title: Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model.
abstract: OBJECTIVE
  RNA interference mediated by small interfering RNAs (siRNAs) is a powerful technology allowing the silencing of mamalian genes with great specificity and potency. The purpose of this study was to demonstrate the feasibility of RNA interference mediated by siRNA in retinal cells in vitro and in the murine retina in vivo.
  
  
  METHODS
  siRNAs specific for enhanced green fluorescent protein (EGFP) and murine and human vascular endothelial growth factor (VEGF) were designed. In vitro studies in human cell lines entailed modulation of endogenous VEGF levels through chemically induced hypoxia. Effects of siRNA treatment on these levels were measured by ELISA. In vivo studies evaluating effects of siRNA on levels of EGFP and VEGF were performed by co-injecting recombinant viruses carrying EGFP or hVEGF cDNAs along with the appropriate siRNAs subretinally in mice. Additional studies aimed at blocking production of endogenous mVEGF were performed using laser-induced choroidal neovascularization (CNV) in mice. Effects of in vivo treatments were evaluated ophthalmoscopically. Retinal/choroidal flat mounts were evaluated after perfusion with dextran-fluorescein. Alternatively, retinas were evaluated in histological sections or VEGF levels were measured in intact eyes using ELISA.
  
  
  RESULTS
  Successful delivery of siRNA to the subretinal space was confirmed by observing significantly reduced levels of EGFP in eyes treated with Ad.CMV.EGFP plus EGFP-directed siRNA. siRNAs directed against hVEGF effectively and specifically inhibit hypoxia-induced VEGF levels in human cell lines and after adenoviral induced hVEGF transgene expression in vivo. In addition, subretinal delivery of siRNA directed against murine Vegf significantly inhibited CNV after laser photocoagulation.
  
  
  CONCLUSIONS
  Delivery of siRNA can be used in vitro and in vivo to target specific RNAs and to reduce the levels of the specific protein product in the targeted cells. This work suggests that RNA interference has potential for application to studies of retinal biology and for the treatment of a variety of retinal diseases, including those involving abnormal blood vessel growth.
authors.affiliation: F. M. Kirby Center for Molecular Ophthalmology, Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia 19104, USA.
authors.forename: Samuel J
authors.initials: SJ
authors.lastname: Reich
authors.forename: Joshua
authors.initials: J
authors.lastname: Fosnot
authors.forename: Akiko
authors.initials: A
authors.lastname: Kuroki
authors.forename: Waixing
authors.initials: W
authors.lastname: Tang
authors.forename: Xiangyang
authors.initials: X
authors.lastname: Yang
authors.forename: Albert M
authors.initials: AM
authors.lastname: Maguire
authors.forename: Jean
authors.initials: J
authors.lastname: Bennett
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Tolentino
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics; D000818:Animals; D002829:Choroid / Q000098:blood supply / Q000601:surgery; D020256:Choroidal Neovascularization / Q000378:metabolism / Q000517:prevention & control*; D003673:Defective Viruses; D004195:Disease Models, Animal; D015536:Down-Regulation; D016228:Endothelial Growth Factors / Q000235:genetics* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D020868:Gene Silencing; D018390:Gene Targeting; D049452:Green Fluorescent Proteins; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D017075:Laser Coagulation; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008222:Lymphokines / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010857:Pigment Epithelium of Eye / Q000378:metabolism / Q000821:virology; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
chemical_list: D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008222:Lymphokines; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D049452:Green Fluorescent Proteins