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Glenn Yiu, Eric Tieu, Brittany Wong, Anthony Nguyen, Zeljka Smit-McBride; Genomic Disruption of VEGF in Human Retinal Pigment Epithelial Cells using CRISPR-Cas9 Endonuclease.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1159. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the use of type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease as a potential form of gene therapy for ocular angiogenesis by genomic disruption of vascular endothelial growth factor (VEGF-A) in human retinal pigment epithelial (RPE) cells.
CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified using 3 different algorithms for predicting on-target and off-target probabilities. Single-guide RNA (sgRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the S. pyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect monolayers of cultured ARPE-19 cells, a human RPE cell line, for 7 days with puromycin to select for transduced cells. Frequency of insertion or deletion (indel) mutations was assessed by T7E1 mismatch detection assay, mRNA levels were measured by quantitative RT-PCR, and protein levels were determined by enzyme-linked immunosorbent assay (ELISA) for secreted VEGF-A. Cells infected with lentivirus expressing SpCas9 only without sgRNA were used as controls. The T7E1 assay was used to determine indel frequency at the top 3 putative off-target sites for the CRISPR sequences with the highest Cas9 activities.
A total of 6 sgRNAs 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 all 6 sgRNAs with SpCas9 resulted in indel formation in the VEGF-A gene, with frequencies ranging from 33.8 ± 5.4 % to 35.6 ± 2.6 %. One sgRNA resulted in a 1.56 ± 0.31 fold reduction in VEGF-A mRNA levels as compared with SpCas9-only control (P=0.005), using GADPH, HPRT1, and beta-2 microtubulin as reference genes. There was a corresponding decrease in secreted VEGF-A protein of 19.9 ± 2.0 % (P = 0.04). No significant indel formation in the top 3 putative off-target sites were detected, suggesting that the CRISPR-Cas9-mediated knockdown of VEGF-A is specific.
The CRISPR-Cas9 endonuclease system may reduce VEGF-A expression and secretion from human RPE cells, supporting the possibility of employing genome engineering for treatment of neovascular retinal diseases.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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