Optimized AAVrh10 and Factor 9 vectors demonstrate improved phenotypic rescue in hemophilia B mice.

Hemophilia B is an X-linked bleeding disorder caused by a functional deficiency of coagulation factor IX (FIX). Adeno-associated virus (AAV) mediated gene therapy based on vector serotypes 5 and 8, has shown promising clinical outcomes. Nonetheless, a high prevalence of neutralizing antibodies in the general population to these serotypes requires the use of an alternate gene transfer system. AAVrh10, a non-human primate-derived serotype, has low pre-existing immunity in humans, making it a promising alternative for hemophilia gene therapy. We reasoned that further rational engineering, particularly at post-translational modification (PTM) sites of AAVrh10 capsid and codon optimization of the Factor 9 (F9) transgene, can enhance therapeutic efficiency. Initially, we evaluated three rationally engineered AAVrh10 capsid mutants (S157A, K169Q, and K709Q) encoding a F9 transgene, regulated by Kozak sequence and liver-specific promoter (LP1) by systemic gene transfer. In the initial screening, AAVrh10K169Q vectors demonstrated a ~ 4.6-fold (p < 0.001) improved FIX clotting activity in hemophilia B mice in comparison to the AAVrh10WT vector, one year after hepatic gene transfer. Subsequently, we performed codon optimization of the hyperactive F9 Padua transgene (CoF9 Padua) to enhance its translational potential. The hepatic gene transfer of AAVrh10WT and AAVrh10K169Q vectors carrying CoF9 Padua transgene exhibited supraphysiological FIX clotting activity (~529.4%-1435.6%) with no immunological complications, ~18 months post gene transfer. Our study highlights the translational potential of optimized AAVrh10-CoF9 vectors for gene therapy in patients with hemophilia B.