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CHM/REP1 cDNA delivery by lentiviral vectors provides functional expression of the transgene in the retinal pigment epithelium of choroideremia mice
Author(s) -
Tolmachova Tanya,
Tolmachov Oleg E.,
WavreShapton Silene T.,
TraceyWhite Dhani,
Futter Clare E.,
Seabra Miguel C.
Publication year - 2012
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1652
Subject(s) - transgene , choroideremia , biology , retinal pigment epithelium , microbiology and biotechnology , transduction (biophysics) , viral vector , genetic enhancement , gene delivery , transfection , retinal , cell culture , gene , genetics , biochemistry , recombinant dna
Background Choroideremia (CHM) is a progressive X‐linked degeneration of three ocular layers: photoreceptors, retinal pigment epithelium (RPE) and choroid, caused by the loss of Rab Escort Protein‐1 (REP1). As a recessive monogenic disorder, CHM is potentially curable by gene addition therapy. The present study aimed to evaluate the potential use of lentiviral vectors carrying CHM/REP1 cDNA transgene for CHM treatment. Methods We generated lentiviral vectors carrying either CHM/REP1 cDNA or EGFP transgene under the control of the elongation factor‐1α promoter (EF‐1α) or its shortened version EFS. We transduced human (HT1080) and dog (D17) cells, CHM patient's fibroblasts and mouse primary RPE cells in vitro , as well as wild‐type and CHM mouse retinas in vivo by subretinal injections. Transgene expression was confirmed by immunoblotting, fluorescence‐activated cell sorting, immunofluorescence and confocal microscopy. CHM/REP1 transgene functionality was assessed by an in vitro prenylation assay. Results Lentiviral vectors with CHM/REP1 and EGFP transgenes efficiently transduced HT1080, D17 and CHM fibroblast cells; CHM/REP1 transgene lead to an increase in prenylation activity. Subretinal injections of lentiviral vectors into mouse retinas resulted in efficient transduction of the RPE (30–35% of total RPE cells transduced after a 1‐µl injection), long‐term expression for at least 6 months and a decrease in amount of unprenylated Rabs in the CHM RPE. Transduction of neuroretinal cells was restricted to the injection site. Conclusions Lentiviral CHM/REP1 cDNA transgene rescues the prenylation defect in CHM mouse RPE and thus could be used to restore REP1 activity in the RPE of CHM patients. Copyright © 2012 John Wiley & Sons, Ltd.