Abstract
Purpose :
Inhibitors such as Lucentis and Eylea have become important therapies for ocular angiogenesis. While these drugs are effective, continuous ocular injections are required. CRISPR-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) system has been demonstrated to be a simple and efficient tool for genome editing and protein depletion in cultured human cells and mice. The goal of this project was to explore a novel approach for blocking the signaling transduction from VEGF to VEGFR2 using CRISPR/Cas9 technology.
Methods :
Four guide RNAs (gRNA) (k11-k14) based on exon 2 of human VEGFR2 genomic sequence (NM_002253.2) were selected (http://crispr.mit.edu/), Control sgRNA sequence was designed to target lacZ gene from E. coli. These were cloned into the lentiv2-Cas9 vector (Addgene:52961), respectively, and were confirmed by DNA sequencing. Lentiviruses produced in293T cells were used to infect porcine aortic endothelial cells overexpressing VEGFR2 (PAEC-KDR). The DNA fragments around the expected Cas9 cleavage site were analyzed by Sanger sequencing, surveyor nuclease assay and next generation sequencing (NGS). The efficiency of CRISPR/Cas9-mediated VEGFR2 depletion and VEGF-stimulated phosphorylation of VEGFR2, Akt and Erk were determined by western blot.
Results :
Sanger DNA sequencingand surveyor nuclease assay demonstrated that VEGFR2 gene was successfully edited by SpCas9 guided by the gRNA k12, and NGS indicated there were two major mutated sequences (42.37% and 22.53%) among the 11 various sequences. Depletion of VEGFR2 in the cells with SpCas9-VEGFR2 gRNA k12 was confirmed by a western blotting analysis. The VEGFR2-depleted cells had lower levels of Akt and Erk phosphorylation than the LacZ control cells treated with Lucentis or Eylea.
Conclusions :
The depletion of VEGFR2 using the CRISPR/Cas9 technology and CRISPR/Cas9 mediated VEGFR2 genomic gene silencing was superior to the anti-VEGF drugs (Lucentis and Eylea) for suppressing VEGF-stimulated signaling pathway. The CRISPR-Cas9 technology provides a novel opportunity to inhibit pathological angiogenesis.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.