Reduction of choroidal neovascularization in mice by adeno-associated virus-delivered anti-vascular endothelial growth factor short hairpin RNA

• Published in: The journal of gene medicine 2012-11, Vol.14 (11), p.632-641
• Main Authors: Askou, Anne Louise, Pournaras, Jean-Antoine C., Pihlmann, Maria, Svalgaard, Jesper D., Arsenijevic, Yvan, Kostic, Corinne, Bek, Toke, Dagnæs-Hansen, Frederik, Mikkelsen, Jacob Giehm, Jensen, Thomas Gryesten, Corydon, Thomas J.
• Format: Article
• Language: English
• Subjects: adeno-associated virus; AMD; angiogenesis; Animals; anti-VEGF gene therapy; Cell Line; choroidal neovascularization; Choroidal Neovascularization - genetics; Choroidal Neovascularization - metabolism; Dependovirus - genetics; Female; Gene therapy; Gene Transfer Techniques; Genetic Vectors - genetics; Humans; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neovascularization, Pathologic - genetics; Neovascularization, Pathologic - metabolism; Retinal Pigment Epithelium - metabolism; Retinal Pigment Epithelium - pathology; RNA, Small Interfering - genetics; short hairpin RNA; Vascular Endothelial Growth Factor A - antagonists & inhibitors; Vascular Endothelial Growth Factor A - genetics; Vascular Endothelial Growth Factor A - metabolism
• ISSN: 1099-498X; 1521-2254
• DOI: 10.1002/jgm.2678

Background Strategies leading to the long‐term suppression of inappropriate ocular angiogenesis are required to avoid the need for repetitive monthly injections for treatment of diseases of the eye, such as age‐related macular degeneration (AMD). The present study aimed to develop a strategy for the sustained repression of vascular endothelial growth factor (VEGF), which is identified as the key player in exudative AMD. Methods We have employed short hairpin (sh)RNAs combined with adeno‐associated virus (AAV) delivery to obtain the targeted expression of potent gene‐regulatory molecules. Anti‐VEGF shRNAs were analyzed in human retinal pigment epithelial (RPE) cells using Renilla luciferase screening. For in vivo delivery of the most potent shRNA, self‐complementary AAV vectors were packaged in serotype 8 capsids (scAAV2/8‐hU6‐sh9). In vivo efficacy was evaluated either by injection of scAAV2/8‐hU6‐sh9 into murine hind limb muscles or in a laser‐induced murine model of choroidal neovascularization (CNV) following scAAV2/8‐hU6‐sh9 subretinal delivery. Results Plasmids encoding anti‐VEGF shRNAs showed efficient knockdown of human VEGF in RPEs. Intramuscular administration led to localized expression and 91% knockdown of endogenous murine (m)VEGF. Subsequently, the ability of AAV2/8‐encoded shRNAs to impair vessel formation was evaluated in the murine model of CNV. In this model, the sizes of the CNV were significantly reduced (up to 48%) following scAAV2/8‐hU6‐sh9 subretinal delivery. Conclusions Using anti‐VEGF vectors, we have demonstrated efficient silencing of endogenous mVEGF and showed that subretinal administration of scAAV2/8‐hU6‐sh9 has the ability to impair vessel formation in an AMD animal model. Thus, AAV‐encoded shRNA can be used for the inhibition of neovascularization, leading to the development of sustained anti‐VEGF therapy. Copyright © 2012 John Wiley & Sons, Ltd.