ALBERT

Description: Hemophilia is an attractive target for gene therapy, since activity levels as low as 1% to 2% of normal are beneficial and levels of ~5% prevent spontaneous bleeding. Our goal was to provide a single treatment that permanently enables hepatic production of therapeutic levels of hFIX activity to decrease or potentially eliminate the need for prophylactic treatment in hemophilia B patients. We performed targeted in vivo genome editing using 1) two zinc finger nucleases (ZFNs) targeting intron 1 of the albumin locus, and 2) a human F9 donor template construct. The ZFNs and donor template are encoded on separate hepatotropic adeno-associated virus serotype 2/6 (AAV2/6) vectors injected intravenously, resulting in targeted insertion of a corrected copy of the hF9 gene into the albumin locus in a proportion of liver hepatocytes. The albumin locus was selected as a "safe harbor" as production of this most abundant plasma protein exceeds 10 g/day, and moderate reductions in those levels are well-tolerated. These genome edited hepatocytes produce normal hFIX in therapeutic quantities, rather than albumin, driven by the highly active albumin enhancer/promoter, to treat hemophilia B; the genetic modification is expected to be sustained even in the face of hepatocyte turnover, making this approach attractive for treating young children with hemophilia before the appearance of significant organ damage. Transformed and primary human hepatocytes transduced in vitro with AAV2/6 encoding human albumin ZFNs and a promoterless hF9 transgene were shown to secrete hFIX. Extensive molecular analyses demonstrated that this was due to targeted integration of the hF9 transgene at the albumin locus and splicing of this gene into the albumin transcript. By employing AAV2/6 delivery of murine-specific ZFNs in vivo, stable levels of hFIX were observed in blood of mice injected with the albumin ZFNs and hF9 transgene donor. C57BL/6 mice were administered vehicle (n=20) or AAV2/6 vectors (n=25) encoding mouse surrogate reagents at 1.0 x1013 vector genome (vg)/kg via tail vein injection. ELISA analysis of plasma hFIX in the treated mice showed peak levels of 50-1053 ng/mL that were sustained for the duration of the 6-month study. Analysis of FIX activity from mouse plasma confirmed bioactivity commensurate with expression levels. Next, we report the feasibility of this approach in non-human primates (NHPs), showing that a single intravenous co-infusion of AAV2/6 vectors encoding the NHP targeted albumin-specific ZFNs and a human F9 donor at 1.2x1013 vg/kg (n=5/group) resulted in 〉50 ng/mL (〉1% of normal) in this large animal model. The use of higher AAV2/6 doses (up to 1.5x1014 vg/kg) yielded plasma hFIX levels up to 1000 ng/ml (or 20% of normal) in several animals and up to 2000 ng/ml (or 50% of normal) in a single animal, for the duration of the study (3 months). The treatment was well tolerated in mice and NHPs, with no significant toxicological findings related to AAV2/6 ZFN + donor treatment in either species at therapeutic doses. Together, these data support a clinical trial to determine if a single co-administration of ZFN and donor AAV vectors is sufficient to enable therapeutic and potentially lifelong production of the clotting factor for the treatment of Hemophilia B. Disclosures Wechsler: Sangamo BioSciences: Employment. Meyer:Sangamo Biosciences Inc: Employment. Spratt:Sangamo Biosciences Inc: Employment. Greengard:Sangamo Biosciences Inc: Employment. Santiago:Sangamo Biosciences Inc: Employment. Sproul:Sangamo Biosciences Inc: Employment. Surosky:Sangamo Biosciences Inc: Employment. Paschon:Sangamo Biosciences Inc: Employment. Dubois-Stringfellow:Sangamo Biosciences Inc: Employment. Ando:Sangamo Biosciences Inc: Employment. Nichol:Sangamo Biosciences Inc: Employment. Rebar:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment.

Description: Hemophilia A, which is caused by a mutation in the Factor 8 (F8) gene resulting in a deficiency or lack of the Factor VIII (FVIII) protein, is the most common inherited bleeding disorder in humans with an estimated worldwide incidence of half a million people. The disorder is X-linked and occurs in approximately 1 in 5,000 males; however there is also a growing appreciation of the impact on carrier females having a single mutant allele, with at least 10% of hemophilia A female carriers having less than normal clotting activity. Even modest increases in Factor V III activity (〉1% of normal) can have a positive impact on patient lives, thus making the disease an ideal candidate for liver-directed gene therapy. Recombinant AAV (rAAV) has been used extensively for nearly 20 years as a gene therapy vector in preclinical and clinical studies where rAAV delivery to non-dividing tissues such as liver, brain and muscle affords stable, long-term transgene expression. However, there has been a lag in the clinical translation of a rAAV gene therapy approach for Hemophilia A/human F8 (hF8) compared to Hemophilia B/human Factor 9 due to poor yields of rAAV encoding a F8 transgene at clinical scale, and a requirement for large doses of rAAV F8 vector to achieve therapeutically relevant levels of circulating human FVIII (hFVIII), with the attendant risk of inducing an AAV-directed immune response requiring transient immunosuppression. To address these issues we optimized a rAAV F8 cDNA vector cassette to improve both virus yields and liver-specific hFVIII expression. The rAAV F8 cDNA vector cassette optimization required multi-factorial modifications to the liver-specific promoter module, hF8 transgene, synthetic polyadenylation signal and vector backbone sequence. This iterative process resulted in improved vector yields at research scale and greater than five-fold improvement in vector yields at clinical scale using our proven manufacturing process. Administration of the optimized rAAV hF8 cDNA packaged in serotype AAV2/6 at a dose of ~7.2E+12 vg/kg to both wild type and Hemophilia A mice resulted in robust circulating hFVIII levels and activity (levels in wild type mice were 241.6% of normal, and activity in Hemophilia A mice were 330.9% of normal). An analysis of hF8 mRNA levels in different tissues following dosage with our optimized vector demonstrated that hF8 expression from the modified promoter module was restricted to the liver. Notably there was a striking impact on hemostasis in the Hemophilia A mice treated with the optimized rAAV hF8 cDNA, with a reduction in bleeding time from 38.3 minutes to 2.5 minutes in treated mice (n = 12, p-value 〈 0.0001), which is in line with bleeding times in wild type mice. Initial studies in non-human primates (NHPs) resulted in supraphysiological levels of circulating hFVIII with mean peak values of 400-600% of normal levels. A follow up dose-ranging study was performed in NHPs with a rAAV2/6 F8 cDNA vector manufactured using our GMP clinical manufacturing process. Administration of vector doses ranging from 6E+11 vg/kg to 6E+12 vg/kg resulted in therapeutic circulating levels of hFVIII that were 5% - 229% of normal levels. The peak circulating hFVIII levels achieved in this dose-ranging study using GMP clinical-scale vector exceeds the levels previously reported in NHPs by several fold on an AAV vector dose basis. The high potency of this enhanced rAAV F8 cDNA cassette could significantly reduce the dose required to achieve therapeutically relevant levels in human subjects and reduce the potential of developing immune responses to AAV capsid requiring immunosuppression. Disclosures Riley: Sangamo BioSciences Inc: Employment. Boonsripisal:Sangamo BioSciences Inc: Employment. Goodwin:Sangamo BioSciences Inc: Employment. Garces:Sangamo BioSciences Inc: Employment. Ballaron:Sangamo BioSciences Inc: Employment. Tran:Sangamo BioSciences Inc: Employment. Kang:Sangamo BioSciences Inc: Employment. Zhang:Sangamo BioSciences Inc: Employment. Meyer:Sangamo BioSciences Inc: Employment. Greengard:Sangamo BioSciences Inc: Employment. Surosky:Sangamo BioSciences Inc: Employment. Ando:Sangamo BioSciences Inc: Employment. Lillicrap:bayer: Research Funding; biogen: Research Funding; CSL: Research Funding; Octapharma: Research Funding; Sangamo Biosciences Inc: Research Funding. Nichol:Sangamo BioSciences Inc: Employment. Holmes:Sangamo BioSciences Inc: Employment.