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Daniel Kampik, Ulrich F O Luhmann, Koji Miura Nishiguchi, Mark Basche, Alexander J Smith, Hong Han, Jennifer Williams, John Greenwood, Stephen E Moss, Frank Larkin, Robin R Ali; Lentiviral gene transfer of E2F2 induces regeneration of retinal pigment epithelium in situ. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2128.
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© ARVO (1962-2015); The Authors (2016-present)
Under normal conditions RPE cells are growth-arrested but retain their proliferative capacity. We test the hypothesis that gene transfer of E2F2, a potent transcriptional regulator of cell proliferation, to RPE cells can induce mitosis and increase RPE cell density.
We used non-integrating lentiviral vectors to deliver E2F2 (LNT-E2F2) or hrGFP (LNT-GFP) as a control to either confluent, serum starved ARPE19 cells (MOI 5) or to the RPE of C57Bl/6J mice by subretinal injection (4 x 105 infectious particles per eye). Transgene expression was verified by immunofluorescence on flatmounted RPE. BrdU was used as a marker for S phase entry. RPE cell density was determined on flatmounts after marking cell outlines by ZO-1 immunostaining. In the RPECreER/DTA mouse model, an inducible RPE-specific Cre recombinase enabled toxic RPE ablation by expression of diphtheria toxin A (DTA).
In vitro, gene transfer of E2F2 to growth-arrested ARPE19 cells induced an increase of Ki67 positive cells (2.3-fold) and uptake of BrdU (3.5-fold) 7 days after transfection. In vivo, 10 days after subretinal injection, LNT-E2F2 caused a 40-fold ±27.2 (median±SD) increase in E2F2 positive RPE cells, and a 10-fold ±4.7 increase in BrdU positive cells in both young (12 weeks) as well as old (18 months) wildtype mice. After LNT-E2F2 treatment, mean RPE cell density increased by 17±12% compared to control vector treatment (p=0.0011, n=15 eyes per group) and by 14±7% compared to untreated eyes (p=0.0071, n=15 eyes).We also tested this approach in a mouse model of RPE thinning. After DTA induction, RPECreER/DTA mice showed a 24±10% reduction in central RPE cell density where pathology was strongest (mean±SEM, n=8 eyes, p=0.0231). LNT-E2F2 subretinal injection led to increased BrdU uptake (9-fold ±3.4) and an increase in cell density (37±12%, mean±SEM) in the central RPE (p=0.0458, n=4 eyes per group).
This data suggests that E2F2 can induce RPE cell proliferation and increase cell density in vivo, especially when RPE density is reduced. Such in situ regeneration may lead to a new treatment concept for retinal degenerations with RPE loss.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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