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Decreased expression of the insulin-like growth factor 1 receptor by ribozyme cleavage
Lynn C Shaw1, Aqeela Afzal, Alfred S Lewin
1Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, Gainesville, Florida 32610, USA.
Purpose:
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.
Insights
Ribozymes targeting insulin-like growth factor 1 receptor (IGF-1R) effectively reduced its expression and function in retinal cells. These findings suggest IGF-1R ribozymes are promising for treating retinal neovascularization.
Area of Science:
- Ophthalmology
- Molecular Biology
- Gene Therapy
Background:
- Insulin-like growth factor (IGF)-1 and its receptor (IGF-1R) play a role in abnormal retinal neovascularization.
- Targeting IGF-1R offers a potential strategy for managing retinal vascular diseases.
Purpose of the Study:
- To design and evaluate ribozymes that selectively decrease IGF-1R expression.
- To assess the efficacy of these ribozymes in inhibiting angiogenesis in vitro and in vivo.
Main Methods:
- Two hammerhead ribozymes targeting human IGF-1R mRNA were designed and cloned into recombinant adeno-associated viral vectors (rAAV).
- rAAV constructs were transfected into human retinal endothelial cells (HRECs) to assess IGF-1R expression and cell migration.
- Intravitreal injection into mice was performed to evaluate the effect on oxygen-induced retinopathy.
Main Results:
- Ribozyme Rz1 significantly reduced IGF-1R mRNA by 40% and protein by 41% in HRECs.
- IGF-1R Rz1 inhibited IGF-1-induced HREC migration by 90%.
- Intravitreal administration of IGF-1R Rz1 reduced preretinal neovascularization by 65% in a mouse model.
Conclusions:
- IGF-1R ribozymes effectively reduce IGF-1R expression and function in retinal cells.
- These ribozymes serve as valuable tools for studying angiogenesis.
- IGF-1R ribozymes show potential as gene therapy agents for pathologic retinal angiogenesis.