Targeted Non-Viral Gene Delivery through Transcutaneous Ultrasound Enables Robust, Redosable, Titratable, and Durable FVIII and FIX Gene Expression in the Liver in Murine Models

I ntroduction: Robust delivery of DNA payloads of Factor VIII and IX to the liver remains a significant challenge for the development of safe and effective nonviral genetic medicines for hematological diseases such Hemophilia A and B and, von Willebrand disease. Current limitations include the ability to 1) achieve durable non-integrating episomal gene expression in a safe, titratable, and redosable manner, 2) deliver large DNA payloads (>5kb), 3) bypass endosomal and cytoplasmic immune recognition and effectively transit the nuclear envelope, and 4) achieve targeted biodistribution in clinically relevant hepatic cell types. Transcutaneous ultrasound-mediated gene delivery (UMGD) is a noninvasive in vivo gene delivery approach that may overcome the key delivery challenges facing genetic medicines. SonoThera is developing a novel ultrasound-guided nonviral gene therapy platform based on UMGD that allows selective targeting of specific organs and tissues within the body in a safe, redosable, durable, and titratable manner. Methods: To evaluate the potential of UMGD for safe and efficient DNA delivery to the liver, we developed a technology platform that utilizes novel acoustic profiles and next-generation genetic payloads with FDA-approved ultrasound components. The delivery process involves intravenous co-administration of DNA payloads and ultrasound contrast agents (a.k.a microbubbles), coupled with targeted application of externally applied ultrasound energy to guide the DNA into specific tissues via UMGD. Wild Type and Hemophilia disease model mouse plasma and liver tissues were analyzed for FVIII and FIX gene and protein presence and expression. Multiple clinical and molecular safety assessments were evaluated using Results:. Studies using a reporter gene show durability that persisted for at least 12 months after a single treatment. RNAscope analyses show successful delivery to hepatocytes with robust FVIII and FIX protein expression. Repeated administrations resulted in significant increases in FVIII expression levels relative to single administrations, and achieved normal levels of circulating FVIII protein. Gene expression levels were titrated effectively in a dose responsive manner. Safety assessments including clinical observations, ALT, AST, and a proinflammatory cytokine panel indicate an excellent tolerability and safety profile following single and repeat treatments in both murine and NHP models. Conclusion: These results position the novel UMGD platform as a promising technology for in vivo noninvasive transgene delivery using next-generation non-viral DNA payloads establishing the foundation for future hematological genetic therapies targeting the liver.