Patent Application
20220133767
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 18, 2020 is named 01948-267WO2_Sequence_Listing_2.18.20_ST25 and is 176,514 bytes in size.
This invention relates to methods and compositions for use in targeting micro RNAs (miRNAs), and methods of treating cancer.
Relapsed disease following conventional treatments remains one of the central problems in cancer management, including epidermal growth factor receptor (EGFR)-based targeted therapy (Kobayashi et al., N. Engl. J. Med. 352(8):786-792, 2005; Paez et al., Science 304(5676):1497-1500, 2004). Tumor cells overcome anti-EGFR treatment by acquisition of drug binding-deficient mutations of EGFR and bypass through other protein tyrosine kinase signaling pathways (Niederst et al., Sci. Signal. 6(294):re6, 2013). For example, a majority of tumors from EGFR-mutant non-smal cell lung cancer (NSCLC) patients acquired resistance mutations such as EGFRT790M or EGFRC797S when the patients were treated with EGFR tyrosine kinase inhibitors (TKIs), gefitinib or erlotinib and osimertinib, respectively (Thress et al., Nat. Med. 21(6):560-562, 2015; Pao et al., PLoS Med. 2(3):e73, 2005). Recently, it has been found that EGFRT790M-positive drug-resistant cells can emerge from EGFRT790M-negative drug-tolerant cells that survive initial drug treatment (Hata et al., Nat. Med. 22(3):262-269, 2016; Ramirez et al., Nat. Commun. 7:10690, 2016). Thus, targeting drug-tolerant cells might be a new strategy to block drug resistance (Sharma et al., Cell 141(1):69-80, 2010; Smith et al., Cancer Cell 29(3):270-284, 2016). With success in applying osimertinib in the first-line treatment of EGFRT790M-positive NSCLC (Soria et al., N. Engl. J. Med. 378(2):113-125, 2018), it is therefore crucial to identify the changes driving drug-tolerance. However, the molecules driving drug-tolerance towards EGFR TKIs are not well studied.
Aberrantly regulated metabolic pathways lead to tumorigenesis and advantageous survival of tumor cells (Go et al., Biochemistry 53(5):947-956, 2014; Ward et al., Cancer Cell 21(3):297-308, 2012; Zhang et al., Cell 148(1-2):259-272, 2012; Jain et al., Science 336(6084):1040-1044, 2012; Vander Heiden et al., Science 324(5960):1029-1033, 2009). The tricarboxylic acid (TCA) cycle is a central pathway in the metabolism of sugars, lipids, and amino acids (Raimundo et al., Trends Mol. Med. 17(11):641-849, 2011). A dysfunctional TCA cycle induces oncogenesis by activating pseudohypoxia responses, which express hypoxia-associated proteins regardless of the oxygen status (Vyas et al., Cell 166(3):555-566, 2016; Sabharwal et al., Nat. Rev. Cancer 14(11):709-721, 2014; MacKenzie et al., Mol. Cell Biol. 27(9):3282-3289, 2007). For example, succinate accumulation caused by functional loss of the TCA cycle enzyme succinate dehydrogenase (SDH) stabilizes hypoxia-inducible factor 1alpha (HIF1alpha) via prolyl-hydroxylase (PHD) inhibition (Selak et al., Cancer Cell 7(1):77-85, 2005; Nowicki et al., FEBS J. 282(15):2796-2805, 2015). In addition, loss of function of Von Hippel-Lindau (VHL) also induces the pseudohypoxia response through decreased ubiquitination and proteasomal degradation of HIF1alpha (Kaelin, Nat. Rev. Cancer 2(9):673-682, 2002). Compared to other cancers, NSCLC is well vascularized and tumor cells depend on high levels of the iron-sulfur cluster biosynthetic enzymes to reduce oxidative damage due to exposure to high oxygen (Alvarez et al., Nature 551(7682):639-643, 2017). Most recently, it was shown that drug-tolerant persistent cancer cells were vulnerable to lipid hydroperoxidase GPX4 inhibition due to a disabled antioxidant program (Hangauer et al., Nature 551(7679):247-250, 2017). However, our understanding of changes conferring drug-tolerance remain limited.
There is a need for approaches to counteract cancer drug tolerance and resistance. Accordingly, we explored which signaling pathways initiate anticancer drug-tolerance and how this shapes cancer metabolism and tumor relapse.
The invention provides methods of treating, reducing, preventing, or delaying tolerance or resistance to anti-receptor tyrosine kinase (RTK) therapy in a subject (e.g., a human patient and/or a subject having cancer), the methods including administration of one or more miR-147b inhibitors to the subject. The invention also provides methods of treating or preventing cancer in a subject (e.g., a human patient and/or a subject having cancer), the methods including administering one or more miR-147b inhibitors to the subject.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, the subject has a cancer selected from the group consisting of kung 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, or one of the cancer types listed elsewhere herein.
In some embodiments, the methods further include administering an anti-RTK therapy to the subject. For example, an anti-EGFR therapy can be administered. In some embodiments, the anti-RTK (e.g., anti-EGFR) therapy includes a tyrosine kinase inhibitor (TKI). In some embodiment, 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. In some embodiments, the anti-EGFR therapy includes an anti-EGFR antibody or fragment thereof, or an anti-EGFR CAR T cell. In some embodiments, the anti-EGFR therapy includes an anti-EGFR antibody selected from the group consisting of cetuximab, necitumumab, panitumumab, nimotuzumab, futuximab, zatuximab, cetugex, and margetuximab. Other antibodies may also be administered, including those listed as follows. Anti-HER2 antibodies include trastuzumab, pertuzumab, 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). In various embodiments, the one or more miR-147b inhibitors are administered before, at the same time as, or after the anti-RTK therapy.
In some embodiments, the subject has or is at risk of developing tolerance or resistance to anti-RTK therapy, e.g., 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, or an anti-RET therapy.
In some embodiments, the anti-RTK therapy to which the subject has or is at risk of developing tolerance or resistance includes a TKI, e.g., 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, or dabrafenib. In some embodiments, the subject has or is at risk of developing tolerance or resistance to an anti-EGFR therapy including an anti-EGFR antibody or fragment thereof, or an anti-EGFR CAR T cell. In some embodiments, the anti-EGFR therapy to which the subject has or is at risk of developing tolerance or resistance includes an anti-EGFR antibody selected from the group consisting of cetuximab, necitumumab, panitumumab, nimotuzumab, futuximab, zatuximab, cetugex, and margetuximab. Other antibodies may also be administered, including those listed as follows. Anti-HER2 antibodies include trastuzumab, pertuzumab, 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).
In some embodiments, the one or more miR-147b inhibitors include one or more 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.
In some embodiments, the one or more miR-147b inhibitors are inhibitors of the production or activity of pri-miR-147b, pre-miR147b, or mature miR-147b.
The invention also provides single-stranded oligonucleotides including a total of 12 to 50 (or 10 to 60, or 8 to 75) interlinked nucleotides and having a nucleobase sequence including at least 6 contiguous nucleobases complementary to an equal-length portion of a miR-147b target nucleic acid.
In some embodiments, the oligonucleotide includes at least one modified nucleobase. In certain embodiments, the at least one modified nucleobase is selected from the group consisting of 5-methylcytosine, 7-deazaguanine, and 6-thioguanine.
In some embodiments, the oligonucleotide includes at least one modified internucleoside linkage. In certain embodiments, the modified internucleoside linkage is a phosphorothioate linkage. In some embodiments, the phosphorothioate linkage is a stereochemically enriched phosphorothioate linkage. In some embodiments, at least 50% or at least 70% of the internucleoside linkages in the oligonucleotide are each independently a modified internucleoside linkage.
In some embodiments, the oligonucleotide includes at least one modified sugar nucleoside. In certain embodiments, the at least one modified sugar nucleoside is a bridged nucleic acid. In some embodiments, the bridged nucleic acid is a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), or a cEt nucleic acid. In some embodiments, the at least one modified sugar nucleoside is a 2′-modified sugar nucleoside, e.g., a sugar with a 2′-modification selected from the group consisting of 2′-fluoro, 2′-methoxy, and 2′-methoxyethoxy.
In some embodiments, the oligonucleotide includes deoxyribonucleotides. In some embodiments, the oligonucleotide includes ribonucleotides. In some embodiments, the oligonucleotide is a morpholino oligonucleotide. In some embodiments, the oligonucleotide is a peptide nucleic acid.
In further embodiments, the oligonucleotide includes a hydrophobic moiety covalently attached at its 5′-terminus, its 3′-terminus, or an internucleoside linkage of the oligonucleotide.
In additional embodiments, the oligonucleotide includes or consists of a sequence selected from the group consisting of SEQ ID NOs: 3 to 736 or a variant thereof (see, e.g., Tables 1 and 3), or the reverse complement thereof. The oligonucleotide may comprise deoxyribonucleotides, ribonucleotides, or a mixture thereof.
In some embodiments, the oligonucleotide includes at least 8 or at least 12 contiguous nucleobases complementary to an equal-length portion of a miR-147b target nucleic acid. In some embodiments, the oligonucleotide includes 20 or fewer contiguous nucleobases complementary to an equal-length portion of a miR-147b target nucleic acid. In some embodiments, the oligonucleotide includes a total of at least 12 interlinked nucleotides. In some embodiments, the oligonucleotide includes a total of 24 or fewer interlinked nucleotides.
In some embodiments, the oligonucleotide is a gapmer, headmer, tailmer, altmer, blockmer, skipmer, or unimer.
In some embodiments, 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, 55-60, 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. In some embodiments, 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. See below for additional, similar variants included in the invention.
The invention also provides double-stranded oligonucleotides including an oligonucleotide as described above hybridized to a complementary oligonucleotide.
Further, the invention provides double-stranded oligonucleotides including a passenger strand hybridized to a guide strand including a nucleobase sequence including 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 includes a total of 12 to 50 (or 10 to 60, or 8 to 75) interlinked nucleotides.
In some embodiments, the passenger strand and/or the guide strand includes at least one modified nucleobase, e.g., 5-methylcytosine, 7-deazaguanine, and 6-thioguanine.
In some embodiments, the passenger strand and/or the guide strand includes at least one modified internucleoside linkage, e.g., a phosphorothioate linkage (such as a stereochemically enriched phosphorothioate linkage).
In some embodiments, at least 50% or at least 70% of the internucleoside linkages in the passenger strand and/or the guide strand are each independently the modified internucleoside linkage.
In some embodiments, the passenger strand and/or the guide strand includes at least one modified sugar nucleoside, e.g., a bridged nucleic acid (such as, e.g., a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), or a cEt nucleic acid). In some embodiments, the at least one modified sugar nucleoside is a 2′-modified sugar nucleoside, e.g., a sugar with a 2′-modification selected from the group consisting of 2′-fluoro, 2′-methoxy, and 2′-methoxyethoxy.
In some embodiments, the passenger strand and/or the guide strand includes deoxyribonucleotides. In some embodiments, the passenger strand and/or the guide strand includes ribonucleotides.
In some embodiments, the passenger strand and/or the guide strand includes a hydrophobic moiety covalently attached at a 5′-terminus, a 3′-terminus, or an internucleoside linkage of the passenger strand.
In some embodiments, the guide strand includes a sequence selected from the group consisting of SEQ ID NOs: 3 to 736 or a variant thereof (or the reverse complement thereof)(see, e.g., Tables 1 and 3). In some embodiments, the passenger strand includes a sequence selected from the group consisting of SEQ ID NOs: 3 to 736 or a variant thereof (or the reverse complement thereof)(see, e.g., Tables 1 and 3). The oligonucleotide may comprise deoxyribonucleotides, ribonucleotides, or a mixture thereof.
In some embodiments, the hybridized oligonucleotide includes at least one 3′-overhang (e.g., two 3′ overhangs). In some embodiments, the hybridized oligonucleotide includes a blunt end.
In some embodiments, the miR-147 target nucleic acid includes pri-miR-147b, pre-miR-147b, or mature miR-147b.
In some embodiments, 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. In some embodiments, 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. See below for additional, similar variants included in the invention.
The invention also includes oligonucleotides that compete 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. In some embodiments, the oligonucleotides include or consists of a sequence selected from SEQ ID NOs: 1, 2, or 737 to 889 (or the reverse complement thereof)(see, e.g., Tables 2 and 4).
The invention further includes vectors including a sequence encoding an oligonucleotide as described herein, wherein the vector optionally further includes a promoter to direct transcription of the sequence. In some embodiments, the vector includes a sequence encoding multiple oligonucleotides, for example, the vector includes a sequence encoding 2, 3, 4, 5, 6, 7, 8, 9, or 10 oligonucleotides. In some embodiments, 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.
The invention also provides pharmaceutical compositions including (i) an oligonucleotide as described herein, a vector as described herein, and/or a small molecule inhibitor of miR-147b, and (ii) a pharmaceutically acceptable excipient or carrier.
The invention additionally provides methods of treating a subject (e.g., a human patient and/or a subject having cancer) in need thereof, the methods including administering to the subject a therapeutically effective amount of an oligonucleotide as described herein, a vector as described herein, and/or a pharmaceutical composition as described herein.
In some embodiments, the methods further include administration of an additional anti-cancer agent, e.g., anti-RTK agent (see, e.g., those anti-RTK agents listed herein).
The invention also provides methods 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 methods including 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.
In these methods, the anti-miR-147 therapy can optionally be selected from an oligonucleotide as described herein, a vector as described herein, and/or a small molecule inhibitor of miR-147b, and/or the anti-RTK therapy can optionally be selected from a TKI, an anti-RTK antibody, and a CAR T cell directed against an RTK. Furthermore, in these methods, determination of the level of miR-147b in the cancer can be carried out by detection of the level of miR-147b in a sample from the subject (e.g., a human patient and/or a subject having cancer) having the cancer. Optionally, the sample includes tumor tissue, tissue swab, sputum, serum, or plasma. The methods further optionally include a step of administering an anti-miR147b therapy to a subject having the cancer (e.g., a human patient and/or a subject having cancer), if it is determined that tolerance or resistance of the cancer to anti-RTK therapy may be effectively treated, reduced, prevented, or delayed by anti-miR-147b therapy.
The invention further provides methods of determining whether a cancer may be effectively treated or prevented with an anti-miR-147b therapy, the methods including 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.
In these methods, the anti-miR-147 therapy can optionally be selected from an oligonucleotide as described herein, a vector as described herein, and/or a small molecule inhibitor of miR-147b, and/or the anti-RTK therapy can optionally be selected from a TKI, an anti-RTK antibody, and a CAR T cell directed against an RTK. Furthermore, in these methods, determination of the level of miR-147b in the cancer can be carried out by detection of the level of miR-147b in a sample from the subject (e.g., a human patient and/or a subject having cancer) having the cancer. Optionally, the sample includes tumor tissue, tissue swab, sputum, serum, or plasma. The methods further optionally include a step of administering an anti-miR147b therapy to a subject having the cancer (e.g., a human patient and/or a subject having cancer), if it is determined that the cancer may be effectively treated with anti-miR147b therapy.
The invention also provides methods of detecting a cancer cell in a sample, the methods including 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.
The invention additionally provides methods of determining whether a cancer cell in a sample may be tolerant or resistant to anti-RTK therapy, the methods including 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.
In some embodiments of these methods, the anti-RTK therapy is anti-EGFR therapy (e.g., as described herein). In some embodiments, the sample includes tumor tissue, tissue swab, sputum, serum, or plasma.
Also provided by the invention are methods of making organoids including lung cells, the methods including the steps of: a. culturing lung cells in a medium including epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and fibroblast growth factor 10 (FGF10); b. maintaining the cells in culture in a medium including Noggin and transforming growth factor-β (TGF-β); and c. differentiating the cells in a medium including fibroblast growth factor 7 (FGF7) and platelet-derived growth factor (PDGF).
In some embodiments, the lung cells are lung epithelial cells obtained from a sample of lung tissue of a subject. In some embodiments, the lung cells are immortalized lung epithelial cells. In some embodiments, the kung cells are cancerous. In some embodiments, the lung cells are non-cancerous. In some embodiments, the lung cells are tolerant or resistant to an anti-RTK agent. In some embodiments, 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. In some embodiments, the organoids show ring-like structures upon treatment with an anti-RTK agent.
The invention further provides three-dimensional organoids including lung cells, wherein the organoid is optionally made by, or has features of organoids made using, the methods described above and elsewhere herein. In some embodiments, the lung cells include lung cancer cells. In some embodiments, the kung cells or lung cancer cells are primary cells, obtained or cultured from the cells of a subject (e.g., a human patient and/or a subject having cancer).
The invention also provides methods 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 methods including contacting a cell with the agent and determining whether the agent decreases the level of miR-147b in the cell. In some embodiments, the cell is included within an organoid, such as an organoid as described herein. In some embodiments, the organoid includes lung cancer cells. In some embodiments, the organoid is an organoid as described herein and/or is made using a method as described herein. In some embodiments, the lung cancer cells are resistant to an anti-RTK therapy. In some embodiments, the cells are primary cells, obtained or cultured from the cells of a subject (e.g., a human patient and/or a subject having cancer). In some embodiments, 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. In some embodiments, 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.
The invention additionally provides kits including one or more agents for detecting the level of miR-147b in a sample. In some embodiments, the agent includes an oligonucleotide, which is optionally an oligonucleotide as described herein. The invention further includes kits including one or more miR-147b inhibitors, which optionally is/are one or more oligonucleotides as described herein, and a second agent for treating cancer (e.g., as described herein).
The invention further provides compositions, as described herein, for use in the methods, as described herein, as well as use of the compositions described herein in the preparation of medicaments for the prevention or treatment of diseases or conditions (e.g., cancer), or for treating, reducing, preventing, or delaying tolerance or resistance to anti-receptor tyrosine kinase (RTK) therapy in a subject, as described herein.
The term “acyl,” as used herein, represents a chemical substituent of formula —C(O)—R, where R is alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heterocyclyl alkyl, heteroaryl, or heteroaryl alkyl. An optionally substituted acyl is an acyl that is optionally substituted as described herein for each group R.
The term “acyloxy,” as used herein, represents a chemical substituent of formula —OR, where R is acyl. An optionally substituted acyloxy is an acyloxy that is optionally substituted as described herein for acyl.
The term “alkanoyl,” as used herein, represents a chemical substituent of formula —C(O)—R, where R is alkyl. An optionally substituted alkanoyl is an alkanoyl that is optionally substituted as described herein for alkyl.
The term “alkoxy,” as used herein, represents a chemical substituent of formula —OR, where R is a C1-6 alkyl group, unless otherwise specified. An optionally substituted alkoxy is an alkoxy group that is optionally substituted as defined herein for alkyl.
The term “alkyl,” as used herein, refers to an acyclic straight or branched chain saturated hydrocarbon group, which, when unsubstituted, has from 1 to 12 carbons, unless otherwise specified. In certain preferred embodiments, unsubstituted alkyl has from 1 to 6 carbons. Alkyl groups are exemplified by methyl; ethyl; n- and iso-propyl; n-, sec-, iso- and tert-butyl; neopentyl, and the like, and may be optionally substituted, valency permitting, with one, two, three, or, in the case of alkyl groups of two carbons or more, four or more substituents independently selected from the group consisting of: alkoxy; acyloxy; amino; aryl; aryloxy; azido; cycloalkyl; cycloalkoxy; halo; heterocyclyl; heteroaryl; heterocyclylalkyl; heteroarylalkyl; heterocyclyloxy; heteroaryloxy; hydroxy; nitro; thiol; silyl; cyano; ═O; ═S; and ═NR′, where R′ is H, alkyl, aryl, or heterocyclyl. In some embodiments, two substituents combine to form a group -L-CO—R, where L is a bond or optionally substituted C1-11 alkylene, and R is hydroxyl or alkoxy. Each of the substituents may itself be unsubstituted or, valency permitting, substituted with unsubstituted substituent(s) defined herein for each respective group.
The term “alkylene,” as used herein, represents a divalent substituent that is an alkyl having one hydrogen atom replaced with a valency. An optionally substituted alkylene is an alkylene that is optionally substituted as described herein for alkyl.
The term “altmer,” as used herein, refers to an oligonucleotide having a pattern of structural features characterized by internucleoside linkages, in which no two consecutive internucleoside linkages have the same structural feature. In some embodiments, an altmer is designed such that it includes a repeating pattern. In some embodiments, an altmer is designed such that it does not include a repeating pattern. In instances, where the “same structural feature” refers to the stereochemical configuration of the internucleoside linkages, the altmer is a “stereoaltmer.”
The term “aryl,” as used herein, represents a mono-, bicyclic, or multicyclic carbocyclic ring system having one or two aromatic rings. Aryl group may include from 6 to 10 carbon atoms. All atoms within an unsubstituted carbocyclic aryl group are carbon atoms. Non-limiting examples of carbocyclic aryl groups include phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl, etc. The aryl group may be unsubstituted or substituted with one, two, three, four, or five substituents independently selected from the group consisting of: alkyl; alkoxy; acyloxy; amino; aryl; aryloxy; azido; cycloalkyl; cycloalkoxy; halo; heterocyclyl; heteroaryl; heterocyclylalkyl; heteroarylalkyl; heterocyclyloxy; heteroaryloxy; hydroxy; nitro; thiol; silyl; and cyano. Each of the substituents may itself be unsubstituted or substituted with unsubstituted substituent(s) defined herein for each respective group.
The term “aryl alkyl,” as used herein, represents an alkyl group substituted with an aryl group. The aryl and alkyl portions may be optionally substituted as the individual groups as described herein.
The term “arylene,” as used herein, represents a divalent substituent that is an aryl having one hydrogen atom replaced with a valency. An optionally substituted arylene is an arylene that is optionally substituted as described herein for aryl.
The term “aryloxy,” as used herein, represents a group —OR, where R is aryl. Aryloxy may be an optionally substituted aryloxy. An optionally substituted aryloxy is aryloxy that is optionally substituted as described herein for aryl.
The term “bicyclic sugar moiety,” as used herein, represents a modified sugar moiety including two fused rings. In certain embodiments, the bicyclic sugar moiety includes a furanosyl ring.
The term “blockmer,” as used herein, refers to an oligonucleotide strand having a pattern of structural features characterized by the presence of at least two consecutive internucleoside linkages with the same structural feature. By same structural feature is meant the same stereochemistry at the internucleoside linkage phosphorus or the same modification at the linkage phosphorus. The two or more consecutive internucleoside linkages with the same structure feature are referred to as a “block.” In instances, where the “same structural feature” refers to the stereochemical configuration of the internucleoside linkages, the blockmer is a “stereoblockmer.”
The expression “Cx-y,” as used herein, indicates that the group, the name of which immediately follows the expression, when unsubstituted, contains a total of from x to y carbon atoms. If the group is a composite group (e.g., aryl alkyl), Cx-y indicates that the portion, the name of which immediately follows the expression, when unsubstituted, contains a total of from x to y carbon atoms. For example, (C5-10-aryl)-C1-6-alkyl is a group, in which the aryl portion, when unsubstituted, contains a total of from 6 to 10 carbon atoms, and the alkyl portion, when unsubstituted, contains a total of from 1 to 6 carbon atoms.
The term “complementary,” as used herein in reference to a nucleobase sequence, refers to the nucleobase sequence having a pattern of contiguous nucleobases that permits an oligonucleotide having the nucleobase sequence to hybridize to another oligonucleotide or nucleic acid to form a duplex structure under physiological conditions. Complementary sequences include Watson-Crick base pairs formed from natural and/or modified nucleobases. Complementary sequences can also include non-Watson-Crick base pairs, such as wobble base pairs (guanosine-uracil, hypoxanthine-uracil, hypoxanthine-adenine, and hypoxanthine-cytosine), and Hoogsteen base pairs.
The term “contiguous,” as used herein in the context of an oligonucleotide, refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other. For example, “contiguous nucleobases” means nucleobases that are immediately adjacent to each other in a sequence.
The term “cycloalkyl,” as used herein, refers to a cyclic alkyl group having from three to ten carbons (e.g., a C3-C10 cycloalkyl), unless otherwise specified. Cycloalkyl groups may be monocyclic or bicyclic. Bicyclic cycloalkyl groups may be of bicyclo[p.q.0]alkyl type, in which each of p and q is, independently, 1, 2, 3, 4, 5, 6, or 7, provided that the sum of p and q is 2, 3, 4, 5, 6, 7, or 8. Alternatively, bicyclic cycloalkyl groups may include bridged cycloalkyl structures, e.g., bicyclo[p.q.r]alkyl, in which r is 1, 2, or 3, each of p and q is, independently, 1, 2, 3, 4, 5, or 6, provided that the sum of p, q, and r is 3, 4, 5, 6, 7, or 8. The cycloalkyl group may be a spirocyclic group, e.g., spiro[p.q]alkyl, in which each of p and q is, independently, 2, 3, 4, 5, 6, or 7, provided that the sum of p and q is 4, 5, 6, 7, 8, or 9. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 1-bicyclo[2.2.1.]heptyl, 2-bicyclo[2.2.1.]heptyl, 5-bicyclo[2.2.1.]heptyl, 7-bicyclo[2.2.1.]heptyl, and decalinyl. The cycloalkyl group may be unsubstituted or substituted (e.g., optionally substituted cycloalkyl) with one, two, three, four, or five substituents independently selected from the group consisting of: alkyl; alkoxy; acyloxy; amino; aryl; aryloxy; azido; cycloalkyl; cycloalkoxy; halo; heterocyclyl; heteroaryl; heterocyclylalkyl; heteroarylalkyl; heterocyclyloxy; heteroaryloxy; hydroxy; nitro; thiol; silyl; cyano; ═O; ═S; ═NR′, where R′ is H, alkyl, aryl, or heterocyclyl. Each of the substituents may itself be unsubstituted or substituted with unsubstituted substituent(s) defined herein for each respective group.
The term “cycloalkylene,” as used herein, represents a divalent substituent that is a cycloalkyl having one hydrogen atom replaced with a valency. An optionally substituted cycloalkylene is a cycloalkylene that is optionally substituted as described herein for cycloalkyl.
The term “cycloalkoxy,” as used herein, represents a group —OR, where R is cycloalkyl. Cycloalkoxy may be an optionally substituted cycloalkoxy. An optionally substituted cycloalkoxy is cycloalkoxy that is optionally substituted as described herein for cycloalkyl.
The term “duplex,” as used herein, represents two oligonucleotides that are paired through hybridization of complementary nucleobases.
The term “gapmer,” as used herein, refers to an oligonucleotide having an RNase H recruiting region (gap) flanked by a 5′ wing and 3′ wing, each of the wings including at least one affinity enhancing nucleoside (e.g., 1, 2, 3, or 4 affinity enhancing nucleosides).
The term “halo,” as used herein, represents a halogen selected from bromine, chlorine, iodine, and fluorine.
The term “headmer,” as used herein, refers to an oligonucleotide 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).
The term “heteroalkyl,” as used herein refers to an alkyl group interrupted one or more times by one or two heteroatoms each time. Each heteroatom is, independently, O, N, or S. None of the heteroalkyl groups includes two contiguous oxygen atoms. The heteroalkyl group may be unsubstituted or substituted (e.g., optionally substituted heteroalkyl). When heteroalkyl is substituted and the substituent is bonded to the heteroatom, the substituent is selected according to the nature and valency of the heteratom. Thus, the substituent bonded to the heteroatom, valency permitting, is selected from the group consisting of ═O, —N(RN2)2, —SO2ORN3, —SO2RN2, —SORN3, —COORN3, an N protecting group, alkyl, aryl, cycloalkyl, heterocyclyl, or cyano, where each RN2 is independently H, alkyl, cycloalkyl, aryl, or heterocyclyl, and each RN3 is independently alkyl, cycloalkyl, aryl, or heterocyclyl. Each of these substituents may itself be unsubstituted or substituted with unsubstituted substituent(s) defined herein for each respective group. When heteroalkyl is substituted and the substituent is bonded to carbon, the substituent is selected from those described for alkyl, provided that the substituent on the carbon atom bonded to the heteroatom is not Cl, Br, or I. It is understood that carbon atoms are found at the termini of a heteroalkyl group. In some embodiments, heteroalkyl is PEG
The term “heteroalkylene,” as used herein, represents a divalent substituent that is a heteroalkyl having one hydrogen atom replaced with a valency. An optionally substituted heteroalkylene is a heteroalkylene that is optionally substituted as described herein for heteroalkyl.
The term “heteroaryl,” as used herein, represents a monocyclic 5-, 6-, 7-, or 8-membered ring system, or a fused or bridging bicyclic, tricyclic, or tetracyclic ring system; the ring system contains one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and at least one of the rings is an aromatic ring. Non-limiting examples of heteroaryl groups include benzimidazolyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, furyl, imidazolyl, indolyl, isoindazolyl, isoquinolinyl, isothiazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, purinyl, pyrrolyl, pyridinyl, pyrazinyl, pyrimidinyl, qunazolinyl, quinolinyl, thiadiazolyl (e.g., 1,3,4-thiadiazole), thiazolyl, thienyl, triazolyl, tetrazolyl, dihydroindolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, etc. The term bicyclic, tricyclic, and tetracyclic heteroaryls include at least one ring having at least one heteroatom as described above and at least one aromatic ring. For example, a ring having at least one heteroatom may be fused to one, two, or three carbocyclic rings, e.g., an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or another monocyclic heterocyclic ring. Examples of fused heteroaryls include 1,2,3,5,8,8a-hexahydroindolizine; 2,3-dihydrobenzofuran; 2,3-dihydroindole; and 2,3-dihydrobenzothiophene. Heteroaryl may be optionally substituted with one, two, three, four, or five substituents independently selected from the group consisting of: alkyl; alkoxy; acyloxy; aryloxy; amino; arylalkoxy; cycloalkyl; cycloalkoxy; halogen; heterocyclyl; heterocyclyl alkyl; heteroaryl; heteroaryl alkyl; heterocyclyloxy; heteroaryloxy; hydroxyl; nitro; thiol; cyano; ═O; —NR2, where each R is independently hydrogen, alkyl, acyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl; —COORA, where RA is hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl; and —CON(RB)2, where each RB is independently hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl. Each of the substituents may itself be unsubstituted or substituted with unsubstituted substituent(s) defined herein for each respective group.
The term “heteroarylene,” as used herein, refers to a heteroaryl in which one hydrogen atom is replaced with a valency. An optionally substituted heteroaryle is a heteroarylene group that is optionally substituted as described herein for heteroaryl.
The term “heteroaryloxy,” as used herein, refers to a structure —OR, in which R is heteroaryl. Heteroaryloxy can be optionally substituted as defined for heteroaryl.
The term “heterocyclyl,” as used herein, represents a monocyclic, bicyclic, tricyclic, or tetracyclic ring system having fused or bridging 4-, 5-, 6-, 7-, or 8-membered rings, unless otherwise specified, the ring system containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. Heterocyclyl may be aromatic or non-aromatic. An aromatic heterocyclyl is heteroaryl as described herein. Non-aromatic 5-membered heterocyclyl has zero or one double bonds, non-aromatic 6- and 7-membered heterocyclyl groups have zero to two double bonds, and non-aromatic 8-membered heterocyclyl groups have zero to two double bonds and/or zero or one carbon-carbon triple bond. Heterocyclyl groups have a carbon count of 1 to 16 carbon atoms unless otherwise specified. Certain heterocyclyl groups may have a carbon count up to 9 carbon atoms. Non-aromatic heterocyclyl groups include pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, homopiperidinyl, piperazinyl, pyridazinyl, oxazolidinyl, isoxazolidiniyl, morpholinyl, thiomorpholinyl, thiazolidinyl, isothiazolidinyl, thiazolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyranyl, dihydropyranyl, dithiazolyl, etc. The term “heterocyclyl” also represents a heterocyclic compound having a bridged multicyclic structure in which one or more carbons and/or heteroatoms bridges two non-adjacent members of a monocyclic ring, e.g., quinuclidine, tropanes, or diaza-bicyclo[2.2.2]octane. The term “heterocyclyl” includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocyclic rings is fused to one, two, or three carbocyclic rings, e.g., a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or another heterocyclic ring. Examples of fused heterocyclyls include 1,2,3,5,8,8a-hexahydroindolizine; 2,3-dihydrobenzofuran; 2,3-dihydroindole; and 2,3-dihydrobenzothiophene. The heterocyclyl group may be unsubstituted or substituted with one, two, three, four, or five substituents independently selected from the group consisting of: alkyl; alkoxy; acyloxy; aryloxy; amino; arylalkoxy; cycloalkyl; cycloalkoxy; halogen; heterocyclyl; heterocyclyl alkyl; heteroaryl; heteroaryl alkyl; heterocyclyloxy; heteroaryloxy; hydroxyl; nitro; thiol; cyano; ═O; ═S; —NR2, where each R is independently hydrogen, alkyl, acyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl; —COORA, where RA is hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl; and —CON(RB)2, where each RB is independently hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl.
The term “heterocyclyl alkyl,” as used herein, represents an alkyl group substituted with a heterocyclyl group. The heterocyclyl and alkyl portions of an optionally substituted heterocyclyl alkyl are optionally substituted as described for heterocyclyl and alkyl, respectively.
The term “heterocyclylene,” as used herein, represents a divalent substituent that is a heterocyclyl having one hydrogen atom replaced with a valency. An optionally substituted heterocyclylene is a heterocyclylene that is optionally substituted as described herein for heterocyclyl.
The term “heterocyclyloxy,” as used herein, refers to a structure —OR, in which R is heterocyclyl. Heterocyclyloxy can be optionally substituted as described for heterocyclyl.
The terms “hydroxyl” and “hydroxy,” as used interchangeably herein, represent —OH.
The term “hydrophobic moiety,” as used herein, represents a monovalent group covalently linked to an oligonucleotide backbone, where the monovalent group is 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. 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. A linker may optionally be used to connect the monovalent group to the oligonucleotide, and 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 C1-12 alkylene, optionally substituted C2-12 heteroalkylene, optionally substituted C6-10 arylene, optionally substituted C3-8 cycloalkylene, optionally substituted C1-9 heteroarylene, optionally substituted C1-9 heterocyclylene, —O—, —S—S—, and —NRN—, where each RN is independently H or optionally substituted C1-12 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.
The term “internucleoside linkage,” as used herein, represents a group or bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide. An internucleoside linkage is an unmodified internucleoside linkage or a modified internucleoside linkage. An “unmodified internucleoside linkage” is a phosphate (—O—P(O)(OH)—O—) internucleoside linkage (“phosphate phosphodiester”). A “modified internucleoside linkage” is an internucleoside linkage other than a phosphate phosphodiester.
The two main classes of modified 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 (—CH2—N(CH)—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)—). Phosphorothioate linkages are phosphodiester linkages and phosphotriester linkages in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. In some embodiments, an internucleoside linkage is a group of the following structure:
where
Z is O, S, or Se;
Y is —X-L-R1;
each X is independently —O—, —S—, —N(-L-R1)—, or L;
each L is independently a covalent bond or a linker (e.g., 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 C1-12 alkylene, optionally substituted C2-12 heteroalkylene, optionally substituted C6-10 arylene, optionally substituted C3-8 cycloalkylene, optionally substituted C1-9 heteroarylene, optionally substituted C1-9 heterocyclylene, —O—, —S—S—, and —NRN—, where each RN is independently H or optionally substituted C1-12 alkyl);
each R1 is independently hydrogen, —S—S—R2, —O—CO—R2, —S—CO—R2, optionally substituted C1-9 heterocyclyl, or a hydrophobic moiety; and
each R2 is independently optionally substituted C1-10 alkyl, optionally substituted C2-10 heteroalkyl, optionally substituted C6-10 aryl, optionally substituted C6-10 aryl C1-6 alkyl, optionally substituted C1-9 heterocyclyl, or optionally substituted C1-9 heterocyclyl C1-6 alkyl.
When L is a covalent bond, R1 is hydrogen, Z is oxygen, and all X groups are —O—, the internucleoside group is known as a phosphate phosphodiester. When L is a covalent bond, R1 is hydrogen, Z is sulfur, and all X groups are —O—, the internucleoside group is known as a phosphorothioate diester. When Z is oxygen, all X groups are —O—, and either (1) L is a linker or (2) R1 is not a hydrogen, the internucleoside group is known as a phosphotriester. When Z is sulfur, all X groups are —O—, and either (1) L is a linker or (2) R1 is not a hydrogen, the internucleoside group is known as a phosphorothioate triester. Non-limiting examples of phosphorothioate triester linkages and phosphotriester linkages are described in US 2017/0037399, the disclosure of which is incorporated herein by reference.
The term “morpholino,” as used herein in reference to a class of oligonucleotides, represents an oligomer of at least 10 morpholino monomer units interconnected by morpholino internucleoside linkages. A morpholino includes a 5′ group and a 3′ group. For example, a morpholino may be of the following structure:
where
n is an integer of at least 10 (e.g., 12 to 30) indicating the number of morpholino units;
each B is independently a nucleobase;
R1 is a 5′ group;
R2 is a 3′ group; and
L is (i) a morpholino internucleoside linkage or, (ii) if L is attached to R2, a covalent bond. A 5′ group in morpholino may be, e.g., hydroxyl, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, disphorodihioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer. A 3′ group in morpholino may be, e.g., hydrogen, 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.
The term “morpholino internucleoside linkage,” as used herein, represents a divalent group of the following structure:
where
Z is O or S;
X1 is a bond, —CH2—, or —O—;
X2 is a bond, —CH2—O—, or —O—; and
Y is —NR2, where each R is independently C1-6 alkyl (e.g., methyl), or both R combine together with the nitrogen atom to which they are attached to form a C2-9 heterocyclyl (e.g., N-piperazinyl); provided that both X1 and X2 are not simultaneously a bond.
The term “nucleobase,” as used herein, represents a nitrogen-containing heterocyclic ring found at the 1′ position of the ribofuranose/2′-deoxyribofuranose of a nucleoside. Nucleobases are unmodified or modified. As used herein, “unmodified” or“natural” 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, or 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.
The term “nucleoside,” as used herein, represents sugar-nucleobase compounds and groups known in the art, as well as modified or unmodified 2′-deoxyribofuranose-nucleobase compounds and groups known in the art. The sugar may be ribofuranose. The sugar may be modified or unmodified. An unmodified ribofuranose-nucleobase is ribofuranose having an anomeric carbon bond to an unmodified nucleobase. Unmodified ribofuranose-nucleobases are adenosine, cytidine, guanosine, and uridine. Unmodified 2′-deoxyribofuranose-nucleobase compounds are 2′-deoxyadenosine, 2′-deoxycytidine, 2′-deoxyguanosine, and thymidine. The modified compounds and groups include one or more modifications selected from the group consisting of nucleobase modifications and sugar modifications described herein. A nucleobase modification is a replacement of an unmodified nucleobase with a modified nucleobase. 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:
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 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.
The term “nucleotide,” as used herein, represents a nucleoside bonded to an internucleoside linkage or a monovalent group of the following structure —X1—P(X2)(R1)2, where X1 is O, S, or NH, and X2 is absent, ═O, or ═S, and each R1 is independently —OH, —N(R2)2, or —O—CH2CH2CN, where each R2 is independently an optionally substituted alkyl, or both R2 groups, together with the nitrogen atom to which they are attached, combine to form an optionally substituted heterocyclyl.
The term “oligonucleotide,” as used herein, represents a structure containing 10 or more contiguous nucleosides covalently bound together by internucleoside linkages. An oligonucleotide includes a 5′ end and a 3′ end. 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. 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 oligonucleotide having a 5′-hydroxyl or 5′-phosphate has an unmodified 5′ terminus. An oligonucleotide having a 5′ terminus other than 5′-hydroxyl or 5′-phosphate has a modified 5′ terminus. An oligonucleotide having a 3′-hydroxyl or 3′-phosphate has an unmodified 3′ terminus. An oligonucleotide having a 3′ terminus other than 3′-hydroxyl or 3′-phosphate has a modified 3′ terminus. Oligonucleotides can be in double- or single-stranded form. Double-stranded oligonucleotide molecules can optionally include one or more single-stranded segments (e.g., overhangs).
The term “oxo,” as used herein, represents a divalent oxygen atom (e.g., the structure of oxo may be shown as ═O).
The term “pharmaceutically acceptable,” as used herein, refers to those compounds, materials, compositions, and/or dosage forms, which are suitable for contact with the tissues of an individual (e.g., a human), without excessive toxicity, irritation, allergic response, and other problem complications commensurate with a reasonable benefit/risk ratio.
The term “pharmaceutical composition,” as used herein, represents a composition containing an oligonucleotide described herein, formulated with a pharmaceutically acceptable excipient, diluent, or carrier, and manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a subject.
The term “protecting group,” as used herein, represents a group intended to protect a functional group (e.g., a hydroxyl, an amino, or a carbonyl) from participating in one or more undesirable reactions during chemical synthesis. The term “O-protecting group,” as used herein, represents a group intended to protect an oxygen containing (e.g., phenol, hydroxyl or carbonyl) group from participating in one or more undesirable reactions during chemical synthesis. The term “N-protecting group,” as used herein, represents a group intended to protect a nitrogen containing (e.g., an amino or hydrazine) group from participating in one or more undesirable reactions during chemical synthesis. Commonly used O- and N-protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 3rd Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference. Exemplary O- and N-protecting groups include alkanoyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl, tri-iso-propylsilyloxymethyl, 4,4′-dimethoxytrityl, isobutyryl, phenoxyacetyl, 4-isopropylpehenoxyacetyl, dimethylformamidino, and 4-nirobenzoyl.
Exemplary O-protecting groups for protecting carbonyl containing groups include, but are not limited to: acetals, acylals, 1,3-dithianes, 1,3-dioxanes, 1,3-dioxolanes, and 1,3-dithiolanes.
Other O-protecting groups include, but are not limited to: substituted alkyl, aryl, and arylalkyl ethers (e.g., trityl; methylthiomethyl; methoxymethyl; benzyloxymethyl; siloxymethyl; 2,2,2,-trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl; ethoxyethyl; 1-[2-(trimethylsilyl)ethoxy]ethyl; 2-trimethylsilylethyl; t-butyl ether; p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl, and nitrobenzyl); silyl ethers (e.g., trimethylsilyl; triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl; t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; and diphenymethylsilyl); carbonates (e.g., methyl, methoxymethyl, 9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl; 2-(trimethylsilyl)ethyl; vinyl, allyl, nitrophenyl; benzyl; methoxybenzyl; 3,4-dimethoxybenzyl; and nitrobenzyl).
Other N-protecting groups include, but are not limited to, chiral auxiliaries such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, phenylalanine, and the like; sulfonyl-containing groups such as benzenesulfonyl, p-toluenesulfonyl, and the like; carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyl oxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydroxy carbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropoxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and the like, arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl, and the like and silyl groups such as trimethylsilyl, and the like.
The term “shRNA,” as used herein, refers to a double-stranded oligonucleotide of the invention having a passenger strand and a guide strand, where the passenger strand and the guide strand are covalently linked by a linker excisable through the action of the Dicer enzyme.
The term “siRNA,” as used herein, refers to a double-stranded oligonucleotide of the invention having a passenger strand and a guide strand, where the passenger strand and the guide strand are not covalently linked to each other.
The term “skipmer,” as used herein, refers a gapmer, in which alternating internucleoside linkages are phosphate phosphodiester linkages and intervening internucleoside linkages are modified internucleoside linkages.
The term “stereochemically enriched,” as used herein, refers to a local stereochemical preference for one enantiomer of the recited group over the opposite enantiomer of the same group. Thus, an oligonucleotide containing a stereochemically enriched internucleoside linkage is an oligonucleotide, in which a phosphorothioate of predetermined stereochemistry is present in preference to a phosphorothioate of stereochemistry that is opposite of the predetermined stereochemistry. This preference can be expressed numerically using a diastereomeric ratio for the phosphorothioate of the predetermined stereochemistry. The diastereomeric ratio for the phosphorothioate of the predetermined stereochemistry is the molar ratio of the diastereomers having the identified phosphorothioate with the predetermined stereochemistry relative to the diastereomers having the identified phosphorothioate with the stereochemistry that is opposite of the predetermined stereochemistry. The diastereomeric ratio for the phosphorothioate of the predetermined stereochemistry may be greater than or equal to 1.1 (e.g., greater than or equal to 4, greater than or equal to 9, greater than or equal to 19, or greater than or equal to 39).
The term “subject,” as used herein, refers to a human or non-human animal (e.g., a mammal) that is suffering from, or is at risk of, disease, disorder, or condition, as determined by a qualified professional (e.g., a physician or a nurse practitioner) with or without known in the art laboratory test(s) of sample(s) from the subject. The subject treated according to the methods of the invention may thus be a human patient, such as an adult patient or a pediatric patient. Non-limiting examples of diseases, disorders, and conditions include cancers. As one example, the cancer may be characterized by a mutant receptor tyrosine kinase (RTK; e.g., mutant 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), or RET). In various embodiments, the cancer may be tolerant or resistant to anti-RTK therapy, or at risk of such tolerance or resistance. Other examples of cancers that the subject may have or be at risk of developing are provided below. A subject treated according to the methods of the invention can optionally be at risk of developing cancer, diagnosed with cancer, in treatment for cancer, or in post-therapy recovery from cancer. The cancer treated according to the methods of the invention can optionally be a primary tumor, locally advanced, or metastatic.
A “sugar” or “sugar moiety” includes naturally occurring sugars having a furanose ring or a structure that is capable of replacing the furanose ring of a nucleoside. 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—CH2-4′ or 2′-O—(CH2)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).
The term “tailmer,” as used herein, refers to an oligonucleotide 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).
“Treatment” and “treating,” as used herein, refer to the medical management of a subject with the intent to improve, ameliorate, stabilize, prevent, or delay a disease, disorder, or condition (e.g., cancer, such as, for example, a cancer characterized by a mutant receptor tyrosine kinase (RTK), which is optionally resistant to RTK-targeted therapy). This term includes active treatment (treatment directed to improve the cancer, or to improve tolerance or resistance to treatment); causal treatment (treatment directed to the cause of the cancer, or to tolerance or resistance to treatment); palliative treatment (treatment designed for the relief of symptoms of the cancer, or for alleviating tolerance or resistance to treatment); preventative treatment (treatment directed to minimizing or partially or completely inhibiting the development of the cancer, or to minimizing or partially or completely inhibiting the development of resistance or tolerance to treatment); and supportive treatment (treatment employed to supplement another therapy).
The term “unimer,” as used herein, refers to an oligonucleotide having a pattern of structural features characterized by all of the internucleoside linkages having the same structural feature. By same structural feature is meant the same stereochemistry at the internucleoside linkage phosphorus or the same modification at the linkage phosphorus. In instances, where the “same structural feature” refers to the stereochemical configuration of the internucleoside linkages, the unimer is a “stereounimer.”
Enumeration of positions within oligonucleotides and nucleic acids, as used herein and unless specified otherwise, starts with the 5′-terminal nucleoside as 1 and proceeds in the 3′-direction.
The compounds described herein, unless otherwise noted, encompass isotopically enriched compounds (e.g., deuterated compounds), tautomers, and all stereoisomers and conformers (e.g. enantiomers, diastereomers, E/Z isomers, atropisomers, etc.), as well as racemates thereof and mixtures of different proportions of enantiomers or diastereomers, or mixtures of any of the foregoing forms as well as salts (e.g., pharmaceutically acceptable salts).
Data are mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001; unpaired t test with Welch's correction (
Data are mean±s.e.m.
Data are mean±s.e.m. *P<0.05; **P<0.01; ***p<0.001; one-way ANOVA (
All data are representative of two separate experiments.
Data are mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed t-test (
Data are mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed t-test (a), Mann-Whitney test (
Data are mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed t-test (
Data are mean±s.e.m. **, P<0.05; ***, P<0.001; ****, P<0.0001; two-tailed t-test (
Data are mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001; NS, not significant (P>0.05); unpaired two-tailed t-test (
Data are mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; unpaired two-tailed t-test (
All figures show mean±s.e.m. *p<0.05; *p<0.01 and **p<0.001. unpaired two-tailed t-test (
Data are mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; NS, not significant; Kruskal-Wallis test (
Data are mean±s.e.m. **P<0.01; NS, not significant; unpaired two-tailed t-test (
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.
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.
The methods, inhibitors, compositions, and organoids of the invention are described further, as follows.
Micro RNAs—miR-147b
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.
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.
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.
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.
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).
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).
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®).
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).
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.
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.
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)).
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+ 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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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).
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).
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
An antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) may be a morpholino.
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.
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).
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′-RPSPSP-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.
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.
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 SP configuration.
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 SP 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 SP 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 RP 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 RP stereochemical configuration.
An oligonucleotide may have, e.g., only one RP stereochemically enriched phosphorothioate internucleoside linkage. An oligonucleotide may have, e.g., multiple RP 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 RP.
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 (SP)mRP or RP(SP)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 (SP)mRP or RP(SP)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 (SP)mRP, 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 RP(SP)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 (SP)mRP or RP(SP)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 SP 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 SP 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 SP 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 RPRPSP and SPSPRP. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is a repeating RPRPSP. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is a repeating SPSPRP.
An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (SP)mRP or RP(SP)m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including RP(SP)m. An oligonucleotide may include a pattern of P-stereogenic centers in the gap region including (SP)mRP. In some embodiments, m is 2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (SP)2RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (RP)2RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including RPSPRP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including SPRPRP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (SP)2RP.
An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (SP)mRP or RP(SP)m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including RP(SP)m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (SP)mRP. In some embodiments, m is 2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (SP)2RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (RP)2RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including RPSPRP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including SPRPRP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (SP)2RP.
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.
A repeating pattern may be, e.g., (SP)m(RP)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 (SP)m(RP)n. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (Sp)m(RP)n. A repeating pattern may be, e.g., (RP)n(SP)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 (RP)n(SP)m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (RP)n(SP)m. In some embodiments, (RP)n(SP)m is (RP)(SP)2. In some embodiments, (SP)n(RP)m is (SP)2(RP).
A repeating pattern may be, e.g., (SP)m(RP)n(SP)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 (SP)m(RP)n(SP)t. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (SP)m(RP)n(SP)t. A repeating pattern may be, e.g., (SP)t(RP)n(SP)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 (SP)t(RP)n(SP)m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (SP)t(RP)n(SP)m.
A repeating pattern is (Np)t(RP)n(SP)m, where each of n and t is independently 1, 2, 3, 4, 5, 6, 7, or 8, Np is independently RP or SP, and m is as described herein. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (Np)t(RP)n(SP)m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (Np)t(Rp)n(SP)m. A repeating pattern may be, e.g., (Np)t(RP)n(SP)m, where each of n and t is independently 1, 2, 3, 4, 5, 6, 7, or 8, Np is independently RP or SP, and m is as described herein. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (Np)t(RP)n(SP)m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (Np)t(RP)n(SP)m. In some embodiments, Np is RP. In some embodiments, Np is SP. All Np may be, e.g., same. All Np may be, e.g., SP. At least one Np may be, e.g., different from another Np. In some embodiments, t is 2.
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.
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.
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.
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, (SP)m(RP)n(SP)t is (SP)2RP(SP)2. In some embodiments, (SP)t(RP)n(SP)m is (SP)2RP(SP)2. In some embodiments, (SP)t(RP)n(SP)m is SPRP(SP)2. In some embodiments, (Np)t(RP)n(SP)m is (Np)t(RP)n(SP)m. In some embodiments, (Np)t(RP)n(SP)m is (Np)2RP(SP)m. In some embodiments, (Np)t(RP)n(SP)m is (RP)2RP(SP)m. In some embodiments, (Np)t(RP)n(SP)m is (SP)2RP(SP)m. In some embodiments, (Np)t(RP)n(SP)m is RPSPRP(SP)m. In some embodiments, (Np)t(RP)n(SP)m is SPRPRP(SP)m.
In some embodiments, (SP)t(RP)n(SP)m is SPRPSPSP. In some embodiments, (SP)t(RP)n(SP)m is (SP)2RP(SP)2. In some embodiments, (SP)t(RP)n(SP)m is (SP)3RP(SP)3. In some embodiments, (SP)t(RP)n(SP)m is (SP)4RP(SP)4. In some embodiments, (SP)t(RP)n(SP)m is (SP)tRP(SP)5. In some embodiments, (SP)t(RP)n(SP)m is SPRP(SP)5. In some embodiments, (SP)t(RP)n(SP)m is (SP)2RP(SP)5. In some embodiments, (SP)t(RP)n(SP)m is (SP)3RP(SP)5. In some embodiments, (SP)t(RP)n(SP)m is (SP)4RP(SP)5. In some embodiments, (SP)t(RP)n(SP)m is (SP)5RP(SP)5.
In some embodiments, (SP)m(RP)n(SP)t is (SP)2RP(SP)2. In some embodiments, (SP)m(RP)n(SP)t is (SP)3RP(SP)3. In some embodiments, (SP)m(RP)n(SP)t is (SP)4RP(SP)4. In some embodiments, (SP)m(RP)n(SP)t is (SP)mRP(SP)5. In some embodiments, (SP)m(RP)n(SP)t is (SP)2RP(SP)5. In some embodiments, (SP)m(RP)n(SP)t is (SP)3RP(SP)5. In some embodiments, (SP)m(RP)n(SP)t is (SP)4RP(SP)5. In some embodiments, (SP)m(RP)n(SP)t is (SP)5RP(SP)5.
The gap region may include, e.g., at least one RP internucleoside linkage. The gap region may include, e.g., at least one RP phosphorothioate internucleoside linkage. The gap region may include, e.g., at least two RP internucleoside linkages. The gap region may include, e.g., at least two RP phosphorothioate internucleoside linkages. The gap region may include, e.g., at least three RP internucleoside linkages. The gap region may include, e.g., at least three RP phosphorothioate internucleoside linkages. The gap region may include, e.g., at least 4, 5, 6, 7, 8, 9, or 10 RP internucleoside linkages. The gap region may include, e.g., at least 4, 5, 6, 7, 8, 9, or 10 RP phosphorothioate internucleoside linkages.
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.
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.
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).
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).
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.
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.
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.
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.
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.
The first internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The first internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The second internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The second internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The third internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The third internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The fifth internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The fifth internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The seventh internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The seventh internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The eighth internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The eighth internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The ninth internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The ninth internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The eighteenth internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The eighteenth internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The nineteenth internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The nineteenth internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The twentieth internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The twentieth internucleoside linkage of the region may be, e.g., an RP internucleoside linkage.
The region may have a length of, e.g., at least 21 bases. The region may have a length of, e.g., 21 bases.
In some embodiments, each stereochemically enriched internucleoside linkage in an oligonucleotide is a phosphorothioate phosphodiester.
An oligonucleotide may have, e.g., at least 25% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 30% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 35% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 40% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 45% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 50% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 55% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 60% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 65% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 70% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 75% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 80% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 85% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 90% of its internucleoside linkages in SP configuration.
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:
[SBn1RBn2SBn3RBn4 . . . SBnxRBny]
where:
each RB independently represents a block of nucleotide units having the RP configuration at the internucleoside linkage phosphorus atom;
each SB independently represents a block of nucleotide units having the SP configuration at the internucleoside linkage phosphorus atom;
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
where the sum of n1 to ny is between 2 and 200.
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.
At least two adjacent n's may be, e.g., equal to one another, so that an oligonucleotide includes adjacent blocks of SP linkages and RP linkages of equal lengths. In some embodiments, an oligonucleotide includes repeating blocks of SP and RP linkages of equal lengths. In some embodiments, an oligonucleotide includes repeating blocks of SP and RP linkages, where at least two such blocks are of different lengths from one another. In some such embodiments, each SP block is of the same length and is of a different length from each RP block, where all RP blocks may optionally be of the same length as one another.
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.
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.
An oligonucleotide of formula [SBn1RBn2SBn3RBn4 . . . SBnxRBny] may be, e.g., a stereoblockmer. An oligonucleotide of formula [SBn1RBn2SBn3RBn4 . . . SBnxRBny] may be, e.g., a stereoskipmer. An oligonucleotide of formula [SBn1RBn2SBn3RBn4 . . . SBnxRBny] may be, e.g., a stereoaltmer. An oligonucleotide of formula [SBn1RBn2SBn3RBn4 . . . SBnxRBny] may be, e.g., a gapmer.
An oligonucleotide of formula [SBn1RBn2SBn3RBn4 . . . SBnxRBny] 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 [SBn1RBn2SBn3RBn4 . . . SBnxRBny] may be, e.g., a stereoskipmer and a P-modification skipmer. An oligonucleotide of formula [SBn1RBn2SBn3RBn4 . . . SBnxRBny] may be, e.g., a stereoblockmer and a P-modification altmer. An oligonucleotide of formula [SBn1RBn2SBn3RBn4 . . . SBnxRBny] may be, e.g., a stereoaltmer and a P-modification blockmer.
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.
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.
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.
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.
An oligonucleotide may be, e.g., a skipmer.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Nucleobase Modifications
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.
Sugar Modifications
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.
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—CH2-4′ or 2′-O—(CH2)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).
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:
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.
Internucleoside Linkage Modifications
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 (—CH2—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.
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 SP or RP to identify the absolute stereochemistry of the phosphorus atom. Within an oligonucleotide, SP phosphorothioate indicates the following structure:
Within an oligonucleotide, RP phosphorothioate indicates the following structure:
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′-CH2—N(CH3)—O-3′), amide-3 (3′-CH2—C(═O)—N(H)-3′), amide-4 (3′-CH2—N(H)—C(═O)-3′), formacetal (3′-O—CH2—O-3′), and thioformacetal (3′-S—CH2—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).
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.
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).
An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (SP)mRP or RP(SP)m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including RP(SP)m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (SP)mRP. In some embodiments, m is 2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (SP)2RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (RP)2RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including RPSPRP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including SPRPRP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (SP)2RP.
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.
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).
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).
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).
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.
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.
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.
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.
The first internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The first internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The second internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The second internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The third internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The third internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The fifth internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The fifth internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The seventh internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The seventh internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The eighth internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The eighth internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The ninth internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The ninth internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The eighteenth internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The eighteenth internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The nineteenth internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The nineteenth internucleoside linkage of the region may be, e.g., an RP internucleoside linkage. The twentieth internucleoside linkage of the region may be, e.g., an SP internucleoside linkage. The twentieth internucleoside linkage of the region may be, e.g., an RP internucleoside linkage.
The region may have a length of, e.g., at least 21 bases. The region may have a length of, e.g., 21 bases.
In some embodiments, each stereochemically enriched internucleoside linkage in an oligonucleotide is a phosphorothioate phosphodiester.
An oligonucleotide may have, e.g., at least 25% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 30% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 35% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 40% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 45% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 50% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 55% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 60% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 65% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 70% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 75% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 80% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 85% of its internucleoside linkages in SP configuration. An oligonucleotide may have, e.g., at least 90% of its internucleoside linkages in SP configuration.
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.
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.
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.
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.
An oligonucleotide may be, e.g., a skipmer.
Terminal Modifications
Oligonucleotides of the invention may include a terminal modification. The terminal modification is a 5′-terminal modification or a 3′-terminal modification.
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.
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.
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 C1-12 alkylene, optionally substituted C2-12 heteroalkylene, optionally substituted C6-10 arylene, optionally substituted C3-8 cycloalkylene, optionally substituted C1-9 heteroarylene, optionally substituted C1-9 heterocyclylene, —O—, —S—S—, and —NRN—, where each RN is independently H or optionally substituted C1-12 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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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).
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.
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).
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
The following examples are meant to illustrate the invention. They are not meant to limit the invention in anyway.
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.
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 (EGFRwt). In a cohort 1, which included frequently studied EGFRmut and EGFRwt lung cancer cell lines (n=15), we found the top six-upregulated miRNAs in a comparison of EGFRmut versus EGFRwt 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.
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 EGFRwt 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 EGFRwt 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.
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.
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 (
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 (
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 (
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%) (
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 (
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 (
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 (
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
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 (
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 (
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
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 (
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 (
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 (
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
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.
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.
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.
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 (R2=0.46) compared with scrambled cells (R2=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.
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 (
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.
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 (
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 (
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 (
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% CO2 atmosphere at 37° C. Cell line identities were confirmed by STR fingerprinting and all were found negative for mycoplasma using the MycoAler Kit (Lonza).
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.
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 mm3 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 ˜1×105 to 1×108 cells, depending on the sample size.
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.
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 NaHCO3, 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.
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.
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).
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.
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.
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).
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.
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.
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).
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.
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).
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.
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).
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.
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).
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.
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−1 polybrene (Sigma). Two days after infection, puromycin was added to the media at 0.5 μg ml−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 (1.6×108 TU ml−1, ABM) or blank control lentiviral particles (2×106 TU ml−1, ABM) together with polybrene. Then the infected cells were passaged and selected by puromycin (Invitrogen) at 0.5 μg ml−1 for 1-2 weeks.
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:
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).
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.
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.
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).
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.
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.
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 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.
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.
4. The method of any one of paragraphs 1 to 3, 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 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.
7. The method of any one of paragraphs 1 to 6, further comprising administering an anti-RTK therapy to the subject.
8. The method of paragraph 7, wherein the anti-RTK therapy is an anti-EGFR therapy.
9. The method of paragraph 8, wherein the anti-EGFR therapy comprises a tyrosine kinase inhibitor (TKI).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 paragraph 22, wherein the oligonucleotide comprises at least one modified nucleobase.
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.
25. The oligonucleotide of any one of paragraphs 22 to 24, wherein the oligonucleotide comprises at least one modified internucleoside linkage.
26. The oligonucleotide of paragraph 25, wherein the modified internucleoside linkage is a phosphorothioate linkage.
27. The oligonucleotide of paragraph 26, wherein the phosphorothioate linkage is a stereochemically enriched phosphorothioate linkage.
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.
29. The oligonucleotide of paragraph 28, wherein at least 70% of the internucleoside linkages in the oligonucleotide are each independently a modified internucleoside linkage.
30. The oligonucleotide of any one of paragraphs 22 to 29, wherein the oligonucleotide comprises at least one modified sugar nucleoside.
31. The oligonucleotide of paragraph 30, wherein the at least one modified sugar nucleoside is a bridged nucleic acid.
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.
33. The oligonucleotide of paragraph 31 or 32, wherein the at least one modified sugar nucleoside is a 2′-modified sugar nucleoside.
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.
35. The oligonucleotide of any one of paragraphs 22 to 34, wherein the oligonucleotide comprises deoxyribonucleotides.
36. The oligonucleotide of any one of paragraphs 22 to 35, wherein the oligonucleotide comprises ribonucleotides.
37. The oligonucleotide of any one of paragraphs 22 to 24, wherein the oligonucleotide is a morpholino oligonucleotide.
38. The oligonucleotide of any one of paragraphs 22 to 24, wherein the oligonucleotide is a peptide nucleic acid.
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.
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.
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.
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.
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.
44. The oligonucleotide of any one of paragraphs 22 to 43, wherein the oligonucleotide comprises a total of at least 12 interlinked nucleotides.
45. The oligonucleotide of any one of paragraphs 22 to 44, wherein the oligonucleotide comprises a total of 24 or fewer interlinked nucleotides.
46. The oligonucleotide of any one of paragraphs 22 to 45, wherein the oligonucleotide is a gapmer, headmer, tailmer, altmer, blockmer, skipmer, or unimer.
47. A double-stranded oligonucleotide comprising the oligonucleotide of any one of paragraphs 22 to 48 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 paragraph 48, wherein the passenger strand comprises at least one modified nucleobase.
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.
51. The oligonucleotide of any one of paragraphs 48 to 50, wherein the passenger strand comprises at least one modified internucleoside linkage.
52. The oligonucleotide of paragraph 51, wherein the modified internucleoside linkage is a phosphorothioate linkage.
53. The oligonucleotide of paragraph 52, wherein the phosphorothioate linkage is a stereochemically enriched phosphorothioate linkage.
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.
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.
56. The oligonucleotide of any one of paragraphs 48 to 55, wherein the passenger strand comprises at least one modified sugar nucleoside.
57. The oligonucleotide of paragraph 56, wherein the at least one modified sugar nucleoside is a bridged nucleic acid.
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.
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.
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.
61. The oligonucleotide of any one of paragraphs 48 to 60, wherein the passenger strand comprises deoxyribonucleotides.
62. The oligonucleotide of any one of paragraphs 48 to 61, wherein the passenger strand comprises ribonucleotides.
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.
64. The oligonucleotide of any one of paragraphs 48 to 63, wherein the guide strand comprises at least one modified nucleobase.
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.
66. The oligonucleotide of any one of paragraphs 48 to 65, wherein the guide strand comprises at least one modified internucleoside linkage.
67. The oligonucleotide of paragraph 66, wherein the modified internucleoside linkage is a phosphorothioate linkage.
68. The oligonucleotide of paragraph 67, wherein the phosphorothioate linkage is a stereochemically enriched phosphorothioate linkage.
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.
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.
71. The oligonucleotide of any one of paragraphs 48 to 70, wherein the guide strand comprises at least one modified sugar nucleoside.
72. The oligonucleotide of paragraph 71, wherein the at least one modified sugar nucleoside is a bridged nucleic acid.
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.
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.
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.
76. The oligonucleotide of any one of paragraphs 48 to 75, wherein the guide strand comprises deoxyribonucleotides.
77. The oligonucleotide of any one of paragraphs 48 to 76, wherein the guide strand comprises ribonucleotides.
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.
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.
80. The oligonucleotide of any one of paragraphs 47 to 79, wherein the hybridized oligonucleotide comprises at least one 3′-overhang.
81. The oligonucleotide of any one of paragraphs 47 to 80, wherein the hybridized oligonucleotide comprises a blunt end.
82. The oligonucleotide of any one of paragraphs 47 to 80, wherein the hybridized oligonucleotide comprises two 3′-overhangs.
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.
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 paragraph 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 paragraph 22, wherein the vector optionally further comprises a promoter to direct transcription of the sequence.
87. The vector of paragraph 86, wherein the vector comprises a sequence encoding multiple oligonucleotides of paragraph 22.
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.
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.
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.
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.
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.
93. The method of any one of paragraphs 1 to 21, 91, or 92, further comprising administration of an additional anti-cancer agent.
94. The method of paragraph 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 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.
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.
99. The method of paragraph 98, wherein the sample comprises tumor tissue, tissue swab, sputum, serum, or plasma.
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.
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 paragraph 102, wherein the anti-RTK therapy is anti-EGFR therapy.
104. The method of paragraph 102 or 103, 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 paragraph 105, wherein the lung cells are lung epithelial cells obtained from a sample of lung tissue of a subject.
107. The method of paragraph 105 or 106, wherein the kung cells are immortalized lung epithelial cells.
108. The method of any one of paragraphs 105 to 107, wherein the lung cells are cancerous.
109. The method of any one of paragraphs 105 to 107, wherein the lung cells are non-cancerous.
110. The method of any one of paragraphs 105 to 109, wherein the lung cells are tolerant or resistant to an anti-RTK agent.
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.
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.
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.
114. The organoid of paragraph 113, wherein the lung cells comprise lung cancer cells.
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.
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 paragraph 116, wherein the cell is comprised within an organoid.
118. The method of paragraph 117, wherein the organoid comprises lung cancer cells.
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.
120. The method of any one of paragraphs 116 to 119, wherein the lung cancer cells are resistant to an anti-RTK therapy.
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.
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.
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.
124. A kit comprising an agent for detecting the level of miR-147b in a sample.
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.
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.
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.
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.
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.
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.
Other embodiments are within the scope of the following claims.
The invention was made with government support under Grant No. CA196530 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/018826 | 2/19/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62807483 | Feb 2019 | US |
