WO2025096807A2 - Nouvelles formes d'adn thérapeutique - Google Patents

Nouvelles formes d'adn thérapeutique Download PDF

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WO2025096807A2
WO2025096807A2 PCT/US2024/053928 US2024053928W WO2025096807A2 WO 2025096807 A2 WO2025096807 A2 WO 2025096807A2 US 2024053928 W US2024053928 W US 2024053928W WO 2025096807 A2 WO2025096807 A2 WO 2025096807A2
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dna
molecule
dsdna
nucleotides
chemically modified
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WO2025096807A3 (fr
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Edward Matthew KENNEDY
Camilo Ayala Breton
Carl Wayne Brown, Iii
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Flagship Pioneering Innovations VII Inc
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/335—Modified T or U
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/336—Modified G
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/532—Closed or circular

Definitions

  • a double stranded DNA (dsDNA) molecule comprising: a) an upstream DNA end form which is a closed end; b) a double stranded region comprising a sense strand and an antisense strand, wherein: the sense strand comprises one or more chemically modified nucleobases, and the antisense strand is substantially free of (e.g., is free of) chemically modified nucleobases; and c) a downstream DNA end form which is a closed end.
  • a double stranded DNA (dsDNA) molecule comprising: a) an upstream DNA end form which is a closed end; b) a double stranded region comprising a sense strand and an antisense strand; and Attorney Docket No.: F2128-7017WO(VL87016-W1) c) a downstream DNA end form which is a closed end, wherein the antisense strand is substantially free of (e.g., is free of) chemically modified nucleobases, and wherein the sense strand comprises one or more (e.g., at least 3) backbone modifications, e.g., phosphorothioate linkages, wherein optionally: the one or more backbone modifications are situated between the 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides adjacent to the upstream DNA end form; and/or the one or more backbone modifications are situated between the 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides adjacent to the downstream DNA end form.
  • the dsDNA molecule of any of the preceding embodiments wherein the closed end of the upstream DNA end form is a single stranded loop. 5. The dsDNA molecule of any of the preceding embodiments, wherein the closed end of the downstream DNA end form is a single stranded loop. 6. The dsDNA molecule of any of the preceding embodiments, wherein the backbone modifications are phosphorothioate linkages. 7. The dsDNA molecule of any of embodiments 1-5, wherein the backbone modifications are boranophosphate linkages. 8. The dsDNA molecule of any of the preceding embodiments, wherein the antisense strand is substantially free of (e.g., is free of) phosphorothioate linkages. 9.
  • the dsDNA molecule of any of the preceding embodiments wherein the 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the sense strand that are adjacent to the downstream end form are substantially free of (e.g., are free of) boranophosphate linkages. 13.
  • the dsDNA molecule of any of the preceding embodiments, wherein the 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the sense strand that are adjacent to the downstream end form are substantially free of (e.g., are free of) backbone modifications.
  • the dsDNA molecule of any of the preceding embodiments which has no chemically modified nucleobases between the downstream-most phosphorothioate linkage and the upstream DNA end form. 15.
  • the dsDNA molecule of any of the preceding embodiments which comprises at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6, and optionally no more than 10, chemically modified backbone modifications adjacent to the upstream DNA end form.
  • the sense strand comprises, in an upstream to downstream direction starting from the nucleotide adjacent to the upstream DNA end form: one backbone modification (e.g., phosphorothioate linkage), one or more canonical phosphodiester linkages, and a second backbone modification (e.g., a second phosphorothioate linkage). 17.
  • the sense strand comprises a backbone modification at a position (the “backbone modification position”) such that there are 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides between the backbone modification position and the upstream DNA end form. 19.
  • the dsDNA molecule of any of the preceding embodiments which is substantially free of (e.g., is free of) backbone modifications in a region of the sense strand that is adjacent to the downstream DNA end form.
  • a double stranded DNA (dsDNA) molecule comprising: a) an upstream DNA end form which is a closed end; b) a double stranded region comprising a sense strand and an antisense strand, wherein: the sense strand comprises one or more chemically modified nucleobases, and the antisense strand is substantially free of (e.g., is free of) chemically modified nucleobases; and c) a downstream DNA end form which is a closed end, wherein the double stranded region encodes a protein, and wherein the dsDNA molecule, when contacted to human cells, results in expression at a level at least 100%, at least 120%, at least 140%, at least 160%, or at least 180% the expression of an unmodified control DNA molecule, wherein the unmodified control DNA molecule comprises the same sequence and same closed end double stranded form as the dsDNA molecule, but comprises no chemically modified nucleobases.
  • dsDNA molecule of any of the preceding embodiments wherein the double stranded region encodes a protein
  • the dsDNA molecule when contacted to human cells, results in expression at a level at least the expression of a modified control DNA molecule, wherein the modified control DNA molecule comprises the same sequence, same closed end double stranded form, and same degree of sense strand nucleobase modification as the dsDNA molecule, but comprises antisense strand nucleobase modification at the same degree as sense strand nucleobase modification.
  • a double stranded DNA (dsDNA) molecule comprising: a) an upstream DNA end form which is a closed end; b) a double stranded region comprising a sense strand and an antisense strand, wherein: the sense strand comprises one or more chemically modified nucleobases, and the antisense strand is substantially free of (e.g., is free of) chemically modified nucleobases; and c) a downstream DNA end form which is a closed end, wherein the double stranded region encodes a protein, and wherein the dsDNA molecule, when contacted to human cells, results in expression at a level at least the expression of a modified control DNA molecule, wherein the modified control DNA molecule comprises the same sequence, same closed end double stranded form, and same degree of sense strand nucleobase modification as the dsDNA molecule, but comprises antisense strand nucleobase modification at the same degree as sense strand nucleobase modification.
  • dsDNA molecule of any of the preceding embodiments wherein the dsDNA molecule, when contacted to human cells, results in a lower level of IL6 or CXCL10 mRNA compared to a control DNA molecule (e.g., lower by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%), wherein the control DNA molecule comprises the same sequence and same closed end double stranded form as the dsDNA molecule, but comprises no chemically modified nucleobases.
  • a double stranded DNA (dsDNA) molecule comprising: a) an upstream DNA end form which is a closed end; b) a double stranded region comprising a sense strand and an antisense strand, wherein: the sense strand comprises one or more chemically modified nucleobases, and the antisense strand is substantially free of (e.g., is free of) chemically modified nucleobases; and c) a downstream DNA end form which is a closed end, wherein the dsDNA molecule, when contacted to human cells, results in a lower level of IL6 or CXCL10 mRNA compared to a control DNA molecule (e.g., lower by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%), wherein the control DNA molecule comprises the same sequence and same closed end double stranded form as the dsDNA molecule, but comprises no chemically modified nucleobases.
  • dsDNA double stranded DNA
  • a population comprising a plurality of double stranded DNA (dsDNA) molecules, each dsDNA molecule of the plurality comprising: a) an upstream DNA end form which is a closed end; b) a double stranded region comprising a sense strand and an antisense strand, wherein: the sense strand comprises one or more chemically modified nucleobases, and the antisense strand is substantially free of (e.g., is free of) chemically modified nucleobases; and c) a downstream DNA end form which is a closed end, wherein: at least 50%, at least 60%, or at least 70% of the dsDNA molecules in the plurality have substantially the same length; at least 50%, at least 60%, or at least 70% of the dsDNA molecules in the plurality have a length in a predetermined range; or at least 50%, at least 60%, or at least 70% of the dsDNA
  • a dsDNA molecule comprising: a) an upstream DNA end form which is a closed end; b) a double stranded region comprising a sense strand and an antisense strand, wherein: the sense strand comprises a first type of chemically modified nucleobase and a second type of chemically modified nucleobase; and the antisense strand is substantially free of (e.g., is free of) chemically modified nucleobases; and c) a downstream DNA end form which is a closed end.
  • the first type of chemically modified nucleobase is a chemically modified cytosine nucleobase.
  • a dsDNA molecule comprising: a) an upstream DNA end form which is a closed end; b) a double stranded region comprising a sense strand and an antisense strand, wherein: the sense strand comprises a uridine nucleotide; and the antisense strand is substantially free of (e.g., is free of) chemically modified nucleobases; and c) a downstream DNA end form which is a closed end.
  • 41. The dsDNA molecule or population of any of embodiments 38-40, wherein the uridine nucleotide is a chemically modified uridine nucleotide.
  • the dsDNA molecule or population of embodiment 41, wherein the chemically modified uridine nucleotide comprises 5-azidomethyluridine, 5-formyluridine, 5-hydroxymethyluridine, or 5- methylthiouridine. 43.
  • 44. The dsDNA molecule or population of any of embodiments 38-43, wherein at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, or at least 99% of thymine or uridine positions in the sense strand of the dsDNA molecule comprise a uridine nucleotide. 45.
  • a dsDNA molecule comprising: a) an upstream DNA end form which is a closed end; b) a double stranded region comprising a sense strand and an antisense strand, wherein: the sense strand comprises an inosine nucleotide; and the antisense strand is substantially free of (e.g., is free of) chemically modified nucleobases; and c) a downstream DNA end form which is a closed end.
  • a dsDNA molecule comprising: a) an upstream DNA end form which is a closed end; b) a double stranded region comprising a sense strand and an antisense strand, wherein: the sense strand comprises a 5-methylthiouridine nucleotide; and the antisense strand is substantially free of (e.g., is free of) chemically modified nucleobases; and Attorney Docket No.: F2128-7017WO(VL87016-W1) c) a downstream DNA end form which is a closed end. 49.
  • a method of making a dsDNA molecule comprising: a) providing a double stranded linear DNA having a sense strand, an antisense strand, an upstream open end, and a downstream open end; b) converting both open ends to closed ends (thereby producing an upstream closed end and downstream closed end) such that the sense strand comprises one or more backbone modifications adjacent to or within 10, 20, or 30 nucleotides of the upstream closed end; c) producing a nick in the sense strand of the DNA adjacent to or within 10, 20, or 30 nucleotides of the downstream closed end and/or the upstream closed end; d) subjecting the DNA of c) to conditions having exonuclease activity, such that at least 90%, at least 95%, at least 99%, or 100% of the region of the sense strand between the nick and the one or more backbone modifications are removed; and e) contacting the DNA of d) with a DNA polymerase, unmodified deoxyribose nucleot
  • a method of making a DNA molecule comprising: a) providing a double stranded linear DNA having a sense strand, an antisense strand, an upstream open end, and a downstream open end; b) converting both open ends to closed ends (thereby producing an upstream closed end and downstream closed end) such that the sense strand comprises one or more backbone modifications adjacent to or within 10, 20, or 30 nucleotides of the upstream closed end; c) producing a nick in the sense strand of the DNA adjacent to or within 10, 20, or 30 nucleotides of the downstream closed end and/or the upstream closed end; and d) subjecting the DNA of c) to conditions having exonuclease activity, such that at least 90%, at least 95%, at least 99%, or 100% of the region of the sense strand between the nick and the one or more backbone modifications are removed; thereby producing the DNA molecule.
  • a) comprises performing a polymerase chain reaction on a composition comprising a DNA template, e.g., a plasmid, a forward primer, a reverse primer, a DNA polymerase, and deoxyribose nucleotides, e.g., unmodified or chemically modified deoxyribose nucleotides, wherein optionally the unmodified deoxyribose nucleotides comprise dATP, dCTP, dTTP, and/or dGTP. 53.
  • a DNA template e.g., a plasmid
  • a forward primer e.g., a reverse primer, a DNA polymerase
  • deoxyribose nucleotides e.g., unmodified or chemically modified deoxyribose nucleotides, wherein optionally the unmodified deoxyribose nucleotides comprise dATP, dCTP, dTTP, and/or dG
  • each open end of the DNA of a) is independently a blunt end or a sticky end.
  • a) comprises incubating a dsDNA molecule with a restriction enzyme that cleaves a restriction enzyme recognition sequence in the dsDNA molecule, thereby making the double stranded linear DNA having a sense strand, an antisense strand, an upstream open end, and a downstream open end.
  • the population of end adaptors comprises: a first sub-population of end adaptors comprising phosphorothioate; and a second sub-population of end adaptors that are free of backbone modifications. 64.
  • the first sub-population of end adaptors is configured to ligate to the upstream open end (e.g., wherein the upstream open end has a sticky end that is compatible with a sticky end of end adaptors of the first sub-population); and the second sub-population of end adaptors is configured to ligate to the downstream open end (e.g., wherein the downstream open end has a sticky end that is compatible with a sticky end of end adaptors of the second sub-population).
  • 65 The method of any of embodiments 49-64, which comprises a step of removing or inactivating the ligase (e.g., heat-inactivating the ligase) between steps b) and c).
  • step c) comprises contacting the DNA of b) with a nickase.
  • the nickase is Nb.BsrDI (e.g., NEB, R0648), Nt.BspQI (e.g., NEB, R0644), or Nb.BtsI (e.g., NEB, R0707).
  • step d) comprises contacting the DNA of c) with an exonuclease. 69.
  • exonuclease is an exonclease that initiates at a 3’ terminus of DNA, e.g., Exonuclease III (e.g., NEB, M0206).
  • Exonuclease III e.g., NEB, M0206
  • the method of embodiment 68, wherein the exonuclease is an exonuclease that initiates at the 5' terminus of DNA, e.g., T7 Exonuclease (e.g., NEB, M0263). 71.
  • any of embodiments 49-70 wherein the DNA polymerase is a KME polymerase or Taq DNA Polymerase (e.g., NEB, M0495).
  • 72. The method of any of embodiments 49-71, wherein the method comprises, after step e), contacting the DNA with a ligase, e.g., to join the chemically modified sense strand to the downstream closed end.
  • 73. The method of any of embodiments 49-72, which further comprises a step of enriching the DNA molecule (e.g., dsDNA molecule), e.g., after step a), b), c), d), or e).
  • a composition comprising a plurality of double stranded DNA (dsDNA) molecules comprising: a) an upstream DNA end form which is a closed end; b) a double stranded region comprising a sense strand and an antisense strand, wherein: the sense strand comprises one or more chemically modified nucleobases, and the antisense strand is substantially free of (e.g., is free of) chemically modified nucleobases; and c) a downstream DNA end form which is a closed end, wherein the composition comprises at least 0.5 mg, at least 1 mg, at least 2 mg, at least 5 mg, at least 10 mg, at least 20 mg, or at least 50 mg of the dsDNA molecules, or wherein the composition comprises 0.5-1, 1-2, 2-5, 5-10, 10-20, 20-50, or 50-100 mg of the dsDNA molecules.
  • dsDNA double stranded DNA
  • dsDNA molecule, population, method, or composition of any of embodiments 1-37 or 49-79 wherein 1%-100% (e.g., 1%-5%, 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%- 40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%- 85%, 85%-90%, 90%-95%, or 95%-100%) of cytosine positions in the sense strand of the dsDNA molecule comprise a chemically modified cytosine nucleotide. 81.
  • a uridine nucleotide e.g., a canonical uridine nucleotide.
  • a uridine nucleotide e.g., a canonical uridine nucleotide
  • 1%-100% e.g., 1%-5%, 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%- 30%, 30%-35%, 35%-40%, 40%-45%,
  • 90. The dsDNA molecule, population, method, or composition of embodiment 89, wherein: (a) wherein at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 50% of guanine and inosine positions in the sense strand of the dsDNA molecule comprise an inosine nucleotide; and (b) wherein at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 50% of thymine and uridine positions in the sense strand of the dsDNA molecule comprise a canonical uridine nucleotide.
  • dsDNA molecule, population, method, or composition of embodiment 89 or 90 wherein: (a) wherein 1%-50% (e.g., 1%-5%, 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%- 35%, 35%-40%, 40%-45%, or 45%-50%) of guanine and inosine positions in the sense strand of the dsDNA molecule comprise an inosine nucleotide; and Attorney Docket No.: F2128-7017WO(VL87016-W1) (b) wherein 1%-50% (e.g., 1%-5%, 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%- 35%, 35%-40%, 40%-45%, or 45%-50%) of thymine and uridine positions in the sense strand of the dsDNA molecule comprise a canonical uridine nucleotide.
  • 1%-50% e.g., 1%
  • a DNA molecule comprising, in an upstream to downstream direction: a) an upstream DNA end form which is a closed end; b) a first double stranded region comprising a first fragment of a sense strand and a first region of an antisense strand, wherein optionally the first fragment of the sense strand comprises one or more backbone modifications; c) a second region of the antisense strand, which region is single stranded; d) a second double stranded region comprising a second fragment of the sense strand and a third region of the antisense strand; and e) a downstream DNA end form which is a closed end.
  • 98. The DNA molecule of any of embodiments 92-97, wherein the closed end of the upstream DNA end form is a single stranded loop, e.g., a loop having a length of 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 nucleotides.
  • the DNA molecule of any of embodiments 92-99, wherein the second region of the antisense strand has a length of 50-100, 100-200, 200-500, 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.
  • a linear DNA molecule comprising, in a 5’ to 3’ direction: a) a first annealing sequence, b) a first looping sequence, c) a second annealing sequence complementary to the first annealing sequence, d) an antisense effector sequence, e) a third annealing sequence, f) a second looping sequence, and g) a fourth annealing sequence complementary to the third annealing sequence.
  • the second annealing sequence comprises one or more (e.g., 2, 3, 4, 5, or 6) nucleotides having backbone modifications. 110.
  • the DNA molecule of any of embodiments 103-111, wherein the third annealing sequence and the fourth annealing sequence have the same length and are perfectly complementary along that length.
  • the fourth annealing sequence comprises one or more (e.g., 2, 3, 4, 5, or 6) nucleotides having backbone modifications.
  • the one or more backbone modifications comprise phosphorothioate linkages or boranophosphate linkages. 117.
  • Attorney Docket No.: F2128-7017WO(VL87016-W1) 118.
  • 121. The DNA molecule of any of embodiments 103-120, wherein the antisense effector sequence is single stranded. 122.
  • a double stranded DNA (dsDNA) molecule comprising an inosine nucleotide. 127.
  • 128. The dsDNA molecule of embodiment 126 or 127, wherein the dsDNA molecule is closed-ended linear.
  • a double stranded DNA (dsDNA) molecule comprising an inosine nucleotide, wherein at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, or at least 75% of guanosine and inosine positions in the dsDNA molecule comprise the inosine nucleotide.
  • dsDNA double stranded DNA
  • a double stranded DNA (dsDNA) molecule comprising an inosine nucleotide, wherein 1%-75% (e.g., 1%-5%, 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, or 70%-75%) of guanosine and inosine positions in the dsDNA molecule comprise the inosine nucleotide.
  • 1%-75% e.g., 1%-5%, 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, or 70%-75% of guanosine and inosine positions in the dsDNA molecule comprise the inosine nucleotide.
  • the dsDNA molecule of any of embodiments 126-132 wherein 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 inosine nucleotides of the dsDNA molecule are deoxyribose sugars.
  • the dsDNA molecule of any of embodiments 126-137, wherein the dsDNA molecule can be concatemerized.
  • the dsDNA molecule of embodiment 140 or 141, wherein the sense strand comprises one or more chemically modified nucleotides. 144.
  • the dsDNA molecule of any of embodiments 126-143 which, when contacted to HEKa cells, e.g., in an assay as described herein, results in one or both of: a lower level of CXCL10 mRNA compared to a control DNA molecule (e.g., at least 50%, at least 40%, at least 30%, at least 20%, or at least 10% lower), or a lower level of IL6 mRNA compared to a control DNA molecule (e.g., at least 40%, at least 30%, at least 20%, or at least 10% lower), wherein the control DNA molecule comprises the same sequence, same strandedness, and same circular or linear character as the dsDNA molecule, but comprises guanosine nucleotides in place of the inosine nucleotides.
  • a lower level of CXCL10 mRNA compared to a control DNA molecule
  • a control DNA molecule e.g., at least 50%, at least 40%, at least 30%, at
  • the dsDNA molecule of embodiment 147 or 148 wherein the upstream DNA end form is double stranded and blunt-ended and comprises a phosphorothioate modification on each strand, and the downstream DNA end form is double stranded and blunt-ended and comprises a phosphorothioate modification on each strand.
  • 152. The dsDNA molecule of embodiment 147, wherein one or both of the upstream exonuclease- resistant DNA end form and downstream exonuclease-resistant DNA end form are closed ends. 153.
  • the upstream exonuclease- resistant DNA end form comprises 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 linkages (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).
  • the upstream exonuclease- resistant DNA end form comprises at least 3 phosphorothioate linkages (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).
  • the upstream exonuclease- resistant DNA end form comprises at least 6 phosphorothioate linkages (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).
  • the upstream and downstream exonuclease-resistant DNA end form each comprises at least 3 phosphorothioate linkages (e.g., between the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 terminal nucleotides of the upstream and downstream exonuclease- resistant DNA end forms, e.g., on the first strand, the second strand, or both of the first and second strands).
  • the dsDNA molecule of embodiment 147, wherein one or both of the upstream exonuclease- resistant DNA end form and the downstream exonuclease-resistant DNA end form comprises a Y-adaptor configuration. 167.
  • the dsDNA molecule of embodiment 166 wherein every nucleotide in the Y-adaptor is a chemically modified nucleotide.
  • the dsDNA molecule of any of embodiments 147-167, wherein one or both of the upstream exonuclease-resistant DNA end form and the downstream exonuclease-resistant DNA end form Attorney Docket No.: F2128-7017WO(VL87016-W1) comprises one or more of: a nuclear targeting sequence, a maintenance sequence, or a sequence that binds an endogenous polypeptide in a target cell. 169.
  • the dsDNA molecule of any of embodiments 147-169, wherein the upstream exonuclease- resistant DNA end form and the downstream exonuclease-resistant DNA end form have different structures. 172.
  • the dsDNA molecule of embodiment 147 wherein one or both of the upstream exonuclease- resistant DNA end form and downstream exonuclease-resistant DNA end form are open ends (e.g., blunt ends, sticky ends, or Y-adaptors). 173.
  • the dsDNA molecule of embodiment 147, wherein one or both of the upstream exonuclease- resistant DNA end form and downstream exonuclease-resistant DNA end form are closed ends (e.g., hairpins). 175.
  • the dsDNA molecule of embodiment 174 wherein the closed end comprises one or more (e.g., 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, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, or at least 50) nucleotides that are not hybridized (e.g., are not part of a double-stranded region). 176.
  • the dsDNA molecule of embodiment 174, wherein the closed end does not comprise any nucleotides that are not hybridized (e.g., wherein all nucleotides of the closed end are hybridized to another nucleotide).
  • ITR inverted terminal repeat
  • the dsDNA molecule of any of embodiments 147-182 wherein one or both of the upstream exonuclease-resistant DNA end form and the downstream exonuclease-resistant DNA end form comprises one or more chemically modified nucleotides (e.g., phosphorothioate modified nucleotides). 184.
  • 185 The dsDNA molecule of any of embodiments 147-184, wherein the double-stranded region comprises an effector sequence encoding an effector, and wherein the antisense strand for the effector sequence comprises one or more chemically modified nucleotides (e.g., phosphorothioate modified nucleotides).
  • the dsDNA molecule of any of embodiments 147-185 wherein the double-stranded region comprises an effector sequence encoding an effector, and wherein the sense strand for the effector sequence comprises one or more chemically modified nucleotides (e.g., phosphorothioate modified nucleotides).
  • the sense strand for the effector sequence comprises one or more chemically modified nucleotides (e.g., phosphorothioate modified nucleotides).
  • the dsDNA molecule of any of embodiments 126-186 wherein at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, or at least 75% of guanosine and inosine positions in the sense strand of the dsDNA molecule comprise the inosine nucleotide.
  • a method of making or manufacturing a double stranded DNA (dsDNA) molecule comprising: (a) providing a composition comprising a DNA template (e.g., a plasmid), a forward primer, a reverse primer, a DNA polymerase, unmodified deoxyribose nucleotides, and inosine nucleotides; and (b) performing a polymerase chain reaction on the composition of (a), thereby making or manufacturing the dsDNA molecule, wherein optionally the dsDNA molecule is a dsDNA molecule of any of enumerated embodiments 126-188. 190.
  • the method of embodiment 189 wherein the method further comprises purification of the dsDNA molecule, e.g., wherein purification comprises use of a DNA purification column or agarose gel purification.
  • the DNA polymerase comprises a KOD polymerase, a KODX polymerase, a Deep Vent polymerase, or a KOD -Multi & Epi- polymerase.
  • the unmodified deoxyribose nucleotides comprise dATP, dCTP, dTTP, and/or dGTP.
  • any of embodiments 189-192 wherein the percentage of guanosine or inosine nucleotides that are inosine nucleotides in the composition of (a) is 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, or 70%-75%.
  • the forward primer, the reverse primer, or both contains a protelomerase recognition sequence, e.g., a TelN protelomerase recognition sequence.
  • the method further comprises (e.g., after step (b)): (c) incubating the dsDNA molecule with a protelomerase, e.g., a TelN protelomerase.
  • a protelomerase e.g., a TelN protelomerase.
  • any of embodiments 189-196 wherein the method further comprises: (i) incubating the dsDNA molecule with a restriction enzyme that cleaves the restriction enzyme recognition sequence, thereby making a cleaved dsDNA molecule; (ii) incubating the cleaved dsDNA molecule with a DNA ligase, e.g., a T3 DNA ligase, thereby making a ligated dsDNA molecule; and/or (iii) optionally, incubating the ligated dsDNA molecule with an exonuclease, e.g., a T5 exonuclease. 198.
  • a restriction enzyme that cleaves the restriction enzyme recognition sequence, thereby making a cleaved dsDNA molecule
  • a DNA ligase e.g., a T3 DNA ligase
  • an exonuclease e.g., a T5 exonucle
  • any of embodiments 189-197 the method further comprising: (c) ligating the dsDNA molecule to a hairpin DNA molecule comprising: a loop region and a double-stranded region comprising one or more chemically modified nucleotides.
  • 199 The method of any of embodiments 189-198, the method further comprising ligating the dsDNA molecule to a self-annealed DNA molecule comprising a first region and a second region, wherein the first region is hybridized to the second region.
  • the self-annealed DNA molecule further comprises a loop between the first region and the second region. 201.
  • the method of embodiment 200 wherein the loop comprises a heterologous functional sequence, e.g., a nuclear targeting sequence (e.g., a CT3 sequence), or a regulatory sequence.
  • a heterologous functional sequence e.g., a nuclear targeting sequence (e.g., a CT3 sequence), or a regulatory sequence.
  • Attorney Docket No.: F2128-7017WO(VL87016-W1) 202.
  • the method of embodiment 199 wherein the self-annealed DNA molecule does not comprise any nucleotides that are not hybridized (e.g., wherein all nucleotides of the self-annealed DNA molecule are hybridized to another nucleotide).
  • the self-annealed DNA molecule does not comprise any nucleotides that are not hybridized (e.g., wherein all nucleotides of the self-annealed DNA molecule are hybridized to another nucleotide).
  • any of embodiments 189-202 which further comprises ligating a second hairpin DNA molecule to the dsDNA molecule, wherein the second hairpin DNA molecule comprises a loop region and a double-stranded region, wherein optionally the second hairpin DNA molecule comprises one or more chemically modified nucleotides in one or both of the loop region or the double stranded region.
  • the second hairpin DNA molecule comprises a loop region and a double-stranded region
  • optionally the second hairpin DNA molecule comprises one or more chemically modified nucleotides in one or both of the loop region or the double stranded region.
  • a method of making or manufacturing a TDSC comprising: a) providing the dsDNA molecule made by a method of any of embodiments 189-203, wherein the dsDNA molecule comprises closed ends; b) incubating the TDSC with a double stranded DNA exonuclease, e.g., Exonuclease III, e.g., e.g., 1 ⁇ L of Exonuclease III per 5 ⁇ g of DNA in 50 ⁇ L, for 1 hour at 37 °C, e.g., as described in Example 2; c) optionally, purifying the TDSC treated in step b), e.g., by Silica membrane column, e.g., as described in Example 2, thereby making or manufacturing the TDSC.
  • a double stranded DNA exonuclease e.g., Exonuclease III, e.g., e.g., 1 ⁇ L of
  • a dsDNA molecule produced by the method of any of embodiments 189-204.
  • the DNA molecule e.g., dsDNA molecule
  • backbone modifications e.g., phosphorothioate linkages
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of any of the preceding embodiments wherein the DNA molecule, when contacted to human cells, results in expression at a level at least 100%, at least 120%, at least 140%, at least 160%, or at least 180% the expression of a control DNA molecule (e.g., an unmodified control DNA molecule), wherein the control DNA molecule comprises the same sequence and same closed end form as the DNA molecule, but comprises no chemically modified nucleobases.
  • a control DNA molecule e.g., an unmodified control DNA molecule
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of any of the preceding embodiments wherein the dsDNA molecule, when contacted to human cells, results in expression at a level at least the expression of a modified control DNA molecule, wherein the modified control DNA molecule comprises the same sequence, same closed end double stranded form, and same degree of sense strand nucleobase modification as the dsDNA molecule, but comprises antisense strand nucleobase modification at the same degree as sense strand nucleobase modification.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the sense strand comprises a first type of chemically modified nucleobase and a second type of chemically modified nucleobase.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the sense strand comprises a uridine nucleotide.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of embodiments 1-209, wherein the sense strand comprises an inosine nucleotide. 212.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of embodiments 1-210, wherein the sense strand comprises a 5-methylthiouridine nucleotide. 213.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the DNA molecule has a length of at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10000, at least 11000, or at least 12000 nucleotides. 214.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the DNA molecule has 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. 215.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the DNA molecule has a length of at least 15 nucleotides, at least 30 nucleotides, at least 50 nucleotides, at least 75 nucleotides, 100 nucleotides, at least 200 nucleotides, at Attorney Docket No.: F2128-7017WO(VL87016-W1) 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,
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the DNA molecule has a length of between 20 and 1000 nucleotides, between 20 and 50 nucleotides, between 100 and 500 nucleotides, between 500 and 50,000 nucleotides, between 1,000 and 50,000 nucleotides, between 2,000 and 40,000 nucleotides, between 5,000 and 50,000 nucleotides, between 500 and 50,000 nucleotides, between 500 and 25,000 nucleotides, between 1,000 and 20,000 nucleotides, between 1,000 and 10,000 nucleotides, between 10,000 and 60,000 nucleotides, between 1,000 and 20,000 nucleotides, between 1,000 and 40,000 nucleotides, between 500 and 1000 nucleotides, between 1000 and 2,000 nucleotides, between 2,000 and 3,000 nucleotides, between 3,000 and 4,000 nucleotides, between 4,000 and 5,000 nucleotides,
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of any of the preceding embodiments wherein one or both of the upstream DNA end form and the downstream DNA end form have one or more of the following characteristics: i) the upstream 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; or ii) the downstream 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. 222.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the upstream DNA end form is resistant to endonuclease digestion. 223.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the upstream DNA end form is resistant to immune sensor recognition.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the downstream DNA end form is resistant to endonuclease digestion. 225.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the downstream DNA end form is resistant to immune sensor recognition. 226.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the double stranded region is resistant to endonuclease digestion. 227.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the upstream DNA end form and the downstream DNA end form have the same nucleotide sequence.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the upstream DNA end form and the downstream DNA end form have different nucleotide sequences. 230.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the upstream DNA end form and the downstream DNA end form have the same length in nucleotides. 231.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the upstream DNA end form and the downstream DNA end form have different lengths in nucleotides. 232.
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of any of the preceding embodiments wherein one or both of the upstream DNA form and downstream DNA form comprises one or more (e.g., 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, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, or at least 50) nucleotides that are not hybridized to another nucleotide.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the one or both of the upstream DNA form and downstream DNA form do not comprise any nucleotides that are not hybridized (e.g., wherein all nucleotides of the closed end are hybridized to another nucleotide).
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the sense strand of the dsDNA molecule comprises one or more phosphorothioate linkages. 235.
  • the DNA molecule e.g., dsDNA molecule
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of any of the preceding embodiments wherein the antisense strand comprises no more than 10, no more than 9, no Attorney Docket No.: F2128-7017WO(VL87016-W1) more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 chemically modified nucleobases. 237.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the antisense strand comprises no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 backbone modifications. 238.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the antisense strand comprises no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 nucleotides having a chemically modified sugar. 239.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein 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.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein all positions in the DNA molecule comprise a deoxyribose sugar.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the DNA molecule comprises a chemical modification of a phosphate group.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the DNA molecule comprises a chemically modified sugar, e.g., a 2’- deoxy-2’-fluoro (2’-F) nucleotide or a 2’-O-methyl (2’-O-Me) nucleotide.
  • a chemically modified sugar e.g., a 2’- deoxy-2’-fluoro (2’-F) nucleotide or a 2’-O-methyl (2’-O-Me
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the DNA molecule further comprises one or more additional chemically modified nucleotide, wherein the additional chemically modified nucleotide comprises a modification in the backbone, sugar, or nucleobase.
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of embodiment 243 wherein one or more of the chemically modified nucleotides is conjugated to a peptide or protein.
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of embodiment 243 or 244 wherein one or more of the chemically modified nucleotides comprises a phosphorothioate linkage. 246.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of embodiments 243-245, wherein the DNA molecule is a dsDNA molecule, wherein each of the first and second strands of the dsDNA molecule comprises one or more chemically modified nucleotides. 247.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of embodiments 243-246, wherein the DNA molecule is a dsDNA molecule, wherein each of the first and second strands of the dsDNA molecule comprises one or more phosphorothioate linkages. 248.
  • the DNA molecule e.g., dsDNA molecule
  • the upstream DNA end form comprises one or more chemically modified nucleotides.
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of any of the preceding embodiments, wherein the downstream DNA end form comprises one or more chemically modified nucleotides. 250.
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of any of the preceding embodiments wherein the sense strand comprises 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 linkages (e.g., between the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides adjacent to the upstream DNA end form).
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of any of the preceding embodiments wherein the sense strand comprises at least 3 phosphorothioate linkages (e.g., between the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides adjacent to the upstream DNA end form).
  • phosphorothioate linkages e.g., between the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides adjacent to the upstream DNA end form.
  • the DNA molecule e.g., dsDNA molecule
  • the DNA molecule e.g., dsDNA molecule
  • an effector e.g., a therapeutic effector
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of embodiment 253, wherein the effector is a polypeptide (e.g., a protein).
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of embodiment 253, wherein the effector is a functional RNA (e.g., a miRNA, siRNA, or tRNA).
  • a functional RNA e.g., a miRNA, siRNA, or tRNA.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the double stranded region or the antisense strand comprises a promoter sequence, e.g., wherein the sequence that encodes an effector is operably linked to the promoter sequence.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein the DNA molecule comprises one or more of: i) a promoter sequence (wherein optionally the promoter sequence is in the double stranded region); ii) an effector sequence that encodes an effector, operably linked to the promoter sequence; iii) a heterologous functional sequence, e.g., a nuclear targeting sequence or a regulatory sequence; iv) a maintenance sequence; and/or v) an origin of replication. 258.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of embodiment 257, which comprises: i, ii, and iii; i, ii, and iv; i, ii, and v; i, ii, iii, and iv; Attorney Docket No.: F2128-7017WO(VL87016-W1) i, ii, iii, and v; i, ii, iv, and v; or i, ii, iii, iv, and v. 259.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of embodiment 257 or 258, wherein the nuclear targeting sequence comprises a CT3 sequence (e.g., a sequence of AATTCTCCTCCCCACCTTCCCCACCCTCCCCA (SEQ ID NO: 40)), or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of embodiment 257 or 258, wherein the nuclear targeting sequence binds to a hnRNPK protein (e.g., a human hnRNPK protein).
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of embodiments 257-260, wherein the effector comprises a polypeptide (e.g., a protein). 262.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of embodiments 257-260, wherein the effector comprises an RNA (e.g., an mRNA, a tRNA, lncRNA, miRNA, rRNA, snRNA, microRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, Y RNA, or hnRNA), wherein optionally the effector comprises a functional RNA (e.g., a miRNA, siRNA, or tRNA).
  • RNA e.g., an mRNA, a tRNA, lncRNA, miRNA, rRNA, snRNA, microRNA, siRNA, piRNA,
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of any of embodiments 1-261 wherein the DNA molecule does not comprise a sequence encoding an RNA.
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of any of the preceding embodiments wherein the DNA molecule can be replicated (e.g., by a DNA polymerase native to a cell comprising the DNA molecule).
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of any of embodiments 1-263 wherein the DNA molecule cannot be replicated. 266.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of embodiments 253-265, wherein the effector is heterologous to a target cell.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein one or both of the upstream DNA end form and the downstream DNA end form comprises one or more of: a nuclear targeting sequence, a maintenance sequence, or a sequence that binds an endogenous polypeptide in a target cell. 268.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of the preceding embodiments, wherein one or both of the upstream DNA end form and the downstream DNA end form does not comprise the nucleic acid sequences TATCAGCACACAATTGCCCATTATACGC (SEQ ID NO: 41) and GCGTATAATGGGCAATTGTGTGCTGATA (SEQ ID NO: 42), 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; and/or the nucleic acid sequences TATCAGCACACAATAGTCCATTATACGC (SEQ ID NO: 43) and GCGTATAATGGACTATTGTGTGCTGATA (SEQ ID NO: 44).
  • TATCAGCACACAATAGTCCATTATACGC SEQ ID NO: 41
  • GCGTATAATGGGCAATTGTGTGCTGATA SEQ ID
  • the DNA molecule e.g., dsDNA molecule
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of embodiment 269 wherein one or more of the protelomerase sequences comprise (e.g., in 5’-to-3’ order) the nucleic acid sequences TATCAGCACACAATTGCCCATTATACGC (SEQ ID NO: 41) and GCGTATAATGGGCAATTGTGTGCTGATA (SEQ ID NO: 42), 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. 271.
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of embodiment 269 or 270 wherein one or more of the protelomerase sequences comprise (e.g., in 5’-to-3’ order) the nucleic acid sequences TATCAGCACACAATAGTCCATTATACGC (SEQ ID NO: 43) and GCGTATAATGGACTATTGTGTGCTGATA (SEQ ID NO: 44), 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. 272.
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of embodiment 269 wherein one or more of the protelomerase sequences comprise (e.g., in 5’-to-3’ order) the nucleic Attorney Docket No.: F2128-7017WO(VL87016-W1) acid sequences ACCTATTTCAGCATACTACGC (SEQ ID NO: 45) and GCGTAGTATGCTGAAATAGGT (SEQ ID NO: 46), 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. 273.
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of embodiment 269 wherein one or more of the protelomerase sequences comprise (e.g., in 5’-to-3’ order) the nucleic acid sequence CACACAATTGCCCATTATACGCGCGTATAATGGGCAATTGTGTG (SEQ ID NO: 47), 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. 274.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of embodiment 269, wherein one or more of the protelomerase sequences comprise (e.g., in 5’-to-3’ order) the nucleic acid sequences: (i) TAAATATAATTTAA (SEQ ID NO: 48) and TTAAATTATATTTA (SEQ ID NO: 49), (ii) AATATATAATCTAA (SEQ ID NO: 50) and TTAGATTATATATT (SEQ ID NO: 51), (iii) TATTTATTATCTTT (SEQ ID NO: 52) and AAAGATAATAAATA (SEQ ID NO: 53), (iv) ATATAATTTTTAATTAGTATAGAATATGTTAA (SEQ ID NO: 54) and TTAACATACTCTATACTAATTAAAAATTATAT (SEQ ID NO: 55), (v) TATAATTTGATATTAGTACAAATCCC (SEQ ID NO: 56) and GGGATTTGTACTAATATCAAATT
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of embodiment 269, wherein 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: 62) and ACAGTTTTTTATACTA (SEQ ID NO: 63), Attorney Docket No.: F2128-7017WO(VL87016-W1) (ii) TAGTATACAAAAGATT (SEQ ID NO: 64) and AATCTTTTGTATACTA (SEQ ID NO: 65), (iii) TAGTATATATATCTCT (SEQ ID NO: 66) and AGAGATATATATACTA (SEQ ID NO: 67), or (iv) TAGTATAAAAAAAATT (SEQ ID NO: 68) and AATTTTTTTTATACTA (SEQ ID NO: 69); or nucleic acid sequences:
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of embodiments 269-275, wherein the protelomerase sequences are not produced by Tel PY54 protelomerase digestion. 279.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of embodiments 269-275, wherein the protelomerase sequences are not produced by TelK protelomerase digestion.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of embodiments 269-275, wherein the protelomerase sequences are not produced by ResT protelomerase digestion. 281.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of embodiments 269-280, wherein the protelomerase sequences are about 28 or 56 nucleotides in length.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of any of embodiments 269-281, wherein 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. 283.
  • the DNA molecule e.g., dsDNA molecule
  • population, method, or composition of any of embodiments 269-283 wherein 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. 285.
  • 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. 285.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of embodiment 269, wherein 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. 286.
  • the DNA molecule (e.g., dsDNA molecule), population, method, or composition of embodiment 269, wherein 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. 287.
  • composition of embodiment 287 or 288, or the plurality of DNA molecules of embodiment 289 wherein: 40%-50%, 50%-60%, or 60%-70% of the DNA molecules in the plurality have substantially the same length; 40%-50%, 50%-60%, or 60%-70% of the DNA molecules in the plurality have a length in a predetermined range; or 40%-50%, 50%-60%, or 60%-70% of the DNA molecules in the plurality have a length of between 100, 200, 300, 400, or 500 nucleotides of each other. 292.
  • a pharmaceutical composition comprising the DNA molecule (e.g., the dsDNA molecule) of any of the preceding embodiments. 293.
  • the pharmaceutical composition of embodiment 292 wherein the DNA molecule lacks a material portion of vector backbone, or does not comprise a non-human (e.g., bacterial) origin of replication. 294.
  • Attorney Docket No.: F2128-7017WO(VL87016-W1) 297 The pharmaceutical composition of any of embodiments 292-296, which is essentially free of viral proteins. 298.
  • a method of expressing an effector in a target cell comprising: (i) introducing into a target cell the DNA molecule (e.g., dsDNA molecule), population, or composition (e.g., pharmaceutical composition) of any of the preceding embodiments, or a DNA molecule (e.g., dsDNA molecule) made by the method of any of the preceding embodiments, wherein the DNA molecule (e.g., the double stranded region or antisense strand of the DNA molecule) comprises an effector sequence encoding an effector; and (ii) maintaining (e.g., incubating) the cell under conditions suitable for expressing the effector from the dsDNA molecule; thereby expressing the effector in the target cell.
  • the DNA molecule e.g., dsDNA molecule
  • population e.g., pharmaceutical composition
  • a DNA molecule e.g., dsDNA molecule
  • a DNA molecule e.g., dsDNA
  • a method of delivering an effector to target cell comprising: introducing into a target cell the DNA molecule (e.g., dsDNA molecule), population, or composition (e.g., pharmaceutical composition) of any of the preceding embodiments, or a DNA molecule (e.g., dsDNA molecule) made by the method of any of the preceding embodiments, wherein the DNA molecule (e.g., the double stranded region or antisense strand of the DNA molecule) comprises an effector sequence encoding an effector; thereby delivering the DNA molecule to the target cell.
  • the DNA molecule e.g., dsDNA molecule
  • population e.g., pharmaceutical composition
  • a method of modulating (e.g., increasing or decreasing) a biological activity in a target cell comprising: (i) providing a target cell comprising the DNA molecule (e.g., dsDNA molecule), population, or composition (e.g., pharmaceutical composition) of any of the preceding embodiments, or a DNA Attorney Docket No.: F2128-7017WO(VL87016-W1) molecule (e.g., dsDNA molecule) made by the method of any of the preceding embodiments, wherein the DNA molecule (e.g., the double stranded region or antisense strand of the DNA molecule) comprises an effector sequence encoding an effector that 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.
  • the DNA molecule e.g., dsDNA molecule
  • population e.
  • the method of embodiment 303, wherein the effector increases the biological activity in the target cell.
  • the method of embodiment 303, wherein the effector decreases the biological activity in the target cell.
  • the biological activity comprises cell growth, cell metabolism, cell signaling, cell movement, specialization, interactions, division, transport, homeostasis, osmosis, or diffusion.
  • the cell is an animal cell, e.g., a mammalian cell, e.g., a human cell. 308.
  • a method of treating a cell, tissue, or subject in need thereof comprising: administering to the cell, tissue, or subject the DNA molecule (e.g., dsDNA molecule), population, or composition (e.g., pharmaceutical composition) of any of the preceding embodiments, or a DNA molecule (e.g., dsDNA molecule) made by the method of any of the preceding embodiments, wherein the DNA molecule (e.g., the double stranded region or antisense strand of the DNA molecule) comprises an effector sequence encoding an effector; thereby treating the cell, tissue, or subject.
  • the DNA molecule e.g., the double stranded region or antisense strand of the DNA molecule
  • 309 The method of any of embodiments 301-308, which is performed ex vivo or in vivo.
  • a first nucleotide being “adjacent” to a second nucleotide means that there are no nucleotides situated between the first and second nucleotide.
  • the two nucleotides may be connected by, Attorney Docket No.: F2128-7017WO(VL87016-W1) for instance, a phosphate linkage or a phosphorothioate linkage.
  • annealing sequence refers to a part of a DNA molecule having a sequence that is complementary to (e.g., perfectly complementary to) another annealing sequence in the same DNA molecule.
  • a first annealing sequence hybridizes to a second annealing sequence to create a hairpin structure.
  • looping sequence refers to a part of a DNA molecule having a sequence that lacks perfect complementarity (e.g., lacks substantial complementarity) to any other region of the DNA molecule, and wherein the looping sequence is situated between an annealing sequence and a second annealing sequence complementary to the first annealing sequence.
  • 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, multispecific, human, humanized, primatized, or chimeric antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), single-domain antibodies (sdAb), epitope-binding fragments, e.g., Fab, Fab' and F(ab').sub.2, Fd, Fvs, single-chain Fvs (scFv), rlgG, single-chain 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.
  • heteroconjugate antibodies e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabod
  • Antibodies described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
  • class e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2
  • subclass of immunoglobulin molecule e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2
  • mAb monoclonal antibody
  • Fab and F(ab')2 fragments lack the Fc fragment of an intact antibody.
  • the term “backbone modification” refers to a chemical modification to the backbone of a DNA molecule.
  • the backbone modification is a chemical modification to a phosphate group, e.g., phosphorothioate.
  • the backbone modification is a chemical modification to deoxyribose.
  • the term “carrier” means a compound, composition, reagent, or molecule that facilitates or promotes the transport or delivery of a composition (e.g., a dsDNA molecule described herein) into a cell.
  • a carrier may be a partially or completely encapsulating agent.
  • 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.
  • a chemically modified nucleotide can be produced directly by chemical synthesis, or by covalently modifying a canonical nucleotide.
  • the term “chemically modified cytosine nucleotide,” as used herein with respect to DNAs, refers to a chemically modified nucleotide wherein the nucleobase comprises a monocyclic 6- member ring in which carbon 4 is covalently bound to a nitrogen that is not one of the six members of the ring, wherein the nucleobase of the chemically modified cytosine nucleotide comprises one or more structural differences relative to canonical cytosine nucleobase.
  • the C-5 position of the nucleobase can have a substitution other than H.
  • the chemically modified cytosine nucleotide further comprises a chemical modification on the sugar or phosphodiester linkage. No particular process of making is implied.
  • the term “chemically modified uridine nucleotide” as used herein with respect to DNAs refers to a chemically modified nucleotide wherein the nucleobase comprises a monocyclic 6- member ring in which carbon 4 is covalently bound to an oxygen through a double bond, wherein the nucleobase of the chemically modified uridine nucleotide comprises one or more structural differences relative to canonical uracil and thymine nucleobases.
  • the C-5 position of the nucleobase can have a substitution other than H or a methyl group.
  • the chemically modified uridine nucleotide further comprises a chemical modification on the sugar or phosphodiester linkage. No particular process of making is implied.
  • the term “uridine nucleotide” as used herein encompasses both canonical uridine nucleotides and chemically modified uridine nucleotides.
  • 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.
  • the DNA end form is simply a covalent bond between the 5’ Attorney Docket No.: F2128-7017WO(VL87016-W1) most nucleotide of the sense strand and the 3’ most nucleotide of the antisense strand, in the case of an upstream closed end, or the 3’ most nucleotide of the sense strand and the 5’ most nucleotide of the antisense strand, in the case of a downstream closed end.
  • a dsDNA molecule 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).
  • 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.
  • the terminal nucleotide comprises a free 5’ phosphate.
  • the terminal nucleotide comprises a free 3’ OH.
  • a dsDNA molecule 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.
  • a dsDNA molecule 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).
  • the open end comprises a blunt end, a sticky end, or a Y- adaptor.
  • the term “DNA” 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.
  • the DNA is a single oligonucleotide chain, while in other embodiments, the DNA comprises a plurality of oligonucleotide chains, while in yet other embodiments the DNA is a portion of an oligonucleotide chain.
  • DNA is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage.
  • the DNA comprises solely canonical nucleotides.
  • the DNA comprises one or more chemically modified nucleotides. 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 are deoxyribose sugars. In some embodiments, the DNA 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.
  • DNA end form refers to a structure comprising a bond between nucleotides (e.g., wherein the DNA end form comprises DNA), that is situated at an end of a dsDNA molecule (e.g., a TDSC).
  • the DNA end form comprises a closed end, and in other embodiments, the DNA end form comprises an open end.
  • the DNA end form is the bond or single stranded DNA region that connects the two strands of the dsDNA molecule (e.g., the DNA end form connects the sense strand to the antisense strand).
  • the DNA end form comprises a loop, a Y-adaptor, a blunt end, or a sticky end.
  • the DNA Attorney Docket No.: F2128-7017WO(VL87016-W1) end form may comprise canonical nucleotides, chemically modified nucleotides, or a combination thereof.
  • the DNA end form comprises between 3-100 nucleotides.
  • the dsDNA molecule comprises a first DNA end form at a first end and a second DNA end form at a second end.
  • the first DNA end form and the second DNA end form of a dsDNA molecule are the same type.
  • the first DNA end form and the second DNA end form of a dsDNA molecule are different types.
  • effector sequence refers to the part of a DNA molecule that exerts a function on a cell, either directly (wherein the effector sequence is a functional DNA sequence) or by encoding a functional RNA or protein.
  • the encoded functional RNA or protein is referred to as the “effector”.
  • end adaptor refers to a DNA molecule that can be joined to a dsDNA molecule to produce a DNA end form.
  • the end adaptor may comprise a hairpin, such that when the hairpin end adaptor is ligated to the dsDNA molecule, the loop of the hairpin becomes a DNA end form which is a closed end, while the stem of the hairpin becomes parts of the sense and antisense strands.
  • an end adaptor may comprise two short DNA strands hybridized to each other, and may be used to produce an DNA end form which is an open end.
  • 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 described in Example 2, and if it comprises an open end (e.g., two open ends), is resistant to the exonuclease assay described in Example 3.
  • 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.
  • 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.
  • a heterologous regulatory sequence e.g., promoter, enhancer
  • 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
  • a heterologous nucleic acid molecule may exist in a native host cell genome, but may have an altered expression level or have a Attorney Docket No.: F2128-7017WO(VL87016-W1) different sequence or both.
  • 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 for more than one generation (e.g., episomal viral vector, plasmid or other self-replicating vector).
  • heterologous functional sequence refers to a nucleic acid sequence that is heterologous to a nearby (e.g., adjacent) nucleic acid sequence and has one or more biological function.
  • the biological function comprises targeting to an organelle, e.g., nuclear targeting.
  • the heterologous functional sequence comprises a nuclear targeting sequence or a regulatory sequence.
  • 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.
  • the amount of the metric described herein 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% relative to the amount of the marker prior to administration, or relative to administration of a control dsDNA molecule, such as a dsDNA molecule comprising chemically modified nucleotides compared to a control dsDNA molecule having only unmodified nucleotides.
  • a control dsDNA molecule such as a dsDNA molecule comprising chemically modified nucleotides compared to a control dsDNA molecule having only unmodified nucleotides.
  • the metric is measured subsequent to administration at a time that the administration has had the recited effect, e.g., at least one day, one week, one month, 3 months, or 6 months, after a treatment regimen has begun.
  • the term “inosine nucleotide,” as used herein with respect to DNAs refers to a nucleotide wherein the nucleobase is hypoxanthine.
  • a linear dsDNA molecule 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 dsDNA molecule comprises a first closed end comprising a first loop and a second closed end comprising a second loop.
  • the term “loop” refers to a nucleic acid sequence that is single stranded.
  • 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.
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a compound comprising amino acid residues covalently linked by 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.
  • a polypeptide comprises a non-canonical amino acid residue.
  • 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.
  • 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).
  • 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 protein, and does not serve as a template for transcription of the protein.
  • an “antisense strand” of a dsDNA is a strand that has a sequence Attorney Docket No.: F2128-7017WO(VL87016-W1) complementary to an mRNA or pre-mRNA which encodes for a functional protein and/or can serve as a template for transcription.
  • double stranded DNA molecule or dsDNA molecule 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.
  • the dsDNA molecule is circular or linear.
  • the dsDNA molecule comprises one or two closed ends.
  • the two complementary chains of deoxyribonucleotides are covalently linked.
  • the dsDNA molecule is a TDSC.
  • terminal nucleotide refers to a nucleotide that is covalently attached to exactly one other nucleotide.
  • the terminal nucleotide comprises a free 5’ phosphate.
  • the terminal nucleotide comprises a free 3’ OH.
  • 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.
  • Treatment 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 disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time Attorney Docket No.: F2128-7017WO(VL87016-W1) 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.
  • 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.
  • FIGS.1A-1B illustrate a looped-end DNA (leDNA) in FIG.1A, and hemi-modified dsDNA in FIG.1B.
  • the leDNA of FIG.1A includes, in an upstream to downstream direction, an upstream DNA end form which is a loop, a short double stranded region comprising phosphorothioate linkages (shown as Xs) on the sense strand and antisense strand, a single stranded antisense region, another double stranded region comprising phosphorothioate linkages on the sense strand and antisense strand, and a downstream DNA end form which is a loop.
  • the antisense strand is substantially free of chemically modified nucleobases.
  • the leDNA shown in this figure has phosphorothioate linkages in four regions, in other embodiments, one or more of those regions may lack phosphorothioate.
  • the leDNA is suitable for administration to a subject, to deliver an effector encoded by the antisense strand.
  • the leDNA is a manufacturing intermediate used to make hemi-modified DNA, by using a polymerase to produce a sense strand having chemically modified nucleobases.
  • one of the looped ends can serve as a primer for synthesis of the sense strand.
  • the top strand is the sense strand and comprises chemically modified nucleobases
  • the bottom line is the antisense strand and is substantially free of chemically modified nucleobases.
  • the hemi-modified DNA achieves both immune stealth and transcription by virtue of the chemically modified nucleobases in the sense strand leading to immune stealth, and the canonical nucleobases in the antisense strand leading to effective transcription.
  • FIGS.2A-2B are a series of diagrams showing exemplary covalently-closed DNA end forms that can be included in a dsDNA molecule, e.g., therapeutic double-stranded construct (TDSC), as described herein (e.g., at one or both ends of the dsDNA molecule).
  • TDSC therapeutic double-stranded construct
  • TDSCs exemplary dsDNA molecules
  • TDSCs comprising no loop ends (e.g., protelomerase sequences), inverted terminal repeats (ITRs), or loops at the ends, which can be made up of unmodified nucleotides (white symbols) or may comprise chemically modified nucleotides (gray symbols).
  • a hairpin configuration is another way to describe a construct in which the closed end is a loop, and the ends of the sense and antisense strands form a stem of the hairpin.
  • Chemically modified nucleotides can include nucleotides modified, for example, in the backbone, sugar, or base, or nucleotides that are conjugated to a peptide or protein.
  • both of the DNA strands are unmodified. In some instances, both of the DNA strands are chemically modified. In some instances, the antisense strand is chemically modified. In some instances, the sense strand is chemically modified.
  • the solid-line box indicates a dsDNA molecule that is covalently closed with hairpins at the ends, e.g., a linear, covalently closed dsDNA molecule with end forms comprising phosphorothioate modifications.
  • the dashed-line box indicates a dsDNA molecule that is covalently closed with no loop ends, e.g., a linear, covalently closed dsDNA molecule with TelN end forms.
  • FIG.3 is a series of diagrams showing double-stranded DNA constructs, including exemplary dsDNA molecules, e.g., TDSCs, comprising exemplary DNA end forms (e.g., at one or both ends) that are not covalently closed.
  • exemplary dsDNA molecules e.g., TDSCs
  • the DNA end forms can, in some instances, be made up of unmodified nucleotides (white symbols). In some instances, the DNA end forms comprise chemically modified nucleotides (gray symbols).
  • Chemically modified nucleotides can include nucleotides modified, for example, in the backbone, sugar, or base, or nucleotides that are conjugated to a peptide or protein.
  • both of the DNA strands are unmodified.
  • both of the DNA strands are chemically modified.
  • the antisense strand is chemically modified.
  • the sense strand is chemically modified.
  • Also shown in the upper right is an exemplary DNA construct lacking DNA end forms or chemical modifications (i.e., an unmodified double-stranded DNA molecule).
  • FIG.4 is a diagram depicting production of covalently closed linear dsDNA molecules with end forms comprising phosphorothioate modifications.
  • FIG.4 discloses SEQ ID NOS 87-88, respectively, in order of appearance.
  • FIG.5 is a diagram depicting production of covalently closed linear dsDNA molecules with TelN end forms.
  • FIG.5 discloses SEQ ID NOS 89-90, respectively, in order of appearance.
  • FIG.6 is a diagram depicting circular dsDNA molecules with or without chemical modifications.
  • FIG.7 is a diagram depicting an exemplary method of production of circular dsDNA molecules.
  • a linear dsDNA molecule may be contacted with a restriction enzyme (e.g., KpnI) that creates compatible sticky ends which may then be joined to each other by ligation, producing a circular dsDNA.
  • a restriction enzyme e.g., KpnI
  • FIG.7 discloses SEQ ID NOS 91-92, respectively, in order of appearance.
  • FIG.8 is a pair of graphs showing the immune steath characteristics of various hemi-modified dsDNAs and looped-end DNA (leDNA), based on IL6 levels (left panel) or CXCL10 levels (right panel).
  • FIG.9 is a pair of graphs showing the function (here, ability to produce a protein, in this case mCherry) of various hemi-modified dsDNAs and looped-end DNA (leDNA), measured as total fluorescence over background (left panel) or % mCherry+ cells (right panel).
  • mCherry hemi-modified dsDNAs and looped-end DNA
  • the effector may be a DNA sequence.
  • the effector sequence encodes a polypeptide, e.g., a therapeutic protein.
  • the effector sequence encodes an RNA, e.g., a regulatory RNA or an mRNA.
  • Chemically modified (e.g., hemi-modified) DNA molecules The dsDNA molecules described herein may have chemical modifications of the nucleobases, sugars, and/or the phosphate backbone (e.g., as shown in FIGS.2A-3). 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.
  • 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”.
  • chemical modifications e.g., one or more modifications
  • the phosphate group (or chemically modified analog thereof) of a nucleotide is considered to be the phosphate 5’ of the sugar of that nucleotide.
  • nucleoside 1 phosphate - nucleoside 1 - phosphorothioate - nucleoside 2
  • the phosphorothioate is considered to be part of the same nucleotide as nucleoside 2
  • the phosphate is considered to be part of the same nucleotide as nucleoside 1. Therefore, when nucleotide X is positioned at the 5’ end of the sense strand and adjacent to an upstream DNA end form, the phosphate (or chemically modified analog thereof) of nucleotide X, which is 5’ of the sugar of nucleotide X, is considered to be part of the sense strand rather than part of the upstream DNA end form.
  • nucleotide Y when nucleotide Y is positioned at the 5’ end of the Attorney Docket No.: F2128-7017WO(VL87016-W1) upstream DNA end form which is a loop, the phosphate (or chemically modified analog thereof) of nucleotide Y, which is 5’ of the sugar of nucleotide Y is considered to be part of the upstream DNA end form rather than part of the antisense strand.
  • chemical modifications to DNA useful in the methods described herein include, e.g., phosphorothioate; or S and R phsophorothioate linkages; . See, e.g., Pu et al.2020.
  • a dsDNA molecule as described herein may comprise a phosphorothioate- modified nucleotide.
  • a DNA end form e.g., an exonuclease-resistant DNA end form
  • the dsDNA molecules described herein may include S and R phosphorothioate modified nucleotide linkages.
  • 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.
  • RP-HPLC reverse-phase high- performance liquid chromatography
  • UPLC/MS Ultraperformance liquid chromatography mass spectrometry
  • a dsDNA molecule described herein, or one strand (e.g., the sense strand or the antisense strand) of the dsDNA molecule comprises between 1%-100% chemically modified nucleotides, between 1%-90% chemically modified nucleotides, between 1%-80% chemically modified nucleotides, between 1%-70% chemically modified nucleotides, between 1%-60% chemically modified nucleotides, between 1%-50% chemically modified nucleotides, between 1%-40% chemically modified nucleotides, between 1%-30% chemically modified nucleotides, between 1%-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.
  • a dsDNA molecule described herein, or one strand (e.g., the sense strand or the antisense strand) of the dsDNA molecule 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 Attorney Docket No.: F2128-7017WO(VL87016-W1) nucleotides
  • chemically modified nucleotides e.g., modifications described herein
  • a dsDNA molecule as described herein comprises chemically modified nucleobases on only one strand (e.g., as shown in FIG.1B or 2A).
  • asymmetrically modified dsDNA molecules may be called “hemi-modified.”
  • the hemi-modified 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 modificaitons (such as backbone modifications) on the antisense strand.
  • a dsDNA molecule as described herein comprises chemically modified nucleotides on the antisense strand.
  • a dsDNA molecule as described herein comprises chemically modified nucleotides on the sense strand.
  • a dsDNA molecule 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).
  • a dsDNA molecule comprises a double-stranded region flanked by non-covalently closed exonuclease-resistant DNA end forms comprising chemically-modified nucleotides, e.g., as described herein (e.g., in FIG.3).
  • a dsDNA molecule described herein has one or more chemical modification that disrupts the ability of a portion of the dsDNA molecule to form a double stranded structure, e.g., a dsDNA molecule described herein has one or more chemical modification on a nucleotide present in a region having intramolecular complementarity. In embodiments, a dsDNA molecule described herein has one or more chemical modification that disrupts base pairing of regions of intramolecular complementarity relative to the unmodified sequence of the dsDNA molecule.
  • 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.
  • a chemically modified dsDNA molecule described herein exhibits decreased recognition by DNA sensors in a host tissue or subject compared to an unmodified dsDNA molecule 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 recognition by DNA Attorney Docket No.: F2128-7017WO(VL87016-W1) sensors in a host tissue or subject compared to an unmodified dsDNA molecule of the same sequence.
  • a chemically modified dsDNA molecule described herein exhibits decreased degradation by DNA nucleases compared to an unmodified dsDNA molecule 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 dsDNA molecule.
  • a chemically modified dsDNA molecule described herein shows decreased activation of the innate immune system in a target/host tissue or subject compared to an unmodified dsDNA molecule 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 dsDNA molecule of the same sequence.
  • a dsDNA molecule comprising chemically modified nucleotides described herein exhibits any of the following properties in a target/host tissue or subject compared to dsDNA 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; or increased binding to DNA binding moieties such as nuclear DNA binding proteins, transcription factors, chaperones, or DNA polymerases.
  • the chemically modified cytosine nucleotide comprises 5- formylcytosine, 5-hydroxycytosine, 5-carboxycytosine, 5-propargylaminocytosine, 5-methylcytosine, 5- hydroxymethylcytosine, or glucosyl-5-hydroxymethylcytosine. Chemically modified cytosine nucleotides are further described in International Application WO/2024/173836, which is herein incorporated by reference in its entirety.
  • a dsDNA molecule described herein comprises a chemically modified uridine nucleotide.
  • the chemically modified uridine nucleotide comprises a substitution other than hydrogen or a methyl group at the carbon 5 (C-5) position of the nucleobase.
  • R 1 is selected from the group consisting of -(CH 2 )OH; -I; -Br; -CHO; -COOH; -aminoallyl; -S-methyl; and - propargylamino.
  • the chemically modified uridine nucleotide comprises 5- hydroxymethyluridine, 5-aminoallyluridine, 5-bromouridine, 5-iodouridine, 5-propargylaminouridine, 5- formyluridine, 5-carboxyuridine, or 5-methylthiouridine.
  • Chemically modified uridine nucleotides are further described in International Application WO2024/173828, which is herein incorporated by reference in its entirety. Attorney Docket No.: F2128-7017WO(VL87016-W1)
  • a DNA molecule, e.g., a dsDNA molecule, described herein may comprise a chemically modified nucleotide, such as an inosine nucleotide.
  • a guanosine nucleotide in a position of a DNA molecule can be replaced with an inosine nucleotide.
  • a deoxyguanosine triphosphate dGTP
  • a reaction e.g., a polymerase chain reaction.
  • an inosine on one strand will pair with a cytosine on the opposite strand.
  • the inosine nucleotide described herein increases the “stealth” of a dsDNA molecule to an immune response, while supporting expression of a gene on the dsDNA molecule.
  • a nucleobase comprised by an inosine nucleotide is shown below as Formula III.
  • the dsDNA molecule comprises at least one chemical modification.
  • a dsDNA molecule as described herein comprises chemically modified nucleotides on only one strand.
  • a dsDNA molecule as described herein comprises chemically modified nucleotides on the antisense strand.
  • a dsDNA molecule as described herein comprises chemically modified nucleotides on the sense strand.
  • a dsDNA molecule described herein, or one strand (e.g., the sense strand or the antisense strand) of the dsDNA molecule comprises inosine at between 0%-100%, 10%-75%, 20%-75%, 30%-75%, 40%-75%, 50%- 75%, 60%-75%, or 10%-50% of guanosine and inosine positions.
  • Other chemically modified (e.g., substantially or fully modified) DNA molecules While many of the DNA molecules described herein are hemi-modified, other configurations are also contemplated.
  • a dsDNA comprises chemically modified nucleotides on both strands.
  • each of the sense strand and the antisense strand comprises some chemically modified nucleotides and other nucleotides that are not chemically modified.
  • the present disclosure provides a DNA molecule (e.g., a dsDNA molecule) comprising two or more chemically modified nucleobases described herein.
  • the first chemically modified nucleobase is an inosine and the second chemically modified Attorney Docket No.: F2128-7017WO(VL87016-W1) nucleobase is a uridine nucleotide (e.g., as described herein) or a chemically modified cytosine nucleotide (e.g., as described herein).
  • the first chemically modified nucleobase comprises a canonical uridine nucleobase and the second chemically modified nucleotide is an inosine nucleotide or a chemically modified cytosine nucleotide (e.g., as described herein).
  • the first chemically modified nucleotide is a 5-methylthiouridine nucleotide and the second chemically modified nucleotide is an inosine nucleotide, a chemically modified cytosine nucleotide (e.g., as described herein), or a different uridine nucleotide (e.g., as described herein).
  • the first chemically modified nucleotide is a 5-azidomethyluridine nucleotide
  • the second chemically modified nucleotide is an inosine nucleotide, a chemically modified cytosine nucleotide (e.g., as described herein), or a different uridine nucleotide (e.g., as described herein).
  • a dsDNA molecule as described herein comprises chemically modified nucleotides on both strands (e.g., as shown in FIGS.2A and 3).
  • 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).
  • the entirety of both strands are composed of chemically modified nucleotides.
  • the two strands of a dsDNA molecule 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).
  • a dsDNA molecule 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 chemically modified. In embodiments, a dsDNA molecule as described herein comprises one or more double-stranded regions in which one strand is chemically modified and the other is not. Elements of DNA constructs The dsDNA molecules or nucleic acids comprising dsDNA described herein 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.
  • the dsDNA molecule drives expression of an effector, e.g., comprises a promoter and a sequence encoding an RNA or a polypeptide, e.g., a therapeutic RNA or polypeptide.
  • the DNA constructs described herein further contain one or both of: a nuclear targeting sequence and a maintenance sequence. While some 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 nucleic acid comprising dsDNA.
  • the dsDNA molecules 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.
  • the DNA end forms described herein comprise an open end comprising at least one nucleotide that is only covalently attached to one other nucleotide of the DNA end form.
  • the DNA end forms are generally exonuclease resistant.
  • a DNA end form comprising a closed end is resistant to the exonuclease assay described in Example 2.
  • a DNA end form comprising an open end e.g., such as a Y adaptor, blunt end, or sticky end, e.g., as described herein
  • every nucleotide of the closed end is hybridized to another nucleotide. Loops
  • a DNA end form comprises a loop.
  • the DNA end form comprising a loop may be produced, e.g., by ligating an end adaptor which is a hairpin to a dsDNA molecule.
  • a hairpin generally comprises a single-stranded loop region covalently attached at both the 5’ and 3’ ends to a double-stranded stalk region.
  • the single- stranded 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.
  • Exemplary loop structures, and exemplary dsDNA molecules comprising loops are shown in FIG.2A.
  • the single-stranded loop region comprises one or more functional elements (e.g., a nuclear import sequence (e.g., a CT3 ssDNA sequence), or a regulatory sequence.
  • 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 dsDNA molecule comprising the DNA end form.
  • the single-stranded loop region of the end form is less than about 5, less than 10, less than 15, less than 20, less than 25, less than 26, less than 27, less than 28, less than 29, or less than 30 nucleotides in length.
  • the single-stranded loop region of the end form 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.
  • the loop is comprised in a dsDNA molecule having a doggybone conformation.
  • the dsDNA molecule comprises a protelomerase sequence (e.g., as described herein).
  • the protelomerase sequence is produced by TelN protelomerase, ResT protelomerase, Tel PY54 protelomerase, or TelK protelomerase digestion.
  • 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 Attorney Docket No.: F2128-7017WO(VL87016-W1) than about 28, less than about 29, or less than about 30 nucleotides in length.
  • 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.
  • 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.
  • a loop can be attached to one or both ends of a double-stranded DNA molecule, for example, by ligation of a hairpin (e.g., as described herein).
  • a dsDNA molecule as described herein comprises, at one or both ends, a loop.
  • the upstream exonuclease-resistant DNA end form of a dsDNA molecule as described herein comprises a loop.
  • the downstream exonuclease-resistant DNA end form of a dsDNA molecule as described herein comprises a loop.
  • a loop comprises one or more unmodified nucleotides.
  • a loop consists entirely of unmodified nucleotides.
  • a loop comprises one or more chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein).
  • a loop consists entirely of chemically modified nucleotides (e.g., phosphorothioate- modified nucleotides, e.g., as described herein).
  • 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).
  • a DNA molecule described herein is produced by a ligating a DNA hairpin loop to a double stranded region.
  • 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).
  • nucleotides in the single-stranded loop region are chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein).
  • the single-stranded loop region of a DNA hairpin loop consists entirely of chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein).
  • 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. Attorney Docket No.: F2128-7017WO(VL87016-W1) 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • nucleotides in the single-stranded loop region are chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein).
  • the single-stranded 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.
  • Y-Adaptors In some embodiments, an exonuclease-resistant DNA end form as described herein comprises a Y-adaptor.
  • a Y-adaptor generally comprises a pair of single-stranded 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).
  • Exemplary Y-adaptor structures and exemplary dsDNA molecules comprising Y-adaptors are shown in FIG.3.
  • a Y-adaptor is produced by attaching a hairpin loop comprising a single- stranded region comprising a cleavable moiety to the end of a double-stranded DNA region (e.g., via ligation). The cleavable moiety can then be cleaved to produce the two single-stranded DNA regions of the Y-adaptor.
  • 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).
  • At least 50%, at least 60%, at least 70%, Attorney Docket No.: F2128-7017WO(VL87016-W1) 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).
  • 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).
  • a single-stranded DNA region (e.g., one or both single-stranded DNA regions) of a Y-adaptor comprises one or more unmodified nucleotides.
  • 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.
  • chemically modified nucleotides e.g., phosphorothioate-modified nucleotides, e.g., as described herein
  • a double-stranded DNA region of the Y-adaptor comprises one or more unmodified nucleotides.
  • nucleotides in the single-stranded DNA region or regions are chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein).
  • 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.
  • No Loop Closed DNA End Forms In some embodiments, a dsDNA molecule as described herein comprises an exonuclease-resistant DNA end form that is covalently closed but does not include a single stranded loop.
  • every nucleotide of a covalently-closed DNA molecule is complementary to another nucleotide.
  • the DNA end form may be a bond between the endmost nucleotide of the sense strand and the nucleotide of the antisense strand which base pairs with the nucleotide of the sense strand.
  • a covalently-closed DNA end form as described herein can be attached to one end of a dsDNA molecule as described herein, e.g., by ligation.
  • Open DNA End Forms In some embodiments, a dsDNA molecule as described herein comprises an exonuclease-resistant DNA end form that is not covalently closed.
  • the DNA end form comprises a blunt end (e.g., a blunt end comprising one or more chemical modifications as described herein) or a sticky end (e.g., a sticky end comprising one or more chemical modifications as described herein).
  • the open DNA end form is produced by nuclease digestion of a covalently closed DNA end form, such as a loop.
  • the dsDNA region near the loop Attorney Docket No.: F2128-7017WO(VL87016-W1) comprises a double-stranded stalk region comprising a cleavable moiety on each strand, and the DNA hairpin is then contacted with an enzyme capable of cleaving the cleavable moieties.
  • a sticky end comprising an overhang.
  • the overhang is digested with an enzyme (e.g., a single-stranded specific nuclease, e.g., a Mung Bean nuclease) to form a blunt end.
  • an enzyme e.g., a single-stranded specific nuclease, e.g., a Mung Bean nuclease
  • 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).
  • 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).
  • the DNA end form comprising a blunt end consists entirely of chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein).
  • 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.
  • 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.
  • 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.
  • 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, e.g., as described herein.
  • 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).
  • 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).
  • the DNA end form comprising a sticky end consists entirely of chemically modified nucleotides (e.g., phosphorothioate-modified nucleotides, e.g., as described herein).
  • a terminal nucleotide of the DNA end form comprising a sticky 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.
  • the Attorney Docket No.: F2128-7017WO(VL87016-W1) 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.
  • Inverted Terminal Repeats ITRs
  • a dsDNA molecule as described herein comprises an exonuclease-resistant DNA end form comprising an inverted terminal repeat (ITR).
  • the ITR is an ITR from a virus, e.g., an adenovirus or an adeno-associated virus (AAV).
  • the ITR comprises a nucleic acid sequence having 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%, 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).
  • the ITR comprises an origin of replication (e.g., a viral origin of replication).
  • a dsDNA molecule as described herein comprises an exonuclease-resistant DNA end form comprising an ITR (e.g., as described herein) at each end.
  • a dsDNA molecule does not comprise an ITR.
  • a DNA molecule (e.g., dsDNA molecule) 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 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.
  • constitutive promoters examples 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 SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1alpha promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • 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.
  • the native promoter for the sequence encoding the effector can be used.
  • the regulatory sequences impart tissue-specific gene expression capabilities.
  • 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.
  • tissue-specific 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) promoter.
  • TSG liver-specific thyroxin binding globulin
  • PY pancreatic polypeptide
  • PPY pancreatic polypeptide
  • Syn synapsin-1
  • MCK creatine kinase
  • DES mammalian desmin
  • a-MHC alpha-myosin heavy chain
  • 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.
  • AFP alpha-fetoprotein
  • Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptoralpha.-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.
  • NSE neuron-specific enolase
  • tissue/cell specific promoters are listed in Table 1: Table 1: Tissue or cell specific promoters Tissue/Cell Promoter Accession Number; Human Genome i t h Attorney Docket No.: F2128-7017WO(VL87016-W1) Tissue/Cell Promoter Accession Number; Human Genome Coordinate (hg38) P rostate Cancer KLK3 NM 001648 hr19 50865760-
  • the constructs described herein may also include other native or heterologous expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences.
  • the effector sequence of a DNA molecule 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).
  • a functional DNA sequence e.g., a therapeutically functional DNA sequence
  • a DNA sequence encoding a therapeutic peptide, polypeptide or protein e.g., a non-coding RNA.
  • 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 may not have a promoter operably linked.
  • a dsDNA molecule described herein may include 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.
  • 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.
  • 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/Cas12a); a Crispr-linked enzyme, e.g.
  • a base editor or prime editor e.g., 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; a clotting factor; a coagulation factor; a structural protein; a
  • a dsDNA molecule 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.
  • a dsDNA molecule described herein may include multiple sequences encoding multiple proteins, e.g., a plurality of proteins in a biological pathway.
  • a dsDNA molecule 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.
  • a dsDNA molecule 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, Attorney Docket No.: F2128-7017WO(VL87016-W1) and a polyA site.
  • a dsDNA molecule 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.
  • the effector comprises a cell penetrating polypeptide.
  • the effector is a fusion protein that comprises a cell penetrating polypeptide and a second amino acid sequence.
  • RNA effectors 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).
  • siRNA short interfering RNA
  • miRNA microRNA
  • piRNA piwi-interacting RNA
  • snoRNA small nucleolar RNA
  • scaRNA small Cajal body-specific RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • RNA aptamer e.g., an RNA aptamer, and a small nuclear RNA (
  • a DNA molecule (e.g., dsDNA molecule) disclosed herein comprises one or more expression sequences that encode a regulatory RNA, e.g., an RNA that modifies expression of an endogenous gene and/or an exogenous gene.
  • the dsDNA molecule or sequence 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, lncRNA, miRNA, rRNA, snRNA, microRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, Y RNA, and hnRNA.
  • 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.
  • the sequence is an miRNA.
  • 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.
  • the DNA molecule e.g., dsDNA molecule
  • 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.
  • sgRNA single guide RNA
  • sgRNA single guide RNA
  • F2128-7017WO(VL87016-W1) tracrRNA for binding the nuclease
  • 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).
  • the gRNA is used as part of a CRISPR system for gene editing.
  • the dsDNA molecule or sequence 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.
  • 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.
  • RNAi molecules include, but are not limited to: short interfering RNAs (siRNAs), double- strand RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), meroduplexes, and dicer substrates (U.S.
  • RNA antisense oligonucleotides RNA ASOs
  • the DNA molecule e.g., dsDNA molecule or sequence disclosed herein comprises a sequence comprising a sense strand of a lncRNA.
  • the dsDNA molecule or sequence disclosed herein comprises a sequence encoding an antisense strand of a lncRNA.
  • the DNA molecule e.g., dsDNA molecule
  • sequence 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.
  • 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.
  • the length of a DNA molecule (e.g., dsDNA molecule) or sequence 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, 12, 13, 14, 15, 16, Attorney Docket No.: F2128-7017WO(VL87016-W1) 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 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%.
  • a DNA molecule e.g., dsDNA molecule or sequence disclosed herein may encode a micro- RNA (miRNA) molecule identical to about 5 to about 30 contiguous nucleotides of a target gene.
  • the 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.
  • the dsDNA molecule or sequence disclosed herein encodes at least one miRNA, e.g., 2, 3, 4, 5, 6, or more.
  • the dsDNA molecule or sequence disclosed herein comprises a sequence that encodes an miRNA having 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% nucleotide sequence identity to any one of the nucleotide sequences or a sequence that is complementary to a target sequence.
  • the dsDNA molecule or sequence 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.
  • 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).
  • the dsDNA molecule or sequence 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 dsDNA molecule or sequence disclosed herein can be designed to target a class of genes with sufficient sequence homology.
  • the dsDNA molecule or sequence 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.
  • the dsDNA molecule or sequence 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.).
  • the dsDNA molecule or sequence Attorney Docket No.: F2128-7017WO(VL87016-W1) disclosed herein can be designed to target a sequence that is unique to a specific RNA sequence of a single gene.
  • 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, 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).
  • 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, less than about 700 bps, less than about 600 bps, less than about 500 bps,
  • the effector sequence has, independently or in addition to, a length greater than 10 bps (e.g., at least 10 bps, at least 20 bps, at least 30 bps, at least 40 bps, at least 50 bps, at least 60 bps, at least 70 bps, at least 80 bps, at least 90 bps, at least 100 bps, at least 200 bps, at least 300 bps, at least 400 bps, at least 500 bps, at least 600 bps, at least 700 bps, at least 800 bps, at least 900 bps, at least 1000 kb, at least 1.1 kb, at least 1.2 kb, at least 1.3 kb, at least 1.4 kb, at least 1.5 kb, at least 1.6 kb, at least 1.7 kb, at least 1.8 kb, at least 1.9 kb, at least 2 kb, at least 2.1 kb, at least 2.2 kb, at least 2.3 kb, at least 2.4 k
  • 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
  • a DNA molecule e.g., dsDNA molecule
  • sequence disclosed herein comprises one or more of the features described hereinabove, e.g., one or more structural DNA sequence, a sequence encoding one or more peptides or proteins, a sequence encoding one or more regulatory Attorney Docket No.: F2128-7017WO(VL87016-W1) 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.
  • a dsDNA molecule 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.
  • the DNA molecule e.g., dsDNA molecule
  • the DNA molecule includes a therapeutically functional, structural DNA sequence.
  • the DNA molecule includes a promoter and a sequence encoding a therapeutic peptide, polypeptide, or protein described herein.
  • the DNA molecule (e.g., dsDNA molecule) includes a promoter and a sequence encoding a regulatory RNA described herein.
  • 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.
  • 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 http://www.kazusa.or.jp/codon/.
  • NTS Nuclear targeting sequences
  • a DNA molecule e.g., dsDNA molecule
  • nucleic acid comprising dsDNA e.g., as disclosed herein
  • NTS nuclear targeting sequence
  • NTS includes binding sites to proteins (e.g., transcription factors, chaperones, etc.) which bind to importin which transports cargo into the nucleus via the nuclear pore complex.
  • proteins e.g., transcription factors, chaperones, etc.
  • an NTS may function generally (e.g. SV40 enhancer NTS).
  • NTS’s may be cell or tissue specific, e.g., containing binding sites for transcription factors expressed in unique cell types that may target a DNA molecule (e.g., dsDNA molecule) described herein to the nucleus in a cell-specific manner (e.g., SRF, Nkx3).
  • NTS can be functional in multiple locations in a dsDNA molecule described herein, e.g., before the promoter and/or after the effector sequence.
  • An NTS may be viral or non-viral derived. NTSs are described, e.g., in Le Guen et al.2021. Nucleic Acids Vol.24: 477-486.
  • NTS nuclear targeting sequences
  • Table 2 Exemplary nuclear targeting sequences Viral/Non-viral Name Sequence V iral SV40 5’-cccaagaagaagaggaaagtc-3’ (SEQ ID NO: 1)
  • the NTS has a sequence according to Table 2, or a functional sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.
  • a DNA molecule (e.g., dsDNA molecule) is capable of being imported into the nucleus, e.g., by a nuclear import protein.
  • the DNA molecule (e.g., dsDNA molecule) can be bound by a nuclear import protein.
  • a DNA molecule (e.g., dsDNA molecule) comprises a recognition sequence for a nuclear import protein.
  • an exonuclease-resistant DNA end form e.g., comprised in a dsDNA molecule) comprises a recognition sequence for a nuclear import protein.
  • Exemplary import proteins include, e.g., basic helix–loop–helix (bHLH) proteins, heterogeneous nuclear ribonucleoprotein (hnRNP) isoforms, or nuclear factor I (NFI) proteins.
  • the import protein comprises an importin.
  • the import protein comprises a Ran binding protein.
  • the import protein comprises a homeobox transcription factor.
  • the import factor specifically binds an E-box, a DTS, a promoter, a telomere, an ATTT motif, a cell cycle regulatory unit (CCRU), a CT3 sequence, an S/MAR, a topoisomerase II consensus sequence, an ARS consensus sequence, a 3NF, a viral ori.
  • Maintenance sequence A DNA molecule (e.g., dsDNA molecule) 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 molecule (e.g., dsDNA molecule) of the invention.
  • 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, Attorney Docket No.: F2128-7017WO(VL87016-W1) 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 molecule (e.g., dsDNA molecule) described herein to facilitate long-term transgene expression and extra-chromosomal maintenance.
  • the maintenance sequence is human interferon-beta MAR (5’tataattcactggaattttttttgtgtatggtatgacatatgggttcccttttattttttacatataaatatatttccctgtttttctaaaaagaaaaagatcatca tttttcccattgtaaaatgccatattttttttcataggtcacttacata-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.
  • the maintenance sequence is human interferon-beta MAR (5’tataattcactggaattttttttgtgtatggtatgacatatgggttcccttttattttttacatataaatatatttccctgtttttctaaaaagaaaaagatcatca tttttcccattgtaaaatgccatattttttttcataggtcacttacata-3’ (SEQ ID NO: 39)), or a functional sequence having at least 80%, 90%, 95%, or 98% identity thereto.
  • a DNA molecule (e.g., dsDNA molecule) described herein is capable of replicating in a mammalian cell, e.g., human cell.
  • a DNA molecule (e.g., dsDNA molecule) described herein is maintained in a host cell, tissue or subject through at least one cell division.
  • a DNA molecule (e.g., dsDNA molecule) 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, or at least 50 cell divisions.
  • a dsDNA molecule described herein is maintained in a host cell, tissue or subject through at least 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 40, 50 or more cell divisions.
  • cell division may be tracked by flow cytometry or microscopy.
  • cell division may be tracked by intravital microscopy.
  • a DNA molecule (e.g., dsDNA molecule) 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.
  • 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.
  • 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 Attorney Docket No.: F2128-7017WO(VL87016-W1) 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.
  • a DNA molecule described herein does not encode a viral protein.
  • the DNA molecule (e.g., dsDNA molecule) 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 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 nucleotides, at least about 12,000 nucleotides, at least about 20,000
  • the DNA molecule (e.g., dsDNA molecule) 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, 11,000-12,000, 10,000-20,000, 20,000-30,000, 30,000-40,000, or 40,000-50,000 nucleotides in length.
  • the size of a DNA molecule (e.g., dsDNA molecule) disclosed herein is a length sufficient to encode useful polypeptides or RNAs.
  • a DNA molecule (e.g., dsDNA molecule) comprises a DNA end form (e.g., as described herein).
  • 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.
  • 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.
  • a DNA molecule e.g., dsDNA molecule
  • the double stranded region is Attorney Docket No.: F2128-7017WO(VL87016-W1) 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 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least about 11,000, at least about 12,000, at least 20,000, at least 30,000, at least 40,000, or at least 50,000 nucleotides in length.
  • the double stranded region is less than 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000, 30,000, 40,000, or 50,000 nucleotides in length.
  • the double stranded 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, less than 12,000, less than 20,000, less than 30,000, less than 40,000, or less than 50,000 nucleotides in length.
  • the double stranded 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, 11,000- 12,000, 10,000-20,000, 20,000-30,000, 30,000-40,000, or 40,000 to 50,000 nucleotides in length.
  • a dsDNA molecule described herein may have less than a threshold level of single stranded structures.
  • the dsDNA molecule 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.
  • double stranded regions formed by a dsDNA molecule described herein is determined as described by Xayaphoummine et al.2005. Kinefold web server for RNA/DNA folding path and structure prediction including pseudoknots and knots. Nucleic Acids Research, Volume 33:W605- 610.
  • the Kinefold website (http://kinefold.curie.fr/cgi-bin/form.pl) is used to predict double stranded regions of a construct described herein, using the following parameters: ⁇ Sequence to fold: enter and select “DNA sequence” ⁇ Stochastic Simulation: Co-transcriptional fold, 3 milliseconds ⁇ Simulated molecular time: default ⁇ Pseudoknots: not allowed ⁇ Entanglements: non crossing Attorney Docket No.: F2128-7017WO(VL87016-W1) ⁇ Random seed: 11453 In some aspects, the present disclosure provides looped-end DNA (leDNA) molecules.
  • a leDNA molecule has a long single stranded region, and each end is folded to produce a hairpin.
  • the leDNA comprises an upstream DNA end form which is a closed end.
  • the leDNA comprises, adjacent to the upstream DNA end form, a first double stranded region comprising a first fragment of a sense strand and a first region of an antisense strand.
  • the first double stranded region comprises backbone modifications in the sense strand, the antisense strand, or both of the sense and antisense strands.
  • the leDNA comprises, adjacent to the first double stranded region, a second region of the antisense strand, which region is single stranded.
  • the leDNA comprises, adjacent to the single stranded region of the antisense strand, a second double stranded region comprising a second fragment of the sense strand and a third region of the antisense strand.
  • the second double stranded region comprises backbone modifications in the sense strand, the antisense strand, or both of the sense and antisense strands.
  • the leDNA comprises, adjacent to the second double stranded region, a downstream DNA end form which is a closed end.
  • 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.
  • the hemi-modified 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., phosphorothioate) on the antisense strand.
  • the hemi-modified DNA molecule comprises chemically modified nucleotides (e.g., nuclotides comprising chemically modified nucleobases) on the sense strand.
  • the hemi-modified DNA molecule comprises inosine nucleotides on the sense strand. Production
  • a hemi-modified dsDNA may be produced as follows.
  • a double stranded linear DNA having a sense strand, an antisense strand, an upstream open end, and a downstream open end is provided, e.g., using routine methods.
  • both open ends are converted to closed ends (thereby producing an upstream closed end and downstream closed end), e.g., by ligating end adaptors.
  • the end adaptors may comprise backbone modifications that are exonuclease-resistant, such as phosphorothioate.
  • the sense strand may comprise one or more backbone modifications adjacent to or within 10, 20, or 30 nucleotides of the upstream closed end and/or the downstream closed end.
  • a nick may be produced in the sense strand of the DNA adjacent to or Attorney Docket No.: F2128-7017WO(VL87016-W1) within 10, 20, or 30 nucleotides of the downstream closed end or the upstream closed end.
  • the nicked DNA may be subjected to conditions having exonuclease activity (e.g., the nicked DNA may be contacted with an exonuclease), such that the region of the sense strand between the nick and the one or more backbone modifications are removed.
  • the leDNA can be converted to dsDNA by contacting the leDNA with a DNA polymerase and nucleotides.
  • a DNA polymerase and nucleotides For instance, a mixture of unmodified deoxyribose nucleotides, and nucleotides comprising chemically modified nucleobases may be used, such that a chemically modified sense strand is produced, and the leDNA is converted into dsDNA.
  • a ligase may be used to connect the newly synthesized sense strand to the neighboring DNA end form. Additional methods of making dsDNAs are described, for instance, in International Application WO/2023/220729, which is hereby incorporated by reference in its entirety. See, for instance, the Examples therein.
  • a dsDNA molecule comprising chemical modifications on one strand is produced by amplification of one strand (e.g., from a plasmid template) using a dNTP mixture comprising one or more chemically modified nucleotides and a primer that can amplify one strand of the dsDNA molecule sequence.
  • the opposite strand (e.g., an unmodified strand or a differently chemically modified strand, e.g., as described herein, for example, in FIGS.2A-3) is produced in a separate amplification reaction, e.g., using a dNTP mixture comprising unmodified nucleotides or a different set of chemically modified nucleotides, and a primer that can amplify the opposite strand of the dsDNA molecule sequence.
  • a dsDNA molecule as described herein is produced from a plasmid assembled to contain the desired elements described herein.
  • the plasmid template can be assembled, for example, using Golden Gate cloning for assembly of multiple DNA fragments in a defined linear order in a recipient vector using a one-pot assembly procedure.
  • Golden Gate cloning is described in Marillonnet & Grützner, 2020, Synthetic DNA assembly using golden gate cloning and the hierarchical modular cloning pipeline, Current Protocols in Molecular Biology, 130:e115.
  • a plasmid template is linearized, for example, by digestion with a nuclease (e.g., a restriction endonuclease) or by PCR amplification of a linear nucleic acid sequence from the plasmid template.
  • a nuclease e.g., a restriction endonuclease
  • a dsDNA molecule comprising the same chemical modification(s) on both strand is produced by amplification of the dsDNA molecule strands (e.g., from a plasmid template) using a dNTP mixture comprising one or more chemically modified nucleotides and primers that can amplify both strand of the dsDNA molecule sequence.
  • a dsDNA molecule sequence e.g., from a plasmid template
  • a dNTP mixture comprising one or more chemically modified nucleotides and primers that can amplify both strand of the dsDNA molecule sequence.
  • an exonuclease-resistant DNA end form e.g., as described herein is introduced (e.g., attached) to one or both ends of a dsDNA molecule.
  • the DNA end form is attached to an end of the dsDNA molecule by ligation.
  • attachment e.g., ligation
  • the DNA end form e.g., a covalently closed DNA end form
  • exonuclease resistance of the attached DNA end form is confirmed, for example, by incubating the dsDNA molecule in the presence of an exonuclease (e.g., Exonuclease III and/or Mung Bean Nuclease), e.g., as described in Examples 2 and 3.
  • an exonuclease e.g., Exonuclease III and/or Mung Bean Nuclease
  • exonuclease resistance of the attached DNA end form is confirmed, for example, by incubating the dsDNA molecule in the presence of Exonuclease III.
  • the DNA end form comprises a blunt end, sticky end, or Y-adaptor (e.g., as described herein), and the exonuclease resistance of the attached DNA end form is confirmed by incubating the dsDNA molecule in the presence of Exonuclease III and (e.g., subsequently, prior to, or concurrently) Mung Bean nuclease.
  • the DNA end form is attached to the end of the dsDNA molecule in a nascent form (e.g., a non-covalently closed DNA end form may be attached to the dsDNA molecule as a hairpin.
  • a nascent form of the DNA end form may be further modified (e.g., cleaved) to produce the final DNA end form.
  • a non-covalently closed DNA end form may be produced by cleavage of a nascent form, e.g., by a nuclease.
  • a nascent form comprising an overhang or sticky end can be converted to a blunt end by digestion with a single strand- specific nuclease, e.g., a Mung Bean nuclease.
  • a nascent form comprising a hairpin comprising a cleavable moiety in its single-stranded loop region is converted to a Y-adaptor by cleavage of the cleavable moiety.
  • the method further comprises formulating the enriched dsDNA molecule for pharmaceutical use, e.g., formulating the dsDNA molecule with a pharmaceutically acceptable excipient and/or with a carrier, e.g., an LNP.
  • a method described herein comprises enriching the dsDNA molecule.
  • the enriching includes substantially removing from the dsDNA molecule one or more impurity selected from: endotoxin, process impurities such as mononucleotides, chemically modified mononucleotides, single stranded DNA, DNA fragments or truncations, and proteins (e.g., enzymes, e.g., ligases, restriction enzymes).
  • the dsDNA molecule may be enriched from impurities or byproducts selected from the group consisting of: endotoxin, process impurities such as mononucleotides, chemically modified mononucleotides, single stranded DNA, circular DNA, proteins (e.g., enzymes, e.g., ligases, restriction enzymes), DNA fragments or truncations.
  • process impurities such as mononucleotides, chemically modified mononucleotides, single stranded DNA, circular DNA, proteins (e.g., enzymes, e.g., ligases, restriction enzymes), DNA fragments or truncations.
  • the enriched dsDNA molecule is substantially free of process byproducts and impurities, e.g., process byproducts or impurities described Attorney Docket No.: F2128-7017WO(VL87016-W1) herein.
  • the pharmaceutical composition is substantially free of impurities or process byproducts, e.g., selected from the group consisting of: endotoxin, mononucleotides, chemically modified mononucleotides, DNA fragments or truncations, and proteins (e.g., enzymes, e.g., ligases, restriction enzymes).
  • the pharmaceutical composition is substantially free of circular DNA.
  • enrichment involves a partial reduction of one or more contaminants.
  • a dsDNA molecule is formulated with a lipid based carrier, e.g., a lipid nanoparticle (LNP), e.g., as described in Example 1.
  • LNP lipid nanoparticle
  • the dsDNA molecule may be sequenced to confirm the desired, designed sequence.
  • other structural analysis of the dsDNA molecule e.g., restriction enzyme analysis
  • a chemically modified dsDNA molecule described herein may be produced by a number of methods, including methods routine in the art. For instance, a chemically modified dsDNA molecule can be produced by performing polymerase chain reaction on a DNA template in the presence of unmodified and chemically modified nucleotides and a suitable polymerase.
  • Exemplary suitable polymerases are described in Example 9 and include KOD polymerase (710864, Sigma Aldrich), KODX polymerase (719753, Sigma Aldrich), Deep Vent polymerase (M0258, NEB), and KOD -Multi & Epi- polymerase (TYB-KME-101, Diagnocine).
  • KOD polymerase 710864, Sigma Aldrich
  • KODX polymerase 719753, Sigma Aldrich
  • Deep Vent polymerase M0258, NEB
  • KOD -Multi & Epi- polymerase TYB-KME-101, Diagnocine
  • a wide variety of polymerases are available, e.g., from commercial sources. Various polymerases can be used so long as they incorporate chemically modified nucleotides with a sufficiently high efficiency.
  • a chemically modified dsDNA molecule may also be produced by a method that does not comprise performing polymerase chain reaction. For instance, direct chemical synthesis may be used.
  • a chemically modified dsDNA molecule may be produced by providing a dsDNA molecule and chemically modifying nucleotides of the dsDNA molecule. For instance, a dsDNA molecule may be contacted with an enzyme, resulting in a chemically modified dsDNA molecule. In some embodiments, the enzyme converts an unmodified nucleotide into a chemically modified nucleotide. In some embodiments, the enzyme converts a chemically modified nucleotide into a differently modified nucleotide.
  • Pharmaceutical compositions The present disclosure includes a DNA molecule (e.g., dsDNA molecule) and related compositions in combination with one or more pharmaceutically acceptable excipients and/or carriers.
  • 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.
  • Attorney Docket No.: F2128-7017WO(VL87016-W1) A DNA molecule (e.g., dsDNA molecule) described herein may be formulated without a carrier, e.g., the DNA molecule (e.g., dsDNA 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.
  • compositions described herein 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.
  • FDA United States Food and Drug Administration
  • Non-limiting 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.
  • solvents e.g., phosphate buffered saline (PBS)
  • PBS phosphate buffered saline
  • Carriers A DNA molecule (e.g., dsDNA 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.
  • 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 dsDNA 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 differentiated reticulocytes), nucleated cells, exosomes, protein carriers (e.g., a protein covalently linked to the DNA molecule (e.g., dsDNA molecule)), peptides (e.g., cell-penetrating peptides), materials (e.g., graphene oxide), single pure lipids (e.g.
  • the DNA molecule e.g., dsDNA molecule
  • 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 Attorney Docket No.: F2128-7017WO(VL87016-W1) 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:10.1155/2011/469679 for review).
  • 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.
  • 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:10.1155/2011/469679 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.
  • 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; https://doi.org/10.1016/j.apsb.2016.02.001.
  • Ex vivo differentiated red blood cells can also be used as a carrier for an agent (e.g., a dsDNA molecule) described herein.
  • 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), Attorney Docket No.: F2128-7017WO(VL87016-W1) and a PEG-modified lipid.
  • the lipid-based carrier (or lipid nanoformulation) contains one or more compounds described herein, or a pharmaceutically acceptable salt thereof.
  • suitable compositions or compounds to be used in the lipid-based carrier include all the isomers and isotopes of the compositions or 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.
  • the lipid-based carrier may further include a second lipid.
  • the second lipid is a cationic lipid, a non-cationic (e.g., neutral, anionic, or zwitterionic) lipid, or an ionizable lipid.
  • the lipid-based carrier may contain positively charged (cationic) lipids, neutral lipids, negatively charged (anionic) lipids, or a combination thereof.
  • Cationic Lipids (Positively Charged) and Ionizable Lipids 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.
  • the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions.
  • exemplary cationic lipids include one or more amine group(s) which bear the positive charge.
  • 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), 3 ⁇ -[N-(N',N'-dimethylaminoethyl)carbamoyl) cholesterol (DC- chol), 1,2-dioleoyloxy-3-[trimethylammonio]-propane (DOTAP), 1,2-dioctadecyloxy-3- [trimethylammonio]-propane (DSTAP), and 1,2-dioleoyl
  • the lipid-based carrier (or lipid nanoformulation) comprises more than one cationic lipid.
  • the lipid-based carrier (or lipid nanoformulation) comprises a cationic lipid having an effective pKa over 6.0.
  • 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.
  • 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.
  • the cationic lipid is an ionizable lipid (e.g., a lipid that is protonated at low pH, but that remains neutral at physiological pH).
  • 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, ,
  • ionizable lipid is a lipid disclosed in Hou, X., et al. Nat Rev Mater 6, 1078–1094 (2021). https://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).
  • 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 2016/0311759; I of US 20150376115 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;
  • the lipid-based carrier (or lipid nanoformulation) further includes biodegradable ionizable lipids, for instance, (9Z,l2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,l2-dienoate, also called 3- ((4,4- bis(octyloxy)butanoyl)oxy)-2-(((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,l2Z)- octadeca-9,l2-dienoate).
  • biodegradable ionizable lipids for instance, (9Z,l2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)
  • Non-Cationic Lipids e.g., Phospholipids
  • the lipid-based carrier or lipid nanoformulation
  • the non-cationic lipid is a phospholipid.
  • the non-cationic lipid is a phospholipid substitute or replacement.
  • the non-cationic lipid is a negatively charged (anionic) lipid.
  • non-cationic lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (
  • 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 include, without limitation, those described in Kim et al. (2020) dx.doi.org/10.1021/acs.nanolett.0c01386, which is incorporated herein by reference.
  • lipids include, in some embodiments, plant lipids found to improve liver transfection with mRNA (e.g., DGTS).
  • 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.
  • DOPC 1,2-Dioleoyl-sn-glycero-3-phosphocholine
  • non-cationic lipids include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl 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.
  • nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl my
  • 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.
  • 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.
  • 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).
  • the lipid-based carrier (or lipid nanoformulation) does not include any phospholipids.
  • 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).
  • the lipid-based carrier (or lipid nanoformulation) described herein may further comprise one or Attorney Docket No.: F2128-7017WO(VL87016-W1) more structural lipids.
  • structural lipid refers to sterols (e.g., cholesterol) and also to lipids containing sterol moieties. Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipid 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.
  • the structural lipid is a sterol.
  • the structural lipid is a steroid.
  • the structural lipid is cholesterol.
  • the structural lipid is an analog of cholesterol.
  • the structural lipid is alpha-tocopherol.
  • structural lipids may be incorporated into the lipid-based carrier at molar ratios ranging from about 0.1 to 1.0 (cholesterol phospholipid).
  • 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.
  • 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.
  • the cholesterol derivative is a polar analogue, e.g., cholesteryl- (4'-hydroxy)-buty1 ether.
  • 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.
  • the lipid-based carrier may include one or more polymers or co-polymers, e.g., poly(lactic-co-glycolic acid) (PFAG) nanoparticles.
  • the lipid-based carrier may include one or more polyethylene glycol (PEG) lipid.
  • PEG-lipids examples include, but are not limited to, 1,2- Diacyl-sn-Glycero-3- Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-350] (mPEG 350 PE); Attorney Docket No.: F2128-7017WO(VL87016-W1) 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); 1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-1000
  • the PEG lipid is a polyethyleneglycol-diacylglycerol (i.e., polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB) conjugate.
  • 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).
  • 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).
  • one or more ionic lipids e.g., non-cationic lipid such as a neutral or anionic, or zwitterionic lipid
  • one or more sterols e.g., cholesterol
  • the PEG conjugate can comprise a PEG-dilaurylglycerol (C12), a PEG-dimyristylglycerol (C14), a PEG-dipalmitoylglycerol (C16), a PEG-disterylglycerol (C18), PEG-dilaurylglycamide (C12), PEG- dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16), and PEG-disterylglycamide (C18).
  • a PEG-dilaurylglycerol C12
  • PEG-dimyristylglycerol C14
  • PEG-dipalmitoylglycerol C16
  • PEG-disterylglycerol C18
  • PEG-dilaurylglycerol C12
  • PEG- dimyristylglycamide C14
  • 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-g
  • DAG P
  • 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 WO 2017/099823, all of which are incorporated herein by reference in their entirety.
  • the PEG-lipid is a compound of Formula III, III-a-I, III-a-2, III-b-1, III-b- 2, or V of US 2018/0028664, which is incorporated herein by reference in its entirety.
  • the PEG-lipid is of Formula II of US 2015/0376115 or US 2016/0376224, both of which Attorney Docket No.: F2128-7017WO(VL87016-W1) are incorporated herein by reference in their entirety.
  • the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl.
  • the PEG-lipid includes one of the following: in place of PEG-lipid.
  • 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.
  • 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.
  • the conjugated lipid e.g., the PEGylated lipid
  • the conjugated 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).
  • the conjugated lipid (e.g., the PEGylated lipid) content is 0.5- 10 mol% or 2-5 mol% of the total lipid components.
  • 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).
  • 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., Attorney Docket No.: F2128-7017WO(VL87016-W1) Working et al.
  • 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.
  • 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.
  • 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; a
  • 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.
  • 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 0-50 mol% phospholipid, about 0-50 mol% sterol, and about 0-10 mol% PEGylated lipid.
  • the encapsulation efficiency of the payload may be at least 70%.
  • 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.
  • 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 Attorney Docket No.: F2128-7017WO(VL87016-W1) 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.
  • the encapsulation efficiency of the payload may be at least 70%.
  • 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.
  • the lipid-based carrier 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
  • 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.
  • a payload e.g., a DNA molecule described herein
  • 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
  • the encapsulation efficiency of the payload may be at least 70%.
  • 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%).
  • 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%).
  • the encapsulation efficiency of the payload may be at least 70%.
  • the lipid-based carrier 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).
  • 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, 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).
  • the encapsulation efficiency of the payload may be at least 70%.
  • the lipid-based carrier (or lipid nanoformulation) comprises (i) a DNA molecule described herein; (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.
  • the lipid-based carrier (or lipid nanoformulation) comprises (i) a DNA molecule described herein; (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 % 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.
  • 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.
  • the lipid-based carrier or lipid nanoformulation
  • 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.
  • the non-ionizable lipid components in the lipid-based carrier 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.
  • the ratio of total lipid components to the payload e.g., an encapsulated therapeutic agent such as a DNA molecule described herein can be varied as desired.
  • the total lipid components to the payload (mass or weight) ratio can be from about 10:1 to about 30:1.
  • the total lipid components to the payload 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.
  • the lipid- based carrier (or lipid nanoformulation’s) overall lipid content can range from about 5 mg/ml to about 30 Attorney Docket No.: F2128-7017WO(VL87016-W1) mg/mL.
  • Nitrogen:phosphate ratios (N:P ratio) is evaluated at values between 0.1 and 100.
  • the efficiency of encapsulation of a payload such as a protein and/or nucleic acid describes the amount of protein and/or nucleic acid 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%, at least 80%, at least 90%, at least 95%, or close to 100%). In some embodiments, the encapsulation efficiency is at least 70%, 80%, 90%, 95%, or close to 100%.
  • the encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid 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 free protein or nucleic acid (e.g., RNA) in a solution.
  • Fluorescence may be used to measure the amount of free protein and/or nucleic acid (e.g., RNA) in a solution.
  • nucleic acid e.g., RNA
  • the encapsulation efficiency of a protein and/or nucleic acid may be 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.
  • the encapsulation efficiency of a protein and/or nucleic acid 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%.
  • the encapsulation efficiency may be at least 70%.
  • the encapsulation efficiency may be at least 80%.
  • the encapsulation efficiency may be at least 90%.
  • the encapsulation efficiency may be at least 95%.
  • a dsDNA molecule described herein is introduced into a cell, tissue or subject by any suitable route.
  • Administration to a target cell or tissue 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).
  • reagents e.g., liposomal, calcium phosphate
  • 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.
  • Administration to a subject may be by parenteral (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, or intracranial) route; by topical administration, transdermal administration or transcutaneous administration.
  • parenteral e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, or intracranial
  • topical administration e.g., transdermal administration or transcutaneous administration.
  • Suitable routes include oral, rectal, transmucosal, intranasal, inhalation (e.g., via an aerosol), buccal (e.g., sublingual), vaginal, intrathecal, Attorney Docket No.: F2128-7017WO(VL87016-W1) 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).
  • direct tissue or organ injection e.g., to liver, eye, skeletal muscle, cardiac muscle, diaphragm, muscle or brain.
  • the DNA molecule (e.g., dsDNA molecule) described herein can be used in therapeutic or health applications for a subject, e.g., a human or non-human animal.
  • a subject e.g., a human or non-human animal.
  • the subject can be any animal, e.g., a mammal, e.g., a human or non-human mammal.
  • the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian).
  • the subject is a human.
  • the method subject is a non-human mammal.
  • 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).
  • 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
  • lagomorph e.g., rabbit
  • 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).
  • avian taxa Galliformes e.g., chickens, turkeys, pheasants, quail
  • Anseriformes e.g., ducks, geese
  • Paleaognathae e.g., ostriches, emus
  • Columbiformes e.g., pigeons, doves
  • Psittaciformes e.g., par
  • the subject is an invertebrate such as an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusk.
  • an arthropod e.g., insects, arachnids, crustaceans
  • a nematode e.g., an annelid, a helminth, or a mollusk.
  • a DNA described herein is provided at a dose of about 0.1-100 mg/kg of the DNA.
  • a DNA molecule (e.g., dsDNA 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, at least 3, at least 4, at least 5, at least 6 days or at least a week; at least 8, at least 9, at least 10, at least 12, at least 14 days or at least two weeks; at least 16, at least 18, at least 20 days or at least 3 weeks; at least 22, at least 24, at least 25, at least 27, at least 28 days or at least 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.
  • a biological effector e.g., expression of a therapeutic polypeptide
  • a dsDNA 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, at least 3, at least 4, at least 5, at least 6 days or at least a week; at least at least 8 days, at least 9 days, at least 10 days, at least 12 days, at least 14 days or two weeks; at least 16 days, at least 18 days, at least 20 days or at least 3 weeks; at least 22 days, at least 24 days, at least 25 days, at least 27 days, at least 28 days or at least a month; at Attorney Docket No.: F2128-7017WO(VL87016-W1) least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months or more; between one week and 6 months, between 1 month to 6 months, between 3 months to 6 months.
  • a DNA molecule (e.g., dsDNA 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.
  • a DNA molecule (e.g., dsDNA molecule) described herein can be used to deliver an effector, e.g., an effector described herein, to a cell, tissue or subject.
  • a DNA molecule (e.g., dsDNA 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.
  • a DNA molecule e.g., dsDNA molecule
  • a DNA molecule described herein can be used to treat a cell, tissue or subject in need thereof by administering a DNA molecule (e.g., dsDNA molecule) described herein to such cell, tissue or subject.
  • the subject has or has been diagnosed with a condition that can be treated with an effector encoded in the dsDNA.
  • the present disclosure provides a method of modulating (e.g., increasing or decreasing) a biological activity in a target cell, the method comprising: (i) providing a target cell comprising a DNA molecule (e.g., dsDNA molecule) as described herein, wherein the DNA molecule (e.g., dsDNA molecule) encodes an effector that modulates a biological activity in the target cell; and (ii) maintaining (e.g., incubating) the cell under conditions suitable for expressing the heterologous effector from the DNA molecule (e.g., dsDNA molecule); thereby modulating the biological activity in the target cell.
  • a DNA molecule e.g., dsDNA molecule
  • the present disclosure provides a method of modulating (e.g., increasing or decreasing) a biological activity in a target cell, the method comprising: (i) providing a target cell comprising a DNA molecule (e.g., dsDNA molecule) as described herein, wherein the DNA molecule (e.g., dsDNA molecule) comprises an effector sequence encoding an effector that 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 (e.g., dsDNA molecule); thereby modulating the biological activity in the target cell.
  • a DNA molecule e.g., dsDNA molecule
  • the DNA molecule (e.g., dsDNA molecule) delivers an effector to a cell, e.g., an epidermal cell (e.g., a keratinocyte).
  • a dsDNA molecule e.g., TDSC
  • LNP Attorney Docket No.: F2128-7017WO(VL87016-W1)
  • Example 2 Determining exonuclease resistance for a dsDNA molecule comprising closed ends
  • Example 3 Determining exonuclease resistance for a dsDNA molecule comprising an open end (e.g., two open ends)
  • Example 4 Design and assembly of a plasmid template for production of double-stranded DNA (dsDNA) molecules
  • Example 5 Quantification of DNA chemical modifications in vitro
  • Example 6 Validation of chemically modified DNA sequences in cells
  • Example 7 Design and assembly of a plasmid template for production of double-stranded
  • Example 1 Formulation of a dsDNA molecule (e.g., TDSC) with LNP
  • a dsDNA molecule e.g., TDSC
  • LNP lipid nanoparticle
  • Nucleic acid constructs are combined with lipid components via microfluidic devices according to the method of Chen et al.2012. J Am Chem Soc. Volume 134, Issue 16:6948-6951. Briefly, the microfluidic devices are fabricated in polydimethylsiloxane (PDMS) according to standard lithographic procedures (McDonald & Whitesides.2002. Accounts Chem Res Volume 35, Issue 7:491-499).
  • PDMS polydimethylsiloxane
  • the lipid components typically containing cationic lipids, cholesterol, helper lipids, polyethylene glycol modified lipids, and lipids facilitating targeting moiety conjugation (optional), are combined and solubilized in 90% ethanol.
  • the nucleic acid constructs are dissolved in buffer.
  • the nucleic acid solution, the lipid solution, and phosphate buffer saline (PBS) are injected into the microfluidic device.
  • the freshly prepared LNPs are dialyzed against PBS buffer using membranes with MWCO of 3.5kD to remove ethanol and exchange buffer.
  • the LNPs are characterized in terms of effective diameter, polydispersity, and zeta potential using dynamic light scattering (DLS) (ZetaPALS, Brookhaven Instruments, NY, 15-mW laser, incident beam 676 nm); and total nucleic acid concentration is determined by lysing the particles and using Quant- iTTM 1X dsDNA Assay Kits, High Sensitivity (HS) and Broad Range (BR) (ThermoFisher Scientific, Q33232).
  • DLS dynamic light scattering
  • HS High Sensitivity
  • BR Broad Range
  • Example 2 Determining exonuclease resistance for a dsDNA molecule comprising closed ends This example describes how to test if a dsDNA molecule comprising closed ends (e.g., an adaptor-ligated linear dsDNA construct) is Exonuclease III (M0206, New England Biolabs Inc.) resistant. The dsDNA molecule is tested next to a non-nuclease control.
  • a dsDNA molecule comprising closed ends e.g., an adaptor-ligated linear dsDNA construct
  • M0206 Exonuclease III
  • the non-nuclease control contains DNA with the identical sequence to the dsDNA molecule of interest except that it underwent the adaptor ligation protocol that is used to add the exonuclease-resistant DNA end form to the dsDNA molecule, but without an adaptor oligonucleotide added to the mixture.1 ⁇ L of Exonuclease III (at a starting concentration of 100 units/uL) is added per 5 ⁇ g of DNA in 50 ⁇ L. The tubes are mixed well and spun down. The tubes are run on the thermocycler for 1 hour at 37 °C, and heat inactivated at 70 °C for 30 minutes.
  • the samples are concentrated via the Nucleospin® Gel and PCR Clean-up kit (catalog # 740609, Macherey-Nagel) using a vacuum manifold. Briefly, the elution buffer is warmed to 70 °C.2x volumes of NTI binding buffer are added to 1x volume of Exo III-treated DNA. The samples are mixed until evenly distributed and left at room temperature for 5 minutes. The column on the vacuum manifold is secured, valve opened, and vacuum turned on.375 ⁇ L DNA-NTI mix is added to 2x columns and allowed to fully pass through each column.700 ⁇ L of NTC wash buffer is added twice. The column is removed from the vacuum manifold and placed into a collection tube.
  • the assembly is centrifuged at 11,000 xg for 1 minute.
  • the column is placed into a new low bind microcentrifuge tube, 25 ⁇ L of prewarmed buffer is added, and the assembly is incubated at 70 °C for 5 min.
  • the assembly is centrifuged at 11,000 xg for 1 min.
  • the incubation and elution steps are repeated a second time.
  • the collected DNA is quantified by dsDNA BR Qubit (Q32850, Thermo Fisher Scientific) on the Qubit 4 Fluorometer (Q33226, Thermo Fisher Scientific).
  • the samples are loaded into E-Gel EX, 1% Agarose Gel (G402021, Thermo Fisher Scientific) in individual wells at an amount of 16 ng of DNA per well.
  • the ladder (10488090, Thermo Fisher Scientific) is loaded at 2 ⁇ l into the left most lane of the gel.
  • the gel is run through the E-Gel Power Snap Electrophoresis System (G8100, G8200, Thermo Fisher Scientific).
  • G8100, G8200, Thermo Fisher Scientific E-Gel Power Snap Electrophoresis System
  • the exonuclease- resistant dsDNA molecule is visible at the molecular weight corresponding to the full-length DNA plus closed-adapter sequence.
  • a dsDNA molecule will be considered exonuclease-resistant in this assay if at least 95% of the product that appears in the gel in that lane corresponds to the full-length dsDNA molecule.
  • Example 3 Determining exonuclease resistance for a dsDNA molecule comprising an open end (e.g., two open ends) This example describes how to test if a dsDNA molecule comprising an open end (e.g., an adaptor-ligated linear dsDNA construct) is Exonuclease III (M0206, New England Biolabs Inc.) resistant. The dsDNA molecule is tested next to a non-nuclease control.
  • the non-nuclease control contains DNA with the identical sequence to the dsDNA molecule of interest except that it underwent the adaptor ligation protocol that is used to add the exonuclease-resistant DNA end form to the dsDNA molecule, but without an adaptor oligonucleotide added to the mixture.2 units of Exonuclease III are added per 200 ng of DNA (at 10 ng/ul), in a 20 ul reaction. The tubes are mixed well and spun down. The tubes are run on the thermocycler for 30 min at 37 °C. The samples are loaded into E-Gel EX, 1% Agarose Gel (G402021, Thermo Fisher Scientific) in individual wells at an amount of 20 ng of DNA per well.
  • the ladder (10488090, Thermo Fisher Scientific) is loaded at 2 ⁇ l into the left most lane of the gel.
  • the gel is run through the E-Gel Power Snap Electrophoresis System (G8100, G8200, Thermo Fisher Scientific).
  • G8100, G8200, Thermo Fisher Scientific E-Gel Power Snap Electrophoresis System
  • the exonuclease- resistant dsDNA molecule is visible at the molecular weight corresponding to the full-length DNA plus closed-adapter sequence.
  • a dsDNA molecule will be considered exonuclease-resistant in this assay if at least 95% of the product that appears in the gel in that lane corresponds to the full-length dsDNA molecule.
  • Example 4 Design and assembly of a plasmid template for production of double-stranded DNA (dsDNA) molecules
  • dsDNA double-stranded DNA
  • This example describes production of a plasmid template for a dsDNA molecule.
  • a construct template was designed with the following specific sequence components.
  • Example 5 Quantification of DNA chemical modifications in vitro This example describes the quantification of modified nucleotides within chemically modified dsDNA molecules. Enriched dsDNA molecules with chemically modified nucleotides are prepared from a plasmid template as described herein. The proportion of modified nucleotides is quantified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) as previously described in Bachman et al., 2014, Nature Chemistry volume 6 issue 12 pages 1049-1055). Briefly, DNA is degraded to nucleosides via incubation with DNA Degradase Plus (Zymo Research).
  • LC-MS/MS tandem mass spectrometry
  • LC-MS/MS analysis is conducted on a mass spectrometer fitted with a liquid chromatography system. Calibration curves are generated using a mixture of synthetic standards in the ranges of 0.01-100 ⁇ M for deoxycytosine and 0.0001-1 ⁇ M for chemically modified nucleotides, respectively. Samples and synthetic standards are spiked with an isotopically labeled mix of deoxycytosine and chemically modified derivatives as an internal standard.
  • the mass spectrometer is operated in multiple reaction monitoring (MRM) mode.
  • the ion source is electrospray in positive mode. Results are expressed as a percentage of total nucleotides of that type.
  • Example 6 Validation of chemically modified DNA sequences in cells
  • Enriched dsDNA constructs with native or chemically modified nucleotides are prepared and delivered to cells as described herein. Following transfection, DNA and RNA are simultaneously extracted from cells (AllPrep DNA/RNA Kit, Qiagen) and converted into libraries for next-generation sequencing (Illumina). Demultiplexed reads are mapped to the sequence of the chemically modified dsDNA constructs as described in Langmead and Salzberg, 2012, Nature Methods volume 9 pages 357- 359. Mutations are identified via standard variant calling programs.
  • Example 7 Design and assembly of a plasmid template for production of double-stranded DNA (dsDNA) molecules
  • dsDNA double-stranded DNA
  • This example describes production of a plasmid template for a dsDNA molecule.
  • a construct template was designed with the following specific sequence components.
  • Example 8 Design of adapter sequences for ligation to dsDNA molecules This Example describes design of end adapters for ligation to dsDNA molecules to create covalently closed DNA with looped ends.
  • End adapters for DNA molecules were designed as shown below: P-Pt6-AvrII-ADR: /5Phos/CTAGTG*C*C*C*G*A*GCAGGATCGAGCCACACGTACTACGCTCGATCCTGC* T*C*G*G*G*CA (SEQ ID NO: 77) P-Pt6-INV-A2-ADR: /5Phos/GATCTG*C*C*C*G*A*GCAGGATCGAGCCACACGTACTACGCTCGATCCTGC*T *C*G*G*G*CA (SEQ ID NO: 78) In these sequences, /5Phos/ indicates that the following base is phosphorylated and the star (*) indicates a phosphorothioate linkage.
  • Example 9 Production of looped-end DNA (leDNA) molecules This Example demonstrates preparation of ssDNA molecules with termini comprising loops, i.e., looped-end DNA (leDNA) (illustrated in FIG.1A).
  • PCR reaction conditions include: a. PCR Buffer for KOD FX (TYB-KFX-1B, Diagnocine) at a 1x concentration. b. Forward and reverse primers at a final concentration of 300 ⁇ M. c.
  • primers contained additional sequences useful in downstream processes: a. Nicking enzyme(s) recognition sequence; b. Restriction enzyme recognition sequence (e.g. BsaI), used to create sticky ends in the DNA after restriction enzyme digestion and facilitate adapter ligation; and c. Additional bases (e.g., 5’- GGTCCTTC-3’) to increase restriction enzyme digestion efficiency.
  • the following primers were used for PCR amplification: o OUT-BsaI-UNIVER-F GGTCCTTCGGTCTCACTAGgctgcttcgcgatgtacgggccag (SEQ ID NO: 79) o OUT-BsaI-INV-A2-R GGTCCTTCGGTCTCAGATCGCAATGAGCCATAGAGCCCACCGCATCCC CAG (SEQ ID NO: 80)
  • the bases specific for the template are in lower case, and the additional bases to create the sticky ends, including the BsaI recognition site, are shown in UPPER CASE. Thermocycling was performed.
  • Example 10 Production of hemi-modified end-closed dsDNA molecules This Example demonstrates preparation of “hemi-modified” dsDNA molecules, i.e., dsDNA molecules containing chemically modified nucleobases on a single strand (illustrated in FIG.1B).
  • the starting material for hemi-modified dsDNA is the looped end DNA (leDNA) generated in Example 9.
  • the polymerase requires a heat activation step.
  • KOD -Multi & Epi- polymerase was incubated at 94°C for two minutes. Thereafter, second-strand synthesis reactions were set up, consisting of 2 units of heat-activated KOD -Multi & Epi- polymerase, 1 microgram of looped-end DNA, dNTPs to a final concentration of 0.2 mM each, and KOD FX polymerase buffer (0.4-1x final concentration).
  • Modified deoxynucleoside triphosphates were added at various ratios with their cognate dNTP, summing to a total of 200 ⁇ M.
  • the cognate dNTP of 5-hydroxymethyluridine triphosphate, 5-formyluridine triphosphate, deoxyuridine triphosphate, 5-azidomethyluridine triphosphate, and 5-methylthiouridine is dTTP
  • the cognate dNTP of deoxyinosine triphosphate is dGTP
  • the cognate dNTP of 5-hydroxycytosine is dCTP.
  • a reaction designed for 25% incorporation would be 50 ⁇ M chemically modified dNTP and 150 ⁇ M cognate dNTP, whereas a reaction designed for 75% incorporation would be 150 ⁇ M chemically modified dNTP and 50 ⁇ M cognate dNTP.100% incorporation entails complete replacement of the cognate dNTP with the modified dNTP.
  • production of hemi-modified dsDNA entails isothermal extension of DNA from a primer, not a thermocycling process akin to polymerase chain reaction. To polymerize DNA starting from Attorney Docket No.: F2128-7017WO(VL87016-W1) the primer, reaction mixtures were incubated for 20 minutes at 68 °C.
  • DNA was concentrated using standard DNA concentration columns. Because KOD -Multi & Epi- polymerase does not have ligase activity, it does not connect the extended DNA to the short double stranded region near the downstream closed end; thus, there will be a nick in the sense DNA strand to be sealed in a ligation reaction. Therefore, ligation reactions consisting of 125 units of T4 DNA ligase (M0202M, New England Biolabs) per microgram of DNA, in a 20 ⁇ l 1x T4 DNA ligase buffer (B0202, New England Biolabs) volume, were incubated for 15 minutes at 25°C.
  • T4 DNA ligase M0202M, New England Biolabs
  • Example 11 Assessment of reporter gene expression in vitro This example demonstrates detection and quantification of gene expression using DNA molecules, e.g., looped end DNA (leDNA) or hemi-modified dsDNA molecules.
  • Experimental DNA molecules and controls were administered via lipid transfection (lipofection). Lipofection for DNA was performed using the Lipofectamine3000 transfection reagent (# L3000001, ThermoFisher) in HEKa cells. A 1:2:3 ratio of DNA:P3000:Lipofectamine3000 was used for all DNA constructs and controls.10,000 to 30,000 cells were pre-seeded into each well of 96-well plates one day before transfection. Transfection was performed when cells reached roughly 80 to 90% confluence.
  • 3X Lipofectamine3000 was first diluted in 5 uL of Opti-MEMTM I Reduced Serum Medium (#31985070, ThermoFisher). DNA was diluted in 5 uL Opti-MEMTM I Reduced Serum Medium with 2X P3000 reagent. The DNA was then added into the Lipofectamine3000 containing Opti- MEMTM I Reduced Serum Medium and mixed gently by pipetting. After incubating for 15 minutes at room temperature, the DNA-Lipofectamine3000 complex was added to target cells with full culture medium in a dropwise manner to different areas of the well.
  • the plate was gently rocked back-and-forth and side-to-side to evenly distribute the DNA-Lipofectamine3000 complex. Following transfection, cells were incubated in a CO 2 tissue culture incubator, and culture medium was changed 6 to 8 hours after transfection. To determine expression of constructs encoding the fluorescent reporter mCherry, cells were first washed with PBS before dissociation with 0.25% Trypsin (#25200056, ThermoFisher) to get single cell suspension.
  • a violet (405 nm) laser with the VL3 (603/48) filter was used.10,000 events were recorded for each sample and data were analyzed using Flowjo V.9.0 software. Cells were first gated on FSC-A and SSC-A plot to remove cell debris. The population was further plotted on an FSC-A and FSC- H plot to circumscribe the single cell population. Cell viability was evaluated based on the signal intensity of the fixable live/dead yellow dye. Cells with low signal intensity were gated as live cells, while the population with high signal intensity was gated as dead cells. Finally, a bivariate plot between the fluorescent signal-expressing and non-expressing cells was used to determine the percentage of expressing cells in the live cell population.
  • FIG.9 shows that leDNA molecules and hemi-modified dsDNA molecules comprising various chemical modifications were functional, as defined by detectable expression of the reporter protein mCherry. Specifically, the proportion of mCherry+ cells and the total fluorescence intensity of cells transfected with leDNA molecules and hemi-modified dsDNA molecules were comparable to, and in some cases greater than, control covalently closed dsDNA (comprising phosphorothioate modifications but not chemically modified nucleobases; “P6 unmodified”) and unmodified circular dsDNA.
  • Example 12 Assessment of innate immune response in cells in vitro. This example demonstrates the effect of looped-end DNA (leDNA) and hemi-modified dsDNA molecules with various chemical modifications on the innate immune response of cultured cells. Experimental constructs were prepared as in Examples 9 and 10 above, then administered to cells as in Example 11 above.
  • qPCR was performed on cells to determine the RNA level of proinflammatory cytokines, including human IL6, CXCL10. Human GAPDH was used as an endogenous control for analysis. Primer sequences can be found in Table 3. Briefly, mRNA was extracted from cells using the PicoPure RNA Isolation Kit (ThermoFisher #KIT0204). cDNA was synthesized using the RNA to cDNA EcoDryTM Premix (Oligo dT) (Takara #639542) kit. The analyses were performed using the QuantStudio7 Flex Real-time PCR System with SYBR Select Master Mix from Life Technologies Attorney Docket No.: F2128-7017WO(VL87016-W1) Corporation.
  • RNA expression was normalized to GAPDH and expressed as fold-change relative to the relevant vehicle control.
  • Table 3 Primer sequences used in qPCR quantification of immune markers.
  • Gene Name SEQ ID NO: 5'-Sequence-3' h-GAPDH-F 81 GTCTCCTCTGACTTCAACAGCG
  • FIG mical modifications led to reduced innate immune response relative to control covalently closed dsDNA (comprising phosphorothioate modifications but not chemically modified nucleobases; “P6 unmodified”) and unmodified circular dsDNA.
  • CXCL10 For two key innate immune markers – CXCL10, a widely used marker of interferon response, and IL6, a prominent pro-inflammatory cytokine) – leDNA and hemi-modified DNAs with various chemical modifications were considerably less immunogenic than control covalently closed DNA.
  • CXCL10 looped-end and hemi-modified DNAs were also considerably less immunogenic than were circular dsDNA molecules.

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Abstract

La divulgation concerne, par exemple, une molécule d'ADN double brin (ADNdb) comportant des nucléotides chimiquement modifiés. Dans certains modes de réalisation, la molécule d'ADNdb comporte une forme d'extrémité d'ADN amont qui est une extrémité fermée, une forme d'extrémité d'ADN aval qui est une extrémité fermée, et une région double brin comprenant un brin sens et un brin antisens, le brin sens présentant une ou plusieurs nucléobases chimiquement modifiées, et le brin antisens étant exempt de nucléobases chimiquement modifiées.
PCT/US2024/053928 2023-10-31 2024-10-31 Nouvelles formes d'adn thérapeutique Pending WO2025096807A2 (fr)

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