Inhibition of Choroidal Neovascularization in Rap1b^-/- Mice by Self complimentary-AAV2-delivered Constitutively Active Rap1a in Retinal Pigment Epithelium

Abstract

Purpose : Loss of the retinal pigment epithelium cell (RPE) barrier integrity is implicated in human AMD. We previously found that pharmacologic activation of a small GTPase, Rap1, improved RPE barrier integrity and reduced choroidal neovascularization (CNV) after laser injury in Rap1b^+/+ and Rap1b^-/- mice compared to vehicle treatment. We tested the hypothesis that activation of Rap1a in RPE reduces CNV formation in Rap1b^-/- mice using self-complementary adeno-associated virus 2 (Sc-AAV2)-delivered gene therapy in a murine laser-induced CNV model.

Methods : Six week old male and female C57Bl/6 wild type (WT) and Rap1b^-/- mice were used. WT mice received subretinal injections of 1 µL 1X10⁸ vp/µL or 5X10⁸ vp/µL sc-AAV2 expressing either GFP (AAV2-RPE65-GFP) or constitutively active human Rap1a (AAV2-RPE65-Rap1aCA-GFP) driven by RPE65 promoter, or phosphate buffered saline (PBS). Virus transduction was assessed by GFP visualization from week 2 to 6 using Micron III imaging. RPE-specific GFP expression was determined by immunostaining (IHC) for GFP and RPE65 colabeling in RPE/choroidal cryosections. Rap1b^-/- mice were injected with 1 µL 5X10⁸ vp/µL AAV2-RPE65-GFP, or AAV2-RPE65-Rap1aCA-GFP into the subretinal space. Five weeks after injection, laser photocoagulation (532 nm diode laser, 400 mW intensity, 100 ms duration) was performed. One week after laser, RPE/choroidal flat mounts were prepared, stained with isolectin and GFP antibody, and imaged with confocal microscopy and summed areas of 3 um optical Z-stacks were analyzed for CNV volume by ImageJ within regions of GFP. Statistics were performed using two-tailed Student’s t-test.

Results : GFP was visualized in WT mice 4-6 weeks after virus injection, and mice injected with 5X10⁸ vp/µL of AAV2 yielded a stronger GFP expression. GFP staining was detected in retinal cryosections and co-localized with RPE65-stained RPE. PBS injected mice did not show GFP expression. AAV2-RPE65-Rap1aCA-GFP injected Rap1b^-/- mice showed a 7.6-fold reduction in CNV volume (0.1299±0.0548) compared to AAV2-RPE65-GFP injected Rap1b^-/- mice (1±0.3023) (p=0.0009 vs. AAV2-RPE65-GFP, n=5-10).

Conclusions : Increased Rap1a activity in RPE by Sc-AAV2 gene therapy effectively reduced CNV following laser injury. Ongoing studies are warranted to determine effects of the AAV2-RPE65-Rap1aCA-GFP on retinal function.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.