354 Rheological Modification of Hydrodynamic Gene Delivery Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy S140 Physical Methods of delivery[.]
Physical Methods of Delivery 352 Improvement of Targeted Gene Transfer Mediated By Ultrasound and Microbubble in a Swine Model Shuxian Song,1 James Harrang,1 Bryn Smith,1 Kyle Morrison,2 George Kielman,2 Carol H Miao.1,3 Seattle Children’s Research Institute, Seattle, WA; 2Sonic Concepts, Inc., Seattle, WA; 3University of Washington, Seattle, WA 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, 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 >104 RLU/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 imagingguided 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 353 New Insights of Microbubbles-Cell Interactions During Sonoporation Process Anthony Delalande, Lucie Pigeon, Patrick Tauc, Eric Deprez, Patrick Midoux, Chantal Pichon Centre de Biophysique Moléculaire-CNRS UPR4301, CNRS and University of Orléans, Orléans, France; 2Laboratoire de Biologie et Pharmacologie Appliquée, CNRS, Institut d’Alembert, CNRS & Ecole Normale Supérieure Cachan, Cachan, France Upon ultrasound (US) exposure, gas microbubbles (MBs) can be expanded, moved and even destroyed These properties offer the opportunity of site-specific local drug/gene delivery Activation of MB under specific US beams induces a transient cell membrane permeabilization with a process known as sonoporation Transient pores formed at the plasma membrane are supposed to be responsible for the intracellular delivery of molecules but also the outward transport of intracellular molecules Endocytosis process has been shown to be involved during sonoporation In the field of gene transfer, several studies including ours have reported an improvement of gene delivery by US assisted MB A key to success of this technique lies in understanding mechanisms governing microbubble-cell interactions Improving our knowledge will allow us to fully exploit this method for gene therapy purpose Here, we investigate how MB and US behave towards cells under optimal conditions that allow an efficient gene transfer Studies were performed on a specific set-up composed of sonoporation chamber mounted on a fluorescence confocal microscopy coupled to a high speed camera Results obtained from our real time sonoporation indicate that, three events types could be recorded according to the acoustic parameters applied: 1) oscillating microbubbles that are stuck on the plasma membrane during the ultrasound stimulation corresponding to the cellular massage; 2) microbubbles entering into cells (translation); 3) microbubbles having a violent interaction with cells All experiments were carried out at 1MHz of frequency The first event occurs at 100 kPa a condition leading to low gene transfer The second event is observed at 150 kPa after 600ms ultrasound stimulation, an optimal efficiency of gene transfer was obtained at this sound pressure range The third event occurs for a sound pressure of 200kPa during which two phenomena were identified: either MBs were driven from the field of view due to excessive radiation force or a violent interaction of MB with the cell was observed At 200kPa the gene transfer efficiency is ten times lower than at 150 kPa due likely to toxicity It is tempting to correlate the entry of microbubbles phenomenon and efficiency of gene transfer meaning that the entry of the microbubble could promote the delivery of the plasmid into the cell MBs were found to attach preferentially on lipid rafts of the plasma membrane Most interestingly, sonoporation was able to modify chromatin compaction and a link between the plasma membrane and the effect on the nucleus has been investigated 354 Rheological Modification of Hydrodynamic Gene Delivery Takeshi Yokoo,1 Tsutomu Kanefuji,1 Takeshi Suda,1 Akiomi Ushida,2 Tomiichi Hasegawa,3 Kenya Kamimura,1 Dexi Liu,4 Shuji Terai.1 Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan; Graduate School of Science and Technology, Niigata University, Niigata, Japan; 3Faculty of Engeering, Niigata University, Niigata, Japan; 4Department of Pharmaceutical and Biochemical Sciences, College of Pharmacy, University of Georgia, Athens, GA The aim of this study is to clarify whether a rheological modification of hydrodynamic gene delivery (HGD) has a potential to sustain gene delivery efficiency with a limited injection volume S140 Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy Physical Methods of Delivery A saline-based DNA solution corresponding to 5% of body weight (NS) instead of 9%, which is used in a canonical HGD, was injected from the mouse tail vein with pCMV-Luc at 10 mg/ml in sec The activities of kinds of solutes were evaluated to enhance gene delivery efficiency Four solutes were contrast media (CM1-4) at 60 mg/ml of iodine concentration, which had various kinds of ionicity and osmolality The others were mannitol (MAN) and glycerin (GLY), which were solubilized as the same concentration as that of a major component in CM1 Liver samples were collected 24 hours after the injections, and luciferase assay and immunohistochemistry (IHC) were conducted The median values of luciferase activities after the delivery with CM1-4, MAN, GLY, and NS, and HGD were 3.2x109, 9.2x108, 3.5x109, 2.5x108, 1.4x109, 7.3x107, 1.7x106, and 1.6x1010 RLU/mg of protein, respectively In IHC, the distributions of luciferase-positive cells after the injections with the solutes were similar to that of HGD The luciferase activity of CM1 was significantly higher than that of NS (n=5, 5, p