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WO2010060030A1 - Nanostructure ligand de tlr-acide nucléique comme nouvel agent immunomodulateur et utilisations de celui-ci - Google Patents

Nanostructure ligand de tlr-acide nucléique comme nouvel agent immunomodulateur et utilisations de celui-ci Download PDF

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WO2010060030A1
WO2010060030A1 PCT/US2009/065508 US2009065508W WO2010060030A1 WO 2010060030 A1 WO2010060030 A1 WO 2010060030A1 US 2009065508 W US2009065508 W US 2009065508W WO 2010060030 A1 WO2010060030 A1 WO 2010060030A1
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tlr
nucleic acid
aptamers
ligand
ligands
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Yung Chang
Hao Yan
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University of Arizona
Arizona's Public Universities
Arizona State University ASU
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/16—Aptamers
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/51—Physical structure in polymeric form, e.g. multimers, concatemers

Definitions

  • the present invention relates to the fields of immunology, nucleic acid-based tiling arrays, nanotechnology, and related fields.
  • TLRs Toll-like receptors
  • PAMP pathogen-associated molecular patterns
  • TLRs tend to form non-covalent dimers in the absence of ligands.
  • TLR2 preferentially forms heterodimers with either TLRl or TLR6, whereas the other TLRs appear to associate as homodimers.
  • the binding of microbial agents to the TLRs triggers a cascade of events that lead to the production of cytokines and anti-microbial peptides.
  • TLRl ten identified functional TLRs
  • TLRl can be categorized to two groups according to their subcellular localization.
  • TLRl, 2, 4, 5, 6, and 10 are expressed on the cell surface and migrate to phagosomes after activation, whereas TLR3, 7, 8 and 9 are expressed in intracellular compartments, mainly in the endosomes and the endoplasmic reticulum, with the ligand binding domain facing the lumen of the vesicle sampling microbial agents.
  • TLR2/TLR1, TLR2/TLR6, and TLR4 ligands lipids and lipopeptides
  • proteins TLR5
  • nucleic acids TLR3, 7, 8, 9
  • TLR3, TLR7, TLR8, and TLR9 are known as microbial sensors because they recognize viral and bacterial nucleic acid, i.e., dsRNA, ssRNA and unmethylated CpG ssDNA.
  • This group of TLRs resides in the endosomes, where self nucleic acids are normally excluded but through which pathogens commonly transit.
  • Unmethylated CpG-containing DNA and ssRNA have been shown to act as TLR ligands and stimulate the activities of the immune cells. (Krieg, A. M. et al. Toll-like receptors 7, 8, and 9: linking innate immunity to autoimmunity. Immunological Rev. 2007, 220:251-269, Kawai, T. et al.
  • Toll-like receptors can function as homodimers or heterodimers.
  • TLR2 forms heterodimers with TLRl and TLR6.
  • TLRs The binding of microbial ligands to TLRs triggers activation of various signal transduction pathways and ultimately leads to activation of mitogen-activated protein (MAP) kinase and nuclear translocation of NF-AB, which result in modulation of expression of cytokines and other immune regulatory molecules.
  • MAP mitogen-activated protein
  • Intracellular TLRs use different pathway(s) that leads to activation of IFN regulatory factors (IRF), and type I IFN production.
  • IRF IFN regulatory factors
  • the invention provides methods of modulating immune response in a cell comprising contacting the cell with a composition of at least one toll-like receptor (TLR) ligand bound to a nucleic acid nanostructure.
  • TLR toll-like receptor
  • the invention provides methods of modulating immune response in a mammal comprising administering to a mammal in need thereof an amount effective to modulate the immune response of a composition comprising at least one TLR ligand bound to a nucleic acid nanostructure.
  • the invention provides methods for treating cancer in a mammal comprising administering to a mammal in need thereof an amount effective to modulate the immune response of a composition comprising at least one TLR ligand bound to a nucleic acid nanostructure.
  • the composition comprises TLR3 and TLR 9 ligands.
  • the invention provides methods of treating microbial infection in a mammal comprising administering to a mammal in need thereof an amount effective to treat the microbial infection of a composition comprising at least one TLR ligand bound to a nucleic acid nanostructure.
  • the composition comprises TLR3 and TLR8 ligands.
  • the composition comprises TLR2 and TLR4 ligands or aptamers.
  • the invention provides methods of treating an autoimmune disease in a mammal comprising administering to a mammal in need thereof an amount effective to treat the autoimmune disease of a composition at least one TLR ligand bound to a nucleic acid nanostructure.
  • the composition comprises at least one TLR9 ligands.
  • the compositions for use in the above aspects are described throughout the application.
  • the invention provides compositions comprising at least one toll-like receptor (TLR) ligand bound to a nucleic acid nanostructure.
  • TLR toll-like receptor
  • the TLR ligand comprises a ligand for TLR 3, TLR7, TLR 8, or TLR 9.
  • the TLR ligand comprises unmethylated CpG- oligodeoxynucleotide, double-stranded RNA, or single-stranded RNA.
  • the ligand-nucleic acid nanostructure is between about 100 nm and about 2000 nm (2 ⁇ m) in length.
  • the TLR ligand comprises a plurality of TLR ligands for one toll-like receptor or a plurality of TLR ligands for different toll-like receptors.
  • the distance between each TLR ligand of the plurality of TLR ligands on the nucleic acid nanostructure is about 10 nm to about 20 nm. In other preferred embodiments, the distance between each TLR ligand of the plurality of TLR ligand on the nucleic acid nanostructure is about 20 nm.
  • the plurality of TLR ligands is present on the ligand-nucleic acid nanostructure at a density of 5-10 TLR ligands per nucleic acid nanostructure.
  • the plurality of TLR ligands is present on the ligand-nucleic acid nanostructure at a density of 8 TLR ligands per nucleic acid nanostructure.
  • the plurality of TLR ligands comprises TLR ligands for at least two or at least three of TLR 3, TLR7, TLR 8, and TLR 9.
  • the plurality of TLR ligands comprises TLR ligands for TLR 3, TLR7, TLR 8, and TLR 9.
  • the plurality of ligands comprises ligands for TLR7 and TLR9; in yet other preferred embodiments, the plurality of ligands comprises ligands for TLR3 and TLR8. In certain other preferred embodiments, the plurality of ligands comprises ligands for TLR3 and TLR9.
  • the TLR ligand comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or octamer of at least one TLR ligand for at least one type of toll-like receptor.
  • the TLR ligand comprises a plurality of TLR ligands for one toll-like receptor or a plurality of TLR ligands for different toll-like receptors.
  • the distance between each TLR ligand of the plurality of TLR ligands on the ligand-nucleic acid nanostructure is about 10 nm to about 20 nm.
  • the distance between each TLR ligand of the plurality of TLR ligand on the nucleic acid nanostructure is about 20 nm.
  • the plurality of TLR ligands is present on the ligand-nucleic acid nanostructure at a density of 5- 10 TLR ligands per ligand-nucleic acid nanostructure.
  • the plurality of TLR ligands is present on the ligand-nucleic acid nanostructure at a density of 8 TLR ligands per nucleic acid nanostructure.
  • the TLR ligand comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or octamer of one or more TLR ligands for at least two or at least three of TLR 3, TLR7, TLR 8, and TLR 9.
  • the TLR ligand comprises a tetramer, pentamer, hexamer, heptamer, or octamer of one or more TLR ligands for TLR 3, TLR7, TLR 8, and TLR 9.
  • the plurality of ligands comprises ligands for TLR7 and TLR9; in yet other preferred embodiments, the plurality of ligands comprises ligands for TLR3 and TLR8. In certain other preferred embodiments, the plurality of ligands comprises ligands for TLR3 and TLR9.
  • the TLR ligand comprises a ligand for TLRl, TLR2, TLR 4, TLR 5, TLR 6 or TLR 10.
  • the TLR ligand comprises an aptamer.
  • the aptamer-nucleic acid nanostructure is between about 10 nm and about 100 nm in length.
  • the aptamer comprises a plurality of aptamers for one toll-like receptor or a plurality of aptamers for different toll-like receptors.
  • the distance between each aptamer of the plurality of aptamers on the nucleic acid nanostructure is about 10 nm to about 20 nm.
  • the distance between each aptamer of the plurality of TLR aptamers on the nucleic acid nanostructure is about 20 nm. In other preferred embodiments, the plurality of aptamers is present on the nucleic acid nanostructure at a density of 5-10 aptamers per nucleic acid nanostructure. In certain preferred embodiments, the plurality of TLR aptamers is present on the aptamer -nucleic acid nanostructure at a density of 8 TLR aptamers per nucleic acid nanostructure.
  • the TLR ligand comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or octamer of at least one TLR ligand for at least one toll-like receptor.
  • the distance between each TLR ligand of the plurality of TLR ligands on the ligand-nucleic acid nanostructure is about 10 nm to about 20 nm. In other preferred embodiments, the distance between each TLR aptamer of the plurality of TLR aptamers on the nucleic acid nanostructure is about 20 nm.
  • the TLR ligand is present on the ligand-nucleic acid nanostructure at a density of 5-10 TLR ligands per ligand-nucleic acid nanostructure.
  • the plurality of TLR aptamers is present on the ligand-nucleic acid nanostructure at a density of 8 TLR aptamers per nucleic acid nanostructure.
  • the plurality of aptamers comprises aptamers for at least two, at least three, at least four, or at least five of TLRl, TLR2, TLR 4, TLR 5, TLR 6 and TLR 10.
  • the plurality of aptamers comprises aptamers for TLRl, TLR2, TLR 4, TLR 5, TLR 6 and TLR 10. In certain preferred embodiments, the plurality of aptamers comprises aptamers for TLRl and TLR2. In other preferred embodiments, the plurality of aptamers comprises aptamers for TLR2 and TLR6. In further preferred embodiments, the plurality of aptamers comprises aptamers for TLR2 and TLR4.
  • the aptamer comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or octamer of one or more aptamers for at least one toll-like receptor.
  • the aptamer comprises a plurality of aptamers for one TLR or a plurality of aptamers for different TLRs.
  • the distance between each aptamer of the plurality of aptamers on the nucleic acid nanostructure is about 10 nm to about 20 nm.
  • the distance between each TLR aptamer of the plurality of TLR aptamers on the nucleic acid nanostructure is about 20 nm. In certain other preferred embodiments, the plurality of aptamers is present on the nucleic acid nanostructure at a density of 5-10 aptamers per nucleic acid nanostructure. In certain preferred embodiments, the plurality of TLR aptamers is present on the aptamer -nucleic acid nanostructure at a density of 8 TLR aptamers per nucleic acid nanostructure.
  • the aptamer comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or octamer of aptamers for at least two, at least three, at least four, or at least five of TLRl, TLR2, TLR 4, TLR 5, TLR 6 and TLR 10.
  • the aptamers comprises a hexamer or a higher order of multimers of aptamers for TLRl, TLR2, TLR 4, TLR 5, TLR 6 and TLR 10.
  • the aptamers comprises a plurality of aptamers.
  • the plurality of aptamers comprises aptamers for TLRl and TLR2. In other preferred embodiments, the plurality of aptamers comprises aptamers for TLR2 and TLR6. In further preferred embodiments, the plurality of aptamers comprises aptamers for TLR2 and TLR4.
  • the ligand/aptamer nucleic acid nanostructures comprise at least one ligand for TLR3, TLR7, TLR8 or TLR9, and at least one aptamer for TLRl, TLR2, TLR4, TLR5, TLR6, and TLRlO.
  • the ligand/aptamer nucleic acid nanostructures comprise at least one TLR9 ligand and at least one TLR4 aptamer.
  • the TLR9 ligand and the TLR4 aptamer are bound to one nucleic acid nanostructure.
  • Certain preferred aspects of the invention further provide methods of treating microbial infection in a mammal comprising administering to a mammal in need thereof of an amount effective to treat the microbial infection of a composition comprising at least one TLR ligand bound to a nucleic acid nanostructure as described herein.
  • Other preferred aspect of the invention provides methods of treating autoimmune disease in a mammal comprising administering to a mammal in need thereof of an amount effective to treat the autoimmune disease of a composition comprising at least one TLR ligand bound to a nucleic acid nanostructure described herein.
  • the composition comprising at least one TLR2 aptamer and at least one TLR4 aptamer bound to a nucleic acid nanostructure. In further preferred embodiments, the composition comprising at least one TLR3 ligand and at least one TLR9 ligand bound to a nucleic acid nanostructure. In certain preferred embodiments, the composition comprising at least one TLR3 ligand and at least one TLR8 ligand bound to a nucleic acid nanostructure. In further preferred embodiments, the composition comprising at least one of TLR3 ligand, TLR7 ligand, TLR8 ligand, and TLR9 ligand bound to a nucleic acid nanostructure.
  • the composition comprising at least one TLR4 aptamer and at least one TLR9 ligand, wherein the TLR4 aptamer and the TLR ligand are bound to one nucleic acid nanostructure or separate nucleic acid nanostructures.
  • the composition comprising at least one TLR4 ligand bound to a first nucleic acid nanostructure and at least one TLR3 ligand and at least one TLR8 ligand bound to a second nucleic acid nanostructure.
  • the invention provides methods of making a composition as described herein comprising contacting at least one TLR ligand, and at least one polynucleotide under conditions suitable for binding of the at least one TLR ligand to the at least one polynucleotide to form a nucleic acid nanostructure, wherein the TLR ligand is bound to the nucleic acid nanostructure.
  • the at least one polynucleotide comprises a plurality of polynucleotides, and wherein the contacting is done under conditions suitable to promote hybridization of the plurality of polynucleotides into at least one nucleic acid tile.
  • the at least one nucleic acid tile comprises a plurality of nucleic acid tiles, and wherein the plurality of nucleic acid tiles forms at least one nucleic acid tiling array.
  • Figure 1 shows results that illustrate the effects of cooperative binding of multivalent aptamers or ligands to a target molecule, in this case, multivalent aptamers binding to thrombin
  • a) Two thrombin aptamers are positioned on a five-helix DNA nanostructure at optimized distance to improve the binding efficiency
  • aptamers are displayed in lines on a rectangular shaped nucleic acid tile. The dual aptamer line possesses stronger binding affinity with protein than each individual aptamer lines.
  • FIG. 2 is a schematic illustration of the immune response of an immune cell triggered by the multivalent TLR7/9 ligand-TLR binding events.
  • Left In the absence of the TLR7/9 ligand, no cytokine is produced from the cell.
  • TLR ligands for example, 2'-deoxy- or 2'-fluoro-modified ss RNA for TLR7 and unmethylated CpG-oligodeoxynucleotide (ODN) for TLR9 linked by a nucleic acid nanostructure
  • This binding event initializes a cascade of signal transduction to the nucleus, resulting in production and secretion of proinflammatory cytokines, such as ILl, monocyte chemoattractant protein-1 (MCPl)_and chemokines.
  • proinflammatory cytokines such as ILl, monocyte chemoattractant protein-1 (MCPl)_and chemokines.
  • MCPl monocyte chemoattractant protein-1
  • FIG. 3 shows a schematic diagram illustrating the effects of a TLR7 and TLR9 ligand-nucleic acid nanostructure on the activation of monocytic cell line THPl (Catalog No. TIB-202, American Type Culture Collection, Manassas, VA) and inhibition of HIV replication. Incorporation onto the nanostructure of other molecules such as HIV-specific RNAi is also indicated. The indicated nanostructures can be released from intracellular compartments such as endosomes to the cytoplasm where the RNAi inhibits HIV replication in HIV-infected CD4+ monocytic cells.
  • Figures 4A and B illustrate exemplary multimeric aptamer-nucleic acid nanostructures.
  • A Aptamer-nucleic acid nanostructures comprising multimeric aptamers of the same kind; B. Aptamer-nucleic acid nanostructures comprising multimeric aptamers of different kinds; and C. selection of receptor-binding multimeric aptamers.
  • Figure 5 shows the results of competition assays of labeled monomeric aptamers binding to a target cell by unlabeled multimeric aptamers.
  • A) A representative profile of flow cytometry histogram indicating fluorescence intensity of the cells incubated with labeled monomeric aptamer in the presence or absence of rigid dimeric aptamers.
  • Figure 6 shows a photograph of image of native gel electrophoresis of Ml dimeric aptamer incubated with or without cells at different temperatures.
  • Ml and M2 are input dimeric and monomeric DNA aptamers, respectively, as illustrated on the right.
  • the number at the bottom of each lane indicates the ratio of the recovered aptamers incubated under test conditions to those under control condition, the control condition being incubation at 4°C in the absence of cells.
  • nucleic acid means one or more nucleic acids.
  • polynucleotide As used herein, the terms "polynucleotide”, “nucleotide”, “oligonucleotide”, and
  • nucleic acid may be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivative thereof, or combination thereof.
  • binding to or “bound to” refers to any of direct binding, indirect binding, covalent binding, or non-covalent binding, unless otherwise specifically indicated.
  • the term "immune cell” refers to cells of the immune systems, including without limitation the innate and adaptive immune systems. Immune cells include, without limitation, monocytes, macrophages, dendritic cells, B lymphocytes, T lymphocytes, myeloid dendritic cells, plasmacytoid dendritic cells, and mast cells.
  • TLR toll-like receptor
  • the term "ligand” is any molecule capable of binding to a target molecule. Such ligands include, but are not limited to, proteins, lipids, carbohydrates, nucleic acids (including, but not limited to, aptamers), and other molecules.
  • the ligand binds to an intracellular toll-like receptor (such as TLR3, TLR7, TLR8, or TLR9); in other preferred embodiments, the ligand, or more preferably aptamer, binds to a toll-like receptor on the cell surface (such as TLRl, TLR2, TLR4, TLR5, TLR6, TLRlO).
  • the ligand Upon binding of the ligand to the receptor, one or more biological or physiological responses are elicited or affected.
  • the ligand is an agonist of a receptor.
  • the ligand is an antagonist of a receptor.
  • a ligand or an aptamer capable of binding, or specific for binding, to a receptor on a target cell indicates that the aptamer specifically binds to a receptor on a target cell under stringent binding conditions as commonly defined or described herein and that the binding of a ligand or an aptamer under the same binding conditions, if any, to other non-target molecules is insubstantial or undetectable as determined by methods commonly used in the immunology or ligand/receptor art.
  • Specific binding can also be determined by competition; for example, a specific binding between a labeled ligand and its receptor under stringent binding conditions can be competed by the unlabeled ligand, but not by other unlabeled irrelevant molecules.
  • the term "agonist” refers to a ligand or molecule that, when binding to its receptor, triggers the downstream signal transduction pathway of the receptor. In one preferred embodiment, the agonist binds to its receptor and triggers the downstream signal transduction pathway of the receptor. In other preferred embodiments, an agonist binds to a receptor and enhances or synergizes the downstream signal transduction pathway of another receptor. [0034] As used herein, the term “antagonist” refers to a ligand or molecule that blocks or dampens an agonist-mediated response. In one preferred embodiment, an antagonist, upon binding to its receptor, diminishes or blocks a certain biological or physiological response that normally would have been triggered by an agonistic ligand binding to the receptor. In certain other preferred embodiments, an antagonist, upon binding to its receptor, diminishes or blocks a certain biological or physiological response that normally would have been triggered by an agonistic ligand binding to another receptor.
  • the ligand is a monovalent ligand; in other preferred embodiments, the ligand is a multivalent ligand.
  • the term "monovalent ligand” refers to a ligand that has one binding site for the target molecule; the term “multivalent ligand” refers to a ligand that has more than one binding site for one or a plurality of target molecules.
  • Multivalent TLR ligand- nucleic acid nanostructure of the invention provides unexpected advantages in fine tuning and modulating immune response that are not possible by using free individual TLR ligands.
  • free or “individual” TLR ligands refer to TLR ligands not bound to a nucleic acid nanostructure.
  • individual TLR ligands either intracellular TLR ligands or cell surface TLR ligands, high doses of the ligands are needed for administering to a mammal. High doses of free TLR ligands are required to achieve sufficient local concentrations of TLR ligands that are necessary for high affinity binding with the receptor.
  • the TLR ligand/aptamer- nucleic acid nanostructures present a high concentration of TLR ligands to a cell and allow high affinity, synergistic binding of ligands to TLRs without the systemic toxicity brought about by high doses of free TLR ligands.
  • the TLR ligand/aptamer-nucleic acid nanostructures comprise a predetermined combination of TLR ligands at predetermined concentrations.
  • the cells in contact with the nucleic acid nanostructures of the invention are exposed to the predetermined combination of TLR ligands at the predetermined concentrations.
  • the ligand can be covalently or non-c ⁇ valently bound to one or more polynucleotides in the nucleic acid nanostmcturc; in other preferred embodiments, the ligand binds to a connector polynucleotide or nucleic acid probe thai is directly or indirectly bound to the one or more polynucleotides of the nucleic acid nanostmcturc
  • Non-coval ⁇ nt binding includes without limitation nucleic acid base pairing of the ligand directly with the polynucleotide of the nucleic acid nanostrueture (one that remains partially unbound after formation of the nucleic acid nanostrueture), by way of base pairing with a connector polynucleotide, or via biotin- streptavidin interaction.
  • TLRl GenBank Accession No. NM_003263, SEQ ID NOs: 1 and 2
  • TLR2 GenBank Accession No. NM_003264, SEQ ID NOs:3 and 4
  • TLR4 GenBank Accession No. NM_138554, SEQ ID NOs:5 and 6
  • TLR5 GenBank Accession No. NM_003268, SEQ ID NOs:7 and 8
  • TLR6 GenBank Accession No. NM_006068, SEQ ID NOs:9 and 10
  • TLRlO GenBank Accession No.
  • NM_030956, SEQ ID NOs: 11 and 12 are expressed on the cell surface, whereas TLR3 (GenBank Accession No. NM_003265, SEQ ID NOs: 13 and 14), TLR7 (GenBank Accession No. NM_016562, SEQ ID NOs: 15 and 16), TLR8 (GenBank Accession No. NM_138636, SEQ ID NOs: 17 and 18) and TLR9 (GenBank Accession No. NM_017442, SEQ ID NOs: 19 and 20) are expressed in intracellular compartments, mainly in the endosomes and the endoplasmic reticulum.
  • TLRs also differ in the pathogen-encoded ligands that each class recognizes: TLR 1, 2, 4, 5, 6, and 10 recognize peptidylglycans, lipids, lipopeptides, lipopolysaccharides, and proteins as ligands, and TLR3, 7, 8, 9 recognize nucleic acids as ligands (nucleic acid ligands).
  • the invention provides a ligand-nucleic acid nanostructure comprising at least one ligand for an intracellular TLR.
  • the invention provides a ligand/aptamer -nucleic acid nanostructure comprising at least one ligand/aptamer for a cell surface TLR; in certain preferred embodiments, the ligand-nucleic acid nanostructure is an aptamer-nucleic acid nanostructure, comprising at least one aptamer for a cell surface TLR.
  • the invention provides compositions comprising a ligand for an intracellular toll-like receptor TLR 3, TLR7, TLR 8, or TLR 9.
  • TLR ligand that binds to this type of intracellular TLRs includes without limitation unmethylated CpG-oligodeoxynucleotide (CpG-ODN), Poly-dI:dC, PoIy-LC, double- stranded RNA, single-stranded RNA, or derivatives thereof.
  • the double-stranded RNA is 40-50 bp double-stranded RNA.
  • the TLR ligand comprises synthetic oligonucleotides with preferential binding motif (such as U-rich sequences) to the intracellular TLRs.
  • the intracellular TLR ligand comprises a plurality of TLR ligands for one toll-like receptor or a plurality of TLR ligands for different toll-like receptors.
  • the plurality of TLR ligands comprises TLR ligands for at least two of TLR 3, TLR7, TLR 8, and TLR 9; in various other preferred embodiments, the plurality of TLR ligands comprises TLR ligands for at least three of TLR 3, TLR7, TLR 8, and TLR 9.
  • the plurality of TLR ligands comprises TLR ligands for TLR 3, TLR7, TLR 8, and TLR 9.
  • the ligand- nucleic acid nanostructure comprises TLR 7 and TLR 9 ligands. In other preferred embodiments, the ligand- nucleic acid nanostructure comprises TLR3 and TLR8 ligands. In certain other preferred embodiments, the ligand- nucleic acid nanostructure comprises TLR7 and TLR8 ligands. In certain other preferred embodiments, the different TLR ligands are present on the nanostructure at a ration of 1 : 1.
  • the TLR ligand comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or higher order of multimer of at least one TLR ligand for at least one of TLR 3, TLR7, TLR 8, and TLR 9.
  • the TLR ligand comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or higher order of multimer of TLR ligands for at least two of TLR 3, TLR7, TLR 8, and TLR 9.
  • the ligand- nucleic acid nanostructure comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or higher order of multimer of TLR 7 and TLR 9 ligands.
  • the ligand- nucleic acid nanostructure comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or higher order of multimer of TLR3 and TLR8 ligands.
  • the ligand- nucleic acid nanostructure comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or higher order of multimer of TLR7 and TLR8 ligands.
  • the TLR ligand comprises a trimer, tetramer, pentamer, hexamer, heptamer, or octamer of TLR ligands for at least three, or a tetramer, pentamer, hexamer, heptamer, or octamer of TLR ligands for all, of TLR 3, TLR7, TLR 8, and TLR 9.
  • the binding unit of a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or higher order of multimer refers to a monomer or monomeric ligand for a TLR.
  • the ligand- nucleic acid nanostructure comprises a trimer, tetramer, pentamer, hexamer, heptamer, octamer, or higher order of multimer of TLR ligands for TLR 3, TLR 7 and TLR 8.
  • the TLR ligand comprises a ligand for TLRl, TLR2, TLR 4, TLR 5, TLR 6 or TLR 10.
  • the TLR ligand comprises an aptamer.
  • the aptamer comprises a plurality of aptamers for one toll-like receptor or a plurality of aptamers for different toll-like receptors.
  • the plurality of aptamers comprises aptamers for at least two, at least three, at least four, or at least five of TLRl, TLR2, TLR 4, TLR 5, TLR 6 and TLR 10.
  • the plurality of aptamers comprises aptamers for TLRl, TLR2, TLR 4, TLR 5, TLR 6 and TLR 10.
  • the aptamer- nucleic acid nanostructure comprises TLR 2 and TLR 4 aptamers.
  • the aptamer- nucleic acid nanostructure comprises TLR 1 and TLR 2 aptamers.
  • the aptamer- nucleic acid nanostructure comprises TLR 2 and TLR 6 aptamers.
  • the different TLR aptamers is present on the nanostructure at a ratio of 1: 1.
  • the aptamer comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or octamer of one or more aptamers for at least one toll-like receptor.
  • the aptamer comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer or higher order of multimer of aptamers for at least one aptamer for TLRl, TLR2, TLR 4, TLR 5, TLR 6 and TLR 10.
  • the aptamer comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer or higher order of multimer of aptamer of at least two, at least three, at least four, or at least five of TLRl, TLR2, TLR 4, TLR 5, TLR 6 and TLR 10.
  • the aptamers comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer or higher order of multimer of aptamers for TLRl, TLR2, TLR 4, TLR 5, TLR 6 and TLR 10.
  • the aptamer- nucleic acid nanostructure comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or higher order of multimer of TLR 1 and TLR 2 aptamers.
  • the aptamer - nucleic acid nanostructure comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or higher order of multimer of TLR2 and TLR4 ligands.
  • the aptamer - nucleic acid nanostructure comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or higher order of multimer of TLR2 and TLR6 ligands.
  • the dimer, trimer, tetramer, pentamer, hexamer, heptamer, or octamer of TLR nucleic acid ligands or aptamers is formed by linking together each monomeric TLR nucleic acid ligand/aptamer either covalently or non-covalently.
  • Non- covalent binding includes without limitation nucleic acid base pairing and biotin-streptavidin interaction.
  • one monomeric ligand binds to another monomeric ligand by direct base pairing or by base pairing with a linker polynucleotide or linker oligonucleotide.
  • the linker polynucleotide has a length between about 5 nucleotides (nt) and about 100 nt; in various other preferred embodiments, 10-30 nt, 20-30 nt, 25-35 nt, 30-50 nt, 40-50 nt, 50-60 nt, 55-65 nt, 50-80 nt, or 80-100 nt. It is within the ability of one of skill in the art to adjust the length of the linker polynucleotide to accommodate each monomeric ligand present on the nucleic acid nanostructure.
  • the TLR nucleic acid ligand or aptamer comprises a plurality of TLR ligands or aptamers, and wherein the distance between each TLR ligand/aptamer of the plurality of TLR ligands/aptamers on the nucleic acid nanostructure is from about 5 nm to 25 nm; in various other preferred embodiments, between 10 nm and 20 nm; 10 nm and 15 nm; 15 nm and 20 nm; 15 nm and 25 nm; 13 nm to 18 nm; 15 nm and 22 nm; 18 nm and 22 nm; 20 nm and 25 nm; 18 nm and 25 nm; or about 20 nm, on the ligand/aptamer-nucleic acid nanostructure.
  • the distance between each ligand/aptamer of the plurality of ligands/aptamers on the nucleic acid nanostructure can be adjusted, for example, by changing the length of the ligand nucleic acid, changing the length of a linker polynucleotide by which each monomeric ligand is bound to each other, and/or by changing the length of a connector polynucleotide by which the ligand/aptamer is bound to the nucleic acid nanostructure.
  • the adjustment of spacing is based on known parameters of B-DNA. For example, it is known that B-DNA is 3.4 angstrom per base pair rise, and 2 nm in diameter.
  • the spacing can be adjusted by lengthening or shortening the ligand/aptamer nucleic acid sequences, the linker polynucleotides, and/or the connector sequences.
  • the spacing can be determined and verified by any suitable methods in the art, including without limitation atomic force microscopy (AFM) and Fluorescent Resonant Energy Transfer (FRET).
  • AFM atomic force microscopy
  • FRET Fluorescent Resonant Energy Transfer
  • the TLR ligand/aptamer- nucleic acid nanostructures further comprise aptamers that specifically bind to cell surface molecules specific for immune cells, i.e., immune cell-specific aptamers.
  • the TLR ligand/aptamer- nucleic acid nanostructures target immune cells through the binding of the immune cell-specific aptamers to the immune cells.
  • the TLR ligand/aptamer- nucleic acid nanostructure further comprise immune cell-specific aptamers for co-stimulatory proteins such as CD80 (GenBank Accession No. NM 005191, SEQ ID NOs: 21 and 22) and CD86 (GenBank Accession No.
  • the ligand-nucleic acid nanostructures comprise ligands for TLR3, TLR7, TLR8, and/or TLR9 and immune cell-specific aptamers specific for CD80 or CD86.
  • the ligand-nucleic acid nanostructures comprise aptamers for TLRl, TLR2, TLR4, TLR5, TLR6, and/or TLRlO and immune cell-specific aptamers specific for CD80 or CD86.
  • the binding affinity of each ligand/aptamer- nucleic acid nanostructure with a defined spatial arrangement of ligands is determined by any suitable methods known in the art.
  • the binding activity of intracellular TLR ligand-nucleic acid nanostructures to the respective TLR in the cells is examined by measuring cytokine production as a result of intracellular TLR activation.
  • Suitable target cytokines that can be used as an indicator of intracellular TLR binding include without limitation IL 12, INF ⁇ / ⁇ , TNF ⁇ and IL6.
  • the expression of certain co-stimulators (e.g., CD83) and certain genes regulated by NF- ⁇ B can be examined as an indicator of TLR activation.
  • binding and activation of TLR2 and TLR4 leads to increased expression of TNF.
  • binding and activation of TLR3 and TLR9 results in increased expression of TNF, IL6, and IL 12.
  • binding and activation of TLR ligand-nucleic acid nanostructure can be determined by comparing with cytokine expression in control cells treated with known free TLR ligands, i.e., TLR ligands not bound to a nucleic acid nanostructure.
  • the effects of nucleic acid nanostructure comprising TLR7 ligand can be evaluated by comparing with the effects of known TLR7 ligands such as synthetic compound imiquimod (marketed by MEDA AB, Solna, Sweden under the trade name ALD ARATM), resiquimod, or isatoribine (Anadys Pharmaceuticals).
  • synthetic compound imiquimod marketed by MEDA AB, Solna, Sweden under the trade name ALD ARATM
  • resiquimod resiquimod
  • isatoribine Anadys Pharmaceuticals.
  • fluorophore-labeled ligand/aptamer-nucleic acid nanostructures are incubated with the cells and their binding activity is examined by flow cytometry.
  • the binding affinity can be determined by the mean fluorescence intensity of target cells bound with fluorophore-labeled aptamers, as described by Tang et al. (Tang, 2007, Selection of aptamers for molecular recognition and characterization of cancer cells. Anal.
  • the spacing between each ligand/aptamer for the same type of TLR or for different types of TLRs on the nucleic acid nanostructure can be adjusted to modulate binding affinity of the ligands to the TLRs by any methods known in the art and described herein.
  • the binding activity of intracellular TLR ligand- nucleic acid nanostructure is examined by fluorescence imaging.
  • cells incubated with fluorescence-labeled TLR ligand-nucleic acid nanostructure are treated with a permeabilizing solution (BD Bioscience, San Jose, CA) and co-stained with fluorochrome-conjugated anti-TLR antibody.
  • a permeabilizing solution BD Bioscience, San Jose, CA
  • fluorochrome-conjugated anti-TLR antibody Co-localization of fluorescence TLR ligands and anti-TLR antibody indicates the binding of TLR ligand- nucleic acid nanostructure to the TLRs.
  • fluorescence- labeled cell surface TLR ligand-nucleic acid nanostructures are incubated with cells and the binding of the ligands to the cell surface TLRs is measured by any methods suitable in the art including without limitation flow cytometry and fluorescence microscopy.
  • the TLR ligands/aptamers are present on the nucleic acid nanostructure at a density of 2-20 ligands/aptamers per nucleic acid nanostructure; in various other preferred embodiments, between 2-15; 2-10; 4-20; 4-15; 4-10; 5-10; 2-9; 4-9, 4-8; 2-8; 4-6; or 2-6 ligands per nucleic acid nanostructure.
  • the TLR ligands/aptamers are present on the nucleic acid nanostructure at a density of 5-10 ligands/aptamers per nucleic acid nanostructure.
  • the ligand-nucleic acid nanostructure comprises TLR3 and TLR 8 ligands at a density of 5-10 ligands per nucleic acid nanostructure.
  • the plurality of TLR ligands/aptamers is present on the ligand/aptamer-nucleic acid nanostructure at a density of 8 TLR ligands/aptamers per nucleic acid nanostructure.
  • nucleic acid ligand can be modified by any suitable methods known in the art. For example, phosphorothioate can be incorporated into the backbone, and substitutions of the 2'-OH groups of the ribose backbone with T- deoxy-NTP, can be incorporated into the RNA molecule to increase their resistance to exo- and endo-nucleases.
  • the resistance of these modified ligands to nuclease can be tested by incubating them with either purified nucleases or nuclease from mouse serum, and the integrity of ligands can be analyzed by gel electrophoresis.
  • the integrity of receptor binding activities of the nucleic acid ligands or aptamers after binding to the nucleic acid nanostructure and/or after being modified to achieve nuclease resistance can be tested by any suitable method in the art, or as described herein, to ensure that binding to the nucleic acid nanostructure and/or modification does not compromise receptor binding activity.
  • nucleic acid nanostracture refers to a nucleic acid structure thai includes at least one nanoscale dimension, wherein the nucleic acid structure comprises single-stranded nucleic acids, which hybridize to form at least a partially double- stranded structure.
  • the TLR ligand comprises an aptamer for cell surface TLRl, 2, 4, 5, 6 and/or 10.
  • aptamer refers to single- stranded nucleic acid molecules with secondary structure(s) that allow binding to a target molecule.
  • the single-stranded nucleic acid is ssDNA, ssRNA or derivatives thereof.
  • the aptamer comprises nucleic acid sequence that does not participate in base-pairing with other polynucleotides within the nucleic acid nanostructure. [0060] Aptamers can be synthesized and screened by any suitable methods in the art.
  • aptamers can be screened and identified from a random aptamer library by SELEX (systematic evolution of ligands by exponential enrichment).
  • SELEX systematic evolution of ligands by exponential enrichment
  • aptamers that bind to a cell surface target molecule can be suitably screened and selected by a modified selection method herein referred to as cell-SELEX or cellular- SELEX, even if the identity of the target molecule is unknown.
  • a suitable nucleotide length for an aptamer ranges from about 25 to 100 nucleotide (nt), and in various other preferred embodiments, 30-100 nt, 30-60 nt, 25- 70 nt, 25-60 nt, 40-60 nt, or 40-70 nt in length.
  • the length of an aptamer is about 2-20 nm, in various other preferred embodiments, 2-15 nm, 5-15 nm, 5-10 nm, or less than 10 nm in size.
  • the monomeric aptamer contains a predetermined sequence that is about 8-10 nm in length (25-30 nt in length).
  • the sequence can be designed with sufficient flexibility such that it can accommodate interactions of aptamers with two targets at the distances described herein.
  • the flexibility of the monomeric aptamer on the nucleic acid nanostructure or between each monomeric binding units of a multimeric aptamer can be monitored by inserting various length of single stranded T linker polynucleotide.
  • the single stranded T linker polynucleotide contains 3-10 nucleotides; in certain other preferred embodiments, the single stranded T linker polynucleotide contains 3-5 nucleotides. In certain preferred embodiments, the single stranded T linker polynucleotide contains 5-10 nucleotides.
  • T polynucleotide provides more flexibility.
  • linker polynucleotides that contain fewer or no Ts result in more rigid multimeric aptamers.
  • the term "rigid dimer” or “rigid tetramer” refers to dimeric ligand/aptamer or tetrameric ligand/aptamer wherein the linker polynucleotide contains fewer or no Ts.
  • the aptamers comprises multimeric aptamers.
  • the term "multimeric aptamer " ' refers to a multimer of aptamers of the same type or different types.
  • the multimcr of aptamers can be covalently linked t ⁇ each other.
  • the multimer of aptamers can be linked to each other by direct base pairing with each other or with a linker polynucleotide, or other n ⁇ n-covalent linkage including without limitation, biotin-streptavidin interaction.
  • Multimeric aptamers provides multivalent binding capacity eitlicr for one type of target molecule or receptor, or for multiple types ⁇ f target molecules or receptors. Multivalent aptamers that bind to two or multiple types of target molecules are referred to as bi-speeiiic or multi-specific multivalent aptamers.
  • the linker polynucleotide has a length between about 5 nucleotides (nt) and about 100 nt; in various other preferred embodiments, 10-30 nt, 20-30 nt, 25-35 nt, 30-50 nt, 40-50 nt, 50-60 nt, 55-65 nt, 50-80 nt, or 80-100 nt. It is within the ability of one of skill in the art to adjust the length of the linker polynucleotide to accommodate each monomeric ligand present on the nucleic acid nanostructure.
  • Multivalent ligands are known to have higher binding avidity for the target molecules as compared to their monovalent counterparts.
  • a multivalent ligand can be built from its known monomeric unit.
  • a monomeric aptamer that binds to a desired target molecule can be screened and identified from any suitable methods in the art, such as SELEX.
  • the monomeric unit aptamers can be chemically linked to one another to form dimeric or multimeric aptamers.
  • the multimeric aptamers can be identified and screened from a random multimeric aptamer library as described herein.
  • the monomeric aptamers are linked to each other by one or a plurality of linker polynucleotides to form multimeric aptamers.
  • Monomeric aptamers can be linked to form multimeric aptamers by any suitable means and in any configurations. Exemplary multimeric aptamers are illustrated in Figure 4.
  • the monomeric aptamer comprises a first portion of a randomized sequence that is about 25 to 100 nucleotides (nt) in length, and in various other preferred embodiments, 30-100 nt, 30-60 nt, 25- 70 nt, 25-60 nt, 40-60 nt, or 40-70 nt in length. In certain preferred embodiments, the randomized sequence is 45 nt in length.
  • the randomized sequence is flanked by at least one, preferably two, predetermined sequences of about 10-50 nt in length, and in various other preferred embodiments, 15-40 nt, 15-30 nt, 20-40 nt, 25-30 nt, or 20-30 nt in length.
  • the predetermined sequence is 20 nt in length.
  • each monomeric aptamer nucleic acid comprises a randomized 45 nt sequence flanked by defined 20 nt sequences both upstream and downstream of the random sequence, i.e., the 5'-arm and 3'-arm, respectively.
  • Computer programs are available to assist in designing the suitable predetermined sequence of the 5'- arm and 3'- arm regions to facilitate hybridization with the linker polynucleotide and to minimize potential secondary structure in the 5'- arm and 3'- arm regions.
  • randomized sequence refers to an undefined nucleic acid molecule that contains degenerative nucleotide residues at one or all positions. Nucleic acid containing randomized sequence can be chemically synthesized by various methods known in the art and described herein.
  • predetermined sequence refers to a defined nucleic acid molecule for which the nucleotide sequence is known. Nucleic acid containing randomized sequence can be chemically synthesized by methods known in the art and described herein or produced recombinantly in a cell.
  • randomized dimeric aptamers are formed wherein a linker polynucleotide comprises sequences complementary to both 5 '-arm and/or 3 '-arm region of random aptamers to form dimeric aptamers.
  • trimeric or tetrameric aptamers are formed when a plurality of linker polynucleotides that hybridize to the 3 '-arm and 5'-arm regions are introduced as illustrated in Figure 4.
  • the linker polynucleotide further comprises a single stranded hinge region situated in between the aptamer-binding motifs as illustrated in Figure 4.
  • the hinge region is 3-10 nt in length; in various other preferred embodiments, the hinge region is 3-8 nt, 3-6 nt or 3-5 nt in length. In other preferred embodiments, the hinge region comprises sequence that is rich in Ts. The additional single stranded hinge region offers flexibility to allow the multimeric aptamers to coordinate and synergize multivalent interactions with target molecules or receptors. [0070] In certain preferred embodiments, the aptamers are further modified to protect the aptamers from nuclease and other enzymatic activities. The aptamer sequence can be modified by any suitable methods known in the art.
  • phosphorothioate can be incorporated into the backbone, and 5 '-modified pyrimidine can be included in 5' end, of ssDNA for DNA aptamer.
  • modified nucleotides such as substitutions of the 2'-OH groups of the ribose backbone, e.g., with 2'-deoxy-NTP or 2'-fluoro-NTP, can be incorporated into the RNA molecule using T7 RNA polymerase mutants (Epicentre Biotech, Madison, WI).
  • nucleic acid means DNA, RNA, peptide nucleic acids ("PNA”), and locked nucleic acids (“LNA”), nucleic acid-like structures, as well as combinations thereof and analogues thereof, unless specifically indicated.
  • Nucleic acid analogues include known analogues of natural nucleotides which have similar or improved binding properties. The term also encompasses nucleic-acid-like structures with synthetic backbones.
  • DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs), methylphosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzylphosphonate linkages, as discussed in US 6,664,057; see also Oligonucleotides and Analogues, a Practical Approach, edited by F.
  • the nucleic acid nanostructure comprises a DNA nanostructure.
  • Synthesis of polynucleotides is well known in the art. See, for example, Yan, H. et al, Science 2003, 301, 1882-1884; US Patent No. 6,255, 469; WO 97/41142; Seeman, N.C., Chem Biol, 2003. 10: p.
  • polynucleotides for the nucleic acid tiles it is highly desirable, but not essential, in making the polynucleotides for the nucleic acid tiles to appropriately design sequences to minimize undesired base pairing and undesired secondary structure formation.
  • Computer programs for such purposes are well known in the art. (See, for example, Seeman, N.C., J Biomol Struct Dyn, 1990. 8: p. 573- 81).
  • the polynucleotides are purified prior to nucleic acid tile assembly. Purification can be by any appropriate means, such as by gel electrophoretic techniques.
  • nucleic acid nanostructurcs A variety of suitable nucleic acid nanostructurcs arc known in the art.
  • the nucleic acid nanostructure comprises a spiral DNA scaffold, a DNA origami, or a DNA tile or tiling array.
  • the nucleic acid nanostruciures are formed by base pairing of single stranded DNA or derivatives thereof or by other non-covalent linkage, such as biotin-streptavidin interaction.
  • the nucleic acid nanostructure comprises a multi-helical bundle to display ligands
  • the multi-helical bundle comprises 2-20 double stranded helices; in various other preferred embodiments, the multi-helical bundle comprise 2-15, 2-12, 2-10, 3-20, 3-12, 3-10, 4-20, 4-10, 4-8 double stranded helices. It is within the skill of one of ordinary skill in the art to determine the suitable number of double stranded helices suitable for use as nucleic acid nanostructure in the present invention.
  • the nucleic acid nanostructure comprises one or more nucleic acid tiles, preferably DNA tiles.
  • Self-assembling nucleic acid tiling lattices represent a versatile system for nanoscale construction. Structure formation using nucleic acid 'smart tiles' begins with the chemical synthesis of single-stranded polynucleotides, which when properly annealed, self-assemble into nucleic acid tile building blocks through Watson-Crick base pairing. DNA tiles bearing complementary sticky ends are then able to further self-assemble into larger arrays with distinct topological and geometric features.
  • a self-assembling, finite nucleic acid -based nanoarray allows a wide variety of discrete molecules to be placed at specific locations with nm-scale accuracy.
  • Various nucleic acid tiles and tiling array have been described in the art. (See for example WO2008/033848 and WO2006/124089, the disclosures of which are incorporated herein by reference in their entirety.)
  • the dimensions of a given nucleic acid tile can be programmed, based on the length of the polynucleotides of the nucleic acid nanostructure (i.e.: those polynucleotides that are integrally involved in the structure of the nucleic acid tile) and their programmed shape and size, the length of the sticky ends (when used), and other design elements. Based on the teachings provided herein and known in the art, those of skill in the art can prepare nucleic acid tiles of any desired size.
  • nucleic acid tiling arrays can also be programmed with the use of boundary tiles (i.e., tiles designed to terminate further assembly of the array), depending on the size of the individual nucleic acid tiles, the number of nucleic acid tiles, the length of the sticky ends (when used), the desired spacing between individual nucleic acid tiles, and other design elements.
  • boundary tiles i.e., tiles designed to terminate further assembly of the array
  • the size of the arrays depends on the purity of the DNA strands, the stoichiometry of the different polynucleotides, and the kinetics (how slow the annealing process is).
  • nucleic acid tiling arrays of any desired size, preferably within a size limit that does not induce internalization of the tiling array by way of endocytosis of the cell.
  • the ligand-nucleic acid nanostructure is of a size sufficient to trigger passive phagocytosis of the ligand-nucleic acid nanostructure by the cells.
  • the phagocytosed vesicles fuse with the endosomal membrane and thereby releasing the ligand-nucleic acid nanostructure to the lumen of the endosome to interact with the endosomal TLRs.
  • the composition comprises an intracellular TLR ligand wherein the ligand-nucleic acid nanostructure is between about 100 nm and about 2 ⁇ m in length (i.e., 100 x 100 nm to 2000 x 2000 nm 2 ); in other preferred embodiments, 100-200 nm, 100-300 nm, 300-500 nm, 300- 800 nm, 800-1000 nm, 1000-1500 nm, 1300- 1800, or 1500-2000 nm in length.
  • the nucleic acid nanostructure is of a size sufficiently small that the nucleic acid nanostructure remains extracellular when in contact with a cell.
  • the nucleic acid nanostructure is 50-200 nm (i.e., 50 x 50 nm to 200 x 200 nm ); in other preferred embodiments, 3-100 nm; 4-60 nm; 10-90 nm; 10-200 nm; 25-200 nm; 25-100 nm; 50-200 nm; 50-150 nm; 100-200 nm; or 50-100 nm in length.
  • the nucleic acid nanostructure is no more than 100 nm in length (i.e.: 100 x 100 nm ).
  • the ligand/aptamer nucleic acid nanostructures comprise at one ligand for TLR3, TLR7, TLR8, or TLR9 and at least one aptamer for TLRl, TLR2, TLR4, TLR5, TLR6, or TLRlO.
  • the ligand/aptamer nucleic acid nanostructures comprise at least one TLR9 ligand and at least one TLR4 aptamer.
  • the TLR9 ligand and TLR4 aptamer bind to the same nucleic acid nanostructure.
  • the nucleic acid nanostructure is about 100-200 nm in length.
  • the ligand/aptamer nucleic acid nanostructure comprises both cell surface and intracellular TLRs wherein the nanostructure binds to cell-surface TLRs and is internalized inside the cell and binds to intracellular TLRs in the lumen of intracellular compartments.
  • the TLR9 ligand and TLR4 aptamer bind to the separate nucleic acid nanostructures.
  • the length and width of individual nucleic acid tiles are between 3 nm and 100 nm; in various other preferred embodiments, widths range from 4 nm to 60 nm and lengths range from 10 nm to 90 nm.
  • the nucleic acid nanostructure comprises of a singe nucleic acid tile or a nucleic acid tiling array that has a dimension consistent with the dimension of a nucleic acid nanostructure as described above suitable for use in the present invention.
  • the intracellular TLR ligands are further modified to facilitate endosome targeting.
  • these ligands are complexed with liposome reagent, such as N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium methyl sulfate (DOTAP) (Roche Applied Science, Indianapolis, IN).
  • DOTAP N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium methyl sulfate
  • 1 ⁇ g of the ligand-nucleic acid nanostructure can be mixed with 6 ⁇ l of DOTAP in culture medium and incubated for 10 min at room temperature, and then the mixture is in
  • the nucleic acid TLR ligand-nucleic acid nanostructure can be delivered to a cell by liposome transfection.
  • Liposome reagent for encapsulating nucleic acid ligand-nucleic acid nanostructure in a liposome is available in the art, for example, DOTAP (Roche Applied Science, Indianapolis, IN).
  • the liposome further contains additional immune modulators.
  • mRNA encoding cytokines can also be included in the liposome to further enhance the immune activation.
  • the mRNA encodes IL 12 or INF ⁇ .
  • the intracellular TLR ligand-nucleic acid nanostructure further comprises a transduction domain.
  • transduction domain means one or more amino acid sequence or any other molecule that can carry the intracellular TLR ligand-nucleic acid nanostructure across cellular membranes.
  • the ligand-nucleic acid nanostructure comprises at least one transduction domain selected from the group consisting of (R) 4- 9 (SEQ ID NO:25); GRKKRRQRRRPPQ (SEQ ID NO:26); AYARAAARQARA (SEQ ID NO:27); DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:28); GWTLNSAGYLLGLLNLKALAALAKKIL (SEQ ID NO:29); PLSSIFSRIGDP (SEQ ID NO:30); AA VALLP A VLLALLAP (SEQ ID NO:31); AAVLLPVLLAAP (SEQ ID NO:32); VTVLALGALAGVGVG (SEQ ID NO:33); GALFLGWLGAAGSTMGAWSQP (SEQ ID NO:34); GWTLNSAGYLLGLLNLKALAALAKKIL (SEQ ID NO:35
  • the TLR ligand-nucleic acid nanostructure is conjugated to a protein to facilitate delivery into a cell, without the side effects of systemic distribution, and thus avoiding systemic toxicity of the ligands.
  • the protein is human serum albumin or IgG.
  • the presence of a nucleic acid nanostructure prevents or reduces the likelihood of the ligand being absorbed into the blood circulation to cause systemic toxicity when the ligand-nucleic acid nanostructure composition is applied locally. It is understood that various means of delivering TLR ligand-nucleic acid nanostructure to a cell either known in the art or as described herein can be used, when desirable, in combination with one another.
  • All embodiments provided herein describing modification of ligands for intracellular TLR ligands to render the nucleic acid ligands resistant to nuclease are applicable to aptamers for cell surface TLR. Further, all embodiments provided herein describing a composition further comprising a pharmaceutical carrier, diluent or excipient can be applied to the composition described in this particular aspect comprising a cell surface TLR aptamer ligand. And all other embodiments described above for intracellular TLR ligands, unless specifically indicated, can be applied to the aspect of cell surface TLR ligands.
  • the nucleic acid nanostructure comprises or consists of a nucleic acid tiling array, comprising a plurality of nucleic acid tiles joined to one another via sticky ends, wherein each nucleic acid tile comprises one or more sticky ends, and wherein a sticky end for a given nucleic acid tile is complementary to a single sticky end of another nucleic acid tile in the nucleic acid tiling array; wherein the nucleic acid tiles are present at predetermined positions within the nucleic acid tiling array as a result of programmed base pairing between the sticky ends of the nucleic acid tiles.
  • one or more tiles in the array comprise nucleic acid probes for binding the ligands to the nanostructure.
  • programmed base pairing means that the sticky ends for the different nucleic acid tiles are designed to ensure interactions of specific nucleic acid tiles through their complementary sticky ends, thus programming the position of the nucleic acid tile within the nucleic acid tiling array.
  • predetermined positions means that the ultimate position of each nucleic acid tile in the self-assembled nucleic acid tiling array is based on the sequence and position of its sticky ends and the sequence and position of the sticky ends of the other nucleic acid tiles in the nucleic acid tiling array, such that the plurality of nucleic acid tiles can only assemble in one specific way.
  • Each "nucleic acid tile” comprises (a) a structural element (also referred to herein as the polynucleotide "core") constructed from a plurality of nucleic acid polynucleotides; and (b) 1 or more "sticky ends" per nucleic acid tile attached to the polynucleotide core.
  • a "plurality" of nucleic acid tiles means 4 or more nucleic acid tiles.
  • the nucleic acid tiling array contains at least 6, 9, 16, 25, 36, 49, 64, 81, 100, 121, 144, 169, 206, 225, 256, 289, 324, 361, or 400 nucleic acid tiles.
  • nucleic acid tiling array is the assembled array of nucleic acid tiles ofthe invention based on specific Watson-Crick base pairing between sticky ends of different nucleic acid tiles.
  • Each nucleic acid tile within the nucleic acid tiling array is located at a pre-determined position in the array, based on the complementarity of its "sticky ends" to sticky ends on a different nucleic acid tile.
  • a given nucleic acid tile may specifically bind to only one other nucleic acid tile in the nucleic acid tiling array (if the given nucleic acid tile is programmed with only a single sticky end), or it may interact with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more other nucleic acid tiles in the nucleic acid tiling array if the given nucleic acid tile has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more sticky ends, respectively.
  • closely packed arrays typically utilize 2-12 sticky ends, but more sticky ends might be used in an array that branched from a central point, as in a dendrimeric nucleic acid tiling array.
  • the nucleic acid tiles in the tiling array include "boundary tiles", nucleic acid tiles that are programmed for self-assembly at the edge of the nucleic acid tiling array based on their sticky end composition.
  • the nucleic acid tiling array is finite.
  • one or more boundary tiles in the nucleic acid tiling array further comprise modification of one or more polynucleotides that terminate further self- assembly.
  • the modification comprises addition of "TTT" (or some other sequence that has no complement within the array) overhangs at the parts of each tile that lies at the edge of the array (or adjacent to holes in it) such that the array must not be continued beyond those points.
  • TTT or some other sequence that has no complement within the array
  • no sticky ends are placed on those sections of the tiles that lie at the edges of the arrays, terminating them instead with blunt-ended nucleic acid, such as double helical DNA (and thus these boundary tiles only have sticky ends to tie into the existing array, but not to extend it).
  • sticky-ends can be added to the edge of the finite size arrays, thus allowing hierarchical assembly of larger arrays with defined dimensions.
  • sticky ends that are not complementary to any of the stick ends on the nucleic acid tiling array are added to the edge of the array to permit complementary binding to any other structure of interest, such as a second finite array.
  • the nucleic acid tiling array comprises an indexing feature to orient the tiling array and thus facilitate identification of each individual nucleic acid tile in the array. Any indexing feature can be used, so long as it is located at some spot on the array that has a lower symmetry than the array itself.
  • indexing features include, but are not limited to: (1) including one or more tiles that impart(s) an asymmetry to the array; (2) including one or more tiles that is/are differentially distinguishable from the other tiles (for example, by a detectable label); for example, a biotin molecule that could later be marked by exposing the array to streptavidin; (3) including any protrusion on an edge of the array that is offset from two edges by unequal amounts, which will serve to index the array even if it is imaged upside down; (4) including a high point on the array that is detectable; (5) introducing one or more gaps in the tiling array that introduce a detectable asymmetry; and (6) making the nucleic acid tiling array of low enough symmetry with respect to rotations and inversions that locations on it could be identified unambiguously; for example, a nucleic acid tiling array in the shape of a letter "L" with unequal sized arms would serve such a purpose.
  • a "sticky end” is a single stranded base sequence attached to the polynucleotide core of a nucleic acid tile. For each sticky end, there is a complementary sticky end on a different nucleic acid tile with which it is designed to bind, via base pairing, within the nucleic acid tiling array.
  • Each nucleic acid tile must contain at least one sticky end (for example, in a boundary nucleic acid tile of certain preferred embodiments), but may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more sticky ends, depending on the design of the nucleic acid tiling array.
  • the sticky ends are incorporated into the nucleic acid tile as a portion of one or more of the core polynucleotides. Such incorporation can be carried out in a variety of ways, in part depending on the type of polynucleotide core used.
  • the length of the sticky ends for each nucleic acid tile can vary, depending on the desired spacing between nucleic acid tiles, the number of nucleic acid tiles in the nucleic acid tiling array, the desired dimensions of the nucleic acid tiling array, and any other design parameters such as the desired distance between ligands attached to the array.
  • the sticky ends do not have to be of identical length for a given nucleic acid tile or relative to other nucleic acid tiles in the nucleic acid tiling array, so long as a complementary sticky end of an identical length is present on the nucleic acid tile to which it is designed to base pair.
  • the sticky ends on all of the nucleic acid tiles can be of identical length.
  • each sticky end for a given nucleic acid tile is (a) different than the other sticky ends for that nucleic acid tile; (b) unique to that nucleic acid tile with respect to all other nucleic acid tiles in the array; and (c) complementary to a single sticky end of one other nucleic acid tile in the nucleic acid tiling array.
  • the polynucleotide structural element of each nucleic acid tile can be identical in this preferred embodiment, so long as the sticky ends are unique.
  • a nucleic acid tiling array with "N” tiles is made by synthesizing "N” different tiles, each containing unique sticky-ends to connect to its neighboring tiles, so that each tile takes up a unique and well defined position in the array.
  • the nucleic acid tiles are not all unique (i.e.: some of the nucleic acid tiles may contain the same sticky ends).
  • the total number of unique sticky end pairs is preferably N*(N-l)/2.
  • each nucleic acid tile comprises an identical polynucleotide structural element, which limits the number of different polynucleotides that must be synthesized and assembled.
  • the nucleic acid tiles differ in their sticky ends, which program the predetermined position of each nucleic acid tile in the nucleic acid tiling array.
  • the nucleic acid tiles in this and all other embodiments may contain further components in addition to the polynucleotide structural element and the sticky ends, and these further components may differ between different nucleic acid tiles.
  • the resulting nucleic acid tiling array is "non- periodic,” meaning that the array does not include simple repetitive nucleic acid tile "patterns," such as ABABAB; ABCDABCD; ABABDCDCABABDCDC. This does not require that all of the tiles in the array be unique.
  • nucleic acid tiling arrays can also be preferably programmed with the use of boundary tiles (i.e., tiles designed to terminate further assembly of the array), depending on the size of the individual nucleic acid tiles, the number of nucleic acid tiles, the length of the sticky ends (when used), the desired spacing between individual nucleic acid tiles, and other design elements.
  • boundary tiles i.e., tiles designed to terminate further assembly of the array
  • the size of the arrays depends on the purity of the DNA strands, the stoichiometry of the different polynucleotides, and the kinetics (how slow the annealing process is).
  • nucleic acid tile or tiling array is not more than 100 nm in length (i.e.: 100 x 100 nm ).
  • the nucleic acid nanostructure comprises or consists of a nucleic acid thread strand-based tile, comprises: (a) a nucleic acid thread strand; (b) a plurality of helper nucleic acid strands that are complementary to parts of the nucleic acid thread strand; wherein a plurality of the helper nucleic acid strands further comprises a nucleic acid probe or a connector polynucleotide; and wherein the nucleic acid thread strand is folded into a desired shape by hybridization to the helper strands; wherein the nucleic acid thread strand is not complementary to any of the nucleic acid probes, and wherein the predetermined size of the array is determined by the length and shape of the nucleic acid thread strand as folded by helper strands.
  • the nucleic acid thread strand is not complementary to any of the nucleic acid probes
  • the nucleic acid probes do not base pair with the thread strand over the length of the nucleic acid probe under the conditions used, and thus the helper strands are available for interactions with a target. In this preferred embodiment, no sticky ends are required for self-assembly.
  • the nucleic acid thread strand can be any suitable polynucleotide of appropriate length and sequence for the desired nucleic acid tile.
  • the nucleic acid thread strand is a genomic nucleic acid strand, or suitable fragments thereof, such as from a virus, bacterium, or indeed any organism from which long DNA can be extracted.
  • the chosen section of genomic nucleic acid should not have sequences that are complementary to the probe sequences, and they should not contain significant amounts of repeated sequences or other sequences that might form structures that interfere with assembly of the array (such the G-rich regions that might form quadruplexes as in telomere DNA).
  • genomic nucleic acid is used as the nucleic acid thread for lengths above about 50bp where synthetic nucleic acid becomes expensive and difficult to make. Lengths up to the full length of an organism's genome (ca. 10 9 bp) are feasible if they met the sequence criteria described above.
  • the nucleic acid helper strands are complementary to regions of the nucleic acid thread and not to each other, and are designed to hybridize to the nucleic acid thread strand so as to constrain the nucleic acid thread strand into a desired shape.
  • a plurality of the nucleic acid helper strands comprises nucleic acid probes.
  • helper strands are between 10 and 50 nucleotides, not including any DNA probe that is part of the helper strand.
  • the nucleic acid thread-based tile further comprises nucleic acid filler strands that hybridize to the nucleic acid thread strand. These strands are not involved in shaping the nucleic acid thread strand, but add further structural integrity to the resulting nucleic acid tile. It is further preferred that a plurality of the nucleic acid filler strands further comprises a nucleic acid probe.
  • one or more of the helper strands can be part of a larger nucleic acid structure.
  • one or more helper strands protrude from one or more nucleic acid tiles. The helper strands fold the thread strand into place, and the nucleic acid tiles (and their nucleic acid probes) comprising the helper strands are thus precisely positioned on the thread strand.
  • one or more of the helper strands may protrude from one or more nucleic acid arrays (formed by combining two or more nucleic acid tiles).
  • one or more helper strands protrude from one or more tiling arrays and fold the thread strand into place, and the tiling arrays (and the nucleic acid tiles they are composed of, including nucleic acid probes) comprising the helper strands are thus precisely positioned on the thread strand.
  • the dimensions of a given nucleic acid thread strand-based tile can be programmed, based on the available length and sequence of thread strand nucleic acid, and other design elements. For example, a 10,000 base thread strand nucleic acid could be formed into a nucleic acid tile covering an area of approximately 2nm x 10,000 x 0.3nm or 6xlO "15 m 2 . This would correspond to a square of about 0.1 ⁇ m on each side. Depending upon the design of the thread strand-based nucleic acid tile, the size of the nucleic acid probe, the specific target, and other design feature, the density of target molecules on the nucleic acid tile can be as high as 10 12 per square cm.
  • the nucleic acid thread-based tile can be assembled in one step.
  • a long template strand of nucleic acid is mixed with shorter 'helper' strands, usually in a large molar excess of the shorter strands.
  • the strand sequences are chosen to fold the long template strand into the desired shape, as described by Yan et al. (Proceedings of the National Academy of Sciences 100, July 8, 2003 pp 8103-8108.)
  • the probe array is then achieved by using one or more helper strands with nucleic acid probes that are not complementary to any part of the template strand or the other helper strands. These will then protrude from the array, forming single stranded probe strands at known locations if the array contains an index feature. General conditions for such hybridization are known in the art.
  • the rigidity and well-defined geometry of nucleic acid nanostructures provide superb spatial and orientational control of the ligands on the array.
  • the spacing of the ligands and their positioning with respect to, for example, a tiling array surface can be precisely controlled to the sub-nanometer scale. This not only allows optimization of geometry for fast kinetics, it also allows efficient rebinding of the receptor to nearby ligands and leads to improved binding efficiency.
  • the well separated positioning of the ligands on the array also allows efficient binding of different receptors to bind corresponding ligands on the tiling array.
  • nucleic acid tiles in a nucleic acid tiling array are required to possess a ligand or aptamer.
  • one or more of the nucleic acid tiles in the nucleic acid tiling array comprises a ligand; more preferably a majority of the nucleic acid tiles in the array comprise a ligand; more preferably all of the nucleic acid tiles comprise a ligand.
  • the invention provides methods of making a random multimeric aptamer library comprising combining at least two monomeric aptamers with at least one linker polynucleotide under conditions suitable for specific hybridization of the monomeric aptamers to the linker polynucleotide, wherein each of the monomeric aptamer comprises a first portion of a randomized sequence and a second portion of a predetermined sequence that is complementary to at least a portion of the sequence of the linker polynucleotide.
  • randomized sequence refers to an undefined nucleic acid molecule that contains degenerative nucleotide residues at one or all positions. Nucleic acid containing randomized sequence can be chemically synthesized by various methods known in the art and described herein.
  • predetermined sequence refers to a defined nucleic acid molecule for which the nucleotide sequence is known. Nucleic acid containing randomized sequence can be chemically synthesized by methods known in the art and described herein or produced recombinantly in a cell. [00121] In certain preferred embodiments, the predetermined sequence is complementary to at least a portion of the sequence of the linker polynucleotide.
  • the predetermined sequence is complementary to at least 10 nt of the sequence of the linker polynucleotide; in various other preferred embodiments, at least 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt or 50 nt of the sequence of the linker polynucleotide.
  • the invention provides methods of making a ligand/aptamer-nucleic acid nanostructure, the method comprising contacting at least one TLR ligand, and at least one polynucleotide under conditions suitable for binding of the at least one TLR ligand/aptamer to the at least one polynucleotide to form a ligand/aptamer- nucleic acid nanostructure, wherein the TLR ligand is bound to the nucleic acid nanostructure.
  • the TLR ligand is a ligand for an intracellular TLR; in other preferred embodiment, the TLR ligand is a ligand for a cell surface TLR.
  • the cell surface TLR ligand comprises an aptamer.
  • the ligand/aptamer directly binds to the polynucleotide, or indirectly through a connector polynucleotide or nucleic acid probe that is bound to the polynucleotide.
  • the ligand/aptamers binds to the polynucleotide non-covalently by ways of, including without limitation, base pairing and biotin-streptavidin interaction.
  • the polynucleotide is part of a nucleic acid tile.
  • the ligand/aptamer-nucleic acid nanostructure comprises a plurality of polynucleotides, and wherein the contacting is done under conditions suitable to promote hybridization of the plurality of polynucleotides to form at least one nucleic acid tile.
  • the plurality of polynucleotides forms a plurality of nucleic acid tiles, and wherein the plurality of nucleic acid tiles forms at least one nucleic acid tiling array.
  • hybridization buffers and other conditions employed can vary depending on the polynucleotide lengths and sequences, and are well within the level of skill in the art based on the teachings provided herein. Any suitable hybridization conditions known in the art can be adopted. Exemplary hybridization conditions are provided as follows. The nucleic acid nanostructures carry a considerable negative charge at low salt, and therefore hybridization in the presence of a significant amount of salt (e.g., 10 mM MgCl 2 or 60OmM or greater monovalent salt like NaCl) is preferred. Other typical annealing conditions include 1 M NaCl, 10 mM NaHPO 4 (pH7).
  • Aptamers when included as ligands typically require 10 mM MgCl 2 to fold properly.
  • General parameters for hybridization conditions for nucleic acids are described in Sambrook et al., supra, and in Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York.
  • the stoichiometric amount of each polynucleotide in a nucleic acid nanostructure is combined under denaturing conditions, such as between 90°C and 99°C, followed by cooling to between 25°C and 50°C in appropriate hybridization buffer, as can be determined by those of skill in the art.
  • denaturing conditions such as between 90°C and 99°C
  • annealing protocols involve a high temperature and low salt denaturing step, followed by a low temperature high salt annealing step.
  • the high salt concentrations are not added to the reaction until the polynucleotides are removed from the heat and placed on ice.
  • polynucleotide concentration used can vary, and those of skill in the art, based on the teachings provided herein and known in the art, can determine appropriate concentrations. In one preferred embodiment, polynucleotide concentration is between 1 nm and 10 ⁇ M.
  • the nucleic acid nanostructure comprises a plurality of nucleic acid tiles that is combined under conditions suitable to promote hybridization of the sticky ends between different nucleic acid tiles.
  • suitable conditions include incubation in appropriate hybridization solution at a beginning temperature of between 25°C and 45°C, followed by cooling in the same hybridization buffer to between 5°C and 25°C over 1 hour to 24 hours.
  • the specific condition chosen need to balance the needs between avoiding disassembly of the tiles, which generally have melting temperatures in the range of 50-65 °C, and to eliminate the possible mismatches among the different sticky ends of the tiles.
  • the buffer condition used comprises 40 mM Tris, 20 mM acetic acid, 2 mM EDTA, and 12.5 mM magnesium acetate, pH 8.0.
  • synthesis of the nucleic acid tiling arrays comprises separating free nucleic acid tiles and/or incompletely hybridized nucleic acid tiles from completely formed nucleic acid tiling arrays. Any appropriate separation method can be used, including but not limited to size exclusion chromatography, sucrose gradient centrifugation, and affinity based separation techniques.
  • the nucleic acid tiling arrays are chemically modified so as to permit affinity-based separation techniques.
  • any chemical modification that permits such affinity -based separation techniques can be used, including but not limited to, chemically modifying the nucleic acid tiling array to contain one or more biotin residues, which can then be used for streptavidin- based affinity separation of the nucleic acid tiles.
  • the ligand/aptamer-nucleic acid nanostructures of the invention can be made and stored as described herein.
  • the ligand/aptamer-nucleic acid nanostructures may be present in solution, or in lyophilized form. All the preferred embodiments of this aspect of methods of making a ligand/aptamer-nucleic acid nanostructure can be used in conjunction with in any other aspects of the invention.
  • the invention provides methods of modulating immune response in a cell comprising contacting the cell with an amount effective to modulate the immune response of a composition comprising at least one toll-like receptor (TLR) ligand bound to a nucleic acid nanostructure.
  • TLR toll-like receptor
  • the TLR ligand is a ligand for an intracellular TLR; in other embodiment, the TLR ligand is a ligand for a cell surface TLR. In further embodiments, the TLR ligand for a cell surface TLR comprises an aptamer. In certain other preferred embodiments, the cell is a dendritic cell or macrophage. It is understood that all the embodiments of TLR ligand/aptamer-nucleic acid nanostructures can be used in this aspect of the invention. All the embodiments of this aspect of methods of modulating immune response in a cell can be used in conjunction with any other aspects of the invention.
  • cells with particular TLR pathways modulated by the ligand/aptamer-nucleic acid nanostructures described herein can be established and used for screening drugs or compounds that inhibit the particular TLR pathways.
  • cells thus established with particular TLR pathways modulated by the ligand/aptamer-nucleic acid nanostructure described herein can be used for transplantation.
  • the invention provides methods of modulating immune response in a mammal comprising administering to a mammal in need thereof an amount effective to modulate the immune response of a composition comprising at least one toll-like receptor (TLR) ligand bound to a nucleic acid nanostructure.
  • TLR toll-like receptor
  • the TLR ligand is for an intracellular TLR.
  • the intracellular TLR is TLR 3, 7, 8, or 9.
  • the TLR ligand is for a cell surface TLR.
  • the cell surface TLR is TLR 1, 2, 4, 5, 6, or 10.
  • the cell surface TLR ligand comprises an aptamer.
  • the mammal is a patient that has, without limitation, the following conditions: cancer, allergy, immune disorders, bacterial infection, viral infection, fungal infection, or immune suppression as a result of other medical complications.
  • TLR3 and TLR9 ligands-nucleic acid nanostructures that can induce ThI -type immunity are used for patients with infectious diseases and cancer.
  • TLR9 ligand-nucleic acid nanostructure will be used to reduce Th2-response for patient with allergies.
  • the binding of the TLR ligand to its receptor activates cytokine expression.
  • the changes in cytokine expression lead to increased or decreased immune response. For example, an increased cytokine production by the combined TLR ligands (such as TLR 3 and TLR9 ligands) can increase anti-tumor immunity. See Whitemore, MM, et al.
  • the binding of TLR ligand to its receptor reduces cytokine expression.
  • the composition comprises one or more TLR7 ligands and one or more TLR8 ligands, wherein the functions of TLR7 and TLR8 antagonize the effects of TLR7 and TLR9. See Ghosh, TK, 2007. International Immunopharmacology. 7: 1111-1121.
  • TLR2 ligands Pre-activation of human peripheral blood cells (PBC) by TLR2 ligands blocks the production of cytokines induced by TLR3 -ligands (such as ILl, IL6, IL8, TNF ⁇ and INF ⁇ ) and therefore reduce ThI -response (Re & Strominger, 2004. J. Immunol, 173: 7548-7555).
  • TLR2-nucleic acid nanostructure can be used to antagonize TLR3 activation and thus reduce ThI response, which is beneficial for treating certain autoimmune diseases.
  • applying a predetermined combination of different TLR ligands or aptamers on the ligand/aptamer-nucleic acid nanostructure can simultaneously activate the predetermined combination of different types of TLR.
  • the ligand/aptamer is an agonist for a TLR.
  • Suitable agonist includes, without limitation, dsRNA, or a ligand/aptamer for a particular TLR that activates the TLR.
  • the ligand/aptamer is an antagonist for a TLR.
  • a TLR2 agonistic ligand/aptamer can be used as an antagonist ligand for TLR3.
  • the ligand/aptamer-nucleic acid nanostructure comprises one or more agonists for one or more TLRs.
  • the ligand/aptamer-nucleic acid nanostructure comprises one or more antagonists for one or more TLRs. In further preferred embodiments, the ligand/aptamer-nucleic acid nanostructure comprises one or more agonists for one or more TLRs, and one or more antagonists for one or more other TLRs. [00138] Depending on the combination of TLR ligands, in certainr preferred embodiments, the multivalent TLR ligand/aptamer-nucleic acid nanostructure leads to down- regulation of immune response. In certain other preferred embodiments, the multivalent TLR ligand-nucleic acid nanostructure leads to up-regulation of immune response.
  • the increased immune responses are achieved by the combination of TLR4 ligands or aptamers and TLR9 ligands on one or more nucleic acid nanostructures.
  • activation of TLR2 and TLR4 by their ligands/aptamers on the ligand/aptamer-nucleic acid nanostructure leads to increased expression of TNF.
  • increased expression of TNF induced by the activation of TLR2 and TLR4 promotes ThI response.
  • activation of TLR 3 and TLR9 by their ligands on the ligand-nucleic acid nanostructure leads to increased TNF, IL6 and IL 12 expression.
  • TNF, IL6 and IL 12 expression promotes ThI response. It is within the skill of one or ordinary skill in the art to decide a combination of TLR ligands for the purpose of eliciting a certain type of immune response. The cooperation of TLR signals in innate immune defense has been reviewed recently by Trinchier & Sher 2007 (Nature Reviews in Immunology.7: 179-190). [00139] In certain preferred embodiments, a multivalent TLR ligand-nucleic acid nanostructure leads to increased production of certain cytokines and decreased production of other cytokines.
  • multivalent TLR 7/9 ligand-nucleic acid nanostructure internalized by a cell triggers the signal transduction pathways downstream of TLR7 and TLR9. Activation of TLR7 and TLR9 leads to increased expression and secretion of cytokines, for example, IL6, IL 12 and monocyte chemoattractant protein- 1 (MCP-I).
  • cytokines for example, IL6, IL 12 and monocyte chemoattractant protein- 1 (MCP-I).
  • MCP-I monocyte chemoattractant protein- 1
  • the cytokines produced can stimulate adaptive immune response and eliminate intracellular pathogens.
  • the multivalent TLR ligand-nucleic acid nanostructure modulates immune response by decreasing cytokine production. For example, the TLR 7/9 ligand-nucleic acid nanostructure down-regulates INF ⁇ production.
  • the TLR ligand-nucleic acid nanostructure induces ThI like cytokine production.
  • the ThI like cytokine is INF ⁇ or IL 12.
  • the TLR ligand-nucleic acid nanostructure reduces the levels of INF ⁇ / ⁇ production. In other preferred embodiments, the reduction of INF ⁇ / ⁇ production reverses pathogenicity of T cell exhaustion as a result of HIV infection.
  • the nucleic acid nanostructure comprises TLR7 and TLR9 ligands.
  • the composition comprising a TLR ligand/aptamer-nucleic acid nanostructure is administered to a mammal in need thereof as an adjuvant.
  • adjuvant refers to an agent that can stimulate the immune system and increase the immune response to a vaccine or a drug.
  • the TLR ligands comprise TLR 7 and TLR3 ligands and the drug comprises an anti-cancer drug.
  • the composition is used as a vaccine adjuvant.
  • the invention provides methods of treating an autoimmune disease in a mammal comprising administering to a mammal in need thereof an amount effective to treat the autoimmune disease of a composition comprising at least one toll-like receptor (TLR) ligand bound to a nucleic acid nanostructure.
  • TLR toll-like receptor
  • the TLR ligand is an antagonist for the TLR.
  • the TLR ligand is an antagonist for an intracellular TLR ligand. In other preferred embodiments, the TLR ligand is an antagonist for a cell surface TLR ligand/aptamer.
  • TLRs have been shown to bind to "self or "endogenous" molecule and are thought to be involved in the development of autoimmune diseases. Activation of TLR2, 3, 4, 7, 8 and 9 by endogenous molecules can cause Thl-type cytokines, leading to autoimmune diseases (Ehlers & Ravetch, 2007. Trends in Immunology. 28:74-79).
  • the TLR ligand comprises an antagonist for TLR2 and/or TLR4; in other preferred embodiments, the TLR ligand comprises an antagonist for TLR3, 7, 8, and/or 9.
  • the invention provides methods of treating microbial infections comprising administering to a mammal in need thereof an amount effective to treat the microbial infection of a composition comprising at least one toll-like receptor (TLR) ligand bound to the nucleic acid nanostructure.
  • TLRs are capable of recognizing conserved microbial patterns like components of the bacterial cell wall, bacterial motility, and microbial nucleic acids, and can quickly mount an innate immune response to combat microbial infection.
  • the microbial infections include without limitation bacterial infection, Mycobacterium tuberculosis infection, viral infection and fungal infection. All the embodiments described in this aspect of the invention can be combined with any other aspects of the invention.
  • the method further comprises a step of administering to the mammal in need thereof an effective amount of an interfering RNA (RNAi) that inhibits viral infection.
  • RNAi interfering RNA
  • the nucleic acid nanostructure further comprises RNAi that inhibits viral infection.
  • the RNAi is enclosed into the DOTAP along with the TLR ligand-nucleic acid nanostructure.
  • the molar ratio of RNAi to TLR ligands is at least 10-100 to 1.
  • RNAi interferes with HIV or other viral replication. Examples of RNAi are known in the art and the sequence of an effective RNAi can be determined by one of skill in the art with the aid of available computer design programs.
  • RNAi design is available at, for example, the IDT website for RNAi design: idtdna.com/Scitools/Applications/RNAi/ RNAi. aspx.
  • the invention provides methods of preventing microbial infection comprising administering to a mammal in need thereof an amount effective to prevent the microbial infection of a vaccine and a composition comprising at least one toll- like receptor (TLR) ligand bound to the nucleic acid nanostructure. It is understood that all the embodiments described above can be combined with any other aspects of the invention. [00147] In another aspect, the invention provides methods of treating cancer in a mammal comprising administering to a mammal in need thereof an amount effective to treat cancer of an anti-cancer drug and a composition comprising at least one TLR ligand bound to a nucleic acid nanostructure.
  • TLR toll- like receptor
  • the TLR ligand comprises TLR7 and TLR3 ligands. In other preferred embodiments, the TLR ligand comprises TLR3 and TLR9 ligands. In certain other preferred embodiments, the TLR7 and TLR3 ligands activate NK cell activity. It is understood that all the embodiments described above can be combined with any other aspects of the invention.
  • the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.
  • Suitable carrier, excipient or diluent includes without limitation water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, and oils. Any other suitable pharmaceutically acceptable carrier excipient or diluent that does not disrupt the stability and binding capacity of the ligand-nucleic acid nanostructure can be used in the present invention.
  • the invention provides pharmaceutical compositions comprising a composition of the invention and at least one pharmaceutically acceptable diluent, carrier and excipient.
  • the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier for injection may be physiological saline solution, or artificial cerebrospinal fluid.
  • Optimal pharmaceutical compositions can be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, desired dosage and recipient tissue. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra. Such compositions may influence the physical state, stability, and effectiveness of the composition.
  • the pharmaceutical composition further comprises NK cells.
  • the pharmaceutical composition to be used for in vivo administration typically is sterile and pyrogen-free. In certain preferred embodiments, this may be accomplished by filtration through sterile filtration membranes. In certain preferred embodiments, where the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution. In certain preferred embodiments, the composition for parenteral administration may be stored in lyophilized form or in a solution. In certain preferred embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical composition of the invention may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.
  • the effective amount of a pharmaceutical composition of the invention to be employed therapeutically depends, for example, upon the therapeutic context and objectives.
  • the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the pharmaceutical composition is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient.
  • a clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • Aptamers delivered at 40-400 ⁇ g/kg have been shown to be effective in vivo. McNamara wt al, 2008. J CUn Invest 118:376-386 [00154]
  • the dosing frequency depends upon the pharmacokinetic parameters of an aptamer-nucleic acid nanostructure in the formulation.
  • compositions may therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data.
  • Administration routes for the pharmaceutical compositions of the invention include topically, orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intra-arterial, intra- portal, subcutaneous, intra-nasal, or intra-lesional routes; by sustained release systems or by implantation devices.
  • the inventive ligand/aptamer nanostructure is administered subcutaneously and intra-nasally.
  • the pharmaceutical compositions may be administered by bolus injection or continuously by infusion, or by implantation device.
  • the pharmaceutical composition also can be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.
  • delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.
  • Example 1 Nanostructure directed assembly of multivalent aptamers for enhanced binding affinity
  • Multivalent interaction plays a large role in biology as many bio-machineries display high affinity multivalent bindings to their targets when sub-domains are arranged accurately with certain spatial and geometric configurations. It was demonstrated that distance dependent multivalent binding effects can be systematically investigated by incorporating multiple aptamers into self-assembled DNA nanostructures with precise control of nano-meter spatial distances. Such multivalent binding events were visualized at the single molecule level with atomic force microscopy (Fig 1).
  • Multimeric aptamer was constructed using a known ssDNA-aptamer (TAGGCAGTGGTTTGACGTCCGCATGTTGGGAATAGCCACGCCT, SEQ ID NO:44) as described by Tang et al. 2007, Anal. Chem. 79: 4900-4907.
  • Dimeric and tetrameric aptamers containing such monomeric aptamer binding unit were constructed by linking monomeric aptamer with linker sequences as shown in Figure 4A. The binding of fluorescence-labeled monomeric aptamer to a target cell was analyzed by flow cytometry.
  • Binding competition assays were performed to assess the binding activities of various aptamer structures, i.e., monomers, dimers, trimers and tetramers to the target cells. Flow cytometry analysis was carried out measuring cell staining with the fluorescence-labeled monomeric aptamer in the presence of unlabeled dimers, trimers, or tetramers. As shown in Figure 5A, the dimeric aptamer competed with labeled monomeric aptamer for binding to the target cells. Among various multivalent aptamers, the tetramer most effectively competed with the labeled-monomeric aptamer for binding to the target cells and thus displayed a much higher binding activity than the monomers. Figure 5B.
  • IC50 the concentration of unlabeled aptamers that resulted in 50% reduction in the binding of fluorescence-labeled monomeric aptamers to the target cells.
  • a nucleic acid nanostructure with multivalent TLR7/TLR9 ligands bound thereto is constructed and tested in a monocytic cell line THPl that expresses TLR 7 and TLR 9 in the endosomes.
  • TLR7 ligand single stranded RNA (20-30 nt) that contains U-rich motif, either synthetic sequence or sequences taken from a virus, such as HIV or Hepatitis C virus, is used (for example, HIV ss-RNA: 5'- GCCCGUCUGUUGUGUGACUC-3' SEQ ID NO:45).
  • TLR9 ligand phosphorothioate CpG oligodeoxynucleotide (5 '-GCTAGACPGTTAGCGT-S ') (SEQ ID NO: 46) is used.
  • the above TLR ligands are extended by additional 10 nucleotides, in which the sequence will be designed to be complementary to a 20-nt connector polynucleotide that is assembled onto a DNA tile, such that TLR ligands can be linked to the DNA-tile.
  • the binding efficiency of TLR7 and TLR9 ligand-nucleic acid nanostructure to TLR7 and TLR9 is determined by the levels of TLR7/9-mediated cytokine production and THPl activation.
  • TLR7 and TLR9 ligand-nucleic acid nanostructure binding to the receptors are measured in several ways: 1) the expression levels and types of cytokines that are produced, such as IL12; 2) expression of co-stimulators on the cell surface, such as CD83; 3) gene expression profiles, such as expression profile of IL6 and TNF ⁇ ; and 4) viral replication, such as HIV replication.
  • Individual TLR ligands are used as controls.
  • the TLR ligand-nucleic acid nanostructure that effectively activates THPl cells is tested for its potential as a treatment or prevention of HIV infection.

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Abstract

La présente invention concerne une composition comprenant au moins un ligand de TLR lié à une nanostructure d'acide nucléique, ses procédés de préparation et ses utilisations.
PCT/US2009/065508 2008-11-24 2009-11-23 Nanostructure ligand de tlr-acide nucléique comme nouvel agent immunomodulateur et utilisations de celui-ci Ceased WO2010060030A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150017201A1 (en) * 2013-03-15 2015-01-15 Regents Of The University Of Minnesota Novel nicotine dna vaccines
WO2015197706A1 (fr) * 2014-06-24 2015-12-30 Aptus Biotech, S.L. Aptamères spécifiques du récepteur tlr-4 et leurs utilisations
WO2017173371A1 (fr) * 2016-04-01 2017-10-05 Colorado State University Research Foundation Compositions et procédés pour stimuler l'immunité innée
US10774107B2 (en) 2014-02-26 2020-09-15 Arizona Board Of Regents On Behalf Of Arizona State University DNA gridiron compositions and methods
US10987373B2 (en) 2017-03-09 2021-04-27 Arizona Board Of Regents On Behalf Of Arizona State University DNA origami nanostructures for treatment of acute kidney injury
US11168320B2 (en) 2016-08-09 2021-11-09 Arizona Board Of Regents On Behalf Of Arizona State University Structure assisted directed evolution of multivalent aptamers
WO2021234453A1 (fr) * 2020-05-20 2021-11-25 Aummune Ltd. Aptamères personnalisés bispécifiques
US11242533B2 (en) 2018-01-10 2022-02-08 Arizona Board Of Regents On Behalf Of Arizona State University RNA-nanostructured double robots and methods of use thereof
US12508279B2 (en) 2022-04-29 2025-12-30 Colorado State University Research Foundation Compositions and methods for enhancing innate immunity in a subject for treatment of infections and cancer and other acute and chronic conditions of the eye

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004064782A2 (fr) * 2003-01-16 2004-08-05 Hybridon, Inc. Modulation des proprietes immunostimulatoires de composes a base d'oligonucleotides, a l'aide de dinucleotides immunostimulatoires modifies
WO2006124089A1 (fr) * 2005-05-12 2006-11-23 The Arizona Board Of Regents, A Body Corporate Acting On Behalf Of Arizona State University Nanomatrices d’acide nucleique auto-assemblees et leur utilisation
WO2008033848A2 (fr) * 2006-09-11 2008-03-20 The Arizona Board Of Regents, A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizona State University Nanomatrices de codage combinatoire auto-assemblées destinées à une biodétection mutliplexe

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004064782A2 (fr) * 2003-01-16 2004-08-05 Hybridon, Inc. Modulation des proprietes immunostimulatoires de composes a base d'oligonucleotides, a l'aide de dinucleotides immunostimulatoires modifies
WO2006124089A1 (fr) * 2005-05-12 2006-11-23 The Arizona Board Of Regents, A Body Corporate Acting On Behalf Of Arizona State University Nanomatrices d’acide nucleique auto-assemblees et leur utilisation
WO2008033848A2 (fr) * 2006-09-11 2008-03-20 The Arizona Board Of Regents, A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizona State University Nanomatrices de codage combinatoire auto-assemblées destinées à une biodétection mutliplexe

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GHOSH ET AL: "TLR-TLR cross talk in human PBMC resulting in synergistic and antagonistic regulation of type-1 and 2 interferons, IL-12 and TNF-alpha", INTERNATIONAL IMMUNOPHARMACOLOGY, vol. 7, no. 8, 11 June 2007 (2007-06-11), pages 1111 - 1121, XP022113162, ISSN: 1567-5769 *
SHUKOOR MOHAMMED IBRAHIM ET AL: "Double-stranded RNA polyinosinic-polycytidylic acid immobilized onto gamma-Fe2O3 nanoparticles by using a multifunctional polymeric linker.", SMALL, vol. 3, no. 8, August 2007 (2007-08-01), pages 1374 - 1378, XP002577359, ISSN: 1613-6829 *
TRINCHIERI GIORGIO ET AL: "Cooperation of Toll-like receptor signals in innate immune defence", NATURE REVIEWS. IMMUNOLOGY, vol. 7, no. 3, 1 March 2007 (2007-03-01), pages 179 - 190, XP002541028, ISSN: 1474-1733 *
ZHENG RONGXIU ET AL: "Paired toll-like receptor agonists enhance vaccine therapy through induction of interleukin-12", CANCER RESEARCH, vol. 68, no. 11, 1 June 2008 (2008-06-01), pages 4045 - 4049, XP009128182, ISSN: 0008-5472 *

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US20150017201A1 (en) * 2013-03-15 2015-01-15 Regents Of The University Of Minnesota Novel nicotine dna vaccines
US10774107B2 (en) 2014-02-26 2020-09-15 Arizona Board Of Regents On Behalf Of Arizona State University DNA gridiron compositions and methods
US11760774B2 (en) 2014-02-26 2023-09-19 Arizona Board Of Regents On Behalf Of Arizona State University DNA gridiron compositions and methods
US10808252B2 (en) 2014-06-24 2020-10-20 Aptatargets, S.L. Aptamers specific for TLR-4 and uses thereof
CN112111495A (zh) * 2014-06-24 2020-12-22 奥普塔目标公司 Tlr-4特异性适配体及其应用
WO2015197706A1 (fr) * 2014-06-24 2015-12-30 Aptus Biotech, S.L. Aptamères spécifiques du récepteur tlr-4 et leurs utilisations
US10196642B2 (en) 2014-06-24 2019-02-05 Aptatargets, S.L. Aptamers specific for TLR-4 and uses thereof
RU2709718C2 (ru) * 2014-06-24 2019-12-19 Аптатархетс, С.Л. Аптамеры, специфические в отношении tlr-4, и их применение
KR102454682B1 (ko) 2014-06-24 2022-10-13 앱타타겟츠 에스.엘. Tlr-4에 특이적인 압타머 및 이의 용도
RU2709718C9 (ru) * 2014-06-24 2020-02-06 Аптатархетс, С.Л. Аптамеры, специфические в отношении tlr-4, и их применение
JP2020127422A (ja) * 2014-06-24 2020-08-27 アプタターゲッツ、エセ．エレ．Ａｐｔａｔａｒｇｅｔｓ，Ｓ．Ｌ． Ｔｌｒ−４特異的アプタマーおよびそれらの使用
KR20170021298A (ko) * 2014-06-24 2017-02-27 앱터스 바이오테크, 에스.엘. Tlr-4에 특이적인 압타머 및 이의 용도
CN106459980A (zh) * 2014-06-24 2017-02-22 奥图视生物技术公司 Tlr‑4特异性适配体及其应用
CN106459980B (zh) * 2014-06-24 2020-11-03 奥普塔目标公司 Tlr-4特异性适配体及其应用
JP2017527260A (ja) * 2014-06-24 2017-09-21 アプタス バイオテック，エス．エル． Ｔｌｒ−４特異的アプタマーおよびそれらの使用
US11400152B2 (en) 2016-04-01 2022-08-02 Colorado State University Research Foundation Compositions and methods for enhancing innate immunity in a subject for treatment of infections and cancer and other acute and chronic conditions of the eye
US10512687B2 (en) 2016-04-01 2019-12-24 Colorado State University Research Foundation Compositions and methods for enhanced innate immunity
WO2017173371A1 (fr) * 2016-04-01 2017-10-05 Colorado State University Research Foundation Compositions et procédés pour stimuler l'immunité innée
US11168320B2 (en) 2016-08-09 2021-11-09 Arizona Board Of Regents On Behalf Of Arizona State University Structure assisted directed evolution of multivalent aptamers
US10987373B2 (en) 2017-03-09 2021-04-27 Arizona Board Of Regents On Behalf Of Arizona State University DNA origami nanostructures for treatment of acute kidney injury
US11242533B2 (en) 2018-01-10 2022-02-08 Arizona Board Of Regents On Behalf Of Arizona State University RNA-nanostructured double robots and methods of use thereof
US12312589B2 (en) 2018-01-10 2025-05-27 Arizona Board Of Regents On Behalf Of Arizona University RNA-nanostructured double robots and methods of use thereof
WO2021234453A1 (fr) * 2020-05-20 2021-11-25 Aummune Ltd. Aptamères personnalisés bispécifiques
JP2023527311A (ja) * 2020-05-20 2023-06-28 オミューン リミテッド 二重特異性個別化アプタマー
JP7753256B2 (ja) 2020-05-20 2025-10-14 オミューン リミテッド 二重特異性個別化アプタマー
US12508279B2 (en) 2022-04-29 2025-12-30 Colorado State University Research Foundation Compositions and methods for enhancing innate immunity in a subject for treatment of infections and cancer and other acute and chronic conditions of the eye
US12509486B2 (en) 2023-07-31 2025-12-30 Arizona Board Of Regents On Behalf Of Arizona State University DNA gridiron compositions and methods

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