Adeno-associated virus-delivered polycistronic microRNA-clusters for knockdown of vascular endothelial growth factor in vivo

J Gene Med. 2012 May;14(5):328-38. doi: 10.1002/jgm.2623.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Dependovirus
  • Gene Expression Regulation*
  • Gene Knockdown Techniques
  • Gene Silencing*
  • Gene Transfer Techniques
  • Genetic Vectors
  • Humans
  • Mice
  • MicroRNAs / genetics*
  • Retinal Pigment Epithelium / cytology
  • Vascular Endothelial Growth Factor A* / genetics
  • Vascular Endothelial Growth Factor A* / metabolism


  • MicroRNAs
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A