WO2025122871A9

WO2025122871A9 - Multi-armed dendrimeric oligonucleotide nanoarchitectures to target the cyclic gmp-amp (cgas)/cyclic gmp-amp receptor stimulator of interferon genes (sting) pathway

Info

Publication number: WO2025122871A9
Application number: PCT/US2024/058866
Authority: WO
Inventors: Chad A. Mirkin; Connor Mackenzie FORSYTH; John CAVALIERE
Original assignee: Northwestern University
Current assignee: Northwestern University
Priority date: 2023-12-07
Filing date: 2024-12-06
Publication date: 2025-08-14
Anticipated expiration: 2026-06-07
Also published as: WO2025122871A1

Abstract

The present disclosure is directed, in some aspects, to oligonucleotide dendrimers comprising a molecular core covalently linked to one or more first oligonucleotide branches, wherein the molecular core is a polyethylene glycol (PEG) core, a polyester (PE) core, or a polyaminoamine (PAMAM) core. The disclosure also provides methods of making and/or using the oligonucleotide dendrimers for, e.g., treating a disease via inducing an immune response.

Description

MULTI-ARMED DENDRIMERIC OLIGONUCLEOTIDE NANOARCHITECTURES TO TARGET THE CYCLIC GMP-AMP (cGAS)ZCYCLIC GMP-AMP RECEPTOR STIMULATOR OF INTERFERON GENES (STING) PATHWAY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/607,497, filed December 7, 2023, which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under grant numbers CA208783 and CA221747 awarded by the National Institutes of Health and grant number FA9550-17-1 - 0348 awarded by the Air Force Office of Scientific Research. The government has certain rights in the invention.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as a text file. The name of the text file containing the Sequence Listing is "2023-237_SeqListing.xml", which was created on December 6, 2024 and is 35,822 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference.

BACKGROUND

[0004] Activation of the Stimulator of Interferon Genes (STING) pathway represents one of the main immune sensing mechanisms that promotes innate and adaptive immune responses against tumors. Tumor-derived DNA is recognized by cyclic GMP-AMP synthase (cGAS) in antigen-presenting cells. Upon nucleic acid recognition, cGAS generates cyclic dinucleotide GMP-AMP (cGAMP). cGAMP in turn binds to and activates the adaptor protein STING and triggers Interferon Regulatory Factor 3 (IRF3) and Nuclear Factor-KB (NF-KB)-dependent transcription, to promote the activation of natural killer (NK) cells, pro-inflammatory macrophages, and T cells.

SUMMARY

[0005] Introducing nucleic acids to the cellular environment faces several challenges, including cell entry, degradation by nucleases, and stimulation of an immune response. Techniques have been developed to overcome these barriers, but they can cause adverse reactions, and research to find effective methods continues. Advances in nanotechnology have led to the development of new materials which, when conjugated to nucleic acids, exhibit unique properties, including cellular uptake, that result from physical characteristics such as their size, shape, surface chemistry, and architecture.

[0006] Immunotherapy has emerged as the fourth pillar of cancer treatment, but current strategies fail to effectively treat cancers that are classified as immunogenically ‘cold,’ exhibiting low mutational burdens and a dearth of infiltrating lymphocytes. New approaches are desperately needed to improve survival outcomes in a diverse array of cancers by targeting novel biological pathways and leveraging emerging delivery platforms like nanoparticles. The structure of molecules and materials (e.g., size, shape, and surface chemistry) is intimately tied to their function. These factors determine how therapeutics interact with cells to execute the desired activity, including interactions with biomolecules, cellular uptake, and intracellular localization. However, it is often difficult to tune each of these parameters independently. Multi- Armed Dendrimeric Oligonucleotide Nanoarchitectures (MADONs) are an attractive platform for the development of nanotherapies as they allow for unprecedented control over nanostructure molecular weight, valency, and chemical ligation. Further, because the constructs are monodisperse (in contrast to lipid nanoparticles, liposomes, and other polymers) and molecularly defined, they offer a high degree of architectural control necessary to formulate precise and effective therapeutics. Additionally, these constructs benefit from their small size, a generalizable ligation strategy that allows for the attachment of diverse homo- and hetero- ODN ligands, and exceptional biocompatibility. Equally important is that these architectures are assembled with covalent chemical bonds that ensures the purity of their preparation and stability in biologically-relevant environment and leverages a convergent synthesis that maximizes scalability.

[0007] Provided herein are MADONs, a novel class of therapeutic that comprise molecularly pure cores that are functionalized with oligonucleotide (ODN) ligands (e.g., DNA, RNA, and modified nucleobases). The core material of these structures is typically comprised of, but not limited to, hyperbranched dendrimers composed of polymers such as polyethylene glycol (PEG), polyester (PE), and polyamidoamine (PAMAM) whose termini are functionalized with chemical moieties that allow for the covalent attachment of oligonucleotides (including but not limited to DNA, RNA, and other modified ODNs) or other therapeutically relevant modalities.

[0008] Critically, multi-armed dendrimeric oligonucleotide architectures offer total control over therapeutic valency. Indeed, scientists are just beginning to understand the importance of polyvalency in the context of biology. Multivalency is an important structural characteristic that is leveraged by biological systems to, for example, improve binding affinity/avidity in the case of IgM pentamers and IgA dimers and tailor the response of sensing mechanisms as in the case of the cGAS-STING and other biological pathways that rely on polyvalent ligand interactions, liquid-liquid phase separation, and the formation of molecular condensates. In developing robust multivalent architectures, we can uniquely target these biological processes in biomedical applications. Additionally, because of the density and orientation of DNA functionality on these particles, they offer a delivery mechanism for synthetic ODNs by imparting properties of high cellular uptake and resistance to nuclease-mediated degradation.

[0009] These architectures offer a versatile and robust nanotherapeutic platform for the development of novel immunotherapies, diagnostics, gene regulation, imaging, small molecule drug delivery, enzyme replacement therapy, and peptide-based therapeutics. MADONs can be formulated as vaccines, wherein ODNs operate as adjuvants that activate or suppress specific immune pathways including but not limited to members of the toll-like receptor (TLR) family (TLRs 3, 7, 8, and 9), the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, NOD-like receptor family, pyrin domain containing 3 (NLRP3), signal transducer and activator of transcription (STAT) 3, and so on. Specifically for the robust activation of cGAS, these particles can be synthesized with ODN ligands that, upon cellular internalization and exposure to chemical stimuli, undergo programmable hybridization chain reaction (HCR) polymerization to assemble artificially elongated ODN molecules. These architectures can act as vaccines through concomitant activation/inhibition of immune pathways and the delivery of antigens through direct covalent attachment or hybridization of peptides, proteins, or mRNA molecules. MADONs can additionally be formulated to present multivalent antibodies (or derivations thereof including nanobodies, single chain variable fragments [scFv], fragment antigen binding [Fab] domains, and bi-specific T-cell engagers [BiTEs]) for the purposes of high-avidity immune checkpoint inhibition and the formation of immune synapses.

[0010] Accordingly, in some aspects the disclosure provides an oligonucleotide dendrimer comprising a molecular core covalently linked to one or more first oligonucleotide branches, wherein the molecular core is a polyethylene glycol (PEG) core, a polyester (PE) core, or a polyaminoamine (PAMAM) core. In further aspects, the disclosure provides an oligonucleotide dendrimer comprising a molecular core covalently linked to one or more first oligonucleotide branches, wherein the molecular core is a polyethylene glycol (PEG) core, or a polyester (PE) core. In some embodiments, at least one of the one or more first oligonucleotide branches comprises a sequence that activates or inhibits a toll-like receptor (TLR), the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, a NOD-like receptor (NLR), pyrin domain-containing 3 (NLRP3), signal transducer and activator of transcription 3 (STAT3), or a combination thereof. In some embodiments, the sequence that inhibits a TLR comprises or consists of a sequence as set out in SEQ ID NO: 16. In some embodiments, the sequence that inhibits a TLR comprises or consists of a sequence as set out in SEQ ID NO: 17. In some embodiments, the sequence that inhibits a TLR comprises or consists of a sequence as set out in SEQ ID NO: 18. In some embodiments, the sequence that inhibits a TLR comprises or consists of a sequence as set out in SEQ ID NO: 19. In some embodiments, the sequence that activates a TLR comprises or consists of a sequence as set out in SEQ ID NO: 20. In some embodiments, the sequence that inhibits STAT3 comprises or consists of a sequence as set out in SEQ ID NO: 21 . In some embodiments, the sequence that inhibits AIM2 comprises or consists of a sequence as set out in SEQ ID NO: 22. In some embodiments, the sequence that activates AIM2 comprises or consists of a sequence as set out in SEQ ID NO: 23. In some embodiments, the sequence that activates DNA-PK comprises or consists of a sequence as set out in SEQ ID NO: 24. In some embodiments, the sequence that activates I Fl 16 comprises or consists of a sequence as set out in SEQ ID NO: 25. In some embodiments, the sequence that activates RIG-I comprises or consists of a sequence as set out in SEQ ID NO: 26. In some embodiments, the sequence that activates RIG-I comprises or consists of a sequence as set out in SEQ ID NO: 27. In various embodiments, at least one of the one or more first oligonucleotide branches comprises: i) a TLR inhibitory sequence comprising a sequence as set out in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19; ii) a TLR agonist sequence comprising a sequence as set out in SEQ ID NO: 20; iii) a STAT3 inhibitory sequence comprising a sequence as set out in SEQ ID NO: 21 ; iv) an AIM2 inhibitory sequence comprising a sequence as set out in SEQ ID NO: 22; v) an AIM2 agonist sequence comprising a sequence as set out in SEQ ID NO: 23; vi) a DNA-PK agonist sequence comprising a sequence as set out in SEQ ID NO: 24; vii) an IFI16 agonist sequence comprising a sequence as set out in SEQ ID NO: 25; viii) a RIG-I agonist sequence comprising a sequence as set out in SEQ ID NO: 26 or SEQ ID NO: 27, or a combination thereof. In some embodiments, the oligonucleotide dendrimer comprises two or more first oligonucleotide branches. In some embodiments, the oligonucleotide dendrimer comprises three or more first oligonucleotide branches. In some embodiments, the oligonucleotide dendrimer comprises four or more first oligonucleotide branches. In further embodiments, the oligonucleotide dendrimer comprises five or more first oligonucleotide branches. In still further embodiments, the oligonucleotide dendrimer comprises six or more first oligonucleotide branches. In various embodiments, the oligonucleotide dendrimer comprises seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve or more first oligonucleotide branches. In some embodiments, each of the one or more first oligonucleotide branches comprise the same nucleotide sequence relative to each other. In further embodiments, at least two of the one or more first oligonucleotide branches have different nucleotide sequences relative to each other. In some embodiments, each of the one or more first oligonucleotide branches comprises a sequence as set out in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In various embodiments, the one or more first oligonucleotide branches comprise DNA, RNA, or a combination thereof. In some embodiments, at least one of the one or more first oligonucleotide branches further comprises a domain comprising at least three (GGX) motifs, wherein X is a nucleotide. In some embodiments, each of the one or more first oligonucleotide branches further comprises a domain comprising at least three (GGX) motifs, wherein X is a nucleotide. In further embodiments, at least one of the one or more first oligonucleotide branches further comprises a domain comprising five or more (GGX) motifs, wherein X is a nucleotide. In further embodiments, each of the one or more first oligonucleotide branches further comprises a domain comprising five or more (GGX) motifs, wherein X is a nucleotide. In further embodiments, the domain comprises five to ten (GGX) motifs, wherein X is a nucleotide. In some embodiments, the nucleotide is a Thymidine. In various embodiments, the domain is located at the 5’ or 3’ end of the at least one of the one or more first oligonucleotide branches. In some embodiments, at least one of the one or more first oligonucleotide branches is at least 20 nucleotides in length. In further embodiments, each of the one or more first oligonucleotide branches is at least 20 nucleotides in length. In some embodiments, at least one of the one or more first oligonucleotide branches is at least 45 nucleotides in length. In further embodiments, each of the one or more first oligonucleotide branches is at least 45 nucleotides in length. In some embodiments, at least one of the one or more first oligonucleotide branches is at least 65 nucleotides in length. In further embodiments, each of the one or more first oligonucleotide branches is at least 65 nucleotides in length. In various embodiments, at least one of the one or more first oligonucleotide branches is hybridized to a second oligonucleotide, thereby creating an at least partially double stranded oligonucleotide dendrimer. In further embodiments, each of the one or more first oligonucleotide branches is hybridized to the second oligonucleotide, thereby creating an at least partially double stranded oligonucleotide dendrimer. In various embodiments, the second oligonucleotide is about 1 to about 90 nucleotides in length. In further embodiments, the second oligonucleotide is about 25 to about 70 nucleotides in length. In some embodiments, the length of the second oligonucleotide is greater than the length of the at least one of the one or more first oligonucleotide branches to which it is hybridized. In various embodiments, the second oligonucleotide comprises DNA or RNA. In further embodiments, the second oligonucleotide comprises a sequence as set out in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In some embodiments, an oligonucleotide dendrimer of the disclosure further comprises a third oligonucleotide that is hybridized to a single-stranded region of the at least partially double stranded oligonucleotide dendrimer. In some embodiments, the at least one of the one or more first oligonucleotide branches and the third oligonucleotide are both hybridized to the second oligonucleotide. In further embodiments, the third oligonucleotide comprises DNA or RNA. In some embodiments, the third oligonucleotide comprises a sequence as set out in SEQ ID NO: 1 1 or SEQ ID NO: 12. In some embodiments, the 3’ end of the third oligonucleotide is ligated to the 5’ end of the first oligonucleotide branch or the 5’ end of the third oligonucleotide is ligated to the 3’ end of the first oligonucleotide branch. In some embodiments, the combined length of the third oligonucleotide and the at least one of the one or more first oligonucleotide branches is greater than or equal to the length of the second oligonucleotide. In further embodiments, the combined length of the third oligonucleotide and the at least one of the one or more first oligonucleotide branches is less than the length of the second oligonucleotide. In some embodiments, the at least partially double stranded oligonucleotide dendrimer comprises an overhang. In further embodiments, the overhang is about 1 to about 25 nucleotides in length. In still further embodiments, the overhang is about 2 to about 5 nucleotides in length. In some embodiments, the oligonucleotide dendrimer comprises one or more cGAS ligands. In some embodiments, the oligonucleotide dendrimer activates cGAS and/or cGAS-STING. In some embodiments, an oligonucleotide dendrimer of the disclosure further comprises an additional agent. In some embodiments, the additional agent is attached to at least one of the one or more first oligonucleotide branches. In further embodiments, the additional agent is attached to the second oligonucleotide. In still further embodiments, the additional agent is attached to the third oligonucleotide. In some embodiments, an oligonucleotide dendrimer of the disclosure comprises a plurality of first oligonucleotide branches and the additional agent is attached to at least two of the plurality of first oligonucleotide branches. In various embodiments, the additional agent is: i) a protein, peptide, or enzyme; ii) a multivalent antibody or derivative thereof; iii) a carbohydrate or a small molecule proteolysis-targeting chimera (PROTAC); iv) a double-stranded DNA molecule; v) an RNA molecule, or a combination thereof. In some embodiments, the multivalent antibody or derivative thereof is a nanobody, a single chain variable fragment, a fragment antigen binding domain, a bi-specific T cell engager, or a combination thereof. In some embodiments, one or more oligonucleotides of the oligonucleotide dendrimer comprise a nucleotide mimetic. In further embodiments, the nucleotide mimetic is a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or a combination thereof. In various embodiments, the multivalent antibody or derivative thereof binds: i) a checkpoint blockade inhibitor protein; ii) a tumor-associated antigen; iii) a blood-brain barrier (BBB) penetration protein, or a combination thereof. In further embodiments, the checkpoint blockade inhibitor protein is PD-1 , PD-L1 , CTLA-4, LAG-3, TIM-3, or TIG IT. In some embodiments, the tumor-associated antigen is EGFR, HER2, VEGFR, CD20, CD19, or PSMA. In further embodiments, the blood-brain barrier (BBB) penetration protein is a transferrin receptor, GLUT 1 , or a Bradykinin B2 receptor.

[0011] In some aspects, the disclosure provides a composition comprising a plurality of oligonucleotide dendrimers of the disclosure. In further aspects, the disclosure provides a pharmaceutical formulation comprising an oligonucleotide dendrimer or composition of the disclosure and a pharmaceutically acceptable carrier, diluent, stabilizer, preservative, or adjuvant. In still further aspects, the disclosure provides an antigenic composition comprising an oligonucleotide dendrimer, composition, or pharmaceutical formulation of the disclosure, wherein the antigenic composition is capable of generating an immune response in a mammalian subject.

[0012] In some aspects, the disclosure provides a method of producing an immune response in a subject, comprising administering to the subject an effective amount of an oligonucleotide dendrimer, composition, pharmaceutical formulation, or antigenic composition of the disclosure, thereby producing an immune response in the subject. In some embodiments, the immune response is a CD86+ dendritic cell-mediated response. In some embodiments, the immune response is a T cell-mediated response. In some embodiments, the immune response is activated via the cGAS-STING pathway. In some embodiments, the immune response is activated at least in part via the cGAS-STING pathway. In some embodiments, administration of the composition induces formation of one or more molecular condensates. In further embodiments, the one or more molecular condensates have a diameter of about 5 to about 25 microns.

[0013] In some aspects, the disclosure provides a method of treating a disease in a subject, comprising administering to the subject an effective amount of an oligonucleotide dendrimer, composition, pharmaceutical formulation, or antigenic composition of the disclosure. In various embodiments, the disease is cancer, an autoimmune disease, an infectious disease, or a combination thereof. In further embodiments, the cancer is breast cancer, peritoneum cancer, cervical cancer, colon cancer, rectal cancer, esophageal cancer, eye cancer, liver cancer, pancreatic cancer, larynx cancer, lung cancer, skin cancer, melanoma, ovarian cancer, prostate cancer, stomach cancer, testicular cancer, thyroid cancer, brain cancer, or a combination thereof.

[0014] In further aspects, the disclosure provides a method of making an oligonucleotide dendrimer, the method comprising contacting a molecular core comprising one or more first oligonucleotide branches with one or more second oligonucleotides, wherein the contacting results in hybridization of a first oligonucleotide branch with a portion of a second oligonucleotide, thereby resulting in an at least partially double stranded oligonucleotide dendrimer comprising an overhang region. In some embodiments, the method further comprises hybridizing a third oligonucleotide to the overhang region. In various embodiments, Na+, Mg2+, urea, DMF, or a combination thereof are present during the contacting. In further embodiments, the stoichiometry of oligonucleotide:molecular core is 12:1 .

[0015] In some aspects, the disclosure provides a molecular condensate comprising an oligonucleotide dendrimer of the disclosure, or a plurality thereof, and a plurality of proteins. In some embodiments, at least one oligonucleotide dendrimer interacts with at least two proteins of the plurality of proteins, and at least one protein of the plurality of proteins binds to more than one oligonucleotide dendrimer or oligonucleotide dendrimer branch, and wherein the plurality of proteins is cGAS-STING effector proteins, pyrin domain-containing 3 (NLRP3) proteins, and/or signal transducer or activator of transcription 3 (STAT3). In further embodiments, the oligonucleotide binds to one or more proteins in the plurality of proteins via hydrogen bonding and/or ionic interaction. In some embodiments, the molecular condensate has a half-life of at least 45 minutes. In various embodiments, the plurality of proteins are cGAS-STING effector proteins that activate a cGAS-STING pathway. In various embodiments, a molecular condensate of the disclosure has a diameter of about 5 to about 25 microns.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 provides the synthesis and characterization of DNA dendrimer therapeutics. A, Abstract schematic of DNA dendrimers detailing their architecture and molecular core chemistry. B, Schematic of the convergent SPAAC click chemistry approach and conditions used in DNA dendrimer conjugation reactions. C, Native PAGE of control DBCO-ISD25 DNA and two crude G1 -ISD25 dendrimer syntheses. D, Denaturing PAGE gel of gel purified G1 DNA dendrimers with 6, 5, and 4 appended ISD25 ligands. E, Single stranded G1-ISD25-6 cores were mixed with increasing equivalents of complementary ISD25, observing saturation of hybridization at 1 x equivalents. F, Denaturing PAGE of gel purified G2 dendrimers with 10 through 4 appended ISD25 ligands. G, Single stranded G2-ISD25-10 cores mixed with increasing equivalents of complementary ISD25. H, DLS of G1 -ISD25-6 and G2-ISD25-10, demonstrating hydrodynamic diameters of 9.66 and 7.14 nm for ss and ds molecules, respectively. Inlay; Zeta potential measurements on ss and ds G1-ISD25-6 and G2-ISD25-10 molecules. I, AFM of double stranded G1 -ISD25-6 dendrimers, exhibiting a diameter of approximately 9 nm. J, Left; Aliquots of dsG1 -ISD25-6 incubated in PBS at 37 °C at time = 0, 1 , and 2 weeks analyzed by native PAGE to determine extent of hydrolytic degradation. Right; Densitometry analysis indicating the relative quantities of G1 -ISD25-6, 5, 4, and 3 over time. K, Left; Aliquots of ISD25 or G1 -ISD25-6 incubated in 1 x PBS with 100 U/mL of DNase-l analyzed by native PAGE. Right; Densitometry analysis showing the relative quantities of fully intact DNA over 24 hours.

[0017] Figure 2 shows characterization of DNA Dendrimers with MALDI MS. A, MALDI MS spectra of purified dendrimers with varying numbers of DNA branches analyzed in linear negative mode from 20-220 kDa. Top, G2-ISD25 products. Middle, G1 -ISD25-Cy3.5 products. Bottom, G1-ISD25 products. B, MALDI MS spectra of DNA dendrimers formulated with different oligonucleotide branches. Top, G1 -AP1 aptameric molecules. Middle, G1 -GGT10 g-quadruplex molecules. Bottom, G1 -5FU molecules.

[0018] Figure 3 provides DNA dendrimers used as templates to append and ligate longer oligonucleotides. A, Denaturing PAGE gel of crude product from reaction between G1 -azide dendrimer cores and either single- or double-stranded (ss and ds, respectively) DBCO-ISD25 ligands. CTL, control; cISD, complementary ISD25. B, Schematic showing the assembly and reaction of G1 -ISD25-X templates to form nicked (N) or ligated (L) G1 -ISD45-X molecules. C, Denaturing PAGE gel of EDC-catalyzed ligations between the 5’-OH groups of G1 -ISD25-6 and 3’-PO4- of ISD20 molecules templated with clSD45 at ligand (ISD20-PO4) to core (G1 -ISD25-6) ratios of 1 :1 and 2:1 , respectively. Note that the reactions produce a ladder-like distribution of products with 1-6 successful ligations. Right; Native PAGE gel of the double-stranded product of G1 -ISD25-6:ISD20 reactions, notated as G1 -ISD45-6 (L, ligated). D, Native PAGE gel of double-stranded DNA molecules. ISD25 (ISD25;clSD25), ISD45 (ISD45:clSD25), ISD45 (N; ISD25:ISD20-PO4:clSD45), G1 -ISD25-6, 5, 4, 3 (G1-ISD25-6:clSD25), G1 -ISD45-6, 5, 4, 3 (N; G1 -ISD25-6: ISD20-PO4:clSD45). E, Double stranded DNA dendrimers with and without Spacer 18 moieties between nucleobases and DBCO functional groups (NS). [0019] Figure 4 shows that DNA dendrimers robustly activate the cGAS enzyme. AJSD25 or ISD45 diluted 2-fold from 250 - 1 .23 nM in solutions containing human [cGAS] = 250 nM. cGAMP screening assays are performed with ISD25, ISD45 (N), G1 -ISD25-6, 5, 4, and 3, or G1 -ISD45-6, 5, 4, and 3 (N) at concentrations from 120 - 1 .88 nM with a final [cGAS] = 30 nM. B, Comparison between the most highly valent DNA dendrimers [G1 -ISD25-6 and G1-ISD45-6 (N)] compared to linear ISD25 and ISD45 (N) controls. C, ISD25-conjugated DNA dendrimers with differential valencies. D, ISD45 (N)-conjugated DNA dendrimers with differential valencies. E, G1 -ISD25-6 through 3 and G1-ISD45-6 through 3 (NS) were diluted 2-fold from 60 - 7.5 nM in wells with a [cGAS] of 60 nM. mP; millipolarization.

[0020] Figure 5 demonstrates that DNA dendrimers engage cGAS with high affinity. A, Electrophoretic Mobility shift assay (EMSA) of ISD25, ISD45, G1 -ISD25-6, and G1 -ISD25-5 incubated with increasing equivalents of human cGAS enzyme (0, 1 , 10, 20, and 40x). B, ITC measurements of mcGAS binding to ISD25 and G1-ISD25-6. Calculated binding parameters are provided on each isotherm.

[0021] Figure 6 demonstrates that DNA dendrimers robustly nucleate the formation of cGAS molecular condensates. A, Timelapse confocal microscopy imaging of cGAS-DNA molecular condensate formation. Assemblies were initiated by mixing 10 pM ISD25 or G1 -ISD25-6 with 5 pM AlexaFluor 488-labeled mcGAS. B, Histogram quantification of molecular condensate area and Feret’s diameter initiated by ISD25 or G1-ISD25-6. Mean area (C) and Feret’s diameter (D) of foci over time. Mean fluorescence intensity (E) and total particle counts (F) over time.

[0022] Figure 7 shows that DNA dendrimers exhibit rapid cellular uptake and robust cGAS- STING pathway activation. A_s Luminescence from supernatant collected from Raw Lucia ISG cells treated with Aduro S100 (ADUS100), ISD45 (L), G1-ISD45-6 (N), and G1 -ISD45-6 (L) for 24 hours. B, Mean fluorescence intensity (MFI) of Cy3.5-labeled ISD25 or G1 -ISD25-6 within DC2.4 cells in a live-cell confocal microscopy experiment where cells were treated at time = 0 and imaged over the course of 30 minutes. C, Viability of Raw Lucia ISGs treated with Aduro S100, ISD25, or G1 -ISD25-6 through 3 for 24 hours. D, Raw Lucia ISG cells treated with ISD25 or G1 -ISD25-6 through 3 with and without spacer 18 moieties (NS; No Spacer).

[0023] Figure 8 shows DNA dendrimer cellular uptake and co-stimulation in dendritic and tumor cells. BMDCs treated with [DNA] = 50 or 250 nM for 1-, 4-, or 8-hour pulses, followed by incubation with fresh media to 24 hours. A, Cy3.5-labeled DNA uptake. B, CD86 co-stimulatory marker expression. DC2.4 cells treated with [DNA] = 50 or 250 nM for an 8-hour pulse, followed by incubation with fresh media to 24 hours. C, Cy3.5-labeled DNA uptake. D, CD86 co- stimulatory marker expression. E, BMDCs treated for an 8-hour pulse with [DNA] = 200 nM, followed by media replacement and incubation to 24 hours. Top, Cy3.5-labeled DNA uptake; Bottom, CD86 co-stimulatory marker expression. F, Left, Cy3.5-labeled DNA uptake in murine glioma CT2A cells. Right, Native PAGE gel of G1-ISD25-6 cores hybridized to clSD25, clSD27 (2 bp overhang), or clSD30 (5 bp overhang) at complement/core ratios of 0.9, 1 .0, and 1 .1 .

DETAILED DESCRIPTION

[0024] The cGAS-STING pathway serves the innate immune system by producing type I interferons (IFNs)¹³. It is initiated by the cytosolic DNA sensor cGAS which produces cyclic CMP-AMP (cGAMP) upon recognition of double-stranded DNA (dsDNA). cGAMP binds to STING, eventually resulting in the production of type I IFNs which shape pro-inflammatory responses through a variety of mechanisms and immune cell types, including dendritic cells (DCs), NK cells, T regulatory (Treg) cells, and MDSCs.¹⁴ For example, activation of the cGAS- STING pathway facilitates the infiltration of cytotoxic T lymphocytes (CTLs) by increasing vascular permeability and inducing chemokine production that promotes CTL trafficking to tumor tissue.¹⁵ In contrast, IFNs negatively regulate Tregs by reducing their differentiation and homing capability^{16 17} and MDSCs by impairing their differentiation and maturation^{18 19}. Exposing tumors directly to type I IFNs enhances their expression of major histocompatibility complex (MHC) class I.²⁰ For example, activating the cGAS-STING pathway in glioblastoma (GBM) is expected to significantly enhance anti-tumor immunity by encouraging a pro-inflammatory tumor microenvironment. Current therapeutics targeting the cGAS-STING axis are limited because to date, all strategies target STING by utilizing analogues of cGAMP or allosteric small molecules, and human trials have shown underwhelming results because of their transient STING activation, complete reliance on type I IFN signaling, and poor biodistribution profiles.^{21 22}

[0025] Given the challenges associated with developing effective STING agonists, targeting the upstream cGAS enzyme is a preferred approach for two reasons: 1) its action is catalytic (i.e., activating one cGAS enzyme results in the sustained production of cGAMP),²³ thereby allowing for prolonged local STING signaling, and 2) it initiates additional inflammatory pathways that are independent of IFN signaling.²⁴ Initial binding interactions between cGAS and DNA form a complex which facilitates cooperative and sequential binding of additional cGAS dimers.²⁵ cGAS activation is dependent on DNA concentration and length but not sequence;²⁶ experiments have shown that affinity of cGAS to dsDNA increases as oligonucleotide (ODN) length increases. Proteins that constrain DNA to a parallel orientation enhance cGAS activation, highlighting the important contribution that DNA arrangement plays in the development of a targeted therapy.²⁵ To that point, cGAS is a multidomain dimer that binds to DNA ligands in a multivalent manner, facilitating the formation of cytoplasmic molecular condensates through liquid-liquid phase separation (LLPS) in which a concentrated cGAS-DNA network efficiently produces cGAMP.²⁷ These observations suggest the importance of DNA architectural design in the development of a potent agonist by presenting DNA of the optimal length, concentration, orientation, and valency for cGAS engagement. Utilizing precision architectural modifications enabled by the Multi-armed dendrimeric oligonucleotide nanoarchitecture (MADONs, interchangeably referred to herein as “oligonucleotide dendrimers”) platforms will determine the extent to which novel classes of therapeutic can be tailored to engage biological targets and processes that rely on polyvalent phase condensation to enhance therapeutic specificity and potency. Such a strategy has already shown promise in a proof of principle study wherein polyvalent STING agonists were engineered to facilitate STING condensation, trafficking to the endoplasmic reticulum-Golgi intermediate compartment, and activation.²⁸

[0026] Oligonucleotide dendrimers are synthesized using phosphoramidite chemistry in which monomers are added to a growing oligonucleotide attached to a solid support.³⁸ However, this strategy is only viable for oligonucleotides with short sequences (<45 bp) because yields exponentially decay with each base. To overcome this limitation, the approach provided herein reacts pre-synthesized and purified core and branch units using click chemistry. The methodology disclosed herein allows for the synthesis of oligonucleotide dendrimers that are physiochemically identical to dendrons but distinguished by their radial symmetry.

[0027] The structure of molecules and materials (e.g., size, shape, and surface chemistry) is intimately tied to their function. These factors determine how therapeutics interact with cells to execute the desired activity, including interactions with biomolecules⁴², cellular uptake⁴³⁴⁴, and intracellular localization⁴⁵. Oligonucleotide dendrimers are an attractive platform with which to study the structure-activity relationships (SARs) between nanoscale architecture and cGAS agonism because these constructs are monodisperse (in contrast to lipid nanoparticles, liposomes, and other polymers) and they offer a high degree of modular architectural control necessary for robust SAR analysis. Evaluating SARs is important for technologies such as oligonucleotide dendrimers; for instance, the valency (i.e., number of branches) of an oligonucleotide dendrimer improves cellular uptake, serum nuclease resistance, and biodistribution³⁸. In some aspects, multivalency improves the ability of oligonucleotide dendrimers to nucleate cGAS:DNA complexes because additional DNA strands enhance local concentrations and favorably orient DNA for cGAS binding. [0028] Disclosed herein, in various aspects, are oligonucleotide dendrimers with high valency and long DNA branches that significantly enhance cGAS-STING activation and generate a pro- inflammatory response. Altering polymer core chemistry, including without limitation polyethylene glycol (PEG), polyester (PE), and polyamidoamine (PAMAM) can impact the efficiency of DNA conjugation reactions and overall stability of particles under physiologic conditions. These polymer cores exhibit different net charges and persistence lengths that will impact reaction yields and differential lability in aqueous conditions, which will affect stability. In some aspects, oligonucleotide dendrimers comprise a molecular core covalently linked to one or more first oligonucleotide branches, wherein the molecular core is a polyethylene glycol (PEG) core, a polyester (PE) core, or a polyaminoamine (PAMAM) core.

[0029] All language such as “from,” “to,” “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can subsequently be broken down into sub-ranges.

[0030] A range includes each individual member. Thus, for example, a group having 1 -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, and so forth.

[0031] As used in this specification and the appended claims, the articles “a” and “an” refer to one or to more than one (for example, to at least one) of the grammatical object of the article.

[0032] “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20-25 percent (%), or, for example, within 20 percent, 10 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent of the stated value or range of values.

[0033] 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.

[0034] The terms “administering”, “administer”, “administration”, and the like, as used herein, refer to any mode of transferring, delivering, introducing, or transporting an oligonucleotide dendrimer to a subject in need of treatment with such an oligonucleotide dendrimer. Such modes include, but are not limited to, oral, topical, intravenous, intraarterial, intraperitoneal, intramuscular, intratumoral, intradermal, intranasal, and subcutaneous administration.

[0035] “Treating” and “treatment” refers to any reduction in the severity and/or onset of symptoms associated with a disease or disorder. Accordingly, “treating” and “treatment” includes therapeutic and prophylactic measures. One of ordinary skill in the art will appreciate that any degree of protection from, or amelioration of, the onset and/or progression of a disease or 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 the appearance of symptoms.

[0036] Dendrimer cores are synthetic and highly branched polymeric molecules. Dendrimers are synthesized by covalently attaching repeating molecular units to a radially symmetric polymer core. As used herein, “Generations” of dendrimer cores refer to the number of layers of repeating molecular units attached to the dendrimer core. Generation 1 , 2, and 3 Bis-MPA azide dendrimer cores (Polyester [PE]) possess exactly 6, 12, or 24 polymeric branches, respectively. Generation 1 , and 2 PEG cores possess exactly 4 or 8 branches, respectively. Generation 1 , 2, and 3 polyamidoamine (PAMAM) dendrimers possess exactly 8, 16, or 32 branches, respectively.

[0037] Oligonucleotide Dendrimers are synthesized by covalently attaching oligonucleotide units to a dendrimer core. First generation polyester core (G1 -PE) dendrimers possess between one and six first oligonucleotide branches, second generation polyester core (G2-PE) dendrimers possess between one and twelve first oligonucleotide branches, and third generation polyester core (G3-PE) dendrimers possess between one and twenty-four first oligonucleotide branches.

[0038] As used herein, “valency” is the number of oligonucleotides covalently attached to a single dendrimer core. For example, an oligonucleotide dendrimer with two first oligonucleotide branches is considered to have a valency of two, and an oligonucleotide dendrimer with three first oligonucleotide branches is considered to have a valency of three. Disclosed herein are oligonucleotide dendrimers comprising one or more first oligonucleotide branches, two or more first oligonucleotide branches, three or more first oligonucleotide branches, four or more first oligonucleotide branches, five or more first oligonucleotide branches, or six or more first oligonucleotide branches.

[0039] As used herein, a “first oligonucleotide branch” refers to an oligonucleotide covalently linked to a dendrimer core. In some embodiments, each of the one or more first oligonucleotide branches that is linked to a dendrimer core is at least about 20nucleotide base pairs in length. In some embodiments, each of the one or more first oligonucleotide branches that is linked to a dendrimer core is at least about 25 nucleotide base pairs in length. In some embodiments, each of the one or more first oligonucleotide branches is at least about 45 nucleotide base pairs in length. In some embodiments, each of the one or more first oligonucleotide branches is at least about 65 nucleotide base pairs in length. In some embodiments, each of the one or more first oligonucleotide branches is at least about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, or about 65 nucleotide base pairs in length. In some embodiments, each of the one or more first oligonucleotide branches is between about 25 to 65 nucleotide base pairs in length. In various embodiments, each of the one or more first oligonucleotide branches is between about 20 to 60, 25 to 60, 30 to 60, 35 to 60, 40 to 60, 45 to

60, 50 to 60, 55 to 60, 20 to 55, 25 to 55, 30 to 55, 35 to 55, to 40 to 55, 45 to 55, 50 to 55, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 20 to 45, 25 to 45, 30 to 45, 35 to 45, 40 to

45, 20 to 35, 25 to 35, 30 to 35, 20 to 30, or 25 to 30 nucleotide base pairs in length.

[0040] In some embodiments, provided herein is an oligonucleotide dendrimer comprising a dendrimer core with seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve first oligonucleotide branches. As an example, an oligonucleotide dendrimer comprising a dendrimer core with eight first oligonucleotide branches is considered to have a valency of eight. In some embodiments, the one or more first oligonucleotide branches of the oligonucleotide dendrimer each comprise the same nucleotide sequence relative to each other. In some aspects, at least two of the one or more first oligonucleotide branches have different nucleotide sequences relative to each other. In various aspects, one or more first oligonucleotide branches each comprise a nucleotide sequence as set out in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0041] In some embodiments, the one or more first oligonucleotide branches comprise DNA or RNA. In some embodiments, at least one of the one or more first oligonucleotide branches further comprises a domain comprising at least three (GGX) motifs, wherein X is a nucleotide. In various embodiments, each of the one or more first oligonucleotide branches further comprises a domain comprising at least three (GGX) motifs, wherein X is a nucleotide. In some embodiments, each of the one or more first oligonucleotide branches further comprises a domain comprising five or more (GGX) motifs, wherein X is a nucleotide. In some embodiments, each of the one or more first oligonucleotide branches further comprises a domain comprising five to ten (GGX) motifs, wherein X is a nucleotide. In some aspects, the nucleotide is a thymidine. In some embodiments, the domain is located at the 5’ end of at least one of the one or more first oligonucleotide branches. In some embodiments, the domain is located at the 5' end of each of the one or more first oligonucleotide branches. In further embodiments, the 3’ end of the at least one of the one or more first oligonucleotide branches is attached to the molecular core. In some embodiments, the domain is located at the 3’ end of at least one of the one or more first oligonucleotide branches. In some embodiments, the domain is located at the 3’ end of each of the one or more first oligonucleotide branches. In further embodiments, the 5’ end of the at least one of the one or more first oligonucleotide branches is attached to the molecular core.

[0042] In some embodiments, at least one of the one or more first oligonucleotide branches comprises a sequence that activates or inhibits a toll-like receptor (TLR), the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, a NOD-like receptor (NLR), pyrin domain-containing 3 (NLRP3), and/or signal transducer and activator of transcription 3 (STAT3). In some aspects, the sequence that inhibits a TLR comprises a sequence as set out in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19. In various aspects, the sequence that activates a TLR comprises a sequence as set out in SEQ ID NO: 20. In some aspects, the sequence that inhibits STAT3 comprises a sequence as set out in SEQ ID NO: 21 . In various aspects, the sequence that inhibits AIM2 comprises a sequence as set out in SEQ ID NO: 22. In some aspects, the sequence that activates AIM2 comprises a sequence as set out in SEQ ID NO: 23. In various aspects, the sequence that activates DNA-PK comprises a sequence as set out in SEQ ID NO: 24. In some aspects, the sequence that activates IFI16 comprises a sequence as set out in SEQ ID NO: 25. In various aspects, the sequence that activates RIG-I comprises a sequence as set out in SEQ ID NO: 26 or SEQ ID NO: 27.

[0043] As disclosed herein, a second oligonucleotide is an oligonucleotide that is capable of hybridizing to a first oligonucleotide branch of an oligonucleotide dendrimer. In some embodiments, the first oligonucleotide branch and the second oligonucleotide are equivalent or about equivalent in length. In various embodiments, the first oligonucleotide branch is 100% complementary to the second oligonucleotide over their entire length. In some embodiments, the length of the second oligonucleotide is greater than that of the first oligonucleotide branch, wherein only a first region of the second oligonucleotide is sufficiently complementary to the first oligonucleotide branch to hybridize to the first oligonucleotide branch. In various embodiments, the first region of the second oligonucleotide has or has at least about 5 base pairs of complementarity with the first oligonucleotide branch. In some embodiments, the first region of the second oligonucleotide has or has at least about 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 35 base pairs, 40 base pairs, 45 base pairs, 50 base pairs, 55 base pairs, 60 base pairs, or 65 base pairs of complementarity with the first oligonucleotide branch. In various embodiments, the first region of the second oligonucleotide has or has at least less than 65 base pairs, 60 base pairs, 55 base pairs, 50 base pairs, 45 base pairs, 40 base pairs, 35 base pairs, 30 base pairs, 25 base pairs, 20 base pairs, 15 base pairs, or 10 base pairs of complementarity with the first oligonucleotide branch. In some embodiments, the first region of the second oligonucleotide has between about 5 to 65 base pairs of complementarity with the first oligonucleotide branch. In various embodiments, the first region of the second oligonucleotide has between about 5 to 65, 10 to 65, 15 to 65, 20 to 65, 25 to 65, 30 to 65, 35 to 65, 40 to 65, 45 to 65, 50 to 65, 55 to 65, 60 to 65, 5 to 55, 10 to 55, 15 to 55, 20 to

55, 25 to 55, 30 to 55, 35 to 55, 40 to 55, 45 to 55, 50 to 55, 5 to 45, 10 to 45, 15 to 45, 20 to 45,

25 to 45, 30 to 45, 35 to 45, 40 to 45, 5 to 35, 10 to 35, 15 to 35, 20 to 35, 25 to 35, 30 to 35, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 5 to 15, or 10 to 15 base pairs of complementarity with the first oligonucleotide branch. In some embodiments, the first region of the second oligonucleotide is at least about 80% complementary to the first oligonucleotide branch. In various aspects, the first region of the second oligonucleotide is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the first oligonucleotide branch. In some aspects, the first region of the second oligonucleotide is 100% complementary to the first oligonucleotide branch. When a second oligonucleotide is hybridized to a first oligonucleotide branch, the resulting oligonucleotide dendrimer is referred to herein as an “at least partially double stranded oligonucleotide dendrimer”, wherein at least a portion of the oligonucleotide attached to the dendrimer core is double-stranded. In some embodiments, the at least partially double stranded oligonucleotide dendrimer comprises a completely double-stranded oligonucleotide branch. In various embodiments, the at least partially double stranded oligonucleotide dendrimer comprises an oligonucleotide branch with a single-stranded region, referred to herein as an “overhang”. In some aspects, the overhang is on the first oligonucleotide branch (i.e. , the overhang is produced due to the first oligonucleotide branch having a greater length than the second oligonucleotide branch to which it is hybridized). In various aspects, the overhang is on the second oligonucleotide (i.e., the overhang is produced due to the second oligonucleotide branch having a greater length than the first oligonucleotide branch to which it is hybridized). In some embodiments, the overhang is about 1 to 25 nucleotides in length. In various aspects, the overhang is about 2 to 5 nucleotides in length. In some aspects, the overhang is about 1 to 25, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 1 to 20, 5 to 20, 10 to 20, 15 to 20, 1 to 15, 5 to 15, 10 to 15, 1 to 10, 5 to 10, 1 to 5, 2 to 25, 2 to 20, 2 to 15, 2 to 10, or 2 to 5 nucleotides in length. In some embodiments, the overhang is or is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 nucleotides in length. In further embodiments, the overhang is less than about 25 nucleotides in length. In still further embodiments, the overhang is less than about 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, or 5 nucleotides in length.

[0044] In various aspects, a second oligonucleotide of the disclosure comprises or consists of a nucleotide sequence as set out in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0045] Percent complementarity is determined over the length of an oligonucleotide. Merely to illustrate, given an first oligonucleotide branch in which 18 of 20 nucleotides of the first oligonucleotide branch are complementary to a 20 nucleotide region in a second oligonucleotide of 100 nucleotides total length, the oligonucleotide would be 90 percent complementary. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleotides. Percent complementarity of a first oligonucleotide branch with a region of a second oligonucleotide can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0046] In some embodiments, at least one of the one or more first oligonucleotide branches is hybridized to a second oligonucleotide, thereby creating an at least partially double stranded oligonucleotide dendrimer. In various embodiments, each of the one or more first oligonucleotide branches is hybridized to the second oligonucleotide, thereby creating an at least partially double stranded oligonucleotide dendrimer.

[0047] In some embodiments, the second oligonucleotide is about 1 to about 90 nucleotides in length. In various aspects, the second oligonucleotide is about 25 to about 70 nucleotides in length. In some aspects, the second oligonucleotide is about 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 10, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 15 to 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 20,

20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, 25 to 90, 25 to 80, 25 to 70,

25 to 60, 25 to 50, 25 to 40, 25 to 30, 30 to 90, 30 to 80, 30 to 70, 30 to 60, 30 to 50, 30 to 40,

40 to 90, 40 to 80, 40 to 70, 40 to 60, 40 to 50, 50 to 90, 50 to 80, 50 to 70, 50 to 60, 60 to 90,

60 to 80, 60 to 70, 70 to 90, 70 to 80, 80 to 90, or 85 to 90 nucleotides in length. In various aspects, the second oligonucleotide is or is at least about 10, 20, 30, 40, 50, 60, 70, 80, or 90 nucleotides in length. In some aspects, the second oligonucleotide is less than about 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides in length. In some embodiments, the length of the second oligonucleotide is greater than the length of the at least one of the one or more first oligonucleotide branches to which it is hybridized. In various embodiments, the second oligonucleotide comprises DNA or RNA.

[0048] As disclosed herein, a third oligonucleotide is an oligonucleotide that is capable of hybridizing to a second oligonucleotide. In various aspects, the third oligonucleotide hybridizes to a second region of the second oligonucleotide. In some embodiments, the third oligonucleotide is hybridized to the second oligonucleotide, wherein the second oligonucleotide is partially hybridized to a first oligonucleotide branch of an oligonucleotide dendrimer. In some embodiments, the third oligonucleotide is hybridized to the second oligonucleotide, wherein the second oligonucleotide is not hybridized to a first oligonucleotide branch. In various aspects, a structure comprising a third oligonucleotide hybridized to a second oligonucleotide is contacted with an oligonucleotide dendrimer, wherein the second oligonucleotide further hybridizes to one or more of the first oligonucleotide branches of the oligonucleotide dendrimer.

[0049] In various embodiments, the second region of the second oligonucleotide has or has at least about 5 base pairs of complementarity with the third oligonucleotide. In some embodiments, the second region of the second oligonucleotide has at least about 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 35 base pairs, 40 base pairs, 45 base pairs, 50 base pairs, 55 base pairs, 60 base pairs, 65 base pairs, 70 base pairs, 75 base pairs, 80 base pairs, 85 base pairs, or 90 base pairs of complementarity with the third oligonucleotide. In various embodiments, the second region of the second oligonucleotide has or has at least less than 90 base pairs, 85 base pairs, 80 base pairs, 75 base pairs, 70 base pairs, 65 base pairs, 60 base pairs, 55 base pairs, 50 base pairs, 45 base pairs, 40 base pairs, 35 base pairs, 30 base pairs, 25 base pairs, 20 base pairs, 15 base pairs, or 10 base pairs of complementarity with the third oligonucleotide. In some embodiments, the second region of the second oligonucleotide has between about 1 to 90 base pairs of complementarity with the third oligonucleotide. In various embodiments, the second region of the second oligonucleotide has between about 1 to 90, 5 to 90, 10 to 90, 15 to 90, 20 to 90, 25 to 90, 30 to 90, 35 to 90, 40 to 90, 45 to 90, 50 to 90, 55 to 90, 60 to 90, 65 to 90, 70 to 90, 75 to 90, 80 to 90, 85 to 90, 5 to 80, 10 to 80, 15 to 80, 20 to 80, 25 to 80, 30 to 80, 35 to 80, 40 to 80, 45 to 80, 50 to 80, 55 to 80, 60 to 80, 65 to 80, 70 to 80, 75 to 80, 5 to 70, 10 to 70, 15 to 70, 20 to 70, 25 to 70, 30 to 70, 35 to 70, 40 to 70, 45 to 70, 50 to 70, 55 to 70, 60 to 70, 65 to 70, 5 to 60, 10 to 60, 15 to 60, 20 to 60, 25 to 60, 30 to 60, 35 to 60, 40 to 60, 45 to 60, 50 to 60, 55 to 60, 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 5 to 40, 10 to 40, 15 to 40, 20 to 40, 25 to 40, 30 to 40, 35 to 40, 5 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 20, 10 to 20, 15 to 20, 20 to 20, or 5 to 10 base pairs of complementarity with the third oligonucleotide. In some embodiments, the second region of the second oligonucleotide is or is at least about 80% complementary to the third oligonucleotide. In various aspects, the second region of the second oligonucleotide is or is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the third oligonucleotide. In some aspects, the second region of the second oligonucleotide is 100% complementary to the third oligonucleotide. In various aspects, a third oligonucleotide comprises a nucleotide sequence as set out in SEQ ID NO: 11 or SEQ ID NO: 12.

[0050] In some embodiments, a third oligonucleotide is hybridized to a single-stranded region of the at least partially double stranded oligonucleotide dendrimer. In some embodiments, at least one of the one or more first oligonucleotide branches and the third oligonucleotide are both hybridized to the second oligonucleotide. In various aspects, the third oligonucleotide comprises DNA or RNA.

[0051] In some embodiments, a phosphate group is attached to the 3’ end of the third oligonucleotide. A phosphate group attached to the 3’ end of an oligonucleotide can serve as the substrate for a 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-catalyzed ligation to the 5’ end another oligonucleotide. EDC ligation forms a zero-length cross-link, with the resulting structure being chemically identical to the enzymatic ligation achieved with T4 DNA ligase (Orun et al., ASC Nanoscience AU, 4(5): 338-348, 2024). In various embodiments, a phosphate group attached to the 5' end of an oligonucleotide can be reacted with a hydroxyl group on the 3’ end of a different oligonucleotide via DNA ligase, producing a covalent bond between the two oligonucleotides (Doherty et al., Nucleic Acids Res, 28(21 ): 4051-4058, 2000). In various embodiments, the 3’ end of the third oligonucleotide is ligated to the 5’ end of the first oligonucleotide branch, or the 5’ end of the third oligonucleotide is ligated to the 3' end of the first oligonucleotide branch, wherein optionally the third oligonucleotide hybridized to the second oligonucleotide prior to the ligation reaction. Ligation may be performed using any method known in the art, such as, but without limitation, using a ligase, such as a DNA ligase or T4 DNA ligase, or via an EDC-catalyzed reaction.

[0052] In some embodiments, the combined length of the third oligonucleotide and at least one of the one or more first oligonucleotide branches is greater than or equal to the length of the second oligonucleotide. In various embodiments, the combined length of the third oligonucleotide and the at least one of the one or more first oligonucleotide branches is less than the length of the second oligonucleotide. [0053] In some embodiments, the at least partially double stranded oligonucleotide dendrimer comprising a third oligonucleotide further comprises an overhang. In some embodiments, the combined length of the first oligonucleotide branch and the third oligonucleotide is greater than the length of the second oligonucleotide. In such an embodiment, the overhang is on the third oligonucleotide. In some embodiments, the combined length of the first oligonucleotide branch and the third oligonucleotide is less than the length of the second oligonucleotide. In such an embodiment, the overhang is on the second oligonucleotide. In some aspects, the overhang is about 1 to 25 nucleotides in length. In various aspects, the overhang is about 2 to 5 nucleotides in length. In some aspects, the overhang is about 1 to 25, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 1 to 20, 5 to 20, 10 to 20, 15 to 20, 1 to 15, 5 to 15, 10 to 15, 1 to 10, 5 to 10, 1 to 5, 2 to 25, 2 to 20, 2 to 15, 2 to 10, or 2 to 5 nucleotides in length. In some embodiments, the overhang is or is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 nucleotides in length. In further embodiments, the overhang is less than about 25 nucleotides in length. In still further embodiments, the overhang is less than about 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, or 5 nucleotides in length.

[0054] In some embodiments, the oligonucleotide dendrimer does not comprise a spacer between any of the one or more first oligonucleotide branches and the molecular core, wherein a spacer is a moiety (e.g., polyethylene glycol (PEG) spacer 18 (hexaethylene glycol)) separating oligonucleotide nucleobases from the moiety (e.g., DBCO) facilitating the attachment between the oligonucleotide and the molecular core. In some embodiments, the oligonucleotide dendrimer comprises one or more spacers between any of the one or more first oligonucleotide branches and the moiety (e.g., DBCO) facilitating the attachment between the oligonucleotide and the molecular core. In some embodiments, the spacer when present is an organic moiety. In some embodiments, 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, an ethylglycol, or a combination thereof. In some embodiments, the spacer is an oligo(ethylene glycol)-based spacer. In various embodiments, a first oligonucleotide branch comprises 1 , 2, 3, 4, 5, or more spacer (e.g., Spacer-18 (hexaethyleneglycol)) moieties. In some embodiments, the spacer is an alkane-based spacer (e.g., C12). In some embodiments, the spacer is an oligonucleotide spacer (e.g., T5). An oligonucleotide spacer may have any sequence that does not interfere with the ability of the first oligonucleotide(s) to become bound to the molecular core or to a target. In various embodiments, 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. In various embodiments, the length of the spacer is or is equivalent 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 greater than 30 nucleotides.

Immune Modulation

[0055] As disclosed herein “cGAS ligands” refer to double stranded DNA or DNA-RNA heteroduplexes. Exemplary double stranded DNA that activates cGAS-STING includes, but is not limited to, self-DNA sources (e.g., non-apoptotic cell-derived DNA, mitochondrial DNA and genomic DNA)(Decout et aL, Nat Rev Immunol, 21 : 548-569, 2021 ) and exogenous DNA sources (e.g., DNA viruses, retroviruses, intracellular bacteria)(Almine et aL, Nat Comm, 8: 14392, 2017). In some embodiments, the oligonucleotide dendrimer comprises one or more cGAS ligands, wherein the oligonucleotides attached to the molecular core comprise the cGAS ligand. In various aspects, the oligonucleotide dendrimer activates cGAS and/or cGAS-STING. In some embodiments, the one or more first oligonucleotide branches comprises a sequence that activates or inhibits toll-like receptors (TLRs).

[0056] In some embodiments, the oligonucleotide dendrimer comprises one or more first oligonucleotide branches, wherein the one or more first oligonucleotide branches comprises a sequence that inhibits the cGAS enzyme, thereby reducing inflammation through inhibition of the cGAS-STING pathway. In some aspects, the disclosure provides oligonucleotide dendrimers comprising at least two DNA ligands. In some embodiments, the at least two DNA ligands comprise a sequence as set out in any one of SEQ ID NOs: 1 -12 that will bind to and inhibit the cGAS enzyme with high affinity, reducing signal amplification through the cGAS-STING pathway. In some embodiments, this interaction inhibits inflammatory immune responses through cGAS-STING pathway inhibition.

[0057] In some embodiments, the oligonucleotide dendrimer comprising one or more first oligonucleotide branches comprises a sequence that activates the Toll-Like Receptor (TLR) pathway, thereby causing inflammation through the activation of the TLR9-MyD88-NF- kB pathway. In some aspects, oligonucleotide dendrimers comprising at least two DNA ligands comprise a sequence as set out in SEQ ID NO: 20, that will bind TLR9 receptors with high affinity, resulting in a conformational change that allows for downstream signal amplification through the MyD88 and NF-kB signaling cascade, and activation of the TLR9 signaling pathway.

[0058] In some embodiments, the oligonucleotide dendrimer comprising one or more first oligonucleotide branches comprises a sequence that activates Absent in Melanoma 2 (AIM2) pathway, resulting in an inflammatory response. In some aspects, the oligonucleotide dendrimer comprises at least two DNA ligands, and in further aspects, the at least two DNA ligands comprise a sequence as set out in SEQ ID NO: 23 that will bind and activate AIM2 with high affinity, resulting in downstream signal amplification through the activation of the AIM2- apoptosis-associated speck-like protein containing a CARD (ASC) pathway. In various aspects, the oligonucleotide dendrimer comprises an inhibitory AIM2 sequence as set out in SEQ ID NO: 22. In some aspects, the inflammatory response comprises IL-1 p and IL-18. In some embodiments, administration of an oligonucleotide dendrimer comprising one or more first oligonucleotide branches comprising a sequence that activates AIM2 leads to the formation of AIM2 molecular condensates, resulting in enhanced activation of AIM2 and the subsequent generation of pro-inflammatory cytokines.

[0059] In some embodiments, the oligonucleotide dendrimer comprising one or more first oligonucleotide branches comprises a sequence that activates the DNA-dependent protein kinase (DNA-PK) pathway, resulting in an inflammatory response. In some aspects, the oligonucleotide dendrimer comprises at least two DNA ligands, and in further aspects, at least two DNA ligands comprise a sequence as set out in SEQ ID NO: 24 that will bind and activate DNA-PK with high affinity through its Ku70/80 subunit, resulting in downstream signal amplification through the activation of the STING pathway, which generates pro-inflammatory cytokines interferon (IFN)-a and IFN-p. In various aspects, the interaction of the oligonucleotide dendrimer with DNA-PK stimulates an inflammatory response through STING pathway activation.

[0060] In some embodiments, the oligonucleotide dendrimer comprising one or more first oligonucleotide branches comprises a sequence that inhibits the signal transducer and activator of transcription 3 (STAT3) pathway, resulting in an inflammatory response. In some aspects, the oligonucleotide dendrimer comprises at least two DNA ligands, and in further aspects, at least two DNA ligands comprise a sequence as set out in SEQ ID NO: 21 , that will bind and sequester STAT3 within the cytosol of cells, preventing its downstream transcriptional activity. In some aspects, the interaction of the oligonucleotide dendrimer with STAT3 enhances an inflammatory response through STAT3 inhibition.

[0061] In some embodiments, the oligonucleotide dendrimer comprising one or more first oligonucleotide branches comprises a sequence that activates the Z-DNA binding protein 1 (ZBP1 ) enzyme, thereby causing cell death and inflammation. In some aspects, the oligonucleotide dendrimer comprises at least two DNA ligands that will bind and activate ZBP1 , resulting in downstream signal amplification through the activation of the interferon regulatory factor (IRF) 3, IRF7, and NF-kB transcription factors, which generate pro-inflammatory cytokines. In further aspects, ZBP1 activation promotes its interaction with receptor-interacting protein kinase 3 (RIPK3) which induces cell death via necroptosis. The interaction between an oligonucleotide dendrimer and ZBP1 enhances inflammation through activation of pro- inflammatory transcription factors, and cell death through necroptosis.

[0062] In some embodiments, the oligonucleotide dendrimer comprising one or more first oligonucleotide branches comprises a sequence that activates the interferon-inducible protein 16 (IFI16), thereby activating the STING pathway and generating inflammation. In some aspects, the oligonucleotide dendrimer comprises at least two DNA ligands, and in further aspects, at least two DNA ligands comprise a sequence as set out in SEQ ID NO: 25, that will bind and activate IFI16 with high affinity, resulting in downstream signal amplification through the activation of the STING pathway, which generates pro-inflammatory cytokines. Such oligonucleotide dendrimers will promote the formation of IFI16 molecular condensates, resulting in the enzyme’s enhanced activation, and subsequent activation of the STING pathway. In some embodiments, administration of an oligonucleotide dendrimer comprising one or more first oligonucleotide branches comprising a sequence that activates IFI16 leads to the formation of IFI16 molecular condensates (e.g., a IFI16 molecular condensate), resulting in enhanced activation of IFI16 and subsequent enhanced activation of the STING pathway.

[0063] In some embodiments, the oligonucleotide dendrimer comprising one or more first oligonucleotide branches comprises a sequence that activates the RNA helicase retinoic acidinducible gene I (RIG-I) , thereby activating the STING pathway and generating inflammation. In some aspects, the oligonucleotide dendrimer comprises at least two RNA ligands, and in further aspects, at least two RNA ligands comprise a sequence as set out in SEQ ID NO: 26, that will bind and activate RIG-I with high affinity, resulting in downstream signal amplification through activation of mitochondrial antivirus signaling protein (MAVS), which will result in the generation of pro-inflammatory cytokines. In various aspects, the oligonucleotide dendrimer comprises a RIG-I agonist sequence as set out in SEQ ID NO: 27. In some aspects, the above interaction stimulates an inflammatory immune response through the MAVS signalosome activation. In some embodiments, administration of an oligonucleotide dendrimer comprising one or more first oligonucleotide branches comprising a sequence that activates RIG-I leads to the formation of RIG-I molecular condensate (e.g., a RIG-1 molecular condensate), resulting in enhanced activation of RIG-I. Additional Agent

[0064] In various aspects, the oligonucleotide dendrimers of the disclosure further comprise an additional agent, or a plurality thereof. The additional agent is, in various embodiments, associated with a first, second, or third oligonucleotide of the oligonucleotide dendrimer, and/or the additional agent is associated with the molecular core of the oligonucleotide dendrimer. In some aspects, the additional agent is attached to at least one of the one or more first oligonucleotide branches. In various aspects, the additional agent is attached to the second oligonucleotide. In some aspects, the additional agent is attached to the third oligonucleotide. In some embodiments, the oligonucleotide dendrimer comprises a plurality of first oligonucleotide branches and the additional agent is attached to at least two of the plurality of first oligonucleotide branches. In some embodiments, the additional agent is associated with the end of an oligonucleotide that is not attached to the molecular core (e.g., if the oligonucleotide is attached to the dendrimer core through its 3’ end, then the additional agent is associated with the 5’ end of the oligonucleotide). Alternatively, in some embodiments, the additional agent is associated with the end of an oligonucleotide that is attached to the molecular core (e.g., if the oligonucleotide is attached to the molecular core through its 3’ end, then the additional agent is associated with the 3’ end of the oligonucleotide). In some embodiments, the additional agent is covalently associated with an oligonucleotide of the oligonucleotide dendrimer that is attached to the molecular core of the oligonucleotide dendrimer. In some embodiments, the additional agent is non-covalently associated with an oligonucleotide of the oligonucleotide dendrimer that is attached to the molecular core of the oligonucleotide dendrimer. However, it is understood that the disclosure provides oligonucleotide dendrimers wherein one or more additional agents are both covalently and non-covalently associated with oligonucleotides of the oligonucleotide dendrimer that are attached to the molecular core of the oligonucleotide dendrimer. It will also be understood that non-covalently associations include hybridization, protein binding, and/or hydrophobic interactions. In some embodiments, an additional agent is administered separately from an oligonucleotide dendrimer of the disclosure. Thus, in some embodiments, an additional agent is administered before, after, or concurrently with an oligonucleotide dendrimer of the disclosure to treat a disease.

[0065] In various embodiments, the additional agent is a) a protein, peptide, or enzyme; b) a multivalent antibody or derivative thereof; c) a carbohydrate or small molecule proteolysistargeting chimera (PROTAC); d) a double-stranded DNA molecule; and/or e) an RNA molecule. In some aspects, the multivalent antibody or derivative thereof is a nanobody, a single chain variable fragment, a fragment antigen binding domain, a bi-specific T cell engager, or a combination thereof.

[0066] In various aspects, one or more oligonucleotides of the oligonucleotide dendrimer comprise a nucleotide mimetic. In some aspects, the nucleotide mimetic is a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or a combination thereof. In some aspects, the multivalent antibody or derivative thereof binds: a) a checkpoint blockade inhibitor protein; b) a tumor- associated antigen; and/or c) a blood-brain barrier (BBB) penetration protein. In some aspects, the checkpoint blockade inhibitor protein is PD-1 , PD-L1 , CTLA-4, LAG-3, TIM-3, or TIG IT. In various aspects, the tumor-associated antigen is EGFR, HER2, VEGFR, CD20, CD19, or PSMA. In some aspects, the blood-brain barrier (BBB) penetration protein is a transferrin receptor, GLUT1 , or a Bradykinin B2 receptor.

Compositions, pharmaceutical formulations, and antigenic compositions

[0067] The disclosure also provides compositions that comprise an oligonucleotide dendrimer of the disclosure, or a plurality thereof. Further disclosed herein are pharmaceutical formulations comprising any of the oligonucleotide dendrimers or compositions disclosed herein, and a pharmaceutically acceptable carrier, diluent, stabilizer, preservative, or adjuvant. The term "carrier" refers to a vehicle within which the oligonucleotide dendrimer as described herein is administered to a subject. Any conventional media or agent that is compatible with the oligonucleotide dendrimers according to the disclosure can be used. The term “carrier” encompasses diluents, excipients, adjuvants and a combination thereof. Pharmaceutically acceptable carriers are well known in the art (see, e.g., Remington's Pharmaceutical Sciences by Martin, 1975, the entire disclosure of which is herein incorporated by reference).

[0068] Exemplary "diluents" include water for injection, saline solution, buffers such as Tris, acetates, citrates or phosphates, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Exemplary "excipients" include but are not limited to stabilizers such as amino acids and amino acid derivatives, polyethylene glycols and polyethylene glycol derivatives, polyols, acids, amines, polysaccharides or polysaccharide derivatives, salts, and surfactants; and pH-adjusting agents. In some embodiments, the oligonucleotide dendrimers provided herein comprise immunostimulatory oligonucleotides (for example and without limitation, a CpG oligonucleotide) as adjuvants. Other adjuvants known in the art may also be used in the compositions of the disclosure. For example, the adjuvant may be aluminum or a salt thereof, mineral oils, Freund adjuvant, vegetable oils, water-in-oil emulsion, mineral salts, small molecules (e.g., imiquimod, resiquimod), bacterial components (e.g., flagellin, monophosphoryl lipid A), or a combination thereof.

[0069] Further disclosed herein are antigenic compositions comprising any of the oligonucleotide dendrimers, compositions, or pharmaceutical formulations disclosed herein, wherein the antigenic composition is capable of generating an immune response in a mammalian subject.

[0070] An oligonucleotide dendrimer of the disclosure can be administered via any suitable route, such as parenteral administration, intramuscular injection, subcutaneous injection, intradermal administration, and/or mucosal administration such as oral or intranasal. Additional routes of administration include but are not limited to intravenous, intraperitoneal, intranasal, intra-vaginal, intra-rectal, and oral administration. A combination of different routes of administration, separately or at the same time, is also contemplated by the disclosure.

Methods of Treating a Disease

[0071] In some embodiments, the oligonucleotide dendrimers of the disclosure are used to treat a disease. Thus, in some aspects, the disclosure provides methods of treating a disorder comprising administering an effective amount of oligonucleotide dendrimers (e.g., compositions, pharmaceutical formulations, antigenic compositions) of the disclosure to a subject (e.g., a human subject) in need thereof, wherein the administering treats the disease. In various embodiments, the disease is a cancer. An “effective amount” of the oligonucleotide dendrimer is an amount sufficient to, for example, treat, ameliorate, and/or prevent the disease. In other embodiments, an “effective” amount of the oligonucleotide dendrimer is an amount effective to induce an immune response in the subject, without causing significant, adverse side effects in the subject. In various embodiments, the cancer is breast cancer, peritoneum cancer, cervical cancer, colon cancer, rectal cancer, esophageal cancer, eye cancer, liver cancer, pancreatic cancer, larynx cancer, lung cancer, skin cancer, ovarian cancer, prostate cancer, stomach cancer, testicular cancer, thyroid cancer, brain cancer, or a combination thereof.

[0072] The disclosure also includes methods of treating, reducing the symptoms of, or ameliorating a disease in a subject comprising administering to the subject an effective amount of the oligonucleotide dendrimers of the disclosure (e.g., administered as a composition, pharmaceutical formulation, or antigenic composition), thereby treating the disease in the subject. Diseases or disorders that are contemplated by the disclosure in such methods include, but are not limited to, cancer. [0073] In some embodiments, the signaling pathway mediating the immune response is the cGAS-STING pathway. cGAS-STING is a major pathway mediating immune responses against diverse classes of pathogens that contain or generate DNA (e.g., viruses), but cGAS can be activated by any double-stranded DNA in a sequence-independent manner, which includes self- DNA (Ablasser et al., Science, 363: eaat8657, 2019). Activation of the cGAS-STING pathway induces inflammatory responses, and this pathway is also implicated in adaptive immunity. For example, cGAS activation by tumor DNA taken up by dendritic cells leads to the production of type I IFNs and chemokines, as well as upregulation of co-stimulatory molecules, such as CD80 and CD86, all of which stimulate tumor-specific T cell proliferation and recruitment to tumors (Zhang et al., Immunity, 53: 43-53, 2020).

Methods of Producing an Immune Response

[0074] Further disclosed herein are methods of producing an immune response in a subject, comprising administering to the subject an effective amount of any of the oligonucleotide dendrimers, compositions, pharmaceutical formulations, or antigenic compositions disclosed herein, thereby producing an immune response in the subject. In various aspects, the immune response is a CD86+ dendritic cell-mediated response. In some aspects, the immune response is a T cell-mediated response. In various aspects, the immune response is activated via the cGAS-STING pathway.

[0075] Antigenic compositions may be used to treat both children and adults, including pregnant women. Thus a subject may be less than 1 year old, 1 -5 years old, 5-15 years old, 15- 55 years old, or at least 55 years old. Preferred subjects for receiving the vaccines are the elderly (e.g., >55 years old, >60 years old, preferably >65 years old), and the young (e.g., <6 years old, 1-5 years old, preferably less than 1 year old).

Molecular Condensate

[0076] Disclosed herein is a molecular condensate comprising any of the oligonucleotide dendrimers disclosed herein, or a plurality thereof, and a plurality of proteins. As used herein, the terms “molecular condensate”, “stress granule”, and “micronuclei” are interchangeable. In some embodiments, the molecular condensate comprises cGAS. cGAS binds to DNA only on the sugar-phosphate backbone, without interacting with bases (Andreeva et al., Nature, 549: 394-398, 2017). cGAS requires a minimum of 18 bp of dsDNA for binding, and longer dsDNA can form a more stable network with cGAS, further increasing the cGAS catalytic activity (Li et al., Immunity, 39: 1019-1031 , 2013). Additionally, the presence of a long DNA sequence leads to a dimeric structure (a 2:2 DNA:cGAS complex) that promotes liquid phase condensation, further stabilizing the active dimeric state through multivalent interactions between nearby cGAS molecules (Du et al., Science, 361 : 704-709, 2018), yielding a molecular condensate. Oligonucleotide binding to cGAS induces a robust phase transition to liquidlike droplets, molecular condensates, which function as microreactors in which the enzyme and reactants are concentrated to greatly enhance the production of cGAMP (Du et aL, Science, 361 : 704-709, 2018). These multivalent interactions drive liquid phase separation (Banani et al., Nat Rev Mol Cell Biol, 18: 285-298, 2017). Long DNA has more binding sites for cGAS than short DNA, and thus dendrimer cores with longer oligonucleotide branches provide more binding sites for cGAS (Zhang et aL, Cell Rep, 6: 421 -430), enhancing formation of oligonucleotide dendrimer-cGAS molecular condensates. Long DNA, for example DNA that is at least about 40 nucleotides, and thus dendrimer cores with oligonucleotide branches that are at least about 40 nucleotides, provide more binding sites for cGAS.

[0077] In some embodiments, at least one oligonucleotide dendrimer interacts with more than one protein of the plurality of proteins comprising the molecular condensate. In further embodiments, at least one protein of the plurality of proteins binds to more than one oligonucleotide dendrimer or oligonucleotide dendrimer branch, wherein the plurality of proteins comprises cGAS-STING effector proteins, pyrin domain-containing 3 (NLRP3) proteins, signal transducer or activator of transcription 3 (STAT3), IFI16, AIM2, or a combination thereof. In some embodiments, at least one oligonucleotide dendrimer interacts with more than one protein of the plurality of proteins, and at least one protein of the plurality of proteins binds to more than one oligonucleotide dendrimer or oligonucleotide dendrimer branch, wherein the plurality of proteins comprises cGAS-STING effector proteins, pyrin domain-containing 3 (NLRP3) proteins, signal transducer or activator of transcription 3 (STAT3), I Fl 16, AIM2, or a combination thereof. In various aspects, the oligonucleotide binds to one or more proteins in the plurality of proteins via hydrogen bonding and/or ionic interaction.

[0078] In some aspects, the molecular condensate has a half-life of at least 45 minutes. In a non-limiting example in which the plurality of proteins comprises cGAS-STING effector proteins, the half-life of the molecular condensate can be evaluated via confocal microscopy with use of an in vitro phase separation assay, wherein labeled recombinant cGAS protein is mixed with labeled DNA of a defined length, in 96-well plates coated with BSA. Mixtures are incubated, and images are captured at various timepoints sufficient for the half-life of the molecular condensate to be captured. For example, a total timecourse of 180 minutes can be used to capture a molecular condensate Phase separation of recombinant cGAS with DNA can be performed in solution determined based on the length of the DNA. For example, phase separation of 45 base pair DNA and cGAS can be performed in performed in 20 mM Tris-HCI, pH 7.5, 150 mM NaCI and 1 mg/ml BSA. As another example, phase separation of 100 base pair DNA and cGAS can be performed 20 mM Tris-HCI, pH 7.5, 300 mM NaCI, 1 mg/ml BSA. Phase diagrams can be generated by mixing recombinant cGAS and DNA in 20 mM Tris-HCI, pH 7.5, 150 mM NaCI and 1 mg/ml BSA, regardless of the length of the DNA. See, for example, Du and Chen, Science, 361 (6403): 704-709, 2018, which is incorporated by reference in its entirety.

[0079] In various aspects, the proteins’ concentration within the molecular condensate is enhanced by at least a factor of 2, compared to a concentration of proteins outside the molecular condensate. In some aspects, the plurality of proteins are cGAS-STING effector proteins that activate a cGAS-STING pathway. In various aspects, the molecular condensate comprises at least 100 proteins. In some aspects, a molecular condensate has a diameter of about 5 to about 25 microns. In various aspects, a molecular condensate has a diameter of about 5 to 25, 10 to 25, 15 to 25, 20 to 25, 5 to 20, 10 to 20, 15 to 20, 5 to 15, or 5 to 10 microns. In some aspects, a molecular condensate has a diameter of at least about 5 microns, 10 microns, 15 microns, 20 microns, or 25 microns. In various aspects, a molecular condensate has a diameter of less than about 30 microns, 25 microns, 20 microns, 15 microns, 10 microns or 5 microns. In some aspects, the concentration of oligonucleotide branches to cGAS-STING effector proteins in the molecular condensate is 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, or 1 :7. In various aspects, the concentration of oligonucleotide branches to cGAS-STING effector proteins in the molecular condensate is 1 :3. Protein concentration within a molecular condensate can be evaluated via purification of the micronuclei from cultured cells via flow cytometry, for example, as described in Toufektchan and Maciejowski, STAR Protoc, 2(1): 100378, 2021 , which is incorporated by reference in its entirety. Briefly, to purify micronuclei, cells (RAW264.7, HEK293T, or NIH3T3) are transfected with stable GFP-cGAS fusion proteins, and are treated for approximately 8 hours with Cy3.5-labeled oligonucleotide dendrimers (or controls). The molecular condensates are isolated with sucrose density gradient centrifugation, and the contents of the molecular condensates are quantified with a combination of flow cytometry and BCA protein quantification assays.

[0080] In various embodiments, the concentration of oligonucleotide branches in the molecular condensate is at least about 0.001 M, 0.01 M, 0.1 M, 1 M, or 10 M. In some embodiments, the concentration of oligonucleotide branches in the molecular condensate is at least less than about 10 M, 1 M, 0.1 M, 0.01 M, or 0.001 M. In various embodiments, the concentration of oligonucleotide branches in the molecular condensate is between about 0.001 M and 10 M. In some embodiments, the concentration of oligonucleotide branches in the molecular condensate is between about 0.001 M to 10 M, 0.01 M to 10 M, 0.1 M to 10 M, 1 M to 10 M, 0.001 M to 1 M, 0.01 M to 1 M, 0.1 M to 1 M, 0.001 M to 0.1 M, 0.01 M to 0.1 M, or 0.001 M to 0.01 M. In various embodiments, the concentration of oligonucleotide branches in the molecular condensate is or is at least about 1 M, 2 M, 3 M, 4 M, 5 M, 6 M, 7 M, 8 M, 9 M, or 10 M. In some embodiments, the concentration of oligonucleotide branches in the molecular condensate is or is at least less than 10 M, 9 M, 8 M, 7 M, 6 M, 5 M, 4 M, 3 M, 2 M, or 1 M. . In various embodiments, the concentration of oligonucleotide branches in the molecular condensate is between about 1 M to 10 M, 2 M to 10 M, 3 M to 10 M, 4 M to 10 M, 5 M to 10 M, 6 M to 10 M, 7 M to 10 M, 8 M to 10 M, 1 M to 9 M, 2 M to 9 M, 3 M to 9 M, 4 M to 9 M, 5 M to 9 M, 6 M to 9 M, 6 M to 9 M, 7 M to 9 M, 8 M to 9 M, 1 M to 8 M, 2 M to 8 M, 3 M to 8 M, 4 M to 8

M, 5 M to 8 M, 6 M to 8 M, 7 M to 8 M, 1 M to 7 M, 2 M to 7 M, 3 M to 7 M, 4 M to 7 M, 5 M to 7

M, 6 M to 7 M, 1 M to 6 M, 2 M to 6 M, 3 M to 6 M, 4 M to 6 M, 5 M to 6 M, 1 M to 5 M, 2 M to 5

M, 3 M to 5 M, 4 M to 5 M, 1 M to 4 M, 2 M to 4 M, 3 M to 4 M, 1 M to 3 M, 2 M to 3 M, or 1 M to

2 M. In some embodiments, the concentration of oligonucleotide branches in the molecular condensate is or is at least about 2.8 M.

[0081] In various embodiments, the concentration of cGAS-STING effector proteins in the molecular condensate is at least about 0.001 M, 0.01 M, 0.1 M, 1 M, or 10 M. In some embodiments, the concentration of cGAS-STING effector proteins in the molecular condensate is at least less than about 10 M, 1 M, 0.1 M, 0.01 M, or 0.001 M. In various embodiments, the concentration of cGAS-STING in the molecular condensate is between about 0.001 M and 10 M. In some embodiments, the concentration of cGAS-STING in the molecular condensate is between about 0.001 M to 10 M, 0.01 M to 10 M, 0.1 M to 10 M, 1 M to 10 M, 0.001 M to 1 M, 0.01 M to 1 M, 0.1 M to 1 M, 0.001 M to 0.1 M, 0.01 M to 0.1 M, or 0.001 M to 0.01 M. In various embodiments, the concentration of oligonucleotide branches in the molecular condensate is or is at least about 1 M, 2 M, 3 M, 4 M, 5 M, 6 M, 7 M, 8 M, 9 M, or 10 M. In some embodiments, the concentration of cGAS-STING effector proteins in the molecular condensate is or is at least less than 10 M, 9 M, 8 M, 7 M, 6 M, 5 M, 4 M, 3 M, 2 M, or 1 M. In various embodiments, the concentration of cGAS-STING effector proteins in the molecular condensate is between about 1 M to 10 M, 2 M to 10 M, 3 M to 10 M, 4 M to 10 M, 5 M to 10 M, 6 M to 10 M, 7 M to 10 M, 8 M to 10 M, 1 M to 9 M, 2 M to 9 M, 3 M to 9 M, 4 M to 9 M, 5 M to 9 M, 6 M to 9 M, 6 M to 9 M, 7 M to 9 M, 8 M to 9 M, 1 M to 8 M, 2 M to 8 M, 3 M to 8 M, 4 M to 8 M, 5 M to 8 M, 6 M to 8 M, 7 M to 8 M, 1 M to 7 M, 2 M to 7 M, 3 M to 7 M, 4 M to 7 M, 5 M to 7 M, 6 M to 7 M, 1 M to 6 M, 2 M to 6 M, 3 M to 6 M, 4 M to 6 M, 5 M to 6 M, 1 M to 5 M, 2 M to 5 M, 3 M to 5 M, 4 M to 5 M, 1 M to 4 M, 2 M to 4 M, 3 M to 4 M, 1 M to 3 M, 2 M to 3 M, or 1 M to 2 M. In some aspects, the concentration of oligonucleotide branches in the molecular condensate is or is at least about 2.8

M, and the concentration of cGAS-STING effector proteins in the molecular condensate is or is at least about 8.4 M.

[0082] In some embodiments, administration of any of the compositions disclosed herein induces formation of one or more molecular condensates, wherein the molecular condensate comprises cGAS-STING proteins, NLRP3 proteins, STAT3 proteins, IFI16 proteins, and/or AIM2 proteins. In various aspects, the one or more molecular condensates have a diameter of about 5 to about 60 microns. In various aspects, a molecular condensate has a diameter of about 5 to 60, 10 to 60, 15 to 60, 20 to 60, 25 to 60, 30 to 60, 35 to 60, 40 to 60, 45 to 60, 50 to 60, 55 to 60, 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 5 to 40, 10 to 40, 15 to 40, 20 to 40, 25 to 40, 30 to 40, 35 to 40, 5 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 20, 10 to 20, 15 to 20, or 5 to 10 microns. In some aspects, a molecular condensate has a diameter of or of at least about 5 microns, 10 microns, 15 microns, 20 microns, 25 microns, 30 microns, 35 microns, 40 microns, 45 microns, 50 microns, 55 microns, or 60 microns. In various aspects, a molecular condensate has a diameter of or of less than about 60 microns, 55 microns, 50 microns, 45 microns, 40 microns, 35 microns, 30 microns, 25 microns, 20 microns, 15 microns, 10 microns or 5 microns.

[0083] Further disclosed herein are methods of treating a disease in a subject, comprising administering to the subject an effective amount of any of the oligonucleotide dendrimers, compositions, pharmaceutical formulations, or antigenic compositions disclosed herein. In various aspects, the disease is cancer, an autoimmune disease, an infectious disease, or a combination thereof. In some aspects, the cancer is breast cancer, peritoneum cancer, cervical cancer, colon cancer, rectal cancer, esophageal cancer, eye cancer, liver cancer, pancreatic cancer, larynx cancer, lung cancer, skin cancer, melanoma, ovarian cancer, prostate cancer, stomach cancer, testicular cancer, thyroid cancer, brain cancer, or a combination thereof.

Method of Making an Oligonucleotide Dendrimer

[0084] Further disclosed herein is a method of making an oligonucleotide dendrimer. In some embodiments, the method comprises contacting a molecular core comprising one or more first oligonucleotide branches with one or more second oligonucleotides, wherein the contacting results in the hybridization of a first oligonucleotide branch with at least a first region of a second oligonucleotide, thereby resulting in an at least partially double stranded oligonucleotide dendrimer with an overhang region. In some aspects, the method further comprises hybridizing a third oligonucleotide to the overhang region of the second oligonucleotide. In various aspects, Na+, Mg2+, urea, and/or DMF are present during the contacting. In some aspects, the stoichiometry of oligonucleotide: molecular core is 12:1 . In various aspects, the stoichiometry of oligonucleotide: molecular core is 8:1 , 9:1 , 10:1 11 :1 , 12:1 , 13:1 , 14:1 , 15:1 , or 16:1 .

[0085] In some embodiments, the method comprises using a convergent strain-promoted azide-alkyne cycloaddition click chemistry approach wherein oligonucleotides with terminal dibenzocyclooctyne (DBCO) moieties are reacted with polymeric azide-terminated dendrimers, forming an at least partially double stranded oligonucleotide dendrimer. In some embodiments, the at least partially double stranded oligonucleotide dendrimer is further reacted with an additional oligonucleotide. In various aspects, the additional oligonucleotide is a second oligonucleotide. In some aspects, the additional oligonucleotide is a second oligonucleotide hybridized to a third oligonucleotide. The skilled artisan would recognize that conditions for reacting distinct oligonucleotide sequences vary based on their physical characteristics (e.g., size, charge, solubility, and second/ tertiary structure), common factors that improve yields include: (1 ) salting reactions to screen negatively charged ODN phosphates, 2) maximization of reactant concentrations, 3) utilization of a mixed solvent system, and 4) inclusion of surfactant to improve reaction kinetics.

[0086] The synthesis of MADONs (e.g., oligonucleotide dendrimers) utilizes a convergent click-chemistry approach to attach oligonucleotides (ODNs) to dendrimeric cores. While a variety of ligation chemistries have been studied, a typical procedure employs ODNs with terminal (either 3 or 5') strained alkyne moieties (principally dibenzoclylooctyne [DBCO]) which are reacted with azide-terminated polymeric dendrimer cores. ODNs can vary in their length (25-65 base pairs), sequence, backbone chemistry (phosphate or phosphorothioate), nucleobase chemistry (DNA, RNA, modified ODN bases), and structure (linear, single or double stranded, loops, aptamers, G-quadruplexes). Dendrimer cores can additionally vary in their chemistry (e.g., polyethylene glycol, polyester, polyamidoamine), valency (i.e., number of terminal branches), and click chemistry moieties. Reactions are performed with at least a 2:1 excess of DBCO:Azide for 16 - 72 hours at 25 - 55 degrees Celsius with high salt concentrations (1 .5 M NaCI or 4 M urea) and volume constriction (2 mM by ODN ligand) in mixed organic/aqueous solutions (50 - 80% organic, dimethylformamide [DMF] or dimethylsulfoxide [DMSO]). Structures are subsequently purified with polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography (SEC), ion exchange chromatography (IEX), high performance liquid chromatography (HPLC), or combinations thereof. Characterization of these particles includes matrix assisted laser desorption/ionization mass spectrometry, analytical PAGE, dynamic light scattering, and zeta potential analysis.

[0087] The following examples are provided merely to illustrate the present disclosure and not in any way to limit its scope.

EXAMPLES

Example 1

[0088] It has been shown that alterations in nanoparticle architecture including their three- dimensional arrangement and multivalency (/.e., the number of conjugated ligands) significantly affect tissue accumulation, elimination, and potency. Unlike SNAs, MADONs are produced in monodisperse populations, facilitating precise structural control and offering a modular, molecularly defined platform for fine tuning architectural parameters to comprehensively understand the valency dependence of cGAS activation. MADONs with high valency and long DNA branches significantly enhance cGAS-STING activation and generate a pro-inflammatory TME that enhances anti-GBM immunity and improves survival in experimental GBM models. The research strategy investigates the premise that architectural modifications can uniquely modulate the DNA structure-dependent cGAS mechanism and that these structure activity relationships (SARs) can be used to develop a novel GBM immunotherapy. The following aims are tested:

[0089] Specific Aim 1 : Establish Conditions for High-Yield MADON Synthesis and Evaluate Structure Activity Relationships (SARs) to Understand cGAS-STING Pathway Activation. A library of MADONs with defined valency, DNA branch length, and core materials (polyester, polyethylene glycol, and polyamide) are synthesized and cell-free and in vitro experiments are performed to quantify their SARs. Generalizable “click” reactions (/.e., azide-alkyne cycloadditions) are used to synthesize a small library of MADONs with variable valency (e.g., 4, 6, 12, etc., DNA branches) and DNA length (e.g., 25, 45, and 65 bp). The library’s degradation kinetics over time is quantified with gel electrophoresis to determine their stability in aqueous conditions and resistance to nucleases. Isothermal titration calorimetry will establish the thermodynamics of cGAS: MADON interactions and a fluorescent cell-free cGAS assay will quantify enzyme activation kinetics, both of which elucidate the extent to which alterations in MADON architecture can leverage the enzyme’s polyvalency-dependent mechanism of action. In vitro studies are performed to measure uptake in myeloid cells, and cGAS-STING pathway activation luciferase reporter cell lines. Additionally, the ability for MADONs to catalyze the formation of intracellular liquid-liquid phase separated molecular condensates is determined with confocal microscopy in immortalized macrophages transfected with GFP-cGAS.

[0090] Specific Aim 2: Determine the MADON Structure with Optimal TME Repolarization and Survival Benefit in Murine GBM. This aim determines how MADON structure affects biodistribution and efficacy in immunocompetent murine models of GBM (e.g., CT2A and QPP4). The accumulation of fluorescently labeled MADONs in various tissues as a function of route of administration (e.g., intravenous and intratumoral) is determined using an in vivo imaging system (I VIS). In these studies, sequential hematologic sampling will inform the pharmacokinetic profiles (e.g., half-life, maximum plasma concentration, and area under the concentration-time curve) of MADON architectures. Animal survival, tumor growth, and tumor rechallenge experiments is performed to determine the features of MADONs that productively modulate the TME and generate anti-GBM immune responses. The specific immune responses, including the transcriptional and phenotypic state of effector cell types is determined using single cell RNA sequencing, the results of which are validated with a comprehensive flow cytometry experiment. These experiments will determine the polarization state of infiltrating immune populations including myeloid cells (e.g., dendritic cells, macrophages, monocytes), T cells, natural killer cells, and microglia and their expression of genes downstream of the cGAS- STING pathway (e.g., NF-KB, IRF, STAT6, etc.).

Example 2

[0091] Synthesis and Characterization of DNA Dendrimers. DNA dendrimers (Fig. 1 A) were synthesized using a convergent strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry approach wherein oligonucleotides (ODNs) with terminal dibenzocyclooctyne (DBCO) moieties were reacted with commercially available polymeric azide-term inated dendrimers (Fig. 1 B). Controlling the solution chemistry of these reactions was essential to achieve high product yields because the conjugation of multiple large, highly negatively charged ODNs to the termini of relatively small polymers is both highly entropically and Coulombically unfavorable. Though the optimal conditions for reacting distinct ODN sequences varies based on their physical characteristics (including size, charge, solubility, and secondary/tertiary structure), there exist several common factors that improve yields: 1 ) salting reactions (using Na+, Mg²⁺, or urea [CO(NH₂)2]) to screen negatively charged ODN phosphates, 2) maximization of reactant concentrations (DBCO-ODNs and polymeric dendrimers), 3) utilization of a mixed solvent system (50-80% DMF v/v in H2O), and 4) inclusion of surfactant to improve reaction kinetics (0- 5 mM DDAB). Additional approaches that were unsuccessful include performing the reactions on solid supports or wholly in the organic phase. Using these insights, 25 base pair (bp) interferon stimulatory DNA (Tables 1-2. ISD DNA; a sequence known to activate cGAS) ligands with 3’-DBCO are appended to generation 1 (G1 ) dimethylolpropionic acid (bis-MPA) dendrimers with six pendant azide-modified branches (Fig. 1 C) with yields of approximately 29%. Notably, this represents a 2.98-, 2.42-, and 1 .13-fold increase in yield for the synthesis of analogous hexavalent molecules.

[0092] Table 1 : DNA Design

[0093] The nomenclature of the disclosed DNA dendrimers is defined as follows: Molecular Core - DNA Ligand - Valency (e.x., G1 -ISD25-6 consists of a generation 1 dendrimer core with six 25 bp ISDs). Purifying reactions using denaturing polyacrylamide gel electrophoresis (PAGE) allowed for the separation of dendrimers by valency, which resulted in the isolation of G1 -ISD25-6, -5, and -4 molecules (Fig. 1 D) and G2-ISD25-10 through -4 molecules (Fig. 1 F). Note that DNA dendrimers exhibited electrophoretic mobility that is inconsistent with that of a linear DNA strand with an equivalent nucleobase content. Rather, the three-dimensional structure of these molecules impedes their mobility. Similarly, given that ISD25 exhibited selfcomplementation in its single-stranded form, the molecules could assemble into higher order structures under native conditions. Single stranded G1-ISD25-6 and G2-ISD25-10 dendrimers mixed with increasing molar ratios of complementary ISD25 DNA (clSD25) exhibited saturated hybridization at 1x equivalent (Fig. 1 E, G). Matrix-assisted laser desorption-ionization (MALDI) confirmed that purified dendrimers align with expected m/z values (Fig. 2A, Table 3). The measured hydrodynamic diameters of single-stranded (ss) and ds dendrimers were 9.66 and 7.14 nm, respectively (Fig. 1 H). Zeta potential measurements demonstrated that ss and ds G1 - ISD25 dendrimers had surface charges of -25 and -27 mV, respectively, while that of ss and dsG2-ISD25-10 were observed to be -31 and -32 mV, respectively (Fig 1 H, Inlay), which confirmed the conjugation of DNA ligands to polymeric cores and complement hybridization to single-stranded cores. Characterization of double-stranded (ds) G1-ISD25-6 with atomic force microscopy (AFM) demonstrated that these molecules were monodisperse and exhibited a diameter of approximately 9 nm (Fig. 11). Previous studies of bare polyester dendrimer cores demonstrated the degradation of these polymers by hydrolysis, which was accelerated by increasing temperatures and pH. To determine the stability of dendrimers in solution, dsG1- ISD25-6 molecules were incubated for 2 weeks at 37 °C. After 2 weeks, analytical PAGE and subsequent densitometric analysis (Fig. 1 J) revealed that G1 -ISD25-6 molecules slowly hydrolyzed in a stepwise manner, demonstrating a 1 .1 , 1.9, and 3.4-fold increase in the quantity of G1 -ISD25-5, 4, and 3 dendrimers, respectively. To determine if the dense packing and radial orientation of DNA dendrimer ligands imparted resistance to nuclease-mediated degradation, ds linear ISD25 or G1 -ISD25-6 molecules were incubated with recombinant DNAse-l nuclease at 37 °C for 24 hours, which showed that dendrimeric architectures abrogated nuclease degradation at early time points (Fig. 1 K). [0094] Table 2: DNA Modifications

[0095] DNA Dendrimers are Utilized as Templates to Produce a Variety of Therapeutics.

Having identified the necessary conditions to produce G1 -ISD25-6 DNA dendrimers in high yields, the next step was to investigate the generalizability of the synthesis to append ODN ligands that vary in their sequence (including the length of sequences and nucleobase composition) and secondary structure (including aptameric hairpins and G-quadruplexes). As such, it was found that these conditions were amenable to conjugating aptameric AP1 (T- SO508) hairpins, G-quadruplex sequences (GGT10), and chemically modified nucleobase (5- fluorouracil [5FU]) to G1 polymeric cores (Fig. 2B, Tables 1 -3).

[0096] Table 3: MALDI MS Analysis

[0097] Utilizing identical reaction conditions, the attachment of 45 bp ISD sequences was found to produce negligible quantities of G1-ISD45-6 dendrimers. It was hypothesized that prehybridizing ODNs to their complementary strands may improve yields by increasing the persistence length of DNA ligands and therefore reducing the impact of steric effects. However, analytical PAGE confirmed that the prehybridization of ISD25 DNA reduced overall reaction yields (Fig. 3A). To circumvent challenges associated with conjugating longer strands to dendrimer cores, G1-ISD25-6 molecules were used as templates to generate a diversity of double stranded therapeutics. G1 -ISD25 dendrimers were hybridized to complementary ISD25 (clSD25) to form dsG1 -ISD25-6, 5, 4, and 3, while those hybridized to both complementary ISD45 (clSD45) and ISD20 (a terminal 20 bp sense strand) form nicked (N) G1 -ISD45-6, 5, 4, and 3 (Fig. 3B, 3D). Incorporating a phosphate group to the 3’ terminus of ISD20 ligands hybridized to G1 -ISD-25-6 and clSD45 allowed for 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDO) -catalyzed ligation that generated a distribution of ISD45-modified DNA dendrimers (Fig. 30, Left) that hybridized to complementary DNA (Fig. 30, Right). Additionally, it was found that DNA dendrimers can be synthesized without polyethylene glycol (PEG) Spacer 18 (hexaethylene glycol) motifs between the nucleobases and DBCO moieties of DNA ligands, and that the resulting purified molecules can efficiently hybridize to complementary DNA (Fig. 3E).

[0098] Defining the Capacity of DNA Dendrimers to Bind and Activate Recombinant cGAS. DNA dendrimers or their linear counterparts were mixed with recombinant human cGAS enzymes with the necessary reactants to quantify cGAMP catalysis in a cell-free environment. Initial experiments that compared the ability of linear ISD25 and ISD45 bp DNA supported the literature precedent that longer DNA ligand more potently activate cGAS across a wide range of enzyme concentrations (Fig. 4A). Comparing the most highly valent molecules, G1 -ISD25-6 and G1 -ISD45-6 (N), to their linear comparators ISD25 and ISD45 (N) revealed two key insights: 1 ) that the length dependency of cGAS was maintained while delivering DNA as dendrimeric architectures, and 2) that the delivery of these ligands as DNA dendrimers substantially improved cGAS agonism over a wide range of concentrations (Fig. 4B). Delivering ISD25 (Fig. 4C) and ISD45 (N) (Fig. 4D) dendrimers with 6, 5, 4, and 3 DNA ligands revealed a stepwise reduction in the effective concentration 50 (EC50) as the valency of these molecules increased. In all comparisons, DNA dendrimers vastly outperformed linear DNA controls. These results illustrate the necessity to develop monodisperse and molecularly precise nanotherapeutic platforms by demonstrating that significantly different biological results were observed between molecules that differ in the conjugation of a single DNA ligand. Additionally, these results represent how the structure and architecture of nanotherapeutics can be rationally designed to develop therapeutics to target enzymes with unique, multivalency-dependent mechanisms of action. Finally, the ability of DNA dendrimers to activate cGAS were compared, with or without (NS) flexible Spacer 18 between the polymeric cores and DNA ligands (Fig. 4E). Results indicated that both sets of DNA dendrimers recapitulated the valency-dependent enzyme activation. Direct comparisons between dendrimers with equivalent valencies suggested that the inclusion of spacers mildly abrogated cGAS agonism in G1 - 4 and 3 molecules, with more substantial differences between dendrimers with shorter 25 bp branches.

[0099] In addition to quantifying the extent of DNA dendrimer-mediated cGAS activation, it was important to determine the relative binding affinity of these molecules to recombinant enzyme. ISD25, ISD45, G1 -ISD25-6, and G1 -ISD25-4 were mixed with increasing stoichiometric ratios of recombinant cGAS (from 0 to 40x), incubated at 37 °C for 30 minutes, and analyzed by PAGE (Fig. 5A). Observing the cGAS/DNA ratio at which DNA bands began to migrate revealed that both DNA dendrimers exhibited an approximate 10-fold greater binding affinity to cGAS than ISD25 and ISD45. These results indicated that structuring DNA ligands around a molecular core improved their ability to engage with target enzymes of interest. Differences in the binding affinity between ISD25 and G1 -ISD25-6 dendrimers were confirmed by comparing isothermal titration calorimetry isotherms generated by injecting equivalent DNA concentrations into a reaction cell with murine cGAS. Fitting the resultant data to an independent binding model showed that the binding affinity (Kd) of DNA dendrimers was approximately 10-fold lower than that of their linear counterparts (Fig. 5B), wherein Kd was measured to be 73.5 and 612 nM for G1-ISD25-6 and ISD25, respectively.

[0100] Quantitative Investigation of DNA Dendrimer-Mediated cGAS Molecular Condensation. The cGAS enzyme is understood to consist of multiple DNA binding sites that coordinate its intramolecular binding interactions with DNA substrate. These multivalent interactions facilitate the formation of phase-separated micronuclei known as molecular condensates that contain dense networks of cGAS-DNA known to potentiate the enzyme’s catalytic activity. The next step was evaluation of whether the presentation of ISD25 DNA as a dendrimer would improve the nucleation and growth of cGAS molecular condensates using confocal microscopy. After mixing AlexaFluor 488-labeled mcGAS and equivalent DNA concentrations of Cy3.5-labeled ISD25 or G1 -ISD25-6, solutions were imaged every 5 minutes for 45 minutes (Fig. 6A). In quantifying the diameter and area of individual condensates, it was observed that the distribution of puncta in ISD25 samples remained largely consistent over 45 minutes, while assemblies initiated with G1 -ISD25-6 dendrimers were observed to continuously grow over time (Fig. B, C, D). Similarly, it was found that the total MFI of images increased over the course of 45 minutes in samples treated with dendrimers (Fig. 6E), suggesting that condensates continued to dynamically recruit DNA dendrimers as the experiment progressed. Image analysis also revealed that G1 -ISD25-6 dendrimers nucleated approximately 2 orders of magnitude more foci compared to ISD25 (Fig. 6 E). These data suggest that DNA dendrimers robustly nucleated comparatively smaller molecular condensates than those formed with ISD25 which may be advantageous for cGAS activation - increasing the surface area to volume ratio of micronuclei can facilitate more rapid mass transfer of cGAS substrate and product.

[0101] Quantifying DNA Dendrimer-Mediated cGAS-STING Pathway Activation and Dendritic Cell Uptake and Co-Stimulation. Using a RAW Lucia Macrophage reporter cell line wherein IRF3 signaling is coupled to the expression of a luciferase gene under the control of an ISG54 promoter, it was found that both nicked and ligated G1 -ISD45-6 dendrimers more potently activated the cGAS-STING axis than the state-of-the-art small molecule STING agonist Aduro S100 and linear ISD45 DNA at equivalent concentrations (Fig. 7A). Additionally, DNA dendrimers, linear DNA, and Aduro S100 were found to have exhibited negligible cytotoxicity in these cells across a wide range of concentrations (Fig. 7B). Treatment of the immortalized DC2.4 cell line with Cyanine 3.5 labeled ISD25 DNA or G1 -ISD25-6 dendrimers at time = 0 and quantifying the extent of cellular uptake over the course of 30 minutes revealed that only DNA delivered as a dendrimer was rapidly and consistently internalized into cells (Fig. 7C). Additionally, it was determined whether Spacer 18 moieties on G1 -ISD25 dendrimers affected their ability to stimulate the cGAS-STING pathway in Raw Lucia ISG cells (Fig. 7D). Results indicated that, while both G1 -ISD25 and G1-ISD25 (NS) exhibited valency-dependent activation across a range of concentrations, there are minimal differences between the two structures that were exaggerated in molecules with low valencies (e.x., G1 -ISD24-4 and 3).

[0102] Next, the degree of cellular uptake and immunostimulatory capacity of DNA dendrimers was characterized in dendritic cells. To that end, Cyanine 3.5-labeled DNA dendrimers were synthesized, and used to treat bone marrow-derived dendritic cells (BMDCs) for 1 , 4, and 8-hour pulses followed by a chase to 24 hours. Flow cytometric analysis revealed that DNA dendrimers were endocytosed significantly more than linear DNA and exhibited valency-dependent cellular uptake (Fig. 8A). Measuring the extent of BMDC activation using the co-stimulatory surface marker CD86, it was similarly found that DNA dendrimers vastly outperformed untreated and control ISD25 samples, with the most highly valent molecules demonstrating the most potent immunostimulation (Fig. 8B). Similar 8-hour pulse chase experiments in DC2.4 cells confirmed the valency dependent cellular uptake (Fig. 8C) and CD86 marker expression (Fig. 8D) of DNA dendrimers, and offered evidence of dosedependency. Comparing dendrimer therapeutics with increasing long DNA ligands [G1 -ISD25-6 vs. G1 -ISD45-6 (N)] in BMDCs, flow cytometry confirmed that molecules with 45 bp ISD DNA branches exhibited a higher degree of cellular uptake and co-stimulatory marker expression (Fig. 8E). These results further exemplified the importance of evaluating the SARs that dictate the biological function of nanoarchitectures; it was shown herein that altering the structure of these molecules by a single DNA ligand yielded significant differences in the extent of DC uptake and activation.

[0103] While cells of the myeloid compartment are the primary targets of cGAS-agonistic DNA dendrimers, it was additionally important to determine how these molecules interact with tumor cells themselves. CT2A murine glioma cells were treated with linear ISD25 DNA or G1 -ISD25-6 dendrimers as single-stranded (ss), double-stranded (ds), or double-stranded with 2 (ds+2) or 5 bp (ds+5) overhangs (Fig. 8F). Results indicated that single-stranded ISD25 and G1 -ISD25-6 molecules demonstrated the highest relative increases in cellular uptake which are abrogated by hybridization to complementary DNA regardless of the length of nucleobase overhang. These findings suggest that, compared to immune cells, tumors cannot achieve substantial internalization of the functional double-stranded DNA dendrimers and that the addition of 2 or 5 overhanging bases does not rescue the uptake behavior of single-stranded molecules.

[0104] In summary, the data presented herein demonstrate the ability to synthesize, purify, and characterize a variety of monodisperse DNA dendrimers varying in their valency and identity of DNA ligands. Delivering ISD DNA as a dendrimeric architecture imparts beneficial therapeutic properties including enhanced resistance to nuclease-mediated degradation, potent cell-free cGAS activation through tight binding interactions and robust nucleation of molecular condensates, rapid cellular uptake, and powerful immunostimulation. From these results, it is apparent that cGAS agonist DNA dendrimers represent a promising emerging class of nanotherapeutic with the potential to improve survival outcomes in a variety of cancers. Example 3: Materials and Methods

[0105] Oligonucleotide synthesis and modification. Oligonucleotides were synthesized using a MerMade 12 oligonucleotide synthesizer (Bio Automation, Texas, USA) and/or an Applied Biosystems 3400 DNA synthesizer on controlled pore glass (CPG) beads. Universal UnyLinker Support CPG (1000 A) were used to synthesize complementary DNA sequences, 3’- PT-Amino-Modifier C6 CPG (Glen Research; 1000 A) were used to synthesize aminoterminated DNA, and 3’-Chemical Phosphorylation Reagent (CPR) II CPG (Glen Research; 1000 A) were used to synthesize DNA with 3’ phosphate moieties. All phosphoramidites and oligonucleotide synthesis reagents were purchased from Glen Research and used according to manufacturer’s instructions. Unlabeled oligonucleotides were deprotected using a 1 :1 solution of 37% ammonium hydroxide/40% methylamine at 55 °C for 25 min. Dye-conjugated oligonucleotides were deprotected with a solution of 37% ammonium hydroxide for 16 hours at room temperature. Oligonucleotides were purified with reverse-phase high-performance liquid chromatography (RP-HPLC; Agilent) with a C18 or C4 (for dye-labeled DNA) column, and peaks were collected as fractions. Dimethoxytrityl (DMT) groups were removed from ODNs with a 1 hour, room temperature incubation in 20% aqueous acetic acid followed by 3 washes with ethyl acetate. Olionucleotides were characterized by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF; Broker RapiFlex) mass spectrometry using 2’, 4’, -dihydroxyacetophenone (Thermo Scientific). DNA concentrations were calculated using the Beer-Lambert law where solution absorbance at A = 260 or 591 nm (for unlabeled and Cyanine 3.5 labeled strands, respectively) was determined with UV-vis spectroscopy (Cary 60 or Cary 5000 UV-vis spectrophotometers; Agilent) and extinction coefficients calculated using the OligoAnalyer tool (Integrated DNA Technologies).

[0106] Amino-terminated DNA strands were conjugated to dibenzocylooctyne-N- hydroxysuccinimide (DBCO-NHS; Lumiprobe) by mixing purified DNA with 30:1 equivalents of DBCO-NHS:DNA in a 50% dimethylformamide (DMF; v/v) solution with 0.1 M NaHCO3 and allowing reactions to proceed overnight at room temperature on an end-over-end rotator. To purify conjugates, 1/1 Oth volume of 1 M MgCI2 was added to the reactions, followed by a 10- fold dilution with 90% EtOH. Resultant solutions were incubated at -30 °C for >1 hour, centrifuged, and decanted. Precipitates were redissolved in water and further purified with three ethyl acetate washes to remove unreacted DBCO-NHS. Confirmation of DBCO-DNA functionalization was confirmed using MALDI-TOF MS as described above. [0107] Dendrimer synthesis and purification. Polymeric dendrimer cores [2,2- bis(hydroxymethyl)propionic acid (bis-MPA) azide, trimethylol propane (TMP) core, generation 1 (G1 ;6 branches)] were purchased from Polymer Factory (Stockholm, Sweden) and diluted to a concentration of 50 mM in DMF. A 2:1 stoichiometric quantity of purified DBCO-DNA:G1 - dendrimer azide was lyophilized and resuspended in a solution containing 0.5 M NaCI and 2 mM DDAB (didodecyldimethylammonium bromide) and mixed with an equivalent volume of Bis- MPA Azide TMP Core, G1 polymer in DMF, achieving a final [DNA] of 0.5 mM and [Bis-MPA Azide TMP Core, G1] of approximately 42 pM. Reactions were incubated at 37 °C and 400 RPM for 48-72 hours. Subsequently, reactions were diluted 10-fold with diH2O and concentrated using Amicon ultra-15 centrifugal filter (3 kDa molecular weight cut-off; Millipore) according to manufacturer’s instructions. In these same filters, products were washed thrice with 15 mL diH2O to remove excess DMF, salt, and detergent. Approximately 200 pL of reaction solution was mixed with an equivalent volume of 8 M urea in 2x Tris-Borate-EDTA (TBE) buffer and purified with 6% 4M urea denaturing polyacrylamide gel electrophoresis (PAGE). Gels were run for 30 min at 175 V followed by ~2 hours at 250 V and imaged with a UV lamp to excise desire bands. Gel bands were mechanically homogenized, resuspended in 5-10 mL of a 0.2% sodium dodecyl sulfate (SDS; Invitrogen), 1 mM ETDA, and incubated at 37 °C and 250 RPM for 48 hours. Extractions were filtered through 70 pm cell strainer (Fisher Scientific), concentrated and washed 3x with diH2O using 3 kDa MWCO spin filters (Millipore), and filtered through a 0.2 pm syringe filter (25 mm GD/X; Whatman). Product identities were confirmed with PAGE and MALDI-TOF MS and DNA concentration determined by UV-vis spectroscopy.

[0108] Dendrimer characterization.

[0109] AFM. DNA dendrons were directly dropped casted onto a freshly cleaved mica disk and incubated for 20 min at room temperature. AFM images were captured using PeakForce Tapping mode in fluid, on a Broker Bioscope Resolve AFM equipped with PeakForce-Hirs-F-B (Broker). The effective imaging force was below 100 pN to minimize sample damage and probe manipulation. The images were flattened using NanoScope Analysis (Broker) to remove tilt and bow.

[0110] DLS and zeta potential. Dynamic light scattering and zeta potential measurements were performed using a Malvern Zetasizer Nano by diluting DNA dendrimers to a final concentration of approximately 10 nM.

[0111] Polyacrylamide gel electrophoresis. DNA Dendrimers were characterized with native and denaturing PAGE gels. Native precast gels (4-20% Mini-PROTEAN TGX Stain-Free gel) were purchased from BioRad. Denaturing gels were prepared using a stock of 40% polyacrylamide/Bis Solution 19:1 (BioRad), 10x TBE (Fisher Scientific), urea (4 M final concentration; Sigma Adrich), and diH2O. The percentage of denaturing gels in this study is 6% unless otherwise noted.

[0112] Wells were typically loaded with 20-50 pmoles of DNA and, for label-free DNA, stained with SYBR Gold (Thermo Scientific). In each case, 1 pL of Tracklt 100 bp DNA Ladder (Thermo Scientific) was used as a reference. Gels were imaged using a ChemiDoc Gel Scanner (BioRad) using the SYBR Gold (for label-free DNA) or Cy3 (for Cy3.5-labeled DNA) channels.

[0113] Stability and nuclease-resistance. Dendrimer stability in aqueous solution was evaluating by incubating 2 pM of pre-hybridized Cy3.5-labeled DNA dendrimers (by DNA concentration) or Cy3.5-labeled ISD25 in 1 x PBS for 2 weeks at 37 °C, taking aliquots of the solutions at time = 0, 1 , and 2 weeks. Aliquots were added at a 1 :1 ratio by volume to 8 M urea in 2x TBE, flash frozen in liquid N2, and stored at -30 °C until the completion of the experiment. 20 pmoles of each aliquot was loaded on a 6% 4 M urea denaturing gel, ran at approximately 120 V for 60 minutes, and imaged using the Cy3 channel of the ChemiDoc Gel Scanner (BioRad). Densitometry analysis was performed to determine the extent of degradation over time.

[0114] Dendrimer stability in the presence of nucleases was determined by incubating 0.83 pM of pre-hybridized Cy3.5-labeled DNA dendrimers (by DNA concentration) or Cy3.5-labeled ISD25 in a 1x PBS solution with 200 U/mL of recombinant, RNAse-free DNase I (New England Biolabs) for 24 hours at 37 °C. Aliquots collected at time = 0, 0.5, 1 , 2, 4, and 24 hours were added at a 1 :1 ratio by volume to 8 M urea in 2x TBE, flash frozen in liquid N2, and stored at -30 °C until the completion of the experiment. 20 pmoles of each aliquot was loaded on a 6% 4 M urea denaturing gel, run at approximately 120 V for 60 minutes, and imaged using the Cy3 channel of the ChemiDoc Gel Scanner (BioRad). Densitometry analysis was performed to determine the extent of degradation over time.

[0115] Template-mediated chemical ligation. To generate DNA dendrimers with branch strands longer than 25 bp, we performed template-mediated chemical ligation of G1-ISD25-6 cores to ISD20-PO4 ligands. Briefly, pre-synthesized G1-ISD25-6, clSD45, and ISD20-PO4 were mixed at various stoichiometric ratios (1 :0:0, 1 :1 :1 , or 1 :1 :2) in the presence of 10 mM MgCI2 and slow cooled from 95 - 4 °C over the course of 2 hours. Reactions were initiated by adding 250 mM of 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; Thermo Scientific) and incubating solutions for 16 hours at 37 °C. Linear ISD45 (L) controls were produced under identical conditions.

[0116] Protein expression and purification. The respective plasmids for protein expression were synthesized by cloning the gene for mcGAS (Table 4, Integrated DNA Technologies) into the pET28a vector backbone using Gibson Assembly. Each plasmid was then transformed into BL21 (DE3) cells (Thermo Fisher) via electroporation. After recovery in S.O.C. medium (Thermo Fisher) for 1 hour at 37 °C with 300 rpm shaking, cells were grown overnight on LB agarose plates containing 50 pg/mL kanamycin. Single colonies were then selected and cultured in 8 mL of LB broth with 50 pg/mL kanamycin overnight at 37 °C with 200 rpm shaking. Glycerol stocks of the cells were prepared and stored at -80 °C for future use. Plasmids were then extracted from the respective cultures using the QIAprep Spin Miniprep Kit (Qiagen) and the plasmid sequences were confirmed via Sanger sequencing using T7 universal primers (ACGT).

[0117] Table 4: murine cGAS amino acid sequence

[0118] Protein expression was initiated by overnight culture of E. coli containing the respective plasmids for mcGAS using an 8 mL LB broth with 50 pg/mL kanamycin at 37 °C with 200 rpm shaking. Afterwards, these cultures were added to 750 mL of 2x YTP broth containing 50 pg/mL kanamycin (Thermo Fisher) and grown at 37 °C with 200 rpm shaking until the OD of the culture at 600 nm was at 1 .1 (~ 3.5 h). The cultures were then induced with 1 mM isopropyl p-d-1 - thiogalactopyranoside (IPTG, Thermo Fisher). After induction, the cultures were then shaken overnight at 18 °C at 200 rpm.

[0119] Subsequently, the cells were pelleted (6,000 g, 25 min, 4 °C, Avanti JXN-30 Ultra Centrifuge from Beckman Coulter), resuspended in buffer A [20 mM Tris-HCI (pH 7.6, 1 M NaCI)], and lysed via homogenization (Emulsiflex-C5 High Pressure Homogenizer, Avestin). The insoluble fraction was then removed with centrifugation (15,000 g, 25 min, 4 °C, Avanti JXN-30 Ultra Centrifuge from Beckman Coulter). The supernatant was then loaded onto a nickel affinity column (EconoFit Nuvia IMAC Column, 5 ml_ Ni-charged, Bio-Rad) for purification of the His6 tagged proteins via the Bio-Rad NGC Quest 10 Plus Chromatography System (Bio-Rad FPLC). The column was then washed with 150 mL of buffer A followed by 150 mL of 2% buffer B [20 mM Tris-HCI (pH 7.6, 1 M NaCI, 0.5 M imidazole)]. His6 tagged proteins were then eluted with 15 mL of 100% buffer B. Afterwards, imidazole was removed from the purified proteins by buffer exchanging via centrifugation with a 30 kDa molecular weight cut-off filter (Amicon Ultra- 15 30 kDa MWCO Centrifugal Filter Units, Sigma) to storage buffer [20 mM Tris-HCI (pH 7.6, 0.1 M NaCI)]. Protein purity was confirmed via SDS-PAGE and MALDI-TOF MS. Protein concentration was determined using a BCA protein assay (Thermo Fisher) and the purified proteins were stored in aliquots at -80 °C.

[0120] High-throughput cGAMP assay. Transcreener cGAMP cGAS Fluorescence Polarization (FP) assays were purchased from BellBrook Labs (3024) along with recombinant human cGAS enzyme (2227/2228). All assays were performed in accordance with the manufacturer’s protocol. Final concentrations of reaction components were as follows unless otherwise described: 100 pM of ATP, 100 pM GTP, and 5 mM MgCL in a 100 mM Tris pH 7.4 buffer. Typical [DNA] ranged from 1000 to 1.95 nM, while [cGAS] remained at 30 (Fig. 3C, 3B) or 60 (Fig. 3E) nM. Reactions were incubated at 37 °C for 1 hour and then quenched with the provided Stop and Detect Buffer for 1 hour at room temperature. FP was measured by a Tecan Infinite M1000 Microplate reader per manufacturer’s instructions. All experiments were performed in technical triplicate.

[0121] Electrophoretic mobility shift assay. Pre-hybridized Cy3.5-labeled ISD25, ISD45, G1 -ISD35-6 and G1 -ISD35-5 were prepared at a concentration of 200 nM by DNA in a 1x PBS solution with 0, 1 , 10, 20, and 40 (0, 0.2, 2, 4, or 8 nM, respectively) equivalents of recombinant hcGAS (BellBrook Labs; 2227/2228). Reactions were incubated at 37 °C for 30 minutes, mixed with 6x MassRuler DNA Loading Dye (Thermo Scientific), and ran on a 4-20% Mini-PROTEAN TGX Stain-Free gel (BioRad) at 120 V for approximately 1 hour.

[0122] Isothermal titration calorimetry. The titration of DNA (ISD25 or G1 -ISD25-6 at 60 pM) to mcGAS (20 pM in the reaction cell) was performed with the TA Instruments Affinity ITC in 1 x PBS using the following parameters: 25 °C, 1 x 0.4 pL + 13 x 3 pL injections, 100 rpm stirring.

[0123] Molecular condensate formation confocal microscopy. Briefly, purified murine cGAS was dye-labeled using a commercially available kit (Thermo A30006) per the manufacturer’s protocol. After labeling, the molecular weight and purity of mcGAS was confirmed by SDS-PAGE and its ability to bind DNA with an EMSA.

[0124] Immediately prior to imaging, solutions containing 10 pM of Cy3.5-labeled DNA (ISD25 or G1 -ISD25-6) with 5 pM A488-mcGAS in a 1x PBS solution with 1 mg/mL of BSA. 100 pL of each mixture was plated in a Lab-Tek coverslip bottomed 8-well chamber slide. Confocal microscopy was performed using a Zeiss LSM800 with the Airyscan module. Image analysis was performed using Imaged.

[0125] Raw lucia reporter cell line experiments. Raw-Lucia ISD cells (InvivoGen) were seeded in a black, clear bottom 96-well plate at a density of 1 .0x10⁵ cells/well and returned to the incubator for at least 30 minutes prior to treatment. Cells were subsequently treated with serial dilutions of Aduro S100, ISD45 (L), G1 -ISD45-6 (N) and G1-ISD45 (L) in 1 x PBS to achieve final concentrations of 160 - 0 nM in technical triplicate. Cells were incubated for 24 hours, at which point, 20 pL of supernatant from each well was transferred to a white, U-bottom 96-well plate. Luciferase luminescence was measured using a BioTek Cytation 5 Multimode Reader with a Dual Reagent Injector Module (Agilent) using QUANTI-Luc reagent (InvivoGen) according to the manufacturer’s protocol.

[0126] Cell viability was measured by adding 20 pL of PrestoBlue HS Cell Viability Reagent (Thermo Scientific) to the black, clear bottom 96-well plate, incubating the cells for 10 minutes, and measuring the fluorescence (Aem/ex = 560/590 nm) with a BioTek Cytation 5 Multimode Reader.

[0127] Flow cytometry. Fluorescent antibodies were purchased from ThermoFisher Scientific (L34962) and BioLegend (117329; 104706; 105028). Fixation Buffer was purchased from BioLegend (420801 ). Flow Cytometry was performed on a BD FACS Symphony A3.

[0128] BMDCs were isolated from the femur and tibia of a C57BL/6 mouse and cultivated in RPMI media supplemented with 40 ng/mL of Granulocyte-macrophage colony-stimulating factor. After 3 days, a further 8 mL of media was added to maintain cell health. Between days 5 and 8 post-extraction, BMDCs were counted and added to 1 .2 mL flow tubes at 1 .25x(10)6 cells/mL in 80 pL (100,000 or 200,000 cells/treatment) and were allowed to recover for approximately 1 h. Immortalized DC2.4 cells were plated under identical conditions using DMEM media. Dendrimers were then delivered to their respective tube as a 20 pL treatment yielding the desired final concentration. After 1 , 4, or 8 hours, the cells were washed twice with 600 pL of PBS and resuspended with 200 pL of media. 24 hours after the initial treatments, 600 pL of PBS was added to each tube. Cells were then centrifuged at 1200 RPM and the supernatant was removed. Treatments were then incubated with fluorophore-conjugated antibodies (Live/Dead Fixable Blue - ThermoFisher Scientific, #L34962; BV421 anti-mouse CD11 c - BioLegend, #117329; FITC anti-mouse CD80 - BioLegend, #104706;

PerCP/Cyanine5.5 anti-mouse CD86 - BioLegend, #105028) for 15 min at 4°C. Cells were then washed with PBS, centrifuged, and resuspended in fixation buffer (Biolegend, #420801) for 30 min at 4°C. Cells were washed, centrifuged, and resuspended in PBS before being analyzed by flow cytometry. All experiments were conducted in triplicate.

Example 4

[0129] Glioblastoma (GBM) remains an incurable cancer with an overall median survival of ~15 months.¹ Among cancers, GBM is uniquely difficult to treat because of challenges including physiological barriers, its infiltrative and heterogeneous nature, and its ability to elicit local and systemic immunosuppression. Tumors are protected by the blood-brain and blood-tumor barriers (BBB and BTB, respectively) which exclude systemically administered therapeutics.² GBM invades surrounding parenchyma, spreading to the brainstem in 82% of patients, causing recurrence at locations distant from the primary tumor.³ Between patients and individual tumors, GBM is characterized by heterogeneity in transcriptional and phenotypic states, rendering monotherapies ineffective because heterogeneous tumors respond differentially to targeted therapies and can transition into resistant populations.^{4 5} These challenges offer rationale for pursuing novel immunotherapeutic strategies to generate broad parenchymal immunosurveillance against heterogenous and transcriptionally plastic tumors as infiltrating cells can access disparate portions of the brain and mount immune responses against dynamic tumor phenotypes.⁶

[0130] The efficacy of first-generation immunotherapies is largely restricted to tumors that are classified as immunogenically ‘hot,’ exhibiting high mutational burdens and extensive tumor infiltrating lymphocytes (TILs).⁷ Comparatively, immunogenically ‘cold’ tumors like GBM foster a tumor microenvironment (TME) that is largely devoid of the pro-inflammatory cytokines and TILs that are necessary to mount potent anti-tumor immune responses.^{8, 9} In GBM, the TME is comprised of exhausted and inactivated immune effector cells (i.e., T and Natural Killer [NK] cells) and immunosuppressive myeloid cells (e.g., myeloid-derived suppressor cells [MDSCs]) that generate chemo- and cytokines which cause immune dysfunction.^9-12 Thus, there is a need to develop the next generation of immunotherapies that function to modulate a broader, myeloid-focused population of immune cells with the capacity to reprogram ‘cold’ TMEs. [0131] The delivery of therapeutic DNA, such as sequences that activate cGAS, is hindered by nuclease degradation, poor bioavailability, and poor cell uptake^{29 30}. To address these limitations, nanotechnologies including spherical nucleic acids (SNAs), wherein oligonucleotides are densely and radially functionalized on the surface of a nanoparticle core, have emerged as promising constructs.^31-33 SNAs exhibit architecture-dependent properties that address the above-mentioned limitations; the ODN corona confers resistance to degradation by serum nucleases³⁴ and mediates rapid Scavenger receptor-A (SR-A) endocytosis.³⁵ In a Phase 0 clinical trial of Bcl2L12 siRNA-loaded gold-SNAs in recurrent GBM (NCT03020017)^{36 37}, SNAs crossed the BBB/BTB and accumulated within tumor cells, tumor-associated macrophages (TAMs), and endothelium. Other SNA formulations have also been evaluated to be safe in Phase I (NCT03086278) and Phase Ib/ll (NCT03684785) clinical trials. However, these SNAs cannot be synthesized with precise architectural modifications to engage cGAS more potently and contain bio-incompatible gold- or relatively unstable liposomal cores. The instant application provides oligonucleotide dendrimers as a molecularly precise subclass of SNAs that recapitulate enhanced cellular uptake and nuclease resistance.^{381 39}

[0132] Oligonucleotide dendrimer structures and routes of administration could affect biodistribution (BD) pharmacokinetics (PK). Based on optimal stability, cellular uptake, and cGAS activation profiles, an oligonucleotide dendrimer library will be narrowed to 3 lead candidates composed of a single polymer chemistry (PEG, PE, or PAMAM) and a single oligonucleotide branch length (25, 45, or 65 nucleotides in length) with variable valency. Based on these results, a lead structure will be identified. Oligonucleotide dendrimers with high valency are hypothesized to exhibit broad BD across organs and lengthy serum half-lives (t1/2). Secondary to improved BD and PK, oligonucleotide dendrimers are hypothesized to be robustly endocytosed by myeloid cells, thereby activating cGAS-STING and reprogramming the TME to a pro-inflammatory state that fosters anti-tumor immunity to extend survival in mouse models.

[0133] To determine an optimal route of administration (RoA), Cyanine dye-labeled oligonucleotide dendrimers will be administered intravenously (IV) or intratumorally (IT) to orthotopically injected CT2A bearing C57BL/6 mice, an established syngeneic model of murine glioma. At 1 -, 2-, 4-, 8-, 12-, and 24-hours post-injection, mice will be imaged using an in vivo imaging system (I VIS) to quantitatively measure BD. Fluorophore concentration in plasma samples acquired through serial submandibular venipuncture will inform PK profiles including t¹/2, maximum plasma concentration (Cmax), and area under the concentration-time curve (AUG).49 A lead RoA for use in the remainder of Aim 2 will be determined based on PK profiles (longest t¹/2, highest Cmax and AUG) and maximal CNS BD.

[0134] Next, C57BU6-wt and C57BL/6-cGAS-/- (null cGAS phenotype available from The Jackson Laboratory) mice aged 6-8 weeks will be inoculated orthotopically with CT2A expressing luciferase (CT2A-Luc) for non-invasive bioluminescence monitoring of tumor establishment and growth. Upon confirmation of progressive tumor growth using bioluminescence imaging (approx. 7 days after implantation), mice will be treated with a single injection of 6 nmoles (by 20 bp increments of dsDNA) of oligonucleotide dendrimers using the lead RoA. After 1 week, single-cell RNA sequencing (scRNA-Seq) analysis will be performed on tumor immune cells/whole tumor to analyze the immune cell populations present in the TME; infiltrating ratios of CD4/CD8+ T cells, pro- and anti-inflammatory TAMs, and the presence of infiltrating Tregs, DCs, and NK cells will be quantified. Seurat cluster identification based on datasets available through the Brain Immune Atlas^50-53 will be used to identify immune cell populations and their differential gene signatures between treatment groups, particularly those downstream of cGAS-STING activation including NF-kB, IRF, and STAT6. These data will be validated with flow cytometry to quantify populations including T cells (CD3+, CD4+, CD8+, CD69+/-), TAMs (CD11 b+, CD45+, Ly6C-), NK cells (CD45+, NK1 .1 +), MDSCs (CD11 b+, CD45+, Ly6C+, Ly6G+/-), microglia (CD45low, CD11 b+), and DCs (CD11c+, MHC II+, CD86+/- ). These experiments will inform the extent to which oligonucleotide dendrimers repolarize GBM TMEs through a cGAS-dependent mechanism. A single oligonucleotide dendrimer that exhibits the most favorable immune, BD, and PK profiles will be used in the final survival experiment. For all experiments, the STING agonist AduroSI 00 and linear dsDNA will be used as positive controls and single stranded oligonucleotide dendrimers and linear ssDNA as negative controls.

[0135] Two GBM mouse models will be utilized to determine the impact of lead oligonucleotide dendrimer administration in survival and tumor challenge experiments. A more thorough analysis of efficacy using the orthotopic wild type CT2A model will be performed with C57BL/6 mice, followed by sequential administration of oligonucleotide dendrimer using the lead RoA. Related experiments will determine the efficacy of treatment in a genetically engineered immune checkpoint inhibitor (ICI)-refractory QPP4 (Nestin-CreERT2, QkiUL, Trp53L/L, PtenUL) tumor neurosphere model in C57BL/6 mice following orthotopic implantation. These tumors harbor deletions of the tumor suppressor genes Quaking (QKI; a STAR-family RNA binding protein)⁵⁴, Phosphatase and Tensin homolog (PTEN) and Tumor Protein p53 (TP53) and closely resemble the histopathologic and transcriptomic profiles of human GBM while additionally demonstrating resistance to treatment with both aCTLA-4 and aPD-1 antibodies.⁵⁵ Long-term survivors of both models will be rechallenged and monitored for survival/tumor rejection.

[0136] It is expected that intratumoral administration will result in the highest proportion of oligonucleotide dendrimers reaching the CNS, and will exhibit the longest serum t¹/z.

Oligonucleotide dendrimers exhibiting potent cGAS activation are expected to result in the most favorable immune responses in vivo, dependent on host expression of cGAS. With respect to oligonucleotide dendrimer-induced tumor microenvironment immunomodulation, it is expected that there will be an increase in populations of inflammatory myeloid cells, infiltrating NK and CD8+ T cells, and a reduction in Tregs and MDSCs. Accordingly, upregulation of gene signatures associated with interferon production (e.g., Tbk1 , Irf3, Ifnbl , Ifnal), anti-tumor macrophage polarization (e.g., Mstl r, Mx1 ), T cell co-stimulation (e.g., Icam, Ncam, Icosl , CD28), immune cell migration (e.g., Ccl3, Cell 4, Cxc1 12, Csf1 ), and NK cell cytotoxicity (e.g., Ncr1 , Klrd 1 , CD247, and Prf 1 ) are expected. It is further expected that there will be concomitant downregulation of genes associated with pro-tumor macrophage polarization (e.g., Arg1 , Arg2, Msr1 , IL-10, CCI17, CCI22) and the suppression of effector T cell functions (e.g., Pdcdl , FoxP3, Vegfa, Hif 1 a). It is possible that transfecting CT2A cells with luciferase results in increased TME inflammation. Identical experiments could be performed with a wild-type cell line, using magnetic resonance imaging to verify tumor establishment, with mice assigned to the various study arms. The principal obstacles for achieving the aforementioned goals are twofold: 1 ) the lead oligonucleotide dendrimer may over stimulate cGAS-STING, resulting in encephalitis and T cell exhaustion, or 2) oligonucleotide dendrimers may inadequately simulate the pathway, leading to insufficient anti-tumor immunity. If treatment is too potent, a dose scheduling and titration experiment can elucidate the therapeutic index of the molecules to ameliorate side effects. On the other hand, if no structure results in potent in vivo immunity, lead candidates can be modified to concomitantly activate cGAS and inhibit STAT3, a potent driver of GBM immunosuppression.⁵⁶’ ⁵⁷ STAT3 inhibition can be achieved by including decoy oligonucleotide sequences that sequester STAT3 in the cytosol of cells, preventing its downstream transcription factor activity. Future experiments will identify changes in TME cytokine levels (e.g., IFNa, p, and y, TNFa, IL-10, IL-6, CXCL10, etc.) and determine if pre-treatment of QPP4 models with MADONs can sensitize tumors to combination therapy immune checkpoint inhibitors.

Example 5 [0137] Synthesizing and characterizing a PAMAM core oligonucleotide dendrimer. PAMAM (Dendritech) terminated in primary amines will first be functionalized with terminal azides by reacting dendrimers with 10-50 equivalents of NHS-Azide in methanol, generating azide-term inated PAMAM dendrimers. Reactions between DBCO-terminated ISD25 DNA (see Table 1 ) and azide-terminated G1 -PAMAM dendrimers (8 functional units per generation), under conditions identical to those described in the application, are expected to produce G1 (PAMAM)-ISD25-8 dendrimers with yields similar to those disclosed in the instant application (i.e. , about 35%). In fact, it is expected that a significantly higher yield will be achieved due to electrostatic interactions between the negatively charged DNA phosphate backbone and the positively charged PAMAM polymer. G1 (PAMAM)- ISD25-8 dendrimers will be characterized using a combination of polyacrylamide gel electrophoresis and MALDI-ToF MS to quantitatively determine their mass and purity.

[0138] Synthesizing and purifying an oligonucleotide dendrimer. Oligonucleotide ligands were activated by reacting amine-terminated oligonucleotides 0.1-2 mM) with NHS-DBCO linkers (2-100 mM) in a 50% v/v DMF solution containing 100 mM NaHCO3 at 25 °C for 4-72 hours, thereby forming an activated oligonucleotide ligand. Activated oligonucleotides were then purified from the bulk solutions via sequential ethanol precipitation and ethyl acetate extraction. Oligonucleotide dendrimers were synthesized by reacting the DBCO groups of the activated oligonucleotide ligands at 0.3-1.2 mM with azide-terminated polymeric molecular cores at 0.025- 0.1 mM in a 25-80% v/v DMF solution for 16 - 72 hours, thereby forming an oligonucleotide dendrimer. Oligonucleotide dendrimers were purified with a first protocol, by reverse-phase high-performance liquid chromatography (RP-HPLC) using an aqueous phase comprising 3- 10% acetonitrile (AON) and 50-100 mM triethylammonium acetate (TEAA) and an organic phase of 100% ACN. Oligonucleotide dendrimers were also purified with a second protocol, by denaturing polyacrylamide gel electrophoresis with polyacrylamide concentrations ranging between 4 and 15% and urea concentrations between 1 and 8M. Oligonucleotide dendrimers were also purified using a combination of the first protocol and the second protocol.

[0139] Covalent elongation of the ligands of an oligonucleotide dendrimer. The first oligonucleotide of an oligonucleotide dendrimer was hybridized to a second oligonucleotide containing at least 3 overhanging nucleotides, which yielded an at least partially double stranded oligonucleotide dendrimer template. The oligonucleotide dendrimer was further hybridized to a third oligonucleotide containing a 3’ phosphate functional group, thereby creating an at least partially double stranded oligonucleotide dendrimer. To ligate the 5’ hydroxyl functional group on the oligonucleotide dendrimer and the 3’ phosphate functional group on the third oligonucleotide, the dendrimers were catalyzed in solutions consisting of 250 mM 1 -ethyl- 3-(3-dimethylaminopropyl)carbodiimide (EDC), 10 mM MgCl2, and 100 mM 2-(N- morpholino)ethanesulfonic acid (MES) for 16 - 24 hours at 25 °C, thereby forming oligonucleotide dendrimers with elongated oligonucleotide branches.

Example 6

[0140] Disclosed herein are materials and methods to synthesize and characterize oligonucleotide dendrimers with pendant oligonucleotide sequences that modulate the function of therapeutically relevant biological targets.

[0141] Relevant Biological Targets. (1 ) cGAS: Oligonucleotide dendrimers will be formulated with oligonucleotides, with the sequences laid out in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and/or SEQ ID NO: 5 with a phosphorothioate backbone. Interferon stimulatory DNA with phosphorothioate backbone chemistry is known to bind and inhibit cGAS. We expect cGAS inhibition to reduce pro-inflammatory immune signaling in autoimmune conditions. (2) Toll Like-Receptors (TLRs): oligonucleotide dendrimers dendrimers formulated with sequences known to bind TLRs (3, 7, and 9) will result in altered immunological signaling through these pathways. Inhibition of TLRs will reduce pro-inflammatory signaling cascades in autoimmune conditions while activation of TLRs will enhance anti-tumor immune responses in cancer. Exemplary inhibitory sequences include, but are not limited to, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. An exemplary agonist sequence includes, but is not limited to, SEQ ID NO: 20 (3) Signal Transducer and Activator of Transcription 3 (STAT3): oligonucleotide dendrimers are formulated with sequences known to bind and sequester STAT3, preventing its immunological signaling through this pathway. STAT3 inhibition will reduce signaling through anti-inflammatory pathways to improve anti-tumor immunity. An exemplary STAT3 inhibitory sequence includes, but is not limited to, SEQ ID NO: 21 . (4) NLRP3: Oligonucleotide dendrimers will be formulated with sequences that bind sensor enzymes (AIM2 or IFI16) upstream of the NLRP3 assembly, modulating the nucleation and polymerization of NLRP3 inflammasomes that initiate pro-inflammatory signaling cascades. Activating AIM2 or IFI16 is expected to enhance NLRP3 signaling, thereby resulting in potent anti-tumor immunity in cancer models. Alternatively, inhibiting the activity of AIM2 or IFI16 is expected to blunt immune responses for the treatment of autoimmune conditions. An exemplary AIM2 inhibitory sequence includes, but is not limited to, SEQ ID NO: 22. An exemplary AIM2 agonist sequence includes, but is not limited to, SEQ ID NO: 23. [0142] Materials and Methods. The synthesis, purification, and characterization of all DNA dendrimers will be performed exactly as described in this application. Solid phase chemical ODN synthesis will be used to generate DNA strands with 3' terminal amine moieties. DNA ligands will be activated by reacting their pendant amines with NHS-DBCO small molecules, generating DBCO-DNA ligands. These activated ligands react with polymeric dendrimer cores to form DNA dendrimers. These are subsequently purified with polyacrylamide gel electrophoresis or high performance liquid chromatography, and characterized using a combination of AGM, DLS, Zeta Potential, analytical PAGE, and MALDI-MS.

[0143] Initial studies will characterize the ability of DNA dendrimers to bind to their target. Recombinantly expressed and purified protein (cGAS, TLR3, TLR7, TLR9, AIM2, or IFI16) will be used to determine DNA dendrimer binding affinity with both an electrophoretic mobility shift assay (EMSA) and isothermal titration calorimetry (ITC). For cGAS, AIM2, and IFI16 targets, we will determine the extent to which DNA dendrimers facilitate molecular condensation or cooperative assembly using timelapse confocal microscopy. Briefly, dye labeled DNA dendrimers and dye labeled recombinant protein will be mixed at various concentrations and the formation of condensates and assemblies quantified over time by colocalization analysis. Subsequently, the ability of DNA dendrimers to activate or inhibit their target of interest will be performed in cell culture using reporter cell lines with luminometric readouts for downstream transcriptional activity (RAW Lucia Macrophages or THP-1 Dual cells for cGAS experiments, THP1 -Dual hTLR3, 7, and 9 for TLR experiments) or genetically manipulated immortalized cell lines (J774A.1 for NLRP3 studies) with coincident qPCR and Western blot analysis to quantify downstream pathway readouts.

[0144] Finally, the extent of DNA dendrimer uptake and immune cell activation will be quantified using flow cytometry analysis on bone marrow derived dendritic cells (BMDCs) treated identically to what is described in this application.

[0145] Expected Results. We expect the results of these experiments to share similar trends across different biological targets. DNA dendrimers are expected to be synthesized in high yield and purified as monodisperse therapeutics. We expect that all DNA dendrimers will bind comparatively more tightly and to more total numbers of enzyme targets compared to linear DNA controls as analyzed by EMSA and ITC. We expect DNA dendrimers to result in the nucleation and growth of molecular condensates and biological assemblies much more rapidly and potently than linear DNA controls. We expect in each case that DNA dendrimers potently and specifically activate or inhibit target enzyme function in the cell lines described above. We expect that each of these constructs will exhibit rapid cellular uptake in BDMCs and (depending on the formulation) lead to potent immune activation or inhibition.

[0146] Table 4: Sequence Listings

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Claims

WHAT IS CLAIMED IS:

1 . An oligonucleotide dendrimer comprising a molecular core covalently linked to one or more first oligonucleotide branches, wherein the molecular core is a polyethylene glycol (PEG) core, a polyester (PE) core, or a polyaminoamine (PAMAM) core.

2. The oligonucleotide dendrimer of claim 1 , wherein at least one of the one or more first oligonucleotide branches comprises a sequence that activates or inhibits a toll-like receptor (TLR), the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, a NOD-like receptor (NLR), pyrin domain-containing 3 (NLRP3), signal transducer and activator of transcription 3 (STAT3), or a combination thereof.

3. The oligonucleotide dendrimer of claim 2, wherein the sequence that inhibits a TLR comprises or consists of a sequence as set out in SEQ ID NO: 16.

4. The oligonucleotide dendrimer of claim 2 or claim 3, wherein the sequence that inhibits a TLR comprises or consists of a sequence as set out in SEQ ID NO: 17.

5. The oligonucleotide dendrimer of any one of claims 2-4, wherein the sequence that inhibits a TLR comprises or consists of a sequence as set out in SEQ ID NO: 18.

6. The oligonucleotide dendrimer of any one of claims 2-5, wherein the sequence that inhibits a TLR comprises or consists of a sequence as set out in SEQ ID NO: 19.

7. The oligonucleotide dendrimer of any one of claims 2-6, wherein the sequence that activates a TLR comprises or consists of a sequence as set out in SEQ ID NO: 20.

8. The oligonucleotide dendrimer of any one of claims 2-7, wherein the sequence that inhibits STAT3 comprises or consists of a sequence as set out in SEQ ID NO: 21 .

9. The oligonucleotide dendrimer of any one of claims 2-8, wherein the sequence that inhibits AIM2 comprises or consists of a sequence as set out in SEQ ID NO: 22.

10. The oligonucleotide dendrimer of any one of claims 2-9, wherein the sequence that activates AIM2 comprises or consists of a sequence as set out in SEQ ID NO: 23.

11 . The oligonucleotide dendrimer of any one of claims 2-10, wherein the sequence that activates DNA-PK comprises or consists of a sequence as set out in SEQ ID NO: 24.

12. The oligonucleotide dendrimer of any one of claims 2-1 1 , wherein the sequence that activates I Fl 16 comprises or consists of a sequence as set out in SEQ ID NO: 25.

13. The oligonucleotide dendrimer of any one of claims 2-12, wherein the sequence that activates RIG-I comprises or consists of a sequence as set out in SEQ ID NO: 26.

14. The oligonucleotide dendrimer of any one of claims 2-13, wherein the sequence that activates RIG-I comprises or consists of a sequence as set out in SEQ ID NO: 27.

15. The oligonucleotide dendrimer of claim 2, wherein at least one of the one or more first oligonucleotide branches comprises: i) a TLR inhibitory sequence comprising a sequence as set out in SEQ ID NO:

16. SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19; ii) a TLR agonist sequence comprising a sequence as set out in SEQ ID NO: 20; iii) a STAT3 inhibitory sequence comprising a sequence as set out in SEQ ID NO: 21 ; iv) an AIM2 inhibitory sequence comprising a sequence as set out in SEQ ID NO: 22; v) an AIM2 agonist sequence comprising a sequence as set out in SEQ ID NO: 23; vi) a DNA-PK agonist sequence comprising a sequence as set out in SEQ ID NO: 24; vii) an IFI16 agonist sequence comprising a sequence as set out in SEQ ID NO: 25; or viii) a RIG-I agonist sequence comprising a sequence as set out in SEQ ID NO:

26 or SEQ ID NO: 27.

16. The oligonucleotide dendrimer of any one of claims claim 1 -15 wherein the oligonucleotide dendrimer comprises two or more first oligonucleotide branches.

17. The oligonucleotide dendrimer of any one of claims 1 -16, wherein the oligonucleotide dendrimer comprises three or more first oligonucleotide branches.

18. The oligonucleotide dendrimer of any one of claims 1 -16, wherein the oligonucleotide dendrimer comprises four or more first oligonucleotide branches.

19. The oligonucleotide dendrimer of any one of claims 1 -16, wherein the oligonucleotide dendrimer comprises five or more first oligonucleotide branches.

20. The oligonucleotide dendrimer of any one of claims 1 -16 , wherein the oligonucleotide dendrimer comprises six or more first oligonucleotide branches.

21 . The oligonucleotide dendrimer of any one of claims 1 -16, wherein the oligonucleotide dendrimer comprises seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve or more first oligonucleotide branches.

22. The oligonucleotide dendrimer of any one of claims 1 -21 , wherein each of the one or more first oligonucleotide branches comprise the same nucleotide sequence relative to each other.

23. The oligonucleotide dendrimer of any one of claims 1 -21 , wherein at least two of the one or more first oligonucleotide branches have different nucleotide sequences relative to each other.

24. The oligonucleotide dendrimer of claim 22, wherein each of the one or more first oligonucleotide branches comprises a sequence as set out in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

25. The oligonucleotide dendrimer of any one of claims 1 -24, wherein the one or more first oligonucleotide branches comprise DNA, RNA, or a combination thereof.

26. The oligonucleotide dendrimer of any one of claims 1 -25, wherein at least one of the one or more first oligonucleotide branches further comprises a domain comprising at least three (GGX) motifs, wherein X is a nucleotide.

27. The oligonucleotide dendrimer of any one of claims 1 -26, wherein each of the one or more first oligonucleotide branches further comprises a domain comprising at least three (GGX) motifs, wherein X is a nucleotide.

28. The oligonucleotide dendrimer of any one of claims 1 -27, wherein each of the one or more first oligonucleotide branches further comprises a domain comprising five or more (GGX) motifs, wherein X is a nucleotide.

29. The oligonucleotide dendrimer of any one of claims 26-28, wherein the domain comprises five to ten (GGX) motifs, wherein X is a nucleotide.

30. The oligonucleotide dendrimer of any one of claims 26-29, wherein the nucleotide is a Thymidine.

31 . The oligonucleotide dendrimer of any one of claims 26-30, wherein the domain is located at the 5’ or 3’ end of the at least one of the one or more first oligonucleotide branches.

32. The oligonucleotide dendrimer of any one of claims 1 -31 , wherein at least one of the one or more first oligonucleotide branches is at least 20 nucleotides in length.

33. The oligonucleotide dendrimer of any one of claims 1 -32, wherein each of the one or more first oligonucleotide branches is at least 20 nucleotides in length.

34. The oligonucleotide dendrimer of claim 32 or claim 33, wherein at least one of the one or more first oligonucleotide branches is at least 45 nucleotides in length.

35. The oligonucleotide dendrimer of any one of claims 32-34, wherein each of the one or more first oligonucleotide branches is at least 45 nucleotides in length.

36. The oligonucleotide dendrimer of any one of claims 32-35, wherein at least one of the one or more first oligonucleotide branches is at least 65 nucleotides in length.

37. The oligonucleotide dendrimer of any one of claims 32-36, wherein each of the one or more first oligonucleotide branches is at least 65 nucleotides in length.

38. The oligonucleotide dendrimer of any one of claims 1 -37, wherein at least one of the one or more first oligonucleotide branches is hybridized to a second oligonucleotide, thereby creating an at least partially double stranded oligonucleotide dendrimer.

39. The oligonucleotide dendrimer of claim 38, wherein each of the one or more first oligonucleotide branches is hybridized to the second oligonucleotide, thereby creating an at least partially double stranded oligonucleotide dendrimer.

40. The oligonucleotide dendrimer of claim 38 or claim 39, wherein the second oligonucleotide is about 1 to about 90 nucleotides in length.

41 . The oligonucleotide dendrimer of any one of claims 38-40, wherein the second oligonucleotide is about 25 to about 70 nucleotides in length.

42. The oligonucleotide dendrimer of claim 41 , wherein the length of the second oligonucleotide is greater than the length of the at least one of the one or more first oligonucleotide branches to which it is hybridized.

43. The oligonucleotide dendrimer of any one of claims 38-42, wherein the second oligonucleotide comprises DNA or RNA.

44. The oligonucleotide dendrimer of any one of claims 38-43, wherein the second oligonucleotide comprises a sequence as set out in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

45. The oligonucleotide dendrimer of any one of claims 38-44, further comprising a third oligonucleotide that is hybridized to a single-stranded region of the at least partially double stranded oligonucleotide dendrimer.

46. The oligonucleotide dendrimer of claim 45, wherein the at least one of the one or more first oligonucleotide branches and the third oligonucleotide are both hybridized to the second oligonucleotide.

47. The oligonucleotide dendrimer of claim 45 or claim 46, wherein the third oligonucleotide comprises DNA or RNA.

48. The oligonucleotide dendrimer of any one of claims 45-47, wherein the third oligonucleotide comprises a sequence as set out in SEQ ID NO: 1 1 or SEQ ID NO: 12.

49. The oligonucleotide dendrimer of any one of claims 45-48, wherein the 3’ end of the third oligonucleotide is ligated to the 5' end of the first oligonucleotide branch or the 5’ end of the third oligonucleotide is ligated to the 3’ end of the first oligonucleotide branch.

50. The oligonucleotide dendrimer of any one of claims 45-49, wherein the combined length of the third oligonucleotide and the at least one of the one or more first oligonucleotide branches is greater than or equal to the length of the second oligonucleotide.

51 . The oligonucleotide dendrimer of any one of claims 45-49, wherein the combined length of the third oligonucleotide and the at least one of the one or more first oligonucleotide branches is less than the length of the second oligonucleotide.

52. The oligonucleotide dendrimer of any one of claims 38-51 , wherein the at least partially double stranded oligonucleotide dendrimer comprises an overhang.

53. The oligonucleotide dendrimer of claim 52, wherein the overhang is about 1 to about 25 nucleotides in length.

54. The oligonucleotide dendrimer of claim 52 or claim 53, wherein the overhang is about 2 to about 5 nucleotides in length.

55. The oligonucleotide dendrimer of any one of claims 1 -54, wherein the oligonucleotide dendrimer comprises one or more cGAS ligands.

56. The oligonucleotide dendrimer of any one of claims 1 -55, wherein the oligonucleotide dendrimer activates cGAS and/or cGAS-STING.

57. The oligonucleotide dendrimer of any one of claims 1 -56, wherein the oligonucleotide dendrimer further comprises an additional agent.

58. The oligonucleotide dendrimer of claim 57, wherein the additional agent is attached to at least one of the one or more first oligonucleotide branches.

59. The oligonucleotide dendrimer of claim 57 or claim 58, wherein the additional agent is attached to the second oligonucleotide.

60. The oligonucleotide dendrimer of any one of claims 57-59, wherein the additional agent is attached to the third oligonucleotide.

61 . The oligonucleotide dendrimer of any one of claims 57-60, wherein the oligonucleotide dendrimer comprises a plurality of first oligonucleotide branches and the additional agent is attached to at least two of the plurality of first oligonucleotide branches.

62. The oligonucleotide dendrimer of any one of claims 57-61 , wherein the additional agent is i) a protein, peptide, or enzyme; ii) a multivalent antibody or derivative thereof; iii) a carbohydrate or a small molecule proteolysis-targeting chimera (PROTAC); iv) a double-stranded DNA molecule; and/or v) an RNA molecule.

63. The oligonucleotide dendrimer of claim 62, wherein the multivalent antibody or derivative thereof is a nanobody, a single chain variable fragment, a fragment antigen binding domain, a bi-specific T cell engager, or a combination thereof.

64. The oligonucleotide dendrimer of claim 62 or claim 63, wherein the multivalent antibody or derivative thereof binds: i) a checkpoint blockade inhibitor protein; ii) a tumor-associated antigen; and/or iii) a blood-brain barrier (BBB) penetration protein.

65. The oligonucleotide dendrimer of claim 64, wherein the checkpoint blockade inhibitor protein is PD-1 , PD-L1 , CTLA-4, LAG-3, TIM-3, or TIGIT.

66. The oligonucleotide dendrimer of claim 64 or claim 65, wherein the tumor- associated antigen is EGFR, HER2, VEGFR, CD20, CD19, or PSMA.

67. The oligonucleotide dendrimer of any one of claims 64-66, wherein the bloodbrain barrier (BBB) penetration protein is a transferrin receptor, GLUT 1 , or a Bradykinin B2 receptor.

68. A composition comprising a plurality of the oligonucleotide dendrimers of any one of claims 1 -67.

69. A pharmaceutical formulation comprising the oligonucleotide dendrimer of any one of claims 1-67, or the composition of claim 68, and a pharmaceutically acceptable carrier, diluent, stabilizer, preservative, or adjuvant.

70. An antigenic composition comprising the oligonucleotide dendrimer of any one of claims 1 -67, the composition of claim 68, or the pharmaceutical formulation of claim 69, wherein the antigenic composition is capable of generating an immune response in a mammalian subject.

71 . A method of producing an immune response in a subject, comprising administering to the subject an effective amount of the oligonucleotide dendrimer of any one of claims 1 -67, the composition of claim 68, the pharmaceutical formulation of claim 69, or the antigenic composition of claim 70, thereby producing an immune response in the subject.

72. The method of claim 71 , wherein the immune response is a CD86+ dendritic cell- mediated response.

73. The method of claim 71 or claim 72, wherein the immune response is a T cell- mediated response.

74. The method of any one of claims 71 -73, wherein the immune response is activated via the cGAS-STING pathway.

75. The method of any one of claims 71 -74, wherein administration of the composition induces formation of one or more molecular condensates.

76. The method of any one of claims 71 -75, wherein the one or more molecular condensates have a diameter of about 5 to about 25 microns.

77. A method of treating a disease in a subject, comprising administering to the subject an effective amount of the oligonucleotide dendrimer of any one of claims 1 -67, the composition of claim 68, the pharmaceutical formulation of claim 69, or the antigenic composition of claim 70.

78. The method of claim 77, wherein the disease is cancer, an autoimmune disease, an infectious disease, or a combination thereof.

79. The method of claim 78, wherein the cancer is breast cancer, peritoneum cancer, cervical cancer, colon cancer, rectal cancer, esophageal cancer, eye cancer, liver cancer, pancreatic cancer, larynx cancer, lung cancer, skin cancer, melanoma, ovarian cancer, prostate cancer, stomach cancer, testicular cancer, thyroid cancer, brain cancer, or a combination thereof.

80. A method of making an oligonucleotide dendrimer, the method comprising contacting a molecular core comprising one or more first oligonucleotide branches with one or more second oligonucleotides, wherein the contacting results in hybridization of a first oligonucleotide branch with a portion of a second oligonucleotide, thereby resulting in an at least partially double stranded oligonucleotide dendrimer comprising an overhang region.

81 . The method of claim 80, further comprising hybridizing a third oligonucleotide to the overhang region.

82. The method of claim 80 or claim 81 , wherein (a) Na+, Mg2+, urea, and/or DMF are present during the contacting; and/or (b) the stoichiometry of oligonucleotide: molecular core is 12:1.

83. A molecular condensate comprising the oligonucleotide dendrimer of any one of claims 1 -67, or a plurality thereof, and a plurality of proteins.

84. The molecular condensate of claim 83, wherein at least one oligonucleotide dendrimer interacts with at least two proteins of the plurality of proteins, and at least one protein of the plurality of proteins binds to more than one oligonucleotide dendrimer or oligonucleotide dendrimer branch, and wherein the plurality of proteins is cGAS-STING effector proteins, pyrin domain-containing 3 (NLRP3) proteins, and/or signal transducer or activator of transcription 3 (STAT3).

85. The molecular condensate of claim 83 or claim 84, wherein the oligonucleotide binds to one or more proteins in the plurality of proteins via hydrogen bonding and/or ionic interaction.

86. The molecular condensate of any one of claims 83-85, wherein the molecular condensate has a half-life of at least 45 minutes.

87. The molecular condensate of any one of claims 83-86, wherein the plurality of proteins are cGAS-STING effector proteins that activate a cGAS-STING pathway.

88. The molecular condensate of any one of claims 83-87 having a diameter of about 5 to about 25 microns.