# US 8,568,976 B2 · Proinflammatory nucleic acids

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- Number: US 8,568,976 B2
- Document ID: US8568976B2
- Status: Granted
- Kind: B2
- Application number: 13/323,461
- Filed: 2011-12-12
- Published: 2012-11-22
- Granted: 2013-10-29
- Earliest claimed: 2005-03-25
- Assignees: Isis Pharmaceuticals, Inc.
- Applicants: Joseph Senn
- Inventors: Joseph Senn
- Web page: https://patentgrep.com/patent/US8568976B2

## CPC

- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 2310/11: Antisense
- C12N 2310/315: Phosphorothioates
- C12N 2310/3341: 5-Methylcytosine
- C12N 2310/341: Gapmers, i.e. of the type ===---===
- C12N 2310/346: having a combination of backbone and sugar modifications
- C12N 2320/53: reducing unwanted side-effects
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides compositions and methods for identifying and utilizing proinflammatory nucleic acids. In particular, this invention relates to compounds, particularly oligonucleotides, which exert their effect through triggering receptors expressed by myeloid cells (TREMs), specifically TREM 2.

## Claims

### Claim 1

A method for identifying a proinflammatory oligonucleotide comprising:
contacting a cell with a test oligonucleotide wherein:
the cell functionally expresses a TREM-2 extracellular domain, an intracellular signaling domain, and a reporter construct comprising a cis-acting DNA consensus element sensitive to activation of the intracellular domain, and wherein the cis-acting DNA consensus element is functionally linked to a reporter gene coding sequence; and
wherein the test oligonucleotide does not comprise a CpG motif, and wherein the oligonucleotide does not specifically hybridizes to a nucleic acid encoding TREM-2;
assaying for activation of the reporter gene; and
identifying test oligonucleotide as being proinflammatory if the reporter gene is activated.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cell is a RAW 264.7 cell.

### Claim 3 (depends on 1)

The method of claim 1, wherein the intracellular signaling domain activates transcription factor nuclear factor of activated T cells (NFAT).

### Claim 4 (depends on 1)

The method of claim 1, wherein the intracellular signaling domain directly activates a transcription factor.

### Claim 5 (depends on 1)

The method of claim 1, wherein the intracellular signaling domain indirectly activates a transcription factor.

### Claim 6 (depends on 1)

The method of claim 1, wherein the cis-acting DNA consensus element is a transcription factor binding site.

### Claim 7 (depends on 6)

The method of claim 6, wherein the transcription factor binding site is an NFAT binding site.

### Claim 8 (depends on 1)

The method of claim 1, wherein the reporter gene is selected from the group consisting of beta-galactosidase, beta-lactamase, luciferase, and alkaline phosphatase.

### Claim 9 (depends on 1)

The method of claim 1, wherein the reporter gene is secreted alkaline phosphatase.

### Claim 10 (depends on 1)

The method of claim 1, wherein the intracellular signaling domain comprises a TREM-2 intracellular signaling domain.

### Claim 11 (depends on 1)

The method of claim 1, wherein the intracellular signaling domain comprises a CD3ζ cytoplasmic domain.

### Claim 12 (depends on 1)

The method of claim 1, wherein the test oligonucleotide consists of a single-stranded modified antisense oligonucleotide.

### Claim 13 (depends on 12)

The method of claim 12, wherein at least one internucleoside linkage of the single-stranded modified antisense oligonucleotide is a modified internucleoside linkage.

### Claim 14 (depends on 13)

The method of claim 13, wherein each internucleoside linkage of the single-stranded modified antisense oligonucleotide is a phosphorothioate internucleoside linkage.

### Claim 15 (depends on 12)

The method of claim 12, wherein the single-stranded modified antisense oligonucleotide comprises at least one modified sugar moiety.

### Claim 16 (depends on 15)

The method of claim 15, wherein at least one modified sugar moiety comprises a 2′-O-methoxyethyl or a 4′-(CH2)n—O-2′ bridge, wherein n is 1 or 2.

### Claim 17 (depends on 12)

The method of claim 12, wherein at least one nucleoside comprises a modified nucleobase.

### Claim 18 (depends on 17)

The method of claim 17, wherein the modified nucleobase is a 5-methylcytosine.

### Claim 19 (depends on 1)

The method of claim 1, wherein the test oligonucleotide comprises:
a gap segment consisting of linked deoxynucleosides;
a 5′ wing segment consisting of linked nucleosides; and
a 3′ wing segment consisting of linked nucleosides;
wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

### Claim 20 (depends on 19)

The method of claim 19, wherein the test oligonucleotide comprises:
a gap segment consisting of ten linked deoxynucleosides;
a 5′ wing segment consisting of five linked nucleosides; and
a 3′ wing segment consisting of five linked nucleosides;
wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; wherein each cytosine in the test oligonucleotide is a 5-methylcytosine, and wherein each internucleoside linkage of the test oligonucleotide is a phosphorothioate linkage.

### Claim 21 (depends on 20)

The method of claim 20, wherein the test oligonucleotide consists of 20 linked nucleosides.