Antisense oligonucleotide inhibition of the ras gene

• US 5,582,986 A
• Filed: 08/19/1994
• Issued: 12/10/1996
• Est. Priority Date: 06/14/1991
• Status: Expired due to Term

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Monia, Brett P., Freier, Susan M., Ecker, David J.
• Primary Examiner(s): Elliott, George C.
• Application Number:
  US08/293,086
• Time in Patent Office: 844 Days
• Field of Search: 435/6, 536/23.1, 536/24.1, 536/24.3, 536/24.5, 514/44
• US Class Current: 435/6.12
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/333 Modified A
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/3527 Other alkyl chain
• CPC Class Codes: C12N 2310/3533 Halogen
• CPC Class Codes: C12N 2310/3535 Nitrogen
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational 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 which are specifically hybridizable with RNA or DNA deriving from the human ras gene, having nucleotide units sufficient in identity and number to effect such specific hybridization. Oligonucleotides or oligonucleotide analogs specifically hybridizable with a translation initiation site or with the codon-12 mutation of activated ras are provided. Such oligonucleotides and oligonucleotide analogs can be used for diagnostics as well as for research purposes. Methods are also disclosed for modulating ras gene expression in cells and tissues using the oligonucleotides or oligonucleotide analogs provided, and for specific modulation of expression of the activated ras gene. Methods for diagnosis, detection and treatment of conditions arising from the activation of the H-ras gene are also disclosed.

Claims

1. An antisense oligonucleotide comprising from 15 to 30 nucleotide units with a phosphodiester or phosphorothioate internucleoside linkage targeted to codon 12 of the human H-ras gene wherein said oligonucleotide is complementary and hybridizes to messenger RNA derived from the human H-ras gene so that the normal role of the messenger RNA is interfered with causing a loss of its function within a cell.

2. A method of modulating the expression of the human H-ras gene comprising contacting tissues or cells containing the gene with an antisense oligonucleotide comprising from 15 to 30 nucleotide units with a phosphodiester or phosphorothioate internucleoside linkage targeted to codon 12 of the human H-ras gene wherein said oligonucleotide is complementary and hybridizes to messenger RNA derived from the human H-ras gene so that the normal role of the messenger RNA is interfered with causing a loss of its function within a cell.

5. The method of claim 2 wherein said oligonucleotide has SEQ ID NO:

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

15 with a phosphodiester or phosphorothioate internucleoside linkage.

3. A method of detecting mutant H-ras based on the differential affinity of particular oligonucleotides for mutant vs. wild-type H-ras comprising contacting cells or tissues suspected of containing it with the oligonucleotide:

5'"'"' CCA-CAC-CGA-CGG-CGC-CC 3'"'"', SEQ ID NO;

3 and contacting an identical sample of cells or tissues with one of the oligonucleotides;

##STR1##

4. An oligonucleotide having SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

15 with a phosphodiester or phosphorothioate internucleoside linkage.

6. A method of modulating the expression of the human H-ras gene comprising contacting tissues or cells containing the gene with an antisense oligonucleotide comprising SEQ ID NO:

1 or SEQ ID NO;

2 having a phosphodiester or phosphorothioate internucleoside linkage.