Optimizing the Cell Efficacy of Synthetic Ribozymes

Abstract

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.Since the discovery that certain naturally occurring RNA motifs were capable of catalytically cleaving other RNA molecules in a sequence-specific manner, extensive studies have defined the sequence and structural characteristics that control the in vitro specificity and kinetics of these RNA enzymes or ribozymes (1-4). Ribozymes have a broad range of potential in vivo applications. These include the use of ribozymes as research tools for probing molecular mechanisms, the use of ribozymes to genetically engineer crops, and the use of ribozymes as therapeutics for human or animal diseases. Each of these applications requires that a ribozyme function efficiently within the intracellular environment. The sequence and structural features that promote optimal intracellular activity of ribozymes are currently under study.Several factors are likely to contribute to the intracellular efficacy of a ribozyme. A ribozyme must colocalize with its molecular target in the appropriate cellular compartment and must be present at sufficiently high concentration to promote hybridization. In addition, its catalytic cleavage rate must be fast enough, and its half-life must be long enough to allow cleavage of a substantial fraction of the target mRNA population. Finally, the cleavage site in the target mRNA must be accessible to ribozyme binding. When the ribozyme is made synthetically, a variety of modifications can be introduced to increase its half-life within the cell, to change its target sequence binding affinity, and possibly also to alter its intracellular trafficking properties. In this study, we have used chemically synthesized hammerhead ribozymes t...

Cleavage of Highly Structured Viral RNA Molecules by Combinatorial Libraries of Hairpin Ribozymes

“…While rapid and simple, sequence scanning has the obvious disadvantage that it ignores the issue of target site accessibility, which can severely limit cleavage efficiency (39). Given funding and personnel sufficient for screening the biological activity of approximately 20 ribozymes, this method has been shown to work (40). Unfortunately, the expense of such a strategy makes this brute force approach impractical in most laboratory settings.…”

“…However, even these sites may become accessible due to RNA dynamics, alternative folding patterns, or repetitive folding of the RNA in vivo, as during ribosomal transit of a site within a mRNA molecule. Prediction of folded structures of candidate ribozymes is quite certainly useful in avoiding the use of some ribozymes that can misfold into inactive structures (40).…”

Efficient Transfer of Synthetic Ribozymes into Cells Using Hemagglutinating Virus of Japan (HVJ)-Cationic Liposomes

“…The half-life of a hammerhead ribozyme in serum is less than 0.1 min (33). However, a combination of modifications, including the introduction of several phosphorothioate linkages at the 5Ј-end of the ribozyme, can substantially increase ribozyme stability (34,35). We therefore used hammerhead ribozymes containing extensive phosphorothioate modifications at the 5Ј-and 3Ј-ends, which have been have been shown to confer higher stability in serum than the 5Ј-end modification alone, without reducing the catalytic efficiency of the ribozyme (36).…”

Myb targeted therapeutics for the treatment of human malignancies

“…Indeed, some groups have argued that inhibition is due to the presence of four contiguous guanosine residues in the oligonucleotide sequence employed and that such`G-quartets' allow for base stacking and tetraplex formation which non-speci®cally inhibits cell growth (Burgess et al, 1995;Castier et al, 1998). Regardless, the physiologic role of myb in regulating cell proliferation in this context seems certain (Brown et al, 1992;Kypreos et al, 1998;Simons et al, 1993Simons et al, , 1995 and myb targeted ribozymes have also resulted in sequence-speci®c inhibition of intimal cell proliferation (Jarvis et al, 1996). For these reasons, e cient targeting of c-myb in vascular tissue may yet prove to be a useful therapeutic maneuver.…”