Non-viral delivery compositions and screening methods

Abstract

The present invention relates to barcode-encoded nucleic acid nanostructure delivery compositions for in vivo screening for use in therapeutic delivery in the body, and methods thereof. More specifically, the present invention relates to nucleic acid nanostructure delivery compositions such as DNA origami structures conjugated with barcodes for high-throughput in vivo screening of nucleic acid nanostructure delivery compositions for use in drug delivery, and methods thereof.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 350,688, filed on June 9, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

[0002] Field of the disclosure

[0003] The present invention relates to barcode - encoded nucleic acid nanostructure delivery compositions for in - vivo screening for in - vivo therapeutic delivery, and methods thereof. More specifically, the present invention relates to nucleic acid nanostructure delivery compositions, such as DNA origami structures conjugated with barcodes for high - throughput in - vivo screening of nucleic acid nanostructure delivery compositions for drug delivery, and methods thereof.

Summary of the Invention

[0004] Background and overview

[0005] Gene therapies (including gene therapy, gene silencing, splicing regulators, and nuclease-based gene editors) are poised to create revolutionary treatments, including vaccines, infectious disease treatments, antibacterial treatments, antiviral treatments, and most notably, hereditary disease treatments. However, delivering the payload of these gene therapies to the specific tissues and cells in the body that need treatment while avoiding tissues and cells that could potentially reduce the efficacy or safety of the gene therapies is a major challenge. Additional challenges include the ability to deliver large gene payloads or multiple payloads. Adeno-associated virus (AAV) is the most widely used tool for gene therapy delivery, but AAV cannot deliver large gene payloads or multiple payloads (such as the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 system), and can cause unwanted immune responses, including the production of anti-AAV antibodies, which are cell-mediated responses. Some of the immune responses caused by AAV in patients can be potentially life-threatening.

[0006] Therapies based on the CRISPR / Cas9 system hold great promise for treating many hereditary diseases due to the precise and programmable gene editing capabilities of this system. There are two main mechanisms for gene editing and repair using the CRISPR / Cas9 system: non-homologous end joining (NHEJ), which induces random indel mutations to repair the cleavage site, and homology-directed repair (HDR), which repairs the cleavage site based on an existing template. Because pre-designed templates can be used for HDR-mediated repair, therapies based on this mechanism can be tailored to the treatment of various hereditary diseases. However, the main challenge is that HDR repair requires the simultaneous delivery of CRISPR / Cas9, small guide RNA (sgRNA), and a donor DNA strand to a specific location. This requirement is particularly limiting for in vivo applications because it is currently impossible to reliably deliver multiple large molecules to the same target site. For example, the Cas9 enzyme sequence and guide RNA complex are too large to fit into AAV.

[0007] There is a need for non-viral delivery systems that are effective not only for gene delivery systems but also for the delivery of small molecule therapeutics. 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. Furthermore, such non-viral gene delivery systems are easily degraded by various enzymes when passing through intracellular or intercellular compartments, and there are also concerns that these systems cannot package multiple large payloads.

[0008] The inventors designed nucleic acid nanostructure delivery compositions (e.g., DNA origami nanostructures). These compositions have the advantages of being biocompatible, non-toxic, and programmable in various ways. For example, nucleic acid nanostructure delivery compositions can be programmed to have functional groups that avoid early degradation, avoid immune responses, and enable intracellular imaging and targeted and controlled delivery of therapeutic genes and small molecule therapeutics. Thus, these non-viral delivery compositions can enhance the stability, safety, and / or efficacy of the payload by providing the ability to avoid immune responses, tissue-directed intracellular delivery, and deliver large gene payloads or multiple payloads, or other gene drug payloads, or small molecule therapeutics.

[0009] The rate-limiting step in the development of such drug delivery vehicles is the in vivo testing of the drug delivery vehicle, which results in delays and non-optimization of the final product and a shortage of new drug delivery vehicle candidates. The inventors demonstrated the utility of nucleic acid nanostructure delivery compositions (e.g., DNA origami nanostructure compositions) for in vivo delivery and also developed a novel method for labeling nucleic acid nanostructure delivery compositions with unique barcodes, administering them to animals, and then extracting and detecting them from animal tissues. This method enables in vivo high-throughput screening of various drug delivery nanoparticles containing DNA origami structures and can be used for the delivery of large gene payloads, multiple payloads, other gene drug payloads, or small molecule therapeutics.

[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 titled "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 vehicle comprising a nucleic acid nanostructure delivery composition and a nucleic acid barcode construct.

[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 any one of clauses 1 to 3, wherein the nucleic acid barcode construct is bound to the nucleic acid nanostructure delivery composition via base pairing.

[0015] 5. The composition of clause 4, wherein the base pairing occurs between the sequence of the single-stranded overhang on the nucleic acid nanostructure delivery composition and the complementary sequence added to the nucleic acid barcode construct.

[0016] 6. The composition according to any one of clauses 1 to 3, wherein the nucleic acid nanostructure delivery composition comprises staples that self-assemble to form the nucleic acid nanostructure delivery composition.

[0017] 7. The composition of clause 6, wherein the staples function as the nucleic acid barcode construct.

[0018] 8. The composition according to any one of clauses 1 to 3, wherein the nucleic acid barcode construct is bound to the nucleic acid nanostructure delivery composition by a high-affinity non-covalent interaction between a biotin molecule at the 5' end and / or 3' end of the nucleic acid barcode construct and a molecule that binds to biotin on the nucleic acid nanostructure delivery composition.

[0019] 9. The composition according to item 8, wherein the molecule that binds to biotin is bound to the nucleic acid nanostructure delivery composition by a covalent phosphoramidate 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.

[0020] 10. The composition according to item 8 or 9, wherein biotin is bound to the nucleic acid barcode construct by a covalent bond.

[0021] 11. The composition according to any one of items 1 to 3, wherein the nucleic acid barcode construct is bound to the nucleic acid nanostructure delivery composition by a covalent bond.

[0022] 12. The composition according to item 11, wherein the covalent bond is 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 amino-terminal nucleotide of the nucleic acid barcode construct.

[0023] 13. The composition according to item 11, wherein the covalent bond is formed through a click chemistry coupling reaction between the azide group on the nucleic acid nanostructure delivery composition and the alkyne group on the nucleic acid barcode construct.

[0024] 14. The composition according to item 11, wherein the covalent bond is formed through a click chemistry coupling reaction between the azide group on the nucleic acid barcode construct and the alkyne group on the nucleic acid nanostructure delivery composition.

[0025] 15. The composition according to any one of items 1 to 3, wherein the nucleic acid barcode construct 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 nucleic acid barcode construct at the 5'-end and / or 3'-end.

[0026] 16. The composition according to any one of claims 1 to 3, wherein the nucleic acid barcode construct comprises two primer binding segments and one or more unique barcode sequences between the two primer binding segments.

[0027] 17. The composition according to claim 16, wherein the length of the primer binding segment ranges from about 15 base pairs to about 30 base pairs.

[0028] 18. The composition according to claim 16 or 17, wherein the primer binding segment is a universal primer binding set.

[0029] 19. The composition according to any one of claims 16 to 18, wherein the one or more unique barcode sequences comprise unique sequences having a length of about 6 to about 20 nucleotides.

[0030] 20. The composition according to any one of claims 16 to 19, wherein the length of the unique barcode sequence is at least twice the length of the primer binding segment.

[0031] 21. The composition according to any one of claims 1 to 20, wherein the nucleic acid barcode construct comprises DNA or RNA.

[0032] 22. The composition according to any one of claims 16 to 21, wherein the unique barcode sequence further comprises a Hamming distance of at least 2 to 6 bases between any two unique barcode sequences.

[0033] 23. The composition according to any one of the preceding claims, wherein the nucleic acid barcode construct further comprises about 6 to about 12 random bases at the 3' end of the unique barcode sequence.

[0034] 24. The composition according to claim 23, wherein the about 6 to about 12 random bases at the 3' end of the unique barcode sequence are for bioinformatics removal of PCR duplicates.

[0035] 25. The composition according to any one of the preceding items, wherein the length of the nucleic acid barcode construct ranges from about 42 nucleotides to about 210 nucleotides.

[0036] 26. A method for screening a desired nucleic acid nanostructure delivery composition in vivo, comprising: (a) preparing a library comprising two or more nucleic acid nanostructure delivery compositions, wherein each nucleic acid nanostructure delivery composition is associated with a nucleic acid barcode construct comprising a different unique barcode sequence; (b) administering the library to an animal; (c) removing cells or tissues from the animal; (d) isolating the nucleic acid barcode construct from the cells or tissues of the animal; (e) detecting the nucleic acid barcode construct in the cells or tissues of the animal; and (f) identifying the desired nucleic acid nanostructure delivery composition for use as a delivery vehicle.

[0037] 27. The method according to item 26, wherein the nucleic acid nanostructure delivery composition is associated with the nucleic acid barcode construct according to any one of items 4 to 15.

[0038] 28. The method according to item 26 or 27, wherein the nucleic acid barcode structure is detected by a method selected from the group consisting of polymerase chain reaction (PCR), isothermal amplification, sequencing, or combinations thereof, and nucleotide sequence data is obtained.

[0039] 29. The method according to any one of items 26 to 28, wherein the nucleic acid nanostructure delivery composition is loaded with a payload.

[0040] 30. The method according to item 29, wherein the payload is a luminescent molecule.

[0041] 31. The method according to item 30, wherein luminescence is used to track the in vivo distribution or cellular uptake of the nucleic acid nanostructure delivery composition via imaging.

[0042] 32. The method according to any one of items 26 to 31, wherein administration to the animal is by intramuscular, intravenous, intraperitoneal, oral, or pulmonary routes.

[0043] 33. The method according to any one of items 26 to 32, wherein the nucleic acid barcode construct is isolated from cells and tissues by mixing with a first organic compound and incubating the organic phase with the aqueous phase of the cell or tissue sample, separating the organic phase from the aqueous phase, mixing the organic phase with a second organic compound, incubating the mixture, precipitating the nucleic acid barcode construct from the mixture, removing the organic phase by evaporation, and resuspending the nucleic acid barcode construct in an aqueous composition.

[0044] 34. The method according to item 33, wherein the organic phase contains phenol chloroform.

[0045] 35. The method according to item 26, wherein the nucleic acid barcode construct is separated from the cationic substances in the cell or tissue by titrating the aqueous composition of the nucleic acid barcode construct to a pH above 7.4.

[0046] 36. The method according to item 26, wherein the nucleic acid barcode construct is separated from the substances in the cell or tissue by binding the nucleic acid barcode construct to a molecule having a higher binding affinity for the nucleic acid barcode construct than for the cell or tissue substance.

[0047] 37. The method according to item 26, wherein the nucleic acid barcode construct is separated from the substances in the cell or tissue by size exclusion chromatography.

[0048] 38. The method according to item 26, wherein the nucleic acid barcode construct is separated from the substances in the cell or tissue by dialysis or diafiltration.

[0049] 39. The method according to item 26, wherein the nucleic acid barcode construct is separated from the substances in the cell or tissue by filtration.

[0050] 40. The method according to item 26, wherein the nucleic acid barcode construct is separated from the substances in the cell or tissue by digesting proteins using an enzyme.

[0051] 41. The method according to item 40, wherein the enzyme is proteinase K.

[0052] 42. The method according to item 26, wherein the nucleic acid barcode construct bound to the nucleic acid nanostructure delivery composition is detected by first diluting the isolated nucleic acid barcode construct by at least 1000-fold and then amplifying the nucleic acid barcode construct by PCR using primers.

[0053] 43. The method according to item 42, wherein the primers from the PCR step are enzymatically digested before detection of the amplicon.

[0054] 44. The method according to item 28, wherein the nucleotide sequence data is converted into a FASTQ file, and the FASTQ file is mapped to a known unique polynucleotide sequence and the unique polynucleotide sequence is enumerated.

[0055] 45. The method according to any one of items 26 to 44, wherein the nucleic acid barcode construct according to any one of items 16 to 25 is used.

[0056] 46. A composition comprising a non-viral delivery medium comprising a nucleic acid nanostructure delivery composition and a payload, wherein the nucleic acid nanostructure delivery composition comprises single-stranded or double-stranded DNA or RNA.

[0057] 47. The composition according to item 46, wherein the nucleic acid nanostructure delivery composition comprises DNA.

[0058] 48. The composition according to item 46, wherein the nucleic acid nanostructure delivery composition comprises RNA.

[0059] 49. The composition according to item 46, wherein the nucleic acid nanostructure delivery composition is single-stranded.

[0060] 50. The composition according to item 46, wherein the nucleic acid nanostructure delivery composition is double-stranded.

[0061] 51. The composition according to any one of claims 46 to 50, wherein the payload contains nucleic acid.

[0062] 52. The composition according to claim 51, wherein the nucleic acid contains DNA or RNA.

[0063] 53. The composition according to claim 51, wherein the payload nucleic acid is used for homology-directed repair or as a transposable element.

[0064] 54. The composition according to claim 51, wherein the payload nucleic acid contains short guide RNA (sgRNA) and a donor DNA strand.

[0065] 55. The composition according to claim 54, wherein the sgRNA is used to target an enzyme to a specific genomic sequence.

[0066] 56. The composition according to any one of claims 46 to 50, wherein the payload contains a CRISPR-related enzyme.

[0067] 57. The composition according to claim 55, wherein the target enzyme is a CRISPR-related enzyme.

[0068] 58. The composition according to claim 51, wherein the payload contains a CRISPR-related enzyme, sgRNA, and a donor DNA strand.

[0069] 59. The composition according to any one of claims 46 to 50, wherein the payload contains CRISPR / Cas9.

[0070] 60. The composition according to claim 51, wherein the payload contains CRISPR / Cas9, sgRNA, and a donor DNA strand.

[0071] 61. The composition according to any one of claims 46 to 50, wherein the payload contains CRISPR / Cas9 and Cas9 is fused with a deaminase.

[0072] 62. The composition according to item 51, wherein the payload comprises a coding sequence of Cas9, an sgRNA, and a donor DNA strand in the form of a plasmid.

[0073] 63. The composition according to item 51, wherein the payload consists of one molecule each of CRISPR / Cas9, sgRNA, and a donor DNA strand.

[0074] 64. The composition according to item 51, wherein the payload comprises an antisense oligonucleotide.

[0075] 65. The composition according to item 51, wherein the payload is a size selected from the group consisting of 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.

[0076] 66. The composition according to any one of items 46 to 51, 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.

[0077] 67. The composition according to any one of items 46 to 65, wherein the payload is bound 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.

[0078] 68. The composition according to item 67, wherein the molecule that binds to biotin is bound to the nucleic acid nanostructure delivery composition by a covalent phosphoramidate 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.

[0079] 69. The composition according to item 67 or 68, wherein biotin is bound to the payload by a covalent bond.

[0080] 70. The composition according to any one of items 46 to 65, wherein the payload is bound to the nucleic acid nanostructure delivery composition by a covalent bond.

[0081] 71. The composition according to item 70, wherein the covalent bond is 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 payload.

[0082] 72. The composition according to item 70, wherein the covalent bond is formed via a click chemistry coupling reaction between the azide group on the nucleic acid nanostructure delivery composition and the alkyne group on the payload.

[0083] 73. The composition according to item 70, wherein the covalent bond is formed via a click chemistry coupling reaction between the azide group on the payload and the alkyne group on the nucleic acid nanostructure delivery composition.

[0084] 74. The composition according to any one of items 46 to 65, wherein the payload 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 amine on the payload.

[0085] 75. The composition according to any one of items 46 to 74, wherein the aspect ratio of the nucleic acid nanostructure delivery composition is about 2.

[0086] 76. The composition according to any one of items 1 to 25 or 46 to 75, wherein the nucleic acid nanostructure delivery composition is coated with one or more polymers.

[0087] 77. The composition according to any one of items 1 to 25 or 46 to 76, wherein the nucleic acid nanostructure delivery composition further comprises a targeting component for targeting cells.

[0088] 78. A method of treating a patient having a disease, comprising administering to the patient a nucleic acid nanostructure delivery composition identified by the in vivo screening method according to any one of paragraphs 26 to 45, or a nucleic acid nanostructure delivery composition according to any one of paragraphs 46 to 77, wherein the nucleic acid nanostructure delivery composition comprises a payload, and treating the disease of the patient.

[0089] 79. The method according to paragraph 78, further comprising administering to the patient a pharmaceutically acceptable carrier.

[0090] 80. The method according to paragraph 79, wherein the pharmaceutically acceptable carrier is for parenteral administration or topical administration.

[0091] 81. The method according to paragraph 78, wherein the patient has a disease or disorder selected from the group consisting of cancer, muscle disease, lung disease, skin disease, neurological disease, neurofibromatosis type 1, and abnormal hemoglobinopathy.

[0092] 82. The method according to paragraph 81, 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.

[0093] 83. The method according to paragraph 81, wherein the skin disease is a Staphylococcus aureus infection.

[0094] 84. The method according to paragraph 81, wherein the muscle disease is muscular dystrophy.

[0095] 85. The method according to paragraph 78, wherein the nucleic acid nanostructure delivery composition is not cytotoxic to the patient's cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Brief Description of the Drawings

[0097]

Figure 1

Figure 2

Figure 3

[0098] Detailed description of exemplary embodiments

[0099] The present invention relates to a barcode - coded nucleic acid nanostructure delivery composition for in - vivo screening for use in the delivery of therapeutic agents in the body, and methods thereof. More specifically, the present invention relates to a nucleic acid nanostructure delivery composition, such as a DNA origami structure conjugated with a barcode for high - throughput in - vivo screening of nucleic acid nanostructure delivery compositions for their subsequent use in drug delivery, and methods thereof.

[0100] The present invention relates to a nucleic acid nanostructure delivery composition for non - viral delivery, and methods thereof. More specifically, the present invention relates to a single - stranded or double - stranded DNA or RNA nanostructure delivery composition, such as a DNA origami structure, for delivering a plurality of payloads, a nucleic acid construct payload of 3 kB or more, other gene - based pharmaceuticals payloads, or small molecule therapeutic agents.

[0101] The following items, 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 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 items.

[0102] 1. A non-viral delivery medium comprising a nucleic acid nanostructure delivery composition, and a composition comprising a nucleic acid barcode construct.

[0103] 2. The composition according to item 1, wherein the nucleic acid nanostructure delivery composition comprises a DNA origami composition.

[0104] 3. The composition according to item 1, wherein the nucleic acid nanostructure delivery composition comprises single-stranded or double-stranded DNA or RNA.

[0105] 4. The composition according to any one of items 1 to 3, wherein the nucleic acid barcode construct is bound to the nucleic acid nanostructure delivery composition via base pairing.

[0106] 5. The composition according to item 4, wherein the base pairing occurs between the sequence of the single-stranded overhang on the nucleic acid nanostructure delivery composition and the complementary sequence added to the nucleic acid barcode construct.

[0107] 6. The composition according to any one of items 1 to 3, wherein the nucleic acid nanostructure delivery composition comprises staples that self-assemble to form the nucleic acid nanostructure delivery composition.

[0108] 7. The composition according to item 6, wherein the staples function as the nucleic acid barcode construct.

[0109] 8. The composition according to any one of items 1 to 3, wherein the nucleic acid barcode construct is bound to the nucleic acid nanostructure delivery composition by a high-affinity non-covalent interaction between a biotin molecule at the 5' end and / or 3' end of the nucleic acid barcode construct and a molecule that binds to biotin on the nucleic acid nanostructure delivery composition.

[0110] 9. The composition according to item 8, wherein the molecule that binds to biotin is bound to the nucleic acid nanostructure delivery composition by a covalent phosphoramidate 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.

[0111] 10. The composition according to item 8 or 9, wherein biotin is bound to the nucleic acid barcode construct by a covalent bond.

[0112] 11. The composition according to any one of items 1 to 3, wherein the nucleic acid barcode construct is bound to the nucleic acid nanostructure delivery composition by a covalent bond.

[0113] 12. The composition according to item 11, wherein the covalent bond is 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 barcode construct.

[0114] 13. The composition according to item 11, wherein the covalent bond is 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 barcode construct.

[0115] 14. The composition according to item 11, wherein the covalent bond is formed via a click chemistry coupling reaction between an azide group on the nucleic acid barcode construct and an alkyne group on the nucleic acid nanostructure delivery composition.

[0116] 15. The composition according to any one of items 1 to 3, wherein the nucleic acid barcode construct 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 nucleic acid barcode construct at the 5' end and / or 3' end.

[0117] 16. The composition according to any one of items 1 to 3, wherein the nucleic acid barcode construct comprises two primer-binding segments and one or more unique barcode sequences between the two primer-binding segments.

[0118] 17. The composition according to item 16, wherein the length of the primer-binding segment ranges from about 15 base pairs to about 30 base pairs.

[0119] 18. The composition according to item 16 or 17, wherein the primer binding segment is a universal primer binding set.

[0120] 19. The composition according to any one of items 16 to 18, wherein one or more unique barcode sequences comprise a unique sequence having a length of about 6 to about 20 nucleotides.

[0121] 20. The composition according to any one of items 16 to 19, wherein the length of the unique barcode sequence is at least twice the length of the primer binding segment.

[0122] 21. The composition according to any one of items 1 to 20, wherein the nucleic acid barcode construct comprises DNA or RNA.

[0123] 22. The composition according to any one of items 16 to 21, wherein the unique barcode sequence further comprises a Hamming distance of at least 2 to 6 bases between any two unique barcode sequences.

[0124] 23. The composition according to any one of the preceding items, wherein the nucleic acid barcode construct further comprises about 6 to about 12 random bases at the 3' end of the unique barcode sequence.

[0125] 24. The composition according to item 23, wherein about 6 to about 12 random bases at the 3' end of the unique barcode sequence are for bioinformatics removal of PCR duplicates.

[0126] 25. The composition according to any one of the preceding items, wherein the length of the acid barcode construct ranges from about 42 nucleotides to about 210 nucleotides.

[0127] 26. A method for screening a desired nucleic acid nanostructure delivery composition in vivo, comprising: (a) preparing a library comprising two or more nucleic acid nanostructure delivery compositions, wherein each nucleic acid nanostructure delivery composition is bound to a nucleic acid barcode construct comprising a different unique barcode sequence; (b) administering the library to an animal; (c) removing cells or tissues from the animal; (d) isolating the nucleic acid barcode construct from the cells or tissues of the animal; (e) detecting the nucleic acid barcode construct in the cells or tissues of the animal; and (f) identifying the desired nucleic acid nanostructure delivery composition for use as a delivery vehicle.

[0128] 27. The method according to claim 26, wherein the nucleic acid nanostructure delivery composition is bound to the nucleic acid barcode construct according to any one of claims 4 to 15.

[0129] 28. The method according to claim 26 or 27, wherein the nucleic acid barcode construct is detected by a method selected from the group consisting of polymerase chain reaction (PCR), isothermal amplification, sequencing, or combinations thereof, and nucleotide sequence data is obtained.

[0130] 29. The method according to any one of claims 26 to 28, wherein the nucleic acid nanostructure delivery composition is loaded with a payload.

[0131] 30. The method according to claim 29, wherein the payload is a luminescent molecule.

[0132] 31. The method according to claim 30, wherein luminescence is used to track the in vivo distribution or cellular uptake of the nucleic acid nanostructure delivery composition via imaging.

[0133] 32. The method according to any one of claims 26 to 31, wherein the administration to the animal is by intramuscular, intravenous, intraperitoneal, oral, or pulmonary routes.

[0134] 33. The method according to any one of items 26 to 32, wherein the nucleic acid barcode construct is isolated from cells and tissues by mixing with a first organic compound and incubating the organic phase with the aqueous phase of the cell or tissue sample, separating the organic phase from the aqueous phase, mixing the organic phase with a second organic compound, incubating the mixture, precipitating the nucleic acid barcode construct from the mixture, removing the organic phase by evaporation, and resuspending the nucleic acid barcode construct in an aqueous composition.

[0135] 34. The method according to item 33, wherein the organic phase contains phenol chloroform.

[0136] 35. The method according to item 26, wherein the nucleic acid barcode construct is separated from the cationic substances in the cell or tissue by titrating the aqueous composition of the nucleic acid barcode construct to a pH above 7.4.

[0137] 36. The method according to item 26, wherein the nucleic acid barcode construct is separated from the substances in the cell or tissue by binding the nucleic acid barcode construct to a molecule having a higher binding affinity for the nucleic acid barcode construct than for the cell or tissue substance.

[0138] 37. The method according to item 26, wherein the nucleic acid barcode construct is separated from the substances in the cell or tissue by size exclusion chromatography.

[0139] 38. The method according to item 26, wherein the nucleic acid barcode construct is separated from the substances in the cell or tissue by dialysis or diafiltration.

[0140] 39. The method according to item 26, wherein the nucleic acid barcode construct is separated from the substances in the cell or tissue by filtration.

[0141] 40. The method according to item 26, wherein the nucleic acid barcode construct is separated from the substances in the cell or tissue by digesting proteins using an enzyme.

[0142] 41. The method according to item 40, wherein the enzyme is proteinase K.

[0143] 42. The method according to item 26, wherein the nucleic acid barcode construct bound to the nucleic acid nanostructure delivery composition is detected by first diluting the isolated nucleic acid barcode construct by at least 1000-fold and then amplifying the nucleic acid barcode construct by PCR using primers.

[0144] 43. The method according to item 42, wherein the primers from the PCR step are enzymatically digested before detection of the amplicon.

[0145] 44. The method according to item 28, wherein the nucleotide sequence data is converted into a FASTQ file, and the FASTQ file is mapped to a known unique polynucleotide sequence and the unique polynucleotide sequence is enumerated.

[0146] 45. The method according to any one of items 26 to 44, wherein the nucleic acid barcode construct according to any one of items 16 to 25 is used.

[0147] 46. A composition comprising a non-viral delivery medium comprising a nucleic acid nanostructure delivery composition and a payload, wherein the nucleic acid nanostructure delivery composition comprises single-stranded or double-stranded DNA or RNA.

[0148] 47. The composition according to item 46, wherein the nucleic acid nanostructure delivery composition comprises DNA.

[0149] 48. The composition according to item 46, wherein the nucleic acid nanostructure delivery composition comprises RNA.

[0150] 49. The composition according to item 46, wherein the nucleic acid nanostructure delivery composition is single-stranded.

[0151] 50. The composition according to item 46, wherein the nucleic acid nanostructure delivery composition is double-stranded.

[0152] 51. The composition according to any one of items 46 to 50, wherein the payload comprises a nucleic acid.

[0153] 52. The composition according to item 51, wherein the nucleic acid comprises DNA or RNA.

[0154] 53. The composition according to item 51, wherein the payload nucleic acid is used as a homology-directed repair or transposable element.

[0155] 54. The composition according to item 51, wherein the payload nucleic acid comprises a short guide RNA (sgRNA) and a donor DNA strand.

[0156] 55. The composition according to item 54, wherein the sgRNA is used to target an enzyme to a specific genomic sequence.

[0157] 56. The composition according to any one of items 46 to 50, wherein the payload comprises a CRISPR-associated enzyme.

[0158] 57. The composition according to item 55, wherein the target enzyme is a CRISPR-associated enzyme.

[0159] 58. The composition according to item 51, wherein the payload comprises a CRISPR-associated enzyme, an sgRNA, and a donor DNA strand.

[0160] 59. The composition according to any one of items 46 to 50, wherein the payload comprises CRISPR / Cas9.

[0161] 60. The composition according to item 51, wherein the payload comprises CRISPR / Cas9, an sgRNA, and a donor DNA strand.

[0162] 61. The composition according to any one of items 46 to 50, wherein the payload comprises CRISPR / Cas9 and Cas9 is fused to a deaminase.

[0163] 62. The composition according to item 51, wherein the payload comprises a coding sequence of Cas9, an sgRNA, and a donor DNA strand in the form of a plasmid.

[0164] 63. The composition according to item 51, wherein the payload consists of one molecule each of CRISPR / Cas9, sgRNA, and a donor DNA strand.

[0165] 64. The composition according to item 51, wherein the payload comprises an antisense oligonucleotide.

[0166] 65. The composition according to item 51, wherein the payload is of a size selected from the group consisting of 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.

[0167] 66. The composition according to any one of items 46 to 51, 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.

[0168] 67. The composition according to any one of items 46 to 65, wherein the payload is bound 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.

[0169] 68. The composition according to item 67, wherein the molecule that binds to biotin is bound to the nucleic acid nanostructure delivery composition by a covalent phosphoramidate 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.

[0170] 69. The composition according to item 67 or 68, wherein biotin is bound to the payload by a covalent bond.

[0171] 70. The composition according to any one of items 46 to 65, wherein the payload is bound to the nucleic acid nanostructure delivery composition by a covalent bond.

[0172] 71. The composition according to item 70, wherein the covalent bond is 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 payload.

[0173] 72. The composition according to item 70, wherein the covalent bond is formed via a click chemistry coupling reaction between the azide group on the nucleic acid nanostructure delivery composition and the alkyne group on the payload.

[0174] 73. The composition according to item 70, wherein the covalent bond is formed via a click chemistry coupling reaction between the azide group on the payload and the alkyne group on the nucleic acid nanostructure delivery composition.

[0175] 74. The composition according to any one of items 46 to 65, wherein the payload 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 amine on the payload.

[0176] 75. The composition according to any one of items 46 to 74, wherein the aspect ratio of the nucleic acid nanostructure delivery composition is about 2.

[0177] 76. The composition according to any one of items 1 to 25 or 46 to 75, wherein the nucleic acid nanostructure delivery composition is coated with one or more polymers.

[0178] 77. The composition according to any one of items 1 to 25 or 46 to 76, wherein the nucleic acid nanostructure delivery composition further comprises a targeting component for targeting cells.

[0179] 78. A method of treating a patient having a disease, comprising administering to the patient a nucleic acid nanostructure delivery composition identified by the in vivo screening method according to any one of items 26 to 45 or the nucleic acid nanostructure delivery composition according to any one of items 46 to 77, wherein the nucleic acid nanostructure delivery composition contains a payload, and treating the patient's disease.

[0180] 79. The method according to item 78, further comprising administering to the patient a pharmaceutically acceptable carrier.

[0181] 80. The method according to item 79, wherein the pharmaceutically acceptable carrier is for parenteral administration or topical administration.

[0182] 81. The method according to item 78, wherein the patient has a disease or disorder selected from the group consisting of cancer, muscle disease, lung disease, skin disease, nerve disease, neurofibromatosis type 1, and abnormal hemoglobinopathy.

[0183] 82. The method according to item 81, 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 system cancer, prostate cancer, leukemia, lymphoma, mesothelioma, bladder cancer, kidney cancer, central nervous system tumor, brain cancer, and adenocarcinoma.

[0184] 83. The method according to item 81, wherein the skin disease is a Staphylococcus aureus infection.

[0185] 84. The method according to item 81, wherein the muscle disease is muscular dystrophy.

[0186] 85. The method according to item 78, wherein the nucleic acid nanostructure delivery composition is not cytotoxic to the patient's cells.

[0187] In various embodiments, the nucleic acid nanostructure delivery compositions described herein can include any non-viral composition for in vivo delivery of a payload. By way of example, the nucleic acid nanostructure delivery compositions described herein can be selected from the group consisting of synthetic virus-like particles, carbon nanotubes, emulsions, and any nucleic acid nanostructure delivery composition, such as a DNA origami structure.

[0188] 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.

[0189] The nucleic acid nanostructure delivery compositions (e.g., DNA origami structures) are programmable as delivery platforms and provide opportunities for accurate scale-up and manufacturing. In this embodiment, the biological and non-viral nature of the nucleic acid nanostructure delivery compositions reduces the likelihood of adverse immune reactions. In this embodiment, control of each nucleotide forming part of the nucleic acid nanostructure delivery composition (e.g., DNA origami nanostructure) enables the precise design and modification of structures containing appropriate chemical moieties that allow for in vivo delivery and endosomal escape. In other embodiments, the nucleic acid nanostructure delivery compositions can include RNA. In various embodiments, the nucleic acid nanostructure delivery compositions are single-stranded or double-stranded and can include DNA or RNA.

[0190] In this embodiment, the nucleic acid nanostructure delivery composition folds into a structure that can undergo self-base pairing (i.e., DNA origami structure) to form a single-stranded or double-stranded backbone capable of encapsulating the payload.

[0191] In this embodiment, the nucleic acid nanostructure delivery composition can include overhangs that bind via complementary base pairing to the payload nucleic acid or nucleic acid barcode construct described herein. In this embodiment, the overhangs are disposed within a cavity within the nucleic acid nanostructure delivery composition backbone, the cavity is covered by a lid and a hinge, and when the lid is closed, the payload or nucleic acid barcode construct is completely encapsulated within the cavity. In this embodiment, the lid can further include an oligonucleotide strand that binds via complementary base pairing to another oligonucleotide strand attached to the nucleic acid nanostructure delivery composition backbone when the lid is in the closed position. DNA nanostructure delivery compositions (e.g., DNA origami structures) are described in U.S. Patent No. 9,765,341, which is incorporated herein by reference.

[0192] 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 when two nucleic acid molecules have "complementary" sequences, they can associate via hydrogen bonds to form a double-stranded nucleic acid molecule. Complementary sequences can be DNA or RNA sequences. Complementary DNA or RNA sequences are referred to as "complements."

[0193] In one aspect, the nucleic acid nanostructure delivery composition of the present invention can include a plurality of payloads for delivery to a target cell, or a nucleic acid payload of 3 kB or more, or another genetic payload, or a small molecule therapeutic for delivery to a target cell. In these embodiments, the nucleic acid payload has a size of 3 kB or more and can be DNA or RNA. In any of the embodiments of the nucleic acid nanostructure delivery composition described herein, the nucleic acid nanostructure can include M13 bacteriophage DNA.

[0194] In an exemplary embodiment, the nucleic acid nanostructure delivery composition further comprises a targeting component for targeting cells. In one aspect, the targeting component can be a nucleotide that is an RNA 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 tuned "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 particular cell, such that the nucleic acid nanostructure delivery composition and payload are targeted to the particular cell. In this embodiment, the polynucleotide that binds to the target cell receptor can bind in combination with a peptide aptamer. In another aspect, the nucleic acid nanostructure delivery composition can be folded such that self base pairs are disrupted, the nucleic acid nanostructure delivery composition is deployed, and release of the payload is induced only in the presence of a particular biomarker, such as a cell receptor, microRNA, DNA, RNA, or antigen. For example, the lock and key mechanism for the induced release 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, more efficient delivery of a payload with fewer side effects is enabled. This is because the nucleic acid nanostructure delivery composition has low immunogenicity and the payload is released only in the presence of, for example, an RNA or peptide biomarker present in the cytoplasm of the target cells and tissues.

[0195] In another embodiment, the cell targeting peptide can be attached to a charge-neutral peptide nucleic acid, a PNA oligonucleotide, instead of a DNA oligonucleotide. 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. Their uncharged backbone has a higher binding affinity to DNA than DNA:DNA, and these molecules are suitable for binding to proteins and peptides.

[0196] In embodiments where a nucleic acid nanostructure delivery composition is used, a computer-aided design tool can predict the nucleotide sequences necessary to produce a highly engineered nucleic acid nanostructure delivery composition. In the case of gene delivery, these nucleic acid nanostructure delivery compositions can adjust the size and shape of the structure to fit the payload, thus providing the advantage of encapsulation efficiency. In another aspect, the loading efficiency can be enhanced by incorporating the payload into the encapsulated nucleic acid nanostructure delivery composition itself.

[0197] 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 the immune response or to enhance endosomal escape. In one 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.

[0198] In various embodiments, the payload can be combined with the nucleic acid nanostructure delivery composition using any or all of covalent bonding, electrostatic interactions, and ligand affinity interactions. In one aspect, the covalent bonding method uses EDC / NHS to form stable amide bonds between the payload and the nucleic acid nanostructure delivery composition, including improving stability (both "shelf" and in vivo), ease of separation and extraction, and sensitive detection. In another exemplary aspect, the electrostatic bonding method involves the use of a cationic nucleic acid nanostructure delivery composition that electrostatically complexes with the payload. In another embodiment, the ligand affinity binding involves the use of ligands such as avidin and biotin, both of which are covalently bound to the nucleic acid nanostructure delivery composition and the payload via EDC / NHS chemistry, resulting in a stable combination of the payload and the nucleic acid nanostructure delivery composition. In another embodiment, a method of binding a payload comprising a nucleic acid barcode construct to a nucleic acid nanostructure delivery composition is provided. This includes the use of a cleavable linker that can reverse the binding with high specificity, such as the inclusion of nuclease-specific oligonucleotide sequences, thereby allowing the payload comprising the nucleic acid barcode construct to be cleaved and extracted as needed. In another embodiment, the pair of cleavable linker and enzyme includes an amide bond and an amidase enzyme.

[0199] In one embodiment, a non-viral delivery vehicle comprising a nucleic acid nanostructure delivery composition and a composition comprising a nucleic acid barcode construct are provided. In embodiments where the nucleic acid nanostructure delivery composition is barcoded, the nucleic acid barcode construct can be bound to the nucleic acid nanostructure delivery composition via base pairing. In this embodiment, base pairing can occur between the sequence of a single-stranded overhang on the nucleic acid nanostructure delivery composition and a complementary sequence added to the nucleic acid barcode construct. In other embodiments, the nucleic acid nanostructure delivery composition can include staples that self-assemble to form the nucleic acid nanostructure delivery composition, and exemplary staples are described in Example 1.

[0200] In other embodiments, the nucleic acid barcode construct can be bound to the nucleic acid nanostructure delivery composition by a high-affinity non-covalent interaction between a biotin molecule at the 5' end and / or 3' end of the nucleic acid barcode construct and a molecule that binds to biotin on the nucleic acid nanostructure delivery composition. In this embodiment, the molecule that binds to biotin can be bound to the nucleic acid nanostructure delivery composition by a covalent phosphoramidate 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 be bound to the nucleic acid barcode construct by a covalent bond.

[0201] In another exemplary embodiment, the nucleic acid barcode construct can be bound to the nucleic acid nanostructure delivery composition by a covalent bond. 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 barcode construct. 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 barcode construct. In yet another embodiment, the covalent bond can be formed via a click chemistry coupling reaction between an azide group on the nucleic acid barcode construct and an alkyne group on the nucleic acid nanostructure delivery composition. In yet another embodiment, the nucleic acid barcode construct can be 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 at the 5' end and / or 3' end of the nucleic acid barcode construct.

[0202] In one aspect, the nucleic acid barcode construct comprises a polynucleotide barcode, the barcode comprising a unique sequence not present in a known genome for identification of the polynucleotide barcode. In another embodiment, a set of different nucleic acid barcode constructs having different polynucleotide barcodes (e.g., 88 or 96 different polynucleotide barcodes) can be used to multiplex samples in a single sequencing run.

[0203] In various embodiments, the length of the barcode can be from about 5 to about 100 base pairs, from about 5 to about 90 base pairs, from about 5 to about 80 base pairs, from about 5 to about 70 base pairs, from about 5 to about 60 base pairs, from about 5 to about 50 base pairs, from about 5 to about 40 base pairs, from about 5 to about 35 base pairs, from about 5 to about 34 base pairs, from about 5 to about 33 base pairs, from about 5 to about 32 base pairs, from about 5 to about 31 base pairs, from about 5 to about 30 base pairs, from about 5 to about 29 base pairs, from about 5 to about 28 base pairs, from about 5 to about 27 base pairs, from about 5 to about 26 base pairs, from about 5 to about 25 base pairs, from about 5 to about 24 base pairs, from about 5 to about 23 base pairs, from about 5 to about 22 base pairs, from about 5 to about 21 base pairs, from about 5 to about 20 base pairs, from about 5 to about 19 base pairs, from about 5 to about 18 base pairs, from about 5 to about 17 base pairs, from about 5 to about 16 base pairs, from about 5 to about 15 base pairs, from about 5 to about 14 base pairs, from about 5 to about 13 base pairs, from about 5 to about 12 base pairs, from about 5 to about 11 base pairs, from about 5 to about 10 base pairs, from about 5 to 9 base pairs, from about 5 to 8 base pairs, from about 6 to 10 base pairs, from about 7 to 10 base pairs, from about 8 to 10 base pairs, or from about 6 to about 20 base pairs.

[0204] Various embodiments of the barcode are shown in Table 1 below (designated "Polynucleotide Barcode"). These barcodes can be used alone in the nucleic acid barcode structure or in combinations such as, for example, two or more barcodes, three or more barcodes, four or more barcodes, etc. In embodiments where two or more barcodes are used, the Hamming distance between the barcodes can be from about 2 to about 6 nucleotides, or any suitable number of nucleotides can form the Hamming distance, or there are no nucleotides between the polynucleotide barcodes.

[0205]

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

[0206] In another embodiment, a random array fragment can be linked to the 5' end and / or 3' end of the barcode, and the random array fragment can be used, for example, for bioinformatics removal of PCR duplicates. The random array fragment can also be used to add length to the nucleic acid construct and can serve as a marker for bioinformatics analysis to identify the start or end of the barcode after sequencing. In another embodiment, the nucleic acid barcode construct includes at least a first and a second random array fragment, the first random array fragment can be linked to the 5' end of the barcode, and the second random array fragment can be linked to the 3' end of the barcode. In another embodiment, one or at least one random array fragment is linked to the 5' end and / or 3' end of the barcode. In one aspect, the random array fragment can be extended as needed to lengthen the nucleic acid barcode construct for various applications such as whole genome sequencing where short insertions may be lost.

[0207] In various embodiments, the random array fragment can be about 5 to about 20 base pairs in length, about 5 to about 19 base pairs in length, about 5 to about 18 base pairs in length, about 5 to about 17 base pairs in length, about 5 to about 16 base pairs in length, about 5 to about 15 base pairs in length, about 5 to about 14 base pairs in length, about 5 to about 13 base pairs in length, about 5 to about 12 base pairs in length, about 5 to about 11 base pairs in length, about 5 to about 10 base pairs in length, about 5 to about 9 base pairs in length, about 5 to about 8 base pairs in length, about 6 to about 10 base pairs in length, about 7 to about 10 base pairs in length, or about 8 to about 10 base pairs in length.

[0208] In another exemplary aspect, the barcode is sandwiched between primer binding segments (i.e., directly or indirectly linked to the barcode), and the nucleic acid barcode construct containing the barcode can be amplified during polymerase chain reaction (PCR) and / or sequencing protocols. In one aspect, the primer binding segment may be useful for binding to one or more universal primers or universal primer sets. In one exemplary embodiment, the universal primer may include an overhang sequence that allows for the attachment of an index adapter for sequencing. In one embodiment, the adapter may be an NGS adapter (e.g., Illumina) located internally towards either the 5' primer or the 3' primer end, rather than as a termination structure, to avoid the formation of primer dimers. In this aspect, the primer can be any primer of interest. In this embodiment, the first primer binding segment is linked at its 3' end to the 5' end of a first random sequence fragment, the second primer binding segment is linked at its 5' end to the 3' end of a second random sequence fragment, and the barcode is positioned between the random sequence fragments. In another embodiment, the first primer binding segment is linked at its 3' end to the 5' end of the barcode, and the second primer binding segment is linked at its 5' end to the 3' end of a random sequence fragment linked to the 3' end of the barcode. In another embodiment, the first primer binding segment is linked at its 3' end to the 5' end of a random sequence fragment, the second primer binding segment is linked at its 5' end to the 3' end of the barcode, and the barcode is linked at its 5' end to the 3' end of the random sequence fragment. In yet another embodiment, the first primer binding segment is linked at its 3' end to the 5' end of the barcode, and the second primer binding segment is linked at its 5' end to the 3' end of the barcode.

[0209] In embodiments where the primer binding segment is included in the nucleic acid barcode construct, the length of the primer binding segment can range from about 15 base pairs to about 30, from about 15 base pairs to about 29 base pairs, from about 15 base pairs to about 28 base pairs, from about 15 base pairs to about 26 base pairs, from about 15 base pairs to about 24 base pairs, from about 15 base pairs to about 22 base pairs, from about 15 base pairs to about 20 base pairs, from 16 base pairs to about 28 base pairs, from about 16 base pairs to about 26 base pairs, from about 16 base pairs to about 24 base pairs, from about 16 base pairs to about 22 base pairs, from about 16 base pairs to about 20 base pairs, from 17 base pairs to about 28 base pairs, from about 17 base pairs to about 26 base pairs, from about 17 base pairs to about 24 base pairs, from about 17 base pairs to about 22 base pairs, from about 17 base pairs to about 20 base pairs, from 18 base pairs to about 28 base pairs, from about 18 base pairs to about 26 base pairs, from about 18 base pairs to about 24 base pairs, from about 18 base pairs to about 22 base pairs, or from about 18 base pairs to about 20 base pairs.

[0210] Exemplary sequences of the nucleic acid barcode construct are shown below. / 5AmMC6 / is a 5’ amine modification for binding to the nucleic acid nanostructure delivery composition. * is a phosphorothioate bond modification for stability. The A*G*A*CGTGTGCTCTTCCGATCT sequence is the 5’ primer binding segment sequence. GCTACATAAT is an example of a barcode sequence. N represents a random sequence fragment. AGATCGGAAGAGCGTCG*T*G*T is the 3’ primer binding segment sequence.

[0211] / 5AmMC6 / A*G*A*CGTGTGCTCTTCCGATCTGCTACATAATNNNNNNNNNNAGATCGGAAGAGCGTCG*T*G*T

[0212] In all of the various embodiments described above, the length of the entire nucleic acid barcode construct can range from about 30 base pairs to about 350 base pairs, from about 30 base pairs to about 300 base pairs, from about 30 base pairs to about 270 base pairs, from about 30 base pairs to about 240 base pairs, from about 30 base pairs to about 230 base pairs, from about 30 base pairs to about 220 base pairs, from about 30 base pairs to about 210 base pairs, from about 30 base pairs to about 200 base pairs, from about 30 base pairs to about 190 base pairs, from about 30 base pairs to about 180 base pairs, from about 30 base pairs to about 170 base pairs, from about 30 base pairs to about 160 base pairs, from about 30 base pairs to about 150 base pairs, from about 30 base pairs to about 140 base pairs, from about 30 base pairs to about 130 base pairs, from about 30 base pairs to about 120 base pairs, from about 30 base pairs to about 110 base pairs, from about 30 base pairs to about 100 base pairs, from about 30 base pairs to about 90 base pairs, from about 30 base pairs to about 80 base pairs, from about 30 base pairs to about 70 base pairs, from about 30 base pairs to about 60 base pairs, from about 30 base pairs to about 50 base pairs, from about 30 base pairs to about 40 base pairs, from 40 base pairs to about 120 base pairs, from about 40 base pairs to about 110 base pairs, from about 40 base pairs to about 100 base pairs, from about 40 base pairs to about 90 base pairs, from about 40 base pairs to about 80 base pairs, from about 40 base pairs to about 70 base pairs, from about 40 base pairs to about 60 base pairs, from about 40 base pairs to about 50 base pairs, from 50 base pairs to about 120 base pairs, from about 50 base pairs to about 110 base pairs, from about 50 base pairs to about 100 base pairs, from about 50 base pairs to about 90 base pairs, from about 50 base pairs to about 80 base pairs, from about 50 base pairs to about 70 base pairs, from about 50 base pairs to about 60 base pairs, or from about 42 base pairs to about 210 base pairs.

[0213] In another embodiment, a method for screening a desired nucleic acid nanostructure delivery composition in vivo is provided. The method includes (a) preparing a library comprising two or more nucleic acid nanostructure delivery compositions, wherein each nucleic acid nanostructure delivery composition is associated with a nucleic acid barcode construct comprising a distinct unique barcode sequence, (b) administering the library to an animal, (c) removing cells or tissue from the animal, (d) isolating the nucleic acid barcode construct from the cells or tissue of the animal, (e) detecting the nucleic acid barcode construct in the cells or tissue of the animal, and (f) identifying the desired nucleic acid nanostructure delivery composition for use as a delivery vehicle. In this embodiment, any nucleic acid nanostructure delivery composition can be used and any nucleic acid barcode construct described herein can be used.

[0214] In various embodiments, any suitable route, such as parenteral administration, can be used to administer a library of nucleic acid nanostructure delivery compositions associated with nucleic acid barcode constructs for in vivo screening of the nucleic acid nanostructure delivery compositions associated with nucleic acid barcode constructs or for the treatment methods described below. Routes suitable for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intracerebroventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular, and subcutaneous delivery. In one embodiment, the means of parenteral administration includes needles (including microneedles) syringes, needleless syringes, and infusion techniques. In other embodiments, oral or pulmonary administration routes can be used.

[0215] In one aspect, the library of nucleic acid nanostructure delivery compositions can be pooled and concentrated before administering the nucleic acid nanostructure delivery compositions associated with nucleic acid barcode constructs to an animal. Methods for preparing and sequencing libraries are described in Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 4th Edition, Cold Spring Harbor Laboratory Press, (2012), which is incorporated herein by reference.

[0216] In various embodiments, a cell or tissue sample can be analyzed for the presence of a nucleic acid nanostructure delivery composition conjugated to a nucleic acid barcode construct described herein. The sample can be any tissue, cell, or body fluid sample derived from an animal, such as urine, nasal secretions, nasal lavage fluid, inner ear fluid, bronchial lavage fluid, bronchoalveolar lavage fluid, cerebrospinal fluid, bone marrow aspirate, sputum, pleural fluid, synovial fluid, pericardial fluid, peritoneal fluid, saliva, tears, gastric secretions, feces, genital tract secretions, lymph fluid, whole blood, serum, plasma, or any tissue or cell sample derived from an animal. Exemplary tissue or cell samples include brain tissue or cells, muscle tissue or cells, skin tissue or cells, heart tissue or cells, kidney tissue or cells, gastric tissue or cells, liver tissue or cells, urinary tract tissue or cells, gastrointestinal tract tissue or cells, head or neck tissue or cells, lung tissue or cells, genital tract tissue or cells, pancreatic tissue or cells, or other tissue or cell types derived from an animal.

[0217] In an exemplary aspect for removing cells or tissues from an animal and isolating a nucleic acid barcode construct from the cells or tissues of the animal, the nucleic acid barcode construct is removed from the cells or tissues of the animal. In various embodiments, a nucleic acid barcode construct (e.g., DNA or RNA) obtained from an animal's tissue or cells can be removed by lysing the cells and isolating the nucleic acid barcode construct from the lysate. Techniques for lysing cells to isolate nucleic acids are well known in the art, and the removal techniques include homogenization by bead beating techniques and the like. In other embodiments, the nucleic acid barcode construct can be isolated by lysing the cells using a solvent such as a detergent or phenol-chloroform. In another aspect, the nucleic acid barcode construct can be separated from the lysate by physical methods including, but not limited to, centrifugation, dialysis, diafiltration, filtration, size exclusion, pressure techniques, digestion of proteins with proteinase K, or by using substances having an affinity for nucleic acids such as beads that bind to nucleic acids.

[0218] In an exemplary embodiment, the nucleic acid barcode construct is removed from the cells or tissue by treatment with a mixture of an organic phase (e.g., phenol chloroform) and an aqueous phase (e.g., water). The organic phase (e.g., phenol chloroform) can be isolated and the nucleic acid barcode construct can be precipitated by raising the pH, for example, to pH 7.4. The organic phase (e.g., phenol chloroform) can be evaporated, the nucleic acid barcode construct can be suspended in water, and diluted to a concentration suitable for PCR and / or sequencing. In one embodiment, the isolated nucleic acid barcode construct is suspended in water or buffer after being thoroughly washed.

[0219] In other embodiments, commercially available kits such as Qiagen®, Nuclisensm®, Wizard® (Promega), QiaAmp 96 DNA Extraction Kit®, and Qiacube HT® instruments, and Promegam® can be used for the isolation of the nucleic acid barcode construct. Methods for preparing nucleic acids for PCR and / or sequencing are also described in Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 4th Edition, Cold Spring Harbor Laboratory Press, (2012), which is incorporated herein by reference.

[0220] The nucleic acid barcode structure can be detected using, for example, polymerase chain reaction (PCR), isothermal amplification, sequencing, and / or imaging. Polymerase chain reaction (PCR) was developed for analyzing nucleic acids in the laboratory. PCR has evolved over the past decade into a new generation of devices and methods known as next-generation sequencing (NGS). NGS provides more inexpensive and faster detection and amplification of nucleic acids. NGS devices and methods enable rapid sequencing because the nucleic acids are amplified on a high-throughput platform of large-scale parallelism.

[0221] In an exemplary embodiment, the nucleic acid barcode construct is sequenced and the polynucleotide barcode is detected using any suitable sequencing method including next-generation sequencing (e.g., using a sequencing platform from Illumina, ThermoFisher, or PacBio or Oxford Nanopore Technologies), sequencing by synthesis, pyrosequencing, nanopore sequencing, or modifications or combinations thereof. In one embodiment, the sequencing can be amplicon sequencing. In another embodiment, the sequencing can be whole-genome sequencing. In another embodiment, the sequencing can be exome / target hybridization sequencing. Methods of sequencing nucleic acids are also well known in the art and are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press, which is incorporated herein by reference.

[0222] In another embodiment, a method of treating a patient having a disease is provided. The method includes administering to the patient a nucleic acid nanostructure delivery composition identified by the in vivo screening method described herein, or any of the nucleic acid nanostructure delivery compositions described herein, wherein the nucleic acid nanostructure delivery composition includes a payload, and treating the patient's disease.

[0223] Exemplary payloads of the nucleic acid nanostructure delivery compositions described herein can include any one or a combination of compositions selected from the group consisting of nucleic acids (e.g., DNA or RNA), pDNA, oligodeoxyribonucleic acid (ODN), dsDNA, ssDNA, antisense oligonucleotides, antisense RNA, siRNA, messenger RNA, guide RNA (e.g., small guide RNA), ribonucleoprotein, donor DNA strands used in the CRISPR / Cas9 system, and enzymes (e.g., CRISPR-related enzymes, e.g., Cas9), enzymes used in other gene editing systems such as ZFNs, custom-designed homing endonucleases, TALENS systems, other gene editing endonucleases, and reverse transcriptase.

[0224] 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 (e.g., a tumor antigen). This targeting results in, for example, cytotoxicity against tumors, and CAR-T cells expressing a CAR can target and kill tumors via a specific tumor antigen. A CAR can include a recognition region (e.g., a single-chain variable fragment (scFv)) obtained from an antibody for recognizing and binding to an antigen expressed by a tumor, 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 structure.

[0225] In another embodiment, the payload can be a nucleic acid (e.g., DNA or RNA) having a size selected from the group consisting of 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 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, 5.31 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.

[0226] In various embodiments, the payload can be any one or more components of a CRISPR RNP system comprising a CRISPR-related enzyme (e.g., Cas9), a short guide RNA (sgRNA), and a donor DNA strand. In embodiments where the payload comprises Cas9, Cas9 can be fused to a deaminase. In yet another embodiment, the payload can comprise an sgRNA used to target an enzyme to a specific genomic sequence. In another aspect, the target enzyme can be a CRISPR-related enzyme. In another exemplary aspect, the payload can comprise 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 as a transposable element. In yet another embodiment, the payload can be any of the payloads described herein in the form of a plasmid construct.

[0227] 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 is the payload and can be used for gene editing. In another embodiment, another gene editing system such as ZFN, custom-designed homing endonucleases, and TALENS system can be the payload. In embodiments where the CRISPR / Cas9 system is the payload, the Cas9 endonuclease can introduce a double-strand break into a DNA target sequence. In this aspect, the Cas9 endonuclease is induced by a guide polynucleotide (e.g., sgRNA), recognizes the double-strand break at a specific target site, and introduces it into the cell's genome as needed. In this exemplary embodiment, the Cas9 endonuclease can unwind the DNA double strand adjacent to 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 approximately oriented towards the 3' end of the target. In this embodiment, the donor DNA strand can be integrated into the genomic target site. The CRISPR / Cas9 system for gene editing is well known in the art.

[0228] In another exemplary embodiment, the payload may include 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 payload can include a nucleotide sequence designed to bind to an endosomal receptor as an aptamer, thereby enhancing intracellular transport of the nucleic acid nanostructure delivery composition.

[0229] In an exemplary embodiment, a nucleic acid nanostructure delivery composition (e.g., a DNA origami) is provided that packages a Cas9 protein, an sgRNA, and a single-stranded donor DNA strand together in one nanostructure to ensure that all components are co-delivered to a specific location simultaneously. In this embodiment, the single-stranded nature of the sgRNA and the donor DNA strand is utilized to convert these components into components of a nucleic acid nanostructure delivery composition (e.g., a DNA origami structure) that, upon reaching the target site (e.g., a target cell), can be co-delivered and dissociate simultaneously from the DNA nanostructure delivery composition. In this embodiment, the DNA nanostructure delivery composition can deliver either the plasmid or the ribonucleoprotein (RNP) form of CRISPR / Cas9.

[0230] In one embodiment, a method for gene therapy is provided. In one aspect, the method includes administering to a patient a nucleic acid nanostructure delivery composition as described herein.

[0231] In one embodiment, the nucleic acid nanostructure delivery composition described herein can be formulated as a pharmaceutical composition for parenteral or topical administration. Such pharmaceutical compositions and methods of 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. Additional active ingredients may be included in the composition.

[0232] In one aspect, the nucleic acid nanostructure delivery composition can be administered directly, for example, to the bloodstream, muscle, or viscera of a patient, or can also be administered in a topical formulation. In various embodiments, suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular, and subcutaneous delivery. In one embodiment, parenteral administration means include needle (including microneedle) syringes, needleless syringes, and infusion techniques.

[0233] In an exemplary embodiment, the parenteral formulation is typically an aqueous solution and may contain carriers or excipients such as salts, carbohydrates, and buffers (preferably pH 3 to 9), but may be more suitably formulated as a sterile non-aqueous solution or in a dry form for use in combination with a suitable medium such as sterile, pyrogen-free water or sterile saline. Preparation under sterile conditions can be readily achieved using standard pharmaceutical techniques well known to those skilled in the art, by manufacturing a sterile lyophilized powder for the parenteral formulation by lyophilization. In one embodiment, the solubility of the composition used in the preparation of the parenteral formulation can be enhanced by using appropriate formulation techniques such as the incorporation of solubility enhancers.

[0234] 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 for providing 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 millimoles. And d) a water-soluble viscosity modifier is provided in a concentration range of about 0.25% to about 10% of the total formulation amount, or in any combination of a), b), c), and d).

[0235] In various exemplary embodiments, the pH buffers used in the compositions and methods described herein are agents known to those skilled in the art and include, for example, acetate, borate, carbonate, citrate, and phosphate buffers, as well as 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.

[0236] 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.

[0237] Useful viscosity regulators include ionic and non-ionic water-soluble polymers, cross-linked acrylic acid polymers (e.g., polymers of the "carbomer" family, such as carboxypolyalkylenes 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, but are not limited thereto. Usually, non-acidic viscosity enhancers such as neutral or basic agents are used to facilitate the achievement of the desired pH of the formulation.

[0238] 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.

[0239] In other embodiments, the compositions described herein can be administered topically. Various dosage forms and bases such as ointments, creams, gels, gel ointments, plasters (e.g., patches, cataplasms), 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.

[0240] For example, petrolatum, higher alcohols, beeswax, vegetable oils, polyethylene glycol, etc. are used. In the preparation of creams, fats and oils, waxes, higher fatty acids, higher alcohols, fatty acid esters, purified water, emulsifiers, etc. are used. In the preparation of gels, ordinary gelling agents such as polyacrylates (e.g., 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 (e.g., liquid paraffin, triglycerides, etc.) are used. By applying the above gel preparations onto a support (e.g., fabric, non-woven fabric), plasters such as poultices and cataplasms can be prepared. In addition to the above components, paraffin, squalane, lanolin, cholesterol esters, higher fatty acid esters, etc. can be optionally used. Furthermore, antioxidants such as BHA, BHT, propyl gallate, pyrogallol, tocopherol can also be incorporated. In addition to the above preparations and components, any other conventional preparations for incorporation with other additives can be optionally used.

[0241] In various embodiments, the dosage of the nucleic acid nanostructure delivery composition can vary widely depending on the patient's condition, or the disease state being treated, the route of administration and tissue distribution, and the possibility of combination with other therapeutic treatments. The effective amount administered to the patient is determined 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 cancer, muscle disorders, lung disorders, skin disorders, 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 cancer, prostate cancer, leukemia, lymphoma, mesothelioma, bladder cancer, kidney cancer, central nervous system tumors, brain tumors, and adenocarcinoma. In another embodiment, the skin disorder is a Staphylococcus aureus infection. In yet another embodiment, the muscle disorder is muscular dystrophy (e.g., Duchenne muscular dystrophy). In yet another embodiment, the nucleic acid nanostructure delivery composition is not cytotoxic to the patient's cells. In another embodiment, gene therapy may result in the inactivation of pathogens (i.e., microorganisms) rather than altering the patient's genome.

[0242] In another embodiment, a diverse set of non-viral gene delivery compositions are synthesized, each non-viral gene delivery composition being different from the diverse set of other non-viral gene delivery compositions with respect to at least one set of compositional characteristics, simultaneously testing one or more quality attributes of each of the diverse set of non-viral gene delivery compositions, and creating a predictive model that correlates the compositional characteristics with the quality attributes from the results of the testing. In this embodiment, the compositional characteristics can include one or more of molecular weight, degree of branching, number of ionizable groups, core-to-corona molecular weight ratio, hydrophilicity, hydrophobicity, aggregability, size, pKa, logP, and surface charge. In this embodiment, the quality attributes can include one or more of cytotoxicity, immunogenicity, transfection efficiency, zeta potential, size, pKa, logP, and loading efficiency. In this embodiment, the diverse set can include hundreds or thousands of non-viral gene delivery compositions. In this embodiment, each of the diverse set of non-viral gene delivery compositions can be a nucleic acid nanostructure delivery composition according to any of the above items. In one embodiment of this method, high-throughput testing and machine learning data analysis can accelerate the design-build-test-learn (DBTL) cycle for the development of CRISPR-based therapeutics.

[0243] In some embodiments, the nucleic acid nanostructure delivery composition can be labeled to enhance downstream separation. For example, this can include covalently attaching the nucleic acid nanostructure delivery composition to magnetic nanoparticles (e.g., superparamagnetic iron oxide), to polyhistidine tags for metal ion chromatography, and / or to fluorescent labels for fluorescence-assisted separation (e.g., by FACS). The label can be used to track the nucleic acid delivery composition in vivo. Possible "endpoints" include, but are not limited to, the quantitative presence in various physiological tissues after administration, measured, for example, via fluorescence.

[0244] In embodiments where a label is used, the label enables rapid in vivo screening of many non-viral delivery vehicle variants involving parallel measurement of quantitative biodistribution, and rapid in vitro screening of many variants regarding stability, cytotoxicity, immunogenicity, and efficacy. This embodiment enables the construction of a large library of non-viral delivery vehicles that can be extracted for use as delivery vehicles for gene pharmaceuticals including gene therapy, gene vaccines, gene editing, gene regulators, and small molecule therapeutics.

[0245] In some exemplary embodiments, a large library of similar but unique nucleic acid nanostructure delivery compositions can be constructed and used in a high-throughput screening process to identify target components that bind to a specific target. This rapid screening platform can quickly determine effective target molecules that can be used for targeted delivery to specific cells or tissues or for use as neutralizing molecules against pathogens.

[0246] References herein to "one embodiment", "an embodiment", "exemplary embodiments", etc., indicate that the embodiments described may include a particular function, structure, or characteristic, but not all embodiments necessarily include that particular function, structure, or characteristic. Further, such phrases do not necessarily refer to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one of ordinary skill in the art to implement such feature, structure, or characteristic in connection with other embodiments, whether or not explicitly described. Further, note that items listed in the form of "at least one of A, B, and C" may 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" may mean (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).

[0247] 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 different way and / or order than 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.

[0248] In the drawings and the foregoing description, some exemplary embodiments have been described in detail, but such descriptions and explanations should be regarded as exemplary and not restrictive, and only the exemplary embodiments are shown and described, and it is desirable to understand that all changes and modifications within the spirit of the present disclosure are 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. Those skilled in the art can easily devise their own implementations of the apparatus, system, and method incorporating one or more of the features of the present disclosure.

[0249] The concept of the present disclosure is capable of various changes and alternative forms, but its specific embodiments are shown by way of example in the accompanying drawings and described in detail herein. However, it is to be understood that there is no intention to limit the concept of the present disclosure to the specific forms disclosed, and on the contrary, it is intended to cover all changes, equivalents, and alternatives consistent with the present disclosure.

Best Mode for Carrying Out the Invention

Examples

[0250] Example 1 Nucleic acid nanostructure delivery composition The following materials and methods illustrate an example where the DNA origami (DNAO) nanostructure is a cuboid structure and the nucleic acid barcode structure is attached to the DNAO via one of the oligonucleotide staples within the DNAO and complementary base pairing of the barcode.

[0251] Barcode design The barcode used in this example contains a unique portion that includes 8 to 10 nucleotides in the center of the polynucleotide, and this unique portion is further characterized by a Hamming distance of at least 3 bases from other barcodes that are pooled. 7 to 10 random bases are included directly at the 3' end of the barcode for bioinformatics removal of PCR duplicates. On both sides of this central sequence, there are universal primer annealing sites that include overhangs for adding index adapters during the preparation of the sequencing library. The polynucleotide barcode of this example was designed to have a biotin functional group at the 5' end.

[0252]

Chemical formula

[0253] Materials for synthesizing nucleic acid nanostructure delivery composition (DNAO) The DNA origami scaffold is single-stranded DNA (ssDNA) isolated from M13 bacteriophage. The oligonucleotide staples are short single-stranded DNAs having the sequences described in Table 2. The barcode is a single-stranded DNA segment described in the barcode design above.

[0254] Preparation of DNA barcode-labeled DNAO structure A reaction mixture was prepared by mixing a DNA scaffold, oligonucleotide staples, and magnesium in TE buffer within a reaction vessel. The amounts were 160 μL of oligonucleotide staples (listed in Table 2) pooled at a total concentration of 500 nM, 80 μL of scaffold (100 nM), 80 μL of water, 40 μL of TE buffer (1.46 g of EDTA, 3.03 g of tris base, 1.46 g of NaCl, 500 mL of water), and 40 μL of 200 mM MgCl₂. The reaction vessel was placed in a thermocycler, and the temperature increase was started at approximately 65 °C and decreased to 24 °C over approximately 67 hours. The product was purified by precipitation using a PEG purification protocol with a PEG solution prepared by the following recipe: 75 g of PEG8000, 50 mL of the above TE buffer, and 62.5 mL of 4 M NaCl made up to 500 mL with water to obtain a DNAO nanostructure in water. The concentration was measured with a nanodrop, and then a barcode was added to the product at a molar ratio of 4:1 (polynucleotide barcode and DNAO nanostructure). This mixture was incubated at 37 °C for 2 to 3 hours. The product was purified by another PEG purification process as described above to obtain the final product of DNA barcode-conjugated DNAO in water. Transmission electron microscope images were taken using an FEI Tecnai G2 Bio Twin TEM on formvar / carbon-coated nickel grids negatively stained with 1% phosphotungstic acid (PTA) (see Figure 1).

[0255] Barcode amplification Dilutions of the above DNA barcode DNAO nanostructures were prepared at concentrations of 13.5, 1.35, 0.135, and 0.0135 nM. The master mix was made with Kapa HiFi 2x master mix, reverse barcode primer, forward barcode primer, DMSO, and nuclease-free water. The master mix (15 μL) was loaded into each well. Each dilution (5 μL) of the DNA barcode DNAO was loaded into each well of a 96-well plate. Nuclease-free water (5 μL) was loaded into the designated NTC wells. A positive control (5 μL) was loaded into the designated positive control wells. The positive control consisted of a solution of polymer nanoparticles in phosphate-buffered saline composed of dimethylaminoethyl methacrylate, polyacrylate, and butyl methacrylate and was labeled with the same barcode as that used for labeling the DNAO as described in U.S. Patent Application 17 / 715784. Each well was covered with a strip cap or adhesive seal and centrifuged at 1,000 x g for approximately 1 minute. Amplification of the barcode was performed by incubating in a thermocycler under general PCR conditions.

[0256] Analysis of PCR Amplification by Gel Electrophoresis Gel electrophoresis was performed on a 4% 12-well ethidium bromide gel using 15 μL of 1 kb E-Gel ladder in the first well. DNA barcode-encoded DNAO dilutions (10 μL) from the above multi-well plate were added to each well of the 12-well gel. E-Gel buffer (10 μL) was added to each well. Nuclease-free water (15 μL) was added to the remaining empty wells as a test-free control (NTC). The power supply of the gel dock was turned on, and current was applied to the gel for approximately 20 to 25 minutes or until the sample buffer line reached the end of the gel. The gel was removed from the base and analyzed with a gel imager (see Figure 2).

[0257] Results Figure 1 is a transmission electron micrograph of the DNA barcoded DNAO nanostructure. This image shows evidence that the nanostructure was successfully folded into a cuboid nanostructure, indicating that the DNA origami folding process was successful. Since the TEM does not provide a resolution sufficient to identify the structural polynucleotide barcode, as shown in Figure 2, PCR amplification was used to prove its presence.

[0258]

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Examples

[0259] Example 2 Nucleic acid nanostructure delivery composition The following materials and methods illustrate an example where the DNA origami (DNAO) nanostructure is a cuboid structure and the nucleic acid barcode structure is attached to the DNAO via complementary base pairing of one of the oligonucleotide staples within the DNAO with the barcode.

[0260] Barcode design The barcode used in this example consists of a unique portion of 8 to 10 nucleotides in the center of the polynucleotide, and this unique portion is further characterized by a Hamming distance of at least 3 bases from other barcodes being pooled. 7 to 10 random bases are included directly at the 3’ end of the barcode for bioinformatics removal of PCR duplicates. On both sides of this central sequence are universal primer annealing sites containing overhangs for adding index adapters during the preparation of the sequencing library. The polynucleotide barcode of this example is designed to have a biotin functional group at the 5’ end.

[0261]

Chemical formula

[0262] Materials for synthesizing nucleic acid nanostructure delivery compositions (DNAO) The DNA origami scaffold is single-stranded DNA (ssDNA) isolated from the M13 bacteriophage. The oligonucleotide staple is short single-stranded DNA having the sequences described in Table 2. The barcode is the single-stranded DNA segment described in the barcode design above.

[0263] Preparation of DNA barcode-conjugated DNAO structures A reaction mixture was prepared by mixing DNA scaffold, oligonucleotide staples, and magnesium in TE buffer in the reaction vessel. The amounts were 160 μL of oligonucleotide staples (listed in Table 2) pooled at a total concentration of 500 nM, 80 μL of scaffold (100 nM), 80 μL of water, 40 μL of TE buffer (1.46 g of EDTA, 3.03 g of Tris base, 1.46 g of NaCl, 500 mL of water), and 40 μL of 200 mM MgCl₂. The reaction vessel was placed in a thermocycler, and the temperature increase was started at about 65 °C and lowered to 24 °C over about 67 hours. The product was purified by precipitation using a PEG purification protocol with a PEG solution prepared by the following recipe: 75 g of PEG8000, 50 mL of the above TE buffer, and 62.5 mL of 4 M NaCl made up to 500 mL with water, and a DNAO nanostructure in water was obtained. The concentration was measured with a nanodrop, and then a barcode was added to the product at a molar ratio of 4:1 (polynucleotide barcode and DNAO nanostructure). This mixture was incubated at 37 °C for 2 to 3 hours. The product was purified by another PEG purification process as described above, and a final product of DNA barcode-labeled DNAO in water was obtained. Transmission electron microscope images were taken using an FEI Tecnai G2 Bio Twin TEM on formvar / carbon-coated nickel grids negatively stained with 1% phosphotungstic acid (PTA) (see Figure 1).

[0264] In vitro transfection HEK293 cells were seeded at 75,000 cells / well in a 48-well plate with 200 μL of complete growth medium 24 hours prior to transfection. The complete growth medium consisted of DMEM supplemented with 10% FBS and 1% Pen-Strep. The cells were dosed in triplicate with DNAO with and without barcode at final concentrations of 10 nM, 5 nM, and 2.5 nM, and a total of 16 wells were dosed. Three wells were used as controls. After 16 hours, the cells were trypsinized and the replicate wells were pooled together. Cell-derived DNA was extracted using a Qiagen DNA extraction kit. These cell extracts were used for PCR amplification.

[0265] Barcode PCR Amplification The master mix was prepared with Kapa HiFi 2x master mix, reverse barcode primer, forward barcode primer, DMSO, and nuclease-free water. The master mix (15 μL) was loaded into each well. Each cell extract (5 μL) was loaded in duplicate into each well of a 96-well plate. Nuclease-free water (5 μL) was loaded into the designated NTC wells. The positive control (5 μL) was loaded into the designated positive control wells. The positive control consisted of a solution of polymer nanoparticles in phosphate-buffered saline composed of dimethylaminoethyl methacrylate, polyacrylate, and butyl methacrylate, and was chemically conjugated with the same barcode as that used for the labeling of DNAO as described above. Each well was covered with a strip cap or an adhesive seal and centrifuged at 1,000 x g for approximately 1 minute. The amplification of the barcode was carried out by incubating in a thermocycler under general PCR conditions.

[0266] Analysis of PCR Amplification by Gel Electrophoresis Gel electrophoresis was performed using a 4% 48-well ethidium bromide gel, with 15 μL of 1 kb E-Gel Ladder in the first well. DNAO and DNA barcode DNAO cell extracts and controls (10 μL) were added to each well of the gel. E-Gel buffer (5 μL) was added to each well. The negative control from the cells was an extract without amplified barcode DNAO. The negative controls for the last two wells were water. Nuclease-free water (15 μL) was added to the remaining empty wells. The power supply of the gel dock was turned on, and current was applied to the gel for about 20 to 25 minutes, or until the sample buffer line reached the end of the gel. The gel was removed from the base and analyzed with a gel imager (see Figure 3).

[0267] Results Figure 3 shows the PCR amplification of DNAO with and without barcode at various transfection concentrations. Positive amplification is indicated by the similarity between a distinct band above the primer band (a bright band spreading across the whole gel towards the bottom of the gel) and the positive control band pattern. The barcode from the cell extract at a 10 nM DNAO dosage was clearly amplified (in the 2nd lane above the primer band), and amplification at a 5 nM DNAO dosage was visually confirmed (in the 2nd lane above the primer band). This indicates that the barcode attached to the DNA origami structure was successfully delivered to the cells and can be read at appropriate concentrations. Note the diffused bands near the wells at the top of the lanes in the "DNAO 10 nM" and "Barcoded DNAO 10 nM" lanes, which are the actual DNA origami nanostructures.

Claims

**Claim 1** A composition comprising a non-viral delivery medium comprising a nucleic acid nanostructure delivery composition and a nucleic acid barcode construct. **Claim 2** The composition according to claim 1, wherein the nucleic acid nanostructure delivery composition comprises a DNA origami composition. **Claim 3** The composition according to claim 1, wherein the nucleic acid nanostructure delivery composition comprises single-stranded or double-stranded DNA or RNA. **Claim 4** The composition according to claim 1, wherein the nucleic acid barcode construct is bound to the nucleic acid nanostructure delivery composition via base pairing. **Claim 5** The composition according to claim 4, wherein the base pairing occurs between the sequence of a single-stranded overhang on the nucleic acid nanostructure delivery composition and a complementary sequence added to the nucleic acid barcode construct. **Claim 6** The composition according to claim 1, wherein the nucleic acid nanostructure delivery composition comprises staples that self-assemble to form the nucleic acid nanostructure delivery composition. **Claim 7** The composition according to claim 6, wherein the staples function as the nucleic acid barcode construct. **Claim 8** The composition according to claim 1, wherein the nucleic acid barcode construct is covalently bound to the nucleic acid nanostructure delivery composition. **Claim 9** The composition according to claim 8, wherein the covalent bond is 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 barcode construct. **Claim 10** The composition according to claim 8, wherein the covalent bond is 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 barcode construct. **Claim 11** The composition according to claim 8, wherein the covalent bond is formed via a click chemistry coupling reaction between an azide group on the nucleic acid barcode construct and an alkyne group on the nucleic acid nanostructure delivery composition. **Claim 12** The composition according to claim 1, wherein the nucleic acid barcode construct 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 at the 5' end and / or 3' end of the nucleic acid barcode construct. **Claim 13** The composition according to claim 1, wherein the nucleic acid barcode construct comprises two primer binding segments and one or more unique barcode sequences between the two primer binding segments. **Claim 14** The composition according to claim 13, wherein the length of the native barcode array is at least twice the length of the primer-binding segment.

15. The composition according to claim 13, wherein the native barcode array further comprises a Hamming distance of at least 2 to 6 bases between any two native barcode arrays.

16. The composition according to claim 13, wherein the nucleic acid barcode construct further comprises from about 6 to about 12 random bases at the 3' end of the native barcode array.

17. The composition according to claim 16, wherein about 6 to about 12 random bases at the 3' end of the native barcode array are for bioinformatics removal of PCR duplicates.

18. A method for screening a desired nucleic acid nanostructure delivery composition in vivo, comprising: (a) preparing a library comprising two or more nucleic acid nanostructure delivery compositions, wherein each nucleic acid nanostructure delivery composition is associated with a nucleic acid barcode construct comprising a different native barcode array; (b) administering the library to an animal; (c) removing cells or tissues from the animal; (d) isolating the nucleic acid barcode construct from the cells or tissues of the animal; (e) detecting the nucleic acid barcode construct in the cells or tissues of the animal; and (f) identifying the desired nucleic acid nanostructure delivery composition for use as a delivery vehicle.

19. The method according to claim 18, wherein the nucleic acid barcode construct is detected by a method selected from the group consisting of polymerase chain reaction (PCR), isothermal amplification, sequencing, or combinations thereof, and nucleotide sequence data is obtained.

20. The method according to claim 18, wherein a payload is loaded onto the nucleic acid nanostructure delivery composition.

21. The method according to claim 20, wherein the payload is a luminescent molecule.

22. The method according to claim 21, wherein luminescence is used to track the in vivo distribution or cellular uptake of the nucleic acid nanostructure delivery composition via imaging.

23. The method according to claim 18, wherein the nucleic acid barcode construct is isolated from cells and tissues by mixing the nucleic acid barcode construct with a first organic compound and incubating the organic phase with the aqueous phase of the cell or tissue sample, separating the organic phase from the aqueous phase, mixing the organic phase with a second organic compound, incubating the mixture, precipitating the nucleic acid barcode construct from the mixture, removing the organic phase by evaporation, and resuspending the nucleic acid barcode construct in an aqueous composition.

24. The method according to claim 23, wherein the organic phase contains phenol chloroform.

25. The method according to claim 18, wherein the nucleic acid barcode construct is separated from cationic substances in cells or tissues by titrating an aqueous composition of the nucleic acid barcode construct to a pH greater than 7.

4.

26. The method according to claim 18, wherein the nucleic acid barcode construct is separated from substances in cells or tissues by binding the nucleic acid barcode construct to a molecule having a binding affinity for the nucleic acid barcode construct greater than the binding affinity for the cell or tissue substance.

27. The method according to claim 18, wherein the nucleic acid barcode construct is separated from substances in cells or tissues by a method selected from the group consisting of size exclusion chromatography, dialysis, diafiltration, and filtration.

28. The method according to claim 18, wherein the nucleic acid barcode construct is separated from substances in cells or tissues by digesting proteins using an enzyme that is proteinase K.

29. The method according to claim 18, wherein a nucleic acid barcode construct bound to a nucleic acid nanostructure delivery composition is detected by first diluting the isolated nucleic acid barcode construct at least 1000-fold and then amplifying the nucleic acid barcode construct by PCR using primers.

30. The method according to claim 29, wherein the primers from the PCR step are enzymatically digested before detection of the amplicon.

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