Full Gene Replacement for Hemophilia A Enabled by the StitchR Technology

ABSTRACT Hemophilia A (HA) is a common sex-linked bleeding disorder affecting ~ 1 in 4,000 male births in the United States, caused by loss-of-function mutations in the Factor VIII (FVIII / F8) gene that disrupt blood coagulation. Conventional treatments, such as recombinant FVIII protein and bispecific antibodies, require frequent administration. Recently, adeno-associated virus (AAV)- mediated gene therapy, which delivers a truncated B domain-deleted FVIII (BDD-FVIII) from the liver, has received FDA approval and shown promise in reducing bleeding events. However, the B domain, while not essential for clotting, is important for FVIII stability and secretion, and BDD- FVIII has been associated with hepatocellular carcinoma (HCC) in some preclinical models. Gene-replacement therapy presents a potentially curative alternative, but AAV's limited cargo capacity has so far restricted clinical testing to truncated FVIII versions, resulting in limited efficacy. To overcome these challenges, we propose the development of a novel dual AAV vector technology, StitchR, which utilizes ribozyme-activated RNA trans-ligation to deliver and express full-length FVIII. This approach addresses AAV's size limitations and aims to provide targeted, stable, and non-mutagenic FVIII expression. Our preliminary data indicate that StitchR can successfully be used to trans-ligate and express full-length FVIII in vitro. The proposed research will advance this technology by engineering robust AAV constructs for FVIII gene delivery, evaluating different tissue-specific promoters to optimize FVIII expression and durability, and assessing therapeutic and unintended host responses using a human-FVIII tolerant mouse model of HA. This project aims to validate a new approach for large gene-replacement therapies for blood disorders, establish a foundation for future research in higher mammals, and provide a best- in-class treatment for HA in patients.