Site-Specific Insertion of Factor VIII Gene Results in Durable Factor VIII Expression in Nonhuman Primates

Hemophilia A is an inherited bleeding disorder arising from a loss of functional factor VIII (FVIII). Exogenous FVIII replacement and bypassing agents have improved outcomes for patients but have not eliminated breakthrough bleeding and are not curative. AAV FVIII gene therapy is limited by a loss of FVIII expression over time. Gene editing technology may overcome these limitations by enabling site specific and permanent integration of a FVIII gene directly into the human genome leading to persistence of the transgene with progressive cell division. We have used metagenomics to identify a novel, highly active and specific CRISPR nuclease (MG29-1) that can cleave DNA and support insertion of a FVIII DNA cassette into a safe harbor location within the first intron of the albumin gene. Our gene editing system is delivered as two components: an AAV delivers a promoterless FVIII DNA cassette and a lipid nanoparticle (LNP) delivers MG29-1 nuclease mRNA and its associated guide RNA. We have demonstrated the feasibility of the FVIII knock-in approach in vitro, in rodents, and in nonhuman primates (NHPs). The strength and constitutive nature of the albumin promoter results in therapeutic levels of FVIII even at low integration rates. Durability of FVIII expression in NHPs over 12+ months was demonstrated using a cynomolgus version of the FVIII gene (cFVIII) to avoid the confounding effects of anti-human FVIII antibodies. Specific quantification of the transgene-derived cFVIII was facilitated through modification of a single amino acid at position 2196 and inactivation of endogenous cFVIII. Three NHPs received an intravenous dose of 2.0E13 vg/kg of the AAV8-cFVIII-F2196K viral vector followed 5 weeks later by a liver-tropic LNP formulation encapsulating the mRNA encoding the MG29-1 nuclease and albumin-targeted guide RNA at 1.0 mg/kg. Plasma was collected starting 14 days post LNP dosing and assayed for safety parameters and/or activity of the transgene derived cFVIII-F2196K using a commercial chromogenic FVIII activity assay with a recombinant human FVIII-BDD protein (Xyntha) as the standard. The cFVIII-F2196K activity was calculated as the mean and standard deviation of at least 3 independent assay runs with each sample run in at least duplicate in each assay. Animals were followed over 12 months, and consistently demonstrated FVIII expression values sufficient for a functional cure. The mean FVIII activity between 3 and 6 months post LNP was 76%, 8.7%, and 29% in animals 1, 2 and 3, respectively. The mean FVIII activity between 9 and 12 months post LNP was 89%, 8.3%, and 37% in animals 1, 2, and 3 respectively, demonstrating that FVIII activity was maintained over 12 months in this ongoing study. The specific albumin-cFVIII-F2196K hybrid mRNA generated by the precise splicing between albumin exon 1 and the integrated cFVIII gene was quantified by ddPCR on total RNA purified from liver tissue biopsies taken pre-LNP, Day 7 post-LNP, and Day 70 post-LNP. An endogenous mRNA was used to normalize between samples. No signal was detected in the liver tissue from the pre-dose biopsy. The albumin-cFVIII-F2196K hybrid mRNA was detected in all 3 animals at both the Day 7 and Day 70 timepoints with no significant change in the mRNA level between Day 7 and Day 70. The FVIII knock-in was achieved with only transient elevation of liver transaminases at the time of LNP administration, no other safety findings, and no impact to circulating albumin levels. Repeat NHP studies using the clinical human FVIII construct have shown consistent efficacy and safety results with both a human B domain deleted FVIII construct, and an engineered construct designed to enhance translation and secretion of FVIII from hepatocytes. To evaluate potential off-target editing, orthogonal assay methods (in silico, biochemical, and cell based) were used to nominate potential off-target sites which were evaluated in primary human hepatocyte donors. To date, no verified off target editing has been observed. Collectively, these results suggest that gene editing may provide a curative approach to hemophilia A patients.