RNA Interference Mediated Inhibition of Gene Expression Using Chemically Modified Short Interfering Nucleic Acid (siNA)

• US 20090023675A1
• Filed: 04/17/2008
• Published: 01/22/2009
• Est. Priority Date: 02/20/2002
• Status: Active Grant

• Current Assignee: Sirna Therapeutics, Inc. (Alnylam Pharmaceuticals Incorporated)
• Original Assignee: Sirna Therapeutics, Inc. (Alnylam Pharmaceuticals Incorporated)
• Inventors: Thompson, James, Wang, Weimin, Morrissey, David, Jamison, Sharon, Chowrira, Bharat, Macejak, Dennis, Fosnaugh, Kathy, Vaish, Narendra, Pavco, Pamela, Beigelman, Leonid, Chen, Tongqian, McSwiggen, James, Vargeese, Chandra, Zinnen, Shawn, Haeberli, Peter, Usman, Nassim
• Granted Patent: US 8,232,383 B2
• Time in Patent Office: Days
• US Class Current: 514/44.R
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/54 the modifying agent being an organic compound
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• 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 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/08 of the prostate
• CPC Class Codes: A61P 13/10 of the bladder
• 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 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 27/16 Otologicals
• 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 3/00 : Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/00 : Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 : Antibacterial agents
• CPC Class Codes: A61P 31/10 : Antimycotics
• CPC Class Codes: A61P 31/12 : Antivirals
• CPC Class Codes: A61P 31/14 : for RNA viruses
• CPC Class Codes: A61P 31/16 : for influenza or rhinoviruses
• CPC Class Codes: A61P 31/18 : for HIV
• CPC Class Codes: A61P 31/20 : for DNA viruses
• CPC Class Codes: A61P 31/22 : for herpes viruses
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/00 : Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/04 : Immunostimulants
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 37/08 : Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 21/02 : with ribosyl as saccharide radical
• 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 15/1131 : against viruses
• CPC Class Codes: C12N 15/1138 : against receptors or cell surface proteins
• CPC Class Codes: C12N 15/8218 : Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 2310/111 : spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/14 : interfering N.A.
• CPC Class Codes: C12N 2310/315 : Phosphorothioates
• CPC Class Codes: C12N 2310/317 : with an inverted bond, e.g. a cap structure
• CPC Class Codes: C12N 2310/318 : where the PO2 is completely replaced, e.g. MMI or formacetal
• CPC Class Codes: C12N 2310/321 : 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 : 2'-R Modification
• CPC Class Codes: C12N 2310/331 : Universal or degenerate base
• CPC Class Codes: C12N 2310/332 : Abasic residue
• CPC Class Codes: C12N 2310/346 : having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/351 : Conjugate
• CPC Class Codes: C12N 2310/3515 : Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2310/3533 : Halogen
• CPC Class Codes: C12N 2310/53 : partially self-complementary or closed
• CPC Class Codes: C12N 2320/11 : for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2320/51 : modulating the chemical stability, e.g. nuclease-resistance
• CPC Class Codes: C12N 2330/30 : chemically synthesised

Abstract

The present invention concerns methods and reagents useful in modulating gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to synthetic chemically modified small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against target nucleic acid sequences. The small nucleic acid molecules are useful in the treatment of any disease or condition that responds to modulation of gene expression or activity in a cell, tissue, or organism.

Claims

1-48. -48. (canceled)

49. A chemically modified nucleic acid molecule, wherein:

(a) the nucleic acid molecule comprises a sense strand and a separate antisense strand, each strand having one or more pyrimidine nucleotides and one or more purine nucleotides;

(b) each strand of the nucleic acid molecule is independently 18 to 27 nucleotides in length;

(c) an 18 to 27 nucleotide sequence of the antisense strand is complementary to a target RNA sequence;

(d) an 18 to 27 nucleotide sequence of the sense strand is complementary to the antisense strand and comprises an 18 to 27 nucleotide sequence of the target RNA sequence; and

(e) 50 percent or more of the nucleotides in each strand comprise a 2′

-sugar modification, wherein the 2′

-sugar modification of any of the pyrimidine nucleotides differs from the 2′

-sugar modification of any of the purine nucleotides.

50. The nucleic acid molecule of claim 49, wherein the 2′

-sugar modification is selected from the group consisting of 2′

-deoxy-2′

-fluoro, 2′

-O-methyl, and 2′

-deoxy.

51. The nucleic acid of claim 50, wherein the 2′

-deoxy sugar modification is a pyrimidine modification.

52. The nucleic acid of claim 50, wherein the 2′

-O-methyl sugar modification is a pyrimidine modification.

53. The nucleic acid of claim 50, wherein the 2′

-deoxy-2′

-fluoro sugar modification is a pyrimidine modification.

54. The nucleic acid molecule according to claims 51, 52, or 53, wherein said pyrimidine modification is in the sense strand.

55. The nucleic acid molecule according to claims 51, 52, or 53, wherein said pyrimidine modification is in the antisense strand.

56. The nucleic acid molecule according to claims 51, 52, or 53, wherein said pyrimidine modification is in the sense strand and the antisense strand.

57. The nucleic acid molecule of claim 50, wherein the 2′

-O-methyl sugar modification is a purine modification.

58. The nucleic acid molecule of claim 50, wherein the 2′

-deoxy sugar modification is a purine modification.

59. The nucleic acid molecule according to claims 57 or 58, wherein said purine modification is in the sense strand.

60. The nucleic acid molecule of according to claims 57 or 58, wherein said purine modification is in the antisense strand.

61. The nucleic acid molecule of claim 58, wherein said purine modification is in the sense strand and the antisense strand

62. The nucleic acid molecule of claim 49, wherein the nucleic acid molecule comprises ribonucleotides.

63. The nucleic acid molecule of claim 49, wherein the sense strand includes a terminal cap moiety at the 5′

-end, the 3′

-end, or both of the 5′

- and 3′

-ends.

64. The nucleic acid molecule of claim 63, wherein the terminal cap moiety is an inverted deoxy abasic moiety.

65. The nucleic acid molecule of claim 49, wherein said nucleic acid molecule includes one or more phosphorothioate internucleotide linkages.

66. The nucleic acid molecule of claim 65, wherein one of the phosphorothioate internucleotide linkages is at the 3′

-end of the antisense strand.

67. The nucleic acid molecule of claim 49, wherein the 5′

-end of the antisense strand includes a terminal phosphate group.

68. The nucleic acid molecule of claim 49, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand include a 3′

-overhang.

69. A composition comprising the nucleic acid molecule of claim 49, in a pharmaceutically acceptable carrier or diluent.

70. A chemically modified nucleic acid molecule, wherein:

(a) the nucleic acid molecule comprises a sense strand and a separate antisense strand, each strand having one or more pyrimidine nucleotides and one or more purine nucleotides;

(b) each strand of the nucleic acid molecule is independently 18 to 27 nucleotides in length;

(c) an 18 to 27 nucleotide sequence of the antisense strand is complementary to a target RNA sequence;

(d) an 18 to 27 nucleotide sequence of the sense strand is complementary to the antisense strand and comprises an 18 to 27 nucleotide sequence of the target RNA sequence; and

(e) 50 percent or more of the nucleotides in each strand comprise a 2′

-sugar modification, wherein the 2′

-sugar modification of any of the purine nucleotides in the sense strand differs from the 2′

-sugar modification of any of the purine nucleotides in the antisense strand.

71. The nucleic acid molecule of claim 70, wherein the 2′

-sugar modification is selected from the group consisting of 2′

-deoxy-2′

-fluoro, 2′

-O-methyl, and 2′

-deoxy.

72. The nucleic acid molecule of claim 71, wherein the 2′

-deoxy-2′

-fluoro sugar modification is a pyrimidine modification.

73. The nucleic acid molecule of claim 72, wherein said pyrimidine modification is in the sense strand.

74. The nucleic acid molecule of claim 72, wherein said pyrimidine modification is in the antisense strand.

75. The nucleic acid molecule of claim 72, wherein said pyrimidine modification is in the sense strand and the antisense strand.

76. The nucleic acid molecule of claim 71, wherein the 2′

-O-methyl sugar modification is a purine modification.

77. The nucleic acid molecule of claim 71, wherein the 2′

-deoxy sugar modification is a purine modification.

78. The nucleic acid molecule of claim 77, wherein said purine modification is in the sense strand.

79. The nucleic acid molecule according to claims 76 or 77, wherein said purine modification is in the antisense strand.

80. The nucleic acid molecule of claim 70, wherein the nucleic acid molecule comprises ribonucleotides.

81. The nucleic acid molecule of claim 70, wherein the sense strand includes a terminal cap moiety at the 5′

-end, the 3′

-end, or both of the 5′

- and 3′

-ends.

82. The nucleic acid molecule of claim 81, wherein the terminal cap moiety is an inverted deoxy abasic moiety.

83. The nucleic acid molecule of claim 70, wherein said nucleic acid molecule includes one or more phosphorothioate internucleotide linkages.

84. The nucleic acid molecule of claim 83, wherein one of the phosphorothioate internucleotide linkages is at the 3′

-end of the antisense strand.

85. The nucleic acid molecule of claim 70, wherein the 5′

-end of the antisense strand includes a terminal phosphate group.

86. The nucleic acid molecule of claim 70, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand include a 3′

-overhang.

87. A composition comprising the nucleic acid molecule of claim 70, in a pharmaceutically acceptable carrier or diluent.