Modified RNAi polynucleotides and uses thereof

• US 10,633,654 B2
• Filed: 10/05/2018
• Issued: 04/28/2020
• Est. Priority Date: 02/11/2008
• Status: Active Grant

• Current Assignee: Phio Pharmaceuticals Corp.
• Original Assignee: Phio Pharmaceuticals Corp.
• Inventors: Pavco, Pamela A., Kamens, Joanne, Woolf, Tod M., Salomon, William, Khvorova, Anastasia
• Primary Examiner(s): McDonald, Jennifer Pitrak
• Application Number:
  US16/153,424
• Publication Number:
  US 20190169608A1
• Time in Patent Office: 571 Days
• Field of Search: None
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• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• 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 15/1136 against growth factors, growth regulators, cytokines, lymphokines or hormones
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/14 interfering N.A.
• 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 2310/341 : Gapmers, i.e. of the type ===---===
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2320/51 : modulating the chemical stability, e.g. nuclease-resistance
• CPC Class Codes: C12N 2320/53 : reducing unwanted side-effects

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

1. A method for inhibiting the expression of a target gene in a mammalian cell, comprising contacting the mammalian cell with a blunt-ended 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.

2. The method of claim 1, wherein said mammalian cell is in culture.

3. The method of claim 1, wherein said mammalian cell is a human cell.

4. The method of claim 1, wherein the mammalian cell is contacted in the presence of a delivery reagent.

5. The method of claim 4, wherein said delivery reagent is a lipid.

6. The method of claim 5, wherein said lipid is a cationic lipid.

7. The method of claim 4, wherein said delivery reagent is a liposome.

9. The method of claim 1, wherein the target gene is SOD1, PPIB, RIP140, PCSK9, TNFα

, AP2 (adipocyte lipid-binding protein), or MAP4K4.

10. The method of claim 1, wherein nucleotides 11, 12, and 13 relative to the 3′

end of the sense strand are purine nucleotides.

11. The method of claim 1, 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.

12. The method of claim 11, wherein the 2′

—

O-alkyl nucleotides are 2′

—

O-methyl nucleotides.

13. The method of claim 1, wherein, compared to a dsRNA construct of a nucleotide sequence of the dsRNA construct of claim 1 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.

14. The method of claim 1, wherein the dsRNA construct is administered to a subject in need thereof.

15. The method of claim 14, 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.

16. The method of claim 15, wherein the inflammatory disease is selected from the group consisting of ICAM-1-related disorders, psoriasis, ulcerative colitis, and Crohn'"'"'s disease.

17. The method of claim 14, wherein the dsRNA construct is formulated for parenteral, oral, or intraperitoneal administration to the subject.

18. The method of claim 17, 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.

19. The method of claim 14, wherein the dsRNA construct is formulated with liposomes, hydrogels, or controlled-release polymers.

20. The method of claim 1, wherein nucleotides 11, 12, and 13 relative to the 3′

end of the sense strand have phosphorothioate linkages.

8. A method for inhibiting the expression of a target gene in a mammalian cell, comprising contacting the mammalian cell with a blunt-ended 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.