Non-viral delivery of low molecular weight therapeutic agents

Abstract

The present invention relates to nucleic acid nanostructure delivery compositions and methods therefor for non-viral delivery. More specifically, the present invention relates to nucleic acid nanostructure delivery compositions, such as DNA origami compositions, and methods therefor, for example for the delivery of small molecule therapeutics.

Description

Technical Field

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 350,650, filed on June 9, 2022, the disclosure of which is hereby expressly incorporated by reference in its entirety into this specification.

[0003] Field of the Disclosure

[0004] The present invention relates to nucleic acid nanostructure delivery compositions and methods for non - viral delivery of small - molecule therapeutics. More specifically, the present invention relates to nucleic acid nanostructure delivery compositions, such as DNA origami compositions, and methods for delivery of small - molecule therapeutics, for example.

Summary of the Invention

[0005] Background and Overview

[0006] The effectiveness of cutting - edge cancer therapies, such as therapies using small - molecule therapeutics, is limited by severe toxicity and the insufficient generation of anti - tumor immunity, which is essential for the long - term survival of patients. The mammalian immune system provides means to recognize and eliminate cancer cells and other pathogenic cells. The immune system usually provides a strong line of defense, but there are numerous examples where cancer cells evade the host immune response and proliferate or persist while being pathogenic to the host. Chemotherapeutic agents and radiation therapy have been developed, for example, to eliminate replicating cancers. However, many of the currently available chemotherapeutic agents and radiation therapy regimens have harmful side effects because they not only destroy cancer but also affect normal host cells, such as cells of the hematopoietic system. The side effects of these anti - cancer agents highlight the need for the development of new cancer - selective therapies with reduced host toxicity and the ability to generate anti - tumor immunity. Furthermore, cancer cells can develop apoptosis - resistant mechanisms and may become less sensitive to conventional chemotherapeutic agents that induce apoptotic cell death.

[0007] Researchers have developed treatment protocols to destroy cancer by targeting cytotoxic compounds to cancer cells such as certain cancer cells. Many of these protocols use toxins conjugated to antibodies that bind to antigens specific to cancer cells or antigens overexpressed by cancer cells in order to minimize the delivery of toxins to normal cells. Another approach to targeting cancer cells within a host is to enhance the host immune response against cancer cells in order to avoid the need to administer compounds that may exhibit independent host toxicity and to provide long-term cancer protection mediated by anti-tumor adaptive immunity. Another strategy is to target immunosuppressive cells such as tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) to enhance the immune response against cancer cells.

[0008] Non-apoptotic immunogenic programmed cell death (PCD) has recently been recognized as having the potential to promote anti-tumor immunity through the activation of innate and adaptive immune cells. As an example, the small molecule talabostat (TLBST) induces a highly inflammatory form of PCD known as pyroptosis. Unlike apoptosis, pyroptosis is associated with the activation of the inflammasome, the maturation of caspase-1-mediated IL-1β and IL-18, lytic cell death, and the release of intracellular contents. Pyroptosis was first identified in myeloid cells, but cancer cells may also undergo pyroptosis. In a mouse cancer model, systemic administration of TLBST demonstrated potent T cell- and NK cell-dependent protection, but the efficacy in phase 2 clinical trials has been variable due to inefficient uptake of TLBST by cancer cells and systemic toxicity.

[0009] The inventors have developed a nucleic acid nanostructure delivery composition (e.g., a DNA origami structure) for delivering small molecule therapeutics to cells including cancer cells and immunosuppressive cells such as TAMs and MDSCs. Compared to other nanocarriers, nucleic acid nanostructure delivery compositions (e.g., DNA origami structures) can be precisely programmed with respect to shape, size, and functionality, form a unique homogeneous population, and have high biocompatibility. Current state-of-the-art non-viral gene delivery systems such as liposomes have many drawbacks, such as low biocompatibility and inability to be easily designed or functionalized. The nucleic acid nanostructure delivery compositions (e.g., DNA origami nanostructures) developed by the inventors have the advantages of being biocompatible, non-toxic, and programmable in many ways. For example, nucleic acid nanostructure delivery compositions can be programmed to have functional groups that avoid premature degradation, avoid immune responses, and enable targeted and controlled delivery of small molecule therapeutics. Thus, these non-viral delivery compositions can enhance the stability, safety, and / or efficacy of small molecule therapeutics by providing immune evasion and tissue-directed intracellular delivery and offering the potential to enhance anti-tumor immunity.

[0010] The following clauses and combinations thereof provide various additional exemplary aspects of the invention described herein. The various embodiments described in other sections of this patent application, including the sections entitled "Detailed Description of Exemplary Embodiments" and "Examples", are applicable to any of the following embodiments of the invention described in the following numbered clauses.

[0011] 1. A composition comprising a non-viral delivery medium comprising a nucleic acid nanostructure delivery composition and a small molecule therapeutic.

[0012] 2. The composition of clause 1, wherein the nucleic acid nanostructure delivery composition comprises a DNA origami composition.

[0013] 3. The composition of clause 1, wherein the nucleic acid nanostructure delivery composition comprises single-stranded or double-stranded DNA or RNA.

[0014] 4. The composition according to item 3, wherein the nucleic acid nanostructure delivery composition contains DNA.

[0015] 5. The composition according to item 3, wherein the nucleic acid nanostructure delivery composition contains RNA.

[0016] 6. The composition according to item 3, wherein the nucleic acid nanostructure delivery composition contains both RNA and DNA.

[0017] 7. The composition according to any one of items 3 and 4 to 6, wherein the nucleic acid nanostructure delivery composition contains both single-stranded and double-stranded regions of nucleic acids.

[0018] 8. The composition according to any one of items 3 to 6, wherein the nucleic acid nanostructure delivery composition is single-stranded.

[0019] 9. The composition according to any one of items 3 to 6, wherein the nucleic acid nanostructure delivery composition is double-stranded.

[0020] 10. The composition according to any one of items 1 to 9, wherein the small molecule therapeutic agent is bound to the nucleic acid nanostructure delivery composition by a high-affinity non-covalent binding interaction between a biotin molecule on the small molecule therapeutic agent and a molecule that binds to biotin on the nucleic acid nanostructure delivery composition.

[0021] 11. The composition according to item 10, wherein the molecule that binds to biotin is bound to the nucleic acid nanostructure delivery composition by a covalent phosphoramidite bond formed through an EDC-NHS coupling reaction between the terminal phosphate group at the 5' end of the overhang on the nucleic acid nanostructure delivery composition and the amine group on the molecule that binds to biotin.

[0022] 12. The composition according to item 10 or 11, wherein biotin is bound to the small molecule therapeutic agent by a covalent bond.

[0023] 13. The composition according to any one of items 1 to 9, wherein the small molecule therapeutic agent is bound to the nucleic acid nanostructure delivery composition by a covalent bond.

[0024] 14. The composition according to item 13, wherein a covalent bond is formed by an EDC-NHS coupling reaction between the terminal phosphate group at the 5'-end of the overhang on the nucleic acid nanostructure delivery composition and the amine group on the small molecule therapeutic agent.

[0025] 15. The composition according to item 13, wherein a covalent bond is formed by a click chemistry coupling reaction between the azide group on the nucleic acid nanostructure delivery composition and the alkyne group on the small molecule therapeutic agent.

[0026] 16. The composition according to item 13, wherein a covalent bond is formed by a click chemistry coupling reaction between the azide group on the small molecule therapeutic agent and the alkyne group on the nucleic acid nanostructure delivery composition.

[0027] 17. The composition according to any one of items 1 to 9, wherein the small molecule therapeutic agent is bound to the nucleic acid nanostructure delivery composition by a covalent bond between the carboxy-terminal molecule on the nucleic acid nanostructure delivery composition and the primary amine on the small molecule therapeutic agent.

[0028] 18. The composition according to any one of items 1 to 9, wherein the small molecule therapeutic agent is bound to the nucleic acid nanostructure delivery composition by an electrostatic interaction between the negatively charged nucleic acid nanostructure delivery composition and the positively charged amine in the small molecule therapeutic agent.

[0029] 19. The composition according to any one of items 1 to 9, wherein the small molecule therapeutic agent is bound to the nucleic acid nanostructure delivery composition by intercalation of the small molecule therapeutic agent into the nucleic acid nanostructure delivery composition.

[0030] 20. The composition according to any one of items 1 to 19, wherein the small molecule therapeutic agent induces pyroptosis.

[0031] 21. The composition according to any one of items 1 to 19, wherein the small molecule therapeutic agent induces apoptosis.

[0032] 22. The composition according to any one of items 1 to 19, wherein the small molecule therapeutic agent induces necroptosis.

[0033] 23. The composition according to any one of items 1 to 18 and item 20, wherein the small molecule therapeutic agent is a post-proline cleaving enzyme inhibitor.

[0034] 24. The composition according to item 23, wherein the small molecule therapeutic agent inhibits a post-proline cleaving dipeptidyl peptidase (DPP) selected from DPP4, DPP8, DPP9, and fibroblast activation protein.

[0035] 25. The composition according to any one of items 1 to 24, wherein the small molecule therapeutic agent induces anti-tumor immunity.

[0036] 26. The composition according to any one of items 1 to 25, wherein the small molecule therapeutic agent induces cytokine production.

[0037] 27. The composition according to any one of items 1 to 26, wherein the small molecule therapeutic agent induces inflammasome activation.

[0038] 28. The composition according to item 26, wherein the small molecule therapeutic agent induces the production of interferon or interleukin.

[0039] 29. The composition according to item 28, wherein the interferon and interleukin are selected from type I interferon, IFN-β, IFN-γ, IL-1β, IL-6, IL-12p70, and IL-18.

[0040] 30. The composition according to item 26, wherein the cytokine is selected from TNF-α and MCP-1 / CCL2.

[0041] 31. The composition according to any one of items 1 to 30, wherein the small molecule therapeutic agent causes cancer cell lysis.

[0042] 32. The composition according to any one of items 1 to 18, 20, and 23 to 31, wherein the small molecule therapeutic agent is talabostat.

[0043] 33. The composition according to any one of claims 1 to 32, wherein the nucleic acid nanostructure delivery medium comprises a cell targeting molecule.

[0044] 34. The composition according to claim 33, wherein the cell targeting molecule is selected from an antibody, an aptamer, a peptide, a PNA, and a small molecule cell targeting molecule.

[0045] 35. The composition according to claim 34, wherein the cell targeting molecule is IL4Pep1.

[0046] 36. The composition according to any one of claims 1 to 35, wherein the nucleic acid nanostructure delivery composition is coated with one or more polymers.

[0047] 37. The composition according to claim 36, wherein the one or more polymers comprise PEG-poly-L-lysine.

[0048] 38. The composition according to claim 32, wherein talabostat modulates the activity of a molecule selected from DPP, NLRP1, CARD8, and gasdermin family members.

[0049] 39. A method for treating a patient having a disease, the method comprising administering to the patient any one of the compositions according to claims 1 to 38 or claim 68 to treat the patient's disease.

[0050] 40. The method according to claim 39, further comprising administering to the patient a pharmaceutically acceptable carrier.

[0051] 41. The method according to claim 40, wherein the pharmaceutically acceptable carrier is for parenteral administration or topical administration.

[0052] 42. The method according to any one of claims 39 to 41, wherein the patient has a disease selected from the group consisting of cancer, muscle diseases, myelodysplastic syndromes and hematological diseases or bone marrow failure states including severe aplastic anemia, lung diseases, skin diseases, neurological diseases, neurofibromatosis 1, and abnormal hemoglobinopathies.

[0053] 43. The method according to claim 42, wherein the cancer is selected from the group consisting of lung cancer, bone cancer, pancreatic cancer, skin cancer, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, gastric cancer, colon cancer, breast cancer, esophageal cancer, endocrine cancer, prostate cancer, leukemia, lymphoma, mesothelioma, bladder cancer, kidney cancer, central nervous system tumor, brain tumor, and adenocarcinoma.

[0054] 44. The method according to claim 42, wherein the skin disease is a Staphylococcus aureus infection.

[0055] 45. The method according to claim 42, wherein the muscle disease is muscular dystrophy.

[0056] 46. The method according to any one of claims 39 to 45, wherein the nucleic acid nanostructure delivery composition is not cytotoxic to the cells of the patient.

[0057] 47. The method according to any one of claims 39 to 46, comprising administering the first composition according to any one of claims 1 to 38 or claim 68, and further comprising administering the second composition according to any one of claims 1 to 31 or claims 33 to 38 or claim 68, wherein the second composition comprises a small molecule therapeutic agent different from the first composition.

[0058] 48. The method according to any one of claims 39 to 46, further comprising administering a nucleic acid nanostructure delivery composition comprising one or more nucleic acid payloads, or another macromolecule selected from an antibody, polypeptide, or antibody-drug conjugate.

[0059] 49. The method according to claim 48, wherein the nucleic acid comprises DNA or RNA.

[0060] 50. The method according to claim 48 or 49, wherein the payload nucleic acid is used as a homology-directed repair or transposable element.

[0061] 51. The method according to any one of claims 48 to 50, wherein the payload nucleic acid comprises a short guide RNA (sgRNA) or a donor DNA strand.

[0062] 52. The method according to item 51, wherein the sgRNA is used to target an enzyme to a specific genomic sequence.

[0063] 53. The method according to any one of items 48 to 52, wherein the payload further comprises a CRISPR-related enzyme.

[0064] 54. The method according to item 52, wherein the target enzyme is a CRISPR-related enzyme.

[0065] 55. The method according to any one of items 48 to 54, wherein the payload comprises a CRISPR-related enzyme, an sgRNA, or a donor DNA strand.

[0066] 56. The method according to any one of items 48 to 55, wherein the payload further comprises a Cas9 nuclease enzyme.

[0067] 57. The method according to any one of items 48 to 56, wherein the payload comprises Cas9, Cas10, an sgRNA, or a donor DNA strand.

[0068] 58. The method according to any one of items 48 to 57, wherein the payload further comprises an inactivated Cas9 enzyme (dCas9) and is fused with a deaminase.

[0069] 59. The method according to any one of items 48 to 58, wherein the payload comprises a coding sequence of Cas9, an sgRNA, or a donor DNA strand in the form of a plasmid.

[0070] 60. The method according to any one of items 48 to 59, wherein the payload consists of one molecule each of CRISPR / Cas9, an sgRNA, or a donor DNA strand.

[0071] 61. The method according to item 48 or 49, wherein the payload comprises an antisense oligonucleotide.

[0072] 62. The method according to item 48 or 49, wherein the payload is a size selected from the group consisting of 0.1 kB or more, 0.2 kB or more, 0.3 kB or more, 0.4 kB or more, 0.5 kB or more, 0.6 kB or more, 0.7 kB or more, 0.8 kB or more, 0.9 kB or more, 1 kB or more, 1.5 kB or more, 2 kB or more, 2.5 kB or more, 3 kB or more, 3.5 kB or more, 4 kB or more, 4.5 kB or more, 5 kB or more, 5.5 kB or more, 6 kB or more, 6.5 kB or more, 7 kB or more, 7.5 kB or more, 8 kB or more, and 8.5 kB or more.

[0073] 63. The method according to any one of items 48 to 62, wherein the nucleic acid nanostructure delivery composition comprises one or more oligonucleotides having overhangs that bind via complementary base pairing to the payload nucleic acid.

[0074] 64. The method according to any one of items 48 to 62, wherein the payload binds to the nucleic acid nanostructure delivery composition by a high-affinity non-covalent binding interaction between a biotin molecule on the payload and a molecule that binds to biotin on the nucleic acid nanostructure delivery composition.

[0075] 65. The method according to item 64, wherein the molecule that binds to biotin is bound to the nucleic acid nanostructure delivery composition by a covalent phosphonamidite bond formed via an EDC-NHS coupling reaction between the terminal phosphate group at the 5' end of the overhang on the nucleic acid nanostructure delivery composition and the amine group on the molecule that binds to biotin.

[0076] 66. The method according to any one of items 1 to 65, wherein the aspect ratio of the nucleic acid nanostructure delivery composition is about 2.

[0077] 67. The method according to any one of items 39 to 66, wherein both the small molecule therapeutic agent and the nucleic acid nanostructure delivery composition induce anti-tumor immunity.

[0078] 68. The composition according to any one of items 1 to 9 or items 19 to 38, wherein a small molecule therapeutic agent is bound to a nucleic acid nanostructure delivery composition by base pairing, wherein the small molecule therapeutic agent contains a nucleic acid covalently bound to the small molecule therapeutic agent, and the nucleic acid covalently bound to the small molecule therapeutic agent is base paired with a complementary nucleic acid on the nucleic acid nanostructure delivery composition.

[0079] 69. The method according to any one of items 39 to 43 or items 46 to 67, wherein the small molecule therapeutic agent targets cancer cells to induce anti-tumor immunity.

[0080] 70. The method according to any one of items 39 to 43 or items 46 to 67, wherein the small molecule therapeutic agent targets immunosuppressive cells to induce anti-tumor immunity.

[0081] 71. The method according to item 70, wherein the immunosuppressive cells are selected from tumor-associated macrophages and myeloid-derived suppressor cells.

Brief Description of the Drawings

[0082] Brief Description of the Drawings

[0083]

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[0084] Detailed Description of Exemplary Embodiments

[0085] The present invention relates to nucleic acid nanostructure delivery compositions and methods for the non-viral delivery of small molecule therapeutics. More specifically, the present invention relates to nucleic acid nanostructure delivery compositions, such as DNA origami compositions, and methods for the delivery of small molecule therapeutics, for example.

[0086] The following clauses and their combinations provide various additional exemplary aspects of the invention described herein. The various embodiments described in other sections of this patent application (including the summary section of the section entitled "Background and Summary", "Examples", and this "Detailed Description of Exemplary Embodiments" section) are applicable to any of the following embodiments of the invention described in the following numbered clauses.

[0087] 1. A composition comprising a non-viral delivery vehicle comprising a nucleic acid nanostructure delivery composition and a small molecule therapeutic agent.

[0088] 2. The composition according to clause 1, wherein the nucleic acid nanostructure delivery composition comprises a DNA origami composition.

[0089] 3. The composition according to clause 1, wherein the nucleic acid nanostructure delivery composition comprises single-stranded or double-stranded DNA or RNA.

[0090] 4. The composition according to clause 3, wherein the nucleic acid nanostructure delivery composition comprises DNA.

[0091] 5. The composition according to clause 3, wherein the nucleic acid nanostructure delivery composition comprises RNA.

[0092] 6. The composition according to clause 3, wherein the nucleic acid nanostructure delivery composition comprises RNA and DNA.

[0093] 7. The composition according to any of clauses 3 to 6, wherein the nucleic acid nanostructure delivery composition comprises both single-stranded and double-stranded regions of the nucleic acid.

[0094] 8. The composition according to any of clauses 3 to 6, wherein the nucleic acid nanostructure delivery composition is single-stranded.

[0095] 9. The composition according to any of clauses 3 to 6, wherein the nucleic acid nanostructure delivery composition is double-stranded.

[0096] 10. The composition according to any one of items 1 to 9, wherein the small molecule therapeutic agent is bound to the nucleic acid nanostructure delivery composition by a high-affinity non-covalent interaction between a biotin molecule on the small molecule therapeutic agent and a molecule that binds to biotin on the nucleic acid nanostructure delivery composition.

[0097] 11. The composition according to item 10, wherein the molecule that binds to biotin is bound to the nucleic acid nanostructure delivery composition by a covalent phosphoramidite bond formed via an EDC-NHS coupling reaction between the terminal phosphate group at the 5' end of the overhang on the nucleic acid nanostructure delivery composition and the amine group on the molecule that binds to biotin.

[0098] 12. The composition according to item 10 or 11, wherein biotin is bound to the small molecule therapeutic agent by a covalent bond.

[0099] 13. The composition according to any one of items 1 to 9, wherein the small molecule therapeutic agent is bound to the nucleic acid nanostructure delivery composition by a covalent bond.

[0100] 14. The composition according to item 13, wherein the covalent bond is formed by an EDC-NHS coupling reaction between the terminal phosphate group at the 5' end of the overhang on the nucleic acid nanostructure delivery composition and the amine group on the small molecule therapeutic agent.

[0101] 15. The composition according to item 13, wherein the covalent bond is formed by a click chemistry coupling reaction between an azide group on the nucleic acid nanostructure delivery composition and an alkyne group on the small molecule therapeutic agent.

[0102] 16. The composition according to item 13, wherein the covalent bond is formed by a click chemistry coupling reaction between an azide group on the small molecule therapeutic agent and an alkyne group on the nucleic acid nanostructure delivery composition.

[0103] 17. The composition according to any one of items 1 to 9, wherein the small molecule therapeutic agent is bound to the nucleic acid nanostructure delivery composition by a covalent bond between a carboxy-terminal molecule on the nucleic acid nanostructure delivery composition and a primary amine on the small molecule therapeutic agent.

[0104] 18. The composition according to any one of items 1 to 9, wherein the small molecule therapeutic agent is bound to the nucleic acid nanostructure delivery composition by electrostatic interaction between the negatively charged nucleic acid nanostructure delivery composition and the positively charged amine in the small molecule therapeutic agent.

[0105] 19. The composition according to any one of items 1 to 9, wherein the small molecule therapeutic agent is bound to the nucleic acid nanostructure delivery composition by intercalation of the small molecule therapeutic agent into the nucleic acid nanostructure delivery composition.

[0106] 20. The composition according to any one of items 1 to 19, wherein the small molecule therapeutic agent induces pyroptosis.

[0107] 21. The composition according to any one of items 1 to 19, wherein the small molecule therapeutic agent induces apoptosis.

[0108] 22. The composition according to any one of items 1 to 19, wherein the small molecule therapeutic agent induces necroptosis.

[0109] 23. The composition according to any one of items 1 to 18 and item 20, wherein the small molecule therapeutic agent is a prolyl peptidase inhibitor.

[0110] 24. The composition according to item 23, wherein the small molecule therapeutic agent inhibits a prolyl peptidase dipeptidyl peptidase (DPP) selected from DPP4, DPP8, DPP9, and fibroblast activation protein.

[0111] 25. The composition according to any one of items 1 to 24, wherein the small molecule therapeutic agent induces anti-tumor immunity.

[0112] 26. The composition according to any one of items 1 to 25, wherein the small molecule therapeutic agent induces cytokine production.

[0113] 27. The composition according to any one of items 1 to 26, wherein the small molecule therapeutic agent induces inflammasome activation.

[0114] 28. The composition according to item 26, wherein the small molecule therapeutic agent induces the production of interferon or interleukin.

[0115] 29. The composition according to item 28, wherein the interferon and interleukin are selected from type I interferon, IFN-β, IFN-γ, IL-1β, IL-6, IL-12p70, and IL-18.

[0116] 30. The composition according to item 26, wherein the cytokine is selected from TNF-α and MCP-1 / CCL2.

[0117] 31. The composition according to any one of items 1 to 30, wherein the small molecule therapeutic agent causes cancer cell lysis.

[0118] 32. The composition according to any one of items 1 to 18, 20, and 23 to 31, wherein the small molecule therapeutic agent is talabostat.

[0119] 33. The composition according to any one of items 1 to 32, wherein the nucleic acid nanostructure delivery medium contains a cell targeting molecule.

[0120] 34. The composition according to item 33, wherein the cell targeting molecule is selected from antibodies, aptamers, peptides, PNAs, and small molecule cell targeting molecules.

[0121] 35. The composition according to item 34, wherein the cell targeting molecule is IL4Pep1.

[0122] 36. The composition according to any one of items 1 to 35, wherein the nucleic acid nanostructure delivery composition is coated with one or more polymers.

[0123] 37. The composition according to item 36, wherein the one or more polymers include PEG-poly-L-lysine.

[0124] 38. The composition according to item 32, wherein talabostat regulates the activity of a molecule selected from DPP, NLRP1, CARD8, and gasdermin family members.

[0125] 39. A method for treating a patient having a disease, comprising administering to the patient any of the compositions according to claims 1 to 38 or claim 68, and treating the disease of the patient.

[0126] 40. The method according to claim 39, further comprising administering to the patient a pharmaceutically acceptable carrier.

[0127] 41. The method according to claim 40, wherein the pharmaceutically acceptable carrier is for parenteral administration or topical administration.

[0128] 42. The method according to any of claims 39 to 41, wherein the patient has a disease selected from the group consisting of cancer, muscle diseases, myelodysplastic syndromes and hematological diseases or bone marrow failure states including severe aplastic anemia, lung diseases, skin diseases, neurological diseases, neurofibromatosis 1, and abnormal hemoglobinopathies.

[0129] 43. The method according to claim 42, wherein the cancer is selected from the group consisting of lung cancer, bone cancer, pancreatic cancer, skin cancer, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, gastric cancer, colon cancer, breast cancer, esophageal cancer, endocrine cancer, prostate cancer, leukemia, lymphoma, mesothelioma, bladder cancer, kidney cancer, central nervous system tumor, brain cancer, and adenocarcinoma.

[0130] 44. The method according to claim 42, wherein the skin disease is a Staphylococcus aureus infection.

[0131] 45. The method according to claim 42, wherein the muscle disease is muscular dystrophy.

[0132] 46. The method according to any of claims 39 to 45, wherein the nucleic acid nanostructure delivery composition is not cytotoxic to the cells of the patient.

[0133] 47. A method according to any one of paragraphs 39 to 46, comprising administering the first composition according to any one of paragraphs 1 to 38 or paragraph 68, and further comprising administering the second composition according to any one of paragraphs 1 to 31 or paragraphs 33 to 38 or paragraph 68, wherein the second composition comprises a small molecule therapeutic agent different from the first composition.

[0134] 48. A method according to any one of paragraphs 39 to 46, further comprising administering a nucleic acid nanostructure delivery composition comprising one or more nucleic acid payloads, or another macromolecule selected from an antibody, polypeptide, or antibody-drug conjugate.

[0135] 49. The method according to paragraph 48, wherein the nucleic acid comprises DNA or RNA.

[0136] 50. The method according to paragraph 48 or 49, wherein the payload nucleic acid is used as a homology-directed repair or transposable element.

[0137] 51. The method according to any one of paragraphs 48 to 50, wherein the payload nucleic acid comprises a short guide RNA (sgRNA) or a donor DNA strand.

[0138] 52. The method according to paragraph 51, wherein the sgRNA is used to target an enzyme to a specific genomic sequence.

[0139] 53. The method according to any one of paragraphs 48 to 52, wherein the payload further comprises a CRISPR-associated enzyme.

[0140] 54. The method according to paragraph 52, wherein the target enzyme is a CRISPR-associated enzyme.

[0141] 55. The method according to any one of paragraphs 48 to 54, wherein the payload comprises a CRISPR-associated enzyme, sgRNA, or donor DNA strand.

[0142] 56. The method according to any one of paragraphs 48 to 55, wherein the payload further comprises a Cas9 nuclease enzyme.

[0143] 57. The method according to any one of claims 48 to 56, wherein the payload comprises Cas9, Cas10, sgRNA, or a donor DNA strand.

[0144] 58. The method according to any one of claims 48 to 57, wherein the payload further comprises an inactivated Cas9 enzyme (dCas9) and is fused with a deaminase.

[0145] 59. The method according to any one of claims 48 to 58, wherein the payload comprises a coding sequence of Cas9, sgRNA, or a donor DNA strand in the form of a plasmid.

[0146] 60. The method according to any one of claims 48 to 59, wherein the payload consists of one molecule each of CRISPR / Cas9, sgRNA, or a donor DNA strand.

[0147] 61. The method according to claim 48 or 49, wherein the payload comprises an antisense oligonucleotide.

[0148] 62. The method according to claim 48 or 49, wherein the payload is a size selected from the group consisting of 0.1 kB or more, 0.2 kB or more, 0.3 kB or more, 0.4 kB or more, 0.5 kB or more, 0.6 kB or more, 0.7 kB or more, 0.8 kB or more, 0.9 kB or more, 1 kB or more, 1.5 kB or more, 2 kB or more, 2.5 kB or more, 3 kB or more, 3.5 kB or more, 4 kB or more, 4.5 kB or more, 5 kB or more, 5.5 kB or more, 6 kB or more, 6.5 kB or more, 7 kB or more, 7.5 kB or more, 8 kB or more, and 8.5 kB or more.

[0149] 63. The method according to any one of claims 48 to 62, wherein the nucleic acid nanostructure delivery composition comprises one or more oligonucleotides having overhangs that bind via complementary base pairing to the payload nucleic acid.

[0150] 64. The method according to any one of items 48 to 62, wherein the payload binds to the nucleic acid nanostructure delivery composition by a high-affinity non-covalent interaction between a biotin molecule on the payload and a molecule that binds to biotin on the nucleic acid nanostructure delivery composition.

[0151] 65. The method according to item 64, wherein the molecule that binds to biotin is bound to the nucleic acid nanostructure delivery composition by a covalent phosphoramidite bond formed through an EDC-NHS coupling reaction between the terminal phosphate group at the 5' end of the overhang on the nucleic acid nanostructure delivery composition and the amine group on the molecule that binds to biotin.

[0152] 66. The method according to any one of items 1 to 65, wherein the aspect ratio of the nucleic acid nanostructure delivery composition is about 2.

[0153] 67. The method according to any one of items 39 to 66, wherein both the small molecule therapeutic agent and the nucleic acid nanostructure delivery composition induce anti-tumor immunity.

[0154] 68. The composition according to any one of items 1 to 9 or items 19 to 38, wherein the small molecule therapeutic agent binds to the nucleic acid nanostructure delivery composition by base pairing, wherein the small molecule therapeutic agent contains a nucleic acid covalently bound to the small molecule therapeutic agent, and the nucleic acid covalently bound to the small molecule therapeutic agent forms base pairs with the complementary nucleic acid on the nucleic acid nanostructure delivery composition.

[0155] 69. The composition according to any one of items 39 to 43 or items 46 to 67, wherein the small molecule therapeutic agent induces anti-tumor immunity by targeting cancer cells.

[0156] 70. The composition according to any one of items 39 to 43 or items 46 to 67, wherein the small molecule therapeutic agent induces anti-tumor immunity by targeting immunosuppressive cells.

[0157] 71. The method according to item 70, wherein the immunosuppressive cells are selected from tumor-associated macrophages and myeloid-derived suppressor cells.

[0158] In various embodiments, the nucleic acid nanostructure delivery compositions described herein can include any non-viral composition for the in vivo delivery of payloads such as small molecule therapeutics. By way of example, the nucleic acid nanostructure delivery compositions described herein can be selected from the group comprising any nucleic acid nanostructure delivery composition such as synthetic virus-like particles, carbon nanotubes, emulsions, and DNA origami structures. DNA origami structures are described in U.S. Patent No. 9,765,341, which is incorporated herein by reference. In any of the embodiments of the nucleic acid nanostructure delivery compositions described herein, the nucleic acid nanostructure can include M13 bacteriophage DNA.

[0159] In these embodiments, the nucleic acid nanostructure delivery compositions have a high degree of tunability of structure and function, an opportunity to protect the payload from adverse reactions or degradation by the immune system, and cell targeting by surface charge, particle size, or binding to various aptamers. These delivery systems are also suitable for computer-aided design and have a suitable pathway to a robust commercial-scale manufacturing process with higher yields and fewer purification steps than the viral manufacturing process.

[0160] Nucleic acid nanostructure delivery compositions (e.g., DNA origami structures) as delivery platforms are programmable and offer opportunities for accurate scale-up and manufacturing. In this embodiment, the biological and non-viral nature of the nucleic acid nanostructure delivery composition reduces the likelihood of harmful immune reactions. In this embodiment, by controlling each nucleotide that forms part of the nucleic acid nanostructure delivery composition (e.g., DNA origami nanostructure), it is possible to accurately design and modify structures that contain appropriate chemical moieties that enable in vivo delivery and endosomal escape. In various embodiments, the nucleic acid nanostructure delivery composition can include DNA or RNA. In various embodiments, the nucleic acid nanostructure delivery composition can be single-stranded or double-stranded or both and can include DNA and RNA.

[0161] In this embodiment, the nucleic acid nanostructure delivery composition folds into a structure that can undergo self-base pairing (i.e., DNA origami structure) and form a single-stranded or double-stranded backbone capable of encapsulating a payload such as a small molecule therapeutic agent, or the backbone can have both single-stranded regions and double-stranded regions.

[0162] In another exemplary embodiment, any of the nucleic acid nanostructure delivery compositions described herein can be coated with one or more polymers to protect the composition from an immune response or enhance endosomal escape. In one embodiment, the one or more polymers include a cationic block copolymer. In another embodiment, the one or more polymers include polyethylene glycol. In another embodiment, the one or more polymers include polyethylene glycol-poly-L-lysine. In yet another embodiment, the one or more polymers include polyethyleneimine. In an additional embodiment, the one or more polymers include polyethylene glycol-poly-L-lysine and polyethyleneimine.

[0163] In one embodiment, the small molecule therapeutic agent can bind to the nucleic acid nanostructure delivery composition, for example, via a biotin-avidin interaction. In one aspect, the molecule that binds to biotin can bind to the nucleic acid nanostructure delivery composition by a covalent phosphoramidite bond formed through an EDC-NHS coupling reaction between the terminal phosphate group at the 5' end of the overhang on the nucleic acid nanostructure delivery composition and the amine group on the molecule that binds to biotin. In this embodiment, biotin can bind to the small molecule therapeutic agent by a covalent bond.

[0164] In another embodiment, the small molecule therapeutic agent can be covalently bound to the nucleic acid nanostructure delivery composition. In this embodiment, a covalent bond can be formed via an EDC-NHS coupling reaction between the terminal phosphate group at the 5' end of the overhang on the nucleic acid nanostructure delivery composition and the amine group on the small molecule therapeutic agent. In another embodiment, a covalent bond can be formed via a click chemistry coupling reaction between the azide group on the nucleic acid nanostructure delivery composition and the alkyne group on the small molecule therapeutic agent. In yet another embodiment, a covalent bond can be formed via a click chemistry coupling reaction between the azide group on the small molecule therapeutic agent and the alkyne group on the nucleic acid nanostructure delivery composition. In yet another aspect, the small molecule therapeutic agent can be bound to the nucleic acid nanostructure delivery composition by a covalent bond between the carboxy-terminal molecule on the nucleic acid nanostructure delivery composition and the primary amine on the small molecule therapeutic agent.

[0165] In another exemplary embodiment, the small molecule therapeutic agent can be bound to the nucleic acid nanostructure delivery composition by electrostatic interaction between the negatively charged nucleic acid nanostructure delivery composition and the positively charged amine in the small molecule therapeutic agent. In yet another embodiment, the small molecule therapeutic agent can be bound to the nucleic acid nanostructure delivery composition by intercalation of the small molecule therapeutic agent into the nucleic acid nanostructure delivery composition.

[0166] In yet another embodiment, the small molecule therapeutic agent can be bound to the nucleic acid nanostructure delivery composition by base pairing, where the small molecule therapeutic agent contains a nucleic acid covalently bound to the small molecule therapeutic agent, and the nucleic acid covalently bound to the small molecule therapeutic agent forms base pairs with the complementary nucleic acid on the nucleic acid nanostructure delivery composition.

[0167] In various embodiments, the small molecule therapeutic agent can be any suitable small molecule therapeutic agent. Exemplary small molecule therapeutic agents include any small molecule therapeutic agent capable of modulating or modifying cell function, including pharmaceutically active compounds. Suitable molecules include peptides, oligopeptides, retro-inverso oligopeptides, proteins, protein analogs in which at least one non-peptide bond is replaced by a peptide bond, apoproteins, glycoproteins, enzymes, coenzymes, enzyme inhibitors, amino acids and their derivatives, receptors and other membrane proteins, antigens and their antibodies, haptens and their antibodies, hormones, lipids, phospholipids, liposomes, toxins, antibiotics, analgesics, bronchodilators, beta blockers, antibacterial agents, antihypertensives, antiarrhythmics, cardiac glycosides, antianginal agents and cardiovascular drugs including vasodilators, stimulants, psychotropic drugs, antimanic drugs and antidepressants including central nervous system drugs, antiviral agents, antihistamines, cancer therapeutic agents including chemotherapeutic agents, tranquilizers, antidepressants, H-2 antagonists, antiepileptic agents, antiemetics, prostaglandins and prostaglandin analogs, muscle relaxants, anti-inflammatory substances, stimulants, decongestants, antiemetics, diuretics, antispasmodics, antiasthmatics, antiparkinsonian agents, minerals and nutritional supplements, and immunomodulators, but are not limited thereto.

[0168] Furthermore, the small molecule therapeutic agent is cytotoxic, enhances tumor permeability, inhibits tumor cell proliferation, induces apoptosis, induces pyroptosis, induces necroptosis, is used for the treatment of diseases caused by infectious agents, or may be any drug known in the art that enhances the endogenous immune response against cancer cells, such as by inhibiting immunosuppressive cells such as TAM or MDSC. Small molecule therapeutic agents suitable for use in accordance with the present invention include adrenal cortical hormones and corticosteroids, alkylating agents, anti-androgens, anti-estrogens, androgens, aclacinomycin and aclacinomycin derivatives, estrogens, cytosine arabinoside, purine analogs, pyrimidine analogs, metabolic antagonists such as methotrexate, busulfan, carboplatin, chlorambucil, cisplatin and other platinum compounds, tamoxifen, taxol, paclitaxel, paclitaxel derivatives, Taxotere (registered trademark), cyclophosphamide, daunomycin, lysocine, T2 toxin, plant alkaloids, prednisone, hydroxyurea, teniposide, mitomycin, discodermolide, microtubule inhibitors, epothilone, tubulysin, cyclopropylbenz[e]indrone, seco-cyclopropylbenz[e]indrone, O-Ac-seco-cyclopropylbenz[e]indrone, bleomycin and other antibiotics, nitrogen mustard, nitrosourea, vincristine, vinblastine, and their analogs and derivatives, such as deacetylvinblastine monohydrazide, colchicine, colchicine derivatives, allocolchicine, thiocolchicine, tritylcysteine, halicondrin B, dastustatin, such as dastustatin 10, amanitin, such as α-amanitin, camptothecin, irinotecan, and other camptothecin derivatives, geldanamycin and geldanamycin derivatives, estramustine, nocodazole, MAP4, colcemid, inflammatory and pro-inflammatory agents, peptides and peptidomimetic signal transduction inhibitors, TLR agonists, such as TLR7 or TLR9 agonists, PI3 kinase inhibitors, microtubule inhibitors, and other technically recognized small molecule therapeutic agents.

[0169] In another embodiment, the small molecule therapeutic agent can be a tyrosine kinase inhibitor selected from the group consisting of crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, capmatinib, tepotinib, gefitinib, erlotinib, lapatinib, icotinib, afatinib, osimertinib, neratinib, dacomitinib, almonertinib, tucatinib, midostaurin, gilteritinib, xidazitinib, pexidartinib, sorafenib, sunitinib, pazopanib, vandetanib, axitinib, cabozantinib, regorafenib, apatinib, lenvatinib, thibozanib, fruquintinib, nintedanib, anlotinib, erdafitinib, pemigatinib, avapritinib, repotrectinib, selpercatinib, pralsetinib, larotrectinib, and larotrectinib.

[0170] In another exemplary embodiment, the small molecule therapeutic agent can be a non-receptor tyrosine kinase inhibitor selected from the group consisting of imatinib, dasatinib, nilotinib, bosutinib, radotinib, ponatinib, ibrutinib, acalabrutinib, zanubrutinib, luxolitinib, and fedratinib.

[0171] In another aspect, the small molecule therapeutic agent can be a small molecule serine / threonine kinase inhibitor selected from the group consisting of vemurafenib, dabrafenib, encorafenib, trametinib, cobimetinib, binimetinib, selumetinib, palbociclib, ribociclib, abemaciclib, idelalisib, copanlisib, duvelisib, alpelisib, temsirolimus, everolimus, and sirolimus. In another embodiment, the small molecule therapeutic agent can be an epigenetic target selected from the group consisting of tazemetostat, vorinostat, romidepsin, belinostat, tucidinostat, panobinostat, enasidenib, and ivosidenib. In yet another aspect, the small molecule therapeutic agent can be a small molecule inhibitor of BCL-2, the hedgehog pathway, the proteasome, or PARP selected from the group consisting of venetoclax, visomodegib, sonidegib, glasdegib, bortezomib, carfilzomib, ixazomib, olaparib, rucaparib, niraparib, and talazoparib.

[0172] In another embodiment, the small molecule therapeutic agent can be a post-proline cleaving enzyme inhibitor. In one aspect, the small molecule therapeutic agent inhibits a post-proline cleaving dipeptidyl peptidase (DPP) selected from DPP4, DPP8, DPP9, and fibroblast activation protein. In this embodiment, the DPP inhibitor can be selected from talabostat, sitagliptin, vildagliptin, alogliptin, saxagliptin, PSN-9301, R1438, TA-6666, PHX1149, GRC 8200, SYR-619, TS-021, SSR 162369, and ALS 2-0426. In one embodiment, the small molecule therapeutic agent can be the immunomodulatory agent talabostat. In another embodiment, talabostat or another small molecule therapeutic agent can modulate the activity of a molecule selected from DPP, NLRP1, CARD8, and members of the gasdermin family.

[0173] In yet other embodiments, the small molecule therapeutic can induce anti-tumor immunity, induce inflammasome activation, inactivate TAMs or MDSCs, induce cancer cell lysis, and / or induce the production of cytokines (e.g., interferon or interleukin). In another embodiment, the small molecule therapeutic can induce the production of interferon and / or interleukin selected from type I interferon, IFN-β, IFN-γ, IL-1β, IL-6, IL-12p70, and IL-18. In another aspect, the small molecule therapeutic can induce the production of cytokines selected from TNF-α and MCP-1 / CCL2.

[0174] In yet another aspect, the nucleic acid nanostructure delivery vehicle can include a cell targeting molecule. In an exemplary embodiment, the cell targeting molecule is selected from an antibody, an aptamer, a peptide, a PNA, and a small molecule cancer cell targeting molecule. In other embodiments, the cell targeting molecule is a vitamin (e.g., folic acid), a peptide ligand identified from library screening, a tumor cell-specific peptide, a tumor cell-specific aptamer, a tumor cell-specific monoclonal or polyclonal antibody, a Fab or scFv (i.e., single-chain variable region) fragment of an antibody, a small organic molecule derived from a combinatorial library, a growth factor such as EGF, FGF, insulin, insulin-like growth factor, and a homologous polypeptide, somatostatin and its analogs, transferrin, steroid hormone, retinoid, various galectins, a delta opioid receptor ligand, a cholecystokinin A receptor ligand, a ligand specific for the angiotensin AT1 or AT2 receptor, and other molecules that specifically bind to receptors preferentially expressed on the surface of cells such as cancer cells. In one embodiment, the cell targeting molecule is IL4Pep1.

[0175] In another aspect, the cell targeting component can be nucleotides that are RNAs forming a stem and loop structure. In this aspect, the nucleic acid nanostructure delivery composition can be designed such that the polynucleotide chain is folded into a three-dimensional structure via a series of highly engineered stem and loop configurations. In this embodiment, the nucleic acid nanostructure delivery composition can have a high affinity for a protein receptor expressed on a specific cell, such that the nucleic acid nanostructure delivery composition and the payload are targeted to the specific cell. In this embodiment, the polynucleotide that binds to the target cell receptor can bind to a peptide aptamer. In another aspect, the nucleic acid nanostructure delivery composition can be folded such that in the presence of a specific biomarker such as a cell receptor, microRNA, DNA, RNA, or antigen, self base pairs are disrupted and the nucleic acid nanostructure delivery composition can be unfolded, such that as a result, trigger release of the payload is effected only in the presence of the specific biomarker. For example, a lock and key mechanism for triggering the opening of a nucleic acid nanostructure delivery composition (e.g., a DNA origami structure) has been previously demonstrated (Andersen, et al., Nature, Vol. 459, pages 73-76(2009), incorporated herein by reference). In these embodiments, by creating a three-dimensional structure that targets cells and tissues using the nucleic acid nanostructure delivery composition, the immunogenicity of the nucleic acid nanostructure delivery composition is low and the payload is released only when there are present RNA or peptide biomarkers present in the cytoplasm of the target cells and tissues, resulting in fewer side effects and more efficient delivery of the payload.

[0176] In another embodiment, a method of treating a patient having a disease is provided. The method includes administering to the patient any of the nucleic acid nanostructure delivery compositions comprising a small molecule therapeutic agent described herein to treat the patient's disease. In this embodiment, the method can further include administering to the patient a pharmaceutically acceptable carrier.

[0177] In various embodiments, administration of the nucleic acid nanostructure delivery composition conjugated to a small molecule therapeutic agent can use any suitable route including parenteral administration. Routes suitable for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, intrasternal, intracranial, intratumoral, intraosseous, intramuscular and subcutaneous delivery. In one embodiment, the means of parenteral administration includes needle (including microneedle) syringes, needleless syringes and infusion techniques. In other embodiments, oral, pulmonary or topical administration routes can be used.

[0178] In one embodiment, the nucleic acid nanostructure delivery composition comprising the small molecule therapeutic agent described herein can be formulated as a pharmaceutical composition for parenteral or topical administration. Such pharmaceutical compositions and methods for their manufacture are known in the art for both human and non-human mammals. See, for example, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, (1995) A. Gennaro, et al., eds., 19th ed., Mack Publishing Co. The composition may contain additional active ingredients.

[0179] In one exemplary aspect, parenteral formulations are typically aqueous solutions and can contain carriers or excipients such as salts, carbohydrates, buffers (preferably pH 3 to 9), but may be more suitably formulated as sterile non-aqueous solutions or in dry forms for use in combination with a suitable vehicle such as sterile, pyrogen-free water, sterile saline. Preparation under sterile conditions by lyophilization to produce a sterile lyophilized powder for parenteral formulations can be readily achieved using standard pharmaceutical techniques well known to those skilled in the art. In one embodiment, the solubility of the composition used in the preparation of parenteral formulations can be enhanced by using suitable formulation techniques such as incorporation of solubility enhancers.

[0180] In an exemplary embodiment, a pharmaceutical composition for parenteral administration comprises a) a pharmaceutically active amount of a nucleic acid nanostructure delivery composition, b) a pharmaceutically acceptable pH buffer to provide a pH in the range of about pH 4.5 to about pH 9, c) an ionic strength modifier in a concentration range of about 0 to about 300 millimolar, and d) a water-soluble viscosity modifier in a concentration range of about 0.25% to about 10% of the total formula weight, or any combination of a), b), c), and d).

[0181] In various exemplary embodiments, the pH buffers used in the compositions and methods described herein are agents known to those of skill in the art and include, for example, acetic acid, borate, carbonate, citrate, and phosphate buffers, and hydrochloric acid, sodium hydroxide, magnesium oxide, monopotassium phosphate, bicarbonate, ammonia, carbonic acid, hydrochloric acid, sodium citrate, citric acid, acetic acid, disodium hydrogen phosphate, borax, boric acid, sodium hydroxide, diethylbarbituric acid, and proteins, as well as various biological buffers such as TAPS, Bicine, Tris, Tricine, HEPES, TES, MOPS, PIPES, cacodylate, or MES.

[0182] In another exemplary embodiment, the ionic strength regulator includes agents known in the art such as, for example, glycerin, propylene glycol, mannitol, glucose, dextrose, sorbitol, sodium chloride, potassium chloride, and other electrolytes.

[0183] Useful viscosity regulators include, but are not limited to, ionic and non-ionic water-soluble polymers; cross-linked acrylic polymers (e.g., polymers of the "carbomer" family, e.g., carboxy polyalkylenes commercially available under Carbopol®); hydrophilic polymers (e.g., polyethylene oxide, polyoxyethylene-polyoxypropylene copolymers, and polyvinyl alcohol); cellulose polymers and cellulose polymer derivatives (e.g., hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, methyl cellulose, carboxymethyl cellulose, and etherified cellulose); gums such as tragacanth gum and xanthan gum; sodium alginate; gelatin, hyaluronic acid and its salts, chitosan, gellan, or any combination thereof. Usually, non-acidic viscosity enhancers such as neutral or basic agents are used to facilitate the achievement of the desired pH of the formulation.

[0184] In one embodiment, the solubility of the compositions described herein used in the preparation of parenteral formulations can be enhanced by the use of appropriate formulation techniques such as the incorporation of solubility enhancers.

[0185] In other embodiments, the compositions described herein can be administered topically. A variety of dosage forms and bases such as ointments, creams, gels, gel ointments, plasters (e.g., patches, poultices), solutions, powders, etc. can be applied to topical formulations. These formulations can be prepared by any conventional method using conventional pharmaceutically acceptable carriers or diluents described below.

[0186] For example, petrolatum, higher alcohols, beeswax, vegetable oils, polyethylene glycol, etc. can be used. In the preparation of cream preparations, fats and oils, waxes, higher fatty acids, higher alcohols, fatty acid esters, purified water, emulsifiers, etc. can be used. In the preparation of gel preparations, conventional gelling substances such as polyacrylates (for example, sodium polyacrylate), hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, purified water, lower alcohols, polyhydric alcohols, polyethylene glycol, etc. are used. In the preparation of gel ointments, in addition to the above gelling agents, emulsifiers (preferably nonionic surfactants), oily substances (for example, liquid paraffin, triglycerides, etc.), etc. are used. Further, by applying the above gel preparation to a support (for example, fabric, non-woven fabric, etc.), adhesive agents such as poultices and cataplasms can be prepared. In addition to the above components, paraffin, squalane, lanolin, cholesterol esters, higher fatty acid esters, etc. may be optionally used. Furthermore, antioxidants such as BHA, BHT, propyl gallate, pyrogallol, tocopherol, etc. may be formulated. In addition to the above preparations and components, other conventional formulations for blending other additives may be optionally used.

[0187] In various embodiments, the dosage of the nucleic acid nanostructure delivery composition can vary significantly depending on the patient's condition, or the disease state being treated, the route of administration and tissue distribution, as well as the possibility of combination with other therapeutic treatments. The effective amount administered to the patient is based on the body surface area, the patient's weight or mass, and the physician's assessment of the patient's condition. In various embodiments, the nucleic acid nanostructure delivery composition can be administered to a patient having a disease or disorder selected from the group consisting of diabetes, cancer, muscle disorders, myelodysplastic syndromes and severe aplastic anemia, blood diseases or bone marrow failure conditions, lung diseases, skin diseases, neurological diseases, neurofibromatosis 1 (NF1), and abnormal hemoglobinopathies. In one embodiment, the cancer is selected from the group consisting of lung cancer, bone cancer, pancreatic cancer, skin cancer, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, gastric cancer, colon cancer, breast cancer, esophageal cancer, endocrine system cancer, prostate cancer, leukemia, lymphoma, mesothelioma, bladder cancer, kidney cancer, central nervous system tumors, brain tumors, and adenocarcinoma. In another embodiment, the skin disease is a Staphylococcus aureus infection. In yet another embodiment, the muscle disease is muscular dystrophy (e.g., Duchenne muscular dystrophy). In yet another embodiment, the nucleic acid nanostructure delivery composition does not exhibit cytotoxicity to the patient's cells.

[0188] In yet another embodiment, the above-described treatment method can include administering any first composition of the nucleic acid nanostructure delivery composition comprising a small molecule therapeutic agent described herein, and further can include administering a second composition comprising a small molecule therapeutic agent different from the first composition, or a macromolecule such as a monoclonal antibody, polypeptide, or antibody-drug conjugate.

[0189] In yet another embodiment, the above-described treatment method can further include administering a nucleic acid nanostructure delivery composition comprising one or more nucleic acid payloads. In this embodiment, the nucleic acid nanostructure delivery composition can include overhangs that bind via complementary base pairing to the payload nucleic acid.

[0190] As used herein, the term "complementary base pairing" refers to the ability of purine and pyrimidine nucleotide sequences to associate via hydrogen bonds to form a double-stranded nucleic acid molecule. Guanine and cytosine, adenine and thymine, and adenine and uracil are complementary and associate by hydrogen bonds to form a double-stranded nucleic acid molecule when two nucleic acid molecules have "complementary" sequences. Complementary sequences are DNA or RNA sequences. A complementary DNA or RNA sequence is called a "complement".

[0191] In one aspect, a nucleic acid nanostructure delivery composition containing a nucleic acid can encapsulate a nucleic acid of 3 kB or more or another gene payload for delivery to a target cell. In these embodiments, the nucleic acid has a size of 3 kB or more and can be DNA or RNA. In other embodiments, the nucleic acid is about 0.1 kB or more, about 0.2 kB or more, about 0.3 kB or more, about 0.4 kB or more, about 0.5 kB or more, about 0.6 kB or more, about 0.7 kB or more, about 0.8 kB or more, about 0.9 kB or more, about 1 kB or more, about 1.5 kB or more, about 2 kB or more, about 2.5 kB or more, about 3 kB or more, about 3.1 kB or more, about 3.2 kB or more, about 3.3 kB or more, about 3.4 kB or more, about 3.5 kB or more, about 3.6 kB or more, about 3.7 kB or more, about 3.8 kB or more, about 3.9 kB or more, about 4 kB or more, about 4.1 kB or more, about 4.2 kB or more, about 4.3 kB or more, about 4.4 kB or more, about 4.5 kB or more, about 4.6 kB or more, about 4.7 kB or more, about 4.8 kB or more, about 4.9 kB or more, about 5 kB or more, about 5.1 kB or more, about 5.2 kB or more, about 5.3 kB or more, about 5.4 kB or more, about 5.5 kB or more, about 5.6 kB or more, about 5.7 kB or more, about 5.8 kB or more, about 5.9 kB or more, about 6 kB or more, about 6.1 kB or more, about 6.2 kB or more, about 6.3 kB or more, about 6.4 kB or more, about 6.5 kB or more, about 6.6 kB or more, about 6.7 kB or more, about 6.8 kB or more, about 6.9 kB or more, about 7 kB or more, about 7.1 kB or more, about 7.2 kB or more, about 7.3 kB or more, about 7.4 kB or more, about 7.5 kB or more, about 7.6 kB or more, about 7.7 kB or more, about 7.8 kB or more, about 7.9 kB or more, about 8 kB or more, about 8.1 kB or more, about 8.2 kB or more, about 8.3 kB or more, about 8.4 kB or more, about 8.5 kB or more in size.

[0192] In embodiments where nucleic acid nanostructure delivery compositions are used, computer-aided design tools can predict the nucleotide sequences required to produce highly engineered nucleic acid nanostructure delivery compositions. In the case of gene delivery, these nucleic acid nanostructure delivery compositions can provide the advantage of encapsulation efficiency because they can be tailored in terms of the size and shape of the structure to the cargo. In another aspect, the loading efficiency can be enhanced by incorporating the nucleic acid payload into the encapsulated nucleic acid nanostructure delivery composition itself.

[0193] In other embodiments, the nucleic acid payload can bind to the nucleic acid nanostructure delivery composition through high-affinity non-covalent binding interactions between biotin molecules at the 5' and / or 3' ends of the nucleic acid payload and molecules that bind to biotin on the nucleic acid nanostructure delivery composition. In this embodiment, the molecule that binds to biotin can bind to the nucleic acid nanostructure delivery composition through a covalent phosphoramidite bond formed via an EDC-NHS coupling reaction between the terminal phosphate group at the 5' end of the overhang on the nucleic acid nanostructure delivery composition and the amine group on the molecule that binds to biotin. In this embodiment, biotin can bind to the nucleic acid payload by a covalent bond.

[0194] In another exemplary embodiment, the nucleic acid payload can be covalently attached to the nucleic acid nanostructure delivery composition. In this embodiment, the covalent bond can be formed via an EDC-NHS coupling reaction between the terminal phosphate group at the 5' end of the overhang on the nucleic acid nanostructure delivery composition and the amine group on the amino-terminal nucleotide of the nucleic acid payload. In another embodiment, the covalent bond can be formed via a click chemistry coupling reaction between an azide group on the nucleic acid nanostructure delivery composition and an alkyne group on the nucleic acid payload. In yet another embodiment, the covalent bond can be formed via a click chemistry coupling reaction between an azide group on the nucleic acid payload and an alkyne group on the nucleic acid nanostructure delivery composition. In yet another embodiment, the nucleic acid payload can be attached to the nucleic acid nanostructure delivery composition by a covalent bond between a carboxy-terminal molecule on the nucleic acid nanostructure delivery composition and a primary amine on the 5' end and / or 3' end of the nucleic acid payload.

[0195] Exemplary nucleic acid payloads of the nucleic acid nanostructure delivery compositions described herein include any one or combination of compositions selected from the group consisting of nucleic acids (e.g., DNA or RNA), pDNA, oligodeoxyribonucleic acids (ODNs), dsDNA, ssDNA, antisense oligonucleotides, antisense RNA, siRNA, messenger RNA, guide RNA (e.g., small guide RNA), ribonucleoproteins, and donor DNA strands used in the CRISPR / Cas9 system, and enzymes such as CRISPR-associated enzymes (e.g., Cas9, Cas10), other Cas enzymes, enzymes used in other gene editing systems such as ZFNs, custom-designed homing endonucleases, TALENS systems, other gene editing endonucleases, and reverse transcriptase can also be delivered.

[0196] Other exemplary payloads include DNA constructs such as chimeric antigen receptor (CAR) constructs. CAR-T cells are T cells that express a chimeric antigen receptor (CAR). A CAR is a genetically engineered receptor designed to target a specific antigen, such as a tumor antigen. This targeting can, for example, result in cytotoxicity against tumors, and CAR-T cells expressing the CAR can target and kill tumors via a specific tumor antigen. A CAR can include a recognition region derived from an antibody for recognizing and binding an antigen expressed by a tumor, such as a single-chain variable fragment (scFv), an activation signaling domain (e.g., the CD3ζ chain of a T cell can function as a T cell activation signal in a CAR), and a co-stimulatory domain (e.g., CD137, CD28, or CD134) for achieving long-term activation of T cells in vivo. In some aspects, a CAR is a large DNA construct.

[0197] In another embodiment, the nucleic acid payload can be a nucleic acid (e.g., DNA or RNA) having a size selected from the group consisting of 0.1 kB or more, 0.2 kB or more, 0.3 kB or more, 0.4 kB or more, 0.5 kB or more, 0.6 kB or more, 0.7 kB or more, 0.8 kB or more, 0.9 kB or more, 1 kB or more, 1.5 kB or more, 2 kB or more, 2.5 kB or more, 3 kB or more, 3.1 kB or more, 3.2 kB or more, 3.3 kB or more, 3.4 kB or more, 3.5 kB or more, 3.6 kB or more, 3.7 kB or more, 3.8 kB or more, 3.9 kB or more, 4.1 kB or more, 4.2 kB or more, 4.3 kB or more, 4.4 kB or more, 4.5 kB or more, 4.6 kB or more, 4.7 kB or more, 4.8 kB or more, 4.9 kB or more, 5 kB or more, 5.1 kB or more, 5.2 kB or more, 5.3 kB or more, 5.4 kB or more, 5.5 kB or more, 5.6 kB or more, 5.7 kB or more, 5.8 kB or more, 5.9 kB or more, 6 kB or more, 6.1 kB or more, 6.2 kB or more, 6.3 kB or more, 6.4 kB or more, 6.5 kB or more, 6.6 kB or more, 6.7 kB or more, 6.8 kB or more, 6.9 kB or more, 7 kB or more, 7.1 kB or more, 7.2 kB or more, 7.3 kB or more, 7.4 kB or more, 7.5 kB or more, 7.6 kB or more, 7.7 kB or more, 7.8 kB or more, 7.9 kB or more, 8 kB or more, 8.1 kB or more, 8.2 kB or more, 8.3 kB or more, 8.4 kB or more, and 8.5 kB or more.

[0198] In various embodiments, the payload can be one or more of the components of a CRISPR RNP system, including a CRISPR-associated enzyme (e.g., Cas9), a short guide RNA (sgRNA), and a donor DNA strand. In embodiments where the payload includes Cas9, Cas9 can be fused to a deaminase. In yet another embodiment, the nucleic acid payload can include an sgRNA that is used to target an enzyme to a specific genomic sequence. In another aspect, the target enzyme can be a CRISPR-associated enzyme. In another exemplary aspect, the payload can include one molecule each of CRISPR / Cas9, sgRNA, and donor DNA strand in the nucleic acid nanostructure delivery composition described herein. In another embodiment, the payload can be a nucleic acid used for homology-directed repair or a nucleic acid used as a transposable element. In yet another embodiment, the payload can be any of the payloads in the form of plasmid constructs described herein.

[0199] In one aspect, the nucleic acid nanostructure delivery composition described herein can encapsulate a payload for use in gene editing. In one aspect, the CRISPR / Cas9 system serves as the payload and can be used for gene editing. In another embodiment, other gene editing systems such as ZFNs, custom-designed homing endonucleases, and TALENS systems can serve as the payload. In embodiments where the CRISPR / Cas9 system is the payload, the Cas9 endonuclease can introduce double-strand breaks into a DNA target sequence. In this aspect, the Cas9 endonuclease is guided by a guide polynucleotide (e.g., sgRNA), recognizes and, if necessary, introduces double-strand breaks at specific target sites within the genome of a cell. In this exemplary embodiment, the Cas9 endonuclease can unwind the DNA double helix near the genomic target site and cleave both target DNA strands upon recognition of the target sequence by the guide polynucleotide (e.g., sgRNA), but only when the correct protospacer adjacent motif (PAM) is oriented approximately towards the 3' end of the target. In this embodiment, donor DNA strands can be integrated into the genomic target site. The CRISPR / Cas9 system for gene editing is well known in the art.

[0200] In another exemplary embodiment, the payload includes a DNA segment that functions as a nuclear localization signal and enhances nuclear delivery of the nucleic acid nanostructure delivery composition upon endosomal escape. In another aspect, the nucleic acid payload includes a nucleotide sequence designed to bind as an aptamer to an endosomal receptor and enhances intracellular trafficking of the nucleic acid nanostructure delivery composition.

[0201] In an exemplary embodiment, a nucleic acid nanostructure delivery composition (e.g., a DNA origami) is provided that packages together a Cas9 protein, an sgRNA, and a single-stranded donor DNA strand into one nanostructure to ensure that all components are co-delivered simultaneously to a specific location. In this embodiment, the single-stranded nature of the sgRNA and the donor DNA strand can be utilized to convert these components into components of the nucleic acid nanostructure delivery composition (e.g., a DNA origami structure), whereby these components are delivered together and dissociate from the DNA nanostructure delivery composition simultaneously upon reaching the target site (e.g., a target cell). In this embodiment, the DNA nanostructure delivery composition can deliver either the plasmid or ribonucleoprotein (RNP) form of CRISPR / Cas9.

[0202] References herein to "one embodiment", "an embodiment", "exemplary embodiment", etc., indicate that the embodiment described may include a particular function, structure, or characteristic, but not every embodiment necessarily includes that particular function, structure, or characteristic. Moreover, such phrases do not necessarily refer to the same embodiment. Further, when a particular function, structure, or characteristic is described in connection with an embodiment, it is contended that it is within the knowledge of one of ordinary skill in the art to implement such function, structure, or characteristic in connection with other embodiments, whether or not explicitly described. Further, it should be understood that items listed in the form of "at least one of A, B, and C" can mean (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C). Similarly, items listed in the form of "at least one of A, B, or C" can mean (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).

[0203] In the drawings, some structural or method features may be shown in a particular arrangement and / or order. However, it should be understood that such a particular arrangement and / or order may not be essential. Rather, in some embodiments, such features may be arranged in a manner and / or order different from that shown in the exemplary figures. Further, the inclusion of a structural or method feature in a particular figure does not mean that such a feature is essential in all embodiments, and in some embodiments, it may be absent or combined with other features.

[0204] In the drawings and the foregoing description, several exemplary embodiments have been described in detail, but such descriptions and explanations should be considered exemplary and not restrictive, and only the exemplary embodiments are shown and described, and it is understood that all changes and modifications within the spirit of the present disclosure are desired to be protected. There are multiple advantages of the present disclosure arising from the various features of the apparatus, system, and method described herein. It should be noted that alternative embodiments of the apparatus, system, and method of the present disclosure may not include all of the described features, but can still benefit from at least some of the advantages of such features. A person skilled in the art can easily devise unique implementations of the apparatus, system, and method incorporating one or more of the features of the present disclosure.

[0205] The concept of the present disclosure allows for various changes and alternative forms, and its specific embodiments are shown by way of example in the accompanying drawings and described in detail herein. However, it is understood that there is no intention to limit the concept of the present disclosure to the specific forms disclosed, and conversely, it is intended to cover all changes, equivalents, and alternatives consistent with the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLES

[0206] Example 1 Self-organization and Characterization of DNA Origami Structures

[0207] DNAO nanostructures were designed using CaDNAno software and self-organized by folding a 7560-nucleotide-long M13mp18 single-stranded scaffold and single-stranded oligonucleotide staples 22 - 58 nucleotides in length. The scaffold and staples were mixed at a ratio of 1:2 in a solution containing 10 mM EDTA, 50 mM TrisBase, 50 mM NaCl, 200 mM MgCl2, and DI water. The mixture was then subjected to a 42-hour thermal annealing process, heated to 65 °C for 1 hour, then cooled at a rate of 1 °C and subsequently held at 4 °C until needed. The excess staple strands after folding were purified using a polyethylene glycol (PEG)-based precipitation method. For this, the target volume of the DNAO nanostructure was mixed with an equal volume of 15% PEG8000 and centrifuged at 16,000 g for 30 minutes. The supernatant was removed and the DNAO nanostructure was resuspended in TrisEDTA buffer containing 20 mM MgCl2.

Example

[0208] Example 2 Functionalization, Loading, and Characterization of Cell-Targeted DNAO-TLBST Nanostructures

[0209] DNA origami (DNAO) can prevent the degradation of drugs by encapsulation and reduce the minimum effective dose by the selective delivery of drugs to target tissues. Therefore, a DNAO nanocarrier loaded with talabostat (TLBST) was designed and synthesized. First, a UV-visible spectroscopy-based method for quantifying the TLBST concentration in solution was developed. Unlike doxorubicin (Dox), which has been well characterized in nanoparticle-based clinical studies, TLBST is delivered only in its free form. Also, unlike Dox, which is a red powder with a fluorescence spectrum in the visible range, talabostat mesylate (MW = 310.18 Da) is a colorless powder with no absorption in the visible spectrum. TLBST was found to exhibit stable absorption in the deep UV region of approximately 208 nm (Figure 1). The calibration curve (Figure 1 (insert)) created at four time points using the peak absorbance value at 208 nm had an R2 > 0.98 and showed a linear and stable correlation.

[0210] For the nanocarrier, a solid rectangular parallelepiped shape with an aspect ratio of 2.3 (50 nm x 21 nm x 16 nm) was selected. This is because it correlates with an increase in cell uptake in vitro. Electrostatic loading of TLBST was performed at various concentrations and durations. The inventors of the present application have previously functionalized rectangular DNAO with biotin and a fluorophore, stabilized it against nuclease degradation using polyethylene glycol (PEG)-poly-L-lysine (PEG-PLL), and demonstrated the cell uptake of Cy5-functionalized DNAO and PEG-PLL DNAO in HEK293T cells (data not shown).

Example

[0211] Example 3 Optimization of rectangular DNAO functionalization with a cell-targeting peptide, confirmation of uptake specificity in vitro, and characterization of drug loading efficiency and structural stability in the presence of serum nuclease

[0212] DNAO nanostructures can be functionalized in multiple ways. In some cases, the molecule of interest is attached to an oligonucleotide and then hybridized to a complementary region of an extended staple strand (handle or overhang) on the DNAO structure. Cell-targeting peptides (CTPs) bind to charge-neutral peptide nucleic acids, PNAs, rather than DNA oligonucleotides. PNA is a synthetic polymer of repeating peptide-like amide units (N-(2-aminoethyl)glycine) that mimics nucleic acids in terms of hybridization affinity and specificity by base pairing and is increasingly being used as a research tool for therapeutic agents. Its uncharged backbone results in a higher binding affinity to DNA than DNA:DNA and is suitable for binding to proteins and peptides. Systematically optimize the binding of IL4RPep1 to PNA and DNA oligonucleotides and compare the functionalization yields of both. PNA-IL4R-pep1 and DNA-IL4R-pep1 conjugates are synthesized by click chemistry to couple azide-modified PNA and DNA oligonucleotides to alkyne-modified IL4R-pep1. Cuboid DNAO is designed using CaDNAno and self-assembled by folding a 7560-nucleotide-long M13 scaffold (tilibit nanosystems) and staple oligonucleotides (Integrated DNA Technologies) using a published protocol (Wagenbauer, K. F. et al. How we make DNA origami. ChemBioChem 18, 1873-1885 (2017)). Staple strands for the functionalization with fluorophores and CTPs are included as needed. The quality and robustness of the PEG-purified structures are evaluated using both agarose gel electrophoresis (AGE) and TEM. The assembly of CTP-DNAO is optimized by incubating PNA-CTP and DNA-CTP with a molar excess (2-fold to 10-fold) of DNA / PNA-CTP at 35 °C for various periods (30 minutes to 2 hours) to hybridize them to complementary overhangs on DNAO. Excess DNA / PNA-CTP is removed by ultracentrifugation.The yield and degree of binding of the product are evaluated by UV-Vis spectroscopy.

[0213] The effect of CTP functionalization on DNAO uptake is examined in THP-1 cells. The characteristics of pyroptosis were first observed in macrophages treated with Bacillus anthracis lethal toxin or macrophages infected with Shigella or Salmonella, and macrophages are the most commonly used cell type in pyroptosis research. Therefore, phorbol myristate acetate (PMA) is used to induce macrophage differentiation of THP-1 cells. THP-1 cells differentiated into macrophages are optimal for cell analysis because they show both cytotoxicity and IL-1β release in response to free TLRBST. The DNAO nanocarrier is targeted to THP-1 cells using the IL4R-pep1 CTP peptide, which is used to localize therapeutic agents to both mouse and human tumor cells and tumor-associated macrophages. Each DNAO cuboid is tagged with the same number of Cy5 fluorophores to visualize cargo delivery in vitro. Differentiated THP-1 cells are seeded in 24-well plates and cultured overnight. Then, the cells are incubated with buffer (untreated), bare DNAO, or DNAO-CTP for 12 - 24 hours. After incubation, the cells are washed and fluorescence is measured by flow cytometry.

[0214] The inventors of the present application have demonstrated a method of electrostatically loading TLBST onto DNAO, but have not characterized the TLBST loading efficiency (LE) at various DNAO concentrations or TLBST concentrations <0.3125 mg / ml. To load TLBST, DNAO (final concentration 10 nM - 30 nM) is mixed with TLBST (final concentration 0.08 - 1.25 mg / ml) and continuously shaken on a stirring plate for 0.5 - 4 hours. The mixture is filtered by ultracentrifugation, and the concentration of excess TLBST is measured using UV-VIS. LE is calculated. Finally, the stability of these structures is tested at 37°C in the presence of nuclease. Cell culture medium supplemented with mammalian serum (fetal bovine serum (FBS)) containing nuclease is used as an alternative to physiological in vivo conditions. DNAO is cultured in RPMI1640 medium containing 10% FBS for 2 - 24 hours. The cultured product is analyzed by AGE to quantify degradation by serum nuclease. Refer to Figure 3 for a schematic diagram of the method of this example.

Example

[0215] Example 4 DNAO-TLBST induces cytotoxicity and the accompanying release of IL-1β, IL-18, and IFNβ in mouse macrophages. Talabostat mesylate (MedChemExpress; TLBST) was reconstituted at 10 mg / mL in deionized water and stored frozen. Before use, TLBST was thawed, incubated at 37 °C for 20 minutes, and sonicated for 10 minutes. TLBST was serially diluted to 5 mg / mL and 2.5 mg / mL in pH 7.4, 40 mM Tris, 10 mM MgCl2 buffer. Free talabostat controls were prepared in triplicate by diluting and serially diluting 10, 5, 2.5 mg / mL TLBST stocks in pH 7.4, 40 mM Tris-HCl, 10 mM MgCl2 buffer to generate 1.25, 0.625, 0.3125 mg / mL free TLBST. DNAO controls (0 mg / mL TLBST) were prepared in triplicate by diluting a 144.5 nM purified DNAO stock in TE to 20 nM concentration in 20 mM MgCl2 buffer and pH 7.4, 40 mM Tris-HCl, 10 mM MgCl2 buffer. DNAO was loaded with TLBST at 20 nM in triplicate experiments by incubating DNAO with 1.25, 0.625, 0.3125 mg / mL TLBST. DNAO samples loaded with 0 - 1.25 mg / mL TLBST and free TLBST controls of 0.3125 - 1.25 mg / mL were incubated at 150 rpm for 2 hours on a stirring table.

[0216] RAW264.7 cells were seeded at 100,000 cells per well in a 96-well plate. After 24 hours, 20 μL of the indicated substance was added to 80 μL of the cells in the growth medium to stimulate the cells. The medium represents TrisTE buffer. DNAO represents cuboid DNAO (assembled using the 7560-nucleotide M13mp18 scaffold of tilibit nanosystems) at 9 nM in TE buffer. 0.9 mM free TLBST represents talabostat mesylate (MedChemExpress) reconstituted with deionized water, stored frozen, and diluted with Tris buffer. DNAO loaded in 1 mM TLBST was incubated for 2 hours with shaking in a solution of 1 mM talabostat mesylate, followed by purification with a 100 kDa cut-off by ultracentrifugation and dilution with TrisTE buffer, and represents cuboid DNAO. Twenty-four hours after stimulation, the cell culture supernatant was collected and stored frozen until quantification of lactate dehydrogenase (LDH) by colorimetric enzyme activity assay, and quantification of interleukin 1β (IL-1β), interleukin 18 (IL-18), and interferon β (IFNβ) by multiplex cytometric bead array assay. The LDH release rate was calculated relative to the maximum LDH released from the lysed cell control. Biomarker concentrations were interpolated from the reference standard curve for each analyte. The MTT signal from the stimulated cells was quantified by colorimetric enzyme activity assay. The viability was calculated relative to the MTT signal measured in the untreated cell control. All enzyme activity and biomarker assays were performed using commercially available kits according to the manufacturer's recommendations.

[0217] Figure 4 shows the effect of DNAO-TLBST on cytotoxicity and cytokine release in mouse macrophages. The results of mouse macrophages treated with non-functionalized DNAO-TLBST suggest that these nanocarriers deliver and release TLBST intracellularly, leading to pyroptosis induction.

Example

[0218] Example 5 In Vitro Effects of CTP-DNAO-TLBST on Human Bone Marrow Cells Pyroptosis is characterized by inflammasome activation, maturation of IL-1β and IL-18 via caspase 1, and release of inflammatory cell contents through pore formation and cell lysis in the cell membrane. Systemic administration of pyroptosis-inducing small molecules for cancer treatment is an area of active research. The inventors have shown that DNAO-TLBST reproduces the effect of free TLBST on cytotoxicity and LDH release in mouse macrophages (Figure 4). This indicates that DNAO delivers biologically active TLBST to cells. In this example, the cytotoxicity and immunogenicity of IL4RPep-1-functionalized DNAO-TLBST (CTP-DNAO-TLBST) against human macrophage cytotoxicity are tested.

[0219] To avoid different cell death responses and cross-talk between different cells, the effect of CTP-DNAO-TLBST described in Example 3 on the cytotoxicity of human macrophages in vitro is evaluated. Differentiated THP-1 cells are treated as shown in Figure 5. After 24 hours of stimulation, the supernatant and cells are collected. A portion of each supernatant sample is used to assay cytotoxicity via a colorimetric enzyme assay that measures lactate dehydrogenase (LDH). Cells collected from each fraction are used to analyze cell viability. Exposed phosphatidylserine (PS; a characteristic of apoptotic cells) is detected by annexin V staining. The integrity of the cell membrane is evaluated using the fluorescent DNA-binding dye 7-AAD. Staining and subsequent flow cytometry acquisition are performed in 96-well plate format to improve throughput and enable quantification of cell numbers using a syringe aspiration acquisition system. Non-apoptotic cytotoxicity is indicated by a decrease in viable cells (annexin V-7-AAD-), an increase in LDH release, and the absence of annexin V+ cells. These characteristics may or may not be accompanied by an increase in dead cells (7-AAD+) depending on the kinetics of cell death. Apoptotic cytotoxicity is indicated by a decrease in viable cells and an increase in annexin V+ cells, regardless of the presence or absence of an increase in dead cells and LDH release. CTP-DNAO-TLBST is predicted to induce greater cytotoxicity compared to non-functionalized DNAO-TLBST. Furthermore, CTP-DNAO-TLBST-induced cytotoxicity is expected to be consistent with non-apoptotic cell death (Table 1).

[0220] Apoptotic cells present and release anti-inflammatory and regenerative mediators such as prostaglandin E2 (PGE2), transforming growth factor beta (TGFβ), and IL-10, promoting cell proliferation and immunosuppression. In addition to cytotoxicity, our analysis has demonstrated that IL-1β and IL-18 are released from DNAO-TLBST-treated mouse macrophages (Figure 4), indicating that DNAO-TLBST induces macrophage pyroptosis. IL-1β and IL-18 promote the maturation and antigen presentation of dendritic cells (DCs), thereby promoting T helper type 1 CD4+ and CD8+ T cell responses, interferon gamma (IFNγ) production, tumor antigen presentation, and the generation of an antitumor immune response.

[0221] DNAO not only delivers the TLBST payload to target cells but is also an endogenous activator of endosomal and cytoplasmic DNA sensors. When activated, these sensors establish an antiviral immune program characterized by the expression of type I interferons (IFNα and IFNβ). Thus, DNA encapsulation functions as an adjuvant that may accelerate T cell-mediated immunity. Indeed, DNAO-TLBST is associated with IFNβ secretion from mouse macrophages, whereas free TLBST is not (Figure 4). Using the supernatant of the same samples outlined above in this example, a multiplex bead-based immunoassay is used to profile the inflammatory secretome of human macrophages after CTP-DNAO-TLBST and control treatments. This panel includes type I IFN, IFNγ, IL-1α, IL-1β, IL-10, IL-18, MCP-1 / CCL2, TNFα, IL-6, and IL-12p70. The concentration of each analyte is quantified individually, and the relationship between pro-inflammatory and anti-inflammatory mediators is examined as the ratio of each pro-inflammatory factor to IL-10. CTP-DNAO-TLBST is expected to induce the secretion of type IIFN, IL-1β, and IL-18, which are major pro-inflammatory endpoints, and IFNγ, MCP-1 / CCL2, TNFα, IL-6, and IL-12p70, which are secondary pro-inflammatory endpoints, may increase, while IL-10, which is an anti-inflammatory endpoint, is hardly or not induced at all. A similar profile is expected for non-functionalized TLBST-DNAO, but the effect is reduced due to a decrease in the extent or rate of cell uptake. Free TLBST is expected to have a relatively specific effect on IL-1β and IL-18, and DNAO on type 1 IFN (Table 2).

[0222]

Table 1

[0223]

Table 2

Example

[0224] Example 6 DNAO-TLBST induces cytotoxicity and the accompanying release of IL-1β, IL-18, and IFNβ in human macrophages. Talabostat mesylate (MedChemExpress; TLBST) was reconstituted to 10 mg / mL in pH 7.4, 40 mM Tris, 10 mM MgCl2 buffer. Before use, TLBST was incubated at 37 °C for 20 minutes and sonicated for 10 minutes. TLBST was serially diluted to 5 mg / mL and 2.5 mg / mL in pH 7.4, 40 mM Tris, 10 mM MgCl2 buffer. Free TLBST controls were prepared by diluting 10, 5, 2.5 mg / mL stock solutions and serial dilutions with pH 7.4, 40 mM Tris-HCl, 10 mM MgCl2 buffer to generate 1.25, 0.625, 0.3125 mg / mL free TLBST. DNAO controls containing 0 mg / mL talabostat were prepared by diluting a 162 nM purified DNAO stock in TE to 20 nM concentration with 20 mM MgCl2 buffer and pH 7.4, 40 mM Tris-HCl, 10 mM MgCl2 buffer. DNAO loading was performed in duplicate by incubating 20 nM DNAO in 1.25, 0.625, 0.3125 mg / mL TLBST. DNAO samples loaded with 0 - 1.25 mg / mL TLBST and free TLBST controls of 0.3125 - 1.25 mg / mL were incubated on a stirring table at 150 rpm for 2 hours.

[0225] THP-1 cells seeded at 100,000 cells per well of a 96-well plate were induced to differentiate into macrophages by administration of 20 ng / mL phorbol myristate acetate (PMA). Differentiation in the presence of PMA was allowed to proceed for 3 days before removing the PMA-containing medium and replacing it with normal growth medium. After 3 days, the cells were stimulated by adding 20 μL of the indicated substance to 80 μL of cells in the growth medium. The medium represents TrisTE buffer. DNAO represents cuboid DNAO (assembled using the 7560 nucleotide M13mp18 scaffold of tilibit nanosystems) at 9 nM in TE buffer. 1 mM free TLBST represents talabostat mesylate (MedChemExpress) reconstituted in deionized water and diluted with Tris buffer. DNAO mounted in 1 mM TLBST represents cuboid DNAO that was incubated for 2 hours with shaking in a solution of 1 mM talabostat mesylate, followed by purification with a 100 kDa cut-off by ultracentrifugation and dilution with TrisTE buffer. Twenty-four hours after stimulation, the cell culture supernatant was collected, clarified by centrifugation, and cryopreserved until quantification of lactate dehydrogenase (LDH) by colorimetric enzyme activity assay and quantification of interleukin 1β (IL-1β), interleukin 18 (IL-18), and interferon β (IFNβ) by multiplex cytometric bead array assay. The LDH release rate was calculated relative to the maximum LDH released from a lysed cell control. Biomarker concentrations were interpolated from the reference standard curves for each analyte. The LDH enzyme activity and biomarker assays were performed using commercially available kits according to the manufacturer's recommendations.

[0226] Figure 6 shows the effects of DNAO-TLBST on the cytotoxicity and cytokine release of human macrophages. The results for human macrophages treated with non-functionalized DNAO-TLBST suggest that these nanocarriers deliver and release TLBST intracellularly, leading to pyroptosis induction.

Example

[0227] Example 7 DNAO-TLBST induces cytotoxicity and the accompanying release of IL-1β, IL-18, and IFNβ in human prostate epithelial cells. Talabostat mesylate (MedChemExpress; TLBST) was reconstituted in deionized water to 10 mg / mL and stored frozen. TLBST was thawed before use, incubated at 37 °C for 20 min, and sonicated for 10 min. TLBST was serially diluted to 5 mg / mL and 2.5 mg / mL in pH 7.4, 40 mM Tris, 10 mM MgCl2 buffer. Free TLBST controls were prepared in triplicate by diluting 10, 5, 2.5 mg / mL TLBST stock and serial dilutions in pH 7.4, 40 mM Tris-HCl, 10 mM MgCl2 buffer to generate 1.25, 0.625, 0.3125 mg / mL solutions. The DNAO control for 0 mg / mL TLBST was prepared in triplicate by diluting a 143 nM purified DNAO stock in TE to 20 nM concentration with 20 mM MgCl2 buffer and pH 7.4, 40 mM Tris-HCl, 10 mM MgCl2 buffer. DNAO was loaded in triplicate reactions by incubation with 1.25, 0.625, 0.3125 mg / mL TLBST at 20 nM DNAO. Subsequently, DNAO samples loaded with 0–1.25 mg / mL TLBST and free TLBST controls of 0.3125–1.25 mg / mL were incubated at 150 rpm for 2 h on a stirring table.

[0228] PC-3 cells were seeded at 10,000 cells per well of a 96-well plate 24 hours before stimulation, and 20 μL of the designated substance was added to 80 μL of cells in the growth medium. The medium represents TrisTE buffer. DNAO represents 9 nM cuboid DNAO (assembled using the 7560 nucleotide M13mp18 scaffold of tilibit nanosystems) in TE buffer. 0.9 mM free TLBST represents talabostat mesylate (MedChemExpress) reconstituted with deionized water and diluted with Tris buffer. DNAO loaded in 1 mM TLBST represents cuboid DNAO incubated for 2 hours in a solution of 1 mM talabostat mesylate with shaking before ultracentrifugation-based 100 kDa cut-off purification and dilution with TrisTE buffer. Twenty-four hours after stimulation, the cell culture supernatant was collected, clarified by centrifugation, and cryopreserved until quantification of lactate dehydrogenase (LDH) by a colorimetric enzyme activity assay was performed according to the manufacturer's recommendations. The LDH release rate was calculated compared to the maximum LDH released from the lysed cell control.

[0229] Figure 7 shows the effect of DNAO-TLBST on cytotoxicity and cytokine release in human prostate epithelial cells. The results of human prostate epithelial cells treated with non-functionalized DNAO-TLBST suggest that these nanocarriers deliver and release TLBST intracellularly, leading to pyroptosis induction.

Claims

1. A composition comprising a nonviral delivery medium containing a nucleic acid nanostructure delivery composition and a small molecule therapeutic agent, wherein the nucleic acid nanostructure delivery composition contains a DNA origami composition and the small molecule therapeutic agent is tarabostat.

2. The composition according to claim 1, wherein the nucleic acid nanostructure delivery composition further comprises a cell target molecule.

3. The composition according to claim 1, wherein tarabostat is bound to the nucleic acid nanostructure delivery composition by electrostatic interaction between the nucleic acid of the negatively charged nucleic acid nanostructure delivery composition and the positively charged amine in the small molecule tarabostat.

4. The composition according to claim 3, wherein the nucleic acid nanostructure delivery composition comprises a single-stranded DNA scaffold containing M13 DNA.

5. The composition according to claim 3, wherein the nucleic acid nanostructure delivery composition comprises a single-stranded DNA scaffold containing M13mp18 DNA; and a plurality of single-stranded oligonucleotide staples that bind to the DNA scaffold and fold the DNA scaffold into a rectangular parallelepiped shape.

6. The composition according to claim 5, wherein the DNA scaffold is an M13mp18 single-stranded scaffold with a length of 7560 nucleotides, and the plurality of single-stranded oligonucleotide staples have lengths selected from 22 to 58 nucleotides.

7. The composition according to claim 1, wherein the small molecule therapeutic agent causes pyroptosis.

8. The composition according to claim 1 for use in the treatment of patients with a disease.

9. The composition for use according to claim 8, wherein the disease is cancer, and tarabostat induces antitumor immunity in the patient.

10. The composition for use according to claim 9, wherein the nucleic acid nanostructure delivery composition is functionalized with IL4RPep-1.

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