Project 457588

Hemophilia A (HA) is an X-linked bleeding disorder caused by a mutation in the factor 8 (F8) gene that codes for FVIII coagulation protein. FVIII is naturally synthesized in the liver and the mutation results in the loss of function of FVIII protein which is critical in the blood clotting cascades. HA occurs is 1 to 5000 male births and it is estimated that there are a total of 3000 patients with HA in Canada. Currently, HA patients are treated with replacement therapy of the deficient factor. However, this approach is transient because of the short half-life of recombinant or plasma derived FVIII and is insufficient due to the formation of anti-drug antibodies. The alternative approach which is curative and promises a long-lasting expression of FVIII protein is gene therapy. In this approach F8 gene is introduced into the liver hepatocytes as a transgene via a viral vector like adeno-associated viruses (AAVs). During the past 25 years of pre-clinical and clinical investigation of AAV gene therapy strategies, it has become increasingly apparent that attaining the most effective and safe protocols for achieving long-term benefit from transgene delivery requires "fine tuning" of the various components of the vector and vector delivery details. In this study, we aim to develop an engineered AAV-FVIII expression cassette that is extremely potent to drive persistent endogenous expression of FVIII in transduced cells. In this project, for the first time, we focus on incorporating a recently identified enhancer sequence of F8 gene in AAV expression cassette to achieve persistent FVIII expression at hemostatic levels. The results derived from this novel study has the potential to address major issues regarding ongoing AAV-FVIII gene delivery trials such as liver toxicity and decline in the FVIII expression following therapy. Additionally, findings of this project contribute to the advancement of AAV-FVIII constructs and ultimately treatment of HA patients.