Engineering Non-Viral Gene Therapy Vector for Simultaneous Transgenic FVIII Protein Expression from Liver Sinusoidal Endothelial Cells (LSECs) and Hepatocytes upon Non-Invasive Transcutaneous Ultrasound-Mediated Gene Delivery

Introduction: Hemophilia A (HemA) results from the absence or deficiency of Factor VIII (FVIII) protein, leading to uncontrolled bleeding. The current standard of care involves lifelong frequent infusions of recombinant FVIII protein. Recently, liver-directed gene therapy for HemA has emerged as a potentially one-time treatment option. However, this approach is limited by high costs, pre-existing immunity to viral vectors, lack of durable FVIII expression, and the inability to redose. Thus, there is a need for alternative gene therapy strategies for HemA. One proposed strategy is the expression of transgenic FVIII in its native production cells, liver sinusoidal endothelial cells (LSECs), which could address issues associated with FVIII production in hepatocytes, such as the development of inhibitors and the lack of durable expression. Transcutaneous ultrasound-mediated gene delivery (UMGD) is a non-invasive in vivo gene delivery method that efficiently targets major liver cell types, making it a promising approach for delivering a large FVIII expression cassette to both liver LSEC and hepatocyte cells.

Methods: Using advanced genetic engineering techniques and screening of next-generation DNA payload designs, we developed an optimized 16-kilobase DNA payload encoding an oversized human FVIII transgene (FVIII-ST) controlled by hepatocyte- and LSEC-specific gene expression cassettes. To assess the potential of UMGD as a non-viral HemA treatment option, we delivered the dual-cassette FVIII-ST payload to mouse liver using novel acoustic profiles developed by SonoThera and FDA-approved ultrasound components and evaluated human FVIII (hFVIII) abundance in plasma and FVIII-ST transgene expression biodistribution in liver tissue. 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 organs of interest via sonoporation.

Results: By fine-tuning of UMGD acoustic profiles for dual-cassette FVIII-ST vector delivery to the liver, we successfully achieved therapeutic levels of hFVIII expression in the plasma of both normal and HemA mice. The level of hFVIII in plasma of the dual-cassette FVIII-ST vector treated mice was up to two-fold higher than in mice treated with hepatocyte only single-cassette FVIII-ST vector. Utilizing ddPCR and RNAscope assays, we confirmed the successful delivery of the dual-cassette FVIII-ST vector to both hepatocytes and LSECs, along with strong FVIII-ST transgene expression in these cell types.

Conclusion: The UMGD delivery platform efficiently delivers a 16-kilobase non-viral gene therapy payload to the liver, effectively targeting LSEC and hepatocyte cells. This supports the use of UMGD as a non-viral gene therapy delivery approach for developing gene-based therapeutics for major blood coagulation disorders.

No relevant conflicts of interest to declare.