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Ana Vanessa Oliveira, Gabriela Silva, Daniel Chung; Sustained Transgene Expression with Non-viral Gene Transfer Following Chitosan Mediated Delivery. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2714.
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A successful ocular gene therapy strategy requires efficient gene transfer and stable transgene expression. Recently, several strategies have been evaluated to promote safe, site-specific integration, long-term gene expression and the ability to mediate large gene transfer. One of the most promising technologies exploits a site-specific recombinase, the phage phiC31 integrase. We aim to develop a novel system for sustained large gene transfer by coupling hybrid polymeric vectors with phiC31-integrase to simultaneously promote enhanced delivery and transgene integration, therefore obtaining sustained transgene expression.
Chitosan-pDNA and chitosan/hyaluronic acid-pDNA nanoparticles were prepared using a NH3:PO4 ratio of 15:1. Particles were produced using either pGFPattB, pCEP290attB (>8kb) with or without pCMVINT at a molecular ratio of 2:1. These nanoparticles were used for pDNA encapsulation and transfection studies on cultured HEK293T cells. Co-transfection with pCMVINT was done by delivering the two plasmids in the same particle or separately. Transgene expression was evaluated by fluorescence microscopy and western blot analysis.
Both types of particles encapsulated pDNA efficiently, even when two plasmids were complexed simultaneously. Transfection studies indicate that transfection efficiency and transgene expression is affected by polymer size and type of polyplex used, as well as the way integrase is delivered with the polyplexes. Transgene expression decreased over time, however GFP expression was still detected 14 weeks after transfection. Over-expression of the large transgene CEP290 following delivery by chitosan polyplexes was detected at least 14 days post transfection.
We have shown that long-term transgene expression can be achieved with a non-viral approach using hybrid polymer polyplexes, as well as its ability to deliver large genes. The combined strategy of polymers and integrase is more efficient than non-integrative strategies. Long-term transgene expression will also be evaluated in vivo in mouse models of retinal pathologies, with further transfection optimization.
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