MODIFIED RNAI POLYNUCLEOTIDES AND USES THEREOF

• US 20110039914A1
• Filed: 02/11/2009
• Published: 02/17/2011
• Est. Priority Date: 02/11/2008
• Status: Active Grant

• Current Assignee: Phio Pharmaceuticals Corp.
• Original Assignee: Rxi Pharmaceuticals Corporation
• Inventors: PAVCO, PAMELA A., Khvorova, Anastasia, Woolf, Tod M., Salomon, William, Kamens, Joanne
• Granted Patent: US 10,131,904 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 17/06 Antipsoriatics
• 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 31/14 for RNA viruses
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• 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 2310/341 : 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 relates 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 preferably 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 is/are modified by four 2′-O-methyl 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 enhanced target specificity.

Claims

1. A double-stranded RNA (dsRNA) construct of 12-35 nucleotides in length, for inhibiting expression of a target gene, comprising:

(1) a sense strand having a 5′

-end and a 3′

-end, wherein the sense strand is highly modified with 2′

-modified ribose sugars, and wherein 3-6 nucleotides in the central portion of the sense strand are not modified with 2′

-modified ribose sugars and,

(2) an antisense strand having a 5′

-end and a 3′

-end, which 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.

8. The dsRNA of claim 1, wherein the dsRNA construct is blunt-ended.

15. The dsRNA of claim 1, wherein the sense strand is 25, 26, 27, 28, 29, or 30 nucleotides in length, and wherein the antisense strand is 25, 26, 27, 28, 29, or 30 nucleotides in length.

16. The dsRNA of claim 1, wherein each end of the sense strand comprises a continuous stretch of 2′

modified ribose sugars, optionally wherein each continuous stretch of 2′

-modified ribose sugars independently starts from the terminal nucleotide, the second nucleotide from the terminal nucleotide, or the third nucleotide from the terminal nucleotide.

23. The dsRNA of claim 1, wherein the antisense strand comprises a 2′

-O-methyl modified nucleotide at the 2^nd nucleotide on the 5′

-end of the antisense strand and no other modified nucleotides or wherein the antisense strand comprises at least four consecutive 2′

-O-methyl modified 3′

-end nucleotides with phosphorothioate linkages.

24. The dsRNA of claim 1, wherein the sense strand of the dsRNA comprises a mismatch nucleotide at the 2^nd nucleotide from the 3′

-end of the sense strand, and/or the last 2^nd-8^th nucleotides at the 3′

-end of the sense strand mis-match their corresponding antisense strand nucleotides.

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

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

36. The dsRNA of claim 1, wherein the sense strand nucleotides between the 2′

-modified ribose nucleotides are purine nucleotides, optionally having phosphorothioate linkages.

38. The dsRNA of claim 1, wherein the sense strand nucleotides between the 2′

-modified ribose nucleotides form one or more bulges of 1-5 nucleotides each.

50. A composition comprising the dsRNA of claim 1, and a pharmaceutically acceptable carrier or diluent.

60. A method for evaluating in vivo delivery of an siRNA construct to a target site, comprising co-delivering the siRNA construct with a dsRNA construct of claim 1 targeting PPIB, and assaying the inhibition of PPIB function at the target site, wherein successful inhibition of the PPIB function at the target site is indicative of successful in vivo delivery of the siRNA construct to the target site.

61. The dsRNA of claim 1 wherein at least 60%, at least 70%, at least 80% or at least 90% of the nucleotides on the sense strand are modified with 2′

-modified ribose sugars and optionally wherein the 2′

-modified ribose sugars are 2′

-O-methyl nucleotides.

62. The dsRNA of claim 1 wherein the antisense strand is unmodified.

63. The dsRNA of claim 1, wherein the 3-6 nucleotides in the central portion of the sense strand that are not modified with 2′

-modified ribose sugars are positioned overlapping nucleotide positions 11-13 relative to the 3′

end of the sense strand.

64. The dsRNA of claim 16, wherein the 5′

end of the sense strand comprises 12-14 consecutive 2′

-modified ribose sugars and the 3′

end of the sense strand comprises 10-12 consecutive 2′

-modified ribose sugars.

65. The dsRNA 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.

66. The dsRNA of claim 65, wherein the 2′

-O-alkyl nucleotides are 2′

-O-methyl nucleotides or 2′

-O-allyl nucleotides.

67. The dsRNA of claim 1, wherein, compared to a similar construct without the 2′

-modification(s) at said position(s), the dsRNA 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.

68. The dsRNA of claim 1, wherein the antisense strand contains one or more 2′

-F modified and/or 2′

-O-methyl-modified nucleotides and/or wherein the 5′

-end of the antisense strand is phosphorylated.

69. An RNA construct for inhibiting expression of a target gene, wherein said construct is identical to the dsRNA of claim 1, except for a single nick on the sense strand.

72. The dsRNA of claim 1, wherein the minimum length of the dsRNA is 18 nucleotides.

73. The dsRNA of claim 61, wherein at least 64%, at least 72%, at least 80% or at least 88% of the nucleotides on the sense strand are modified with 2′

-modified ribose sugars and optionally wherein the 2′

-modified ribose sugars are 2′

-O-methyl nucleotides.

2-7. -7. (canceled)

9-14. -14. (canceled)

17-22. -22. (canceled)

25-34. -34. (canceled)

37. (canceled)

39-49. -49. (canceled)

51. A method for inhibiting the expression of a target gene in a mammalian cell, comprising contacting the mammalian cell with a double-stranded RNA (dsRNA) construct of 12-35 nucleotides in length, comprising a sense strand having a 5′

-end and a 3′

-end, wherein the sense strand is highly modified with 2′

-modified ribose sugars and wherein 3-6 nucleotides in the central portion of the sense strand are not modified with 2′

-modified ribose sugars, and an antisense strand having a 5′

-end and a 3′

-end, wherein the antisense strand hybridizes to the sense strand and to mRNA of the target gene, and wherein the dsRNA inhibits expression of the target gene in a sequence-dependent manner.

52-58. -58. (canceled)

59. A method for improving the gene silencing effect of a small interference RNA (siRNA), comprising modifying the siRNA to become a double-stranded RNA (dsRNA) construct of 12-35 nucleotides in length, comprising a sense strand having a 5′

-end and a 3′

-end, wherein the sense strand is highly modified with 2′

-modified ribose sugars and wherein 3-6 nucleotides in the central portion of the sense strand are not modified with 2′

-modified ribose sugars, and an antisense strand having a 5′

-end and a 3′

-end, wherein the antisense strand hybridizes to the sense strand and to mRNA of the target gene, and wherein the dsRNA inhibits expression of the target gene in a sequence-dependent manner.

70. A 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, for inhibiting expression of a target gene, wherein the sense strand is completely modified with 2′

-modified ribose sugars except for 3 nucleotides in the central portion of the sense strand, corresponding to nucleotides 11, 12 and 13 relative to the 3′

end of the sense strand.

71. A double-stranded RNA (dsRNA) construct of 12-35 nucleotides in length, comprising a sense strand having a 5′

-end and a 3′

-end, wherein at least 60%, at least 70%, at least 80% or at least 90% of the nucleotides on the sense strand are modified with 2′

-modified ribose sugars and wherein 3-6 nucleotides in the central portion of the sense strand are not modified with 2′

-modified ribose sugars, and an antisense strand having a 5′

-end and a 3′

-end, for inhibiting expression of a target gene, wherein the antisense strand hybridizes to the sense strand and to mRNA of the target gene, and wherein the dsRNA inhibits expression of the target gene in a sequence-dependent manner.

74. A double-stranded RNA (dsRNA) construct of 12-35 nucleotides in length, comprising a sense strand having a 5′

-end and a 3′

-end, and an antisense strand having a 5′

-end and a 3′

-end, for inhibiting expression of a target gene, wherein the sense strand comprises a 5′

region, a central region and a 3′

region, wherein the 5′

region starts at the 5′

most nucleotide of the sense strand and the majority of nucleotides within the 5′

region are modified with 2′

-modified ribose sugars, wherein the 5′

region ends with a 2′

-modified ribose sugar, wherein the central region has consecutive nucleotides without 2′

-modified ribose sugars, wherein the 3′

region begins with a 2′

-modified sugar and has a majority of 2′

modified ribose sugars, and wherein the 3′

region ends at the 3′

most nucleotide of the sense strand.