Ultrasound-mediated gene delivery of factor VIII plasmids for hemophilia A gene therapy in mice.

Gene therapy offers great promises for a cure of hemophilia A resulting from factor VIII (FVIII) gene deficiency. We have developed and optimized a non-viral ultrasound-mediated gene delivery (UMGD) strategy. UMGD of reporter plasmids targeting mice livers achieved high levels of transgene expression predominantly in hepatocytes. Following UMGD of a plasmid encoding human FVIII driven by a hepatocyte-specific promoter/enhancer (pHP-hF8/N6) into the livers of hemophilia A mice, a partial phenotypic correction was achieved in treated mice. In order to achieve persistent and therapeutic FVIII gene expression, we adopted a plasmid (pHP-hF8-X10) encoding an FVIII variant with significantly increased FVIII secretion. By employing an optimized pulse-train ultrasound condition and immunomodulation, the treated hemophilia A mice achieved 25%-150% of FVIII gene expression on days 1-7 with very mild transient liver damage, as indicated by a small increase of transaminase levels that returned to normal within 3 days. Therapeutic levels of FVIII can be maintained persistently without the generation of inhibitors in mice. These results indicate that UMGD can significantly enhance the efficiency of plasmid DNA transfer into the liver. They also demonstrate the potential of this novel technology to safely and effectively treat hemophilia A.

Transcutaneous Ultrasound-Mediated Nonviral Gene Delivery to the Liver in a Porcine Model.

Ultrasound (US)-mediated gene delivery (UMGD) of nonviral vectors was demonstrated in this study to be an effective method to transfer genes into the livers of large animals via a minimally invasive approach. We developed a transhepatic venous nonviral gene delivery protocol in combination with transcutaneous, therapeutic US (tUS) to facilitate significant gene transfer in pig livers. A balloon catheter was inserted into the pig hepatic veins of the target liver lobes via jugular vein access under fluoroscopic guidance. tUS exposure was continuously applied to the lobe with simultaneous infusion of pGL4 plasmid (encoding a luciferase reporter gene) and microbubbles. tUS was delivered via an unfocused, two-element disc transducer (H105) or a novel focused, single-element transducer (H114). We found applying transcutaneous US using H114 and H105 with longer pulses and reduced acoustic pressures resulted in an over 100-fold increase in luciferase activity relative to untreated lobes. We also showed effective UMGD by achieving focal regions of >105 relative light units (RLUs)/mg protein with minimal tissue damage, demonstrating the feasibility for clinical translation of this technique to treat patients with genetic diseases.

Prolonging pulse duration in ultrasound-mediated gene delivery lowers acoustic pressure threshold for efficient gene transfer to cells and small animals.

While ultrasound-mediated gene delivery (UMGD) has been accomplished using high peak negative pressures (PNPs) of 2 MPa or above, emerging research showed that this may not be a requirement for microbubble (MB) cavitation. Thus, we investigated lower-pressure conditions close to the MB inertial cavitation threshold and focused towards further increasing gene transfer efficiency and reducing associated cell damage. We created a matrix of 21 conditions (n = 3/cond.) to test in HEK293T cells using pulse durations spanning 18 µs-36 ms and PNPs spanning 0.5-2.5 MPa. Longer pulse duration conditions yielded significant increase in transgene expression relative to sham with local maxima between 20 J and 100 J energy curves. A similar set of 17 conditions (n = 4/cond.) was tested in mice using pulse durations spanning 18 µs-22 ms and PNPs spanning 0.5-2.5 MPa. We observed local maxima located between 1 J and 10 J energy curves in treated mice. Of these, several low pressure conditions showed a decrease in ALT and AST levels while maintaining better or comparable expression to our positive control, indicating a clear benefit to allow for effective transfection with minimized tissue damage versus the high-intensity control. Our data indicates that it is possible to eliminate the requirement of high PNPs by prolonging pulse durations for effective UMGD in vitro and in vivo, circumventing the peak power density limitations imposed by piezo-materials used in US transducers. Overall, these results demonstrate the advancement of UMGD technology for achieving efficient gene transfer and potential scalability to larger animal models and human application.