FIX variant CB 2679d-GT significantly outperformed the R338L-Padua variant after gene therapy.
Increase in the overall potency of the gene therapy vectors may allow for lower and potentially safer vector doses in future human trials.
Sustained expression of therapeutic factor IX (FIX) levels has been achieved after adeno-associated viral (AAV) vector-based gene therapy in patients with hemophilia B. Nevertheless, patients are still at risk of vector dose-limiting toxicity, particularly liver inflammation justifying the need for more efficient vectors and a lower dosing regimen. A novel increased potency FIX (designated as CB 2679d-GT), containing three amino acid substitutions (R318Y, R338E, T343R), significantly outperformed the R338L-Padua variant after gene therapy. CB 2679d-GT demonstrated a statistically significant ~3-fold improvement in clotting activity when compared to R338L-Padua after AAV-based gene therapy in hemophilic mice. Moreover, CB 2679d-GT gene therapy showed a significantly reduced bleeding time (~5 to 8-fold) and total blood loss volume (~4-fold) compared with mice treated with the R338L-Padua, thus achieving a more rapid and robust hemostatic correction. FIX expression was sustained for at least 20 weeks with both CB 2679d-GT and R338L-Padua while immunogenicity was not significantly increased. This is a novel gene therapy study demonstrating the superiority of CB 2679d-GT highlighting its potential to obtain higher FIX activity levels and superior hemostatic efficacy following AAV directed gene therapy in hemophilia B patients than what is currently achievable with the R338L-Padua variant.