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Miguel C. Seabra, Oleg Tolmachov, Robert E. MacLaren, Tanya Tolmachova; Pre-clinical Gene Therapy Studies In Choroideremia Mouse Models Using AAV Vectors. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1926.
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© ARVO (1962-2015); The Authors (2016-present)
Choroideremia (CHM) is a progressive degeneration of the photoreceptors, retinal pigment epithelium (RPE) and choriocapillaris caused by defects in the CHM/REP1 gene. CHM/REP1 gene encodes Rab Escort Protein-1 (REP1), which participates in the lipid modification (prenylation) of Rab GTPases. We showed previously that correction of prenylation defect in CHM cells could be achieved with lentiviral vectors carrying CHM/REP1 cDNA transgene. The aim of the present work was to perform preclinical gene therapy study using AAV vectors.
We generated AAV2/2 and AAV2/5 vectors expressing human CHM/REP1 cDNA or EGFP under control of EFS (elongation factor-1 short) or CAG (CMV-enhanced chicken beta-actin) promoter. WPRE was present on all vectors. We transduced D17 cell line and primary fibroblasts derived from CHM patients. REP1 expression was verified by Western blot and functionality was confirmed by in vitro prenylation assay. AAV vectors expressing REP1 and GFP were injected subretinally into eyes of 4-week old wild type and choroideremia mice (Chm null/WT). The contralateral eye was not injected. Expression of EGFP transgene in the RPE and neuroretina was observed by immunofluorescence. RPE from injected and non-injected eyes was collected, and REP1 expression was verified by Western blot.
We observed expression of REP1 and GFP transgenes in D17 line and CHM fibroblasts. Expression level was proportionally reduced with each cell division. In D17 cells, expression from 2 promoters and 2 serotypes was similar, while in CHM fibroblasts CAG vectors provided much higher level of transgene expression, which was estimated as a 10-fold increase in comparison to the REP1 level in fibroblasts from controls. Extracts from the cells transduced with AAV2/2-CAG-REP1 had increased prenylation activity in comparison to AAV2/2-CAG-GFP-transduced and untransduced cells. In the mouse eyes injected with AAV2/2-CAG-GFP, we observed signal in the RPE and neuroretina. In the RPE isolated from the mouse eyes injected with AAV2/2-CAG-REP1, expression of REP1 was increased in comparison to non-injected eye.
The CAG promoter provided stronger expression of the CHM/REP1 transgene in comparison to the EFS promoter. CHM/REP1 transgene delivered by AAV vectors was functional and thus the AAV2/2-CAG-REP1 vector is suitable for use in choroideremia human trials.
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