Background: Adeno-associated-virus (AAV) based gene transfer has shown potential to provide sustained Factor IX (FIX) activity at low levels in severe hemophilia B, preventing spontaneous bleeding and reducing FIX consumption. However, further enhancement was hampered by dose-dependent liver toxicity, most likely due to cytotoxic T-cells directed against transduced hepatocytes or cellular stress by high protein expression. The hyperactive FIX Padua variant enhanced efficacy but the issue of dose-limiting liver toxicity remained.  

Aims: Two FIX variants, based on FIX-Padua, with further enhanced intrinsic activity and one of them with decreased collagen IV binding were evaluated for their potential to enhance efficacy after AAV-based gene transfer and to allow novel strategies of recombinant FIX substitution in hemophilia B.

Methods: Recombinant FIX variants were tested in-vivo for pharmacokinetic and pharmacodynamic parameters and their immunogenic potential. The FIX variants were further tested in AAV-based gene transfer for efficacy and safety, particularly with regard to thrombotic events.

Results: Both novel FIX variants showed 1.5-fold increased activity compared to FIX Padua. Collagen type IV binding had no impact on enzymatic or functional activity of the protein. Instead, reduction of collagen IV binding dramatically increased the bioavailability in hemophilia B mice after intravenous or subcutaneous injection but also enhanced the elimination of the protein. In AAV gene therapy, the reduced collagen type IV binding variant has no additional effect. Risk assessments of the new hyperactive variants in hemophilia B mice showed neither increased immunogenicity due to the introduced mutations, nor increased propensity to thrombosis due to further increased intrinsic activity.

Conclusions: Our new hyperactive FIX variants harbor increased activity compared to FIX-Padua, promising enhanced efficiency in AAV-based gene therapy for hemophilia B without increasing therapy-related toxicity. In addition, decreased collagen type IV binding improves bioavailability and bears potential for alternative substitution strategies, such as subcutaneous application.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/novel-hyperactive-fix-variants-and-their-potential-for-hemophilia-b-therapy/