Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease

Abstract

PurposeTo employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells.MethodsCRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcus pyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay.ResultsFive gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% ± 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% ± 7.4% (P < 0.001), and reduction of endothelial tube formation up to 39.4% ± 9.8% (P = 0.02). No significant indel formation in the top three putative off-target sites tested was detected.ConclusionsThe CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases.

In Vivo Knockout of the Vegfa Gene by Lentiviral Delivery of CRISPR/Cas9 in Mouse Retinal Pigment Epithelium Cells

“…Recently, genomic disruption of VEGFA in human RPE (ARPE-19) cells using LV-delivered CRISPR/Cas9 demonstrated indel formation in Vegfa at frequencies up to 37.0% with corresponding decreases in secreted VEGFA protein up to 41.2%. 39 In the present study using HEK293-VEGFA cells LV-delivered CRISPR/Cas9 induced indel formation in Vegfa at frequencies up to 93% with corresponding decreases in secreted VEGFA protein up to 78%. The observed differences obtained in the two model systems may reflect variations in transduction efficiency.…”

“…Our study is the first to report LV-based retinal delivery of CRISPR/Cas9 generating genomic knockout indel formation. LVs have previously been applied for in vitro screening of various targets including Vegfa 36 , 37 , 38 , 39 but only sparsely for in vivo CRISPR/Cas9 delivery. 21 In this study, we show (1) indel formation in Vegfa , (2) functional knockout of genomic Vegfa in vitro following LV transduction, (3) delivery of LVs expressing sgRNA with SpCas9 to the RPE cells following a subretinal injection in mice, and (4) in vivo targeting of Vegfa as result of LV transduction of Cas9 in combination with three different sgRNAs.…”

A Critical Analysis of the AvailableIn VitroandEx VivoMethods to Study Retinal Angiogenesis

“…RPE secretes essential factors for the structural integrity of the retina, and it is well established that RPE has a crucial role in the development of many retinopathies through the production of many proangiogenic factors [ 12 ]. The authors used these cells either in coculture with ECs [ 88 , 129 ] or independently [ 45 , 59 , 70 , 81 , 96 , 109 , 116 , 119 , 124 , 125 , 155 , 156 ]. When in coculture, it is possible to assess direct cell-cell interactions.…”

“…The reported incubation period varies between 4 hours and 72 hours. The minimal incubation period in standard matrigel was 4 hours [ 94 , 124 ], while in GF-reduced matrigel was 6 hours [ 72 , 77 ]. Some studies used different matrices from several angiogenesis assays since nowadays there are several laboratories purchasing angiogenesis kits, all based on the matrix method.…”

CRISPR-LbCpf1 prevents choroidal neovascularization in a mouse model of age-related macular degeneration

“…The advent of anti-VEGF agents (e.g., aflibercept, bevacizumab, and ranibizumab) has led to significantly reduced retinal and choroidal neovascularizations (CNV) in the clinic 3 , 4 . However, currently used anti-VEGF therapies require frequent repetitive injections over time to sustain the therapeutic effect on ocular neovascularization 5 – 7 . Therefore, it is ideal to suppress pathologic angiogenesis with a single treatment over the long-term.…”