CHM/REP1 cDNA delivery by lentiviral vectors provides functional expression of the transgene in the retinal pigment epithelium of choroideremia mice.
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.
Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, London, UK.
Tanya
T
Tolmachova
Oleg E
OE
Tolmachov
Silene T
ST
Wavre-Shapton
Dhani
D
Tracey-White
Clare E
CE
Futter
Miguel C
MC
Seabra
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015794:Choroideremia / Q000235:genetics / Q000378:metabolism* / Q000628:therapy*; D018076:DNA, Complementary / Q000235:genetics; D005347:Fibroblasts; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D016086:Lentivirus; D051379:Mice; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D014161:Transduction, Genetic
D048868:Adaptor Proteins, Signal Transducing; C081787:CHM protein, human; D018076:DNA, Complementary