625. Optimization of High Intensity Therapeutic Ultrasound and Microbubbles for Gene Delivery into Rat Liver

PHYSICAL METHODS OF DELIVERY gene therapy treatment regimens. We set out to develop a method for gene modification of host tissues that would yield sustainable and clinically detectable production of gene products by targeted tissues with minimal effect on non-targeted tissues. Methods: Gene delivery was carried out using a recombinant adeno-associated viral vector, termed rAAVrec2 (previously derived using a PCR shuffling technique from human and non-human primate viral isolates.) This vector was used to deliver the luciferase gene into the pectoralis major muscle in wild-type C57BL/6 mice. Viral vector was delivered by one of two surgical methods: (1) viral vector suspended in saline was directly injected into the belly of the pectoralis muscle using a micro syringe and 30 gauge hypodermic needle and (2) autologous fat tissue, obtained from a donor mouse, was injected with the same viral vector and allowed to incubate ex vivo for 15 minutes before it was processed into smaller fragments and surgically implanted into the belly of the pectoralis muscle. Following the procedure, animals were serially imaged to detect luciferase activity using the in vivo, real-time imaging system IVIS 100 (Caliper Life Sciences, MA). Tissues were collected and processed at 4-5 weeks post injection of viral vector and histologically examined with immunohistochemical staining techniques. Results: Direct injection of viral vectors into muscle tissues lead to clinically detectable luciferase activity on IVIS imaging that was confirmed by histological analysis; however, these animals also demonstrated strong enzymatic activity in the mid-abdominal region that was shown by histology to be consistent with hepatic luciferase expression. Mice whose muscles were genemodified using autologous fat grafts as a vehicle for delivery of the viral vectors also showed luciferase activity on IVIS imaging, but had no evidence of signal on whole-body imaging. Histological analysis of liver sections from these mice confirmed absence of hepatic luciferase expression. Further examination of histological sections of gene modified muscle indicated that both the adipose and nearby muscle cells were responsible for the luciferase activity. Conclusions: Using fat grafts as a vehicle, we have developed a method for gene delivery into muscle tissue that leads to highly specific gene modification of targeted tissues with minimal infection of non-target tissues.

625. Optimization of High Intensity Therapeutic Ultrasound and Microbubbles for Gene Delivery into Rat Liver

Shuxian Song,1 Misty L. Noble,1 Samuel Sun,1 Liping Chen,1 Andrew A. Brayman,3 Carol H. Miao.1,2,3 1 Seattle Children’s Research Institute, Seattle, WA; 2Department of Pediatrics, University of Washington, Seattle, WA; 3Center for Industrial and Medical Ultrasound, Applied Physics Laboratory, University of Washington, Seattle, WA.

The use of ultrasound (US) combined with microbubbles (MBs) has been explored widely in vitro and in vivo as a potentially effective and relatively safe physical method for gene delivery. We previously reported efficient gene transfer with US exposure and MBs in a mouse model. We subsequently investigated scaling-up gene delivery in the rat model using an unfocused high intensity therapeutic ultrasound system and targeted injection of plasmid and MBs into a specific liver lobe. For developing safe and effective application to larger animal models with the new system, we evaluated both the luciferase reporter gene transfer efficacy as well as liver damages under various MB concentrations and different acoustic parameters. In studies of MB doses using Definity® contrast agent and an acoustic pressure amplitude of 2.7 MPa at 1 MHz, the luciferase expression levels were significantly enhanced over the range of 0-30 Vol% MB concentrations (15∼350 fold vs. plasmid only negative control) with a plateau rapidly being reached between 0.5 and 30 Vol%. These results differed from those of earlier mouse experiments obtained using a focused US system, in which much higher threshold MB concentrations (∼5%) were needed for significant enhancement of S240

gene transfer efficiency. In US-treated rats, a maximum level was observed at the MB concentration of 10 Vol%, while in the mouse model, the peak was at 15 Vol%. This nominal difference is mostly likely due to more efficient acoustic exposure from the bigger unfocused transducer used in the rat experiments. Application of pulse-train US exposure regimens improved tissue distribution of plasmid DNA/MBs and produced reporter gene expression levels comparable to the positive control treated with standard US condition even though the acoustic energy was reduced 30-50%. We evaluated the liver damages by transaminase assays and histological staining. Only minor increases in transaminase levels were observed after US treatment under various conditions, indicating that US treatment of one specific liver lobe produced minimum impact on total liver function. More detailed analysis of microscopic damages carried out by H&E staining showed hemorrhage and focal coagulative necrosis in the treated lobe, which resolved over time. Less liver damage was produced with lower MB concentrations and lower ‘extents’ of acoustic exposure; e.g., lower acoustic pressures, lower acoustic pulse repetition frequency and shorter overall exposure time. These attempts on optimization of the new US/MB system for rat livers indicated that efficient gene transfer and minimum liver damage could be achieved by using lower MB concentrations and pulse-train US procedures with lower acoustic energy and sustained plasmid DNA/ MB distribution.

626. Gene Delivery Using a Novel Physical Mode of Transfection Based on Electrospray

Michael Maguire,1 Heather Kavanagh,1 Eoin Judge,2 Natasha McCormack,1 James Egan,2 Shirley O’Dea.1 1 Institute of Immunology, Biology Department, National University of Ireland Maynooth, Maynooth, Kildare, Ireland; 2National Heart and Lung Transplant Unit, Mater Misericordiae University Hospital, Dublin, Ireland. BACKGROUND: Gene delivery to many cell types in vitro and to tissues in vivo remains a significant challenge. Limitations of lipidand virus-mediated transfection methods include toxicity, high cost and complexity in vitro and efficacy, targeting and immunogenicity in vivo. Our group is pioneering ‘electrospray’ as a physical means of transfection that overcomes many of these issues in vitro and in vivo. Electrospray is a method of generating a very fine spray of a fluid by electrostatic charging. We have developed a system, ‘ProFector’, capable of creating a continuous electrospray that is controlled by software. The electrospray is formed from colloids of a solution containing plasmid DNA. The colloids attain high velocities and sufficient force to penetrate through the cell membrane and transfect the cell. ProFector comprises a spray head (patent pending) and a conduit. The spray head is capable of generating the electrospray and can be deployed both in vitro and in vivo. Here we determined the ability of ProFector to transfect lung cells in vitro. METHODS: Human lung cell lines (BEAS-2B and DLKP) or primary mouse airway epithelial cells (MAECs) were seeded into 24-well cell culture plates. Cells were electrosprayed with solutions containing either green fluorescent protein (pMGFP) or Gaussia luciferase (pGLuc) reporter plasmids. Typically 750 ng DNA was delivered per well. After 48 hrs, GFP or luciferase expression were determined by flow cytometry or luminometry respectively. Cell viability was determined by ethidium bromide/acridine orange (EBAO) staining. RESULTS: In BEAS-2B and DLKP cells electrosprayed with pMGFP, analysis of GFP expression at 48 hrs by flow cytometry demonstrated that transfection efficiencies of 30-65% were achieved. Efficiencies of 15% were achieved with pMGFP in primary MAECs. With the pGluc plasmid, luciferase acitivity at 48 hrs was increased 42-fold in DLKP cells and 10-fold in primary MAECs. Cell viability throughout was similar to controls and typically >95%. CONCLUSIONS: Profector currently delivers molecules into cells cultured in a multi-well (24Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy