September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Inactivation of VEGFR2 using CRISPR/Cas9 provides superior inhibition to the anti-VEGF drugs
Author Affiliations & Notes
  • Xionggao Huang
    Schepens Eye Research Institute, Boston, Massachusetts, United States
  • Gaoen Ma
    Schepens Eye Research Institute, Boston, Massachusetts, United States
  • Yewlin Chee
    Ophthalmology, Massachusetts Eye & Ear, Boston, Massachusetts, United States
  • Shizuo Mukai
    Ophthalmology, Massachusetts Eye & Ear, Boston, Massachusetts, United States
  • Patricia A. D’Amore
    Schepens Eye Research Institute, Boston, Massachusetts, United States
  • Hetian Lei
    Schepens Eye Research Institute, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Xionggao Huang, None; Gaoen Ma, None; Yewlin Chee, None; Shizuo Mukai, None; Patricia A. D’Amore, None; Hetian Lei, None
  • Footnotes
    Support  none
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 5024. doi:
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      Xionggao Huang, Gaoen Ma, Yewlin Chee, Shizuo Mukai, Patricia A. D’Amore, Hetian Lei; Inactivation of VEGFR2 using CRISPR/Cas9 provides superior inhibition to the anti-VEGF drugs. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5024.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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