Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1994-08-31
2002-04-30
Martinell, James (Department: 1633)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
Reexamination Certificate
active
06380169
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the selective cleavage of a target nucleic acid using a cleavage compound. Inhibition of messenger RNA translation can be achieved using an anti-sense oligonucleoside conjugated to a cleavage-enhancing metal complex, which hybridizes to the target nucleic acid to effect cleavage at a target site in the 5′-cap structure of the nucleic acid.
BACKGROUND OF THE INVENTION
The possibility of developing therapeutic agents which bind to critical regions of RNA, for example mRNA, and selectively inhibit the function, replication or survival of abnormal cells or foreign organisms is an exciting concept. See, e.g., Dervan,
Science
1988; 232:464-471. Various laboratories have pursued the design and development of molecules which interact with DNA in a sequence-specific manner. Such molecules have been proposed to have far-reaching implications for the diagnosis and treatment of diseases involving foreign genetic materials (such as viruses) or alterations to genomic DNA (such as cancer).
Anti-sense oligonucleotides are one type of sequence-specific molecule that has been demonstrated to be effective for inhibition of virus and human genes. In one application of this technology, anti-sense oligonucleotides are complementary to at least a portion of the messenger RNA (MRNA) transcribed from the target gene and can hybridize with the mRNA, thereby preventing ribosomal translation and subsequent protein synthesis. Anti-sense oligonucleotides have been shown to mediate inhibition of the Rous Sarcoma virus in tissue cultures (Zamecnik and Stephenson,
Proc. Natl. Sci. U.S.A.
1978; 75:280-284) as well as the HTLV-III (HIV-1) virus (Zamecnik, et al.,
Proc. Natl. Acad. Sci. U.S.A.
1986; 83:4145-4146). Anti-sense oligonucleotides also have been shown to suppress the expression of selected non-viral genes in vitro, such as rabbit-globin (Goodchild, et al.,
Arch. Biochem. Biophys.
1988; 264:401-409) and human c-myb (Anfossi, et al.,
Proc. Natl. Acad. Sci. U.S.A.
1989; 86:3379).
Naturally-occurring oligonucleotides are subject to degradation or inactivation by cellular endogenous nucleases. Since anti-sense oligonucleotides must remain intact to be effective, some researchers have modified oligonucleotides to make them resistant to degradation or inactivation by nucleases. These modified oligonucleotides typically contain altered internucleoside linkages in which one or more of the naturally occurring phosphodiester linkages has been replaced. Oligonucleosides having phosphoroamidate or phosphorothioate linkages have been shown to increase the inhibition of HIV-1 in tissue cultures. See, e.g. Agarwal, et al.,
Proc. Natl. Acad. Sci. U.S.A.
1988; 85:7079-7083.
Nuclease-resistant nonionic oligonucleosides having methylphosphonate linkages have also been studied in vitro and in vivo as potential anticancer, antiviral and antibacterial agents. Miller, et al., Anti-Cancer Drug Design, 2:117-128 (1987). For example, anti-sense oligonucleosides containing methylphosphonate linkages have been demonstrated to inhibit HIV-induced syncytium formation. Sarin, et al.,
Proc. Natl. Acad. Sci. U.S.A.
1988; 85:7448-7451. The internucleoside bonds of these analogs are said to approximate the conformation of phosphodiester bonds in nucleic acids. It has been noted that the nucleic acid phosphate backbone in a methylphosphonate linkage is rendered neutral by methyl substitution of one anionic phosphoryl oxygen. This substitution is thought to decrease inter- and intra-strand repulsion attributable to charged phosphate groups. Miller, et al., Anti-Cancer Drug Design 2:117-128 (1987).
Oligonucleotide analogs with a methylphosphonate backbone are believed to be capable of penetrating living cells and have been reported to inhibit mRNA translation in globin synthesis and vesicular stomatitis viral protein synthesis and to inhibit herpes simplex virus replication by preventing splicing of pre-mRNA. Blake, et al.,
Biochemistry
1985; 24:6132-6138; Blake, et al.,
Biochemistry
1985; 24:6139-6145; Murakami, et al.,
Biochemistry
1985; 24:40414046; Miller, et al.,
Biochimie
1985; 67:769-776; Agris, et al.,
Biochemistry
1986; 23:6268-6275. Mechanisms of action for inhibition by the methylphosphonate analogs include formation of stable complexes with RNA and/or DNA having a substantially complementary nucleic acid sequence.
Nonionic oligonucleotide alkyl- and aryl-phosphonate analogs complementary to a selected single stranded foreign nucleic acid sequence are reported to be able to selectively inhibit the function or expression of that particular nucleic acid by binding to or interfering with that nucleic acid, without disturbing the function or expression of other nucleic acid present in the cell. See, e.g., U.S. Pat. Nos. 4,469,863 and 4,511,713. The use of complementary nuclease-resistant nonionic oligonucleoside methylphosphonates which are taken up by mammalian cells to inhibit viral protein synthesis in certain contexts, including herpes simplex virus-1, is described in U.S. Pat. No. 4,757,055.
The inhibition of infection of cells by HTLV-III by administration of oligonucleotides complementary to highly conserved regions of the HTLV-III genome necessary for HTLV-III replication and/or expression is reported in U.S. Pat. No. 4,806,463. The oligonucleotides were said to affect viral replication and/or gene expression as assayed by reverse transcriptase activity (replication) and production of viral proteins p15 and p24 (gene expression).
Anti-sense oligonucleotides or phosphorothioate or other analogs complementary to a sequence of viral RNA theoretically may be employed to interrupt the transcription and translation of viral mRNA into protein. The anti-sense constructs can bind to viral mRNA and obstruct ribosomes from moving along the mRNA, thereby halting the translation of mRNA into protein. This process is called “translation arrest” or “ribosomal-hybridization arrest.” Yarochan, et al., “AIDS Therapies”, Scientific American, pages 110-119 (October, 1988).
However, in practice, the use of an anti-sense hybridizing sequence to obstruct the ribosome from reading along the mRNA is not generally useful in the coding portion of the target mRNA, since an anti-sense sequence targeted to the coding portion is often removed from hybridization with the target sequence during the course of translation, even where the binding constant is high. In contrast, an anti-sense sequence targeted to the 5′-untranslated position of a mRNA molecule may achieve translation arrest through a blocking type mechanism.
One approach to selective targeting of coding sequences is to rely on the ability of RNaseH to cleave duplexed RNA strands. In theory, by utilizing an anti-sense sequence which hybridizes to a target coding sequence on RNA, RNaseH cleavage of the target RNA could be achieved in this manner. However, in practice, cleavage by RNaseH requires that the strands in the target duplex sequence have 2′-deoxy sugar portions as well as charged (e.g., phosphodiester) backbone linkages. This means that uncharged-backbone anti-sense sequences such as methylphosphonate oligonucleosides (which are particularly useful because they are less subject to in vivo degradation) would not be expected to activate RNaseH activity against target RNA sequences. As a result, the increases in potency which can be achieved using modified oligonucleosides such as the methylphosphonates may not be realized (especially with respect to coding sequence targets) if RNaseH or translation arrest is relied upon to inhibit expression.
In an effort to increase the interference with protein synthesis of target genes and thereby increase potency, various agents can be bound to the anti-sense oligonucleotides which enhance the inactivation of the target nucleic acid. Such inactivating agents include alkylating agents, crosslinking agents and cleaving agents. These agents typically are capable of chemically modifying nucleic acid nonspecifically. By linking these agents to an anti-sense oligonucle
Adams Thomas H.
Reynolds Mark A.
ISIS Pharmaceuticals Inc.
Martinell James
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