Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-05-27
2003-11-11
LeGuyader, John L. (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S091100, C435S325000, C435S366000, C435S375000, C536S023100, C536S024310, C536S024330, C536S024500
Reexamination Certificate
active
06645943
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the control of gene expression. More particularly, this invention relates to the use of synthetic, modified oligonucleotides to down-regulate the expression of a gene in an animal.
The potential for the development of an antisense oligonucleotide therapeutic approach was first suggested in three articles published in 1977 and 1978. Paterson et al. (
Proc. Natl. Acad. Sci
. (
USA
) (1977) 74:4370-4374) discloses that cell-free translation of mRNA can be inhibited by the binding of an oligonucleotide complementary to the mRNA. Zamecnik et al. (
Proc. Natl. Acad. Sci
. (
USA
) (1978) 75:280-284 and 285-288) discloses that a 13mer synthetic oligonucleotide that is complementary to a part of the Rous sarcoma virus (RSV) genome inhibits RSV replication in infected chicken fibroblasts and inhibits RSV-mediated transformation of primary chick fibroblasts into malignant sarcoma cells.
These early indications that synthetic oligonucleotides can be used to inhibit virus propagation and neoplasia have been followed by the use of synthetic oligonucleotides to inhibit a wide variety of viruses, such as HIV (see, e.g., U.S. Pat. No. 4,806,463); influenza (see, e.g., Leiter et al. (1990) (
Proc. Natl. Acad. Sci
. (
USA
) 87:3430-3434); vesicular stomatitis virus (see, e.g., Agris et al. (1986)
Biochem
. 25:6268-6275); herpes simplex (see, e.g., Gao et al. (1990).
Antimicrob. Agents Chem
. 34:808-812); SV40 (see, e.g., Birg et al. (1990) (
Nucleic Acids Res
. 18:2901-2908); and human papilloma virus (see, e.g., Storey et al. (1991) (
Nucleic Acids Res
. 19:4109-4114). The use of synthetic oligonucleotides and their analogs as antiviral agents has recently been extensively reviewed by Agrawal (
Trends in Biotech
. (1992) 10:152-158).
In addition, synthetic oligonucleotides have been used to inhibit a variety of non-viral pathogens, as well as to selectively inhibit the expression of certain cellular genes. Thus, the utility of synthetic oligonucleotides as agents to inhibit virus propagation, propagation of non-viral, pathogens and selective expression of cellular genes has been well established.
Improved oligonucleotides have more recently been developed that have greater efficacy in inhibiting such viruses, pathogens and selective gene expression. Some of these oligonucleotides having modifications in their internucleotide linkages have been shown to be more effective than their unmodified counterparts. For example, Agrawal et al. (
Proc. Natl. Acad. Sci
. (
USA
) (1988) 85:7079-7083) teaches that oligonucleotide phosphorothioates and certain oligonucleotide phosphoramidates are more effective at inhibiting HIV-1 than conventional phosphodiester-linked oligodeoxynucleotides. Agrawal et al. (
Proc. Natl. Acad. Sci
. (
USA
) (1989) 86:7790-7794) discloses the advantage of oligonucleotide phosphorothioates in inhibiting HIV-1 in early and chronically infected cells.
In addition, chimeric oligonucleotides having more than one type of internucleotide linkage within the oligonucleotide have been developed. Pederson et al. (U.S. Pat. Nos. 5,149,797 and 5,220,007 discloses chimeric oligonucleotides having an oligonucleotide phosphodiester or oligonucleotide phosphorothioate core sequence flanked by nucleotide methylphosphonates or phosphoramidates. Furdon et al. (
Nucleic Acids Res
. (1989) 17:9193-9204) discloses chimeric oligonucleotides having regions of oligonucleotide phosphodiesters in addition to either oligonucleotide phosphorothioate or methylphosphonate regions. Quartin et al. (
Nucleic Acids Res
. (1989) 17:7523-7562) discloses chimeric oligonucleotides having regions of oligonucleotide phosphodiesters and oligonucleotide methylphosphonates. Inoue et al. (
FEBS Lett
. (1987) 215:237-250) discloses chimeric oligonucleotides having regions of deoxyribonucleotides and 2′-O-methyl-ribonucleotides.
Many of these modified oligonucleotides have contributed to improving the potential efficacy of the antisense oligonucleotide therapeutic approach. However, certain deficiencies remain in the known oligonucleotides, and these deficiencies can limit the effectiveness of such oligonucleotides as therapeutic agents. For example, Wickstrom (
J. Biochem. Biophys. Meth
. (1986) 13:97-102) teaches that oligonucleotide phosphodiesters are susceptible to nuclease-mediated degradation, thereby limiting their bioavailability in vivo. Agrawal et al. (
Proc. Natl. Acad. Sci
. (
USA
) (1990) 87:1401-1405) teaches that oligonucleotide phosphoramidates or methylphosphonates when hybridized to RNA do not activate RNase H, the activation of which can be important to the function of antisense oligonucleotides. Thus, a need for methods of controlling gene expression exists which uses oligonucleotides with improved therapeutic characteristics.
Several reports have been published on the development of phosphorothioate-linked oligonucleotides as potential anti-AIDS therapeutic agents. Although extensive studies on chemical and molecular mechanisms of oligonucleotides have demonstrated the potential value of this novel therapeutic strategy, little is known about the pharmacokinetics and metabolism of these compounds in vivo.
Recently, several preliminary studies on this topic have been published. Agrawal et al. (
Proc. Natl. Acad. Sci
. (
USA
) (1991) 88:7595-7599) describes the intravenous and intraperitoneal administration to mice of a 20mer phosphorothioate linked-oligonucleotide. In this study, approximately 30% of the administered dose was excreted in the urine over the first 24 hours with accumulation preferentially in the liver and kidney. Plasma half-lives ranged from about 1 hour t
½&agr;
,) and 40 hours (t
½&bgr;
), respectively. Similar results have been reported in subsequent studies (Iversen (1991)
Anti
-
Cancer Drug Design
6:531-538; Iversen (1994)
Antisense Res. Devel
. 4:43-52; and Sands (1994)
Mol. Pharm
. 45:932-943). However, stability problems may exist when oligonucleotides are administered intravenously and intraperitoneally.
Thus, there remains a need to develop more effective therapeutic methods of down-regulating the expression of genes which can be easily manipulated to fit the animal and condition to be treated, and the gene to be targeted. Preferably, these methods should be simple, painless, and precise in effecting the target gene.
SUMMARY OF THE INVENTION
The present invention provides a method of down-regulating the expression of a gene in an animal which involves the administration of an oligonucleotide complementary to the gene via an oral route, thereby bypassing the complications which may be experienced during intravenous and other modes of in vivo administration.
It has been discovered that hybrid oligonucleotides with other than phosphodiester bonds and having at least one 2′-substituted ribonucleotide and chimeric oligonucleotides with at least two different types of internucleotide linkages are relatively stable in vivo following oral administration to an animal, and that these molecules are successfully absorbed from the gastrointestinal tract and distributed to various body tissues. This discovery has been exploited to develop the present invention, which is a method of down-regulating the expression of a gene in an animal.
This method is also a means of examining the function of various genes in an animal, including those essential to animal development. Presently, gene function can only be examined by the arduous task of making a “knock out” animal such as a mouse. This task is difficult, time-consuming and cannot be accomplished for genes essential to animal development since the “knock out” would produce a lethal phenotype. The present invention overcomes the shortcomings of this model.
In the method of the invention, a pharmaceutical formulation containing an oligonucleotide complementary to the targeted gene is orally administered in a pharmaceutically acceptable carrier to the animal harboring the gene. The oligonucleotide inhibits the expression of the gene, thereby down-regulating
Agrawal Sudhir
Diasio Robert B.
Zhang Ruiwen
Hale and Dorr LLP
Hybridon, Inc.
LeGuyader John L.
Schmidt M
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