Non-viral ultrasound-mediated delivery of an episomal FVIII expression vector enables durable FVIII protein production at therapeutic levels in non-human primates

Abstract Hemophilia A (HemA) is a monogenic bleeding disorder caused by mutations in the F8 gene, which encodes coagulation Factor VIII (FVIII). Current treatments rely on frequent administration of recombinant FVIII or bypassing agents. While recently approved AAV-based gene therapies offer a potential one-time treatment, their clinical utility is limited by variable expression, uncertain long-term durability, and an inability to redose due to immune responses. To address these limitations, we have developed ultrasound-mediated gene delivery (UMGD) as a non-viral, liver-targeted, redosable technology for delivery of a FVIII therapeutic transgene to liver cells. Our previous work demonstrated that UMGD enables efficient hepatic delivery of an oversized vector encoding a human FVIII transgene (STx-FVIII), resulting in supratherapeutic FVIII expression and full phenotypic rescue in a murine HemA model without triggering safety endpoints. In this study, we optimized UMGD conditions for robust and safe delivery of DNA expression vectors to the nonhuman primate liver resulting in highly efficient transfection of the majority of the liver following a single dose. To evaluate efficiency of ultrasound-mediated delivery of FVIII expressing transgene to the nonhuman primate liver, we developed a cynomolgus version of the STx-FVIII transgene (cSTx-FVIII) to avoid the inhibitory effects of anti-human FVIII antibodies. Additionally, the F2196K mutation was introduced to allow specific transgenic FVIII quantification by providing resistance to a FVIII inhibitory antibody. UMGD treatment of the NHP liver resulted in durable protein expression of up to 40% of normal circulating cSTx-FVIII level in blood, as quantified by ELISA (total FVIII protein enrichment over endogenous background level) as well as an inhibitory chromogenic activity assay. Evaluation of safety endpoints including ALT, AST, and proinflammatory cytokinerevealed no significant elevations post-treatment. Overall, the data suggest UMGD is an efficient, safe, and redosable delivery method for FVIII vectors, achieving therapeutic and durable expression in mouse and nonhuman primate preclinical models, supporting the use of the technology for the development of a non-viral genetic medicine for Hemophilia A patients.