Host immune responses from AAV-CRISPR-mediated gene editing of VEGFA in nonhuman primate eyes.

Abstract

Purpose : CRISPR-based gene editing enables permanent suppression of VEGFA as a potential treatment for choroidal neovascularization (CNV), but early studies using subretinal AAV-mediated SpCas9 delivery in nonhuman primate (NHP) eyes showed evidence of retinal disruption. Here, we examine the local and systemic immune responses of these NHPs up to 12 weeks after gene editing therapy.

Methods : We subretinally injected AAV8 vectors expressing SpCas9 and gRNA targeting VEGFA or empty control with a GFP reporter into contralateral eyes of 4 adult rhesus macaques, followed by laser-induced CNV 4 weeks later. Multimodal imaging including fundus photography (FP), fundus autofluorescence (FAF), fluorescein angiography (FA), and optical coherence tomography (OCT) were performed at weeks 2, 4, 8, and 12 after injections. We collected serum and PBMCs monthly to measure neutralizing antibodies (NAbs), binding antibodies (BAbs), and T-cell responses against AAV8 and transgenes using ELISpot assays. Retinal tissues were collected at 12 weeks for deep sequencing, ELISA, and immunohistochemistry (IHC) to analyze genome ablation rate, VEGF protein levels, and retinal structural changes. Tissues from brain, heart, liver, kidney, spleen, and gonad were collected to assess systemic biodistribution by quantitative PCR against GFP.

Results : Eyes that received low dose AAV8-CRISPR-Cas9 with gRNA targeting VEGFA demonstrated a trend toward reduced VEGF protein level and CNV severity. However, both eyes receiving active gRNAs and controls generated unexpected concentric macular rings, subfoveal hyperreflective material, and outer retinal disruption that were more prominent at the higher dose. IHC showed disrupted rod and cone structure, increased GFAP+ cells, and loss of ramified Iba-1+ cells, suggesting glial and microglial activation. Subretinal AAV8 did not trigger an increase in NAbs or BAbs against AAV8, SpCas9, and GFP. Finally, peripheral tissues showed minimal systemic biodistribution of AAV8, except one animal that showed some vector distribution to cardiac tissues.

Conclusions : Subretinal AAV-CRISPR-mediated ablation of VEGFA in NHP eyes enabled suppression of VEGF and CNV, but also demonstrated local inflammation and photoreceptor disruption, with minimal systemic immune responses. Future studies are needed to determine the mechanism underlying local inflammation and injury.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.