Efficiency of lentiviral transduction during development innormal and rd mice

[摘要] Purpose: To compare the transduction efficiency of a lentiviralvector in the retina of normal mice and retinal degenerative (rd)mice following subretinal injection at various postnatal ages.Methods: Subretinal injections of lentiviral vector (pHR-CMV-GFP,107IU/ml) were performed in normal (C57/6J) and rd mice onpostnatal days P1 to P7 using a trans-scleral method and on days P14-P35by a trans-corneal method. One to six weeks later the eyes were preparedfor histological analysis. GFP positive cells were identified in retinalsections and retinal whole mounts to determine the overall extent anddistribution of lentiviral transduction.Results: Expression of GFP was observed adjacent to the injectionsite starting about 1 week after injection in both normal and rdmice and lasted 6 weeks (the longest period examined). In normal mice,GFP expression continued to increase and peaked around 2-3 weeks afterinjection with expression varying from approximately one quarter to theentire retina. GFP expression peaked earlier in rd mice injectedfrom P1 to P7 compared to normal mice. Lenti-GFP expression decreasedrapidly in rd mice older than P15. This was attributed to a periodof intensive photoreceptor (PR) degeneration characteristic to thismutant. Retinal GFP expression was virtually absent in eyes injectedafter P14 in both normal and rd mice. Histological sections from P3injected eyes showed GFP expression 9 days post-injection in bothretinal pigment epithelium (RPE) and photoreceptor (PR) cells. GFPexpression in RPE cells was stronger than that in PR cells. Both rodsand cones expressed the lenti-GFP. GFP expression was limited to the RPEof normal mice if injections were performed at P14 or later. In rdmice, GFP expression in RPE was observed one week after injection at P1;GFP+-PR and -RPE cells were first detected 9 days after injectionat P1, and 7 days after injection at P3-P7; RPE cells and occasionalMuller cells around the injection site were GFP+ when theinjection was performed at P14 or later.Conclusions: Lentiviral-mediated GFP transduction of RPE wasefficient and sustained at all ages examined in both the normal andrd mouse. Trans-scleral, subretinal injection of lenti-GFP duringthe first postnatal week produced age-dependent transduction of PR cellsin both mouse strains. Lenti-GFP expression was absent in both mousestrains if injections occurred after P14. There was a dramatic decreasein the transduction efficiency in rd mouse retinas corresponding tothe degeneration of PR cells. However, the early stages of retinaldegeneration in rd mice appeared to increase the transductionefficiency of PR cells. These data suggest that both age and degree ofPR degeneration are important parameters to consider when designing genetherapy experiments or protocols.

[学科分类] 生物化学/生物物理