VMD2 Promoter-mediated Gene Therapy Optimizes Active Rap1a Expression in the Retinal Pigment Epithelium of Wild Type Mice

Abstract

Purpose : Our previous studies found that subretinal injections of self-complementary adeno-associated viral vector 2 (scAAV2) with a retinal RPE65 promoter (scAAV2-RPE65) to drive constitutively active Rap1a (CARap1a) expression in the RPE significantly reduced choroidal neovascularization in Rap1b^-/- mice but not in wild type (WT) mice. We interpreted the different effects of scAAV2-RPE65-CARap1a in Rap1b^-/- vs. WT mice to be due to the weak RPE65 promoter activity. Here, we compared the RPE65 promoter in scAAV2-RPE65 to the VMD2 promoter in AAV2-VMD2 at driving CARap1a expression in the retinal pigment epithelium (RPE) of WT mice.

Methods :
AAV2-VMD2-CARap1a, scAAV2-RPE65-CARap1a and respective controls, AAV2-VMD2-GFP and scAAV2-RPE65-GFP vectors, were created. Six-week old male and female C57Bl/6 WT mice received bilateral subretinal injections of 1 µL 5X10⁸ vp/µL of the same virus: AAV2-VMD2-CARap1a, scAAV2-RPE65-CARap1a, AAV2-VMD2-GFP or scAAV2-RPE65-GFP. Vector transduction was assessed by GFP visualization with Micron IV live imaging. Five weeks after injections, each eye received 4 spots of laser photocoagulation (532 nm diode laser, 400 mW intensity, 100 ms duration) 2 disc diameters from the optic nerve, avoiding major vessels. Seven days later, euthanized mice had one eye processed for protein by western blots of Rap1, LC3A/B and β-actin (n=6 eyes) and the other eye for immunohistochemistry (IHC) of GFP and/or RPE65 (n=3 eyes). Statistics were performed using two-tailed Student’s t-test.

Results :
GFP was visualized in WT mice 5 weeks after either AAV2-VMD2 or scAAV2-RPE65 injection. RPE specific vector transduction by IHC of GFP colabeled with RPE65-stained RPE was greater in AAV2-VMD2-injected eyes compared to scAAV2-RPE65-injected eyes. IHC of GFP in scAAV2-RPE65-injected eyes was also seen in retinal ganglion cells. Compared to eyes injected with scAAV2-RPE65-CARap1a, eyes with AAV2-VMD2-CARap1a had greater expression of Rap1a in RPE/choroids (p=0.005). However, an autophagy regulator, LC3A/B protein in RPE/choroid was not increased in AAV2-VMD2-CARap1a injected eyes compared to either AAV2-VMD2-GFP or scAAV2-RPE65-CARap1a.

Conclusions :
The VMD2 promoter has greater specificity for vector mediated expression in the RPE than the RPE65 promoter and increased expression of CARap1a without increasing autophagy.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.