Efficiency of lentiviral transduction during development in normal and rd mice

To compare the transduction efficiency of a lentiviral vector in the retina of normal mice and retinal degenerative (rd) mice following subretinal injection at various postnatal ages.

Subretinal injections of lentiviral vector (pHR-CMV-GFP, 107IU/ml) were performed in normal (C57/6J) and rd mice on postnatal days P1 to P7 using a trans-scleral method and on days P14-P35 by a trans-corneal method. One to six weeks later the eyes were prepared for histological analysis. GFP positive cells were identified in retinal sections and retinal whole mounts to determine the overall extent and distribution of lentiviral transduction.

Expression of GFP was observed adjacent to the injection site starting about 1 week after injection in both normal and rd mice and lasted 6 weeks (the longest period examined). In normal mice, GFP expression continued to increase and peaked around 2-3 weeks after injection with expression varying from approximately one quarter to the entire retina. GFP expression peaked earlier in rd mice injected from P1 to P7 compared to normal mice. Lenti-GFP expression decreased rapidly in rd mice older than P15. This was attributed to a period of intensive photoreceptor (PR) degeneration characteristic to this mutant. Retinal GFP expression was virtually absent in eyes injected after P14 in both normal and rd mice. Histological sections from P3 injected eyes showed GFP expression 9 days post-injection in both retinal pigment epithelium (RPE) and photoreceptor (PR) cells. GFP expression in RPE cells was stronger than that in PR cells. Both rods and cones expressed the lenti-GFP. GFP expression was limited to the RPE of normal mice if injections were performed at P14 or later. In rd mice, GFP expression in RPE was observed one week after injection at P1; GFP+-PR and -RPE cells were first detected 9 days after injection at P1, and 7 days after injection at P3-P7; RPE cells and occasional Muller cells around the injection site were GFP+ when the injection was performed at P14 or later.

Lentiviral-mediated GFP transduction of RPE was efficient and sustained at all ages examined in both the normal and rd mouse. Trans-scleral, subretinal injection of lenti-GFP during the first postnatal week produced age-dependent transduction of PR cells in both mouse strains. Lenti-GFP expression was absent in both mouse strains if injections occurred after P14. There was a dramatic decrease in the transduction efficiency in rd mouse retinas corresponding to the degeneration of PR cells. However, the early stages of retinal degeneration in rd mice appeared to increase the transduction efficiency of PR cells. These data suggest that both age and degree of PR degeneration are important parameters to consider when designing gene therapy experiments or protocols.