361. Metallic Particles Generated During Electric Pulses Enhance Adenovirus-Mediated Gene Transfer

Physical Methods of Delivery function. We previously showed that electroporation-mediated gene transfer of the b1 subunit of the Na,K-ATPase to the lung not only increased alveolar fluid clearance, but also improved epithelium/ endothelium barrier functions via up-regulation of tight junction proteins to provide partial treatment of lipopolysaccaride- (LPS-) injured lungs. To further investigate if we could improve treatment, we co-delivered plasmids expressing occludin, which is the one of major constituents of tight junctions located at the plasma membrane. Lung injury was induced 1 day before gene delivery by intratracheal administration of LPS (5 mg/kg) and assessed by wet-to-dry ratios, histological analysis, myeloperoxidase (MPO) measurement, and bronchoalveolar lavage (BAL) protein levels and cellularity. Delivery of b1-Na,K-ATPase and occludin alone or in combination using electroporation treated previously injured lungs in a mouse model, compared to the control plasmid pCDNA3. However, combined gene transfer of b1-Na,K-ATPase and occludin only provided similar treatment benefit to either gene delivery of b1-Na,K-ATPase or occludin alone although the expression of tight junction proteins had been increased. These results demonstrate that both b1-Na,K-ATPase and occludin regulate formation of tight junctions in severely injured lungs and thus may play a critical role in the treatment of ALI/ARDS. It indicates that epithelial or endothelial barrier functions may be involved in the treatment of ALI/ARDS.

360. Magnetofection: A Versatile Approach for Messenger RNA Delivery

Zohreh Sadat Badieyan,1 Manish K. Aneja,2 Christian Plank.1,2 Institute of Experimental Oncology and Therapy Research, Technical University Munich, Munich, Germany; 2Ethris GmbH, Martinsried, Germany.

1

Transcript therapy, using stabilized non-immunogenic messenger RNA (SNIM RNA), overcomes the potential risks of mutagenicity and immunogenicity, normally associated with DNA based gene delivery and recombinant protein therapy, respectively. Magnetofection, as a delivery system, has been shown to enhance DNA based viral and non-viral gene delivery. In this method, viral or non-viral gene carriers containing nucleic acids are associated with magnetic nanoparticles and a gradient magnetic field is applied to pull them on to the target cell surface. In this study, we established a magnetofection based protocol for enhanced mRNA delivery, using non-viral carriers. Firstly, we compared magnetofection, using different magnetic nanoparticles, to lipofection and polyfection, to find the best protocol for in vitro mRNA delivery. Then, the established protocol was generalized for co-transfection of up to three reporter SNIM RNAs (Luc, eGFP and RFP) in mono-culture of Primary Mouse Embryonic Fibroblasts (PMEF), or co-culture of PMEF and Porcine Fetal Fibroblast (PFF). In parallel, expression kinetics post magnetofection of d2eGFP SNIM RNA were determined. Magnetofection significantly improved transfection efficiency in mono culture as well as co-culture of applied cells. Using this protocol, co-transfection of different SNIM RNAs did not inhibit the transfection of every single SNIM RNA into PMEF or PFF cells. Moreover, rapid and higher peak of expression was observed with using magnetic nanoparticles. To conclude, magnetofection of mRNA presents itself as an efficient mRNA delivery system, thereby bringing transcript therapies a step closer to their clinical uses.

Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy

361. Metallic Particles Generated During Electric Pulses Enhance Adenovirus-Mediated Gene Transfer

Thierry Ragot,1,2,3 Antoine Azan,1,2,3 Lluis M. Mir.1,2,3 Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses - UMR 8203, Univ Paris-Sud, Villejuif, France; 2Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses - UMR 8203, CNRS, Villejuif, France; 3Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses - UMR 8203, Gustave Roussy, Villejuif, France. 1

In vitro and in vivo gene transfer into cells and tissues using electric pulse application is more and more attractive due to its safety, versatility and efficiency improvement of protocols. However, the DNA penetration across the plasmic membrane during electrotransfer is not fully understood, and even large uncertainties remain about which mechanism(s) are actually involved. To learn more about the process(es) implicated for another large molecular object, we have applied electric pulses during recombinant adenovirus infection of cells in suspension. We observed an important increase of recombinant adenovirusmediated GFP gene transfer when low voltage millisecond pulses were applied on poorly adenovirus-infectable cells. High quantities of metallic particles generated from the electrodes during this type of electropulsation were probably responsible of the observed effect. Indeed, if infection solution is pulsed separately and later on added to the cells, the same increase in adenovirus-mediated transfer is observed. Moreover, at a given multiplicity of infection of the virus, the effect is proportional to the quantities of metallic particles generated. After centrifugation of the pulsed infection solution, the virus was found mainly associated with metallic particles in the pellet. We are currently investigating the structure of adenovirus/metallic particle complex and the process involved in the enhancement of adenovirus entry into cells.

362. Utilization of Magnetic Field To Redirect Oncolytic Adenovirus Complexed With Iron Oxide for Augmented Therapeutic Efficacy

Joung-Woo Choi,1,2 Ji Won Park,2 You Jin Na,1 Soo-Jung Jung,1 Dayananda Kasala,1 June Kyu Hwang,1 Chae-Ok Yun.1 1 Department of Bioengineering, C ollege of Engineering, Hanyang University, Seoul, Korea; 2Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT. Adenovirus (Ad) is a widely used vector for cancer gene therapy but its therapeutic efficacy is limited on the lacked expression of the coxsackievirus and adenovirus receptor (CAR) in tumor tissues as well as specificity of targeted infection. Ad infectivity and specificity can be markedly improved by creating Ad-magnetic nanoparticles cluster complexes and subjecting them to an external magnetic field. First, we characterized the PEGylated and cross-linked iron oxide nanoparticles (PCION) through NMR, transmission electron microscope, and cytotoxicity assay. Then, we electrostatically complexed GFP-expressing replication-incompetent Ad (dE1/GFP, or dAd) with PCION, generating dAd/PCION complex. dAd/PCION showed increased transduction efficiency, independent of CAR in the absence or presence of an external magnetic field (MGF). The size distribution and zeta potential of the polyelectrolyte complex (dAd/PCION) were shown 182.3 and 24.6, respectively, compared with naked Ad (74.6 and -22.6). Due to the magnetic properties of the clustered magnetic nanoparticles, we attributed the markedly enhanced transduction efficiency to be primarily mediated by the MGF. Cancer cell killing effect was significantly increased Hmt/ PCION+MGF system compared to that of HmT or HmT/PCION, independent of CAR expression. Importantly, in MCF7 tumor S143