CoChat
Sign Up
Cited by 20CRISPR and Genetic EngineeringRetinal Diseases and TreatmentsRetinal Development and Disorders

CRISPR/Cas9 mediated specific ablation of vegfa in retinal pigment epithelium efficiently regresses choroidal neovascularization

Open access
Park, Jinkyu, Cui, Gang, Lee, Hyundong, Jeong, Han, Kwak, Jay Jiyong, Lee, Junwon, Byeon, Suk Ho·2023·Scientific Reports
DOI 10.1038/s41598-023-29014-zPMC PMC9988861
Open this paper in CoChat — free accountOpen PDF

Abstract

The CRISPR/Cas9 system easily edits target genes in various organisms and is used to treat human diseases. In most therapeutic CRISPR studies, ubiquitously expressed promoters, such as CMV, CAG, and EF1α, are used; however, gene editing is sometimes necessary only in specific cell types relevant to the disease. Therefore, we aimed to develop a retinal pigment epithelium (RPE)-specific CRISPR/Cas9 system. We developed a CRISPR/Cas9 system that operates only in retinal pigment epithelium (RPE) by expressing Cas9 under the RPE-specific vitelliform macular dystrophy 2 promoter (pVMD2). This RPE-specific CRISPR/pVMD2-Cas9 system was tested in human retinal organoid and mouse model. We confirmed that this system works specifically in the RPE of human retinal organoids and mouse retina. In addition, the RPE-specific Vegfa ablation using the novel CRISPR-pVMD2-Cas9 system caused regression of choroidal neovascularization (CNV) without unwanted knock-out in the neural retina in laser-induced CNV mice, which is a widely used animal model of neovascular age-related macular degeneration. RPE-specific Vegfa knock-out (KO) and ubiquitous Vegfa KO were comparable in the efficient regression of CNV. The promoter substituted, cell type-specific CRISPR/Cas9 systems can be used in specific ‘target cell’ therapy, which edits genes while reducing unwanted off- ‘target cell’ effects.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1038/s41598-023-29014-z

Cites

5 of 5 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • CRISPR/Cas9-mediated disruption of VEGF in hiPS-derived RPE has been reported using lentiviral delivery.

    “In the case of hiPS-derived RPE, CRISPR/Cas9-mediated disruption of VEGF has been reported using lentiviral delivery [10].”

    From Precise CRISPR/Cas9 and Cas12 Correction Using Lipoplexes in Retinal Models Derived from Patients with Inherited Retinal Dystrophies (2026) · cites this paper as [10]

    The Results report lentiviral CRISPR/Cas9 editing of Vegfa in mouse RPE and editing of AAVS1, not VEGF, in human retinal organoids. The Methods section on retinal organoid differentiation says those organoids came from human embryonic stem cells, not hiPS cells. Thus the paper supports lentiviral RPE editing, but not VEGF disruption in hiPS-derived RPE. (LLM, conf=0.89)

    Experimental — accuracy not yet measured

  • Previous studies have focused on specific cell types believed to be major contributors to VEGFA expression, like RPE and Müller cells.

    “previous studies have focused on specific cell types that are believed to be major contributors to VEGFA expression, like RPE and Müller cells (4, 43).”

    From Characterization of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina (2024) · cites this paper as [43]

  • Recent efforts to address the problem of frequent intravitreal injections have employed gene editing strategies like targeting VEGFA using CRISPR-Cas9.

    “Recent efforts to address this problem have employed gene editing strategies like targeting VEGFA using CRISPR-Cas9 (43, 45).”

    From Characterization of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina (2024) · cites this paper as [43]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

Flagged first, then by citations

Research works printed but not linked: 29

  1. [2]Fleckenstein M Age-related macular degeneration Nat. Rev. Dis. Primers 2021 7 31 10.1038/s41572-021-00265-2 33958600 PMC12878645 · DOI
  2. [3]Ferrara N Vascular endothelial growth factor and age-related macular degeneration: from basic science to therapy Nat. Med. 2010 16 1107 1111 10.1038/nm1010-1107 20930754 · DOI
  3. [4]Leung DW Cachianes G Kuang WJ Goeddel DV Ferrara N Vascular endothelial growth-factor is a secreted angiogenic mitogen Sci. 1979 1989 246 1306 1309 10.1126/science.2479986 2479986 · DOI
  4. [6]Group CR Ranibizumab and bevacizumab for neovascular age-related macular degeneration N. Engl. J. Med. 2011 364 1897 1908 10.1056/NEJMoa1102673 21526923 PMC3157322 · DOI · Paper page
  5. [8]Funk M Neovascular age-related macular degeneration: intraocular cytokines and growth factors and the influence of therapy with ranibizumab Ophthalmology 2009 116 2393 2399 10.1016/j.ophtha.2009.05.039 19815292 · DOI
  6. [9]Sawada O Aqueous vascular endothelial growth factor after intravitreal injection of pegaptanib or ranibizumab in patients with age-related macular degeneration Retina 2010 30 1034 1038 10.1097/IAE.0b013e3181ce74c8 20616682 · DOI
  7. [10]Yiu G Tieu E Nguyen AT Wong B Smit-McBride Z Genomic disruption of VEGF-A expression in human retinal pigment epithelial cells using CRISPR-Cas9 endonuclease Invest. Ophthalmol. Vis. Sci. 2016 57 5490 5497 10.1167/iovs.16-20296 27768202 PMC5072539 · DOI
  8. [11]Esumi N Oshima Y Li Y Campochiaro PA Zack DJ Analysis of the VMD2 promoter and implication of E-box binding factors in its regulation J. Biol. Chem. 2004 279 19064 19073 10.1074/jbc.M309881200 14982938 · DOI
  9. [12]Askou AL Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors Mol. Ther. Methods Clin. Dev. 2015 2 14064 10.1038/mtm.2014.64 26052532 PMC4449022 · DOI
  10. [13]Koo T CRISPR-LbCpf1 prevents choroidal neovascularization in a mouse model of age-related macular degeneration Nat. Commun. 2018 9 1855 10.1038/s41467-018-04175-y 29748595 PMC5945874 · DOI
  11. [14]Kim K Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration Genome Res. 2017 27 419 426 10.1101/gr.219089.116 28209587 PMC5340969 · DOI
  12. [15]Lambert V Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice Nat. Protoc. 2013 8 2197 2211 10.1038/nprot.2013.135 24136346 · DOI
  13. [16]Eldred KC Thyroid hormone signaling specifies cone subtypes in human retinal organoids Science 2018 1979 362 10.1126/science.aau6348 PMC6249681 30309916 · DOI
  14. [17]Wahlin KJ Photoreceptor outer segment-like structures in long-term 3D retinas from human pluripotent stem cells Sci. Rep. 2017 7 766 10.1038/s41598-017-00774-9 28396597 PMC5429674 · DOI
  15. [18]Park J Lim K Kim JS Bae S Cas-analyzer: An online tool for assessing genome editing results using NGS data Bioinformatics 2017 33 286 288 10.1093/bioinformatics/btw561 27559154 PMC5254075 · DOI
  16. [19]Fortmann SD Lorenc VE Shen J Hackett SF Campochiaro PA Mousetap, a novel technique to collect uncontaminated vitreous or aqueous and expand usefulness of mouse models Sci. Rep. 2018 8 6371 10.1038/s41598-018-24197-2 29686307 PMC5913258 · DOI
  17. [20]Auricchio A Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: The retina as a model Hum. Mol. Genet. 2001 10 3075 3081 10.1093/hmg/10.26.3075 11751689 · DOI
  18. [21]Bejjani RA Nanoparticles for gene delivery to retinal pigment epithelial cells Mol. Vis. 2005 11 124 132 15735602
  19. [22]Mitra RN Synthesis and characterization of glycol chitosan DNA nanoparticles for retinal gene delivery ChemMedChem 2014 9 189 196 10.1002/cmdc.201300371 24203490 PMC4019313 · DOI
  20. [23]Sun D Targeted multifunctional lipid ECO plasmid DNA nanoparticles as efficient non-viral gene therapy for leber's congenital amaurosis Mol. Ther. Nucleic Acids 2017 7 42 52 10.1016/j.omtn.2017.02.005 28624218 PMC5363681 · DOI · Paper page
  21. [25]Wang H Kunz E Stoddard GJ Hauswirth WW Hartnett ME Optimal inhibition of choroidal neovascularization by scAAV2 with VMD2 promoter-driven active Rap1a in the RPE Sci. Rep. 2019 9 15732 10.1038/s41598-019-52163-z 31673119 PMC6823539 · DOI
  22. [26]Kim E In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni Nat. Commun. 2017 8 14500 10.1038/ncomms14500 28220790 PMC5473640 · DOI · Paper page
  23. [27]Holmgaard A In vivo knockout of the vegfa gene by lentiviral delivery of CRISPR/Cas9 in mouse retinal pigment epithelium cells Mol. Ther. Nucleic Acids 2017 9 89 99 10.1016/j.omtn.2017.08.016 29246327 PMC5626917 · DOI
  24. [28]Ling S Lentiviral delivery of co-packaged Cas9 mRNA and a Vegfa-targeting guide RNA prevents wet age-related macular degeneration in mice Nat. Biomed. Eng. 2021 5 144 156 10.1038/s41551-020-00656-y 33398131 · DOI
  25. [30]Chung SH CRISPR-based VEGF suppression using paired guide RNAs for treatment of choroidal neovascularization Mol. Ther. Nucleic Acids 2022 28 613 622 10.1016/j.omtn.2022.04.015 35614998 PMC9114159 · DOI · Paper page
  26. [31]Kwak N Okamoto N Wood JM Campochiaro PA VEGF is major stimulator in model of choroidal neovascularization Invest. Ophthalmol. Vis. Sci. 2000 41 3158 3164 10967078
  27. [32]Robbins SG Conaway JR Ford BL Roberto KA Penn JS Detection of vascular endothelial growth factor (VEGF) protein in vascular and non-vascular cells of the normal and oxygen-injured rat retina Growth Factors 1997 14 229 241 10.3109/08977199709021522 9386988 · DOI
  28. [33]Saint Geniez M Endogenous VEGF is required for visual function: evidence for a survival role on müller cells and photoreceptors PLoS ONE 2008 3 e3554 e3554 10.1371/journal.pone.0003554 18978936 PMC2571983 · DOI
  29. [34]Beck M Munk MR Ebneter A Wolf S Zinkernagel MS Retinal ganglion cell layer change in patients treated with anti-vascular endothelial growth factor for neovascular age-related macular degeneration Am. J. Ophthalmol. 2016 167 10 17 10.1016/j.ajo.2016.04.003 27084000 · DOI

Cited by

Sorted by citations

Authors
Park, J., Cui, G., Lee, H., Jeong, H., Kwak, J. J., Lee, J., & Byeon, S. H.
Affiliations
Yonsei University (KR)
Corresponding author
  • Lee, Junwon
  • Byeon, Suk Ho
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2023-03-06
Volume
13
Issue
1
Pages
3715-3715
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-023-29014-z
PMC
PMC9988861
OpenAlex
W4323313645
Cited by
20
References
5
Citations by year
2026: 4 · 2025: 10 · 2024: 5 · 2023: 1
FWCI
1.78
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • YUHSpace (Yonsei University Medical Library) (submittedVersion, cc-by-nc-nd)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Research Foundation of Korea (2021R1I1A1A01045648, 2019R1A2C2086729)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Health Sciences › Medicine › Ophthalmology › Retinal Diseases and Treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinal Development and Disorders
Keywords
CRISPR/Cas9, retinal pigment epithelium, VEGF-A, choroidal neovascularization, retinal organoids, neovascular age-related macular degeneration, laser-induced CNV, off-target effects, gene ablation
MeSH terms
Animals, Disease Models, Animal, Craniocerebral Trauma, Humans, Retina, Choroidal Neovascularization, Vascular Endothelial Growth Factor A, Mice, Retinal Pigment Epithelium, CRISPR-Cas Systems

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review