STAT3 targeting oligonucleotides and uses thereof

Abstract

The subject matter disclosed herein relates to modulating STAT3 gene expression using siRNA compositions and methods that involve affecting key cell populations that support cancer growth and metastasis to achieve beneficial treatment, alleviation or removal of potential tumors in a patient.

Description

Cross - related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 425,861, filed on Nov. 16, 2022. The entire content of the U.S. Provisional Application is incorporated herein by reference. Background of the Invention

[0002] Currently, chemotherapy is the most prevalent cancer treatment globally, often combined with surgery or a combination of surgery and radiotherapy, depending on the tumor type and stage (Abbas et al., AN OVERVIEW OF CANCER TREATMENT MODALITIES / INTECHOPEN, 2018). Since the discovery of several important mutations that lead to carcinogenesis (e.g., epidermal cell alterations (Yamaoka et al., INT. J. MOL. SCI. (2017) 18(11):2420)), these mutations and the proteins they represent have been widely used as targets for developing more selective drugs and drug combinations to treat cancer patients. Although these drugs are effective, multi-drug resistance (MDR) is often observed in patients, which frequently leads to tumor recurrence, limited treatment options, and poor patient quality of life. Additionally, cancer research has often focused on tumor cells, although it has been demonstrated that the role of the tumor microenvironment and the "normal" or non-cancerous cells within it play a key role in tumor progression, development, and MDR (Klemm et al., TRENDS CELL BIOL (2015) 25(4):198-213). There is a need for new therapies that target different aspects of the TME that promote tumor growth. Summary of the Invention

[0003] The present disclosure is based in part on the discovery of oligonucleotides that target STAT3 mRNA and reduce its expression. The present disclosure is further based on the finding that the combination of STAT3 oligonucleotides and a PD-L1 inhibitor provides synergistic anti-tumor efficacy against tumors in different tumor microenvironments. Specifically, as demonstrated herein, lipid-conjugated STAT3 oligonucleotides, when co-delivered with an anti-PD-L1 antibody, reduced tumor volume in vivo in immunosuppressive and inflamed tumor models. Additionally, as shown herein, the combination of STAT3 oligonucleotides and a PD-L1 inhibitor induced an anti-tumor memory response, as no tumors formed when the mice were rechallenged with cancer cells. Furthermore, the efficacy of STAT3 oligonucleotides and a PD-L1 inhibitor is dependent on the presence of CD8+ T cells.

[0004] Accordingly, in one aspect, the present disclosure provides an oligonucleotide for reducing STAT3 expression, the oligonucleotide comprising an antisense strand having a length of 15 to 30 nucleotides and a sense strand having a length of 15 to 40 nucleotides, wherein the sense strand and the antisense strand form a duplex region, wherein the antisense strand has a complementary region complementary to the target sequence of STAT3 shown in SEQ ID NO: 140, and wherein the sense strand comprises at least one lipid moiety conjugated to the 5'-terminal nucleotide of the sense strand.

[0005] In some or any of the foregoing or related aspects, the antisense strand has a length of 19 to 27 nucleotides. In some aspects, the antisense strand has a length of 21 to 27 nucleotides; optionally wherein the antisense strand has a length of 22 nucleotides.

[0006] In some or any of the foregoing or related aspects, the sense strand has a length of 19 to 40 nucleotides, optionally wherein the sense strand has a length of 36 nucleotides.

[0007] In some or any of the foregoing or related aspects, the duplex region has a length of at least 19 nucleotides. In some aspects, the duplex region has a length of at least 20 nucleotides, optionally wherein the duplex region has a length of 21 nucleotides. In some aspects, the complementary region complementary to STAT3 has a length of at least 19 consecutive nucleotides. In some aspects, the complementary region complementary to STAT3 has a length of at least 21 consecutive nucleotides.

[0008] In some or any of the foregoing or related aspects, the antisense strand comprises the sequence shown in SEQ ID NO: 965.

[0009] In some or any of the foregoing or related aspects, the sense strand comprises the sequence shown in SEQ ID NO: 875.

[0010] In some or any of the foregoing or related aspects, the sense strand comprises a stem-loop at its 3'-end as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop having a length of 3 to 5 nucleotides between S1 and S2.

[0011] In some aspects, the present disclosure provides oligonucleotides for reducing STAT3 expression, the oligonucleotides comprising an antisense strand and a sense strand, wherein the antisense strand has a length of 21 to 27 nucleotides and has a complementary region complementary to the target sequence of STAT3 shown in SEQ ID NO: 140, wherein the sense strand comprises a stem-loop at its 3' end as shown below: S1-L-S2, wherein S1 is complementary to S2, wherein L forms a loop having a length of 3 to 5 nucleotides between S1 and S2, wherein the antisense strand and the sense strand form a duplex structure having a length of at least 19 nucleotides, and wherein the sense strand comprises a lipid moiety conjugated to the 5'-terminal nucleotide of the sense strand.

[0012] In some aspects, the present disclosure provides double-stranded oligonucleotides for reducing STAT3 expression, the oligonucleotides comprising: (i) an antisense strand having a length of 19-30 nucleotides, wherein the antisense strand comprises a nucleotide sequence having a complementary region complementary to the STAT3 mRNA target sequence, wherein the complementary region is shown in SEQ ID NO: 140, and (ii) a sense strand having a length of 19-50 nucleotides, which comprises a complementary region complementary to the antisense strand, wherein the sense strand comprises a lipid moiety conjugated to the 5'-terminal nucleotide of the sense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0013] In some or any of the foregoing or related aspects, L is a tetraloop, optionally wherein L has a length of 4 nucleotides. In some aspects, L comprises the sequence represented as GAAA.

[0014] In some or any of the foregoing or related aspects, the antisense strand has a length of 27 nucleotides and the sense strand has a length of 25 nucleotides, optionally wherein the antisense strand has a length of 22 nucleotides and the sense strand has a length of 36 nucleotides. In some aspects, the antisense strand and the sense strand form a duplex region having a length of 25 nucleotides, optionally wherein the length of the duplex is 20 nucleotides. In some aspects, the antisense strand comprises a 3'-overhang sequence having a length of one or more nucleotides, optionally wherein the length of the 3'-overhang sequence is 2 nucleotides, optionally wherein the 3'-overhang sequence is GG.

[0015] In some or any of the foregoing or related aspects, the oligonucleotide comprises at least one modified nucleotide. In some aspects, the modified nucleotide comprises a 2'-modification. In some aspects, 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 aspects, about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand comprise a 2'-fluoro modification. In some aspects, 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 aspects, 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.

[0016] In some or any of the foregoing or related aspects, the sense strand comprises 36 nucleotides from position 1-36 from 5' to 3', wherein positions 8-11 comprise a 2'-fluoro modification. In some aspects, the antisense strand comprises 22 nucleotides from position 1-22 from 3' to 5', and wherein positions 2, 3, 4, 5, 7, 10, and 14 comprise a 2'-fluoro modification. In some aspects, the remaining nucleotides comprise a 2'-O-methyl modification.

[0017] In some or any of the foregoing or related aspects, the oligonucleotide comprises at least one modified internucleotide linkage. In some aspects, the at least one modified internucleotide linkage is a phosphorothioate bond. In some aspects, the sense strand comprises a phosphorothioate bond between positions 1 and 2 of the sense strand. In some aspects, the antisense strand comprises 22 nucleotides from position 1-22 from 3' to 5', wherein the antisense strand comprises phosphorothioate bonds between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22. In some aspects, the sense strand comprises a phosphorothioate bond between positions 1 and 2 of the sense strand, and the antisense strand comprises 22 nucleotides from position 1-22 from 3' to 5', wherein the antisense strand comprises phosphorothioate bonds between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22.

[0018] In some or any of the foregoing or related aspects, the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphonate analogue. In some aspects, the phosphonate analogue is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

[0019] In some or any of the foregoing or related aspects, the lipid moiety is a saturated or unsaturated fatty acid moiety. In some aspects, the lipid moiety is a saturated fatty acid moiety having a length in the range of C10 to C24.

[0020] In some or any of the foregoing or related aspects, the lipid moiety is a C16 saturated fatty acid moiety. In some aspects, the C16 saturated fatty acid moiety is represented by the following formula:

[0021] In some or any of the foregoing or related aspects, the lipid moiety is a C18 saturated fatty acid moiety. In some aspects, the C18 saturated fatty acid moiety is represented by the following formula:

[0022] In some or any of the foregoing or related aspects, the lipid moiety is selected from:

[0023] In some or any of the foregoing or related aspects, the lipid moiety is conjugated to the 2'-carbon of the ribose ring of the 5'-terminal nucleotide.

[0024] In some or any of the foregoing or related aspects, the sense strand comprises the sequence shown in SEQ ID NO: 1222. In some aspects, the antisense strand comprises the sequence shown in SEQ ID NO: 1145. In some or any of the foregoing or related aspects, the sense strand comprises the sequence shown in SEQ ID NO: 1222, and wherein the antisense strand comprises the sequence shown in SEQ ID NO: 1145.

[0025] In some aspects, the present disclosure provides a double-stranded oligonucleotide for reducing STAT3 expression, wherein the oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprises the sequence shown in SEQ ID NO: 1222, the antisense strand comprises the sequence shown in SEQ ID NO: 1145, wherein the sense strand and the antisense strand form an asymmetric duplex region having a length of 20 nucleotides and having a 2-nucleotide overhang at the 3'-end of the antisense strand.

[0026] In some or any of the foregoing or related aspects, the complementary region is fully complementary to the STAT3 target sequence. In some aspects, the complementary region is partially complementary to the STAT3 target sequence. In some aspects, the complementary region contains no more than 4 mismatches with the STAT3 target sequence. In some aspects, the complementary region is fully complementary to the STAT3 target sequence at nucleotide positions 2-8 or 2-11 of the antisense strand, wherein the nucleotide positions are numbered from 5' to 3'.

[0027] In some or any of the foregoing or related aspects, the oligonucleotide is a nuclease substrate that, upon processing by an endogenous nuclease, generates a double-stranded nucleic acid having a length of 19-21 nucleotides that is capable of reducing STAT3 mRNA expression in mammalian cells.

[0028] In some or any of the foregoing or related aspects, the oligonucleotide reduces STAT3 mRNA expression in one or more immune cells associated with the tumor microenvironment.

[0029] In some aspects, the present disclosure provides a pharmaceutical composition comprising an oligonucleotide of any of the foregoing or related aspects and a pharmaceutically acceptable carrier, delivery agent, or excipient.

[0030] In some embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an oligonucleotide or pharmaceutical composition of any of the foregoing or related aspects.

[0031] In some or any of the foregoing or related aspects, a PD-L1 inhibitor is administered to the subject.

[0032] In some aspects, 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 to the subject an oligonucleotide or pharmaceutical composition of any of the foregoing or related aspects, thereby treating the cancer in the subject.

[0033] In some aspects, the present disclosure provides a method of treating cancer in a subject who has received or is receiving an oligonucleotide targeting STAT3, wherein the oligonucleotide targeting STAT3 is an oligonucleotide or pharmaceutical composition of any of the foregoing or related aspects, the method comprising administering to the subject a PD-L1 inhibitor, thereby treating the cancer in the subject.

[0034] In some aspects, the present disclosure provides a method of treating a disease, disorder, or condition associated with STAT3 expression in a subject, the method comprising administering to the subject an effective amount of an oligonucleotide or pharmaceutical composition of any of the foregoing or related aspects.

[0035] In some or any of the foregoing or related aspects, a PD-L1 inhibitor is administered to the subject.

[0036] In some aspects, the present disclosure provides a method of treating a disease, disorder, or condition associated with STAT3 expression in a subject who has received or is receiving a PD-L1 inhibitor, the method comprising administering to the subject an oligonucleotide or pharmaceutical composition of any of the foregoing or related aspects, thereby treating the cancer in the subject.

[0037] In some aspects, the present disclosure provides methods for treating a disease, disorder, or condition associated with STAT3 expression in a subject who has received or is receiving an oligonucleotide targeting STAT3, wherein the oligonucleotide targeting STAT3 is an oligonucleotide or pharmaceutical composition of any of the foregoing or related aspects, and the method comprises administering a PD-L1 inhibitor to the subject, thereby treating cancer in the subject.

[0038] In some or any of the foregoing or related aspects, the disease, disorder, or condition associated with STAT3 expression is cancer. In some aspects, 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 aspects, the cancer comprises an immunosuppressive tumor microenvironment. In some aspects, the cancer comprises an inflamed tumor microenvironment. In some aspects, the inflamed tumor microenvironment comprises infiltrating T cells.

[0039] In some or any of the foregoing or related aspects, the PD-L1 inhibitor is an antibody. In some aspects, the antibody is an anti-PD-L1 antibody. In some aspects, the anti-PDL1 antibody is selected from FAZ053, atezolizumab, avelumab, durvalumab, envafolimab, and BMS-936559.

[0040] In some or any of the foregoing or related aspects, the antibody is an anti-PD-1 inhibitor antibody. In some aspects, the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, and cemiplimab.

[0041] In some or any of the foregoing or related aspects, treating cancer comprises reducing or inhibiting tumor growth in a subject.

[0042] In some aspects, the present disclosure provides a method for reducing the expression of STAT3 mRNA in a cell, which comprises contacting the cell with an oligonucleotide of any of the foregoing or related aspects.

[0043] In some aspects, the present disclosure provides a kit comprising a container containing an oligonucleotide of any of the foregoing or related aspects, an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder, or condition associated with STAT3 expression.

[0044] In some aspects, the disease, disorder, or condition associated with STAT3 expression is cancer.

[0045] In some aspects, the present disclosure provides a kit comprising a container containing an oligonucleotide of any of the foregoing or related aspects, an optional pharmaceutically acceptable carrier, and a package insert including instructions for administration to a cancer subject who has received or is receiving a PD-L1 inhibitor.

[0046] In some aspects, the present disclosure provides a kit comprising a container containing a PD-L1 inhibitor, an optional pharmaceutically acceptable carrier, and a package insert including instructions for administration to a cancer subject who has received or is receiving an oligonucleotide of any of the foregoing or related aspects.

[0047] In some aspects, the present disclosure provides a kit comprising an oligonucleotide, an optional pharmaceutically acceptable carrier, and a package insert including instructions for administration of the oligonucleotide to a subject in need who has received or is receiving a PD-L1 inhibitor, wherein the oligonucleotide is an oligonucleotide of any of the foregoing or related aspects.

[0048] In some aspects, the present disclosure provides a kit comprising a PD-L1 inhibitor, an optional pharmaceutically acceptable carrier, and a package insert including instructions for administration of the inhibitor to a subject in need who has received or is receiving an oligonucleotide, wherein the oligonucleotide is an oligonucleotide of any of the foregoing or related aspects.

[0049] In some or any of the foregoing or related aspects, the subject has a disease, disorder, or condition associated with activated STAT3 expression. In some aspects, the subject has cancer.

[0050] In some aspects, the present disclosure provides a method for determining the responsiveness of a cancer subject who has received or is receiving treatment, the method comprising detecting the presence of a marker of myeloid-derived suppressor cells (MDSC) or MDSC activity in a biological sample of the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, and wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject is responsive to the treatment.

[0051] In some aspects, the present disclosure provides a method for determining the responsiveness of a cancer subject who has received or is receiving treatment, which comprises: (i) obtaining a biological sample from the subject; and (ii) detecting the presence of a marker of MDSC or MDSC activity in the biological sample, wherein the treatment is administration of an oligonucleotide targeting STAT3, and wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject is responsive to the treatment.

[0052] In some or any of the foregoing or related aspects, the detection comprises determining the amount of MDSCs or the amount of a marker of MDSC activity.

[0053] In some aspects, the reduction in MDSCs or the marker of MDSC activity is relative to the amount or level of MDSCs or the marker of MDSC activity in the subject prior to treatment.

[0054] In some aspects, the reduction in MDSCs or the marker of MDSC activity is relative to the amount or level of MDSCs or the marker of MDSC activity in a patient population not receiving the treatment. In some aspects, the reduction in MDSCs or the marker of MDSC activity is based on the amount or level of MDSCs or the marker of MDSC activity in a patient population responsive to the treatment.

[0055] In some aspects, the MDSCs are granulocytic-MDSCs (G-MDSCs). In some aspects, the MDSCs are monocytic-MDSCs (M-MDSCs). In some aspects, the MDSCs express Arg1.

[0056] In some aspects, the MDSCs express IDO. In some aspects, the presence of MDSCs or a marker of MDSC activity is determined by flow cytometry.

[0057] In some aspects, the biological sample is a blood or serum sample.

[0058] In some aspects, the response to treatment includes a decrease or inhibition of tumor growth and / or tumor size.

[0059] In some aspects, the oligonucleotide targeting STAT3 is the oligonucleotide of any of the foregoing or related aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1A The structure of an RNAi oligonucleotide molecule having chemical modifications is provided, wherein GalNAc or a lipid (e.g., a C18 hydrocarbon chain) is conjugated to the oligonucleotide molecule to generate an oligonucleotide-ligand conjugate.

[0061] Figure 1B The structure of a lipid tail suitable for conjugation to an RNAi oligonucleotide molecule is provided.

[0062] Figure 2A and Figure 2B is a graph showing the remaining murine Stat3 mRNA levels in the livers of mice treated with GalXC-STAT3-conjugates (GalNAc conjugates) targeting different regions of Stat3 mRNA. A single dose (3 mg / kg) was administered to the mice ( Figure 2A) and / or different doses (0.3, 1.0 or 3.0 mg / kg) to determine dose responsiveness ( Figure 2B ). Arrows indicate constructs selected for further study.

[0063] Figure 3A and Figure 3B are graphs showing mouse Stat3 mRNA expression in G-MDSC and M-MDSC from Pan02 xenografts in implanted mice, 3 days after treatment with the GalXC-STAT3-C18 conjugate. Tumors were dosed at 25 mg / kg ( Figure 3A ) and 50 mg / kg ( Figure 3B ).

[0064] Figure 4A and Figure 4B are graphs showing mouse Stat3 mRNA expression in the tumor mass (TME) ( Figure 4A ) and tumor-draining lymph nodes (TdLN) ( Figure 4B ) of Pan02 xenograft mice after treatment with the GalXC-STAT3-C18 conjugate at doses of 25 and 50 mg / kg.

[0065] Figure 5A The graphs provided show the effects of GalXC-STAT3-C18-4123 on Stat3 and Pdl1 mRNA levels in G / M-MDSC in the TME and TdLN of Pan02 xenograft mice 3 days after conjugated oligonucleotide dosing at 25 or 50 mg / kg.

[0066] Figure 5B The graphs provided show the effects of GalXC-STAT3-C18-4123 on Stat3 and Pdl1 mRNA levels in the TdLN of Pan02 xenograft mice 7 days after conjugated oligonucleotide dosing at 25 mg / kg.

[0067] Figure 6A and Figure 6B are graphs showing the in vivo effects 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 murine pancreatic tumors. Mice were treated with four 12.5 mg / kg ( Figure 6A ) or two 25 mg / kg ( Figure 6B ) doses of the conjugated oligonucleotide. Lines show the mean for all tested animals.

[0068] Figure 7The provided graph depicts the percentage (%) of human STAT3 mRNA remaining in Huh7 cells endogenously expressing human STAT3 after 24 hours of treatment with 1 nM of DsiRNAs targeting different regions of the STAT3 gene. 192 DsiRNAs were designed and screened. Two primer pairs were used. Expression between samples was normalized using the HPRT and SFRS9 housekeeping genes (Forward 1 - SEQ ID NO:1219, Reverse 1 - SEQ ID NO:1220; Probe 1 - SEQ ID NO:1221; Forward 2 - SEQ ID NO:1, Reverse 2 - SEQ ID NO:2; Probe 2 - SEQ ID NO:3).

[0069] Figure 8A and Figure 8B The provided graph depicts the percentage (%) of human STAT3 mRNA remaining in Huh7 cells endogenously expressing human STAT3 after 24 hours of treatment with 0.05 nM, 0.3 nM, or 1 nM of DsiRNAs targeting different regions of the STAT3 gene. 48 GalNAc-conjugated STAT3 oligonucleotides were assayed in Figure 8A and 34 of these oligonucleotides were selected for further in vivo testing ( Figure 8B ).

[0070] Figure 9A and Figure 9B The provided graph depicts the percentage (%) of human STAT3 mRNA remaining in the livers of mice (hydrodynamic injection model) exogenously expressing human STAT3 after treatment with GalNAc-conjugated STAT3 oligonucleotides. Mice were administered 1 mg / kg of the indicated GalNAc-STAT3 oligonucleotide formulated in PBS subcutaneously. Three days after administration, mice were given a hydrodynamic injection (HDI) of a DNA plasmid encoding human STAT3. Levels of human STAT3 mRNA were assayed in livers collected 18 hours after injection. Arrows indicate the 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.

[0071] Figure 10Graphs depicting the dose response of GalNAc-conjugated STAT3 oligonucleotides are provided. The percentage (%) of human STAT3 mRNA remaining in the livers of mice (HDI model) that exogenously express STAT3 was measured after treatment with human GalNAc-conjugated STAT3 oligonucleotides at two different doses (0.3 mg / kg or 1 mg / kg). The levels of human STAT3 mRNA were determined in livers collected 18 hours after injection of the plasmid encoding human STAT3. The arrows indicate the oligonucleotides selected for dose-response analysis. Hs / Mf = human / monkey consensus sequence; Hs / Mm = human / mouse consensus sequence.

[0072] Figure 11 The provided graph depicts the normalized relative mouse STAT3 mRNA remaining (relative to Ppib) in the livers of mice that endogenously express mouse STAT3 after treatment with GalNAc-conjugated STAT3 oligonucleotides. The indicated GalNAc-STAT3 oligonucleotides formulated in PBS were administered subcutaneously to mice at 3 mg / kg. Livers were collected five days after administration and the levels of mouse STAT3 mRNA were determined. The arrows indicate the top-ranked oligonucleotides and the oligonucleotides selected for the dose-response study.

[0073] Figure 12 The provided graph depicts the normalized relative mouse STAT3 mRNA remaining (relative to Ppib) in the livers of mice that endogenously express mouse STAT3 after treatment with GalNAc-conjugated STAT3 oligonucleotides. The indicated GalNAc-STAT3 oligonucleotides formulated in PBS were administered subcutaneously to mice at 3 mg / kg. Livers were collected five days after administration and the levels of mouse STAT3 mRNA were determined. The arrows indicate the oligonucleotides selected for the dose-response study.

[0074] Figure 13A and Figure 13B Graphs depicting the dose response of GalNAc-conjugated STAT3 oligonucleotides are provided. The percentage (%) of mouse STAT3 mRNA remaining in the livers of mice that endogenously express STAT3 was measured after treatment with human GalNAc-conjugated STAT3 oligonucleotides at three doses (0.3 mg / kg, 1 mg / kg, and 3 mg / kg). The levels of mouse STAT3 mRNA were determined in livers collected 5 days later. TC = triple consensus (mouse / human / monkey); Hs_Mm = human / mouse.

[0075] Figure 14The provided figure depicts the percentage (%) of remaining human STAT3 mRNA in the liver of mice (hydrodynamic injection model) that ectopically express human STAT3 after treatment with GalNAc-conjugated STAT3 oligonucleotides. Mice were administered 1 mg / kg of the indicated GalNAc-STAT3 oligonucleotides formulated in PBS subcutaneously. Three days after administration, the mice were subjected to hydrodynamic injection (HDI) of a DNA plasmid encoding human STAT3. The level of human STAT3 mRNA was determined in the liver collected 18 hours after injection. The arrows indicate the oligonucleotides selected for the dose-response study.

[0076] Figure 15 A figure depicting the dose-response of GalNAc-conjugated STAT3 oligonucleotides is provided. The percentage (%) of remaining human STAT3 mRNA in the liver of mice (hydrodynamic injection model) that ectopically express human STAT3 after treatment with GalNAc-conjugated STAT3 oligonucleotides. Three doses (0.3 mg / kg, 1 mg / kg, and 3 mg / kg) of the indicated GalNAc-STAT3 oligonucleotides formulated in PBS were administered to mice subcutaneously. Three days after administration, the mice were subjected to hydrodynamic injection (HDI) of a DNA plasmid encoding human STAT3. The level of human STAT3 mRNA was determined in the liver collected 18 hours after injection. TC = triple common (mouse / human / monkey); Hs_Mm = human / mouse; Hs = human.

[0077] Figure 16 A figure depicting the dose-response of GalNAc-conjugated STAT3 oligonucleotides is provided. The percentage (%) of remaining human STAT3 mRNA in the liver of mice (hydrodynamic injection model) that ectopically express human STAT3 after treatment with GalNAc-conjugated STAT3 oligonucleotides. Two doses (0.3 mg / kg and 1 mg / kg) of the indicated GalNAc-STAT3 oligonucleotides formulated in PBS were administered to mice subcutaneously. Three days after administration, the mice were subjected to hydrodynamic injection (HDI) of a DNA plasmid encoding human STAT3. The level of human STAT3 mRNA was determined in the liver collected 18 hours after injection.

[0078] Figure 17 The provided figure depicts the percentage (%) of remaining human STAT1 mRNA in Huh7 cells that endogenously express STAT3 and STAT1 after treatment with GalNAc-conjugated STAT3 oligonucleotides. The cells were treated with three doses (0.05 nM, 0.3 nM, and 1 nM) of the oligonucleotides for 24 hours.

[0079] Figure 18AThe provided figures depict tumor volume after administration of GalXC-STAT3-C18 oligonucleotide either alone or in combination with anti-PD-L1 mAb. Immunocompetent mice bearing Pan02 murine pancreatic tumors were given 25 mg / kg of GalXC-STAT3-C18-4123 subcutaneously (s.c.), and were treated intraperitoneally (i.p.) with 10 mg / kg of anti-PD-L1 mAb. Controls included GalXC-placebo (an HBV siRNA with the same chemical properties and lipid conjugation as GalXC-STAT3 oligonucleotide), 25 mg / kg of GalXC-STAT3-C18-4123, or 25 mg / kg of GalXC-placebo in combination with 10 mg / kg of anti-PD-L1 mAb. Mice were first administered two doses three days apart, and then two more doses three days apart two weeks later [(q3dx2)x2]. Arrows indicate the days of dose administration.

[0080] Figure 18B The provided figures depict tumor volume after administration of GalXC-STAT3-C18 oligonucleotide in combination with anti-PD-L1 mAb. Immunocompetent mice bearing Pan02 murine pancreatic tumors were given 25 mg / kg of GalXC-STAT3-C18-4123 subcutaneously (s.c.), and were treated intraperitoneally (i.p.) with 10 mg / kg of anti-PD-L1 mAb. Mice were administered GalXC-placebo on days 42 and 45 after transplantation, and then GalXC-STAT3 in combination with anti-PD-L1 mAb on days 60 and 63.

[0081] Figures 19A - 19C The provided figures depict tumor volume in tumors with different immunophenotypes after administration of GalXC-STAT3-C18 oligonucleotide either alone or in combination with anti-PD-L1 mAb, or GalXC-placebo either alone or in combination with anti-PD-L1 mAb. 4T1 (triple-negative breast cancer, checkpoint resistant) ( Figure 19A )、MC-38 (colon cancer, partially checkpoint sensitive) ( Figure 19B ) or Hepa1-6 (hepatocellular carcinoma, checkpoint sensitive) ( Figure 19C) The cells were implanted into mice. Intraperitoneal treatment with 25 mg / kg of GalXC-STAT3-C18-4123 and 10 mg / kg of anti-PD-L1 mAb was given to tumor-bearing mice subcutaneously. Controls included GalXC-placebo, 25 mg / kg of GalXC-STAT3-C18-4123, or 25 mg / kg of GalXC-placebo combined with 10 mg / kg of anti-PD-L1 mAb. MC-38 and Hepa1-6 tumor-bearing mice were administered two doses at 25 mg / kg every three days and the same treatment regimen was repeated in the following week. 4T1 tumor-bearing mice were administered three doses, every three days (q3d x 3). Arrow (5 / 5CR) = all treated mice had a complete response.

[0082] Figure 20 The provided figure depicts the effect of Hepa1-6 rechallenge on tumors that have been completely eradicated. After treatment with GalXC-STAT3-C18 (25 mg / kg, s.c.) and anti-PD-L1 mAb (10 mg / kg, i.p.) in Figure 19C resulted in complete tumor regression in all 5 mice, the mice were rechallenged on day 51 with Hepa1-6 cells (2e6 cells / mouse) in the contralateral flank of the mice, and tumor volume ([[]] Figure 20 ) was monitored. Arrow (5 / 5CR) = all mice remained tumor-free even after rechallenge.

[0083] Figure 21A and Figure 21B The provided figure depicts tumor volume after administration of GalXC-STAT3-C18 oligonucleotides alone or in combination with anti-PD-L1 mAb in immunocompetent mice ([[]] Figure 21A ) with functional CD8+ T cells and immunocompromised mice ([[]] Figure 21B ) without functional CD8+ T cells. GalXC-STAT3-C18-4123 (25 mg / kg, three times per dose, every three days (q3d x 3)) was administered subcutaneously to 4T1 tumor-bearing mice (immunocompetent or immunocompromised), and anti-PD-L1 mAb (10 mg / kg, q3d x 3) was administered intraperitoneally. Controls included GalXC-placebo, 25 mg / kg of GalXC-STAT3-C18-4123, or 25 mg / kg of GalXC-placebo combined with 10 mg / kg of anti-PD-L1 mAb.

[0084] Figure 22 The provided images show the appearance of the tumors (with cell death) in mice measured in [[[]] Figure 21A and perforin staining for positive cytotoxic CD8+ T cells in the tumors at the end of the study.

[0085] Figure 23 The provided figures depict tumor volume and images showing lung tumor metastasis after administration of GalXC-STAT3-C18-4123 oligonucleotide alone or in combination with anti-PD-L1 mAb. GalXC-STAT3-C18-4123 (50 mg / kg, q3dx 3) was administered subcutaneously to 4T1 tumor-bearing mice (immunocompetent or immunocompromised), and anti-PD-L1 mAb (10 mg / kg, q3d x 3) was administered intraperitoneally. Controls included GalXC-vehicle, 50 mg / kg of GalXC-STAT3-C18-4123, or 50 mg / kg of GalXC-vehicle combined with 10 mg / kg of anti-PD-L1 mAb.

[0086] Figure 24 The provided heatmap shows the modulation of targets involved in immune regulation observed in CT26 tumors after combination treatment with GalXC-STAT3-C18-4123 (s.c, 25 mg / kg, q3d x 3) and anti-PD-L1 mAb (i.p., 10 mg / kg, q3d x 3) compared to controls including GalXC-vehicle, 25 mg / kg of GalXC-STAT3-C18-4123, or 25 mg / kg of GalXC-vehicle combined with 10 mg / kg of anti-PD-L1 mAb.

[0087] Figure 25 The structure of an RNAi oligonucleotide molecule with chemical modifications is provided, wherein a C18 lipid is conjugated to the 5'-terminal nucleotide of the sense strand to generate an oligonucleotide-ligand conjugate.

[0088] Figures 26A - 26CThe provided figures depict tumor volumes after administration of DCR-STAT3 (a human-specific STAT3 sequence with a C18 lipid conjugation at the 5' end of the passenger strand, corresponding to SEQ ID NO: 1222 and 1145) or GalXC-placebo (a chemically matched irrelevant sequence that does not bind to the Stat3 / STAT3 mRNA target sequence) either alone or in combination with an anti-PD-L1 antibody. Immunocompetent mice bearing B16F10 (murine melanoma), Pan02 (murine pancreatic cancer), and MC-38 (murine colorectal cancer) tumors were treated with three or four subcutaneous (s.c.) doses of 25 mg / kg conjugated oligonucleotide either alone or in combination with 10 mg / kg intraperitoneal (i.p.) anti-PD-L1 antibody. B16F10 tumor-bearing mice received three doses at three-day intervals; Pan02 tumor-bearing mice received two doses at three-day intervals first, and then two more doses at three-day intervals one week later. MC-38 tumor-bearing mice received two doses at three-day intervals first, and then two more doses at three-day intervals four days later. Arrows indicate the days of dose administration. Detailed Description

[0089] The present disclosure will now be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the disclosure are shown. However, the disclosure may be embodied in many different forms and should not be construed as 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. Definitions

[0090] Publications discussed throughout this text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

[0091] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Further, the singular forms as well as the article "a", "an", and "the" are also intended to include the plural forms unless expressly stated otherwise. It should also be understood that the terms "comprises" and / or "comprising", when used in this specification, specify the presence of the 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. Additionally, it should 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 can be directly connected or coupled to the other element, or intervening elements may be present.

[0092] 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. Exemplary methods and materials are described herein, but methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions.

[0093] General texts describing molecular biological 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, Vol. 152, (Academic Press, Inc., San Diego, Calif.) ("Berger"); Sambrook et al., MOLECULAR CLONING--A LABORATORY MANUAL, 2nd ed., Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989 ("Sambrook") and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F.M. Ausubel et al. eds., CURRENT PROTOCOLS, A JOINT VENTURE BETWEEN GREENE PUBLISHING ASSOCIATES, INC. AND JOHN WILEY AND SONS, INC., (Supplemented through 1999) ("Ausubel").Examples of protocols sufficient to direct one of ordinary skill in the art to perform in vitro amplification methods (including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplification, and other RNA polymerase-mediated techniques (e.g., NASBA), e.g., methods for generating the homologous nucleic acids of the present disclosure) can be found in Berger, Sambrook, and Ausubel, and Mullis et al., U.S. Patent 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) C&EN 36-47; J. NIH RES. (1991) 3:81-94; Kwoh et 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 13:563-564. An improved method for cloning in vitro amplified nucleic acids is described in U.S. Patent No. 5,426,039 to Wallace et al. Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng et al., (1994) NATURE 369:684-85 and the references cited therein, where PCR amplicons of up to 40 kb were generated.

[0094] Unless the context clearly dictates otherwise, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0095] Ranges can be expressed in this document as from “about” one value and / or to “about” another value. When expressing such a range, another embodiment includes from one value and / or to another value. Similarly, when a value is expressed as an approximation by use of the antecedent “about”, it should be understood that the value constitutes another embodiment. Further, it should be understood that each endpoint of a range is significant both in relation to and independent of the other endpoint. It should also be understood that several values are disclosed herein, and each value is also disclosed herein as “about” that value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It should also be understood that when a value is disclosed, “less than or equal to” that value, “greater than or equal to” that value, and the possible ranges between values are also disclosed, as would be appropriately understood by a person skilled in the art. For example, if the value “10” is disclosed, then “less than or equal to 10” and “greater than or equal to 10” are also disclosed. It should also be understood that throughout the application, data is provided in several different formats, and the data represents ranges of endpoints and starting points and any combination of data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, then it should be understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 and the range between 10 and 15 are considered to be disclosed. It should also be understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0096] In this specification and the appended claims, several terms will be referenced, and these terms shall be defined to have the following meanings:

[0097] The terms “cancer” or “tumor” include, but are not limited to, solid tumors and hematogenous tumors. These terms include diseases of the skin, tissues, organs, bones, cartilage, blood, and blood vessels. These terms further encompass primary cancer and metastatic cancer.

[0098] The term “PD-1” refers to a protein found on T cells that helps control the immune response. When PD-1 binds to another protein called PD-L1, it helps prevent T cells from killing other cells, including cancer cells. Some 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 be used to kill cancer cells.

[0099] 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 RefSeq#, or NM_139276.3). STAT3 mediates the expression of multiple genes in response to cellular stimuli and thus plays a key role in many cellular processes such as cell growth and apoptosis, as well as the growth and progression of cancer.

[0100] As used herein, the term "cold tumor" or "non-inflamed tumor" refers to a tumor or tumor microenvironment in which there are minimal or no anti-tumor immune cells, such as tumor-infiltrating lymphocytes (TIL), and / or contains cell subsets associated with immunosuppression, including regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC), and M2 macrophages. Specifically, in some embodiments, a cold tumor is characterized by a low or even absent infiltration of anti-tumor immune cells, such cells may be present but still remain in the surrounding stroma and thus unable to colonize the tumor microenvironment to provide their anti-tumor function.

[0101] As used herein, "complementary" refers to the 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, a purine nucleotide of one nucleic acid complementary to a pyrimidine nucleotide of an opposing nucleic acid can base pair together by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides can base pair in a Watson-Crick manner or in any other manner that allows for the formation of a stable duplex. In some embodiments, two nucleic acids may have regions of multiple nucleotides that are complementary to each other to form complementary regions, as described herein.

[0102] As used herein, a "species cross-reactive oligonucleotide" refers to an oligonucleotide capable of inhibiting the expression of a target mRNA in more than one species. For example, in some embodiments, a species cross-reactive oligonucleotide is capable of inhibiting the expression of a target mRNA in humans and non-human primates. Exemplary species include, but are not limited to, humans, non-human primates, mice, and rats. In some embodiments, a species cross-reactive oligonucleotide is capable of targeting and inhibiting mRNA in at least two, at least three, or at least four species.

[0103] As used herein, a "deoxyribonucleotide" refers to a nucleotide that has a hydrogen rather than a hydroxyl group at the 2'-position of its pentose sugar compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide having one or more modifications or substitutions of atoms other than the 2'-position, including modifications or substitutions of the sugar, phosphate group, or base, or modifications or substitutions among them.

[0104] As used herein, "double-stranded RNA" or "dsRNA" refers to an RNA oligonucleotide that is substantially in a duplex form. In some embodiments, the complementary base pairing of the duplex region of the dsRNA oligonucleotide forms between the antiparallel nucleotide sequences of covalently separated nucleic acid strands. In some embodiments, the complementary base pairing of the duplex region of the dsRNA forms between the antiparallel nucleotide sequences of covalently linked nucleic acid strands. In some embodiments, the complementary base pairing of the duplex region of the dsRNA is formed by a single nucleic acid strand that folds (e.g., by a hairpin) to provide complementary antiparallel nucleotide sequences that base pair together. In some embodiments, the dsRNA comprises two covalently separated nucleic acid strands that are fully duplexed with each other. However, in some embodiments, the dsRNA comprises two covalently separated nucleic acid strands that are partially duplexed (e.g., having overhangs at one or both ends). In some embodiments, the dsRNA comprises partially complementary antiparallel nucleotide sequences and thus can have one or more mismatches, which can include internal mismatches or terminal mismatches.

[0105] As used herein, "duplex" with respect to a nucleic acid (e.g., an oligonucleotide) refers to a structure formed by the complementary base pairing of two antiparallel nucleotide sequences.

[0106] As used herein, "excipient" refers to a non-therapeutic agent that can be included in a composition, e.g., to provide or contribute to a desired consistency or stabilizing effect.

[0107] As used herein, the term "hot tumor" or "inflamed tumor" refers to a tumor or tumor microenvironment in which there is a large abundance of anti-tumor immune cells, particularly TIL, and thus is generally immunostimulatory.

[0108] As used herein, "loop" refers to an unpaired region of a nucleic acid (e.g., an oligonucleotide) that is flanked by two antiparallel regions of the nucleic acid that are sufficiently complementary to each other such that under appropriate hybridization conditions (e.g., in phosphate buffer, in a cell), the two antiparallel regions flanking the unpaired region hybridize to form a duplex (referred to as the "stem"). A loop that contains four nucleotides can be referred to as a tetraloop (tetraL). A loop that contains three nucleotides can be referred to as a triloop (triL).

[0109] As used herein, "modified internucleotide linkage" refers to an internucleotide linkage having one or more chemical modifications compared to a reference internucleotide linkage that includes a phosphodiester bond. In some embodiments, the modified internucleotide linkage is a non-naturally occurring linkage. Generally, a modified internucleotide linkage confers one or more desired properties to a nucleic acid in which the modified internucleotide linkage is present. For example, a modified internucleotide linkage can improve thermal stability, degradation resistance, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, and the like.

[0110] As used herein, "modified nucleotide" refers to a nucleotide having one or more chemical modifications compared to a corresponding reference nucleotide selected from adenosine ribonucleotide, guanosine ribonucleotide, cytidine ribonucleotide, uridine ribonucleotide, deoxyadenosine ribonucleotide, deoxyguanosine ribonucleotide, deoxycytidine ribonucleotide, and thymidine ribonucleotide. 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. Generally, a modified nucleotide confers one or more desired properties to a nucleic acid in which the modified nucleotide is present. For example, a modified nucleotide can improve thermal stability, degradation resistance, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, and the like.

[0111] As used herein, "nicked tetraloop structure" refers to the structure of an RNAi oligonucleotide characterized by separate sense (passenger) and antisense (guide) strands, wherein the sense strand has a complementary region that is complementary to the antisense strand, and wherein at least one strand, typically the sense strand, has a tetraloop configured to stabilize adjacent stem regions formed within that at least one strand.

[0112] As used herein, "oligonucleotide" refers to a short nucleic acid (e.g., having a length shorter than about 100 nucleotides). Oligonucleotides can be single-stranded (ss) or double-stranded (ds). Oligonucleotides can have or lack a duplex region. Oligonucleotides can comprise deoxyribonucleotides, ribonucleosides, or a combination of both. In some embodiments, a double-stranded oligonucleotide comprising ribonucleotides is referred to as "dsRNA". As a non-limiting group of examples, oligonucleotides can be, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), dicer-substrate interfering RNA (dsiRNA), antisense oligonucleotides, short siRNA, or ss siRNA. In some embodiments, double-stranded RNA (dsRNA) is an RNAi oligonucleotide.

[0113] 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.

[0114] As used herein, “overhang” refers to terminal non-base paired nucleotides created by the extension of one strand or region beyond the end of the complementary strand that forms a duplex with that one strand or region. In some embodiments, the overhang comprises one or more unpaired nucleotides extending 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 or sense strand of the dsRNA.

[0115] As used herein, “phosphonate analogue” refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphonate analogue is located at the 5’-terminal nucleotide of the oligonucleotide instead of the 5’-phosphate that is normally readily removed enzymatically. In some embodiments, the 5’ phosphonate analogue contains a phosphatase-resistant linkage. Examples of phosphonate analogues include, but are not limited to, 5’-phosphonates such as 5’-methylenephosphonate (5’-MP) and 5’-(E)-vinylphosphonate (5’-VP). In some embodiments, the oligonucleotide has a phosphonate analogue at the 4’-carbon position of the sugar at the 5’-terminal nucleotide (referred to as a “4’-phosphonate analogue”). An example of a 4’-phosphonate analogue is oxy-methylenephosphonate, where the oxygen atom of the oxy-methyl group is bonded to the sugar moiety (e.g., at its 4’-carbon) or an analogue thereof. See, e.g., U.S. Provisional Patent Application No. 62 / 383,207, filed Sep. 2, 2016, and U.S. Provisional Patent Application No. 62 / 393,401, filed Sep. 12, 2016. Other modifications have been developed for the 5’-end of oligonucleotides (see, e.g., International Patent Application No. WO2011 / 133871; U.S. Patent No. 8,927,513; and Prakash et al. (2015) NUCLEIC ACIDS RES. 43:2993-3011).

[0116] As used herein, "reduced expression" of a gene (e.g., STAT3) refers to a decrease in the amount or level of an RNA transcript (e.g., STAT3 mRNA) or a protein encoded by the gene, and / or a decrease in the amount or level of the activity of the gene, in a cell, cell population, sample, or subject as compared to an appropriate reference (e.g., a reference cell, cell population, sample, or subject). For example, the act of contacting a cell with an oligonucleotide of the present invention (e.g., an oligonucleotide comprising an antisense strand having a nucleotide sequence complementary to the nucleotide sequence of a STAT3 mRNA) can result in a decrease in the amount or level of STAT3 mRNA, protein, and / or activity (e.g., via degradation of STAT3 mRNA by the RNAi pathway) as compared to a cell not treated with dsRNA. Similarly, and as used herein, "reducing expression" refers to an act that results in reduced expression of a gene (e.g., STAT3). As used herein, "reduction of 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 as compared to an appropriate reference (e.g., a reference cell, cell population, sample, or subject).

[0117] As used herein, "complementary region" refers to a nucleotide sequence of a nucleic acid (e.g., dsRNA) that is sufficiently complementary to an antiparallel nucleotide sequence to allow the two nucleotide sequences to hybridize under appropriate hybridization conditions (e.g., in phosphate buffer, in a cell, etc.). In some embodiments, the oligonucleotides of the present invention comprise a targeting sequence having a complementary region that is complementary to an mRNA target sequence.

[0118] As used herein, "ribonucleotide" refers to a nucleotide having ribose as its pentose and containing a hydroxyl group at its 2'-position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms other than at the 2'-position, including modifications or substitutions of ribose, phosphate groups, or bases, or combinations thereof.

[0119] As used herein, "RNAi oligonucleotide" refers to (a) a dsRNA having a sense (passenger) strand and an antisense (guide) strand, wherein the antisense strand or a portion of the antisense strand is used by the Argonaute2 (Ago2) endonuclease in the cleavage of a target mRNA, or (b) a single-stranded oligonucleotide having a single antisense strand, wherein the antisense strand (or a portion of the antisense strand) is used by the Ago2 endonuclease in the cleavage of a target mRNA.

[0120] As used herein, a "strand" refers to a single, continuous nucleotide sequence joined together by inter-nucleotide linkages (e.g., phosphodiester or phosphorothioate linkages). In some embodiments, the strand has two free ends (e.g., a 5' end and a 3' end).

[0121] As used herein, a "subject" refers to any mammal, including mice, rabbits, non-human primates (NHPs), and humans. In one embodiment, the subject is a human or an NHP. Additionally, "individual" or "patient" may be used interchangeably with "subject".

[0122] As used herein, "synthetic" refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine such as a solid-phase nucleic acid synthesizer) or otherwise not derived from a natural source (e.g., a cell or organism) that normally produces the molecule.

[0123] As used herein, a "targeting ligand" refers to a molecule or "moiety" (e.g., a carbohydrate, an amino sugar, cholesterol, a polypeptide, or a 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 to target that other substance to the tissue or cell of interest. For example, in some embodiments, a targeting ligand can be conjugated to an oligonucleotide to target the oligonucleotide to a specific tissue or cell of interest. In some embodiments, the targeting ligand selectively binds to a cell surface receptor. Thus, in some embodiments, when the targeting ligand is conjugated to an oligonucleotide, delivery of the oligonucleotide into a specific cell is facilitated by selective binding to a receptor expressed on the cell surface and endocytosis of the complex comprising the oligonucleotide, targeting ligand, and receptor. In some embodiments, the targeting ligand is conjugated to the oligonucleotide via a linker that is cleaved after or during cell internalization such that the oligonucleotide is released from the targeting ligand in the cell.

[0124] As used herein, a "loop", "triloop", or "tetraloop" refers to a loop that increases the stability of adjacent duplexes formed by hybridization of nucleotide flanking sequences. The increase in stability can be detected as an increase in the melting temperature (T m ) of the adjacent stem duplexes, which T m is higher than the T m of adjacent stem duplexes expected on average for a set of loops of comparable length composed of randomly selected nucleotide sequences. For example, a loop (e.g., a tetraloop or triloop) can confer on a hairpin containing a duplex of at least 2 base pairs (bp) a T m。In some embodiments, a loop (e.g., a tetraloop) can stabilize the bp in adjacent stem duplexes through stacking interactions. Additionally, interactions between nucleotides in the tetraloop include, but are not limited to, non-Watson-Crick base pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al., (1990) NATURE 346:680-82; Heus and Pardi (1991) SCIENCE 253:191-94). In some embodiments, the loop contains or consists of 3 to 6 nucleotides, and typically 4 to 5 nucleotides. In certain embodiments, the loop contains or consists of 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., it may or may not be conjugated to a targeting moiety). In some embodiments, the tetraloop contains or consists of 3 to 6 nucleotides, and typically 4 to 5 nucleotides. In certain embodiments, the tetraloop contains or consists of 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., it may or may not be conjugated to a targeting moiety). In one embodiment, a loop consisting of 4 nucleotides is a tetraloop. Any nucleotide can be used in the loop (e.g., tetraloop), and the standard IUPAC-IUB symbols for such nucleotides can be used as described by Cornish-Bowden ((1985) NUCLEIC ACIDS RES. 13:3021-3030). For example, the letter "N" can be used to indicate that any base can be at that position, the letter "R" can be used to indicate that A (adenine) or G (guanine) can be at that position, and "B" can be used to indicate that C (cytosine), G (guanine), or T (thymine) can be at that position. Examples of tetraloops include tetraloops of the UNCG family (e.g., UUCG), tetraloops of the GNRA family (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 tetraloops of the d(GNNA) family (e.g., d(GTTA)), tetraloops of the d(GNRA) family, tetraloops of the d(GNAB) family, tetraloops of the d(CNNG) family, and tetraloops of the d(TNCG) family (e.g., d(TTCG)). (See, e.g., Nakano et al., (2002) BIOCHEM. 41:4281-92; Shinji et al., (2000) NIPPON KAGAKKAI KOENYOKOSHU 78:731). In some embodiments, the tetraloop is contained within a nicked tetraloop structure.

[0125] As used herein, "treatment" or "treating" refers to the act of providing medical care to a subject in need, e.g., by administering a therapeutic agent (e.g., an oligonucleotide of the present disclosure) to the subject for improving the health and / or well-being of the subject with respect to an existing condition (e.g., a disease, disorder), or for 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., a disease, disorder) experienced by the subject.

[0126] As used herein, the term "tumor microenvironment" refers to the cellular environment in which any given tumor exists, including the tumor stroma, surrounding blood vessels, immune cells, fibroblasts, other cells, signaling molecules, and ECM. It should be understood that the tumor microenvironment harbors and / or surrounds the tumor cells with which it interacts. Method of Use Combination of STAT3 Oligonucleotide and PD-L1 Inhibitor

[0127] In some embodiments, the present disclosure provides STAT3 oligonucleotides for use in or suitable for treating a subject (e.g., a human having a disease, disorder, or condition associated with STAT3 expression) who has received or is receiving a PD-L1 inhibitor.

[0128] In some embodiments, the methods described herein include selecting a subject having or predisposed to a disease, disorder, or condition associated with STAT3 expression and / or PD-L1 expression. In some cases, the method may include selecting an individual having a marker of a disease such as cancer or other chronic lymphoproliferative disorders associated with STAT3 expression and / or PD-L1 expression.

[0129] Similarly, and as detailed herein, the method may further include steps such as 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 evaluate the effectiveness of the treatment.

[0130] In some embodiments, the present disclosure provides a method of treating a subject having, suspected of having, or at risk of developing a disease, disorder, or condition with a STAT3 oligonucleotide of the present disclosure, wherein the subject has received or is receiving a PD-L1 inhibitor. In some embodiments, the present disclosure provides a method 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, wherein the subject has received or is receiving a STAT3 oligonucleotide described herein.

[0131] In some aspects, the present disclosure provides methods of treating a disease, disorder, or condition associated with STAT3 expression or attenuating the onset or progression thereof using the STAT3 oligonucleotides herein in combination with a PD-L1 inhibitor. In other aspects, the present disclosure provides methods of achieving one or more therapeutic benefits in a subject having a disease, disorder, or condition associated with STAT3 expression using the STAT3 oligonucleotides herein in combination with a PD-L1 inhibitor. In some embodiments of the methods herein, a 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, a subject is treated by administering a therapeutically effective amount of the STAT3 oligonucleotides herein to a subject who has received or is receiving a PD-L1 inhibitor. In some embodiments of the methods herein, a subject is treated by administering a therapeutically effective amount of a PD-L1 inhibitor to a subject who has received or is receiving the STAT3 oligonucleotides herein. In some embodiments, a subject is treated therapeutically. In some embodiments, a subject is treated prophylactically.

[0132] In some aspects, the present disclosure provides methods of treating or attenuating the onset or progression of a disease, disorder, or condition associated with STAT3 expression using STAT3 oligonucleotides comprising a sense strand and an antisense strand, wherein the sense strand comprises the sequence set forth in SEQ ID NO: 875 and the antisense strand comprises the sequence set forth in SEQ ID NO: 965. In some aspects, the present disclosure provides methods of treating or attenuating the onset or progression of a disease, disorder, or condition associated with STAT3 expression using STAT3 oligonucleotides comprising a sense strand and an antisense strand, wherein the sense strand comprises the sequence set forth in SEQ ID NO: 1222 and the antisense strand comprises the sequence set forth in SEQ ID NO: 1145. In other aspects, the present disclosure provides methods of achieving one or more therapeutic benefits in a subject having a disease, disorder, or condition associated with STAT3 expression using STAT3 oligonucleotides comprising a sense strand and an antisense strand in combination with a PD-L1 inhibitor, wherein the sense strand comprises the sequence set forth in SEQ ID NO: 875 and the antisense strand comprises the sequence set forth in SEQ ID NO: 965. In other aspects, the present disclosure provides methods of achieving one or more therapeutic benefits in a subject having a disease, disorder, or condition associated with STAT3 expression using STAT3 oligonucleotides comprising a sense strand and an antisense strand in combination with a PD-L1 inhibitor, wherein the sense strand comprises the sequence set forth in SEQ ID NO: 1222 and the antisense strand comprises the sequence set forth in SEQ ID NO: 1145. In some embodiments of the methods herein, a subject is treated by administering a therapeutically effective amount of STAT3 oligonucleotides comprising a sense strand and an antisense strand in combination with a PD-L1 inhibitor, wherein the sense strand comprises the sequence set forth in SEQ ID NO: 875 and the antisense strand comprises the sequence set forth in SEQ ID NO: 965. In some embodiments of the methods herein, a subject is treated by administering a therapeutically effective amount of STAT3 oligonucleotides comprising a sense strand and an antisense strand in combination with a PD-L1 inhibitor, wherein the sense strand comprises the sequence set forth in SEQ ID NO: 1222 and the antisense strand comprises the sequence set forth in SEQ ID NO: 1145. In some embodiments of the methods herein, a subject is treated by administering a therapeutically effective amount of STAT3 oligonucleotides comprising a sense strand and an antisense strand to a subject who has received or is receiving a PD-L1 inhibitor, wherein the sense strand comprises the sequence set forth in SEQ ID NO: 875 and the antisense strand comprises the sequence set forth in SEQ ID NO: 965. In some embodiments of the methods herein, a subject is treated by administering a therapeutically effective amount of STAT3 oligonucleotides comprising a sense strand and an antisense strand to a subject who has received or is receiving a PD-L1 inhibitor, wherein the sense strand comprises the sequence set forth in SEQ ID NO: 1222 and the antisense strand comprises the sequence set forth in SEQ ID NO: 1145.In some embodiments of the methods herein, a subject is treated by administering to the subject a therapeutically effective amount of a PD-L1 inhibitor who has received or is receiving a STAT3 oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises the sequence set forth in SEQ ID NO: 875 and the antisense strand comprises the sequence set forth in SEQ ID NO: 965. In some embodiments of the methods herein, a subject is treated by administering to the subject a therapeutically effective amount of a PD-L1 inhibitor who has received or is receiving a STAT3 oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises the sequence set forth in SEQ ID NO: 1222 and the antisense strand comprises the sequence set forth in SEQ ID NO: 1145. In some embodiments, the subject is treated therapeutically. In some embodiments, the subject is treated prophylactically.

[0133] In some embodiments of the methods herein, one or more of the STAT3 oligonucleotides herein or a pharmaceutical composition 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 in the subject is reduced, thereby treating the subject. In some embodiments of the methods herein, a STAT3 oligonucleotide comprising a sense strand and an antisense strand (the sense strand comprising the sequence set forth in SEQ ID NO:875 and the antisense strand comprising the sequence set forth in SEQ ID NO:965) or a pharmaceutical composition comprising the STAT3 oligonucleotide is 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 in the subject is reduced, thereby treating the subject. In some embodiments of the methods herein, a STAT3 oligonucleotide comprising a sense strand and an antisense strand (the sense strand comprising the sequence set forth in SEQ ID NO:1222 and the antisense strand comprising the sequence set forth in SEQ ID NO:1145) or a pharmaceutical composition comprising the STAT3 oligonucleotide is 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 in the subject is reduced, thereby treating the subject. In some embodiments, the amount or level of STAT3 mRNA in the subject is reduced. In some embodiments, the amount or level of STAT3 and / or protein in the subject is reduced. In some embodiments of the methods herein, one or more of the STAT3 oligonucleotides herein or a pharmaceutical composition 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 in the subject are reduced, thereby treating the subject. In some embodiments of the methods herein, a STAT3 oligonucleotide comprising a sense strand and an antisense strand (the sense strand comprising the sequence set forth in SEQ ID NO:875 and the antisense strand comprising the sequence set forth in SEQ ID NO:965) or a pharmaceutical composition comprising the STAT3 oligonucleotide is 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 in the subject are reduced, thereby treating the subject.In some embodiments of the methods herein, a STAT3 oligonucleotide comprising a sense strand and an antisense strand (the sense strand comprising the sequence set forth in SEQ ID NO: 1222 and the antisense strand comprising the sequence set forth in SEQ ID NO: 1145) or a pharmaceutical composition comprising the STAT3 oligonucleotide is 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 in the subject are reduced, thereby treating the subject. In some embodiments, the amount or level of STAT3 mRNA and PD-L1 signaling in the subject is reduced. In some embodiments, the amount or level of STAT3 and / or protein in the subject is reduced and PD-L1 signaling in the subject is reduced.

[0134] In some embodiments, a therapeutically effective amount of the STAT3 oligonucleotide and / or a PD-L1 inhibitor is administered to the subject. A therapeutically acceptable amount can be an amount capable of therapeutically treating a disease or disorder. The appropriate dosage for any given subject will depend on certain factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredients in the composition, the time and route of administration, general health, and other drugs being co-administered.

[0135] In some embodiments, any of the compositions herein is administered to the subject enterally (e.g., orally, via a gastric feeding tube, via a duodenal feeding tube, via gastrostomy or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intraarterial injection or infusion, intraosseous infusion, intramuscular injection, intracerebral injection, intraventricular injection, intrathecal), topically (e.g., epidermally, by inhalation, via eye drops or via mucous membranes) or by direct injection into a target organ (e.g., the liver of the subject). Generally, the oligonucleotides herein are administered intravenously or subcutaneously.

[0136] As a group of non-limiting examples, the oligonucleotides herein are typically administered quarterly (once every three months), bi-monthly (once every two months), monthly or weekly. For example, the oligonucleotide can be administered weekly or at two- or three-week intervals. Alternatively, the oligonucleotide can be administered daily. In some embodiments, one or more loading doses of the oligonucleotide are administered to the subject, followed by one or more maintenance doses of the oligonucleotide.

[0137] In some embodiments, the PD-L1 inhibitor herein (e.g., an anti-PD-L1 antibody) is administered quarterly (once every three months), bi-monthly (once every two months), monthly or weekly. For example, the inhibitor is administered weekly or at two- or three-week intervals. Alternatively, the inhibitor is administered daily.

[0138] In some embodiments, the oligonucleotides herein are administered in combination with a PD-L1 inhibitor. In some embodiments, the oligonucleotide and the inhibitor are administered in combination simultaneously, sequentially (in any order), or intermittently. For example, the oligonucleotide and the inhibitor can be co-administered simultaneously. Alternatively, the oligonucleotide can be administered and the inhibitor can be administered at any length of time (e.g., one hour, one day, one week, or one month) later, and vice versa.

[0139] In some embodiments, the subject to be treated is a human or non-human primate or other mammalian subject. Other exemplary subjects include domestic 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. Cancer

[0140] In some embodiments, the 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, II, III, or IV tumor. In some embodiments, the tumor is a solid tumor. A solid tumor refers to the condition where cancer forms a mass.

[0141] 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 cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal cancer, glioblastoma, cervical cancer, bladder cancer, hepatoma, metastatic breast cancer, colon cancer, rectal cancer, endometrial cancer or uterine cancer, salivary gland adenocarcinoma, kidney cancer, prostate cancer, vulvar cancer, liver cancer, anal cancer, penile cancer, Merkel cell carcinoma, testicular cancer, esophageal cancer, or biliary tract tumor. In some embodiments, the cancer is refractory to anti-PD1, anti-PDL1, and / or anti-CTLA4 therapies. In some embodiments, the cancer is pancreatic cancer or lung cancer. In some embodiments, the cancer includes tumors 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.

[0142] In some embodiments, the STAT3 oligonucleotides and the PD-L1 inhibitor reduce tumor volume. Tumor volume is measured using methods known to those of skill in the art. For example, the excised tumor is manually measured using calipers. Other methods include imaging methods such as ultrasound and MRI. In some embodiments, the oligonucleotide conjugate reduces the 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. Treatment response

[0143] In some embodiments, the present disclosure provides methods of monitoring the treatment response of a subject. In some embodiments, the treatment includes any of the STAT3-targeting oligonucleotides described herein. In some embodiments, the treatment includes any of the STAT3-targeting oligonucleotides described herein in combination with a PD-L1 inhibitor.

[0144] In some embodiments, the present disclosure provides a method of monitoring the treatment response of a subject having a tumor, the method comprising detecting the amount of myeloid-derived suppressor cells (MDSC) in a biological sample of the subject who has received or is receiving treatment with a STAT3-targeting oligonucleotide to treat the subject's tumor, wherein a decrease in the amount of MDSC in the biological sample indicates that the subject is responsive to the treatment with the oligonucleotide.

[0145] In some embodiments, the present disclosure provides a method of monitoring the treatment response of a subject having a tumor, comprising: (i) obtaining a biological sample from a subject who has received or is receiving treatment with a STAT3-targeting oligonucleotide; (ii) detecting the amount of MDSC in the biological sample; and (iii) comparing the amount of MDSC in the biological sample with a pre-determined amount of MDSC, wherein a decrease in the amount of MDSC in the biological sample indicates that the subject is responsive to the treatment with the oligonucleotide.

[0146] In some embodiments, the present disclosure provides a method of determining the responsiveness of a subject having cancer to treatment. In some embodiments, the treatment includes any of the STAT3-targeting oligonucleotides described herein. In some embodiments, the treatment includes any of the STAT3-targeting oligonucleotides described herein in combination with a PD-L1 inhibitor.

[0147] In some embodiments, the present disclosure provides a method for determining the responsiveness of a cancer subject who has received or is receiving treatment, the method comprising detecting the presence of a marker of myeloid-derived suppressor cells (MDSC) or MDSC activity in a biological sample of the subject. In some embodiments, the method for determining the responsiveness of a cancer subject who has received or is receiving treatment comprises detecting the presence of a marker of myeloid-derived suppressor cells (MDSC) or MDSC activity in a biological sample of the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, and wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject is responsive to the treatment.

[0148] In some embodiments, the present disclosure provides a method for determining the responsiveness of a cancer subject who has received or is receiving treatment, which comprises: (i) obtaining a biological sample from the subject; and (ii) detecting the presence of a marker of MDSC or MDSC activity in the biological sample, wherein the treatment is administration of an oligonucleotide targeting STAT3, and wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject is responsive to the treatment.

[0149] In some embodiments, the detecting comprises determining the amount of MDSC or the amount of the marker of MDSC activity. In some embodiments, the decrease in MDSC or the marker of MDSC activity is relative to the amount or level of MDSC or the marker of MDSC activity in the subject prior to treatment. In some embodiments, the decrease in MDSC or the marker of MDSC activity is relative to the amount or level of MDSC or the marker of MDSC activity in the subject prior to treatment. In some embodiments, the decrease in MDSC or the marker of MDSC activity is based on the amount or level of MDSC or the marker of MDSC activity in a population of patients responsive to the treatment.

[0150] In some embodiments, the pre-determined amount of MDSC is the amount of MDSC detected in the subject prior to treatment with the oligonucleotide. In some embodiments, the pre-determined amount of MDSC is based on the average amount of MDSC in a population of patients not receiving oligonucleotide treatment. In some embodiments, the population of patients is a population of healthy patients. In some embodiments, the population of patients is a population not suffering from cancer. In some embodiments, the population of patients is a population receiving placebo oligonucleotide treatment. In some embodiments, the population of patients is a population of patients receiving oligonucleotide treatment and in whom tumor growth and / or tumor size has decreased or been inhibited.

[0151] In some embodiments, the MDSC is granulocytic-MDSC (G-MDSC). In some embodiments, the MDSC is monocytic-MDSC (M-MDSC). In some embodiments, the MDSC expresses Arg1. In some embodiments, the MDSC expresses IDO. In some embodiments, the MDSC is Arg1+ M-MDSC. In some embodiments, the MDSC is Arg1+ G-MDSC. In some embodiments, the MDSC is IDO+ M-MDSC. In some embodiments, the MDSC is IDO+ G-MDSC. In some embodiments, the MDSC is G-MDSC, M-MDSC, Arg1+ M-MDSC, Arg1+ G-MDSC, IDO+ M-MDSC, IDO+ G-MDSC, or a combination thereof.

[0152] In some embodiments, the amount of MDSC is determined using methods known to those of skill in the art. In some embodiments, flow cytometry is used to determine the amount of MDSC.

[0153] In some embodiments, MDSC is measured from a biological sample. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a serum sample.

[0154] In some embodiments, the response to treatment includes a decrease or inhibition of tumor growth and / or tumor size. In some embodiments, the response to treatment includes a decrease or inhibition of tumor growth. In some embodiments, the response to treatment includes a decrease or inhibition of tumor size.

[0155] In some embodiments, a method for determining the responsiveness of a cancer subject who has received or is receiving treatment, the method comprising detecting the presence of a marker of myeloid-derived suppressor cells (MDSC) or MDSC activity in a biological sample of the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject responds to the treatment, and wherein the oligonucleotide targeting STAT3 comprises a sense strand and an antisense strand, the sense strand comprising a sequence selected from SEQ ID NOs: 857-946, and the antisense strand comprising a sequence selected from SEQ ID NOs: 947-1036.

[0156] In some embodiments, a method of determining the responsiveness of a cancer subject who has received or is receiving treatment, the method comprising detecting the presence of a marker of myeloid-derived suppressor cells (MDSC) or MDSC activity in a biological sample of the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject is responsive to the treatment, and wherein the oligonucleotide targeting STAT3 comprises a sense strand and an antisense strand, the sense strand comprising a sequence selected from SEQ ID NOs: 1037-1126, and the antisense strand comprising a sequence selected from SEQ ID NOs: 1127-1216.

[0157] In some embodiments, a method of determining the responsiveness of a cancer subject who has received or is receiving treatment, the method comprising detecting the presence of a marker of myeloid-derived suppressor cells (MDSC) or MDSC activity in a biological sample of the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject is responsive to the treatment, and wherein the oligonucleotide targeting STAT3 comprises a sense strand and an antisense strand, the sense strand comprising a sequence selected from SEQ ID NOs: 9, 37, 65 and 69, and the antisense strand comprising a sequence selected from SEQ ID NOs: 10, 38, 66 and 70.

[0158] In some embodiments, a method of determining the responsiveness of a cancer subject who has received or is receiving treatment, the method comprising detecting the presence of a marker of myeloid-derived suppressor cells (MDSC) or MDSC activity in a biological sample of the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject is responsive to the treatment, and wherein the oligonucleotide targeting STAT3 comprises a sense strand and an antisense strand, the sense strand comprising a sequence selected from SEQ ID NOs: 11, 39, 67 and 71, and the antisense strand comprising a sequence selected from SEQ ID NOs: 12, 40, 68 and 72.

[0159] In some embodiments, a method of determining the responsiveness of a cancer subject who has received or is receiving treatment, the method comprising detecting the presence of a marker of myeloid-derived suppressor cells (MDSC) or MDSC activity in a biological sample of the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, wherein a decrease in MDSC or a decrease in a marker of MDSC activity in the biological sample indicates that the subject is responsive to the treatment, and wherein the oligonucleotide targeting STAT3 comprises a sense strand and an antisense strand, the sense strand comprising a sequence selected from SEQ ID NO: 9, 37, 65, and 69, and the antisense strand comprising a sequence selected from SEQ ID NO: 10, 38, 66, 70.

[0160] In some embodiments, a method of determining the responsiveness of a cancer subject who has received or is receiving treatment, the method comprising detecting the presence of a marker of myeloid-derived suppressor cells (MDSC) or MDSC activity in a biological sample of the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, wherein a decrease in MDSC or a decrease in a marker of MDSC activity in the biological sample indicates that the subject is responsive to the treatment, and wherein the oligonucleotide targeting STAT3 comprises a sense strand and an antisense strand, the sense strand comprising SEQ ID NO: 875, and the antisense strand comprising SEQ ID NO: 965.

[0161] In some embodiments, a method of determining the responsiveness of a cancer subject who has received or is receiving treatment, the method comprising detecting the presence of a marker of myeloid-derived suppressor cells (MDSC) or MDSC activity in a biological sample of the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, wherein a decrease in MDSC or a decrease in a marker of MDSC activity in the biological sample indicates that the subject is responsive to the treatment, and wherein the oligonucleotide targeting STAT3 comprises a sense strand and an antisense strand, the sense strand comprising SEQ ID NO: 1145, and the antisense strand comprising SEQ ID NO: 1222.

[0162] In some embodiments, the present disclosure provides a method of determining the responsiveness of a cancer subject who has received or is receiving treatment, comprising: (i) obtaining a biological sample from the subject; and (ii) Detect the presence of a marker of MDSC or MDSC activity in the biological sample, wherein the treatment is administration of an oligonucleotide targeting STAT3, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand comprising a sequence selected from SEQ ID NOs: 857 - 946, the antisense strand comprising a sequence selected from SEQ ID NOs: 947 - 1036, and wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject responds to the treatment.

[0163] In some embodiments, the present disclosure provides a method for determining the responsiveness of a cancer subject who has received or is receiving treatment, comprising: (i) Obtaining a biological sample from the subject; and (ii) Detecting the presence of a marker of MDSC or MDSC activity in the biological sample, wherein the treatment is administration of an oligonucleotide targeting STAT3, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand comprising a sequence selected from SEQ ID NOs: 1037 - 1126, the antisense strand comprising a sequence selected from SEQ ID NOs: 1127 - 1216, and wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject responds to the treatment.

[0164] In some embodiments, the present disclosure provides a method for determining the responsiveness of a cancer subject who has received or is receiving treatment, comprising: (i) Obtaining a biological sample from the subject; and (ii) Detecting the presence of a marker of MDSC or MDSC activity in the biological sample, wherein the treatment is administration of an oligonucleotide targeting STAT3, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand comprising a sequence selected from SEQ ID NOs: 11, 39, 67, and 71, the antisense strand comprising a sequence selected from SEQ ID NOs: 12, 40, 68, and 72, and wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject responds to the treatment.

[0165] In some embodiments, the present disclosure provides a method for determining the responsiveness of a cancer subject who has received or is receiving treatment, comprising: (i) Obtaining a biological sample from the subject; and (ii) Detecting the presence of a marker of MDSC or MDSC activity in the biological sample, wherein the treatment is administration of an oligonucleotide targeting STAT3, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand comprising a sequence selected from SEQ ID NO: 9, 37, 65, and 69, the antisense strand comprising a sequence selected from SEQ ID NO: 10, 38, 66, 70, and wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject responds to the treatment.

[0166] In some embodiments, the present disclosure provides a method for determining the responsiveness of a cancer subject who has received or is receiving treatment, comprising: (i) Obtaining a biological sample from the subject; and (ii) Detecting the presence of a marker of MDSC or MDSC activity in the biological sample, wherein the treatment is administration of an oligonucleotide targeting STAT3, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand comprising SEQ ID NO: 875, the antisense strand comprising SEQ ID NO: 965, and wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject responds to the treatment.

[0167] In some embodiments, the present disclosure provides a method for determining the responsiveness of a cancer subject who has received or is receiving treatment, comprising: (i) Obtaining a biological sample from the subject; and (ii) Detecting the presence of a marker of MDSC or MDSC activity in the biological sample, wherein the treatment is administration of an oligonucleotide targeting STAT3, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand comprising SEQ ID NO: 1145, the antisense strand comprising SEQ ID NO: 1222, and wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject responds to the treatment. Oligonucleotide inhibitors of STAT3

[0168] In some aspects, the present disclosure particularly provides oligonucleotides that reduce or inhibit STAT3 expression. In some embodiments, the oligonucleotides that inhibit STAT3 expression herein target STAT3 mRNA. The sequence of human STAT3 mRNA (NM_001369512.1) is shown as SEQ ID NO: 85 or NM_139276.3 (SEQ ID NO: 1217). STAT3 is a known target of conventional cancer therapies.

[0169] 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 in a range of cancer types as well as in preclinical models in vitro and in vivo (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 regulation 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 because of its well-defined contribution to the immunosuppressive tumor microenvironment. STAT3 contributes to the immunosuppressive tumor microenvironment by upregulating inhibitory receptors expressed by T cells and by expression of its ligands (PD-1 / PD-L1) through increased IFNγ secretion (Bu et al., Journal of Dental Research, 96(9):1027–34 (2017)). It has long been known that inhibition of STAT3 signaling in antigen-presenting cells (APCs) leads to priming of antigen-specific CD4+ T cells in response to other tolerogenic stimuli (Cheng et al., Immunity, 19:425–36 (2003)). Additionally, phosphorylated STAT3 on MDSCs directly contributes to the regulation of the inhibitory tumor microenvironment by regulating inhibitory components such as the amino acid arginine through transcriptional control (Vasques-Dunndel et al., J. Clin. Invest., 15(3):668–79 (2013)). Over the years, several approaches have been explored to therapeutically target STAT3. While directly targeting the protein is appealing, the true target is the protein–protein interaction, which has been considered a paradigm of an “undruggable” target due to historical data showing that multiple classes of compounds have failed to potently inhibit its activity (Lau et al., Cancers (2019) 11(11):1681, Zou et al., Mol Cancer (2020) 19:145). Additionally, the ubiquitous expression of STAT3 in several tissues has raised concerns about 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).

[0170] In some embodiments, a suitable assay or technique for evaluating one or more properties or characteristics of a cell or cell population associated with STAT3 can be used (e.g., using a STAT3 expression biomarker), or a reduction in STAT3 expression can be determined 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 an 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 not contacted with the oligonucleotide or contacted with a control oligonucleotide). In some embodiments, a suitable control level of mRNA expression to protein after delivery of an RNAi molecule can be a pre-determined level or value such that it is not necessary to measure the control level each time. The pre-determined level or value can take various forms. In some embodiments, the pre-determined level or value can be a single cut-off value, such as a median or an average.

[0171] In some embodiments, administration of an 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 a suitable control level of mRNA. The suitable control level can be the level of mRNA expression and / or protein translation in a cell or cell population that has not been contacted with an oligonucleotide herein. In some embodiments, the effect of delivering an oligonucleotide to a cell according to the methods herein is evaluated after a limited period of time. For example, the level of mRNA in the cell can be analyzed at least about 8 hours, about 12 hours, about 18 hours, about 24 hours after introducing the oligonucleotide into the cell; or at least about 1, 2, 3, 4, 5, 6, 7 days or even up to 14 days.

[0172] In some embodiments, the oligonucleotide is delivered in the form of a transgene that is engineered to express the oligonucleotide or the strands that make up the oligonucleotide (e.g., its sense and antisense strands) in a cell. In some embodiments, a transgene engineered to express any oligonucleotide disclosed herein is used to deliver the oligonucleotide. 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 a subject. STAT3 target sequence

[0173] In some embodiments, the oligonucleotide targets a target sequence comprising STAT3 mRNA. In some embodiments, the oligonucleotide or a portion, fragment, or strand thereof (e.g., the antisense strand or guide strand of a dsRNA) binds or anneals to a target sequence comprising STAT3 mRNA, thereby inhibiting STAT3 expression. In some embodiments, for the purpose of inhibiting STAT3 expression in vivo, the oligonucleotide targets the STAT3 target sequence. In some embodiments, the amount or degree to which the oligonucleotide targeting the STAT3 target sequence inhibits STAT3 expression is related to the potency of the oligonucleotide. In some embodiments, the amount or degree to which the oligonucleotide targeting the STAT3 target sequence inhibits STAT3 expression is related to the amount or degree of therapeutic benefit in a subject or patient suffering from a disease, disorder, or condition associated with STAT3 expression and treated with the oligonucleotide.

[0174] By examining the nucleotide sequences of mRNAs encoding STAT3, including the nucleotide sequences of mRNAs from multiple 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), certain nucleotide sequences of STAT3 mRNA have been found to be more susceptible to oligonucleotide-based inhibition than other sequences and can thus be used as target sequences for the oligonucleotides herein. In some embodiments, the sense strand of the oligonucleotide (e.g., dsRNA) described herein comprises the STAT3 target sequence. In some embodiments, a portion or region of the sense strand of the dsRNA described herein comprises the STAT3 target sequence. In some embodiments, the STAT3 mRNA target sequence comprises the sequence of SEQ ID NO:85, or consists of the sequence of SEQ ID NO:85. In some embodiments, the STAT3 mRNA target sequence comprises the sequence of SEQ ID NO:1217, or consists of the sequence of SEQ ID NO:1217. In some embodiments, the STAT3 mRNA target sequence comprises the sequence shown in SEQ ID NO:140, or consists of the sequence shown in SEQ ID NO:140. STAT3 targeting sequence

[0175] In some embodiments, the oligonucleotides herein have a complementary region that is complementary to STAT3 mRNA (e.g., within the target sequence of STAT3 mRNA) for the purpose of targeting mRNA in a cell and reducing or inhibiting its expression. In some embodiments, the oligonucleotides herein comprise a STAT3 targeting sequence (e.g., the antisense or guide strand of a dsRNA) that has a complementary region that binds or anneals to the STAT3 target sequence by complementary (Watson-Crick) base pairing. The targeting sequence or complementary region generally has a suitable length and base content such that the oligonucleotide (or its strand) can bind or anneal to the STAT3 mRNA, thereby achieving the purpose of inhibiting its expression. In some embodiments, the length of the targeting sequence or complementary region 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 some embodiments, the length of the targeting sequence or complementary region 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 some embodiments, the length of the targeting sequence or complementary region is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 18 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 19 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 20 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 21 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 22 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 23 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 24 nucleotides. In some embodiments, the oligonucleotide comprises a target sequence or complementary region that is complementary to the sequence of SEQ ID NO:140, and the length of the targeting sequence or complementary region is 18 nucleotides. In some embodiments, the oligonucleotide comprises a target sequence or complementary region that is complementary to the sequence of SEQ ID NO:140, and the length of the targeting sequence or complementary region is 19 nucleotides. In some embodiments, the oligonucleotide comprises a target sequence or complementary region that is complementary to the sequence of SEQ ID NO:524, and the length of the targeting sequence or complementary region is 20 nucleotides.In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is complementary to the sequence of SEQ ID NO: 524, and the targeting sequence or complementary region is 21 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is complementary to the sequence of SEQ ID NO: 524, and the targeting sequence or complementary region is 22 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is complementary to the sequence of SEQ ID NO: 524, and the targeting sequence or complementary region is 23 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is complementary to the sequence of SEQ ID NO: 524, and the targeting sequence or complementary region is 24 nucleotides in length.

[0176] In some embodiments, the oligonucleotides herein comprise a targeting sequence or complementary region that is fully complementary to a STAT3 target sequence (e.g., the antisense or guide strand of a double-stranded oligonucleotide). In some embodiments, the targeting sequence or complementary region is partially complementary to the STAT3 target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is fully complementary to the sequence of STAT3 or STAT3. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is partially complementary to the sequence of STAT3 or STAT3.

[0177] In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is fully complementary to the sequence of SEQ ID NO: 140. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is partially complementary to the sequence of SEQ ID NO: 140.

[0178] In some embodiments, the oligonucleotides herein comprise a targeting sequence or complementary region that is complementary to a contiguous nucleotide sequence that makes up STAT3 mRNA, wherein the contiguous nucleotide sequence is about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 20, 12 to 18, 12 to 16, 14 to 22, 16 to 20, 18 to 20, or 18 to 19 nucleotides in length). In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is complementary to a contiguous nucleotide sequence that makes up STAT3 mRNA, wherein the contiguous nucleotide sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is complementary to a contiguous nucleotide sequence that makes up STAT3 mRNA, wherein the contiguous nucleotide sequence is 19 nucleotides in length.

[0179] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise a targeting sequence or complementary region that is complementary to a contiguous nucleotide sequence of SEQ ID NO: 140, optionally wherein the contiguous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is complementary to a contiguous nucleotide sequence of SEQ ID NO: 524, wherein the contiguous nucleotide sequence is 20 nucleotides in length.

[0180] In some embodiments, the targeting sequence or complementary region of an oligonucleotide that is complementary to a contiguous nucleotide of STAT3 or a STAT3 target sequence spans the entire length of the antisense strand. In some embodiments, the complementary region of an oligonucleotide that is complementary to a contiguous nucleotide of STAT3 or a STAT3 target sequence spans a portion of the entire length of the antisense strand. In some embodiments, the oligonucleotides herein comprise a complementary region (e.g., on the antisense strand of a dsRNA) that is at least partially (e.g., fully) complementary to a contiguous nucleotide segment spanning nucleotides 1-20 of STAT3 or a target sequence of STAT3.

[0181] In some embodiments, the targeting sequence or complementary region of the oligonucleotides herein (e.g., RNAi oligonucleotides) is complementary to a contiguous nucleotide sequence of SEQ ID NO: 140 and spans the entire length of the antisense strand. In some embodiments, the targeting sequence or complementary region of the oligonucleotide is complementary to a contiguous nucleotide sequence of SEQ ID NO: 140 and spans a portion of the entire length of the antisense strand. In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise a complementary region (e.g., on the antisense strand of a dsRNA) that is at least partially (e.g., fully) complementary to a contiguous nucleotide segment spanning nucleotides 1-19 or 1-20 of the sequence shown in SEQ ID NO: 524.

[0182] In some embodiments, the oligonucleotides herein comprise a targeting sequence or complementary region having one or more bp mismatches with the corresponding STAT3 target sequence. In some embodiments, the targeting sequence or complementary region 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, provided that the ability of the targeting sequence or complementary region to bind or anneal to STAT3 mRNA under appropriate hybridization conditions and / or the ability of the oligonucleotide to inhibit STAT3 expression is maintained. Alternatively, the targeting sequence or complementary region 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, provided that the ability of the targeting sequence or complementary region to bind or anneal to STAT3 mRNA under appropriate hybridization conditions and / or the ability of the oligonucleotide to inhibit STAT3 expression is maintained. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region having 1 mismatch with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region having 2 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region having 3 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region having 4 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region having 5 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region having more than one mismatch (e.g., 2, 3, 4, 5, or more mismatches) with the corresponding target sequence, wherein at least 2 (e.g., all) of the mismatches are consecutively positioned (e.g., consecutive 2, 3, 4, 5, or more mismatches), or wherein the mismatches are dispersed throughout the targeting sequence or complementary region. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region complementary to the contiguous nucleotide sequence of SEQ ID NO:140, wherein the targeting sequence or complementary region 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 complementary region complementary to the contiguous nucleotide sequence of SEQ ID NO:140, wherein the targeting sequence or complementary region 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. Types of oligonucleotides

[0183] A variety of oligonucleotide types and / or structures can be used to target a target sequence in the methods herein, including but not limited to RNAi oligonucleotides, antisense oligonucleotides, miRNAs, etc. Any oligonucleotide type described herein or elsewhere is contemplated as a framework to incorporate the targeting sequences herein.

[0184] In some embodiments, the oligonucleotides herein inhibit the expression of a target sequence by engaging the RNA interference (RNAi) pathway either upstream or downstream of the involvement of Dicer. For example, RNAi oligonucleotides have been developed that are each about 19-25 nucleotides in size and have at least one 3'-overhang consisting of 1 to 5 nucleotides (see, e.g., U.S. Patent No. 8,372,968). Longer oligonucleotides have also been developed that are processed by Dicer to generate active RNAi products (see, e.g., U.S. Patent No. 8,883,996). Further work has resulted in extended dsRNAs where at least one end of at least one strand extends beyond the duplex targeting region, including structures where one strand contains a thermodynamically stable tetraloop structure (see, e.g., U.S. Patents Nos. 8,513,207 and 8,927,705, and International Patent Application Publication No. WO 2010 / 033225). Such structures can include single-stranded extensions (on one or both sides of the molecule) as well as double-stranded extensions.

[0185] In some embodiments, the oligonucleotides herein engage the RNAi pathway downstream of the involvement of Dicer (e.g., Dicer cleavage). In some embodiments, the oligonucleotides described herein are Dicer substrates. In some embodiments, after endogenous Dicer processing, a double-stranded nucleic acid of 19-23 nucleotides in length that is capable of reducing target mRNA expression is produced. 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 the two strands anneal to form a 19-bp duplex and a 2-nucleotide overhang at either or both 3'-ends. Longer oligonucleotide designs can also be employed, including oligonucleotides having a 23-nucleotide guide strand and a 21-nucleotide passenger strand, where there is 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 consisting of two 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 21-bp duplex region. See, e.g., U.S. Patents Nos. 9,012,138, 9,012,621, and 9,193,753.

[0186] In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand each having a length in the range of about 17 to 26 (e.g., 17 to 26, 20 to 25, or 21 - 23) nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand each having a length in the range of about 17 to 36 (e.g., 17 to 36, 20 to 25, or 21 - 23) nucleotides. In some embodiments, the oligonucleotides described herein comprise an antisense strand having a length of 19 - 30 nucleotides and a sense strand having a length of 19 - 50 nucleotides, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having a 1 - 4 nucleotide overhang at the 3' end of the antisense strand. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand each having a length in the range of about 19 - 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 a 3'-overhang is present on the sense strand or the antisense strand, or on both the sense strand and the antisense strand. In some embodiments, for an oligonucleotide having a sense strand and an antisense strand each having a length in the range of about 21 - 23 nucleotides, the 3'-overhang on the sense strand, the antisense strand, or both the sense strand and the antisense strand has a length of 1 or 2 nucleotides. In some embodiments, the oligonucleotide has a guide strand of 22 nucleotides and a passenger strand of 20 nucleotides, wherein there is 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 consisting 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 duplex region.

[0187] Other oligonucleotide designs for use with the compositions and methods of the present invention include: 16-mer siRNAs (see, e.g., NUCLEIC ACIDS IN CHEMISTRY AND BIOLOGY, Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., having a stem of 19 bp or shorter; see, e.g., Moore et al. (2010) METHODS MOL. BIOL. 629:141-58), blunt-end 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 duplex siRNAs (see, e.g., Chang et al. (2009) Mol. Ther. 17:725-32), fork siRNAs (see, e.g., Hohjoh (2004) FEBS Lett. 557:193-98), single-stranded 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 internally segmented interfering RNAs (siRNAs; see, e.g., Bramsen et al. (2007) Nucleic Acids Res. 35:5886-97). Other non-limiting examples of oligonucleotide constructs that can be used in some embodiments to reduce or inhibit STAT3 expression 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).

[0188] In addition, in some embodiments, the oligonucleotides used herein to reduce or inhibit the expression of a target sequence are single-stranded. Such constructs can include, but are not limited to, single-stranded RNAi molecules. Recent efforts have demonstrated the activity of single-stranded RNAi molecules (see, e.g., Matsui et al., (2016) Mol. Ther. 24:946-55). However, in some embodiments, the oligonucleotides herein are antisense oligonucleotides (ASOs). Antisense oligonucleotides are single-stranded oligonucleotides having a nucleobase sequence that, when written in the 5' to 3' direction, contains the reverse complementary sequence of the targeted segment of a particular nucleic acid and is appropriately modified (e.g., as a gapmer) to induce RNase H-mediated cleavage of its target RNA in cells, or (e.g., as a mixmer) to inhibit translation of the target mRNA in cells. The ASOs for use herein can be modified in any suitable manner known in the art, including, for example, as shown in U.S. Patent No. 9,567,587 (including, e.g., alterations in length, sugar moiety of the nucleobases (pyrimidines, purines), and heterocyclic moiety of the nucleobases). In addition, ASOs have been used for decades to reduce the expression of specific target genes (see, e.g., Bennett et al., (2017) Annu. Rev. Pharmacol. 57:81-105).

[0189] In some embodiments, the antisense oligonucleotide shares a complementary region 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. Double-stranded oligonucleotide

[0190] In some embodiments, the present disclosure provides double-stranded dsRNAs for targeting and inhibiting the expression of a target sequence (e.g., via the RNAi pathway), which comprise a sense strand (also referred to herein as the passenger strand) and an antisense strand (also referred to herein as the 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 duplex region, wherein the sense strand and the antisense strand or portions thereof bind to each other in a complementary manner (e.g., via Watson-Crick base pairing).

[0191] In some embodiments, the sense strand has a first region (R1) and a second region (R2), where R2 comprises a first sub-region (S1), a loop (L) such as a tetraloop (tetraL) or a triloop (triL), and a second sub-region (S2), where L, tetraL or triL is located between S1 and S2, and where S1 and S2 form a second duplex (D2). D2 can have different lengths. In some embodiments, the length of D2 is about 1-6 bp. In some embodiments, the length of D2 is 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5 or 4-5 bp. In some embodiments, the length of D2 is 1, 2, 3, 4, 5 or 6 bp. In some embodiments, the length of D2 is 6 bp.

[0192] In some embodiments, R1 of the sense strand and the antisense strand form a first duplex (D1). In some embodiments, the length of 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 some embodiments, the length of D1 ranges from about 12 to 30 nucleotides (e.g., the length is 12 to 30, 12 to 27, 15 to 22, 18 to 22, 18 to 25, 18 to 27, 18 to 30 or 21 to 30 nucleotides). In some embodiments, the length of D1 is at least 12 nucleotides (e.g., the length is at least 12, at least 15, at least 20, at least 25 or at least 30 nucleotides). In some embodiments, the length of D1 is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In some embodiments, the length of D1 is 20 nucleotides. In some embodiments, D1 comprising the sense strand and the antisense strand does not span the entire length of the sense strand and / or the antisense strand. In some embodiments, D1 comprising the sense strand and the antisense strand spans the entire length of the sense strand or the antisense strand or both. In certain embodiments, D1 comprising the sense strand and the antisense strand spans the entire length of both the sense strand and the antisense strand.

[0193] It should be understood that in some embodiments, when describing the structure of an oligonucleotide or other nucleic acid, reference may be made to the sequences presented in the sequence listing. In such embodiments, compared to the specified sequence, the actual oligonucleotide or other nucleic acid can have one or more alternative nucleotides (e.g., the RNA counterpart of a DNA nucleotide or the DNA counterpart of an RNA nucleotide) and / or one or more modified nucleotides and / or one or more modified internucleotide linkages and / or one or more other modifications, while still retaining substantially the same or similar complementary properties as the specified sequence.

[0194] In some embodiments, the double-stranded RNA (dsRNA) herein comprises a sense strand of 25 nucleotides and an antisense strand of 27 nucleotides, which, when acted upon by a nuclease, produces an antisense strand that incorporates into the mature RISC. In some embodiments, the sense strand of the 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 the 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 (e.g., 26, 27, 28, 29, or 30 nucleotides).

[0195] 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 length of the 3'-overhang on the antisense strand is about 1-8 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length). Generally, oligonucleotides for RNAi have a two-nucleotide overhang at the 3' end of the antisense (guide) strand. However, other overhangs are possible. In some embodiments, the overhang is a 3'-overhang that is 1 to 6 nucleotides in length, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides in length. However, in some embodiments, the overhang is a 5'-overhang that is 1 to 6 nucleotides in length, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides in length.

[0196] 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 on the 3' end of the antisense strand of the oligonucleotide herein include 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 on each 3' end of the oligonucleotide in the gapped tetracyclic 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. Typically, one or both of the two terminal GG nucleotides on each 3' end of the oligonucleotide are not complementary to the target.

[0197] In some embodiments, there are one or more (e.g., 1, 2, 3, 4, or 5) mismatches between the sense strand and the antisense strand. If there are more than one mismatch between the sense strand and the antisense strand, they can be positioned consecutively (e.g., 2, 3, or more consecutively), or scattered throughout the complementary region. In some embodiments, the 3' end of the sense strand contains one or more mismatches. In one embodiment, two mismatches are incorporated at the 3' end of the sense strand. In some embodiments, base mismatches or segment destabilization at the 3' end of the sense strand of the oligonucleotide improves the effectiveness of synthesizing duplexes in RNAi, which may be achieved by promoting the processing of the enzyme. a. Antisense strand

[0198] In some embodiments, the dsRNA comprises an antisense strand that is 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 oligonucleotides herein (e.g., RNAi oligonucleotides) comprise an antisense strand that is 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 can have an antisense strand that is 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, the oligonucleotide can have an antisense strand that ranges in length from about 12 to about 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 22, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides. In some embodiments, the oligonucleotide comprises an antisense strand that is 15 to 30 nucleotides in length. In some embodiments, the oligonucleotide can 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.

[0199] In some embodiments, the antisense strand of the oligonucleotide can be referred to as the "guide strand". For example, if the antisense strand is capable of engaging with an RNA-induced silencing complex (RISC) and binding to an Argonaute protein such as Ago2, or engaging or binding with one or more similar factors and guiding the silencing of a target gene, it can be referred to as the guide strand. In some embodiments, the sense strand complementary to the guide strand can be referred to as the "passenger strand".

[0200] In some embodiments, the oligonucleotides for targeting STAT3 disclosed herein comprise an antisense strand that comprises the sequence shown in SEQ ID NO: 333 or consists of the sequence shown in SEQ ID NO: 333. In some embodiments, the oligonucleotides 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 shown in SEQ ID NO: 333. In some embodiments, the oligonucleotides for targeting STAT3 disclosed herein (e.g., RNAi oligonucleotides) comprise an antisense strand that comprises the sequence shown in SEQ ID NO: 716 or consists of the sequence shown in SEQ ID NO: 716. In some embodiments, the oligonucleotides 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 shown in SEQ ID NO: 716. In some embodiments, the oligonucleotides for targeting STAT3 disclosed herein comprise an antisense strand that comprises the sequence shown in SEQ ID NO: 965 or consists of the sequence shown in SEQ ID NO: 965. In some embodiments, the oligonucleotides 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 shown in SEQ ID NO: 965. In some embodiments, the oligonucleotides for targeting STAT3 disclosed herein comprise an antisense strand that comprises the sequence shown in SEQ ID NO: 333 or consists of the sequence shown in SEQ ID NO: 333. In some embodiments, the oligonucleotides 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 shown in SEQ ID NO: 333. b. Sense strand

[0201] In some embodiments, the oligonucleotides (e.g., RNAi oligonucleotides) for targeting STAT3 mRNA and inhibiting STAT3 expression disclosed herein comprise a sense strand sequence as set forth in SEQ ID NO: 140. 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 as set forth in SEQ ID NO: 140. In some embodiments, the oligonucleotides (e.g., RNAi oligonucleotides) for targeting STAT3 mRNA and inhibiting STAT3 expression disclosed herein comprise a sense strand sequence as set forth in SEQ ID NO: 524. 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 as set forth in SEQ ID NO: 524. In some embodiments, the oligonucleotides for targeting STAT3 mRNA and inhibiting STAT3 expression disclosed herein comprise a sense strand sequence as set forth in SEQ ID NO: 875. 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 as set forth in SEQ ID NO: 875.

[0202] In some embodiments, the oligonucleotide comprises a sense strand (or passenger strand) having a length of up to about 40 nucleotides (e.g., a length of 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 some embodiments, the oligonucleotide can have a sense strand having a length of at least about 12 nucleotides (e.g., a length of 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 some embodiments, the oligonucleotide can have a sense strand having a length in the range of about 12 to about 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand having a length of 15 to 50 nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand having a length of 18 to 36 nucleotides. In some embodiments, the oligonucleotide can have a sense strand having a length of 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 some embodiments, the oligonucleotide comprises a sense strand having a length of 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 some embodiments, the oligonucleotides herein comprise a sense strand having a length of 36 nucleotides.

[0203] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a sense strand that comprises a stem-loop structure at its 3' end. 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.

[0204] In some embodiments, the stem-loop provides protection of the oligonucleotide from degradation (e.g., enzymatic degradation), promotes or improves targeting and / or delivery to a target cell, tissue, or organ (e.g., the liver), or both. For example, in some embodiments, the loop of the stem-loop consists of one or more modified nucleotides that promote, improve, or increase targeting to a target, inhibition of target gene expression, and / or delivery, uptake, and / or penetration into a target cell, tissue, or organ (e.g., the liver), or a combination thereof. In some embodiments, the stem-loop itself or a modification to the stem-loop does not affect or substantially does not affect the inherent gene expression inhibitory activity of the oligonucleotide, but promotes, improves, or increases the stability of the oligonucleotide (e.g., provides protection against degradation) and / or its delivery, uptake, and / or penetration into a target cell, tissue, or organ. In certain embodiments, the oligonucleotides herein comprise a sense strand that comprises (e.g., at its 3' end) a stem-loop as shown below: S1-L-S2, where S1 is complementary to S2, and where L forms a single-stranded loop of linked nucleotides having a length of up to about 10 nucleotides (e.g., a length of 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) between S1 and S2. In some embodiments, the loop (L) has a length of 3 nucleotides (referred to herein as a "three-loop"). In some embodiments, the loop (L) has a length of 4 nucleotides (referred to herein as a "four-loop"). In some embodiments, the loop (L) has a length of 5 nucleotides. In some embodiments, the loop (L) has a length of 6 nucleotides. In some embodiments, the loop (L) has a length of 7 nucleotides. In some embodiments, the loop (L) has a length of 8 nucleotides. In some embodiments, the loop (L) has a length of 9 nucleotides. In some embodiments, the loop (L) has a length of 10 nucleotides.

[0205] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a targeting sequence or complementary region that is complementary to a contiguous nucleotide sequence of SEQ ID NO: 140, and the oligonucleotide comprises a sense strand that comprises (e.g., at its 3' end) a stem-loop as shown below: S1-L-S2, where S1 is complementary to S2, and where L forms a single-stranded loop of linked nucleotides having a length of up to about 10 nucleotides (e.g., a length of 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) between S1 and S2. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is complementary to a contiguous nucleotide sequence of SEQ ID NO: 140, and the oligonucleotide comprises a sense strand that comprises (e.g., at its 3' end) a stem-loop as shown below: S1-L-S2, where S1 is complementary to S2, and where L forms a single-stranded loop of linked nucleotides having a length of 4 nucleotides between S1 and S2.

[0206] In some embodiments, the four - loop contains the sequence 5'-GAAA-3'. In some embodiments, the stem - loop contains the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQ ID NO:86).

[0207] In some embodiments, the sense strand contains a stem - loop structure at its 3' end. In some embodiments, the sense strand contains a stem - loop structure at its 5' end. In some embodiments, the stem is a duplex of length 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 bp. In some embodiments, the stem - loop provides protection of the molecule from degradation (e.g., enzymatic degradation) and promotes the targeting properties for delivery to target cells. For example, in some embodiments, the loop provides added nucleotides on which modifications can be made with substantially no effect on the gene expression inhibitory activity of the oligonucleotide. In certain embodiments, oligonucleotides are provided herein, wherein the sense strand contains (e.g., at its 3' end) a stem - loop as shown below: S1 - L - S2, where S1 is complementary to S2, and where L forms a loop of up to about 10 nucleotides in length (e.g., a loop of length 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) between S1 and S2. Figure 1 depicts a non - limiting example of such an oligonucleotide.

[0208] In some embodiments, the loop (L) of the stem - loop having the structure S1 - L - S2 as described herein is a three - loop. In some embodiments, the three - loop contains ribonucleotides, deoxyribonucleotides, modified nucleotides, ligands (e.g., delivery ligands), and combinations thereof.

[0209] In some embodiments, the loop of the stem - loop is a four - loop (e.g., within a nicked four - loop structure). The four - loop can contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Generally, the four - loop has 4 to 5 nucleotides. Duplex length

[0210] In some embodiments, the length of 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 some embodiments, the length of the duplex formed between the sense strand and the antisense strand ranges from 12 to 30 nucleotides (e.g., the length is 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 some embodiments, the length of 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 some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 12 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 13 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 14 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 15 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 16 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 17 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 18 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 19 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 20 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 21 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 22 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 23 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 24 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 25 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 26 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 27 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 28 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 29 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 30 nucleotides.In some embodiments, the duplex formed between the sense strand and the antisense strand does not span the entire length of the sense strand and / or the antisense strand. In some embodiments, the duplex between the sense strand and the antisense strand spans the entire length of the sense strand or the antisense strand. In some embodiments, the duplex between the sense strand and the antisense strand spans the entire length of both the sense strand and the antisense strand.

[0211] In some embodiments, the duplex between the sense strand and the antisense strand spans the entire length of both the sense strand and the antisense strand. In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO:875 and 965, respectively, wherein the length of the duplex formed between the sense strand and the antisense strand ranges from 12 to 30 nucleotides (e.g., a length of 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). Oligonucleotide termini

[0212] In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, wherein the terminus of either or both strands comprises a blunt end. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, which are separate strands that form an asymmetric duplex region having an overhang at the 3'-end of the antisense strand. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the terminus of either or both strands comprises an overhang containing one or more nucleotides. In some embodiments, the one or more nucleotides constituting the overhang are unpaired nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the 3'-end of the sense strand and the 5'-end of the antisense strand comprise blunt ends. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the 5'-end of the sense strand and the 3'-end of the antisense strand comprise blunt ends.

[0213] In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the 3'-end of either or both strands comprises a 3'-overhang containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the sense strand comprises a 3'-overhang containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the antisense strand comprises a 3'-overhang containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein both the sense strand and the antisense strand comprise 3'-overhangs containing one or more nucleotides.

[0214] In some embodiments, the length of the 3'-overhang is from about one (1) to twenty (20) nucleotides (e.g., from 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 some embodiments, the length of the 3'-overhang is from about one (1) to nineteen (19), one (1) to eighteen (18), one (1) to seventeen (17), one (1) to sixteen (16), one (1) to fifteen (15), one (1) to fourteen (14), one (1) to thirteen (13), one (1) to twelve (12), one (1) to eleven (11), one (1) to ten (10), one (1) to nine (9), one (1) to eight (8), one (1) to seven (7), one (1) to six (6), one (1) to five (5), one (1) to four (4), one (1) to three (3), or from about one (1) to two (2) nucleotides. In some embodiments, the length of the 3'-overhang is one (1) nucleotide. In some embodiments, the length of the 3'-overhang is two (2) nucleotides. In some embodiments, the length of the 3'-overhang is three (3) nucleotides. In some embodiments, the length of the 3'-overhang is four (4) nucleotides. In some embodiments, the length of the 3'-overhang is five (5) nucleotides. In some embodiments, the length of the 3'-overhang is six (6) nucleotides. In some embodiments, the length of the 3'-overhang is seven (7) nucleotides. In some embodiments, the length of the 3'-overhang is eight (8) nucleotides. In some embodiments, the length of the 3'-overhang is nine (9) nucleotides. In some embodiments, the length of the 3'-overhang is ten (10) nucleotides. In some embodiments, the length of the 3'-overhang is eleven (11) nucleotides. In some embodiments, the length of the 3'-overhang is twelve (12) nucleotides. In some embodiments, the length of the 3'-overhang is thirteen (13) nucleotides. In some embodiments, the length of the 3'-overhang is fourteen (14) nucleotides. In some embodiments, the length of the 3'-overhang is fifteen (15) nucleotides. In some embodiments, the length of the 3'-overhang is sixteen (16) nucleotides. In some embodiments, the length of the 3'-overhang is seventeen (17) nucleotides. In some embodiments, the length of the 3'-overhang is eighteen (18) nucleotides. In some embodiments, the length of the 3'-overhang is nineteen (19) nucleotides. In some embodiments, the length of the 3'-overhang is twenty (20) nucleotides.

[0215] In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the 5'-end of either or both strands comprises a 5'-overhang containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the sense strand comprises a 5'-overhang containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the antisense strand comprises a 5'-overhang containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein both the sense strand and the antisense strand comprise a 5'-overhang containing one or more nucleotides.

[0216] In some embodiments, the length of the 5'-overhang is from about one (1) to twenty (20) nucleotides (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 length of the 5'-overhang is from about one (1) to nineteen (19), one (1) to eighteen (18), one (1) to seventeen (17), one (1) to sixteen (16), one (1) to fifteen (15), one (1) to fourteen (14), one (1) to thirteen (13), one (1) to twelve (12), one (1) to eleven (11), one (1) to ten (10), one (1) to nine (9), one (1) to eight (8), one (1) to seven (7), one (1) to six (6), one (1) to five (5), one (1) to four (4), one (1) to three (3), or about one (1) to two (2) nucleotides. In some embodiments, the length of the 5'-overhang is one (1) nucleotide. In some embodiments, the length of the 5'-overhang is two (2) nucleotides. In some embodiments, the length of the 5'-overhang is three (3) nucleotides. In some embodiments, the length of the 5'-overhang is four (4) nucleotides. In some embodiments, the length of the 5'-overhang is five (5) nucleotides. In some embodiments, the length of the 5'-overhang is six (6) nucleotides. In some embodiments, the length of the 5'-overhang is seven (7) nucleotides. In some embodiments, the length of the 5'-overhang is eight (8) nucleotides. In some embodiments, the length of the 5'-overhang is nine (9) nucleotides. In some embodiments, the length of the 5'-overhang is ten (10) nucleotides. In some embodiments, the length of the 5'-overhang is eleven (11) nucleotides. In some embodiments, the length of the 5'-overhang is twelve (12) nucleotides. In some embodiments, the length of the 5'-overhang is thirteen (13) nucleotides. In some embodiments, the length of the 5'-overhang is fourteen (14) nucleotides. In some embodiments, the length of the 5'-overhang is fifteen (15) nucleotides. In some embodiments, the length of the 5'-overhang is sixteen (16) nucleotides. In some embodiments, the length of the 5'-overhang is seventeen (17) nucleotides. In some embodiments, the length of the 5'-overhang is eighteen (18) nucleotides. In some embodiments, the length of the 5'-overhang is nineteen (19) nucleotides. In some embodiments, the length of the 5'-overhang is twenty (20) nucleotides.

[0217] In some embodiments, one or more (e.g., 2, 3, 4, 5, or more) nucleotides at the 3' or 5' end of the sense strand and / or the 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 contains a 2' modification or it contains 2'-O-methoxyethyl. In some embodiments, the last or last two terminal nucleotides at the 3' end of the antisense strand are complementary to the target. In some embodiments, the last or last two nucleotides at the 3' end of the antisense strand are not complementary to the target.

[0218] In some embodiments, the oligonucleotides (e.g., RNAi oligonucleotides) disclosed herein comprise a sense strand and an antisense strand, wherein the 3' end of the sense strand comprises a stem-loop as described herein and the 3' end of the antisense strand comprises a 3'-overhang as described herein. In some embodiments, the oligonucleotides (e.g., RNAi oligonucleotides) herein comprise a sense strand and an antisense strand that form a nicked tetraloop structure as described herein, wherein the 3' end of the sense strand comprises a stem-loop, wherein the loop is the tetraloop as described herein, and wherein the 3' end of the antisense strand comprises a 3'-overhang as described herein. In some embodiments, the length of the 3'-overhang is two (2) nucleotides. In some embodiments, both of the two (2) nucleotides that make up the 3'-overhang comprise guanine (G) nucleobases. Typically, one or both of the nucleotides that make up the 3'-overhang of the antisense strand are not complementary to the target mRNA. Oligonucleotide Modifications a. Sugar Modifications

[0219] In some embodiments, the modified sugars (also referred to herein as sugar analogs) include modified deoxyribose or ribose moieties, wherein, for example, one or more modifications occur at the 2', 3', 4', and / or 5'-carbon positions of the sugar. In some embodiments, the modified sugars may also include non-natural alternative carbon structures such as those present in locked nucleic acids ("LNA"; see, e.g., Koshkin et al., (1998) TETRAHEDON 54:3607-3630), unlocked nucleic acids ("UNA"; see, e.g., Snead et al., (2013) MOL. THER-NUCL. ACIDS 2:e103), and bridged nucleic acids ("BNA"; see, e.g., Imanishi and Obika (2002) CHEM COMMUN. (CAMB) 21:1653-1659).

[0220] In some embodiments, the nucleotide modification in the sugar includes 2'-modification. In some embodiments, the 2'-modification can be 2'-O-propargyl, 2'-O-propylamino, 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 in the sugar includes modification of the sugar ring, which can include modification of one or more carbons of the sugar ring. For example, the modification of the sugar of the nucleotide can include connection of the 2'-oxygen of the sugar to the 1'-carbon or 4'-carbon of the sugar, or connection of the 2'-oxygen to the 1'-carbon or 4'-carbon via an ethylene or methylene bridge. In some embodiments, the modified nucleotide has an acyclic sugar lacking the 2'-carbon to 3'-carbon bond. In some embodiments, the modified nucleotide has a thiol group, for example at the 4'-position of the sugar.

[0221] In some embodiments, the oligonucleotides described herein contain at least about 1 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 contains at least about 1 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 contains at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more).

[0222] 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 nucleotide contains a 2'-modification (e.g., 2'-F or 2'-OMe, 2'-MOE, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid). In some embodiments, the modified nucleotide contains a 2'-modification (e.g., 2'-F or 2'-OMe).

[0223] In some embodiments, the present disclosure provides oligonucleotides having different modification patterns. In some embodiments, the oligonucleotides herein comprise a sense strand having a modification pattern as set forth in the Examples and Sequence Listing and an antisense strand having a modification pattern as set forth in the Examples and Sequence Listing.

[0224] In some embodiments, the oligonucleotides (e.g., RNAi oligonucleotides) disclosed herein comprise an antisense strand having nucleotides modified with 2'-F. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising nucleotides modified with 2'-F and 2'-OMe. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand having nucleotides modified with 2'-F. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand comprising nucleotides modified with 2'-F and 2'-OMe.

[0225] 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 comprise a 2'-fluoro modification. In some embodiments, about 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 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, about 15-25%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the nucleotides of the oligonucleotide comprise a 2'-fluoro modification. In some embodiments, about 19% of the nucleotides in the dsRNAi oligonucleotide comprise a 2'-fluoro modification.

[0226] In some embodiments, the modified oligonucleotides comprise a sense strand sequence having a modification pattern as set forth in FIG. 1 or Example 7 and an antisense strand having a modification pattern as set forth in FIG. 1 or Example 7. In some embodiments, for these oligonucleotides, one or more of positions 8, 9, 10, or 11 of the sense strand are modified with a 2'-F group. In other embodiments, for these oligonucleotides, the sugar moiety at each nucleotide at positions 1-7 and 12-20 in the sense strand is modified with 2'-OMe.

[0227] In some embodiments, the antisense strand has 3 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 of the antisense strand and optionally up to 1 nucleotide at positions 3, 3, 7, and 10 are modified with 2'-F. In some embodiments, the sugar moieties at positions 2, 5, and 14 of the antisense strand and optionally up to 3 nucleotides at positions 3, 4, 7, and 10 are modified with 2'-F. In other embodiments, the sugar moiety at each of positions 2, 5, and 14 of the antisense strand is modified with 2'-F. In other embodiments, the sugar moiety at each of positions 1, 2, 5, and 14 of the antisense strand is modified with 2'-F. In other embodiments, the sugar moiety at each of positions 2, 4, 5, and 14 of the antisense strand is modified with 2'-F. In still other embodiments, the sugar moiety at each of positions 1, 2, 3, 5, 7, and 14 of the antisense strand is modified with 2'-F. In other embodiments, the sugar moiety at each of positions 2, 3, 4, 5, 7, and 14 of the antisense strand is modified with 2'-F. In yet another embodiment, the sugar moiety at each of positions 1, 2, 3, 5, 10, and 14 of the antisense strand is modified with 2'-F. In other embodiments, the sugar moiety at each of positions 2, 3, 4, 5, 10, and 14 of the antisense strand is modified with 2'-F. In another embodiment, the sugar moiety at each of positions 2, 3, 5, 7, 10, and 14 of the antisense strand is modified with 2'-F. In yet another embodiment, the sugar moiety at each of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand is modified with 2'-F.

[0228] In some embodiments, the oligonucleotides provided herein comprise an antisense strand, wherein the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 of the antisense strand is modified with 2'-OMe.

[0229] In some embodiments, the oligonucleotides provided herein comprise an antisense strand, wherein the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 of the antisense strand is modified with 2'-F.

[0230] In some embodiments, the oligonucleotides provided herein comprise an antisense strand, wherein the sugar moiety at position 1, position 6, position 8, position 9, position 11, position 12, position 13, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 of the antisense strand is modified with 2'-OMe.

[0231] In some embodiments, the oligonucleotides provided herein comprise an antisense strand, wherein the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 of the antisense strand is modified with a modification selected from 2'-O-propargyl, 2'-O-propylamino, 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).

[0232] In some embodiments, the oligonucleotides provided herein comprise a sense strand, wherein the sugar moiety at positions 8-11 of the sense strand is modified with 2'-F. In some embodiments, the oligonucleotides provided herein comprise a sense strand, wherein the sugar moieties at positions 3, 8, 9, 10, 12, 13 and 17 of the sense strand are modified with 2'-F. In some embodiments, the oligonucleotides provided herein comprise a sense strand, wherein the sugar moieties at positions 1-7 and 12-17 or 12-20 of the sense strand are modified with 2'-OMe. In some embodiments, the oligonucleotides provided herein comprise a sense strand, wherein the sugar moieties at positions 1-7, 12-27 and 31-36 of the sense strand are modified with 2'-OMe. In some embodiments, the oligonucleotides provided herein comprise a sense strand, wherein the sugar moieties at positions 1-7 and 12-36 of the sense strand are modified with 2'-OMe. In some embodiments, the oligonucleotides provided herein comprise a sense strand, wherein the sugar moiety of each nucleotide at positions 1-7 and 12-17 or 12-20 is modified with a modification selected from 2'-O-propargyl, 2'-O-propylamino, 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, wherein the sugar moieties at positions 1-2, 4-7, 11, 14-16 and 18-20 of the sense strand are modified with 2'-OMe. In some embodiments, the oligonucleotides provided herein comprise a sense strand, wherein the sugar moiety of each nucleotide at positions 1-2, 4-7, 11, 14-16 and 18-20 is modified with a modification selected from 2'-O-propargyl, 2'-O-propylamino, 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).

[0233] In some embodiments, the oligonucleotides provided herein comprise a sense strand, wherein the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 of the sense strand is modified with 2'-F.

[0234] In some embodiments, the oligonucleotides provided herein comprise a sense strand, wherein the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 of the sense strand is modified with 2'-OMe.

[0235] In some embodiments, the oligonucleotides provided herein comprise a sense strand, wherein the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 of the sense strand is modified with a modification selected from 2'-O-propargyl, 2'-O-propylamino, 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).

[0236] In some embodiments, the oligonucleotides provided herein comprise a sense strand and an antisense strand, wherein the sense strand has the sugar moiety at positions 8-11 modified with 2'-F and the sugar moieties at positions 1-7 and 12-36 modified with 2’-OMe, and the antisense strand has the sugar moiety at each of positions 2, 3, 4, 5, 7, 10, and 14 modified with 2'-F and the sugar moieties at positions 1, 6, 8, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, and 22 modified with 2'-OMe. b. 5'-terminal phosphate

[0237] 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 by phosphatases or other enzymes, which may limit their bioavailability in vivo. In some embodiments, the oligonucleotide includes a 5'-phosphate analog that is resistant to such degradation. In some embodiments, the phosphate analog can be an oxy-methylphosphonate, a vinylphosphonate, or a malonylphosphonate. In certain embodiments, the 1'-end of the oligonucleotide chain is attached to a chemical moiety ("phosphate mimic") that mimics the electrostatic and steric properties of the native 5'-phosphate group.

[0238] 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. WO 2018 / 045317. In some embodiments, the oligonucleotides herein include a 4'-phosphate analog at the 5'-terminal nucleotide. In some embodiments, the phosphate analog is an oxy-methylphosphonate, wherein the oxygen atom of the oxy-methyl group is bonded to the sugar moiety (e.g., at its 4'-carbon) or an analog thereof. In other embodiments, the 4'-phosphate analog is a thio-methylphosphonate or an amino-methylphosphonate, wherein the sulfur atom of the thio-methyl group or the nitrogen atom of the amino-methyl group is bonded to the 4'-carbon of the sugar moiety or an analog thereof. In certain embodiments, the 4'-phosphate analog is an oxy-methylphosphonate. In some embodiments, the oxy-methylphosphonate is represented by the formula –O–CH2–PO(OH)2 or –O–CH2–PO(OR)2, wherein R is independently selected from H, CH3, alkyl, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3, or a protecting group. In certain embodiments, the alkyl is CH2CH3. More typically, R is independently selected from H, CH3, or CH2CH3.

[0239] In some embodiments, the oligonucleotides provided herein comprise an antisense strand that includes a 4'-phosphate analog at the 5'-terminal nucleotide, wherein the 5'-terminal nucleotide has the following structure: 4'-O-Monomethylphosphonate-2'-O-methyluridine phosphorothioate [MePhosphonate-4O-mUs]. Chemical Formula 1 c. Modified internucleotide linkages

[0240] In some embodiments, the oligonucleotide may comprise modified internucleoside linkages. In some embodiments, phosphonate modifications or substitutions may result in an oligonucleotide comprising at least about 1 (e.g., at least 1, at least 2, at least 3, or at least 5) modified internucleotide linkages. In some embodiments, any of the oligonucleotides disclosed herein comprise from about 1 to about 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3, or 1 to 2) modified internucleotide linkages. In some embodiments, any of the oligonucleotides disclosed herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages.

[0241] The modified internucleotide linkages can be dithiophosphate linkages, 4'-O-methylenephosphonate linkages, phosphorothioate linkages, phosphotriester linkages, thioalkylphosphonate linkages, thioalkylphosphotriester linkages, phosphoramidate linkages, phosphonate linkages, or boranophosphate linkages. In some embodiments, at least one modified internucleotide linkage of any of the oligonucleotides disclosed herein is a phosphorothioate linkage. In some embodiments, at least one modified internucleotide linkage of any of the oligonucleotides disclosed herein is a 4'-O-methylenephosphonate linkage.

[0242] In some embodiments, the oligonucleotides described herein have phosphorothioate linkages 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 linkages between each 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 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand. d. Base modifications

[0243] In some embodiments, the oligonucleotides herein have one or more modified nucleobases. In some embodiments, the modified nucleobases (also referred to herein as base analogs) are attached at the 1'-position of the nucleotide sugar moiety. In certain embodiments, the modified nucleobases are nitrogenous bases. In certain embodiments, the modified nucleobases do not contain nitrogen atoms. See, e.g., U.S. Patent Application Publication No. 2008 / 0274462. In some embodiments, the modified nucleotides contain universal bases. However, in certain embodiments, the modified nucleotides do not contain nucleobases (abasic).

[0244] In some embodiments, the universal base is a heterocyclic moiety that is located at the 1'-position of the nucleotide sugar moiety in the modified nucleotide or at an equivalent position in a nucleotide sugar moiety substitution such that when present in a duplex, it can base-pair opposite more than one type of base with substantially no change in the structure of the duplex. In some embodiments, a single-stranded nucleic acid containing a universal base forms a duplex with a target nucleic acid that has a lower T m than a duplex formed with a complementary nucleic acid, compared to a reference single-stranded nucleic acid (e.g., an oligonucleotide) that is fully complementary to the target nucleic acid. However, in some embodiments, a single-stranded nucleic acid containing a universal base forms a duplex with a target nucleic acid that has a higher T m than a duplex formed with a nucleic acid containing the mismatched base, compared to a reference single-stranded nucleic acid in which the universal base has been replaced with a base to create a single mismatch.

[0245] 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). e. Reversible Modifications

[0246] Although certain modifications can be made to protect oligonucleotides from the in vivo environment before reaching the target cell, once the oligonucleotides reach the cytosol of the target cell, they may reduce the potency or activity of the oligonucleotides. Reversible modifications can be made such that the molecule retains the desired properties extracellularly and then the modification is removed upon entry into the cytoplasmic environment of the cell. For example, the reversible modification can be removed by the action of intracellular enzymes or by chemical conditions within the cell (e.g., by reduction of intracellular glutathione).

[0247] In some embodiments, the reversibly modified nucleotides comprise a glutathione-sensitive moiety. Typically, nucleic acid molecules have been chemically modified with a cyclic disulfide moiety to mask the negative charge generated by the internucleotide phosphorodiester bond and to improve cellular uptake and nuclease resistance. See U.S. Patent Application Publication No. 2011 / 0294869, International Patent Application Publication Nos. WO 2014 / 088920 and WO 2015 / 188197, and Meade et al., (2014) Nat. Biotechnol. 32:1256-63. This reversible modification of the internucleotide phosphorodiester bond is designed to be cleaved intracellularly by the reducing environment of the cytosol (e.g., glutathione). Early examples include neutralizing phosphotriester modifications reported to be cleavable intracellularly (see Dellinger et al., (2003) J. Am. Chem. Soc. 125:940-50).

[0248] In some embodiments, such reversible modifications allow for protection during in vivo administration (e.g., transport through the bloodstream and / or the lysosomal / endosomal compartments of cells), in which the oligonucleotides will be exposed to nucleases and other harsh environmental conditions (e.g., pH). When released into the cytosol of a cell where the glutathione level is higher than in the extracellular space, the modification is reversed, resulting in the unmodified oligonucleotide. Compared to available options using irreversible chemical modifications, the use of a reversible glutathione-sensitive moiety may allow for the introduction of larger chemical groups into the oligonucleotide of interest. This is because these larger chemical groups will be removed in the cytosol and thus will not interfere with the biological activity of the oligonucleotide within the cytosol of the cell. Thus, 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 alter its release kinetics.

[0249] In some embodiments, the glutathione-sensitive moiety is attached to the sugar of a nucleotide. In some embodiments, the glutathione-sensitive moiety is attached to the 2'-carbon of the sugar of a modified nucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 5'-carbon of the sugar, particularly when the modified nucleotide is the 5'-terminal nucleotide of an oligonucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 3'-carbon of the sugar, particularly when the modified nucleotide is the 3'-terminal nucleotide of an oligonucleotide. In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group. See, for example, U.S. Provisional Patent Application No. 62 / 378,635, filed Aug. 23, 2016, entitled Compositions Comprising Reversibly Modified Oligonucleotides and Uses Thereof. Targeting ligand

[0250] 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 can help avoid adverse effects in other organs or avoid excessive loss of the oligonucleotide to cells, tissues, or organs that will not benefit from the oligonucleotide. Targeting the oligonucleotide to one or more cells or one or more organs can be achieved by a variety of methods. Conjugation of the oligonucleotide with a tissue- or cell-specific antibody, small molecule, or targeting ligand can facilitate delivery of the oligonucleotide to one or more target cells or tissues and alter the accumulation of the oligonucleotide in one or more target cells or tissues (Chernolovskaya et al., (2019) FRONT PHARMACOL. 10:444). For example, conjugation of the oligonucleotide with a saturated fatty acid (e.g., C22) can facilitate delivery to cells or tissues such as adipose tissue or immune cells, and such ligands are more readily taken up than conventional oligonucleotide ligands. Thus, in some embodiments, the oligonucleotides disclosed herein are modified to facilitate targeting and / or delivery to a tissue, cell, or organ (e.g., to facilitate delivery of the oligonucleotide to the liver). In certain embodiments, the oligonucleotides disclosed herein are modified to facilitate delivery of the oligonucleotide to cells of the immune system. In certain embodiments, the oligonucleotides disclosed herein are 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 to one or more targeting ligands.

[0251] In some embodiments, the targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a peptide, a polypeptide, a protein or a portion of a protein (e.g., an antibody or an antibody fragment), or a lipid. In some embodiments, the targeting ligand is an aptamer. For example, the targeting ligand can be an RGD peptide for targeting tumor vasculature or glioma cells, a CREKA peptide for targeting tumor vasculature or stoma, transferrin, lactoferrin or an aptamer for targeting the transferrin receptor expressed on the CNS vasculature, or an anti-EGFR antibody for targeting EGFR on glioma cells. In certain embodiments, the targeting ligand is one or more GalNAc moieties.

[0252] 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 to 4 nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, the targeting ligand is conjugated to 2 to 4 nucleotides at either end of the sense or antisense strand (e.g., the targeting ligand is conjugated to the overhang or extension of 2 to 4 nucleotides at the 5' or 3' end of the sense or antisense strand), such that the targeting ligand is analogous to the bristles of a toothbrush and the oligonucleotide is analogous to the toothbrush handle. For example, the oligonucleotide can comprise a stem-loop at the 5' or 3' end of the sense strand, and 1, 2, 3 or 4 nucleotides of the loop of the stem can be individually conjugated to the targeting ligand. In some embodiments, the oligonucleotide provided by the present disclosure (e.g., dsRNA) comprises a stem-loop at the 3' end of the sense strand, wherein the loop of the stem-loop comprises a tricyclic or tetracyclic ring, and wherein 3 or 4 nucleotides constituting the tricyclic or tetracyclic ring are individually conjugated to the targeting ligand, respectively. In some embodiments, the oligonucleotide provided by the present disclosure (e.g., an RNAi oligonucleotide) comprises a stem-loop at the 3' terminus of the sense strand, wherein the loop of the stem-loop comprises a tetracyclic ring, and wherein 3 nucleotides of the tetracyclic ring are individually conjugated to the targeting ligand.

[0253] GalNAc is a high-affinity ligand for ASGPR, which is mainly expressed on the sinusoidal surface of hepatocytes and plays a major role in the binding, internalization and subsequent clearance of circulating glycoproteins (asialoglycoproteins) containing terminal galactose or GalNAc residues. The conjugation (either indirect or direct) of GalNAc moieties to the 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 to at least one or more GalNAc moieties, wherein the GalNAc moieties target the oligonucleotides to ASGPR expressed on human liver cells (e.g., human hepatocytes). In some embodiments, the GalNAc moieties target the oligonucleotides to the liver.

[0254] In some embodiments, the oligonucleotides of the present disclosure are conjugated directly or indirectly to monovalent GalNAc. In some embodiments, the oligonucleotide is conjugated directly or indirectly to more than one monovalent GalNAc (i.e., conjugated to 2, 3, or 4 monovalent GalNAc moieties, and typically conjugated to 3 or 4 monovalent GalNAc moieties). In some embodiments, the oligonucleotide is conjugated to one or more divalent GalNAc, trivalent GalNAc, or tetravalent GalNAc moieties.

[0255] 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 to 4 nucleotides of the tetraloop are each conjugated to a separate GalNAc. In some embodiments, 1 to 3 nucleotides of the triloop are each conjugated to a separate GalNAc. In some embodiments, the targeting ligand is conjugated to 2 to 4 nucleotides at either end of the sense or antisense strand (e.g., the ligand is conjugated to the overhang or extension of 2 to 4 nucleotides at the 5' or 3' end of the sense or antisense strand), such that the GalNAc moieties are analogous to the bristles of a toothbrush and the oligonucleotide is analogous to the 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, where each GalNAc moiety is conjugated to 1 nucleotide.

[0256] In some embodiments, the tetraloop is any combination of adenine and guanine nucleotides.

[0257] In some embodiments, the tetraloop (tetraL) has a monovalent GalNAc moiety attached via any of the linkers described herein to any one or more guanine nucleotides of the tetraloop, as shown in Chemical Formula 2 below (X = heteroatom):

[0258] In some embodiments, the tetraloop (tetraL) has a monovalent GalNAc attached via any of the linkers described herein to any one or more adenine nucleotides of the tetraloop, as shown in Chemical Formula 3 below (X = heteroatom):

[0259] In some embodiments, the oligonucleotides herein comprise a monovalent GalNAc attached to a guanine nucleotide, designated [ademG-GalNAc] or 2'-aminodiethoxymethanol-guanine-GalNAc, as shown in Chemical Formula 4 below:

[0260] 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 in Chemical Formula 5 below:

[0261] An example of such a conjugation is shown below for a loop comprising the nucleotide sequence GAAA from 5' to 3' (Chemical Formula 6) (L = linker, X = heteroatom), showing the stem attachment points. For example, such a loop can be present at positions 27-30 of the sense strand as shown in FIG. 1. In the chemical formula, is used to describe the attachment point to the oligonucleotide chain (Chemical Formula 6).

[0262] A targeting ligand can be attached to the nucleotide using a suitable method or chemistry (e.g., click chemistry). In some embodiments, a click linker is used to conjugate the targeting ligand to the nucleotide. In some embodiments, an acetal-based linker is used to conjugate the targeting ligand to the nucleotide of any of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. WO 2016 / 100401. In some embodiments, the linker is an unstable linker. However, in other embodiments, the linker is stable. Examples of loops comprising the nucleotide GAAA from 5' to 3' are shown below, where the GalNAc moiety is attached to the nucleotide of the loop using an acetal linker (Chemical Formulas 7 and 8). For example, such a loop can be present at positions 27-30 of any of the sense strands as shown in FIG. 1. In the chemical formula, is the attachment point to the oligonucleotide chain (Chemical Formulas 7 and 8).

[0263] As mentioned, a targeting ligand can be attached to the nucleotide using various suitable methods or chemical synthesis techniques (e.g., click chemistry). In some embodiments, a click linker is used to conjugate the targeting ligand to the nucleotide. In some embodiments, an acetal-based linker is used to conjugate the targeting ligand to the nucleotide of any of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. WO 2016 / 100401. In some embodiments, the linker is an unstable linker. However, in other embodiments, the linker is a stable linker.

[0264] In some embodiments, a duplex 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 oligonucleotides herein do not have GalNAc conjugated thereto. Structure of conjugated STAT3 targeting oligonucleotide

[0265] In some embodiments, the STAT3 targeting oligonucleotides described herein comprise a nucleotide sequence having a complementary region complementary to a STAT3 mRNA target sequence and one or more targeting ligands, wherein the nucleotide sequence comprises one or more nucleosides (nucleic acids) conjugated to one or more targeting ligands represented by Formula I-a: or a pharmaceutically acceptable salt thereof, wherein: 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 1 and R 2 on the same carbon 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 an optionally substituted group selected from C 1-6 aliphatic groups, phenyl, 4-7 membered saturated or partially unsaturated heterocycles having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and 5-6 membered heteroaryl rings having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; each R is independently hydrogen, a suitable protecting group or an optionally substituted group selected from C 1-6 aliphatic groups, phenyl, 4-7 membered saturated or partially unsaturated heterocycles having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and 5-6 membered heteroaryl rings 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 lipid conjugate moieties (LC), carbohydrates, amino sugars or GalNAc; and each LC is independently a lipid conjugate moiety comprising a saturated or unsaturated straight-chain or branched-chain C 1-50 hydrocarbon chain, wherein 0-10 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 an optionally substituted divalent ring selected from phenylene, 8-10 membered bicyclic arylene, 4-7 membered saturated or partially unsaturated carbocyclic group, 4-11 membered saturated or partially unsaturated spirocarbocyclic group, 8-10 membered bicyclic saturated or partially unsaturated carbocyclic group, 4-7 membered saturated or partially unsaturated heterocyclic group having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur, 4-11 membered saturated or partially unsaturated spiroheterocyclic group having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, 8-10 membered bicyclic saturated or partially unsaturated heterocyclic group having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, 5-6 membered heteroarylene having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or 8-10 membered bicyclic heteroarylene having 1-5 heteroatoms independently selected from nitrogen, oxygen or sulfur; n is 1-10; L is a covalent bond or a divalent saturated or unsaturated straight-chain or branched-chain C 1-50 hydrocarbon chain, wherein 0-10 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-, -V 1 CR 2 W 1 - or replaced by; m is 1-50; 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, R 3 is hydrogen, a suitable protecting group, a suitable prodrug or an optionally substituted group selected from C 1-6 aliphatic group, phenyl, 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; X 2is 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'-terminus of a nucleoside, nucleotide or oligonucleotide; Y 2 is hydrogen, a suitable protecting group, a phosphoramidite analogue, an internucleotide linking group attached to the 5'-terminus of a nucleoside, nucleotide or oligonucleotide, or a linking group attached to a solid support; and Z is -O-, -S-, -NR- or -CR2-.

[0266] In some embodiments, the STAT3-targeting oligonucleotide comprises one or more nucleic acids conjugated to a targeting ligand and represented by Formula II-a: or a pharmaceutically acceptable salt thereof.

[0267] In some embodiments, the STAT3-targeting oligonucleotide comprises one or more nucleic acids conjugated to a targeting ligand and represented by Formula II-b or II-c: or a pharmaceutically acceptable salt thereof, wherein: L 1 is a covalent bond, a monovalent or divalent saturated or unsaturated straight-chain or branched-chain C 1-50 hydrocarbon chain, wherein 0-10 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- or replaced; R 4 is hydrogen, R A or a suitable amine protecting group; and R 5 is adamantyl, or a saturated or unsaturated straight-chain or branched-chain C 1-50 hydrocarbon chain, wherein 0-10 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.

[0268] In some embodiments, R 5 is selected from

[0269] In some embodiments, R 5 is selected from:

[0270] In some embodiments, R 5 is In some embodiments, R 5 is In some embodiments, R 5 is In some embodiments, R 5 is In some embodiments, R 5 is In some embodiments, R 5 is In some embodiments, R 5 is In some embodiments, R 5 is In some embodiments, R 5 is In some embodiments, R 5 is In some embodiments, R 5 is In some embodiments, R 5 is In some embodiments, R 5 is In some embodiments, R 5 is

[0271] In some embodiments, the STAT3-targeting oligonucleotide comprises one or more nucleic acids conjugated to a targeting ligand and represented by Formula II-Ib or II-Ic: or a pharmaceutically acceptable salt thereof; wherein B is a nucleobase or hydrogen; m is 1 - 50; X 1 is -O- or -S-; Y is hydrogen, R 3is 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 analogue, an internucleotide linking group attached to the 5'-end of a nucleoside, nucleotide or oligonucleotide, or a linking group attached to a solid support; R 5 is adamantyl, or a saturated or unsaturated straight-chain or branched C 1-50 hydrocarbon chain, wherein 0-10 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-; and R is hydrogen, a suitable protecting group or an optionally substituted group selected from C 1-6 aliphatic group, 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.

[0272] In some embodiments, R 5 is selected from

[0273] In some embodiments, R 5 is

[0274] In some embodiments, R 5 is

[0275] In some embodiments, the nucleotide sequence of the STAT3-targeting oligonucleotide comprises 1-10 targeting ligands. In some embodiments, the nucleotide sequence comprises 1, 2 or 3 targeting ligands.

[0276] In some embodiments, the STAT3-targeting oligonucleotide is a double-stranded molecule. In some embodiments, the STAT3-targeting oligonucleotide is an RNAi molecule.

[0277] In some embodiments, the STAT3-targeting oligonucleotide comprises a sense strand of 36 nucleotides, numbered 1-36 from 5' to 3'.

[0278] In some embodiments, the STAT3-targeting oligonucleotide comprises a lipid conjugated to the 5'-terminal nucleotide of the sense strand. In some embodiments, the STAT3-targeting oligonucleotide comprises a C16 lipid conjugated to the 5'-terminal nucleotide of the sense strand. In some embodiments, the STAT3-targeting oligonucleotide comprises a C18 lipid conjugated to the 5'-terminal nucleotide of the sense strand.

[0279] In some embodiments, any STAT3-targeting oligonucleotide sequence described herein comprises a lipid conjugated to the 5'-terminal nucleotide of the sense strand. In some embodiments, any STAT3-targeting oligonucleotide sequence described herein comprises a C16 lipid conjugated to the 5'-terminal nucleotide of the sense strand. In some embodiments, any STAT3-targeting oligonucleotide sequence described herein comprises a C18 lipid conjugated to the 5'-terminal nucleotide of the sense strand.

[0280] In some embodiments, the STAT3-targeting oligonucleotide comprises a lipid conjugated to the 5'-terminal nucleotide of the sense strand, wherein the lipid is

[0281] In some embodiments, the STAT3-targeting oligonucleotide comprises a lipid conjugated to the 5'-terminal nucleotide of the sense strand, wherein the lipid is

[0282] In some embodiments, the STAT3-targeting oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising the sequence shown in SEQ ID NO:140, and the antisense strand comprising the sequence shown in SEQ ID NO:333, wherein the sense strand comprises a lipid conjugated to the 5'-terminal nucleotide. In some embodiments, the STAT3-targeting oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising the sequence shown in SEQ ID NO:140, and the antisense strand comprising the sequence shown in SEQ ID NO:333, wherein the sense strand comprises a C16 lipid conjugated to the 5'-terminal nucleotide. In some embodiments, the STAT3-targeting oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising the sequence shown in SEQ ID NO:140, and the antisense strand comprising the sequence shown in SEQ ID NO:333, wherein the sense strand comprises a C18 lipid conjugated to the 5'-terminal nucleotide.

[0283] In some embodiments, the STAT3-targeting oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprises the sequence shown in SEQ ID NO:875, and the antisense strand comprises the sequence shown in SEQ ID NO:965, wherein the sense strand comprises a lipid conjugated to the 5'-terminal nucleotide. In some embodiments, the STAT3-targeting oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprises the sequence shown in SEQ ID NO:875, and the antisense strand comprises the sequence shown in SEQ ID NO:965, wherein the sense strand comprises a C16 lipid conjugated to the 5'-terminal nucleotide. In some embodiments, the STAT3-targeting oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprises the sequence shown in SEQ ID NO:875, and the antisense strand comprises the sequence shown in SEQ ID NO:965, wherein the sense strand comprises a C18 lipid conjugated to the 5'-terminal nucleotide.

[0284] In some embodiments, the STAT3-targeting oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprises the sequence shown in SEQ ID NO:140, and the antisense strand comprises the sequence shown in SEQ ID NO:333, wherein the sense strand comprises a lipid conjugated to the 5'-terminal nucleotide, and wherein the lipid is

[0285] In some embodiments, the STAT3-targeting oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprises the sequence shown in SEQ ID NO:140, and the antisense strand comprises the sequence shown in SEQ ID NO:333, wherein the sense strand comprises a lipid conjugated to the 5'-terminal nucleotide, and wherein the lipid is

[0286] In some embodiments, the STAT3-targeting oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprises the sequence shown in SEQ ID NO:875, and the antisense strand comprises the sequence shown in SEQ ID NO:965, wherein the sense strand comprises a lipid conjugated to the 5'-terminal nucleotide, and wherein the lipid is In some embodiments, the STAT3-targeting oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprises the sequence shown in SEQ ID NO:875, and the antisense strand comprises the sequence shown in SEQ ID NO:965, wherein the sense strand comprises a lipid conjugated to the 5'-terminal nucleotide, and wherein the lipid is

[0287] In some embodiments, the STAT3-targeting oligonucleotide comprises an antisense strand of 15 to 30 nucleotides and a sense strand of 15 to 40 nucleotides, wherein the sense strand and the antisense strand form a duplex region, wherein the antisense strand comprises a complementary region complementary to the STAT3 mRNA target sequence expressed in immune cells associated with the tumor microenvironment, wherein the sense strand comprises a stem-loop at its 3'-end, the stem-loop comprising a tetraloop containing 4 nucleosides, and wherein the 5'-terminal nucleotide of the sense strand is represented by Formula II-Ib: wherein B is selected from adenine and guanine nucleobases, and wherein R 5 is a hydrocarbon chain. In some embodiments, m is 1, X1 is O, Y2 is an internucleotide linking group attached to the 5'-end of the nucleoside, Y is represented by 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.

[0288] 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 In some embodiments, the hydrocarbon chain is a C18 hydrocarbon chain. In some embodiments, the C18 hydrocarbon chain is represented by In some embodiments, the oligonucleotide comprises a sense strand that comprises the sequence of SEQ ID NO:140, wherein the sense strand comprises a C18 lipid.

[0289] Exemplary STAT3-targeting oligonucleotides In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises the sense strand and the antisense strand described herein, wherein the sense strand and the antisense strand are modified based on the following pattern

[0291] Sense strand: [ademXs-C18][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][mX][mX][mX][mX][mX][mX][mX][mX][mX] Hybridizes to Antisense strand: ​[MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][mX][fX] [mX][mX][mX][fX][mX][mX][mX][mX][mX][mXs][mXs][mX] (Keywords are provided in Table 7). In some embodiments, C# is C16 or C18.

[0292] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises the sense strand and the antisense strand described herein, wherein the sense strand and the antisense strand are modified based on the following pattern Sense strand: [ademXs-C#][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][mX][mX][mX][mX][mX][mX][mX][mX][mX] Hybridizes to antisense strand: [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][mX][fX][mX][mX][mX][fX][mX][mX][mX][mX][mX][mXs][mXs][mX] (Keywords are provided in Table 7).

[0293] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises a sense strand and an antisense strand, the sense strand and the antisense strand respectively comprising SEQ ID NO:875 and 965, wherein the sense strand and the antisense strand are modified based on the following pattern Sense strand: [ademXs-C18][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][mX][mX][mX][mX][mX][mX][mX][mX][mX] Hybridizes to Antisense strand: [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][mX][fX] [mX][mX][mX][fX][mX][mX][m]X[mX][mX][mXs][mXs][mX] (The keywords are provided in Table 7). In some embodiments, C# is C16 or C18.

[0294] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises a sense strand and an antisense strand, which respectively comprise SEQ ID NO: 875 and 965, wherein the sense strand and the antisense strand are modified in the following pattern Sense strand: [ademXs-C#][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][mX][mX][mX][mX][mX][mX][mX][mX][mX] Hybridize to Antisense strand: [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][mX][fX][mX][mX][mX][fX][mX][mX][mX][mX][mX][mXs][mXs][mX](The keywords are provided in Table 7).

[0295] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises a sense strand and an antisense strand, which respectively comprise SEQ ID NO: 1222 and 1145.

[0296] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 140. In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 875.

[0297] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises the antisense strand sequence of SEQ ID NO: 333. In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises the antisense strand sequence of SEQ ID NO: 965.

[0298] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO:875 and the antisense strand of SEQ ID NO:965.

[0299] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO:1222.

[0300] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises the antisense strand sequence of SEQ ID NO:1145.

[0301] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO:1222 and the antisense strand sequence of SEQ ID NO:1145.

[0302] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA described herein exhibits minimal off-target effects. For example, in some embodiments, the oligonucleotide described herein reduces STAT3 expression without reducing STAT1 expression, or reduces STAT1 expression to a lesser extent than it reduces STAT3 expression. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence shown in SEQ ID NO:875, and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO:965, wherein the oligonucleotide reduces STAT3 expression without reducing STAT1 expression, or reduces STAT1 expression to a lesser extent than it reduces STAT3 expression. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence shown in SEQ ID NO:1222, and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO:1145, wherein the oligonucleotide reduces STAT3 expression without reducing STAT1 expression, or reduces STAT1 expression to a lesser extent than it reduces STAT3 expression.

[0303] 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%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

[0304] In some embodiments, the oligonucleotide for reducing the 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 human STAT3 mRNA.

[0305] In some embodiments, the oligonucleotide for reducing the 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%.

[0306] In some embodiments, the oligonucleotide for reducing the 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 at the 5'-terminal nucleotide of the sense strand.

[0307] In some embodiments, the oligonucleotide for reducing the 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 at the 5'-terminal nucleotide of the sense strand.

[0308] In some embodiments, the oligonucleotide for reducing the 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 at the 5'-terminal nucleotide of the sense strand and reduces human STAT3 mRNA.

[0309] In some embodiments, the oligonucleotide for reducing the 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 at the 5'-terminal nucleotide of the sense strand and reduces human STAT3 mRNA by at least 75%.

[0310] In some embodiments, the oligonucleotide for reducing the 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 at the 5'-terminal nucleotide of the sense strand and reduces human STAT3 mRNA by at least 75%.

[0311] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO:1222 and the antisense strand sequence of SEQ ID NO:1145, wherein the oligonucleotide reduces human STAT3 mRNA.

[0312] In some embodiments, the oligonucleotide for reducing the expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO:1222 and the antisense strand sequence of SEQ ID NO:1145, wherein the oligonucleotide reduces STAT3 mRNA by at least 75%. Formulation

[0313] A variety of formulations have been developed to facilitate the use of oligonucleotides. For example, formulations that minimize degradation, promote delivery and / or uptake, or provide another beneficial property to the oligonucleotide in the formulation can be used to deliver the oligonucleotide to a subject or a cellular environment. In some embodiments, the oligonucleotide is formulated in a buffered solution such as phosphate buffered saline, liposomes, micellar structures, and shells.

[0314] Oligonucleotide formulations with 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.

[0315] 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 can otherwise be formulated for administration to the cells, tissues, organs, or body of a subject in need (see, e.g., Remington: THE SCIENCE AND PRACTICE OF PHARMACY, 22nd Edition, Pharmaceutical Press, 2013).

[0316] In some embodiments, the formulations herein contain excipients. In some embodiments, the excipients confer improved stability, improved absorption, improved solubility, and / or therapeutic enhancement of the active ingredient to the composition. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, tris base, or sodium hydroxide) or a vehicle (e.g., a buffer solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotide is lyophilized to extend its shelf life and then made into a solution prior to use (e.g., administration to a subject). Thus, the excipient in a composition containing any of the oligonucleotides described herein can be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature modifier (e.g., dextran, Ficoll TM or gelatin).

[0317] In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.

[0318] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (when water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. In many cases, it is preferred to include an isotonic agent, such as sugar, polyols like mannitol, sorbitol, sodium chloride, in the composition. A sterile injectable solution can be prepared by introducing the required amount of the oligonucleotide, as needed, into a selected solvent with one or a combination of the ingredients listed above, followed by filtration sterilization.

[0319] In some embodiments, the composition can contain at least about 0.1% or more of the therapeutic agent, although the percentage of the active ingredient can be between about 1% and about 80% or more of the total weight or volume of the composition. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and thus, a variety of dosages and treatment regimens may be desirable.

[0320] Although several embodiments relate to hepatic-targeted delivery of any of the oligonucleotides herein, targeting of other tissues is also contemplated. Programmed death ligand 1 (PD-L1) inhibitor

[0321] 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 inhibits the association of PD-L1 and PD-1. In some embodiments, the PD-L1 inhibitor is specific for PD-L1. 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.

[0322] In some embodiments, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the anti-PD-L1 antibody is avelumab. In some embodiments, the anti-PD-L1 antibody is envafolimab. In some embodiments, the anti-PD-L1 antibody is durvalumab.

[0323] In some embodiments, the anti-PD-L1 antibody is any anti-PD-L1 antibody known in the art, including but not limited to the anti-PD-L1 antibodies disclosed 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. 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.

[0324] In some embodiments, the anti-PD-1 antibody is nivolumab. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, the anti-PD-1 antibody is cemiplimab.

[0325] In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of from 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.

[0326] In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of from 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.

[0327] In some embodiments, the antibodies are generated using display technologies. Display technologies for generating antibody polypeptides include any display technology (e.g., display library screening technologies). In some embodiments, synthetic antibodies are designed, selected, or optimized by screening target antigens using display technologies (e.g., phage display technology). Phage display libraries can contain millions to billions of phage vectors, each expressing a unique antibody fragment on its viral coat. Such libraries can provide a rich and diverse resource for selecting potentially hundreds of antibody fragments with different affinity levels for one or more antigens of interest (McCafferty et al., 1990. Nature. 348:552-4; Edwards, B.M. 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). Typically, the antibody fragments present in such libraries include scFv antibody fragments, which comprise a fusion protein of V H and V L antibody domains linked by a flexible linker. In some cases, the scFv may contain identical sequences, except for the unique sequences of the variable loops encoding the CDRs. In some cases, the scFv is expressed as a fusion protein linked to a viral coat protein (e.g., the N-terminus of the viral capsid protein). The VL chain can be expressed separately for assembly with the VH chain in the periplasm, after which the complex is incorporated into the viral coat. The precipitated library members can be sequenced from the bound phage to obtain the cDNA encoding the desired scFv. The antibody variable domains or CDRs from such sequences can be directly incorporated into antibody sequences for recombinant antibody production, or mutated by in vitro affinity maturation and used for further optimization.

[0328] In some embodiments, yeast surface display technology is utilized to generate the sequences of polypeptides to be encoded in the viral genome. In some embodiments, recombinant antibodies are developed by displaying antibody fragments of interest as fusions on the yeast surface, where the proteins interact with proteins and small molecules in solution. Magnetic separation and flow cytometry can be used to isolate scFvs with affinity for the desired receptor from the yeast surface. Several cycles of yeast surface display and isolation can be performed to obtain scFvs with desired properties by directed evolution.

[0329] Methods for determining the affinity of an antibody for its antigen are known in the art. Exemplary methods for determining binding affinity employ surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that allows analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, such as using a BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further description, see Jonsson, U. et al., (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., (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. Kit

[0330] In some embodiments, the present disclosure provides a kit that includes the STAT3 oligonucleotides herein, and instructions for administering the STAT3 oligonucleotides to a subject. In some embodiments, the present disclosure provides a kit that includes the STAT3 oligonucleotides herein, and instructions for administering the STAT3 oligonucleotides to a subject who has received or is receiving a PD-L1 inhibitor. In some embodiments, the kit includes the oligonucleotides 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 includes at least one vial, well, test tube, flask, bottle, syringe, or other container device in which the oligonucleotides are placed and, in some cases, appropriately aliquoted. In some embodiments where additional components are provided, the kit includes additional containers for holding the components. The kit may also include means for containing the oligonucleotides, and any other reagents that are tightly restricted for commercial sale. Such containers can include injection-molded or blow-molded plastic containers that hold the required vials. These containers and / or kits can include labels with instructions for use and / or warnings.

[0331] In some embodiments, the kit comprises the STAT3 oligonucleotides herein, and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the oligonucleotides and instructions for treating a disease, disorder or condition associated with STAT3 expression or delaying its progression in a subject in need thereof. In some embodiments, the kit comprises the oligonucleotides herein, and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the oligonucleotides and instructions for treating a disease, disorder or condition associated with STAT3 expression or delaying its progression in a subject in need thereof, wherein the subject has received or is receiving a PD-L1 inhibitor. In some embodiments, the kit comprises the STAT3 oligonucleotides herein, and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the oligonucleotides and instructions for treating cancer in a subject in need thereof or delaying cancer progression. In some embodiments, the kit comprises the oligonucleotides herein, and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the oligonucleotides and instructions for treating cancer in a subject in need thereof or delaying cancer progression, wherein the subject has received or is receiving a PD-L1 inhibitor.

[0332] In some embodiments, the kit comprises a PD-L1 inhibitor, and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the oligonucleotides and instructions for treating a disease, disorder or condition in a subject in need thereof or delaying its progression, wherein the subject has received or is receiving the STAT3 oligonucleotides herein. In some embodiments, the kit comprises a PD-L1 inhibitor, and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the oligonucleotides and instructions for treating cancer in a subject in need thereof or delaying cancer progression, wherein the subject has received or is receiving the STAT3 oligonucleotides herein. Examples

[0333] While the present disclosure has been described with reference to specific embodiments set forth in the following examples, those skilled in the art will understand that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the present disclosure. Furthermore, the following examples are provided by way of illustration and are not intended to limit the scope of the present disclosure in any way. Additionally, modifications may be made to adapt a situation, material, composition of matter, process, or a process step to 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 techniques specifically described below were used. Abbreviations Ac: Acetyl AcOH: Acetic acid ACN: Acetonitrile Ad: Adamantyl AIBN: 2,2’-Azobisisobutyronitrile Anhyd: Anhydrous Aq: Aqueous B2Pin2: Bis(pinacolato)diboron - 4,4,4’,4’,5,5,5’,5’-Octamethyl-2,2’-bis(1,3,2-dioxaborolane) BINAP: 2,2’-Bis(diphenylphosphino)-1,1’-binaphthyl BH3: Borane Bn: Benzyl Boc: tert-Butyloxycarbonyl Boc2O: Di-tert-butyl dicarbonate BPO: Benzoyl peroxide BuOH: n-Butanol CDI: Carbonyldiimidazole COD: Cyclooctadiene d: Day DABCO: 1,4-Diazabicyclo[2.2.2]octane DAST: Diethylaminosulfur trifluoride dba: Dibenzylideneacetone DBU: 1,8-Diazabicyclo[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’-Dimethoxytriphenylmethyl 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 hr: Hour 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-Chloroperoxybenzoic acid M: Molar concentration MeCN: Acetonitrile MeOH: Methanol Me2S: Dimethyl sulfide MeONa: Sodium methoxide Mel: Iodomethane min: Minute mL: Milliliter mM: Millimolar concentration mmol: Millimole MPa: Megapascal MOMCl: Methyl chloromethyl ether MsCl: Methanesulfonyl chloride MTBE: Methyl tert-butyl ether nBuLi: n-Butyllithium 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 ℃: Degree Celsius Pd / C: Palladium on carbon Pd(OAc)2: Palladium(II) acetate PBS: Phosphate Buffered Saline PE: Petroleum ether POCl3: Phosphorus oxychloride PPh3: Triphenylphosphine PyBOP: (Benzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexafluorophosphate Rel: Relative R.T. or rt: Room temperature s or sec: Second sat: Saturated SEMCl: Chloromethyl 2-(trimethylsilyl)ethyl ether SFC: Supercritical Fluid Chromatography SOCl2: Thionyl chloride 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: p-Toluenesulfonic acid UPLC: Ultra Performance Liquid Chromatography wt: Weight Xantphos: 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene Example 1: Preparation of double-stranded RNAi oligonucleotides General synthetic method

[0334] The following examples are intended to illustrate the disclosure and should not be construed as limiting it. Temperatures are given in degrees Celsius (°C). If not otherwise mentioned, all evaporations are carried out under reduced pressure, preferably between about 15 mmHg and 100 mmHg (= 20 - 133 mbar). The structures of the final products, intermediates, and starting materials are confirmed by standard analytical methods, such as microanalysis and spectral characteristics, such as MS, IR, NMR. The abbreviations used are conventional in the art.

[0335] All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts used to synthesize the nucleic acids or analogs thereof of the present disclosure are commercially available or can be generated by organic synthesis methods known to those of ordinary skill in the art (METHODS OF ORGANIC SYNTHESIS, Thieme, Volume 21 (Houben-Weyl 4th Edition, 1952)). In addition, the nucleic acids or analogs thereof of the present disclosure can be generated by organic synthesis methods known to those of ordinary skill in the art, as shown in the following examples.

[0336] Unless otherwise stated, all reactions are carried out under nitrogen or argon.

[0337] Proton NMR ( 1 H NMR) is carried out in deuterated solvents. In some of the nucleic acids or analogs thereof disclosed herein, one or more 1 H shifts overlap with the residual proton (proteo) solvent signals; these signals have not been reported in the experiments provided below. As described in the following examples, in certain exemplary embodiments, nucleic acids or analogs thereof are prepared according to the following general procedure. It should be understood that while the general method describes the synthesis of certain nucleic acids or analogs thereof of the present disclosure, the following general method and other methods known to those of ordinary skill in the art can be applied to all nucleic acids or analogs thereof and to each subclass and species of these nucleic acids or analogs thereof, as described herein. Example 1a: Synthesis of 2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxocane-9-yl)oxy)methoxy)ethoxy)ethan-1-aminium formate (1-6)

[0338] 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 tetraisopropyl disiloxane 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 X 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.

[0339] Compound 1-2 (37.00 g, 60.33 mmol) in DMSO (20 mL) 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 from ACN (30 mL) to give compound 1-3 (15.65 g, 38.4%) as a white solid.

[0340] Compound 1-3 (20.00 g, 29.72 mmol) in DCM (120 mL) was treated with Fmoc-amino-ethoxyethanol (11.67 g, 35.66 mmol) at 25 °C. The mixture was stirred to give a clear solution, then treated with 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 indicated >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.

[0341] 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. Then the organic layer was separated, washed with water (100 mL) and diluted with DCM (130 mL). The solution was treated in four portions with fumaric acid (7.05 g, 60.76 mmol) and molecular sieves (26.34 g). 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. 11H NMR (400 MHz, d6-DMSO) δ 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). 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)

[0342] 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 at 0 °C with hexanoic acid (10.33 mL, 82.61 mmol), HATU (33.66 g, 88.52 mmol), and DMAP (10.81 g, 147.52 mmol). 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 column chromatography on silica gel (1:1 hexanes / acetone) gave compound 2-1a as a white solid (34.95 g, 71.5%).

[0343] 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 dropwise with triethylamine trihydrofluoride (20.61 mL, 126.58 mmol) 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 x 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. The 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, concentrated, and the crude product was obtained as a slurry. Flash column chromatography on silica gel (1:1 hexane / acetone) gave compound 2-3a (30.05 g, 33.8 mmol, 79.9%) as a white solid.

[0344] Under a nitrogen atmosphere, the 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). Bis(diisopropylamino)chlorophosphine (9.02 g, 33.80 mmol) was added dropwise to this 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 (3×50 mL). The combined organic layers were washed with saturated NaHCO3 (50 mL), concentrated, and the crude solid was obtained, 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: 1 H NMR (400 MHz, 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 - 2.80 (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 (162 MHz, d6-DMSO) 149.43, 149.18.

[0345] Compound 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%): 1 H NMR (400 MHz, 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 - 6.80 (m, 4H), 6.21 - 6.15 (m, 1H), 5.23 - 5.17 (m, 1H), 4.80 - 4.69 (m, 3H), 4.40 - 4.21 (m, 2H), 3.91 - 3.80 (m, 1H), 3.74 (s, 6H), 3.74 - 3.52 (m, 3H), 3.50 - 3.20 (m, 6H), 3.14 - 3.09 (m, 2H), 3.09 (s, 1H), 2.83 - 2.79 (m, 1H), 2.68 - 2.62 (m, 1H), 2.05 - 1.97 (m, 2H), 1.50 - 1.38 (m, 2H), 1.31 - 1.10 (m, 18H), 1.08 - 1.05 (m, 2H), 0.85 - 0.78 (m, 3H); 31 P NMR (162 MHz, d6-DMSO) 149.43, 149.19.

[0346] Compound 2-4c was obtained as an off-white solid (36.60 g, 66.3%): 11H NMR (400 MHz, d6-DMSO) δ 11.22 (s, 1H), 8.64 - 8.59 (m, 2H), 8.05 - 8.00 (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 - 6.80 (m, 4H), 6.21 - 6.15 (m, 1H), 5.25 - 5.17 (m, 1H), 4.80 - 4.69 (m, 3H), 4.40 - 4.21 (m, 2H), 3.91 - 3.80 (m, 1H), 3.74 (s, 6H), 3.74 - 3.50 (m, 3H), 3.50 - 3.20 (m, 6H), 3.14 - 3.09 (m, 2H), 3.09 (s, 1H), 2.83 - 2.79 (m, 1H), 2.68 - 2.62 (m, 1H), 2.05 - 1.99 (m, 2H), 1.50 - 1.38 (m, 2H), 1.33 - 1.12 (m, 38H), 1.08 - 1.05 (m, 2H), 0.86 - 0.80 (m, 3H); 31 31P NMR (162 MHz, d6-DMSO) δ 149.42, 149.17.

[0347] Compound 2-4d was obtained as an off-white solid (26.60 g, 72.9%): 1 1H NMR (400 MHz, d6-DMSO) δ 11.22 (s, 1H), 8.64 - 8.59 (m, 2H), 8.05 - 8.00 (m, 2H), 7.73 - 7.70 (m, 1H), 7.67 - 7.60 (m, 1H), 7.59 - 7.51 (m, 2H), 7.38 - 7.33 (m, 2H), 7.30 - 7.25 (m, 7H), 6.89 - 6.80 (m, 4H), 6.21 - 6.15 (m, 1H), 5.22 - 5.17 (m, 1H), 4.80 - 4.69 (m, 3H), 4.40 - 4.21 (m, 2H), 3.91 - 3.80 (m, 1H), 3.74 (s, 6H), 3.74 - 3.52 (m, 3H), 3.50 - 3.20 (m, 6H), 3.14 - 3.09 (m, 2H), 3.09 (s, 1H), 2.83 - 2.79 (m, 1H), 2.68 - 2.62 (m, 1H), 2.05 - 1.99 (m, 2H), 1.50 - 1.38 (m, 2H), 1.35 - 1.08 (m, 38H), 1.08 - 1.05 (m, 2H), 0.85 - 0.79 (m, 3H); 31P NMR (162 MHz, d6-DMSO) 149.47, 149.22.

[0348] Compound 2-4e was obtained as a white solid (38.10 g, 54.0%): 1 H NMR (400 MHz, d6-DMSO) 11.21 (s, 1H), 8.64 - 8.59 (m, 2H), 8.05 - 8.00 (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 - 6.80 (m, 4H), 6.21 - 6.15 (m, 1H), 5.23 - 5.17 (m, 1H), 4.80 - 4.69 (m, 3H), 4.40 - 4.21 (m, 2H), 3.91 - 3.80 (m, 1H), 3.73 (s, 6H), 3.74 - 3.52 (m, 3H), 3.47 - 3.22 (m, 6H), 3.14 - 3.09 (m, 2H), 3.09 (s, 1H), 2.83 - 2.79 (m, 1H), 2.68 - 2.62 (m, 1H), 2.05 - 1.99 (m, 2H), 1.50 - 1.38 (m, 2H), 1.35 - 1.06 (m, 46H), 1.08 - 1.06 (m, 2H), 0.85 - 0.77 (m, 3H); 31 P NMR (162 MHz, d6-DMSO) 149.41, 149.15. Example 2. Synthesis of GalXC RNAi Oligonucleotide-Lipid Conjugates Scheme 1. Synthesis of GalXC RNAi Oligonucleotide-Lipid Conjugates with Mono-Lipids (Linear and Branched) Conjugated to Tetracyclic. The conjugation was achieved by amide coupling reaction after synthesis.

[0349] The R1COOH group represents fatty acids C8:0, C10:0, C11:0, C12:0, C14:0, C16:0, C17:0, C18:0, C18:1, C18:2, C22:5, C22:0, C24:0, C26:0, C22:6, C24:1, diacyl C16:0 or diacyl C18:1. Synthetic sense 1 and antisense 1 were prepared by solid-phase synthesis. Synthesis of conjugated sense 1a - 1i.

[0350] Conjugated sense 1a was synthesized by the synthetic suffixation method. In an Eppendorf tube 1, a solution of octanoic acid (0.58 mg, 4 μmol) in DMA (0.75 mL) was treated with HATU (1.52 mg, 4 μmol) at room temperature. In an Eppendorf tube 2, a solution of oligo sense 1 (10.00 mg, 0.8 μmol) in H2O (0.25 mL) was treated with DIPEA (1.39 μL, 8 μmol). The solution in Eppendorf tube 1 was added to Eppendorf tube 2 and mixed using a Thermomixer at room temperature. After LC-MS analysis indicated completion of the reaction, the reaction mixture was diluted with 5 mL of water and purified by reverse-phase XBridge C18 column using a 5-95% gradient of 100 mM TEAA in ACN and H2O. The product fractions were concentrated under reduced pressure using a Genevac. The combined residual solvents were dialyzed against water (1X), brine (1X), and water (3X) using an Ultra-15 Centrifugal (3K). The Amicon membrane was washed with water (3X 2 mL), and then the combined solvents were lyophilized to obtain an amorphous white solid of conjugated sense 1a (6.43 mg, 64% yield). The combined residual solvents were dialyzed against water (1X), brine (1X), and water (3X) using an Ultra-15 Centrifugal (3K). The Amicon membrane was washed with water (3X 2 mL), and then the combined solvents were lyophilized to obtain an amorphous white solid of conjugated sense 1a (6.43 mg, 64% yield).

[0351] Conjugated sense 1b - 1i were prepared using a procedure similar to that described for the synthesis of conjugated sense 1a and obtained in yields of 42% - 69%.

[0352] Annealing of duplexes 1a - 1j.

[0353] Conjugated sense 1a (10 mg, measured by weight) was dissolved in 0.5 mL of deionized water to prepare a 20 mg / mL solution. Antisense 1 (10 mg, measured by OD) was dissolved in 0.5 mL of deionized water to prepare a 20 mg / mL solution, which was used for titration of the conjugated sense strand and quantification of the duplex amount. Based on the calculation of the molar amounts of the conjugated sense and antisense strands, a certain proportion of the required antisense 1 was added to the conjugated sense 1a solution. The resulting mixture was stirred at 95 °C for 5 min and cooled to room temperature. The annealing process was monitored by ion-exchange HPLC. According to the annealing progress, several portions of antisense 1 were further added to complete the annealing with a purity > 95%. The solution was lyophilized to obtain duplex 1a (C8), and its amount was calculated based on the molar amount of the antisense strand consumed in the annealing.

[0354] Duplexes 1b - 1i were prepared using the same procedure as described for the annealing of duplex 1a (C8).

[0355] The following Scheme 1-2 depicts the synthesis of a nicked tetracyclic GalXC conjugate with a single lipid on the ring. The post-synthetic conjugation was achieved by Cu-catalyzed alkyne-azide cycloaddition reaction.

[0356] Sense 1B and antisense 1B were prepared by solid-phase synthesis.

[0357] Synthesis of conjugated sense 1j.

[0358] In Eppendorf tube 1, a solution of oligomer (10.00 mg, 0.8 umol) in a 3:1 mixture of DMA / H2O (0.5 mL) was treated with lipid linker azide (11.26 mg, 4 umol). In Eppendorf tube 2, CuBr dimethyl sulfide (1.64 mg, 8 umol) was dissolved in ACN (0.5 mL). Both solutions were degassed by bubbling N2 through them for 10 min. Then the ACN solution of CuBrSMe2 was added to tube 1, and the resulting mixture was stirred at 40 °C. After LC-MS analysis indicated completion of the reaction, the reaction mixture was diluted with 0.5 M EDTA (2 mL) and dialyzed against water (2X) using Ultra-15 Centrifugal (3K). The crude reaction was purified by reverse-phase XBridge C18 column using a 5-95% gradient of 100 mM TEAA in ACN (adulterated with 30% IPA) and H2O. The product fractions were concentrated under reduced pressure using Genevac. The combined residual solvent was dialyzed against water (1X), brine (1X), and water (3X) using Ultra-15 Centrifugal (3K). The Amicon membrane was washed with water (3X 2 mL), and the combined solvent was lyophilized to give an amorphous white solid of conjugated sense 1j (6.90 mg, 57% yield).

[0359] Duplex 1j (PEG2K-diacyl C18) was prepared using the same procedure as described for annealing of duplex 1a (C8).

[0360] The following Scheme 1-3 depicts the synthesis of a nicked tetracyclic GalXC conjugate with two lipids on the ring using a post-synthetic conjugation method. Sense 2 and antisense 2 were prepared by solid-phase synthesis.

[0361] Conjugated sense 2a and 2b were prepared using a procedure similar to that described for the synthesis of conjugated sense 1a, but using 10 eq of lipid, 10 eq of HATU, and 20 eq of DIPEA.

[0362] Duplex 2a (2XC11) and 2b (2XC22) were prepared using the same procedure as described for annealing of duplex 1a (C8).

[0363] The following Schemes 1-4 depict the synthesis of GalXC with a fully thiophosphorylated stem-loop conjugated to a single lipid using the synthetic suffix conjugation method. Sense 3 and Antisense 3 were prepared by solid-phase synthesis.

[0364] Conjugated Sense 3a was prepared using a procedure similar to that described for synthetically conjugated Sense 1a and obtained in 65% yield.

[0365] Duplex 3a (PS-C22) was prepared using the same procedure as described for annealing of duplex 1a (C8).

[0366] The following Schemes 1-5 depict the synthesis of GalXC with a short sense strand conjugated to a single lipid using the synthetic suffix conjugation method. Sense 4 and Antisense 4 were prepared by solid-phase synthesis.

[0367] Conjugated Sense 4a was prepared using a procedure similar to that described for synthetically conjugated Sense 1a and obtained in 74% yield.

[0368] Duplex 4a (SS-C22) was prepared using the same procedure as described for annealing of duplex 1a (C8).

[0369] The following Schemes 1-6 depict the synthesis of a nicked tetracyclic GalXC conjugated to a tricyclohexylamine moiety on the loop using the synthetic suffix conjugation method. Sense 5 and Antisense 5 were prepared by solid-phase synthesis.

[0370] Conjugated Sense 5a and 5b were prepared using a procedure similar to that described for synthetically conjugated Sense 1a and obtained in 42%-73% yield.

[0371] Duplex 5a (3X adamantane) and duplex 5b (3X acetyladamantane) were prepared using the same procedure as described for annealing of duplex 1a (C8).

[0372] The following Schemes 1-7 depict an example of the solid-phase synthesis of a nicked tetracyclic GalXC conjugated to a lipid on the loop. Synthesis of conjugated Sense 6.

[0373] The conjugated sense 6 was prepared by solid-phase synthesis using a commercial oligonucleotide synthesizer. Oligonucleotides were synthesized using 2'-modified nucleoside phosphoramidites such as 2'-F or 2'-OMe and fatty acid amide nucleoside phosphoramidites linked with 2'-diethoxymethanol. Oligonucleotide synthesis was carried out in the 3' to 5' direction on a solid support using standard oligonucleotide synthesis protocols. In these efforts, 5-ethylthio-1H-tetrazole (ETT) was used as the activator for the coupling reaction. An iodine solution was used for the oxidation of phosphite triesters. 3-(Dimethylaminomethylene)amino-3H-1,2,4-dithiazole-3-thione (DDTT) was used to form phosphorothioate bonds. The synthesized oligonucleotide was treated with concentrated aqueous ammonium solution for 10 h. Ammonia was removed from the suspension and the solid support residue was removed by filtration. The crude oligonucleotide was treated with TEAA, analyzed, and purified by strong anion exchange high performance liquid chromatography (SAX-HPLC). The fractions were combined and dialyzed against water (3X), brine (1X), and water (3X) using Ultra-15 Centrifugal (3K). The remaining solvent was then lyophilized to obtain the desired conjugated sense 6.

[0374] Duplex 6 was prepared using the same procedure as described for annealing against duplex 1a (C8). Scheme 8. Synthesis of nicked tetracyclic GalXC conjugated with one adamantane unit on the ring by a post-synthesis conjugation method. Synthesis of conjugated sense 7a and 7b

[0375] Conjugated sense 7a and sense 7b were obtained using the same method or a substantially similar method as for the synthesis of conjugated sense 5. Synthesis example of duplex 7a and 7b

[0376] Duplex 7a and duplex 7b were obtained using the same method or a substantially similar method as for the synthesis of duplex 5.

[0377] Scheme 9. Synthesis of nicked tetracyclic GalXC conjugated with two adamantane units on the ring by a post-synthesis conjugation method. Synthesis of conjugated sense 8a and 8b

[0378] Conjugated sense 8a and sense 8b were obtained using the same method or a substantially similar method as for the synthesis of conjugated sense 5. Synthesis example of duplex 8a and 8b

[0379] Duplex 8a and duplex 8b were obtained using the same method or a substantially similar method as for the synthesis of duplex 5.

[0380] Schemes 1-10 below depict the synthesis of GalXC with short sense and short stem-loop conjugated to single lipid using synthetic suffixation method. Synthesis of sense 9a

[0381] The conjugated sense 9a is obtained using the same method or a substantially similar method as that for the synthesis of conjugated sense 5. Synthesis example of duplex 9a

[0382] The duplex 9a is obtained using the same method or a substantially similar method as that for the synthesis of duplex 5.

[0383] Schemes 1-11 below depict the synthesis of GalXC conjugated to single lipid at the 5'-end using synthetic suffixation method. Synthesis of conjugated sense 10a

[0384] The conjugated sense 10a is obtained using the same method or a substantially similar method as that for the synthesis of conjugated sense 5. Synthesis example of duplex 10a

[0385] The duplex 10a is obtained using the same method or a substantially similar method as that for the synthesis of duplex 5.

[0386] Schemes 1-12a and 1-12b below depict the synthesis of GalXC with blunt ends conjugated to single lipid at the 3'-end or 5'-end using synthetic suffixation method. Synthesis of conjugated sense 11a and 12a

[0387] The conjugated sense 11a and 12a are obtained using the same method or a substantially similar method as that for the synthesis of conjugated sense 5. Synthesis example of duplex 11a and 12a

[0388] The duplex 11a and 12a are obtained using the same method or a substantially similar method as that for the synthesis of duplex 5.

[0389] The conjugated duplex 8D and duplex 9D are obtained using the same method or a substantially similar method as that for the synthesis of duplex 5.

[0390] Subsequently, an acyl chain was conjugated to a nucleic acid inhibitor molecule targeting the STAT3 gene, which is a gene expressed in the tissue of interest. A passenger strand with a 2'-amine linker [ademA] was used for solid-phase post-conjugation. The same chemistry was used to conjugate different types of lipids to generate a series of conjugates ( Figure 1A and Figure 1B ). SAR studies were performed to identify lipid conjugates that can be used to deliver the payload to the tissue of interest for mediating target knockdown. Example 3: Tissue-specific targets in MDSC cell populations and tumor-draining lymph nodes.

[0391] STAT3 is involved in immunosuppression, and there are numerous reported examples in the literature. Targeting STAT3 transcription through the RNAi mechanism may overcome challenges in the development of pharmacological STAT3 inhibitors. For these reasons, STAT3 was selected as a proof-of-concept target to demonstrate tissue-specific activity in tissues of interest such as myeloid-derived suppressor cells (MDSC). The STAT3 sequence was designed in the GalXC format with the described modification pattern and screened for target knockdown in liver tissue in normal CD-1 mice. 18 STAT3-GalXC conjugates (Table 1) were administered subcutaneously once at 3 mg / kg. Table 1: Candidate GalXC compounds for identifying tool compounds for proof-of-concept studies in mice:

[0392] Five days after injection, the livers were collected and mRNA analysis was performed by qPCR. As a result of the screening, four sequences (GalXC -STAT3-838, GalXC-STAT3-1402, GalXC-STAT3-4110, and GalXC-STAT3-4123) that showed >85% target knockdown in the liver were selected for further evaluation ( Figure 2A ). Three of these sequences were identified as mouse-specific, while one was identified as human-mouse cross-reactive. These 4 sequences were further screened in CD-1 mice at 3 different doses (0.3, 1, and 3 mg / kg) to evaluate the dose response. GalXC-STAT3-4110 and 4123 were identified as the most effective sequences after the dose-response screening, each with an ED 50 of 0.3 mg / kg, and thus these molecules were selected for further studies ( Figure 2B)。Both GalXC-STAT3-4110 and 4123 were conjugated with C18 lipids for proof-of-concept studies (Table 2). Table 2: GalXC-STAT3 Lipid Conjugates Table 3: GalXC-STAT3 Lipid Conjugates

[0393] To evaluate the performance of the GalXC-STAT3-C18 conjugate, Pan02 tumors were implanted into nude mice, and after sufficient tumor volume was reached, the mice were randomly grouped as described above. The mice received a single subcutaneous dose of 25 mg / kg, 50 mg / kg of GalXC-STAT3-C18 4110 and 4123, or PBS. Three days after injection, the excised tumors were collected and the MDSC subsets were isolated. Overall, MDSCs are characterized by the co-expression of the cell surface or mRNA markers CD11b (a marker of myeloid cells of the macrophage lineage) and Gr-1 (a marker of myeloid lineage differentiation antigens), and are expressed as CD11b + Gr-1 + cells. Gr-1 is further composed of two components, Ly6G and Ly6C. MDSCs consist of two subsets: granulocytic MDSCs (G-MDSCs), which are further characterized as CD11b + Ly6G + Ly6C lo ; and monocytic MDSCs (M-MDSCs), which are characterized as CD11b + Ly6G - Ly6C hi . To isolate CD11b-positive cells, a single-cell suspension of the tumor was prepared using a gentle MACS dissociator. The CD11b-positive cells in the single-cell suspension were then magnetically labeled with MACS microbeads and enriched through a MACS column, and the labeled cells retained in the column were subsequently eluted as the positively selected fraction (CD11b MicroBeads UltraPure, mouse kit catalog number 130-126-725). For tumor cell isolation, the non-target cells in the cell suspension were magnetically labeled with a microbead mixture and passed through a MACS column. During this process, the unwanted labeled cells were retained in the column, while the unlabeled target cells (tumor cells) were collected as a pure fraction in the flow-through (Tumor Cell Isolation Kit, human catalog number 130-108-339). After cell isolation, mRNA was analyzed by qPCR ( Figure 3A and Figure 3B ). GalXC-STAT3-C18-4123 reduced the Stat3 mRNA levels in G-MDSC and M-MDSC by approximately 40%. GalXC-STAT3-C18-4110 reduced the Stat3 mRNA levels by only 20% in these two MDSC subsets. To understand how the dose levels of GalXC-STAT3-C18 conjugates function in the trafficking of these molecules to different tissues and cell subsets, subsequent studies were conducted using the same tumor model as described previously. Pan02 tumor-bearing mice were treated with a single subcutaneous dose of 50 mg / kg of GalXC-STAT3-C18-4123 or PBS, and Stat3 mRNA levels were measured 3 days later. Compared to the knockdown observed at the 25 mg / kg dose, the Stat3 knockdown in G-MDSC was not significantly altered, but there was a significant improvement in Stat3 silencing observed in the M-MDSC subset at this same dose level. In a parallel study conducted as described previously, Stat3 knockdown was evaluated in bulk tumors and TdLNs at day 7 ( Figure 4A and Figure 4B ). Dose-dependent Stat3 mRNA knockdown was observed in bulk tumors using two GalXC-STAT3-C18 sequences. In TdLNs, GalXC-STAT3-C18-4123 reduced the Stat3 mRNA levels by approximately 60 - 65%, and two doses of GalXC-STAT3-C18-4110 reduced the Stat3 mRNA levels by approximately 25 - 30%, suggesting a saturation effect at these dose levels. Based on these data, GalXC-STAT3-C18-4123 was selected for further efficacy evaluation in immunocompetent mice. Example 4: STAT3 inhibition reduces PD-L1 levels in MDSC and mediates acute tumor effects

[0394] The transcriptional signature of phosphorylated STAT3 has been positively correlated with PD-L1 expression in tumors (Song et al., JOURNAL OF CELL PHYSIOLOGY (2020), Zerdes et al., CANCERS (2019), Song et al., BLOOD (2018)). To infer this correlation with STAT3 expressed by MDSC, Pdl1 mRNA assays were performed on isolated MDSC populations treated with PBS or GalXC-STAT3 conjugates. In both G-MDSC and M-MDSC populations treated with 25 or 50 mg / kg of GalXC-STAT3, the Pdl1 mRNA levels were reduced by approximately 80% ( Figure 5A)。After treatment with GalXC-STAT3 conjugates, especially GalXC-STAT3-C18-4123, the Pdl1 level in TdLN also decreased significantly( Figure 5B )。These data suggest the potential for downstream immunomodulation of PD-L1 after STAT3 knockdown.

[0395] In a separate study, Pan02 (murine pancreatic syngeneic model)-bearing C57BL / 6 mice (n = 4 per group) were treated subcutaneously with GalXC-STAT3-C18 conjugate according to a fractionated dosing model, in which all animals received a total dose of 50 mg / kg, administered as 25 mg / kg x 2 doses or 12.5 mg / kg x 4 doses. Tumors treated with the 25 mg / kg split dose showed acute tumor regression even after the first dose( Figure 6B )。After the second dose of 25 mg / kg, the tumors of 3 out of 4 mice regressed to a size too small to be collected for further processing. The antitumor effect of GalXC-STAT3 treatment was also observed in mice receiving the 12.5 mg / kg split dose( Figure 6A )。These data suggest that in immunocompetent Pan02-bearing mice, STAT3-mediated regulation of PD-L1 results in an acute and significant effect on tumor growth. Example 5: Preparation of double-stranded RNAi oligonucleotides Oligonucleotide synthesis and purification

[0396] The double-stranded RNAi (dsRNA) oligonucleotides described in the previous examples were chemically synthesized using the methods described herein. Generally, in addition to using known phosphoramidite synthesis (see, e.g., Hughes and Ellington (2017) Cold Spring Harb Perspect Biol. 9(1):a023812; Beaucage S.L., Caruthers M.H. Studies on Nucleotide Chemistry V: Deoxynucleoside Phosphoramidites—A New Class of Key Intermediates for Deoxypolynucleotide Synthesis. Tetrahedron Lett. 1981; 22:1859-1862. doi:10.1016 / S0040-4039(01)90461-7), the solid-phase oligonucleotide synthesis methods described for 19-23-mer siRNAs were also used (see, e.g., Scaringe et al. (1990) Nucleic Acids Res. 18:5433-5441 and Usman et al. (1987) J. Am. Chem. Soc. 109:7845-7845; see also U.S. Pat. Nos. 5,804,683, 5,831,071, 5,998,203, 6,008,400, 6,111,086, 6,117,657, 6,353,098, 6,362,323, 6,437,117, and 6,469,158) to synthesize the dsRNAi oligonucleotides. The dsRNAi oligonucleotides having a 19-mer core sequence were formatted as constructs having a 25-mer sense strand and a 27-mer antisense strand to allow processing by the RNAi machinery. The 19-mer core sequence is complementary to a region in the STAT3 mRNA.

[0397] Each RNA strand was synthesized according to standard methods (Integrated DNA Technologies; Coralville, IA) and purified by HPLC. For example, RNA oligonucleotides were synthesized using solid-phase phosphoramidite chemistry and deprotected and desalted on a NAP-5 column (Amersham Pharmacia Biotech; Piscataway, NJ) using standard techniques (Damha & Olgivie (1993) Methods Mol. Biol. 20:81-114; Wincott et al. (1995) Nucleic Acids Res. 23:2677-2684). Oligomers were purified using ion-exchange high-performance liquid chromatography (IE-HPLC) on an Amersham Source 15Q column (1.0 cm × 25 cm; Amersham Pharmacia Biotech) using a 15-min step linear gradient. The gradient was changed from 90:10 buffer A:B to 52:48 buffer A:B, where buffer A was 100 mM Tris pH 8.5 and buffer B was 100 mM Tris pH 8.5, 1 M NaCl. Samples were monitored at 260 nm, and the peaks corresponding to the full-length oligonucleotide species were collected, combined, desalted on a NAP-5 column, and lyophilized.

[0398] The purity of each oligomer was determined by capillary electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc.; Fullerton, CA). The CE capillary had an inner diameter of 100 μm and contained ssDNA 100R Gel (Beckman-Coulter). Typically, approximately 0.6 nmol of oligonucleotide was injected into the capillary, run in an electric field of 444 V / cm, and detected based on UV absorbance at 260 nm. Denaturing Tris-borate-7M-urea electrophoresis buffer was purchased from Beckman-Coulter. Oligoribonucleotides were obtained with a purity of at least 90% as assessed by CE and were used in the experiments described below. On a Voyager DE TM Biospectometry Work Station (Applied Biosystems; Foster City, CA), the identity of the compounds was verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry according to the manufacturer's recommended protocol. The relative molecular masses of all oligomers were obtained, typically within 0.2% of the expected molecular mass. Preparation of duplexes

[0399] Resuspend single-stranded RNA oligomers (e.g., at a concentration of 100 μM) in duplex buffer (pH 7.5) consisting of 100 mM potassium acetate and 30 mM HEPES. Mix the complementary sense and antisense strands in equimolar amounts to produce a final solution of, for example, 50 μM duplex. Heat the sample to 100 °C for 5' in RNA buffer (IDT) and cool to room temperature before use. Store dsRNA oligonucleotides at -20 °C. Store single-stranded RNA oligomers as a lyophilized product or in nuclease-free water at -80 °C. Example 6 : Generation of double-stranded RNAi oligonucleotides targeting STAT3 Identification of STAT3 mRNA target sequences

[0400] Signal transducer and activator of transcription 3 (STAT3) is a transcription factor involved in several developmental and disease functions. To generate RNAi oligonucleotide inhibitors against STAT3 expression, a computer-based algorithm was used to computationally determine STAT3 mRNA target sequences suitable for assaying inhibition of STAT3 expression via the RNAi pathway. This algorithm provides RNAi oligonucleotide guide (antisense) strand sequences, each having a complementary region complementary to a suitable STAT3 target sequence of human STAT3 mRNA (e.g., SEQ ID NO: 1217; Table 4). Some of the guide strand sequences determined by this algorithm are also complementary to the corresponding STAT3 target sequences of monkey STAT3 mRNA (SEQ ID NO: 1218, Table 4) and / or mouse STAT3 mRNA. STAT3 RNAi oligonucleotides containing complementary regions complementary to homologous STAT3 mRNA target sequences with nucleotide sequence similarity are expected to have the ability to target homologous STAT3 mRNA. Table 4: Sequences of human and monkey STAT3 mRNA Species Reference sequence # SEQ ID NO Human (Hs) NM_139276.3 1217 Cynomolgus macaque (Mf) XM_005584240.2 1218 Mouse (Mus Musculus) (Mm) NM_213659.3 8

[0401] Generate RNAi oligonucleotides (formatted as DsiRNA oligonucleotides) for in vitro evaluation as described in Example 5. Each DsiRNA was generated with the same modification pattern and each had a unique guide strand having a complementary region complementary to the STAT3 target sequences represented by SEQ ID NO: 89 - 280. Modifications of sense and antisense DsiRNAs include the following (X - any nucleotide; m - 2'-O-methyl modified nucleotide; r - ribosyl modified nucleotide): Sense strand: rXmXrXmXrXrXrXrXrXrXrXrXrXmXrXmXrXrXrXrXrXrXXX Antisense strand: mXmXmXmXrXrXrXrXrXrXmXrXmXrXrXrXrXrXrXrXrXrXmXrXmXmXmX

[0402] The ability of each modified DsiRNA in Table 5 to reduce STAT3 mRNA was measured using cell-based in vitro assays. Briefly, human hepatocytes (Huh7) expressing the endogenous human STAT3 gene were transfected with each DsiRNA listed in Table 5 at 1 nM in separate wells of a multi-well cell culture plate. After transfection with the modified DsiRNA, the cells were maintained for 24 hours and then a -based qPCR assay was used to determine the amount of remaining STAT3 mRNA in the transfected cells. Two qPCR assays were used, a 3' assay and a 5' assay (forward 1 - SEQ ID NO:1219, reverse 1 - SEQ ID NO:1220, probe 1 - SEQ ID NO:1221; forward 2 - SEQ ID NO:1, reverse 2 - SEQ ID NO:2, probe 2 - SEQ ID NO:3) to measure STAT3 mRNA levels as measured using a PCR probe conjugated to 6-carboxy-fluorescein (FAM). The percentage of remaining RNA was determined for each primer pair as shown in Table 5 and Figure 7 . A DsiRNA that resulted in less than or equal to 10% of the remaining STAT3 mRNA in the DsiRNA-transfected cells compared to mock-transfected cells was considered a DsiRNA "hit". A Huh7 cell-based assay evaluating the ability of the DsiRNAs listed in Table 5 to inhibit STAT3 expression identified several candidate DsiRNAs. In summary, these results demonstrate that DsiRNAs designed to target human STAT3 mRNA inhibit STAT3 expression in cells as determined by the reduction in STAT3 mRNA in DsiRNA-transfected cells relative to control cells. These results demonstrate that nucleotide sequences containing DsiRNAs can be used to generate RNAi oligonucleotides to inhibit STAT3 expression. Additionally, these results demonstrate that multiple STAT3 mRNA target sequences are suitable for RNAi-mediated inhibition of STAT3 expression. Table 5. Analysis of STAT3 mRNA in Huh7 cells

[0403] After initial in vitro screening, 48 constructs were selected for dosing studies. Huh7 cells were treated with 0.05 nM, 0.3 nM, or 1 nM of oligonucleotide for 24 hours. mRNA was isolated and measured to determine the effective dose ( Figure 8A ). Among the oligonucleotides tested, 34 sequences were selected for further in vivo testing (Table 6 and Figure 8B ). Table 6. Analysis of STAT3 mRNA in Huh7 dosing studies Example 7: In vivo RNAi oligonucleotide inhibition of STAT3

[0404] The in vitro screening assay in Example 6 verified the ability of STAT3-targeted DsiRNA to knockdown target mRNA. To confirm the ability of RNAi oligonucleotides to knockdown STAT3 in vivo, an HDI mouse model was used. A subset of the DsiRNAs determined in Example 6 was used to generate the corresponding double-stranded RNAi oligonucleotides, which comprised a nicked tetraloop GalNAc-conjugated structure with a 36-mer passenger strand and a 22-mer guide strand (referred to herein as "GalNAc-conjugated STAT3 oligonucleotides" or "GalNAc-STAT3 oligonucleotides") (Table 8 and Table 9). In addition, the nucleotide sequences of the passenger and guide strands had a unique pattern of modified nucleotides and phosphorothioate bonds. Three of the nucleotides that make up the tetraloop were each conjugated to a GalNAc moiety (CAS#14131-60-3). The modification pattern used was as follows: Pattern 1 Sense strand: 5’ mX-S-mX-mX-mX-mX-mX-mX-fX-fX-fX-fX[-mX-] 16 -[ademX-GalNAc]-[ademX-GalNAc]-[ademX-GalNAc]-mX-mX-mX-mX-mX-mX 3’. Hybridizes to: Antisense strand: 5’ [MePhosphonate-4O-mX]-S-fX-S-fX-fX-fX-mX-fX-mX-mX-fX-mX-mX-mX-fX-mX-mX-mX-mX-mX-mX-S-mX-S-mX 3’. Or, expressed as: 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][adem A-GalNAc][ademA-GalNAc][ademA-GalNAc][mX][mX][mX][mX][mX][mX] Hybridize to: Antisense strand: [MePhosphonate-4O-mXs][fXs][fX][fX][fX][mX][fX][mX] [mX][fX][mX][mX][mX][fX][mX][mX][mX][mX][mX][mXs][mXs][mX] Pattern 2 Sense strand: 5’ mX-S-mX-mX-mX-mX-mX-mX-fX-fX-fX-fX[-mX-] 16 -[ademX-GalNAc]-[ademX-GalNAc]-[ademX-GalNAc]-mX-mX-mX-mX-mX-mX 3’. Hybridize to: Antisense strand: 5’ [MePhosphonate-4O-mX]-S-fX-S-fX-S-fX-fX-mX-fX-mX-mX-fX-mX-mX-mX-fX-mX-mX-mX-mX-mX-mX-S-mX-S-mX 3’. Or, expressed as: 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][adem A-GalNAc][ademA-GalNAc][ademA-GalNAc][mX][mX][mX][mX][mX][mX] Hybridize to: Antisense strand: [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX] [mX][fX][mX][mX][mX][fX][mX][mX][mX][mX][mX][mXs][mXs][mX](Modified keyword: Table 7).

[0405] The oligonucleotides in Tables 8 and 9 were evaluated in mice engineered to transiently express human STAT3 mRNA in hepatocytes of the mouse liver. Briefly, the indicated GalNAc-conjugated STAT3 oligonucleotides formulated in PBS were administered subcutaneously to 6-8-week-old female CD-1 mice (n = 4-5) at a dose of 1 mg / kg. Control mice (n = 3-4) received only PBS. Three days later (72 hours), the mice were given a hydrodynamic injection (HDI) of a DNA plasmid (25 μg) encoding the full-length human STAT3 gene under the control of the ubiquitous cytomegalovirus (CMV) promoter sequence. One day after the introduction of the DNA plasmid, liver samples were collected from the HDI mice. As described in Example 6, qRT-PCR analysis was performed on the total RNA obtained from these HDI mice to determine the STAT3 mRNA levels. The mRNA levels of human mRNA were measured. These values were normalized to the transfection efficiency using the NeoR gene contained on the DNA plasmid. Benchmark controls (STAT3-1388) containing different modification patterns were used for both assays (sense strand SEQ ID NO: 1100; antisense strand SEQ ID NO: 1190). Table 8. GalNAc-conjugated STAT3 RNAi oligonucleotides for HDI screening Table 9. GalNAc-conjugated STAT3 RNAi oligonucleotides for HDI screening

[0406] Figure 9A and Figure 9B The results in and demonstrated that the GalNAc-conjugated STAT3 oligonucleotides designed to target human STAT3 mRNA inhibited human STAT3 mRNA expression in HDI mice, as determined by the decrease in the amount of human STAT3 mRNA expression in liver samples from HDI mice treated with GalNAc-conjugated STAT3 oligonucleotides relative to control HDI mice treated with only PBS.

[0407] Further verified in the dosing study in Figure 9A andFigure 9B Subset of GalNAc-conjugated STAT3 oligonucleotides tested. Specifically, dosing studies were conducted using nine GalNAc-conjugated STAT3 oligonucleotides (STAT3-715, STAT3-716, STAT3-717, STAT3-720, STAT3-721, STAT3-1145, STAT3-1286, STAT3-1287, and STAT3-1287). Mice were hydrodynamically injected as described above and treated with oligonucleotides at 0.1 mg / kg, 0.3 mg / kg, or 1 mg / kg. Livers were harvested one day later and STAT3 expression was measured to determine the effective dose ( Figure 10 ). All GalNAc-conjugated STAT3 oligonucleotides were able to reduce STAT3 expression at the 1 mg / kg dose, while STAT3-1286 was able to reduce expression at the 0.3 mg / kg dose. Overall, the HDI study identified several potential GalNAc-conjugated STAT3 oligonucleotides for inhibiting STAT3 expression in the liver. Example 8: In Vivo Species-Specific RNAi Oligonucleotide Inhibition of STAT3

[0408] To confirm the ability of RNAi oligonucleotides to knockdown STAT3 in vivo, several cross-species and species-specific GalNAc-conjugated STAT3 oligonucleotides were generated. Specifically, triple-common (targeting human, non-human primate, and mouse; Hs / Mf / Mm), human / mouse (Hs / Mm), and human-specific (Hs) oligonucleotides were evaluated. Hs / Mf / Mm and Hs / Mm common

[0409] GalNAc-conjugated STAT3 oligonucleotides shown in Table 10 were subcutaneously injected into mice expressing endogenous mouse STAT3 in the liver at a dose of 3 mg / kg. Livers were harvested five days later and STAT3 expression was measured. Overall, this study identified several potential Hs / Mf / Mm GalNAc-conjugated STAT3 oligonucleotides for inhibiting STAT3 expression in the liver ( Figure 11 ). Table 10. GalNAc-conjugated human / monkey / mouse STAT3 RNAi oligonucleotides for endogenous STAT3 screening.

[0410] The human / mouse GalNAc-conjugated STAT3 oligonucleotides shown in Table 11 were tested in mice that endogenously express mouse STAT3. As described above, the oligonucleotides were injected subcutaneously into the mice at a dose of 3 mg / kg. Five days later, the livers were collected and mouse STAT3 expression was measured. Overall, this study identified several potential Hs / Mm GalNAc-conjugated STAT3 oligonucleotides for inhibiting STAT3 expression in the liver( Figure 12 ). Table 11. GalNAc-conjugated human / mouse STAT3 RNAi oligonucleotides for endogenous STAT3 screening.

[0411] A subset of the GalNAc-conjugated STAT3 oligonucleotides tested in Figure 11 and Figure 12 was further validated in a dosing study. Specifically, a dosing study was performed using ten GalNAc-conjugated STAT3 oligonucleotides (STAT3-2626, STAT3-2627, STAT3-2408, STAT3-2412, STAT3-2139, STAT3-4909, STAT3-461, STAT3-678, STAT3-2148, and STAT3-2144). The oligonucleotides were injected subcutaneously into mice that endogenously express mouse STAT3 at doses of 0.3 mg / kg, 1 mg / kg, or 3 mg / kg. Five days later, the livers were collected and mouse STAT3 expression was measured to determine the effective dose( Figure 13A and Figure 13B ). Overall, the endogenous mouse STAT3 expression study identified several potential GalNAc-conjugated STAT3 oligonucleotides for inhibiting STAT3 expression in the mouse liver. Hs-specific

[0412] Using the HDI model described in Example 7, human-specific GalNAc-conjugated STAT3 oligonucleotides were evaluated. Specifically, the indicated GalNAc-conjugated STAT3 oligonucleotides (Table 12) formulated in PBS were administered subcutaneously to 6-8-week-old female CD-1 mice (n = 4-5) at a dose of 1 mg / kg. Control mice (n = 3-4) received only PBS. Three days later (72 hours), the mice were given a hydrodynamic injection (HDI) of a DNA plasmid (25 μg) encoding the full-length human STAT3 gene under the control of the ubiquitous cytomegalovirus (CMV) promoter sequence. One day after the introduction of the DNA plasmid, liver samples were collected from the HDI mice. Total RNA obtained from these HDI mice was analyzed by qRT-PCR to determine STAT3 mRNA levels. Table 12. GalNAc-conjugated human STAT3 RNAi oligonucleotides for exogenous STAT3 screening.

[0413] Figure 14 The results in demonstrate that GalNAc-conjugated STAT3 oligonucleotides designed to target human STAT3 mRNA inhibit human STAT3 mRNA expression in HDI mice, as determined by a reduction in the amount of human STAT3 mRNA expression in liver samples from HDI mice treated with the GalNAc-conjugated STAT3 oligonucleotides relative to control HDI mice treated with PBS alone.

[0414] In the drug administration study, it was further verified that Figure 14 A subset of GalNAc-conjugated STAT3 oligonucleotides tested in . Specifically, dosing studies were performed using five GalNAc-conjugated STAT3 oligonucleotides (STAT3-426, STAT3-432, STAT3-1068, STAT3-1388, and STAT3-2404). Mice were hydrodynamically injected as described above and treated with 0.3 mg / kg, 1 mg / kg, or 3 mg / kg of oligonucleotides. The liver was collected one day later, and human STAT3 expression was measured to determine the effective dose ( Figure 15 ). A dose of 1 mg / kg was able to reduce STAT3 mRNA by about 75%, thereby identifying several potential GalNAc-conjugated STAT3 oligonucleotides for inhibiting STAT3 expression in the liver. Figure 23 The best two sequences from and the best sequence from Figure 28 were tested in the final HDI screen ( Figure 16 ). Example 9: Specific STAT3 inhibition by GalNAc-conjugated STAT3 oligonucleotides

[0415] The specificity of GalNAc-conjugated STAT3 oligonucleotides to inhibit STAT3 rather than family members (e.g., STAT1) was measured. Specifically, Huh7 cells expressing endogenous STAT1 were treated with 0.05 nM, 0.3 nM, or 1 nM of GalNAc-conjugated STAT3 oligonucleotides (STAT3-721, STAT3-1286, and STAT3-1388) using lipofectamine as a transfection agent for 24 hours. The percentage (%) of remaining mRNA was measured compared to mock controls (PBS; no lipofectamine or siRNA) and UTR (untransfected; treated with lipofectamine but no siRNA) (Table 13 and Figure 17 ). STAT3721 and 1286 did not downregulate human STAT1, but STAT31388 downregulated human STAT1 (Table 13). The oligonucleotides did not downregulate STAT1 expression, demonstrating specificity for STAT3 while having limited off-target effects on STAT1. Table 13. STAT1 expression Example 10: STAT3 inhibition in combination with checkpoint inhibition significantly improves anti-tumor efficacy

[0416] To evaluate the performance of the GalXC-STAT3-C18 conjugate as a single agent or in combination with the checkpoint inhibitor anti-PD-L1 mAb, Pan02 tumors (2x10 6 cells) were implanted into 6-8-week-old C57BL / 6 mice and the mice were randomized after the tumors reached a volume of 300-400 mm 3 . Mice received a single subcutaneous dose of 25 mg / kg of GalXC-STAT3-C18-4123, either as a single agent or in combination with 10 mg / kg (i.p.) of anti-PD-L1 mAb (anti-mouse PD-L1 mAb (B7-H1), clone 10F.9G2). Mice were first administered two doses at three-day intervals and then two more doses at three-day intervals two weeks later [(q3dx2)x2]. Control groups were treated with GalXC-placebo as a single agent or in combination with anti-PD-L1 mAb as described for the GalXC-STAT3-C18-4123 compound. Two weeks after the last dose, the same dosing regimen was repeated. Tumor size was measured twice a week throughout the study.

[0417] As Figure 18AAs shown, tumors treated with GalXC-placebo or GalXC-placebo + mAb continued to grow to the same extent. However, the group receiving GalXC-STAT3 showed antitumor efficacy after the first round of treatment, but their tumors continued to grow despite receiving a second dose. Compared to monotherapy, the group receiving the combination of GalXC-STAT3 and mAb showed significantly more tumor regression. This indicates that combination therapy with checkpoint inhibitors can achieve improved antitumor efficacy.

[0418] In a separate study, Pan02 tumors (2x10 6 cells) were implanted into 6-8-week-old C57BL / 6 mice, and when they reached a volume of 300-400 mm 3 , GalXC-placebo (25 mg / kg) was administered to the mice in two doses at three-day intervals (days 42 and 45). Two weeks later, the mice received two subcutaneous doses of 25 mg / kg of GalXC-STAT3-C18-4123 at three-day intervals, in combination with 10 mg / kg (i.p.) of anti-PD-L1 mAb (anti-mouse PD-L1 mAb (B7-H1), clone 10F.9G2). Tumor size was measured twice a week throughout the study. Figure 18B The regression of tumor size shown after administration of the GalXC-STAT3 / PD-L1 mAb combination therapy further demonstrates that combination therapy can achieve enhanced antitumor efficacy. Example 11: Correlation between combination therapy of GalXC-STAT3 and PD-L1 mAb and tumor immune phenotype

[0419] To determine whether the combination efficacy pattern corresponded to the tumor immune phenotype, tumor types with different phenotypes were selected for implantation into mice. The selected tumor types included Pan02 ( Figure 18A , checkpoint-resistant tumor), 4T1 (triple-negative breast cancer, checkpoint-resistant tumor), MC-38 (colon cancer, partially checkpoint-sensitive tumor), and Hepa1-6 (hepatocellular carcinoma, checkpoint-sensitive tumor). Pan02 (5e6 cells + Matrigel, Figure 18A)、MC-38 (5e6 cells) and Hepa1-6 tumors (2e6 cells) grew in C57BL / 6 mice (7 - 8 weeks old), while 4T1 tumors (7 - 8 weeks old) grew in Balb / c mice. When each tumor reached a sufficient tumor volume, they were sorted and treated as described in Example 5 (4T1 tumors were treated three times with a combination of subcutaneous GalXC-STAT3-C18-4123 and anti-PD-L1 mAb or with monotherapy GalXC-placebo, GalXC-STAT3-C18-4123 or GalXC-placebo combined with mAb, with a 3-day interval between each administration (q3dx3), as Figure 19A shown. During the entire study period, tumor volume was measured twice a week. MC-38 and Hepa1-6 tumors were treated with a combination of subcutaneous GalXC-STAT3-C18-4123 and anti-PD-L1 mAb or with monotherapy GalXC-placebo, GalXC-STAT3-C18-4123 or GalXC-placebo combined with mAb (2 doses, 3 days apart, for 2 weeks), as Figure 19B and Figure 19C shown.

[0420] Combination therapy showed synergistic efficacy in resistant tumor types, in which tumors were expected to have no or very little CD8+ T cell infiltration in the TME, and a larger MDSC population (CD8 低 MDSC 高 )(Figure 18 and Figure 19A ). In partially sensitive tumors, combination therapy showed improved efficacy compared to checkpoint therapy alone, in which these tumors had a slightly higher level of CD8+ T cell infiltration and a larger MDSC population (CD8 中 MDSC 高 )( Figure 19B ). Intriguingly, combination therapy led to complete regression of sensitive tumors (CD8 高 MDSC 高 )( Figure 19C ). Tumors with higher CD8+ T cell infiltration and MDSC levels were completely eradicated when treated with the GalXC-STAT3-C18-4123 + anti-PD-L1 mAb combination. Example 12: Treatment-mediated tumor regression and generation of tumor-specific memory

[0421] To evaluate whether combination therapy that showed complete regression also led to the generation of memory T cells in treated mice, on day 51, the tumors that had completely regressed in the mice were rechallenged with Hep1-6 cells (2e6 cells) in the contralateral flank of the mice Figure 19C as Figure 20As shown, even after a secondary attack, all the mice remained tumor-free and survived during the maintenance and sustenance period (about 2 months). These data demonstrate that the combination therapy has a potent anti-tumor therapeutic effect, leading to long-term immune memory. Example 13: CD8+ T cell-mediated combined efficacy also depends on perforin

[0422] To evaluate whether the efficacy mediated by the combination therapy is CD8+ T cell-mediated, as described in Example 7, a efficacy study was conducted using 4T1 tumors (2e6 cells) in immunocompetent Balb / c mice (7-8 weeks old). This experiment was repeated on immune-compromised nude mice bearing 4T1 tumors. As Figure 21A shown, the GalXC-STAT3-C18-4123 + anti-PD-L1 mAb combination therapy had a synergistic efficacy in tumor-bearing immunocompetent mice, but no efficacy was observed in 4T1 tumor-bearing nude mice ( Figure 21B ), suggesting that CD8+ T cells play a key role in mediating anti-tumor efficacy. To confirm that the efficacy is mediated by cytotoxic CD8+ T cells, perforin staining was performed on tumor samples at the end time point of the study. As Figure 22 shown, there was a significantly larger population of perforin-positive cells in the tumors that received the combination therapy, indicating that the T cells involved in mediating the efficacy are cytotoxic in nature. Example 14: Effect of combination therapy on spontaneous tumor metastasis in a highly metastatic tumor model

[0423] To evaluate whether the combination therapy reduces metastasis in a spontaneous metastatic tumor model, as described in Example 7, 4T1 tumors (2e6 cells / mouse) were implanted into Balb / c mice (7-8 weeks old). When the tumors reached a size of 500 mm 3 , they were treated with GalXC-placebo, GalXC-STAT3-C18-4123, GalXC-placebo + anti-PD-L1 mAb, or GalXC-STAT3 + anti-PD-L1 mAb (q3d x 3, GalXC oligonucleotide was administered at 50 mg / kg, anti-PD-L1 mAb was administered at 10 mg / kg), and tumor growth was monitored. Twelve days after the last dose, the mice were sacrificed and lung photographs were taken. As Figure 23 shown, the lungs treated with single agent or placebo showed tumor metastases throughout the organ, while the mice treated with the combination therapy (GalXC-STAT3-C18-4123 + anti-PD-L1 mAb) showed no visible metastases in the lungs of all five mice, suggesting that the treatment not only reduced local tumor growth as shown, but also reduced spontaneous metastasis to the lungs. In Figure 23It also shows that the same experiment was repeated on nude mice. Tumor metastases occurred in all lungs, including those of mice receiving combination therapy, further confirming the role of CD8+ T cells in anti-tumor efficacy. Example 15: Treatment-Mediated Immunomodulation in Tumors

[0424] To understand how the combination therapy of GalXC-STAT3-C18-4123 and anti-PD-L1 mAb alters the immune profile in tumors, CT26 tumors were implanted into Balb / c mice. These tumors are partially sensitive to checkpoint inhibitors and have a profile similar to that of MC38 (CD8 中 MDSC 中 / 高 ). When the tumors reached a sufficient size, they were treated with GalXC-placebo, GalXC-STAT3-C18-4123, GalXC-placebo + anti-PD-L1 mAb, or GalXC-STAT3-C18-4123 + anti-PD-L1 mAb (q3dx 2, 25 mg / kg or 10 mg / kg). Seven days after the last dose, the tumors were collected, homogenized, and subjected to nanowire analysis (mRNA was extracted from paraffin-embedded samples and mRNA expression was performed by ncounter R Mouse Pancancer IO360 TM Panel (Nanostring Technologies, Seattle, WA)).

[0425] Analysis showed that compared with monotherapy or treatment with GalXC-placebo and anti-PD-L1 mAb, after combination therapy, inhibitory genes (checkpoints, STAT3-mediated genes, inhibitory cytokines / chemokines, angiogenesis, and matrix remodeling-related genes) were reduced, while genes favoring T cell activation (genes involved in T cell migration, activation, memory, and cytotoxicity) were increased, suggesting that combination therapy is transforming the TME from an inhibitory TME to a TME favorable for T cell infiltration ( Figure 24 ). Example 16: STAT3 Oligonucleotides for Treating Diseases

[0426] To study the efficacy of STAT3 oligonucleotides used alone or in combination with anti-PD-L1 mAb, STAT3 oligonucleotides or STAT3 oligonucleotides in combination with anti-PD-L1 mAb were administered to subjects. Specifically, STAT3 oligonucleotides were administered to subjects, wherein the sense strand contains the sequence shown in SEQ ID NO: 1222 and wherein the antisense strand contains the sequence shown in SEQ ID NO: 1145, as shown below (depicted in Figure 25 ): Sense strand: [ademAs-C18][mA][mU][mU][mA][mU][mC][fA][fG][fC][fU][mU] [mA][mA][mA][mA][mU][mU][mA][mA][mG][mC][mA][mG][mC][mC][mG][mA][mA][mA][mG][mG][mC][mU][mG][mC] Hybridizes to: Antisense strand: [MePhosphonate-4O-mUs][fUs][fAs][fA][fU][mU][fU][mU][mA][fA][mG][mC][mU][fG][mA][mU][mA][mA][mU][mUs][mGs][mG] (Keywords are provided in Table 7).

[0427] The STAT3 oligonucleotides described above are administered alone or in combination with an anti-PD-L1 antibody. The STAT3 oligonucleotides are administered before, simultaneously with, or after the administration of the anti-PD-L1 antibody. After administration, tumor size and subject survival are measured. Example 17: STAT3 inhibition combined with checkpoint inhibition significantly improves anti-tumor efficacy

[0428] Studies were conducted in three different murine tumor models: B16F10, Pan02, and MC-38. B16F10 and Pan02 are murine melanoma and pancreatic cancer models that are considered resistant to checkpoint inhibitors (CPI) due to the presence of a large number of myeloid-derived suppressor cells (MDSC) and few or no CD8+ T cells in the tumor microenvironment (TME). The MC-38 tumor model is a murine colon cancer model that is known to be partially sensitive to CPI and contains moderate levels of MDSC and CD8+ T cells in its TME. The experiments described in this example were designed to evaluate the efficacy of DCR-STAT3 (a human-specific STAT3 sequence with a C18 lipid conjugation at the 5' end of the passenger strand, corresponding to SEQ ID NO: 1222 and 1145, "DCR-STAT3") in preclinical models of CPI resistance and sensitivity.

[0429] Mice were administered GalXC - placebo or DCR - STAT3, with and without anti - PD - L1 mouse antibody. GalXC - placebo and DCR - STAT3 were administered subcutaneously at 25 mg / kg, while the anti - PD - L1 antibody was administered intraperitoneally at 10 mg / kg. In the B16F10 tumor model, doses were administered on day 6 (6 days after tumor implantation), day 9, and day 12. In the Pan02 model, doses were administered on day 38 (38 days after tumor implantation), day 41, day 48, and day 51. In the MC - 38 tumor model, doses were administered on day 5 (5 days after tumor implantation), day 8, day 12, and day 15.

[0430] In the CPI - resistant B16F10 model, after 3 doses of DCR - STAT3 or DCR - STAT3 + anti - PD - L1 antibody, the tumor size on day 13 was reduced by 36% (p < 0.01) and 64% (p < 0.0001), respectively, compared to the GalXC - placebo group. The anti - PD - L1 antibody alone had no effect on tumor growth, and the tumors grew to the same size as the GalXC - placebo group. The tumor size of the combination group (DCR - STAT3 + anti - PD - L1 antibody) was reduced by 43% (p < 0.05) compared to DCR - STAT3 alone and by 64% (p < 0.0001) compared to the anti - PD - L1 antibody alone. A similar pattern was observed in the Pan02 study. After 4 doses of DCR - STAT3 or DCR - STAT3 + anti - PD - L1 antibody, the tumor size on day 58 was reduced by 39% (p < 0.01) and 75% (p < 0.0001), respectively, compared to the control group. The anti - PD - L1 antibody alone had no effect on tumor growth, and the tumors grew to the same size as the GalXC - placebo group. The tumor size of the combination group was reduced by 59% (p < 0.01) compared to DCR - STAT3 alone and by 76% (p < 0.0001) compared to the anti - PD - L1 antibody alone, suggesting that DCR - STAT3 has activity as a single agent and that in this CPI - resistant tumor model, its single - agent activity is further enhanced when combined with the antibody.

[0431] In the CPI partial sensitivity MC-38 model, after 4 doses of anti-PD-L1 antibody or DCR-STAT3, the tumor size on day 18 decreased by 57% (p<0.01) and 45% (p<0.01), respectively, compared with the GalXC-placebo group. On day 18, after 4 doses of DCR-STAT3 + anti-PD-L1 antibody, the tumor size decreased by 95% (p<0.0001) compared with the GalXC-placebo group. Compared with anti-PD-L1 antibody or DCR-STAT3, in the DCR-STAT3 + anti-PD-L1 antibody group, the tumor size decreased by 89% (p<0.05) and 91% (p<0.01), respectively. Administration of anti-PD-L1 antibody or DCR-STAT3 as monotherapy was active, but the combination of the two further enhanced the efficacy of the monotherapy.

[0432] Data from these 3 experiments demonstrated that in CPI-resistant tumors where anti-PD-L1 antibody was inactive, DCR-STAT3 was active as monotherapy, and when DCR-STAT3 was combined with anti-PD-L1 antibody, synergistic anti-tumor activity was produced. In CPI-sensitive tumors where anti-PD-L1 also showed monotherapy activity, DCR-STAT3 was also active, and when used in combination, most tumors regressed by nearly 100%. Sequence Listing

Claims

1. An oligonucleotide for reducing STAT3 expression, the oligonucleotide comprising an antisense strand having a length of 15 to 30 nucleotides and a sense strand having a length of 15 to 40 nucleotides, wherein the sense strand and the antisense strand form a duplex region, wherein the antisense strand has a complementary region complementary to the target sequence of STAT3 shown in SEQ ID NO: 140, and wherein the sense strand comprises at least one lipid moiety conjugated to the 5'-terminal nucleotide of the sense strand.

2. The oligonucleotide according to claim 1, wherein the length of the antisense strand is 19 to 27 nucleotides.

3. The oligonucleotide according to any one of claims 1-2, wherein the length of the antisense strand is 21 to 27 nucleotides, optionally wherein the length of the antisense strand is 22 nucleotides.

4. The oligonucleotide according to any one of claims 1-3, wherein the length of the sense strand is 19 to 40 nucleotides, optionally wherein the length of the sense strand is 36 nucleotides.

5. The oligonucleotide according to any one of claims 1-4, wherein the length of the duplex region is at least 19 nucleotides.

6. The oligonucleotide according to any one of claims 1-5, wherein the length of the duplex region is at least 20 nucleotides, optionally wherein the length of the duplex region is 21 nucleotides.

7. The oligonucleotide according to any one of claims 1-6, wherein the length of the complementary region complementary to STAT3 is at least 19 consecutive nucleotides.

8. The oligonucleotide according to any one of claims 1-7, wherein the length of the complementary region complementary to STAT3 is at least 21 consecutive nucleotides.

9. The oligonucleotide according to any one of claims 1-8, wherein the antisense strand comprises the sequence shown in SEQ ID NO:

965.

10. The oligonucleotide according to any one of claims 1-9, wherein the sense strand comprises the sequence shown in SEQ ID NO:

875.

11. The oligonucleotide according to any one of claims 1-10, wherein the sense strand comprises a stem-loop at its 3'-end: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop having a length of 3 to 5 nucleotides between S1 and S2.

12. An oligonucleotide for reducing STAT3 expression, the oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand has a length of 21 to 27 nucleotides and has a complementary region complementary to the target sequence of STAT3 shown in SEQ ID NO: 140, wherein the sense strand comprises a stem-loop at its 3'-end: S1-L-S2, wherein S1 is complementary to S2, wherein L forms a loop having a length of 3 to 5 nucleotides between S1 and S2, wherein the antisense strand and the sense strand form a duplex structure having a length of at least 19 nucleotides, and wherein the sense strand comprises a lipid moiety conjugated to the 5'-terminal nucleotide of the sense strand.

13. A double-stranded oligonucleotide for reducing STAT3 expression, the oligonucleotide comprising: (i) An antisense strand having a length of 19-30 nucleotides, wherein the antisense strand comprises a nucleotide sequence containing a complementary region that is complementary to a STAT3 mRNA target sequence, wherein the complementary region is shown in SEQ ID NO: 140, and (ii) A sense strand having a length of 19-50 nucleotides, which comprises a complementary region complementary to the antisense strand, wherein the sense strand comprises a lipid moiety conjugated to the 5'-terminal nucleotide of the sense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang at the 3'-end of the antisense strand consisting of 1-4 nucleotides.

14. The oligonucleotide according to any one of claims 11-13, wherein L is a tetraloop, optionally wherein the length of L is 4 nucleotides.

15. The oligonucleotide according to any one of claims 11-14, wherein L comprises a sequence represented as GAAA.

16. The oligonucleotide according to any one of claims 1-15, wherein the antisense strand has a length of 27 nucleotides and the sense strand has a length of 25 nucleotides, optionally wherein the antisense strand has a length of 22 nucleotides and the sense strand has a length of 36 nucleotides.

17. The oligonucleotide according to claim 16, wherein the antisense strand and the sense strand form a duplex region having a length of 25 nucleotides, optionally wherein the length of the duplex is 20 nucleotides.

18. The oligonucleotide according to any one of claims 1-17, wherein the antisense strand comprises a 3'-overhang sequence having a length of one or more nucleotides, optionally wherein the length of the 3'-overhang sequence is 2 nucleotides, optionally wherein the 3'-overhang sequence is GG.

19. The oligonucleotide according to any one of the foregoing claims, wherein the oligonucleotide comprises at least one modified nucleotide.

20. The oligonucleotide according to claim 19, wherein the modified nucleotide comprises a 2'-modification.

21. The oligonucleotide according to claim 20, wherein 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.

22. The oligonucleotide according to any one of claims 19-21, wherein about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand comprise a 2'-fluoro modification.

23. The oligonucleotide according to any one of claims 19-22, wherein 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.

24. The oligonucleotide according to any one of claims 19 - 23, wherein 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.

25. The oligonucleotide according to any one of claims 19 - 24, wherein the sense strand comprises 36 nucleotides from position 1 - 36 from 5' to 3', wherein positions 8 - 11 comprise a 2'-fluoro modification.

26. The oligonucleotide according to any one of claims 19 - 25, wherein the antisense strand comprises 22 nucleotides from position 1 - 22 from 3' to 5', and wherein positions 2, 3, 4, 5, 7, 10 and 14 comprise a 2'-fluoro modification.

27. The oligonucleotide according to any one of claims 22 - 26, wherein the remaining nucleotides comprise a 2'-O-methyl modification.

28. The oligonucleotide according to any one of the preceding claims, wherein the oligonucleotide comprises at least one modified internucleotide linkage.

29. The oligonucleotide according to claim 28, wherein the at least one modified internucleotide linkage is a phosphorothioate bond.

30. The oligonucleotide according to claim 29, wherein the sense strand comprises a phosphorothioate bond between positions 1 and 2 of the sense strand.

31. The oligonucleotide according to claim 29, wherein the antisense strand comprises 22 nucleotides from position 1 - 22 from 3' to 5', wherein the antisense strand comprises phosphorothioate bonds between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22.

32. The oligonucleotide according to any one of claims 29 - 31, wherein the sense strand comprises a phosphorothioate bond between positions 1 and 2 of the sense strand, and the antisense strand comprises 22 nucleotides from position 1 - 22 from 3' to 5', wherein the antisense strand comprises phosphorothioate bonds between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22.

33. The oligonucleotide according to any one of the preceding claims, wherein the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphonate analogue.

34. The oligonucleotide according to claim 33, wherein the phosphonate analogue is an oxy-methylphosphonate, vinylphosphonate or malonylphosphonate.

35. The oligonucleotide according to any one of the preceding claims, wherein the lipid moiety is a saturated or unsaturated fatty acid moiety.

36. The oligonucleotide according to any one of the preceding claims, wherein the lipid moiety is a saturated fatty acid moiety having a length in the range of C10 to C24.

37. The oligonucleotide according to claim 36, wherein the lipid moiety is a C16 saturated fatty acid moiety.

38. The oligonucleotide according to claim 37, wherein the C16 saturated fatty acid moiety is represented by the following formula:

39. The oligonucleotide according to claim 36, wherein the lipid moiety is a C18 saturated fatty acid moiety.

40. The oligonucleotide according to claim 39, wherein the C18 saturated fatty acid moiety is represented by the following formula:

41. The oligonucleotide according to any one of the preceding claims, wherein the lipid moiety is selected from:

42. The oligonucleotide according to any one of the preceding claims, wherein the lipid moiety is conjugated to the 2'-carbon of the ribose ring of the 5'-terminal nucleotide.

43. The oligonucleotide according to any one of claims 1-42, wherein the sense strand comprises the sequence shown in SEQ ID NO: 1222.

44. The oligonucleotide according to any one of claims 1-43, wherein the antisense strand comprises the sequence shown in SEQ ID NO: 1145.

45. The oligonucleotide according to any one of claims 1-44, wherein the sense strand comprises the sequence shown in SEQ ID NO: 1222, and wherein the antisense strand comprises the sequence shown in SEQ ID NO: 1145.

46. A double-stranded oligonucleotide for reducing STAT3 expression, wherein the oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprises the sequence shown in SEQ ID NO: 1222, the antisense strand comprises the sequence shown in SEQ ID NO: 1145, and wherein the sense strand and the antisense strand form an asymmetric duplex region having a length of 20 nucleotides and having an overhang consisting of 2 nucleotides at the 3'-end of the antisense strand.

47. The oligonucleotide according to any one of claims 1-45, wherein the complementary region is fully complementary to the STAT3 target sequence.

48. The oligonucleotide according to any one of claims 1-45, wherein the complementary region is partially complementary to the STAT3 target sequence.

49. The oligonucleotide according to claim 48, wherein the complementary region comprises no more than 4 mismatches with the STAT3 target sequence.

50. The oligonucleotide according to any one of claims 1-49, wherein the complementary region is fully complementary to the STAT3 target sequence at nucleotide positions 2-8 or 2-11 of the antisense strand, wherein the nucleotide positions are numbered from 5' to 3'.

51. The oligonucleotide according to any one of claims 1-50, wherein the oligonucleotide is a nuclease substrate, and after endogenous nuclease processing, it generates a double-stranded nucleic acid having a length of 19-21 nucleotides that can reduce the expression of STAT3 mRNA in mammalian cells.

52. The oligonucleotide according to any one of claims 1-51, wherein the oligonucleotide reduces the expression of STAT3 mRNA in one or more immune cells associated with the tumor microenvironment.

53. A pharmaceutical composition comprising the oligonucleotide according to any one of claims 1-52 and a pharmaceutically acceptable carrier, delivery agent, or excipient.

54. A method for treating cancer in a subject, the method comprising administering to the subject an effective amount of the oligonucleotide according to any one of claims 1-52 or the pharmaceutical composition according to claim 53.

55. The method according to claim 54, which comprises administering a PD-L1 inhibitor to a subject.

56. A method of treating cancer in a subject who has received or is receiving a PD-L1 inhibitor, the method comprising administering to the subject an oligonucleotide according to any one of claims 1-52 or a pharmaceutical composition according to claim 53, thereby treating the cancer in the subject.

57. A method of treating cancer in a subject who has received or is receiving an oligonucleotide targeting STAT3, wherein the oligonucleotide targeting STAT3 is an oligonucleotide according to any one of claims 1-52, the method comprising administering a PD-L1 inhibitor to the subject, thereby treating the cancer in the subject.

58. A method of treating a disease, disorder or condition associated with STAT3 expression in a subject, the method comprising administering to the subject an effective amount of an oligonucleotide according to any one of claims 1-52 or a pharmaceutical composition according to claim 53.

59. The method according to claim 58, which comprises administering a PD-L1 inhibitor to the subject.

60. A method of treating a disease, disorder or condition associated with STAT3 expression in a subject who has received or is receiving a PD-L1 inhibitor, the method comprising administering to the subject an oligonucleotide according to any one of claims 1-52 or a pharmaceutical composition according to claim 53, thereby treating the cancer in the subject.

61. A method of treating a disease, disorder or condition associated with STAT3 expression in a subject who has received or is receiving an oligonucleotide targeting STAT3, wherein the oligonucleotide targeting STAT3 is an oligonucleotide according to any one of claims 1-52, the method comprising administering a PD-L1 inhibitor to the subject, thereby treating the cancer in the subject.

62. The method according to any one of claims 58-61, wherein the disease, disorder or condition associated with STAT3 expression is cancer.

63. The method according to any one of claims 54-57 and 62, 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.

64. The method according to any one of claims 54-58 and 62-63, wherein the cancer comprises an immunosuppressive tumor microenvironment.

65. The method according to any one of claims 54-58 and 62-63, wherein the cancer comprises an inflamed tumor microenvironment.

66. The method according to claim 65, wherein the inflamed tumor microenvironment comprises infiltrating T cells.

67. The method according to any one of claims 55-57, 59-66, wherein the PD-L1 inhibitor is an antibody.

68. The method according to claim 67, wherein the antibody is an anti-PD-L1 antibody.

69. The method according to claim 68, wherein the anti-PDL1 antibody is selected from FAZ053, atezolizumab, avelumab, durvalumab, envolimab, and BMS-936559.

70. The method according to claim 67, wherein the antibody is an anti-PD-1 antibody.

71. The method according to claim 70, wherein the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, and cemiplimab.

72. The method according to any one of claims 54-57 and 62-71, wherein treating cancer comprises reducing or inhibiting tumor growth in a subject.

73. A method of reducing the expression of STAT3 mRNA in a cell, which comprises contacting the cell with an oligonucleotide according to any one of claims 1-52.

74. A kit, which comprises a container containing an oligonucleotide according to any one of claims 1-52, optionally a pharmaceutically acceptable carrier, and a package insert, the package insert comprising instructions for administration to a subject having a disease, disorder, or condition associated with STAT3 expression.

75. The kit according to claim 74, wherein the disease, disorder, or condition associated with STAT3 expression is cancer.

76. A kit, which comprises a container containing an oligonucleotide according to any one of claims 1-52, optionally a pharmaceutically acceptable carrier, and a package insert, the package insert comprising instructions for administration to a cancer subject who has received or is receiving a PD-L1 inhibitor.

77. A kit, which comprises a container containing a PD-L1 inhibitor, optionally a pharmaceutically acceptable carrier, and a package insert, the package insert comprising instructions for administration to a cancer subject who has received or is receiving an oligonucleotide according to any one of claims 1-52.

78. A kit, which comprises an oligonucleotide, optionally a pharmaceutically acceptable carrier, and a package insert, the package insert comprising instructions for administration of the oligonucleotide to a subject in need who has received or is receiving a PD-L1 inhibitor, wherein the oligonucleotide is an oligonucleotide according to any one of claims 1-52.

79. A kit, which comprises a PD-L1 inhibitor, optionally a pharmaceutically acceptable carrier, and a package insert, the package insert comprising instructions for administration of the inhibitor to a subject in need who has received or is receiving an oligonucleotide, wherein the oligonucleotide is an oligonucleotide according to any one of claims 1-52.

80. The kit according to claim 78 or 79, wherein the subject has a disease, disorder, or condition associated with activated STAT3 expression.

81. The kit according to any one of claims 78-80, wherein the subject has cancer.

82. A method for determining the responsiveness of a cancer subject who has received or is receiving treatment, the method comprising detecting the presence of a marker of myeloid-derived suppressor cells (MDSC) or MDSC activity in a biological sample of the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, and wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject is responsive to the treatment.

83. A method for determining the responsiveness of a cancer subject who has received or is receiving treatment, comprising: (i) obtaining a biological sample from the subject; and (ii) detecting the presence of a marker of MDSC or MDSC activity in the biological sample, wherein the treatment is administration of an oligonucleotide targeting STAT3, and wherein a decrease in MDSC or a decrease in the marker of MDSC activity in the biological sample indicates that the subject is responsive to the treatment.

84. The method according to any one of claims 82-83, wherein the detection comprises determining the amount of MDSC or the amount of the marker of MDSC activity.

85. The method according to any one of claims 82-84, wherein the decrease in MDSC or the marker of MDSC activity is relative to the amount or level of MDSC or the marker of MDSC activity in the subject prior to treatment.

86. The method according to any one of claims 82-84, wherein the decrease in MDSC or the marker of MDSC activity is relative to the amount or level of MDSC or the marker of MDSC activity in a patient population not receiving the treatment.

87. The method according to any one of claims 82-86, wherein the decrease in MDSC or the marker of MDSC activity is based on the amount or level of MDSC or the marker of MDSC activity in a patient population responsive to the treatment.

88. The method according to any one of claims 82-87, wherein the MDSC is granulocyte-MDSC (G-MDSC).

89. The method according to any one of claims 82-88, wherein the MDSC is monocyte-MDSC (M-MDSC).

90. The method according to any one of claims 82-89, wherein the MDSC expresses Arg1.

91. The method according to any one of claims 82-90, wherein the MDSC expresses IDO.

92. The method according to any one of claims 82-91, wherein the presence of MDSC or the marker of MDSC activity is determined by flow cytometry.

93. The method according to any one of claims 82-92, wherein the biological sample is a blood or serum sample.

94. The method according to any one of claims 82-93, wherein the response to the treatment comprises a decrease or inhibition of tumor growth and / or tumor size.

95. The method according to any one of claims 82-94, wherein the oligonucleotide targeting STAT3 is the oligonucleotide according to any one of claims 1-52.

Images