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Cited by 142CRISPR and Genetic EngineeringAngiogenesis and VEGF in CancerRetinal Development and Disorders

The DOI matches its Crossref record; 42 of the 42 references it cites were found in a registry or an index.

Genome editing abrogates angiogenesis in vivo

Open access
Huang, Xionggao, Zhou, Guohong, Wu, Wenyi, Duan, Yajian, Ma, Gaoen, Song, Jingyuan, Xiao, Ru, Vandenberghe, Luk, Zhang, Feng, D’Amore, Patricia A., Lei, Hetian·2017·Nature Communications
DOI 10.1038/s41467-017-00140-3PMC PMC5524639
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Abstract

Angiogenesis, in which vascular endothelial growth factor receptor (VEGFR) 2 plays an essential role, is associated with a variety of human diseases including proliferative diabetic retinopathy and wet age-related macular degeneration. Here we report that a system of adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) is used to deplete VEGFR2 in vascular endothelial cells (ECs), whereby the expression of SpCas9 is driven by an endothelial-specific promoter of intercellular adhesion molecule 2. We further show that recombinant AAV serotype 1 (rAAV1) transduces ECs of pathologic vessels, and that editing of genomic VEGFR2 locus using rAAV1-mediated CRISPR/Cas9 abrogates angiogenesis in the mouse models of oxygen-induced retinopathy and laser-induced choroid neovascularization. This work establishes a strong foundation for genome editing as a strategy to treat angiogenesis-associated diseases.

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Fig. 1 AAV-CRISPR/Cas9-mediated depletion of VEGFR2 in vitro. a Schematic of AAV-SpGuide (V1)15. Graphical representation of the mouse VEGFR2-targeted locus. The oligos of mK22 and its compliment were annealed and cloned into the V1 vector by SapI. The PAM is marked in blue. ITR inverted terminal repeat, U6 a promoter of polymerase III, CMV a promoter of cytomegalovirus, GFP green fluorescent protein. b Schematic of AAV-SpCas9 (V3). pMecp2: a neuron-specific promoter for methyl CpG-binding protein in V0 was substituted for pICAM219 by XbaI/AgeI. c Transduction of cultured cells with rAAV1. HRECs, HUVECs, and hPRPE cells in a 48-well plate to 50% confluence were infected with rAAV1-CMV-GFP (2 μl/well, 3.75 × 1012 viral genome-containing particles (vg)/ml). Three days later, the cells were photographed under an immunofluorescence microscope. Three independent experiments showed rAAV1 transduction efficiency in HRECs, HUVECs and hPRPE cells of 85.6 ± 2.2, 88.5 ± 2.3 and 86.8 ± 2.6%, respectively. Scale bar: 200 μm. d pICAM2-driven expression of SpCas9 in ECs. After transduction with rAAV1-CMV-GFP (GFP) or rAAV1-pICAM2-SpCas9 (SpCas9) (2 μl/well, 3.75 × 1012vg/ml) in a 48-well plate for 4 days, cell lysates were subjected to western blot analysis with antibodies against Cas9 and β-actin. Data shown are representative of three independent experiments. e Sanger DNA sequencing was conducted on PCR products amplified from the genomic VEGFR2 loci of MVECs, which were transduced by rAAV1-SpCas9 plus rAAV1-lacZ (lacZ) or rAAV1-mK22 (mK22). f Depletion of VEGFR2 expression using AAV-CRISPR/Cas9. Total cell lysates from the transduced MVECs were subjected to western blot analysis with antibodies against VEGFR2 and β-actin. The bar graphs are mean ± SD of three independent experiments. “*” indicates a significant difference between the compared two groups using an unpaired t-test. p < 0.05
Fig. 1 AAV-CRISPR/Cas9-mediated depletion of VEGFR2 in vitro. a Schematic of AAV-SpGuide (V1)15. Graphical representation of the mouse VEGFR2-targeted locus. The oligos of mK22 and its compliment were annealed and cloned into the V1 vector by SapI. The PAM is marked in blue. ITR inverted terminal repeat, U6 a promoter of polymerase III, CMV a promoter of cytomegalovirus, GFP green fluorescent protein. b Schematic of AAV-SpCas9 (V3). pMecp2: a neuron-specific promoter for methyl CpG-binding protein in V0 was substituted for pICAM219 by XbaI/AgeI. c Transduction of cultured cells with rAAV1. HRECs, HUVECs, and hPRPE cells in a 48-well plate to 50% confluence were infected with rAAV1-CMV-GFP (2 μl/well, 3.75 × 1012 viral genome-containing particles (vg)/ml). Three days later, the cells were photographed under an immunofluorescence microscope. Three independent experiments showed rAAV1 transduction efficiency in HRECs, HUVECs and hPRPE cells of 85.6 ± 2.2, 88.5 ± 2.3 and 86.8 ± 2.6%, respectively. Scale bar: 200 μm. d pICAM2-driven expression of SpCas9 in ECs. After transduction with rAAV1-CMV-GFP (GFP) or rAAV1-pICAM2-SpCas9 (SpCas9) (2 μl/well, 3.75 × 1012vg/ml) in a 48-well plate for 4 days, cell lysates were subjected to western blot analysis with antibodies against Cas9 and β-actin. Data shown are representative of three independent experiments. e Sanger DNA sequencing was conducted on PCR products amplified from the genomic VEGFR2 loci of MVECs, which were transduced by rAAV1-SpCas9 plus rAAV1-lacZ (lacZ) or rAAV1-mK22 (mK22). f Depletion of VEGFR2 expression using AAV-CRISPR/Cas9. Total cell lysates from the transduced MVECs were subjected to western blot analysis with antibodies against VEGFR2 and β-actin. The bar graphs are mean ± SD of three independent experiments. “*” indicates a significant difference between the compared two groups using an unpaired t-test. p < 0.05CC BY · PubMed Central
Fig. 2 Transduction of ECs with rAAV1 in vivo. a, b On P7, C57BL/6J litters were exposed to 75% oxygen until P1223, 37 when the pups were injected intravitreally with rAAV1-CMV-GFP (1 μl, 3.75 × 1012 vg/ml). After return to room air (21% oxygen) for 5 days, and whole-mount retinas from the killed mice were stained with IB4 (red). Images were taken under TxRed channel (a, c), GFP channel (b, d). e Merged image of c and d. Scale bar: 200 μm. f–i Four lesions were induced in an eight-week-old mouse on the Bruch’s membrane using a 532-nm green laser. rAAV1-CMV-GFP (1 μl, 3.75 × 1012 vg/ml) was injected intravitreally into the mouse. Seven days later, the whole-mount choroid was stained with IB4, and images were taken under GFP channel (f, g) and Txred channel (h). i A merged image of g and h. Scale bar: 200 μm. Each figure represents at least six ones from different mice
Fig. 2 Transduction of ECs with rAAV1 in vivo. a, b On P7, C57BL/6J litters were exposed to 75% oxygen until P1223, 37 when the pups were injected intravitreally with rAAV1-CMV-GFP (1 μl, 3.75 × 1012 vg/ml). After return to room air (21% oxygen) for 5 days, and whole-mount retinas from the killed mice were stained with IB4 (red). Images were taken under TxRed channel (a, c), GFP channel (b, d). e Merged image of c and d. Scale bar: 200 μm. f–i Four lesions were induced in an eight-week-old mouse on the Bruch’s membrane using a 532-nm green laser. rAAV1-CMV-GFP (1 μl, 3.75 × 1012 vg/ml) was injected intravitreally into the mouse. Seven days later, the whole-mount choroid was stained with IB4, and images were taken under GFP channel (f, g) and Txred channel (h). i A merged image of g and h. Scale bar: 200 μm. Each figure represents at least six ones from different miceCC BY · PubMed Central
Fig. 3 Editing genomic VEGFR2 abrogated hypoxia-induced angiogenesis. a Litters of P12 mice that had been exposed to 75% oxygen for 5 days were injected intravitreally with 1 μl (3.75 × 1012 vg/ml) containing equal rAAV1-SpCas9 and rAAV1-lacZ (lacZ) or rAAV1-mK22 (mK22). On P17, whole-mount retinas were stained with IB4. lacZ and mK22 indicate retinas from the rAAV1-SpCas9/lacZ and mK22-injected mice, respectively. b Analysis of avascular areas from the IB4-stained retinas (n = 6). c Analysis of NV areas from the IB4-stained retinas (n = 6). d NGS analysis of indels. The DNA fragments around the PAM sequences were PCR amplified from genomic DNA of the rAAV1-SpCas9/lacZ or -mK22-injected retinas, and then subjected to NGS. e, f The lysates of the rAAV1-SpCas9/lacZ or -mK22-injected retinas were subjected to western blot analysis using indicated antibodies. The bar graph data are mean ± SD of three retinas. “*” indicates significant difference using an unpaired t-test. p < 0.05
Fig. 3 Editing genomic VEGFR2 abrogated hypoxia-induced angiogenesis. a Litters of P12 mice that had been exposed to 75% oxygen for 5 days were injected intravitreally with 1 μl (3.75 × 1012 vg/ml) containing equal rAAV1-SpCas9 and rAAV1-lacZ (lacZ) or rAAV1-mK22 (mK22). On P17, whole-mount retinas were stained with IB4. lacZ and mK22 indicate retinas from the rAAV1-SpCas9/lacZ and mK22-injected mice, respectively. b Analysis of avascular areas from the IB4-stained retinas (n = 6). c Analysis of NV areas from the IB4-stained retinas (n = 6). d NGS analysis of indels. The DNA fragments around the PAM sequences were PCR amplified from genomic DNA of the rAAV1-SpCas9/lacZ or -mK22-injected retinas, and then subjected to NGS. e, f The lysates of the rAAV1-SpCas9/lacZ or -mK22-injected retinas were subjected to western blot analysis using indicated antibodies. The bar graph data are mean ± SD of three retinas. “*” indicates significant difference using an unpaired t-test. p < 0.05CC BY · PubMed Central
Fig. 4 AAV-CRISPR/Cas9-targeting genomic VEGFR2 suppressed NV in laser-induced choroid NV in mice. After laser injury of Bruch’s membrane, fundus images (day 0) were taken using the Micron III system, and the mice were injected intravitreally with 1 μl (3.75 × 1012 vg/ml) containing equal rAAV1-SpCas9 and rAAV1-lacZ or -mK22 right immediately after the laser injury (a) or 7 days of the laser injury (d). Seven days after AAV1 injection, the mice were injected intraperitoneally with fluorescein, and the FA images were taken using the Micron III system. Subsequently, whole mounts of choroids were stained with IB4, and the images were taken under an immunofluorescence microscope. Areas of NV were analyzed based on the images of FA (b, e) and IB staining (c, f) (n = 6). “*” indicates significant difference between the compare two groups using an unpaired t-test. p < 0.05
Fig. 4 AAV-CRISPR/Cas9-targeting genomic VEGFR2 suppressed NV in laser-induced choroid NV in mice. After laser injury of Bruch’s membrane, fundus images (day 0) were taken using the Micron III system, and the mice were injected intravitreally with 1 μl (3.75 × 1012 vg/ml) containing equal rAAV1-SpCas9 and rAAV1-lacZ or -mK22 right immediately after the laser injury (a) or 7 days of the laser injury (d). Seven days after AAV1 injection, the mice were injected intraperitoneally with fluorescein, and the FA images were taken using the Micron III system. Subsequently, whole mounts of choroids were stained with IB4, and the images were taken under an immunofluorescence microscope. Areas of NV were analyzed based on the images of FA (b, e) and IB staining (c, f) (n = 6). “*” indicates significant difference between the compare two groups using an unpaired t-test. p < 0.05CC BY · PubMed Central

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Metadata

Authors
Huang, X., Zhou, G., Wu, W., Duan, Y., Ma, G., Song, J., Xiao, R., Vandenberghe, L., Zhang, F., D’Amore, P. A., & Lei, H.
Affiliations
  • Massachusetts Eye and Ear Infirmary (US)
  • Broad Institute (US)
Corresponding author
Lei, Hetian
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2017-07-24
Volume
8
Issue
1
Pages
112-112
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-017-00140-3
PMC
PMC5524639
OpenAlex
W2735017428
Cited by
142
References
42
Citations by year
2026: 6 · 2025: 14 · 2024: 14 · 2023: 17 · 2022: 20 · 2021: 19 · 2020: 19 · 2019: 8 · 2018: 21 · 2017: 4
FWCI
5.81
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
  • Nature
Funders
  • National Institutes of Health
  • National Eye Institute (R01EY012509, P30EY003790)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Angiogenesis and VEGF in Cancer
  • Health Sciences › Medicine › Ophthalmology › Retinal Development and Disorders
Keywords
angiogenesis, VEGFR2, CRISPR/Cas9 genome editing, vascular endothelial cells, oxygen-induced retinopathy, diabetic retinopathy, age-related macular degeneration, gene silencing
MeSH terms
Gene Editing, Dependovirus, Animals, Cells, Cultured, Female, Humans, Male, Mice, Inbred C57BL, Microscopy, Fluorescence, Neovascularization, Pathologic, Blotting, Western, Retinal Neovascularization, Choroidal Neovascularization, Vascular Endothelial Growth Factor Receptor-2, Endothelial Cells, CRISPR-Cas Systems

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