Generation of a Unique Cohort of Hemophilia A Dogs Tolerant to Human FVIII for Evaluating the Safety and Efficacy of AAV Delivery of Wild Type and Variant Human FVIII

The hemophilia A (HA) dogs have been a valuable model for evaluating the efficacy of novel hemophilia therapeutics. These dogs have a mutation that is analogous to the most common mutation in humans with severe disease, the intron 22 inversion. The advantages of the hemophilia dog model include: (1) it was predictive of the therapeutic adeno-associated viral (AAV) vector dose in human factor IX (hFIX) clinical trials, (2) it is an outbred immunocompetent species, (3) it demonstrates a clinical bleeding phenotype consistent with patients, including hemarthrosis, and (4) it permits long-term investigation of both efficacy and safety. Our previous studies delivering AAV-canine factor VIII (cFVIII) in the HA dogs demonstrated long-term (up to 10 years of follow-up) dose-dependent cFVIII expression without evidence of an immune response to the cFVIII protein. In hemophilia B preclinical studies, the comparable biological characteristics between canine FIX and hFIX allows preclinical results using cFIX to be translated to clinical studies. In contrast, for hemophilia A, our studies of recombinant cFVIII and human FVIII (hFVIII) proteins demonstrate that cFVIII is a more stable protein that has higher biological activity and is secreted better than hFVIII (Sabatino et al. 2009). Thus, expression of cFVIII following AAV delivery does not accurately predict the therapeutic dose of AAV-hFVIII which is relevant for translation to clinical trials. The challenge of administering AAV-hFVIII to the HA dogs is that this expressed xenoprotein (hFVIII) results in inhibitor formation that precludes the ability to measure transgene (hFVIII) expression. We hypothesized that tolerizing HA dogs to hFVIII will (1) permit accurate evaluation of hFVIII expression and thus predict the therapeutic vector dose and (2) allow the evaluation of the potential immune response of AAV8-hFVIII versus a novel hFVIII variant that has increased activity and secretion (Nguyen et al. 2017). In this study we used a neonatal retroviral (RV) delivery approach to tolerize the HA dogs (n=5) to B-domain deleted hFVIII (hFVIII-BDD). HA neonatal male dogs (S28, S29, V06, V26, V27) were treated with the retrovirus (RV-hAAT-hFVIII-BDD-WPRE) (3x10e9 TU/kg) on day 2 of life. The levels of hFVIII expression after RV delivery were 0.3-6% 4 weeks after RV delivery and plateaued after 6 months to 0.8% (S28), 0.3% (S29), 0% (V06), 1.5% (V26) and 1.7% (V27) based on Coatest assay. At 5-6 months of life the dogs (S28, S29, V06) were challenged with hFVIII-BDD (Xyntha; 25IU/kg; I.V.) weekly for 6 consecutive weeks. Samples were collected before and 15 minutes after each protein challenge to demonstrate the successful infusion of the protein. At 4 weeks after the final challenge, no anti-hFVIII IgG1 or IgG2 antibodies were detected consistent with no evidence of an inhibitor. At 4.5 years of age, S28 and S29 had stable expression of 0.5-1% of hFVIII and were rechallenged with hFVIII-BDD (Xyntha; 25IU/kg per week x 6 wks). No anti-hFVIII IgG1 or IgG2 antibodies were detected 4 weeks after the final protein challenge. Thus, these data demonstrate that all of the RV-hAAT-hFVIII-BDD-WPRE treated dogs that have been challenged (n=3) have been tolerant to hFVIII-BDD. Since the goal of this study is to generate a cohort of HA dogs tolerant to hFVIII-BDD to address the efficacy and safety of AAV8-hFVIII-BDD versus a hFVIII-BDD variant, we treated the first dog (S29) 12 weeks after the second series of protein challenges with an optimized AAV vector cassette containing a codon-optimized hFVIII sequence with a modified transthyretin (TTRm) promoter, AAV8-TTRm-hFVIII-BDD (2x10e12 vg/kg). Prior to AAV administration the hFVIII activity was 0.2%. At 8 weeks post AAV administration, the hFVIII activity increased to 3.5%. No anti-hFVIII-BDD IgG1 or IgG2 was detected after AAV-hFVIII administration. These data demonstrate that low levels of sustained hFVIII expression of 0.2-2% up to 4 years post-retroviral delivery were able to induce and maintain tolerance to hFVIII. Overall, these studies demonstrate that the neonatal HA dogs treated with a retrovirus targeting hFVIII expression to the liver are tolerant to hFVIII and provide a unique large animal model to evaluate both efficacy as well as potential immunogenicity of our novel FVIII variants. Disclosures Sabatino:Spark Therapeutics: Patents & Royalties.