# US 10,370,659 B2 · Compounds and methods for increasing antisense activity

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- Number: US 10,370,659 B2
- Document ID: US10370659B2
- Status: Granted
- Kind: B2
- Application number: 15/552,725
- Filed: 2016-02-23
- Published: 2018-05-31
- Granted: 2019-08-06
- Earliest claimed: 2015-02-23
- Assignees: Ionis Pharmaceuticals, Inc.
- Applicants: Ionis Pharmaceuticals, Inc.
- Inventors: Xue-hai Liang; Wen Shen; Stanley T. Crooke
- Web page: https://patentgrep.com/patent/US10370659B2

## CPC

- 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)}
- A61K 31/7125: Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
- C07K 14/47: from mammals
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites}
- C12N 2310/11: Antisense
- C12N 2310/113: targeting other non-coding nucleic acids, e.g. antagomirs
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/315: Phosphorothioates
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/3341: 5-Methylcytosine
- C12N 2310/335: Modified T or U
- C12N 2310/341: Gapmers, i.e. of the type ===---===
- C12N 2310/351: Conjugate
- C12N 2310/3517: Marker; Tag
- C12N 2320/50: Methods for regulating/modulating their activity
- C12Q 2600/158: Expression markers
- G01N 2500/04: Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

In certain embodiments, the present disclosure provides compounds and methods for increasing the antisense activity of an antisense compound in a cell. In certain embodiments, the present disclosure provides methods for identifying antisense compounds having high activity. In certain embodiments, the present disclosure provides methods for identifying antisense compounds that bind to enhancer or repressor proteins.

## Claims

### Claim 1

A method for increasing the antisense activity of an antisense compound in a cell, comprising:
contacting the cell with an inhibitor of the amount or activity of a repressor protein that binds antisense compounds comprising a plurality of phosphorothioate internucleoside linkages in the cell;
and subsequently contacting the cell with the antisense compound, wherein the target nucleic acid of the antisense compound does not code for the repressor protein, and wherein the antisense compound comprises a modified oligonucleotide that comprises a plurality of phosphorothioate internucleoside linkages.

### Claim 2 (depends on 1)

The method of claim 1, wherein the inhibitor comprises a second antisense compound targeting a nucleic acid that codes for the repressor protein.

### Claim 3 (depends on 1)

The method of claim 1, wherein the repressor protein is selected from Ku70, Ku80, hnRNPK, P54nrb, PSF, and PSPC1.

### Claim 4 (depends on 1)

The method of claim 1, wherein the repressor protein is selected from Ku70 and Ku80.

### Claim 5 (depends on 1)

The method of claim 1, wherein the repressor protein is selected from hnRNPK, P54nrb, PSF, and PSPC1.

### Claim 6 (depends on 1)

The method of claim 1, wherein all of the internucleoside linkages of the antisense compound are phosphorothioate internucleoside linkages.

### Claim 7 (depends on 1)

The method of claim 1, wherein the antisense compound comprises a gapmer.

### Claim 8 (depends on 1)

The method of claim 1, wherein the antisense activity is reduction of the amount of a target nucleic acid of the antisense compound.

### Claim 9 (depends on 8)

The method of claim 8, wherein the reduction of the amount of a target nucleic acid of the antisense compound is increased relative to the reduction that occurs when a cell is contacted with the antisense compound in the absence of the inhibitor of the amount or activity of a repressor protein in the cell.

### Claim 10 (depends on 1)

The method of claim 1, wherein the antisense compound comprises at least one conjugate.

### Claim 11 (depends on 1)

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

### Claim 12 (depends on 11)

The method of claim 11, wherein the animal is a human.