268. Enhanced Gene Expression of Factor VIII by Ultrasound-Mediated Gene Delivery in Dogs

Abstract

Previously we demonstrated that ultrasound (US)-mediated gene delivery (UMGD) can significantly enhance reporter gene transfer into the mouse and rat livers. This nonviral gene transfer strategy can bypass many obstacles encountered by viral gene therapy. Most significantly, we have achieved therapeutic levels of FVIII following UMGD into hemophilia A mice. Recently we have successfully developed prototype US systems including several unfocused and semi-focused transducers to treat large tissue volumes in canine and swine. In order to facilitate the translation of this technology to treat hemophilia, we have recently treated 2 normal dogs with UMGD of FVIII plasmids using an open surgery procedure. Four mg of a high-expressing, liver-specific pBS-HCRHPI-FVIIIA plasmid and 3 ml of Definity® MBs in 8 ml total PBS solution were injected via the segmental portal vein branch with simultaneous exposure of the target liver lobe to therapeutic US (1.1 MHz frequency, 20 cycle pulses, 50 Hz pulse repetition frequency) for 4 minutes using the large diameter transducers. A sham-treated dog received an equivalent pGL4/MB dose, but was not exposed to tUS. We used an apodized dual element unfocused transducer H105 at 2.5Mpa peak negative pressure (PNP) in the first dog experiment and observed low levels of hFVIII gene expression in the treated liver lobes. The second dog experiment was performed using a cylindrically 6.2 MPa at the focal area. One day following treatment, the treated liver lobes were sectioned, and representative sections were fixed and stained for FVIII expression by histochemical staining using a polyclonal anti-FVIII antibody. Untreated normal dog liver and human liver were used as negative and positive controls. Significant hFVIII gene expression was obtained in both treated liver lobes with the expression levels slightly lower than control human liver lobe. Furthermore, fairly homogeneous distribution of FVIII gene expression was observed in hepatocytes. Next, we performed Western blot analysis to confirm if the staining is specific to human FVIII protein. A heavy chain band specific to hFVIII was observed in treated dog plasma and positive hFVIII control, but not in control normal dog plasma and pre-bleed plasma sample from the dog before treatment. Significant enhancement of FVIII-specific clotting activity was also obtained in the second treated dog compared to pre-treatment and the first treated dog. In addition, transaminase levels and histology analysis indicated minimal tissue damage in treated dog livers. Enhancement of FVIII gene expression by UMGD is currently evaluated in hemophilia dogs. Our results sugges that UMGD has great potential for therapeutic treatment of hemophilia A.