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Updated: Feb 17, 2026
In Vitro Model of Coronary Angiogenesis
Published on: March 10, 2020
AAV-CRISPR/Cas9-Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro
Wenyi Wu1,2, Yajian Duan1,3, Gaoen Ma1,4
1Schepens Eye Research Institute of Massachusetts Eye and Ear; Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
Purpose:
Pathologic 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.
Methods:
The 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.
Results:
AAV5 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.
Conclusions:
AAV-CRISRP/Cas9-mediated depletion of VEGFR2 is a potential therapeutic strategy for pathologic angiogenesis.
Insights
CRISPR-Cas9 gene editing via adeno-associated virus (AAV) effectively reduced vascular endothelial growth factor receptor 2 (VEGFR2) in human cells. This approach shows promise for treating diseases driven by pathologic angiogenesis.
Area of Science:
- Biotechnology
- Gene Editing
- Ophthalmology
Background:
- Pathologic angiogenesis contributes to diseases like wet age-related macular degeneration, diabetic retinopathy, and cancer metastasis.
- Vascular endothelial growth factor receptor 2 (VEGFR2) plays a critical role in angiogenesis.
Purpose of the Study:
- To investigate the efficacy of adeno-associated virus (AAV)-mediated CRISPR-Cas9 gene editing to deplete VEGFR2 expression in human vascular endothelial cells.
- To assess the impact of VEGFR2 depletion on downstream signaling and cellular functions.
Main Methods:
- A dual AAV system utilizing Streptococcus pyogenes CRISPR/Cas9 (AAV-SpGuide and -SpCas9) was adapted for genomic VEGFR2 editing.
- An endothelial-specific ICAM2 promoter drove SpCas9 and GFP expression in recombinant AAV serotype 5 (rAAV5) vectors.
- Primary human retinal microvascular endothelial cells (HRECs) were infected with rAAV5 for VEGFR2 depletion, followed by Western blot and functional assays.
Main Results:
- rAAV5 successfully infected HRECs and retinal pigment epithelial cells, with ICAM2 promoter driving expression specifically in HRECs.
- CRISPR/Cas9 mediated by rAAV5 achieved an 80% depletion of VEGFR2 in HRECs.
- VEGF-induced Akt activation, proliferation, migration, and tube formation were completely blocked.
Conclusions:
- Adeno-associated virus-CRISPR/Cas9-mediated VEGFR2 depletion is a viable strategy for inhibiting pathologic angiogenesis.
- This gene-editing approach holds potential for therapeutic applications in angiogenesis-related diseases.
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