. toll-like receptor, TLR, agonists) have been studied in depth, and some of the findings Fig 2. Scanning electron micrograph of dendritic cells (e.g. will facilitate the generation of more potent im- the cell with ‘veils’ at the right hand side) interacting with munostimulatory DC for clinical use. resting T cells and an activated T cell (small round cells and large round cell, respectively) Cluster 3, Global Responses, comprises two Expected outcome: WPs: DC as adjuvants in vivo (WP7) and pre-clinical studies (WP8). In WP7, two approaches have DC-THERA will evolve into a centre of excellence been taken to enhance immune responses infor DC Biology, with a lasting and global impact. duced by DC, either by targeting particular receptors on DC themselves, or by targeting dif- Main findings: ferent cell types, such as natural killer (NK) cells, NKT cells and gamma-delta T cells, to exploit By the end of its second year, the network has their bidirectional interactions with DCs. In WP8, made considerable progress in all areas. Some a variety of preclinical models of infectious dis- highlights are as follows: ease and cancer have been used, respectively, to Cluster 1, Genes and Proteins, comprises three investigate the role of DCs in immunity against work packages (WPs): genomics (WP1), proteom- viruses (LCMV and HSV-1, the latter in neonates) ics (WP2) and bioinformatics (WP3). In WP1, tran- and bacteria (Mycobacterium tuberculosis and scriptional profiling of DCs exposed to different listeria), as well as to explore various strategies microbial stimuli has resulted in the observation to enhance anti-tumour responses that include that surprisingly similar patterns of gene expres- depletion of regulatory T cells. sion may be involved in cellular responses to different agents. In WP2, initial proteomic mapping Cluster 4, Therapeutic Applications, comprises of subcellular organelles of DC, including phago- three WPs: clinical trials (WP9), immunomonitor- somes and exosomes, is in progress. And in WP3, ing (WP10) and regulatory affairs (WP11). In WP9, testing of a database that will ultimately link ge- clinical trials of DC-based immunotherapy for a nomic and proteomic information is under way. variety of cancers including chronic lymphocytic leukaemia, melanoma and renal cell carcinoma Cluster 2, Molecular Cell Biology, also comprises are continuing, while others are in advanced three WPs: advanced imaging (WP4), intracellu- stages of planning, and a retrospective critical re- lar signalling (WP5) and in vitro activation of DC view of published trials will be undertaken in the (WP6). In WP4, a variety of imaging techniques, next period. In WP10, a critical review of immu- including confocal and two photon microscopy, nomonitoring approaches has also been under-

160 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

taken, and initial consideration has been given to the future standardisation of immunomonitoring approaches. Finally, in WP11, standard operating procedures for DC generation and quality control, and templates for clinical trial protocols and case report forms have been prepared. These will be made available to different centres as models before the preparation of master standard operating procedures and templates commences, in the next period.

As an NoE, DC-THERA seeks to enhance not only the integration of joint research activities, as exemplified by the four S&T clusters, but also the infrastructure underpinning the fields of DC immunobiology, vaccinology and immunotherapy in Europe. Cluster 5 has been structured to meet the latter goal, and it comprises four WPs. The first, ‘promoting integration’ (WP12), is designed to enhance the research capabilities of the network, and to remove obstacles hampering progress, by establishing core technological platforms and promoting the development of Fig 3. Magnetic resonance (MR) imaging of lymph node shared tools and protocols. The second, ‘ensuring region before and after DC vaccination. The patient was injected with superparamagnetic iron oxide(SPIO)-la- excellence’ (WP13), focuses on the development beled DC in a lymph node of the inguinal lymph node of education and training activities, the sharing region under ultrasound guidance. MR imaging of this and dissemination of expertise and resources region was performed before (fig 1a,c,e) and 48 hours after (fig 1b,d,f) the injection of the DC. Arrows indicate with groups from outside of the network, and lymph nodes that show a decreased signal intensity after the forging of collaborative links with other vaccination indicating the presence of SPIO-DC. networks. workshops on gene expression profiling and bio- Three technological platforms have now been informatics: DC migration and dynamic imaging established, as part of WP12, for genomics, imag- techniques; good manufacturing practice (GMP) ing and cell therapeutics in order to enhance the and good clinical practice (GCP). research infrastructure for S&T clusters 1, 2 and 4, respectively. For example, the genomics platform As part of its education activities in WP13, DC- has provided microarray analyses as a service to THERA has created 13 new PhD positions for other partners who would not necessarily nor- young researchers who target collaborative re- mally have access to such specialised expertise search projects between 21 of the network’s 32 and resources. The platforms are also responsible, partners. These positions are fincanced for a total as part of WP13, for delivering the training activi- of 37 person years. It has also organised an annual ties for the network, which include theoretical Graduate School for Young Investigators, the first courses, practical workshops and individual train- of which was held in Celerina, St Moritz of Swit- ing for specific purposes. To date, the network zerland, in March 2006. The remaining activities has provided a total of six high quality courses or in WP13 include an associated partner scheme in

New Therapies – Immunotherapy and Transplantation 161 DC-THERA

which specific groups from outside the network dendritic cells in melanoma patients for moni- are invited to participate in its activities for the toring of cellular therapy’, Nat Biotechnol, 2005, mutual benefit of both, and for the exploration Nov;23(11):1407-13. of potential collaborative interactions with other networks in related fields. Van Hall, T., Wolpert, E.Z., van Veelen, P., Laban, S., van der Veer, M., Roseboom, M., Bres, S., Grufman, To date, a total of ~40 additional groups and SMEs P., de Ru, A., Meiring, H., de Jong, A., Franken, K., working in the area have been integrated as as- Teixeira, A., Valentijn, R., Drijfhout, J.W., Koning, sociated partners, including several from new F., Camps, M., Ossendorp, F., Karre, K., Ljunggren, and candidate member states. Moreover, close H.G., Melief, C.J., Offringa, R., ‘Selective cytotoxic interactions with a STREP in the area, DC-VACC, T-lymphocyte targeting of tumor immune es- has resulted in two joint meetings between the cape variants’, Nat Med, 2006, Apr;12(4):417-24. networks. WP13 also deals with communications issues and includes the development of the net- Sporri, R., Reis e Sousa, C., ‘Inflammatory media- work website at www.dc-thera.org, which is now tors are insufficient for full dendritic cell activa- integrated with a new management and com- tion and promote expansion of CD4+ T cell pop- munications software tool for all partners and ulations lacking helper function’, Nat Immunol, associated partners. The remaining two WPs of 2005, Feb;6(2):163-70. Cluster 5, ‘exploiting results’ (WP14) and ‘maintaining infrastructures’ (WP15) deal with man- Frentsch, M., Arbach, O., Kirchhoff, D., Moewes, B., agement issues per se and look forward to the Worm, M., Rothe, M., Scheffold, A., Thiel, A., ‘Direct future sustainability and continued impact of the access to CD4+ T cells specific for defined anti- network after the end of the contract. gens according to CD154 expression’, Nat Med, 2005, Oct;11(10):1118-24. Major publications Napolitani, G., Rinaldi, A., Bertoni, F., Sallusto, Savina, A., Jancic, C., Hugues, S., Guermonprez, P., F., Lanzavecchia, A., ‘Selected Toll-like receptor Vargas, P., Moura, I.C., Lennon-Dumenil, A.M., Sea- agonist combinations synergistically trigger a bra, M.C., Raposo, G., Amigorena, S., ‘NOX2 controls T helper type 1-polarizing program in dendritic phagosomal pH to regulate antigen processing cells’, Nat Immunol, 2005, Aug;6(8):769-76. during crosspresentation by dendritic cells’, Cell, 2006, Jul 14;126(1):205-18. Taieb, J., Chaput, N., Menard, C., Apetoh, L., Ullrich, E., Bonmort, M., Pequignot, M., Casares, N., Terme, Koch, M., Stronge, V.S., Shepherd, D., Gadola, M., Flament, C., Opolon, P., Lecluse, Y., Metivier, S.D., Mathew, B., Ritter, G., Fersht, A.R., Besra, G.S., D., Tomasello, E., Vivier, E., Ghiringhelli, F., Martin, Schmidt, R.R., Jones, E.Y., Cerundolo, V., ‘The crys- F., Klatzmann, D., Poynard, T., Tursz, T., Raposo, G., tal structure of human CD1d with and without Yagita, H., Ryffel, B., Kroemer, G., Zitvogel, L., ‘A alpha-galactosylceramide’, Nat Immunol, 2005, novel dendritic cell subset involved in tumor im- Aug;6(8):819-26. munosurveillance’, Nat Med, 2006, Feb;12(2):214-9.

De Vries, I.J., Lesterhuis, W.J., Barentsz, J.O., Ver- dijk, P., van Krieken, J.H., Boerman, O.C., Oyen, W.J., Bonenkamp, J.J., Boezeman, J.B., Adema, G.J., Bulte, J.W., Scheenen, T.W., Punt, C.J., Heerschap, A., Figdor, C.G., ‘Magnetic resonance tracking of

162 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

Coordinator Andrea Splendiani Leaf Bioscience S.R.L. Jonathan M Austyn Milan, Italy University of Oxford John Radcliffe Hospital Charles Nicolette Nuffield Department of Surgery Argos Biosciences, Inc Headington Durham, US Oxford OX3 9DU, UK E-mail (Project Manager): [email protected] Filippo Petralia Sekmed S.R.L. Partners Milan, Italy

Carl G. Figdor Sebastian Amigorena University Hospital Institut Curie Medical Centre Nijmegen Paris, France Nijmegen, Netherlands Thierry Boon and Pierre Coulie Muriel Moser Christian de Duve Instite of Cellular Pathology Université Libre de Bruxelles Brussels, Belgium Brussels, Belgium Duccio Cavalieri Anne O’Garra Preclinica e Clinica Università degli studi di Firenze Medical Research Council Dipartimento Di Farmacologia London, UK Florence, Italy

Francesca Granucci Sandra Gessani University of Milano-Bicocca Istituto Superiore di Sanita Milan, Italy Rome, Italy

Gerold Schuler and Alexander Steinkasserer Nicolas Glaichenhaus Friedrich Alexander Universitat Universite Nice-Sophia Antipolis Erlangen, Germany INSERM Valbonne, France Robert Coffin Biovex Rolf Kiessling, Hakan Mellstedt, Pavel Pisa Abingdon, UK Karolinska Institutet Stockholm, Sweden Catherine De Greef BruCells SA Cornelis J. M. Melief Brussels, Belgium Leiden University Medical Centre Leiden, Netherlands Ugo D’Oro Chiron Vaccines S.R.L. Philippe Pierre Siena, Italy Centre CNRS-INSERM d’Immunologie de Marseille-Luminy Marseille, France

New Therapies – Immunotherapy and Transplantation 163 DC-THERA

Maria Pia Protti Matthias Mann Fondazione Centro San Raffaele Del Monte Tabor Max-Planck Institute of Biochemistry Milan, Italy Martinsried, Germany

Andreas Radbruch, Alexander Scheffold, Alberto Mantovani Andreas Thiel Istituto Clinico Humanitas Deutsche Rheuma-Forschungszentrum Berlin Milan, Italy Berlin, Germany

Caetano Reis e Sousa Cancer Research UK London, UK

Benedita Rocha Université René Descartes Faculté de Médecine Necker Paris, France

Antonio Lanzavechia, Markus Manz, Federica Sallusto, Mariagrazia Uguccioni Insitute for Research in Biomedicine Bellinzona, Switzerland

Mark Suter University of Zurich Zurich, Switzerland

Kris Thielemans Vrije Universiteit Brussel Brussels, Belgium

Hermann Wagner Technische Universitaet Muenchen Munich, Germany

Laurence Zitvogel Institut Gustave Roussy Paris, France

Paola Ricciardi-Castagnoli Genopolis – Consortium for Functional Genomics Milan, Italy

164 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation DC-VACC Therapeutic in vivo DNA repair by site-specific double-strand breaks

Contract No LSHB-CT-2003-503037 Project type Specific Targeted Research Project EC contribution e 2 000 000 Starting date 1 January 2004 Duration 36 months

Background and objectives: of Vaccine Technologies Targeted to Dendritic Cells’, proposed as a STREP for the Framework The immune system of vertebrate animals Programme 6. The basis of this proposal was to evolved to respond to different types of per- develop in situ DC targeting for use as vaccines turbations, such as pathogens, whilst limiting in infectious diseases and cancer. self-tissue damage. Initiation of the immune response is accomplished by unique antigen pre- Approach and methodology: senting cells called dendritic cells (DC) –highly phagocytic sentinels of the immune system, rest- The consortium has effectively generated im- ing until they encounter foreign microorganisms proved reagents and protocols for antigen de- or inflammatory stimuli. livery and targeting, which improved antigen processing and presentation by DC and can be Early-activated DC trigger innate immune re- used for vaccine and therapeutic technology. sponses that represent the first line of defence They have also defined optimal reagents and against pathogens and provide effective anti- protocols for maturation and activation of mouse tumour immunity. Activated DC subsequently and human DC in vitro for use in vaccination and/ prime antigen-specific immune responses (T or therapeutic intervention. Optimisation of pro- and B lymphocytes) to clear the infections and tocols was compared across both species, which give rise to immunological memory. Activation is essential for use in preclinical models and clini- of Natural Killer (NK) cells is also key during an cal trials in future NoEs and/or IPs. anti-pathogen response. A direct involvement of NK cells has also been shown in anti-tumour re- Main findings: sponses in different systems and unequivocally observed in patients with cancer. DC-VACC targeted two specific objectives in Work Packages (WP) 1 and 2. The aim of DC-VACC, a Specific Targeted Research project (STREP), was to develop novel vaccine WP1 generated the tools and methods required technologies and to use DC as natural adjuvants for appropriate and efficient targeting and anti- with specificity and minimum side effects. Early gen delivery for the development of DC vaccine clinical trials have shown that antigen-pulsed DC technology. This included the construction of have potential in the treatment of cancer – also viral and bacterial vectors, modification of RNA, applicable in the eradication of infectious dis- peptides and proteins, and antibody develop- eases. The objective of the proposed project was ment for specific targeting of DC receptor rep- part of the priority area 1.2.4-6: ‘Development ertoire. Excellent research progress was made in

New Therapies – Immunotherapy and Transplantation 165 DC-VACC

Time (min)

the following: The humanised antibody to DC-SIGN hD1 G2/G4 (hD1) • microbial vectors development (bac- was cross-linked to a model antigen, keyhole limpet hemocyanin (KLH). Using confocal microscopy it was terial and viral); observed that the chimeric antibody-protein complex • modification of RNA, peptides and (hD1-KLH).bound specifically to DC-SIGN, was rapidly proteins; internalised and translocated to the lysosomal compartment as shown in the above figure over time (minutes). • antibody development for specific targeting of DC receptor repertoire There has been great productivity with respect WP2 defined optimal reagents and protocols for to the development of microbial vectors, and the maturation and activation of mouse and human ability to deliver antigen and target DC, as well as DC in vitro for use in vaccination and/or thera- approaches. These include the discovery of new peutic intervention, such that optimisation of pathways to target the enhancement of anti-tu- protocols for both species are compared so as to mour therapy and therapeutic intervention, as promptly facilitate information from preclinical well as for vaccination in infectious diseases. models being transferred rapidly to clinical trials in future NoEs and/or IPs. The DC-VACC consor- The knowledge from this project has been widely tium determined the following: disseminated via publications and international • signals for activation/maturation of immunology conferences. During the duration mouse and human DC; of the project, meetings (held together with an • optimised human DC-vaccines: rea- NoE, DC-THERA) were held at various sites to ex- gents and procedures; change scientific knowledge and via the website • transcriptome analysis of mouse and created by Partner Biopolo (http://www.biopolo. human DC; it/), where the list of publications generated from • DC-based bioassays. DC-VACC are displayed.

Ultimately, the studies from DC-VACC have resulted in the discovery of new adjuvant approaches.

166 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

Major publications Boonstra, A., Rajsbaum, R., Holman, M., Marques, R., Asselin-Paturel, C., Pereira, J.P., Bates, E.M., Akira, Smith, C.L., Dunbar, P.R., Mirza, F., Palmowski, M.J., S., Vieira, P., Liu, Y-J., Trinchieri, G., O’Garra, A. ‘Mac- Shepherd, D., Gilbert, S.C., Coulie, P., Schneider, J., rophages and myeloid DC, but not plasmacytoid Hoffman, E., Hawkins, R., Harris, A.L., Cerundolo, V., DC, produce IL-10 in response to MyD88- and ‘Recombinant modified vaccinia Ankara primes TRIF- dependent TLR signals, and TLR-independ- functionally activated CTL specific for a melano- ent signals’, J.Immunol, 2006, 177, 7551 - 7558. ma tumor antigen epitope in melanoma patients with a high risk of disease recurrence’, Int J Cancer, 2005, Jan 10;113(2):25.

Hermans, I., Silk, J., Gileadi, U., Masri, H.S., Shepherd, D., Farrand, K.J., Salio, M., Cerundolo, V., ‘Dendritic cell function can be modulated through co-operative actions of TLR ligands and invariant NKT cells’, J. of Immunology, 2007, Mar 1; 178(5):2721-9.

Granucci, F., Zanoni, I., Pavelka, N., van Dommelen, S.L.H., Andoniou, C.E., Belardelli, F., Degli Esposti, M.A., Ricciardi-Castagnoli, P., ‘A Contribution of Mouse Dendritic Cell-Derived IL-2 for NK Cell Ac- tivation’, J. Exp. Med., Aug 2004; 200: 287-295.

Foti, M., Granucci, F., Pelizzola, M., Beretta, O., Ric- ciardi-Castagnoli, P., ‘Dendritic cells in pathogen recognition and induction of immune responses: a functional genomics approach’, J Leukoc Biol, 2006, May;79(5):913-6. Review.

Schaft, N., Dorrie, J., Thumann, P., Beck, V.E., Mull- er, L., Schultz, E.S., Kampgen, E., Dieckmann, D., Schuler, G., ‘Generation of an optimized polyva- lent monocyte-derived dendritic cell vaccine by transfecting defined RNAs after rather than before maturation’, J. Immunol, 2005, 174 (5):3087- 3097.

Prechtel, A.T., Turza, N.M., Kobelt, D.J., Eisemann, J.I., Coffin, R.S., McGrath, Y., Hacker, C., Ju, X., Zenke, M., Steinkasserer, A., ‘Infection of mature dendritic cells with herpes simplex virus type 1 dramati- cally reduces lymphoid chemokine-mediated migration’, J Gen Virol, 2005, Jun;86(Pt 6):1645-57.

New Therapies – Immunotherapy and Transplantation 167 DC-VACC

Coordinator

Anne O’Garra National Institute for Medical Research Council The Ridgeway Mill Hill London NW7 1AA, UK E-mail: [email protected]

Partners

Leonardo Biondi Biopolo SCRL Milan, Italy

Vicenzo Cerundolo University of Oxford Oxford, UK

Carl Figdor University Medical Centre Nijmegen, Netherlands

Muriel Moser Université Libre de Bruxelles Brussels, Belgium

Paola Ricciardi Castagnoli University of Milano-Bicocca Milan, Italy

Gerold Schuler University Hospital of Erlangen Erlangen, Germany

Filippo Petralia Sekmed Srl Milan, Italy

Kai Zwingenberger Brucells SA Brussels, Belgium

168 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation THERAVAC Optimised delivery system for vaccines targeted to dendritic cells

Contract No LSHB-CT-2004-503582 Project type Specific Targeted Research Project EC contribution e 2 267 000 Starting date 1 March 2004 Duration 54 months

Background and objectives:

In response to urgent medical and societal needs for novel immunotherapies for cancer and chronic infections, as well as for prophylactic vaccination, optimised delivery systems for vaccines targeting dendritic cells will be developed and clinically evaluated. The approach relies on two new antigen delivery vectors: the detoxified adenylate cyclase toxoid (ACT) and the porcine parvovirus-like Electron microscopy image of PPV-VLP particles, one of particles (PPV-VLP), which were recently shown to the two vaccine delivery vehicles developed by THERAVAC target dendritic cells very efficiently and specifically, allowing for a highly effective presentation in-depth analysis of the cellular and molecular of delivered antigens to T cells. mechanisms, as well as of the structural basis of ACT interaction with dendritic cells will be con- These vaccine vectors enable the induction of ducted. Particular emphasis will be placed on strong, specific and protective immune respons- gaining new knowledge for furthering the de- es, and have an established record of safety and livery capacity of the ACT molecule towards en- efficacy in preclinical animal models. Under this hanced efficiency and broader versatility in clini- project, academic experts in immunology, vac- cal use. The PPV-VLP vector will be developed in cinology, and molecular and structural biology parallel, by defining its cellular receptor and traf- have joined forces with toxicologists, clinicians ficking inside dendritic cells, preclinical efficacy and vaccine production experts from two com- and toxicology, in order to bring this alternative panies, to translate these novel vaccine technolo- vaccine carrier to the level of clinical trial maturity. gies from research into clinical application. Approach and methodology: Based on the preclinical record of ACT-based vaccines in animal models, the step into the THERAVAC combines basic research into the safety and efficacy phase I/II human trial will be mechanism of function of these novel vaccine made with an ACT-based construct delivering targeting systems with their preclinical applica- the tyrosinase A.2 epitope as a therapeutic vac- tion, including a phase I/II trial. The first two work cine for metastasic melanoma. Simultaneously packages form a demonstration activity centred with Good Manufacturing Practice (GMP) batch on the production of the GMP lot and the han- production, development and clinical testing, an dling of the phase I/II trial. The remaining four

New Therapies – Immunotherapy and Transplantation 169 THERAVAC

work packages correspond to the research and These various activities associated with the ACT development component of the project, aimed molecule synergise to bring about the potency at understanding the basic mechanisms at the of ACT as an antigen delivery vector into den- molecular level. The latter work package employs dritic cells and induction of specific T cell medi- cell biology, immunology, biochemistry and bio- ated responses against delivered antigens. The physics, including structural biology, to address complex activity of ACT is dissected into indi- the questions of specificity and efficacy of the vidual contributions by characterising properties vaccine vehicle molecules used. of ACT variants, which are selectively blocked by specific mutations in individual steps of toxoid Expected outcome: penetration into cells and in channel-forming and signalling properties of ACT. A novel mecha- THERAVAC aims to achieve: nism of calcium mobilisation into myeloid cells • production of a GMP batch of ACT car- by ACT was discovered, and shown to be linked rying the melanoma tyrosinase epitope to AC domain translocation across cytoplasmic suitable for a phase I/II trial; membrane of cells. Over the past 36 months, • the detailed toxicology assessment of important progress was made in understanding this GMP batch of ACT; the mechanisms underlying the toxoid interac- • a phase I/II trial in melanoma patients tion with CD11b, signalling activity on myeloid of this GMP batch of ACT carrying the ty- cells and the capacity to deliver the AC domain rosinase melanoma CD8+ T cell epitope; with antigens into cells. This was efficiently used • the understanding of the interaction to manipulate the antigen delivery potency of of ACT with dendritic cells at molecular toxoid forms of ACT. and atomic details; • the detailed understanding of the Important progress was also made in the under- mechanism of PPV-VLP specificity and standing of the mechanisms of how PPV-VLPs efficacy; interact with dendritic cells. Importantly, these • the engineering of improved vac- VLPs were shown to possess a strong adjuvant cine delivery molecules based on our im- activity. THERAVAC demonstrates that the bacu- proved understanding of their functional lovirus (BV) is responsible for this adjuvant effect mechanism. and that it plays a major role in the strong immu-

Main findings: Scheme of antigen delivery by recombinant ACT. The toxoid inserts into cellular membrane of antigen The Bordetella adenylate cyclase toxin (ACT) presenting cells in two different conformations that give binds the complement receptor 3 (CD11b/CD18, rise to molecules with the AC domain translocated into cell cytosol and to molecules forming oligomeric membrane CR3 or Mac-1) of host myeloid phagocytes, pen- channels with the AC domain stuck at the external face of etrates their cytoplasmic membrane, and upon cellular membrane. The translocated AC domain bearing the inserted antigen is processed inside the cytosol of antigen translocation into cytosol and binding of cal- presenting cells by proteasome. The peptides are then modulin, it delivers into the MHC Class I/II anti- transported by TAP into the endoplasmic reticulum and bind gen presentation pathway its AC domain. Fur- to newly synthesised MHC I molecules. The MHC I – peptide complexes are transported to the cell surface and pre- thermore, ACT can form small cation selective sented to CD8+ T cells, thereby promoting their activation. channels in target membranes, bind calcium, Endocytosis of membrane associated CyaA may also occur promote its influx into cells and induce a cascade and CyaA molecules can be processed to antigenic peptides that bind to MHC II molecules. These MHC II complexes are of signalling events leading to the maturation of presented to CD4+ T cells. Reviewed in Simsova et al. (2004) dendritic cells. Int. J. Med. Microbiol. 293, 571-576. © Urban & Fischer Verlag.

170 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

nogenicity of PPV-VLPs produced in the baculo- Basler, M., Masin, J., Osicka, R., Sebo, P., ‘Pore-form- virus-insect cell expression system. This adjuvant ing and enzymatic activities of Bordetella ade- behaviour of BVs is mediated primarily by IFNα/β, nylate cyclase synergise in promoting lysis of although mechanisms independent of type I in- monocytes’, Infect Immun, 2006, 74, 2207-14. terferon signalling are also involved. Mascarell, L., Bauche, C., Fayolle, C., Diop, O.M., The first pre-GMP batch has been prepared on a Dupuy, M., Nougarede, N., Perraut R., Ladant, D., small scale by CellGenix, GmbH, the subcontrac- Leclerc, C., ‘Delivery of the HIV-1 Tat protein to tor chosen for this task. Once this batch is vali- dendritic cells by the CyaA vector induces spe- dated, a GMP-like batch will be provided for toxi- cific Th1 responses and high affinity neutralising cology, as well as a larger-scale GMP preparation antibodies in non human primates’, Vaccine, 2006, for the clinical trial, starting in 2008. 24, 3490-9.

Major publications Prior, S., Fleck, R. A., Gillett, M., Rigsby, P., Corbel, M. a) Publications in peer-reviewed journals J., Stacey, G., Xing, D. K. L., ‘Evaluation of adenylate

New Therapies – Immunotherapy and Transplantation 171 THERAVAC

cyclase toxin constructs from Bordetella pertus- Coordinator sis as vaccine candidate antigens in an in vitro complement-dependent opsonophagocytic-kill- Claude Leclerc ing model’, Vaccine, 2006, 24:4794-4803. Institut Pasteur 25 rue du Dr. Roux Cheung, Y.C.G., Xing, D., Prior, S., Corbel, M.J., Par- 75014 Paris, France ton, R., Coote, J.G., ‘Adjuvant effect of different E-mail: [email protected] forms of adenylate cyclase toxin of Bordetella pertussis on protection afforded by acellular per- Partners tussis vaccine in a murine model. Infect. Immu, 2006, 74:6797-6805. Anita Lewit-Bentley CNRS Bauche, C., Chenal, A., Knapp, O., Bodenreider, C., Cachan, France Benz, R., Chaffotte, A., Ladant, D., ‘Structural and functional characterisation of an essential RTX Rino Rappuoli sub-domain of Bordetella pertussis adenylate cy- Novartis clase toxin’, J Biol Chem, 2006, 281:16914-16926. Sienna, Italy

Fiser, R., Masin, J., Basler, M., Krusek, J., Spulakova, Paloma Rueda V., Konopasek, I., Sebo, P., ‘Third Activity of Bor- INGENASA detella Adenylate Cyclase (AC) Toxin-Hemolysin: Madrid, Spain Membrane translocation of AC domain polypeptide promotes calcium influx into CD11b+ mono- Peter Sebo cytes independently of the catalytic and hemo- Institute of Microbiology lytic activities’, J. Biol. Chem, 2007, 282 2808-2820. Prague, Czech Republic

Vojtova, J., Kamanova, J., Sebo, P., Bordetella ade- Daniel Ladant nylate cyclase toxin: a swift saboteur of host de- Institut Pasteur fense. Curr. Op. Microbiol, 2006, 9, 69-75. Paris, France

Hervas-Stubbs, S., Rueda, P., Lopez, L., Leclerc, C., Benoît Van den Eynde Insect baculovirus strongly potentiate adap- ICP tive immune responses through the induc- Brussels, Belgium tion of type 1 interferon. J. Immunol, 2007, Feb 15;178(4):2361-9. Dorothy Xing NIBSC b) Patents Potters Bar, UK

Leclerc, C., Boisgerault, F., Rueda, P., ‘Empleo de partículas pseudovirales como adyuvante. No. P200403077’ Presented in the Spanish Patent and Trademark Office, 23/12/2004.

Leclerc, C., Hervas-Stubbs, S., Rueda, P., Lopez, L.. Viral adjuvants. EP 06380240.9, 1.09.06

172 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation DENDRITOPHAGES Therapeutic cancer vaccines

Contract No LSHB-CT-2003-503583 Project type Specific Targeted Research Project EC contribution e 1 999 940 Starting date 1 January 2004 Duration 42 months

Background and objectives: transformed into effector monocyte-derived DCs (dendritophages) which fight the patient’s own Cancer has been considered as a fatal disease disease. The therapeutic cell drug comprises DCs for many years and has been selected as a pri- loaded with cancer-specific antigens to activate ority disease throughout successive European the patient’s immune system after re-injection. research programmes. Cancer is a group of over This ex vivo approach is compared to the in vivo 100 related diseases characterised by uncon- injection of the tumour antigen plus adjuvant. trolled cell growth that expands locally by invasion and systemically by metastases. Cancer is the This project aims to demonstrate on a second most common cause of death in western multinational level, the immunogenicity and countries accounting for 2.5 million deaths in the efficacy, as well as the reproducibility and developed world. feasibility of anticancer vaccine by sequential steps: choosing the best DC vaccination strategy Existing therapies have been able to increase through adequate pre-clinical studies (DC the survival rate, though only marginally. Cur- differentiation and maturation, tumour antigens rent cancer treatments allow for the treatment selection and loading, dose delivered, and site of approximately 50% of the patients, but these and vaccination schedule); and monitoring the therapies are associated with potentially serious immune response in correlation to the clinical or intolerable adverse side effects. Thus, the qual- response after defining the most relevant ity of life of cancer patients is presently not sat- immuno-monitoring techniques. isfactorily preserved with surgery, chemotherapy and radiotherapy. However, the advent of new This requires the establishment of quality control therapies, such as cancer vaccines, is expected criteria, the design for the production of the cel- to enhance the quality of life since the dendritic lular product, as well as for the proteic tumour cell (DC) vaccines present an alternative immu- antigen and its formulation, and to optimise a notherapy approach with very few side effects. GMP process. DENDRITOPHAGES initiated a clini- Quality of life is therefore preserved during treat- cal trial to evaluate the vaccines on progressing ment and no hospitalisation is required. prostate cancer patients.

The goal of cancer therapeutic cell vaccine is On a short-term basis, the new treatments direct- to prevent progression and tumour recurrence. ed to residual cancer diseases resistant to con- Adoptive therapy in adjuvant settings will com- ventional treatments will be used in an adjuvant plement classical anti-cancer treatments. In this setting as second line therapy. On a long-term technology the patient’s blood monocytes are basis, these patient-oriented therapeutic strate-

New Therapies – Immunotherapy and Transplantation 173 DENDRITOPHAGES

Investigations in mouse models and recent evidence from the use of the cell therapeutic vaccine to treat and prevent cancer recurrence will therefore be comprised of adequately differentiated and maturated DCs derived from the patient’s blood monocytes loaded in vitro with tumour antigens. The resulting cellular vaccine is re-injected into the patient to stimulate an immune response against their cancer. In a clinical setting, DC should be differentiated ex vivo, loaded with the chosen antigenic formulation, and finally, maturation should be initiated by a short treatment.

DENDRITOPHAGES anticipates that, once injected, DC will migrate to T cell areas during the sponta- Dendritophage Copyright: IDM Author: J. Davous neous progression of their activation and repro- duce the physiological process of maturation. gies should replace some first line conventional treatments of cancer. The goal of these therapeutic vaccines is to prevent metastasis development and to provide Dendritic cells long-term protection. This immunotherapeutic approach can be applied to diseases other than Discovered in the early 1970s by Steinman and cancer, in particular to stimulate an immune re- Collin, DCs, key actors of the immune system, action against one or several antigens associated are now considered as being the most potent with virus-infected cells. adjuvant for cancer immunotherapy to present and stimulate specific T cell responses. Capable In view of the urgent need to provide adapted of taking up the antigen at the pathogen entry therapy to treat cancer, improved standardised site, to migrate into secondary lymphoid tissues DC protocols will enable rationally designed and to activate both helper and cytotoxic T cells, clinical studies to give cancer vaccine immuno- DC can also interact with B cells, and probably therapy further credibility and cancer patients a natural killer cells, and thus direct the specificity new therapeutic option associated with a better of the immune response. quality of life. Sequential steps and objectives are required. However, it is only upon maturation that they ac- quire full expression of costimulatory molecules Approach and methodology: and major histocompatibility peptide complexes; increase cytokine production; and migrate The methodology developed and optimised dur- to draining lymphoid organs. Despite a growing the project is adoptive cancer vaccine where ing interest in the use of autologous DC for the DCs are differentiated from apheresis of mono- management of cancer and infectious diseases, nuclear cells, then matured and loaded with tu- the relation between the type of DC infused in mour antigen before being purified, frozen, aliq- humans and the type of immune response ob- uoted and re-injected into the patients. tained, is not yet clear. The participating teams compared a number of

174 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

differentiation and maturation methods, and se- • injection site: The migration of imma- lected IL-13, GM-CSF and then IFNg + FMKp as ture versus mature DCs were followed in GMP processes, thus producing more than one patients according to the injection route, billion DCs. using Cu64 labelled DCs. Intradermal injection delivers a better accumulation in KSA (Epcam) was chosen as a tumour proteic an- lymph nodes. The tracking and migration tigen and loaded on DCs as a protein, encaplu- of DCs were also studied in mice for the lated in liposomes or in virosomes. The stimula- different DC types produced. They were tion of KSA specific cytotoxic T lymphocytes by labelled with Ittrium 86. these DCs was achieved. Such loaded DCs have • the development of an emulsion with already been injected in prostate cancer patients adjuvant: Loading with liposomes also in Vienna, and will be compared to the KSA-lipo- containing lipid A as adjuvant and resus- somes subcutaneous vaccine. pended in water in oil emulsion appeared the most effective, in comparison with Main findings: KSA alone or in virosomes. • immunological monitoring of the DENDRITOPHAGES has already finalised many response to the vaccine: Specific CD8 T steps required for the evaluation of an antican- cell and antibody responses to KSA are cer vaccine: measured. • standardised preparation, differentia- • generation of a GMP process accept- tion and maturation of DCs: three distinct able by regulatory authorities: Based on processes for DC differentiation (IL-13 + the GMP industrial cellular process de- GM-CSF 7 days; IL-4 + GM; IFN α 3 days) and veloped by IDM, DENDRITOPHAGES de- maturation factors were compared by five termined the optimal strategy of DCs im- partners for differentiated DCs, matured munotherapy against prostate cancer. The or not. DCs differentiated in the presence DC vaccine was elaborated and tested in of IL-13 appear to be the most robust in a phase I/II studies on prostate cancer using GMP process and the only ones to secrete the selected autologous DCs loaded with high IL-12. Maturation is induced within the KSA antigen. The researchers evalu- six hours of being in the presence of IFNg ated its tolerance and immune response and of Klepsiella bacterial membrane generated against KSA, and looked for fraction. signs of clinical vaccine efficiency, prob- • KSA is the choice of the adequate anti- ably based on serum PSA level and evolu- gen: The KSA (EpCam) antigen was chosen tion. A second study with liposomal KSA as a pancarcinoma tumour Ag protected plus adjuvant was initiated in prostate by IDM I.P. cancer patients for comparison. • antigen loading: Loading of immature DCs with KSA liposomes/KSA, virosomes/ GMP processes and clean room for cell therapy KSA, and following a 24-hour stimula- were achieved in Paris, Rome, Melbourne and Vi- tion period of T8 expansion, the findings enna. A GMP lot of KSA incorporated in liposomes showed that virosomes were effective was prepared, qualified and released for clinical only in stimulating CD4 T cells. KSA lipo- use. A phase I clinical trial initiated in Vienna by somes were the most effective in inducing the end of 2005 on advanced prostate cancer pa- a CD8 T cell response. They were therefore tients compared the immune response obtained selected for clinical approach. after vaccination with DCs loaded with free KSA

New Therapies – Immunotherapy and Transplantation 175 DENDRITOPHAGES

or with a liposomal KSA-lipid A emulsion. A sec- Coordinator ond clinical trial was initiated in Regensburg on less-advanced prostate cancer patients vac- Jacques Bartholeyns cinated with liposomal KSA emulsion plus lipid IDM SA Immuno-Designed Molecules A. The duration of the project was extended by 172 rue de Charonne six months to launch this clinical protocol after 75011 Paris, France regulatory approval. E-mail: [email protected]

Partners

Miles Prince Centre for Blood Cell Therapies c/o Peter MacCallum Cancer Centre Victoria, Australia

Rheinard Glueck Etnabiotech c/o Università di Catania Facoltà di Medicina Dipartimento di Farmacologia Sperimentale e Clinica Catania, Italy

Thomas Felzmann Children’s Cancer Research Institute Vienna, Austria

Filippo Belardelli Istituto Superiore di Sanità Laboratori di Virologia Rome, Italy

Andreas Mackensen University of Regensburg Department of Haematology/Oncology Regensburg, Germany

Anne-Cécile de Giacomoni Alma Consulting Group Lyon, France

176 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation Genomes To Vaccines Translating genome and proteome information into immune recognition

Contract No LSHB-CT-2003-503231 Project type Specific Targeted Research Project EC contribution e 2 000 000 Starting date 1 January 2004 Duration 48 months Website www.cbs.dtu.dk/researchgroups/ immunology/Genomes2vaccines/index.php

Background and objectives: and predicting how the immune system handles proteins; or more specifically, of how it gener- Genome information is accumulating rapidly and ates, selects and recognises peptides. Generating it encompasses a wide range of species, includ- these predictive tools amounts to translating ge- ing humans and many pathogens, among others. nomes/proteomes into immunogens, and it will It is now possible to completely sequence micro- enable a new and rational approach for vaccina- bial genomes within a period ranging from days tion and immunotherapy. to months, and genomics can therefore be used as a powerful starting point to investigate the bi- Approach and methodology: ology of any pathogen of interest. The translated protein sequence information constitutes a sig- Genomes To Vaccines focuses on MHC class I nificant input to the immune system. mediated antigen presentation to cytolytic T cells. While several mechanisms involved in an- Indeed, the immune system considers peptides tigen processing and presentation have been as one of its key targets, and it has devoted an en- described in general terms, only a few of them tire arm — that of T cells, which essentially control have been described in sufficient detail to allow specific immune responsiveness — to peptide for the accurate prediction of their outcome; it is recognition. T cells are specific for peptides pre- a key requirement so as to achieve an overall (in- sented in the context of major histocompatibil- tegrated) prediction of the final result of a series ity complexes (MHC). Prior to their presentation, of processing events. these peptides were generated in the cytosol by limited proteolytic fragmentation of all available To produce ample and accurate data, Genomes protein antigens, then translocated to the endo- To Vaccines will generate biochemical high- plasmic reticulum (ER) and specifically sampled throughput assays, representing the most im- by the MHC for subsequent presentation. portant events in antigen processing and presentation. Focused on peptides and peptide The MHC is extremely polymorphic and the pep- recognition, the research team seeks to develop tide binding specificity varies for the different a high throughput peptide-array technology, polymorphic MHC molecules. The net effect of and it aims to generate accurate and quantita- this complicated system is that each individual tive predictions through the following data-driv- presents to their T cells a unique and highly di- en approach: verse peptide imprint of the ongoing protein • build and extend databases contain- metabolism. The scientific rationale of Genomes ing information on natural ligands and To Vaccines is one of understanding, describing immune epitopes;

New Therapies – Immunotherapy and Transplantation 177 Genomes To Vaccines

• use these databases to generate pre- ing one of the bottlenecks in the current state- liminary predictions; of-the-art search for vaccine candidates and • use these predictions to select a set of targets for immunotherapy. Only with computa- data points that will complement existing tional bioinformatics tools and concurrent high data (i.e. the most information-rich new throughput life-science tools will it be possible data); to search entire genomes for the presence of im- • perform the experiment to actually munogenic epitopes and validate them. Eventu- generate the new quantitative data; ally, it will enable a rational approach to popula- • add the new data to the existing tion-wide vaccination, as well as individualised databases; immunotherapy. • update and improve the bioinformatics resources. Main findings:

The approach constitutes an iterative and in- Genomes To Vaccines has generated efficient bi- teractive loop between bioinformatics and wet ochemical assays for three different major steps biochemistry, leading to successively improved in the generation of T cell epitopes. This includes predictions. Eventually, this will allow Genomes_ the proteolytic fragmentation of protein antigens To_Vaccines to search the entire sequence space (proteasome), the peptide translocation event (more than 1012 members) computationally (fast (TAP), and the peptide selection and presenta- and cheap), while reserving the experimental tion event (MHC). New biochemical assays were work (slow and costly) to the most information- used to generate data representing the above rich data points. The researchers believe that this events. Significant progress in generating a new integrated and iterative approach will generate and improved method for the generation of accurate prediction algorithms with a minimum high-density peptide arrays was also made, and of effort (i.e. in the most cost-efficient manner). the researchers hope to implement this technology in the generation of orders of magnitude for Expected outcome: more biochemical data in the future.

Genomes To Vaccines believes that the inte- The data obtained so far has been used to gen- gration of experiments and bioinformatics will erate bioinformatics tools capable of predict- lead to an overall tool-generation process that ing the outcome of these events in silico and requires fewer and more pointed experiments, to initiate the iterative process leading to im- with more accurate predictions. The resulting proved predictions. For validation purposes, the predictors will be able to computationally (i.e. researchers have generated a set of data using rapidly and exhaustively) translate and evalu- peptides extracted from cellular MHC, as repre- ate genomic data for the presence of immuno- sentative examples of how the immune system genic epitopes. These resources will be hosted at works under natural conditions. They integrated the Genomes To Vaccines website and be made the prediction tools and demonstrated that such publicly available. This will enable any interested an integrated predictor is more successful in pre- basic or clinical scientist to perform searches for dicting natural epitopes. immune targets from any microbial organism, or tumour, of their choice. Genomes To Vaccines has made these tools generally available as web-based services, and will The researchers expect that this tool will become eventually also host the data itself. Finally, the an essential part of medicine in the future, solv- utility of these tools was demonstrated through

178 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

the prediction of human T cell responses. Initially, transport efficiency, and proteasomal cleavage the researchers showed the speed of these tools predictions’, Eur J Immunol, 2005, Aug;35(8):2295- by performing a complete epitope scanning of 303. the SARS virus. Sylvester-Hvid, C., Nielsen, M., Lamberth, K., Roder, Subsequently, they performed a complete G., Justesen, S., Lundegaard, C., Worning, P., Tho- epitope scanning of Influenza A isolates, and madsen, H., Lund, O, Brunak, S., Buus, S., ‘SARS CTL tested the predicted peptides both in biochemi- vaccine candidates; HLA supertype-, genome- cal MHC binding assays, but, more importantly, in wide scanning and biochemical validation’, Tissue healthy influenza convalescents. The latter study Antigens, 2004, May;63(5):395-400. identified 13 new influenza epitopes, which covered virtually the entire population. These Lund, O., Nielsen, M., Kesmir, C., Petersen, A.G., epitopes were selected for being highly con- Lundegaard, C., Worning, P., Sylvester-Hvid, C., served, and notably, they all encompassed the Lamberth, K., Roder, G., Justesen, S., Buus, S., Bru- current bird flu isolates. nak, S., ‘Definition of supertypes for HLA molecules using clustering of specificity matrices’, The ability to translate genomic information Immunogenetics, 2004, Mar;55(12):797-810. Epub into immune recognition will form the basis for 2004 Feb 13. a rational approach to immunotherapy (including vaccination), which the researchers believe Nielsen, M., Lundegaard, C., Worning, P., Hvid, will serve the policy objectives of enhancing the C.S., Lamberth, K., Buus, S., Brunak, S., Lund, O., quality of life for EU citizens. ‘Improved prediction of MHC class I and class II epitopes using a novel Gibbs sampling approach’, Major publications Bioinformatics, 2004, Jun 12;20(9):1388-97. Epub 2004 Feb 12. Wang, M., Lamberth, K., Harndahl, M., Roder, G., Stryhn, A., Larsen, M.V., Nielsen, M., Lundegaard, C., Tang, S.T., Dziegiel, M.H., Rosenkvist, J., Ped- ersen, A.E., Buus, S., Claesson, M.H., Lund, O., ‘CTL epitopes for influenza A including the H5N1 bird flu; genome-, pathogen-, and HLA-wide screening’, Vaccine, 2006, Dec 29; [Epub ahead of print]

Peters, B., Bui, H.H., Frankild, S., Nielson, M., Lunde- gaard, C., Kostem, E., Basch, D., Lamberth, K., Harn- dahl, M., Fleri, W., Wilson, S.S., Sidney, J., Lund, O., Buus, S., Sette, A., ‘A community resource benchmarking predictions of peptide binding to MHC-I molecules’, PLoS Comput Biol, 2006, Jun 9;2(6):e65. Epub 2006 Jun 9.

Larsen, M.V., Lundegaard, C., Lamberth, K., Buus, S., Brunak, S., Lund, O., Nielsen, M., ‘An integrative approach to CTL epitope prediction: a combined algorithm integrating MHC class I binding, TAP

New Therapies – Immunotherapy and Transplantation 179 Genomes To Vaccines

Coordinator

Søren Buus University of Copenhagen Institute of Medical Microbiology and Immunology Panum 18.3.12 Blegdamsvej 3 DK-2200 Copenhagen N, Denmark E-mail: [email protected]

Partners

Stefan Stevanovic University of Tübingen Tübingen, Germany

Peter van Endert INSERM U25 Hôpital Necker Paris, France

Hansjörg Schild University of Mainz Mainz, Germany

Søren Brunak Technical University of Denmark Lyngby, Denmark

Claus Schafer-Nielsen Symbion Science Park Copenhagen, Denmark

180 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation CompuVac Rational design and standardised evaluation of genetic vaccines

Contract No LSHB-CT-2004-005246 Project type Integrated Project EC contribution e 7 969 442 Starting date 1 January 2005 Duration 48 months Website www.compuvac.org

Background and objectives:

CompuVac has assembled 16 European teams from both academia and industry, in 9 European countries, with multidisciplinary expertise in the fields of vaccine development, immunology, virology, vectorology, biomathematics and computer sciences. Recombinant viral vectors and virus- like particles are considered the most promising vehicles to deliver antigens in prophylactic and therapeutic vaccines against infectious diseases CompuVac members : The CompuVac 2nd Annual and cancer. Several potential vaccine designs ex- Meeting, November 2006, Athens, Greece ist, but their cost-effective development cruelly lacks a standardised evaluation system. Approach and methodology:

CompuVac is devoted to the standardisation of As end products, the consortium’s vector plat- vaccine evaluation and the rational development form and ‘gold standard’ tools, methods and of a novel platform of recombinant vaccines, with algorithms will be made available to the scien- HCV as a target disease. The CompuVac consor- tific and industrial communities as a toolbox and tium has several primary objectives: interactive database. CompuVac seeks to apply • to standardise the qualitative and these vectors, tools and methods to the develop- quantitative evaluation of genetic vac- ment of a preventive and/or therapeutic vaccine cines using defined ‘gold standard’ anti- against Hepatitis C Virus, incorporating one or gens and methods; more of the project’s platform vectors expressing • to rationally develop a platform of the HCV envelope protein. novel genetic vaccines using genomic and proteomic information, together with CompuVac recognises that a uniform method the project’s gold standards; for side-by-side qualitative and quantitative vac- • to generate and make available to the cine evaluation is lacking. Thus, standardising scientific community a ‘tool box’ and an the evaluation of vaccine efficacy and molecu- ‘interactive database’ allowing for a com- lar signature by using ‘gold standard’ tools, and parative assessment of future vaccines molecular and cellular methods in virology and to be developed with the project’s gold immunology, as well as algorithms based on ge- standards. nomic information, is key.

New Therapies – Immunotherapy and Transplantation 181 CompuVac

Main findings: iii. Measles virus/Measles-VLP, iv. MVA/MVA-VLP, Within the first 2 years of its development, 4 ma- v. Polyoma virus; jor platforms have been set up by CompuVac. • BCG; These are presented below: • DNA/DNA-VLP.

1. The CompuVac vector provider platform. This There are two selected ‘gold standard’ antigens: platform is dedicated to the engineering and/or • T-cell antigen: GP33-41 epitope from optimisation of vectors expressing and/or con- LCMV, to study the T cell response. The taining the chosen model epitopes. A rational LCMV glycoprotein G1 peptide p33-41 is vector design is carried out based on up-to-date an extremely well-characterised model genomic and proteomic information. This plat- antigen that has been widely used in nu- form assembles a wide range of viral vectors and merous studies for evaluating humoral virus-like particles that are among today’s most response. In addition, this is a virus that is promising vaccine candidates: mainly controlled by cellular immune re- • inert particle vaccines — antigen display: sponses in vivo. i. VLPs derived from retrovirus, polyoma- • B-cell antigen: VSV-G protein, to study viruses, hepatitis B virus, bacteriophage, the B cell response. The VSV envelop pro- ii. Polyepitope peptides; tein is the viral envelop that is best pseu- • gene expression vectors — antigen/trans- dotyped on retroviral-based particles gene/VLP expression: used for neutralisation assays. In addition, i. Adenovirus/adeno-VLP, this is a virus that is mainly controlled by ii. Herpes Simplex Virus 1/HSV-VLP, humoral immune responses in vivo.

182 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

Fig 1 & 2: Radar and Bar charts - CompuVac, Genetic These ‘gold standard’ antigens have been intro- Vaccines Decision Support System : Results duced in the set of vectors of CompuVac. of the system has already been created. Scientific 2. The CompuVac immunological testing platforms for immunisation protocols, animal models form. Once produced, the immunological re- and T cell responses were created, and the immu- sponse will be studied by the immunological nisation protocols were then analysed and im- testing platform regarding T and B cell response plemented in GeVaDSs. A public test of GeVaDSs and molecular signature. Data have been gener- took place at the CompuVac’s second executive ated and entered in the ‘Genetic Vaccine Decision committee meeting in Stockholm in June 2006. Support system’ (GeVaDSs). ‘Gold standard’ algo- There were 2 reports generated by GeVaDSs: rithms for the intelligent interpretation of vac- • The T cell response project report: in cine efficacy and molecular signature are built this project, CompuVac compared the into CompuVac’s interactive GeVaDSs, which following: (i) interferon g production, (ii) generates the following: memory phenotype induction, (iii) clonal • vector classification according to in- expansion, (iv) peak of clonal response, duced immune response quality, account- and (v) cytotoxic T lymphocytes response; ing for gender and age; • The molecular signature report. • vector combination counsel for prime- boost immunisations; 3. A CompuVac training and dissemination plat- • vector molecular signature according form. The exchange programme was effectively to genomic analysis. launched, allowing CompuVac’s young scientists to learn new technologies and benefit from the A GeVaDSs user manual for the current version partners’ expertise. An expert immunologist and

New Therapies – Immunotherapy and Transplantation 183 CompuVac

mathematician were hired, to train and connect management of the scientists, the administrative the scientists involved in the building-up of Ge- and financial management, and the intellectual VaDSs. property management, with a major feature being the study of the future of GaVaDSs. Four training sessions dedicated to the use of GeVaDSs by the data producers and the Compu- Major publications Vac team leaders, were coordinated by the computer scientists in order to improve data integra- Bellier, B., Dalba. C., Clerc, B., Desjardins, D., Drury, tion into GeVaDSs. A dissemination meeting was R., Cosset, F.L., Collins, M., Klatzmann, D., ‘DNA vac- organised by the CompuVac management team cines encoding retrovirus-based virus-like parti- at the 2006 annual meeting of the European So- cles induce efficient immune responses without ciety of Gene Therapy on ‘Perspectives in Vaccine adjuvant’, Vaccine, 2006, Mar 24;24(14):2643-55. Development’. Dalba, C., Bellier, B., Kasahara, N., Klatzmann, D., The CompuVac tool for dissemination is a 26- ‘Replication-competent Vectors and Empty Vi- minute film dedicated to the project and new rus-like Particles: New Retroviral Vector Designs vaccine development. A film team (Actes&Avril for Cancer Gene Therapy or Vaccines’, Mol. Ther, Productions) was contracted for the CompVac 2007, Jan 23. film, and sequences were shot between November and December 2006 in the following Dreux, M., Pietschmann, T., Granier, C., Voisset, C., locations: Ricard-Blum, S., Mangeot, P.E., Keck, Z., Foung, S., • Hellenic Pasteur Institute, Athens, Vu-Dac, N., Dubuisson, J., Bartenschlager, R., Lavil- Greece (CompuVac annual meeting, ESGT lette, D., Cosset, F.L., ‘High density lipoprotein dissemination meeting, and participating inhibits hepatitis C virus-neutralizing antibod- institute/immunological testing platform); ies by stimulating cell entry via activation of • University of Munich, Munich, Germa- the scavenger receptor BI’ J Biol Chem, 2006, Jul ny (participating institute/immunological 7;281(27):18285-95. testing platform); • University of Zürich, Zürich, Switzer- Tegerstedt, K., Franzen, A., Ramqvist, T., Dalianis, land (participating institute/immunologi- T., ‘Dendritic cells loaded with polyomavirus VP1/ cal testing platform); VP2Her2 virus-like particles (VLPs) efficiently • CNRS – Pierre et Marie Curie University, prevent outgrowth of a Her2/neu expressing Paris, France (participating institute/scien- tumour’, Cancer Immunology Immuntherapy, In tific coordination/management platform); press, 2007. • Poznan University of Technology, Poznan, Poland (participating institute/ Sominskaya, I., Alekseeva, E., Skrastina, D., GeVaDSs platform); Mokhonov, V., Starodubova, E., Jansons, J., Levi, • Cytos Biotec, Zürich, Switzerland (par- M., Prilipov, A., Kozlovska, T., Smirnov, V., Pumpens, ticipating biotechnology company/vector P., Isaguliants, M.G., ‘Signal sequences modu- platform), late the immunogenic performance of human • Epixis SA, Lyon, France, (participating hepatitis C virus E2 gene’, Mol Immunol, 2006, biotechnology company/vector platform). May;43(12):1941-1952.

4. The CompuVac management platform has Bartosch, B., Cosset, F.L., ‘Cell entry of hepatitis C been set up, and is dedicated to the day-to-day Virus’, Virology, 2006, Apr 25;348(1):1-12.

184 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

Lavillette, D., Bartosch, B., Nourrisson, D., Verney, Charlotte Dalba G., Cosset, F.L., Penin, F., Pecheur, E.I., ‘Hepatitis C Epixis SA virus glycoproteins mediate low pH-dependent Paris, France membrane fusion with liposomes’, J Biol Chem, 2006, Feb 17;281(7):3909-17. Alberto Epstein Université Claude Bernard Lyon 1 Villeurbanne, France Coordinator Stefan Kochanek David Klatzmann Universitätsklinikum Ulm CNRS and Université Pierre et Marie Curie Ulm, Germany Laboratoire de Biologie et Thérapeutique des Pathogies Immunitaires Penelope Mavromara UMR 7087 – UPMC-CNRS Hellenic Pasteur Institute Groupe Hospitalier Pitié-Salpêtrière Athens, Greece Bat. CERVI, 83, boulevard de l’Hôpital 75651 Paris Cedex 13 - France Albertus Osterhaus E-mail : [email protected] Erasmus MC Rotterdam Rotterdam, Netherlands Partners Paul Pumpens Anatoly Sharipo BioMedical Research and Study Centre ASLA Biotech Ltd Riga, Latvia Riga, Latvia Kestutis Sasnauskas Jacek Blazewicz Institute of Biotechnology Poznan University of Technology Vilnius, Lithuania Poznan, Poland Rudolf Martin Zinkernagel Thomas Brocker University of Zurich Institute of Immunology - Ludwig-Maximilians-Univerität Institute of Experimental Immunology München Zurich, Switzerland Munich, Germany

François-Loïc Cosset INSERM Lyon, France

Gary Thomas Jennings Cytos Biotechnology AG Zurich-Schlieren, Switzerland

Tina Dalianis Karolinska Institutet Stockholm, Sweden

New Therapies – Immunotherapy and Transplantation 185 HEPACIVAC New preventative and therapeutic Hepatitis C vaccines: from preclinical to phase I

Contract No LSHB-CT-2007-037435 Project type Integrated Project EC contribution e 8 800 000 Starting date 1 January 2007 Duration 60 months

Background and objectives: models. The consortium will focus on the skills of the individual participants towards the develop- Liver disease caused by Hepatitis C Virus (HCV) ment of new preventative and therapeutic strat- infection is a major medical problem, affecting egies against HCV infection, integrating different an estimated 123 million people worldwide. No approaches and disciplines. vaccine is available and the best antiviral therapy — a combination of interferon alpha and ribavi- The HEPACIVAC proposed work is focused on the rin — is only effective in a minority of patients. development of two HCV vaccine candidates, An increasing body of data suggests that vi- independently identified by IRBM (Istituto di rus-specific T cell responses are associated with Ricerche di Biologia Molecolare) and Novartis. clearance of HCV in acutely infected humans and This study will be performed with a strong com- chimpanzees. mitment to translate the results into effective approaches for the prevention and therapy of Based on these observations, the long-term HCV. The first vaccine candidate is gene-based, objective of the HCV vaccine programme is encodes for the 2 000 amino acid-long HCV Non to develop both prophylactic and therapeutic Structural region (from NS3 to NS5B) and uti- vaccines that elicit antiviral CD4+ and CD8+ re- lises adenoviral vectors for delivery. These vec- sponses capable of one of the following: tors have been shown to elicit potent CD4+ and • reducing the rate of incidence and/or CD8+ T cell responses in rodents and primates. persistence of HCV infection following exposure (for prophylactic vaccine); Recently, a proof-of-concept vaccination and het- • increasing the rate of clearance of erologous challenge experiment in chimpanzees HCV infection as a monotherapy, and/or showed that potent, broad and long-lived T-cell in combination with current therapy or responses to HCV were elicited in vaccinated ani- novel anti-viral therapy (for therapeutic mals. The gene-based vaccine protected against vaccine). acute and chronic disease induced by challenge with a high dose of an heterologous HCV strain. Approach and methodology: The second vaccine candidate consists of recombinant HCV glycoproteins, gpE1- and gpE2- The HEPACIVAC project is being carried out by a associated, to resemble a pre-virion envelope large consortium, which includes two major vac- structure. Protection against homologous and cine manufacturers, and several academic and heterologous challenge, mediated by CD4+ T cell research groups with expertise in clinical vac- response and antibodies, was observed in exper- cine development, human immunity and animal iments in chimpanzees.

186 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

It follows that both vaccine candidates have the • demonstration of immunogenicity of potential to protect humans from a large number the gene-based vaccine, in HCV chroni- of viral strains. The complementary action of these cally infected chimpanzees; vaccines might be highly instrumental in making • demonstration of immunogenicity of a vaccine against HCV, which is able to stimulate the two vaccines administered in combi- several components of the immune system and nation in macaques; to elicit efficacious immune responses, both in • standardisation of the immunological preventative and therapeutic vaccination set- assays; tings. Furthermore, since the two vaccines target • evaluation of safety and immuno- different arms of the acquired immune response, genicity of each of the two candidate vac- HEPACIVAC will offer the opportunity to analyse cines, in healthy volunteers and in chroni- in detail immune correlates of protection from cally infected individuals. HCV infection, as well as to standardise immunogenicity assays, and to establish benchmark ref- erences for future preclinical and clinical studies of HCV candidate vaccines.

During the first part of the project and after the production of GLP & GMP vaccines, the work will focus on several areas: animal safety and tolerability studies for the gene-based vaccine; and a preclinical evaluation of compatibility, synergistic or antagonistic actions of the two vaccines administered in combination in macaques. These studies, aimed at verifying the possibility of using this vaccine in humans, will be accompanied by specific activities on preclinical and clinical samples, in order to identify and standardise suitable immunological assays to be used in the following HCV vaccine clinical trials.

In the second part of the project, HEPACIVAC plans to perform Phase I trials with the vaccines separately, to assess safety, tolerability and immunogenicity in healthy volunteers and in chronically infected individuals.

Expected outcome:

HEPACIVAC anticipates the following results:

• production of vaccine lots in sufficient amounts to conduct preclinical and clinical studies; demonstration of safety in animals, of the gene-based vaccine;

New Therapies – Immunotherapy and Transplantation 187 HEPACIVAC

Coordinator Krystina Bienkowska-Szewczyk University of Gdansk Riccardo Cortese Gdansk, Poland CeInge Biotecnologie avanzate s.c.a.r.l. via Pansini 5 Sayed F. Abdelwahab 80131 Naples, Italy The Egyptian Company for Diagnostics E-mail: [email protected] Agouza Giza, Egypt Cristiana Tozzi Scientific coordinator ALTA Srl Siena, Italy Sergio Abrignani Novartis Vaccine & Diagnostics Adrian Hill via Fiorentina 1 University of Oxford 53100 Siena, Italy Oxford, UK E-mail: [email protected]

Partners

Alfredo Nicosia Istituto di Ricerche di Biologia Molecolare (IRBM) P.Angeletti Pomezia (RM), Italy

Ferruccio Bonino Fondazione IRCCS Ospedale Maggiore Mangiagalli e Regina Elena Milan, Italy

Albert D. M. E. Osterhaus Erasmus Medical Centre Rotterdam, Netherlands

Geert Leroux Roels University of Ghent Ghent, Belgium

Jane McKeating University of Birmingham Birmingham, UK

Stefan Zeuzem University of Saarland Saarland, Germany

188 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation BacAbS Assessment of structural requirements in complement-mediated bactericidal events: ‘Towards a global approach to the selection of new vaccine candidates’

Contract No LSHB-CT-2006-037325 Project type SME-Specific Targeted Research Project EC contribution e 2 269 999 Starting date 1 January 2007 Duration 36 months Website www.bacabs.org

Background and objectives: project, and five academic partners that are internationally recognised for their experimental High throughput cloning and expression of large and theoretical studies of protein structure and sets of genomic open reading frames (ORFs) has interactions. become the preferred industrial strategy for genome-wide searches of new vaccine candidates. The development of antibiotic resistance consti- For invasive infections in particular, the aim is tutes one of the most potentially serious threats to find proteins eliciting antibodies capable of in modern medicine. One approach to minimis- binding to the bacterial cell surface, and through ing the use of antibiotics, is to vaccinate against interaction with the complement system effec- pathogenic strains of bacteria. A clear candidate tively killing the bacteria. for this approach is Neisseria meningitidis, a major cause of bacterial septicemia and meningi- However, current data from reverse vaccinology tis against which no effective vaccine exists. N. studies (the targeting of possible vaccine can- meningitidis is a Gram-negative bacterium that didates, beginning with genomic information) is capsulated in its invasive form, and classified show that only a small fraction of surface-ex- into five major pathogenic serogroups on the posed proteins appears to elicit antibodies with basis of the chemical composition of distinctive bactericidal activity. Using information gener- capsular polysaccharides. Although a promising ated by reverse vaccinology projects within the candidate has entered clinical development, no consortium, the BacAbs project will apply a novel effective vaccine against serogroup-B N. menin- multidisciplinary approach that will help to sin- gitidis (MenB), which is responsible for more than gle out the structural requirements for viable 50% of all meningococcal diseases in Europe, has bactericidal vaccine candidates, and will develop reached the market yet. The capsular polysac- bioinformatics tools to predict compliance with charide of MenB is identical to that of a widely such structural requirements. distributed human carbohydrate, making its use as the basis of a vaccine for prevention of MenB To this end, a systematic analysis of the sequence, diseases problematic. Consequently, most efforts structure, dynamics and interactions of selected have turned to the development of vaccines protein targets will be conducted, using the based on surface-exposed or exported proteins. serogroup-B Neisseria meningitidis as a model system. The consortium consists of an industrial A bactericidal response, i.e. one that leads to partner with extensive experience in vaccine bacterial-cell death, can be triggered through a development, three SMEs with strong expertise variety of methods. For meningococcal infections, in several key technological aspects of the in vitro bactericidity assays with immune sera,

New Therapies – Immunotherapy and Transplantation 189 BacAbS

correlate with protection in humans. Although of non-bactericidal antibodies. in vivo protection against MenB may not be solely achieved by complement-dependent To achieve this goal, the consortium will inves- bacteriolysis, an antigen that elicits (murine) tigate the requirements for a productive Ag- antibodies capable of triggering bacterial-cell Ab-C1q complex formation. It proposes to find death in vitro in a complement-dependent relevant answers using a multidisciplinary and manner, is normally considered a candidate for comparative approach to study the structure of human vaccine development. a number of these complexes. The MenB vaccine development project of Novartis Vaccines & Di- In this context, one major obstacle to vaccine de- agnostics is to serve as a model, while a source velopment, besides sequence and antigenic vari- of useful data and reagent molecules will be ability, is the difficulty to identify antigens that employed as well. To single out possible struc- will generate a bactericidal response. Typically, tural determinants, the focus is being placed on only a very small fraction of the antibodies raised groups of antigens with similar size, abundance, in large-scale antigen-screening studies are bac- solubility, etc. (both eliciting and not eliciting tericidal. Thus, while potential antigens can be bactericidal antibodies). readily identified, this information is of little use unless we are also able to predict the antigens Although initially centred around group-B N. that can lead to the production of bactericidal meningitidis, the specific target of the project antibodies. is the development of tools that can be applied effectively to the genome-wide identification of In principle, the capacity of a protein antigen to vaccine candidates against any bacterial patho- raise bactericidal antibodies may depend on (a gen susceptible to complement-mediated lysis. combination of) the following factors: • size, shape, structural complexity, Role of SMEs abundance, solubility, propensity and oli- gomerisation; The three SMEs involved in the BacAbs project • sequence, structure, dynamics and have a principal technological role. The IT com- location of specific protein regions pany INFOCIENCIA SL will be operating the man- (epitopes). agement and dissemination web servers of the Consortium, carrying out bioinformatics analysis These factors may in turn modulate the properties of antigen and epitope sequences, implementing of the Ag-Ab complex and its susceptibility to be algorithms, protocols and data emerging from recognised by the C1q component. The BacAbs the Consortium’s work into computational tools project aims at deciphering possible correlations and databases within a web-based technological between these factors and bactericidity. platform, and evaluating the commercial interest of this platform via demonstration. Approach and methodology: Two biotechs will be working on sample prepara- Following the framework outlined above, the tion and protein-structure determination. ASLA BacAbs project is concerned with the identifi- Biotech Ltd will be performing protein expres- cation of (surface-exposed or exported) protein sion and labelling; screening and optimisation of antigens that may elicit complement-mediated sample conditions for NMR analysis; monoclonal bactericidity in vitro. This includes early discrimi- antibody generation; and sequential backbone nation of antigens that may induce production assignment and structure determination via NMR.

190 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

Bio-Xtal SA will be performing protein expres- Potential applications: sion and purification, especially in connection with derivatives (seleno-methionine labelled), The results of the BacAbs project could eventu- for X-ray analysis, expression and solubilisation ally be applied in the selection of new vaccine screens on highly hydrophobic or poorly soluble candidates against group-B Neisseria meningi- targets. The consortium will also explore protein tidis and other bacterial pathogens. crystallogenesis using nanodrop scale robot- ics techniques based on commercially available and proprietary screening approaches; develop and optimise robotic processes for crystallisation plate storage and drop visualization; refine crystal growth conditions for selected hits to yield diffraction quality crystals; and collect X-ray data and structure solutions.

Expected outcome:

The BacAbs project will provide the following: • Structural information on a set of proteins that are components of the cell surface (the bacterial organ for interaction with eukaryotic host cells) of a major human pathogen; • Improved experimental protocols and techniques, bioinformatics tools and databases to assist the development of vaccines against human bacterial pathogens in general, and group-B Neisseria meningitidis in particular; • A framework in which experimental and in silico methods for determining protein structure and studying macro- molecular recognition, immunological response mechanisms, and sequence-structure-function relationships can be further developed; • A web-based technological platform integrating this knowledge, with the potential to improve the effectiveness and reducing the costs of vaccine-candidate searches.

New Therapies – Immunotherapy and Transplantation 191 BacAbS

Coordinator

Xavier Daura Universitat Autònoma de Barcelona Campus UAB s/n Institución Catalana de Investigaciòn y Estudios Avanzados 08193 Bellaterra (Cerdanyola del Vallès), Spain E-mail: [email protected]

Partners

Guido Grandi Novartis Vaccines and Diagnostics Siena, Italy

Anatoly Sharipo ASLA BIOTECH, Ltd. Riga, Latvia

Gherici Hassaine Bio-Xtal SA Mundolsheim, France

Giorgio Colombo Consiglio Nazionale delle Ricerche Istituto di Chimica del Riconoscimento Molecolare Milan, Italy

Martin Zacharias International University Bremen GmbH School of Engineering and Science Bremen, Germany

Martino Bolognesi Università degli Studi di Milano Milan, Italy

Alexandre M.J.J. Bonvin Universiteit Utrecht Department of Chemistry Utrecht, Netherlands

José Manuel Mas Benavente INFOCIENCIA SL Barcelona, Spain

192 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation MimoVax Alzheimer’s disease-treatment targeting truncated Aß 40/42 by active immunisation

Contract No LSHB-CT-2006-037702 Project type SME-Specific Targeted Research Project EC contribution e 2 370 155 Starting date 1 October 2006 Duration 36 months Website www.mimovax.eu

Background and objectives: brain is predicted to slow down or halt disease progression, and could also stop cognitive de- Alzheimer’s disease (AD) is the most common cline in AD patients. form of dementia in humans. The Alzheimer As- sociation has observed that there are currently Indeed, immunotherapeutic treatment using 12 million patients worldwide suffering from this active and passive immunisation strategies to disease, with estimated social costs for every pa- target full length Aß led to the reduction of Aß tient reaching €40 000 per year. Currently, there plaques and had a beneficial impact on disease is no effective treatment available to stop the progression in animal models of AD. However, progressive neurodegeneration and associated the first phase II vaccination trial in AD patients cognitive decline in human patients. The current using full length Aß42 as an antigen had to be cases are expected to almost double within the discontinued, due to severe neuroinflammatory next 20 years. All the treatment strategies pres- side effects, including brain infiltration by auto- ently being applied are focusing on the use of reactive T cells. small molecular drugs that inhibit the activity of cholinesterase, to alleviate disease symptoms. MimoVax — Alzheimer’s vaccine — is a specific These drugs, however, have not been proven to targeted research project (STREP) for the devel- effectively halt or to revert disease progression opment and optimisation of a first treatment to after prolonged treatment. stop the progression of AD. This project aims at developing a vaccine against modified forms of Alzheimer’s disease is caused by the deposition BA. The immune system of AD patients will be ac- of Beta-Amyloide (BA) into plaques within the tivated to attack and remove BA, and will be able patients’ brain. The main constituent of BA is pro- to fight the cause of the disease directly. tein fragments, of 40-42 amino acids. These fragments derive from the Amyloid Precursor Protein The innovative technology presented in MimoVax (APP), which is expressed on various cell types in has been developed to create antigens mimicking the nervous system. In humans, the majority of the structure of neo-epitopes which do not con- amyloid plaque material is formed by Aß40/42 tain sequences of the native Aß peptide. A Mimo- derivatives which are frequently truncated and tope-based AD vaccine would induce antibody modified. Aß peptides are considered to be di- responses, exclusively reacting with the patholog- rectly involved in the pathogenesis and progres- ical Aß molecules but not with parental structures sion of AD. like APP. Furthermore, Mimotopes do not contain potential T cell self-epitopes and they avoid induc- Consequently, reduction of Aß burden in the tion of detrimental autoreactive T cells.

New Therapies – Immunotherapy and Transplantation 193 MimoVax

didates will be tested for efficacy in animal models of AD, altering disease progression.

This evaluation will include analysis of AD-pathology in the brain, as well as the testing of memory and learning in these animals by cognitive tests in vivo. The successful candidates will then be tested for tolerability and safety in a first phase I trial in AD patients. Additionally, new diagnostic methods will be developed and evaluated in order to monitor treatment efficacyin vivo in animal models.

Amyloid plaque staining in the brain of an AD mouse. Expected outcome: One of the hallmarks of Alzheimer’s disease is the accumulation of amyloid plaques in the brain. These amyloid plaques can be visualised on sections of these brains by The expected outcome of the project will com- staining with amyloid specific antibodies. This staining prise a proof of concept for active immunisation reaction results in a red amyloid plaque (indicated by a using MimoVax vaccines in animal models. Suc- white arrow) surrounded by blue nuclei of surrounding cells like neurons or astrocytes. A Mimotope-based AD cessful treatment should result in a reduction of vaccine as described in this abstract would induce such pathologic alterations in the brain, as well as in specific antibody responses reacting with the pathologi- improved learning and memory of the treated cal Aβ molecules and could therefore be a safe treatment regimen to efficiently fight AD in patients. animals. Furthermore, the researchers expect to demonstrate the safety and tolerability of the Despite the fact that similar to full length Aß, vaccine in patients, in initial phase I testing. truncated and modified Aß peptides seem to be involved in the pathogenesis and progression of In summary, vaccines based on Mimotope pep- AD, no relevant development programme has tides mimicking neo-epitopes which are derived been initiated to date. The goal of MimoVax is from the highly pathological and abundant Amy- the development of a safe and efficacious Alzhe- loid-β derivatives, provide an innovative, inexpen- imer vaccine, which can prevent or even revert sive and efficient way to treat this widespread cognitive decline in AD patients. In addition, new disease with its severe personal and economic diagnostic methods will be developed in order impact on patients, their families and society. to monitor treatment efficacy.

Approach and methodology:

This novel vaccine-technology is based on Mim- otope peptides mimicking neo-epitopes which are present on the above-mentioned highly pathological and abundant Amyloid-ß derivatives, but not on the parental Amyloid Precursor Protein (APP). The first step in this project will be the identification and evaluation of novel mimotopes mimicking epitopes present on different Aß derivatives. Subsequently, these vaccine can-

194 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

Coordinator

Frank Mattner Affiris GmbH Campus Vienna Biocentre Viehmarktgasse 2a A-1030 Vienna, Austria E-mail: [email protected]

Scientific coordinator

Markus Mandler Affiris GmbH Campus Vienna Biocentre Viehmarktgasse 2a A-1030 Vienna, Austria E-mail: [email protected]

Partners

Iris Grünert Biolution gruenert & co KEG Vienna, Austria

Antón Alvarez EUROESPES, SA Department of Neuropharmacology A Coruna, Spain

Manfred Windisch JSW – Research GmbH Graz, Austria

Fritz Andreae piCHEM research development Graz, Austria

Richard Dodel Philipps-Universität Marburg Marburg, Germany

Alexander Drzezga Technische Universität München – Klinikum rechts der Isar Munich, Germany

New Therapies – Immunotherapy and Transplantation 195 Pharma-Planta Recombinant pharmaceuticals from plants for human health

Contract No LSHB-CT-2003-503565 Project type Integrated Project EC contribution e 12 million Starting date 1 February 2004 Duration 60 months Website www.pharma-planta.org

Background and objectives: biotechnology, risk assessment and IP management. This is a unique opportunity to make an Pharma-Planta aims to build a plant-based pro- impact on EU and global health, through the reduction platform for pharmaceuticals in Europe sponsible development of plant biotechnology. and to enter the first candidate pharmaceuticals into clinical trials. The programme will develop Approach and methodology: robust risk-assessment and risk-management practices based on health and environmental im- The Pharma-Planta project is divided into 6 work pact and will work with EU regulatory authorities packages (WPs), focusing on distinct aspects of to ensure safety and acceptance. pharmaceutical production in plants. WP1 examines targets and is responsible for producing Plants have enormous potential for the produc- expression constructs and assays for the disease tion of recombinant pharmaceutical proteins as target proteins to be expressed in plants. In this, they are inexpensive and versatile, in addition to the third year of the programme, the role of them being amenable to rapid and economical WP1 is all but complete. WP2 is concerned with scale-up. A major goal will be to address the nec- regulatory guidance and engagement with the essary biosafety and regulatory requirements regulatory authorities. WP3 focuses on the major for the use of plant-derived pharmaceuticals production crops — maize and tobacco — which through a process of engagement and consulta- were chosen early in the project after consultation with the regulatory bodies involved in GM tions carried out in WP2. plants, as well as new medicines. The project addresses pharmaceuticals for the prevention of Of the 8 targets, 2 were chosen in WP1, and were HIV, rabies, tuberculosis and diabetes, which re- ‘fast-tracked’ in WP3, which means they were main significant health problems, both in Europe selected for breeding, scale-up and eventual and the developing world. production under good manufacturing practice (GMP) conditions. It was decided at the start of The Pharma-Planta consortium partners repre- the project that these targets would be 2 anti- sent many of the major laboratories in Europe fo- HIV antibodies. However, WP3 is concerned only cusing on the creation of transgenic plants that with the upstream production and the genera- express important pharmaceuticals for human tion of plant material ready for extraction and health. Collectively, the consortium has a wide processing of these antibodies. WP3 also looks at range of expertise spanning the areas of molecu- alternative expression platforms, including plas- lar and plant biology, immunology, recombinant tid transformation in tobacco, tomato and let- protein expression technology, vaccinology, plant tuce. WP4 is called the development loop, and its

196 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

role is to examine various different strategies to but also through a significant number of interac- improve production yield and product quality. tions between WP2 and the regulatory authorities in Europe and South Africa. Different workgroups within WP4 are look- ing at fusion proteins to enhance stability, pro- Major publications tease knockouts, genetic modification of glycan structures, protein targeting and even the use Sparrow, P.A.C., Irwin, J.A., Dale, P.J., Twyman, R.M., of plant viruses as expression vectors. Novel dis- Ma, J.K.C., ‘Pharma-Planta: Road testing the de- coveries and innovations in WP4 will be benefi- veloping regulatory guidelines for plant-made cial to molecular farming as a whole and will be pharmaceuticals’, Transgenic Res., 2007, Feb 7, exploited in future programmes. WP5 and WP6 16(2):147-61. Epub. have gained importance as we enter the second half of the project. WP5 is concerned with down- Van Droogenbroeck, B., Cao, J., Stadlmann, J., Alt- stream processing – extraction, clarification, pu- mann, F., Colanesi, S., Hillmer, S., Robinson, D.G., rification and formulation of the antibodies as a Van Lerberge, E., Terryn, N., Van Montagu, M., clinical batch under GMP conditions. WP6, which Liang, M., Depicker, A., Jaeger, G.D., ‘Aberrant lo- will run until the end of the project, is the clinical calization and underglycosylation of highly accu- trial itself. mulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds’, Proc Natl Acad Sci USA, Expected outcome: 2007, 104(4):1430-1435.

The overall aim of the project is to complete a Dunkley, T.P., Hester, S., Shadforth, I.P., Runions, phase I trial in humans, using either or both of J., Weimar, T., Hanton, S.L., Griffin, J.L., Bessant, C., the 2 anti-HIV antibodies selected for fast-track- Brandizzi, F., Hawes, C., Watson, R.B., Dupree, P., Lil- ing through production, processing and regula- ley, K.S. ‘Mapping the Arabidopsis organelle pro- tory compliance. At this stage of the project, the teome’, Proc Natl Acad Sci USA, 2006, 103(17):6518- Pharma-Plant consortium remains convinced 65123. that this outcome will be achieved. Ma, J.K., Barros, E., Bock, R., Christou, P., Dale, P.J., Main findings: Dix, P.J., Fischer, R., Irwin, J., Mahoney, R., Pezzotti, M., Schillberg, S., Sparrow, P., Stoger, E., Twyman, In the first year of the project, the 2 anti-HIV anti- R.M., ‘European Union Framework 6 Pharma- bodies were chosen as fast-track molecules and Planta Consortium Molecular farming for new the other 6 targets were consigned to the devel- drugs and vaccines. Current perspectives on the opment loop. Another significant decision was to production of pharmaceuticals in transgenic focus on maize and tobacco as dual production plants. EMBO Rep., 2005, 6(7):593-599. systems. Pharma-Plant has achieved high levels of antibody expression in both systems, and fully Ma, J.K., Chikwamba, R., Sparrow, P., Fischer, R., expects to achieve a clinical batch of antibody for Mahoney, R., Twyman, R.M., ‘Plant-derived phar- clinical trials by 2009. The project partners have maceuticals - the road forward’, Trends Plant Sci., achieved significant progress in the area of proc- 2005, 10(12):580-585. ess development, to allow the extraction and purification of antibody molecules from plant tissue Nuttall, J., Ma, J.K., Frigerio, L., ‘A functional an- under GMP conditions. This has been facilitated tibody lacking N-linked glycans is efficiently not only by process development work in WP5, folded, assembled and secreted by tobacco

New Therapies – Immunotherapy and Transplantation 197 Pharma-Planta

mesophyll protoplasts’, Plant Biotechnol J.,,2005, Philip J. Dale and George Lomonossoff 3(5):497-504. John Innes Centre Norwich, UK Twyman, R.M., Schillberg, S., Fischer, R., ‘Transgen- ic plants in the biopharmaceutical market’, Expert Laurence Dedieu and Roger Frutos Opin Emerg Drugs., 2005, 10(1):185-218. Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD) Paris, France

Coordinator Chris Hawes Oxford Brookes University Rainer Fischer Oxford, UK Fraunhofer Gesellschaft Hansastrasse 27c Lorenzo Frigerio D-80686 Munich, Germany University of Warwick E-mail: [email protected] Coventry, UK

Scientific coordinator Friedrich Altmann Universität für Bodenkultur Julian Ma Vienna, Austria St. George’s Hospital Medical School Department of Cellular and Molecular Medicine (CMM) Friedemann Hesse Cranmer Terrace Polymun Scientific Immunbiologische Forschungs GmbH London SW17 0RE, UK Vienna, Austria E-mail: [email protected] Mario Pezzotti Partners Università Degli Studi di Verona Verona, Italy Rainer Fischer, Juergen Drossard and Stefan Schillberg Fraunhofer Institute of Molecular Biology and Applied Eugenio Benvenuto Ecology Ente per le Nuove Technologie, l’Energia e l’Ambriente Aachen, Germany Rome, Italy

Julian Ma and David Lewis Christian Vivares St George’s Hospital Medical School Université Blaise Pascal Clermont-Ferrand II London, UK Aubiere, France

Eva Stoger and Rainer Fischer Henri Salmon Rheinisch-Westfälische Technische Hochschule (RWTH) Institut National de la Recherche Agronomique Aachen, Germany Paris, France

Philip J. Dix and Jackie Nugent John C. Gray and Kathryn Lilley National University of Ireland University of Cambridge Maynooth, Republic of Ireland Cambridge, UK

198 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

Paul Garside Ann Depicker University of Glasgow Vlaams Interuniversitair Instituut voor Biotechnologie Glasgow, UK VZW Zwijnaarde, Belgium David Robinsion Ruprecht-Karls-Universität Heidelberg Nikos Lambrou Heidelberg, Germany Agricultural University of Athens Athens, Greece Marc Boutry Université Catholique de Louvain Rob Eiss and Harry Thangaraj Lovain-la-Neuve, Belgium Centre for the Management of Intellectual Property in Health Research and Development (MIHR) Jurgen Denecke London, UK University of Leeds Leeds, UK Eric van Wijk Mosaic Systems BV Alessandro Vitale Prinsenbeek, Netherlands Consiglio Nazionale Delle Ricerche Rome, Italy Paul Christou Univeritat de Lleida Rachel Chikwamba and Eugenia Barros Lleida, Spain Council for Scientific and Industrial Research (CSIR) Pretoria, South Africa Oscar Reif Sartorius AG Johnathan A. Napier Gottingen, Germany Rothamstead Research Ltd Harpenden, UK

Jean-Marc Neuhaus Université de Neuchatel Neuchatel, Switzerland

Ralph Bock Max Planck Institute Postdam, Germany

Tony Kavanagh Trinity College Dublin, Republic of Ireland

Udo Conrad Institut für Pflanzengenetik und Kulturpflanzenforschung Gatersleben, Germany

New Therapies – Immunotherapy and Transplantation 199 SAGE SME-led antibody glycol-engineering

Contract No LSHB-CT-2007-037241 Project type SME-Specific Targeted Research Project EC contribution e 1 843 427 Starting date 1 April 2007 Duration 36 months

Background and objectives: The partners, part of an international consortium of four research organisations, one small The use of plant systems for the production of and medium-sized enterprise (SME) and two pharmaceutical glycoproteins (including anti- companies, will produce an antibody as a panel bodies) offers an attractive alternative to the cur- of different glycoforms, which will be tested for rent state-of-the-art in protein production, given the following: their cost efficiency and overall safety. Producing • stability; large quantities of antibodies for use in cancer • efficacy (e.g. in Fc-receptor binding diagnostic and therapeutic systems using plant assays, tumour cell binding assays and tu- systems could therefore prove a viable and finan- mour grafting); cially sound approach. • pharmacokinetic properties, such as serum half life and antibody-dependent However, the differences in glycan structures cellular cytoxicity (ADCC). added by plants in comparison to those found in humans pose significant obstacles and in fact re- The project partners will use the results to de- searchers recognise that the plant species used velop safer and more active glycoform varieties as well as the tissue and cell type and the age for therapeutic applications. have a huge impact on the final glycoform of an antibody. Approach and methodology:

The SAGE project targets an improved plant pro- By using the plant expression systems and BY-2 duction platform for pharmaceutical glycopro- cells to generate the same recombinant antibody, teins. The partners, a high-calibre team of experts the consortium targets the production of H10, a in plant-based production technology and im- human full-length immunoglobulin G (IgG) that munology, will use four plant-based expression recognises the CEA. systems (including transgenic plants, virus-in- SAGE will also conduct a comprehensive, com- fected plants and transformed plant cell lines) parative study to establish how the structural, and mammalian cells as a control to generate a functional and clinical properties of the anti- therapeutic antibody that recognises the well- body are influenced by the glycan structures. The characterised carcinoembryonic antigen (CEA), project partners will compare the properties of as well as their experience and know-how to the H10 antibodies with those of a control H10 launch protein therapeutics on the market. molecule generated in Chinese Hamster Ovary (CHO) cells.

200 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

Expected outcome: Coordinator

The use of advanced plant-based expression Stefan Schillberg technologies will help SAGE generate innovative Fraunhofer-Institut für Molekularbiologie und Ange- therapeutic and diagnostic antibodies. The con- wandte Oekologie IME sortium will establish which best plant-based Department of Plant Biotechnology expression platform should be used to produce Forckenbeckstrasse 6 therapeutic antibodies, as well as identify the 52074 Aachen, Germany glycan structures that give antibodies superior E-mail: [email protected] properties in a clinical setting. Partners The team will also determine the effects the various plant-derived glycans have on the physical Yuri Gleba and functional properties of antibodies. This ac- ICON Genetics tion will support SAGE’s intention to produce im- Halle, Germany proved antibody-based therapeutics with superior performance, as well as to provide treatment Gilbert Gorr for many more people who need it. Ultimately, greenovation Biotech GmbH SAGE will be instrumental in improving health Freiburg, Germany care for everyone and in bringing down the costs for treatment. Gerben van Eldik Bayer BioScience With strong business support, SAGE will convert Gent, Belgium any new knowledge or product developed during the duration of the three-year project direct- Dirk Bosch, Dion Florack, Gerard Rouwendal ly into strategic advantage. The project partner Plant Research International BV Bayer BioScience will utilise IP management sup- Wageningen, Netherlands port to conduct IP searches and secure extra IP protection, if needed. Eva Stoger and Rainer Fischer RWTH SAGE will also transfer the acquired knowledge to Aachen, Germany other scientific groups working in the same field. Results and information will be disseminated via Hardev S Pandha and David Lewis electronic mail, peer-reviewed articles, abstracts St George’s Hospital Medical School and posters. The SAGE consortium website will London, UK also make key findings available to the public.

New Therapies – Immunotherapy and Transplantation 201 BMC Bispecific monoclonal antibody technology concept

Contract No LHSB-CT-2005-518185 Project type STREP EC contribution e 2 450 000 Starting date 1 November 2005 Duration 36 months

Background and objectives: of the cell, an event known to enhance the signal transduction pathway of apoptosis; Among protein-based drugs, monoclonal an- • modify the carbohydrate moiety of tibodies (mAb) have a particular characteristic, bispecific antibodies and the tumour tar- acting both as a drug and as a targeted delivery geting the complement regulator mol- system. MAb have recently shown great poten- ecule, properdin, to trigger the activation tial in the treatment of human cancer because of the complement enzymatic cascade at they are much more specific than conventional the tumour site; chemotherapy. • establish the chemical conjugation of antibodies of different specificities on Antibodies are large protein molecules compris- emulsion nanoparticles. ing two parts: First, the two variable binding sites confer the unique specificity to the molecule, and Approach and methodology: can send a signal of growth inhibition or of pro- grammed death, called apoptosis. Second, a con- The development of novel approaches to opti- stant part can trigger the activation of host de- mise the targeting and destruction of tumoural fence cytotoxic molecules or killer lymphocytes, cells is ensured in the following ways: by engi- directed against the cancer cells. However, a great neering new constructs of recombinant bispe- deal of progress is needed before we can under- cific or bifunctional antibodies reacting with stand the mechanism of binding and the activity two different antigens on the same tumour cells; of antibodies, in order to improve their targeting by developing new strategies for the induction capabilities and their efficiency. of complement-dependant cytotoxicity (CDC), either through modifications of the glycosyla- BMC aims to do the following: tion of bispecific monoclonal antibodies, or by • develop a recombinant bispecific mAb genetically fusing properdin to recombinant with tetrameric binding sites, directed monoclonal antibodies; and by creating a chemi- against two different antigens expressed cal conjugation of antibodies of different specifi- on the same target tumour cell, which will cities on emulsion nanoparticles. add more specificity and also double its chances to target tumour cells and to re- The consortium will evaluate and validate the cruit more effector natural killer cells; novel tumour targeting strategies proposed in • develop new bispecific or bifunctional the project, on tumour cells from patients with molecules with the property of cross-link- CD5+ B-cell lymphoproliferative diseases, as well ing two different receptors on the surface as in an experimental model of SCID mice graft-

202 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

ed with CD5+ B-cell lines. The optimisation of the Coordinator benefit/risk ratio in B CD5+ B-cell leukaemias and lymphomas will also constitute a key approach. Jean Kadouche Monoclonal Antibodies Therapeutics Expected outcome: Génopole Campus 1 5, rue Henri Desbruères A doubling of the avidity of bispecific antibodies 91030 Evry cedex, France for the BCLL targets tumour cells, in comparison E-mail: [email protected] with state-of-the-art monospecific monoclonal antibodies, can be made possible with through Partners the following activities: • induction of apoptosis in B-CLL cells Jean-Pierre Mach previously unresponsive to monomeric University of Lausanne anti- CD5 mAb; Bruno Robert Institute of Biochemistry • induction of lysis of at least 50% of Lausanne, Switzerland BCLL cells in the presence of human complement and specific mAbs with carbohy- Christian Berthoud drate modification or fused properdin; Université de Brest • induction of C3b opsonisation in 100% Brest, France of BCLL cells in the presence of the same reagents; Martine Cerutti • increase of vascular permeability in Centre National de la Recherche Scientifique subcutaneous tumour xenografts of hu- Montpellier, France man BCLL in SCID mice, after systemic injection of mAb with a complement ac- Simon Benita tivating property; Hebrew University of Jerusalem • inhibit tumour growth in a model of School of Pharmacy subcutaneous xenografts of human B- Jerusalem, Israel CLL in SCID mice, by systemic injection of selected bispecific or bifunctional mol- Josée Golay ecules. Ospedali Riuniti di Bergamo Bergamo, Italy

Jurgen Borlak Fraunhofer – Institut Toxikologie und Experimentelle Medizin Hannover, Germany

Candice Ouinon ALMA Consulting Group Lyon, France

Patrick Henno MABGENE S.A. Alès, France

New Therapies – Immunotherapy and Transplantation 203 AutoCure Curing autoimmune diseases. A translational approach to autoimmune diseases in the post-gnomic era using inflammatory arthritis and myositis as prototypes and learning examples Contract No LSHB-CT-2006-018661 Project type Integrated Project EC contribution e 11 000 000 Starting date 1 March 2006 Duration 60 months Website www.autocure.org

Background and objectives: combinations of novel molecular tools, and precise definition of disease phenotypes. The objective of the AutoCure project is to transform knowledge obtained from molecular re- AutoCure’s aims involve producing the following: search, particularly within genomics, into a cure • an increased understanding of the for the increasing number of patients suffering causes of RA and myositis, enabling better from inflammatory rheumatic diseases. Rheu- prevention; matoid arthritis (RA) is used as a prototype, since • new potential targets for therapy in ar- this disease offers unique opportunities to define thritis and myositis, which can be further and evaluate new therapies. tested in other rheumatic inflammatory diseases; Development of the first generation of targeted • a prototype system for translational therapies (anti-TNF and anti-IL-1) in chronic in- research in Europe, enabling collabora- flammatory disease used RA as the prototype tive development of targeted therapies in disease, following work by European investiga- many inflammatory diseases and allowing tors included in the current project. This work European SMEs to rapidly develop ideas demonstrated the following: and patents into targeted therapies in in- • that targeted therapies can be flammatory diseases. efficient; • that a cure, while not yet achieved, is Approach and methodology: within the reach of a strong international consortium covering translational re- The project targets available knowledge in genet- search and molecular technology. ics and genomics, to gain a better understanding of rheumatoid arthritis and myositis. This will be Potential key molecular mechanisms determin- achieved through the appropriate use of large- ing the course of RA and myositis are defined scale genetic investigations to characterise sub- from genetic studies in humans, from relevant sets of rheumatoid arthritis and myositis, and animal models and from basic cell and molecu- the use of genetic, as well as other molecular lar biology. Predictors of disease development characteristics, to predict disease development and therapeutic responses, which could lead to in these different subsets. Subsequently, knowl- future individualised therapies, are developed edge about molecular mechanisms in different with the help of the consortium’s unique large genetically defined subsets would be used to patient group and biobanks. Development and develop new targeted therapies, suitable for dif- evaluation of new therapies is performed using ferent subsets of these diseases.

204 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

Expected outcome:

The AutoCure consortium expects to deliver new and original knowledge concerning molecular pathways used for different kinds of rheumatic diseases, and also to provide a platform for the development of new preventive measures and therapies.

Main findings:

Findings have been made within the areas of eti- ology and pathogenesis of arthritis. Using large biobanks with information on genetics and environmental factors, and immunity, the consortium has been able to define new risk factors for RA, such as smoking and other environmental risk factors, how they interact with genes, and how they give rise to immune reactions that may cause arthritis. Immunity against citrullinated antigens were initially described by scientists within this consortium, and studies on how such immunity can cause arthritis is now very actively ongoing within the consortium. • 21 academic institution Factors triggering the development of myositis • 5 SME’s are investigated in one work package, and new • The Karolinska Institute (Co-ordinating institution) findings have been made concerning the molec- • Prof Lars Klareskog (Coordinating Investigator) ular mechanisms of development of the symptoms of myositis, such as the characteristic weak- Who are AutoCure ness that causes disability. Using experimental models for arthritis, new modes of using siRNa A major effort is being made apropos the harmo- for inhibiting cytokine production have been nisation of guidelines for collection, and the use used both in vitro and in vivo. Many groups are of large clinical data bases and biobanks in rela- now collaborating on the greater use of siRNa for tion to bioethical rules and regulations in Europe. therapy of arthritis. The guidelines to be developed will effectively help scientists — both within rheumatology and The development of therapies continues in many in other fields — to work efficiently together of the clinics that are participating in the project. while adhering strictly to guidelines and ethi- In particular, studies on the individualisation of cal rules. Collaboration is ongoing between the therapy are ongoing, where biomarkers and ge- clinical and experimental academic laboratories, netic determinants are used to determine the ef- as well as the companies contributing to the con- ficacy, as well as the adverse effects of targeted sortium. Here, in particular, Genmab has been de- therapies against arthritis and myositis. veloping active new therapies with support from scientists active in the consortium.

New Therapies – Immunotherapy and Transplantation 205 AutoCure

Grundtman, C., Salomonsson, S., Dorph, C., Bruton, J., Andersson, U., Lundberg, I.E., ‘Immunolocaliza- tion of interleukin-1 receptors in the sarcolemma and nuclei of skeletal muscle in patients with idiopathic inflammatory myopathies’, Arthritis Rheum, 2007, Jan 30;56(2):674-687

Hultqvist, M., Olofsson, P., Gelderman, K.A., Hol- mberg, J., Holmdahl, R., ‘A new arthritis therapy with oxidative burst inducers’, PLoS Med, 2006, Sep;3(9):e348. Overview Canete, J.D., Santiago, B., Cantaert, T., Sanmarti, Major publications R., Palacin, A., Celis, R., Graell, E., Gil-Torregrosa, B., Baeten, D., Pablos, J.L., ‘Ectopic lymphoid neogen- Van der Pouw Kraan, T.C., Wijbrandts, C.A., van esis in psoriatic arthritis’, Ann Rheum Dis, 2007, Jan Baarsen, L.G., Voskuyl, A.E., Rustenburg, F., Baggen, 12; J.M., Ibrahim, S.M., Fero, M., Dijkmans, B.A., Tak, P.P., Verweij, C.L., ‘Rheumatoid Arthritis subtypes iden- Wesoly, J., Hu X., Thabet, M.M., Chang, M., Uh, H., tified by genomic profiling of peripheral blood Allaart, C.F., Toes, R.E., Houwing-Duistermaat, J.J., cells: Assignment of a type I interferon signature Begovich, A.B., Huizinga, T.W., ‘The 620W allele is in a subpopulation of patients’, Ann Rheum Dis., the PTPN22 genetic variant conferring suscepti- 2007, Jan 18; bility to RA in a Dutch population’, Rheumatology, Oxford, 2006, Nov 29. Michou, L., Croiseau, P., Petit-Teixeira, E., du Mont- cel, S.T., Lemaire, I., Pierlot, C., Osorio, J., Frigui, W., Bendtzen, K., Geborek, P., Svenson, M., Larsson, L., Lasbleiz, S., Quillet, P., Bardin, T., Prum, B., Clerget- Kapetanovic, M.C., Saxne, T., ‘Individualized moni- Darpoux, F., Cornelis, F., ‘European Consortium toring of drug bioavailability and immunogenic- on Rheumatoid Arthritis Families. Validation of ity in rheumatoid arthritis patients treated with the reshaped shared epitope HLA-DRB1 classifi- the tumor necrosis factor alpha inhibitor inflixi- cation in rheumatoid arthritis’, Arthritis Res Ther, mab’, Arthritis Rheum, 2006, Dec; 54(12):3782-9. 2006, 8(3):R79. Michou, L., Lasbleiz, S., Rat, A.C., Migliorini, P., Balsa, A., Westhovens, R., Barrera, P., Alves, H., Pier- Working model lot, C., Glikmans, E., Garnier, S., Dausset, J., Vaz, C., Fernandes, M., Petit-Teixeira, E., Lemaire, I., Pas- cual-Salcedo, D., Bombardieri, S., Dequeker, J., Radstake, T.R., Van Riel, P., van de Putte, L., Lopes- Vaz, A., Prum, B., Bardin, T., Dieude, P., Cornelis, F., ‘European Consortium on Rheumatoid Arthritis Families. Linkage proof for PTPN22, a rheumatoid arthritis susceptibility gene and a human autoimmunity gene’, Proc Natl Acad Sci USA, 2007, Jan 30;104(5):1649-54.

206 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

Coordinator Renate Gay Lars Klareskog University of Zurich Karolinska Institutet Centre of Experimental Rheumatology Department of Medicine and WHO Collaborating Centre Rheumatology Unit Zurich, Switzerland Stockholm, Sweden E-mail [email protected] Tore Saxne and Rikard Holmdahl or [email protected] Lund University Lund, Sweden Partners Solbritt Rantapää-Dahlqvist Tom Huizinga Umeå University Leiden University Medical Centre Umeå, Sweden Leiden, Netherlands Jiri Vencovsky Gerd Burmester and Andreas Radbruch Institute of Rheumatology Charité Universitätsmedizin Berlin Prague, Czech Republic Berlin, Germany Jane Worthington Josef Smolen University of Manchester Medical University of Vienna Manchester, UK Vienna, Austria Jan van de Winkel Barry Bresnihan GENMAB A/S St Vincent’s University Hospital Copenhagen, Denmark Dublin, Ireland Christopher Buckley Paul Peter Tak and Dominique Baeten University of Birmingham Univesrity of Amsterdam Birmingham, UK Amsterdam, Netherlands François Cornelis Andrew Cope Univeristé d’Evry Imperial College Evry, France London, UK Wlodzimierz Maslinski Wim van den Berg and Walther van Venrooij Institute of Rheumatology University Medical Centre Warsaw, Poland Nijmegen, Netherlands Dimitrios Boumpas Christian Jorgensen University of Crete Institut de la santé et de la recherche medicale Heraklion, Crete, Greece Paris, France Gerd Burmester EULAR Genomics Study Group Zurich, Switzerland

New Therapies – Immunotherapy and Transplantation 207 AutoCure

Cor Verweij VU University Medical Centre Amsterdam, Netherlands

Peter Olofsson Biovitrum Gothenburg, Sweden

Karine Mignon-Godefroy BMD Marne-la-Vallée, France

Margriet Vervoordeldonk Arthrogen B.V. Amsterdam, Netherlands

208 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation INNOCHEM Innovative chemokine-based therapeutic strategies for autoimmunity and chronic inflammation

Contract No LSHB-CT-2005-518167 Project type Integrated Project Innochem EC contribution e 11 730 696 Starting date 1 November 2005 Duration 60 months Website www.altaweb.eu/innochem

Background and objectives: overlapping approaches to target the chemokine system with recombinant and low molecular The general objective of INNOCHEM is to develop weight molecules. innovative chemokine-based therapeutic strategies for autoimmunity and chronic inflammation. INNOCHEM is expected to conduct a ‘proof-of- The project is based on the scientific excellence principle’ clinical study in volunteers. The ambi- of the applicants, which have made major rection of this project is to re-establish European ognised contributions to the field since the very leadership in basic and applied chemokine re- beginning of chemokine discovery, and on the search, by integrating academic and industrial construction of shared technological platforms. cutting-edge groups, to develop innovative ther- This includes the following: apeutic strategies against autoimmunity and • proteomics; chronic inflammatory disorders. • transcriptional profiling for the outline of the ‘chemokinome’ in pathophysiologi- Approach and methodology: cal conditions and identification of new antagonists; The approach of INNOCHEM is based on ‘plat- • molecular modelling of agonist/antag- forms’, a ‘pipeline’ and the ‘integration of excel- onist receptor or agonist/inhibitor interac- lence’. The hard core ‘platforms’ on which INNO- tion, for pharmacology and drug design; CHEM is based, include the following: • gene modified mice for target valida- • proteomics and whole genome tran- tion in autoimmune disorders. scriptional profiling for the definition of the chemokinome in pathophysiological Genetic, structural, biological, and immun- conditions, and for the identification of opathological studies will provide a framework new modulators; for the development of innovative chemokine- • gene modified mice, including double based therapeutic strategies. The therapeutic ap- knock outs (KO) and KO mice in autoim- proaches to be investigated are innovative and mune backgrounds for pathophysiology, not limited to conventional antagonists. These target identification and validation, and include decoy receptors, agonist binders, and preclinical evaluation; non-competitive allosteric inhibitors. In addition • molecular modelling for agonist/an- to academic groups, therapy-oriented research tagonist-receptor interaction or agonist- includes 3 biotech SMEs, 1 medium- and 2 large- inhibitor interaction and pharmacology sized pharmaceutical companies. The companies for the design and optimisation of innova- involved are developing complementary non- tive strategies.

New Therapies – Immunotherapy and Transplantation 209 INNOCHEM

The ‘pipeline’ characteristics are presented Expected outcome: below: • Development of original, anti-chem- The goal of INNOCHEM is to develop innovative okine strategies including receptor an- chemokine inhibitors as a novel therapeutic ap- tagonists, agonist blockers, competitors of proach for the treatment of autoimmune and agonist-GAG interaction, decoy receptors, chronic inflammatory diseases up to one ‘proof- and non-competitive allosteric inhibitors. of-concept’ study in humans. The development These strategies are original inasmuch as of innovative therapeutic strategies will be based they extend beyond (but include) conven- on a definition of pathophysiological significance tional antagonists. and target validation (Fig 2). The therapeutic ap- • Pathophysiology of the chemokine proaches to be investigated are innovative, and system in preclinical and clinical condi- not limited to conventional antagonists. These tions, as a basis for rational, targeted inter- will include decoy receptors, agonist binders, and vention. non-competitive allosteric inhibitors. • Preclinical, multistep evaluation, including target validation in gene modified A unique non-competitive allosteric inhibitor, mice and by genetic analysis in humans Reparixin, which has completed phase I and has • Proof-of-principle clinical studies. been awarded orphan drug status by FDA and EMEA, is available for proof-of-concept studies in The entire pipeline has been activated from the humans. very beginning of the study (Fig 1). ‘Integration of excellence’ represents a major characteristic The results obtained in this project will open new of the INNOCHEM approach. Not only does IN- perspectives in therapeutic intervention against NOCHEM have the ambition of collecting and at- autoimmunity and chronic inflammation, with tracting major European groups in the field, but social impacts measurable as improved health its components also have a proven strong track and reduced health societal costs, (including di- record of bilateral interactions. In the context of rect and indirect costs for patient assistance, as INNOCHEM, a quantum leap of integrating Euro- well as loss of manpower), emerge. pean efforts in the field will be possible, on the solid basis of a tradition of collaboration.

Figure 1. – INNOCHEM pipeline.

210 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

Main findings:

During the first year of activity, the INNOCHEM consortium focused its efforts along the lines originally identified, albeit with some adjustments. In general, the lines of activity encompassed diverse fields, ranging from the basic biology of the chemokine system to translational efforts in the clinical phase. Potential technological approaches have included the use of whole genome transcriptional profiling, the use of gene-modified mice, and original reagents generated by the group. A major thrust of the INNO- CHEM efforts has been devoted to strengthen- Figure 2. - Outline of INNOCHEM targeting chemokine/re- ing integration and collaboration. As an example, ceptor interactions, emphasising the implementation of therapeutic strategies. one could emphasise the strengthening of Euro- pean leadership in the field of silent receptors for chemokines which can act as decoys. In terms of tokines and chemokines in prostate inflamma- translational efforts, a major result obtained dur- tion: Interleukin 8 as a predictive biomarker in ing the first year is the identification of a chem- chronic prostatitis/chronic pelvic pain syndrome okine (CXCL8) as a surrogate endpoint for the and benign prostatic hyperplasia’, Eur Urol, 2007; activity in patients in inflammatory prostatitis of Feb;51(2):524-33. a compound currently being developed by one of the participating SMEs. Schmid, H., Boucherot, A., Yasuda, Y., Henger, A., Brunner, B., Eichinger, F., Nitsche, A., Kiss, E., Bleich, Major publications M., Grone, H. J., Nelson, P. J., Schlondorff, D., Cohen, C. D., Kretzler, M., ‘Modular activation of Nuclear Pello, O.M., Moreno-Ortiz, M.D., Rodriguez-Frade, Factor-κB transcriptional programs in human J.M., Martinez-Munoz, L., Lucas, D., Gomez, L., Lu- diabetic nephropathy’, Diabetes, 2006, 55:2993- cas, P., Samper, E., Aracil, M., Martinez, A., Bernad, 3003. A., Mellado, M., ‘SOCS upregulation mobilises autologous stem cells through CXCR4 blockade’, Proost, P., Struyf, S., Loos, T., Gouwy, M., Schutyser, Blood, 2006, 108:3928-3937. E., Conings, R., Ronsse, I., Parmentier, M., Grillet, B., Opdenakker, G., Balzarini, J., Van Damme, J., ‘Coex- Rosenkilde, M.M., David, R., Oerlecke, I., ned- pression and interaction of CXCL10 and CD26 in Jensen, T., Geumann, U., Beck-Sickinger, A. G., mesenchymal cells by synergising inflammatory Schwartz, T.W., ‘Conformational constraining of cytokines: CXCL8 and CXCL10 are discriminative inactive and active states of a 7TM receptor by markers for autoimmune arthropathies’, Arthritis metal-ion site engineering in the extracellular Res Ther, 2006, 8:R107. end of transmembrane segment V (TM-V)’, Mol Pharmacol, 2006, 70:1892-1901. Springael, J. Y., Le Minh, P. N., Urizar, E., Costagli- ola, S., Vassart, G., Parmentier, M., ‘Allosteric mod- Penna, G., Mondaini, N., Amuchastegui, S., Degli ulation of binding properties between units of Innocenti, S., Carini, M., Giubilei, G., Fibbi, B., Colli, chemokine receptor homo- and hetero-oligom- E., Maggi, M., Adorini, L., ‘Seminal plasma cy- ers’, Mol Pharmacol, 2006, 69:1652-1661.

New Therapies – Immunotherapy and Transplantation 211 INNOCHEM

Sironi, M., Martinez, F.O., D’Ambrosio, D., Gattorno, Coordinator M., Polentarutti, N., Locati, M., Gregorio, A., Iellem, A., Cassatella, M.A., Van Damme, J., Sozzani, S., Alberto Mantovani Martini, A., Sinigaglia, F., Vecchi, A., Mantovani, A., Humànitas Mirasole S.p.A ‘Differential regulation of chemokine production Via Manzoni 56 by Fcγ receptor engagement in human mono- 20089 Rozzano-Milano, Italy cytes: association of CCL1 with a distinct form of E-mail: [email protected] M2 monocyte activation (M2b, type 2)’, J Leukoc Biol, 2006, 80:342-349. Scientific coordinator

Mariagrazia Uguccioni Istituto di Ricerca in Biomedicina Via Vela 6 6500 Bellinzona, Switzerland E-mail: [email protected]

Partners

Christophe Combadiere Université Pierre et Marie Curie-Paris INSERM U543 Paris, France

Dominique Emilie INSERM Paris, France

Andrzej Glabinski Uniwersytet Medyczny w Łodzi, Department of Neurology Lodz, Poland

Martin Lipp Max-Delbrueck-Centrum für Moleckulare Medizin Berlin, Germany

Carlos Martinez Centro Nacional de Biotecnologia, CSIC Madrid, Spain

Bernhard Moser Universität Bern, Institute of Cell Biology Bern, Switzerland

212 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

Peter J. Nelson Antal Rot Ludwig-Maximilians-University Münich Novartis Institutes for Biomedical Research Münich, Germany Vienna, Austria

Marc Parmentier Amanda Proudfoot Université Libre de Bruxelles, IRIBHM Serono International SA Brussels, Belgium Plan les Outas, Switzerland

Costantino Pitzalis Aldo Tagliabue King’s College London Alta S.r.l. London, UK Siena, Italy

Sergio Romagnani Università degli Studi di Firenze, “MCIDNENT” Florence, Italy

Mette M. Rosenkilde University of Copenhagen The Panum Institute Department of Pharmacology Copenhagen, Denmark

Jozef van Damme Katholieke Universiteit Leuven, Belgium

Timothy Williams Imperial College of Science, Technology and Medicine London, UK

Amnon Peled BioKine Therapeutics Ltd. Rehovot, Israel

Luciano Adorini BioXell Milan, Italy

Marcello Allegretti Dompe pha.r.ma Spa L’Aquila, Italy

Jan W. Vrijbloed Polyphor Ltd.- PEMB Unit Allschwill, Switzerland

New Therapies – Immunotherapy and Transplantation 213 CELLAID European symposia for the evaluation of potentials and perspectives of curative cell therapies for autoimmune diseases

Contract No LSHB-CT-2004-005094 Project type Specific Support Action EC contribution e 198 800 Starting date 1 January 2005 Duration 30 months Website www.cellaid-eu.org

Background and objectives: Such networking of European research institutions and clinics will enhance research expertise, Autoimmune diseases, such as inflammatory the standardisation of clinical interventions and rheumatic diseases, are currently impossible to quality assessment, e.g. immune monitoring. Pa- cure with either conventional therapies or mod- tient cohorts will grow, even for rare autoimmune ern biologics therapies. Existing therapies are diseases. The translation of concepts into clinics immune-suppressive, systemic and/or accom- will involve extensive industry cooperation: joint panied by serious side effects, which drastically efforts will boost the competitiveness of small, reduce quality of life and life expectancy. The so- medium and large biotech and pharmaceutical cioeconomic impact of inflammatory rheumatic companies in Europe. diseases is approximately €15 billion per year in Germany alone, with a trend for costs to increase Approach and methodology: rapidly due to the market prices of emerging biologics. The first two CELLAID symposia were held in Berlin in June 2005, and in Brussels in April 2006. The CELLAID project will establish a European Cellular concepts of immune therapies for au- consortium to organise three symposia on im- toimmune diseases and European perspectives munocyte-based therapies for autoimmune towards immunocyte-based therapies were ad- diseases. The new treatment concepts should dressed by an international selection of expert target pathomechanisms, especially the involved speakers, chairmen and participants. Results immune cells and messenger biomolecules in (programmes and summaries) of the symposia the course of the disease. CELLAID also aims at are published on the website of the project, www. providing a communication platform for Euro- cellaid-eu.org. The third symposium is scheduled pean experts to evaluate the existing potential, to take place in Florence, Italy, on 20-22 February and develop integrated pan-European strategies. 2007. Joint European efforts are required to strengthen the European lead in respective technologies, Expected outcome: and also to combine experience in pan-Europe- an clinical trials. Currently, no cure is available for autoimmune diseases, which require a life-long immune sup- The European Research Area can maintain excel- pressive treatment with a high risk of adverse lence, and scientific, technological and economic events, and lack a regenerative perspective. Im- competitiveness with innovative therapies for munocyte-based therapies — supported by re- autoimmune diseases through networking. cent advances made in genomics, immunology

214 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

CellAid 2005 - Andreas Radbruch (Coordinator) and cytometry — aim for an autoimmunity cure and a reset of tolerance, which is a prerequisite for regenerative therapies. They will have high trans- symposia. Currently, B cell (e.g. CD 20)-targeted lational potential for other immune diseases. biologics do achieve the most convincing therapeutic effects. However, it remains to be seen Main findings: whether remission or a cure can be achieved without long-term side effects. While research highlights of the two symposia were numerous, only a selection is mentioned in With regard to genomics in multi-factorial diseas- this report. Detailed summaries are available at es, such as rheumatoid arthritis, the identification www.cellaid-eu.org. The function and dysfunc- of genes other than the major histocompatibility tion of regulatory T cells were the areas of focus complex that regulate autoimmune diseases was in a number of presentations. Cytokines such as discussed. One regulator of arthritis severity in IL-6 and its transsignaling mechanisms were pre- rats was identified and then presented at the first sented, representing potential new future thera- CELLAID symposium. In experimental models, a peutic targets. Many other relevant cytokines single gene such as AIRE has provided important and their functions were discussed, highlighting insight into the regulation of autoimmunity. Dis- the role of special cytokine networks that per- cussions at the two CELLAID symposia highlight- petuate chronic inflammation, particularly in au- ed that the power and impact of genetic analy- toimmunity. B cells and their roles as plasma and sis is undisputed, and that the translation from memory cells were intensively discussed in both animal models to genetic analysis for diagnosis,

New Therapies – Immunotherapy and Transplantation 215 CELLAID

prediction and therapy of autoimmune diseases Coordinator patients is in progress. Andreas Radbruch One of the experimental therapies gaining ap- Deutsches Rheuma-Forschungszentrum proval is autologous stem cell transplantation, Charitéplatz 1 performed for many autoimmune diseases. In 10117 Berlin, Germany Europe, they are registered through the EULAR E-mail: [email protected] and EBMT working parties. They do in some in- stances (e.g. Systemic Lupus Erythematosis) have Scientific coordinator the potential to induce full remission, even in patients with a long disease history. As discussed in Jutta Steinkötter the presentations at both meetings, this is seem- Competence network rheumatology ingly based on the combination of a drastic de- Luisenstr. 41 pletion of pathogenic memory and effector cells, 10117 Berlin, Germany and the development of a new and tolerant im- E-mail: [email protected] mune system. So far, however, autologous stem cell transplantation is only applicable for standard treatment of refractory patients, as the side effects can be hazardous.

Finally, mesenchymal stem cells were introduced as ‘targets’ and they have been applied successfully in experimental animal models, where they show they can provide strong immunosuppressive effects. Results from the first European trials in human were presented at the CELLAID symposia.

216 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation STEMDIAGNOSTICS The development of new diagnostic tests, new tools and non-invasive methods for the prevention, early diagnosis and monitoring for haematopoietic stem cell transplantation

Contract No LSHB-CT-2007-037703 Project type SME-Specific Targeted Research Project EC contribution e 2 500 000 Starting date 1 June 2007 Duration 36 months

Background and objectives: • exploit the new tools for commercial use. Over 7 000 allogeneic haematopoietic stem cell transplants (HSCT) are carried out each year in STEMDIAGNOSTICS will develop diagnostic tests Europe alone, as a treatment for leukaemia and using single nucleotide polymorphism (SNP) lymphoma. Techniques and cure rates are im- analyses (IMGM), based on results from previous proving, but the overall survival rate remains be- EC-funded research (Eurobank, Transeurope). tween 40% and 60%. It will use proteomics via mass spectrometry STEMDIAGNOSTICS will develop new proteomic, evaluation/development of diagnostic patterns biological and genomic tests and tools for early (Mosaiques), ELISA kits (Apotech) and protein diagnosis and monitoring of patient response to biochip prototypes (Orla) for the development of novel therapeutics for the most severe complica- fast, high throughput technologies. The consorti- tion of HSCT - graft versus host disease (GvHD). um will also develop novel reagents for monitor- It will bring to the clinic a new generation of ing graft versus leukaemia, GvHD and targeted diagnostics that will significantly improve HSCT therapy (Multimune; Nascacell). Finally, compara- therapy and patient outcome. tive studies in an autoimmune disease model of inflammation — rheumatoid arthritis — will be The consortium unites 5 European SMEs with ex- made. pertise and markets in genomic and proteomic testing, diagnostic assay development and bio- Adult stem cells are starting to be used for the chips, with clinical partners selected for their therapy of diverse disorders including autoim- world leading research in HSCT and access to mune disease and cancer. The potential differ- clinical samples and patient groups. The project entiation of such stem cells into various types will focus on the role of relevant genes and bi- of adult tissue is also a new and exciting area of omarkers associated with acute and chronic research. It will create opportunities for the in- GvHD, using retrospective samples from estab- novation of a new generation of medical diag- lished biobanks and prospective clinical trials to: nostic tools, tests and therapies; for improving • identify novel bio and genomic mark- healthcare services and patient outcomes; and ers for diagnostics; for reducing expenditure on healthcare systems. • develop novel diagnostic tools using It offers Europe’s biotech SMEs the opportunity genomics, proteomics, in vitro bioassays to strengthen their competitiveness and suc- and biochips; cessfully help meet the growing demands of the • test the new diagnostics in animal healthcare sector. models and on clinical samples; Stem cell research is essential due to the lack,

New Therapies – Immunotherapy and Transplantation 217 STEMDIAGNOSTICS

over the past 30 years, of any general cure for is an important predictor of transplant survival. various illnesses, such as cancer, heart disease or The strongest predictor of GvHD is the degree of autoimmune disorders, among others. mismatching between patient and donor; however, there is a significant incidence of acute and Haematopoietic stem cell transplantation (HSCT), chronic GVHD even in patients undergoing HLA however, is one area of stem cell research where identical sibling transplants, thus clearly indicat- major advances in the cure of haematological dis- ing a role of genetic risk factors other than major orders, like leukaemia and lymphoma, inherited HLA differences There are also no reliable predic- immune disorders, and aplastic anaemia, have tive or diagnostic indicators to date for either been made. Transplantation of stem cells in this acute or chronic GvHD and no reliable markers context, where immature cells develop into white that distinguish GvHD from viral or other inflam- and red blood cells and platelets (i.e. haematopoi- matory manifestations. esis) from related or unrelated donors leads to immune reconstitution and can be a life saving The most reliable clinical predictors of GvHD are therapy. Currently over 7 000 transplants are car- the donor recipient sex-mismatch (female donor ried out each year in Europe. Despite this success to male recipient) but no other clinical factors the overall survival rate after HSCT is poor. can predict acute GvHD. Research therefore has focused on the biology of GvHD and the involve- A 40-60% survival rate is the norm for such ment of cytokines in the “cytokine storm”). Serum transplants, which involve the use of bone mar- levels of cytokines, mRNA expression of candidate row, peripheral blood stem cells and umbilical cytokines in peripheral blood or target tissue, or cord blood as the stem cell source. The cure of as in the case of several of the current partners, patients is limited by clinical complications that a study of non HLA genetic polymorphisms for arise post-transplant. These are largely due to a candidate cytokines using pretransplant recipi- lack of understanding of acceptance and rejec- ent and donor DNA have been analysed. How- tion mechanisms and genetic differences that ever, none have as yet been tested effectively exist between a given patient and donor and for commercial use as novel diagnostic tests). inaccurate/lack of diagnostic tests for early com- These studies will form the basis of further devel- plications. opment of novel diagnostics based on the fact that early detection of GvHD and its severity is The application of HSCT is also hampered by the urgently needed and that its progression needs lack of suitably matched donors. Only 25-30% of to be monitored during the course of HSCT. patients will find a HLA matched sibling donor and, therefore, more matched unrelated donors Approach and methodology: within International Bone Marrow Transplant Registries are needed. There is therefore an ur- The project will focus on the role of relevant gent need to improve patient-donor matching at genes and biomarkers associated with acute and both the biological response and genomic level, chronic GvHD, using retrospective samples from which will increase the pool of both suitable and established biobanks and prospective clinical tri- more appropriate donors and reduce the most als to: severe complication of acute and chronic graft • identify novel bio and genomic mark- versus host disease (GvHD). ers for diagnostics; • develop novel diagnostic tools using It is essential to predict the development, the genomics proteomics, in vitro bioassays severity and treatment outcome of GvHD, which and biochips;

218 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

• test the new diagnostics in animal Coordinator models and on clinical samples; • exploit the new tools for commercial Anne Dickinson use; University of Newcastle upon Tyne • undertake comparative studies in an School of Clinical and Laboratory Sciences autoimmune disease model of inflamma- The Medical School tion; rheumatoid arthritis. Newcastle upon Tyne, UK E-mail: [email protected] The SMEs’ specific aims include: • development of new tools for novel Partners diagnostics, novel drug targets and therapeutics for use in both the transplant and Ernst Holler autoimmune settings; Klinikum der Universität Regensburg • access to important clinical and bio- Dept. Hamatology und Internistiche Onkologie logical samples, as well as data for use in Regensburg, Germany accurate assessment of results and con- firmatory studies for the development of Harald Mischak novel diagnostics; Mosaiques Diagnostics GmbH • testing and evaluation of new diag- Hannover, Germany nostics on independent cohorts and correlation of data across HSCT centres; Gabriele Multhoff • testing of new prototypes against Multimmune GmbH current assays via collaboration between Munich, Germany SMEs; • use of new target molecules in the Ralph Oehlmann monitoring of response to therapy and IMGM Laboratories during the post transplant period to as- Martinstried, Germany sess acute and chronic GvHD. Lars French Expected outcome: Zurich University Hospital Department of Dermatology STEMDIAGNOSTICS will identify new diagnos- Zürich, Switzerland tics in the form of novel proteins associated with graft versus host disease (GvHD) compared to Olivier Donzé viral disease. The consortium will also develop Apotech Corporation (Headquarters) early diagnostic tools for GvHD and rheumatoid Epalinges, Switzerland arthritis using gene profiling. The fast throughput development of novel monoclonal antibodies Hans-Jochem Kolb and ELISA kits for research, diagnostics, and po- Clinical Cooperation Group Hematopoietic Cell tentially therapeutic use emerge as well. STEMDI- Transplantation, Institute of Molecular Immunology AGNOSTICS will develop novel peptides for use Forschungszentrum fuer Umwelt und Gesundheit in monitoring GvHD and graft versus leukaemia Munich, Germany effects in transplant patients, and it will identify new single nucleotide polymorphisms (SNPs) for analysis in prognostic/diagnostic indices.

New Therapies – Immunotherapy and Transplantation 219 STEMDIAGNOSTICS

Dale Athey Orla Protein Technologies Ltd Nanotechnology Centre University of Newcastle upon Tyne, Newcastle, UK

Gerard Socie Association de Recherche sur la Greffe de CSP Aplosies et HPN Paris, France

Hildegard Greinix University Hospital of Vienna Bone Marrow Transplantation Unit Vienna, Austria

Ilona Hromadníková Charles University 3rd Medical Faculty Prague, Czech Republic

Amanda McMurray CENAMPS The Fabriam Centre Atmel Way Newcastle upon Tyne, UK

220 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation RISET Reprogramming the immune system for the establishment of tolerance

Contract No LSHB-CT-2005-512090 Project type Integrated Project EC contribution e 10 000 000 Starting date 1 March 2005 Duration 60 months Website www.risetfp6.org

Background and objectives:

RISET aims at inducing tolerance, which is defined as the permanent acceptance of the transplant in the absence of continuous immunosuppression. It is based on the translation of recent advances in post-genomic immunology that are focused on the development of new biotechnology products to promote long-term transplant acceptance in preclinical models. Université Libre de Bruxelles- IMI. Gosselies. Belgique. In order to achieve this objective, the RISET programme anticipates developing diagnostic to induce long-term tolerance in transplanted tests to identify transplanted patients for whom patients, in the absence of any immunosuppres- immunosuppressive treatment could be safely sion. This ambitious objective will be achieved minimised or withdrawn. Related ethical and through a multistep approach involving inter- societal questions will be specifically addressed, mediate milestones: in addition to communication with patient • the development of biological tests organisations and regulatory bodies. In parallel, predictive of transplant tolerance or ‘near- relevant models of tolerance will be used to tolerance’; identify new genes, molecules or cell types that • the clinical and biological assessment will form the basis for novel diagnostic and of the outcome of patients enrolled in therapeutic approaches. clinical investigations, targeting the complete withdrawal of immunosuppression; The use of knowledge will be facilitated by the di- • the establishment of ethical guidelines rect involvement of SMEs in the consortium, the for tolerance induction protocols and edu- building of an industry platform, and the yearly cational programmes on tolerance induc- monitoring of external reviewers with both sci- tion for patients and their families; entific and industrial backgrounds in the field. • the establishment of educational programmes on tolerance induction for phy- Expected outcome: sicians, scientists and nurses; • the identification of new molecular The ultimate goal of this project is to develop targets for tolerance induction in preclini- safe and efficient diagnostic tools, and therapies cal models.

New Therapies – Immunotherapy and Transplantation 221 RISET

ary 2006, 2 more patients were transplanted, and relevant data will be provided in the next report. Identification and recruitment of additional patients to be involved over the second period is under way. In parallel, the use of a combination of anti-CD3 and anti-CD7 immunotoxins to induce long-standing remission of severe high-grade graft-versus-host disease, has been investigated (drug components have been produced and purified, draft protocols and inform consent prepared, phase I/II study has been registered and a preclinical evaluation has been realised). A study using monkeys to perform cytotoxicity tests has been prepared.

Ethical aspects have been monitored through Participants WP4 and further research is ongoing for key societal, organisation, ethical, regulatory and legal Main findings: issues related to activities performed in RISET.

A first series of diagnostic tests have been de- Several dissemination supports have been defined and validated in-house in WP1 (work veloped (website, leaflet, newsletter, and presen- package 1), to identify transplanted patients for tations to scientific and large public audiences). whom immunosuppressive treatment could be Training actions related to RISET activities and safely minimised or withdrawn. The validation of research objectives have taken place at local, na- these tests and markers in clinical protocols has tional and international level. A feasibility study started, with first samples of tolerance induction to examine the opportunity of setting up an in- pilot trials already collected and distributed un- dustry platform to foster exploitation aspects of der strict quality control. the project has been performed, and has led to recommendations for RISET’s future activities. Basic research performed in WP2 has contributed to the characterisation at molecular level of important actors and mediators of tolerance (mark- Coordinator ers of regulatory T cells, tolerogenic DCs). Models of endothelial cell damage to test the effect of Michel Goldman endothelial cell protection on allograft survival Université Libre de Bruxelles were initiated. Antibody therapy in human CD3 Rue Adrienne Bolland, 8 or CD52 transgenic animals was tested, with a 6041 Charleroi (Gosselies), Belgium view to optimising anti-CD3 and anti-CD52 treat- E-mail:[email protected] ment in clinical settings. Partners Clinical trials have started. In total, 12 patients have been included in RISET clinical trials so far. Hans-Dieter Volk Safety, efficacy and biological tests have been Charite, University Medicine Berlin performed for 10 patients. In January and Febru- Berlin, Germany

222 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

M. C. Cuturi M. Daha and F. Claas CHU Nantes Leids Universitair Medisch Centrum Nantes, France Leiden, Netherlands

L. Chatenoud U. Janssen Université René Descartes Paris Miltenyi Biotec GmbH Hospital Necker Cologne, Germany Paris, France H. Waldmann K. Wood Sir William Dunn School of Pathology Oxford University Oxford, UK John Radcliffe Hospital Nuffield Department of Surgery, level 6, Headington M.G. Roncarolo Oxford, UK San Raffaele Telethon Institute for Gene Therapy Milan, Italy R. Lechler Kings College M. Guillet Hodgkin Building, Guy’s Campus TC Land London, UK Nantes, France

R. Rieben U. Kunzendorf Bern University Hospital University of Schleswig-Holstein Bern, Switzerland Kiel, Germany

F. Fandrich B. Miranda Blasticon Gmb Organización Nacional de Transplantes Kiel, Germany Madrid, Spain

B. Arnold Y. Reisner Deutsches Krebsforschungszentrum Weizmann Institute of Science Heidelberg, Germany Rehovot, Israel

A. Cambon-Thomsen A. Wendel INSERM U558 Université Konstanz Toulouse, France Konstanz, Germany

J. P. Soulillou N. Schwabe CHU Nantes ProImmune Ltd Nantes, France Oxford, UK

O. Vilkicky S. Houard Institute for Clinical and Experimental Medicine Henogen Prague, Czech Republic Gosselies, Belgium

New Therapies – Immunotherapy and Transplantation 223 XENOME Engineering of the porcine genome for xenotransplantation studies in primates: a step towards clinical application

Contract No LSHB-CT-2006-037377 Project type Integrated Project EC contribution e 9 876 546 Starting date 1 November 2006 Duration 60 months Website www.xenome.eu

Background and objectives: tail the development of technologies enabling the timely diagnosis of infection, the design of The ultimate goal of XENOME is to generate the a safety plan for an efficacious containment of necessary data to allow xenotransplantation to an untoward infectious event, the breeding of a advance towards its initial clinical phase. The data herd of ‘clean’ source pigs, and the provision of generated in this project will encompass both ef- safety-related data derived from long-term in ficacy and safety aspects of xenotransplantation. vivo studies in primate xenograft recipients. Fi- Tools that will be used to reach this ambitious nally, the project will also offer a strong ethical, objective include state-of-the-art biomolecular social (especially regarding public communica- technologies and in vivo models. tion) and regulatory framework for xenotransplantation research (and possibly for clinical ap- XENOME aims to produce a ‘super-engineered’ plication too). pig, i.e. a pig with a newly generated genotype that will improve the efficacy and safety pro- Xenotransplantation addresses the growing file of xenotransplantation. Assessments of ef- problem of the shortage of human organs, which ficacy will first take advantage of existing pig prevents many patients with terminal organ fail- lines expressing human complement regulators, ure from receiving a human organ. As a result of thrombomodulin (TM) and knock-out for α-Gal improved medical and technological interven- transferase (α-GalT KO). Using the most suitable tions in the field of transplantation, an increasing background, further engineering of the pig ge- number of people are now short-listed for trans- nome will be undertaken. Additional transgenes plants. The waiting lists continue to grow due able to control immune responses, endothelial to a limited organ supply; it is, for instance, esti- cell activation and subsequent microangiopathy, mated that approximately 45 000 people across will be added. the EU are currently on waiting lists for a kidney transplant. The ultimate pig strain will thus combine the The fundamental objectives of this project are already available background with novel mol- presented below: ecules exhibiting anticoagulant and immuno- • Bring the EU back to the forefront of suppressive properties. In addition, an effective xenotransplantation research, a field with immuno-suppression regimen will be defined, a huge potential for the treatment of end- and new pharmaceutical agents will be tested. A stage organ failure. At the end of the pro- strong safety framework will also be established, gramme, XENOME will deliver a European that may allow, at some stage, the progression of ‘product’ for xenotransplantation. This will xenotransplantation into the clinic. This will en- consist of a ‘super-engineered’ pig with

224 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation ©Shutterstock, 2007 ©Shutterstock,

European intellectual property rights. Approach and methodology: • Generate in vivo efficacy data: experimental evidence that the ‘engineered’ pig, Addressing safety and the efficacy of xenotrans- combined with new immunosuppressive plantation is the major endeavour of this initia- reagents, allows long-term survival in tive. Novel constructs that could confer advan- non-human primates. tages to the xenograft will be passed on to the • Assess new immunosuppressive regi- pig-engineering team whose principal objective mens and agents for the efficient inhibi- will be to modify the pig characteristic profile tion of complement activation and acute according to the desired traits. The biomolecu- vascular rejection. lar tools applied will allow complete abrogation • Gain a better understanding of the (knock-out), reduced expression (knock-down), physiology of long-term transplanted or over-expression of candidate genes of interest kidney xenografts in the pig-to-primate in porcine endothelial cells (transgenesis). model. • Establish the necessary safety frame- The pig-engineering team in XENOME will en- work that would allow the progression of sure that the in vitro findings generated are ade- xenotransplantation into the clinic. quately utilised and translated to in vivo work, re- • Provide a strong ethical, social, educa- sulting in novel and stable pig lines. It will also be tional (especially apropos communication part of the responsibilities of the same team to with the public) and regulatory frame- ensure that animals are appropriately screened, work, within which xenotransplantation bred and provided in sufficient number to gener- research (and possibly clinical application) ate the proof-of-concept data in in vivo primate should take place. studies. Indeed, when new lines of engineered

New Therapies – Immunotherapy and Transplantation 225 XENOME

pigs are made available to this network, the ef- Coordinator ficacy and safety of the organs will be tested by transplanting them into primates. Emanuele Cozzi Azienda Ospedaliera di Padova All the activities undertaken will be tightly moni- Ospedale Giustinianeo tored and supervised by an ethical and legal Via Giustiniani, 2 team, whose responsibilities will include ensur- Padova, Italy ing that all the initiatives conducted within the E-mail: [email protected] scope of this programme are fully compliant with existing European ethical and legal frameworks. Partners Furthermore, this team will target the need to bridge the gaps between laboratories and soci- Jean-Paul Soulillou ety, and between scientists, citizens and institu- ITERT tions, both by creating and maintaining a con- Nantes, France tinuous communication flow with the scientific partners, as well as by establishing a connection Pierre Gianello with the general public. This will be made possi- Université Catholique de Louvain ble via diverse means, such as the preparation of Leuven, Belgium videos and leaflets, and the organisation of open laboratory workshops. Public consultation activi- Carlos Romeo-Casabona ties will also be conducted. Universidad del Pais Vasco Bilbao, Spain Expected outcome: Marialuisa Lavitrano XENOME is expected to generate the neces- University of Milan sary set of data that may allow the transition of Milan, Italy xenotransplantation from the current preclinical phase to its initial clinical phase, within the next Rainer de Martin 10 years. This is an ambitious goal and the data Medical University of Vienna that will be generated during the five- year dura- Vienna, Austria tion of this project will address both the efficacy and safety aspects of xenotransplantation. Yasuhiro Takeuchi University College London London, UK

Heiner Niemann Institute for Animal Breeding, Federal Agricultural Research Centre Neustadt, Germany

Linda Scobie University of Glasgow Glasgow, UK

226 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

Cesare Galli Mohamed R. Daha Consorzio per l’incremento Zootecnico srl Leiden University Medical Centre Cremona, Italy Leiden, Netherlands

Jiri Hejner Institute of Molecular Genetics, Academy of Sciences of the Czech Republic Prague, Czech Republic

Reinhard Schwinzer Hannover Medical School Hannover, Germany

Paolo Simoni University of Padua Padua, Italy

Roger Barker Cambridge Centre for Brain Repair Cambridge, UK

Johannes Regenbogen GATC Biotech AG Konstanz, Germany

Ermanno Ancona Consorzio per la Ricerca sul trapianto d’Organi Padova, Italy

Olle Korsgren Corline Systems AB Uppsala, Sweden

Guilherme De Oliveira Biomedical Law Centre Coimbra, Portugal

Mariachiara Tallachini Università Cattolica S.C. Milano Milan, Italy

Miguel Che Parreira Soares Instituto Gulbenkian de Ciência Oeiras, Portugal

New Therapies – Immunotherapy and Transplantation 227 CLINT Facilitating international prospective clinical trials in stem cell transplantation

Contract No LSHB-CT-2007-037662 Project type Specific Support Action EC contribution e 500 000 Starting date 1 April 2007 Duration 24 months

Background and objectives: severely curtailed by the recently introduced requirement to carry out these studies in accord- Autologous and allogeneic stem cell transplan- ance with the EU Directive on Clinical Trials. tation (SCT) is the treatment of choice for many haematological diseases. Within healthcare pro- Although introduced with laudable intentions, vision, SCT is not only one of the most costly but the effect of the directive has been to increase it is also one of the most risky procedures for pa- the resources and therefore the expense in- tients, with transplant related mortalities of up to volved in clinical trials, whilst at the same time 50%. SCT has always been on the cutting edge national differences in the interpretation of the of translational medicine, and over the past dec- new legislation have rendered international ade there have been many changes to the way studies extremely difficult. The objective of CLINT in which transplant is performed, such as the in- is to support the EBMT to further develop the introduction of new drugs and technologies. While frastructure necessary to perform academically- these innovations have the potential to improve initiated international prospective studies in SCT patient outcome, they can also increase the cost throughout Europe. This will hasten the evalua- considerably. At the same time, many new devel- tion of new treatment strategies and improve opments have emerged with respect to targeted the outcome for European citizens. The CLINT drug therapies; leave supportive care in these dis- consortium will be assisted in this endeavour, by eases and therapies may replace or delay trans- advice provided from a similar initiative in the plant for some patients. These new treatments United States. are also expensive and require urgent evaluation. It is essential that they and SCT are used wisely and economically.

There has always been willingness for the SCT community to critically evaluate the role of transplants, as is exemplified by the transmission of outcome data from individual centres to a central database held by the European Bone Marrow Transplant Group (EBMT) for further analysis and reporting. SCT physicians have also been enthu- siastic exponents of clinical trials, and are ready to test new hypotheses, and to compare SCT with other treatments. Their ability to do this has been

228 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

Coordinator

Jane Apperley Imperial College Department of Haematology Hammersmith Hospital Ducane Road London W12 0NN, UK E-mail: [email protected]

Partners

Fiona MacDonald European Group for Blood and Marrow Transplantation Barcelona, Spain

Doris Schroeder University of Central Lancashire Preston, UK

New Therapies – Immunotherapy and Transplantation 229 TRIE Transplantation research integration across Europe

Contract No LSHB-CT-2007-037540 Project type Specific Support Action EC contribution e 424 732 Starting date 1 March 2007 Duration 24 months

Background and objectives: consultations with working groups and members of the advisory council: The primary objectives of the project are out- - biomarkers and pharmacogenomics, lined below: to tailor immunosuppression, - how to promote living donation 1. To identify current opportunities and chal- - regulatory aspects of novel cell-based lenges in the field of transplantation research, immunotherapies, focusing on themes common to cell and solid - standardisation and validation of im- organ transplantation. In order to achieve this munomonitoring tests, overall objective, the following sub-objectives - training programmes in transplanta- are required: tion medicine; • The establishment of a respected advisory council which includes the following: 2. To prepare a set of recommendations regard- - recognised scientists in the field of ing priority actions to be implemented, so as to transplantation research, selected on the foster the integration of research activities on basis of ‘peer recognition’, themes common to cell and solid organ trans- - representatives of the key organisa- plantation. In order to achieve this overall objec- tions involved in transplantation research tive, the following sub-objectives are required: in Europe, i.e. the European Society for Or- • to form working groups on different gan Transplantation (ESOT) and the Euro- research themes, to facilitate discussion pean Group for Blood and Marrow Trans- and consultation in order to reach a con- plant Society (EBMT), sensus on existing ‘gaps’; - representatives of relevant EU-funded • to prepare detailed recommendations research projects in the field of transplan- on optimal measures to address the gaps; tation (e.g. RISET, Allostem, Alliance-O, • based on the output of the working DOPKA etc), groups, to prepare a consensual position - a representative of the European paper that will present in an articulated Agency for Evaluation of Medicinal Prod- manner the challenges, opportunities and ucts (EMEA), recommendations in terms of priority ac- - representatives of industries active in tions to integrate research activities on the field (both big pharmas and SMEs); cell and solid organ transplantation. • To identify common opportunities and challenges on different transplantation 3. Organisation of a public ‘dissemination’ event research themes, through discussions and in close collaboration with media experts and

230 New Therapies – Immunotherapy and Transplantation Immunotherapy and Transplantation

patients’ representatives. This event would have Coordinator the following two sub-objectives: • project coordinators and participants Michel Goldman would present concrete results of past Université Libre de Bruxelles and current research efforts — supported Institute for Medical Immunology namely by the EU Commission — in the Rue Adrienne Bolland, 8 field of cell and organ transplantation; B-6041 Charleroi (Gosselies), Belgium • to present, discuss and debate the out- E-mail: [email protected] put of TRIE with stakeholders. Partners

Kathryn Wood University of Oxford Nuffield Department of Surgery - Immunology Oxford, UK

Alejandro Madrigal The Antony Nolan Trust London, UK

Blanca Miranda Organización Nacional de Trasplantes Madrid, Spain

Siobhàn McQuaid Abu International Project Management Ltd Dublin, Ireland

New Therapies – Immunotherapy and Transplantation 231 NEW THERAPIES

232 New Therapies – Index of Projects NEW THERAPIES INDEX OF PROJECTS

AlloStem 155 STEMDIAGNOSTICS 217 ANGIOSKIN 144 STEM-HD 72 AutoCure 204 STEMS 64 BacAbS 189 STEMSTROKE 61 BACULOGENES 102 STROKEMAP 67 BetaCellTherapy 47 SyntheGeneDelivery 138 BMC 202 THERADPOX 107 CELLAID 214 THERAPEUSKIN 44 CLINIGENE 85 THERAVAC 169 CLINT 228 TherCord 30 COMPUVAC 181 THOVLEN 104 CONSERT 92 TRIE 230 CRYSTAL 80 Ulcer Therapy 37 DC-THERA 159 XENOME 224 DC-VACC 165 ZNIP 115 DENTRITOPHAGES 173 EPISTEM 32 EPI-VECTOR 129 EuroSTEC 52 EuroStemCell 17 Genomes To Vaccines 177 GENOSTEM 22 GIANT 98 HEPACIVAC 186 Improved Precision 121 IndustryVectorTrain 150 INNOCHEM 209 INTHER 124 InVivoVectorTrain 147 Magselectofection 135 MimoVax 193 MOLEDA 141 Myoamp 77 NEUROscreen 75 OsteoCord 27 Pharma-Planta 196 PolExGene 133 RESCUE 69 RIGHT 110 RISET 221 SAGE 200 SC&CR 57 Skintherapy 41 SNIPER 118

New Therapies – Index of Projects 233 NEW THERAPIES INDEX OF COORDINATORS NETWORK OF EXCELLENCE

Austyn, Jonathan M (DC-THERA) 163 Cohen-Haguenauer, Odile (CLINIGENE) 89

INTEGRATED PROJECTS

Cortese, Riccardo (HEPACIVAC) 188 Cozzi, Emanuele (XENOME) 226 Feitz, Wouter (EUROSTEC) 55 Fischer, Rainer (Pharma-Planta) 196 Goldman, Michel (RISET) 222 Jorgensen, Christian (GENOSTEM) 25 Klareskog, Lars (AutoCure) 207 Klatzmann, David (COMPUVAC) 185 Ma, Julian (Pharma-Planta, scientific coordinator) 198 Madrigal, Alejandro (AlloStem) 157 Maitland, Norman J. (GIANT) 101 Meyer, Thomas F. (RIGHT) 113 Mantovani, Alberto (INNOCHEM) 213 Pipeleers, Daniel (BetaCellTherapy) 50 Schenk-Braat, Ellen (GIANT, scientific coordinator) 101 Smith, Austin (EuroStemCell) 21 Vandenabeele, Peter (EPISTEM) 35 Wagemaker, Gerard (CONSERT) 96

SPECIFIC SUPPORT ACTION

Apperley, Jane (CLINT) 229 Goldman, Michel (TRIE) 231 Mezzina, Mauro (InVivoVectorTrain) 149 (IndustryVectorTrain) 151 Radbruch Andreas (CELLAID) 216

234 New Therapies – Index of Coordinators NEW THERAPIES INDEX OF COORDINATORS SPECIFIC TARGETED RESEARCH

Allsopp, Tim (NEUROscreen) 75 Bartholeyns, Jacques (DENDRITOPHAGES) 176 Bigot, Yves (SyntheGeneDelivery) 140 Buus, Søren (Genomes To Vaccines) 180 Capogrossi, Maurizio C. (SC&CR) 60 Daura, Xavier (BacAbS) 192 Dickinson, Anne (STEMDIAGNOSTICS) 219 Epstein, Alberto (THOVLEN) 106 Genever, Paul (OsteoCord) 29 Hescheler, Jürgen (CRYSTAL) 82 Hook, Lilian (NEUROscreen, scientific coordinator) 75 Hovnanian, Alain (THERAPEUSKIN) 46 Izsvák, Zsuzsanna (INTHER) 128 Jackson, Dean A. (EpiVector) 132 Kadouche, Jean (BMC) 203 Kokaia, Zaal (StemStroke) 63 Krauss, Stefan (SNIPER) 120 Krauss, Stefan (ZNIP) 117 Leclerc, Claude (THERAVAC) 172 Lusky, Monika (THERADPOX) 109 Martin, John (BACULOGENES) 103 Mattner, Frank (MimoVax) 195 Meneguzzi, Guerrino (Skintherapy) 43 Mir, Lluis M. (ANGIOSKIN) 145 Mouly, Vincent (myoamp, scientific coordinator) 79 Onteniente, Brigitte (STEMS) 66 Peschanski, Marc (STEM-HD) 74 Plank, Christian (Magselectofection) 137 Privat, Alain (RESCUE) 71 Rosenecker, Joseph (Improved Precision) 123 Schacht, Etienne (PolExGene) 134 Scherman, Daniel (MOLEDA) 143 Schillberg, Stefan (SAGE) 201 Verfaillie, Catherine (STROKEMAP) 68 Zambruno, Giovanna (Ulcer Therapy) 40

New Therapies – Index of Coordinators 235 236 New Therapies Directorate F – Health Unit F5 – Health Biotechnology

Head of Unit Arnd HOEVELER ([email protected]) Secretary: Andrea JAEGER ([email protected])

New Therapies Charles KESSLER ([email protected]) Fatiha SADALLAH ([email protected]) Juergen SAUTTER([email protected]) Secretary: Caroline RICHMOND ([email protected]) KI-NA-22841-EN-C

New Therapies Modern biomedical research is opening up many novel approaches to therapy. These include gene and cell therapy, immunotherapy, tissue engineering and regenerative medicine. These approaches offer hope for therapy of diseases which are currently untreatable, where life is at stake, and for regenerating diseased, damaged or defective tissues and organs. This is a very active research area in Europe, involving scientists, clinicians and industry, as well as patient organisations, ethical committees and regulatory authorities. Research in new therapies formed part of the EU’s Sixth Framework Programme (2002- 2006) and the purpose of this catalogue is to demonstrate the activities initiated over the duration of the programme and the initial results obtained.