Novel plasmid constructs with angiogenic growth factors genes - human VEGF, HGF and angiopoietin-1 for therapeutic angiogenesis



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Abstract

Efficacy of critical limb ischemia gene therapy can be improved by application of novel plasmid vectors with higher transgene expression. The goal of this study is to evaluate in vitro and in vivo expression of angiogenic growth factors after gene transfer using a novel plasmid vector PC4W.
Plasmid constructs with genes of human VEGF185 CpC4W-hHGFopt), HGF CpC4W-hHGFopt) and angiopoietin-1 (pC4W-hAng-1optJ were tested in vitro in a HEK293T cell culture. Cells were subjected to calcium-phosphate transfection and conditioning medium samples were assayed for transgene levels using Western blot and ELISA. Results were compared with commercially available pcDNA3 based vectors encoding the same growth factors. Reverse transcription PCR was used to assay transgene expression in BALB-c mice ischemic muscle.
ELISA and Western blotting data suggest that PC4W based constructs give a higher protein output of about 2-2,5 fold compared with pcDNA3 based plasmids. Optimization of nucleotide sequence in growth factors cDNA results in additional increase in transgene expression. RT-PCR data shows that expression of human HGF persists in murine ischemic skeletal muscle up to 14 days after gene transfer.
Our results indicate that novel plasmid constructs for angiogenic growth factors expression have a good efficacy in vitro and in vivo and can be used for VEGF1B5, HGF and angiopoietin-1 expression in human cell culture and in experimental animals' tissue. At the moment all developed constructs pass through a series of experiments in animal ischemia models and will be used for combined gene therapy development.

About the authors

P I Makarevich

Faculty of Basic medicine, Lomonosov Moscow State University, Moscow

Faculty of Basic medicine, Lomonosov Moscow State University, Moscow

A Ya Shevelev3

LLC «MONA», Moscow

LLC «MONA», Moscow

I N Rybalkin

LLC «MONA», Moscow

LLC «MONA», Moscow

N M Kashirina

LLC «MONA», Moscow

LLC «MONA», Moscow

L N Lipatova

LLC «MONA», Moscow

LLC «MONA», Moscow

Z I Tsokolaeva

Institute of experimental cardiology, Russian cardiology research center, Moscow

Institute of experimental cardiology, Russian cardiology research center, Moscow

E K Shevchenko

Institute of experimental cardiology, Russian cardiology research center, Moscow

Institute of experimental cardiology, Russian cardiology research center, Moscow

I B Beloglazova

Institute of experimental cardiology, Russian cardiology research center, Moscow

Institute of experimental cardiology, Russian cardiology research center, Moscow

M A Boldyreva

Institute of experimental cardiology, Russian cardiology research center, Moscow

Institute of experimental cardiology, Russian cardiology research center, Moscow

К A Rubina

LLC «Gene and Cell therapy», Moscow

LLC «Gene and Cell therapy», Moscow

T N Vlasik

LLC «MONA», Moscow

LLC «MONA», Moscow

Ye V Parfyonova

Institute of experimental cardiology, Russian cardiology research center, Moscow

Institute of experimental cardiology, Russian cardiology research center, Moscow

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