Reduced size self-delivering RNAi compounds

• US 10,041,073 B2
• Filed: 03/23/2011
• Issued: 08/07/2018
• Est. Priority Date: 09/22/2008
• Status: Active Grant

• Current Assignee: Phio Pharmaceuticals Corp.
• Original Assignee: Rxi Pharmaceuticals Corporation
• Inventors: Khvorova, Anastasia, Salomon, William, Kamens, Joanne, Samarsky, Dmitry, Woolf, Tod M., Cardia, James
• Primary Examiner(s): ANGELL, JON E
• Application Number:
  US13/069,780
• Publication Number:
  US 20110263680A1
• Time in Patent Office: 2,694 Days
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• 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 2310/3231 : having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/3341 : 5-Methylcytosine
• CPC Class Codes: C12N 2310/3515 : Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2310/3519 : Fusion with another nucleic acid
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2320/32 : Special delivery means, e.g. tissue-specific
• 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 present invention relates to RNAi constructs with minimal double-stranded regions, and their use in gene silencing. RNAi constructs associated with the invention include a double stranded region of 8-14 nucleotides and a variety of chemical modifications, and are highly effective in gene silencing. The RNAi constructs may be, for instance, miRNA constructs that are miRNA modulators.

Claims

1. An isolated nucleic acid molecule comprising

a guide strand of 18-23 nucleotides in length that has complementarity to a miRNA sequence, and

a passenger strand of 8-16 nucleotides in length,

wherein the guide strand and the passenger strand form the nucleic acid molecule such that the nucleic acid molecule has a double stranded region of 10-15 nucleotides in length and a single stranded region, wherein the single stranded region is at the 3′

end of the guide strand and is 2-13 nucleotides in length and comprises at least two phosphorothioate modifications, wherein at least 50% of the pyrimidines in the nucleic acid molecule are modified, and wherein the isolated nucleic acid molecule modulates miRNA-mediated gene expression in a mammalian cell.

2. The nucleic acid molecule of claim 1, wherein the nucleotide in position one of the guide strand has a 2′

-O-methyl modification.

3. The nucleic acid molecule of claim 2, wherein at least 60%, at least 80%, at least 90% or wherein 100% of the pyrimidines in the nucleic acid molecule are modified.

4. The nucleic acid molecule of claim 2, wherein modified pyrimidines are 2′

fluoro or 2′

O methyl modified.

5. The nucleic acid molecule of claim 2, wherein at least one U or C includes a hydrophobic modification.

6. The nucleic acid molecule of claim 5, wherein the hydrophobic modification is a methyl or ethyl hydrophobic base modification.

7. The nucleic acid molecule of claim 2, wherein the guide strand contains 6-8 phosphorothioate modifications.

8. The nucleic acid molecule of claim 2, wherein the guide strand includes 4-14 phosphate modifications.

9. The nucleic acid molecule of claim 2, wherein the single stranded region of the guide strand is 6 nucleotides long or 8 nucleotides long.

10. The nucleic acid molecule of claim 2, wherein the double stranded region is 13 nucleotides long.

11. The nucleic acid molecule of claim 2, wherein the double stranded nucleic acid molecule has one end that is blunt or includes a one nucleotide overhang.

12. The nucleic acid molecule of claim 2, wherein the passenger strand is linked at the 3′

end to a sterol.

13. The nucleic acid molecule of claim 2, wherein the isolated double stranded nucleic acid molecule is an miRNA mimic and wherein the miRNA sequence to which the guide strand is complementary is a miRNA recognition element.

14. The nucleic acid molecule of claim 2, wherein the isolated double stranded nucleic acid molecule is an miRNA inhibitor and wherein the miRNA sequence to which the guide strand is complementary is an antisense strand of a mature miRNA.

15. The nucleic acid molecule of claim 14, wherein the guide strand is at least 50% chemically modified.

16. The nucleic acid molecule of claim 14, wherein the mature miRNA is miR 17-92.

17. A method for modulating miRNA-mediated gene expression in a mammalian cell, comprising contacting the mammalian cell with an isolated double stranded nucleic acid molecule of claim 1 in an effective amount to modulate miRNA-mediated gene expression.

18. The method of claim 17, wherein the mammalian cell is contacted with the isolated nucleic acid in vivo or ex vivo.

19. A method for modulating miRNA-mediated gene expression in a stem cell, comprising contacting the stem cell with an isolated double stranded nucleic acid molecule of claim 1 in an effective amount to modulate miRNA-mediated gene expression in the stem cell.

20. The nucleic acid molecule of claim 2, wherein the 2′

-O-methyl modification is a 5P-2′

O-methyl U modification.

21. The nucleic acid molecule of claim 2, wherein a plurality of U'"'"'s and/or C'"'"'s include a hydrophobic modification.

22. The nucleic acid molecule of claim 2, wherein the 3′

terminal 10 nucleotides of the guide strand include at least eight phosphorothioate modifications.

23. The nucleic acid molecule of claim 12, wherein the sterol is cholesterol.

24. The nucleic acid molecule of claim 13, wherein the miRNA mimic is a mimic of a miRNA selected from the group consisting of miR21, miR 139, miR 7, miR29, miR 122, miR 302-367 cluster, miR 221, miR-96, miR 126, miR 225 and miR 206.