# US 2019/0169608 A1 · Modified rnai polynucleotides and uses thereof

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- Number: US 2019/0169608 A1
- Document ID: US20190169608A1
- Status: Published application
- Kind: A1
- Application number: 16/153,424
- Filed: 2018-10-05
- Published: 2019-06-06
- Earliest claimed: 2008-02-11
- Assignees: Phio Pharmaceuticals Corp.
- Applicants: Phio Pharmaceuticals Corp.
- Inventors: Pamela A. Pavco; Joanne Kamens; Tod M. Woolf; William Salomon; Anastasia Khvorova
- Web page: https://patentgrep.com/patent/US20190169608A1

## CPC

- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 17/06: Antipsoriatics
- A61P 27/02: Ophthalmic agents
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/12: Antivirals
- A61P 31/14: for RNA viruses
- A61P 31/18: for HIV
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- 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)}
- C12N 15/1136: {against growth factors, growth regulators, cytokines, lymphokines or hormones}
- C12N 15/1137: {against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138)}
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/315: Phosphorothioates
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/322: 2'-R Modification
- C12N 2310/341: Gapmers, i.e. of the type ===---===
- C12N 2320/51: modulating the chemical stability, e.g. nuclease-resistance
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The invention related to improved RNAi constructs and uses thereof. The construct has a double stranded region of 19-49 nucleotides, preferably 25, 26, or 27 nucleotides, and preferable blunt-ended. The construct has selective minimal modifications to confer an optimal balance of biological activity, toxicity, stability, and target gene specificity. For example, the sense strand may be modified (e.g., one or both ends of the sense strand it/are modified by four 2′—O-mehtyl groups), such that the construct is not cleaved by Dicer or other RNAse III, and the entire length of the antisense strand is loaded into RISC. In addition, the antisense strand may also be modified by 2′—O-methyl group at the 2nd 5′-end nucleotide to greatly reduce off-target silencing. The constructs of the invention largely avoids the interferon response and sequence-independent apoptosis in mammalian cells, exhibits better serum stability, and enhance target specificity.

## Claims

### Claim at position 1

1-50. (canceled)

### Claim 51

A method for inhibiting the expression of a target gene in a mammalian cell, comprising contacting the mammalian cell with a blunt double-stranded RNA (dsRNA) construct comprising a sense strand of 25, 26, 27, 28, 29, or 30 nucleotides in length, having a 5′-end and a 3′-end, wherein the sense strand consists of 2′-modified ribose sugars except for nucleotides 11, 12, and 13 relative to the 3′-end of the sense strand, which prevents processing of the double-stranded RNA construct by Dicer, and an antisense strand of 25, 26, 27, 28, 29, or 30 nucleotides in length having a 5′-end and a 3′-end, wherein the antisense strand is unmodified, wherein the antisense strand hybridizes to the sense strand and to mRNA of the target gene, wherein the dsRNA inhibits expression of the target gene in a sequence-dependent manner, and wherein the dsRNA does not form a hairpin.

### Claim 52 (depends on 51)

The method of claim 51, wherein said mammalian cell is in culture.

### Claim 53 (depends on 51)

The method of claim 51, wherein said mammalian cell is a human cell.

### Claim 54 (depends on 51)

The method of claim 51, wherein the mammalian cell is contacted in the presence of a delivery reagent.

### Claim 55 (depends on 54)

The method of claim 54, wherein said delivery reagent is a lipid.

### Claim 56 (depends on 55)

The method of claim 55, wherein said lipid is a cationic lipid.

### Claim 57 (depends on 54)

The method of claim 54, wherein said delivery reagent is a liposome.

### Claim at position 9

58-60. (canceled)

### Claim 61

A method for inhibiting the expression of a target gene in a mammalian cell, comprising contacting the mammalian cell with a blunt double-stranded RNA (dsRNA) construct comprising a sense strand having a 5′-end and a 3′-end, wherein the sense strand is 25, 26, or 27 nucleotides in length, and an antisense strand having a 5′-end and a 3′-end, wherein the antisense strand is 25, 26 or 27 nucleotides in length, wherein the sense strand consists of 2′-modified ribose sugars except for nucleotides 11, 12 and 13 relative to the 3′ end of the sense strand, which prevents processing of the double-stranded RNA construct by Dicer, and wherein the dsRNA does not form a hairpin.

### Claim 62 (depends on 51)

The method of claim 51, wherein the target gene is SOD1, PPIB, RIP140, PCSK9, TNFα, AP2 (adipocyte lipid-binding protein), or MAP4K4.

### Claim 63 (depends on 51)

The method of claim 51, wherein nucleotides 11, 12, and 13 relative to the 3′end of the sense strand are purine nucleotides.

### Claim 64 (depends on 51)

The method of claim 51, wherein the 2′-modified ribose sugars are 2′—O-alkyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy nucleotides, 2′-H (deoxyribonucleotides), or a combination thereof.

### Claim 65 (depends on 64)

The method of claim 64, wherein the 2′—O-alkyl nucleotides are 2′—O-methyl nucleotides.

### Claim 66 (depends on 51)

The method of claim 51, wherein, compared to a dsRNA construct of a nucleotide sequence of the dsRNA construct of claim 51 but wherein the sense strand does not include 2′-modifications, the dsRNA construct exhibits:
(i) enhanced target specificity;
(ii) reduced off-target silencing;
(iii) improved stability in serum and/or cerebral spinal fluid; and/or
(iv) reduced induction of interferon response in primary cells.

### Claim 67 (depends on 51)

The method of claim 51, wherein the dsRNA construct is administered to a subject in need thereof.

### Claim 68 (depends on 67)

The method of claim 67, wherein the dsRNA construct is administered for treatment of a disease selected from the group consisting of: cancers, retinopathies, autoimmune diseases, inflammatory diseases, viral diseases, and cardiovascular diseases.

### Claim 68 (depends on 67)

The method of claim 67, wherein the dsRNA construct is formulated for parenteral, oral, or intraperitoneal administration to the subject.

### Claim 69 (depends on 68)

The method of claim 68, wherein the inflammatory disease is selected from the group consisting of ICAM-1-related disorders, psoriasis, ulcerative colitis, and Crohn's disease.

### Claim 69 (depends on 68)

The method of claim 68, wherein the parenteral administration is selected from the group consisting of: intravenous, intramuscular, interstitial, intraarterial, subcutaneous, intra ocular, intrasynovial, trans epithelial, pulmonary, ophthalmic, sublingual and buccal, topical, dermal, ocular, rectal, and nasal inhalation.

### Claim 70 (depends on 67)

The method of claim 67, wherein the dsRNA construct is formulated with liposomes, hydrogels, or controlled-release polymers.

### Claim 71 (depends on 51)

The method of claim 51, wherein nucleotides 11, 12, and 13 relative to the 3′end of the sense strand have phosphorothioate linkages.