AAV-CRISPR/Cas9–Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro

Abstract

PurposePathologic angiogenesis is a component of many diseases, including neovascular age-related macular degeneration, proliferation diabetic retinopathy, as well as tumor growth and metastasis. The purpose of this project was to examine whether the system of adeno-associated viral (AAV)–mediated CRISPR (clustered regularly interspaced short palindromic repeats)–associated endonuclease (Cas)9 can be used to deplete expression of VEGF receptor 2 (VEGFR2) in human vascular endothelial cells in vitro and thus suppress its downstream signaling events.MethodsThe dual AAV system of CRISPR/Cas9 from Streptococcus pyogenes (AAV-SpGuide and -SpCas9) was adapted to edit genomic VEGFR2 in primary human retinal microvascular endothelial cells (HRECs). In this system, the endothelial-specific promoter for intercellular adhesion molecule 2 (ICAM2) was cloned into the dual AAV vectors of SpGuide and SpCas9 for driving expression of green fluorescence protein (GFP) and SpCas9, respectively. These two AAV vectors were applied to production of recombinant AAV serotype 5 (rAAV5), which were used to infect HRECs for depletion of VEGFR2. Protein expression was determined by Western blot; and cell proliferation, migration, as well as tube formation were examined.ResultsAAV5 effectively infected vascular endothelial cells (ECs) and retinal pigment epithelial (RPE) cells; the ICAM2 promoter drove expression of GFP and SpCas9 in HRECs, but not in RPE cells. The results showed that the rAAV5-CRISPR/Cas9 depleted VEGFR2 by 80% and completely blocked VEGF-induced activation of Akt, and proliferation, migration as well as tube formation of HRECs.ConclusionsAAV-CRISRP/Cas9–mediated depletion of VEGFR2 is a potential therapeutic strategy for pathologic angiogenesis.

Precise CCM1 gene correction and inactivation in patient‐derived endothelial cells: Modeling Knudson's two‐hit hypothesis in vitro

“…The delivery of the CRISPR/Cas9 components into primary endothelial and other hard-to-transfect cells has been a major obstacle for cardiovascular research. Plasmids, lenti-, adeno-or adeno-associated viral vectors have most often been used to achieve sufficient gene knockout rates in ECs (Abrahimi et al, 2015;Cullere, Plovie, Bennett, MacRae, & Mayadas, 2015;Gong et al, 2017;Miao et al, 2018;Wu et al, 2017). Just recently, we have demonstrated that gene knockouts in human ECs can be established with a crR-NA:tracrRNA:Cas9 RNP approach (Schwefel et al, 2019).…”

The Role of Inflammation in Age-Related Macular Degeneration

“…Drugs such as Lampalizumb, Eculizumab, Zimura and Iluvien have initially shown potential effectiveness in clinical trials, and need to be further verified [142][143][144]. As some popular emerging technologies, small interfering RNAs (siRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPRassociated protein 9 (Cas9) could selectively disrupt the VEGF gene [145][146][147][148][149]. Several inflammatoryrelated cytokines also act on VEGF or VEGF-related mechanisms, so we hypothesized whether can use CRISPR/ Cas9 or siRNAs to target these cytokines and achieved the therapeutic effect on AMD.…”

β‑elemene inhibits oxygen‑induced retinal neovascularization via promoting miR‑27a and reducing VEGF expression

“…No effective treatment for retinal neovascularization (RNV) is available to date. Recent studies have shown that retinal angiogenesis may due to complex interactions among multiple angiogenic stimulators ( 4 , 5 ).…”