121. In Vivo Gene Delivery from Modified PLG Scaffolds

CHEMICAL AND MOLECULAR CONJUGATES I blots, flow cytometry and immuno-fluorescence staining). We conrmed that HeLa cells strongly expressed FR whereas the other cell lines expressed this receptor at a much lower extent. In free folate medium, the transfection ability of the various formulations was then tested in vitro, using the lipofectamine (Charge Ratio 2) as a positive control. We observed that the MM18/FA-PEG570-diether (90:10) (CR 1) was the most efcient formulation to transfect HeLa cells (5,8 fold higher than control). On A549 cells, this formulation used at CR 2 reached equivalent efciency as the control. On 16HBE14o(-) cells, the MM18/FA-PEG570-diether formulation (95:5; CR 1) showed the best efciency (35 fold higher than control). Moreover, competitive inhibition experiments (by adding free folate in the medium) showed that the transfection efciency and specicity of the carriers equipped with FA pattern was drastically decreased, from 10 nM of free folate for the cell lines that poorly expressed FR such as A549, and 25 nM for the HeLa cells that strongly expressed FR. This is clearly demonstrating that transfection was depending on the number of FR present at the membrane. These data show headway in cell-targeting systems of gene transfer making folate-linked particles of great interest for future therapeutic applications, especially for aerosol in vivo delivery.

121. In Vivo Gene Delivery from Modied PLG Scaffolds

Misael O. Aviles, Lonnie D. Shea. Chemical and Biological Engineering, Northwestern University, Evanston, IL. Tissue engineering strategies are used to enhance the native ability of the body to heal. Many strategies are based on the use of polymeric scaffolds to provide mechanical support and promote cell inltration, combined with the delivery of tissue inductive factors. Gene delivery from scaffolds has the potential to target one or more processes that limit regeneration by delivering DNA encoding for inductive proteins in tissue repair. Releasing DNA from biomaterials often results in transgene expression adjacent to the material, yet scaffolds aimed at regenerating lost tissue should maintain expression within the scaffold. Towards this goal, PLG scaffolds were modied with cationic polymers to reversibly associate with naked plasmid and delay release. Multiple cationic polymers, including PEI, PLL, and dopamine were used to modify the microspheres used to make the scaffolds. The scaffolds were implanted in the abdominal fat of mice and the quantity and length of transgene expression was measured using a luciferase vector as a reporter gene. Changes in release prole of plasmid did not signicantly impact the quantity and length of transgene expression, suggesting that the amount of DNA initially available for transfection is critical to achieve gene expression. Transfected cells were observed within the scaffold during the rst few weeks; however, at later times, transfected cells were observed primarily outside the scaffold. An alternative strategy to retain DNA within the scaffold involved lling the pores with a brin hydrogel, which provides an additional barrier to diffusion from the scaffold. Initial studies with plasmid did not achieve signicant transgene expression, and we subsequently employed delivery of a lentiviral vector for more efcient delivery. Initial in vivo studies had demonstrated that transgene expression occurs at a higher level and persists for extended times. Localization of transgene expression within the scaffold is under investigation. These results have multiple implications in the design of scaffolds for gene delivery, and the gene expression obtained from these scaffolds have potential for cell transplantation and gene therapy applications.

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122. The Inuence of Charge Density and Substituent Composition on ImidazoliumContaining Polymers for Mediated DNA Delivery Michael H. Allen,1 Matthew D. Green,1 Timothy E. Long.1 1 Chemistry, Virginia Tech, Blacksburg, VA.

To improve non-viral gene delivery for more effective gene therapy, the need to synthesize novel therapeutics exists to better establish the structure-property-transfection relationship. One vehicle for non-viral gene delivery widely investigated is cationic polymers. Here, we explore the use of novel, cationic imidazolium-based homopolymers and copolymers as efcient gene delivery therapeutics. Found in the amino acid histidine, the imidazole ring acquires a cationic charge below a pH of 6, and the accumulation of the histidine residues inside an endosome causes endosome destabilization and rupture through a proton sponge effect. We determined the inuence of varying the substituent attached to the tertiary nitrogen of the imidazole ring and the resulting difference in charge density of the copolymers on cytotoxicity, DNA binding, and in vitro plasmid DNA delivery efciency in COS-7 cells. The copolymers were synthesized using conventional free radical polymerization and were modied postpolymerization creating a series of copolymers containing different charge-densities. MTT assays revealed lower charge densities were less cytotoxic; however, heparin competitive binding experiments revealed the higher charge density copolymers bound DNA more effectively. We have found the difference in charge density of these copolymers signicantly affected transfection efciency, and luciferase reporter gene expression increased as charge density decreased. Also, varying the substituent on the imidazole ring also greatly affected the cellular uptake of the polymer/DNA complexes. In conclusion, our data suggested the difference in charge density of the novel imidazolium copolymers dramatically affected transfection efciency as well as varying the substituent on the imidazole ring, providing a better understanding of structure-property-transfection relationships.

123. Concentrated PEI Formulations Retain Biological Efcacy Following Long-Term Storage at 4°C

Lee A. Davies,1,2 Graciela A. Nunez-Alonso,1,2 Stephen C. Hyde,1,2 Deborah R. Gill.1,2 1 Gene Medicine Research Group, University of Oxford, Oxford, United Kingdom; 2UK Cystic Fibrosis Gene Therapy Consortium, Oxford, London, Edinburgh, United Kingdom.

Aerosol delivery of gene transfer agents is likely to be the most practical route for routine vector administration to the lung. When complexed with plasmid DNA (pDNA), the cationic polymer, 25kDa branched polyethyleneimine (PEI) retains transfection efciency following aerosolisation and consequently is a popular choice for lung gene transfer. Until recently, progress towards the clinical use of PEI was limited by the low pDNA concentration achievable in standard PEI formulations. However, we have shown that concentrated PEI (cPEI) formulations containing up to 8 mg/ml pDNA can be prepared by controlled ultraltration of pDNA/PEI complexes, leading to robust levels of gene expression in the lungs of mice and sheep following aerosol delivery. We have capitalised on this important development to further exploit the potential of cPEI aerosol delivery for use in the clinic, focusing on formulations that are physically stable and suitable for long-term storage following manufacture, an absolute requirement for clinical utility. Plasmid DNA encoding rey luciferase reporter gene was complexed with branched 25kDa PEI (N:P of 10:1) at an initial pDNA concentration of 0.2 mg/ml in sterile water and concentrated to 2.0 ± 0.2 mg/ml using a pressurised ultraltration cell incorporating a 100kDa cut-off cellulose membrane. Complexes were lter sterilised and aliquots Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy