Inhibition of corneal neovascularization by recombinant adenovirus mediated antisense VEGF RNA.
The expression of vascular endothelial growth factor has been strongly implicated in the pathogenesis of conditions leading to inappropriate blood vessel growth in the eye. As such, vascular endothelial growth factor is an attractive target for anti-angiogenic therapies designed to treat neovascular eye diseases. One such therapy, antisense gene therapy, is a technique based on the ability of single-stranded DNA or RNA sequences to alter the expression of targeted genes. Recombinant adenoviruses have demonstrated efficient ocular cell transduction with a high level of transgene production. Cauterization of the normally avascular rat cornea results in a strong neovascular response, making it an ideal animal model for the testing of anti-angiogenic therapies. In this study, a recombinant adenovirus system was assessed for the ability to express biologically relevant antisense RNA to reduce vascular endothelial growth factor expression in a rat model of corneal neovascularization. Recombinant adenovirus constructs expressing short and long antisense and sense vascular endothelial growth factor cDNA, under the control of cytomegalovirus major immediate early promoter or the RNA polymerase III promoter, VA1, were constructed. The expression of short and long antisense RNAs was demonstrated by Northern blot hybridization. All constructs were capable of producing RNA, and the highest level of antisense RNA production was detected in retinal pigment epithelial cells which had been transduced with the longer antisense cDNA construct under the control of the VA1 promoter. This construct was also the most efficient in reducing in vitro vascular endothelial growth factor production (P<0.05) and human endothelial cell proliferation. This construct was subsequently injected into rat eyes 24hr prior to cauterization of the cornea and antisense vascular endothelial growth factor expression was demonstrated by in situ hybridization. The resulting neovascular response was clearly inhibited at 4, 7 and 14 days post-cautery, compared to the control injections which demonstrated an intense neovascular response. Only one out of six eyes injected with the long antisense cDNA construct under the control of the VA1 promoter demonstrated any vascular response to cautery. The reduction in the neovascular response was correlated, with significantly lower amounts of vascular endothelial growth factor protein in the corneas (P=0.006). These observations suggest that the specific down-regulation of vascular endothelial growth factor production is sufficient to reduce the corneal neovascular response and that recombinant adenovirus might be a useful vehicle to produce antisense RNA in situ to down-regulate ocular gene expression.
Lai
Chooi-May
CM
Centre for Ophthalmology and Vision Science, The University of Western Australia, Perth, Australia.
Spilsbury
Katrina
K
Brankov
Meliha
M
Zaknich
Tammy
T
Rakoczy
P Elizabeth
PE
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000867:Anterior Chamber / Q000378:metabolism; D018929:Cell Culture Techniques; D002455:Cell Division; D003315:Cornea / Q000378:metabolism; D016510:Corneal Neovascularization / Q000517:prevention & control*; D004195:Disease Models, Animal; D015536:Down-Regulation; D016228:Endothelial Growth Factors / Q000096:biosynthesis / Q000235:genetics*; D004730:Endothelium, Vascular / Q000166:cytology; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D017403:In Situ Hybridization; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics*; D008222:Lymphokines / Q000096:biosynthesis / Q000235:genetics*; D016372:RNA, Antisense / Q000235:genetics*; D051381:Rats; D020318:Rats, Long-Evans; D014161:Transduction, Genetic; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D016372:RNA, Antisense; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors