Spherical nucleic acids for modulation of the cGAS-STING and STAT3 pathways for the immunotherapeutic treatment of cancer

Abstract

The present disclosure is generally directed to spherical nucleic acids (SNAs), which are nanostructures having a core surrounded by radial presentation of oligonucleotides capable of activating cytoplasmic DNA sensors, including but not limited to, cyclic GMP-AMP synthase (cGAS). In some embodiments, the SNA also inactivates transcription factors, including but not limited to, signal transduction and activator of transcription 3 (STAT3). Methods of making and using SNAs are also provided herein. In some aspects, the present disclosure provides spherical nucleic acids (SNAs), comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides bound to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a double-stranded or single-stranded stem-loop DNA oligonucleotide that activates cyclic GMP-AMP synthase (cGAS) and is at least 15 base pairs in length.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 280,499, filed November 17, 2021, which is incorporated herein by reference in its entirety.

[0002] Statement of Government Interest This invention was made with Government support under Grant Nos. 1R01CA208783-01A1 and 5U54CA199091-05 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Submitted Materials A sequence listing, which is part of this disclosure, is submitted as a text file concurrently with the specification. The text file containing the sequence listing is named "2021-221_sequence_listing.xml", was created on November 17, 2022, and is 9,693 bytes in size. The contents of the sequence listing are incorporated herein by reference.

[0004] The present disclosure generally relates to spherical nucleic acids (SNAs), which are nanostructures having a core surrounded by radial presentation of oligonucleotides capable of activating cyclic GMP-AMP synthase (cGAS). In some embodiments, the SNAs also inactivate signal transducer and activator of transcription 3 (STAT3). Methods of making and using the SNAs are also provided herein. [Background technology]

[0005] Activation of the stimulator of interferon genes (STING) pathway represents one of the major immune sensing mechanisms promoting innate and adaptive immune responses against tumors 1Tumor-derived DNA is recognized by cyclic GMP-AMP synthase (cGAS) in antigen-presenting cells. Upon nucleic acid recognition, cGAS generates the cyclic dinucleotide GMP-AMP (cGAMP). cGAMP then binds to and activates the adaptor protein STING, inducing interferon regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB)-dependent transcription to promote the activation of natural killer (NK) cells, proinflammatory macrophages, and T cells. 1 . Summary of the Invention

[0006] Intratumoral administration of STING-antagonistic cyclic dinucleotides (CDNs) antagonizes tumor progression in multiple cancer models, including an orthotopic glioblastoma (GBM) model. 2 Currently being tested in Phase I clinical trials in patients with advanced non-CNS cancers. However, limited stability and bioavailability combined with poor lipophilicity limit clinical CDN development, especially considering non-invasive therapeutic approaches.

[0007] Spherical nucleic acids (SNAs) are modular structures that contain a nanoparticle core densely functionalized with a shell of radially oriented oligonucleotides. 3 The unique three-dimensional architecture confers enhanced resistance to nuclease-mediated degradation, allowing for reliable cell penetration and bioavailability without the need for auxiliary delivery vehicles. siRNA ) 4 , and CpG-rich DNA oligonucleotides (SNA) in solid tumors CpG ) bearing toll-like receptor (TLR) 9 antagonistic SNA 5、6 Early clinical trials have provided the first clinical evidence that SNAs represent a safe, brain-penetrant therapy for gene regulation and immune stimulation in GBM and other solid tumors.

[0008] Advantages of the technology provided herein include, but are not limited to: Interferon-stimulating (ISD) oligonucleotides (SNAs) were used to directly activate cGAS. ISD ) conjugated to SNAs are superior to STING-targeting CDNs currently under clinical development, especially when considering non-invasive delivery approaches. cGAS and STAT3 proteins (SNA ISD / STAT3i ), concomitantly targeting orthogonal signaling pathways to achieve more effective antitumor immunity.

[0009] Thus, in some aspects, the present disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a double-stranded DNA oligonucleotide or a single-stranded stem-loop DNA oligonucleotide that activates cyclic GMP-AMP synthase (cGAS) and is at least 15 base pairs in length. In some aspects, the present disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a double-stranded DNA oligonucleotide that activates cyclic GMP-AMP synthase (cGAS) and is at least 15 base pairs in length. In some aspects, the present disclosure provides a spherical nucleic acid (SNA), comprising: (a) a nanoparticle core; and (b) an oligonucleotide shell attached to the outer surface of the nanoparticle core, the oligonucleotide shell comprising a single-stranded stem-loop DNA oligonucleotide that activates cyclic GMP-AMP synthase (cGAS) and is at least 15 base pairs in length. In some embodiments, the oligonucleotide shell comprises a plurality of double-stranded DNA oligonucleotides and / or single-stranded stem-loop DNA oligonucleotides, each of which activates cGAS and is at least 15 base pairs in length. In some embodiments, the oligonucleotide shell comprises a plurality of double-stranded DNA oligonucleotides, single-stranded stem-loop DNA oligonucleotides, or combinations thereof, each of which activates cGAS and is at least 15 base pairs in length. In some embodiments, the oligonucleotide shell consists of a plurality of double-stranded DNA oligonucleotides and / or single-stranded stem-loop DNA oligonucleotides, each of which activates cGAS and is at least 15 base pairs in length.In some embodiments, the oligonucleotide shell is comprised of a plurality of double-stranded DNA oligonucleotides, single-stranded stem-loop DNA oligonucleotides, or combinations thereof, each of which activates cGAS and is at least 15 base pairs in length. In some embodiments, the oligonucleotide shell is comprised of a plurality of double-stranded DNA oligonucleotides, each of the plurality of double-stranded DNA oligonucleotides comprising one strand comprising SEQ ID NO:3 and another strand comprising SEQ ID NO:4. In some embodiments, the oligonucleotide shell is comprised of a plurality of double-stranded DNA oligonucleotides, each of the plurality of double-stranded DNA oligonucleotides comprising one strand comprising SEQ ID NO:3 and another strand comprising SEQ ID NO:4. In some embodiments, the double-stranded DNA oligonucleotides and / or single-stranded stem-loop DNA oligonucleotides inactivate signal transducer and activator of transcription 3 (STAT3). In some embodiments, the double-stranded DNA oligonucleotides inactivate signal transducer and activator of transcription 3 (STAT3). In some embodiments, the single-stranded stem-loop DNA oligonucleotides inactivate signal transducer and activator of transcription 3 (STAT3). In some embodiments, the shell of the oligonucleotide comprises a plurality of double-stranded DNA oligonucleotides and / or single-stranded stem-loop DNA oligonucleotides, each of which activates cGAS and inactivates signal transduction and activator of transcription 3 (STAT3) and is at least 15 base pairs in length. In some embodiments, the shell of the oligonucleotide comprises a plurality of double-stranded DNA oligonucleotides, each of which activates cGAS and inactivates signal transduction and activator of transcription 3 (STAT3) and is at least 15 base pairs in length. In some embodiments, the shell of the oligonucleotide comprises a plurality of single-stranded stem-loop DNA oligonucleotides, each of which activates cGAS and inactivates signal transduction and activator of transcription 3 (STAT3) and is at least 15 base pairs in length.In some embodiments, the oligonucleotide shell is comprised of a plurality of double-stranded DNA oligonucleotides and / or single-stranded stem-loop DNA oligonucleotides, each of which activates cGAS and inactivates signal transduction and activator of transcription 3 (STAT3) and is at least 15 base pairs in length. In further embodiments, the oligonucleotide shell is comprised of a plurality of double-stranded DNA oligonucleotides, each of which activates cGAS and inactivates signal transduction and activator of transcription 3 (STAT3) and is at least 15 base pairs in length. In some embodiments, the oligonucleotide shell is comprised of a plurality of single-stranded stem-loop DNA oligonucleotides, each of which activates cGAS and inactivates signal transduction and activator of transcription 3 (STAT3) and is at least 15 base pairs in length. In various embodiments, the nanoparticle core is a metal core, a semiconductor core, an insulator core, an upconverting core, a micelle core, a dendrimer core, a liposome core, a polymer core, a metal-organic framework core, a lipid nanoparticle core, a protein core, or a combination thereof. In further embodiments, the polymer is polylactide, polylactide-polyglycolide copolymer, polycaprolactone, polyacrylate, alginate, albumin, polypyrrole, polythiophene, polyaniline, polyethyleneimine, poly(methyl methacrylate), poly(lactic-co-glycolic acid) (PLGA), or chitosan. In various embodiments, the nanoparticle core comprises gold, silver, platinum, aluminum, palladium, copper, cobalt, indium, cadmium selenide, iron oxide, fullerene, metal-organic framework, silica, zinc sulfide, or nickel. In some embodiments, the lipid nanoparticle core comprises an ionizable lipid, a phospholipid, a sterol, and a lipid-polyethylene glycol (lipid-PEG) conjugate. In further embodiments, each oligonucleotide within the oligonucleotide shell is covalently attached to the exterior of the lipid nanoparticle core through a lipid-PEG conjugate. In some embodiments, the liposome core comprises a plurality of lipid groups.In further embodiments, the plurality of lipid groups comprises lipids selected from the group consisting of the phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine families of lipids. In various embodiments, the lipids are selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-dioleo ... 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE). In some embodiments, at least one oligonucleotide in the oligonucleotide shell is attached to the exterior of the liposome core or lipid nanoparticle core through a lipid anchor group. In some embodiments, the lipid anchor group is attached to the 5' terminus or 3' terminus of at least one oligonucleotide. In further embodiments, the lipid anchor group is tocopherol, DOPE lipid, or cholesterol. In further embodiments, the oligonucleotide shell comprises one or more additional oligonucleotides. In various embodiments, the one or more additional oligonucleotides comprise DNA, RNA, or a combination thereof. In further embodiments, the one or more additional oligonucleotides comprise single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, or a combination thereof. In still further embodiments, the one or more additional oligonucleotides are immunostimulatory oligonucleotides, inhibitory oligonucleotides, oligonucleotides that inactivate signal transducer and activator of transcription 3 (STAT3), or a combination thereof.In some embodiments, the inhibitory oligonucleotide is an antisense oligonucleotide, a small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme. In some embodiments, the immunostimulatory oligonucleotide is a CpG motif-containing oligonucleotide, a double-stranded DNA oligonucleotide, or a single-stranded RNA oligonucleotide. In some embodiments, the immunostimulatory oligonucleotide is a toll-like receptor (TLR) agonist. In various embodiments, the TLR is selected from the group consisting of toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 5 (TLR5), toll-like receptor 6 (TLR6), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), toll-like receptor 9 (TLR9), toll-like receptor 10 (TLR10), toll-like receptor 11 (TLR11), toll-like receptor 12 (TLR12), and toll-like receptor 13 (TLR13). In further embodiments, the inhibitory oligonucleotide is an antisense oligonucleotide, a small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme. In some embodiments, the SNA of the present disclosure further comprises an antigen. In some embodiments, the antigen is bound to one or more oligonucleotides within the shell of the oligonucleotide. In some embodiments, the antigen is bound to the surface of the SNA. In some embodiments, the antigen is encapsulated in the nanoparticle core. In various embodiments, the antigen is a tumor-associated antigen, a tumor-specific antigen, a neoantigen, or a combination thereof. In further embodiments, the antigen is OVA1, MSLN, P53, Ras, mutant IDH1 (IDH1R132H), a melanoma-associated antigen, an HPV-associated antigen, a prostate cancer-associated antigen, a glioblastoma antigen, a grade IV astrocytoma antigen, an ovarian cancer-associated antigen, a breast cancer-associated antigen, a hepatocellular carcinoma-associated antigen, a colon cancer-associated antigen, or a human papillomavirus (HPV) E7 nucleoprotein. In various embodiments, the SNA is about 1 to about 150 nanometers (nm) in diameter.In further embodiments, the oligonucleotide shell comprises from about 4 to about 250 oligonucleotides. In yet further embodiments, each oligonucleotide within the oligonucleotide shell is from about 15 to about 100 base pairs in length.

[0010] In some aspects, the present disclosure provides a composition comprising a plurality of spherical nucleic acids (SNAs) of the present disclosure.

[0011] In a further aspect, the present disclosure provides a method of generating an immune response against cancer in a subject, comprising administering to the subject an effective amount of a spherical nucleic acid (SNA) of the present disclosure, a composition of the present disclosure, or a combination thereof, thereby generating an immune response against cancer in the subject. In yet a further aspect, the present disclosure provides a method of treating and / or ameliorating cancer in a subject, comprising administering to the subject an effective amount of a SNA of the present disclosure, a composition of the present disclosure, or a combination thereof. In various embodiments, the cancer is breast cancer, peritoneal cancer, cervical cancer, colon cancer, rectal cancer, esophageal cancer, eye cancer, liver cancer, pancreatic cancer, laryngeal cancer, lung cancer, skin cancer, ovarian cancer, prostate cancer, gastric cancer, testicular cancer, thyroid cancer, brain cancer, or a combination thereof. In some embodiments, the cancer is glioblastoma. In various embodiments, the administering is by oral administration, topical administration, intravenous administration, intraarterial administration, mucosal administration, intraperitoneal administration, intramuscular administration, intratumoral administration, parenteral administration, intradermal administration, intranasal administration, subcutaneous administration, or a combination thereof. In some embodiments, the administering is by direct intracranial / intramyocellular administration. In some embodiments, the administering is combined with focused ultrasound (FUS), co-administration of microbubbles to temporarily open the blood-brain barrier, or a combination thereof. In some embodiments, the subject is female. In some embodiments, the subject is male. In some embodiments, the administering is by intranasal administration.

[0012] In a further aspect, the present disclosure provides a spherical nucleic acid (SNA), comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides bound to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a double-stranded DNA oligonucleotide or a single-stranded stem-loop DNA oligonucleotide that activates a cytoplasmic DNA sensor and is at least 15 base pairs in length. In some embodiments, the shell of oligonucleotides comprises a plurality of double-stranded DNA oligonucleotides and / or single-stranded stem-loop DNA oligonucleotides, each of which activates a cytoplasmic DNA sensor and is at least 15 base pairs in length. In some embodiments, the shell of oligonucleotides comprises a plurality of double-stranded DNA oligonucleotides and / or single-stranded stem-loop DNA oligonucleotides, each of which activates a cytoplasmic DNA sensor and is at least 15 base pairs in length. In further embodiments, the double-stranded DNA oligonucleotides and / or single-stranded stem-loop DNA oligonucleotides inactivate a transcription factor. In some embodiments, the shell of oligonucleotides comprises a plurality of double-stranded DNA oligonucleotides and / or single-stranded stem-loop DNA oligonucleotides, each of which activates a cytoplasmic DNA sensor and inactivates a transcription factor, and is at least 15 base pairs in length. In some embodiments, the shell of oligonucleotides comprises or consists of one or more G-quartet oligonucleotides. In further embodiments, the shell of oligonucleotides consists of a plurality of double-stranded DNA oligonucleotides and / or single-stranded stem-loop DNA oligonucleotides, each of which activates a cytoplasmic DNA sensor and inactivates a transcription factor, and is at least 15 base pairs in length. In various embodiments, the cytoplasmic DNA sensor is cyclic GMP-AMP synthase (cGAS), AIM2 (absent in melanoma-2), RNA polymerase III, DAI (DNA-dependent activator of IFN-regulatory factors), IFI16 (interferon-gamma-inducible protein 16), or a combination thereof. In any of the aspects or embodiments of the present disclosure, the transcription factor promotes cancer progression.In various embodiments, the transcription factor is signal transducer and activator of transcription 3 (STAT3), cMyc, NANOG, SOX2, OCT4, or a combination thereof. In some embodiments, the oligonucleotide shell comprises a plurality of double-stranded DNA oligonucleotides, each of the plurality of double-stranded DNA oligonucleotides comprising one strand comprising SEQ ID NO:3 and another strand comprising SEQ ID NO:4. In some embodiments, the oligonucleotide shell is comprised of a plurality of double-stranded DNA oligonucleotides, each of the plurality of double-stranded DNA oligonucleotides comprising one strand comprising SEQ ID NO:3 and another strand comprising SEQ ID NO:4. In various embodiments, the nanoparticle core is a metal core, a semiconductor core, an insulator core, an upconverting core, a micelle core, a dendrimer core, a liposome core, a polymer core, a metal-organic framework core, a lipid nanoparticle core, a protein core, or a combination thereof. In various embodiments, the polymer is polylactide, polylactide-polyglycolide copolymer, polycaprolactone, polyacrylate, alginate, albumin, polypyrrole, polythiophene, polyaniline, polyethyleneimine, poly(methyl methacrylate), poly(lactic-co-glycolic acid) (PLGA), or chitosan. In various embodiments, the nanoparticle core comprises gold, silver, platinum, aluminum, palladium, copper, cobalt, indium, cadmium selenide, iron oxide, fullerene, metal-organic framework, silica, zinc sulfide, or nickel. In some embodiments, the lipid nanoparticle core comprises an ionizable lipid, a phospholipid, a sterol, and a lipid-polyethylene glycol (lipid-PEG) conjugate. In some embodiments, each oligonucleotide in the oligonucleotide shell is covalently attached to the exterior of the lipid nanoparticle core through a lipid-PEG conjugate. In some embodiments, the liposome core comprises a plurality of lipid groups. In a further embodiment, the plurality of lipid groups comprises lipids selected from the group consisting of the phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine families of lipids.In still further embodiments, the lipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1 The oligonucleotide may be selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE). In various embodiments, at least one oligonucleotide in the oligonucleotide shell is attached to the exterior of the liposome core or lipid nanoparticle core through a lipid anchor group. In some embodiments, the lipid anchor group is attached to the 5' terminus or 3' terminus of at least one oligonucleotide. In some embodiments, the lipid anchor group is tocopherol, DOPE lipid, or cholesterol. In further embodiments, the oligonucleotide shell comprises one or more additional oligonucleotides. In various embodiments, the one or more additional oligonucleotides comprise DNA, RNA, or a combination thereof. In further embodiments, the one or more additional oligonucleotides comprise single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, or a combination thereof. In some embodiments, the one or more additional oligonucleotides are immunostimulatory oligonucleotides, inhibitory oligonucleotides, oligonucleotides that inactivate signal transducer and activator of transcription 3 (STAT3), or a combination thereof. In some embodiments, the inhibitory oligonucleotides are antisense oligonucleotides, small interfering RNA (siRNA), aptamers, short hairpin RNA (shRNA), DNAzymes, or aptazymes.In further embodiments, the immunostimulatory oligonucleotide is a CpG motif-containing oligonucleotide, a double-stranded DNA oligonucleotide, or a single-stranded RNA oligonucleotide. In some embodiments, the immunostimulatory oligonucleotide is a toll-like receptor (TLR) agonist. In various embodiments, the TLR is selected from the group consisting of toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 5 (TLR5), toll-like receptor 6 (TLR6), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), toll-like receptor 9 (TLR9), toll-like receptor 10 (TLR10), toll-like receptor 11 (TLR11), toll-like receptor 12 (TLR12), and toll-like receptor 13 (TLR13). In various embodiments, the inhibitory oligonucleotide is an antisense oligonucleotide, a small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme. In some embodiments, the SNA of the present disclosure further comprises an antigen. In various embodiments, the antigen is attached to one or more oligonucleotides within the shell of the oligonucleotide. In some embodiments, the antigen is attached to the surface of the SNA. In some embodiments, the antigen is encapsulated in the nanoparticle core. In various embodiments, the antigen is a tumor-associated antigen, a tumor-specific antigen, a neoantigen, or a combination thereof. In further embodiments, the antigen is OVA1, MSLN, P53, Ras, mutant IDH1 (IDH1R132H), mutant telomerase reverse transcriptase, melanoma-associated antigen, HPV-associated antigen, prostate cancer-associated antigen, glioblastoma antigen, grade IV astrocytoma antigen, ovarian cancer-associated antigen, breast cancer-associated antigen, hepatocellular carcinoma-associated antigen, colon cancer-associated antigen, or human papillomavirus (HPV) E7 nuclear protein. In various embodiments, the mutant telomerase reverse transcriptase is TERT;C228T, C250T, or a combination thereof. In some embodiments, the SNA is about 1 to about 150 nanometers (nm) in diameter. In some embodiments, the shell of oligonucleotides comprises about 4 to about 250 oligonucleotides.In some embodiments, each oligonucleotide within the shell of the oligonucleotide is about 15 to about 100 base pairs in length. In some aspects, the disclosure provides a composition comprising a plurality of spherical nucleic acids (SNAs) of the disclosure. In some aspects, the disclosure provides a method of generating an immune response against cancer in a subject, comprising administering to the subject an effective amount of a spherical nucleic acid (SNAs) of the disclosure, a composition of the disclosure, or a combination thereof, thereby generating an immune response against cancer in the subject. In some aspects, the disclosure provides a method of treating and / or ameliorating cancer in a subject, comprising administering to the subject an effective amount of a SNA of the disclosure, a composition of the disclosure, or a combination thereof. In various embodiments, the cancer is breast cancer, peritoneal cancer, cervical cancer, colon cancer, rectal cancer, esophageal cancer, eye cancer, liver cancer, pancreatic cancer, laryngeal cancer, lung cancer, skin cancer, ovarian cancer, prostate cancer, gastric cancer, testicular cancer, thyroid cancer, brain cancer, or a combination thereof. In some embodiments, the cancer is glioblastoma. In various embodiments, the administering is by direct intracranial / intramyocellular administration, oral administration, topical administration, intravenous administration, intraarterial administration, mucosal administration, intraperitoneal administration, intramuscular administration, intratumoral administration, parenteral administration, intradermal administration, intranasal administration, subcutaneous administration, or a combination thereof. In some embodiments, the subject is female. In some embodiments, the subject is male. In some embodiments, the administering is by intranasal administration. In further embodiments, the administering is combined with focused ultrasound (FUS), co-administration of microbubbles to temporarily open the blood-brain barrier, or a combination thereof. [Brief description of the drawings]

[0013] [Figure 1] We show that ISD45-SNA directly engages cGAS. (A) Schematic of SNA architecture. (B) Cell-free cGAS activation assay. (C) Reporter assay quantifying median IRF induction + / - standard deviation is shown. *p<0.05. [Diagram 2]Figure 1 shows that ISD45 SNA induces IRFs and proinflammatory cytokines. (A) ICP-MS to quantify cellular levels of gold in cells treated with ISD45-SNA. (B-D) Luciferase reporter assays to quantify IRF induction in RAW-Lucia™ macrophages (B), THP cells (C), and WT, cGAS-, STING-, and IRF-deficient RAW-Lucia™ macrophages. TFX, transfection agent. Median + / - standard deviation is shown. *p<0.05. (E) Multiplex ELISA-based and (F) antibody array-based cytokine profiling in RAW-Lucia™ macrophages. [Diagram 3] (A) Schematic of PLGA-SNAs showing that cGAS agonism is mediated by the oligonucleotide shell independent of core material and size. (B) Uptake of PLGACy5-based ISD45-SNAs in RAW Lucia™ macrophages pre-treated with cytochalasin D or fucoidan. (C) Luciferase reporter assay in RAW Lucia™ macrophages. (D) Dose-dependent IRF3 activation in response to PLGA-based ISD45-SNAs and AduroS100. [Figure 4]Figure 2 shows that ISD45-SNA promotes M1 macrophage polarization, tumor cell death, and survival in GBM-bearing mice. (A) Immunoblot of macrophage lysates showing the abundance of the indicated cGAS-STING-pathway components. (B) Nitrite levels determined in cell supernatants are shown at the bottom. (C) Growth curves of CT2A tumor cells co-cultured with macrophages or left untreated or exposed to supernatants from macrophages treated with ssDNA45-SNA or ISD45-SNA. (D) MTT viability assay of CT2A tumor cells incubated with macrophage supernatants. Median + / - standard deviation is shown. (E) H&E staining and MRI scan of the brain of tumor-bearing mice injected (via direct intratumoral administration) with Gd(III)-labeled SNA. (F) Kaplan-Meier survival curves of female BL6 / C57 mice inoculated with CT2A glioma tumor cells and treated with ssDNA45- or ISD45-SNAs via direct intratumoral administration. *p<0.05. [Diagram 5] Nose-to-brain delivery of ISD45-SNAs. (A) Schematic of the nasal cavity and olfactory / trigeminal pathways for drug delivery to the CNS. (B) ICP-MS analysis of gold in different organs including lung, liver, kidney, spleen, and nasal / oral cavity shows that gold ISD45-SNAs accumulate in the brain / brain tumor parenchyma. (C) Gold-based ISD45-SNAs labeled with Cy5. (D-E) Fluorescence microcopy of tumor cell or trigeminal nerve cross sections shows accumulation of Cy5-labeled ISD45-SNAs in the tumor and localized within the epineurium of the trigeminal nerve. (F) Schematic of Cy5-labeled PLGA-SNAs. Note, Cy5 is attached to the core. (G) IVIS images of the head (including nasal cavity) and brain. (H) Fluorescence microscopy of cross sections shows PLGA-SNACy5 accumulation in the olfactory bulb and in different tumor sections. (I) IVIS and fluorescence images of an isolated trigeminal nerve. Transverse and longitudinal sections are shown. (A) Adapted from Balyasnikova and colleagues, Expert Opinion Drug Delivery, 2018. [Figure 6]Figure 1 shows that the antitumor effect of intranasally delivered ISD45-SNA is STING-dependent and can be enhanced by checkpoint inhibition. (A-B) Quantification of bioluminescence in CT2A-bearing C57BL / 6wt mice (A) and STINGgt / gt mice (B). (C) Quantification of bioluminescence in CT2A-bearing C57BL / 6-wt mice left untreated or treated with either ssDNA45-SNA, ISD45-SNA, or CDN AduroS100. Mean + / - standard deviation is shown. (D-G) Kaplan-Meier survival analysis in CT2A-bearing C57BL / 6-wt mice (F), STINGgt / gt mice (E), and CT2A-bearing C57BL / 6-wt mice co-treated with SNA and checkpoint inhibitors (G). In panel D, overall survival time and survival time in female and male animal subjects are shown. In Panel E, median survival times are shown in days. [Figure 7] Figure 1 shows that ISD45-SNA antitumor effects can be enhanced by co-treatment with checkpoint inhibitors (CPIs). (A) Kaplan-Meier survival curves of CT2A tumor-bearing mice (total, female and male animal subjects). (B) Long-term survivors (females, identified in the experiment in panel A) were retested with CT2A tumor cells implanted in the contralateral hemisphere. Survival times were analyzed using the Kaplan-Meier method. [Figure 8] Immune profiles upon nose-to-brain delivery of ISD45-SNA. (A-B) Heatmaps comparing effector T cell (A) and TAM content (B) in tumors isolated from mice left untreated or treated intranasally with ssDNA45- and ISD45-SNA. (C-F) Minimum weight spanning trees (C) and histograms (D-F) showing enrichment of NK cell populations in deep cervical draining lymph nodes from mice treated with ISD45-SNA. Means + / - standard deviations are shown. *p<0.05. [Figure 9]Bimodal SNAISD / STAT3i. (A) Schematic of SNA architectures designed for bimodal cGAS and STAT3 modulation compared to monofunctional dsISD45-SNA. (B) STAT3 reporter assay to monitor STAT3 transactivation activity. (C) IRF reporter assay in RAW-Lucia macrophages. (D) Immunofluorescence microscopy showing subcellular distribution of phosphorylated STAT3. DAPI, nuclear counterstain. (E) Assessment of glioma stem cell sphere size upon treatment with different SNA architectures. (F) Kaplan-Meier survival curves of CT2A-bearing BL / 6 mice treated via ic administration of the indicated SNAs. Mean + / - standard deviation is shown. *p<0.05. [Figure 10]Figure 1 shows the development of a bimodal SNA for concomitant cGAS activation and STAT3 inhibition. (A) Sequences of oligonucleotides conjugated to gold nanoparticle cores for cGAS activation and STAT3 inhibition. ssDNA45 oligonucleotide was used as a negative control. Sequences: ssDNAT45=5'-TACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACA-3' (SEQ ID NO:5); ISD45 sense=5'-TACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACA-3' (SEQ ID NO:10); ISD45 antisense=5'-ACATCTAGTACATGTCTAGTCAGTATCTAGTGATCATCAGACA-3' (SEQ ID NO:6); STAT3i sense=5'-CTAAATGCCCTTTAC-3' (SEQ ID NO:7); STAT3i antisense=5'-GTAAAGGGCATTTAG-3' (SEQ ID NO:8); ISDG5STAT3i=5'-TACAACATTTCCCGTAAATCGGGGGGATTTA CGGGAAATGTTGTA-3' (SEQ ID NO: 9) (B-E) Modified gel shift assays and Hill-Langmuir computing to assess SNA binding to human recombinant cGAS (B-C) and STAT3 (D-E). (F-G) Cell-free and cell-based IRF3 reporter assays showing that all SNAs, i.e., ISD45-SNA, ISDG5STAT3i, and STAT3i15SNA architectures, induced IRF3 activation. (H) IL6 reporter assay in HEK-Blue IL6 reporter cells showing that ISD15STAT3i and ISDG5STAT3i-SNA, but not ISD45-SNA, suppress STAT3-driven IL-6 transactivation. [Figure 11]Sexual dimorphism underlying the antitumor effect of unimodal or bimodal SNAs is shown. (A) CT2A-bearing, (B) QPP7-bearing, or (C) QPP4-bearing C57BL / 6 male and female mice were treated with PBS, ssDNA45-SNA, ISD45-SNA, ISD15STAT3i, or ISDG5STAT3i-SNA via intracranial administration (15 μmol Au) dosed 8 days after tumor cell implantation. Keplan-Meier survival curves are shown. P values ​​were calculated using the log-rank (Mantel-Cox) test. *p<0.05. [Figure 12] Nanostring expression profiling reveals immune cell activation in female but not male animal subjects bearing QPP4 tumors following SNA treatment. Expression profiling was performed using the Nanostring nCounter PanCancer Immune Profiling Panel. Total RNA was isolated from tumor-infiltrating immune cells 30 days after tumor cell implantation. (A,B) Heatmap showing unsupervised clustering of z-score data for all 730 genes in treatment groups PBS, ssDNA45-SNA, ISD45-SNA, STAT3i15-SNA, ISDG5STAT3i-SNA in female and male hosts. Selected genes listed in the heatmap represent distinct clusters responsible for immune activation or immune suppression. (C,D) Nanostring gene set pathway analysis for male and female immune cell populations. (E) Heatmap of gene expression ranked by z-score for male vs. female mice (n=3 biological replicates) treated with ISDG5STAT3i-SNA. (F) Volcano plot of differentially expressed genes in females vs. males for ISDG5STAT3i-SNA treatment group (Log2 FC>1.5 and p-value<0.05). Key upregulated genes in females compared to males for ISDG5STAT3i-SNA treatment are shown. (G) Gene ontology analysis revealed enrichment of biological pathways in females compared to males for ISDG5STAT3i-SNA treatment group. Data were analyzed using ShinyGO tool (Ge SX, Jung D & Yao R, Bioinformatics 2020). [Figure 13] Figure 1 shows that cGAS protein is expressed at higher levels in female versus male mice. (A) Western blot analysis of spleen and (B) BMDM lysates from male or female mice. β-actin is shown as a loading control. (C) Flow cytometry-based quantification of cGAS protein levels in tumor-associated bone marrow cells (TAMCs) isolated from male or female PBS-, ssDNA45-SNA-, or ISD45-SNA-treated CT2A-bearing mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Vaccines, drugs, and engineered human cells that activate the immune system against tumors can improve outcomes and extend the lives of patients diagnosed with certain cancers, but fail to provide a survival benefit to patients with glioblastoma (GBM). Activation of the stimulator of interferon genes (STING) pathway represents one of the main innate immune sensing pathways that enables natural killer (NK) and T cell priming against tumors.

[0015] This disclosure shows that incorporation of oligonucleotides into SNA structures (e.g., presentation of oligonucleotides at high density on the surface of nanoparticles) leads to biochemical and biological properties that are fundamentally different from those of linear ("free") oligonucleotides. These properties include cellular uptake of SNAs by a wide variety of cells, gene regulatory activity of SNAs functionalized with siRNA or antisense DNA oligonucleotides, and TLR agonist activity of SNAs conjugated with immunostimulatory oligonucleotides. Clinical trials have recently been completed with first generation siRNA-based SNAs (NCT03020017; GBM) and toll-like receptor 9 (TLR9) agonist SNAs (NCT03086278; solid cancers).

[0016] Thus, in various aspects, the present disclosure provides cGAS agonist immunotherapy by targeting cGAS, a sensor of cytoplasmic dsDNA upstream of STING, with SNAs that present interferon-stimulating DNA (ISD) oligonucleotides at high surface density, and identifies SNAs as a novel class of immune stimulatory therapy for use in clinical neuro-oncology. ISD This approach is distinct from other current approaches that target the STING pathway with small molecules (including CDNs). Without wishing to be bound by theory, it is believed that targeting cGAS may enhance the ability to inhibit SNA. ISDThe strategy of using SNAs exploits the ability of cGAS to deliver dsDNA and trigger a STING response by inducing catalytic production of endogenous CDNs. The use of SNAs as provided herein addresses the challenge of therapeutic nucleic acid delivery through enhanced uptake of nucleic acids formulated as SNAs, and further exploits the multivalent presentation of oligonucleotides at high density on nanoparticle templates. Thus, as shown herein, binding of closely spaced adjacent dsDNA molecules on the surface of SNAs leads to strong cGAS activation. In some aspects, the present disclosure provides bimodal cGAS-activating and STAT3-inhibiting SNAs that can both induce a strong type of IFN response and sequester / inactivate STAT3 in the cytosol of cells to achieve effective anti-tumor immunity. In various embodiments, such bimodal SNAs include one or more cGAS-activating oligonucleotides, one or more oligonucleotides that inactivate STAT3, one or more oligonucleotides that activate cGAS and inactivate STAT3, or a combination thereof. In a further aspect, the present disclosure provides bimodal cytoplasmic DNA sensor-activating and transcription factor-inhibiting SNAs that are capable of both inducing an effective type of IFN response and sequestering / inactivating transcription factors (e.g., STAT3) in the cytosol of cells to achieve effective anti-tumor immunity. In various embodiments, such bimodal SNAs include one or more cytoplasmic DNA sensor-activating oligonucleotides, one or more oligonucleotides that inactivate transcription factors (e.g., STAT3), one or more oligonucleotides that activate cytoplasmic DNA sensors and inactivate transcription factors, or combinations thereof.

[0017] Terminology Language such as "from," "to," "up to," "at least," "greater than," "less than," and the like all refer to ranges that include the recited numbers and can then be broken down into sub-ranges.

[0018] Ranges include each individual member. Thus, for example, a group having 1 to 3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members.

[0019] As used in this specification and the appended claims, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

[0020] "About" and "approximately" are generally intended to mean an acceptable degree of error for the quantity measured given the nature or precision of the measurement. Exemplary degrees of error are within 20-25 percent (%), e.g., within 20 percent, within 10 percent, within 5 percent, within 4 percent, within 3 percent, within 2 percent, or within 1 percent of the stated value or range of values.

[0021] The terms "polynucleotide" and "oligonucleotide" are interchangeable when used herein.

[0022] A "subject" is a vertebrate organism. The subject can be a non-human mammal (e.g., a mouse, a rat, or a non-human primate), or the subject can be a human subject.

[0023] As used herein, the terms "administering," "administer," "administration," and the like refer to any mode of transferring, delivering, introducing, or transporting an SNA to a subject in need of treatment with such an agent. Such modes include, but are not limited to, oral, topical, intravenous, intraarterial, mucosal, intraperitoneal, intramuscular, intratumoral, parenteral, intradermal, intranasal, and subcutaneous administration. Combinations of different administration routes, either separately or simultaneously, are also contemplated by the present disclosure, as are combinations of different administration routes in combination with focused ultrasound (FUS).

[0024] As used herein, "treating" and "treatment" refer to any reduction in the severity and / or onset of symptoms associated with a disorder. Thus, "treating" and "treatment" include therapeutic and prophylactic measures. One skilled in the art will appreciate that any degree of protection or amelioration from a disorder is beneficial to a subject, such as a human patient. The quality of life of a patient is improved by reducing to any degree the severity of symptoms in a subject and / or delaying or preventing the onset of symptoms.

[0025] An "effective amount" or "sufficient amount" of a substance is the amount necessary to produce a beneficial or desired result, including a clinical result, and thus "effective amount" depends on the context in which it is applied. For example, but not by way of limitation, an effective amount of an SNA of the present disclosure is an amount that is sufficient to induce an immune response, inhibit gene expression, and / or treat cancer. An effective amount may be administered in one or more doses. Efficacy may be demonstrated in experimental or clinical trials, for example, by comparing the results achieved with the substance of interest compared to an experimental control.

[0026] The term "dose" as used herein with respect to the SNAs of the present disclosure refers to a measured portion of the SNA (e.g., as a pharmaceutical formulation) taken by a subject (administered to a subject or ingested by a subject) at any one time.

[0027] An "antigenic composition" is a composition of matter suitable for administration to a human or animal subject (e.g., in an experimental or clinical setting) that is capable of eliciting a specific immune response. In some embodiments, the immune response is elicited against an antigen, such as a cancer-associated antigen. Thus, in some embodiments, the antigenic composition comprises one or more antigens or antigenic epitopes. The antigenic composition may also, in some embodiments, comprise one or more additional components that are capable of eliciting or enhancing an immune response, such as an excipient, carrier, and / or adjuvant.

[0028] An "immune response" is a response of the immune system (e.g., an interferon response) or a cell of the immune system, such as a B cell, a T cell, or a monocyte, to a stimulus, such as an antigen (e.g., formulated as an antigenic composition). The immune response resulting from treatment with a SNA of the present disclosure may elicit one or more of: (1) infiltration and activation of CD8+ T cells within the glioma tumor microenvironment; (2) M2 / M0 to M1 reeducation of macrophages; and (3) activation of NK and NKT cells. The immune response may be a B cell response, which results in the production of specific antibodies, such as antigen-specific neutralizing antibodies. The immune response may also be a T cell response, such as a CD4+ response or a CD8+ response. B cell and T cell responses are aspects of a "cellular" immune response. The immune response may also be an antibody-mediated "humoral" immune response. In some cases, the response is specific to a particular antigen (i.e., an "antigen-specific response").

[0029] As used herein, an "immunostimulatory oligonucleotide" is an oligonucleotide that can stimulate (e.g., induce or enhance) an immune response (e.g., an interferon response). As described herein, the present disclosure provides SNAs that include an immunostimulatory oligonucleotide that activates cGAS. In some embodiments, the present disclosure provides SNAs that include an immunostimulatory oligonucleotide that activates a cytoplasmic DNA sensor. In some embodiments, the SNAs of the present disclosure include an oligonucleotide that activates cGAS and one or more additional immunostimulatory oligonucleotides that target additional DNA sensors or pattern recognition receptors. Exemplary examples of immunostimulatory oligonucleotides are CpG motif-containing oligonucleotides, single-stranded RNA oligonucleotides, double-stranded RNA oligonucleotides, toll-like receptor (TLR) agonists, and double- or single-stranded DNA or RNA oligonucleotides that target additional DNA sensors, including, but not limited to, AIM2 (absent in melanoma-2), RNA polymerase III, DAI (DNA-dependent activator of IFN-regulatory factors), IFI16 (interferon-gamma inducible protein 16), or combinations thereof. In various embodiments, the TLR is selected from the group consisting of toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 5 (TLR5), toll-like receptor 6 (TLR6), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), toll-like receptor 9 (TLR9), toll-like receptor 10 (TLR10), toll-like receptor 11 (TLR11), toll-like receptor 12 (TLR12), and toll-like receptor 13 (TLR13). A "CpG motif" is a cytosine-guanine dinucleotide sequence. In various embodiments, one or more immunostimulatory oligonucleotides are encapsulated in the nanoparticle core of the SNA, attached to the outer surface of the nanoparticle core, or a combination thereof. In some embodiments, one or more immunostimulatory oligonucleotides are encapsulated within the nanoparticle core and / or attached to the exterior surface of the nanoparticle core.In some embodiments, the one or more immunostimulatory oligonucleotides are not associated with the SNA and are administered separately, either in the same composition as the SNA or in a separate composition.

[0030] The term "inhibitory oligonucleotide" refers to an oligonucleotide that reduces the production or expression of a protein, for example, by preventing the translation of mRNA into a protein in a ribosome, or that is sufficiently complementary to either a gene or mRNA encoding one or more target proteins to specifically bind (hybridize) to one or more target genes or mRNAs, thereby reducing the expression or biological activity of the target protein. Inhibitory oligonucleotides include, but are not limited to, isolated or synthetic short hairpin RNA (shRNA or DNA), antisense oligonucleotides (e.g., antisense RNA or DNA, chimeric antisense DNA or RNA), miRNA and miRNA mimics, small interfering RNA (siRNA), single guide RNA (sgRNA combined with Cas9 delivery), DNA or RNA inhibitors of innate immune receptors, aptamers, DNAzymes, or aptazymes. In some embodiments, an inhibitory oligonucleotide is an oligonucleotide that binds to a receptor but does not activate the receptor, thereby inhibiting the receptor from further binding to and activating a ligand.

[0031] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter cited, and may, in some cases, be incorporated herein in its entirety.

[0032] Spherical nucleic acid (SNA) As used herein, a "spherical nucleic acid" (SNA) comprises a spherical or substantially spherical nanoparticle core functionalized with a highly oriented oligonucleotide shell. In any of the aspects or embodiments of the present disclosure, the SNA comprises (a) a nanoparticle core and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides that activate cyclic GMP-AMP synthase (cGAS) and are at least 15 base pairs in length. In some aspects, the SNA of the present disclosure comprises (a) a nanoparticle core and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides that activate a cytoplasmic DNA sensor and are at least 15 base pairs in length. In any of the aspects or embodiments of the present disclosure, the SNA comprises (a) a nanoparticle core and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides that (i) activate cyclic GMP-AMP synthase (cGAS), (ii) inactivate signal transducer and activator of transcription 3 (STAT3), and (iii) are at least 15 base pairs in length. In some aspects, the SNA of the present disclosure comprises (a) a nanoparticle core and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides that (i) activate a cytoplasmic DNA sensor, (ii) inactivate a transcription factor, and (iii) are at least 15 base pairs in length. In some embodiments, the SNA comprises (a) a nanoparticle core and (b) an oligonucleotide shell attached to the outer surface of the nanoparticle core, the oligonucleotide shell comprising a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates cyclic GMP-AMP synthase (cGAS) and is at least 15 base pairs in length.In some embodiments, the SNA comprises (a) a nanoparticle core and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates a cytoplasmic DNA sensor and is at least 15 base pairs in length. In some embodiments, the SNA comprises (a) a nanoparticle core and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which (i) activates cyclic GMP-AMP synthase (cGAS), (ii) inactivates signal transducer and activator of transcription 3 (STAT3), and (iii) is at least 15 base pairs in length. In some embodiments, the SNA comprises (a) a nanoparticle core and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which (i) activates a cytoplasmic DNA sensor, (ii) inactivates a transcription factor, and (iii) is at least 15 base pairs in length. In some embodiments, the SNA comprises (a) a nanoparticle core and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which (i) activates a cyclic GMP-AMP synthase (cGAS) or other DNA sensor, (ii) inactivates a signal transducer and activator of transcription 3 (STAT3) or other transcription factor, and (iii) is at least 15 base pairs in length.

[0033] In some aspects, the disclosure provides bimodal cGAS-activating and STAT3-inhibiting SNAs that are capable of both activating cGAS and inactivating STAT3. In various embodiments, such bimodal SNAs include one or more cGAS-activating oligonucleotides, one or more oligonucleotides that inactivate STAT3, one or more oligonucleotides that activate cGAS and inactivate STAT3, or a combination thereof. In some aspects, the disclosure provides bimodal cytoplasmic DNA sensor-activating and transcription factor-inhibiting SNAs that are capable of both activating a cytoplasmic DNA sensor and inactivating a transcription factor. In various embodiments, such bimodal SNAs include one or more cytoplasmic DNA sensor-activating oligonucleotides, one or more oligonucleotides that inactivate a transcription factor, one or more oligonucleotides that activate a cytoplasmic DNA sensor and inactivate a transcription factor, or a combination thereof.

[0034] In some embodiments, the nanoparticle core is a protein core. In various embodiments, the protein core comprises or consists of a CRISPR-associated protein (e.g., Cas9). In some embodiments, the protein core comprises or consists of a CRISPR-associated protein (e.g., Cas9) that comprises one or more single guide RNAs (sgRNAs) bound thereto.

[0035] In some embodiments, the nanoparticle core is a liposome core. The liposome core of the present disclosure has an at least substantially spherical shape, an interior and an exterior, and comprises a lipid bilayer. The lipid bilayer, in various embodiments, comprises a plurality of lipid groups. The plurality of lipid groups, in various embodiments, comprises lipids from the phosphatidylcholine, phosphatidylglycerol, and / or phosphatidylethanolamine families of lipids. While not meant to be limiting, in various embodiments, the lipids include 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-dioleo ... 1,2-di-(9Z-octadecanoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), monophosphoryl lipid A (MPLA), or a combination thereof.

[0036] The nanoparticle core of the SNA may be from about 1 nanometer (nm) to about 500 nm, from about 1 nm to about 400 nm, from about 1 nm to about 300 nm, from about 1 nm to about 200 nm, from about 1 nm to about 100 nm, from about 1 nm to about 50 nm, from about 10 nm to about 500 nm, from about 10 nm to about 400 nm, from about 10 nm to about 300 nm, from about 10 nm to about 200 nm, from about 10 nm to about 100 nm, from about 10 nm to about 50 nm, from about 10 nm to about 40 nm, from about 20 nm to about 50 nm, from about 20 nm to about 40 nm, from about 20 nm to about 30 nm, The nanoparticle core may range in size from about 10 nm to about 150 nm in diameter, about 10 nm to about 140 nm in diameter, about 10 nm to about 130 nm in diameter, about 10 nm to about 120 nm in diameter, about 10 nm to about 110 nm in diameter, about 10 nm to about 100 nm in diameter, about 10 nm to about 90 nm in diameter, about 10 nm to about 80 nm in diameter, about 10 nm to about 70 nm in diameter, about 10 nm to about 60 nm in diameter, about 10 nm to about 50 nm in diameter, about 10 nm to about 40 nm in diameter, about 10 nm to about 30 nm in diameter, or about 10 nm to about 20 nm in diameter. In some embodiments, the nanoparticle core is a metal core (e.g., gold) and has a diameter of about 14 nm. In some embodiments, the nanoparticle core is a polymer core (e.g., PLGA) and has a diameter of about 70 nm. In other aspects, the present disclosure provides a plurality of SNAs each comprising a nanoparticle core. In these embodiments, the size of the multiple nanoparticle cores is about 10 nm to about 150 nm (average diameter), an average diameter of about 10 nm to about 140 nm, an average diameter of about 10 nm to about 130 nm, an average diameter of about 10 nm to about 120 nm, an average diameter of about 10 nm to about 110 nm, an average diameter of about 10 nm to about 100 nm, an average diameter of about 10 nm to about 90 nm, an average diameter of about 10 nm to about 80 nm, an average diameter of about 10 nm to about 70 nm, an average diameter of about 10 nm to about 60 nm, an average diameter of about The average diameter is about 10 nm to about 50 nm, an average diameter of about 10 nm to about 40 nm, an average diameter of about 10 nm to about 30 nm, an average diameter of about 10 nm to about 20 nm, or an average diameter of about 20 nm to about 100 nm, an average diameter of about 20 nm to about 90 nm, an average diameter of about 20 nm to about 80 nm, an average diameter of about 20 nm to about 70 nm, an average diameter of about 20 nm to about 60 nm, an average diameter of about 20 nm to about 50 nm, an average diameter of about 20 nm to about 40 nm, or an average diameter of about 20 nm to about 30 nm.In some embodiments, the diameter of the nanoparticle core (or the average diameter for a plurality of nanoparticle cores) is about 10 nm to about 150 nm, about 30 to about 100 nm, or about 40 to about 80 nm. In some embodiments, the size of the nanoparticle cores used in the methods is varied as needed depending on their particular use or application. Variation in size is advantageously used, for example, to optimize a particular physical characteristic of the nanoparticle, such as an optical property or amount of surface area that can be functionalized as described herein. In further embodiments, a plurality of SNAs are produced, the SNAs in the plurality having an average diameter of about 150 nanometers or less (e.g., about 10 nanometers to about 150 nanometers), or about 100 nanometers or less (e.g., about 10 nanometers to about 100 nanometers), or about 80 nanometers or less (e.g., about 10 nanometers to about 80 nanometers). In further embodiments, the nanoparticle cores in the plurality produced by the methods of the present disclosure have a diameter or average diameter of about 20 nanometers or less, or about 25 nanometers or less, or about 30 nanometers or less, or about 35 nanometers or less, or about 40 nanometers or less, or about 45 nanometers or less, or about 50 nanometers or less, or about 55 nanometers or less, or about 60 nanometers or less, or about 65 nanometers or less, or about 70 nanometers or less, or about 75 nanometers or less, or about 80 nanometers or less, or about 85 nanometers or less, or about 90 nanometers or less, or about 95 nanometers or less, or about 100 nanometers or less, or about 100 nanometers or less, or about 120 nanometers or less, or about 130 nanometers or less, or about 140 nanometers or less, or about 150 nanometers or less. It will be understood that any of the aforementioned diameters of the nanoparticle core can apply to the diameter of the nanoparticle core itself or to the diameter of the SNA (i.e., the nanoparticle core and the shell of oligonucleotides attached to the outer surface of the nanoparticle core).

[0037] As described herein, in some embodiments, the nanoparticle core of the SNA is a liposome core. Thus, the present disclosure also provides liposome particles, for example as disclosed in International Patent Application No. PCT / US2014 / 068429 (incorporated herein by reference in its entirety). The liposome particles of the present disclosure have at least a substantially spherical shape, an inner side and an outer side, and comprise a plurality of lipid groups. In various embodiments, the plurality of lipid groups comprises lipids selected from the group consisting of the phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine families of lipids. Lipids contemplated by the present disclosure include 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DSPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DSPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DSPG ...PG), 1,2-dipalmitoyl-sn-glycero-3-phosphocho These include, but are not limited to, toyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), cardiolipin, lipid A, monophosphoryl lipid A (MPLA), and combinations thereof. In various embodiments, at least one oligonucleotide in the oligonucleotide shell is attached to the exterior surface of the liposome core through a lipid anchor group. In further embodiments, the lipid anchor group is attached to the 5' or 3' terminus of at least one oligonucleotide. In yet further embodiments, the lipid anchor group is tocopherol or cholesterol.Thus, in various embodiments, at least one of the oligonucleotides in the oligonucleotide shell is an oligonucleotide-lipid conjugate containing a lipid anchor group, which is adsorbed to the lipid bilayer. In some embodiments, all of the oligonucleotides in the oligonucleotide shell are oligonucleotide-lipid conjugates containing a lipid anchor group, which is adsorbed to the lipid bilayer. In various embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the oligonucleotides in the oligonucleotide shell are bound (e.g., adsorbed) to the outer surface of the liposome core through lipid anchor groups. The lipid anchor group, in various embodiments, includes tocopherol, palmitoyl, dipalmitoyl, stearyl, distearyl, or cholesterol. The lipid anchor can be attached to the oligonucleotide using any chemistry known to those skilled in the art, including, but not limited to, amide bonds or click chemistry. Methods for making liposomal SNAs are generally known (see, e.g., Wang, S.; Qin, L.; Yamankurt, G.; Skakuj, K.; Huang, Z.; Chen, P.-C.; Dominguez, D.; Lee, A.; Zhang, B.; Mirkin, C. Regulation Vaccinology with Spherical Nucleic Acids. Proc. Natl. Acad. Sci. 2019, 116(21), 10473-10481, the entire contents of which are incorporated herein by reference).

[0038] In some embodiments, the nanoparticle core is a lipid nanoparticle core. The lipid nanoparticle (LNP) spherical nucleic acid comprises a lipid nanoparticle core (e.g., a solid lipid nanoparticle core) decorated with a shell of oligonucleotides. The lipid nanoparticle core comprises an ionizable lipid, a phospholipid, a sterol, and a lipid-polyethylene glycol (lipid-PEG) conjugate. The shell of oligonucleotides is attached to the outer surface of the lipid nanoparticle core and, in any of the aspects or embodiments of the present disclosure, comprises one or more cGAS-activating oligonucleotides. In some embodiments, the shell of oligonucleotides attached to the outer surface of the lipid nanoparticle core comprises one or more oligonucleotides that activate cGAS and inactivate STAT3. The spherical architecture of the oligonucleotide shell provides unique advantages over conventional nucleic acid delivery methods, including entry into nearly all cells independent of transfection agents, resistance to nuclease degradation, sequence-based function, targeting, and diagnostics.

[0039] In various embodiments, the ionizable lipid is dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), C12-200, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), similar lipid / lipidoid structures, or combinations thereof. In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dihexadecanoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), monophosphoryl lipid A (MPLA), or combinations thereof. In a further embodiment, the sterol is 3β-hydroxycholest-5-ene (cholesterol), 9,10-secocholesta-5,7,10(19)-trien-3β-ol (vitamin D3), 9,10-secoegosta-5,7,10(19),22-tetraen-3β-ol (vitamin D2), calcipotriol, 24-ethyl-5,22-cholestadien-3β-ol (stigmasterol), 22,23-dihydrostigmasterol (β-sitosterol), 3,28-dihydroxy-lupeol (beta-sitosterol), 5,5-dihydroxy-1,1,2,3,4,4-trimethylol, 5,5-dihydro ... Turin), lupeol, ursolic acid, oleanolic acid, 24α-methylcholesterol (campesterol), 24-ethylcholesta-5,24(28)E-dien-3β-ol (fucosterol), 24-methylcholesta-5,22-dien-3β-ol (brassicasterol), 24-methylcholesta-5,7,22-trien-3β-ol (ergosterol), 9,11-dehydroergosterol, daucosterol, or any of the foregoing sterols modified with one or more amino acids. In some embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate comprises a 2000 Dalton (Da) polyethylene glycol. In further embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate is a lipid-PEG-maleimide.In still further embodiments, the lipid-PEG-maleimide is 1,2-dipalmitryl-sn-glycero-3-phosphoethanolamine (DPPE) conjugated to a 2000 Da polyethylene glycol maleimide, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) conjugated to a 2000 Da polyethylene glycol maleimide, or a combination thereof.

[0040] In any of the aspects or embodiments of the present disclosure, the oligonucleotides are attached to the exterior of the lipid nanoparticle core via covalent attachment to the lipid-polyethylene glycol (lipid-PEG) conjugate of the oligonucleotide. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the oligonucleotides in the oligonucleotide shell are covalently attached to the exterior of the lipid nanoparticle core through the lipid-PEG conjugate. In various embodiments, one or more oligonucleotides in the oligonucleotide shell are attached (or adsorbed) to the exterior of the lipid nanoparticle core through a lipid anchor group as described herein. In various embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the oligonucleotides in the oligonucleotide shell are attached (or adsorbed) to the exterior of the lipid nanoparticle core through a lipid group as described herein. The lipid anchor group is attached to the 5' or 3' end of the oligonucleotide in various embodiments. In various embodiments, the lipid anchor group is tocopherol, palmitoyl, dipalmitoyl, stearyl, distearyl, or cholesterol.

[0041] Oligonucleotides In some aspects, the present disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a double-stranded or single-stranded stem-loop DNA oligonucleotide that activates a cyclic GMP-AMP synthase (cGAS) and is at least 15 base pairs in length. In some aspects, the present disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a double-stranded or single-stranded stem-loop DNA oligonucleotide that activates a cytoplasmic DNA sensor and is at least 15 base pairs in length. In some aspects, the present disclosure provides a spherical nucleic acid (SNA), comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides bound to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides that activate a cytoplasmic DNA sensor and are at least 15 base pairs in length. In various embodiments, the cytoplasmic DNA sensor is cyclic GMP-AMP synthase (cGAS), AIM2 (absent in melanoma-2), RNA polymerase III, DAI (DNA-dependent activator of IFN-regulatory factors), IFI16 (interferon-γ-inducible protein 16), or a combination thereof. In some aspects, the present disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; and (b) an oligonucleotide shell attached to the outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides that activate cyclic GMP-AMP synthase (cGAS) and are at least 15 base pairs in length.In some aspects, the present disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides that (i) activate cyclic GMP-AMP synthase (cGAS), (ii) inactivate signal transduction and activator of transcription 3 (STAT3), and (iii) are at least 15 base pairs in length. In some aspects, the present disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides that (i) activate a cytoplasmic DNA sensor, (ii) inactivate a transcription factor, and (iii) are at least 15 base pairs in length. In any of the aspects or embodiments of the present disclosure, the transcription factor promotes cancer progression. In various embodiments, the transcription factor is signal transducer and activator of transcription 3 (STAT3), cMyc, NANOG, SOX2, OCT4, or a combination thereof. In some embodiments, the oligonucleotide shell comprises a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates cGAS and inactivates STAT3, and is at least 15 base pairs in length. In some embodiments, the oligonucleotide shell comprises a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates cGAS and inactivates STAT3, and is at least 15 base pairs in length. In further embodiments, the oligonucleotide shell consists of a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates cGAS and inactivates STAT3, and is at least 15 base pairs in length. In yet further embodiments, the oligonucleotide shell consists of a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates cGAS and inactivates STAT3, and is at least 15 base pairs in length.In some embodiments, the oligonucleotide shell comprises a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates a cytoplasmic DNA sensor and is at least 15 base pairs long. In some embodiments, the oligonucleotide shell comprises a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates a cytoplasmic DNA sensor and inactivates a transcription factor, and is at least 15 base pairs long. In further embodiments, the oligonucleotide shell comprises a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates a cytoplasmic DNA sensor and is at least 15 base pairs long. In yet further embodiments, the oligonucleotide shell comprises a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates a cytoplasmic DNA sensor and inactivates a transcription factor, and is at least 15 base pairs long. In various embodiments, the oligonucleotide shell comprises one or more additional oligonucleotides. In some embodiments, the SNA of the present disclosure comprises one or more additional oligonucleotides encapsulated in the nanoparticle core. In some embodiments, the oligonucleotide shell comprises one or more additional oligonucleotides, and the one or more additional oligonucleotides are encapsulated in the nanoparticle core. In some embodiments, the one or more additional oligonucleotides each have the same nucleotide sequence. In some embodiments, the one or more additional oligonucleotides comprise at least two oligonucleotides having different nucleotide sequences. In various embodiments, the additional oligonucleotides are immunostimulatory oligonucleotides, inhibitory oligonucleotides, oligonucleotides that inactivate signal transduction and activator of transcription 3 (STAT3), or combinations thereof. The oligonucleotides in the oligonucleotide shell may be attached to the nanoparticle core through any means (e.g., covalent or non-covalent). The oligonucleotides in the oligonucleotide shell may be attached to the nanoparticle core via their 5' or 3' termini.

[0042] An oligonucleotide that "activates cyclic GMP-AMP synthase (cGAS)" or a "cGAS-activating oligonucleotide" or the like refers to an oligonucleotide that activates cGAS and can lead to activation of stimulator of interferon genes (STING), downstream IRF3, and NF-KB signaling, and release of proinflammatory mediators and type I interferons (IFNs). Such oligonucleotides generally comprise double-stranded or single-stranded stem-loop DNA oligonucleotides that are at least about 15 base pairs (bp) in length and optionally do not contain CpG-rich motifs. Thus, in any of the aspects or embodiments of the present disclosure, the shell of oligonucleotides on the surface of the SNA comprises one or more oligonucleotides that activate cGAS, where the one or more oligonucleotides comprise a double-stranded DNA oligonucleotide that includes a G-quartet oligonucleotide, a single-stranded stem-loop DNA oligonucleotide, or a combination thereof. In some embodiments, the shell of oligonucleotides on the surface of the SNA comprises a plurality of oligonucleotides that activate cGAS, where the plurality of oligonucleotides comprise a double-stranded DNA oligonucleotide, a single-stranded stem-loop DNA oligonucleotide, or a combination thereof. In some embodiments, one or more or all of the oligonucleotides on the exterior surface of the SNA activate cGAS and inactivate STAT3. Assays for identifying cGAS-activating oligonucleotides are known in the art (e.g., Transcreener® assays (BellBrook Labs, Madison, WI; see also Herzner AM. et al., 2015. Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA. Nat Immunol. 16(10):1025-33) and described herein.

[0043] An oligonucleotide that "inactivates signal transducer and activator of transcription 3 (STAT3)" is one that can inactivate or inhibit the transcription factor STAT3. In various embodiments, the oligonucleotide that inactivates STAT3 is a transcription factor decoy, a G-quartet oligonucleotide, an aptamer, an inhibitory oligonucleotide, or a combination thereof. A transcription factor decoy comprises a nucleotide sequence derived from a conserved genomic regulatory element that is recognized and bound by the transcription factor in question (i.e., STAT3). The decoy acts by competitively inhibiting the binding of the transcription factor to the corresponding cis-element in genomic DNA, preventing expression of the target gene. Assays for identifying oligonucleotides capable of inactivating STAT3 are generally known in the art (e.g., Leong PL, Andrews GA, Johnson DE, et al. Targeted inhibition of Stat3 with a decoy oligonucleotide abrogates head and neck cancer cell growth. Proc Natl Acad Sci US A. 2003; 100(7):4138-4143. doi:10.1073 / pnas.0534764100; K. Lau YT, Ramaiyer M, E. Johnson D, R. Grandis J. Targeting STAT3 in Cancer with Nucleotide Therapeutics. Cancers. 2019; 11(11):1681). Non-limiting examples of STAT3 decoys that can be used in accordance with the present disclosure are as follows: [Table 1]

[0044] In various embodiments, the oligonucleotides of the present disclosure include DNA oligonucleotides, RNA oligonucleotides, modified forms thereof, or combinations thereof. In any aspect or embodiment described herein, the oligonucleotide is single-stranded, double-stranded, or partially double-stranded.

[0045] Modified forms of oligonucleotides, including those with at least one modified internucleotidic linkage as described herein, are also contemplated by the present disclosure. In some embodiments, the oligonucleotide is all or partly a peptide nucleic acid. Other modified internucleoside linkages include at least one phosphorothioate linkage. Still other modified oligonucleotides include those that include one or more universal bases. A "universal base" refers to a molecule that can replace a linkage to any one of A, C, G, T, and U in a nucleic acid by forming a hydrogen bond without significant structural destabilization. Oligonucleotides incorporating universal base analogs can function, for example, as probes in hybridization. Examples of universal bases include, but are not limited to, 5'-nitroindole-2'-deoxyriboside, 3-nitropyrrole, inosine, and hypoxanthine.

[0046] The term "nucleotide" or its plural forms as used herein are interchangeable with the variants discussed herein and otherwise known in the art. The term "nucleobase" or its plural forms as used herein are interchangeable with the variants discussed herein and otherwise known in the art. Nucleotides or nucleobases include the naturally occurring nucleobases A, G, C, T, and U. Non-naturally occurring nucleobases include, but are not limited to, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosine, N',N'-ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C3-C6)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, and the "non-naturally occurring" nucleobases described in Benner et al., U.S. Pat. No. 5,432,272 and Susan M. Freier and Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp 4429-4443. The term "nucleobase" includes the known purine and pyrimidine heterocycles, as well as their heterocyclic analogs and tautomers.Additionally, for naturally occurring and non-naturally occurring nucleobases, see U.S. Pat. No. 3,687,808 (Merigan, et al.), Chapter 15 of Antisense Research and Application, Ed. S.T. Crooke and B. Lebleu, CRC Press, 1993, Englisch et al., 1991, Angewandte Chemie, International Edition, 30:613-722 (see especially pages 622 and 623), and the Concise Encyclopedia of Polymer Science and Engineering, J.I. Kroschwitz Ed., John Wiley & Sons, 1990, pages 858-859, Cook, Anti-Cancer Drug Design 1991, 6, 585-607, each of which is incorporated herein by reference in its entirety). In various embodiments, oligonucleotides also contain one or more "nucleoside bases" or "base units", which are a category of non-naturally occurring nucleotides that contain compounds such as heterocyclic compounds that are not nucleoside bases in the most classical sense, but can function as nucleoside bases, including certain "universal bases". Universal bases include 3-nitropyrrole, optionally substituted indoles (e.g., 5-nitroindoles), and optionally substituted hypoxanthines. Other desirable universal bases include pyrrole, diazole, or triazole derivatives that contain universal bases known in the art.

[0047] Examples of oligonucleotides include those containing modified backbones or non-natural internucleoside linkages. Oligonucleotides with modified backbones include those that have a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are considered to be within the meaning of "oligonucleotide."

[0048] Modified oligonucleotide backbones containing a phosphorus atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phototriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs thereof, as well as those having reverse polarity in which one or more internucleotide linkages are 3' to 3', 5' to 5', or 2' to 2' linkages. Oligonucleotides having reverse polarity, including a single 3' to 3' linkage at the 3'-most internucleotide linkage, i.e., a single reverse nucleoside residue, which may be abasic (either missing a nucleotide or having a hydroxyl group in its place), are also contemplated. Salts, mixed salts, and free acid forms are also contemplated. Representative United States patents which teach the preparation of the above phosphorus-containing linkages include U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, Nos. 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361, 5,194,599, 5,565,555, 5,527,899, 5,721,218, 5,672,697 and 5,625,050, the disclosures of which are incorporated herein by reference.

[0049] Modified oligonucleotide backbones that do not contain phosphorus atoms therein have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include backbones with morpholino linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and methylenethioformacetyl backbones; riboacetyl backbones; alkene-containing backbones; sulfamic acid backbones; methyleneimino and methylenehydrazino backbones; sulfonic acid and sulfonamide backbones; amide backbones; and N, O, S and CH 2 Other scaffolds exist that have a mixture of component parts, e.g., U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, 5,59 See Nos. 6,086, 5,602,240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, 5,792,608, 5,646,269 and 5,677,439, the disclosures of which are incorporated herein by reference.

[0050] In yet further embodiments, there are oligonucleotide mimetics in which one or more sugars and / or one or more internucleotide bonds of nucleotide units are both replaced with "non-naturally occurring" groups. The bases of the oligonucleotide are maintained for hybridization. In some aspects, this embodiment contemplates peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced with an amide-containing backbone. See, for example, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, and Nielsen et al., Science, 1991, 254, 1497-1500, the disclosures of which are incorporated herein by reference.

[0051] In still further embodiments, oligonucleotides having phosphorothioate backbones and oligonucleosides having heteroatom backbones are provided, including those having the -CH 2 -NH-O-CH 2 -, -CH 2 -N(CH 3 )-O-CH 2 -, -CH 2 -ON(CH 3 )-CH 2 -, -CH 2 -N(CH 3 )-N(CH 3 )-CH 2 - and -ON(CH 3 )-CH 2 -CH 2 Also contemplated are oligonucleotides having morpholino backbone structures, as described in U.S. Patent No. 5,034,506.

[0052] In various forms, the bond between two consecutive monomers in an oligonucleotide is -CH 2 -, -O-, -S-, -NR H -, >C=O, >C=NR H , >C=S, -Si(R”) 2 -, -SO-, -S(O) 2 -, -P(O) 2-, -PO(BH 3 )-, -P(O,S)-, -P(S) 2 -, -PO(R")-, -PO(OCH 3 )- and -PO(NHR H ) , wherein R H is hydrogen and C 1-4 -alkyl, R″ is selected from C 1-6 -alkyl and phenyl. An illustrative example of such a bond is -CH 2 -CH 2 -CH 2 -, -CH 2 -CO-CH 2 -, -CH 2 -CHOH-CH 2 -, -O-CH 2 -O-, -O-CH 2 -CH 2 -, -O-CH 2 -CH=(R when used as a bond to a subsequent monomer 5 ), -CH 2 -CH 2 -O-, -NR H -CH 2 -CH 2 -, -CH 2 -CH 2 -NR H -, -CH 2 -NR H -CH 2 - -, -O-CH 2 -CH 2 -NR H -, -NR H -CO-O-, -NR H -CO-NR H -, -NR H -CS-NR H -, -NR H -C(=NR H )-NR H -, -NR H -CO-CH 2 -NR H -O-CO-O-, -O-CO-CH 2 -O-, -O-CH 2 -CO-O-, -CH 2-CO-NR H -, -O-CO-NR H -, -NR H -CO-CH 2 -, -O-CH 2 -CO-NR H -, -O-CH 2 -CH 2 -NR H -, -CH=NO-, -CH 2 -NR H -O-, -CH 2 -ON=(R when used as a bond to a subsequent monomer) 5 ), -CH 2 -O-NR H -, -CO-NR H -CH 2 -, -CH 2 -NR H -O-, -CH 2 -NR H -CO-, -O-NR H -CH 2 -, -O-NR H , -O-CH 2 -S-, -S-CH 2 -O-, -CH 2 -CH 2 -S-, -O-CH 2 -CH 2 -S-, -S-CH 2 -CH=(R when used as a bond to a subsequent monomer 5 ), -S-CH 2 -CH 2 -,-S-CH 2 -CH 2 --O-, -S-CH 2 -CH 2 -S-, -CH 2 -S-CH 2 -, -CH 2 -SO-CH 2 -, -CH 2 -SO 2 -CH 2 -, -O-SO-O-, -OS(O) 2 -O-, -OS(O) 2 -CH 2 -, -OS(O) 2 -NRH -,-NR H -S(O) 2 -CH 2 -;-O-S(O) 2 -CH 2 -,-O-P(O) 2 -O-,-O-P(O,S)-O-,-O-P(S) 2 -O-,-S-P(O) 2 -O-,-S-P(O,S)-O-,-S-P(S) 2 -O-,-O-P(O) 2 -S-,-O-P(O,S)-S-,-O-P(S) 2 -S-,-S-P(O) 2 -S-,-S-P(O,S)-S-,-S-P(S) 2 -S-,-O-PO(R”)-O-,-O-PO(OCH 3 )-O-,-O-PO(O CH 2 CH 3 )-O-,-O-PO(O CH 2 CH 2 S-R)-O-,-O-PO(BH 3 )-O-,-O-PO(NHR N )-O-,-O-P(O) 2 -NR H H-,-NR H -P(O) 2 -O-,-O-P(O,NR H )-O-,-CH 2 -P(O) 2 -O-,-O-P(O) 2 -CH 2 -、and -O-Si(R”) 2 -O- and among these, especially -CH 2 -CO-NR H -,-CH 2 -NR H -O-,-S-CH 2 -O-,-O-P(O) 2 -O-O-P(-O,S)-O-,-O-P(S) 2 -O-,-NR H P(O) 2 -O-,-O-P(O,NR H )-O-,-O-PO(R”)-O-,-O-PO(CH3 )-O- and -O-PO(NHR N )-O-(wherein, R H is hydrogen and C 1-4 -alkyl, R″ is selected from C 1-6 -alkyl and phenyl) are contemplated. Further examples are provided in Mesmaeker et.al., Current Opinion in Structural Biology 1995, 5, 343-355 and Susan M. Freier and Karl-Heinz Altmann, Nucleic Acids Research, 1997, vol 25, pp 4429-4443.

[0053] Still other modified forms of oligonucleotides are described in detail in US Patent Application Publication No. 2004 / 0219565, the disclosure of which is incorporated herein by reference in its entirety.

[0054] Modified oligonucleotides may also contain one or more substituted sugar moieties. In certain embodiments, the oligonucleotide comprises one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C 1 ~C 10 Alkyl or C 2 ~C 10 It can be alkenyl and alkynyl. In other embodiments, O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH2 ) n ON[(CH 2 ) n CH 3 ] 2 where n and m are from 1 to about 10. Other oligonucleotides include one of the following at the 2' position: 1 ~C 10 Lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , S.O. 2 CH 3 , O.N.O. 2 , NO 2 , N 3 , NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, or an RNA cleaving group. In one aspect, the modification includes 2'-methoxyethoxy (also known as 2'-O-(2-methoxyethyl) or 2'-MOE, 2'-O-CH 2 CH 2 OCH 3 ) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., alkoxyalkoxy groups. Other modifications include O(CH), also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE. 2 ) 2 ON(CH 3 ) 2 groups, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH 2 -O-CH 2 -N(CH 3 ) 2 Examples include:

[0055] Yet another modification is 2'-methoxy (2'-O-CH 3 ), 2'-aminopropoxy (2'-OCH2 CH 2 CH 2 NH 2 ), 2'-allyl (2'-CH 2 -CH=CH 2 ), 2'-O-allyl (2'-O-CH 2 -CH=CH 2 ), and 2'-fluoro (2'-F). The 2' modification can be at the arabino (up) or ribo (down) position. In one aspect, the 2'-arabino modification is 2'-F. Similar modifications can also be made at other positions on oligonucleotides, such as the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and at the 5' position of 5' terminal nucleotide. Oligonucleotides can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. See, for example, U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,576,427, 5,591,7 22, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, 5,792,747, and 5,700,920, the disclosures of which are incorporated herein by reference in their entireties.

[0056] In some embodiments, sugar modifications include Locked Nucleic Acids (LNAs), in which a 2'-hydroxyl group is attached to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. In certain embodiments, the linkage is a methylene (-CH 2 -) n where n is 1 or 2. LNAs and their preparation are described in WO98 / 39352 and WO99 / 14226.

[0057] Modified nucleotides are described in EP 1072679 and WO 97 / 12896, the disclosures of which are incorporated herein by reference. Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (succinimide), ... douracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Further modified bases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases can also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, Englisch et al., 1991, Angewandte Chemie, International Edition, 30:613, and Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993. Some of these bases are useful for increasing binding affinity and include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C, and in certain embodiments are combined with 2'-O-methoxyethyl sugar modifications. See U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, and 5,55 See, for example, Nos. 2,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,830,653, 5,763,588, 6,005,096, 5,750,692, and 5,681,941, the disclosures of which are incorporated herein by reference.

[0058] Methods for making polynucleotides of a given sequence are well known. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press, New York, 1991). Solid-phase synthesis methods are suitable for both polyribonucleotides and polydeoxyribonucleotides (the well-known methods for DNA synthesis are also useful for RNA synthesis). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases can also be incorporated into polynucleotides. See, e.g., U.S. Pat. No. 7,223,833; Katz, J. Am. Chem. Soc., 74:2238 (1951); Yamane, et al., J. Am. Chem. Soc., 83:2599 (1961); Kosturko, et al., Biochemistry, 13:3949 (1974); Thomas, J. Am. Chem. Soc., 76:6032 (1954); Zhang, et al., J. Am. Chem. Soc., 127:74-75 (2005); and Zimmermann, et al., J. Am. Chem. Soc., 124:13684-13685 (2002).

[0059] In various aspects, the oligonucleotides of the disclosure (e.g., cGAS-activating oligonucleotides) or modified forms thereof are generally from about 5 nucleotides to about 5000 nucleotides in length. In various embodiments, the oligonucleotides of the disclosure are from about 5 to about 4000 nucleotides in length, from about 5 to about 3000 nucleotides in length, from about 5 to about 2000 nucleotides in length, from about 5 to about 1000 nucleotides in length, from about 5 to about 900 nucleotides in length, from about 5 to about 800 nucleotides in length, from about 5 to about 700 nucleotides in length, from about 5 to about 600 nucleotides in length, from about 5 to about 500 nucleotides in length, from about 5 to about 450 nucleotides in length, from about 5 to about 400 nucleotides in length, from about 5 to about 350 nucleotides in length, from about 5 to about 300 nucleotides in length, from about 5 to about 250 nucleotides long, about 5 to about 200 nucleotides long, about 5 to about 150 nucleotides long, about 5 to about 100, about 5 to about 90 nucleotides long, about 5 to about 80 nucleotides long, about 5 to about 70 nucleotides long, about 5 to about 60 nucleotides long, about 5 to about 50 nucleotides long, about 5 to about 45 nucleotides long, about 5 to about 40 nucleotides long, about 5 to about 35 nucleotides long, about 5 to about 30 nucleotides long, about 5 to about 25 nucleotides long, about 5 to about 20 nucleotides long, about 5 to about 15 nucleotides long, about 5 to about 10 nucleotides long long, about 10 to about 4000 nucleotides long, about 10 to about 3000 nucleotides long, about 10 to about 2000 nucleotides long, about 10 to about 1000 nucleotides long, about 10 to about 900 nucleotides long, about 10 to about 800 nucleotides long, about 10 to about 700 nucleotides long, about 10 to about 600 nucleotides long, about 10 to about 500 nucleotides long, about 10 to about 450 nucleotides long, about 10 to about 400 nucleotides long, about 10 to about 350 nucleotides long, about 10 to about 300 nucleotides long, about 10 to about 250 nucleotides long , about 10 to about 200 nucleotides long, about 10 to about 150 nucleotides long, about 10 to about 100 nucleotides long, about 10 to about 90 nucleotides long, about 10 to about 80 nucleotides long, about 10 to about 70 nucleotides long, about 10 to about 60 nucleotides long, about 10 to about 50 nucleotides long, about 10 to about 45 nucleotides long, about 10 to about 40 nucleotides long, about 10 to about 35 nucleotides long, about 10 to about 30 nucleotides long, about 10 to about 25 nucleotides long, about 10 to about 20 nucleotides long, about 10 to about 15 nucleotides long,From about 18 to about 28 nucleotides in length, from about 15 to about 26 nucleotides in length, and all oligonucleotide lengths intermediate to the specifically disclosed sizes such that the oligonucleotide achieves the desired result. Thus, in various embodiments, the oligonucleotides of the present disclosure are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or more nucleotides in length, or at least thereof. In further embodiments, the oligonucleotides of the disclosure are 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 110, 1 3, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000 or more nucleotides in length. In further embodiments, the oligonucleotides of the present disclosure are from about 5 to about 250 nucleotides in length, from about 5 to about 200 nucleotides in length, from about 5 to about 150 nucleotides in length, from about 5 to about 100 nucleotides in length, from about 5 to about 90 nucleotides in length, from about 5 to about 80 nucleotides in length,about 5 to about 70 nucleotides long, about 5 to about 60 nucleotides long, about 5 to about 50 nucleotides long, about 5 to about 45 nucleotides long, about 5 to about 40 nucleotides long, about 5 to about 35 nucleotides long, about 5 to about 30 nucleotides long, about 5 to about 25 nucleotides long, about 5 to about 20 nucleotides long, about 5 to about 15 nucleotides long, about 5 to about 10 nucleotides long, about 10 to about 250 nucleotides long, about 10 to about 200 nucleotides long, about 10 to about 150 nucleotides long, about 10 to about 100 nucleotides long, about 10 to about 90 nucleotides long, about 10 to about 80 nucleotides long, about 10 to about 60 nucleotides long, about 10 to about 50 nucleotides long, about 10 to about 45 nucleotides long, about 10 to about 40 nucleotides long, about 10 to about 35 nucleotides long, about 10 to about 30 nucleotides long long, about 10 to about 25 nucleotides long, about 10 to about 20 nucleotides long, about 10 to about 15 nucleotides long, about 15 to about 250 nucleotides long, about 15 to about 200 nucleotides long, about 15 to about 150 nucleotides long, about 15 to about 100 nucleotides long, about 15 to about 90 nucleotides long, about 15 to about 80 nucleotides long, about 15 to about 70 nucleotides long, about 15 to about 60 nucleotides long, about 15 to about 50 nucleotides long, about 15 to about 45 nucleotides long, about 15 to about 40 nucleotides long, about 15 to about 35 nucleotides long, about 15 to about 30 nucleotides long, about 15 to about 25 nucleotides long, about 15 to about 20 nucleotides long, and all oligonucleotide lengths intermediate to the specifically disclosed sizes to the extent that the oligonucleotide achieves the desired result. In some embodiments, the oligonucleotides of the present disclosure are, or are at least, 15 nucleotides long. It will be understood that each of the foregoing lengths may refer to a length of nucleotides (e.g., when referring to a single-stranded nucleic acid) or a length of base pairs (e.g., when referring to a double-stranded nucleic acid such as a cGAS-activating oligonucleotide). In various embodiments, the shell of oligonucleotides attached to the exterior surface of the nanoparticle core of the SNA comprises a plurality of oligonucleotides all having the same length / sequence, and in some embodiments, the plurality of oligonucleotides comprises one or more oligonucleotides having a different length and / or sequence relative to at least one other oligonucleotide in the plurality. In some embodiments,The oligonucleotide shell comprises a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates cGAS or other cytoplasmic DNA sensors, including but not limited to AIM2 (absent in melanoma-2), RNA polymerase III, DAI (DNA-dependent activator of IFN-regulatory factors), or IFI16 (interferon-gamma inducible protein 16), and is at least 15 base pairs in length. In some embodiments, the oligonucleotide shell comprises a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates cGAS and is at least 15 base pairs in length, and further comprises one or more additional oligonucleotides. In some embodiments, the oligonucleotide shell is comprised of a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates cGAS and is at least 15 base pairs in length. In some embodiments, one or more of the oligonucleotides in the oligonucleotide shell are dual-function oligonucleotides that activate cGAS and also inactivate signal transducer and activator of transcription 3 (STAT3). In some embodiments, one or more of the oligonucleotides in the shell of oligonucleotides are dual-function oligonucleotides that activate a cytoplasmic DNA sensor (such as, but not limited to, cyclic GMP-AMP synthase (cGAS), AIM2 (absent in melanoma 2), RNA polymerase III, DAI (DNA-dependent activator of IFN regulatory factors), IFI16 (interferon gamma-inducible protein 16), or a combination thereof) and inactivate a transcription factor (such as, but not limited to, signal transducer and activator of transcription 3 (STAT3), cMyc, NANOG, SOX2, OCT4, or a combination thereof). In some embodiments, the shell of oligonucleotides comprises (i) one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates cyclic GMP-AMP synthase (cGAS) and is at least 15 base pairs in length, and (ii) one or more oligonucleotides that inactivate signal transducer and activator of transcription 3 (STAT3). In various embodiments,The SNAs of the present disclosure comprise a shell of oligonucleotides comprising (i) one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides that activate cyclic GMP-AMP synthase (cGAS) and are at least 15 base pairs in length, (ii) one or more oligonucleotides that inactivate signal transducer and activator of transcription 3 (STAT3), (iii) one or more oligonucleotides that each activate cGAS and inactivate signal transducer and activator of transcription 3 (STAT3), or (iv) a combination thereof. In some embodiments, the shell of oligonucleotides comprises (i) one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides that each activate a cytoplasmic DNA sensor and are at least 15 base pairs in length, and (ii) one or more oligonucleotides that inactivate a transcription factor. In various embodiments, the SNA of the present disclosure comprises a shell of oligonucleotides comprising: (i) one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides that activate a cytoplasmic DNA sensor and are at least 15 base pairs in length; (ii) one or more oligonucleotides that inactivate a transcription factor; (iii) one or more oligonucleotides that each activate a cytoplasmic DNA sensor and inactivate a transcription factor; or (iv) a combination thereof.

[0060] Spacer. In some aspects and embodiments, one or more oligonucleotides in the shell of oligonucleotides bound to the nanoparticle core of the SNA comprise a spacer. In some aspects and embodiments, each oligonucleotide in the shell of oligonucleotides bound to the nanoparticle core of the SNA comprises a spacer. As used herein, "spacer" refers to a moiety that serves to increase the distance between the nanoparticle core and the oligonucleotide, or to increase the distance between individual oligonucleotides when bound to the nanoparticle core in multiple copies, or to improve the synthesis of the SNA. Thus, it is contemplated that the spacer is located between the oligonucleotide and the nanoparticle core. In some embodiments, the oligonucleotide encapsulated in the nanoparticle core of the SNA comprises a spacer.

[0061] In some aspects, the spacer, when present, is an organic moiety. In some aspects, the spacer is a polymer, including but not limited to a water-soluble polymer, a nucleic acid, a polypeptide, an oligosaccharide, a carbohydrate, a lipid, ethyl glycol, or a combination thereof. In any of the aspects or embodiments of the present disclosure, the spacer is an oligo(ethylene glycol)-based spacer. In various embodiments, the oligonucleotide comprises one, two, three, four, five, or more spacer (e.g., spacer-18 (hexaethylene glycol)) moieties. In further embodiments, the spacer is an alkane-based spacer (e.g., C12). In some embodiments, the spacer is an oligonucleotide spacer (e.g., T5). The oligonucleotide spacer can have any sequence that does not interfere with the ability of the oligonucleotide to become bound to the nanoparticle core or the target. In certain aspects, the bases of the oligonucleotide spacer are all adenylic acids, all thymidylic acids, all cytidylic acids, all guanylic acids, all uridylic acids, or all some other modified base.

[0062] In various embodiments, the length of the spacer is or is equal to at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, 5-10 nucleotides, 10 nucleotides, 10-30 nucleotides, or even more than 30 nucleotides.

[0063] Density. The number of oligonucleotides in the shell of oligonucleotides on the external surface of the SNA can vary. In general, the appropriate surface density to stabilize the nanoparticles and the conditions necessary to obtain it for a desired combination of nanoparticles and oligonucleotides can be determined experimentally. In some embodiments, one or more oligonucleotides are also encapsulated in the nanoparticle core. The number and length of the oligonucleotides encapsulated in the nanoparticle core can be determined in part by the diameter of the nanoparticle core. Similarly, the number of oligonucleotides on the external surface of the SNA can be determined in part by the diameter of the nanoparticle core and / or the nucleotide sequence of the oligonucleotides. In general, at least about 2 pmoles / cm 2 A surface density of 15 pmoles / cm is suitable for providing a stable nanoparticle-oligonucleotide composition. In some embodiments, the surface density is 15 pmoles / cm 2 Also, the oligonucleotide has a concentration of at least 2 pmol / cm 2 , at least 3 pmol / cm 2 , at least 4 pmol / cm 2 , at least 5 pmol / cm 2 , at least 6 pmol / cm 2 , at least 7 pmol / cm 2 , at least 8 pmol / cm 2 , at least 9 pmol / cm 2 , at least 10 pmol / cm 2 , at least about 15 pmol / cm 2 , at least about 19 pmol / cm 2 , at least about 20 pmol / cm 2 , at least about 25 pmol / cm 2 , at least about 30 pmol / cm 2 , at least about 35 pmol / cm 2 , at least about 40 pmol / cm 2 , at least about 45 pmol / cm 2 , at least about 50 pmol / cm 2 , at least about 55 pmol / cm 2 , at least about 60 pmol / cm 2, at least about 65 pmol / cm 2 , at least about 70 pmol / cm 2 , at least about 75 pmol / cm 2 , at least about 80 pmol / cm 2 , at least about 85 pmol / cm 2 , at least about 90 pmol / cm 2 , at least about 95 pmol / cm 2 , at least about 100 pmol / cm 2 , at least about 125 pmol / cm 2 , at least about 150 pmol / cm 2 , at least about 175 pmol / cm 2 , at least about 200 pmol / cm 2 , at least about 250 pmol / cm 2 , at least about 300 pmol / cm 2 , at least about 350 pmol / cm 2 , at least about 400 pmol / cm 2 , at least about 450 pmol / cm 2 , at least about 500 pmol / cm 2 , at least about 550 pmol / cm 2 , at least about 600 pmol / cm 2 , at least about 650 pmol / cm 2 , at least about 700 pmol / cm 2 , at least about 750 pmol / cm 2 , at least about 800 pmol / cm 2 , at least about 850 pmol / cm 2 , at least about 900 pmol / cm 2 , at least about 950 pmol / cm 2 , at least about 1000 pmol / cm 2 Also provided is a method for bonding the nanoparticles to the exterior surface of the nanoparticle core at a surface density of 0.1 to 100 nm, or greater.

[0064] Alternatively, the density of oligonucleotides on the external surface of the SNA is measured by the number of oligonucleotides on the external surface of the SNA. With respect to the surface density of oligonucleotides on the external surface of the SNA of the present disclosure, it is contemplated that the SNA described herein comprises from about 1 to about 250 oligonucleotides on its external surface. In various embodiments, the SNA comprises from about 10 to about 200, or from about 10 to about 190, or from about 10 to about 180, or from about 10 to about 170, or from about 10 to about 160, or from about 10 to about 150, or from about 10 to about 140, or from about 10 to about 130, or from about 10 to about 120, or from about 10 to about 110, or from about 10 to about 10 or about 10 to about 90, or about 10 to about 80, or about 10 to about 70, or 10 to about 60, or about 10 to about 50, or about 10 to about 40, or about 10 to about 30, or about 10 to about 20, or about 50 to about 200, or about 50 to about 150, or about 50 to about 100, or about 50 to about 80 oligonucleotides. In further embodiments, the SNA has on its surface about 4 to about 250, or about 4 to about 200, or about 4 to about 190, or about 4 to about 180, or about 4 to about 170, or about 4 to about 160, or about 4 to about 150, or about 4 to about 140, or about 4 to about 130, or about 4 to about 120, or about 4 to about 110, or 4 to about 100 or about 4 to about 90, or about 4 to about 80, or about 4 to about 70, or 4 to about 60, or about 4 to about 50, or about 4 to about 40, or about 4 to about 30, or about 4 to about 20, or about 4 to about 10, or about 50 to about 200, or about 50 to about 150, or about 50 to about 100, or about 50 to about 80 oligonucleotides. In some embodiments, the SNA comprises about 80 to about 140 oligonucleotides on its surface. In further embodiments, the SNA comprises at least about 5, 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, or 250 polynucleotides on its surface.In a further embodiment the SNA consists of 5, 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240 or 250 polynucleotides on its surface. In some embodiments, an SNA comprising a liposome or lipid nanoparticle core (which in various embodiments may be at or less than about 150 nanometers in diameter, or at or less than about 100 nanometers in diameter, or at or less than about 80 nanometers in diameter, or at or less than about 70 nanometers in diameter) comprises about 10 to about 2,000 oligonucleotides on its surface, or about 10 to about 1,000 oligonucleotides, or about 10 to about 80 oligonucleotides, or about 10 to about 40 oligonucleotides. In some embodiments, an SNA comprising a metal (e.g., gold) core comprises about 70 to about 120 oligonucleotides on its surface. In some embodiments, an SNA comprising a polymer (e.g., PLGA) core comprises about 70 oligonucleotides on its surface.

[0065] composition In some aspects, the disclosure provides compositions comprising one or more spherical nucleic acids (SNAs), comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides that activate cyclic GMP-AMP synthase (cGAS) and are at least 15 base pairs in length. In some aspects, the disclosure provides compositions comprising one or more spherical nucleic acids (SNAs), comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides that activate cytoplasmic DNA sensors and are at least 15 base pairs in length. In some aspects, the disclosure provides a composition comprising one or more spherical nucleic acids (SNAs), comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides that (i) activate cyclic GMP-AMP synthase (cGAS), (ii) inactivate signal transducer and activator of transcription 3 (STAT3), and (iii) are at least 15 base pairs in length. In some aspects, the disclosure provides a composition comprising one or more spherical nucleic acids (SNAs), comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides that (i) activate a cytoplasmic DNA sensor, (ii) inactivate a transcription factor, and (iii) are at least 15 base pairs in length.In some aspects, the disclosure provides a composition comprising one or more spherical nucleic acids (SNAs), comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates cyclic GMP-AMP synthase (cGAS) and is at least 15 base pairs in length. In some aspects, the disclosure provides a composition comprising one or more spherical nucleic acids (SNAs), comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which (i) activates cyclic GMP-AMP synthase (cGAS), (ii) inactivates signal transducer and activator of transcription 3 (STAT3), and (iii) is at least 15 base pairs in length. In some aspects, the disclosure provides a composition comprising one or more spherical nucleic acids (SNAs), comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which activates a cytoplasmic DNA sensor and is at least 15 base pairs in length. In some aspects, the disclosure provides a composition comprising one or more spherical nucleic acids (SNAs), comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a plurality of double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which (i) activates a cytoplasmic DNA sensor, (ii) inactivates a transcription factor, and (iii) is at least 15 base pairs in length.In various embodiments, the cytoplasmic DNA sensor is cyclic GMP-AMP synthase (cGAS), AIM2 (absent in melanoma-2), RNA polymerase III, DAI (DNA-dependent activator of IFN-regulatory factors), IFI16 (interferon-γ-inducible protein 16), or a combination thereof. In any of the aspects or embodiments of the present disclosure, the transcription factor promotes cancer progression. In various embodiments, the transcription factor is signal transducer and activator of transcription 3 (STAT3), cMyc, NANOG, SOX2, OCT4, or a combination thereof.

[0066] In some embodiments, the composition is an antigenic composition. In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier. The term "carrier" refers to a vehicle in which the SNA described herein is administered to a mammalian subject. The term carrier includes diluents, excipients, adjuvants, and combinations thereof. Pharmaceutically acceptable carriers are well known in the art (see, e.g., Remington's Pharmaceutical Sciences by Martin, 1975).

[0067] Exemplary "diluents" include sterile liquids such as sterile water, saline solution, and buffers (e.g., phosphate, tris, borate, succinate, or histidine). Exemplary "excipients" are inert substances that may enhance vaccine stability, including, but not limited to, polymers (e.g., polyethylene glycol), carbohydrates (e.g., starch, glucose, lactose, sucrose, or cellulose), and alcohols (e.g., glycerol, sorbitol, or xylitol). Adjuvants include vaccine delivery systems (e.g., emulsions, microparticles, immune stimulating complexes (ISCOMS), or liposomes) that target associated antigens to antigen-presenting cells (APCs), and immunostimulatory adjuvants.

[0068] antigen In various aspects, the present disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a double-stranded or single-stranded stem-loop DNA oligonucleotide that activates a cyclic GMP-AMP synthase (cGAS) and is at least 15 base pairs in length. The present disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising a double-stranded or single-stranded stem-loop DNA oligonucleotide that activates a cytoplasmic DNA sensor described herein and is at least 15 base pairs in length. In some embodiments, the SNA of the present disclosure comprises an antigen. In various embodiments, the antigen is a peptide, a protein, a DNA or RNA molecule encoding the antigen, or a combination thereof. In some embodiments, the antigen is a cancer-associated antigen. In further embodiments, the antigen is a tumor-associated antigen, a tumor-specific antigen, a neoantigen, or a combination thereof. In various embodiments, the cancer is breast cancer, peritoneal cancer, cervical cancer, colon cancer, rectal cancer, esophageal cancer, eye cancer, liver cancer, pancreatic cancer, laryngeal cancer, lung cancer, skin cancer, ovarian cancer, prostate cancer, gastric cancer, testicular cancer, thyroid cancer, brain cancer, or a combination thereof. In various embodiments, the antigen is OVA1, MSLN, P53, Ras, mutant IDH1 (IDH1R132H), mutant telomerase reverse transcriptase (TERT; C228T or C250T), melanoma-associated antigen, HPV-associated antigen, prostate cancer-associated antigen, glioblastoma antigen, grade IV astrocytoma antigen, ovarian cancer-associated antigen, breast cancer-associated antigen, hepatocellular carcinoma-associated antigen, colon cancer-associated antigen, or human papillomavirus (HPV) E7 nucleoprotein.

[0069] In some embodiments, the antigen is encapsulated in the nanoparticle core. In some embodiments, the antigen is a DNA or RNA molecule encoding the antigen encapsulated in the nanoparticle core. In some embodiments, the antigen is bound to the surface of the nanoparticle core, the antigen is bound to an oligonucleotide in the shell of the oligonucleotide, or both (see, e.g., U.S. Patent Application Publication No. 2020 / 0384104, which is incorporated by reference in its entirety). In further embodiments, the antigen is (i) encapsulated in the nanoparticle core, (ii) bound to the surface of the nanoparticle core, and / or bound to an oligonucleotide in the shell of the oligonucleotide. In some embodiments, the antigen is bound to the surface of the nanoparticle core via a linker.

[0070] Use of SNAs to treat disorders In some embodiments, the SNAs of the present disclosure are used to treat, alleviate, or ameliorate a disorder. Thus, in some aspects, the present disclosure provides a method of treating or ameliorating a disorder (e.g., cancer), comprising administering an effective amount of the SNA or composition of the present disclosure to a subject (e.g., a human subject) in need thereof, where the administering treats or ameliorates the disorder. In various embodiments, the cancer is breast cancer, peritoneal cancer, cervical cancer, colon cancer, rectal cancer, esophageal cancer, eye cancer, liver cancer, pancreatic cancer, laryngeal cancer, lung cancer, skin cancer, ovarian cancer, prostate cancer, stomach cancer, testicular cancer, thyroid cancer, brain cancer, or a combination thereof.

[0071] Administration of the SNAs and compositions of the present disclosure may involve a single dose or a multiple dose schedule, in which the various doses may be given by the same or different routes.

[0072] Methods of Gene Regulation In some aspects of the disclosure, the oligonucleotides associated with the SNAs of the disclosure inhibit gene expression. Thus, in some embodiments, the SNAs of the disclosure activate cGAS, optionally inactivate STAT3, and also have gene-inhibiting function. Thus, in some embodiments, the shell of oligonucleotides attached to the outer surface of the nanoparticle core activates cyclic GMP-AMP synthase (cGAS), comprises one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which is at least 15 base pairs in length, and further comprises one or more inhibitory oligonucleotides designed to inhibit target gene expression. In some embodiments, the SNAs of the disclosure activates a cytoplasmic DNA sensor, optionally inactivates a transcription factor, and also has gene-inhibiting function. In further embodiments, the shell of oligonucleotides attached to the outer surface of the nanoparticle core activates a cytoplasmic DNA sensor as described herein, comprises one or more double-stranded and / or single-stranded stem-loop DNA oligonucleotides, each of which is at least 15 base pairs in length, and further comprises one or more inhibitory oligonucleotides designed to inhibit target gene expression. In some embodiments, one or more inhibitory oligonucleotides designed to inhibit target gene expression are encapsulated in the nanoparticle core. In some embodiments, one or more inhibitory oligonucleotides designed to inhibit target gene expression are both attached to the exterior surface of the nanoparticle core and encapsulated in the nanoparticle core.

[0073] Methods of inhibiting expression of a gene product include those in which expression of the target gene product is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% compared to expression of the gene product in the absence of SNA. In other words, the methods provided encompass essentially any degree of inhibition of expression of the target gene product.

[0074] The degree of inhibition is determined in vivo from bodily fluid or biopsy samples, or by imaging techniques well known in the art, or, alternatively, in cell culture assays as a predictable measure of the degree of inhibition that can generally be expected in vivo resulting from the use of a particular type of SNA and a particular oligonucleotide.

[0075] In various embodiments, the methods involve the use of oligonucleotides that are 100% complementary, i.e., a perfect match, to the target oligonucleotide, while in other embodiments, the oligonucleotides include the use of oligonucleotides that are at least (meaning greater than or equal to) about 95% complementary to the polynucleotide over the length of the oligonucleotide, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20% complementary to the polynucleotide over the length of the oligonucleotide to the extent that the oligonucleotide is capable of achieving the desired degree of inhibition of the target gene product.

[0076] Percent complementarity is determined over the length of the oligonucleotide. For example, consider an antisense compound in which 18 of the 20 nucleotides of an inhibitory oligonucleotide are complementary to a 20 nucleotide region of a target polynucleotide of full length 100 nucleotides, and the oligonucleotide will be 90 percent complementary. In this example, the remaining non-complementary nucleotides can be clustered or interspersed with complementary nucleotides, and do not have to be adjacent to each other or to complementary nucleobases. Percent complementarity of an inhibitory oligonucleotide with a region of a target nucleic acid can be routinely determined using BLAST (basic local alignment search tool) and PowerBLAST programs (Altschul et al., J.Mol.Biol., 1990,215,403-410; Zhang and Madden, Genome Res., 1997,7,649-656) known in the art.

[0077] The oligonucleotides utilized in such methods are either RNA or DNA. The RNA may be an inhibitory oligonucleotide, such as an inhibitory RNA (RNAi) that performs a regulatory function, and in various embodiments is selected from the group consisting of a small inhibitory RNA (siRNA), a single guide RNA (sgRNA), a single stranded RNA (ssRNA), and a ribozyme. Alternatively, the RNA is a microRNA that performs a regulatory function. The DNA, in some embodiments, is antisense-DNA. In some embodiments, the RNA is a piwi-interacting RNA (piRNA).

[0078] Therapeutic Agents The technology provided herein has broad applicability. ISD and SNA ISD / STAT3i ) are a class of oligonucleotide-based drugs that can be used as generalizable antitumor agents against a wide range of tumor types.

[0079] Therapeutic agents contemplated by the present disclosure include, but are not limited to, a protein (e.g., a therapeutic protein), a growth factor, a hormone, an interferon, an interleukin, an antibody or antibody fragment, a small molecule, a peptide, an antibiotic, an antifungal agent, an antiviral agent, a chemotherapeutic agent, or a combination thereof.

[0080] The term "small molecule" as used herein refers to a chemical compound or drug, or any other low molecular weight organic compound, either natural or synthetic. By "low molecular weight" we mean a compound having a molecular weight of less than 1500 Daltons, typically between 100 and 700 Daltons.

[0081] In some embodiments, SNAs are used in combination with one or more standard of care therapeutic agents, such as radiation, and existing immunotherapies, including but not limited to checkpoint inhibitors, such as anti-PD1, and inhibitors of immunosuppressive adenosine signaling, such as anti-CD73 (Ty / 23) and the A2aR inhibitor SCH58261.

[0082] Thus, in some embodiments, the SNA of the present disclosure further comprises a therapeutic agent, or agents. The therapeutic agent, in various embodiments, is simply associated with an oligonucleotide in the shell of the oligonucleotide bound to the external surface of the nanoparticle core of the SNA, and / or the therapeutic agent is associated with the nanoparticle core of the SNA, and / or the therapeutic agent is encapsulated in the SNA. In some embodiments, the therapeutic agent is associated with the terminus of an oligonucleotide in the shell of an oligonucleotide that is not bound to a nanoparticle core (e.g., if the oligonucleotide is bound to the nanoparticle core through its 3' terminus, the therapeutic agent is associated with the 5' terminus of the oligonucleotide). Alternatively, in some embodiments, the therapeutic agent is associated with the terminus of an oligonucleotide in the shell of an oligonucleotide that is bound to a nanoparticle core (e.g., if the oligonucleotide is bound to the nanoparticle core through its 3' terminus, the therapeutic agent is associated with the 3' terminus of the oligonucleotide). In some embodiments, the therapeutic agent is covalently associated with an oligonucleotide in the shell of an oligonucleotide that is bound to the external surface of the nanoparticle core of the SNA. In some embodiments, the therapeutic agent is covalently associated with a linker or spacer that is bound to the external surface of the nanoparticle core of the SNA. In some embodiments, the therapeutic agent is non-covalently associated with the oligonucleotides in the shell of oligonucleotides bound to the outer surface of the nanoparticle core of the SNA. However, it should be understood that the present disclosure provides SNAs in which one or more therapeutic agents are both covalently and non-covalently associated with the oligonucleotides in the shell of oligonucleotides bound to the outer surface of the nanoparticle core of the SNA. It should also be understood that non-covalent association includes hybridization, protein binding, and / or hydrophobic interactions. In some embodiments, the therapeutic agent is administered separately from the SNA of the present disclosure. Thus, in some embodiments, the therapeutic agent is administered before, after, or simultaneously with the SNA of the present disclosure to treat a disorder.

[0083] The following examples illustrate various embodiments contemplated by the present disclosure. The examples are illustrative in nature and are not intended to be limiting in any way. EXAMPLES

[0084] Example 1 In this example, the gold nanoparticle core (SNA ISD We have synthesized and characterized cGAS-activated SNAs that display interferon-stimulated double-stranded (ds)DNA (ISD) at high surface density on . The data provided herein demonstrate that SNAs ISD We showed that SNA activated the cGAS-STING pathway more potently than CDN, culminating in the downstream induction of IRF- and NF-κB-dependent transcription. When administered intratumorally or intranasally to immune-competent GBM-bearing mice, SNA ISD is the free ISD 45 It antagonized tumor progression more robustly than oligonucleotides or CDNs and promoted long-term survival in animal subjects through specific activation of the cGAS-STING signaling pathway.

[0085] In addition to monofunctional cGAS-activating SNAs, we generated bimodal SNAs that can activate cGAS and concomitantly inhibit the transactivation activity of the STAT3 master transcription factor. To this end, we replaced the standard ISD DNA oligonucleotides with oligonucleotides shown to bind to, sequester and inactivate the master transcription factor STAT3, since DNA recognition by cGAS occurs independently of the oligonucleotide sequence. Bimodal SNAs ISD / STAT3i SNA was shown to activate cGAS, block the nuclear translocation and transcriptional activity of STAT3 in vitro, and antagonize glioma progression in vivo.

[0086] Development and characterization of ISD45-SNA Because 45-bp ISD oligonucleotides represent the most used and extensively characterized cGAS activators, to exploit the SNA architecture for cGAS activation, we synthesized ISD45-SNA by conjugating 13-nm gold nanoparticles with a 45-base pair (bp) dsISD oligonucleotide (ISD45-SNA, Figure 1A). 7 "dsISD 45 The terms "ISD45" and "ISD45" are interchangeable as used herein. Citrate-stabilized gold nanoparticles were prepared using the Frens method. 4、8、9 Thiolated dsDNA oligonucleotides were synthesized, purified using reversed-phase high-performance liquid chromatography, and covalently attached to gold nanoparticle surfaces by salt aging with sodium chloride. 10、11 Electron microscopy and dynamic light scattering confirmed the SNA ISD-45 The shape and size were confirmed (13 nm gold core, 72 nm diameter including DNA corona). In addition, oligonucleotide incorporation was quantified by generating melting curves and determining zeta potential using the OliGreen fluorescence assay. 4、8、9 .SNA ISD To assess the direct involvement of the cGAS DNA-sensing enzyme in the activation of GFP, we developed a cell-free cGAS activation assay using human recombinant cGAS enzyme (Figure 1B). 2 A mixture of ATP and GTP, ssDNA 45 , ISD45, cGAMP, ssDNA 45 The mixture was incubated with either IRF-SNAs, ISD45-SNAs, or ISD45-SNAs. Upon inactivation of the cGAS enzyme, the mixture was added to cGAS-deficient RAW-Lucia™ macrophages, carrying stable integration of an IRF-inducible Lucia luciferase reporter, to luminometrically quantitate IRF induction. This macrophage line is unable to synthesize cGAMP due to cGAS deficiency. As shown in Figure 1C, ISD45-SNAs activated IRFs to similar levels compared to free ISD45 oligonucleotides or cGAMP-transfected cultures, but not ssDNA. 45ISD45-SNA-treated macrophages did not activate IRF to similar levels. In both RAW-Lucia™ macrophages and THP-1 cells, a human monocytic cell line, ISD45-SNA potently activated IRF, reflecting robust cellular uptake, particularly in macrophages (Figure 2A), when compared with free oligonucleotides and when compared with similar levels achieved with free oligonucleotides delivered via lipoplex (Lipofectamine 3000) transfection (TFX) (Figures 2B-C). The dependency of IRF induction on functional cGAS-STING pathway components was then assessed in isogenic cGAS-, STING-, and IRF-deficient macrophages (Figure 2D), demonstrating that SNA strongly activated IRF in the absence of STING, cGAS, or IRF. ISD-45 In addition, multiplex cytokine profiling using ELISA and antibody arrays demonstrated that IRF induction in response to SNA or ISD-45 treatment was blunted. ISD In response to treatment, we revealed robust activation of NF-κB- and STAT6-induced proinflammatory cytokines, including IL-6, TNF-α, and CCL2 (Figure 2E-F). Importantly, as reported for gene-regulatory and TLR9-activating SNAs, the cGAS-activating properties of ISD45-SNAs arise not from the nanoparticle core but from the densely functionalized and highly oriented nucleic acid shell. 12~14 Similar to the 13 nm gold-based architecture (Figures 1 and 2), the ISD45-SNA with an 80 nm PLGA-based core (Figure 3A) entered macrophages via phagocytosis and scavenger receptor-dependent endocytosis (Figure 3B), promoted IRF activation, unlike ssDNA45-SNA (Figure 3C), and showed a higher EC than AduroS100, a CDN currently in clinical trials for non-CNS solid cancers. 50 The values ​​are shown (Figure 3D).

[0087] ISD45-SNA induced M1 macrophage polarization that reduced tumor cell viability and antagonized tumor progression in vivo. In addition to inducing adaptive T cell responses against tumors, several studies have shown that activation of the cGAS-STING pathway antagonizes tumor growth by promoting the activation of proinflammatory M1-polarized macrophages. Because GBM is mostly composed of tumor-associated macrophages with an anti-inflammatory M0 / 2-like phenotype, we investigated whether ISD45-SNA could re-educate M0 macrophages to an M1 phenotype. In RAW-Blue™ macrophages, ISD45-SNA treatment caused robust TBK1 phosphorylation, as well as induction of cytokine-inducible nitric oxide synthase (iNOS), a classic marker of a proinflammatory macrophage state, but not ssDNA phosphorylation. 45 -SNA did not induce this induction (Fig. 4A). ISD-45 increased nitrite levels in macrophage supernatants (Figure 4B), and SNA ISD-45 or treated with conditioned medium from macrophages exposed to SNA ISD-45 Tumor cells co-cultured with macrophages exposed to cGAS reduced tumor cell proliferation and viability (Figure 4C-D). When injected intratumorally, SNAs infiltrated intracranial tumors extensively, as shown by MR imaging of SNAs functionalized with Gd(III)-labeled DNA oligonucleotides (Figure 4E). To investigate whether cGAS agonist SNAs reduce GBM tumor progression in vivo, a syngeneic luciferase-modified CT2A orthotopic engraftment model was developed in which macrophages functionalized with ssDNA were injected 7 days after tumor cell inoculation. 45 - or ISD45-SNA (corresponding to 0.15 mg / kg dsDNA) administered intratumorally. Analysis of subject survival using the Kaplan-Meier method demonstrated that ISD45-SNA significantly delayed GBM tumor progression and promoted long-term survival in more than 50% of treated female subjects, whereas ssDNA 45 This was not the case in the control SNA ( Fig. 4<em>F ).

[0088] Non-invasive nose-to-brain delivery of ISD45-SNAs Localized ISD45-SNA delivery to the tumor bed avoids the blood-brain barrier and circumvents toxicity due to systemic exposure, and therefore remains an attractive and transferable option for GBM treatment. Therefore, we explore and optimize intratumoral administration of cGAS agonist SNAs, as described below. In addition to direct intratumoral administration of cGAS agonist SNAs, we explore non-invasive nose-to-brain delivery of SNAs, taking advantage of the increased stability and bioavailability of SNA-formulated oligonucleotides, as well as their tendency to infiltrate the tumor parenchyma. Similar to intratumoral delivery, intranasal delivery bypasses the blood-brain barrier and minimizes systemic exposure. Additionally, the emergence of novel intranasal delivery technologies, coupled with recent preclinical and clinical studies, have demonstrated the safety and efficacy of nose-to-brain delivery for the delivery of a broad spectrum of therapeutic agents, including perillyl alcohol (NEO100) [currently being evaluated in a Phase I / II clinical trial] for the treatment of recurrent GBM, making intranasal delivery a promising, non-invasive delivery method for the treatment of GBM, motivating the studies outlined in the grant application to comprehensively evaluate and optimize nose-to-brain delivery of SNAs.

[0089] Intranasally administered drugs reach the CNS through a pathway involving the olfactory and trigeminal nerves that connect the nasal meatus to the CNS (Figure 5A). ICP-MS analysis of Au content showed that approximately 10% of the total administered ISD45-SNA dose accumulated in the tumor-bearing brain when administered intranasally (Figure 5B). Similarly, using gold-based ISD45-SNAs labeled with Cy5 (Figure 5C), fluorescence microscopy imaging revealed SNA accumulation within tumor elements (Figure 5D) and within the epineurium of the trigeminal nerve (Figure 5E). Similarly, PLGA-based SNAs labeled with Cy5 (Figure 5F) showed brain accumulation, with Cy5-labeled structures detectable both in the olfactory bulb and within intracranial tumors (Figure 5G-H). ISD45-SNAs were also found within the epineurium, perineurium, and, to a lesser extent, endoneurium of the trigeminal nerve (Figure 5I). These data suggested that nose-to-brain delivery of ISD45-SNAs occurred, at least in part, along the extracellular components of the trigeminal nerve.

[0090] Intranasal administration of ISD45-SNA reduces GBM progression in a STING-dependent manner and promotes long-term survival in combination with immune-mediated checkpoint inhibition. Luciferase-labeled CT2A tumor cells were implanted into either C57BL / 6-wt or C57BL / 6-STING Golden Ticket (gt / gt) syngeneic hosts. ISD45-SNA delivered via a single intranasal administration reduced tumor progression and increased animal survival in CT2A-bearing C57BL / 6 wild-type mice (Figure 6A) and did not affect tumor progression in STING-deficient gt / gt mice (Figure 6B). The benefit of therapeutic SNA was significantly higher compared to ADU-S100, a cyclic dinucleotide currently in clinical trials (Figure 6C). Kaplan-Meier survival analysis revealed that ISD45-SNA extended animal subject survival in C57BL / 6 wild-type animals, particularly in female but not male animals (Figure 6D), but did not extend survival in C57BL / 6 hosts lacking functional STING in the tumor microenvironment when compared to untreated or ssDNA45-SNA-treated mice (Figure 6E-F). When co-treated with ISD45-SNA and immune-mediated checkpoint inhibition (anti-PD1 + anti-CTLA4), a single intranasal delivery abolished GBM tumor development in nearly all female animals (Figure 6G), but had no effect on male mice (Figure 7A). Re-examination of long-term surviving female mice injected with tumor cells in the contralateral hemisphere resulted in long-term survival in all mice indicating long-term anti-tumor memory (Figure 7B).

[0091] Analysis of tumor-associated and peripheral immune system composition in mice injected with SNA through direct intratumoral administration was compared with untreated, ssDNA 45We revealed enrichment of effector T cell (Figure 8A), M1 polarized macrophages (Figure 8B), and activated NK cell populations in ISD45-SNA-treated mice compared to ISD45-SNA-treated animal subjects (Figure 8C-F). These data indicated that the antitumor activity of ISD45-SNA was greater than that of CDNs, given non-invasive nose-to-brain delivery, required functional STING in the tumor microenvironment, and could be enhanced by combining with immune checkpoint inhibition selectively in female animal subjects, but not in males.

[0092] Example 2 Development of bimodal ISD45 / STAT3i-SNA Signal Transducer and Activator of Transcription 3 (STAT3) as a Critical Transcriptional Regulator of Innate and Adaptive Immunity and an Actionable Immune Target 15 Recent studies have identified STAT3 inhibitors as a novel class of immunotherapeutic agents that can enhance antitumor immunity in response to activation of the STING pathway. 15 In the absence of strict sequence and structural parameters guiding dsDNA engagement with cGAS, we investigated SNA constructs formulated with oligonucleotides that, in addition to cGAS activation, could function as STAT3 decoy oligonucleotides that sequester STAT3 in the cytoplasm and prevent it from translocating to the nucleus and activating transcription there. Recent studies defining the interaction of ISD oligonucleotides with cGAS suggested that a minimum length of 15 bp is required to promote the synthesis of cGAMP1 and identified a palindrome flanked by unpaired guanosine trimers (G3s) that potentiates the immune stimulatory effects of otherwise inactive blunted sequences in a cGAS-STING-dependent manner.

[0093] To increase the antitumor immune response of the prototype ISD45-SNA, we designed SNAs conjugated with a 15 bp ISD oligonucleotide derived from a hairpin structure containing a G5 repeat element adjacent to a STAT3 decoy sequence (ISD15STAT3i-SNA) and a STAT3 decoy palindrome (ISDG5STAT3i-SNA, Figure 9A). Modified gel shift assays and Hill-Langmuir computing showed that all SNAs formed complexes with cGAS with KDs ranging from 0.17 to 0.24 nM (Figure 9B,C), and both ISD15STAT3i and ISDG5STAT3i-SNAs bound directly to STAT3 with KDs of 0.045 and 0.07 nM, but ISD45-SNA did not bind directly to it (Figure 9D,E). Consistent with the cell-free binding profiles, all SNA architectures induced IRF3 activation as assessed by both cell-free and cell-based IRF3 reporter assays (Figure ​ (Figure9F,G), while only ISD15STAT3i- and ISDG5STAT3i-SNAs, but not ISD45-SNAs, suppressed IL-6 levels as assessed in HEK-Blue IL6 reporter cells (Figure ​ (Figure9H).9H).

[0094] Bimodal ISD compared with monofunctional ISD45-SNA in CT2A- and advanced syngeneic orthotopic glioblastoma models using tumor cell spheres derived from genetically engineered glioblastoma mouse models 15 STAT3i and ISD G5 The therapeutic effect of STAT3i-SNA was evaluated. In this model, glioma formation was regulated by the expression of Quaking, a STAR family RNA-binding protein that regulates pluripotency and self-renewal in neural and glioma stem cells, as well as the expression of p53 and PTEN tumor suppressors (genotype: Nestin-CreER). T2 ;Qki L / L ;Pten L / L ;Trp53 L / LThe checkpoint inhibitor-sensitive CT2A and QPP7 models are driven by tamoxifen-induced CNS-specific deletion of ISD45-SNA and ISD5-SNA (referred to as the QPP model). Tumor neurospheres isolated from QPP tumors and orthotopically implanted into syngeneic hosts led to glioblastoma tumor formation with a latency of 40–70 days. Tumor neurosphere cultures derived from different QPP tumors showed a spectrum of sensitivity to checkpoint inhibition. QPP4 tumors were resistant to checkpoint inhibitor treatment, whereas explant tumor models derived from QPP7 cells were sensitive to anti-CTLA4 and showed a trend towards sensitivity to anti-PD1 blockade. In the checkpoint inhibitor-sensitive CT2A and QPP7 models, ISD45-SNA and ISD 15 STAT3i-SNAs potently antagonized tumor progression and extended survival in female animal subjects, with 60-80% of mice (but not males) showing long-term survival, but not ISD G5 STAT3i-SNA did not show this (Figure 10A, B). In the checkpoint blockade-resistant QPP4 model, we used only female mice to investigate the ISD G5 Treatment with STAT3i provided a significant but smaller benefit with 30% of mice showing long-term survival, whereas ISD45-SNA and ISD 15 This was not observed with STAT3i-SNA (Fig. 10C).

[0095] Immune-mediated changes in response to SNA treatment are gender specific. To define immune-mediated changes in SNA- vs. control-treated male and female animal subjects, gene expression analysis of the TME was performed using the Nanostring PanCancer Immune Profiling Panel. QPP4 tumor-bearing C57BL / 6 mice were treated with PBS, ssDNA45-SNA, ISD45-SNA, ISD45-SNA, or ISD45-SNA via intracranial administration 8 days after tumor cell inoculation. 15 STAT3i-SNA or ISD G5Following treatment with STAT3i-SNA, RNA was isolated from tumor-infiltrating immune cells 30 days after tumor implantation. In females, unsupervised clustering of gene expression ranked by z-score revealed a clear separation between treatment and control groups, with SNA treatment inducing immune activating and down-regulating immune suppressive genes (Figure 11A). Up-regulated genes included inducers of interferon (Tbk1, Irf3, Ifnb1, Ifna1), M1 polarization (Mst1r, Mx1), T cell costimulation (Icam, Ncam, Icosl, CD28), T cell activation (CD69), and immune cell migration (Ccl3, Ccl4, Cxcl12, Csf1). In addition, immune suppressive genes expressed by activated M2 macrophages and myeloid cells (Arg1, Arg2, Msr1, IL-10, CCl17, CCl22), as well as genes responsible for suppressing CD4+ and CD8+ effector T cell function (Pdcd1, FoxP3, Vegfa, Hif1a), were significantly downregulated with SNA treatment compared to PBS and ssDNA45-SNA controls. In stark contrast, SNA treatment in male mice downregulated a broad array of genes, including immune checkpoint receptors (Ctla4, Havcr2, Icos), and genes required for activation of innate immunity (Isg20, Isg15, Cxcl10), and T cells (CD28, CD3, Grmb, CD8) (Figure 11B). Nanostring gene set pathway analysis demonstrated that in the female QPP4 glioma model, ISD G5 -STAT3i-SNA upregulates 25 / 30 immune cell pathways involved in macrophage, microglia, dendritic cells, T cells, and leukocyte function, inflammation, and responses to chemokines and cytokines and pathogens, and ISD45-SNA and ISD 15 Compared with STAT3i SNA, ISD G5 We found that STAT3i-SNA showed stronger pro-inflammatory and anti-tumor activity in male QPP4 mice (Fig. 11C). G5 -STAT3i-SNA and ISD45-SNA did not induce any immune response and downregulated all innate and adaptive immune mechanisms (30 / 30) (Figure 11D).

[0096] In particular, ISD G5 To characterize the sexually dimorphic gene signature induced in response to -STAT3i-SNA, we performed unsupervised clustering on all female (n = 3) and male (n = 4) samples (Figure 11E-G), followed by gene ontology pathway analysis. As shown in Figure 11F, ISD G5 STAT3i-SNA treatment led to a 15-fold enrichment of the adaptive immune response.

[0097] SNA ISD / STAT3i To begin to dissect the molecular basis for sexually dimorphic responses to treatment, cGAS protein expression was analyzed using Western blot and flow cytometry analysis in whole spleens (FIG. 12A), BMDMs (FIG. 12B), and TAMCs isolated from tumor-bearing mice treated with either PBS, ssDNA45-SNA, or ISD45-SNA (FIG. 12C). Results showed higher cGAS expression in female versus male immune cells. Thus, these data suggest cGAS expression levels as a putative predictive biomarker for cGAS agonist SNA response.

[0098] Consideration Stimulation of the cGAS-STING pathway increases T cell activation and tracking into tumors and reverses the immunosuppressive phenotype of myeloid cells. Direct targeting of STING receptors using synthetic cyclic dinucleotide (CDN) ligands represents an attractive immunotherapeutic strategy for the treatment of lymphopenic and myeloid cell-rich tumors such as glioblastoma (GBM). However, this approach is limited by the metabolic instability of CDNs as well as the presence of immune evasive and STING inhibitory mechanisms in the tumor microenvironment (TME), including those orchestrated by the master transcriptional regulator of inflammation and immunity, STAT3 (signal transducer and activator of transcription 3). To activate cGAS antitumor responses and simultaneously inhibit STAT3-driven immune suppression in the GBM TME, we generated spherical nucleic acids (SNAs) consisting of a nanoparticle core densely functionalized with a shell of radially oriented DNA oligonucleotides containing a STAT3 decoy sequence. The bimodal SNA architecture, via multivalent and high affinity binding of cGAS and STAT3 proteins, induced type I interferon (IFN), blocked STAT3 nuclear translocation and transactivation, reduced tumor cell viability, promoted immune activation in the GBM tumor microenvironment, and induced strong antitumor activity. A remarkable sexually dimorphic response to SNA treatment was observed, with female animal subjects having therapeutic effects of bimodal SNAs, but males did not. These studies established multifunctional SNAs as first-in-class single entity nucleic acid therapeutic agents for targeting multiple immune pathways in GBM.

[0099] Because DNA recognition by cGAS is sequence-independent, we tested whether replacing the canonical cGAS agonist oligonucleotide sequence with a palindromic sequence known to bind and sequester STAT3 would result in an SNA architecture that could promote cGAS-STING pathway activation and simultaneously inhibit STAT3 function (Figure 9A). Indeed, ISD G5 and ISD G5 / STAT3i-SNA blocked STAT3 transactivation (Figure 9B) and promoted IRF activation (Figure 9C) as assessed by STAT3 reporter assay. Consistent with the reduced STAT3 transactivation activity in response to SNA treatment, bimodal SNA sequestered pSTAT3 in the cytoplasm (Figure 9D), reduced sphere size of glioma stem cell cultures (Figure 9E), and extended survival of animal subjects when administered via intracranial administration (Figure 9F).

[0100] Materials and Methods Cell lines. Raw-Lucia™ ISG, Raw-Lucia™ ISG cGAS KO, ​​and HEK-blue STAT3 reporter cell lines were purchased from InvivoGen (San Diego, CA, USA) and cultured according to the manufacturer's protocol. QPP4, QPP7 cell lines were generated and donated by the laboratory of Dr. Amy Heimberger. QPP tumor neurospheres were cultured in a genetically engineered mouse model (genotype: Nestin-Cre-ERT2; Quaking L / L ;p53 L / L ;PTEN L / L ) were isolated from GBM tumors arising in the NeuroCult Proliferation Kit (Media and Supplements, Stem Cells) supplemented with EGF (20 μL / 10 mL of media) and FGF (10 μL / 10 mL of media). GL261-luc, GL261, and CT2A cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM, Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Hyclone). All media were supplemented with 1% penicillin / streptomycin (Invitrogen) and cell lines were cultured at 37°C in a humidified environment with 5% CO2. All cell lines were tested negative for mycoplasma either in-house using the MycoAlert Plus Mycoplasma detection kit (Lonza) or by Charles River Research Animal Diagnostic Services (CR RADS, MA USA).

[0101] Mouse tumor model for in vivo SNA testing. All animals were used under approved animal research protocols, maintained in the Northwestern University animal facility, and treated in accordance with the Institutional Animal Care and Use Committee (IACUC) regulations and guidelines. 6-8 week-old female or male C57BL / 6J mice (The Jackson Laboratory) were inoculated with CT2A (8 × 10 4 cells / mouse), QPP7, or QPP4 cells (100 × 10 4 Cell lines (cells / mouse) were implanted intracranially. Each mouse was anesthetized using an intraperitoneal (ip) injection of ketamine / xylazine, and the surgical area was cleaned with alcohol and betadine. After an incision was made on the scalp, a 0.7 mm burr hole was created using a superfine surgical drill, 2 mm lateral to the right of the sagittal suture and 4 mm behind the coronal suture. Cell lines loaded into a 25 μL Hamilton syringe were implanted using a stereotaxic frame (Stoelting). After surgery, the skin was closed with sutures. After tumor implantation, mice were randomly assigned to control or treatment groups (n=6-10 / group). Approximately 100 mm 3 When the tumors reached an average size of 100 μg / mL, they were cultured in PBS, ssDNA, T45 -SNA, ISD 45 -SNAs, STAT3i 15 -SNA, ISD G5 Mice were treated via intratumoral or intranasal injection of either STAT3i-SNA nanoparticle formulations. Mice were sacrificed upon observation of neurological dysfunction (lethargy, loss of locomotion, lack of food intake, or >20% weight loss), which typically occurred 48 hours before death. Survival analysis was performed using the Kaplan-Meier method, and statistical significance was assessed using the log-rank (Mantel-Cox) test.

[0102] Intranasal delivery. ISD 45 -The therapeutic efficacy of SNA was also improved by CT2A (8 × 10 4The effect was determined upon intranasal (in) administration in a mouse model (100 μg / mouse). A single dose of sterile saline or SNA (15 μmol Au / kg mouse weight) was given 7 days after intracranial tumor cell implantation. 2 μL aliquots of saline or SNA were given to each nostril every 5 min.

[0103] Cannula implantation and intracranial SNA injection. Mice were anesthetized with ketamine / xylazine as described above. A 26-gauge sterile plastic guide cannula was inserted into the skull at a depth of 2 mm through the burr hole. The implanted cannula was secured and the skin was closed by applying surgical adhesive. Standard post-operative care was given according to IACUC approved protocols. Cells were implanted or therapeutics diluted in sterile 0.9% saline were injected using a 33-gauge sterile Hamilton syringe with a sleeve designed to extend 1 mm beyond the tip of the guide cannula during injection. After injection, the cannula was covered with a 33-gauge dummy cannula or cap.

[0104] NanoString nCounter analysis. 1×10 6 QPP4 cells were implanted intracranially via a cannula into 6-8 week old male and female C57BL / 6 mice as described above. Mice were then re-injected with sterile PBS, ssDNA, 100 mM NaCl ... T45 -SNAs, ISDs 45 -SNA or ISD G5 Mice were treated intracranially with STAT3i-SNA (n=4 per group). The dose of AuNPs was 15 μmol Au per kg. Thirty days after SNA treatment, mice were euthanized and perfused intracardially with 15 mL of DPBS. The perfused brain tissue was further processed with "Mouse Brain Tumor Dissociation Kit" (Miltenyi Biotec, #130-096-730) to obtain isolated single-cell tumor cells and tumor-infiltrating immune cell suspensions. Immune cells were separated from the rest of the brain tissue by 30 / 70 Percoll gradient separation (GE Healthcare).

[0105] For Nanostring analysis, total RNA was extracted using a kit (Invitrogen™ PureLink™ RNA Mini Kit) according to the manufacturer's protocol. Quality control of the isolated RNA was performed using an Agilent Bioanalyzer and a NanoDrop 8000 (Thermo Scientific). RNA isolated from immune cells was analyzed by NanoString nCounter using the PanCancer Immune Profiling Panel. Briefly, 150 ng of RNA was hybridized for 16 hours using capture and reporter probes. Samples were then immobilized in cartridges using an nConter™ prep-station. Digital images were processed in the nCounter™ Digital Analyzer instrument and 555 fields of view (fov) were collected for each sample. Reporter probe counts (i.e., the number of times the color-coded barcode for that gene is detected) were analyzed using the nSolver™ package (version 3.0), the Advanced nSolver Analysis module (version 1.0.36), and ROSALIND software. QC, normalization, differential expression, and pathway analysis were performed using the nSolver Advanced Analysis module and ROSALIND software according to the guidance provided by the manufacturer. Fold changes were calculated by comparing against the average normalized gene expression value of PBS-treated tumors.

[0106] In vitro experiments to assess the involvement of cGAS and STAT3 Electrophoretic mobility shift assay. Recombinant human cGAS protein (Cayman) or recombinant human STAT3 protein was incubated with SNA in buffer (20 mM Tris buffer pH 7.2, sample volume 50 uL) for 1 hour at room temperature. Reactions contained 15 umoles of Au and cGAS / STAT3 protein (0.1ug-2ug). Reactions were analyzed by electrophoresis on a 1% agarose gel in 1x TAE buffer. Gel images were scanned using a Biorad geldoc system using FAST blast selection and analyzed for Au band intensity using FIJI.

[0107] IRF3 luciferase reporter assay. To measure IRF3 activation, Raw-Lucia™ ISG (InvivoGen) cells were seeded in 96-well plates at a density of 3.0×104 cells / well. The next day, cells were treated with SNA or PBS at a concentration of 15 umol Au. After 24 hours of incubation, the supernatant was transferred to a new white clear-bottom 96-well plate. Luciferase luminescence was then recorded on a Synergy H2 Microplate Reader (BioTek) using QUANTI-Luc luciferase reagent (Invivogen) according to the manufacturer's recommended protocol.

[0108] IL-6 reporter assay. To measure STAT3 activation, HEK-Blue™ IL-6 cells were cultured at 3.0 × 10 4 The cells were seeded in 96-well plates at a density of 100 cells / well and cultured overnight. The next day, the cells were incubated with SNA or PBS at a concentration of 15 umol Au for 4 hours, followed by IL-6 (1.5 ng / mL) treatment for 12 hours. Upon IL-6 stimulation, HEK-Blue™ IL-6 cells induce the activation of STAT3 and subsequent secretion of SEAP. The level of STAT3-induced SEAP was monitored using QUANTI-Blue™ solution.

[0109] cGAS cell-free activity assay. For evaluation of cGAS activity in cell-free assays, 60 nM recombinant human cGAS protein (Cayman) was mixed with ATP (5 mM), GTP (300 μM), and SNA (15 umoles Au) in reaction buffer (20 mM Tris-HCl, pH 7.5, 1.5 mM MgCl2). After incubation at 37°C for 2 h, the reaction was terminated by heating the reaction mixture at 95°C for 5 min to denature the proteins, which were removed by centrifugation at 20,000 x g for 5 min. The supernatant was transfected into Raw-Lucia™ ISG cGAS KO Raw Lucia Blue macrophages (InvivoGen). Cells were cultured at 37°C for 24 h, and then luminescence was recorded on a Synergy H2 Microplate Reader (BioTek) using QUANTI-Luc luciferase reagent (Invivogen) according to the manufacturer's recommended protocol.

[0110] Statistical analysis. All statistical analyses were performed using GraphPad Prism software, version 9.0. Nonparametric unpaired Student's t-test and Mann-Whitney U-test were used to assess the significance of differences between two groups. All data were reported as mean ± SEM. Multiple groups were analyzed by one-way ANOVA with Tukey's multiple comparison post-hoc test. Kaplan-Meier plots were generated to determine the relative survival of glioma-bearing mice under different treatment courses. The log-rank method was used to calculate p-values ​​for curve comparisons, followed by Bonferroni correction.

[0111] cGAS expression analysis. CT-2A-bearing mice treated with PBS, ssDNA45-SNA, or ISD45-SNA were incubated with CO on day 15. 2The animals were euthanized by centrifugation and perfused intracardially with 5 mL of DPBS. Brain / tumor single cell suspensions were obtained in Hank's Balanced Salt Solution (HBSS, Gibco) using a tissue homogenizer (Potter-Elvehjem PTFE pestle), followed by removal of myelin and debris by 30 / 70 Percoll gradient separation (GE Healthcare). Glioma-infiltrating immune cells were collected in complete RPMI for phenotyping or ex vivo studies and stained for cGAS expression. For this intracellular staining, cells were treated with STIM cocktail (BD Biosciences) for 4 hours before staining. Cells were washed and stained with Zombie NIR live / dead viability dye (ThermoFisher Scientific) in PBS, washed two more times, and stained for 30 minutes at 4°C with a cocktail of surface antibodies for CD11b and CD45 diluted 1:200 (final concentration) in PBS. For intracellular staining of cGAS, cells were fixed and permeabilized overnight in permeabilization buffer (eBioscience), incubated with cGAS anti-rabbit primary antibody for 1 h at room temperature, and then stained with goat anti-rabbit AF488 (Thermofisher A-11008) at a concentration of 1:2000 for 30 min. Samples were washed, acquired on a Cytek Aurora (Northern Lights), and analyzed using Cytobank V7.3.0.

[0112] For cGAS Western blot analysis, homogenized whole spleens or mouse BMDMs were lysed in RIPA buffer to generate whole cell lysates and subjected to Western blot analysis for cGAS and β-actin (loading control). The sequences disclosed herein: [Table 2] References 1 Corrales,L.,McWhirter,S.M.,Dubensky,T.W.,Jr. & Gajewski,T.F.The host STING pathway at the interface of cancer and immunity.The Journal of clinical investigation 126,2404-2411,doi:10.1172 / JCI86892(2016).4922692 2 Ohkuri,T.,Ghosh,A.,Kosaka,A.,Sarkar,S.N. & Okada,H.Protective role of STING against gliomagenesis:Rational use of STING agonist in anti-glioma immunotherapy.Oncoimmunology 4,e999523,doi:10.1080 / 2162402X.2014.999523 (2015).4485761 3 Mirkin,C.A. & Stegh,A.H.Spherical nucleic acids for precision medicine.Oncotarget 5,9-10(2014).3960185 4 Jensen,S.A.et al.Spherical Nucleic Acid Nanoparticle Conjugates as an RNAi-Based Therapy for Glioblastoma.Science translational medicine 5,209ra152,doi:10.1126 / scitranslmed.3006839(2013) 5 Radovic-Moreno,A.F.et al.Immunomodulatory spherical nucleic acids.Proceedings of the National Academy of Sciences of the United States of America 112,3892-3897,doi:10.1073 / pnas.1502850112(2015).4386353 6 Skakuj,K.et al.Conjugation Chemistry-Dependent T-Cell Activation with Spherical Nucleic Acids.Journal of the American Chemical Society 140,1227-1230,doi:10.1021 / jacs.7b12579(2018).5831183 7 Stetson,D.B. & Medzhitov,R.Recognition of cytosolic DNA activates an IRF3-dependent innate immune response.Immunity 24,93-103,doi:10.1016 / j.immuni.2005.12.003(2006) 8 Giljohann,D.A.,Seferos,D.S.,Prigodich,A.E.,Patel,P.C. & Mirkin,C.A.Gene regulation with polyvalent siRNA-nanoparticle conjugates.Journal of the American Chemical Society 131,2072-2073,doi:10.1021 / ja808719p(2009).2843496 9 Kouri,F.M.et al.miR-182 integrates apoptosis,growth,and differentiation programs in glioblastoma.Genes & development 29,732-745,doi:10.1101 / gad.257394.114(2015).4387715 10 Mirkin,C.A.,Letsinger,R.L.,Mucic,R.C. & Storhoff,J.J.A DNA-based method for rationally assembling nanoparticles into macroscopic materials.Nature 382,607-609,doi:10.1038 / 382607a0(1996) 11 Rosi,N.L.et al.Oligonucleotide-modified gold nanoparticles for intracellular gene regulation.Science 312,1027-1030,doi:10.1126 / science.1125559(2006) 12 Calabrese,C.M.et al.Biocompatible infinite-coordination-polymer nanoparticle-nucleic-acid conjugates for antisense gene regulation.Angewandte Chemie 54,476-480,doi:10.1002 / anie.201407946(2015).PMC4314394 13 Cutler,J.I. et al.Polyvalent nucleic acid nanostructures.Journal of the American Chemical Society 133,9254-9257,doi:10.1021 / ja203375n(2011).PMC3154250 14 Morris,W.,Briley,W.E.,Auyeung,E.,Cabezas,M.D. & Mirkin,C.A.Nucleic acid-metal organic framework (MOF) nanoparticle conjugates.Journal of the American Chemical Society 136,7261-7264,doi:10.1021 / ja503215w(2014) 15 Tripathi,S.,Najem,H.,Mahajan,A.S.,Zhang,P.,Low,J.T.,Stegh,A.H.,Curran,M.A.,Ashley,D.M.,James,C.D.,Heimberger,A.B.cGAS-STING pathway targeted therapies and their applications in the treatment of high-grade glioma.F1000Res.11:1010(2022).

Claims

1. Spherical nucleic acids (SNAs) comprising: (a) a nanoparticle core; (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising double-stranded DNA oligonucleotides or single-stranded stem-loop DNA oligonucleotides that activate cyclic GMP-AMP synthase (cGAS) and are at least 15 base pairs in length.

2. 2. The SNA of claim 1, wherein the shell of oligonucleotides comprises a plurality of double-stranded DNA oligonucleotides and / or single-stranded stem-loop DNA oligonucleotides, each of which activates cGAS and is at least 15 base pairs in length.

3. 2. The SNA of claim 1, wherein the shell of oligonucleotides comprises a plurality of double-stranded DNA oligonucleotides, each of the plurality of double-stranded DNA oligonucleotides comprising one strand comprising SEQ ID NO: 5 and another strand comprising a sequence complementary to SEQ ID NO:

5.

4. The SNA described in claim 1, wherein the oligonucleotide shell comprises a plurality of double-stranded DNA oligonucleotides, each of the plurality of double-stranded DNA oligonucleotides comprising one strand comprising SEQ ID NO: 1 and another strand comprising SEQ ID NO:

2.

5. The SNA described in claim 1, wherein the oligonucleotide shell consists of a plurality of double-stranded DNA oligonucleotides, each of the plurality of double-stranded DNA oligonucleotides comprising one strand containing sequence number 1 and another strand containing sequence number 2.

6. The SNA described in claim 1, wherein the shell of the oligonucleotide comprises a plurality of single-stranded stem-loop DNA oligonucleotides, each of the plurality of single-stranded stem-loop DNA oligonucleotides comprising a sequence represented by sequence number 9.

7. The SNA described in claim 1, wherein the shell of the oligonucleotide consists of a plurality of single-stranded stem-loop DNA oligonucleotides, each of the plurality of single-stranded stem-loop DNA oligonucleotides comprising a sequence represented by sequence number 9.

8. 2. SNA according to claim 1, wherein said double-stranded DNA oligonucleotide and / or single-stranded stem-loop DNA oligonucleotide inactivates signal transducer and activator of transcription 3 (STAT3).

9. 2. The SNA of claim 1, wherein the nanoparticle core is a metal core, a semiconductor core, an insulator core, an upconverting core, a micelle core, a dendrimer core, a liposome core, a polymer core, a metal-organic framework core, a lipid nanoparticle core, a protein core, or a combination thereof.

10. 2. The SNA of claim 1, wherein the shell of oligonucleotides comprises one or more additional oligonucleotides.

11. 11. The SNA of claim 10, wherein the one or more additional oligonucleotides are immunostimulatory oligonucleotides, inhibitory oligonucleotides, oligonucleotides that inactivate signal transducer and activator of transcription 3 (STAT3), or combinations thereof.

12. 12. The SNA of claim 11, wherein the immunostimulatory oligonucleotide is a toll-like receptor (TLR) agonist.

13. 10. The SNA of claim 1 further comprising an antigen.

14. 14. The SNA of claim 13, wherein the antigen is (i) bound to one or more oligonucleotides within the shell of the oligonucleotide, (ii) bound to the surface of the SNA, and / or (iii) encapsulated in the nanoparticle core.

15. 14. The SNA of claim 13, wherein the antigen is a tumor-associated antigen, a tumor-specific antigen, a neoantigen, or a combination thereof.

16. 2. The SNA of claim 1, wherein the SNA has a diameter of about 1 to about 200 nanometers (nm), the oligonucleotide shell comprises about 4 to about 250 oligonucleotides, and / or each oligonucleotide within the oligonucleotide shell is about 15 to about 100 bases in length.

17. A composition comprising a plurality of spherical nucleic acids (SNAs) according to any one of claims 1 to 16.

18. 19. A pharmaceutical composition for generating an immune response against cancer in a subject, comprising an effective amount of a spherical nucleic acid (SNA) according to any one of claims 1 to 16, a composition according to claim 17, or a combination thereof.

19. 17. A pharmaceutical composition for treating and / or ameliorating cancer in a subject, comprising an effective amount of an SNA according to any one of claims 1 to 15, a composition according to claim 16, or a combination thereof.

20. 20. The pharmaceutical composition of claim 19, wherein the cancer is breast cancer, peritoneal cancer, cervical cancer, colon cancer, rectal cancer, esophageal cancer, eye cancer, liver cancer, pancreatic cancer, laryngeal cancer, lung cancer, skin cancer, ovarian cancer, prostate cancer, stomach cancer, testicular cancer, thyroid cancer, brain cancer, or a combination thereof.

21. 20. The pharmaceutical composition of claim 19, wherein the cancer is glioblastoma.

22. 20. The pharmaceutical composition of claim 19, wherein the subject is a female.

23. The pharmaceutical composition of claim 19, which is administered by intranasal administration.

24. The pharmaceutical composition of claim 18, administered in combination with focused ultrasound (FUS).

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