title: Adeno-associated virus-delivered polycistronic microRNA-clusters for knockdown of vascular endothelial growth factor in vivo.
abstract: BACKGROUND
  Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that plays a critical role in several diseases, including cancer, rheumatoid arthritis and diseases of the eye. Persistent regulation of VEGF by expression of small interfering RNAs targeting VEGF represents a potential future strategy for treatment of such diseases. As a step toward this goal, the present study combines the potency of VEGF-targeted miRNA mimics, produced from a miRNA cluster, with delivery by adeno-associated virus (AAV)-based vectors.
  
  
  METHODS
  Nine different engineered tri-cistronic miRNA clusters encoding anti-VEGF effectors were generated and tested in adult human retinal pigment epithelial (ARPE-19) cells using Renilla luciferase screening, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), western blotting and immunostaining analysis. In vivo efficacy was tested by the injection of scAAV2/8 vectors expressing the most effective miRNA cluster into murine hindlimb muscles, followed by quantitative RT-PCR.
  
  
  RESULTS
  Plasmids containing anti-VEGF miRNA clusters showed efficient silencing of VEGF and demonstrated a combined gene silencing effect for miRNA clusters composed of multiple miRNA-mimicked RNA interference effectors. The most potent molecule, miR-5,10,7, resulted in a knockdown of VEGF by approximately 75%. Injection of scAAV2/8 vectors expressing miR-5,10,7 into murine hindlimb muscles, resulted in a 44% reduction of endogenous VEGF.
  
  
  CONCLUSIONS
  We have developed miRNA clusters encoding anti-VEGF effectors and shown, in a mouse model, that VEGF is efficiently down-regulated by scAAV2/8-delivered miRNA clusters, allowing potent attenuation of VEGF. These findings may contribute to the development of gene therapy based on AAV-mediated delivery of miRNA clusters.
authors.affiliation: Department of Biomedicine, Aarhus University, and Department of Ophthalmology, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Maria
authors.initials: M
authors.lastname: Pihlmann
authors.forename: Anne Louise
authors.initials: AL
authors.lastname: Askou
authors.forename: Lars
authors.initials: L
authors.lastname: Aagaard
authors.forename: Gitte Hoffmann
authors.initials: GH
authors.lastname: Bruun
authors.forename: Jesper Dyrendom
authors.initials: JD
authors.lastname: Svalgaard
authors.forename: Marie Hebsgaard
authors.initials: MH
authors.lastname: Holm-Nielsen
authors.forename: Frederik
authors.initials: F
authors.lastname: Dagnaes-Hansen
authors.forename: Toke
authors.initials: T
authors.lastname: Bek
authors.forename: Jacob Giehm
authors.initials: JG
authors.lastname: Mikkelsen
authors.forename: Thomas Gryesten
authors.initials: TG
authors.lastname: Jensen
authors.forename: Thomas Juhl
authors.initials: TJ
authors.lastname: Corydon
mesh_terms: D000818:Animals; D000229:Dependovirus; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D055213:Retinal Pigment Epithelium / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: D035683:MicroRNAs; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A