Engineering FVIII Protein for Enhancing Expression and Secretion Profile to Achieve Normal Level of FVIII in Plasma Utilizing Noninvasive Transcutaneous Ultrasound Mediated Gene Delivery
Authors: Krivega Ivan , Krivega Margarita , Poniatowski Julia , Kim David , Del Greco Elizabeth , Rao Surabhi , Foster Victor , Frederich Bert , Arauz Glenda , Ortiz Celia , Chen Oliver , Ghosh Tanaya , Bole Dhruv , Benthall Katelyn N , Rao Anagha , Satyadi David , Gulzman-Poltorak Zoya , Feinstein Steve , Greenberg Kenneth 
Published: December 2024

Introduction: Hemophilia A (HemA) is caused by the absence or deficiency of Factor VIII (FVIII) protein resulting in uncontrolled bleeding. The current standard of care for HemA consists of lifelong infusions of recombinant FVIII protein on a frequent basis. The recent approval of viral gene therapy for HemA provides an alternative, potentially once-a-lifetime treatment option. However, several limitations including high product cost, pre-existing immunity to viral vectors, lack of durable FVIII expression and inability to redose suggest the need to develop alternative non-viral HemA gene therapy treatments to overcome these major obstacles. Transcutaneous ultrasound-mediated gene delivery (UMGD) is a non-invasive in vivo gene delivery approach that may overcome the key delivery challenges facing HemA 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: Employing advanced genetic engineering techniques and screening of next-generation DNA payload designs, we developed an optimized episomal DNA payload encoding an oversized FVIII transgene (FVIII-ST). Transgenic FVIII expression was measured in vitro in human and mouse cells and in vivo in mouse plasma to compare the activity of FVIII-ST with the clinically validated FVIII-V3. To evaluate the potential of UMGD as a nonviral HemA treatment option, we delivered an optimized HemA payload using novel ultrasound acoustic profiles, developed by SonoThera, and FDA-approved ultrasound components. The UMGD 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 DNA into specific tissues via sonoporation.
Results: The FVIII-ST demonstrated superior performance compared to FVIII-V3, with significantly higher levels of human FVIII expression and secretion observed in both in vitro and in vivo models. By fine-tuning the UMGD delivery acoustic profiles in vivo, we successfully delivered the FVIII-ST payload to the livers of both normal and HemA mice, achieving sustained therapeutic FVIII levels in mouse plasma without causing liver toxicity. Multiple UMGD treatments resulted in a marked increase in transgenic FVIII levels in plasma, reaching normal physiological levels, indicating the potential for a redosing regimen to maintain or elevate FVIII levels.
Conclusion: The promising efficacy and safety outcomes of the novel FVIII-ST payload and the UMGD delivery platform support further clinical development of this approach for the treatment of Hemophilia A.