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S Iwai, LC Shaw, S Caballero, K Oguchi, EA Ellis, MB Grant; The Development of Hammerhead Ribozymes that Specifically Cleave the mRNA of alpha1, alpha3, alpha5 and alphaV Subunits of Integrin . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3712.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR) are characterized by the abnormal growth of blood vessels on the retina. Tissue hypoxia and ischemia initiate a series of events that lead to angiogenesis. The integrins play a critical role in the formation of new blood vessels. The integrins consist of a large family of cell adhesion receptors that facilitate cell-extracellular matrix and cell-cell interactions. A functional integrin consists of two transmembrane glycoprotein subunits (alpha and beta). Several integrins (e.g. alpha1beta1, alpha5beta1, alphaVbeta3, alphaVbeta5) have been shown to be upregulated in endothelial vascular cells in diabetic retinopathy, and down regulation of integrins by specific inhibitors reduces angiogenesis. Our goal was to produce a hammerhead ribozyme that would specifically cleave a subunits of integrin mRNAs and selectively reduce the expression of these proteins in human retinal endothelial cells and in in vivo angiogenesis models in order to study the function of these proteins in angiogenesis. Methods:We have designed pairs of hammerhead ribozymes to specifically cleave the mRNA of the human integrin subunits alpha1, alpha3, alpha5 and alphaV. We have performed in vitro time course of cleavage analysis and multiple turnover kinetic analysis all ribozymes using RNA oligonucleotides for both the ribozymes and targets. The more promising ribozymes have been cloned into recombinant Adeno Associated Viral vectors (rAAV) for subsequent packaging into AAV and for cell transfection and in vivo experimental analysis. Results:In vitro kinetic analysis demonstrated that the more efficient ribozymes we designed varied by as much as 100-fold with a kcat varying from 0.2 min-1 to 21.5 min-1. One ribozyme from each pair was selected and cloned into a rAAV vector. The kcat of these ribozymes was 3.8 min-1 for alpha1Rz1, 0.4 min-1 for alpha3Rz1, 21.5 min-1 for alpha5Rz2 and 19.1 min-1 for alphaVRz2. These values are similar than values we have obtained with other ribozymes (Shaw, et al., 200l), and suggest that these ribozymes would be effective ribozymes in vivo. We are now in the process of testing these AAV-ribozyme constructs in both HREC tissue culture transfection experiments and in the ROP mouse model (Smith, et al. 1994). Conclusion:Our in vitro data suggests that theses ribozyme will effectively reduce the target mRNAs in vivo and provide a tool for the selective inhibition of these a integrin subunits.
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