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KA Drenser, D Bok, MM LaVail; Ribozyme-mediated Inhibition of Apoptosisin the Treatment of Retinal Disease . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4587.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: This project aims to slow the progression of retinal disease, such as retinitis pigmentosis, glaucoma and ischemic disease, using ribozymes, catalytic RNA molecules, to inhibit apoptosis in affected retinal cells. Ribozymes have been engineered to target and cleave the RNA encoding the Bax protein. Bax, a member of the Bcl-2 family, plays an important role in the induction of apoptsis. Bcl-2, as well as other Bcl-2 family proteins, forms heterodimers with Bax. With cell stress the level of Bax protein increases and Bax homodimers begin to form. The Bax homodimers alter the cell membrane integrity and induce apoptosis by activating downstream events. Decreasing the concentration of Bax protein within the cell by ribozyme-mediated destruction will reduce the concentration of Bax homodimers, inhibiting programmed cell death by and thereby allowing for cell survival. Methods: Ribozymes were designed to target and cleave the RNA encoding the Bax protein. The ribozymes and synthetic Bax coding sequences were generated by mutually primed synthesis and cloned into an in vitro expression plasmid. Radiolabeled synthetic RNAs of both ribozymes and target Bax molecules were made by in vitro transcription. Inactive ribozymes and mutated targets were also generated and serve as controls. Ribozymes were tested for activity and specificity by incubating targets and ribozymes together in 20mM MgCl2 buffers at 37 degrees C for one hour. Cleavage products were separated by denaturing gel electrophoresis and autoradiogram exposure. Animal models of apoptosis consist of a peripherin mutation on a Rds mouse background and a transgenic rat carrying a rhodopsin mutation. Results: Two ribozymes, one hairpin and one hammerhead, were designed and tested in vitro for activity against the Bax RNA. Both ribozymes were found to cleave the synthetic Bax RNA target while leaving the control target intact. The control target differs from the Bax target by one base pair, representing significant target discrimination by the ribozymes. The target was cleaved in excess of ribozyme molecules, demonstrating catalyitic activity. The control ribozymes did not cleave either the synthetic Bax target or the control target. Conclusion: Ribozymes are able to selectively and catalytically cleave the Bax RNA. The correlating decrease in Bax protein may alter the Bax to Bcl-2 concentration in vivo, thereby inhibiting Bax dimerization and the induction of apoptosis. This represents a possible primary or adjuvant therapy in the treatment of retinal diseases where apoptosis plays a role in vision loss.
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