215. Investigating Urokinase Plasminogen Activator Receptor (CD87), as a Potential Target for Non-Viral Gene Transfer

MOLECULAR CONJUGATES 215. Investigating Urokinase Plasminogen Activator Receptor (CD87), as a Potential Target for Non-Viral Gene Transfer Maya Thanou,1 Thomas Kean.2 Genetic Therapies Centre, Chemistry, Imperial College London, London, United Kingdom; 2Centre for Polymer Therapeutics, Pharmacy, Cardiff University, Cardiff, United Kingdom.

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The identification of cell membrane receptors for targeted delivery is important for non-viral gene therapy. The cell surface-associated urokinase-type plasminogen activator (uPA) system plays an important role in tumour cell invasion and metastasis. uPA receptor is a glycosylphosphatidyl inositol linked proteoglycan, overexpressed in malignant tumours compared to normal tissues. We selected two peptide sequences from the amino terminal fragment (ATF), responsible for receptor (uPAR) binding. These peptides, u7 (CLNGGTC) and u11 (VSNKYFSNIHW) are derived from the growth factor module of the ATF. The peptide ligands u7 (with or without protection on Cys) and u11 were synthesised. Peptides were tested in competitive (FITCuPA) binding studies on uPAR over-expressing cell lines. Peptide ligands were allowed to be bound onto polyplexes such as those made with polyethylene-imine (PEI): pDNA. FITC-uPA binding competition studies showed that FITC-uPA uptake was inhibited more by u11 compared to u7, whereas scrambled peptides showed no inhibition. However, transfection experiments showed that PEI/u7-polyplexes were more efficient in gene transfer compared to PEI/u11-polyplexes. PEI/u7 polyplexes showed a substantial increase in the gene transfer of PEI/DNA. Results also showed that u7 was more efficient in increasing the gene transfer of PEI/DNA polyplexes when it was bearing protecting groups on the cysteins. It is postulated that conformational changes of the free u11 on the polyplex limited its binding affinity to the receptor.

PS-HVJ-E or CG-HVJ-E, respectively. In case of in vivo study, we tried intravenous administration at first. In conclusion, the luciferase gene expression in liver mediated by intravenous administration of CG-HVJ-E was much higher than the luciferase gene expression mediated by PS-HVJ-E or PS-CG-HVJ-E and approximately 100 times higher than that mediated by HVJ-E alone. Next, we tried intraperitoneal administration of CG-HVJ-E containing luciferase gene to the peritoneal disseminated colon tumor model mice. As shown in Fig.2, the luciferase gene expression was specifically detected at tumor nodules. Thus, cationized gelatin-conjugated HVJE enhanced gene transfection efficiency both in vitro and in vivo. These results suggest that the conjugation with low molecular weight cationized gelatin may increase in vivo transfection efficiency of various recombinant viral vectors such as retrovirus, herpes virus and lentivirus vector.

216. Enhancement of Gene Transfection Both In Vitro and In Vivo Using Polymer Conjugated HVJ-Envelope Vector Hidetoshi Mima,1,3 Ryuji Tomoshige,2 Yasuhiko Tabata,2 Seiji Yamamoto,1 Susumu Ito,3 Katsuto Tamai,1 Yasufumi Kaneda.1 1 Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Suita City, Osaka, Japan; 2Department of Biomaterials, Fields of Tissue Engineering Institute for Medical Science, Kyoto University, Kyoto City, Kyoto, Japan; 3Department of Digestive and Cardiovascular Medicine, University of Tokushima Graduate School, Tokushima City, Tokushima, Japan. Vector development is critical for the advancement of human gene therapy. However, the use of viral vectors raises many safety concerns and most non-viral methods are less efficient for gene transfer. One of the breakthroughs in vector technology is the combination of the vector with various polymers. HVJ (hemagglutinating virus of Japan) envelope vector (HVJ-E) has been developed as a versatile gene transfer vector. In this study, we combined HVJ-E with cationized gelatin(CG) to make it a more powerful tool and assessed its transfection efficiency in vitro and in vivo. CG is one of the most suitable polymers for the complex formation with HVJ-E because of the positive charge and biocompatibility. As shown in Fig.1, we combined it with HVJ-E and used it in vitro and in vivo experiments. In addition, we investigated the mechanism of the gene transfer by means of the inhibition of fusion or endocytosis. The combination of both protamine sulfate(PS) and CG with HVJ-E, referred to as PS-CGHVJ-E, further enhanced the in vitro transfection efficiency. In CT26 cells, the luciferase gene expression of PS-CG-HVJ-E was approximately 10 times higher than that of the combination of PS with HVJ-E or the combination of CG with HVJ-E, referred to as Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy

217. Synthesis and Characterization of a Smart Triblock Copolymer Carrier for siRNA Tatiana Segura,1,2 Jeffrey A. Hubbell.1 School of Life Sciences, Swiss Federal Institute of Technology, Lausanne, Switzerland; 2Chemical Engineering, University of California, Los Angeles, CA.

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The ability to specifically down-regulate gene expression using the RNAi pathway in mammalian cells has tremendous potential as a therapeutic and as a tool in basic science. For example, after abdominopelvic surgery, the accumulation of fibrin between adjacent sides of the wound prevents the regeneration of healthy functional tissue, creating an adhesion between the two sites. Small interfering RNA (siRNA) targeted against key proteins in the blood clotting cascade that prevent the degradation of the accumulating fibrin could lead to a decrease of abdominal adhesions. However, a delivery system capable of efficiently and specifically delivering siRNA to the target cells is currently not available. Cationic polymers are some of the most widely studied and commonly utilized gene delivery vehicles, which can self-assemble with nucleic acids through electrostatic interacionts. Limitations of cationic-based gene delivery systems include toxicity, aggregation, and unpacking of the siRNA. S85