STAT3-Targeted Oligonucleotides and Uses Thereof

Abstract

The subject matter disclosed herein is directed to modulating STAT3 gene expression using siRNA compositions and methods directed to affecting key cell populations that support cancer growth and metastasis to affect beneficial treatment, remission, or elimination of the underlying tumor in a patient.

Description

[Technical Field]

[0001] Interrelated Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 425,861, filed November 16, 2022, the entire contents of which are incorporated herein by this reference. [Background technology]

[0002] Currently, chemotherapy, often combined with surgery or surgery and radiation therapy depending on the tumor type and stage, is the primary cancer therapy worldwide (Abbas et al., An Overview of Cancer Treatment Modalities / IntechOpen, 2018). Since the discovery of several key mutations that contribute to carcinogenesis (e.g., alterations in epidermal cells (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 the development of more selective drugs and drug combinations to treat cancer patients. Despite the effectiveness of these drugs, multidrug resistance (MDR) is often observed in patients, which often leads to tumor recurrence, limited treatment options, and a reduced quality of life for patients. Furthermore, cancer research has often focused on tumor cells, despite the effects of the tumor microenvironment and "normal" or non-cancerous cells within the tumor microenvironment, which have been shown to play a key role in tumor progression, development, and MDR (Klemm et al., TRENDS CELL BIOL (2015) 25(4):198-213). Novel therapies that target different aspects of the TME responsible for tumor growth are needed. Summary of the Invention

[0003] The present disclosure is based, in part, on the discovery of oligonucleotides that target and reduce STAT3 mRNA expression. The present disclosure is further based, in part, on the discovery that the combination of a STAT3 oligonucleotide and a PD-L1 inhibitor provides synergistic antitumor efficacy against tumors in various tumor microenvironments. Specifically, as demonstrated herein, lipid-conjugated STAT3 oligonucleotides, when delivered in combination with an anti-PD-L1 antibody, reduced tumor volume in vivo in immunosuppressive and inflammatory tumor models. Furthermore, as shown herein, the combination of a STAT3 oligonucleotide and a PD-L1 inhibitor induced an antitumor memory response in mice when tumors were not established upon rechallenge with cancer cells. Furthermore, the efficacy of the STAT3 oligonucleotide and the PD-L1 inhibitor depended on the presence of CD8+ T cells.

[0004] Thus, in some aspects, the present disclosure provides oligonucleotides for reducing STAT3 expression, the oligonucleotides comprising an antisense strand 15-30 nucleotides in length and a sense strand 15-40 nucleotides in length, wherein the sense strand and the antisense strand form a double-stranded region, the antisense strand having a region of complementarity to a target sequence of STAT3 set forth in SEQ ID NO: 140, and 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 embodiments, the antisense strand is 19 to 27 nucleotides in length. In some embodiments, the antisense strand is 21 to 27 nucleotides in length, and optionally, the antisense strand is 22 nucleotides in length.

[0006] In some or any of the aforementioned or related embodiments, the sense strand is 19-40 nucleotides in length, and optionally, the sense strand is 36 nucleotides in length.

[0007] In some or any of the foregoing or related embodiments, the double-stranded region is at least 19 nucleotides in length. In some embodiments, the double-stranded region is at least 20 nucleotides in length, and optionally, the double-stranded region is 21 nucleotides in length. In some embodiments, the region of complementarity to STAT3 is at least 19 contiguous nucleotides in length. In some embodiments, the region of complementarity to STAT3 is at least 21 contiguous nucleotides in length.

[0008] In some embodiments, the antisense strand comprises the sequence set forth in SEQ ID NO:965.

[0009] In some or any of the aforementioned or related embodiments, the sense strand comprises the sequence set forth in SEQ ID NO:875.

[0010] In some or any of the aforementioned or related embodiments, the sense strand comprises at its 3' end a stem-loop described as S1-L-S2, where S1 is complementary to S2 and L forms a loop 3-5 nucleotides long between S1 and S2.

[0011] In some aspects, the disclosure provides oligonucleotides for reducing STAT3 expression, the oligonucleotides comprising an antisense strand and a sense strand, wherein the antisense strand is 21 to 27 nucleotides in length and has a region of complementarity to a target sequence of STAT3 set forth in SEQ ID NO: 140, the sense strand comprises at its 3' end a stem-loop described as S1-L-S2, wherein S1 is complementary to S2 and L forms a loop 3 to 5 nucleotides in length between S1 and S2, the antisense strand and the sense strand form a duplex structure at least 19 nucleotides in length, and 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 a double-stranded oligonucleotide for reducing STAT3 expression, the oligonucleotide comprising: (i) an antisense strand having a length of 19 to 30 nucleotides, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a STAT3 mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 140; (ii) a sense strand 19 to 50 nucleotides in length that includes a region of complementarity to the antisense strand, wherein the sense strand includes a lipid moiety conjugated to the 5'-terminal nucleotide of the sense strand; The antisense and sense strands are separate strands that form an asymmetric duplex region with an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand.

[0013] In some or any of the aforementioned or related embodiments, L is a tetraloop, and optionally L is 4 nucleotides in length. In some embodiments, L comprises the sequence shown as GAAA.

[0014] In some or any of the foregoing or related embodiments, the antisense strand is 27 nucleotides in length, and the sense strand is 25 nucleotides in length, optionally the antisense strand is 22 nucleotides in length, and the sense strand is 36 nucleotides in length. In some embodiments, the antisense strand and the sense strand form a double-stranded region 25 nucleotides in length, optionally the double-stranded region is 20 nucleotides in length. In some embodiments, the antisense strand includes a 3' overhang sequence of one or more nucleotides in length, optionally the 3' overhang sequence is 2 nucleotides in length, and optionally the 3' overhang sequence is GG.

[0015] In some or any of the foregoing or related embodiments, the oligonucleotide comprises at least one modified nucleotide. In some embodiments, the modified nucleotide comprises a 2'-modification. In some embodiments, the 2'-modification is selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid. In some embodiments, about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides in the sense strand comprise a 2'-fluoro modification. In some embodiments, about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides in the antisense strand comprise a 2'-fluoro modification. In some embodiments, about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the oligonucleotide comprise 2'-fluoro modifications.

[0016] In some or any of the aforementioned or related embodiments, the sense strand comprises 36 nucleotides, having positions 1-36 from 5' to 3', with positions 8-11 comprising 2'-fluoro modifications. In some embodiments, the antisense strand comprises 22 nucleotides, having positions 1-22 from 3' to 5', with positions 2, 3, 4, 5, 7, 10, and 14 comprising 2'-fluoro modifications. In some embodiments, the remaining nucleotides comprise 2'-O-methyl modifications.

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

[0018] In some or any of the aforementioned or related embodiments, the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analog, hi some embodiments, the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

[0019] In some or any of the foregoing or related embodiments, the lipid moiety is a saturated or unsaturated fatty acid moiety. In some embodiments, the lipid moiety is a saturated fatty acid moiety ranging in size from C10 to C24 in length.

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

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

[0022] In some or any of the aforementioned or related embodiments, the lipid moiety is selected from the following: [ka]

[0023] In some or any of the foregoing or related embodiments, 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 aforementioned or related embodiments, the sense strand comprises the sequence set forth in SEQ ID NO: 1222. In some embodiments, the antisense strand comprises the sequence set forth in SEQ ID NO: 1145. In some or any of the aforementioned or related embodiments, 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.

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

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

[0027] In some or any of the aforementioned or related embodiments, the oligonucleotide is a Dicer substrate that, upon endogenous Dicer processing, produces a double-stranded nucleic acid 19-21 nucleotides in length that is capable of reducing STAT3 mRNA expression in mammalian cells.

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

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

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

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

[0032] In some aspects, the disclosure provides methods for treating cancer in a subject who has received or is receiving a PD-L1 inhibitor, the method comprising administering to the subject the oligonucleotide or pharmaceutical composition of any of the preceding or related aspects, thereby treating cancer in the subject.

[0033] In some aspects, the disclosure provides methods for treating cancer in a subject who has received or is receiving an oligonucleotide that targets STAT3, where the oligonucleotide that targets STAT3 is the oligonucleotide or pharmaceutical composition of any of the preceding or related aspects, and the method comprises administering a PD-L1 inhibitor to the subject, thereby treating cancer in the subject.

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

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

[0036] In some aspects, the disclosure provides a method for 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 the oligonucleotide or pharmaceutical composition of any of the preceding or related aspects, thereby treating cancer in the subject.

[0037] In some aspects, the 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, where the oligonucleotide targeting STAT3 is the oligonucleotide or pharmaceutical composition of any of the preceding 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 embodiments, the disease, disorder, or condition associated with STAT3 expression is cancer. In some embodiments, the cancer is selected from carcinoma, sarcoma, melanoma, lymphoma, and leukemia, prostate cancer, breast cancer, hepatocellular carcinoma (HCC), colorectal cancer, pancreatic cancer, and glioblastoma. In some embodiments, the cancer comprises an immunosuppressive tumor microenvironment. In some embodiments, the cancer comprises an inflammatory tumor microenvironment. In some embodiments, the inflammatory tumor microenvironment comprises infiltrating T cells.

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

[0040] In some or any of the aforementioned or related embodiments, the antibody is an anti-PD-1 antibody. In some embodiments, 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 includes reducing or inhibiting tumor growth in the subject.

[0042] In some aspects, the disclosure provides methods of reducing expression of STAT3 mRNA in a cell, the method comprising contacting the cell with the oligonucleotide of any of the preceding or related aspects.

[0043] In some aspects, the present disclosure provides kits comprising a container comprising the oligonucleotide of any of the preceding or related aspects, optionally a 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 embodiments, the disease, disorder, or condition associated with STAT3 expression is cancer.

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

[0046] In some embodiments, the disclosure provides kits that include a container containing a PD-L1 inhibitor, an optional pharmaceutically acceptable carrier, and a package insert that includes instructions for administration to a subject with cancer who has received or is receiving an oligonucleotide of any of the preceding or related embodiments.

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

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

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

[0050] In some aspects, the disclosure provides methods of determining responsiveness in a subject having cancer who has undergone or is undergoing treatment, the method comprising detecting the presence of myeloid-derived suppressor cells (MDSCs) or a marker of MDSC activity in a biological sample from the subject, wherein the treatment is administration of an oligonucleotide that targets STAT3, and wherein a reduction in MDSCs or a reduction in the marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment.

[0051] In some aspects, the present disclosure provides a method for determining responsiveness in a subject having cancer who has undergone or is undergoing treatment, the method comprising: (i) obtaining a biological sample from a subject; (ii) detecting the presence of MDSCs or markers of MDSC activity in the biological sample, wherein the treatment is administration of an oligonucleotide that targets STAT3, and wherein a reduction in MDSCs or a reduction in the markers of MDSC activity in the biological sample indicates that the subject is responding to the treatment.

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

[0053] In some embodiments, the reduction in MDSCs or markers of MDSC activity is relative to an amount or level of MDSCs or markers of MDSC activity before treatment of the subject.

[0054] In some embodiments, the reduction in MDSCs or markers of MDSC activity is relative to an amount or level of MDSCs or markers of MDSC activity in a population of patients who did not receive the treatment. In some embodiments, the reduction in MDSCs or markers of MDSC activity is based on an amount or level of MDSCs or markers of MDSC activity in a population of patients who responded to the treatment.

[0055] In some embodiments, the MDSC is a granulocytic MDSC (G-MDSC). In some embodiments, the MDSC is a monocytic MDSC (M-MDSC). In some embodiments, the MDSC expresses Arg1.

[0056] In some embodiments, the MDSCs express IDO. In some embodiments, the markers of MDSC presence or activity are determined by flow cytometry.

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

[0058] In some embodiments, responding to treatment includes a reduction or inhibition of tumor growth and / or tumor size.

[0059] In some embodiments, the oligonucleotide targeting STAT3 is the oligonucleotide of any of the preceding or related embodiments. [Brief explanation of the drawings]

[0060] [Figure 1A] FIG. 1A provides the structure of an RNAi oligonucleotide molecule having a chemical modification with GalNAc or a lipid (e.g., a C18 hydrocarbon chain) conjugated to the oligonucleotide molecule to generate an oligonucleotide-ligand conjugate. [Figure 1B] FIG. 1B provides the structure of a lipid tail suitable for conjugation to an RNAi oligonucleotide molecule. [Figure 2A-2B] Figures 2A and 2B are graphs showing residual mouse Stat3 mRNA levels in the livers of mice treated with GalXC-STAT3 conjugates (GalNAc conjugates) targeting different regions of Stat3 mRNA. Mice were administered a single dose (3 mg / kg) (Figure 2A) and / or various doses (0.3, 1.0, or 3.0 mg / kg) (Figure 2B) to determine dose response. Arrows indicate constructs selected for further study. [Figure 3A-3B] Figures 3A and 3B are graphs showing mouse Stat3 mRNA expression in G-MDSCs and M-MDSCs from Pan02 xenografts implanted in mice after 3 days of treatment with GalXC-STAT3-C18 conjugate administered at 25 mg / kg (Figure 3A) and 50 mg / kg (Figure 3B) into tumors. [Figure 4A-4B] Figures 4A and 4B are graphs showing mouse Stat3 mRNA expression after treatment of Pan02 xenograft mice with GalXC-STAT3-C18 conjugate at doses of 25 and 50 mg / kg in the bulk tumor (TME) (Figure 4A) and tumor-draining lymph node (TdLN) (Figure 4B). [Figure 5A] Figure 5A provides a graph showing the effect of GalXC-STAT3-C18-4123 on Stat3 and Pdl1 mRNA levels in G / M-MDSCs in the TME and TdLN of Pan02 xenograft mice 3 days after administration of 25 or 50 mg / kg of the conjugated oligonucleotide. [Figure 5B] FIG. 5B provides a graph showing the effect of GalXC-STAT3-C18-4123 on Stat3 and Pdl1 mRNA levels in the TdLN of Pan02 xenograft mice 7 days after a 25 mg / kg dose of the conjugated oligonucleotide. [Figures 6A-6B]Figures 6A and 6B are graphs showing the in vivo effect of subcutaneous treatment with GalXC-STAT3-C18-4123 at a total dose of 50 mg / kg on tumor volume over time in immunocompetent mice bearing Pan02 murine pancreatic tumors. Mice were treated with either four doses of 12.5 mg / kg (Figure 6A) or two doses of 25 mg / kg (Figure 6B) of the conjugated oligonucleotide. The lines represent the average of all animals tested. [Figure 7] Figure 7 provides a graph showing the percent (%) of human STAT3 mRNA remaining in Huh7 cells that endogenously express human STAT3 after 24 hours of treatment with 1 nM DsiRNAs targeting various regions of the STAT3 gene. 192 DsiRNAs were designed and screened. Two primer pairs were used. Expression was normalized between samples using the housekeeping genes HPRT and SFRS9 (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). [Figure 8A-8B] Figures 8A and 8B provide graphs showing the percent (%) of human STAT3 mRNA remaining in Huh7 cells that endogenously express human STAT3 after 24 hours of treatment with 0.05 nM, 0.3 nM, or 1 nM DsiRNAs targeting various regions of the STAT3 gene. 48 GalNAc-conjugated STAT3 oligonucleotides were assayed in Figure 8A, and 34 of these oligonucleotides were selected for further testing in vivo (Figure 8B). [Figure 9A-9B]Figures 9A and 9B provide graphs showing the percent (%) of human STAT3 mRNA remaining in the livers of mice exogenously expressing human STAT3 (hydrodynamic injection model) after treatment with GalNAc-conjugated STAT3 oligonucleotides. Mice were administered 1 mg / kg of the indicated GalNAc-STAT3 oligonucleotides subcutaneously formulated in PBS. Three days after administration, mice were hydrodynamically injected (HDI) with a DNA plasmid encoding human STAT3. Human STAT3 mRNA levels were determined from livers harvested 18 hours after injection. Arrows indicate oligonucleotides selected for dose-response analysis. Hs / Mf = human / monkey consensus sequence; Hs / Mm = human / mouse consensus sequence; Hs / Mf / Mm = human / monkey / mouse triple consensus sequence. [Figure 10] Figure 10 provides a graph showing the dose response of GalNAc-conjugated STAT3 oligonucleotides. The percent (%) of human STAT3 mRNA remaining in the liver of mice exogenously expressing human STAT3 (HDI model) after treatment with two different doses (0.3 mg / kg or 1 mg / kg) of human GalNAc-conjugated STAT3 oligonucleotides was measured. Human STAT3 mRNA levels were determined from livers harvested 18 hours after injection with a plasmid encoding human STAT3. Arrows indicate the oligonucleotides selected for dose-response analysis. Hs / Mf = human / monkey consensus sequence, Hs / Mm = human / mouse consensus sequence. [Figure 11] Figure 11 provides a graph showing normalized (relative to Ppib) mouse STAT3 mRNA remaining in the liver of mice endogenously expressing mouse STAT3 after treatment with GalNAc-conjugated STAT3 oligonucleotides. Mice were subcutaneously administered 3 mg / kg of the indicated GalNAc-STAT3 oligonucleotide formulated in PBS. Five days after administration, livers were harvested and mouse STAT3 mRNA levels were determined. Arrows indicate the top oligonucleotide and the oligonucleotide selected for dose-response studies. [Figure 12]Figure 12 provides a graph showing normalized (relative to Ppib) mouse STAT3 mRNA remaining in the liver of mice endogenously expressing mouse STAT3 after treatment with GalNAc-conjugated STAT3 oligonucleotides. Mice were subcutaneously administered 3 mg / kg of the indicated GalNAc-STAT3 oligonucleotide formulated in PBS. Five days after administration, livers were harvested and mouse STAT3 mRNA levels were determined. Arrows indicate the oligonucleotides selected for dose-response studies. [Figures 13A-13B] Figures 13A and 13B provide graphs showing the dose response of GalNAc-conjugated STAT3 oligonucleotides. The percent (%) of mouse STAT3 mRNA remaining in the livers of mice endogenously expressing human STAT3 was measured after treatment with three doses (0.3 mg / kg, 1 mg / kg, and 3 mg / kg) of human GalNAc-conjugated STAT3 oligonucleotides. Mouse STAT3 mRNA levels were determined from livers harvested 5 days later. TC = triple common (mouse / human / monkey); Hs_Mm = human / mouse. [Figure 14] Figure 14 provides a graph showing the percent (%) of human STAT3 mRNA remaining in the liver of mice exogenously expressing human STAT3 (hydrodynamic injection model) after treatment with GalNAc-conjugated STAT3 oligonucleotides. Mice were subcutaneously administered 1 mg / kg of the indicated GalNAc-STAT3 oligonucleotide formulated in PBS. Three days after administration, mice were hydrodynamically injected (HDI) with a DNA plasmid encoding human STAT3. Human STAT3 mRNA levels were determined from livers harvested 18 hours after injection. The arrow indicates the oligonucleotide selected for the dose-response study. [Figure 15]Figure 15 provides a graph showing the dose response of GalNAc-conjugated STAT3 oligonucleotides. Percentage (%) of human STAT3 mRNA remaining in the liver of mice exogenously expressing human STAT3 (hydrodynamic injection model) after treatment with GalNAc-conjugated STAT3 oligonucleotides. Mice were subcutaneously administered three doses (0.3 mg / kg, 1 mg / kg, and 3 mg / kg) of the indicated GalNAc-STAT3 oligonucleotide formulated in PBS. Three days after administration, mice were hydrodynamically injected (HDI) with a DNA plasmid encoding human STAT3. Human STAT3 mRNA levels were determined from livers harvested 18 hours after injection. TC = triple common (mouse / human / monkey); Hs_Mm = human / mouse; Hs = human. [Figure 16] Figure 16 provides a graph showing the dose response of GalNAc-conjugated STAT3 oligonucleotides. Percentage (%) of human STAT3 mRNA remaining in the liver of mice exogenously expressing human STAT3 (hydrodynamic injection model) after treatment with GalNAc-conjugated STAT3 oligonucleotides. Mice were subcutaneously administered two doses (0.3 mg / kg and 1 mg / kg) of the indicated GalNAc-STAT3 oligonucleotide formulated in PBS. Three days after administration, mice were hydrodynamically injected (HDI) with a DNA plasmid encoding human STAT3. Human STAT3 mRNA levels were determined from livers harvested 18 hours after injection. [Figure 17] 17 provides a graph showing the percent (%) of human STAT1 mRNA remaining in Huh7 cells, which endogenously express STAT3 and STAT1, treated with GalNAc-conjugated STAT3 oligonucleotides. Cells were treated with three doses (0.05 nM, 0.3 nM, and 1 nM) of oligonucleotide for 24 hours. [Figure 18A]Figure 18A provides a graph showing tumor volume following administration of GalXC-STAT3-C18 oligonucleotide alone or in combination with anti-PD-L1 mAb. Immunocompetent mice bearing Pan02 murine pancreatic tumors were treated intraperitoneally (ip) with 10 mg / kg of anti-PD-L1 mAb followed by subcutaneous (sc) administration of 25 mg / kg of GalXC-STAT3-C18-4123. Controls included GalXC-placebo (HBV siRNA with the same chemical and lipid conjugation as the 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 initially administered two doses, 3 days apart, and then two weeks later received two more doses, 3 days apart [(q3d × 2) × 2]. Arrows indicate the days on which doses were administered. [Figure 18B] Figure 18B provides a graph showing tumor volume after administration of GalXC-STAT3-C18 oligonucleotide in combination with anti-PD-L1 mAb. Immunocompetent mice bearing Pan02 murine pancreatic tumors were treated intraperitoneally (ip) with 10 mg / kg of anti-PD-L1 mAb followed by subcutaneous (sc) administration of 25 mg / kg of GalXC-STAT3-C18-4123. Mice were administered GalXC-placebo on days 42 and 45 post-implantation, followed by GalXC-STAT3 in combination with anti-PD-L1 mAb on days 60 and 63. [Figures 19A-19C]Figures 19A-19C provide graphs showing tumor volume following administration of GalXC-STAT3-C18 oligonucleotide alone or in combination with anti-PD-L1 mAb, or GalXC-placebo alone or in combination with anti-PD-L1 mAb in tumors with different immunophenotypes. Mice were implanted with 4T1 (triple-negative breast, checkpoint-resistant) (Figure 19A), MC-38 (clonal carcinoma, partially checkpoint-sensitive) (Figure 19B), or Hepa1-6 (hepatocellular carcinoma, checkpoint-sensitive) (Figure 19C). Tumor-bearing mice were administered 25 mg / kg of GalXC-STAT3-C18-4123 sc, followed by 10 mg / kg of anti-PD-L1 mAb i.p. treatment. Controls included 25 mg / kg GalXC-placebo, GalXC-STAT3-C18-4123, or 25 mg / kg GalXC-placebo in combination with 10 mg / kg anti-PD-L1. Mice bearing MC-38 and Hepa1-6 tumors were administered two doses of 25 mg / kg, three days apart, with the same regimen repeated the following week. Mice bearing 4T1 tumors were administered three doses, each three days apart (q3d x 3). Arrows (5 / 5 CR) indicate all treated mice were complete responders. [Figure 20] Figure 20 provides a graph showing the effect of Hepa1-6 rechallenge on completely eradicated tumors. After tumors in all five mice were completely regressed with treatment with GalXC-STAT3-C18 (25 mg / kg, sc) and anti-PD-L1 mAb (10 mg / kg, ip) in Figure 19C, the mice were rechallenged with Hepa1-6 cells (2e6 cells / mouse) on the opposite flank on day 51, and tumor volume was monitored (Figure 20). Arrows (5 / 5 CR) = All mice remained tumor-free even after rechallenge. [Figures 21A-21B]Figures 21A and 21B provide graphs showing tumor volume after administration of GalXC-STAT3-C18 oligonucleotide alone or in combination with anti-PD-L1 mAb in immunocompetent mice with functional CD8+ T cells (Figure 21A) and immunodeficient mice without functional CD8+ T cells (Figure 21B). Mice bearing 4T1 tumors (immunocompetent or immunodeficient) were administered sc with GalXC-STAT3-C18-4123 (25 mg / kg, three doses at 3-day intervals (q3d x 3)) and i.p. with anti-PD-L1 mAb (10 mg / kg, q3d x 3). Controls included 25 mg / kg GalXC-placebo, GalXC-STAT3-C18-4123, or 25 mg / kg GalXC-placebo in combination with 10 mg / kg anti-PD-L1. [Figure 22] FIG. 22 provides images showing the appearance of tumors (with cell death) from the mice assayed in FIG. 21A, and perforin staining for positive cytotoxic CD8+ T cells in the tumors at the end of the study. [Figure 23] Figure 23 provides a graph showing tumor volume and images showing lung tumor metastases after administration of GalXC-STAT3-C18-4123 oligonucleotide alone or in combination with anti-PD-L1 mAb. Mice (immunocompetent or immunodeficient) bearing 4T1 tumors were administered sc with GalXC-STAT3-C18-4123 (50 mg / kg, q3d x 3) and i.p. with anti-PD-L1 mAb (10 mg / kg, q3d x 3). Controls included 50 mg / kg GalXC-placebo, GalXC-STAT3-C18-4123, or 50 mg / kg GalXC-placebo in combination with 10 mg / kg anti-PD-L1. [Figure 24]Figure 24 provides a heat map showing the regulation of targets involved in immune modulation observed in CT26 tumors following combined treatment with GalXC-STAT3-C18-4123 (sc, 25 mg / kg, q3d x 3) and anti-PD-L1 mAb (ip, 10 mg / kg, q3d x 3) compared to a control including 25 mg / kg GalXC-placebo, GalXC-STAT3-C18-4123, or 25 mg / kg GalXC-placebo in combination with 10 mg / kg anti-PD-L1 mAb. [Figure 25] FIG. 25 provides the structure of an RNAi oligonucleotide molecule having a chemical modification with a C18 lipid conjugated to the 5' terminal nucleotide of the sense strand to generate an oligonucleotide-ligand conjugate. [Figures 26A-26C] Figures 26A-26C provide graphs showing tumor volume after administration of DCR-STAT3 (a human-specific STAT3 sequence with a C18 at the 5' position of the passenger strand corresponding to SEQ ID NOs: 1222 and 1145) or GalXC-placebo (a chemically matched, unrelated sequence that does not bind to the Stat3 / STAT3 mRNA target sequence) alone or in combination with an anti-PD-L1 antibody. Immunocompetent mice bearing B16F10 (murine melanoma), Pan02 (murine pancreatic), and MC-38 (murine colorectal) tumors were treated with either three or four subcutaneous (sc) doses of 25 mg / kg of conjugated oligonucleotide alone or in combination with 10 mg / kg of intraperitoneal (ip) anti-PD-L1 antibody. B16F10 tumor-bearing mice received three doses at 3-day intervals, while Pan02 tumor-bearing mice received two doses initially at 3-day intervals, followed one week later by two additional doses at 3-day intervals. MC-38 tumor-bearing mice were initially administered two doses, three days apart, and four days later, two more doses, three days apart. Arrows indicate the days on which doses were administered. DETAILED DESCRIPTION OF THE INVENTION

[0061] The present disclosure will now be described in more detail below with reference to the accompanying drawings, in which exemplary embodiments of the present disclosure are shown. However, the present disclosure may be embodied in many different forms and should not be construed as 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.

[0062] definition Publications discussed throughout the text are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

[0063] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Furthermore, the singular and the articles "a," "an," and "the" are intended to include the plural unless expressly stated otherwise. Furthermore, it should be understood that as used herein, the terms "includes," "comprises," "including," and / or "comprising" specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Furthermore, 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 may be directly connected or coupled to the other element, or intervening elements may be present.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the methods and compositions of the present disclosure, exemplary methods and materials are described herein.

[0065] General texts describing molecular biology techniques useful herein, including the use of vectors, promoters, and many other related topics, include Berger and Kimmel, Guide to Molecular Cloning Techniques, METHODS IN ENZYMOLOGY, volume 152, (Academic Press, Inc., San Diego, Calif.) (“Berger”), Sambrook et al., MOLECULAR CLONING—A LABORATORY MANUAL, 2nd ed., Vol. 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 skill in the art through in vitro amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Q. beta.-replicase amplification, and other RNA polymerase-mediated techniques (e.g., NASBA) for the production of homologous nucleic acids of the present disclosure, can be found in Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987) 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) Cand 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 Wallace et al., U.S. Patent No. 5,426,039. Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng et al., (1994) Nature 369:684-85, and references cited therein, where PCR amplicons of up to 40 kb are generated.

[0066] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0067] Ranges may be expressed herein as from "about" one value and / or to "about" another value. When such a range is expressed, another embodiment includes from the one value and / or to the other value. Similarly, it will be understood that when values ​​are expressed as approximations, the use of "about" the preceding term causes the value to form another embodiment. It will further be understood that each endpoint of a range is significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are several values ​​disclosed herein, and that each value is also disclosed herein as "about" that value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. It is also understood that when a value is disclosed, "less than or equal to," "greater than or equal to," and possible ranges between values ​​are also disclosed, as appropriately understood by one of ordinary skill in the art. For example, if the value "10" is disclosed, "less than or equal to 10" as well as "greater than or equal to 10" are also disclosed. It is also understood that throughout this application, data are provided in several different formats, and that this data represents endpoints and starting points, as well as ranges for any combination of the data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, then values ​​greater than 10 and 15, greater than or equal to 10 and 15, less than 10 and 15, less than or equal to 10 and 15, and equal to 10 and 15 are considered to be disclosed, as are values ​​between 10 and 15. It is also understood that each unit between two specified units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0068] In this specification and the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0069] The terms "cancer" or "tumor" include, but are not limited to, solid tumors and blood-borne tumors. These terms include diseases of the skin, tissues, organs, bone, cartilage, blood, and blood vessels. These terms further encompass primary and metastatic cancers.

[0070] The term "PD-1" refers to a protein found on T cells that helps keep the immune response in check. When PD-1 is bound to another protein called PD-L1, this helps prevent T cells from killing other cells, including cancer cells. Several anti-cancer drugs called immune checkpoint inhibitors are used to block PD-1. When this protein is prevented from acting on T cells, they can act to kill cancer cells.

[0071] The term "STAT3" refers to signal transducer and activator of transcription 3 (STAT3), a transcription factor encoded in humans by the STAT3 gene (STAT3 human (Hs) NM_001369512.1 Genbank reference sequence number or NM_139276.3). STAT3 mediates the expression of various genes in response to cellular stimuli and thus plays an important role in many cellular processes, such as cell proliferation and apoptosis, as well as cancer growth and progression.

[0072] As used herein, the terms "cold tumor" or "non-inflammatory tumor" refer to a tumor or tumor microenvironment that has minimal or no anti-tumor immune cells, such as tumor-infiltrating lymphocytes (TILs), and / or contains cell subsets associated with immunosuppression, including regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and M2 macrophages. Specifically, in some embodiments, cold tumors are characterized by low numbers or even absence of infiltrating anti-tumor immune cells; such cells may be present but remain adherent to the surrounding stroma, thus unable to colonize the tumor microenvironment and provide their anti-tumor function.

[0073] As used herein, "complementary" refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand) that allows the two nucleotides to base pair with each other. For example, purine nucleotides of one nucleic acid that are complementary to pyrimidine nucleotides of an opposing nucleic acid can base pair together by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides can base pair by Watson-Crick or any other method that allows the formation of a stable duplex. In some embodiments, two nucleic acids can have a region of multiple nucleotides that are complementary to each other and form a region of complementarity, as described herein.

[0074] As used herein, " species cross-reactive oligonucleotide " refers to an oligonucleotide that can inhibit the expression of target mRNA in two or more species.For example, in some embodiments, species cross-reactive oligonucleotide can inhibit the expression of target mRNA in human and non-human primates.Examples of species include, but are not limited to, human, non-human primates, mouse, and rat.In some embodiments, species cross-reactive oligonucleotide can target and inhibit the mRNA in at least two, at least three, or at least four species.

[0075] As used herein, "deoxyribonucleotide" refers to a nucleotide that, compared to a ribonucleotide, has a hydrogen instead of a hydroxyl at the 2' position of its pentose sugar. Modified deoxyribonucleotides are deoxyribonucleotides that have one or more modifications or substitutions of atoms other than the 2' position, including in or modifications or substitutions of the sugar, phosphate group, or base.

[0076] As used herein, "double-stranded RNA" or "dsRNA" refers to an RNA oligonucleotide that is substantially double-stranded. In some embodiments, the complementary base pairing in the double-stranded region of a dsRNA oligonucleotide is formed between antiparallel sequences of nucleotides in covalently separated nucleic acid strands. In some embodiments, the complementary base pairing in the double-stranded region of a dsRNA oligonucleotide is formed between antiparallel sequences of nucleotides in covalently linked nucleic acid strands. In some embodiments, the complementary base pairing in the double-stranded region of a dsRNA oligonucleotide is formed from a single nucleic acid strand that folds back (e.g., via a hairpin) to provide a complementary antiparallel sequence of base-pairing nucleotides together. In some embodiments, a dsRNA comprises two covalently separated nucleic acid strands that are fully double-stranded with each other. However, in some embodiments, a dsRNA comprises two covalently separated nucleic acid strands that are partially double-stranded (e.g., with overhangs at one or both ends). In some embodiments, the dsRNA comprises antiparallel sequences of partially complementary nucleotides and thus may have one or more mismatches, which may include internal or terminal mismatches.

[0077] As used herein, "duplex" with respect to a nucleic acid (eg, an oligonucleotide) refers to the structure formed through complementary base pairing of two antiparallel sequences of nucleotides.

[0078] As used herein, "excipient" refers to a non-therapeutic agent that may be included in a composition, for example, to provide or contribute a desired consistency or stabilizing effect.

[0079] As used herein, the term "hot tumor" or "inflammatory tumor" refers to a tumor or tumor microenvironment that has a significant presence of anti-tumor immune cells, particularly TILs, and is therefore typically immunostimulatory.

[0080] As used herein, a "loop" refers to an unpaired region of a nucleic acid (e.g., an oligonucleotide) adjacent to two antiparallel regions of the nucleic acid that are sufficiently complementary to each other so that under appropriate hybridization conditions (e.g., intracellularly, in a phosphate buffer), the two antiparallel regions adjacent to the unpaired region hybridize to form a duplex (called a "stem"). A loop containing four nucleotides may be referred to as a tetraloop (tetraL). A loop containing three nucleotides may be referred to as a triloop (triL).

[0081] As used herein, "modified internucleotide linkage" refers to an internucleotide linkage that has one or more chemical modifications compared to a reference internucleotide linkage containing a phosphodiester bond. In some embodiments, the modified nucleotide is a non-naturally occurring linkage. Typically, the modified internucleotide linkage imparts one or more desirable properties to the nucleic acid in which the modified internucleotide linkage is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0082] As used herein, a "modified nucleotide" refers to a nucleotide having one or more chemical modifications compared to a corresponding reference nucleotide selected from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides, and thymidine deoxyribonucleotides. In some embodiments, the modified nucleotide is a non-naturally occurring nucleotide. In some embodiments, the modified nucleotide has one or more chemical modifications in its sugar, nucleobase, and / or phosphate group. In some embodiments, the modified nucleotide has one or more chemical moieties conjugated to the corresponding reference nucleotide. Typically, the modified nucleotide confers one or more desirable properties to the nucleic acid in which the modified nucleotide is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0083] As used herein, "nicked tetraloop structure" refers to a structure of an RNAi oligonucleotide characterized by separate sense (passenger) and antisense (guide) strands, where the sense strand has a region of complementarity with the antisense strand, and at least one of the strands, generally the sense strand, has a tetraloop configured to stabilize an adjacent stem region formed in at least one strand.

[0084] As used herein, "oligonucleotide" refers to a short nucleic acid (e.g., less than about 100 nucleotides in length). An oligonucleotide can be single-stranded (ss) or double-stranded (ds). An oligonucleotide may or may not have a double-stranded region. An oligonucleotide may contain deoxyribonucleotides, ribonucleosides, or a combination of both. In some embodiments, a double-stranded oligonucleotide containing ribonucleotides is referred to as "dsRNA." As a non-limiting example, the oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a dicer substrate interfering RNA (dsiRNA), an antisense oligonucleotide, a short siRNA, or a ss siRNA. In some embodiments, the double-stranded RNA (dsRNA) is an RNAi oligonucleotide.

[0085] 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.

[0086] As used herein, " overhang " refers to the terminal unpaired nucleotides that arise from one strand or region that extends beyond the end of the complementary strand that forms a double strand.In some embodiments, overhang comprises one or more unpaired nucleotides that extend from the double-stranded region at the 5'-end or 3'-end of dsRNA.In certain embodiments, overhang is the 3' or 5' overhang on the antisense strand or sense strand of dsRNA.

[0087] As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is positioned at the 5'-terminal nucleotide of an oligonucleotide in place of the 5'-phosphate, which is often susceptible to enzymatic removal. In some embodiments, the 5'-phosphate analog comprises a phosphatase-resistant linkage. Examples of phosphate analogs include, but are not limited to, 5'-phosphonates, such as 5'-methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). In some embodiments, an oligonucleotide has a phosphate analog at the 4'-carbon position of the sugar (referred to as a "4'-phosphate analog") at the 5'-terminal nucleotide. An example of a 4'-phosphate analog is an oxymethyl phosphonate, in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., its 4' carbon) or an analog thereof. See, e.g., U.S. Provisional Patent Application Nos. 62 / 383,207, filed September 2, 2016, and 62 / 393,401, filed September 12, 2016. Other modifications to the 5' end of oligonucleotides have been developed (see, e.g., International Patent Application No. 2011 / 133871, U.S. Patent No. 8,927,513, and Prakash et al., (2015) Nucleic Acids Res. 43:2993-3011).

[0088] As used herein, "reduced expression" of a gene (e.g., STAT3) refers to the reduction in the amount or level of RNA transcript (e.g., STAT3 mRNA) or protein encoded by the gene in a cell, cell population, sample, or subject, compared with an appropriate reference (e.g., reference cell, reference cell population, reference sample, or reference subject).For example, contacting a cell with the oligonucleotide herein (e.g., an oligonucleotide comprising an antisense strand having a nucleotide sequence that is complementary to the nucleotide sequence comprising STAT3 mRNA) can result in a reduction in the amount or level of STAT3 mRNA, protein, and / or activity (e.g., through the degradation of STAT3 mRNA by the RNAi pathway) compared with cells that are not treated with dsRNA.Similarly, as used herein, "reducing expression" refers to the action of reducing the expression of a gene (e.g., STAT3). As used herein, "reduced STAT3 expression" refers to a decrease in the amount or level of STAT3 mRNA, STAT3 protein, and / or STAT3 activity in a cell, cell population, sample, or subject compared to an appropriate reference (e.g., a reference cell, a reference cell population, a reference sample, or a reference subject).

[0089] As used herein, "region of complementarity" refers to a nucleotide sequence of a nucleic acid (e.g., dsRNA) that is sufficiently complementary to an antiparallel sequence of nucleotides to allow hybridization between the two sequences of nucleotides under suitable hybridization conditions (e.g., in a phosphate buffer, in a cell, etc.). In some embodiments, the oligonucleotide herein comprises a target sequence that has a region of complementarity to an mRNA target sequence.

[0090] As used herein, "ribonucleotide" refers to a nucleotide having ribose as its pentose sugar, containing a hydroxyl group at its 2' position. A modified ribonucleotide is a ribonucleotide with one or more modifications or substitutions of atoms other than the 2' position, including in or modifications or substitutions of the ribose, phosphate group, or base.

[0091] As used herein, "RNAi oligonucleotide" refers to either (a) a dsRNA having a sense strand (passenger) and an antisense strand (guide), where the antisense strand, or a portion of the antisense strand, is used by Argonaute 2 (Ago2) endonuclease in cleaving a target mRNA, or (b) an ss oligonucleotide having a single antisense strand, where the antisense strand (or a portion of the antisense strand) is used by Ago2 endonuclease in cleaving a target mRNA.

[0092] As used herein, a "strand" refers to a single, contiguous sequence of nucleotides linked together through internucleotide bonds (e.g., phosphodiester or phosphorothioate bonds). In some embodiments, the strand has two free ends (e.g., a 5' end and a 3' end).

[0093] As used herein, "subject" refers to any mammal, including mice, rabbits, non-human primates (NHPs), and humans. In one embodiment, the subject is a human or an NHP. Furthermore, "individual" or "patient" can be used interchangeably with "subject."

[0094] As used herein, "synthetic" refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine (e.g., a solid-state nucleic acid synthesizer)) or is not otherwise derived from a natural source (e.g., a cell or organism) that normally produces the molecule.

[0095] As used herein, a "targeting ligand" refers to a molecule or "moiety" (e.g., a carbohydrate, amino sugar, cholesterol, polypeptide, or lipid) that selectively binds to a cognate molecule (e.g., a receptor) in a tissue or cell of interest and / or can be conjugated to another substance for the purpose of targeting other substances to the tissue or cell of interest. For example, in some embodiments, a targeting ligand can be conjugated to an oligonucleotide for the purpose of targeting the oligonucleotide to a specific tissue or cell of interest. In some embodiments, the targeting ligand selectively binds to a cell surface receptor. Thus, in some embodiments, the targeting ligand, when conjugated to the oligonucleotide, promotes delivery of the oligonucleotide to a specific cell through selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of a complex comprising the oligonucleotide, targeting ligand, and receptor. In some embodiments, the targeting ligand is conjugated to the oligonucleotide via a linker that is cleaved after cellular internalization or during cellular internalization, such that the oligonucleotide is released from the targeting ligand within the cell.

[0096] As used herein, "loop," "triloop," or "tetraloop" refers to a loop that increases the stability of adjacent duplexes formed by hybridization of adjacent sequences of nucleotides. The increase in stability is measured by the average predicted melting temperature (T) of adjacent stem duplexes from a set of loops of equivalent length consisting of randomly selected sequences of nucleotides. m ) higher than the T of the adjacent stem duplex m For example, the loop can be detected as an increase in T of at least about 50° C., at least about 55° C., at least about 56° C., at least about 58° C., at least about 60° C., at least about 65° C., or at least about 75° C. in 10 mM NaHPO. mIn some embodiments, a loop (e.g., a tetraloop) can stabilize adjacent bp of the stem duplex by stacking interactions. Additionally, interactions between nucleotides in a tetraloop include, but are not limited to, non-Watson-Crick base pairing, stacking interactions, hydrogen bonds, and contact interactions (Cheong et al., (1990) Nature 346:680-82; Heus and Pardi (1991) Science 253:191-94). In some embodiments, the loop comprises or consists of 3 to 6 nucleotides, typically 4 to 5 nucleotides. In certain embodiments, the loop comprises or consists of 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., conjugated to a targeting moiety). In some embodiments, the tetraloop comprises or consists of 3 to 6 nucleotides, typically 4 to 5 nucleotides. In certain embodiments, a tetraloop comprises or consists of 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., conjugated to a targeting moiety). In one embodiment, a loop consisting of 4 nucleotides is a tetraloop. Any nucleotide may be used in the loop (e.g., a tetraloop), and the standard IUPAC-IUB symbols for such nucleotides may be used as described in Cornish-Bowden ((1985) Nucleic Acids Res. 13:3021-3030). For example, the letter "N" may be used to indicate that any base can be at that position, the letter "R" may be used to indicate that A (adenine) or G (guanine) can be at that position, and "B" may be used to indicate that C (cytosine), G (guanine), or T (thymine) can be at that position.Examples of tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop (Woese et al., (1990) Proc. Natl. Acad. Sci. USA 87:8467-71; Antao et al., (1991) Nucleic Acids Res. 19:5901-05). Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA), d(GNRA)) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)). (See, e.g., Nakano et al., (2002) Biochem. 41:4281-92; Shinji et al., (2000) Nippon Kagakkai Koen Yokoshu 78:731.) In some embodiments, the tetraloop is contained within a nicked tetraloop structure.

[0097] As used herein, "treat" or "treating" refers to the act of providing care to a subject in need of treatment, for example, by administering a therapeutic agent (e.g., an oligonucleotide herein) to the subject, with the intent of improving the subject's health and / or well-being with respect to an existing condition (e.g., a disease, a disorder) or preventing or reducing the likelihood of the condition occurring. 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, a disorder) experienced by the subject.

[0098] As used herein, the term "tumor microenvironment" refers to the cellular environment in which any given tumor resides, including the tumor stroma, surrounding blood vessels, immune cells, fibroblasts, other cells, signaling molecules, and ECM. It is understood that the tumor microenvironment harbors and / or surrounds the tumor cells with which it interacts.

[0099] How to use Combination of STAT3 oligonucleotide and PD-L1 inhibitor In some embodiments, the present disclosure provides STAT3 oligonucleotides for use or adaptable for use to treat a subject who has received or is receiving PD-L1 (e.g., a human having a disease, disorder, or condition associated with STAT3 expression).

[0100] In some embodiments, the methods described herein include selecting a subject having or prone to a disease, disorder, or condition associated with STAT3 expression and / or PD-L1 expression. In some cases, the methods may include selecting an individual with markers for a disease associated with STAT3 expression and / or PD-L1 expression, such as cancer or other chronic lymphoproliferative disorder.

[0101] Similarly, as detailed herein, the methods may also include steps such as measuring or obtaining baseline values ​​of markers of, for example, STAT3 expression and / or PD-L1 expression, and then comparing the values ​​so obtained with one or more other baseline values ​​or values ​​obtained after administration of the oligonucleotide to assess the effectiveness of the treatment.

[0102] In some embodiments, the present disclosure provides methods of treating a subject having, suspected of having, or at risk of developing a disease, disorder, or condition with a STAT3 oligonucleotide described herein, wherein the subject has received or has received a PD-L1 inhibitor. In some embodiments, the present disclosure provides methods of treating a subject having, suspected of having, or at risk of developing a disease, disorder, or condition with a PD-L1 inhibitor described herein, wherein the subject has received or has received a STAT3 oligonucleotide described herein.

[0103] In some aspects, the present disclosure provides methods of using a STAT3 oligonucleotide herein in combination with a PD-L1 inhibitor to treat or ameliorate the onset or progression of a disease, disorder, or condition associated with STAT3 expression. In other aspects, the present disclosure provides methods of using a STAT3 oligonucleotide herein in combination with a PD-L1 inhibitor to achieve one or more therapeutic benefits in a subject having a disease, disorder, or condition associated with STAT3 expression. In some embodiments of the methods herein, the subject is treated by administering a therapeutically effective amount of a STAT3 oligonucleotide herein in combination with a PD-L1 inhibitor. In some embodiments of the methods herein, the subject is treated by administering a therapeutically effective amount of a STAT3 oligonucleotide herein to a subject who has received or is receiving a PD-L1 inhibitor. In some embodiments of the methods herein, the subject is treated by administering a therapeutically effective amount of a PD-L1 inhibitor to a subject who has received or is receiving a STAT3 oligonucleotide herein. In some embodiments, the subject is treated therapeutically. In some embodiments, the subject is treated prophylactically.

[0104] In some embodiments, the present disclosure provides methods for treating or ameliorating the onset or progression of a disease, disorder, or condition associated with STAT3 expression using a STAT3 oligonucleotide comprising a sense strand comprising the sequence set forth in SEQ ID NO: 875 and an antisense strand comprising the sequence set forth in SEQ ID NO: 965, in combination with a PD-L1 inhibitor. In some embodiments, the present disclosure provides methods for treating or ameliorating the onset or progression of a disease, disorder, or condition associated with STAT3 expression using a STAT3 oligonucleotide comprising a sense strand comprising the sequence set forth in SEQ ID NO: 1222 and an antisense strand comprising the sequence set forth in SEQ ID NO: 1145, in combination with a PD-L1 inhibitor. In other embodiments, the present disclosure provides methods for achieving one or more therapeutic benefits in a subject having a disease, disorder, or condition associated with STAT3 expression using a STAT3 oligonucleotide comprising a sense strand comprising the sequence set forth in SEQ ID NO: 875 and an antisense strand comprising the sequence set forth in SEQ ID NO: 965, in combination with a PD-L1 inhibitor. In other aspects, the present disclosure provides methods for using a STAT3 oligonucleotide comprising a sense strand comprising the sequence set forth in SEQ ID NO: 1222 and an antisense strand comprising the sequence set forth in SEQ ID NO: 1145, in combination with a PD-L1 inhibitor, to achieve one or more therapeutic benefits in a subject having a disease, disorder, or condition associated with STAT3 expression. In some embodiments of the methods herein, a subject is treated by administering a therapeutically effective amount of a STAT3 oligonucleotide comprising a sense strand comprising the sequence set forth in SEQ ID NO: 875 and an antisense strand comprising the sequence set forth in SEQ ID NO: 965, 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 a STAT3 oligonucleotide comprising a sense strand comprising the sequence set forth in SEQ ID NO: 1222 and an antisense strand comprising the sequence set forth in SEQ ID NO: 1145, 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 a STAT3 oligonucleotide comprising a sense strand comprising the sequence set forth in SEQ ID NO: 875 and an antisense strand comprising the sequence set forth in SEQ ID NO: 965 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 STAT3 oligonucleotide comprising a sense strand comprising the sequence set forth in SEQ ID NO: 1222 and an antisense strand comprising the sequence set forth in SEQ ID NO: 1145 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 a STAT3 oligonucleotide comprising a sense strand comprising the sequence set forth in SEQ ID NO: 875 and an antisense strand comprising 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 a PD-L1 inhibitor to a subject who has received or is receiving a STAT3 oligonucleotide comprising a sense strand comprising the sequence set forth in SEQ ID NO: 1222 and an antisense strand comprising 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.

[0105] In some embodiments of the methods herein, one or more 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 is reduced in the subject, thereby treating the subject. In some embodiments of the methods herein, a STAT3 oligonucleotide comprising a sense strand comprising the sequence set forth in SEQ ID NO: 875 and an antisense strand comprising the sequence set forth in SEQ ID NO: 965, or a pharmaceutical composition comprising a 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 is reduced in the subject, thereby treating the subject. In some embodiments of the methods herein, a STAT3 oligonucleotide comprising a sense strand comprising the sequence set forth in SEQ ID NO: 1222 and an antisense strand comprising the sequence set forth in SEQ ID NO: 1145, or a pharmaceutical composition comprising a 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 is reduced in the subject, thereby treating the subject. In some embodiments, an amount or level of STAT3 mRNA is reduced in the subject. In some embodiments, an amount or level of STAT3 and / or protein is reduced in the subject. In some embodiments of the methods herein, one or more STAT3 oligonucleotides herein, or a pharmaceutical composition comprising one or more STAT3 oligonucleotides, 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 are reduced in the subject, thereby treating the subject.In some embodiments of the methods herein, a STAT3 oligonucleotide comprising a sense strand comprising the sequence set forth in SEQ ID NO: 875 and an 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 are reduced in the subject, thereby treating the subject. In some embodiments of the methods herein, a STAT3 oligonucleotide comprising a sense strand comprising the sequence set forth in SEQ ID NO: 1222 and an 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 are reduced in the subject, thereby treating the subject. In some embodiments, an amount or level of STAT3 mRNA and PD-L1 signaling is reduced in the subject. In some embodiments, the amount or level of STAT3 and / or protein is reduced in a subject and PD-L1 signaling is reduced in a subject.

[0106] In some embodiments, a therapeutically effective amount of a STAT3 oligonucleotide and / or a PD-L1 inhibitor is administered to a subject.A therapeutically acceptable amount can be an amount that can treat a disease or disorder therapeutically.The appropriate dose for any one subject will depend on certain factors, including the subject's size, body surface area, age, the specific composition administered, the active ingredient in the composition, the time and route of administration, general health, and other drugs administered concomitantly.

[0107] In some embodiments, the subject is administered any one of the compositions herein enterally (e.g., orally, by gastric feeding tube, by duodenal feeding tube, via gastrostomy, or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intraarterial injection or infusion, intraosseous injection, intramuscular injection, intracerebral injection, intraventricular injection, intrathecal injection), topically (e.g., transdermally, inhalation, via eye drops, or via mucosa), or by direct injection into target organ (e.g., the subject's liver). Typically, the oligonucleotide herein is administered intravenously or subcutaneously.

[0108] As a non-limiting example of a series, the oligonucleotide herein will typically be administered quarterly (every three months), bimonthly (every two months), monthly, or weekly.For example, oligonucleotide can be administered every week, or every two weeks or three weeks.Alternatively, oligonucleotide can be administered every day.In some embodiments, the subject is administered one or more loading doses of oligonucleotide, followed by one or more maintenance doses of oligonucleotide.

[0109] In some embodiments, the PD-L1 inhibitors (e.g., anti-PD-L1 antibodies) herein are administered quarterly (every three months), bimonthly (every two months), monthly, or weekly. For example, the inhibitors are administered weekly, or at two-week or three-week intervals. Alternatively, the inhibitors are administered daily.

[0110] In some embodiments, the oligonucleotide herein is administered in combination with a PD-L1 inhibitor. In some embodiments, the oligonucleotide and the inhibitor are administered in parallel, sequentially (in any order), or intermittently in combination. For example, the oligonucleotide and the inhibitor can be co-administered in parallel. Alternatively, the oligonucleotide can be administered, followed by the inhibitor after any period (for example, 1 hour, 1 day, 1 week, or 1 month), or vice versa.

[0111] In some embodiments, the subject to be treated is a human or non-human primate or other mammalian subject. Other exemplary subjects include domesticated animals such as dogs and cats, farm animals such as horses, cows, pigs, sheep, goats, and chickens, and animals such as mice, rats, guinea pigs, and hamsters.

[0112] cancer In some embodiments, STAT3 oligonucleotides and PD-L1 inhibitor targets are used to treat cancer or tumors. In some embodiments, the tumor is a primary tumor. In some embodiments, the tumor is a metastatic tumor. In some embodiments, the tumor is a refractory tumor. In some embodiments, the tumor is a stage I, II, III, or IV tumor. In some embodiments, the tumor is a solid tumor. A solid tumor refers to a condition in which the cancer forms a mass.

[0113] In some embodiments, the cancer is thyroid cancer, papillary thyroid cancer, head and neck cancer, liver cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, carcinoma, blastoma, medulloblastoma, retinoblastoma, sarcoma, liposarcoma, synovial cell sarcoma, neuroendocrine tumor, carcinoid tumor, gastrinoma, islet cell carcinoma, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, lymphoid malignancy, squamous cell carcinoma, epithelial squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastric cancer, or stomach cancer. In some embodiments, the cancer is refractory to anti-PD1, anti-PDL1, and / or anti-CTLA4 therapy. In some embodiments, the cancer is pancreatic cancer or lung cancer. In some embodiments, the cancer comprises a tumor with an immunosuppressive tumor microenvironment. In some embodiments, the cancer is resistant to immune checkpoint therapy. In some embodiments, the cancer is partially resistant to immune checkpoint therapy. In some embodiments, the cancer is sensitive to immune checkpoint therapy.

[0114] In some embodiments, the STAT3 oligonucleotide and PD-L1 inhibitor reduce tumor volume. Tumor volume is measured using methods known to those skilled in the art. For example, extracted tumors are measured manually using calipers. Other methods include imaging methods such as ultrasound and MRI. In some embodiments, the oligonucleotide conjugate reduces tumor volume by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to an untreated tumor.

[0115] Treatment response In some embodiments, the present disclosure provides a method for monitoring a therapeutic response in a subject. In some embodiments, the treatment comprises any of the STAT3-targeting oligonucleotides described herein. In some embodiments, the treatment comprises any of the STAT3-targeting oligonucleotides described herein in combination with a PD-L1 inhibitor.

[0116] In some embodiments, the present disclosure provides a method for monitoring a therapeutic response in a subject having a tumor, the method comprising detecting an amount of myeloid-derived suppressor cells (MDSCs) in a biological sample from the subject that has been or is being treated with an oligonucleotide targeting STAT3 to treat the tumor in the subject, wherein a reduced amount of MDSCs in the biological sample indicates that the subject is responding to treatment with the oligonucleotide.

[0117] In some embodiments, the present disclosure provides a method for monitoring a therapeutic response in a subject having a tumor, the method comprising: (i) obtaining a biological sample from a subject who has been or is currently undergoing treatment with an oligonucleotide targeting STAT3; (ii) detecting an amount of MDSCs in a biological sample; and (iii) comparing the amount of MDSCs in the biological sample with a predetermined amount of MDSCs, wherein a reduced amount of MDSCs in the biological sample indicates that the subject is responding to treatment with the oligonucleotide.

[0118] In some embodiments, the present disclosure provides a method for determining responsiveness to a treatment in a subject with cancer. In some embodiments, the treatment comprises any of the STAT3-targeting oligonucleotides described herein. In some embodiments, the treatment comprises any of the STAT3-targeting oligonucleotides described herein in combination with a PD-L1 inhibitor.

[0119] In some embodiments, the present disclosure provides methods of determining responsiveness in a subject with cancer who has undergone or is undergoing a treatment, the method comprising detecting the presence of myeloid-derived suppressor cells (MDSCs) or a marker of MDSC activity in a biological sample from the subject. In some embodiments, methods of determining responsiveness in a subject with cancer who has undergone or is undergoing a treatment, the method comprising detecting the presence of myeloid-derived suppressor cells (MDSCs) or a marker of MDSC activity in a biological sample from the subject, wherein the treatment is administration of an oligonucleotide that targets STAT3, and wherein a reduction in MDSCs or a reduction in the marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment.

[0120] In some embodiments, the present disclosure provides a method for determining responsiveness in a subject having cancer who has undergone or is undergoing treatment, the method comprising: (i) obtaining a biological sample from a subject; (ii) detecting the presence of MDSCs or a marker of MDSC activity in the biological sample; The treatment is administration of an oligonucleotide that targets STAT3, and a reduction in MDSCs or a reduction in a marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment.

[0121] In some embodiments, detecting comprises determining an amount of MDSC or an amount of a marker of MDSC activity. In some embodiments, the reduction in MDSC or marker of MDSC activity is relative to an amount or level of MDSC or marker of MDSC activity before treatment of the subject. In some embodiments, the reduction in MDSC or marker of MDSC activity is relative to an amount or level of MDSC or marker of MDSC activity before treatment of the subject. In some embodiments, the reduction in MDSC or marker of MDSC activity is relative to an amount of the level of MDSC or marker of MDSC activity in a population of patients who responded to treatment.

[0122] In some embodiments, the predetermined amount of MDSCs is a certain amount of MDSCs detected in a subject before treatment with an oligonucleotide. In some embodiments, the predetermined amount of MDSCs is the average amount of MDSCs based on a patient population that has not been treated with an oligonucleotide. In some embodiments, the patient population is a healthy population of patients. In some embodiments, the patient population is a population that does not have cancer. In some embodiments, the patient population is a population that has been treated with a placebo oligonucleotide. In some embodiments, the patient population is a population of patients that has been treated with an oligonucleotide and has experienced a reduction or inhibition of tumor growth and / or tumor size.

[0123] In some embodiments, the MDSCs are granulocytic MDSCs (G-MDSCs). In some embodiments, the MDSCs are monocytic MDSCs (M-MDSCs). In some embodiments, the MDSCs express Arg1. In some embodiments, the MDSCs express IDO. In some embodiments, the MDSCs are Arg1+ M-MDSCs. In some embodiments, the MDSCs are Arg1+G-MDSCs. In some embodiments, the MDSCs are IDO+ M-MDSCs. In some embodiments, the MDSCs are IDO+G-MDSCs. In some embodiments, the MDSCs are G-MDSCs, M-MDSCs, Arg1+M-MDSCs, Arg1+G-MDSCs, IDO+M-MDSCs, IDO+G-MDSCs, or combinations thereof.

[0124] In some embodiments, the amount of MDSCs is determined using methods known to those skilled in the art. In some embodiments, the amount of MDSCs is determined using flow cytometry.

[0125] In some embodiments, MDSCs are 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.

[0126] In some embodiments, responding to treatment comprises a reduction or inhibition of tumor growth and / or tumor size. In some embodiments, responding to treatment comprises a reduction or inhibition of tumor growth. In some embodiments, responding to treatment comprises a reduction or inhibition of tumor size.

[0127] In some embodiments, a method of determining responsiveness in a subject having cancer who has undergone or is undergoing treatment, the method comprising detecting the presence of myeloid-derived suppressor cells (MDSCs) or a marker of MDSC activity in a biological sample from the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, and wherein a reduction in MDSCs or a reduction in the marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment, and wherein the oligonucleotide targeting STAT3 comprises a sense strand comprising a sequence selected from SEQ ID NOs: 857-946 and an antisense strand comprising a sequence selected from SEQ ID NOs: 947-1036.

[0128] In some embodiments, a method of determining responsiveness in a subject having cancer who has undergone or is undergoing treatment, the method comprising detecting the presence of myeloid-derived suppressor cells (MDSCs) or a marker of MDSC activity in a biological sample from the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, and wherein a reduction in MDSCs or a reduction in the marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment, and wherein the oligonucleotide targeting STAT3 comprises a sense strand comprising a sequence selected from SEQ ID NOs: 1037-1126 and an antisense strand comprising a sequence selected from SEQ ID NOs: 1127-1216.

[0129] In some embodiments, a method of determining responsiveness in a subject with cancer who has undergone or is undergoing treatment, the method comprising detecting the presence of myeloid-derived suppressor cells (MDSCs) or a marker of MDSC activity in a biological sample from the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, and wherein a reduction in MDSCs or a reduction in the marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment, and wherein the oligonucleotide targeting STAT3 comprises a sense strand comprising a sequence selected from SEQ ID NOs: 9, 37, 65, and 69, and an antisense strand comprising a sequence selected from SEQ ID NOs: 10, 38, 66, and 70.

[0130] In some embodiments, there is provided a method of determining responsiveness in a subject having cancer who has undergone or is undergoing treatment, the method comprising detecting the presence of myeloid-derived suppressor cells (MDSCs) or a marker of MDSC activity in a biological sample from the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, and wherein a reduction in MDSCs or a reduction in the marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment, and wherein the oligonucleotide targeting STAT3 comprises a sense strand comprising a sequence selected from SEQ ID NOs: 11, 39, 67, and 71, and an antisense strand comprising a sequence selected from SEQ ID NOs: 12, 40, 68, and 72.

[0131] In some embodiments, there is provided a method of determining responsiveness in a subject having cancer who has undergone or is undergoing treatment, the method comprising detecting the presence of myeloid-derived suppressor cells (MDSCs) or a marker of MDSC activity in a biological sample from the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, and wherein a reduction in MDSCs or a reduction in the marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment, and wherein the oligonucleotide targeting STAT3 comprises a sense strand comprising a sequence selected from SEQ ID NOs: 9, 37, 65, and 69, and an antisense strand comprising a sequence selected from SEQ ID NOs: 10, 38, 66, 70.

[0132] In some embodiments, a method of determining responsiveness in a subject having cancer who has undergone or is undergoing treatment, the method comprising detecting the presence of myeloid-derived suppressor cells (MDSCs) or a marker of MDSC activity in a biological sample from the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, and wherein a reduction in MDSCs or a reduction in the marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment, and wherein the oligonucleotide targeting STAT3 comprises a sense strand comprising SEQ ID NO: 875 and an antisense strand comprising SEQ ID NO: 965.

[0133] In some embodiments, a method of determining responsiveness in a subject having cancer who has undergone or is undergoing treatment, the method comprising detecting the presence of myeloid-derived suppressor cells (MDSCs) or a marker of MDSC activity in a biological sample from the subject, wherein the treatment is administration of an oligonucleotide targeting STAT3, and wherein a reduction in MDSCs or a reduction in the marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment, and wherein the oligonucleotide targeting STAT3 comprises a sense strand comprising SEQ ID NO: 1145 and an antisense strand comprising SEQ ID NO: 1222.

[0134] In some embodiments, the present disclosure provides a method for determining responsiveness in a subject having cancer who has undergone or is undergoing treatment, the method comprising: (i) obtaining a biological sample from a subject; (ii) detecting the presence of MDSCs or a marker of MDSC activity in the biological sample; The treatment involves administration of an oligonucleotide targeting STAT3, the oligonucleotide comprising a sense strand comprising a sequence selected from SEQ ID NOs: 857-946 and an antisense strand comprising a sequence selected from SEQ ID NOs: 947-1036, and a reduction in MDSCs or a reduction in a marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment.

[0135] In some embodiments, the present disclosure provides a method for determining responsiveness in a subject having cancer who has undergone or is undergoing treatment, the method comprising: (i) obtaining a biological sample from a subject; (ii) detecting the presence of MDSCs or a marker of MDSC activity in the biological sample; The treatment involves administration of an oligonucleotide targeting STAT3, the oligonucleotide comprising a sense strand comprising a sequence selected from SEQ ID NOs: 1037-1126 and an antisense strand comprising a sequence selected from SEQ ID NOs: 1127-1216, and a reduction in MDSCs or a reduction in a marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment.

[0136] In some embodiments, the present disclosure provides a method for determining responsiveness in a subject having cancer who has undergone or is undergoing treatment, the method comprising: (i) obtaining a biological sample from a subject; (ii) detecting the presence of MDSCs or a marker of MDSC activity in the biological sample; The treatment is administration of an oligonucleotide targeting STAT3, the oligonucleotide comprising a sense strand comprising a sequence selected from SEQ ID NOs: 11, 39, 67, and 71, and an antisense strand comprising a sequence selected from SEQ ID NOs: 12, 40, 68, and 72, and a reduction in MDSCs or a reduction in a marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment.

[0137] In some embodiments, the present disclosure provides a method for determining responsiveness in a subject having cancer who has undergone or is undergoing treatment, the method comprising: (i) obtaining a biological sample from a subject; (ii) detecting the presence of MDSCs or a marker of MDSC activity in the biological sample; The treatment is administration of an oligonucleotide targeting STAT3, the oligonucleotide comprising a sense strand comprising a sequence selected from SEQ ID NOs: 9, 37, 65, and 69, and an antisense strand comprising a sequence selected from SEQ ID NOs: 10, 38, 66, and 70, and a reduction in MDSCs or a reduction in a marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment.

[0138] In some embodiments, the present disclosure provides a method for determining responsiveness in a subject having cancer who has undergone or is undergoing treatment, the method comprising: (i) obtaining a biological sample from a subject; (ii) detecting the presence of MDSCs or a marker of MDSC activity in the biological sample; The treatment is administration of an oligonucleotide targeting STAT3 comprising a sense strand comprising SEQ ID NO: 875 and an antisense strand comprising SEQ ID NO: 965, and a reduction in MDSCs or a reduction in a marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment.

[0139] In some embodiments, the present disclosure provides a method for determining responsiveness in a subject having cancer who has undergone or is undergoing treatment, the method comprising: (i) obtaining a biological sample from a subject; (ii) detecting the presence of MDSCs or a marker of MDSC activity in the biological sample; The treatment is administration of an oligonucleotide targeting STAT3 comprising a sense strand comprising SEQ ID NO: 1145 and an antisense strand comprising SEQ ID NO: 1222, and a reduction in MDSCs or a reduction in a marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment.

[0140] Oligonucleotide inhibitors of STAT3 In some aspects, the present disclosure provides, inter alia, oligonucleotides that reduce or inhibit STAT3 expression. In some embodiments, the oligonucleotides herein that inhibit STAT3 expression target STAT3 mRNA. The sequence of human STAT3 mRNA (NM_001369512.1) is listed as SEQ ID NO: 85 or NM_139276.3 (SEQ ID NO: 1217). STAT3 is a known target of conventional cancer therapy.

[0141] The tolerogenic activity of MDSCs is regulated by the oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) (Su et al., Int J. Mol Sci (2018) 19(6):1803). STAT3 is also known to be highly expressed across a wide range of cancer types and in vitro and in vivo preclinical models (Huynh et al., NAT. REV. CANCER (2019) 19:82-96). Inhibition of STAT3 leads to selective apoptosis of tumor cells and tumor growth inhibition through modulation of downstream target genes (Wang et al., International Journal of Biological Sciences, 15(3):668-79 (2019)). STAT3 is of particular interest in immuno-oncology due to its well-documented contribution to the immunosuppressive tumor microenvironment. STAT3 inhibits IFN by upregulating inhibitory receptors expressed by T cells and through the expression of its ligands (PD-1 / PD-L1).

number

[0142] In some embodiments, the reduction of STAT3 expression can be determined by a suitable assay or technique for assessing one or more properties or characteristics of a cell or cell population associated with STAT3 expression (e.g., using a STAT3 expression biomarker), or by an assay or technique for assessing a molecule that directly indicates STAT3 expression (e.g., STAT3 mRNA or STAT3 protein). In some embodiments, the extent to which the oligonucleotides herein reduce STAT3 expression is assessed by comparing STAT3 expression in a cell or cell population contacted with the oligonucleotide to an appropriate control (e.g., an appropriate 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 into protein after delivery of an RNAi molecule can be a predetermined level or value, such that the control level does not need to be measured every time. The predetermined level or value can take various forms. In some embodiments, the predetermined level or value can be a single cutoff value, such as a median or mean value.

[0143] In some embodiments, administration of the oligonucleotides herein results in a reduction in STAT3 expression in a cell or cell population. In some embodiments, the reduction in STAT3 or STAT3 expression is about 1% or less, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less compared to an appropriate control level of mRNA. An appropriate control level may be the level of mRNA expression and / or protein translation in a cell or cell population that has not been contacted with the oligonucleotides herein. In some embodiments, the effect of delivering an oligonucleotide to a cell according to the methods herein is evaluated after a finite period of time. For example, mRNA levels may be analyzed in cells at least about 8 hours, about 12 hours, about 18 hours, about 24 hours, or at least about 1, 2, 3, 4, 5, 6, 7, or up to 14 days after introduction of the oligonucleotide into the tumor.

[0144] In some embodiments, the oligonucleotide is delivered in the form of a transgene engineered to express the oligonucleotide or a strand containing the oligonucleotide (e.g., its sense and antisense strands) in cells. In some embodiments, the oligonucleotide is delivered using a transgene engineered to express any of the oligonucleotides disclosed herein. The transgene can be delivered using a viral vector (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus) or a non-viral vector (e.g., plasmid or synthetic mRNA). In some embodiments, the transgene can be directly injected into the subject.

[0145] STAT3 target sequence In some embodiments, the oligonucleotide is targeted to a target sequence containing STAT3 mRNA. In some embodiments, the oligonucleotide, or a portion, fragment, or strand thereof (e.g., the antisense strand or guide strand of dsRNA) binds to or anneals to a target sequence containing STAT3 mRNA, thereby inhibiting STAT3 expression. In some embodiments, the oligonucleotide is targeted to a STAT3 target sequence for the purpose of inhibiting STAT3 expression in vivo. In some embodiments, the amount or degree of inhibition of STAT3 expression by an oligonucleotide targeted to a STAT3 target sequence correlates with the efficacy of the oligonucleotide. In some embodiments, the amount or degree of inhibition of STAT3 expression by an oligonucleotide targeted to a STAT3 target sequence correlates with the amount or degree of therapeutic effect in a subject or patient with a disease, disorder, or condition associated with STAT3 expression who is treated with the oligonucleotide.

[0146] Through examination of the nucleotide sequences of mRNAs encoding STAT3, including mRNAs from several different species (e.g., human, cynomolgus monkey, mouse, and rat; see, e.g., Example 6), and as a result of in vitro and in vivo testing (see, e.g., Examples 7 and 8), it has been discovered that certain nucleotide sequences of STAT3 mRNA are more susceptible to oligonucleotide-based inhibition than others and are therefore useful as target sequences for the oligonucleotides herein. In some embodiments, the sense strand of an oligonucleotide (e.g., dsRNA) described herein comprises a STAT3 target sequence. In some embodiments, a portion or region of the sense strand of a dsRNA described herein comprises a STAT3 target sequence. In some embodiments, the STAT3 mRNA target sequence comprises or consists of the sequence of SEQ ID NO:85. In some embodiments, the STAT3 mRNA target sequence comprises or consists of the sequence of SEQ ID NO:1217. In some embodiments, the STAT3 mRNA target sequence comprises or consists of the sequence set forth in SEQ ID NO:140.

[0147] STAT3 targeting sequence In some embodiments, the oligonucleotides herein have a region of complementarity to STAT3 mRNA (e.g., within the target sequence of STAT3 mRNA) for the purpose of targeting the mRNA in a cell and reducing or inhibiting its expression. In some embodiments, the oligonucleotides herein comprise a STAT3 target sequence (e.g., the antisense strand or guide strand of a dsRNA) with a region of complementarity that binds or anneals to the STAT3 target sequence by complementary (Watson-Crick) base pairing. The targeting sequence or region of complementarity generally has a suitable length and base content to allow binding or annealing of the oligonucleotide (or a strand thereof) to it for the purpose of inhibiting expression of STAT3 mRNA. In some embodiments, the targeting sequence or region of complementarity is at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is about 12 to about 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30) nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 18 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 19 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 20 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 21 nucleotides in length.In some embodiments, the targeting sequence or region of complementarity is 22 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 23 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 24 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or region of complementarity complementary to the sequence of SEQ ID NO: 140, and the targeting sequence or region of complementarity is 18 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or region of complementarity complementary to the sequence of SEQ ID NO: 140, and the targeting sequence or region of complementarity is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or region of complementarity complementary to the sequence of SEQ ID NO: 524, and the targeting sequence or region of complementarity is 20 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or region of complementarity complementary to the sequence of SEQ ID NO: 524, and the targeting sequence or region of complementarity is 21 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity complementary to the sequence of SEQ ID NO: 524, and the targeting sequence or region of complementarity is 22 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity complementary to the sequence of SEQ ID NO: 524, and the targeting sequence or region of complementarity is 23 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity complementary to the sequence of SEQ ID NO: 524, and the targeting sequence or region of complementarity is 24 nucleotides in length.

[0148] In some embodiments, the oligonucleotides herein comprise a targeting sequence or region of complementarity that is fully complementary to a STAT3 target sequence (e.g., the antisense strand or guide strand of a double-stranded oligonucleotide). In some embodiments, the targeting sequence or region of complementarity is partially complementary to a STAT3 target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is fully complementary to a STAT3 sequence or STAT3. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is partially complementary to a STAT3 sequence or STAT3.

[0149] In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is fully complementary to the sequence of SEQ ID NO: 140. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is partially complementary to the sequence of SEQ ID NO: 140.

[0150] In some embodiments, the oligonucleotides herein comprise a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides comprising STAT3 mRNA, wherein the contiguous sequence of nucleotides is about 12 to about 30 nucleotides in length (e.g., 12-30, 12-28, 12-26, 12-24, 12-20, 12-18, 12-16, 14-22, 16-20, 18-20, or 18-19 nucleotides in length). In some embodiments, the oligonucleotides comprise a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides comprising STAT3 mRNA, wherein the contiguous sequence of nucleotides is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the oligonucleotides comprise a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides comprising STAT3 mRNA, wherein the contiguous sequence of nucleotides is 19 nucleotides in length.

[0151] In some embodiments, the oligonucleotides (e.g., RNAi oligonucleotides) herein comprise a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 140, where optionally, the contiguous sequence of nucleotides is 19 nucleotides in length. In some embodiments, the oligonucleotides comprise a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 524, where the contiguous sequence of nucleotides is 20 nucleotides in length.

[0152] In some embodiments, the targeting sequence or region of complementarity of an oligonucleotide that is complementary to consecutive nucleotides of STAT3 or a STAT3 target sequence spans the entire length of the antisense strand. In some embodiments, the region of complementarity of an oligonucleotide that is complementary to consecutive nucleotides 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 region of complementarity (e.g., on the antisense strand of a dsRNA) that is at least partially (e.g., completely) complementary to a STAT3 target sequence or a consecutive stretch of nucleotides spanning nucleotides 1 to 20 of STAT3.

[0153] In some embodiments, the targeting sequence or region of complementarity of an oligonucleotide (e.g., an RNAi oligonucleotide) herein is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 140 and spans the entire length of the antisense strand. In some embodiments, the targeting sequence or region of complementarity of an oligonucleotide is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 140 and spans a portion of the entire length of the antisense strand. In some embodiments, an oligonucleotide (e.g., an RNAi oligonucleotide) herein comprises a region of complementarity (e.g., on the antisense strand of a dsRNA) that is at least partially (e.g., fully) complementary to a contiguous stretch of nucleotides spanning nucleotides 1-19 or 1-20 of the sequence set forth in SEQ ID NO: 524.

[0154] In some embodiments, the oligonucleotides herein comprise a targeting sequence or region of complementarity that has one or more bp mismatches with the corresponding STAT3 target sequence. In some embodiments, the targeting sequence or region of complementarity may have up to about one, two, three, four, or five mismatches with the corresponding STAT3 target sequence, provided that the ability of the targeting sequence or region of complementarity to bind to or anneal with STAT3 mRNA and / or the ability of the oligonucleotide to inhibit STAT3 expression under appropriate hybridization conditions is maintained. Alternatively, the targeting sequence or region of complementarity may have no more than one, no more than two, no more than three, no more than four, or no more than five mismatches with the corresponding STAT3 target sequence, provided that the ability of the targeting sequence or region of complementarity to bind to or anneal with STAT3 mRNA and / or the ability of the oligonucleotide to inhibit STAT3 expression under appropriate hybridization conditions is maintained. In some embodiments, the oligonucleotides comprise a targeting sequence or region of complementarity that has one mismatch with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity with two mismatches to the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity with three mismatches to the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity with four mismatches to the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity with five mismatches to the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity with two or more mismatches (e.g., 2, 3, 4, 5, or more mismatches) to the corresponding target sequence, wherein at least two (e.g., all) of the mismatches are positioned contiguously (e.g., 2, 3, 4, 5, or more mismatches side by side) or the mismatches are scattered throughout the targeting sequence or region of complementarity.In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 140, and the targeting sequence or region of complementarity may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding STAT3 target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 140, and the targeting sequence or region of complementarity may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding STAT3 target sequence.

[0155] Oligonucleotide Types A variety of oligonucleotide types and / or structures are useful for targeting target sequences in the methods herein, including, but not limited to, RNAi oligonucleotides, antisense oligonucleotides, miRNA, etc. Any of the oligonucleotide types described herein or elsewhere are contemplated for use as a framework for incorporating the targeting sequences herein.

[0156] In some embodiments, the oligonucleotides herein inhibit the expression of target sequences by participating in the RNA interference (RNAi) pathway upstream or downstream of Dicer intervention. For example, RNAi oligonucleotides have been developed with a size of approximately 19-25 nucleotides, each with at least one 3' overhang of 1-5 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 research has produced extended dsRNAs in which at least one end of at least one strand extends beyond the double-stranded targeting region, and one of the strands contains a thermodynamically stabilizing tetraloop structure (see, e.g., U.S. Patent Nos. 8,513,207 and 8,927,705, and International Patent Application Publication No. 2010 / 033225). Such structures may contain ss extensions as well as ds extensions (on one or both sides of the molecule).

[0157] In some embodiments, the oligonucleotides described herein participate in the RNAi pathway downstream of Dicer intervention (e.g., Dicer cleavage). In some embodiments, the oligonucleotides described herein are Dicer substrates. In some embodiments, endogenous Dicer processing produces a double-stranded nucleic acid 19-23 nucleotides in length that can reduce target mRNA expression. In some embodiments, the oligonucleotide has an overhang (e.g., 1, 2, or 3 nucleotides in length) at the 3' end of the sense strand. In some embodiments, the oligonucleotide (e.g., siRNA) comprises a 21-nucleotide guide strand that is antisense to the target RNA and a complementary passenger strand, and both strands anneal to form a 19-bp duplex and a 2-nucleotide overhang at either or both 3' ends. Longer oligonucleotide designs are also available, including the oligonucleotide with a guide strand of 23 nucleotides and a passenger strand of 21 nucleotides, with a blunt end on the right side of the molecule (the 3' end of the passenger strand / the 5' end of the guide strand), and a 3'-guide strand overhang of 2 nucleotides on the left side of the molecule (the 5' end of the passenger strand / the 3' end of the guide strand).In such molecules, there is a 21bp double-stranded region.See, for example, U.S. Patent No. 9,012,138, U.S. Patent No. 9,012,621 and U.S. Patent No. 9,193,753.

[0158] In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, both ranging in length from about 17 to 26 (e.g., 17 to 26, 20 to 25, or 21 to 23) nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, both ranging in length from about 17 to 36 (e.g., 17 to 36, 20 to 25, or 21 to 23) nucleotides. In some embodiments, the oligonucleotides herein comprise an antisense strand 19 to 30 nucleotides in length and a sense strand 19 to 50 nucleotides in length, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region with an overhand of 1 to 4 nucleotides at the 3' end of the antisense strand. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, both ranging in length from about 19 to 22 nucleotides. In some embodiments, the sense strand and the antisense strand are of equal length. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, such that a 3' overhang is present on either the sense strand or the antisense strand, or on both the sense strand and the antisense strand. In some embodiments, for oligonucleotides having a sense strand and an antisense strand, both of which are in the range of about 21-23 nucleotides in length, the 3' overhang on the sense strand, the antisense strand, or both the sense strand and the antisense strand is 1 or 2 nucleotides in length. In some embodiments, the oligonucleotide has a 22-nucleotide guide strand and a 20-nucleotide passenger strand, with a blunt end on the right side of the molecule (3' end of the passenger strand / 5' end of the guide strand) and a 2-nucleotide 3'-guide strand overhang on the left side of the molecule (5' end of the passenger strand / 3' end of the guide strand). Such molecules have a 20-bp double-stranded region.

[0159] Other oligonucleotide designs for use in conjunction with the compositions and methods herein include 16-mer siRNAs (see, e.g., NUCLEIC ACIDS IN CHEMISTRY AND BIOLOGY. Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., with stems of 19 bp or less; see, e.g., Moore et al., (2010) Methods Mol. Biol. 629:141-58), blunt siRNAs (e.g., 19 bp in length; see, e.g., Kraynack and Baker (2006) RNA 12:163-76), asymmetric siRNAs (aiRNAs; see, e.g., Sun et al., (2008) Nat. Biotechnol. 26:1379-82), asymmetric shorter double-stranded siRNAs (see, e.g., Chang et al., (2008) Nat. Biotechnol. 26:1379-82), and other siRNAs with shorter duplexes (see, e.g., Chang et al., (2008) Nat. Biotechnol. 26:1379-82). al., (2009) Mol. Ther. 17:725-32), forked siRNA (see, e.g., Hohjoh (2004) FEBS Lett. 557:193-98), ss siRNA (Elsner (2012) Nat. Biotechnol. 30:1063), dumbbell-shaped circular siRNA (see, e.g., Abe et al., (2007) J. Am. Chem. Soc. 129:15108-09), and small internal segmented interfering RNA (siRNA; see, e.g., Bramsen et al., (2007) Nucleic Acids Res. 35:5886-97). Further non-limiting examples of oligonucleotide structures that may be used in some embodiments to reduce or inhibit the expression of STAT3 are microRNAs (miRNAs), short hairpin RNAs (shRNAs), and short siRNAs (see, e.g., Hamilton et al., (2002) EMBO J. 21:4671-79; see also U.S. Patent Application Publication No. 2009 / 0099115).

[0160] Furthermore, in some embodiments, the oligonucleotide for reducing or inhibiting the expression of a target sequence herein is ss. Such structures may include, but are not limited to, ss RNAi molecules. Recent efforts have demonstrated the activity of ss RNAi molecules (see, for example, Matsui et al., (2016) Mol. Ther. 24:946-55). However, in some embodiments, the oligonucleotide herein is an antisense oligonucleotide (ASO). An antisense oligonucleotide is an ss oligonucleotide having a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of a specific nucleic acid targeting segment and is suitably modified to induce RNase H-mediated cleavage of its target RNA in cells (e.g., as a gapmer) or inhibit the translation of target mRNA in cells (e.g., as a mixmer). ASOs for use herein can be modified in any suitable manner known in the art, including, for example, those described in U.S. Patent No. 9,567,587 (e.g., including modifications of length, sugar moieties of nucleobases (pyrimidines, purines), and heterocyclic moieties of nucleobases). Furthermore, ASOs have been used for decades to reduce the expression of specific target genes (see, for example, Bennett et al., (2017) Annu. Rev. Pharmacol. 57:81-105).

[0161] In some embodiments, the antisense oligonucleotide shares a region of complementarity with the target mRNA. In some embodiments, the antisense oligonucleotide is 15-50 nucleotides in length. In some embodiments, the antisense oligonucleotide is 15-25 nucleotides in length. In some embodiments, the antisense oligonucleotide is 22 nucleotides in length. In some embodiments, the antisense oligonucleotide is at least 15 contiguous nucleotides in length. In some embodiments, the antisense oligonucleotide is at least 19 contiguous nucleotides in length. In some embodiments, the antisense oligonucleotide is at least 20 contiguous nucleotides in length. In some embodiments, the antisense oligonucleotide differs from the target sequence by 1, 2, or 3 nucleotides.

[0162] double-stranded oligonucleotides In some embodiments, the present disclosure provides a double-stranded dsRNA for targeting a target sequence and inhibiting its expression (e.g., via the RNAi pathway), comprising a sense strand (also referred to herein as a passenger strand) and an antisense strand (also referred to herein as a guide strand). In some embodiments, the sense strand and the antisense strand are separate strands and are not covalently linked. In some embodiments, the sense strand and the antisense strand are covalently linked. In some embodiments, the sense strand and the antisense strand form a double-stranded region, and the sense strand and the antisense strand, or portions thereof, are bound to each other in a complementary manner (e.g., by Watson-Crick base pairing).

[0163] In some embodiments, the sense strand has a first region (R1) and a second region (R2), where R2 includes a first subregion (S1), a loop (L), such as a tetraloop (tetraL) or triloop (triL), and a second subregion (S2), where L, tetraL, or triL is located between S1 and S2, and S1 and S2 form a second duplex (D2). D2 can have a variety of lengths. In some embodiments, D2 is about 1-6 bp in length. In some embodiments, D2 is 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, or 4-5 bp in length. In some embodiments, D2 is 1, 2, 3, 4, 5, or 6 bp in length. In some embodiments, D2 is 6 bp in length.

[0164] In some embodiments, R1 of the sense strand and the antisense strand form a first duplex (D1). In some embodiments, D1 is at least about 15 nucleotides in length (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21). In some embodiments, D1 is in the range of about 12-30 nucleotides in length (e.g., 12-30, 12-27, 15-22, 18-22, 18-25, 18-27, 18-30, or 21-30 nucleotides in length). In some embodiments, D1 is at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 20, at least 25, or at least 30 nucleotides in length). In some embodiments, D1 is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, D1 is 20 nucleotides in length. In some embodiments, D1, including 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, including the sense strand and the antisense strand, spans the entire length of either the sense strand or the antisense strand or both. In certain embodiments, D1, including the sense strand and the antisense strand, spans the entire length of both the sense strand and the antisense strand.

[0165] Of course, in some embodiments, when describing the structure of an oligonucleotide or other nucleic acid, reference can be made to the sequence listed in the sequence listing.In such embodiments, the actual oligonucleotide or other nucleic acid can have one or more alternative nucleotides (such as 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 bonds and / or one or more other modifications compared to the designated sequence, while retaining essentially the same or similar complementary properties as the designated sequence.

[0166] In some embodiments, the double-stranded RNA (dsRNA) herein comprises a sense strand of 25 nucleotides and an antisense strand of 27 nucleotides, and when acted by Dicer enzyme, the antisense strand is incorporated into mature RISC.In some embodiments, the sense strand of dsRNA is longer than 27 nucleotides (for example, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides).In some embodiments, the sense strand of dsRNA is longer than 27 nucleotides (for example, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides). In some embodiments, the sense strand of the dsRNA is longer than 25 nucleotides (eg, 26, 27, 28, 29, or 30 nucleotides).

[0167] In some embodiments, the oligonucleotides herein have one 5' end that is thermodynamically less stable than the other 5' end. In some embodiments, asymmetric oligonucleotides are provided that include a blunt end at the 3' end of the sense strand and a 3' overhang at the 3' end of the antisense strand. In some embodiments, the 3' overhang on the antisense strand is about 1 to 8 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length). Typically, RNAi oligonucleotides have a two-nucleotide overhang on the 3' end of the antisense (guide) strand. However, other overhangs are possible. In some embodiments, the overhang is 1 to 6 nucleotides, 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 comprising 1 to 6 nucleotides, 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.

[0168] In some embodiments, the two terminal nucleotides on the 3' end of the antisense strand are modified. In some embodiments, the two terminal nucleotides on the 3' end of the antisense strand are complementary to the target mRNA. In some embodiments, the two terminal nucleotides on the 3' end of the antisense strand are not complementary to the target mRNA. In some embodiments, the two terminal nucleotides at the 3' end of the antisense strand comprise unpaired GG. In some embodiments, the two (2) terminal nucleotides on the 3' end of the antisense strand of the oligonucleotide herein are not complementary to the target mRNA. In some embodiments, the two terminal nucleotides at each 3' end of the oligonucleotide in the nicked tetraloop structure are GG. In some embodiments, one or both of the two (2) terminal GG nucleotides on each 3' end of the oligonucleotide herein are not complementary to the target mRNA. In some embodiments, one or both of the two (2) terminal GG nucleotides on each 3' end of the oligonucleotide are not complementary to the target.

[0169] 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. When there are two or more mismatches between the sense strand and the antisense strand, they can be positioned consecutively (e.g., two, three, or more in a row) 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 into the 3'-end of the sense strand. In some embodiments, base mismatches or destabilization of the segment at the 3'-end of the sense strand of the oligonucleotide improve the efficacy of synthetic duplexes in RNAi, possibly by facilitating Dicer processing.

[0170] a. antisense strand In some embodiments, the dsRNA comprises an antisense strand of up to about 40 nucleotides in length (e.g., up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the oligonucleotides (e.g., RNAi oligonucleotides) herein comprise an antisense strand of up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the oligonucleotides may have an antisense strand of at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27, at least 30, at least 35, or at least 38 nucleotides in length). In some embodiments, oligonucleotides can have antisense strands ranging in length from about 12 to about 40 nucleotides (e.g., 12-40, 12-36, 12-32, 12-28, 15-40, 15-36, 15-32, 15-28, 17-22, 17-25, 19-27, 19-30, 20-40, 22-40, 25-40, or 32-40). In some embodiments, oligonucleotides include antisense strands 15-30 nucleotides in length. In some embodiments, the oligonucleotide may have an antisense strand that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

[0171] In some embodiments, the antisense strand of oligonucleotide can be called " guide strand ".For example, if antisense strand can participate in RNA-induced silencing complex (RISC), bind to Argonaute protein such as Ago2, or participate in or bind to one or more similar factors, and direct the silencing of target gene, it can be called guide strand.In some embodiments, the sense strand that is complementary to guide strand can be called " passenger strand ".

[0172] In some embodiments, oligonucleotides disclosed herein for targeting STAT3 comprise an antisense strand that comprises or consists of the sequence set forth in SEQ ID NO: 333. In some embodiments, 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 set forth in SEQ ID NO: 333. In some embodiments, oligonucleotides (e.g., RNAi oligonucleotides) disclosed herein for targeting STAT3 comprise an antisense strand that comprises or consists of the sequence set forth in SEQ ID NO: 716. In some embodiments, 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 set forth in SEQ ID NO: 716. In some embodiments, oligonucleotides disclosed herein for targeting STAT3 comprise an antisense strand that comprises or consists of the sequence set forth in SEQ ID NO: 965. In some embodiments, 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 set forth in SEQ ID NO: 965. In some embodiments, oligonucleotides disclosed herein for targeting STAT3 comprise an antisense strand that comprises or consists of the sequence set forth in SEQ ID NO: 333.In some embodiments, the oligonucleotides herein comprise an antisense strand comprising at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence set forth in SEQ ID NO: 333.

[0173] B sense strand In some embodiments, oligonucleotides (e.g., and RNAi oligonucleotides) disclosed herein for targeting STAT3 mRNA and inhibiting STAT3 expression comprise a sense strand sequence set forth in SEQ ID NO: 140. In some embodiments, the oligonucleotides herein have a sense strand comprising at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence set forth in SEQ ID NO: 140. In some embodiments, oligonucleotides (e.g., and RNAi oligonucleotides) disclosed herein for targeting STAT3 mRNA and inhibiting STAT3 expression comprise a sense strand sequence set forth in SEQ ID NO: 524. In some embodiments, the oligonucleotides herein have a sense strand comprising at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence set forth in SEQ ID NO: 524. In some embodiments, oligonucleotides disclosed herein for targeting STAT3 mRNA and inhibiting STAT3 expression comprise a sense strand sequence set forth in SEQ ID NO: 875. In some embodiments, the oligonucleotides herein have a sense strand composed of at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence set forth in SEQ ID NO: 875.

[0174] In some embodiments, an oligonucleotide comprises a sense strand (or passenger strand) up to about 40 nucleotides in length (e.g., up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, an oligonucleotide may have a sense strand at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36, or at least 38 nucleotides in length). In some embodiments, oligonucleotides can have a sense strand ranging in length from about 12 to about 40 nucleotides (e.g., 12-40, 12-36, 12-32, 12-28, 15-40, 15-36, 15-32, 15-28, 17-21, 17-25, 19-27, 19-30, 20-40, 22-40, 25-40, or 32-40). In some embodiments, oligonucleotides herein comprise a sense strand that is 15-50 nucleotides in length. In some embodiments, oligonucleotides herein comprise a sense strand that is 18-36 nucleotides in length. In some embodiments, the oligonucleotide may have a sense strand that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In some embodiments, the oligonucleotide comprises a sense strand that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the oligonucleotides herein comprise a sense strand that is 36 nucleotides in length.

[0175] In some embodiments, oligonucleotides (e.g., RNAi oligonucleotides) provided herein comprise a sense strand comprising a stem-loop structure at the 3' end of the sense strand. In some embodiments, the stem-loop is formed by intrastrand base pairing. In some embodiments, the sense strand comprises a stem-loop structure at its 5' end. In some embodiments, the stem of the stem-loop comprises a duplex that is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 2 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 3 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 4 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 5 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 6 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 7 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 8 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 9 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 10 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 11 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 12 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 13 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 14 nucleotides in length.

[0176] In some embodiments, the stem-loop provides oligonucleotide protection against degradation (e.g., enzymatic degradation), facilitates or improves targeting and / or delivery to target cells, tissues, or organs (e.g., the liver), or both. For example, in some embodiments, the loop of the stem-loop is comprised of nucleotides that include one or more modifications that facilitate, improve, or increase targeting to the target, inhibition of target gene expression, and / or delivery, uptake, and / or penetration into target cells, tissues, or organs (e.g., the liver), or a combination thereof. In some embodiments, the stem-loop itself or modifications to the stem-loop do not affect or substantially affect the intrinsic gene expression inhibitory activity of the oligonucleotide, but facilitate, improve, or increase stability (e.g., providing protection against degradation) and / or delivery, uptake, and / or penetration of the oligonucleotide into target cells, tissues, or organs. In certain embodiments, the oligonucleotides herein comprise a sense strand comprising (e.g., at its 3' end) a stem-loop described as S1-L-S2, where S1 is complementary to S2, and L forms a single-stranded loop of linked nucleotides between S1 and S2 up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length). In some embodiments, loop (L) is 3 nucleotides in length (referred to herein as a "triloop"). In some embodiments, loop (L) is 4 nucleotides in length (referred to herein as a "tetraloop"). In some embodiments, loop (L) is 5 nucleotides in length. In some embodiments, loop (L) is 6 nucleotides in length. In some embodiments, loop (L) is 7 nucleotides in length. In some embodiments, loop (L) is 8 nucleotides in length. In some embodiments, loop (L) is 9 nucleotides in length. In some embodiments, loop (L) is 10 nucleotides in length.

[0177] In some embodiments, an oligonucleotide (e.g., an RNAi oligonucleotide) provided herein comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 140, wherein the oligonucleotide comprises a sense strand comprising a stem-loop (e.g., at its 3' end) designated as S1-L-S2, wherein S1 is complementary to S2, and L forms a single-stranded loop between S1 and S2 up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length). In some embodiments, an oligonucleotide comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 140, wherein the oligonucleotide comprises a sense strand comprising a stem-loop (e.g., at its 3' end) designated as S1-L-S2, wherein S1 is complementary to S2, and L forms a single-stranded loop between S1 and S2 up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length).

[0178] In some embodiments, the tetraloop comprises the sequence 5'-GAAA-3'. In some embodiments, the stem loop comprises the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQ ID NO: 86).

[0179] In some embodiments, the sense strand comprises a stem-loop structure at its 3'-end. In some embodiments, the sense strand comprises a stem-loop structure at its 5'-end. In some embodiments, the stem is double-stranded and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 bp in length. In some embodiments, the stem-loop provides molecular protection against degradation (e.g., enzymatic degradation) and promotes targeting characteristics for delivery to target cells. For example, in some embodiments, the loop provides additional nucleotides that can be modified without substantially affecting the gene expression inhibitory activity of the oligonucleotide. In certain embodiments, the oligonucleotide is one in which the sense strand comprises a stem-loop (e.g., at its 3'-end), described herein as S1-L-S2, where S1 is complementary to S2 and L forms a loop between S1 and S2 up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length). FIG. 1 shows a non-limiting example of such an oligonucleotide.

[0180] In some embodiments, the loop (L) of the stem-loop having the structure S1-L-S2 described herein is a triloop, hi some embodiments, the triloop comprises a ribonucleotide, a deoxyribonucleotide, a modified nucleotide, a ligand (e.g., a delivery ligand), and combinations thereof.

[0181] In some embodiments, the loop of the stem-loop is a tetraloop (e.g., in a nicked tetraloop structure). The tetraloop can contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, the tetraloop has 4 to 5 nucleotides.

[0182] Double-strand length In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 12 nucleotides in length (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21). In some embodiments, the duplex formed between the sense strand and the antisense strand is in the range of 12-30 nucleotides in length (e.g., 12-30, 12-27, 12-22, 15-25, 18-30, 18-22, 18-25, 18-27, 18-30, 19-30, or 21-30 nucleotides in length). In some embodiments, the duplex formed between the sense strand and the antisense strand is 12, 13, 14, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 12 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 13 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 14 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 15 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 16 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 17 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 18 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 19 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 20 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 21 nucleotides in length.In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 22 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 23 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 24 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 25 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 26 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 27 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 28 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 29 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 30 nucleotides in length. In some embodiments, the duplex formed between sense strand and antisense strand does not span the entire length of sense strand and / or antisense strand.In some embodiments, the duplex formed between sense strand and antisense strand spans the entire length of either sense strand or antisense strand.In some embodiments, the duplex formed between sense strand and antisense strand spans the entire length of both sense strand and antisense strand.

[0183] In some embodiments, the duplex between the sense and antisense strands spans the entire length of both the sense and antisense strands. In some embodiments, the sense and antisense strands of the oligonucleotide are (a) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 875 and 965, respectively; The duplex formed between the sense and antisense strands ranges from 12 to 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length).

[0184] Oligonucleotide ends In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, and the ends of either or both strands comprise a blunt end. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand and an antisense strand, and the sense strand and the antisense strand are separate strands that form an asymmetric double-stranded region with an overhang at the 3' end of the antisense strand. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand and an antisense strand, and the ends of either or both strands comprise an overhang comprising one or more nucleotides. In some embodiments, one or more nucleotides comprising the overhang are unpaired nucleotides. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand and an antisense strand, and the 3' end of the sense strand and the 5' end of the antisense strand comprise a blunt end. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand and an antisense strand, and the 5' end of the sense strand and the 3' end of the antisense strand comprise a blunt end.

[0185] In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and the 3'-end of either or both strands comprises a 3' overhang comprising one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and the sense strand comprises a 3' overhang comprising one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and the antisense strand comprises a 3' overhang comprising one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and both the sense strand and the antisense strand comprise a 3' overhang comprising one or more nucleotides.

[0186] In some embodiments, the 3' overhang is about 1 to 20 nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length). In some embodiments, the 3' overhang is about 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or about 1 to 2 nucleotides in length. In some embodiments, the 3' overhang is 1 nucleotide in length. In some embodiments, the 3' overhang is 2 nucleotides in length. In some embodiments, the 3' overhang is 3 nucleotides in length. In some embodiments, the 3' overhang is 4 nucleotides in length. In some embodiments, the 3'-overhang is 5 nucleotides in length. In some embodiments, the 3'-overhang is 6 nucleotides in length. In some embodiments, the 3'-overhang is 7 nucleotides in length. In some embodiments, the 3'-overhang is 8 nucleotides in length. In some embodiments, the 3'-overhang is 9 nucleotides in length. In some embodiments, the 3'-overhang is 10 nucleotides in length. In some embodiments, the 3'-overhang is 11 nucleotides in length. In some embodiments, the 3'-overhang is 12 nucleotides in length. In some embodiments, the 3'-overhang is 13 nucleotides in length. In some embodiments, the 3'-overhang is 14 nucleotides in length. In some embodiments, the 3'-overhang is 15 nucleotides in length. In some embodiments, the 3'-overhang is 16 nucleotides in length. In some embodiments, the 3'-overhang is 17 nucleotides in length. In some embodiments, the 3'-overhang is 18 nucleotides in length.In some embodiments, the 3'-overhang is 19 nucleotides in length. In some embodiments, the 3' overhang is 20 nucleotides in length.

[0187] In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and the 5'-end of either or both strands comprises a 5'-overhang comprising one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and the sense strand comprises a 5'-overhang comprising one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and the antisense strand comprises a 5'-overhang comprising one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, and both the sense strand and the antisense strand comprise a 5'-overhang comprising one or more nucleotides.

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

[0189] In some embodiments, one or more (e.g., 2, 3, 4, 5, or more) nucleotides comprising the 3'-end or 5'-end of the sense and / or antisense strand are modified. For example, in some embodiments, one or two terminal nucleotides at the 3'-end of the antisense strand are modified. In some embodiments, the last nucleotide at the 3'-end of the antisense strand is modified so that it contains a 2'-modification or a 2'-O-methoxyethyl. In some embodiments, the last one or two terminal nucleotides at the 3'-end of the antisense strand are complementary to the target. In some embodiments, the last one or two nucleotides at the 3'-end of the antisense strand are not complementary to the target.

[0190] 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 step-loop as described herein, and the 3' end of the antisense strand comprises a 3' overhang as described herein. In some embodiments, the oligonucleotides (e.g., RNAi oligonucleotides) disclosed 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, the loop being a tetraloop as described herein, and the 3' end of the antisense strand comprises a 3' overhang as described herein. In some embodiments, the 3' overhang is two nucleotides in length. In some embodiments, both of the two nucleotides comprising the 3' overhang comprise a guanine (G) nucleobase. Typically, one or both of the nucleotides comprising the 3' overhang of the antisense strand are not complementary to the target mRNA.

[0191] Oligonucleotide Modification a. Sugar modification In some embodiments, modified sugars (also referred to herein as sugar analogs) comprise modified deoxyribose or ribose moieties, e.g., where one or more modifications occur at the 2', 3', 4', and / or 5' carbon positions of the sugar. In some embodiments, modified sugars can also comprise unnatural alternative carbon structures, such as those found in locked nucleic acids ("LNAs," see e.g., Koshkin et al., (1998) Tetrahedon 54:3607-3630), non-locked nucleic acids ("UNAs," see e.g., Snead et al., (2013) Mol. Ther-Nucl. Acids 2:e103), and bridged nucleic acids ("BNAs," see e.g., Imanishi and Obika (2002) Chem Commun. (Camb) 21:1653-1659).

[0192] In some embodiments, the nucleotide modification in the sugar comprises a 2'-modification. In some embodiments, the 2'-modification may be 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-fluoro (2'-F), 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), or 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA). In some embodiments, the modification is 2'-F, 2'-OMe, or 2'-MOE. In some embodiments, the modification in the sugar comprises a modification of the sugar ring, which may include modification of one or more carbons of the sugar ring. For example, modifications of the sugar of a nucleotide can include linking the 2'-oxygen of the sugar to the 1'- or 4'-carbon of the sugar, or linking the 2'-oxygen to the 1'- or 4'-carbon via an ethylene or methylene bridge. In some embodiments, the modified nucleotide has an acyclic sugar that lacks a 2'-carbon to 3'-carbon bond. In some embodiments, the modified nucleotide has, for example, a thiol group at the 4'-position of the sugar.

[0193] In some embodiments, the oligonucleotides described herein comprise at least about one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, or more). In some embodiments, the sense strand of the oligonucleotide comprises at least about one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or more). In some embodiments, the antisense strand of the oligonucleotide comprises at least about one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more).

[0194] In some embodiments, all nucleotides in the sense strand of the oligonucleotide are modified. In some embodiments, all nucleotides in the antisense strand of the oligonucleotide are modified. In some embodiments, all nucleotides in the oligonucleotide (i.e., both the sense strand and the antisense strand) are modified. In some embodiments, the modified nucleotides include 2'-modifications (e.g., 2'-F or 2'-OMe, 2'-MOE, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid). In some embodiments, the modified nucleotides include 2'-modifications (e.g., 2'-F or 2'-OMe).

[0195] In some embodiments, the present disclosure provides oligonucleotides having different modification patterns. In some embodiments, the oligonucleotides herein include a sense strand having the modification pattern shown in the Examples and Sequence Listing, and an antisense strand having the modification pattern shown in the Examples and Sequence Listing.

[0196] In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise an antisense strand having 2'-F modified nucleotides. In some embodiments, the oligonucleotides disclosed herein comprise an antisense strand comprising 2'-F and 2'-OMe modified nucleotides. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand having 2'-F modified nucleotides. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand comprising 2'-F and 2'-OMe modified nucleotides.

[0197] 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 in the sense strand comprise a 2'-fluoro modification. In some embodiments, 11% of the nucleotides in 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 in the antisense strand comprise a 2'-fluoro modification. In some embodiments, about 32% of the nucleotides in the antisense strand comprise a 2'-fluoro modification. In some embodiments, the oligonucleotides have about 15-25%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of their nucleotides comprise a 2'-fluoro modification. In some embodiments, about 19% of the nucleotides in the dsRNAi oligonucleotide comprise a 2'-fluoro modification.

[0198] In some embodiments, the modified oligonucleotides comprise a sense strand sequence having a modification pattern as described in Figure 1 or Example 7, and an antisense strand having a modification pattern as described in Figure 1 or Example 7. In some embodiments, these oligonucleotides have one or more of positions 8, 9, 10, or 11 of the sense strand modified with a 2'-F group. In other embodiments, these oligonucleotides have the sugar moiety of each of nucleotides 1-7 and 12-20 of the sense strand modified with a 2'-OMe.

[0199] In some embodiments, the antisense strand has three nucleotides modified with 2'-F at the 2' position of the sugar moiety. In some embodiments, the sugar moieties at positions 2, 5, and 14, and optionally up to three nucleotides at positions 1, 3, 7, and 10 of the antisense strand, are modified with 2'-F. In some embodiments, the sugar moieties at positions 2, 5, and 14, and optionally up to three nucleotides at positions 3, 4, 7, and 10 of the antisense strand, are modified with 2'-F. In other embodiments, the sugar moieties at positions 2, 5, and 14 of the antisense strand are modified with 2'-F. In other embodiments, the sugar moieties at positions 1, 2, 5, and 14 of the antisense strand are modified with 2'-F. In other embodiments, the sugar moieties at positions 2, 4, 5, and 14 of the antisense strand are modified with 2'-F. In yet another embodiment, the sugar moieties at each of positions 1, 2, 3, 5, 7, and 14 of the antisense strand are modified with 2'-F. In another embodiment, the sugar moieties at each of positions 2, 3, 4, 5, 7, and 14 of the antisense strand are modified with 2'-F. In yet another embodiment, the sugar moieties at each of positions 1, 2, 3, 5, 10, and 14 of the antisense strand are modified with 2'-F. In another embodiment, the sugar moieties at each of positions 2, 3, 4, 5, 10, and 14 of the antisense strand are modified with 2'-F. In another embodiment, the sugar moieties at each of positions 2, 3, 5, 7, 10, and 14 of the antisense strand are modified with 2'-F. In yet another embodiment, the sugar moieties at each of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand are modified with 2'-F.

[0200] In some embodiments, the oligonucleotides provided herein comprise an antisense strand having a sugar moiety at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 that is modified with 2'-F.

[0201] In some embodiments, the oligonucleotides provided herein comprise an antisense strand having a 2'-OMe modified sugar moiety at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.

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

[0203] In some embodiments, the oligonucleotides provided herein comprise an antisense strand having a sugar moiety at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA).

[0204] In some embodiments, the oligonucleotides provided herein comprise a sense strand having 2'-F modified sugar moieties at positions 8-11. In some embodiments, the oligonucleotides provided herein comprise a sense strand having 2'-F modified sugar moieties at positions 3, 8, 9, 10, 12, 13, and 17. In some embodiments, the oligonucleotides provided herein comprise a sense strand having 2'OMe modified sugar moieties at positions 1-7 and 12-17 or 12-20. In some embodiments, the oligonucleotides provided herein comprise a sense strand having 2'OMe modified sugar moieties at positions 1-7, 12-27, and 31-36. In some embodiments, the oligonucleotides provided herein comprise a sense strand having 2'OMe modified sugar moieties at positions 1-7 and 12-36. In some embodiments, the oligonucleotides provided herein comprise a sense strand having the sugar moieties at each of the nucleotides at positions 1-7 and 12-17 or 12-20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA). In some embodiments, the oligonucleotides provided herein comprise a sense strand having the sugar moieties at positions 1-2, 4-7, 11, 14-16, and 18-20 modified with 2'OMe.In some embodiments, the oligonucleotides provided herein comprise a sense strand having the sugar moieties of each of the nucleotides at positions 1-2, 4-7, 11, 14-16, and 18-20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid (2'-FANA).

[0205] In some embodiments, the oligonucleotides provided herein comprise a sense strand having a sugar moiety at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 that is modified with 2'-F.

[0206] In some embodiments, the oligonucleotides provided herein comprise a sense strand having a 2'-OMe modified sugar moiety at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36.

[0207] In some embodiments, the oligonucleotides provided herein are selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinanyl. and a sense strand having a sugar moiety at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 modified with a modification selected from the group consisting of: 2'-FANA, ...

[0208] In some embodiments, the oligonucleotides provided herein comprise a sense strand having 2'-F modified sugar moieties at positions 8-11 and 2'-OMe modified sugar moieties at positions 1-7 and 12-36, and an antisense strand having 2'-F modified sugar moieties at positions 2, 3, 4, 5, 7, 10, and 14, and 2'-OMe modified sugar moieties at positions 1, 6, 8, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, and 22.

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

[0210] In some embodiments, oligonucleotides have a phosphate analog at the 4' carbon position of the sugar (referred to as a "4' phosphate analog"). See, e.g., International Patent Application Publication No. 2018 / 045317. In some embodiments, oligonucleotides herein include a 4' phosphate analog at the 5'-terminal nucleotide. In some embodiments, the phosphate analog is an oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., its 4' carbon) or an analog thereof. In other embodiments, the 4'-phosphate analog is a thiomethylphosphonate or aminomethylphosphonate, in which the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is attached to the 4'-carbon of the sugar moiety or an analog thereof. In certain embodiments, the 4'-phosphate analog is an oxymethylphosphonate. In some embodiments, the oxymethylphosphonate is represented by the formula -O-CH-PO(OH) or -O-CH-PO(OR), where R is independently selected from H, CH, an alkyl group, CHCHCN, CHOCOC(CH), CHOCHCHSi(CH), or a protecting group. In particular embodiments, the alkyl group is CHCH. More typically, R is independently selected from H, CH, or CHCH.

[0211] In some embodiments, the oligonucleotides provided herein comprise an antisense strand that comprises a 4'-phosphate analog at the 5'-terminal nucleotide, wherein the 5'-terminal nucleotide comprises the following structure: [ka] 4'-O-monomethylphosphonate-2'-O-methyluridine phosphorothioate [Mephosphonate-4O-mUs] chemical formula 1

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

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

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

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

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

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

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

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

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

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

[0222] Targeting Ligands In some embodiments, it may be desirable to target the STAT3-targeting oligonucleotides of the present disclosure to one or more cells or one or more organs. Such strategies may be useful for avoiding undesirable effects in other organs or for avoiding unnecessary loss of the oligonucleotide to cells, tissues, or organs that would not benefit from the oligonucleotide. Targeting of oligonucleotides to one or more cells or one or more organs can be achieved through various approaches. Conjugation of oligonucleotides to tissue- or cell-specific antibodies, small molecules, or targeting ligands can facilitate delivery to one or more target cells or tissues and alter oligonucleotide accumulation (Chernolovskaya et al., (2019) Front Pharmacol. 10:444). For example, conjugation of oligonucleotides to saturated fatty acids (e.g., C22) may facilitate delivery to cells or tissues, such as adipose tissue or immune cells, that take up such ligands more readily than conventional oligonucleotide ligands. Thus, in some embodiments, the oligonucleotides disclosed herein are modified to facilitate targeting and / or delivery to tissues, cells, or organs (e.g., to facilitate delivery of oligonucleotides to the liver). In certain embodiments, the 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 with one or more targeting ligands.

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

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

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

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

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

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

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

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

[0231] 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 below in Formula 4. [ka] chemical formula 4

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

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

[0234] The targeting ligand can be attached to the nucleotide using an appropriate method or chemistry (e.g., click chemistry). In some embodiments, the targeting ligand is conjugated to the nucleotide using a click linker. In some embodiments, the targeting ligand is conjugated to any one of the nucleotides of the oligonucleotides described herein using an acetal-based linker. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. 2016 / 100401. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is stable. An example is shown below (Formula 7 and Formula 8) for a loop containing the nucleotide GAAA from 5' to 3', in which a GalNAc moiety is attached to the nucleotide of the loop using an acetal linker. Such a loop can be present, for example, at positions 27-30 of any one of the sense strands shown in Figure 1. In the formula: [ka] is the point of attachment to the oligonucleotide chain (Formula 7 and Formula 8). [ka] Formula 6, or [ka] chemical formula 7

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

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

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

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

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

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

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

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

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

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

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

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

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

[0248] In some embodiments, the STAT3-targeting oligonucleotide is a double-stranded molecule. In some embodiments, the STAT3-targeting oligonucleotide is an RNAi molecule.

[0249] In some embodiments, the STAT3 targeting oligonucleotide comprises a sense strand of 36 nucleotides with positions numbered 1 to 36 from 5' to 3'.

[0250] 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.

[0251] 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.

[0252] In some embodiments, the STAT3-targeting oligonucleotide comprises a lipid conjugated to the 5'-terminal nucleotide of the sense strand, wherein the lipid is [ka] is.

[0253] In some embodiments, the STAT3-targeting oligonucleotide comprises a lipid conjugated to the 5'-terminal nucleotide of the sense strand, wherein the lipid is: [ka]

[0254] In some embodiments, the STAT3 targeting oligonucleotide comprises a sense strand comprising the sequence set forth in SEQ ID NO: 140 and an antisense strand comprising the sequence set forth 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 comprising the sequence set forth in SEQ ID NO: 140 and an antisense strand comprising the sequence set forth 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 comprising the sequence set forth in SEQ ID NO: 140 and an antisense strand comprising the sequence set forth in SEQ ID NO: 333, wherein the sense strand comprises a C18 lipid conjugated to the 5'-terminal nucleotide.

[0255] In some embodiments, the STAT3 targeting oligonucleotide comprises a sense strand comprising the sequence set forth in SEQ ID NO: 875 and an antisense strand comprising the sequence set forth 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 comprising the sequence set forth in SEQ ID NO: 875 and an antisense strand comprising the sequence set forth 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 comprising the sequence set forth in SEQ ID NO: 875 and an antisense strand comprising the sequence set forth in SEQ ID NO: 965, wherein the sense strand comprises a C18 lipid conjugated to the 5'-terminal nucleotide.

[0256] In some embodiments, the STAT3 targeting oligonucleotide comprises a sense strand comprising the sequence set forth in SEQ ID NO: 140 and an antisense strand comprising the sequence set forth in SEQ ID NO: 333, wherein the sense strand comprises a lipid conjugated to the 5' terminal nucleotide, the lipid comprising [ka] is.

[0257] In some embodiments, the STAT3 targeting oligonucleotide comprises a sense strand comprising the sequence set forth in SEQ ID NO: 140 and an antisense strand comprising the sequence set forth in SEQ ID NO: 333, wherein the sense strand comprises a lipid conjugated to the 5' terminal nucleotide, the lipid comprising [ka] is.

[0258] In some embodiments, the STAT3 targeting oligonucleotide comprises a sense strand comprising the sequence set forth in SEQ ID NO: 875 and an antisense strand comprising the sequence set forth in SEQ ID NO: 965, wherein the sense strand comprises a lipid conjugated to the 5' terminal nucleotide, the lipid comprising [ka] is. In some embodiments, the STAT3 targeting oligonucleotide comprises a sense strand comprising the sequence set forth in SEQ ID NO: 875 and an antisense strand comprising the sequence set forth in SEQ ID NO: 965, wherein the sense strand comprises a lipid conjugated to the 5' terminal nucleotide, the lipid comprising [ka] is.

[0259] In some embodiments, the STAT3-targeting oligonucleotide comprises an antisense strand of 15-30 nucleotides and a sense strand of 15-40 nucleotides, wherein the sense and antisense strands form a double-stranded region, wherein the antisense strand comprises a region of complementarity to a STAT3 mRNA target sequence expressed in immune cells associated with the tumor microenvironment, wherein the sense strand comprises at its 3' end a stem-loop comprising a tetraloop containing four nucleosides, and wherein the 5'-terminal nucleotide of the sense strand is represented by Formula II-Ib: [ka] wherein B is selected from adenine and guanine nucleobases; R 5 is a hydrocarbon chain. In some embodiments, m is 1, X1 is O, and Y2 is an internucleotide linkage group attached to the 5' end of the nucleoside; Y is [ka] wherein Y1 is a linking group attached to the 2' or 3' end of the nucleotide, X2 is O, X3 is O, and R3 is H.

[0260] In some embodiments, the hydrocarbon chain is a C8-C30 hydrocarbon chain. In some embodiments, the hydrocarbon chain is a C16 hydrocarbon chain. In some embodiments, the C16 hydrocarbon chain is represented by: [ka] In some embodiments, the hydrocarbon chain is a C hydrocarbon chain. In some embodiments, the C hydrocarbon chain is represented by: [ka]

[0261] In some embodiments, the oligonucleotide comprises a sense strand comprising the sequence of SEQ ID NO: 140, wherein the sense strand comprises a C18 lipid.

[0262] Exemplary STAT3-Targeting Oligonucleotides In some embodiments, oligonucleotides for reducing expression of STAT3 mRNA comprise the sense and antisense strands described herein, wherein the sense and antisense strands are modified according to 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] This hybridizes to Antisense strand: [Mephosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][fX][mX][mX][fX][mX][mX][mX][mX][mXs][mXs][mXs][mX] (Key is provided in Table 7). In some embodiments, C# is C16 or C18.

[0263] In some embodiments, oligonucleotides for reducing expression of STAT3 mRNA comprise the sense and antisense strands described herein, wherein the sense and antisense strands are modified according to the following pattern: Sense strand: [ademXs-C#][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX][mX][mX][mX][m [mX] This hybridizes to Antisense strand: [Mephosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][fX][mX][mX][fX][mX][mX][mX][mX][mXs][mXs][mXs][mX] (Key is provided in Table 7).

[0264] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises a sense strand and an antisense strand comprising SEQ ID NOs: 875 and 965, respectively. The sense and antisense strands are modified according to the following patterns: 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] This hybridizes to Antisense strand: [Mephosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][mX][fX] [mX][mX][mX][fX][mX][m]X[mX][mX][mXs][mXs][mX] (Key is provided in Table 7.) In some embodiments, C# is C16 or C18.

[0265] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises a sense strand and an antisense strand comprising SEQ ID NOs: 875 and 965, respectively. The sense and antisense strands are modified according to the following patterns: 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] This hybridizes to Antisense strand: [Mephosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][mX][fX][mX][mX][mX][mX][mXs][mXs][mXs] (key provided in Table 7).

[0266] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises a sense strand and an antisense strand comprising SEQ ID NOs: 1222 and 1145, respectively.

[0267] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 140. In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 875.

[0268] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises the antisense strand sequence of SEQ ID NO: 333. In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises the antisense strand sequence of SEQ ID NO: 965.

[0269] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO:875 and a selected antisense strand of SEQ ID NO:965.

[0270] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO:1222.

[0271] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises the antisense strand sequence of SEQ ID NO:1145.

[0272] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO:1222 and the antisense strand sequence of SEQ ID NO:1145.

[0273] In some embodiments, the oligonucleotides described herein for reducing STAT3 mRNA expression have minimal off-target effects. For example, in some embodiments, the oligonucleotides described herein reduce STAT3 expression and do not reduce STAT1 expression, or reduce STAT1 expression less than STAT3 expression. In some embodiments, the oligonucleotides comprise a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 875 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 965, and the oligonucleotides reduce STAT3 expression and do not reduce STAT1 expression, or reduce STAT1 expression less than STAT3 expression. In some embodiments, the oligonucleotides comprise a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1222 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1145, and the oligonucleotides reduce STAT3 expression and do not reduce STAT1 expression, or reduce STAT1 expression less than STAT3 expression.

[0274] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA reduces STAT3 mRNA by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0275] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 875 and the antisense strand sequence of SEQ ID NO: 965, wherein the oligonucleotide reduces STAT3 mRNA in humans.

[0276] In some embodiments, the oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 875 and the antisense strand sequence of SEQ ID NO: 965, wherein the oligonucleotide reduces STAT3 mRNA by at least 75%.

[0277] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 875 and the antisense strand sequence of SEQ ID NO: 965, wherein the oligonucleotide is conjugated to a lipid on the 5'-terminal nucleotide of the sense strand.

[0278] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 875 and the antisense strand sequence of SEQ ID NO: 965, wherein the oligonucleotide is conjugated to a C18 lipid on the 5'-terminal nucleotide of the sense strand.

[0279] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 875 and the antisense strand sequence of SEQ ID NO: 965, wherein the oligonucleotide is conjugated to a lipid on the 5'-terminal nucleotide of the sense strand, and reduces STAT3 mRNA in humans.

[0280] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 875 and the antisense strand sequence of SEQ ID NO: 965, wherein the oligonucleotide is conjugated to a lipid on the 5'-terminal nucleotide of the sense strand, and reduces STAT3 mRNA by at least 75% in humans.

[0281] In some embodiments, an oligonucleotide for reducing expression of STAT3 mRNA comprises the sense strand sequence of SEQ ID NO: 875 and the antisense strand sequence of SEQ ID NO: 965, wherein the oligonucleotide is conjugated to a C18 lipid on the 5'-terminal nucleotide of the sense strand, and reduces STAT3 mRNA by at least 75% in humans.

[0282] In some embodiments, the oligonucleotide for reducing 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 in humans.

[0283] In some embodiments, the oligonucleotide for reducing 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%.

[0284] formulation In order to facilitate the use of oligonucleotide, various formulations have been developed.For example, oligonucleotide can be delivered to subject or cellular environment using formulations that minimize degradation, promote delivery and / or uptake, or provide other beneficial properties to the oligonucleotide in the formulation.In some embodiments, oligonucleotide is formulated in buffer solution such as phosphate buffered saline, liposome, micelle structure, and capsid.

[0285] Formulations of oligonucleotides containing cationic lipids can be used to facilitate 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.

[0286] Thus, in some embodiments, the formulation comprises a lipid nanoparticle. In some embodiments, the excipient may comprise a liposome, lipid, lipid complex, microsphere, microparticle, nanosphere, or nanoparticle, or may be otherwise formulated for administration to a cell, tissue, organ, or body of a subject in need thereof (see, e.g., Remington: THE SCIENCE AND PRACTICE OF PHARMACY, 22nd edition, Pharmaceutical Press, 2013).

[0287] In some embodiments, the formulations herein include an excipient. In some embodiments, the excipient confers 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 put into solution before use (e.g., administration to a subject). Thus, the excipient in a composition comprising any one of the oligonucleotides described herein can be a cryoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a disintegration temperature regulator (e.g., dextran, Ficoll™, or gelatin).

[0288] In some embodiments, a pharmaceutical composition is formulated to be compatible with its intended route of administration, which includes parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.

[0289] Pharmaceutical compositions suitable for injection use include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In many cases, it will be preferable to include an isotonic agent in the composition, such as a sugar, polyalcohol, e.g., mannitol, sorbitol, or sodium chloride. Sterile injectable solutions can be prepared by incorporating the required amount of oligonucleotide in a selected solvent, optionally containing one or a combination of the ingredients listed above, followed by filtered sterilization.

[0290] In some embodiments, the composition may contain at least about 0.1% or more of the therapeutic agent, although the percentage of active ingredient may be from about 1% to about 80% or more by weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be taken into account by those skilled in the art when preparing such pharmaceutical formulations, and various dosages and treatment regimens may be desirable accordingly.

[0291] Some embodiments are directed to liver-targeted delivery of any of the oligonucleotides herein, although targeting of other tissues is also contemplated.

[0292] Programmed death-ligand 1 (PD-L1) inhibitors In some embodiments, the 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.

[0293] 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 emvafolimab. In some embodiments, the anti-PD-L1 antibody is durvalumab.

[0294] 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 described in Akinleye & Rasool, "Immune checkpoint inhibitors of PD-L1 as cancer therapeutics," J. of Hematology & Oncology. 12(92):2019. In some embodiments, the anti-PD-L1 antibody is BMS-936559. 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.

[0295] 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.

[0296] In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 30 nM to about 100 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 30 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 40 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 50 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 60 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 70 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 80 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 90 nM. In some embodiments, the anti-PD-L1 antibodies described herein bind to PD-L1 with an affinity of about 100 nM.

[0297] In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 30 nM to about 100 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 30 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 40 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 50 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 60 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 70 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 80 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 90 nM. In some embodiments, the anti-PD-1 antibodies described herein bind to PD-1 with an affinity of about 100 nM.

[0298] In some embodiments, antibodies are generated using display technologies. Display technologies used to generate antibody polypeptides include any of the display techniques (e.g., display library screening techniques). In some embodiments, synthetic antibodies are designed, selected, or optimized by screening target antigens using display technologies (e.g., phage display technologies). Phage display libraries may contain millions to billions of phage vectors, each expressing a unique antibody fragment on their viral coat. Such libraries can provide a sufficiently diverse resource to be used to select potentially hundreds of antibody fragments with varying levels of affinity for one or more antigens of interest (McCafferty, et al., 1990. Nature. 348:552-4; Edwards, BM et al., 2003. JMB. 334:103-18; Schofield, D. et al., 2007. Genome Biol. 8, R254; and Pershad, K. et al., 2010. Protein Engineering Design and Selection. 23:279-88; the contents of each of which are incorporated herein by reference in their entirety). Often, the antibody fragments present in such libraries comprise V-type antibody fragments linked by flexible linkers. H Antibody domains and V LThese include scFv antibody fragments, including fusion proteins of antibody domains. In some cases, scFvs may contain the same sequence except for the unique sequence encoding the variable loop of the CDR. In some cases, scFvs are expressed as fusion proteins linked to a viral coat protein (e.g., the N-terminus of the viral coat protein). The VL chain can be expressed separately for assembly with the VH chain in the periplasm before complex incorporation into the viral coat. Precipitated library members can be sequenced from bound phage to obtain 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 can be mutated and utilized for further optimization through in vitro affinity maturation.

[0299] In some embodiments, the sequence of a polypeptide encoded by a viral genome is produced using yeast surface display technology. In some embodiments, recombinant antibodies are developed by displaying the antibody fragment of interest as a fusion on the surface of yeast, where the protein interacts with proteins and small molecules in solution. ScFvs with affinity for the desired receptor can be isolated from the yeast surface using magnetic separation and flow cytometry. Several cycles of yeast surface display and isolation can be performed to achieve scFvs with desired properties through directed evolution.

[0300] Methods for determining the affinity of an antibody to its antigen are known in the art. An exemplary method for determining binding affinity uses surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that allows for the analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further explanation, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627; Johnson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0301] kit In some embodiments, the present disclosure provides a kit comprising a STAT3 oligonucleotide described herein and instructions for administering the STAT3 oligonucleotide to a subject. In some embodiments, the present disclosure provides a kit comprising a STAT3 oligonucleotide described herein and instructions for administering the STAT3 oligonucleotide to a subject who has received or is receiving a PD-L1 inhibitor. In some embodiments, the kit comprises, in a suitable container, the oligonucleotide described herein, one or more controls, and various buffers, reagents, enzymes, and other standard components known in the art. In some embodiments, the container comprises at least one vial, well, test tube, flask, bottle, syringe, or other container means into which the oligonucleotide is placed, and in some cases, suitably aliquoted. In some embodiments in which additional components are provided, the kit comprises an additional container into which this component is placed. The kit may also include means for securely closing the oligonucleotide and any other reagents for commercial sale. Such containers may include injection-molded or blow-molded plastic containers into which the desired vials are held. The container and / or kit may include a label with instructions and / or warnings for use.

[0302] In some embodiments, the kit comprises a STAT3 oligonucleotide described herein and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the oligonucleotide, and instructions for treating or delaying the progression of a disease, disorder, or condition associated with STAT3 expression in a subject in need thereof. In some embodiments, the kit comprises a STAT3 oligonucleotide described herein and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the oligonucleotide, and instructions for treating or delaying the progression of a disease, disorder, or condition associated with STAT3 expression in a subject in need thereof, wherein the subject has received or is receiving a PD-L1 inhibitor. In some embodiments, the kit comprises a STAT3 oligonucleotide described herein and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the oligonucleotide, and instructions for treating or delaying the progression of cancer in a subject in need thereof. In some embodiments, the kit comprises a STAT3 oligonucleotide described herein and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the oligonucleotide, and instructions for treating or delaying the progression of cancer in a subject in need thereof, wherein the subject has received or is receiving a PD-L1 inhibitor.

[0303] In some embodiments, the kit comprises a pharmaceutical composition comprising a PD-L1 inhibitor and a pharmaceutically acceptable carrier or oligonucleotide described herein and instructions for treating or delaying the progression of a disease, disorder, or condition in a subject in need thereof, where the subject has received or has received a STAT3 oligonucleotide described herein. In some embodiments, the kit comprises a pharmaceutical composition comprising a PD-L1 inhibitor and a pharmaceutically acceptable carrier or oligonucleotide described herein and instructions for treating or delaying the progression of cancer in a subject in need thereof, where the subject has received or has received a STAT3 oligonucleotide described herein. [Example]

[0304] While the present disclosure has been described with reference to specific embodiments illustrated in the following examples, those skilled in the art should understand that various changes can be made and equivalents can be substituted without departing from the true 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. In addition, modifications can be made to the circumstances, materials, compositions of matter, processes, process steps, or steps to adapt them to the objective, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the present disclosure. Standard techniques known in the art or those specifically described below were utilized. Abbreviation 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'-bi(1,3,2-dioxaborolane) BINAP: 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl BH3: Boran Bn: Benzyl Boc: tert-butoxycarbonyl Boc2O: di-tert-butyl dicarbonate BPO: Benzoyl peroxide BuOH: n-butanol CDI: carbonyldiimidazole COD: Cyclooctadiene d:day DABCO: 1,4-diazobicyclo[2.2.2]octane DAST: Diethylaminosulfur trifluoride dba: dibenzylidene acetone DBU: 1,8-diazobicyclo[5.4.0]undec-7-ene DCE: 1,2-dichloroethane DCM: dichloromethane DEA: Diethylamine DHP: dihydropyran DIBAL-H: Diisobutylaluminum hydride DIPA: Diisopropylamine DIPEA or DIEA: N,N-diisopropylethylamine DMA: N,N-dimethylacetamide DME: 1,2-dimethoxyethane DMAP: 4-dimethylaminopyridine DMF: N,N-dimethylformamide DMP: Dess-Martin periodinane DMSO - Dimethyl sulfoxide DMTr: 4,4'-dimethyoxytrityl DPPA: Diphenylphosphoryl azide dppf: 1,1'-bis(diphenylphosphino)ferrocene EDC or EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ee: enantiomeric excess ESI: electrospray ionization EA: Ethyl acetate EtOAc: ethyl acetate EtOH: ethanol FA: Formic acid h or hrs: hours HATU: N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate HCl: Hydrochloric acid HPLC: High-performance liquid chromatography HOAc: acetic acid IBX: 2-iodoxybenzoic acid IPA: Isopropyl alcohol KHMDS: potassium hexamethyldisilazide K2CO3: Potassium carbonate LAH: Lithium aluminum hydride LDA: lithium diisopropylamide L-DBTA: Dibenzoyl-L-tartaric acid m-CPBA: metachloroperbenzoic acid M: mole MeCN: acetonitrile MeOH: Methanol Me2S: dimethyl sulfide MeONa: Sodium methylate MeI: iodomethane min:minutes mL: milliliter mM: millimolar mmol: millimolar MPa: Megapascals MOMCl: methyl chloromethyl ether MsCl: methanesulfonyl chloride MTBE: Methyl tert-butyl ether nBuLi: n-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 o C: Celsius Pd / C: Palladium on carbon Pd(OAc)2: Palladium acetate PBS: phosphate buffered saline PE: Petroleum ether POCl3: Phosphorus oxychloride PPh3: Triphenylphosphine PyBOP: (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate Rel: Relative RT or rt: room temperature s or sec: seconds sat: saturation SEMCl: chloromethyl-2-trimethylsilyl ethyl ether SFC: Supercritical Fluid Chromatography SOCl2: sulfur dichloride tBuOK: potassium tert-butoxide TBAB: tetrabutylammonium bromide TBAF: Tetrabutylammonium fluoride TBAI: Tetrabutylammonium iodide TEA: Triethylamine Tf: Trifluoromethanesulfonate TfAA, TFMSA or TfO: Trifluoromethanesulfonic anhydride TFA: Trifluoroacetic acid TIBSCl: 2,4,6-triisopropylbenzenesulfonyl chloride TIPS: Triisopropylsilyl THF: tetrahydrofuran THP: tetrahydropyran TLC: Thin Layer Chromatography TMEDA: Tetramethylethylenediamine pTSA: para-toluenesulfonic acid UPLC: Ultra-high performance liquid chromatography wt: weight Xantphos: 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

[0305] Example 1: Preparation of double-stranded RNAi oligonucleotides General synthesis method The following examples are intended to illustrate the present disclosure and should not be construed as limiting the present disclosure. Temperatures are given in degrees Celsius (C). Unless otherwise noted, all evaporations are carried out under reduced pressure, preferably at about 15 mm Hg to 100 mm Hg (= 20 to 133 mbar). The structures of final products, intermediates, and starting materials were confirmed by standard analytical methods, e.g., microanalysis and spectroscopic properties, e.g., MS, IR, NMR. Abbreviations used are those commonly used in the art.

[0306] All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts utilized to synthesize the nucleic acids or analogs thereof of the present disclosure are commercially available or can be produced by organic synthesis methods known to those skilled in the art (Methods of Organic Synthesis, Thieme, Volume 21 (Houben-Weyl 4th Ed. 1952)). Furthermore, the nucleic acids or analogs thereof of the present disclosure can be produced by organic synthesis methods known to those skilled in the art, as shown in the examples below.

[0307] Unless otherwise specified, all reactions are carried out under nitrogen or argon.

[0308] Proton NMR ( 1 1 H NMR) was performed in a deuterated solvent. Certain nucleic acids or analogs thereof disclosed herein may contain one or more 1 The H shifts overlap with residual proteosolvent signals, and these signals are not reported in the experiments provided below. As shown in the Examples below, in certain exemplary embodiments, nucleic acids or analogs thereof were prepared according to the following general procedures: It will be understood that while the general methods refer to the synthesis of specific nucleic acids or analogs thereof of the present disclosure, the following general methods, and others known to those of skill in the art, can be applied to all nucleic acids or analogs thereof, as well as each subclass and species of these nucleic acids or analogs thereof, as described herein. Example 1a: Synthesis of 2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-fluoro[3,2-f][1,3,5,2,4]trioxadisirosin-9-yl)oxy)methoxy)ethoxy)ethane-1-ammonium formate (1-6) [ka]

[0309] A solution of compound 1-1 (25.00 g, 67.38 mmol) in 20 mL of DMF was treated with pyridine (11 mL, 134.67 mmol) and tetraisopropyldisiloxane dichloride (22.63 mL, 70.75 mmol) at 10 °C. The resulting mixture was stirred at 25 °C for 3 hours and quenched with 20% citric acid (50 mL). The aqueous layer was extracted with EtOAc (3 × 50 mL), and the combined organic layers were concentrated in vacuo. The crude residue was recrystallized from a mixture of MTBE and n-heptane (1:15, 320 mL) to give compound 1-2 (37.20 g, 90%) as a white oily solid.

[0310] A solution of compound 1-2 (37.00 g, 60.33 mmol) in 20 mL of DMSO was treated with AcOH (20 mL, 317.20 mmol) and AcO (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 KCO (50 mL). The aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic layers were concentrated and recrystallized with ACN (30 mL) to give compound 1-3 (15.65 g, 38.4%) as a white solid.

[0311] A solution of compound 1-3 (20.00 g, 29.72 mmol) in 120 mL of DCM was treated with Fmoc-amino-ethoxyethanol (11.67 g, 35.66 mmol) at 25° C. The mixture was stirred to obtain a clear solution and then treated with 4 Å molecular sieves (20.0 g), N-iodosuccinimide (8.02 g, 35.66 mmol), and TfOH (5.25 mL, 59.44 mmol). The mixture was stirred at 30° C. until HPLC analysis showed >95% consumption of compound 1-3. The reaction was quenched with TEA (6 mL) and filtered. The filtrate was diluted with EtOAc, washed with saturated NaHCO3 (2 × 100 mL), saturated Na2SO3 (2 × 100 mL), and water (2 × 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.

[0312] A solution of compound 1-4 (26.34 g, 27.62 mmol) in a mixture of DCM / water (10:7, 170 mL) was treated with DBU (7.00 mL, 45.08 mmol) at 5 °C. The mixture was stirred at 5 to 25 °C for 1 h. The organic layer was then separated, washed with water (100 mL), and diluted with DCM (130 mL). The solution was treated with fumaric acid (7.05 g, 60.76 mmol) and 4 Å molecular sieves (26.34 g) in four portions. The mixture was stirred for 1 h, concentrated, and recrystallized from a mixture of MTBE and DCM (5:1) to give compound 1-6 (14.74 g, 62.9%) as a white solid. 1 H NMR(400MHz,d6-DMSO)8.73(s,1H),8.58(s,1H),8.15-8.02(m,2H),7.65-7.60(m,1H),7.59-7.51(m,2H),6.52(s,2H),6.15 (s,1H),5.08-4.90(m,3H),4.83-4.78(m,1H),4.15-3.90(m,3H),3.79-3.65(m,2H),2.98-2.85(m,6H),1.20-0.95(m,28H). Example 1b: Synthesis of (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((2-(2-[lipid]-amidoethoxy)ethoxy)methoxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (2-4a to 2-4e) [ka]

[0313] A solution of compound 1-6 (50.00 g, 59.01 mmol) in 150 mL of 2-methyltetrahydrofuran was washed with ice-cold aqueous KHPO (6%, 100 mL) and brine (20%, 2 × 100 mL). The organic layer was separated and treated with hexanoic acid (10.33 mL, 82.61 mmol), HATU (33.66 g, 88.52 mmol), and DMAP (10.81 g, 147.52 mmol) at 0 °C. The resulting mixture was warmed to 25 °C and stirred for 1 h. The solution was washed with water (2 × 100 mL), brine (100 mL), and concentrated in vacuo to give a crude residue. Flash chromatography on silica gel (1:1 hexane / acetone) afforded compound 2-1a (34.95 g, 71.5%) as a white solid.

[0314] 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 NaHCO (5 × 20 mL) and brine (50 mL). The organic layer was concentrated in vacuo to give crude compound 2-2a (24.72 g, 99%), which was used directly in the next step without further purification. A solution of compound 2-2a (24.72 g, 42.18 mmol) in 50 mL of DCM was treated with N-methylmorpholine (18.54 mL, 168.67 mmol) and DMTr-Cl (15.69 g, 46.38 mmol). The mixture was stirred at 25 °C for 2 h and quenched with saturated NaHCO (50 mL). The organic layer was separated, washed with water, and concentrated to give a crude slurry. Flash chromatography on silica gel (1:1 hexane / acetone) afforded compound 2-3a (30.05 g, 33.8 mmol, 79.9%) as a white solid.

[0315] A solution of compound 2-3a (25.00 g, 28.17 mmol) in 50 mL of DCM was treated with N-methylmorpholine (3.10 mL, 28.17 mmol) and tetrazole (0.67 mL, 14.09 mmol) under a nitrogen atmosphere. Bis(diisopropylamino)chlorophosphine (9.02 g, 33.80 mmol) was added dropwise to the solution, and the resulting mixture was stirred at 25 °C for 4 h. The reaction was quenched with water (15 mL), and the aqueous layer was extracted with DCM (3 × 50 mL). The combined organic layers were washed with saturated NaHCO (50 mL) and concentrated to give a crude solid, which was recrystallized from a mixture of DCM / MTBE / n-hexane (1:4:40) to give compound 2-4a (25.52 g, 83.4%) as a white solid. 1H NMR(400MHz,d6-DMSO)11.25(s,1H),8.65-8.60(m,2) H),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),3.9 0-3.78(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,2 H),0.85-0.79(m,3H); 31 P NMR (162MHz, d6-DMSO) 149.43,149.18.

[0316] Compounds 2-4b, 2-4c, 2-4d, and 2-4e were prepared using a procedure similar to that described above for compound 2-4a. Compound 2-4b was obtained as a white solid (25.50 g, 85.4%). 1H NMR(400MHz,d6--DMSO)11.23(s,1H),8.65-8.60(m,2 H),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(162MHz,d6-DMSO)149.43,149.19.

[0317] Compound 2-4c was obtained as a solid product (36.60 g, 66.3%). 1 H NMR(400MHz,d6-DMSO)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); 31P NMR(162MHz,d6-DMSO)149.42,149.17.

[0318] Compound 2-4d was obtained as a solid product (26.60 g, 72.9%). 1 H NMR(400MHz,d6-DMSO)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); 31 P NMR(162MHz,d6-DMSO)149.47,149.22.

[0319] Compound 2-4e was obtained as a white solid (38.10 g, 54.0%). 1H NMR(400MHz,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 (162MHz, 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 monolipids (linear and branched) conjugated to a tetraloop. Post-synthetic conjugation was achieved via an amide coupling reaction. [ka]

[0320] The R1COOH group represents a fatty acid 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. [ka] Synthetic sense 1 and antisense 1 were prepared by solid phase synthesis. Synthesis of conjugated Senses 1a–1i.

[0321] Conjugated Sense 1a was synthesized via a post-syntenic conjugation approach. In 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 Eppendorf tube 2, a solution of oligoSense 1 (10.00 mg, 0.8 μmol) in HO (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 at room temperature using a Thermomixer. After LC-MS analysis showed the reaction was complete, the reaction mixture was diluted with 5 mL of water and purified on a reverse-phase XBridge C18 column using a 5–95% gradient of 100 mM TEAA in ACN and HO. The product fractions were concentrated under reduced pressure using a Genevac. The combined residual solvent was dialyzed against water (1×), saline (1×), and water (3×) using an Amicon® Ultra-15 Centrifugal (3K) membrane. The Amicon membrane was washed with water (3×2 mL), and the combined solvent was then lyophilized to give an amorphous white solid of conjugated Sense 1a (6.43 mg, 64% yield).

[0322] Conjugated Sense 1b-1i were prepared using a procedure similar to that described for the synthesis of conjugated Sense 1a and were obtained in yields ranging from 42% to 69%.

[0323] Annealing of duplexes 1a–1j.

[0324] 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. This solution was used for titration of the conjugated sense and quantification of the amount of double stranded DNA. Based on the calculation of the molar amounts of both the conjugated sense and antisense DNA, the required proportion of antisense 1 was added to the conjugated sense 1a solution. The resulting mixture was stirred at 95°C for 5 minutes and cooled to room temperature. The progress of annealing was monitored by ion-exchange HPLC. Depending on the progress of annealing, additional proportions of antisense 1 were added to complete the annealing with a purity of >95%. The solution was lyophilized to obtain double stranded DNA 1a ​​(C8), the amount of which was calculated based on the molar amount of antisense DNA consumed in annealing.

[0325] Duplexes 1b-1i were prepared using the same procedure as described for the annealing of duplex 1a (C8).

[0326] Schemes 1-2 below show the synthesis of a nicked tetraloop GalXC conjugate with a monolipid on the loop. Post-synthetic conjugation was achieved via a Cu-catalyzed alkyne-azide cycloaddition reaction. [ka] Scheme 1-2

[0327] Sense 1B and antisense 1B were prepared by solid phase synthesis.

[0328] Synthesis of conjugated Sense1j.

[0329] In Eppendorf tube 1, a solution of oligo (10.00 mg, 0.8 μmol) in a 3:1 mixture of DMA / HO (0.5 mL) was treated with lipid linker azide (11.26 mg, 4 μmol). In Eppendorf tube 2, CuBr dimethyl sulfide (1.64 mg, 8 μmol) was dissolved in ACN (0.5 mL). Both solutions were degassed for 10 min by bubbling N through them. The ACN solution of CuBrSMe was then added to tube 1, and the resulting mixture was stirred at 40 °C. After LC-MS analysis showed the reaction was complete, the reaction mixture was diluted with 0.5 M EDTA (2 mL) and dialyzed against water (2×) using an Amicon® Ultra-15 Centrifugal (3K). The crude reaction product was purified on a reverse-phase XBridge C18 column using a 5-95% gradient of 100 mM TEAA in ACN (spiked with 30% IPA) and HO. The product fractions were concentrated under reduced pressure using a Genevac. The combined residual solvents were dialyzed against water (1×), saline (1×), and water (3×) using an Amicon® Ultra-15 Centrifugal (3K). The Amicon membrane was washed with water (3×2 mL), and the combined solvents were lyophilized to give an amorphous white solid of conjugated Sense 1j (6.90 mg, 57% yield).

[0330] Duplex 1j (PEG2K-diacyl C18) was prepared using the same procedure as described for the annealing of duplex 1a (C8).

[0331] Schemes 1-3 below show the synthesis of a nicked tetraloop GalXC conjugate with a di-lipid on the loop using a post-synthetic conjugation approach. [ka] Schemes 1-3 Sense 2 and antisense 2 were prepared by solid phase synthesis.

[0332] Conjugated Sense 2a and 2b were prepared using a procedure similar to that described for the synthesis of conjugated Sense 1a, except that 10 equivalents of lipid, 10 equivalents of HATU, and 20 equivalents of DIPEA were used.

[0333] Duplexes 2a (2XC11) and 2b (2XC22) were prepared using the same procedure as described for the annealing of duplex 1a (C8).

[0334] Schemes 1-4 below show the synthesis of a fully phosphorothioated stem-loop GalXC conjugated with a monolipid using a post-synthetic conjugation approach. [ka] Schemes 1-4 Sense 3 and antisense 3 were prepared by solid phase synthesis.

[0335] Conjugated Sense 3a was prepared using a procedure similar to that described for the synthesis of conjugated Sense 1a and was obtained in 65% yield.

[0336] Duplex 3a (PS-C22) was prepared using the same procedure as described for the annealing of duplex 1a (C8).

[0337] Schemes 1-5 below show the synthesis of short-sense GalXC conjugated with monolipids using a post-synthetic conjugation approach. [ka] Schemes 1-5 Sense 4 and antisense 4 were prepared by solid phase synthesis.

[0338] Conjugated Sense 4a was prepared using a procedure similar to that described for the synthesis of conjugated Sense 1a and was obtained in 74% yield.

[0339] Duplex 4a (SS-C22) was prepared using the same procedure as described for the annealing of duplex 1a (C8).

[0340] Schemes 1-6 below show the synthesis of a nicked tetraloop GalXC conjugated with a triadamantane moiety on the loop using a post-synthetic conjugation approach. [ka] Schemes 1-6 Sense 5 and antisense 5 were prepared by solid phase synthesis.

[0341] Conjugated Sense 5a and 5b were prepared using a procedure similar to that described for the synthesis of conjugated Sense 1a and were obtained in yields ranging from 42% to 73%.

[0342] Duplex 5a (3× adamantane) and duplex 5b (3× acetyladamantane) were prepared using the same procedure as described for the annealing of duplex 1a (C8).

[0343] Schemes 1-7 below show examples of solid-phase synthesis of a nicked tetraloop GalXC with a lipid conjugated on the loop. [ka] Schemes 1-7 Synthesis of conjugated sense 6.

[0344] Conjugated Sense 6 was prepared by solid-phase synthesis using a commercially available oligosynthesizer. Oligonucleotides were synthesized using 2'-modified nucleoside phosphoramidites, such as 2'-F or 2'-OMe, and 2'-diethoxymethanol-linked fatty acid amide nucleoside phosphoramidites. Oligonucleotide synthesis was performed in the 3' to 5' direction on solid support using standard oligonucleotide synthesis protocols. For these, 5-ethylthio-1H-tetrazole (ETT) was used as an activator for the coupling reaction. Iodine solution was used for phosphite triester oxidation. 3-(dimethylaminomethylidene)amino-3H-1,2,4-dithiazole-3-thione (DDTT) was used for phosphorothioate bond formation. The synthesized oligonucleotide was treated with concentrated aqueous ammonium hydroxide for 10 hours. The 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 combined fractions were dialyzed against water (3x), saline (1x), and water (3x) using an Amicon® Ultra-15 Centrifugal (3K) column. The remaining solvent was then lyophilized to obtain the desired conjugated Sense 6.

[0345] Duplex 6 was prepared using the same procedure as described for the annealing of duplex 1a (C8). Scheme 8. Synthesis of nicked tetraloop GalXC with one adamantane unit conjugated on the loop via a post-synthetic conjugation approach. [ka] N=0: Adamantanecarboxylic acid, n=1: Adamantaneacetic acid Schemes 1-8

[0346] Synthesis of conjugated Sense 7a and 7b Conjugated Sense 7a and Sense 7b were obtained using the same or substantially similar methods as for the synthesis of conjugated Sense 5.

[0347] Synthesis of duplexes 7a and 7b Duplex 7a and duplex 7b were obtained using the same or substantially similar methods as for the synthesis of duplex 5.

[0348] Scheme 9. Synthesis of nicked tetraloop GalXC with two adamantane units conjugated on the loop via a post-synthetic conjugation approach. [ka] Schemes 1-9

[0349] Synthesis of conjugated Sense 8a and 8b Conjugated Sense 8a and Sense 8b were obtained using the same or substantially similar methods as for the synthesis of conjugated Sense 5.

[0350] Synthesis of duplexes 8a and 8b Duplex 8a and duplex 8b were obtained using the same or substantially similar methods as for the synthesis of duplex 5.

[0351] Schemes 1-10 below show the synthesis of short sense and short stem-loop GalXCs conjugated with monolipids using a post-synthetic conjugation approach. [ka] Schemes 1-10 Synthesis of Sense 9a

[0352] Conjugated Sense 9a was obtained using the same or substantially similar methods as for the synthesis of conjugated Sense 5.

[0353] Synthesis example of double-stranded 9a Duplex 9a was obtained using the same or substantially similar methods as for the synthesis of duplex 5.

[0354] Schemes 1-11 below show the synthesis of GalXC conjugated with a monolipid at the 5' end using a post-synthetic conjugation approach. [ka] Schemes 1-11

[0355] Synthesis of conjugated Sense 10a Conjugated Sense 10a was obtained using the same or substantially similar methods as for the synthesis of conjugated Sense 5.

[0356] Synthesis example of double strand 10a Duplex 10a was obtained using the same or substantially similar methods as for the synthesis of duplex 5.

[0357] Schemes 1-12a and 1-12b below show the synthesis of blunt-ended GalXC conjugated with a monolipid at the 3' or 5' end using a post-synthetic conjugation approach. [ka] Scheme 1-12a [ka] Scheme 1-12b

[0358] Synthesis of conjugated Sense 11a and 12a Conjugated Sense 11a and 12a were obtained using the same or substantially similar methods as for the synthesis of conjugated Sense 5.

[0359] Synthesis of duplexes 11a and 12a Duplexes 11a and 12a were obtained using the same or substantially similar methods as for the synthesis of duplex 5.

[0360] Conjugate duplexes 8D and 9D were obtained using the same or substantially similar methods as for the synthesis of duplex 5.

[0361] The acyl chain was then conjugated to a nucleic acid inhibitor molecule targeting the STAT3 gene, a gene expressed in the tissue of interest. A passenger chain with a 2'-amine linker [ademA] was used for solid-phase post-conjugation. Different types of lipids were conjugated using the same chemistry to generate a series of conjugates (Figures 1A and 1B). SAR studies were performed to identify lipid conjugates that could be used to deliver payloads to tissues of interest to mediate targeted knockdown.

[0362] Example 3: MDSC cell populations and tissue-specific targeting in tumor-draining lymph nodes. STAT3 is involved in immune suppression, and numerous examples have been reported in the literature. Targeting STAT3 transcription through the RNAi mechanism could potentially overcome challenges in developing 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 (MDSCs). STAT3 sequences were designed in the GalXC format with the described modification pattern, and screening for targeted knockdown in liver tissue was performed in normal CD-1 mice. Eighteen STAT3-GalXC conjugates (Table 1) were administered subcutaneously once at 3 mg / kg. [Table 1-1] [Table 1-2]

[0363] Five days after injection, livers were harvested and subjected to mRNA analysis 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 greater than 85% target knockdown in the liver were selected for further evaluation (Figure 2A). Of these sequences, three were identified as mouse-specific, and one was identified as human-mouse cross-reactive. These four sequences were further screened in CD-1 mice at three different doses (0.3, 1, and 3 mg / kg) to assess dose response. GalXC-STAT3-4110 and 4123 were identified as the most potent sequences after dose-response screening, each with an ED of 0.3 mg / kg. 50 Therefore, these molecules were selected for further study (Figure 2B). For proof-of-concept studies, C18 lipid conjugation was performed on both GalXC-STAT3-4110 or 4123 (Table 2). [Table 2] [Table 3]

[0364] To evaluate the performance of the GalXC-STAT3-C18 conjugate, Pan02 tumors were implanted into nude mice. Once sufficient tumor volume was reached, mice were randomized as previously described. Mice received a single subcutaneous injection of either 25 mg / kg or 50 mg / kg of GalXC-STAT3-C18 4110 and 4123, or PBS. Three days after injection, bulk tumors were harvested and MDSC subsets were isolated. Collectively, MDSCs are characterized by co-expression of the cell surface or mRNA markers CD11b (a marker for myeloid cells of the macrophage lineage) and Gr-1 (a marker for myeloid lineage differentiation antigen), and CD11b + Gr-1 + Gr-1 cells are further composed of two components, Ly6G and Ly6C. MDSCs are divided into two subsets: CD11b+ Ly6G + Ly6C lo Granulocytic MDSCs (G-MDSCs), which are further characterized as CD11b + Ly6G - Ly6C hi The tumor consists of monocytic MDSCs (M-MDSCs), characterized as CD11b+ cells. To isolate CD11b+ cells, a single-cell suspension of tumor cells was generated using a gentle MACS dissociator. The CD11b+ cells in the single-cell suspension were then magnetically labeled with MACS microbeads and passed through a MACS column. The retained labeled cells were then enriched by elution from the column as a positively selected fraction (CD11b MicroBeads UltraPure, mouse kit catalog number 130-126-725). For tumor cell isolation, non-target cells in the cell suspension were magnetically labeled with a cocktail of microbeads and passed through a MACS column. During this process, unwanted labeled cells were retained within the column, and unlabeled target cells (tumor cells) were collected in the flow-through as a pure fraction (Tumor Cell Isolation Kit, human catalog number 130-108-339). After cell isolation, mRNA was analyzed by qPCR (Figures 3A and 3B). Stat3 mRNA levels were reduced by approximately 40% in G-MDSCs and M-MDSCs by GalXC-STAT3-C18-4123. GalXC-STAT3-C18-4110 reduced Stat3 mRNA levels by only 20% in both MDSC subsets. To understand the role of the dose level of the GalXC-STAT3-C18 conjugate in the transport of these molecules to different tissues and cell subsets, a follow-up study was performed using the same tumor model as described above. Pan02 tumor-bearing mice were treated with a single subcutaneous dose of either 50 mg / kg GalXC-STAT3-C18-4123 or PBS, and Stat3 mRNA levels were measured 3 days later. Stat3 knockdown in G-MDSCs was not significantly altered compared to the knockdown observed at the 25 mg / kg dose, but at this same dose level, significant improvement in Stat3 silencing was observed in the M-MDSC subset. In parallel studies performed as described above, Stat3 knockdown was assessed in bulk tumors and TdLNs at day 7 (Figures 4A and 4B). Dose-dependent Stat3 mRNA knockdown was observed in bulk tumors bearing both GalXC-STAT3-C18 sequences. In TdLN, Stat3 mRNA levels were reduced by approximately 60-65% by GalXC-STAT3-C18-4123 and by approximately 25-30% by GalXC-STAT3-C18-4110 at therapeutic doses, suggesting a saturable effect at these dose levels. Based on the data, GalXC-STAT3-C18-4123 was selected for further efficacy evaluation in immunocompetent mice.

[0365] Example 4: STAT3 inhibition reduces PD-L1 levels in MDSCs and mediates acute tumor effects The transcriptional signature of phosphorylated STAT3 positively correlates 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 extrapolate this correlation to STAT3 expressed by MDSCs, isolated populations of MDSCs treated with either PBS or GalXC-STAT3 conjugate were assayed for Pdl1 mRNA. mRNA levels were reduced by approximately 80% in both G- and M-MDSC populations treated with either 25 or 50 mg / kg GalXC-STAT3 (Figure 5A). Pdl1 levels were also dramatically reduced in TdLNs after treatment with GalXC-STAT3 conjugates, particularly GalXC-STAT3-C18-4123 (Figure 5B). These data suggest the possibility of downstream immunoregulation of PD-L1 following STAT3 knockdown.

[0366] In a separate study, Pan02 (a mouse pancreatic syngeneic model) tumor-bearing C57BL / 6 mice (n = 4 per group) were subcutaneously treated with the GalXC-STAT3-C18 conjugate following a split-dose model, with all animals receiving a total dose of 50 mg / kg administered at either two doses of 25 mg / kg or four doses of 12.5 mg / kg. Tumors treated using a split 25 mg / kg dose exhibited acute tumor regression even after the first dose (Figure 6B). After a second dose of 25 mg / kg, tumors from three of four mice regressed to a size too small to be harvested for further processing. The antitumor effect of GalXC-STAT3 treatment was also observed in mice receiving a split 12.5 mg / kg dose (Figure 6A). These data suggest that STAT3-mediated modulation of PD-L1 has an acute and dramatic effect on tumor growth in Pan02 tumor-bearing immunocompetent mice.

[0367] Example 5: Preparation of double-stranded RNAi oligonucleotides Oligonucleotide synthesis and purification The double-stranded RNAi (dsRNA) oligonucleotides described in the preceding examples were chemically synthesized using methods described herein. Generally, dsRNAi oligonucleotides can be prepared using known phosphoramidite synthesis (see, e.g., Hughes and Ellington (2017) Cold Spring Harb Perspect Biol. 9(1):a023812; Beaucage SL, Caruthers MH 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), as well as solid-phase oligonucleotide synthesis methods described for 19-23 mer siRNAs (see, e.g., Scaringe et al. (1990) Nucleic Acids Res. 18:5433-5441 and Usman et al. (See, for example, et al. (1987) J.Am.Chem.Soc.109:7845-7845, and also see, for example, U.S. Patent 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) are synthesized using the dsRNAi oligonucleotide with 19mer core sequence is formatted into a construct with 25mer sense strand and 27mer antisense strand, allowing it to be processed by RNAi mechanism.19mer core sequence is complementary to the region of STAT3 mRNA.

[0368] Individual RNA strands were synthesized and purified by HPLC according to standard methods (Integrated DNA Technologies; Coralville, IA). 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 x 25 cm; Amersham Pharmacia Biotech) using a 15-minute linear step gradient. The gradient varied 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 peaks corresponding to full-length oligonucleotide species were collected, pooled, desalted on a NAP-5 column, and lyophilized.

[0369] 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 nmoles of oligonucleotide was injected into the capillary, run at an electric field of 444 V / cm, and detected by UV absorbance at 260 nm. Denaturing Tris-borate-7M-urea running buffer was purchased from Beckman-Coulter. Oligoribonucleotides that were at least 90% pure, as assessed by CE, were obtained for use in the experiments described below. Compound identity was confirmed by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry on a Voyager DE™ Biospectometry Work Station (Applied Biosystems; Foster City, CA) according to the manufacturer's recommended protocol. The relative molecular masses of all oligomers were obtained, in most cases within 0.2% of the predicted molecular mass.

[0370] Preparation of double strands Single-stranded RNA oligomers were resuspended (e.g., at a concentration of 100 μM) in a duplex buffer consisting of 100 mM potassium acetate, 30 mM HEPES, pH 7.5. Complementary sense and antisense strands were mixed in equimolar amounts to obtain a final solution of, e.g., 50 μM duplex. Samples were heated 5' to 100°C in RNA buffer (IDT) and allowed to cool to room temperature before use. dsRNA oligonucleotides were stored at -20°C. Single-stranded RNA oligomers were lyophilized or stored at -80°C in nuclease-free water.

[0371] Example 6: Generation of STAT3-targeting double-stranded RNAi oligonucleotides Identification of STAT3 mRNA target sequence Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor involved in several developmental and disease functions. To generate RNAi oligonucleotide inhibitors of STAT3 expression, a computer-based algorithm was used to identify suitable STAT3 mRNA target sequences for assaying inhibition of STAT3 expression by the RNAi pathway. The algorithm provided RNAi oligonucleotide guide (antisense) strand sequences, each of which had a region of complementarity to a suitable STAT3 target sequence in human STAT3 mRNA (e.g., SEQ ID NO: 1217; Table 4). Some of the guide strand sequences identified by the algorithm were also complementary to the corresponding STAT3 target sequence in monkey STAT3 mRNA (SEQ ID NO: 1218, Table 4) and / or mouse STAT3 mRNA. STAT3 RNAi oligonucleotides containing a region of complementarity to homologous STAT3 mRNA target sequences with nucleotide sequence similarity are predicted to have the ability to target the homologous STAT3 mRNA. [Table 4]

[0372] RNAi oligonucleotides (formatted as DsiRNA oligonucleotides) were generated as described in Example 5 for in vitro evaluation. Each DsiRNA was generated with the same modification pattern, each with a unique guide strand having a region of complementarity to the STAT3 target sequence identified by SEQ ID NOs: 89-280. Modifications for sense and antisense DsiRNAs included the following (X - any nucleotide; m - 2'-O-methyl modified nucleotide, r - ribosyl modified nucleotide): Sense strand: rXmXrXmXrXrXrXrXrXrXrXrXrXmXrXmXrXrXrXrXrXrXrXXX Antisense strand: mXmXmXmXrXrXrXrXrXrXmXrXmXrXrXrXrXrXrXrXrXrXmXrXmXmXmX

[0373] The ability of each of the modified DsiRNAs in Table 5 to reduce STAT3 mRNA was measured using an in vitro cell-based assay. Briefly, human hepatocytes (Huh7) expressing the endogenous human STAT3 gene were transfected with each of the DsiRNAs listed in Table 5 at 1 nM in separate wells of a multi-well cell culture plate. The cells were maintained for 24 hours after transfection with the modified DsiRNA, and then the amount of remaining STAT3 mRNA from the transfected cells was determined using a TAQMAN®-based qPCR assay. Two qPCR assays, 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), were used to determine STAT3 mRNA levels, measured using a PCR probe conjugated to 6-carboxy-fluorescein (FAM). Each primer pair was assayed for % RNA remaining, as shown in Table 5 and Figure 7. DsiRNAs that resulted in 10% or less STAT3 mRNA remaining in DsiRNA-transfected cells compared to mock-transfected cells were considered "hits." Huh7 cell-based assays evaluating the ability of the DsiRNAs listed in Table 5 to inhibit STAT3 expression identified several candidate DsiRNAs. Taken together, these results show that DsiRNAs designed to target human STAT3 mRNA inhibit STAT3 expression in cells, as determined by a reduction in the amount of STAT3 mRNA in DsiRNA-transfected cells compared to control cells. These results demonstrate that nucleotide sequences containing DsiRNAs are useful for generating RNAi oligonucleotides that inhibit STAT3 expression. Furthermore, these results demonstrate that multiple STAT3 mRNA target sequences are suitable for RNAi-mediated inhibition of STAT3 expression. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9]

[0374] After initial in vitro screening, 48 constructs were selected for dose 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). Of the tested oligonucleotides, 34 sequences were selected for further in vivo testing (Table 6 and Figure 8B). [Table 6-1] [Table 6-2]

[0375] Example 7: RNAi oligonucleotide inhibition of STAT3 in vivo The in vitro screening assay of Example 6 verified the ability of STAT3-targeting DsiRNAs to knock down target mRNA. To confirm the ability of RNAi oligonucleotides to knock down STAT3 in vivo, an HDI mouse model was used. A subset of the DsiRNAs identified in Example 6 was used to generate corresponding double-stranded RNAi oligonucleotides containing nicked tetraloop GalNAc-conjugated structures with a 36-mer passenger strand and a 22-mer guide strand (referred to herein as "GalNAc-conjugated STAT3 oligonucleotides" or "GalNAc-STAT3 oligonucleotides") (Tables 8 and 9). Furthermore, the nucleotide sequences containing the passenger strand and the guide strand have distinctly different patterns of modified nucleotides and phosphorothioate linkages. Three of the tetraloop-containing nucleotides were each conjugated with a GalNAc moiety (CAS#14131-60-3). The modification patterns used are shown below. Pattern 1 Sense strand: 5'mX-S-mX-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'. This hybridizes to: Antisense strand: 5'[Mephosphonate-4O-mX]-S-fX-S-fX-fX-fX-mX-fX-mX-fX-mX-mX-fX-mX-mX-mX-mX-mX-mX-S-mX-S-mX 3'. or as follows: 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][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mX][mX][mX][mX][mX][mX] This hybridizes to: Antisense strand: [Mephosphonate-4O-mXs][fXs][fX][fX][fX][mX][fX][mX][mX][fX][mX][mX][fX][mX][mX][mX][mX][mXs][mXs][mXs] Pattern 2 Sense strand: 5'mX-S-mX-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'. This hybridizes to: Antisense strand: 5'[Mephosphonate-4O-mX]-S-fX-S-fX-fX-mX-fX-mX-fX-mX-mX-fX-mX-mX-mX-mX-mX-mX-mX-S-mX-S-mX 3'. or as follows: 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][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mX][mX][mX][mX][mX][mX] This hybridizes to: Antisense strand: [Mephosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][fX][mX][mX][fX][mX][mX][mX][mX][mXs][mXs][mXs][mX] [Table 7]

[0376] 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, 6-8 week-old female CD-1 mice (n=4-5) were subcutaneously administered the indicated GalNAc-conjugated STAT3 oligonucleotide at a dose of 1 mg / kg formulated in PBS. A control group of mice (n=3-4) received PBS alone. Three days (72 hours) later, mice were hydrodynamically injected (HDI) with a DNA plasmid (25 μg) encoding the complete human STAT3 gene under the control of the ubiquitous cytomegalovirus (CMV) promoter sequence. One day after DNA plasmid transfer, liver samples from HDI mice were harvested. Total RNA from these HDI mice was subjected to qRT-PCR analysis to determine STAT3 mRNA levels, as described in Example 6. mRNA levels were measured for human mRNA. Values ​​were normalized for transfection efficiency using the NeoR gene contained in the DNA plasmid. A benchmark control (STAT3-1388) containing a different modification pattern was used in both assays (sense strand SEQ ID NO: 1100, antisense strand SEQ ID NO: 1190). [Table 8] [Table 9-1] [Table 9-2]

[0377] The results in Figures 9A and 9B show that GalNAc-conjugated STAT3 oligonucleotides designed to target human STAT3 mRNA inhibited human STAT3 mRNA expression in HDI mice, as determined by the reduced amount of human STAT3 mRNA expression in liver samples from HDI mice treated with GalNAc-conjugated STAT3 oligonucleotides compared to control HDI mice treated with PBS only.

[0378] A subset of the GalNAc-conjugated STAT3 oligonucleotides tested in Figures 9A and 9B was further validated in a dose study. Specifically, a dose study was 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 0.1 mg / kg, 0.3 mg / kg, or 1 mg / kg of oligonucleotide. 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 a dose of 1 mg / kg, and STAT3-1286 was able to reduce expression at a dose of 0.3 mg / kg. Overall, HDI studies identified several promising GalNAc-conjugated STAT3 oligonucleotides for inhibiting STAT3 expression in the liver.

[0379] Example 8: Species-specific RNAi oligonucleotide inhibition of STAT3 in vivo To confirm the ability of RNAi oligonucleotides to knockdown STAT3 in vivo, we generated several cross-species and species-specific GalNAc-conjugated STAT3 oligonucleotides. Specifically, we evaluated triple-consensus (human, nonhuman primate, and mouse; Hs / Mf / Mm), human / mouse (Hs / Mm), and human-specific (Hs) oligonucleotides.

[0380] Hs / Mf / Mm and Hs / Mm common Mice expressing endogenous mouse STAT3 in the liver were subcutaneously injected with the GalNAc-conjugated STAT3 oligonucleotides listed in Table 10 at a dose of 3 mg / kg. Five days later, livers were harvested and STAT3 expression was measured. Overall, the study identified several potential Hs / Mf / Mm GalNAc-conjugated STAT3 oligonucleotides for inhibiting STAT3 expression in the liver (FIG. 11). [Table 10-1] [Table 10-2]

[0381] The human / mouse GalNAc-conjugated STAT3 oligonucleotides listed in Table 11 were tested in mice that endogenously express mouse STAT3. As described above, mice were subcutaneously injected with the oligonucleotides at a dose of 3 mg / kg. Five days later, livers were harvested and mouse STAT3 expression was measured. Overall, the study identified several potential Hs / Mm GalNAc-conjugated STAT3 oligonucleotides for inhibiting STAT3 expression in the liver ( FIG. 12 ). [Table 11]

[0382] A subset of the GalNAc-conjugated STAT3 oligonucleotides tested in Figures 11 and 12 was further validated in a dose study. Specifically, a dose study was conducted using 10 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). Mice endogenously expressing mouse STAT3 were subcutaneously injected with 0.3 mg / kg, 1 mg / kg, or 3 mg / kg of oligonucleotide. Livers were harvested 5 days later, and mouse STAT3 expression was measured to determine the effective dose (Figures 13A and 13B). Overall, the endogenous mouse STAT3 expression study identified several potential GalNAc-conjugated STAT3 oligonucleotides for inhibiting mouse STAT3 expression in the liver.

[0383] Hs specific Human-specific GalNAc-conjugated STAT3 oligonucleotides were evaluated using the HDI model described in Example 7. Specifically, 6- to 8-week-old female CD-1 mice (n = 4-5) were subcutaneously administered the indicated GalNAc-conjugated STAT3 oligonucleotides (Table 12) formulated in PBS at a dose of 1 mg / kg. A control group of mice (n = 3-4) received PBS alone. Three days later (72 hours), mice were hydrodynamically injected (HDI) with a DNA plasmid (25 μg) encoding the complete human STAT3 gene under the control of the ubiquitous cytomegalovirus (CMV) promoter sequence. One day after DNA plasmid transfer, liver samples from HDI mice were harvested. Total RNA from these HDI mice was subjected to qRT-PCR analysis to determine STAT3 mRNA levels. [Table 12-1] [Table 12-2]

[0384] The results in Figure 14 show that GalNAc-conjugated STAT3 oligonucleotides designed to target human STAT3 mRNA inhibited human STAT3 mRNA expression in HDI mice, as determined by the reduced amount of human STAT3 mRNA expression in liver samples from HDI mice treated with GalNAc-conjugated STAT3 oligonucleotides compared to control HDI mice treated with PBS only.

[0385] A subset of the GalNAc-conjugated STAT3 oligonucleotides tested in Figure 14 was further validated in a dose study. Specifically, a dose study was conducted 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 oligonucleotide. Livers were harvested 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 approximately 75%, thereby identifying several potential GalNAc-conjugated STAT3 oligonucleotides for inhibiting STAT3 expression in the liver. The best two sequences from Figure 23 and the best sequence from Figure 28 will be tested in a final HDI screen (Figure 16).

[0386] Example 9: Specific STAT3 inhibition by GalNAc-conjugated STAT3 oligonucleotides The specificity of GalNAc-conjugated STAT3 oligonucleotides, which inhibit STAT3 but not family members (e.g., STAT1), was determined. Specifically, Huh7 cells expressing endogenous STAT1 were treated with 0.05 nM, 0.3 nM, or 1 nM GalNAc-conjugated STAT3 oligonucleotides (STAT3-721, STAT3-1286, and STAT3-1388) for 24 hours using lipofectamine as the transfection agent. The percent (%) of remaining mRNA was measured compared to a mock control (PBS; no lipofectamine or siRNA) and UTR (untransfected; treated with lipofectamine but not siRNA) (Table 13 and Figure 17). STAT3 721 and 1286 did not downregulate human STAT1, whereas STAT3 1388 did (Table 13). The oligonucleotides did not downregulate STAT1 expression, demonstrating specificity for STAT3 and limited off-target effects on STAT1. [Table 13]

[0387] Example 10: STAT3 inhibition in combination with checkpoint inhibition significantly improves anti-tumor efficacy To evaluate the performance of the GalXC-STAT3-C18 conjugate as a single agent or in combination with a checkpoint inhibitor, anti-PD-L1 mAb, Pan02 tumors (2 × 10 6 The cells were transplanted into 6-8 week old C57BL / 6 mice, and 300-400 mm 3When volume reached this level, mice were randomized. Mice received a single 25 mg / kg subcutaneous dose of GalXC-STAT3-C18-4123 either as a single agent or in combination with 10 mg / kg (ip) of anti-PD-L1 mAb (anti-mouse PD-L1 mAb (B7-H1), clone 10F.9G2). Mice were initially administered two doses at 3-day intervals, followed two weeks later by two additional doses at 3-day intervals [(q3d x 2) x 2]. A control group was treated with GalXC-placebo either 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 final dose, the same dosing regimen was repeated. Tumor size was measured twice weekly throughout the study period.

[0388] As shown in Figure 18A, 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 treatment, but continued to grow despite receiving a second dose. The group receiving the combination of GalXC-STAT3 and mAb showed significantly greater tumor regression than single-agent treatment. This demonstrates that combination therapy with checkpoint inhibitors can achieve improved antitumor efficacy.

[0389] In another study, Pan02 tumors (2 × 10 6 The cells were transplanted into 6- to 8-week-old C57BL / 6 mice, and 300-400 mm 3Upon reaching this volume, mice were administered two doses of GalXC-placebo (25 mg / kg) three days apart (days 42 and 45). Two weeks later, mice received two doses of 25 mg / kg of GalXC-STAT3-C18-4123 subcutaneously three days apart in combination with 10 mg / kg (ip) of anti-PD-L1 mAb (anti-mouse PD-L1 mAb (B7-H1), clone 10F.9G2). Tumor size was measured twice weekly throughout the study period. Figure 18B shows the regression of tumor size following administration of GalXC-STAT3 / PD-L1 mAb combination treatment, further demonstrating that combination therapy can achieve improved anti-tumor efficacy.

[0390] Example 11: Correlation between treatment with a combination of GalXC-STAT3 and PD-L1 mAb and tumor immunophenotype To confirm whether the combination efficacy pattern coincided with the tumor immunophenotype, tumor types with distinct phenotypes were selected for implantation in mice. The tumor types selected included Pan02 (Figure 18A, checkpoint-resistant tumor), 4T1 (triple-negative breast, checkpoint-resistant tumor), MC-38 (colon carcinoma, 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) were grown in C57BL / 6 mice (7-8 weeks old), and 4T1 tumors (7-8 weeks old) were grown in Balb / c mice. When each tumor reached a sufficient tumor volume, they were separated and subjected to treatment as described in Example 5. (4T1 tumors were treated with subcutaneous GalXC-STAT3-C18-4123, GalXC-STAT3-C18-4123, or GalXC-placebo in combination with anti-PD-L1 mAb or single-agent GalXC-placebo, as shown in Figure 19A. Each dose was treated three times at 3-day intervals (q3d x 3). Tumor volumes were measured twice weekly throughout the study period. MC-38 tumors and Hepa1-6 tumors were treated with subcutaneous anti-PD-L1 mAb or single-agent GalXC-placebo, as shown in Figure 19B and Figure 19C.) Patients were treated with subcutaneous GalXC-STAT3-C18-4123 with mAb or single-agent GalXC-placebo, GalXC-STAT3-C18-4123, or GalXC-placebo with mAb (two doses, 3 days apart for 2 weeks).

[0391] Combination treatment was expected to result in tumors with little or no CD8+ T cell infiltration in the TME and a larger population of MDSCs (CD8 低 MDSC 高 ), which showed synergistic efficacy in resistant tumor types (Figures 18 and 19A). Combination treatment also demonstrated that tumors had slightly higher levels of CD8+ T cell infiltration and a large population of MDSCs (CD8 中 MDSC 高) showed improved efficacy compared to checkpoint monotherapy in partially sensitive tumors (Figure 19B). Interestingly, the combination treatment also showed improved efficacy in sensitive tumors (CD8 高 MDSC 高 ) resulted in complete regression (Figure 19C). Tumors with higher levels of CD8+ T cell infiltration and MDSCs were completely eradicated when treated with the combination of GalXC-STAT3-C18-4123 + anti-PD-L1 mAb.

[0392] Example 12: Therapy-mediated tumor regression and generation of tumor-specific memory To assess whether the combination treatment demonstrating complete regression also led to the generation of memory T cells in treated mice, tumors that completely regressed in Figure 19C were rechallenged with Hep1-6 cells (2e6 cells) on the opposite flank of the mice on day 51. As shown in Figure 20, even after rechallenge, all mice were tumor-free and survived for the duration that the mice were retained and maintained (approximately 2 months). These data demonstrate the potent therapeutic antitumor efficacy of the combination treatment, leading to long-term immunological memory.

[0393] Example 13: CD8+ T cell-mediated combination efficacy is also perforin-dependent To assess whether the efficacy mediated by the combination treatment was CD8+ T cell-mediated, efficacy studies were performed using 4T1 tumors (2e6 cells) in immunocompetent Balb / c mice (7-8 weeks old) as described in Example 7. The experiments were repeated in immunodeficient nude mice bearing 4T1 tumors. As shown in Figure 21A, there was synergistic efficacy with combination treatment of GalXC-STAT3-C18-4123 and anti-PD-L1 mAb in tumor-bearing immunocompetent mice, but no efficacy was observed in nude mice bearing 4T1 tumors (Figure 21B), suggesting an important role for CD8+ T cells in mediating antitumor efficacy. To confirm that efficacy was mediated by cytotoxic CD8+ T cells, tumor samples from the final time point of the study were stained for perforin. As shown in Figure 22, the significantly larger population of perforin-positive cells in tumors receiving the combination treatment indicates that the T cells involved in mediating efficacy were cytotoxic in nature.

[0394] Example 14: Effect of combination treatment on spontaneous tumor metastasis in a highly metastatic tumor model To evaluate whether combination treatment reduces metastasis in a spontaneous metastatic tumor model, 4T1 tumors (2e6 cells / mouse) were implanted into Balb / c mice (7-8 weeks old) as described in Example 7. Tumors were grown to 500 mm 3When tumors reached a size of approximately 100 mm, 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 administered at 50 mg / kg, and anti-PD-L1 mAb administered at 10 mg / kg), and the tumors were monitored for tumor growth. Twelve days after the final dose, mice were sacrificed and the lungs were photographed. As shown in Figure 23, lungs from single-agent or placebo treatments showed tumor metastases throughout all organs, whereas mice receiving combination treatment (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 in the figure, but also reduced spontaneous metastases to the lungs. The same experiment was repeated in nude mice, which is also shown in Figure 23. All lungs, including those from mice receiving the combination treatment, had tumor metastases, further confirming the role of CD8+ T cells in anti-tumor efficacy.

[0395] Example 15: Treatment-mediated immunomodulation in tumors To understand how combined treatment with 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 are associated with MC38 (CD8 中 MDSC 中 / 高) have a similar profile. When 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 (q3d x 2, 25 mg / kg or 10 mg / kg). Seven days after the final dose, tumors were harvested, homogenized, and nanostring analysis was performed (mRNA extracted from paraffin-embedded samples was analyzed for mRNA expression via the ncounter® Mouse Pancancer IO 360™ Panel (Nanostring Technologies, Seattle, WA)).

[0396] Analysis showed that genes that are inherently inhibitory (checkpoint, STAT3-mediated genes, inhibitory cytokines / chemokines, angiogenesis and matrix remodeling-related genes) were reduced and genes that favor T cell activation (genes involved in T cell migration, activation, memory, and cytotoxicity) were increased after combination treatment compared to single-agent, GalXC-placebo, or anti-PD-L1 mAb treatment, suggesting that combination treatment shifts the TME from inhibitory to favorable for T cell infiltration (Figure 24).

[0397] Example 16: STAT3 Oligonucleotides for the Treatment of Disease To investigate the efficacy of STAT3 oligonucleotide alone or in combination with anti-PD-L1 mAb, subjects are administered a STAT3 oligonucleotide or a STAT3 oligonucleotide in combination with anti-PD-L1 mAb. Specifically, subjects are administered a STAT3 oligonucleotide, where 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 (shown in Figure 25), as shown below. 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] This 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] (Key provided in Table 7)

[0398] The STAT3 oligonucleotide described above is administered alone or in combination with an anti-PD-L1 antibody. The STAT3 oligonucleotide is administered before, simultaneously with, or after the administration of the anti-PD-L1 antibody. After administration, tumor size and subject survival rate are measured. Example 17: STAT3 inhibition in combination with checkpoint inhibition significantly improves anti-tumor efficacy

[0399] Studies were conducted in three different mouse tumor models: B16F10, Pan02, and MC-38. B16F10 and Pan02 are mouse melanoma and pancreatic cancer models that are thought to be resistant to checkpoint inhibitors (CPIs) due to the presence of a large population of myeloid-derived suppressor cells (MDSCs) and little or no CD8+ T cells in the tumor microenvironment (TME). The MC-38 tumor model is a mouse colon cancer model known to be partially sensitive to CPIs and harbors moderate levels of MDSCs 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 NOs: 1222 and 1145, "DCR-STAT3") in preclinical models of CPI resistance and sensitivity.

[0400] Mice were administered either GalXC-placebo or DCR-STAT3 with or without anti-PD-L1 mouse antibody. GalXC-placebo and DCR-STAT3 were administered subcutaneously at 25 mg / kg, and anti-PD-L1 antibody was administered intraperitoneally at 10 mg / kg. In the B16F10 tumor model, doses were administered on days 6 (6 days after tumor implantation), 9, and 12. In the Pan02 model, doses were administered on days 38 (38 days after tumor implantation), 41, 48, and 51. In the MC-38 tumor model, doses were administered on days 5 (5 days after tumor implantation), 8, 12, and 15.

[0401] In the CPI-resistant B16F10 model, after three doses of DCR-STAT3 or DCR-STAT3 + anti-PD-L1 antibody, tumor size at day 13 was reduced by 36% (p<0.01) and 64% (p<0.0001), respectively, compared with the GalXC-placebo group. Anti-PD-L1 antibody alone had no effect on tumor growth, with tumors growing to the same size as in the GalXC-placebo group. Tumor size in the combination group (DCR-STAT3 + anti-PD-L1 antibody) was reduced by 43% (p<0.05) compared with DCR-STAT3 alone and by 64% (p<0.0001) compared with the anti-PD-L1 antibody alone. A similar pattern was observed in the Pan02 study. After four doses of DCR-STAT3 or DCR-STAT3 plus anti-PD-L1 antibody, tumor size at 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 had no effect on tumor growth, with tumors growing to the same size as in the GalXC-placebo group. Tumor size in the combination group was reduced by 59% (p<0.01) compared to DCR-STAT3 alone and 76% (p<0.0001) compared to the anti-PD-L1 antibody alone, suggesting that DCR-STAT3 is active as a single agent and that single-agent activity was further enhanced when combined with the antibody in this CPI-resistant tumor model.

[0402] In the CPI partially sensitive MC-38 model, after four doses of anti-PD-L1 antibody or DCR-STAT3, tumor size at day 18 was reduced by 57% (p<0.01) and 45% (p<0.01), respectively, compared with the GalXC placebo group. At day 18, after four doses of DCR-STAT3 + anti-PD-L1 antibody, tumor size was reduced by 95% (p<0.0001) compared with the GalXC-placebo group. Compared with anti-PD-L1 antibody or DCR-STAT3, tumor size was reduced by 89% (p<0.05) and 91% (p<0.01) in the DCR-STAT3 + anti-PD-L1 antibody group, respectively. While administration of either anti-PD-L1 antibody or DCR-STAT3 was active as single agents, the combination of both further enhanced the efficacy of either single agent.

[0403] Data from these three experiments provide evidence that DCR-STAT3 was active as a CPI-resistant tumor in which anti-PD-L1 antibodies were inactive and that DCR-STAT3, when combined with anti-PD-L1 antibodies, resulted in synergistic anti-tumor activity. DCR-STAT3 was also active in CPI-sensitive tumors in which anti-PD-L1 also showed single-agent activity, and when used in combination, the majority of tumors regressed by nearly 100%. [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5] [Table 14-6] [Table 14-7] [Table 14-8] [Table 14-9] [Table 14-10] [Table 14-11] [Table 14-12] [Table 14-13] Table 14-14 Table 14-15 Table 14-16 Table 14-17 Table 14-18 Table 14-19 Table 14-20 Table 14-21 Table 14-22 Table 14-23 Table 14-24 Table 14-25 Table 14-26 Table 14-27 Table 14-28 Table 14-29 Table 14-30 Table 14-31 Table 14-32 Table 14-33 Table 14-34 Table 14-35 Table 14-36 Table 14-37 Table 14-38 Table 14-39 Table 14-40 Table 14-41 Table 14-42 Table 14-43 Table 14-44 Table 14-45 Table 14-46 Table 14-47 Table 14-48 Table 14-49 Table 14-50 Table 14-51 Table 14-52 Table 14-53 Table 14-54 Table 14-55 Table 14-56 Table 14-57 Table 14-58 Table 14-59 Table 14-60 Table 14-61 Table 14-62 Table 14-63 Table 14-64 Table 14-65 Table 14-66 Table 14-67 Table 14-68 Table 14-69 Table 14-70 Table 14-71 Table 14-72 Table 14-73 Table 14-74 Table 14-75 Table 14-76 Table 14-77 Table 14-78 Table 14-79 Table 14-80

Claims

1. 1. An oligonucleotide for reducing STAT3 expression, the oligonucleotide comprising an antisense strand 15-30 nucleotides in length and a sense strand 15-40 nucleotides in length, the sense strand and the antisense strand forming a double-stranded region, the antisense strand having a region of complementarity to a target sequence of STAT3 set forth in SEQ ID NO: 140, and the sense strand comprising at least one lipid moiety conjugated to the 5'-terminal nucleotide of the sense strand.

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

3. 3. The oligonucleotide of claim 1 or 2, wherein the antisense strand is 21 to 27 nucleotides in length, optionally the antisense strand is 22 nucleotides in length.

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

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

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

7. The oligonucleotide of any one of claims 1 to 6, wherein the region of complementarity to STAT3 is at least 19 contiguous nucleotides in length.

8. The oligonucleotide of any one of claims 1 to 7, wherein the region of complementarity to STAT3 is at least 21 contiguous nucleotides in length.

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

965.

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

875.

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

12. 1. An oligonucleotide for reducing STAT3 expression, the oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand is 21 to 27 nucleotides in length and has a region of complementarity to a target sequence of STAT3 set forth in SEQ ID NO: 140, the sense strand comprises at its 3' end a stem-loop described as S1-L-S2, wherein S1 is complementary to S2 and L forms a loop 3 to 5 nucleotides in length between S1 and S2, the antisense strand and the sense strand form a double-stranded structure at least 19 nucleotides in length, and 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 of 19 to 30 nucleotides in length, said antisense strand comprising a nucleotide sequence comprising a region of complementarity to a STAT3 mRNA target sequence, said region of complementarity being set forth in SEQ ID NO: 140; and (ii) a sense strand 19 to 50 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the sense strand comprises a lipid moiety conjugated to the 5'-terminal nucleotide of the sense strand; A double-stranded oligonucleotide, wherein the antisense strand and the sense strand are separate strands forming an asymmetric duplex region with an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand.

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

15. The oligonucleotide of any one of claims 11 to 14, wherein L comprises the sequence written as GAAA.

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

17. 17. The oligonucleotide of claim 16, wherein the antisense strand and the sense strand form a double-stranded region 25 nucleotides in length, and optionally, the duplex is 20 nucleotides in length.

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

19. The oligonucleotide of any one of claims 1 to 18, wherein the oligonucleotide comprises at least one modified nucleotide.

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

21. 21. The oligonucleotide of 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-P-d-arabinonucleic acid.

22. 22. The oligonucleotide of any one of claims 19 to 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. 23. The oligonucleotide of 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. 24. The oligonucleotide of any one of claims 19 to 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. 25. The oligonucleotide of any one of claims 19 to 24, wherein the sense strand comprises 36 nucleotides having positions 1 to 36 from 5' to 3', and positions 8 to 11 comprise 2'-fluoro modifications.

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

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

28. The oligonucleotide of any one of claims 1 to 27, wherein the oligonucleotide comprises at least one modified internucleotide bond.

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

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

31. 30. The oligonucleotide of claim 29, wherein the antisense strand comprises 22 nucleotides having positions 1 to 22 from 3' to 5', and the antisense strand comprises phosphorothioate linkages between positions 1 and 2, between positions 2 and 3, between positions 3 and 4, between positions 20 and 21, and between positions 21 and 22.

32. 32. The oligonucleotide of claim 29, wherein the sense strand comprises a phosphorothioate bond between positions 1 and 2 of the sense strand, and the antisense strand comprises 22 nucleotides having positions 1 to 22 from 3' to 5', and the antisense strand comprises phosphorothioate bonds between positions 1 and 2, between positions 2 and 3, between positions 3 and 4, between positions 20 and 21, and between positions 21 and 22.

33. The oligonucleotide of any one of claims 1 to 32, wherein the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analog.

34. 34. The oligonucleotide of claim 33, wherein the phosphate analog is an oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

35. The oligonucleotide of any one of claims 1 to 34, wherein the lipid moiety is a saturated or unsaturated fatty acid moiety.

36. 36. The oligonucleotide of any one of claims 1 to 35, wherein the lipid moiety is a saturated fatty acid moiety ranging in size from C10 to C24 in length.

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

38. 38. The oligonucleotide of claim 37, wherein the C16 saturated fatty acid moiety is represented by: 【Chemistry 1】

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

40. 40. The oligonucleotide of claim 39, wherein the C18 saturated fatty acid moiety is represented by: 【Chemistry 2】

41. The oligonucleotide of any one of claims 1 to 40, wherein the lipid moiety is selected from: 【Chemistry 3-1】 【Chemistry 3-2】

42. 42. The oligonucleotide of any one of claims 1 to 41, wherein the lipid moiety is conjugated to the 2' carbon of the ribose ring of the 5' terminal nucleotide.

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

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

45. The oligonucleotide of any one of claims 1 to 44, 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.

46. A double-stranded oligonucleotide for reducing STAT3 expression, wherein the oligonucleotide comprises a sense strand comprising the sequence set forth in SEQ ID NO: 1222 and an antisense strand comprising the sequence set forth in SEQ ID NO: 1145; A double-stranded oligonucleotide, wherein the sense strand and the antisense strand form an asymmetric duplex region that is 20 nucleotides in length and has a two-nucleotide overhang at the 3' end of the antisense strand.

47. 46. ​​The oligonucleotide of any one of claims 1 to 45, wherein the region of complementarity is perfectly complementary to the STAT3 target sequence.

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

49. 49. The oligonucleotide of claim 48, wherein the region of complementarity contains no more than four mismatches to the STAT3 target sequence.

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

51. 51. The oligonucleotide of any one of claims 1 to 50, wherein the oligonucleotide is a Dicer substrate that, upon endogenous Dicer processing, produces a double-stranded nucleic acid of 19 to 21 nucleotides in length that is capable of reducing STAT3 mRNA expression in mammalian cells.

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

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

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

55. 55. The method of claim 54, comprising administering to the subject a PD-L1 inhibitor.

56. 1. A method of treating cancer in a subject who has received or is receiving a PD-L1 inhibitor, comprising:

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

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

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

59. 59. The method of claim 58, comprising administering to the subject a PD-L1 inhibitor.

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

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

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

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

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

66. 66. The method of claim 65, wherein the inflammatory tumor microenvironment comprises infiltrating T cells.

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

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

69. the anti-PDL1 antibody is FAZ053, 69. The method of claim 68, wherein the therapeutic agent is selected from atezolizumab, avelumab, durvalumab, and BMS-936559.

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

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

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

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

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

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

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

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

78. 53. A kit comprising an oligonucleotide, an optional pharmaceutically acceptable carrier, and a package insert containing instructions for administering said oligonucleotide to a subject in need thereof who has received or is receiving a PD-L1 inhibitor, wherein said oligonucleotide is the oligonucleotide of any one of claims 1 to 52.

79. 53. A kit comprising a PD-L1 inhibitor, an optional pharmaceutically acceptable carrier, and a package insert containing instructions for administering said inhibitor to a subject in need thereof who has received or is receiving an oligonucleotide, wherein said oligonucleotide is the oligonucleotide of any one of claims 1 to 52.

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

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

82. 1. A method of determining responsiveness in a subject with cancer who has undergone or is undergoing treatment, the method comprising detecting the presence of myeloid-derived suppressor cells (MDSCs) or a marker of MDSC activity in a biological sample from the subject, wherein the treatment is administration of an oligonucleotide that targets STAT3, and wherein a reduction in MDSCs or a reduction in the marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment.

83. 1. A method for determining responsiveness in a subject with cancer who has undergone or is undergoing treatment, comprising: (i) obtaining a biological sample from said subject; (ii) detecting the presence of MDSCs or a marker of MDSC activity in said biological sample; The method, wherein the treatment is administration of an oligonucleotide targeting STAT3, and a reduction in MDSCs or a reduction in a marker of MDSC activity in the biological sample indicates that the subject is responding to the treatment.

84. 84. The method of claim 82 or 83, wherein detecting comprises determining an amount of MDSCs or an amount of a marker of MDSC activity.

85. 85. The method of any one of claims 82 to 84, wherein the reduction of MDSCs or markers of MDSC activity is relative to the amount or level of MDSCs or markers of MDSC activity before treatment of the subject.

86. 85. The method of any one of claims 82 to 84, wherein said reduction of MDSCs or markers of MDSC activity is relative to the amount or level of MDSCs or markers of MDSC activity in a population of patients who did not receive said treatment.

87. 87. The method of any one of claims 82 to 86, wherein said reduction of MDSCs or markers of MDSC activity is based on the amount or level of MDSCs or markers of MDSC activity in a population of patients that responded to said treatment.

88. The method of any one of claims 82 to 87, wherein the MDSCs are granulocytic MDSCs (G-MDSCs).

89. The method of any one of claims 82 to 88, wherein the MDSCs are monocytic MDSCs (M-MDSCs).

90. The method of any one of claims 82 to 89, wherein the MDSCs express Argl.

91. The method of any one of claims 82 to 90, wherein the MDSCs express IDO.

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

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

94. 94. The method of any one of claims 82 to 93, wherein responding to treatment comprises a reduction or inhibition of tumor growth and / or tumor size.

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

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