Abstract
Purpose :
CRISPR-based genome editing enables permanent suppression of pro-angiogenic factors like vascular endothelial growth factor (VEGF) and overcomes the burden of frequent intravitreal injections in patients with neovascular age-related macular degeneration (nAMD). We previously designed adeno-associated viral (AAV) vectors with S. pyogenes Cas 9 (SpCas9) and single guide RNAs (gRNAs) to target conserved regions in exon 1 of VEGFA across mice, rhesus macaques, and humans, with successful suppression of laser-induced choroidal neovascularization (CNV) in mouse eyes. Here we report the preclinical safety and efficacy of adapting this AAV-CRISPR system to target VEGFA in nonhuman primate eyes.
Methods :
We subretinally injected dual AAV8 vectors (6E11 - 6E12 vg/eye) to express SpCas9 and gRNAs targeting VEGFA or empty control into 6 adult rhesus macaque eyes followed by induction of laser CNV 4 weeks later. Clinical examination and multimodal imaging including fundus photography (FP), fundus autofluorescence (FAF), fluorescein angiography (FA), and optical coherence tomography (OCT) were performed at 2-week intervals. Aqueous, serum, and PBMCs were collected monthly to measure neutralizing antibodies (NAbs) and T-cells against AAV8 and transgenes. Retinal tissues were collected at 3 months for deep sequencing and ELISA to analyze genome editing rate and VEGFA protein levels.
Results :
Submacular injections of the dual AAV8 vectors did not trigger clinical signs of anterior chamber or vitreous inflammation at all doses, but generated concentric macular rings on FAF imaging, which corresponded to outer retinal atrophy on OCT and was more prominent at 6E12 vg/eye, intraretinal hyperreflective foci, and subfoveal accumulation of hyperreflective material, starting as early as 2 weeks after injection and persisted until the terminal point at 12 weeks, with no clear difference between the two groups. Masked grading of CNV severity from FA images showed a non-significant trend toward lower CNV severity in eyes that received SpCas9 with gRNAs targeting VEGFA.
Conclusions :
AAV-mediated CRISPR-based gene ablation of VEGFA demonstrated no significant suppression of laser-induced CNV in rhesus macaque eyes, with evidence of retinal thinning and photoreceptor disruption that may be associated with retinal inflammation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.