Inhibition of choroidal neovascularization by adenovirus-mediated delivery of short hairpin RNAs targeting VEGF as a potential therapy for AMD.
OBJECTIVE
Choroidal neovascularization (CNV) is the leading cause of blindness in age-related macular degeneration (AMD). Several lines of evidence implicate increased levels of vascular endothelial growth factor (VEGF) in retinal pigment epithelium (RPE) from patients with AMD. Current approaches to attenuate VEGF or its receptors, including the use of small interfering (si)RNA, show significant promise, but still have limited efficacy and require repeat administrations, using procedures associated with multiple complications. The goal of this study was to develop an approach for long-term endogenous expression of short hairpin (sh)RNA that would significantly attenuate VEGF and hence act as a potential therapy for AMD.


METHODS
Several shRNAs expressed from recombinant adenovirus were developed. These shRNAs were expressed in human RPE cells in the presence of adenovirus vectors overexpressing VEGF, and the amount of VEGF attenuation was evaluated. Adenovirus vectors expressing VEGF were subsequently injected into the subretinal space of mice, and induction of CNV was measured in the presence of adenovirus vectors expressing shRNA targeting VEGF.


RESULTS
Potent shRNA sequences were identified that were able to silence VEGF in human RPE cells. When expressed from adenovirus backbones, these shRNA constructs silenced VEGF by 94% at a 1:5 molar ratio (VEGF to shRNA) and 64% at a 1:0.05 molar ratio. Adenovirus vectors expressing high levels of VEGF could induce CNV in mice within 5 days. Co-injection of VEGF-expressing viruses into mice with shRNA targeting VEGF led to a substantial (84%) reduction in CNV.


CONCLUSIONS
shRNA targeting VEGF from adenovirus vectors allows potent attenuation of VEGF and prevents CNV. This approach shows promise as a therapy for AMD.
Cashman
Siobhan M
SM
Department of Ophthalmology, Tufts University School of Medicine, Boston, MA 02111, USA.
Bowman
Lisa
L
Christofferson
Jason
J
Kumar-Singh
Rajendra
R
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D018929:Cell Culture Techniques; D020256:Choroidal Neovascularization / Q000517:prevention & control*; D005451:Fluorescein Angiography; D020868:Gene Silencing; D018390:Gene Targeting; D015316:Genetic Therapy; D005822:Genetic Vectors; D008268:Macular Degeneration / Q000628:therapy*; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D010857:Pigment Epithelium of Eye / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D034741:RNA, Small Interfering; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A