Antisense inhibition of ras gene with chimeric and alternating oligonucleotides

• US 5,965,722 A
• Filed: 04/30/1997
• Issued: 10/12/1999
• Est. Priority Date: 05/21/1991
• Status: Expired due to Term

• Current Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Monia, Brett P., Freier, Susan M., Cook, Phillip Dan, Ecker, David J., Sanghvi, Yogesh S.
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/848,840
• Time in Patent Office: 895 Days
• Field of Search: 435/6, 435/91.1, 536/27.21, 536/27.6, 536/27.8, 536/27.81, 536/28.4, 536/28.5, 536/28.53, 536/28.54, 536/28, 536/29.5, 536/23.1, 514/44
• US Class Current: 536/23.1
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/318 where the PO2 is completely replaced, e.g. MMI or formacetal
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/341 Gapmers, i.e. of the type ===---===
• CPC Class Codes: C12N 2310/345 having at least two different backbone modifications
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3525 MOE, methoxyethoxy
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

Compositions and methods are provided for the modulation of expression of the human ras gene in both the normal and activated forms. Oligonucleotides are provided that have methylene(methylimino) linkages alternating with phosphorothioate or phosphodiester linkages. Further oligonucleotides are provide that have a first region having a methylene(methylimino) linkage alternating with a phosphorothioate or phosphodiester linkage and a second region having phosphorothioate linkages. Such oligonucleotides can be used for diagnostics as well as for research purposes including methods for diagnosis, detection and treatment of conditions arising from the activation of the H-ras gene.

Claims

1. An oligonucleotide having at least one portion comprising at least one CH₂ --NH--O--CH₂, CH₂ --N(CH₃)--O--CH₂, CH₂ --O--N(CH₃)--CH₂, CH₂ --N(CH), --N(CH)₃ --CH₂ or O--N(CH)₂ --CH₂ CH₂ linkage alternating with a phosphorothioate or phosphodiester linkage.

2. The oligonucleotide of claim 1 wherein said at least one CH₂ --NH--O--CH₂, CH₂ --N(CH₃)--O--CH₂, CH₂ --O--N(CH₂, CH₂ --N (CH₃)--N(CH₃)--CH₂ or O--N(CH)--CH--₃ CH linkage, is a methylene (methylimino) linkage.

3. The oligonucleotide of claim 1 comprising 8 to 30 nucleotide units hybridizable with selected DNA or mRNA deriving from the human H-ras gene.

4. The oligonucleotide of claim 3 hybridizable with a translation initiation site or codon 12 of the human H-ras gene.

5. The oligonucleotide of claim 3 comprising SEQ ID No:

1, SEQ ID No;

2 or SEQ ID No;

3.

6. The oligonucleotide of claim 1 wherein at least one of the nucleosidic units of said oligonucleotide is modified at the 2'"'"'position.

7. The oligonucleotide of claim 6 wherein said 2'"'"'modification is 2'"'"'-fluoro, 2'"'"'-O-alkyl or 2'"'"'-O-substituted alkyl.

8. The oligonucleotide of claim 7 wherein said 2'"'"'modification is 2'"'"'-substituted alkyl.

9. The oligonucleotide of claim 8 wherein said 2'"'"'substituted alkyl is 2'"'"'-O-methoxyethyl.

10. The oligonucleotide of claim 2 wherein said methylene(methylimino) linkage is alternating with a phosphorothioate linkage.

11. The oligonucleotide of claim 2 wherein said methylene(methylimino) linkage is alternating with a phosphodiester linkage.

12. The oligonucleotide of claim 2 comprising one, two or three methylene(methylimino) linkages alternating with phosphorothioate or phosphodiester linkages.

13. The oligonucleotide of claim 12 comprising one or two methylene(methylimino) linkages alternating with phosphorothioate linkages.

14. An oligonucleotide comprising from 8 to 30 nucleotide units hybridizable with selected DNA or mRNA and being a substrate for RNAse H and having at least one methylene(methylimino) linkage.

15. The oligonucleotide of claim 14 hybridizable with DNA or mRNA deriving from the human H-ras gene.

16. The oliqonucleotide of claim 15 comprising SEQ ID No:

1, SEQ ID No;

2 and SEQ ID No;

3.

21. The chimeric oligonucleotide of claim 15 wherein said second region comprises 2'"'"'-deoxynucleotides.

22. The chimeric oligonucleotide of claim 21 wherein said second region is at least four nucleotides long.

23. The chimeric oligonucleotide of claim 21 wherein said second region is five to nine nucleotides long.

17. A chimeric oligonucleotide comprising from 8 to 30 nucleotide units hybridizable with selected DNA or mRNA and containing a first region having at least one methylene(methylimino) linkage and a second region having at least one phosphorothioate linkage.

18. The chimeric oligonucleotide of claim 17 wherein said second region is flanked by two of said first regions each of which includes at least one methylene(methylimino) linkage.

19. The chimeric oligonucleotide of claim 17 wherein said first region includes at least one nucleosidic unit modified at the 2'"'"' position.

20. The chimeric oligonucleotide of claim 19 wherein the modification at the 2'"'"'position is a 2'"'"'-O-alkyl, 2'"'"'-O-substituted alkyl or a 2'"'"'-fluoro modification.

24. The chimeric oligonucleotide of claim 17 wherein said first region includes at least one methylene(methylimino) linkage alternating with a phosphorothioate or phosphodiester linkage.

25. The chimeric oligonucleotide of claim 24 wherein said first region includes one, two or three methylene(methylimino) linkages alternating with phosphorothioate or phosphodiester linkages.

26. The chimeric oligonucleotide of claim 25 wherein said first region includes one, two or three methylene(methylimino) linkages alternating with phosphorothioate linkages.

27. The chimeric oligonucleotide of claim 25 wherein said first region includes one, two or three methylene(methylimino) linkages alternating with phosphodiester linkages.

28. An oligonucleotide comprising from 8 to 30 nucleotide units hybridizable with selected DNA or mRNA deriving from the human H-ras gene and having at least one methylene(methylimino) internucleoside linkage.

29. The oligonucleotide of claim 28 wherein said oligonucleotide is hybridizable with a translation initiation site or codon 12 of the human H-ras gene.