Gene Therapy Intervention in Neovascular Eye Disease: A Recent Update

Aberrant growth of blood vessels (neovascularization) is a key feature of severe eye diseases that can cause legal blindness, including neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR). The development of anti-vascular endothelial growth factor (VEGF) agents has revolutionized the treatment of ocular neovascularization. Novel proangiogenic targets, such as angiopoietin and platelet-derived growth factor (PDGF), are under development for patients who respond poorly to anti-VEGF therapy and to reduce adverse effects from long-term VEGF inhibition. A rapidly advancing area is gene therapy, which may provide significant therapeutic benefits. Viral vector-mediated transgene delivery provides the potential for continuous production of antiangiogenic proteins, which would avoid the need for repeated anti-VEGF injections. Gene silencing with RNA interference to target ocular angiogenesis has been investigated in clinical trials. Proof-of-concept gene therapy studies using gene-editing tools such as CRISPR-Cas have already been shown to be effective in suppressing neovascularization in animal models, highlighting the therapeutic potential of the system for treatment of aberrant ocular angiogenesis. This review provides updates on the development of anti-VEGF agents and novel antiangiogenic targets. We also summarize current gene therapy strategies already in clinical trials and those with the latest approaches utilizing CRISPR-Cas gene editing against aberrant ocular neovascularization. Aberrant growth of blood vessels (neovascularization) is a key feature of severe eye diseases that can cause legal blindness, including neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR). The development of anti-vascular endothelial growth factor (VEGF) agents has revolutionized the treatment of ocular neovascularization. Novel proangiogenic targets, such as angiopoietin and platelet-derived growth factor (PDGF), are under development for patients who respond poorly to anti-VEGF therapy and to reduce adverse effects from long-term VEGF inhibition. A rapidly advancing area is gene therapy, which may provide significant therapeutic benefits. Viral vector-mediated transgene delivery provides the potential for continuous production of antiangiogenic proteins, which would avoid the need for repeated anti-VEGF injections. Gene silencing with RNA interference to target ocular angiogenesis has been investigated in clinical trials. Proof-of-concept gene therapy studies using gene-editing tools such as CRISPR-Cas have already been shown to be effective in suppressing neovascularization in animal models, highlighting the therapeutic potential of the system for treatment of aberrant ocular angiogenesis. This review provides updates on the development of anti-VEGF agents and novel antiangiogenic targets. We also summarize current gene therapy strategies already in clinical trials and those with the latest approaches utilizing CRISPR-Cas gene editing against aberrant ocular neovascularization. Angiogenesis is the formation of new blood vessels from preexisting vasculature, a process regulated by a dynamic balance between endogenous proangiogenic and antiangiogenic factors. Stimuli, such as biomechanical stress, hypoxia, ischemia, immune or inflammatory responses, and genetic variations, can disturb the balance in favor of angiogenesis.1Carmeliet P. Jain R.K. Angiogenesis in cancer and other diseases.Nature. 2000; 407: 249-257Crossref PubMed Scopus (6654) Google Scholar Ocular angiogenesis can occur in the retina, choroid, as well as cornea, and unchecked pathological blood vessel formation (neovascularization) can lead to severe visual impairment. These newly formed blood vessels are exudative, resulting in the accumulation of extracellular fluid subsequent to impairment of retinal function. Moreover, the aberrant growth of new vessels interferes with normal tissue structure and corneal transparency.2Sharif Z. Sharif W. Corneal neovascularization: updates on pathophysiology, investigations & management.Rom. J. Ophthalmol. 2019; 63: 15-22Crossref PubMed Google Scholar,3Witmer A.N. Vrensen G.F. Van Noorden C.J. Schlingemann R.O. Vascular endothelial growth factors and angiogenesis in eye disease.Prog. Retin. Eye Res. 2003; 22: 1-29Crossref PubMed Scopus (699) Google Scholar Neovascularization is associated with a range of ocular disorders, including neovascular age-related macular degeneration (nAMD), diabetic retinopathy (DR), retinopathy of prematurity (ROP), corneal neovascularization, retinal vessel occlusion, and neovascular glaucoma (reviewed in Campochiaro4Campochiaro P.A. Ocular neovascularization.J. Mol. Med. (Berl.). 2013; 91: 311-321Crossref PubMed Scopus (0) Google Scholar and Usui et al.5Usui Y. Westenskow P.D. Murinello S. Dorrell M.I. Scheppke L. Bucher F. Sakimoto S. Paris L.P. Aguilar E. Friedlander M. Angiogenesis and eye disease.Annu. Rev. Vis. Sci. 2015; 1: 155-184Crossref PubMed Google Scholar). Among these neovascularization-related diseases, nAMD and DR are leading causes of visual impairment in the developed world.6Flaxman S.R. Bourne R.R.A. Resnikoff S. Ackland P. Braithwaite T. Cicinelli M.V. Das A. Jonas J.B. Keeffe J. Kempen J.H. et al.Vision Loss Expert Group of the Global Burden of Disease StudyGlobal causes of blindness and distance vision impairment 1990–2020: a systematic review and meta-analysis.Lancet Glob. Health. 2017; 5: e1221-e1234Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar AMD occurs in patients ≥65 years of age. It is predicted that the number of individuals affected globally by AMD will reach 288 million by 2040.7Wong W.L. Su X. Li X. Cheung C.M. Klein R. Cheng C.Y. Wong T.Y. Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis.Lancet Glob. Health. 2014; 2: e106-e116Abstract Full Text Full Text PDF PubMed Scopus (1193) Google Scholar AMD can be classified as non-neovascular (dry form AMD [dAMD]) or neovascular forms (nAMD). nAMD, an advanced stage of AMD, is characterized by the pathologic growth of blood vessels from the choroid, beneath the macula, which is also termed choroidal neovascularization (CNV). The vessel outgrowth from the choroidal lacks normal vascular structure and function, thus resulting in fluid leakage and ultimately hemorrhagic or exudative retinal detachment. The growth of new vessels is often accompanied by fibrosis, which causes further damage to retina cells, particularly photoreceptors.8Solomon S.D. Lindsley K. Vedula S.S. Krzystolik M.G. Hawkins B.S. Anti-vascular endothelial growth factor for neovascular age-related macular degeneration.Cochrane Database Syst. Rev. 2019; 3: CD005139PubMed Google Scholar,9Green W.R. Enger C. Age-related macular degeneration histopathologic studies. The 1992 Lorenz E. Zimmerman Lecture.Ophthalmology. 1993; 100: 1519-1535Abstract Full Text PDF PubMed Google Scholar Despite a variety of available treatments, nearly half of patients develop retinal pigment epithelium (RPE) atrophy or macular scarring within 2 years, a major cause of permanent vision loss.10Martin D.F. Maguire M.G. Fine S.L. Ying G.S. Jaffe G.J. Grunwald J.E. Toth C. Redford M. Ferris 3rd, F.L. Comparison of Age-related Macular Degeneration Treatments Trials (CATT) Research GroupRanibizumab and bevacizumab for treatment of neovascular age-related macular degeneration: two-year results.Ophthalmology. 2012; 119: 1388-1398Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar The most susceptible genetic loci relevant to AMD are CFH (complement factor H) and ARMS2 (age-related maculopathy susceptibility 2). Non-genetic risk factors for AMD include smoking and deficiency in dietary intake of antioxidants such as carotenoids and zinc.11Vavvas D.G. Small K.W. Awh C.C. Zanke B.W. Tibshirani R.J. Kustra R. CFH and ARMS2 genetic risk determines progression to neovascular age-related macular degeneration after antioxidant and zinc supplementation.Proc. Natl. Acad. Sci. USA. 2018; 115: E696-E704Crossref PubMed Scopus (0) Google Scholar,12Awh C.C. Lane A.M. Hawken S. Zanke B. Kim I.K. CFH and ARMS2 genetic polymorphisms predict response to antioxidants and zinc in patients with age-related macular degeneration.Ophthalmology. 2013; 120: 2317-2323Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar During the last two decades, therapeutic intervention for nAMD has shifted from laser photocoagulation to block leaky vessels to pharmacotherapeutic intervention, particularly anti-vascular endothelial growth factor (VEGF) therapy.13Emerson M.V. Lauer A.K. Current and emerging therapies for the treatment of age-related macular degeneration.Clin. Ophthalmol. 2008; 2: 377-388PubMed Google Scholar Anti-VEGF therapy has also become a mainstay for the treatment of neovascularization and vascular hyperpermeability in DR. DR is a leading cause of legal blindness in those of working age (20–65 years).14Simó-Servat O. Hernández C. Simó R. Diabetic retinopathy in the context of patients with diabetes.Ophthalmic Res. 2019; 62: 211-217Crossref PubMed Scopus (4) Google Scholar According to the International Diabetes Federation (IDF), in 2017 there were an estimated 451 million people with diabetes mellitus (DM), a number projected to increase to 693 million by 2045.15Cho N.H. Shaw J.E. Karuranga S. Huang Y. da Rocha Fernandes J.D. Ohlrogge A.W. Malanda B. IDF Diabetes Atlas: global estimates of diabetes prevalence for 2017 and projections for 2045.Diabetes Res. Clin. Pract. 2018; 138: 271-281Abstract Full Text Full Text PDF PubMed Scopus (1375) Google Scholar The prevalence of any form of retinal pathology (retinopathy) in DM patients is approximately 35%, and the number of people with DR is estimated to increase to 191 million in 2030.16Zheng Y. He M. Congdon N. The worldwide epidemic of diabetic retinopathy.Indian J. Ophthalmol. 2012; 60: 428-431Crossref PubMed Scopus (206) Google Scholar,17Yau J.W. Rogers S.L. Kawasaki R. Lamoureux E.L. Kowalski J.W. Bek T. Chen S.J. Dekker J.M. Fletcher A. Grauslund J. et al.Meta-Analysis for Eye Disease (META-EYE) Study GroupGlobal prevalence and major risk factors of diabetic retinopathy.Diabetes Care. 2012; 35: 556-564Crossref PubMed Scopus (1681) Google Scholar Around 7%–10% patients with DM will develop vision-threatening (proliferative) retinopathy, an advanced stage of DR.17Yau J.W. Rogers S.L. Kawasaki R. Lamoureux E.L. Kowalski J.W. Bek T. Chen S.J. Dekker J.M. Fletcher A. Grauslund J. et al.Meta-Analysis for Eye Disease (META-EYE) Study GroupGlobal prevalence and major risk factors of diabetic retinopathy.Diabetes Care. 2012; 35: 556-564Crossref PubMed Scopus (1681) Google Scholar,18Maniadakis N. Konstantakopoulou E. Cost effectiveness of treatments for diabetic retinopathy: a systematic literature review.Pharmacoeconomics. 2019; 37: 995-1010Crossref PubMed Scopus (6) Google Scholar Diabetes-associated hyperglycemia damages the retinal vasculature vascular, including endothelial cells, basement membrane, and supporting pericytes.19Abcouwer S.F. Gardner T.W. Diabetic retinopathy: loss of neuroretinal adaptation to the diabetic metabolic environment.Ann. N Y Acad. Sci. 2014; 1311: 174-190Crossref PubMed Scopus (96) Google Scholar Non-proliferative DR (NPDR) is the initial stage of DR characterized by mild changes such as microaneurysms, microhemorrhages, hard exudates, and cotton wool spots, which can be detected clinically using ophthalmoscopy or fundus imaging. Diabetic macular edema (DME) is a frequent cause of vision loss, resulting from the breakdown of the outer blood-retinal barrier due to damaged endothelial tight junctions that allow salts, proteins, and water to accumulate within the retina.20Stitt A.W. Lois N. Medina R.J. Adamson P. Curtis T.M. Advances in our understanding of diabetic retinopathy.Clin. Sci. (Lond.). 2013; 125: 1-17Crossref PubMed Scopus (0) Google Scholar,21Sacconi R. Giuffrè C. Corbelli E. Borrelli E. Querques G. Bandello F. Emerging therapies in the management of macular edema: a review.F1000Res. 2019; 8: F1000Crossref PubMed Scopus (3) Google Scholar DME can occur at any stage of DR. Proliferative DR (PDR), or advanced DR, is characterized by neovascularization with or without pre-retinal or vitreal hemorrhages. Retinal neovascularization along with fibrosis can promote tractional retinal detachment, a severe complication of PDR that requires surgical interference. Retinal neovascularization can also occur on the iris and fluid drainage angle at the anterior eye, promoting the risk of neovascular glaucoma (reviewed in Rodríguez et al.22Rodríguez M.L. Pérez S. Mena-Mollá S. Desco M.C. Ortega A.L. Oxidative stress and microvascular alterations in diabetic retinopathy: future therapies.Oxid. Med. Cell. Longev. 2019; 2019: 4940825Crossref PubMed Scopus (4) Google Scholar). For these reasons, those patients that progress to the PDR stage are at high risk of vision loss. Anti-VEGF therapies, along with laser photocoagulation, are currently the first-line treatment for DR.21Sacconi R. Giuffrè C. Corbelli E. Borrelli E. Querques G. Bandello F. Emerging therapies in the management of macular edema: a review.F1000Res. 2019; 8: F1000Crossref PubMed Scopus (3) Google Scholar,22Rodríguez M.L. Pérez S. Mena-Mollá S. Desco M.C. Ortega A.L. Oxidative stress and microvascular alterations in diabetic retinopathy: future therapies.Oxid. Med. Cell. Longev. 2019; 2019: 4940825Crossref PubMed Scopus (4) Google Scholar A vasoactive molecule that drives neovascularization common to corneal, retinal, and choroidal vascular beds is VEGF.23Kvanta A. Ocular angiogenesis: the role of growth factors.Acta Ophthalmol. Scand. 2006; 84: 282-288Crossref PubMed Scopus (98) Google Scholar,24Rubio R.G. Adamis A.P. Ocular angiogenesis: vascular endothelial growth factor and other factors.Dev. Ophthalmol. 2016; 55: 28-37Crossref PubMed Scopus (23) Google Scholar VEGF is considered to be the most critical regulator of ocular angiogenesis, where it regulates cellular proliferation, survival, motility, and vascular permeability.25Campochiaro P.A. Aiello L.P. Rosenfeld P.J. Anti-vascular endothelial growth factor agents in the treatment of retinal disease: from bench to bedside.Ophthalmology. 2016; 123: S78-S88Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar,26Cross M.J. Dixelius J. Matsumoto T. Claesson-Welsh L. VEGF-receptor signal transduction.Trends Biochem. Sci. 2003; 28: 488-494Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar VEGF is released either as soluble or extracellular matrix-bound forms to interact with VEGF receptor 2 (VEGFR2), which are found on vascular endothelial cells.27Chen T.T. Luque A. Lee S. Anderson S.M. Segura T. Iruela-Arispe M.L. Anchorage of VEGF to the extracellular matrix conveys differential signaling responses to endothelial cells.J. Cell Biol. 2010; 188: 595-609Crossref PubMed Scopus (213) Google Scholar Activation of VEGFR2 modulates a number of downstream signaling cascades, one of which is receptor tyrosine kinases. Engagement of VEGFR2 by VEGF activates phosphatidylinositol 3-kinase (PI3K), phospholipase C γ (PLCγ), focal adhesion kinase (FAK), and p38 mitogen-activated protein kinase (p38MAPK) (Figure 1). PI3K, in turn, activates AKT and Rac, resulting in inhibition of apoptotic signaling (promote survival) and decreased cellular adhesion (increase vascular permeability), respectively. PLCγ activates protein kinase C (PKC) and subsequently the MAPK cascade, which promotes cell proliferation and cytoskeletal reorganization (increase motility).26Cross M.J. Dixelius J. Matsumoto T. Claesson-Welsh L. VEGF-receptor signal transduction.Trends Biochem. Sci. 2003; 28: 488-494Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar Both FAK and p38MAPK are known as the key mediators of endothelial cell adhesion and migration (increase motility).28Yoshizuka N. Chen R.M. Xu Z. Liao R. Hong L. Hu W.Y. Yu G. Han J. Chen L. Sun P. A novel function of p38-regulated/activated kinase in endothelial cell migration and tumor angiogenesis.Mol. Cell. Biol. 2012; 32: 606-618Crossref PubMed Scopus (42) Google Scholar The combined effects of increased VEGF contribute to several important steps in angiogenesis such as endothelial cell mitogenesis, migration, sprouting, and tube formation.29Ucuzian A.A. Gassman A.A. East A.T. Greisler H.P. Molecular mediators of angiogenesis.J. Burn Care Res. 2010; 31: 158-175Crossref PubMed Scopus (155) Google Scholar Clinical studies have shown that abnormally high VEGF levels are found in several ocular diseases, including nAMD, DR, DME, ROP, and neovascular glaucoma.4Campochiaro P.A. Ocular neovascularization.J. Mol. Med. (Berl.). 2013; 91: 311-321Crossref PubMed Scopus (0) Google Scholar Intravitreal injection of anti-VEGF agents significantly improves visual function in patients with nAMD and DR (Table 1).8Solomon S.D. Lindsley K. Vedula S.S. Krzystolik M.G. Hawkins B.S. Anti-vascular endothelial growth factor for neovascular age-related macular degeneration.Cochrane Database Syst. Rev. 2019; 3: CD005139PubMed Google Scholar,30Solomon S.D. Chew E. Duh E.J. Sobrin L. Sun J.K. VanderBeek B.L. Wykoff C.C. Gardner T.W. Diabetic retinopathy: a position statement by the American Diabetes Association.Diabetes Care. 2017; 40: 412-418Crossref PubMed Scopus (184) Google Scholar, 31Spooner K. Fraser-Bell S. Hong T. Chang A.A. Five-year outcomes of retinal vein occlusion treated with vascular endothelial growth factor inhibitors.BMJ Open Ophthalmol. 2019; 4: e000249Crossref PubMed Scopus (5) Google Scholar, 32Kang H.G. Choi E.Y. Byeon S.H. Kim S.S. Koh H.J. Lee S.C. Kim M. Anti-vascular endothelial growth factor treatment of retinopathy of prematurity: efficacy, safety, and anatomical outcomes.Korean J. Ophthalmol. 2018; 32: 451-458Crossref PubMed Google Scholar, 33Petsoglou C. Balaggan K.S. Dart J.K. Bunce C. Xing W. Ali R.R. Tuft S.J. Subconjunctival bevacizumab induces regression of corneal neovascularisation: a pilot randomised placebo-controlled double-masked trial.Br. J. Ophthalmol. 2013; 97: 28-32Crossref PubMed Scopus (37) Google Scholar, 34Puliafito C.A. Wykoff C.C. Looking ahead in retinal disease management: highlights of the 2019 angiogenesis, exudation and degeneration symposium.Int. J. Retina Vitreous. 2019; 5: 22Crossref PubMed Google ScholarTable 1Summary of Available Anti-VEGF TherapiesGeneric NamePegaptanibBevacizumabRanibizumabAfliberceptBrolucizumabBrand nameMacugenAvastinLucentisEyleaBeovuTargetsonly one VEGF-A isoform (VEGF165)all VEGF-A isoformall VEGF-A isoformall VEGF-A/VEGF-B/PlGF isoformsall VEGF-A isoformFormataptamerfull monoclonal antibodyantibody fragmentVEGFR1/2 recombinant fusion proteinsingle-chain antibody fragmentFunctionVEGF inhibitoranti-VEGF antibodyanti-VEGF antibodyVEGF trapanti-VEGF antibodyMolecular mass49 kDa149 kDa48 kDa115 kDa26 kDaFDA-approved indicationsnAMDno FDA approval for ophthalmic usenAMD; DR; DME; macular edema after RVO; mCNVnAMD; DR; DME; macular edema after RVOnAMDClinical dosage regimen0.3 mg every 6 weeks35Rosina C. Bottoni F. Staurenghi G. Clinical experience with pegaptanib sodium.Clin. Ophthalmol. 2008; 2: 485-488PubMed Google Scholar1.25 mg every 4 weeks36Holekamp N.M. Review of neovascular age-related macular degeneration treatment options.Am. J. Manag. Care. 2019; 25: S172-S181PubMed Google Scholar0.5 mg every 4 weeks for nAMDaMay give every 12 weeks after 3 or 4 monthly injections for nAMD, but is less effective than once monthly dosing. or 0.3 mg every 4 weeks for DR or DME37Stewart M.W. A review of ranibizumab for the treatment of diabetic retinopathy.Ophthalmol. Ther. 2017; 6: 33-47Crossref PubMed Scopus (16) Google Scholar,36Holekamp N.M. Review of neovascular age-related macular degeneration treatment options.Am. J. Manag. Care. 2019; 25: S172-S181PubMed Google Scholar2.0 mg every 4 weeks for first three injections, then every 8 weeks for nAMDbMay give every 12 weeks for selected patients after first year. or 2.0 mg every 4 weeks for the first five injections, then every 8 weeks for DR or DME36Holekamp N.M. Review of neovascular age-related macular degeneration treatment options.Am. J. Manag. Care. 2019; 25: S172-S181PubMed Google Scholar6.0 mg every 4 weeks for the first three injections, then every 8–12 weeks38Yannuzzi N.A. Freund K.B. Brolucizumab: evidence to date in the treatment of neovascular age-related macular degeneration.Clin. Ophthalmol. 2019; 13: 1323-1329Crossref PubMed Scopus (7) Google ScholarIntraocular half-lives4 days39Drolet D.W. Nelson J. Tucker C.E. Zack P.M. Nixon K. Bolin R. Judkins M.B. Farmer J.A. Wolf J.L. Gill S.C. Bendele R.A. Pharmacokinetics and safety of an anti-vascular endothelial growth factor aptamer (NX1838) following injection into the vitreous humor of rhesus monkeys.Pharm. Res. 2000; 17: 1503-1510Crossref PubMed Scopus (164) Google Scholar6.7 days40Zhu Q. Ziemssen F. Henke-Fahle S. Tatar O. Szurman P. Aisenbrey S. Schneiderhan-Marra N. Xu X. Grisanti S.; Tübingen Bevacizumab Study Group Vitreous levels of bevacizumab and vascular endothelial growth factor-A in patients with choroidal neovascularization.Ophthalmology. 2008; 115: 1750-1755.e1Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar7.19 days41Krohne T.U. Liu Z. Holz F.G. Meyer C.H. Intraocular pharmacokinetics of ranibizumab following a single intravitreal injection in humans.Am. J. Ophthalmol. 2012; 154: 682-686.e2Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar11 days42Do D.V. Rhoades W. Nguyen Q.D. Pharmacokinetic study of intravitreal aflibercept in humans with neovascular age-related macular degeneration.Retina. 2020; 40: 643-647Crossref PubMed Scopus (3) Google Scholar4.3 days43Caruso A. Füth M. Alvarez-Sánchez R. Belli S. Diack C. Maass K.F. Schwab D. Kettenberger H. Mazer N.A. Ocular half-life of intravitreal biologics in humans and other species: meta-analysis and model-based prediction.Mol. Pharm. 2020; 17: 695-709PubMed Google Scholar4.9 days44Moisseiev E. Waisbourd M. Ben-Artsi E. Levinger E. Barak A. Daniels T. Csaky K. Loewenstein A. Barequet I.S. Pharmacokinetics of bevacizumab after topical and intravitreal administration in human eyes.Graefes Arch. Clin. Exp. Ophthalmol. 2014; 252: 331-337Crossref PubMed Scopus (49) Google Scholar9 days45Xu L. Lu T. Tuomi L. Jumbe N. Lu J. Eppler S. Kuebler P. Damico-Beyer L.A. Joshi A. Pharmacokinetics of ranibizumab in patients with neovascular age-related macular degeneration: a population approach.Invest. Ophthalmol. Vis. Sci. 2013; 54: 1616-1624Crossref PubMed Scopus (108) Google ScholarMolecular structureVEGF, vascular endothelial growth factor; PlGF, placental growth factor; nAMD, neovascular age-related macular degeneration; DR, diabetic retinopathy; DME, diabetic macular edema; mCNV, myopic choroidal neovascularization; RVO, retinal vein occlusion.a May give every 12 weeks after 3 or 4 monthly injections for nAMD, but is less effective than once monthly dosing.b May give every 12 weeks for selected patients after first year. Open table in a new tab VEGF, vascular endothelial growth factor; PlGF, placental growth factor; nAMD, neovascular age-related macular degeneration; DR, diabetic retinopathy; DME, diabetic macular edema; mCNV, myopic choroidal neovascularization; RVO, retinal vein occlusion. The VEGF aptamer pegaptanib (Macugen; Eyetech Pharmaceuticals/Pfizer) was the first antiangiogenic agent approved at a recommended dosage of 0.3 mg, administered by intravitreal injection every 6 weeks by the US Food and Drug Administration (FDA) for the treatment of nAMD.46Schmidt-Erfurth U. Pollreisz A. Mitsch C. Bolz M. Antivascular endothelial growth factors in age-related macular degeneration.Dev. Ophthalmol. 2010; 46: 21-38Crossref PubMed Scopus (0) Google Scholar,35Rosina C. Bottoni F. Staurenghi G. Clinical experience with pegaptanib sodium.Clin. Ophthalmol. 2008; 2: 485-488PubMed Google Scholar Pegaptanib specifically inhibits VEGF165, the dominant isoform of VEGF-A. This selectivity limits its efficacy compared with non-selective VEGF inhibitors (e.g., bevacizumab) for nAMD treatment.8Solomon S.D. Lindsley K. Vedula S.S. Krzystolik M.G. Hawkins B.S. Anti-vascular endothelial growth factor for neovascular age-related macular degeneration.Cochrane Database Syst. Rev. 2019; 3: CD005139PubMed Google Scholar,47Ng E.W. Shima D.T. Calias P. Cunningham Jr., E.T. Guyer D.R. Adamis A.P. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease.Nat. Rev. Drug Discov. 2006; 5: 123-132Crossref PubMed Scopus (912) Google Scholar Due to its long-term safety profile, pegaptanib has been suggested for patients who are prone to the development of thromboembolic events (e.g., diabetes, cardiovascular disease) or geographic atrophy.48Grunwald J.E. Pistilli M. Daniel E. Ying G.S. Pan W. Jaffe G.J. Toth C.A. Hagstrom S.A. Maguire M.G. Martin D.F. Comparison of Age-Related Macular Degeneration Treatments Trials Research GroupIncidence and growth of geographic atrophy during 5 years of comparison of age-related macular degeneration treatments trials.Ophthalmology. 2017; 124: 97-104Abstract Full Text Full Text PDF PubMed Google Scholar,49Dombi T. Kwok K.K. Sultan M.B. A retrospective, pooled data analysis of the safety of pegaptanib sodium in the treatment of age-related macular degeneration in subjects with or without diabetes mellitus.BMC Ophthalmol. 2012; 12: 37Crossref PubMed Scopus (0) Google Scholar Other anti-VEGF agents, including ranibizumab, aflibercept, and bevacizumab, are widely used to inhibit ocular neovascularization.50Cabral T. Mello L.G.M. Lima L.H. Polido J. Regatieri C.V. Belfort Jr., R. Mahajan V.B. Retinal and choroidal angiogenesis: a review of new targets.Int. J. Retina Vitreous. 2017; 3: 31Crossref PubMed Scopus (40) Google Scholar Bevacizumab (Avastin) and ranibizumab (Lucentis) are immunoglobulin antibodies (monoclonal and fragment, respectively) both targeting VEGF-A. Ranibizumab is 5- to 30-fold more potent at neutralizing VEGF-A than is bevacizumab.51Chen Y. Wiesmann C. Fuh G. Li B. Christinger H.W. McKay P. de Vos A.M. Lowman H.B. Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen.J. Mol. Biol. 1999; 293: 865-881Crossref PubMed Scopus (360) Google Scholar Ranibizumab was shown to improve vision and was approved at a dose of 0.5 mg every 4 weeks for nAMD treatment in 2006,52Heier J.S. Antoszyk A.N. Pavan P.R. Leff S.R. Rosenfeld P.J. Ciulla T.A. Dreyer R.F. Gentile R.C. Sy J.P. Hantsbarger G. Shams N. Ranibizumab for treatment of neovascular age-related macular degeneration: a phase I/II multicenter, controlled, multidose study.Ophthalmology. 2006; 113: 633.e1-4Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar,53Frennesson C. Nilsson U.L. Peebo B.B. Nilsson S.E. Significant improvements in near vision, reading speed, central visual field and related quality of life after ranibizumab treatment of wet age-related macular degeneration.Acta Ophthalmol. 2010; 88: 420-425Crossref PubMed Scopus (31) Google Scholar and 0.3 mg every 4 weeks for DR and DME in 2012.37Stewart M.W. A review of ranibizumab for the treatment of diabetic retinopathy.Ophthalmol. Ther. 2017; 6: 33-47Crossref PubMed Scopus (16) Google Scholar Bevacizumab was originally approved for treatment of metastatic colorectal cancer but has been used since 2005 as an off-label treatment for nAMD at a dose of 1.25 mg every 4 weeks.54Stewart M.W. Off-label drug use: the bevacizumab story.Mayo Clin. Proc. 2013; 88: 305Abstract Full Text Full Text PDF PubMed Google Scholar Because of its lower cost and greater availability, bevacizumab has become the first option for many patients.55Hussain R.M. Ciulla T.A. Emerging vascular endothelial growth factor antagonists to treat neovascular age-related macular degeneration.Expert Opin. Emerg. Drugs. 2017; 22: 235-246Crossref PubMed Scopus (38) Google Scholar,56Rosenfeld P.J. Moshfeghi A.A. Puliafito C.A. Optical coherence tomography findings after an intravitreal injection of bevacizumab (avastin) for neovascular age-related macular degeneration.Ophthalmic Surg. Lasers Imaging. 2005; 36: 331-335Crossref PubMed Google Scholar Aflibercept (Eylea) is a recombinant fusion protein that forms a VEGF trap, which targets VEGF-A, VEGF-B, and placental growth factor (PlGF).57Malik D. Tarek M. Caceres del Carpio J. Ramirez C. Boyer D. Kenney M.C. Kuppermann B.D. Safety profiles of anti-VEGF drugs: bevacizumab, ranibizumab, aflibercept and ziv-aflibercept on human retinal pigment epithelium cells in culture.Br. J. Ophthalmol. 2014; 98: i11-i16Crossref PubMed Scopus (76) Google Scholar PlGF is another member of the VEGF family that activates VEGFR1.58Park J.E. Chen H.H. Winer J. Houck K.A. Ferrara N. Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR.J. Biol. Chem. 1994; 269: 25646-25654PubMed Google Scholar PlGF was reported to potentiate angiogenic signaling by forming heterodimers with VEGF-A, or by displacing VEGF-A from VEGFR1, and contributes to the development of neovascularization.59Rakic J.M. Lambert V. Devy L. Luttun A. Carmeliet P. Claes C. Nguyen L. Foidart J.M. Noël A. Munaut C. Placental growth factor, a member of the VEGF family, contributes to the development of choroidal neovascularization.Invest. Ophthalmol. Vis. Sci. 2003; 44: 3186-3193Crossref PubMed Scopus (255) Google Scholar Aflibercept is 100-fold higher than both bevacizumab and ranibizumab,60Papadopoulos N. Martin J. Ruan Q. Rafique A. Rosconi M.P. Shi E. Pyles E.A. Yancopoulos G.D. Stahl N. Wiegand S.J. Binding