Reduced size self-delivering RNAi compounds

• US 8,796,443 B2
• Filed: 09/22/2009
• Issued: 08/05/2014
• 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): MCDONALD, JENNIFER SUE PITRAK
• Application Number:
  US13/120,342
• Publication Number:
  US 20120040459A1
• Time in Patent Office: 1,778 Days
• Field of Search: None
• US Class Current: 536/24.5
• 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.

Claims

1. An isolated double stranded nucleic acid molecule comprising a guide strand and a passenger strand,

wherein the isolated double stranded nucleic acid molecule includes a double stranded region and a single stranded region, wherein the double stranded region is from 8-15 nucleotides long, wherein the single stranded region is at the 3′

end of the guide strand and is 4-12 nucleotides long, wherein the single stranded region contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, wherein at least 40% of the nucleotides of the isolated double stranded nucleic acid molecule are modified, wherein a hydrophobic conjugate is attached to the isolated double stranded nucleic acid molecule, and wherein the isolated double stranded nucleic acid molecule does not form a hairpin.

2. The isolated double stranded nucleic acid molecule of claim 1, wherein the double stranded region is 11, 12, 13, or 14 nucleotides long and/or wherein the single stranded region is at least 6 or at least 7 nucleotides long.

3. The isolated double stranded nucleic acid molecule of claim 1, wherein each nucleotide within the single stranded region has a phosphorothioate modification.

4. The isolated double stranded nucleic acid molecule of claim 1, wherein at least one of the nucleotides of the isolated double stranded nucleic acid molecule that is modified comprises a 2′

O-methyl or a 2′

-fluoro modification and/or wherein at least one of the nucleotides of the isolated double stranded nucleic acid molecule that is modified comprises a hydrophobic modification.

5. The isolated double stranded nucleic acid molecule of claim 1, wherein the guide strand of the double stranded nucleic acid molecule exhibits complementarity to a gene encoding for Osteopontin (SPP1), SOD1 or MAP4K4, optionally wherein the guide strand comprises SEQ ID NO:

170, SEQ ID NO;

40 or SEQ ID NO;

25.

18. The isolated double stranded nucleic acid molecule of claim 1, wherein each of the guide strand and the passenger strand has a 5′

end and a 3′

end, and wherein the hydrophobic conjugate is attached to;

i) the 3′

end of the passenger strand;

ii) the 3′

end of the guide strand;

or

iii) the 5′

end of the passenger strand.

6. An isolated asymmetric nucleic acid molecule comprising:

a first polynucleotide wherein the first polynucleotide is complementary to a second polynucleotide and a target gene; and

a second polynucleotide,

wherein the second polynucleotide is at least 6 nucleotides shorter than the first polynucleotide, wherein the first polynucleotide includes a single stranded region of 6, 7, 8, 9, 10, 11 or 12 nucleotides, wherein the single stranded region of the first polynucleotide contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, wherein the asymmetric nucleic acid molecule also includes a double stranded region of 8-15 nucleotides long, wherein at least 50% of C and U nucleotides in the double stranded region are 2′

O-methyl modified or 2′

-fluoro modified, and wherein a hydrophobic conjugate is attached to the asymmetric nucleic acid molecule.

7. The isolated asymmetric nucleic acid molecule of claim 6, wherein the single stranded region is 6 or 7 nucleotides long and/or wherein each nucleotide within the single stranded region has a phosphorothioate modification.

8. The isolated asymmetric nucleic acid molecule of claim 6, wherein each of the first polynucleotide and the second polynucleotide has a 5′

end and a 3′

end, wherein the hydrophobic conjugate is attached to;

i) the 3′

end of the second polynucleotide;

ii) the 3′

end of the first polynucleotide;

or

iii) the 5′

end of the second polynucleotide.

9. An isolated double stranded nucleic acid molecule comprising:

a guide strand of 17-21 nucleotides in length that has complementarity to a target gene, and a passenger strand of 8-16 nucleotides in length,

wherein the isolated double stranded nucleic acid molecule includes a double stranded region of 8-15 nucleotides long and a single stranded region, wherein the guide strand and the passenger strand form the double stranded nucleic acid molecule having the double stranded region and the single stranded region, wherein the single stranded region is at the 3′

end of the guide strand and is 4-12 nucleotides in length, wherein the single stranded region comprises 2-12 phosphorothioate modifications, wherein at least 40% of the nucleotides of the isolated double stranded nucleic acid molecule are modified, and wherein the double stranded nucleic acid molecule is linked to a hydrophobic conjugate, and wherein the isolated double stranded nucleic acid molecule does not form a hairpin.

10. The isolated double stranded nucleic acid molecule of claim 9, wherein the isolated double stranded nucleic acid molecule contains at least one hydrophobic base modification and wherein the hydrophobic base modification comprises a hydrophobic modification of a pyrimidine base, optionally at position 4 or 5, optionally wherein the hydrophobic base modification is selected from the group consisting of a phenyl, 4-pyridyl, 2-pyridyl, indolyl, isobutyl, tryptophanyl (C₈H₆N)CH₂CH(NH₂)CO), methyl, butyl, aminobenzyl, and naphthyl modification of a uridine or cytidine.

11. The isolated double stranded nucleic acid molecule of claim 9, wherein the hydrophobic conjugate is a small molecule, optionally wherein the small molecule is a sterol-type molecule, optionally wherein the sterol-type molecule is cholesterol.

12. The isolated double stranded nucleic acid molecule of claim 9, wherein the hydrophobic conjugate is attached to the double stranded nucleic acid molecule through a linker, optionally wherein the linker is a TEG linker.

13. A method for inhibiting the expression of a target gene in a mammalian cell, comprising contacting the mammalian cell with an isolated double stranded nucleic acid molecule comprising a guide strand and a passenger strand,

wherein the isolated double stranded nucleic acid molecule includes a double stranded region and a single stranded region, wherein the double stranded region is from 8-15 nucleotides long, wherein the single stranded region is at the 3′

end of the guide strand and is 4-12 nucleotides long, wherein the single stranded region of the guide strand contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, wherein at least 40% of the nucleotides of the isolated double stranded nucleic acid molecule are modified, and wherein a hydrophobic conjugate is attached to the isolated double stranded nucleic acid molecule, and wherein the isolated double stranded nucleic acid molecule does not form a hairpin.

14. The method of claim 13, wherein the double stranded region is 11, 12, 13, or 14 nucleotides long and/or wherein the single stranded region is at least 6 or at least 7 nucleotides long.

15. The method of claim 13, wherein each nucleotide within the single stranded region has a phosphorothioate modification.

16. The method of claim 13, wherein at least one of the nucleotides of the isolated double stranded nucleic acid molecule that is modified comprises a 2′

O-methyl or a 2′

-fluoro modification and/or wherein at least one of the nucleotides of the isolated double stranded nucleic acid molecule that is modified comprises a hydrophobic modification.

17. The method of claim 13, wherein the double stranded nucleic acid molecule exhibits complementarity to a gene encoding for Osteopontin (SPP1), SOD1 or MAP4K4, optionally wherein the guide strand comprises SEQ ID NO:

170, SEQ ID NO;

40 or SEQ ID NO;

25.

19. The method of claim 13, wherein each of the guide strand and the passenger strand has a 5′

end and a 3′

end, and wherein the hydrophobic conjugate is attached to;

i) the 3′

end of the passenger strand;

ii) the 3′

end of the guide strand;

or

iii) the 5′

end of the passenger strand.