Compounds and methods for modulating interaction between proteins and target nucleic acids

• US 9,518,259 B2
• Filed: 06/15/2011
• Issued: 12/13/2016
• Est. Priority Date: 06/15/2010
• Status: Active Grant

• Current Assignee: Cold Spring Harbor Laboratory, Ionis Pharmaceuticals, Inc.
• Original Assignee: Ionis Pharmaceuticals, Inc.
• Inventors: Rigo, Frank, Bennett, C. Frank, Krainer, Adrian R., Hua, Yimin
• Primary Examiner(s): SHIN, DANA H
• Application Number:
  US13/703,322
• Publication Number:
  US 20130289092A1
• Time in Patent Office: 2,008 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/341 Gapmers, i.e. of the type ===---===
• CPC Class Codes: C12N 2310/3525 MOE, methoxyethoxy
• CPC Class Codes: C12N 2310/3533 Halogen
• CPC Class Codes: C12N 2320/33 Alteration of splicing

Abstract

Provided herein are antisense compounds and methods for recruiting one or more non-cleaving protein to a target nucleic acid in a cell. In certain instances such recruitment of a non-cleaving protein alters the function or activity of the target nucleic acid. In certain such instances, the target nucleic acid a pre-mRNA and the recruitment of the non-cleaving protein results in a change in splicing of the pre-mRNA.

Claims

1. A method of inducing exon skipping in a target pre-mRNA in a cell comprising contacting the cell with an antisense compound comprising a chemically modified oligonucleotide consisting of 18 linked nucleosides;

and thereby inducing exon skipping in the pre-mRNA in the cell;

wherein the antisense compound comprises;

a 5′

-wing region consisting of 2 linked duplex stabilizing nucleosides;

a 3′

-wing region consisting of 2 linked duplex stabilizing nucleosides; and

a central gap region located between the 5′

-wing region and the 3′

-wing region

and consisting of 14 contiguous nucleosides comprising 2′

-F modifications;

wherein each duplex stabilizing nucleoside is a 2′

-methoxyethyl (2′

-MOE) nucleoside, and

wherein the antisense compound is complementary to an intron of the target pre-mRNA.

2. The method of claim 1, wherein the antisense compound binds to the target pre-mRNA and recruits at least one non-cleaving nucleic acid binding protein to the antisense compound/target pre-mRNA duplex.

3. The method of claim 2, wherein at least one of the at least one non-cleaving nucleic acid binding proteins recruited to the compound/target pre-mRNA duplex is Interleukin Enhancer Binding Factor 2 or Interleukin Enhancer Binding Factor 3.

4. The method of claim 1, wherein the target pre-mRNA is associated with a disease or disorder.

5. The method of claim 4, wherein the target pre-mRNA is Bcl-x.

6. The method of claim 4, wherein the disease or disorder is cancer.

7. The method of claim 4, wherein the disease or disorder is Duchenne muscular dystrophy.

8. The method of claim 1, wherein the cell is in vitro.

9. The method of claim 1, wherein the cell is in an animal.

10. The method of claim 1 comprising performing an assay to determine whether the exon has been skipped.

11. The method of claim 1 comprising performing an assay to determine whether a protein has been recruited to the antisense compound/target pre-mRNA duplex.

12. The method of claim 11 comprising performing an assay to determine the identity of one or more protein recruited to the antisense compound/target pre-mRNA duplex.

13. The method of claim 6, wherein the cell is in an animal.

14. The method of claim 7, wherein the cell is in an animal.