Vascular endothelial growth factor receptor (VEGFR)-2 is a key switch for angiogenesis, which is observed in various human diseases. In this study, a novel system for advanced prime editing (PE), termed PE6h, is developed, consisting of dual lentiviral vectors: (1) a clustered regularly interspaced palindromic repeat-associated protein 9 (H840A) nickase fused with reverse transcriptase and an enhanced PE guide RNA and (2) a dominant negative (DN) MutL homolog 1 gene with nicking guide RNA. PE6h was used to edit VEGFR2 (c.18315T>A, 50.8%) to generate a premature stop codon (TAG from AAG), resulting in the production of DN-VEGFR2 (787 aa) in human retinal microvascular endothelial cells (HRECs). DN-VEGFR2 impeded VEGF-induced phosphorylation of VEGFR2, Akt, and extracellular signal-regulated kinase-1/2 and tube formation in PE6h-edited HRECs in vitro. Overall, our results highlight the potential of PE6h to inhibit angiogenesis in vivo.
Get full access to this article
View all access options for this article.
References
1.
StahlA, ConnorKM, SapiehaP, et al.The mouse retina as an angiogenesis model. Invest Ophthalmol Vis Sci, 2010; 51(6):2813–2826; doi: 10.1167/iovs.10-5176
2.
ChakravarthyU, HardingSP, RogersCA, et al.Alternative treatments to inhibit VEGF in age-related choroidal neovascularisation: 2-year findings of the IVAN randomised controlled trial. Lancet, 2013; 382(9900):1258–1267; doi: 10.1016/S0140-6736(13)61501-9
3.
WilliamsR, AireyM, BaxterH, et al.Epidemiology of diabetic retinopathy and macular oedema: A systematic review. Eye (Lond), 2004; 18(10):963–983; doi: 10.1038/sj.eye.6701476
4.
Fraser-BellS, KainesA, HykinPG. Update on treatments for diabetic macular edema. Curr Opin Ophthalmol, 2008; 19(3):185–189; doi: 10.1097/ICU.0b013e3282fb7c45
5.
Mintz-HittnerHA, KennedyKA, ChuangAZ, et al.Efficacy of intravitreal bevacizumab for stage 3+ retinopathy of prematurity. N Engl J Med, 2011; 364(7):603–615; doi: 10.1056/NEJMoa1007374
6.
HuangX, ZhouG, WuW, et al.Genome editing abrogates angiogenesis in vivo. Nat Commun, 2017; 8(1):112; doi: 10.1038/s41467-017-00140-3
SuzukiM, NagaiN, Izumi-NagaiK, et al.Predictive factors for non-response to intravitreal ranibizumab treatment in age-related macular degeneration. Br J Ophthalmol, 2014; 98(9):1186–1191; doi: 10.1136/bjophthalmol-2013-304670
9.
UludagG, HassanM, MatsumiyaW, et al.Efficacy and safety of intravitreal anti-VEGF therapy in diabetic retinopathy: What we have learned and what should we learn further? Expert Opin Biol Ther, 2022; 22(10):1275–1291; doi: 10.1080/14712598.2022.2100694
10.
ArrigoA, AragonaE, BandelloF. VEGF-targeting drugs for the treatment of retinal neovascularization in diabetic retinopathy. Ann Med, 2022; 54(1):1089–1111; doi: 10.1080/07853890.2022.2064541
11.
AnzaloneAV, RandolphP, DavisJR, et al.Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 2019; 576(7785):149–157.
12.
RaguramA, BanskotaS, LiuDR. Therapeutic in vivo delivery of gene editing agents. Cell, 2022; 185(15):2806–2827; doi: 10.1016/j.cell.2022.03.045
13.
ChenPJ, LiuDR. Prime editing for precise and highly versatile genome manipulation. Nat Rev Genet, 2023; 24(3):161–177; doi: 10.1038/s41576-022-00541-1
14.
DavisJR, BanskotaS, LevyJM, et al.Efficient prime editing in mouse brain, liver and heart with dual AAVs. Nat Biotechnol, 2023; 42(2):253–264; doi: 10.1038/s41587-023-01758-z
15.
NelsonJW, RandolphPB, ShenSP, et al.Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol, 2022; 40(3):402–410; doi: 10.1038/s41587-021-01039-7
16.
DomanJL, SousaAA, RandolphPB, et al.Designing and executing prime editing experiments in mammalian cells. Nat Protoc, 2022; 17(11):2431–2468; doi: 10.1038/s41596-022-00724-4
17.
DomanJL, PandeyS, NeugebauerME, et al.Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell, 2023; 186(18):3983–4002 e3926; doi: 10.1016/j.cell.2023.07.039
18.
LiuY, LiX, HeS, et al.Efficient generation of mouse models with the prime editing system. Cell Discov, 2020; 6:27; doi: 10.1038/s41421-020-0165-z
19.
LiuP, LiangS-Q, ZhengC, et al.Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. Nat Commun, 2021; 12(1):2121; doi: 10.1038/s41467-021-22295-w
20.
JiangT, ZhangXO, WengZ, et al.Deletion and replacement of long genomic sequences using prime editing. Nat Biotechnol, 2022; 40(2):227–234; doi: 10.1038/s41587-021-01026-y
21.
AvrutskyMI, ChenCW, LawsonJM, et al.Caspase-9 inhibition confers stronger neuronal and vascular protection compared to VEGF neutralization in a mouse model of retinal vein occlusion. Front Neurosci, 2023; 17:1209527; doi: 10.3389/fnins.2023.1209527
22.
ChoiJ, ChenW, SuiterCC, et al.Precise genomic deletions using paired prime editing. Nat Biotechnol, 2022; 40(2):218–226; doi: 10.1038/s41587-021-01025-z
23.
Ferreira da SilvaJ, OliveiraGP, Arasa-VergeEA, et al.Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair. Nat Commun, 2022; 13(1):760; doi: 10.1038/s41467-022-28442-1
24.
ChowRD, ChenJS, ShenJ, et al.A web tool for the design of prime-editing guide RNAs. Nat Biomed Eng, 2021; 5(2):190–194; doi: 10.1038/s41551-020-00622-8
25.
MatthewsW, JordanCT, GavinM, et al.A receptor tyrosine kinase cDNA isolated from a population of enriched primitive hematopoietic cells and exhibiting close genetic linkage to c-kit. Proc Natl Acad Sci U S A, 1991; 88(20):9026–9030.
26.
SwiechL, HeidenreichM, BanerjeeA, et al.In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol, 2015; 33(1):102–106.
27.
HuangX, ZhouG, WuW, et al.Editing VEGFR2 blocks VEGF-induced activation of Akt and tube formation. Invest Ophthalmol Vis Sci, 2017; 58(2):1228–1236; doi: 10.1167/iovs.16-20537
28.
WuW, XiaX, TangL, et al.Normal vitreous promotes angiogenesis via activation of Axl. FASEB J, 2021; 35(1):e21152; doi: 10.1096/fj.201903105R
29.
SanjanaNE, ShalemO, ZhangF. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods, 2014; 11(8):783–784; doi: 10.1038/nmeth.3047
30.
ChenPJ, HussmannJA, YanJ, et al.Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell, 2021; 184(22):5635–5652 e5629; doi: 10.1016/j.cell.2021.09.018
StewartSA, DykxhoornDM, PalliserD, et al.Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA, 2003; 9(4):493–501; doi: 10.1261/rna.2192803
33.
RuanGX, KazlauskasA. Axl is essential for VEGF-A-dependent activation of PI3K/Akt. EMBO J, 2012; 31(7):1692–1703; doi: 10.1038/emboj.2012.21
34.
WuW, ZhouG, HanH, et al.PI3Kdelta as a novel therapeutic target in pathological angiogenesis. Diabetes, 2020; 69(4):736–748; doi: 10.2337/db19-0713
35.
LiangCC, ParkAY, GuanJL. In vitro scratch assay: A convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc, 2007; 2(2):329–333; doi: 10.1038/nprot.2007.30
36.
ImE, VenkatakrishnanA, KazlauskasA. Cathepsin B regulates the intrinsic angiogenic threshold of endothelial cells. Mol Biol Cell, 2005; 16(8):3488–3500; doi: 10.1091/mbc.E04-11-1029
37.
SchröderARW, ShinnP, ChenH, et al.HIV-1 integration in the human genome favors active genes and local hotspots. Cell, 2002; 110(4):521–529; doi: 10.1016/s0092-8674(02)00864-4
38.
PhilippeS, SarkisC, BarkatsM, et al.Lentiviral vectors with a defective integrase allow efficient and sustained transgene expression in vitro and in vivo. Proc Natl Acad Sci U S A, 2006; 103(47):17684–17689; doi: 10.1073/pnas.0606197103
39.
GrüterO, KosticC, CrippaSV, et al.Lentiviral vector-mediated gene transfer in adult mouse photoreceptors is impaired by the presence of a physical barrier. Gene Ther, 2005; 12(11):942–947; doi: 10.1038/sj.gt.3302485
40.
PuppoA, CesiG, MarroccoE, et al.Retinal transduction profiles by high-capacity viral vectors. Gene Ther, 2014; 21(10):855–865; doi: 10.1038/gt.2014.57
41.
WangXJ, GreenhalghDA, BickenbachJR, et al.Expression of a dominant-negative type II transforming growth factor beta (TGF-beta) receptor in the epidermis of transgenic mice blocks TGF-beta-mediated growth inhibition. Proc Natl Acad Sci U S A, 1997; 94(6):2386–2391; doi: 10.1073/pnas.94.6.2386
42.
IkunoY, KazlauskasA. An in vivo gene therapy approach for experimental proliferative vitreoretinopathy using the truncated platelet-derived growth factor alpha receptor. Invest Ophthalmol Vis Sci, 2002; 43:2406–2411.
43.
NarayanV, Barber-RotenbergJS, JungI-Y, et al.PSMA-targeting TGFβ-insensitive armored CAR T cells in metastatic castration-resistant prostate cancer: A phase 1 trial. Nat Med, 2022; 28(4):724–734; doi: 10.1038/s41591-022-01726-1
44.
MacheinMR, RisauW, PlateKH. Antiangiogenic gene therapy in a rat glioma model using a dominant-negative vascular endothelial growth factor receptor 2. Hum Gene Ther, 1999; 10(7):1117–1128; doi: 10.1089/10430349950018111
45.
StratmannA, AckerT, BurgerAM, et al.Differential inhibition of tumor angiogenesis by tie2 and vascular endothelial growth factor receptor-2 dominant-negative receptor mutants. Int J Cancer, 2001; 91(3):273–282; doi: 10.1002/1097-0215(200002)9999:9999<::aid-ijc1054>3.0.co;2-q
46.
TsouR, FathkeC, WilsonL, et al.Retroviral delivery of dominant-negative vascular endothelial growth factor receptor type 2 to murine wounds inhibits wound angiogenesis. Wound Repair Regen, 2002; 10(4):222–229; doi: 10.1046/j.1524-475x.2002.10405.x
47.
FrankeTF, KaplanDR, CantleyLC, et al.Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science, 1997; 275(5300):665–668.
48.
SarbassovDD, GuertinDA, AliSM, et al.Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science, 2005; 307(5712):1098–1101; doi: 10.1126/science.1106148
49.
ChenJ, SomanathPR, RazorenovaO, et al.Akt1 regulates pathological angiogenesis, vascular maturation and permeability in vivo. Nat Med, 2005; 11(11):1188–1196; doi: 10.1038/nm1307
50.
ElyZA, Mathey-AndrewsN, NaranjoS, et al.A prime editor mouse to model a broad spectrum of somatic mutations in vivo. Nat Biotechnol, 2024; 42(3):424–436; doi: 10.1038/s41587-023-01783-y
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
