Ultrasound-Mediated Gene Therapy in Swine Livers Using Single-Element, Multi-lensed, High-Intensity Ultrasound Transducers.

We have achieved significant enhancement of gene delivery into livers of large animals using ultrasound (US)-targeted microbubble (MB) destruction methods. An infusion of pGL4 (encoding a luciferase reporter gene) plasmid DNA (pDNA) and MBs into a portal-vein segmental branch of a porcine liver was exposed to US for 4 min. Therapeutic US induced cavitation of MBs to temporarily permeabilize the vascular endothelium and cell membranes, allowing entry of pDNA. We obtained a 64-fold enhancement in luciferase expression in pig livers compared to control without US using an unfocused, dual-element transducer (H105, center frequency [fc] = 1.10 MHz) at 2.7 MPa peak negative pressure (PNP). However, input electrical energy was limited, and modified transducers were designed to have spherical (H185A, fc = 1.10 MHz) or cylindrical foci (H185B, fc = 1.10 MHz; H185D, fc = 1.05 MHz) to enhance PNP output. The revised transducers required less electrical input to achieve 2.7 MPa PNP compared to H105, thereby allowing PNP outputs of up to 6.2 MPa without surpassing the piezo-material limitations. Subsequently, luciferase expression significantly improved up to 9,000-fold compared to controls with minor liver damage. These advancements will allow us to modify our current protocols toward minimally invasive US gene therapy.

Ultrasound-targeted microbubble destruction-mediated gene delivery into canine livers.

Ultrasound (US) was applied to a targeted canine liver lobe simultaneously with injection of plasmid DNA (pDNA)/microbubble (MB) complexes into a portal vein (PV) segmental branch and occlusion of the inferior vena cava (IVC) to facilitate DNA uptake. By using a 1.1 MHz, 13 mm diameter transducer, a fivefold increase in luciferase activity was obtained at 3.3 MPa peak negative pressure (PNP) in the treated lobe. For more effective treatment of large tissue volumes in canines, a planar unfocused transducer with a large effective beam diameter (52 mm) was specifically constructed. Its apodized dual element configuration greatly reduced the near-field transaxial pressure variations, resulting in a remarkably uniform field of US exposure for the treated tissues. Together with a 15 kW capacity US amplifier, a 692-fold increase of gene expression was achieved at 2.7 MPa. Transaminase and histology analysis indicated minimal tissue damage. These experiments represent an important developmental step toward US-mediated gene delivery in large animals and clinics.