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.

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 >10 5 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.

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Physical Methods of delivery and cheMical/Molecular conjugates of the oxygen saturation of the Hb in the blood of the injected lobe from 30 to 0 unit upon the injection and recovered smoothly after the injection. Serum analysis showed a transient, 10-to 20-fold increase in hepatobiliary enzymes, including aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase after hydrodynamic injections, which recovered within 4 days. We also explored for the first time, the levels of the cytokines following the hydrodynamic injections to the large animals. There was no increase in the systemic inflammatory cytokines of IFN-γ, IL-8, IL-18, and IL-4, although an increase in serum levels of TNF-α, IL-10, MCP-1, canine KC, and IL-6, which were related to vascular stretching representing the sinusoidal expansion was observed. No impacts on their respiratory, cardiovascular conditions, or long-term body weight changes were observed throughout the study. These results of preclinical assessments support the clinical applications of image-guided, livertargeted hydrodynamic gene delivery.