580. Vitreous - A Barrier to Non-Viral Ocular Gene Therapy

LIPID MEDIATED GENE TRANSFER: NON-VIRAL DELIVERY SYSTEM 579. In-Vivo Time Course of Reporter Gene Expression after Nucleic Acid Delivery to the Lateral Ventricle in Rat Brain Ellen S. Hauck,1 Shaomin Zou,1 Michael H. Nantz,2 James G. Hecker.1 1 Anesthesia, University of Pennsylvania, Philadelphia, PA; 2 Chemistry, University of California-Davis, Davis, CA. Transient expression of a therapeutic protein can be accomplished by delivery of nonviral nucleic acid-cationic lipid complexes to target tissue(s). We have previously reported the successful wide spread CNS expression of Hsp70 as well as the reporter genes eGFP and firefly luciferase after delivery of nucleic acid-cationic lipid complexes to the lateral ventricle in the rat brain(1, 2 ). We now report on the in-vivo time course of protein activity following nonviral gene delivery. Nucleic acid (cDNA or mRNA) encoding the reporter gene firefly luciferase complexed with the cationic lipid MLRI was injected into the CSF in the lateral ventricle. Time course of luciferase expression was followed using optical imaging (Xenogen IVIS). Our results show wide spread CNS expression consistent with our previously reported results. In addition, the time course of protein activity after injection of a plasmid carrying luciferase cDNA shows peak expression approximately 72 hours after vector delivery. In vitro data from CHO, NIH3T3, and primary neuronal cells transfected with the same vectors demonstrate a peak somewhat earlier, at approximately 48 hours (unpublished results). Luciferase activity in vivo drops off rapidly after 72 hours, but residual activity has been detected up to 18 days after delivery of the vector. Preliminary data after injection of mRNA-cationic lipid complex into the lateral ventricle demonstrates a more rapid increase and decay of CNS expression of luciferase. Results of the time course of expression of luciferase after cDNA or mRNA delivery will be presented as a series of sequential in vivo images, with region-of-interest (ROI) analysis. References. 1. Hecker JG, Hall LL, Irion VR. Non-viral gene delivery to the lateral ventricles in rat brain: Initial evidence for distribution and expression in the CNS. Molecular Therapy (2001), 3(3): 375-384. 2. Anderson DM, Hall LL, Ayyalapu AR, Irion VR, Nantz MH, Hecker JG. Stability of mRNA/cationic lipid lipoplexes in human and rat cerebrospinal fluid: methods and evidence for non-viral mRNA gene delivery to the CNS. Human Gene Therapy (2003)14(3):1-12.

580. Vitreous - A Barrier to Non-Viral Ocular Gene Therapy Liesbeth Peeters,1 Niek N. Sanders,1 Kevin Braeckmans,1 Stefaan C. De Smedt,1 Joseph Demeester.1 1 Laboratory of General Biochemistry, University Ghent, Ghent, Oost-Vlaanderen, Belgium. Purpose. Intravitreal injection of therapeutic DNA, complexed to non-viral carriers like cationic liposomes, may be promising to treat many severe retinal eye diseases. However, after intravitreal injection such DNA/cationic liposome complexes, so called lipoplexes, which are typically some hundreds of nanometers in size, must first diffuse through the vitreous before they can reach the retina. The aim of this study was both to elucidate whether vitreous is a barrier for the lipoplexes as well as to evaluate strategies to overcome this barrier. Methods. Fluorescent polystyrene nanospheres and lipoplexes were mixed with vitreous and their mobility was monitored by fluorescence recovery after photobleaching (FRAP), a microscopy based technique. The stability of lipoplexes and naked pDNA in vitreous was studied by gel electrophoresis. S224

Results. We showed that polystyrene nanospheres, in our first experiments used as a model for the lipoplexes, do not diffuse freely into the vitreous but adhere to fibrillar structures in the vitreous, most likely to collagen fibers. Making the surface of the polystyrene nanospheres hydrophillic by attaching hydrophillic polyethyleneglycol (PEG) chains at their surface circumvented the binding to fibrillar structures in the vitreous. FRAP revealed that “pegylated” polystyrene nanospheres, as long as they are smaller than 500 nm in size, are indeed mobile in the vitreous. It was further demonstrated that lipoplexes severely aggregate in vitreous and strongly bind to biopolymers in the vitreous which immobilizes them completely. However, as observed for the polystyrene nanopheres, coating of the lipoplexes with PEG avoided both their aggregation in the vitreous as well as their binding to fibrillar structures. Conclusions. Modifying the surface of lipoplexes with hydrophylic PEG chains prevents them from aggregation in vitreous. In this way lipoplexes are obtained which can freely move in vitreous, an absolute request to be able to reach the retina after intravitreal injection.

581. Development of a Non-Viral DNA Vector Multifunctional Envelop-type Nano Device Modified with Octaarginine Peptide Kentaro Kogure,1 Ikramy A. Khalil,1,2 Rumiko Moriguchi,1,2 Shiroh Futaki,3,4 Hideyoshi Harashima.1,2 1 Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido, Japan; 2CREST, Japan Science and Technology Agency(JST), Kawaguchi, Saitama, Japan; 3 Institute for Chemical Research, Kyoto University, Uji, Kyoto, Japan; 4PRESTO, Japan Science and Technology Agency(JST), Kawaguchi, Saitama, Japan. For efficient gene delivery to the nucleus, non-viral vectors need to overcome several barriers such as the plasma membrane, the endosomal membrane and the nuclear membrane. To overcome these obstacles, it is necessary to equip the delivery system with various functional devices [Kamiya H., et al., Drug Discov. Today 21 (2003) 990]. However, it is difficult to package all these functional devices into a single system to exert each of their functions at the appropriate time and at the correct location. Thus, we proposed a new packaging concept, “Programmed Packaging”. This concept consists of three components: (1) A program to overcome all barriers. (2) Development of functional devices and their three dimensional assignment. (3) Nano-technology for assembling all devices into a nano-size structure. In the present study, we constructed an original non-viral vector MEND based on Programmed Packaging. The MEND consists of a condensed core of plasmid DNA and lipid envelope. The packaging method is comprised of three steps, i) plasmid DNA condensation with poly-L-lysine (PLL) at a nitrogen/phosphate ratio of 2.4, ii) hydration of lipid film composed of dioleoyl phosphoatidylethanolamine (DOPE)/ cholesteryl hemisuccinate (CHEMS) for the electrostatic binding of DNA/polycation complex (DPC), and iii) sonication to package the DPC with lipids as MEND [Kogure K., et al., J. Control. Release 98 (2004) 317]. The packaging was confirmed by sucrose density gradient fractionation. The encapsulation efficiency of DNA was 73.2%. Transfection activity of MEND was 10-fold higher than DPC in NIH3T3 cells, indicating that the packaging of DPC with pHsensitive fusogenic lipid (DOPE/CHEMS) improved the transfection activity, passively due to their enhanced endosomal escape. The modification of MEND with R8 peptide, which is a promising device for efficient cellular uptake and intracellular delivery, enhanced transfection activities by two-orders of magnitude compared to MEND without R8. Furthermore, the transfection activity of the

Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy