Comparison of CRISPR-based Genome Editing of VEGF-A as Treatment Strategy for Choroidal Neovascularization.

Abstract

Purpose : Frequent intravitreal injections of anti-vascular endothelial growth factor (VEGF) for choroidal neovascularization (CNV) pose a significant clinical burden for patients with exudative age-related macular degeneration (AMD). Clustered regularly interspaced palindromic repeats (CRISPR)-based genome editing holds the promise for permanent VEGF suppression, but the optimal approach is unclear. Here, we compare the efficacy of Cas9 endonucleases to target the VEGF-A gene and suppress laser-induced CNV in mouse.

Methods : Guide RNAs (gRNAs) for S. pyogenes Cas9 (SpCas9) and S. aureus (SaCas9) were computationally identified based on predicted on-target and off-target scores, then co-transfected with their corresponding Cas9 endonucleases into NIH-3T3 cells to assess insertion-deletion (indel) mutations in vitro using the T7E1 mismatch detection assay. The gRNAs with highest indel frequencies were packaged into AAV8 and injected subretinally into mouse eyes using a dual-vector approach for SpCas9 and single-vector system for SaCas9. Viral transduction was confirmed on fundus imaging and histology. In vivo indel frequencies were evaluated by deep sequencing and Tracking Indels by DEcomposition (TIDE) analysis. VEGF levels were assessed by ELISA. Laser-induced CNV was created at 6 weeks after AAV injection, and CNV suppression was determined by fluorescein angiography (FA), optical coherence tomography (OCT) and OCT-angiography (OCT-A) 1-week later.

Results : Using the 2 gRNAs with the highest on-target score for each ortholog, indel frequencies in vitro were similar for dual-vector SpCas9 (30-32%) and single-vector SaCas9 (33-34%) (P>0.05). AAV8-mediated delivery of Cas9 orthologs showed robust transduction of the RPE and photoreceptors. Genomic DNA extracted from RPE-choroid tissues confirmed accurate genome editing of the VEGF-A gene in vivo, with indel frequencies up to 4.3% for SpCas9 and 1.5% for SaCas9 (P<0.05), as compared with scrambled gRNA controls. Eyes that received dual-vector SpCas9+gRNA showed decreased VEGF protein and reduction of laser-induced CNV in vivo.

Conclusions : CRISPR-based genome editing of VEGF may be effective in suppressing CNV. Dual-vector AAV8 delivery of SpCas9 appears superior to single-vector SaCas9 in vivo, despite similar cutting efficiency in vitro.

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