Abstract
Purpose :
Frequent intravitreal injections of anti-vascular endothelial growth factor (VEGF) for choroidal neovascularization (CNV) pose a significant clinical burden for patients with neovascular age-related macular degeneration (AMD). We previously demonstrated successful genomic ablation of Vegfa using S. pyogenes Cas9 (SpCas9) and partial suppression of laser-induced CNV in mouse eyes. Here, we evaluate the use of paired guide RNAs (gRNAs) to simultaneously target 2 loci in the Vegfa gene to enhance VEGF suppression.
Methods :
We identified paired gRNAs for SpCas9 to target conserved sequences in exon 1 of Vegfa in mice, macaques, and humans using computational methods in silico. DNA vectors expressing SpCas9 and respective gRNA pairs were transfected into human HEK293T cells to measure gene editing events by deep sequencing, and VEGF-A suppression by ELISA. Dual AAV8 vectors expressing SpCas9 and paired gRNAs were subretinally injected into mouse eyes, followed by isolation of retinal pigmented epithelial (RPE) cells 3 weeks later to measure gene editing events by deep sequencing and VEGF-A suppression by ELISA.
Results :
In HEK293T cells, expression of SpCas9 with paired gRNAs generated 95±3% truncation, 0.5±0.84% inversion, and 0.15±0.25 % indel mutations, whereas SpCas9 with single gRNAs generated 35±16% indels. Genomic ablation of Vegfa using SpCas9 with paired gRNAs resulted in 26±6.7% VEGF protein suppression, compared with 17±8.1% with single gRNAs. In mouse eyes, AAV8-mediated expression of SpCas9 with paired gRNAs produced 29±11.1% truncation, 18.45±9.3% indel mutation and, with a 35±22% reduction in VEGF expression in RPE cells in vivo. In contrast, SpCas9 with single gRNAs received eyes induced 7.1±6.4% indel mutation and 10±7.3% reduction in VEGF expression in RPE cells.
Conclusions :
CRISPR-based genome editing of Vegfa using paired gRNAs produces higher rates of gene ablation and greater VEGF suppression than single gRNA platforms, and may have greater translational potential for treatment of neovascular AMD.
This is a 2020 ARVO Annual Meeting abstract.