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Subrata K. Das, xiaohui zhang, Ling Luo, Hironori Uehara, Nirbhai Singh, Bonnie Archer, Balamurali K. Ambati; A Novel Approach For Sustained Treatment Of Neovascularization By Flt23k Integration Into Genome. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1909.
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Vascular endothelial growth factor A (VEGF-A) and its receptors play important roles in neovascularization. VEGFR1 (also known as Flt-1) is the most potent receptor among others. Flt23K receptor (FLT domains 2 and 3 tagged with KDEL) is able to bind and sequester VEGF-A and can be used to suppress neovascularization. The delivery and long-term incorporation of the Flt23K is a major hurdle. Our objective is incorporation of the Flt23K gene into the genome by transpositional insertion, leading to long term reduction of neovascularization in response to an injury without the need for repeated intraocular injections.
To achieve genomic integration of Flt23K gene, we utilized transposon based helper-independent piggybac plasmid (piGENIE). Flt23K and DSRed Express 2 cDNA were cloned in piGENIE. The plasmid lacking transposase (pBt gene) gene was used as a control. To examine genomic integration and expression of transgenes in vitro, we transfected the plasmids into HeLa cells and cultured up to 10 passages. Fluorescence microscopy was done to check the transfection efficiency with DS Red fluorescence. Also Flt23K expression was examined by western blot with Anti-VEGF Receptor 1 antibody. Genomic integration was analyzed using PCR based genome walking approach. For in-vivo study we used laser induced retina model. Plasmids with 10% neuroporter were injected into the vitreous space of C57BL/6J mice. Choroidal neovascularization (CNV) was induced by laser photocoagulation one month after plasmid injection. CNV was stained with Isolectin GS-IB4 one week after laser injury and CNV volume was calculated with the confocal microscope software.
Genome walking study in transfected HeLa cell indicated insertion of the transgenes into genome. HeLa cells transfected with control plasmid did not indicate any insertion of transgenes. Flt23K protein (25kDa) was detected only in transfected HeLa cell lysate but not in control. CNV volume was sharply reduced by 53% in piggybac injected eyes compared to control.
Our results indicate that nonviral gene therapeutic approach based on Flt23K expression in invitro cell model was significantly pronounced and showed distinctly reducing effective in choroidal neovascularization in C56BL/6J Mouse model. Our approach can be extrapolated as novel way to treat pathological neovascularization by long term expression of Flt23K.
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