Occupancy OnlyMediated Mechanisms

Classic competitive antagonists are thought to alter biological activities because they bind to receptors, thereby preventing agonists from binding the inducing normal biological processes. Binding of oligonucleotides to specific sequences may inhibit the interaction of the RNA with proteins, other nucleic acids, or other factors required for essential steps in the intermediary metabolism of the RNA or its use by the cell.

To create antisense inhibitors that clearly wik through non-RNase H mechanisms, the antisense agents must be modified sufficiently rot to support RNase H cleavage. Fortunately, numerous analogs have been identified that do not support RNase H cleavage. These can be classified into modifications of sugar moiety, or the phosphate, or replacement of the sugar-phosphate backbone (98). Unfortunately, in a number of earlier publications, conclusions about mechanisms of action were drawn without using appropriately modified oligonucleotides.

4.1.1 lnhibition of 5'-Capping. Conceptually, inhibition of 5'-capping of mRNA could be an effective antisense mechanism. 5'-Cap-ping is critical in stabilizing mRNA and in enabling the translation of mRNA (99). To date, however, no reports of antisense inhibitors of capping have appeared, which may be attributable to the inaccessibility of the 5'-end of mRNA before capping.

4.1.2 lnhibition of Splicing. A key step in the intermediary metabolism of most mRNA molecules is the excision of introns. These "splicing" reactions are sequence specific and require the concerted action of spliceosomes. Consequently, oligonucleotides that bind to sequences required for splicing may prevent binding of necessary factors or physically prevent the required cleavage reactions. This would then result in inhibition of the production of the mature mRNA. Activities have been reported for anti-c-rnyc and antiviral oli-gonucleotides with phosphodiester, meth-ylphosphonate, and phosphorothioate backbones (31, 99-101). Kole and colleagues (102-104)were the first to use modified oligo-nucleotides to inhibit splicing. They showed that 2'-MOE phosphorothioate oligonucleo-tides could correct aberrant beta-globin splicing in a cell-free system. Similar observations were recorded in a cellular system (104).

In our laboratory, we have attempted to characterize the factors that determine whether splicing inhibition is effected by an antisense drug (105). To this end, a number cf luciferase-reporter plasmids containing various introns were constructed and transfected into HeLa cells. Then the effects of antisense drugs designed to bind to various sites were characterized. The effects of RNase H-compe-tent oligonucleotides were compared to those of oligonucleotides that do not serve as RNase H substrates. The major conclusions from this study were, first, that most of the earlier stud ies in which splicing inhibition was reported were probably the result of nonspecific effects. Second, less effectively spliced introns are better targets than those with strong consensus splicing signals. Third, the 3'-splice site and branchpoint are usually the best sites to which to target the oligonucleotide to inhibit splicing.

Several studies have now demonstrated antisense-mediated redirection of slicing of an endogenous cellular mRNA. In one study, 2'-

oligonucleotides redirected splicing of IL-5 receptor pre-mRNA (106). Similar results were reported for antisense agents designed to bind to Bclx pre-mRNA (107, 108). Thus, antisense-mediated alternative splicing is a potentially powerful tool with which to investigate this important source of biological diversity, and to create focused therapies for diseases caused by disorders is splicing.

4.1.3 Translational Arrest. A mechanism for which many oligonucleotides have been designed is translational arrest by binding to the translation initiation codon. The positioning of the initiation codon within the area of complementarity of the oligonucleotide and the length of oligonucleotide used have varied considerably. Again, unfortunately, in only a relatively few studies have the oligonucleo-tides been shown to bind to the sites for which they were designed, and other data that support translation arrest as the mechanism have not been reported.

Target RNA species that have been reported to be inhibited include HIV (28), vesicular stomatitis virus (VSV) (76), n-myc(109), and a number of normal cellular genes (110113). However, to demonstrate that RNase H is not involved in effects observed, it is necessary to use antisense drugs that do not form duplexes that are RNase H substrates (e.g., fully modified 2'-oligonucleotides).

Studies with PNA analogs have shown that these analogs can inhibit translation in cellfree systems, but to date no data have been reported from cellular studies (114, 115). For morpholino oligomers, antisense activity has been reported in both cell-free and cellular assays (116, 117). Numerous oligonucleotides with 2'-modifications have also been studied and have been shown to inhibit translation when targeted to 5'-UTR or the translation initiation codon (118). However,optimal inhibition is effected by binding at the 5'-cap in RNAs that have significant 5'-untranslated regions (119). In conclusion, translation arrest represents an important mechanism of action for antisense drugs. A number of examples purporting to employ this mechanism have been reported, and recent studies on several compounds have provided data that unambiguously demonstrate that this mechanism can result in potent antisense drugs.

4.1.4 Disruption of Necessary RNA Structure. RNA adopts a variety of three-dimensional structures induced by intramolecular hybridization, the most common of which is the stem loop. These structures play crucial roles in a variety of functions. They are used to provide additional stability for RNA and as recognition motifs for a number of proteins, nucleic acids, and ribonucleoproteins that participate in the intermediary metabolism and activities of RNA species. Thus, given the potential general activity of the mechanism, it is surprising that occupancy-based disruption RNA has not been more extensively exploited.

As an example, we designed a series of oligonucleotides that bind to the important stem loop present in all RNA species in HIV, the TAR element. We synthesized a number of oligonucleotides designed to disrupt TAR, showed that several indeed did bind to TAR, disrupt the structure, and inhibit TAR-mediated production of a reporter gene (66). Furthermore, general rules useful in disrupting stem-loop structures were developed as well (84).

Although designed to induce relatively nonspecific cytotoxic effects, two other examples are noteworthy. Oligonucleotides designed to bind to a 17-nucleotide loop in Xeno-pus 28 S RNA required for ribosome stability and protein synthesis inhibited protein synthesis when injected into Xenopus oocytes (120). Similarly, oligonucleotides designed to bind to highly conserved sequences in 5.8 S RNA inhibited protein synthesis in rabbit re-ticulocyte and wheat germ systems (121).

Transcription

Capping/ CAP

polyadenyMon

Splicing

Transport \

Degradation

Translation

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