Modified DNA compositions and related methods

Abstract

The disclosure provides, for example, DNA molecules comprising at least one nucleotide with a macromolecule, small molecule, or reactive handle (e.g., click handle) appended to a phosphorothioate backbone. The disclosure also provides methods for making such DNA molecules, for example using click chemistry. In some embodiments, the DNA molecule comprises a sequence that encodes an effector (e.g., a therapeutic effector).

Description

[0001] Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0002] MODIFIED DNA COMPOSITIONS AND RELATED METHODS

[0003] RELATED APPLICATIONS

[0004] This application claims priority to U.S. Serial No.: 63 / 691,659, filed September 6, 2024, the entire contents of which are incorporated herein by reference.

[0005] SEQUENCE LISTING

[0006] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 14, 2025, is named F2128-7027WO SL.xml and is 68,241 bytes in size.

[0007] BACKGROUND

[0008] There is a need for novel therapeutic modalities to address unmet medical need.

[0009] SUMMARY OF THE INVENTION

[0010] Described herein are pharmaceutical DNA compositions, constructs, preparations, methods of using such compositions, constructs and preparations, and methods of making the same.

[0011] Enumerated Embodiments

[0012] 1. A DNA molecule comprising: a) a DNA region that comprises one or more phosphorothioate and has a length of at least 200 nucleotides; and b) an organic moiety linked to the DNA region via a phosphorothioate of the DNA backbone.

[0013] 2. A DNA molecule comprising: a) a DNA region comprising a sequence that encodes an effector (e.g., a therapeutic effector), wherein the DNA region comprises one or more phosphorothioate; and

[0014] 1601838421.1 1 Atorney Docket No.: F2128-7027WO(VL87026-W1) b) an organic moiety linked to the DNA region via a phosphorothioate of the DNA backbone.

[0015] 3. The DNA molecule of embodiment 2, which has a length of at least 50, at least 100, or at least 200 nucleotides.

[0016] 4. The DNA molecule of any of the preceding embodiments, wherein the phosphorothioate is situated between two nucleotides of the DNA molecule.

[0017] 5. The DNA molecule of any of the preceding embodiments, wherein the organic moiety is linked to the phosphorothioate via a trifunctional linker, wherein the trifunctional linker is linked to a second organic moiety.

[0018] 6. The DNA molecule of any of the preceding embodiments, wherein the organic moiety is linked to the phosphorothioate via a multifunctional linker, wherein the multifunctional linker is linked to at least a second organic moiety.

[0019] 7. A DNA molecule comprising a plurality of nucleotides, wherein: at least one nucleotide of the DNA molecule comprises a structure according to Formula Formula (I), wherein Su is a sugar moiety, B is a nucleobase or hydrogen; G is a bond or a linker group L; and R is an organic moiety; and the DNA molecule has a length of at least 200 nucleotides.

[0020] 8. The DNA molecule of any of embodiments 1-7, which comprises a promoter sequence operatively linked to an effector sequence that encodes an effector (e.g., a therapeutic effector).

[0021] 1601838421.1 2 Attorney Docket No.: F2128-7027WO(VL87026-W1)

[0022] 9. A DNA molecule comprising a plurality of nucleotides, wherein: at least one nucleotide of the DNA molecule comprises a structure according to Formula (I): Formula (I), wherein Su is a sugar moiety; B is a nucleobase or hydrogen; G is a bond or a linker group L; and R is an organic moiety, and the DNA molecule comprises a promoter sequence operatively linked to an effector sequence that encodes an effector (e.g., a therapeutic effector).

[0023] 10. The DNA molecule of any of embodiments 7-9, wherein the sugar moiety is deoxyribose.

[0024] 11. The DNA molecule of any of embodiments 7-10, wherein G is a bond.

[0025] 12. The DNA molecule of any of embodiments 7-10, wherein G is a linker group L, and R-

[0026] 1601838421.1 3 Atorney Docket No.: F2128-7027WO(VL87026-W1) wherein, for both Formula (II) and Formula (Ila), m = 0-10; n=0-10; z=0-4; Li is a peptide bond of -CONH- or -NHCO-; RL, if present, is each independently a reactive linker; and Ri is the organic moiety, and wherein, for Formula (Ila), p= 1 -10 and L2 is -O-CH2-CH2- or -CH2-CH2-O-.

[0027] 13. The DNA molecule of embodiment 12, wherein m = 2-10.

[0028] 14. The DNA molecule of any of embodiments 7-10, wherein G is a linker group L, and R- G- comprises Formula (III): Formula (III), wherein o=0 or 1; p=0 or 1, wherein o^p; q = 0-10; z=0-4; RL, if present, is each independently a reactive linker; and R2 is an organic moiety.

[0029] 15. The DNA molecule of any of embodiments 7-10, wherein G is a linker group L, and R-

[0030] G- comprises Formula (IV): Formula (IV), wherein u = 0-10; z=0-4; RL, if present, is each independently a reactive linker; and R3 is an organic moiety.

[0031] 1601838421.1 4 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0032] 16. The DNA molecule of embodiment 15, wherein u = 3-10.

[0033] 17. The DNA molecule of any of embodiments 1-16, wherein the organic moiety comprises a macromolecule, a small molecule, or a reactive handle (e.g., a click handle).

[0034] 18. The DNA molecule of embodiment 17, wherein the macromolecule comprises (1) a polypeptide; (2) a nucleic acid; (3) a glycan; or (4) a lipid.

[0035] 19. The DNA molecule of any of embodiments 1-18, wherein the DNA molecule has a length of at least 200, at least 300, at least 400, at least 500, at least 1000, at least 1500, at least 2000, at least 5000, or at least 10,000 nucleotides, or wherein the DNA molecule has a length of 200-300, 300-400, 400-500, 500-1000, 1000-1500, 1500-2000, 2000-5000, or 5000-10,000 nucleotides.

[0036] 20. The DNA molecule of any of embodiments 1-19, wherein the DNA molecule is single stranded or double stranded.

[0037] 21. The DNA molecule of any of embodiments 1-20, wherein the DNA molecule is circular or linear.

[0038] 22. The DNA molecule of any of embodiments 1-21, wherein the DNA molecule is closed- ended, linear, and double stranded.

[0039] 23. The DNA molecule of any of embodiments 1-21, which comprises a plasmid or minicircle.

[0040] 24. The DNA molecule of any of embodiments 1-23, which comprises a second nucleotide having a chemically modified nucleobase.

[0041] 25. The DNA molecule of any of embodiments 1-24, which comprises a second nucleotide having a chemically modified sugar.

[0042] 1601838421.1 5 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0043] 26. The DNA molecule of any of embodiments 1-25, which comprises a second nucleotide having a backbone modification (e.g., phosphorothioate).

[0044] 27. The DNA molecule of any of embodiments 7-26, wherein B is H.

[0045] 28. The DNA molecule of any of embodiments 7-26, wherein B is a nucleobase.

[0046] 29. The DNA molecule of any of embodiments 7-26, wherein B comprises a canonical nucleobase, wherein optionally the canonical nucleobase is A, C, G, or T.

[0047] 30. The DNA molecule of any of embodiments 7-26, wherein B comprises a chemically modified nucleobase.

[0048] 31. The DNA molecule of any of embodiments 7-30, wherein at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, or at least 500 nucleotides of the DNA molecule each independently comprises a nucleotide according to Formula (I), or wherein 2-5, 5-10, 10- 20, 20-50, 50-100, 100-200, or 200-500 nucleotides of the DNA molecule each independently comprises a nucleotide according to Formula (I).

[0049] 32. The DNA molecule of any of embodiments 7-31, wherein 1-2%, 2-5%, 5-10%, 10-20%, or 20-50% of the nucleotides of the DNA molecule are each independently a nucleotide according to Formula (I).

[0050] 33. The DNA molecule of any of embodiments 1-32, wherein one or more nucleotides of the DNA molecule comprise a canonical phosphate group.

[0051] 34. The DNA molecule of any of embodiments 7-33, wherein all nucleotides of the DNA molecule comprise either a canonical phosphate group or a nucleotide of Formula (I).

[0052] 1601838421.1 6 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0053] 35. The DNA molecule of any of embodiments 1 -34, which comprises a sense strand and an antisense strand.

[0054] 36. The DNA molecule of embodiment 35, wherein the nucleotide according to Formula (I) is in the sense strand.

[0055] 37. The DNA molecule of embodiment 35, wherein all nucleotides according to Formula (I) in the DNA molecule are in the sense strand.

[0056] 38. The DNA molecule of any of embodiments 35-37, wherein the antisense strand is substantially free of (e.g., is free of) chemically modified nucleobases.

[0057] 39. The DNA molecule of any of embodiments 35-38, wherein the antisense strand is substantially free of (e.g., is free of) chemically modified nucleotides.

[0058] 40. The DNA molecule of any of embodiments 8-39, wherein the nucleotide according to Formula (I) is inside the promoter sequence or outside the promoter sequence.

[0059] 41. The DNA molecule of any of embodiments 8-40, wherein the nucleotide according to Formula (I) is inside the effector sequence or outside the effector sequence.

[0060] 42. The DNA molecule of any of embodiments 8-41, which comprises an origin of replication, and wherein the nucleotide according to Formula (I) is inside the origin of replication or outside the origin of replication.

[0061] 43. The DNA molecule of any of embodiments 7-42, wherein the nucleotide according to Formula (I) is situated in a single stranded region of the DNA molecule.

[0062] 44. The DNA molecule of any of embodiments 7-42, wherein the nucleotide according to Formula (I) is situated in a double stranded region of the DNA molecule.

[0063] 1601838421.1 7 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0064] 45. The DNA molecule of any of embodiments 1 -22 or 24-44, which comprises: a) an upstream DNA end form which is a closed end; b) a double stranded region; and c) a downstream DNA end form which is a closed end.

[0065] 46. The DNA molecule of embodiment 45, wherein the nucleotide according to Formula (I) is situated in the upstream DNA end form.

[0066] 47. The DNA molecule of embodiment 45, wherein the nucleotide according to Formula (I) is situated in the downstream DNA end form.

[0067] 48. The DNA molecule of embodiment 45, wherein the nucleotide according to Formula (I) is situated in the double stranded region.

[0068] 49. The DNA molecule of any of embodiments 2-6 or 8-48, wherein the effector comprises a polypeptide (e.g., a DNA binding protein; an epigenetic modifying factor; an antigen; a hormone; an enzyme; a CRISPR-linked enzyme; a mobile genetic element protein; a gene writer; an antibody; a signaling peptide; a receptor ligand; a receptor (e.g., a chimeric antigen receptor (CAR) or a T cell receptor); or a clotting factor).

[0069] 50. The DNA molecule of any of embodiments 2-6 or 8-48, wherein the effector comprises an RNA (e.g., an mRNA, miRNA, or siRNA).

[0070] 51. The DNA molecule of any of embodiments 2-6 or 8-50, which further encodes a second effector.

[0071] 52. The DNA molecule of embodiment 51, which further comprises a second promoter operatively linked to a second effector sequence that encodes the second effector.

[0072] 53. The DNA molecule of embodiment 51 or 52, wherein the effector and the second effector have the same sequence or different sequences.

[0073] 1601838421.1 8 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0074] 54. The DNA molecule of any of embodiments 2-6, 8-49, or 51-53, wherein the effector has a length of at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1000 amino acids.

[0075] 55. The DNA molecule of any of embodiments 2-6, 8-49, or 51-53, wherein the effector has a length of 5-10, 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 amino acids.

[0076] 56. The DNA molecule of any of embodiments 7-55, wherein R is a polypeptide, and the polypeptide has a length of 2-10, 10-20, 20-30, 30-40, or 40-50 amino acids.

[0077] 57. The DNA molecule of any of embodiments 7-55, wherein R is a polypeptide, and the polypeptide has a length of at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, or at least 50 amino acids.

[0078] 58. The DNA molecule of any of embodiments 7-57, wherein every R in the DNA molecule is a polypeptide having the same polypeptide sequence.

[0079] 59. The DNA molecule of any of embodiments 7-57, wherein at least 90%, at least 95%, or at least 99% of instances of R in the DNA molecule are polypeptides having the same polypeptide sequence.

[0080] 60. The DNA molecule of any of embodiments 7-10 or 12-59, wherein R is a polypeptide, and the polypeptide binds to linker group L via the N-terminus of the polypeptide.

[0081] 61. The DNA molecule of any of embodiments 7-10 or 12-59, wherein R is a polypeptide, and the polypeptide binds to linker group L via the C-terminus of the polypeptide.

[0082] 62. The DNA molecule of any of embodiments 7-61, wherein R is a polypeptide comprising a cell penetrating peptide, an albumin protein, an antibody, a transferrin, insulin, a targeting peptide, or a nuclear localization sequence (NLS).

[0083] 1601838421.1 9 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0084] 63. The DNA molecule of any of embodiments 7-62, wherein R is a polypeptide other than a myoglobin or other than swMb (sperm whale myoglobin B).

[0085] 64. The DNA molecule of any of embodiments 7-55, wherein R is a nucleic acid, and the nucleic acid comprises an aptamer, an adjuvanting sequence (e.g., CpG oligodeoxynucleotide), an immune suppressing sequence (e.g., GpC oligodeoxynucleotide), or a homopolymer.

[0086] 65. The DNA molecule of any of embodiments 7-55, wherein R is a glycan, and the glycan comprises amino sugars (e.g., N-acetylgalactosamine), uronic sugars, aldohexoses (e.g., glucose, galactose, or mannose), ketohexoses (e.g., fructose), sialic acids, or a combination thereof.

[0087] 66. The DNA molecule of any of embodiments 7-55, wherein R is a small molecule, and the small molecule comprises a monosaccharide (e.g., glucose, fructose, galactose, mannose, or N- acetylgalactosamine).

[0088] 67. The DNA molecule of any of embodiments 7-55, wherein R is a small molecule, and the small molecule comprises a disaccharide (e.g., lactose).

[0089] 68. The DNA molecule of any of embodiments 7-55, wherein R is a lipid, and the lipid comprises saturated fatty acids, unsaturated fatty acids, triglycerides, diglycerides, monoglycerides, phospholipids, sphingolipids, steroids, glycolipids, or a combination thereof.

[0090] 69. The DNA molecule of any of embodiments 7-10 or 12-68, wherein L comprises a reactive linker, e.g., a click linker.

[0091] 70. The DNA molecule of any of embodiments 1-69, wherein the DNA molecule lacks a material portion of vector backbone (e.g., plasmid backbone).

[0092] 71. The DNA molecule of any of embodiments 1-70, which does not comprise a non-human (e.g., bacterial) origin of replication.

[0093] 1601838421.1 10 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0094] 72. The DNA molecule of any of embodiments 1-71, which does not comprise an antibiotic resistance selectable marker.

[0095] 73. The DNA molecule of any of embodiments 2-6 or 8-72, wherein the effector sequence does not encode a viral protein.

[0096] 74. The DNA molecule of any of embodiments 1-73, which is unencapsidated.

[0097] 75. The DNA molecule of any of embodiments 1-74, which does not comprise a viral packaging signal.

[0098] 76. The DNA molecule of any of embodiments 1-75, which does not comprise a viral ITR.

[0099] 77. The DNA molecule of any of embodiments 1-76, which is essentially free of viral proteins.

[0100] 78. The DNA molecule of any of embodiments 1-77, which lacks a photoreactive moiety.

[0101] 79. The DNA molecule of any of embodiments 1-78, which is free of biotin.

[0102] 80. The DNA molecule of any of embodiments 1-79, which is free of streptavidin.

[0103] 81. The DNA molecule of any of embodiments 1-80, which is free of a radioactive isotope.

[0104] 82. The DNA molecule of any of embodiments 2-6 or 8-81, which expresses the effector when present in a cell.

[0105] 83. The DNA molecule of any of embodiments 1-82, which is transcribed when present in a cell.

[0106] 1601838421.1 11 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0107] 84. The DNA molecule of any of embodiments 1 -83, wherein when the DNA molecule is introduced to a cell, the cell exhibits an interferon beta mRNA level that is no more than 50% greater than that of a control cell of the same type that was contacted with an otherwise similar DNA molecule having the same sequence as the DNA molecule at the same molar amount as the DNA molecule, but comprising all canonical phosphodiester backbone(s).

[0108] 85. The DNA molecule of any of embodiments 1-83, wherein when the DNA molecule is introduced to a cell, the cell exhibits an interferon beta mRNA level that is no more than 50% greater than that of a control cell of the same type that was contacted with an otherwise similar DNA molecule having the same sequence as the DNA molecule at the same molar amount as the DNA molecule, but comprising only canonical nucleotides.

[0109] 86. The DNA molecule of any of embodiments 1-85, wherein when the DNA molecule is introduced to a cell, the cell expresses the effector at a level no less than 50% of that of a control cell of the same cell type that was contacted with an otherwise similar DNA molecule having the same sequence as the DNA molecule at the same molar amount as the DNA molecule, but comprising all canonical phosphodiester backbones.

[0110] 87. The DNA molecule of any of embodiments 1-85, wherein when the DNA molecule is introduced to a cell, the cell expresses the effector at a level no less than 50% of that of a control cell of the same cell type that was contacted with an otherwise similar DNA molecule having the same sequence as the DNA molecule at the same molar amount as the DNA molecule, but comprising only canonical nucleotides.

[0111] 88. The DNA molecule of any of embodiments 1-87, wherein the DNA molecule exhibits a half-life in 0.1% FBS at a temperature of 37 C wherein the half life is no less than 50% of that of an otherwise similar DNA molecule having the same sequence as the DNA molecule at the same molar amount as the DNA molecule, but comprising all canonical phosphodi ester backbones.

[0112] 89. The DNA molecule of any of embodiments 1-87, wherein the DNA molecule exhibits a half-life in 0.1% FBS at a temperature of 37 C wherein the half life is no less than 50% of that of

[0113] 1601838421.1 12 Atorney Docket No.: F2128-7027WO(VL87026-W1) an otherwise similar DNA molecule having the same sequence as the DNA molecule at the same molar amount as the DNA molecule, but comprising only canonical nucleotides.

[0114] 90. A composition comprising a plurality of copies of the DNA molecule of any of embodiments 1-89, wherein all DNA molecules in the composition have substantially the same length in nucleotides (e.g., all DNA molecules in the composition have the same length in nucleotides).

[0115] 91. A composition comprising a plurality of copies of the DNA molecule of any of embodiments 2-6 or 8-89, wherein the effector region of all DNA molecules in the composition have substantially the same length in nucleotides (e.g., the effector region of all DNA molecules in the composition have the same length in nucleotides).

[0116] 92. A composition comprising a plurality of copies of the DNA molecule of any of embodiments 1-89, wherein all DNA molecules in the composition have a length of between 100, 200, 500, or 1000 nucleotides of each other.

[0117] 93. A composition comprising a plurality of copies of the DNA molecule of any of embodiments 1-89, wherein all DNA molecules in the composition have a length of between 500-1000, 1000-2000, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, 7000-8000, 8000-9000, 9000-10000, 10000-11000, or 11000-12000 nucleotides.

[0118] 94. A composition comprising a plurality of copies of the DNA molecule of any of embodiments 2-6 or 8-89, wherein all DNA molecules in the composition encode substantially the same effector (e.g., all DNA molecules in the composition encode the same effector).

[0119] 95. A composition comprising a plurality of copies of the DNA molecule of any of embodiments 1-89, wherein all DNA molecules in the composition have substantially the same sequence (e.g., all DNA molecules in the composition have the same sequence).

[0120] 96. A compound comprising a nucleotide having Formula (1):

[0121] 1601838421.1 13 Attorney Docket No.: F2128-7027WO(VL87026-W1) Formula (1), wherein Bl is H or a nucleobase; Su is a sugar moiety; m=0-10; n=0-10; z=0-4; L is a peptide bond of -CONH- or -NHCO-; RL, if present, is each independently a reactive linker; and Ri is an organic moiety.

[0122] 97. The compound of embodiment 96 represented by Formula (la): Formula (la).

[0123] 98. The compound of embodiment 96 or 97, wherein m = 2-10.

[0124] 99. A compound comprising a nucleotide having Formula (2): Formula (2),

[0125] 1601838421.1 14 Attorney Docket No.: F2128-7027WO(VL87026-W1) wherein B2 is H or a nucleobase; Su is a sugar moiety; o=0 or 1; p=0 or 1, wherein o^p; q = 0- 10; z=0-4; RL, if present, is each independently a reactive linker; and R2 is an organic moiety.

[0126] 100. The compound of embodiment 99 represented by Formula (2a): Formula (2a).

[0127] 101. The compound of embodiment 99 or 100, wherein q = 3-10.

[0128] 102. A compound comprising a nucleotide having Formula (3): Formula (3), wherein B3 is H or a nucleobase; Su is a sugar moiety; u=0-10; z=0-4; RL, if present, is each independently a reactive linker; and R3 is an organic moiety.

[0129] 103. The compound of embodiment 102 represented by Formula (3a):

[0130] 1601838421.1 15 Atorney Docket No.: F2128-7027WO(VL87026-W1) Formula (3a).

[0131] 104. The compound of embodiment 102 or 103, wherein u = 3-10.

[0132] 105. The compound of any of embodiments 96-104, wherein the organic moiety comprises a macromolecule, a small molecule, or a reactive handle (e.g., a click handle).

[0133] 106. The compound of embodiment 105, wherein the macromolecule comprises any of (1) a polypeptide; (2) a nucleic acid; (3) a glycan; or (4) a lipid.

[0134] 107. The compound of any of embodiments 96-106, which is a single nucleotide.

[0135] 108. The compound of any of embodiments 96-107, wherein the phosphorothioate group is covalently bound to a phosphate group, e.g., the nucleotide is a diphosphate or triphosphate.

[0136] 109. The compound of any of embodiments 96-106 or 108, wherein the nucleotide is part of a DNA molecule.

[0137] 110. The compound of any of embodiments 105 or 107-109, wherein the reactive handle comprises dibenzocyclooctyne, azide, maleimide, thiol, amine, or N-hydroxy succinimide (NHS) ester.

[0138] 111. A DNA molecule comprising at least one of nucleotides of embodiments 96-106 or 108-

[0139] 110, wherein the DNA molecule has a length of at least 5, at least 10, at least 15, or at least 20 nucleotides.

[0140] 1601838421.1 16 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0141] 112. A DNA molecule comprising at least one of nucleotides of embodiments 96-106 or 108- 110, wherein the DNA molecule has a length of 5-10, 10-15, 15-20, 20-50, 50-100, or 100-200 nucleotides.

[0142] 113. A pharmaceutical composition comprising a DNA molecule of any of embodiments 1-89 or 111-112, and a pharmaceutically acceptable carrier or excipient.

[0143] 114. A pharmaceutical composition comprising a DNA molecule that comprises a compound of any of embodiments 96-106 or 108-110, and a pharmaceutically acceptable carrier or excipient.

[0144] 115. The pharmaceutical composition of embodiment 113 or 114, wherein the DNA molecule is comprised in a lipid nanoparticle (LNP).

[0145] 116. The pharmaceutical composition of embodiment 113 or 114, which is substantially free of (e.g., is free of) LNPs.

[0146] 117. The pharmaceutical composition of embodiment 113 or 114, which is substantially free of (e g., free of) nanoparticles.

[0147] 118. The pharmaceutical composition of embodiment 113 or 114, which is substantially free of (e.g., is free) of lipids.

[0148] 119. The pharmaceutical composition of any of embodiments 113-118, which is free of a solid support.

[0149] 120. The pharmaceutical composition of any of embodiments 113-119, which is free of a solid material.

[0150] 121. The pharmaceutical composition of any of embodiments 113-120, which is in a solution.

[0151] 1601838421.1 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0152] 122. The pharmaceutical composition of any of embodiments 113-121, which is not a suspension.

[0153] 123. A method of modulating (e.g., increasing or decreasing) a biological activity in a target cell, the method comprising:

[0154] (i) contacting a target cell with the DNA molecule of any of embodiments 2-6 or 8-89, the composition of any of embodiments 90-95, or the pharmaceutical composition of any of embodiments 113-122, wherein the effector modulates a biological activity in the target cell; and

[0155] (ii) maintaining (e g., incubating) the cell under conditions suitable for expressing the effector from the DNA molecule; thereby modulating the biological activity in the target cell.

[0156] 124. A method of modulating (e.g., increasing or decreasing) a biological activity in a target cell, the method comprising:

[0157] (i) providing a target cell comprising the DNA molecule of any of embodiments 2-6 or 8- 89, the composition of any of embodiments 90-95, or the pharmaceutical composition of any of embodiments 113-122, wherein the effector modulates a biological activity in the target cell; and

[0158] (ii) maintaining (e.g., incubating) the cell under conditions suitable for expressing the effector from the DNA molecule; thereby modulating the biological activity in the target cell.

[0159] 125. The method of embodiment 124, wherein (i) comprises contacting the target cell with the DNA molecule, composition, or pharmaceutical composition.

[0160] 126. The method of any of embodiments 123-125, wherein the biological activity comprises cell growth, cell metabolism, cell signaling, cell movement, specialization, interactions, division, transport, homeostasis, osmosis, or diffusion.

[0161] 127. The method of any of embodiments 123-126, wherein the cell is an animal cell, e.g., a mammalian cell, e.g., a human cell.

[0162] 1601838421.1 18 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0163] 128. A method of treating a cell, tissue, or subject in need thereof, the method comprising: administering to the cell, tissue, or subject the DNA molecule of any of embodiments 1-

[0164] 89, 111, or 112, the composition of any of embodiments 90-95, or the pharmaceutical composition of any of embodiments 113-122; thereby treating the cell, tissue, or subject.

[0165] 129. The method of any of embodiments 123-128, which is performed ex vivo or in vivo.

[0166] 130. A method of making a DNA molecule, the method comprising: providing a PS DNA molecule comprising Formula (a): Formula (a), wherein B is a nucleobase and Su is a sugar moiety; and providing a halide agent having Formula (b) Formula (b), wherein m = 0-10; n=0-10; z=0-4; Li is a peptide bond of -CONH- or -NHCO-; RL, if present, is each independently a reactive linker; X = halo (e.g., I, Br or Cl); and Ri is an organic moiety, in a solution; and reacting the PS DNA molecule and the halide agent to form the DNA molecule.

[0167] 131. A method of making a DNA molecule, the method comprising: providing a PS DNA molecule comprising Formula (a)

[0168] 1601838421.1 19 Attorney Docket No.: F2128-7027WO(VL87026-W1) Formula (a), wherein B is a nucleobase and Su is a sugar moiety, and providing a maleimide agent having Formula (d) Formula (d), wherein u = 0-10; z=0-4; RL, if present, is each independently a reactive linker; R3 is an organic moiety, in a solution; and reacting the PS DNA molecule and the maleimide agent to form the DNA molecule. 132. The method of embodiment 130 or 131, wherein the PS DNA molecule comprises

[0169] Formula (c) Formula (c).

[0170] 133. The method of any of embodiments 130-132, wherein the organic moiety comprises a macromolecule, a small molecule, or a reactive handle (e.g., a click handle).

[0171] 1601838421.1 20 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0172] 134. The method of any of embodiments 130-133, wherein the organic moiety is a reactive handle, and the method further comprises contacting the DNA molecule with a macromolecule or small molecule comprising a second, compatible reactive handle, under conditions that allow the reactive handle and the second reactive handle to react.

[0173] 135. The method of embodiment 134, wherein the reactive handle is a click handle, and the second reactive handle is a second click handle.

[0174] 136. A DNA molecule produced by the method of any of embodiments 130-135.

[0175] Definitions

[0176] As used herein, the term "antibody" refers to a molecule that specifically binds to, or is immunologically reactive with, a particular antigen and includes at least the variable domain of a heavy chain, and normally includes at least the variable domains of a heavy chain and of a light chain of an immunoglobulin. Antibodies and antigen-binding fragments, variants, or derivatives thereof include, but are not limited to, polyclonal, monoclonal, multi specific, human, humanized, primatized, or chimeric antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), single-domain antibodies (sdAb), epitopebinding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), rlgG, singlechain antibodies, disulfide-linked Fvs (sdFv), nanobody, fragments including either a VL or VH domain, fragments produced by an Fab expression library, and anti -idiotypic (anti-Id) antibodies. Antibodies described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. Moreover, unless otherwise indicated, the term "monoclonal antibody" (mAb) is meant to include both intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) that are capable of specifically binding to a target protein. Fab and F(ab')2 fragments lack the Fc fragment of an intact antibody.

[0177] As used herein, the term “carrier” means a compound, composition, reagent, or molecule that facilitates or promotes the transport or delivery of a composition (e.g., a DNA molecule

[0178] 1601838421.1 21 Atorney Docket No.: F2128-7027WO(VL87026-W1) described herein) into a cell. For example, a carrier may be a partially or completely encapsulating agent.

[0179] As used herein, the term “chemically modified nucleotide,” as used herein with respect to DNAs, refers to a nucleotide comprising one or more structural differences relative to the canonical deoxyribonucleotides (i.e., G, T, C, and A). A chemically modified nucleotide may have (relative to a canonical nucleotide) a chemically modified nucleobase, a chemically modified sugar, a chemically modified phosphodiester linkage, or a combination thereof. No particular process of making is implied; for instance, a chemically modified nucleotide can be produced directly by chemical synthesis, or by covalently modifying a canonical nucleotide.

[0180] A “click handle,” as that term is used herein, refers to a chemical moiety that is capable of reacting with a second click handle in a click reaction to produce a click linker.

[0181] A “click linker,” as that term is used herein, refers to a plurality of atoms disposed between and covalently linking entity A and entity B, wherein the click linker is formed as the product of a click reaction that links entity A and entity B. In some embodiments, the click linker has the structure of a click linker that is formed as the product of a click reaction that links entity A and entity B, but is not limited to a click linker made by any particular process. For example, a click linker may be formed by a click reaction, but a click linker can also be formed or provided by a process other than a click reaction. In an embodiment, the click linker is an alkyne / azide click linker, e.g., the click linker comprises a triazole.

[0182] A “click reaction”, as that term is used herein, refers to a range of reactions used to covalently link a first moiety and a second moiety, for convenient production of linked products. It typically has one or more of the following characteristics: it is fast, is specific, is high-yield, is efficient, is spontaneous, does not significantly alter biocompatibility of the linked entities, has a high reaction rate, produces a stable product, favors production of a single reaction product, has high atom economy, is chemoselective, is modular, is stereoselective, is insensitive to oxygen, is insensitive to water, is high purity, generates only inoffensive or relatively non-toxic byproducts that can be removed by nonchromatographic methods (e.g., crystallization or distillation), needs no solvent or can be performed in a solvent that is benign or physiologically compatible, e.g., water, stable under physiological conditions. Examples include an alkyne / azide reaction, a diene / dienophile reaction, or a thiol / alkene reaction. Other reactions can be used. In some embodiments, the click reaction is fast, specific, and high-yield. For instance, in embodiments, a

[0183] 1601838421.1 22 Atorney Docket No.: F2128-7027WO(VL87026-W1) fast click reaction has a second order forward rate constant of 10-200 least 1, 2, 3, 5, 10, 20, 50, 60, 100, 200, 500, 1E3, 2E3, 5E3, 1E4, 2E4, 5E4, 1E5, 2E5, 5E5, or 1E6 M^s'1, e.g., at 20°C in PBS. In some embodiments, a high-yield click reaction is one which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% yield, e.g., for a reaction time of 1 hour at 20°C in PBS.

[0184] A “reactive handle,” as that term is used herein, refers to a chemical moiety that is capable of reacting with a second reactive handle to produce a reactive linker. In some embodiments, the reactive handle comprises NHS ester, amine, pyridyl dithiol, or thiol. In some embodiments, the reactive handle is a click handle.

[0185] As used herein, the term “reactive linker” refers to an atom or a group of atoms disposed between and covalently linking entity A and entity B, wherein the reactive linker is formed as the product of a reaction that links entity A and entity B. Reactive linkers include click linkers. The reactive linker is not limited to being made by any particular process.

[0186] As used herein, the term “closed end” refers to a portion of a DNA molecule positioned at one end of a double-stranded region, in which all nucleotides within the portion of the DNA molecule are covalently attached to adjacent nucleotides on either side. A closed end may, in some embodiments, include a loop comprising one or more nucleotides that are not hybridized to another nucleotide. In some embodiments, every nucleotide of the closed end is hybridized to another nucleotide. In some embodiments, a double-stranded DNA region comprises a first closed end (e.g., upstream of a heterologous object sequence) and a second closed end (e g., downstream of a heterologous object sequence).

[0187] As used herein, the term “open end” refers to a portion of a DNA molecule positioned at one end of a double-stranded region, in which at least one nucleotide (a “terminal nucleotide”) is covalently attached to exactly one other nucleotide. In some embodiments, the terminal nucleotide comprises a free 5’ phosphate. In some embodiments, the terminal nucleotide comprises a free 3’ OH. In some embodiments, in a double-stranded DNA region comprising a first DNA strand and a second DNA strand, the open end comprises a first terminal nucleotide on the first DNA strand and a second terminal nucleotide on the second DNA strand. In some embodiments, a DNA region comprises a first open end (e.g., upstream of a heterologous object sequence) and a second open end (e.g., downstream of a heterologous object sequence). In some embodiments, the open end comprises a blunt end, a sticky end, or a Y-adaptor.

[0188] 1601838421.1 23 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0189] As used herein, the term “DNA molecule” refers to any compound and / or substance that comprises at least two (e.g., at least 10, at least 20, at least 50, at least 100) covalently linked deoxyribonucleotides. In some embodiments, the DNA molecule is a single oligonucleotide chain, while in other embodiments, the DNA molecule comprises a plurality of oligonucleotide chains (e.g., the DNA molecule is a double stranded circular DNA molecule comprising a first single stranded chain base paired with a a second single stranded chain), while in yet other embodiments the DNA molecule is a portion of a larger molecule, e.g., the DNA molecule may be a portion of a longer DNA molecule. In some embodiments, the DNA molecule is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage, phosphorothioate linkage or a boranophosphate linkage. In some embodiments, the DNA molecule comprises solely canonical nucleotides. In some embodiments, the DNA comprises one or more chemically modified nucleotides. In some embodiments, the DNA molecule comprises one or more noncanonical nucleobases or abasic sites. In some embodiments, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of the sugars of the DNA molecule are deoxyribose sugars. In some embodiments, the DNA molecule was prepared by one or more of: isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, the DNA molecule comprises a non-nucleotide conjugate.

[0190] As used herein, the term “DNA region” refers to a portion of a DNA molecule, wherein the portion comprises (e.g., consists of) DNA, and wherein the DNA molecule further comprises an organic moiety. The DNA region does not comprise the organic moiety. In some embodiments, the DNA comprises solely canonical nucleotides. In some embodiments, the DNA comprises one or more chemically modified nucleotides. In some embodiments, the DNA region is single-stranded. In some embodiments, the DNA region is double-stranded.

[0191] As used herein, the term “DNA end form” refers to a structure comprising DNA that is situated at an end of a double-stranded DNA region. In some embodiments, the DNA end form comprises a closed end. In other embodiments, the DNA end form comprises an open end. In some embodiments, the DNA end form comprises a hairpin, a loop, a Y-adaptor, a blunt end, or a sticky end. The DNA end form may comprise one or both of a single stranded region and a double stranded region. The DNA end form may comprise canonical nucleotides, chemically

[0192] 1601838421.1 24 Atorney Docket No.: F2128-7027WO(VL87026-W1) modified nucleotides, or a combination thereof. In some embodiments, the DNA end form comprises between 3-100 nucleotides. In some embodiments, the double-stranded DNA region comprises a first DNA end form at a first end and a second DNA end form at a second end. In some embodiments, the first DNA end form and the second DNA end form of a DNA region are the same type. In some embodiments, the first DNA end form and the second DNA end form of a DNA region are different types.

[0193] As used herein, the term “exonuclease-resistant”, when used to describe a DNA, means that the DNA, if it comprises closed ends, is resistant to the exonuclease assay as described in Example 10 or Example 11 of PCT / US2023 / 066950.

[0194] As used herein, the term “heterologous”, when used to describe a first element in reference to a second element means that the first element and second element do not exist in nature disposed as described. For example, a heterologous NLS, relative to a particular DNA region, does not occur in nature linked to that DNA region. For example, a heterologous polypeptide, nucleic acid molecule, construct or sequence refers to (a) a polypeptide, nucleic acid molecule or portion of a polypeptide or nucleic acid molecule sequence that is not native to a cell in which it is expressed, (b) a polypeptide or nucleic acid molecule or portion of a polypeptide or nucleic acid molecule that has been altered or mutated relative to its native state, or (c) a polypeptide or nucleic acid molecule with an altered expression as compared to the native expression levels under similar conditions. For example, a heterologous regulatory sequence (e.g., promoter, enhancer) may be used to regulate expression of a gene or a nucleic acid molecule in a way that is different than the gene or a nucleic acid molecule is normally expressed in nature. In another example, a heterologous domain of a polypeptide or nucleic acid sequence (e.g., a DNA binding domain of a polypeptide or nucleic acid encoding a DNA binding domain of a polypeptide) may be disposed relative to other domains or may be a different sequence or from a different source, relative to other domains or portions of a polypeptide or its encoding nucleic acid. In certain embodiments, a heterologous nucleic acid molecule may exist in a native host cell genome, but may have an altered expression level or have a different sequence or both. In other embodiments, heterologous nucleic acid molecules may not be endogenous to a host cell or host genome but instead may have been introduced into a host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently (e.g., mRNA) or semi-stably

[0195] 1601838421.1 25 Atorney Docket No.: F2128-7027WO(VL87026-W1) for more than one generation (e.g., episomal viral vector, plasmid or other self-replicating vector).

[0196] As used herein, the term “heterologous functional sequence” refers to a nucleic acid sequence that is heterologous to an adjacent (e.g., directly adjacent) nucleic acid sequence and has one or more biological function.

[0197] As used herein, the terms "increasing" and "decreasing" refer to modulating resulting in, respectively, greater or lesser amounts, of function, expression, or activity of a metric relative to a reference. For example, subsequent to administration of a DNA molecule in a method described herein, the amount of the metric described herein (e.g., the level of gene expression, or a marker of innate immunity) may be increased or decreased in a subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or more relative to the amount of the marker prior to administration, or relative to administration of a control DNA molecule. Generally, the metric is measured subsequent to administration at a time that the administration has had the recited effect, e.g., at least one day, at least one week, at least one month, at least 3 months, or at least 6 months, after a treatment regimen has begun.

[0198] As used herein the term “linear” in reference to a double-stranded DNA region described herein, means a nucleic acid comprising two DNA strands or portions of strands which hybridize with each other (thereby forming a double stranded region), wherein the structure comprises two ends. An end may be a closed end or an open end. The two strands that hybridize with each other may be partially or completely complementary. In some embodiments, a linear doublestranded DNA region consists of a single strand of DNA that is circular under denaturing conditions, wherein under physiological conditions a first portion of the strand hybridizes to a second portion of the strand (thereby forming a double stranded region), and the linear doublestranded DNA region comprises a first closed end comprising a first loop and a second closed end comprising a second loop.

[0199] As used herein, when two entities are “linked”, the two entities are physically connected by means of one or more covalent or noncovalent bond. In some embodiments, the two entities are directly linked, i .e., an atom of the first entity forms a covalent or noncovalent bond with an atom of the second entity. In some embodiments, the two entities are indirectly linked through a

[0200] 1601838421.1 26 Atorney Docket No.: F2128-7027WO(VL87026-W1) third entity; for example A is linked to C by virtue of A being directly linked to B and B being directly linked to C.

[0201] As used herein, the terms “linker” refers to a moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. The linker may be a reactive linker, e.g., a click linker.

[0202] As used herein, the term “loop” refers to a nucleic acid sequence that is single stranded. A loop is connected at both ends by a double stranded region referred to as a “stem”, to form a “stem-loop”.

[0203] As used herein, the term “macromolecule” is a molecule of high relative molecular mass (e.g., greater than 500 daltons, greater than 750 daltons, greater than 1000 daltons, or greater than 5000 daltons). The macromolecule may be covalently conjugated to a DNA molecule. The macromolecule may be natural and / or synthetic polymeric molecule. In some embodiments, the macromolecule comprises a plurality of monomers. The macromolecule can be a biopolymer. The macromolecule can comprise a nucleic acid, polypeptide, glycan, or lipid. The macromolecule can also comprise a conjugate of a biopolymer, including but not limited to, protein-protein conjugates.

[0204] As used herein, the term “maintenance sequence” is a DNA sequence or motif that enables or facilitates retention of a DNA molecule in the nucleus through cell division. A maintenance sequence typically enables replication and / or transcription of DNA in the nucleus by interacting with proteins that facilitate chromatin looping. An example of a maintenance sequence is a scaffold / matrix atached region (S / MAR element).

[0205] As used herein, a "pharmaceutical composition" or "pharmaceutical preparation" is a composition or preparation which is indicated for animal, e.g., human or veterinary pharmaceutical use, for example, non-human animal or human prophylactic or therapeutic use. A pharmaceutical preparation comprises an active agent having a biological effect on a cell or tissue of a subject, e.g., having pharmacological activity or an effect in the mitigation, treatment, or prevention of disease, in combination with a pharmaceutically acceptable excipient or diluent. A pharmaceutical composition also means a finished dosage form or formulation of a prophylactic or therapeutic composition.

[0206] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a compound comprising amino acid residues covalently linked by

[0207] 1601838421.1 27 Atorney Docket No.: F2128-7027WO(VL87026-W1) peptide bonds, or by means other than peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or by means other than peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. In some embodiments, a polypeptide comprises a non-canonical amino acid residue.

[0208] As used herein, the term “protelomerase sequence” refers to a nucleotide sequence capable of being generated by a protelomerase that joins a first protelomerase recognition sequence (PRS) to a second PRS. In some embodiments, the protelomerase sequence was produced by a process involving protelomerase, and in other embodiments the protelomerase sequence was produced by a process that does not involve protelomerase (e.g., by solid phase synthesis).

[0209] As used herein, a “sense strand” of a dsDNA is a strand which has the same sequence as an mRNA or pre-mRNA which encodes for a functional RNA or protein, and does not serve as a template for transcription. An “antisense strand” of a dsDNA is a strand that has a sequence complementary to an mRNA or pre-mRNA which encodes for a functional RNA or protein and / or can serve as a template for transcription.

[0210] As used herein, the term “double-stranded DNA” or dsDNA means a DNA composition comprising two complementary chains of deoxyribonucleotides that base pair to each other. The two complementary strands may have perfect complementarity or may have one or more mismatches, e.g., forming bulges. Either of the two strands may, in some embodiments, have paired regions of self-complementarity that form intramolecular / intrastrand double stranded motifs in a folded configuration, for example, may form hairpin loops, junctions, bulges or internal loops. In some embodiments, the dsDNA comprises one or two closed ends. In some embodiments, the dsDNA molecule is circular or linear. In some embodiments (e.g., in a dsDNA molecule with closed ends) the two complementary chains of deoxyribonucleotides are covalently linked.

[0211] As used herein, the term “spacer domain” refers to a portion of a linker, the portion comprising one or more atoms. The spacer domain increases the length of the linker relative to

[0212] 1601838421.1 28 Atorney Docket No.: F2128-7027WO(VL87026-W1) an otherwise similar linker that lacks the spacer domain. In some embodiments, the spacer domain comprises a PEG moiety. In some embodiments, the PEG moiety has a structure of -(O- CH2-CH2)n, wherein n=2-24, e.g., wherein n=2-8 or n=2-4. In some embodiments, the spacer domain comprises a PEG2 moiety.

[0213] As used herein, the term “therapeutic double stranded construct” (“TDSC”) refers to a linear construct comprising DNA, wherein the construct is at least partially double stranded. A TDSC does not comprise a plasmid backbone sequence (e.g., does not comprise a bacterial origin of replication). A TDSC does not comprise a viral capsid or a viral envelope. In some embodiments, the TDSC comprises a closed end or an open end (e.g., a blunt end or a sticky end). In some embodiments, the TDSC is suitable for administration to a human subject.

[0214] As used herein, the term “second strand motif’ or SSM is a sequence or structural motif in a ssDNA that promotes or enables second strand synthesis. An SSM may comprise a binding site(s) for proteins that initiate DNA synthesis of a second strand, and / or places the DNA in the correct orientation for DNA polymerase binding.

[0215] As used herein, the term “terminal nucleotide” refers to a nucleotide that is covalently attached to exactly one other nucleotide. In some embodiments, the terminal nucleotide comprises a free 5’ phosphate. In some embodiments, the terminal nucleotide comprises a free 3’ OH.

[0216] As used herein, "treatment" and "treating" refer to the medical management of a subject with the intent to improve, ameliorate, stabilize (i.e., not worsen), prevent or cure a disease, pathological condition, or disorder. This term includes active treatment (treatment directed to improve the disease, pathological condition, or disorder), causal treatment (treatment directed to the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed for the relief of symptoms), preventative treatment (treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder); and supportive treatment (treatment employed to supplement another therapy). Treatment also includes diminishment of the extent of the disease or condition; preventing spread of the disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. "Ameliorating" or "palliating" a disease or condition means that the extent and / or undesirable clinical manifestations of the

[0217] 1601838421.1 29 Atorney Docket No.: F2128-7027WO(VL87026-W1) disease, disorder, or condition are lessened and / or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0218] As used herein, the term “Y-adaptor” refers to a nucleic acid structure comprising a first nucleic acid region and a second nucleic acid region which are complementary (e.g., perfectly complementary) to each other; the first and second regions may hybridize to form a double stranded region. The first nucleic acid region is covalently linked to a third nucleic acid region, and the second nucleic acid region is covalently linked to a fourth nucleic acid region, and the third and fourth nucleic acid regions are not substantially complementary to each other; the third and fourth regions may be single stranded. The first nucleic acid region is 3’ of the third nucleic acid region and the second nucleic acid region is 5’ of the fourth nucleic acid region. As a result, the third and fourth regions may be situated on the same side of the double stranded regions. The Y-adaptor may be part of a double-stranded DNA region.

[0219] BRIEF DESCRIPTION OF THE DRAWINGS

[0220] FIGS. 1A and IB are a schematic and agarose gel, respectively, showing that an azide moiety present on PS-ssDNA allows for a click reaction to occur with DBCO-Cy3. PS-ssDNA is first reacted with iodoacetamide azide or iodoacetamide-PEG3 -azide, yielding PS-ssDNA with an azide click handle which is then reacted with DBCO-Cy3, as shown conceptually in FIG. 1A. The zig-zag line in the conjugated DNA of FIG. 1 A generally represents a linker, and various linkers are described herein. The gel (FIG. IB) shows the PS-ssDNA before and after reaction with DBCO-Cy3. Lane a shows unreacted PS-ssDNA, lane b shows PS-ssDNA that was reacted with iodoacetamide azide, and lane c shows PS-ssDNA that was reacted with iodoacetamide- PEG3-azide. The change in electrophoretic mobility of lanes b and c following DBCO-Cy3 treatment indicates successful click reactions.

[0221] FIGS. 2A and 2B are a schematic (FIG. 2A) and two agarose gel images (FIG. 2B) showing that PS-dsDNA can be reacted with iodoacetamide azide or iodoacetamide-PEG3 -azide to append an azide click handle onto PS-DNA. Schematic (FIG. 2A) shows the different

[0222] 1601838421.1 30 Atorney Docket No.: F2128-7027WO(VL87026-W1) chemistries tested to append either an azide click handle (using iodoacetamide azide, shown in structure b), or iodoacetamide-PEG3 -azide, shown in structure c), resulting in the DNA conjugated to azide which is shown in the right hand side of the reaction. FIG. 2A also shows that PS-dsDNA can be reacted with maleimide-sulfo-Cy3, shown in structure d), to append a Cy3 dye onto PS-DNA, in order to produce DNA appended to Cy3 (not shown). The zig-zag line in the conjugated DNA of FIG. 2A generally represents a linker, and various linkers are described herein. Gel (FIG. 2B, left) shows the change in electrophoretic mobility between PS- dsDNA after various reactions, indicating a successful reaction. Lane a shows unreacted dsDNA, lane b shows dsDNA after reaction with iodoacetamide azide, lane c shows dsDNA after reaction with iodoacetamide-PEG3 -azide, and lane d shows dsDNA after reaction with maleimide-sulfo- Cy3. Gel (FIG. 2B, right) shows that no change is observed when a control (phosphodiester- dsDNA) is used instead of PS-dsDNA.

[0223] FIGS. 3A and 3B are a schematic (FIG. 3A) and a graph (FIG. 3B) showing that a Cy3 dye molecule can be appended onto PS-dsDNA through a click reaction. Reaction scheme in FIG. 3 A (top) shows the chemical reactions of PS-dsDNA with iodoacetamide azide or iodoacetamide-PEG3 -azide followed by DBCO-Cy3 treatment. Reaction scheme in FIG. 3A (bottom) shows treatment of PS-dsDNA with maleimide-sulfo-Cy3. The zig-zag line in the conjugated DNA of FIG. 3 A generally represents a linker, and various linkers are described herein. Graph (FIG. 3B) indicates relative levels of Cy3 detected from the tested reactions. Cy3 signal was detected for all three reactions.

[0224] FIG. 4 is two images of an agarose gel showing that maleimide-sulfo-Cy3 successfully conjugates with PS-dsDNA via a click reaction. Lane L shows DNA ladder, lane 1 shows unreacted PS-dsDNA, and lane 2 shows PS-dsDNA after reaction with maleimide-sulfo-Cy3. Cy3 signal is only present for the PS-dsDNA sample that was reacted with maleimide-sulfo-Cy3.

[0225] FIG. 5 is a schematic showing chemical functionalization of a PS-DNA with an azide- containing molecule. The chemical structure on the right hand side of the reaction shows the structure after the PS-DNA has reacted with iodoacetamide-PEG3-azide.

[0226] DETAILED DESCRIPTION

[0227] This disclosure relates to compositions and methods for providing an effector, e.g., a therapeutic effector, to a cell, tissue or subject, e.g., in vivo or in vitro. The effector may be a

[0228] 1601838421.1 31 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0229] DNA sequence, a polypeptide, e.g., a therapeutic protein, or an RNA, e.g., a regulatory RNA or an mRNA.

[0230] Elements of DNA molecules

[0231] The DNA molecules described herein can contain elements sufficient to deliver an effector sequence to a target cell, tissue or subject. In some embodiments, the effector sequence is a DNA sequence. In some embodiments, the DNA molecule drives expression of an effector, e.g., the DNA molecule comprises a promoter and a sequence encoding an RNA or a polypeptide, e.g., a therapeutic RNA or polypeptide. In some embodiments, the DNA molecules described herein further contain a maintenance sequence.

[0232] In some embodiments, the DNA molecule comprises a double-stranded DNA region. In some embodiments, the DNA region comprises a circular double-stranded DNA, in which the circular double-stranded DNA is a plasmid or a minicircle. In some embodiments, the DNA region comprises a linear double-stranded DNA (e.g., TDSC). While many of the embodiments herein refer to a TDSC, it is understood that as applicable an embodiment that refers to a TDSC may also apply to a DNA molecule or the DNA region of a DNA molecule.

[0233] In some embodiments, the DNA molecule comprises a circular single-stranded DNA (ssDNA), in which the ssDNA lacks a free end. A circular ssDNA may be covalently closed or may form a closed structure without free DNA ends through non-covalent interactions, e.g., the ssDNA may be closed through a splint, e.g., a nucleic acid (e.g., DNA or RNA) splint, through a moiety such as a protein that binds and brings together both ends of a linear ssDNA, or through binding of a plurality of proteins, each of two of the plurality binding to a different ssDNA end, and then binding to each other or a third moiety to close the DNA structure. In the case of circular ssDNA, the term circular does not imply that the ssDNA structure lacks all intramolecular structure; rather, a circular ssDNA may have short regions of intramolecular double stranded regions or other structures.

[0234] In some embodiments, a DNA region disclosed herein is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 500 nucleotides, at least about 1000 nucleotides, at least about 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, at least about

[0235] 1601838421.1 32 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0236] 5000 nucleotides, at least about 6000 nucleotides, at least about 7000 nucleotides, at least about 8000 nucleotides, at least about 9000 nucleotides, at least about 10,000 nucleotides, at least about 11,000, or at least about 12,000 nucleotides in length. In some embodiments, the DNA disclosed herein is between 20-30, 30-40, 40-50, 50-75, 75-100, 100-200, 200-300, 300-500, 500-1000, 1000-2000, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, 7000-8000, 8000-9000, 9000-10,000, 10,000-11,000, or 11,000-12,000 nucleotides in length. In some embodiments, the size of a DNA region disclosed herein is a length sufficient to encode useful polypeptides or

[0237] RNAs.

[0238] In some embodiments, a DNA molecule described herein is resistant to endonuclease digestion and / or resistant to immune sensor recognition. In some embodiments, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of the sugars of a DNA molecule described herein are deoxyribose sugars.

[0239] In some embodiments, a DNA region described herein can be replicated (e g., by a DNA polymerase native to a cell comprising the DNA region). In some embodiments, a DNA region described herein cannot be replicated. In some embodiments, a DNA region or a portion thereof can be integrated into the genome. In some embodiments, a DNA region or a portion thereof cannot be integrated into the genome.

[0240] In some embodiments, a compound described herein comprises a nucleotide having Formula (lb): Formula (lb), wherein Bl is H or a nucleobase; Su is a sugar moiety; m=0-10; n=0-10; z=0-4; L is a peptide bond of -CONH- or -NHCO-; RL, if present, is each independently a reactive linker; L2 is -O-CH2-CH2- or -CH2-CH2-O-, and Ri is an organic moiety. In some embodiments, the nucleotide has Formula (1c):

[0241] 1601838421.1 33 Atorney Docket No.: F2128-7027WO(VL87026-W1) Formula (1c).

[0242] Structural elements of double-stranded DNA regions

[0243] In some embodiments, a double-stranded DNA region comprises an exonuclease-resistant DNA end form (e.g., as described herein). In some embodiments, the DNA end form is at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides in length. In some embodiments, the DNA end form is less than 10, less than 15, less than 20, less than 25, less than 30, less than 40, less than 50, less than 60, less than 70, less than 80, less than 90, or less than 100 nucleotides in length. In some embodiments, the DNA end form is 2-5, 5-10, 10- 15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-70, 70-80, 80-90, or 90- 100 nucleotides in length. It is understood that when the length of a linear closed-ended dsDNA molecule is discussed herein, the length refers to the number of nucleotides starting with and including the upstream end, through the downstream end. For example, a no-loop dsDNA molecule having 100 base pairs would have a length of 100 nucleotides.

[0244] In some embodiments, a double-stranded DNA region encodes an effector (e.g., a polypeptide or RNA, e.g., as described herein), e.g., positioned between two exonucleaseresistant DNA end forms.

[0245] In some embodiments, a double-stranded DNA region is at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least

[0246] 1601838421.1 34 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0247] 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, or at least 12,000 nucleotides in length. In some embodiments, a double-stranded DNA region is less than 50, less than 60, less than 70, less than 80, less than 90, less than 100, less than 200, less than 300, less than 400, less than 500, less than 600, less than 700, less than 800, less than 900, less than 1000, less than 2000, less than 3000, less than 4000, less than 5000, less than 6000, less than 7000, less than 8000, less than 9000, less than 10,000, less than 11,000, or less than 12,000 nucleotides in length. In some embodiments, the double stranded DNA region is 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-300, 300- 400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-2000, 2000-3000, 3000- 4000, 4000-5000, 5000-6000, 6000-7000, 7000-8000, 8000-9000, 9000-10,000, 10,000-11,000, or 11,000-12,000 nucleotides in length.

[0248] A double-stranded DNA region described herein may have less than a threshold level of single stranded structures. In one embodiment, the double-stranded DNA region does not comprise more than 20, more than 18, more than 16, more than 14, more than 12, more than 10, more than 8, more than 7, more than 5, more than 4, more than 3, more than 2, or more than 1 single stranded region longer than 100, longer than 80, longer than 70, longer than 60, longer than 50, longer than 40, longer than 30, longer than 20 or longer than 10 bases, e.g., does not comprise single stranded regions longer than 100, longer than 80, longer than 70, longer than 60, longer than 50, longer than 40, longer than 30, longer than 20 or longer than 10 bases.

[0249] In some embodiments, a double-stranded DNA region comprises a sense strand and an antisense strand.

[0250] In some embodiments, a DNA region comprises (a) an upstream end form (e.g., upstream exonuclease-resistant DNA end form); (b) double-stranded DNA; and (c) a downstream end form (e.g., exonuclease-resistant DNA end form). In some embodiments, the upstream DNA end form (e.g., upstream exonuclease-resistant DNA end form) comprises one or more chemically modified nucleotides. In some embodiments, the downstream DNA end form (e.g., downstream exonuclease-resistant DNA end form) comprises one or more chemically modified nucleotides. In some embodiments, one or both of the upstream exonuclease-resistant DNA end form and the downstream exonuclease-resistant DNA end form does not comprise the nucleic acid sequences TATCAGCACACAATTGCCCATTATACGC (SEQ ID NO: 55) and GCGTATAATGGGCAATTGTGTGCTGATA (SEQ ID NO: 56), or nucleic acid sequences

[0251] 1601838421.1 35 Atorney Docket No.: F2128-7027WO(VL87026-W1) having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, one or both of the upstream exonuclease-resistant DNA end form and the downstream exonuclease-resistant DNA end form does not comprise the nucleic acid sequences TATCAGCACACAATAGTCCATTATACGC (SEQ ID NO: 57) and GCGTATAATGGACTATTGTGTGCTGATA (SEQ ID NO: 58). In some embodiments, the upstream exonuclease-resistant DNA end form has a loop size of less than about 28 or less than about 56 nucleotides in length or greater than about 28 or greater than about 56 nucleotides in length. In some embodiments, the downstream exonuclease-resistant DNA end form has a loop size of less than about 28 or less than about 56 nucleotides in length or greater than about 28 or greater than about 56 nucleotides in length. In some embodiments, every nucleotide in the DNA region binds another nucleotide in the DNA region.

[0252] In some embodiments, the upstream DNA end form and the downstream DNA end form have the same nucleotide sequence. In some embodiments, the upstream DNA end form and the downstream DNA end form have different nucleotide sequences. In some embodiments, the upstream exonuclease-resistant DNA end form and the downstream exonuclease-resistant DNA end form have the same structure. In some embodiments, the upstream exonuclease-resistant DNA end form and the downstream exonuclease-resistant DNA end form have different structures.

[0253] In some embodiments, a double-stranded DNA region described herein is linear and can be circularized. In some embodiments, a double- stranded DNA region described herein is linear and cannot be circularized. In some embodiments, a double-stranded DNA region described herein can be concatemerized. In some embodiments, a double-stranded DNA region described herein cannot be concatemerized.

[0254] In some embodiments, a dsDNA form described herein is asymmetrically modified, where one strand comprises chemically modified nucleobases and the other strand is substantially free of chemically modified nucleobases. In some embodiments, the hemimodified DNA may be completely free of chemically modified nucleotides on the antisense strand, and in other embodiments, the hemi-modified DNA may comprise a few chemical modifications (such as backbone modifications, e.g., phosphor othioate) on the antisense strand.

[0255] 1601838421.1 36 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0256] In some embodiments, the hemi-modified DNA molecule comprises chemically modified nucleotides (e.g., nuclotides comprising chemically modified nucleobases) on the sense strand.

[0257] In some embodiments, a DNA molecule described herein comprises a hemi-modified DNA that is linked to an organic moiety via a reactive linker, e.g., a click linker.

[0258] In some embodiments, a hemi-modified DNA comprises a nucleotide in the sense strand, wherein the nucleotide in the sense strand is linked, e.g., directly linked, to an avidin moiety or a biotin moiety. In some embodiments, a DNA molecule described herein comprises a hemi- modified DNA molecule that is linked to an organic moiety via binding between a biotin moiety and an avidin moiety.

[0259] Exomiclease-re si slant DNA end forms

[0260] In some embodiments, a double-stranded DNA region described herein comprise a DNA end form at each end of the double-stranded DNA molecule. The DNA end forms described herein can, in some instances, comprise a closed end, wherein every nucleotide of the DNA end form is covalently attached to two other nucleotides of the DNA end form. In other instances, the DNA end forms described herein comprise an open end comprising at least one nucleotide that are only covalently atached to one other nucleotide of the DNA end form. The DNA end forms are generally exonuclease resistant.

[0261] In some embodiments, a DNA region described herein comprises an upstream DNA end form which is a closed end; (b) a double stranded DNA; and (c) a downstream DNA end form which is a closed end.

[0262] Exemplary exonuclease-resistant DNA end forms, the production of exonucleaseresistant DNA end forms, and assessment of exonuclease resistance can be found, for example, in WO / 2023 / 220729, incorporated herein by reference in its entirety.

[0263] Closed Ends, e.g.. Hairpins

[0264] In some embodiments, an exonuclease-resistant DNA end form comprises a DNA hairpin. A hairpin generally comprises a single-stranded loop region covalently attached at both the 5’ and 3’ ends to a double-stranded stalk region. In certain embodiments, the single-stranded

[0265] 1601838421.1 37 Atorney Docket No.: F2128-7027WO(VL87026-W1) loop region comprises one or more nucleotides (e.g., 1-2, 2-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, or 35-40 nucleotides) that are not hybridized to another nucleotide.

[0266] In certain embodiments, the single-stranded loop region comprises one or more functional elements or a regulatory sequence. In embodiments, a functional element comprised in the single-stranded loop region is heterologous to one or more other elements of the DNA end form and / or a double-stranded DNA region comprising the DNA end form. In certain embodiments, the single-stranded loop region of a hairpin loop is less than about 5, less than about 10, less than about 15, less than about 20, less than about 25, less than about 26, less than about 27, less than about 28, less than about 29, or less than about 30 nucleotides in length.

[0267] In embodiments, the hairpin is comprised in a DNA region having a doggybone conformation. In embodiments, the hairpin comprises a protelomerase sequence (e.g., as described herein). In embodiments, the protelomerase sequence is produced by TelN protelomerase, ResT protelomerase, Tel PY54 protelomerase, or TelK protelomerase digestion. In embodiments, the protelomerase sequence is less than about 15, less than about 20, less than about 25, less than about 26, less than about 27, less than about 28, less than about 29, or less than about 30 nucleotides in length. In embodiments, the protelomerase sequences are between about 28 (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides and about 56 (e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60) nucleotides in length. In embodiments, the protelomerase sequences are greater than about 56 (e.g., greater than 50, greater than 51, greater than 52, greater than 53, greater than 54, greater than 55, greater than 56, greater than 57, greater than 58, greater than 59, greater than 60, greater than 65, greater than 70, greater than 75, greater than 80, greater than 90, or greater than 100) nucleotides in length.

[0268] A hairpin can be attached to one or both ends of a double-stranded DNA region, for example, by ligation (e.g., as described herein). In some embodiments, a double-stranded DNA region as described herein comprises, at one or both ends, a DNA hairpin loop. In some embodiments, the upstream exonuclease-resistant DNA end form of a double-stranded DNA region as described herein comprises a DNA hairpin loop. In some embodiments, the downstream exonuclease-resistant DNA end form of a double-stranded DNA region as described herein comprises a DNA hairpin loop.

[0269] In certain embodiments, a DNA hairpin loop comprises one or more unmodified nucleotides. In embodiments, a DNA hairpin loop consists entirely of unmodified nucleotides.

[0270] 1601838421.1 38 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0271] In certain embodiments, a DNA hairpin loop comprises one or more chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein). In embodiments, a DNA hairpin loop consists entirely of chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein).

[0272] In certain embodiments, the single-stranded loop region of a DNA hairpin loop comprises one or more chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein). In embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 99% of the nucleotides in the single-stranded loop region are chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein). In embodiments, the singlestranded loop region of a DNA hairpin loop consists entirely of chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein). In certain embodiments, the single-stranded loop region of a DNA hairpin loop comprises one or more unmodified nucleotides. In embodiments, the single-stranded loop region of a DNA hairpin loop consists entirely of unmodified nucleotides.

[0273] In certain embodiments, the double-stranded stalk region of a DNA hairpin loop comprises one or more unmodified nucleotides. In embodiments, the double-stranded stalk region of a DNA hairpin loop consists entirely of unmodified nucleotides. In certain embodiments, the double-stranded stalk region of a DNA hairpin loop comprises one or more chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein). In embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 99% of the nucleotides in the double-stranded stalk region are modified nucleotides (e.g., phosphorothioate- modified nucleotides, e.g., as described herein). In embodiments, the double-stranded stalk region of a DNA hairpin loop consists entirely of chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein).

[0274] In embodiments, the single-stranded loop region of a DNA hairpin loop comprises one or more chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein) and the double-stranded stalk region comprises one or more unmodified nucleotides. In embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 99% of the

[0275] 1601838421.1 39 Atorney Docket No.: F2128-7027WO(VL87026-W1) nucleotides in the single-stranded loop region are chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein). In embodiments, the singlestranded loop region of a DNA hairpin loop consists entirely of chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein) and the double-stranded stalk region consists entirely of unmodified nucleotides.

[0276] In some embodiments, a double-stranded DNA region described herein comprises an upstream DNA end form and a downstream DNA end form, wherein the upstream DNA end form, the downstream DNA end form, or both, does not comprise a protelomerase sequence. In some embodiments, a double-stranded DNA region described herein comprises an upstream DNA end form and a downstream DNA end form, wherein the upstream DNA end form, the downstream DNA end form, or both, comprises a protelomerase sequence. In some embodiments, one or more of the protelomerase sequences comprise (e.g., in 5’-to-3’ order) the nucleic acid sequences TATCAGCACACAATTGCCCATTATACGC (SEQ ID NO: 55) and GCGTATAATGGGCAATTGTGTGCTGATA (SEQ ID NO: 56), or nucleic acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, one or more of the protelomerase sequences comprise (e.g., in 5’-to-3’ order) the nucleic acid sequences TATCAGCACACAATAGTCCATTATACGC (SEQ ID NO: 57) and GCGTATAATGGACTATTGTGTGCTGATA (SEQ ID NO: 58), or nucleic acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, one or more of the protelomerase sequences comprise (e.g., in 5’-to-3’ order) the nucleic acid sequences ACCTATTTCAGCATACTACGC (SEQ ID NO: 60) and GCGTAGTATGCTGAAATAGGT (SEQ ID NO: 61), or nucleic acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, one or more of the protelomerase sequences comprise (e.g., in 5’-to-3’ order) the nucleic acid sequence CACACAATTGCCCATTATACGCGCGTATAATGGGCAATTGTGTG (SEQ ID NO: 62), or a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0277] 1601838421.1 40 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0278] In some embodiments, one or more of the protelomerase sequences comprise (e.g., in 5’- to-3’ order) the nucleic acid sequences: (i) TAAATATAATTTAA (SEQ ID NO: 63) and TTAAATTATATTTA (SEQ ID NO: 64), (ii) AATATATAATCTAA (SEQ ID NO: 65) and TTAGATTATATATT (SEQ ID NO: 66), (iii) TATTTATTATCTTT (SEQ ID NO: 67) and AAAGATAATAAATA (SEQ ID NO: 68), (iv) ATATAATTTTTAATTAGTATAGAATATGTTAA (SEQ ID NO: 69) and TTAACATACTCTATACTAATTAAAAATTATAT (SEQ ID NO: 70), (v) TATAATTTGATATTAGTACAAATCCC (SEQ ID NO: 71) and GGGATTTGTACTAATATCAAATTATA (SEQ ID NO: 72), (vi) ATATAATATTTATTTAGTACAAAGTTC (SEQ ID NO: 73) and GAACTTTGTACTAAATAAATATTATAT (SEQ ID NO: 74), (vii) ATATAATTTTTTATTAGTATAGAGTAT (SEQ ID NO: 75) and ATACTCTATACTAATAAAAAATTATAT (SEQ ID NO: 76), or (viii) TAAATATAATTTAA (SEQ ID NO: 63) and TTAAATTATATTTA (SEQ ID NO: 64); or nucleic acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0279] In some embodiments, one or more of the protelomerase sequences further comprise (e.g., in 5’-to-3’ order) the nucleic acid sequences: (i) TAGTATAAAAAACTGT (SEQ ID NO: 77) and ACAGTTTTTTATACTA (SEQ ID NO: 78), (ii) TAGTATACAAAAGATT (SEQ ID NO: 79) and AATCTTTTGTATACTA (SEQ ID NO: 80), (iii) TAGTATATATATCTCT (SEQ ID NO: 81) and AGAGATATATATACTA (SEQ ID NO: 82), or (viii) TAGTATAAAAAAAATT (SEQ ID NO: 83) and AATTTTTTTTATACTA (SEQ ID NO: 84); or nucleic acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0280] In some embodiments, the protelomerase sequences are produced by TelN protelomerase, ResT protelomerase, Tel PY54 protelomerase, or TelK protelomerase digestion. In some embodiments, the protelomerase sequences are not produced by TelN protelomerase digestion. In some embodiments, the protelomerase sequences are not produced by Tel PY54 protelomerase digestion. In some embodiments, the protelomerase sequences are not produced by TelK protelomerase digestion. In some embodiments, the protelomerase sequences are not produced by ResT protelomerase digestion.

[0281] 1601838421.1 41 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0282] In some embodiments, the protelomerase sequences are about 28 or about 56 nucleotides in length. In some embodiments, the protelomerase sequences are less than 28 (e.g., less than 15, less than 20, less than 25, less than 26, less than 27, or less than 28) nucleotides in length. In some embodiments, the protelomerase sequences are between about 28 (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides and about 56 (e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60) nucleotides in length. In some embodiments, the protelomerase sequences are greater than about 56 (e.g., greater than 50, greater than 51, greater than 52, greater than 53, greater than 54, greater than 55, greater than 56, greater than 57, greater than 58, greater than 59, greater than 60, greater than 65, greater than 70, greater than 75, greater than 80, greater than 90, or greater than 100) nucleotides in length.

[0283] In some embodiments, the protelomerase sequence is produced from a first protelomerase recognition sequence (PRS) and a second PRS that are recognized by a TelN protelomerase or ResT protelomerase. In some embodiments, the protelomerase sequence is produced from a first protelomerase recognition sequence (PRS) and a second PRS that are recognized by a Tel PY54 protelomerase or TelK protelomerase.

[0284] Y-Adaptors

[0285] In some embodiments, an exonuclease-resistant DNA end form as described herein comprises a Y-adaptor. As described herein, a Y-adaptor generally comprises a pair of singlestranded DNA regions, each attached at one end to a strand of a double-stranded DNA region, thereby forming a “Y” shape (wherein the base of the “Y” represents the double-stranded DNA region, and each of the upper prongs of the “Y” represents the two single-stranded DNA region).

[0286] In certain embodiments, a single-stranded DNA region (e.g., one or both single- stranded DNA regions) of a Y-adaptor comprises one or more chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein). In embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 99% of the nucleotides in the single-stranded DNA region are chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein). In embodiments, a single- stranded DNA region (e.g., one or both single-stranded DNA regions) of a Y-adaptor consists entirely of chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein). In certain embodiments, a

[0287] 1601838421.1 42 Atorney Docket No.: F2128-7027WO(VL87026-W1) single-stranded DNA region (e.g., one or both single-stranded DNA regions) of a Y-adaptor comprises one or more unmodified nucleotides.

[0288] In embodiments, a single-stranded DNA region (e.g., one or both single-stranded DNA regions) of a Y-adaptor comprises one or more chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein) and a double-stranded DNA region of the Y-adaptor comprises one or more unmodified nucleotides. In embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 99% of the nucleotides in the single- stranded DNA region or regions are chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein). In embodiments, a single-stranded DNA region (e.g., one or both single-stranded DNA regions) of a Y-adaptor consists entirely of chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e g., as described herein) and the double-stranded DNA region of the Y-adaptor consists entirely of unmodified nucleotides.

[0289] No Loop Closed DNA End Forms

[0290] In some embodiments, a double-stranded DNA region as described herein comprises an exonuclease-resistant DNA end form that is covalently closed but does not include a hairpin loop. For example, in certain embodiments, every nucleotide of a covalently-closed DNA end form is hybridized to another nucleotide. In certain embodiments, the covalently-closed DNA end form comprises a first region and a second region, wherein the first region is capable of hybridizing in its entirety to the second region (e.g., wherein the first region is complementary to the second region) and wherein the 3’ end of the first region is covalently attached to the 5’ end of the second region. In embodiments, a covalently-closed DNA end form as described herein can be attached to one end of a double-stranded DNA region as described herein, e.g., by ligation.

[0291] Open DNA End Forms

[0292] In some embodiments, a double-stranded DNA region as described herein comprises an exonuclease-resistant DNA end form that is not covalently closed. In certain embodiments, the DNA end form comprises a blunt end (e.g., a blunt end comprising one or more chemical

[0293] 1601838421.1 43 Atorney Docket No.: F2128-7027WO(VL87026-W1) modifications as described herein) or a sticky end (e.g., a sticky end comprising one or more chemical modifications as described herein).

[0294] In certain embodiments, a DNA end form comprising a blunt end comprises one or more chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein). In embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 99% of the nucleotides in the DNA end form comprising a blunt end are chemically modified nucleotides (e g., phosphorothioate-modified nucleotides, e.g., as described herein). In embodiments, the DNA end form comprising a blunt end consists entirely of chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein). In embodiments, the terminal base pair of the DNA end form comprising a blunt end comprises a chemically modified nucleotide (e.g., one or both nucleotides of the base pair are chemically modified), e.g., a phosphorothioate-modified nucleotide, e.g., as described herein. In embodiments, a plurality of base pairs (e.g., 2, 3, 4, 5, or 6 base pairs) at the terminal end of the DNA end form comprise chemically modified nucleotides (e.g., one or both nucleotides of the base pair are chemically modified), e.g., phosphorothioate-modified nucleotides, e.g., as described herein. In an embodiment, the three base pairs at the terminal end of the DNA end form comprise chemically modified nucleotides (e.g., one or both nucleotides of the base pair are chemically modified), e.g., phosphorothioate-modified nucleotides, e.g., as described herein. In an embodiment, the six base pairs at the terminal end of the DNA end form comprise chemically modified nucleotides (e.g., one or both nucleotides of the base pair are chemically modified), e g., phosphorothioate- modified nucleotides.

[0295] In certain embodiments, a DNA end form comprising a sticky end comprises one or more chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein). In embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 99% of the nucleotides in the DNA end form comprising a sticky end are chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein). In embodiments, the DNA end form comprising a sticky end consists entirely of chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein). In embodiments, a terminal nucleotide of the DNA end form comprising a sticky end comprises a chemically

[0296] 1601838421.1 44 Atorney Docket No.: F2128-7027WO(VL87026-W1) modified nucleotide (e.g., one or both nucleotides of the base pair are chemically modified), e.g., a phosphorothioate-modified nucleotide, e.g., as described herein. In embodiments, the overhang region of the sticky end of a DNA end form comprises one or more chemically modified nucleotide, e.g., phosphorothioate-modified nucleotides, e.g., as described herein.

[0297] Inverted Terminal Repeats (ITRs)

[0298] In some embodiments, a double-stranded DNA region as described herein comprises an exonuclease-resistant DNA end form comprising an inverted terminal repeat (ITR). In some embodiments, the ITR is an ITR from a virus, e.g., an adenovirus or an adeno-associated virus (AAV). In some embodiments, the ITR comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an ITR sequence from a virus, e.g., an adenovirus or an adeno-associated virus (AAV). In certain embodiments, the ITR comprises an origin of replication (e.g., a viral origin of replication). In embodiments, a double-stranded DNA region as described herein comprises an exonuclease-resistant DNA end form comprising an ITR (e.g., as described herein) at each end. In some embodiments, a double-stranded DNA region does not comprise an ITR.

[0299] Structural elements of single-stranded DNA regions

[0300] A single-stranded DNA (ssDNA) region described herein may be circular, e.g., covalently closed. Exemplary production of ssDNA constructs may be found, for example, in WO / 2023 / 069948, incorporated herein by reference in its entirety.

[0301] A single-stranded DNA region described herein may have less than a threshold level of intramolecular complementarity or double stranded structures. In one embodiment, the ssDNA does not comprise more than 50, more than 40, more than 30, more than 20, more than 18, more than 16, more than 14, more than 12, more than 10, more than 8, more than 7, more than 5, more than 4, more than 3, more than 2, or more than 1 double stranded region longer than 100, longer than 80, longer than 70, longer than 60, longer than 50, longer than 40, longer than 30, longer than 20 or longer than 10 base pairs. In some embodiments, the ssDNA does not comprise any regions of intramolecular complementarity longer than 100, longer than 80, longer than 70, longer than 60, longer than 50, longer than 40, longer than 30, longer than 20 or longer than 10

[0302] 1601838421.1 45 Atorney Docket No.: F2128-7027WO(VL87026-W1) base pairs. In some embodiments, the ssDNA comprises 1, 2, 3, 4, 5, 7, 8, 10, 21, 14, 15, 18, or 20 double stranded regions, e g., wherein the double stranded regions are no more than 100, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30, no more than 20 or no more than 10 base pairs. For example, the single-stranded DNA region is not a doggybone structure, i.e., it is not a primarily double stranded, closed ended construct.

[0303] In some embodiments, the single-stranded DNA region does not form a double stranded structure longer than 100 base pairs. In some embodiments, the single-stranded DNA region does not form a double stranded structure longer than 80 base pairs. In some embodiments, the single-stranded DNA region does not form a double stranded structure longer than 60 base pairs. In some embodiments, the single-stranded DNA region does not form a double stranded structure longer than 50 base pairs. In some embodiments, the single-stranded DNA region does not form a double stranded structure longer than 45 base pairs. In some embodiments, the single-stranded DNA region does not form a double stranded structure longer than 40 base pairs. In some embodiments, the single-stranded DNA region does not form a double stranded structure longer than 35 base pairs. In some embodiments, the single-stranded DNA region does not form a double stranded structure longer than 30 base pairs. In some embodiments, the single-stranded DNA region does not form a double stranded structure longer than 25 base pairs. In some embodiments, the single-stranded DNA region does not form a double stranded structure longer than 20 base pairs. In some embodiments, the single-stranded DNA region does not comprise a length of double strandedness that is recognized by cyclic GMP-AMP synthase (cGAS) in a cell. Without wishing to be bound by theory, cGAS is thought to mediate innate immunity to foreign double stranded DNA.

[0304] In some embodiments, the single-stranded DNA region does not comprise a first sequence that hybridizes with a second sequence, wherein the first sequence and the second sequence are at least 5, at least 10, at least 15, at least 20, or at least 25 nt long, and wherein the first sequence and the second sequence are positioned less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 nucleotides apart from each other.

[0305] In some aspects, the present disclosure provides a pharmaceutical formulation comprising: an LNP comprising a DNA molecule, wherein the DNA molecule comprises a single-stranded DNA region. In some embodiments, the single-stranded DNA region: (a)

[0306] 1601838421.1 46 Atorney Docket No.: F2128-7027WO(VL87026-W1) encodes a therapeutic protein, (b) is covalently closed, (c) does not form a double stranded structure longer than 100 base pairs, (d) is more than 200 nucleotides in length, and (e) does not comprise a protelomerase target sequence. In some embodiments, the pharmaceutical formulation is substantially free of linear DNA and protein. In some embodiments, the single stranded DNA region does not form a double stranded structure longer than 40 base pairs.

[0307] In some embodiments, the ssDNA region has a GC content of 30-40%, 40-50%, 50-60%, or 60-70%. In some embodiments, the ssDNA region lacks one or both of a bacteriophage packaging site and a bacteriophage origin of replication, or wherein the ssDNA region does not encode a bacteriophage capsid gene. In some embodiments, the ssDNA region was not produced by rolling circle amplification. In some embodiments, the ssDNA region was not produced by strand displacement amplification. In some embodiments, the ssDNA region comprises between 200-3,000 nucleotides. In some embodiments, the ssDNA region comprises between 500 and 2,000 nucleotides. In some embodiments, the ssDNA region is a sense ssDNA strand. In some embodiments, the DNA region is an antisense ssDNA strand.

[0308] In one aspect the invention features a composition, e.g., a pharmaceutical composition, that includes a single stranded DNA region comprising an effector sequence, the single stranded DNA having one, two or three of the following characteristics: the ssDNA region is covalently closed; the ssDNA region does not form a double stranded structure longer than 100 base pairs; or the ssDNA region comprises at least one covalent modification.

[0309] In an embodiment, the single stranded DNA region has at least 15 nucleotides, at least 30 nucleotides, at least 50 nucleotides, at least 75 nucleotides, 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 500 nucleotides, at least 750 nucleotides, at least 1,000 nucleotides, at least 2,000 nucleotides, at least 3,000 nucleotides, at least 4,000 nucleotides, at least 5,000 nucleotides, at least 6,000 nucleotides, at least 7,000 nucleotides, at least 8,000 nucleotides, at least 9,000 nucleotides, at least 10,000 nucleotides, at least 11,000 nucleotides, or at least 12,000 nucleotides.

[0310] In an embodiment, the single stranded DNA region has between 20 and 1000 nucleotides, between 20 and 50 nucleotides, between 100 and 500 nucleotides, between 500 and 12,000 nucleotides, between 500 and 10,000 nucleotides, between 1,000 and 12,000 nucleotides, between 2,000 and 12,000 nucleotides, between 5,000 and 12,000 nucleotides, between 5,000 and 10,000 nucleotides, between 1,000 and 10,000 nucleotides, between 200 and 1,000

[0311] 1601838421.1 47 Atorney Docket No.: F2128-7027WO(VL87026-W1) nucleotides, between 200 and 2,000 nucleotides, between 200 and 3,000 nucleotides, between 500 and 1,000 nucleotides, between 500 and 2,000 nucleotides, between 500 and 3,000 nucleotides, between 1,000 and 2,000 nucleotides, between 1,000 and 3,000 nucleotides, or between 2,000 and 3,000 nucleotides. In some embodiments, the ssDNA region comprises between 20-12,000 nucleotides. In some embodiments the ssDNA region comprises between SO- 12, 000 nucleotides.

[0312] In an embodiment, the single stranded DNA region is a sense ssDNA strand. In an embodiment, the single stranded DNA region is an antisense ssDNA strand.

[0313] In some embodiments, a ssDNA described herein is provided as a sense strand or an antisense strand. The single stranded antisense strand has a sequence complementary to an mRNA or pre-mRNA which encodes an effector (e.g., an effector protein) and / or can serve as a template for transcription. The single stranded sense strand has the same sequence as an mRNA or pre-mRNA which encodes an effector (e.g., an effector protein), and does not serve as a template for transcription. Rather, without wishing to be bound by theory, in some embodiments, the cell converts the single stranded sense strand into double stranded DNA via replication machinery, and the newly formed antisense strand may serve as a template for transcription. Thus, a sense strand may “encode” an effector by providing the sequence necessary for the cell to produce the complementary DNA strand which may then be transcribed.

[0314] In some embodiments, when the DNA molecule is introduced to a cell, the cell exhibits a lower cytokine mRNA level increase (e.g., normalized to GAPDH mRNA levels) compared to a control cell of the same type that was contacted with a dsDNA having the same sequence as the single stranded DNA region at the same molar amount as the DNA molecule. In some embodiments, the cytokine comprises cytokine IFN-b, IL-6, IL-lb, TNF-a, or CXCL10. In some embodiments, the cytokine increase in the control cells is less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the cytokine increase in the control cells.

[0315] In some embodiments, a DNA molecule described herein has an A260 / A280 ratio of 1.6- 1.7, 1.7-1.8. or 1.63-1.76. In some embodiments, a DNA molecule described herein has an A230 / A260 ratio of 0.3-1, 1-1.5, 1.5-1.8, or 0.34-1.79.

[0316] In any embodiment described herein, the single stranded DNA region may be covalently closed, e.g., the ssDNA is circularized.

[0317] 1601838421.1 48 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0318] Sequence elements of DNA regions

[0319] In some embodiments, a DNA region described herein comprises a promoter sequence. In some embodiments, a DNA region described herein comprises an effector sequence (e.g., a therapeutic effector sequence) operably linked to the promoter sequence. In some embodiments, a DNA region described herein comprises a heterologous functional sequence. In some embodiments, a DNA region described herein comprises a maintenance sequence. In some embodiments, a DNA region described herein comprises an origin of replication. In some embodiments, the DNA region comprises one, two, three, four, or all of a promoter sequence, an effector sequence, a heterologous functional sequence, a maintenance sequence, or an origin of replication. In some embodiments, the DNA region comprises a promoter sequence, an effector sequence, and a heterologous functional sequence. In some embodiments, the DNA region comprises a promoter sequence, an effector sequence, and a maintenance sequence. In some embodiments, the DNA region comprises a promoter sequence, an effector sequence, and an origin of replication. In some embodiments, the DNA region comprises a promoter sequence, an effector sequence, a heterologous functional sequence, and a maintenance sequence. In some embodiments, the DNA region comprises a promoter sequence, an effector sequence, a heterologous functional sequence, and an origin of replication. In some embodiments, the DNA region comprises a promoter sequence, an effector sequence, a maintenance sequence, and an origin of replication. In some embodiments, the DNA region comprises a promoter sequence, an effector sequence, a heterologous functional sequence, a maintenance sequence, and an origin of replication. In the case of double-stranded DNA, in some embodiments, the promoter and / or effector sequence is in the double-stranded region.

[0320] In some embodiments, the effector sequence encodes a polypeptide (e.g., a protein). In some embodiments, the effector sequence encodes a functional RNA (e.g., a miRNA, siRNA, or tRNA). In some embodiments, the effector sequence is heterologous to a target cell.

[0321] Promoters and Other Regulatory Sequences

[0322] The DNA region described herein may contain a promoter (a DNA sequence at which RNA polymerase and transcription factors bind to, directly or indirectly, to initiate transcription) operably linked to an effector sequence. A promoter may be found in nature operably linked to the effector sequence, or may be heterologous to the effector sequence. A promoter described

[0323] 1601838421.1 49 Atorney Docket No.: F2128-7027WO(VL87026-W1) herein may be native to the target cell or tissue, or heterologous to the target cell or tissue. A promoter may be constitutive, inducible and / or tissue-specific.

[0324] Examples of constitutive promoters include the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, Cell, 41 :521-530 (1985), the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl alpha promoter.

[0325] Inducible promoters allow regulation of expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of sources. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc- inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al., Science, 268: 1766-1769 (1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486-inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Then, 4:432-441 (1997)) and the rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)).

[0326] In some embodiments, the native promoter for the sequence encoding the effector can be used.

[0327] In some embodiments, the regulatory sequences impart tissue-specific gene expression capabilities. In some cases, the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are known in the art. Exemplary tissuespecific regulatory sequences include, but are not limited to the following tissue specific promoters: a liver-specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, a alpha-myosin heavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT)

[0328] 1601838421.1 50 Attorney Docket No.: F2128-7027WO(VL87026-W1) promoter. Other exemplary promoters include Beta-actin promoter, hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7: 1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24: 185-96 (1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11 :654-64 (1996)), CD2 promoter (Hansal et al., J. Immunol., 161 : 1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptor alpha-chain promoter, neuronal such as neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), among others which will be known to the skilled artisan.

[0329] Examples of tissue / cell specific promoters are listed in Table 1 :

[0330] Table 1: Tissue or cell specific promoters

[0331] 1601838421.1 51 Attorney Docket No.: F2128-7027WO(VL87026-W1)

[0332] The DNA molecules described herein may also include other native or heterologous expression control elements, such as enhancer elements, poly adenylation sites or Kozak consensus sequences.

[0333] Effector sequence

[0334] The effector sequence of a DNA region described herein may be, e.g., a functional DNA sequence, e.g., a therapeutically functional DNA sequence; a DNA sequence encoding a therapeutic peptide, polypeptide or protein; or a DNA sequence encoding a therapeutic RNA (e.g., a non-coding RNA).

[0335] DNA effectors:

[0336] A therapeutically functional DNA sequence may be a DNA sequence that forms a functional structure, e.g., a DNA sequence comprising a DNA aptamer, DNAzyme or allele- specific oligonucleotide (a DNA ASO). A therapeutically functional DNA sequence typically lacks a promoter operably linked. In embodiments, a DNA region described herein may include

[0337] 1601838421.1 52 Atorney Docket No.: F2128-7027WO(VL87026-W1) one or a plurality of functional DNA sequences, e.g., 2, 3, 4, 5, 6, or more sequences, which may be the same or different.

[0338] Polypeptide effectors:

[0339] A DNA sequence encoding a therapeutic polypeptide may be a DNA sequence encoding one or more effector which is a peptide, protein, or combinations thereof. For example, the DNA sequence encodes an mRNA. The peptide or protein may be: a DNA binding protein; an RNA binding protein; a transporter; a transcription factor; a translation factor; a ribosomal protein; a chromatin remodeling factor; an epigenetic modifying factor; an antigen; a hormone; an enzyme (such as a nuclease, e.g., an endonuclease, e g., a nuclease element of a CRISPR system, e.g., a Cas9, dCas9, aCas9-nickase, Cpf / Casl2a); a Crispr-linked enzyme, e.g. a base editor or prime editor; a mobile genetic element protein (e.g., a transposase, a retrotransposase, a recombinase, an integrase); a gene writer; a polymerase; a methylase; a demethylase; an acetylase; a deacetylase; a kinase; a phosphatase; a ligase; a deubiquitinase; a protease; an integrase; a recombinase; a topoisomerase; a gyrase; a helicase; a lysosomal acid hydrolase); an antibody (e.g., an intact antibody, a fragment thereof, or a nanobody); a signaling peptide; a receptor ligand; a receptor (e.g., a chimeric antigen receptor (CAR) or a T cell receptor); a clotting factor; a coagulation factor; a structural protein; a caspase; a membrane protein; a mitochondrial protein; a nuclear protein; or an engineered binder such as a centyrin, darpin, or adnectin. See, e.g., Gebauer & Skerra. 2020. Annual Review of Pharmacology and Toxicology 60:1, 391-415.

[0340] In embodiments, a DNA region described herein may include one or a plurality of sequences encoding a polypeptide, e.g., 2, 3, 4, 5, 6, or more sequences encoding a polypeptide. Each of the plurality may encode the same or different protein. For example, a sequence described herein may include multiple sequences encoding multiple proteins, e.g., a plurality of proteins in a biological pathway.

[0341] In some embodiments, a DNA region or sequence described herein may include a plurality of sequences encoding a polypeptide, e.g., 2, 3, 4, 5, 6, or more sequences encoding a polypeptide, separated by a self-cleaving peptide, e.g., P2A, T2A, E2A or F2A. self-cleaving peptides are 18-22 amino acids long, and can induce ribosomal skipping during protein translation so that two polypeptides can be encoded in the same transcript. Each of the polypeptides may encode the same or different protein. In one embodiment, a DNA region or

[0342] 1601838421.1 53 Atorney Docket No.: F2128-7027WO(VL87026-W1) sequence described herein may include a promoter followed by a sequence encoding a first polypeptide of interest, a sequence encoding a 2A self-cleaving peptide, a sequence encoding a second polypeptide of interest, and a polyA site. In another embodiment, a DNA region or sequence described herein may include a promoter followed by a sequence encoding the first polypeptide of interest, a first 2A self-cleaving peptide, a second polypeptide of interest, a sequence encoding a second 2A self-cleaving peptide, a sequence encoding a third polypeptide of interest, and a polyA site.

[0343] In some embodiments, the effector comprises a cell penetrating polypeptide. In some embodiments, the effector is a fusion protein that comprises a cell penetrating polypeptide and a second amino acid sequence. In some embodiments, the DNA molecule does not comprise a cell penetrating polypeptide. For example, in some embodiments, the DNA molecule does not comprise a fusion protein that comprises a cell penetrating polypeptide.

[0344] In some embodiments, an effector described herein comprises an immunogen. In some embodiments, an effector described herein comprises a viral antigen, a bacterial antigen, a fungal antigen, or a tumor antigen. In some embodiments, an effector described herein comprises a peptide antigen. In some embodiments, a composition described herein is administered to a subject as a vaccine. In some aspects, the present disclosure provides a method of vaccinating a subject, comprising administering to the subject a composition described herein.

[0345] In some embodiments, an effector sequence described herein does not encode viral protein.

[0346] RNA effectors:

[0347] An effector sequence may be a DNA sequence encoding a non-coding RNA, e.g., one or more of a short interfering RNA (siRNA), a microRNA (miRNA), long non-coding RNA, a piwi-interacting RNA (piRNA), a small nucleolar RNA (snoRNA), a small Cajal body-specific RNA (scaRNA), a transfer RNA (tRNA), a ribosomal RNA (rRNA), an RNA aptamer, and a small nuclear RNA (snRNA). In some embodiments, a DNA region described herein comprises a sequence encoding an RNA (e.g., an mRNA, siRNA, or miRNA). In some embodiments, a DNA region described herein does not comprise a sequence encoding an RNA.

[0348] In some embodiments, the DNA region disclosed herein comprises one or more expression sequences that encode a regulatory RNA, e.g., an RNA that modifies expression of an

[0349] 1601838421.1 54 Atorney Docket No.: F2128-7027WO(VL87026-W1) endogenous gene and / or an exogenous gene. In some embodiments, the DNA region disclosed herein can comprise a sequence that is antisense to a regulatory nucleic acid like a non-coding RNA, such as, but not limited to, tRNA, IncRNA, miRNA, rRNA, snRNA, microRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, Y RNA, and hnRNA. In one embodiment, the regulatory nucleic acid targets a host gene. A regulatory nucleic acid may include, but is not limited to, a nucleic acid that hybridizes to an endogenous gene, e.g., an antisense RNA, a guide RNA, a nucleic acid that hybridizes to an exogenous nucleic acid such as a viral DNA or RNA, nucleic acid that hybridizes to an RNA, nucleic acid that interferes with gene transcription, nucleic acid that interferes with RNA translation, nucleic acid that stabilizes RNA or destabilizes RNA such as through targeting for degradation, and nucleic acid that modulates a DNA or RNA binding factor. In one embodiment, the sequence is an miRNA. In some embodiments, the regulatory nucleic acid targets a sense strand of a host gene. In some embodiments, the regulatory nucleic acid targets an antisense strand of a host gene.

[0350] In some embodiments, the DNA region disclosed herein encodes a guide RNA. Guide RNA sequences are generally designed to have a sequence having a length of between 15-30 nucleotides (e.g., 17, 19, 20, 21, 24 nucleotides) that is complementary to the targeted nucleic acid sequence, and a region that facilitates complex formation (e.g., with a tracrRNA or a nuclease). Custom gRNA generators and algorithms are available commercially for use in the design of effective guide RNAs. Gene editing has also been achieved using a chimeric "single guide RNA" ("sgRNA"), an engineered (synthetic) single RNA molecule that mimics a naturally occurring crRNA-tracrRNA complex and contains both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing). Chemically modified sgRNAs have also been demonstrated to be effective in genome editing; see, for example, Hendel et al. (2015) Nature Biotechnol., 985-991. The gRNA may recognize specific DNA sequences (e.g., sequences adjacent to or within a promoter, enhancer, silencer, or repressor of a gene). In one embodiment, the gRNA is used as part of a CRISPR system for gene editing. For the purposes of gene editing, the DNA region disclosed herein may be designed to include one or multiple sequences encoding guide RNA sequences corresponding to a desired target DNA sequence; see, for example, Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308.

[0351] 1601838421.1 55 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0352] A DNA region disclosed may encode certain regulatory nucleic acids that can inhibit gene expression through the biological process of RNA interference (RNAi). RNAi molecules comprise RNA or RNA-like structures typically containing 15-50 base pairs (such as about 18- 25 base pairs) and having a nucleobase sequence identical (complementary) or nearly identical (substantially complementary) to a coding sequence in an expressed target gene within the cell. Such RNAi molecules include, but are not limited to: short interfering RNAs (siRNAs), doublestrand RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), meroduplexes, dicer substrates (U.S. Pat. Nos. 8,084,599 8,349,809 and 8,513,207), and RNA antisense oligonucleotides (RNA ASOs).

[0353] In one embodiment, the DNA region disclosed herein comprises a sequence comprising a sense strand of a IncRNA. In one embodiment, the DNA region or sequence disclosed herein comprises a sequence encoding an antisense strand of a IncRNA.

[0354] The DNA region disclosed herein may encode a regulatory nucleic acid substantially complementary, or fully complementary, to a fragment of an endogenous gene or gene product (e.g., mRNA). The regulatory nucleic acids may complement sequences at the boundary between introns and exons, in between exons, or adjacent to exon, to prevent the maturation of newly- generated nuclear RNA transcripts of specific genes into mRNA for transcription. The regulatory nucleic acids that are complementary to specific genes can hybridize with the mRNA for that gene and prevent its translation. The antisense regulatory nucleic acid can be DNA, RNA, or a derivative or hybrid thereof. In some embodiments, the regulatory nucleic acid comprises a protein-binding site that can bind to a protein that participates in regulation of expression of an endogenous gene or an exogenous gene.

[0355] The length of a DNA region disclosed herein that may encode a regulatory nucleic acid that hybridizes to a transcript of interest and may be, for instance, between about 5 to 30 nucleotides, between about 10 to 30 nucleotides, or about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or more nucleotides. The degree of identity of the regulatory nucleic acid to the targeted transcript should be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0356] A DNA region disclosed herein may encode a micro-RNA (miRNA) molecule identical to about 5 to about 30 contiguous nucleotides of a target gene. In some embodiments, the

[0357] 1601838421.1 56 Atorney Docket No.: F2128-7027WO(VL87026-W1) miRNA sequence targets a mRNA and commences with the dinucleotide AA, comprises a GC- content of about 30-70% (about 30-60%, about 40-60%, or about 45%-55%), and does not have a high percentage identity to any nucleotide sequence other than the target in the genome of the mammal in which it is to be introduced, for example as determined by standard BLAST search. In some embodiments, the DNA regione disclosed herein encodes at least one miRNA, e.g., 2, 3, 4, 5, 6, or more. In some embodiments, the DNA region disclosed herein comprises a sequence that encodes an miRNA having at least about 75%, at least about 80%, at least about 85%, at least about 90% at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% nucleotide sequence identity to any one of the nucleotide sequences or a sequence that is complementary to a target sequence. Lists of known miRNA sequences can be found in databases maintained by research organizations, such as Wellcome Trust Sanger Institute, Penn Center for Bioinformatics, Memorial Sloan Kettering Cancer Center, and European Molecule Biology Laboratory, among others. Known effective siRNA sequences and cognate binding sites are also well represented in the relevant literature. RNAi molecules are readily designed by technologies known in the art. In addition, there are computational tools that increase the chance of finding effective and specific sequence motifs (see, e.g., Lagana et al., Methods Mol. Bio., 2015, 1269:393-412).

[0358] The DNA region disclosed herein may modulate expression of RNA encoded by a gene. Because multiple genes can share some degree of sequence homology with each other, in some embodiments, the DNA region disclosed herein can be designed to target a class of genes with sufficient sequence homology. In some embodiments, the DNA region disclosed herein can contain a sequence that has complementarity to sequences that are shared amongst different gene targets or are unique for a specific gene target. In some embodiments, the DNA region disclosed herein can be designed to target conserved regions of an RNA sequence having homology between several genes thereby targeting several genes in a gene family (e.g., different gene isoforms, splice variants, mutant genes, etc.). In some embodiments, the DNA region disclosed herein can be designed to target a sequence that is unique to a specific RNA sequence of a single gene.

[0359] In embodiments, the effector sequence encoding a regulatory RNA has a length less than 5000 bps (e.g., less than about 5000 bps, less than about 4000 bps, less than about 3000 bps, less than about 2000 bps, less than about 1000 bps, less than about 900 bps, less than about 800 bps,

[0360] 1601838421.1 57 Atorney Docket No.: F2128-7027WO(VL87026-W1) less than about 700 bps, less than about 600 bps, less than about 500 bps, less than about 400 bps, less than about 300 bps, less than about 200 bps, less than about 100 bps, less than about 50 bps, less than about 40 bps, less than about 30 bps, less than about 20 bps, less than about 10 bps, or less). In some embodiments, the effector sequence has, independently or in addition to, a length greater than 10 bps (e.g., at least about 10 bps, at least about 20 bps, at least about 30 bps, at least about 40 bps, at least about 50 bps, at least about 60 bps, at least about 70 bps, at least about 80 bps, at least about 90 bps, at least about 100 bps, at least about 200 bps, at least about 300 bps, at least about 400 bps, at least about 500 bps, at least about 600 bps, at least about 700 bps, at least about 800 bps, at least about 900 bps, at least about 1000 kb, at least about 1.1 kb, at least about 1.2 kb, at least about 1.3 kb, at least about 1.4 kb, at least about 1.5 kb, at least about 1.6 kb, at least about 1.7 kb, at least about 1.8 kb, at least about 1.9 kb, at least about 2 kb, at least about 2.1 kb, at least about 2.2 kb, at least about 2.3 kb, at least about 2.4 kb, at least about 2.5 kb, at least about 2.6 kb, at least about 2.7 kb, at least about 2.8 kb, at least about 2.9 kb, at least about 3 kb, at least about 3.1 kb, at least about 3.2 kb, at least about 3.3 kb, at least about 3.4 kb, at least about 3.5 kb, at least about 3.6 kb, at least about 3.7 kb, at least about 3.8 kb, at least about 3.9 kb, at least about 4 kb, at least about 4.1 kb, at least about 4.2 kb, at least about 4.3 kb, at least about 4.4 kb, at least about 4.5 kb, at least about 4.6 kb, at least about 4.7 kb, at least about 4.8 kb, at least about 4.9 kb, or at least about 5 kb or greater).

[0361] In some embodiments, a DNA region disclosed herein comprises one or more of the features described herein, e.g., one or more structural DNA sequence, a sequence encoding one or more peptides or proteins, a sequence encoding one or more regulatory element, a sequence encoding one or more regulatory nucleic acids, e.g., one or more non-coding RNAs, other expression sequences, and any combination of the aforementioned. A construct described herein may have one or a plurality of effector sequences, e.g., 2, 3, 4, 5 or more effector sequences. In the case of a plurality of effector sequences in a single construct, the effector sequences may be the same or different.

[0362] In one embodiment, the DNA region includes a therapeutically functional, structural DNA sequence. In one embodiment, the DNA region includes a promoter and a sequence encoding a therapeutic peptide, polypeptide, or protein described herein. In one embodiment, the DNA region includes a promoter and a sequence encoding a regulatory RNA described herein.

[0363] 1601838421.1 58 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0364] In some embodiments, the effector sequence that encodes a polypeptide or protein is codon optimized, e.g., codon optimized for expression in a mammal, e.g., a human. In general, codon optimization means modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., one or more, e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons; e.g., at least 1%, at least 5%, at least 10%, at least 20%, at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Codon usage tables are available, for example, at the "Codon Usage Database" available at www.kazusa.or.jp / codon / . These tables can be adapted in a number of ways, see, e.g., Nakamura et al., 2000, Nucl. Acids Res. 28:292. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge.

[0365] Maintenance sequence

[0366] A DNA region disclosed herein may include a maintenance sequence that supports or enables sustained gene expression through successive rounds of cell division and / or progenitor differentiation in a host cell for a DNA described herein. In embodiments, a maintenance sequence is a nuclear scaffold / matrix attachment region (S / MAR). S / MAR elements are diverse, AT-rich sequences ranging from 60-500 bp that are conserved across species, thought to anchor chromatin to nuclear matrix proteins during interphase (Bode et al. 2003. Chromosome Res 11, 435-445). An S / MAR can be incorporated into a DNA region described herein to facilitate longterm transgene expression and extra-chromosomal maintenance. In one embodiment, the maintenance sequence is human interferon-beta MAR (5’tataattcactggaattttttgtgtgtatggtatgacatatgggttcccttttattttttacatataaatatatttccctgtttttctaaaaaagaaaa agatcatcattttcccatgtaaaatgccatattttttcataggtcacttacata-3’ (SEQ ID NO: 39)), or a functional sequence having at least 80%, at least 90%, at least 95%, or at least 98% identity thereto. In embodiments, S / MARs useful in the constructs described herein can be found by searching the MARome at bioinfo. net. in / MARome, described also by Narwade et al. 2019. Nucleic Acids Research. Volume 47, Issue 14: 7247-7261.

[0367] In embodiments, a DNA region described herein is capable of replicating in a mammalian cell, e.g., human cell. In some embodiments, a DNA region described herein is maintained in a

[0368] 1601838421.1 59 Attorney Docket No.: F2128-7027WO(VL87026-W1) host cell, tissue or subject through at least one cell division. For example, a DNA region described herein is maintained in a host cell, tissue or subject through at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10, at least 15, at least 20, at least 40, at least 50 or more cell divisions. In vitro, cell division may be tracked by flow cytometry or microscopy. In vivo, cell division may be tracked by intravital microscopy.

[0369] Second strand motif

[0370] A ssDNA region disclosed herein may also include a second strand motif (SSM). An example of a SSM is derived from a virus or mobile genetic element. In some embodiments the SSM is an inverted repeat or hairpin sequence, e.g., an inverted terminal repeat (ITR) from a virus, e.g., from an AAV, or a conserved 8-nucleotide hairpin in the anellovirus origin of replication. Examples of SSM’s are listed below in Table 2:

[0371] Table 2: Exemplary second strand motifs

[0372] In some embodiments, an SSM is a short sequence of RNA or DNA that is complementary to a region of the ssDNA, e.g., an RNA primer or a DNA primer. In some embodiments, the primer is a splint sequence connecting ends of a ssDNA described herein. In some embodiments the RNA or DNA primer is less than 100, less than 75, less than 50, less than

[0373] 1601838421.1 60 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0374] 40, less than 30, less than 25, less than 20, less than 15, less than 10, or less than 5 nucleotides. In some embodiments the RNA or DNA primer is between 5-100 nucleotides, between 10-100 nucleotides, between 20-80 nucleotides, between 20-60 nucleotides.

[0375] Other elements

[0376] A DNA region disclosed herein may also include other control elements operably linked to the effector sequence, e.g., the sequence encoding an effector, in a manner which permits its transport, localization, transcription, translation and / or expression in a target cell, or which promotes its degradation or repression of expression in a non-target cell. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the sequence encoding the effector and expression control sequences that act in trans or at a distance to control the sequence encoding the effector. The precise nature of regulatory sequences needed for gene expression in host cells may vary between species, tissues or cell types, but in general may include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, enhancer elements and the like. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The constructs described herein may optionally include 5' leader or signal sequences. In some embodiments, the DNA region may comprise a sequence encoding a 5’ untranslated region and / or a sequence encoding a 3’ untranslated region.

[0377] The DNA region may comprise a non-coding region. In some embodiments, the noncoding region is completely free of predicted ORFs. In some embodiments, the non-coding region does not encode a protein sequence. In some embodiments, the non-coding region is not translated or is not translated at a substantial level.

[0378] Sequence elements of ssDNA constructs

[0379] In an embodiment, an ssDNA region described herein comprises an effector sequence and one or both of a maintenance sequence and a second strand motif (SSM). In one embodiment the ssDNA region comprises a DNA effector sequence. In one embodiment, the ssDNA region comprises a DNA effector sequence and a SSM. In one embodiment, the ssDNA region comprises a DNA effector sequence and a maintenance sequence. In one embodiment,

[0380] 1601838421.1 61 Atorney Docket No.: F2128-7027WO(VL87026-W1) the ssDNA region comprises a DNA effector sequence, an SSM and a maintenance sequence. In one embodiment, the ssDNA region comprises a promoter operably linked to a sequence encoding an RNA or protein (peptide or polypeptide) effector. In one embodiment, the ssDNA region comprises a promoter operably linked to a sequence encoding an RNA or protein (peptide or polypeptide) effector and a SSM. In one embodiment, the ssDNA region comprises a promoter operably linked to a sequence encoding an RNA or protein (peptide or polypeptide) effector and a maintenance sequence. In one embodiment, the ssDNA region comprises a promoter operably linked to a sequence encoding an RNA or protein (peptide or polypeptide) effector, an SSM and a maintenance sequence.

[0381] In some embodiments, an ssDNA region described herein comprises an enhancer. In some embodiments, an ssDNA region comprises two enhancers. In some embodiments, an ssDNA region described herein comprises a SSM (e.g., an Anellovirus hairpin). In some embodiments, an ssDNA region described herein comprises a polyA signal, e.g., a bGH polyA signal. In some embodiments, an ssDNA region described herein comprises a promoter, e.g., an EFla promoter.

[0382] In some embodiments, the enhancer is situated upstream of the promoter. In some embodiments, the SSM (e.g., Anellovirus hairpin) is situated upstream of the promoter.

[0383] In some embodiments, the promoter is situated between the enhancer and the effector sequence. In some embodiments, the SSM (e.g., anellovirus hairpin) is situated between the enhancer and the promoter. In some embodiments, the polyA signal is situated between the effector sequence and the enhancer. In some embodiments, the polyA signal is situated between the SSM (e.g., anellovirus hairpin) and the effector sequence.

[0384] In embodiments, the ssDNA can include a plurality of effector sequences. The plurality may be the same or different types, e.g., an ssDNA can include an effector sequence that is a structural DNA and a second effector sequence that is a DNA sequence encoding a functional RNA or polypeptide. In some embodiments, the ssDNA comprises a second effector sequence which is the same as or different than the first effector sequence. An ssDNA can include an effector sequence that is a DNA sequence encoding a functional RNA and a second effector sequence that is a DNA sequence encoding a functional polypeptide. The plurality of effector sequences may be the same or different sequences of the same type.

[0385] 1601838421.1 62 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0386] Organic moi eties

[0387] A DNA molecule described herein can be functionalized with various organic moieties. In some embodiments, the organic moiety is a reactive handle, e.g., as described in the section herein entitled “Reactive handles, e.g., click handles.” In some embodiments, the organic moiety is a macromolecule. In some embodiments, the organic moiety is a small molecule.

[0388] Macromolecules

[0389] In some embodiments, a macromolecule described herein is a polypeptide, e.g. a polypeptide comprising a cell penetrating peptide, an albumin protein, an antibody, a transferrin, insulin, a targeting peptide, or an NLS (e.g., as described in the section herein entitled “Nuclear Localization Sequence”. In some embodiments, a macromolecule described herein is a nucleic acid, e.g. a nucleic acid comprising an aptamer, an adjuvanting sequence (e.g., CpG oligodeoxynucleotide), an immune suppressing sequence (e.g., GpC oligodeoxynucleotide), or a homopolymer. In some embodiments, a macromolecule described herein is a glycan, e.g. a glycan comprising amino sugars (e.g. N-acetylgalactosamine), uronic sugars, aldohexoses (e.g. glucose or galactose), ketohexoses (e.g. fructose), sialic acids, or a combination thereof. In some embodiments, a macromolecule described herein is a lipid, e.g. a lipid comprising saturated fatty acids, unsaturated faty acids, triglycerides, diglycerides, monoglycerides, phospholipids, sphingolipids, steroids, glycolipids, or a combination thereof. In some embodiments, a macromolecule described herein is streptavidin.

[0390] Small molecules

[0391] In some embodiments, a small molecule described herein is a dye, e.g., Cy3. In some embodiments, a small molecule described herein is a small molecule (e.g., biotin moiety) that binds a protein. In some embodiments, a small molecule described herein is a monosaccharide (e.g., glucose, fructose, galactose, mannose, or N-acetylgalactosamine) or a disaccharide (e.g., lactose). In some embodiments, a small molecule described herein is an immune modulating small molecule (e.g., a cyclic GMP-AMP synthase (cGAS) inhibitor). In some embodiments, the cGAS inhibitor is RU.521, G140, or G150. In some embodiments, a small molecule described herein comprises a hydroxyquinoline. In some embodiments, a small molecule described herein is hydroxychloroquine.

[0392] 1601838421.1 63 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0393] Nuclear Localization Sequence

[0394] The macromolecule may be a nuclear localization sequence (NLS). Consequently, in some embodiments, a DNA molecule, e.g., as described herein, is linked to a peptide comprising an NLS, e.g., as described in Table 3. In some embodiments, the NLS has a sequence according to Table 3 or a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto. In some embodiments, the NLS has a sequence according to Table 3 or a sequence with no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 sequence differences (e.g., substitutions, insertions, or deletions) relative thereto. Without being bound by theory, a nuclear localization sequence is an amino acid sequence that mediates transport of a molecule, e.g., a DNA described herein, into the nucleus.

[0395] In some embodiments, the NLS is a human NLS or an NLS having sequence similarity to a human NLS. In some embodiments, the NLS is a viral NLS or an NLS having sequence similarity to a viral NLS. In some embodiments, the NLS is a classical NLS. In some embodiments, the NLS is a non-classical NLS. In some embodiments, the NLS is a monopartite NLS. In some embodiments, the NLS is a bipartite NLS. In some embodiments, the NLS is a PY-NLS. In some embodiments, the NLS is other than a monopartite NLS, bipartite NLS, or PY-NLS. In some embodiments, the NLS has a predicted net charge at pH 7 of -2-0, 0-2, 2-4, 4- 6, 6-8, 8-10, 10-12, or 12-14. The predicted net charge is measured using a peptide calculator (www.bachem.com / knowledge-center / peptide-calculator / ), assuming an “H” at the N termini and a “NH2” at the C termini. In some embodiments, the NLS has a predicted net negative charge at pH 7. In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the residues of the NLS are basic residues.

[0396] Without wishing to be bound by theory, in some embodiments, a positively charged NLS may bind nonspecifically and non-covalently to negatively charged DNA. Accordingly, in some embodiments, an NLS with a negative charge, neutral charge, or weaker positive charge may reduce nonspecific binding.

[0397] A DNA molecule described herein may comprise a plurality of NLSs, e.g., a first NLS and a second NLS. In some embodiments, the first NLS and the second NLS have the same amino acid sequence. In some embodiments, the first NLS and the second NLS have different amino acid sequences.

[0398] 1601838421.1 64 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0399] Table 3: Exemplary NLS sequences

[0400] 1601838421.1 65

[0401] Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0402] 1601838421.1 66

[0403] Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0404] Reactive handles and reactive linkers

[0405] In some embodiments, a DNA region described herein is linked to an organic moiety through one or more covalent bonds. In some embodiments, a DNA region described herein is linked to an organic moiety through a reactive linker, e.g., via a click linker. In some embodiments, the click linker is formed as a product of a click reaction between a first click handle and a second click handle. A variety of reactions that fulfill the criteria for click chemistry are known, a wide range of commercially available reagents for click chemistry can be used, and one skilled in the art could use (for example) any one of a number of published methodologies (see, e.g., pubs.acs.org / doi / 10.1021 / acs.chemrev. lc00469 or www.ncbi.nlm.nih.gov / pmc / articles / PMC2562613 / , which are herein incorporated by reference in their entirety). In some embodiments, conjugation is performed using click chemistry.

[0406] Reactive handles, e.g., click handles

[0407] For example, in some embodiments, a first reactive handle (e g., click handle) is affixed to a DNA region. For example, the first reactive handle (e.g., click handle) may be covalently bound (e.g., directly bound) to a nucleotide of the DNA region. In some embodiments, a second reactive handle (e.g., click handle) is bound to an organic moiety, e.g., macromolecule or small molecule. For example, the second reactive handle may be covalently bound (e.g., directly bound) to an amino acid of a polypeptide, such as a lysine. The DNA and first reactive handle may be contacted with the organic moiety and second reactive handle under conditions that allow the first reactive handle to react with the second reactive handle, e.g., in a click reaction.

[0408] In some embodiments, the first reactive handle is incorporated into DNA by using PCR to produce the DNA region or a fragment thereof, wherein a nucleotide in the PCR reaction comprises a reactive handle. In some embodiments, the second reactive handle is incorporated into the organic moiety (e.g., polypeptide or nucleic acid) by solid phase synthesis, wherein an amino acid or nucleic acid in the solid phase synthesis reaction comprises the second reactive handle.

[0409] A variety of molecules that comprise a click handle can be used. For instance, in some embodiments, the molecule is chosen from:

[0410] 1601838421.1 67 Attorney Docket No.: F2128-7027WO(VL87026-W1)

[0411] SCO-Lysine, e.g., for use in a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction or a strain -promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reaction;

[0412] Cyclopropene lysine, e.g., for use in a SPIEDAC reaction;

[0413] TCO*A-Lysine, e.g., for use in a SPIEDAC reaction;

[0414] Exo-BCN-Lysine, e.g., for

[0415] 1601838421.1 68 Attorney Docket No.: F2128-7027WO(VL87026-WI)

[0416] NBO-Lysine, e.g., for use in a SPIED AC reaction; rac-BCN-Lysine, e.g., for use

[0417] TCO-Lysine, e.g., for use in a SPIED AC reaction;

[0418] Endo-BCN-Lysine, e.g., for u

[0419] 1601838421.1 69 Attorney Docket No.: F2128-7027WO(VL87026-W1)

[0420] PrK-HCl-salt, e.g. for use in a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction; N3-Lysine, e.g., for use p-acetylphenyl alanine, e.g., for use with a site-specific oxime ligation (e.g., mediated by DBCO- amine), followed by SPAAC; p-azidomethylphenylalanine, e.g., for use in a SPAAC or SPIED AC reaction;

[0421] 1601838421.1 70 Attorney Docket No.: F2128-7027WO(VL87026-W1) seleno-cysteine, e.g., for reaction with maleimide; or methyltetrazine-PEG3-maleimide, e.g. for use in SPIED AC or reaction with thiol;

[0422] BCN-PEG3-oxyamine, e.g. for use in SPAAC or SPIEDAC reaction or an oxyamine ligation. A click handle may comprise an azide or an alkyne. In some embodiments, a DNA region described herein is linked to a first click handle that reacts specifically with a second click handle linked to an organic moiety, thereby producing a click linker between the DNA region and the organic moiety. In some embodiments, the first click handle comprises an azide moiety,

[0423] 1601838421.1 71 Atorney Docket No.: F2128-7027WO(VL87026-W1) and the second click handle comprises an alkyne moiety. In some embodiments, the first click handle comprises an alkyne moiety, and the second click handle comprises an azide moiety.

[0424] In some embodiments, the click handle comprises an alkyne moiety. In some embodiments, the alkyne moiety comprises a propargyl moiety or a cyclooctynyl moiety. Exemplary alkyne moieties include diarylcyclooctyne (DBCO)-sulfo-NHS-ester, diarylcyclooctyne (DBCO)-PEG-NHS-ester, diarylcyclooctyne (DBCO)-C6-NHS-ester, diarylcyclooctyne (DBCO)-NHS-ester, diarylcyclooctyne (DBCO)-amine, diarylcyclooctyne (DBCO)-acid, sulfo diarylcyclooctyne (DBCO)-maleimide, diarylcyclooctyne (DBCO)- maleimide, bis-sulfone-PEG-diarylcyclooctyne (DBCO), propargyl-NHS ester, propargyl- maleimide, alkyne-PEG-NHS ester, alkyne-PEG-maleimide, or a derivative thereof. In some embodiments, the alkyne moiety comprises DBCO provided as a dibenzocyclooctyne-acid (CAS 1353016-70-2).

[0425] In some embodiments, the click handle comprises an azide moiety. In some embodiments, the azide moiety comprises an azidoalkyl moiety, azidoaryl moiety, or an azidoheteroaryl moiety. Exemplary azide moieties include 3-azidopropionic acid sulfo-NHS ester, azidoacetic acid NHS ester, azido-PEG-NHS ester, azidopropylamine, azido-PEG-amine, azido-PEG-maleimide, bis-sulfone-PEG-azide, or a derivative thereof.

[0426] Click handles may also comprise an alkene moiety, e.g., a transcycloalkene moiety, an oxanorb ornadiene moiety, or a tetrazine moiety. Additional click handles can be found in Click Chemistry Tools (clickchemistrytools.com / ), Lahann, J (ed) (2009) Click Chemistry for Biotechnology and Materials Science, McKay et al, “Click chemistry in complex mixtures: bioorthogonal bioconjugation” Chem Biol. 2014 Sep 18;21(9): 1075-101, Becer et al. “Click chemistry beyond metal -catalyzed cycloaddition” Angew Chem Int Ed Engl. 2009;48(27):4900- 8., and Hein et al. “Click chemistry, a powerful tool for pharmaceutical sciences” Pharm Res. 2008 Oct;25(10):2216-30, each of which is incorporated herein by reference in its entirety.

[0427] In embodiments, the click handle comprises a tetrazine moiety, e.g., for reaction with an alkene moiety. For instance, in embodiments, the tetrazine is a 1,2, 4, 5 tetrazine and the alkene is a strained alkene. In embodiments, the alkene moiety comprises a trans-cyclooctene, (E)- Cyclooct-4-enol, (E)-Cyclooct-4-enyl 2,5-dioxo-l-pyrrolidinyl carbonate, 5-Norbornene-2-acetic acid succinimidyl ester, 5-Norbornene-2-endo-acetic acid, TCO PEG4 succinimidyl ester, TCO- amine, or TCO-PEG3-maleimide. In embodiments, the tetrazine click handle comprises (4-

[0428] 1601838421.1 72 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0429] (l ,2,4,5-Tetrazin-3-yl)phenyl)methanamine or 2,5-Dioxo-l-pyrrolidinyl 5-[4-(l,2,4,5-tetrazin-3- yl)benzylamino]-5-oxopentanoate, 5-[4-(l,2,4,5-Tetrazin-3-yl)benzylamino]-5-oxopentanoic acid. In embodiments, the tetrazine and alkene react in a Diels-Alder cycloaddition to yield a stable covalent linkage. In embodiments, a catalyst is not needed. In embodiments, the only byproduct is dinitrogen. In embodiments, the reaction at least one order of magnitude faster than azide-cyclooctyne based click chemistry. Without wishing to be bound by theory, tetrazine / alkene reactions can be used with low concentrations of reactant.

[0430] In some embodiments, the click handles react via an azide-alkyne Huisgen cycloaddition. In some embodiments, an azide-alkyne Huisgen cycloaddition comprises a copper(I)-catalyzed azide-alkyne cycloaddition or a strain-promoted azide-alkyne cycloaddition.

[0431] In some embodiments, the click handles react to form a heteroaryl, e.g., a triazole. In some embodiments, the triazole comprises a 1,2,3-triazole, e.g., a 1,4-di substituted 1,2,3-triazole or a 1,5 -di substituted 1,2,3-triazole.

[0432] In some embodiments, the click handle comprises an alkyne and reacts with an amine. In some embodiments, the click handle comprises a cyclooctyne and reacts with an amine. In some embodiments, the click handle comprises diarylcyclooctyne (DBCO)-sulfo-NHS-ester or di aryl cy cl oocty ne (DB CO )-PEG5 -NH S -ester.

[0433] In some embodiments, the click handle comprises an azide and reacts with an amine. In some embodiments, the click handle is 3-azidopropionic acid sulfo-NHS ester or azido-PEG4- NHS-ester.

[0434] In an embodiment, the click handle is water soluble. In an embodiment, the click handle is membrane impermeable, e.g., has sufficient charge to render it membrane impermeable. In an embodiment the click handle is charged, e.g., positively charged or negatively charged. In an embodiment, the click handle comprises a cationic moiety or an anionic moiety, e.g., a SO3 moiety.

[0435] In some embodiments, the click handle comprises a detection agent, e.g., useful for detection of the DNA molecule. Exemplary detection agents may include a fluorescent molecule (e.g., a cyanine dye, e.g., Cy3, Cy 3.5, Cy5, Cy5.5, Cy7, or Cy7.5), a metal chelate, a contrast agent, a radionuclide, a positron emission tomography (PET) imaging agent, an infrared imaging agent, a near-IR imaging agent, a computer assisted tomography (CAT) imaging agent, a photon

[0436] 1601838421.1 73 Atorney Docket No.: F2128-7027WO(VL87026-W1) emission computerized tomography imaging agent (e.g., DIBO-DFO, where DFO chelates Zirconium-89), an X-ray imaging agent, or a magnetic resonance imaging (MRI) agent.

[0437] Suitable click handles may comprise, for example, an amine, sulfate, thiol, hydroxyl, azide, alkyne, alkene, carboxyl groups aldehyde groups, sulfone groups, vinylsulfone groups, isocyanate groups, acid anhydride groups, epoxide groups, aziridine groups, episulfide groups, groups such as -CO2N(COCH2)2, -CO2N(COCH2)2, -CO2H, -CHO, -CHOCH2, - N.dbd.C.dbd.O, --SO2CH.dbd.CH2, — N(COCH)2, — S— S— (C5H4N) and groups of the following structures wherein X is halogen and R is hydrogen or Ci to C4 alkyl:

[0438] In an embodiment, a reactive handle is a GMP grade material.

[0439] Reactive linkers, e.g., click linkers

[0440] Linkage of the two substrates (e.g., DNA region and organic moiety) typically results in a residual linker between the first and second substrate. For example, in the case of click handles comprising an azide and an alkyne, a click linker may be formed comprising a triazole (e.g., a 1,2,3-triazole).

[0441] In some embodiments, the reactive linker is situated between the organic moiety and a nucleotide of a DNA sequence that comprises a promoter, a DNA end form, a maintenance sequence, or an origin of replication. In some embodiments, the reactive linker is situated between the organic moiety and a nucleotide of the upstream DNA end form. In some embodiments, the reactive linker is situated between the organic moiety and a nucleotide of the downstream DNA end form. In some embodiments, the reactive linker is situated between the organic moiety and a nucleotide of the effector sequence that encodes an effector. In some embodiments, the reactive linker is situated between the organic moiety and a nucleotide that is

[0442] 1601838421.1 74 Attorney Docket No.: F2128-7027WO(VL87026-W1) outside of the effector sequence that encodes an effector. In some embodiments, the reactive linker is situated between the organic moiety and a nucleotide of the promoter. In some embodiments, the reactive linker is situated between the organic moiety and a nucleotide that is outside of the promoter. In some embodiments, the reactive linker is situated between the organic moiety and a nucleotide of the maintenance sequence. In some embodiemnts, the reactive linker is situated between the organic moiety and a nucleotide that is outside of the maintenance sequence. In some embodiments, the reactive linker is situated between the organic moiety and a nucleotide of the origin of replication. In some embodiments, the reactive linker is situated between the organic moiety and a nucleotide that is outside of the origin of replication. In some embodiments, the reactive linker is situated between the organic moiety and a nucleotide of a single stranded region of the DNA region. In some embodiments, the reactive linker is situated between the organic moiety and a nucleotide of a double stranded region of the DNA region. In some embodiments, the reactive linker is situated between the organic moiety and a nucleotide of a region encoding a 5’ UTR. In some embodiments, the reactive linker is situated between the organic moiety and a nucleotide of a region encoding a 3’ UTR. In some embodiments, the reactive linker is situated between the organic moiety and a nucleotide of a polyA site.

[0443] Exemplary click linkers suitable for use in the composition and methods described herein include, for example

[0444] 1601838421.1 75 Attorney Docket No.: F2128-7027WO(VL87026-W1) wherein R is H, a hydrocarbon or heteroatom which may be further optionally substituted, and A is a C3-C12 cycloalkane, cycloalkene, cycloalkyne, or a heterocycle, all of which may be further optionally substituted;

[0445] 1601838421.1 76 Attorney Docket No.: F2128-7027WO(VL87026-W1) wherein R is H, a hydrocarbon or heteroatom which may be further optionally substituted, and A is a C3-C12 cycloalkane, cycloalkene, cycloalkyne, or a heterocycle, all of which may be further optionally substituted, wherein A is a C3-C20 cycloalkene, cycloalkyne, or a heterocycle, all of which may be further optionally substituted,

[0446] 1601838421.1 77 Atorney Docket No.: F2128-7027WO(VL87026-W1) wherein R is a hydrocarbon which is further optionally substituted.

[0447] In an embodiment, the click linker is an alkyne / azide click linker (e.g., wherein the alkyne is a cyclooctyne, activated alkyne, or electron-deficient alkyne), e.g., the click linker comprises a triazole, e.g., a 1,2, 3 -triazole and / or a disubstituted triazole. In an embodiment, the click linker is a diene / dienophile click linker (e.g., wherein the dienophile comprises an alkene moiety), e g., the click linker comprises a cycloalkene, e.g., a disubstituted alkene. In some embodiments, the click linker is a tetrazine / alkene click linker, e.g., the click linker comprises a dihydropyrazine, e.g., a 1,2-dihydropyrazine. In some embodiments, the click linker is a tetrazole / alkene click linker, e.g., the click linker comprises a diazole. In some embodiments, the click linker is a dithioester / diene click linker, e.g., the click linker comprises a sulfur-containing ring, e.g., a tetrahdrothiophene, e.g., a disubstituted tetrahdrothiophene. In some embodiments, the click linker is a dithioester / diene linker, e.g., the click linker comprises a sulfur-containing ring, e.g., a thiopyran. In some embodiments, the click linker is a thiol / alkene click linker, e.g., the click linker comprises an alkyl sulfide.

[0448] Click reactions

[0449] In some embodiments, a method described herein comprise a step of performing a click reaction. In some embodiments, a DNA molecule described herein is produced using a click reaction.

[0450] In some embodiments, the click reaction is a cycloaddition (e.g., a 1,3-dipolar cycloaddition or hetero-Diels- Alder cycloaddition), nucleophilic ring-opening (e.g., openings of strained heterocyclic electrophiles such as aziridines, epoxides, cyclic sulfates, aziridinium ions, and episulfonium ions), carbonyl chemistry of no-aldol type (e.g., formation of ureas, thioureas,

[0451] 1601838421.1 78 Atorney Docket No.: F2128-7027WO(VL87026-W1) hydrazones, oxime ethers, amides, or aromatic heterocycles), or an addition to a carbon-carbon multiple bond (e.g., epoxidation, aziridination, dihydroxylation, sulfenyl halide addition, nitosyl halide addition, or Michael addition). Examples of these types of click reaction are described in greater detail in Hein et al., Pharm. Res. 2008 October; 25(10):2216-2230, which is herein incorporated by reference in its entirety. In embodiments, the click reaction is a metal-free [3+2] cycloaddition reaction, Diels-Alder reaction, or thiol-alkene radical reaction. Examples of these types of click reaction are described in greater detail in Becer et al., Angew. Chem. Int. Ed. 2009, 48, 4900-4908, which is herein incorporated by reference in its entirety.

[0452] In embodiments, the click reaction does not require a catalyst. In embodiments, the click reaction does not require copper ions, e.g., proceeds at substantially the same rate in the absence of copper ions as in the presence of copper ions, e.g., under conditions described in Tornoe, C. W. et al (2002). "Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)- Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides". In embodiments, the click reaction proceeds efficiently at a temperature of about 10-40, 20-40, 20-30, 20-25, 30-40, or 35-40, or about 37 °C. In embodiments, the click reaction proceeds efficiently at a temperature of below 50, 45, 40, 35, 30, 25, or 20°C.

[0453] In embodiments, the activation barrier for a click reaction is 24-30, 25-29, or 26-28 kcal / mol, e g., about 27.8 kcal / mol or 26 kcal / mol. In embodiments, the activation barrier for a click reaction is the same as or no less than 50%, 40%, 30%, 20%, or 10%, different from the activation barrier of a Huisgen Cu-catalyzed cycloaddition reaction between an azide and a terminal alkene, e.g., as described in Hein et al. Click chemistry, a powerful tool for pharmaceutical sciences” Pharm Res. 2008 Oct;25(10):2216-30.

[0454] In embodiments, the click reaction is exergonic, e.g., having a AG° of between -10 and - 100, -20 and -90, -30 and -70, -40 and -70, -50 and -60, or about -61 kcal / mol. In embodiments, the AG° for a click reaction is the same as or no less than 50%, 40%, 30%, 20%, or 10%, different from the AG° of a Huisgen Cu-catalyzed cycloaddition reaction between an azide and a terminal alkene.

[0455] In embodiments, the click reaction has a AG° of between -30 and -140, -40 and -130, -50 and -120, -60 and -110, -70 and -100, -80 and -90, or about 84 kl / mol.

[0456] One example of a cycloaddition reaction is the Huisgen 1,3-dipolar cycloaddition of a dipolarophile with a 1,3 dipolar component that produce five membered (hetero)cycles.

[0457] 1601838421.1 79 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0458] Examples of dipolarophiles are alkenes, alkynes, and molecules that possess related heteroatom functional groups, such as carbonyls and nitriles. Specifically, another example is the 2+3 cycloaddition of alkyl azides and acetylenes. Other cycloaddition reactions include Diels-Alder reactions of a conjugated diene and a dienophile (such as an alkyne or alkene). Examples of cycloaddition reactions are described, e.g., in US Pat. 9,517,291, which is herein incorporated by reference in its entirety.

[0459] Other examples of click reactions include a hydrosilation reaction of H— Si and simple non-activated vinyl compounds, urethane formation from alcohols and isocyanates, Menshutkin reactions of tertiary amines with alkyl iodides or alkyl trifluoromethanesulfonates, Michael additions, e.g., the very efficient maleimide-thiol reaction, atom transfer radical addition reactions between — SO2CI and an olefin (R1, R2— C=C — R3, R4), metathesis, Staudinger reaction of phosphines with alkyl azides, oxidative coupling of thiols, nucleophilic substitution, especially of small strained rings like epoxy and aziridine compounds, carbonyl chemistry like formation of ureas, and addition reactions to carbon-carbon double bonds like dihydroxylation. Therefore, attached functionality may be chosen from acetylene bond, an azido-group, a nitrile group, acetylenic, amino group, phosphino group. The click chemistry reaction may result in the addition of a functional group selected from amino, primary amino, hydroxyl, sulfonate, benzotri azole, bromide, chloride, chloroformate, trimethylsilane, phosphonium bromide or bio- responsive functional group including polypeptides, proteins and nucleic acids.

[0460] In some embodiments, a click reaction forms very energy-efficient carbon-heteroatom bonds, in particular a ring opening nucleophilic reaction or a cycloaddition reaction. A type of reaction which is widely represented in click chemistry is the abovementioned alkyne-azide cycloaddition catalyzed with Cu(I). Examples of click reactions are also described, e.g., in US Pat. 9,453,843, which is herein incorporated by reference in its entirety.

[0461] Click chemistry may generate substances quickly and reliably by joining small modular units together (see, e.g., Kolb et al. (2001) Angewandte Chemie Inti. Ed. 40:2004-2011; Evans (2007) Australian J. Chem. 60:384-395; Carlmark et al. (2009) Chem. Soc. Rev. 38:352-362; each herein incorporated by reference in its entirety).

[0462] 1601838421.1 80 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0463] Non-covalent bonds, e.g.. biotin-avidin interactions

[0464] In some embodiments, a DNA molecule described herein may be complexed non- covlaently with another molecule. For example, the DNA molecule may be covalently linked to a small molecule (e.g., a biotin moiety), and the small molecule may be non-covalently complexed to another molecule (e.g., a polypeptide, e.g., avidin). The other molecule (e.g., avidin) may be bound covalently or non-covalently to an organic moiety.

[0465] In some embodiments, the small molecule (e.g., a biotin moiety), and the organic moiety is linked to another molecule (e.g., a polypeptide, e g., an avidin). In some embodiments, the DNA region is linked to an avidin moiety, and the organic moiety is linked to a small molecule (e.g., a biotin moiety). In some embodiments, the DNA region is linked to a plurality of small molecule (e.g., biotin) moieties, e.g., two biotin moieties.

[0466] In some embodiments, the avidin moiety comprises a streptavidin, e.g., a wild-type strepatividin. In some embodiments, the streptavidin comprises an amino acid sequence of SEQ ID NO: 85, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the avidin moiety comprises a monovalent strepatividin.

[0467] MRKIVVAAIAVSLTTVSITASASADPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGAD GALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTW SGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNN GNPLDAVQQ (SEQ ID NO: 85)

[0468] Chemically modified nucleotides

[0469] The DNA regions described herein may have chemical modifications of the nucleobases, sugars, and / or the phosphate backbone. While not wishing to be bound by theory, such modifications can be useful for protecting a DNA from degradation (e.g., from exonucleases) or from the immune system of a host tissue or subject. In general, a chemically modified nucleotide has the same base-pairing specificity as the unmodified nucleotide, e.g., a chemically modified adenine “A” can base-pair with thymine “T”. One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain

[0470] 1601838421.1 81 Atorney Docket No.: F2128-7027WO(VL87026-W1) embodiments, chemical modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage.

[0471] Examples of chemical modifications to DNA useful in the methods described herein include, e.g., N6-Methyladenosine (m6A, 6mA); 5-formylcytosine (5-formyl-2’-deoxycytosine, 5fC, f5C); 5-carboxylcytosine (5-carboxyl-2’-deoxycytosine, 5-carboxycytosine, ca5C, 5caC); 5- hydroxymethylcytosine (5 -hydroxymethyl-2 ’-deoxy cytosine, 5hmC, hm5C); 5- methyldeoxycytosine (5-methylcytosine; 5 -methyl-2’ -deoxy cytosine; m5dC; 5mC, m5C); 5’- methylcytosine; 3 -methylcytosine (m3C); 2'-fluoro-2'deoxynucleoside; 5- glucosylmethylcytosine; 5-methyl pyrimidine; 8-oxoguanine (8-oxoG); phosphorothioate; S and R phsophorothioate linkages; methylthymine; N3’-P5’ Phosphoroamidate (NP); cyclohexane nucleic acid (CeNA); tricyclo-DNA (tcDNA). See, e.g., Pu et al. 2020. AH in-vitro DNA phosphorothioate modification reaction. Mol Microbiol. 113: 452 463; Zheng & Sheng. 2021. Synthesis of N4-methylcytidine (m4C) and N4,N4-dimethylcytidine (m42C) modified RNA. Current Protocols, 1, e248; Ohkubo et al. 2021. Chemical synthesis of modified oligonucleotides containing 5’-amino-5'-deoxy-5'-hydroxymethylthymidine residues. Current Protocols, 1, e70; Bao & Xu. 2021. Observation ofZ-DNA structure via the synthesis of oligonucleotide DNA containing 8-trifluoromethyl-2-deoxyguanosine. Current Protocols, 1, e28; Skakujet al. 2020. Automated synthesis and purification of guanidine -backbone oligonucleotides. Current Protocols in Nucleic Acid Chemistry, 81, el 10.

[0472] In some embodiments, a DNA region described herein comprises a nucleotide comprising a chemically modified cytosine nucleobase.

[0473] In some embodiments, a DNA region comprises a sense strand and an antisense strand, wherein the antisense strand comprises one or more chemically modified nucleotides. In some embodiments, a DNA region comprises a sense strand and an antisense strand, wherein the sense strand does not comprise any chemically modified nucleotides. In some embodiments, a DNA region comprises a sense strand and an antisense strand, wherein the sense strand comprises one or more chemically modified nucleotides.

[0474] In some embodiments, a DNA region as described herein may comprise a phosphorothioate-modified nucleotide. In some embodiments, a DNA end form (e.g., an exonuclease-resistant DNA end form) as described herein may comprise a phosphorothioate- modified nucleotide. In some embodiments, the DNA region described herein may include S

[0475] 1601838421.1 82 Atorney Docket No.: F2128-7027WO(VL87026-W1) and R phosphorothioate modified nucleotide linkages. In one embodiment, the phosphorothioate linkages are made according to Iwamoto et al, 2017, Nature Biotechnology, Volume 35:845-851. Briefly, monomers of nucleoside 3’-oxazaphospholidine derivates undergo stereocontrolled oligonucleotide synthesis with iterative capping and sulfurization to create stereocontrolled phosphorothioate linkages. The final sample is analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) and Ultraperformance liquid chromatography mass spectrometry (UPLC / MS) to determine stereochemistry of the modification. Nucleic acids containing phosphorothioate linkages are also commercially available. In some embodiments, a DNA region comprises an upstream exonuclease-resistant DNA end form comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 phosphorothioate bonds (e.g., between the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 terminal nucleotides of the upstream exonuclease-resistant DNA end form, e g., on the first strand, the second strand, or both of the first and second strands). In some embodiments, a DNA region comprises a downstream exonuclease-resistant DNA end form comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 phosphorothioate bonds (e g., between the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 terminal nucleotides of the downstream exonuclease-resistant DNA end form, e.g., on the first strand, the second strand, or both of the first and second strands).

[0476] In some embodiments, a DNA region described herein may include boranophosphate modified nucleotides, e.g., following the methods in Sergueev and Shaw, 1998, J Am Chem Soc, Volume 120, Issue 37:9417-9427. Briefly, H-phosphonate chain elongation is followed by boronation to substitute a borano group for a nonbridging oxygen in the phosphate backbone. The final sample is purified and analyzed by RP-HPLC to determine stereochemistry of the modification. Boranophosphate modified nucleotides are also commercially available.

[0477] In some embodiments, a DNA region described herein may include 5-methylcytosine modified nucleotides, e.g., made following the methods in Lin et al, 2002, Mol Cell Biol, Volume 22, Issue 3:704-723. Briefly, cytosine or the sequence containing cytosine is incubated with glutathione S-transferase fusion of wild-type Dnmt3a (GST-3 a) protein using unlabeled S- adenosylmethionine (AdoMet). The nucleotides are purified and analyzed by HPLC to determine that the nucleotides are methylated at the correct position. 5-methylcytosine modified nucleotides are also available commercially.

[0478] 1601838421.1 83 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0479] In some embodiments, a DNA region described herein may include 7-methylguanine modified nucleotides. In one embodiment, 7-methylguanine modified nucleotides are made following the methods in Jones and Robins, 1963, Purine nucleosides. III. Methylation studies of certain naturally occurring purine nucleosides, J Am Chem Soc, Volume 85: 193. Briefly, 2’- deoxyguanosine in dimethyl sulfoxide is treated with methyl iodide. The nucleotides are purified and analyzed by HPLC to determine that the nucleotides are methylated at the correct position. In another embodiment, 7-methylguanine modified nucleotides are made according to the methods described in Hendler et al, 1970, Volume 9, Issue 21 :4141 :4153, and Kore and Parmar, 2006, Biochemistry, Volume 25, Issue 3:337-340. Briefly, instead of guanosine 5 ’-diphosphate, guanine 5 ’-diphosphate in water is added to dimethyl sulfate to yield 7-methyl GDP. The nucleotides are purified and analyzed by HPLC to determine that the nucleotides are methylated at the correct position. 7-methylguanine modified nucleotides are also available commercially.

[0480] In some embodiments, a DNA region described herein comprises methylation at one or more CpG or GpC dinucleotide. In some embodiments, a DNA region described herein comprises a methylation introduced by an Alul methyltransferase. In some embodiments, a DNA region described herein comprises a methylation introduced by a BamHI methyltransferase. In some embodiments, a DNA region described herein comprises a methylation introduced by a CpG methyltransferase (M.Sssl). In some embodiments, a DNA region described herein comprises a methylation introduced by a dam methyltransferase. In some embodiments, a DNA region described herein comprises a methylation introduced by an EcoGII methyltransferase. In some embodiments, a DNA region described herein comprises a methylation introduced by an EcoRI methyltransferase. In some embodiments, a DNA region described herein comprises a methylation introduced by a GpC methyltransferase (M.CviPI). In some embodiments, a DNA region described herein comprises a methylation introduced by an Haelll methyltransferase. In some embodiments, a DNA region described herein comprises a methylation introduced by an Hhal methyltransferase. In some embodiments, a DNA region described herein comprises a methylation introduced by an Hpall methyltransferase. In some embodiments, a DNA region described herein comprises a methylation introduced by a MspI methyltransferase. In some embodiments, a DNA region described herein comprises a methylation introduced by a TaqI methyltransferase. In some embodiments, a method described herein comprises contacting a dsDNA with an Alul methyltransferase, a BamHI

[0481] 1601838421.1 84 Atorney Docket No.: F2128-7027WO(VL87026-W1) methyltransf erase, M.Sssl, a dam methyltransferase, an EcoGII methyltransf erase, an EcoRI methyltransferase, M.CviPI, an Haelll methyltransferase, an Hhal methyltransferase, an Hpall methyltransferase, a MspI methyltransferase, or a TaqI methyltransferase.

[0482] In some embodiments, a DNA region described herein comprises a carboxyl modification or a formyl modification.

[0483] In embodiments, a DNA region described herein, or one strand (e.g., the sense strand or the antisense strand) of the DNA region, comprises between 1-100% chemically modified nucleotides, between 1 %-90% chemically modified nucleotides, between l%-80% chemically modified nucleotides, between l%-70% chemically modified nucleotides, between l%-60% chemically modified nucleotides, between l%-50% chemically modified nucleotides, between l%-40% chemically modified nucleotides, between l%-30% chemically modified nucleotides, between l%-20% chemically modified nucleotides, between 1%-15% chemically modified nucleotides, between 1 %- 10% chemically modified nucleotides, between 20%-90% chemically modified nucleotides, between 20%-80% chemically modified nucleotides. In embodiments, a DNA region described herein, or one strand (e.g., the sense strand or the antisense strand) of the DNA region, comprises at least 1% chemically modified nucleotides, at least 5% chemically modified nucleotides; at least 10% chemically modified nucleotides; at least 15% chemically modified nucleotides; at least 20% chemically modified nucleotides; at least 25% chemically modified nucleotides; at least 30% chemically modified nucleotides; at least 40% chemically modified nucleotides; at least 50% chemically modified nucleotides; at least 60% chemically modified nucleotides; at least 70% chemically modified nucleotides; at least 80% chemically modified nucleotides; at least 85% chemically modified nucleotides; at least 90% chemically modified nucleotides; at least 92% chemically modified nucleotides; at least 95% chemically modified nucleotides; at least 97% chemically modified nucleotides. In embodiments, a DNA region described herein, or one strand (e.g., the sense strand or the antisense strand) of the DNA region, comprises chemically modified nucleotides at between 0%-100% of each distinct nucleotide, e.g., 0%-100% chemically modified T nucleotides, 0%-100% chemically modified A nucleotides, 0%-100% chemically modified C nucleotides, and 0%-100% chemically modified G nucleotides for each construct. In embodiments, a DNA region described herein, or one strand (e.g., the sense strand or the antisense strand) of the double-stranded DNA region, comprises chemically modified nucleotides at between 0-100%, 10%-100%, 20%-100%, 30%-100%, 40%-

[0484] 1601838421.1 85 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0485] 100%, 50%-100%, 60%-100%, 10%-50% of each distinct nucleotide, e.g., between 0-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 10%-50% of chemically modified T nucleotides; between 0-100%, 10%-100%, 20%-100%, 30%-100%, 40%- 100%, 50%-100%, 60%-100%, 10%-50% of chemically modified A nucleotides; between 0- 100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 10%-50% of chemically modified C nucleotides; or between 0-100%, 10%- 100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 10%-50% of chemically modified G nucleotides. For example, a DNA region could contain 100% chemically modified T nucleotides, 50% chemically modified A nucleotides, 0% chemically modified C nucleotides, and 25% chemically modified G nucleotides.

[0486] In embodiments, chemically modified nucleotides, e.g., modifications described herein, can be introduced in the DNA regions described herein throughout the entire sequence; within an element of a sequence, e.g., an element described herein; at a 5'- or 3'- end; and / or between the last 10, 8, 6, 5, 4, 3, or 2 nucleotides at the 5’- or 3’- end.

[0487] In some embodiments, a double-stranded DNA region as described herein comprises chemically modified nucleotides on only one strand. In some embodiments, a double-stranded DNA region as described herein comprises chemically modified nucleotides on the antisense strand. In some embodiments, a double-stranded DNA region as described herein comprises chemically modified nucleotides on the sense strand.

[0488] In some embodiments, a double-stranded DNA region as described herein comprises chemically modified nucleotides on both strands. In certain embodiments, both strands comprise chemical modifications at the same positions (e.g., chemically modified nucleotides on one strand are base-paired with chemically modified nucleotides on the opposite strand, and / or non- chemically modified nucleotides on one strand are base-paired with non-chemically modified nucleotides on the opposite strand). In embodiments, the entirety of both strands are composed of chemically modified nucleotides. In other embodiments, the two strands of a double-stranded DNA region as described herein comprise different chemical modification patterns (e.g., one or more chemically modified nucleotides on one strand are base-paired with non-chemically modified nucleotides on the other strand). In embodiments, a double-stranded DNA region as described herein comprises one or more double-stranded regions in which both strands are chemically modified, and / or one or more double-stranded regions in which neither strand is

[0489] 1601838421.1 86 Atorney Docket No.: F2128-7027WO(VL87026-W1) chemically modified. In embodiments, a double-stranded DNA region as described herein comprises one or more double-stranded regions in which one strand is chemically modified and the other is not.

[0490] In embodiments, a double-stranded DNA region as described herein comprises one or more DNA end forms (e.g., exonuclease-resistant DNA end forms, e.g., covalently closed DNA end forms or non-covalently closed DNA end forms, e.g., as described herein) that each comprise one or more chemically-modified nucleotides (e.g., on one or both strands of the DNA end form). In embodiments, a double-stranded DNA region comprises a double-stranded region flanked by non-covalently closed exonuclease-resistant DNA end forms comprising chemically- modified nucleotides, e.g., as described herein.

[0491] In embodiments, a double-stranded DNA region described herein has one or more chemical modification that disrupts the ability of a portion of the double-stranded DNA region to form a double stranded structure, e g., a double-stranded DNA region described herein has one or more chemical modification on a nucleotide present in a region having intramolecular complementarity. In embodiments, a double-stranded DNA region described herein has one or more chemical modification that disrupts base pairing of regions of intramolecular complementarity relative to the unmodified sequence of the double-stranded DNA region. In some embodiments the chemically modified nucleotides used herein have a reduced propensity to base-pair with chemically modified nucleotides compared to the propensity of unmodified nucleotides to base pair with unmodified nucleotides. In some embodiments the chemically modified nucleotides used herein have an increased propensity to base-pair with unmodified nucleotides compared to modified nucleotides.

[0492] Other modifications are also contemplated. For example, ends of a linear doublestranded DNA region described herein can be chemically modified, e.g., to protect them from exonucleases. For example, one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and / or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang, et al. (1987) Proc. Nail. Acad. Sci. USA 84:4959- 4963; Nehls, et at (1996) Science 272:886-889.

[0493] In some embodiments, a chemically modified DNA region described herein exhibits decreased recognition by DNA sensors in a host tissue or subject compared to an unmodified DNA region of the same sequence, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at

[0494] 1601838421.1 87 Atorney Docket No.: F2128-7027WO(VL87026-W1) least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more decreased recognition by DNA sensors in a host tissue or subject compared to an unmodified DNA region of the same sequence. In some embodiments, a chemically modified DNA region described herein exhibits decreased degradation by DNA nucleases compared to an unmodified DNA region of the same sequence, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more decreased degradation by DNA nucleases in a host tissue or subject compared to an unmodified DNA region. In some embodiments, a chemically modified DNA region described herein shows decreased activation of the innate immune system in a target / host tissue or subject compared to an unmodified DNA region of the same sequence, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more decreased activation of the innate immune system in a target / host tissue or subject compared to an unmodified DNA region of the same sequence.

[0495] In some embodiments, a DNA region comprising chemically modified nucleotides described herein exhibits any of the following properties in a target / host tissue or subject compared to a DNA region of the same sequence that does not comprise chemically modified nucleotides (unmodified dsDNA): increased integration of exogenous construct in genome of target cell; increased retention in a target cell through replication; reduced secondary or tertiary structure formation; reduced interaction with innate immune sensors; reduced interaction with nucleases; enhanced stability; enhanced longevity; reduced toxicity; enhanced delivery; increased expression; increased transport across membranes; increased binding to DNA binding moieties such as nuclear DNA binding proteins, transcription factors, chaperones, DNA polymerases. In embodiments, any of the above listed properties is modulated at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more in a target / host tissue or subject compared to an unmodified DNA region of the same sequence.

[0496] Production

[0497] In some aspects, the present disclosure provides methods of reacting PS-DNA (e.g., dsDNA) with a reagent described herein such as a halo-acetamide reagent or maleimide reagent. In some embodiments, the reaction is carried out at a temperature greater than 37 °C, for

[0498] 1601838421.1 88 Atorney Docket No.: F2128-7027WO(VL87026-W1) example 40-50 °C, 50-60 °C, 60-70 °C, 70-80 °C, or 80-90 °C. Without wishing to be bound by theory, in some embodiments a higher temperature promotes denaturation of double stranded DNA, into a form more amenable for reaction. In some embodiments, the reaction is allowed to proceed for at least 1 hour, at least 2 hours at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, or at least 36 hours. In some embodiments, the reaction is allowed to proceed for 1-2 hours, 2-6 hours 6-12 hours, 12-24 hours, 24-48 hours, or 48-36 hours. In some embodiments, the reaction is allowed to reach substantial completion.

[0499] In some aspects, the present disclosure provides methods of making a DNA molecule comprising a DNA region linked to an organic moiety. In some embodiments, the method comprises (a) providing a DNA region covalently linked to a first reactive handle (e.g., click handle), (b) providing an organic moiety covalently linked to a second reactive handle (e.g., click handle) that is capable of reacting with the first handle; (c) contacting (a) with (b) under conditions that allow for reaction of the first reactive handle with the second reactive handle, thereby producing a reactive linker (e.g., click linker) between the DNA region and the organic moiety, thereby making the DNA molecule. In some embodiments, the DNA region is a hemi- modified DNA, as described herein.

[0500] In some embodiments, a second click handle linked to an organic moiety, e.g., an organic moiety described herein, is produced. In some embodiments, a PEG spacer is situated between the organic moiety and the click handle. In some embodiments, the PEG spacer is a PEG2 spacer, optionally wherein PEG2 is a 8-amino-3,6-dioxaoctanoic acid. PEG can contain various numbers of ethylene oxide units, for instance two units in the case of PEG2. In some embodiments, a DNA region comprising a first click handle is contacted with the organic moiety comprising the second click handle under conditions that allow for a click reaction (e.g., strain promoted alkyne-azide cycloaddition), thereby producing a click linker between the DNA region and the organic moiety.

[0501] In some embodiments, the method comprises making or manufacturing a DNA molecule, the method comprising (a) providing a DNA molecule described herein, and (b) determining whether the structure of the DNA molecule matches a reference structure, thereby making or manufacturing the DNA molecule. In some embodiments, the determining of (b) comprises sequencing the DNA molecule, (e.g., sequencing all of or a part of a DNA molecule). In some embodiments, the determining of (b) comprises digesting the DNA molecule with a restriction

[0502] 1601838421.1 89 Atorney Docket No.: F2128-7027WO(VL87026-W1) enzyme. In some embodiments, the structure of the DNA molecule that matches the reference structure is identical to the reference structure. In some embodiments, the structure of the DNA molecule that matches the reference structure has the same sequence as the reference structure. In some embodiments, the structure of the DNA molecule that matches the reference structure has the same length as the reference structure.

[0503] The DNA molecule may be enriched or purified from impurities or byproducts selected from the group consisting of: endotoxin, mononucleotides, chemically modified mononucleotides, single stranded DNA, circular DNA, proteins (e.g., enzymes, e.g., ligases, restriction enzymes), DNA fragments or truncations. In some embodiments, the purified DNA molecule is substantially free of process byproducts and impurities, e.g., process byproducts or impurities described herein.

[0504] In some embodiments, a DNA molecule is formulated with a lipid based carrier, e.g., a lipid nanoparticle (LNP).

[0505] The DNA molecule may be sequenced to confirm the desired, designed sequence. In embodiments, other structural analysis of the DNA molecule (e.g., restriction enzyme analysis) may be performed to confirm or verify its sequence.

[0506] In some aspects, a method of making a DNA molecule described herein comprises providing a PS DNA molecule comprising Formula (a):

[0507] Su"Bs- P-O 11 o Formula (a), wherein B is a nucleobase and Su is a sugar moiety; and providing a halide agent having Formula (bl) wherein m = 0-10; n=0-10; p=l - 10; z=0-4; Li is a peptide bond of -CONH- or -NHCO-;

[0508] L2 is -O-CH2-CH2- or -CH2-CH2-O-; RL, if present, is each independently a reactive linker; X = halo (e.g., I, Br or Cl); and Ri is an organic moiety, in a solution; and

[0509] 1601838421.1 90 Atorney Docket No.: F2128-7027WO(VL87026-W1) reacting the PS DNA molecule and the halide agent to form the DNA molecule.

[0510] Enrichment

[0511] A composition described herein is typically enriched to remove process impurities and / or contaminants. In some embodiments, a composition comprising a DNA molecule described herein is enriched. For instance, in some embodiments, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% by mass of total DNA in the composition may be the DNA molecule. As an example, the composition may also comprise other forms of DNA, e.g., as a process impurity, for instance host cell DNA. As an example, the composition may comprise a contaminant, such as bacterial or viral or fungal agents.

[0512] In some embodiments, a composition described herein (e.g., a composition comprising a DNA molecule, e.g., a pharmaceutical composition comprising a DNA molecule, or a manufacturing intermediate) is free of or is substantially free of one or more process impurity or contaminant, e.g., as described in this section. In some embodiments, a method described herein results in a composition that is free of or is substantially free of one or more process impurity or contaminant, e.g., as described in this section. In some embodiments, a method described herein comprises a step of assaying for one or more process impurity or contaminant, e.g., as described in this section. In some embodiments, the method comprises approving or releasing a batch if the batch is free of or substantially free of the process impurity or contaminant or meets a release criterion for that process impurity or contaminant.

[0513] In some embodiments, the process impurity comprises a nonhuman animal serum (e.g., fetal bovine serum); an enzyme, e.g., a ligase, a polymerase, or a digestive enzyme (e.g., a trypsin, a collagenase, a DNase, a RNase, an exonuclease, or an endonuclease, e.g., a restriction endonuclease); a growth factor; a cytokine; an antibody (e.g., a monoclonal antibody); a bead (e.g., an antibody-coated bead); an antibiotic; a cell culture medium; a component of a cell culture medium; a detergent; a protein, e.g., a host cell protein; an extraneous nucleic acid sequence (e.g., a mononucleotide (e.g., a modified mononucleotide), or a DNA fragment or truncation); helper virus contaminant (e.g., infectious virus, viral DNA, or viral proteins); a solvent; a cellular debris; a cell; a pyrogen; a fungus; or any combination thereof, or a portion of any of the foregoing. In some embodiments, the contaminant was a component introduced

[0514] 1601838421.1 91 Atorney Docket No.: F2128-7027WO(VL87026-W1) during a manufacturing process. In some embodiments, the contaminant comprises a viral protein.

[0515] In some embodiments, the contaminant comprises an agent for transmissible spongiform encephalopathy (TSE). In some embodiments, a test for this contaminant is performed on a composition for which a bovine material was used in manufacturing.

[0516] In some embodiments, the contaminant comprises a zoonotic virus, a porcine circovirus 1, a porcine circovirus 2, or a porcine parvovirus; or any combination thereof, or a portion of any of the foregoing. In some embodiments, a test for this contaminant is performed on a composition for which non-human animal material, e.g., a porcine material, was used in manufacturing.

[0517] In some embodiments, the contaminant comprises a virus or portion thereof, e.g., a human virus; human immunodeficiency virus (HIV); HIV-1; HIV-2; hepatitis B virus (HBV); hepatitis C virus (HCV); human TSE, including Creutzfeldt-Jakob disease (CJD); variant CJD (vCJD); Treponema pallidum (syphilis); human T-lymphotropic virus (HTLV), HTLV-1, HTLV-2; or cytomegalovirus, human herpesvirus (e.g., human herpesvirus -6, -7 or -8 (HHV-6, - 7, or -8)), JC virus, BK virus, Epstein-Barr virus (EBV), human parvovirus B 19, human papillomavirus (HPV); an adenovirus, e.g., adenovirus El; SV40 Large T antigen sequence; HPV E6 or E7 DNA; or any combination thereof, or a portion of any of the foregoing. In some embodiments, a test for this contaminant is performed on a composition for which human donor cells (e.g., leukocyte-rich cells) were used in manufacturing. In some embodiments, a test for this contaminant is performed on a cell bank.

[0518] In some embodiments, the contaminant comprises a microbe or a portion thereof; a bacterium (e.g., a Gram-negative bacterium); mycoplasma; spiroplasma (e.g., when insect cells are used); bacterial toxin (e.g., endotoxin); or an adventitious agent, e.g., an adventitious viral agent or a non-viral adventitious agent, or any combination thereof, or a portion of any of the foregoing. In some embodiments, the contaminant comprises a simian virus, e.g., simian polyomavirus SV40 or simian retrovirus, or any combination thereof, or a portion of any of the foregoing. In some embodiments, the contaminant comprises an arbovirus. In some embodiments, the contaminant comprises a bacteriophage. In some embodiments, a test for this contaminant is performed on a cell bank, e.g., a cell bank of bacterial cells.

[0519] 1601838421.1 92 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0520] In some embodiments, the contaminant or process impurity comprises DNA from a host cell, e.g., wherein the host cell is a non-tumorigenic cell. In some embodiments, the DNA is present at a level of less than 10 ng / dose. In some embodiments, the DNA size is below about 200 nucleotides in length.

[0521] In some embodiments, the contaminant is an endotoxin. In some embodiments, a level of the endotoxin is less than 5 Endotoxin Unit (EU) / kg body weight / hour, e.g., wherein the composition is formulated for parenteral administration. In some embodiments, a level of the endotoxin is less than 0.2 EU / kg body weight / hour, e.g., wherein the composition is formulated for intrathecal administration. In some embodiments, a level of the endotoxin is not more than 2.0 EU / dose / eye, e.g., wherein the composition is formulated for injection or implantation into the eye, or not more than 0.5 EU / mL, e.g., wherein the composition is formulated for intraocular administration.

[0522] In some embodiments, a process impurity comprises an organic solvent, e.g., an aromatic organic solvent, e.g., phenol or chloroform.

[0523] In some embodiments, the contaminant or process impurity is described in Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs) - Guidance for Industry (U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologies Evaluation and Research, January 2020), which is herein incorporated by reference in its entirety.

[0524] In some embodiments, the composition is substantially free of (e.g., is free of) a polymerase. In some embodiments, the composition is substantially free (e.g., is free of) lipids, e.g., LNPs. In some embodiments, the composition is substantially free (e.g., is free of) nanoparticles.

[0525] In some embodiments, the composition is substantially free of (e.g., is free of) agarose. In some embodiments, the composition is substantially free of (e.g., is free of) acrylamide.

[0526] In some embodiments, the composition is substantially free of (e.g., is free of) polypeptides.

[0527] Pharmaceutical compositions

[0528] The present disclosure includes a DNA molecule and related compositions in combination with one or more pharmaceutically acceptable excipients and / or carriers.

[0529] 1601838421.1 93 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0530] Pharmaceutical compositions may optionally comprise one or more additional active substances, e.g., therapeutically and / or prophylactically active substances. Pharmaceutical compositions of the present invention are generally sterile and / or pyrogen-free.

[0531] A DNA molecule described herein may be formulated without a carrier, e.g., the DNA molecule described herein may be administered to a host cell, tissue or subject “naked”. A naked formulation may include pharmaceutical excipients or diluents but lacks a carrier.

[0532] Pharmaceutically acceptable excipients or diluents may comprise an inactive substance that serves as a vehicle or medium for the compositions described herein, such as any one of the inactive ingredients approved by the United States Food and Drug Administration (FDA) and listed in the Inactive Ingredient Database, which is incorporated by reference herein. Nonlimiting examples of pharmaceutically acceptable excipients or diluents include solvents, aqueous solvents, non-aqueous solvents, tonicity agents, dispersion media, cryoprotectants, diluents, suspension aids, surface active agents, isotonic agents, thickening agents, emulsifying agents, preservatives, hyaluronidases, dispersing agents, preservatives, lubricants, granulating agents, disintegrating agents, binding agents, antioxidants, buffering agents (e.g., phosphate buffered saline (PBS)), lubricating agents, oils, and mixtures thereof.

[0533] General considerations in the formulation and / or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).

[0534] Carriers

[0535] A DNA molecule described herein may also be formulated, or included, with a carrier. General considerations of carriers and delivery of pharmaceutical agents may be found, for example, in “Delivery Technologies for Biopharmaceuticals: Peptides, Proteins, Nucleic Acids and Vaccines” (Lene Jorgensen and Hanne Morck Nielson, Eds.) Wiley; 1st edition (December 21, 2009); and Vargason et al. 2021. Nat Biomed Eng 5, 951-967.

[0536] Non-limiting examples of carriers include carbohydrate carriers (e.g., an anhydride- modified phytoglycogen or glycogen-type material, GalNAc), nanoparticles (e.g., a nanoparticle that encapsulates or is covalently linked to the DNA molecule, gold nanoparticles, silica nanoparticles), lipid particles (e.g., liposomes, lipid nanoparticles), cationic carriers (e.g., a cationic lipopolymer or transfection reagent), fusosomes, non-nucleated cells (e.g., ex vivo

[0537] 1601838421.1 94 Atorney Docket No.: F2128-7027WO(VL87026-W1) differentiated reticulocytes), nucleated cells, exosomes, protein carriers (e.g., a protein covalently linked to the DNA molecule), peptides (e.g., cell-penetrating peptides), materials (e.g., graphene oxide), single pure lipids (e.g., cholesterol), DNA origami (e.g., DNA tetrahedron).

[0538] In one embodiment, the DNA molecules, compositions, constructs and systems described herein can be formulated in liposomes or other similar vesicles. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes may be anionic, neutral or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:l 0.1155 / 2011 / 469679 for review).

[0539] Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparation of multilamellar vesicle lipids are known in the art (see for example U.S. Pat. No. 6,693,086, the teachings of which relating to multilamellar vesicle lipid preparation are incorporated herein by reference). Although vesicle formation can be spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: l 0.1155 / 2011 / 469679 for review). Extruded lipids can be prepared by extruding through filters of decreasing size, as described in Templeton et al., Nature Biotech, 15:647-652, 1997, the teachings of which relating to extruded lipid preparation are incorporated herein by reference.

[0540] Exosomes can also be used as drug delivery vehicles for the compositions and systems described herein. For a review, see Ha et al. July 2016. Acta Pharmaceutica Sinica B. Volume 6, Issue 4, Pages 287-296; doi.org / 10.1016 / j.apsb.2016.02.001.

[0541] Ex vivo differentiated red blood cells can also be used as a carrier for an agent (e.g., a DNA molecule) described herein. See, e.g., WO2015073587; WO2017123646; WO2017123644; W02018102740; WO2016183482; W02015153102; WO2018151829; W02018009838; Shi et al. 2014. Proc Natl Acad Sci USA. 111(28): 10131-10136; US Patent

[0542] 1601838421.1 95 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0543] 9,644,180; Huang et al. 2017. Nature Communications 8: 423; Shi et al. 2014. Proc Natl Acad Sci USA. 111(28): 10131-10136.

[0544] Fusosome compositions, e.g., as described in WO2018208728, can also be used as carriers to deliver the DNA molecules described herein.

[0545] Lipid Nanoformiilalions Lipid-based carriers

[0546] In some embodiments, compounds, e.g., DNA molecules, described herein are formulated into a lipid-based carrier (or lipid nanoformulation). In some embodiments, the lipid- based carrier (or lipid nanoformulation) is a liposome or a lipid nanoparticle (LNP). In one embodiment, the lipid-based carrier is an LNP.

[0547] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises a cationic lipid (e.g., an ionizable lipid), a non-cationic lipid (e.g., phospholipid), a structural lipid (e.g., cholesterol), and a PEG-modified lipid. In some embodiments, the lipid-based carrier (or lipid nanoformulation) contains one or more compounds described herein, or a pharmaceutically acceptable salt thereof.

[0548] As described herein, suitable compounds to be used in the lipid-based carrier (or lipid nanoformulation) include all the isomers and isotopes of the compounds described above, as well as all the pharmaceutically acceptable salts, solvates, or hydrates thereof, and all crystal forms, crystal form mixtures, and anhydrides or hydrates.

[0549] In addition to one or more compounds described herein, the lipid-based carrier (or lipid nanoformulation) may further include a second lipid. In some embodiments, the second lipid is a cationic lipid, a non-cationic e.g., neutral, anionic, or zwitterionic) lipid, or an ionizable lipid.

[0550] One or more naturally occurring and / or synthetic lipid compounds may be used in the preparation of the lipid-based carrier (or lipid nanoformulation).

[0551] The lipid-based carrier (or lipid nanoformulation) may contain positively charged (cationic) lipids, neutral lipids, negatively charged (anionic) lipids, or a combination thereof.

[0552] Cationic Lipids (Positively Charged) and Ionizable Lipids

[0553] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises one or more cationic lipids, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated. In some

[0554] 1601838421.1 96 Atorney Docket No.: F2128-7027WO(VL87026-W1) embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions.

[0555] Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Examples of positively charged (cationic) lipids include, but are not limited to, N,N'- dimethyl-N,N'-dioctacyl ammonium bromide (DDAB) and chloride DDAC), N-(l-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 3P-[N-(N',N'- dimethylaminoethyl)carbamoyl) cholesterol (DC-chol), l,2-dioleoyloxy-3-[trimethylammonio]- propane (DOTAP), l,2-dioctadecyloxy-3-[trimethylammonio]-propane (DSTAP), and 1,2- dioleoyloxypropyl-3-dimethyl-hydroxy ethyl ammonium chloride (DORI), N, N-di oleyl -N,N- dimethylammonium chloride (DODAC), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), l,2-Dioleoyl-3-Dimethylammonium-propane (DODAP), l,2-Dioleoylcarbamyl-3- Dimethylammonium-propane (DOCDAP), l,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP), 3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12- octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3- dimethyl-l-(cis, cis-9',12'-octadecadienoxy)propane (CpLin DMA), N,N-Dimethyl-3,4- dioleyloxybenzylamine (DMOBA), and the cationic lipids described in e.g. Martin et al., Current Pharmaceutical Design, pages 1-394, which is herein incorporated by reference in its entirety. In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises more than one cationic lipid.

[0556] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises a cationic lipid having an effective pKa over 6.0. In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa) than the first cationic lipid.

[0557] In some embodiments, cationic lipids that can be used in the lipid-based carrier (or lipid nanoformulation) include, for example those described in Table 4 of WO 2019 / 217941, which is incorporated by reference.

[0558] In some embodiments, the cationic lipid is an ionizable lipid (e.g., a lipid that is protonated at low pH, but that remains neutral at physiological pH). In some embodiments, the lipid-based carrier (or lipid nanoformulation) may comprise one or more additional ionizable lipids, different than the ionizable lipids described herein. Exemplary ionizable lipids include, but are not limited to,

[0559] 1601838421.1 97 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0560] 1601838421.1 98 Atorney Docket No.: F2128-7027WO(VL87026-W1) (see WO 2017 / 004143A1, which is incorporated herein by reference in its entirety).

[0561] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more compounds described by WO 2021 / 113777 (e.g., a lipid of Formula (3) such as a lipid of Table 3 of WO 2021 / 113777), which is incorporated herein by reference in its entirety.

[0562] In one embodiment, the ionizable lipid is a lipid disclosed in Hou, X., et al. Nat Rev Mater 6, 1078-1094 (2021). doi.org / 10.1038 / s41578-021-00358-0 (e.g., L319, C12-200, and DLin-MC3-DMA), (which is incorporated by reference herein in its entirety).

[0563] Examples of other ionizable lipids that can be used in lipid-based carrier (or lipid nanoformulation) include, without limitation, one or more of the following formulas: X of US 2016 / 031 1759; I of US 201503761 1 or in US 2016 / 0376224; Compound 5 or Compound 6 in US 2016 / 0376224; I, IA, or II of US 9,867,888; I, II or III of US 2016 / 0151284; I, IA, II, or IIA of US 2017 / 0210967; I-c of US 2015 / 0140070; A of US 2013 / 0178541; I of US 2013 / 0303587 or US 2013 / 0123338; I of US 2015 / 0141678; II, III, IV, or V of US 2015 / 0239926; I of US 2017 / 0119904; I or II of WO 2017 / 117528; A of US 2012 / 0149894; A of US 2015 / 0057373; A of WO 2013 / 116126; A of US 2013 / 0090372; A of US 2013 / 0274523; A of US 2013 / 0274504; A of US 2013 / 0053572; A of WO 2013 / 016058; A of WO 2012 / 162210; I of US 2008 / 042973;

[0564] I, II, III, or IV of US 2012 / 01287670; I or II of US 2014 / 0200257; I, II, or III of US 2015 / 0203446; I or III of US 2015 / 0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or IILXXIV of US 2014 / 0308304; of US 2013 / 0338210; I, II, III, or IV of WO 2009 / 132131; A of US

[0565] 1601838421.1 99 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0566] 2012 / 01011478; I or XXXV of US 2012 / 0027796; XIV or XVII of US 2012 / 0058144; of US 2013 / 0323269; I of US 2011 / 0117125; I, II, or III of US 2011 / 0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US 2012 / 0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US 2011 / 0076335; I or II of US 2006 / 008378; I of WO2015 / 074085 (e.g., ATX-002); I of US 2013 / 0123338; I or X-A-Y-Z of US 2015 / 0064242; XVI, XVII, or XVIII of US 2013 / 0022649; I, II, or III of US 2013 / 0116307; I, II, or III of US 2013 / 0116307; I or II of US 2010 / 0062967; I-X of US 2013 / 0189351; I of US 2014 / 0039032; V of US 2018 / 0028664; I of US 2016 / 0317458; I of US 2013 / 0195920; 5, 6, or 10 of US 10,221,127; III-3 of WO 2018 / 081480; 1-5 or 1-8 of WO 2020 / 081938; I of WO 2015 / 199952 (e.g, compound 6 or 22) and Table 1 therein; 18 or 25 of US 9,867,888; A of US 2019 / 0136231; II of WO 2020 / 219876;

[0567] 1 of US 2012 / 0027803; OF-02 of US 2019 / 0240349; 23 of US 10,086,013; CKK-E12 / A6 of Miao et al (2020); C12-200 of WO 2010 / 053572; 7C1 of Dahlman et al (2017); 304-013 or 503- 013 of Whitehead et al; TS-P4C2 of U S9, 708, 628; I of WO 2020 / 106946; I of WO 2020 / 106946; (1), (2), (3), or (4) of WO 2021 / 113777; and any one of Tables 1-16 of WO 2021 / 113777, all of which are incorporated herein by reference in their entirety.

[0568] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further includes biodegradable ionizable lipids, for instance, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3- ((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate). See, e.g, lipids of WO 2019 / 067992, WO 2017 / 173054, WO 2015 / 095340, and WO 2014 / 136086, which are incorporated herein by reference in their entirety.

[0569] Non-Cationic Lipids (e.g., Phospholipids)

[0570] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more non-cationic lipids. In some embodiments, the non-cationic lipid is a phospholipid. In some embodiments, the non-cationic lipid is a phospholipid substitute or replacement. In some embodiments, the non-cationic lipid is a negatively charged (anionic) lipid.

[0571] Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine

[0572] 1601838421.1 100 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0573] (DOPC), dipalmitoylphosphatidylcholine (DPPC), di oleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), di oleoyl-phosphatidyl ethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, l-stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidyl serine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoylphosphatidylethanolamine (DEPE), 1,2-dilauroyl- sn-glycero-3 -phosphocholine (DLPC), Sodium 1,2- ditetradecanoyl-sn-glycero-3 -phosphate (DMPA), phosphatidylcholine (lecithin), phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl. Additional exemplary lipids, in certain embodiments, include, without limitation, those described in Kim et al. (2020) dx.doi.org / 10.1021 / acs.nanolett.0c01386, which is incorporated herein by reference. Such lipids include, in some embodiments, plant lipids found to improve liver transfection with mRNA (e.g, DGTS).

[0574] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may comprise a combination of distearoylphosphatidylcholine / cholesterol, dipalmitoylphosphatidylcholine / cholesterol, dimyrystoylphosphatidylcholine / cholesterol, 1 ,2- Dioleoyl-sn-glycero-3-phosphocholine (DOPC) / cholesterol, or egg sphingomyelin / cholesterol.

[0575] Other examples of suitable non-cationic lipids include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl

[0576] 1601838421.1 101 Atorney Docket No.: F2128-7027WO(VL87026-W1) palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like. Other noncationic lipids are described in WO 2017 / 099823 or US 2018 / 0028664, which are incorporated herein by reference in their entirety.

[0577] In one embodiment, the lipid-based carrier (or lipid nanoformulation) further comprises one or more non-cationic lipid that is oleic acid or a compound of Formula I, II, or IV of US 2018 / 0028664, which is incorporated herein by reference in its entirety.

[0578] The non-cationic lipid content can be, for example, 0-30% (mol) of the total lipid components present. In some embodiments, the non-cationic lipid content is 5-20% (mol) or 10- 15% (mol) of the total lipid components present.

[0579] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a neutral lipid, and the molar ratio of an ionizable lipid to a neutral lipid ranges from about 2: 1 to about 8: 1 (e.g., about 2: 1, 3: 1, 4:1, 5: 1, 6: 1, 7: 1, or 8: 1).

[0580] In some embodiments, the lipid-based carrier (or lipid nanoformulation) does not include any phospholipids.

[0581] In some embodiments, the lipid-based carrier (or lipid nanoformulation) can further include one or more phospholipids, and optionally one or more additional molecules of similar molecular shape and dimensions having both a hydrophobic moiety and a hydrophilic moiety (e.g, cholesterol).

[0582] Structural Lipids

[0583] The lipid-based carrier (or lipid nanoformulation) described herein may further comprise one or more structural lipids. As used herein, the term “structural lipid” refers to sterols (e.g., cholesterol) and also to lipids containing sterol moi eties.

[0584] Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol or cholesterol derivative, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural

[0585] 1601838421.1 102 Atorney Docket No.: F2128-7027WO(VL87026-W1) lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha-tocopherol.

[0586] In some embodiments, structural lipids may be incorporated into the lipid-based carrier at molar ratios ranging from about 0.1 to 1.0 (cholesterol phospholipid).

[0587] In some embodiments, sterols, when present, can include one or more of cholesterol or cholesterol derivatives, such as those described in WO 2009 / 127060 or US 2010 / 0130588, which are incorporated herein by reference in their entirety. Additional exemplary sterols include phytosterols, including those described in Eygeris et al. (2020), Nano Lett. 2020;20(6):4543- 4549, incorporated herein by reference.

[0588] In some embodiments, the structural lipid is a cholesterol derivative. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 53- coprostanol, cholesteryl -(2’ -hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy)-butyl ether, and 6- ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p- cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue, e.g., cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in WO 2009 / 127060 and US 2010 / 0130588, each of which is incorporated herein by reference in its entirety.

[0589] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises sterol in an amount of 0-50 mol% (e.g., 0-10 mol %, 10-20 mol %, 20-50 mol%, 20- 30 mol %, 30-40 mol %, or 40-50 mol %) of the total lipid components.

[0590] Polymers and Polyethylene Glycol (PEG) - Lipids

[0591] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may include one or more polymers or co-polymers, e.g., poly(lactic-co-glycolic acid) (PF AG) nanoparticles.

[0592] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may include one or more polyethylene glycol (PEG) lipid. Examples of useful PEG-lipids include, but are not limited to, l,2-Diacyl-sn-Glycero-3- Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)- 350] (mPEG 350 PE); 1,2-Diacyl-sn- Glycero-3-Phosphoethanolamine-N- [Methoxy(Polyethylene glycol)-550] (mPEG 550 PE); 1,2- Diacyl-sn-Glycero-3- Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-750] (mPEG 750 PE); 1,2-Diacyl-sn- Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-1000] (mPEG 1000 PE);

[0593] 1601838421.1 103 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0594] 1 ,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000] (mPEG 2000 PE); l,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N- [Methoxy(Poly ethylene glycol)- 3000] (mPEG 3000 PE); l,2-Diacyl-sn-Glycero-3- Phosphoethanolamine-N- [Methoxy (Poly ethylene glycol)-5000] (mPEG 5000 PE); N-Acyl- Sphingosine- 1- [Succinyl(Methoxy Polyethylene Glycol) 750] (mPEG 750 Ceramide); N-Acyl- Sphingosine- 1- [Succinyl(Methoxy Polyethylene Glycol) 2000] (mPEG 2000 Ceramide); and N- Acyl- Sphingosine-l-[Succinyl(Methoxy Polyethylene Glycol) 5000] (mPEG 5000 Ceramide). In some embodiments, the PEG lipid is a polyethyleneglycol-diacylglycerol (i.e., polyethyleneglycol di acylglycerol (PEG-DAG), PEG-chol esterol, or PEG-DMB) conjugate.

[0595] In some embodiments, the lipid-based carrier (or nanoformulation) includes one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of WO 2019 / 217941, which is incorporated herein by reference in its entirety). In some embodiments, the one or more conjugated lipids is formulated with one or more ionic lipids (e.g., non-cationic lipid such as a neutral or anionic, or zwitterionic lipid); and one or more sterols (e.g, cholesterol).

[0596] The PEG conjugate can comprise a PEG-dilaurylglycerol (C12), a PEG- dimyristylglycerol (C14), a PEG-dipalmitoylglycerol (C16), a PEG-disterylglycerol (C18), PEG- dilaurylglycamide (C 12), PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C 16), and PEG-disterylglycamide (Cl 8).

[0597] In some embodiments, conjugated lipids, when present, can include one or more of PEG- diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypolyethylene glycol 2000)- 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those described in Table 2 of WO 2019 / 051289 (which is herein incorporated by reference in its entirety), and combinations of the foregoing.

[0598] Additional exemplary PEG-lipid conjugates are described, for example, in US 5,885,613, US 6,287,591, US 2003 / 0077829, US 2003 / 0077829, US 2005 / 0175682, US 2008 / 0020058, US 2011 / 0117125, US 2010 / 0130588, US 2016 / 0376224, US 2017 / 0119904, US 2018 / 0028664, and

[0599] 1601838421.1 104 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0600] WO 2017 / 099823, all of which are incorporated herein by reference in their entirety.

[0601] In some embodiments, the PEG-lipid is a compound of Formula III, III-a-I, III-a-2, Ill-b- 1, III-b-2, or V of US 2018 / 0028664, which is incorporated herein by reference in its entirety. In some embodiments, the PEG-lipid is of Formula II of US 2015 / 0376115 or US 2016 / 0376224, both of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. In some embodiments, the PEG-lipid includes one of the following:

[0602] In some embodiments, lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.

[0603] Exemplary conjugated lipids, e.g., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids, include those described in Table 2 of WO 2019 / 051289A9, which is incorporated herein by reference in its entirety.

[0604] 1601838421.1 105 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0605] In some embodiments, the conjugated lipid (e.g, the PEGylated lipid) can be present in an amount of 0-20 mol% of the total lipid components present in the lipid-based carrier (or lipid nanoformulation). In some embodiments, the conjugated lipid (e.g., the PEGylated lipid) content is 0.5-10 mol% or 2-5 mol% of the total lipid components.

[0606] When needed, the lipid-based carrier (or lipid nanoformulation) described herein may be coated with a polymer layer to enhance stability in vivo (e.g., sterically stabilized LNPs).

[0607] Examples of suitable polymers include, but are not limited to, poly(ethylene glycol), which may form a hydrophilic surface layer that improves the circulation half-life of liposomes and enhances the amount of lipid nanoformulations (e.g., liposomes or LNPs) that reach therapeutic targets. See, e.g., Working et al. J Pharmacol Exp Ther, 289: 1128-1133 (1999); Gabizon et al., J Controlled Release 53: 275-279 (1998); Adlakha Hutcheon et al., Nat Biotechnol 17: 775-779 (1999); and Koning et al., Biochim Biophys Acta 1420: 153-167 (1999), which are incorporated herein by reference in their entirety.

[0608] Percentages of Lipid Nanoformulation Components

[0609] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises one of more of the compounds described herein, optionally a non-cationic lipid (e.g., a phospholipid), a sterol, a neutral lipid, and optionally conjugated lipid (e.g., a PEGylated lipid) that inhibits aggregation of particles. In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a payload (e g., a DNA molecule described herein). The amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, the ionizable lipid including the lipid compounds described herein is present in an amount from about 20 mol% to about 100 mol% (e.g., 20-90 mol%, 20-80 mol%, 20-70 mol%, 25-100 mol%, 30-70 mol%, 30-60 mol%, 30-40 mol%, 40-50 mol%, or 50- 90 mol%) of the total lipid components; a non-cationic lipid (e.g., phospholipid) is present in an amount from about 0 mol% to about 50 mol% (e.g., 0-40 mol%, 0-30 mol%, 5-50 mol%, 5-40 mol%, 5-30 mol%, or 5-10 mol%) of the total lipid components, a conjugated lipid (e.g., a PEGylated lipid) in an amount from about 0.5 mol% to about 20 mol% (e.g., 1-10 mol% or 5- 10%) of the total lipid components, and a sterol in an amount from about 0 mol % to about 60 mol% (e.g., 0-50 mol%, 10-60 mol%, 10-50 mol%, 15-60 mol%, 15-50 mol%, 20-50 mol%, 20- 40 mol%) of the total lipid components, provided that the total mol% of the lipid component

[0610] 1601838421.1 106 Atorney Docket No.: F2128-7027WO(VL87026-W1) does not exceed 100%.

[0611] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises about 25-100 mol% of the ionizable lipid including the lipid compounds described herein, about 0-50 mol% phospholipid, about 0-50 mol% sterol, and about 0-10 mol% PEGylated lipid.

[0612] In some embodiments, the lipid-based carrier comprises a payload (e.g., a DNA molecule described herein, etc.) that is formulated in a lipid nanoparticle, wherein the lipid nanoparticle comprises about 25-100 mol% of the ionizable lipid including the lipid compounds described herein, about 0-50 mol% phospholipid, about 0-50 mol% sterol, and about 0-10 mol% PEGylated lipid. In some embodiments, the encapsulation efficiency of the payload may be at least 70%.

[0613] In one embodiment, the lipid-based carrier (or lipid nanoformulation) comprises about 25-100 mol% of the ionizable lipid including the lipid compounds described herein; about 0-40 mol% phospholipid e.g., DSPC), about 0-50 mol% sterol e.g., cholesterol), and about 0-10 mol% PEGylated lipid.

[0614] In some embodiments, the lipid-based carrier comprises a payload (e.g., a DNA molecule described herein) that is formulated in a lipid nanoparticle, wherein the lipid nanoparticle comprises about 25-100 mol% of the ionizable lipid including the lipid compounds described herein; about 0-40 mol% phospholipid (e.g., DSPC), about 0-50 mol% sterol (e.g., cholesterol), and about 0-10 mol% PEGylated lipid. In some embodiments, the encapsulation efficiency of the payload may be at least 70%.

[0615] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises about 30-60 mol% (e.g., about 35-55 mol%, or about 40-50 mol%) of the ionizable lipid including the lipid compounds described herein, about 0-30 mol% (e.g., 5-25 mol%, or 10-20 mol%) phospholipid, about 15-50 mol% (e.g., 18.5-48.5 mol%, or 30-40 mol%) sterol, and about 0-10 mol% (e.g., 1-5 mol%, or 1.5-2.5 mol%) PEGylated lipid.

[0616] In some embodiments, the lipid-based carrier comprises a payload (e.g., a DNA molecule described herein) that is formulated in a lipid nanoparticle, wherein the lipid nanoparticle comprises about 30-60 mol% (e.g., about 35-55 mol%, or about 40-50 mol%) of the ionizable lipid including the lipid compounds described herein, about 0-30 mol% (e.g., 5-25 mol%, or 10- 20 mol%) phospholipid, about 15-50 mol% (e.g., 18.5-48.5 mol%, or 30-40 mol%) sterol, and about 0-10 mol% (e.g., 1-5 mol%, or 1.5-2.5 mol%) PEGylated lipid. In some embodiments, the

[0617] 1601838421.1 107 Atorney Docket No.: F2128-7027WO(VL87026-W1) encapsulation efficiency of the payload may be at least 70%.

[0618] In some embodiments, molar ratios of ionizable lipid / sterol / phospholipid (or another structural lipid) / PEG-lipid / additional components is varied in the following ranges: ionizable lipid (25-100%); phospholipid (DSPC) (0-40%); sterol (0-50%); and PEG lipid (0-5%).

[0619] In some embodiments, the lipid-based carrier comprises a payload (e.g., a DNA molecule described herein) that is formulated in a lipid nanoparticle, wherein the lipid nanoparticle comprises molar ratios of ionizable lipid / sterol / phospholipid (or another structural lipid) / PEG- lipid / additional components in the following ranges: ionizable lipid (25-100%); phospholipid (DSPC) (0-40%); sterol (0-50%); and PEG lipid (0-5%). In some embodiments, the encapsulation efficiency of the payload may be at least 70%.

[0620] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises, by mol% or wt% of the total lipid components, 50-75% ionizable lipid (including the lipid compound as described herein), 20-40% sterol (e.g., cholesterol or derivative), 0 to 10% non- cationic-lipid, and 1-10% conjugated lipid (e.g., the PEGylated lipid).

[0621] In some embodiments, the lipid-based carrier comprises a payload (e.g., a DNA molecule described in) that is formulated in a lipid nanoparticle, wherein the lipid nanoparticle comprises, by mol% or wt% of the total lipid components, 50-75% ionizable lipid (including the lipid compound as described herein), 20-40% sterol (e.g., cholesterol or derivative), 0 to 10% non- cationic-lipid, and 1-10% conjugated lipid (e.g., the PEGylated lipid). In some embodiments, the encapsulation efficiency of the payload may be at least 70%.

[0622] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises (i) a DNA molecule; (ii) a cationic lipid comprising from 50 mol% to 65 mol% of the total lipid present in the lipid-based carrier; (iii) a non-cationic lipid comprising a mixture of a phospholipid and a cholesterol derivative thereof, wherein the phospholipid comprises from 3 mol% to 15 mol% of the total lipid present in the lipid-based carrier and the cholesterol or derivative thereof comprises from 30 mol% to 40 mol% of the total lipid present in the lipid- based carrier; and (iv) a conjugated lipid comprising 0.5 mol% to 2 mol% of the total lipid present in the particle.

[0623] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises (i) a DNA molecule; (ii) a cationic lipid comprising from 50 mol % to 85 mol % of the total lipid present in the lipid-based carrier; (iii) a non-cationic lipid comprising from 13 mol % to 49.5 mol

[0624] 1601838421.1 108 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0625] % of the total lipid present in the lipid-based carrier; and (d) a conjugated lipid comprising from 0.5 mol % to 2 mol % of the total lipid present in the lipid-based carrier.

[0626] In some embodiments, the phospholipid component in the mixture may be present from 2 mol% to 20 mol%, from 2 mol% to 15 mol%, from 2 mol% to 12 mol%, from 4 mol% to 15 mol%, from 4 mol% to 10 mol%, from 5 mol% to 10 mol%, (or any fraction of these ranges) of the total lipid components. In some embodiments, the lipid-based carrier (or lipid nanoformulation) is phospholipid-free.

[0627] In some embodiments, the sterol component (e.g. cholesterol or derivative) in the mixture may comprise from 25 mol% to 45 mol%, from 25 mol% to 40 mol%, from 25 mol% to 35 mol%, from 25 mol% to 30 mol%, from 30 mol% to 45 mol%, from 30 mol% to 40 mol%, from 30 mol% to 35 mol%, from 35 mol% to 40 mol%, from 27 mol% to 37 mol%, or from 27 mol% to 35 mol% (or any fraction of these ranges) of the total lipid components.

[0628] In some embodiments, the non-ionizable lipid components in the lipid-based carrier (or lipid nanoformulation) may be present from 5 mol% to 90 mol%, from 10 mol% to 85 mol%, or from 20 mol% to 80 mol% (or any fraction of these ranges) of the total lipid components.

[0629] The ratio of total lipid components to the payload (e.g., an encapsulated therapeutic agent such as a DNA molecule) can be varied as desired. For example, the total lipid components to the payload (mass or weight) ratio can be from about 10:1 to about 30: 1. In some embodiments, the total lipid components to the payload ratio (mass / mass ratio; w / w ratio) can be in the range of from about 1 : 1 to about 25:1, from about 10:1 to about 14: 1, from about 3: 1 to about 15: 1, from about 4 : 1 to about 10: 1, from about 5 : 1 to about 9: 1, or about 6 : 1 to about 9: 1. The amounts of total lipid components and the payload can be adjusted to provide a desired N / P ratio, for example, N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or higher. Generally, the lipid-based carrier (or lipid nanoformulation’s) overall lipid content can range from about 5 mg / ml to about 30 mg / mL. Nitrogen: phosphate ratios (N:P ratio) is evaluated at values between 0.1 and 100.

[0630] The efficiency of encapsulation of a payload such as a DNA molecule, describes the amount of the DNA molecule that is encapsulated or otherwise associated with a lipid nanoformulation (e.g., liposome or LNP) after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., at least 70%, 80%, 90%, 95%, or close to 100%). The encapsulation efficiency may be measured, for example, by comparing the

[0631] 1601838421.1 109 Atorney Docket No.: F2128-7027WO(VL87026-W1) amount of DNA molecule in a solution containing the liposome or LNP before and after breaking up the liposome or LNP with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of DNA molecule in a solution. Fluorescence may be used to measure the amount of DNA molecule in a solution. For the lipid- based carrier (or lipid nanoformulation) described herein, the encapsulation efficiency of a DNA molecule may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 70%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0632] Route of administration

[0633] A DNA molecule described herein is introduced into a cell, tissue or subject by any suitable route.

[0634] Administration to a target cell or tissue (e.g., ex vivo) may be by methods known in the art such as transfection, e.g., transient or stable transfection using reagents (e.g., liposomal, calcium phosphate) or physical means (e.g., electroporation, gene gun, microinjection, microfluidic fluid shear, cell squeezing). Other methods are described, e.g., in Rad et al. 2021. Adv. Mater. 33:2005363, which is incorporated herein by reference.

[0635] Administration to a subject, e.g., a mammal, e.g., a human subject, may be by parenteral (e g., intravenous, intramuscular, intraperitoneal, subcutaneous, or intracranial) route; by topical administration, transdermal administration or transcutaneous administration. Other suitable routes include oral, rectal, transmucosal, intranasal, inhalation (e.g., via an aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intraendothelial, in utero (or in ovo), intrapleural, intracerebral, intraarticular, topical, intralymphatic. Also included is direct tissue or organ injection (e.g., to liver, eye, skeletal muscle, cardiac muscle, diaphragm, muscle or brain).

[0636] Applications

[0637] The DNA molecule described herein can be used in therapeutic or health applications for a subject, e.g., a human or non-human animal. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are

[0638] 1601838421.1 110 Atorney Docket No.: F2128-7027WO(VL87026-W1) suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal. The subject can be any animal, e.g., a mammal, e.g., a human or non-human mammal. In embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In embodiments, the subject is a human. In embodiments, the method subject is a non-human mammal. In embodiments, the subject is a non-human mammal is such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit). In embodiments, the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In embodiments, the subject is an invertebrate such as an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusk.

[0639] In some embodiments, a DNA molecule described herein is provided at a dose of about 0.1-100 mg / kg ofDNA.

[0640] In some embodiments, a DNA molecule described herein imparts a biological effect of the effector, e.g., expression of a therapeutic polypeptide, on a host cell, tissue or subject over a time period of at least 2, 3, 4, 5, 6 days or a week; at least 8, 9, 10, 12, 14 days or two weeks; at least 16, 18, 20 days or 3 weeks; at least 22, 24, 25, 27, 28 days or a month; at least 2 months, 3 months, 4 months, 5 months, 6 months or more; between one week and 6 months, between 1 month to 6 months, between 3 months to 6 months.

[0641] In some embodiments, a DNA molecule described herein imparts a biological effect of the effector, e.g., expression of a therapeutic polypeptide, on a host cell, tissue or subject over a time period of at least 1 cell divisions of the host cell.

[0642] In some embodiments, a DNA molecule described herein can be used to deliver an effector, e.g., an effector described herein, to a cell, tissue or subject.

[0643] In some embodiments, a DNA molecule described herein can be used to modulate (e.g., increase or decrease) a biological parameter in a cell, tissue or subject. The biological parameter may be an increase or decrease in gene expression of a subject gene in a target cell, tissue or subject. In some embodiments, a DNA molecule described herein increases or decreases a biological activity in a target cell, wherein the biological activity comprises cell growth, cell

[0644] 1601838421.1 111 Atorney Docket No.: F2128-7027WO(VL87026-W1) metabolism, cell signaling, cell movement, specialization, interactions, division, transport, homeostasis, osmosis, or diffusion. In some embodiments, the cell is an animal cell, e.g., a mammalian cell, e.g., a human cell.

[0645] In some embodiments, a DNA molecule described herein can be used to treat a cell, tissue or subject in need thereof by administering the DNA molecule described herein to such cell, tissue or subject.

[0646] In some embodiments, the DNA molecule delivers an effector to a cell.

[0647] EXAMPLES

[0648] Example 1: Modification of PS DNA with halo-acetamide-containing molecules This example demonstrates the post-synthetic modification of PS DNA (dsDNA or ssDNA) with halo-acetamide-containing molecules.

[0649] Halo-acetamide reagents (iodoacetamide azide or iodoacetamide-PEG3-azide) were added to a solution of PS DNA construct, as shown in FIG. 1A for ssDNA and FIG. 2A for dsDNA, (10: 1 molar ratio of reagent / phosphorothioate handle) in 200 mM HEPES buffer at pH 7.3. For dsDNA, the PS DNA construct was 2328 bp in length, encoded eGFP under the control of the EFla promoter, and comprised a PS as part of every T nucleotide (901 T nucleotides in total). For ssDNA, the PS DNA construct was 48 nucleotides in length, consisted of the sequence CACACGTCCCGAGGTCTCAGGGGGCCATAGAGCCCACCGCATCCCCAG (SEQ ID NO: 114), and comprised a PS as part of the first 8 nucleotides. The reaction had 0.006 nmol of DNA for a total of 5.6 nmol of PS nucleotides. 56.0 nmol of the haloacetamide reagent was also present.

[0650] In the reaction, DMSO was <5%, for example 1-2% (v / v) may be used. The reaction was incubated in a thermocycler at 80C for 1 hr. The reaction was then diluted 3 -fold in nuclease-free water and enriched using Sephadex ProbeQuant resins in spin column. Modified PS DNA was then eluted in RNase free water in the same volume as the initial reaction. Successful modification was observed and characterized by gel electrophoresis (FIG. IB, FIG. 2B).

[0651] Example 2: Modification of PS DNA with maleimide-containing molecules

[0652] This example demonstrates the post-synthetic modification of phosphorothioate-modified (PS) DNA with maleimide-containing molecules.

[0653] 1601838421.1 112 Atorney Docket No.: F2128-7027WO(VL87026-W1)

[0654] Maleimide-sulfo-Cy3 was added to a solution of PS DNA construct, as shown in FIG. 3 A, (10: 1 molar ratio of reagent / phosphorothioate handle) in 200 mM HEPES buffer at pH 7.3. The PS DNA construct was the same construct as described in Example 1. The reaction had 0.006 nmol of DNA for a total of 5.6 nmol of PS nucleotides. 56.0 nmol of maleimide-sulfo-Cy3 was also present.

[0655] In the reaction, DMSO was <5%, for example 1-2% (v / v) may be used. The reaction was incubated in a thermocycler at 80C for 1 hr. The reaction was then diluted 3 -fold in nuclease-free water and enriched using Sephadex ProbeQuant resins in spin column. Modified PS DNA was then eluted in RNase free water in the same volume as the initial reaction. Successful modification was observed and characterized by gel electrophoresis (as shown in FIG. 2B, FIG. 3B, and FIG. 4)

[0656] Example 3: Conjugation ofCy3 to modified PS DNA using click chemistr

[0657] This example demonstrates the conjugation of an organic moiety, in this case a Cy3 dye, to PS DNA that had been successfully modified with iodoacetamide azide or iodoacetamide- PEG3-azide such that the PS DNA now has an azide click handle attached. The DNA having the click handle was produced according to Example 1. This DNA was reacted with DBCO-Cy3. Reagents were added in 200 mM HEPES buffer at pH 7.3 to a final concentration of 70 ng / pL of modified PS DNA and DBCO-Cy3 was added in a 1 :50 molar ratio per PS handle. The reaction mixture contained modified PS DNA and DBCO-Cy3.

[0658] The reaction was incubated in a thermocycler at 80 °C for 1 hr. The reaction was then diluted two-fold in nuclease-free water and enriched using ProbeQuant G-50 columns (Cytiva). Conjugated DNA was then eluted in RNAse free water in the same reaction volume. Successful conjugation was observed and characterized by gel electrophoresis, as shown in FIG. 3B.

[0659] Example 4: Conjugation to modified PS DNA

[0660] This example describes the conjugation of organic moieties to modified PS DNA. Organic moieties include, but are not limited to, small molecules, dyes, polypeptides, nucleic acids, lipids, and glycans. Organic moieties are prepared for conjugation reactions by first

[0661] 1601838421.1 113 Atorney Docket No.: F2128-7027WO(VL87026-W1) functionalizing them with a reactive handle (e.g., dibenzocyclooctyne, azide, maleimide, thiol, amine, and NHS ester).

[0662] The DNA starting material is PS DNA that has been previously modified through one or more of the methods outlined in prior examples, such that the PS backbone now contains a reactive handle attached. Modified PS DNA is reacted with an organic moiety by mixing the two components in a salted aqueous solution (e.g., PBS, Tris, HEPES). Organic moieties are added at a significant molar excess to PS DNA (e.g., organic moiety:PS-base molar ratios of 1 :1, 5: 1, 10: 1, 50: 1, 500: 1, 1000: 1). To identify suitable reaction conditions, variables, such as pH, temperature, reaction time, concentration, and solvent composition are all systematically modulated. Reaction efficiency is determined using gel electrophoresis, MALDLMS, and / or HPLC. Excess organic moiety is removed using various chromatography techniques including reverse phase ion pairing and size exclusion chromatography.

[0663] For all patents, applications, or other reference cited herein, such as non-patent literature and reference sequence information, it should be understood that they are incorporated by reference in their entirety for all purposes as well as for the proposition that is recited. Where any conflict exists between a document incorporated by reference and the present application, this application will control. All information associated with reference gene sequences disclosed in this application, such as GenelDs or accession numbers (typically referencing NCBI accession numbers), including, for example, genomic loci, genomic sequences, functional annotations, allelic variants, and reference mRNA (including, e g., exon boundaries or response elements) and protein sequences (such as conserved domain structures), as well as chemical references (e.g., PubChem compound, PubChem substance, or PubChem Bioassay entries, including the annotations therein, such as structures and assays, et cetera), are hereby incorporated by reference in their entirety.

[0664] Headings used in this application are for convenience only and do not affect the interpretation of this application.

[0665] 1601838421.1 114

Claims

1. Attorney Docket No.: F2128-7027WO(VL87026-W1)CLAIMS1. A DNA molecule comprising a plurality of nucleotides, wherein: at least one nucleotide of the DNA molecule comprises a structure according to FormulaFormula (I), wherein Su is a sugar moiety, B is a nucleobase or hydrogen; G is a bond or a linker group L; and R is an organic moiety; and the DNA molecule has a length of at least 200 nucleotides.

2. The DNA molecule of claim 1, which comprises a promoter sequence operatively linked to an effector sequence that encodes an effector (e.g., a therapeutic effector).

3. A DNA molecule comprising a plurality of nucleotides, wherein: at least one nucleotide of the DNA molecule comprises a structure according to Formula (I):Formula (I), wherein Su is a sugar moiety; B is a nucleobase or hydrogen; G is a bond or a linker group L; and R is an organic moiety, and the DNA molecule comprises a promoter sequence operatively linked to an effector sequence that encodes an effector (e.g., a therapeutic effector).

4. The DNA molecule of any of claims 1-3, wherein the sugar moiety is deoxyribose.

5. The DNA molecule of any of claims 1-4, wherein G is a linker group L, and R-G- comprises Formula (II):1601838421.1 115Attorney Docket No.: F2128-7027WO(VL87026-W1)Formula (II), wherein m = 0-10; n=0-10; z=0-4; Li is a peptide bond of -CONH- or -NHCO-; RL, if present, is each independently a reactive linker; and Ri is the organic moiety.

6. The DNA molecule of any of claims 1-4, wherein G is a linker group L, and R-G- comprises Formula (IV):Formula (IV), wherein u = 0-10; z=0-4; RL, if present, is each independently a reactive linker; and R3 is an organic moiety.

7. The DNA molecule of any of claims 1-6, wherein the organic moiety comprises a macromolecule, a small molecule, or a reactive handle (e.g., a click handle).

8. The DNA molecule of claim 7, wherein the macromolecule comprises (1) a polypeptide; (2) a nucleic acid; (3) a glycan; or (4) a lipid.

9. The DNA molecule of any of claims 1-8, wherein the DNA molecule is single stranded or double stranded.

10. The DNA molecule of any of claims 1-9, wherein the DNA molecule is circular or linear.

11. The DNA molecule of any of claims 1-10, wherein the DNA molecule is closed-ended, linear, and double stranded.1601838421.1 116Attorney Docket No.: F2128-7027WO(VL87026-W1)12. The DNA molecule of any of claims 2-11, wherein the effector comprises a polypeptide (e.g., a DNA binding protein; an epigenetic modifying factor; an antigen; a hormone; an enzyme; a CRISPR-linked enzyme; a mobile genetic element protein; a gene writer; an antibody; a signaling peptide; a receptor ligand; a receptor; or a clotting factor).

13. The DNA molecule of any of claims 2-11, wherein the effector comprises an RNA (e.g., an mRNA, miRNA, or siRNA).

14. The DNA molecule of any of claims 2-13, which expresses the effector when present in a cell.

15. A compound comprising a nucleotide having Formula (1):wherein Bl is H or a nucleobase; Su is a sugar moiety; m=0-10; n=0-10; z=0-4; L is a peptide bond of -CONH- or -NHCO-; RL, if present, is each independently a reactive linker; and Ri is an organic moiety.

16. A compound comprising a nucleotide having Formula (3):Formula (3), wherein Ba is H or a nucleobase; Su is a sugar moiety; u=0-10; z=0-4; RL, if present, is each independently a reactive linker; and Ra is an organic moiety.1601838421.1 117Attorney Docket No.: F2128-7027WO(VL87026-W1)17. A DNA molecule comprising: a) a DNA region that comprises one or more phosphorothioate and (i) has a length of at least 200 nucleotides or (ii) comprises a sequence that encodes an effector; and b) an organic moiety linked to the DNA region via a phosphorothioate of the DNA backbone.

18. A pharmaceutical composition comprising a DNA molecule of any of claims 1-14 or 17.

19. A pharmaceutical composition comprising a DNA molecule that comprises a compound of claim 15 or 16.

20. A method of modulating (e.g., increasing or decreasing) a biological activity in a target cell, the method comprising:(i) contacting a target cell with the DNA molecule of any of claims 2-14 or 17 or the pharmaceutical composition of claim 18 or 19, wherein the effector modulates a biological activity in the target cell; and(ii) maintaining (e.g., incubating) the cell under conditions suitable for expressing the effector from the DNA molecule; thereby modulating the biological activity in the target cell.

21. A method of treating a cell, tissue, or subject in need thereof, the method comprising: administering to the cell, tissue, or subject the DNA molecule of any of claims 1-14 or 17 or the pharmaceutical composition of claims 18 or 19; thereby treating the cell, tissue, or subject.

22. A method of making a DNA molecule, the method comprising: providing a PS DNA molecule comprising Formula (a):1601838421.1 118Attorney Docket No.: F2128-7027WO(VL87026-W1)Formula (a), wherein B is a nucleobase and Su is a sugar moiety; and providing a halide agent having Formula (b)Formula (b), wherein m = 0-10; n=0-10; z=0-4; Li is a peptide bond of -CONH- or -NHCO-; RL, if present, is each independently a reactive linker; X = halo (e g., I, Br or Cl); and Ri is an organic moiety, in a solution; and reacting the PS DNA molecule and the halide agent to form the DNA molecule.

23. A method of making a DNA molecule, the method comprising: providing a PS DNA molecule comprising Formula (a)Formula (a), wherein B is a nucleobase and Su is a sugar moiety, and providing a maleimide agent having Formula (d)1601838421.1 119Attorney Docket No.: F2128-7027WO(VL87026-W1)Formula (d), wherein u = 0-10; z=0-4; RL, if present, is each independently a reactive linker; R3 is an organic moiety, in a solution; and reacting the PS DNA molecule and the maleimide agent to form the DNA molecule.1601838421.1 120

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