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Daniel C. Chung, Hadassah Janumala; Sustained In Vitro Gene Expression Following Non-Viral Gene Transfer of Large Transgene. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1923.
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To demonstrate stable, long-term gene expression of large retinal degeneration gene following non-viral phi C31 integrase mediated gene transfer in cultured HEK293T cells.
Plasmid construct was developed carrying the full-length cDNA for the Centrosomal Protein 290 (CEP290) gene, a large 8.2 kb gene that is responsible for Leber Congenital Amaurosis (LCA) type 10, with an additional integrase attachment site (attB) and marker gene (pCEP290.eGFP.attB). Human embryonic kidney 293T (HEK293T) cells were plated on 2 chamber Tissue Tek slides and 100mm tissue culture dishes. Cultured cells were co-transfected with the CEP290 construct and an integrase containing plasmid (pInt) and controls without DNA transfection and pCEP290.eGFP.attB only. All transfections were done using 1.5ug/ul of Lipofectamine 2000, following standard methods. Cell lysates were obtained at 1, 2, 4 and 12-week time points, and Western blot analysis was performed to evaluate CEP290 protein expression following phi C31 integrase mediated gene transfer.
Western blot analysis showed robust protein expression in both the lysate of co-tranfected cells (pCEP290.eGFP.attb with pInt) and those of therapeutic vector without integrase at week 1 and 2. At week 4, expression was limited to cells that were co-transfected, whereas those transfected without integrase returned to background levels. At 12 weeks after initial transfection, following multiple passages of the transfected cells, protein expression for co-transfected cells did not diminish. Non-transfected cells lysates showed background expression at all time points.
Phi C3 Integrase is a uni-directional, site-specific integrating recombinase that results in stable, efficient gene expression. It has a capacity to accommodate large transgenes that some viral based vectors cannot package. PhiC integrase mediated gene transfer of pCEP290.eGFP.attb to HEK 293T cells demonstrates the ability for stable protein expression. Future studies for therapeutic efficacy in animal models are being pursued.
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