Review of the Environmental Risks from Marketing GM Veterinary and Human Medicines

Review of GMOs in medicines – final report

Review of the environmental risks from marketing GM veterinary and human medicines

Final report

August 2008

Review of GMOs in medicines – final report

EXECUTIVE SUMMARY

Objectives and scope of this study

1.1 The purpose of this report is to review current and future applications of live genetically modified organisms (GMOs) in veterinary and human medicines, and provide an assessment of the potential risks of such applications to the environment and wider public health. The application of genetic modification (GM) technology in medicine is very diverse and increasing. The use of this technology encompases the growth of GM plants and microorganisms to produce compounds for use as medicines, as well as the use of live GM microorganisms as part of the medical treatments themselves. This report reviews this latter area of application, namely the use of live GMOs in medicines.

Applications of live GMOs in medicines

1.2 Whilst the use of live GMOs in medicines is diverse, the applications have been categorised into four areas: gene therapy, vaccination, direct action, and drug delivery and probiotic-type treatments. However there is considerable overlap between these areas, with GMOs designed to offer roles in more than one area; these areas are defined below:

♦ Gene therapy – the transfer of genetic material into a cell nucleus, tissue, or whole organ, with the goal of curing a disease or at least improving the clinical status of a patient. Both GM bacteria and viruses have been developed as gene therapy vectors to deliver the genetic material for the treatment of cancers; cardiovascular, monogenic, infectious, neurological and ocular diseases; and deafness. GMOs offer new treatments on their own, or as complimentary therapies for existing conventional (non-GM) treatments such as chemotherapy or radiotherapy. ♦ Vaccination – the process of administering weakened or dead pathogens to a healthy person or animal, with the intent of conferring immunity against a targeted form of a related disease agent. Genetic modification is applied in this area in the attenuation of pathogens, and also in the development of new recombinant vaccines where an unrelated organism (typically non-pathogenic) is

Review of GMOs in medicines – final report

modified to express the relevant antigens to induce immunity against a particular pathogen. ♦ Direct action – the term used in this report to describe treatments where the therapeutic effect is conferred through the replication and growth of the GMO. Whilst the GMOs used may also be modified to provide a gene therapy or drug delivery role, direct action refers essentially to the effects caused by the presence of the GMO at the target area. Examples include GM oncolytic viruses and anaerobic bacteria used to target cancer. The potential application of GM bacteriophages to target pathogenic bacteria has also been proposed. ♦ Drug delivery and probiotic-type treatments – the use of GM bacteria as drug delivery vectors to mucosal surfaces, especially the gastrointestinal (GI) and the female urogenital tracts. Whilst GMOs are currently not present in commercially available probiotic treatments, there are a number of developments reported involving the genetic modification of probiotic strains to target enteric pathogens and toxins.

1.3 Applications from all four areas have been evaluated in clinical trials (for use as human medicines) or field trials (for use in veterinary treatments), with some applications in later stage (Phase III) trials or commercial use.

1.4 The development and application of live GMOs in medicine is broadly the same for human and veterinary applications. Consequently the uses in these two areas have been considered together within each of the four groups of application. Differences in the use of a particular application, for example gene therapy is relatively uncommon in veterinary medicine, are a consequence of difference in demand because of cost or ethical considerations. There is little technical basis for these differences.

1.5 Most of the GMOs identified are either viruses or bacteria, although the genetic modification of the parasites Plasmodium sp. and Leishmania sp. (causative agents of malaria and leishmaniasis respectively) has been reported as part of the development of live attenuated vaccines against these two diseases.

Potential risks posed to the environment and human health

1.6 The use of live GMOs in medicines poses a number of potential risks to the environment and human health. These risks are a consequence of the realisation of various hazards such as pathogenicity and the production of non-target effects. In considering the risks posed it is important to recognise that the presence of a live GMO in a medicine does not always pose a risk to the environment or human health per se , and will depend on the characteristics of the GMOs present, the intended application of the medicine and the effectiveness of any management strategies that

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are applied. A hazard such as the adverse response of the recipient to a gene therapy vector (vector-induced immune response) is for example equally relevant to GM and non-GM vectors as it is linked to the presence of the vector, rather than the expression of the transgenes.

1.7 Risks may be realised to both the immediate recipient of the GMO (i.e. the human or animal patient), and/or to the wider environment and general public. Whilst the purpose of the risk assessment in this report is the evaluation of the effects to the general public and the wider environment, the two areas are linked and cannot be considered in isolation. No significant adverse effects have been reported from the use of live GMOs in human and veterinary medicines to the general public and wider environment. This is a consequence of careful selection of the microorganism to be modified (for example the use of non-pathogenic strains) or design of the GMO (to ensure replication deficiency for example). Whilst some of the GMOs described have been found to be released from the patient into the wider environment, no subsequent adverse effects have been reported or are assessed as likely to occur.

1.8 The potential for the GMO to cause adverse effects is not desirable both in terms of compliance with the regulatory framework, and from a functional perspective as the effects will impair the action of the GMO. The potential for such effects is therefore expected to be avoided during the design and development of the GMO. All such GM-based medicines should therefore pose negligible risk to the environment and human health. Where particularly significant adverse effects from treatments involving GMOs have occurred, such as the onset of T cell leukaemia and a very small number of deaths, these have been to patients receiving the GMO and not to the general public or animals.

1.9 The level of risk posed by a GMO is affected by any containment or management strategies that are in place. With respect to GM viruses the most effective of these is ensuring that the virus is unable to replicate. This means that following administration to the patient, no further release and spread of the GM virus can occur. In this situation the risk to the wider environment and public health is restricted to a release of the GM virus pre-administration or exposure to contaminated materials such as needles or swabs.

1.10 In summary, the review of the uses of live GMOs in medicines has identified many applications in which GM technology has provided the means to develop treatments that would not be possible through conventional (non-GM) processes. These have highlighted the potential advantages offered by this technology and the role it can play in both the development of new medicines and to complement existing treatments. Whilst subject to a strong regulatory framework, genetic modification may also offer strategies to reduce the potential risks associated with the development and use of conventional medicines. A key feature of GM technology as

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the tool to alter a microorganism for use in medicines, compared to modifications achieved through non-GM processes, is that the resulting changes are more clearly defined and characterised. Whilst the ability of genetic modification to make very specific changes should increase regulatory confidence in the effectiveness and stability of the modification, unwanted non-target effects may still occur. The likelihood of such effects occurring can be minimised through correct selection and design of the GMO.

Recommendation for further work

1.11 The one area identified as requiring further investigation is the effectiveness of auxotrophic modifications as a strategy for the biological containment of GM bacteria. These modifications are used in a number of the GM bacteria to limit or prevent the survival of the GM bacteria in the environment. Whilst laboratory studies show that auxotrophic modification is effective in controlling survival, quantitative information on survival of such GMOs in the environment is much more limited. This may be significant from a risk assessment perspective given the recent number of studies investigating the genetic modification of probiotic bacteria for use as treatments against enteric pathogens and diseases. Some auxotrophic modifications likely to be more suitable than others.

Review of GMOs in medicines – final report

EXECUTIVE SUMMARY I

1. INTRODUCTION 1

Content of the Report 1

2. CHAPTER 2 – REVIEW OF CURRENT AND POTENTIAL FUTURE APPLICATIONS 2-3

2.1 Overview of the areas of application 2-4 Gene Therapy 2-4 Gene therapy vectors 2-5 Retrovirus (includes lentivirus and foamy virus) 2-6 Adenovirus 2-8 Adeno-associated virus 2-10 Herpes Simplex virus 2-11 Vaccinia Virus 2-12 Baculovirus 2-12 Bacterial vectors 2-13 Vaccination 2-13 Direct Action 2-15 Drug delivery and Probiotic-type treatments 2-15 2.2 Review of current and potential future applications in medicine – Gene therapy 2-16 Gene therapy treatments for cancer 2-16 Restoration of tumour suppressor gene function 2-17 Inhibition of oncogene function 2-18 Gene-directed enzyme prodrug therapy 2-19 Targeted expression of cytotoxic/pro-apoptotic genes 2-22 Immunogene therapy 2-22 Anti-angiogenic gene therapy 2-24 Gene therapy treatments for cardiovascular diseases 2-25 Therapeutic angiogenesis 2-26 Restenosis following balloon angioplasty 2-27 Gene therapy for the treatment of monogenic diseases 2-27 Gene therapy for the treatment of infectious diseases 2-28 Gene therapy for the treatment of neurological diseases 2-29 Treatment of neurotrophic insults 2-30 Treatment of psychiatric diseases 2-31 Gene therapy for the treatment of ocular diseases 2-31 Outer retina / retinal pigment epithelium 2-32 Retinal ganglion cells 2-32 Retinoblastoma 2-33 Optic nerve 2-33 Retinal and choroidal vasculature 2-33 Lens 2-34

Review of GMOs in medicines – final report

Conjunctival and corneal epithelia 2-34 Gene therapy for the treatment of deafness 2-34 Gene therapy treatments in clinical trials 2-34 Current limitations to the use of gene therapy 2-37 Future developments in gene therapy 2-38 2.3 Review of current and potential future applications in medicine – Vaccination 2-40 Attenuated vaccines 2-41 Attenuated virus-based vaccines 2-41 Attenuated bacteria-based vaccines 2-42 Attenuated parasite-based vaccines 2-45 Recombinant vaccines 2-48 Recombinant virus-based vaccines 2-49 Recombinant virus vaccines for immunocontraception 2-55 Recombinant bacterial vaccines 2-56 Recombinant bacterial spores as vaccine vectors 2-58 Recombinant vaccines for the treatment of parasite mediated disease 2-59 Summary of GM-based vaccines 2-60 Approved GM-based vaccines 2-60 GM-based vaccines in research trials 2-62 Proposed GM-based vaccines 2-63 2.4 Review of current and potential future applications in medicine – Direct action 2-64 Direct action applications for the treatment of cancer 2-64 Oncolytic viruses 2-64 Bacteria-based applications 2-67 Direct action applications for the treatment of dental caries 2-68 Direct action applications using bacteriophage 2-69 Direct action applications in clinical trials 2-70 2.5 Review of current and potential future applications in medicine – Drug delivery and probiotic-type treatments 2-72 Gastrointestinal tract treatments 2-72 Treatment of inflammatory bowel disease 2-73 Application of lactic acid bacteria expressing IL-10 2-74 Application of lactic acid bacteria expressing trefoil factors 2-75 Female urogenital tract treatments 2-76 Commercialisation of GM bacteria as drug delivery vectors 2-77 Modification of probiotics 2-77 Designer probiotics 2-78

3. CHAPTER 3 – ASSESSMENT OF THE POTENTIAL RISKS TO THE ENVIRONMENT AND WIDER PUBLIC HEALTH 3-80 Risk assessment framework for GMOs 3-80 Generic hazards associated with GMOs in medicines 3-81 General Comment 3-83 3.1 Assessment of the potential risks associated with GM viruses in medicines 3-86

Review of GMOs in medicines – final report

Pathogenicity (GM viruses) 3-86 Pathogenicity of the unmodified (wildtype) strain 3-86 Application or environment in which the GMO is used 3-87 Presence of attenuations 3-87 Production of biologically active and/or toxic products (GM viruses) 3-89 Production of non-target effects (GM viruses) 3-89 Untargeted delivery and expression of the transgene(s) by the GM virus 3-90 Vector-induced immune response (GM viruses) 3-91 Insertional mutagenesis / oncogenes activation (GM viruses) 3-94 Genetic stability (GM viruses) 3-96 Changes in cell, tissue and host tropism (GM viruses) 3-96 Gene transfer (GM viruses) 3-97 Horizontal gene transfer (GM viruses) 3-97 Vertical gene transfer (GM viruses) 3-98 Survival and dissemination of the GMO (GM viruses) 3-98 Shedding (GM viruses) 3-100 Dissemination as a consequence of mechanism of delivery (GM viruses) 3-101 Arthropod transmission (GM viruses) 3-102 Disposal of contaminated materials (GM viruses) 3-102 3.2 Assessment of the potential risks associated with GM bacteria in medicines 3-104 Pathogenicity (GM bacteria) 3-104 Production of biologically active and/or toxic products (GM bacteria) 3-107 Production of non-target effects (GM bacteria) 3-108 Vector-induced immune response (GM bacteria) 3-109 Genetic stability (GM bacteria) 3-109 Plasmid stability (GM bacteria) 3-110 Plasmid conjugation (GM bacteria) 3-111 Horizontal gene transfer (GM bacteria) 3-112 Survival and dissemination of the GMO (GM bacteria) 3-113 Shedding – the release of microorganisms into the environment (GM bacteria) 3-115 Dissemination other than shedding (GM bacteria) 3-117 Indirect effects on survival and dissemination (GM bacteria) 3-117 Replication competence (GM bacteria) 3-118 Auxotrophic attenuation (GM bacteria) 3-118 Self-destruction as a containment system (GM bacteria) 3-120 3.3 Assessment of the potential risks associated with GM Parasites in medicines 3-121 Conclusion 3-122

4. CHAPTER 4 – RECOMMENDATIONS FOR FUTURE RESEARCH 4-124

5. APPENDIX 1 – GENE THERAPY APPLICATIONS IN CLINICAL TRIALS 5-126

6. REFERENCES 6-132

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1. INTRODUCTION

1.2 GMOs are defined as organisms in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination [7]. Their use in medicines is a rapidly developing area of application of genetic modification technology, with a number of products in clinical trials [1][2]. Defra’s responsibilities under the Deliberate Release Directive (2001/18/EC) mean that they require a good understanding of the many developments in this area and the potential risks posed to the environment and wider public health. This report has therefore been written to provide Defra with an information resource to support their regulatory responsibilities, and also to provide a document that can be used as a basis to identify gaps in the current understanding of risk assessment and risk management of these applications.

1.3 The review has been compiled through a collaboration between Dr Colin Cartwright at Atkins, Dr Linda Scobie of the Department of Biological and Biomedical Sciences (Glasgow Caledonian University), Dr Stephen Dunham of the Faculty of Veterinary Medicine (University of Glasgow), Professor Erik Remaut of the Department for Molecular Biomedical Research (Ghent University, Belgium), and Dr Gabi Dachs of the Angiogenesis Research Group (University of Otago, Christchurch, New Zealand).

CONTENT OF THE REPORT

1.4 The report is comprised of three chapters:

♦ Chapter 2 – reviewing the current and potential future applications of live GMOs in medicines and an assessment of the likelihood of the commercialisation of each product. Medicines that use products derived from GMOs, but do not contain live GMOs, for example plants that have been genetically modified to express cholera toxin for use as oral vaccines have not addressed in this report. A review of these products is available in a previous Defra publication ‘The effects of compositional traits on the survivability and persistence of GM crops’ (2004, Defra reference EPG 1/5/197).

Review of GMOs in medicines – final report

♦ Chapter 3 – assessing the potential risks of each of the applications or products identified in Chapter 2 to the environment and wider public health.

♦ Chapter 4 - recommendations for future research into the use or potential risks of GMOs in medicines, to inform future risk assessment and risk management decisions.

Review of GMOs in medicines – final report

2. CHAPTER 2 – REVIEW OF CURRENT AND POTENTIAL FUTURE APPLICATIONS

2.1 The aim of Chapter 2 is to review the current and potential future applications of genetic modification technologies in human and veterinary medicines worldwide. The review has been compiled in four sections, each addressing a particular area of medicines in which GMOs are used, or are in development. The areas of application are gene therapy, vaccination, direct action and drug delivery and probiotic treatments (further information and definitions are provided in the following sections).

2.2 The review recognises that whilst these applications have been described in separate sections, this does not mean they are used in isolation. There is considerable overlap between the applications, both in terms of how they are used and how they are defined. As described in this report, treatments for particular diseases often involve approaches from more than one application area, and the GMOs used may have multiple roles, for example as a drug delivery vector and a direct action agent. Where particular combinations are used this will be highlighted in the report. Because the science and the general use of GMOs in medicine are applicable to both human and veterinary medicines then these have been considered together within the review of use of GMOs in each area of application. Differences in the use of particular application in veterinary or human medicines are in most cases a consequence of differences in demand because of cost or ethical considerations. There is little technical basis for the greater use of vaccination than gene therapy in veterinary medicine for example. There are differences in the potential risk to the environment and human health between use of a GMO in a veterinary versus a human medicine. These differences are considered in Chapter 3.

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2.1 OVERVIEW OF THE AREAS OF APPLICATION

Gene Therapy Gene therapy vectors Retrovirus (includes lentivirus and foamy virus) Adenovirus Adeno-associated virus Herpes Simplex virus Vaccinia Virus Baculovirus Vaccination Direct Action Drug delivery and Probiotic-type treatments

Gene Therapy

2.3 Gene therapy is defined broadly as the transfer of genetic material into a cell nucleus, tissue, or whole organ, with the goal of curing a disease or at least improving the clinical status of a patient 1 [3]. This may be achieved in vitro where the cells are modified outside the patient’s body and then transplanted back into the patient, or in vivo where the alteration is achieved within the patient.

2.4 Both in vivo and in vitro (also referred to as ex vivo in this context) approaches may involve the use of a live virus or bacterium to deliver the gene(s) of interest (the transgene(s)) into the target cell(s) [94]. The alteration of the vector (a virus or bacterium) for this purpose means that the vector is likely to be classified as a live genetically modified organism (GMO). Both in vivo and some ex vivo approaches involving a GMO are of relevance to this report. The ex vivo applications that are not of relevance are those where the GMO delivery vector is not present in the patient’s cells when they are transplanted back into the patient. Other ex vivo applications where the GMO is present in the transplanted cells are of relevance and may pose similar risks to in vivo applications. An ex vivo approach is generally limited to a few cell types, such as blood cells that are easy to remove and then return to the patient [299]. The use of viruses as the delivery vector utilises their natural function to insert their own genetic material into the target cell. Some viral vectors, namely retrovirus, lentivirus, adeno-associated virus and non lytic herpesvirus (such as Epstein Barr virus) are able to insert their genetic material into the target cell’s chromosome (chromosomal integration). Adenovirus and herpes simplex virus do not [78].

2.5 Bacterial vectors do not have the same natural abilities as viral vectors in terms of the insertion of their genetic material, but offer other advantages, particularly the specific targeting of solid tumours. Bacterial vectors can home into the target site and either stay in the extracellular space, or enter target cells. Intracellular bacterial vectors such as Salmonella sp. can be phagocytosed and their genetic material,

1 The patient is the direct recipient of the GMO, i.e. the person or animal to whom the medicine containing the GMO is administered. 2-4

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including plasmids, becomes incorporated into target cell nucleus, resulting in gene transfer. A common requirement of all vectors (bacterial and viral) is that they pose limited or no toxicity to the patient, outside their vector function.

2.6 Numerous human clinical trials have been conducted using gene therapy in the treatment of monogenic deficiency diseases (caused by a deficiency in one gene) and cancers. Edelstein (2004)[4] for example reported a total of 900 clinical trials involving gene therapy underway or completed worldwide, although not all of these involved a GMO. However, gene therapy applications using GM viruses in particular represent a significant area of application of genetic modification technology in both human [5] and veterinary medicine [6][8]. The use of viral vectors offers the advantage of a higher transfection efficiency compared with non-viral gene delivery systems [14]. The use of live GMOs in veterinary medicine in a gene therapy application is limited. This is a consequence of the relatively high cost of gene therapy (compared to other treatments), rather than any limitations to the use of gene therapy in animals. The relative cost involved means that gene therapy is most likely to be applied to companion animals such as dogs, or higher value livestock such as thoroughbred horses and pedigree bulls or rams.

Gene therapy vectors 2.7 The viruses used as vectors in clinical trials include retrovirus, adenovirus, adeno- associated virus (AAV), herpes simplex virus (HSV), vaccinia virus, and poxviruses (including canarypox), with the first two the most commonly used [14][81]. Preclinical tests have been conducted to characterise the gene delivery properties of lentivirus based on HIV-1, feline immunodeficiency virus (FIV) and equine infectious anaemia virus (EIAV); as well as human cytomegalovirus (CMV); Epstein-Barr virus; alphaviruses; negative-stranded RNA viruses such as influenza virus, herpesvirus saimiri and foamy virus; as well as hybrid adenovirus/retrovirus and hybrid alphavirus/retrovirus [81]. There has also been increasing interest in the use of baculoviruses as gene delivery vectors for mammalian cells, although none have reached clinical trials stage to date (2007) [244]. Baculoviruses have also been proposed as vaccine vectors [277].

2.8 Each of these has distinct characteristics and therefore may offer particular advantages and disadvantages as a viral vector for a particular gene therapy application. No single vector is optimal for all applications [106]. The diverse nature of the applications currently under investigation (as illustrated in this chapter) means there is also a demand for vectors that are optimised for that application (to target a specific cell for example), hence the large number of different vector systems used. Hybrid vectors, for example herpes simplex virus / adenovirus, herpes simplex / adeno-associated virus, and the triple herpes simplex / Epstein Barr virus / retrovirus (HER) hybrid have been constructed to provide the advantages of two or three viruses, and thereby allow for an even greater variety of potential vectors [102][100]. The HER hybrid for example allows the advantages of the retrovirus as a vector to be utilised in non-dividing cells [102].

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2.9 In order to improve the range of recipient cells to which GM viruses can be used as vectors, further modification of the virus has been conducted to alter the proteins on the surface of the virus so that they are able to interact favourably with a wider range of recipient cell surface proteins (and thereby infect a wider range of recipient cells). The host range can be increased, restricted or redirected by changing surface receptors. Envelope proteins of the host virus have been replaced by envelope proteins from other viruses or by chimeric proteins. Viruses whose envelope proteins have been altered in this way are referred to as pseudotyped viruses. Of particular note to gene therapy are lentiviruses pseudotyped with the G-protein envelope protein from Vesicular Stomatitis virus. Such GM viral vectors, referred to as VSV-G pseudotyped lentiviruses, infect an almost universal set of cells.

2.10 The characteristics, advantages and disadvantages of the more commonly used viruses are described below [81][14][55][78][82].

Retrovirus (includes lentivirus and foamy virus) 2.11 The retroviruses used as delivery vectors are based mainly on the Moloney murine leukaemia virus (MoMuLV), with the newer ones based on HIV-1, FIV or EIAV. Biosafety concerns have been raised regarding the use of HIV-1 as a gene therapy vector (in relation to conversion of the vector to replication competent HIV-1), and over the requirement to produce large quantities of the HIV-1 vector in category 3 facilities [81]. These concerns were described as less significant by O’Connor (2006) and GM HIV-1 (VRX496 third generation vector) has been used as a vector ex vivo in human cells as part of a Phase I clinical trial against HIV-1 infection [96]. Replacing the native HIV-1 viral coat protein with a heterologous envelope protein such as VSV-G, and removing the tat gene (which is essential for replication) reduces the potential for the generation of replication competent virus [284]. The vector used in the Phase I trial (VRX496) was a completely gutted lentivirus that does not code for any viral proteins. Data from in vitro studies has indicated that HIV vectors such as VRX496 could reduce viral loads in HIV-infected individuals when used to stimulate anti-viral immunity, potentially delaying the onset of AIDS [96].

2.12 The development of FIV-based vectors, to which the biosafety issues associated with HIV-1 do not apply, may render the use of HIV-1 vectors obsolete. To date (2007), no in vivo clinical trials have been conducted using FIV as the viral vector 2 [214]. Foamyviruses (and spumaviruses) in contrast to HIV offer the advantage of not being linked to any specific pathogenic state [237].

♦ Retrovirus Vector Characteristics o Stable expression of the transgene due to integration of viral genome into the cell chromosome. This is of particular benefit for the treatment of diseases that require long-term, stable expression of the transgene, such as inherited or acquired monogenic diseases, neurological disorders and

2 No reference to such clinical trials listed in PubMed or Gene Medicine Clinical Trials. 2-6

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cardiovascular diseases. The therapeutic effect is potentially sustained life-long. o No toxic effect on infected cells. o Total insertional capacity of a maximum 10kb (transgene + transgene vector). o Transduction of dividing cells only. Means the vector will target dividing tumour cells. This characteristic is of particularly relevance to brain tumours, where the tumour cells are the only dividing cells in the brain. Lentiviral vectors can infect non-dividing cells and are an alternative to MoMuLV based vectors when this is required [234]. o No induction of an innate immune response to the vector. ♦ Disadvantages/Limitations of Retrovirus as a Vector o Insertion of the viral genome into the chromosome of the recipient cell. Whilst this confers a stable chromosomal integration it may result in insertional mutagenesis 3. Although integration into the genome does not necessarily result in the generation of a neoplasia4, such adverse effects have been reported, with children in a trial for X-linked severe combined immunodeficiency (SCID) in France subsequently developing leukaemia [218] 5. It may also make the cell more susceptible to undergo neoplastic transformation at a later date. The long-term effects that may occur following insertional mutagenesis will be most significant with younger patients and/or those surviving the disease 6. The adverse issues caused by random insertion may be reduced through the use of tissue- or cell- specific vectors. o Potential for the formation of replication competent virus through homologous recombination with endogenous virus. The likelihood of this occurring is minimised through correct design of the vector and in the choice of packaging cell line used to produce the virus. o Possible recombination with human endogenous retroviruses (HERVs). Approximately 1% of the human genome is estimated to comprise of HERV-related sequences (Larsson et al. 1998; cited by [81]) although most of these are defective genes. Studies of possible recombination between HERV and porcine endogenous retrovirus (PERV) have reported a low potential for recombination between the two retroviruses

3 Insertional mutagenesis is the mutagenesis of DNA through the insertion of new genetic material. This can lead to the development of tumours where cellular DNA (containing the viral insert) undergoes repeated replication. Mammalian studies have reported a ratio of ‘mutations versus insertional events’ of between 10 -9 to 10 -3 [81]. 4 Neoplasia is an abnormal proliferation of cells in a tissue or organ. 5 Self-inactivating (SIN) gammaretroviral vectors have been reported to offer reduced potential for insertional mutagenesis in cellular and in vivo models of SCID-X1 [105]. SIN viruses have promoter elements which are inactivated upon integration so should not lead to proviral activation of cellular oncogenes. 6 A longer survival time following treatment increases the likelihood of sufficient random mutations arising leading to neoplastic transformation. 2-7

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and the subsequent generation of novel viruses [294]. Quantitative analysis found that less than 1 in 10 4-10 7 PERV particles might contain HERV sequences. Cross-packaging levels between murine leukaemia virus (MLV) and PERV were higher at one MLV vector transcript occurring in every 10 4 PERV particles. o Vector particles are rapidly degraded by complement7, although this may be avoided by expressing hybrid amphotropic envelope on the viral membrane. o Transduction of dividing cells only, does mean retroviruses cannot be used as a vector (in vivo ) in neurons for the treatment of neurological disorders. Lentiviral vectors (HIV-1 and FIV) however, can infect non- dividing cells, which means they can be used for gene delivery to the central nervous system and liver, unlike MoMuLV based retroviral vectors. This is also listed as a disadvantage as it limits the application of retroviruses against neurological diseases (although in some applications that may not be disadvantageous). o Reduced range of cell tropism compared to adenovirus and AAV. Cell tropism can be widened or modified through pseudotyping with the G glycoprotein of Vesicular Stomatitis virus (VSV) or tissue specific receptors. o Only moderate transfer efficiency ( in vivo ), although lentiviral vectors are slightly more efficient. o The lowest level of expression of the viral vectors, although this may be altered, for example with different promoters. Lentiviral vectors have slightly higher expression levels, with an equivalent level of expression to AAV and HSV, but lower than adenovirus.

Adenovirus 2.13 Adenovirus is a double stranded DNA, non-enveloped virus. Improvements in adenoviral vector design have increased their use over other viral vectors in gene therapy trials. Most adenoviruses in use as gene therapy vectors are unable to replicate as a consequence of a deletion of one or more of the E genes 8. The E genes can also be modified to generate replication competent 9 and conditionally

7 Groups of plasma enzymes and regulatory proteins that function in innate immunity and that are activated in a cascading fashion to promote cell lysis. 8 The adenovirus genome comprises early (E) and late (L) genes which are expressed, respectively, before and after replication of the viral genome. E1 gene products are involved in the control of viral gene transcription; the E2 gene codes for proteins involved in viral replication; the E3 genes code for proteins that interfere with the host’s immune response against the viral infection; and the E4 genes are involved in the transition from early to late gene expression, the shutoff of host cell gene expression, viral replication and assembly of the virion. Deletion of any combination of the E1, E1+E3, E2, E4, E2+E4 or E1+E2+E4 genes renders the adenovirus replication deficient. 9 As <40% of the E3 gene is essential for viral replication, then the transgenes can be inserted within the virus’s E3 gene without affecting replication ability. The adenovirus is described as replication competent. 2-8

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replication competent 10 adenoviral vectors [174]. These have also been used more recently. Adenoviruses with an E1 or E1+E3 deletion are routinely described as first generation adenovirus vectors; those with E1 or E1+E3, and E2 or E2+E4 deletion as second generation vectors; whilst the helper dependent (HD) or gutless vectors are described as third generation [174]. Second generation adenovirus vectors are reported to induce a lower innate immune response than first generation vectors [175].

♦ Adenovirus Vector Characteristics o Many human and animal adenoviruses are non-pathogenic for their natural hosts. o Can be produced at very high titres (10 10 pfu/ml, compared to 10 7 pfu/ml for retrovirus vectors). o Transiently high levels of gene expression. This may be a limitation for the delivery of genes which require continuous expression to achieve their therapeutic effect (for example some anti-angiogenic genes). o Able to transfect virtually all cell types (although they have a natural tropism towards liver cells via the Coxsackie and adenovirus receptor (CAR). Because adenovirus binds to target cells through epitopes in its fibre and penton bases, modification of these can alter cell tropism. o Can infect non-dividing cells. o An absence of germline transmission. o Good transfection efficiency. Melo (2004)[78] described adenovirus vectors having the highest transfer efficiency ( in vivo ) of the viral vectors. o Adenovirus serotypes 2 and 5 have been used as vectors against monogenic diseases. Adenovirus serotype 5 is preferentially localised in the liver, making this serotype potentially useful for liver-directed gene therapy applications 11 . o Transgene capacity of 35kb. ♦ Disadvantages/Limitation of Adenovirus as a Vector o Highly immunogenic in the recipient, causing adverse recipient immune responses (inflammatory and toxic reactions), and the depletion of transduced cells. This is partly a consequence of having been exposed to wildtype adenovirus at some point (or exposure to repeated doses of a therapeutic vector). Recipient responses can be minimised through deletion of E1, E2 and E4 genes to avoid expression of immunogenic viral proteins in transduced cells, and overexpression (with a strong constitutive promoter) of the E3-encoded 19kDa glycoprotein. This

10 Conditionally replication competent adenoviruses are those in which the E1A gene is under the control of a tissue or cancer antigen or tumour condition- specific promoter.

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glycoprotein inhibits transport of the major histocompatibility complex (MHC) class I molecules to the cell membrane, thereby impairing the cell’s antigen-presenting cell mechanism. Other approaches to minimise the recipient’s response are to optimise transgene expression to allow a reduction in viral titre administered, to deliver the vector directly to the target organ rather than into the blood stream, and to modify the adenovirus capsid proteins (in effect to alter the visibility of the adenovirus to the recipient’s innate immune system) [240][241]. o Recipient’s humoral immune responses may neutralise the vector particles during, or even before, the gene transfer process has occurred. o No integration into the recipient genome and therefore not suitable for long-term expression. Described as having the most transient expression of the commonly used viral vectors [81]. This is not an issue with cancer therapies. o Complicated vector genome. o An absence of germline transmission.

Adeno-associated virus 2.14 Adeno-associated virus (AAV) is a non-enveloped single-stranded DNA human virus. There are five human AAV serotypes, and AAV-based vectors are usually based on serotypes 1 or 2, although the serotype 1 or 2 may be pseudotyped with the capsid of another serotype that provides greater specificity for the target organ, for example serotype 8 capsid for liver, and serotype 5 capsid for lung epithelium. Significantly, AAV is not associated with any human disease.

♦ Adeno-associated virus Vector Characteristics o Able to transduce a wide variety of cells, including both dividing and non- dividing cells. AAV is of particular relevance as a vector for organs whose cells do not turnover rapidly, such as brain, liver, heart, pleura (lung) and retina. o Requires the assistance of an auxiliary virus (such as adenovirus or herpes simplex virus) for productive infection 12 . o Integrates into recipient cell genome. o Long term (life-long) expression. o Can be produced at high titres (10 10 pfu/ml). o The viral genome of AAVs used as a delivery vector contains no expressed genes, and therefore the issue of recipient immunogenic

11 Waddington et al. (2008) [304] reported that the adenovirus hexon is as important in mediating liver tropism. Modification of the hexon is likely to be undertaken to alter virus tropism, allowing the virus to be delivered intravascularly to solid tumours for example. 12 The autonomous parvoviruses which are the other main subgroup of parvoviruses along with AAVs do not require the assistance of an auxiliary virus, and do not integrate into the host cell genome [238]. 2-10

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responses is less significant compared to adenovirus (almost zero [241]). Some antibody response against the AAV capsid may occur. o Relatively low expression of the transgene(s). Whilst this may make AAV vectors suitable for the treatment of conditions/disease requiring a long- term effect, it makes this vector type less suitable against acute conditions. ♦ Disadvantages/Limitation of Adeno-associated virus as a Vector o Difficult to grow to high titres. o Risk of insertional mutagenesis due to integration into recipient cell genome. o Causes recipient immunological response, albeit less significant than adenovirus. o Relatively small insertional capacity of <4kb. A solution for this small size has been to deliver multiple transgenes in separate vectors [82].

Herpes Simplex virus 2.15 Herpes Simplex virus (HSV) is a double stranded DNA enveloped virus. There are two types of herpes simplex virus vector described in the literature; amplicon and recombinant [104]. Amplicons are plasmids modified to contain a HSV-1 origin of replication, an HSV-1 packaging site and a bacterial origin of replication. The amplicon vector does exhibit low vector titres and a high probability of recombination leading to the production of replication competent virus. No amplicon HSV vectors have been reported as being used in clinical trials [214]. The recombinant vectors are modified wildtype virus, described below.

♦ Herpes Simplex virus Vector Characteristics o Broad cell tropism, including central nervous system (CNS) cells. Herpes simplex virus is particularly suitable as a vector for CNS cells. o Can be grown to high titres (10 10 transducing units (TU) per ml) 13 . o Large insertional capacity (30kb), allowing for the insertion (and delivery) of multiple transgenes and heterologous promoters. o Long term expression is feasible (although not as long as with retroviral and AAV vectors), although the vector does not insert into the recipient cell genome. No issues therefore with insertional mutagenesis. o The ability to enter a state of latency in neurons. In latency the virus genome persists as a non-integrated concatemeric or circular molecule in the neuron’s nucleus. During latency no viral proteins are synthesised, and the neurons appear to function normally [102]. ♦ Disadvantages/Limitation of Herpes Simplex virus as a Vector

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o Difficult to manipulate due to complex life cycle. o Limited promoter selection, with just the cytomegalovirus (CMV) promoter available. This may limit the ability to target the expression of the herpes simplex virus delivered genes. o Risk of reversion to wild-type.

Vaccinia Virus 2.16 Vaccinia virus (a poxvirus) is a double-stranded DNA enveloped virus. It has a long history of therapeutic use, being used as the first widely applied vaccine in the eradication of smallpox. GM vaccinia virus has applications as a vector for tumour directed gene therapy, as well as a vaccine vector.

♦ Vaccinia virus Vector Characteristics o Broad cell tropism across a wide range of mammalian cells. o Large (nearly 200kb) genome allows for the delivery of large transgene sequences. o Replicates only in the cytoplasm of the recipient cell, outside the nucleus, although the transgenes enter the nucleus for transcription. ♦ Disadvantages/Limitation of Vaccinia virus as a Vector o Elicits a rigorous immune response, although this can be exploited to augment recipient immunity against tumour cells.

Baculovirus 2.17 Baculoviruses are double stranded DNA viruses of invertebrates and have been used extensively for the expression of recombinant proteins in insect cells [244]. Although they are unable to replicate in mammalian cells, they can be used as a gene delivery agent to mammals if the gene transferred by the baculovirus is under the control of a promoter that is active in mammalian cells [277]. Recent research has shown that the vectors can be used in vivo [279].

2.18 The use of baculoviruses as a vector for mammalian cells offers a number of advantages [244][279]:

o Unable to initiate a replicate cycle and produce infectious virus in mammalian cells. They are therefore biologically contained within the mammalian cells post-administration. o Easy to manipulate.

13 TU per ml is a measure of the ability of the virus to infect cells effectively and lead to target protein production. PFU per ml just measures viable virus particles. For an efficient vector the two measures are likely to be comparable. 2-12

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o Able to accommodate large insertions of DNA. o Able to confer expression in a wide variety of mammalian cell lines [297].

Bacterial vectors 2.19 Whilst bacteria have fewer applications as gene therapy vectors than viruses they have been used in human and veterinary treatments against cancers [128] (Table 2.2).

Vaccination

2.20 Vaccination is the process of administering weakened or dead pathogens to a healthy person or animal, with the intent of conferring immunity (humoral, mucosal or cell mediated) 14 against a related disease agent. Vaccination represents a key method for the long-term management of many human and animal diseases. Management of animal diseases through vaccination may often be more humane than other strategies such as eradication.

2.21 A number of studies have been reported in which GM viruses, bacteria and parasites have been used in vaccination, as attenuated or recombinant vaccines [8][9].

♦ Attenuated vaccines – involves the use of a less pathogenic (an attenuated) form of a pathogen to induce an immune response in the recipient. Genetic modification can be used to introduce specific attenuations into the target pathogen. (Microorganisms attenuated by genetic modification are often also described as recombinant attenuated vaccines). Whilst conferring strong immunity and therefore a reduced need for multiple inoculations, attenuated vaccines are limited in that (1) they cannot be used by immunocompromised individuals, (2) they are generally not recommended for use in pregnant humans or animals, (3) there are a limited number of pathogens that can be attenuated successfully, and (4) there is a risk of conversion back to the wildtype form (i.e. the pathogenic form) of the microorganism. (These issues are the basis for some of the potential risks posed by attenuated GM vaccines to the environment and human health). The recombinant vaccinia-based vaccine for rabies was developed in part because of concerns over the potential for the non-GM attenuated rabies virus vaccine to revert to virulence [199]. The live attenuated H5N1 influenza vaccine has been developed in response to a global requirement for a vaccine against the wildtype H5N1 avian influenza potential pandemic. Genetic modification provides the ability to produce such a vaccine rapidly on a large scale and low cost [263]. The vaccine is a modified version of

14 Humoral immunity is protective against extracellular infections and therefore against the pathogens that reside and replicate outside host cells in the alimentary, urogenital and respiratory tracts, blood and extracellular fluids. Humoral immunity involves the production of specific antibodies and the activation of the complement system. Mucosal immunity is important for the protection against infection that occurs at mucosal surfaces. Cell mediated immunity does not involve the production of antibodies, and is protective against intracellular pathogens such as viruses, certain bacteria and parasites residing in membrane bound vesicles (e.g. salmonella and mycobacteria) or in the cytoplasm of the host cell (e.g. listeria and most viruses). 2-13

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the non-pathogenic human vaccine strain PR8 with the haemagglutinin and neuraminidase genes (H and N) replaced by those from a Hong Kong avian strain H5N1. The H5 gene is further modified to reduce pathogenicity of the vaccine strain by removal of the polybasic cleavage site [251]. ♦ Recombinant vaccines – involve the use of a microorganism as the vector to deliver the antigen to the recipient and thereby induce immunity. The vector microorganism may be an existing vaccine strain (for example BCG 15 ), with the modification resulting in a vaccine that confers protection against more than one pathogen; or it may be a non-pathogenic organism. The use of recombinant- based vaccines based on non-pathogenic vectors avoids the issues associated with reversion to a pathogenic wildtype that may occur with attenuated strains. Recombinant vaccines allow for a vaccine strategy to be used when attenuation of the target pathogen is not possible, or when the necessary quantities of the target pathogen or attenuated vaccine cannot be produced easily or safely. Both attenuated and recombinant vaccines are capable of inducing humoral and cell mediated immunity.

2.22 In addition to these two vaccine types, vaccination programmes are also described as using inactivated (or killed) vaccines 16 , bacterial ghost (BG) vaccines 17 , virus-like particles (VLPs) 18 , DNA vaccines 19 and subunit vaccines 20 . However, as none of

15 Bacille Calmette Guerin (BCG) is the attenuated strain of Mycobacterium bovis . It has a long history of safe use as a vaccine in both humans and animals against tuberculosis. Genetic modification technology allows well characterised and safe vaccines to be modified for use against other pathogens, for example the BCG strain modified to express outer surface protein A (OspA) of Borrelia burgdorferi (causative agent of Lyme disease)[194]. 16 Inactivated vaccines – based on killed rather than live microorganism. Killed whole cell vaccines may be relatively less effective as they contain both immunogenic and non-immunogenic components. Immune responses against the non-immunogenic components are not relevant to the purpose of the vaccination and may consequently diminish the overall effect. The non-immunogenic components may also cause unwanted side effects. Inactivated vaccines are relatively inexpensive to produce. The majority of the viral vaccines licensed for use in fish (as of 2005) for example are inactivated vaccines (with a small number of recombinant protein-based vaccines)[147]. No live attenuated microbal (bacterial or viral) vaccines are currently licensed for fish [146]. 17 Bacterial ghosts are non-living cell envelope preparations from Gram-negative cells, developed for use as delivery vectors. They are devoid of cytoplasmic contents, although their cellular morphology and native antigenic structures are maintained [195]. 18 Virus-like particles (VLPs) are constructed particles, usually consisting of the virus structural proteins with or without a lipid bilayer [223]. They are very immunogenic and used as vaccine vectors. They differ from ‘gutless’ viral vectors in that they are constructed particles rather than ‘stripped down’ viruses. VLPs are incapable of replication as they possess no nucleic acid and are therefore viewed as outside the scope of this report. 19 DNA vaccines – involve the injection of naked DNA containing the relevant antigen genes to confer the immune response. DNA vaccines offer a number of theoretical advantages over other vaccine strategies. These include an absence of a live organism and the associated risks of adverse immune response and reversion to wildtype and latency; the ease of production and preparation of plasmid DNA; the expression of antigens in their native form, which leads to the efficient generation of both cytotoxic and helper T cells; the potential to reduce the number of doses of vaccine required to generate a protective immune response; and that the cells need not be the target cells that are normally infected by the infectious agent. The potential disadvantages of DNA vaccination include accidental introduction of the plasmid DNA into cells other than the intended cell types; generation of anti-DNA antibodies to the plasmid used for the vaccination; and random integration of the injected DNA into the target cells [85]. DNA vaccines are also reported to achieve lower levels of expression compared to whole cell vaccines [151]. 2-14

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these involve the delivery of live GMOs they are outside the scope of this report and have therefore not been addressed. The exception is where DNA vaccines are delivered using a GM bacterial vector, such as Shigella flexneri [161].

Direct Action

2.23 Direct action is the term used in this report to describe treatments where the therapeutic effect is conferred through the replication and growth of the GMO. Whilst the GMOs used may also be modified to provide a gene or drug delivery role, direct action refers essentially to the effects caused by the presence of the GMO at the target area.

2.24 Examples of microorganisms with potential direct action applications include the anaerobic bacteria which are able to germinate and grow in the hypoxic or anaerobic regions within solid tumours [54], and oncolytic viruses that preferentially replicate and kill cancer cells whilst leaving surrounding non-cancerous cells relatively intact [55]. Although many of the direct action applications developed to date have focused on the treatment of tumours using both GM viruses and bacteria [95][94][55], there are applications in other areas such as the treatment of dental caries [124] and the prevention of infections of the female urogenital tract [123] have also been reported.

Drug delivery and Probiotic-type treatments

2.25 This section of report reviews the application of GM bacteria as drug delivery vectors in the gastrointestinal (GI) tract. The bacteria used are either common residents of a healthy GI tract or used in food production. They are non-pathogenic. The application of GM bacteria in this way is characterised by the type of bacteria used, and the specific targeting of the intestinal mucosa with no invasion of the patient’s tissues and an absence of infection.

2.26 The probiotic-type treatments now widely available commercially as yoghurts and dry supplements are designed to confer a beneficial effect on the consumer by improving the intestinal microbial balance [97]. None of the commercial products currently available contain GM bacteria. However the similarities between the bacterial species used, the mode of delivery (ingestion) and target area (GI microflora and GI mucosa) between drug delivery applications and probiotic treatments means that it is appropriate to consider these applications together.

20 Subunit vaccines – are comprised of just the antigen itself. Examples include the feline leukaemia virus p45 subunit vaccine which is licensed, and the GM birch pollen derivatives used for the treatment of pollen allergy [145]. Subunit vaccines by definition do not involve a whole organism and are therefore potentially safer than live attenuated vaccines. They are however not strongly immunogenic and are therefore less effective [142]; although this is countered by the low manufacturing costs [199]. 2-15

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2.2 REVIEW OF CURRENT AND POTENTIAL FUTURE APPLICATIONS IN MEDICINE – GENE THERAPY

Gene therapy treatments for cancer Restoration of tumour suppressor gene function Inhibition of oncogene functions Gene-directed enzyme prodrug therapy Targeted expression of cytotoxic/pro-apoptotic genes Immunogene therapy Anti-angiogenic gene therapy Gene therapy treatments for cardiovascular diseases Therapeutic angiogenesis Restenosis following balloon angioplasty Gene therapy for the treatment of monogenic diseases Gene therapy for the treatment of infectious diseases Gene therapy for the treatment of neurological diseases Treatment of neurotrophic insults Treatment of psychiatric diseases Gene therapy for the treatment of ocular diseases Outer retina / retinal pigment epithelium Retinal ganglion cells Retinoblastoma Optic nerve Retinal and choroidal vasculature Lens Conjunctival and corneal epithelia Gene therapy for the treatment of deafness Gene therapy treatments in clinical trials Current limitations to the use of gene therapy Future developments in gene therapy

2.27 Gene therapy is the transfer of genetic material into a cell nucleus, tissue, or whole organ, with the goal of curing a disease or at least improving the clinical status of a patient. The applications for which gene therapy treatments have been developed are diverse, covering the treatment of cancers, cardiovascular diseases, monogenic diseases, infectious diseases, neurological diseases, ocular diseases, and deafness. These are reviewed in more detail in the following sections.

Gene therapy treatments for cancer

2.28 Cancer is the most frequent application of gene therapy treatments. Strategies for the treatment of cancers using gene therapy include the following (some of these have applications as gene therapy treatments in other areas such as the treatment of cardiovascular and ocular diseases):

♦ restoration of tumour suppression gene function

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♦ inhibition of oncogene function ♦ GDEPT, or suicide gene therapy ♦ targeted expression of cytotoxic/pro-apoptotic genes ♦ immunogene therapy ♦ anti-angiogenic gene therapy

2.29 These strategies have been used on their own and in combination with each other, and also with conventional surgery, chemotherapy and radiotherapy approaches. Gene therapy offers the advantages of being a non-surgical approach, and therefore particularly suited to cancers which are difficult to remove surgically, such as brain tumours, diffuse tumours or metastatic disease [64]; as well as being tumour specific and thereby having minimal toxic effect on non-cancerous tissue.

2.30 Where gene therapy has been tested in combination with other treatments (GM and conventional), the multi-treatment approach has been reported to be more successful in treating the cancer than each treatment (conventional or GM) on its own. Gene therapy is therefore seen as an additional approach for the treatment of cancers, alongside existing conventional treatments, rather than a replacement for existing approaches. In addition to providing a better more effective response, combined treatments (GM and conventional) are also reported to reduce the potential for the emergence of drug resistant tumour cells [55].

Restoration of tumour suppressor gene function 2.31 Cancers are characterised by a malignant transformation of the affected cells, with a loss of control of cell proliferation and apoptosis (cell death) 21 . These transformations are associated with the loss of function of certain genes through deletions, mutations, promoter inactivation and other epigenetic (genome function) changes [64][21]. In non-cancerous tissues, genome integrity, cell proliferation and apoptosis are controlled by tumour suppressor genes (such as p53) 22 so that an equilibrated turnover of cells is maintained [22]. In the absence of these genes cell proliferation is not halted and apoptosis not triggered in response to DNA damage, with the cells therefore continuing to accumulate mutations, leading to tumour formation, and resistance to conventional treatments. GMOs may be used as vectors to restore these mutated genes.

2.32 The observation that mutations in tumour suppressor gene p53, or alterations in its pathway (such as the downstream effectors p21, E2F1 and p16), occur in >90% of human tumours (including colon, lung, oesophagus, breast, liver, brain and

21 Apoptosis is a tightly controlled, multistep, multipathway cell death program that is inherent in every cell [62]. Apoptosis is described as occurring in two signalling cascades involving receptors belonging to the TNF superfamily (the extrinsic pathway) and mitochondria (the intrinsic pathway). The cascades are not mutually exclusive and may interact at many levels. 22 The gene p53 is only expressed in cells during cell cycle progression or in response to genetic damage. If a genetic abnormality occurs, p53 acts to stop the cell division cycle and monitors the repair process. If the damage to the DNA is too great, p53 may induce apoptosis of the cell [64]. 2-17

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reticuloendothelial tissues) [26][27][64], led to the development of the world’s first commercially licensed gene therapy product (Gendicine). This involved a GM adenoviral vector expressing the p53 cDNA under the control of a CMV promoter, with the p53 expression cassette replacing the adenovirus’s E1 gene [114]. Gendicine was licensed in China for the treatment of head and neck squamous cell carcinoma [25]. Information on the success of the Gendicine in China is limited [266], although it is reported to have been administered to >4,000 patients from various ethnic backgrounds with fifty different cancer indications [114]. Gendicine is described as an effective treatment against cancer with a significant synergistic effect when combined with conventional therapies. Adverse side effects are reportedly limited, with the most commonly observed effect being fever (grade I or II) which occurs ~2-4 hours post injection, lasting for 2-6 hours and then disappearing [114].

2.33 Overexpression of p53 is also reported to increase tumour sensitivity to chemotherapeutic drugs and radiotherapy [64]. The use of viral vectors to express the downstream effectors p16 and p21 was found to be more effective than p53 in animal studies (Wang et al. 2001; cited by [64]).

2.34 Whilst the use of tumour suppression genes (including p53) has shown some success in the treatment of cancer [23][24], it is limited in part by the requirement to introduce the tumour suppressor gene (and ensure its expression) in virtually all the cancer cells. This is described as technically impossible with current gene therapy vectors and with solid tumours [21].

Inhibition of oncogene function 2.35 The rationale of the inhibition of oncogenes is similar to the restoration of tumour suppressor genes, in that it seeks to alter the activity of genes with a known role in the development of cancers. Oncogenes, such as Bcl-2, are those genes involved in the maintenance of cell proliferation and the acquisition of metastatic phenotype (spread of cancer cells through the lymphatic and blood systems away from its primary site in the body to other areas) [21]. The Bcl-2 gene is overexpressed in a variety of human epithelial malignant tumours [118]. Treatments in this area are therefore focused on inhibiting the action of these genes through antisense RNA [28] or RNA interference (RNAi) [118], thereby decreasing cell proliferation and restoring the sensitivity of the cells to apoptopic stimuli. As with approaches targeting tumour suppressor genes, this approach also requires the target gene (in this case the oncogene) to have a dominant role in the malignant transformation process [21].

♦ Antisense RNA – use of artificial antisense nucleotides that are complimentary to the mRNA transcribed from the target gene) [28]. ♦ RNAi – utilises the cellular mechanism in which double stranded RNA triggers the silencing of the corresponding cellular gene [118]. Studies with mammalian cell lines reported that the transfection of synthetic small interfering RNAs (siRNAs) induced sequence-specific gene silencing and reduced expression of

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the oncogenic protein. Studies in mice reported that the repeated administration of a GM adenovirus that induces the production of siRNA against p28-GANK caused a dramatic decrease in the growth of human liver cancer grafts [302]. The human gankyrin gene product p28-GANK is an oncoprotein which induces hyperphosphorylation and increases degradation of pRB (retinoblastoma) and has found to be overexpressed in the majority of hepatocellular carcinoma (HCC) [29]. HCC accounts for 80% of primary liver tumours in adult humans [21].

Gene-directed enzyme prodrug therapy 2.36 GDEPT, or suicide gene therapy, is a two stage process for the treatment of cancers. The first stage involves the delivery of the transgene to the tumour and its uptake/expression by the tumour or stromal cells. In the second stage a harmless prodrug is administered to the patient. This prodrug is converted by the enzyme that is transcribed and translated from the introduced transgene, to produce a cytotoxic metabolite, and resulting in the death of the transfected cells [32][30][31]. In some cases the process also causes the destruction of surrounding cancer cells (described as the ‘bystander effect’) [64][21].

2.37 The advantage of GDEPT compared to conventional chemotherapy treatments for cancer is that in targeted GDEPT the cytotoxic chemical is confined to the tumour, whereas in conventional chemotherapy the whole patient is exposed to the chemical. Conventional treatments are therefore limited by systemic toxic effects which restrict the total dose administered. Systemic toxic effects can be minimised in GDEPT systems (providing the vector is delivered selectively to the tumour and the cytotoxic chemical has a short half-life) [38].

2.38 GDEPT systems are applicable to the treatment of most types of cancer, with the transgene expression, delivery and action of the process determined by the vectors and promoters used, and the route of administration. Their efficacy is reliant on a large bystander effect as the delivery vectors currently available only result in a relatively low cell transfection efficiency. Therefore only a small fraction of the tumour cells are modified to produce the cytotoxic compound [31]. Increasing the bystander effect may avoid this limitation [64].

2.39 The most common approach to GDEPT uses viral genes [37]. The most widely used and well characterised systems is the HSV-TK/GCV system which uses the herpes simplex virus thymidine kinase gene (HSV-TK) and the antiviral agent (prodrug) ganciclovir (GCV) [31][32][64]. The HSV thymidine kinase converts GCV into the monophosphate intermediate which is subsequently converted into the triphosphate by cellular enzymes. The ganciclovir-triphosphate is incorporated into the cell’s DNA, disrupting DNA synthesis and causing apoptosis [21]. However, as the triphosphate form is highly polar, it cannot diffuse outside the transfected cell, thereby limiting the bystander effect. (Some bystander effect is reported to occur through gap junction transfer of the triphosphate, and phagocytosis by neighbouring cells) [33]. Fusion of the TK with the transport protein VP22 is reported to improve

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the bystander effect by transferring the enzyme to neighbouring cells [34], although the efficacy of this approach in vivo has been questioned in more recent studies [35].

2.40 An improved bystander effect is reported with the yeast cytosine deaminase (CD) system, which converts the antifungal drug 5-fluorocytosine (5-FC) into the cytotoxic thymidylate synthetase inhibitor 5-fluorouracyl (5-FU) [37]. This causes cell death through the inhibition of DNA synthesis. The greater bystander effect is achieved as the 5-FU metabolite can diffuse to surrounding cells more readily than ganciclovir- triphosphate [36]. The significance of this greater bystander effect with the CD/5-FC system was reported by Huber et al. (1994) (cited by [64]) who found that the transduction of as little as 2-4% of tumour cells with the CD gene still achieved significant regression of the colorectal tumour in xenografted tumour murine models.

2.41 The CD/5-FC system has shown good results against primary and metastasic liver cancer in animal models (rat) using an adenoviral vector [37], and also against breast cancer tumours (also in rats) using a bacterial ( Bifidobacterium longum ) vector 23 [60]. A limitation with viral vectors is that they have limited access to solid tumours (such as those that characterise primary tumours of the breast and uterine cervix), due to the poor vascularisation of these tumours. As described in the later section on ‘Direct Action’, anaerobic bacteria such as B. longum are able to exploit the conditions within the solid tumours and therefore offer greater success as delivery vectors to these types of tumour [69].

2.42 Combinations of the HSV-TK/GCV and CD/5-FC systems with other GM and conventional treatments have been reported, with many identifying improved efficacy against the target tumours. The use of the CD/5-FC system against brain tumours for example is enhanced when used in combination with conventional radiation therapy (Kambara et al. 2002; cited by [64]). The application of the HSV-TK/GCV and CD/5-FC systems together has also been investigated in animal studies, with a faster and more complete regression of the targeted tumour described [65]. The CD/5-FC system has been used in a small human pilot trial with Salmonella sp. as the vector [128]. The significance of using salmonella is that the bacterium presents an additional ‘direct action’ impact to the target tumour.

2.43 Both the HSV-TK/GCV and 5-FC/5-FU systems are described as cell-cycle dependent systems in that they are only able to kill dividing cells (and not quiescent cells). Cell-cycle independent GDEPT systems include the mammalian cytochrome P450/cyclophosphamide (CPA) [39] and bacterial nitroreductase/dinitrobenzamide [38] systems. A more comprehensive list of GDEPT systems used in gene therapy, and their current stage of development are presented in Appendix 1 (Table A.1).

♦ cytochrome P450/CPA –cytochrome P450 converts the CPA into a mustard toxin which triggers DNA crosslinking and protein alkylation [1]. Many different

23 Strategy named the BifidobactErial Selective Targeting-Cytosine Deaminase (BEST-CD) therapy [60]. 2-20

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isoforms of P450 exist and only a small number have been tested for GDEPT [214]. This system has also been reported to have greater efficacy when combined with conventional chemotherapy (Chen et al. 1995; cited by [64]). The bystander effect is cell type and CYP isoform dependent and has been reported to kill 90-95% of cells when 14-20% of cells expressed CYP2B1 [284]. ♦ nitroreductase/dinitrobenzamide – uses the bacterial (Escherichia coli ) enzyme nitroreductase to activate CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) to a short lived highly toxic DNA cross-linking agent. The system, which has a high bystander effect, has been used in a Phase I clinical trial using an adenoviral vector (dose of 1x10 8-5x10 11 viral particles) injected intra-tumourally to target liver cancer [38]. The trial, which was the first use of a nitroreductase GDEPT system for the treatment of resectable primary or secondary (colorectal) liver cancer, found no evidence of virus shedding and no adverse effects reported by the patients (apart from pain at the injection site). ♦ carboxypeptidase G2 (CPG2) – the CPG2 is found in bacteria where it removes glutamic acid moieties from folic acid, thereby inhibiting cell growth. The cell- surface tethered version works best as the prodrug does not readily enter the cell. When combined with the prodrug 4-benzoyl-L-glutamic acid (CMDA) it releases a mustard like compound with similar effect to the cytochrome P450/CPA system [64]. The CPG2/CMDA bystander effect kills between 50- 100% of breast cancer cells following the transduction of just 10-12% of the tumour cells (Stribbling et al. 2000; cited by [64]). This system (delivered by adenoviral vector) has also been shown to kill 70% of brain tumour cells that were resistant to conventional chemotherapeutic drugs and also not killed by the HSV-TK/GCV system [66]. ♦ E.coli purine nucleoside phosphorylase (PNP) – the E.coli PNP converts non- toxic purine nucleoside analogs into toxic adenine analogs which block both mRNA and protein synthesis [64]. It has been used with the prodrugs 6- methylpurine and F-araAMP, and exhibits a high level of bystander activity.

2.44 An approach similar to GDEPT has also been reported for the treatment of cancer in which the GM virus is used to deliver a gene that enables conventional radio-iodine- based therapy 24 to be used against the tumour [40]. The sodium iodide symporter (NIS) is a specialised active iodide transporter that co-transports a sodium and an iodide ion across the plasma membrane of thyroid follicular cells. The transfection of tumour cells with the gene for NIS, coupled with conventional radioiodine therapy results in the transfected cells accumulating 131 iodine, resulting in cell cycle blockade and cell death. Faivre et al. (2004) [41] reported a study with rats in which a GM adenovirus was used to transfect liver cancer cells with the NIS gene (under the control of a CMV promoter). The study reported specific accumulation of 131 I in the injected tumour and a significant reduction in tumour volume.

24 Radioiodine therapy is an existing well-recognised approach for the treatment of human thyroid cancer [41]. 2-21

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Targeted expression of cytotoxic/pro-apoptotic genes 2.45 The targeted expression of cytotoxic/pro-apoptopic genes involves the selective transfer of genes that will cause the destruction of cancer cells through a range of mechanisms. The approach relies primarily on the targeting of gene transfer and expression into cancer cells, using specific surface ligands or promoters [21].

2.46 One area of investigation involves the TNF-related apoptosis inducing ligand (TRAIL) which induces apoptosis preferentially in cancer cells and may therefore offer a tumour specific treatment therapy [45]. Studies with an adeno-associated (AAV) vector expressing sTRAIL (the extracellular domain of TRAIL that works as a soluble cytokine) have reported potent antitumour effects in subcutaneous liver cancer xenografts in mice, following oral or intra-peritioneal administration of the vector [44]. Toxic effects to mice and non-human primates have not been found following systemic administration of sTRAIL [45]. The application of TRAIL with conventional chemotherapy agents has reported positive results [45].

Immunogene therapy 2.47 Immunogene therapy is used extensively as a gene therapy application against cancer. It involves the transfer of genes designed to elicit an immune response against the tumours, and the consequent control of the primary tumour and prevention of metastasis [21]. The success of conventional (non-GM) immunogenic therapies has been limited as the cancer cells evolve mechanisms to evade immune detection. Gene therapy has been described as offering the means to overcome this limitation [56].

2.48 Three areas of immunogene therapy involving GMOs that have shown promising results are:

♦ Tumour antigens delivered through adenoviral expression – GM viral vectors have been used to express tumour proteins, and thereby prime a patient’s immune system against that tumour. Preclinical trials have been conducted against renal cell carcinoma using GM adenovirus expressing the tumour antigen carbonic anhydrase IX protein (also referred to as the G250 protein) [67]. ♦ Mobilising dendritic cells – interest in dendritic cells is based on the understanding that dendritic cells are the principal antigen presenting cells of the immune system, and a required component for the development of an antigen-dependent immune response. They are also highly effective inducers of tumour specific killer T cells. Dendritic cells differentiate from the precursor cells in response to the expression of Fms-like tyrosine kinase 3 ligand (Flt3L) [64]. Studies into the use of dendritic cells for the treatment of cancer focused initially on the pre-exposure of the cells to tumour antigen in vitro, followed by intra-tumoural injection. This essentially sensitised the patient’s immune system to the cancer cells [56]. However, in vitro manipulation was found to reduce the effectiveness of the cells in vivo . More recent studies in rats have involved intra- 2-22

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tumoural injection of GM adenovirus expressing Flt3L (the dendritic growth factor). This has proved more successful, with tumour rejection achieved in 60- 80% of the animals studied [68]. ♦ Enhancement of immune response using interferons and cytokines.

2.49 Cytokines (such as various interleukins (IL), interferon 25 , tumour necrosis factor (TNF) and granulocyte-macrophage colony stimulating factor (GM-CSF)) are key mediators in the function of the immune system and have therefore been used to stimulate the immune system against tumours [21]. Although trials have been reported involving the direct administration of cytokines [46], these are not of direct relevance to this report as they do not involve the use of live GMOs. Direct administration based approaches may also be limited by severe toxic effects to the patients [47].

2.50 The use of vectors for the delivery of the cytokine encoding gene allows for the localisation of the effects to the tumour, and should reduce the systemic toxic effects described above [49]. Three clinical trials illustrating this approach are described below:

♦ A Phase I trial using a GM adenoviral vector expressing IL-12 injected intra- tumourally [48]. The trial involved patients with advanced pancreatic, colorectal, or primary liver malignancies, with patients receiving intra-tumoural injections over a three month period at a dose of 2.5x10 10 to 3x10 12 viral particles. No systemic toxic effects were observed, and remission of the injected tumour mass was observed in a patient with hepatocellular carcinoma. ♦ A Phase I trial using a non-replicating adenovirus to deliver the human TNF-α gene under the control of an early growth response 1 promoter which is activated by ionising radiation [49]. The adenovirus was delivered by intratumoural injection (dose of 4x10 11 viral particles) to patients over a five week period, followed by exposure to ionising radiation. Four patients had complete regression of the injected tumour. ♦ A Phase I trial using a replication incompetent adenovirus to inject the gene encoding the cytokine MDA-7 (IL-24) directly into tumour tissue [56]. The cytokine acts by inducing apoptosis. In the 28 patient trial, 22 patients exhibited systemic immune activation as well as local apoptosis. A Phase II trial is reported to be underway.

2.51 Approaches involving a combination of cytokines may have the potential to be more effective and less toxic than single cytokine based treatments, although the cytokines may not necessarily be delivered on the same vector [50]. The combination of immunogene approaches (TNF α and IL-4 for example) with the HSV- TK/GCV prodrug system has also been reported to be successful in the treatment of brain tumours, and may be more efficient than the GDEPT system on its own [64].

25 Interferon gamma and IL-4 are reported to have anti-angiogenic properties (see next section), as well as an immunogenic effect (Saleh et al. 2000; cited by [64]). 2-23

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2.52 Immunogenic therapies have also been used as the basis for the treatment of diseases other than cancer. Studies using adenoviral vectors to deliver the equine IL-1Ralpha gene intra-articularly (into the joint) in horses have been conducted for the treatment of equine osteoarthritis [108]. These reported significant improvements in clinical parameters of pain and disease activity, and the preservation of articular (joint) cartilage. The therapy may also have application for the treatment of osteoarthritis in humans [108].

Table 2.1 - Selected recent immunotherapy clinical trials [56]

Clinical Cancer Stimulating genes Description phase Mouse protein-sugars are expressed on Murine α(1,3)- Prostate allogeneic prostate cells to induce a I galactosyltransferase hyperacute rejection response. Replication incompetent vaccinia and fowlpox viruses engineered to produce CEA Pancreatic CEA and MUC-1 and MUC-1 given subcutaneously to II produce an immune response to pancreatic cancer. Allogenic prostate cells expressing the GM- CSF gene are used to induce immune Prostate GM-CSF I/II response following chemotherapy and peripheral blood mononuclear cells infusion. Autologous tumour cells are combined with allogenic cells that express GM-CSF and Lymphoma GM-CSF and CD40L II CD40L and incorporated into a vaccine with low doses of IL-2. Autologous tumour cells engineered to Melanoma IL-2 express IL-2 are incorporated into a II vaccine. A modified replication incompetent adenovirus containing the tumour antigen Kidney CD-80 CD-80 is injected subcutaneously along with II the cytokine IL-2 to produce an immune response to the prostate cancer.

Anti-angiogenic gene therapy 2.53 Angiogenesis is a normal process in both growth and development, and wound healing, involving the rapid proliferation of endothelial vascular cells, culminating in the formation of new blood vessels [64]. The process is tightly regulated in adults and is coordinated by the expression of both activators and inhibitors. Angiogenesis is also a fundamental stage in the development of tumours from a dormant to a malignant stage. As tumours grow they promote angiogenesis to obtain their blood supply and ensure sufficient oxygen and nutrients are available to support their growth. The basis of anti-angiogenic gene therapy is therefore to block or inhibit this cancer-induced formation of new blood vessels, thereby limiting the growth of the tumour [51]. This strategy is potentially safe with few side-effects as angiogenesis in healthy adults usually only occurs in response to damage from wounds (and during female menstrual cycle) or hypoxia [64], and the anti-angiogenic factors (or angiogenic inhibitors) such as endostatin and angiostatin do not affect mature vessels of normal tissues [21]. Non-GM angiogenesis inhibitors for cancer have

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been approved by the USFDA 26 in the USA, and in 29 countries worldwide. The two least toxic and most broad spectrum of these inhibitors are Caplostatin and endostatin [51]. Endostatin for example inhibits the growth of 65 different tumour types [51]. Many of the anti-angiogenic approaches conducted to date have focused on blocking the VEGF (vascular endothelial growth factor) receptor, which is an important mediator of angiogenesis [51][21].

2.54 Because the angiogenesis inhibitor peptides such as endostatin are difficult to generate in sufficient quantities for clinical use in vitro , their use in clinical treatments is much more suited to use in gene therapy [64].

2.55 Both viruses (adenovirus, AAV) and bacteria ( Bifidobacterium longum and Salmonella choleraesuis ) have been reported as vectors for the delivery of anti- angiogenic agents (cited by [21]). The Bifidobacterium longum system has been used in animal studies (mice) to deliver endostatin for the treatment of solid liver cancer [52]. The study reported strong inhibition of the solid liver tumour and prolonged survival times. Inhibition was enhanced when the mice were also given selenium. This effect is due to the selenium improving the activity of the NK and T cells and stimulating the activity of IL-2 and TNF-α [52].

Gene therapy treatments for cardiovascular diseases

2.56 The application of gene therapy for the treatment of cardiovascular disease has followed increased understanding of myocardial disease and the availability of vectors with an enhanced tropism for the myocardia [78]. The key requirements for gene delivery vectors against cardiovascular disease are sustained, targeted and regulated expression of the therapeutic agent [76]. Adeno-associated virus (AAV) vectors have this characteristic, and, along with adenovirus vectors have been used in a number of pre-clinical and clinical trials against cardiovascular diseases [72]. To date (2007) no gene therapies have been approved for clinical use in cardiovascular applications [76].

2.57 With respect to delivery of the vector, direct administration is reported to be more efficient than the less-targeted intra-arterial or intra-venous application route [78][76]. For so-called global myocardial diseases such as heart failure and cardiomyopathy 27 , intra-coronary delivery is reported as the most efficient direct delivery approach, whereas for the treatment of more regional conditions intra-myocardial injection is the most appropriate. This is due in part to the selective nature of the coronary endothelium 28 which may restrict the diffusion of some vectors and therefore limit distribution and uptake of the transgene [78].

26 United States Food and Drug Administration 27 Cardiomyopathy is the deterioration of the function of the mycocardium (the muscular tissue of the heart). 28 The coronary endothelium is the thin layer of squamous cells lining the inside of the heart. As with the endeothelium lining throughout the circulatory system, the coronary endothelium cells control the 2-25

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2.58 Gene therapy involving live GMOs is reported to be used in the treatment of two areas of cardiovascular disease, namely therapeutic angiogenesis, and restenosis following balloon angioplasty. Gene therapy using naked plasmids or liposomes for the gene transfer has also been investigated for use in cardiovascular diseases [70]. However as these applications do not involve live GMOs they are outside the scope of this report and have not been considered further.

2.59 Whilst acute myocardial infarction (heart attack) is a relatively common heart manifestation of heart disease it is relatively difficult to treat using gene therapy. This is a consequence of the time taken by delivery, transcription and translation of the transgene to the therapeutic product being longer than the time available for successful intervention and prevention of the attack. An alternative approach that has been proposed is the use of vectors to achieve a long term expression of antioxidant or cytoprotective genes, and thereby protect the heart from future attacks [78]. This approach has been tested with some success in rat models using AAV vectors to deliver haem oxygenase-1 (HO-1).

Therapeutic angiogenesis 2.60 Applications of gene therapy in relation to angiogenesis and cancer have been discussed earlier in this report. Whereas the applications in cancer have focused on the inhibition of tumour-mediated angiogenesis, the applications in therapeutic angiogenesis (for cardiovascular treatments) have addressed the promotion of angiogenesis.

2.61 The reasoning behind the approach is that damage to blood vessels, through coronary artery disease (CAD) or peripheral arterial disease (PAD) for example, can restrict the blood flow to tissues and organs. If the subsequent development of collateral blood vessels (through angiogenesis) around the restriction, blockage or damage is insufficient to allow for the required perfusion of the affected tissue, a condition called ischaemia occurs. This relatively quickly results in the necrosis of the affected areas.

2.62 Human clinical trials of gene therapy for the treatment of diseases such as CAD and PAD that can cause ischemia have been underway since 1994 [74]. Some of these trials 29 have used a GM adenovirus as the vector for one of several angiogenic growth factors such as VEGF121 30 , VEGF-2 and fibroblast growth factor (FGF) [70][71]. Hepatocyte growth factor (HGF) has also been identified as a candidate for therapeutic angiogenesis, although its successful use to date in clinical trials has only been with naked plasmid or liposome mediated transfer [70]. The gene therapy based trials have been reported to be more successful than protein therapy trials passage of materials into and out of the bloodstream. The blood-brain barrier described earlier in this report is an example of a highly specialised endothelial control system. 29 Other trials have used naked plasmids as the vector. 30 VEGF is vascular endothelial growth factor, an important mediator of angiogenesis. The use of gene therapy to block the VEGF receptor has been described in a previous section for the treatment of cancer. 2-26

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(where the growth factor proteins have been administered directly), probably because transfer of the gene allows for the sustained production of the required angiogenic protein [75].

2.63 Adenovirus-mediated transfer of VEGF121 has been used in a Phase I clinical trial. A multi-centre Phase I/IIa trial involving intra-coronary infusion of an adenovirus encoding FGF has also been conducted. The latter trial (named Angiogenic GENe Therapy (AGENT)) achieved sustained in situ production of the FGF protein over a 12 week period following a single intra-coronary infusion of the replication defective adenovirus vector, and was reported to improve cardiac function without severe toxicity [75].

2.64 The gene therapy applications described above may be viewed as treatments used in response to the occurrence of diseases such as CAD. However, protective/preventative applications have also been investigated involving the overexpression of antioxidant enzymes such as HO-1, SOD, catalase, and GPx [78]. These are designed to counter the prominent role of oxidative stress in CAD, and therefore to protect the heart from future injury. This minimises the need for acute intervention and subsequent problems with restenosis for example (see below).

Restenosis following balloon angioplasty 2.65 Balloon angioplasty (the mechanical widening of a narrowed or blocked blood vessel) is one of the major therapeutic approaches to coronary artery stenosis (the narrowing of the coronary arteries). However, its effectiveness long term is limited by the development of restenosis (recurring stenosis).

2.66 Vascular endothelial growth factors such as VEGF165 have been tested in clinical trials (using GM adenovirus) for the treatment of restenosis [70]. The potential role of gene therapy for the treatment of restenosis is significant as traditional pharmco- therapeutic approaches have been unsuccessful in eliminating this condition [77].

Gene therapy for the treatment of monogenic diseases

2.67 Monogenic diseases are those resulting from a mutation in a single gene, and are estimated to include over 10,000 diseases (World Health Organisation data). The concept of ‘gene therapy’ originated as a potential strategy for the treatment of monogenic diseases (Baltimore, 1978; cited by [80]), although as evident from this report, gene therapy is now used for the treatment of a much wider range of conditions and diseases.

2.68 Three approaches for the application of gene therapy to treat monogenic diseases are/have been investigated; namely somatic stem cells, gene transfer and RNA modification (involving antisense oligonucleotides for example) [82]. Of these, only gene transfer involving modified viral vectors is of relevance to this report, and successful treatment (abnormal phenotype corrected) of the several monogenic

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diseases31 has been reported in animal studies using modified AAV and retrovirus. AAV has also been evaluated in humans for the treatment of cystic fibrosis, muscular dystrophy, α1-antitrypsin deficiency, haemophilia with factor IX deficiency (haemophilia B), and two paediatric neurodegeneration disorders (aspartoacylase deficiency (Canavan disease) and late infantile ceroid lipofuscinosis (a form of Batten disease)) [80][82].

2.69 Human clinical trials using such GMOs have been conducted against four monogenic diseases [80]. Clinical trials have also been conducted using non-viral delivery, for example for the treatment of cystic fibrosis. Whilst these trials do not involve a live GMO they are mentioned here as they also pose potential risks to the patient and wider community. The potential risks posed by these gene therapy treatments are not just a consequence of the GMO:

♦ Ornithine transcarbamylase (OTC) deficiency: o One trial using E1 + E4 deleted adenovirus to transfer OTC cDNA. However, no clinical benefit was reported in a study of 18 patients, and one patient died [80]. ♦ Haemophilia caused by deficiency in Factor IX: o Modified AAV to transfer a modified Factor IX gene. No sustained clinical benefit in either of the two trials conducted [80].

♦ Severe combined immunodeficiency (SCID): o Modified retrovirus, but ex vivo transduction of cells. Study involved ten patients, of which nine patients developed a functional immune system [218]. However, four patients later developed T cell lymphoblastic leukaemia, and one patient died [86]. o In two further studies [236][11], also using ex vivo retroviral transduction of cells, all patients in the trials developed a functional immune system and no adverse effects were reported [13][12]. ♦ Chronic granulomatous disease (CGD): o Study reported a positive result for both patients involved. Study conducted by ex vivo retroviral transduction of the cells [80].

Gene therapy for the treatment of infectious diseases

2.70 Gene therapy for the treatment of infectious diseases can be divided into four broad categories [85], namely gene therapies based on nucleic acid moieties, protein approaches (such as transdominant negative proteins (TNPs) and single chain antibodies, GDEPT (gene-directed enzyme prodrug treatment)32 , and

31 Haemophilia A and haemophilia B, mucopolysaccharidosis I and mucopolysaccharidosis IIIb, Niemann–Pick A, and glycogen storage disease type II [82]. 32 Described in more detail in the GDEPT section reviewing applications to treat cancer. 2-28

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immunotherapeutic approaches using genetic vaccines or pathogen-specific lymphocytes [85]. Only the last two (GDEPT and genetic vaccines) are of relevance to this report as they may involve the use of live GM vectors. Vaccination based approaches are described in a later section ‘Review of current and potential future applications in Human Medicine – Vaccination’.

2.71 A significant area (in terms of research and investigation) of application of gene therapy against infectious diseases is in the treatment of HIV-1. In 2000 for example there were 27 active gene therapy protocols for the treatment of HIV-1 infection underway in the USA [81], currently 40 trials open worldwide [17][286]. Other infectious diseases that have been investigated as potential targets for gene therapy include malaria, T-cell leukaemia, influenza, tuberculosis, hepatitis (A, B and C), Ebola virus and La Crosse virus [85][81]. The approaches against influenza, malaria, tuberculosis and hepatitis have been vaccination based (and are therefore reviewed in the Vaccination section of this report).

♦ T-cell leukaemia – strategies developed using retrovirus vectors to inhibit expression of key genes required for the replication of human T-cell lymphotropic virus type 1 (HTLV-1), a causative agent of adult T-cell leukaemia. HTLV-1 genes targeted include those encoding the Rex protein and Tax protein [85]. ♦ HIV-1 – the use of antisense RNA to block transcripts of the viral genes tat, rev, vpu and gag has been proposed [85]. Although antisense technology does not always require a live GMO, a modified retroviral vector was used in this application to ensure a long-term and high level of expression of the antisense sequences. Whilst antisense technology requires a minimum of one antisense sequence for each target sequence, more effective inhibition is achieved at an antisense : target ratio of 5:1 or 10:1.

2.72 Treatments have also been investigated against the neurovirological diseases (human and veterinary) caused by viral infections of the nervous system. Examples include poliovirus, rabies virus, Nipah virus and West Nile virus, and those causing demyelinating diseases such as Theiler’s virus, Semliki Forest virus, coronaviruses and maedi-visna virus [103]. No successful gene therapy specific treatments have been identified for the treatment of these to date.

Gene therapy for the treatment of neurological diseases

2.73 Whilst a number of applications of gene therapy for the treatment of brain tumours have been described in previous sections, the use of viral vectors for the treatment of neurological diseases is much less developed. Limitations in stability and regulation of transgene expression, and the safety of the vector and expressed transgene have still to be fully addressed [101]. However, as described below some advances in this area have been achieved, and studies at a laboratory and animal model level suggest that further developments may be achieved in the future [109].

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2.74 With respect to the application of gene therapy for the treatment neurological diseases there is a distinction between diseases caused as a result of neural insult, such as the loss of neurones or endogenous factors through congenital or environmental reasons, and psychiatric disorders [100]. Examples of diseases caused by neural insult are Alzheimer’s, Parkinson’s, Fabry disease and impairment(s) following seizures.

2.75 The key issue in the application of gene therapy for the treatment of both groups of neurological diseases is the identification of vectors capable of transferring genes into the nervous system. Transfer is restricted by the presence of the blood / brain barrier which prevents application through systemic injection, and also because (in adult brains) the neurons are primarily post-mitotic 33 . This latter point means that viral vectors such as retroviruses that are only able to transduce dividing cells cannot be used [100]. Vectors that are suitable are those derived from neurotrophic viruses such as herpes simplex, adenovirus, adeno-associated virus and lentivirus that are capable of infecting non-dividing cells.

2.76 Delivery of the vector into the brain is also challenging. Direct injection often only results in the cells in close proximity to the injection point being transfected, which may not be suitable when targeting larger tumours. An easier approach that can achieve a greater spread of delivery is direct infusion of the ventricles. However, limited penetration of the vector from the ventricles means that it is typically the ependymal cells and not the neurons that are transfected. Such limitations require transgenes whose protein products are effective extracellularly, rather than within the neurons [100]. This approach has been successful in decreasing neurotoxicity after neurotic insults.

2.77 The limited spread of the vector from the point of injection has resulted in further modification of vectors to express a fluorescent marker gene so that their spread can be easily monitored [100]. The marker should have no effect on the function of the vector and should pose negligible risk.

Treatment of neurotrophic insults 2.78 The treatment approach to neurotrophic insults targets the enhancement of either a cellular process that increases the likelihood of neuron survival such as the reducing neuronal loss through apoptosis, or an endogenous process such as the overexpression of antioxidants [104][100]. The use of viral vectors to enable overexpression of glucose transporters is reported to enhance neuron survival in the face of a range of neurological insults, as these ultimately represent energy crises within the cells, either through impairing the neuron’s ability to generate adequate energy (as in hypoxia-ischemia or hypoglycaemia) or pathologically consuming energy (as in prolonged seizure) [104].

33 Refers to cells that are no longer undergoing cell division. 2-30

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Treatment of psychiatric diseases 2.79 The difficulty in the treatment of psychiatric diseases is that the genetic defects do not always result in an occurrence of the disease. Therefore whilst treatments such as the use of viral vectors to overexpress benzodiazepine receptors to ameliorate anxiety, or antisense to a dopamine receptor to counter schizophrenia, may in theory seem appropriate; in practice they are likely to be extremely simplistic with limited effectiveness. As with the other gene therapy approaches described in this report, these more simplistic approaches involve the constitutive expression of the transgene, whereas in the treatment of psychiatric diseases expression may need to be inducible and therefore activated by the same conditions (environmental or biological for example) that increase the risk of onset of the disease. Such inducible systems are in development, although it is unlikely that the application of gene therapy as a psychiatric treatment will be available clinically in the near future.

2.80 Addictive type diseases, such as cocaine addiction have been successfully treated in a rat model using viral vectors to overexpress the dopamine signal transduction molecule CREB [100].

Gene therapy for the treatment of ocular diseases

2.81 The complex structure of the eye presents unique challenges for agent delivery [89], and has limited the effectiveness of treatment strategies for many ocular conditions, such as glaucoma and retinitis pigmentosa (degeneration of photoreceptor cells in the retina) which affect the back of the eye [89], and herpes simplex keratitis (a disease of the cornea) [90]. Gene therapy may offer a more successful strategy to existing treatments for the following reasons [91][90][89]:

♦ The eye’s well-defined anatomy, accessibility, and the transparent nature of its media means that the transgenes can be delivered to the target area with a high degree of accuracy. The small size of the eye may limit the quantity of material that is introduced. ♦ Immunoprivileged status of the eye. The eye is not protected by the body’s immune system in the same way as other tissues. However, inflammation can still be triggered by viral vectors. ♦ Cell division is minimal in the mature human eye and is limited to discrete cell types. The use of the appropriate viral vector can therefore target the transgene to the relevant cell(s) with a high degree of accuracy. Retroviral vectors could for example only be used to transfect ocular cells where cell division does occur. ♦ The blood / ocular barrier within the eye separates it from the rest of the body. Therefore any vector delivered within the eye is unlikely to be released into the rest of the body, thereby minimising the risk of systemic effects. ♦ The retina requires relatively low virus titres (~1/1000 th the level used for systemic diseases) to achieve the required therapeutic effect. This is significant

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as the relatively small size of the eye prohibits large injection volumes, and demands an efficient gene delivery system.

2.82 Gene therapy applications have been developed in the following areas within the eye. AAV vectors have achieved efficient gene delivery to photoreceptors, corneal epithelial cells and pigmented epithelial cells following subretinal injection [89], particularly where well-defined promoters are used to focus expression of the transgene in the target cells. Derivatives of the rhodopsin kinase (RK) promoter have been used for example with AAV to target transgene expression in rod and cone photoreceptor cells [15]. Lentiviral vectors have been used to transfect corneal endothelial cells, trabecular meshwork (TM) cells and cultured retinal pigment epithelial (RPE) cells [91]. Some of these applications are described in more detail below.

Outer retina / retinal pigment epithelium 2.83 The RPE is the pigmented cell layer that supports the retinal visual cells. The epithelium also serves as the limiting transport factor that maintains the retinal environment and acts as a barrier to blood borne substances. All of the approaches in this area have sought to improve or prolong the survival of photoreceptor cells through the introduction of angiogenic growth factors such as ciliary neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF), pigmented epithelium- derived factor (PEDF) and basic fibroblast growth factors (bFGF, FGF2); and the alteration of apoptosis pathways [92]. The introduction of a calcium-channel blocker has also been reported to confer enhanced survival of photoreceptors.

2.84 The introduction of CNTF and FGF factors using AAV or adenovirus vectors is reported to confer two advantages over direct (intra-ocular) injection of the protein into the eye. CNTF protein is toxic to the retina and brain if injected directly, and the use of AAV as the delivery vector is proposed to minimise this toxicity. Delivery of FGF factors by adenovirus vector is reported to confer prolonged photoreceptor survival of at least two months compared to direct injection.

2.85 Human clinical trials are currently underway (2007/2008) at University College London using an AAV vector to deliver functioning copies of the RPE65 gene. Mutations in RPE65 result in the condition Leber’s congenital amaurosis (LCA) which results in a progressive deterioration in vision. There are currently no effective treatments available. This gene therapy strategy has been tested successfully in dogs [91] and is now being trialled with young adult patients who developed LCA as children [16][18].

Retinal ganglion cells 2.86 Damage to RGCs is a causative factor of glaucoma, which is a leading cause of blindness worldwide [92]. Damage to these cells is thought to occur through long- term elevation in intra-ocular pressure, or the presence of genetic factors that

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predispose a patient to loss of RGCs in the absence of increased intra-ocular pressure.

2.87 Until the genetic basis of glaucoma is understood, gene therapy applications in this area have focused on altering the biochemical manifestations of glaucoma, or helping RGCs to avoid apoptosis (which occurs as a consequence of these biochemical changes). The identification of the trabecular meshwork as a key target tissue for pressure induced glaucoma may direct further investigation in this area.

2.88 Studies have been reported that used AAV as the delivery vector for the treatment of glaucoma, and lentivirus and adenovirus for the treatment of ocular diseases where neovascularisation plays a key role in pathogenesis. As described in the antiangiogenesis review of cancer treatment, the delivery of anti-angiogenesis factors such as endostatin reduces levels of VEGF and inhibits the development of diseases such as diabetic retinopathy and neovascular age-related macular degeneration [89]. HSV vectors have been used successfully to transduce trabecular meshwork (TM) cells, as well as ciliary body epithelial cells and RGCs, providing a possible effective treatment for glaucoma [89].

Retinoblastoma 2.89 Retinoblastoma (cancer of the retina) is the most common primary ocular malignancy of childhood. Although treatable with radiotherapy, there is the increased risk of secondary cancers caused by the radiation. Gene therapy offers an alternative strategy for the treatment of the tumour, and animal studies using the HSV/TK/GCV system have been successful in reducing the tumour burden [92].

Optic nerve 2.90 Successful results against optic nerve conditions have been achieved with intra- vitreal injection 34 of AAV vectors near the optic nerve head in animal models [92]. Because the AAV system achieved long-term gene expression (>1 year for the reporter gene used), the system may also be suitable for the treatment of chronic or recurrent optic neuropathies. The use of an adenovirus vector ensured more rapid transduction of the transgene, suggesting that this vector may be more suitable for the treatment of acute conditions, rather than the AAV [92].

Retinal and choroidal vasculature 2.91 Choroidal neovascularisation (CNV) is the major cause of severe loss of vision in patients with age-related macular degeneration (AMD), whilst retinal neovascularisation is the prime cause of vision loss in diabetic retinopathy and in retinopathy of prematurity. AMD and diabetic retinopathy are the most frequent causes of untreatable blindness in the world [92]. Both are chronic diseases and therefore any gene therapy approach needs to ensure long-term expression of the

34 Injection directly into the vitreous humour. Administration of therapeutic compounds by this route bypasses the blood / ocular barrier and reduces systemic side effects. 2-33

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transgene(s). Some success in animal models has been reported, although no human clinical trials have been identified in this area.

Lens 2.92 Successful animal studies have been conducted involving intra-cameral injection (into the eye) of the HSV/TK/GCV system (with adenovirus vector) to prevent posterior capsule opacification (PCO). PCO is a common late complication of cataract surgery, the existing treatment of this can have serious complications [92].

Conjunctival and corneal epithelia 2.93 Adenovirus vectors have been used to transfect cells in both the corneal and conjuctival epithelia [92]. The rapid turnover of cells in both epithelia means than any in vivo expression achieved only lasts for a few days. Whilst this may be advantageous in preventing long-term expression, it does mean that any treatments requiring prolonged expression will need to transfect the corneal or conjunctival stem cells (localised at the limbus and fornix respectively). This may be achievable with a retroviral vector [92].

Gene therapy for the treatment of deafness

2.94 The application of gene therapy to treat deafness used an adenoviral vector to deliver gene Atoh1 which is a key regulator of hair cell development [280]. In a study with guinea pigs the treatment resulted in the regrowth of hair cells in the cochlea (in the inner ear) with the animals regaining up to 80% of their hearing. Loss of these cells is a key cause of deafness. This trial represented the first successful biological treatment of hearing in a mammal.

Gene therapy treatments in clinical trials

2.95 The most comprehensive database of gene therapy clinical trials is held by the Journal of Gene Medicine (Clinical Trial Site)[17]. This reports a total of 1283 gene therapy trials conducted since 1989 to January 2007 in 28 countries. The majority of these trials were focused on the treatment of cancers (67% of trials), followed by cardiovascular diseases (9.1%) and inherited monogenic diseases (8.4%). Viral vectors are the most frequently used delivery system (Table 2.2). Most of these viral vectors (highlighted bold in Table 2.2) are likely to be genetically modified and therefore of relevance to this report.

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Table 2.2 – Gene therapy clinical trials (1989-2007) – vector system used [17] (trials involving viral or bacterial vectors, which are likely to be genetically modified are highlighted in blue) Gene Therapy Clinical Vector Trials

Number % Adenovirus 319 24.9 Retrovirus 296 23.1 Naked/Plasmid DNA 232 18.1 Lipofection 102 8 Vaccinia virus 64 5 Poxvirus 60 4.7 Adeno-associated virus 47 3.7 Herpes simplex virus 43 3.4 Unknown 40 3.1 Poxvirus + Vaccinia virus 26 2 RNA transfer 16 1.2 Lentivirus 8 0.6 Flavivirus 5 0.4 Gene gun 5 0.4 Adenovirus + Retrovirus 3 0.2 Measles virus 3 0.2 Naked/Plasmid DNA + 2 0.2 Adenovirus Saccharomyces cerevisiae 2 0.2 Salmonella typhimurium 2 0.2 Listeria monocytogenes 1 0.1 Newcastle disease virus 1 0.1 Poliovirus 1 0.1 Semliki forest virus 1 0.1 Sendai virus 1 0.1 Simian virus 40 1 0.1 Streptococcus mutans 1 0.1 Vibrio cholerae 1 0.1 Total 1283

2.96 Of the 1283 clinical trials reported, the majority are Phase I trials (787 trials (61.3% of the total)), with 256 (20%) Phase I/II, 198 (15%) Phase II, 13 (1%) Phase II/III, and 29 (2.3%) Phase III [17]. The Phase III trials are for gene therapy treatments at the final stage of testing prior to marketing 35 . Information on clinical trials in the USA

35 Phase I: Researchers test a new drug or treatment in a small group of people (20-80) for the first time to evaluate its safety, determine a safe dosage range, and identify side effects. Whilst most Phase I trials use healthy volunteers, some trials (particularly cancer and HIV drug trials) are usually only conducted on patients. Phase I trials of new cancer drugs typically involve patients with advanced (metastatic) cancer; Phase II: The drug or treatment is given to a larger group of people (20-300) to assess the activity of the drug or treatment and to further evaluate its safety, usually in combination with the current best treatment option; Phase III: The drug or treatment is given to large groups of people (300-3000) to confirm its effectiveness, monitor side effects, compare it to commonly used treatments, and collect information that will allow the drug or treatment to be used safely. Phase III trials are the most time-consuming of the four phases to run. New drugs or treatments typically have 2-35

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is held by the US National Institutes of Health The U.S. National Institutes of Health resource for public access to information on clinical research studies

2.97 Further information on gene therapy applications that have undergone clinical trials is presented in Appendix 1 (Tables A1- A3).

2.98 The following bullets highlight clinical trials that have either reached Phase II or III, or offer particularly innovative methods [56]:

♦ The TNFerade system. This is in late Phase II trials and involves a replication incompetent adenovirus to deliver the cytokine TNF-α, under the transcriptional control of a radiation inducible promoter. The patient receives the adenovirus by injection, but the system is only activated following a dose of radiation therapy. The system is being tested against pancreatic, oesophageal, rectal and melanoma cancers. ♦ Rexin-G, is a retroviral system designed to interfere with the cyclin G1 gene causing cell death. The system is the first injectable gene therapy agent to achieve orphan drug status 36 from the USFDA for the treatment of pancreatic cancer. Rexin-G is currently undergoing Phase I and II trials against pancreatic cancer. ♦ The HSV-GCV system. HSV-TK/GCV has reached Phase III clinical trials, targeting brain tumours. The trial treated (248) patients with glioblastoma multiforme using retroviral transfer [159]. Although the safety of the procedure was demonstrated, there was a poor rate of delivery of the HSV-TK gene to just those cells in the vicinity of the needle track. Overall, the results of the trial were disappointing and showed no difference in time to tumour progression, or overall survival time. In comparison, Immonen et al. (2004)[215] treated patients with malignant glioma with adenoviral-delivered HSV-TK/GCV. They reported a significant increase in survival of the patients (time until death or surgery for recurrence) from 39 to 70 weeks. ♦ Adenovirus systems to deliver the p53 gene to a range of cancer cells are currently in Phase II and III trials. The Gendicine system (replication incompetent (E1-deleted) adenovirus vector to deliver p53 gene) has received regulatory approval in China and is therefore to date the only gene therapy product to obtain regulatory approval in any global market. Many patients are reported to have received treatment for head and neck squamous cell

to undergo two successful Phase III trials before approval from the regulatory agencies is given. Phase III are usually done as comparative studies with other treatments (double-blind, placebo- controlled); Phase IV: Studies are done after the drug or treatment has been marketed to gather information on the drug's effect in various populations and any side effects associated with long-term use. The identification of adverse effects at this stage may result in subsequent restrictions of use being imposed. (Definitions from United States National Library of Medicine). The clinical trials system only applies to treatments for human use. 36 Orphan drugs are those developed under the Orphan Drug Act and applies to drugs for diseases with a low incidence within the population (defined as those affecting <200,000 people, or <5 per 10,000 in the community in the USA). 2-36

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carcinoma 37 with Gendicine, although results from these clinical trials are limited. This is due to the findings being hard to evaluate with the quality of the data questioned by some reviewers [266]. The limited results are also a consequence of the small number of patients tested (no more than 120 people 38 , although a much larger number of people have received Gendicine to date (2007)). In vivo animal studies have reported that the GM adenovirus does not replicate in infected cells and is incapable of multi-cycle infection and spread to neighbouring cells. Although the GMO’s DNA was detectable up to 14 days post administration (by local or systemic injection). When injected locally, no DNA was detected in excrements of urine, stool or bile [296].

Current limitations to the use of gene therapy

2.99 A large number of gene therapy systems involving GMOs have been reported for treatment of a wide range of diseases. These have been developed for use on their own, or in the case of cancer treatment, in combination with the conventional techniques of surgery, chemotherapy and radiotherapy. Many of these gene therapy treatments show (and have shown) promising results in animal studies and are currently undergoing (or have undergone) human clinical trials. However, a number of limitations remain for this technology [83][21]:

♦ In vector selection and development: o The availability of the appropriate vector, that is able to ensure efficient delivery of the transgene(s) with no adverse effect to the patient. Such vectors have been developed for some but not all potential applications. o Cost and large scale production of the vectors. The more targeted the vector system the lower quantity of vector required per treatment. ♦ In cancer treatment: o Transfer of genes to the majority of cancer cells in solid tumours. This is due to increased distance from nearest blood vessel due to lower vessel density compared to normal tissue in most tumours; and increased interstitial fluid pressure. o Transfer of vectors to treat cancers of the brain and the eye is limited by the presence of the barriers between these organs and the body’s blood system. In treatment therapies for brain tumours the ability to inject only a relatively low dose of vector has been addressed through the use of virus producing cells (VPCs). VPCs are short-lived and incapable of migration but are able to produce replication deficient retrovirus vectors [64].

37 Head and neck carcinomas are particularly suited to a p53 treatment approach as 60% have a mutated p53 gene. This type of carcinoma is also common in China. 38 This number is much smaller than would be used in a Phase III trial in the USA or Europe. 2-37

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o The comparison of adenovirus and retrovirus vectors against human brain glioma has identified adenovirus vectors as able to achieve a higher gene transfer efficiency and improved patient survival [64]. ♦ In the application of oncolytic viruses: o Lack of understanding of the detailed genetic basis of diseases and the regulatory mechanisms involved. This has restricted the development of efficient vectors to target those diseases. o Untargeted delivery and expression of the transgene(s) leading to systemic toxicity and other side effects. o Immune reaction to the viral vector. Adverse immune reactions to the viral vector have been reported, particularly with adenovirus vectors. In some cases these have been severe. ♦ Oncogenic mutagenesis caused by chromosomal integration of the therapeutic gene [218]. Studies of cancer development (cited by [80]) have reported that progression to oncogenesis requires four to six genetic lesions within a single cell. Genetic mutation in somatic cells means that the acquisition of genetic lesions is a continuous process and most people develop enough ‘hits’ in a single cell by the age of 80 to develop cancer. Gene therapy vectors such as retrovirus-based vectors that involve the integration of the therapeutic gene therefore involve hundreds of millions of cells receiving a genetic ‘hit’. Although this is not proposed to result in cancer in the short-term it may be expected to increase the cancer risk over the patient’s lifetime. Whilst this may not be a consideration with current gene therapy trials that are used to treat life- threatening diseases, and that are often (at present) a ‘last-gasp’ treatment, it may become significant with more preventative type treatments that are administered earlier in a patient’s life. The risks posed can be reduced by improving vector efficiency and specificity, thereby reducing both the potential for oncogenic mutagenesis and the number of cells that are infected. ♦ Non-selective expression of the gene responsible for the conversion of the prodrug into the toxic compound in GDEPT-based, cytotoxic and pro-apoptotic therapies.

Future developments in gene therapy

2.100 Gene therapy as a therapeutic procedure is in development for a wide range of diseases and conditions. The number of these undergoing advanced stage clinical trials suggests increasing application of gene therapy in mainstream healthcare. The key limitations with the technology have been described above, and some of these relate to the characteristics of the GM vector used. Future developments in gene therapy are expected to address these limitations. As commented by O’Connor and Crystal (2006)[82], the biggest remaining challenges are how to achieve expression that is sufficient to correct the clinical phenotype without inducing the recipient’s defences that compromise safety and, for the integrating vectors how to minimise the risk of insertional mutagenesis, particularly if the corrected cells have

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a subsequent selective advantage and are continuing to proliferate. As the vectors are further developed, particularly with identification of serotypes and modifications of coat proteins that enhance gene transfer, the doses that are required to gain adequate expression will be reduced, with consequent enhancement of safety and efficacy.

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2.3 REVIEW OF CURRENT AND POTENTIAL FUTURE APPLICATIONS IN MEDICINE – VACCINATION

Attenuated vaccines Attenuated virus-based vaccines Attenuated bacteria-based vaccines Attenuated parasite-based vaccines Recombinant vaccines Recombinant virus-based vaccines Recombinant virus vaccines for immunocontraception Recombinant bacterial vaccines Recombinant bacterial spores as vaccine vectors Recombinant vaccines for the treatment of parasite mediated disease Summary of GM-based vaccines Approved GM-based vaccines GM-based vaccines in research trials Proposed GM-based vaccines

2.101 The primary application of vaccination has been the management of disease through the enhancement of an organism’s immune system against a particular disease agent. More recently, the use of vaccination has been extended to include the treatment of cancer and autoimmune responses, and birth control [6][219][177]. Vaccination confers an enhanced resistance to disease for both the recipient of the vaccine and the wider human or animal population through herd immunity 39 . As vaccinated individuals often disseminate or shed a significantly lower quantity of the pathogen than unvaccinated individuals then vaccination should effectively reduce the spread of a pathogen. Vaccination of animals may also provide indirect benefits to human health through reduced exposure of people to zoonotic diseases 40 ; and a reduction in the use of other veterinary drugs, and possible residues in food animal products [6].

2.102 With respect to the use of live GMOs in vaccines, genetic modification offers the potential to improve existing vaccines and to develop new ones which cannot be produced in a safe and/or effective form through conventional (non-GM) processes. The variability of RNA virus pathogens in particular means that genetic modification may offer the most effective approach to generate a vaccine that is active against the strain of pathogenic virus that is currently circulating in the field [255]. Both attenuated and recombinant GM vaccines have been developed [6][8][9]. The vaccines described in the following sections cover viral, bacterial, and parasite-based systems.

39 Herd immunity (also referred to as community immunity) describes the immunity that occurs when the vaccination of a portion of a population provides protection to unvaccinated individuals, as there is an insufficient number of unvaccinated individuals to sustain an epidemic. 40 Zoonotic diseases (or zoonoses), are diseases that can be transmitted from animals to humans. Examples include bubonic plague and rabies. 2-40

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Attenuated vaccines

2.103 Attenuated vaccines comprise a modified version of the target microorganism 41 that is less pathogenic but equally immunogenic as the wildtype target strain. The ideal attenuated vaccine is therefore entirely avirulent with no change in its immunogenicity, and 100% effective in preventing infection 42 [6][142]. To prevent reversion to wildtype two or more attenuating deletions are usually required. Although the modification (or attenuation) of the target microorganism can be achieved through both conventional and genetic modification techniques, only microorganisms that have been genetically modified are reviewed in this section. The development of the attenuation through genetic modification is reported to offer the advantage of a more defined attenuation than conventional approaches [200]. This has implications to understanding the potential for adverse non-target effects. The key feature of all attenuated vaccines is the balance between enough attenuation to prevent disease from occurring but not too much attenuation so that an immune response is not induced and protection is not conferred. Attenuated vaccines may be further modified to express additional functions (for example a marker protein). Whilst these may be considered both attenuated and recombinant, they have been described in the following attenuated section.

Attenuated virus-based vaccines 2.104 Attenuated virus-based vaccines have been investigated against a wide range of diseases. These include pseudorabies, bovine herpes virus type 1, bovine viral diarrhoea virus, Lassa fever, foot and mouth disease, infectious bursal disease virus (IBDV) 43 , and bovine respiratory syncytial virus [6][178][255]. The vaccine against pseudorabies virus (PRV) represents the first GM attenuated viral vaccine to be licensed by the USFDA (1986). This GM PRV vaccine was developed by the removal of the PRV gene encoding glycoprotein I and/or glycoprotein X. A subsequent PRV vaccine was developed with an additional marker to enable serological differentiation between livestock (pigs) that had been infected with wildtype PRV and those that had been vaccinated (Kit, 1990; cited by [6]). The presence of such a marker is viewed as a key characteristic of any veterinary vaccine 44 . The Vaxxitek HVT+IBD vaccine is a licensed GM attenuated herpesvirus designed to protect chickens against both Marek’s disease and infectious bursal disease.

41 For the purposes of this report ‘target microorganism’ refers to the microorganism for which the vaccine is designed to induce immunity against. 42 Realistically a level of >90% effectiveness is sought from vaccines, with the vaccine becoming effective at preventing infection within a few days to several weeks post administration, and for extended periods after that. 43 IBDV is a pathogen of major importance to the poultry industry. Because the virus targets B- lymphoid cells it can cause immunosuppression. This has wider implications as it exposes the poultry to infection by other pathogens, and also allows the failure of other vaccinations. The vaccination against IBDV is therefore important for reasons other than protection against the direct effects of IBDV. 44 An example of the significance of a differentiating marker is with foot and mouth disease (FMD). FMD-free countries generally restrict the use of the existing inactivated vaccine due to difficulties in distinguishing between a vaccinated and infected animal. 2-41

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2.105 The Lassa fever vaccine reported by Bergthaler et al. (2006)[178] was developed through the modification of the Lassa fever virus 45 to express an envelope glycoprotein from another arenavirus (lymphocytic choriomeningitis virus) in place of its own glycoprotein (a process termed envelope exchange). The attenuating modification made the Lassa virus avirulent, but still viable and with no change in its immunogenicity.

2.106 Although these GM attenuated virus vaccines have been reported, there are relatively few GM attenuated vaccines compared to recombinant ones 46 . This is probably because whilst developing the GM attenuated strain, additional sequences are likely to be introduced, for example cytokines. Such additional changes would make the GM vaccines recombinant rather than just attenuated [213].

Attenuated bacteria-based vaccines 2.107 Live attenuated non-GM bacteria-based vaccines are available commercially using Salmonella enterica serovar Typhi Ty21a and Mycobacterium bovis BCG strains [194]. Attenuated GM bacteria vaccines have been developed from these as well as the GM Vibrio cholerae CVD 103-HgR [226], and strains of Shigella sp. and Listeria monocytogenes 47 . The bacteria have been attenuated through the genetic modification of virulence, regulatory or metabolic genes. The attenuated bacteria replicate within the vaccinated person (or animal) and reversion to a pathogenic wildtype is a potential risk posed by these vaccines. (Reversion to pathogenic wildtype is a risk posed by both GM and non-GM attenuated vaccines).

2.108 The development of bacteria-based vaccines (attenuated or recombinant) has been pursued as they offer a number of particular advantages. They are easy and relatively inexpensive to produce on a large scale, and are potentially stable without refrigeration. They may also be controlled if required post release with antibiotics [194]. Where they are suitable for oral (or nasal) administration, for example if they are attenuated enteropathogenic bacteria, they provide the added advantage of inducing mucosal immunity [199]. Oral administration of a vaccine confers better mucosal immunity than parenteral administration (administration by injection or infusion). This is significant as 90% of human infections are initiated at mucosal surfaces [246], and oral rather than parenteral administration is better at protecting against them. (A similarly high percentage may be expected in other animals). Vaccination at a mucosal surface also allows for protection against infection, i.e. against colonisation as well as against disease [246].

45 Although primarily a disease of Africa, increased global air travel has allowed the movement of Lassa fever into Europe and the USA. 46 This may be a consequence of the definition of the terms ‘attenuated’ and ‘recombinant’ in the literature. In this report, attenuated vaccines which have had further functions added (for example production of cytokines) have still been described as attenuated. However, they may be described in the literature as either attenuated or recombinant. 47 Listeria monocytogenes replicates in the cytoplasm without coming into contact with the extracellular environment, and therefore does not elicit a strong humoral immune response. L. monocytogenes based vaccines are therefore more appropriate for diseases that do not require significant humoral immunity. 2-42

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2.109 Increased understanding of the genetic profile of bacteria such as Salmonella sp. has enabled the vaccine strains to be generated with a high degree of accuracy (in terms of the genetic modifications made)[203]. Salmonella strains have been genetically modified in a number of ways to produce avirulent but immunogenic strains for use as vaccines [154][204]. Each of these attenuated strains has been shown to be genetically stable, clinically safe and potent for their intended application. However, as of 2005, none had been used commercially [6] (although not for any specific reason):

♦ galE deficient mutants of Salmonella (Ty21a) have been licensed for use as an oral vaccine for typhoid fever [155]. This bacterium requires an external source of galactose as a consequence of the modification. ♦ deletions in the synthesis of aromatic compounds including amino acids has been used to attenuate salmonella serotypes such as Salmonella typhimurium, S. typhi , and S. enteritidis (cited by [6]). The S. typhimurium strain is reported to have been tested as an oral vaccine in calves against the wildtype strain. ♦ deletions in global regulatory proteins such as adenylate cyclase, cyclic AMP receptor proteins, and the expression of outer membrane protein genes.

2.110 The development of Salmonella vaccines in poultry is an area of significant commercial interest, although to date only the non-GM attenuated strain of S. enteritidis has been licensed (Merial’s Gallivac SE vaccine).

2.111 Clinical trials (Phase I) have been conducted with GM Shigella flexneri strain 2a, with the bacteria attenuated through the deletion of two genes encoding enterotoxins (ShETs 1 and 2), and also modified for guanine dependence. The modified shigella was reported to be immunogenic and well tolerated by the recipient patients [156]. The shigella strains used (CVD 1204 and CVD 1208) are two of the most widely evaluated shigella vaccine strains, both as attenuated and recombinant vaccines [194].

2.112 In Salmonella , regulatory genes such as the phoP/phoQ locus have been modified to alter the bacterium’s ability to survive within macrophages and its resistance to defensin and reduced pH [194]. In shigella, the modification of the virB/icsA, iuc and iut genes alters the ability of the GMO to move both intra- and intercellularly in the infected host and to survive in tissues [194]. The shigella strain SC602 expressing these modifications has been reported to provide protection against virulent Shigella flexneri in a Phase 1 trial following vaccination of 15 people with a single dose of 10 4 CFU [285].

2.113 The V. cholerae CVD 103-HgR strain does not express the active subunit of the cholera toxin, thereby rendering the strain non-pathogenic. It also possesses a mercury resistance gene ( hgR) which allows differentiation of the attenuated and pathogenic strains [194]. Further genetic modification of V. cholerae strains has been reported [194], including the complete removal of the cholera toxin genetic

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element (the filamentous phage Ctx ϕ) and its site of re-integration ( attRS1 ). The removal of attRS1 decreases the likelihood that the modified vaccine strains will reacquire Ctx ϕ in the environment and thereby become pathogenic. Attenuation of L. monocytogenes through genetic modification has been achieved through the deletion of actA and plcB which prevent cell to cell spread and escape from the phagosome, and escape from secondary vacuole respectively [194]. A small scale human trial (20 volunteers) vaccinated with an attenuated L. monocytogenes with deletions in actA and plcB found an immune response following administration of a dose of 10 9 CFU (no immune response elicited at 10 6 CFU dose) [194]. Aro mutations were also developed for Listeria monocytogenes [213].

2.114 The deletion or mutation of genes encoding for metabolic enzymes that are essential for survival of the GMO in the environment, or if required, within the mammalian vaccinated host (auxotrophic modifications) 48 , render the GM bacterium unable to survive and therefore biologically contained within the laboratory or within the recipient of the vaccine. Mutation of the purA and aro genes are two of the commonly used auxotrophic modifications [194]. The deletion of the Da gene and dat gene are also used, and render the GMO auxotrophic for D-alanine and D-amino acid transferase 49 respectively. S.enterica Typhi strain CVD 908-htrA, which has deletions of the aroC and aroD genes, as well as the stress response gene ( htrA ) has been used in multiple human and animal studies as both an attenuated vaccine against salmonella and as a recombinant vector for the protection against other pathogens [194]. It is not clear whether the aro and cya 50 deletions are truly bacteriocidal. However, bacteria modified with these deletions have a very low survival rate in the human body [308].

2.115 Deletion of aroA is the biological containment mechanism used in the Equilis StrepE ® vaccine to protect horses against strangles 51 [197][196]. The auxotrophic deletion prevents propagation of the GM Streptococcus equi in the vaccinated animal and is not reported to shed into the environment. The Equilis StrepE ® vaccine is the first and (as of 2007) the only licensed live GM bacterial vaccine for veterinary application [196]. The Equilis StrepE ®vaccine is designed to be administered submucosally to the upper lip as a single dose of 0.2ml containing 10 9 to 10 9.4 cfu of the GM S. equi [198].

2.116 A more extreme form of containment has been described for an attenuated Shigella flexneri that, in addition to its vaccine role, was modified through deletion of the aspartate-semi-aldehyde dehydrogenase gene ( asd ) to lyse in the absence of

48 Auxotrophic describes the inability of organism to synthesise a particular organic compound required for its growth. 49 D-amino acid transferase is necessary for cell wall biosynthesis. D-alanine is an essential component in the biosynthesis of bacterial cell wall peptidoglycan, as well as a major component of bacterial cell wall teichoic acids and lipoteichoic acids [308]. 50 The cya gene encodes adenylate cyclase. Deletion of cya affects the cyclic AMP pathway (adenosine monophosphate) which is involved in many essential metabolic functions.

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diaminopimelate. This compound is an essential component of the peptidoglycan of the cell wall of Gram-negative bacteria [194]. The absence of diaminopimelate in mammalian cells means that the GM Shigella flexneri vector lyses on entering the cells [157]. This lysis has the additional benefit of enhancing delivery of the antigen into the cells, as well as preventing dissemination of the live organism.

2.117 GM Shigella flexneri has also been reported as a vector for the delivery of DNA plasmids that, on being delivered to the target cells by the shigella are able to direct cell synthesis of the antigen [160][161]. The use of DNA plasmids in this way is a variation in the use of DNA vaccines. The advantage of using Shigella flexneri as a vector is that the DNA plasmid can be directed to the mucosal surface and promote mucosal immunity. Salmonella sp. and Listeria sp. have also been developed for this application.

Attenuated parasite-based vaccines 2.118 Parasite-induced diseases such as malaria and leishmania are significant causes of death across the world 52,53 . The observation that people who survive the infection subsequently express a life-long immunity against further infection (in the case of leishmania), or who develop immunity through repeated lower level exposure through childhood (as with malaria), is seen as evidence of the potential for vaccination to prevent these diseases [205][193].

2.119 Both malaria and leishmania are endemic in developing countries within the tropics, and increased global movements of people mean that both diseases are being seen increasingly in developed countries [200]. There is no effective human vaccine for leishmania, and prolonged treatment by chemotherapy is limited because of the high cost and development of drug resistance by the parasite [200][205]. With respect to a malaria vaccine, there is no licensed vaccine currently available [193], although clinical trials (up to Phase III) have been conducted with several subunit 54 and recombinant vaccines.

2.120 The development of a vaccine for each disease is viewed as the most appropriate means of tackling and preventing such widespread conditions [207][206]. With respect to leishmania, vaccination may be the only effective long-term approach to treatment as a low level of persistence of the parasite in vivo has been found to be needed to maintain the immunological memory against re-infection, and thereby

51 Strangles, also referred to as equine distemper, is a contagious upper respiratory tract infection of equines. It is caused by Streptococcus equi and is endemic in domesticated horse populations worldwide. 52 World Health Organisation reports that 3.2 billion people are at risk from malaria worldwide, with 300-500 million cases and >1 million deaths annually. 53 Trypanosomatid parasites of the genus Leishmania infect about 12 million people worldwide, with an annual death toll of 50,000 people/year. 54 Phase I trials have been conducted with FMP1 and Phase III with RTS/S based vaccines [9][2][262], and a Phase III trial with the SPff66 subunit vaccine [193]. These do not however contain live GMOs and therefore have not been reviewed in more detail in this report. 2-45

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ensure life-long protection. Such a requirement may only be possible with a GM attenuated vaccine to ensure that reversion to wildtype does not occur [200].

∗ Leishmania 2.121 There are two major clinical forms of leishmania, cutaneous and visceral, which are caused by infection of different species of the parasite. Visceral leishmania is caused by Leishmania donovani, L. infantum and L. chagasi , and is fatal if not treated. Cutaneous leishmania is caused by L. major, L. tropica, L. aethiopica, L. mexicana or L. braziliensis [200]. Although both forms are a disease of humans, dogs can be an important reservoir for the pathogen in some countries (for example foxhounds in the USA [224]).

2.122 The infection of people occurs when the person is bitten by a sandfly carrying the motile promastigote form of the leishmania parasite in its alimentary canal. Following transmission, the parasite is ingested by the recipient’s macrophages where it then differentiates into the non-motile amastigote form. The parasite is able to survive in the macrophage in this form [200].

2.123 The genetic modification of leishmania to overexpress a mutant form of a particular protein (and therefore not express the required protein in its correct form), has been reported to cause a reduction in the growth of the parasite in both its promastigote and amastigote forms and reduced survival in macrophages [200]. Other GM-based approaches have sought to knock out particular genes through homologous recombination [200], and have reported a successful reduction in virulence of the leishmania 55 . As of 2006, the gene knock-out approach has achieved an attenuated phenotype for L. major, L. mexicana, and L. donovani (Table 2.3) [200]. These GM leishmania exhibited either reduced survival in macrophages or in the sandfly vector, or avirulence in a mouse model.

55 Because Leishmania sp. is diploid throughout its life cycle it is necessary for any genetic modification to disrupt both the alleles of the particular gene. 2-46

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Table 2.3 Gene knock-out studies for the generation of GM attenuated Leishmania sp. (adapted from [200]) Leishmania Gene(s) knocked Effect of modification on Mutant phenotype species out growth/survival of the leishmania L. major Dihydrofolate Auxotrophic for Survival in macrophage is thymidine reductase – thymidine. dependent, with the GM leishmania thymidylate incapable of causing disease in mice and synthase ( dhfr-ts ) rhesus monkeys. The GM leishmania did not protect monkeys on challenge with virulent parasite. L. major Leishmanolysin Deficient in Showed normal development in sandfly, (gp63 genes 1-7) leishmanolysin; no but delayed lesion formation in mice. change in growth in vitro . L. major Galactofuranosyl Deficient in LPG but Did not infect sandfly, mouse or transferase ( lpg1 ) contained normal levels macrophages. of related glycoconjugates and GPI- anchored proteins. L. major Golgi GDP-Man Mutant lacked all Unable to survive in sandfly, persisted transporter ( lpg2 ) phosphoglycans. indefinitely in mice but with no disease, provided protection from challenge with virulent parasites in the absence of a strong Th 1 response. L. mexicana Glucose Promastigotes showed Reduced growth rate in sandfly mid gut, transporter genes reduced growth rate in and reduced infectivity in macrophages. (LmGT1, LmGT2, vitro . LmGT3 ) L. mexicana Cysteine Deficient in cysteine Reduced infectivity in macrophages, proteases ( cpa, protease, no change in attenuated virulence in mice and provided cpb, cpc ) growth in vitro . protection upon challenge with virulent parasite. L. donovani Partial knockout of The proliferation of Attenuated virulence in mice. A2-A2rel gene mutants in culture clusters compromised. L. donovani Biopterin Biopterin transport Reduced infectivity, generation of parasite transporter ( BTI ) abolished. specific production of IFN-γ. Provide protection following challenge with virulent parasite. L. donovani Centrin ( Ldcen ) Defects in cytokinesis in Reduced parasite survival in macrophage. amastigote form.

∗ Malaria 2.124 Malaria occurs following the transmission of infectious Plasmodium sporozoites from an infected female Anopheles mosquito to a person during a blood meal. Following infection the sporozoites move to the liver where they multiply in the hepatocytes (liver cells) and subsequently infect erythrocytes (red blood cells). Whilst malaria- associated pathology is restricted exclusively to the asexual replication of the parasite within the erythrocytes [193], it is a complex disease and questions have been raised as to whether the development of a whole parasite attenuated vaccine is worthwhile, considering the progress being made with the more economically viable subunit vaccines [193].

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2.125 A GM attenuated P. falciparum has been developed that is deficient in the gene uis3 [208]. The uis3 gene (upregulated in infective sporozoites gene 3) is essential for the early liver-stage development of the parasite. The GMO is able to infect the hepatocytes but is then unable to establish a blood-stage infection. Consequently it is unable to cause disease. Vaccination of rats with the GMO was found to confer complete protection against an infectious sporozoite challenge, thereby demonstrating the potential for GM attenuated vaccines for malaria.

2.126 Genetically attenuated parasites (GAPs) such as the uis3 and uis4 deficient Plasmodium sp. described above have a number of advantages over γ-irradiated parasites that have also been used as the basis for a whole-parasite attenuated vaccine. These include the production of a more consistently attenuated organism, better genetic stability and higher potency [209]. The attenuation may also ensure the biological containment of the GM Plasmodium within the recipient, as failure to establish a blood-stage infection means the parasite will not complete its erythocytic cycle and become available to a mosquito (for subsequent re-infection).

Recombinant vaccines

2.127 Recombinant vaccines are designed to provide the same function as attenuated vaccines, namely avirulence, ability to induce a strong immunogenic effect, and achievement of a high level of protection. For the purposes of this report, recombinant and attenuated vaccines are differentiated in most cases by recombinant vaccines operating as a vector for the delivery of an antigen(s) to induce protection against a different microorganism; for example Salmonella enterica genetically modified to express the tetanus toxin fragment C (TTFC) from Clostridium tetani, or Newcastle Disease virus (NDV) as the GM vector to express antigens of the avian virus IBDV [167]. However, recombinant vaccines also include bacteria or viruses which are genetically modified to improve their immunogenicity to the patient, through the insertion of cytokines or intracellular trafficking molecules for example. In this case the GMO is a modified version of the microorganism for which the vaccination is designed to induce protection to.

2.128 The recombinant vector microorganism may be an attenuated pathogen or completely non-pathogenic. Where the vector microorganism is an attenuated pathogen then the recombinant vaccine may induce protection against more than one pathogen. The non-pathogenic bacteria used are typically organisms used in the food industry, e.g. lactic acid bacteria 56 that have a long history of safe use [148].

56 The lactic acid bacteria are a taxonomically diverse group of non-pathogenic Gram-positive bacteria. They are found in a variety of environments such as plant surfaces and as part of the resident microflora in the GI tract of vertebrates. They have a long history of safe use as key components for the manufacture of fermented dairy products (cheese, yoghurts and sour milks) [10]. Lactococcus lactis for example is regarded as ‘safe’ by the USFDA with regard to its use in food products [250]. The well characterised and non-pathogenic nature of these microorganisms makes them strong candidates as medical/veterinary vectors [171]. 2-48

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Recombinant virus-based vaccines 2.129 Most of the recombinant virus-based vaccines developed to date have used poxvirus, adenovirus or herpesvirus as the vector, although other viruses such as NDV 57 (a paramyxovirus) and vesicular stomatitis virus (an arbovirus) are also under consideration [181]. Adenovirus is for example particularly effective as a vaccine vector for the development of mucosal immunity [169]. The non-GM LaSota strain of NDV is used widely as a live vaccine for poultry diseases and confers long-lasting humoral, cellular and mucosal immunity. NDV has a simple genome encoding few proteins, and this is reported to assist in minimising a non-specific immune response to the vector [167]. Recombinant viral vaccines have consequently been developed using the LaSota NDV strain as the base.

2.130 The virus vectors are either replication-defective or replication competent. Ability to replicate has implications to their use as a vector, and the potential risks they may pose to the environment and human health. Replication-defective vectors are in general likely to pose a lower risk to the environment and wider public health by virtue of the biological containment conferred by their inability to replicate and therefore survive in the environment. Replication-defective viruses however, may be less immunogenic and therefore less able to confer protection compared to replication competent strains.

2.131 Poxviruses in particular have been used as the basis for live recombinant vaccines for livestock (cattle, pigs, poultry for example) and companion animals (dogs and cats)[136]. The commercially available RABORAL V-RG58 rabies vaccine for example is based on a thymidine kinase negative vaccinia Copenhagen strain, and is used in both Europe and North America for the oral immunisation of wildlife against rabies [136] 59 . The TROVAC-AIV H5 is a live recombinant fowlpox licensed for use against avian influenza in Mexico, Guatemala and El Salvador (over 2 billion doses administered) 60 . As well as protecting chickens against avian influenza induced mortality for at least 20 weeks, the TROVAC vaccine also causes a significant reduction in shedding of the influenza virus post infection [182]. Additional reported advantages of the TROVAC vaccine are that vaccinated poultry can be differentiated from influenza-infected birds, and that it is immunogenic in cats, thereby indicating the potential for use in mammalian vaccines [182].

57 Elimination of expression of the V protein of Newcastle Disease virus (NDV) causes a significant reduction in viral pathogenesis but has no effect on its immunogenicity [167]. 58 Produced by Merial. 59 Vaccinia virus is used as the basis for vaccines against rabies environmentally robust and will retain viability post release. Other orthopoxviruses, such as racoon poxvirus are used as the vector for plague vaccine for similar reasons. However, under certain environmental conditions even robust virus vectors may not be suitable. Whilst GM vaccinia virus is used as the vaccine of choice for the immunisation of wild red foxes against rabies in Europe, concerns over the ability of the virus to survive freezing temperatures means that it is not used as the rabies vaccine for arctic foxes. Freeze- dried attenuated rabies virus is used instead [199]. Other GMOs, for example the GM bovine herpesvirus designed for the vaccination of wild pigs against porcine herpesvirus is more labile and is distributed in a protective lipid matrix to assist survival before ingestion. 60 TROVAC-AIV H5 was also licensed in 1998 for emergency use in the USA. Reported to protect against a wide panel of H5-subtype avian influenza strains regardless of neuraminidase subtype. 2-49

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2.132 A particular advantage of poxviruses from a biosafety standpoint, is that they are unable to replicate in the vaccinated animal, either as a consequence of attenuation or species specificity. Attenuated poxviruses such as the vaccinia Ankara strain 61 for example is even unable to replicate in immunosuppressed animals, although its production of immunogenic proteins, and therefore its immunogenic effect is unaltered (Sutter and Moss, 1992; cited by [6]). Avian poxviruses such as fowlpox virus and canarypox virus offer the advantage in that they replicate poorly in mammalian cells but with no concomitant effect on the expression of foreign antigens [183][136]. Of the avian poxviruses, canarypox virus is reported to be a better vaccine candidate [137] and offers the advantages of being able to induce both humoral and cell mediated immunity, acting in the absence of an adjuvant 62 , and being non-pathogenic to birds (including canaries)[136].

2.133 The recombinant canarypox ALVAC ® virus system 63 has been used as the basis for commercially available vaccines for a variety of companion animals and horses (Table 2.4). Its use is also being assessed in sheep and pigs, and as a vector against HIV in human clinical trials 64 :

♦ sheep as a vector for the VP2 and VP5 genes of Blue Tongue virus (BTV) to vaccinate against BTV-induced viraemia; ♦ pigs as a vector for the envelope fusion protein or glycoprotein of Nipah virus. This work has the double aim of inducing immunity against Nipah virus, and also to prevent nasal and pharyngeal shedding 65 of the Nipah virus and subsequent exposure of people [141]; ♦ humans as the vector for the vCP1521 HIV-1 vaccine (AIDSVAX). The vaccine which incorporates envelope antigens from the predominant strain of HIV in Thailand, is undergoing Phase III trials in Thailand [318]. The AIDSVAX vaccine is the only HIV vaccine to have entered Phase III trials [287].

2.134 Fowlpox virus has been used as the base for a poultry vaccine against Newcastle disease and avian influenza H5. The fowlpox virus does replicate in chickens but is reported not to be shed in the environment after vaccination [138]. Fowlpox virus does not replicate in mammalian cells [184].

61 Safety of the attenuated vaccinia virus Ankara strain (MVA or modified vaccinia Ankara) has been demonstrated through the use of this virus in the eradication of smallpox. The Ankara strain lacks ~10% of the vaccinia virus genome. 62 Adjuvants are substances and formulations in vaccines that non-specifically raise immune responses to an antigen; although the mechanism of action of most adjuvants is poorly understood. The use of adjuvants is more widespread in veterinary medicines, with only three adjuvants reported to be licensed for use in human medicine [255]. The absence of an adjuvant in the canarypox-based vaccine is significant in that it negates the possibility of inflammatory side effects of the adjuvant. 63 The canarypox strain used in the ALVAC system was isolated originally from a single pox lesion in a canary and serially passaged 200 times in chicken embryo fibroblasts and serially plaque purified under agarose [255]. 64 The canarypox ALVAC system is also being used as a gene therapy tool for the delivery of IL-2 to target tumours [140] following successful trials in cats against fibrosarcoma (using feline IL-2). 65 As Nipah virus is a zoonotic virus transmitted to humans from pigs, then the vaccination of pigs should prevent subsequent exposure of people. 2-50

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Table 2.4 – Canarypox virus vector vaccines licensed for veterinary application (adapted from [136]) Species vaccinated Target disease Modification (insertion of) Dog, ferret Canine distemper Haemagglutinin and fusion protein Cat Rabies Glycoprotein Envelope glycoprotein, group specific Cat Feline leukaemia antigen / protease Horse Equine influenza Haemagglutinin Horse West-Nile Pre-membrane and envelope proteins 2.135 Non-replicating virus vaccines may be limited in terms of the duration of immunity (DOI) that is induced by the vaccine. However studies with canarypox-based vaccines against feline leukaemia, and West Nile virus in cats and horses respectively reported a DOI of at least one year [136].

∗ Replication deficient recombinant viral vaccines 2.136 Replication deficient human adenovirus is described as one of the most efficient recombinant vector systems for the delivery of vaccine antigens to humans [142]. The human adenovirus (HAd) used in most vaccine studies conducted to date is HAd serotype 5 (HAd5). This serotype is the best characterised of all human adenoviruses, and has been used extensively as a vector in gene therapy applications [144]. Preclinical and clinical trials involving HAd5 as a vaccine vector are currently being conducted for:

♦ Ebola virus. Study being conducted in primates, with the adenovirus modified to express Ebola virus glycoprotein and nucleoprotein [119]. ♦ HIV, with the HAd5 modified to express HIV-1 gag gene. Study being conducted in monkeys and baboons [149]. ♦ Anthrax. Studies being conducted in a mouse model [121]. ♦ Severe acute respiratory syndrome (SARS). Study being conducted in monkeys [303].

2.137 HAd5 has also been used successfully as the vector for a number of animal vaccine trials (described below):

♦ the rabies vaccine, with the HAd5 (replication deficient, and replication competent) expressing rabies glycoprotein. Administration of the vaccine by intramuscular, subcutaneous or intranasal routes induced immunity in a range of animals including rodents, dogs, foxes and skunks. Oral administration failed to induce a similar response [142]. ♦ a vaccine for porcine respiratory and reproductive syndrome virus (PRRSV). Developed through the modification of HAd5 to express the PRRSV envelope protein GP5 [158]. ♦ a vaccine for foot and mouth disease (FMD). The most recent version of this vaccine, based on HAd5, has been shown completely to protect pigs within 24 hours of vaccination. This is significantly quicker than the seven days achieved

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with the existing inactivated vaccine, and could therefore provide a more effective strategy for dealing rapidly with outbreaks of FMD [142]. ♦ a vaccine for swine influenza virus (SIV). The development of an effective vaccine against SIV is important both in economic terms for the protection of pigs, and also in relation to wider public health. Pigs have a significant role in the global ecology of influenza A viruses, and consequently in the development of potentially pandemic influenza strains. Whilst commercially available SIV vaccines (inactivated, attenuated and sub-unit) are used, they do not provide complete protection. Two recombinant HAd5 vaccines have been developed, expressing the influenza virus H3 haemagglutinin, and the nucleoprotein (cited by [142]). Pigs vaccinated with both recombinants in a mixture were found to be completely protected. ♦ a vaccine for bovine herpes virus 1 (BHV-1). Developed using both replication- defective (E1 deleted) and replication competent (E3 deleted) HAd5 expressing the glycoprotein D (gD) gene (full length and truncated) from the BHV-1 envelope [162]. Whilst both the replication competent and replication deficient versions induced an immune response to BHV-1, the full length gD induced the higher immune response. A similar study using bovine adenovirus (serotype 3) to express BHV-1 gD glycoprotein found the replication competent virus induced a more effective immune response [163]. ♦ a vaccine for rinderpest virus (RP) and peste des petits ruminants virus (PPR). Both viruses are highly contagious and cause significant economic impact on cattle farming in infected areas. Although attenuated tissue culture vaccines, delivered by subcutaneous injection, are available, an orally administered vaccine would be cheaper and also suitable to the treatment of wildlife (which comprise an important reservoir for the virus and thus an obstacle to global eradication of these viruses). A recombinant HAd5 expressing two different capsid proteins from the viruses are currently undergoing veterinary trials in cattle [142].

2.138 With regard the non-target effects of these adenoviral vaccines, animal adenoviruses are typically species specific and therefore present negligible risk to humans or to other animal species [169].

∗ Replication competent recombinant viral vaccines 2.139 Whilst most of the vaccines tested in clinical trials for human use are based on vectors that do not replicate in humans; replication competent viruses are under investigation as vaccine vectors in human and also veterinary medicine [136]. An advantage of replication competent vectors is that they may induce better mucosal immunity. This is of particular importance for the immunisation against pathogens that infect through the mucosal membranes, such as those in the respiratory or genital tracts [142].

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2.140 Examples of replication competent viruses that have been used as viral vectors include:

♦ a canine distemper virus (CDV) vaccine. Based on canine adenovirus (serotype 2), modified to express haemagglutinin or fusion proteins of CDV in the E3 region of the adenovirus [216]. ♦ a vaccine for emerging strains of feline calicivirus, for example those which cause “virulent systemic disease”. A recombinant attenuated myxoma virus (poxvirus) modified to express the capsid proteins of two strains (F9 and LS015) of feline calcivirus (FCV) (causative agent of the acute oral and upper respiratory tract disease in cats) [202]. Although non-GM vaccines (both live attenuated and inactivated preparations) are available, there are concerns that antigenic variation in wild strains of FCV may lead to the emergence of new strains which are not covered by the vaccine. A bivalent vaccine is viewed as the most appropriate means of protecting against new strains. The myxoma virus was modified through the insertion of the two FCV capsid proteins into the myxoma’s virulence factor gene loci. This produced a significantly attenuated myxoma virus without a concomitant effect on its growth. The authors of the study [202] commented that the strong possibility of unwanted recombination events with poxviruses made it difficult to obtain the bivalent construct required 66 . ♦ a classical swine fever (CSFV) vaccine. CSFV is an important disease of pigs (in economic terms) in areas of intensive pig farming 67 . A replication competent recombinant porcine adenovirus modified to express the CSFV envelope glycoprotein E2 is reported to provide complete protection following two oral 6 68 doses of 10 TCID 50 /animal , or one dose administered subcutaneously [164]. ♦ a vaccine for avian infectious bronchitis virus (IBV). Recombinant replication competent fowl adenovirus expressing the IBV glycoprotein S1 was found to confer mucosal immunity to chickens following a single dose of 10 6

TCID 50 /animal [143]. Avian adenovirus has a much larger genome than mammalian adenovirus. ♦ a vaccine for infectious bursal disease virus (IBDV). A recombinant replication competent fowl adenovirus expressing the VP2 gene from IBDV has been developed for this disease of chickens. Protection has been achieved following a dose of 107 pfu/bird [166]. A reported advantage of a GM recombinant vaccine for IBDV is that conventional live vaccines (based on attenuated IBDV) may contribute to the emergence of antigenic variant viruses through reassortment between vaccines and field strains of IBDV. Using a recombinant GM vaccine (with adenovirus or NDV as the vector) avoids any reassortment issues as the vaccine does not contain live IBDV [167].

66 Whilst they may not pose a risk per se , unwanted recombination events are also not desirable from a risk assessment perspective. 67 Classical swine fever was last reported in the UK in 2000. 68 TCID 50 is 50% of the tissue culture infective dose. 2-53

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♦ a vaccine for transmissible gastroenteritis (corona) virus (TGEV). Infection with TGEV in a fully susceptible herd results in almost 100% mortality of newborn piglets, and existing commercial vaccines (both attenuated and inactivated) do not fully protect piglets. A recombinant HAd5 expressing the spike (S) protein has been reported to protect pigs without side effects [168]. Subsequent studies have shown that a porcine adenovirus (PAd5) with the same modifications is more effective than HAd [169]. The GM porcine adenovirus was detected in rectal swabs until 6-7 days post oral administration [169]. The use of porcine or bovine adenoviruses as vaccine vectors is reported not to require an adjuvant to induce the required immunogenic response (see earlier footnote with respect to adverse effects associated with adjuvants) [142]. ♦ a vaccine for Lassa fever [181]. Vesicular stomatitis virus (VSV) modified to express Lassa fever glycoprotein. A single intramuscular vaccination induced strong humoral and cellular immunity in a monkey model, with no detectable shedding of the VSV. However, the protection was only short-term and therefore unsuitable in endemically-infected areas [178].

2.141 The potential generic hazard of replication competent viruses in terms of risks to the environment and human health is of course their ability to replicate, as this improves their potential to persist in the environment if released. Concerns over potential risks from replication competence can be reduced/negated if the attenuated virus is well characterised with a history of safe use. Such viruses are those organisms already used in live vaccination programmes, such as yellow fever virus, vaccinia and influenza A [179]. The genetic modification of these vaccine strains utilises a well characterised base to generate a vaccine against different pathogens. Three particular examples of the use of non-GM vaccines with a history of safe use as the base for recombinant vaccines are:

♦ the West Nile virus vaccine that is based on the yellow fever vaccine strain 17D genetically modified to express the envelope genes of the West Nile virus. A key characteristic of the West Nile vaccine is that the genes encoding the pre- membrane and envelope structural proteins of the yellow fever vaccine strain were replaced with corresponding genes from wildtype West Nile virus. The modified chimeric virus therefore contains the antigens responsible for protection against West Nile virus whilst retaining the replication efficiency of yellow fever strain 17D. The modified yellow fever strain is being marketed commercially as the ChimeriVax system, and has been used in a similar way to develop vaccines against other flaviviruses such as Japanese Encephalitis virus and Dengue virus [170][272]. ♦ the LaSota strain of NDV currently used commercially as a live attenuated vaccine against Newcastle Disease in poultry. This has been genetically modified to express the VP2 protein from IBDV, thereby providing protection against two avian diseases [167]. The recombinant vaccine has been tested successfully in chicks [167]. Multivalent vaccines such as this are particularly attractive to the poultry industry on cost grounds (as the value of an individual bird is relatively small). NDV replicates preferentially in avian cells, but is

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replication deficient in mammalian cells [167]. (NDV is for example under development as an ‘armed’ oncolytic vector for gene therapy/cancer treatment in humans)[61]. ♦ the attenuated Influenza A (H1N1) 69 virus modified to express antigens to induce immunity against the bacterial pathogen Chlamydia trachomatis [180]. The modification involved the insertion of the immunodominant T-cell epitopes from chlamydial major outer membrane protein into the stalk region of the influenza’s neuraminidase gene. C. trachomatis is a pathogen of the genital mucosa and therefore any vaccine must be able to induce mucosal immunity. GM attenuated influenza A virus delivered intranasally in mice is reported to also confer immunity to the genital mucosa. The vaccinated mice exhibited protective immunity to chlamydial infection, and significantly also shed less chlamydiae.

2.142 As described previously genetic modification provides a means to introduce specific alterations into a microorganism. Attenuations produced by genetic modification are therefore better characterised than those produced by conventional means. As a consequence GM attenuated vaccines should in general be safer than non-GM attenuated vaccines with respect to reversion to wildtype.

Recombinant virus vaccines for immunocontraception 2.143 The previous text on the applications of recombinant viruses as vaccine vectors considered vaccines as a therapy for the prevention of disease. Recombinant virus- based vaccines have also been reported as a contraception tool. This application, termed virally vectored immunocontraception (VVIC) or immunoneutering (VVIN) [186][255], is viewed as sufficiently different to warrant separate consideration to the other viral vaccine applications. VVIC is designed to prevent either fertilisation of the oocycte by sperm or implantation of the fertilised egg whilst retaining the animal’s sexual behaviour patterns and competition in mating. VVIN is designed to prevent all sexual behaviour in both male and female animals. VVIC is most suited to the control of feral animal pests and native wildlife, whereas VVIN is proposed as more appropriate for companion animals, livestock, and in some cases feral animal pests [255]. For companion animals and livestock the vector can be delivered by injection, whereas for wild animals delivery through treated bait is a more appropriate strategy. A further approach uses the natural ability of the virus to spread through the target population [187].

2.144 The basis of VVIC is that the immune system of the vaccinated animal is stimulated to attack its own reproductive cells and thereby rendering it sterile 70 [186]. The reported applications include population control of captive and wild animals, the control of aggressive behaviour (through consequent reduction in fertility hormones), and the improvement of meat quality in livestock [177]. This approach provides a

69 Low pathogenic H1N1 strains of influenza virus exist in the wild (community) and were responsible for approximately half of all flu infections in 2006 (United States Center for Disease Control). 70 Immunocontraception strategies are also being developed without live GM vectors. Such strategies are outside the scope of this report. 2-55

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humane alternative to methods relying on surgical or chemical sterilisation, or lethal control.

2.145 Laboratory studies have confirmed the proof of concept for VVIC in mouse, rabbit and fox [210]. The virus vectors used are species specific, with the mouse cytomegalovirus (MCMV) and ectromelia virus (ECTV) used to express the mouse ovarian glycoprotein zona pellucida 3 (mZP3) in mice [211][212], and the myxoma virus in rabbits [187]. These three viruses are all considered species specific [19][187]. Evaluation of VVIC for foxes using canine herpesvirus (CHV-1) reported that the virus was less species specific than MCMV, ECTV and myxoma viruses, although only canids appeared susceptible to infection [20]. European Red Foxes that ate treated bait containing unmodified CHV-1 did not become ill and did not shed infectious virus into the environment [42]. This study supported the suitability of CHV-1 as a vector for VVIC in foxes.

2.146 Field trials using surgical sterilisation to mimic immunocontraception have reported that sterility in ≥70-80% of the target population is required to reduce population sizes significantly [187].

2.147 Whilst studies with mice using GM MCMV reported long-term infertility in up to 100% of female BALB/c mice, trials with rabbits and foxes have been found to be less successful with the contraceptive effects not lasting as long as the reproductive lifespan of the animal [187]. The technology may therefore be more suitable for the treatment of companion animals or livestock where repeat treatments (by injection) may be administered. Field trials in Australia, where much of the VVIC work has been conducted are currently on hold.

2.148 Where the VVIC strategy is designed to control wild populations, for example a rodent pest, the virus vector needs to be replication competent to ensure sufficient spread, and therefore control through the pest population. This of course has implications to the environmental containment of the GM virus.

Recombinant bacterial vaccines 2.149 A wide range of bacteria are reported to date to have been genetically modified for use as potential vaccine vectors, although none has (as of 2006) reached the market [148]. Recombinant bacterial vectors are genetically modified either to express particular protein antigens, or to deliver DNA encoding the required antigens [199]. Bacteria modified to date for these applications include Shigella flexneri, Salmonella enterica, Yersinia enterocolitica, Listeria monocytogenes, Bordetella bronchiseptica, Erysipelothrix rhusiopathiae, Mycobacterium bovis, and Brucella abortus (Table 2.5). These bacteria are either attenuated pathogens, or strains with a low pathogenicity, that have been genetically modified to express additional antigens. Whilst there are potential risks with using pathogenic organisms as vaccines (see Chapter 3), the inherent invasiveness of these bacteria does enable them to enter the recipient’s cells and deliver the antigen to the recipient’s antigen presenting cells. This is more

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difficult to achieve with non-pathogenic vectors such as the lactic acid bacteria, although it has not prevented their use as vaccine vectors.

2.150 The different mechanisms of pathogenesis between microorganisms can be utilised to deliver the target antigen to the required area. Shigella flexneri for example normally does not translocate beyond the intestinal mucosa and therefore its use as a vaccine vector generates an exclusively mucosal response. In contrast Salmonella enterica can induce both a mucosal and systemic response as it can translocate beyond the mucosal membrane [199].

Table 2.5 – Summary of attenuated recombinant bacteria used as vaccine vectors (adapted from [148]) Attenuation (genes Animal Bacterial vector Foreign insert deleted) model ∆asd pCMV β guinea pig ∆asd CS3 and LTB/STm mouse Shigella flexneri ∆rfbF HIV-1 SF2Gag mouse ∆dapA, ∆dapB Β-gal, gene vaccine in vitro ∆aroA, ∆iscA gp120, gene vaccine mouse pCMV β, pCMV octA and in vitro , ∆aroA pCMV hly mouse ∆aroA, ∆aroD Clostridium tetani TTFC mouse Salmonella enterica ∆aroA, ∆htrA TTFC mouse ∆aroA and others GFP and cytokines mouse ∆cya, ∆crp, ∆asd SP10 cDNA mouse GalE and unspecified Helicobacter pylori, ure AB human pYV- Brucella abortus, P39 mouse Yersinia enterocolitica pYV- Ova mouse ∆actA Leishmania major mouse ∆actA LCM virus mouse Listeria monocytogenes ∆dal, ∆dat HIV-1 gag gene vaccine mouse Mycobacterium bovis gene ∆2 mouse vaccine Bordetella ∆aroA TTFC mouse bronchiseptica Erysipelothrix Mycobacterium Tn916- mouse, pig rhusiopathiae hyopneumonie Plasmodium falciparum, Mycobacterium bovis Unspecified mouse CSP Brucella abortus Rough mutant (O -) lacZ or HSP65 mouse

2.151 Although they are non-invasive, lactic acid bacteria offer a number of characteristics that make them good candidates for use as oral vaccine vectors. These include an adhesive ability to mucosal surfaces, tolerance to gastric acid and bile salts, and the production of antagonistic substances against pathogenic microorganisms. They are also suitable for use in immunocompromised individuals.

2.152 Prototype vaccines using Lactobacillus sp. as the vector have or are being developed against Brucella abortis (expression of antigen L7/L12), Heliobacter pylori (urease subunit B), anthrax, rotavirus (bovine rotavirus non-structural protein 4) and tetanus (tetanus toxic fragment C). Similar vaccines have also been developed

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against HIV (V3 domain of the HIV type 1 glycoprotein 120) and measles using Streptococcus gordonii as the vector (cited by [116]). Some of the studies have reported that lactic acid bacteria-based vaccines do not require an adjuvant [116][150].

2.153 GM Lactobacillus sp. has also been used as a vector for the delivery of preformed antibodies. This so called passive vaccination 71 approach has been used in a rat model for the treatment of dental caries. The rats were treated orally with GM Lactobacillus zeae expressing a single-chain antibody fragment (scFv) recognising the adhesion molecule of Streptococcus mutans. The treatment reduced the number of S. mutans present and consequently the development of caries [171]. Constitutive expression (rather than inducible expression) of the scFV conferred a greater level of protection [172]. The application of GM lactic acid bacteria in a passive vaccination role was further supported by the modification of:

♦ Streptococcus gordonii to express a microbiocidal single-chain antibody for the treatment of vaginal candidiasis (also in a rat model) [217]; and ♦ Lactobacillus plantarum to express the birch pollen allergen Der p1 [219]. ♦ Lactobacillus paracasei to express constitutively the neutralising variable domain of llama heavy-chain (VHH) antibody fragments against rotavirus [173]. Oral administration of the GM lactobacillus shortened disease duration, severity and viral load in a mouse with rotaviral-induced diarrhoea. The findings are described as applicable to other diarrhoeal diseases [173].

2.154 With the exception of the IL-10 expressing Lactococcus lactis [10], little information is available on the specific biological containment measures applied in the bacterial vectors described above. One general containment approach that may be applied involves the insertion of the new antigen into the vector’s chromosome in place of a metabolic essential gene, thereby making the GMO auxotrophic for the gene product. If that essential compound is not readily available then the GM bacterium is unable to survive in the environment. However, should it reacquire the deleted gene (through horizontal gene transfer) 72 then it will lose the transgene encoding the antigen, and consequently be no different to the non-GM strain.

Recombinant bacterial spores as vaccine vectors 2.155 Some work has been reported into the use of bacterial endospores as vaccine vectors [220][222]. The potential advantages offered by endospores are hardiness of the spore, ease of genetic manipulation, cost-effective manufacturing and long- term storage. Bacillus subtilis endospores are also non-pathogenic and reported to have a history of safe use as a probiotic for both humans and animals [221].

71 Delivery of pathogen- or toxin-neutralising agents, most commonly immunoglobulins. However, because complete immunoglobulins are difficult to produce in bacteria, delivery has focused on just the binding domain of the immunoglobulin [116]. Described as ‘passive vaccination’ as it involves the transfer of antibodies (i.e. the generation of ‘passive immunity’). 72 Horizontal gene transfer is defined as the nonsexual transfer of genetic information between genomes or between different organs in the same or different species [153]. 2-58

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2.156 Recombinant B. subtilis spores displaying tetanus toxin fragment C (TTFC) conferred protection in a mouse model to an otherwise lethal challenge of tetanus toxin following oral administration [220]. The TTFC fragment is non-toxic and immunogenic. In the treated mice, a dose of 10 9 spores for five consecutive days found the B. subtilis in faeces for at least four days after the final dose (1.44x10 3 total bacilli, and 1.08x10 3 spores). Counts in faeces were higher during the dosing period (1.68x10 6 total bacilli, and 1.73x10 6 spores on day 1 for example). These results demonstrated that dissemination of the bacilli may occur, and that some germination of the endospores occurred in the intestine. Duc et al. (2003)[220] also commented that the vegetative bacilli arising from the germinated spores do not survive long term in faecal matter.

2.157 The hardiness of endospores may be an issue for risks to the environment as the GM endospores will be able to survive for a significant period of time (years) in the environment should any release occur.

Recombinant vaccines for the treatment of parasite mediated disease 2.158 Some research has been conducted into the development of recombinant parasite vaccines, using GM bacterial or viral vectors to express Plasmodium sp. or Leishmania sp. antigens [201]. Results with these applications have been mixed. With respect to the leishmania vaccines, some success has been reported in animals, although the immunogenic response in humans has been inconsistent.

2.159 Poxvirus, adenovirus and alphavirus are reported to have been used as viral vectors for the delivery of malaria antigens [188][189][190]. Some of these vectors have been found to induce malaria-specific immunity in animal models and human trials. The development of immunogenicity is suggested to be affected both by the viral vector used and the presentation of the plasmodium genes (in terms of codon optimisation 73 , de-glycosylation and removal of toxic domains)[190]. These variations may be expected to result in further developments in this area.

2.160 Phase I clinical trials have been reported using a GM E. coli as the vector to deliver the Plasmodium sp. apical membrane antigen (AMA-1) 74 , and the falciparum malaria protein (FMP1) [191][192]. The FMP1 based vaccine was reported to be well- tolerated by the recipients with no serious adverse effects. The AMA-1 vaccine elicited both humoral and cellular immunity in the human recipients.

∗ Saliva-directed recombinant vaccines 2.161 GM recombinant bacteria or viruses have also been proposed as vectors to vaccinate at risk people against components of the saliva of the arthropods that are the disease vectors, namely the female Anopholes sp. mosquito (for malaria) and various species of sandfly (for leishmania). The saliva of the arthropods that transmit these two diseases is known to enhance the infectivity of the pathogens

73 Codon optimisation involves the alteration of the gene sequence but not the protein sequence.

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transmitted. Vaccinating the recipient can protect that person from infection [176]. This approach may not be sufficient on its own, with a more effective approach being a multi-subunit vaccine combining the anti-saliva approach with specific targeting of the pathogen following infection (but prior to any disease) [176].

Summary of GM-based vaccines

Approved GM-based vaccines 2.162 The following veterinary vaccines containing a live GMO have been approved for use [198][199][255][270][272][274][276]:

♦ ProteqFlu: suspension for injection for horses. One dose contains Influenza A/equi-2/Kentucky/94 [H 3N8] recombinant Canarypox virus (vCP1529) 10 ≥5.2 log FAID 50 ; Influenza A/equi-2/Newmarket/2/93 [H 3N8] recombinant 10 Canarypox virus (vCP1533) ≥ 5.2 log FAID 50 . The vaccine is described to protect horses against equine influenza and to eliminate wildtype virus excretion post-infection (i.e. no shedding). Authorised for use in the European Union (ref. EU/2/03/037/005) as a prescription only medicine to be administered by a veterinarian. ♦ Equilis StrepE: one dose (0.2 ml) contains a live, modified strain of Streptococcus equi TW928 109.0 to 10 9.4 cfu. Equine vaccine intended for use in horses for which a risk of Streptococcus equi infection has been clearly identified, due to contact with horses from areas where this pathogen is known to be present, e.g. stables with horses that travel to shows and/or competitions in such areas, or stables that obtain or have livery horses from such areas . Authorised for use in the European Union (ref. EU/2/04/043/001) as a prescription only medicine to be administered by a veterinarian. ♦ RABORAL V-RG: thymidine kinase negative vaccinia virus modified to express rabies virus glycoprotein G and used as a rabies vaccine for the inoculation of wild animals, primarily mesocanines (red fox, arctic fox in Northern Europe; and racoon, gray and red foxes, coyote, shunk and arctic fox in North America). Over 8.5 million field doses since the late 1980’s. ♦ TROVAC-AIV H5: recombinant fowlpox virus expressing the haemagglutin gene of an avian influenza H5 subtype isolate. The vaccine is licensed for use against avian influenza in Latin America and the USA. Over 2 billion doses have been administered since 1998. ♦ Purevax FeLV: one vial of suspension for injection containing ≥ 10 7.2 FeLV

recombinant Canarypox virus (vCP97) CCID 50 . Administered by subcutaneous injection. Canarypox-based Purevax vaccines have also been produced for protection of cats against rabies (Purevax Feline Rabies) and fur animals against distemper virus (Purevax Ferret Distemper). Authorised for use in the

74 AMA-1 is expressed by the blood stage plasmodium parasite. 2-60

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European Union (ref. EU/2/00/019/005) as a prescription only medicine to be administered by a veterinarian. ♦ PreveNile: modified virus based on the ChimeriVax system for the protection of horses against West Nile virus. Studies with horses found no transfer between vaccinated and non-vaccinated individuals. Vaccine registered in the USA in 2006. ♦ RECOMBITEK Equine WNV: modified canarypox based vaccine for the protection of horses against West Nile virus. Licensed for use in the USA for administration by veterinarians. ♦ RECOMBITEK Canine Parvo, and RECOMBITEK Corona MLV: modified virus for the protection of dogs (from 6 weeks old) against canine parvovirus, and canine coronavirus respectively. Licensed for use in the USA. ♦ RECOMBITEK rDistemper: canarypox-based vaccine for the protection of dogs against canine distemper virus. In use throughout North and South America. ♦ Vaxxitek HVT+IBD: modified herpesvirus of turkey (HVT) expressing the VP2 protein on infectious bursal disease virus (IBDV). Vaccination at 10 5 pfu achieved 100% protection against an IBDV challenge. Designed for the vaccination of poultry in ovo, and licensed for use in the USA . ♦ Vectormune FP-ND: modified fowlpox virus for the protection of poultry against Newcastle Disease virus. Designed for the vaccination of poultry. ♦ Avian influenza virus (H5N1): modified Newcastle Disease virus expressing avian influenza haemagglutinin subtype H5. Inoculation of 1 day old chickens with the GM vaccine conferred protection against both NDV and avian influenza. Shedding of the avian influenza virus (AIV) was not observed. As the vaccine and wildtype AIV can be distinguished serologically then the vaccine may be used as a marker vaccine for the control of avian influenza. Field trials of this vaccine are reported to commence in 2007 in the USA [255][276]. ♦ MeganVax 1: double gene-deleted Salmonellla enterica serovar Typhimurium for the vaccination of chickens. Licensed for use in the USA in broilers and hens in 1998 and 2003 respectively.

2.163 The following three vaccines which have also been approved for use as veterinary vaccines have been listed separately as the viruses have been modified by processes that mean they are not classed as GMOs [7]. Whilst the modifications listed (gE and tk deletions) could have been generated using molecular techniques, and thereby classify the virus as a GMO, these four vaccines were developed through traditional multiple passages or chemical means [271]. The risks posed by these non-GM vaccines are however the same as those for a GM vaccine. They have therefore been included in this report for completeness.

♦ Bovilis IBR Marker live: is a live, attenuated marker vaccine, containing per dose 10 - at least 5.7 log TCID 50 of gE BHV-1 strain GK/D. Vaccine reduces the intensity and duration of symptoms caused by BHV-1, and also nasal excretion

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of the BHV-1 pathogen. Vaccine may spread intranasally to in-contact cattle. Described as having no implications to milk or meat quality. ♦ Rispoval IBR Marker live: a live, freeze-dried, Infectious Bovine Rhinotracheitis (IBR) Marker (gE negative) vaccine. One dose (2 ml) contains: Bovine herpes virus type 1 (BHV-1), attenuated strain of IBR-Marker virus gE negative min. 5.0 10 CCID 50 plus stabiliser 6.0 mg. Vaccination (administered intranasally or intramuscularly) reduces clinical signs and shedding of wildtype IBR post- infection.

5.5 ♦ Porcillis Begonia DF: a live freeze-dried vaccine containing at least 10 TCID 50 6.5 and maximum 10 TCID 50 of Aujeszky’s disease (pseudorabies) virus strain Begonia per dose. The diluent contains 75 mg dl-α-tocopherol acetate as an adjuvant. The freeze-dried pellet must be reconstituted an adjuvant (Diluvac Forte) before use. The virus strain is thymidine kinase and glycoprotein gE negative (tk -, gE -), genetically stable and does not persist in the pigs. Vaccination allows the discrimination from field infections (marker vaccine), prevents mortality and clinical signs of pseudorabies and reduces replication of the pseudorabies virus post-infection. ♦ Suvaxyn Aujeszky: a gE - and thymidine kinase-deleted pseudorabies virus (PRV) as a vaccination against PRV mediated Aujeszky’s disease. The vaccinated pigs exhibited lower levels of shedding of wildtype PRV following subsequent exposure.

2.164 Vaccines that are being developed by commercial organisations, such as the recombinant myxoma virus as a vaccine for feline virulent systemic disease [202], may be more likely to reach commercialisation.

2.165 The shigella vaccine strain Shigella flexneri 2a SC602 being developed at the Pasteur Institute (Paris, France) has been tested in clinical studies, and is currently undergoing further human safety trials [226]. The strain, which exhibits deletions of the plasmid encoded virulence genes icsA and virG , and the chromosomal iucA-iut locus which is involved in iron uptake is described as substantially attenuated. Regulatory approval of strain SC602 may be prevented by the long period of excretion of the bacteria (average of 11.6 days in 34 recipients, with a maximum of 33 days) [226].

2.166 The FluMist vaccine (live attenuated influenza virus) has been approved in the USA for use as a human vaccine against avian influenza. Although the vaccine contains a live attenuated virus, the version approved by the USFDA is not genetically modified as the attenuation is achieved by cold adaptation of the master donor virus followed by reassortment with conventional vaccine strains. However, should the vaccine strain be produced by reverse genetics rather than reassortment (which is slower and less predictable [306], then the vaccine vector is considered a GM virus [307].

GM-based vaccines in research trials

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2.167 The following GM-based vaccines are listed by the Joint Research Centre [295] as undergoing (or having undergone) field trials (veterinary vaccines). Details on Phase III gene therapy trials are present in Table A.3 (Appendix 1). No Phase III trials are reported for applications other than gene therapy.

♦ Field trial of the GM IBR vaccine, modified to be gE and tk negative. No adverse effects to the environment identified for this GMO which is very host specific and not reported to affect animals other than bovines. The vaccine strain (CEDDEL) is also reported not to be shed from the vaccinated animal and not spread from inoculated to non-inoculated animals. Field trial conducted in 2007 in Spain (ref. B/ES/07/44). ♦ Field trial of the GM feline herpes virus, administered as an intranasal vaccination to cats. Virus modified to be tk negative and therefore non-virulent. Field trial conducted between 2004-2008 in the Netherlands (ref. B/NL/04/001). ♦ Field trial of a GM vaccinia virus as a vaccine for dogs against canine leishmaniasis. Field trial conducted between 2005-2006 in Spain (ref. B/ES/05/01). ♦ Laboratory preclinical studies of avian influenza virus (H5N1): modified fowlpox virus for use as a veterinary vaccine. Laboratory tests in China reported that after one dose of immunisation of this vaccine, chickens could develop a >40 weeks protective immune response against H5N1 virus challenge [305].

Proposed GM-based vaccines 2.168 Candidates for wildlife vaccination programmes include fruit bats in Australia to reduce the occurrence of variant lyssaviruses; corvids (crow family) in the USA to reduce the spread of West Nile Virus; and migrating anseriforms (ducks, swans and geese) to control H5N1 avian influenza [199]. The Menangle, Nipah, Hendra and Tioman viruses that are spread by fruit bats and have been reported to cause disease in both animals and humans are also possible candidates for GM vaccines.

2.169 Other areas where future developments in veterinary vaccines (and possibly GM vaccines) may be expected are welfare and geriatric applications for companion animals [255]. Interest in these areas is viewed as increasingly lucrative which may well promote the commercialisation of appropriate vaccines.

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2.4 REVIEW OF CURRENT AND POTENTIAL FUTURE APPLICATIONS IN MEDICINE – DIRECT ACTION

Direct action applications for the treatment of cancer Oncolytic viruses Bacteria-based applications Direct action applications for the treatment of dental caries Direct action applications using bacteriophage Direct action applications in clinical trials

2.170 Direct action is the term used in this report to describe treatments where the therapeutic effect is conferred through the replication and growth of the GMO. Whilst the GMOs used may also be modified to provide a gene or drug delivery role, direct action refers essentially to the effects caused by the presence of the GMO at the target area.

Direct action applications for the treatment of cancer

2.171 Applications for the treatment of cancer describe the use of GM viruses and bacteria to target the micro-environments within solid tumours, or the tumour cells themselves. This results in the disruption of the growth and development of the tumour through the depletion of essential nutrients and/or an alteration of the tumour’s microenvironment as a result of the colonisation [126], also induction of cell death via apoptosis or other type of cell death. Significantly, hypoxic regions of solid tumours are insensitive to both radiation and chemotherapy [129].

Oncolytic viruses 2.172 Oncolytic viruses are viruses that preferentially replicate and kill cancer cells whilst leaving surrounding non-cancerous cells relatively intact [55]. Non-modified viruses that have been investigated to date for their oncolytic potential include Vesicular Stomatitis virus (VSV), measles virus and Newcastle Disease virus (NDV) [21][62]. Tissue-attenuated (non-GM) NDV has been shown to exhibit potent oncolytic activity for example against colon, lung, breast and prostate cancer [87], and viable virus was recovered from the patient’s urine and, less commonly, their sputum [88].

2.173 Of relevance to this report is the genetic modification of viruses (existing oncolytic and other) to target tumour cells. Approaches to achieve this include:

♦ The use of single-stranded RNA (ssRNA) rather than DNA viruses 75 [87]. A key stage in the replication of RNA viruses is the conversion of their ssRNA to double-stranded RNA (dsRNA). The presence of dsRNA stimulates the

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formation of protein kinase R (PKR) by the infected cell, which promotes apoptosis and limits the spread of the viral infection in a normal cell environment. However tumour cells are frequently defective in their PKR pathway, and therefore provide a more permissible environment for RNA viruses [87]. ♦ Modification of viral envelope glycoproteins or capsid proteins (depending on whether it is an enveloped virus or not) to promote virus entry through cell surface receptors expressed at high levels on tumours cells [87]. ♦ The removal the viral genes required for replication in normal cells, whilst retaining the genes conferring the ability to replicate in cancer cells. The deletion of the E1A and E1B functions in adenovirus for example results in a GM virus that replicates preferentially in tumours. ♦ Use of tumour specific promoters to control the transcription of viral genes important for replication, for example the E1A and E4 genes in adenovirus. This approach has been applied in a Phase I/II trial involving a GM adenovirus where the E1A gene (the expression of which is critical for viral replication) was under the control of the tumour specific prostate specific antigen gene promoter- enhancer element (Rodriguez et al. 1997; cited by [55]). The GM adenovirus therefore replicated preferentially in tissues with a high level of expression of prostate-specific antigen. ♦ Modification to limit replication in dividing cells or in cells with mutated cellular functions. For example: o the modification of vaccinia virus so that the thymidine-kinase (TK) gene is inactive, rendering GM virus unable to replicate in normal non-dividing cells, but still capable of replication in tumour cells [55]; and o Deletion of the SPI-1 and SPI-2 genes which encode serine proteases and are required for viral replication. However, as tumour cells overexpress homologous proteins the modified vaccinia virus is still able to replicate in these cells, whereas it is unable to do so in normal cells [55]. ♦ The use of broad-specificity tumour specific promoters also allows for a wider application of the oncolytic virus against cancers.

2.174 The application of GM oncolytic viruses has been enhanced by combining their direct action role with strategies such as GDEPT or the delivery on immunostimulatory cytokines to produce a multifunctional treatment. These so called ‘armed viruses’ offer a number of advantages over the use of non-GM oncolytic viruses and ‘unarmed’ GM oncolytic viruses, as the viral oncolysis usually acts in parallel with the secondary ‘armed’ function. For example Vesicular Stomatitis virus genetically modified to produce thymidine kinase or the cytokine IL-4 was found to have greater oncolytic activity against breast and melanoma tumours compared to the wild-type

75 RNA viruses, such as mumps virus, Newcastle disease virus, measles virus, Vesicular Stomatitis virus, human reovirus, poliovirus, and influenza virus, have their genetic material as RNA rather than DNA. 2-65

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(which also has known oncolytic activity) [87]. The additional function can also be used to confer greater specificity of the virus to the tumour (through the use of particular promoter sequences), or to minimise non-target effects.

2.175 The relatively large genome capacity of adenovirus, Vesicular Stomatitis virus, and Herpes Simplex virus makes these viruses particularly applicable as ‘armed’ viruses [21]. A GM Herpes simplex virus (HSV-1716) for example has been used successfully in clinical trials in Glasgow since 1997 for the treatment of grade IV brain glioma [103].

2.176 Bischoff et al. (1996) [57] reported the first oncolytic adenovirus, modified with a deletion in the E1B gene to confer preferential replication in cancer cells. Injection of the GM adenovirus into cervical carcinomas in mice caused a significant reduction in tumour size and a complete regression of 60% of the tumours. This GM adenovirus has subsequently been applied in human clinical trials, although only limited therapeutic effects have been reported [21]. However, greater success has been found when the system has been used in combination with other cancer treatment techniques (such as chemotherapy) [58], possibly due to synergistic effects with the conventional treatment [21][55].

2.177 Although some success with GM oncolytic viruses has been reported in clinical trials, the technology does have a number of inherent weaknesses that need to be considered [56]:

♦ Immune-rejection and destruction of oncolytic virus, leading to poor survival of oncolytic viruses in the body and limiting their effective distribution within the tumour. Oncolytic viruses were envisioned originally as autonomous agents that following injection into the patient, would seek out and destroy cancer cells. However, most people have antibodies to the viruses that are commonly used as oncolytic agents. An immune response is therefore often mounted by the patient which clears the virus before it has time to infect cells, thereby leading to the failure of the treatment. This immune response also neutralises the effect of subsequent injections. The effective window for these viruses is therefore relatively narrow. ♦ Shedding of replication competent virus as a result of the use of replication competent viruses as oncolytic agents. Whilst these are more attractive therapeutically than replication deficient viruses as they offer a higher therapeutic efficiency [64], the use of replication competent viruses does present problems if shedding from the patient occurs, as this could result in the dissemination of a genetically modified virus capable of replication. The use of replication competent viruses also makes clinical trials potentially cumbersome and expensive. ♦ The need for viral particle production rates in the infected cancer cells to outstrip the growth rate of the uninfected cancer cells. If this is not achieved then the effectiveness of the treatment diminishes. Achieving this may require large

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doses of virus in situations where systemic injection is used. This in turn can have safety/environmental implications particularly when the oncolytic virus is replication competent. Where sufficient growth rates are difficult to achieve, for example in large or fast growing tumours, the oncolytic treatment will need to be combined with conventional approaches such as surgery to reduce the number of cancer cells. However, where replication competent viruses are used, the ability of the virus to multiply in vivo (in the tumour cells) may actually reduce the initial quantities that need to be administered.

Bacteria-based applications 2.178 Bacteria-based direct action treatments utilise the existence of hypoxic-necrotic regions within solid tumours. These are not present within normal tissues [53]. Obligate anaerobic bacteria such as Bifidobacterium longum and Clostridium oncolyticum , and facultative anaerobes such as Salmonella typhimurium , have been shown to be able to localise and proliferate selectively within solid tumours following systemic injection [54][127][53]. Investigations with salmonella for example reported a 1000 fold higher level of the bacterium within the tumour compared to normal tissue post injection. Proof of concept using non-GM anaerobic bacteria against tumours has been reported in animal and human studies [54]. The application of GM technology to this approach has been to reduce or remove the pathogenicity of the strains used, and also to confer a dual role on the anaerobic bacterium so that it also acts as a vector for the prodrug in a GDEPT application 76 .

2.179 The use of GM salmonella as drug delivery vectors for the treatment of tumours is being promoted commercially in the form of the TAPET ® system 77 (Vion Pharmaceutical Inc).

2.180 The application of anaerobic bacteria for the treatment of solid tumours, either on their own or in combination with other approaches, offers the advantage that the bacteria (as spores) can be injected systemically, rather than directly into the tumour. This enables greater ease of application. The obligate anaerobic nature of the bacteria means that even when injected systemically they only replicate in the tumour, due to the oxygen tension in normal tissues being too high 78 for spore germination to occur. This provides a degree of biological containment of the bacteria within the patient. The tumour-specificity was demonstrated with Clostridium tetani (the causative agent of tetanus) (Malmgren and Flanigen, 1955; cited by [54]). Two groups of mice, one with existing solid tumours, and one without were injected intravenously with C. tetani spores. The tumour-free group were unaffected by the injection, whereas the group with tumours developed tetanus as the tumours provided an environment in which the C. tetani could germinate and release the tetanus toxin systemically. It should be noted that C. tetani has no actual

76 See section on gene-directed enzyme prodrug therapy in the Gene Therapy chapter. 77 TAPET ® – tumour amplified protein expression therapy. 78 Median range of oxygen partial pressure of 10-30 mm Hg in tumours, with a significant proportion of readings <2.5 mm Hg. Oxygen partial pressure of normal tissues ranges from 24-66 mm Hg. 2-67

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therapeutic application as a direct action agent, with the Malmgren and Flanigen (1955) study conducted to demonstrate proof of purpose.

2.181 Subsequent work with non-pathogenic GM strains, for example Clostridium oncolyticum, C. beijerinckii or B. longum , also reported no adverse effects following systemic injection, as well as no release of the transgene product from the tumour into other tissues through the bloodstream [54]. Studies with B. longum have shown that following injection the bacteria are able to maintain a steady population within the tumour (maintenance rate of 84% after 30 generations) [53]. These three points are all significant advantages of this treatment system. The sensitivity of the microorganisms to antibiotics confers an additional level of control [126].

2.182 GM Salmonella typhimurium has also been reported to be effective in a direct action role against both small metastatic lesions and larger tumours 79 in dogs [126]. The GM salmonella were modified for attenuated pathogenicity by partial deletion of both the msbB gene and the purl gene. Modification of the purl gene makes the GMO dependent on an external source of purines. These may be present in higher concentrations in the interstitial fluids of the tumour microenvironment (thereby providing a further level of biological containment of the GMO within the tumour). Dogs were dosed weekly or biweekly at doses ranging from 1.5x10 5 to 1x108 cfu/kg, with 3x10 7 cfu/kg being found to be the maximum tolerable dose. A major antitumour response was found in 15% of the treated dogs.

Direct action applications for the treatment of dental caries

2.183 One of the causes of dental caries is the presence of microorganisms within the dental microflora that convert sugar in food to lactic acid [125]. Applications of genetic modification technology in this area have involved the use of dental microorganisms that are unable to produce lactic acid. The application works on the basis that the GM lactic acid deficient bacteria out compete the lactic acid producing strains, thereby reducing the production of lactic acid.

2.184 The modification of a Streptococcus mutans to delete the gene encoding lactate dehydrogenase resulted in the GMO being unable to produce lactic acid. The lactic acid dehydrogenase gene was replaced with the gene encoding alcohol dehydrogenase B from Zymomonas mobilis [309]. When the strain was tested in rats, the modification was found to be stable with no deleterious side effects reported, and a reduction in cariogenesis 80 . Colonisation of the GMO over non-GM lactic acid producing S. mutans strains was enhanced by the GM S. mutans also being modified to produce elevated levels of the antibiotic mutacin 1140 [124]. The colonisation of the GMO within the dental microflora was reported to be sufficiently

79 The status of S. typhimurium has a facultative anaerobe, rather than an obligate anaerobe (as with Clostridium sp. and Bifidobacterium sp.) means that it can survive in both oxygenated and hypoxic conditions, and therefore colonise small metastatic lesions which may have a limited hypoxic environment, as well as the larger tumours [126]. 80 The occurrence of dental caries. 2-68

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effective to indicate that only a single application should be sufficient in humans to ensure permanent establishment and the eventual displacement of the lactic acid producing S. mutans strains [124].

2.185 Biological containment of the GM S. mutans (strain JH1140) was achieved by deleting the dal gene (a gene analogous to alr which encodes alanine racemase), and making the GMO auxotrophic for D-alanine [150]. In vitro studies reported that the dal deficient bacteria start to lyse within two hours in the absence of D-alanine [309]. In vivo studies with rats found that a population of the GMO could be maintained in the oral cavity at levels similar to wild type if D-alanine was provided in drinking water. Removal of the D-alanine from the drinking water resulted in a significant reduction in the level of colonisation, but did not result in complete eradication of the GMO (four months post infection). Further experiments indicated that normal turnover of bacterial flora in the oral cavity may permit a dal mutant to scavenge sufficient D-alanine to survive and maintain a low level of colonisation [309]. Other investigations are also being conducted to determine the potential for transfer of the GMO in human to human contact such as kissing [260].

2.186 The GM S. mutans system has been granted approval by the USFDA for early stage clinical trials. The system is being promoted by the Oragenics company as part of their trademarked ‘SMaRT Replacement Therapy’.

Direct action applications using bacteriophage

2.187 Bacteriophages (or phages) are viruses that specifically infect bacteria, with infection leading to the death of the bacterial cell. Bacteriophage therapy for the treatment of bacterial infections in humans was commonplace in the 1930s and 1940s [283]. Their use in Western medicine declined 81 as a consequence of a greater emphasis on antibiotics which offered a broad spectrum of action and greater ease of manufacture, storage and prescription.

2.188 The development of antibiotic resistance in many strains of pathogenic bacteria which are typically treated with antibiotics has led to renewed interest in bacteriophages as a treatment strategy. Recent animal studies have confirmed that bacteriophages can offer a highly effective strategy for the treatment of many different types of bacterial infections in both human and veterinary medicine [282]. Bacteriophages are specific to individual species of bacteria, and even particular strains in some cases. This reduces the potential for non-target effects to occur, for example through damage to the patient’s gut microflora.

2.189 As bacteriophages infect and kill specific species of bacteria then they maybe considered as having a direct action role in the treatment of bacterial infection. Whilst information on the genetic modification of bacteriophages to alter their direct

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action role has not been identified, GM technology has enabled extra functionality to added to the bacteriophage to confer a drug delivery role [281].

Direct action applications in clinical trials

2.190 The following bullets highlight clinical trials that have either reached Phase II or III, or offer particularly innovative methods [56]:

♦ Two notable oncolytic virus systems currently undergoing clinical trials are the adenovirus based ONYX-015 system, and the G207 and NV1020 herpes simplex based systems. The adenovirus in the ONYX-015 system is modified to lack the E1B protein and consequently is unable to replicate in cells with a normal p53 pathway. As described earlier, because many cancer cells lack a functional p53 pathway the ONYX-015 virus preferentially infects these p53 mutant cells, replicates in them and lyses them. ONYX-015 has been tested on its own in Phase I and II level trials, and in combination with conventional chemotherapy in a Phase III trial. The Phase I and II trials demonstrated the efficacy of the system against squamous cell carcinoma of the head and neck, with the effectiveness correlating with the p53 status of the tumour. The tumours with an inactive pathway were more susceptible than those with a partially functioning p53 pathway. The combination treatment investigated in the Phase III trial was found to be more effective than the ONYX-015 system on its own [56]. The ONYX-015 system is also being tested as a preventative treatment (in a mouthwash) 82 for precancerous oral tissue, on the hypothesis that cells may exhibit mutations in their p53 pathway before they become cancerous [98].

The herpes simplex virus based systems contain a series of mutations that cause the virus to replicate only in cancerous cells. G207 for example is mutated for attenuated neurovirulence and cannot replicate in nondividing cells and those lacking normal protein synthesis controls [64][56], whereas NV1020 is an ‘armed’ oncolytic virus package using the HSV-TK/GCV system. G207 has undergone successful Phase I trials for the treatment of malignant glioma, with a viral dose of 3x10 9 plaque forming units (pfu) being well tolerated by patients [64]. G207 has also been tested successfully in vitro against hepatocellular carcinoma, and all known human hepatocellular carcinomas have been found to be sensitive to this system [99]. NV1020 has undergone a Phase I/II trial against colorectal cancer metastases to the liver [56]. Pre-clinical studies with replication competent retrovirus against glioma have shown promising results, and it is likely that Phase I trials involving these vectors may be conducted [64].

♦ The oncolytic adenovirus H101, which has undergone Phase III trials in the USA and has received commercial approval in China [114][61][59]. H101 has partial deletions in E1B (similar to the ONYX system) and E3, and has demonstrated

82 The potential implications of the use of a live GM virus in a mouthwash are reviewed in Chapter 3 (assessment of risks). 2-70

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good efficacy against various cancers in clinical trials, particularly when combined with conventional chemotherapy. Crompton and Kirn (2007)[61] described the effectiveness of H101 and ONYX-015 as being limited beyond the locality of injection. The target of the second generation of these oncolytic adenovirus systems, as well as other viral ‘platforms’ based on herpes simplex virus, Newcastle Disease virus, reovirus and vaccinia virus, is improved systemic delivery and efficacy [61].

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2.5 REVIEW OF CURRENT AND POTENTIAL FUTURE APPLICATIONS IN MEDICINE – DRUG DELIVERY AND PROBIOTIC-TYPE TREATMENTS

Gastrointestinal tract treatments Treatment of inflammatory bowel disease Application of lactic acid bacteria expressing IL-10 Application of lactic acid bacteria expressing trefoil factors Female urogenital tract treatments Commercialisation of GM bacteria as drug delivery vectors Modification of probiotics Designer probiotics

2.191 This section of the report reviews the applications of GM bacteria as drug delivery vectors to the mucosal membrane of the gastrointestinal (GI) and female urogenital tracts. Because many of the bacteria used in this type of application are GM versions of bacteria used in probiotic treatments, then possible applications of GMOs in probiotic treatments are also addressed within this section.

2.192 It is important to note that probiotic products that are currently commercially available do not contain GM bacteria.

2.193 The applications of GMOs described in this section are distinct to the vaccination treatments described in a previous section in that the GMO only targets the intestinal mucosa (for example) and does not invade the patient’s tissues and lead to subsequent infection. They differ from gene therapy applications in that the purpose of the GMO is to deliver the therapeutic compound as an active protein, rather than delivering the relevant gene which is subsequently transcribed and translated by the patient’s own cells into the therapeutic protein.

Gastrointestinal tract treatments

2.194 The important role of the GI tract in many physiological processes, such as food intake, and the regulation of immunity, energy balance and metabolism, makes the intestine an important target for therapeutic treatments designed to affect these processes and alleviate a variety of important diseases. Treatments involving the direct use of protein or peptide based therapeutics, either through ingestion or systemic injection, have been found to have only a limited beneficial effect, and problems with side effects [10]. Systemic injection is also limited as only a fraction of the therapeutic agent reaches the intended target of the intestinal membrane.

2.195 The use of microoganisms as delivery vectors has been found to overcome these limitations. The more targeted and more stable method of delivery provided by microbial vectors means that the treatment requires a lower dose of therapeutic

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agent to be administered. This has advantages of reducing side effects and also cost. For example the application of GMOs for the treatment of intestinal conditions such as IBD, in particular Crohn’s Disease is reported to be more effective and without the side effects of existing systemic treatments [254]. With the use of GM Lactococcus lactis expressing IL-10 for example [247], the GM system required a 10,000 fold lower dose compared with a systemically administered recombinant IL- 10 protein. (These data were obtained in animal models exhibiting a pathology very similar to Crohn’s Disease).

2.196 Although a gene therapy treatment using a GM adenovirus to deliver a gene encoding interleukin 4 (IL-4) has been developed against inflammatory bowel disease (IBD) (Hogaboam et al. 1997; cited by [117])83 , it is GM bacteria rather than GM viruses which have shown most promise in this area, due to their important role as part of the gut microflora.

2.197 Many strains of bacteria have been described as effective against gastrointestinal conditions. However, most of those described are probiotic microorganisms 84 , and are not genetically modified. The application of GM bacteria for the treatment of GI tract conditions has to date focused on the treatment of inflammatory bowel disease (IBD).

Treatment of inflammatory bowel disease 2.198 IBD is a chronic intestinal inflammatory disease which may manifest itself in the form of ulcerative colitis (an inflammation of the large intestine) and Crohn’s disease (which can affect any part of the GI tract). In Western countries the incidence of IBD (currently 1-2 per every 1000 individuals) is increasing [10]. The actual cause of IBD is unknown, although pro-inflammatory cytokines such as (tumour necrosis factor) TNF-α and (interferon) IFN-γ, as well as the anti-inflammatory cytokines such as (interleukin) IL-10 and (tumour growth factor) TFG-β are reported to be involved [10].

2.199 With respect to conventional treatment of IBD, the application of anti-inflammatory corticosteroids is not effective in all patients. Treatments such as the systemic injection with anti-TNF monoclonal antibodies, and injection of recombinant IL-10 have proved successful against Crohn’s disease, albeit with side effects occurring in many patients (including the reactivation of tuberculosis with the anti-TNF treatment)[10].

83 Studies in rats reported that two injections of the GM adenovirus (one had no effect) significantly inhibited tissue damage in trinitrobenzene sulphonic acid (TNBS) induced colitis. High expression of the transgene was however reported in the liver and spleen, indicating poor organ specificity and the increased potential for adverse immune response. 84 Probiotic microorganisms have been defined as bacteria that impart clinically verified beneficial effects on the health of the host when consumed orally [120]. To date, most probiotics are either lactic acid bacteria or bifidobacteria. 2-73

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2.200 GM lactic acid bacteria are reported to offer an alternative strategy for the treatment of IBD, as they enable the topical delivery of the therapeutic agent [93][115][122]. These GM bacteria are able to overexpress the therapeutic product continuously without any effect on their growth rate (Steidler et al. 1996; cited by [115]). Two specific applications of GM lactic acid bacteria have been described for the therapeutic treatment of IBD:

♦ expression of interleukin-10 (IL-10). ♦ expression of trefoil factors (TFF).

Application of lactic acid bacteria expressing IL-10 2.201 The modification of Lactococcus lactis to express murine IL-10 in a mouse model reported both a reduction in the inflammation of an existing intestinal colitis and the prevention of the development of colitis [247]. Significantly, the GM system required a 10,000 fold lower dose, compared to a systemically administered recombinant IL- 10 to achieve the same effect [247]. The success of this system in a mouse model demonstrated the application of GM lactic acid bacteria as vectors for the delivery of drugs to the intestinal mucosa.

2.202 Because the proposed use of the GM L. lactis involves the ingestion of live bacteria that may ultimately be excreted in the patient’s stools, then a biological containment system was required to prevent the survival of the GMO in the environment post- excretion. Biological containment was achieved through the replacement of the genomic thymidylate synthase gene thyA by the expression cassette for IL-10 . Thymidylate synthase is required for the synthesis of a precursor for dTTP (thymidine triphosphate) and is essential for the growth of L. lactis. An absence of thymine or thymidine results in the activation of the SOS repair system in a thyA- deficient strain resulting in DNA fragmentation, and ultimately cell death. The absence of the gene therefore makes the bacterium dependent on an external source of thymidine or thymine.

2.203 Bacteria modified for the deletion of thyA (thymine auxotrophy) are reported to require external thymine at a concentration ~20µg/ml [253], although Deutsch and Pauling (1971)[258] reported a lower figure of ≥2µg/ml 85 . A reduction in the thymine concentration below this level leads to a concomitant decrease in viability of the GMOs. No net growth occurs at a thymine concentration of 0.2µg/ml. A complete absence of external thymine is therefore not required to halt growth of the GMOs. The GM L. lactis described by Steidler et al. (2003) [248] is thyA deleted and requires ~20µg/ml of thymine/thymidine to grow ( in vitro ). Reduction of the thymidine concentration to 2.4µg/ml led to the viability of the culture dropping from 1x10 7 cfu/ml to 1x10 3 cfu/ml after 60 hours, with no viable cells detectable after 250 hours. Reducing the concentration further to 0.1µg/ml caused viability to decrease

85 The lower figure of ≥2µg/ml may have be a consequence of the modified bacteria exhibiting additional modifications. Mutations of the deoB or deoC genes, or the deoB / deoC and deoR (as well at thyA ) is reported to reduce the quantity of thymine required for growth down to 2-5µg/ml and 0.2- 0.5µg/ml respectively [253]. 2-74

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to 1x10 1 cfu/ml after 60 hours, with no viable cells detected after 175 hours. In the absence of thymidine no viable cells were detectable after 72 hours. A thymidine level in the ileum section of the porcine GI tract has been determined at 0.025µg/ml [248]. Applying this figure to the in vitro results indicates that that the thyA deficient GM bacteria will not survive in vivo for longer than 175 hours.

2.204 In the unlikely event that the GMO was to regain the thyA gene through recombination, this would result in the elimination of the IL-10 sequence, and the resulting strain would no longer be a GMO. Evaluation of the survival of the GM thyA deficient (thyA -) IL-10 producing L. lactis in pigs, relative to the thyA + strain found that the viability of the thyA - strain decreased approximately 20-fold more rapidly after passage through the intestine. The thyA modification therefore confers a reduction in viability of the GM L. lactis , and the limited availability of thymidine and thymine in the environment should reduce survival still further [248][10].

2.205 A small scale human clinical trial has been conducted (Netherlands) with the GM thyA - L. lactis , with ten patients with moderate to severe Crohn’s Disease [122]. Patients took ten enteric-coated capsules containing 2x10 10 cfu GM lactococcus daily for seven days. The patients were under physical containment during the study 86 . The presence of the GM lactococcus in the patient’s stools was determined by DNA analysis (which detected both live and dead bacteria), with the highest counts (16x10 7 genome equivalents) detected on day 4. Approximately 6% of the GM bacteria present at this time were deduced as being live. No GMOs (live or dead) were detected two days after termination of the seven day trial 87 . Growth of the GMOs was dependent on the addition of thymidine. Positive clinical benefits were reported in eight of the ten patients with limited side effects.

Application of lactic acid bacteria expressing trefoil factors 2.206 Trefoil factors (TFFs) play an important role in the protection and repair of the intestinal epithelium. In normal intestine TFFs are expressed in the GI tract in a tissue specific manner. As the manifestations of Crohn’s Disease include a breakdown of the intestinal epithelial barrier, then TFFs have been identified as a possible therapeutic agent against Crohn’s Disease [10]. However, TFFs cannot be administered on their own as they stick to the mucus of the small intestine.

2.207 L. lactis has been genetically modified to express TFFs [249], and has been found to protect against acute colitis in a mouse model by reducing intestinal epithelial damage and inflammation. The GMO has also been reported to ameliorate established chronic colitis (in mice) [249].

86 Patients were kept in an isolation ward for the duration of the study, and all stool material was collected and decontaminated. 87 The limit of detection of the DNA analysis gave a detection limit of 10 4 genome equivalents per g stool. 2-75

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Female urogenital tract treatments

2.208 As with the GI tract, the female urogenital tract is densely populated with commensal bacteria (primarily certain lactobacillus species), which play an important role in protecting the host from infection. Disruption of the microflora is known to lead to adverse health effects including an increased incidence of urinary tract infection (UTI), and greater potential risk of acquiring a sexually transmitted infection, including HIV [123].

2.209 A number of studies have been reported using GM bacteria to reduce the risk of vaginal HIV infection through the delivery of compounds to the vaginal mucosa:

♦ Modification of Lactobacillus reuteri RC-14 to express HIV-1 inhibitors that target and block the first four sequential steps by which HIV-1 enters cells [123]. ♦ Modification of L. jensenii Xna to express two-domain CD4 proteins which inhibited entry of HIV-1 into target cells [131]. ♦ Modification of L. lactis MG1363 to express the virucidal compound cyanovirin [132]. ♦ Modification of L. jensenii 1153 to express cyanovirin [130]. The study reported stable integration of the cyanovirin expression cassette into the bacterial chromosome, and the effective colonisation of the vagina in mouse studies. ♦ Modification of E. coli Nissle 1917 to secrete the HIV-gp41-hemolysin A hybrid peptides that block HIV fusion and entry into target cells [133]. E. coli Nissle 1917 is a highly colonising probiotic strain 88 , and the GMO was found to be capable of colonising mucosal surfaces in mice for periods of weeks to months.

2.210 These applications offer the advantage over the direct administration of antimicrobial agents in that they can confer long-term production of the antimicrobial at the required area. The Lactobacillus reuteri and L. jensenii strains described are strains known to exist as part of the human vaginal microflora, whereas the L. lactis and Escherichia coli are not (although E. coli Nissle 1917 is found as part of the human GI microflora and has been used as an over the counter probiotic).

2.211 No information on the biological containment for these GMOs has been identified, although this is probably a consequence of the reported studies being early stage proof-of concept investigations. Auxotrophic modifications may provide an effective biological containment strategy for these GM bacteria.

88 The non-GM strain E. coli Nissle 1917 is marketed by Ardeypharm as their Mutaflor product for use as a probiotic for the treatment of ulcerative colitis and diarrhoea. Studies with healthy volunteers found that live E. coli Nissle 1917 was present in faeces after ingestion of the probiotic [134]. This release of the live microorganism has implications to the authorisation of use of a GM version. 2-76

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Commercialisation of GM bacteria as drug delivery vectors

2.212 Many of the applications described above are being promoted by various research institutes and start-up companies for commercialisation. These include:

♦ the TopAct TM delivery system marketed by ActoGeniX. TopAct TM is the GM L. lactis thyA - system described for the treatment of IBD. The L. lactis modified to express IL-10 has completed Phase I clinical trials as a treatment for Crohn’s disease [122]. ActoGeniX intends to conduct a multicenter Phase IIa study with IL-10 secreting thyA L. lactis in ulcerative colitis patients in Europe and North America in the course of 2008. ActoGeniX also plans to start a Phase II study with the same products in Crohn’s disease patients in the course of 2009 [313]. Other variations of the system are in the product pipeline with L. lactis modified to express TFFs for the treatment of ulcerative colitis and mucositis having completed a proof of concept in animal studies and currently undergoing strain optimisation prior to clinical assessment. Variations to treat allergies, autoimmune disorders [250] and obesity are at the proof of concept stage in animal studies. ♦ the MucoCept HIV product marketed by Osel. This is based on the Lactobacillus jensenii 1153 modified to express the antiviral compound cyanovirin.

2.213 There are of course a large number of commercial probiotic products based on non- GM bacteria, such as Ardeypharm’s Mutaflor, and Alimentary Health’s Bifantis®89 products. It is recognised that any of the organisms used in these products could be genetically modified at some future date to alter or enhance the intended benefit of the product.

Modification of probiotics

2.214 GMOs are currently not present in commercially available probiotic treatments. However, as described in the previous sections on ‘direct action’ and ‘drug delivery’ there is considerable overlap between the GM-based and the non-GM probiotic treatments in the type of bacterial species used and the purpose of their application. This applies to both human and veterinary applications [135]. Many of the GMOs described are themselves modified probiotic strains.

2.215 The use of GM strains as part of probiotic treatments may therefore be expected if there is a commercial willingness to do so. There is little technical reason for existing commercial products not to be genetically modified, if some improvement of the probiotic strain is needed and genetic modification can assist with that improvement. An example of such an area is the modification of strains to express bacteriocidal compounds to improve their competitiveness towards other bacterial species and thereby their colonisation ability.

89 Bifantis® is based on Bifidobacterium infantis 35624 2-77

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2.216 In that trials with non-GM microorganisms for probiotic applications have reported the release of live bacteria (in faeces for example) post ingestion [134], then the biological containment of any GM version would be required before clinical trials and commercialisation could proceed.

Designer probiotics 2.217 ‘Designer probiotics’ is the term given to an emerging application of GM probiotic bacteria in which strains are modified to target certain pathogens and/or toxins in vivo [264][265]. The GMOs are designed to bind to particular receptors that are expressed on the surface of pathogens. These receptors typically bind to corresponding oligosaccharide structures on intestinal epithelia cells and are used by microbial pathogens to assist in the colonisation of the mucosal surface or the entry of a toxin (or the pathogen itself) into the cell [264]. The modification of probiotic bacteria to bind to the pathogens, and thereby block their ability to bind to the mucosal surface represents a novel strategy for the prevention of disease.

2.218 The approach is particularly applicable for the prevention of enteric diseases [264]. These diseases are currently targeted with antimicrobial compounds (including antibiotics). However, increased resistance to these compounds by human and veterinary pathogens has highlighted the need for alternative treatment strategies. In veterinary applications, concerns over the presence of antibiotics in food have also led to a reduction in their use as a preventative treatment.

2.219 Studies with mice administered with non-pathogenic E. coli R1 genetically modified to express the receptor for the Shiga toxin 90 was found to be 100% effective at protecting the mice from highly pathogenic Shiga toxin-producing E. coli (STEC) [333]. A similar study in mice has also been reported where a GM E.coli expressed the receptor to bind cholera toxin [264][269]. No clinical trials have been reported. The GM E. coli used in the two animal studies described carried antibiotic resistance genes and would therefore not have been suitable from a regulatory perspective.

2.220 Potential regulatory issues with this application are the release of live GM bacteria from the intestine post-administration and the development of an autoimmune response. The GM E. coli expressing Shiga toxin receptor administered to mice were eliminated from the intestine within two days of cessation of treatment [333] suggesting that excretion does occur. No biological containment strategies were proposed for the GM E. coli, apart from the use of a non-pathogenic strain.

2.221 The approach offers the advantages that it does not apply a selective pressure for the evolution of resistance by the pathogen (as in the case of antimicrobial compounds), and the widespread use of such ‘designer’ probiotics should have negligible adverse effects in the long-term [264]. It is also particularly suited to the treatment of enteric pathogens as it can be administered orally. The identification of

90 Shiga toxin is produced by Shigella dysenteriae and the Shigatoxigenic group of Escherichia coli (STEC), which includes serotype O157:H7 and other enterohaemorrhagic E. coli. 2-78

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the oligosaccharide structures that are the receptors for the toxins produced by Vibrio cholerae, E. coli and Shigella dysenteriae (Shiga toxin), Clostridium difficile (toxin A), E. coli (heat labile toxin), C. perfringens (δ toxin) and C. botulinum (neurotoxin) means that this approach could provide a strategy for the prevention of a number of significant enteric diseases [264].

2.222 As some oligosaccharide compounds are known to reduce or suppress cell- mediated immunity [275], then the generation of GM bacteria expressing these compounds may provide a strategy to prevent or treat inflammatory conditions of the GI tract.

2.223 A limitation with the application of GM probiotic bacteria in this manner is the inactivation of the GMOs as they pass through the acid environment of the stomach [264]. The genetic modification of the probiotic strain Bifidobacterium breve to express the listerial betaine uptake system BetL (from Listeria monocytogenes ) improves tolerance to acid conditions in the stomach [268]. Such a modification has been described as ‘patho-biotechnology’ [278][267], as it utilises the stress survival strategies of pathogenic bacteria (such as L. monocytogenes ) to improve the survival of the probiotic strains [268]. The generation of GM strains with enhanced survival does have implications to the risk assessment.

2.224 Mice fed with the GM BetL expressing B. breve exhibited higher viable levels of the GMO in their intestine compared to the non-GM control, and also higher viable levels in faeces [268].

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3. CHAPTER 3 – ASSESSMENT OF THE POTENTIAL RISKS TO THE ENVIRONMENT AND WIDER PUBLIC HEALTH

3.1 The purpose of Chapter 3 is to assess the potential risks to the environment and wider public health as a consequence of the uses (current and future) of live GMOs in medicines. The assessment in Chapter 3 has been conducted on the basis of the information collated in the previous Chapter, and has reviewed the likelihood and consequences of the particular hazards that may be associated with the identified GMOs, and the strategies available to prevent such hazards from being realised. Due to the large number of potential applications of GMOs in medicines it has not been possible in the risk assessment to review the individual risk(s) posed by each one. The risk assessment has therefore reviewed the risks associated with each of the generic hazards posed by the use of live GMOs in medicines. In making the assessment consideration has been given to the likelihood of occurrence, the severity of the consequences and the availability and effectiveness of any risk management strategies. Where any risks described have been reported for specific applications of GMOs in medicines then these have been highlighted in the text. Although the hazards posed by GMOs in medicine apply to all uses, differences in the use and containment of viruses and bacteria means that it is appropriate to review these separately. The small number of applications reported for GM parasites to date, are described at the end of Chapter 3.

Risk assessment framework for GMOs

3.2 The potential risks posed to the environment and/or human health by the use of live GMOs in medicines are determined through the following. In determining the level of risk consideration is given to the relative risk posed by the unmodified parent organism and whether the genetic modification increases or decreases that risk:

♦ Identification of the hazard posed by the genetic modification or the GMO. This includes the hazard posed by the organism, and also by the method of use/application; ♦ Consequence(s) of the hazard to the environment and/or human health; ♦ Likelihood of that hazard occurring; ♦ The level of resulting risk (determined by severity of the hazard multiplied by frequency of the occurrence); and

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♦ The existence and effectiveness of control measures to manage the risk (through reduction of consequence or frequency).

Generic hazards associated with GMOs in medicines

3.3 The particular risks posed by live GMOs in medicines are a consequence of the realisation of one or more of the following seven groups of hazards. The presence of a live GMO does not pose a risk per se . Depending on the characteristics of the GMO, including the nature of the modification(s) and the intended use; all of the following hazards may not apply to each GMO. Whilst these hazards are described separately in the following text it is recognised that they cannot be considered on their own when determining the level of overall risk, as there are considerable linkages between all of them. Poor genetic stability for example can lead to a reversion to virulence, increased pathogenicity and the occurrence of non-target effects [226].

♦ Pathogenicity of the GMO - the ability of the GMO to cause disease, either within the recipient, or to the wider environment following any release of the GMO; ♦ Production of biologically active and/or toxic products by the GMO, or by the recipient in response to the presence of the GMO; ♦ Production of non-target effects by the GMO, or as a consequence of the presence of the GMO; ♦ Genetic instability of the modifications, both deletions and additions; ♦ Changes in cell, tissue and host tropism of the GMO; ♦ Gene transfer (horizontal and vertical) of the added genes (the transgenes); and ♦ Survival and dissemination of the GMO in the environment.

3.4 The risk(s) posed is therefore a consequence of a combination of some or all of the seven hazards listed. Whilst Chapter 3 has sought to review the potential risks of the GMO to the wider environment and human health, and not potential risks to the recipient of the GMO, the two areas are closely linked and cannot be considered in isolation. In all cases (but one) the risk to the environment and human health is a consequence of what happens to the GMO after it has been administered to the recipient. If it is unable to survive and be released from the recipient then subsequent exposure of the environment and people should not occur, and the level of risk posed should be negligible.

3.5 The exception is the potential exposure occurring during the administration of the live GMO to the recipient. This is described by the EMEA (European Medicines Agency) [225] as occurring through:

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♦ dispersal of portions of product during production, normal handling and use. This includes storage prior to use; ♦ accidental dissemination during handling and use (including storage prior to use); and ♦ disposal of unused product or waste products (including used needles and swabs for example). Environmental exposure may also occur post- administration following release of the GMO in the patient’s excreta.

3.6 The significance of this exposure route will vary according to the nature of the medicine (containing the GMO) and the method of administration. Medicines containing GMOs that are administered parenterally for example will generate needles, swabs and dressings that are contaminated with the GMO. Such waste materials would need to be disposed of correctly to avoid dissemination of the GMO into the environment. GM medicines that are administered orally should generate a much smaller waste stream (no needles, swabs or dressings), particularly where they exist in a solid pill (rather than a liquid) form. Oral administration also reduces the potential for needlestick injury and the resulting possible dissemination of the GMO to the injured person. Compared to parenteral administration, oral delivery therefore poses a lower level of risk with respect to inadvertent administration to other people (through needlestick injury) and the generation of contaminated materials. An exception is oral administration in doped bait or feed where animals other than the target species may consume the GMO. In this circumstance, oral administration may also not provide guaranteed delivery to the target animal.

3.7 Delivery of a live GM virus in doped bait resulted in one of the few reported incidences of a live GMO in a medicine causing an adverse effect to a person who was not the intended recipient of the GMO. This occurred with the recombinant vaccinia rabies glycoprotein virus vaccine prepared for wild racoons and foxes 91 . A 28 year old woman with epidermolytic hyperkeratosis (a skin condition in which the skin is relatively fragile and prone to blistering) reported being bitten by her dog whilst trying to remove a bait (containing the oral rabies vaccine) from the dog’s mouth. The woman exhibited adverse health effects which required hospital treatment with antibiotics. The effects stopped 34 days post infection and she recovered completely. Samples taken from the woman exhibited 100% homology with those expected for the recombinant vaccina-rabies glycoprotein virus, indicating that she had been infected as a consequence of the dog bite [252].

3.8 GM medicines that are administered during clinical trials are likely to be given under relatively controlled conditions (in a hospital or clinic for example) by trained staff and according to an agreed standard operating procedure. The risks associated with the disposal of waste material(s), unused product and patient’s excreta may be

91 The non-target effect was documented as baits doped with the rabies vaccine in Ohio, USA are also marked with a telephone number linked to a rabies information line. The Ohio programme has documented 160 incidences of human contact with the baits, with only one case of an adverse effect to the person. 3-82

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considered to be less significant under these conditions. In situations where there is less confidence that any waste will be disposed of properly then changes to the GMO may need to be re-considered to minimise the higher risks posed. For example if excreta cannot be collected and disposed of in a manner that will inactivate any GMOs present, then the GMO may require further modification so that either excretion does not occur, or any GMO that is excreted is unable to survive or disseminate following release into the environment. In the clinical trial for patients with oral dysplasia where the ONYX-015 GM adenovirus was administered as an oral rinse [98] no information is available on the disposal of the mouthwash. However, as the trial was supervised then it is likely that the solution would have been decontaminated prior to disposal, thereby minimising potential risks to the environment. Trials where the mouthwash is taken in an unsupervised situation (at home for example) may pose a greater risk to the environment if the expectorated mouthwash is not treated to remove any GM virus before disposal (to the drain).

3.9 The potential risk(s) posed to the environment and/or human health from GMOs that are administered as a dose will be reduced where measures have been taken to reduce the level of dose of GMO that is used. This can be achieved through the use of the correct vector with appropriate promoter sequences, and the most efficient delivery system.

General Comment

3.10 All medicines containing a live GMO that have reached late stage clinical trials (or commercialisation) have been designed and produced under a regulatory framework that places a strong emphasis on minimising potential risks to the wider environment and human health 92 . Although such a clinical trials system does not exist for veterinary medicines, these products are still evaluated prior to commercial use to determine their effectiveness and the potential for adverse effects. Veterinary medicines, for example, are typically evaluated at a high dose (or overdose) and at repeated doses. A high dose is equivalent to 10x the maximum administered dose of the vaccine. In order to evaluate potential worst case effects, the assessments are conducted using the most susceptible animals (typically young animals at the minimum age recommended for vaccination).

3.11 The potential for the GMO to cause adverse effects is not desirable both in terms of compliance with the regulatory framework, and from a functional perspective as the effects will impair the action of the GMO. The potential for such effects is therefore expected to be avoided during the design and development of the GMO. All such

92 In the UK guidance on compliance with the regulations on the use of GMOs as a deliberate release or in contained conditions are provided by Defra, and the Scientific Advisory Committee on Genetic Modification (SACGM) in consultation with HSE, respectively. The guidance produced by both organisations is considered as best practice on the development and use of GMOs. Less information is available on the regulatory frameworks in other countries, for example China. 3-83

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GM-based medicines should therefore pose negligible risk to the environment and human health.

3.12 The presence of a live GMO in a medicine for human or veterinary application does not constitute a risk to the environment per se . In considering the potential risk(s) posed it is important to consider the characteristics of the GMOs present, the intended application of the medicine and the effectiveness of any management strategies that are applied. The GMO can only pose a risk to the wider environment if it is released prior to administration, or from the recipient post-administration.

3.13 The potential risk posed by the medicine may not be a consequence of the GMO and may be applicable to both GM and non-GM based medicines. An example is the use of a live attenuated pathogen as a vaccine where the risk of pathogenesis would apply irrespective of whether the attenuated pathogen was genetically modified or not. The potential risks assessed in this Chapter are therefore those posed by the genetic modification and the GMO, rather than the therapy or treatment itself. Where possible the risk(s) posed by the GMO has been assessed against the unmodified or wildtype organism 93 .

3.14 The key feature of genetic modification technology as the tool to alter a microorganism for use in medicines, compared to modifications achieved through non-GM processes, is that the resulting changes are more clearly defined and characterised [228]. Conventional (non-GM) modification, achieved through physical, chemical or ultraviolet mutagenesis for example results in many changes to the microorganism in addition to the desired changes [196]. Such uncharacterised mutations may cause unwanted effects. The ability of genetic modification to make very specific changes [196] should increase regulatory confidence in the effectiveness and stability of the modification, as well as leading to a more targeted safety testing programme on volunteers and a potential reduction in the use of animal experimentation [196].

3.15 The vaccination of animals provides a number of side effects of relevance to this risk assessment. Vaccinated animals tend to spread much lower levels of the wildtype pathogen relative to unvaccinated animals [226]. Vaccination therefore provides advantages both to the vaccinated animal (by reducing the chances of infection and disease) and the wider environment (by reducing the overall pathogen load). Where the vaccination is conducted against zoonotic or food-borne pathogens it reduces the risk to the wider human population of the related disease [255]. Examples include the vaccination of wildlife against rabies and the vaccination of poultry against Salmonella sp. to protect consumers from salmonellosis. Where the vaccination is conducted as an alternative to the treatment of disease with pharmaceuticals it will result in a reduction in the use of veterinary pharmaceuticals and hormones. This

93 As per the principles set out by the European Commission Decision 2002/623/EC on guidance on environmental risk assessment for GMOs. 3-84

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should reduce the likelihood of these compounds (or residues of them) occurring in the human food chain [255].

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3.1 ASSESSMENT OF THE POTENTIAL RISKS ASSOCIATED WITH GM VIRUSES IN MEDICINES

Pathogenicity (GM viruses) Pathogenicity of the unmodified (wildtype) strain Application or environment in which the GMO is used Presence of attenuations Production of biologically active and/or toxic products (GM viruses) Production of non-target effects (GM viruses) Untargeted delivery and expression of the transgene(s) by the GM virus Non-specific expression of GM viruses in GDEPT applications Vector-induced immune response (GM viruses) Insertional mutagenesis / oncogenes activation (GM viruses) Genetic stability (GM viruses) Changes in cell, tissue and host tropism (GM viruses) Gene transfer (GM viruses) Horizontal gene transfer (GM viruses) Vertical gene transfer (GM viruses) Survival and dissemination of the GMO (GM viruses) Shedding (GM viruses) Dissemination as a consequence of mechanism of delivery (GM viruses) Arthropod transmission (GM viruses) Disposal of contaminated materials (GM viruses)

Pathogenicity (GM viruses)

3.16 Pathogenicity refers to the ability of the GMO to cause disease, either within the recipient, or to the wider environment following any release of the GMO. With the exception of a limited number of ‘direct action’ applications, a GM virus in a medicine is required to be much less pathogenic than the unmodified wildtype, or if possible completely non-pathogenic. From a risk assessment perspective, zero or low pathogenicity of the GMO is preferable as it reduces the importance of other factors in minimising the overall risk posed by the GMO.

3.17 The pathogenicity of the GMO to the recipient and the wider environment is determined by the following factors.

Pathogenicity of the unmodified (wildtype) strain 3.18 The modification of a pathogenic wildtype organism will result in a pathogenic GMO, unless the GMO is attenuated to negate the pathogenic characteristics. Conversely, the modification of a non-pathogenic organism should result in a non-pathogenic GMO, unless the modification is designed specifically to introduce pathogenic determinants. However, this latter assumption does not always apply and

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consideration should be given to any inadvertent introduction of pathogenic traits [293].

Application or environment in which the GMO is used 3.19 The use of a pathogenic organism in an environment in which it is unable to spread, replicate or survive should prevent the onset of disease and negate the pathogenic characteristics of the GMO. An example is the use of fowlpox virus as a vaccine vector in mammalian cells [183]. Although wildtype fowlpox virus is pathogenic to birds it is unable to replicate successfully in mammalian cells. Therefore, when it is used as a vaccine vector to mammals it will not replicate and cause disease. Baculoviruses (which typically infect insects) have been proposed as vectors for mammalian cells for a similar reason [244].

3.20 Whilst using GM viruses from one species as a vector for an unrelated species can reduce the potential for pathogenic effects to the recipient animal (and for gene transfer through homologous recombination) the approach does pose a risk to other animals should a release into the wider environment occur. Examples include the potential risks of GM fowlpox virus to birds, and GM myxoma virus to rabbits. The use of GM myxoma virus as a vaccine vector against feline calcivirus (FCV) [202] should pose minimal risk to cats as it cannot replicate in this species. However it can infect rabbits. If the rabbit is also infected with wildtype myxoma virus (the causative agent of myxomatosis) then recombination between the GM and wildtype myxoma viruses could occur. The risks posed by such an event will depend on the nature of the modification, and whether it might pose any selective advantage to the wildtype virus. The GM myxoma virus reported by McCabe and Spibey (2005)[202] is likely to pose a very low risk as the FCV capsid protein will not confer a selective advantage to the myxoma virus and therefore will not be maintained should gene transfer occur. The risk posed however may be different should the transgene(s) encode for cytokines or other immunomodulatory genes [213]. With respect to GM avian poxviruses (such as fowlpox and canarypox), whilst fowlpox can replicate in chickens [137], particular avia poxvirus strains are non-pathogenic to birds [136]. No pathogenicity will be a minimum requirement to minimise the risk to birds posed by the use of these GM viruses.

Presence of attenuations 3.21 The presence of attenuating modifications will reduce the pathogenicity of the GMO. This can be achieved through the attenuation of pathogenic traits, or the alteration of other characteristics of the virus rendering it unable to survive for a sufficient period of time, replicate to a sufficient level, or spread through the recipient sufficiently to cause disease. As viruses are obligate pathogens then survival, replication and spread are linked. Inability to replicate means that the GM virus will not persist within the recipient organism beyond the initial infection. The virus will also not be able to spread to other animals or people. Targeted delivery of the GM virus through the use of particular regulatory elements can provide further biological containment of

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the virus within the recipient. Targeted delivery can involve altering the tropism of the GM virus through changes to cell surface receptors so that is can only infect certain cells, and altering gene regulatory elements to restrict gene expression to the target cell.

3.22 The success of any genetic modification to reduce (attenuate) pathogenicity and thereby reduce risk, depends on the effectiveness and stability of the attenuation(s). If the attenuations are ineffective then there may not be the sufficient reduction in pathogenicity required. Likewise, if the attenuations are not genetically stable then they could be lost by the microorganism, resulting in the reversion to the pathogenic wildtype. However, GMOs whose attenuations are not effective or stable would not be put forward as candidates for deliberate release (or even contained use) trials or commercialisation as they would not function as required (in addition to the regulatory restrictions).

3.23 Maintaining the attenuated form of the microorganism is improved if multiple attenuations are used. Pathogenic attenuations may also be strengthened by restricting the ability of the GMO to reacquire the deleted gene(s). This may be achieved by attenuating the microorganism through whole or partial gene deletions instead of point mutations or transposon insertions [226], inserting a new gene into the genome in the place of the deleted gene, or by also removing any integration or re-acquisition mechanisms:

♦ Deletion rather than point mutations – removal of entire genes, or large sections of the genes reduces the potential for reversion to pathogenic wildtype. Single point mutation as a strategy for attenuation is particularly ineffective for microorganisms such as RNA viruses that exhibit a high rate of spontaneous mutation. ♦ Replacement – inserting the transgene(s) in the place of the deleted gene. Therefore should the GMO reacquire the missing gene it will lose the transgene (by recombination).

3.24 Whilst a higher number of attenuations will reduce the chances of reversion to a pathogenic form, the level of attenuation may have implications to the behaviour of the GMO and its effectiveness [226]. For example with GMOs used as vaccine vectors excessive attenuation can reduce the immunogenicity of the vector and consequently its effectiveness as a vaccine [194]. No specific occurrence of pathogenesis has been identified for GM viruses used as veterinary vaccines.

3.25 The determination of the success of any attenuation may not be straightforward. Some studies for example have reported difficulties in applying the results from animal studies to humans, with modifications demonstrating good attenuation in animals but only limited attenuation in humans [194]. This is an important point and

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should be considered in the risk assessment when extrapolating results from animal studies to potential effects to human health.

Production of biologically active and/or toxic products (GM viruses)

3.26 The production of biologically active and/or toxic products may be a required function of the GMO, for example in the case of vaccination and gene therapy. Where this is the case then the risk assessment should assess the intended role of that compound, the potential for it to be transferred to other microorganisms and the implications of that transfer to the behaviour of those microorganisms. An inability of the trait to be transferred, either through gene transfer or spread of the GM virus will reduce the risk posed by the GMO to other organisms.

3.27 Production of the compound should be tailored to its intended function with consideration given to whether too much or too little compound is produced. Overproduction may give rise to non-target effects, whilst insufficient production may render the modification ineffective.

3.28 The gene therapy applications where the GM virus is not designed to produce a biologically active or toxic product are gene-directed enzyme prodrug therapy (GDEPT), and the two-step transcriptional amplification system (TSTA). With GDEPT, allthough the treatment involves the generation of a cytotoxic compound, this is only produced following the administration of both the GMO and the harmless prodrug. The administration of the GMO on its own does not generate any toxic compound. TSTA is employed to enhance the promoter strengths of weak tissue- specific promoters [79].

3.29 None of the applications identified in Chapter 2 are assessed as posing an adverse risk to the wider environment or general public as a consequence of the production of biologically active products.

Production of non-target effects (GM viruses)

3.30 The production of non-target effects by the GMO is not desirable from a regulatory perspective and should be avoided wherever possible. The defined changes that are achieved with genetic modification relative to conventional techniques should minimise the potential for non-target effects to occur. The use of GMOs provides the opportunity to deliver the transgene precisely to the required location. Such targeted delivery poses a much lower risk of non-target effects occurring, particularly when compared to the non-target effects that may arise following the systemic administration. The systemic injection of cytokines for example generates a severe toxic reaction from the patient which does not occur when the same cytokine is delivered by a targeted GM vector [47].

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3.31 An additional indirect benefit of a more targeted treatment is less medicine needing to be delivered. Therefore if a release to the environment does occur then there should be less material to be released. With GDEPT for example the use of GM systems with a high bystander effect should also reduce the quantities administered.

3.32 Non-target effects may occur as a consequence of the expression of genetic modifications that have been made in addition to those conferring the intended function. The presence of superfluous marker transgenes (such as GFP (green fluorescent protein) and antibiotic resistance genes) should therefore be avoided. Such transgenes do not include the marker genes incorporated into vaccines for veterinary applications to assist in the differentiation between vaccinated and infected animals (DIVA vaccines).

3.33 The greatest potential risks of incurring non-target effects are from the use of GM viruses in applications involving repeated administration of the GMO. Such applications include most gene therapy applications94 , and some vaccination strategies that require repeat injections. Of the GM veterinary virus-based vaccines described in Chapter 2, non-target effects have only been reported for the canarypox 9.4 (ALVAC) system (albeit at very high doses of 10 CCID 50 /dose). At these doses the ALVAC vaccine system has been found to cause mild, localised and transient side- effects in cats, dogs, ferrets and horses [136].

3.34 A high incidence of sarcomas (soft tissue tumours) has been reported in cats at the points where the animals have been vaccinated. The occurrence of ‘feline injection- site sarcoma’ is most commonly associated rabies and feline leukaemia virus (FeLV) vaccination. Whilst the exact aetiology of the sarcomas is not clear [43], the adjuvant used in the vaccine may be a factor [49]. The GM canarypox-based rabies vaccine (which does not contain an adjuvant) may therefore offer an alternative to adjuvanted vaccines with a lower potential for the occurrence of sarcomas.

Untargeted delivery and expression of the transgene(s) by the GM virus 3.35 Untargeted delivery and expression of the transgene(s) is reported to lead to non- target effects to the recipient through:

♦ Problems with the expression of the transgene(s) outside the target cells. Where cells other than the target cells are infected this essentially reduces the number of target cells infected and consequently diminishes the effectiveness of the gene therapy treatment. An important safety implication of a reduction in effectiveness is that the treatment consequently requires a higher viral dose, or a greater number of injections. ♦ Sub-optimum expression of the transgene(s). Too little expression may result in the failure to correct the disorder, and too much may result in adverse effects.

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♦ Inadvertent modification of germline cells in the gonads (germline transduction). Only one incident of inadvertent germline modification has been reported [110], indicating that the risk posed is very low (although this does assume that adequate surveillance in other trials has taken place). ♦ Difficulty in the delivery of the transgene (and vector) to target organ-wide conditions such as cystic fibrosis. In this case, although the disease is caused by a mutation in one gene (CFTR), the use of viral vectors to deliver a correctly functioning version and achieve an effective treatment has been limited by a failure in ensuring transgene expression of sufficient magnitude and distribution throughout the lungs.

3.36 Ensuring effective delivery should therefore reduce the occurrence of non-target effects and the level of risk posed. Apart from the single germline incident, no adverse effects occurring as a consequence of untargeted delivery have been reported. The adverse effects of vector-induced immune response are a consequence of the presence of the vector rather than the expression of the transgene. These have been discussed in a separate section below.

3.37 Non-selective expression of the gene, encoding the enzyme responsible for the conversion of the prodrug into the toxic compound in GDEPT-based, cytotoxic and pro-apoptotic therapies can result in damage to normal cells. The limitation can be overcome by placing the relevant genes, for example TK (in the HSV-TK-GCV system), and γCD (in the CD/5-FC system), under the control of tumour-associated regulators so that expression is tumour specific [37]. Tumour specificity can be made to either all tumours using a general regulator such as a mutated p53 pathway, or more specific to a particular type of tumour, such as prostate cancer [55]. Further reduction of the potential for such non-target effects can be achieved through the use of non-invasive imaging (via PET) 95 to confirm correct location of transgene prior to prodrug administration.

3.38 This non-target effect could only pose a risk to animals or people other than the recipient patient if they were exposed to both the prodrug and the GMO. The likelihood of this occurring is negligible and means that the potential risk is effectively zero.

Vector-induced immune response (GM viruses) 3.39 Non-target effects as a consequence of an immune response to the GMO may be expected with GMOs to which the recipient has had some prior exposure, either from previous administrations of the virus vector or prior contact with the wildtype. Such non-target effects are therefore most likely to occur in treatment therapies, such as gene therapy and some vaccination applications that involve multiple injections of the

94 Not all gene therapy applications pose such non-target effects. Anti-angiogenic treatments are reported not to pose non-target effects [64].

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GMO. Vector-induced immune response is described as a non-target effect as it occurs as a response to the presence of the viral vector, and not to its transcription. Occurrence of the immune response is therefore not unique to GM viruses and may also occur with non-GM strains [242].

3.40 The issue of vector-induced immunity is of greater relevance to gene therapy than vaccination, as the former usually involves multiple doses of the gene therapy agent over a relatively short period of time; and because vaccination by design is intended to generate an immune response. In addition to the adverse effects to the recipient, this non-target response also limits the uptake of the vector, the duration in which expression of the transgene(s) can occur and the effectiveness of subsequent inoculations (including booster vaccinations as part of the vaccination programme)[144][174].

3.41 The viral vectors most likely to induce a non-target recipient immune response are those such as adenovirus which express immunogenic epitopes within the virus [241]. The use of adenoviruses as in vivo gene delivery vectors, both in gene therapy and vaccination applications is reported to result in an innate immune response by the recipient to the vector [240][239][241]. In some cases, depending on the particular vector and the dose, a severe acute inflammatory response can occur, and one death has been reported. The fatality occurred in a human clinical trial following administration of 6x10 11 human adenovirus serotype 5 particles modified to express the ornithine transcarbamylase (OTC) gene. The GM adenovirus was replication deficient with both E1 and E4 genes deleted [259]. This illustrates the worst case scenario in a trial of this nature.

3.42 The adverse effects posed can be minimised, although not avoided altogether through the following [144][174]. The success of these reduces for treatments requiring repeated administration of the GMO [214]:

♦ the administration of the vector via mucosal or intramuscular routes rather than intravenous delivery. Mucosal or intramuscular delivery are not suitable for the treatment of solid tumours; ♦ the use of a different serotype (serotype switching). Some viral serotypes are more prevalent in the environment than others. Using a less common serotype should therefore reduce the likelihood of the recipient having had prior exposure to the wildtype. For human adenovirus for example there are more than fifty serotypes identified, with HAd11 and HAd35 described as having particularly low seroprevalence, and therefore less likely to cause a vector-induced immune response; ♦ the use of a virus strain from a different species. For example because adenoviruses are species specific then nonhuman adenovirus are not prevalent

95 PET – positron emission tomography is a medical imaging technique which produces a three dimensional image or map of functional processes in the body. 3-92

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in humans. A simian adenovirus as a vector for a human application should therefore evade any preexisting recipient immunity to human adenovirus; ♦ the use of gutless (helper-dependent) vectors. These are viral vectors in which the majority of the virus genes have been deleted, thereby reducing (but not eliminating) the potential for the virus to induce an immune response. Gutless vectors also offer a greater carrying capacity (up to 36kb) [175]. The extra capacity of gutless vectors may itself have implications to the recipient immune response. Because the DNA carried by these vectors is usually much lower than 36kb, ‘stuffer DNA’ is used to complete the excess. This stuffer DNA can elicit an immune response, although this can be avoided if human intronic sequences are used (for human applications) [175]; ♦ co-administration of the vector with immunosuppressive agents such as cyclosporine, cyclophosphamide and deoxyspergualin that are designed to inhibit the recipient’s humoral and cell-mediated immune responses; ♦ vector microencapsulation, in which the virus vector is coated in a polyethylene glycol-cationic lipid. Where the encapsulating material used is biodegradable then virus viability is reported to be unaffected by the process, although transgene expression may be 50-70% lower, leading to higher quantities of vector needing to be administered; and ♦ modification of the vector, through the alteration of: o replication competence through (in the case of adenovirus) deletion of the E (early) genes. Second generation adenovirus vectors for example are reported to induce a lower recipient immune response than first generation vectors 96 , although not as low as third generation vectors [175]. E3 gene products in particular are reported to be involved in the modulation of recipient immune response to the virus. o immunodominant epitopes of the vector’s capsid proteins through the covalent attachment of polymers such as HPMA 97 or polyethylene glycol (PEG).

3.43 A similar, albeit less severe recipient immune response is reported to occur with the use of vaccinia virus as a vector. This is a consequence of pre-existing immunity within the human population following the use of vaccinia virus in the vaccination against smallpox. This adverse response to vaccinia virus is avoidable through the use of fowlpox or canarypox viruses as an alternative poxvirus vector [185].

3.44 Non-target effects have also been reported due to the particular adjuvant used in the vaccine [255]. The majority of attenuated live vaccines do not require adjuvants (whereas killed/subunit vaccines do).

96 First generation adenovirus vectors have deletions of their E1 or E1+E3 genes, whereas second generation vectors are E1 or E1+E3 and E2 or E2+E4 deleted. Third generation vectors are the gutless (helper-dependent) vectors. 97 N-(2-hydroxypropyl) methacrylamide. 3-93

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Insertional mutagenesis / oncogenes activation (GM viruses) 3.45 The ability of some viral vectors (particularly retroviruses and adeno-associated viruses) to integrate into the chromosome can result in insertional mutagenesis and the activation of oncogenes [237]. The likelihood of insertional mutagenesis occurring increases with repeated administrations of the GM virus. Gene therapy and some vaccination applications therefore pose the greatest risk of insertional mutagenesis.

3.46 Oncogenesis occurs as a consequence of viral integration, either through the virus delivering its own oncogene, or by activating a recipient’s oncogenes post- integration:

♦ the delivery of the oncogenes by the virus is associated with induction of cancers with a relatively short development period. ♦ the activation of the recipient’s oncogenes(s) is associated with a much longer development period, indicating that there is a much lower probability of oncogenesis occurring in this way. However, the use of gene therapy agents in young people as a preventative treatment (rather than use in older people as a reactive treatment against ‘ageing’ diseases such as cancer) means that the recipient has an increased likelihood of still being alive at the end of the latency period. Examples of preventative treatments include the use of GM viruses to deliver antioxidant or cytoprotective genes to protect against future heart attacks [78].

3.47 The potential hazards (to the immediate recipient) were demonstrated in the gene therapy trial for X-linked severe combined immunodeficiency (X-SCID) in which some of the patients developed T cell leukaemia [218][80]. It is however unclear if insertional mutagenesis was responsible for the severity of the effects that occurred with this trial. It is possible that the therapeutic principle is itself part of the problem, as the selective advantage conferred on cells carrying the therapeutic gene is very strong and there is effectively no competition from normal lymphoid cells due to the gross deficit in untreated X-SCID patients [301].

3.48 The potential hazards of insertional mutagenesis and oncogenesis are only of relevance to the wider environment and public health if the GM virus is released from the recipient and infects other people or animals. This can only occur with replication competent viruses, although not all replication competent viruses are overtly oncogenic [237]. Replication competent retroviruses have been used as the gene therapy vector in a Phase I clinical trial against glioma [64].

3.49 The major determinants of whether integration can lead to oncogenesis are the oncogenic potential of the virus itself and the viral long terminal repeat (LTR) [237]. In the X-SCID clinical trial where oncogene activation was observed [218] both risk factors were present. The pMFG viral vector used was a MoMuLV (a strongly

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oncongenic murine retrovirus), and the virus’s LTR was also used to control transcription of the transgene. As with other non-defective oncogenic retroviruses the LTR is the primary pathological determinant of MoMuLV. The virus has been reported to induce oncogenesis via activation of any one of a number of cellular genes 98 [237].

3.50 Concerns have also been raised over the oncogenic potential of X protein sequences contained in the post-transcriptional regulatory element of the woodchuck hepatitis virus (WPRE). The significance of this is that WPRE has been incorporated into a number of viral vectors (including retrovirus, adenovirus, AAV and lentivirus) where it confers significant beneficial effects on transgene expression [84]. Although the X protein is not directly oncogenic, studies have reported that under some circumstances it can act as a weak cofactor for oncogenesis. The potential for oncogenesis may be minimised by modifying the WPRE to prevent any expression of the X protein fragments [84].

3.51 The occurrence of oncogenesis and the potential risk posed may therefore be minimised through the following [237][235]:

♦ the selection of non-oncogenic retroviral and AAV vectors. The use of self- inactivating (SIN) retroviral vectors should pose a lower risk as the LTRs are deleted post-transduction of the cell [237][105]. However, limitations on gene transfer efficiency mean that retroviral SIN vectors have not been used widely [237]; ♦ the use of a promoter for the transgene that does not have highly active enhancer elements. If high levels of gene product are required then consideration should be given to strategies other than enhanced promotion, such as codon-optimisation; ♦ the prevention of genes adjacent to the point of viral integration also being activated. This can be achieved through the incorporation of strong transcription termination/polyadenylation signals and gene isolator sequences; and ♦ avoidance of viral vectors using the WPRE regulatory element.

3.52 If possible it would also be advantageous to direct the insertion of the retrovirus so that it only occurs at specific sites within the recipient cell chromosome [243]. This would minimise the potential for activation of the recipient’s oncogenes. Although some strategies designed to achieve this have been reported they have not been successful to date [235].

98 Activation of the any one of the following MoMuLV cellular genes can induce oncogenesis - Ahi1, Bla1, Bmi1, Cyclin D2, Dsi1, Emi1, Ets1, Evi1, Gfi1, c-Ha-ras, Lck, Mis2, Mlvi2, 3 and 4, c-myb, c-myc, N-myc, Notch1, Pal1, Pim1 and 2, prolactin receptor, Pvt1, Tiam1 and Tpl2 [237]. 3-95

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3.53 Whilst integration poses a potential hazard, it does offer the advantages of stable and efficient insertion of the gene into the recipient’s chromosome (leading potentially to lifelong expression of the gene), and the subsequent transcription of the gene as part of the normal cellular process. It is therefore not straightforward just to avoid using integrating vectors because of insertional mutagenesis. If integrating vectors are used then the risks posed can be minimised through the careful selection, design and modification of the virus vectors [237]. Applications involving replication competent retrovirus where these steps have been taken should have a lower risk of oncogenesis occurring. In a therapeutic context the risk of oncogenesis should be considered against the potential for terminal disease should treatment not take place.

Genetic stability (GM viruses)

3.54 The stability of the genetic modification is important as it ensures that the GMO continues to behave as intended through the production process and during its intended use. With GM attenuated viruses in particular, genetic stability is important to prevent reversion to a pathogenic wildtype. As described, pathogenic attenuations are enhanced through large and multiple deletions and/or replacement strategies.

Changes in cell, tissue and host tropism (GM viruses)

3.55 The genetic modification of microorganisms by definition results in the alteration of the organism’s genetic material. These changes increase the risk posed by the GMO where they confer an increased ability to survive and/or replicate in the environment, or greater pathogenicity compared to the wildtype strain. Should the modification confer any of these changes then the GMO will pose a potentially greater risk to the environment and/or human health.

3.56 Some of the modifications described in Chapter 2, such as the alteration of virus coat proteins are designed to change the host tropism of the virus. Where these changes are an intended characteristic of the GMO then the risk assessment should consider the new hosts that could be infected and the implications to them 99 . The modification of lentiviruses with the G-protein envelope protein from Vesicular Stomatitis virus for example extends the host range of the GM lentivirus so that it can infect an almost universal set of cells, creating a greater risk should the virus contaminate the environment.

99 Where the changes are not intended, then further work with the GMO should not be conducted until the potential for such changes is removed. 3-96

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Gene transfer (GM viruses)

3.57 Transfer of the modified genes has the potential to occur through horizontal and vertical gene transfer. These provide a mechanism for effects to the wider environment and human health to occur, and therefore increase the potential risk posed by the GM virus.

Horizontal gene transfer (GM viruses) 3.58 Horizontal gene transfer involves the nonsexual transfer of genetic information between genomes or between different organisms of the same or different species [153]. The transfer of genetic material between GM and non-GM viruses does not necessarily pose a risk to the environment and wider public health. The key consideration is the function of the gene(s) transferred and the changes it may confer on other viruses. Such transfer is significant from a risk assessment perspective where the GM virus is able to obtain new unwanted characteristics such as virulence traits or the reversion to a pathogenic wildtype.

3.59 In viruses horizontal gene transfer occurs as a consequence of homologous recombination between replication competent viruses occupying the same cell 100 . Where viruses with segmented genomes (e.g. orthomyxoviruses 101 ) co-infect the same cell, a novel virus can be generated through the daughter virus acquiring genetic information from multiple parent viruses.

3.60 Homologous recombination can be minimised through the use of replication deficient strains; the careful design of replication competent vectors to minimise homologous sequences; and the use of GM viruses as vectors in animals of a different species to their natural host. This latter point, in which a human adenovirus could be used as a veterinary vaccine vector ensures a negligible likelihood of a natural reservoir of the wildtype virus (i.e. the human adenovirus) existing within the recipient animal. Although no examples of non-human viruses being used as GM vaccine vectors for humans have been reported, the canarypox ALVAC strain has been used as a gene therapy vector to deliver IL-2 to target colorectal cancer in humans [140]. GM human viruses and GM animal viruses are being used as vectors in veterinary applications [136] (see Chapter 2).

3.61 With regard to recombination between GM vaccine vectors and wildtype viruses, it has been reported that cats can be infected with cowpox virus [139]. If the cat has also been vaccinated with a canarypox-based vaccine (such as the Purevax Feline Rabies vaccine) then there is the potential for recombination between the cowpox and canarypox viruses. No such recombination has been reported, indicating that the potential risk of gene transfer between these viruses is low. The reported issues

100 Homologous recombination can also occur where only one of the viruses is replication competent, and the other replication deficient. 101 Includes influenza viruses. 3-97

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with recombination are more of a developmental problem rather than something which might be expected in the field, and should therefore not be considered a risk with commercial GM vaccines.

Vertical gene transfer (GM viruses) 3.62 Vertical gene transfer involves the movement of genes ‘downwards’ (or ‘vertically’) through generations of organisms, thereby providing a mechanism for the dissemination of the genes into the wider environment. Such transfer to germline cells is a potential risk following the systemic administration of viral vectors [113]. However, the incidence of occurrence involving a GMO is very low [111], with only one such event reported [110]. In this case the transgene delivered using a GM adeno-associated virus (AAV) vector was identified in the patient’s semen. The clinical trial involving AAV serotype 2 administered to the liver via the hepatic artery in adult men with haemophilia B was halted immediately [110][112].

3.63 Further studies in a surrogate animal model (rabbit) indicated that the occurrence of vector sequences in semen are dose and time dependent [113][112]. Vector sequences were only detected within four days after administration of the AAV2 and were undetectable for hundreds of spermatogenesis cycles thereafter [112]. This finding was supported by Arruda et al. (2001)[113] who found no vector sequences in semen samples in rabbit at time points ranging from 7-90 days post-intramuscular injection of 1x10 13 vector genomes (vg) of GM AAV per kg. The same research also reported an absence of vector sequences in semen following intramuscular injection of AAV at doses up to 2x10 12 vg/kg (also in rabbit).

3.64 The severity of consequence, and therefore the risk of vertical gene transfer is higher where recipients of the GM vectors are of a reproductive age, as they may disseminate the transferred gene [112].

Survival and dissemination of the GMO (GM viruses)

3.65 The survival and subsequent spread of the GMO is a key consideration of the regulatory risk assessment process. With viruses, dissemination is dependent on the virus being able to replicate, and then being released from the infected animal. Survival is determined by a virus’s resistance to environmental factors such as ultraviolet and desiccation, as well as its ability to infect a suitable host. Viruses that are both replication-incompetent and have limited ability to survive in the environment outside a suitable host therefore pose a lower risk to the environment and human health. They will not be spread from the recipient person or animal and the transgene(s) will be contained biologically within that person or animal. Ability to survive in the environment is therefore less significant. With replication-incompetent GM viruses, survival is only important from a risk assessment perspective should stocks of the GM virus be spilt or released prior to their intended use.

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3.66 Replication competent GM viruses are able to replicate within the recipient’s cells post-administration. Consequently the recipient person or animal is able to spread GM virus particles into the environment or directly to other organisms. Although therefore posing a greater risk, replication competent viruses offer particular advantages in terms of therapeutic efficiency (as gene therapy vectors)[64] and immunogenicity (as vaccine vectors)[163]. It is therefore not appropriate to restrict the use of replication competent GMOs purely on the basis of the potential risks posed by their ability to replicate. The consequence(s) of any replication, in terms of potential pathogenesis or the effect of the transgene(s) must be considered.

3.67 Table 3.1 illustrates the types of factors that influence the risk posed by replication competent GM viruses. Although the examples of the modifications to the GM human adenovirus (serotype 5) refer to gene therapy applications, the points made are relevant to all applications of GM viruses. In the table the replication competent wildtype adenovirus is assigned a relative risk of zero. The modification of the virus to reduce its host range is assessed to reduce the potential risk to the environment and human health, whereas expanding the host range with additional modification is assessed to pose the greatest increase in risk. These points are discussed in more detail in the SACGM’s Compendium of Guidance [298]. As they are applicable to all GMOs, and not just GM viruses in medicines they have not been described further here.

Table 3.1 – Relative risk of the properties of GM HAd5 relative to a replication competent wildtype (adapted from [152]) Risk relative to Property Example of GM HAd5 virus replication competent wildtype Rep. competent + host range CRAd-HSVtk-RGD +2 expansion + transgene Rep. competent + host range CRAd-RGD +1 expansion Rep. defective + transgene + host HAd5 HSVtk-RGD +1 range expanding modification Rep. competent + host range ONYX-tk 0 restriction + transgene Rep. defective + transgene HAd5 HSVtk 0 Rep. competent wildtype HAd5 0a Rep. defective HAd5 dl 312 (E1A deleted) -1 Rep. defective + transgene + host HAd5 HSVtk-CAR ablated -1 range restricting modification Rep. competent + host range ONYX-015 (E1B deleted, -1 restriction CRAd) b a the ‘zero’ assigned here does not mean that replication competent HAd5 poses no risk to human health and the environment. The purpose of the risk values is to present the potential risk posed by each general modification relative to the replication competent wildtype. b CRAd – conditionally replicating adenovirus

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3.68 The replication competence of GM viral vectors can be altered either through the genetic modification of the virus itself to prevent it from being able to replicate, or by using the GM virus in an environment in which it is unable to replicate (or at least replicate poorly).

3.69 The genetic modification of viruses to reduce or negate their ability to replicate is achieved through the deletion of the particular genes required for replication. As with other attenuating modifications these deletions are only effective if the GM virus is unable to reacquire the missing genes. Although reacquisition may occur through homologous recombination with a wildtype version of the GM virus, it requires both viruses to be present in the same cell. As described, the potential for homologous recombination can be reduced through the correct design of the vector. Separating the vector and packaging functions for a particular virus onto multiple plasmids will reduce the potential for the generation of replication competent strains [284]. In the case of retroviruses the generation of replication competent retroviruses (RCRs) can be further avoided through the use of self-inactivating vectors (SINs) or the complete removal of the coding sequences for gag, pol, and env genes [243] 102 .

3.70 The spread of replication competent GM viruses to other organisms may occur through shedding, contact transmission, (the transfer during physical contact, or contact of contaminated materials, between people or animals), or arthropod vector transmission. These dissemination pathways do not apply to all replication competent viruses.

3.71 The potential risk posed by the release of replication competent GM viruses through shedding or other process may be reduced if the GM virus is administered in a situation in which it can be physically contained. Such uses are restricted to medical or veterinary hospital based administration of GMOs in a treatment where the patient remains within the hospital for the period of the therapy and releases of the GMO from the recipient can be contained and disposed of correctly.

3.72 Where oral delivery involves the use of laced bait, consideration must be given to the ability of the GMO to persist for a period of time in the environment from the point of release in bait to ingestion by the target animal. Some survival in the environment is required so that the GMO is still viable when ingested. However, extended persistence in the environment increases the likelihood of non-target organisms coming into contact with the GMO.

Shedding (GM viruses) 3.73 Shedding has been reported for GM viruses used in veterinary medicines. GM porcine adenovirus for example has been isolated from nasal swabs of some pigs

102 The inadvertent production of replication competent retrovirus (RCR) constitutes the principal safety concern for the use of lentiviral vectors in human clinical protocols [300] as a consequence of the potential for insertional mutagenesis. 3-100

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following vaccination with the GM adenovirus expressing gD gene from pseudorabies virus [165]. Nasal isolation suggests that some aerosol release may be possible. Although such findings indicate that the use of the GM adenovirus as a vaccine vector in pigs might result in the release of GM virus to the environment, the implication to the environment is the spread of immunity to pseudorabies virus to other pigs. This may only be significant if it is confused with a case of actual pathogenic pseudorabies (i.e. where clinical symptoms cannot be differentiated).

3.74 GM oncolytic viruses used in clinical (human) trials may also be shed from the patient. Although no such results have been identified, a Phase I clinical trial with non-GM Newcastle Disease Virus (NDV) 103 found the virus in the patient’s urine up to three weeks after the first injection [63]. Similar results could occur with GM versions of the virus.

Dissemination as a consequence of mechanism of delivery (GM viruses) 3.75 Applications of GMOs as veterinary vaccines have the potential for release to the environment as a consequence of their mechanism of delivery. The vaccination of wild animals for the control of diseases such as rabies through the distribution of doped bait may result in the dissemination of the GMO if the bait is not eaten by the target animal.

3.76 The use of GMOs in veterinary vaccinology in a contraceptive capacity (virally vectored immunocontraception (VVIC)) is an application that was intended at its inception to be released into the wider environment and survive for a sufficient period of time to infect the target animal [177]. Assessment of the potential risks posed by this application must therefore address the host specificity of the released GMO and ensure that the GMO can only infect the target pest, with negligible effect(s) to other organisms.

3.77 However, initial studies found that for the approach to be successful it needed to infect a significant proportion of a target pest population ( ≥70-80%). The combination of this with the temporary nature of the contraceptive effect has meant that this technology may be too costly for use against wild pest animals (with the exception of mice) 104 [187]. The technology is more suited for use in companion animals and livestock, with the vector administered parenterally. Delivery to each animal by injection negates any requirement for the vector to be able to spread through the animal population (as envisaged originally for wild pest populations). The vector can then be designed to be contained biologically within the vaccinated animal. No shedding of infectious canine herpesvirus (CHV-1) was found from foxes

103 The NDV used in the clinical trial was replication competent and naturally attenuated. It was used in the trial as an oncolytic virus to lyse various human cancer cells in vitro. 104 As the reproductive life-span of a mouse does not exceed the period of the contraceptive effect means that VVIC can provide a permanent contraceptive effect in mice, unlike other animals such as rabbits and foxes. 3-101

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post-infection for example. Changes in the intended use of VVIC technology should therefore reduce the potential risks of this application to the environment.

3.78 The dissemination of GMOs used as vaccine vectors for fish into the water environment is expected to be limited. This is a consequence of the limited use of GMOs in aquaculture, and the method of administration (see subsequent section on Survial and Dissemination). None of the studies describing the use of GMOs in fish used live GMOs [146]. (There is one report of the successful use of human adenovirus (HAd5) as a GM vector for fish (rainbow trout) [107]. Vector delivery was however only successful by intramuscular injection, and even then at low infection efficiency). The use of GMOs as vaccine vectors for fish therefore poses a negligible risk to the environment.

Arthropod transmission (GM viruses) 3.79 Arthropods may act as the vectors for the transmission of arthropod-borne viruses (arboviruses). Arboviruses reported to have been genetically modified are vesicular stomatitis virus, West Nile virus and yellow fever virus. The transmission through arthropod vectors is dependent on presence of the relevant vector. Yellow fever and West Nile viruses are spread by mosquitoes, and vesicular stomatitis virus spread by mosquito and sandfly. In the eastern USA and Europe the vector for West Nile virus is the mosquito Culex pipiens. In Europe there are genetically distinct populations of the mosquito, one which bites birds and one humans. Whilst birds are able to transmit the virus to other mosquitoes (birds are an amplifying host), the infection of mammals is a ‘dead end’ infection and does not result in the further infection of other organisms. In the eastern USA 40% of the C. pipiens population bites both species [73]. This is proposed as the explanation for the greater spread of West Nile virus in the USA.

3.80 An absence of the arthropod vector will prevent dissemination of the GM arbovirus, and therefore limit the risk of spread through the environment. However, changes in the movements and survival of arthropod vectors as a consequence of climate change means that areas previously free of particular vectors may not remain that way.

Disposal of contaminated materials (GM viruses) 3.81 The risk of release through incorrect disposal of contaminated materials or needlestick injury is dependent on the mechanism of delivery for the vaccine (and the incorrect application of biosafety regulations). Oral delivery does not involve the use of needles (or other sharps), thereby avoiding risk of needlestick injury whereas of course parenteral administration does. Oral delivery is also likely to result in the potential contamination of a smaller quantity of materials, such as swabs and dressings, as well as dispensing vials and needles than parenteral administration. Incorrect disposal of contaminated materials represents a pathway for the release of the GMO into the environment. Adherence to the biosafety regulations produced by

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organisations such as the UK’s Health and Safety Executive (HSE) and the Gene Therapy Advisory Committee (GTAC) mean that release of GMOs to the environment through incorrect disposal of contaminated materials should not happen.

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3.2 ASSESSMENT OF THE POTENTIAL RISKS ASSOCIATED WITH GM BACTERIA IN MEDICINES

Pathogenicity (GM bacteria) Production of biologically active and/or toxic products (GM bacteria) Production of non-target effects (GM bacteria) Vector-induced immune response (GM bacteria) Genetic stability (GM bacteria) Plasmid stability (GM bacteria) Plasmid conjugation (GM bacteria) Horizontal gene transfer (GM bacteria) Survival and dissemination of the GMO (GM bacteria) Shedding – the release of microorganisms into the environment (GM bacteria) Dissemination other than shedding (GM bacteria) Indirect effects on survival and dissemination (GM bacteria) Replication competence (GM bacteria) Auxotrophic attenuation (GM bacteria) Self-destruction as a containment system (GM bacteria)

Pathogenicity (GM bacteria)

3.82 From a risk assessment perspective, zero or low pathogenicity of the GM bacteria is preferable as it reduces the importance of other factors in minimising the overall risk posed by the GMO. The non-pathogenic and non-invasive nature of the GM bacteria used as drug delivery vectors means that pathogenic and non-target effects should not occur or be expected from these strains. The wildtype strains from which the GMOs are derived are approved food-grade microorganisms and therefore non- pathogenic.

3.83 The factors determining the pathogenicity of GM bacteria to the recipient and the wider environment are as follows:

♦ application or environment in which the GMO is used. For example the use of obligate anaerobic bacteria as gene delivery vectors [54] restricts the pathogenic effect of the bacteria to the anaerobic environment of the solid tumour. ♦ presence of attenuations to the microorganism’s pathogenic characteristics so that it is rendered less or non-pathogenic. Examples include both the direct and indirect modification of virulence genes. A direct modification would be the alteration of the ctxAB gene of Vibrio cholerae which encodes the cholera toxin. An indirect modification would be the alteration of genes involved in the regulation of virulence genes, e.g. phoP (the phosphorylated transcriptional activator) [196].

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♦ presence of attenuations to other characteristics of the microorganism that render it unable to survive for a sufficient period of time and replicate to a sufficient level, or spread through the recipient sufficiently to cause disease. Examples include: o Survive - auxotrophic modification of an invasive strain of Escherichia coli to render it dependent on the presence of diaminopimelate [157]. This is not present in mammalian cells and consequently the E. coli is not able to persist for longer than required and cause disease. The effectiveness of the auxotrophic modification in preventing disease will of course depend on the type of modification and the availability of the missing compound. o Spread - the deletion of virG, kcpA or icsA genes in Shigella flexneri, or the actA gene in Listeria monocytogenes rendering it unable to spread from cell to cell in the recipient [194]. Modifications to restrict expression to either specific tissues within the recipient, or to particular conditions such as hypoxia, oxidative stress or inflammation [78]. The GMO is therefore effectively biologically contained within those tissues or environments.

3.84 The success of any genetic modification to reduce (attenuate) pathogenicity depends on the effectiveness and stability of the attenuation(s). Whilst not reported for GM bacteria, adverse effects caused by a reversion to virulence have been reported for a non-GM attenuated vaccine. Eleven cases of systemic Pasteurella haemolytica infection in cattle were reported following vaccination with a live avirulent culture of the bacterium 105 [261].

3.85 The introduction of multiple attenuations, and/or restricting the ability of the GMO to reacquire the deleted gene(s) improves the likelihood of maintaining the attenuated form of the microorganism. Attenuated pathogenic GM bacteria that exhibit one or more of these strategies should therefore present a lower risk of pathogenesis. This may be achieved by:

♦ Deletion rather than point mutations – removal of entire genes, or large sections of the genes, rather than the modification of small sections such as the mutation of single amino acids. Deletion of larger sections of the genes reduces the potential for reversion to pathogenic wildtype. Point mutations may also be overcome by secondary mutations of the microorganism, leading to a reversion to a wildtype strain. Modification of the diphtheria toxin ( Corynebacterium diphtheriae ) with a site directed mutation of Glu-148 is reported to be overcome by subsequent mutations substituting the glutamic acid for Val-147 or by a five codon deletion [196]. The potential for reversion through secondary mutation therefore needs to be considered as part of the risk assessment process.

105 Interestingly several thousand doses of the vaccine had previously been administered without such adverse effects being reported. The effects reported [261] were thought to have arisen because of other factors, including stress. 3-105

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♦ Replacement – inserting the transgene(s) in the place of the deleted gene. Therefore should the GMO reacquire the missing gene it will lose the transgene. ♦ Prevention of re-acquisition – for example the removal of the entire cholera toxin genetic element (the filamentous phage Ctx ϕ) is used to attenuate pathogenic Vibrio cholerae for use as a live vaccine. A further deletion of the attRS1 gene, which is the site of reintegration of Ctx ϕ (also referred to as the phage attachment site) decreases the likelihood that the attenuated V. cholerae can reacquire Ctx ϕ in the environment and revert to a pathogenic strain [194][226]. In a slight variation on this, V. cholerae CVD 103-HgR has been attenuated to contain defective copies of the Ctx ϕ element. Whilst rendering the strain non- pathogenic, the presence of the defective copies of Ctx ϕ prevents the bacterium acquiring a functional version [226]. At a more general level, removing the ability of the GM bacteria to undergo conjugation should block re-acquisition of the gene through this process.

3.86 For GM bacteria used as vaccine vectors, excessive attenuation can reduce the immunogenicity of the vector and consequently its effectiveness as a vaccine [194]. Therefore some balance needs to be made so that the GMO retains the antigens necessary to induce the immune response, without retaining too much virulence. With toxin genes for example this can be achieved by:

♦ modifiying the toxin gene to produce just a truncated toxin peptide without the enzymatically active domain; ♦ introducing a point mutation to render the enzymatic domain inactive; ♦ deletion of nearly all the toxin gene(s) leaving only the genes encoding accessory toxin subunits such as adhesions or translocation domains.

3.87 The first two approaches are reported to be suitable and effective for subunit toxins such as tetanus and botulinum ( Clostridium tetani and C. botulinum respectively). The third approach is the one used with Orochol ® the GM V. cholerae strain CVD 103-HgR that has received commercial approval for use as a live cholera vaccine [196]. CVD 103-HgR has been modified so that almost all the ctxA genes that encode the active part of the cholera toxin have been removed, leaving just the genes encoding the protective antigenic CtxB subunits that are responsible for the docking of the receptor to the host cells. Such a deletion makes reacquisition of the toxin genes impossible by reversion, although it can still theoretically occur through horizontal gene transfer from wildtype V. cholerae . Rigorous assessments prior to commercial approval for CVD 103-HgR reported no such transfer [228].

3.88 Studies have reported difficulties in applying the results from animal studies to humans, with modifications demonstrating good attenuation in animals but only limited attenuation in humans. Examples include variations of the galE deficient

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Salmonella typhi Ty21a strain 106 , and the aroA and virG deleted Shigella flexneri 2a strain. Both bacteria were highly attenuated in a mouse and guinea pig model respectively. However when administered to humans the volunteers became ill with a typhoid-like fever (when administered GM S. typhi ) or fever/dysentery (when administered GM S. flexneri ) [194]. Care should therefore be exercised in predicting the responses in other species from results in heterologous models.

Production of biologically active and/or toxic products (GM bacteria)

3.89 Where the production of a biologically active and/or toxic compound is the desired function of the GMO then the risk assessment should assess the intended role of that compound, the potential for it to be transferred to other microorganisms and the implications of that transfer to the behaviour of those microorganisms. Production of the compound should be tailored to its intended function with consideration given to whether too much or too little compound is produced. Overproduction may give rise to non-target effects, whilst insufficient production may render the modification ineffective.

3.90 Overexpression of the antigen by the vaccine vector, for example as a result of a high copy number plasmid, a strong promoter, or constant expression can lead to a reduction in immunogenicity, as well as decreased vector fitness and increased vector attenuation [194]. Overexpression can be minimised through the use of in vivo inducible promoters such as the anaerobically inducible nirB and dmsA, and iron-regulated irgA, rather than strong constitutive promoters [194]. Achieving the correct level of expression will ensure that the optimum amount of GMO is administered. Underexpression would require more GMO to be administered, leading potentially to a greater risk of non-target effects post-administration. Overexpression leading to adverse effects to the general public or wider environment has not been reported for any application of a GM bacteria-based medicine.

3.91 Any expression of a toxin by a vaccine vector should only be a fragment of the complete toxin i.e. that sufficient to elicit an immune response, and should therefore not be biologically active.

3.92 The genetic modification of a microorganism to produce a novel product should also consider the implications to the behaviour of the modified strain. Particular consideration should be given to changes that alter the pathogenicity, and host or tissue tropism of the GMO. Such changes alter the ability of the GMO to compete with other microorganisms in the environment, and therefore enhance their survival.

3.93 Some of the modifications described in Chapter 2 are intended to alter these characteristics of the GMO. An example is the modification of Streptococcus mutans

106 Although S. typhi Ty21a is an effective attenuated vaccine, three to four doses are often required to induce significant protective immunity. Therefore efforts are ongoing to improve the vaccine strain. 3-107

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to produce the antibiotic mutacin 1140, thereby enhancing its ability to out-colonise wildtype strains within the dental microflora [124]. Where such changes occur the level of risk posed must also consider the potential for the GMOs to be released into the wider environment or to transfer the modification(s) to other microorganisms. With the mutacin 1140 expressing S. mutans for example, the bacterium was also modified for auxotrophic dependence on D-alanine. The GM S. mutans is therefore modified to survive in the environment (in dental microflora) for a significant period of time, and outcompete lactic acid producing S. mutans present. If the modifications have no other effects on the behaviour of the GMO or the function of the dental microflora, then the risk posed by the use of this GM bacteria is assessed as relatively low. The potential risk would be lower if the auxotrophic D-alanine modification provides an effective control of the GM bacteria. Information from studies with rats suggests that such a modification may not confer complete control over the GMO, as GM S. mutans modified to be auxotrophic for D-alanine did persist at a low level in the oral cavity of rats in the absence of D-alanine supplements [309].

Production of non-target effects (GM bacteria)

3.94 GMOs causing non-target effects pose a potentially greater risk to the environment and human health. Non-target effects may occur as a consequence of the expression of genetic modifications that have been made in addition to those conferring the intended function. These include marker genes, for example antibiotic resistance genes, that are used during the development of the GMO to assist in the identification and selection of the correct genotype. Their presence in a commercial product should be avoided. An exception is the inclusion of marker genes in DIVA vaccines for veterinary applications [255]. The key issue with these DIVA marker systems is that they do not pose any effect of their own and do not provide the GM vector with any selective advantage (that could be realised in the environment and confer improved survival). For example the mercury resistance operon is expressed chromosomally by the V. cholerae CVD 103-HgR attenuated vaccine as an identification marker. The marker does not confer a selective advantage to the GMO

in the environment as the mercury concentration required to trigger it ( ≥20µM HgCl 2) is higher than the highest mercury concentrations occurring in the environment (1- 10nM in mercury contaminated waters) [228][226]. Many of the DIVA vaccines are null mutants and therefore do not pose such effects. The likelihood of non-target effects occurring as a consequence of marker sequences in GM DIVA vaccines is therefore assessed as negligible.

3.95 Of the GM bacteria-based veterinary vaccines described in Chapter 2, non-target effects have only been reported for the Equilis StrepE ® equine vaccine. This vaccine was designed originally to be administered intranasally. However, in early stage studies only intramuscular administration was found to confer adequate protection [255]. This administration route incurred significant adverse effects to the vaccinated horse (inflammation, abscess formation at the injection site, and muscle soreness). Subsequent investigations found that these could be avoided, with no effect on

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efficacy of protection, by administration submucosally into the horse’s upper lip. Equilis StrepE ® was subsequently licensed with this route of application. The vaccine does require administration every three months and can still cause abscess formation in the upper lip.

3.96 Non-target effects may also occur as a consequence of errors during the transcription and translation of the genetic modification. This can be affected by the choice of promoter, the localisation of the transgene(s) or protein (if the GMO is being used as a vector to deliver the protein rather than the DNA) within the vector, codon optimisation for a bacterial host, and plasmid copy number [194].

Vector-induced immune response (GM bacteria) 3.97 Non-target effects as a consequence of an immune response to the GMO may be expected with GMOs to which the recipient has had some prior exposure. The prior exposure may be to the GMO or the wildtype microorganism. Such effects are most likely to occur in gene therapy treatments and some vaccination applications involving multiple injections of the GMO. The adverse effects reported following intramuscular administration of the Equilis StrepE ® vaccine are a consequence of such a response.

Genetic stability (GM bacteria)

3.98 If the genetic modification is not stable then it may be lost from the GMO or become mutated, leading to the occurrence of unwanted effects or even a reversion to a pathogenic wildtype. The non-GM Salmonella typhi Ty21a strain for example has been found to be genetically stable and extremely safe with spontaneous occurrences of adverse events being reported in only 0.002% of vaccinated people between 1991 and 2001 [227]. GMOs constructed from strain Ty21a may also exhibit such stability. The IL-10 expressing GM L. lactis for example is designed so that the IL-10 gene cassette is stably incorporated in the bacterial chromosome [10], and the bacterium further modified to reduce the potential for gene transfer. These characteristics should ensure good genetic stability. GMOs in which the genetic modification is not stable pose a greater risk to the environment and human health as they may revert to a pathogenic state (if attenuated) or transfer the modification to other organisms. The risks posed by poor genetic stability are therefore higher if the GMO is an attenuated pathogen, or if the modification could provide a selective advantage to other organisms.

3.99 The stability of the modification is enhanced if it is constructed on genomic loci that are known to be stable and are not flanked by known active mobile elements (such as transposons) [196]. The presence of multiple copies or dormant versions of the relevant genes should be determined, and, if present, measures should be taken to prevent reactivation or recombination with them. For example:

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♦ V. cholerae contains two copies of the ctxA gene (encoding the active subunit of cholera toxin), both of which are deleted in the vaccine strain CVD 103-HgR [196]. The vaccine strain CVD 103-HgR is reported to be genetically stable [226]. ♦ Mycoplasma mycoides subsp. mycoides (causative agent of contagious bovine pleuropneumonia) contains the genes (whose deletion would result in an attenuated microorganism) as duplicate copies on tandem repeats on the genome. Although only one copy is active, the deletion of both copies is required for a stable and effective vaccine (Westberg et al. 2004; cited by [196]).

Plasmid stability (GM bacteria) 3.100 Although genetic stability is enhanced if the transgene(s) is carried chromosomally [246], in some cases it may be impossible, unnecessary or even disadvantageous to do so. Chromosomal integration is suitable and effective if the modification involves just a single gene copy under the control of a strong promoter [194]. However, if just a single copy of the gene is insufficient to confer the desired response then the insertion of the gene on a multicopy plasmid may be more effective. Plasmids also offer the advantages of relative ease of manipulation.

3.101 If the transgene is carried on a plasmid then in order for the GMO to operate as intended the plasmid (with transgene) must be maintained by the GMO. The ideal plasmid (in addition to conferring the level of expression needed for the required function), should be stable in vivo, not compromise the growth rate or metabolism of the vector, be non-transmissible 107 and have a narrow host range [246]. High copy number plasmids are often unstable in vivo [246].

3.102 A common mechanism to achieve plasmid stability in vivo is described as the ‘balanced lethal host-vector’ system. In this the plasmid contains genes whose products are essential for the survival of the GMO, as well as the genes required to confer the required modification [194][246]. The system means that if the plasmid is lost then the GMO dies. Balanced lethal systems have been developed on the basis of:

♦ Auxotrophies in biosynthetic pathways and cell wall synthesis - o the asd (aspartate-semial-dehyde dehydrogenase) system which confers an absolute requirement for diaminopimelate (an essential component of the peptidoglycan of the cell wall of Gram-negative and Gram-positive bacteria) [194]. The deletion of asd renders the bacterium unable to maintain its cell wall structure causing it to lyse. Asd-stabilised plasmid systems encoding additional antigens have been widely evaluated in

107 For the purposes of this report non-transmissible plasmids are defined as those plasmids which are unable to move from their host cell to another bacterial cell, either on their own through conjugation, or with the assistance or another plasmid. 3-110

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Salmonella typhimurium and were found to ensure stable gene expression. The system has also been reported to be safe in humans [194]. o thymidine deficiency, with the thyA gene conferring the ability to synthesise thymidine. The thyA modification has also been used as an additional modification of Salmonella Ty21a strain in a Phase I clinical trial against cholera and Helicobacter pylori , and also in a GM attenuated V. cholerae strain for a vaccine trial. o the purB system developed for Salmonella sp. The gene encodes an adenylosuccinate lyase which catalyses an essential step in the de novo synthesis of adenosine monophosphate. o the alr gene, encoding alanine racemase. Deletion of this gene renders the GMO auxotrophic for D-alanine [150]. o the glnA system used in V. cholerae [229]. The glnA gene (conferring the ability to synthesise the essential amino acid glutamine) is provided to the V. cholerae on the same plasmid as the toxin antigen. Studies in mice however have found that plasmid-free bacteria could be isolated up to eight days post oral administration of V. cholerae expressing a glnA + plasmid. This indicated that there was sufficient glutamine in the intestinal lumen of the mice for the glnA + plasmid not to be required by the GM bacteria. The glnA system may therefore not provide effective biological containment. o the infA system in E. coli [246]. The infA gene encodes translation initiation factor 1 (IF1), a small intracellular protein essential for the viability of the cell. The system is reported to be stable for at least 120 generations and may be applicable to other bacteria. ♦ Toxin-antitoxin modules - o the hok-sok system [230]. Identified originally from the R1 plasmid of E. coli the hok-sok system comprises three genes ( hok (host killing) , mok (modulation of killing) and sok (suppression of killing)). Whilst the plasmid is retained, Sok-RNA binds to the mature form of the hok mRNA and represses translation. However, if the plasmid is lost the Sok-RNA pool is depleted through rapid decay (half-life 30 seconds) allowing translation of the hok mRNA to a highly toxic trans-membrane protein that irreversibly damages the cell membrane killing the cell. However, further studies with this system have found that >50% of the bacteria tested remained viable even after they had lost the plasmid, suggesting that the system may not be that effective [231].

Plasmid conjugation (GM bacteria) 3.103 Conjugation is the direct transfer of genetic material between bacteria, and is mediated by conjugative plasmids (such as the F plasmid in E. coli and other enteric bacteria) or transposons (for example Tn 916 which can be transferred between a 3-111

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range of Gram positive bacteria)[233]. Chromosomal genes can also be moved during the transfer of a conjugative plasmid, but not with a conjugative transposon [233]. The potential for gene transfer from a GMO would therefore be enhanced if the GM bacteria contained conjugative elements. As gene transfer through the movement of conjugative plasmids provides a key pathway through which the genetic modification can affect the wider environment and human health, then the presence of such plasmids in the GMO means that it poses a potential greater risk to these areas. No GM bacteria with conjugative plasmids have been identified in Chapter 2.

Horizontal gene transfer (GM bacteria)

3.104 Horizontal gene transfer provides a mechanism for effects to the wider environment and human health to occur, and therefore increases the potential risk posed. In bacteria such transfer can occur via phages or mobile genetic elements (plasmids or transposons), and there is therefore an increased likelihood that any changes to phages, plasmids or transposons may be transferred to other microorganisms. Where horizontal gene transfer is assessed to pose a risk to the wider environment and/or public health then the modification of genetic material on structures such as conjugative plasmids and transducing phages that have a higher likelihood of being transferred should be avoided [226]. The potential for transfer may also be reduced by avoiding the introduction of recombination-prone sequence duplications, and/or by disabling essential DNA recombination functions such as reactivation of the recA locus in E. coli [226].

3.105 Gene transfer can of course occur both from and to the GMO. The significance of each depends on the nature of the genetic modification (deletion, mutation or addition of genetic material) and the function of the altered gene(s). Gene transfer from the GMO has the potential to alter the characteristics of other microorganisms in the environment, and vice versa. Where the GMO has been modified to reduce or eliminate pathogenic traits then gene transfer to the GMO, through phage-mediated conversion or by plasmid/transposon-mediated cis or trans -complementation, has the potential to generate a pathogenic microorganism.

3.106 The significance of such transfer to the GMO depends on whether the relevant genes are present within the wider microbial community on mobile genetic elements or phages, and if so whether they can be acquired by the GMO. Uptake of naked DNA from the extracellular environment by the oral streptococci for example is regulated by the com operon (comprising comC, comD and comD ) [310]. The lactic acid deficient GM Streptococcus mutans was also modified to inactivate the comE gene [309], thereby reducing the ability of the GMO to undertake horizontal gene transfer and improving the stability of the genetic modifications.

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3.107 The IL-10 expressing, thyA - L. lactis for example is designed so that the potential for horizontal gene transfer is very unlikely [248]. These modifications consist of:

♦ the stable incorporation of the IL-10 transgene in the bacterial chromosome, rather than on a plasmid; ♦ the absence of any plasmids; ♦ the development of the GMO from a strain of Lactococcus lactis subspecies cremoris MG1363 which lacks a host factor required for conjugative transposition. The GMO is therefore also unable to re-acquire thyA through conjugation; and ♦ as a consequence of the thyA deletion, the severe impairment of phage replication, thereby disabling phage mediated transduction.

3.108 The IL-10 gene cassette is also assessed not to pose any particular selective advantage to another microorganism should horizontal transfer occur. This further reduces the likelihood of gene transfer occurring with this GMO.

3.109 Studies conducted to determine whether a thyA deficient L. lactis can acquire a thyA gene through horizontal gene transfer reported that even in mixed cultures of thyA - L. lactis and thyA + L. lactis, Lactobacillus casei , E. coli or Salmonella choleraesuis (high population density of 5x10 11 cfu/ml) there was no acquisition of the thyA gene [248]. The likelihood of reacquisition of the thyA is therefore assessed as very low.

Survival and dissemination of the GMO (GM bacteria)

3.110 The survival and subsequent spread of the GMO is a key consideration of the regulatory risk assessment process. Survival and replication of a microorganism are linked, as without replication most microorganisms are unable to survive for a significant period of time (although some microorganisms may persist in a non- replicative state, as spores for example). From a risk assessment perspective, the potential risk(s) posed by the GMO relative to the wildtype will be lower if the GMO is less able to survive in the wider environment. If the genetic modification confers a selective advantage to the GMO then the microorganism is more likely to retain the transgene(s) should it be released into the environment. As bacteria are not obligate parasites like viruses, then mechanisms must be in place to prevent the survival or external spread of the bacteria and minimse the spread of the transgene(s) [298].

3.111 The use of bacterial spores as vaccine vectors [220] and also as gene therapy and direct action agents [54] poses a particular issue in terms of survival of the GMO as spores are very hardy and capable of survival in the environment for long periods of time (years). In considering the risks posed by the use of GM spores the survival of the microorganism in the environment should be viewed as likely with the risk assessment focused on the potential effects posed by the bacteria (following germination of the spore) and the nature of the modification. With the GM Bacillus

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subtilis described by le Duc et al. (2003)[220] the unmodified bacterium is non- pathogenic with a history of safe use as a probiotic, and expressing a fragment of the tetanus toxin which should confer no selective advantage. Whilst the likelihood of survival in the environment is high, the risk posed by this particular application of a GM spore in terms of pathogenesis or causing non-target effects is assessed as low.

3.112 The development of aquacultural applications of GMOs may pose a particular risk of dissemination into the environment if the GMO cannot be contained within the fish. The use of GMOs in this aquaculture is limited with only a few applications of recombinant bacteria based vaccines reported [147]. Oral administration of the GMO in feed is limited, with the GMO delivered either by intra-peritoneal injection (for salmonid fish) or by immersion in a contained tank [147]. The risk of release of the GMO into the wider environment during administration is therefore assessed as low.

3.113 Delivery of the GMO by routes other than parenteral injection may result in <100% of the GMO being delivered to the recipient. In veterinary applications in particular this is likely to result in some release of the GMO into the environment. Parenteral administration is more expensive than oral delivery and is therefore often not used for treatments involving many individuals and where cost is important. In contrast to large scale human vaccination programmes, in veterinary vaccination there is a cost associated with both the medicine and the recipient of the vaccine. In the case of poultry for example the chicken or duck is of relatively little value (although breeding stock are of relatively greater value). This has implications to the cost (per dose) of the vaccine, and usually results in an oral delivery method being used. A limitation with the vaccination of poultry is that poultry bred for meat typically takes ~40 days to grow to slaughter weight. Vaccination of chicks or adult birds may therefore not provide protection until well into the bird’s lifespan and may therefore not be suitable treatment strategies. In such cases in ovo vaccination is required (if effective for that disease). In veterinary applications parenteral vaccination is usually only appropriate when there are a small number of animals to be vaccinated and guaranteed delivery is required, for example the vaccination of cheetahs and rhinos against anthrax in Etosha National Park (Namibia) [199].

3.114 Biological containment of the GMO to limit or prevent its survival and replication can be achieved through a number of processes, both passive and active 108 . The potential risks posed by the GMO should be lower if one or more containment strategies exist and are effective. The effectiveness of these biological containment measures will of course also depend on the stability of the genetic modification

108 Passive biological containment refers to metabolic or other gene defects which are supplemented with either the intact gene or the essential metabolite (for example thyA auxotrophy). Active containment provides control through the conditional production of a toxic compound whose expression is tightly controlled by an environmental factor or suppressed by internal elements (for example the hok/sok system)[246]. 3-114

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conferring the containment, and in the case of auxotrophic modification, the availability of the missing essential compound:

♦ Replication competence; ♦ Auxotrophic modification; ♦ Targeted expression - use of tumour specific promoters or condition-specific expression to ensure that the transgenes are only expressed at a specific location within the recipient. Should subsequent release of the GMO occur from the patient (through shedding for example) the potential effects to the wider environment will be negligible unless the GMO can find another environment in which expression can occur. The GMO will not survive if such an environment is not available. ♦ Intercellular spread; ♦ Self-destruction – a system developed for Listeria monocytogenes where the bacteria self-destruct following entry into the recipient cell’s cytosol.

Shedding – the release of microorganisms into the environment (GM bacteria) 3.115 Shedding of GMOs used as vaccine vectors from pigs and cattle has been reported. The V. cholerae vaccine CVD 103-HgR for example has been reported to be shed at levels of <200 live vibrios per gram faeces by 20-30% of vaccinees for a maximum of seven days. This figure is significantly lower than the 10 7 vibrios per gram faeces for wildtype V. cholerae [226]. The vaccine strain is also reported to die off within 20 days under various environmental conditions [226]. The difference between the quantities of GMO shed versus the wildtype should be viewed as a positive benefit of the GM vaccine in the risk assessment process.

3.116 Salmonella enterica Typhi strain CVD 908-htrA ( aroC, aroD and htrA deleted) has been used in multiple human and animal studies and has shown good results as an orally-administered attenuated vaccine against salmonella and as a recombinant vector for the protection against other pathogens [194][226]. Some shedding of the bacterium has been reported:

♦ When administered at 5x10 7 cfu the salmonella was shed by 15% of recipients for more than 2 days, but by only 3% for more than 3 days. Maximum quantity shed was 2.8x10 6 cfu/g stool. ♦ At a higher dose of 4.8x10 8 cfu 33% of recipients shed the bacteria for more than 2 days, and 13% for more than 3 days. Maximum quantity shed was 8x10 6 cfu/g stool. ♦ Because both studies reported high levels of excretion, microcosm studies were conducted to assess the ability of the GMO to survive in soil and stream water. The GM bacteria died off rapidly in stream water and soil, with no bacteria detected (detection limit <10 cfu/ml) after 7 and 5 days respectively.

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3.117 Whilst shedding of the GM S. enterica Typhi strain CVD 908-htrA will result in a release of the GM bacteria into the environment, the risk with respect to effects on the wider environment and public health is relatively low. The GMO is an attenuated strain of salmonella with a history of safe use as a vaccine vector in human and animal studies. The auxotrophic deletions ( aroC and aroD ) should reduce the likelihood of the GM bacteria surviving in the environment (in the absence of an external supply of the required aromatic compounds. The deletion of the stress response gene htrA should also mean that the GMO is outcompeted in the environment by unmodified strains, thereby also reducing its ability to survive.

3.118 The GM Shigella flexneri 2a SC602 vaccine vector is reported to be shed into the environment for a relatively long period of time post administration (average of 11.6 days in a 34 patient study) [226]. However, the attenuating modifications of the GM bacteria (rendering it unable to spread intercelullarly) should prevent the occurrence of any significant infection and the potential risk posed.

3.119 The clinical trial with the GM thyA - L. lactis in ten patients with moderate to severe Crohn’s Disease [122], for example, reported the presence of the GM bacterium in patient’s stools up to two days after termination of a seven day trial with daily administration of 2x10 10 live bacteria. The GMO was not detected in stools after this time. The thyA deletion should prevent the survival of the GM bacteria in the environment.

3.120 The reported shedding of GM Bacillus subtilis spores (used as vaccine vectors) into faeces [220] provides an example where the released GMO is likely to persist in the environment. In the application reported by Duc et al. (2003), the risk to the environment was likely to be low as the genetic modification was for a fragment of the tetanus toxin. This is unlikely to confer a selective advantage to the GM B. subtilis and therefore not increase the survival of any germinated spores in the environment. The survival of ungerminated spores will be unaffected and they may be expected to persist. The combination of enhanced survival (through spore generation) and a release pathway means that the risk of the release of these GM bacteria into the environment is high. Although the genetic modification may not provide the GM B. subtilis with a selective advantage, it is not known whether the additional genetic load will reduce its ability to survive. The reported shedding into faeces indicates that other GM bacilli spores would also be released if used in a similar application.

3.121 The number of studies reporting the presence of live GMOs in faeces following oral administration means that excretion of any GM bacteria used in such an application should be expected. From a risk assessment perspective, particular consideration therefore should be made to the biological containment mechanisms applied and the potential for gene transfer and long term survival of the GMO in the environment.

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3.122 In contrast the non-GM bacterial vector Ty21a is reported to exhibit minimal or no shedding in human volunteers [226]. Such characteristics may be expected to be maintained where Ty21a is genetically modified as a vector.

Dissemination other than shedding (GM bacteria) 3.123 The GM lactic acid deficient Streptococcus mutans designed to prevent the occurrence of dental caries [124] is one human application of GM bacteria that could be disseminated to other people through contact transmission (kissing for example). The GMO is intended to be applied to the teeth to prevent the growth of lactic acid producing bacteria, and to persist in dental microflora for a significant period of time (a year at least). Although the USFDA granted approval (in 2005) for the GMO to undergo Phase I clinical trials, concerns have been raised as to the potential for person to person dissemination of the GMO. The initial trials that have been approved have only involved people with dentures (i.e. without their own teeth) so that the GMO can be more easily removed from the recipient if required. The GMO has an auxotrophic attenuation for alanine designed to prevent its survival should wider dissemination occur. Recipients of the GMO would be required to provide the bacteria with a regular supply of alanine, possibly as an oral rinse. As described earlier the potential risks posed by this GM bacteria, in terms of dissemination and survival in the environment, will be lower if the D-alanine auxotrophy can be shown to be effective in controlling growth of the GMO. Studies in rats showed that withdrawal of D-alanine supplements reduced, but did not eliminate the population of the GM bacteria present [309]. On the basis of the rat study, D-alanine auxotrophy can be considered effective only in limiting the growth and colonisation of the GMO. It does not provide complete control in that the GMOs were not completely removed from the oral cavity four months post administration. A further reduction in the population of the GMO was achieved through the daily administration of chlorhexidene (mouth wash) 109 . The combination of the genetic modifications ( dal deletion and comE inactivation) was described by Hillman et al. (2007)[309] as sufficient to make the GMO suitable for safe use in human clinical trials.

3.124 The latest situation with this application (August 2007) is that the USFDA will not approve Phase IB clinical trials until further clinical information is provided [260].

Indirect effects on survival and dissemination (GM bacteria) 3.125 The genetic modification itself is reported to have an effect on the ability of the GMO to survive and disseminate in the environment [194]. GMOs are typically less able to survive in the environment than wildtype strains. This effect is independent of the trait(s) conferred by the modification, but is likely to be enhanced if the microorganism is modified so that any characteristics that provide a selective advantage in the environment are removed. Such characteristics include metabolic properties and virulence traits.

109 Chlorhexidene reduced the entire dental microflora, and was not specific to the GMO. 3-117

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3.126 An increase in genetic load (through the addition of chromosomal genes, plasmids or tranposons) may cause a reduction in the fitness of the GMO and therefore its ability to survive in the environment. Studies with E. coli for example have shown that plasmid-bearing bacteria grow more slowly than plasmid-less strains, and that growth rate is reduced as plasmid copy number increases [194]. A similar result is reported following the addition of chromosomal genes under a strong promoter [232], with growth rate decreasing with increased expression of the gene(s) [194]. A slower growth rate means that the GMO is likely to be outcompeted in a particular environment by faster growing wildtype strains, thereby reducing the likelihood of it surviving.

Replication competence (GM bacteria) 3.127 GM bacteria used in medicines are replication competent, although their actual ability to replicate in a particular environment (either within the recipient or the wider environment) may be prevented by incompatible temperature, oxygen or nutrient requirements. The Bifidobacterium longum anaerobic bacteria described as a vector to target solid tumours for example [53] are capable of replication, but are only able to do so in anaerobic environments.

Auxotrophic attenuation (GM bacteria) 3.128 Auxotrophic attenuation provides the ability to contain the GM bacterial vector biologically and prevent its proliferation, either within the recipient of the vaccine or in the wider environment. The effectiveness of the biological containment depends on the type of auxotrophic mutation (conferring a bacteriostatic or bacteriocidal effect) and the availability of the missing compound. For example the auxotrophic attenuation based on diaminopimelate [157] should provide a high level of containment within the mammalian recipient as diaminopimelate is not present in mammalian cells. Salmonella enterica CVD 908-htrA modified for auxotrophic deletions of aroC and aroD are reported to have limited survival in the environment with detection levels in water of <10 cfu/ml after 7 days, and no detection in soil after 5 days [226]. Any release of this GM bacterial vector into the environment should therefore not pose a risk with respect to the long term survival of the microorganism [226], assuming that the GMO cannot be rescuscitated if the required aromatic compounds become available.

3.129 Although quantitative information on the survival of GMOs expressing auxotrophic attenuations in the environment is limited, the information from laboratory studies with a range of auxotrophic mutants and field work with Salmonella enterica CVD 908-htrA indicates that such modifications do provide an effective biological containment strategy for GM bacteria. The removal of characteristics that provide the wildtype strain with an ability to survive in the environment will result in the GMO being less able to compete and survive.

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3.130 The alr and thyA auxotrophic modifications are bacteriocidal [253][308] with bacteria expressing these modifications being unable to persist longterm in the environment in the absence of the auxotrophic compound [248]. The effectiveness of the alr modification is supported by D-alanine not being present in large amounts, if at all, in the environment and GI tract. Studies with an alr deleted Lactobacillus plantarum reported that the bacterium could not be grown in the absence of D-alanine (2.25mM concentration in complex growth (MRS) medium). After withdrawal of the D-alanine growth of the L. plantarum stopped with cfu numbers dropping a hundredfold after 25 hours. No cell lysis was observed, although the cells appeared severely damaged under scanning electron micrographs [311]. The results with the L. plantarum suggest that the alr deletion had a bacteriostatic, rather than a bacteriocidal effect on the bacteria. However, in similar studies with E. coli and B. subtilis deprivation of D- alanine results in rapid and extensive cell lysis, i.e. in a bactericidal effect [311].

3.131 The modification of the aromatic acid synthesis pathway (alteration or deletion of genes aroA, aroC, and aroD ) rendering the microorganism unable to produce aromatic amino acids was one of the first attenuating modifications of a central metabolic pathway investigated. The modification means the GMO is unable to replicate (and therefore survive longterm) unless it is provided with the essential aromatic amino acids. The survival of these auxotrophic mutants in the environment (in the absence of the required compounds) is reported to be significantly lower than wildtype strains [196]. Auxotrophic attenuations may also cause a reduction in virulence when applied to pathogenic organisms, with purA deleted (adenine auxotrophy) Salmonella dublin and Shigella flexneri reported to be less virulent than wildtype strains [257][256].

3.132 The genes, such as aroA , involved in central metabolic pathways are located stably on the chromosome and are generally not carried on or in the vicinity of mobilisable genetic elements (plasmids and transposons). This makes any large deletion (i.e. the removal of entire genes or large segments of the genes) genetically stable. Reacquisition of the missing gene(s) is also limited due to an absence of any horizontal transfer of these so-called housekeeping genes [196]. As described, increased stability and decreased ability to reacquire the deleted genes improves the effectiveness of the attenuation and reduces potential risks posed. The stability of these deletions has been demonstrated in studies with GMOs exhibiting auxotrophic modifications grown alongside populations of wildtype strains (without the deletions). The GMOs did not reacquire the deleted genes [196]. The thyA deletion reported by Steidler et al. (2003)[248] to confer thymine auxotrophy on Lactococcus lactis was reinforced by inserting the transgene cassette encoding IL-10 in its place. Therefore, the GM bacterium can only reacquire the deleted thyA gene at the expense of the transgene, leaving a bacterium that was no different to the wildtype. Such applications should pose negligible risk to the environment with respect to the survival of the GMO. Whilst the GM bacterium retains the IL-10 gene it cannot regain the thyA gene and consequently undergoes thymineless death. If it regains

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the thyA gene and survives, it loses the IL-10 gene and is no longer genetically modified.

3.133 The deletion of aroA, aroC and aroD genes has been reported to provide effective biological containment in a range of bacterial species, including Aeromonas hydrophila, Listeria monocytogenes, Salmonella typhimurium, Shigella glexneri, Pseudomonas aeruginosa, Shigella dysenteriae, Bordetella pertussis, Neisseria gonorrhoeae, Bacillus anthracis, avian pathogenic Escherichia coli and Salmonella abortusovis (studies cited by [196]).

3.134 Other auxotrophic attenuations include:

♦ the guaBA attenuation in Shigella flexneri 2a which confers dependence on purine enzymes, and is the most extensively evaluated shigella vaccine strain [194]; ♦ thymidine ( thyA ); ♦ glutamine synthetase ( glnA ). Described as not having great potential as an effective auxotrophic modification [308]; and ♦ the purA attenuation which renders the GMO auxotrophic for adenine [257].

Self-destruction as a containment system (GM bacteria) 3.135 The self-destruction system developed for L. monocytogenes works by the modification of the bacterium with a L. monocytogenes-specific endolysin (phage lysine 118) under the control of a cytosolically activated listerial promoter. The modification results in cell wall degradation (and therefore death of the L. monocytogenes vector) as soon as the endolysin is released, which is likely to occur after the death of some listerial cells [245]. The endolysin causes cell wall degradation of the bacterial vector through cleavage between L-alanine and D- glutamate of the cell wall peptidoglycan. However, for it to be successful, degradation of some cells of the GM bacteria must be achieved through other processes in order for the endolysin to be released. This initial degradation is for example more likely in spleen than liver cells [245], indicating that the system as a biological containment strategy may not be effective in all applications of L. monocytogenes .

3.136 The modification is also reported to result in the attenuation of the pathogenicity of 8 the bacterium, with an LD 50 dose for a mouse in excess of 1x10 cfu, compared to 1x10 4 cfu for the wildtype L. monocytogenes [245]. Because the endolysin targets peptiodglycan that is not present in mammalian cells it should not affect the recipient.

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3.3 ASSESSMENT OF THE POTENTIAL RISKS ASSOCIATED WITH GM PARASITES IN MEDICINES

3.137 The development of GM parasites for medical applications has been reported for GM Plasmodium berghei (the causative agent of malaria)[209] and GM Leishmania major, L. mexicana, and L. donovani (causative agent of leishmaniasis) [200], for use as live attenuated vaccines for malaria and leishmaniasis respectively. The GM leishmania are modified for reduced virulence, and the GM plasmodium to render the GM parasite unable to establish a blood-stage infection, thereby containing the GMO biologically within the recipient. Should a vaccinated person be bitten by the arthropod vector there would be no GM plasmodium available in their bloodstream to be taken up by the vector and disseminated.

3.138 Both management strategies employed for these GMOs should reduce the risk posed relative to the wildtype. The biological containment approach used for the GM plasmodium should, if effective, result in negligible risk to the environment and the general public as the GMO would be contained within the patient, and not transmitted by the mosquito vector. The negligible level of risk posed would therefore be unaffected by the presence of a suitable mosquito vector in the environment. The presence or absence of a suitable arthropod vector is usually an important question in the risk assessment process for GMOs that are spread by arthropod vectors.

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CONCLUSION

3.139 The risks posed by the use of live GMOs in human and veterinary medicines to the environment and human health are largely restricted to particular GMOs used in specific applications. No general areas that are likely to give rise to adverse effects to the wider environment and general public have been identified. Of the adverse effects that have been reported, the most significant of these, such as adverse immune responses to the vector or insertional mutagenesis, are to the immediate recipient of the of the GMO and not to the wider general public or environment. Adverse effects such as vector-induced immune reponse are not a consequence of the genetic modification. As described there are a number of strategies available to minimise their significance and therefore the risk posed. Careful design of the GMO and consideration of how it is applied should reduce the level of risk.

3.140 Whilst no general areas likely to give rise to adverse effects have been reported, a number of studies have demonstrated that some release of the GMO into the environment does occur. Therefore where release is likely, for example treatments involving the oral administration of enteric GM bacteria then some form of biological control strategy to limit the survival (and therefore the spread) of the GMOs in the environment should be in place. With GM bacteria, auxotrophic modification has been identified as a generic strategy to reduce risk to the environment and human health by linking the survival of the GM bacteria to the provision of a particular compound that is either absent or limiting both in the environment and preferably also in the treated patient. With respect to GM viruses the most significant biological containment strategy is replication competence, as viruses that are modified to lack this ability will pose negligible risk to the environment and human health as they will not be spread beyond the immediate recipient of the GM virus (assuming the GM virus is unable to regain its replication competence). With both strategies, the effectiveness of the approach is dependent on the inability of the GMO to overcome the modification. Consideration should also be given to the survival of the GMO as a whole in the environment, in addition to the effectiveness conferred by the modifications. Many microorganisms have specific requirements that restrict their survival to particular environments, and to particular host species. These requirements, which may vary down to the subspecies level 110 , should be considered in the risk assessment process.

3.141 The review of the use of live GMOs in human and veterinary medicines has identified a diverse array of applications. Whilst many of these are still in development, some have entered clinical trials or field trials programmes for use in human or veterinary medicines respectively. Whilst particular hazards have been identified, careful design of the GMO and how it is administered and used will reduce the level of risk posed to the wider environment and public health. Given the diverse array of

110 Lactococcus lactis subspecies lactis has been isolated from milk as well as plants; whereas Lactococcus lactis subspecies cremoris has never been isolated from plant material [312]. 3-122

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applications and the unique characteristics of each, the current regulatory approach to risk assessment of a case by case evaluation of each application is viewed as the most appropriate system to address the potential risks.

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4. CHAPTER 4 – RECOMMENDATIONS FOR FUTURE RESEARCH

4.1 The purpose of Chapter 4 is to make recommendation for future research into the use or the potential risks posed the use of live GMOs in medicines, to inform future risk assessment and risk management decisions.

4.2 The one area identified as requiring further investigation is the effectiveness of auxotrophic modifications as a strategy for the biological containment of GM bacteria. These modifications are used in a number of the GM bacteria described in Chapter 2 to limit or prevent the survival of the GM bacteria in the environment. As described in Chapter 3, the effectiveness of auxotrophic modification as a biological containment strategy is determined by the type of modification introduced and the availability of the missing compound on which the GM bacteria depends.

♦ Information from laboratory studies with a range of auxotrophic mutants shows that growth of the GM bacteria does not occur in the absence of the missing compound or if the compound is below a threshold level. For the auxotrophic modifications of thymine ( thyA -) and diaminopimelate ( asd -) deficiency the absence of the compound causes a lethal effect on the GM bacteria. However, for other auxotrophic modifications the removal of the compound only causes a biostatic effect. Therefore the GM bacteria may be able to persist until a further source of the compound becomes available. ♦ Whilst the laboratory information shows that auxotrophic modification is effective in controlling survival, quantitative information on survival of such GMOs in the environment is much more limited [308]. With respect to thyA auxotrophy, there is also considerable variability amoung bacterial species (modified to lack thyA ) in the kinetics of cell death and the lag period between withdrawal of the thymine and loss of viability [253]. This variability is expected to apply to other auxotrophic modifications. The effectiveness and variability of response to auxotrophic modification may be significant from a risk assessment perspective given the recent number of studies investigating the genetic modification of probiotic bacteria for use as treatments against enteric pathogens and diseases. The nature of the use of these GM bacteria means that some shedding of the GMO is expected in faeces. Auxotrophic modification, if effective, would be an appropriate strategy to ensure the biological containment of these GM bacteria.

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♦ However, further information is required for each of the auxotrophic modifications used on their effectiveness in limiting the survival of the GMO, both in the GI tract and in the environment post-excretion. ♦ For biostatic modifications such as aromatic acid synthesis deficiency, information is also required on how long the GM bacteria can persist in the absence of the compound and still be ‘rescusitated’ should a fresh supply of the compound become available. A study with GM Vibrio cholerae with a deleted glnA gene in mice, found that glnA deficient bacteria could be isolated from the GI tract of the mice up to eight days post oral administration of GM V. cholerae. Therefore, whilst the glnA modification might be effective in vitro, there is sufficient extraneous glutamine in the intestinal lumen of the mice for the GM bacteria to survive the auxotrophic modification. A similar finding has been reported for the GM dal deficient Streptococcus mutans designed to protect against dental caries [124]. However, the human clinical trial with thyA deficient Lactococcus lactis reported no detectable survival of the GM bacteria two days after termination of a seven day trial (involving daily administration of 2x10 10 live bacteria) [122]. Similar studies would be useful to assess the effectiveness of the other auxotrophic modifications described.

4.3 Of the other applications and potential risks described in Chapters 2 and 3 there are no other general areas identified where further investigation would be particularly useful. As is the approach under the existing regulatory framework, applications to develop and use live GMOs in medicines are assessed on a case by case basis. Although the specifics of any new application are likely to differ slightly to the applications described in this report, there should be considerable similarities in terms of the general purpose of the modification, the GMO used and the risk management strategies employed between the new application and applications already reported. The information provided in this report, and other documents such as SACGM Guidance [298] should enable these similarities to be identified and an understanding made as to the significance of the risks posed.

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5. APPENDIX 1 – GENE THERAPY APPLICATIONS IN CLINICAL TRIALS

Table A.1 – GDEPT gene therapy applications in clinical trials (table adapted from [292][291][290])

Reference Released metabolite or Stage of Enzyme (origin) Prodrug (for the cytotoxin development clinical trials) Cytochrome P450 Oxazaphosphorines: cyclophosphamide Alkylating agents (4-hydroxy (Human: CYP2B1, 2B6, 2C8, 2C9, (CPA) and ifosfamide (IFO) forms) Phase I/II 2C18, 3A), Ipomeanol, 2-aminoantracene, Toxic metabolites (CYP2B1/IFO, [287][288] (Rat: CYP2B10), dacarbazine, procarbazine CYP2B6/CPA) (Rabbit: CYP4B1 ±CYPOR), N-acetyl benzoquinone imine Acetaminophen (Dog: CYP2B11) (NABQI) Cytosine deaminase (CD) (±UPRT) ( E. Phase I/II (CD/5- 5-fluorocytosine (5-FC) 5-fluorouracil (5-FU) [128][289] coli , S. cerevisiae ) FC) Alkylating agents (N-acetoxy CB1954 and analogues derivatives) Phase I Nitroreductase (NTR) ( E. coli ) [38] Alkylating agents, pyrazolidines, (NTR/CB1954) Self-immolative prodrugs enediynes Modified purine and pyrimidine Monophosphorylated nucleotide Thymidine kinase (TK) ( Herpes nucleosides: GCV, E-GCV, ACV, analogues Phase III (HSV- simplex virus, Varicella zoster virus, valacyclovir, araM, araT, BVDU [215][159] TK/GCV) Equine herpes virus) FIAU, purine and pyrimidine Monophosphorylated nucleotide nucleosides, araM analogues

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Table A.2 – Gene therapy applications in clinical trials in China, Japan and Korea (table adapted from [114])

Date of commencement Indication Gene Vector Current status of clinical trial Cancer p53 Adenovirus Commercialized 1998 Cancer H101 Adenovirus Commercialized 2000 Selective oncolytic Cancer Adenovirus Phase II ongoing 2003 adenovirus Cancer TK Adenovirus Phase I ongoing 2004 Cancer IL-2 Adenovirus Phase I/II ongoing 2003 Ischemic disease Endostatin Adenovirus Phase I ongoing 2004 Ischemic disease HGF Adenovirus Phase I ongoing 2005 Cardiovascular disease VEGF Adenovirus Phase I completed 2001 Adeno-vaccine + DNA AIDS Adenovirus Phase I ongoing 2004 vaccine Cytokine-activated Leukemia Retrovirus ( ex vivo ) Phase I completed 1997 lymphocyte Cancer Activated dendritic cell Retrovirus ( ex vivo ) Phase I completed 2001 IL-2-modified allogenic Late stage gastric cancer Retrovirus ( ex vivo ) Phase I 2001 gastric cancer Hemophilia cell line vaccine AAV-2 Phase I completed 1994 Hemophilia Factor IX AAV-2 Phase I ongoing 2003 Hemophilia Factor IX Retrovirus ( ex vivo ) Phase I completed 1991 pLTKcSN/VPC (HSV- Glioma Retrovirus ( ex vivo ) Phase I completed 1996 tk/GCV) Lung cancer (NSCC) p53 Adenovirus Phase I/II completed 1998 Esophageal cancer p53 Adenovirus Phase I/II completed 2000 Lung cancer (NSCC) p53 Adenovirus Phase I/II completed 2000 Lung cancer (NSCC) p53 Adenovirus Phase I/II completed 2000 Prostate cancer HSV-tk Adenovirus Phase I/II completed 2000 Lung cancer (NSCC) p53 Adenovirus Phase I/II completed 2000

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Lung cancer (NSCC) p53 Adenovirus Phase I/II completed 2000 Prostate cancer HSV-tk Adenovirus Phase I/II ongoing 2003 ADA deficiency ADA Retrovirus ( ex vivo ) Phase I/II completed 1995 ADA deficiency ADA Retrovirus ( ex vivo ) Phase I/II on going 2002 Renal carcinoma GM-CSF Retrovirus ( ex vivo ) Phase I completed 1998 Mammary cancer MDR1 Retrovirus ( ex vivo ) Phase I/II ongoing 2000 Leukemia HSV-tk Retrovirus ( ex vivo ) Phase I/II ongoing 2002 ASO/Beurger FGF-2 Sendai virus Phase I/II ongoing 2006 Aromatic l-amino acid Parkinson’s disease AAV-2 Phase I/II ongoing 2007 decarboxylase (AADC) Skin fibroblasts transduced Melanoma, breast cancer, with retroviral vectors Retrovirus ( ex vivo ) Phase I completed 1998 head-and-neck cancer expressing IL-12 Oncolytic vaccinia virus Liver cancer Vaccinia virus Phase I 2006 expressing GM-CSF Chronic granulomatous gp91 Retrovirus ( ex vivo ) Phase I/II 2007 disease Osteoarthritis TGF-β Retrovirus ( ex vivo ) Phase I 2006 Prostate cancer TK, CD Adenovirus Phase II 2005

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Review of GMOs in medicines – final report Table A.3 – Gene therapy applications involving live GMOs in Phase III clinical trials reported by the Journal of Gene Medicine [17](Information complete as of July 2007); US National Institutes for Health, National Cancer Institute [286].

Trial Current Country Description Vector Reference status AU-001 [17] Australia Multidrug treatment followed by randomisation to HIV vaccination Poxvirus Open

BE-002 [17] Belgium Prospective, open-label, parallel-group, randomized, multicenter trial comparing the efficacy of surgery, radiation, and injection of murine cells producing herpes simplex thymidine kinase vector followed by intravenous ganciclovir against the efficacy of Retrovirus Closed surgery and radiation in the treatment of newly diagnosed, previously untreated gliogblastoma

BE-010 [17] Belgium A phase III open-label, comparative, multicentre trials to test the concept of durable virologic suppresion in subjects with primary HIV-1 infection after intensive induction of quadruple HAART followed by double blind randomization to HIV vaccination with Poxvirus Open ALVAC-HIV (vCP-1452) and remune or placebo while maintaining optimal therapeutic viral suppression

BE-013 [17] Belgium A phase III, multi centre, open-label, randomised study to compare the overall survival and safety of bi-weekly intratumoural administration of INGN 201 versus weekly Adenovirus Cancelled methotrexate in 240 patients with refractory squamous cell carcinoma of the head and neck (SCCHN)

BE-014 [17] Belgium A phase III, multi centre, open-label, randomised study to compare the effectiveness and safety of intratumoural administration of INGN 201 in combination with chemotherapy Adenovirus Cancelled versus chemotherapy alone in 288 patients with recurrent squamous cell carcinoma of the head and neck (SCCHN)

CH-023 [17] Switzerland Gene therapy in patients with HIV infection Poxvirus Closed

DE-037 [17] Germany A Phase III, Multi-Center, open-label, randomized study to compare the Overall Survival and Safety of Bi-weekly intratumoral administration of INGN-201 versus weekly adenovirus closed Methotrexate in 240 patients with chemotherapy refractory Squamous Cell Carcinoma of the Head and Neck (SCCHN) - INGN-201 Trial 301

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DE-040 [17] Germany Gene therapy in patients with HIV infection Not stated Open

DE-072 [17] Germany A Phase III, multi-center, open-label, randomized study to compare the Effectiveness and Safety of intratumoral administration of INGN-201 in combination with chemotherapy adenovirus Open versus chemotherapy alone in 288 patients with recurrent Squamous Cell Carcinoma of the Head and Neck (SCCHN) - INGN-201 Trial 302

US-635 [17] USA A Phase III Randomized, Controlled Study to Evaluate the Safety and Efficcacy of PANVAC-VF in Combination with GM-CSF Versus Best Supportive Care of Palliative Poxvirus and Open Chemotherapy in Patients with Metastatic (Stage IV) Adenocarcinoma of the Pancreas vaccinia virus Who Have Failed a Gemcitabine-Containing Chemotherapy Regimen

US-653 [17] USA A Phase III Randomized, Open-Label Study of CG1940 and CG8711 Versus Docetaxel and Estramustine in Patients with Metastatic Hormone-Refractory Prostate Cancer Who Adeno- are Chemotherapy-naive. Modification of the cells is conducted ex vivo. Study also associated Open conducted in the Netherlands under various references including B/NL/06/003 [295]. The virus AAV vector is considered not to be genetically modified by the UK, Belgium, Swedish and Polish competent authorities [295].

US-708 [17] USA A Phase 3 Randomized, Open-Label Study of Docetaxel in Combination with CG1940 Adeno- and CG8711 Versus Docetaxel and Prednisone in Taxane-Naive Patients with Metastatic associated Open Hormone-Refractory Prostate Cancer with Pain. Modification of the cells is conducted ex virus vivo. Study also conducted in the Netherlands under reference B/NL/07/010 [295].

US-751 [17] USA A Phase III Study of a PSA Vaccine in Androgen Ablation Refractory Prostate Cancer Poxvirus and Open with Absence of Metastatic Disease and GM-CSF vaccinia virus

US-821 [17] USA A Randomized, Double-Blind, Placebo-Controlled, Parallel Group, Multicenter Study to Evaluate the Efficacy and Safety of Ad5FGF-4 in Female Patients with Stable Angina Adenovirus Open Pectoris Who Are Not Candidates for Revascularization

US-842 [17] USA A Randomized, Controlled Phase III Trial of Replication-Competent Adenovirus-Mediated Under Suicide Gene Therapy in Combination with IMRT Versus IMRT Alone for the Treatment adenovirus review of Newly-Diagnosed Intermediate-Risk Prostate Cancer

US-854 [17] USA A Phase III, Randomized, Controlled, Open Label, Multicentre Study of the Efficacy and Adenovirus Open Safety of Trinam(r) (EG004); an Assessment of Vascular Access Graft Survival in

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XX-003 [17] Multi- A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and country ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with Retrovirus Closed previously untreated glioblastoma multiforme

XX-005 [17] Multi- Multidrug treatment followed by randomisation to HIV vaccination Poxvirus Open country

[286] USA A Randomized, Controlled Trial of Replication-Competent Adenovirus-Mediated Suicide Gene Therapy in Combination With IMRT Versus IMRT Alone for the Treatment of Adenovirus Open Newly-Diagnosed Intermediate-Risk Prostate Cancer

[286] USA Phase III Randomized Study of Ad5CMV-p53 Gene Therapy (INGN 201) Versus Methotrexate in Patients With Refractory Squamous Cell Carcinoma of the Head and Adenovirus Open Neck (T301)

[286] USA Phase III Randomized Study of Cisplatin and Fluorouracil With Versus Without Ad5CMV- p53 Gene Therapy (INGN 201) in Patients With Unresectable Recurrent Squamous Cell Adenovirus Open Carcinoma of the Head and Neck (T302)

[286] USA A Study of TNFerade Biologic With 5-FU and Radiation Therapy for First-Line Treatment Adenovirus Open of Unresectable Locally Advanced Pancreatic Cancer

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6. REFERENCES

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