200. A Novel Non-Viral Polymer for Plasmid Delivery to Bone Marrow Stromal Cells (BMSC)

Bone marrow stromal cells (BMSC) are multipotent stem cells, which can readily be isolated from the bones of donors and used in the treatment of many diseases due to their ability to regenerate and differentiate. BMSC can be genetically modified ex-vivo to produce therapeutic proteins, which can be transplanted back into the patient delivering therapeutic genes to many tissues. Delivery of corrective genetic material to BMSC has been achieved by using several viral vectors, but oncogenic and immunogenicity-related concerns limit the use of viral carriers. Non-viral gene delivery to BMSC can eliminate these virus-related problems. The objective of the current study is to demonstrate the BMSC transfection efficiency of a new carrier, poly-L-lysine-Palmitic Acid (PLL-PA), by using a Enhanced Green Fluorescent Protein expressing plasmid (pEGFP). We hypothesized that Palmitic Acid (PA) conjugation could enhance the gene delivery capacity of the cationic polymer PLL due to increased membrane interactions created by the lipophilic PA. Initial experiments were performed to compare the transfection efficiency and toxicity of the PLL-PA conjugate to commercial transfection agents (Fugene and Lipofectamine), an adenoviral vector, and two polymeric vectors (PLL and Polyethyleneimine, PEI). To prepare PLL-PA, PLL was reacted with palmitic acid-N-Hydroxysuccinimide (PA-NHS). The reaction was carried under N2 in dimethylformamide-dimethylsulfoxide for 2 hours, followed by precipitation in an excess of ethyl ether. Polymers were characterized by 1H-NMR. Primary BMSC were isolated from rat femurs, expanded on tissue culture flasks, and seeded on 6-well tissue culture plates. The cells were incubated for 24 hours with the complexes or the adenoviral vector expressing GFP. The expression of EGFP was measured by flow cytometry subsequently (FACS; BD FACScan). The novel PLL-PA polymer gave the highest transfection efficiency with 10-12% of BMSC expressing GFP, 1 and 3 days after transfection. The transfection efficiency of other vectors was increased from day 1 to day 3, but the efficacy of PLL-PA was superior to all vectors. Cell count data indicated that the given dose of PLL-PA was not toxic to the BMSC. Further studies explored whether the addition of Lipofectamine improved the transfection efficiency of PLL and PLL-PA. PLL-PA was again found to be most beneficial in transferring pEGFP as compared to unmodified PLL and Lipofectamine (13% vs. 2% and 7%, respectively). Adding Lipofectamine to PLL and PLL-PA resulted in slight improvement (2-4%) in the transfection efficiency. These results indicated the beneficial effect of having both a DNA-condensing polymer and a membrane-penetrating lipid as part of a designed carrier. In conclusion, PA-modified PLL was found to be an efficacious transfection agent as compared to commercially available reagents. Future efforts will be directed towards further improving the transfection efficiency of the novel PLL-PA polymer, and visualizing the intracellular path of the DNA/carrier complexes using fluorescent microscopy.

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