TARGETING MICRORNAS TO OVERCOME DRUG TOLERANCE AND RESISTANCE
20220133767 · 2022-05-05
Inventors
Cpc classification
C12N2310/20
CHEMISTRY; METALLURGY
A61K31/7088
HUMAN NECESSITIES
A61K31/7088
HUMAN NECESSITIES
C12N2501/06
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
A61K31/5377
HUMAN NECESSITIES
A61K31/506
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
G01N2800/52
PHYSICS
C12N2310/3231
CHEMISTRY; METALLURGY
A61K31/5377
HUMAN NECESSITIES
C12Q2600/106
CHEMISTRY; METALLURGY
A61K2300/00
HUMAN NECESSITIES
C12N2310/113
CHEMISTRY; METALLURGY
A61K2300/00
HUMAN NECESSITIES
A61K31/506
HUMAN NECESSITIES
International classification
A61K31/7088
HUMAN NECESSITIES
A61K31/506
HUMAN NECESSITIES
A61K31/5377
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
Abstract
The invention provides methods and compositions for use in targeting micro RNAs (miRNAs), as well as methods and compositions for use in treating, reducing, inhibiting, or delaying resistance or tolerance to anti-cancer treatment, and methods and compositions for use in treating or preventing cancer.
Claims
1. A method of treating, reducing, preventing, or delaying tolerance or resistance to anti-receptor tyrosine kinase (RTK) therapy in a subject, the method comprising administering a miR-147b inhibitor to the subject.
2. The method of claim 1, wherein the RTK is selected from the group consisting of epidermal growth factor receptor (EGFR), human EGFR2 (HER2), HER3, anaplastic lymphoma kinase (ALK), ROS1, ERBB2/3/4, KIT, MET/hepatocyte growth factor receptor (HGFR), RON, platelet derived growth factor receptor (PDGFR), vascular endothelial cell growth factor receptor (VEGFR), VEGFR1, VEGFR2, fibroblast growth factor receptor (FGFR), insulin-like growth factor 1 receptor (IGF1R), and RET.
3. The method of claim 1, wherein the miR-147b inhibitor reduces a Von Hippel-Lindau (VHL)-pseudohypoxia response or counteracts metabolic changes in the tricarboxylic acid (TCA) cycle associated with drug tolerance in the subject.
4. The method of claim 1, wherein the subject has cancer.
5. A method of treating or preventing cancer in a subject, the method comprising administering a miR-147b inhibitor to the subject.
6. The method of claim 4, wherein the subject has a cancer selected from the group consisting of lung cancer, non-smal cell lung cancer, colorectal cancer, anal cancer, glioblastoma, squamous cell carcinoma, squamous cell carcinoma of the head and neck, pancreatic cancer, breast cancer, renal cell carcinoma, thyroid cancer, gastroesophageal adenocarcinoma, and gastric cancer.
7. The method claim 1, further comprising administering an anti-RTK therapy to the subject.
8. The method of claim 7, wherein the anti-RTK therapy is an anti-EGFR therapy.
9. The method of claim 8, wherein the anti-EGFR therapy comprises a tyrosine kinase inhibitor (TKI).
10. The method of claim 9, wherein the TKI is selected from the group consisting of gefitinib, erlotinib, afatinib, lapatinib, neratinib, osimertinib, vandetanib, crizotinib, dacomitinib, regorafenib, ponatinib, vismodegib, pazopanib, cabozantinib, bosutinib, axitinib, vemurafenib, ruxolitinib, nilotinib, dasatinib, imatinib, sunitinib, sorafenib, trametinib, cobimetanib, and dabrafenib.
11. The method of claim 8, wherein the anti-EGFR therapy comprises an anti-EGFR antibody or fragment thereof, or an anti-EGFR CAR T cell.
12. The method of claim 11, wherein the anti-EGFR therapy comprises an anti-EGFR antibody selected from the group consisting of cetuximab, necitumumab, panitumumab, nimotuzumab, futuximab, zatuximab, cetugex, and margetuximab.
13. The method of claim 7, wherein the miR-147b inhibitor is administered before, at the same time as, or after the anti-RTK therapy.
14. The method of claim 1, wherein the subject has or is at risk of developing tolerance or resistance to anti-RTK therapy.
15. The method of claim 14, wherein the anti-RTK therapy to which the subject has or is at risk of developing tolerance or resistance is an anti-EGFR therapy, an anti-AKL therapy, an anti-ROS1 therapy, an anti-ERBB2/3/4 therapy, an anti-KIT therapy, an anti-MET/hepatocyte growth factor receptor (HGFR) therapy, an anti-platelet derived growth factor receptor (PDGFR) therapy, an anti-vascular endothelial cell growth factor receptor (VEGFR) therapy, an anti-fibroblast growth factor receptor (FGFR) therapy, and an anti-RET therapy.
16. The method of claim 15, wherein the anti-RTK therapy to which the subject has or is at risk of developing tolerance or resistance comprises a TKI.
17. The method of claim 16, wherein the subject has or is at risk of developing tolerance or resistance to an anti-EGFR therapy selected from the group consisting of gefitinib, erlotinib, afatinib, lapatinib, neratinib, osimertinib, vandetanib, crizotinib, dacomitinib, regorafenib, ponatinib, vismodegib, pazopanib, cabozantinib, bosutinib, axitinib, vemurafenib, ruxolitinib, nilotinib, dasatinib, imatinib, sunitinib, sorafenib, trametinib, cobimetanib, and dabrafenib.
18. The method of claim 15, wherein the subject has or is at risk of developing tolerance or resistance to an anti-EGFR therapy comprising an anti-EGFR antibody or fragment thereof, or an anti-EGFR CAR T cell.
19. The method of claim 18, wherein the anti-EGFR therapy to which the subject has or is at risk of developing tolerance or resistance comprises an anti-EGFR antibody selected from the group consisting of cetuximab, necitumumab, panitumumab, nimotuzumab, futuximab, zatuximab, cetugex, and margetuximab.
20. The method of claim 1, wherein the miR-147b inhibitor comprises an inhibitory molecule selected from the group consisting of an antisense oligonucleotide, an antagomir, an anti-miRNA sponge, a competitive inhibitor, a triplex-forming oligonucleotide, a double-stranded oligonucleotide, a short interfering RNA, an siRNA, an shRNA, a guide sequence for RNAse P, a small molecule, a catalytic RNA, and a ribozyme; or the inhibition is carried out by the use of a gene editing approach, such as CRISPR-cas9.
21. The method of claim 1, wherein the miR-147b inhibitor is an inhibitor of the production or activity of pri-miR-147b, pre-miR147b, or mature miR-147b.
22. A single-stranded oligonucleotide comprising a total of 12 to 50 interlinked nucleotides and having a nucleobase sequence comprising at least 6 contiguous nucleobases complementary to an equal-length portion of a miR-147b target nucleic acid.
23. The oligonucleotide of claim 22, wherein the oligonucleotide comprises at least one modified nucleobase.
24. The oligonucleotide of claim 23, wherein the at least one modified nucleobase is selected from the group consisting of 5-methylcytosine, 7-deazaguanine, and 6-thioguanine.
25. The oligonucleotide of claim 22, wherein the oligonucleotide comprises at least one modified internucleoside linkage.
26. The oligonucleotide of claim 25, wherein the modified internucleoside linkage is a phosphorothioate linkage.
27. The oligonucleotide of claim 26, wherein the phosphorothioate linkage is a stereochemically enriched phosphorothioate linkage.
28. The oligonucleotide of claim 25, wherein at least 50% of the internucleoside linkages in the oligonucleotide are each independently a modified internucleoside linkage.
29. The oligonucleotide of claim 28, wherein at least 70% of the internucleoside linkages in the oligonucleotide are each independently a modified internucleoside linkage.
30. The oligonucleotide of claim 22, wherein the oligonucleotide comprises at least one modified sugar nucleoside.
31. The oligonucleotide of claim 30, wherein the at least one modified sugar nucleoside is a bridged nucleic acid.
32. The oligonucleotide of claim 31, wherein the bridged nucleic acid is a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), or a cEt nucleic acid.
33. The oligonucleotide of claim 31, wherein the at least one modified sugar nucleoside is a 2′-modified sugar nucleoside.
34. The oligonucleotide of claim 33, wherein the at least one 2′-modified sugar nucleoside comprises a 2′-modification selected from the group consisting of 2′-fluoro, 2′-methoxy, and 2′-methoxyethoxy.
35. The oligonucleotide of claim 22, wherein the oligonucleotide comprises deoxyribonucleotides.
36. The oligonucleotide of claim 22, wherein the oligonucleotide comprises ribonucleotides.
37. The oligonucleotide of claim 22, wherein the oligonucleotide is a morpholino oligonucleotide.
38. The oligonucleotide of claim 22, wherein the oligonucleotide is a peptide nucleic acid.
39. The oligonucleotide of claim 22, wherein the oligonucleotide comprises a hydrophobic moiety covalently attached at its 5′-terminus, its 3′-terminus, or an internucleoside linkage of the oligonucleotide.
40. The oligonucleotide of claim 22, wherein the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 3 to 736 or a variant thereof.
41. The oligonucleotide of claim 22, wherein the oligonucleotide comprises at least 8 contiguous nucleobases complementary to an equal-length portion of a miR-147b target nucleic acid.
42. The oligonucleotide of claim 22, wherein the oligonucleotide comprises at least 12 contiguous nucleobases complementary to an equal-length portion of a miR-147b target nucleic acid.
43. The oligonucleotide of claim 22, wherein the oligonucleotide comprises 20 or fewer contiguous nucleobases complementary to an equal-length portion of a miR-147b target nucleic acid.
44. The oligonucleotide of claim 22, wherein the oligonucleotide comprises a total of at least 12 interlinked nucleotides.
45. The oligonucleotide of claim 22, wherein the oligonucleotide comprises a total of 24 or fewer interlinked nucleotides.
46. The oligonucleotide of claim 22, wherein the oligonucleotide is a gapmer, headmer, tailmer, altmer, blockmer, skipmer, or unimer.
47. A double-stranded oligonucleotide comprising the oligonucleotide of claim 22 hybridized to a complementary oligonucleotide.
48. A double-stranded oligonucleotide comprising a passenger strand hybridized to a guide strand comprising a nucleobase sequence comprising at least 6 contiguous nucleobases complementary to an equal-length portion of a miR-147b target nucleic acid, wherein each of the passenger strand and the guide strand comprises a total of 12 to 50 interlinked nucleotides.
49. The oligonucleotide of claim 48, wherein the passenger strand comprises at least one modified nucleobase.
50. The oligonucleotide of claim 49, wherein the at least one modified nucleobase is selected from the group consisting of 5-methylcytosine, 7-deazaguanine, and 6-thioguanine.
51. The oligonucleotide of claim 48, wherein the passenger strand comprises at least one modified internucleoside linkage.
52. The oligonucleotide of claim 51, wherein the modified internucleoside linkage is a phosphorothioate linkage.
53. The oligonucleotide of claim 52, wherein the phosphorothioate linkage is a stereochemically enriched phosphorothioate linkage.
54. The oligonucleotide of claim 51, wherein at least 50% of the internucleoside linkages in the passenger strand are each independently the modified internucleoside linkage.
55. The oligonucleotide of claim 54, wherein at least 70% of the internucleoside linkages in the passenger strand are each independently the modified internucleoside linkage.
56. The oligonucleotide of claim 48, wherein the passenger strand comprises at least one modified sugar nucleoside.
57. The oligonucleotide of claim 56, wherein the at least one modified sugar nucleoside is a bridged nucleic acid.
58. The oligonucleotide of claim 57, wherein the bridged nucleic acid is a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), or a cEt nucleic acid.
59. The oligonucleotide of claim 56, wherein the at least one modified sugar nucleoside is a 2′-modified sugar nucleoside.
60. The oligonucleotide of claim 59, wherein the at least one 2′-modified sugar nucleoside comprises a 2′-modification selected from the group consisting of 2′-fluoro, 2′-methoxy, and 2′-methoxyethoxy.
61. The oligonucleotide of claim 48, wherein the passenger strand comprises deoxyribonucleotides.
62. The oligonucleotide of claim 48, wherein the passenger strand comprises ribonucleotides.
63. The oligonucleotide of claim 48, wherein the passenger strand comprises a hydrophobic moiety covalently attached at a 5′-terminus, a 3′-terminus, or an internucleoside linkage of the passenger strand.
64. The oligonucleotide of claim 48, wherein the guide strand comprises at least one modified nucleobase.
65. The oligonucleotide of claim 64, wherein the at least one modified nucleobase is selected from the group consisting of 5-methylcytosine, 7-deazaguanine, and 6-thioguanine.
66. The oligonucleotide of claim 48, wherein the guide strand comprises at least one modified internucleoside linkage.
67. The oligonucleotide of claim 66, wherein the modified internucleoside linkage is a phosphorothioate linkage.
68. The oligonucleotide of claim 67, wherein the phosphorothioate linkage is a stereochemically enriched phosphorothioate linkage.
69. The oligonucleotide of claim 66, wherein at least 50% of the internucleoside linkages in the guide strand are each independently the modified internucleoside linkage.
70. The oligonucleotide of claim 69, wherein at least 70% of the internucleoside linkages in the guide strand are each independently the modified internucleoside linkage.
71. The oligonucleotide of claim 48, wherein the guide strand comprises at least one modified sugar nucleoside.
72. The oligonucleotide of claim 71, wherein the at least one modified sugar nucleoside is a bridged nucleic acid.
73. The oligonucleotide of claim 72, wherein the bridged nucleic acid is a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), or a cEt nucleic acid.
74. The oligonucleotide of claim 71, wherein the at least one modified sugar nucleoside is a 2′-modified sugar nucleoside.
75. The oligonucleotide of claim 74, wherein the at least one 2′-modified sugar nucleoside comprises a 2′-modification selected from the group consisting of 2′-fluoro, 2′-methoxy, and 2′-methoxyethoxy.
76. The oligonucleotide of claim 48, wherein the guide strand comprises deoxyribonucleotides.
77. The oligonucleotide of claim 48, wherein the guide strand comprises ribonucleotides.
78. The oligonucleotide of claim 48, wherein the guide strand comprises a hydrophobic moiety covalently attached at a 5′-terminus, a 3′-terminus, or an internucleoside linkage of the guide strand.
79. The oligonucleotide of claim 48, wherein the guide strand comprises a sequence selected from the group consisting of SEQ ID NOs: 3 to 736 or a variant thereof.
80. The oligonucleotide of claim 47, wherein the hybridized oligonucleotide comprises at least one 3′-overhang.
81. The oligonucleotide of claim 47, wherein the hybridized oligonucleotide comprises a blunt end.
82. The oligonucleotide of claim 47, wherein the hybridized oligonucleotide comprises two 3′-overhangs.
83. The oligonucleotide of claim 22, wherein the miR-147 target nucleic acid comprises pri-miR-147b, pre-miR-147b, or mature miR-147b.
84. An oligonucleotide that competes with miR-147b for binding to a target mRNA or pre-mRNA sequence, thereby inhibiting or reducing the effects of miR-147b on the mRNA or pre-mRNA.
85. The oligonucleotide of claim 84, comprising a sequence selected from SEQ ID NOs: 1, 2, or 737 to 889.
86. A vector comprising a sequence encoding an oligonucleotide of claim 22, wherein the vector optionally further comprises a promoter to direct transcription of the sequence.
87. The vector of claim 86, wherein the vector comprises a sequence encoding multiple oligonucleotides as described herein.
88. The vector of claim 87, wherein the vector comprises a sequence encoding 2, 3, 4, 5, 6, 7, 8, 9, or 10 oligonucleotides as described herein.
89. The vector of claim 86, wherein the vector is a virus, such as a lentivirus, an adenovirus, or an adeno-associated virus; or is a plasmid, a cosmid, or a phagemid.
90. A pharmaceutical composition comprising (i) an oligonucleotide of claim 22 or a vector comprising said oligonucleotide, or a small molecule inhibitor of miR-147b, and (ii) a pharmaceutically acceptable excipient or carrier.
91. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide of claim 22 or a vector comprising said oligonucleotide, or a pharmaceutical composition as described herein.
92. The method of claim 1, wherein the miR-147b inhibitor comprises an oligonucleotide as described herein.
93. The method of claim 1, further comprising administration of an additional anti-cancer agent.
94. The method of claim 93, wherein the additional anti-cancer agent is an anti-RTK agent.
95. A method of determining whether tolerance or resistance of a cancer to anti-RTK therapy may be effectively treated, reduced, prevented, or delayed by anti-miR-147b therapy, the method comprising determining the level of miR-147b in the cancer, wherein detection of an increased level of miR-147b, relative to a control, indicates that tolerance or resistance of the cancer to anti-RTK therapy may be effectively treated, reduced, prevented, or delayed with anti-miR-147b therapy, optionally in combination with anti-RTK therapy.
96. A method of determining whether a cancer may be effectively treated or prevented with an anti-miR-147b therapy, the method comprising determining the level of miR-147b in the cancer, wherein detection of an increased level of miR-147b in the cancer, relative to a control, indicates that the cancer may effectively be treated or prevented with anti-miR-147b therapy, optionally in combination with anti-RTK therapy.
97. The method of claim 95, wherein the anti-miR-147 therapy is selected from an oligonucleotide as described herein, a vector comprising the oligonucleotide, and a small molecule inhibitor of miR-147b and/or the anti-RTK therapy is selected from a TKI, an anti-RTK antibody, and a CAR T cell directed against an RTK.
98. The method of claim 95, wherein determination of the level of miR-147b in the cancer is carried out by detection of the level of miR-147b in a sample from the subject having the cancer.
99. The method of claim 98, wherein the sample comprises tumor tissue, tissue swab, sputum, serum, or plasma.
100. The method of claim 95, further comprising administering an anti-miR147b therapy to a subject having the cancer, if it is determined that (i) tolerance or resistance of the cancer to anti-RTK therapy may be effectively treated, reduced, prevented, or delayed by anti-miR-147b therapy, or (ii) the cancer may be effectively treated with anti-miR147b therapy.
101. A method of detecting a cancer cell in a sample, the method comprising determining the level of miR-147b in the sample, wherein detection of an increased level of miR-147b in the sample, relative to a control, indicates the presence of a cancer cell in the sample.
102. A method of determining whether a cancer cell in a sample may be tolerant or resistant to anti-RTK therapy, the method comprising determining the level of miR-147b in the sample, wherein detection of an increased level of miR-147b, relative to a control, indicates that the cancer cell may be tolerant or resistant to anti-RTK therapy.
103. The method of claim 102, wherein the anti-RTK therapy is anti-EGFR therapy.
104. The method of claim 102, wherein the sample comprises tumor tissue, tissue swab, sputum, serum, or plasma.
105. A method of making an organoid comprising lung cells, the method comprising the steps of: a. culturing lung cells in a medium comprising epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and fibroblast growth factor 10 (FGF10); b. maintaining the cells in culture in a medium comprising Noggin and transforming growth factor-β (TGF-β); and c. differentiating the cells in a medium comprising fibroblast growth factor 7 (FGF7) and platelet-derived growth factor (PDGF).
106. The method of claim 105, wherein the lung cells are lung epithelial cells obtained from a sample of lung tissue of a subject.
107. The method of claim 105, wherein the lung cells are immortalized lung epithelial cells.
108. The method of claim 105, wherein the lung cells are cancerous.
109. The method of claim 105, wherein the lung cells are non-cancerous.
110. The method of claim 105, wherein the lung cells are tolerant or resistant to an anti-RTK agent.
111. The method of claim 105, wherein the maintaining step is carried out on days 0-3 of the method, maintenance is carried out on days 4-6, and differentiation is carried out on days 7-24.
112. The method of claim 105, wherein the organoids show ring-like structures upon treatment with an anti-RTK agent.
113. A three-dimensional organoid comprising lung cells, wherein the organoid is optionally made by, or has features of organoids made using, the method of claim 105.
114. The organoid of claim 113, wherein the lung cells comprise lung cancer cells.
115. The organoid of claim 113, wherein the lung cells or lung cancer cells are primary cells, obtained or cultured from the cells of a subject.
116. A method for identifying an agent that may be used (i) to treat, reduce, prevent, or delay tolerance or resistance to anti-RTK therapy, or (ii) in the treatment or prevention of cancer, the method comprising contacting a cell with the agent and determining whether the agent decreases the level of miR-147b in the cell.
117. The method of claim 116, wherein the cell is comprised within an organoid.
118. The method of claim 117, wherein the organoid comprises lung cancer cells.
119. The method of claim 117, wherein the organoid is an organoid as described herein, or is made by a method as described herein.
120. The method of claim 116, wherein the lung cancer cells are resistant to an anti-RTK therapy.
121. The method of claim 116, wherein the cells are primary cells, obtained or cultured from the cells of a subject.
122. The method of claim 116, wherein the agent is a candidate compound, not previously known to be effective at treating, reducing, preventing, or delaying tolerance or resistance to anti-RTK therapy, or at treating or preventing cancer.
123. The method of claim 116, wherein the method is carried out to determine an optimal approach to treat, reduce, prevent, or delay tolerance or resistance of a cancer to anti-RTK therapy in a subject, or to treat or prevent a cancer in a subject.
124. A kit comprising an agent for detecting the level of miR-147b in a sample.
125. The kit of claim 124, wherein the agent comprises an oligonucleotide, which is optionally an oligonucleotide as described herein.
126. A kit comprising a miR-147b inhibitor, which optionally is an oligonucleotide as described herein, and a second agent for treating cancer.
127. The oligonucleotide of claim 22, wherein the oligonucleotide targets a sequence comprising or consisting of nucleotides 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 7-12, 8-13, 9-14, 10-15, 11-16, 12-17, 13-18, 14-19, 15-20, 16-21, 17-22, 18-23, 19-24, 20-25, 21-26, 22-27, 23-28, 24-29, 25-30, 26-31, 27-32, 28-33, 29-34, 30-35, 31-36, 32-37, 33-38, 34-39, 35-40, 36-41, 37-42, 38-43, 39-44, 40-45, 41-46, 42-47, 43-48, 44-49, 45-50, 48-51, 47-52, 48-53, 49-54, 50-55, 51-56, 52-57, 53-58, 54-59, 55-80, 56-61, 57-62, 58-63, 59-64, 60-65, 61-66, 62-67, 63-68, 64-69, 65-70, 66-71, 67-72, 68-73, 69-74, 70-75, 71-76, 72-77, 73-78, 74-79, or 75-80 of SEQ ID NO: 1.
128. The oligonucleotide of claim 127, wherein the oligonucleotide targets said sequence and additionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74 additional nucleotides of SEQ ID NO: 1, whether all on one side of the indicated fragment or wherein the fragment is between the one or more additional nucleotides.
129. The oligonucleotide of claim 48, wherein the oligonucleotide targets a sequence comprising or consisting of nucleotides 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 7-12, 8-13, 9-14, 10-15, 11-16, 12-17, 13-18, 14-19, 15-20, 16-21, 17-22, 18-23, 19-24, 20-25, 21-26, 22-27, 23-28, 24-29, 25-30, 26-31, 27-32, 28-33, 29-34, 30-35, 31-36, 32-37, 33-38, 34-39, 35-40, 36-41, 37-42, 38-43, 39-44, 40-45, 41-46, 42-47, 43-48, 44-49, 45-50, 48-51, 47-52, 48-53, 49-54, 50-55, 51-56, 52-57, 53-58, 54-59, 55-80, 56-61, 57-62, 58-63, 59-64, 60-65, 61-66, 62-67, 63-68, 64-69, 65-70, 66-71, 67-72, 68-73, 69-74, 70-75, 71-76, 72-77, 73-78, 74-79, or 75-80 of SEQ ID NO: 1.
130. The oligonucleotide of claim 129, wherein the oligonucleotide targets said sequence and additionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74 additional nucleotides of SEQ ID NO: 1, whether all on one side of the indicated fragment or wherein the fragment is between the one or more additional nucleotides.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0241] The invention is based, in part, on our discovery that miR-147b plays a role in tolerance and resistance of receptor tyrosine kinase (RTK) (e.g., epidermal growth factor (EGFR))-mutated cancer to RTK-targeted therapies, such as tyrosine kinase inhibitors (TKIs). We have also found that miR-147b inhibition can be used to treat cancer. Accordingly, the invention includes methods for treating, reducing, preventing, or delaying tolerance or resistance of cancer to RTK (e.g., EGFR)-targeted therapy by administration of one or more inhibitors of miR-147b, as well as methods of treating or preventing cancer using one or more of these inhibitors. As explained further below, these methods can optionally be carried out in combination with other therapies, such as anti-cancer therapies (e.g., TKIs or anti-RTK antibody therapy; also see below). The invention also provides miR-147b inhibitors, compositions including them (optionally in combination with other agents), diagnostic methods, and screening methods.
[0242] The invention is also based, in part, on our discovery of methods to prepare and use three-dimensional organoids including lung-derived cells, e.g., lung cancer cells. Accordingly, the invention also provides such organoids, as well as methods of their use.
[0243] The methods, inhibitors, compositions, and organoids of the invention are described further, as follows.
Micro RNAs—miR-147b
[0244] Micro RNAs (miRNAs) are small, non-coding RNA modulators of gene activity, which act primarily by base pairing to the 3′-untranslated regions of target RNAs (e.g., mRNAs and pre-mRNAs), leading to target RNA degradation or mRNA translation inhibition. MiRNAs are typically produced as follows. First, an initial transcript, pri-miRNA, is cleaved in the nucleus to generate pre-miRNA, which comprises a stem-loop structure. This molecule is then exported from the nucleus to the cytoplasm, where it is processed by Dicer to generate an miRNA duplex lacking a connecting loop. The reverse-complement of the mature miRNA sequence is then removed from the duplex, and the mature miRNA is incorporated into a multi-component RNA-induced silencing complex (RISC). The mature miRNA, in the context of RISC, can then act by base pairing to a target RNA, as noted above.
[0245] MiRNAs play critical roles in many biological processes, and their dysregulation accordingly plays roles in many different diseases. We have found that increased miR-147b levels are associated with tolerance and resistance to anti-RTK therapies, as described herein. We have also found that decreasing miR-147b levels is effective to counter these effects, and also to directly treat cancer. Accordingly, the present invention establishes miR-147b as a therapeutic target for treating, reducing, preventing, or delaying tolerance or resistance to anti-RTK therapy, as well as a target for anti-cancer treatment and prevention.
Therapeutic Methods
[0246] MiR-147b inhibitors, such as those described herein, can be used in therapeutic methods, as noted above. In some examples, the inhibitors are used to treat, reduce, inhibit, or delay tolerance or resistance to an anti-cancer treatment. In particular, the inhibitors can be used in the context of tolerance or resistance of cancer to RTK-targeted therapies including, for example, TKIs and/or anti-RTK immunotherapies (e.g., antibody- or CAR T-based therapies). In other examples, the inhibitors are used to treat or prevent cancer directly.
[0247] Examples of RTKs, with respect to which a miR-147b inhibitor of the invention can be used to treat, reduce, inhibit, or delay tolerance or resistance to targeting thereof, include, e.g., epidermal growth factor receptor (EGFR), human EGFR2 (HER2), HER3, anaplastic lymphoma kinase (ALK), ROS1, ERBB2/3/4, KIT, MET/hepatocyte growth factor receptor (HGFR), RON, platelet derived growth factor receptor (PDGFR), vascular endothelial cell growth factor receptor (VEGFR), VEGFR1, VEGFR2, fibroblast growth factor receptor (FGFR), insulin-like growth factor 1 receptor (IGF1R), and RET.
[0248] The miR-147b inhibitors can be administered as sole therapeutic agents or, optionally, can be administered in combination with each other or one or more additional therapeutic agents (e.g., one or more anti-RTK therapy). MiR-147b inhibitors can be administered to a subject before, at the same time as, or after another therapeutic agent (e.g., an anti-RTK-targeted therapy), or after multiple rounds of another agent (e.g., an anti-RTK-targeted therapy), as determined to be appropriate by those of skill in the art. Accordingly, in some embodiments, the invention includes combination therapy methods, in which one or more miR-147b inhibitor is administered in combination with one or more other agents (e.g., anti-RTK therapy), and optionally one or more further anti-cancer treatments (see, e.g., below).
[0249] In addition to the above, miR-147b inhibitors can also be used to treat or prevent cancer, due to direct anti-cancer effects of the inhibitors. In these methods, the inhibitors can be used alone or in combination with each other or other anti-cancer treatments including (in addition to anti-RTK-targeted therapies), for example, the anti-cancer agents listed below, as well as other treatments (e.g., radiotherapy and surgery).
[0250] As noted above, examples of anti-RTK therapies include TKIs, anti-RTK antibodies, and anti-RTK CAR T cells. Examples of TKIs include gefitinib (Iressa®), erlotinib (Tarceva®), afatinib (Gilotrif®), lapatinib (Tykerb®), neratinib (Nertynx®), osimertinib (Tagrisso®), vandetanib (Caprelsa®), crizotinib (Xalcori®), dacomitinib (Vizimpro®), regorafenib (Stivarga®), ponatinib (Iclusig®), vismodegib (Erivedge®), pazopanib (Votrient®), cabozantinib (Cabozantinib®), bosutinib (Bosulif®), axitinib (Inlyta®), vemurafenib (Zelboraf®), ruxolitinib (Jakafi®), nilotinib (Tasigna®), dasatinib (Sprycel®), imatinib (Gleevec®), sunitinib (Sutent®), sorafenib (Nexavar®), trametinib (Mekinist®), cobimetanib (Cotellic®), and dabrafenib (Tafinlar®).
[0251] As is known in the art, TKIs such as these vary with respect to the RTKs that they target, and therefore also the cancer types targeted. Selection of a particular TKI for administration to a subject, in the context of a miR-147b inhibitor, can thus be carried out by those of skill in the art depending upon the particular cancer to be treated (see, e.g., Jeong et al., Curr. Probl. Cancer 37(3):110-144, 2013).
[0252] Examples of anti-RTK antibodies that can be used in the invention include anti-EGFR antibodies such as, for example, cetuximab (Erbitux®), nimotuzumab (TheraCIM®), necitumumab (Portrazza®), panitumumab (Vedibix®), futuximab, zatuximab, CetuGEX™, and margetuximab. Anti-HER2 antibodies include trastuzumab (Herceptin®), pertuzumab (Perjeta®), trasGEX™. seribantumab, and patritumab. Antibodies against additional RTKs include the following: onartuzumab (HER3), namatumab (RON), ganitumab (RON), cixutumumab (RON), dalotuzumab (IGF1R), teprotumumab (IGF1R), icrucumab (VEGFR1), ramucirumab (VEGFR1), tanibirumab (VEGFR2), and olaratumab (PDGFR) (Fauvel et al., Mabs 6(4):838-851, 2014). Accordingly, miR-147b inhibitors, such as those described herein, can be used to treat, reduce, inhibit, or delay tolerance or resistance to therapies such as these. They can also be administered with such therapies, in order to treat, reduce, inhibit, or delay tolerance, as well as to optionally provide a separate anti-cancer effect.
[0253] As noted above, the methods of the invention can also include administration of one or more additional anti-cancer agents. For example, agents such as antimetabolites (e.g., methotrexate, pemetrexed, purine antagonists (e.g., mercaptopurine, thioguanine, fludarabine phosphate, cladribine, or pentostatin), or pyrimidine antagonists (e.g., gemcitabine, capecitabine, fluoropyrimidines, fluorouracil, 5-fluorouracil, cytarabine, or azacitidine)), antibiotics (e.g., anthracyclines (e.g., doxorubicin, epirubicin, daunorubicin, or idarubicin), adriamycin, dactinomycin, idarubincin, plicamycin, mitomycin, bleomycin, or mitoxantrone), alkylating agents (e.g., cyciophosphamide, temozolomide, procarbazine, dacarbazine, altretamine, cisplatin, carboplatin, oxaliplatin, or nitrosoureas), plant alkaloids (e.g., vinblastine, vincristine, etoposide, teniposide, topotecan, irinotecan, paclitaxel, nab-paclitaxel, ABRAXA E® (protein-bound paclitaxel), or docetaxel), anti-tubulin agents (e.g., eribulin, ixabepilone, vinorelbine, or vincristine), anticoagulants (e.g., heparin or warfarin), biological agents (e.g., hormonal agents, cytokines, interleukins, interferons, granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), or chemokines), and/or anti-angiogenic agents (e.g., angiostatin or endostatin) can be used.
[0254] The methods of the invention can further be carried out in combination with immunotherapeutic approaches to treating cancer. These include, for example, anti-CTLA-4 antagonist antibodies (e.g., ipilimumab, Yervoy®, BMS), anti-VEGF antibodies (e.g., bevacizumab, Avastin®), anti-OX40 agonist antibodies (e.g., Medi6469, MedImmune, and MOXR0916/RG7888, Roche), and PD-1 and/or PD-L1 targeted therapies (e.g., nivolumab (Opdivo®, BMS-936558, MDX-1106, and ONO-4538) and pembrolizumab (Keytruda®, MK-3475)). Further immunotherapeutic approaches include anti-TIGIT antagonist antibodies (e.g., BMS-986207, Bristol-Myers Squibb/Ono Pharmaceuticals), IDO inhibitors (see, e.g., US 2016/0060237 and US 2015/0352206; Indoximod, New Link Genetics), RORγ agonists (e.g., LYC-55716 (Lycera/Celgene) and INV-71 (Innovimmune)), and cancer vaccines (e.g., MAGE3 vaccine (e.g., for melanoma and bladder cancer), MUC1 vaccine (e.g., for breast cancer), EGFRv3 (such as Rindopepimut, e.g., for brain cancer, such as glioblastoma multiforme), or ALVAC-CEA (e.g., for CEA+ cancers)).
[0255] Further, as noted above, the miR-147b inhibitors of the invention can be used in the context of CAR T cell therapy, e.g., anti-RTK CAR T cell therapy. For example, CAR T cells directed against EGFR, which are useful against, e.g., gliomas and other EGFR.sup.+ solid tumors, can be used. In another example, CAR T cells directed against EGFRvIII, which are useful against, e.g., glioblastoma multiforme and gliomas, such as EGFRvIII+ gliomas, can be used.
[0256] Examples of cancers that can be treated according to the methods of the invention include lung cancer (e.g., adenocarcinoma of the lung; non-small cell lung cancer), colorectal cancer, anal cancer, glioblastoma, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), pancreatic cancer, breast cancer, renal cell carcinoma, squamous cell carcinoma, thyroid cancer, gastroesophageal adenocarcinoma, and gastric cancer.
[0257] In further examples, the cancer can be selected from the group consisting of stomach cancer, colon cancer, liver cancer, biliary tract cancer, gallbladder cancer, rectal cancer, renal cancer, bladder cancer, endometrial cancer, ovarian cancer, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, prostate cancer, testicular cancer, pelvic cancer, brain cancer, esophageal cancer, bronchus cancer, oral cancer, oropharyngeal cancer, larynx cancer, thyroid cancer, skin cancer, cancer of the central nervous system, cancer of the respiratory system, and cancer of the urinary system.
[0258] In still further examples, the cancer can be selected from the group consisting basal cell carcinoma, large cell carcinoma, small cell carcinoma, non-small cell lung carcinoma, renal carcinoma, hepatocarcinoma, gastric carcinoma, choriocarcinoma, adenocarcinoma, hepatocellular carcinoma, giant (or oat) cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, adrenocortical carcinoma, cholangiocarcinoma, Merkel cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, hepatoblastoma, medulloblastoma, nephroblastoma, neuroendocrine tumors, pheochromocytoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma, leukemia, B-cell leukemia, T-cell leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic (lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), erythroleukemia, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), thyoma, multiple myeloma, plasmacytoma, localized myeloma, extramedullary myeloma, melanoma, superficial spreading melanoma, nodular melanoma, lentigno maligna melanoma, acral lentiginous melanoma, amelanotic melanoma, ganglioneuroma, Pacinian neuroma, acoustic neuroma, astrocytoma, oligoastrocytoma, ependymoma, glioma, glioblastoma multiforme, brainstem glioma, optic nerve glioma, oligoastrocytoma, pheochromocytoma, meningioma, malignant mesothelioma, and a virally induced cancer.
[0259] In additional examples, the cancer is a sarcoma, for example, a sarcoma selected from the group consisting of angiosarcoma, hemangiosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, malignant fibrous cytoma, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, synovial sarcoma, vascular sarcoma, Kaposi's sarcoma, dermatofibrosarcoma, epithelioid sarcoma, leiomyosarcoma, and neurofibrosarcoma.
[0260] In further examples, the cancer is a breast cancer selected from the group consisting of triple-negative breast cancer, triple-positive breast cancer, HER2-negative breast cancer, HER2-positive breast cancer, estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, progesterone receptor-positive breast cancer, progesterone receptor-negative breast cancer, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, Paget disease of the nipple, and phyllodes tumor.
[0261] Anti-cancer therapies, including miR-147b inhibitors and other anti-cancer therapies, such as those described above, are administered in the practice of the methods of the invention as is known in the art (e.g., according to FDA-approved regimens or other regimens determined to be appropriate by those of skill in the art). In some embodiments, anti-cancer therapies of the invention are administered in amounts effective to treat, reduce, inhibit, or delay resistance or tolerance to anti-RTK therapy, as described herein, or to treat or prevent cancer. The therapeutically effective amount is typically dependent upon the weight of the subject being treated, his or her physical or health condition, the extensiveness of the condition to be treated, the age of the subject being treated, pharmaceutical formulation methods, and/or administration methods (e.g., administration time and administration route).
[0262] In some embodiments, anti-cancer therapies such as those described above are administered by various mutes, including, but not limited to, oral, intravenous, intra-arterial, parenteral, intratumoral, intraperitoneal, and subcutaneous mutes. The appropriate formulation and mute of administration can be selected by those of skill in the art according to the intended application.
MIR-147b Inhibitors
[0263] Inhibitors of miR-147b, according to the invention, can target the miRNA at any stage in the process of its production or action. Thus, for example, an inhibitor can block transcription of the pri-miRNA, formation of pre-miRNA, export of the pre-miRNA from the nucleus, Dicer cleavage to generate an miRNA duplex, formation of miRNA/RISC, or binding of miRNA/RISC to its target.
[0264] Several different types of molecules and approaches can be used to inhibit miR-147b, according to the invention. These include, for example, single-stranded antisense oligonucleotide (e.g., antagomir and anti-miR miRNA sponge), double-stranded oligonucleotide (e.g., short interfering RNA, such as siRNA and shRNA), small molecule, decoy, aptamer, catalytic RNA (e.g., ribozyme), and gene editing (e.g., CRISPR-cas9) based approaches. Descriptions of examples of molecules and approaches such as these, in the context of inhibiting miR-147b, are provided below.
Antisense
[0265] In one approach, the invention provides antisense molecules that include sequences that are complementary to a target miR-147b sequence, which includes mature miR-147b or a precursor (i.e., pri-miR-147b or pre-miR-147b) or fragment thereof. These molecules are, in general, referred to herein as antisense molecules or antisense oligonucleotides. Specific examples of these types of molecules include antagomirs, miRNA sponges, and competitive inhibitors (see below).
[0266] Accordingly, the invention provides single-stranded oligonucleotides having nucleobase sequences with at least 6 contiguous nucleobases complementary to an equal-length portion within a miR-147b target sequence, as noted above (including pri-miR-147b, pre-miR-147b, mature mi-147b, as well as fragments thereof). This approach is typically referred to as an antisense approach, and the corresponding oligonucleotides of the invention are referred to as antisense oligonucleotides (ASO). Without wishing to be bound by theory, this approach involves hybridization of an oligonucleotide of the invention to a target miR-147b sequence, followed by ribonuclease H (RNase H) mediated cleavage of the target miR-147b nucleic acid. Alternatively, and without wishing to be bound by theory, this approach involves hybridization of an oligonucleotide of the invention to a target miR-147b sequence, thereby sterically blocking the target miR-147b nucleic acid from binding to its target mRNA or pre-mRNA. Alternatively, in some embodiments, the single-stranded oligonucleotide may be delivered to a patient as a double stranded oligonucleotide, where the oligonucleotide of the invention is hybridized to another oligonucleotide (e.g., an oligonucleotide having a total of 6 to 30 nucleotides).
[0267] An antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) includes a nucleobase sequence having at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30) contiguous nucleobases complementary to, e.g., an equal-length portion within a miR-147b sequence. The equal-length portion may be disposed within the sequence at any position.
[0268] An antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) may be a gapmer, headmer, or tailmer. Gapmers are oligonucleotides having an RNase H recruiting region (gap) flanked by a 5′ wing and 3′ wing, each of the wings optionally including at least one affinity enhancing nucleoside (e.g., 1, 2, 3, or 4 affinity enhancing nucleosides). Headmers are oligonucleotides having an RNase H recruiting region (gap) flanked by a 5′ wing including at least one affinity enhancing nucleoside (e.g., 1, 2, 3, or 4 affinity enhancing nucleosides). Tailmers are oligonucleotides having an RNase H recruiting region (gap) flanked by a 3′ wing including at least one affinity enhancing nucleoside (e.g., 1, 2, 3, or 4 affinity enhancing nucleosides). In certain embodiments, each wing includes 1-5 nucleosides. In some embodiments, each nucleoside of each wing is a modified nucleoside. In particular embodiments, the gap includes 7-12 nucleosides. Typically, the gap region includes a plurality of contiguous, unmodified deoxyribonucleotides. For example, all nucleotides in the gap region are unmodified deoxyribonucleotides (2′-deoxyribofuranose-based nucleotides). In some embodiments, an antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) is a gapmer, headmer, or tailmer.
[0269] The 5′-wing may consist of, e.g., 1 to 8, 1 to 7, 1 to 6, 1 to 5, 2 to 5, 3 to 5, 4 or 5, 1 to 4, 1 to 3, 1 or 2, 2 to 4, 2 or 3, 3 or 4, 1, 2, 3, 4, 5, or 6 linked nucleosides. The 3′-wing may consists of, e.g., 1 to 8, 1 to 7, 1 to 6, 1 to 5, 2 to 5, 3 to 5, 4 or 5, 1 to 4, 1 to 3, 1 or 2, 2 to 4, 2 or 3, 3 or 4, 1, 2, 3, 4, 5, or 6 linked nucleosides.
[0270] The 5′-wing may include, e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 bridged nucleosides. The 5′-wing may include, e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 constrained ethyl (cEt) nucleosides. The 5′-wing may include, e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 LNA nucleosides. Each nucleoside of the 5′-wing may be, e.g., a bridged nucleoside. Each nucleoside of the 5′-wing may be, e.g., a constrained ethyl (cEt) nucleoside. Each nucleoside of the 5′-wing may be, e.g., a LNA nucleoside.
[0271] The 3′-wing may include, e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 bridged nucleosides. The 3′-wing may include, e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 constrained ethyl (cEt) nucleosides. The 3′-wing may include, e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 LNA nucleosides. Each nucleoside of the 3′-wing may be, e.g., a bridged nucleoside. Each nucleoside of the 3′-wing may be, e.g., a constrained ethyl (cEt) nucleoside. Each nucleoside of the 3′-wing may be, e.g., a LNA nucleoside.
[0272] The 5′-wing may include, e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 non-bicyclic modified nucleosides. The 5′-wing may include, e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 2′-substituted nucleosides. The 5′-wing may include, e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 2′-MOE nucleosides. The 5′-wing may include, e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 2′-OMe nucleosides. Each nucleoside of the 5′-wing may be, e.g., a non-bicyclic modified nucleoside. Each nucleoside of the 5′-wing may be, e.g., a 2′-substituted nucleoside. Each nucleoside of the 5′-wing may be, e.g., a 2′-MOE nucleoside. Each nucleoside of the 5′-wing may be, e.g., a 2′-OMe nucleoside.
[0273] The 3′-wing may include, e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 non-bicyclic modified nucleosides. The 3′-wing may include, e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 2′-substituted nucleosides. The 3′-wing may include, e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 2′-MOE nucleosides. The 3′-wing may include, e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 2′-OMe nucleosides. Each nucleoside of the 3′-wing may be, e.g., a non-bicyclic modified nucleoside. Each nucleoside of the 3′-wing may be, e.g., a 2′-substituted nucleoside. Each nucleoside of the 3′-wing may be, e.g., a 2′-MOE nucleoside. Each nucleoside of the 3′-wing may be, e.g., a 2′-OMe nucleoside.
[0274] The gap may consist of, e.g., 6 to 20 linked nucleosides. The gap may consist of, e.g., 6 to 15, 6 to 12, 6 to 10, 6 to 9, 6 to 8, 6 or 7, 7 to 10, 7 to 9, 7 or 8, 8 to 10, 8 or 9, 6, 7, 8, 9, 10, 11, or 12 linked nucleosides. Each nucleoside of the gap may be, e.g., a 2′-deoxynucleoside. The gap may include, e.g., one or more modified nucleosides. Each nucleoside of the gap may be, e.g., a 2′-deoxynucleoside or may be, e.g., a modified nucleoside that is “DNA-like.” In such embodiments, “DNA-like” means that the nucleoside has similar characteristics to DNA, such that a duplex including the gapmer and an RNA molecule is capable of activating RNase H. For example, under certain conditions, 2′-(ara)-F may support RNase H activation, and thus is DNA-like. In certain embodiments, one or more nucleosides of the gap is not a 2′-deoxynucleoside and is not DNA-like. In certain such embodiments, the gapmer nonetheless supports RNase H activation (e.g., by virtue of the number or placement of the non-DNA nucleosides).
[0275] In certain embodiments, gaps include a stretch of unmodified 2′-deoxynucleoside interrupted by one or more modified nucleosides, thus resulting in three sub-regions (two stretches of one or more 2′-deoxynucleosides and a stretch of one or more interrupting modified nucleosides). In certain embodiments, no stretch of unmodified 2′-deoxynucleosides is longer than 5, 6, or 7 nucleosides. In certain embodiments, such short stretches is achieved by using short gap regions. In certain embodiments, short stretches are achieved by interrupting a longer gap region.
[0276] The gap may include, e.g., one or more modified nucleosides. The gap may include, e.g., one or more modified nucleosides selected from among cEt, FHNA, LNA, and 2-thio-thymidine. The gap may include, e.g., one modified nucleoside. The gap may include, e.g., a 5′-substituted sugar moiety selected from the group consisting of 5′-Me and 5′-(R)-Me. The gap may include, e.g., two modified nucleosides. The gap may include, e.g., three modified nucleosides. The gap may include, e.g., four modified nucleosides. The gap may include, e.g., two or more modified nucleosides and each modified nucleoside is the same. The gap may include, e.g., two or more modified nucleosides and each modified nucleoside is different.
[0277] The gap may include, e.g., one or more modified internucleoside linkages. The gap may include, e.g., one or more methyl phosphonate linkages. In certain embodiments the gap may include, e.g., two or more modified internucleoside linkages. The gap may include, e.g., one or more modified linkages and one or more modified nucleosides. The gap may include, e.g., one modified linkage and one modified nucleoside. The gap may include, e.g., two modified linkages and two or more modified nucleosides.
[0278] An antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) may include one or more mismatches. For example, the mismatch may be specifically positioned within a gapmer, headmer, or tailmer. The mismatch may be, e.g., at position 1, 2, 3, 4, 5, 6, 7, or 8 (e.g., at position 1, 2, 3, or 4) from the 3′-end of the gap region. Alternatively, or additionally, the mismatch may be, e.g., at position 9, 8, 7, 6, 5, 4, 3, 2, or 1 (e.g., at position 4, 3, 2, or 1) from the 3′-end of the gap region. In some embodiments, the 5′ wing and/or 3′wing do not include mismatches.
[0279] An antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) may be a morpholino.
[0280] An antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) may include a total of X to Y interlinked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number nucleosides in the range. In these embodiments, X and Y are each independently selected from the group consisting of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X<Y. For example, an oligonucleotide of the invention may include a total of 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 interlinked nucleosides.
[0281] In some embodiments, an antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) includes at least one modified internucleoside linkage. A modified internucleoside linkage may be, e.g., a phosphorothioate internucleoside linkage (e.g., a phosphorothioate diester or phosphorothioate triester).
[0282] In some embodiments, an antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) includes at least one stereochemically enriched phosphorothioate-based internucleoside linkage. In some embodiments, an antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) includes a pattern of stereochemically enriched phosphorothioate internucleoside linkages described herein (e.g., a 5′-R.sub.PS.sub.PS.sub.P-3′). These patterns may enhance target miR-147b cleavage by RNase H relative to a stereorandom corresponding oligonucleotide. In some embodiments, inclusion and/or location of particular stereochemically enriched linkages within an oligonucleotide may alter the cleavage pattern of a target nucleic acid, when such an oligonucleotide is utilized for cleaving the nucleic acid. For example, a pattern of internucleoside linkage P-stereogenic centers may increase cleavage efficiency of a target nucleic acid. A pattern of internucleoside linkage P-stereogenic centers may provide new cleavage sites in a target nucleic acid. A pattern of internucleoside linkage P-stereogenic centers may reduce the number of cleavage sites, for example, by blocking certain existing cleavage sites. Moreover, in some embodiments, a pattern of internucleoside linkage P-stereogenic centers may facilitate cleavage at only one site within the target sequence that is complementary to an oligonucleotide utilized for the cleavage. Cleavage efficiency may be increased by selecting a pattern of internucleoside linkage P-stereogenic centers that reduces the number of cleavage sites in a target nucleic acid.
[0283] Purity of an oligonucleotide may be expressed as the percentage of oligonucleotide molecules that are of the same oligonucleotide type within an oligonucleotide composition. At least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% of the oligonucleotides may be, e.g., of the same oligonucleotide type.
[0284] An oligonucleotide may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more stereochemically enriched internucleoside linkages. An oligonucleotide may include at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% stereochemically enriched internucleoside linkages. Exemplary stereochemically enriched internucleoside linkages are described herein. An oligonucleotide may include at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% stereochemically enriched internucleoside linkages in the S.sub.P configuration.
[0285] A stereochemically enriched internucleoside linkage may be, e.g., a stereochemically enriched phosphorothioate internucleoside linkage. A provided oligonucleotide may comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% stereochemically enriched phosphorothioate internucleoside linkages. All internucleoside linkages may be, e.g., stereochemically enriched phosphorothioate internucleoside linkages. In some embodiments, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% stereochemically enriched phosphorothioate internucleoside linkages have the S.sub.P stereochemical configuration. In some embodiments, less than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% stereochemically enriched phosphorothioate internucleoside linkages have the S.sub.P stereochemical configuration. In some embodiments, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% stereochemically enriched phosphorothioate internucleoside linkages have the R.sub.P stereochemical configuration. In some embodiments, less than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% stereochemically enriched phosphorothioate internucleoside linkages have the R.sub.P stereochemical configuration.
[0286] An oligonucleotide may have, e.g., only one R.sub.P stereochemically enriched phosphorothioate internucleoside linkage. An oligonucleotide may have, e.g., multiple R.sub.P stereochemically enriched phosphorothioate internucleoside linkages, where all internucleoside linkages are stereochemically enriched phosphorothioate internucleoside linkages. A stereochemically enriched phosphorothioate internucleoside linkage may be, e.g., a stereochemically enriched phosphorothioate diester linkage. In some embodiments, each stereochemically enriched phosphorothioate internucleoside linkage is independently a stereochemically enriched phosphorothioate diester linkage. In some embodiments, each internucleoside linkage is independently a stereochemically enriched phosphorothioate diester linkage. In some embodiments, each internucleoside linkage is independently a stereochemically enriched phosphorothioate diester linkage, and only one is R.sub.P.
[0287] The gap region may include, e.g., a stereochemically enriched internucleoside linkage. The gap region may include, e.g., stereochemically enriched phosphorothioate internucleoside linkages. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is (S.sub.P).sub.mR.sub.P or R.sub.P(S.sub.P).sub.m, where m is 2, 3, 4, 5, 6, 7, or 8. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is (S.sub.P).sub.mR.sub.P or R.sub.P(S.sub.P).sub.m, where m is 2, 3, 4, 5, 6, 7, or 8. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is (S.sub.P).sub.mR.sub.P, where m is 2, 3, 4, 5, 6, 7, or 8. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is R.sub.P(S.sub.P).sub.m, where m is 2, 3, 4, 5, 6, 7, or 8. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is (S.sub.P).sub.mR.sub.P or R.sub.P(S.sub.P).sub.m, where m is 2, 3, 4, 5, 6, 7, or 8. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is a motif including at least 33% of internucleoside linkages with the S.sub.P stereochemical identify. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is a motif including at least 50% of internucleoside linkages with the S.sub.P stereochemical identify. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is a motif including at least 66% of internucleoside linkages with the S.sub.P stereochemical identify. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is a repeating triplet motif selected from R.sub.PR.sub.PS.sub.P and S.sub.PS.sub.PR.sub.P. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is a repeating R.sub.PR.sub.PS.sub.P. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is a repeating S.sub.PS.sub.PR.sub.P.
[0288] An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (S.sub.P).sub.mR.sub.P or R.sub.P(S.sub.P).sub.m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including R.sub.P(S.sub.P).sub.m. An oligonucleotide may include a pattern of P-stereogenic centers in the gap region including (S.sub.P).sub.mR.sub.P. In some embodiments, m is 2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including R.sub.P(S.sub.P).sub.2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (S.sub.P).sub.2R.sub.P(S.sub.P).sub.2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (R.sub.P).sub.2R.sub.P(S.sub.P).sub.2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including R.sub.PS.sub.PR.sub.P(S.sub.P).sub.2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including S.sub.PR.sub.PR.sub.P(S.sub.P).sub.2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (S.sub.P).sub.2R.sub.P.
[0289] An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (S.sub.P).sub.mR.sub.P or R.sub.P(S.sub.P).sub.m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including R.sub.P(S.sub.P).sub.m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (S.sub.P).sub.mR.sub.P. In some embodiments, m is 2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including R.sub.P(S.sub.P).sub.2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (S.sub.P).sub.2R.sub.P(S.sub.P).sub.2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (R.sub.P).sub.2R.sub.P(S.sub.P).sub.2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including R.sub.PS.sub.PR.sub.P(S.sub.P).sub.2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including S.sub.PR.sub.PR.sub.P(S.sub.P).sub.2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (S.sub.P).sub.2R.sub.P.
[0290] In the embodiments of internucleoside P-stereogenic center patterns, m is 2, 3, 4, 5, 6, 7, or 8, unless specified otherwise. In some embodiments of internucleoside P-stereogenic center patterns, m is 3, 4, 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 4, 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 2. In some embodiments of internucleoside P-stereogenic center patterns, m is 3. In some embodiments of internucleoside P-stereogenic center patterns, m is 4. In some embodiments of internucleoside P-stereogenic center patterns, m is 5. In some embodiments of internucleoside P-stereogenic center patterns, m is 6. In some embodiments of internucleoside P-stereogenic center patterns, m is 7. In some embodiments of internucleoside P-stereogenic center patterns, m is 8.
[0291] A repeating pattern may be, e.g., (S.sub.P).sub.m(R.sub.P).sub.n, where n is independently 1, 2, 3, 4, 5, 6, 7, or 8, and m is independently as described herein. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (S.sub.P).sub.m(R.sub.P).sub.n. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (S.sub.p).sub.m(R.sub.P).sub.n. A repeating pattern may be, e.g., (R.sub.P).sub.n(S.sub.P).sub.m, where n is independently 1, 2, 3, 4, 5, 6, 7, or 8, and m is independently as described herein. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (R.sub.P).sub.n(S.sub.P).sub.m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (R.sub.P).sub.n(S.sub.P).sub.m. In some embodiments, (R.sub.P).sub.n(S.sub.P).sub.m is (R.sub.P)(S.sub.P).sub.2. In some embodiments, (S.sub.P).sub.n(R.sub.P).sub.m is (S.sub.P).sub.2(R.sub.P).
[0292] A repeating pattern may be, e.g., (S.sub.P).sub.m(R.sub.P).sub.n(S.sub.P).sub.t, where each of n and t is independently 1, 2, 3, 4, 5, 6, 7, or 8, and m is as described herein. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (S.sub.P).sub.m(R.sub.P).sub.n(S.sub.P).sub.t. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (S.sub.P).sub.m(R.sub.P).sub.n(S.sub.P).sub.t. A repeating pattern may be, e.g., (S.sub.P).sub.t(R.sub.P).sub.n(S.sub.P).sub.m, where each of n and t is independently 1, 2, 3, 4, 5, 6, 7, or 8, and m is as described herein. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (S.sub.P).sub.t(R.sub.P).sub.n(S.sub.P).sub.m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (S.sub.P).sub.t(R.sub.P).sub.n(S.sub.P).sub.m.
[0293] A repeating pattern is (Np).sub.t(R.sub.P).sub.n(S.sub.P).sub.m, where each of n and t is independently 1, 2, 3, 4, 5, 6, 7, or 8, Np is independently R.sub.P or S.sub.P, and m is as described herein. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (Np).sub.t(R.sub.P).sub.n(S.sub.P).sub.m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (Np).sub.t(R.sub.p).sub.n(S.sub.P).sub.m. A repeating pattern may be, e.g., (Np).sub.t(R.sub.P).sub.n(S.sub.P).sub.m, where each of n and t is independently 1, 2, 3, 4, 5, 6, 7, or 8, Np is independently R.sub.P or S.sub.P, and m is as described herein. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (Np).sub.t(R.sub.P).sub.n(S.sub.P).sub.m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (Np).sub.t(R.sub.P).sub.n(S.sub.P).sub.m. In some embodiments, Np is R.sub.P. In some embodiments, Np is S.sub.P. All Np may be, e.g., same. All Np may be, e.g., S.sub.P. At least one Np may be, e.g., different from another Np. In some embodiments, t is 2.
[0294] In the embodiments of internucleoside P-stereogenic center patterns, n is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, n is 2, 3, 4, 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, n is 3, 4, 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, n is 4, 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, n is 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, n is 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, n is 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, n is 1. In some embodiments of internucleoside P-stereogenic center patterns, n is 2. In some embodiments of internucleoside P-stereogenic center patterns, n is 3. In some embodiments of internucleoside P-stereogenic center patterns, n is 4. In some embodiments of internucleoside P-stereogenic center patterns, n is 5. In some embodiments of internucleoside P-stereogenic center patterns, n is 6. In some embodiments of internucleoside P-stereogenic center patterns, n is 7. In some embodiments of internucleoside P-stereogenic center patterns, n is 8.
[0295] In the embodiments of internucleoside P-stereogenic center patterns, t is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, t is 2, 3, 4, 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, t is 3, 4, 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, t is 4, 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, t is 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, t is 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, t is 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, t is 1. In some embodiments of internucleoside P-stereogenic center patterns, t is 2. In some embodiments of internucleoside P-stereogenic center patterns, t is 3. In some embodiments of internucleoside P-stereogenic center patterns, t is 4. In some embodiments of internucleoside P-stereogenic center patterns, t is 5. In some embodiments of internucleoside P-stereogenic center patterns, t is 6. In some embodiments of internucleoside P-stereogenic center patterns, t is 7. In some embodiments of internucleoside P-stereogenic center patterns, t is 8.
[0296] At least one of m and t may be, e.g., greater than 2. At least one of m and t may be, e.g., greater than 3. At least one of m and t may be, e.g., greater than 4. At least one of m and t may be, e.g., greater than 5. At least one of m and t may be, e.g., greater than 6. At least one of m and t may be, e.g., greater than 7. In some embodiments, each of m and t is greater than 2. In some embodiments, each of m and t is greater than 3. In some embodiments, each of m and t is greater than 4. In some embodiments, each of m and t is greater than 5. In some embodiments, each of m and t is greater than 6. In some embodiments, each of m and t is greater than 7.
[0297] In some embodiments of internucleoside P-stereogenic center patterns, n is 1, and at least one of m and t is greater than 1. In some embodiments of internucleoside P-stereogenic center patterns, n is 1 and each of m and t is independent greater than 1. In some embodiments of internucleoside P-stereogenic center patterns, m>n and t>n. In some embodiments, (S.sub.P).sub.m(R.sub.P).sub.n(S.sub.P).sub.t is (S.sub.P).sub.2R.sub.P(S.sub.P).sub.2. In some embodiments, (S.sub.P).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is (S.sub.P).sub.2R.sub.P(S.sub.P).sub.2. In some embodiments, (S.sub.P).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is S.sub.PR.sub.P(S.sub.P).sub.2. In some embodiments, (Np).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is (Np).sub.t(R.sub.P).sub.n(S.sub.P).sub.m. In some embodiments, (Np).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is (Np).sub.2R.sub.P(S.sub.P).sub.m. In some embodiments, (Np).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is (R.sub.P).sub.2R.sub.P(S.sub.P).sub.m. In some embodiments, (Np).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is (S.sub.P).sub.2R.sub.P(S.sub.P).sub.m. In some embodiments, (Np).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is R.sub.PS.sub.PR.sub.P(S.sub.P).sub.m. In some embodiments, (Np).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is S.sub.PR.sub.PR.sub.P(S.sub.P).sub.m.
[0298] In some embodiments, (S.sub.P).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is S.sub.PR.sub.PS.sub.PS.sub.P. In some embodiments, (S.sub.P).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is (S.sub.P).sub.2R.sub.P(S.sub.P).sub.2. In some embodiments, (S.sub.P).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is (S.sub.P).sub.3R.sub.P(S.sub.P).sub.3. In some embodiments, (S.sub.P).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is (S.sub.P).sub.4R.sub.P(S.sub.P).sub.4. In some embodiments, (S.sub.P).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is (S.sub.P).sub.tR.sub.P(S.sub.P).sub.5. In some embodiments, (S.sub.P).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is S.sub.PR.sub.P(S.sub.P).sub.5. In some embodiments, (S.sub.P).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is (S.sub.P).sub.2R.sub.P(S.sub.P).sub.5. In some embodiments, (S.sub.P).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is (S.sub.P).sub.3R.sub.P(S.sub.P).sub.5. In some embodiments, (S.sub.P).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is (S.sub.P).sub.4R.sub.P(S.sub.P).sub.5. In some embodiments, (S.sub.P).sub.t(R.sub.P).sub.n(S.sub.P).sub.m is (S.sub.P).sub.5R.sub.P(S.sub.P).sub.5.
[0299] In some embodiments, (S.sub.P).sub.m(R.sub.P).sub.n(S.sub.P).sub.t is (S.sub.P).sub.2R.sub.P(S.sub.P).sub.2. In some embodiments, (S.sub.P).sub.m(R.sub.P).sub.n(S.sub.P).sub.t is (S.sub.P).sub.3R.sub.P(S.sub.P).sub.3. In some embodiments, (S.sub.P).sub.m(R.sub.P).sub.n(S.sub.P).sub.t is (S.sub.P).sub.4R.sub.P(S.sub.P).sub.4. In some embodiments, (S.sub.P).sub.m(R.sub.P).sub.n(S.sub.P).sub.t is (S.sub.P).sub.mR.sub.P(S.sub.P).sub.5. In some embodiments, (S.sub.P).sub.m(R.sub.P).sub.n(S.sub.P).sub.t is (S.sub.P).sub.2R.sub.P(S.sub.P).sub.5. In some embodiments, (S.sub.P).sub.m(R.sub.P).sub.n(S.sub.P).sub.t is (S.sub.P).sub.3R.sub.P(S.sub.P).sub.5. In some embodiments, (S.sub.P).sub.m(R.sub.P).sub.n(S.sub.P).sub.t is (S.sub.P).sub.4R.sub.P(S.sub.P).sub.5. In some embodiments, (S.sub.P).sub.m(R.sub.P).sub.n(S.sub.P).sub.t is (S.sub.P).sub.5R.sub.P(S.sub.P).sub.5.
[0300] The gap region may include, e.g., at least one R.sub.P internucleoside linkage. The gap region may include, e.g., at least one R.sub.P phosphorothioate internucleoside linkage. The gap region may include, e.g., at least two R.sub.P internucleoside linkages. The gap region may include, e.g., at least two R.sub.P phosphorothioate internucleoside linkages. The gap region may include, e.g., at least three R.sub.P internucleoside linkages. The gap region may include, e.g., at least three R.sub.P phosphorothioate internucleoside linkages. The gap region may include, e.g., at least 4, 5, 6, 7, 8, 9, or 10 R.sub.P internucleoside linkages. The gap region may include, e.g., at least 4, 5, 6, 7, 8, 9, or 10 R.sub.P phosphorothioate internucleoside linkages.
[0301] A gapmer may include a wing-gap-wing motif that is a 5-10-5 motif, where the nucleosides in each wing region are Z-MOE-modified nucleosides. A wing-gap-wing motif of a gapmer may be, e.g., a 5-10-5 motif where the nucleosides in the gap region are 2′-deoxyribonucleosides. A wing-gap-wing motif of a gapmer may be, e.g., a 5-10-5 motif, where all internucleoside linkages are phosphorothioate internucleoside linkages. A wing-gap-wing motif of a gapmer may be, e.g., a 5-10-5 motif, where all internucleoside linkages are stereochemically enriched phosphorothioate internucleoside linkages. A wing-gap-wing motif of a gapmer may be, e.g., a 5-10-5 motif, where the nucleosides in each wing region are Z-MOE-modified nucleosides, the nucleosides in the gap region are 2′-deoxyribonucleosides, and all internucleoside linkages are stereochemically enriched phosphorothioate internucleoside linkages.
[0302] In certain embodiments, a wing-gap-wing motif is a 5-10-5 motif where the residues at each wing region are not Z-MOE-modified residues. In certain embodiments, a wing-gap-wing motif is a 5-10-5 motif where the residues in the gap region are Z-deoxyribonucleotide residues. In certain embodiments, a wing-gap-wing motif is a 5-10-5 motif, where all internucleosidic linkages are phosphorothioate internucleosidic linkages. In certain embodiments, a wing-gap-wing motif is a 5-10-5 motif, where all internucleoside linkages are stereochemically enriched phosphorothioate internucleoside linkages. In certain embodiments, a wing-gap-wing motif is a 5-10-5 motif where the residues at each wing region are not Z-MOE-modified residues, the residues in the gap region are Z-deoxyribonucleotide, and all internucleoside linkages are stereochemically enriched phosphorothioate internucleoside linkages.
[0303] An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being a P-stereogenic linkage (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least two of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages are stereogenic. At least three of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least four of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least five of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least six of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). At least seven of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). At least eight of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). At least nine of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). One of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). Two of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Three of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). Four of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Five of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). Six of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Seven of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). Eight of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Nine of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). Ten of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester).
[0304] An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least two of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). At least three of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least four of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). At least five of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). At least six of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least seven of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). One of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). Two of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). Three of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Four of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). Five of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). Six of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Seven of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester). Eight of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester).
[0305] An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester), and at least one internucleoside linkage being non-stereogenic. An oligonucleotide may include a region in which at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotiester), and at least one internucleoside linkage being non-stereogenic. At least two internucleoside linkages may be, e.g., non-stereogenic. At least three internucleoside linkages may be, e.g., non-stereogenic. At least four internucleoside linkages may be, e.g., non-stereogenic. At least five internucleoside linkages may be, e.g., non-stereogenic. At least six internucleoside linkages may be, e.g., non-stereogenic. At least seven internucleoside linkages may be, e.g., non-stereogenic. At least eight internucleoside linkages may be, e.g., non-stereogenic. At least nine internucleoside linkages may be, e.g., non-stereogenic. At least 10 internucleoside linkages may be, e.g., non-stereogenic. At least 11 internucleoside linkages may be, e.g., non-stereogenic. At least 12 internucleoside linkages may be, e.g., non-stereogenic. At least 13 internucleoside linkages may be, e.g., non-stereogenic. At least 14 internucleoside linkages may be, e.g., non-stereogenic. At least 15 internucleoside linkages may be, e.g., non-stereogenic. At least 16 internucleoside linkages may be, e.g., non-stereogenic. At least 17 internucleoside linkages may be, e.g., non-stereogenic. At least 18 internucleoside linkages may be, e.g., non-stereogenic. At least 19 internucleoside linkages may be, e.g., non-stereogenic. At least 20 internucleoside linkages may be, e.g., non-stereogenic. In some embodiments, one internucleoside linkage is non-stereogenic. In some embodiments, two internucleoside linkages are non-stereogenic. In some embodiments, three internucleoside linkages are non-stereogenic. In some embodiments, four internucleoside linkages are non-stereogenic. In some embodiments, five internucleoside linkages are non-stereogenic. In some embodiments, six internucleoside linkages are non-stereogenic. In some embodiments, seven internucleoside linkages are non-stereogenic. In some embodiments, eight internucleoside linkages are non-stereogenic. In some embodiments, nine internucleoside linkages are non-stereogenic. In some embodiments, 10 internucleoside linkages are non-stereogenic. In some embodiments, 11 internucleoside linkages are non-stereogenic. In some embodiments, 12 internucleoside linkages are non-stereogenic. In some embodiments, 13 internucleoside linkages are non-stereogenic. In some embodiments, 14 internucleoside linkages are non-stereogenic. In some embodiments, 15 internucleoside linkages are non-stereogenic. In some embodiments, 16 internucleoside linkages are non-stereogenic. In some embodiments, 17 internucleoside linkages are non-stereogenic. In some embodiments, 18 internucleoside linkages are non-stereogenic. In some embodiments, 19 internucleoside linkages are non-stereogenic. In some embodiments, 20 internucleoside linkages are non-stereogenic. An oligonucleotide may include a region in which all internucleoside linkages, except at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages which is P-stereogenic, are non-stereogenic.
[0306] An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least one internucleoside linkage being phosphate phosphodiester. An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least one internucleoside linkage being phosphate phosphodiester. At least two internucleoside linkages may be, e.g., phosphate phosphodiesters. At least three internucleoside linkages may be, e.g., phosphate phosphodiesters. At least four internucleoside linkages may be, e.g., phosphate phosphodiesters. At least five internucleoside linkages may be, e.g., phosphate phosphodiesters. At least six internucleoside linkages may be, e.g., phosphate phosphodiesters. At least seven internucleoside linkages may be, e.g., phosphate phosphodiesters. At least eight internucleoside linkages may be, e.g., phosphate phosphodiesters. At least nine internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 10 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 11 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 12 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 13 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 14 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 15 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 16 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 17 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 18 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 19 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 20 internucleoside linkages may be, e.g., phosphate phosphodiesters. In some embodiments, one internucleoside linkage is phosphate phosphodiesters. In some embodiments, two internucleoside linkages are phosphate phosphodiesters.
[0307] In some embodiments, three internucleoside linkages are phosphate phosphodiesters. In some embodiments, four internucleoside linkages are phosphate phosphodiesters. In some embodiments, five internucleoside linkages are phosphate phosphodiesters. In some embodiments, six internucleoside linkages are phosphate phosphodiesters. In some embodiments, seven internucleoside linkages are phosphate phosphodiesters. In some embodiments, eight internucleoside linkages are phosphate phosphodiesters. In some embodiments, nine internucleoside linkages are phosphate phosphodiesters.
[0308] In some embodiments, 10 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 11 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 12 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 13 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 14 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 15 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 16 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 17 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 18 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 19 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 20 internucleoside linkages are phosphate phosphodiesters. An oligonucleotide may include a region with all internucleoside linkages, except at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, being phosphate phosphodiesters.
[0309] An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least 10% of all internucleoside linkages in the region being non-stereogenic. An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least 10% of all internucleoside linkages in the region being non-stereogenic. At least 20% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 30% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 40% of all the internucleoside linkages in the region may be, e.g., non-stereogenic.
[0310] At least 50% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 60% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 70% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 80% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 90% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 50% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. A non-stereogenic internucleoside linkage may be, e.g., a phosphate phosphodiester. In some embodiments, each non-stereogenic internucleoside linkage is a phosphate phosphodiester.
[0311] The first internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The first internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The second internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The second internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The third internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The third internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The fifth internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The fifth internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The seventh internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The seventh internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The eighth internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The eighth internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The ninth internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The ninth internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The eighteenth internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The eighteenth internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The nineteenth internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The nineteenth internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The twentieth internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The twentieth internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage.
[0312] The region may have a length of, e.g., at least 21 bases. The region may have a length of, e.g., 21 bases.
[0313] In some embodiments, each stereochemically enriched internucleoside linkage in an oligonucleotide is a phosphorothioate phosphodiester.
[0314] An oligonucleotide may have, e.g., at least 25% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 30% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 35% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 40% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 45% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 50% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 55% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 60% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 65% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 70% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 75% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 80% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 85% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 90% of its internucleoside linkages in S.sub.P configuration.
[0315] An oligonucleotide may include at least two internucleoside linkages having different stereochemical configuration and/or different P-modifications relative to one another. The oligonucleotide may have a structure represented by the following formula:
[S.sup.B.sub.n1R.sup.B.sub.n2S.sup.B.sub.n3R.sup.B.sub.n4 . . . S.sup.B.sub.nxR.sup.B.sub.ny]
[0316] where:
[0317] each R.sup.B independently represents a block of nucleotide units having the R.sub.P configuration at the internucleoside linkage phosphorus atom;
[0318] each S.sup.B independently represents a block of nucleotide units having the S.sub.P configuration at the internucleoside linkage phosphorus atom;
[0319] each of n1 to ny is zero or an integer, provided that at least one odd n and at least one even n must be non-zero so that the oligonucleotide includes at least two internucleoside linkages with different stereochemistry relative to one another; and
[0320] where the sum of n1 to ny is between 2 and 200.
[0321] In some embodiments, the sum of n1 to ny is between a lower limit selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and more, and the upper limit selected from the group consisting of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200, the upper limit being greater than the lower limit. In some of these embodiments, each n has the same value. In some embodiments, each even n has the same value as each other even n. In some embodiments, each odd n has the same value each other odd n. At least two even n's may have, e.g., different values from one another. At least two odd n's may have, e.g., different values from one another.
[0322] At least two adjacent n's may be, e.g., equal to one another, so that an oligonucleotide includes adjacent blocks of S.sub.P linkages and R.sub.P linkages of equal lengths. In some embodiments, an oligonucleotide includes repeating blocks of S.sub.P and R.sub.P linkages of equal lengths. In some embodiments, an oligonucleotide includes repeating blocks of S.sub.P and R.sub.P linkages, where at least two such blocks are of different lengths from one another. In some such embodiments, each S.sub.P block is of the same length and is of a different length from each R.sub.P block, where all R.sub.P blocks may optionally be of the same length as one another.
[0323] At least two skip-adjacent n's may be, e.g., equal to one another, so that a provided oligonucleotide includes at least two blocks of internucleoside linkages of a first stereochemistry that are equal in length to one another and are separated by a separating block of internucleoside linkages of the opposite stereochemistry, where the separating block may be of the same length or a different length from the blocks of first stereochemistry.
[0324] In some embodiments, n's associated with linkage blocks at the ends of an oligonucleotide are of the same length. In some embodiments, an oligonucleotide has terminal blocks of the same linkage stereochemistry. In some such embodiments, the terminal blocks are separated from one another by a middle block of the opposite linkage stereochemistry.
[0325] An oligonucleotide of formula [S.sup.B.sub.n1R.sup.B.sub.n2S.sup.B.sub.n3R.sup.B.sub.n4 . . . S.sup.B.sub.nxR.sup.B.sub.ny] may be, e.g., a stereoblockmer. An oligonucleotide of formula [S.sup.B.sub.n1R.sup.B.sub.n2S.sup.B.sub.n3R.sup.B.sub.n4 . . . S.sup.B.sub.nxR.sup.B.sub.ny] may be, e.g., a stereoskipmer. An oligonucleotide of formula [S.sup.B.sub.n1R.sup.B.sub.n2S.sup.B.sub.n3R.sup.B.sub.n4 . . . S.sup.B.sub.nxR.sup.B.sub.ny] may be, e.g., a stereoaltmer. An oligonucleotide of formula [S.sup.B.sub.n1R.sup.B.sub.n2S.sup.B.sub.n3R.sup.B.sub.n4 . . . S.sup.B.sub.nxR.sup.B.sub.ny] may be, e.g., a gapmer.
[0326] An oligonucleotide of formula [S.sup.B.sub.n1R.sup.B.sub.n2S.sup.B.sub.n3R.sup.B.sub.n4 . . . S.sup.B.sub.nxR.sup.B.sub.ny] may be, e.g., of any of the above described patterns and may further include, e.g., patterns of P-modifications. For instance, an oligonucleotide of formula [S.sup.B.sub.n1R.sup.Bn.sub.2S.sup.B.sub.n3R.sup.B.sub.n4 . . . S.sup.B.sub.nxR.sup.B.sub.ny] may be, e.g., a stereoskipmer and a P-modification skipmer. An oligonucleotide of formula [S.sup.B.sub.n1R.sup.B.sub.n2S.sup.B.sub.n3R.sup.B.sub.n4 . . . S.sup.B.sub.nxR.sup.B.sub.ny] may be, e.g., a stereoblockmer and a P-modification altmer. An oligonucleotide of formula [S.sup.B.sub.n1R.sup.B.sub.n2S.sup.B.sub.n3R.sup.B.sub.n4 . . . S.sup.B.sub.nxR.sup.B.sub.ny] may be, e.g., a stereoaltmer and a P-modification blockmer.
[0327] An oligonucleotide may include, e.g., at least one phosphate phosphodiester and at least two consecutive modified internucleoside linkages. An oligonucleotide may include, e.g., at least one phosphate phosphodiester and at least two consecutive phosphorothioate triesters.
[0328] An oligonucleotide may be, e.g., a blockmer. An oligonucleotide may be, e.g., a stereoblockmer. An oligonucleotide may be, e.g., a P-modification blockmer. An oligonucleotide may be, e.g., a linkage blockmer.
[0329] An oligonucleotide may be, e.g., an altmer. An oligonucleotide may be, e.g., a stereoaltmer. An oligonucleotide may be, e.g., a P-modification altmer. An oligonucleotide may be, e.g., a linkage altmer.
[0330] An oligonucleotide may be, e.g., a unimer. An oligonucleotide may be, e.g., a stereounimer. An oligonucleotide may be, e.g., a P-modification unimer. An oligonucleotide may be, e.g., a linkage unimer.
[0331] An oligonucleotide may be, e.g., a skipmer.
[0332] In addition to the above, an antisense oligonucleotide may be generated in vivo in a cell (e.g., in a cell of a subject, such as a cancer patient) expressing the oligonucleotide. Thus, for example, an miRNA sponge including multiple sequences that are antisense to a miR-147b sequence can be expressed in a cell. This can be achieved, for example, by introduction of a vector into the cell. Optionally, the vector includes a promoter to direct transcription of the oligonucleotide, which may include, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sequences (e.g., tandem repeated sequences) that are antisense to, and thus soak up and deplete or reduce the miR-147b of the cell. The miRNA binding sites in such miRNA sponges can be either perfectly antisense or contain mismatches, e.g., in the middle positions. Thus, for example, sponges can include bulged nucleotides that are mispaired opposite miRNA positions, e.g., positions 9-12, as is known in the art. These miRNA binding sites can be placed, for example, in the 3′-UTR of a nontoxic gene expressed in the cell. An miRNA sponge can be used to achieve stable inhibition, as well as inducible or tissue-specific inhibition, of a target miRNA, as needed. In various examples, a vector, such as a viral vector, e.g., a lentivirus, an adenovirus, or an adeno-associated virus is used to achieve expression of the miRNA sponge. In other examples, the vector is a plasmid, a cosmid, a phagemid, or a P element. Expression of miRNA sponges can be transient or stable, as is known in the art. See, e.g., Ebert et al., Nat. Methods 4:721-726, 2007; Ebert et al., RNA 16:2043-2050, 2010; Chen et al., Oncol. Rep. 31:1573-1580, 2014, for additional information regarding miRNA sponges.
[0333] Antisense molecules can also be competitive inhibitors of miR-147b with respect to binding to miR-147b targets. Accordingly, such inhibitors hybridize to targets of miR-147b, thus blocking the binding of miR-147b to these targets. In some embodiments, such inhibitors do not facilite the activity of RNAse H. In some embodiments, the affinity of such inhibitors for the targets is sufficient to block the activity of miR-147b, but does not block functional processing of the target (e.g., translation of the target).
[0334] In addition to the antisense molecules described above, the invention includes peptide nucleic acids (PNAs), which are synthetic molecules having certain characteristics analogous to characteristics of typical naturally occurring nucleic acids. In particular, typical naturally occurring nucleic acids include a sugar-phosphate backbone, together with nitrogenous nucleobases. PNA molecules, by contrast, can include a pseudo-peptide backbone including N-(2-aminoethyl) glycine units (rather than, e.g., a sugar-phosphate backbone), together with nitrogenous nucleobases (as described, for example, in U.S. Pat. No. 9,193,758. See also Nielsen et al., Science 254: 1497-1500, 1991). In such PNA molecules, repeating N-(2-aminoethyl)-glycine units can be linked by amide bonds. The PNA pseudo-peptide backbone can be acyclic, achiral, and neutrally charged. Nucleobases can be attached to the PNA pseudo-peptide backbone through methylene carbonyl linkages. Due at least in part to their distinct, hybrid composition, PNAs are resistant to both nucleases and proteases. Accordingly, the invention includes PNA molecules targeted against miR-147b, as described herein.
RNAi
[0335] In another approach, the invention provides a double-stranded oligonucleotide including a passenger strand hybridized to a guide strand having a nucleobase sequence with at least 6 contiguous nucleobases complementary to an equal-length portion within a target miR-147b sequence, which includes mature miR-147b or a precursor (i.e., pri-miR-147b or pre-miR-147b) or fragment thereof. This approach is typically referred to as an RNAi approach, and the corresponding oligonucleotides of the invention are referred to as siRNA. Without wishing to be bound by theory, this approach involves incorporation of the guide strand into an RNA-induced silencing complex (RISC), which can identify and hybridize to a miR-147b sequence in a cell through complementarity of a portion of the guide strand and the target nucleic acid. Upon identification (and hybridization), RISC may either remain on the target nucleic acid thereby sterically blocking translation or cleave the target nucleic acid.
[0336] A double-stranded oligonucleotide of the invention may be an siRNA of the invention. An siRNA of the invention includes a guide strand and a passenger strand that are not covalently linked to each other. Alternatively, a double-stranded oligonucleotide of the invention may be an shRNA. An shRNA of the invention includes a guide strand and a passenger strand that are covalently linked to each other by a linker. Without wishing to be bound by theory, shRNA is processed by the Dicer enzyme to remove the linker and produce a corresponding siRNA. A double-stranded oligonucleotide of the invention (e.g., an siRNA of the invention) includes a nucleobase sequence having at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) contiguous nucleobases complementary to an equal-length portion of a miR-147b target nucleic acid, as described herein.
[0337] Typically, a guide strand includes a seed region, a slicing site, and 5′- and 3′-terminal residues. The seed region—typically, a six nucleotide-long sequence from position 2 to position 7—are involved in the target nucleic acid recognition. The slicing site are the nucleotides (typically at positions 10 and 11) that are complementary to the target nucleosides linked by an internucleoside linkage that undergoes a RISC-mediated cleavage. The 5′- and 3′ terminal residues typically interact with or are blocked by the Ago2 component of RISC.
[0338] A double-stranded oligonucleotide of the invention (e.g., an siRNA of the invention) may include one or more mismatches. For example, the one or more mismatches may be included outside the seed region and the slicing site. Typically, the one or more mismatches may be included among the 5′- and/or 3′-terminal nucleosides.
[0339] A double-stranded oligonucleotide of the invention (e.g., an siRNA of the invention) may include a guide strand having total of X to Y interlinked nucleosides and a passenger strand having a total of X to Y interlinked nucleosides, where each X represents independently the fewest number of nucleosides in the range and each Y represents independently the largest number nucleosides in the range. In these embodiments, X and Y are each independently selected from the group consisting of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X<Y. For example, a strand (e.g., a guide strand or a passenger strand) in a double-stranded oligonucleotide of the invention may include a total of 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 interlinked nucleosides.
Complementarity
[0340] Oligonucleotides of the invention, such as antisense oligonucleotides and siRNA, can optionally be 100% complementary to a target sequence (e.g., miR-147b, or a precursor or fragment thereof, or a target of miR-147b). However, it is possible to introduce mismatch bases without eliminating activity. Accordingly an oligonucleotide of the invention may include (i) a nucleobase sequence having at least 6 contiguous nucleobases complementary to an equal-length portion within a target miR-147b sequence, which includes mature miR-147b or a precursor (i.e., pri-miR-147b or pre-miR-147b) or fragment thereof, and (ii) a nucleobase sequence having a plurality of nucleobases including one or more nucleobases complementary to a target miR-147b sequence (including mature miR-147b or a precursor (i.e., pri-miR-147b or pre-miR-147b) or fragment thereof) and one or more mismatches.
[0341] In some embodiments, oligonucleotides of the invention are complementary to a miR-147b target nucleic acid over the entire length of the oligonucleotide. In other embodiments, oligonucleotides can be variants that are at least 80%, 85%, 90%, 95%, 99%, or 100% complementary to the miR-147b target nucleic acid. In further embodiments, oligonucleotides are at least 80% complementary to the miR-147b target nucleic acid over the entire length of the oligonucleotide and include a nucleobase sequence that is fully complementary to a miR-147b target nucleic acid. The nucleobase sequence that is fully complementary may be, e.g., 6 to 20, 10 to 18, or 18 to 20 contiguous nucleobases in length.
[0342] An oligonucleotide of the invention may include one or more mismatched nucleobases relative to a target nucleic acid. In certain embodiments, an antisense or RNAi activity against the target is reduced by such mismatch, but activity against a non-target is reduced by a greater amount. Thus, the off-target selectivity of the oligonucleotides may be improved.
Oligonucleotide Modifications
[0343] An oligonucleotide of the invention may be a modified oligonucleotide. A modified oligonucleotide of the invention includes one or more modifications, e.g., a nucleobase modification, a sugar modification, an internucleoside linkage modification, or a terminal modification.
[0344] Nucleobase Modifications
[0345] Oligonucleotides of the invention may include one or more modified nucleobases. Unmodified nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines, as well as synthetic and natural nucleobases, e.g., 5-methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl) adenine and guanine, 2-alkyl (e.g., 2-propyl) adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 5-trifluoromethyl uracil, 5-trifluoromethyl cytosine, 7-methyl guanine, 7-methyl adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine. Certain nucleobases are particularly useful for increasing the binding affinity of nucleic acids, e g., 5-substituted pyrimidines; 6-azapyrimidines; N2-, N6-, and/or 06-substituted purines. Nucleic acid duplex stability can be enhanced using, e.g., 5-methylcytosine. Non-limiting examples of nucleobases include: 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (—C≡C—CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deazaadenine, 7-deazaguanine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in Merigan et al., U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442-443.
[0346] Sugar Modifications
[0347] Oligonucleotides of the invention may include one or more sugar modifications in nucleosides. Nucleosides having an unmodified sugar include a sugar moiety that is a furanose ring as found in ribonucleosides and 2′-deoxyribonucleosides.
[0348] Sugars included in the nucleosides of the invention may be non-furanose (or 4′-substituted furanose) rings or ring systems or open systems. Such structures include simple changes relative to the natural furanose ring (e.g., a six-membered ring). Alternative sugars may also include sugar surrogates wherein the furanose ring has been replaced with another ring system such as, e.g., a morpholino or hexitol ring system. Non-limiting examples of sugar moieties useful that may be included in the oligonucleotides of the invention include β-D-ribose, β-D-2′-deoxyribose, substituted sugars (e.g., 2′, 5′, and bis substituted sugars), 4′-S-sugars (e.g., 4′-S-ribose, 4′-S-2′-deoxyribose, and 4′-S-2′-substituted ribose), bicyclic sugar moieties (e.g., the 2′-O—CH.sub.2-4′ or 2′-O—(CH.sub.2).sub.2-4′ bridged ribose derived bicyclic sugars) and sugar surrogates (when the ribose ring has been replaced with a morpholino or a hexitol ring system).
[0349] Typically, a sugar modification may be, e.g., a 2′-substitution, locking, carbocyclization, or unlocking. A 2′-substitution is a replacement of 2′-hydroxyl in ribofuranose with 2′-fluoro, 2′-methoxy, or 2′-(2-methoxy)ethoxy. Alternatively, a 2′-substitution may be a 2′-(ara) substitution, which corresponds to the following structure:
##STR00005##
where B is a nucleobase, and R is a 2′-(ara) substituent (e.g., fluoro). 2′-(ara) substituents are known in the art and can be same as other 2′-substituents described herein. In some embodiments, 2′-(ara) substituent is a 2′-(ara)-F substituent (R is fluoro). A locking modification is an incorporation of a bridge between 4′-carbon atom and 2′-carbon atom of ribofuranose. Nucleosides having a sugar with a locking modification are known in the art as bridged nucleic acids, e.g., locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA), and cEt nucleic acids. The bridged nucleic acids are typically used as affinity enhancing nucleosides.
[0350] Internucleoside Linkage Modifications
[0351] Oligonucleotides of the invention may include one or more internucleoside linkage modifications. The two main classes of internucleoside linkages are defined by the presence or absence of a phosphorus atom. Non-limiting examples of phosphorus-containing internucleoside linkages include phosphodiester linkages, phosphotriester linkages, phosphorothioate diester linkages, phosphorothioate triester linkages, morpholino internucleoside linkages, methylphosphonates, and phosphoramidate. Non-limiting examples of non-phosphorus internucleoside linkages include methylenemethylimino (—CH.sub.2—N(CH3)-O—CH2-), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—), siloxane (—O—Si(H)2-O—), and N,N′-dimethylhydrazine (—CH2-N(CH3)-N(CH3)-). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are known in the art.
[0352] Internucleoside linkages may be stereochemically enriched. For example, phosphorothioate-based internucleoside linkages (e.g., phosphorothioate diester or phosphorothioate triester) may be stereochemically enriched. The stereochemically enriched internucleoside linkages including a stereogenic phosphorus are typically designated S.sub.P or R.sub.P to identify the absolute stereochemistry of the phosphorus atom. Within an oligonucleotide, S.sub.P phosphorothioate indicates the following structure:
##STR00006##
Within an oligonucleotide, R.sub.P phosphorothioate indicates the following structure:
##STR00007##
[0353] The oligonucleotides of the invention may include one or more neutral internucleoside linkages. Non-limiting examples of neutral internucleoside linkages include phosphotriesters, phosphorothioate triesters, methylphosphonates, methylenemethylimino (3′-CH.sub.2—N(CH.sub.3)—O-3′), amide-3 (3′-CH.sub.2—C(═O)—N(H)-3′), amide-4 (3′-CH.sub.2—N(H)—C(═O)-3′), formacetal (3′-O—CH.sub.2—O-3′), and thioformacetal (3′-S—CH.sub.2—O-3′). Further neutral internucleoside linkages include nonionic linkages including siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester, and amides (see, for example, Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65).
[0354] Oligonucleotides may include, e.g., modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. Oligonucleotides may include, e.g., a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides of the present disclosure include a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide may include, e.g., a region that is uniformly linked by phosphorothioate internucleoside linkages. The oligonucleotide may be, e.g., uniformly linked by phosphorothioate internucleoside linkages. Each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. Each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate. The oligonucleotide may include, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphorothioate internucleoside linkages.
[0355] The oligonucleotide may include, e.g., at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least one block of at least 7 consecutive phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least one block of at least 9 consecutive phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least one block of at least 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide. The oligonucleotide may include, e.g., fewer than 15 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 14 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 13 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 12 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 11 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 10 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 9 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 8 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 7 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 6 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 5 phosphorothioate internucleoside linkages. In some embodiments, at least one phosphorothioate internucleoside linkage is a phosphorothioate diester. In some embodiments, each phosphorothioate internucleoside linkage is a phosphorothioate diester. In some embodiments, at least one phosphorothioate internucleoside linkage is a phosphorothioate triester. In some embodiments, each phosphorothioate internucleoside linkage is a phosphorothioate triester. In some embodiments, each internucleoside linkage is independently a phosphodiester (e.g., phosphate phosphodiester or phosphorothioate diester).
[0356] An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (S.sub.P).sub.mR.sub.P or R.sub.P(S.sub.P).sub.m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including R.sub.P(S.sub.P).sub.m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (S.sub.P).sub.mR.sub.P. In some embodiments, m is 2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including R.sub.P(S.sub.P).sub.2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (S.sub.P).sub.2R.sub.P(S.sub.P).sub.2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (R.sub.P).sub.2R.sub.P(S.sub.P).sub.2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including R.sub.PS.sub.PR.sub.P(S.sub.P).sub.2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including S.sub.PR.sub.PR.sub.P(S.sub.P).sub.2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (S.sub.P).sub.2R.sub.P.
[0357] In the embodiments of internucleoside P-stereogenic center patterns, m is 2, 3, 4, 5, 6, 7, or 8, unless specified otherwise. In some embodiments of internucleoside P-stereogenic center patterns, m is 3, 4, 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 4, 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 5, 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 6, 7, or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 2. In some embodiments of internucleoside P-stereogenic center patterns, m is 3. In some embodiments of internucleoside P-stereogenic center patterns, m is 4. In some embodiments of internucleoside P-stereogenic center patterns, m is 5. In some embodiments of internucleoside P-stereogenic center patterns, m is 6. In some embodiments of internucleoside P-stereogenic center patterns, m is 7. In some embodiments of internucleoside P-stereogenic center patterns, m is 8.
[0358] An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being a P-stereogenic linkage (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least two of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages are stereogenic. At least three of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least four of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least five of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least six of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least seven of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least eight of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least nine of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). One of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Two of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Three of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Four of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Five of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Six of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Seven of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Eight of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Nine of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Ten of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester).
[0359] An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages being P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least two of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least three of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least four of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least five of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least six of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least seven of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). One of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Two of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Three of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Four of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Five of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Six of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester).
[0360] Seven of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Eight of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester).
[0361] An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester), and at least one internucleoside linkage being non-stereogenic. An oligonucleotide may include a region in which at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester), and at least one internucleoside linkage being non-stereogenic. At least two internucleoside linkages may be, e.g., non-stereogenic. At least three internucleoside linkages may be, e.g., non-stereogenic. At least four internucleoside linkages may be, e.g., non-stereogenic. At least five internucleoside linkages may be, e.g., non-stereogenic. At least six internucleoside linkages may be, e.g., non-stereogenic. At least seven internucleoside linkages may be, e.g., non-stereogenic. At least eight internucleoside linkages may be, e.g., non-stereogenic. At least nine internucleoside linkages may be, e.g., non-stereogenic. At least 10 internucleoside linkages may be, e.g., non-stereogenic. At least 11 internucleoside linkages may be, e.g., non-stereogenic. At least 12 internucleoside linkages may be, e.g., non-stereogenic. At least 13 internucleoside linkages may be, e.g., non-stereogenic. At least 14 internucleoside linkages may be, e.g., non-stereogenic. At least 15 internucleoside linkages may be, e.g., non-stereogenic. At least 16 internucleoside linkages may be, e.g., non-stereogenic. At least 17 internucleoside linkages may be, e.g., non-stereogenic. At least 18 internucleoside linkages may be, e.g., non-stereogenic. At least 19 internucleoside linkages may be, e.g., non-stereogenic. At least 20 internucleoside linkages may be, e.g., non-stereogenic. In some embodiments, one internucleoside linkage is non-stereogenic. In some embodiments, two internucleoside linkages are non-stereogenic. In some embodiments, three internucleoside linkages are non-stereogenic. In some embodiments, four internucleoside linkages are non-stereogenic. In some embodiments, five internucleoside linkages are non-stereogenic. In some embodiments, six internucleoside linkages are non-stereogenic. In some embodiments, seven internucleoside linkages are non-stereogenic. In some embodiments, eight internucleoside linkages are non-stereogenic. In some embodiments, nine internucleoside linkages are non-stereogenic. In some embodiments, 10 internucleoside linkages are non-stereogenic. In some embodiments, 11 internucleoside linkages are non-stereogenic. In some embodiments, 12 internucleoside linkages are non-stereogenic. In some embodiments, 13 internucleoside linkages are non-stereogenic. In some embodiments, 14 internucleoside linkages are non-stereogenic. In some embodiments, 15 internucleoside linkages are non-stereogenic. In some embodiments, 16 internucleoside linkages are non-stereogenic. In some embodiments, 17 internucleoside linkages are non-stereogenic. In some embodiments, 18 internucleoside linkages are non-stereogenic. In some embodiments, 19 internucleoside linkages are non-stereogenic. In some embodiments, 20 internucleoside linkages are non-stereogenic. An oligonucleotide may include a region in which all internucleoside linkages, except at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages which is P-stereogenic, are non-stereogenic.
[0362] An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least one internucleoside linkage being phosphate phosphodiester. An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least one internucleoside linkage being phosphate phosphodiester. At least two internucleoside linkages may be, e.g., phosphate phosphodiesters. At least three internucleoside linkages may be, e.g., phosphate phosphodiesters. At least four internucleoside linkages may be, e.g., phosphate phosphodiesters. At least five internucleoside linkages may be, e.g., phosphate phosphodiesters. At least six internucleoside linkages may be, e.g., phosphate phosphodiesters. At least seven internucleoside linkages may be, e.g., phosphate phosphodiesters. At least eight internucleoside linkages may be, e.g., phosphate phosphodiesters. At least nine internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 10 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 11 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 12 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 13 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 14 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 15 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 16 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 17 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 18 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 19 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 20 internucleoside linkages may be, e.g., phosphate phosphodiesters. In some embodiments, one internucleoside linkage is phosphate phosphodiesters. In some embodiments, two internucleoside linkages are phosphate phosphodiesters.
[0363] In some embodiments, three internucleoside linkages are phosphate phosphodiesters. In some embodiments, four internucleoside linkages are phosphate phosphodiesters. In some embodiments, five internucleoside linkages are phosphate phosphodiesters. In some embodiments, six internucleoside linkages are phosphate phosphodiesters. In some embodiments, seven internucleoside linkages are phosphate phosphodiesters. In some embodiments, eight internucleoside linkages are phosphate phosphodiesters. In some embodiments, nine internucleoside linkages are phosphate phosphodiesters. In some embodiments, 10 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 11 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 12 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 13 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 14 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 15 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 16 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 17 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 18 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 19 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 20 internucleoside linkages are phosphate phosphodiesters. An oligonucleotide may include a region with all internucleoside linkages, except at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, being phosphate phosphodiesters.
[0364] An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least 10% of all internucleoside linkages in the region being non-stereogenic. An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least 10% of all internucleoside linkages in the region being non-stereogenic. At least 20% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 30% of al the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 40% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 50% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 60% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 70% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 80% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 90% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 50% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. A non-stereogenic internucleoside linkage may be, e.g., a phosphate phosphodiester. In some embodiments, each non-stereogenic internucleoside linkage is a phosphate phosphodiester.
[0365] The first internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The first internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The second internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The second internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The third internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The third internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The fifth internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The fifth internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The seventh internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The seventh internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The eighth internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The eighth internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The ninth internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The ninth internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The eighteenth internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The eighteenth internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The nineteenth internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The nineteenth internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage. The twentieth internucleoside linkage of the region may be, e.g., an S.sub.P internucleoside linkage. The twentieth internucleoside linkage of the region may be, e.g., an R.sub.P internucleoside linkage.
[0366] The region may have a length of, e.g., at least 21 bases. The region may have a length of, e.g., 21 bases.
[0367] In some embodiments, each stereochemically enriched internucleoside linkage in an oligonucleotide is a phosphorothioate phosphodiester.
[0368] An oligonucleotide may have, e.g., at least 25% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 30% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 35% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 40% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 45% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 50% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 55% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 60% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 65% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 70% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 75% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 80% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 85% of its internucleoside linkages in S.sub.P configuration. An oligonucleotide may have, e.g., at least 90% of its internucleoside linkages in S.sub.P configuration.
[0369] An oligonucleotide may include, e.g., at least one phosphate phosphodiester and at least two consecutive modified internucleoside linkages. An oligonucleotide may include, e.g., at least one phosphate phosphodiester and at least two consecutive phosphorothioate triesters.
[0370] An oligonucleotide may be, e.g., a blockmer. An oligonucleotide may be, e.g., a stereoblockmer. An oligonucleotide may be, e.g., a P-modification blockmer. An oligonucleotide may be, e.g., a linkage blockmer.
[0371] An oligonucleotide may be, e.g., an altmer. An oligonucleotide may be, e.g., a stereoaltmer. An oligonucleotide may be, e.g., a P-modification altmer. An oligonucleotide may be, e.g., a linkage altmer.
[0372] An oligonucleotide may be, e.g., a unimer. An oligonucleotide may be, e.g., a stereounimer. An oligonucleotide may be, e.g., a P-modification unimer. An oligonucleotide may be, e.g., a linkage unimer.
[0373] An oligonucleotide may be, e.g., a skipmer.
[0374] Terminal Modifications
[0375] Oligonucleotides of the invention may include a terminal modification. The terminal modification is a 5′-terminal modification or a 3′-terminal modification.
[0376] The 5 end of an oligonucleotide may be, e.g., hydroxyl, a hydrophobic moiety, 5′ cap, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, diphosphrodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer. An unmodified 5′-terminus is hydroxyl or phosphate. An oligonucleotide having a 5′ terminus other than 5′-hydroxyl or 5′-phosphate has a modified 5′ terminus.
[0377] The 3 end of an oligonucleotide may be, e.g., hydroxyl, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, disphorodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer (e.g., polyethylene glycol). An unmodified 3′-terminus is hydroxyl or phosphate. An oligonucleotide having a 3′ terminus other than 3′-hydroxyl or 3′-phosphate has a modified 3′ terminus.
[0378] The terminal modification (e.g., 5′-terminal modification) may be, e.g., a hydrophobic moiety. Advantageously, an oligonucleotide including a hydrophobic moiety may exhibit superior cellular uptake, as compared to an oligonucleotide lacking the hydrophobic moiety. Oligonucleotides including a hydrophobic moiety may therefore be used in compositions that are substantially free of transfecting agents. A hydrophobic moiety is a monovalent group (e.g., a bile acid (e.g., cholic acid, taurocholic acid, deoxycholic acid, oleyl lithocholic acid, or oleoyl cholenic acid), glycolipid, phospholipid, sphingolipid, isoprenoid, vitamin, saturated fatty acid, unsaturated fatty acid, fatty acid ester, triglyceride, pyrene, porphyrine, texaphyrine, adamantine, acridine, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl, t-butydimethylsilyl, t-butyldiphenylsilyl, cyanine dye (e.g., Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen) covalently inked to the terminus of the oligonucleotide backbone (e.g., 5′-terminus). Non-limiting examples of the monovalent group include ergosterol, stigmasterol, β-sitosterol, campesterol, fucosterol, saringosterol, avenasterol, coprostanol, cholesterol, vitamin A, vitamin D, vitamin E, cardiolipin, and carotenoids. The linker connecting the monovalent group to the oligonucleotide may be a linker consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 monomers independently selected from the group consisting of optionally substituted C.sub.1-12 alkylene, optionally substituted C.sub.2-12 heteroalkylene, optionally substituted C.sub.6-10 arylene, optionally substituted C.sub.3-8 cycloalkylene, optionally substituted C.sub.1-9 heteroarylene, optionally substituted C.sub.1-9 heterocyclylene, —O—, —S—S—, and —NR.sup.N—, where each R.sup.N is independently H or optionally substituted C.sub.1-12 alkyl. The linker may be bonded to an oligonucleotide through, e.g., an oxygen atom attached to a 5′-terminal carbon atom, a 3′-terminal carbon atom, a 5′-terminal phosphate or phosphorothioate, a 3′-terminal phosphate or phosphorothioate, or an internucleoside linkage.
Preparation of Oligonucleotides
[0379] Oligonucleotides of the invention may be prepared using techniques and methods known in the art for the oligonucleotide synthesis. For example, oligonucleotides of the invention may be prepared using a phosphoramidite-based synthesis cycle. This synthesis cycle includes the steps of (1) de-blocking a 5-protected nucleotide to produce a 5′-deblocked nucleotide, (2) coupling the 5′-deblocked nucleotide with a 5-protected nucleoside phosphoramidite to produce nucleosides linked through a phosphite, (3) repeating steps (1) and (2) one or more times, as needed, (4) capping the 5′-terminus, and (5) oxidation or sulfurization of internucleoside phosphites. The reagents and reaction conditions useful for the oligonucleotide synthesis are known in the art.
[0380] The oligonucleotides disclosed herein may be linked to solid support as a result of solid-phase synthesis. Cleavable solid supports that may be used with the oligonucleotides are known in the art. Non-limiting examples of the solid support include, e.g., controlled pore glass or macroporous polystyrene bonded to a strand through a cleavable linker (e.g., succinate-based linker) known in the art (e.g., UnyLinker™). An oligonucleotide linked to solid support may be removed from the solid support by cleaving the linker connecting an oligonucleotide and solid support.
[0381] The oligonucleotides may further be synthesized such that they include any of the modifications described above and elsewhere herein including, e.g., 5′ and/or 3′ end modifications, or internucleoside modifications, used to facilitate targeting, delivery, and/or cell uptake. Also, as noted above, in certain instances, an oligonucleotide of the invention is synthesized in vivo. In such instances, an oligonucleotide (e.g., an miRNA sponge) may be generated from a vector (see above).
Small Molecules and Other Inhibitors
[0382] As used herein, the term “smal molecule” refers to a molecule having a low molecular weight, typically less than 1000 Da. A small molecule may be naturally occurring or synthetic, and organic or inorganic. Smal molecule inhibitors of miR-147b can be identified, for example, using high throughput screening methods, which are optionally carried out in combination with bioinformatics-based analyses (see, e.g., Haga et al., Methods Mol. Biol. 1517:179-198, 2017). Furthermore, platforms for sequence-based design of small molecules targeting RNAs case be used (e.g., Infoma; Disney et al., ACS Chem. Biol. 11:1720-1728,2016). Also see, e.g., Xiao et al., Drug Target miRNA: Methods and Protocols, Schmidt, Ed., Springer, New York, N.Y., p. 169-178, 2017; and Vo et al., ACS Chem. Biol. 9:711-721, 2014; for additional information. Analyses of nucleic acid sequences, secondary structures, and effects of mutations, together with computer-aided drug design, can further be carried out to identify candidate small molecule inhibitors of miR-147b.
[0383] Small molecule inhibitors of the invention can act at any stage of miR-147b (or precursor) synthesis or affect its action, as described above. Thus, small molecule inhibitors can, for example, inhibit at the level of transcription pri-miR-147b, processing of pri-miR-147b to form pre-miR-147b, export of pre-miR-147b from the nucleus, processing of pre-miR-147b to form mature miR-147b, formation of miR-147b/RISC, and/or binding of miR-147b/RISC to its targets. Accordingly, small molecules can be screened for their activities at any one or more of these stages. In some embodiments, a small molecule inhibitor may target the narrow groove of the secondary structure of pre-miR-147b.
[0384] Other miR-147b inhibitors of the invention include, e.g., catalytic RNAs (e.g., ribozymes), aptamers, decoy oligonucleotides (see e.g., Wu et al., PlosOne 8(12):e82167, 2013; and Haraguchi et al., Nuc. Acids Res. 37:e43, 2009), and antibodies (e.g., antibodies that recognize RNA:RNA duplexes). In addition, gene editing approaches (e.g., CRISPR-cas9) can be used to knock-out miR147b or related molecules, as is known in the art. Small molecules and other miR-147b inhibitors identified using methods such as those described above can further be screened, for example, by use of organoids and related methods, such as those described herein.
Pharmaceutical Compositions
[0385] An oligonucleotide, small molecule, decoy, or other miR-147b inhibitor of the invention (see, e.g., above) may be included in a pharmaceutical composition, optionally in combination with one or more additional miR-147b inhibitor or other therapeutic agent (see, e.g., above). A pharmaceutical composition typically includes a pharmaceutically acceptable diluent or carrier. A pharmaceutical composition may include (e.g., consist of), e.g., a sterile saline solution and an oligonucleotide of the invention. The sterile saline is typically a pharmaceutical grade saline. A pharmaceutical composition may include (e.g., consist of), e.g., sterile water and an oligonucleotide of the invention. The sterile water is typically a pharmaceutical grade water. A pharmaceutical composition may include (e.g., consist of), e.g., phosphate-buffered saline (PBS) and an oligonucleotide of the invention. The sterile PBS is typically a pharmaceutical grade PBS.
[0386] In certain embodiments, pharmaceutical compositions include one or more oligonucleotides and one or more excipients. In certain embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.
[0387] In certain embodiments, oligonucleotides may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, e.g., route of administration, extent of disease, or dose to be administered.
[0388] In certain embodiments, pharmaceutical compositions including an oligonucleotide encompass any pharmaceutically acceptable salts of the oligonucleotide, esters of the oligonucleotide, or salts of such esters. In certain embodiments, pharmaceutical compositions including an oligonucleotide, upon administration to a subject (e.g., a human), are capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of oligonucleotides, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, e.g., sodium and potassium salts. In certain embodiments, prodrugs include one or more conjugate group attached to an oligonucleotide, wherein the conjugate group is cleaved by endogenous nucleases within the body.
[0389] Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid, such as an oligonucleotide, is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
[0390] In certain embodiments, pharmaceutical compositions include a delivery system. Examples of delivery systems include, e.g., liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those including hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.
[0391] In certain embodiments, pharmaceutical compositions include one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.
[0392] In certain embodiments, pharmaceutical compositions include a co-solvent system. Certain of such co-solvent systems include, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol including 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
[0393] In certain embodiments, pharmaceutical compositions are prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration. In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, intrathecal, intracerebroventricular, intracranial, intraocular etc.). In certain examples of such embodiments, a pharmaceutical composition includes a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers, such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, e.g., lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.
Organoids
[0394] Conventional two-dimensional (2D) monolayer cell culture has been widely applied in vitro for screening small molecules targeting oncogenic signaling in cancers, including of EGFR TKIs against EGFR in lung cancer. However, cell lines typically grown in a 2D monolayer fail to represent the native architecture and cellular heterogeneity observed in the tumors from which these lines are derived. In recent years, evidence has accumulated pointing to the existence of a new dimension of intratumor heterogeneity and a hitherto-unappreciated subclass of neoplastic cells within tumors, termed tumor-initiating cells (TICs). The concept of TICs has significant clinical implications in that TICs are more resistant to current therapeutics including chemotherapy and radiotherapy. Thus, the current 2D monolayer cell culture might not be the ideal model to find new vulnerabilities for treatment resistance.
[0395] The present invention provides organoids, which are three-dimensional (3D) collections of organ-specific cell types that develop from stem cells or organ progenitors and self-organize through cell sorting and spatially restricted lineage commitment in a manner similar to that seen in vivo. The organoids of the present invention, which are based on lung cells (including, e.g., lung cancer cells) are designed to represent the native architecture of patient-derived tumors and treatment response towards current therapeutics. Accordingly, the invention provides methods for culturing lung cells (including lung cancer cells) as organoids from both primary tissues and cell lines. In one embodiment, the present invention provides methods for culturing lung tissue that maintains the differentiated state of the alveolar epithelial cells of the lung, or recapitulates the phenotype of lung tumors.
[0396] In some embodiments, methods for obtaining organoids according to the invention include the following steps: (a) obtaining a sample of lung tissue from a subject; (b) dissociating the sample of lung tissue; (c) isolating dissociated lung epithelial cells from the sample of lung tissue; and (d) culturing the dissociated lung epithelial cells. In some embodiments, the lung tissue is non-cancerous. In other embodiments, the lung tissue is cancerous. In further embodiments, the organoids are used as lung cancer xenografts in animal models, e.g., patient-derived xenograft (PDX)-containing mice.
[0397] In more detail, a stepwise method to establish lung organoids ex vivo, according to the invention, mimics the dynamic process of benign and malignant lung tissues formation, and includes stages of initiation (days 0-3), maintenance (days 4-6), and differentiation (days 7-24). The protocol first uses factors such as, e.g., EGF, FGF2, FGF10, and other niche factors to promote self-renewal of stem-like cells in the lung organoid. Then, factors such as FGF7 and PDGF are used during the differentiation stage to induce the differentiation of stem-like cells. Details of specific methods that can be used to generate organoids are found below in the Examples.
Diagnostic and Screening Methods
[0398] The invention provides diagnostic methods that can be used to determine whether a subject has a cancer that may be (or be at risk of becoming) tolerant or resistant to anti-RTK therapy (e.g., anti-EGFR therapy; also see above) and, if so, if the resistance or tolerance may effectively be treated, reduced, prevented, or delayed by administration of a miR-147b inhibitor, as described herein. The invention also includes diagnostic methods that can be used to determine whether a subject has a cancer that may be effectively treated by administration of a miR-147b inhibitor, as described herein.
[0399] According to these methods, a sample from a subject (e.g., a human patient) is obtained and the sample is assayed for the presence of miR-147b (or a precursor or fragment thereof). Samples that can be used in these methods include, e.g., tumor tissues, tissue swabs, sputum, or blood samples (e.g., serum or plasma). Detection of miR-147b (or a precursor or fragment thereof) can carried out using standard methods including, e.g., hybridization assays, RNA-Seq, RT-PCR, and microarray-based assays. In both methods, detection of an increased level of miR-147b (or a precursor or fragment thereof), relative to a control (e.g., cells from a tissue-matched cancer that is not anti-RTK-therapy resistant or normal tissue-matched cells, as determined to be appropriate by those of skill in the art), indicates that miR-147b-targeted treatment may be effective. The level of increase that is diagnostic can be determined by those of skill in the art and may be, e.g., an increase of 25%, 50%, 100%, 150%, 200%, 300%, 500%, or more. Optionally, these diagnostic methods can also include a step of administering a miR-147b inhibitor to a subject identified as potentially benefiting from such treatment.
[0400] The invention further provides screening methods, which can be used to identify or characterize new miR-147b inhibitors, and also to select treatment that may be effective for a particular subject (e.g., a human patient having cancer). In these methods, a cell expressing miR-147b is contacted with a candidate inhibitor and the effects of the inhibitor on miR-147b expression or activity is determined (e.g., by RNA-Seq, etc.). A candidate inhibitor that is found to decrease the expression level or activity of miR-147b, relative to a control, can be considered as a potential miR-147b inhibitor that can be subject to further analysis, as needed. According to theses methods, the cells can be cultured cells (e.g., lung cancer-derived cell lines or primary cells) or the cells can be present in animal models (e.g., PDX-animal models, such as mice). Advantageously, the cells are lung cells (e.g., lung cancer cells) that are cultured to form organoids, as described above. As explained above, these structures model certain aspects of lung structure in vivo, and thus can provide for more accurate characterization of a candidate therapeutic agent (e.g., a miR-147b inhibitor). Moreover, if an organoid is derived from cells of a particular patient (e.g., cancer cells from a particular patient), the organoid can advantageously be used to test various treatments (e.g., miR-147b inhibitors, anti-RTK therapies, and/or other treatments), in order to identify a treatment protocol and regimen that may be particularly well-suited to the patient from whom the cells are derived. In addition to the above, the screening methods can be used to test combinations of therapies, e.g., combinations of miR-147b inhibitors of the invention with each other and other agents, such as other agents and treatments listed herein (e.g., carboplatin-base chemotherapy, radiotherapy, EGFR-based targeted therapy, and PD-1/PD-L1 based immunotherapy).
Kits
[0401] The invention also provides kits for use in carrying out the methods of the invention. In some embodiments, a kit of the invention includes one or more agents (e.g., antisense oligonucleotides) for use in detecting the level of miR-147b (or a precursor or fragment thereof) in a sample (e.g., a patient sample, such as tumor tissue, tissue swab, sputum, or blood (e.g., serum or plasma)). In some embodiments, a kit of the invention includes multiple miR-147b inhibitors, as described herein, optionally in combination with one or more other therapeutic agent (e.g., a TKI, such as a TKI as described herein). In other, related embodiments, the kits include a miR-147b inhibitor in combination with one or more other therapeutic agent (e.g., a TKI, such as a TKI as described herein).
Sequences
[0402] The sequence of pri-miR-147b is as follows: UAUAAAUCUAGUGGAAACAUUUCUGCACAAACUAGAUUCUGGACACCAGUGUGCGGAAAUGCUUC UGCUACAUUUUUAGG (SEQ ID NO: 1), while the sequence of mature miR-147b is: GUGUGCGGAAAUGCUUCUGCUA (SEQ ID NO: 2). Sequences that are antisense to these molecules can be used in the invention. Examples of such sequences, which can be used to target miR-147b (or a precursor or fragment thereof), according to the invention, include those comprising or consisting of the sequences in Tables 1 and 3 (e.g., SEQ ID NOs: 3-735). These sequences are various fragments of the reverse complement of SEQ ID NO: 1 (CCUAAAAAUGUAGCAGAAGCAUUUCCGCACACUGGUGUCCAGAAU CUAGUUUGUGCAGAAAUGUUUCCACUAGAUUUAUA; SEQ ID NO: 736). The sequences can comprise or be components of, e.g., antisense molecules described herein, or fragments thereof (e.g., a gap, 5′-wing, or 3′-wing). The sequences can further be present in molecules in single-stranded form or in double-stranded form. Furthermore, the sequences can be encoded in vectors, as described herein, for in vivo expression. As explained above, such sequences can optionally be present for expression as tandem multimers.
[0403] Sequences that can be used as competitive inhibitors, to compete with miR-147b for binding to an mRNA or pre-mRNA target, include the mature miR-147b sequence itself (SEQ ID NO: 2), or fragments or variants thereof. Examples of sequences that can be included in molecules that target miR-147b binding sites, according to the invention, include those comprising or consisting of the sequences in Tables 2 and 4 (e.g., SEQ ID NOs: 737-889).
[0404] For all the sequences listed herein, it is to be understood that U's are replaced with T's in the context of deoxyribonucleic acid molecules, and T's are to be replaced with U's in the context of ribonucleic acid molecules. Accordingly, even if a sequence is listed herein including U's, the sequence can be considered as including T's in their place, if appropriate in the context of the type of molecule under consideration. Similarly, if a sequence is listed herein including T's, the sequence can be considered as including U's in their place, if appropriate under the circumstances. Accordingly, if reference is made to a sequence identification number herein, then whether a T or U is to be considered in the sequence, regardless of the indicator in the sequence identifier, is based on the type of molecule intended. Mixed sequences, including both U's and T's are also included in the invention. Such molecules may include, e.g., T's in the gap region of an antisense molecule and then T's and/or U's in the wing(s). Such mixed sequences are included in the invention based on, e.g., the sequences listed in Tables 1-4, wherein one or more (e.g., all) U's are replaced with one or more T's. In addition, those of skill in the art can readily determine the sequence of a reference strand to utilize, relative to the miRNA sequences described herein, in the various contexts described herein. Furthermore, as is understood in the art, the length of each of these sequences may vary by the addition or deletion of 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more) nucleotides on either or both ends. Also, as described above, additional sequences included in the invention are variants having sequence identity to these sequences (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%). Variants having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) deletions or substations are also included in the invention. All sequences listed herein are in 5 to 3 orientation, unless otherwise indicated. In Tables 1-4, sequence identifiers are listed in one column, with the corresponding sequence in the next column. Sequences such as those listed in the Tables below, as well as in the Examples, below, can be included within the context of various molecules described above and elsewhere herein (e.g., antisense and siRNA molecules).
[0405] In some embodiments, the methods of the invention include targeting of sequences of or within SEQ ID NO: 1, e.g., sequences comprising or consisting of nucleotides 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 7-12, 8-13, 9-14, 10-15, 11-16, 12-17, 13-18, 14-19, 15-20, 16-21, 17-22, 18-23, 19-24, 20-25, 21-26, 22-27, 23-28, 24-29, 25-30, 26-31, 27-32, 28-33, 29-34, 30-35, 31-36, 32-37, 33-38, 34-39, 35-40, 36-41, 37-42, 38-43, 39-44, 40-45, 41-46, 42-47, 43-48, 44-49, 45-50, 48-51, 47-52, 48-53, 49-54, 50-55, 51-56, 52-57, 53-58, 54-59, 55-80, 58-61, 57-62, 58-63, 59-64, 60-65, 61-66, 62-67, 63-68, 64-69, 65-70, 68-71, 67-72, 68-73, 69-74, 70-75, 71-76, 72-77, 73-78, 74-79, or 75-80 of SEQ ID NO: 1. In some embodiments, the methods of the invention include targeting sequences that consist of one of the sequence fragments listed immediately above and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74 additional nucleotides of SEQ ID NO: 1, whether all on one side of the indicated fragment or wherein the fragment is between the one or more additional nucleotides. In some embodiments the sequence targeted consists of or comprises SEQ ID NO: 2.
[0406] Accordingly, for example, sequences comprising or consisting of nucleotides 1-7, 2-8, 3-9, 4-10, 5-11, 6-12, 7-13, 8-14, 9-15, 10-16, 11-17, 12-18, 13-19, 14-20, 15-21, 16-22, 17-23, 18-24, 19-25, 20-26, 21-27, 22-28, 23-29, 24-30, 25-31, 26-32, 27-33, 28-34, 29-35, 30-36, 31-37, 32-38, 33-39, 34-40, 35-41, 36-42, 37-43, 38-44, 39-45, 40-46, 41-47, 42-48, 43-49, 44-50, 45-51, 48-52, 47-53,48-54, 49-55, 50-56, 51-57, 52-58, 53-59, 54-60, 55-61, 56-62, 57-63, 58-84, 59-65, 60-66, 61-67, 62-68, 63-69, 64-70, 65-71, 66-72, 67-73, 68-74, 69-75, 70-76, 71-77, 72-78, 73-79, or 74-80 of SEQ ID NO: 1, optionally having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73 additional nucleotides of SEQ ID NO: 1, whether all on one side of the indicated fragment or wherein the fragment is between the one or more additional nucleotides, can be targeted. In some embodiments the sequence targeted consists of or comprises SEQ ID NO: 2.
[0407] In other embodiments, for example, sequences comprising or consisting of nucleotides 1-8, 2-9, 3-10, 4-11, 5-12, 6-13, 7-14, 8-15, 9-16, 10-17, 11-18, 12-19, 13-20, 14-21, 15-22, 16-23, 17-24, 18-25, 19-26, 20-27, 21-28, 22-29, 23-30, 24-31, 25-32, 26-33, 27-34, 28-35, 29-36, 30-37, 31-38, 32-39, 33-40, 34-41, 35-42, 36-43, 37-44, 38-45, 39-46, 40-47, 41-48, 42-49, 43-50, 44-51, 45-52, 46-53, 47-54, 48-55, 49-56, 50-57, 51-58, 52-59, 53-60, 54-61, 55-62, 56-63, 57-64, 58-65, 59-66, 60-67, 61-68, 62-69, 63-70, 64-71, 65-72, 66-73, 67-74, 68-75, 69-76, 70-77, 71-78, 72-79, or 73-80 of SEQ ID NO: 1, optionally having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 additional nucleotides of SEQ ID NO: 1, whether all on one side of the indicated fragment or wherein the fragment is between the one or more additional nucleotides, can be targeted. In some embodiments the sequence targeted consists of or comprises SEQ ID NO: 2.
[0408] In other embodiments, for example, sequences comprising or consisting of nucleotides 1-10, 2-11, 3-12, 4-13, 5-14, 6-15, 7-16, 8-17, 9-18, 10-19, 11-20, 12-21, 13-22, 14-23, 15-24, 16-25, 17-26, 18-27, 19-28, 20-29, 21-30, 22-31, 23-32, 24-33, 25-34, 26-35, 27-36, 28-37, 29-38, 30-39, 31-40, 32-41, 33-42, 34-43, 35-44, 36-45, 37-46, 38-47, 39-48, 40-49, 41-50, 42-51, 43-52, 44-53, 45-54, 48-55, 47-56, 48-57, 49-58, 50-59, 51-40, 52-61, 53-62, 54-63, 55-64, 58-65, 57-66, 58-67, 59-88, 60-69, or 61-70 of SEQ ID NO: 1, optionally having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 additional nucleotides of SEQ ID NO: 1, whether all on one side of the indicated fragment or wherein the fragment is between the one or more additional nucleotides, can be targeted. In some embodiments the sequence targeted consists of or comprises SEQ ID NO: 2.
[0409] In other embodiments, for example, sequences comprising or consisting of nucleotides 1-12, 2-13, 3-14, 4-15, 5-16, 6-17, 7-18, 8-19, 9-20, 10-21, 11-22, 12-23, 13-24, 14-25, 15-26, 16-27, 17-28, 18-29, 19-30, 20-31, 21-32, 22-33, 23-34, 24-35, 25-36, 26-37, 27-38, 28-39, 29-40, 30-41, 31-42, 32-43, 33-44, 34-45, 35-46, 36-47, 37-48, 38-49, 39-50, 40-51, 41-52, 42-53, 43-54, 44-55, 45-56, 48-57, 47-58, 48-59, 49-80, 50-61, 51-62, 52-63, 53-64, 54-65, 55-66, 58-67, 57-88, 58-69, or 59-70 of SEQ ID NO: 1, optionally having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 68 additional nucleotides of SEQ ID NO: 1, whether all on one side of the indicated fragment or wherein the fragment is between the one or more additional nucleotides, can be targeted. In some embodiments the sequence targeted consists of or comprises SEQ ID NO: 2.
[0410] In other embodiments, for example, sequences comprising or consisting of nucleotides 1-15, 2-16, 3-17, 4-18, 5-19, 6-20, 7-21, 8-22, 9-23, 10-24, 11-25, 12-26, 13-27, 14-28, 15-29, 16-30, 17-31, 18-32, 19-33, 20-34, 21-35, 22-36, 23-37, 24-38, 25-39, 26-40, 27-41, 28-42, 29-43, 30-44, 31-45, 32-46, 33-47, 34-48, 35-49, 36-50, 37-51, 38-52, 39-53, 40-54, 41-55, 42-56, 43-57, 44-58, 45-59, 48-80, 47-61, 48-62, 49-63, 50-84, 51-65, 52-66, 53-67, 54-68, 55-69, 58-70, 57-71, 58-72, 59-73, 60-74, 61-75, 62-76, 63-77, 64-78, 65-79, or 66-80 of SEQ ID NO: 1, optionally having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 additional nucleotides of SEQ ID NO: 1, whether all on one side of the indicated fragment or wherein the fragment is between the one or more additional nucleotides, can be targeted. In some embodiments the sequence targeted consists of or comprises SEQ ID NO: 2.
[0411] In other embodiments, for example, sequences comprising or consisting of nucleotides 1-18, 2-19, 3-20, 4-21, 5-22, 6-23, 7-24, 8-25, 9-26, 10-27, 11-28, 12-29, 13-30, 14-31, 15-32, 16-33, 17-34, 18-35, 19-36, 20-37, 21-38, 22-39, 23-40, 24-41, 25-42, 26-43, 27-44, 28-45, 29-46, 30-47, 31-48, 32-49, 33-50, 34-51, 35-52, 36-53, 37-54, 38-55, 39-56, 40-57, 41-58, 42-59, 43-60, 44-61, 45-62, 48-63, 47-64, 48-65, 49-66, 50-67, 51-68, 52-69, 53-70, 54-71, 55-72, 58-73, 57-74, 58-75, 59-76, 60-77, 61-78, 62-79, or 63-80 of SEQ ID NO: 1, optionally having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62 additional nucleotides of SEQ ID NO: 1, whether all on one side of the indicated fragment or wherein the fragment is between the one or more additional nucleotides, can be targeted. In some embodiments the sequence targeted consists of or comprises SEQ ID NO: 2.
[0412] In other embodiments, for example, sequences comprising or consisting of nucleotides 1-20, 2-21, 3-22, 4-23, 5-24, 6-25, 7-26, 8-27, 9-28, 10-29, 11-30, 12-31, 13-32, 14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39, 21-40, 22-41, 23-42, 24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49, 31-50, 32-51, 33-52, 34-53, 35-54, 36-55, 37-56, 38-57, 39-58, 40-59, 41-60, 42-61, 43-62, 44-63, 45-64, 48-65, 47-66, 48-67, 49-68, 50-69, 51-70, 52-71, 53-72, 54-73, 55-74, 58-75, 57-76, 58-77, 59-78, 60-79, or 61-80 of SEQ ID NO: 1, optionally having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 additional nucleotides of SEQ ID NO: 1, whether all on one side of the indicated fragment or wherein the fragment is between the one or more additional nucleotides, can be targeted. In some embodiments the sequence targeted consists of or comprises SEQ ID NO: 2.
[0413] As noted above, the sequences of Tables 1 and 2, below, can each be considered to include one or more T's in place of one or more noted U, depending upon the use. Accordingly, the following tables describe the specifically listed sequences, as well as variants in which one or more U is replaced with a T. Furthermore, the sequences of Tables 1 and 2 can be considered as DNA, RNA, mixed DNA and RNA, or modifications thereof, and each of these different types of molecules is thus described herein. Tables 3 and 4 include the same sequences as Tables 1 and 2, respectively, but with U's replaced with T's. The same sequence identifiers are used to show the corresponding sequences.
TABLE-US-00001 TABLE 1 3 CCUAAA 52 UUUGUG 101 UCCGCAC 150 AUUUAUA 4 CUAAAA 53 UUGUGC 102 CCGCACA 151 CCUAAAAA 5 UAAAAA 54 UGUGCA 103 CGCACAC 152 CUAAAAAU 6 AAAAAU 55 GUGCAG 104 GCACACU 153 UAAAAAUG 7 AAAAUG 56 UGCAGA 105 CACACUG 154 AAAAAUGU 8 AAAUGU 57 GCAGAA 106 ACACUGG 155 AAAAUGUA 9 AAUGUA 58 CAGAAA 107 CACUGGU 156 AAAUGUAG 10 AUGUAG 59 AGAAAU 108 ACUGGUG 157 AAUGUAGC 11 UGUAGC 60 GAAAUG 109 CUGGUGU 158 AUGUAGCA 12 GUAGCA 61 AAAUGU 110 UGGUGUC 159 UGUAGCAG 13 UAGCAG 62 AAUGUU 111 GGUGUCC 160 GUAGCAGA 14 AGCAGA 63 AUGUUU 112 GUGUCCA 161 UAGCAGAA 15 GCAGAA 64 UGUUUC 113 UGUCCAG 162 AGCAGAAG 16 CAGAAG 65 GUUUCC 114 GUCCAGA 163 GCAGAAGC 17 AGAAGC 66 UUUCCA 115 UCCAGAA 164 CAGAAGCA 18 GAAGCA 67 UUCCAC 116 CCAGAAU 165 AGAAGCAU 19 AAGCAU 68 UCCACU 117 CAGAAUC 166 GAAGCAUU 20 AGCAUU 69 CCACUA 118 AGAAUCU 167 AAGCAUUU 21 GCAUUU 70 CACUAG 119 GAAUCUA 168 AGCAUUUC 22 CAUUUC 71 ACUAGA 120 AAUCUAG 169 GCAUUUCC 23 AUUUCC 72 CUAGAU 121 AUCUAGU 170 CAUUUCCG 24 UUUCCG 73 UAGAUU 122 UCUAGUU 171 AUUUCCGC 25 UUCCGC 74 AGAUUU 123 CUAGUUU 172 UUUCCGCA 26 UCCGCA 75 GAUUUA 124 UAGUUUG 173 UUCCGCAC 27 CCGCAC 76 AUUUAU 125 AGUUUGU 174 UCCGCACA 28 CGCACA 77 UUUAUA 126 GUUUGUG 175 CCGCACAC 29 GCACAC 78 CCUAAAA 127 UUUGUGC 176 CGCACACU 30 CACACU 79 CUAAAAA 128 UUGUGCA 177 GCACACUG 31 ACACUG 80 UAAAAAU 129 UGUGCAG 178 CACACUGG 32 CACUGG 81 AAAAAUG 130 GUGCAGA 179 ACACUGGU 33 ACUGGU 82 AAAAUGU 131 UGCAGAA 180 CACUGGUG 34 CUGGUG 83 AAAUGUA 132 GCAGAAA 181 ACUGGUGU 35 UGGUGU 84 AAUGUAG 133 CAGAAAU 182 CUGGUGUC 36 GGUGUC 85 AUGUAGC 134 AGAAAUG 183 UGGUGUCC 37 GUGUCC 86 UGUAGCA 135 GAAAUGU 184 GGUGUCCA 38 UGUCCA 87 GUAGCAG 136 AAAUGUU 185 GUGUCCAG 39 GUCCAG 88 UAGCAGA 137 AAUGUUU 186 UGUCCAGA 40 UCCAGA 89 AGCAGAA 138 AUGUUUC 187 GUCCAGAA 41 CCAGAA 90 GCAGAAG 139 UGUUUCC 188 UCCAGAAU 42 CAGAAU 91 CAGAAGC 140 GUUUCCA 189 CCAGAAUC 43 AGAAUC 92 AGAAGCA 141 UUUCCAC 190 CAGAAUCU 44 GAAUCU 93 GAAGCAU 142 UUCCACU 191 AGAAUCUA 45 AAUCUA 94 AAGCAUU 143 UCCACUA 192 GAAUCUAG 46 AUCUAG 95 AGCAUUU 144 CCACUAG 193 AAUCUAGU 47 UCUAGU 96 GCAUUUC 145 CACUAGA 194 AUCUAGUU 48 CUAGUU 97 CAUUUCC 146 ACUAGAU 195 UCUAGUUU 49 UAGUUU 98 AUUUCCG 147 CUAGAUU 196 CUAGUUUG 50 AGUUUG 99 UUUCCGC 148 UAGAUUU 197 UAGUUUGU 51 GUUUGU 100 UUCCGCA 149 AGAUUUA 198 AGUUUGUG 199 GUUUGUGC 248 CCGCACACU 297 CCUAAAAAUG 346 UUUGUGCAGA 200 UUUGUGCA 249 CGCACACUG 298 CUAAAAAUGU 347 UUGUGCAGAA 201 UUGUGCAG 250 GCACACUGG 299 UAAAAAUGUA 348 UGUGCAGAAA 202 UGUGCAGA 251 CACACUGGU 300 AAAAAUGUAG 349 GUGCAGAAAU 203 GUGCAGAA 252 ACACUGGUG 301 AAAAUGUAGC 350 UGCAGAAAUG 204 UGCAGAAA 253 CACUGGUGU 302 AAAUGUAGCA 351 GCAGAAAUGU 205 GCAGAAAU 254 ACUGGUGUC 303 AAUGUAGCAG 352 CAGAAAUGUU 206 CAGAAAUG 255 CUGGUGUCC 304 AUGUAGCAGA 353 AGAAAUGUUU 207 AGAAAUGU 256 UGGUGUCCA 305 UGUAGCAGAA 354 GAAAUGUUUC 208 GAAAUGUU 257 GGUGUCCAG 306 GUAGCAGAAG 355 AAAUGUUUCC 209 AAAUGUUU 258 GUGUCCAGA 307 UAGCAGAAGC 356 AAUGUUUCCA 210 AAUGUUUC 259 UGUCCAGAA 308 AGCAGAAGCA 357 AUGUUUCCAC 211 AUGUUUCC 260 GUCCAGAAU 309 GCAGAAGCAU 358 UGUUUCCACU 212 UGUUUCCA 261 UCCAGAAUC 310 CAGAAGCAUU 359 GUUUCCACUA 213 GUUUCCAC 262 CCAGAAUCU 311 AGAAGCAUUU 360 UUUCCACUAG 214 UUUCCACU 263 CAGAAUCUA 312 GAAGCAUUUC 361 UUCCACUAGA 215 UUCCACUA 264 AGAAUCUAG 313 AAGCAUUUCC 362 UCCACUAGAU 216 UCCACUAG 265 GAAUCUAGU 314 AGCAUUUCCG 363 CCACUAGAUU 217 CCACUAGA 266 AAUCUAGUU 315 GCAUUUCCGC 364 CACUAGAUUU 218 CACUAGAU 267 AUCUAGUUU 316 CAUUUCCGCA 365 ACUAGAUUUA 219 ACUAGAUU 268 UCUAGUUUG 317 AUUUCCGCAC 366 CUAGAUUUAU 220 CUAGAUUU 269 CUAGUUUGU 318 UUUCCGCACA 367 UAGAUUUAUA 221 UAGAUUUA 270 UAGUUUGUG 319 UUCCGCACAC 368 CCUAAAAAUGU 222 AGAUUUAU 271 AGUUUGUGC 320 UCCGCACACU 369 CUAAAAAUGUA 223 GAUUUAUA 272 GUUUGUGCA 321 CCGCACACUG 370 UAAAAAUGUAG 224 CCUAAAAAU 273 UUUGUGCAG 322 CGCACACUGG 371 AAAAAUGUAGC 225 CUAAAAAUG 274 UUGUGCAGA 323 GCACACUGGU 372 AAAAUGUAGCA 226 UAAAAAUGU 275 UGUGCAGAA 324 CACACUGGUG 373 AAAUGUAGCAG 227 AAAAAUGUA 276 GUGCAGAAA 325 ACACUGGUGU 374 AAUGUAGCAGA 228 AAAAUGUAG 277 UGCAGAAAU 326 CACUGGUGUC 375 AUGUAGCAGAA 229 AAAUGUAGC 278 GCAGAAAUG 327 ACUGGUGUCC 376 UGUAGCAGAAG 230 AAUGUAGCA 279 CAGAAAUGU 328 CUGGUGUCCA 377 GUAGCAGAAGC 231 AUGUAGCAG 280 AGAAAUGUU 329 UGGUGUCCAG 378 UAGCAGAAGCA 232 UGUAGCAGA 281 GAAAUGUUU 330 GGUGUCCAGA 379 AGCAGAAGCAU 233 GUAGCAGAA 282 AAAUGUUUC 331 GUGUCCAGAA 380 GCAGAAGCAUU 234 UAGCAGAAG 283 AAUGUUUCC 332 UGUCCAGAAU 381 CAGAAGCAUUU 235 AGCAGAAGC 284 AUGUUUCCA 333 GUCCAGAAUC 382 AGAAGCAUUUC 236 GCAGAAGCA 285 UGUUUCCAC 334 UCCAGAAUCU 383 GAAGCAUUUCC 237 CAGAAGCAU 286 GUUUCCACU 335 CCAGAAUCUA 384 AAGCAUUUCCG 238 AGAAGCAUU 287 UUUCCACUA 336 CAGAAUCUAG 385 AGCAUUUCCGC 239 GAAGCAUUU 288 UUCCACUAG 337 AGAAUCUAGU 386 GCAUUUCCGCA 240 AAGCAUUUC 289 UCCACUAGA 338 GAAUCUAGUU 387 CAUUUCCGCAC 241 AGCAUUUCC 290 CCACUAGAU 339 AAUCUAGUUU 388 AUUUCCGCACA 242 GCAUUUCCG 291 CACUAGAUU 340 AUCUAGUUUG 389 UUUCCGCACAC 243 CAUUUCCGC 292 ACUAGAUUU 341 UCUAGUUUGU 390 UUCCGCACACU 244 AUUUCCGCA 293 CUAGAUUUA 342 CUAGUUUGUG 391 UCCGCACACUG 245 UUUCCGCAC 294 UAGAUUUAU 343 UAGUUUGUGC 392 CCGCACACUGG 246 UUCCGCACA 295 AGAUUUAUA 344 AGUUUGUGCA 393 CGCACACUGGU 247 UCCGCACAC 296 CCUAAAAAUG 345 GUUUGUGCAG 394 GCACACUGGUG 395 CACACUGGUGU 443 AAAUGUAGCAGA 491 UGCAGAAAUGUU 396 ACACUGGUGUC 444 AAUGUAGCAGAA 492 GCAGAAAUGUUU 397 CACUGGUGUCC 445 AUGUAGCAGAAG 493 CAGAAAUGUUUC 398 ACUGGUGUCCA 446 UGUAGCAGAAGC 494 AGAAAUGUUUCC 399 CUGGUGUCCAG 447 GUAGCAGAAGCA 495 GAAAUGUUUCCA 400 UGGUGUCCAGA 448 UAGCAGAAGCAU 496 AAAUGUUUCCAC 401 GGUGUCCAGAA 449 AGCAGAAGCAUU 497 AAUGUUUCCACU 402 GUGUCCAGAAU 450 GCAGAAGCAUUU 498 AUGUUUCCACUA 403 UGUCCAGAAUC 451 CAGAAGCAUUUC 499 UGUUUCCACUAG 404 GUCCAGAAUCU 452 AGAAGCAUUUCC 500 GUUUCCACUAGA 405 UCCAGAAUCUA 453 GAAGCAUUUCCG 501 UUUCCACUAGAU 406 CCAGAAUCUAG 454 AAGCAUUUCCGC 502 UUCCACUAGAUU 407 CAGAAUCUAGU 455 AGCAUUUCCGCA 503 UCCACUAGAUUU 408 AGAAUCUAGUU 456 GCAUUUCCGCAC 504 CCACUAGAUUUA 409 GAAUCUAGUUU 457 CAUUUCCGCACA 505 CACUAGAUUUAU 410 AAUCUAGUUUG 458 AUUUCCGCACAC 506 ACUAGAUUUAUA 411 AUCUAGUUUGU 459 UUUCCGCACACU 507 CCUAAAAAUGUAGCA 412 UCUAGUUUGUG 460 UUCCGCACACUG 508 CUAAAAAUGUAGCAG 413 CUAGUUUGUGC 461 UCCGCACACUGG 509 UAAAAAUGUAGCAGA 414 UAGUUUGUGCA 462 CCGCACACUGGU 510 AAAAAUGUAGCAGAA 415 AGUUUGUGCAG 463 CGCACACUGGUG 511 AAAAUGUAGCAGAAG 416 GUUUGUGCAGA 464 GCACACUGGUGU 512 AAAUGUAGCAGAAGC 417 UUUGUGCAGAA 465 CACACUGGUGUC 513 AAUGUAGCAGAAGCA 418 UUGUGCAGAAA 466 ACACUGGUGUCC 514 AUGUAGCAGAAGCAU 419 UGUGCAGAAAU 467 CACUGGUGUCCA 515 UGUAGCAGAAGCAUU 420 GUGCAGAAAUG 468 ACUGGUGUCCAG 516 GUAGCAGAAGCAUUU 421 UGCAGAAAUGU 469 CUGGUGUCCAGA 517 UAGCAGAAGCAUUUC 422 GCAGAAAUGUU 470 UGGUGUCCAGAA 518 AGCAGAAGCAUUUCC 423 CAGAAAUGUUU 471 GGUGUCCAGAAU 519 GCAGAAGCAUUUCCG 424 AGAAAUGUUUC 472 GUGUCCAGAAUC 520 CAGAAGCAUUUCCGC 425 GAAAUGUUUCC 473 UGUCCAGAAUCU 521 AGAAGCAUUUCCGCA 426 AAAUGUUUCCA 474 GUCCAGAAUCUA 522 GAAGCAUUUCCGCAC 427 AAUGUUUCCAC 475 UCCAGAAUCUAG 523 AAGCAUUUCCGCACA 428 AUGUUUCCACU 476 CCAGAAUCUAGU 524 AGCAUUUCCGCACAC 429 UGUUUCCACUA 477 CAGAAUCUAGUU 525 GCAUUUCCGCACACU 430 GUUUCCACUAG 478 AGAAUCUAGUUU 526 CAUUUCCGCACACUG 431 UUUCCACUAGA 479 GAAUCUAGUUUG 527 AUUUCCGCACACUGG 432 UUCCACUAGAU 480 AAUCUAGUUUGU 528 UUUCCGCACACUGGU 433 UCCACUAGAUU 481 AUCUAGUUUGUG 529 UUCCGCACACUGGUG 434 CCACUAGAUUU 482 UCUAGUUUGUGC 530 UCCGCACACUGGUGU 435 CACUAGAUUUA 483 CUAGUUUGUGCA 531 CCGCACACUGGUGUC 436 ACUAGAUUUAU 484 UAGUUUGUGCAG 532 CGCACACUGGUGUCC 437 CUAGAUUUAUA 485 AGUUUGUGCAGA 533 GCACACUGGUGUCCA 438 CCUAAAAAUGUA 486 GUUUGUGCAGAA 534 CACACUGGUGUCCAG 439 CUAAAAAUGUAG 487 UUUGUGCAGAAA 535 ACACUGGUGUCCAGA 440 UAAAAAUGUAGC 488 UUGUGCAGAAAU 536 CACUGGUGUCCAGAA 441 AAAAAUGUAGCA 489 UGUGCAGAAAUG 537 ACUGGUGUCCAGAAU 442 AAAAUGUAGCAG 490 GUGCAGAAAUGU 538 CUGGUGUCCAGAAUC 539 UGGUGUCCAGAAUCU 587 AGAAGCAUUUCCGCACACUG 540 GGUGUCCAGAAUCUA 588 GAAGCAUUUCCGCACACUGG 541 GUGUCCAGAAUCUAG 589 AAGCAUUUCCGCACACUGGU 542 UGUCCAGAAUCUAGU 590 AGCAUUUCCGCACACUGGUG 543 GUCCAGAAUCUAGUU 591 GCAUUUCCGCACACUGGUGU 544 UCCAGAAUCUAGUUU 592 CAUUUCCGCACACUGGUGUC 545 CCAGAAUCUAGUUUG 593 AUUUCCGCACACUGGUGUCC 546 CAGAAUCUAGUUUGU 594 UUUCCGCACACUGGUGUCCA 547 AGAAUCUAGUUUGUG 595 UUCCGCACACUGGUGUCCAG 548 GAAUCUAGUUUGUGC 596 UCCGCACACUGGUGUCCAGA 549 AAUCUAGUUUGUGCA 597 CCGCACACUGGUGUCCAGAA 550 AUCUAGUUUGUGCAG 598 CGCACACUGGUGUCCAGAAU 551 UCUAGUUUGUGCAGA 599 GCACACUGGUGUCCAGAAUC 552 CUAGUUUGUGCAGAA 600 CACACUGGUGUCCAGAAUCU 553 UAGUUUGUGCAGAAA 601 ACACUGGUGUCCAGAAUCUA 554 AGUUUGUGCAGAAAU 602 CACUGGUGUCCAGAAUCUAG 555 GUUUGUGCAGAAAUG 603 ACUGGUGUCCAGAAUCUAGU 556 UUUGUGCAGAAAUGU 604 CUGGUGUCCAGAAUCUAGUU 557 UUGUGCAGAAAUGUU 605 UGGUGUCCAGAAUCUAGUUU 558 UGUGCAGAAAUGUUU 606 GGUGUCCAGAAUCUAGUUUG 559 GUGCAGAAAUGUUUC 607 GUGUCCAGAAUCUAGUUUGU 560 UGCAGAAAUGUUUCC 608 UGUCCAGAAUCUAGUUUGUG 561 GCAGAAAUGUUUCCA 609 GUCCAGAAUCUAGUUUGUGC 562 CAGAAAUGUUUCCAC 610 UCCAGAAUCUAGUUUGUGCA 563 AGAAAUGUUUCCACU 611 CCAGAAUCUAGUUUGUGCAG 564 GAAAUGUUUCCACUA 612 CAGAAUCUAGUUUGUGCAGA 565 AAAUGUUUCCACUAG 613 AGAAUCUAGUUUGUGCAGAA 566 AAUGUUUCCACUAGA 614 GAAUCUAGUUUGUGCAGAAA 567 AUGUUUCCACUAGAU 615 AAUCUAGUUUGUGCAGAAAU 568 UGUUUCCACUAGAUU 616 AUCUAGUUUGUGCAGAAAUG 569 GUUUCCACUAGAUUU 617 UCUAGUUUGUGCAGAAAUGU 570 UUUCCACUAGAUUUA 618 CUAGUUUGUGCAGAAAUGUU 571 UUCCACUAGAUUUAU 619 UAGUUUGUGCAGAAAUGUUU 572 UCCACUAGAUUUAUA 620 AGUUUGUGCAGAAAUGUUUC 573 CCUAAAAAUGUAGCAGAAGC 621 GUUUGUGCAGAAAUGUUUCC 574 CUAAAAAUGUAGCAGAAGCA 622 UUUGUGCAGAAAUGUUUCCA 575 UAAAAAUGUAGCAGAAGCAU 623 UUGUGCAGAAAUGUUUCCAC 576 AAAAAUGUAGCAGAAGCAUU 624 UGUGCAGAAAUGUUUCCACU 577 AAAAUGUAGCAGAAGCAUUU 625 GUGCAGAAAUGUUUCCACUA 578 AAAUGUAGCAGAAGCAUUUC 626 UGCAGAAAUGUUUCCACUAG 579 AAUGUAGCAGAAGCAUUUCC 627 GCAGAAAUGUUUCCACUAGA 580 AUGUAGCAGAAGCAUUUCCG 628 CAGAAAUGUUUCCACUAGAU 581 UGUAGCAGAAGCAUUUCCGC 629 AGAAAUGUUUCCACUAGAUU 582 GUAGCAGAAGCAUUUCCGCA 630 GAAAUGUUUCCACUAGAUUU 583 UAGCAGAAGCAUUUCCGCAC 631 AAAUGUUUCCACUAGAUUUA 584 AGCAGAAGCAUUUCCGCACA 632 AAUGUUUCCACUAGAUUUAU 585 GCAGAAGCAUUUCCGCACAC 633 AUGUUUCCACUAGAUUUAUA 586 CAGAAGCAUUUCCGCACACU 634 CCUAAAAAUGUAGCAGAAGCAUUU 635 CUAAAAAUGUAGCAGAAGCAUUUC 678 UCUAGUUUGUGCAGAAAUGUUUCC 636 UAAAAAUGUAGCAGAAGCAUUUCC 679 CUAGUUUGUGCAGAAAUGUUUCCA 637 AAAAAUGUAGCAGAAGCAUUUCCG 680 UAGUUUGUGCAGAAAUGUUUCCAC 638 AAAAUGUAGCAGAAGCAUUUCCGC 681 AGUUUGUGCAGAAAUGUUUCCACU 639 AAAUGUAGCAGAAGCAUUUCCGCA 682 GUUUGUGCAGAAAUGUUUCCACUA 640 AAUGUAGCAGAAGCAUUUCCGCAC 683 UUUGUGCAGAAAUGUUUCCACUAG 641 AUGUAGCAGAAGCAUUUCCGCACA 684 UUGUGCAGAAAUGUUUCCACUAGA 642 UGUAGCAGAAGCAUUUCCGCACAC 685 UGUGCAGAAAUGUUUCCACUAGAU 643 GUAGCAGAAGCAUUUCCGCACACU 686 CCUAAAAAUGUAGCAGAAGCAUUUCCGCAC 644 UAGCAGAAGCAUUUCCGCACACUG 687 CUAAAAAUGUAGCAGAAGCAUUUCCGCACA 645 AGCAGAAGCAUUUCCGCACACUGG 688 UAAAAAUGUAGCAGAAGCAUUUCCGCACAC 646 GCAGAAGCAUUUCCGCACACUGGU 689 AAAAAUGUAGCAGAAGCAUUUCCGCACACU 647 CAGAAGCAUUUCCGCACACUGGUG 690 AAAAUGUAGCAGAAGCAUUUCCGCACACUG 648 AGAAGCAUUUCCGCACACUGGUGU 691 AAAUGUAGCAGAAGCAUUUCCGCACACUGG 649 GAAGCAUUUCCGCACACUGGUGUC 692 AAUGUAGCAGAAGCAUUUCCGCACACUGGU 650 AAGCAUUUCCGCACACUGGUGUCC 693 AUGUAGCAGAAGCAUUUCCGCACACUGGUG 651 AGCAUUUCCGCACACUGGUGUCCA 694 UGUAGCAGAAGCAUUUCCGCACACUGGUGU 652 GCAUUUCCGCACACUGGUGUCCAG 695 GUAGCAGAAGCAUUUCCGCACACUGGUGUC 653 CAUUUCCGCACACUGGUGUCCAGA 696 UAGCAGAAGCAUUUCCGCACACUGGUGUCC 654 AUUUCCGCACACUGGUGUCCAGAA 697 AGCAGAAGCAUUUCCGCACACUGGUGUCCA 655 UUUCCGCACACUGGUGUCCAGAAU 698 GCAGAAGCAUUUCCGCACACUGGUGUCCAG 656 UUCCGCACACUGGUGUCCAGAAUC 699 CAGAAGCAUUUCCGCACACUGGUGUCCAGA 657 UCCGCACACUGGUGUCCAGAAUCU 700 AGAAGCAUUUCCGCACACUGGUGUCCAGAA 658 CCGCACACUGGUGUCCAGAAUCUA 701 GAAGCAUUUCCGCACACUGGUGUCCAGAAU 659 CGCACACUGGUGUCCAGAAUCUAG 702 AAGCAUUUCCGCACACUGGUGUCCAGAAUC 660 GCACACUGGUGUCCAGAAUCUAGU 703 AGCAUUUCCGCACACUGGUGUCCAGAAUCU 661 CACACUGGUGUCCAGAAUCUAGUU 704 GCAUUUCCGCACACUGGUGUCCAGAAUCUA 662 ACACUGGUGUCCAGAAUCUAGUUU 705 CAUUUCCGCACACUGGUGUCCAGAAUCUAG 663 CACUGGUGUCCAGAAUCUAGUUUG 706 AUUUCCGCACACUGGUGUCCAGAAUCUAGU 664 ACUGGUGUCCAGAAUCUAGUUUGU 707 UUUCCGCACACUGGUGUCCAGAAUCUAGUU 665 CUGGUGUCCAGAAUCUAGUUUGUG 708 UUCCGCACACUGGUGUCCAGAAUCUAGUUU 666 UGGUGUCCAGAAUCUAGUUUGUGC 709 UCCGCACACUGGUGUCCAGAAUCUAGUUUG 667 GGUGUCCAGAAUCUAGUUUGUGCA 710 CCGCACACUGGUGUCCAGAAUCUAGUUUGU 668 GUGUCCAGAAUCUAGUUUGUGCAG 711 CGCACACUGGUGUCCAGAAUCUAGUUUGUG 669 UGUCCAGAAUCUAGUUUGUGCAGA 712 GCACACUGGUGUCCAGAAUCUAGUUUGUGC 670 GUCCAGAAUCUAGUUUGUGCAGAA 713 CACACUGGUGUCCAGAAUCUAGUUUGUGCA 671 UCCAGAAUCUAGUUUGUGCAGAAA 714 ACACUGGUGUCCAGAAUCUAGUUUGUGCAG 672 CCAGAAUCUAGUUUGUGCAGAAAU 715 CACUGGUGUCCAGAAUCUAGUUUGUGCAGA 673 CAGAAUCUAGUUUGUGCAGAAAUG 716 ACUGGUGUCCAGAAUCUAGUUUGUGCAGAA 674 AGAAUCUAGUUUGUGCAGAAAUGU 717 CUGGUGUCCAGAAUCUAGUUUGUGCAGAAA 675 GAAUCUAGUUUGUGCAGAAAUGUU 718 UGGUGUCCAGAAUCUAGUUUGUGCAGAAAU 676 AAUCUAGUUUGUGCAGAAAUGUUU 719 GGUGUCCAGAAUCUAGUUUGUGCAGAAAUG 677 AUCUAGUUUGUGCAGAAAUGUUUC 720 GUGUCCAGAAUCUAGUUUGUGCAGAAAUGU 721 GUCCAGAAUCUAGUUUGUGCAGAAAUGUUU 722 UCCAGAAUCUAGUUUGUGCAGAAAUGUUUC 723 CCAGAAUCUAGUUUGUGCAGAAAUGUUUCC 724 CAGAAUCUAGUUUGUGCAGAAAUGUUUCCA 725 AGAAUCUAGUUUGUGCAGAAAUGUUUCCAC 726 GAAUCUAGUUUGUGCAGAAAUGUUUCCACU 727 AAUCUAGUUUGUGCAGAAAUGUUUCCACUA 728 AUCUAGUUUGUGCAGAAAUGUUUCCACUAG 729 UCUAGUUUGUGCAGAAAUGUUUCCACUAGA 730 CUAGUUUGUGCAGAAAUGUUUCCACUAGAU 731 UAGUUUGUGCAGAAAUGUUUCCACUAGAUU 732 AGUUUGUGCAGAAAUGUUUCCACUAGAUUU 733 GUUUGUGCAGAAAUGUUUCCACUAGAUUUA 734 UUUGUGCAGAAAUGUUUCCACUAGAUUUAU 735 UUGUGCAGAAAUGUUUCCACUAGAUUUAUA
TABLE-US-00002 TABLE 2 737 GUGUGC 785 GUGUGCGGA 833 AAUGCUUCUGCU 738 UGUGCG 786 UGUGCGGAA 834 AUGCUUCUGCUA 739 GUGCGG 787 GUGCGGAAA 835 GUGUGCGGAAAUG 740 UGCGGA 788 UGCGGAAAU 836 UGUGCGGAAAUGC 741 GCGGAA 789 GCGGAAAUG 837 GUGCGGAAAUGCU 742 CGGAAA 790 CGGAAAUGC 838 UGCGGAAAUGCUU 743 GGAAAU 791 GGAAAUGCU 839 GCGGAAAUGCUUC 744 GAAAUG 792 GAAAUGCUU 840 CGGAAAUGCUUCU 745 AAAUGC 793 AAAUGCUUC 841 GGAAAUGCUUCUG 746 AAUGCU 794 AAUGCUUCU 842 GAAAUGCUUCUGC 747 AUGCUU 795 AUGCUUCUG 843 AAAUGCUUCUGCU 748 UGCUUC 796 UGCUUCUGC 844 AAUGCUUCUGCUA 749 GCUUCU 797 GCUUCUGCU 845 GUGUGCGGAAAUGC 750 CUUCUG 798 CUUCUGCUA 846 UGUGCGGAAAUGCU 751 UUCUGC 799 GUGUGCGGAA 847 GUGCGGAAAUGCUU 752 UCUGCU 800 UGUGCGGAAA 848 UGCGGAAAUGCUUC 753 CUGCUA 801 GUGCGGAAAU 849 GCGGAAAUGCUUCU 754 GUGUGCG 802 UGCGGAAAUG 850 CGGAAAUGCUUCUG 755 UGUGCGG 803 GCGGAAAUGC 851 GGAAAUGCUUCUGC 756 GUGCGGA 804 CGGAAAUGCU 852 GAAAUGCUUCUGCU 757 UGCGGAA 805 GGAAAUGCUU 853 AAAUGCUUCUGCUA 758 GCGGAAA 806 GAAAUGCUUC 854 GUGUGCGGAAAUGCU 759 CGGAAAU 807 AAAUGCUUCU 855 UGUGCGGAAAUGCUU 760 GGAAAUG 808 AAUGCUUCUG 856 GUGCGGAAAUGCUUC 761 GAAAUGC 809 AUGCUUCUGC 857 UGCGGAAAUGCUUCU 762 AAAUGCU 810 UGCUUCUGCU 858 GCGGAAAUGCUUCUG 763 AAUGCUU 811 GCUUCUGCUA 859 CGGAAAUGCUUCUGC 764 AUGCUUC 812 GUGUGCGGAAA 860 GGAAAUGCUUCUGCU 765 UGCUUCU 813 UGUGCGGAAAU 861 GAAAUGCUUCUGCUA 766 GCUUCUG 814 GUGCGGAAAUG 862 GUGUGCGGAAAUGCUU 767 CUUCUGC 815 UGCGGAAAUGC 863 UGUGCGGAAAUGCUUC 768 UUCUGCU 816 GCGGAAAUGCU 864 GUGCGGAAAUGCUUCU 769 UCUGCUA 817 CGGAAAUGCUU 865 UGCGGAAAUGCUUCUG 770 GUGUGCGG 818 GGAAAUGCUUC 866 GCGGAAAUGCUUCUGC 771 UGUGCGGA 819 GAAAUGCUUCU 867 CGGAAAUGCUUCUGCU 772 GUGCGGAA 820 AAAUGCUUCUG 868 GGAAAUGCUUCUGCUA 773 UGCGGAAA 821 AAUGCUUCUGC 869 GUGUGCGGAAAUGCUU C 774 GCGGAAAU 822 AUGCUUCUGCU 870 UGUGCGGAAAUGCUUC U 775 CGGAAAUG 823 UGCUUCUGCUA 871 GUGCGGAAAUGCUUCU G 776 GGAAAUGC 824 GUGUGCGGAAA 872 UGCGGAAAUGCUUCUG C 777 GAAAUGCU 825 UGUGCGGAAAU 873 GCGGAAAUGCUUCUGC G U 778 AAAUGCUU 826 GUGCGGAAAUG 874 CGGAAAUGCUUCUGCU C A 779 AAUGCUUC 827 UGCGGAAAUGC 875 GUGUGCGGAAAUGCUU U CU 780 AUGCUUCU 828 GCGGAAAUGCU 876 UGUGCGGAAAUGCUUC U UG 781 UGCUUCUG 829 CGGAAAUGCUU 877 GUGCGGAAAUGCUUCU C GC 782 GCUUCUGC 830 GGAAAUGCUUC 878 UGCGGAAAUGCUUCUG U CU 783 CUUCUGCU 831 GAAAUGCUUCU 879 GCGGAAAUGCUUCUGC G UA 784 UUCUGCUA 832 AAAUGCUUCUG 880 GUGUGCGGAAAUGCUU C CUG 881 UGUGCGGAAAUGCUUCUGC 882 GUGCGGAAAUGCUUCUGCU 883 UGCGGAAAUGCUUCUGCUA 884 GUGUGCGGAAAUGCUUCUGC 885 UGUGCGGAAAUGCUUCUGCU 886 GUGCGGAAAUGCUUCUGCUA 887 GUGUGCGGAAAUGCUUCUGCU 888 UGUGCGGAAAUGCUUCUGCUA 889 GUGUGCGGAAAUGCUUCUGCUA
TABLE-US-00003 TABLE 3 3 CCTAAA 52 TTTGTG 101 TCCGCAC 150 ATTTATA 4 CTAAAA 53 TTGTGC 102 CCGCACA 151 CCTAAAAA 5 TAAAAA 54 TGTGCA 103 CGCACAC 152 CTAAAAAT 6 AAAAAT 55 GTGCAG 104 GCACACT 153 TAAAAATG 7 AAAATG 56 TGCAGA 105 CACACTG 154 AAAAATGT 8 AAATGT 57 GCAGAA 106 ACACTGG 155 AAAATGTA 9 AATGTA 58 CAGAAA 107 CACTGGT 156 AAATGTAG 10 ATGTAG 59 AGAAAT 108 ACTGGTG 157 AATGTAGC 11 TGTAGC 60 GAAATG 109 CTGGTGT 158 ATGTAGCA 12 GTAGCA 61 AAATGT 110 TGGTGTC 159 TGTAGCAG 13 TAGCAG 62 AATGTT 111 GGTGTCC 160 GTAGCAGA 14 AGCAGA 63 ATGTTT 112 GTGTCCA 161 TAGCAGAA 15 GCAGAA 64 TGTTTC 113 TGTCCAG 162 AGCAGAAG 16 CAGAAG 65 GTTTCC 114 GTCCAGA 163 GCAGAAGC 17 AGAAGC 66 TTTCCA 115 TCCAGAA 164 CAGAAGCA 18 GAAGCA 67 TTCCAC 116 CCAGAAT 165 AGAAGCAT 19 AAGCAT 68 TCCACT 117 CAGAATC 166 GAAGCATT 20 AGCATT 69 CCACTA 118 AGAATCT 167 AAGCATTT 21 GCATTT 70 CACTAG 119 GAATCTA 168 AGCATTTC 22 CATTTC 71 ACTAGA 120 AATCTAG 169 GCATTTCC 23 ATTTCC 72 CTAGAT 121 ATCTAGT 170 CATTTCCG 24 TTTCCG 73 TAGATT 122 TCTAGTT 171 ATTTCCGC 25 TTCCGC 74 AGATTT 123 CTAGTTT 172 TTTCCGCA 26 TCCGCA 75 GATTTA 124 TAGTTTG 173 TTCCGCAC 27 CCGCAC 76 ATTTAT 125 AGTTTGT 174 TCCGCACA 28 CGCACA 77 TTTATA 126 GTTTGTG 175 CCGCACAC 29 GCACAC 78 CCTAAAA 127 TTTGTGC 176 CGCACACT 30 CACACT 79 CTAAAAA 128 TTGTGCA 177 GCACACTG 31 ACACTG 80 TAAAAAT 129 TGTGCAG 178 CACACTGG 32 CACTGG 81 AAAAATG 130 GTGCAGA 179 ACACTGGT 33 ACTGGT 82 AAAATGT 131 TGCAGAA 180 CACTGGTG 34 CTGGTG 83 AAATGTA 132 GCAGAAA 181 ACTGGTGT 35 TGGTGT 84 AATGTAG 133 CAGAAAT 182 CTGGTGTC 36 GGTGTC 85 ATGTAGC 134 AGAAATG 183 TGGTGTCC 37 GTGTCC 86 TGTAGCA 135 GAAATGT 184 GGTGTCCA 38 TGTCCA 87 GTAGCAG 136 AAATGTT 185 GTGTCCAG 39 GTCCAG 88 TAGCAGA 137 AATGTTT 186 TGTCCAGA 40 TCCAGA 89 AGCAGAA 138 ATGTTTC 187 GTCCAGAA 41 CCAGAA 90 GCAGAAG 139 TGTTTCC 188 TCCAGAAT 42 CAGAAT 91 CAGAAGC 140 GTTTCCA 189 CCAGAATC 43 AGAATC 92 AGAAGCA 141 TTTCCAC 190 CAGAATCT 44 GAATCT 93 GAAGCAT 142 TTCCACT 191 AGAATCTA 45 AATCTA 94 AAGCATT 143 TCCACTA 192 GAATCTAG 46 ATCTAG 95 AGCATTT 144 CCACTAG 193 AATCTAGT 47 TCTAGT 96 GCATTTC 145 CACTAGA 194 ATCTAGTT 48 CTAGTT 97 CATTTCC 146 ACTAGAT 195 TCTAGTTT 49 TAGTTT 98 ATTTCCG 147 CTAGATT 196 CTAGTTTG 50 AGTTTG 99 TTTCCGC 148 TAGATTT 197 TAGTTTGT 51 GTTTGT 100 TTCCGCA 149 AGATTTA 198 AGTTTGTG 199 GTTTGTGC 248 CCGCACACT 297 CCTAAAAATG 346 TTTGTGCAGA 200 TTTGTGCA 249 CGCACACTG 298 CTAAAAATGT 347 TTGTGCAGAA 201 TTGTGCAG 250 GCACACTGG 299 TAAAAATGTA 348 TGTGCAGAAA 202 TGTGCAGA 251 CACACTGGT 300 AAAAATGTAG 349 GTGCAGAAAT 203 GTGCAGAA 252 ACACTGGTG 301 AAAATGTAGC 350 TGCAGAAATG 204 TGCAGAAA 253 CACTGGTGT 302 AAATGTAGCA 351 GCAGAAATGT 205 GCAGAAAT 254 ACTGGTGTC 303 AATGTAGCAG 352 CAGAAATGTT 206 CAGAAATG 255 CTGGTGTCC 304 ATGTAGCAGA 353 AGAAATGTTT 207 AGAAATGT 256 TGGTGTCCA 305 TGTAGCAGAA 354 GAAATGTTTC 208 GAAATGTT 257 GGTGTCCAG 306 GTAGCAGAAG 355 AAATGTTTCC 209 AAATGTTT 258 GTGTCCAGA 307 TAGCAGAAGC 356 AATGTTTCCA 210 AATGTTTC 259 TGTCCAGAA 308 AGCAGAAGCA 357 ATGTTTCCAC 211 ATGTTTCC 260 GTCCAGAAT 309 GCAGAAGCAT 358 TGTTTCCACT 212 TGTTTCCA 261 TCCAGAATC 310 CAGAAGCATT 359 GTTTCCACTA 213 GTTTCCAC 262 CCAGAATCT 311 AGAAGCATTT 360 TTTCCACTAG 214 TTTCCACT 263 CAGAATCTA 312 GAAGCATTTC 361 TTCCACTAGA 215 TTCCACTA 264 AGAATCTAG 313 AAGCATTTCC 362 TCCACTAGAT 216 TCCACTAG 265 GAATCTAGT 314 AGCATTTCCG 363 CCACTAGATT 217 CCACTAGA 266 AATCTAGTT 315 GCATTTCCGC 364 CACTAGATTT 218 CACTAGAT 267 ATCTAGTTT 316 CATTTCCGCA 365 ACTAGATTTA 219 ACTAGATT 268 TCTAGTTTG 317 ATTTCCGCAC 366 CTAGATTTAT 220 CTAGATTT 269 CTAGTTTGT 318 TTTCCGCACA 367 TAGATTTATA 221 TAGATTTA 270 TAGTTTGTG 319 TTCCGCACAC 368 CCTAAAAATGT 222 AGATTTAT 271 AGTTTGTGC 320 TCCGCACACT 369 CTAAAAATGTA 223 GATTTATA 272 GTTTGTGCA 321 CCGCACACTG 370 TAAAAATGTAG 224 CCTAAAAAT 273 TTTGTGCAG 322 CGCACACTGG 371 AAAAATGTAGC 225 CTAAAAATG 274 TTGTGCAGA 323 GCACACTGGT 372 AAAATGTAGCA 226 TAAAAATGT 275 TGTGCAGAA 324 CACACTGGTG 373 AAATGTAGCAG 227 AAAAATGTA 276 GTGCAGAAA 325 ACACTGGTGT 374 AATGTAGCAGA 228 AAAATGTAG 277 TGCAGAAAT 326 CACTGGTGTC 375 ATGTAGCAGAA 229 AAATGTAGC 278 GCAGAAATG 327 ACTGGTGTCC 376 TGTAGCAGAAG 230 AATGTAGCA 279 CAGAAATGT 328 CTGGTGTCCA 377 GTAGCAGAAGC 231 ATGTAGCAG 280 AGAAATGTT 329 TGGTGTCCAG 378 TAGCAGAAGCA 232 TGTAGCAGA 281 GAAATGTTT 330 GGTGTCCAGA 379 AGCAGAAGCAT 233 GTAGCAGAA 282 AAATGTTTC 331 GTGTCCAGAA 380 GCAGAAGCATT 234 TAGCAGAAG 283 AATGTTTCC 332 TGTCCAGAAT 381 CAGAAGCATTT 235 AGCAGAAGC 284 ATGTTTCCA 333 GTCCAGAATC 382 AGAAGCATTTC 236 GCAGAAGCA 285 TGTTTCCAC 334 TCCAGAATCT 383 GAAGCATTTCC 237 CAGAAGCAT 286 GTTTCCACT 335 CCAGAATCTA 384 AAGCATTTCCG 238 AGAAGCATT 287 TTTCCACTA 336 CAGAATCTAG 385 AGCATTTCCGC 239 GAAGCATTT 288 TTCCACTAG 337 AGAATCTAGT 386 GCATTTCCGCA 240 AAGCATTTC 289 TCCACTAGA 338 GAATCTAGTT 387 CATTTCCGCAC 241 AGCATTTCC 290 CCACTAGAT 339 AATCTAGTTT 388 ATTTCCGCACA 242 GCATTTCCG 291 CACTAGATT 340 ATCTAGTTTG 389 TTTCCGCACAC 243 CATTTCCGC 292 ACTAGATTT 341 TCTAGTTTGT 390 TTCCGCACACT 244 ATTTCCGCA 293 CTAGATTTA 342 CTAGTTTGTG 391 TCCGCACACTG 245 TTTCCGCAC 294 TAGATTTAT 343 TAGTTTGTGC 392 CCGCACACTGG 246 TTCCGCACA 295 AGATTTATA 344 AGTTTGTGCA 393 CGCACACTGGT 247 TCCGCACAC 296 CCTAAAAATG 345 GTTTGTGCAG 394 GCACACTGGTG 395 CACACTGGTGT 443 AAATGTAGCAGA 491 TGCAGAAATGTT 396 ACACTGGTGTC 444 AATGTAGCAGAA 492 GCAGAAATGTTT 397 CACTGGTGTCC 445 ATGTAGCAGAAG 493 CAGAAATGTTTC 398 ACTGGTGTCCA 446 TGTAGCAGAAGC 494 AGAAATGTTTCC 399 CTGGTGTCCAG 447 GTAGCAGAAGCA 495 GAAATGTTTCCA 400 TGGTGTCCAGA 448 TAGCAGAAGCAT 496 AAATGTTTCCAC 401 GGTGTCCAGAA 449 AGCAGAAGCATT 497 AATGTTTCCACT 402 GTGTCCAGAAT 450 GCAGAAGCATTT 498 ATGTTTCCACTA 403 TGTCCAGAATC 451 CAGAAGCATTTC 499 TGTTTCCACTAG 404 GTCCAGAATCT 452 AGAAGCATTTCC 500 GTTTCCACTAGA 405 TCCAGAATCTA 453 GAAGCATTTCCG 501 TTTCCACTAGAT 406 CCAGAATCTAG 454 AAGCATTTCCGC 502 TTCCACTAGATT 407 CAGAATCTAGT 455 AGCATTTCCGCA 503 TCCACTAGATTT 408 AGAATCTAGTT 456 GCATTTCCGCAC 504 CCACTAGATTTA 409 GAATCTAGTTT 457 CATTTCCGCACA 505 CACTAGATTTAT 410 AATCTAGTTTG 458 ATTTCCGCACAC 506 ACTAGATTTATA 411 ATCTAGTTTGT 459 TTTCCGCACACT 507 CCTAAAAATGTAGCA 412 TCTAGTTTGTG 460 TTCCGCACACTG 508 CTAAAAATGTAGCAG 413 CTAGTTTGTGC 461 TCCGCACACTGG 509 TAAAAATGTAGCAGA 414 TAGTTTGTGCA 462 CCGCACACTGGT 510 AAAAATGTAGCAGAA 415 AGTTTGTGCAG 463 CGCACACTGGTG 511 AAAATGTAGCAGAAG 416 GTTTGTGCAGA 464 GCACACTGGTGT 512 AAATGTAGCAGAAGC 417 TTTGTGCAGAA 465 CACACTGGTGTC 513 AATGTAGCAGAAGCA 418 TTGTGCAGAAA 466 ACACTGGTGTCC 514 ATGTAGCAGAAGCAT 419 TGTGCAGAAAT 467 CACTGGTGTCCA 515 TGTAGCAGAAGCATT 420 GTGCAGAAATG 468 ACTGGTGTCCAG 516 GTAGCAGAAGCATTT 421 TGCAGAAATGT 469 CTGGTGTCCAGA 517 TAGCAGAAGCATTTC 422 GCAGAAATGTT 470 TGGTGTCCAGAA 518 AGCAGAAGCATTTCC 423 CAGAAATGTTT 471 GGTGTCCAGAAT 519 GCAGAAGCATTTCCG 424 AGAAATGTTTC 472 GTGTCCAGAATC 520 CAGAAGCATTTCCGC 425 GAAATGTTTCC 473 TGTCCAGAATCT 521 AGAAGCATTTCCGCA 426 AAATGTTTCCA 474 GTCCAGAATCTA 522 GAAGCATTTCCGCAC 427 AATGTTTCCAC 475 TCCAGAATCTAG 523 AAGCATTTCCGCACA 428 ATGTTTCCACT 476 CCAGAATCTAGT 524 AGCATTTCCGCACAC 429 TGTTTCCACTA 477 CAGAATCTAGTT 525 GCATTTCCGCACACT 430 GTTTCCACTAG 478 AGAATCTAGTTT 526 CATTTCCGCACACTG 431 TTTCCACTAGA 479 GAATCTAGTTTG 527 ATTTCCGCACACTGG 432 TTCCACTAGAT 480 AATCTAGTTTGT 528 TTTCCGCACACTGGT 433 TCCACTAGATT 481 ATCTAGTTTGTG 529 TTCCGCACACTGGTG 434 CCACTAGATTT 482 TCTAGTTTGTGC 530 TCCGCACACTGGTGT 435 CACTAGATTTA 483 CTAGTTTGTGCA 531 CCGCACACTGGTGTC 436 ACTAGATTTAT 484 TAGTTTGTGCAG 532 CGCACACTGGTGTCC 437 CTAGATTTATA 485 AGTTTGTGCAGA 533 GCACACTGGTGTCCA 438 CCTAAAAATGTA 486 GTTTGTGCAGAA 534 CACACTGGTGTCCAG 439 CTAAAAATGTAG 487 TTTGTGCAGAAA 535 ACACTGGTGTCCAGA 440 TAAAAATGTAGC 488 TTGTGCAGAAAT 536 CACTGGTGTCCAGAA 441 AAAAATGTAGCA 489 TGTGCAGAAATG 537 ACTGGTGTCCAGAAT 442 AAAATGTAGCAG 490 GTGCAGAAATGT 538 CTGGTGTCCAGAATC 539 TGGTGTCCAGAATCT 587 AGAAGCATTTCCGCACACTG 540 GGTGTCCAGAATCTA 588 GAAGCATTTCCGCACACTGG 541 GTGTCCAGAATCTAG 589 AAGCATTTCCGCACACTGGT 542 TGTCCAGAATCTAGT 590 AGCATTTCCGCACACTGGTG 543 GTCCAGAATCTAGTT 591 GCATTTCCGCACACTGGTGT 544 TCCAGAATCTAGTTT 592 CATTTCCGCACACTGGTGTC 545 CCAGAATCTAGTTTG 593 ATTTCCGCACACTGGTGTCC 546 CAGAATCTAGTTTGT 594 TTTCCGCACACTGGTGTCCA 547 AGAATCTAGTTTGTG 595 TTCCGCACACTGGTGTCCAG 548 GAATCTAGTTTGTGC 596 TCCGCACACTGGTGTCCAGA 549 AATCTAGTTTGTGCA 597 CCGCACACTGGTGTCCAGAA 550 ATCTAGTTTGTGCAG 598 CGCACACTGGTGTCCAGAAT 551 TCTAGTTTGTGCAGA 599 GCACACTGGTGTCCAGAATC 552 CTAGTTTGTGCAGAA 600 CACACTGGTGTCCAGAATCT 553 TAGTTTGTGCAGAAA 601 ACACTGGTGTCCAGAATCTA 554 AGTTTGTGCAGAAAT 602 CACTGGTGTCCAGAATCTAG 555 GTTTGTGCAGAAATG 603 ACTGGTGTCCAGAATCTAGT 556 TTTGTGCAGAAATGT 604 CTGGTGTCCAGAATCTAGTT 557 TTGTGCAGAAATGTT 605 TGGTGTCCAGAATCTAGTTT 558 TGTGCAGAAATGTTT 606 GGTGTCCAGAATCTAGTTTG 559 GTGCAGAAATGTTTC 607 GTGTCCAGAATCTAGTTTGT 560 TGCAGAAATGTTTCC 608 TGTCCAGAATCTAGTTTGTG 561 GCAGAAATGTTTCCA 609 GTCCAGAATCTAGTTTGTGC 562 CAGAAATGTTTCCAC 610 TCCAGAATCTAGTTTGTGCA 563 AGAAATGTTTCCACT 611 CCAGAATCTAGTTTGTGCAG 564 GAAATGTTTCCACTA 612 CAGAATCTAGTTTGTGCAGA 565 AAATGTTTCCACTAG 613 AGAATCTAGTTTGTGCAGAA 566 AATGTTTCCACTAGA 614 GAATCTAGTTTGTGCAGAAA 567 ATGTTTCCACTAGAT 615 AATCTAGTTTGTGCAGAAAT 568 TGTTTCCACTAGATT 616 ATCTAGTTTGTGCAGAAATG 569 GTTTCCACTAGATTT 617 TCTAGTTTGTGCAGAAATGT 570 TTTCCACTAGATTTA 618 CTAGTTTGTGCAGAAATGTT 571 TTCCACTAGATTTAT 619 TAGTTTGTGCAGAAATGTTT 572 TCCACTAGATTTATA 620 AGTTTGTGCAGAAATGTTTC 573 CCTAAAAATGTAGCAGAAGC 621 GTTTGTGCAGAAATGTTTCC 574 CTAAAAATGTAGCAGAAGCA 622 TTTGTGCAGAAATGTTTCCA 575 TAAAAATGTAGCAGAAGCAT 623 TTGTGCAGAAATGTTTCCAC 576 AAAAATGTAGCAGAAGCATT 624 TGTGCAGAAATGTTTCCACT 577 AAAATGTAGCAGAAGCATTT 625 GTGCAGAAATGTTTCCACTA 578 AAATGTAGCAGAAGCATTTC 626 TGCAGAAATGTTTCCACTAG 579 AATGTAGCAGAAGCATTTCC 627 GCAGAAATGTTTCCACTAGA 580 ATGTAGCAGAAGCATTTCCG 628 CAGAAATGTTTCCACTAGAT 581 TGTAGCAGAAGCATTTCCGC 629 AGAAATGTTTCCACTAGATT 582 GTAGCAGAAGCATTTCCGCA 630 GAAATGTTTCCACTAGATTT 583 TAGCAGAAGCATTTCCGCAC 631 AAATGTTTCCACTAGATTTA 584 AGCAGAAGCATTTCCGCACA 632 AATGTTTCCACTAGATTTAT 585 GCAGAAGCATTTCCGCACAC 633 ATGTTTCCACTAGATTTATA 586 CAGAAGCATTTCCGCACACT 634 CCTAAAAATGTAGCAGAAGCATTT 635 CTAAAAATGTAGCAGAAGCATTTC 678 TCTAGTTTGTGCAGAAATGTTTCC 636 TAAAAATGTAGCAGAAGCATTTCC 679 CTAGTTTGTGCAGAAATGTTTCCA 637 AAAAATGTAGCAGAAGCATTTCCG 680 TAGTTTGTGCAGAAATGTTTCCAC 638 AAAATGTAGCAGAAGCATTTCCGC 681 AGTTTGTGCAGAAATGTTTCCACT 639 AAATGTAGCAGAAGCATTTCCGCA 682 GTTTGTGCAGAAATGTTTCCACTA 640 AATGTAGCAGAAGCATTTCCGCAC 683 TTTGTGCAGAAATGTTTCCACTAG 641 ATGTAGCAGAAGCATTTCCGCACA 684 TTGTGCAGAAATGTTTCCACTAGA 642 TGTAGCAGAAGCATTTCCGCACAC 685 TGTGCAGAAATGTTTCCACTAGAT 643 GTAGCAGAAGCATTTCCGCACACT 686 CCTAAAAATGTAGCAGAAGCATTTCCGCAC 644 TAGCAGAAGCATTTCCGCACACTG 687 CTAAAAATGTAGCAGAAGCATTTCCGCACA 645 AGCAGAAGCATTTCCGCACACTGG 688 TAAAAATGTAGCAGAAGCATTTCCGCACAC 646 GCAGAAGCATTTCCGCACACTGGT 689 AAAAATGTAGCAGAAGCATTTCCGCACACT 647 CAGAAGCATTTCCGCACACTGGTG 690 AAAATGTAGCAGAAGCATTTCCGCACACTG 648 AGAAGCATTTCCGCACACTGGTGT 691 AAATGTAGCAGAAGCATTTCCGCACACTGG 649 GAAGCATTTCCGCACACTGGTGTC 692 AATGTAGCAGAAGCATTTCCGCACACTGGT 650 AAGCATTTCCGCACACTGGTGTCC 693 ATGTAGCAGAAGCATTTCCGCACACTGGTG 651 AGCATTTCCGCACACTGGTGTCCA 694 TGTAGCAGAAGCATTTCCGCACACTGGTGT 652 GCATTTCCGCACACTGGTGTCCAG 695 GTAGCAGAAGCATTTCCGCACACTGGTGTC 653 CATTTCCGCACACTGGTGTCCAGA 696 TAGCAGAAGCATTTCCGCACACTGGTGTCC 654 ATTTCCGCACACTGGTGTCCAGAA 697 AGCAGAAGCATTTCCGCACACTGGTGTCCA 655 TTTCCGCACACTGGTGTCCAGAAT 698 GCAGAAGCATTTCCGCACACTGGTGTCCAG 656 TTCCGCACACTGGTGTCCAGAATC 699 CAGAAGCATTTCCGCACACTGGTGTCCAGA 657 TCCGCACACTGGTGTCCAGAATCT 700 AGAAGCATTTCCGCACACTGGTGTCCAGAA 658 CCGCACACTGGTGTCCAGAATCTA 701 GAAGCATTTCCGCACACTGGTGTCCAGAAT 659 CGCACACTGGTGTCCAGAATCTAG 702 AAGCATTTCCGCACACTGGTGTCCAGAATC 660 GCACACTGGTGTCCAGAATCTAGT 703 AGCATTTCCGCACACTGGTGTCCAGAATCT 661 CACACTGGTGTCCAGAATCTAGTT 704 GCATTTCCGCACACTGGTGTCCAGAATCTA 662 ACACTGGTGTCCAGAATCTAGTTT 705 CATTTCCGCACACTGGTGTCCAGAATCTAG 663 CACTGGTGTCCAGAATCTAGTTTG 706 ATTTCCGCACACTGGTGTCCAGAATCTAGT 664 ACTGGTGTCCAGAATCTAGTTTGT 707 TTTCCGCACACTGGTGTCCAGAATCTAGTT 665 CTGGTGTCCAGAATCTAGTTTGTG 708 TTCCGCACACTGGTGTCCAGAATCTAGTTT 666 TGGTGTCCAGAATCTAGTTTGTGC 709 TCCGCACACTGGTGTCCAGAATCTAGTTTG 667 GGTGTCCAGAATCTAGTTTGTGCA 710 CCGCACACTGGTGTCCAGAATCTAGTTTGT 668 GTGTCCAGAATCTAGTTTGTGCAG 711 CGCACACTGGTGTCCAGAATCTAGTTTGTG 669 TGTCCAGAATCTAGTTTGTGCAGA 712 GCACACTGGTGTCCAGAATCTAGTTTGTGC 670 GTCCAGAATCTAGTTTGTGCAGAA 713 CACACTGGTGTCCAGAATCTAGTTTGTGCA 671 TCCAGAATCTAGTTTGTGCAGAAA 714 ACACTGGTGTCCAGAATCTAGTTTGTGCAG 672 CCAGAATCTAGTTTGTGCAGAAAT 715 CACTGGTGTCCAGAATCTAGTTTGTGCAGA 673 CAGAATCTAGTTTGTGCAGAAATG 716 ACTGGTGTCCAGAATCTAGTTTGTGCAGAA 674 AGAATCTAGTTTGTGCAGAAATGT 717 CTGGTGTCCAGAATCTAGTTTGTGCAGAAA 675 GAATCTAGTTTGTGCAGAAATGTT 718 TGGTGTCCAGAATCTAGTTTGTGCAGAAAT 676 AATCTAGTTTGTGCAGAAATGTTT 719 GGTGTCCAGAATCTAGTTTGTGCAGAAATG 677 ATCTAGTTTGTGCAGAAATGTTTC 720 GTGTCCAGAATCTAGTTTGTGCAGAAATGT 721 GTCCAGAATCTAGTTTGTGCAGAAATGTTT 722 TCCAGAATCTAGTTTGTGCAGAAATGTTTC 723 CCAGAATCTAGTTTGTGCAGAAATGTTTCC 724 CAGAATCTAGTTTGTGCAGAAATGTTTCCA 725 AGAATCTAGTTTGTGCAGAAATGTTTCCAC 726 GAATCTAGTTTGTGCAGAAATGTTTCCACT 727 AATCTAGTTTGTGCAGAAATGTTTCCACTA 728 ATCTAGTTTGTGCAGAAATGTTTCCACTAG 729 TCTAGTTTGTGCAGAAATGTTTCCACTAGA 730 CTAGTTTGTGCAGAAATGTTTCCACTAGAT 731 TAGTTTGTGCAGAAATGTTTCCACTAGATT 732 AGTTTGTGCAGAAATGTTTCCACTAGATTT 733 GTTTGTGCAGAAATGTTTCCACTAGATTTA 734 TTTGTGCAGAAATGTTTCCACTAGATTTAT 735 TTGTGCAGAAATGTTTCCACTAGATTTATA
TABLE-US-00004 TABLE 4 737 GTGTGC 785 GTGTGCGGA 833 AATGCTTCTGCT 738 TGTGCG 786 TGTGCGGAA 834 ATGCTTCTGCTA 739 GTGCGG 787 GTGCGGAAA 835 GTGTGCGGAAATG 740 TGCGGA 788 TGCGGAAAT 836 TGTGCGGAAATGC 741 GCGGAA 789 GCGGAAATG 837 GTGCGGAAATGCT 742 CGGAAA 790 CGGAAATGC 838 TGCGGAAATGCTT 743 GGAAAT 791 GGAAATGCT 839 GCGGAAATGCTTC 744 GAAATG 792 GAAATGCTT 840 CGGAAATGCTTCT 745 AAATGC 793 AAATGCTTC 841 GGAAATGCTTCTG 746 AATGCT 794 AATGCTTCT 842 GAAATGCTTCTGC 747 ATGCTT 795 ATGCTTCTG 843 AAATGCTTCTGCT 748 TGCTTC 796 TGCTTCTGC 844 AATGCTTCTGCTA 749 GCTTCT 797 GCTTCTGCT 845 GTGTGCGGAAATGC 750 CTTCTG 798 CTTCTGCTA 846 TGTGCGGAAATGCT 751 TTCTGC 799 GTGTGCGGAA 847 GTGCGGAAATGCTT 752 TCTGCT 800 TGTGCGGAAA 848 TGCGGAAATGCTTC 753 CTGCTA 801 GTGCGGAAAT 849 GCGGAAATGCTTCT 754 GTGTGCG 802 TGCGGAAATG 850 CGGAAATGCTTCTG 755 TGTGCGG 803 GCGGAAATGC 851 GGAAATGCTTCTGC 756 GTGCGGA 804 CGGAAATGCT 852 GAAATGCTTCTGCT 757 TGCGGAA 805 GGAAATGCTT 853 AAATGCTTCTGCTA 758 GCGGAAA 806 GAAATGCTTC 854 GTGTGCGGAAATGCT 759 CGGAAAT 807 AAATGCTTCT 855 TGTGCGGAAATGCTT 760 GGAAATG 808 AATGCTTCTG 856 GTGCGGAAATGCTTC 761 GAAATGC 809 ATGCTTCTGC 857 TGCGGAAATGCTTCT 762 AAATGCT 810 TGCTTCTGCT 858 GCGGAAATGCTTCTG 763 AATGCTT 811 GCTTCTGCTA 859 CGGAAATGCTTCTGC 764 ATGCTTC 812 GTGTGCGGAAA 860 GGAAATGCTTCTGCT 765 TGCTTCT 813 TGTGCGGAAAT 861 GAAATGCTTCTGCTA 766 GCTTCTG 814 GTGCGGAAATG 862 GTGTGCGGAAATGCTT 767 CTTCTGC 815 TGCGGAAATGC 863 TGTGCGGAAATGCTTC 768 TTCTGCT 816 GCGGAAATGCT 864 GTGCGGAAATGCTTCT 769 TCTGCTA 817 CGGAAATGCTT 865 TGCGGAAATGCTTCTG 770 GTGTGCGG 818 GGAAATGCTTC 866 GCGGAAATGCTTCTGC 771 TGTGCGGA 819 GAAATGCTTCT 867 CGGAAATGCTTCTGCT 772 GTGCGGAA 820 AAATGCTTCTG 868 GGAAATGCTTCTGCTA 773 TGCGGAAA 821 AATGCTTCTGC 869 GTGTGCGGAAATGCTT T 774 GCGGAAAT 822 ATGCTTCTGCT 870 TGTGCGGAAATGCTTC T 775 CGGAAATG 823 TGCTTCTGCTA 871 GTGCGGAAATGCTTCT G 776 GGAAATGC 824 GTGTGCGGAAA 872 TGCGGAAATGCTTCTG T C 777 GAAATGCT 825 TGTGCGGAAAT 873 GCGGAAATGCTTCTGC G T 778 AAATGCTT 826 GTGCGGAAATG 874 CGGAAATGCTTCTGCT C A 779 AATGCTTC 827 TGCGGAAATGC 875 GTGTGCGGAAATGCTT T CT 780 ATGCTTCT 828 GCGGAAATGCT 876 TGTGCGGAAATGCTTC T TG 781 TGCTTCTG 829 CGGAAATGCTT 877 GTGCGGAAATGCTTCT C GC 782 GCTTCTGC 830 GGAAATGCTTC 878 TGCGGAAATGCTTCTG T CT 783 CTTCTGCT 831 GAAATGCTTCT 879 GCGGAAATGCTTCTGC G TA 784 TTCTGCTA 832 AAATGCTTCTG 880 GTGTGCGGAAATGCTT C CTG 881 TGTGCGGAAATGCTTCTGC 882 GTGCGGAAATGCTTCTGCT 883 TGCGGAAATGCTTCTGCTA 884 GTGTGCGGAAATGCTTCTGC 885 TGTGCGGAAATGCTTCTGCT 886 GTGCGGAAATGCTTCTGCTA 887 GTGTGCGGAAATGCTTCTGCT 888 TGTGCGGAAATGCTTCTGCTA 889 GTGTGCGGAAATGCTTCTGCTA
[0414] The following examples are meant to illustrate the invention. They are not meant to limit the invention in anyway.
EXAMPLES
[0415] Drug-tolerance is an acute defense response prior to a fully drug-resistant state and tumor relapse. There are few therapeutic agents targeting drug-tolerance in the clinic. Here we show that miR-147b initiates a reversible tolerant-state to the EGFR inhibitor osimertinib in non-small cell lung cancer. MiR-147b was the most upregulated non-coding RNA in osimertinib-tolerant and EGFR mutated lung cancer cells by miRNA-seq analysis. Whole transcriptome analysis of single-cell derived clones revealed a link between osimertinib-tolerance and pseudohypoxia responses irrespective of oxygen levels. Further metabolomics and genetic studies demonstrated that osimertinib-tolerance is driven by miR-147b repression of VHL and succinate dehydrogenase linked to the tricarboxylic acid cycle and pseudohypoxia pathways. Locked nucleic acid miR-147b inhibitor pretreatment delayed osimertinib-associated drug tolerance in patient-derived organoids. The link between miR-147b and tricarboxylic acid cycle may provide promising targets for preventing tumor relapse.
MiRNA Expression in Lung Cancer Cell Lines
[0416] We analyzed a publicly available RNA sequencing dataset in an unbiased way for 122 human lung cancer cell lines. Eight of the cell lines contained EGFR mutations (sensitive and resistant to TKI); 72 of the cell lines were wild type EGFR (EGFR.sup.wt). In a cohort 1, which included frequently studied EGFRmut and EGFR.sup.wt lung cancer cell lines (n=15), we found the top six-upregulated miRNAs in a comparison of EGFRmut versus EGFR.sup.wt include miR-147b, miR-936, miR-614, miR-222, miR-433, and miR-127 (p<0.05). Several miRNAs in the set up upregulated miRNAs were reported previously to be associated with the EGFR signaling pathway, including miR-222 and miR-127. We focused our study on miR-147b because miR-147b is the most upregulated miRNA in EGFRmut lung cancer cells from our analysis and because the function of miR-147b is not well known.
[0417] In addition to acquiring additional EGFR mutations such as T790M or C797S, lung cancer cells also activate alternative RTKs via bypass mechanisms to promote cancer cell survival and proliferation. To understand whether miR-147b is associated with mutations in other RTKs, we analyzed miR-147b expression in cancer cells of cohort 2 with mutations in other RTKs, including BRAF, ALK, ROS1, and ERBB2/3/4. As expected, cancer cells with those RTKs mutations also expressed higher levels of miR-147b compared with EGFR.sup.wt cancer cells. Then we asked whether miR-147b expression is linked to tolerance and resistance to EGFR inhibition. To address this question, we derived a number of gefitinib-resistant lung cancer cell lines upon continuous gefitinib treatment in parental sensitive cancer cells in vitro. Consistent with RNA seq analysis, our qPCR results showed that miR-147b was expressed ˜20 fold higher in EGFRmut lung cancer cell lines (n=7) compared with EGFR.sup.wt cell lines (n=5). Moreover, the expression levels of miR-147b in gefitinib-resistant cancer cells (PC9ER, H1975, and HCC827GR, n=3) were up to three-fold higher than gefitinib-sensitive cancer cells (H1650, PC9, H3255, and HCC827). These results indicate that upregulation of miR-147b correlates with an activated EGFR signaling pathway and increased resistance to EGFR TKIs.
[0418] In addition, we tested whether miR-147b expression could distinguish tumor cells from normal cells. To address this question, we included one immortalize lung epithelial cell line AALE in our study. Our results demonstrated that expression levels of miR-147b were up to 700-fold up-regulated in a comparison of EGFRmut cancer cells versus normal cells. Analysis of a separate qRT-PCR dataset showed that the expression level of miR-147b was 2.4-fold higher in EGFRmut lung cancer tissues than normal lung tissues. Thus, we have found a new miRNA, miR-147b, which is linked to tumorigenesis and increased resistance to current EGFR-based targeted therapy.
Lung Cancer Cells Adopt a Tolerance Strategy to EGFR Inhibitors
[0419] Due to an advantage for visualizing in vivo-like structures in organoids, we established 3D lung organoids in immortalized tracheobronchial epithelial AALE cells and EGFR mutated lung cancer HCC827 cells (
[0420] Using both organoid and monolayer cultures, we treated HCC827 cells with serially diluted osimertinib for three days to observe their acute treatment responses. We found that a subpopulation of tumor cells survived cytotoxic doses (0.01-2 μM) of osimertinib treatment initially (
[0421] To better understand the transcriptomic changes and tumor heterogeneity conferring osimertinib or gefitinib tolerance in lung cancer, we developed single cell-derived clones in PC9 (
[0422] To exclude the possibility that pre-existing cellular heterogeneity could be responsible for this tolerance, we made single cell clones first followed by exposure to 2 μM gefitinib. In parallel, as in the previous experiment, PC9 cells were cloned in the same concentration of gefitinib (parental clones) as control. All tested single cell-derived clones generate gefitinib-tolerant clones at a frequency of 1.9˜2.1% (n=4 clones), which is comparable to that in parental PC9 clones (2.2 t 0.1%) (
MicroRNA-147b Initiates Anticancer Drug Tolerance
[0423] To test which microRNAs (miRNAs) are linked to osimertinib-tolerance, we performed miRNA-seq analysis in two paired osimertinib-tolerant cells and osimertinib-sensitive parental cells from HCC827 and PC9. A list of differentially expressed miRNAs (n=45) relevant to osimertinib-tolerance was derived from this analysis. The top upregulated miRNAs included miR-181a-2-3p, miR-147b, miR-574-5p and the top downregulated miRNAs included miR-7641-1, miR-4454, and miR-125b-1-3p (
[0424] As expected, our qRT-PCR analysis validated the up to five-fold upregulation of miR-147b expression in gefitinib- and osimertinib-tolerant cells compared with parental cells in both PC9 and HCC827 (
[0425] EGFR and KRAS mutations are widely known as mutually exclusive in lung cancer patients, and mutations in KRAS are associated with a lack of sensitivity to gefitinib (Pao et al., PloS Med. 2(1):e17, 2005). EGFR-TKI tolerant cells still respond to EGFR inhibitors at higher concentrations (
[0426] Furthermore, to study the functional roles of miR-147b in regulating drug-tolerance, we overexpressed lentiviral miR-147b in HCC827 cells. We found that the enforced overexpression of miR-147b enhanced drug-tolerance by 60-fold and 30-fold at the half-maximum inhibitory concentration (IC50) of osimertinib and gefitinib, respectively (
miR-147b-VHL Axis Confers Drug-Tolerance
[0427] To study which genes are repressed by miR-147b directly, we performed sequence-based target prediction using the TargetScan tool. The predicted targets were then analyzed to match the signaling pathways for drug-tolerance (
[0428] Next, we designed a dual-luciferase assay based on the VHL 3′UTR, wild-type and mutant in those predicted 3′UTR miR-147b binding sites (
[0429] Furthermore, we checked the VHL protein level in miR-147b overexpressing cells in AALE. The VHL protein levels decreased only two-fold when miR-147b was overexpressed in AALE cells (
Tricarboxylic Acid Pathways Mediate Drug Tolerance and Depend on miR-147b
[0430] In addition to the functional roles of VHL-mediated “pseudohypoxia gene signature” in drug-tolerance, we hypothesized that another predicted target of miR-147b, SDH might also mediate drug-tolerance induced by miR-147b through its impact on the TCA cycle. To test this hypothesis, we first designed a dual-luciferase assay based on the SDHD 3′UTR, wild-type and mutant in the predicted miR-147b 3′UTR binding sites (
[0431] SDHD, one of the subunits of SDH complex, catalyzes the conversion of succinate to fumarate and regulates both the TCA cycle and the ETC. We asked whether miR-147b-SDHD axis mediated drug-tolerance could impact on the metabolite changes in metabolic pathways. To answer this question, the human lung adenocarcinoma cell line H1975 harboring with EGFR T790M; L858R mutations was used for a metabolomics study. Cells with either EGFR L858R or EGFR T790M are sensitive to osimertinib. The osimertinib-tolerant cells (H1975OTR) were derived from parental H1975 treated with 100 nM osimertinib for 21 days in monolayer cultures. H1975OTR cells are stable and continue to proliferate even in the presence of 100 nM osimertinib. As a control, H1975 cells were treated with vehicle for 21 days. Then we performed a LC/MS metabolomics study using the paired H1975 and H1975OTR cells (
[0432] Then we asked whether the perturbed metabolite changes could be rescued by blocking miR-147b in drug-tolerant cells. To address this question, we knocked down miR-147b in drug-tolerant cells in H1975 and analyzed the metabolic changes with LC/MS tool. As expected, the increased levels of succinate and 2-oxoglutarate, as well as the decreased levels of metabolites such as fumarate, malate, NAD+, and GSH, were partially rescued by knocking down miR-147b on osimertinib-tolerant cells (
[0433] Then we asked whether the metabolomic changes in the monolayer cultures are reproducible in the 3D organoid models. To address this question, we established drug-tolerant organoids and parental organoids by continuous treatments with 100 nM osimertinib or vehicle for 21 days on H1975 cells and performed a LC/MS metabolomics study. Consistently, the levels of fumarate, malate, and NAD+ were reduced in osimertinib-tolerant organoids. Knockdown of miR-147b rescued the decreased levels of the above metabolites in those tolerant organoids (
Blocking miR-147b Overcomes Drug Tolerance
[0434] To understand the functional roles of miR-147b in driving EGFR-TKI tolerance and resistance, we perturbed miR-147b expression using lentiviral inhibitors against miR-147b in H1975 organoids that are partially sensitive to osimertinib. We found that knocking down miR-147b alone or osimertinib administration alone decreased the total number of organoids by two-fold (p<0.01). Following co-treatment with osimertinib and miR-147b inhibition, the number of organoid was decreased by up to 18-fold compared with the control group (p<0.01). This suggests that miR-147b inhibition is synergistic with osimertinib in overcoming TKI-tolerance. Then we treated H1975 cells with serially diluted doses of osimertinib. Our data demonstrated that the IC50 value decreased 166-fold in H1975 cells with miR-147b knockdown compared with the control group. This shows that blocking miR-147b sensitizes H1975 cells towards osimertinib. Unexpectedly, miR-147b knockdown did not decrease 2D-monolayer cell proliferation at high cell-density except at clonal cell-density in H1975 cells. Clonal tumor initiation capacity and clonal long-term repopulation are the principal properties of TICs in cancers.
[0435] We hypothesized that functions of miR-147b in driving EGFR-TKI tolerance and resistance are conferred by cancer sternness and TICs. To test this hypothesis, we applied cancer-stemness assays including spheroid-formation assay and limiting-dilution analysis here. Knocking down miR-147b decreased the TICs frequency by seven-fold from 1/11.8 (8.5%) to 1/83.1 (1.2%) (p<0.01). As expected, combinational therapy with miR-147b inhibition and osimertinib almost abolished all those osimertinib-tolerant tumor spheroids and tumor colonies. Consistently, RNA-seq analysis demonstrated that miR-147b knockdown decreased expression levels of stemness-related genes, including activated leukocyte cell adhesion molecule (ALCAM), glycine decarboxylase (GLDC), thyroid transcription factor 1 (TTF1), and AXL receptor tyrosine kinase (AXL). Thus, the osimertinib-tolerance is conferred by miR-147b and cancer-stemness in lung cancer.
[0436] Furthermore, we found that mir-147b overexpression enhances malignant transformation and EGFR-TKI tolerance and resistance. First, to understand whether overexpression of miR-147b is linked to lung cancer patient survival, we performed prognosis analysis using the TCGA data portal (http://cancergenome.nih.gov/) and oncoLnc resource. The hazard ratio of miR-147b-high/low was 1.5 (95% confidence interval 1.1-2.2) (p<0.05). Next, we asked whether overexpression of miR-147b would make TKI-sensitive cells more tolerant towards TKIs. To address this question, we used lentiviral vectors with miR-147b to enforce the overexpression of miR-147b. The expression level of miR-147b increased 15-fold in HCC827 cells by qRT-PCR analysis. Using a colony-formation assay, we found that miR-147b overexpression increased colony formation by three-fold. As expected, the frequency of stem-like cells in HCC827 cells increased three-fold from 1/12.69 to 1/4.67 by limiting-dilution assay. To quantify the potential effects of miR-147b overexpression on tolerance to osimertinib treatment, we performed an IC50 assay and found that enforced expression of miR-147b enhanced osimertinib-tolerance in H1975 cells. Further, a clonogenicity assay with osimertinib or gefitinib treatment demonstrated that miR-147b overexpression rescued the osimertinib/gefitinib-induced reduction of colony formation. Thus, it suggests that miR-147b overexpression is important for enhanced osimertinib-tolerance via enhancing cancer sternness.
[0437] Next, to understand the potential oncogenic roles of miR-147b, we utilized lung patient-derived xenograft (PDX) tumors directly to analyze the expression of miR-147b. We demonstrated that the miR-147b expression levels in PDX tumors were up to 160-fold higher than normal lung tissues. Then we overexpressed miR-147b using a lentiviral vector in an immortalized human normal lung epithelial cell AALE with undetectable expression level of miR-147b. Overexpression of miR-147b was seen at 43-fold, and enhanced AALE cell proliferation by 1.4-fold on day six. Measuring DNA synthesis is the most precise way to detect changes in cell proliferation. An image-based proliferation assay demonstrated that EdU-positive cells were up two-fold in miR-147b-overexpressing cells compared with scrambled control cells. More interestingly, AALE cells with miR-147b overexpression that were grown for three consecutive passages grew in spherical colonies containing approximately 100 cells spontaneously, while cells expressing the scrambled control grew in flat monolayers. This indicates that miR-147b may enhance the anchorage-independent growth in AALE cells. Then we hypothesized that miR-147b might also decrease the dependence on growth factors for cell growth. EGF is the crucial growth factor for epithelial cells development and growth. To support this hypothesis, we starved the cells in EGF-free media overnight and then tested the growth of cells in media containing various concentrations of EGF. Our data demonstrated that the growth of miR-147-overexpressing cells was less dependent on EGF (R.sup.2=0.46) compared with scrambled cells (R.sup.2=0.72) significantly (p<0.05). It suggests that miR-147b overexpression could rescue EGF-withdrawal-induced proliferation reduction. Consistently, RNA sequencing analysis showed that miR-147b overexpression increased proliferation-promoting genes including EGFR, MYC, ID1, and NOTCH1 and decreased proliferation-inhibitory genes such as BMP4. The apoptosis-inhibitory genes such as RIPK3 were elevated and the apoptosis-promoting genes such as CD40 were decreased in cells with miR-147b overexpression compared with control cells.
[0438] We asked whether miR-147b is a druggable target in lung cancer. First, we knocked down miR-147b with a lentiviral miRNA inhibitor in H1975 cells and transplanted those cells into nude mice. The tumor growth in the cohort with miR-147b knockdown was up to two-fold slower compared with that in the control group (
[0439] Additionally, analysis of GTEx (https://www.gtexportal.org/home/) RNA-seq in 53 tissues from 570 human healthy donors demonstrated that cells and tissues expressing the highest levels of miR-147b are transverse colon, small intestine, and esophagus. The remaining tissues, including lung tissue, express low levels of miR-147b. VHL is moderately expressed in normal lung tissues and other normal tissues indicating that miR-147b-VHL axis might be therapeutic targets that are crucial for tumor initiation and maintenance.
[0440] Furthermore, to understand functional roles of miR-147b in regulating drug-tolerance via regulation of a pseudohypoxia signaling pathway, we blocked miR-147b by administration of locked nucleic acid (LNA) miRNA inhibitors, as well as perturbing pseudohypoxia signaling with small molecule activators and inhibitors. First, LNA-miR-147b inhibitor treatment increased the sensitivity of drug-tolerant organoids to osimertinib by 30-fold compared with the control group in H1975 (
[0441] To understand roles of HIF-1 or HIF-2 in the osimertinib tolerant state, we knocked down HIF1A and HIF2A/EPAS1 (endothelial PAS domain protein 1) using lentiviral shRNAs in H1975 cells and investigated their effect on osimertinib response. Our results showed that HIF1A knockdown increased cell sensitivity up to 2.6-fold towards osimertinib (
[0442] Lastly, we asked whether we could delay drug-tolerance to EGFR-TKIs by targeting miR-147b. To address this question, organoids obtained from PDX lung tumors were tested. Among these PDX lung tumors, one EGFR T790M mutated PDX tumor-derived organoid (PDX_LU_10) at passage two was tested in the following functional study (
Methods
[0443] Cell Culture. Human lung EGFR-wild type cell lines H358, H460, A549, H1299, and H69 (ATCC) as well as EGFR-mutant cell lines H1650, H1975, HCC827, PC9, PC9ER, and H3255 (provided by S.K.) were cultured in DMEM (high glucose) (GIBCO) with 10% FBS, 2 mM L-glutamine and 1% penicillin-streptomycin. Immortalized tracheobronchial epithelial AALE cells (provided by W.C.H.) were derived as previously described (Lundberg et al., Oncogene 21(29):4577-4586, 2002) and maintained in SAGM media (Lonza). Each cell line was maintained in a 5% CO.sub.2 atmosphere at 37° C. Cell line identities were confirmed by STR fingerprinting and all were found negative for mycoplasma using the MycoAler Kit (Lonza).
[0444] Mice. All research involving animals complied with protocols approved by the BIDMC Biological Resource Center Institutional Animal Care and Use Committee. 4-6 weeks old female nude immunodeficient mice (Jackson Laboratory) were used for subcutaneous injections. For subcutaneous xenograft tumor assay, 100,000 cells in serum-free medium and growth factor reduced Matrigel (BD) (1:1) were inoculated into the flank of nude mice. The xenograft tumor formation was monitored by calipers twice a week. The recipient mice were monitored and euthanized when the tumors reached 1 cm in diameter.
[0445] Patient-derived Xenograft Tumor Specimens. Tumor samples from patient-derived xenografts (PDXs) were generated at The Jackson Laboratory and the Yale Cancer Center by subcutaneous implantation of previously passaged tumors in up to 5 female NSG mice. When tumor samples reached 1000 mm.sup.3 they were shipped to the laboratory in frozen media of DMEM with 90% FBS and 10% DMSO in dry ice. Samples were washed with cold phosphate buffer saline (PBS) with antibiotics (Sigma-Aldrich, St. Louis, Mo.) three times, chopped with a sterile blade, and incubated in 0.001% deoxyribonuclease (DNase) (Sigma-Aldrich, St. Louis, Mo.), 1 mg/ml collagenase/dispase (Roche, Indianapolis, Ind.), 200 U/ml penicillin, 200 μg/ml streptomycin, 0.5 μg/ml amphotericin B (2% antibiotics, Sigma) in DMEM/F12 medium (GIBCO, Grandlsland, N.Y.) at 37° C. water bath for 3 hours with intermittent shaking. After incubation, the suspensions were repeatedly triturated, passed through 70 μm and 40 μm cell-strainers (BD Falcon, San Jose, Calif.), and centrifuged at 122 g for 5 minutes at 4° C. Cells were resuspended in red blood cell lysis buffer (eBioscience, San Diego, Calif.) for 4 minutes at room temperature with intermittent shaking, before resuspension in serum-free medium. After lysis, cell viability was evaluated by trypan blue dye exclusion. Live single cells accounted for 90% of the whole population and dead cells accounted for less than 10%. Each tumor sample yielded ˜1×10.sup.5 to 1×10.sup.8 cells, depending on the sample size.
[0446] Antibodies. For immunofluorescence staining, primary mouse anti-human ZO-1 (1:100, cat #339100) was from Thermo Fisher Scientific. Secondary goat anti-mouse IgG conjugated with Alexa Fluor 488 (1:500, cat #A-11055) was from Invitrogen. For western blot, primary rabbit anti-VHL antibody (1:100, Cat #PA5-27322) was from Thermo Fisher Scientific. Mouse anti-β-actin (1:5,000, clone C4, Santa Cruz, sc-47778) was used as loading control. IRDye 680RD goat anti-rabbit (1:20,000, LI-COR926-68171, LI-COR Biosciences) and IRDye 800CW goat-anti-mouse (1:20,000, LI-COR827-08364, LI-COR Biosciences) were used as secondary antibodies.
[0447] 3D Spheroids and Organoids. For 3D spheroid formation, single-cell suspensions (10,000 cells/well) were plated in 6-well ultra-low attachment (Corning) or non-treated cell culture plates (Nunc) in DMEM/F12 medium containing 2 mM L-glutamine, 15 mM HEPES, 1 mg/ml NaHCO.sub.3, 0.6% Glucose, 1% NEAA, 4 mg/nl BSA (Sigma), ITS (0.05 mg/ml insulin/transferrin/selenous acid, BD Biosciences), 1% antibiotics (Sigma), 50 ng/ml EGF, and 20 ng/ml FGF2 (Invitrogen). Fresh medium was replenished every 3 days. Spheroids were cultured for 10-14 days and then quantified. For passaging, spheroids were digested by accutase (Chemicon) into single cells and re-plated into the above plates. For limiting dilution assays, 200, 600, and 1800 cells were plated to assess spheroid formation.
[0448] For 3D organoid formation, single-cell suspensions (2000 cells/well/20 μl) were co-plated with geltrex (25 μl) in 96-well non-treated clear plates (Corning, cat #08-772-53). The plate was incubated for 20 minutes at 37° C. followed by adding 100 μl complete growth media. The complete growth media was advanced DMEM/F12 with glutamax (1×), HEPES (1×), 1.25 mM N-Acetylcysteine, 10 mM Nicotinamide, 10 μM Forskolin, B27 (1×), 5 ng/ml Noggin, 100 ng/ml FGF10, 20 ng/ml FGF2, 50 ng/ml EGF, 10 ng/ml PDGFA, 10 ng/ml FGF7, 1% penicillin-streptomycin, and 10 μM Y-27632. Y-27632 was used only for the initial three days because Y27632 is a rock inhibitor preventing apoptosis of single cells (Watanabe et al., Nat. Biotechnol. 25(6):681-686, 2007). PDGFA and FGF7 were not used until day 7 in organoid cultures because they are important for alveolarization during late lung development (Padela et al., Pediatr. Res. 63(3):232-8, 2008; Bostrom et al., Cell 85(6):863-873, 1996). FGF10 is essential for maintenance of lung progenitor cells and branching morphogenesis as well as tissue homeostasis in the adult lung (Sekine et al., Nat. Genet. 21(1):138-141, 1999). EGF and FGF2 are mitogens for growth of epithelial cells and used for maintaining lung tumor-initiating cells previously by us (Zhang et al., Cell 148(1-2):259-272, 2012). Noggin binds and inactivates bone morphogenetic protein-4 and is involved in the development of the lungs (Krause et al., Int. J. Biochem. Cell Biol. 43(4):478-481, 2011). The media was changed every three days in 24 days. The organoids were photographed with a microscope (Evos Fla., Life Technology) and their size was measured by ImageJ.
[0449] Colony Formation Assay in Plate. Single cells were plated in 10 cm dish in triplicates with 20, 40, 80, or 300 cells per dish. Fresh medium was replenished every 3 days. The cells were incubated for 10-12 days followed by Giemsa (Sigma) staining. The plates were air-dried, photos taken, and the total number of colonies was analyzed by openCFU (opencfu.sourceforge.net).
[0450] Single Cell-Derived Clones of PC9 and HCC827 Cells. In PC9 and HCC827 cells, a single cell was sorted into a 96-well plate at one cell per well by fluorescence-activated cell sorting (FACS) using a FACSAria (BD). The single cell in each well was confirmed under a microscope 12 hours after sorting. Gefitinib or osimertinib were administrated to both parental clones and single-cell clones. In parental clones, the single cells were treated immediately with 0.1, 0.4, and 2 μM gefitinib, osimertinib, or vehicle for 14 days on the second day (n=192 wells per group). In single-cell clones, clones were made first, and then exposed to 0.1-2 μM gefitinib, osimertinib, or vehicle for 14 days. Drug responses of the surviving clones were determined by measuring an IC50. The frequency of colony formation was calculated as a ratio of the total number of colonies (consisting of more than 50 cells) to the total number of wells plated with a single cell. Medium and smal molecule inhibitors were replenished every three days. One parental single-cell derived clone treated with vehicle that was sensitive to gefitinib and two gefitinib-tolerant single-cell derived clones were randomly selected and applied for the following whole transcriptome analysis by microarray. Four single-cell clones from PC9 and HCC827 were established from the above were used for drug-tolerance assay.
[0451] Compounds. Osimertinib (S7297) and gefitinib (S1025) were purchased from Selleck Chemicals. DMOG (Jaakkola et al., Science 292(5516):468-472, 2001) (Cat #400091) was from Calbiochem. R59949 (Temes et al., J. Biol. Chem. 280(25):24238-24244, 2005) (Cat #D5794) and dimethyl malonate (Mills et al., Cell 167(2):457-470, 2016; Dervartanian et al., Biochim. Biophys. Acta. 92:233-247, 1964) (DMM, Cat #136441) were purchased from Sigma-Aldrich.
[0452] Compound Treatment. Cell viability experiments were performed in 96-well format using opaque white plates (Corning). For 2D monolayer cell cultures and organoids, cells were plated into 96-well plates with 100-2000 cells per well in three-four replicates on day 0. Twenty-four hours after seeding, cells or organoids were exposed to compounds at indicated concentrations for 72 hours. Cellular ATP levels (as a surrogate for viability) were measured using CeliTiter-Glo (Cat #G7570, Promega) or CeliTiter-Glo 3D (Cat #G9681, Promega). For co-treatment experiments, spent medium was removed 24 hours after cell seeding and replaced with medium containing a single concentration of the modulator of interest (for example, osimertinib).
[0453] To establish gefitinib and osimertinib tolerant cells, PC9 single-cells were treated with 20 nM osimertinib and 40 nM gefitinib for 12-14 days, HCC827 cell monolayers and organoids were treated with 20-160 nM osimertinib for 12-21 days, and H1975 cell monolayers and organoids were treated with 25 nM-1 μM osimertinib for 12-21 days. To study effects of organoid culture stages on outcome of drug-tolerance, both single-cells (grown for 1 day) and established organoids from HCC827 cells (grown for 24 days) were made first followed by 100 nM osimertinib treatment for additional 21 days. Medium was replenished every three days.
[0454] RNA Extraction and Real-Time PCR. Total RNA was extracted from solid tissues and cultured cells using mirVana™ miRNA Isolation Kit (Ambion #AM1561) according to the manufacturer's instructions. A total of 10 ng RNA each sample was input for consecutive reactions including Poly(A) Tail reaction, Ligation reaction, Reverse Transcription reaction, and miR-Amp reaction using the Taqman Advanced miRNA cDNA synthesis kit (Applied Biosystems #A28007). Then miRNA expression was assessed by Taqman Advanced microRNA Assay and the Taqman Fast Advanced miRNA master mix (Applied Biosystems #4444557). The PCR reaction plate was run in a real-time PCR instrument (Roche Lightcycler 480 system) according to the manufacturer's instructions. Three biological replicates were applied for each sample. MiRNA expression was assessed by Taqman Fast Advanced MicroRNA Assay, and the gene expression of mRNAs was evaluated by Taqman Probes (Applied Biosystems). Taqman miRNA probes were as follow: hsa-miR-147b (478717_mir) and hsa-miR-423-5p (478090_mir). hsa-miR-423-5p was used as endogenous control. Taqman gene-expression probes were as follow: ID2 (Hs04187239_m1), SFTPC (Hs00951326_g1), HOPX (Hs05028646_s1), NKX2.1 (Hs00968940_m1), CEACAM5 (Hs00944025_m1), LIN28B (Hs01013729_m1), EPAS1 (Hs01026149_m1), VHL (Hs03046964_s1), KRT17 (Hs00356958_m1), CA9 (Hs00154208_m1), WNT5A (Hs00998537_m1), WNT4 (Hs01573505_m1), EGLN3 (Hs00222966_m1), SLC2A1 (Hs00892681_m1), SLC2A3 (Hs00359840_m1), LOX (Hs00942483_m1), CS (Hs02574374_s1), TCEB1 (Hs00855349_g1), CAD (Hs00983188_m1), CDKN1A (Hs00355782_m1), IDH3A (Hs00194253_m1), SPRY4 (Hs01935412_s1), FZD7 (Hs00275833_s1), FZD2 (Hs00361432_s1), UBC (Hs05002522_g1), RAC1 (Hs01902432_s1), P4HA1 (Hs00914594_m1), P4HA2 (Hs00990001_m1), ADM (Hs00969450_g1), BNIP3L (Hs00188949_m1), ANKRD37 (Hs00699180_m1), NDRG1 (Hs00608387_m1), DCBLD1 (Hs00543575_m1), KCTD11 (Hs00922550_s1), BNIP3 (Hs00969291_m1), VEGFA (Hs00900055_m1), ALDOA (Hs00605108_g1), PFAS (Hs00389822_m1), GLS (Hs01014020_m1), GLUD1 (Hs03989560_s1), ASNSD1 (Hs00219383_m1), GMPS (Hs00269500_m1), NIT2 (Hs00252405_m1), ACLY (Hs00982738_m1), ACO2 (Hs00426616_g1), PDHA1 (Hs01049345_g1), OGDH (Hs01081865_m1), FH (Hs00264683_m1), SDHA (Hs00417200_m1), SDHB (Hs01042478_g1), SDHC (Hs01698067_s1), SDHD (Hs00829723_g1), SDHAF2 (Hs00215235_m1), DLAT (Hs00898876_m1), DLST (Hs04276516_g1), ISCU (Hs00384510_m1), TCEA3 (Hs00957468_m1), SLC1A4 (Hs00983079_m1), CDC14B (Hs00372920_m1), CDCA4 (Hs00937497_s1), GSTO2 (Hs01598184_m1), NDUFA4 (Hs00800172_s1), NDUFA11 (Hs00418300_m1), ACTB (Hs01060665_g1) and GAPDH (Hs02786624_g1). ACTB was used as endogenous control.
[0455] Pyrosequencing for quantitative analysis of sequence variations. The parental cells, gefitinib-tolerant cells, and gefitinib-resistant cells in PC9 were extracted for DNA (QIAamp DNA blood mini kit, Cat #51104, Qiagen) and analyzed for pyrosequencing. The methods were described as previously (Koontz et al., BMC Med. Genet. 10:80, 2009).
[0456] High-Throughput Sequencing. The total RNA samples (1 μg) were processed by LC Sciences for microRNA sequencing (miRNA-seq). All RNA samples were analyzed for quality on an Agilent 2100 Bioanalyzer.
[0457] For miRNA-seq, paired osimertinib-tolerant cells and parental cells (treated with 20 nM osimertinib or vehicle for 14 days) from HCC827 and PC9 cells were applied. The RNA samples were processed utilizing Illumina's TruSeq small RNA sample preparation protocol for small RNA library generation (Part #15004197 Rev. F, Cat #RS-200-9002DOC). The subsequent sequencing was performed on the HiSeq 2500 platform for 1×50-nt single-end sequencing and the sequencing adaptor was trimmed from the raw reads. The reads were then mapped to the miRBase v21 (http://www.mirbase.org/) and the human genome (GRCh37) using Bowtie (Langmead et al., Genome Biol. 10(3):R25, 2009). The mapping results were summarized using an in-house script to estimate the number of reads mapped to each miRNA. Normalization was done using the median of the ratio of the read count to the geometric mean of read counts across samples as implemented in DESeq (Anders et al., Genome Biol. 11(10):R106, 2010).
[0458] Whole Transcriptome Analysis by microarray. The Illumina Whole Human Genome Microarray Kit (HumanHT-12 v4 Expression BeadChip, Cat #BD-103-0204) was used to identify differentially expressed genes in single-cell clones from PC9. Amplification of RNA, hybridization, image processing, and raw data extraction: The Illumina TotalPrep RNA Amplification kit (Ambion, UK) was used for all samples using 200 ng of total RNA as starting material. Briefly, the procedure consisted of a reverse transcription step using an oligo (Dt) primer bearing a T7 promoter and the high yield ArrayScript™ reverse transcriptase. The cDNA then underwent second strand synthesis and clean-up to become a template for in vitro transcription with T7 RNA Polymerase and biotin-NTP mix. Labelled cRNA was then cleaned up and 1.5 μg were hybridized to humanHT12_V4 beadarrays (Illumina, CA, USA) for 16 hours at 55° C. Following hybridization, beadarrays were washed and stained with streptavidin-Cy3 (GE Healthcare, UK). Fluorescent images were obtained with a Beadarray reader and processed with the BeadScan software (Illumina, CA, USA). The whole transcriptome raw data were obtained from the GenomeStudio software with the subtraction of the background. All mRNA raw data were normalized based on the Cross-Correlation method (Chua et al., Nucleic Acids Res. 34(5):e38, 2006). Significantly changed mRNAs were identified based on average fold change cutoff of 1.5 and the cutoff of the p value cross all replicates at 0.05.
[0459] Two-Color Western Blot and Chemical Reagents. Cells were harvested and lysed with RIPA buffer (Radio Immuno Precipitation Assay buffer) supplemented with protease and phosphatase inhibitor cocktail (Roche). Protein concentrations of the extracts were measured using BCA assay (Pierce) and equalized with the extraction reagent. Equal amount of the extracts was loaded and subjected to SDS-PAGE, transferred onto Immobilon-FL PVDF membranes. The PVDF membranes were air-dried for 1 hour at room temperature followed by rehydration. The membranes were blocked with Odyssey Blocking Buffer for 1 hour and then incubated with primary antibodies in cold room overnight. Then IRDye 680RD goat anti-rabbit (1:20,000, LI-COR926-68171) and IRDye 800CW goat-anti-mouse (1:20,000, LI-COR827-08364) were used as secondary antibodies. Then the images were scanned with Odyssey Family Imaging System (LI-COR Biosciences). Western blot quantification was performed by Image Studio Lite (LI-COR Biosciences).
[0460] Transfection by LNAs in vitro. Tumor cells were plated at 2,000 cells in complete growth medium in a 96 well plate to reach 50-60% confluence. 0˜120 nM of fluorescein-conjugated LNA anti-miR-147b (Sequence: AGCAGAAGCATTTCCGCACA; SEQ ID NO: 890) (Cat #4100977-011) or negative control (Sequence: TAACACGTCTATACGCCCA; SEQ ID NO: 891) (Cat #199006-011, Exiqon) with PureFection (System Biosciences) were applied for transfection. The transfected cells were harvested after culturing for 48 and 72 hours.
[0461] HIF1A and EPAS1 shRNAs and cDNA transfection. H1975 cells were seeded in a 6-well plate at 100,000 cells per well one day prior to transfection. A mixture of 2.5 μg pGFP-C-shLenti vector targeting HIF1A (OrGene, Cat #320380), EPAS1 (OriGene, Cat #TL315484), scrambled negative control (Cat #TR30021), lentiviral vector targeting HIF1A mutantA588T (OrGene, Cat #RC402571), control vector and 7.5 μL of PureFection (System Biosciences, Cat #LV750A-1) were used for transfection. The transfected cells were selected and maintained in 0.5 μg/ml puromycin (for shRNAs) or 600 μg/ml neomycin (for HIF1A A5887) in DMEM containing with 10% FBS for 9 days. Then the stable cells were passaged into 96-well plate at 3,000 cells per well followed by treatment with 100 nM osimertinib for 3 days. hsa-HIF1A targeting sequences: shRNA 1: AGCTTGCTCATCAGTTGCCACTTCCACAT (SEQ ID NO: 892), shRNA 2: AGGCCACATTCACGTATATGATACCAACA (SEQ ID NO: 893), shRNA 3: TACGTTGTGAGTGGTATTATTCAGCACGA (SEQ ID NO: 894), shRNA 4: ACAAGAACCTACTGCTAATGCCACCACTA (SEQ ID NO: 895). hsa-EPAS1 targeting sequences: shRNA 1: GTATGAAGAGCAAGCCTTCCAGGACCTGA (SEQ ID NO: 896), shRNA 2: AGCACTGCTTCAGTGCCATGACAAACATC (SEQ ID NO: 897), shRNA 3: CCTGGTGGCAGCACCTCACATTTGATGTG (SEQ ID NO: 898), shRNA 4: GGCTGTGTCTGAGAAGAGTAACTTCCTAT (SEQ ID NO: 899).
[0462] Transient Transfection and Dual-Luciferase Assay. PureFection (System Biosciences) was used for transient transfection. 100 ng of wild-type or mutant 3′UTR reporter constructs of VHL or SDHD constructs (GeneCopoeia) were transfected into H1975 cells with 120 nM of LNA anti-miR-147b or negative control. Firefly and Renilla luciferase activities were measured 48 hours post-transfection using Dual-Luciferase Reporter System (Promega). The firefly luminescence was normalized to Renilla luminescence as an internal control for transfection efficiency. MiR-147b binding site CGCAC (SEQ ID NO: 900) was substituted with GCGTG (SEQ ID NO: 901) in mutated VHL and binding site CGCACA (SEQ ID NO: 28) was substituted with GCGTGT in mutated SDHD.
[0463] Lentiviral-mediated miRNA and VHL Overexpression or Knockdown Infection. For lentiviral overexpression or knockdown of miR-147b, cells (AALE, HCC827, H1975, and PC9ER) were infected with the lentiviral particles (Applied Biological Material Inc., ABM) for 48 hours in the presence of 1:100 Viralplus transduction enhancer (ABM) and 8 μg ml.sup.−1 polybrene (Sigma). Two days after infection, puromycin was added to the media at 0.5 μg ml.sup.−1, and cell populations were selected for 1-2 weeks. For lentiviral overexpression of VHL, cells (HCC827) at 70% confluence were transduced with VHL lentiviral particles (1.6×10.sup.8 TU ml.sup.−1, ABM) or blank control lentiviral particles (2×10.sup.6 TU ml.sup.−1, ABM) together with polybrene. Then the infected cells were passaged and selected by puromycin (Invitrogen) at 0.5 μg ml.sup.−1 for 1-2 weeks.
[0464] crRNA:tracrRNA transfection. H1975-Cas9 cells were generated with plenti-EF1a-Cas9 lentiviral particles (ABM, Cat #K003) and maintained in 0.5 μg/ml puromycin in DMEM containing with 10% FBS. H1975-Cas9-intergrated cells were seeded in a 98-well plate at 3,000 cells per well one day prior to transfection. Edit-R-synthetic crRNA (CRISPR RNA) targeting MIR147B (GE Healthcare Dharmacon, Cat #crRNA-413428, 413429, 413430 and 413431), non-targeting control (Cat #U-007501-01-20) and tracrRNA (trans-activating CRISPR RNA) (Cat #U-002005-20) were individually resuspended in 10 mM Tris-HCl pH7.5 to a concentration of 100 uM. crRNA and tracrRNA were obtained at equimolar ratio and diluted to 2.5 μM using 10 mM Tris-HCl pH7.5. A final concentration of 50 nM crRNA-tracrRNA complex was used for transfection. Cells were transfected using 0.4 μL/well of DharmaFECT Duo transfection reagent (GE Healthcare Dharmacon, Cat #T-2010-02). hsa-miR-147b targeting sequences:
TABLE-US-00005 crRNA 1: (SEQ ID NO: 902) 5′ AGAGTACTCTATAAATCTAG 3′, crRNA 2: (SEQ ID NO: 903) 5′ TTTCTGCACAAACTAGATTC 3′, crRNA 3: (SEQ ID NO: 904) 5′ AGATTCTGGACACCAGTGTG 3′, and crRNA 4: (SEQ ID NO: 905) 5′ GCAGAAGCATTTCCGCACAC 3′.
[0465] H&E Staining and Immunofluorescence. Samples were formalin-fixed, paraffin-embedded, sectioned, and stained with hematoxylin-eosin (H&E) according to standard histopathological techniques. For immunofluorescence, organoids were fixed and then incubated with mouse anti-ZO-1 (Thermo Fisher Scientific), washed, then incubated with anti-mouse IgG-Alexa Fluor 488 (Invitrogen). The organoids were counterstained with Hoechst 33342. Z-stack images were acquired with 2 μm slice interval and 3-D projection was created with a confocal microscope (Zeiss LSM 880).
[0466] Metabolite extraction. For collecting adherent cells from 10-cm dishes, the metabolomics samples were prepared according to a previous method (Yuan et al., Nat. Protoc. 7(5):872-881, 2012). Briefly, the growing cells at 80% confluence were incubated with 80% methanol at −80° C. for 15 minutes. The cell lysate/methanol mixture were transferred to 15 mL conical tubes and centrifuged at 4500 g at 4° C. for 15 minutes in cold room to pellet cell debris and proteins. The centrifugation was repeated twice, and all three extractions were pooled together. The supernatants were completely dried by speedVac and were further processed for LC-MS analysis. Five biological replicates were used in each group and the analysis was normalized with the same number of cells of each group.
[0467] For collecting organoids, the above method was modified. Briefly, single cells mixed with geltrex were plated into six-well low attachment plates (Nunc) and incubated with complete media for 21 days. Next, the organoids/geltrex mixtures were incubated with TrypLE Express (Gibco) at 37° C. for 5 minutes to separate geltrex from organoids. The supernatants were aspirated after centrifuge at 188 g for 5 minutes. Then the organoid pellets were incubated with 80% methanol at −80° C. for 30 minutes. The cell lysate/methanol mixture were transferred to 15 mL conical tubes and centrifuged at 4500 g at 4° C. for 15 minutes to pellet cell debris and proteins. The centrifugation was repeated twice, and all three extractions were pooled. The supernatants were completely dried by speedVac and were further processed for LC-MS analysis. Five biological replicates were used in each group and the analysis was normalized with the same number of cells of each group.
[0468] Targeted Mass Spectrometry. Samples were re-suspended using 20 mL HPLC grade water for mass spectrometry. 5-7 μL were injected and analyzed using a hybrid 5500 QTRAP triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC system (Shimadzu) via selected reaction monitoring (SRM) of a total of 274 unique endogenous water-soluble metabolites for steady-state analyses of samples. Some metabolites were targeted in both positive and negative ion mode for a total of 306 SRM transitions using positive/negative ion polarity switching. ESI voltage was +4900V in positive ion mode and −4500V in negative ion mode. The dwell time was 3 ms per SRM transition and the total cycle time was 1.65 seconds. Approximately 9-13 data points were acquired per detected metabolite. Samples were delivered to the mass spectrometer via hydrophilic interaction chromatography (HILIC) using a 4.6 mm i.d.×10 cm Amide XBridge column (Waters) at 400 μL/minute. Gradients were run starting from 85% buffer B (HPLC grade acetonitrile) to 42% B from 0-5 minutes; 42% B to 0% B from 5-16 minutes; 0% B was held from 16-24 minutes; 0% B to 85% B from 24-25 minutes; 85% B was held for 7 minutes to re-equilibrate the column. Buffer A was comprised of 20 mM ammonium hydroxide/20 mM ammonium acetate (pH=9.0) in 95:5 water acetonitrile. Peak areas from the total ion current for each metabolite SRM transition were integrated using MultiQuant v2.1 software (AB/SCIEX). Further informatics analysis was performed with online MetaboAnalyst 3.0 software (Xia et al., Curr. Protoc. Bioinformatics 55:14.10.1-14.10.91, 2016).
[0469] Statistical Analysis. No statistical methods were used to predetermine sample size. For mouse experiments, the mice were not randomized. The investigators performing tumor volume measurements were blinded. All experiments were performed in two to five biological replicates, and independently reproduced as indicated in figure legends. Data are presented as the means t SEM. Unless otherwise stated, statistical significance was determined by a Student's two-tailed t-test by GraphPad Prism 6. P<0.05 was considered statistically significant. For two samples that are not normally distributed, Mann-Whitney test was applied for a comparison. Fisher's exact test was applied for an association analysis between miR-147b expression levels and EGFR/KRAS mutations in lung adenocarcinoma tissues from the TCGA dataset. Spearman correlation test was used for a correlation analysis between VHL and its upstream candidate VHL-regulating miRNAs emerging from the TargetScan analysis. The TIC frequencies were estimated using ELDA software (Hu et al., J. Immunol. Methods 347(1-2):70-78, 2009). The survival curves and hazard ratios were compared by log-rank test. The enrichment of Gene Ontology (version: releases/2016-09-30) functional annotations using DAVID Bioinformatics tool (v6.8, October 2016) was performed by modified Fisher's exact test on the microarray data from PC9 single-cell clones. The enrichments were based on all evidence codes.
OTHER EMBODIMENTS
[0470] Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. Some embodiments are within the scope of the following numbered paragraphs.
[0471] 1. A method of treating, reducing, preventing, or delaying tolerance or resistance to anti-receptor tyrosine kinase (RTK) therapy in a subject, the method comprising administering a miR-147b inhibitor to the subject.
[0472] 2. The method of paragraph 1, wherein the RTK is selected from the group consisting of epidermal growth factor receptor (EGFR), human EGFR2 (HER2), HER3, anaplastic lymphoma kinase (ALK), ROS1, ERBB2/3/4, KIT, MET/hepatocyte growth factor receptor (HGFR), RON, platelet derived growth factor receptor (PDGFR), vascular endothelial cell growth factor receptor (VEGFR), VEGFR1, VEGFR2, fibroblast growth factor receptor (FGFR), insulin-like growth factor 1 receptor (IGF1R), and RET.
[0473] 3. The method of paragraph 1, wherein the miR-147b inhibitor reduces a Von Hippel-Lindau (VHL)-pseudohypoxia response or counteracts metabolic changes in the tricarboxylic acid (TCA) cycle associated with drug tolerance in the subject.
[0474] 4. The method of any one of paragraphs 1 to 3, wherein the subject has cancer.
[0475] 5. A method of treating or preventing cancer in a subject, the method comprising administering a miR-147b inhibitor to the subject.
[0476] 6. The method of paragraph 4 or 5, wherein the subject has a cancer selected from the group consisting of lung cancer, non-small cell lung cancer, colorectal cancer, anal cancer, glioblastoma, squamous cell carcinoma, squamous cell carcinoma of the head and neck, pancreatic cancer, breast cancer, renal cell carcinoma, thyroid cancer, gastroesophageal adenocarcinoma, and gastric cancer.
[0477] 7. The method of any one of paragraphs 1 to 6, further comprising administering an anti-RTK therapy to the subject.
[0478] 8. The method of paragraph 7, wherein the anti-RTK therapy is an anti-EGFR therapy.
[0479] 9. The method of paragraph 8, wherein the anti-EGFR therapy comprises a tyrosine kinase inhibitor (TKI).
[0480] 10. The method of paragraph 9, wherein the TKI is selected from the group consisting of gefitinib, erlotinib, afatinib, lapatinib, neratinib, osimertinib, vandetanib, crizotinib, dacomitinib, regorafenib, ponatinib, vismodegib, pazopanib, cabozantinib, bosutinib, axitinib, vemurafenib, ruxolitinib, nilotinib, dasatinib, imatinib, sunitinib, sorafenib, trametinib, cobimetanib, and dabrafenib.
[0481] 11. The method of paragraph 8, wherein the anti-EGFR therapy comprises an anti-EGFR antibody or fragment thereof, or an anti-EGFR CAR T cell.
[0482] 12. The method of paragraph 11, wherein the anti-EGFR therapy comprises an anti-EGFR antibody selected from the group consisting of cetuximab, necitumumab, panitumumab, nimotuzumab, futuximab, zatuximab, cetugex, and margetuximab.
[0483] 13. The method of any one of paragraphs 7 to 12, wherein the miR-147b inhibitor is administered before, at the same time as, or after the anti-RTK therapy.
[0484] 14. The method of any one of paragraphs 1 to 13, wherein the subject has or is at risk of developing tolerance or resistance to anti-RTK therapy.
[0485] 15. The method of paragraph 14, wherein the anti-RTK therapy to which the subject has or is at risk of developing tolerance or resistance is an anti-EGFR therapy, an anti-AKL therapy, an anti-ROS1 therapy, an anti-ERBB2/3/4 therapy, an anti-KIT therapy, an anti-MET/hepatocyte growth factor receptor (HGFR) therapy, an anti-platelet derived growth factor receptor (PDGFR) therapy, an anti-vascular endothelial cell growth factor receptor (VEGFR) therapy, an anti-fibroblast growth factor receptor (FGFR) therapy, and an anti-RET therapy.
[0486] 16. The method of paragraph 15, wherein the anti-RTK therapy to which the subject has or is at risk of developing tolerance or resistance comprises a TKI.
[0487] 17. The method of paragraph 16, wherein the subject has or is at risk of developing tolerance or resistance to an anti-EGFR therapy selected from the group consisting of gefitinib, erlotinib, afatinib, lapatinib, neratinib, osimertinib, vandetanib, crizotinib, dacomitinib, regorafenib, ponatinib, vismodegib, pazopanib, cabozantinib, bosutinib, axitinib, vemurafenib, ruxolitinib, nilotinib, dasatinib, imatinib, sunitinib, sorafenib, trametinib, cobimetanib, and dabrafenib.
[0488] 18. The method of paragraph 15, wherein the subject has or is at risk of developing tolerance or resistance to an anti-EGFR therapy comprising an anti-EGFR antibody or fragment thereof, or an anti-EGFR CAR T cell.
[0489] 19. The method of paragraph 18, wherein the anti-EGFR therapy to which the subject has or is at risk of developing tolerance or resistance comprises an anti-EGFR antibody selected from the group consisting of cetuximab, necitumumab, panitumumab, nimotuzumab, futuximab, zatuximab, cetugex, and margetuximab.
[0490] 20. The method of any one of paragraphs 1 to 19, wherein the miR-147b inhibitor comprises an inhibitory molecule selected from the group consisting of an antisense oligonucleotide, an antagomir, an anti-miRNA sponge, a competitive inhibitor, a triplex-forming oligonucleotide, a double-stranded oligonucleotide, a short interfering RNA, an siRNA, an shRNA, a guide sequence for RNAse P, a small molecule, a catalytic RNA, and a ribozyme; or the inhibition is carried out by the use of a gene editing approach, such as CRISPR-cas9.
[0491] 21. The method of any one of paragraphs 1 to 20, wherein the miR-147b inhibitor is an inhibitor of the production or activity of pri-miR-147b, pre-miR147b, or mature miR-147b.
[0492] 22. A single-stranded oligonucleotide comprising a total of 12 to 50 interlinked nucleotides and having a nucleobase sequence comprising at least 6 contiguous nucleobases complementary to an equal-length portion of a miR-147b target nucleic acid.
[0493] 23. The oligonucleotide of paragraph 22, wherein the oligonucleotide comprises at least one modified nucleobase.
[0494] 24. The oligonucleotide of paragraph 23, wherein the at least one modified nucleobase is selected from the group consisting of 5-methylcytosine, 7-deazaguanine, and 6-thioguanine.
[0495] 25. The oligonucleotide of any one of paragraphs 22 to 24, wherein the oligonucleotide comprises at least one modified internucleoside linkage.
[0496] 26. The oligonucleotide of paragraph 25, wherein the modified internucleoside linkage is a phosphorothioate linkage.
[0497] 27. The oligonucleotide of paragraph 26, wherein the phosphorothioate linkage is a stereochemically enriched phosphorothioate linkage.
[0498] 28. The oligonucleotide of any one of paragraphs 25 to 27, wherein at least 50% of the internucleoside linkages in the oligonucleotide are each independently a modified internucleoside linkage.
[0499] 29. The oligonucleotide of paragraph 28, wherein at least 70% of the internucleoside linkages in the oligonucleotide are each independently a modified internucleoside linkage.
[0500] 30. The oligonucleotide of any one of paragraphs 22 to 29, wherein the oligonucleotide comprises at least one modified sugar nucleoside.
[0501] 31. The oligonucleotide of paragraph 30, wherein the at least one modified sugar nucleoside is a bridged nucleic acid.
[0502] 32. The oligonucleotide of paragraph 31, wherein the bridged nucleic acid is a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), or a cEt nucleic acid.
[0503] 33. The oligonucleotide of paragraph 31 or 32, wherein the at least one modified sugar nucleoside is a 2′-modified sugar nucleoside.
[0504] 34. The oligonucleotide of paragraph 33, wherein the at least one 2′-modified sugar nucleoside comprises a 2′-modification selected from the group consisting of 2′-fluoro, 2′-methoxy, and 2′-methoxyethoxy.
[0505] 35. The oligonucleotide of any one of paragraphs 22 to 34, wherein the oligonucleotide comprises deoxyribonucleotides.
[0506] 36. The oligonucleotide of any one of paragraphs 22 to 35, wherein the oligonucleotide comprises ribonucleotides.
[0507] 37. The oligonucleotide of any one of paragraphs 22 to 24, wherein the oligonucleotide is a morpholino oligonucleotide.
[0508] 38. The oligonucleotide of any one of paragraphs 22 to 24, wherein the oligonucleotide is a peptide nucleic acid.
[0509] 39. The oligonucleotide of any one of paragraphs 22 to 38, wherein the oligonucleotide comprises a hydrophobic moiety covalently attached at its 5′-terminus, its 3′-terminus, or an internucleoside linkage of the oligonucleotide.
[0510] 40. The oligonucleotide of any one of paragraphs 22 to 39, wherein the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 3 to 736 or a variant thereof.
[0511] 41. The oligonucleotide of any one of paragraphs 22 to 40, wherein the oligonucleotide comprises at least 8 contiguous nucleobases complementary to an equal-length portion of a miR-147b target nucleic acid.
[0512] 42. The oligonucleotide of any one of paragraphs 22 to 41, wherein the oligonucleotide comprises at least 12 contiguous nucleobases complementary to an equal-length portion of a miR-147b target nucleic acid.
[0513] 43. The oligonucleotide of any one of paragraphs 22 to 42, wherein the oligonucleotide comprises 20 or fewer contiguous nucleobases complementary to an equal-length portion of a miR-147b target nucleic acid.
[0514] 44. The oligonucleotide of any one of paragraphs 22 to 43, wherein the oligonucleotide comprises a total of at least 12 interlinked nucleotides.
[0515] 45. The oligonucleotide of any one of paragraphs 22 to 44, wherein the oligonucleotide comprises a total of 24 or fewer interlinked nucleotides.
[0516] 46. The oligonucleotide of any one of paragraphs 22 to 45, wherein the oligonucleotide is a gapmer, headmer, tailmer, altmer, blockmer, skipmer, or unimer.
[0517] 47. A double-stranded oligonucleotide comprising the oligonucleotide of any one of paragraphs 22 to 48 hybridized to a complementary oligonucleotide.
[0518] 48. A double-stranded oligonucleotide comprising a passenger strand hybridized to a guide strand comprising a nucleobase sequence comprising at least 6 contiguous nucleobases complementary to an equal-length portion of a miR-147b target nucleic acid, wherein each of the passenger strand and the guide strand comprises a total of 12 to 50 interlinked nucleotides.
[0519] 49. The oligonucleotide of paragraph 48, wherein the passenger strand comprises at least one modified nucleobase.
[0520] 50. The oligonucleotide of paragraph 49, wherein the at least one modified nucleobase is selected from the group consisting of 5-methylcytosine, 7-deazaguanine, and 6-thioguanine.
[0521] 51. The oligonucleotide of any one of paragraphs 48 to 50, wherein the passenger strand comprises at least one modified internucleoside linkage.
[0522] 52. The oligonucleotide of paragraph 51, wherein the modified internucleoside linkage is a phosphorothioate linkage.
[0523] 53. The oligonucleotide of paragraph 52, wherein the phosphorothioate linkage is a stereochemically enriched phosphorothioate linkage.
[0524] 54. The oligonucleotide of any one of paragraphs 51 to 53, wherein at least 50% of the internucleoside linkages in the passenger strand are each independently the modified internucleoside linkage.
[0525] 55. The oligonucleotide of paragraph 54, wherein at least 70% of the internucleoside linkages in the passenger strand are each independently the modified internucleoside linkage.
[0526] 56. The oligonucleotide of any one of paragraphs 48 to 55, wherein the passenger strand comprises at least one modified sugar nucleoside.
[0527] 57. The oligonucleotide of paragraph 56, wherein the at least one modified sugar nucleoside is a bridged nucleic acid.
[0528] 58. The oligonucleotide of paragraph 57, wherein the bridged nucleic acid is a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), or a cEt nucleic acid.
[0529] 59. The oligonucleotide of any one of paragraphs 56 to 58, wherein the at least one modified sugar nucleoside is a 2′-modified sugar nucleoside.
[0530] 60. The oligonucleotide of paragraph 59, wherein the at least one 2′-modified sugar nucleoside comprises a 2′-modification selected from the group consisting of 2′-fluoro, 2′-methoxy, and 2′-methoxyethoxy.
[0531] 61. The oligonucleotide of any one of paragraphs 48 to 60, wherein the passenger strand comprises deoxyribonucleotides.
[0532] 62. The oligonucleotide of any one of paragraphs 48 to 61, wherein the passenger strand comprises ribonucleotides.
[0533] 63. The oligonucleotide of any one of paragraphs 48 to 62, wherein the passenger strand comprises a hydrophobic moiety covalently attached at a 5′-terminus, a 3′-terminus, or an internucleoside linkage of the passenger strand.
[0534] 64. The oligonucleotide of any one of paragraphs 48 to 63, wherein the guide strand comprises at least one modified nucleobase.
[0535] 65. The oligonucleotide of paragraph 64, wherein the at least one modified nucleobase is selected from the group consisting of 5-methylcytosine, 7-deazaguanine, and 6-thioguanine.
[0536] 66. The oligonucleotide of any one of paragraphs 48 to 65, wherein the guide strand comprises at least one modified internucleoside linkage.
[0537] 67. The oligonucleotide of paragraph 66, wherein the modified internucleoside linkage is a phosphorothioate linkage.
[0538] 68. The oligonucleotide of paragraph 67, wherein the phosphorothioate linkage is a stereochemically enriched phosphorothioate linkage.
[0539] 69. The oligonucleotide of any one of paragraphs 66 to 68, wherein at least 50% of the internucleoside linkages in the guide strand are each independently the modified internucleoside linkage.
[0540] 70. The oligonucleotide of paragraph 69, wherein at least 70% of the internucleoside linkages in the guide strand are each independently the modified internucleoside linkage.
[0541] 71. The oligonucleotide of any one of paragraphs 48 to 70, wherein the guide strand comprises at least one modified sugar nucleoside.
[0542] 72. The oligonucleotide of paragraph 71, wherein the at least one modified sugar nucleoside is a bridged nucleic acid.
[0543] 73. The oligonucleotide of paragraph 72, wherein the bridged nucleic acid is a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), or a cEt nucleic acid.
[0544] 74. The oligonucleotide of any one of paragraphs 71 to 73, wherein the at least one modified sugar nucleoside is a 2′-modified sugar nucleoside.
[0545] 75. The oligonucleotide of paragraph 74, wherein the at least one 2′-modified sugar nucleoside comprises a 2′-modification selected from the group consisting of 2′-fluoro, 2′-methoxy, and 2′-methoxyethoxy.
[0546] 76. The oligonucleotide of any one of paragraphs 48 to 75, wherein the guide strand comprises deoxyribonucleotides.
[0547] 77. The oligonucleotide of any one of paragraphs 48 to 76, wherein the guide strand comprises ribonucleotides.
[0548] 78. The oligonucleotide of any one of paragraphs 48 to 77, wherein the guide strand comprises a hydrophobic moiety covalently attached at a 5′-terminus, a 3′-terminus, or an internucleoside linkage of the guide strand.
[0549] 79. The oligonucleotide of any one of paragraphs 48 to 78, wherein the guide strand comprises a sequence selected from the group consisting of SEQ ID NOs: 3 to 736 or a variant thereof.
[0550] 80. The oligonucleotide of any one of paragraphs 47 to 79, wherein the hybridized oligonucleotide comprises at least one 3′-overhang.
[0551] 81. The oligonucleotide of any one of paragraphs 47 to 80, wherein the hybridized oligonucleotide comprises a blunt end.
[0552] 82. The oligonucleotide of any one of paragraphs 47 to 80, wherein the hybridized oligonucleotide comprises two 3′-overhangs.
[0553] 83. The oligonucleotide of any one of paragraphs 22 to 82, wherein the miR-147 target nucleic acid comprises pri-miR-147b, pre-miR-147b, or mature miR-147b.
[0554] 84. An oligonucleotide that competes with miR-147b for binding to a target mRNA or pre-mRNA sequence, thereby inhibiting or reducing the effects of miR-147b on the mRNA or pre-mRNA.
[0555] 85. The oligonucleotide of paragraph 84, comprising a sequence selected from SEQ ID NOs: 1, 2, or 737 to 889.
[0556] 86. A vector comprising a sequence encoding an oligonucleotide of paragraph 22, wherein the vector optionally further comprises a promoter to direct transcription of the sequence.
[0557] 87. The vector of paragraph 86, wherein the vector comprises a sequence encoding multiple oligonucleotides of paragraph 22.
[0558] 88. The vector of paragraph 87, wherein the vector comprises a sequence encoding 2, 3, 4, 5, 6, 7, 8, 9, or 10 oligonucleotides of paragraph 22.
[0559] 89. The vector of any one of paragraphs 86 to 88, wherein the vector is a virus, such as a lentivirus, an adenovirus, or an adeno-associated virus; or is a plasmid, a cosmid, or a phagemid.
[0560] 90. A pharmaceutical composition comprising (i) an oligonucleotide of any one of paragraphs 22 to 85, a vector of any one of paragraphs 86-89, or a small molecule inhibitor of miR-147b, and (ii) a pharmaceutically acceptable excipient or carrier.
[0561] 91. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide of any one of paragraphs 22 to 85, a vector of any one of paragraphs 86 to 89, or a pharmaceutical composition of paragraph 90.
[0562] 92. The method of any one of paragraphs 1 to 21, wherein the miR-147b inhibitor comprises an oligonucleotide of any one of paragraphs 22 to 85.
[0563] 93. The method of any one of paragraphs 1 to 21, 91, or 92, further comprising administration of an additional anti-cancer agent.
[0564] 94. The method of paragraph 93, wherein the additional anti-cancer agent is an anti-RTK agent.
[0565] 95. A method of determining whether tolerance or resistance of a cancer to anti-RTK therapy may be effectively treated, reduced, prevented, or delayed by anti-miR-147b therapy, the method comprising determining the level of miR-147b in the cancer, wherein detection of an increased level of miR-147b, relative to a control, indicates that tolerance or resistance of the cancer to anti-RTK therapy may be effectively treated, reduced, prevented, or delayed with anti-miR-147b therapy, optionally in combination with anti-RTK therapy.
[0566] 96. A method of determining whether a cancer may be effectively treated or prevented with an anti-miR-147b therapy, the method comprising determining the level of miR-147b in the cancer, wherein detection of an increased level of miR-147b in the cancer, relative to a control, indicates that the cancer may effectively be treated or prevented with anti-miR-147b therapy, optionally in combination with anti-RTK therapy.
[0567] 97. The method of paragraph 95 or 96, wherein the anti-miR-147 therapy is selected from an oligonucleotide of any one of paragraphs 22 to 85, a vector of any one of paragraphs 86-89, and a small molecule inhibitor of miR-147b and/or the anti-RTK therapy is selected from a TKI, an anti-RTK antibody, and a CAR T cell directed against an RTK.
[0568] 98. The method of any one of paragraphs 95 to 97, wherein determination of the level of miR-147b in the cancer is carried out by detection of the level of miR-147b in a sample from the subject having the cancer.
[0569] 99. The method of paragraph 98, wherein the sample comprises tumor tissue, tissue swab, sputum, serum, or plasma.
[0570] 100. The method of any one of paragraphs 95 to 99, further comprising administering an anti-miR147b therapy to a subject having the cancer, if it is determined that (i) tolerance or resistance of the cancer to anti-RTK therapy may be effectively treated, reduced, prevented, or delayed by anti-miR-147b therapy, or (ii) the cancer may be effectively treated with anti-miR147b therapy.
[0571] 101. A method of detecting a cancer cell in a sample, the method comprising determining the level of miR-147b in the sample, wherein detection of an increased level of miR-147b in the sample, relative to a control, indicates the presence of a cancer cell in the sample.
[0572] 102. A method of determining whether a cancer cell in a sample may be tolerant or resistant to anti-RTK therapy, the method comprising determining the level of miR-147b in the sample, wherein detection of an increased level of miR-147b, relative to a control, indicates that the cancer cell may be tolerant or resistant to anti-RTK therapy.
[0573] 103. The method of paragraph 102, wherein the anti-RTK therapy is anti-EGFR therapy.
[0574] 104. The method of paragraph 102 or 103, wherein the sample comprises tumor tissue, tissue swab, sputum, serum, or plasma.
[0575] 105. A method of making an organoid comprising lung cells, the method comprising the steps of:
[0576] a. culturing lung cells in a medium comprising epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and fibroblast growth factor 10 (FGF10);
[0577] b. maintaining the cells in culture in a medium comprising Noggin and transforming growth factor-β (TGF-β); and
[0578] c. differentiating the cells in a medium comprising fibroblast growth factor 7 (FGF7) and platelet-derived growth factor (PDGF).
[0579] 106. The method of paragraph 105, wherein the lung cells are lung epithelial cells obtained from a sample of lung tissue of a subject.
[0580] 107. The method of paragraph 105 or 106, wherein the kung cells are immortalized lung epithelial cells.
[0581] 108. The method of any one of paragraphs 105 to 107, wherein the lung cells are cancerous.
[0582] 109. The method of any one of paragraphs 105 to 107, wherein the lung cells are non-cancerous.
[0583] 110. The method of any one of paragraphs 105 to 109, wherein the lung cells are tolerant or resistant to an anti-RTK agent.
[0584] 111. The method of any one of paragraphs 105 to 110, wherein the maintaining step is carried out on days 0-3 of the method, maintenance is carried out on days 4-6, and differentiation is carried out on days 7-24.
[0585] 112. The method of any one of paragraphs 105 to 111, wherein the organoids show ring-like structures upon treatment with an anti-RTK agent.
[0586] 113. A three-dimensional organoid comprising lung cells, wherein the organoid is optionally made by, or has features of organoids made using, the method of any one of paragraphs 105 to 112.
[0587] 114. The organoid of paragraph 113, wherein the lung cells comprise lung cancer cells.
[0588] 115. The organoid of paragraph 113 or 114, wherein the lung cells or lung cancer cells are primary cells, obtained or cultured from the cells of a subject.
[0589] 116. A method for identifying an agent that may be used (i) to treat, reduce, prevent, or delay tolerance or resistance to anti-RTK therapy, or (ii) in the treatment or prevention of cancer, the method comprising contacting a cell with the agent and determining whether the agent decreases the level of miR-147b in the cell.
[0590] 117. The method of paragraph 116, wherein the cell is comprised within an organoid.
[0591] 118. The method of paragraph 117, wherein the organoid comprises lung cancer cells.
[0592] 119. The method of paragraph 117 or 118, wherein the organoid is an organoid of any one of paragraphs 113 to 115, or is made by a method of any one of paragraphs 105 to 112.
[0593] 120. The method of any one of paragraphs 116 to 119, wherein the lung cancer cells are resistant to an anti-RTK therapy.
[0594] 121. The method of any one of paragraphs 116 to 120, wherein the cells are primary cells, obtained or cultured from the cells of a subject.
[0595] 122. The method of any one of paragraphs 116 to 121, wherein the agent is a candidate compound, not previously known to be effective at treating, reducing, preventing, or delaying tolerance or resistance to anti-RTK therapy, or at treating or preventing cancer.
[0596] 123. The method of any one of paragraphs 116 to 121, wherein the method is carried out to determine an optimal approach to treat, reduce, prevent, or delay tolerance or resistance of a cancer to anti-RTK therapy in a subject, or to treat or prevent a cancer in a subject.
[0597] 124. A kit comprising an agent for detecting the level of miR-147b in a sample.
[0598] 125. The kit of paragraph 124, wherein the agent comprises an oligonucleotide, which is optionally an oligonucleotide of any one of paragraphs 22 to 85.
[0599] 126. A kit comprising a miR-147b inhibitor, which optionally is an oligonucleotide of any one of paragraphs 22 to 85, and a second agent for treating cancer.
[0600] 127. The oligonucleotide of paragraph 22, wherein the oligonucleotide targets a sequence comprising or consisting of nucleotides 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 7-12, 8-13, 9-14, 10-15, 11-16, 12-17, 13-18, 14-19, 15-20, 16-21, 17-22, 18-23, 19-24, 20-25, 21-26, 22-27, 23-28, 24-29, 25-30, 26-31, 27-32, 28-33, 29-34, 30-35, 31-36, 32-37, 33-38, 34-39, 35-40, 36-41, 37-42, 38-43, 39-44, 40-45, 41-46, 42-47, 43-48, 44-49, 45-50, 48-51, 47-52, 48-53, 49-54, 50-55, 51-56, 52-57, 53-58, 54-59, 55-80, 58-61, 57-62, 58-63, 59-64, 60-65, 61-66, 62-67, 63-68, 64-69, 65-70, 66-71, 67-72, 68-73, 69-74, 70-75, 71-76, 72-77, 73-78, 74-79, or 75-80 of SEQ ID NO: 1.
[0601] 128. The oligonucleotide of paragraph 127, wherein the oligonucleotide targets said sequence and additionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74 additional nucleotides of SEQ ID NO: 1, whether all on one side of the indicated fragment or wherein the fragment is between the one or more additional nucleotides.
[0602] 129. The oligonucleotide of paragraph 48, wherein the oligonucleotide targets a sequence comprising or consisting of nucleotides 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 7-12, 8-13, 9-14, 10-15, 11-16, 12-17, 13-18, 14-19, 15-20, 16-21, 17-22, 18-23, 19-24, 20-25, 21-26, 22-27, 23-28, 24-29, 25-30, 26-31, 27-32, 28-33, 29-34, 30-35, 31-36, 32-37, 33-38, 34-39, 35-40, 36-41, 37-42, 38-43, 39-44, 40-45, 41-46, 42-47, 43-48, 44-49, 45-50, 46-51, 47-52, 48-53, 49-54, 50-55, 51-56, 52-57, 53-58, 54-59, 55-40, 58-61, 57-62, 58-63, 59-64, 60-65, 61-66, 62-67, 63-68, 64-69, 65-70, 66-71, 67-72, 68-73, 69-74, 70-75, 71-76, 72-77, 73-78, 74-79, or 75-80 of SEQ ID NO: 1.
[0603] 130. The oligonucleotide of paragraph 129, wherein the oligonucleotide targets said sequence and additionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74 additional nucleotides of SEQ ID NO: 1, whether all on one side of the indicated fragment or wherein the fragment is between the one or more additional nucleotides.
[0604] Other embodiments are within the scope of the following claims.