Lentiviral gene transfer of E2F2 induces regeneration of retinal pigment epithelium in situ

Abstract

Purpose : 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.

Methods : 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 RPE^CreER/DTA mouse model, an inducible RPE-specific Cre recombinase enabled toxic RPE ablation by expression of diphtheria toxin A (DTA).

Results : 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, RPE^CreER/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).

Conclusions : 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.