Decreased expression of the insulin-like growth factor 1 receptor by ribozyme cleavage.
OBJECTIVE
Insulin-like growth factor (IGF)-1 and its receptor (IGF-1R) are associated with abnormal retinal neovascularization. Ribozymes were designed that selectively decreased the expression of the IGF-1R and these ribozymes were tested in angiogenesis models in vitro and in vivo.


METHODS
Two hammerhead ribozymes were designed that cleave the human IGF-1R mRNA. The ribozymes were cloned into recombinant adeno-associated viral vectors (rAAV). The rAAV constructs were transfected into human retinal endothelial cells (HRECs). IGF-1R mRNA and protein levels were examined and the modified Boyden chamber assay used to examine ribozyme effects on cell migration. These constructs were injected intravitreally into mice to determine the effect of the ribozymes on retinal neovascularization in a mouse model of oxygen-induced retinopathy.


RESULTS
Relative quantitative RT-PCR analysis showed that IGF-1R Rz1 reduced IGF-1R mRNA levels by 40% +/- 10% (P = 0.003), and Western blot analysis showed a 41% +/- 5% (P = 4.6 x 10(-5)) reduction of IGF-1R protein, confirming that this ribozyme reduces IGF-1R expression. IGF-1R Rz1 also reduced IGF-1-induced cell migration by 90% +/- 5% (P = 2.9 x 10(-9)) showing that IGF-1R Rz1 reduces IGF-1R function in HRECs. IGF-1R Rz1 also reduced the amount of preretinal neovascularization by 65% +/- 6% (P = 2.7 x 10(-5)), as measured by the average number of endothelial preretinal nuclei per section.


CONCLUSIONS
These studies demonstrate that the IGF-1R ribozymes are effective at reducing the expression and function of the IGF-1R in vitro and in vivo. Therefore, the IGF-1R ribozymes are an effective method for studying the process of angiogenesis and may ultimately be effective as gene therapy tools for the reduction of pathologic retinal angiogenesis.
Shaw
Lynn C
LC
Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, Gainesville, Florida 32610, USA.
Afzal
Aqeela
A
Lewin
Alfred S
AS
Timmers
Adrian M
AM
Spoerri
Polyxenie E
PE
Grant
Maria B
MB
D000818:Animals; D015153:Blotting, Western; D002465:Cell Movement; D003001:Cloning, Molecular; D000229:Dependovirus / Q000235:genetics; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids; D011247:Pregnancy; D016337:RNA, Catalytic / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000235:genetics* / Q000378:metabolism; D015861:Retinal Neovascularization / Q000378:metabolism / Q000517:prevention & control*; D012171:Retinal Vessels / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1