Background: Gene therapy using Adeno-associated virus (AAV) vectors has emerged as a promising alternative to frequent coagulation factor IX(FIX) infusion during replacement therapy. AAV5 and AAV8 serotype based approved gene therapy products, have shown clinical success in hemophiila B patients. However, challenges such as capsid-specific immune responses and lack of long-term FIX expression in patients, remain. 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 the FIX transgene, can enhance vector stability and therapeutic efficiency.

Aim: To develop AAVrh10 capsid mutants with enhanced transduction efficiency and a modified codon optimized transgene encoding human coagulation FIX with Padua variant and under the control of a novel Kozak sequence and to assess their combined effect on phenotypic correction in hemophilia B mouse mice.

Methods: Capsid mutations targeting PTM sites were identified by in-silico analysis and liquid chromatography-mass spectroscopy (LC-MS). Mutants were generated by site-directed mutagenesis (SDM) at the predicted and identified capsid sites. Hemophilia B mice were injected via tail vein either with PBS, wild-type, or mutant AAVrh10 vectors (1 × 10¹⁰ vg/mouse) containing human FIX, and circulating FIX levels were assessed by ELISA. FIX-specific clotting-based assays were performed to evaluate FIX activity. The Bethesda assay was performed to further evaluate the development of inhibitors against human FIX. Transgene FIX expression in liver was analyzed by immunohistochemistry (IHC). Immunological assessment included ELISPOT assay and T/B/Treg cell enumeration. The most effective mutant AAVrh10K169Q, along with control AAVrh10WT vectors, were further packaged with Kozak codon-optimized FIX Padua variant and administered at a dose of 1 × 10¹¹ vg/mouse. FIX activity was assessed at 4-, 8-, and 34-weeks using clotting-based assays. Enumeration of T/B and Treg cells were conducted after 80 weeks to assess the safety of the developed therapeutic vector.

Results: In the first set of studies, we evaluated the therapeutic potential of mutant AAVrh10 capsids (S157A, K169Q, and K709Q) containing LP1- FIX transgene at a low dose of 1×10¹⁰ vg per mouse (n = 6 animals per group) at 5 and 51 weeks, after gene therapy. The hex-acylation site mutant vector AAVrh10S157A achieved average FIX activity of 38.6 % (3.6-fold) and 27.3 % (1.9-fold); the SUMOylation mutant K709Q showed levels of 10.8–8.0% and the Neddylation mutant vector K169Q demonstrated the highest FIX levels at 83.8 % (7.9-fold) and 66.9 % (4.6-fold), respectively, in comparison to AAVrh10WT (10.5–14.5%) vectors. These data were further corroborated by FIX antigen and immunohistochemistry of liver tissue. Bethesda assay did not detect any FIX inhibitors in treated mice and immune profiling (T cell and B cell, T regulatory (Treg) cells, Interferon gamma ELISPOT assay) did not identify any vector-induced immune activation in mice that received gene therapy.

Subsequently, we evaluated the engineered FIX codon optimized transgene containing an optimized kozak sequence along with hyperactive FIX-Padua variant, packaged under WT and K169Q vectors and delivered at a high dose (1×10¹¹ vg/mouse, n=7-10 animals). We observed supraphysiological FIX activity between 4 to 34 weeks after hepatic gene therapy with these vectors [529%-1435%]. Long-term immune cell (B and T cells and Treg) enumeration confirmed vector safety and absence of immunogenicity.

Conclusions: Our data reveals that the combination of optimized FIX transgene with either AAVrh10WT or the neddylation-site mutant capsid sustains supraphysiological FIX activity after liver directed gene therapy. These findings highlight their potential as a promising candidate for clinical gene therapy in patients with hemophilia B.

Funding support: Pfizer 2019 Global Haemophilia ASPIRE program

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2025
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