Protocol. This trial protocol has been provided by the authors to give readers additional information about their work.

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1 Protocol This trial protocol has been provided by the authors to give readers additional information about their work. Protocol for: Nathwani AC, Tuddenham EGD, Rangarajan S, et al. Adenovirus-associated virus vector mediated gene transfer in hemophilia B. N Engl J Med 2011;365: DOI: /NEJMoa

2 SJCRH AGT4HB Initial version, dated: ; resubmitted to IRB: (IRB approved: ), Submitted Date: 10 March-09; Submitted to IRB: -----(IRB approved: ) Amendment 2.0, Submitted Date: Submitted to IRB: (IRB approved: ) Activation Date: Amendment 3.0, Submitted Date: (IRB approved 4/19/2010) Amendment 4.0, Submitted Date: (IRB approved 1/24/11) AN OPEN LABEL DOSE-ESCALATION STUDY OF A SELF COMPLEMENTARY ADENO- ASSOCIATED VIRAL VECTOR (scaav2/8-lp1-hfixco) FOR GENE TRANSFER IN SUBJECTS WITH HEMOPHILIA B Sponsor: St. Jude Children s Research Hospital Legal Representative in the UK: University College London EUDRACT NO: Amendment 2.0 IND # SJCRH Principal Investigator of Record: Arthur W. Nienhuis, M.D. 1 Clinical Principal Investigator: Ulrike Reiss, M.D. 1 SJCRH Co-Investigators: Andrew M. Davidoff, M.D. 2, John Gray, Ph.D. 4, Scott Howard, M.D. 3, Wing Leung, M.D., Ph.D. 3, Amendment 3.0 Song Wu, Ph.D. 5, Shane Cross, Pharm.D 6 Departments of Hematology 1, Surgery 2, Oncology 3 Vector Development and Production Shared Resource 4 Biostatistics 5, and Pharmaceutical Sciences 6 St. Jude Children s Research Hospital 262 Danny Thomas Place Memphis, TN USA (901) External Co-Investigators: Mark Kay, M.D., Ph.D. 7, Bert Glader, M.D. 7 Department of Pediatrics 7 Stanford University School of Medicine Stanford, CA , USA Katherine A. High, M.D. 8 Investigator, Howard Hughes Medical Institute, Department of Pediatrics 8 Children s Hospital of Philadelphia Philadelphia, PA , USA External UK Principal Investigator: Amit C. Nathwani, MBChB, FRCP, FRCPath, PhD 9 University College London & Royal Free Hospital School of Medicine 9 Department of Haematology 72 Huntley Street, London, WC1E 6BT, U.K.

3 External UK Co- Investigators: David C. Linch, MD, FRCP, FRCPath 9 University College London & Royal Free Hospital School of Medicine 9 Department of Haematology 72 Huntley Street, London, WC1E 6BT, U.K. Amendment 3.0 Edward G.D. Tuddenham, MD, FRCP, FRCPath 10 The Katharine Dormandy Haemophilia Centre & Thrombosis Unit 10 Royal Free Hospital, UK John Pasi, MD, FRCP, FRCPath 11 Centre for Haematology, Institute of Cell and Molecular Science 11 Barts and the London, Queen Mary s School of Medicine 4 Newark Street, London, E1 2AT, U.K.

4 Table of Contents 1. STUDY SUMMARY 1 2. BACKGROUND Hemophilia, a good model for gene therapy Previous experience with AAV Vectors 4 3. PRECLINICAL DATA AND RATIONALE FOR THE STUDY Nonhuman primate model allows evaluation of AAV in a manner relevant to humans Evaluation of vectors based on alternative serotypes in rhesus macaques Self-complementary vectors Peripheral vein administration of low doses of scaav in nonhuman primates Rationale for the proposed study RESEARCH PARTICIPANT ELIGIBILITY CRITERIA AND STUDY ENROLLMENT Participant Eligibility Criteria Screening, Informed Consent and Trial Enrollment Management of Adult Participants in a Pediatric Hospital STUDY PLAN Pre-gene transfer evaluations Description and production of study agent Protocol for infusion of vector Evaluation to document toxicity, efficacy and vector biodistribution Dose escalation criteria Follow-up schedule and summary of evaluations after vector administration Concomitant Medications/Therapies Alternative therapy for Hemophilia B Qualification of Hemophilia Treatment Centers for Participation in Follow-up Study Steering Committee ADDITIONAL INFORMATION REGARDING THE STUDY AGENT Quality control assays Vector release criteria Storage, label, shipment and administration POTENTIAL RISKS AND BENEFITS Information regarding risks Potential risks and solutions Data Safety Monitoring Board Medical Advisory Committee Efficacy Importance of the knowledge to be gained OFF STUDY CRITERIA SAFETY AND ADVERSE EVENT REPORTING REQUIREMENTS Adverse event and serious adverse event Safety data collection and collection time period Medical Coordinating Center, Medical Monitor and Reporting Requirements of AEs and SAEs - collaborating sites Reporting requirements in the event of pregnancy Reporting arrangement for the UK sites Reporting requirements to the St. Jude Institutional Review Board (IRB) Reporting requirements to the St. Jude Institutional Biosafety Committee (IBC) Reporting requirements to the FDA 52

5 9.9. Reporting to the National Heart, Lung and Blood Institute (NHLBI) Reporting to the NIH Office of Biotechnology Activities (OBA) Post-one year late effects reporting Summary of reporting mechanism by the St. Jude Office of Regulatory Affairs St. Jude continuing review reports Stopping Rules DATA COLLECTION, MONITORING, AND CONFIDENTIALITY Enrollment on study Data collection and management Data submission from HTC/HC to infusion site Quality assurance monitoring Confidentiality STATISTICAL CONSIDERATIONS OBTAINING INFORMED CONSENT STUDY ADMINISTRATION Study agent accountability Study amendments Record retention Safety issues for healthcare workers Environment Termination of the study SIGNED AGREEMENT OF THE STUDY PROTOCOL TABLES 64 APPENDIX A1: SCHEDULE OF CLINICAL AND LABORATORY EVALUATIONS 67 A2: DETAILS OF LABORATORY EVALUATION 68 APPENDIX B: MODIFIED SYMPTOM-SPECIFIC TOXICITY SCALE 69 APPENDIX C: FDA FORM APPENDIX D: TREATMENT OF IMMUNE HEPATITIS REFERENCES 74 Acronyms used in the protocol AAV = adeno-associated virus CBC =Complete Blood Count CRO = Contract Research Organization CRP = C-Reactive Protein DHHS = Department of Health and Human Services DSMB = Data and Safety Monitoring Board EUDRACT = European clinical trials database FDA = Food and Drug Administration FIX = Coagulation Factor IX

6 Acronyms used in the protocol (Continued) GMP = Good Manufacturing Practices GTAC = Gene Therapy Advisory Committee HC = Haemophilia Centre HTC = Hemophilia Treatment Center IBC = Institutional Biosafety Committee IRB = Institutional Review Board INR = International Normalized Ratio ITR = Inverted Terminal Repeat LFT = Liver Function Tests LREC and MREC = Local and Multicenter Research Ethics Committees, respectively MAC = Medical Advisory Committee MHRA = Medicines and Health-care products Regulatory Authority NAB = Neutralizing Antibody NIH = National Institutes of Health OBA = Office of Biotechnology Activities raav = Recombinant AAV scaav = Self Complementary AAV UCL = University College London and Royal Free Hospital School of Medicine WHO = World Health Organization

7 1. Study Summary Primary Objective: To assess the safety of systemic administration of a novel self complementary AAV vector (scaav2/8-lp1-hfixco) in adults with severe hemophilia B at up to three different dose levels. We predict that systemic administration of scaav2/8-lp1-hfixco vector pseudotyped with AAV8 capsid protein and encoding the human FIX (hfix) gene will be safe with absence of persistent Grade III or greater dose limiting toxicity. Any Grade I-II toxicity is likely to be dose dependent and reversible. To test this hypothesis we have developed a comprehensive clinical monitoring plan that will detect and document any adverse events following gene transfer. Secondary Objectives: Amendment 3.0 a. To estimate the dose of scaav required to achieve stable expression of hfix at or above 3% of normal ( 3u/dl), a level which would significantly ameliorate the severe bleeding phenotype. The kinetics, duration and magnitude of scaav-mediated hfix expression in individuals with hemophilia B will be evaluated and related to vector dose. b. To describe the immune responses to the hfix transgene product and AAV capsid proteins following systemic administration of scaav2/8-lp1-hfixco. c. To assess viral shedding in various body fluids after systemic administration of scaav2/8- LP1-hFIXco. Research Subject Population: Recruitment will be limited to adults ( 18 years) with a confirmed diagnosis of hemophilia B (HB), resulting from a missense mutation in the coagulation factor IX (FIX) gene or a nonsense mutation that has not been associated with an inhibitor. Only subjects who have no evidence of active hepatitis or anti-hfix antibodies, and who have been treated/exposed to Factor IX concentrates for at least ten years and have had an average of 3 bleeding episodes per year requiring FIX administration will be enrolled. Patients will be recruited within the United States for treatment at one of two centers and patients will be recruited in England and other countries for treatment in London by our British collaborators. Amendment 3.0 Study Centers: This study will involve three main sites: (1) St Jude Children s Research Hospital, Memphis, TN, USA, (2) Katharine Dormandy Haemophilia Centre and Thrombosis Unit (KDHCTU) which is part of the University College London and Royal Free Hospital School of Medicine, London, UK, and (3) Stanford Medical School, Stanford, CA, USA. Several sites, usually established Hemophilia Treatment Centers, will be identified across the USA and UK whose role will be to help with local monitoring of the participant according to the study protocol. These sites will be involved in identification of potential participants but will not be involved in the consent process or vector infusion. Number of research participants: 18 (maximum). Trial Design: In this open label dose-escalation, Phase I/II study, a single dose of a novel self complementary AAV (scaav2/8-lp1-hfixco) vector will be administered into a peripheral vein of adult subjects with severe HB. We are proposing to test up to three dose levels, 2 x 10 10, 6 x and 2 x vector genomes per kilogram (vg/kg) of body weight. A comprehensive monitoring schedule has been established to assess the primary end point of safety which includes an array of clinical and laboratory evaluations including liver biochemistry, semen analysis for vector genomes, and 1

8 immunological response to hfix and AAV capsid. Enrollment of each subject will proceed only after the previous subject has been observed for at least 42 days. Accrual will be suspended if dose limiting toxicity, including any Grade III-IV adverse events or any Grade II adverse events that persist for more than 7 days and are at least possibly related to the vector product occur in any of the enrolled subjects. If two subjects at a given dose level show no evidence of dose limiting toxicity within 42 days of vector infusion and Factor IX levels are less than 3% at last follow-up in one or both subjects, we will proceed to the next dose level. All enrolled participants will continue to be monitored for toxicity and FIX levels according to our monitoring plan and if the average of two FIX levels obtained within 3 weeks of the next participant being enrolled has fallen below 3% in one or more participants, the newly enrolled participant will receive the next highest vector dose. This design minimizes the number of individuals who may receive a sub-therapeutic infusion. Efficacy, a secondary endpoint, will be defined as expression of biologically relevant levels of hfix (> 3% = 3u/dl = 150ng/ml) in the peripheral blood. Individual participants will be evaluated for toxicity and efficacy, and new participants enrolled sequentially as outlined in detail in a later section of this protocol (Section 5.5). Consideration will be given to amending the protocol to allow a higher dose level if no toxicity has been observed, even if all participants enrolled at a given dose level have Factor IX levels of 3%. Such an amendment will be predicated on the desire to achieve higher Factor IX expression to achieve greater therapeutic efficacy if the vector proves safe. Distinct features of this study: This study differs from previous HB clinical trials with AAV vectors in three important aspects. Firstly, AAV8 pseudotyped vectors will be used instead of AAV2 primarily because of the substantially lower prevalence of pre-existing humoral immunity to this AAV serotype in humans. The second difference relates to the use of a vector containing a self complementary genome which, because of its ability to rapidly form stable, transcriptionally active double stranded linear molecules in target tissues, offers a unique opportunity to mediate efficient therapeutic gene transfer potentially at a low dose of vector. This will further enhance the safety of our approach without compromising efficacy. Finally, because the biodistribution of vector predominantly to the liver is the same regardless of the route of administration, scaav particles will be administered via a peripheral vein. Our preclinical data in nonhuman primates indicates that this will not compromise efficacy or safety. Indeed administration of vector into a peripheral vein dispenses with the need for invasive surgical procedures, making this a safer and more desirable route of vector delivery for patients with severe HB. Because of the potential risk of a serotype cross reactive, cytotoxic T- lymphocyte response to hepatocytes transduced with raav8, we are prepared to initiate immunosuppression, with guidance from a Medical Advisory Board, if transaminitis is detected. A separate Medical Advisory Board composed of physicians with expertise in Hematology, Hepatology and Immunology will be assembled at each treatment center to consult about treatment of individual patients. Study duration: 1 year for each patient to assess the study objectives followed by regular follow-up for fifteen years. Endpoints: The primary safety endpoint is the development of dose limiting toxicity including any Grade III-IV adverse events or any Grade II adverse events that persist for more than 7 days and are at least possibly related to the study agent, according to the modified symptom specific NCI toxicity scale in Appendix B which is derived from the NCI Common Terminology Criteria for Adverse Events, v3.0 ( ). We have developed a comprehensive plan to enable careful assessment of safety. Efficacy is a secondary objective of this study and is defined as persistent expression of functional hfix at > 3% of normal levels in plasma. This target level is 2

9 significantly above the 1% concentration of hfix (=1u/dl = 50ng/ml) that is necessary for amelioration of spontaneous bleeding and allows for some variation in expression that has been occasionally observed longitudinally in our preclinical studies. 2. BACKGROUND 2.1. Hemophilia, a good model for gene therapy Hemophilia B (HB) is an X-linked recessive bleeding disorder that affects approximately 1 in 30,000 males. This disorder results from a defect in the factor IX (FIX) gene, which encodes a serine protease, critical for appropriate fibrin clot formation. The majority of individuals with HB have a severe disorder characterized by functional FIX levels of less than 1% of normal (<1u/dl, = 50ng/ml). 1 Clinically the disease is characterised by frequent spontaneous bleeding into joints and soft tissues which, without adequate treatment, causes a chronic debilitating arthropathy. Spontaneous bleeding into closed spaces such as the brain occurs in a significant proportion of patients with catastrophic manifestations including death. Current treatment for HB in the Western World involves intravenous infusions of plasma derived or recombinant factor concentrates at the time of a bleed ( on demand therapy). This is highly effective at arresting hemorrhage but cannot totally abrogate chronic damage that ensues after a bleed. In severely affected patients, bleeding episodes can be dramatically reduced when plasma FIX levels are maintained at or above only 1% of normal by prophylactic administration of hfix protein concentrates. 2 The relatively short halflife of FIX necessitates frequent intravenous administration of factor concentrates (2-3 times a week) which is invasive, inconvenient and highly problematic for children. Central lines are often required in children which may cause significant morbidity due to catheter-related complications. The safety of plasma derived factor concentrates has been greatly improved by viral screening and inactivation measures. However there remains an ever-present concern about blood borne infections including new variant Creutzfeldt-Jakob disease (nvcjd). 3 Recombinant factor concentrates may offer freedom from blood borne infections. However, availability is still an issue as there is only one worldwide manufacturer. Additionally the cost of prophylactic treatment for an adult is in excess of $150,000 per year, a significant cost burden for all healthcare systems even within the developed world. In the context of hemophilia worldwide, the cheaper plasma derived FIX concentrates, if used for treatment alone rather than prophylaxis, still remain unaffordable by the majority of the world s hemophiliacs. 4 Formation of neutralizing antibodies (inhibitors) which occur in 3% of HB patients resulting in poor response to factor concentrates, often with fatal outcomes, is a rare but significant complication of protein replacement therapy. Additionally the significant psychological burden associated with the constant but unpredictable risk of bleeding in this chronic disorder is not fully appreciated. Therefore replacement therapy with FIX products continues to be less than ideal both in the context of the West as well as the developing world. These unmet needs have fuelled interest in gene therapy of HB which has several fundamental advantages over other single gene disorders. The clinical manifestations represent a simple cause and effect relationship which is attributable to the lack of a single gene product that circulates in minute amounts in the plasma. In addition, tightly regulated control of gene expression is not essential since a wide range of FIX is expected to be beneficial and nontoxic. The availability of animal models including FIX-knockout mice 5-7 and HB dogs 8;9 has facilitated extensive preclinical evaluation of gene therapy strategies. Perhaps the most important single aspect of HB that makes it such an excellent target for gene therapy is the fact that even a small rise (1% of physiological levels) in 3

10 circulating clotting factor would have a significant beneficial therapeutic effect which could transform the lives of patients with severe hemophilia. Small changes in FIX can be easily assessed using both clinical and laboratory evaluation. Continuous synthesis of FIX by the host cells after gene therapy provides a real opportunity to prevent bleeding episodes rather than simply treating the bleeds after they have occurred. Additionally the risks and the inconvenience of regular infusion of protein concentrates would be avoided following gene therapy. Importantly endogenous synthesis of FIX within hepatocytes after liver targeted delivery of vector may also lead to immunological tolerance to the transgene product thereby potentially reducing the risk of neutralising FIX antibody formation Because of the relatively small size of the gene for FIX several gene therapy approaches for HB have been evaluated in animal models, including retroviral vectors, recombinant adenovirus, direct hydrodynamic injection of plasmid DNA and Sleeping Beauty transposase technology (reviewed in 13 ). At present, however, recombinant adeno-associated virus (raav) vectors show the greatest promise for HB gene therapy and other disorders affecting the liver Previous experience with AAV Vectors Most of the preclinical and clinical experience to date has been with AAV2, the first serotype to be isolated and fully characterized. We and others have established in a number of different in-vivo settings that AAV2 vectors are particularly efficient at transducing terminally differentiated cells such as neurons 14;15, myofibres 16;17, retinal photoreceptor cells 18;19 bronchial epithelium and hepatocytes 10;23;24 resulting in long-term transgene expression in animal models. Correction of the bleeding diathesis (reviewed in 13 ) following a single administration of AAV2 into either muscle or liver has been consistently observed in murine, 10;25-27 and canine 17;23;28;29 models of hemophilia without significant toxicity. These preclinical studies have resulted in clinical evaluation of AAV2 vectors in over a hundred patients suffering from cystic fibrosis, 21;30-33 Canavan disease, 34 limbgirdle muscular dystropy, 35 and alpha 1-antitrypsin deficiency. 36 Collectively, these early studies suggest that AAV2 vectors are safe in humans although efficacy has been limited. Two clinical trials of AAV-mediated FIX gene transfer have been performed in adults with severe HB. The first study involved intramuscular delivery of a AAV2 which contained the hfix gene under the control of the constitutively active CMV promoter. 37,38 Eight adult men with severe HB were enrolled over 3 dose levels ranging from 2x10 11 to 1.8x10 12 vg/kg. Intramuscular administration of vector into multiple sites (ranging from sites) was well tolerated. Transduction of myocytes around the injection sites was confirmed by immunostaining and Southern blot detection of AAV vector genomes. However therapeutic levels of hfix were only transiently detected in the plasma of just one patient in the low-dose cohort. Thus although intramuscular administration of AAV2 vectors encoding hfix was safe in patients with HB, efficacy was quite limited. Although anti-hfix antibodies were not detected at any time after AAV mediated gene transfer, plans to extend this study with higher doses of vector were not pursued because of the potential risk of evoking an immunological response to hfix following intramuscular injection. 10 Attention in the second clinical trial focused on liver targeted delivery of the vector. 39 Liver is the natural site for FIX synthesis, and expression of transgene within hepatocytes is less likely to provoke a neutralising antibody response to FIX when compared to intramuscular injections Seven patients with severe HB received AAV2 via hepatic artery injection. Three dose levels were tested in the initial 6 subjects, 8x10 10 vg/kg, 4x10 11 vg/kg and 2x10 12 vg/kg. FIX was not detected in the serum of the 4 individuals treated at the 2 lower dose levels but was present transiently in the 4

11 serum of the patients (E and F) who received the highest dose. Patient E had a low neutralizing antibody (NAB) titer of 1:2 to AAV2 prior to vector administration. He exhibited an increase in FIX to 12% at week 2 post infusion which persisted at 10% at week 4. However, beginning at that time, he had a subclinical elevation in liver transaminases which coincided with a decline in FIX levels over the next 6 weeks to pre-treatment levels. The transaminitis gradually resolved completely by week 14. Subject F, also treated at the higher dose, had a higher AAV2 NAB titer of 1:17. He had a transient rise in factor IX to 3% at week 2 which returned to baseline by week 4 and no transaminitis. A seventh subject (G) was treated at the lower vector dose of 4x10 11 vg/kg which had proved subtherapeutic in the previous 2 patients. This individual had a low pretreatment NAB titer. No measurable FIX was present in the plasma. However, this individual experienced transient transaminitis beginning at week 4 with transaminases returning to baseline by week 10. Cytotoxic T-lymphocytes (CTL) specific for selected AAV capsid peptides were detected in the blood of patient G two weeks after vector infusion. Such CTLs were not present prior to infusion and receded to undetectable levels by week 12 following infusion. In a subsequent study, expansion of the specific CTLs in vitro to 25% of the total by stimulation with an AAV capsid peptide was possible. 40 The investigators interpreted these data to indicate that even modest NAB titers precluded liver transduction and avoided subsequent transaminitis. 39 However, individuals previously sensitized to AAV2 with lower or undetectable antibody titers had successful liver transduction but experienced reawakening of memory T-cells to generate CTLs resulting in immune mediated destruction of transduced liver cells and elimination of therapeutic FIX production. Also, the AAV genome was transiently detected in the semen of 6 participants, regardless of vector dose. As summarized in detail in Section 7.2.2, evidence has accumulated which suggests that the CTL response to capsid epitopes which are conserved among AAV serotypes may complicate the use of most raav vectors for liver targeted gene therapy. Many humans have demonstrable memory T- cells in the spleen which can be expanded by an AAV2 peptide challenge in culture 40 and which exhibit CTL activity against a human hepatocyte line transduced with vector particles of various pseudotypes including raav8. 41 These considerations have prompted us to plan to monitor carefully for any signs of immune hepatitis by measuring liver enzymes and hfix levels frequently, and by assaying for CTLs using available reagents. 39,40 If evidence of hepatitis is detected in the absence of another potential cause, we will initiate immunosuppression with guidance by a local Medical Advisory Committee (MAC) composed of physicians with expertise in Hematology, Hepatology and Immunology. If any patient develops immune hepatitis and requires immunosuppressive drugs, accrual will be suspended and the Data Safety and Monitoring Board (DSMB) consulted. If judged appropriate, the protocol will be amended to provide for immunosuppression beginning before and for several weeks after vector administration in subsequent patients in an effort to prevent memory T-cell expansion and the emergence of liver targeted CTLs. 3. PRECLINICAL DATA AND RATIONALE FOR THE STUDY 3.1. Nonhuman primate model allows evaluation of AAV in a manner relevant to humans Over the last 7 years we have extensively evaluated AAV vectors derived from different serotypes in a rhesus macaque model using a sensitive assay that enables detection of minute amounts of hfix in rhesus plasma 24, This large outbred animal model, unlike the murine and canine models of hemophilia used to date for preclinical safety studies, is a natural host for AAV and is 5

12 potentially better suited for modelling future clinical trials as we begin to fully appreciate the importance of vector immunity and tropism. Nonhuman primates have proven to be more reliable in predicting outcomes in humans when assessing drugs, vaccines and novel therapeutics including gene transfer vectors Indeed, they had correctly predicted the toxicity of adenoviral vectors and the poor efficacy of oncoretroviral vectors in humans. 52,53 Their longer life span provides a unique opportunity to study long-term toxicity in a context relevant to humans. Indeed our experience with AAV2 in nonhuman primates has highlighted many of the difficulties encountered in the clinical trials with AAV2. Consistent with data from clinical trials of HB gene therapy, virus shedding, as demonstrated by PCR, occurred transiently in the plasma and, to a lesser extent, the urine and saliva, of all the macaques for up to one week following liver targeted delivery of AAV2. 24,43-45 However this period of viremia was not associated with an increase in inflammatory markers such as interleukin-6 (IL-6). 24 Limited tissue biodistribution studies using PCR on tissue samples obtained at various time points after gene transfer indicated that the hfix transgene was localized predominantly in the liver but with some spill-over in a dose related manner to other organs including the testis. 24,43 Importantly, expression of hfix at therapeutic levels was achieved in 5 out of 8 macaques in our initial studies and has persisted for at least 5 years after gene transfer. 24,43 As in humans, poor and inconsistent transduction was observed in animals with detectable titers of anti-aav2 antibody prior to vector administration Evaluation of vectors based on alternative serotypes in rhesus macaques Transduction efficiency of AAV2 vectors in humans has been impaired by pre-existing immunity. The animal models previously used to support clinical trials are not natural hosts for AAV2. In contrast this serotype is endemic in humans resulting in the presence of neutralizing antibodies (NAB) against AAV2 in over 70% of individuals. 54,55 Such antibodies have been shown to eliminate or greatly reduce subsequent AAV2 mediated transgene expression in animal models. 20,24,43-45 Needed, therefore, is a model that enables evaluation of new strategies with AAV vectors in a context that is relevant to humans. Among the myriad of AAV serotypes isolated recently, 56,57 eight isolates have been extensively characterised and shown to have different tissue tropism and to elicit distinct humoral responses. We have focused our attention on vectors based on serotypes 5 and 8 which have proven to be superior to AAV2 in directing transgene expression in several tissues including the liver in mice. 43 Our sero-epidemiological survey of 170 volunteer blood donors and 23 HB patients indicates that the prevalence of immunity to AAV8 resulting from wild type viral infection is low, as only 5% of humans have NAB to AAV8 compared with >70% to AAV2. This confirms similar findings reported by others. 55 Additionally AAV8 vectors have distinct biological properties that enable them to uncoat and release their genome more rapidly than other AAV serotypes, 42 suggesting that AAV8 capsid proteins may persist in hepatocytes for a significantly shorter time than AAV2 capsid proteins thereby reducing the potential for cell mediated immune response to the transduced cells. 42,58 Methods that enable scale-up and purification of vectors based on AAV5 and 8 to levels that would be suitable for clinical use have been developed by our group to enable a critical analysis of alternative serotypes in nonhuman primates. 43,59 Liver directed administration of AAV5 in 2 naïve macaques resulted in hfix expression of between 2-4%. Elevation of liver enzymes was not observed in either animal. However, biodistribution studies revealed the presence of vector 6

13 genome in the testis of one animal. 43 Liver targeted administration of AAV2/8 vectors (AAV2 genome pseudotyped with AAV8 capsid protein) into a macaque at our then standard dose of 4x10 12 vp/kg resulted in 8% circulating levels of hfix for more than four years following vector administration. Transgene expression in another animal treated with AAV2/8 vector was, however, subtherapeutic. In this animal anti-aav8 antibodies were detectable at low levels prior to injection with AAV8 highlighting, once again, the impact of pre-existing serotype-specific immunity on AAV mediated gene transfer in primates. Our studies indicate that in nonhuman primates AAV serotypes 2, 5 and 8 appear to mediate comparable levels of gene transfer despite their disparate transduction efficiencies in mice, emphasizing once again the importance of animal model selection in preclinical evaluation of AAV. In animals with pre-existing immunity to AAV2 or 8, following natural infection with wild type virus, we have successfully affected gene transfer with AAV5 pseudotyped particles to levels observed in naïve animals. This is an important consideration as most humans have immunity to AAV2 and suggested that this may be overcome by using vectors based on alternative serotypes of AAV. 43,44 Figure 1: Schematic of scaav LP1-hFIX. Each vector is shown schematically as it is packaged in the virion, with scaav vectors shown as dimers. Top: ssaav-hcr-haat-fix consisting of HCR enhancer and haat promoter driving expression from the 1.6 kb human FIX cdna (hfix) followed by the bovine growth hormone polyadenylation signal (BGpA) flanked by the AAV internal terminal repeats (ITR shown as hairpin loop). Bottom: Self 3.3. Self-complementary vectors To further improve the transduction efficiency of AAV vectors for HB gene therapy without having to increase the vector dose, we have constructed a liverrestricted, small hfix expression cassette (scaav-lp1-hfixco) which can be packaged as a single stranded but selfcomplementary genome within individual AAV particles (Figure 1). This vector contains an intact AAV2 5 ITR including the terminal resolution site (trs). The AAV2 3 ITR however contains a deletion within the trs to facilitate efficient packaging of the tail-to-tail selfcomplementary genome (see Figure 1) within a single scaav virion. 44 The expression cassette contains the LP1 enhancer/promoter, which was derived from the human apolipoprotein hepatic control region (HCR), and the human alpha-1-antitrypsin (haat) gene promoter from which sequences not essential for liver specific expression had been removed. Downstream of this regulatory region is a small SV40 intron followed by a modified hfix transgene in which the coding sequences were reconstructed (hfixco) using a subset of codons most frequently found in highly expressed eukaryotic genes ( codon optimization ), and further modified to reduce the potential for inappropriate splicing and CpG methylation. In addition, the 3 untranslated region of the hfix cdna has been deleted. These vectors overcome the need to convert the single-stranded (ss) AAV genome into transcriptionally active double-stranded (ds) forms in target cells, a process that is dependent on host cell mediated DNA synthesis of the leading strand 60 and/or annealing of complementary genomes derived from separate virions. 61 After uncoating the sc genome, it rapidly anneals to form stable transcriptionally active ds-linear molecules. 7

14 Figure 2: Correction of clotting times and expression of hfix:c in 129/sv HB mice. Following tail vein injection of either 1x10 10 (low dose cohort, n=6, 4x10 11 vg/kg) or 5x10 10 (high dose cohort, n=4, 2 x10 12 vg/kg) scaav2/8-lp1-hfixco into 129/sv HB mice. The clotting time (left hand panel at 4 weeks) and biologically active hfix:c levels (right hand panel) over 16 weeks were determined scaav are highly efficient in rodents The unique genome conformation of scaav enabled hfix expression at supraphysiological levels (>150µg/ml) in mice after systemic administration of only 1x10 11 AAV8 pseudotyped vector genomes without any toxicity. 44 This is a 20-fold improvement over ssaav2/8 vectors and 3 logs greater than levels achieved with conventional ssaav2 vectors in mice. A 50-fold reduction of scaav2/8 dose still generated hfix (>3.2µg/ml) approaching 100% of physiological levels. Indeed, 1x10 10 scaav2/8 particles resulted in expression of biologically active hfix at 8 IU/ml (8x normal) leading to correction of the bleeding diathesis in HB mice (Figure 2). We have evaluated scaav vectors in a variety of rodent models at doses ranging from 4x10 10 to 5x10 13 vg/kg (Table 1, section 14). A relatively linear vector dosetransgene expression profile was observed in these rodent models with no evidence of saturation kinetics at the doses examined. The transgene expression profile was identical at all dose levels and in all strains with hfix being detectable within a week and reaching peak levels after a lag phase of less than 4 weeks. Toxicity has not been observed at any dose levels despite careful evaluation of these animals over a period of months. Importantly even at the lowest dose level expression of hfix was in the therapeutic range Episomal state of scaav Detection of AAV integration into host chromosomes has been confounded by the persistence of the provirus as large episomally retained concatemers. To distinguish between transgene expression from integrated and extrachromosomal vector, we induced hepatocellular regeneration by performing a two-thirds hepatectomy in HB mice, 16 weeks after administration of 1x10 10 scaav2/8-lp1-hfixco. Though hemostasis was easily achieved, there was a sharp decline (~90%) in hfix:c levels which did not recover after complete restoration of liver cell mass within 4 weeks of surgery. In addition a concomitant decline in the vector copy number (~86%) was also observed post-hepatectomy (Figure 3) indicating that extrachromosomal and not integrated scaav genomes are primarily responsible for transgene expression in mice with this potent vector system. These results are similar to previous results using single stranded AAV vectors suggesting that the probability of integration is not increased by use of a scaav vector genome. 8

15 Liver targeted delivery of scaav in rhesus macaques Our standard dose for effecting therapeutic transduction in nonhuman primates using other serotypes was initially estimated at 4x10 12 vg/kg (summarized in Table 2, section 14). 24,43 The higher potency of scaav in mice prompted the evaluation of two lower doses of scaav2/8-lp1- hfixco in macaques (Table 2). The first group of macaques (M1-sc and M2-sc) received 1x10 12 vp/kg of vector which was infused into a mesenteric vein. scaav2/8-lp1-hfixco was well Figure 3: Genome analysis of murine liver. Southern blot analysis of liver DNA isolated pre- and post-partial hepatectomy at 16 and 20 weeks respectively after tail vein administration of 1x10 10 scaav2/8-lp1-hfixco into 129/sv HB mice (HBM1 and 2). Each lane contains 10 g of DNA digested with EcoRI and PstI which releases a 1.1kb fragment. Proviral copy number (shown at the bottom) was deduced from standards, which consisted of serial dilutions of vector DNA (0.13 to 13 copies) in 10 g genomic DNA from naïve mice. tolerated without perturbation of serum IL6 levels or liver transaminases over a period of 6 weeks after gene transfer. 44 Within 24 hours of vector administration, hfix was detectable at 5% and 24%, peaking at 18% and 30% of normal in M1 and M2-sc respectively within 2 weeks of gene transfer. These levels were stably maintained in M1-sc for 9 weeks before being abrogated by anti-hfix antibodies whose titer increased over a period of 4 weeks prior to reaching stable level of 15 BIAU/ml. The coagulation screen in this animal was normal consistent with selective targeting of hfix and not its rhesus cognate by the rhesus anti-fix antibodies. Epitope mapping studies using hfix domain swap variants demonstrated that the rhesus antibodies were exclusively directed against the serine protease domain. Of the 12 amino acid differences between hfix and its rhesus cognate, 6 are located in the catalytic domain, spread over surface loops that border the reactive site cleft. Using a panel of loop substitution mutants, antibody binding could be attributed predominantly to hfix loop , which differs from its rhesus cognate by Ala instead of Thr in position 261. This suggests that the humoral response was provoked by species specific differences in the FIX protein. 44 Treatment with Rituximab and daily oral cyclosporine was commenced to eradicate the neutralizing anti-hfix antibody and within 4 weeks the inhibitor became undetectable. Human FIX was once again detectable in rhesus plasma at approximately 16% of normal. The inhibitor re-emerged after 16 weeks and the animal received 4 monthly courses of Rituximab and cyclophosphamide which resulted in a more durable response which has lasted more than 61 weeks. Two additional macaques (M3-sc and M4-sc) received an estimated dose of 4x10 11 vp/kg of scaav2/8-lp1-hfixco, via a mesenteric vein, which represents a log reduction of our standard dose. 44 Transgene expression in one animal (M3-sc) has been maintained at between 1-3% level for the duration of the study (>9 months). The second macaque in this group (M4-sc) had a low, 9

16 but detectable pre-existing anti-aav8 antibody titer (3.4 compared to a range of relative units respectively in M1, M2 and M3-sc). This macaque was not successfully transduced after liver targeted administration of 4x10 11 vg/kg, suggesting that even modest levels of pre-existing humoral immunity are sufficient to block successful transduction of the liver. However, switching capsid proteins resulted in successful transduction in this animal when challenged with delivery of 1x10 12 vg/kg of scaav2/5-lp1-hfixco, despite a high titer of anti-aav8 antibodies (>27 relative units) at the time of vector administration. The kinetics of hfix expression were identical to that observed in M1 and M2 given scaav-2/8 with a rapid increase in expression to peak levels of 25% (1190ng/ml) of normal within 2 weeks after vector administration. Similar results have been obtained in another macaque with pre-existing antibodies to AAV8 resulting from natural infection with wild type virus that blocked gene transfer with a conventional single stranded AAV8 vector. In this animal mesenteric vein administration of 1x10 12 vg/kg of scaav2/5-lp1- hfixco resulted in hfix expression at approximately 23% of normal levels. 3.4 Peripheral vein administration of low doses of scaav in nonhuman primates We have previously demonstrated that AAV8 has a remarkable tropism for the liver such that tail vein administration of AAV8 results in transduction of murine hepatocytes at levels that were comparable to those achieved following intraportal injection of Peripheral vein delivery of scaav2/8 in nonhuman primates results in stable hfix expression 1x10 12 vg/kg into saphenous vein hfix ( g/ml) Days scaav2/ % 30% 20% 10% Figure 4: hfix levels in three rhesus macaques after peripheral vein injection of scaav2/8 LP-1 hfixco. the same vector dose. 43,44 The safety and efficacy of tail vein administration of scaav over a range of different doses has been described above (summarized in Table 1, section 14). Recently we have tested the safety and efficacy of a simple bolus infusion of scaav2/8-lp1-hfixco into the peripheral vein of 3 nonhuman primates at a dose initially estimated to be 1 x vg/kg using a single vector stock. 45 One of these animals had high titer antibodies to AAV5 although this animal was immunologically naïve to AAV8. In addition, we administered the same dose of AAV5 pseudotyped scaav-lp1-hfixco to two macaques with modest titers of anti-aav8 antibodies to determine if naturally acquired immunity to AAV8 can be overcome by switching serotypes. AAV8 is endemic in macaques and so the answer to this question will shed light on whether antibodies to AAV2, which are endemic in humans, can be circumvented by switching AAV serotypes. As with AAV2 administration in humans, the scaav viral genome was detectable in rhesus plasma for 24 hours after vector administration but not in other bodily secretions including urine or saliva. Despite this period of viremia, vector administration by the peripheral venous route was well tolerated without perturbation of acute phase reactants or liver transaminases over a period of 6 weeks after gene transfer, even in animals with pre-existing immunity. Transgene expression profiles in all 5 macaques were similar with mean steady state hfix levels of 23±3 and 22.4±1% of normal in the scaav8 (Figure 4) and scaav5 cohorts, respectively,

17 which is comparable to that achieved after liver targeted administration. This is substantially higher than the levels required for amelioration of the bleeding diathesis in HB patients. Similarly transduction of the liver did not appear to be significantly influenced by route of administration or vector pseudotype. Figure 5: Limited biodistribution analysis. 1 g of genomic DNA was isolated at 1 month from the indicated organs following either liver targeted (top two panels) or peripheral vein (lower three panels) administration of 1x10 12 vg/kg of scaav2/8-lp1- hfixco and subjected to PCR amplification using primers unique to hfixco designed to amplify a 617bp product. Integrity of DNA was determined by amplifying a 604bp region of the rhesus actin gene and is shown at the bottom of each panel. Limited biodistribution studies demonstrated that the vast majority of the vector genomes were in the liver, although almost all other tissues contained detectable levels of vector genomes. This pattern of vector distribution is similar to that observed after delivery of vector into the mesenteric vein (Figure 5). A sensitive RT-PCR assay revealed that FIX transcript was only detectable in the liver, suggesting that the LP1 promoter in our vector restricts expression to the liver. Extensive biodistribution studies were performed in an animal that was sacrificed almost 1.5 years after mesenteric vein delivery of 1x10 12 vg/kg of scaav2/5-lp1-hfixco. As shown in Figure 6 the AAV transgene was detected in most organs analysed by qpcr with the liver harbouring the highest number of AAV genomes at 15 vector genomes/cell. This was between fold higher than that observed in the small group of organs with intermediate transgene copy number and substantially higher (>400 fold) than that observed in the other organs. Our recent results have shown that the hybridizing signal in an in situ DNA hybridization analysis with our codon optimized hfix probe is localized in a single spot within the nucleus within 5 to 6 weeks following vector injection. This pattern differs from that observed in mouse liver in that at 1 month following vector injection we find multiple hybridizing spots dispersed throughout the nucleus in mouse specimens. Using a rhesus bacterial artificial chromosome (BAC) probe, we find 2 hybridizing signals in approximately two-thirds of hepatocytes and 4 hybridizing signals in the remaining third, suggesting that these cells have progressed through S phase. The same proportions of hepatocyte nuclei have a single or double hybridizing signal with the hfix probe, respectively. We have been able to develop a modification of the ligationmediated PCR technique to study concatameric and integrated forms of AAV following delivery of vector into murine and macaque liver in the absence of selection. We have analyzed over 100 clones thus far. As in mice, 62 the majority of these clones reveal only vector sequences in various concatameric forms, including complete head-to-tail and tail-to-tail junctions. Putative AAV host integration junctions were identified in 5% of the clones. These integration sites appear to be 11

18 located on chromosomes 1, 4, 9 and 11. Therefore based on our initial results and the published results of others, 63 it appears that AAV integration in primates is uncommon. Copy number (vg/dc) High Intermediate Low Copy number (vg/dc) Liver Spleen lymph node thymus adrenal kidney bladder prostate testis gall bladder pancreas colon adrenal Ileum Spleen Jejun heart stom lungs brian lymph heart node skl thymus muscle Copy number (others) (vg/dc) kidney brian bladder prostate testis gall bladder pancreas colon Ileum lymph node thymus kidney Jejun bladder stom prostate lungs There are important practical implications of our results. 1) Since peripheral vein infusion of vector is a technically less complex and risky procedure than selective catheterization of the hepatic artery or mesenteric vein, but equally effective at targeting our vector to the liver, peripheral vein administration is the preferred method for delivering vector in patients with a severe bleeding diathesis. 2) In the context of a clinical trial, these results indicate that there is little cross-reactivity between serotypes, as we have achieved efficient gene transfer in macaques with pre-existing anti-aav antibodies by switching serotypes. Additionally in participants who develop neutralising anti-aav antibodies after receiving a subtherapeutic dose of scaav8, our results suggest that successful and efficient transduction can be achieved with scaav5, should our approach ultimately prove to be successful. 3.5 Rationale for the proposed study Copy number (vg/dc) skl muscle testis gall bladder pancreas This study is designed to evaluate the safety and potential efficacy of a single bolus infusion of scaav2/8-lp1-hfixco into the peripheral vein of patients with severe HB. Successful gene therapy for hemophilia offers the potential of effective lifetime prevention of bleeding and its complications, which can transform the life experience for all sufferers of this disorder, not just the fortunate 20% who live where replacement therapy is affordable and available, but throughout the world. At least 6 clinical trials of gene therapy for hemophilia have already been carried out on this basis. 13,37-39 Importantly the therapeutic goal for HB is modest as stable expression of FIX at 1% of physiological levels would ameliorate the bleeding phenotype. This goal is clearly potentially achievable as a result of our recent advances. The virtues of our vector system together with rationale for vector dose range and route of administration are summarized below Rationale for use of AAV serotype Figure 6: Biodistribution of vector genomes 22 months after administration of raav vector particles. colon Ileum Jejun stom lungs skl muscle AAV is currently the vector of choice for gene therapy of HB and other disorders affecting the liver because: 12

19 Amendment It is able to mediate high level stable transduction of hepatocytes following a single intravenous administration of vector in a variety of animal models including nonhuman primates It has the best safety profile amongst vectors of viral origin as: a. Wild type AAV is not associated with any disease pathology in humans. b. AAV vectors are completely devoid of viral genes and therefore the risk of inducing an immunological response to the transduced cells is, in principle, reduced. c. Despite the high prevalence of wild type AAV in humans and its ability to integrate in a site specific 64,65 as well as random manner, infection with this virus is not associated with oncogenesis. Instead, the published data suggest that wild-type AAV may protect against the effects of certain onocogenes Additionally, recombinant AAV vectors are thought to mediate expression mainly from episomally retained transgene copies 43,44 and therefore the potential risk of insertional mutagenesis following AAV mediated gene transfer in patients with hemophilia may be further reduced. 3. AAV8 capsid pseudotyped vectors are preferred for gene therapy of HB because: a. AAV8 was isolated from a non-human primate 57 and therefore it seems less likely that preexisting antibodies will limit liver transduction in humans by a raaa8 vector. We have screened 40 Factor IX deficient individuals and found only one with AAV8 neutralizing antibodies. We have found an incidence of AAV8 antibody of 5% in 650 blood donors or hospitalized individuals. However, a recent report from the Wilson lab suggests that use of a more sensitive, in vitro assay than that used in that lab previously, results in the detection of neutralizing antibodies in 55% of volunteers 69. In vitro assays to detect neutralizing antibodies are inherently problematic because of the low capacity for AAV8 to transduce cultured cells. In our studies, we have relied on detection of neutralizing antibodies by injection of human serum into mice followed by injection of a challenged dose of an raav8 vector. This assay has reliably allowed us to select 22 rhesus macaques without neutralizing antibodies that responded appropriately to our raav2/8 FIX vector b. AAV8 capsid uncoats more rapidly after gene transfer 42 which may, in principal, reduce the opportunity for evoking a T cell immune response as AAV8 capsid protein may not be available for as long as AAV2 capsid for presentation by antigen presenting cells after vector administration. However, early exposure of capsid peptides and/or a higher concentration on the hepatocyte surface as a consequence of more rapid uncoating may make AAV8 more immunogenic than AAV2. It is important to note in this respect that we have not seen delayed transaminitis (at 4-10 weeks after gene transfer) or significant decline of hfix expression in any of the macaques successfully transduced with AAV8 based vectors despite the endemic nature of this virus in nonhuman primates. 4. Importantly our modified vector design restricts expression to the liver while enabling packaging of self complementary genomes in a single capsid. This feature of our vector has significantly improved the potency of AAV without compromising safety. Based on our preclinical data, there is real opportunity to effect therapeutic gene transfer using lower doses of vectors which has significant practical and safety advantages of its own: 13

20 a. Pressure on vector production would be considerably reduced, b. Inadvertent gene transfer into nonhepatic tissues, as occurred in HB patients after liver targeted delivery of AAV2, 39 would be minimized as the degree of extra-hepatic spill-over is directly proportional to the dose of vector administered c. Direct and possibly immune mediated vector related toxicity such as the transient hepatitis observed in humans in a recent HB study 39 may be reduced because of exposure to a smaller capsid protein load. 5. Finally to facilitate transition of AAV 8 vectors to the clinic we have developed a scaleable method that enables purification of vector particles to a level suitable for use in human. 59 This combined with the dedicated GMP vector production facility at St Jude Children s Research Hospital provides a real opportunity to fulfil our goals Rationale for the vector doses proposed We propose to test three dose levels, 2 x 10 10, 6 x and 2 x vg/kg body weight. These doses reflect our best current estimate of the minimum level necessary to potentially achieve therapeutic levels (2 x vg/kg). We seek to minimize the risk of significant toxicity by giving the initial cohort the lowest dose that may, in principle, be therapeutic. Selection of the minimal dose is based primarily on results obtained in a cohort of 3 monkeys described in our recently published article in Blood 45 and several animals injected subsequently. At the time the single lot that was used for these studies was titered, we were using a slot blot method. The lot had an estimated titer of 6.7 x /ml and this measurement was used to give the estimated dose of 1 x vg/kg. The monkeys 2 x 10 had an average Factor IX level of 23%. 12 vp/kg Since these studies were completed 2 x vp/kg three years ago, we have switched to a qpcr method for titering because it has 6 x vp/kg 2 x vp/kg Figure 7: Dose finding study with clinical grade vector in rhesus. At least three macaques were treated in each dose level. Note that expression is reported using a log scale. Also shown are the standard errors for expression at each time point. been more reproducible. The qpcr titer of the lot given to the three monkeys was 8.5 x /ml, a value which is relatively close to the estimated slot blot titer of 6.7 x /ml on which the administered dose was calculated. However, at the time that this measurement was made, we were using a super coiled plasmid as the standard for the qpcr assay. We have learned in the interim that a linear plasmid gives more reproducible standard curves although the signal intensity per μg of standard is significantly higher when the plasmid is linearized compared to closed circular plasmid. Based on the current standard curves, the estimated titer of the lot used for the 3 monkeys would have been 1 x /ml had a linearized standard been used. If one accepts this value, then the animals received approximately 2 x vg/kg, based on our current titering methodology. We have now completed production of a dose finding lot in the GMP facility using exactly the same methodology that we propose to use for production of the clinical lot. Four additional monkeys have been given an estimated dose of 2 x vg/kg based on the qpcr assay. As shown in Figure 7, all animals had levels of hfix of between 20-40%. Four monkeys have been given a 14