Platelet-Targeted Hyperfunctional Factor IX (FIX) Gene Therapy for Hemophilia B Mice with Pre-Existing Anti-FIX Immunity

Hemophilia B (HB) is a prime model for gene therapy. While data from current clinical trials using AAV-mediated liver-targeted FIX gene therapy are very encouraging, this protocol can only be applied to adults without liver disease or anti-AAV antibodies. Thus, developing another gene therapy protocol is desired. Our previous studies have demonstrated that platelet-targeted FIX expression driven by the platelet-specific αIIb promoter (2bF9) restores hemostasis and induces immune tolerance in HB mice (Chen et al. Mol Ther 2014). To improve the efficacy, we used a codon-optimized hyperfunctional FIX Padua (2bCoF9R338L) to replace the normal FIX expression cassette. We showed a 5.8-fold higher platelet-FIX antigen (plt-F9:Ag) and a 28-fold activity (plt-F9:C) levels in HB mice (the non-inhibitor model), respectively, in the 2bCoF9R338L group compared to the 2bF9 group. Here we evaluate 2bCoF9R338L gene therapy in HB mice with preexisting anti-F9 immunity (the inhibitor model). Both donor and recipient HB mice were immunized with rhF9 in the presence of Incomplete Freund's Adjuvant to induce inhibitor development. Plt-F9 expression was introduced by 2bCoF9R338L lentivirus transduction of Sca-1+ cells followed by syngeneic transplantation into primed recipients preconditioned with either a lethal 11Gy or a sub-lethal 6.6Gy total body irradiation. Animals were analyzed starting at 4 weeks after transplantation. FACS analysis showed that there was 21.4 ± 11.4% (n = 6) transduced platelets in the 11Gy group, which was not significantly different compared to the 6.6Gy group (12.5 ± 10.9%, n = 7) or the 6.6Gy non-inhibitor model (17.7 ± 11.7%, n = 4). Plt-F9:C levels in the 11Gy and 6.6Gy groups were 18.68 ± 8.61 and 9.17 ± 12.22 mU/108 platelets, respectively. Plt-F9:Ag levels in the two groups were 3.96 ± 1.57 and 1.70 ± 1.64 mU/108 platelets, respectively, which were not significantly different compared to that obtained in the non-inhibitor model. To determine if plt-F9 is γ-carboxylated, BaSO4 precipitation was performed on platelet lysates from 2bCoR338L-transduced recipients. Diluted platelet lysate samples were treated with or without BaSO4 , and the remaining unprecipitated FIX (meaning un-γ-carboxylated) in supernatants was determined by FIX ELISA. No F9:Ag remained in the supernatants of BaSO4-treated platelet lysates, suggesting that FIX can fully undergo functional γ-carboxylation in platelets. Of note, no anaphylaxis occurred in primed HB mice after pla