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J Pang, M Cheng, S Day, V Planelles, JC Blanks; Lentiviral Mediated Gene Transfer Into Retinal Epithelium and Photoreceptor Cells by Subretinal Injection in Neonatal Mice . Invest. Ophthalmol. Vis. Sci. 2002;43(13):704.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To compare the transduction efficiency of a lentiviral vector in the retina of retinal degenerative (rd) to normal mice following subretinal injection at various postnatal ages. Methods: Subretinal injections of lentiviral vector (pHR-CMV-GFP, 107IU/ml) were performed in rd and C57 normal mice on postnatal days (P) 1, 3, 5 and 7. After sacrifice from 1-6 weeks, eyes were enucleated, fixed and prepared for examination. Eyecups were divided into two groups at each age to evaluate the distribution of GFP expression in retinal whole mounts (Group I) and to determine the extent and the cell type of GFP expression in sections (Group II). In Group I, retinal whole mounts were prepared by removing the sclera, choroid and retinal pigment epithelium (RPE); in Group II, eyecups were embedded and sectioned for light microscopy. Results: Group I: Expression of GFP was observed in retinal whole mounts from normal and rd mice starting 1 week after injection at P1 to P7, with more expression in rd compared to normals. In both strains, less GFP expression was observed around the injection site at P1 or P3 (less than 1/4 of retina) than at P5 or P7 (1/4 to entire retina). GFP expression was at its peak 2 weeks after injection in normal mice with variable expression in 1/3 to the entire retina. In rd mice, GFP expression decreased after age P15. GFP expression lasted 6 weeks (the longest period examined) in both strains of mice. Group II: Histological examination showed fluorescence due to GFP expression in both RPE and photoreceptor (PR) cells at all ages. More GFP+ cells were observed 1 week after injection at P5 and P7 in rd and 2 weeks after injection at the same age in the normal retina. The number of GFP+ cells remained stable in the normal for at least 6 weeks following injection from P1 to P7. In rd retinas, GFP expression became stable in RPE 2-6 weeks after injection at P1-P7; GFP+ PR cells decreased dramatically after P15 so that few GFP+ PR cells were present in retinas from rd animals older than 3 weeks. Conclusion: Lentiviral-mediated GFP expression occurred in RPE and PR cells after subretinal injection, especially at P5 to P7 in rd mice. The transduction efficiency in rd decreased as the degeneration of PR cells increased. Transduction effciency in rd mice was higher when lentiviral vector was delivered by subretinal injection in older (P5-P7) compared to younger mice (P1-P3). These results correspond to our previous in vitro results.
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