Gene Therapy for Pediatric Diseases

10/27/2014

DHHS-NIH

ORDR, NINDS, NIAMS, Coalition of Patient NICHD, NHLBI, NIDDK, Advocacy Groups Gene Therapy for Pediatric NIDCR, NIAID, NCI (CPAG) Diseases

The Data Management and Chester B. Whitley, Ph.D., M.D. Coordinating Center

Gene Therapy Center • Collaborative Clinical Research • Public Resources and Education University of Minnesota • Centralized Data Coordination and Technology Development Minneapolis, MN, USA • Training

American Academy of Pediatrics San Diego, October 12, 2014

Disclosure Statement Rare Diseases Clinical Research Network (RDCRN) Actelion –Speakers Bureau Amicus Therapeutics – Meeting Sponsor Relationship NIH BioStrategies – Research Grant

BioMarin – Research Grant NIAID ORDR / NCATS NIAMS Genzyme‐Sanofi – Research Grant, Consultant and Meeting Sponsor Program Coordination Fairview –Speakers Bureau Pfizer –Speakers Bureau NIDCR NHLBI

Protalix –Speakers Bureau NCI NICHD National Institutes of Health NINDS NIDDK • Lysosomal Disease Network • Gene Therapy for Metabolic Disorders RDCRN Consortia, Steering Committee OSMBs/DSMBs ReGenX – Meeting Sponsor DMCC and CPAG Sangamo BioSciences

Shire – Research Grant, Consultant and Meeting Sponsor Angelman, Rett and Prader‐ Genetic Disorders of Mucociliary Urea Cycle Disorders Porphyrias Consortium Synageva – Meeting Sponsor Willi Syndromes Consortium Clearance Consortium Consortium University of Minnesota Autonomic Rare Diseases Inherited Neuropathies Primary Immune Deficiency Vasculitis Clinical Research • Department of Pediatrics Clinical Research Consortium Consortium Treatment Consortium Consortium • Center for Translational Science Institute (CTSI) Brain Vascular Malformation Rare Kidney Stone Data Management and Lysosomal Disease Network Coordinating Center (DMCC) Zebraic d.b.a. WORLD Symposia (Feb 9‐13, 2015, Orlando, FL, USA) Consortium Consortium “We’re Organizing Research on Lysosomal Diseases” Chronic Graft Versus Host Nephrotic Syndrome Study Salivary Gland Carcinomas Coalition of Patient Advocacy Disease Consortium Network Consortium Groups (CPAG) (> 95 PAG)

North American Mitochondrial Sterol and Isoprenoid Dystonia Coalition This presentation may discuss off‐label use of FDA‐approved medications. Disease Consortium Diseases Consortium 5

Assignment: The sequencing of the human Lysosomal Diseases genome has led to hope and expectation of • Aspartylglucosaminuria • Hunter syndrome • Multiple sulfatase gene therapy for genetic disorders. Reports of • Wolman disease • Sanfilippo syndrome type deficiency • Cystinosis A, B, C and D • Batten disease success in treating hemophilia, Leber • Danon disease • Galactosialidosis types I • Tay‐Sachs disease • Fabry disease and II • Pompe disease congenital amaurosis, leukemia, and severe • Farber disease • Krabbe disease • Batten diseases combined immunodeficiency will lead to the • Fucosidosis • Sandhoff disease • Late infantile Northern • Gaucher disease • Vogt‐Spielmeyer disease epilepsy expectation that this will soon be available for • GM1‐Gangliosidosis types • Hurler syndrome • Pycnodysostosis all genetic disorders. I/II/III • Niemann‐Pick disease • Schindler disease • GM2‐Gangliosidosis • I‐cell disease • Sialuria This session will provide the pediatrician • ‐Mannosidosis types I • Pseudo‐Hurler • Salla disease and II polydystrophy with a solid understanding of the success, and • ‐Mannosidosis • Morquio syndrome • Metachromatic • Maroteaux‐Lamy limitations, of gene therapy thus far and which leukodystrophy syndrome types of disorders will be most likely to be • Sialidosis types I and II • Sly syndrome • Mucolipidosis type IV • Mucopolysaccharidosis treated in this novel manner in the near future. • Scheie syndrome type IX

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Genetic Engineering and Gene Therapy c. 1972 “…gene therapy may ameliorate some human genetic diseases in the future. For this reason, we believe that research directed at the development of techniques for gene therapy should continue. For the foreseeable future, however, we oppose any further attempts at gene therapy in human patients because: (i) Our understanding of such basic processes as gene regulation and genetic recombination in human cells is inadequate; (ii) Our understanding of the details of the relation between the molecular defect and the disease state is rudimentary for essentially all genetic diseases; and (iii) We have no information on the short‐range and long‐term side effects of gene therapy Friedmann T, Roblin R. Gene therapy for human genetic disease? Science. 1972 Mar 3;175(4025):949‐55.

Milestones in Gene Therapy Genetic Engineering and Gene Therapy c. 1972

1972 “Gene therapy” coined by Ted Freidmann We therefore propose that a sustained effort be made to formulate a complete set of ethicoscientific criteria to guide the development and clinical application of gene therapy techniques. Such an endeavor could go a long way toward ensuring that gene therapy is used in humans only in those instances where it will prove beneficial, and toward preventing its misuse through premature application.

Friedmann T, Roblin R. Gene therapy for human genetic disease? Science. 1972 Mar 3;175(4025):949‐55.

Gene Therapy Milestones in Gene Therapy

(AAVrh.10-SGSH) 1972 “Gene therapy” coined by Ted Friedmann 1973 Self-imposed moratorium on recombinant DNA research by the molecular biology scientific community 1975 Asilomar Conference on Recombinant DNA leading to establishment of the NIH Recombinant DNA Committee (RAC)

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NIH Recombinant DNA Advisory Committee (RAC) NIH Recombinant DNA Advisory Committee (RAC) Human Gene Therapy Protocols Human Gene Therapy Protocols 9007-002 9409‐087 Blaese, R. Michael; National Institutes of Health, Bethesda, Maryland; Treatment of Severe Combined Immune Deficiency Whitley, Chester B.; University of Minnesota, (SCID) due to Adenosine Deaminase (ADA) Deficiency with Minneapolis, Minnesota; Retroviral‐Mediated Transfer Autologous Lymphocytes Transduced with the Human ADA of the Iduronate‐2‐Sulfatase Gene into Lymphocytes for Gene: An Experimental Study Treatment of Mild Hunter Syndrome *RAC Recommends Approval: 7-31-90/NIH Approval: 9-6-90 (Mucopolysaccharidosis Type II)

Gene Therapy/Phase I/Monogenic Disease/Severe Combined Immune Deficiency due to Adenosine Deaminase Deficiency/In Vitro/Autologous Peripheral Blood Cells/CD34+ Autologous Peripheral Blood Cells/Cord Blood/Placenta Gene Therapy/Phase I/Monogenic Disease/Hunter Cells/Retrovirus/Adenosine Deaminase cDNA/Neomycin Syndrome/In Vitro/Autologous Peripheral Blood Phosphotransferase cDNA/Intravenous Cells/Retrovirus/Iduronate‐2‐Sulfatase cDNA/Intravenous

Gene Therapy for Hunter Syndrome NIH Recombinant DNA Advisory Committee (RAC) (Mucopolysaccharidosis type II) Human Gene Therapy Protocols 9512-139 1. Jonsson JJ, Aronovich EL, Braun SE, Whitley CB: Molecular diagnosis of mucopolysaccharidosis type II (Hunter syndrome) by automated Batshaw, Mark; Institute for Human Gene Therapy, sequencing and computer‐assisted analysis: toward mutation mapping of University of Pennsylvania Medical Center, Philadelphia, the iduronate‐2‐sulfatase gene, Am J Hum Genet 56:597‐607, 1995. Pennsylvania; A Phase I Study of Adenoviral Vector Mediated Gene Transfer to Liver in Adults with Partial 1. Braun, SE, Pan, D Aronovich, EL Jonsson, JJ, McIvor, RS, Whitley, CB: Ornithine Transcarbamylase Deficiency Preclinical studies of lymphocyte gene therapy for mild Hunter syndrome (mucopolysaccharidosis type II), Hum Gene Ther 7(3):283‐290, 1996. Gene Therapy/Phase I/Monogenic Disease/Partial Ornithine 2. Braun SE, Aronovich EL, Anderson RA, Crotty PL, McIvor RS, Whitley CB: Transcarbamylase (OTC) Deficiency/In Vivo/Autologous Correction of mucopolysaccharidosis type II (Hunter syndrome) by Peripheral Blood Cells/Adenovirus/Type 5 (E2a retroviral‐mediated gene transfer and expression of human iduronate‐2‐ Temperature-Sensitive Mutant)/Ornithine Transcarbamylase sulfatase in lymphoblastoid cell lines. Proc Natl Acad Sci USA 90:11830‐ cDNA/Intravenous 11834, 1993.

NIH Recombinant DNA Advisory Committee (RAC) NIH Recombinant DNA Advisory Committee (RAC) Human Gene Therapy Protocols Human Gene Therapy Protocols 0312‐619 9409‐087 Crystal, Ronald, Weill Medical College of Cornell University, Whitley, Chester B.; University of Minnesota, New York, New York; Administration of a Replication Minneapolis, Minnesota; Retroviral‐Mediated Transfer Deficient Adeno‐Associated Virus Gene Transfer Vector of the Iduronate‐2‐Sulfatase Gene into Lymphocytes for Expressing the Human CLN2 cDNA to the Brain of Children Treatment of Mild Hunter Syndrome with Late Infantile Neuronal Ceroid Lipofuscinosis (Mucopolysaccharidosis Type II) Gene Therapy/Monogenic Disease/ Late Infantile Neuronal Gene Therapy/Phase I/Monogenic Disease/Hunter Ceroid Lipofuscinosis/In Vivo/Brain/Adeno‐Associated Syndrome/In Vitro/Autologous Peripheral Blood Virus 2/CLN2 (Tripeptidyl Peptidase) Cells/Retrovirus/Iduronate‐2‐Sulfatase cDNA/Intraparenchymal Injection cDNA/Intravenous

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NIH Recombinant DNA Advisory Committee (RAC) Human Gene Therapy Protocols

0904‐977 Crystal, Ronald G.; Weill Cornell Medical College; New York, New York; Direct CNS Administration of a Replication Deficient Adeno‐ Associated Virus Gene Transfer Vector Serotype rh.10 Expressing the Human CLN2 cDNA to Children with Late Infantile Neuronal Ceroid Lipofuscinosis

(Open; RAC reviewed with recommendations) Gene Therapy/Phase I/Monogenic Disease/Late Infantile Neuronal Ceroid Lipofuscinosis/In Vivo/Brain/Adeno‐Associated Virus 10/CLN2 cDNA/Intraparenchymal Injection

Emerging Trends in Gene Therapy Leber Congenital Amaurosis • Glybera approval by European Commission The first retinal gene therapy in human blindness from • Leber congenital amaurosis RPE65 mutations has focused on safety and efficacy, as • Lentiviral-mediated hematopoeitic cell defined by improved vision. The disease component not transplantation (ADA-deficient SCID, Don studied, however, has been the fate of photoreceptors in Kohn) this progressive retinal degeneration. We show that gene • Intravenous lentiviral gene therapy for Hurler therapy improves vision for at least 3 y, but photoreceptor syndrome degeneration progresses unabated in humans … The study • Sanfilippo syndrome type A, AAV injection into shows the need for combinatorial therapy to improve the brain, Lysogene vision in the short term but also slow retinal degeneration • Giant axonal neuropathy, AAV intrathecal in the long term. injection

NIH Recombinant DNA Advisory Committee (RAC) Human Gene Therapy Protocols 0910‐1006 Kohn, Donald; University of California, Los Angeles; Los Angeles, California; and Candotti, Fabio; National Institutes of Health; Bethesda, Maryland; Treatment of Subjects with Adenosine Deaminase (ADA) Deficient Severe Combined Immunodeficiency (SCID) with Autologous Bone Marrow CD34+ Stem/Progenitor Cells after Addition of a Normal Human ADA cDNA by the EFS‐ADA Lentiviral Vector

Gene Therapy/Gene Therapy/Phase I/II/Monogenic Disease/Severe Combined Immune Deficiency due to Adenosine Deaminase Deficiency (ADA)/In Vitro/Autologous CD34+ Cells from Bone Marrow/Lentivirus/Adenosine Deaminase cDNA/Intravenous Infusion

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Hematopoietic Stem Cell Differentiation The EFS-ADA Lentiviral Vector

Normal gene Ψ EFS Hu ADA WPRE

Clinical Trial Experimental Time-Line

Active Long - term Phase: Recruit Screen Intervention Follow - up Follow - up Years 0 -2 Years 3 - 15

End - Point Monitor BM Harvest Evaluations Safety Process Cells (separate study) Consent D/C Event: IV Busulfan CD34+ Completed ERT Eligibility Infuse Final Confirmed Cell Product

Gene Therapy Using Hematopoietic Stem Cells Phase I/II Trial: EFS-ADA Lentiviral Vector Busulfan Months Age at CD34+ ID Gender Race VCN AUC on entry x106/kg (mmol/L*min) Protocol

501U 7 mos F H 11.1 2.9 5,673 11 mos

502U 3.6 y F AA 5.6 2.4 4,518 9 mos

503U 3.0 y M H n.d. (<1) 1.7 n.d. N/A

504U 4 mos M C 15.0 1.6 5.199 4 mos

505U 9 mos M AA 9.0 6.3 3,673 3 mos

506N 2 y M H 3.0 2.8 4,055 2 mos

507U 10 mos F C 15.0 Pend 3,247 0

508U 6.5 mos F C Pend Pend Pend pend

First and Second Generation Retroviral and Lentiviral Vectors Diagnosed by CA NBS Final Cell Product: Busulfan dose: 4mg/kg Adagen started @ 1 m/o 11.1 X 10^6 CD34+/kg Busulfan AUC: 5673 umol/L min Age at Transplant: 8 m/o EFS-ADA Vector copy/cell: 2.9 Adagen stopped day +30 ADA enzyme activity: 6974 (U)

LLN CD3+

LLN CD19+

Shaw K and Kohn DB A Tale of Two SCIDS. Sci Trans Med, 2011

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Gene Therapy for Hurler Syndrome Murine MPS I is Indistinguishable in Newborn Pups but Readily Differentiated in Older Mice • Hematopoietic stem cell transplant stabilizes disease (1982) • Donor engraftment halts IQ loss of 1.6 IQ points/month • Complicated by significant morbidity and mortality MPS I homozygote Normal

Self-inactivating Lentiviral Vector CSP1 for Expression of Human -L-iduronidase

 CMV R U5 PGK IDUA U3 R U5

Four Plasmids for Production of a Third- Hypothesis generation Self-inactivating HIV-based Lentiviral Vector

Would simple, early intravenous GARRE VSVG polyA RSV cIDUA CMV administration of a lentiviral vector to a pRRL.SIN-18 SD SA newborn achieve high levels of gene SD pMD.G transfer and gene expression, resulting GAG RRE in correction of MPS I? CMV polyA RSV REV polyA PRO POL SD SA RSV-Rev pMDLg/pRRE

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Correction of Craniofacial Dysmorphology in MPS I -L-Iduronidase Activity in Plasma Mice 100 Days After Neonatal Treatment After IV Lentiviral Gene Therapy

1000 54 55

56 100 61

Activity 62

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/ml/hr) 10 63 64

nmol 1 ( Enzyme 66

67 0.1 IDUA IDUA

T hi 40 50 60 70 80 90 100 s Carrier level Normal Treated MPS I MPS I Duration After Injection (day)

-L-Iduronidase Activity in MPS I Mice Correction of GM2-Ganglioside 100 Days After IV Lentiviral Gene Therapy Accumulation in the Hippocampus of Mice Liver Spleen Brain Gonad Murine Hurler Syndrome 54 15.1 1.0 0.10 0.23 55 1.12 1.3 0.09 UD Murine MPS 56 0.85 1.1 0.06 0.29 I Normal 61 50.5 2.7 0.16 0.08 62 67.6 2.9 0.26 0.16 63 44.3 2.2 0.22 0.07 64 47.3 2.2 0.13 0.11 65 0.29 0.26 0.15 0.20 66 0.82 0.71 0.14 0.12 MPS I 67 52 2.47 0.09 0.32 Carrier 4.0 + 0.42 14.9 + 2.4 3.7 + 0.55 15.8 + 5.7 treated

66 UD, undetectable, < 0.01 nmol/mg/hr.

Habituation: Locomotor Activity

Murine Hurler Syndrome Liver Pathology 0

-10 MPS I MPS I (50+100) -20 Heterozygotes -30

-40 * -50

-60

Untreated Lentiviral Treatment -70

% Change Between First andTrial Final Change Between% First -80 *p = 0.064 vs. MPS I *p = 0.014 vs. Carrier

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‐Iduronidase Expression in the Brain of Mice iht Translation from bench to bedside Hurler Syndrome after Intravenous Administration of 2007 2008 2009 2010 2011in < 2012 5 YEARS 2013 Lentiviral Vector Activities 2007 2008 2009 2010 2011 2012 2013 Proof of concept and vector design

Selection of manufacturer

Production of GLP batches / tox. batches

Production, import, QA & release of GMP vector

Efficacy studies

Toxicology & Biodistribution studies

CTA submission, review & approval (AFSSAPS + ERB)

CT phase I/II

Sanfilippo Syndrome Type A Phase I/II Study Design (Mucopolysaccharidosis Type III A) • Primary objective: to assess tolerance and safety of SAF‐301 • Progressive • Secondary: Definition of clinical endpoints for future efficacy neurodegenerative studies (brain imaging, neuropsychological tests and biological disease markers) • Open‐label non‐controlled monocentric study • Normal at birth – 4 patients treated in a single neurosurgical procedure with one year • Peak developmental follow‐up Aug 2011 –May 2013 – Immunosuppressive treatment to prevent transduced cells elimination age of 28 months • Survival to 15 ‐ 30 Patient 1 Patient 2 Patient 3 Patient 4 Age at 6y 7m 6y 5m 5y 10m 2y 8m years of age inclusion

Lysogene MPS IIIA development overview Phase I/II safety results

• AAV vector serotype rh10 Tardieu et al. (2014) Human Gene Safety (primary endpoint) – Transduces a large area of the CNS with high level Therapy of transgene expression (Swain et al. Gene Ther. 2013) • Good tolerance – Favorable immunogenicity profile (Chirmule • Neurosurgery itself was 1999, Halbert 2006) uneventful • Carrying a normal copy of the N‐sulfoglycosamine • Absence of adverse events sulfohydrolase (SGSH) gene related to the injected • Intracerebral administered gene therapy using product frameless stereotactic guidance: the most advanced • 1 No increase in the number and proven strategy in humans (Batten , Canavan of infectious events trials2, Metachromatic Leukodystrophy3 and Sanfilippo B4) • No biological sign of • Orphan drug status in EU and US toxicity related to immunosuppressive drugs Concept: Correction of defect should reduce/reverse toxic storage, improve functions and health status 1Souweidane et al. J Neur Ped 2010 2Leone et al. Sci Transl Med 2012 3ClinicalTrials.gov number: NCT01801709 4Institut Pasteur

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Phase I/II Efficacy Results

Efficacy (secondary endpoint) • Neuropsychological evaluations suggested improvement, although moderate, in behaviour and attention in the 3 older patients who showed cognitive decline at inclusion. • Neurocognitive benefit was more marked in the youngest patient • Improvement in behavioural disorders, hyperactivity and sleep, reported by the parents (stopped symptomatic medications)

Tardieu et al. (2014) Human Gene Therapy Nalani et al., European Journal of Medical Genetics, 2008

Lessons learned Giant Axonal Neuropathy (GAN)

• Rare autosomal recessive disease of the central and peripheral nervous Younger patients most likely system caused by loss of gigaxonin gene (GAN) to benefit from new therapy GFAP Improved dosing and Cul3 Rbx1 Keratin administration: cerebellar Ubiquitin Ligase NF‐H injections Vimentin

gigaxonin Peripherin Need quantified validated data Map1B Alpha‐Internexin Ub Ub on hyperactivity, sleeping Map8 Ub Other IFs disorders and quality of life TBC‐B Ub gigaxonin

•Loss of gigaxonin protein results in intermediate filament (IF) accumulation •Axonal accumulation of IFs causes the most severe disease symptoms

Phase II/III Study design Phase I Trial to Treat Giant Axonal Neuropathy

• Single‐arm, non‐randomised open‐label study including 12 patients JeT promoter worldwide Gigaxonin coding, 1897 bp AAV9 Capsid • Total dose increased versus SAF‐301 synth. polyA • Additional injection site (cerebellum) self‐complementary genome • Anticipate minimum three clinical trial sites in Europe & US • Duration: average 2 year follow up post dosing per patient depending Targets: on age of patient at inclusion • Primary targets will be spinal cord and brainstem motor neurons, DRG (to treat • Start: Q4 2015 peripheral motor and sensory disease) • Secondary targets will be the brain (to address white matter abnormalities and protein • Immunosuppressive treatment aggregations / inclusion bodies) • European Medicines Agency (EMA) protocole assistance completed 09/14 Preliminary Clinical Approach • USA: Pre‐IND (investigational new drug) procedure • Agent: GMP scAAV9/JeT‐GANopt vector manufactured by the UNC Vector Core Human Applications Lab • Dose = 3.5x1013 vg per person (~2.5x1011 vg per mL CSF) • Route = intrathecal (1 administration) • Patient Age = 5 yrs and older • Phase I (safety). Approved by the RAC, IRB, and FDA.

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NIH Recombinant DNA Advisory Committee (RAC) Human Gene Therapy Protocols 1304‐1231 Bonnemann, Carsten; National Institutes of Health; Bethesda, Maryland; A Phase I Study of Intrathecal Administration of scAAV9/JeT‐GAN for Gigaxonin the Treatment of Giant Axonal Neuropathy. expression Sponsor: Hannah’s Hope Fund

Giant Axonal Neuropathy/n Vivo/Adenovirus Associated Virus Serotype 9/GAN Gene, Encodes Peripherin aggregates the Protein Gigaxonin/Intrathecal Administration

Summary Gene Therapy for Pediatric Diseases • Convergence of the human genome project, and gene therapy technolgy, have offered hope for the treatment of many genetic disorders for more than 40 years. • Recent successes in the past few years predict steady, and accelerating progress in the CONCLUSIOS: • Intrathecal injection of AAV9 in juvenile pigs resulted future. in 50‐100% of spinal cord motor neuron transduction across the entire spinal cord. • The successes − and limitaons − of gene therapy promise novel treatments particularly for serious, lethal disorders.

Gene Therapy, 2013

CONCLUSIONS: • Intrathecal administration of AAV9 leads to global vector distribution and transgene expression to the CNS. • Intrathecal administration allows for a lower dose, avoidance of anti‐capsid antibodies, and reduced peripheral organ biodistribution. • This approach translates effectively from rodents to non‐human primates