Double-stranded oligonucleotides and methods of use

Abstract

The present disclosure provides, inter alia, compounds comprising a phosphorothioated CpG oligodeoxynucleotide linked to a DNA oligonucleotide that is hybridized to another DNA oligonucleotide containing a constrained nucleotide, pharmaceutical compositions containing the compounds, and methods of treating cancer using the compounds.

Description

[Background technology]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 414,160, filed October 7, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] STATEMENT OF RIGHTS TO INVENTIONS MADE IN FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under Grant No. W81XWH-19-1-0852 awarded by the Medical Research and Development Command and Grant No. R01 CA215183 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] (Reference to a "Sequence Listing", table, or computer program listing appendix submitted as an ASCII file) The sequence listing entitled "048440-850001WO-Sequence Listing", written in XML format and having 92,140 bytes, created on October 3, 2023, is incorporated by reference.

[0004] The immune system can function as an extrinsic tumor suppressor. However, the microenvironment of established tumors typically contains tumor-specific CD8+ cells, in addition to an excess of suppressive regulatory T cells and myeloid-derived suppressor cells that promote tumor immune evasion. +Tumors are characterized by a lack of T cells. Myeloid cells and other immune cells in the tumor microenvironment also produce growth factors and angiogenesis / metastasis factors important for tumor progression. Signal Transducer and Activator of Transcription 3 (STAT3) is a key oncogenic molecule. The integration of these processes in the tumor microenvironment is highly dependent on the oncogenic transcription factor STAT3. In particular, STAT3 plays a key role in mediating tumor immune evasion. Activated STAT3 in myeloid cells inhibits the expression of numerous immunostimulatory molecules associated with Th1-type responses while promoting the production of several important immunosuppressive and angiogenic factors. Furthermore, by mediating the signaling of certain cytokines and growth factors, particularly IL-6, STAT3 activation in myeloid cells activates STAT3 in tumor cells, enhancing their proliferation and survival. There is a medical need for novel compounds and methods for treating cancer that are safe and effective. The present disclosure is directed to these and other important objectives. Summary of the Invention

[0005] Provided herein are compounds comprising a phosphorothioated CpG oligodeoxynucleotide (ODN) linked to a first DNA oligonucleotide, the first DNA oligonucleotide hybridizing to a second DNA oligonucleotide, the first DNA oligonucleotide comprising from about 5 to about 70 unmodified nucleotides, and the second DNA oligonucleotide comprising (i) from about 5 to about 70 nucleotides, and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end. In embodiments, the constrained nucleotide is an LNA-, cMOE-, or cET-modified nucleotide.

[0006] Provided herein are methods for treating cancer by administering to a patient an effective amount of a compound described herein. In embodiments, the method further comprises administering to the patient an effective amount of an immune checkpoint inhibitor, e.g., a PD-1 pathway inhibitor.

[0007] These and other embodiments and aspects of the present disclosure are described herein. [Brief explanation of the drawings]

[0008] [Figure 1A] The serum stability and activity of double-stranded antisense oligonucleotides are shown. Figure 1A: Double-stranded STAT3 ASO exhibits higher serum stability than single-stranded oligonucleotides. Oligonucleotides were resolved by 15% PAGE before and after incubation in 50% human serum. Referring to Figures 1A-1D, the STAT3 ASO is SEQ ID NO: 4, the STAT3 dsASO is SEQ ID NO: 3 hybridized to SEQ ID NO: 4, and the AR-ASO is 5'accAAGTTTCTTCagc (SEQ ID NO: 35, bold lowercase letters indicate LNA-modified nucleotides) (described by Zhang et al., Nucleic Acids Research, 46(7):3612-3624 (2018)), a ds-AR-ASO (SEQ ID NO: 35) hybridized to 5'gctgaagaaacttggt (SEQ ID NO: 36). [Figure 1B]Figure 1B-1C shows serum stability and activity of double-stranded antisense oligonucleotides. Double-stranded STAT3-specific ASOs (Figures 1B, 1D) or androgen receptor (AR)-specific ASOs show comparable or improved knockdown of their respective targets compared with their respective single-stranded ASOs within 48 hours after transfection (Figures 1B-1C; 100 ng each) or spontaneous / gymnotic uptake (Figure 1D; 1 μM) in human LnCaP prostate cancer, A431 carcinoma, U118 glioma, or murine GL261 glioma cells. Referring to Figures 1A to 1D, the STAT3 ASO is SEQ ID NO: 4, the STAT3 dsASO is SEQ ID NO: 3 hybridized to SEQ ID NO: 4, and the AR-ASO is 5'accAAGTTTCTTCagc (SEQ ID NO: 35, bold lowercase letters indicate LNA-modified nucleotides) (described by Zhang et al., Nucleic Acids Research, 46(7):3612-3624 (2018)), which is a ds-AR-ASO (SEQ ID NO: 35) hybridized to 5'gctgaagaaacttggt (SEQ ID NO: 36). [Figure 1C]Figure 1B-1C shows serum stability and activity of double-stranded antisense oligonucleotides. Double-stranded STAT3-specific ASOs (Figures 1B, 1D) or androgen receptor (AR)-specific ASOs show comparable or improved knockdown of their respective targets compared with their respective single-stranded ASOs within 48 hours after transfection (Figures 1B-1C; 100 ng each) or spontaneous / gymnotic uptake (Figure 1D; 1 μM) in human LnCaP prostate cancer, A431 carcinoma, U118 glioma, or murine GL261 glioma cells. Referring to Figures 1A to 1D, the STAT3 ASO is SEQ ID NO: 4, the STAT3 dsASO is SEQ ID NO: 3 hybridized to SEQ ID NO: 4, and the AR-ASO is 5'accAAGTTTCTTCagc (SEQ ID NO: 35, bold lowercase letters indicate LNA-modified nucleotides) (described by Zhang et al., Nucleic Acids Research, 46(7):3612-3624 (2018)), which is a ds-AR-ASO (SEQ ID NO: 35) hybridized to 5'gctgaagaaacttggt (SEQ ID NO: 36). [Figure 1D]Figure 1B-1C shows serum stability and activity of double-stranded antisense oligonucleotides. Double-stranded STAT3-specific ASOs (Figures 1B, 1D) or androgen receptor (AR)-specific ASOs show comparable or improved knockdown of their respective targets compared with their respective single-stranded ASOs within 48 hours after transfection (Figures 1B-1C; 100 ng each) or spontaneous / gymnotic uptake (Figure 1D; 1 μM) in human LnCaP prostate cancer, A431 carcinoma, U118 glioma, or murine GL261 glioma cells. Referring to Figures 1A to 1D, the STAT3 ASO is SEQ ID NO: 4, the STAT3 dsASO is SEQ ID NO: 3 hybridized to SEQ ID NO: 4, and the AR-ASO is 5'accAAGTTTCTTCagc (SEQ ID NO: 35, bold lowercase letters indicate LNA-modified nucleotides) (described by Zhang et al., Nucleic Acids Research, 46(7):3612-3624 (2018)), which is a ds-AR-ASO (SEQ ID NO: 35) hybridized to 5'gctgaagaaacttggt (SEQ ID NO: 36). [Figure 2A]

[0033] Figure 2A shows the design, serum stability, and in vitro activity of CpG-conjugated double-stranded STAT3 antisense oligonucleotides. Figure 2A: Schematic diagram of double-stranded CpG-STAT3 dsASO. Referring to Figures 2A-2D, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via a portion of formula (A), which is hybridized to SEQ ID NO: 4, the CpG-passenger is SEQ ID NO: 15 linked to SEQ ID NO: 3 via a portion of formula (A), the STAT3 ASO is SEQ ID NO: 4, and (in this figure and throughout this application) the portion of formula (A) is

[0009] [ka] is. [Figure 2B]Figure 2B shows the design, serum stability, and in vitro activity of CpG-conjugated double-stranded STAT3 antisense oligonucleotides. Figure 2B: Serum stability of double-stranded CpG-STAT3 dsASO compared to each individual oligonucleotide strand (ASO alone and CpG-passenger strand). Oligonucleotides were incubated in 50% human serum at 37°C for the indicated times and then resolved by 15% PAGE. Referring to Figures 2A-2D, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via a portion of formula (A), which is hybridized to SEQ ID NO: 4; the CpG-passenger is SEQ ID NO: 15 linked to SEQ ID NO: 3 via a portion of formula (A); the STAT3 ASO is SEQ ID NO: 4; and (in this figure and throughout this application) the portion of formula (A) is

[0010] [ka] is. [Figure 2C] Figures 2C-2D show the design, serum stability, and in vitro activity of CpG-conjugated double-stranded STAT3 antisense oligonucleotides. Figures 2C-2D: Double-stranded CpG-STAT3 dsASOs retain comparable target knockdown potency and kinetics to the STAT3 ASO alone. Human A431 cells (Figure 2C) and mouse GL261 cells (Figure 2D) were incubated with 1 μM of the indicated oligonucleotides for various periods of time. Western blot analysis of STAT3 protein normalized to the level of β-actin, which was used as a control. Referring to Figures 2A-2D, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via a portion of formula (A), which is hybridized to SEQ ID NO: 4; the CpG-passenger is SEQ ID NO: 15 linked to SEQ ID NO: 3 via a portion of formula (A); the STAT3 ASO is SEQ ID NO: 4; and (in this figure and throughout this application) the portion of formula (A) is

[0011] [ka] is. [Figure 2D] Figures 2C-2D show the design, serum stability, and in vitro activity of CpG-conjugated double-stranded STAT3 antisense oligonucleotides. Figures 2C-2D: Double-stranded CpG-STAT3 dsASOs retain comparable target knockdown potency and kinetics to the STAT3 ASO alone. Human A431 cells (Figure 2C) and mouse GL261 cells (Figure 2D) were incubated with 1 μM of the indicated oligonucleotides for various periods of time. Western blot analysis of STAT3 protein normalized to the level of β-actin, which was used as a control. Referring to Figures 2A-2D, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via a portion of formula (A), which is hybridized to SEQ ID NO: 4; the CpG-passenger is SEQ ID NO: 15 linked to SEQ ID NO: 3 via a portion of formula (A); the STAT3 ASO is SEQ ID NO: 4; and (in this figure and throughout this application) the portion of formula (A) is

[0012] [ka] is. [Figure 3A]We demonstrate that CpG-STAT3 dsASOs exhibit better efficacy in target gene knockdown than single-stranded CpG-STAT3 ASO variants in different human and mouse target cells. Human prostate cancer cells (DU145, LAPC4, and ENZR42D) (Figures 3A-C) were incubated with 1 μM of the indicated 2'-O-methyl or LNA-modified oligonucleotides for 72 hours. Total STAT3 protein levels were quantified by Western blotting using β-actin normalization and are shown as quantification of band intensity. Referring to Figures 3A to 3F, the CpG-STAT3 ASO(2'OMe) is SEQ ID NO: 15 linked to SEQ ID NO: 33 via the portion of formula (A), the CpG-passenger (LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), the STAT3 ASO(LNA) is SEQ ID NO: 4, the CpG-STAT3 dsASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the portion of formula (A). [Figure 3B]We demonstrate that CpG-STAT3 dsASOs exhibit better efficacy in target gene knockdown than single-stranded CpG-STAT3 ASO variants in different human and mouse target cells. Human prostate cancer cells (DU145, LAPC4, and ENZR42D) (Figures 3A-C) were incubated with 1 μM of the indicated 2'-O-methyl or LNA-modified oligonucleotides for 72 hours. Total STAT3 protein levels were quantified by Western blotting using β-actin normalization and are shown as quantification of band intensity. Referring to Figures 3A to 3F, the CpG-STAT3 ASO(2'OMe) is SEQ ID NO: 15 linked to SEQ ID NO: 33 via the portion of formula (A), the CpG-passenger (LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), the STAT3 ASO(LNA) is SEQ ID NO: 4, the CpG-STAT3 dsASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the portion of formula (A). [Figure 3C]We demonstrate that CpG-STAT3 dsASOs exhibit better efficacy in target gene knockdown than single-stranded CpG-STAT3 ASO variants in different human and mouse target cells. Human prostate cancer cells (DU145, LAPC4, and ENZR42D) (Figures 3A-C) were incubated with 1 μM of the indicated 2'-O-methyl or LNA-modified oligonucleotides for 72 hours. Total STAT3 protein levels were quantified by Western blotting using β-actin normalization and are shown as quantification of band intensity. Referring to Figures 3A to 3F, the CpG-STAT3 ASO(2'OMe) is SEQ ID NO: 15 linked to SEQ ID NO: 33 via the portion of formula (A), the CpG-passenger (LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), the STAT3 ASO(LNA) is SEQ ID NO: 4, the CpG-STAT3 dsASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the portion of formula (A). [Figure 3D]This shows that the CpG-STAT3 dsASO exhibits better efficacy in target gene knockdown than single-stranded CpG-STAT3 ASO variants in different human and mouse target cells. Human (U251) cells (Figure 3D) were incubated with 1 μM of the indicated 2'-O-methyl or LNA-modified oligonucleotides for 72 hours. Total STAT3 protein levels were quantified by Western blotting using β-actin normalization and are shown as quantification of band intensity. Referring to Figures 3A to 3F, the CpG-STAT3 ASO(2'OMe) is SEQ ID NO: 15 linked to SEQ ID NO: 33 via the portion of formula (A), the CpG-passenger (LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), the STAT3 ASO(LNA) is SEQ ID NO: 4, the CpG-STAT3 dsASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the portion of formula (A). [Figure 3E]We show that CpG-STAT3 dsASOs exhibit better efficacy in target gene knockdown than single-stranded CpG-STAT3 ASO variants in different human and mouse target cells. Mouse (GL261) glioma cells (Figure 3E) were incubated with 1 μM of the indicated 2'-O-methyl or LNA-modified oligonucleotides for 72 hours. Total STAT3 protein levels were quantified by Western blotting using β-actin normalization and are shown as quantification of band intensity. Referring to Figures 3A to 3F, the CpG-STAT3 ASO(2'OMe) is SEQ ID NO: 15 linked to SEQ ID NO: 33 via the portion of formula (A), the CpG-passenger (LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), the STAT3 ASO(LNA) is SEQ ID NO: 4, the CpG-STAT3 dsASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the portion of formula (A). [Figure 3F]We demonstrate that CpG-STAT3 dsASOs exhibit better efficacy in target gene knockdown than single-stranded CpG-STAT3 ASO variants in different human and mouse target cells. Mouse macrophages (RAW264.7) (Figure 3F) were incubated with 1 μM of the indicated 2'-O-methyl or LNA-modified oligonucleotides for 72 hours. Total STAT3 protein levels were quantified by Western blotting using β-actin normalization and are shown as quantification of band intensity. Referring to Figures 3A to 3F, the CpG-STAT3 ASO(2'OMe) is SEQ ID NO: 15 linked to SEQ ID NO: 33 via the portion of formula (A), the CpG-passenger (LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), the STAT3 ASO(LNA) is SEQ ID NO: 4, the CpG-STAT3 dsASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the portion of formula (A). [Figure 4A] A comparison of time- and dose-dependent ASO uptake by human and mouse target cells is provided. Human cells (U251, LN229, T98G) (Figures 4A-4C) were incubated with fluorescently labeled double-stranded CpG-STAT3 dsASOCy3, single-stranded CpG-STAT3 ssASOCy3, or unconjugated STAT3 ssASOCy3 at concentrations of 100 nM or 500 nM for 1 hour or 4 hours. Oligonucleotide internalization was assessed using flow cytometry. Referring to Figures 4A-3F, the STAT3 ASO is SEQ ID NO:4, the CpG-STAT3 dsASO is SEQ ID NO:15 linked to SEQ ID NO:3 via the moiety of formula (A), SEQ ID NO:3 is hybridized to SEQ ID NO:4, and the CpG-STAT3 ssASO is SEQ ID NO:15 linked to SEQ ID NO:4 via the moiety of formula (A). [Figure 4B]A comparison of time- and dose-dependent ASO uptake by human and mouse target cells is provided. Human cells (U251, LN229, T98G) (Figures 4A-4C) were incubated with fluorescently labeled double-stranded CpG-STAT3 dsASOCy3, single-stranded CpG-STAT3 ssASOCy3, or unconjugated STAT3 ssASOCy3 at concentrations of 100 nM or 500 nM for 1 hour or 4 hours. Oligonucleotide internalization was assessed using flow cytometry. Referring to Figures 4A-3F, the STAT3 ASO is SEQ ID NO:4, the CpG-STAT3 dsASO is SEQ ID NO:15 linked to SEQ ID NO:3 via the moiety of formula (A), SEQ ID NO:3 is hybridized to SEQ ID NO:4, and the CpG-STAT3 ssASO is SEQ ID NO:15 linked to SEQ ID NO:4 via the moiety of formula (A). [Figure 4C] A comparison of time- and dose-dependent ASO uptake by human and mouse target cells is provided. Human cells (U251, LN229, T98G) (Figures 4A-4C) were incubated with fluorescently labeled double-stranded CpG-STAT3 dsASOCy3, single-stranded CpG-STAT3 ssASOCy3, or unconjugated STAT3 ssASOCy3 at concentrations of 100 nM or 500 nM for 1 hour or 4 hours. Oligonucleotide internalization was assessed using flow cytometry. Referring to Figures 4A-3F, the STAT3 ASO is SEQ ID NO:4, the CpG-STAT3 dsASO is SEQ ID NO:15 linked to SEQ ID NO:3 via the moiety of formula (A), SEQ ID NO:3 is hybridized to SEQ ID NO:4, and the CpG-STAT3 ssASO is SEQ ID NO:15 linked to SEQ ID NO:4 via the moiety of formula (A). [Figure 4D]A comparison of time- and dose-dependent ASO uptake by human and mouse target cells is provided. Mouse (GL261, K-luc) glioma cells (Figures 4D-4E) were incubated with fluorescently labeled double-stranded CpG-STAT3 dsASOCy3, single-stranded CpG-STAT3 ssASOCy3, or unconjugated STAT3 ssASOCy3 at concentrations of 100 nM or 500 nM for 1 hour or 4 hours. Oligonucleotide internalization was assessed using flow cytometry. Referring to Figures 4A-4F, the STAT3 ASO is SEQ ID NO: 4, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the moiety of formula (A). [Figure 4E] A comparison of time- and dose-dependent ASO uptake by human and mouse target cells is provided. Mouse (GL261, K-luc) glioma cells (Figures 4D-4E) were incubated with fluorescently labeled double-stranded CpG-STAT3 dsASOCy3, single-stranded CpG-STAT3 ssASOCy3, or unconjugated STAT3 ssASOCy3 at concentrations of 100 nM or 500 nM for 1 hour or 4 hours. Oligonucleotide internalization was assessed using flow cytometry. Referring to Figures 4A-4F, the STAT3 ASO is SEQ ID NO: 4, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the moiety of formula (A). [Figure 4F]A comparison of time- and dose-dependent ASO uptake by human and mouse target cells is provided. Mouse (GL261, K-luc) glioma cells (Figures 4D-4E) were incubated with fluorescently labeled double-stranded CpG-STAT3 dsASOCy3, single-stranded CpG-STAT3 ssASOCy3, or unconjugated STAT3 ssASOCy3 at concentrations of 100 nM or 500 nM for 1 hour or 4 hours. Oligonucleotide internalization was assessed using flow cytometry. Referring to Figures 4A-4F, the STAT3 ASO is SEQ ID NO: 4, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the moiety of formula (A). [Figure 5A] Figure 5 shows a comparison of time- and dose-dependent ASO uptake by primary human immune cells. Human PBMCs were incubated with fluorescently labeled double-stranded CpG-STAT3 dsASOCy3, single-stranded CpG-STAT3 ssASOCy3, or unconjugated STAT3 ssASOCy3 at concentrations of 100 nM or 500 nM for 1 hour or 4 hours. Oligonucleotide internalization was assessed using flow cytometry. Histograms showing oligonucleotide uptake by primary human CD1c+ myeloid dendritic cells (mDCs) (Figure 5A) are shown. Referring to Figures 5A-5E, the STAT3 ASO is SEQ ID NO: 4, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the moiety of formula (A). [Figure 5B]Figure 5B shows a comparison of time- and dose-dependent ASO uptake by primary human immune cells. Human PBMCs were incubated with fluorescently labeled double-stranded CpG-STAT3 dsASOCy3, single-stranded CpG-STAT3 ssASOCy3, or unconjugated STAT3 ssASOCy3 at concentrations of 100 nM or 500 nM for 1 hour or 4 hours. Oligonucleotide internalization was assessed using flow cytometry. Figure 5B shows a histogram showing oligonucleotide uptake by CD303+ plasmacytoid DCs (pDCs). Referring to Figures 5A-5E, the STAT3 ASO is SEQ ID NO: 4, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the moiety of formula (A). [Figure 5C] Figure 5 shows a comparison of time- and dose-dependent ASO uptake by primary human immune cells. Human PBMCs were incubated with fluorescently labeled double-stranded CpG-STAT3 dsASOCy3, single-stranded CpG-STAT3 ssASOCy3, or unconjugated STAT3 ssASOCy3 at concentrations of 100 nM or 500 nM for 1 hour or 4 hours. Oligonucleotide internalization was assessed using flow cytometry. Histograms showing oligonucleotide uptake by CD14+ monocytes (Figure 5C) are shown. Referring to Figures 5A-5E, the STAT3 ASO is SEQ ID NO: 4, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the moiety of formula (A). [Figure 5D]Figure 5 shows a comparison of time- and dose-dependent ASO uptake by primary human immune cells. Human PBMCs were incubated with fluorescently labeled double-stranded CpG-STAT3 dsASOCy3, single-stranded CpG-STAT3 ssASOCy3, or unconjugated STAT3 ssASOCy3 at concentrations of 100 nM or 500 nM for 1 hour or 4 hours. Oligonucleotide internalization was assessed using flow cytometry. A histogram showing oligonucleotide uptake by CD19+ B cells (Figure 5D) is shown. Referring to Figures 5A-5E, the STAT3 ASO is SEQ ID NO: 4, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the moiety of formula (A). [Figure 5E] Figure 5A shows a comparison of time- and dose-dependent ASO uptake by primary human immune cells. Human PBMCs were incubated with fluorescently labeled double-stranded CpG-STAT3 dsASOCy3, single-stranded CpG-STAT3 ssASOCy3, or unconjugated STAT3 ssASOCy3 at concentrations of 100 nM or 500 nM for 1 hour or 4 hours. Oligonucleotide internalization was assessed using flow cytometry. Figure 5E shows a histogram showing oligonucleotide uptake by CD3+ T cells. Referring to Figures 5A-5E, the STAT3 ASO is SEQ ID NO: 4, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the moiety of formula (A). [Figure 6A]Figure 6 shows a comparison of dose-dependent ASO uptake by primary mouse immune cells. Mouse splenocytes were incubated with fluorescently labeled double-stranded CpG-STAT3 dsASOCy3, single-stranded CpG-STAT3 ssASOCy3, or unconjugated STAT3 ssASOCy3 at concentrations of 100 nM or 500 nM for 4 hours. Oligonucleotide internalization was assessed using flow cytometry. Histograms showing oligonucleotide uptake by CD11b+CD11c- macrophages (Figure 6A) are shown. Referring to Figures 6A-6D, the STAT3 ASO is SEQ ID NO: 4, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the moiety of formula (A). [Figure 6B] Figure 6 shows a comparison of dose-dependent ASO uptake by primary mouse immune cells. Mouse splenocytes were incubated with fluorescently labeled double-stranded CpG-STAT3 dsASOCy3, single-stranded CpG-STAT3 ssASOCy3, or unconjugated STAT3 ssASOCy3 at concentrations of 100 nM or 500 nM for 4 hours. Oligonucleotide internalization was assessed using flow cytometry. Histograms showing oligonucleotide uptake by CD11b+CD11c+ dendritic cells (DCs) (Figure 6B) are shown. Referring to Figures 6A-6D, the STAT3 ASO is SEQ ID NO: 4, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the moiety of formula (A). [Figure 6C]Figure 6 shows a comparison of dose-dependent ASO uptake by primary mouse immune cells. Mouse splenocytes were incubated with fluorescently labeled double-stranded CpG-STAT3 dsASOCy3, single-stranded CpG-STAT3 ssASOCy3, or unconjugated STAT3 ssASOCy3 at concentrations of 100 nM or 500 nM for 4 hours. Oligonucleotide internalization was assessed using flow cytometry. Histograms showing oligonucleotide uptake by CD19+ B cells (Figure 6C) are shown. Referring to Figures 6A-6D, the STAT3 ASO is SEQ ID NO: 4, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the moiety of formula (A). [Figure 6D] Figure 6 shows a comparison of dose-dependent ASO uptake by primary mouse immune cells. Mouse splenocytes were incubated with fluorescently labeled double-stranded CpG-STAT3 dsASOCy3, single-stranded CpG-STAT3 ssASOCy3, or unconjugated STAT3 ssASOCy3 at concentrations of 100 nM or 500 nM for 4 hours. Oligonucleotide internalization was assessed using flow cytometry. A histogram showing oligonucleotide uptake by CD3+ T cells (Figure 6D) is shown. Referring to Figures 6A-6D, the STAT3 ASO is SEQ ID NO: 4, the CpG-STAT3 dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), SEQ ID NO: 3 is hybridized to SEQ ID NO: 4, and the CpG-STAT3 ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the moiety of formula (A). [Figure 7A]Biodistribution of intratumorally injected CpG-STAT3 ASO in the mouse brain is shown. C57 / BL6 mice bearing intracranial GL261 glioma tumors were injected with 1 mg / kg of single- or double-chain CpG-STAT3 ASOCy3 or unconjugated STAT3 ASOCy3. After 3 or 5 days, mice were euthanized and brains were harvested. The percentage of Cy3+ M1-like macrophages (CD11b+ CD45high CD86+) (Figure 7A) was assessed using flow cytometry in single-cell suspensions from tumor-free and tumor-bearing brain hemispheres. Results are from an experiment with 3 to 4 mice per group; mean ± SEM. Referring to Figures 7A to 7F, CpG-STAT3ASO(2'OMe) is sequence number 15 linked to sequence number 33 via the portion of formula (A), STAT3ASO-h / m#3(LNA) is sequence number 4, and CpG-STAT3ASO h / m#3(LNA) is sequence number 15 linked to sequence number 4 via the portion of formula (A). [Figure 7B] Biodistribution of intratumorally injected CpG-STAT3 ASO in the mouse brain is shown. C57 / BL6 mice bearing intracranial GL261 glioma tumors were injected with 1 mg / kg of single-chain or double-chain CpG-STAT3 ASOCy3 or unconjugated STAT3 ASOCy3. After 3 or 5 days, mice were euthanized and brains were harvested. The percentage of M2-like macrophages (CD11b+CD45highCD206+) (Figure 7B) was assessed using flow cytometry in single-cell suspensions from tumor-free and tumor-bearing brain hemispheres. Results are from an experiment with 3 to 4 mice per group; mean ± SEM. Referring to Figures 7A to 7F, CpG-STAT3ASO(2'OMe) is sequence number 15 linked to sequence number 33 via the portion of formula (A), STAT3ASO-h / m#3(LNA) is sequence number 4, and CpG-STAT3ASO h / m#3(LNA) is sequence number 15 linked to sequence number 4 via the portion of formula (A). [Figure 7C]Biodistribution of intratumorally injected CpG-STAT3 ASO in the mouse brain is shown. C57 / BL6 mice bearing intracranial GL261 glioma tumors were injected with 1 mg / kg of single- or double-stranded CpG-STAT3 ASOCy3 or unconjugated STAT3 ASOCy3. After 3 or 5 days, mice were euthanized and brains were harvested. The percentage of M1-like microglia (CD11b+CD45lowCD86+) (Figure 7C) was assessed using flow cytometry in single-cell suspensions from tumor-free and tumor-bearing brain hemispheres. Results are from an experiment with 3-4 mice per group; mean ± SEM. Referring to Figures 7A to 7F, CpG-STAT3ASO(2'OMe) is sequence number 15 linked to sequence number 33 via the portion of formula (A), STAT3ASO-h / m#3(LNA) is sequence number 4, and CpG-STAT3ASO h / m#3(LNA) is sequence number 15 linked to sequence number 4 via the portion of formula (A). [Figure 7D] Biodistribution of intratumorally injected CpG-STAT3 ASO in the mouse brain is shown. C57 / BL6 mice bearing intracranial GL261 glioma tumors were injected with 1 mg / kg of single- or double-stranded CpG-STAT3 ASOCy3 or unconjugated STAT3 ASOCy3. After 3 or 5 days, mice were euthanized and brains were harvested. The percentage of M2-like microglia (CD11b+CD45highCD206+) (Figure 7D) was assessed using flow cytometry in single-cell suspensions from tumor-free and tumor-bearing brain hemispheres. Results are from an experiment with 3-4 mice per group; mean ± SEM. Referring to Figures 7A to 7F, CpG-STAT3ASO(2'OMe) is sequence number 15 linked to sequence number 33 via the portion of formula (A), STAT3ASO-h / m#3(LNA) is sequence number 4, and CpG-STAT3ASO h / m#3(LNA) is sequence number 15 linked to sequence number 4 via the portion of formula (A). [Figure 7E]Biodistribution of intratumorally injected CpG-STAT3 ASO in the mouse brain is shown. C57 / BL6 mice bearing intracranial GL261 glioma tumors were injected with 1 mg / kg of single- or double-stranded CpG-STAT3 ASOCy3 or unconjugated STAT3 ASOCy3. After 3 or 5 days, mice were euthanized and brains were harvested. The percentage of DCs (CD11b+CD11c+) (Figure 7E) was assessed using flow cytometry in single-cell suspensions from tumor-free and tumor-bearing brain hemispheres. Results are from an experiment with 3-4 mice per group; mean ± SEM. Referring to Figures 7A to 7F, CpG-STAT3ASO(2'OMe) is sequence number 15 linked to sequence number 33 via the portion of formula (A), STAT3ASO-h / m#3(LNA) is sequence number 4, and CpG-STAT3ASO h / m#3(LNA) is sequence number 15 linked to sequence number 4 via the portion of formula (A). [Figure 7F] Biodistribution of intratumorally injected CpG-STAT3 ASO in the mouse brain is shown. C57 / BL6 mice bearing intracranial GL261 glioma tumors were injected with 1 mg / kg of single- or double-stranded CpG-STAT3 ASOCy3 or unconjugated STAT3 ASOCy3. After 3 or 5 days, mice were euthanized and brains were harvested. The percentage of MDSCs (CD11b+Gr1+) (Figure 7F) was assessed using flow cytometry in single-cell suspensions from tumor-free and tumor-bearing brain hemispheres. Results are from an experiment using 3 to 4 mice per group; mean ± SEM. Referring to Figures 7A to 7F, CpG-STAT3ASO(2'OMe) is sequence number 15 linked to sequence number 33 via the portion of formula (A), STAT3ASO-h / m#3(LNA) is sequence number 4, and CpG-STAT3ASO h / m#3(LNA) is sequence number 15 linked to sequence number 4 via the portion of formula (A). [Figure 8A]These results demonstrate that double-stranded CpG-STAT3 dsASOs are safer than single-stranded CpG-STAT3 ssASOs and are well tolerated by naive mice. Figure 8A: Determination of the maximum tolerated dose (MTD) of CpG-STAT3 ASO variants in naive mice. C57BL / 6 mice were intracranially injected with 0.1, 0.3, or 1 mg / kg CpG-STAT3 dsASO or CpG-STAT3 ssASO twice weekly and euthanized after 2 weeks. Each treatment group was weighed; mean ± SEM values ​​are shown. Referring to Figures 8A-8F, the dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the formula (A) moiety, which is hybridized to SEQ ID NO: 4. The ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the formula (A) moiety. [Figure 8B] The double-stranded CpG-STAT3 dsASO is safer than the single-stranded CpG-STAT3 ssASO and is well tolerated by naive mice. Figures 8B-8C: Mouse behavior, assessed using the acute tolerability scoring system (ATSS) (Figure 8B), was significantly altered by a low dose of single-stranded CpG-STAT3 ASO but not by double-stranded CpG-STAT3 ASO (Figure 8C). Assessment of acute CNS toxicity based on mouse phenotypic behavior 1 and 4 hours after IC injection of CpG-STAT3 ASO. Referring to Figures 8A-8F, the dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the formula (A) moiety, which is hybridized to SEQ ID NO: 4, and the ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the formula (A) moiety. [Figure 8C]The double-stranded CpG-STAT3 dsASO is safer than the single-stranded CpG-STAT3 ssASO and is well tolerated by naive mice. Figures 8B-8C: Mouse behavior, assessed using the acute tolerability scoring system (ATSS) (Figure 8B), was significantly altered by a low dose of single-stranded CpG-STAT3 ASO but not by double-stranded CpG-STAT3 ASO (Figure 8C). Assessment of acute CNS toxicity based on mouse phenotypic behavior 1 and 4 hours after IC injection of CpG-STAT3 ASO. Referring to Figures 8A-8F, the dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the formula (A) moiety, which is hybridized to SEQ ID NO: 4, and the ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the formula (A) moiety. [Figure 8D] The results show that double-stranded CpG-STAT3 dsASOs are safer than single-stranded CpG-STAT3 ssASOs and are well tolerated by naive mice. Figures 8D-8F: Single-stranded CpG-STAT3 ASOs induce significant changes in white blood cells and platelets in treated mice, whereas double-stranded CpG-STAT3 ASOs do not. Hematological parameters, including red blood cells (RBCs), white blood cells (WBCs), and platelets (PLTs), were evaluated in blood collected from mice treated as described above. The gray area indicates the expected normal range for each parameter. Results from an experiment using a total of 3-4 mice from each group; mean ± SEM. Referring to Figures 8A-8F, the dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the formula (A) moiety, which is hybridized to SEQ ID NO: 4, and the ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the formula (A) moiety. [Figure 8E]The results show that double-stranded CpG-STAT3 dsASOs are safer than single-stranded CpG-STAT3 ssASOs and are well tolerated by naive mice. Figures 8D-8F: Single-stranded CpG-STAT3 ASOs induce significant changes in white blood cells and platelets in treated mice, whereas double-stranded CpG-STAT3 ASOs do not. Hematological parameters, including red blood cells (RBCs), white blood cells (WBCs), and platelets (PLTs), were evaluated in blood collected from mice treated as described above. The gray area indicates the expected normal range for each parameter. Results from an experiment using a total of 3-4 mice from each group; mean ± SEM. Referring to Figures 8A-8F, the dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the formula (A) moiety, which is hybridized to SEQ ID NO: 4, and the ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the formula (A) moiety. [Figure 8F] The results show that double-stranded CpG-STAT3 dsASOs are safer than single-stranded CpG-STAT3 ssASOs and are well tolerated by naive mice. Figures 8D-8F: Single-stranded CpG-STAT3 ASOs induce significant changes in white blood cells and platelets in treated mice, whereas double-stranded CpG-STAT3 ASOs do not. Hematological parameters, including red blood cells (RBCs), white blood cells (WBCs), and platelets (PLTs), were evaluated in blood collected from mice treated as described above. The gray area indicates the expected normal range for each parameter. Results from an experiment using a total of 3-4 mice from each group; mean ± SEM. Referring to Figures 8A-8F, the dsASO is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the formula (A) moiety, which is hybridized to SEQ ID NO: 4, and the ssASO is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the formula (A) moiety. [Figure 9A]We demonstrate that local administration of both LNA-modified single-stranded and double-stranded CpG-STAT3 ASOs produces similar direct antitumor effects against intracranial human glioma xenografts in immunocompromised mice. NSG mice bearing human U251 gliomas were intratumorally / IC injected every other day with 1 mg / kg of 2'-O-methyl or LNA-modified CpG-STAT3 ASOs, as indicated by the red arrows. Figure 9A: Tumor progression and occasional extracranial spread were monitored using bioluminescent imaging (BLI). Referring to Figures 9A and 9B, the CpG-STAT3 ASO(2'OMe) is sequence number 15 linked to sequence number 34 via the portion of formula (A), the CpG-STAT3 dsASO(LNA) is sequence number 43 linked to sequence number 1 via the portion of formula (A), which is hybridized to sequence number 2, and the CpG-STAT3-ssASO(LNA) is sequence number 43 linked to sequence number 2 via the portion of formula (A). [Figure 9B] Local administration of both LNA-modified single-stranded and double-stranded CpG-STAT3 ASOs results in similar direct antitumor effects against intracranial human glioma xenografts in immunocompromised mice. NSG mice bearing human U251 gliomas were intratumorally / IC injected every other day with 1 mg / kg of 2'-O-methyl or LNA-modified CpG-STAT3 ASOs, as indicated by the red arrows. Figure 9B: Both forms of CpG-STAT3 ASO significantly prolonged animal survival. Kaplan-Meier survival curves (n = 5-6) are shown. Referring to Figures 9A and 9B, the CpG-STAT3 ASO(2'OMe) is sequence number 15 linked to sequence number 34 via the portion of formula (A), the CpG-STAT3 dsASO(LNA) is sequence number 43 linked to sequence number 1 via the portion of formula (A), which is hybridized to sequence number 2, and the CpG-STAT3-ssASO(LNA) is sequence number 43 linked to sequence number 2 via the portion of formula (A). [Figure 10A]Local administration of single-stranded and double-stranded CpG-STAT3 ASOs improved animal survival and induced immune activation in immunocompetent mice. C57BL / 6 mice bearing established GL261 gliomas were intratumorally / IC injected twice weekly with 0.25 mg / kg of 2'-O-methyl or LNA-modified CpG-STAT3 ASO. Figure 10A: CpG-STAT3 ASO treatment significantly improved animal survival. Kaplan-Meier survival curves (n = 10-11) are shown. Referring to Figures 10A to 10I, CpG-STAT3 ASO(2'OMe) is SEQ ID NO: 15 linked to SEQ ID NO: 33 via the portion of formula (A), CpG-STAT3 dsASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), which is hybridized to SEQ ID NO: 4, and CpG-STAT3-ssASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the portion of formula (A). [Figure 10B]We demonstrate that local administration of single- and double-stranded CpG-STAT3 ASOs improves animal survival and induces immune activation in immunocompetent mice. C57BL / 6 mice bearing established GL261 gliomas were intratumorally / IC injected twice weekly with 0.25 mg / kg of 2'-O-methyl or LNA-modified CpG-STAT3 ASO. Figures 10B-10F: All three types of CpG-STAT3 ASO injections induced maturation / activation of intratumoral DCs, macrophages, and microglia, but reduced the number of tumor-associated M2 macrophages and resting microglia as assessed by flow cytometry. The percentages of mature DCs (CD11b+CD11c+MHCII+CD80+), mature macrophages (CD11b+CD11c-MHCII+CD80+MHCII+CD80+), M2-like macrophages (CD11b+F4 / 80+CD206+), activated microglia (CD11b+CD45lowMHCII+CX3CR1+), and resting microglia (CD11b+CD45lowMHCII-CX3CR1+) are shown in Figure 10A-10C. Referring to 01, CpG-STAT3 ASO(2'OMe) is SEQ ID NO: 15 linked to SEQ ID NO: 33 via a portion of formula (A), CpG-STAT3 dsASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via a portion of formula (A), which is hybridized to SEQ ID NO: 4, and CpG-STAT3-ssASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 4 via a portion of formula (A). [Figure 10C]We demonstrate that local administration of single- and double-stranded CpG-STAT3 ASOs improves animal survival and induces immune activation in immunocompetent mice. C57BL / 6 mice bearing established GL261 gliomas were intratumorally / IC injected twice weekly with 0.25 mg / kg of 2'-O-methyl or LNA-modified CpG-STAT3 ASO. Figures 10B-10F: All three types of CpG-STAT3 ASO injections induced maturation / activation of intratumoral DCs, macrophages, and microglia, but reduced the number of tumor-associated M2 macrophages and resting microglia as assessed by flow cytometry. The percentages of mature DCs (CD11b+CD11c+MHCII+CD80+), mature macrophages (CD11b+CD11c-MHCII+CD80+MHCII+CD80+), M2-like macrophages (CD11b+F4 / 80+CD206+), activated microglia (CD11b+CD45lowMHCII+CX3CR1+), and resting microglia (CD11b+CD45lowMHCII-CX3CR1+) are shown in Figure 10A-10C. Referring to 01, CpG-STAT3 ASO(2'OMe) is SEQ ID NO: 15 linked to SEQ ID NO: 33 via a portion of formula (A), CpG-STAT3 dsASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via a portion of formula (A), which is hybridized to SEQ ID NO: 4, and CpG-STAT3-ssASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 4 via a portion of formula (A). [Figure 10D]We demonstrate that local administration of single- and double-stranded CpG-STAT3 ASOs improves animal survival and induces immune activation in immunocompetent mice. C57BL / 6 mice bearing established GL261 gliomas were intratumorally / IC injected twice weekly with 0.25 mg / kg of 2'-O-methyl or LNA-modified CpG-STAT3 ASO. Figures 10B-10F: All three types of CpG-STAT3 ASO injections induced maturation / activation of intratumoral DCs, macrophages, and microglia, but reduced the number of tumor-associated M2 macrophages and resting microglia as assessed by flow cytometry. The percentages of mature DCs (CD11b+CD11c+MHCII+CD80+), mature macrophages (CD11b+CD11c-MHCII+CD80+MHCII+CD80+), M2-like macrophages (CD11b+F4 / 80+CD206+), activated microglia (CD11b+CD45lowMHCII+CX3CR1+), and resting microglia (CD11b+CD45lowMHCII-CX3CR1+) are shown in Figure 10A-10C. Referring to 01, CpG-STAT3 ASO(2'OMe) is SEQ ID NO: 15 linked to SEQ ID NO: 33 via a portion of formula (A), CpG-STAT3 dsASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via a portion of formula (A), which is hybridized to SEQ ID NO: 4, and CpG-STAT3-ssASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 4 via a portion of formula (A). [Figure 10E]We demonstrate that local administration of single- and double-stranded CpG-STAT3 ASOs improves animal survival and induces immune activation in immunocompetent mice. C57BL / 6 mice bearing established GL261 gliomas were intratumorally / IC injected twice weekly with 0.25 mg / kg of 2'-O-methyl or LNA-modified CpG-STAT3 ASO. Figures 10B-10F: All three types of CpG-STAT3 ASO injections induced maturation / activation of intratumoral DCs, macrophages, and microglia, but reduced the number of tumor-associated M2 macrophages and resting microglia as assessed by flow cytometry. The percentages of mature DCs (CD11b+CD11c+MHCII+CD80+), mature macrophages (CD11b+CD11c-MHCII+CD80+MHCII+CD80+), M2-like macrophages (CD11b+F4 / 80+CD206+), activated microglia (CD11b+CD45lowMHCII+CX3CR1+), and resting microglia (CD11b+CD45lowMHCII-CX3CR1+) are shown in Figure 10A-10C. Referring to 01, CpG-STAT3 ASO(2'OMe) is SEQ ID NO: 15 linked to SEQ ID NO: 33 via a portion of formula (A), CpG-STAT3 dsASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via a portion of formula (A), which is hybridized to SEQ ID NO: 4, and CpG-STAT3-ssASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 4 via a portion of formula (A). [Figure 10F]We demonstrate that local administration of single- and double-stranded CpG-STAT3 ASOs improves animal survival and induces immune activation in immunocompetent mice. C57BL / 6 mice bearing established GL261 gliomas were intratumorally / IC injected twice weekly with 0.25 mg / kg of 2'-O-methyl or LNA-modified CpG-STAT3 ASO. Figures 10B-10F: All three types of CpG-STAT3 ASO injections induced maturation / activation of intratumoral DCs, macrophages, and microglia, but reduced the number of tumor-associated M2 macrophages and resting microglia as assessed by flow cytometry. The percentages of mature DCs (CD11b+CD11c+MHCII+CD80+), mature macrophages (CD11b+CD11c-MHCII+CD80+MHCII+CD80+), M2-like macrophages (CD11b+F4 / 80+CD206+), activated microglia (CD11b+CD45lowMHCII+CX3CR1+), and resting microglia (CD11b+CD45lowMHCII-CX3CR1+) are shown in Figure 10A-10C. Referring to 01, CpG-STAT3 ASO(2'OMe) is SEQ ID NO: 15 linked to SEQ ID NO: 33 via a portion of formula (A), CpG-STAT3 dsASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via a portion of formula (A), which is hybridized to SEQ ID NO: 4, and CpG-STAT3-ssASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 4 via a portion of formula (A). [Figure 10G]These results demonstrate that local administration of single- and double-stranded CpG-STAT3 ASOs improves animal survival and induces immune activation in immunocompetent mice. C57BL / 6 mice bearing established GL261 gliomas were intratumorally / IC injected with 0.25 mg / kg of 2'-O-methyl or LNA-modified CpG-STAT3 ASO twice weekly. Figures 10G-10I: CpG-STAT3 ASO injection improves the ratio of intratumoral CD8 T cells to Tregs. The percentages of CD8+ T cells (CD3+CD8+) and regulatory T cells (CD3+CD4+FOXP3+), as well as their ratios, are shown. Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA with Tukey's multiple comparison post-hoc test. Referring to Figures 10A to 10I, CpG-STAT3 ASO(2'OMe) is SEQ ID NO: 15 linked to SEQ ID NO: 33 via the portion of formula (A), CpG-STAT3 dsASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), which is hybridized to SEQ ID NO: 4, and CpG-STAT3-ssASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the portion of formula (A). [Figure 10H]These results demonstrate that local administration of single- and double-stranded CpG-STAT3 ASOs improves animal survival and induces immune activation in immunocompetent mice. C57BL / 6 mice bearing established GL261 gliomas were intratumorally / IC injected with 0.25 mg / kg of 2'-O-methyl or LNA-modified CpG-STAT3 ASO twice weekly. Figures 10G-10I: CpG-STAT3 ASO injection improves the ratio of intratumoral CD8 T cells to Tregs. The percentages of CD8+ T cells (CD3+CD8+) and regulatory T cells (CD3+CD4+FOXP3+), as well as their ratios, are shown. Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA with Tukey's multiple comparison post-hoc test. Referring to Figures 10A to 10I, CpG-STAT3 ASO(2'OMe) is SEQ ID NO: 15 linked to SEQ ID NO: 33 via the portion of formula (A), CpG-STAT3 dsASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), which is hybridized to SEQ ID NO: 4, and CpG-STAT3-ssASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the portion of formula (A). [Figure 10I]These results demonstrate that local administration of single- and double-stranded CpG-STAT3 ASOs improves animal survival and induces immune activation in immunocompetent mice. C57BL / 6 mice bearing established GL261 gliomas were intratumorally / IC injected with 0.25 mg / kg of 2'-O-methyl or LNA-modified CpG-STAT3 ASO twice weekly. Figures 10G-10I: CpG-STAT3 ASO injection improves the ratio of intratumoral CD8 T cells to Tregs. The percentages of CD8+ T cells (CD3+CD8+) and regulatory T cells (CD3+CD4+FOXP3+), as well as their ratios, are shown. Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA with Tukey's multiple comparison post-hoc test. Referring to Figures 10A to 10I, CpG-STAT3 ASO(2'OMe) is SEQ ID NO: 15 linked to SEQ ID NO: 33 via the portion of formula (A), CpG-STAT3 dsASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the portion of formula (A), which is hybridized to SEQ ID NO: 4, and CpG-STAT3-ssASO(LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 4 via the portion of formula (A). [Figure 11A] These results show that the combination of CpG-STAT3 dsASO and PD1 blockade resulted in glioma regression in the majority of treated mice. C57BL / 6 mice bearing established GL261 gliomas were injected twice weekly with 200 μg of PD1-specific or control antibody intraperitoneally, 0.25 mg / kg of LNA-modified CpG-STAT3 dsASO intratumorally / IC injections, or a combination of both treatments. Figures 11A-11E: Tumor progression was monitored using BLI. Referring to Figures 11A-11F, the IgG is polyclonal rat IgG (Bio X Cell, catalog number: BE0094), the anti-PD-1 is an anti-mouse PD-1 antibody (Bio X Cell, clone: ​​29F.1A12, catalog number: BE0273), the CpG-STAT3 dsASO (LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), and SEQ ID NO: 3 is hybridized to SEQ ID NO: 4. [Figure 11B] These results show that the combination of CpG-STAT3 dsASO and PD1 blockade resulted in glioma regression in the majority of treated mice. C57BL / 6 mice bearing established GL261 gliomas were injected twice weekly with 200 μg of PD1-specific or control antibody intraperitoneally, 0.25 mg / kg of LNA-modified CpG-STAT3 dsASO intratumorally / IC injections, or a combination of both treatments. Figures 11A-11E: Tumor progression was monitored using BLI. Referring to Figures 11A-11F, the IgG is polyclonal rat IgG (Bio X Cell, catalog number: BE0094), the anti-PD-1 is an anti-mouse PD-1 antibody (Bio X Cell, clone: ​​29F.1A12, catalog number: BE0273), the CpG-STAT3 dsASO (LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), and SEQ ID NO: 3 is hybridized to SEQ ID NO: 4. [Figure 11C] These results show that the combination of CpG-STAT3 dsASO and PD1 blockade resulted in glioma regression in the majority of treated mice. C57BL / 6 mice bearing established GL261 gliomas were injected twice weekly with 200 μg of PD1-specific or control antibody intraperitoneally, 0.25 mg / kg of LNA-modified CpG-STAT3 dsASO intratumorally / IC injections, or a combination of both treatments. Figures 11A-11E: Tumor progression was monitored using BLI. Referring to Figures 11A-11F, the IgG is polyclonal rat IgG (Bio X Cell, catalog number: BE0094), the anti-PD-1 is an anti-mouse PD-1 antibody (Bio X Cell, clone: ​​29F.1A12, catalog number: BE0273), the CpG-STAT3 dsASO (LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), and SEQ ID NO: 3 is hybridized to SEQ ID NO: 4. [Figure 11D]These results show that the combination of CpG-STAT3 dsASO and PD1 blockade resulted in glioma regression in the majority of treated mice. C57BL / 6 mice bearing established GL261 gliomas were injected twice weekly with 200 μg of PD1-specific or control antibody intraperitoneally, 0.25 mg / kg of LNA-modified CpG-STAT3 dsASO intratumorally / IC injections, or a combination of both treatments. Figures 11A-11E: Tumor progression was monitored using BLI. Referring to Figures 11A-11F, the IgG is polyclonal rat IgG (Bio X Cell, catalog number: BE0094), the anti-PD-1 is an anti-mouse PD-1 antibody (Bio X Cell, clone: ​​29F.1A12, catalog number: BE0273), the CpG-STAT3 dsASO (LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), and SEQ ID NO: 3 is hybridized to SEQ ID NO: 4. [Figure 11E] These results show that the combination of CpG-STAT3 dsASO and PD1 blockade resulted in glioma regression in the majority of treated mice. C57BL / 6 mice bearing established GL261 gliomas were injected twice weekly with 200 μg of PD1-specific or control antibody intraperitoneally, 0.25 mg / kg of LNA-modified CpG-STAT3 dsASO intratumorally / IC injections, or a combination of both treatments. Figures 11A-11E: Tumor progression was monitored using BLI. Referring to Figures 11A-11F, the IgG is polyclonal rat IgG (Bio X Cell, catalog number: BE0094), the anti-PD-1 is an anti-mouse PD-1 antibody (Bio X Cell, clone: ​​29F.1A12, catalog number: BE0273), the CpG-STAT3 dsASO (LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), and SEQ ID NO: 3 is hybridized to SEQ ID NO: 4. [Figure 11F]Figure 11F shows that the combination of CpG-STAT3 dsASO and PD1 blockade resulted in glioma regression in the majority of treated mice. C57BL / 6 mice bearing established GL261 gliomas were injected twice weekly with 200 μg of PD1-specific or control antibody intraperitoneally, 0.25 mg / kg of LNA-modified CpG-STAT3 dsASO intratumorally / IC, or a combination of both treatments. Figure 11F: The combination of CpG-STAT3 ASO / anti-PD1 treatment resulted in complete glioma regression in 5 / 6 treated mice. Kaplan-Meier survival curves (n = 6-7 / group) are shown. Referring to Figures 11A-11F, the IgG is polyclonal rat IgG (Bio X Cell, catalog number: BE0094), the anti-PD-1 is an anti-mouse PD-1 antibody (Bio X Cell, clone: ​​29F.1A12, catalog number: BE0273), the CpG-STAT3 dsASO (LNA) is SEQ ID NO: 15 linked to SEQ ID NO: 3 via the moiety of formula (A), and SEQ ID NO: 3 is hybridized to SEQ ID NO: 4. [Figure 12] This figure shows that double-stranded CpG-STAT3 dsASO (lower right panel) is more potent against RM9 prostate tumors than single-stranded CpG-STAT3 ssASO (upper right panel) or CpG-psODN (CpG passenger strand only) (lower left panel). C57BL / 6 mice bearing established double RM9 tumors were treated with local intratumoral injections of the indicated oligonucleotides. Tumor growth progression was monitored by caliper measurements at a secondary, uninjected tumor site (n = 4-5 per group). [Figure 13]This shows that local administration of LNA-modified single-stranded or double-stranded CpG-STAT3 ASO improved survival in mice bearing syngeneic intracranial QPP8 gliomas. C57BL / 6 mice bearing established QPP8 gliomas (Qk / Tp53 / Pten-del) were intracranially / IC-injected with 0.25 mg / kg of LNA-modified CpG-STAT3 ASO twice weekly. CpG STAT3 ASO treatment significantly improved animal survival. Kaplan-Meier survival curves (n=6 / group) are shown. Median survival for PBS was 49 days. Median survival for LNA-modified STAT3 ASO was 52 days. Median survival for LNA-modified CpG-STAT3 double-stranded ASO (CpG-STAT3dsASOLNA) was 66.5 days. The median survival time for the LNA-modified CpG-STAT3-single-stranded ASO (CpG-STAT3ssASOLNA) was 61.5 days. [Figure 14] This shows that the combination of CpG-STAT3 dsASO and PD1 blockade resulted in QPP8 glioma regression in the majority of treated mice. C57BL / 6 mice bearing established QPP8 gliomas were injected twice weekly with 200 μg of PD1-specific antibody intraperitoneally / IP, 0.25 mg / kg of LNA-modified CpG-STAT3 ASO intracranially / IC, or a combination of both treatments. The CpG-STAT3 dsASO / anti-PD1 combination resulted in glioma regression in 4 / 5 treated mice. Kaplan-Meier survival curves (n=5 / group) are shown. [Figure 15]Surviving mice were protected from rechallenge with the same tumor. Mice surviving anti-PD1 / CpG-STAT3 dsASO combination treatment were rechallenged with an intracranial injection of 10 GL261 cells, and tumor progression was monitored using bioluminescent imaging (BLI). Most of the previously treated surviving mice survived the glioma rechallenge, whereas naive mice did not. Kaplan-Meier survival curves (n = 4–5 per group) are shown. The median survival time for the control group was 21 days. The median survival time for mice treated with anti-PD1 and LNA-modified CpG-STAT3 dsASO combination exceeded 152 days. Referring to Figures 12 to 15, the LNA-modified STAT3 ASO is sequence number 4, the LNA-modified CpG-STAT3-double-stranded ASO (CpG-STAT3dsASOLNA) is sequence number 15 linked to sequence number 3 via the portion of formula (A), sequence number 3 is hybridized to sequence number 4, and the LNA-modified CpG-STAT3-single-stranded ASO (CpG-STAT3ssASOLNA) is sequence number 15 linked to sequence number 4 via the portion of formula (A). DETAILED DESCRIPTION OF THE INVENTION

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the present disclosure. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not intended to limit the scope of the present disclosure.

[0014] The term "CpG oligodeoxynucleotide" or "CpG ODN" refers to a 5' C nucleotide linked to a 3' G nucleotide via a phosphodiester internucleotide linkage or a phosphodiester derivative internucleotide linkage. In embodiments, the CpG ODN comprises a phosphodiester internucleotide linkage. In embodiments, the CpG ODN comprises a phosphodiester derivative internucleotide linkage.

[0015] "Class A CpG ODN" or "A-class CpG ODN" or "D-type CpG ODN" or "class A CpG DNA sequence" refers to a CpG motif comprising an oligodeoxynucleotide containing one or more poly-G sequences at the 5', 3', or both ends, an internal palindromic sequence containing a CpG motif, or one or more phosphodiester derivatives linking the deoxynucleotides. In embodiments, the class A CpG ODN comprises a poly-G sequence at the 5', 3', or both ends, an internal palindromic sequence containing a CpG motif, and one or more phosphodiester derivatives linking the deoxynucleotides. In embodiments, the phosphodiester derivative is phosphorothioate. Examples of class A CpG ODNs include ODN D19, ODN 1585, ODN 2216, and ODN 2336, the sequences of which are known in the art.

[0016] "Class B CpG ODN" or "B class CpG ODN" or "K-type CpG ODN" or "class B CpG DNA sequence" refers to a CpG motif comprising one or more 6-mer motifs containing a CpG motif, oligodeoxynucleotides comprising phosphodiester derivatives linking all deoxynucleotides. In embodiments, the class B CpG ODN comprises one or more copies of a 6-mer motif containing a CpG motif and phosphodiester derivatives linking all deoxynucleotides. In embodiments, the phosphodiester derivative is phosphorothioate. In embodiments, the class B CpG ODN comprises one 6-mer motif containing a CpG motif. In embodiments, the class B CpG ODN comprises two copies of a 6-mer motif containing a CpG motif. In embodiments, the class B CpG ODN comprises three copies of a 6-mer motif containing a CpG motif. In embodiments, the class B CpG ODN comprises four copies of a 6-mer motif containing a CpG motif. Examples of class B CpG ODNs include ODN 1668, ODN 1826, ODN 2006, ODN 2007, ODN BW006, and ODN D-SL01, the sequences of which are known in the art.

[0017] The term "class C CpG ODN" or "C-class CpG ODN" or "C-type CpG DNA sequence" refers to an oligodeoxynucleotide containing a palindromic sequence containing a CpG motif and phosphodiester derivatives (phosphorothioates) linking all deoxynucleotides. Examples of class C CpG ODNs include ODN 2395, ODN M362, and ODN D-SL03, the sequences of which are known in the art.

[0018] The term "STAT" or "STAT transcription factor" refers to "Signal Transducer and Activator of Transcription" proteins and their homologs (e.g., STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6, STAT7, STAT8, STAT7 / 8, STAT9). In embodiments, "STAT transcription factor" refers to a human protein. The term "STAT transcription factor" includes wild-type and mutant forms of the protein. In embodiments, "STAT transcription factor" refers to a wild-type protein. In embodiments, "STAT transcription factor" refers to a mutant protein. "Phosphorylated STAT" refers to a STAT protein that is phosphorylated and activated by phosphorylation. In embodiments, activation of a STAT transcription factor means that the STAT is able to activate transcription.

[0019] "STAT3" or "STAT3 protein" refers to either a recombinant or naturally occurring form of the Signal Transducer and Activator of Transcription 3 (STAT3) protein, or a variant or homolog thereof that maintains STAT3 protein activity (within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to STAT3). In embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring STAT3 polypeptide. In embodiments, the STAT3 protein is substantially identical to the protein identified by NCBI Reference Number GI:47458820, or a variant or homolog having substantial identity thereto. In embodiments, the STAT3 protein is substantially identical to the protein identified by NCBI reference number GI:1610577068, or a variant or homolog having substantial identity thereto. In embodiments, the STAT3 protein is substantially identical to the protein identified by NCBI reference number GI:1610577050, or a variant or homolog having substantial identity thereto. "Phosphorylated STAT3" refers to a STAT3 protein that has been phosphorylated and activated by phosphorylation. In embodiments, phosphorylated STAT3 is phosphorylated on tyrosine 705 or a residue corresponding to tyrosine 705 in a homolog. In embodiments, activation of STAT3 means that STAT3 is able to activate transcription.

[0020] As used herein, the term "STAT3 gene" or "STAT3 sequence" refers to a gene encoding a STAT3 polypeptide or a variant thereof that is capable of maintaining the activity of a STAT3 polypeptide (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the STAT3 polypeptide). In embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity compared to the STAT3 sequence over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous nucleic acid portion). In embodiments, the STAT3 is substantially identical to the nucleic acid sequence identified by Accession No. NG007370, or a variant or homolog having substantial identity thereto.

[0021] The term "STAT4" refers to the "Signal Transducer and Activator of Transcription 4" protein and its homologs. In embodiments, "STAT4" refers to the protein associated with Entrez Gene 6775, OMIM 600558, UniProt Q14765, and / or RefSeq(protein) NP001230764. In embodiments, the immediately preceding reference numbers refer to proteins and related nucleic acids known as of the filing date of this application.

[0022] The term "STAT5A" refers to the "Signal Transducer and Activator of Transcription 5A" protein and its homologs. In embodiments, "STAT5A" refers to the protein associated with Entrez Gene 6776, OMIM 601511, UniProt P42229, and / or RefSeq(protein) NP003143. In embodiments, the reference numbers immediately above refer to proteins and related nucleic acids known as of the filing date of this application.

[0023] The term "STAT5B" refers to the "Signal Transducer and Activator of Transcription 5B" protein and its homologs. In embodiments, "STAT5B" refers to the protein associated with Entrez Gene 6777, OMIM 604260, UniProt P51692, and / or RefSeq(protein) NP036580. In embodiments, the immediately preceding reference numbers refer to proteins and related nucleic acids known as of the filing date of this application.

[0024] The term "STAT6" refers to the "Signal Transducer and Activator of Transcription 6" protein and its homologs. In embodiments, "STAT6" refers to the protein associated with Entrez Gene 6778, OMIM 601512, UniProt P42226, and / or RefSeq(protein) NP001171549. In embodiments, the reference numbers immediately above refer to proteins and related nucleic acids known as of the filing date of this application.

[0025] The term "linked" or "conjugated" when referring to two moieties (e.g., a phosphorothioated CpG ODN linked to a first DNA oligonucleotide) means that the two moieties are joined, and the bond connecting the two moieties is a covalent or non-covalent bond. In embodiments, the two moieties are covalently linked to one another (e.g., directly or via a linking group). Exemplary linking groups include a covalent bond, a nucleic acid sequence, a DNA sequence, a substituted or unsubstituted alkylene, a substituted or unsubstituted heteroalkylene, a substituted or unsubstituted cycloalkylene, a substituted or unsubstituted heterocycloalkylene, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene, or a combination of two or more of these.

[0026] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof, or their complements, or nucleosides (e.g., deoxyribonucleosides or ribonucleosides), in either single-, double-, or multi-stranded form. In embodiments, "nucleic acid" does not include nucleosides. The terms "polynucleotide," "oligonucleotide," "oligo," and the like refer, in their normal and customary sense, to a linear sequence of nucleotides. The term "nucleoside" refers, in its normal and customary sense, to a glycosylamine containing a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. The term "nucleotide," in its normal and customary sense, refers to a single polynucleotide unit, i.e., monomer. A nucleotide can be a ribonucleotide, a deoxyribonucleotide, or a modified form thereof. Examples of polynucleotides contemplated herein include single- and double-stranded DNA, single- and double-stranded RNA, and hybrid molecules containing a mixture of single- and double-stranded DNA and RNA. Examples of nucleic acids, e.g., polynucleotides, contemplated herein include all types of RNA, such as mRNA, siRNA, miRNA, and guide RNA, as well as all types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term "double-stranded" in the context of a polynucleotide refers to double-strandedness in the usual and customary sense. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides, or they can be branched, e.g., the nucleic acid contains one or more arms or branches of nucleotides. Optionally, branched nucleic acids are repeatedly branched to form higher-order structures such as dendrimers.

[0027] For example, nucleic acids, including nucleic acids with phosphothioate backbones, can contain one or more reactive moieties. As used herein, the term reactive moiety includes any group that can react with another molecule, such as a nucleic acid or a polypeptide, through a covalent bond, a non-covalent bond, or other interaction. For example, a nucleic acid can contain an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide through a covalent bond, a non-covalent bond, or other interaction.

[0028] The term also encompasses synthetic, naturally occurring, and non-naturally occurring nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphoamidates, phosphorodiamidates, phosphorothioates (also known as phosphorothioates where the oxygen in the phosphate is replaced with a double-bonded sulfur), phosphorodithioates, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acids, phosphonoformic acids, methyl phosphonates, boron phosphonates, or O-methyl phosphoramidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), as well as phosphodiester derivatives including 2'O-methyl, 5'fluoro, 2'-deoxy-2'fluoro, 2'-deoxy, universal base nucleotides, 5-C methyl nucleotides, incorporation of inverted deoxy base residues, modifications to the nucleotide base such as 5-methylcytidine or pseudouridine, and peptide nucleic acid backbones and linkages. Other nucleic acid analogs include those with positively charged backbones, non-ionic backbones, modified sugars, and non-ribose backbones (e.g., phosphorodiamidate morpholino oligos or locked nucleic acids (LNAs) known in the art). Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acid. Modifications of the ribose-phosphate backbone may be made for various reasons, for example, to increase the stability and half-life of such molecules in physiological environments or as probes on biochips. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0029] A nucleic acid may contain a non-specific sequence. As used herein, the term "non-specific sequence" refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to any other nucleic acid sequence or that are only partially complementary to any other nucleic acid sequence. For example, a non-specific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.

[0030] An "unmodified nucleotide" refers to a nucleotide that is not modified from its natural state.

[0031] "Modified nucleotide" refers to a nucleotide that has been modified from its natural state. Modifications to the nucleotide can be to the base, sugar, phosphate, or two or more of these. The nucleotide can be modified to include, for example, a 2'-O-aminopropyl group, a 2'-O-ethyl group, a 2'-fluoro group, a 2'-O-methyl group, a 2'-deoxy-2'fluoro group, a 2'-O-methoxyethyl group, a 2'-O-allyl group, a 2'-O-propyl group, a 2'-O-pentyl group, or a constrained nucleotide.

[0032] "Constrained nucleotide" refers to a nucleotide that has been modified to maintain a rigid backbone structure. In embodiments, a constrained nucleotide refers to a nucleotide in which the pentose has been modified to maintain a rigid backbone structure. Exemplary modifications to nucleotides that maintain a rigid structure include locked nucleic acids (e.g., LNA-modified nucleotides), cMOE-modified nucleotides, cEt-modified nucleotides, and the like. Constrained nucleotides are described, for example, in Pallan et al., Chem Commun (Camb), 48(66):8195-8197 (2012), the disclosure of which is incorporated herein by reference in its entirety.

[0033] "LNA modified nucleotide" refers to a locked or bridged nucleotide in which the pentose moiety is modified with an extra bridge connecting the 2' oxygen and the 4' carbon, as shown in the structure below.

[0034] [ka] where Bx is a base. See Braasch et al, Chemistry & Biology, 8:1-7 (2001).

[0035] "cMOE modified nucleotide" or "2',4'-constrained 2'-O-methoxyethyl modified nucleotide" refers to a nucleotide having the structure:

[0036] [ka] wherein Bx is a base. In an embodiment, the cMOE-modified nucleotide is an (R)-cMOE-modified nucleotide. In an embodiment, the cMOE-modified nucleotide is an (S)-cMOE-modified nucleotide. See Pallan et al., Chem Commun (Camb), 48(66):8195-8197 (2012).

[0037] "2'-O-ethyl modified nucleotide" or "2',4'-constrained 2'-O-ethyl modified nucleotide" or "cEt modified nucleotide" refers to a nucleotide having the structure:

[0038] [ka] wherein Bx is a base. In an embodiment, the cEt modified nucleotide is an (R)-cEt modified nucleotide. In an embodiment, the cEt modified nucleotide is an (S)-cEt modified nucleotide. See Pallan et al, Chem Commun (Camb), 48(66):8195-8197 (2012).

[0039] A "spacer modification" refers to a moiety that does not contain a nucleobase. Exemplary spacer modifications include an abasic spacer, a spacer phosphoramidite, an abasic phosphoramidite, a hexadecane phosphoramidite, an octadecane phosphoramidite, a C6 disulfide phosphoramidite, and the like. In embodiments, the spacer phosphoramidite is a C3 spacer phosphoramidite, a C6 spacer phosphoramidite, or a C12 spacer phosphoramidite.

[0040] A "C3 spacer phosphoramidite" is a spacer modification represented by the following structure:

[0041] [ka]

[0042] An "abasic spacer" or "d-spacer" is a 1',2'-dideoxyribose with no nucleobase attached.

[0043] As referred to herein, an "antisense nucleic acid" is a nucleic acid (e.g., a DNA or RNA molecule) that is complementary to at least a portion of a specific target nucleic acid and can reduce transcription of the target nucleic acid (e.g., mRNA from DNA), reduce translation of the target nucleic acid (e.g., mRNA), alter transcript splicing (e.g., single-stranded morpholino oligos), or interfere with the endogenous activity of the target nucleic acid. Typically, synthetic antisense nucleic acids (e.g., oligonucleotides) are generally 15 to 25 bases in length. Thus, the antisense nucleic acid can hybridize (e.g., selectively hybridize) to the target nucleic acid. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid in vitro. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid inside a cell. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid in an organism. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid under physiological conditions. Antisense nucleic acids can contain naturally occurring or modified nucleotides, such as phosphorothioates, methylphosphonates, and anomeric sugar-phosphate, backbone-modified nucleotides.

[0044] In cells, antisense nucleic acids hybridize to corresponding RNA to form double-stranded molecules. Antisense nucleic acids interfere with the endogenous behavior of RNA and inhibit its function compared to the absence of antisense nucleic acids. Furthermore, double-stranded molecules can be degraded through the RNAi pathway. The use of antisense methods to inhibit in vitro translation of genes is well known in the art. Furthermore, antisense molecules that directly bind to DNA can be used. Antisense nucleic acids can be single-stranded or double-stranded nucleic acids. Non-limiting examples of antisense nucleic acids include siRNAs (including their derivatives or precursors, such as nucleotide analogs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), saRNAs (small activating RNAs), and small nucleolar RNAs (snoRNAs), or portions of their derivatives or precursors.

[0045] A polynucleotide generally consists of a specific sequence of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T) (if the polynucleotide is RNA, uracil (U) is substituted for thymine (T)). Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be used for the polynucleotide molecule itself. This alphabetical representation may be input into a data system of a computer with a central processing unit and used in bioinformatics applications such as functional genomics and homology searching. A polynucleotide may optionally contain one or more non-standard nucleotides, nucleotide analogs, and / or modified nucleotides.

[0046] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to nucleic acids that encode the same or essentially the same amino acid sequence. Due to the degeneracy of the genetic code, many nucleic acid sequences encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. Those skilled in the art will understand that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0047] As used herein, the term "complement" refers to a nucleotide (e.g., RNA or DNA) or sequence of nucleotides that can base-pair with a complementary nucleotide or sequence of nucleotides. As described herein and generally known in the art, the complementary (matching) nucleotide of adenosine is thymidine, and the complementary (matching) nucleotide of guanidine is cytosine. Thus, a complement may comprise a sequence of nucleotides that base-pair with the corresponding complementary nucleotides of a second nucleic acid sequence. The complementary nucleotides may partially or perfectly match the nucleotides of the second nucleic acid sequence. When the complementary nucleotides perfectly match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. When the complementary nucleotides partially match the nucleotides of the second nucleic acid sequence, only a portion of the complementary nucleotides form base pairs with the nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding sequences and non-coding sequences, where the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence. Another example of complementary sequences is a sense sequence and an antisense sequence, and the sense sequence comprises the complementary nucleotide of the antisense sequence, thus forming the complement of the antisense sequence.When the sequence is partially complementary, only a part of the nucleic acid is matched according to base pairing, or when it is complete, all of the nucleic acid is matched according to base pairing.Therefore, two sequences that are complementary to each other may have a certain percentage of identical nucleotides (i.e., about 60% identity over a certain region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity).

[0048] The term "gene" refers to a segment of DNA involved in producing a protein. This includes regions preceding and following the coding region (leader and trailer), as well as intervening sequences (introns) between individual coding segments (exons). The leader, trailer, and introns contain regulatory elements required during transcription and translation of a gene. Furthermore, a "protein gene product" is a protein expressed from a particular gene.

[0049] The term "expression" or "expressed" as used herein with respect to a gene refers to the transcription and / or translation product of that gene. The expression level of a DNA molecule in a cell can be determined based on either the amount of corresponding mRNA present in the cell or the amount of protein encoded by that DNA produced by the cell. The expression level of a non-coding nucleic acid molecule (e.g., siRNA) can be detected by standard PCR or Northern blotting techniques well known in the art.

[0050] The phrases "specifically (or selectively) bind" or "specifically (or selectively) immunoreact" with an antibody, when referring to a protein or peptide, often refer to a binding reaction that determines the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under specified immunoassay conditions, a particular antibody will bind to a particular protein at least twice as high as background, and more typically 10-100 times higher than background. Specific binding to an antibody under such conditions requires that the antibody be selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with a selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein.

[0051] The terms "isolate" and "isolated," when applied to nucleic acids, viruses, or proteins, mean that the nucleic acid, virus, or protein is essentially free from other cellular components with which it is naturally associated. It can be, for example, in a homogeneous state, either dry or in an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein present as the predominant species in a preparation is substantially purified.

[0052] "Percentage sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence for optimal alignment of the two sequences (which does not contain additions or deletions). This percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue is present in both sequences to generate the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to generate the percentage sequence identity.

[0053] The terms "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that have amino acid residues or nucleotides that are identical or that are identical by a specified percentage (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region), as measured using the BLAST or BLAST 2.0 sequence comparison algorithm or by manual alignment and visual inspection (see, e.g., http: / / www.ncbi.nlm.nih.gov / BLAST / ). Such sequences are therefore said to be "substantially identical." This definition may also refer to or be applied to the complement of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as sequences that have substitutions. As explained below, preferred algorithms can account for gaps, etc. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, and more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0054] The "position" of an amino acid or nucleotide base is indicated by a number that ordinal identifies each amino acid (or nucleotide base) in a reference sequence based on its position relative to the N-terminus (or 5'-terminus). Due to deletions, insertions, truncations, fusions, etc., which must be considered when determining optimal alignment, the number of amino acid residues in a test sequence, determined simply by counting from the N-terminus, is generally not necessarily the same as the number of corresponding positions in the reference sequence. For example, if a variant has a deletion compared to the aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of the deletion. If there is an insertion in the aligned reference sequence, the insertion does not correspond to a numbered amino acid position in the reference sequence. In the case of a truncation or fusion, there may be a stretch of amino acids in either the reference sequence or the aligned sequence that does not correspond to any amino acid in the corresponding sequence.

[0055] When used in the context of the numbering of a given amino acid or polynucleotide sequence, the term "numbered with reference to" or "corresponding to" refers to the numbering of residues in a particular reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.

[0056] The term "about" refers to a range of values ​​that includes the specified value and that one of ordinary skill in the art would consider to be reasonably similar to the specified value. In embodiments, about refers to within a standard deviation using measurements generally accepted in the art. In embodiments, about refers to a range that covers + / - 10% of the specified value. In embodiments, about refers to a range that covers + / - 5% of the specified value. In embodiments, about refers to a range that covers + / - 1 of the specified value. In embodiments, about includes the stated value.

[0057] The terms "control" or "control experiment" are used according to their plain and ordinary meaning to refer to an experiment in which the experimental subject or reagent is treated the same as in a parallel experiment, except for the omission of an experimental procedure, reagent, or variable. In embodiments, a control is used as a standard of comparison when evaluating experimental efficacy. In embodiments, a control is a measurement of protein activity in the absence of a compound described herein (including the embodiments and examples). One of ordinary skill in the art will understand which standard control is most appropriate in a given situation and will be able to analyze data based on comparison to the standard control value. Standard controls are also useful for determining the significance (e.g., statistical significance) of data. For example, if the value of a given parameter varies widely in the standard control, the variation in the test sample will not be considered significant.

[0058] Substituents, when specified by their conventional chemical formula written from left to right, equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to -OCH2-.

[0059] The term "alkyl," by itself or as part of another substituent, unless otherwise stated, can include divalent and polyvalent groups that may be fully saturated, monounsaturated, or polyunsaturated, and has the specified number of carbon atoms (e.g., C1-C6). 10means 1 to 10 carbons), linear (i.e., unbranched) or branched acyclic carbon chains (or carbons), or combinations thereof. Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, and homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl groups are those containing one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (—O—).

[0060] The term "alkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited to, by -CHCHCH-. Typically, an alkyl (or alkylene) group has from 1 to 24 carbon atoms, with groups having 10 or fewer carbon atoms being preferred in the present invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having 8 or fewer carbon atoms. The term "alkenylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.

[0061] The term "heteroalkyl," by itself or in combination with other terms, means, unless otherwise specified, a stable acyclic, straight or branched chain, or combination thereof, containing at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -(CH2)2-O-CH3, -(CH2)2-NH-CH3, -(CH2)3-OH, -CH2-NH2, -CH2-NO2, -(CH2)2-N(CH3)-CH3, -CH2-S-CH2-CH3, -S(O)-CH3, -(CH2)2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -O-CH3, -CH=CH-N(CH3)-CH3, -O-CH-2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0062] The term "heteroalkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, including, but not limited to, -CH-CH-S-CH-CH-, -O-CH-CH-NH-CH-, -O-(CH)-O-PO-, -O-(CH)-O-PO-, -O-(CH)-O-PO-, -O-(CH)-O-PO-, -O-(CH)-O-PO-, and the like. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)R'- represents both -C(O)R'- and -R'C(O)-. As noted above, heteroalkyl groups as used herein include groups attached to the remainder of the molecule via a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or -SOR'. When "heteroalkyl" is recited followed by a specific heteroalkyl group, such as -NR'R'', it is understood that the terms heteroalkyl and -NR'R'' are not redundant or mutually exclusive. Rather, the specific heteroalkyl group is recited for clarity. Thus, the term "heteroalkyl" should not be interpreted as excluding specific heteroalkyl groups, such as -NR'R''.

[0063] The terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, mean, unless otherwise specified, cyclic, non-aromatic versions of "alkyl" and "heteroalkyl," respectively, in which the carbons comprising the ring are not necessarily bonded to hydrogen, since all carbon valencies participate in bonds with non-hydrogen atoms. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, etc. "Cycloalkylene" and "heterocycloalkylene," alone or as part of another substituent, mean a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

[0064] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0065] The term "acyl," unless otherwise specified, means -C(O)R, where R is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0066] The term "aryl," unless otherwise specified, refers to a polyunsaturated, aromatic, hydrocarbon substituent, which may be a single ring or multiple rings (preferably 1 to 3 rings) fused together (i.e., fused-ring aryl) or covalently linked (e.g., biphenyl). A fused-ring aryl refers to multiple rings fused together, at least one of which is an aryl ring. The term "heteroaryl" refers to an aryl group (or ring) containing at least one heteroatom, such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. Thus, the term "heteroaryl" includes fused-ring heteroaryl groups (i.e., multiple rings fused together, at least one of which is an aromatic heterocycle). A 5,6-fused-ring heteroarylene refers to two rings fused together, one ring having five members and the other having six members, and at least one ring being a heteroaryl ring. Similarly, a 6,6-fused ring heteroarylene refers to two rings fused together, one ring having 6 members and the other ring having 6 members, and at least one ring being a heteroaryl ring. Also, a 6,5-fused ring heteroarylene refers to two rings fused together, one ring having 6 members and the other ring having 5 members, and at least one ring being a heteroaryl ring. The heteroaryl group can be bonded to the rest of the molecule via a carbon atom or a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, Examples include 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. "Arylene" and "heteroarylene," by themselves or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively. Non-limiting examples of heteroaryl groups include pyridinyl, pyrimidinyl, thiophenyl, thienyl, furanyl, indolyl, benzoxadiazolyl, benzodioxolyl, benzodioxanyl, thianaphthalyl, pyrrolopyridinyl, indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl, quinazolinonyl, benzisoxazolyl, imidazopyridinyl, benzofuranyl, benzothienyl, benzothiophenyl, phenyl, naphthyl, biphenyl, and pyrrolopyridinyl. Examples of heteroaryl include aryl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furylthienyl, pyridyl, pyrimidyl, benzothiazolyl, purinyl, benzimidazolyl, isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl, diazolyl, triazolyl, tetrazolyl, benzothiadiazolyl, isothiazolyl, pyrazolopyrimidinyl, pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl, and quinolyl. The above examples can be substituted or unsubstituted, and the divalent radical of each of the above heteroaryl examples is a non-limiting example of heteroarylene.

[0067] A fused-ring heterocycloalkyl-aryl is an aryl fused to a heterocycloalkyl. A fused-ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl. A fused-ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl. A fused-ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl. Each fused-ring heterocycloalkyl-aryl, fused-ring heterocycloalkyl-heteroaryl, fused-ring heterocycloalkyl-cycloalkyl, or fused-ring heterocycloalkyl-heterocycloalkyl can independently be unsubstituted or substituted with one or more of the substituents described herein.

[0068] The term "oxo" means an oxygen that is double bonded to a carbon atom.

[0069] As used herein, the term "alkylsulfonyl" refers to a moiety having the formula -S(O2)-R', where R' is a substituted or unsubstituted alkyl group as defined above. R' can have a specified number of carbon atoms (e.g., "C1-C4 alkylsulfonyl").

[0070] Each of the above terms (e.g., "alkyl," "heteroalkyl," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0071] Substituents for alkyl and heteroalkyl groups (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can range in number from zero to (2m'+1), where m' is the total number of atoms in such group, and can include -OR', ═O, ═NR', ═N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CON It can be one or more of a variety of groups selected from, but not limited to, R'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NRSO2R', -NR'NR''R''', -ONR'R'', -NR'C=(O)NR''NR'''R'''', -CN, -NO2. R, R', R'', R''', and R'''' each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1 to 3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy group, or arylalkyl group. When a compound of the invention includes more than one R group, e.g., when two or more of these groups are present, each of the R groups is independently selected as each R', R'', R''', and R'''' group. When R' and R'' are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R'' includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups that contain carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF and -CHCF) and acyl (e.g., -C(O)CH, -C(O)CF, -C(O)CHOCH, etc.).

[0072] Similar to the substituents described for the alkyl group, substituents for the aryl and heteroaryl groups are varied and include, for example, —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —COR′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)R′, —NR—C(NR′R″R′″)═NR′″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)R′, —S(O)NR′R″, —NRSOR′, —NR′NR″R′″, — and R', R'', R''', R''', and R'''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound of the invention includes more than one R group, e.g., when more than one of these groups is present, each of the R groups is independently selected as the R', R'', R''', and R'''' group, respectively.

[0073] Two or more substituents may optionally be linked to form an aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group. Such so-called ring-forming substituents are typically, but not necessarily, found attached to a cyclic base structure. In embodiments, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic base structure form a fused ring structure. In embodiments, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure. In embodiments, the ring-forming substituents are attached to non-adjacent members of the base structure.

[0074] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be of the formula -TC(O)-(CRR') q -U-, where T and U are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -A-(CH2) r wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer from 1 to 4. One of the single bonds in the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR') s -X'-(C''R''R'') dwherein s and d are independently integers from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituents R, R', R", and R'" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0075] As used herein, the term "heteroatom" or "ring heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0076] As used herein, a "substituent" means a group selected from the following moieties: (A) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SON2NH2, -NHNH2, -ONH2, -NHC=(O)NHNH2, -NHC=(O)NH2, -NHS02H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, -NHSO2CH3, -N3, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from the following: (i) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SON2NH2, -NHNH2, -ONH2, -NHC=(O)NHNH2, -NHC=(O)NH2, -NHS02H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, -NHSO2CH3, -N3, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from the following: (a) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SON2NH2, -NHNH2, -ONH2, -NHC=(O)NHNH2, -NHC=(O)NH2, -NHS02H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, -NHSO2CH3, -N3, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl substituted with at least one substituent selected from oxo, halogen, —CF3, —CN, —OH, —NH2, —COOH, —CONH2, —NO2, —SH, —S02Cl, —S03H, —S04H, —SON2NH2, —NHNH2, —ONH2, —NHC═(O)NHNH2, —NHC═(O)NH2, —NHS02H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF3, —OCHF2, —NHSO2CH3, —N3, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

[0077] As used herein, a "size-limited substituent" or "size-limited substituent group" refers to a group selected from all of the substituents described above for "substituent," and each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C 20 each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 20-membered heteroalkyl; each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl; each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3- to 8-membered heterocycloalkyl; and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C8 10 aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5- to 10-membered heteroaryl.

[0078] As used herein, a "lower substituent" or "lower substituent group" means a group selected from all of the substituents described above for "substituent group," wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3-7 membered heterocycloalkyl, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C8 10 aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5- to 9-membered heteroaryl.

[0079] In embodiments, each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene described in the compounds herein is substituted with at least one substituent group. In embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. In embodiments, at least one or all of these groups are substituted with at least one lower-rank substituent group.

[0080] In the compound embodiments herein, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C 20 each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 20-membered heteroalkyl; each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl; each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3- to 8-membered heterocycloalkyl; and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C8 10 aryl, and / or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5-10 membered heteroaryl. In embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C 20 each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2- to 20-membered heteroalkylene; each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene; each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3- to 8-membered heterocycloalkylene; and each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C8 10 arylene, and / or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5- to 10-membered heteroarylene.

[0081] In an embodiment, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3-7 membered heterocycloalkyl, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C8 10 In an embodiment, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2-8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3-7 membered heterocycloalkylene, and each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C8 10 arylene, and / or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5- to 9-membered heteroarylene. In embodiments, the compound is of a species described in the Examples section below.

[0082] The terms "activation," "activate," "activating," "activator," and the like, with respect to protein-inhibitor interactions, refer to positively affecting (e.g., increasing) the activity or function of a protein compared to the activity or function of the protein in the absence of the activator. In aspects, activation refers to positively affecting (e.g., increasing) the concentration or level of a protein compared to the concentration or level of the protein in the absence of the activator. These terms can refer to activating, activating, sensitizing, or upregulating signal transduction or enzymatic activity or the amount of a protein that is decreased in a disease. Thus, activation can include, at least in part, partially or completely increasing the stimulus, increasing, enabling activation, or activating, sensitizing, or upregulating signal transduction or enzymatic activity or the amount of a protein associated with a disease (e.g., a protein that is decreased in a disease compared to an unaffected control). Activation can include, at least in part, partially or completely increasing the stimulus, increasing, enabling activation, or activating, sensitizing, or upregulating signal transduction or enzymatic activity or the amount of a protein.

[0083] The terms "agonist," "activator," "up-regulator," and the like refer to a substance capable of detectably increasing the expression or activity of a given gene or protein. An agonist can increase expression or activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control in the absence of the agonist. In certain instances, expression or activity is greater than expression or activity in the absence of the agonist.

[0084] With respect to protein-inhibitor interactions, terms such as "inhibit," "inhibit," "inhibiting," and the like refer to negatively affecting (e.g., decreasing) the activity or function of a protein compared to the activity or function of the protein in the absence of the inhibitor. In aspects, inhibition refers to adversely affecting (e.g., decreasing) the concentration or level of a protein compared to the concentration or level of the protein in the absence of the inhibitor. In aspects, inhibition refers to the reduction of a disease or disease symptom. In embodiments, inhibition refers to the reduction of the activity of a specific protein target. Thus, inhibition includes at least partially or completely blocking a stimulus, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating signaling or enzymatic activity or the amount of a protein. In embodiments, inhibition refers to the reduction of the activity of a target protein due to a direct interaction (e.g., an inhibitor binds to the target protein). In embodiments, inhibition refers to the reduction of the activity of a target protein due to an indirect interaction (e.g., an inhibitor binds to a protein that activates the target protein, thereby preventing activation of the target protein).

[0085] The terms "inhibitor," "repressor," or "antagonist," or "down-regulator" refer interchangeably to a substance that can detectably reduce the expression or activity of a given gene or protein. Antagonists can reduce expression or activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control in the absence of the antagonist. In embodiments, expression or activity is lower than expression or activity in the absence of the antagonist.

[0086] compound Provided herein are compounds comprising a first DNA oligonucleotide and a second DNA oligonucleotide. The first DNA oligonucleotide comprises from about 5 to about 70 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 5 to about 70 nucleotides, and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end. In embodiments, the first DNA oligonucleotide comprises from about 6 to about 60 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 6 to about 60 nucleotides, and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end. In embodiments, the first DNA oligonucleotide comprises from about 6 to about 50 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 6 to about 50 nucleotides, and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end. In an embodiment, the first DNA oligonucleotide comprises from about 8 to about 40 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 8 to about 40 nucleotides, and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In an embodiment, the first DNA oligonucleotide comprises from about 8 to about 30 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 8 to about 30 nucleotides, and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In an embodiment, the first DNA oligonucleotide comprises from about 8 to about 26 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 8 to about 26 nucleotides, and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide.In an embodiment, the first DNA oligonucleotide comprises from about 10 to about 30 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 10 to about 30 nucleotides, and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In an embodiment, the first DNA oligonucleotide comprises from about 10 to about 28 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 10 to about 28 nucleotides, and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In an embodiment, the first DNA oligonucleotide comprises from about 10 to about 24 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 10 to about 24 nucleotides, and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In an embodiment, the first DNA oligonucleotide comprises from about 10 to about 22 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 10 to about 22 nucleotides, and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In an embodiment, the first DNA oligonucleotide comprises from about 10 to about 20 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 10 to about 20 nucleotides, and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In an embodiment, the first DNA oligonucleotide comprises from about 12 to about 20 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 12 to about 20 nucleotides, and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide.In embodiments, the first DNA oligonucleotide comprises about 14 to about 18 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) about 14 to about 18 nucleotides, and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end. In embodiments, the constrained nucleotide is an LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. The constrained nucleotides in the second DNA oligonucleotide can be the same or different. In embodiments, the constrained nucleotide at the 3' end is different from the constrained nucleotide at the 5' end. In embodiments, the constrained nucleotide at the 3' end is the same as the constrained nucleotide at the 5' end. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification.

[0087] In an embodiment, the first DNA oligonucleotide comprises about 10 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) about 10 nucleotides and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In an embodiment, the first DNA oligonucleotide comprises about 11 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) about 11 nucleotides and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In an embodiment, the first DNA oligonucleotide comprises about 12 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) about 12 nucleotides and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In an embodiment, the first DNA oligonucleotide comprises about 13 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) about 13 nucleotides and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In an embodiment, the first DNA oligonucleotide comprises about 14 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) about 14 nucleotides and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end. In an embodiment, the first DNA oligonucleotide comprises about 15 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) about 15 nucleotides and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end. In an embodiment, the first DNA oligonucleotide comprises about 16 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) about 16 nucleotides and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end. In an embodiment, the first DNA oligonucleotide comprises about 17 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) about 17 nucleotides and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end.In an embodiment, the first DNA oligonucleotide comprises about 18 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) about 18 nucleotides and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end. In an embodiment, the first DNA oligonucleotide comprises about 19 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) about 19 nucleotides and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end. In an embodiment, the first DNA oligonucleotide comprises about 20 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) about 20 nucleotides and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end. In an embodiment, the first DNA oligonucleotide comprises about 21 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) about 21 nucleotides and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end. In embodiments, the first DNA oligonucleotide comprises about 22 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) about 22 nucleotides and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end. In embodiments, the constrained nucleotide is an LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. The constrained nucleotides in the second DNA oligonucleotide can be the same or different. In embodiments, the constrained nucleotide at the 3' end is different from the constrained nucleotide at the 5' end. In embodiments, the constrained nucleotide at the 3' end is the same as the constrained nucleotide at the 5' end. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification.

[0088] In embodiments of the compounds described herein, the second DNA oligonucleotide comprises one constrained nucleotide at the 3'-end and one constrained nucleotide at the 5'-end. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-end and one constrained nucleotide at the 5'-end. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-end and one constrained nucleotide at the 5'-end, where the two constrained nucleotides are consecutive. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-end and one constrained nucleotide at the 5'-end, where the two constrained nucleotides are alternating. In embodiments, the second DNA oligonucleotide comprises one constrained nucleotide at the 3'-end and two constrained nucleotides at the 5'-end. In embodiments, the second DNA oligonucleotide comprises one constrained nucleotide at the 3'-end and two constrained nucleotides at the 5'-end, where the two constrained nucleotides are consecutive. In embodiments, the second DNA oligonucleotide comprises one constrained nucleotide at the 3'-end and two constrained nucleotides at the 5'-end, the two constrained nucleotides alternating. In embodiments, the constrained nucleotide is an LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. The constrained nucleotides in the second DNA oligonucleotide can be the same or different. In embodiments, the constrained nucleotide at the 3'-end is different from the constrained nucleotide at the 5'-end. In embodiments, the constrained nucleotide at the 3'-end is the same as the constrained nucleotide at the 5'-end. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification.

[0089] In embodiments, the second DNA oligonucleotide comprises one constrained nucleotide at the 3'-end and three constrained nucleotides at the 5'-end. In embodiments, the second DNA oligonucleotide comprises one constrained nucleotide at the 3'-end and three constrained nucleotides at the 5'-end, where the three constrained nucleotides are consecutive. In embodiments, the second DNA oligonucleotide comprises one constrained nucleotide at the 3'-end and three constrained nucleotides at the 5'-end, where the three constrained nucleotides alternate. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides at the 3'-end and one constrained nucleotide at the 5'-end. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides at the 3'-end and one constrained nucleotide at the 5'-end, where the three constrained nucleotides are consecutive. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides at the 3'-end and one constrained nucleotide at the 5'-end, where the three constrained nucleotides alternate. In embodiments, the constrained nucleotide is an LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. The constrained nucleotides in the second DNA oligonucleotide can be the same or different. In embodiments, the constrained nucleotide at the 3'-end is different from the constrained nucleotide at the 5'-end. In embodiments, the constrained nucleotide at the 3'-end is the same as the constrained nucleotide at the 5'-end. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification.

[0090] In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-end and two constrained nucleotides at the 5'-end. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-end and two constrained nucleotides at the 5'-end, where the two constrained nucleotides at the 3'-end are contiguous and the two constrained nucleotides at the 5'-end are contiguous. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-end and two constrained nucleotides at the 5'-end, where the two constrained nucleotides at the 3'-end are alternating and the two constrained nucleotides at the 5'-end are alternating. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-end and two constrained nucleotides at the 5'-end, where the two constrained nucleotides at the 3'-end are contiguous and the two constrained nucleotides at the 5'-end are alternating. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-terminus and two constrained nucleotides at the 5'-terminus, wherein the two constrained nucleotides at the 3'-terminus are alternating and the two constrained nucleotides at the 5'-terminus are consecutive. In embodiments, the constrained nucleotides are LNA-modified nucleotides, cMOE-modified nucleotides, or cET-modified nucleotides. In embodiments, the constrained nucleotides are LNA-modified nucleotides. In embodiments, the constrained nucleotides are cMOE-modified nucleotides. In embodiments, the constrained nucleotides are cET-modified nucleotides. The constrained nucleotides in the second DNA oligonucleotide may be the same or different. In embodiments, the constrained nucleotide at the 3'-terminus is different from the constrained nucleotide at the 5'-terminus. In embodiments, the constrained nucleotide at the 3'-terminus is the same as the constrained nucleotide at the 5'-terminus. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification.

[0091] In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-end and three constrained nucleotides at the 5'-end. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-end and three constrained nucleotides at the 5'-end, where the two constrained nucleotides at the 3'-end are consecutive and the three constrained nucleotides at the 5'-end are consecutive. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-end and three constrained nucleotides at the 5'-end, where the two constrained nucleotides at the 3'-end are alternating and the three constrained nucleotides at the 5'-end are alternating. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-end and three constrained nucleotides at the 5'-end, where the two constrained nucleotides at the 3'-end are consecutive and the three constrained nucleotides at the 5'-end are alternating. In embodiments, the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-end and three constrained nucleotides at the 5'-end, wherein the two constrained nucleotides at the 3'-end are alternating and the three constrained nucleotides at the 5'-end are consecutive. In embodiments, the constrained nucleotides are LNA-modified nucleotides, cMOE-modified nucleotides, or cET-modified nucleotides. In embodiments, the constrained nucleotides are LNA-modified nucleotides. In embodiments, the constrained nucleotides are cMOE-modified nucleotides. In embodiments, the constrained nucleotides are cET-modified nucleotides. The constrained nucleotides in the second DNA oligonucleotide may be the same or different. In embodiments, the constrained nucleotide at the 3'-end is different from the constrained nucleotide at the 5'-end. In embodiments, the constrained nucleotide at the 3'-end is the same as the constrained nucleotide at the 5'-end. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification.

[0092] In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides at the 3'-end and two constrained nucleotides at the 5'-end. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides at the 3'-end and two constrained nucleotides at the 5'-end, where the three constrained nucleotides at the 3'-end are consecutive and the two constrained nucleotides at the 5'-end are consecutive. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides at the 3'-end and two constrained nucleotides at the 5'-end, where the three constrained nucleotides at the 3'-end are alternating and the two constrained nucleotides at the 5'-end are alternating. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides at the 3'-end and two constrained nucleotides at the 5'-end, where the three constrained nucleotides at the 3'-end are consecutive and the two constrained nucleotides at the 5'-end are alternating. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides at the 3'-terminus and two constrained nucleotides at the 5'-terminus, wherein the three constrained nucleotides at the 3'-terminus are alternating and the two constrained nucleotides at the 5'-terminus are consecutive. In embodiments, the constrained nucleotides are LNA-modified nucleotides, cMOE-modified nucleotides, or cET-modified nucleotides. In embodiments, the constrained nucleotides are LNA-modified nucleotides. In embodiments, the constrained nucleotides are cMOE-modified nucleotides. In embodiments, the constrained nucleotides are cET-modified nucleotides. The constrained nucleotides in the second DNA oligonucleotide may be the same or different. In embodiments, the constrained nucleotide at the 3'-terminus is different from the constrained nucleotide at the 5'-terminus. In embodiments, the constrained nucleotide at the 3'-terminus is the same as the constrained nucleotide at the 5'-terminus. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification.

[0093] In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides at the 3'-end and three constrained nucleotides at the 5'-end. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides at the 3'-end and three constrained nucleotides at the 5'-end, where the three constrained nucleotides at the 3'-end are consecutive and the three constrained nucleotides at the 5'-end are consecutive. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides at the 3'-end and three constrained nucleotides at the 5'-end, where the three constrained nucleotides at the 3'-end are alternating and the three constrained nucleotides at the 5'-end are alternating. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides at the 3'-end and three constrained nucleotides at the 5'-end, where the three constrained nucleotides at the 3'-end are consecutive and the three constrained nucleotides at the 5'-end are alternating. In embodiments, the second DNA oligonucleotide comprises three constrained nucleotides at the 3'-terminus and three constrained nucleotides at the 5'-terminus, wherein the three constrained nucleotides at the 3'-terminus are alternating and the three constrained nucleotides at the 5'-terminus are consecutive. In embodiments, the constrained nucleotides are LNA-modified nucleotides, cMOE-modified nucleotides, or cET-modified nucleotides. In embodiments, the constrained nucleotides are LNA-modified nucleotides. In embodiments, the constrained nucleotides are cMOE-modified nucleotides. In embodiments, the constrained nucleotides are cET-modified nucleotides. The constrained nucleotides in the second DNA oligonucleotide can be the same or different. In embodiments, the constrained nucleotide at the 3'-terminus is different from the constrained nucleotide at the 5'-terminus. In embodiments, the constrained nucleotide at the 3'-terminus is the same as the constrained nucleotide at the 5'-terminus. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification.

[0094] In the second DNA oligonucleotide embodiments described herein, the term "3' end" means that the terminal nucleotide is adjacent to the 3' end. Similarly, the term "5' end" means that the terminal nucleotide is adjacent to the 5' end. For example, the structure:

[0095] [Table 1] For example, the phrase "the 3'-terminal constrained nucleotide" refers to the 3'-terminal lowercase underlined nucleotide.

[0096] [Table 2] With respect to a nucleotide having the formula: ##STR00001## the phrase "5'-terminal constrained nucleotide" refers to the 5'-terminal lowercase underlined nucleotide.

[0097] When two or more constrained nucleotides are at the 3' or 5' end, the two or more nucleotides may be consecutive. For example, the structure:

[0098] [Table 3] For example, the three contiguous constrained nucleotides at the 3' end refer to the three contiguous nucleotides at the 3' end that are underlined and in lower case.

[0099] [Table 4] With respect to a nucleotide having the formula: "A" or "B", the two contiguous constrained nucleotides at the 3'-terminus refer to the two contiguous lowercase underlined nucleotides at the 5'-terminus. Thus, the term "contiguous" has its plain and ordinary meaning of "next to" or "in order together."

[0100] When two or more constrained nucleotides are at the 3' or 5' end, the two or more nucleotides may alternate. As used herein, the term "alternating" means that the terminal nucleotide is constrained, the next consecutive nucleotide is not constrained, and the next consecutive nucleotide is constrained. For example,

[0101] [Table 5] For example, the two alternating constrained nucleotides at the 5' end refer to the two lowercase underlined nucleotides at the 5' end that alternate between locked nucleic acids, nucleic acids without LNA modifications, and locked nucleic acids.

[0102] [Table 6] With respect to the nucleotide having the formula (I), the three alternating LNA-modified nucleotides at the 3'-end refer to the locked nucleic acid, the nucleic acid without the LNA modification, and the three lowercase underlined nucleotides at the 3'-end that alternate between the two locked nucleic acids. Thus, the term "alternating" can also be described as consecutive, except that the second nucleotide from the 3'-end or 5'-end does not have an LNA modification.

[0103] In the embodiment of the compound described herein, the first DNA oligonucleotide and the second DNA oligonucleotide do not have the same number of nucleotides.For example, the first DNA oligonucleotide can have 18 unmodified nucleotides, and the second DNA oligonucleotide can have 16 nucleotides.As another example, the first DNA oligonucleotide can have 16 unmodified nucleotides, and the second DNA oligonucleotide can have 18 nucleotides.

[0104] In the embodiment of the compound described herein, the first DNA oligonucleotide and the second DNA oligonucleotide have the same number of nucleotides.For example, the first DNA oligonucleotide can have 16 unmodified nucleotides, and the second DNA oligonucleotide can have 16 nucleotides.

[0105] In embodiments of the compounds described herein, the second DNA oligonucleotide comprises nucleotides having a phosphorothioate modification. In embodiments, at least 10% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 20% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 30% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 40% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 50% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 60% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 70% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 80% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, at least 90% of the nucleotides in the second DNA oligonucleotide have a phosphorothioate modification. In embodiments, all nucleotides in the second DNA oligonucleotide have a phosphorothioate internucleotide linkage.

[0106] In embodiments, the second DNA oligonucleotide further comprises a nucleotide having a modification. The modification can be a spacer modification or a nucleotide modification. In embodiments, the second DNA oligonucleotide further comprises a nucleotide having a modification to the base or sugar. In embodiments, the second DNA oligonucleotide comprises a nucleotide having a modification selected from the group consisting of a 2'-O-aminopropyl group, a 2'-O-ethyl group, a 2'-fluoro group, a 2'-O-methyl group, a 2'-deoxy-2'fluoro group, a 2'-O-methoxyethyl group, a 2'-O-allyl group, a 2'-O-propyl group, a 2'-O-pentyl group, and a constrained nucleotide. In embodiments, the second DNA oligonucleotide further comprises a nucleotide having a 2'-O-methyl group. In embodiments, the second DNA oligonucleotide further comprises a nucleotide having a 2'-fluoro group. In embodiments, the second DNA oligonucleotide further comprises a nucleotide having a 2'-deoxy-2'fluoro group. In embodiments, the second DNA oligonucleotide further comprises a nucleotide having a constrained nucleic acid modification, wherein the constrained nucleic acid modification is added to the 3'- and 5'-terminal constrained nucleotide. In embodiments, the additional nucleotide has a constrained nucleic acid modification that is contiguous with the 3'- or 5'-terminal constrained nucleotide. In embodiments, the additional nucleotide has a constrained nucleic acid modification and is separated from the 3'- or 5'-terminal constrained nucleotide by one or more nucleotides that do not have the constrained modification. In embodiments, the constrained nucleotide is an LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide.

[0107] In embodiments, the second DNA oligonucleotide further comprises a spacer modification. In embodiments, the spacer modification is an abasic spacer, a spacer phosphoramidite, an abasic phosphoramidite, a hexadecane phosphoramidite, an octadecane phosphoramidite, a C6 disulfide phosphoramidite, or a combination of two or more thereof. In embodiments, the spacer phosphoramidite is a C3 spacer phosphoramidite, a C6 spacer phosphoramidite, or a C12 spacer phosphoramidite. In embodiments, the second DNA oligonucleotide further comprises an abasic spacer modification. In embodiments, the second DNA oligonucleotide further comprises a C3 spacer phosphoramidite.

[0108] In embodiments, the second DNA oligonucleotide is an antisense oligonucleotide. In embodiments, the second DNA oligonucleotide is a STAT antisense oligonucleotide. In embodiments, the second DNA oligonucleotide is a STAT1 antisense oligonucleotide. In embodiments, the second DNA oligonucleotide is a STAT2 antisense oligonucleotide. In embodiments, the second DNA oligonucleotide is a STAT3 antisense oligonucleotide. In embodiments, the second DNA oligonucleotide is a STAT4 antisense oligonucleotide. In embodiments, the second DNA oligonucleotide is a STAT5 antisense oligonucleotide. In embodiments, the second DNA oligonucleotide is a STAT6 antisense oligonucleotide.

[0109] In embodiments, the second DNA oligonucleotide is a STAT3 antisense oligonucleotide. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:35, or SEQ ID NO:36. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:2. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:4. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:6. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:8. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:10. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:12. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:14. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:35. In embodiments, the STAT3 antisense oligonucleotide comprises SEQ ID NO:36.

[0110] [Table 7]

[0111] In Table 1, bold lowercase letters refer to LNA modified nucleotides, underlines refer to phosphorothioated nucleotides (phosphorothioated internucleotide linkages), single quotes (e.g., G') indicate 2'O-methyl modifications, and X 1 is an abasic spacer (" / idSp / "), and X 2 is a C3 spacer phosphoramidite (" / iSpC3 / ").

[0112] In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 1. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 3. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 5. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 7. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 9. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 11. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO: 13.

[0113] [Table 8]

[0114] In Table 2, all nucleotides are unmodified.

[0115] In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:1 and the second DNA oligonucleotide comprises SEQ ID NO:2. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:3 and the second DNA oligonucleotide comprises SEQ ID NO:4. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:5 and the second DNA oligonucleotide comprises SEQ ID NO:6. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:7 and the second DNA oligonucleotide comprises SEQ ID NO:8. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:9 and the second DNA oligonucleotide comprises SEQ ID NO:10. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:11 and the second DNA oligonucleotide comprises SEQ ID NO:12. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:13 and the second DNA oligonucleotide comprises SEQ ID NO:14. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:1 and the second DNA oligonucleotide comprises SEQ ID NO:35. In embodiments, the first DNA oligonucleotide comprises SEQ ID NO:1 and the second DNA oligonucleotide comprises SEQ ID NO:36.

[0116] The phrase "a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide" is equivalent to the phrase "a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide, where the first DNA oligonucleotide is hybridized to a second DNA oligonucleotide."

[0117] The compounds described herein include phosphorothioated CpG oligodeoxynucleotides (ODNs). In embodiments, the CpG ODNs are CpG-A ODNs, CpG-B ODNs, CpG-C ODNs, or a combination of two or more thereof. In embodiments, the CpG ODNs are CpG-A ODNs. In embodiments, the CpG ODNs are CpG-B ODNs. In embodiments, the CpG ODNs are CpG-C ODNs. In embodiments, the CpG ODN is CpG ODN 1585, CpG ODN 2216, CpG ODN 2336, CpG ODN 1668, CpG ODN 1826, CpG ODN 2006, CpG ODN 2007, CpG ODN BW006, CpG ODN D-SL01, CpG ODN 2395, CpG ODN M362, CpG ODN D-SL03, CpG ODN D19, or a combination of two or more thereof. In embodiments, the CpG ODN is CpG ODN 1585. In embodiments, the CpG ODN is CpG ODN 2216. In embodiments, the CpG ODN is CpG ODN 2336. In embodiments, the CpG ODN is CpG ODN 1668. In embodiments, the CpG ODN is CpG ODN 1826. In embodiments, the CpG ODN is CpG ODN 2006. In embodiments, the CpG ODN is CpG ODN 2007. In embodiments, the CpG ODN is CpG ODN BW006. In embodiments, the CpG ODN is CpG ODN D-SL01. In embodiments, the CpG ODN is CpG ODN 2395. In embodiments, the CpG ODN is CpG ODN M362. In embodiments, the CpG ODN is CpG ODN D-SL03. In embodiments, the CpG ODN is CpG ODN D19.

[0118] In embodiments, the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. In embodiments, the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21. In embodiments, the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. In embodiments, the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21. In embodiments, the CpG ODN is SEQ ID NO:43 or SEQ ID NO:15. In embodiments, the CpG ODN is SEQ ID NO: 43. In embodiments, the CpG ODN is SEQ ID NO: 15. In embodiments, the CpG ODN is SEQ ID NO: 16. In embodiments, the CpG ODN is SEQ ID NO: 17. In embodiments, the CpG ODN is SEQ ID NO: 18. In embodiments, the CpG ODN is SEQ ID NO: 19. In embodiments, the CpG ODN is SEQ ID NO: 20. In embodiments, the CpG ODN is SEQ ID NO: 21. In embodiments, the CpG ODN is SEQ ID NO: 22. In embodiments, the CpG ODN is SEQ ID NO: 23. In embodiments, the CpG ODN is SEQ ID NO: 24. In embodiments, the CpG ODN is SEQ ID NO: 25. In embodiments, the CpG ODN is SEQ ID NO: 26. In embodiments, the CpG ODN is SEQ ID NO: 27. In embodiments, the CpG ODN is SEQ ID NO: 28. In embodiments, the CpG ODN is SEQ ID NO: 29. In embodiments, the CpG ODN is SEQ ID NO: 30. In embodiments, the CpG ODN is SEQ ID NO: 31. In embodiments, the CpG ODN is SEQ ID NO: 32.

[0119] In embodiments, the compounds described herein comprise a phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO: 43 covalently linked to a first DNA oligonucleotide comprising SEQ ID NO: 1, wherein SEQ ID NO: 1 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO: 2, and the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide. In embodiments, a phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO: 15 is covalently linked to a first DNA oligonucleotide comprising SEQ ID NO: 1, wherein SEQ ID NO: 1 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO: 2, and the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide. In embodiments, the phosphorothioated CpG oligodeoxynucleotide is covalently linked to the first DNA oligonucleotide via a moiety of formula (A).

[0120] In embodiments, the compounds described herein comprise a phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO: 43 covalently linked to a first DNA oligonucleotide comprising SEQ ID NO: 1, wherein SEQ ID NO: 1 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO: 35, and the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide. In embodiments, a phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO: 15 is covalently linked to a first DNA oligonucleotide comprising SEQ ID NO: 1, wherein SEQ ID NO: 1 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO: 35, and the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide. In embodiments, the phosphorothioated CpG oligodeoxynucleotide is covalently linked to the first DNA oligonucleotide via a moiety of formula (A).

[0121] In embodiments, the compounds described herein comprise a phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO: 43 covalently linked to a first DNA oligonucleotide comprising SEQ ID NO: 1, wherein SEQ ID NO: 1 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO: 36, and the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide. In embodiments, a phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO: 15 is covalently linked to a first DNA oligonucleotide comprising SEQ ID NO: 1, wherein SEQ ID NO: 1 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO: 36, and the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide. In embodiments, the phosphorothioated CpG oligodeoxynucleotide is covalently linked to the first DNA oligonucleotide via a moiety of formula (A).

[0122] In embodiments, the compounds described herein comprise a phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO: 15 covalently linked to a first DNA oligonucleotide comprising SEQ ID NO: 3, wherein SEQ ID NO: 3 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO: 4, and the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide. In embodiments, a phosphorothioated CpG oligodeoxynucleotide comprising SEQ ID NO: 43 is covalently linked to a first DNA oligonucleotide comprising SEQ ID NO: 3, wherein SEQ ID NO: 3 is hybridized to a second DNA oligonucleotide comprising SEQ ID NO: 4, and the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide. In embodiments, the phosphorothioated CpG oligodeoxynucleotide is covalently linked to the first DNA oligonucleotide via a moiety of formula (A).

[0123] [Table 9]

[0124] In Table 3, underlining indicates phosphorothioated nucleotides (phosphorothioated internucleotide linkages).

[0125] The phosphorothioated CpG ODN is linked to the first DNA oligonucleotide by any linking group known in the art. In embodiments, the linking group comprises a bond, a nucleic acid sequence, a DNA sequence, a substituted or unsubstituted alkylene, a substituted or unsubstituted heteroalkylene, a substituted or unsubstituted cycloalkylene, a substituted or unsubstituted heterocycloalkylene, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene, or a combination of two or more thereof. In embodiments, the linking group comprises a bond, a nucleic acid sequence, an unsubstituted alkylene, an unsubstituted heteroalkylene, or a combination of two or more thereof. In embodiments, the linking group is a covalent bond. In embodiments, the linking group is a nucleic acid sequence. In embodiments, the linking group is a DNA sequence. In embodiments, the linking group comprises a nucleic acid sequence and a substituted or unsubstituted alkylene. In embodiments, the linking group comprises a nucleic acid sequence and an unsubstituted alkylene. In embodiments, the linking group comprises a nucleic acid sequence and a substituted or unsubstituted heteroalkylene. In embodiments, the linking group comprises a nucleic acid sequence and an unsubstituted heteroalkylene. In embodiments, the linking group comprises a nucleic acid sequence and an unsubstituted heteroalkylene. In embodiments, the linking group comprises a substituted or unsubstituted heteroalkylene. In embodiments, the linking group comprises a substituted heteroalkylene.

[0126] In embodiments, the linking group comprises a substituted heteroalkylene. In embodiments, the linking group is a substituted 6- to 60-membered heteroalkylene. In embodiments, the linking group is a substituted 6- to 54-membered heteroalkylene. In embodiments, the linking group is a substituted 12- to 48-membered heteroalkylene. In embodiments, the linking group is a substituted 18- to 42-membered heteroalkylene. In embodiments, the linking group is a substituted 24- to 36-membered heteroalkylene. In embodiments, the linking group comprises a substituted 30-membered heteroalkylene. In embodiments, the heteroalkylene comprises an oxygen atom, a phosphorus atom, or a combination thereof. In embodiments, the substituent on the substituted heteroalkylene is oxo, —OH, —O, or —O. - In embodiments, the linking group is a substituted 18-42 membered heteroalkylene, wherein the heteroalkylene comprises an oxygen atom, a phosphorus atom, or a combination thereof, and the substituents are oxo, -OH, and -O. - are independently selected from the group consisting of:

[0127] In embodiments, the linking group comprises any one of the following structures:

[0128] [ka] In the formula, z1, z2, z3, and z4 are independently an integer of 0 to 20, and each X is independently -OH or -O - In an embodiment, z1 is an integer from 0 to 5. In an embodiment, z1 is an integer from 2 to 4. In an embodiment, z2 is an integer from 0 to 5. In an embodiment, z2 is an integer from 2 to 4. In an embodiment, z3 is an integer from 0 to 5. In an embodiment, z1 is an integer from 2 to 4. In an embodiment, z4 is an integer from 3 to 7. In an embodiment, z4 is an integer from 4 to 6. In an embodiment, each X is -OH.

[0129] In embodiments, the linking group comprises the following structure:

[0130] [ka] In the formula, n is an integer from 1 to 10. In an embodiment, n is an integer from 2 to 8. In an embodiment, n is an integer from 3 to 7. In an embodiment, n is an integer from 4 to 6. In an embodiment, n is 1. In an embodiment, n is 2. In an embodiment, n is 3. In an embodiment, n is 4. In an embodiment, n is 5. In an embodiment, n is 6. In an embodiment, n is 7. In an embodiment, n is 8. In an embodiment, n is 9. In an embodiment, n is 10.

[0131] In embodiments, the compound further comprises a detectable moiety. In embodiments, the phosphorothioated CpG ODN, the first DNA oligonucleotide, the second DNA oligonucleotide, or any combination thereof comprises a detectable moiety. In embodiments, the phosphorothioated CpG ODN comprises a detectable moiety. In embodiments, the first DNA oligonucleotide comprises a detectable moiety. In embodiments, the second DNA oligonucleotide comprises a detectable moiety.

[0132] A compound comprising a detectable moiety is one that is covalently bound to the detectable moiety via a linker or chemical bond, or non-covalently bound to the detectable moiety via ionic, van der Waals, electrostatic, or hydrogen bonds, such that the presence of the nucleic acid can be detected by detecting the presence of the detectable moiety bound to the nucleic acid. Alternatively, methods using high affinity interactions can achieve the same result when one member of a pair of binding partners binds to the other, e.g., a detectable moiety. In embodiments of the compounds described herein, phosphorothioate nucleic acids or phosphorothioate polymer backbones comprise detectable agents disclosed herein and known in the art.

[0133] A "detectable agent" or "detectable moiety" is a compound or composition detectable by appropriate means, such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means. A detectable moiety is a monovalent detectable agent or a detectable agent attached (e.g., covalently and directly or via a linking group) to another compound, e.g., a nucleic acid.Exemplary detectable agents / moieties for use in the present disclosure include antibody ligands, peptides, nucleic acids, radioisotopes, paramagnetic metal ions, fluorophores (e.g., fluorescent dyes), electron-dense reagents, enzymes (e.g., commonly used in ELISAs), biotin, biotin-avidin complexes, biotin-streptavidin complexes, digoxigenin, magnetic beads (e.g., DYNABEADS® by ThermoFisher, DYNABEADS® by ThermoFisher, and the like). BEADS® M-270 amine and other functionalized magnetic beads), paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic iron oxide nanoparticles, ultrasmall superparamagnetic iron oxide nanoparticle aggregates, superparamagnetic iron oxide nanoparticles, superparamagnetic iron oxide nanoparticle aggregates, single crystalline iron oxide nanoparticles, single crystalline iron oxide, nanoparticle contrast agents, liposomes or other delivery vehicles containing gadolinium chelating molecules, gadolinium, radionuclides (e.g., carbon-11, nitrogen-13, oxygen-15, fluorine-18, rubidium-82), Fluorodeoxyglucose (e.g., fluorine-18 labeled), any gamma-emitting radionuclide, positron-emitting radionuclide, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biocolloids, microbubbles (including, for example, a microbubble shell comprising albumin, galactose, lipids, and / or polymers; a microbubble gas core comprising air, heavy gases, perfluorocarbons, nitrogen, octafluoropropane, perflexan lipid microspheres, perflutren, etc.), iodinated contrast agents (e.g., iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate), barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores, or haptens and proteins, or other substances that can be made detectable, for example, by incorporating a radiolabel into a peptide or an antibody that specifically reacts with a target peptide.

[0134] Pharmaceutical Compositions In embodiments, the present disclosure provides a pharmaceutical composition comprising a compound described herein and a pharmaceutically acceptable excipient. A "pharmaceutically acceptable excipient" refers to a substance that aids in the administration and absorption of an active agent by a subject and can be included in a composition of the present disclosure without causing significant adverse toxic effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solution, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, saline (such as Ringer's solution), alcohol, oil, gelatin, carbohydrates such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and coloring agents. Such preparations may be sterilized and, if desired, mixed with auxiliary substances such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, and / or aromatic substances that do not deleteriously react with the compounds of the present disclosure. Those skilled in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0135] Provided herein is a pharmaceutical composition comprising: (1) an immune checkpoint inhibitor; and (2) a compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide, wherein the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end. In embodiments, compound (2) is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the constrained nucleotide is an LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor.

[0136] An "effective amount" is an amount of a compound sufficient to achieve a desired purpose (e.g., achieve the effect for which it is administered, treat a disease) compared to the absence of the compound. An example of an "effective amount" is an amount sufficient to contribute to the treatment of a disease, which may also be referred to as a "therapeutically effective amount." "Relief" of symptoms (and grammatical equivalents of this phrase) refers to a reduction in the severity or frequency of symptoms, or the elimination of symptoms. The exact amount depends on the purpose of treatment and can be determined by one skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). For any compound described herein, the therapeutically effective amount can be initially determined from cell culture assays. The target concentration will be the concentration of active compound that is capable of achieving the methods described herein when measured using methods described herein or known in the art. As is known in the art, the therapeutically effective amount for use in humans can also be determined from animal models. For example, a human dose can be formulated to achieve a concentration found to be effective in animals. The dosage in humans can be adjusted by monitoring the effectiveness of the compound or composition described herein and adjusting the dosage upward or downward. Adjusting the dosage to achieve maximum efficacy in humans based on the above and other methods is well within the capabilities of one skilled in the art.

[0137] Dosage may vary depending on the patient's requirements and the compound used. In light of the present disclosure, the dose administered to a patient should be sufficient to produce a beneficial therapeutic response in the patient over time. The size of the dose will also be determined by the existence, nature, and extent of any adverse side effects. Determining the appropriate dosage for a particular situation is within the ability of one of ordinary skill in the art. Generally, treatment is initiated with smaller dosages that are less than the optimal dose of the compound. Thereafter, the dosage is increased by small increments until the optimal effect under the circumstances is reached. Dosage amount and interval can be individually adjusted to provide a level of administered compound that is effective for the particular clinical indication being treated. This will provide a treatment regimen appropriate to the severity of the individual's condition.

[0138] The term "administering" refers to oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or implantation of a sustained-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intratumoral, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. In embodiments, a compound or pharmaceutical composition described herein is administered parenterally to a patient. In embodiments, a compound or pharmaceutical composition described herein is administered intratumorally to a patient. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. In embodiments, administration does not include administration of any active agents other than the listed active agents.

[0139] Treatment method In embodiments, the present disclosure provides a method of treating cancer in a patient in need thereof. The method of treating cancer comprises administering to the patient an effective amount of a compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide hybridized to a second DNA oligonucleotide. Pharmaceutical compositions are provided in which the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end. In embodiments, the constrained nucleotide is an LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification. In embodiments, the compound is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the method further comprises administering to the patient an effective amount of an immune checkpoint inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In embodiments, the method further comprises administering to the patient an effective amount of an anti-cancer agent. In embodiments, the cancer is a central nervous system cancer, leukemia, lymphoma, solid tumor cancer, or epidermoid cancer. In embodiments, the cancer is a central nervous system cancer. In embodiments, the cancer is a solid tumor cancer. In embodiments, the cancer is an epidermoid cancer. In embodiments, the cancer is a leukemia. In embodiments, the cancer is a lymphoma. In embodiments, the cancer is a B-cell lymphoma.

[0140] In embodiments, the present disclosure provides a method of treating central nervous system cancer in a patient in need thereof by administering to the patient an effective amount of a compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide. Pharmaceutical compositions are provided in which the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification. In embodiments, the compound is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the method further comprises administering to the patient an effective amount of an immune checkpoint inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In embodiments, the method further comprises administering to the patient an effective amount of an anti-cancer agent. In embodiments, the central nervous system cancer is a glioma, cranial primitive neuroectodermal tumor, ependymoma tumor, hemangiopericytoma, germ cell tumor, pineal tumor, or primary central nervous system lymphoma. In embodiments, the cancer is a glioma. In embodiments, the glioma is an astrocytoma, glioblastoma, or oligodendroglioma. In embodiments, the glioma is an astrocytoma. In embodiments, the glioma is a glioblastoma. In embodiments, the glioma is an oligodendroglioma. In embodiments, the glioma is a brainstem glioma. In embodiments, the glioma is a mixed glioma.In embodiments, the glioma is an optic pathway glioma. In embodiments, the cancer is a cranial primitive neuroectodermal tumor. In embodiments, the cranial primitive neuroectodermal tumor is a medulloblastoma, a cranial neuroblastoma, a pineoblastoma, or a nasal neuroblastoma. In embodiments, the cranial primitive neuroectodermal tumor is a medulloblastoma. In embodiments, the cranial primitive neuroectodermal tumor is a cranial neuroblastoma. In embodiments, the cranial primitive neuroectodermal tumor is a pineoblastoma. In embodiments, the cranial primitive neuroectodermal tumor is a neuroblastoma. In embodiments, the cancer is an ependymoma tumor. In embodiments, the ependymoma tumor is an ependymoma, a myxopapillary ependymoma, or a subependymoma. In embodiments, the ependymoma tumor is an ependymoma. In embodiments, the ependymoma tumor is a myxopapillary ependymoma. In embodiments, the ependymoma tumor is a subependymoma. In embodiments, the cancer is hemangiopericytoma. In embodiments, the cancer is a germ cell tumor. In embodiments, the cancer is a pineal tumor. In embodiments, the cancer is a primary central nervous system lymphoma.

[0141] In embodiments, the present disclosure provides a method of treating glioma in a patient in need thereof by administering to the patient an effective amount of a compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide. Pharmaceutical compositions are provided in which the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification. In embodiments, the compound is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the method further comprises administering to the patient an effective amount of an immune checkpoint inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In embodiments, the method further comprises administering to the patient an effective amount of an anti-cancer agent.

[0142] In embodiments, the present disclosure provides a method of treating leukemia in a patient in need thereof by administering to the patient an effective amount of a compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide. Pharmaceutical compositions are provided in which the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification. In embodiments, the compound is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the cancer is acute myeloid leukemia. In embodiments, the method further comprises administering to the patient an effective amount of an immune checkpoint inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In embodiments, the method further comprises administering to the patient an effective amount of an anti-cancer agent.

[0143] In embodiments, the present disclosure provides a method of treating solid tumor cancer in a patient in need thereof by administering to the patient an effective amount of a compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide hybridized to a second DNA oligonucleotide. Pharmaceutical compositions are provided in which the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification. In embodiments, the compound is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the method further comprises administering to the patient an effective amount of an immune checkpoint inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In embodiments, the method further comprises administering to the patient an effective amount of an anti-cancer agent. In embodiments, the solid tumor cancer is prostate cancer, breast cancer, colorectal cancer, bladder cancer, lung cancer, liver cancer, pancreatic cancer, kidney cancer, gastric cancer, or melanoma. In embodiments, the solid tumor cancer is prostate cancer. In embodiments, the solid tumor cancer is breast cancer. In embodiments, the solid tumor cancer is colorectal cancer. In embodiments, the solid tumor cancer is bladder cancer. In embodiments, the solid tumor cancer is lung cancer. In embodiments, the solid tumor cancer is liver cancer. In embodiments, the solid tumor cancer is pancreatic cancer.In embodiments, the solid tumor cancer is renal cancer. In embodiments, the cancer is gastric cancer. In embodiments, the cancer is melanoma.

[0144] In embodiments, the present disclosure provides a method of treating prostate cancer in a patient in need thereof by administering to the patient an effective amount of a compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide. Pharmaceutical compositions are provided in which the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In embodiments, the compound is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the constrained nucleotide is an LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification. In embodiments, the method further comprises administering to the patient an effective amount of an immune checkpoint inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In embodiments, the method further comprises administering to the patient an effective amount of an anti-cancer agent.

[0145] In embodiments, the present disclosure provides a method of treating epidermoid carcinoma in a patient in need thereof by administering to the patient an effective amount of a compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide. Pharmaceutical compositions are provided in which the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides, and the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide. In embodiments, the constrained nucleotide is an LNA-modified nucleotide. In embodiments, the constrained nucleotide is a cMOE-modified nucleotide. In embodiments, the constrained nucleotide is a cET-modified nucleotide. In embodiments, the second DNA oligonucleotide comprises a nucleotide with a phosphorothioate modification. In embodiments, the compound is any compound described herein, including all embodiments of the compounds described herein. In embodiments, the method further comprises administering to the patient an effective amount of an immune checkpoint inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In embodiments, the method further comprises administering to the patient an effective amount of an anti-cancer agent. In embodiments, the epidermoid cancer is thyroid cancer, esophageal cancer, vaginal cancer, anal cancer, cervical cancer, or head and neck cancer. In embodiments, the epidermoid cancer is thyroid cancer. In embodiments, the epidermoid cancer is esophageal cancer. In embodiments, the cancer is anal cancer. In embodiments, the cancer is cervical cancer. In embodiments, the cancer is head and neck cancer. In embodiments, the cancer is vaginal cancer.

[0146] In embodiments of the methods described herein, the PD-1 inhibitor is pembrolizumab, nivolumab, cemiplimab, dostarlimab, camrelizumab, sintilimab, tislelizumab, toripalimab, spartalizumab, retifanlimab, pimivalimab (JTX-4014), AMP-224, or MEDI0680 (AMP-514). In embodiments, the PD-1 inhibitor is pembrolizumab, nivolumab, cemiplimab, dostarlimab, camrelizumab, sintilimab, tislelizumab, toripalimab, retifanlimab, or spartalizumab. In embodiments, the PD-1 inhibitor is pembrolizumab. In embodiments, the PD-1 inhibitor is nivolumab. In embodiments, the PD-1 inhibitor is cemiplimab. In embodiments, the PD-1 inhibitor is dostarlimab. In embodiments, the PD-1 inhibitor is camrelizumab. In embodiments, the PD-1 inhibitor is sintilimab. In embodiments, the PD-1 inhibitor is tislelizumab. In embodiments, the PD-1 inhibitor is toripalimab. In embodiments, the PD-1 inhibitor is spartalizumab. In embodiments, the PD-1 inhibitor is pimivalimab. In embodiments, the PD-1 inhibitor is retifanlimab. In embodiments, the PD-1 inhibitor is AMP-224. In embodiments, the PD-1 inhibitor is MEDI0680.

[0147] In embodiments of the methods described herein, the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, embafolimab, cosibelimab, AUNP12, CA-170, or BMS-986189. In embodiments, the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, embafolimab, cosibelimab, AUNP12, CA-170, or BMS-986189. In embodiments, the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, embafolimab, or cosibelimab. In embodiments, the PD-L1 inhibitor is atezolizumab. In embodiments, the PD-L1 inhibitor is avelumab. In embodiments, the PD-L1 inhibitor is durvalumab. In embodiments, the PD-L1 inhibitor is envafolimab. In embodiments, the PD-L1 inhibitor is cosibelimab. In embodiments, the PD-L1 inhibitor is AUNP12. In embodiments, the PD-L1 inhibitor is CA-170. In embodiments, the PD-L1 inhibitor is BMS-986189.

[0148] The term "cancer" refers to all types of cancer, neoplasm, or malignant tumors found in mammals (e.g., humans), including leukemia, lymphoma, carcinoma, and sarcoma. Exemplary cancers that may be treated with the compounds and pharmaceutical compositions provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head cancer, Hodgkin's disease, and non-Hodgkin's lymphoma. Exemplary cancers that may be treated with the compounds and pharmaceutical compositions provided herein include thyroid cancer, endocrine system cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, rectal cancer, gastric cancer, and uterine cancer. Further examples of cancers that may be treated with the compounds and pharmaceutical compositions provided herein include thyroid cancer, bile duct cancer, pancreatic adenocarcinoma, cutaneous melanoma of the skin, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, head and neck squamous cell carcinoma, invasive breast cancer, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocytopenia, primary macroglobulinemia, and primary thrombocytopenia. cancer, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, premalignant skin lesion, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, endocrine or exocrine pancreatic neoplasm, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer.

[0149] The terms "treating" and "treatment" refer to clinical success of a therapy or any indication of improvement of a disease (e.g., cancer), including any objective or subjective parameter, such as relief, remission, reduction of symptoms, or making the disease more tolerable to the patient, slowing the rate of degeneration or decline, making the end point of degeneration less debilitating, improving the patient's physical or mental well-being, etc. The treatment or amelioration of symptoms can be based on objective or subjective parameters, including the results of a physical exam. The term "treating" does not include prevention.

[0150] "Patient" or "subject in need" refers to an organism suffering from or susceptible to a disease that can be treated by administration of a compound or pharmaceutical composition herein. Non-limiting examples include humans, other mammals, cows, rats, mice, dogs, cats, monkeys, goats, sheep, cattle, and other non-mammals. In an embodiment, the patient is a human.

[0151] As used herein, a cancer model organism is an organism that exhibits a phenotype that indicates cancer or the activity of cancer-causing factors within the organism. The term cancer is defined above. A wide variety of organisms can serve as cancer model organisms, including, for example, cancer cells and mammalian organisms such as rodents (e.g., mice or rats) and primates (e.g., humans). A cancer cell line is generally understood by those skilled in the art as a cell that exhibits a phenotype or genotype similar to in vivo cancer. As used herein, a cancer cell line includes cell lines derived from animals (e.g., mice) and humans.

[0152] The term "immune checkpoint inhibitor" refers to a compound (e.g., an antibody) that can bind to or disrupt the interaction between an inhibitory receptor and its ligand, which is essential for balancing costimulatory receptor activity and limiting T cell activation. Thus, immune checkpoint inhibitors target immune system checkpoints, such as the PD-1 pathway.

[0153] A "PD-1 pathway inhibitor" refers to a substance that can detectably reduce the expression or activity level of the PD-1 signaling pathway compared to a control. An "inhibitor" is a compound or small molecule that inhibits the PD-1 signaling pathway, for example, by binding to, partially or completely blocking stimulation of the PD-1 pathway, reducing, preventing, or delaying activation of the PD-1 pathway, or inactivating, desensitizing, or downregulating signaling, gene expression, or enzymatic activity of the PD-1 pathway. In embodiments, the PD-1 pathway inhibitor is a programmed death-ligand 1 (PD-L1) inhibitor or a PD-1 inhibitor. A PD-L1 inhibitor is a substance that at least partially, partially, or completely blocks stimulation of PD-1, reduces, prevents, or delays activation of PD-1, or inactivates, desensitizes, or downregulates PD-1 signaling. A PD-1 inhibitor is a substance that at least partially, partially, or completely blocks stimulation of PD-1, reduces, prevents, or delays activation of PD-1, or inactivates, desensitizes, or downregulates PD-1 signaling.

[0154] 111 In, 90 Y, or 131 Anti-CD20 monoclonal antibodies conjugated to IFN-γ, triptolide, homoharringtonine, dactinomycin, doxorubicin, epirubicin, topotecan, itraconazole, vindesine, cerivastatin, vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan, clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib, gefitinib, EGFR inhibitors, epidermal growth factor receptor (EGFR) inhibitors, receptor, EGFR) targeted therapy or therapeutic agent (e.g., gefitinib, erlotinib, cetuximab, lapatinib, panitumumab, vandetanib, afatinib / BIBW2992, CI-1033 / canertinib, neratinib / HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, These include dacomitinib / PF299804, OSI-420 / desmethylerlotinib, AZD8931, AEE788, pelitinib / EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib, dasatinib, and hormone therapy.

[0155] Embodiment Embodiment 1. A compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide hybridized to a second DNA oligonucleotide, wherein (a) the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides, and (b) the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end.

[0156] Embodiment 2. The compound of embodiment 1, wherein the second DNA oligonucleotide comprises one constrained nucleotide at the 3' end and one constrained nucleotide at the 5' end.

[0157] Embodiment 3. The compound of embodiment 1, wherein the second DNA oligonucleotide comprises one constrained nucleotide at the 3' end and two constrained nucleotides at the 5' end.

[0158] Embodiment 4. The compound of embodiment 1, wherein the second DNA oligonucleotide comprises two constrained nucleotides at the 3' end and one constrained nucleotide at the 5' end.

[0159] Embodiment 5. The compound of embodiment 1, wherein the second DNA oligonucleotide comprises two constrained nucleotides at the 3' end and two constrained nucleotides at the 5' end.

[0160] Embodiment 6 The compound of embodiment 1, wherein the second DNA oligonucleotide comprises two constrained nucleotides at the 3' end and three constrained nucleotides at the 5' end.

[0161] Embodiment 7 The compound of embodiment 1, wherein the second DNA oligonucleotide comprises three constrained nucleotides at the 3' end and two constrained nucleotides at the 5' end.

[0162] Embodiment 8. The compound of any one of embodiments 3-7, wherein the two constrained nucleotides are consecutive.

[0163] Embodiment 9. The compound of any one of embodiments 3-7, wherein the two constrained nucleotides alternate.

[0164] Embodiment 10 The compound of embodiment 1, wherein the second DNA oligonucleotide comprises three constrained nucleotides at the 3' end and three constrained nucleotides at the 5' end.

[0165] Embodiment 11. The compound of embodiment 6, 7, or 9, wherein the three constrained nucleotides are consecutive.

[0166] Embodiment 12 The compound of embodiment 6, 7, or 9, wherein the constrained nucleotides are alternating.

[0167] Embodiment 13 The compound of any one of embodiments 1 to 12, wherein the constrained nucleotide is an LNA modified nucleotide.

[0168] Embodiment 14 The compound of any one of embodiments 1 to 12, wherein the constrained nucleotide is a cMOE-modified nucleotide.

[0169] Embodiment 15. The compound of any one of embodiments 1 to 12, wherein the constrained nucleotide is a cET-modified nucleotide.

[0170] Embodiment 16 The compound of any one of embodiments 1 to 12, wherein the constrained nucleotide is an LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide.

[0171] Embodiment 17. The compound of any one of embodiments 1 to 16, wherein the first DNA oligonucleotide comprises from about 10 unmodified nucleotides to about 22 unmodified nucleotides, and the second DNA oligonucleotide comprises from about 10 nucleotides to about 22 nucleotides.

[0172] Embodiment 18. The compound of embodiment 17, wherein the first DNA oligonucleotide comprises from about 14 unmodified nucleotides to about 18 unmodified nucleotides, and the second DNA oligonucleotide comprises from about 14 nucleotides to about 18 nucleotides.

[0173] Embodiment 19. The compound of any one of embodiments 1 to 18, wherein the first DNA oligonucleotide and the second DNA oligonucleotide contain the same number of nucleotides.

[0174] Embodiment 20. The compound of any one of embodiments 1 to 19, wherein the second DNA oligonucleotide comprises nucleotides with phosphorothioate modifications.

[0175] Embodiment 21. The compound of any one of embodiments 1 to 20, wherein the second DNA oligonucleotide further comprises a nucleotide having a modification selected from the group consisting of a 2'-O-aminopropyl group, a 2'-O-ethyl group, a 2'-fluoro group, a 2'-O-methyl group, a 2'-deoxy-2'fluoro group, a 2'-O-methoxyethyl group, a 2'-O-allyl group, a 2'-O-propyl group, a 2'-O-pentyl group, and a constrained nucleotide.

[0176] Embodiment 22 The compound of any one of embodiments 1 to 21, wherein the second DNA oligonucleotide further comprises a spacer modification.

[0177] Embodiment 23 The compound of any one of embodiments 1 to 22, wherein the second DNA oligonucleotide is an antisense oligonucleotide.

[0178] Embodiment 24 The compound of embodiment 23, wherein the antisense oligonucleotide is a STAT3 antisense oligonucleotide.

[0179] Embodiment 25 The compound of embodiment 24, wherein the STAT3 antisense oligonucleotide comprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:35, or SEQ ID NO:36.

[0180] Embodiment 26 The compound of embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NO:1 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:2.

[0181] Embodiment 27 The compound of embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NO:3 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:4.

[0182] Embodiment 28 The compound of embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NO:5 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:6.

[0183] Embodiment 29 The compound of embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NO:7 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:8.

[0184] Embodiment 30 The compound of embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NO:9 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:10.

[0185] Embodiment 31 The compound of embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NO:11 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:12.

[0186] Embodiment 32 The compound of embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NO: 13 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 14.

[0187] Embodiment 33 The compound of embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NO: 1 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 35.

[0188] Embodiment 34 The compound of embodiment 24, wherein the first DNA oligonucleotide comprises SEQ ID NO:1 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:36.

[0189] Embodiment 35 The compound of any one of embodiments 1 to 34, wherein the phosphorothioated CpG oligodeoxynucleotide is a class A CpG oligodeoxynucleotide, a class B CpG oligodeoxynucleotide, or a class C CpG oligodeoxynucleotide.

[0190] Embodiment 36. The compound of any one of embodiments 1 to 34, wherein the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32.

[0191] Embodiment 37 The compound of embodiment 36, wherein the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO: 43.

[0192] Embodiment 38 The compound of embodiment 36, wherein the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO: 15.

[0193] Embodiment 39 The compound of any one of embodiments 1 to 38, wherein the first DNA oligonucleotide is a passenger strand and the second DNA oligonucleotide is a guide strand.

[0194] Embodiment 40 The compound of any one of embodiments 1 to 39, wherein the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide.

[0195] Embodiment 41 The compound of any one of embodiments 1 to 39, wherein the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 3' end of the first DNA oligonucleotide.

[0196] Embodiment 42. The compound of embodiment 1, wherein the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO: 43, the first DNA oligonucleotide comprises SEQ ID NO: 1, and the second DNA oligonucleotide comprises SEQ ID NO: 2, and the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide.

[0197] Embodiment 43 The compound of embodiment 1, wherein the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO: 15, the first DNA oligonucleotide comprises SEQ ID NO: 3, and the second DNA oligonucleotide comprises SEQ ID NO: 4, and the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide.

[0198] Embodiment 44. The compound of any one of embodiments 1 to 43, wherein the linking group comprises a bond, a nucleic acid, a substituted or unsubstituted alkylene, a substituted or unsubstituted heteroalkylene, a substituted or unsubstituted cycloalkylene, a substituted or unsubstituted heterocycloalkylene, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene, or a combination of two or more thereof.

[0199] Embodiment 45. The compound of embodiment 44, wherein the linking group comprises a substituted 6- to 60-membered heteroalkylene.

[0200] Embodiment 46. The linking group has the formula:

[0201] [ka] 46. ​​The compound of embodiment 45, comprising a substituted heteroalkylene of the formula: wherein n is an integer from 1 to 10.

[0202] Embodiment 47. The compound of embodiment 46, wherein n is 5.

[0203] Embodiment 48. A pharmaceutical composition comprising a compound of any one of embodiments 1 to 47 and a pharmaceutically acceptable excipient.

[0204] Embodiment 49. A method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a compound of any one of embodiments 1 to 47, or a pharmaceutical composition of embodiment 48.

[0205] Embodiment 50. The method of embodiment 49, wherein the cancer is a glioma, a cranial primitive neuroectodermal tumor, an ependymoma tumor, a hemangiopericytoma, a germ cell tumor, a pineal tumor, or a primary central nervous system lymphoma.

[0206] Embodiment 51. The method of embodiment 50, wherein the central nervous system cancer is a glioma, a cranial primitive neuroectodermal tumor, an ependymoma tumor, a hemangiopericytoma, a germ cell tumor, a pineal tumor, or a primary central nervous system lymphoma.

[0207] Embodiment 52. The method of embodiment 51, wherein the glioma is an astrocytoma, glioblastoma, or oligodendroglioma, the cranial primitive neuroectodermal tumor is a medulloblastoma, cerebral neuroblastoma, pineoblastoma, or nasal neuroblastoma, and the ependymoma tumor is an ependymoma, myxopapillary ependymoma, or subependymoma.

[0208] Embodiment 53 The method of embodiment 49, wherein the cancer is glioma.

[0209] Embodiment 54. The method of embodiment 49, wherein the cancer is leukemia.

[0210] Embodiment 55 The method of embodiment 49, wherein the cancer is a solid tumor cancer.

[0211] Embodiment 56. The method of embodiment 49, wherein the cancer is prostate cancer, breast cancer, colorectal cancer, bladder cancer, lung cancer, liver cancer, pancreatic cancer, kidney cancer, gastric cancer, or melanoma.

[0212] Embodiment 57. The method of embodiment 49, wherein the cancer is prostate cancer.

[0213] Embodiment 58. The method of embodiment 49, wherein the cancer is epidermoid carcinoma.

[0214] Embodiment 59. The method of embodiment 58, wherein the epidermoid cancer is thyroid cancer, esophageal cancer, vaginal cancer, anal cancer, cervical cancer, or head and neck cancer.

[0215] Embodiment 60 The method of any one of embodiments 49-59, further comprising administering to the patient an effective amount of an immune checkpoint inhibitor.

[0216] Embodiment 61 The method of embodiment 60, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

[0217] Embodiment 62. The method of embodiment 61, wherein the PD-1 inhibitor is pembrolizumab, nivolumab, cemiplimab, dostallimab, camrelizumab, sintilimab, tislelizumab, toripalimab, spartalizumab, retifanlimab, pimivalimab, AMP-224, or MEDI0680.

[0218] Embodiment 63 The method of embodiment 60, wherein the immune checkpoint inhibitor is a PD-L1 inhibitor.

[0219] Embodiment 64. The method of embodiment 63, wherein the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, embafolimab, cosibelimab, AUNP12, CA-170, or BMS-986189.

[0220] Embodiment 65. A pharmaceutical composition comprising a compound of any one of embodiments 1 to 47 and an immune checkpoint inhibitor.

[0221] Embodiment 66 The pharmaceutical composition of embodiment 65, wherein the immune checkpoint inhibitor is a PD inhibitor or a PD-L1 inhibitor.

[0222] Embodiment 67. A STAT3 antisense oligonucleotide comprising SEQ ID NO:4.

[0223] Embodiments N1 to N23 Embodiment N1. A compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide hybridized to a second DNA oligonucleotide, wherein (a) the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides, and (b) the second DNA oligonucleotide comprises (i) from about 5 nucleotides to about 70 nucleotides, and (ii) a constrained nucleotide at the 3' end and a constrained nucleotide at the 5' end.

[0224] Embodiment N2. The compound of embodiment N1 wherein the second DNA oligonucleotide comprises one constrained nucleotide at the 3' end and one constrained nucleotide at the 5' end.

[0225] Embodiment N3. The compound of embodiment N1, wherein (i) the second DNA oligonucleotide comprises one constrained nucleotide at the 3'-terminus and two constrained nucleotides at the 5'-terminus, (ii) the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-terminus and one constrained nucleotide at the 5'-terminus, (iii) the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-terminus and two constrained nucleotides at the 5'-terminus, (iv) the second DNA oligonucleotide comprises two constrained nucleotides at the 3'-terminus and three constrained nucleotides at the 5'-terminus, (v) the second DNA oligonucleotide comprises three constrained nucleotides at the 3'-terminus and two constrained nucleotides at the 5'-terminus, or (vi) the second DNA oligonucleotide comprises three constrained nucleotides at the 3'-terminus and three constrained nucleotides at the 5'-terminus.

[0226] Embodiment N4. The compound of embodiment N3, wherein the two or three constrained nucleotides are consecutive or alternating.

[0227] Embodiment N5. The compound of any one of Embodiments N1-N4, wherein the constrained nucleotide is an LNA-, cMOE-, or cET-modified nucleotide.

[0228] Embodiment N6. The compound of any one of embodiments N1 to N5, wherein the first DNA oligonucleotide comprises from about 10 unmodified nucleotides to about 22 unmodified nucleotides and the second DNA oligonucleotide comprises from about 10 nucleotides to about 22 nucleotides.

[0229] Embodiment N7. The compound of any one of embodiments N1-N6, wherein the second DNA oligonucleotide comprises nucleotides with phosphorothioate modifications.

[0230] Embodiment N8. The compound of any one of embodiments N1 to N7, wherein the second DNA oligonucleotide further comprises a nucleotide having a modification selected from the group consisting of a 2'-O-aminopropyl group, a 2'-O-ethyl group, a 2'-fluoro group, a 2'-O-methyl group, a 2'-deoxy-2'fluoro group, a 2'-O-methoxyethyl group, a 2'-O-allyl group, a 2'-O-propyl group, a 2'-O-pentyl group, and a constrained nucleotide.

[0231] Embodiment N9. The compound of any one of embodiments N1-N8, wherein the second DNA oligonucleotide is an antisense oligonucleotide.

[0232] Embodiment N10. The compound of any one of embodiments N1-N8, wherein the second DNA oligonucleotide is a STAT3 antisense oligonucleotide.

[0233] Embodiment N11. The compound of embodiment N10, wherein the STAT3 antisense oligonucleotide comprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:35, or SEQ ID NO:36.

[0234] Embodiment N12. (a) the first DNA oligonucleotide comprises SEQ ID NO:1 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:2; (b) the first DNA oligonucleotide comprises SEQ ID NO:3 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:4; (c) the first DNA oligonucleotide comprises SEQ ID NO:5 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:6; (d) the first DNA oligonucleotide comprises SEQ ID NO:7 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:8; or (e) the first DNA oligonucleotide comprises SEQ ID NO:9 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:10. The compound of any one of embodiments N1 to N11, wherein the nucleotide comprises SEQ ID NO: 10; (f) the first DNA oligonucleotide comprises SEQ ID NO: 11 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 12; (g) the first DNA oligonucleotide comprises SEQ ID NO: 13 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 14; (h) the first DNA oligonucleotide comprises SEQ ID NO: 1 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 35; or (i) the first DNA oligonucleotide comprises SEQ ID NO: 1 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 36.

[0235] Embodiment N13. The compound of any one of embodiments N1-N12, wherein the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32.

[0236] Embodiment N14. The compound of any one of embodiments N1 to N13, wherein the first DNA oligonucleotide is the passenger strand and the second DNA oligonucleotide is the guide strand.

[0237] Embodiment N15. The compound of any one of embodiments N1 to N14, wherein the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide, or the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 3' end of the first DNA oligonucleotide.

[0238] Embodiment N16. The compound of embodiment N1, wherein (a) the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43, the first DNA oligonucleotide comprises SEQ ID NO:1, and the second DNA oligonucleotide comprises SEQ ID NO:2, and the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide, or (b) the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:15, the first DNA oligonucleotide comprises SEQ ID NO:3, and the second DNA oligonucleotide comprises SEQ ID NO:4, and the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide.

[0239] Embodiment N17. A pharmaceutical composition comprising a compound of any one of embodiments N1-N16 and a pharmaceutically acceptable excipient.

[0240] Embodiment N18. A method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a compound of any one of embodiments N1 to N16, or a pharmaceutical composition of embodiment N17.

[0241] Embodiment N19. The method of embodiment N18, wherein the cancer is glioma.

[0242] Embodiment N20. The method of embodiment N18, wherein the cancer is central nervous system cancer, leukemia, prostate cancer, breast cancer, colorectal cancer, bladder cancer, lung cancer, liver cancer, pancreatic cancer, kidney cancer, gastric cancer, melanoma, or epidermoid cancer.

[0243] Embodiment N21. The method of any one of embodiments N18-N20, further comprising administering to the patient an effective amount of an immune checkpoint inhibitor.

[0244] Embodiment N22. The method of embodiment N21, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

[0245] Embodiment N23. The method of embodiment N22, wherein (i) the PD-1 inhibitor is pembrolizumab, nivolumab, cemiplimab, dostallimab, camrelizumab, sintilimab, tislelizumab, toripalimab, spartalizumab, retifanlimab, pimivalimab, AMP-224, or MEDI0680, and (ii) the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, embafolimab, cosibelimab, AUNP12, CA-170, or BMS-986189. [Example]

[0246] It is understood that the examples described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the appended claims.

[0247] The application of immunostimulatory oligonucleotides to brain tumors is limited by the potential for immunotoxicity. Here, we developed a TLR9-targeting double-stranded STAT3 antisense oligonucleotide (CpG-STAT3dsASO) with optimized stability and efficacy for cancer immunotherapy.

[0248] Example 1 To enhance STAT3 knockdown in glioma cells, we used locked nucleic acid (LNA) chemistry within the ASO portion of the molecule. As shown in Figure 1, double-stranded ASO molecules containing gapmer ASOs hybridized to complementary, non-phosphorothioate DNA strands were stable in human serum (Figure 1A) and resulted in comparable target gene knockdown as single-stranded ASOs in various cancer cell lines (Figure 1B-1D). Similar effects were observed with dsASOs targeting STAT3 and a different gene specific for the androgen receptor in prostate cancer cells (Figure 1C).

[0249] Using this design, we generated a TLR9-targeting CpG-dsASO conjugate (Figure 2A). The conjugate exhibited a half-life of more than 2 days in human serum and remained detectable after 5 days of incubation (Figure 2B). We confirmed that the CpG-STAT3 dsASO had similar target gene knockdown potency and kinetics to that of the single-stranded STAT3 ASO alone in cancer cells, including prostate cancer, epidermoid carcinoma, glioma cells, and mouse macrophages (Figures 2C-D, 3). All chemically modified and fluorescently labeled CpG-STAT3 ASO variants were rapidly internalized by human and mouse glioma and bone marrow cells in vitro (Figures 4-6). While none of the STAT3 ASO variants was internalized by human T cells, mouse T cells showed low internalization at high oligonucleotide concentrations only at longer incubation times. Biodistribution experiments in mice bearing intracranial gliomas using intratumoral oligonucleotide injections confirmed highly efficient uptake by various myeloid cells in the brain, including macrophages, microglia, DCs, and MDSCs (Figure 7).

[0250] Example 2 We compared the potential toxicity of intracranial administration of single-stranded and double-stranded CpG-STAT3 ASOs (Figure 8). Repeated four injections of CpG-STAT dsASO (0.1–1 mg / kg) were well tolerated, with approximately 10% weight loss at the highest dose of 1 mg / kg. In contrast, administration of 0.3 mg / kg CpG-STAT3 ssASO already resulted in approximately 25% weight loss in mice, which is considered an adverse effect (Figure 8A). Assessment of acute neurotoxicity based on mouse phenotypic behavior confirmed that IC injection of CpG-STAT dsASO was significantly better tolerated than the standard single-stranded conjugate (Figure 8B). These observations were also associated with a significant increase in platelet levels in mice treated with the single-stranded CpG-STAT3 ASO conjugate but not with the double-stranded CpG-STAT3 ASO conjugate (Figure 8C).

[0251] Example 3 The antitumor efficacy of the benchmark 2'O-methyl-modified CpG-STAT3 ASO was compared with single-stranded and double-stranded LNA-modified CpG-STAT3 ASOs. As shown in Figure 9, both LNA-modified CpG-STAT3 ASOs reduced the progression of orthotopic human U251 gliomas and improved survival in immunocompromised mice, whereas the benchmark molecule did not. In the absence of an immune system, mice well tolerated 1 mg / kg injections of single-stranded and double-stranded LNA-modified CpG-STAT3 ASOs. The antitumor efficacy of treatment was evaluated in a syngeneic GL261 model in immunocompetent mice (Figure 10A). Local administration of all tested single-stranded and double-stranded CpG-STAT3 ASOs improved animal survival and induced immune activation in immunocompetent mice. All three types of CpG-STAT3 ASO injections induced the maturation / activation of intratumoral DCs, macrophages, and microglia, but reduced the number of tumor-associated M2 macrophages and resting microglia as assessed by flow cytometry (Figure 10B). Importantly, CpG-STAT3 ASO injections improved the ratio of intratumoral CD8 T cells to Tregs (Figure 10C).

[0252] Example 4 To further improve therapeutic efficacy, LNA-modified CpG-STAT3dsASO treatment was combined with systemic administration of a PD1-blocking antibody. As shown in Figure 11, LNA-modified CpG-STAT3dsASO sensitized GL261 glioma-bearing mice to immune checkpoint inhibition, resulting in synergistic effects and complete tumor regression in the majority of treated mice after 2 weeks of treatment. Combined treatment resulted in long-term tumor-free survival of 5 / 6 treated mice for over 150 days. Furthermore, locally injected LNA-modified CpG-STAT3dsASO also demonstrated efficacy against RM9 prostate tumors in mice, with evidence of an abscopal effect (Figure 12).

[0253] Example 5 The activity of LNA-modified CpG-STAT3 dsASO was validated in a novel model of neural stem cell-derived immune checkpoint-resistant QPP8 (Qk / Tp53 / Pten-del) glioma. As shown in Figure 13, LNA-modified CpG-STAT3 dsASO demonstrated improved antitumor efficacy as a single agent compared with single-chain LNA-modified CpG-STAT3 ASO. When combined with a PD1-specific antibody, LNA-modified CpG-STAT3 dsASO resulted in complete regression of QPP8 glioma in the majority of treated mice (Figure 14). Neither anti-PD1 alone nor LNA-modified CpG-STAT3 dsASO resulted in eradication of glioma, but both improved mouse survival. To confirm that surviving mice developed protective antitumor immunity, we used mice that rejected tumors in the study shown in Figure 11. The majority of these mice resisted rechallenge with the same type of glioma, whereas all age-matched untreated mice developed tumors (FIG. 15).

[0254] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application are expressly incorporated herein by reference in their entirety for all purposes.

Claims

1. 1. A compound comprising a phosphorothioated CpG oligodeoxynucleotide linked to a first DNA oligonucleotide that is hybridized to a second DNA oligonucleotide, (a) the first DNA oligonucleotide comprises from about 5 unmodified nucleotides to about 70 unmodified nucleotides; (b) the second DNA oligonucleotide is (i) from about 5 nucleotides to about 70 nucleotides, and (ii) A compound comprising a 3'-terminally constrained nucleotide and a 5'-terminally constrained nucleotide.

2. The compound of claim 1 , wherein the second DNA oligonucleotide comprises one constrained nucleotide at the 3′ end and one constrained nucleotide at the 5′ end.

3. (i) the second DNA oligonucleotide comprises one constrained nucleotide at the 3' end and two constrained nucleotides at the 5' end; (ii) the second DNA oligonucleotide comprises two constrained nucleotides at the 3' end and one constrained nucleotide at the 5' end; (iii) the second DNA oligonucleotide comprises two constrained nucleotides at the 3' end and two constrained nucleotides at the 5' end; (iv) the second DNA oligonucleotide comprises two constrained nucleotides at the 3' end and three constrained nucleotides at the 5' end; (v) the second DNA oligonucleotide comprises three constrained nucleotides at the 3' end and two constrained nucleotides at the 5' end; or (vi) The compound of claim 1, wherein the second DNA oligonucleotide comprises three constrained nucleotides at the 3' end and three constrained nucleotides at the 5' end.

4. The compound of claim 3 , wherein the two or three constrained nucleotides are consecutive or alternating.

5. 2. The compound of claim 1, wherein the constrained nucleotide is an LNA-modified nucleotide, a cMOE-modified nucleotide, or a cET-modified nucleotide.

6. 2. The compound of claim 1, wherein the first DNA oligonucleotide comprises from about 10 unmodified nucleotides to about 22 unmodified nucleotides and the second DNA oligonucleotide comprises from about 10 nucleotides to about 22 nucleotides.

7. The compound of claim 1 , wherein the second DNA oligonucleotide comprises nucleotides having phosphorothioate modifications.

8. 2. The compound of claim 1, wherein the second DNA oligonucleotide further comprises a nucleotide having a modification selected from the group consisting of a 2'-O-aminopropyl group, a 2'-O-ethyl group, a 2'-fluoro group, a 2'-O-methyl group, a 2'-deoxy-2'fluoro group, a 2'-O-methoxyethyl group, a 2'-O-allyl group, a 2'-O-propyl group, a 2'-O-pentyl group, and a constrained nucleotide.

9. The compound of claim 1 , wherein the second DNA oligonucleotide is an antisense oligonucleotide.

10. The compound of claim 1 , wherein the second DNA oligonucleotide is a STAT3 antisense oligonucleotide.

11. 11. The compound of claim 10, wherein the STAT3 antisense oligonucleotide comprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:35, or SEQ ID NO:

36.

12. (a) the first DNA oligonucleotide comprises SEQ ID NO: 1 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 2; or (b) the first DNA oligonucleotide comprises SEQ ID NO: 3 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 4; or (c) the first DNA oligonucleotide comprises SEQ ID NO:5 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:6; or (d) the first DNA oligonucleotide comprises SEQ ID NO: 7 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 8; or (e) the first DNA oligonucleotide comprises SEQ ID NO: 9 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 10; or (f) the first DNA oligonucleotide comprises SEQ ID NO: 11 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 12; or (g) the first DNA oligonucleotide comprises SEQ ID NO: 13 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 14; or (h) the first DNA oligonucleotide comprises SEQ ID NO: 1 and the STAT3 antisense oligonucleotide comprises SEQ ID NO: 35; or (i) The compound of claim 1, wherein the first DNA oligonucleotide comprises SEQ ID NO: 1 and the STAT3 antisense oligonucleotide comprises SEQ ID NO:

36.

13. 2. The compound of claim 1, wherein the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:

32.

14. The compound of claim 1 , wherein the first DNA oligonucleotide is a passenger strand and the second DNA oligonucleotide is a guide strand.

15. 2. The compound of claim 1, wherein the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide, or the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 3' end of the first DNA oligonucleotide.

16. (a) the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO:43, the first DNA oligonucleotide comprises SEQ ID NO:1, the second DNA oligonucleotide comprises SEQ ID NO:2, and the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide; or (b) The compound of claim 1, wherein the phosphorothioated CpG oligodeoxynucleotide comprises SEQ ID NO: 15, the first DNA oligonucleotide comprises SEQ ID NO: 3, the second DNA oligonucleotide comprises SEQ ID NO: 4, and the 3' end of the phosphorothioated CpG oligodeoxynucleotide is linked to the 5' end of the first DNA oligonucleotide.

17. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable excipient.

18. 10. A method of treating cancer in a patient in need thereof, comprising administering to said patient an effective amount of a compound of claim 1.

19. 19. The method of claim 18, wherein the cancer is glioma.

20. 19. The method of claim 18, wherein the cancer is central nervous system cancer, leukemia, prostate cancer, breast cancer, colorectal cancer, bladder cancer, lung cancer, liver cancer, pancreatic cancer, kidney cancer, gastric cancer, melanoma, or epidermoid cancer.

21. 20. The method of claim 18, further comprising administering to the patient an effective amount of an immune checkpoint inhibitor.

22. The method of claim 21, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

23. (i) the PD-1 inhibitor is pembrolizumab, nivolumab, cemiplimab, dostallimab, camrelizumab, sintilimab, tislelizumab, toripalimab, spartalizumab, retifanlimab, pimivalimab, AMP-224, or MEDI0680; (ii) The method of claim 22, wherein the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, embafolimab, cosibelimab, AUNP12, CA-170, or BMS-986189.

24. A STAT3 antisense oligonucleotide comprising SEQ ID NO:4.

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