title: Adeno-associated virus type 8 vector-mediated expression of siRNA targeting vascular endothelial growth factor efficiently inhibits neovascularization in a murine choroidal neovascularization model.
abstract: OBJECTIVE
  To assess the feasibility of a gene therapeutic approach to treating choroidal neovascularization (CNV), we generated an adeno-associated virus type 8 vector (AAV2/8) encoding an siRNA targeting vascular endothelial growth factor (VEGF), and determined the AAV2/8 vector's ability to inhibit angiogenesis.
  
  
  METHODS
  We initially transfected 3T3 cells expressing VEGF with the AAV2/8 plasmid vector psiRNA-VEGF using the H1 promoter and found that VEGF expression was significantly diminished in the transfectants. We next injected 1 μl (3 × 10(14) vg/ml) of AAV2/8 vector encoding siRNA targeting VEGF (AAV2/8/SmVEGF-2; n = 12) or control vector encoding green fluorescent protein (GFP) (AAV2/8/GFP; n = 14) into the subretinal space in C57BL/6 mice. One week later, CNV was induced by using a diode laser to make four separate choroidal burns around the optic nerve in each eye. After an additional 2 weeks, the eyes were removed for flat mount analysis of the CNV surface area.
  
  
  RESULTS
  Subretinal delivery of AAV2/8/SmVEGF-2 significantly diminished CNV at the laser lesions, compared to AAV8/GFP (1597.3 ± 2077.2 versus 5039.5 ± 4055.9 µm(2); p<0.05). Using an enzyme-linked immunosorbent assay, we found that VEGF levels were reduced by approximately half in the AAV2/8/SmVEGF-2 treated eyes.
  
  
  CONCLUSIONS
  These results suggest that siRNA-VEGF can be expressed across the retina and that long-term suppression of CNV is possible through the use of stable AAV2/8-mediated siRNA-VEGF expression. In vivo gene therapy may thus be a feasible approach to the clinical management of CNV in conditions such as age-related macular degeneration.
authors.affiliation: Department of Ophthalmology, Nippon Medical School, Tokyo, Japan ; Department of Biochemistry and Molecular Biology, Division of Gene Therapy Research Center for Advanced Medical Technology, Nippon Medical School, Tokyo, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Igarashi
authors.affiliation: Department of Biochemistry and Molecular Biology, Division of Gene Therapy Research Center for Advanced Medical Technology, Nippon Medical School, Tokyo, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Miyake
authors.affiliation: Department of Ophthalmology, Nippon Medical School, Tokyo, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Fujimoto
authors.affiliation: Department of Ophthalmology, Nippon Medical School, Tokyo, Japan.
authors.forename: Chiemi
authors.initials: C
authors.lastname: Yaguchi
authors.affiliation: Department of Biochemistry and Molecular Biology, Division of Gene Therapy Research Center for Advanced Medical Technology, Nippon Medical School, Tokyo, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Iijima
authors.affiliation: Department of Biochemistry and Molecular Biology, Division of Gene Therapy Research Center for Advanced Medical Technology, Nippon Medical School, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Shimada
authors.affiliation: Department of Ophthalmology, Nippon Medical School, Tokyo, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: Department of Biochemistry and Molecular Biology, Division of Gene Therapy Research Center for Advanced Medical Technology, Nippon Medical School, Tokyo, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Miyake
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002829:Choroid / Q000378:metabolism / Q000473:pathology; D020256:Choroidal Neovascularization / Q000473:pathology / Q000628:therapy*; D000229:Dependovirus / Q000378:metabolism*; D004195:Disease Models, Animal; D005822:Genetic Vectors / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000378:metabolism / Q000473:pathology; D014161:Transduction, Genetic; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000627:therapeutic use*
chemical_list: D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D049452:Green Fluorescent Proteins
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