352. Improvement of Targeted Gene Transfer Mediated By Ultrasound and Microbubble in a Swine Model

Abstract

Ultrasound (US) combined with microbubble (MB) represents a promising non-viral gene delivery strategy. Previously we successfully developed US/MB mediated reporter gene delivery system in murine models and scaled up into large animal models. In order to further pursue non-invasive gene therapy strategy for clinical application, in this study we investigated alternative surgical operation and different acoustic transducers/parameters.Firstly, we investigated the optimal US protocol under transcutaneous US treatment using intraoperative procedures. Following midline incision, the plasmid/MB was injected into the portal vein branch with inferior vena cava occluded during treatment. Simultaneously, transcutaneous US treatment on the skin was applied towards the target liver lobe (20 cycle pulses, 50 Hz PRF, 6 MPa PNP). Compared with the control liver lobe, luciferase expression of the treated liver showed a significant enhancement (≈2000 RLU /mg protein, ≈200 fold higher vs. control). Previously we observed that higher acoustic pressure was required for large animals than that for mice at short pulse durations. In addition, even higher pressure will be needed to overcome the attenuation of US intensities across skin tissue layers. However, higher pressure can induce more severe liver damages and is limited by power output capacity of the transducers. Recently we found that by prolonging pulse durations, efficient gene transfer with minimized liver damage can be achieved at lower acoustic pressures in cell culture and mice. Similar strategy was employed in pigs by applying US on target liver lobe at various parameters (50μs-4ms pulse duration, 0.4-3 MPa). Significant enhancement of gene expression was achieved with 3000 RLU/mg protein at very low PNP (0.4 MPa) and >104RLU/mg protein at 1MPa PNP. Our studies indicated a targetable range of millisecond pulse durations which is useful across small and large animal models. These new US conditions will allow for more effective transcutaneous treatment in the pig livers where attenuation across multiple intervening tissue layers poses a significant barrier to achieving high PNPs. Next, we evaluated gene transfer without laparotomy using a US imaging-guided procedure. Plasmid/MB was infused into a specific portal vein branch via a catheter guided by diagnostic US through the skin. The target liver was simultaneously treated by transcutaneous US on the skin. Although the liver damages were significantly minimized after treatment, large variations in the gene expression levels among different animals were obtained due to varied degrees of plasmid/MB distribution and retention. We are currently developing a minimally invasive interventional radiologic technique to deliver plasmid/MB into the liver combined with transcutaneous US procedure.In conclusion, the exploration of different treatment protocols and acoustic parameters for gene delivery into pig livers paves the way to potentially efficient and non-invasive clinical application of US/MB mediated gene therapy.