584. Improvement of Gene Delivery By Inhibition of Endonucleases

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One of the fundamental problems for gene therapy is poor gene transfer/expression efficiency resulting from DNase/endonuclease activity that plays important role of the host cell defense by degrading foreign DNA. The bulk of endonuclease activity in cells and biological fluids is formed by apoptotic endonucleases which depolymerize DNA by hydrolyzing phosphodiester bonds within the polynucleotide chain. Among the identified nine apoptotic endonucleases, deoxyribonuclease 1 (DNase I) and endonuclease G (EndoG) are the most abundant and most active in mammals. However, there is small number of available inhibitors for DNase I and EndoG, and they are mostly non-specific and inapplicable in vivo. Our in vitro experiments comparing blood plasma of DNase I or EndoG knockout mice with wild-type mice showed that DNase I and EndoG are responsible for degradation of DNA in blood plasma. The genetic inactivation of endonucleases resulted in an improvement of CFP gene expression in HCC1954 cell line after exposure with pECFP plasmid DNA with blood plasma. However using chemical inhibitors would be more translation in this regard. To search for endonuclease inhibitors, we have developed a high-throughput DNase assay using fluorophore-labeled oligonucleotide to screen a home-made chemical library and identify two inhibitors: IG-17 (1, 3-phenylene-bis-aminoguanidine hydrochloride) for DNase I and PNR-3-82 (5-((1-(2-naphthoyl)-5-methoxy-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H, 5H)-dione) for EndoG. The inhibitors are found to be specific in vitro and non-toxic in cellular assays. They significantly reduced the in vitro and in vivo (total body) endonuclease activity. In addition, we found that CFP gene delivery to various mouse organs measured by real-time RT-PCR was increased if the inhibitors were administered 2 days before intravenously injection of pECFP plasmid DNA compared to the vehicle-injected mice. This finding can be utilized for further improvement of gene delivery-based therapeutic strategies.