Abstract Gene transfer for hemophilia A offers the potential for a one-time disease altering treatment, eliminating the risk of bleeds while freeing patients from the burden of lifelong chronic therapy. SPK-8011 consists of a bioengineered AAV capsid expressing B domain-deleted factor VIII (FVIII) under the control of a liver-specific promoter. In pre-clinical studies, we showed a dose-dependent increase in circulating FVIII levels in non-human primates infused with SPK-8011. We conducted a Phase I/II study of SPK-8011 in 12 men (ages 18-52 years) with severe (n=11) or moderately severe (n=1) hemophilia A. Prior to gene therapy, 8/12 subjects were on prophylaxis, and 4/12 received on-demand treatment. Subjects were enrolled in 1 of 3 dose cohorts, 5E11 vg/kg (n=2), 1E12(n=3), or 2E12(N=7). Safety analysis showed no inhibitor formation. A single serious adverse event (SAE) was reported, associated with an immune response to AAV capsid characterized by simultaneous decline in FVIII, transaminase elevation peaking at Grade 2, and development of positive IFN-g ELISPOTs to capsid was observed beginning at week 6.5 after vector infusion. The asymptomatic transaminase elevation did not respond promptly to initiation of oral steroids and the subject received two infusions of IV methylprednisolone in hospital, thereby fulfilling SAE criteria. The SAE has resolved. All vector doses led to expression of FVIII levels adequate to prevent bleeding and allow cessation of prophylaxis. Across the 12 subjects at 3 doses, there was a 97% reduction in annualized bleeding rate (ABR), and a 97% reduction in annualized infusion rate (AIR). In the 5E11 dose cohort, mean FVIII levels beginning 12 weeks post vector infusion are 13%, with no bleeding events, no elevated transaminase levels, no use of steroids, and stable FVIII expression out to 66 weeks (ongoing). In the 1E12 dose cohort, mean FVIII levels are 15% beginning at 12 weeks post-infusion and stable out to 46 weeks (ongoing). The first subject in the 1E12 dose infused a single dose of factor concentrate for a spontaneous joint bleed at day 159, and the second received multiple infusions for a traumatic bleed beginning at day 195. Declining FVIII levels triggered initiation of a course of tapering steroids in both subjects, at 12 and 7 weeks post vector infusion respectively, which led to stabilization of FVIII levels. The third subject has had no bleeding and did not receive factor infusions or steroids. In the 2E12 (highest) dose cohort, 5/7 subjects currently have FVIII levels 16-49%; their mean FVIII level beginning 12 weeks post-infusion is 30%. No bleeds have been reported among these subjects beginning 4 weeks post vector infusion. Additionally, 5/7 subjects in the 2E12 dose cohort received a course of steroids, initiated at 6-11 weeks post vector infusion, for one or more of the following: declining FVIII levels, rise in ALT above subject baseline, or elevated IFN-g ELISPOTs to AAV capsid. Steroid initiation normalized ALT levels and extinguished the ELISPOT signal in all cases; 2 subjects showed limited stabilization of FVIII levels, which fell to <6% likely due to the immune response. For one of these, no bleeds have been reported through 12 weeks of follow up; the other has had 4 bleeds through 37 weeks of observation. Our data indicate that the kinetics of SPK-8011 expression are similar to those observed with investigational SPK-9001 for hemophilia B. All subjects demonstrated durable transgene expression for up to 66 weeks post vector administration (data cutoff 7/13/18). On cumulative follow up of 345 weeks, SPK-8011 demonstrated a favorable safety profile with no evidence of FVIII inhibitor formation, a single SAE, and 2/12 subjects who experienced ALT elevation above the upper limit of normal that resolved with steroid initiation. Data from the 5E11 (lowest) dose cohort are consistent with published natural history data indicating FVIII:C 12% is adequate to prevent spontaneous bleeding events. Given that 2 subjects in the 2E12 dose cohort lost some FVIII expression, which then stabilized on steroids, and 5/7 subjects in this cohort required steroids, prophylactic steroids may be warranted. We conclude that infusion of SPK-8011 in 12 subjects with severe or moderately severe hemophilia A resulted in safe, durable, dose-dependent FVIII expression resulting in an excellent preliminary efficacy profile with an overall 97% reduction in ABR and AIR. Disclosures High: Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. George:University of Pennsylvania: Equity Ownership; Pfizer: Consultancy. Ragni:CSL Behring: Research Funding; Alnylam: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sangamo: Research Funding; Shire: Research Funding; Biomarin: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Research Funding; Bioverativ: Consultancy, Research Funding; MOGAM: Membership on an entity's Board of Directors or advisory committees; SPARK: Consultancy, Research Funding. Croteau:Novo Nordisk: Consultancy; Octapharma: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Spark Therapeutics: Research Funding; Tremeau Pharmaceuticals: Consultancy; Genetech: Consultancy, Research Funding; CSL-Behring: Consultancy; Catalyst Biosciences: Consultancy; Bioveritiv: Consultancy; Biomarin: Consultancy; Bayer: Consultancy; Baxalta/Shire: Consultancy, Research Funding. Joseney-Antoine:Spark Therapeutics: Employment. Macdougall:Spark Therapeutics: Employment. Tompkins:Spark Therapeutics: Employment. Hait:Spark Therapeutics: Employment. Couto:Spark Therapeutics: Employment. Bassiri:Spark Therapeutics: Employment. Valentino:Spark Therapeutics: Employment. Carr:Spark Therapeutics: Employment. Hui:Spark Therapeutics: Employment. Wachtel:Spark Therapeutics: Employment. Takefman:Spark Therapeutics: Employment. Mingozzi:Spark Therapeutics, Inc.: Employment. Anguela:Spark Therapeutics, Inc.: Employment. Reape:Spark Therapeutics: Employment.

BACKGROUND The prevention of bleeding with adequately sustained levels of clotting factor, after a single therapeutic intervention and without the need for further medical intervention, represents an important goal in the treatment of hemophilia. METHODS We infused a single-stranded adeno-associated viral (AAV) vector consisting of a bioengineered capsid, liver-specific promoter and factor IX Padua (factor IX-R338L) transgene at a dose of 5x10(11) vector genomes per kilogram of body weight in 10 men with hemophilia B who had factor IX coagulant activity of 2% or less of the normal value. Laboratory values, bleeding frequency, and consumption of factor IX concentrate were prospectively evaluated after vector infusion and were compared with baseline values. RESULTS No serious adverse events occurred during or after vector infusion. Vector-derived factor IX coagulant activity was sustained in all the participants, with a mean (+/- SD) steady-state factor IX coagulant activity of 33.7 +/- 18.5% (range, 14 to 81). On cumulative follow-up of 492 weeks among all the participants (range of follow-up in individual participants, 28 to 78 weeks), the annualized bleeding rate was significantly reduced (mean rate, 11.1 events per year [range, 0 to 48] before vector administration vs. 0.4 events per year [range, 0 to 4] after administration; P = 0.02), as was factor use (mean dose, 2908 IU per kilogram [range, 0 to 8090] before vector administration vs. 49.3 IU per kilogram [range, 0 to 376] after administration; P = 0.004). A total of 8 of 10 participants did not use factor, and 9 of 10 did not have bleeds after vector administration. An asymptomatic increase in liver-enzyme levels developed in 2 participants and resolved with short-term prednisone treatment. One participant, who had substantial, advanced arthropathy at baseline, administered factor for bleeding but overall used 91% less factor than before vector infusion. CONCLUSIONS We found sustained therapeutic expression of factor IX coagulant activity after gene transfer in 10 participants with hemophilia who received the same vector dose. Transgene-derived factor IX coagulant activity enabled the termination of baseline prophylaxis and the near elimination of bleeding and factor use.

Background : Hemophilia A therapy is currently based on intravenous administration of exogenous FVIII protein either on demand to treat bleeding or prophylactically to prevent bleeding. Prophylaxis has revolutionized health outcomes in hemophilia by significantly reducing the frequency of bleeding, preventing the development of arthropathy, improving health-related quality of life and enabling affected individuals to increase participation in physical activities enhancing psychosocial outcomes. The burden of repetitive infusions and the time required to perform them may be partially responsible for a strikingly high rate of non-compliance. A recent multi-center study revealed only 43% of individuals with hemophilia adhered to their prophylactic regimen (Schrijvers 2016) putting improved outcomes in jeopardy. Recombinant adeno-associated viral (rAAV) vectors have been in development for >25 years. No major safety concerns have emerged in the >100 previously conducted rAAV gene transfer clinical trials. A single administration of rAAV vector encoding human coagulation F8 or F9 gene may result in sustained expression of therapeutic factor activity levels sufficient to reduce or eliminate the need for exogenous factor infusions (Pasi 2017; George 2017). Objectives: 1) To safely obtain consistent, predictable and sustained FVIII activity (FVIII:C) >12% adequate to prevent spontaneous bleeding without the need for prophylactic FVIII infusions, manipulation of normal coagulant or anticoagulant pathways or increased thrombotic risk; 2) Minimize a dose-dependent capsid immune response by using the lowest possible vector dose that produces clinically relevant improvements in FVIII:C. Methods : SPK-8011 is a recombinant AAV vector composed of a bio-engineered capsid (AAV-Spark200) with liver specific enhanced tropism and a codon-optimized expression cassette that encodes the SQ-FVIII variant of a B-domain-deleted (BDD) human F8 gene (Lind 1995). This Phase 1/2 study is an open-label, non-randomized, dose-escalation study of SPK-8011 with a starting dose of 5x10 11 vg/kg. The study is evaluating the safety, tolerability and efficacy of a single intravenous infusion of SPK-8011 in up to 18 adult men with hemophilia A (endogenous FVIII activity levels ≤2% of normal). Data on bleeding and factor infusions in the year prior to enrollment are retrospectively compiled. Laboratory values, bleeding frequency, and FVIII consumption are prospectively evaluated following vector infusion. Results : As of 8/1/17, we infused 3 subjects with SPK-8011, two at a dose of 5 x10 11 vg/kg and one at a dose of 1x10 12 vg/kg. Infused subjects were adult males ages 34-52 years with baseline FVIII:C 11 vg/kg have FVIII:C of 11% and 14% at 23 and 12 weeks, respectively, after infusion (Figure 1). Subject 3 is two-weeks post infusion with 1x10 12 vg/kg and has not yet reached peak steady-state transgene expression. No subjects have experienced hepatic transaminase elevation, a decline in FVIII:C or required steroid intervention. ELISPOT reactivity to Spark200 capsid peptides revealed a transient low-level positivity to two different AAV peptides at week 3 and 4 in the second subject. By week 5 all ELISPOTs were negative. The positive ELISPOTs were not associated with a decrease in FVIII:C or increase in hepatic transaminases. No participants have developed a FVIII inhibitor. All subjects have discontinued FVIII prophylaxis. In the cumulative 258 days of follow up, there have been no vector or procedure-related adverse events. Conclusion : Our preliminary results from the ongoing Phase 1/2 study of SPK-8011 demonstrate FVIII:C levels in the range of 12%, sufficient in the trial to date to prevent spontaneous bleeding without the need for exogenous factor infusions, and with no evidence of a cellular immune response to transduced hepatocytes. Therapeutic transgene-derived FVIII:C was achieved at a 120-fold lower vector dose than that previously reported (Pasi 2017) in another ongoing AAV gene transfer trial for hemophilia A. Transduction in the participant dosed at 1x10 12 vg/kg tracks at higher levels than observed at the same time point for participants in the low dose cohort, providing early evidence of a dose-response. No adverse events or safety concerns have been observed in the first 258 days of subject exposure to SPK-8011. Disclosures George: Spark Therapeutics: Other: Principal Investigator of Ongoing Phase I/II Gene Therapy Trials for Hemophilia A and B; Pfizer: Consultancy. Ragni: Alnylam, CSL Behring, BAYER, Biomarin, Biomarin, Bioverativ, Genetech/Roche, Pfizer, Shire, SPARK: Research Funding; A Anylam, Biomarin, Bioverativ, Shire: Honoraria. Cuker: Spark Therapeutics: Research Funding; T2 Biosystems: Research Funding. Cole: Spark Therapeutics: Employment. Wright: Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. Chen: Spark Therapeutics: Employment. Hui: Spark Therapeutics: Employment, Equity Ownership. Wachtel: Spark Therapeutics: Employment. Takefman: Spark Therapeutics: Employment. Couto: Spark Therapeutics: Employment. Reape: Spark Therapeutics: Employment. Carr: Spark Therapeutics: Employment. Anguela: Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. High: Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Abstract Background: Earlier data demonstrated long-term expression of factor IX (mean FIX:C ~5.1%) following AAV8-mediated gene transfer at 2 x1012 vg/kg in hemophilia B (Nathwani et al., 2014). While the clinical improvement imparted by stable FIX levels is clear, these levels of expression fall short of trough values obtained by long-acting FIX prophylaxis (Santagostino et al. 2016), and of natural history data suggesting that levels of ~12% are required to eliminate spontaneous hemarthroses (den Uijl et al. 2011). Achieving higher levels of FIX:C with dose escalation has not been possible without eliciting a dose-dependent, capsid-specific immune response that may prevent sustained expression and efficacy (Mingozzi et al. 2007, Monahan et al. 2015). We sought to develop a highly efficient vector capsid and expression cassette that could be administered at low doses to achieve hemostatic FIX expression without need for immunosuppression. Methods: The investigational product, SPK-9001, utilizes a bioengineered AAV capsid (Spark100) with liver specific tropism. The prevalence of neutralizing antibodies (NAb) to Spark100 among sampled hemophilia B sera was 40% (Anguela et al. 2015). The expression cassette is a codon-optimized, single-stranded transgene encoding FIX Padua, a naturally occurring variant with a single amino acid substitution (R338L) that confers ~8-fold greater specific activity compared to wild-type FIX (Simioni et al. 2009). Data on bleeding and factor infusions in the year prior to enrollment were retrospectively compiled. Laboratory values, bleeding frequency, FIX consumption, changes in activity and quality-of-life via Haem-A-QoL were prospectively evaluated after vector infusion. Results: We enrolled 9 subjects, of whom 2 failed screening for liver fibrosis and 7 were infused with SPK-9001 at a dose of 5 x1011 vg/kg. Infused subjects were adult males ages 18-52 years with baseline FIX:C =2% and Spark100 NAb titer of <1:1 or 1:1. Table 1 outlines infused subject data with a follow up interval of >2-34 weeks after vector infusion. Figure 1 outlines subject vector-derived FIX:C for the first 12 weeks. There have been no vector or procedure related adverse events. Steady-state FIX expression is reached by 12 weeks after vector infusion, resulting in a mean FIX:C of 32.3% ±6.5%. To date, no subjects required immunosuppression or demonstrated evidence of a cytotoxic immune response (characterized by loss of FIX activity, elevation of transaminase values >/=1.5-times the upper limit of normal, and positive IFN-gammaELISPOT response to capsid peptides). No subjects developed a FIX inhibitor or demonstrated ELISPOT reactivity to the FIX (R338L) gene product. Subject 3 infused with FIX concentrate for a suspected ankle bleed 2 days after vector infusion. Beyond this, no subjects required factor or experienced any bleeding events. The 4 subjects previously maintained on prophylaxis safely stopped without break-through bleeding. As of today (cumulative 724 days post vector infusion), total factor consumption was reduced by 543,589 IU, tantamount to a cumulative savings of $1,182,298 USD.Six of 7 subjects report increased physical activity and improved quality of life. Conclusion: As of 8/4/2016, we report the highest and most consistent levels of sustained vector-derived FIX:C following FIX gene transfer. Levels of FIX:C achieved by SPK-9001 permitted termination of prophylaxis, prevention of bleeding, and nearly complete cessation of factor use. Despite the heterogeneity in subjects with respect to presence and extent of hemophilic arthropathy, age, and co-morbidities, consistency of transgene expression and clinical outcomes have been observed in all participants studied to date. A vector dose of 5x1011 vg/kg is the lowest dose currently reported in hemophilia gene transfer trials; the absence of any observed CD8+ T cell immune response supports the hypothesis that lowering the dose can reduce or eliminate the risk of a capsid-specific immune response and maximize efficacy. In summary, preliminary data suggest SPK-9001 safely and consistently produces sustained elevation in FIX:C levels sufficient to prevent spontaneous hemarthroses without the need for factor consumption or immunosuppression. Disclosures Ducore: Octapharama: Membership on an entity's Board of Directors or advisory committees; LFB: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Baxalta (Shire): Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees. Cuker:Biogen-Idec: Consultancy, Research Funding; T2 Biosystems: Research Funding; Genzyme: Consultancy; Stago: Consultancy; Amgen: Consultancy. McGuinn:Spark: Research Funding; Biogen: Research Funding; Novo Nordisk: Research Funding; Baxalta: Research Funding. Luk:Spark Therapeutics, Inc.: Employment. Wright:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties: SPK-9001. Chen:Spark Therapeutics, Inc.: Employment. Hui:Spark Therapeutics, Inc.: Employment. Wachtel:Spark Therapeutics, Inc.: Employment. Urich:Spark Therapeutics, Inc.: Employment. Takefman:Spark Therapeutics, Inc.: Employment. Couto:Spark Therapeutics, Inc.: Employment. Carr:Pfizer, Inc.: Research Funding. Anguela:Spark Therapeutics, Inc.: Employment, Patents & Royalties: SPK-9001. High:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties: SPK-9001.

Clinical development of gene therapy began over 20 years ago. Currently there are more than 320 ongoing gene therapy clinical trials that are regulated by the Office of Cellular, Tissue, and Gene Therapies (OCTGT) in the Center for Biologics Evaluation and Research of the US Food and Drug Administration (FDA). The FDA’s mission is to advance the public health by helping to speed innovations that make medicines safer and more effective. The diverse gene therapy products in clinical development for a wide range of disorders offer an opportunity for the FDA to fulfill that mission. The recent history of gene therapy has been a mixture of promise and disappointment. Gene therapy products often initially appear promising because they reflect strong scientific rationale when they are proposed as treatments for specific, well-defined genetic disorders. In addition, some gene therapy products have been well publicized because they offer hope for the treatment of rare, disabling, or life-threatening disorders. Despite strong scientific rationale and publicity-generated enthusiasm, the field has not yet produced any products that have gone through clinical development and provided the evidence of safety and effectiveness necessary for FDA marketing approval. In addition, the death of Jesse Gelsinger in 1999 resulted in a period of circumspection in the field of gene therapy. Subsequent enthusiasm generated by exciting scientific results from a trial for treatment of X-linked severe combined immunodeficiency (X-SCID) was tempered by reports of insertional mutagenesis.1 More recently, the field has been rejuvenated by several reports of encouraging results in clinical trials. Many of these results were presented at the National Institutes of Health Gene Therapy Symposium that took place in September 2011.2 The FDA shares the field’s optimism based on the scientific data reported from these recent clinical trials. The OCTGT is committed to working with sponsors as these new products make their way from early-phase trials into confirmatory phase III trials and to marketing approval. Gene therapies are novel and complex products that can offer unique challenges in product development. Hence, ongoing communication between the FDA and stakeholders is essential to meet these challenges. For sponsors of clinical trials, the OCTGT encourages both formal meetings (e.g., at the end of phase I, end of phase II, or pre-biologics license application) and informal communications. Such communications are necessary to ensure that the overall clinical development program and specific clinical trials are designed to provide the necessary evidence of safety and effectiveness. Moreover, such communications are critical to ensuring that product development addresses the good manufacturing practice requirements for a licensed product. Investigators and trial sponsors are encouraged to visit the FDA website on the regulation of gene therapies (http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/). This site provides general information about the OCTGT and the regulatory process, as well as a link to OCTGT Learn, a series of educational “webinars” specific to products regulated by the OCTGT. The FDA recognizes that many gene therapy products are being developed for rare disorders and that clinical development of a product for these indications can present unique challenges. The agency’s regulations provide the flexibility necessary to meet these challenges and facilitate the development of therapeutics for rare disorders.3,4,5 However, consistent communication between sponsors and the OCTGT throughout drug development, as outlined above, is essential to optimizing the development of gene therapies for rare disorders. In addition, manufacturers of gene therapy products for rare disorders are eligible to apply for orphan drug designation by the FDA’s Office of Orphan Product Development; such designation comes with tax credits, marketing incentives, and the opportunity to apply for support through the Orphan Products Grants Program. Because gene therapy products are being developed around the world, the FDA is engaged in a number of international harmonization activities in this area. The agency works with the International Conference on Harmonisation on Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). Specifically, the FDA has participated in the ICH Gene Therapy Discussion Group and published three relevant documents (on inadvertent germline integration, oncolytic viruses, and virus and vector shedding), which are available on the ICH website (http://www.ich.org/products/consideration-documents.html). There is also a formal cooperation and confidentiality arrangement between the FDA and the European Medicines Agency (EMA). Through this arrangement, the FDA and EMA have bimonthly meetings to share views on regulatory approaches to both general and specific issues in gene therapies (referred to as “advanced-therapy medicinal products” in Europe). Additionally, the FDA and the EMA participate in a program to provide parallel scientific advice. Its goal is to provide a mechanism for EMA and FDA assessors and sponsors to exchange views on scientific issues during development of new medicinal products. The OCTGT will continue to work with sponsors, investigators, and other regulatory bodies to navigate the challenges of therapeutic development. Despite the setbacks of the past, the OCTGT shares the enthusiasm of the field and is confident that ongoing clinical investigations will lead to commercially available gene therapy products that are safe and effective and advance the public health.