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Mark Basche, Daniel Kampik, Alexander Smith, Frank Larkin, Robin Ali; A Strategy To Induce Corneal Endothelial Cell Replication Using A Reverse Transcription Defective Lentiviral Vector. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6461.
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The osmotic activity of corneal endothelial cells (CECs) maintains the clarity of the eye’s cornea. CECs are non-replicative in humans and any decrease of their density due to aging, injury, surgical trauma etc can lead to corneal swelling and a loss of transparency. Thus transduction of CECs with an oncogene such as YAP1, which has described roles regulating cell cycle progression and contact inhibition, could be a potential therapy. However, such gene transfer carries substantial risks. We investigate whether modifications to the lentiviral lifecycle could improve the safety profile of gene delivery by mediating a low-level, temporary gene expression. Additionally we characterised the endogenous expression pattern of YAP1 in the CECs as a candidate therapeutic gene.
We modified our Lentiviral vectors by deleting the tRNA Primer Binding Site (dPBS) from the genome thus rendering the vectors incapable of undergoing reverse transcription. No promoter was necessary in these constructs. Proof of concept was carried out using a vector encoding Luciferase (dPBS-LUC2) with a PBS intact SFFV-LUC2 vector serving as the control. Luciferase expression was quantified using Promega’s Bright-Glo reagents and a BMG Labtech Luminometer. Human corneal tissue with a normal endothelium was obtained from the eye bank and maintained in ex-vivo culture. Endogenous YAP1 expression in the endothelium was investigated by immunohistochemistry.
Transduction of HEK293T cells with dPBS-LUC2 resulted in detectable amounts of LUC2 ~2-logs above background maintained for >70 hours. Transduction compared to control was >4-logs reduced. The expression of YAP1 in CECs is almost exclusively confined to the cytoplasm; a pattern consistent with a strongly contact inhibited tissue.
Lentiviral vectors carrying the dPBS mutation are capable of delivering quantifiable amounts of protein to cells. Genes carried by such vectors should never be encoded as DNA and thus integration and sustained expression are highly unlikely. Nuclear overexpression of YAP1 in CECs could provide a means to break their contact inhibition and initiate cell replication.
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