PB0207 - Enhancing FVIII Gene Therapy: Noninvasive Transcutaneous Ultrasound-Mediated Gene Delivery of Human Factor VIII Plasmid in Hemophilia A Mice

Background: Hemophilia A (HemA) is an X-linked clotting disorder characterized by a deficiency of functional factor VIII (FVIII) protein. The field of gene therapy holds significant promise in treating this condition. Ultrasound-mediated gene delivery (UMGD) has emerged as a promising non-viral method that utilizes therapeutic ultrasound (US) and microbubbles (MBs) to facilitate gene transfer.

Aims: To develop a noninvasive intravenous non-viral gene delivery protocol in combination with transcutaneous UMGD in HemA mouse models to achieve efficient transfection of FVIII plasmids in the liver.

Methods: The FVIII plasmid, combined with RC2K MBs, was intravenously injected into mice via retro-orbital injection, accompanied by transcutaneous UMGD to the liver. The US parameters used in our experiments include: a center frequency of 1.1 MHz, a pulse repetition frequency of 14 Hz, and peak negative pressure between 1.2 and 1.5 MPa, with a pulse duration of 150 µs in a 1-second ON and 2-seconds OFF pulse train for a total treatment time of 60 seconds. Weekly blood samples were obtained to monitor human FVIII activity and alanine levels.

Results: The immunodeficient HemA mice, treated with 50µg FVIII plasmid under specific US conditions, initially achieved approximately 40% FVIII activity, sustaining around 18% for a month. A subsequent treatment aimed to enhance FVIII activity in these mice, resulting in an initial 35% activity, which gradually stabilized at 10-15% for two months. Following a third treatment, a similar trend in FVIII activity was observed. Slightly elevated ALT levels post-treatment normalized within a week.

Conclusion(s): In conclusion, UMGD of plasmid DNA in animals achieved highly efficient, stable, and redosable gene expression. Viral gene therapy is promising for treating hemophilia, but the safety concerns and challenges remain due to the pre-existing antibodies against viral vectors. The minimally invasive, non-viral, and effective UMGD method holds promise for translating into future treatments for hemophilia A patients.