Combination of STAT3-targeting oligonucleotides and PD-L1 inhibitors

Abstract

The subject matter disclosed herein is directed to modulating gene expression using siRNA compositions and methods directed to affecting key cell populations that support cancer growth and metastasis in order to affect beneficial treatment, remission, or elimination of the underlying tumor in a patient.

Description

[Technical field]

[0001] REFERENCE TO CROSS-REFERENCED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63,320,163, filed March 15, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing Reference The contents of the electronic sequence listing (DICN_021_001WO_SeqList_ST26.xml, size: 4,153,073 bytes, and creation date: March 8, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] Currently, chemotherapy is the main cancer therapy worldwide, often combined with surgery or surgery and radiation therapy, depending on the type and stage of the tumor (Non-Patent Document 1). Since the discovery of several important mutations that contribute to carcinogenesis (e.g., epidermal cell alterations (Non-Patent Document 2)), these mutations and the proteins they represent have been widely used as targets for the development of more selective drugs and drug combinations to treat cancer patients. Despite the effectiveness of these drugs, multidrug resistance (MDR) is often found in patients, which often leads to tumor recurrence, limited treatment options, and reduced quality of life for patients. Furthermore, cancer research is often focused on tumor cells, despite the effects of the tumor environment and "normal" or non-cancerous cells within the tumor environment, which have been shown to play important roles in tumor progression, development, and MDR (Non-Patent Document 3). Novel therapies that target different aspects of the TME responsible for tumor growth are needed. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Abbas et al.,AN OVERVIEW OF CANCER TREATMENT MODALITIES / INTECHOPEN,2018 [Non-Patent Document 2] Yamaoka et al.,INT.J.MOL.SCI.(2017)18(11):2420) [Non-Patent Document 3] Klemm et al.,TRENDS CELL BIOL(2015)25(4):198-213 Summary of the Invention

[0005] The present disclosure is based in part on the discovery that the combination of STAT3 oligonucleotide and PD-L1 inhibitor provides synergistic antitumor efficacy against tumors in various tumor microenvironments.Specifically, as demonstrated herein, lipid-conjugated STAT3 oligonucleotides, when delivered in combination with anti-PD-L1 antibodies, reduced tumor volume in vivo in immunosuppressive and inflammatory tumor models.Furthermore, as shown herein, the combination of STAT3 oligonucleotide and PD-L1 inhibitor induced antitumor memory responses when tumors were not established when mice were rechallenged with cancer cells.Furthermore, the efficacy of STAT3 oligonucleotide and PD-L1 inhibitor was dependent on the presence of CD8+ T cells.

[0006] Accordingly, in some aspects, the disclosure provides a method of treating cancer in a subject that has received or is receiving a PD-L1 inhibitor, the method comprising administering an RNAi oligonucleotide comprising an antisense strand 15-30 nucleotides in length and a sense strand 15-40 nucleotides in length, wherein the antisense strand and the sense strand form a double-stranded region, and the antisense strand comprises a region of complementarity to a STAT3 mRNA target sequence, and the region of complementarity is at least 15 contiguous nucleotides in length, thereby treating cancer in the subject.

[0007] In another aspect, the disclosure provides a method of treating cancer in a subject that has received or is receiving an RNAi oligonucleotide, the method comprising administering a PD-L1 inhibitor, wherein the oligonucleotide comprises an antisense strand 15-30 nucleotides in length and a sense strand 15-40 nucleotides in length, wherein the antisense strand and the sense strand form a double-stranded region, wherein the antisense strand comprises a region of complementarity to a STAT3 mRNA target sequence, wherein the region of complementarity is at least 15 contiguous nucleotides in length, thereby treating cancer in the subject.

[0008] In yet another aspect, the disclosure provides a method of treating a disease, disorder, or condition associated with activated STAT3 expression, comprising administering to a subject in need thereof an RNAi oligonucleotide, wherein the subject has received or has received a PD-L1 inhibitor, and the oligonucleotide comprises an antisense strand 15-30 nucleotides in length and a sense strand 15-40 nucleotides in length, wherein the antisense strand and the sense strand form a double-stranded region, and the antisense strand comprises a region of complementarity to a STAT3 mRNA target sequence, and the region of complementarity is at least 15 contiguous nucleotides in length, thereby treating the disease, disorder, or condition associated with activated STAT3 expression.

[0009] In a further aspect, the disclosure provides a kit comprising an RNAi oligonucleotide, an optional pharma- ceutically acceptable carrier, and a package insert containing instructions for administering the oligonucleotide to a subject in need thereof who has received or is receiving a PD-L1 inhibitor, wherein the oligonucleotide comprises an antisense strand 15-30 nucleotides in length and a sense strand 15-40 nucleotides in length, wherein the antisense strand and the sense strand form a double-stranded region, and wherein the antisense strand comprises a region of complementarity to a STAT3 mRNA target sequence, and wherein the region of complementarity is at least 15 contiguous nucleotides in length.

[0010] In another aspect, the disclosure provides a kit comprising a PD-L1 inhibitor, an optional pharma- ceutically acceptable carrier, and a package insert containing instructions for administering the antibody to a subject in need thereof who has or has received an RNAi oligonucleotide, wherein the RNAi oligonucleotide comprises an antisense strand 15-30 nucleotides in length and a sense strand 15-40 nucleotides in length, wherein the antisense strand and the sense strand form a double-stranded region, wherein the antisense strand comprises a region of complementarity to a STAT3 mRNA target sequence, and wherein the region of complementarity is at least 15 contiguous nucleotides in length.

[0011] In some or any of the foregoing or related embodiments, the subject has a disease, disorder, or condition associated with activated STAT3 expression. In some embodiments, the disease, disorder, or condition associated with activated STAT3 expression is cancer. In some embodiments, the cancer is selected from carcinoma, sarcoma, melanoma, lymphoma, and leukemia, prostate cancer, breast cancer, hepatocellular carcinoma (HCC), colorectal cancer, pancreatic cancer, and glioblastoma. In some embodiments, the cancer comprises an immunosuppressive tumor microenvironment. In some embodiments, the immunosuppressive tumor microenvironment has low levels of CD8+ T cell infiltration and high levels of MDSC. In other embodiments, the cancer comprises an inflammatory tumor microenvironment. In some embodiments, the inflammatory tumor microenvironment comprises infiltrating T cells. In some embodiments, the inflammatory tumor microenvironment comprises high levels of CD8+ T cell infiltration and high levels of MDSC. In some embodiments, the cancer comprises a tumor that is resistant to immune checkpoint therapy. In some embodiments, the cancer comprises a tumor that is partially resistant to immune checkpoint therapy. In some aspects, the cancer comprises a tumor that is susceptible to immune checkpoint therapy.

[0012] In any or all of the foregoing or related aspects, the methods described herein treat spontaneous tumor metastasis.

[0013] In some or any of the above or related embodiments, the method described herein reduces immunosuppressive genes and enhances immunoactivating genes.In some embodiments, the immunosuppressive genes include checkpoint inhibitors, STAT3-mediated genes, suppressive cytokines, suppressive chemokines, and angiogenesis and matrix remodeling related genes.In some embodiments, the immunoactivating genes include genes related to T cell migration, T cell activation, T cell memory, and / or T cell cytotoxicity.

[0014] In some or any of the foregoing or related embodiments, the PD-L1 inhibitor is an antibody. In some embodiments, the antibody is an anti-PD-L1 antibody. In some embodiments, the anti-PDL1 antibody is selected from FAZ053, atezolizumab, avelumab, durvalumab, embafolimab, and BMS-936559. In some embodiments, the antibody is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, and cemiplimab.

[0015] In some embodiments, the PD-L1 inhibitor is a small molecule inhibitor. In some embodiments, the PD-L1 inhibitor is a peptide. In some embodiments, the PD-L1 inhibitor is a nucleic acid molecule. In some embodiments, the nucleic acid molecule is selected from an antisense oligonucleotide, an siRNA, or an miRNA.

[0016] In some or any of the foregoing or related embodiments, the STAT3 mRNA target sequence comprises any one of SEQ ID NOs: 89-280. In some embodiments, the region of complementarity is fully complementary to the STAT3 mRNA target sequence. In some embodiments, the region of complementarity comprises no more than four mismatches to the STAT3 mRNA target sequence.

[0017] In some or any of the foregoing or related embodiments, the antisense strand is 19-27 nucleotides in length. In some embodiments, the antisense strand is 21-27 nucleotides in length, and optionally, the antisense strand is 22 nucleotides in length.

[0018] In some or any of the aforementioned or related embodiments, the sense strand is 19-40 nucleotides in length, and optionally, the sense strand is 36 nucleotides in length.

[0019] In some or any of the foregoing or related embodiments, the double-stranded region is at least 19 nucleotides in length. In some embodiments, the double-stranded region is at least 20 nucleotides in length, and optionally, the double-stranded region is 21 nucleotides in length.

[0020] In some or any of the foregoing or related embodiments, the region of complementarity to STAT3 is at least 19 contiguous nucleotides in length. In some embodiments, the region of complementarity to STAT3 is at least 21 contiguous nucleotides in length.

[0021] In some or any of the aforementioned or related embodiments, the sense strand comprises at its 3' end a stem-loop depicted as S1-L-S2, where S1 is complementary to S2, and L forms a loop between S1 and S2 that is 3-5 nucleotides in length. In some embodiments, L is a tetraloop, and optionally, L is 4 nucleotides in length. In some embodiments, L comprises the sequence depicted as GAAA.

[0022] In some or any of the aforementioned or related embodiments, the antisense strand includes a 3' overhang sequence one or more nucleotides in length, optionally, the 3' overhang sequence is 2 nucleotides in length, and optionally, the 3' overhang sequence is GG.

[0023] In some or any of the foregoing or related embodiments, the oligonucleotide comprises at least one modified nucleotide. In some embodiments, the modified nucleotide comprises a 2'-modification. In some embodiments, the 2'-modification is a modification selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid. In some embodiments, about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand comprise a 2'-fluoro modification. In some embodiments, about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the antisense strand comprise a 2'-fluoro modification. In some embodiments, about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the oligonucleotide comprise a 2'-fluoro modification. In some embodiments, the sense strand comprises 36 nucleotides having positions 1-36 from 5' to 3', and positions 8-11 comprise a 2'-fluoro modification. In some embodiments, the antisense strand comprises 22 nucleotides having positions 1-22 from 3' to 5', and positions 2, 3, 4, 5, 7, 10, and 14 comprise a 2'-fluoro modification. In some embodiments, the remaining nucleotides comprise a 2'-O-methyl modification. In some embodiments, all of the nucleotides of the oligonucleotide are modified.

[0024] In some or any of the foregoing or related embodiments, the oligonucleotide comprises at least one modified internucleotide linkage. In some embodiments, the at least one modified internucleotide linkage is a phosphorothioate linkage.

[0025] In some or any of the foregoing or related embodiments, the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analog, hi some embodiments, the phosphate analog is an oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

[0026] In some or any of the foregoing or related embodiments, at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands. In some embodiments, the nucleotide is conjugated to two or more targeting ligands, and the targeting ligands are the same or different. In some embodiments, the one or more targeting ligands are selected from carbohydrates, amino sugars, cholesterol, polypeptides, or lipids. In some embodiments, the one or more targeting ligands are saturated or unsaturated fatty acid moieties. In some embodiments, the targeting ligands are saturated fatty acid moieties ranging in size from C10 to C24 in length. In some embodiments, the targeting ligands are C16 saturated fatty acid moieties. In some embodiments, the targeting ligands are C18 saturated fatty acid moieties. In some embodiments, the targeting ligands are C22 saturated fatty acid moieties. In some embodiments, the targeting ligands include an N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the GalNAc moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety, hi some embodiments, the L of up to four nucleotides of the stem-loop are each conjugated to a monovalent GalNAc moiety.

[0027] In some or any of the foregoing or related embodiments, the sense strand comprises a sequence set forth in SEQ ID NOs: 857-946. In some embodiments, the antisense strand comprises a sequence set forth in SEQ ID NOs: 947-1036.

[0028] In any or all of the above or related embodiments, the sense strand and the antisense strand are (a) SEQ ID NOs: 9 and 10, respectively; (b) SEQ ID NOs: 37 and 38, respectively; (c) SEQ ID NOs: 65 and 66, respectively, and (d) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 69 and 70, respectively.

[0029] In any or all of the above or related embodiments, the sense strand and the antisense strand are (a) SEQ ID NOs: 861 and 951, respectively; (b) SEQ ID NOs: 857 and 947, respectively; (c) SEQ ID NOs: 858 and 948, respectively; (d) SEQ ID NOs: 859 and 949, respectively; (e) SEQ ID NOs: 860 and 950, respectively; (f) SEQ ID NOs: 862 and 952, respectively; (g) SEQ ID NOs: 863 and 953, respectively; (h) SEQ ID NOs: 864 and 954, respectively; (i) SEQ ID NOs: 865 and 955, respectively; (j) SEQ ID NOs: 866 and 956, respectively; (k) SEQ ID NOs: 867 and 957, respectively; (l) SEQ ID NOs: 868 and 958, respectively; (m) SEQ ID NOs: 869 and 959, respectively; (n) SEQ ID NOs: 870 and 960, respectively; (o) SEQ ID NOs: 871 and 961, respectively; (p) SEQ ID NOs: 872 and 962, respectively; (q) SEQ ID NOs: 873 and 963, respectively; (r) SEQ ID NOs: 874 and 964, respectively; (s) SEQ ID NOs: 875 and 965, respectively; (t) SEQ ID NOs: 876 and 966, respectively; (u) SEQ ID NOs: 877 and 967, respectively; (v) SEQ ID NOs: 878 and 968, respectively; (w) SEQ ID NOs: 879 and 969, respectively; (x) SEQ ID NOs: 880 and 970, respectively; (y) SEQ ID NOs: 881 and 971, respectively; (z) SEQ ID NOs: 882 and 972, respectively; (aa) SEQ ID NOs: 883 and 973, respectively; (bb) SEQ ID NOs: 884 and 974, respectively; (cc) SEQ ID NOs: 885 and 975, respectively; (dd) SEQ ID NOs: 886 and 976, respectively; (ee) SEQ ID NOs: 887 and 977, respectively; (ff) SEQ ID NOs: 888 and 978, respectively; (gg) SEQ ID NOs: 940 and 1030, respectively; (hh) SEQ ID NOs: 896 and 986, respectively; and (ii) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 920 and 1010, respectively.

[0030] In some or any of the aforementioned or related embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:862 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:952. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:875 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:965. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:876 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:966. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:920 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:1010.

[0031] In any or all of the above or related embodiments, the sense strand and the antisense strand are (a) SEQ ID NOs: 901 and 991, respectively; (b) SEQ ID NOs: 910 and 1000, respectively; (c) SEQ ID NOs: 899 and 989, respectively; (d) SEQ ID NOs: 896 and 986, respectively; (e) SEQ ID NOs: 892 and 982, respectively; (f) SEQ ID NOs: 890 and 980, respectively; and (g) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 889 and 979, respectively.

[0032] In any or all of the above or related embodiments, the sense strand and the antisense strand are (a) SEQ ID NOs: 940 and 1030, respectively; (b) SEQ ID NOs: 937 and 1027, respectively, and (c) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 939 and 1029, respectively.

[0033] In any or all of the above or related embodiments, the sense strand and the antisense strand are (a) SEQ ID NOs: 915 and 1005, respectively; (b) SEQ ID NOs: 924 and 1014, respectively; (c) SEQ ID NOs: 913 and 1003, respectively; and (d) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 920 and 1010, respectively.

[0034] In some or any of the aforementioned or related embodiments, the sense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 11, 39, 67, and 71. In some embodiments, the antisense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 12, 40, 68, and 72.

[0035] In any or all of the above or related embodiments, the sense strand and the antisense strand are (a) SEQ ID NOs: 11 and 12, respectively; (b) SEQ ID NOs: 39 and 40, respectively; (c) SEQ ID NOs: 67 and 68, respectively, and (d) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 71 and 72, respectively.

[0036] In some or any of the aforementioned or related embodiments, the sense strand comprises a nucleotide sequence selected from SEQ ID NOs: 1042, 1055, 1056, and 1100. In some embodiments, the antisense strand comprises a nucleotide sequence selected from SEQ ID NOs: 1132, 1145, 1146, and 1190.

[0037] In any or all of the above or related embodiments, the sense strand and the antisense strand are (a) SEQ ID NOs: 1041 and 1131, respectively; (b) SEQ ID NOs: 1037 and 1127, respectively; (c) SEQ ID NOs: 1038 and 1128, respectively; (d) SEQ ID NOs: 1039 and 1129, respectively; (e) SEQ ID NOs: 1040 and 1130, respectively; (f) SEQ ID NOs: 1042 and 1132, respectively; (g) SEQ ID NOs: 1043 and 1133, respectively; (h) SEQ ID NOs: 1044 and 1134, respectively; (i) SEQ ID NOs: 1045 and 1135, respectively; (j) SEQ ID NOs: 1046 and 1136, respectively; (k) SEQ ID NOs: 1047 and 1137, respectively; (l) SEQ ID NOs: 1048 and 1138, respectively; (m) SEQ ID NOs: 1049 and 1139, respectively; (n) SEQ ID NOs: 1050 and 1140, respectively; (o) SEQ ID NOs: 1051 and 1141, respectively; (p) SEQ ID NOs: 1052 and 1142, respectively; (q) SEQ ID NOs: 1053 and 1143, respectively; (r) SEQ ID NOs: 1054 and 1144, respectively; (s) SEQ ID NOs: 1055 and 1145, respectively; (t) SEQ ID NOs: 1056 and 1146, respectively; (u) SEQ ID NOs: 1057 and 1147, respectively; (v) SEQ ID NOs: 1058 and 1148, respectively; (w) SEQ ID NOs: 1059 and 1149, respectively; (x) SEQ ID NOs: 1060 and 1150, respectively; (y) SEQ ID NOs: 1061 and 1151, respectively; (z) SEQ ID NOs: 1062 and 1152, respectively; (aa) SEQ ID NOs: 1063 and 1153, respectively; (bb) SEQ ID NOs: 1064 and 1154, respectively; (cc) SEQ ID NOs: 1065 and 1155, respectively; (dd) SEQ ID NOs: 1066 and 1156, respectively; (ee) SEQ ID NOs: 1067 and 1157, respectively; (ff) SEQ ID NOs: 1068 and 1158, respectively; (gg) SEQ ID NOs: 1120 and 1210, respectively; (hh) SEQ ID NOs: 1076 and 1166, respectively; and (ii) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1100 and 1190, respectively.

[0038] In some or any of the aforementioned or related embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO: 1042 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 1132. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO: 1055 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 1145. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO: 1056 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 1146. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO: 1100 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 1190.

[0039] In any or all of the above or related embodiments, the sense strand and the antisense strand are (a) SEQ ID NOs: 1081 and 1171, respectively; (b) SEQ ID NOs: 1090 and 1180, respectively; (c) SEQ ID NOs: 1079 and 1169, respectively; (d) SEQ ID NOs: 1076 and 1166, respectively; (e) SEQ ID NOs: 1072 and 1162, respectively; (f) SEQ ID NOs: 1070 and 1160, respectively; and (g) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1069 and 1159, respectively.

[0040] In any or all of the above or related embodiments, the sense strand and the antisense strand are (a) SEQ ID NOs: 1120 and 1210, respectively; (b) SEQ ID NOs: 1117 and 1207, respectively, and (c) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1119 and 1209, respectively.

[0041] In any or all of the above or related embodiments, the sense strand and the antisense strand are (a) SEQ ID NOs: 1095 and 1185, respectively; (b) SEQ ID NOs: 1104 and 1194, respectively; (c) SEQ ID NOs: 1093 and 1183, respectively; and (d) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1100 and 1190, respectively.

[0042] In some aspects, the disclosure provides methods of treating cancer in a subject who has received or is receiving an anti-PD-L1 antibody, the method comprising administering an RNAi oligonucleotide comprising an antisense strand comprising a nucleotide sequence set forth in SEQ ID NO: 84, the antisense strand comprising a region of complementarity to a STAT3 mRNA target sequence, and a sense strand comprising a nucleotide sequence set forth in SEQ ID NO: 83, thereby treating cancer in the subject.

[0043] In another aspect, the disclosure provides a method of treating cancer in a subject who has received or is receiving an RNAi oligonucleotide, the method comprising administering an anti-PD-L1 antibody, where the oligonucleotide comprises an antisense strand comprising a nucleotide sequence set forth in SEQ ID NO: 84, the antisense strand comprising a region of complementarity to the STAT3 mRNA target sequence, and a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 83, thereby treating cancer in the subject.

[0044] In yet another aspect, the disclosure provides a method of treating a disease, disorder, or condition associated with activated STAT3 expression, comprising administering to a subject in need thereof an RNAi oligonucleotide, where the subject has received or has received an anti-PD-L1 antibody, and the oligonucleotide comprises an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 84, where the antisense strand comprises a region of complementarity to the STAT3 mRNA target sequence, and a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 83, thereby treating the disease, disorder, or condition associated with activated STAT3 expression.

[0045] In a further aspect, the disclosure provides a kit comprising an RNAi oligonucleotide, an optional pharma- ceutically acceptable carrier, and a package insert containing instructions for administering the oligonucleotide to a subject in need thereof who has received or is receiving an anti-PD-L1 antibody, wherein the oligonucleotide comprises an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO:84 (the antisense strand comprises a region of complementarity to the STAT3 mRNA target sequence) and a sense strand comprising the nucleotide sequence set forth in SEQ ID NO:83.

[0046] In other aspects, the disclosure provides a kit comprising an anti-PD-L1 antibody, an optional pharma- ceutically acceptable carrier, and a package insert containing instructions for administering the antibody to a subject in need thereof who has received or is receiving an RNAi oligonucleotide, wherein the RNAi oligonucleotide comprises an antisense strand comprising a nucleotide sequence set forth in SEQ ID NO:84 (the antisense strand comprises a region of complementarity to the STAT3 mRNA target sequence) and a sense strand comprising a nucleotide sequence set forth in SEQ ID NO:83.

[0047] In some aspects, the disclosure provides methods of treating cancer in a subject who has received or is receiving an anti-PD-L1 antibody, the method comprising administering an RNAi oligonucleotide comprising an antisense strand comprising a nucleotide sequence set forth in SEQ ID NO:70, the antisense strand comprising a region of complementarity to a STAT3 mRNA target sequence, and a sense strand comprising a nucleotide sequence set forth in SEQ ID NO:69, thereby treating cancer in the subject.

[0048] In another aspect, the disclosure provides a method of treating cancer in a subject who has received or is receiving an RNAi oligonucleotide, the method comprising administering an anti-PD-L1 antibody, where the oligonucleotide comprises an antisense strand comprising a nucleotide sequence set forth in SEQ ID NO: 70, the antisense strand comprising a region of complementarity to the STAT3 mRNA target sequence, and a sense strand comprising a nucleotide sequence set forth in SEQ ID NO: 69, thereby treating cancer in the subject.

[0049] In yet another aspect, the disclosure provides a method of treating a disease, disorder, or condition associated with activated STAT3 expression, comprising administering to a subject in need thereof an RNAi oligonucleotide, where the subject has received or has received an anti-PD-L1 antibody, and the oligonucleotide comprises an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 70, where the antisense strand comprises a region of complementarity to the STAT3 mRNA target sequence, and a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 69, thereby treating the disease, disorder, or condition associated with activated STAT3 expression.

[0050] In a further aspect, the disclosure provides a kit comprising an RNAi oligonucleotide, an optional pharma- ceutically acceptable carrier, and a package insert containing instructions for administering the oligonucleotide to a subject in need thereof who has received or is receiving an anti-PD-L1 antibody, wherein the oligonucleotide comprises an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO:70 (the antisense strand comprises a region of complementarity to the STAT3 mRNA target sequence) and a sense strand comprising the nucleotide sequence set forth in SEQ ID NO:69.

[0051] In other aspects, the disclosure provides a kit comprising an anti-PD-L1 antibody, an optional pharma- ceutically acceptable carrier, and a package insert containing instructions for administering the antibody to a subject in need thereof who has received or has received an RNAi oligonucleotide, wherein the RNAi oligonucleotide comprises an antisense strand comprising a nucleotide sequence set forth in SEQ ID NO:70 (the antisense strand comprises a region of complementarity to the STAT3 mRNA target sequence) and a sense strand comprising a nucleotide sequence set forth in SEQ ID NO:69. [Brief description of the drawings]

[0052] [Figure 1A] FIG. 1A provides structures of RNAi oligonucleotide molecules having chemical modifications with GalNAc (top) or lipids (e.g., C18 hydrocarbon chains) (bottom) conjugated to the oligonucleotide molecule to generate oligonucleotide-ligand conjugates. [Figure 1B] FIG. 1B provides the structure of a lipid tail suitable for conjugation to an RNAi oligonucleotide molecule. [Figure 2A-2B] 2A and 2B are graphs showing the remaining mouse Stat3 mRNA levels in the liver of mice treated with GalXC-STAT3-conjugates (GalNAc conjugates) targeting different regions of Stat3 mRNA. Mice were administered a single dose (3 mg / kg) (FIG. 2A) or various doses (0.3, 1.0, or 3.0 mg / kg) (FIG. 2B) to determine dose response. Arrows indicate constructs selected for further study. [Figure 3A-3B] 3A and 3B are graphs showing mouse Stat3 mRNA expression in G-MDSCs and M-MDSCs from Pan02 xenografts implanted in mice after 3 days of treatment with GalXC-STAT3-C18 conjugate administered at 25 mg / kg (FIG. 3A) and 50 mg / kg (FIG. 3B) into tumors. [Figure 4A-4B]4A and 4B are graphs showing mouse Stat3 mRNA expression following treatment of Pan02 xenograft mice with GalXC-STAT3-C18 conjugate at doses of 25 and 50 mg / kg in the bulk tumor (TME) (FIG. 4A) and tumor-draining lymph nodes (TdLN) (FIG. 4B). [Figure 5A] FIG. 5A provides a graph showing the effect of GalXC-STAT3-C18-4123 on Stat3 and Pdl1 mRNA levels in G / M-MDSCs in the TME and TdLN of Pan02 xenograft mice 3 days after administration of 25 or 50 mg / kg of conjugated oligonucleotide. [Figure 5B] FIG. 5B provides a graph showing the effect of GalXC-STAT3-C18-4123 on Stat3 and Pdl1 mRNA levels in the TdLNs of Pan02 xenograft mice 7 days after a 25 mg / kg dose of conjugated oligonucleotide. [Figure 6A-6B] 6A and 6B are graphs showing the in vivo effect of subcutaneous treatment with GalXC-STAT3-C18-4123 at a total dose of 50 mg / kg on tumor volume over time in immunocompetent mice bearing Pan02 mouse pancreatic tumors. Mice were treated with either four doses of 12.5 mg / kg (FIG. 6A) or two doses of 25 mg / kg (FIG. 6B) of the conjugated oligonucleotide. [Figure 7] Figure 7 provides a graph showing the percent (%) of human STAT3 mRNA remaining in Huh7 cells that endogenously express human STAT3 after 24 hours of treatment with 1 nM DsiRNAs targeting various regions of the STAT3 gene. 192 DsiRNAs were designed and screened. Two primer pairs were used. Expression was normalized between samples using housekeeping genes HPRT and SFRS9 (forward 1-sequence number 1219, reverse 1-sequence number 1220; probe 1-sequence number 1221; forward 2-sequence number 1222, reverse 2-sequence number 1223; probe 2-sequence number 1224). [Figure 8A-8B]Figures 8A and 8B provide graphs showing the percent (%) of human STAT3 mRNA remaining in Huh7 cells that endogenously express human STAT3 after 24 hours of treatment with 0.05 nM, 0.3 nM, or 1 nM DsiRNAs targeting various regions of the STAT3 gene. Forty-eight GalNAc-conjugated STAT3 oligonucleotides were assayed in Figure 8A, and 34 of those oligonucleotides were selected for further testing in vivo (Figure 8B). [Figure 9A-9B] 9A and 9B provide graphs showing the percent (%) of human STAT3 mRNA remaining in the liver of mice exogenously expressing human STAT3 (hydrodynamic injection model) following treatment with GalNAc-conjugated STAT3 oligonucleotides. Mice were administered 1 mg / kg of the indicated GalNAc-STAT3 oligonucleotides subcutaneously formulated in PBS. Three days after administration, mice were hydrodynamically injected (HDI) with a DNA plasmid encoding human STAT3. Human STAT3 mRNA levels were determined from livers harvested 18 hours after injection. Arrows indicate oligonucleotides selected for dose-response analysis. Hs / Mf=human / monkey consensus sequence; Hs / Mm=human / mouse consensus sequence; Hs / Mf / Mm=human / monkey / mouse triple consensus sequence. [Figure 10] Figure 10 provides a graph showing the dose response of GalNAc-conjugated STAT3 oligonucleotides. The percent (%) of human STAT3 mRNA remaining in the liver of mice exogenously expressing human STAT3 (HDI model) after treatment with two different doses (0.3 mg / kg or 1 mg / kg) of human GalNAc-conjugated STAT3 oligonucleotides was measured. Human STAT3 mRNA levels were determined from livers harvested 18 hours after injection with a plasmid encoding human STAT3. Arrows indicate oligonucleotides selected for dose response analysis. Hs / Mf = human / monkey consensus sequence, Hs / Mm = human / mouse consensus sequence. [Figure 11]Figure 11 provides a graph showing normalized (relative to Ppib) mouse STAT3 mRNA remaining in the liver of mice that endogenously express mouse STAT3 after treatment with GalNAc-conjugated STAT3 oligonucleotides. Mice were subcutaneously administered 3 mg / kg of the indicated GalNAc-STAT3 oligonucleotides formulated in PBS. Livers were harvested 5 days after administration and mouse STAT3 mRNA levels were determined. Arrows indicate the top oligonucleotide and the oligonucleotide selected for dose-response studies. [Figure 12] Figure 12 provides a graph showing normalized (relative to Ppib) mouse STAT3 mRNA remaining in the liver of mice that endogenously express mouse STAT3 after treatment with GalNAc-conjugated STAT3 oligonucleotides. Mice were subcutaneously administered 3 mg / kg of the indicated GalNAc-STAT3 oligonucleotides formulated in PBS. Livers were harvested 5 days after administration and mouse STAT3 mRNA levels were determined. Arrows indicate the oligonucleotides selected for dose-response studies. [Figure 13A-13B] Figures 13A and 13B provide graphs showing the dose response of GalNAc-conjugated STAT3 oligonucleotides. The percent (%) of mouse STAT3 mRNA remaining in the liver of mice that endogenously express human STAT3 was measured after treatment with three doses (0.3 mg / kg, 1 mg / kg, and 3 mg / kg) of human GalNAc-conjugated STAT3 oligonucleotides. Mouse STAT3 mRNA levels were determined from livers harvested 5 days later. TC = triple common (mouse / human / monkey); Hs_Mm = human / mouse. [Figure 14]Figure 14 provides a graph showing the percent (%) of human STAT3 mRNA remaining in the liver of mice exogenously expressing human STAT3 (hydrodynamic injection model) after treatment with GalNAc-conjugated STAT3 oligonucleotides. Mice were subcutaneously administered 1 mg / kg of the indicated GalNAc-STAT3 oligonucleotides formulated in PBS. Three days after administration, mice were hydrodynamically injected (HDI) with a DNA plasmid encoding human STAT3. Human STAT3 mRNA levels were determined from livers harvested 18 hours after injection. Arrows indicate oligonucleotides selected for dose-response studies. [Figure 15] Figure 15 provides a graph showing the dose response of GalNAc-conjugated STAT3 oligonucleotides. Percentage (%) of human STAT3 mRNA remaining in liver of mice exogenously expressing human STAT3 (hydrodynamic injection model) after treatment with GalNAc-conjugated STAT3 oligonucleotides. Mice were subcutaneously administered three doses (0.3 mg / kg, 1 mg / kg, and 3 mg / kg) of the indicated GalNAc-STAT3 oligonucleotides formulated in PBS. Three days after administration, mice were hydrodynamically injected (HDI) with a DNA plasmid encoding human STAT3. Human STAT3 mRNA levels were determined from livers harvested 18 hours after injection. TC = triple common (mouse / human / monkey); Hs_Mm = human / mouse; Hs = human. [Figure 16]Figure 16 provides a graph showing the dose response of GalNAc-conjugated STAT3 oligonucleotides. Percentage (%) of human STAT3 mRNA remaining in the liver of mice exogenously expressing human STAT3 (hydrodynamic injection model) after treatment with GalNAc-conjugated STAT3 oligonucleotides. Mice were subcutaneously administered two doses (0.3 mg / kg and 1 mg / kg) of the indicated GalNAc-STAT3 oligonucleotides formulated in PBS. Three days after administration, mice were hydrodynamically injected (HDI) with a DNA plasmid encoding human STAT3. Human STAT3 mRNA levels were determined from livers harvested 18 hours after injection. [Figure 17] 17 provides a graph showing the percent (%) of human STAT1 mRNA remaining in Huh7 cells that endogenously express STAT3 and STAT1 and were treated with GalNAc-conjugated STAT3 oligonucleotides. Cells were treated with three doses (0.05 nM, 0.3 nM, and 1 nM) of oligonucleotide for 24 hours. [Figure 18A] Figure 18A provides a graph showing tumor volume following administration of GalXC-STAT3-C18 oligonucleotides alone or in combination with anti-PD-L1 mAb. Immunocompetent mice bearing Pan02 mouse pancreatic tumors were subcutaneously (sc) administered 25 mg / kg GalXC-STAT3-C18-4123 with intraperitoneal (ip) treatment of 10 mg / kg anti-PD-L1 mAb. Controls included GalXC-placebo (HBV siRNA with identical chemical and lipid conjugation to GalXC-STAT3 oligonucleotides), 25 mg / kg GalXC-STAT3-C18-4123, or 25 mg / kg GalXC-placebo in combination with 10 mg / kg anti-PD-L1 mAb. Mice were initially administered two doses spaced 3 days apart and then two weeks later received two additional doses spaced 3 days apart [(q3d×2)×2]. Arrows indicate the days on which doses were administered. [Figure 18B]Figure 18B provides a graph showing tumor volumes following administration of GalXC-STAT3-C18 oligonucleotide in combination with anti-PD-L1 mAb. Placebo-treated mice from Figure 18A were administered 25 mg / kg GalXC-STAT3-C18-4123 subcutaneously (sc) along with intraperitoneal (ip) treatment with 10 mg / kg anti-PD-L1 mAb on day 59. [Figures 19A-19C] Figures 19A-19C provide graphs showing tumor volumes following administration of GalXC-STAT3-C18 oligonucleotide alone or in combination with anti-PD-L1 mAb, or GalXC-placebo alone or in combination with anti-PD-L1 mAb, in tumors with different immunophenotypes. Mice were implanted with 4T1 (triple negative breast, checkpoint resistant) (Figure 19A), MC-38 (colon cancer, partially checkpoint sensitive) (Figure 19B), or Hepa1-6 (hepatocellular carcinoma, checkpoint sensitive) (Figure 19C) cells. Tumor-bearing mice were administered 25 mg / kg GalXC-STAT3-C18-4123 sc, accompanied by 10 mg / kg ip treatment of anti-PD-L1 mAb. Controls included 25mg / kg GalXC-placebo, GalXC-STAT3-C18-4123, or 25mg / kg GalXC-placebo in combination with 10mg / kg anti-PD-L1. Mice bearing MC-38 and Hepa1-6 tumors were administered two doses at 25mg / kg, three days apart, and the same regimen was repeated the following week. Mice bearing 4T1 tumors were administered three doses each, three days apart (q3dx3). Arrows (5 / 5 CR) = all treated mice were complete responders. [Figure 20]Figure 20 provides a graph showing the effect of Hepa1-6 rechallenge on completely eradicated tumors. After tumors in all five mice were completely regressed with GalXC-STAT3-C18 (25 mg / kg, sc) and anti-PD-L1 mAb (10 mg / kg, ip) treatment in Figure 19C, the mice were rechallenged with Hepa1-6 cells (2e6 cells / mouse) on the opposite flank on day 51 and tumor volume was monitored (Figure 20). Arrow (5 / 5 CR) = all mice remained tumor-free even after rechallenge. [Figure 21] Figure 21 provides a graph showing tumor volume after administration of GalXC-STAT3-C18 oligonucleotide alone or in combination with anti-PD-L1 mAb in immunodeficient mice without functional CD8+ T cells. Mice bearing 4T1 tumors were administered GalXC-STAT3-C18-4123 (25 mg / kg, each dose three times at 3-day intervals (q3d×3)) sc and anti-PD-L1 mAb (10 mg / kg, q3d×3) ip. Controls included 25 mg / kg GalXC-placebo, GalXC-STAT3-C18-4123, or 25 mg / kg GalXC-placebo in combination with 10 mg / kg anti-PD-L1. [Figure 22] FIG. 22 provides images showing the emergence of tumors (with cell death) from the mice assayed in FIG. 19A, and perforin staining for positive cytotoxic CD8+ T cells in the tumors at the end of the study. [Diagram 23]Figure 23 provides images showing lung tumor metastasis following administration of GalXC-STAT3-C18-4123 oligonucleotide alone or in combination with anti-PD-L1 mAb as treated in Figures 19A and 21. Mice bearing 4T1 tumors (immunocompetent or immunodeficient) were administered GalXC-STAT3-C18-4123 (50mg / kg, q3dx3) sc and anti-PD-L1 mAb (10mg / kg, q3dx3) ip. Controls included 50mg / kg GalXC-placebo, GalXC-STAT3-C18-4123, or 50mg / kg GalXC-placebo in combination with 10mg / kg anti-PD-L1. [Figure 24] Figure 24 provides a heat map showing the regulation of targets involved in immune modulation observed in CT26 tumors following combined treatment with GalXC-STAT3-C18-4123 (sc, 25 mg / kg, q3d x 3) and anti-PD-L1 mAb (ip, 10 mg / kg, q3d x 3) compared to controls including GalXC-placebo at 25 mg / kg, GalXC-STAT3-C18-4123, or GalXC-placebo at 25 mg / kg in combination with anti-PD-L1 mAb at 10 mg / kg. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The present disclosure will now be described in more detail below with reference to the accompanying drawings, in which exemplary embodiments of the present disclosure are shown. However, the present disclosure may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0054] definition Publications discussed throughout the text are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

[0055] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Furthermore, the singular form and the articles "a," "an," and "the" are intended to include the plural, unless expressly specified otherwise. Furthermore, it is to be understood that as used herein, the terms "includes," "comprises," "including," and / or "comprising" specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Furthermore, it is to be understood that when an element, including a component or subsystem, is referred to and / or shown as being connected or coupled to another element, it may be directly connected or coupled to the other element, or there may be intervening elements.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the methods and compositions of the present disclosure, exemplary methods and materials are described herein.

[0057] General texts describing molecular biology techniques useful herein, including the use of vectors, promoters, and many other related topics include Berger and Kimmel, GUIDE TO MOLECULAR CLONING TECHNIQUES, METHODS IN ENZYMOLOGY, volume 152, (Academic Press, Inc., San Diego, Calif.) ("Berger"), Sambrook et al., MOLECULAR CLONING--A LABORATORY MANUAL, 2d ed., Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989 ("Sambrook"), and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, FMAusubel et al., eds., CURRENT PROTOCOLS, A JOINT VENTURE BETWEEN GREENE PUBLISHING ASSOCIATES, INC. AND JOHN WILEY AND SONS, INC., (through 1999 Supplement) (“Ausubel”).Examples of protocols sufficient to instruct one of skill in the art through in vitro amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Q. beta.-replicase amplification, and other RNA polymerase-mediated techniques (e.g., NASBA), for the production of homologous nucleic acids of the present disclosure, can be found in Berger, Sambrook, and Ausubel, as well as in Mullis et al., (1987) U.S. Pat. No. 4,683,202, Innis et al., eds. (1990), PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS (Academic Press Inc. San Diego, Calif.) ("Innis"), Arnheim and Levinson (Oct. 1, 1990) Cand EN 36-47, J. NIH RES. (1991) 3:81-94, Kwoh et al. al.,(1989)PROC.NATL.ACAD.SCI.USA 86:1173, Guatelliet et al.,(1990)PROC.NAT'L.ACAD.SCI.USA 87:1874, Lomell et al.,(1989)J.CLIN.CHEM 35:1826, Landegren et al.,(1988)SCIENCE 241:1077-80, Van Brunt (1990) BIOTECHNOLOGY 8:291-94, Wu and Wallace (1989) GENE 4:560, Barringer et al., (1990) GENE 89:117, and Sooknanan and Malek (1995) BIOTECHNOLOGY Found in 13:563-564. Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Patent No. 5,426,039. Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng et al., (1994) NATURE 369:684-85, and references cited therein, where PCR amplicons of up to 40 kb are generated.

[0058] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0059] Ranges may be expressed herein as from "about" one value and / or to "about" another value. When such a range is expressed, another embodiment includes from the one value and / or to the other value. Similarly, it will be understood that when values ​​are expressed as approximations, the use of the preceding "about" causes the value to form another embodiment. Further, it will be understood that each endpoint of a range is significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are several values ​​disclosed herein, and that each value is also disclosed herein as "about" that value in addition to the value itself. For example, when the value "10" is disclosed, "about 10" is also disclosed. It is also understood that when a value is disclosed, "less than or equal to," "greater than or equal to," and possible ranges between the values ​​are also disclosed, as would be well understood by one of ordinary skill in the art. For example, when the value "10" is disclosed, "less than or equal to 10" as well as "greater than or equal to 10" are also disclosed. It is also understood that throughout the application, data is provided in several different formats, and that this data represents endpoints and starting points, as well as ranges of any combination of data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, then 10 and greater than 15, greater than or equal to 10 and 15, less than 10 and 15, less than or equal to 10 and 15, and equal to 10 and 15 are considered to be disclosed as well as 10 to 15. It is also understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0060] In this specification and the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0061] The term "cancer" or "tumor" includes, but is not limited to, solid tumors and blood-borne tumors. These terms include diseases of the skin, tissues, organs, bone, cartilage, blood, and blood vessels. These terms further encompass primary and metastatic cancers.

[0062] The term "PD-1" refers to a protein found on T cells that helps keep the immune response in check. When PD-1 is bound to another protein called PD-L1, this helps prevent T cells from killing other cells, including cancer cells. Several anti-cancer drugs called immune checkpoint inhibitors are used to block PD-1. When this protein is prevented from acting on T cells, they can act to kill cancer cells.

[0063] The term "STAT3" refers to signal transducer and activator of transcription 3 (STAT3), a transcription factor encoded in humans by the STAT3 gene (STAT3 human (Hs) NM_001369512.1 Genbank reference sequence or NM_139276.3). STAT3 mediates the expression of various genes in response to cellular stimuli and thus plays an important role in many cellular processes, such as cell proliferation and apoptosis, as well as cancer proliferation and progression.

[0064] As used herein, the term "cold tumor" or "non-inflammatory tumor" refers to a tumor or tumor microenvironment with minimal or no presence of anti-tumor immune cells, such as tumor-infiltrating lymphocytes (TILs), and / or containing cell subsets associated with immune suppression, including regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and M2 macrophages. Specifically, in some embodiments, cold tumors are characterized by low numbers or even absence of infiltration of anti-tumor immune cells, and such cells may be present but remain attached to the surrounding stroma and thus are unable to colonize the tumor microenvironment and provide their anti-tumor function.

[0065] As used herein, "complementary" refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand) that allows the two nucleotides to base pair with each other. For example, purine nucleotides of one nucleic acid that are complementary to pyrimidine nucleotides of an opposing nucleic acid may base pair together by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides may base pair by Watson-Crick or any other method that allows the formation of a stable duplex. In some embodiments, two nucleic acids may have a region of multiple nucleotides that are complementary to each other and form a region of complementarity, as described herein.

[0066] As used herein, "species cross-reactive oligonucleotide" refers to an oligonucleotide that can inhibit the expression of target mRNA in two or more species.For example, in some embodiments, species cross-reactive oligonucleotide can inhibit the expression of target mRNA in human and non-human primates.Examples of species include, but are not limited to, human, non-human primates, mouse, and rat.In some embodiments, species cross-reactive oligonucleotide can target and inhibit mRNA in at least two, at least three, or at least four species.

[0067] As used herein, "deoxyribonucleotide" refers to a nucleotide that, compared to a ribonucleotide, has a hydrogen instead of a hydroxyl at the 2' position of its pentose sugar. Modified deoxyribonucleotides are deoxyribonucleotides that have one or more modifications or substitutions of atoms other than the 2' position, including in or modifications or substitutions of the sugar, phosphate group, or base.

[0068] As used herein, "double-stranded RNA" or "dsRNA" refers to an RNA oligonucleotide that is in a substantially double-stranded form. In some embodiments, the complementary base pairing of the double-stranded region of the dsRNA oligonucleotide is formed between the antiparallel sequence of nucleotides of the covalently separated nucleic acid strands. In some embodiments, the complementary base pairing of the double-stranded region of the dsRNA oligonucleotide is formed between the antiparallel sequence of nucleotides of the covalently linked nucleic acid strands. In some embodiments, the complementary base pairing of the double-stranded region of the dsRNA is formed from a single nucleic acid strand that folds back (e.g., via a hairpin) to provide a complementary antiparallel sequence of nucleotides that base pair together. In some embodiments, the dsRNA comprises two covalently separated nucleic acid strands that are fully double-stranded with each other. However, in some embodiments, the dsRNA comprises two covalently separated nucleic acid strands that are partially double-stranded (e.g., with an overhang at one or both ends). In some embodiments, the dsRNA comprises antiparallel sequences of nucleotides that are partially complementary and thus may have one or more mismatches, which may include internal or terminal mismatches.

[0069] As used herein, "duplex" with respect to a nucleic acid (eg, an oligonucleotide) refers to the structure formed through complementary base pairing of two antiparallel sequences of nucleotides.

[0070] As used herein, "excipient" refers to a non-therapeutic agent that may be included in a composition, for example, to provide or contribute a desired consistency or stabilizing effect.

[0071] As used herein, the term "hot tumor" or "inflammatory tumor" refers to a tumor or tumor microenvironment that has a significant presence of anti-tumor immune cells, particularly TILs, and is therefore typically immunostimulatory.

[0072] As used herein, a "loop" refers to an unpaired region of a nucleic acid (e.g., an oligonucleotide) that is adjacent to two antiparallel regions of the nucleic acid that are sufficiently complementary to each other such that under suitable hybridization conditions (e.g., in a cell, in a phosphate buffer), the two antiparallel regions adjacent to the unpaired region hybridize to form a duplex (called a "stem"). A loop that contains four nucleotides may be referred to as a tetraloop (tetraL). A loop that contains three nucleotides may be referred to as a triloop (triL).

[0073] As used herein, "modified internucleotide bond" refers to an internucleotide bond that has one or more chemical modifications compared to a reference internucleotide bond that includes a phosphodiester bond. In some embodiments, the modified nucleotide is a non-naturally occurring bond. Typically, the modified internucleotide bond confers one or more desirable properties to the nucleic acid in which the modified internucleotide bond is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, resistance to nuclease, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0074] As used herein, "modified nucleotide" refers to a nucleotide that has one or more chemical modifications compared to a corresponding reference nucleotide selected from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. In some embodiments, the modified nucleotide is a non-naturally occurring nucleotide. In some embodiments, the modified nucleotide has one or more chemical modifications in its sugar, nucleobase, and / or phosphate group. In some embodiments, the modified nucleotide has one or more chemical moieties conjugated to the corresponding reference nucleotide. Typically, the modified nucleotide confers one or more desirable properties to the nucleic acid in which the modified nucleotide is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0075] As used herein, "nicked tetraloop structure" refers to a structure of an RNAi oligonucleotide characterized by separate sense (passenger) and antisense (guide) strands, where the sense strand has a region complementary to the antisense strand, and at least one of the strands, typically the sense strand, has a tetraloop configured to stabilize an adjacent stem region formed in at least one strand.

[0076] As used herein, "oligonucleotide" refers to a short nucleic acid (e.g., less than about 100 nucleotides in length). An oligonucleotide can be single-stranded (ss) or double-stranded (ds). An oligonucleotide may or may not have a double-stranded region. An oligonucleotide can contain deoxyribonucleotides, ribonucleosides, or a combination of both. In some embodiments, a double-stranded oligonucleotide containing ribonucleotides is referred to as "dsRNA". As a set of non-limiting examples, an oligonucleotide can be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a dicer substrate interfering RNA (dsiRNA), an antisense oligonucleotide, a short siRNA, or a ss siRNA. In some embodiments, the double-stranded RNA (dsRNA) is an RNAi oligonucleotide.

[0077] The terms "RNAi oligonucleotide conjugate" and "oligonucleotide-ligand conjugate" are used interchangeably and refer to an oligonucleotide comprising one or more nucleotides conjugated to one or more targeting ligands.

[0078] As used herein, "overhang" refers to the terminal unpaired nucleotides that originate from one strand or region that extends beyond the end of the complementary strand that forms a duplex.In some embodiments, the overhang comprises one or more unpaired nucleotides that extend from the duplex region at the 5'-end or 3'-end of the dsRNA.In certain embodiments, the overhang is a 3' or 5' overhang on the antisense strand or sense strand of the dsRNA.

[0079] As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is located at the 5'-terminal nucleotide of an oligonucleotide in place of the 5'-phosphate, which is often susceptible to enzymatic removal. In some embodiments, the 5' phosphate analog comprises a phosphatase-resistant linkage. Examples of phosphate analogs include, but are not limited to, 5' phosphonates, such as 5' methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). In some embodiments, an oligonucleotide has a phosphate analog at the 4' carbon position of the sugar (referred to as a "4' phosphate analog") at the 5'-terminal nucleotide. An example of a 4'-phosphate analog is an oxymethyl phosphonate, in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., its 4' carbon) or an analog thereof. See, e.g., U.S. Provisional Patent Application Nos. 62 / 383,207, filed September 2, 2016, and 62 / 393,401, filed September 12, 2016. Other modifications to the 5' end of oligonucleotides have been developed (see, e.g., International Patent Application No. 2011 / 133871, U.S. Patent No. 8,927,513, and Prakash et al., (2015) NUCLEIC ACIDS RES. 43:2993-3011).

[0080] As used herein, "reduced expression" of a gene (e.g., STAT3) refers to a reduction in the amount or level of RNA transcript (e.g., STAT3 mRNA) or protein encoded by the gene, and / or a reduction in the amount or level of the activity of the gene in a cell, cell population, sample, or subject, compared to an appropriate reference (e.g., a reference cell, a reference cell population, a reference sample, or a reference subject). For example, the act of contacting a cell with an oligonucleotide herein (e.g., an oligonucleotide comprising an antisense strand having a nucleotide sequence that is complementary to a nucleotide sequence comprising STAT3 mRNA) can result in a reduction in the amount or level of STAT3 mRNA, protein, and / or activity, compared to a cell that is not treated with dsRNA (e.g., via the degradation of STAT3 mRNA by the RNAi pathway). Similarly, as used herein, "reducing expression" refers to an action that results in a reduction in the expression of a gene (e.g., STAT3). As used herein, "reduced STAT3 expression" refers to a decrease in the amount or level of STAT3 mRNA, STAT3 protein, and / or STAT3 activity in a cell, cell population, sample, or subject compared to an appropriate reference (e.g., a reference cell, a reference cell population, a reference sample, or a reference subject).

[0081] As used herein, "region of complementarity" refers to a nucleotide sequence of a nucleic acid (e.g., dsRNA) that is sufficiently complementary to an antiparallel sequence of nucleotides to allow hybridization between the two sequences of nucleotides under suitable hybridization conditions (e.g., in a phosphate buffer, in a cell, etc.). In some embodiments, the oligonucleotide herein comprises a target sequence that has a region that is complementary to an mRNA target sequence.

[0082] As used herein, "ribonucleotide" refers to a nucleotide having ribose as its pentose sugar containing a hydroxyl group at its 2' position. A modified ribonucleotide is a ribonucleotide that has one or more modifications or substitutions of atoms other than the 2' position, including in or modifications or substitutions of the ribose, phosphate group, or base.

[0083] As used herein, "RNAi oligonucleotide" refers to either (a) a dsRNA having a sense strand (passenger) and an antisense strand (guide), where the antisense strand, or a portion of the antisense strand, is used by Argonaute 2 (Ago2) endonuclease in cleaving a target mRNA, or (b) a ss oligonucleotide having a single antisense strand, where the antisense strand (or a portion of the antisense strand) is used by Ago2 endonuclease in cleaving a target mRNA.

[0084] As used herein, a "strand" refers to a single, contiguous sequence of nucleotides linked together through internucleotide bonds (e.g., phosphodiester or phosphorothioate bonds). In some embodiments, a strand has two free ends (e.g., a 5' end and a 3' end).

[0085] As used herein, "subject" refers to any mammal, including mice, rabbits, non-human primates (NHPs), and humans. In one embodiment, the subject is a human or NHP. Furthermore, "individual" or "patient" may be used interchangeably with "subject."

[0086] As used herein, "synthetic" refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine (e.g., a solid-state nucleic acid synthesizer)) or is not otherwise derived from a natural source (e.g., a cell or organism) that normally produces the molecule.

[0087] As used herein, a "targeting ligand" refers to a molecule or "moiety" (e.g., carbohydrate, amino sugar, cholesterol, polypeptide, or lipid) that selectively binds to a cognate molecule (e.g., a receptor) of a tissue or cell of interest and / or can be conjugated to another substance for the purpose of targeting other substances to the tissue or cell of interest. For example, in some embodiments, a targeting ligand can be conjugated to an oligonucleotide for the purpose of targeting the oligonucleotide to a particular tissue or cell of interest. In some embodiments, a targeting ligand selectively binds to a cell surface receptor. Thus, in some embodiments, a targeting ligand, when conjugated to an oligonucleotide, facilitates delivery of an oligonucleotide to a particular cell through selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of a complex comprising the oligonucleotide, the targeting ligand, and the receptor. In some embodiments, the targeting ligand is conjugated to the oligonucleotide via a linker that is cleaved after cellular internalization or during cellular internalization, such that the oligonucleotide is released from the targeting ligand within the cell.

[0088] As used herein, a "loop," "triloop," or "tetraloop" refers to a loop that increases the stability of adjacent duplexes formed by hybridization of adjacent sequences of nucleotides. The increase in stability is measured by the increase in the average expected melting temperature (T) of adjacent stem duplexes from a set of loops of equivalent length composed of randomly selected sequences of nucleotides. m ) higher than the T of the adjacent stem duplex m For example, the loop can be detected as an increase in T of at least about 50° C., at least about 55° C., at least about 56° C., at least about 58° C., at least about 60° C., at least about 65° C., or at least about 75° C. in 10 mM NaHPO4, in a hairpin comprising a duplex of at least 2 base pairs (bp) in length. mIn some embodiments, the loop (e.g., tetraloop) can stabilize the bp of the adjacent stem duplex by stacking interactions. Additionally, interactions between nucleotides in the tetraloop include, but are not limited to, non-Watson-Crick base pairing, stacking interactions, hydrogen bonds, and contact interactions (Cheong et al., (1990) NATURE 346:680-82, Heus and Pardi (1991) SCIENCE 253:191-94). In some embodiments, the loop comprises or consists of 3-6 nucleotides, typically 4-5 nucleotides. In certain embodiments, the loop comprises or consists of 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., conjugated to a targeting moiety). In some embodiments, the tetraloop comprises or consists of 3-6 nucleotides, typically 4-5 nucleotides. In certain embodiments, the tetraloop comprises or consists of 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., conjugated to a targeting moiety). In one embodiment, a loop consisting of 4 nucleotides is a tetraloop. Any nucleotide may be used for the loop (e.g., tetraloop), and the standard IUPAC-IUB symbols for such nucleotides may be used as described in Cornish-Bowden ((1985) NUCLEIC ACIDS RES. 13:3021-3030). For example, the letter "N" may be used to mean that any base may be at that position, the letter "R" may be used to indicate that A (adenine) or G (guanine) may be at that position, and "B" may be used to indicate that C (cytosine), G (guanine), or T (thymine) may be at that position.Examples of tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop (Woese et al., (1990) PROC. NATL. ACAD. SCI. USA 87:8467-71; Antao et al., (1991) NUCLEIC ACIDS RES. 19:5901-05). Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA), d(GNRA)) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)). (See, e.g., Nakano et al., (2002) BIOCHEM. 41:4281-92; Shinji et al., (2000) NIPPON KAGAKKAI KOEN YOKOSHU 78:731.) In some embodiments, the tetraloop is contained within a nicked tetraloop structure.

[0089] As used herein, "treat" or "treating" refers to the act of providing care to a subject in need of treatment, for example, by administering a therapeutic agent (e.g., an oligonucleotide herein) to the subject for the purpose of improving the health and / or well-being of the subject with respect to an existing condition (e.g., disease, disorder) or preventing or reducing the likelihood of the occurrence of a condition. In some embodiments, treatment involves reducing the frequency or severity of at least one sign, symptom, or contributing factor of a condition (e.g., disease, disorder) experienced by the subject.

[0090] As used herein, the term "tumor microenvironment" refers to the cellular environment in which any given tumor resides, including the tumor stroma, surrounding blood vessels, immune cells, fibroblasts, other cells, signaling molecules, and ECM. It is understood that the tumor microenvironment harbors and / or surrounds the tumor cells with which it interacts.

[0091] How to use Combination of STAT3 oligonucleotide and PD-L1 inhibitor In some embodiments, the disclosure provides STAT3 oligonucleotides for use or adaptable for use to treat a subject who has been or is receiving PD-L1 (e.g., a human having a disease, disorder, or condition associated with STAT3 expression).

[0092] In some embodiments, the methods described herein include selecting a subject having or prone to a disease, disorder, or condition associated with STAT3 expression and / or PD-L1 expression. In some cases, the methods may include selecting an individual having a marker for a disease associated with STAT3 expression and / or PD-L1 expression, such as cancer or other chronic lymphoproliferative disorder.

[0093] Similarly, as detailed herein, the methods may also include steps such as, for example, measuring or obtaining a baseline value of a marker of STAT3 expression and / or PD-L1 expression and then comparing the value so obtained with one or more other baseline values ​​or values ​​obtained after administration of the oligonucleotide to assess the effectiveness of the treatment.

[0094] In some embodiments, the disclosure provides methods of treating a subject having, suspected of having, or at risk of developing a disease, disorder, or condition with a STAT3 oligonucleotide herein, where the subject has received or has received a PD-L1 inhibitor.In some embodiments, the disclosure provides methods of treating a subject having, suspected of having, or at risk of developing a disease, disorder, or condition with a PD-L1 inhibitor described herein, where the subject has received or has received a STAT3 oligonucleotide described herein.

[0095] In some aspects, the disclosure provides methods of using the STAT3 oligonucleotides herein in combination with a PD-L1 inhibitor to treat or reduce the onset or progression of a disease, disorder, or condition associated with STAT3 expression. In other aspects, the disclosure provides methods of using the STAT3 oligonucleotides herein in combination with a PD-L1 inhibitor to achieve one or more therapeutic benefits in a subject having a disease, disorder, or condition associated with STAT3 expression. In some embodiments of the methods herein, the subject is treated by administering a therapeutically effective amount of the STAT3 oligonucleotides herein in combination with a PD-L1 inhibitor. In some embodiments of the methods herein, the subject is treated by administering a therapeutically effective amount of the STAT3 oligonucleotides herein to a subject that has received or is receiving a PD-L1 inhibitor. In some embodiments of the methods herein, the subject is treated by administering a therapeutically effective amount of the STAT3 oligonucleotides herein to a subject that has received or is receiving a PD-L1 inhibitor. In some embodiments, the subject is treated therapeutically. In some embodiments, the subject is treated prophylactically.

[0096] In some embodiments of the methods herein, one or more STAT3 oligonucleotides herein, or pharmaceutical compositions comprising one or more STAT3 oligonucleotides, are administered to a subject having a disease, disorder, or condition associated with STAT3 expression, who has received or is receiving a PD-L1 inhibitor, such that STAT3 expression is reduced in the subject, thereby treating the subject. In some embodiments, the amount or level of STAT3 mRNA is reduced in the subject. In some embodiments, the amount or level of STAT3 and / or protein is reduced in the subject. In some embodiments of the methods herein, one or more STAT3 oligonucleotides herein, or pharmaceutical compositions comprising one or more STAT3 oligonucleotides, are administered to a subject having a disease, disorder, or condition associated with STAT3 expression, who has received or is receiving a PD-L1 inhibitor, such that STAT3 expression and PD-L1 signaling are reduced in the subject, thereby treating the subject. In some embodiments, the amount or level of STAT3 mRNA and PD-L1 signaling are reduced in the subject. In some embodiments, the amount or level of STAT3 and / or protein is reduced in the subject, and PD-L1 signaling is reduced in the subject.

[0097] In some embodiments, a therapeutically effective amount of STAT3 oligonucleotide and / or PD-L1 inhibitor is administered to the subject.A therapeutically acceptable amount can be an amount that can treat a disease or disorder therapeutically.The appropriate dose for any one subject will depend on certain factors, including the subject's size, body surface area, age, the specific composition administered, the active ingredient in the composition, the time and route of administration, general health condition, and other drugs administered in parallel.

[0098] In some embodiments, the subject is administered any one of the compositions herein enterally (e.g., orally, by gastric feeding tube, by duodenal feeding tube, via gastrostomy, or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intraarterial injection or infusion, intraosseous injection, intramuscular injection, intracerebral injection, intraventricular injection, intrathecal injection), locally (e.g., transdermally, inhalation, via eye drops, or via mucosa), or by direct injection into a target organ (e.g., the liver of the subject).Typically, the oligonucleotide herein is administered intravenously or subcutaneously.

[0099] As a non-limiting series of examples, the oligonucleotide herein will typically be administered quarterly (every three months), bimonthly (every two months), monthly, or weekly.For example, the oligonucleotide can be administered weekly, or at two-week or three-week intervals.Alternatively, the oligonucleotide can be administered daily.In some embodiments, the subject is administered one or more loading doses of oligonucleotide, followed by one or more maintenance doses of oligonucleotide.

[0100] In some embodiments, the PD-L1 inhibitors (e.g., anti-PD-L1 antibodies) herein are administered quarterly (every three months), bimonthly (every two months), monthly, or weekly. For example, the inhibitors are administered weekly, or at two- or three-week intervals. Alternatively, the inhibitors are administered daily.

[0101] In some embodiments, the oligonucleotide herein is administered in combination with a PD-L1 inhibitor. In some embodiments, the oligonucleotide and the inhibitor are administered in parallel, sequentially (in any order), or intermittently in combination. For example, the oligonucleotide and the inhibitor can be co-administered in parallel. Alternatively, the oligonucleotide can be administered, followed by the inhibitor after any period (e.g., 1 hour, 1 day, 1 week, or 1 month), or vice versa.

[0102] In some embodiments, the subject to be treated is a human or non-human primate or other mammalian subject. Other exemplary subjects include domesticated animals such as dogs and cats, livestock such as horses, cows, pigs, sheep, goats, and chickens, and animals such as mice, rats, guinea pigs, and hamsters. In some embodiments, the present disclosure provides a method of treating cancer in a subject who has received or is receiving a PD-L1 inhibitor, the method comprising administering an RNAi oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are (a) SEQ ID NOs: 9 and 10, respectively; (b) SEQ ID NOs: 37 and 38, respectively; (c) SEQ ID NOs: 65 and 66, respectively, and (d) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 69 and 70, respectively; Thereby, treating the cancer in the subject.

[0103] In some embodiments, the disclosure provides a method of treating cancer in a subject who has received or is receiving a PD-L1 inhibitor, the method comprising administering an RNAi oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are (a) SEQ ID NOs: 9 and 10, respectively; (b) SEQ ID NOs: 37 and 38, respectively; (c) SEQ ID NOs: 65 and 66, respectively, and (d) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 69 and 70, respectively; The lipid moiety is conjugated to a nucleotide of the sense strand, thereby treating cancer in the subject.

[0104] In some embodiments, the disclosure provides a method of treating cancer in a subject who has received or is receiving a PD-L1 inhibitor, the method comprising administering an RNAi oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 9 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 10, thereby treating cancer in the subject.

[0105] In some embodiments, the disclosure provides a method of treating cancer in a subject who has received or is receiving a PD-L1 inhibitor, the method comprising administering an RNAi oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 9 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 10, and a lipid moiety is conjugated to a nucleotide of the sense strand, thereby treating cancer in the subject.

[0106] In some embodiments, the disclosure provides a method of treating cancer in a subject who has received or is receiving a PD-L1 inhibitor, the method comprising administering an RNAi oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 37 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 38, thereby treating cancer in the subject.

[0107] In some embodiments, the disclosure provides a method of treating cancer in a subject who has received or is receiving a PD-L1 inhibitor, the method comprising administering an RNAi oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 37, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 38, and a lipid moiety is conjugated to a nucleotide of the sense strand, thereby treating cancer in the subject.

[0108] In some embodiments, the disclosure provides a method of treating cancer in a subject who has received or is receiving a PD-L1 inhibitor, the method comprising administering an RNAi oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 65 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 66, thereby treating cancer in the subject.

[0109] In some embodiments, the disclosure provides a method of treating cancer in a subject who has received or is receiving a PD-L1 inhibitor, the method comprising administering an RNAi oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 65 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 66, and a lipid moiety is conjugated to a nucleotide of the sense strand, thereby treating cancer in the subject.

[0110] In some embodiments, the disclosure provides a method of treating cancer in a subject who has received or is receiving a PD-L1 inhibitor, the method comprising administering an RNAi oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 69 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 70, thereby treating cancer in the subject.

[0111] In some embodiments, the disclosure provides a method of treating cancer in a subject who has received or is receiving a PD-L1 inhibitor, the method comprising administering an RNAi oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 69 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 70, and a lipid moiety is conjugated to a nucleotide of the sense strand, thereby treating cancer in the subject.

[0112] In some embodiments, the disclosure provides a method of treating cancer in a subject who has received or is receiving an RNAi oligonucleotide, the method comprising administering a PD-L1 inhibitor, wherein the oligonucleotide comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising: (a) SEQ ID NOs: 9 and 10, respectively; (b) SEQ ID NOs: 37 and 38, respectively; (c) SEQ ID NOs: 65 and 66, respectively, and (d) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 69 and 70, respectively; Thereby, treating the cancer in the subject.

[0113] In some embodiments, the disclosure provides a method of treating cancer in a subject who has received or is receiving an RNAi oligonucleotide, the method comprising administering a PD-L1 inhibitor, wherein the oligonucleotide comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising: (a) SEQ ID NOs: 9 and 10, respectively; (b) SEQ ID NOs: 37 and 38, respectively; (c) SEQ ID NOs: 65 and 66, respectively, and (d) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 69 and 70, respectively; The lipid moiety is conjugated to a nucleotide of the sense strand, thereby treating cancer in the subject.

[0114] In some embodiments, the disclosure provides a method of treating cancer in a subject that has received or is receiving an RNAi oligonucleotide, the method comprising administering a PD-L1 inhibitor, wherein the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 9 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 10, thereby treating cancer in the subject.

[0115] In some embodiments, the disclosure provides a method of treating cancer in a subject that has received or is receiving an RNAi oligonucleotide, the method comprising administering a PD-L1 inhibitor, wherein the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 9 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 10, and wherein a lipid moiety is conjugated to a nucleotide of the sense strand, thereby treating cancer in the subject.

[0116] In some embodiments, the disclosure provides a method of treating cancer in a subject that has received or is receiving an RNAi oligonucleotide, the method comprising administering a PD-L1 inhibitor, wherein the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 37 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 38, thereby treating cancer in the subject.

[0117] In some embodiments, the disclosure provides a method of treating cancer in a subject that has received or is receiving an RNAi oligonucleotide, the method comprising administering a PD-L1 inhibitor, wherein the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 37, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 38, and a lipid moiety is conjugated to a nucleotide of the sense strand, thereby treating cancer in the subject.

[0118] In some embodiments, the disclosure provides a method of treating cancer in a subject that has received or is receiving an RNAi oligonucleotide, the method comprising administering a PD-L1 inhibitor, wherein the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 65 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 66, thereby treating cancer in the subject.

[0119] In some embodiments, the disclosure provides a method of treating cancer in a subject that has received or is receiving an RNAi oligonucleotide, the method comprising administering a PD-L1 inhibitor, wherein the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 65, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 66, and a lipid moiety is conjugated to a nucleotide of the sense strand, thereby treating cancer in the subject.

[0120] In some embodiments, the disclosure provides a method of treating cancer in a subject that has received or is receiving an RNAi oligonucleotide, the method comprising administering a PD-L1 inhibitor, wherein the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 69 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 70, thereby treating cancer in the subject.

[0121] In some embodiments, the disclosure provides a method of treating cancer in a subject that has received or is receiving an RNAi oligonucleotide, the method comprising administering a PD-L1 inhibitor, wherein the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 69, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 70, and wherein a lipid moiety is conjugated to a nucleotide of the sense strand, thereby treating cancer in the subject.

[0122] In some embodiments, the disclosure provides a method of treating a disease, disorder, or condition associated with activated STAT3 expression, comprising administering to a subject in need thereof an RNAi oligonucleotide, wherein the subject has received or has received a PD-L1 inhibitor, and the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are (a) SEQ ID NOs: 9 and 10, respectively; (b) SEQ ID NOs: 37 and 38, respectively; (c) SEQ ID NOs: 65 and 66, respectively, and (d) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 69 and 70, respectively; Thereby, treating the cancer in the subject.

[0123] In some embodiments, the disclosure provides a method of treating a disease, disorder, or condition associated with activated STAT3 expression, comprising administering to a subject in need thereof an RNAi oligonucleotide, wherein the subject has received or has received a PD-L1 inhibitor, and the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are (a) SEQ ID NOs: 9 and 10, respectively; (b) SEQ ID NOs: 37 and 38, respectively; (c) SEQ ID NOs: 65 and 66, respectively, and (d) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 69 and 70, respectively; The lipid moiety is conjugated to a nucleotide of the sense strand, thereby treating cancer in the subject.

[0124] In some embodiments, the disclosure provides a method of treating a disease, disorder, or condition associated with activated STAT3 expression, comprising administering an RNAi oligonucleotide to a subject in need thereof, where the subject has received or has received a PD-L1 inhibitor, and the oligonucleotide comprises a sense strand and an antisense strand, where the sense strand comprises the nucleotide sequence of SEQ ID NO: 9 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 10, thereby treating cancer in the subject.

[0125] In some embodiments, the disclosure provides a method of treating a disease, disorder, or condition associated with activated STAT3 expression, comprising administering an RNAi oligonucleotide to a subject in need thereof, where the subject has received or has received a PD-L1 inhibitor, and the oligonucleotide comprises a sense strand and an antisense strand, where the sense strand comprises the nucleotide sequence of SEQ ID NO:9, and the antisense strand comprises the nucleotide sequence of SEQ ID NO:10, and a lipid moiety is conjugated to a nucleotide of the sense strand, thereby treating cancer in the subject.

[0126] In some embodiments, the disclosure provides a method of treating a disease, disorder, or condition associated with activated STAT3 expression, comprising administering an RNAi oligonucleotide to a subject in need thereof, where the subject has received or has received a PD-L1 inhibitor, and the oligonucleotide comprises a sense strand and an antisense strand, where the sense strand comprises the nucleotide sequence of SEQ ID NO: 37 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 38, thereby treating cancer in the subject.

[0127] In some embodiments, the disclosure provides a method of treating a disease, disorder, or condition associated with activated STAT3 expression, comprising administering an RNAi oligonucleotide to a subject in need thereof, where the subject has received or has received a PD-L1 inhibitor, and the oligonucleotide comprises a sense strand and an antisense strand, where the sense strand comprises the nucleotide sequence of SEQ ID NO: 37, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 38, and a lipid moiety is conjugated to a nucleotide of the sense strand, thereby treating cancer in the subject.

[0128] In some embodiments, the disclosure provides a method of treating a disease, disorder, or condition associated with activated STAT3 expression, comprising administering an RNAi oligonucleotide to a subject in need thereof, where the subject has received or has received a PD-L1 inhibitor, and the oligonucleotide comprises a sense strand and an antisense strand, where the sense strand comprises the nucleotide sequence of SEQ ID NO: 65 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 66, thereby treating cancer in the subject.

[0129] In some embodiments, the disclosure provides a method of treating a disease, disorder, or condition associated with activated STAT3 expression, comprising administering an RNAi oligonucleotide to a subject in need thereof, where the subject has received or has received a PD-L1 inhibitor, and the oligonucleotide comprises a sense strand and an antisense strand, where the sense strand comprises the nucleotide sequence of SEQ ID NO: 65, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 66, and a lipid moiety is conjugated to a nucleotide of the sense strand, thereby treating cancer in the subject.

[0130] In some embodiments, the disclosure provides a method of treating a disease, disorder, or condition associated with activated STAT3 expression, comprising administering an RNAi oligonucleotide to a subject in need thereof, where the subject has received or has received a PD-L1 inhibitor, and the oligonucleotide comprises a sense strand and an antisense strand, where the sense strand comprises the nucleotide sequence of SEQ ID NO: 69 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 70, thereby treating cancer in the subject.

[0131] In some embodiments, the disclosure provides a method of treating a disease, disorder, or condition associated with activated STAT3 expression, comprising administering an RNAi oligonucleotide to a subject in need thereof, where the subject has received or has received a PD-L1 inhibitor, and the oligonucleotide comprises a sense strand and an antisense strand, where the sense strand comprises the nucleotide sequence of SEQ ID NO: 69, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 70, and a lipid moiety is conjugated to a nucleotide of the sense strand, thereby treating cancer in the subject.

[0132] In some embodiments, the methods disclosed herein increase the expression of immune activation genes. In some embodiments, administering an RNAi oligonucleotide described herein with a PD-L1 inhibitor increases the expression of immune activation genes. In some embodiments, the methods described herein increase the expression of one or more of CD8B1, IL2Rα, TNFRSF9, CCL19, CCL27a, CD40Ig, CXCR5, CD44, STAT4, GZMB, PFR1, FASL, and HMGB1.

[0133] In some embodiments, the method of the present disclosure reduces the expression of immunosuppressive genes. In some embodiments, administering the RNAi oligonucleotides described herein with a PD-L1 inhibitor reduces the expression of immunosuppressive genes. In some embodiments, the method described herein reduces the expression of one or more of CD274, LAG3, TIGIT, PDSD1IG2, CD163, PIK3R5, FOXP3, TGFBR2, IDO1, CD200R1, CCL22, CCL8, CCR9, IL10, IL33, CXCL3, CSF2RD, TNFRSF18, MMP9, LOX12, FGF18, and FAP.

[0134] cancer In some embodiments, STAT3 oligonucleotides and PD-L1 inhibitor targets are used to treat cancer or tumors. In some embodiments, the tumor is a primary tumor. In some embodiments, the tumor is a metastatic tumor. In some embodiments, the tumor is a refractory tumor. In some embodiments, the tumor is a stage I, stage II, stage III, or stage IV tumor. In some embodiments, the tumor is a solid tumor. A solid tumor refers to a condition in which the cancer forms a mass.

[0135] In some embodiments, the cancer is thyroid cancer, papillary thyroid cancer, head and neck cancer, liver cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, carcinoma, blastoma, medulloblastoma, retinoblastoma, sarcoma, liposarcoma, synovial cell sarcoma, neuroendocrine tumor, carcinoid tumor, gastrinoma, islet cell carcinoma, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, lymphoid malignancy, squamous cell carcinoma, epithelial squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastric cancer or stomach cancer. In some embodiments, the cancer is refractory to anti-PD1, anti-PDL1, and / or anti-CTLA4 therapy. In some embodiments, the cancer is pancreatic or lung cancer. In some embodiments, the cancer comprises a tumor with an immunosuppressive tumor microenvironment. In some embodiments, the cancer is resistant to immune checkpoint therapy. In some embodiments, the cancer is partially resistant to immune checkpoint therapy. In some embodiments, the cancer is sensitive to immune checkpoint therapy.

[0136] In some embodiments, the STAT3 oligonucleotide and the PD-L1 inhibitor reduce tumor volume. Tumor volume is measured using methods known to those skilled in the art. For example, the extracted tumor is measured manually using a caliper. Other methods include imaging methods such as ultrasound and MRI. In some embodiments, the oligonucleotide conjugate reduces tumor volume by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to an untreated tumor.

[0137] Oligonucleotide inhibitors of STAT3 In some aspects, the present disclosure provides, inter alia, oligonucleotides that reduce or inhibit STAT3 expression.In some embodiments, the oligonucleotides herein that inhibit STAT3 expression target STAT3 mRNA.The sequence of human STAT3 mRNA (NM_001369512.1) is listed as SEQ ID NO:85 or NM_139276.3 (SEQ ID NO:1217).STAT3 is a known target of conventional cancer therapy.

[0138] The tolerogenic activity of MDSCs is controlled by the oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) (Su et al., INT J. MOL SCI (2018) 19(6): 1803). STAT3 is also known to be highly expressed across a wide range of cancer types and in vitro and in vivo preclinical models (Huynh et al., NAT. REV. CANCER (2019) 19: 82-96). Inhibition of STAT3 leads to selective apoptosis of tumor cells and tumor growth inhibition through modulation of downstream target genes (Wang et al., INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES, 15(3): 668-79 (2019)). STAT3 is of particular interest in immuno-oncology due to its well-documented contribution to the immunosuppressive tumor microenvironment. STAT3 contributes to the immunosuppressive tumor microenvironment by upregulating inhibitory receptors expressed by T cells and through the expression of its ligands (PD-1 / PD-L1) through increased secretion of IFNγ (Bu et al., JOURNAL OF DENTAL RESEARCH, 96(9):1027-34(2017)). It has been known for many years that inhibition of STAT3 signaling in antigen-presenting cells (APCs) results in the priming of antigen-specific CD4+ T cells in response to other tolerogenic stimuli (Cheng et al., IMMUNITY, 19:425-36(2003)). In addition, phosphorylated STAT3 on MDSCs directly contributes to the regulation of the suppressive tumor microenvironment by controlling suppressive components such as the amino acid arginine through transcriptional regulation (Vasques-Dunndel et al., J. CLIN. INVEST., 15(3):668-79(2013)). Over the years, several methodologies have been investigated to therapeutically target STAT3.Although direct targeting of proteins is attractive, the true target is a protein-protein interaction that has been taken as an example of an “undruggable” target due to past data showing that multiple classes of compounds failed to effectively inhibit its activity (Lau et al., CANCERS (2019) 11(11): 1681; Zou et al., MOL CANCER (2020) 19: 145). Furthermore, the ubiquitous expression of STAT3 across several tissues has raised concerns regarding severe on-target toxicity (Wong et al., EXPERT OPINION ON INVESTIGATIONAL DRUGS, 26(8): 883-87 (2017); (Kortylewski et al., CANCER IMMUNOL IMMUNOTHER (2017) 66(8): 979-88).

[0139] In some embodiments, the reduction of STAT3 expression can be determined by a suitable assay or technique for evaluating one or more characteristics or features of a cell or cell population associated with STAT3 expression (e.g., using a STAT3 expression biomarker), or by an assay or technique that evaluates a molecule that directly indicates STAT3 expression (e.g., STAT3 mRNA or STAT3 protein). In some embodiments, the extent to which the oligonucleotide herein reduces STAT3 expression is evaluated by comparing STAT3 expression in a cell or cell population contacted with the oligonucleotide to a suitable control (e.g., a suitable cell or cell population that has not been contacted with the oligonucleotide or that has been contacted with a control oligonucleotide). In some embodiments, a suitable control level of mRNA expression into protein after delivery of an RNAi molecule may be a predetermined level or value, such that the control level does not need to be measured every time. The predetermined level or value can take various forms. In some embodiments, the predetermined level or value can be a single cutoff value, such as a median or mean value.

[0140] In some embodiments, administration of the oligonucleotide herein results in a reduction in STAT3 expression in a cell or cell population. In some embodiments, the reduction in STAT3 or STAT3 expression is about 1% or less, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less, compared to an appropriate control level of mRNA. An appropriate control level may be the level of mRNA expression and / or protein translation in a cell or cell population that is not contacted with the oligonucleotide herein. In some embodiments, the effect of delivery of the oligonucleotide to a cell according to the method herein is evaluated after a finite period of time. For example, mRNA levels may be analyzed in cells at least about 8 hours, about 12 hours, about 18 hours, about 24 hours, or at least about 1, 2, 3, 4, 5, 6, 7, or up to 14 days after introduction of the oligonucleotide into the tumor.

[0141] In some embodiments, the oligonucleotide is delivered in the form of a transgene engineered to express the oligonucleotide or a strand containing the oligonucleotide (e.g., its sense and antisense strands) in cells. In some embodiments, the oligonucleotide is delivered using a transgene engineered to express any of the oligonucleotides disclosed herein. The transgene can be delivered using a viral vector (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus), or a non-viral vector (e.g., plasmid or synthetic mRNA). In some embodiments, the transgene can be directly injected into the subject.

[0142] STAT3 target sequence In some embodiments, the oligonucleotide is targeted to a target sequence that includes STAT3 mRNA. In some embodiments, the oligonucleotide, or a part, fragment, or strand thereof (e.g., the antisense strand or guide strand of dsRNA) binds or anneals to a target sequence that includes STAT3 mRNA, thereby inhibiting STAT3 expression. In some embodiments, the oligonucleotide is targeted to a STAT3 target sequence for the purpose of inhibiting STAT3 expression in vivo. In some embodiments, the amount or degree of inhibition of STAT3 expression by the oligonucleotide targeted to the STAT3 target sequence correlates with the efficacy of the oligonucleotide. In some embodiments, the amount or degree of inhibition of STAT3 expression by the oligonucleotide targeted to the STAT3 target sequence correlates with the amount or degree of therapeutic effect in subjects or patients with diseases, disorders, or conditions related to STAT3 expression that are treated with the oligonucleotide.

[0143] Through examination of the nucleotide sequences of mRNAs encoding STAT3, including mRNAs from several different species (e.g., human, cynomolgus monkey, mouse, and rat; see, e.g., Example 6), and as a result of in vitro and in vivo testing (see, e.g., Examples 7 and 8), it has been discovered that certain nucleotide sequences of STAT3 mRNA are more susceptible to oligonucleotide-based inhibition than others, and thus are useful as target sequences for the oligonucleotides herein. In some embodiments, the sense strand of an oligonucleotide (e.g., dsRNA) described herein comprises a STAT3 target sequence. In some embodiments, a portion or region of the sense strand of a dsRNA described herein comprises a STAT3 target sequence. In some embodiments, the STAT3 mRNA target sequence comprises or consists of the sequence of SEQ ID NO:85. In some embodiments, the STAT3 mRNA target sequence comprises or consists of the sequence of SEQ ID NO:1217. In some embodiments, the STAT3 mRNA target sequence comprises or consists of any one of the sequences of SEQ ID NO:89-280. In some embodiments, the STAT3 mRNA target sequence comprises or consists of the sequence set forth in SEQ ID NO:108. In some embodiments, the STAT3 mRNA target sequence comprises or consists of the sequence set forth in SEQ ID NO: 140. In some embodiments, the STAT3 mRNA target sequence comprises or consists of the sequence set forth in SEQ ID NO: 141. In some embodiments, the STAT3 mRNA target sequence comprises or consists of the sequence set forth in SEQ ID NO: 147.

[0144] STAT3 targeting sequence In some embodiments, the oligonucleotides herein have a region of complementarity to STAT3 mRNA (e.g., within the target sequence of STAT3 mRNA) for the purpose of targeting the mRNA in a cell and reducing or inhibiting its expression. In some embodiments, the oligonucleotides herein comprise a STAT3 target sequence (e.g., the antisense strand or guide strand of a dsRNA) with a region of complementarity that binds or anneals to the STAT3 target sequence by complementary (Watson-Crick) base pairing. The targeting sequence or region of complementarity generally has a suitable length and base content to allow binding or annealing of the oligonucleotide (or a strand thereof) thereto for the purpose of inhibiting expression of STAT3 mRNA. In some embodiments, the targeting sequence or region of complementarity is at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is about 12 to about 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30) nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 18 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 19 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 20 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 21 nucleotides in length.In some embodiments, the targeting sequence or region of complementarity is 22 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 23 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 24 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or region of complementarity complementary to any one of SEQ ID NOs: 89-280, and the targeting sequence or region of complementarity is 18 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or region of complementarity complementary to any one of SEQ ID NOs: 89-280, and the targeting sequence or region of complementarity is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or region of complementarity complementary to any one of SEQ ID NOs: 473-664, and the targeting sequence or region of complementarity is 20 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity complementary to any one of SEQ ID NOs: 473-664, and the targeting sequence or region of complementarity is 21 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity complementary to any one of SEQ ID NOs: 473-664, and the targeting sequence or region of complementarity is 22 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity complementary to any one of SEQ ID NOs: 473-664, and the targeting sequence or region of complementarity is 23 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity complementary to any one of SEQ ID NOs: 473-664, and the targeting sequence or region of complementarity is 24 nucleotides in length.

[0145] In some embodiments, the oligonucleotide herein comprises a targeting sequence or a region of complementarity that is fully complementary to a STAT3 target sequence (e.g., the antisense strand or guide strand of a double-stranded oligonucleotide). In some embodiments, the targeting sequence or a region of complementarity is partially complementary to a STAT3 target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity that is fully complementary to a STAT3 sequence or STAT3. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity that is partially complementary to a STAT3 sequence or STAT3.

[0146] In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity that is fully complementary to any one of SEQ ID NOs: 89-280. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity that is fully complementary to any one of SEQ ID NOs: 108, 140, 141, and 147. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity that is partially complementary to any one of SEQ ID NOs: 89-280. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity that is partially complementary to any one of SEQ ID NOs: 108, 140, 141, and 147.

[0147] In some embodiments, the oligonucleotides herein comprise a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides comprising STAT3 mRNA, the contiguous sequence of nucleotides being about 12 to about 30 nucleotides in length (e.g., 12-30, 12-28, 12-26, 12-24, 12-20, 12-18, 12-16, 14-22, 16-20, 18-20, or 18-19 nucleotides in length). In some embodiments, the oligonucleotides comprise a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides comprising STAT3 mRNA, the contiguous sequence of nucleotides being 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the oligonucleotides comprise a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides comprising STAT3 mRNA, the contiguous sequence of nucleotides being 19 nucleotides in length.

[0148] In some embodiments, the oligonucleotides (e.g., RNAi oligonucleotides) herein comprise a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 89-280, and optionally, the contiguous sequence of nucleotides is 19 nucleotides in length. In some embodiments, the oligonucleotides comprise a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 108, 140, 141, and 147, and the contiguous sequence of nucleotides is 19 nucleotides in length. In some embodiments, the oligonucleotides comprise a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 473-664, and the contiguous sequence of nucleotides is 20 nucleotides in length. In some embodiments, the oligonucleotides comprise a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 492, 524, 525, and 531, and the contiguous sequence of nucleotides is 20 nucleotides in length.

[0149] In some embodiments, the targeting sequence or region of complementarity of the oligonucleotide that is complementary to consecutive nucleotides of STAT3 or STAT3 target sequence spans the entire length of the antisense strand. In some embodiments, the region of complementarity of the oligonucleotide that is complementary to consecutive nucleotides of STAT3 or STAT3 target sequence spans a portion of the entire length of the antisense strand. In some embodiments, the oligonucleotide herein comprises a region of complementarity (e.g., on the antisense strand of dsRNA) that is at least partially (e.g., completely) complementary to the target sequence of STAT3 or a consecutive stretch of nucleotides spanning nucleotides 1-20 of STAT3.

[0150] In some embodiments, the targeting sequence or region of complementarity of the oligonucleotide (e.g., RNAi oligonucleotide) herein is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 89-280 and spans the entire length of the antisense strand. In some embodiments, the targeting sequence or region of complementarity of the oligonucleotide is complementary to a contiguous sequence of nucleotides of SEQ ID NOs: 89-280 and spans a portion of the entire length of the antisense strand. In some embodiments, the oligonucleotide (e.g., RNAi oligonucleotide) herein comprises a region of complementarity (e.g., on the antisense strand of a dsRNA) that is at least partially (e.g., completely) complementary to a contiguous stretch of nucleotides spanning nucleotides 1-19 or 1-20 of the sequence set forth in any one of SEQ ID NOs: 473-664.

[0151] In some embodiments, the oligonucleotides herein comprise a targeting sequence or a region of complementarity that has one or more bp mismatches with the corresponding STAT3 target sequence. In some embodiments, the targeting sequence or the region of complementarity can have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding STAT3 target sequence, provided that the ability of the targeting sequence or the region of complementarity to bind or anneal to STAT3 mRNA under suitable hybridization conditions and / or the ability of the oligonucleotide to inhibit STAT3 expression is maintained. Alternatively, the targeting sequence or the region of complementarity can have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding STAT3 target sequence, provided that the ability of the targeting sequence or the region of complementarity to bind or anneal to STAT3 mRNA under suitable hybridization conditions and / or the ability of the oligonucleotide to inhibit STAT3 expression is maintained. In some embodiments, the oligonucleotides comprise a targeting sequence or a region of complementarity that has one mismatch with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity with two mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity with three mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity with four mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity with five mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity with two or more mismatches (e.g., 2, 3, 4, 5, or more mismatches) with the corresponding target sequence, with at least two (e.g., all) of the mismatches being contiguously positioned (e.g., 2, 3, 4, 5, or more mismatches side by side) or the mismatches being interspersed throughout the targeting sequence or region of complementarity.In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 89-280, and the targeting sequence or region of complementarity may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding STAT3 target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 89-280, and the targeting sequence or region of complementarity may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding STAT3 target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of any one of SEQ ID NOs: 108, 140, 141, and 147, and the targeting sequence or region of complementarity may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding STAT3 target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of any one of SEQ ID NOs: 108, 140, 141, and 147, and the targeting sequence or region of complementarity may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding STAT3 target sequence.

[0152] Oligonucleotide types A variety of oligonucleotide types and / or structures are useful for targeting target sequences in the methods herein, including, but not limited to, RNAi oligonucleotides, antisense oligonucleotides, miRNAs, etc. Any of the oligonucleotide types described herein or elsewhere are contemplated for use as a framework for incorporating the targeting sequences herein.

[0153] In some embodiments, the oligonucleotides herein inhibit the expression of target sequences by participating in the RNA interference (RNAi) pathway upstream or downstream of Dicer intervention. For example, RNAi oligonucleotides have been developed with a size of about 19-25 nucleotides, with at least one 3' overhang of 1-5 nucleotides on each strand (see, for example, U.S. Pat. No. 8,372,968). Longer oligonucleotides have also been developed that are processed by Dicer to generate active RNAi products (see, for example, U.S. Pat. No. 8,883,996). Further research has produced extended dsRNAs, in which at least one end of at least one strand is extended beyond the double-stranded targeting region, and one of the strands includes a structure that includes a thermodynamically stabilizing tetraloop structure (see, for example, U.S. Pat. Nos. 8,513,207 and 8,927,705, and International Patent Application Publication No. WO 2010 / 033225). Such structures may contain ss extensions (on one or both sides of the molecule) as well as ds extensions.

[0154] In some embodiments, the oligonucleotides described herein participate in the RNAi pathway downstream of Dicer intervention (e.g., Dicer cleavage). In some embodiments, the oligonucleotides described herein are Dicer substrates. In some embodiments, endogenous Dicer processing produces a double-stranded nucleic acid of 19-23 nucleotides in length that can reduce target mRNA expression. In some embodiments, the oligonucleotide has an overhang (e.g., 1, 2, or 3 nucleotides in length) at the 3' end of the sense strand. In some embodiments, the oligonucleotide (e.g., siRNA) comprises a 21 nucleotide guide strand that is antisense to the target RNA, and a complementary passenger strand, where both strands anneal to form a 19 bp duplex and a 2 nucleotide overhang at either or both 3' ends. Longer oligonucleotide designs are also available, including oligonucleotides with 23 nucleotides of guide strand and 21 nucleotides of passenger strand, with blunt end (3' end of passenger strand / 5' end of guide strand) on the right side of the molecule and 2 nucleotides of 3'-guide strand overhang (5' end of passenger strand / 3' end of guide strand) on the left side of the molecule.In such molecules, there is a 21bp double-stranded region.See, for example, U.S. Patent Nos. 9,012,138, 9,012,621 and 9,193,753.

[0155] In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, both ranging in length from about 17 to 26 (e.g., 17 to 26, 20 to 25, or 21 to 23) nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, both ranging in length from about 17 to 36 (e.g., 17 to 36, 20 to 25, or 21 to 23) nucleotides. In some embodiments, the oligonucleotides herein comprise an antisense strand that is 19 to 30 nucleotides long, and a sense strand that is 19 to 50 nucleotides long, where the antisense strand and the sense strand are separate strands that form an asymmetric duplex region with an overhand of 1 to 4 nucleotides at the 3' end of the antisense strand. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, both ranging in length from about 19 to 22 nucleotides. In some embodiments, the sense strand and the antisense strand are of equal length. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand such that there is a 3' overhang on either the sense strand or the antisense strand, or on both the sense strand and the antisense strand. In some embodiments, for oligonucleotides having a sense strand and an antisense strand that are both in the range of about 21-23 nucleotides in length, the 3' overhang on the sense strand, the antisense strand, or both the sense strand and the antisense strand is 1 or 2 nucleotides in length. In some embodiments, the oligonucleotide has a guide strand of 22 nucleotides and a passenger strand of 20 nucleotides, with a blunt end on the right side of the molecule (3' end of the passenger strand / 5' end of the guide strand) and a 3'-guide strand overhang of 2 nucleotides on the left side of the molecule (5' end of the passenger strand / 3' end of the guide strand). In such molecules, there is a 20 bp double-stranded region.

[0156] Other oligonucleotide designs for use in conjunction with the compositions and methods herein include 16-mer siRNAs (see, e.g., NUCLEIC ACIDS IN CHEMISTRY AND BIOLOGY. Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., with stems of 19 bp or less; see, e.g., Moore et al., (2010) METHODS MOL. BIOL. 629:141-58), blunt siRNAs (e.g., 19 bp in length; see, e.g., Kraynack and Baker (2006) RNA 12:163-76), asymmetric siRNAs (aiRNAs; see, e.g., Sun et al., (2008) NAT. BIOTECHNOL. 26:1379-82), asymmetric shorter double-stranded siRNAs (see, e.g., Chang et al., (2008) NAT. BIOTECHNOL. 26:1379-82), and the like. al., (2009) MOL. THER. 17:725-32), forked siRNAs (see, e.g., Hohjoh (2004) FEBS LETT. 557:193-98), ss siRNAs (Elsner (2012) NAT. BIOTECHNOL. 30:1063), dumbbell-shaped circular siRNAs (see, e.g., Abe et al., (2007) J. AM. CHEM. SOC. 129:15108-09), and small internal segmented interfering RNAs (siRNAs; see, e.g., Bramsen et al., (2007) NUCLEIC ACIDS RES. 35:5886-97). Further non-limiting examples of oligonucleotide structures that may be used in some embodiments to reduce or inhibit expression of STAT3 are microRNAs (miRNAs), short hairpin RNAs (shRNAs), and short siRNAs (see, e.g., Hamilton et al., (2002) EMBO J. 21:4671-79; see also U.S. Patent Application Publication No. 2009 / 0099115).

[0157] Moreover, in some embodiments, the oligonucleotide for reducing or inhibiting the expression of the target sequence herein is ss. Such structures can include, but are not limited to, ss RNAi molecules. Recent efforts have demonstrated the activity of ss RNAi molecules (see, for example, Matsui et al., (2016) MOL.THER.24:946-55). However, in some embodiments, the oligonucleotide herein is an antisense oligonucleotide (ASO). An antisense oligonucleotide is an ss oligonucleotide having a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of a targeting segment of a specific nucleic acid and is suitably modified to induce RNaseH-mediated cleavage of its target RNA in a cell (e.g., as a gapmer) or inhibit the translation of a target mRNA in a cell (e.g., as a mixmer). ASOs for use herein may be modified in any suitable manner known in the art, including, for example, those set forth in U.S. Patent No. 9,567,587 (e.g., including modifications of length, sugar moieties of nucleobases (pyrimidines, purines), and heterocyclic moieties of nucleobases). Additionally, ASOs have been used for decades to reduce the expression of specific target genes (see, for example, Bennett et al., (2017) ANNU. REV. PHARMACOL. 57:81-105).

[0158] In some embodiments, the antisense oligonucleotide shares a region of complementarity with the target mRNA. In some embodiments, the antisense oligonucleotide is 15-50 nucleotides in length. In some embodiments, the antisense oligonucleotide is 15-25 nucleotides in length. In some embodiments, the antisense oligonucleotide is 22 nucleotides in length. In some embodiments, the antisense oligonucleotide is at least 15 contiguous nucleotides in length. In some embodiments, the antisense oligonucleotide is at least 19 contiguous nucleotides in length. In some embodiments, the antisense oligonucleotide is at least 20 contiguous nucleotides in length. In some embodiments, the antisense oligonucleotide differs from the target sequence by 1, 2, or 3 nucleotides.

[0159] Double-stranded oligonucleotides In some embodiments, the present disclosure provides a double-stranded dsRNA for targeting and inhibiting expression of a target sequence (e.g., via the RNAi pathway), comprising a sense strand (also referred to herein as a passenger strand) and an antisense strand (also referred to herein as a guide strand). In some embodiments, the sense strand and the antisense strand are separate strands and are not covalently linked. In some embodiments, the sense strand and the antisense strand are covalently linked. In some embodiments, the sense strand and the antisense strand form a double-stranded region, and the sense strand and the antisense strand, or portions thereof, are linked to each other in a complementary manner (e.g., by Watson-Crick base pairing).

[0160] In some embodiments, the sense strand has a first region (R1) and a second region (R2), R2 includes a first subregion (S1), a loop (L), such as a tetraloop (tetraL) or a triloop (triL), and a second subregion (S2), where L, tetraL, or triL is located between S1 and S2, and S1 and S2 form a second duplex (D2). D2 can have a variety of lengths. In some embodiments, D2 is about 1-6 bp in length. In some embodiments, D2 is 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, or 4-5 bp in length. In some embodiments, D2 is 1, 2, 3, 4, 5, or 6 bp in length. In some embodiments, D2 is 6 bp in length.

[0161] In some embodiments, R1 of the sense strand and the antisense strand form a first duplex (D1). In some embodiments, D1 is at least about 15 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length. In some embodiments, D1 is in the range of about 12-30 nucleotides in length (e.g., 12-30, 12-27, 15-22, 18-22, 18-25, 18-27, 18-30, or 21-30 nucleotides in length). In some embodiments, D1 is at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 20, at least 25, or at least 30 nucleotides in length). In some embodiments, D1 is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, D1 is 20 nucleotides in length. In some embodiments, D1 comprising sense and antisense strands does not span the entire length of sense and / or antisense strands. In some embodiments, D1 comprising sense and antisense strands spans the entire length of either or both of sense and antisense strands. In certain embodiments, D1 comprising sense and antisense strands spans the entire length of both sense and antisense strands.

[0162] Of course, in some embodiments, when describing the structure of oligonucleotide or other nucleic acid, reference can be made to the sequence presented in sequence listing.In such embodiments, the actual oligonucleotide or other nucleic acid can have one or more alternative nucleotides (e.g., the RNA counterpart of DNA nucleotide or the DNA counterpart of RNA nucleotide) and / or one or more modified nucleotides and / or one or more modified internucleotide bonds and / or one or more other modifications compared to the designated sequence, while retaining essentially the same or similar complementary properties as the designated sequence.

[0163] In some embodiments, the double-stranded RNA (dsRNA) herein comprises a sense strand of 25 nucleotides and an antisense strand of 27 nucleotides, and when acted upon by dicer enzyme, the antisense strand is incorporated into mature RISC.In some embodiments, the sense strand of dsRNA is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides).In some embodiments, the sense strand of dsRNA is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides). In some embodiments, the sense strand of the dsRNA is longer than 25 nucleotides (eg, 26, 27, 28, 29, or 30 nucleotides).

[0164] In some embodiments, the oligonucleotides herein have one 5' end that is less thermodynamically stable compared to the other 5' end. In some embodiments, asymmetric oligonucleotides are provided that include a blunt end at the 3' end of the sense strand and a 3' overhang at the 3' end of the antisense strand. In some embodiments, the 3' overhang on the antisense strand is about 1-8 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length). Typically, RNAi oligonucleotides have a 2 nucleotide overhang on the 3' end of the antisense (guide) strand. However, other overhangs are possible. In some embodiments, the overhang is a 3' overhang that includes 1-6 nucleotides, optionally 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, 5-6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides in length. However, in some embodiments, the overhang is a 5' overhang comprising 1-6 nucleotides, optionally 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, 5-6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides in length.

[0165] In some embodiments, the two terminal nucleotides on the 3' end of the antisense strand are modified. In some embodiments, the two terminal nucleotides on the 3' end of the antisense strand are complementary to the target mRNA. In some embodiments, the two terminal nucleotides on the 3' end of the antisense strand are not complementary to the target mRNA. In some embodiments, the two terminal nucleotides at the 3' end of the antisense strand comprise unpaired GG. In some embodiments, the two (2) terminal nucleotides on the 3' end of the antisense strand of the oligonucleotide herein are not complementary to the target mRNA. In some embodiments, the two terminal nucleotides at each 3' end of the oligonucleotide in the nicked tetraloop structure are GG. In some embodiments, one or both of the two (2) terminal GG nucleotides on each 3' end of the oligonucleotide herein are not complementary to the target mRNA. In some embodiments, one or both of the two terminal GG nucleotides on each 3' end of the oligonucleotide herein are not complementary to the target.

[0166] In some embodiments, there is one or more (e.g., 1, 2, 3, 4, or 5) mismatches between the sense strand and the antisense strand. When there are two or more mismatches between the sense strand and the antisense strand, they can be located consecutively (e.g., 2, 3 or more side by side) or can be scattered throughout the region of complementarity. In some embodiments, the 3' end of the sense strand contains one or more mismatches. In one embodiment, two mismatches are incorporated into the 3' end of the sense strand. In some embodiments, base mismatches or destabilization of segments at the 3' end of the sense strand of the oligonucleotide improves the efficacy of synthetic duplexes in RNAi, possibly by facilitating processing by Dicer.

[0167] A antisense strand In some embodiments, the dsRNA comprises an antisense strand of up to about 40 nucleotides in length (e.g., up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the oligonucleotide (e.g., RNAi oligonucleotide) herein comprises an antisense strand of up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the oligonucleotide may have an antisense strand of at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27, at least 30, at least 35, or at least 38 nucleotides in length). In some embodiments, oligonucleotides can have antisense strands ranging in length from about 12 to about 40 nucleotides (e.g., 12-40, 12-36, 12-32, 12-28, 15-40, 15-36, 15-32, 15-28, 17-22, 17-25, 19-27, 19-30, 20-40, 22-40, 25-40, or 32-40). In some embodiments, oligonucleotides include antisense strands that are 15-30 nucleotides in length. In some embodiments, oligonucleotides may have an antisense strand that is 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, or 40 nucleotides in length.

[0168] In some embodiments, the antisense strand of oligonucleotide can be called "guide strand".For example, if antisense strand can participate in RNA-induced silencing complex (RISC), bind to Argonaute protein such as Ago2, or participate in or bind to one or more similar factors, and direct the silencing of target gene, it can be called guide strand.In some embodiments, the sense strand that is complementary to guide strand can be called "passenger strand".

[0169] In some embodiments, the oligonucleotides disclosed herein for targeting STAT3 comprise an antisense strand that comprises or consists of a sequence set forth in any one of SEQ ID NOs: 281-472. In some embodiments, the oligonucleotides disclosed herein comprise an antisense strand that comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence set forth in any one of SEQ ID NOs: 281-472. In some embodiments, the oligonucleotides disclosed herein for targeting STAT3 (e.g., RNAi oligonucleotides) comprise an antisense strand that comprises or consists of a sequence set forth in any one of SEQ ID NOs: 665-856. In some embodiments, the oligonucleotides disclosed herein comprise an antisense strand that comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence set forth in any one of SEQ ID NOs: 665-856. In some embodiments, the oligonucleotides disclosed herein for targeting STAT3 comprise an antisense strand that comprises or consists of a sequence set forth in any one of SEQ ID NOs: 947-1036. In some embodiments, the oligonucleotides disclosed herein comprise an antisense strand that comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence set forth in any one of SEQ ID NOs: 947-1036.In some embodiments, the oligonucleotides disclosed herein for targeting STAT3 comprise an antisense strand that comprises or consists of a sequence set forth in any one of SEQ ID NOs: 10, 38, 66, and 70. In some embodiments, the oligonucleotides disclosed herein comprise an antisense strand that comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence set forth in any one of SEQ ID NOs: 10, 38, 66, and 70. In some embodiments, the oligonucleotides disclosed herein for targeting STAT3 comprise an antisense strand that comprises or consists of a sequence set forth in any one of SEQ ID NOs: 281-472. In some embodiments, the oligonucleotides disclosed herein comprise an antisense strand that comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence set forth in any one of SEQ ID NOs: 281-472. In some embodiments, the oligonucleotides disclosed herein for targeting STAT3 comprise an antisense strand that comprises or consists of a sequence set forth in any one of SEQ ID NOs: 952, 965, 966, and 1010. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence set forth in any one of SEQ ID NOs: 952, 965, 966, and 1010.In some embodiments, the oligonucleotides disclosed herein for targeting STAT3 comprise an antisense strand that comprises or consists of the sequence set forth in any one of SEQ ID NOs: 991, 1000, 989, 986, 982, 980, and 979. In some embodiments, the oligonucleotides disclosed herein comprise an antisense strand that comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 991, 1000, 989, 986, 982, 980, and 979. In some embodiments, the oligonucleotides disclosed herein for targeting STAT3 comprise an antisense strand that comprises or consists of the sequence set forth in any one of SEQ ID NOs: 1030, 1027, and 1029. In some embodiments, the oligonucleotides disclosed herein comprise an antisense strand that comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 1030, 1027, and 1029. In some embodiments, the oligonucleotides disclosed herein for targeting STAT3 comprise an antisense strand that comprises or consists of the sequence set forth in any one of SEQ ID NOs: 1005, 1014, 1003, and 1010. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence set forth in any one of SEQ ID NOs: 1005, 1014, 1003, and 1010.

[0170] B sense strand In some embodiments, the oligonucleotides disclosed herein for targeting STAT3 mRNA and inhibiting STAT3 expression (e.g., and RNAi oligonucleotides) comprise a sense strand sequence set forth in any one of SEQ ID NOs: 89-280. In some embodiments, the oligonucleotides herein have a sense strand comprising at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence set forth in any one of SEQ ID NOs: 89-280. In some embodiments, the oligonucleotides disclosed herein for targeting STAT3 mRNA and inhibiting STAT3 expression (e.g., and RNAi oligonucleotides) comprise a sense strand sequence set forth in any one of SEQ ID NOs: 473-664. In some embodiments, the oligonucleotides herein have a sense strand that comprises at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 473-664. In some embodiments, the oligonucleotides disclosed herein for targeting STAT3 mRNA and inhibiting STAT3 expression comprise a sense strand sequence set forth in any one of SEQ ID NOs: 857-946. In some embodiments, the oligonucleotides herein have a sense strand that is composed of at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 857-946.In some embodiments, the oligonucleotide disclosed herein for targeting STAT3 mRNA and inhibiting STAT3 expression comprises a sense strand sequence as set forth in any one of SEQ ID NOs: 9, 37, 65, and 69. In some embodiments, the oligonucleotide disclosed herein has a sense strand that is composed of at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence as set forth in any one of SEQ ID NOs: 9, 37, 65, and 69. In some embodiments, the oligonucleotide disclosed herein for targeting STAT3 mRNA and inhibiting STAT3 expression comprises a sense strand sequence as set forth in any one of SEQ ID NOs: 862, 875, 876, and 920. In some embodiments, the oligonucleotide herein has a sense strand comprising at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 862, 875, 876, and 920. In some embodiments, the oligonucleotide disclosed herein for targeting STAT3 mRNA and inhibiting STAT3 expression comprises a sense strand sequence set forth in any one of SEQ ID NOs: 901, 910, 899, 896, 892, 890, and 889. In some embodiments, the oligonucleotides herein have a sense strand that includes at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 901, 910, 899, 896, 892, 890, and 889.In some embodiments, the oligonucleotides disclosed herein for targeting STAT3 comprise an antisense strand that comprises or consists of the sequence set forth in any one of SEQ ID NOs: 940, 937, and 939. In some embodiments, the oligonucleotides disclosed herein comprise an antisense strand that comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 940, 937, and 939. In some embodiments, the oligonucleotides disclosed herein for targeting STAT3 comprise an antisense strand that comprises or consists of the sequence set forth in any one of SEQ ID NOs: 915, 924, 913, and 920. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 915, 924, 913, and 920.

[0171] In some embodiments, an oligonucleotide comprises a sense strand (or passenger strand) of up to about 40 nucleotides in length (e.g., up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, an oligonucleotide may have a sense strand of at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36, or at least 38 nucleotides in length). In some embodiments, the oligonucleotides may have a sense strand ranging in length from about 12 to about 40 nucleotides (e.g., 12-40, 12-36, 12-32, 12-28, 15-40, 15-36, 15-32, 15-28, 17-21, 17-25, 19-27, 19-30, 20-40, 22-40, 25-40, or 32-40). In some embodiments, the oligonucleotides herein comprise a sense strand that is 15-50 nucleotides in length. In some embodiments, the oligonucleotides herein comprise a sense strand that is 18-36 nucleotides in length. In some embodiments, the oligonucleotides may have a sense strand that is 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, or 40 nucleotides in length. In some embodiments, the oligonucleotides include a sense strand that is 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, or 50 nucleotides in length. In some embodiments, the oligonucleotides herein comprise a sense strand that is 36 nucleotides in length.

[0172] In some embodiments, oligonucleotides (e.g., RNAi oligonucleotides) provided herein comprise a sense strand that comprises a stem-loop structure at the 3' end of the sense strand. In some embodiments, the stem-loop is formed by intrastrand base pairing. In some embodiments, the sense strand comprises a stem-loop structure at its 5' end. In some embodiments, the stem of the stem-loop comprises a duplex that is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 2 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 3 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 4 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 5 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 6 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 7 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 8 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 9 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 10 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 11 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 12 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 13 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 14 nucleotides in length.

[0173] In some embodiments, the stem-loop provides oligonucleotide protection against degradation (e.g., enzymatic degradation), facilitates or improves targeting and / or delivery to target cells, tissues, or organs (e.g., liver), or both. For example, in some embodiments, the loop of the stem-loop is comprised of nucleotides that include one or more modifications that facilitate, improve, or increase targeting to the target, inhibition of target gene expression, and / or delivery, uptake, and / or penetration into target cells, tissues, or organs (e.g., liver), or a combination thereof. In some embodiments, the stem-loop itself or modifications to the stem-loop do not affect or do not substantially affect the intrinsic gene expression inhibitory activity of the oligonucleotide, but facilitate, improve, or increase stability (e.g., provides protection against degradation), and / or delivery, uptake, and / or penetration of the oligonucleotide into target cells, tissues, or organs. In certain embodiments, the oligonucleotides herein comprise a sense strand that comprises (e.g., at its 3' end) a stem-loop described as S1-L-S2, where S1 is complementary to S2, and L forms a single-stranded loop of linked nucleotides between S1 and S2 up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length). In some embodiments, loop (L) is 3 nucleotides in length (referred to herein as a "tri-loop"). In some embodiments, loop (L) is 4 nucleotides in length (referred to herein as a "tetra-loop"). In some embodiments, loop (L) is 5 nucleotides in length. In some embodiments, loop (L) is 6 nucleotides in length. In some embodiments, loop (L) is 7 nucleotides in length. In some embodiments, loop (L) is 8 nucleotides in length. In some embodiments, loop (L) is 9 nucleotides in length. In some embodiments, loop (L) is 10 nucleotides in length.

[0174] In some embodiments, an oligonucleotide (e.g., an RNAi oligonucleotide) provided herein comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of any one of SEQ ID NOs: 89-280, the oligonucleotide comprises a sense strand comprising a stem-loop (e.g., at its 3' end) as S1-L-S2, S1 being complementary to S2, and L forming a single-stranded loop between S1 and S2 up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length). In some embodiments, an oligonucleotide comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of any one of SEQ ID NOs: 89-280, the oligonucleotide comprises a sense strand comprising a stem-loop (e.g., at its 3' end) as S1-L-S2, S1 being complementary to S2, and L forming a single-stranded loop between S1 and S2 up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length).

[0175] In some embodiments, the tetraloop comprises the sequence 5'-GAAA-3'. In some embodiments, the stem loop comprises the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQ ID NO:86).

[0176] In some embodiments, the sense strand comprises a stem-loop structure at its 3' end. In some embodiments, the sense strand comprises a stem-loop structure at its 5' end. In some embodiments, the stem is double stranded with a length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 bp. In some embodiments, the stem-loop provides molecular protection against degradation (e.g., enzymatic degradation) and promotes targeting characteristics for delivery to target cells. For example, in some embodiments, the loop provides additional nucleotides that can be modified without substantially affecting the gene expression inhibitory activity of the oligonucleotide. In certain embodiments, the oligonucleotide is one in which the sense strand comprises a stem-loop (e.g., at its 3' end) described herein as S1-L-S2, where S1 is complementary to S2, and L forms a loop between S1 and S2 with a length of up to about 10 nucleotides (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides). FIG. 1A shows a non-limiting example of such an oligonucleotide.

[0177] In some embodiments, the loop (L) of the stem-loop having the structure S1-L-S2 described herein is a triloop, hi some embodiments, the triloop comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, ligands (e.g., delivery ligands), and combinations thereof.

[0178] In some embodiments, the loop of the stem-loop is a tetraloop (e.g., in a nicked tetraloop structure). The tetraloop can contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, the tetraloop has 4-5 nucleotides.

[0179] Length of the double strand In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is in the range of 12-30 nucleotides in length (e.g., 12-30, 12-27, 12-22, 15-25, 18-30, 18-22, 18-25, 18-27, 18-30, 19-30, or 21-30 nucleotides in length). In some embodiments, the duplex formed between the sense strand and the antisense strand is 12, 13, 14, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 12 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 13 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 14 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 15 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 16 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 17 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 18 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 19 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 20 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 21 nucleotides in length.In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 22 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 23 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 24 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 25 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 26 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 27 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 28 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 29 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 30 nucleotides in length. In some embodiments, the duplex formed between sense strand and antisense strand does not cover the entire length of sense strand and / or antisense strand.In some embodiments, the duplex formed between sense strand and antisense strand covers the entire length of either sense strand or antisense strand.In some embodiments, the duplex formed between sense strand and antisense strand covers the entire length of both sense strand and antisense strand.

[0180] In some embodiments, the duplex between the sense and antisense strands spans the entire length of both the sense and antisense strands. In some embodiments, the sense and antisense strands of the oligonucleotide are (a) SEQ ID NOs: 861 and 951, respectively; (b) SEQ ID NOs: 857 and 947, respectively; (c) SEQ ID NOs: 858 and 948, respectively; (d) SEQ ID NOs: 859 and 949, respectively; (e) SEQ ID NOs: 860 and 950, respectively; (f) SEQ ID NOs: 862 and 952, respectively; (g) SEQ ID NOs: 863 and 953, respectively; (h) SEQ ID NOs: 864 and 954, respectively; (i) SEQ ID NOs: 865 and 955, respectively; (j) SEQ ID NOs: 866 and 956, respectively; (k) SEQ ID NOs: 867 and 957, respectively; (l) SEQ ID NOs: 868 and 958, respectively; (m) SEQ ID NOs: 869 and 959, respectively; (n) SEQ ID NOs: 870 and 960, respectively; (o) SEQ ID NOs: 871 and 961, respectively; (p) SEQ ID NOs: 872 and 962, respectively; (q) SEQ ID NOs: 873 and 963, respectively; (r) SEQ ID NOs: 874 and 964, respectively; (s) SEQ ID NOs: 875 and 965, respectively; (t) SEQ ID NOs: 876 and 966, respectively; (u) SEQ ID NOs: 877 and 967, respectively; (v) SEQ ID NOs: 878 and 968, respectively; (w) SEQ ID NOs: 879 and 969, respectively; (x) SEQ ID NOs: 880 and 970, respectively; (y) SEQ ID NOs: 881 and 971, respectively; (z) SEQ ID NOs: 882 and 972, respectively; (aa) SEQ ID NOs: 883 and 973, respectively; (bb) SEQ ID NOs: 884 and 974, respectively; (cc) SEQ ID NOs: 885 and 975, respectively; (dd) SEQ ID NOs: 886 and 976, respectively; (ee) SEQ ID NOs: 887 and 977, respectively; (ff) SEQ ID NOs: 888 and 978, respectively; (gg) SEQ ID NOs: 940 and 1030, respectively; (hh) SEQ ID NOs: 896 and 986, respectively; and (ii) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 920 and 1010, respectively; The duplex formed between the sense and antisense strands ranges from 12 to 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length).

[0181] In some embodiments, the duplex between the sense and antisense strands spans the entire length of both the sense and antisense strands. In some embodiments, the sense and antisense strands of the oligonucleotide are (a) SEQ ID NOs: 857 and 947, respectively; (b) SEQ ID NOs: 858 and 948, respectively; (c) SEQ ID NOs: 859 and 949, respectively; (d) SEQ ID NOs: 860 and 950, respectively; (e) SEQ ID NOs: 862 and 952, respectively; (f) SEQ ID NOs: 867 and 957, respectively; (g) SEQ ID NOs: 875 and 965, respectively; and (h) a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 876 and 966, respectively; The duplex formed between the sense and antisense strands ranges from 12 to 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length).

[0182] In some embodiments, the duplex between the sense and antisense strands spans the entire length of both the sense and antisense strands. In some embodiments, the sense and antisense strands of the oligonucleotide are (a) SEQ ID NOs: 9 and 10, respectively; (b) SEQ ID NOs: 37 and 38, respectively; (c) SEQ ID NOs: 65 and 66, respectively, and (d) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 69 and 70, respectively. The duplex formed between the sense and antisense strands ranges from 12 to 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length).

[0183] In some embodiments, the duplex between the sense and antisense strands spans the entire length of both the sense and antisense strands. In some embodiments, the sense and antisense strands of the oligonucleotide are (a) SEQ ID NOs: 901 and 991, respectively; (b) SEQ ID NOs: 910 and 1000, respectively; (c) SEQ ID NOs: 899 and 989, respectively; (d) SEQ ID NOs: 896 and 986, respectively; (e) SEQ ID NOs: 892 and 982, respectively; (f) SEQ ID NOs: 890 and 980, respectively; and (g) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 889 and 979, respectively. The duplex formed between the sense and antisense strands ranges from 12 to 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length).

[0184] In some embodiments, the duplex between the sense and antisense strands spans the entire length of both the sense and antisense strands. In some embodiments, the sense and antisense strands of the oligonucleotide are (a) SEQ ID NOs: 940 and 1030, respectively; (b) SEQ ID NOs: 937 and 1027, respectively, and (c) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 939 and 1029, respectively. The duplex formed between the sense and antisense strands ranges from 12 to 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length).

[0185] In some embodiments, the duplex between the sense and antisense strands spans the entire length of both the sense and antisense strands. In some embodiments, the sense and antisense strands of the oligonucleotide are (a) SEQ ID NOs: 915 and 1005, respectively; (b) SEQ ID NOs: 924 and 1014, respectively; (c) SEQ ID NOs: 913 and 1003, respectively; and (d) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 920 and 1010, respectively. The duplex formed between the sense and antisense strands ranges from 12 to 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length).

[0186] Oligonucleotide End In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, and the ends of either or both strands comprise a blunt end. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand and an antisense strand, and the sense strand and the antisense strand are separate strands that form an asymmetric double-stranded region with an overhang at the 3' end of the antisense strand. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand and an antisense strand, and the ends of either or both strands comprise an overhang comprising one or more nucleotides. In some embodiments, the one or more nucleotides comprising the overhang are unpaired nucleotides. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand and an antisense strand, and the 3' end of the sense strand and the 5' end of the antisense strand comprise a blunt end. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand and an antisense strand, and the 5' end of the sense strand and the 3' end of the antisense strand comprise a blunt end.

[0187] In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and the 3' end of either or both strands comprises a 3' overhang comprising one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and the sense strand comprises a 3' overhang comprising one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and the antisense strand comprises a 3' overhang comprising one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and both the sense strand and the antisense strand comprise a 3' overhang comprising one or more nucleotides.

[0188] In some embodiments, the 3' overhang is about 1-20 nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length). In some embodiments, the 3' overhang is about 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or about 1-2 nucleotides in length. In some embodiments, the 3'-overhang is one nucleotide in length. In some embodiments, the 3' overhang is two nucleotides in length. In some embodiments, the 3' overhang is three nucleotides in length. In some embodiments, the 3' overhang is four nucleotides in length. In some embodiments, the 3'-overhang is 5 nucleotides in length. In some embodiments, the 3'-overhang is 6 nucleotides in length. In some embodiments, the 3'-overhang is 7 nucleotides in length. In some embodiments, the 3'-overhang is 8 nucleotides in length. In some embodiments, the 3'-overhang is 9 nucleotides in length. In some embodiments, the 3'-overhang is 10 nucleotides in length. In some embodiments, the 3'-overhang is 11 nucleotides in length. In some embodiments, the 3'-overhang is 12 nucleotides in length. In some embodiments, the 3'-overhang is 13 nucleotides in length. In some embodiments, the 3'-overhang is 14 nucleotides in length. In some embodiments, the 3'-overhang is 15 nucleotides in length. In some embodiments, the 3'-overhang is 16 nucleotides in length. In some embodiments, the 3'-overhang is 17 nucleotides in length. In some embodiments, the 3'-overhang is 18 nucleotides in length.In some embodiments, the 3'-overhang is 19 nucleotides in length. In some embodiments, the 3' overhang is 20 nucleotides in length.

[0189] In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and the 5' end of either or both strands comprises a 5' overhang comprising one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and the sense strand comprises a 5'-overhang comprising one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and the antisense strand comprises a 5'-overhang comprising one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and both the sense strand and the antisense strand comprise a 5' overhang comprising one or more nucleotides.

[0190] In some embodiments, the 5' overhang is about 1-20 nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length). In some embodiments, the 5' overhang is about 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or about 1-2 nucleotides in length. In some embodiments, the 5'-overhang is (1) nucleotide in length. In some embodiments, the 5'-overhang is 2 nucleotides in length. In some embodiments, the 5'-overhang is 3 nucleotides in length. In some embodiments, the 5'-overhang is 4 nucleotides in length. In some embodiments, the 5'-overhang is 5 nucleotides in length. In some embodiments, the 5'-overhang is 6 nucleotides in length. In some embodiments, the 5'-overhang is 7 nucleotides in length. In some embodiments, the 5'-overhang is 8 nucleotides in length. In some embodiments, the 5'-overhang is 9 nucleotides in length. In some embodiments, the 5'-overhang is 10 nucleotides in length. In some embodiments, the 5'-overhang is 11 nucleotides in length. In some embodiments, the 5'-overhang is 12 nucleotides in length. In some embodiments, the 5'-overhang is 13 nucleotides in length. In some embodiments, the 5'-overhang is 14 nucleotides in length. In some embodiments, the 5'-overhang is 15 nucleotides in length. In some embodiments, the 5'-overhang is 16 nucleotides in length. In some embodiments, the 5'-overhang is 17 nucleotides in length. In some embodiments, the 5'-overhang is 18 nucleotides in length.In some embodiments, the 5'-overhang is 19 nucleotides in length. In some embodiments, the 5' overhang is 20 nucleotides in length.

[0191] In some embodiments, one or more (e.g., 2, 3, 4, 5, or more) nucleotides, including the 3'-end or 5'-end of the sense and / or antisense strand, are modified. For example, in some embodiments, one or two terminal nucleotides at the 3'-end of the antisense strand are modified. In some embodiments, the last nucleotide at the 3'-end of the antisense strand is modified such that it comprises a 2' modification or that it comprises a 2'-O-methoxyethyl. In some embodiments, the last one or two terminal nucleotides at the 3'-end of the antisense strand are complementary to the target. In some embodiments, the last one or two nucleotides at the 3'-end of the antisense strand are not complementary to the target.

[0192] In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, the 3' end of the sense strand comprises a step-loop as described herein, and the 3' end of the antisense strand comprises a 3' overhang as described herein. In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand that form a nicked tetraloop structure as described herein, the 3' end of the sense strand comprises a stem-loop, the loop being a tetraloop as described herein, and the 3' end of the antisense strand comprises a 3' overhang as described herein. In some embodiments, the 3' overhang is two nucleotides in length. In some embodiments, the two nucleotides that comprise the 3' overhang both comprise a guanine (G) nucleobase. Typically, one or both of the nucleotides that comprise the 3' overhang of the antisense strand are not complementary to the target mRNA.

[0193] Oligonucleotide Modification a. Sugar modification In some embodiments, modified sugars (also referred to herein as sugar analogs) include modified deoxyribose or ribose moieties, e.g., where one or more modifications occur at the 2', 3', 4', and / or 5' carbon positions of the sugar. In some embodiments, modified sugars can also include unnatural alternative carbon structures, such as those found in locked nucleic acids ("LNAs", see e.g., Koshkin et al., (1998) TETRAHEDON 54:3607-3630), non-locked nucleic acids ("UNAs", see e.g., Snead et al., (2013) MOL. THER-NUCL. ACIDS 2:e103), and bridged nucleic acids ("BNAs", see e.g., Imanishi and Obika (2002) CHEM COMMUN. (CAMB) 21:1653-1659).

[0194] In some embodiments, the nucleotide modification at the sugar comprises a 2' modification. In some embodiments, the 2'-modification may be 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-fluoro (2'-F), 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), or 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA). In some embodiments, the modification is 2'-F, 2'-OMe, or 2'-MOE. In some embodiments, the modification at the sugar comprises a modification of the sugar ring, which may include a modification of one or more carbons of the sugar ring. For example, modifications of the sugar of a nucleotide may include linking the 2'-oxygen of the sugar to the 1'-carbon or 4'-carbon of the sugar, or linking the 2'-oxygen to the 1'-carbon or 4'-carbon via an ethylene or methylene bridge. In some embodiments, modified nucleotides have acyclic sugars that lack a 2'-carbon to 3'-carbon bond. In some embodiments, modified nucleotides have a thiol group, for example, at the 4' position of the sugar.

[0195] In some embodiments, the oligonucleotides described herein comprise at least about one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, or more). In some embodiments, the sense strand of the oligonucleotide comprises at least about one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or more). In some embodiments, the antisense strand of the oligonucleotide comprises at least about one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more).

[0196] In some embodiments, all nucleotides of the sense strand of the oligonucleotide are modified. In some embodiments, all nucleotides of the antisense strand of the oligonucleotide are modified. In some embodiments, all nucleotides of the oligonucleotide (i.e., both the sense strand and the antisense strand) are modified. In some embodiments, the modified nucleotides include 2'-modifications (e.g., 2'-F or 2'-OMe, 2'-MOE, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid). In some embodiments, the modified nucleotides include 2'-modifications (e.g., 2'-F or 2'-OMe).

[0197] In some embodiments, the present disclosure provides oligonucleotides with different modification patterns.In some embodiments, the oligonucleotides herein include a sense strand with the modification patterns shown in the examples and sequence listing, and an antisense strand with the modification patterns shown in the examples and sequence listing.

[0198] In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise an antisense strand having 2'-F modified nucleotides. In some embodiments, the oligonucleotides disclosed herein comprise an antisense strand comprising 2'-F and 2'-OMe modified nucleotides. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand having 2'-F modified nucleotides. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand comprising 2'-F and 2'-OMe modified nucleotides.

[0199] In some embodiments, the oligonucleotides described herein comprise a sense strand in which about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand comprise a 2'-fluoro modification. In some embodiments, 11% of the nucleotides of the sense strand comprise a 2'-fluoro modification. In some embodiments, the oligonucleotides described herein comprise an antisense strand in which about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the antisense strand comprise a 2'-fluoro modification. In some embodiments, about 32% of the nucleotides of the antisense strand comprise a 2'-fluoro modification. In some embodiments, the oligonucleotides have about 15-25%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of their nucleotides comprise a 2'-fluoro modification. In some embodiments, about 19% of the nucleotides in the dsRNAi oligonucleotide contain a 2'-fluoro modification.

[0200] In some embodiments, the modified oligonucleotides comprise a sense strand sequence having a modification pattern as described in FIG. 1A or Example 7, and an antisense strand having a modification pattern as described in FIG. 1A or Example 7. In some embodiments, these oligonucleotides have one or more of positions 8, 9, 10, or 11 of the sense strand modified with a 2'-F group. In other embodiments, these oligonucleotides have the sugar moiety of each of the nucleotides at positions 1-7 and 12-20 in the sense strand modified with a 2'-OMe.

[0201] In some embodiments, the antisense strand has three nucleotides modified with 2'-F at the 2' position of the sugar moiety. In some embodiments, the sugar moieties at positions 2, 5, and 14, and optionally up to three nucleotides at positions 1, 3, 7, and 10 of the antisense strand are modified with 2'-F. In some embodiments, the sugar moieties at positions 2, 5, and 14, and optionally up to three nucleotides at positions 3, 4, 7, and 10 of the antisense strand are modified with 2'-F. In other embodiments, the sugar moieties at positions 2, 5, and 14 of the antisense strand are each modified with 2'-F. In other embodiments, the sugar moieties at positions 1, 2, 5, and 14 of the antisense strand are each modified with 2'-F. In other embodiments, the sugar moieties at positions 2, 4, 5, and 14 of the antisense strand are each modified with 2'-F. In yet another embodiment, the sugar moieties at each of positions 1, 2, 3, 5, 7, and 14 of the antisense strand are modified with 2'-F. In another embodiment, the sugar moieties at each of positions 2, 3, 4, 5, 7, and 14 of the antisense strand are modified with 2'-F. In yet another embodiment, the sugar moieties at each of positions 1, 2, 3, 5, 10, and 14 of the antisense strand are modified with 2'-F. In another embodiment, the sugar moieties at each of positions 2, 3, 4, 5, 10, and 14 of the antisense strand are modified with 2'-F. In another embodiment, the sugar moieties at each of positions 2, 3, 5, 7, 10, and 14 of the antisense strand are modified with 2'-F. In yet another embodiment, the sugar moieties at each of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand are modified with 2'-F.

[0202] In some embodiments, the oligonucleotides provided herein comprise an antisense strand having a sugar moiety at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 that is modified with 2'-F.

[0203] In some embodiments, the oligonucleotides provided herein comprise an antisense strand having a sugar moiety at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 that is 2'-OMe modified.

[0204] In some embodiments, the oligonucleotides provided herein comprise an antisense strand having a sugar moiety at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA).

[0205] In some embodiments, the oligonucleotides provided herein comprise a sense strand having sugar moieties at positions 8-11 modified with 2'-F. In some embodiments, the oligonucleotides provided herein comprise a sense strand having sugar moieties at positions 3, 8, 9, 10, 12, 13, and 17 modified with 2'-F. In some embodiments, the oligonucleotides provided herein comprise a sense strand having sugar moieties at positions 1-7 and 12-17 or 12-20 modified with 2'OMe. In some embodiments, the oligonucleotides provided herein comprise a sense strand having sugar moieties at positions 1-7, 12-27, and 31-36 modified with 2'OMe. In some embodiments, the oligonucleotides provided herein comprise a sense strand having sugar moieties at each of nucleotides 1-7 and 12-17 or 12-20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA). In some embodiments, the oligonucleotides provided herein comprise a sense strand having sugar moieties at positions 1-2, 4-7, 11, 14-16, and 18-20 modified with 2'OMe. In some embodiments, the oligonucleotides provided herein comprise a sense strand having sugar moieties at each of the nucleotides at positions 1-2, 4-7, 11, 14-16, and 18-20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA).

[0206] In some embodiments, the oligonucleotides provided herein comprise a sense strand having a sugar moiety at position 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, or 36 that is modified with 2'-F.

[0207] In some embodiments, the oligonucleotides provided herein comprise a sense strand having a sugar moiety at position 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, or 36 that is 2'-OMe modified.

[0208] In some embodiments, the oligonucleotides provided herein include 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinyl. and a sense strand having sugar moieties at positions 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, or 36 modified with a modification selected from the group consisting of: binonucleic acid (2'-FANA).

[0209] b. 5'-terminal phosphate In some embodiments, the 5'-terminal phosphate group of the oligonucleotide enhances the interaction with Ago2. However, oligonucleotides containing a 5'-phosphate group may be susceptible to degradation via phosphatases or other enzymes, which may limit their performance and / or bioavailability in vivo. In some embodiments, the oligonucleotide comprises an analog of the 5' phosphate that is resistant to such degradation. In some embodiments, the phosphate analog may be an oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In certain embodiments, the 1' end of the oligonucleotide chain is attached to a chemical moiety ("phosphate mimetic") that mimics the electrostatic and steric properties of the natural 5' phosphate group.

[0210] In some embodiments, the oligonucleotide has a phosphate analog at the 4' carbon position of the sugar (referred to as a "4' phosphate analog"). See, for example, International Patent Application Publication No. 2018 / 045317. In some embodiments, the oligonucleotide herein comprises a 4' phosphate analog at the 5' terminal nucleotide. In some embodiments, the phosphate analog is an oxymethylphosphonate in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., its 4' carbon) or an analog thereof. In other embodiments, the 4'-phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate in which the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is attached to the 4'-carbon of the sugar moiety or an analog thereof. In certain embodiments, the 4'-phosphate analog is an oxymethylphosphonate. In some embodiments, the oxymethylphosphonate is represented by the formula -O-CH2-PO(OH)2 or -O-CH2-PO(OR)2, where R is independently selected from H, CH3, an alkyl group, CH2CH2CN, CH2OCOC(CH3)3, CHOCH2CH2Si(CH3)3, or a protecting group. In certain embodiments, the alkyl group is CH2CH3. More typically, R is independently selected from H, CH3, or CH2CH3.

[0211] In some embodiments, the oligonucleotides provided herein comprise an antisense strand that comprises a 4'-phosphate analog at the 5' terminal nucleotide, wherein the 5' terminal nucleotide comprises the following structure (Formula 1): [ka]

[0212] C modified internucleotide linkage In some embodiments, the oligonucleotide may include modified internucleoside linkages. In some embodiments, the phosphate modification or substitution may result in an oligonucleotide that includes at least about one (e.g., at least one, at least two, at least three, or at least five) modified internucleotidic linkages. In some embodiments, any one of the oligonucleotides disclosed herein includes about one to about ten (e.g., 1-10, 2-8, 4-6, 3-10, 5-10, 1-5, 1-3, or 1-2) modified internucleotidic linkages. In some embodiments, any one of the oligonucleotides disclosed herein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotidic linkages.

[0213] Modified internucleotide bond can be phosphorodithioate bond, 4'-O-methylene phosphonate bond, phosphorothioate bond, phosphotriester bond, thionoalkylphosphonate bond, thionealkylphosphotriester bond, phosphoramidite bond, phosphonate bond or boranophosphate bond.In some embodiments, at least one modified internucleotide bond of any one of the oligonucleotides disclosed herein is phosphorothioate bond.In some embodiments, at least one modified internucleotide bond of any one of the oligonucleotides disclosed herein is 4'-O-methylene phosphonate bond.

[0214] In some embodiments, the oligonucleotides described herein have phosphorothioate bonds between one or more of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand. In some embodiments, the oligonucleotides described herein have phosphorothioate bonds between positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.

[0215] D. Base modification In some embodiments, the oligonucleotide herein has one or more modified nucleobases. In some embodiments, the modified nucleobase (also referred to herein as base analog) is attached to the 1' position of the nucleotide sugar moiety. In certain embodiments, the modified nucleobase is a nitrogenous base. In some embodiments, the modified nucleobase does not contain a nitrogen atom. See, for example, US Patent Application Publication No. 2008 / 0274462. In some embodiments, the modified nucleotide comprises a universal base. However, in certain embodiments, the modified nucleotide does not contain a nucleobase (abasic).

[0216] In some embodiments, a universal base is a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety in a modified nucleotide, or at an equivalent position in a nucleotide sugar moiety substitution, and when present in a duplex, can be positioned opposite more than one type of base without substantially altering the structure of the duplex. In some embodiments, compared to a reference single-stranded nucleic acid (e.g., an oligonucleotide) that is fully complementary to a target nucleic acid, a single-stranded nucleic acid containing a universal base has a lower T than a duplex formed with a complementary nucleic acid. mHowever, in some embodiments, when compared to a reference single-stranded nucleic acid in which the universal base is replaced with a base to create a single mismatch, the single-stranded nucleic acid containing the universal base has a higher T than the duplex formed with the nucleic acid containing the mismatched base. m It forms a duplex with a target nucleic acid having the formula:

[0217] Non-limiting examples of universal binding nucleotides include, but are not limited to, inosine, 1-β-D-ribofuranosyl-5-nitroindole, and / or 1-β-D-ribofuranosyl-3-nitropyrrole (see U.S. Patent Application Publication No. 2007 / 0254362; Van Aerschot et al., (1995) NUCLEIC ACIDS RES. 23:4363-4370; Loakes et al., (1995) NUCLEIC ACIDS RES. 23:2361-66; and Loakes and Brown (1994) NUCLEIC ACIDS RES. 22:4039-43).

[0218] e. Reversible modification Certain modifications can be made to protect oligonucleotide from the in vivo environment before reaching target cells, but they can reduce the efficacy or activity of oligonucleotide when it reaches the cytosol of target cells.Reversible modifications can be made so that the molecule retains desirable properties outside the cell, and then is removed when it enters the cytoplasmic environment of the cell.Reversible modifications can be removed, for example, by the action of intracellular enzymes or by intracellular chemical conditions (e.g., through reduction by intracellular glutathione).

[0219] In some embodiments, the reversibly modified nucleotide comprises a glutathione-sensitive moiety.Typically, nucleic acid molecules are chemically modified with cyclic disulfide moieties to mask the negative charge created by internucleotide diphosphate bonds and improve cellular uptake and nuclease resistance.See US Patent Application Publication No. 2011 / 0294869, International Patent Publication No. 2014 / 088920 and International Patent Publication No. 2015 / 188197, and Meade et al., (2014) NAT.BIOTECHNOL.32:1256-63.This reversible modification of internucleotide diphosphate bonds is designed to be cleaved intracellularly by the reducing environment of cytosol (e.g., glutathione). Previous examples include neutralizing phosphotriester modifications that have been reported to be cleavable intracellularly (see Dellinger et al., (2003) J. AM. CHEM. SOC. 125:940-50).

[0220] In some embodiments, such reversible modifications allow protection during in vivo administration (e.g., passage through the blood and / or lysosomal / endosomal compartments of cells), where the oligonucleotide is exposed to nucleases and other harsh environmental conditions (e.g., pH). Upon release into the cytosol of cells, where the level of glutathione is higher compared to the extracellular space, the modifications are reversed, resulting in cleaved oligonucleotides. Compared to the options available using irreversible chemical modifications, it is possible to introduce sterically larger chemical groups into the oligonucleotide of interest using reversible glutathione-sensitive moieties. This is because these larger chemical groups are removed in the cytosol and therefore should not interfere with the biological activity of the oligonucleotide inside the cytosol of the cell. As a result, these larger chemical groups can be engineered to confer various advantages to the nucleotide or oligonucleotide, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, and reduced immunogenicity. In some embodiments, the structure of the glutathione-sensitive moiety can be engineered to modify the kinetics of its release.

[0221] In some embodiments, the glutathione-sensitive moiety is attached to the sugar of the nucleotide. In some embodiments, the glutathione-sensitive moiety is attached to the 2'-carbon of the sugar of the modified nucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 5'-carbon of the sugar, especially when the modified nucleotide is the 5'-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 3'-carbon of the sugar, especially when the modified nucleotide is the 3'-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group. See, e.g., U.S. Provisional Patent Application No. 62 / 378,635, filed August 23, 2016, entitled Compositions Comprising Reversibly Modified Oligonucleotides and Uses Thereof.

[0222] Targeting Ligands In some embodiments, it is desirable to target the STAT3 targeting oligonucleotides of the present disclosure to one or more cells or one or more organs. Such a strategy may help to avoid undesirable effects in other organs or to avoid unnecessary loss of oligonucleotides to cells, tissues or organs that would not benefit from the oligonucleotide. Targeting of oligonucleotides to one or more cells or one or more organs may be achieved through a variety of approaches. Conjugation of oligonucleotides to tissue or cell specific antibodies, small molecules, or targeting ligands can facilitate delivery to one or more target cells or tissues and modify accumulation of oligonucleotides (Chernolovskaya et al., (2019) FRONT PHARMACOL. 10:444). For example, conjugation of oligonucleotides to saturated fatty acids (e.g., C22) may facilitate delivery to cells or tissues, such as adipose tissue or immune cells, that take up such ligands more readily than conventional oligonucleotide ligands. Thus, in some embodiments, the oligonucleotides disclosed herein are modified to facilitate targeting and / or delivery to tissues, cells, or organs (e.g., to facilitate delivery of oligonucleotides to the liver). In certain embodiments, the oligonucleotide disclosed herein is modified to facilitate delivery of the oligonucleotide to cells of the immune system.In certain embodiments, the oligonucleotide disclosed herein is modified to facilitate delivery of the oligonucleotide to myeloid-derived suppressor cells.In some embodiments, the oligonucleotide comprises at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6 or more nucleotides) conjugated with one or more targeting ligands.

[0223] In some embodiments, the targeting ligand comprises carbohydrate, amino sugar, cholesterol, peptide, polypeptide, protein, or protein part (e.g., antibody or antibody fragment), or lipid. In some embodiments, the targeting ligand is an aptamer. For example, the targeting ligand may be RGD peptide used to target tumor vasculature or glioma cells, CREKA peptide to target tumor vasculature or stoma, transferring, lactoferrin, or aptamer to target transferrin receptor expressed in CNS vasculature, or anti-EGFR antibody to target EGFR on glioma cells. In certain embodiments, the targeting ligand is one or more GalNAc moieties.

[0224] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, 2-4 nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, the targeting ligand is conjugated to 2-4 nucleotides at either the sense or antisense end, such that the targeting ligand resembles toothbrush bristles and the oligonucleotide resembles a toothbrush (e.g., the targeting ligand is conjugated to an overhang or extension of 2-4 nucleotides at the 5' or 3' end of the sense or antisense strand). For example, the oligonucleotide may include a stem-loop at either the 5' or 3' end of the sense strand, and 1, 2, 3, or 4 nucleotides of the stem loop may be individually conjugated to a targeting ligand. In some embodiments, the oligonucleotide (e.g., dsRNA) provided by the present disclosure comprises a stem-loop at the 3' end of the sense strand, the loop of the stem-loop comprises a triloop or a tetraloop, and the three or four nucleotides of the triloop or the tetraloop are respectfully conjugated to a targeting ligand.In some embodiments, the oligonucleotide (e.g., RNAi oligonucleotide) provided by the present disclosure comprises a stem-loop at the 3' end of the sense strand, the loop of the stem-loop comprises a tetraloop, and the three nucleotides of the tetraloop are conjugated to a targeting ligand individually.

[0225] GalNAc is a high affinity ligand for ASGPR, which is mainly expressed on the sinusoidal surface of hepatocytes and plays a major role in binding, internalization, and subsequent removal of circulating glycoproteins that contain terminal galactose or GalNAc residues (asialoglycoproteins).Conjugation (either indirectly or directly) of GalNAc moiety with oligonucleotides of the present disclosure can be used to target these oligonucleotides to ASGPR expressed on cells.In some embodiments, the oligonucleotides of the present disclosure are conjugated with at least one or more GalNAc moieties, and the GalNAc moiety targets the oligonucleotide to ASGPR expressed on human liver cells (e.g., human hepatocytes).In some embodiments, the GalNAc moiety targets the oligonucleotide to liver.

[0226] In some embodiments, the oligonucleotide of the present disclosure is directly or indirectly conjugated with monovalent GalNAc moiety.In some embodiments, the oligonucleotide is directly or indirectly conjugated with more than one monovalent GalNAc moiety (i.e., is conjugated with 2, 3 or 4 monovalent GalNAc moieties, typically is conjugated with 3 or 4 monovalent GalNAc moieties).In some embodiments, the oligonucleotide is conjugated with one or more divalent GalNAc, trivalent GalNAc or tetravalent GalNAc moieties.

[0227] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide are each conjugated to a GalNAc moiety. In some embodiments, 2-4 nucleotides of the tetraloop are each conjugated to a separate GalNAc. In some embodiments, 1-3 nucleotides of the triloop are each conjugated to a separate GalNAc. In some embodiments, the targeting ligand is conjugated to 2-4 nucleotides at both ends of the sense or antisense strand (e.g., the ligand is conjugated to an overhang or extension of 2-4 nucleotides on the 5' or 3' end of the sense or antisense strand) such that the GalNAc moieties resemble toothbrush bristles and the oligonucleotide resembles a toothbrush. In some embodiments, the GalNAc moiety is conjugated to a nucleotide of the sense strand. For example, 4 GalNAc moieties can be conjugated to nucleotides in the tetraloop of the sense strand, with each GalNAc moiety being conjugated to one nucleotide.

[0228] In some embodiments, the tetraloop is any combination of adenine and guanine nucleotides.

[0229] In some embodiments, the tetraloop (tetraL) has a monovalent GalNAc moiety attached to any one or more guanine nucleotides of the tetraloop via any linker described herein, as shown below in Formula 2 (X = heteroatom). [ka]

[0230] In some embodiments, the tetraloop (tetraL) has a monovalent GalNAc moiety attached to any one or more adenine nucleotides of the tetraloop via any linker described herein, as shown below in Formula 3 (X = heteroatom). [ka]

[0231] In some embodiments, the oligonucleotides herein comprise a monovalent GalNAc attached to a guanine nucleotide, referred to as [ademG-GalNAc] or 2'-aminodiethoxymethanol-guanine-GalNAc, as shown below in Formula 4. [ka]

[0232] In some embodiments, the oligonucleotides herein comprise a monovalent GalNAc attached to an adenine nucleotide, referred to as [ademA-GalNAc] or 2'-aminodiethoxymethanol-adenine-GalNAc, as shown below in Formula 5. [ka]

[0233] One example of such a conjugation is shown below (Formula 6) for a loop containing the nucleotide sequence GAAA (L=linker, X=heteroatom) from 5' to 3', with the stem attachment points indicated. Such a loop may be present, for example, at positions 27-30 of the sense strand shown in FIG. 1A. In the formula: [ka] is used to describe the point of attachment to the oligonucleotide chain (Equation 6). [ka]

[0234] The targeting ligand can be attached to the nucleotide using a suitable method or chemistry (e.g., click chemistry). In some embodiments, the targeting ligand is conjugated to the nucleotide using a click linker. In some embodiments, the targeting ligand is conjugated to any one of the nucleotides of the oligonucleotides described herein using an acetal-based linker. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. 2016 / 100401. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is stable. An example is shown below (Formula 7 and Formula 8) for a loop containing the nucleotide GAAA at 5' to 3', with a GalNAc moiety attached to the nucleotide of the loop using an acetal linker. Such a loop can be present, for example, at positions 27-30 of any one of the sense strands shown in FIG. 1. In the formula: [ka] is the point of attachment to the oligonucleotide chain (Formula 7 and Formula 8). [ka]

[0235] As mentioned above, the targeting ligand can be attached to the nucleotide using various suitable methods or chemical synthesis techniques (e.g., click chemistry). In some embodiments, the targeting ligand is conjugated to the nucleotide using a click linker. In some embodiments, the targeting ligand is conjugated to any one of the nucleotides of the oligonucleotides described herein using an acetal-based linker. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. 2016 / 100401. In some embodiments, the linker is an unstable linker. However, in other embodiments, the linker is a stable linker.

[0236] In some embodiments, a double-stranded extension (e.g., up to 3, 4, 5, or 6 bp in length) is provided between the targeting ligand (e.g., GalNAc moiety) and the dsRNA. In some embodiments, the oligonucleotide herein does not have GalNAc conjugated thereto.

[0237] Structure of conjugated STAT3-targeting oligonucleotides In some embodiments, the STAT3 targeting oligonucleotides described herein comprise a nucleotide sequence having a region of complementarity to a STAT3 mRNA target sequence and one or more targeting ligands, the nucleotide sequence being represented by Formula Ia: [ka] or a pharma- ceutically acceptable salt thereof (In the formula, B is a nucleobase or hydrogen; R 1 and R 2 are independently hydrogen, halogen, R A , -CN, -S(O)R, -S(O)2R, -Si(OR)2R, -Si(OR)R2, or -SiR3, or R on the same carbon 1 and R 2 together with their intervening atoms form a 3-7 membered saturated or partially unsaturated ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R A is independently 1-6 an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R is independently hydrogen, a suitable protecting group, or C 1-6an optionally substituted group selected from aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two R groups on the same atom together with their intervening atoms form a 4-7 membered saturated, partially unsaturated, or heteroaryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, silicon, and sulfur; Each targeting ligand is selected from a lipid conjugate moiety (LC), a carbohydrate, an amino sugar, or GalNAc, and each LC is independently a saturated or unsaturated, linear, or branched C 1-50 a lipid conjugate moiety comprising a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-; Each -Cy- is independently selected from phenylenyl; 8-10 membered bicyclic arylenyl; 4-7 membered saturated or partially unsaturated carbocyclylenyl; 4-11 membered saturated or partially unsaturated spirocarbocyclylenyl; 8-10 membered bicyclic saturated or partially unsaturated carbocyclylenyl; 4-7 membered saturated or partially unsaturated heterocyclylenyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; 4-11 membered saturated or partially unsaturated heterocyclylenyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. an optionally substituted bivalent ring selected from: a saturated or partially unsaturated spiroheterocyclylenyl; an 8-10 membered bicyclic saturated or partially unsaturated heterocyclylenyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 5-6 membered heteroarylenyl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-10 membered bicyclic heteroarylenyl having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; n is 1 to 10; L is a covalent bond or a divalent saturated or unsaturated, linear or branched C 1-50 It is a hydrocarbon chain, and 0 to 10 methylene units of the hydrocarbon chain are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, -V 1 CR 2 W 1 -,or [ka] has been replaced by m is 1 to 50; X 1 , V 1 , and W 1 are independently -C(R)2-, -OR, -O-, -S-, -Se-, or -NR-; Y is hydrogen, a suitable hydroxyl protecting group, [ka] and R 3 is hydrogen, a suitable protecting group, a suitable prodrug, or C 1-6 an optionally substituted group selected from a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from aliphatic, phenyl, nitrogen, oxygen, and sulfur, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; X 2 is O, S, or NR; X 3 is -O-, -S-, -BH2-, or a covalent bond, Y 1 is a linking group attached to the 2' or 3' end of a nucleoside, nucleotide, or oligonucleotide, Y 2is hydrogen, a suitable protecting group, a phosphoramidite analog; an internucleotide linkage attached to the 5' end of a nucleoside, nucleotide, or oligonucleotide, or a linkage attached to a solid support; Z comprises one or more nucleosides (nucleic acids) conjugated to one or more targeting ligands represented by -O-, -S-, -NR-, or -CR2-.

[0238] In some embodiments, the STAT3 targeting oligonucleotide has the formula II-a: [ka] or a pharma- ceutically acceptable salt thereof, conjugated to a targeting ligand.

[0239] In some embodiments, the STAT3 targeting oligonucleotide has the formula II-b or II-c: [ka] or a pharma- ceutically acceptable salt thereof, L 1 is a covalent bond, monovalent or divalent, saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain, 0 to 10 methylene units of which are independently -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, or [ka] has been replaced by R 4 is hydrogen, R A or a suitable amine protecting group, R 5 is adamantyl or saturated or unsaturated, linear or branched C 1-50and a hydrocarbon chain, wherein 0 to 10 methylene units of the hydrocarbon chain are independently replaced by -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR, conjugated to a targeting ligand.

[0240] In some embodiments, R 5 is selected from the following: [ka]

[0241] In some embodiments, R 5 is selected from the following: [ka]

[0242] In some embodiments, R 5 teeth, [ka] In some embodiments, R 5 teeth, [ka] In some embodiments, R 5 teeth, [ka] In some embodiments, R 5 teeth, [ka] In some embodiments, R 5 teeth, [ka] In some embodiments, R5 teeth, [ka] In some embodiments, R 5 teeth, [ka] In some embodiments, R 5 teeth, [ka] In some embodiments, R 5 teeth, [ka] In some embodiments, R 5 teeth, [ka] In some embodiments, R 5 teeth, [ka] In some embodiments, R 5 teeth, [ka] In some embodiments, R 5 teeth, [ka] In some embodiments, R 5 teeth, [ka] It is.

[0243] In some embodiments, the STAT3 targeting oligonucleotide has the formula II-Ib or II-Ic: [ka] or a pharma- ceutically acceptable salt thereof, B is a nucleobase or hydrogen; m is 1 to 50; X 1 is -O- or -S-; Y is hydrogen, [ka] and R 3 is hydrogen or a suitable protecting group, X 2 is O or S, X 3 is -O-, -S-, or a covalent bond; Y 1 is a linking group attached to the 2' or 3' end of a nucleoside, nucleotide, or oligonucleotide, Y 2 is hydrogen, a phosphoramidite analog; an internucleotide linkage attached to the 5' end of a nucleoside, nucleotide, or oligonucleotide, or a linkage attached to a solid support; R 5 is adamantyl or saturated or unsaturated, linear or branched C 1-50 a hydrocarbon chain in which 0 to 10 methylene units are independently replaced by -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-; R is hydrogen, a suitable protecting group, or C 1-6 aliphatic; phenyl; a 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, which are optionally substituted groups.

[0244] In some embodiments, R 5 is selected from the following: [ka]

[0245] In some embodiments, R 5 teeth, [ka] It is.

[0246] In some embodiments, R 5 teeth, [ka] It is.

[0247] In some embodiments, the nucleotide sequence of the STAT3 targeting oligonucleotide comprises between 1 and 10 targeting ligands, in some embodiments, the nucleotide sequence comprises 1, 2, or 3 targeting ligands.

[0248] In some embodiments, the STAT3 targeting oligonucleotide is a double-stranded molecule. In some embodiments, the STAT3 targeting oligonucleotide is an RNAi molecule. In some embodiments, the STAT3 targeting double-stranded oligonucleotide comprises a stem loop. In some embodiments, the ligand is conjugated to any of the nucleotides in the stem loop. In some embodiments, the ligand is conjugated to the first nucleotide 5' to 3' in the stem loop. In some embodiments, the ligand is conjugated to the second nucleotide 5' to 3' in the stem loop. In some embodiments, the ligand is conjugated to the third nucleotide 5' to 3' in the stem loop. In some embodiments, the ligand is conjugated to the fourth nucleotide 5' to 3' in the stem loop. In some embodiments, the ligand is conjugated to one, two, three, or four of the nucleotides in the stem loop. In some embodiments, the ligand is conjugated to three of the nucleotides in the stem loop.

[0249] In some embodiments, the STAT3 targeting double-stranded oligonucleotide comprises a stem loop, and one or more lipids are conjugated to one or more nucleotides of the stem loop.In some embodiments, the STAT3 targeting double-stranded oligonucleotide comprises a stem loop, and one or more C16 lipids are conjugated to one or more nucleotides of the stem loop.In some embodiments, the STAT3 targeting double-stranded oligonucleotide comprises a stem loop, and one or more C18 lipids are conjugated to one or more nucleotides of the stem loop.

[0250] In some embodiments, the STAT3 targeting oligonucleotide comprises a 36 nucleotide sense strand with positions numbered 1-36 from 5' to 3'. In some embodiments, the STAT3 targeting oligonucleotide comprises a lipid conjugated to position 27 of the 36 nucleotide sense strand. In some embodiments, the STAT3 targeting oligonucleotide comprises a lipid conjugated to position 28 of the 36 nucleotide sense strand. In some embodiments, the STAT3 targeting oligonucleotide comprises a lipid conjugated to position 29 of the 36 nucleotide sense strand. In some embodiments, the STAT3 targeting oligonucleotide comprises a lipid conjugated to position 30 of the 36 nucleotide sense strand. In some embodiments, the 36 nucleotide sense strand forms a stem loop with the loop having positions 27-30. In some embodiments, lipids are conjugated to more than one position of the loop (e.g., positions 27 and 28 of the 36-nucleotide sense strand).

[0251] In some embodiments, the STAT3 targeting oligonucleotide comprises a C16 lipid conjugated to position 27 of the 36-nucleotide sense strand. In some embodiments, the STAT3 targeting oligonucleotide comprises a C16 lipid conjugated to position 28 of the 36-nucleotide sense strand. In some embodiments, the STAT3 targeting oligonucleotide comprises a C16 lipid conjugated to position 29 of the 36-nucleotide sense strand. In some embodiments, the STAT3 targeting oligonucleotide comprises a C16 lipid conjugated to position 30 of the 36-nucleotide sense strand. In some embodiments, the 36-nucleotide sense strand forms a stem-loop with the loop having positions 27-30. In some embodiments, the C16 lipid is conjugated to more than one position of the loop (e.g., positions 27 and 28 of the 36-nucleotide sense strand).

[0252] In some embodiments, the STAT3 targeting oligonucleotide comprises a C18 lipid conjugated to position 27 of the 36-nucleotide sense strand. In some embodiments, the STAT3 targeting oligonucleotide comprises a C18 lipid conjugated to position 28 of the 36-nucleotide sense strand. In some embodiments, the STAT3 targeting oligonucleotide comprises a C18 lipid conjugated to position 29 of the 36-nucleotide sense strand. In some embodiments, the STAT3 targeting oligonucleotide comprises a C18 lipid conjugated to position 30 of the 36-nucleotide sense strand. In some embodiments, the 36-nucleotide sense strand forms a stem-loop with the loop having positions 27-30. In some embodiments, the C18 lipid is conjugated to two or more positions of the loop (e.g., positions 27 and 28 of the 36-nucleotide sense strand).

[0253] In some embodiments, the STAT3 targeted oligonucleotide comprises an antisense strand of 15-30 nucleotides and a sense strand of 15-40 nucleotides, wherein the sense and antisense strands form a double-stranded region, wherein the antisense strand comprises a region of complementarity to a STAT3 mRNA target sequence expressed in immune cells associated with the tumor microenvironment, and wherein the sense strand comprises at its 3' end a stem-loop comprising a tetraloop comprising four nucleosides, wherein one or more of the four nucleosides are represented by the formula II-Ib: [ka] wherein B is selected from adenine and guanine nucleobases; R 5 is a hydrocarbon chain. In some embodiments, m is 1, X1 is O, and Y2 is an internucleotide linkage group attached to the 5' end of the nucleoside; Y is [ka] wherein Y1 is a linking group attached to the 2' or 3' end of the nucleotide, X2 is O, X3 is O, and R3 is H.

[0254] In some embodiments, the hydrocarbon chain is a C8-C30 hydrocarbon chain. In some embodiments, the hydrocarbon chain is a C16 hydrocarbon chain. In some embodiments, the C16 hydrocarbon chain is represented by: [ka] In some embodiments, the hydrocarbon chain is a C18 hydrocarbon chain. In some embodiments, the C18 hydrocarbon chain is represented by: [ka]

[0255] In some embodiments, the oligonucleotide comprises a sense strand comprising a sequence selected from SEQ ID NOs: 89-280, wherein the sense strand comprises a C18 lipid. In some embodiments, the 4 nucleosides of the tetraloop are numbered 1-4 from 5' to 3', and position 1 is represented by formula II-Ib. In some embodiments, position 2 is represented by formula II-Ib. In some embodiments, position 3 is represented by formula II-Ib. In some embodiments, position 4 is represented by formula II-Ib. In some embodiments, the sense strand is 36 nucleotides having positions numbered 1-36 from 5' to 3', the stem loop comprises nucleotides at positions 21-36, and one or more nucleosides at positions 27-30 are represented by formula II-Ib. In some embodiments, the antisense strand is 22 nucleotides.

[0256] Exemplary STAT3 Targeting Oligonucleotides In some embodiments, the oligonucleotides targeting STAT3 comprise a sense strand and an antisense strand as set forth in Tables 1, 2, 3, 8, 9, 10, 11, and 12, the oligonucleotides comprise a stem-loop structure having a double-stranded stem of about 2-6 base pairs and a loop of 3-4 nucleotides, and the sense strand and the antisense strand comprise a modification pattern as set forth in FIG. 1A or Example 7. In some embodiments, the oligonucleotides targeting STAT3 comprise a sense strand and an antisense strand as set forth in Tables 1, 2, 3, 8, 9, 10, 11, and 12, the oligonucleotides comprise a stem-loop structure having a double-stranded stem of about 2-6 base pairs and a loop of 3-4 nucleotides, the sense strand and the antisense strand comprise a modification pattern as set forth in FIG. 1A, and the antisense strand is modified with an oxymethylphosphonate at the 4' carbon of the 5' terminal nucleotide. In some embodiments, the oligonucleotides comprise a stem-loop comprising the nucleotide sequence of SEQ ID NO:86. In some embodiments, the oligonucleotide comprises a 6 base pair double stranded stem and a 4 nucleotide stem loop comprising 1, 2, 3 or 4 GalNAc conjugated nucleotides, In some embodiments, the GalNAc conjugated nucleotide comprises a monovalent GalNAc conjugated to an adenine nucleotide, referred to as [ademA-GalNAc] or 2'-aminodiethoxymethanol-adenine-GalNAc, as shown below. [ka]

[0257] In some embodiments, the stem loop comprises a double-stranded stem of six base pairs and a loop comprising the nucleotide sequence GAAA, wherein each adenine nucleotide is ademA-GalNAc.

[0258] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 9 and 10, respectively; (b) SEQ ID NOs: 37 and 38, respectively; (c) SEQ ID NOs: 65 and 66, respectively, and (d) an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 69 and 70, respectively.

[0259] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 9 and 10, respectively; (b) SEQ ID NOs: 37 and 38, respectively; (c) SEQ ID NOs: 65 and 66, respectively, and (d) a sense strand and an antisense strand each comprising a nucleotide sequence selected from SEQ ID NOs: 69 and 70, The sense and antisense strands are modified according to the following patterns: Sense strand: [mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX][mX][mX][mX][mX][mX] [mX][mX][mX][mX][mX][mX][mX][mX][mX][ademX-C#][mX][mX][mX][mX][mX][mX][mX][mX] This hybridizes to Antisense strand: [Mephosphonate-4O-mXs][fXs][fX][fX][fX][mX][fX][mX][fX][mX][mX][fX][mX][mX][mX][mX][mXs][mXs][mXs] (Key is provided in Table 7.) In some embodiments, C# is C16 or C18.

[0260] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 862 and 952, respectively; (b) SEQ ID NOs: 875 and 965, respectively; (c) SEQ ID NOs: 876 and 966, respectively; and (d) a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 920 and 1010, respectively; The sense and antisense strands are modified according to the following patterns: Sense strand: [mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX][mX][mX][mX][mX][mX] [mX][mX][mX][mX][mX][mX][mX][mX][mX][ademX-C#][mX][mX][mX][mX][mX][mX][mX][mX] This hybridizes to Antisense strand: [Mephosphonate-4O-mXs][fXs][fX][fX][fX][mX][fX][mX][fX][mX][mX][fX][mX][mX][mX][mX][mXs][mXs][mXs] (Key is provided in Table 7.) In some embodiments, C# is C16 or C18.

[0261] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 862 and 952, respectively; (b) SEQ ID NOs: 875 and 965, respectively; (c) SEQ ID NOs: 876 and 966, respectively; and (d) a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 920 and 1010, respectively; The sense and antisense strands are modified according to the following patterns: Sense strand: [mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX][mX][mX][mX][mX][mX] [mX][mX][mX][mX][mX][mX][mX][mX][mX][ademX-C#][mX][mX][mX][mX][mX][mX][mX][mX] This hybridizes to Antisense strand: [Mephosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][fX][mX][mX][fX][mX][mX][mX][mX][mXs][mXs][mXs] (Key is provided in Table 7.) In some embodiments, C# is C16 or C18.

[0262] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 11 and 12, respectively; (b) SEQ ID NOs: 39 and 40, respectively; (c) SEQ ID NOs: 67 and 68, respectively, and (d) comprising a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 71 and 72, respectively.

[0263] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises the sequence set forth in SEQ ID NO: 81. In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises the sequence set forth in SEQ ID NO: 82. In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises the sequence set forth in SEQ ID NO: 83. In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises the sequence set forth in SEQ ID NO: 84.

[0264] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises a sense strand and an antisense strand having the nucleotide sequences set forth in SEQ ID NOs: 87 and 68, respectively. In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises a sense strand and an antisense strand having the nucleotide sequences set forth in SEQ ID NOs: 88 and 71, respectively.

[0265] In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises a sense strand sequence selected from SEQ ID NOs: 89-280. In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises a sense strand sequence selected from SEQ ID NOs: 857-946. In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises a sense strand sequence selected from SEQ ID NOs: 857-888. In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises a sense strand sequence selected from SEQ ID NOs: 889-912. In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises a sense strand sequence selected from SEQ ID NOs: 913-934. In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises a sense strand sequence selected from SEQ ID NOs: 935-946.

[0266] In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises an antisense strand sequence selected from SEQ ID NOs: 947-1036. In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises an antisense strand sequence selected from SEQ ID NOs: 947-978. In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises an antisense strand sequence selected from SEQ ID NOs: 979-1002. In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises an antisense strand sequence selected from SEQ ID NOs: 1003-1024. In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises an antisense strand sequence selected from SEQ ID NOs: 1025-1036.

[0267] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises a sense strand sequence selected from SEQ ID NOs: 857-946, and an antisense strand selected from SEQ ID NOs: 947-1036. In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises a sense strand sequence selected from SEQ ID NOs: 857-888, and an antisense strand selected from SEQ ID NOs: 947-978. In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises a sense strand sequence selected from SEQ ID NOs: 889-912, and an antisense strand selected from SEQ ID NOs: 979-1002. In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises a sense strand sequence selected from SEQ ID NOs: 913-934, and an antisense strand selected from SEQ ID NOs: 1003-1024. In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises a sense strand sequence selected from SEQ ID NOs: 935-946, and an antisense strand selected from SEQ ID NOs: 1025-1036.

[0268] In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises a sense strand sequence selected from SEQ ID NOs: 1037-1126. In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises a sense strand sequence selected from SEQ ID NOs: 1037-1068. In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises a sense strand sequence selected from SEQ ID NOs: 1069-1092. In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises a sense strand sequence selected from SEQ ID NOs: 1093-1114. In some embodiments, the oligonucleotide for reducing STAT3 mRNA expression comprises a sense strand sequence selected from SEQ ID NOs: 1115-1126.

[0269] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises an antisense strand sequence selected from SEQ ID NOs: 1127-1216. In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises an antisense strand sequence selected from SEQ ID NOs: 1127-1158. In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises an antisense strand sequence selected from SEQ ID NOs: 1159-1182. In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises an antisense strand sequence selected from SEQ ID NOs: 1183-1204. In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises an antisense strand sequence selected from SEQ ID NOs: 1205-1216.

[0270] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises a sense strand sequence selected from SEQ ID NOs: 1037-1126, and an antisense strand selected from SEQ ID NOs: 1127-1216. In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises a sense strand sequence selected from SEQ ID NOs: 1037-1068, and an antisense strand selected from SEQ ID NOs: 1127-1182. In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises a sense strand sequence selected from SEQ ID NOs: 1069-1092, and an antisense strand selected from SEQ ID NOs: 1159-1182. In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises a sense strand sequence selected from SEQ ID NOs: 1093-1114, and an antisense strand selected from SEQ ID NOs: 1183-1204. In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises a sense strand sequence selected from SEQ ID NOs: 1115-1126, and an antisense strand selected from SEQ ID NOs: 1205-1216.

[0271] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 857 and 947, respectively; (b) SEQ ID NOs: 858 and 948, respectively; (c) SEQ ID NOs: 859 and 949, respectively; (d) SEQ ID NOs: 860 and 950, respectively; (e) SEQ ID NOs: 862 and 952, respectively; (f) SEQ ID NOs: 867 and 957, respectively; (g) SEQ ID NOs: 875 and 965, respectively; and (h) comprising a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 876 and 966, respectively.

[0272] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 901 and 991, respectively; (b) SEQ ID NOs: 910 and 1000, respectively; (c) SEQ ID NOs: 899 and 989, respectively; (d) SEQ ID NOs: 896 and 986, respectively; (e) SEQ ID NOs: 892 and 982, respectively; (f) SEQ ID NOs: 890 and 980, respectively; and (g) comprising a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 889 and 979, respectively.

[0273] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 940 and 1030, respectively; (b) SEQ ID NOs: 937 and 1027, respectively, and (c) comprising a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 939 and 1029, respectively.

[0274] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 915 and 1005, respectively; (b) SEQ ID NOs: 924 and 1014, respectively; (c) SEQ ID NOs: 913 and 1003, respectively; and (d) comprising a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 920 and 1010, respectively.

[0275] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 862 and 952, respectively; (b) SEQ ID NOs: 875 and 965, respectively; (c) SEQ ID NOs: 876 and 966, respectively; and (d) comprising a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 920 and 1010, respectively.

[0276] In some embodiments, the sense strand comprises the sequence of SEQ ID NO:862 and the antisense strand comprises the sequence of SEQ ID NO:952.

[0277] In some embodiments, the sense strand comprises the sequence of SEQ ID NO:875 and the antisense strand comprises the sequence of SEQ ID NO:965.

[0278] In some embodiments, the sense strand comprises the sequence of SEQ ID NO:876 and the antisense strand comprises the sequence of SEQ ID NO:966.

[0279] In some embodiments, the sense strand comprises the sequence of SEQ ID NO:920 and the antisense strand comprises the sequence of SEQ ID NO:1010.

[0280] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 1037 and 1127, respectively; (b) SEQ ID NOs: 1038 and 1128, respectively; (c) SEQ ID NOs: 1039 and 1129, respectively; (d) SEQ ID NOs: 1040 and 1130, respectively; (e) SEQ ID NOs: 1042 and 1132, respectively; (f) SEQ ID NOs: 1047 and 1137, respectively; (g) SEQ ID NOs: 1055 and 1145, respectively; and (h) comprising a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 1056 and 1146, respectively.

[0281] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 1081 and 1171, respectively; (b) SEQ ID NOs: 1090 and 1180, respectively; (c) SEQ ID NOs: 1079 and 1169, respectively; (d) SEQ ID NOs: 1076 and 1166, respectively; (e) SEQ ID NOs: 1072 and 1162, respectively; (f) SEQ ID NOs: 1070 and 1160, respectively; and (g) comprising a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 1069 and 1159, respectively.

[0282] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 1120 and 1210, respectively; (b) SEQ ID NOs: 1117 and 1207, respectively, and (c) comprising a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 1119 and 1209, respectively.

[0283] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 1095 and 1185, respectively; (b) SEQ ID NOs: 1104 and 1194, respectively; (c) SEQ ID NOs: 1093 and 1183, respectively; and (d) comprising a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 1100 and 1190, respectively.

[0284] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 1042 and 1132, respectively; (b) SEQ ID NOs: 1055 and 1145, respectively; (c) SEQ ID NOs: 1056 and 1146, respectively; and (d) comprising a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 1100 and 1190, respectively.

[0285] In some embodiments, the sense strand comprises the sequence of SEQ ID NO:1042 and the antisense strand comprises the sequence of SEQ ID NO:1132.

[0286] In some embodiments, the sense strand comprises the sequence of SEQ ID NO:1055 and the antisense strand comprises the sequence of SEQ ID NO:1145.

[0287] In some embodiments, the sense strand comprises the sequence of SEQ ID NO:1056 and the antisense strand comprises the sequence of SEQ ID NO:1146.

[0288] In some embodiments, the sense strand comprises the sequence of SEQ ID NO:1100 and the antisense strand comprises the sequence of SEQ ID NO:1190.

[0289] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 1042 and 4, respectively; (b) SEQ ID NOs: 1055 and 5, respectively; (c) SEQ ID NOs: 1056 and 6, respectively, and (d) comprising a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 1100 and 7, respectively.

[0290] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA described herein comprises minimal off-target effects. For example, in some embodiments, the oligonucleotide described herein reduces STAT3 expression and does not reduce STAT1 expression, or reduces STAT1 expression less than STAT3 expression. In some embodiments, the oligonucleotide comprises a sense strand comprising the nucleotide sequence described in SEQ ID NO: 862, and an antisense strand comprising the nucleotide sequence described in SEQ ID NO: 952, and the oligonucleotide reduces STAT3 expression and does not reduce STAT1 expression, or reduces STAT1 expression less than STAT3 expression. In some embodiments, the oligonucleotide comprises a sense strand comprising the nucleotide sequence described in SEQ ID NO: 1042, and an antisense strand comprising the nucleotide sequence described in SEQ ID NO: 1132, and the oligonucleotide reduces STAT3 expression and does not reduce STAT1 expression, or reduces STAT1 expression less than STAT3 expression. In some embodiments, the oligonucleotide comprises a sense strand comprising the nucleotide sequence described in SEQ ID NO: 875, and an antisense strand comprising the nucleotide sequence described in SEQ ID NO: 965, and the oligonucleotide reduces STAT3 expression and does not reduce STAT1 expression, or reduces STAT1 expression less than STAT3 expression. In some embodiments, the oligonucleotide comprises a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1055 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1145, and the oligonucleotide reduces STAT3 expression and does not reduce STAT1 expression or reduces STAT1 expression less than STAT3 expression.

[0291] In some embodiments, the oligonucleotides for reducing the expression of STAT3 mRNA described herein are species cross-reactive oligonucleotides. In some embodiments, the oligonucleotides described herein can reduce the expression of STAT3 mRNA in at least two different species. In some embodiments, the oligonucleotides described herein can reduce the expression of STAT3 mRNA in at least two different species, but do not cross-react with non-STAT3 mRNA (e.g., STAT1). In some embodiments, the oligonucleotides for reducing the expression of STAT3 mRNA are cross-reactive between at least two species. In some embodiments, the oligonucleotides for reducing the expression of STAT3 cross-react with human, non-human primate, and mouse STAT3 mRNA. In some embodiments, the oligonucleotides for reducing the expression of STAT3 mRNA cross-react with human and mouse STAT3 mRNA. In some embodiments, the oligonucleotides for reducing the expression of STAT3 mRNA cross-react with human and non-human primate STAT3 mRNA.

[0292] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA reduces STAT3 mRNA by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0293] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA reduces STAT3 mRNA by at least 50% to at least 75% in humans, non-human primates, and mice (i.e., the oligonucleotide is a species cross-reactive oligonucleotide). In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA reduces STAT3 mRNA by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% in humans, non-human primates, and mice (i.e., the oligonucleotide is a species cross-reactive oligonucleotide). In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA reduces STAT3 mRNA by at least 80%, at least 85%, at least 90%, or at least 95% in humans, non-human primates, and mice (i.e., the oligonucleotide is a species cross-reactive oligonucleotide).

[0294] In some embodiments, the oligonucleotides for reducing the expression of STAT3 mRNA reduce STAT3 mRNA by at least 50% to at least 75% in humans and non-human primates (i.e., the oligonucleotides are species cross-reactive oligonucleotides). In some embodiments, the oligonucleotides for reducing the expression of STAT3 mRNA reduce STAT3 mRNA by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% in humans and non-human primates (i.e., the oligonucleotides are species cross-reactive oligonucleotides). In some embodiments, the oligonucleotides for reducing the expression of STAT3 mRNA reduce STAT3 mRNA by at least 80%, at least 85%, at least 90%, or at least 95% in humans and non-human primates (i.e., the oligonucleotides are species cross-reactive oligonucleotides).

[0295] In some embodiments, the oligonucleotides for reducing STAT3 mRNA expression reduce STAT3 mRNA by at least 50% to at least 75% in humans and mice (i.e., the oligonucleotides are species cross-reactive oligonucleotides). In some embodiments, the oligonucleotides for reducing STAT3 mRNA expression reduce STAT3 mRNA by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% in humans and mice (i.e., the oligonucleotides are species cross-reactive oligonucleotides). In some embodiments, the oligonucleotides for reducing STAT3 mRNA expression reduce STAT3 mRNA by at least 80%, at least 85%, at least 90%, or at least 9 ... are (a) SEQ ID NOs: 901 and 991, respectively; (b) SEQ ID NOs: 910 and 1000, respectively; (c) SEQ ID NOs: 899 and 989, respectively; (d) SEQ ID NOs: 896 and 986, respectively; (e) SEQ ID NOs: 892 and 982, respectively; (f) SEQ ID NOs: 890 and 980, respectively; and (g) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 889 and 979, respectively; The oligonucleotides reduce STAT3 mRNA in humans, non-human primates, and mice (ie, the oligonucleotides are species cross-reactive oligonucleotides).

[0296] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 940 and 1030, respectively; (b) SEQ ID NOs: 937 and 1027, respectively, and (c) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 939 and 1029, respectively; The oligonucleotides reduce STAT3 mRNA in humans, non-human primates, and mice (ie, the oligonucleotides are species cross-reactive oligonucleotides).

[0297] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 915 and 1005, respectively; (b) SEQ ID NOs: 924 and 1014, respectively; (c) SEQ ID NOs: 913 and 1003, respectively; and (d) sense and antisense strands selected from SEQ ID NOs: 920 and 1010, respectively; The oligonucleotides reduce STAT3 mRNA in humans, non-human primates, and mice (ie, the oligonucleotides are species cross-reactive oligonucleotides).

[0298] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 862 and the antisense strand sequence of SEQ ID NO: 952, and the oligonucleotide reduces STAT3 mRNA in humans and non-human primates (i.e., the oligonucleotide is a species cross-reactive oligonucleotide).

[0299] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 875 and the antisense strand sequence of SEQ ID NO: 965, wherein the oligonucleotide reduces STAT3 mRNA in humans.

[0300] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 876 and the antisense strand sequence of SEQ ID NO: 966, wherein the oligonucleotide reduces STAT3 mRNA in humans.

[0301] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 920 and the antisense strand sequence of SEQ ID NO: 1010, wherein the oligonucleotide reduces STAT3 mRNA in humans.

[0302] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 857 and 947, respectively; (b) SEQ ID NOs: 858 and 948, respectively; (c) SEQ ID NOs: 859 and 949, respectively; (d) SEQ ID NOs: 860 and 950, respectively; (e) SEQ ID NOs: 862 and 952, respectively; (f) SEQ ID NOs: 867 and 957, respectively; (g) SEQ ID NOs: 875 and 965, respectively; and (h) a sense strand and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 876 and 966, respectively; The oligonucleotides reduce STAT3 mRNA by at least 75%.

[0303] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 901 and 991, respectively; (b) SEQ ID NOs: 910 and 1000, respectively; (c) SEQ ID NOs: 899 and 989, respectively; (d) SEQ ID NOs: 896 and 986, respectively; (e) SEQ ID NOs: 892 and 982, respectively; (f) SEQ ID NOs: 890 and 980, respectively; and (g) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 889 and 979, respectively; The oligonucleotides reduce STAT3 mRNA by at least 75%.

[0304] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 940 and 1030, respectively; (b) SEQ ID NOs: 937 and 1027, respectively, and (c) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 939 and 1029, respectively; The oligonucleotides reduce STAT3 mRNA by at least 75%.

[0305] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 915 and 1005, respectively; (b) SEQ ID NOs: 924 and 1014, respectively; (c) SEQ ID NOs: 913 and 1003, respectively; and (d) sense and antisense strands selected from SEQ ID NOs: 920 and 1010, respectively; The oligonucleotides reduce STAT3 mRNA by at least 75%.

[0306] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 862 and 952, respectively; (b) SEQ ID NOs: 875 and 965, respectively; (c) SEQ ID NOs: 876 and 966, respectively; and (d) sense and antisense strands selected from SEQ ID NOs: 920 and 1010, respectively; The oligonucleotides reduce STAT3 mRNA by at least 75%.

[0307] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO:862 and the antisense strand sequence of SEQ ID NO:952, wherein the oligonucleotide reduces STAT3 mRNA by at least 75%.

[0308] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO:875 and the antisense strand sequence of SEQ ID NO:965, wherein the oligonucleotide reduces STAT3 mRNA by at least 75%.

[0309] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO:876 and the antisense strand sequence of SEQ ID NO:966, wherein the oligonucleotide reduces STAT3 mRNA by at least 75%.

[0310] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 920 and the antisense strand sequence of SEQ ID NO: 1010, wherein the oligonucleotide reduces STAT3 mRNA by at least 75%.

[0311] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 857 and 947, respectively; (b) SEQ ID NOs: 858 and 948, respectively; (c) SEQ ID NOs: 859 and 949, respectively; (d) SEQ ID NOs: 860 and 950, respectively; (e) SEQ ID NOs: 862 and 952, respectively; (f) SEQ ID NOs: 867 and 957, respectively; (g) SEQ ID NOs: 875 and 965, respectively; and (h) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 876 and 966, respectively; The oligonucleotide is conjugated to a lipid.

[0312] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 901 and 991, respectively; (b) SEQ ID NOs: 910 and 1000, respectively; (c) SEQ ID NOs: 899 and 989, respectively; (d) SEQ ID NOs: 896 and 986, respectively; (e) SEQ ID NOs: 892 and 982, respectively; (f) SEQ ID NOs: 890 and 980, respectively; and (g) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 889 and 979, respectively; The oligonucleotide is conjugated to a lipid on the sense strand.

[0313] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 940 and 1030, respectively; (b) SEQ ID NOs: 937 and 1027, respectively, and (c) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 939 and 1029, respectively; The oligonucleotide is conjugated to a lipid on the sense strand.

[0314] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 915 and 1005, respectively; (b) SEQ ID NOs: 924 and 1014, respectively; (c) SEQ ID NOs: 913 and 1003, respectively; and (d) sense and antisense strands selected from SEQ ID NOs: 920 and 1010, respectively; The oligonucleotide is conjugated to a lipid on the sense strand.

[0315] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 862 and 952, respectively; (b) SEQ ID NOs: 875 and 965, respectively; (c) SEQ ID NOs: 876 and 966, respectively; and (d) sense and antisense strands selected from SEQ ID NOs: 920 and 1010, respectively; The oligonucleotide is conjugated to a lipid on the sense strand.

[0316] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 862 and the antisense strand sequence of SEQ ID NO: 952, and the oligonucleotide is conjugated to a lipid on the sense strand.

[0317] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises a sense strand sequence of SEQ ID NO: 875 and an antisense strand sequence of SEQ ID NO: 965, and the oligonucleotide is conjugated to a lipid on the sense strand.

[0318] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 876 and the antisense strand sequence of SEQ ID NO: 966, and the oligonucleotide is conjugated to a lipid on the sense strand.

[0319] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises a sense strand sequence of SEQ ID NO: 920 and an antisense strand sequence of SEQ ID NO: 1010, and the oligonucleotide is conjugated to a lipid on the sense strand.

[0320] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 857 and 947, respectively; (b) SEQ ID NOs: 858 and 948, respectively; (c) SEQ ID NOs: 859 and 949, respectively; (d) SEQ ID NOs: 860 and 950, respectively; (e) SEQ ID NOs: 862 and 952, respectively; (f) SEQ ID NOs: 867 and 957, respectively; (g) SEQ ID NOs: 875 and 965, respectively; and (h) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 876 and 966, respectively; The oligonucleotide is conjugated to a C18 lipid on the sense strand.

[0321] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 901 and 991, respectively; (b) SEQ ID NOs: 910 and 1000, respectively; (c) SEQ ID NOs: 899 and 989, respectively; (d) SEQ ID NOs: 896 and 986, respectively; (e) SEQ ID NOs: 892 and 982, respectively; (f) SEQ ID NOs: 890 and 980, respectively; and (g) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 889 and 979, respectively; The oligonucleotide is conjugated to a C18 lipid on the sense strand.

[0322] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 940 and 1030, respectively; (b) SEQ ID NOs: 937 and 1027, respectively, and (c) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 939 and 1029, respectively; The oligonucleotide is conjugated to a C18 lipid on the sense strand.

[0323] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 915 and 1005, respectively; (b) SEQ ID NOs: 924 and 1014, respectively; (c) SEQ ID NOs: 913 and 1003, respectively; and (d) sense and antisense strands selected from SEQ ID NOs: 920 and 1010, respectively; The oligonucleotide is conjugated to a C18 lipid on the sense strand.

[0324] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 862 and 952, respectively; (b) SEQ ID NOs: 875 and 965, respectively; (c) SEQ ID NOs: 876 and 966, respectively; and (d) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 920 and 1010, respectively; The oligonucleotide is conjugated to a C18 lipid on the sense strand.

[0325] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises a sense strand sequence of SEQ ID NO: 862 and an antisense strand sequence of SEQ ID NO: 952, wherein the oligonucleotide is conjugated to a C18 lipid on the sense strand.

[0326] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises a sense strand sequence of SEQ ID NO: 875 and an antisense strand sequence of SEQ ID NO: 965, wherein the oligonucleotide is conjugated to a C18 lipid on the sense strand.

[0327] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises a sense strand sequence of SEQ ID NO: 876 and an antisense strand sequence of SEQ ID NO: 966, wherein the oligonucleotide is conjugated to a C18 lipid on the sense strand.

[0328] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises a sense strand sequence of SEQ ID NO: 920 and an antisense strand sequence of SEQ ID NO: 1010, and the oligonucleotide is conjugated to a C18 lipid on the sense strand.

[0329] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 901 and 991, respectively; (b) SEQ ID NOs: 910 and 1000, respectively; (c) SEQ ID NOs: 899 and 989, respectively; (d) SEQ ID NOs: 896 and 986, respectively; (e) SEQ ID NOs: 892 and 982, respectively; (f) SEQ ID NOs: 890 and 980, respectively; and (g) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 889 and 979, respectively; The oligonucleotides reduce STAT3 mRNA by at least 75% in humans, non-human primates, and mice (ie, the oligonucleotides are species cross-reactive oligonucleotides).

[0330] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 940 and 1030, respectively; (b) SEQ ID NOs: 937 and 1027, respectively, and (c) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 939 and 1029, respectively; The oligonucleotides reduce STAT3 mRNA by at least 75% in humans and mice (ie, the oligonucleotides are species cross-reactive oligonucleotides).

[0331] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 915 and 1005, respectively; (b) SEQ ID NOs: 924 and 1014, respectively; (c) SEQ ID NOs: 913 and 1003, respectively; and (d) sense and antisense strands selected from SEQ ID NOs: 920 and 1010, respectively; The oligonucleotides reduce STAT3 mRNA by at least 75% in humans.

[0332] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 862 and the antisense strand sequence of SEQ ID NO: 952, and the oligonucleotide reduces STAT3 mRNA by at least 75% in humans and non-human primates (i.e., the oligonucleotide is a species cross-reactive oligonucleotide).

[0333] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 875 and the antisense strand sequence of SEQ ID NO: 965, wherein the oligonucleotide reduces STAT3 mRNA by at least 75% in humans.

[0334] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 876 and the antisense strand sequence of SEQ ID NO: 966, wherein the oligonucleotide reduces STAT3 mRNA by at least 75% in humans.

[0335] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 920 and the antisense strand sequence of SEQ ID NO: 1010, wherein the oligonucleotide reduces STAT3 mRNA by at least 75% in humans.

[0336] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 901 and 991, respectively; (b) SEQ ID NOs: 910 and 1000, respectively; (c) SEQ ID NOs: 899 and 989, respectively; (d) SEQ ID NOs: 896 and 986, respectively; (e) SEQ ID NOs: 892 and 982, respectively; (f) SEQ ID NOs: 890 and 980, respectively; and (g) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 889 and 979, respectively; The oligonucleotide is conjugated to a lipid on the sense strand and reduces STAT3 mRNA in humans, non-human primates, and mice (ie, the oligonucleotide is a species cross-reactive oligonucleotide).

[0337] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 940 and 1030, respectively; (b) SEQ ID NOs: 937 and 1027, respectively, and (c) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 939 and 1029, respectively; The oligonucleotide is conjugated to a lipid on the sense strand and reduces STAT3 mRNA in humans and mice (ie, the oligonucleotide is a species cross-reactive oligonucleotide).

[0338] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 915 and 1005, respectively; (b) SEQ ID NOs: 924 and 1014, respectively; (c) SEQ ID NOs: 913 and 1003, respectively; and (d) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 920 and 1010, respectively; The oligonucleotide is conjugated to a lipid on the sense strand and reduces STAT3 mRNA in humans.

[0339] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 862 and the antisense strand sequence of SEQ ID NO: 952, wherein the oligonucleotide is conjugated to a lipid on the sense strand and reduces STAT3 mRNA in humans and non-human primates (i.e., the oligonucleotide is a species cross-reactive oligonucleotide).

[0340] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 875 and the antisense strand sequence of SEQ ID NO: 965, wherein the oligonucleotide is conjugated to a lipid on the sense strand and reduces STAT3 mRNA in humans.

[0341] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 876 and the antisense strand sequence of SEQ ID NO: 966, wherein the oligonucleotide is conjugated to a lipid on the sense strand and reduces STAT3 mRNA in humans.

[0342] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 920 and the antisense strand sequence of SEQ ID NO: 1010, wherein the oligonucleotide is conjugated to a lipid on the sense strand and reduces STAT3 mRNA in humans.

[0343] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 901 and 991, respectively; (b) SEQ ID NOs: 910 and 1000, respectively; (c) SEQ ID NOs: 899 and 989, respectively; (d) SEQ ID NOs: 896 and 986, respectively; (e) SEQ ID NOs: 892 and 982, respectively; (f) SEQ ID NOs: 890 and 980, respectively; and (g) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 889 and 979, respectively; The oligonucleotide is conjugated to a C18 lipid on the sense strand and reduces STAT3 mRNA in humans, non-human primates, and mice (ie, the oligonucleotide is a species cross-reactive oligonucleotide).

[0344] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 940 and 1030, respectively; (b) SEQ ID NOs: 937 and 1027, respectively, and (c) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 939 and 1029, respectively; The oligonucleotide is conjugated to a C18 lipid on the sense strand and reduces STAT3 mRNA in humans and mice (ie, the oligonucleotide is a species cross-reactive oligonucleotide).

[0345] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 915 and 1005, respectively; (b) SEQ ID NOs: 924 and 1014, respectively; (c) SEQ ID NOs: 913 and 1003, respectively; and (d) sense and antisense strands selected from SEQ ID NOs: 920 and 1010, respectively; The oligonucleotide is conjugated to a C18 lipid on the sense strand and reduces STAT3 mRNA in humans.

[0346] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 862 and the antisense strand sequence of SEQ ID NO: 952, wherein the oligonucleotide is conjugated to a C18 lipid on the sense strand and reduces STAT3 mRNA in humans and non-human primates (i.e., the oligonucleotide is a species cross-reactive oligonucleotide).

[0347] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 875 and the antisense strand sequence of SEQ ID NO: 965, wherein the oligonucleotide is conjugated to C18 on the sense strand lipid, and reduces STAT3 mRNA in humans.

[0348] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 876 and the antisense strand sequence of SEQ ID NO: 966, wherein the oligonucleotide is conjugated to a C18 lipid on the sense strand and reduces STAT3 mRNA in humans.

[0349] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 920 and the antisense strand sequence of SEQ ID NO: 1010, wherein the oligonucleotide is conjugated to a C18 lipid on the sense strand and reduces STAT3 mRNA in humans.

[0350] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 901 and 991, respectively; (b) SEQ ID NOs: 910 and 1000, respectively; (c) SEQ ID NOs: 899 and 989, respectively; (d) SEQ ID NOs: 896 and 986, respectively; (e) SEQ ID NOs: 892 and 982, respectively; (f) SEQ ID NOs: 890 and 980, respectively; and (g) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 889 and 979, respectively; The oligonucleotide is conjugated to a lipid on the sense strand and reduces STAT3 mRNA by at least 75% in humans, non-human primates, and mice (ie, the oligonucleotide is a species cross-reactive oligonucleotide).

[0351] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 940 and 1030, respectively; (b) SEQ ID NOs: 937 and 1027, respectively, and (c) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 939 and 1029, respectively; The oligonucleotide is conjugated to a lipid on the sense strand and reduces STAT3 mRNA by at least 75% in humans and mice (ie, the oligonucleotide is a species cross-reactive oligonucleotide).

[0352] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 915 and 1005, respectively; (b) SEQ ID NOs: 924 and 1014, respectively; (c) SEQ ID NOs: 913 and 1003, respectively; and (d) sense and antisense strands selected from SEQ ID NOs: 920 and 1010, respectively; The oligonucleotide is conjugated to a lipid on the sense strand and reduces STAT3 mRNA by at least 75% in humans.

[0353] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 862 and the antisense strand sequence of SEQ ID NO: 952, wherein the oligonucleotide is conjugated to a lipid on the sense strand and reduces STAT3 mRNA by at least 75% in humans and non-human primates (i.e., the oligonucleotide is a species cross-reactive oligonucleotide).

[0354] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 875 and the antisense strand sequence of SEQ ID NO: 965, wherein the oligonucleotide is conjugated to a lipid on the sense strand, and reduces STAT3 mRNA by at least 75% in humans.

[0355] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 876 and the antisense strand sequence of SEQ ID NO: 966, wherein the oligonucleotide is conjugated to a lipid on the sense strand, and reduces STAT3 mRNA by at least 75% in humans.

[0356] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 920 and the antisense strand sequence of SEQ ID NO: 1010, wherein the oligonucleotide is conjugated to a lipid on the sense strand, and reduces STAT3 mRNA by at least 75% in humans.

[0357] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 901 and 991, respectively; (b) SEQ ID NOs: 910 and 1000, respectively; (c) SEQ ID NOs: 899 and 989, respectively; (d) SEQ ID NOs: 896 and 986, respectively; (e) SEQ ID NOs: 892 and 982, respectively; (f) SEQ ID NOs: 890 and 980, respectively; and (g) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 889 and 979, respectively; The oligonucleotide is conjugated to a C18 lipid on the sense strand and reduces STAT3 mRNA by at least 75% in humans, non-human primates, and mice (ie, the oligonucleotide is a species cross-reactive oligonucleotide).

[0358] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 940 and 1030, respectively; (b) SEQ ID NOs: 937 and 1027, respectively, and (c) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 939 and 1029, respectively; The oligonucleotide is conjugated to a C18 lipid on the sense strand and reduces STAT3 mRNA by at least 75% in humans and mice (ie, the oligonucleotide is a species cross-reactive oligonucleotide).

[0359] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises: (a) SEQ ID NOs: 915 and 1005, respectively; (b) SEQ ID NOs: 924 and 1014, respectively; (c) SEQ ID NOs: 913 and 1003, respectively; and (d) comprising a sense strand and an antisense strand selected from SEQ ID NOs: 920 and 1010, respectively; The oligonucleotide is conjugated to a C18 lipid on the sense strand and reduces STAT3 mRNA by at least 75% in humans.

[0360] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 862 and the antisense strand sequence of SEQ ID NO: 952, wherein the oligonucleotide is conjugated to a C18 lipid on the sense strand and reduces STAT3 mRNA by at least 75% in humans and non-human primates (i.e., the oligonucleotide is a species cross-reactive oligonucleotide).

[0361] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 875 and the antisense strand sequence of SEQ ID NO: 965, wherein the oligonucleotide is conjugated to a C18 lipid on the sense strand, and reduces STAT3 mRNA by at least 75% in humans.

[0362] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 876 and the antisense strand sequence of SEQ ID NO: 966, wherein the oligonucleotide is conjugated to a C18 lipid on the sense strand, and reduces STAT3 mRNA by at least 75% in humans.

[0363] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 920 and the antisense strand sequence of SEQ ID NO: 1010, wherein the oligonucleotide is conjugated to a C18 lipid on the sense strand, and reduces STAT3 mRNA by at least 75% in humans.

[0364] formulation In order to facilitate the use of oligonucleotide, various formulations have been developed.For example, oligonucleotide can be delivered to subject or cellular environment using formulations that minimize degradation, facilitate delivery and / or uptake, or provide other beneficial properties to the oligonucleotide in the formulation.In some embodiments, oligonucleotide is formulated in buffer such as phosphate buffered saline, liposome, micelle structure, and capsid.

[0365] Formulations of oligonucleotides containing cationic lipids can be used to facilitate the transfection of oligonucleotides into cells. For example, cationic lipids such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine) can be used. Suitable lipids include oligofectamine, lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche), all of which can be used according to the manufacturer's instructions.

[0366] Thus, in some embodiments, the formulation comprises lipid nanoparticles. In some embodiments, the excipient comprises liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, or nanoparticles, or may be otherwise formulated for administration to the subject's cell, tissue, organ, or body in need of administration (see, for example, Remington: THE SCIENCE AND PRACTICE OF PHARMACY, 22nd edition, Pharmaceutical Press, 2013).

[0367] In some embodiments, the formulation herein comprises an excipient. In some embodiments, the excipient provides the composition with improved stability, improved absorption, improved solubility, and / or therapeutic enhancement of the active ingredient. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, Tris base, or sodium hydroxide), or a vehicle (e.g., buffer solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotide is lyophilized to extend its shelf life, and then put into solution before use (e.g., administration to a subject). Thus, the excipient in the composition comprising any one of the oligonucleotides described herein can be a cryoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone), or a disintegration temperature regulator (e.g., dextran, Ficoll™, or gelatin).

[0368] In some embodiments, a pharmaceutical composition is formulated to be compatible with its intended route of administration. Exemplary routes of administration include parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.

[0369] Pharmaceutical compositions suitable for injection use include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). Carriers can be, for example, solvents or dispersion media containing water, ethanol, polyols (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols, for example, mannitol, sorbitol, sodium chloride. Sterile injectable solutions can be prepared by incorporating the required amount of oligonucleotide into a selected solvent containing one or a combination of the above-listed ingredients as needed, followed by filtered sterilization.

[0370] In some embodiments, the composition may contain at least about 0.1% or more of a therapeutic agent, although the percentage of active ingredient may be from about 1% to about 80% or more by weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimens may be desirable.

[0371] Some embodiments are directed to liver-targeted delivery of any of the oligonucleotides herein, although targeting of other tissues is also contemplated.

[0372] Programmed Death-Ligand 1 (PD-L1) Inhibitors In some embodiments, the present disclosure provides a PD-L1 inhibitor for use in combination with the oligonucleotides described herein. In some embodiments, the PD-L1 inhibitor is a small molecule, a peptide, a protein, an antibody, or a nucleic acid molecule such as siRNA, miRNA, or antisense RNA. In some embodiments, the PD-L1 inhibitor inhibits the association of PD-L1 and PD-1. In some embodiments, the PD-L1 inhibitor is specific for PD-L1.

[0373] In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the PD-L1 inhibitor is specific for PD-1. In some embodiments, the PD-L1 inhibitor is an anti-PD-1 antibody. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an antibody fragment. In some embodiments, the PD-L1 inhibitor is a small molecule.

[0374] In some embodiments, the anti-PD-L1 antibody is atezolizumab. Atezolizumab (MPDL3280A) is a fully human, engineered IgG1 monoclonal antibody against PD-L1. In some embodiments, the anti-PD-L1 antibody is avelumab. Avelumab (MSB0010718C) is a fully human, engineered IgG1 monoclonal antibody against PD-L1. In some embodiments, the anti-PD-L1 antibody is emvafolimab. In some embodiments, the anti-PD-L1 antibody is durvalumab. Duvalumab (MEDI4736) is a human monoclonal antibody against PD-L1. In some embodiments, the anti-PD-L1 antibody is TSR-042. TSR-042 refers to an engineered chimeric antibody directed against the PD-1 / PD-L1 pathway. In some embodiments, the anti-PD-L1 antibody is KD-033. KD-033 refers to a bifunctional anti-PD-L1 / IL-15 fusion protein, where the anti-PD-L1 antibody binds to the cytokine IL-15 through the sushi domain of the IL-15 receptor connected by the tail. In some embodiments, the anti-PD-L1 antibody is STI-1014. STI-1014 refers to an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is KY-1003. KY-1003 is a monoclonal antibody against PD-L1. In some embodiments, the anti-PD-L1 antibody is YW243.55.S70. The antibody YW243.55.S70 is an anti-PDL1 antibody described in U.S. Patent No. 9,920,123, the contents of which are incorporated herein in their entirety. In some embodiments, the anti-PD-L1 antibody is MDX-1106. MDX-1106, also known as MDX-1106-04, ONO-4538, or BMS-936558, is an anti-PD1 antibody described in U.S. Patent No. 8,008,449, the contents of which are incorporated herein in their entirety. In some embodiments, the anti-PD-L1 antibody is Merck 3745. Merck 3745, also known as MK-3475 or SCH-900475, is an anti-PD1 antibody described in U.S. Patent No. 8,168,757, the contents of which are incorporated herein in their entirety.In some embodiments, the anti-PD-L1 antibody is CT-011. CT-011, also known as hBAT or hBAT-1, is an anti-PD1 antibody described in U.S. Patent No. 8,747,847, the contents of which are incorporated herein in their entireties. In some embodiments, the anti-PD-L1 antibody is AMP-224. AMP-224, also known as B7-DC1g, is a PDL2-Fc fusion soluble receptor described in U.S. Publication No. 2011 / 0223188 and U.S. Publication No. 2013 / 0017199, the contents of which are incorporated herein in their entireties.

[0375] In some embodiments, the anti-PD-L1 antibody is any anti-PD-L1 antibody known in the art, including but not limited to those described in Akinleye & Rasool “Immune checkpoint inhibitors of PD-L1 as cancer therapeutics” J. of Hematology & Oncology. 12(92):2019. In some embodiments, the anti-PD-L1 antibody is BMS-936559. BMS-936559 (MDX-1105) is a fully human IgG4 monoclonal antibody against PD-L1 and is described in U.S. Patent No. 7,943,743, the contents of which are incorporated herein in their entirety. In some embodiments, the anti-PD-L1 antibody is CK-301. In some embodiments, the anti-PD-L1 antibody is CS-1001. In some embodiments, the anti-PD-L1 antibody is SHR-1316. In some embodiments, the anti-PD-L1 antibody is BG-A333. In some embodiments, the anti-PD-L1 antibody is an antibody described in WO 2021 / 231741, the contents of which are incorporated herein in their entirety.

[0376] In some embodiments, the anti-PD-1 antibody is nivolumab. Nivolumab (BMS-936558) is an IgG4 monoclonal antibody described in U.S. Patent No. 8,008,449, the contents of which are incorporated herein in their entirety. In some embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is an IgG4 monoclonal antibody described in U.S. Patent No. 8,354,509, the contents of which are incorporated herein in their entirety. In some embodiments, the anti-PD-1 antibody is cemiplimab. Cemiplimab (REGN2810) is an IgG4 monoclonal antibody described in U.S. Patent No. 9,987,500, the contents of which are incorporated herein in their entirety.

[0377] In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 30 nM to about 100 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 30 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 40 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 50 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 60 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 70 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 80 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 90 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 100 nM.

[0378] In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 30 nM to about 100 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 30 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 40 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 50 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 60 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 70 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 80 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 90 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 100 nM.

[0379] In some embodiments, the antibodies are generated using display techniques. Display techniques used to generate antibody polypeptides include any of the display techniques (e.g., display library screening techniques). In some embodiments, synthetic antibodies are designed, selected, or optimized by screening target antigens using display techniques (e.g., phage display techniques). Phage display libraries may contain millions to billions of phage vectors, each expressing a unique antibody fragment on their viral coat. Such libraries can provide a sufficiently diverse resource to be used to select potentially hundreds of antibody fragments with varying levels of affinity for one or more antigens of interest (McCafferty, et al., 1990. Nature. 348:552-4; Edwards, BM et al., 2003. JMB. 334:103-18; Schofield, D. et al., 2007. Genome Biol. 8, R254, and Pershad, K. et al., 2010. Protein Engineering Design and Selection. 23:279-88; the contents of each of which are incorporated herein by reference in their entirety). Often, the antibody fragments present in such libraries are comprised of V-linkers linked by flexible linkers. H Antibody domains and V LThe scFv antibody fragments include fusion proteins of antibody domains. In some cases, the scFvs may contain the same sequence, except for the unique sequence encoding the variable loop of the CDR. In some cases, the scFvs are expressed as fusion proteins bound to a viral coat protein (e.g., the N-terminus of the viral pill coat protein). The VL chain may be expressed separately for assembly with the VH chain in the periplasm before complex incorporation into the viral coat. The precipitated library members may be sequenced from the bound phage to obtain the cDNA encoding the desired scFv. The antibody variable domains or CDRs from such sequences may be directly incorporated into the antibody sequence for recombinant antibody production, or may be mutated and utilized for further optimization through in vitro affinity maturation.

[0380] In some embodiments, the sequence of the polypeptide encoded by the viral genome is produced using yeast surface display technology. In some embodiments, recombinant antibodies are developed by displaying the antibody fragment of interest as a fusion to the surface of yeast, where the protein interacts with proteins and small molecules in solution. The scFv with affinity for the desired receptor can be isolated from the yeast surface using magnetic separation and flow cytometry. Several cycles of yeast surface display and isolation may be performed to achieve the scFv with the desired properties through directed evolution.

[0381] Methods for determining the affinity of an antibody to its antigen are known in the art. An exemplary method for determining binding affinity uses surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that allows the analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further explanation, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnsson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0382] In some embodiments, the PD-L1 inhibitor is an siRNA molecule. In some embodiments, the PD-L1 inhibitor is ALN-PDL. In some embodiments, the PD-L1 inhibitor is a PD-L1 inhibitor described in U.S. Patent No. 10,889,813, U.S. Patent No. 10,745,704, U.S. Publication No. 2021 / 0277403, U.S. Publication No. 2006 / 0276422A1, U.S. Publication No. 2021 / 0277403, International Publication No. 2019 / 000149, or International Publication No. 2019 / 000149, the contents of which are incorporated herein in their entirety. In some embodiments, the PD-L1 inhibitor is a siRNA molecule described in Barati, M., et al. A review of PD-1 / PD-L1 siRNA delivery systems in immune T cells and cancer cells, International Immunopharmacology, 2022, vol. 111. In some embodiments, the siRNA molecule for inhibition of PD-L1 is an siRNA molecule generated using methods known to those of skill in the art.

[0383] In some embodiments, the PD-L1 inhibitor is an antisense oligonucleotide. In some embodiments, the PD-L1 inhibitor is an antisense oligonucleotide described in U.S. Patent No. 10,745,480, U.S. Patent No. 10,982,215, U.S. Publication No. 2021 / 0269797A1, or U.S. Publication No. US2022 / 0220485A1, the contents of which are incorporated herein in their entirety. In some embodiments, the antisense oligonucleotide for inhibition of PD-L1 is an antisense oligonucleotide generated using methods known to those skilled in the art.

[0384] In some embodiments, the PD-L1 inhibitor is a small molecule. In some embodiments, the small molecule is CA-170. CA-170 is a small molecule antagonist of PD-L1 and VISTA. In some embodiments, the small molecule is CA-327. CA-327 refers to a small molecule antagonist of PD-L1 and TIM3. In some embodiments, the small molecule is BMS-1001. In some embodiments, the small molecule is BMS-1166. In some embodiments, the small molecule is BMS-8. In some embodiments, the small molecule is BMS-37. In some embodiments, the small molecule is BMS-202. In some embodiments, the small molecule is BMS-200. In some embodiments, the small molecule is Incyte-001. In some embodiments, the small molecule is Incyte-011. In some embodiments, the small molecule is INCB086550. In some embodiments, the small molecule is LH1306. In some embodiments, the small molecule is LH1307. In some embodiments, the small molecule is ARB-272572. In some embodiments, the PD-L1 inhibitor is a small molecule described in Sasikumar, P. et al. Small Molecule Agents Targeting PD-1 Checkpoint Pathway for Cancer Immunotherapy: Mechanisms of Action and Other Considerations for Their Advanced Development, Front. Immunol. 2022, 13:752065. In some embodiments, the PD-L1 inhibitor is a small molecule described in U.S. Pat. No. 11,130,740, U.S. Pat. No. 10,590,105, WO 2019 / 076343, U.S. Pat. No. 11,555,029, or U.S. Publication No. 2018 / 0305315, the contents of which are incorporated herein in their entirety.In some embodiments, the small molecule is a small molecule described in Wu, Q. et al. Small molecule inhibitors targeting the PD-1 / PD-L1 signaling pathway, Acta Pharmacologica Sinica. 2021, 42:1-9. In some embodiments, the small molecule is a small molecule described in Wang, Y. et al. A Small Molecule Antagonist of PD-1 / PD-L1 Interactions Acts as an Immune Checkpoint Inhibitor for NSCLC and Melanoma Immunotherapy, Front. Immuno. 2021. Vol. 12. In some embodiments, the small molecule inhibitor is a stereoisomer, tautomer, pharma- ceutically acceptable salt, hydrate, or solvate of a small molecule described herein. In some embodiments, the small molecule for inhibition of PD-L1 is a small molecule generated using methods known to those skilled in the art.

[0385] In some embodiments, the PD-L1 inhibitor is a peptide. In some embodiments, the PD-L1 inhibitor is a peptide inhibitor described in U.S. Pat. No. 9,422,339 or U.S. Pat. No. 9,850,283, the contents of which are incorporated herein in their entirety. In some embodiments, the PD-L1 inhibitor is a peptide inhibitor described in Yin, H. et al. Rational Design of Potent Peptide Inhibitors of the PD-1:PD-L1 Interaction for Cancer Immunotherapy, J.Am.Chem.Soc.2021,143:44,18536-18547. In some embodiments, the PD-L1 inhibitor is a peptide inhibitor described in Lin, X., et al. Progress in PD-1 / PD-L1 pathway inhibitors:From biomacromolecules to small molecules, Euro.J.Med.Chem.2020,vol.186. In some embodiments, inhibitor peptides for inhibition of PD-L1 are peptides generated using methods known to those of skill in the art.

[0386] Those skilled in the art know how to determine whether a compound is a PD1 inhibitor and / or PDL1 inhibitor by testing the compound in a suitable assay. The binding of an inhibitor to PD1 and / or PDL1 and / or PDL2 can be measured, for example, in an ELISA-type assay well known in the art. Bioassays for measuring the biological effect of PD1 and / or PDL1 and / or PDL2 inhibition are well known to those skilled in the art.

[0387] kit In some embodiments, the disclosure provides a kit comprising a STAT3 oligonucleotide herein and instructions for administering the STAT3 oligonucleotide to a subject who has received or is receiving a PD-L1 inhibitor. In some embodiments, the kit comprises the oligonucleotide herein, one or more controls, and various buffers, reagents, enzymes, and other standard components known in the art in a suitable container. In some embodiments, the container comprises at least one vial, well, test tube, flask, bottle, syringe, or other container means into which the oligonucleotide is placed, and in some cases, suitably aliquoted. In some embodiments where additional components are provided, the kit comprises an additional container into which the component is placed. The kit may also include a means for tightly closing the oligonucleotide and any other reagents for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are held. The container and / or kit may include a label with instructions and / or warnings for use.

[0388] In some embodiments, the kit comprises a STAT3 oligonucleotide and a pharma- ceutically acceptable carrier, or a pharmaceutical composition comprising the oligonucleotide, and instructions for treating or delaying the progression of a disease, disorder, or condition associated with STAT3 expression in a subject in need thereof, where the subject has received or is receiving a PD-L1 inhibitor. In some embodiments, the kit comprises a STAT3 oligonucleotide and a pharma- ceutically acceptable carrier, or a pharmaceutical composition comprising the oligonucleotide, and instructions for treating or delaying the progression of a cancer in a subject in need thereof, where the subject has received or is receiving a PD-L1 inhibitor.

[0389] In some embodiments, the kit comprises a pharmaceutical composition comprising a PD-L1 inhibitor and a pharma- ceutically acceptable carrier or oligonucleotide as described herein, and instructions for treating or delaying the progression of a disease, disorder, or condition in a subject in need thereof, where the subject has received or has received a STAT3 oligonucleotide as described herein.In some embodiments, the kit comprises a pharmaceutical composition comprising a PD-L1 inhibitor and a pharma- ceutically acceptable carrier or oligonucleotide as described herein, and instructions for treating or delaying the progression of a cancer in a subject in need thereof, where the subject has received or has received a STAT3 oligonucleotide as described herein. EXAMPLES

[0390] Although the present disclosure has been described with reference to specific embodiments shown in the following examples, those skilled in the art should understand that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the present disclosure. Moreover, the following examples are provided as illustrations and are not intended to limit the scope of the present disclosure in any manner. In addition, modifications can be made to the circumstances, materials, compositions of matter, processes, process steps, or steps to suit the purpose, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the present disclosure. Standard techniques known in the art or those specifically described below were utilized.

[0391] Abbreviation Ac: Acetyl AcOH: acetic acid ACN: Acetonitrile Ad: Adamantil AIBN: 2,2'-azobisisobutyronitrile Anhydride: Anhydrous Aq: Water-based B2Pin2: Bis(pinacolato)diboron-4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) BINAP: 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl BH3: Volan Bn: Benzyl Boc: tert-butoxycarbonyl Boc2O: Di-tert-butyl dicarbonate BPO: Benzoyl peroxide BuOH: n-butanol CDI: Carbonyldiimidazole COD: Cyclooctadiene d:day DABCO: 1,4-diazobicyclo[2.2.2]octane DAST: Diethylaminosulfur trifluoride dba: dibenzylideneacetone DBU: 1,8-diazobicyclo[5.4.0]undec-7-ene DCE: 1,2-dichloroethane DCM: dichloromethane DEA: Diethylamine DHP: Dihydropyran DIBAL-H: Diisobutylaluminum hydride DIPA: Diisopropylamine DIPEA or DIEA: N,N-Diisopropylethylamine DMA: N,N-Dimethylacetamide DME: 1,2-dimethoxyethane DMAP: 4-dimethylaminopyridine DMF: N,N-dimethylformamide DMP: Dess-Martin periodinane DMSO - Dimethyl sulfoxide DMTr: 4,4'-Dimethyloxytrityl DPPA: Diphenylphosphoryl azide dppf: 1,1'-bis(diphenylphosphino)ferrocene EDC or EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ee: enantiomeric excess ESI: electrospray ionization EA: Ethyl acetate EtOAc: ethyl acetate EtOH: Ethanol FA: Formic acid h or hrs: hours HATU: N,N,N',N'-Tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate HCl: Hydrochloric acid HPLC: High-performance liquid chromatography HOAc: acetic acid IBX: 2-iodoxybenzoic acid IPA: Isopropyl alcohol KHMDS: Potassium hexamethyldisilazide K2CO3: Potassium carbonate LAH: Lithium aluminum hydride LDA: Lithium diisopropylamide L-DBTA: Dibenzoyl-L-tartaric acid m-CPBA: meta-chloroperbenzoic acid M: mole MeCN: Acetonitrile MeOH: Methanol Me2S: Dimethyl sulfide MeONa: Sodium methylate MeI: Iodomethane min:minutes mL: milliliter mM: millimolar mmol: millimolar MPa: Megapascals MOMCl: Methyl chloromethyl ether MsCl: methanesulfonyl chloride MTBE: Methyl tert-butyl ether nBuLi: n-butyl lithium NaNO2: Sodium nitrite NaOH: Sodium hydroxide Na2SO4: Sodium sulfate NBS: N-bromosuccinimide NCS: N-chlorosuccinimide NFSI: N-fluorobenzenesulfonimide NMO: N-methylmorpholine N-oxide NMP: N-methylpyrrolidine NMR: nuclear magnetic resonance ℃: Celsius degree Pd / C: Palladium on carbon Pd(OAc)2: Palladium acetate PBS: Phosphate-buffered saline PE: Petroleum ether POCl3: Phosphorus oxychloride PPh3: Triphenylphosphine PyBOP: (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate Rel:Relative RT or rt: room temperature s or sec: seconds sat: saturation SEMCl: Chloromethyl-2-trimethylsilyl ethyl ether SFC: Supercritical Fluid Chromatography SOCl2: Sulfur dichloride tBuOK: Potassium tert-butoxide TBAB: Tetrabutylammonium bromide TBAF: Tetrabutylammonium fluoride TBAI: Tetrabutylammonium iodide TEA: Triethylamine Tf: Trifluoromethanesulfonate TfAA, TFMSA or Tf2O: Trifluoromethanesulfonic anhydride TFA: Trifluoroacetic acid TIBSCl: 2,4,6-triisopropylbenzenesulfonyl chloride TIPS: Triisopropylsilyl THF: tetrahydrofuran THP: Tetrahydropyran TLC: Thin Layer Chromatography TMEDA: Tetramethylethylenediamine pTSA: para-toluenesulfonic acid UPLC: Ultra-high performance liquid chromatography wt: weight Xantphos: 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

[0392] Example 1: Preparation of double-stranded RNAi oligonucleotides General synthesis method The following examples are intended to illustrate the present disclosure and should not be construed as limiting the present disclosure. Temperatures are given in degrees Celsius (C). Unless otherwise stated, all evaporations are carried out under reduced pressure, preferably at about 15 mm Hg to 100 mm Hg (=20 to 133 mbar). The structures of final products, intermediates and starting materials were confirmed by standard analytical methods, e.g., microanalysis and spectroscopic characteristics, e.g., MS, IR, NMR. The abbreviations used are those commonly used in the art.

[0393] All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts utilized to synthesize the nucleic acids or analogs thereof of the present disclosure are commercially available or can be produced by organic synthesis methods known to those skilled in the art (METHODS OF ORGANIC SYNTHESIS, Thieme, Volume 21 (Houben-Weyl 4th Ed. 1952)). In addition, the nucleic acids or analogs thereof of the present disclosure can be produced by organic synthesis methods known to those skilled in the art, as shown in the following examples.

[0394] Unless otherwise specified, all reactions are carried out under nitrogen or argon.

[0395] Proton NMR ( 1 1 H NMR) was performed in a deuterated solvent. Certain nucleic acids or analogs thereof disclosed herein include one or more 1 The H shifts overlap with residual proteosolvent signals, which are not reported in the experiments presented below.

[0396] As shown in the Examples below, in certain exemplary embodiments, nucleic acids or analogs thereof were prepared according to the following general procedures: While the general methods are shown to synthesize specific nucleic acids or analogs thereof of the present disclosure, it will be understood that the following general methods, as well as other methods known to those of skill in the art, can be applied to all nucleic acids or analogs thereof, as well as each subclass and species of these nucleic acids or analogs thereof, as described herein.

[0397] Example 1a: Synthesis of 2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-fluoro[3,2-f][1,3,5,2,4]trioxadisilosin-9-yl)oxy)methoxy)ethoxy)ethane-1-ammonium formate (1-6) [ka] A solution of compound 1-1 (25.00 g, 67.38 mmol) in 20 mL of DMF was treated with pyridine (11 mL, 134.67 mmol) and tetraisopropyldisiloxane dichloride (22.63 mL, 70.75 mmol) at 10° C. The resulting mixture was stirred at 25° C. for 3 h and quenched with 20% citric acid (50 mL). The aqueous layer was extracted with EtOAc (3×50 mL) and the combined organic layers were concentrated in vacuo. The crude residue was recrystallized from a mixture of MTBE and n-heptane (1:15, 320 mL) to give compound 1-2 (37.20 g, 90%) as a white oily solid.

[0398] A solution of compound 1-2 (37.00 g, 60.33 mmol) in 20 mL of DMSO was treated with AcOH (20 mL, 317.20 mmol) and Ac2O (15 mL, 156.68 mmol). The mixture was stirred at 25 °C for 15 h. The reaction was diluted with EtOAc (100 mL) and quenched with saturated K2CO3 (50 mL). The aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic layers were concentrated and recrystallized with ACN (30 mL) to give compound 1-3 (15.65 g, 38.4%) as a white solid.

[0399] A solution of compound 1-3 (20.00 g, 29.72 mmol) in 120 mL of DCM was treated with Fmoc-amino-ethoxyethanol (11.67 g, 35.66 mmol) at 25° C. The mixture was stirred to obtain a clear solution and then treated with 4 Å molecular sieves (20.0 g), N-iodosuccinimide (8.02 g, 35.66 mmol), and TfOH (5.25 mL, 59.44 mmol). The mixture was stirred at 30° C. until HPLC analysis showed >95% consumption of compound 1-3. The reaction was quenched with TEA (6 mL) and filtered. The filtrate was diluted with EtOAc, washed with saturated NaHCO3 (2 x 100 mL), saturated Na2SO3 (2 x 100 mL), and water (2 x 100 mL), and concentrated in vacuo to give crude compound 1-4 (26.34 g, 93.9%) as a yellow solid, which was used directly in the next step without further purification.

[0400] A solution of compound 1-4 (26.34 g, 27.62 mmol) in a mixture of DCM / water (10:7, 170 mL) was treated with DBU (7.00 mL, 45.08 mmol) at 5 °C. The mixture was stirred at 5-25 °C for 1 h. The organic layer was then separated, washed with water (100 mL) and diluted with DCM (130 mL). The solution was treated with fumaric acid (7.05 g, 60.76 mmol) and 4 Å molecular sieves (26.34 g) in four portions. The mixture was stirred for 1 h, concentrated and recrystallized from a mixture of MTBE and DCM (5:1) to give compound 1-6 (14.74 g, 62.9%) as a white solid. 1H NMR(400MHz,d6-DMSO)8.73(s,1H),8.58(s,1H),8.15-8.02(m,2H),7.65-7.60(m,1H),7.59-7.51(m,2H),6.52(s,2H),6.15 (s,1H),5.08-4.90(m,3H),4.83-4.78(m,1H),4.15-3.90(m,3H),3.79-3.65(m,2H),2.98-2.85(m,6H),1.20-0.95(m,28H).

[0401] Example 1b: Synthesis of (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((2-(2-[lipid]-amidoethoxy)ethoxy)methoxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (2-4a to 2-4e) [ka] A solution of compound 1-6 (50.00 g, 59.01 mmol) in 150 mL of 2-methyltetrahydrofuran was washed with ice-cold aqueous K2HPO4 (6%, 100 mL) and brine (20%, 2 x 100 mL). The organic layer was separated and treated with hexanoic acid (10.33 mL, 82.61 mmol), HATU (33.66 g, 88.52 mmol), and DMAP (10.81 g, 147.52 mmol) at 0 °C. The resulting mixture was warmed to 25 °C and stirred for 1 h. The solution was washed with water (2 x 100 mL), brine (100 mL), and concentrated in vacuo to give a crude residue. Flash chromatography on silica gel (1:1 hexane / acetone) afforded compound 2-1a (34.95 g, 71.5%) as a white solid.

[0402] A mixture of compound 2-1a (34.95 g, 42.19 mmol) and TEA (9.28 mL, 126.58 mmol) in 80 mL of THF was treated with triethylamine trihydrofluoride (20.61 mL, 126.58 mmol) dropwise at 10° C. The mixture was warmed to 25° C. and stirred for 2 h. The reaction was concentrated, dissolved in DCM (100 mL), and washed with saturated NaHCO3 (5×20 mL), and brine (50 mL). The organic layer was concentrated in vacuo to give crude compound 2-2a (24.72 g, 99%), which was used directly in the next step without further purification.

[0403] A solution of compound 2-2a (24.72 g, 42.18 mmol) in 50 mL of DCM was treated with N-methylmorpholine (18.54 mL, 168.67 mmol) and DMTr-Cl (15.69 g, 46.38 mmol). The mixture was stirred at 25° C. for 2 h and quenched with saturated NaHCO3 (50 mL). The organic layer was separated, washed with water, and concentrated to give a crude slurry. Flash chromatography on silica gel (1:1 hexane / acetone) afforded compound 2-3a (30.05 g, 33.8 mmol, 79.9%) as a white solid.

[0404] A solution of compound 2-3a (25.00 g, 28.17 mmol) in 50 mL of DCM was treated with N-methylmorpholine (3.10 mL, 28.17 mmol) and tetrazole (0.67 mL, 14.09 mmol) under nitrogen atmosphere. Bis(diisopropylamino)chlorophosphine (9.02 g, 33.80 mmol) was added dropwise to the solution and the resulting mixture was stirred at 25 °C for 4 h. The reaction was quenched with water (15 mL) and the aqueous layer was extracted with DCM (3 x 50 mL). The combined organic layers were washed with saturated NaHCO3 (50 mL) and concentrated to give a crude solid, which was recrystallized from a mixture of DCM / MTBE / n-hexane (1:4:40) to give compound 2-4a (25.52 g, 83.4%) as a white solid. 1H NMR(400MHz,d6-DMSO)11.25(s,1H),8.65-8.60(m,2H),8.09-8.02(m,2H),7.71(s,1H),7.67-7.60(m,1H),7.59-7.51(m,2H) ),7.38-7.34(m,2H),7.30-7.25(m,7H),6.85-6.79(m,4H),6.23-6.20(m,1H),5.23-5.14(m,1H),4.80-4.69(m,3H),4.33-4 .23(m,2H),3.90-3.78(m,1H),3.75(s,6H),3.74-3.52(m,3H),3.50-3.20(m,6H),3.14-3.09(m,2H),3.09(s,1H),2.82-2.8 0(m,1H),2.65-2.60(m,1H),2.05-1.96(m,2H),1.50-1.39(m,2H),1.31-1.10(m,14H),1.08-1.05(m,2H),0.85-0.79(m,3H); 31 P NMR (162MHz, d6-DMSO) 149.43,149.18.

[0405] Compounds 2-4b, 2-4c, 2-4d, and 2-4e were prepared using a procedure similar to that described above for compound 2-4a. Compound 2-4b was obtained as a white solid (25.50 g, 85.4%). 1H NMR(400MHz,d6--DMSO)11.23(s,1H),8.65-8.60(m,2H),8.05-8.02(m,2H),7.73-7.70(m,1H),7.67-7.60(m,1H),7.59-7.51 (m,2H),7.38-7.34(m,2H),7.30-7.25(m,7H),6.89-6....

Claims

1. 1. An RNAi oligonucleotide for use in treating cancer in a subject who has received or is receiving a PD-L1 inhibitor, said oligonucleotide comprising an antisense strand 15 to 30 nucleotides in length and a sense strand 15 to 40 nucleotides in length, said antisense strand and sense strand forming a double-stranded region, said antisense strand comprising a region of complementarity to a STAT3 mRNA target sequence, said region of complementarity being at least 15 contiguous nucleotides in length.

2. 2. The RNAi oligonucleotide of claim 1, wherein the cancer is selected from carcinoma, sarcoma, melanoma, lymphoma, and leukemia, prostate cancer, breast cancer, hepatocellular carcinoma (HCC), colorectal cancer, pancreatic cancer, and glioblastoma.

3. The RNAi oligonucleotide of claim 1 or 2, wherein the cancer comprises an immunosuppressive tumor microenvironment or an inflammatory tumor microenvironment.

4. The RNAi oligonucleotide of claim 3, wherein the inflammatory tumor microenvironment comprises infiltrating T cells.

5. The RNAi oligonucleotide of claim 1, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands.

6. The RNAi oligonucleotide described in claim 5, wherein the targeting ligand is a C18 saturated fatty acid moiety.

7. The RNAi oligonucleotide of claim 1, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 1055 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 1145.

8. The RNAi oligonucleotide described in claim 1, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 875 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:

965.

9. The RNAi oligonucleotide of claim 1, wherein the PD-L1 inhibitor is an antibody.

10. 10. The RNAi oligonucleotide of claim 9, wherein the antibody is an anti-PD-L1 antibody selected from FAZ053, atezolizumab, avelumab, durvalumab, embafolimab, and BMS-936559.

11. The RNAi oligonucleotide of claim 9, wherein the antibody is an anti-PD-1 antibody selected from nivolumab, pembrolizumab, and cemiplimab.

12. The RNAi oligonucleotide of claim 1, wherein the PD-L1 inhibitor is a small molecule inhibitor or a peptide.

13. The RNAi oligonucleotide of claim 1, wherein the PD-L1 inhibitor is a nucleic acid molecule.

14. The RNAi oligonucleotide of claim 13 , wherein the nucleic acid molecule is selected from an antisense oligonucleotide, an siRNA, or an miRNA.

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