Long-Term AAV-Mediated Factor VIII Expression in Nine Hemophilia A Dogs: A 10 Year Follow-up Analysis on Durability, Safety and Vector Integration

Abstract

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.

Long-Term Delivery of an Anti-SIV Monoclonal Antibody With AAV

“…Remarkably our animal 84-05 never received any immunosuppressant. The hemophilia gene therapy arena also has good examples of long-term delivery with AAV for up to a decade of Factor IX in hemophilic humans (59) and of Factor VIII in hemophilic dogs (60). For more examples on long-term delivery with AAV of hemophilia factors, please see the following references (61)(62)(63)(64)(65)(66)(67)(68).…”

Guest Editor: G. Castaman GENE THERAPY FOR HEMOPHILIA: FACTS AND QUANDARIES IN THE 21ST CENTURY

“…Although the vector infusion is given only once, patients will need to be followed for recognition of potential unexpected findings, given the lack of preclinical models that recapitulate the transient liver toxicity due to cellular immune response to vector in humans. In contrast to the loss of transgene activity in the BMN-270 trial, increasing gene expression has been seen in long-term follow-up of a canine HA model, 90 the reasons for these discrepancies continue to be determined. Further, previous retroviral and AAV studies using a CMV promoter in mice and dogs were complicated by gene silencing events of transgene expression; 91 , 92 this has not been reported with the use of liver-specific promoters.…”

A non-viral genome editing platform for site-specific insertion of large transgenes

“…AAV and other vectors have been widely used as a carrier for the truncated version of the F8 gene; however, certain safety issues persist [ 64 , 66 – 68 ]. An example of remaining adverse virus-mediated oncogenic effects has been concretely pointed in a canine model of hemophilia administered with AAV gene therapy in a decade long follow-up study, wherein DNA payload insertion was evidenced near genes that regulate cell growth [ 69 , 70 ]. Many precedented ex vivo pioneering studies [ 71 – 76 ] have also been attempted to either genetically correct or introduce a separate functional copy of truncated F8 into different types of cells by lentiviral, transposons and CRISPR Cas systems with a fair degree of success, yet still requiring significant improvements.…”