Nine hemophilia A dogs were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII (AAV-cFVIII) corrected the FVIII deficiency to 1.9%−11.3% of normal FVIII levels. In two of nine dogs, FVIII activity increased gradually starting about four years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of these integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A while emphasizing the importance of long-term monitoring for potential genotoxicity.

Hemophilia is an X-linked bleeding disorder caused by a deficiency in clotting factor VIII (FVIII)(hemophilia A, HA) or factor IX (FIX)(hemophilia B, HB). While early clinical trials of AAV delivery of FIX for HB have demonstrated stable FIX expression for >8 years, an ongoing clinical trial of AAV-FVIII delivery for HA achieved high levels of transgene expression that unexpectedly declined after 1 year. Here we describe preclinical studies of AAV-canine FVIII (cFVIII) delivery in nine HA dogs with sustained FVIII expression for the duration of the study, as long as 10 years. FVIII was delivered using two delivery approaches: (1) co-administration of two AAV vectors encoding separate cFVIII heavy and light chains driven by the thyroxine binding globulin (TBG) promoter (Two chain approach)(TC) (n=5) at two AAV doses (2.5 x 1013vg/kg; F24, Woodstock, J60) and (1.2 x 1013vg/kg; Linus, H19) or (2) delivery of cFVIII as a single chain driven by the human alpha-1 anti-trypsin (hAAT) promoter (Single chain approach)(SC)(n=4) at two AAV doses (4 x 1013 vg/kg; M50, M06) and (2 x 1013vg/kg; M66, L51) (Sabatino 2011). We demonstrated that both strategies were efficacious; preventing >95% of spontaneous bleeding episodes without toxicity. We now report the long-term follow-up of between 2.2 and 10.1 years for these treated dogs. Dose-dependent cFVIII:C (Coatest SP4 FVIII) was observed. At the final time point, the cFVIII:C was 2.7% (F24), 7.1% (Woodstock), 4.5% (J60), 11.3% (Linus) and 2.5% (H19) for TC dogs. For the SC dogs, the cFVIII:C was 9.4% (M06), 10.3% (M50), 1.9% (L51) and 3.7% (M66). Stable FVIII expression was maintained for seven of the dogs over the course of the study. Two dogs (Linus, M50) had a gradual increase in FVIII:C that began about three years after vector administration and continued for an additional seven years (Linus) and four years (M50), until the termination of the study. Liver function tests, serum alpha-fetoprotein concentrations, fibrinogen levels as well as liver pathology did not suggest altered liver function or tumor development in Linus and M50 compared to the other dogs. Clinically, there was no evidence of malignancy and no tumors were detected at the time of necropsy in any dog. One of the safety concerns for AAV-mediated gene therapy approaches is the potential for AAV integration events to be genotoxic and lead to tumorigenesis. While recombinant AAV primarily remains as an episome, integration events have been observed in mouse models and hepatocellular carcinoma has been observed after neonatal delivery of AAV vectors. In addition, the increase in FVIII expression in Linus and M50 prompted us to investigate integration and clonal expansion as a potential mechanism for these observations. Vector copy number (VCN) analysis was performed on liver samples (5-29 per dog, n=8 dogs) by Q-PCR and detected DNA copy numbers between 0.0 and 7.8 per diploid genome (Fig 1A). We performed integration target site analysis on liver samples (n=3/dog) from six of the AAV-treated HA dogs and naïve HA dogs (n=2) by ligation-mediated PCR, Ilumina paired-end sequencing and analysis using the custom software pipeline, AAVenger. Analysis of the 20 samples identified >2,000 unique AAV integration events (IE). There was a correlation between the DNA copy number and the number of integration events detected. Clonal abundances were estimated by counting the unique genome breaks associated with integration positions, which showed that the maximum clonal abundance ranged from 1 to 138. The integration events were distributed across the canine genome. Clonal expansions were observed with integration near genes previously associated with growth control and transformation in humans (Fig 1B) with the most abundant clones located in DLEU2L (Linus), PEBP4 (J60) and EGR3 (M50). Integration events in EGR3, EGR2, CCND1, LTO1 and ZNF365 were detected in multiple dogs. Validation of integration sites in the most abundant clones was performed using targeted PCR to isolate junction fragments followed by Sanger sequencing. While AAV integration and clonal expansion was observed, the dogs had no evidence for tumorigenesis and it is not clear if the increase in FVIII expression is associated with the clonal expansions detected. Overall, these studies demonstrate long-term sustained FVIII expression for up to 10 years with clonal expansion, but without clinical adverse events after AAV-mediated gene therapy for hemophilia. Disclosures Sabatino: Spark Therapeutics: Patents & Royalties.

Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) has highly increased the feasibility of producing GM mice in research laboratories. However, genetic modification in inbred mouse strains of interest such as C57BL/6 (B6) is still challenging because of their low fertility and embryo fragility. We have successfully generated multiple novel GM mouse strains in the B6 background while attempting to optimize i-GONAD. We found that i-GONAD reduced the litter size in superovulated pregnant females but did not impact pregnancy rates. Natural mating or low-hormone dose did not increase the low fertility rate observed in superovulated B6 females. However, diet enrichment had a positive effect on pregnancy success. We also optimized breeding conditions to increase the survival of small litters by co-housing i-GONAD-treated pregnant B6 females with synchronized pregnant FVB/NJ companion mothers. Thus, GM mice generation was increased by an enriched diet and shared pup rearing with highly fertile females such as FVB/NJ. In the present study, we generated 16 GM mice using a CRISPR/Cas system to target individual and multiple loci simultaneously or consecutively. We also compared homology-directed repair efficiency using different methods for LoxP insertion for conditional knockout mouse production. We found that a two-step serial LoxP insertion, in which each LoxP sequence was inserted individually in different i-GONAD procedures, was a low-risk high-efficiency method for generating floxed mice.

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The molecules RIPK3 and MLKL are required for necroptotic cell death, which is widely thought of as an antiviral mechanism. Here we show that C57BL/6 (B6) mice deficient in RIPK3 or MLKL are as susceptible as WT B6 mice to ECTV lethality after a low-dose intraperitoneal infection and are as resistant as WT B6 mice after ECTV infection through the natural footpad route.