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Volume 271, Number 46, Issue of November 15, 1996 pp. 29107-29112
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Optimizing the Cell Efficacy of Synthetic Ribozymes
SITE SELECTION AND CHEMICAL MODIFICATIONS OF RIBOZYMES TARGETING THE PROTO-ONCOGENE c-myb

(Received for publication, July 24, 1996, and in revised form, September 3, 1996)

From Ribozyme Pharmaceuticals, Incorporated, Boulder, Colorado 80301

Expression of the proto-oncogene c-myb is necessary for proliferation of vascular smooth muscle cells. We have developed synthetic hammerhead ribozymes that recognize and cleave c-myb RNA, thereby inhibiting cell proliferation. Herein, we describe a method for the selection of hammerhead ribozyme cleavage sites and optimization of chemical modifications that maximize cell efficacy. In vitro assays were used to determine the relative accessibility of the ribozyme target sites for binding and cleavage. Several ribozymes thus identified showed efficacy in inhibiting smooth muscle cell proliferation relative to catalytically inactive controls. A combination of modifications including several phosphorothioate linkages at the 5-end of the ribozyme and an extensively modified catalytic core resulted in substantially increased cell efficacy. A variety of different 2-modifications at positions U4 and U7 that confer nuclease resistance gave comparable levels of cell efficacy. The lengths of the ribozyme binding arms were varied; optimal cell efficacy was observed with relatively short sequences (13-15 total nucleotides). These synthetic ribozymes have potential as therapeutics for hyperproliferative disorders such as restenosis and cancer. The chemical motifs that give optimal ribozyme activity in smooth muscle cell assays may be applicable to other cell types and other molecular targets.

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