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M. C. Seabra, D. C. Tracey-White, O. Tolmachov, H. J. Graham, T. Tolmachova; Correction of the Prenylation Defect in Choroideremia by Viral Delivery of the Choroideremia Gene. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4300.
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Choroideremia (CHM) is a currently non-treatable progressive degeneration of photoreceptors, retinal pigment epithelium (RPE) and choriocapillaris. It is caused by defects in the CHM/REP1 gene positioned on chromosome Xq21.2. Pathological changes develop gradually starting with a night blindness and tunnel vision in teenage patients followed by slow demise of vision within next 2-3 decades and eventually blindness. CHM/REP1 gene encodes Rab Escort Protein-1 (REP1), which participates in the lipid modification of Rab GTPases. The resulting dysfunction of Rabs is believed to be the trigger for retinal degeneration in CHM. Gene therapy is an attractive approach to combat monogenic recessive disorders and was successfully used for treatment of ocular diseases in animal models and recently in patients. CHM is a suitable candidate for the gene therapy since patients can be diagnosed before major pathological changes occur and slow progress of the disease provides a time window for the treatment. Recently we created a conditional knock-out mouse model of CHM that mimics the human condition. The aim of our current investigation is to perform preclinical gene therapy studies in CHM mouse models.
We generated several HIV-based vectors expressing REP1 and GFP that were used to transducer a canine cell line D17 and primary fibroblasts derived from CHM patients. REP1 expression was verified by Western blot and functionality was confirmed by in vitro prenylation assays. Lentiviral vectors expressing REP1 and GFP were injected subretinally into one eye of 3-week old mice, and the contralateral eye was not injected. EGFP expression was confirmed by immunofluorescence and Western blot. RPE from injected and non-injected eyes was collected, and REP1 expression in the RPE was analysed by Western blot. Correction of prenylation defect was confirmed by an in vitro prenylation assay using RPE cytosolic extracts as a source of unprenylated Rabs.
We observed persistent expression of the transgenes in the cell lines and in situ (RPE) for as long as 6 months post injection (latest time point tested). Rab prenylation defect in the CHM fibroblasts and in the RPE of CHM mice was rescued by the lentiviral vectors. Photoreceptors were not transduced.
Delivery of the lentiviral vectors carrying CHM/REP1 cDNA corrects the prenylation defect in the RPE of CHM mice and cell lines. A similar study involving adeno-associated viral (AAV) vectors is underway.
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