OC 12.4 - An Enhanced Hemostatic Factor VIII Variant for Hemophilia A Gene Therapy: Prothrombotic and Immunological Risk Assessment

Background: Despite phenotypic improvement in clinical trials, universally sustained AAV-mediated FVIII expression at levels necessary to eliminate bleeding has not been achieved. Like the success of FIX-Padua for HB gene therapy, we hypothesized an enhanced hemostatic-function FVIII-variant transgene for HA gene transfer may achieve hemostatic efficacy at lower AAV vector doses thereby overcoming vector dose-dependent efficacy limitations while eliminating bleeding. We described a FVIII variant (FVIII-R336Q/R562Q; FVIII-QQ) resistant to APC-mediated inactivation with ~5-fold enhanced hemostatic function in vivo (Wilhelm et al. Blood 2021).

Aims: We investigated the safety and efficacy of FVIII-QQ for HA AAV-mediated gene therapy.

Methods: AAV-hFVIII-WT and AAV-hFVIII-QQ vectors were administered to HA/CD4KO mice; FVIII:C was determined by COAMATIC chromogenic assay and AAV-treated cohorts were followed for survival or analyzed by tail-clip assay. WT mice zygotes underwent CRISPR/cas9 editing to generate FVIII-QQ mice. Recombinant hFVIII-WT or hFVIII-QQ, both produced in our lab, was intravenously administered to immunocompetent HA mice 6 times weekly at 0.2 µg/mouse followed by 5 µg/mouse on week 7 and Bethesda titer was determined.

Results: The vector dose EC50 and EC80 of blood loss post tail-clip for AAV-hFVIII-QQ was 4.7 and 10-fold lower, respectively, than AAV-hFVIII-WT supporting a 5-10-fold benefit of the hFVIII-QQ transgene. Ongoing studies of HA/CD4KO mice expressing hFVIII-WT and hFVIII-QQ in the mild and normal ranges of FVIII:C thus far demonstrate no significant survival differences for at least 3 months post-vector (Figure1). Homozygous female/hemizygous male mice endogenously expressing FVIII-QQ via CRISPR-mediated gene editing are viable, fertile and the same weight as littermate controls. Immunocompetent HA mice treated with recombinant hFVIII-WT or hFVIII-QQ demonstrated expected inhibitor development that did not statistically differ (Figure2).

Conclusion(s): These data demonstrate a hemostatic advantage of using the FVIII-QQ transgene for AAV-mediated gene transfer. Preliminary murine safety studies did not demonstrate evidence of FVIII-QQ enhanced prothrombotic or immunological risk relative to FVIII-WT.