DNA molecule composition, method for preparing the same, and method for using the same

Abstract

Provided herein are double-stranded DNA molecules comprising an inverted repeat, an expression cassette, and one or more restriction sites for a nicking endonuclease, methods of their use, and methods of their production.

Description

[Technical Field]

[0001] (Priority) This application claims the benefit of priority to U.S. Serial No. 63 / 057,179, filed on July 27, 2020, and U.S. Serial No. 63 / 139,486, filed on January 20, 2021, each of which is incorporated herein by reference in its entirety.

[0002] (Reference to Electronically Submitted Sequence Listing) This application incorporates by reference the Sequence Listing submitted herewith as a text file named "14497-005-228_Seqlisting.txt", created on July 26, 2021 and having a size of 134 kilobytes.

[0003] (1. Field) Provided herein are double-stranded DNA molecules comprising one or more restriction sites for inverted repeats, expression cassettes, and nicking endonucleases, methods of use thereof, and methods of making thereof. [Background Art]

[0004] (2. Background) Gene therapy aims to treat or prevent disease by introducing genes into target cells. By supplying transcription cassettes (sometimes called transgenes) containing active gene products, gene therapy can improve clinical outcomes. This is because the gene product may acquire beneficial functional effects, lose harmful functional effects, or produce other results; for example, in cancer patients, it may have an oncolytic effect. Delivery and expression of modified genes in the patient's target cells can be carried out by many methods, including non-viral delivery (e.g., via liposomes) or viral delivery methods, including the use of engineered viruses and viral gene delivery vectors. Among the available virus-derived vectors (e.g., recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, etc.), also known as viral particles, the AAV system has become popular as a versatile vector in gene therapy.

[0005] However, there are several significant drawbacks to using viral particles as gene delivery vectors. One significant drawback is that packaging the transcription cassette within the viral particle depends on the viral life cycle and viral proteins. As a result, the use of viral vectors is limited in terms of the size of the transgene (e.g., protein-coding capacity of less than 150,000 Da in AAV) or the need for certain viral sequences (e.g., Rep-binding elements) that can destabilize the expression cassette to ensure efficient replication and packaging. Therefore, delivering large transgenes (e.g., transgenes encoding proteins larger than 150,000 Da, or transgenes longer than approximately 4.7 Kb) may require two or more viral particles. The use of two or more AAV constructs may increase the risk of AAV genome reactivation. Furthermore, the use of viral Rep or non-structural protein-binding elements may increase the risk of vector mobilization in patients.

[0006] A second drawback is that the viral particles used in gene therapy often originate from wild-type viruses to which a portion of the population may have been exposed during their lifetime. These patients are known to possess neutralizing antibodies that may impair the effectiveness of gene therapy, as further described in the literature "Adeno-associated virus: methods and protocols" by Snyder, Richard O., and Philippe Moullier, Totowa, NJ: Humana Press, 2011. For the remaining seronegative patients, the capsid of the viral vector is often immunogenic, which can hinder the re-administration of viral vector therapy if the initial dose is insufficient or if the therapy gradually becomes ineffective.

[0007] Therefore, there remains an unmet need for non-viral, capsid-free AAV-based gene therapies as a substitute for viral particles, particularly for therapies requiring the delivery of large transgenes. There is also a need for capsid-free AAV vectors that offer greater intranuclear stability, enabling extended expression compared to circular plasmid DNA. Furthermore, there is an unmet need for methods to produce these capsid-free vectors in host cells without coexisting plasmids or DNA sequences encoding the viral replication mechanism (e.g., the AAV Rep gene). This is because these viral proteins, or the viral DNA sequences encoding them, may contaminate the isolated DNA of capsid-free viral vectors. Additionally, there remains a significant unmet need for recombinant DNA vectors with improved manufacturability and / or expressivity. There is also an unmet need for DNA-based vectors that do not induce antiviral (e.g., viral capsid, Toll-like receptor activation) immune responses, enabling repeated administration without loss of efficacy (e.g., due to neutralizing antibodies) or loss of transgene-expressing cells. [Overview of the project]

[0008] (3. Overview) In one embodiment, provided herein, the top chain is in the 5' to 3' direction: a. A first reverse repeat, wherein, when the top chain separates from the bottom chain of the first reverse repeat, the nicking results in a top chain 5' overhang containing the first reverse repeat or a fragment thereof, the first and second limiting sites for the nicking endonuclease are located on opposing chains near the first reverse repeat; b. Expression cassette; and c. The second reverse repeat, wherein, when the top chain separates from the bottom chain of the second reverse repeat, the nicking results in a top chain 3' overhang containing the second reverse repeat or a fragment thereof, the third and fourth restricting sites for the nicking endonuclease are located on opposing chains near the second reverse repeat. It is a double-stranded DNA molecule containing [the specified element].

[0009] In one embodiment of this model, a. The first nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the first reverse repeat; b. The second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the first reverse repeat; c. The third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the second reverse repeat; and / or d. The fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the second reverse repeat; The first nick and the fourth nick are two nicks that occur on the top chain, the first nick is the nick closer to the 5' end of the top chain, and the fourth nick is the nick closer to the 3' end of the top chain, and The second and third nicks are two nicks that occur on the bottom chain, the third nick being the one closer to the 5' end of the bottom chain, and the second nick being the one closer to the 3' end of the bottom chain.

[0010] In one embodiment, provided herein, the top chain is in the 5' to 3' direction: a. A first reverse repeat, wherein when the top chain separates from the bottom chain of the first reverse repeat, the nicking results in a bottom chain 3' overhang containing the first reverse repeat, the first and second restricting regions for the nicking endonuclease are located on opposing chains near the first reverse repeat; b. Expression cassette; and c. The second reverse repeat, wherein when the top chain separates from the bottom chain of the second reverse repeat, the nicking results in a bottom chain 5' overhang containing the second reverse repeat, the third and fourth restricting regions for the nicking endonuclease are located on the opposing chain near the second reverse repeat. It is a double-stranded DNA molecule containing [the specified element].

[0011] In one embodiment of this model, a. The first nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the first reverse repeat; b. The second nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the first reverse repeat; c. The third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the second reverse repeat; and / or d. The fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the second reverse repeat; The first nick and the fourth nick are two nicks that occur on the top chain, the first nick is the nick closer to the 5' end of the top chain, and the fourth nick is the nick closer to the 3' end of the top chain, and The second and third nicks are two nicks that occur on the bottom chain, the third nick being the one closer to the 5' end of the bottom chain, and the second nick being the one closer to the 3' end of the bottom chain.

[0012] In one embodiment, provided herein, the top chain is in the 5' to 3' direction: a. A first reverse repeat, wherein when the top chain separates from the bottom chain of the first reverse repeat, the nicking results in a top chain 5' overhang containing the first reverse repeat, the first and second restricting regions for the nicking endonuclease are located on opposing chains near the first reverse repeat; b. Expression cassette; and c. The second reverse repeat, wherein when the top chain separates from the bottom chain of the second reverse repeat, the nicking results in a bottom chain 5' overhang containing the second reverse repeat, the third and fourth restricting regions for the nicking endonuclease are located on the opposing chain near the second reverse repeat. It is a double-stranded DNA molecule containing [the specified element].

[0013] In one embodiment of this embodiment, a. The first nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the first reverse repeat; b. The second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the first reverse repeat; c. The third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the second reverse repeat; and / or d. The fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the second reverse repeat; The first nick and the fourth nick are two nicks that occur on the top chain, the first nick is the nick closer to the 5' end of the top chain, and the fourth nick is the nick closer to the 3' end of the top chain, and The second and third nicks are two nicks that occur on the bottom chain, the third nick being the one closer to the 5' end of the bottom chain, and the second nick being the one closer to the 3' end of the bottom chain.

[0014] In one aspect, provided herein is, in the 5' to 3' direction of the top strand: a. a first inverted repeat, wherein first and second restriction sites for a nicking endonuclease are positioned on opposite strands near the first inverted repeat, such that when the top strand separates from the bottom strand of the first inverted repeat, nicking results in a 3' overhang on the bottom strand comprising the first inverted repeat; said first inverted repeat; b. an expression cassette; and c. a second inverted repeat, wherein third and fourth restriction sites for a nicking endonuclease are positioned on opposite strands near the second inverted repeat, such that when the top strand separates from the bottom strand of the second inverted repeat, nicking results in a 3' overhang on the top strand comprising the second inverted repeat; said second inverted repeat a double-stranded DNA molecule comprising

[0015] In one embodiment of this aspect, a. a first nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closing base pair of said first inverted repeat; b. a second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of said ITR closing base pair of said first inverted repeat; c. a third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closing base pair of said second inverted repeat; and / or d. a fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of said ITR closing base pair of said second inverted repeat; The first nick and the fourth nick are two nicks that occur on the top chain, the first nick is the nick closer to the 5' end of the top chain, and the fourth nick is the nick closer to the 3' end of the top chain, and The second and third nicks are two nicks that occur on the bottom chain, the third nick being the one closer to the 5' end of the bottom chain, and the second nick being the one closer to the 3' end of the bottom chain.

[0016] In one embodiment, the first nick, the second nick, the third nick, and / or the fourth nick are inside the reverse iteration. In one embodiment, the first nick, the second nick, the third nick, and / or the fourth nick are outside the reverse iteration.

[0017] In one embodiment, the double-stranded DNA molecule is an isolated DNA molecule.

[0018] In one embodiment, the first, second, third, and fourth restriction sites for the nickel endonuclease are all the same restriction site for the nickel endonuclease.

[0019] In one embodiment, the first and second reverse repeats are identical. In one embodiment, the first and / or second reverse repeats are an ITR of a parvovirus. In one embodiment, the first and / or second reverse repeats are a modified ITR of a parvovirus. In a specific embodiment, the parvovirus is a dependent parvovirus, bocaparvovirus, erythroparvovirus, protoparvovirus, or tetraparvovirus. In a specific embodiment, the nucleotide sequence of the modified ITR is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% identical to the ITR of the parvovirus.

[0020] In one embodiment, the double-stranded DNA molecule is a plasmid.

[0021] In a specific embodiment, the plasmid further includes a bacterial origin of replication.

[0022] In a specific embodiment, the plasmid further includes a restriction enzyme site in the region 5' relative to the first reverse repeat and 3' relative to the second reverse repeat, wherein the restriction enzyme site is not located in the first reverse repeat, the second reverse repeat, or the region between the first and second reverse repeats. In a specific embodiment, cleavage with the restriction enzyme results in a single-stranded overhang that does not anneal at a detectable level under conditions suitable for annealing of the first and / or second reverse repeats. In a specific embodiment, the plasmid further includes an open reading frame encoding the restriction enzyme. In a specific embodiment, the expression of the restriction enzyme is under the control of an inducible promoter.

[0023] In one embodiment, the foregoing provides a method for preparing hairpin-terminated DNA: a. Culturing host cells containing the double-stranded DNA molecule described in the previous paragraph under conditions that result in amplification of the double-stranded DNA molecule; b. To cause the double-stranded DNA molecule to be released from the host cell; c. Incubating the double-stranded DNA molecule with one or more nicking endonucleases that recognize the four restriction sites and produce four nicks; d. To perform denaturation, thereby generating a DNA fragment that includes the expression cassette and is adjacent to the two single-stranded DNA overhangs; e. To cause intramolecular annealing of the single-stranded DNA overhang, thereby generating hairpinned reverse repeats at both ends of the DNA fragment obtained as a result of step 77.d; f. Incubating the double-stranded DNA molecule or the fragment obtained as a result of step 77.d with the restriction enzyme, and thereby cleaving the double-stranded DNA molecule or the fragment of the double-stranded DNA molecule; and g. Incubate the fragment of the double-stranded DNA molecule with an exonuclease to digest the fragment of the double-stranded DNA molecule, excluding the fragment obtained as a result of step 77.e. The method includes the above.

[0024] In a specific embodiment, the plasmid further includes fifth and sixth restriction sites for nicking endonucleases in a region that is 5' relative to the first reverse repeat and 3' relative to the second reverse repeat, wherein the fifth and sixth restriction sites for nicking endonucleases: a. are on opposite strands; and b. cause the cleavage to occur within the double-stranded DNA molecule such that the single-stranded overhang of the cleavage does not undergo intermolecular or molecular annealing at a level detectable under conditions suitable for annealing of the first and / or the second reverse repeat. In a specific embodiment, the fifth and sixth nicks are separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In a specific embodiment, the first, second, third, fourth, fifth, and sixth restriction sites for the nickel endonuclease are all the same nickel endonuclease target sequence. In a specific embodiment, the plasmid further includes an open reading frame encoding a nickel endonuclease that recognizes the first, second, third, fourth, fifth, and / or sixth restriction sites for the nickel endonuclease. In a specific embodiment, the expression of the nickel endonuclease is under the control of an inducible promoter. In one embodiment, the nickel endonucleases that recognize the fifth and sixth restriction sites for nickel endonucleases are Nt.BsmAI;Nt.BtsCI;N.ALwl;N.BstNBI;N.BspD6I;Nb.Mva1269I;Nb.BsrDI;Nt.BtsI;Nt.BsaI;Nt.Bpu10I;Nt.BsmBI;Nb.BbvCI;Nt.BbvCI; or Nt.BspQI.

[0025] In one embodiment, the foregoing provides a method for preparing hairpin-terminated DNA: a. Culturing host cells containing the double-stranded DNA molecule described in the previous paragraph under conditions that result in amplification of the double-stranded DNA molecule; b. To cause the double-stranded DNA molecule to be released from the host cell; c. Incubating the double-stranded DNA molecule with one or more nicking endonucleases that recognize the first, second, third, and fourth restriction sites and produce four nicks; d. To perform denaturation, thereby generating a DNA fragment that includes the expression cassette and is adjacent to the two single-stranded DNA overhangs; e. To cause intramolecular annealing of the single-stranded DNA overhang, thereby generating hairpinned reverse repeats at both ends of the DNA fragment obtained as a result of step 78.d; f. Incubating the double-stranded DNA molecule or the fragment obtained as a result of step 78.d with one or more nickel endonucleases that recognize the fifth and sixth restriction sites resulting in cleavage in the double-stranded DNA molecule; and g. Incubate the fragment of the double-stranded DNA molecule with an exonuclease to digest the fragment of the double-stranded DNA molecule, excluding the fragment obtained as a result of step 78.e. The method includes the above.

[0026] In one embodiment, one or more of the nickel endonuclease sites are target sequences of endogenous nickel endonucleases.

[0027] In one embodiment, the nickel endonucleases that recognize the first, second, third, and / or fourth restriction sites for nickel endonucleases are Nt.BsmAI;Nt.BtsCI;N.ALwl;N.BstNBI;N.BspD6I;Nb.Mva1269I;Nb.BsrDI;Nt.BtsI;Nt.BsaI;Nt.Bpu10I;Nt.BsmBI;Nb.BbvCI;Nt.BbvCI; or Nt.BspQI.

[0028] In various embodiments, the expression cassette includes a promoter operatively linked to a transcription unit. In some embodiments, the transcription unit includes an open reading frame. In some embodiments, the expression cassette further includes a post-transcriptional regulator. In some embodiments, the expression cassette further includes polyadenylation and termination signals. In some embodiments, the size of the expression cassette is at least 4kb, at least 4.5kb, at least 5kb, at least 5.5kb, at least 6kb, at least 6.5kb, at least 7kb, at least 7.5kb, at least 8kb, at least 8.5kb, at least 9kb, at least 9.5kb, or at least 10kb.

[0029] In one embodiment, the method for preparing hairpin-terminated DNA molecules is provided herein: a. Culturing host cells containing the double-stranded DNA molecule described above under conditions that result in amplification of the double-stranded DNA molecule; b. To cause the double-stranded DNA molecule to be released from the host cell; c. Incubating the double-stranded DNA molecule with one or more nicking endonucleases that recognize the four restriction sites and produce four nicks; d. To perform denaturation, thereby generating a DNA fragment that includes the expression cassette and is adjacent to the two single-stranded DNA overhangs; e. The method comprising intramolecular annealing of the single-stranded DNA overhang and thereby generating hairpinned reverse repeats at both ends of the DNA fragment obtained as a result of step d.

[0030] In one embodiment of the method for preparing hairpin-terminated DNA molecules described herein, the method further comprises repairing the nick using a ligase to generate circular DNA.

[0031] In one embodiment of the method for preparing hairpin-terminated DNA molecules described herein, the steps are performed in the order in which they appear in the embodiment.

[0032] In one embodiment of the method for preparing hairpin-terminated DNA molecules described herein, the hairpin-terminated DNA consists of two hairpin ends.

[0033] In one embodiment of the method for preparing hairpin-terminated DNA molecules described herein, the hairpin-terminated DNA is a viral genome. In a specific embodiment, the viral genome is a parvovirus genome. In a specific embodiment, the parvovirus is dependent parvovirus, bocaparvovirus, erythroparvovirus, protoparvovirus, or tetraparvovirus.

[0034] In one embodiment, provided herein, the top chain is in the 5' to 3' direction: a. First hairpin reverse iteration; b. Nicks in the bottom chain; c. Expression cassette; d. Nicks of the bottom chain; and e. Second hairpin transformation reverse iteration It is a double-stranded DNA molecule containing [the specified element].

[0035] In one embodiment, provided herein, the top chain is in the 5' to 3' direction: a. First hairpin reverse iteration; b. Nicks in the top chain; c. Expression cassette; d. Nicks on the top chain; and e. Second hairpin transformation reverse iteration It is a double-stranded DNA molecule containing [the specified element].

[0036] In one embodiment, provided herein, the top chain is in the 5' to 3' direction: a. First hairpin reverse iteration; b. Nicks in the bottom chain; c. Expression cassette; d. Nicks on the top chain; and e. Second hairpin transformation reverse iteration It is a double-stranded DNA molecule containing [the specified element].

[0037] In one embodiment, provided herein, the top chain is in the 5' to 3' direction: a. First hairpin reverse iteration; b. Nicks in the top chain; c. Expression cassette; d. Nicks in the bottom chain; and e. Second hairpin transformation reverse iteration It is a double-stranded DNA molecule containing [the specified element].

[0038] In one embodiment, the double-stranded DNA molecule is an isolated DNA molecule.

[0039] In one embodiment, the first and / or second reverse repeats are an ITR of a parvovirus. In one embodiment, the first and second reverse repeats are identical. In one embodiment, the first and / or second reverse repeats are a modified ITR of a parvovirus. In a specific embodiment, the parvovirus is a dependent parvovirus, bocaparvovirus, erythroparvovirus, protoparvovirus, or tetraparvovirus. In a specific embodiment, the nucleotide sequence of the modified ITR is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% identical to the ITR of the parvovirus.

[0040] In various embodiments, the expression cassette includes a promoter operatively linked to a transcription unit. In some embodiments, the transcription unit includes an open reading frame. In some embodiments, the expression cassette further includes a post-transcriptional regulator. In some embodiments, the expression cassette further includes polyadenylation and termination signals. In some embodiments, the size of the expression cassette is at least 4kb, at least 4.5kb, at least 5kb, at least 5.5kb, at least 6kb, at least 6.5kb, at least 7kb, at least 7.5kb, at least 8kb, at least 8.5kb, at least 9kb, at least 9.5kb, or at least 10kb.

[0041] In one embodiment, the double-stranded DNA molecule is protected from the exonuclease. In a specific embodiment, the exonuclease is RecBCD exonuclease.

[0042] In one embodiment, the double-stranded DNA molecule lacks at least one replication-associated protein binding sequence ("RABS"). In one embodiment, the double-stranded DNA molecule lacks a replication-associated protein ("RAP") coding sequence. In one embodiment, the double-stranded DNA molecule lacks a viral capsid protein coding sequence. In a specific embodiment, the first reverse repeat lacks at least one RABS. In a specific embodiment, the second reverse repeat lacks at least one RABS. In a specific embodiment, the DNA sequence between the ITR closed base pair of the first reverse repeat and the ITR closed base pair of the second reverse repeat lacks at least one RABS. In a specific embodiment, the first reverse repeat lacks at least one RABS, and the second reverse repeat lacks at least one RABS.

[0043] In one embodiment, the double-stranded DNA molecule lacks a terminal separation site (TRS). In a specific embodiment, the first reverse repeat lacks a TRS. In a specific embodiment, the second reverse repeat lacks a TRS. In a specific embodiment, the DNA sequence between the ITR closed base pair of the first reverse repeat and the ITR closed base pair of the second reverse repeat lacks a TRS. In a specific embodiment, the first reverse repeat lacks a TRS, and the second reverse repeat lacks a TRS.

[0044] In one embodiment, the risk of mobilization of the double-stranded DNA molecule when administered to a host is 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% lower than that of a control DNA molecule having at least one RABS and / or the TRS.

[0045] In one embodiment in which the double-stranded DNA molecule lacks TRS, the risk of mobilization of the double-stranded DNA molecule when administered to a host is 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% lower than that of a control DNA molecule having TRS.

[0046] In one embodiment in which the double-stranded DNA molecule lacks at least one RABS and lacks a TRS, the risk of mobilization of the double-stranded DNA molecule when administered to a host is 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% lower than that of a control DNA molecule having at least one RABS and the TRS.

[0047] In one embodiment, the herein provides an isolated double-stranded DNA molecule as described herein, which does not contain any fragments of the double-stranded DNA molecule.

[0048] In one embodiment, provided herein are isolated double-stranded DNA molecules, wherein the fragments of the double-stranded DNA molecule do not exceed 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the isolated double-stranded DNA molecule.

[0049] In one embodiment, the herein provides an isolated double-stranded DNA molecule that is free from nucleic acid contaminants that are not fragments of the double-stranded DNA molecule.

[0050] In one embodiment, provided herein are isolated double-stranded DNA molecules wherein nucleic acid contaminants that are not fragments of the double-stranded DNA molecule constitute less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the isolated double-stranded DNA molecule.

[0051] In one embodiment, the herein provides an isolated double-stranded DNA molecule that does not contain baculovirus DNA.

[0052] In one embodiment, provided herein are isolated double-stranded DNA molecules wherein the baculovirus DNA constitutes less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the isolated double-stranded DNA molecule.

[0053] In one embodiment, the herein provides a delivery vehicle comprising a double-stranded DNA molecule as described herein. In one embodiment, the delivery vehicle comprises a hybridosome, a liposome, or lipid nanoparticles.

[0054] (3.1 Exemplary Embodiment Set 1) (Embodiment 1) From the 5' to 3' direction of the top chain: a. A first reverse repeat, wherein, when the top chain separates from the bottom chain of the first reverse repeat, the nicking results in a top chain 5' overhang containing the first reverse repeat or a fragment thereof, the first and second limiting sites for the nicking endonuclease are located on opposing chains near the first reverse repeat; b. Expression cassette; and c. The second reverse repeat, wherein, when the top chain separates from the bottom chain of the second reverse repeat, the nicking results in a top chain 3' overhang containing the second reverse repeat or a fragment thereof, the third and fourth restricting sites for the nicking endonuclease are located on opposing chains near the second reverse repeat. A double-stranded DNA molecule containing [the specified element].

[0055] (Embodiment 2) From the 5' to 3' direction of the top chain: a. A first reverse repeat, wherein when the top chain separates from the bottom chain of the first reverse repeat, the nicking results in a bottom chain 3' overhang containing the first reverse repeat, the first and second restricting regions for the nicking endonuclease are located on opposing chains near the first reverse repeat; b. Expression cassette; and c. The second reverse repeat, wherein when the top chain separates from the bottom chain of the second reverse repeat, the nicking results in a bottom chain 5' overhang containing the second reverse repeat, the third and fourth restricting regions for the nicking endonuclease are located on the opposing chain near the second reverse repeat. A double-stranded DNA molecule containing [the specified element].

[0056] (Embodiment 3) From the 5' to 3' direction of the top chain: a. A first reverse repeat, wherein when the top chain separates from the bottom chain of the first reverse repeat, the nicking results in a top chain 5' overhang containing the first reverse repeat, the first and second restricting regions for the nicking endonuclease are located on opposing chains near the first reverse repeat; b. Expression cassette; and c. The second reverse repeat, wherein when the top chain separates from the bottom chain of the second reverse repeat, the nicking results in a bottom chain 5' overhang containing the second reverse repeat, the third and fourth restricting regions for the nicking endonuclease are located on the opposing chain near the second reverse repeat. A double-stranded DNA molecule containing [the specified element].

[0057] (Embodiment 4) From the 5' to 3' direction of the top chain: a. A first reverse repeat, wherein when the top chain separates from the bottom chain of the first reverse repeat, the nicking results in a bottom chain 3' overhang containing the first reverse repeat, the first and second restricting regions for the nicking endonuclease are located on opposing chains near the first reverse repeat; b. Expression cassette; and c. The second reverse repeat, wherein when the top chain separates from the bottom chain of the second reverse repeat, the nicking results in a top chain 3' overhang containing the second reverse repeat, the third and fourth restricting regions for the nicking endonuclease are located on the opposing chain near the second reverse repeat. A double-stranded DNA molecule containing [the specified element].

[0058] (Embodiment 5) The double-stranded DNA molecule according to any one of Embodiments 1 to 4, wherein the double-stranded DNA molecule is an isolated DNA molecule.

[0059] (Embodiment 6) A double-stranded DNA molecule according to any one of embodiments 1 to 5, wherein the first, second, third, and fourth restriction sites for nickel endonucleases are all the same restriction site for nickel endonucleases.

[0060] (Embodiment 7) A double-stranded DNA molecule according to any one of Embodiments 1 to 5, wherein the first and second reverse repeats are identical.

[0061] (Embodiment 8) A double-stranded DNA molecule according to any one of embodiments 1 to 5, wherein the first and / or second reverse repeats are the parvovirus ITR.

[0062] (Embodiment 9) A double-stranded DNA molecule according to any one of embodiments 1 to 5, wherein the first and / or second reverse repeats are modified parvovirus ITRs.

[0063] (Embodiment 10) The double-stranded DNA molecule according to Embodiment 8 or 9, wherein the parvovirus is dependent parvovirus, boca parvovirus, erythroparvovirus, protoparvovirus, or tetraparvovirus.

[0064] (Embodiment 11) The double-stranded DNA molecule according to Embodiment 9, wherein the nucleotide sequence of the modified ITR is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% identical to the ITR of the parvovirus.

[0065] (Embodiment 12) a. The first nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the first reverse repeat; b. The second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the first reverse repeat; c. The third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the second reverse repeat; and / or d. The fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the second reverse repeat; The first nick and the fourth nick are two nicks that occur on the top chain, the first nick is the nick closer to the 5' end of the top chain, and the fourth nick is the nick closer to the 3' end of the top chain, and The second and third nicks are two nicks that occur on the bottom chain, the third nick being the nick closer to the 5' end of the bottom chain, and the second nick being the nick closer to the 3' end of the bottom chain. A double-stranded DNA molecule according to Embodiment 1 or 5.

[0066] (Embodiment 13) a. The first nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the first reverse repeat; b. The second nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the first reverse repeat; c. The third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the second reverse repeat; and / or d. The fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the second reverse repeat; The first nick and the fourth nick are two nicks that occur on the top chain, the first nick is the nick closer to the 5' end of the top chain, and the fourth nick is the nick closer to the 3' end of the top chain, and The second and third nicks are two nicks that occur on the bottom chain, the third nick being the nick closer to the 5' end of the bottom chain, and the second nick being the nick closer to the 3' end of the bottom chain. A double-stranded DNA molecule according to Embodiment 2 or 5.

[0067] (Embodiment 14) a. The first nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the first reverse repeat; b. The second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the first reverse repeat; c. The third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the second reverse repeat; and / or d. The fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the second reverse repeat; The first nick and the fourth nick are two nicks that occur on the top chain, the first nick is the nick closer to the 5' end of the top chain, and the fourth nick is the nick closer to the 3' end of the top chain, and The second and third nicks are two nicks that occur on the bottom chain, the third nick being the nick closer to the 5' end of the bottom chain, and the second nick being the nick closer to the 3' end of the bottom chain. A double-stranded DNA molecule according to Embodiment 3 or 5.

[0068] (Embodiment 15) a. The first nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the first reverse repeat; b. The second nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the first reverse repeat; c. The third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the second reverse repeat; and / or d. The fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the second reverse repeat; The first nick and the fourth nick are two nicks that occur on the top chain, the first nick is the nick closer to the 5' end of the top chain, and the fourth nick is the nick closer to the 3' end of the top chain, and The second and third nicks are two nicks that occur on the bottom chain, the third nick being the nick closer to the 5' end of the bottom chain, and the second nick being the nick closer to the 3' end of the bottom chain. A double-stranded DNA molecule according to Embodiment 4 or 5.

[0069] (Embodiment 16) A double-stranded DNA molecule according to any one of embodiments 12 to 15, wherein the first nick, the second nick, the third nick, and / or the fourth nick are located within the reverse repeat.

[0070] (Embodiment 17) A double-stranded DNA molecule according to any one of embodiments 12 to 15, wherein the first nick, the second nick, the third nick, and / or the fourth nick are located outside the reverse repeat.

[0071] (Embodiment 18) A plasmid, a double-stranded DNA molecule as described in any one of embodiments 1 to 17.

[0072] (Embodiment 19) The double-stranded DNA molecule according to Embodiment 18, wherein the plasmid further comprises a bacterial origin of replication.

[0073] (Embodiment 20) The double-stranded DNA molecule according to Embodiment 18, wherein the plasmid further includes a restriction enzyme site in the region 5' with respect to the first reverse repeat and 3' with respect to the second reverse repeat, and the restriction enzyme site is not located in the first reverse repeat, the second reverse repeat, or the region between the first and second reverse repeats.

[0074] (Embodiment 21) A double-stranded DNA molecule according to Embodiment 20, wherein cleavage with the restriction enzyme results in a single-stranded overhang that does not anneal at a detectable level under conditions suitable for annealing of the first reverse repeat and / or the second reverse repeat.

[0075] (Embodiment 22) The double-stranded DNA molecule according to Embodiment 20, wherein the plasmid further comprises an open reading frame encoding the restriction enzyme.

[0076] (Embodiment 23) The double-stranded DNA molecule according to Embodiment 22, wherein the expression of the restriction enzyme is under the control of an inducible promoter.

[0077] (Embodiment 24) The plasmid further includes fifth and sixth restriction sites for a nickel endonuclease in a region that is 5' relative to the first reverse repeat and 3' relative to the second reverse repeat, wherein the fifth and sixth restriction sites for the nickel endonuclease are: a. On opposing chains; and b. The break occurs within the double-stranded DNA molecule such that the single-stranded overhang of the break does not undergo intermolecular or molecular annealing at a detectable level under conditions suitable for annealing of the first reverse repeat and / or the second reverse repeat. A double-stranded DNA molecule according to Embodiment 18.

[0078] (Embodiment 25) The double-stranded DNA molecule according to Embodiment 24, wherein the fifth and sixth nicks are separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0079] (Embodiment 26) A double-stranded DNA molecule according to Embodiment 24, wherein the first, second, third, fourth, fifth, and sixth restriction sites for nickel endonucleases are all the same nickel endonuclease target sequence.

[0080] (Embodiment 27) A double-stranded DNA molecule according to any one of embodiments 1 to 26, wherein one or more of the aforementioned nickel endonuclease sites are target sequences of endogenous nickel endonucleases.

[0081] (Embodiment 28) A double-stranded DNA molecule according to any one of embodiments 24 to 27, wherein the plasmid further comprises an open reading frame encoding a nickeling endonuclease that recognizes the first, second, third, fourth, fifth, and / or sixth restriction sites for the nickeling endonuclease.

[0082] (Embodiment 29) The double-stranded DNA molecule according to Embodiment 28, wherein the expression of the aforementioned nickel endonuclease is under the control of an inducible promoter.

[0083] (Embodiment 30) A double-stranded DNA molecule according to any one of embodiments 1 to 29, wherein the nickeling endonuclease that recognizes the first, second, third, and / or fourth restriction sites for the nickeling endonuclease is Nt.BsmAI;Nt.BtsCI;N.ALwl;N.BstNBI;N.BspD6I;Nb.Mva1269I;Nb.BsrDI;Nt.BtsI;Nt.BsaI;Nt.Bpu10I;Nt.BsmBI;Nb.BbvCI;Nt.BbvCI; or Nt.BspQI.

[0084] (Embodiment 31) A double-stranded DNA molecule according to embodiment 24, wherein the nickeling endonuclease that recognizes the fifth and sixth restriction sites for nickeling endonuclease is Nt.BsmAI;Nt.BtsCI;N.ALwl;N.BstNBI;N.BspD6I;Nb.Mva1269I;Nb.BsrDI;Nt.BtsI;Nt.BsaI;Nt.Bpu10I;Nt.BsmBI;Nb.BbvCI;Nt.BbvCI; or Nt.BspQI.

[0085] (Embodiment 32) A double-stranded DNA molecule according to any one of Embodiments 1 to 31, wherein the expression cassette includes a promoter operatively linked to a transcription unit.

[0086] (Embodiment 33) The double-stranded DNA molecule according to Embodiment 32, wherein the transcription unit includes an open reading frame.

[0087] (Embodiment 34) The double-stranded DNA molecule according to embodiment 32 or 33, wherein the expression cassette further comprises a post-transcriptional regulatory factor.

[0088] (Embodiment 35) The double-stranded DNA molecule according to Embodiment 32 or 33, wherein the expression cassette further comprises polyadenylation and a termination signal.

[0089] (Embodiment 36) A double-stranded DNA molecule according to any one of embodiments 32 to 35, wherein the size of the expression cassette is at least 4kb, at least 4.5kb, at least 5kb, at least 5.5kb, at least 6kb, at least 6.5kb, at least 7kb, at least 7.5kb, at least 8kb, at least 8.5kb, at least 9kb, at least 9.5kb, or at least 10kb.

[0090] (Embodiment 37) From the 5' to 3' direction of the top chain: a. First hairpin reverse iteration; b. Nicks in the bottom chain; c. Expression cassette; d. Nicks of the bottom chain; and e. Second hairpin transformation reverse iteration A double-stranded DNA molecule containing [the specified element].

[0091] (Embodiment 38) From the 5' to 3' direction of the top chain: a. First hairpin reverse iteration; b. Nicks in the top chain; c. Expression cassette; d. Nicks on the top chain; and e. Second hairpin transformation reverse iteration A double-stranded DNA molecule containing [the specified element].

[0092] (Embodiment 39) From the 5' to 3' direction of the top chain: a. First hairpin reverse iteration; b. Nicks in the bottom chain; c. Expression cassette; d. Nicks on the top chain; and e. Second hairpin transformation reverse iteration A double-stranded DNA molecule containing [the specified element].

[0093] (Embodiment 40) From the 5' to 3' direction of the top chain: a. First hairpin reverse iteration; b. Nicks in the top chain; c. Expression cassette; d. Nicks in the bottom chain; and e. Second hairpin transformation reverse iteration A double-stranded DNA molecule containing [the specified element].

[0094] (Embodiment 41) An isolated DNA molecule, a double-stranded DNA molecule as described in any one of embodiments 37 to 40.

[0095] (Embodiment 42) A double-stranded DNA molecule according to any one of embodiments 37 to 41, wherein the first and / or second reverse repeats are the parvovirus ITR.

[0096] (Embodiment 43) A double-stranded DNA molecule according to any one of embodiments 37 to 41, wherein the first and second reverse repeats are identical.

[0097] (Embodiment 44) A double-stranded DNA molecule according to any one of embodiments 37 to 41, wherein the first and / or second reverse repeats are modified parvovirus ITRs.

[0098] (Embodiment 45) The double-stranded DNA molecule according to Embodiment 42 or 44, wherein the parvovirus is dependent parvovirus, boca parvovirus, erythroparvovirus, protoparvovirus, or tetraparvovirus.

[0099] (Embodiment 46) The double-stranded DNA molecule according to Embodiment 45, wherein the nucleotide sequence of the modified ITR is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% identical to the ITR of the parvovirus.

[0100] (Embodiment 47) A double-stranded DNA molecule according to any one of embodiments 37 to 46, wherein the expression cassette includes a promoter operatively linked to a transcription unit.

[0101] (Embodiment 48) The double-stranded DNA molecule according to Embodiment 47, wherein the transcription unit includes an open reading frame.

[0102] (Embodiment 49) The double-stranded DNA molecule according to Embodiment 47 or 48, wherein the expression cassette further comprises a post-transcriptional regulatory factor.

[0103] (Embodiment 50) The double-stranded DNA molecule according to Embodiment 47 or 48, wherein the expression cassette further comprises polyadenylation and a termination signal.

[0104] (Embodiment 51) A double-stranded DNA molecule according to any one of embodiments 37 to 50, wherein the size of the expression cassette is at least 4kb, at least 4.5kb, at least 5kb, at least 5.5kb, at least 6kb, at least 6.5kb, at least 7kb, at least 7.5kb, at least 8kb, at least 8.5kb, at least 9kb, at least 9.5kb, or at least 10kb.

[0105] (Embodiment 52) A double-stranded DNA molecule protected from exonucleases, as described in any one of embodiments 37 to 51.

[0106] (Embodiment 53) The double-stranded DNA molecule according to Embodiment 52, wherein the exonuclease is RecBCD exonuclease.

[0107] (Embodiment 54) The double-stranded DNA molecule according to any one of Embodiments 1 to 53, wherein the double-stranded DNA molecule lacks at least one replication-associated protein-binding sequence ("RABS").

[0108] (Embodiment 55) The double-stranded DNA molecule according to any one of Embodiments 1 to 54, wherein the double-stranded DNA molecule lacks a replication-associated protein ("RAP") coding sequence.

[0109] (Embodiment 56) The double-stranded DNA molecule according to any one of Embodiments 1 to 55, wherein the double-stranded DNA molecule lacks a viral capsid protein coding sequence.

[0110] (Embodiment 57) A double-stranded DNA molecule according to any one of embodiments 1 to 56, wherein the first reverse repeat lacks at least one RABS.

[0111] (Embodiment 58) A double-stranded DNA molecule according to any one of embodiments 1 to 57, wherein the second reverse repeat lacks at least one RABS.

[0112] (Embodiment 59) A double-stranded DNA molecule according to any one of Embodiments 1 to 58, wherein the DNA sequence between the ITR closed base pair of the first reverse repeat and the ITR closed base pair of the second reverse repeat lacks at least one RABS.

[0113] (Embodiment 60) A double-stranded DNA molecule according to any one of embodiments 1 to 59, wherein the first reverse repeat lacks at least one RABS, and the second reverse repeat lacks at least one RABS.

[0114] (Embodiment 61) The double-stranded DNA molecule according to any one of Embodiments 1 to 60, wherein the double-stranded DNA molecule lacks a terminal separation site (TRS).

[0115] (Embodiment 62) The first reverse repeat is a double-stranded DNA molecule according to any one of embodiments 1 to 61, wherein the first reverse repeat lacks a TRS.

[0116] (Embodiment 63) The double-stranded DNA molecule according to any one of embodiments 1 to 62, wherein the second reverse repeat lacks a TRS.

[0117] (Embodiment 64) A double-stranded DNA molecule according to any one of embodiments 1 to 63, wherein the DNA sequence between the ITR closed base pair of the first reverse repeat and the ITR closed base pair of the second reverse repeat lacks a TRS.

[0118] (Embodiment 65) A double-stranded DNA molecule according to any one of embodiments 1 to 64, wherein the first reverse repeat lacks a TRS and the second reverse repeat also lacks a TRS.

[0119] (Embodiment 66) A double-stranded DNA molecule according to any one of embodiments 53 and 56-64, wherein the risk of mobilization of the double-stranded DNA molecule when administered to a host is 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% lower than that of a control DNA molecule having at least one RABS and / or the TRS.

[0120] (Embodiment 67) A double-stranded DNA molecule according to any one of embodiments 53 and 56-64, lacking TRS, wherein the risk of mobilization of the double-stranded DNA molecule when administered to a host is 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% lower than that of a control DNA molecule having TRS.

[0121] (Embodiment 68) A double-stranded DNA molecule according to any one of embodiments 53 and 56-64, lacking at least one RABS and lacking a TRS, wherein the risk of mobilization of the double-stranded DNA molecule when administered to a host is 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% lower than a control DNA molecule having the at least one RABS and the TRS.

[0122] (Embodiment 69) An isolated double-stranded DNA molecule according to any one of embodiments 5 to 67, which does not contain the fragments of the double-stranded DNA molecule.

[0123] (Embodiment 70) An isolated double-stranded DNA molecule according to any one of embodiments 5 to 68, wherein the fragments of the double-stranded DNA molecule do not exceed 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the isolated double-stranded DNA molecule.

[0124] (Embodiment 71) An isolated double-stranded DNA molecule according to any one of embodiments 5 to 69, which does not contain nucleic acid impurities other than fragments of the double-stranded DNA molecule.

[0125] (Embodiment 72) An isolated double-stranded DNA molecule according to any one of embodiments 5 to 70, wherein nucleic acid contaminants that are not fragments of the double-stranded DNA molecule constitute less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the isolated double-stranded DNA molecule.

[0126] (Embodiment 73) An isolated double-stranded DNA molecule according to any one of embodiments 5 to 71, which does not contain baculovirus DNA.

[0127] (Embodiment 74) An isolated double-stranded DNA molecule according to any one of embodiments 5 to 72, wherein the baculovirus DNA makes up less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the isolated double-stranded DNA molecule.

[0128] (Embodiment 75) A delivery vehicle comprising a double-stranded DNA molecule according to any one of embodiments 37 to 73.

[0129] (Embodiment 76) A delivery vehicle according to Embodiment 74, comprising hybridosomes, liposomes, or lipid nanoparticles.

[0130] (Embodiment 77) A method for preparing hairpin-terminated DNA molecules: a. Culturing host cells containing a double-stranded DNA molecule according to any one of Embodiments 1 to 35 under conditions that result in amplification of the double-stranded DNA molecule; b. To cause the double-stranded DNA molecule to be released from the host cell; c. Incubating the double-stranded DNA molecule with one or more nicking endonucleases that recognize the four restriction sites and produce four nicks; d. To perform denaturation, thereby generating a DNA fragment that includes the expression cassette and is adjacent to the two single-stranded DNA overhangs; e. To perform intramolecular annealing on the single-stranded DNA overhang, thereby generating hairpinned reverse repeats at both ends of the DNA fragment obtained as a result of step 76.d. The method comprising the above.

[0131] (Embodiment 78) A method for preparing hairpin-end DNA: a. Culturing host cells containing the double-stranded DNA molecule described in Embodiment 20 under conditions that result in amplification of the double-stranded DNA molecule; b. To cause the double-stranded DNA molecule to be released from the host cell; c. Incubating the double-stranded DNA molecule with one or more nicking endonucleases that recognize the four restriction sites and produce four nicks; d. To perform denaturation, thereby generating a DNA fragment that includes the expression cassette and is adjacent to the two single-stranded DNA overhangs; e. To cause intramolecular annealing of the single-stranded DNA overhang, thereby generating hairpinned reverse repeats at both ends of the DNA fragment obtained as a result of step 77.d; f. Incubating the double-stranded DNA molecule or the fragment obtained as a result of step 77.d with the restriction enzyme, and thereby cleaving the double-stranded DNA molecule or the fragment of the double-stranded DNA molecule; and g. Incubate the fragment of the double-stranded DNA molecule with an exonuclease to digest the fragment of the double-stranded DNA molecule, excluding the fragment obtained as a result of step 77.e. The method, including the method described above.

[0132] (Embodiment 79) A method for preparing hairpin-end DNA: a. Culturing host cells containing the double-stranded DNA molecule described in Embodiment 24 under conditions that result in amplification of the double-stranded DNA molecule; b. To cause the double-stranded DNA molecule to be released from the host cell; c. Incubating the double-stranded DNA molecule with one or more nicking endonucleases that recognize the first, second, third, and fourth restriction sites and produce four nicks; d. To perform denaturation, thereby generating a DNA fragment that includes the expression cassette and is adjacent to the two single-stranded DNA overhangs; e. To cause intramolecular annealing of the single-stranded DNA overhang, thereby generating hairpinned reverse repeats at both ends of the DNA fragment obtained as a result of step 78.d; f. Incubating the double-stranded DNA molecule or the fragment obtained as a result of step 78.d with one or more nickel endonucleases that recognize the fifth and sixth restriction sites resulting in cleavage in the double-stranded DNA molecule; and g. Incubate the fragment of the double-stranded DNA molecule with an exonuclease to digest the fragment of the double-stranded DNA molecule, excluding the fragment obtained as a result of step 78.e. The method, including the method described above.

[0133] (Embodiment 80) The method according to embodiment 76, 77, or 78, further comprising repairing the nick using h. ligase to generate circular DNA.

[0134] (Embodiment 81) The method according to any one of embodiments 76 to 79, wherein the steps are performed in the order in which they appear in the embodiment.

[0135] (Embodiment 82) The method according to any one of embodiments 76 to 80, wherein the hairpin-terminal DNA consists of two hairpin terminals.

[0136] (Embodiment 83) The method according to any one of embodiments 76 to 81, wherein the hairpin terminal DNA is a viral genome.

[0137] (Embodiment 84) The method according to embodiment 82, wherein the viral genome is a parvovirus genome.

[0138] (Embodiment 85) The method according to Embodiment 83, wherein the parvovirus is dependent parvovirus, bocaparvovirus, erythroparvovirus, protoparvovirus, or tetraparvovirus.

[0139] (3.2 Exemplary Embodiments Set 2) (Embodiment 1) From the 5' to 3' direction of the top chain: a. A first reverse repeat, wherein when the top chain separates from the bottom chain of the first reverse repeat, the nicking results in a top chain 5' overhang containing the first reverse repeat, the first and second restricting regions for the nicking endonuclease are located on opposing chains near the first reverse repeat; b. Expression cassette; and c. The second reverse repeat, wherein when the top chain separates from the bottom chain of the second reverse repeat, the nicking results in a top chain 3' overhang containing the second reverse repeat, the third and fourth restricting regions for the nicking endonuclease are located on the opposing chain near the second reverse repeat. A double-stranded DNA molecule containing [the specified element].

[0140] (Embodiment 2) From the 5' to 3' direction of the top chain: a. A first reverse repeat, wherein when the top chain separates from the bottom chain of the first reverse repeat, the nicking results in a bottom chain 3' overhang containing the first reverse repeat, the first and second restricting regions for the nicking endonuclease are located on opposing chains near the first reverse repeat; b. Expression cassette; and c. The second reverse repeat, wherein, when the top chain separates from the bottom chain of the second reverse repeat, the nicking results in a bottom chain 5' overhang containing the second reverse repeat, and the third and fourth restricting regions for the nicking endonuclease are located on opposing chains near the second reverse repeat. A double-stranded DNA molecule containing [the specified element].

[0141] (Embodiment 3) From the 5' to 3' direction of the top chain: a. A first reverse repeat, wherein when the top chain separates from the bottom chain of the first reverse repeat, the nicking results in a top chain 5' overhang containing the first reverse repeat, the first and second restricting regions for the nicking endonuclease are located on opposing chains near the first reverse repeat; b. Expression cassette; and c. The second reverse repeat, wherein, when the top chain separates from the bottom chain of the second reverse repeat, the nicking results in a bottom chain 5' overhang containing the second reverse repeat, and the third and fourth restricting regions for the nicking endonuclease are located on opposing chains near the second reverse repeat. A double-stranded DNA molecule containing [the specified element].

[0142] (Embodiment 4) From the 5' to 3' direction of the top chain: a. A first reverse repeat, wherein when the top chain separates from the bottom chain of the first reverse repeat, the nicking results in a bottom chain 3' overhang containing the first reverse repeat, the first and second restricting regions for the nicking endonuclease are located on opposing chains near the first reverse repeat; b. Expression cassette; and c. The second reverse repeat, wherein when the top chain separates from the bottom chain of the second reverse repeat, the nicking results in a top chain 3' overhang containing the second reverse repeat, the third and fourth restricting regions for the nicking endonuclease are located on the opposing chain near the second reverse repeat. A double-stranded DNA molecule containing [the specified element].

[0143] (Embodiment 5) An isolated DNA molecule, the DNA molecule described in any one of Embodiments 1 to 4.

[0144] (Embodiment 6) A DNA molecule according to any one of embodiments 1 to 5, wherein the first, second, third, and fourth restriction sites for nickel endonucleases are all the same restriction site for nickel endonucleases.

[0145] (Embodiment 7) A DNA molecule according to any one of Embodiments 1 to 5, wherein the first and second reverse repeats are identical.

[0146] (Embodiment 8) A DNA molecule according to any one of embodiments 1 to 5, wherein the first and / or second reverse repeats are the parvovirus ITR.

[0147] (Embodiment 9) A DNA molecule according to any one of embodiments 1 to 5, wherein the first and / or second reverse repeats are modified parvovirus ITRs.

[0148] (Embodiment 10) The DNA molecule according to Embodiment 8 or 9, wherein the parvovirus is dependent parvovirus, boca parvovirus, erythroparvovirus, protoparvovirus, or tetraparvovirus.

[0149] (Embodiment 11) The DNA molecule according to Embodiment 9, wherein the nucleotide sequence of the modified ITR is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% identical to the ITR of the parvovirus.

[0150] (Embodiment 12) a. The first nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the first reverse repeat; b. The second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the first reverse repeat; c. The third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the second reverse repeat; and / or d. The DNA molecule according to Embodiment 1 or 5, wherein the fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the second reverse repeat.

[0151] (Embodiment 13) a. The first nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the first reverse repeat; b. The second nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the first reverse repeat; c. The third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the second reverse repeat; and / or d. The fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the second reverse repeat. A DNA molecule according to Embodiment 2 or 5.

[0152] (Embodiment 14) a. The first nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the first reverse repeat; b. The second nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the first reverse repeat; c. The third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the second reverse repeat; and / or d. The fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the second reverse repeat. A DNA molecule according to Embodiment 3 or 5.

[0153] (Embodiment 15) a. The first nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the first reverse repeat; b. The second nick is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the first reverse repeat; c. The third nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 5' nucleotide of the ITR closed base pair of the second reverse repeat; and / or d. The fourth nick is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' nucleotide of the ITR closed base pair of the second reverse repeat. A DNA molecule according to Embodiment 4 or 5.

[0154] (Embodiment 16) The DNA molecule according to any one of embodiments 12 to 15, wherein the nick is located inside the reverse repeat.

[0155] (Embodiment 17) A DNA molecule according to any one of embodiments 12 to 15, wherein the nick is located outside the reverse repeat.

[0156] (Embodiment 18) A plasmid, which is a DNA molecule described in any one of embodiments 1 to 17.

[0157] (Embodiment 19) The DNA molecule according to Embodiment 18, wherein the plasmid further comprises a bacterial origin of replication.

[0158] (Embodiment 20) The plasmid further comprises restriction enzyme sites in the 5' region relative to the first reverse repeat and the 3' region relative to the second reverse repeat, The restriction enzyme site is not located in the first reverse repeat, the second reverse repeat, or the region between the first reverse repeat and the second reverse repeat. The DNA molecule described in Embodiment 18.

[0159] (Embodiment 21) The DNA molecule according to Embodiment 20, wherein cleavage with the restriction enzyme results in a single-stranded overhang that does not anneal at a detectable level under conditions suitable for annealing the first reverse repeat and / or the second reverse repeat.

[0160] (Embodiment 22) The DNA molecule according to Embodiment 20, wherein the plasmid further comprises an open reading frame encoding the restriction enzyme.

[0161] (Embodiment 23) The DNA molecule according to Embodiment 22, wherein the expression of the restriction enzyme is under the control of an inducible promoter.

[0162] (Embodiment 24) The plasmid further includes fifth and sixth restriction sites for nickel end nucleases in a region that is 5' relative to the first reverse repeat and 3' relative to the second reverse repeat, The fifth and sixth limiting regions for the nickel end nuclease are: a. On opposing chains; and b. The break occurs within the double-stranded DNA molecule such that the single-stranded overhang of the break does not undergo intermolecular or molecular annealing at a detectable level under conditions suitable for annealing of the first reverse repeat and / or the second reverse repeat. The DNA molecule described in Embodiment 18.

[0163] (Embodiment 25) The DNA molecule according to Embodiment 24, wherein the fifth and sixth nicks are separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0164] (Embodiment 26) The DNA molecule according to Embodiment 24, wherein the first, second, third, fourth, fifth, and sixth restriction sites for nickel endonucleases are all the same nickel endonuclease target sequence.

[0165] (Embodiment 27) A DNA molecule according to any one of embodiments 1 to 26, wherein one or more of the aforementioned nickel endonuclease sites are target sequences of endogenous nickel endonucleases.

[0166] (Embodiment 28) The DNA molecule according to any one of embodiments 24 to 27, wherein the plasmid further comprises an open reading frame encoding a nickeling endonuclease that recognizes the first, second, third, fourth, fifth, and / or sixth restriction sites for the nickeling endonuclease.

[0167] (Embodiment 29) The DNA molecule according to Embodiment 28, wherein the expression of the aforementioned nickel endonuclease is under the control of an inducible promoter.

[0168] (Embodiment 30) A DNA molecule according to any one of embodiments 1 to 29, wherein the nickeling endonuclease that recognizes the first, second, third, and / or fourth restriction sites for the nickeling endonuclease is Nt.BsmAI;Nt.BtsCI;N.ALwl;N.BstNBI;N.BspD6I;Nb.Mva1269I;Nb.BsrDI;Nt.BtsI;Nt.BsaI;Nt.Bpu10I;Nt.BsmBI;Nb.BbvCI;Nt.BbvCI; or Nt.BspQI.

[0169] (Embodiment 31) The DNA molecule according to embodiment 24, wherein the nickeling endonuclease that recognizes the fifth and sixth restriction sites for nickeling endonuclease is Nt.BsmAI;Nt.BtsCI;N.ALwl;N.BstNBI;N.BspD6I;Nb.Mva1269I;Nb.BsrDI;Nt.BtsI;Nt.BsaI;Nt.Bpu10I;Nt.BsmBI;Nb.BbvCI;Nt.BbvCI; or Nt.BspQI.

[0170] (Embodiment 32) The DNA molecule according to any one of Embodiments 1 to 26, wherein the expression cassette includes a promoter operatively linked to a transcription unit.

[0171] (Embodiment 33) The DNA molecule according to embodiment 32, wherein the transcription unit includes an open reading frame.

[0172] (Embodiment 34) The DNA molecule according to embodiment 32 or 33, wherein the expression cassette further comprises a post-transcriptional regulatory factor.

[0173] (Embodiment 35) The DNA molecule according to embodiment 32 or 33, wherein the expression cassette further comprises polyadenylation and a termination signal.

[0174] (Embodiment 36) The DNA molecule according to any one of embodiments 32 to 35, wherein the size of the expression cassette is at least 4kb, at least 4.5kb, at least 5kb, at least 5.5kb, at least 6kb, at least 6.5kb, at least 7kb, at least 7.5kb, at least 8kb, at least 8.5kb, at least 9kb, at least 9.5kb, or at least 10kb.

[0175] (Embodiment 37) From the 5' to 3' direction of the top chain: a. First hairpin reverse iteration; b. Nick on the bottom strand; c. Expression cassette; d. Said nick on the bottom strand; and e. A second hairpin-forming inverted repeat A double-stranded DNA molecule comprising the above.

[0176] (Embodiment 38) In the 5' to 3' direction of the top strand: a. A first hairpin-forming inverted repeat; b. Said nick on the top strand; c. Expression cassette; d. Said nick on the top strand; and e. A second hairpin-forming inverted repeat A double-stranded DNA molecule comprising the above.

[0177] (Embodiment 39) In the 5' to 3' direction of the top strand: a. A first hairpin-forming inverted repeat; b. Nick on the bottom strand; c. Expression cassette; d. Said nick on the top strand; and e. A second hairpin-forming inverted repeat A double-stranded DNA molecule comprising the above.

[0178] (Embodiment 40) In the 5' to 3' direction of the top strand: a. A first hairpin-forming inverted repeat; b. Said nick on the top strand; c. Expression cassette; d. Nick on the bottom strand; and e. A second hairpin-forming inverted repeat A double-stranded DNA molecule comprising the above.

[0179] (Embodiment 41) The double-stranded DNA molecule according to any one of Embodiments 37 to 40, which is an isolated DNA molecule.

[0180] (Embodiment 42) A double-stranded DNA molecule according to any one of embodiments 37 to 41, wherein the first and / or second reverse repeats are the parvovirus ITR.

[0181] (Embodiment 43) A double-stranded DNA molecule according to any one of embodiments 37 to 41, wherein the first and second reverse repeats are identical.

[0182] (Embodiment 44) A DNA molecule according to any one of embodiments 37 to 41, wherein the first and / or second reverse repeats are modified parvovirus ITRs.

[0183] (Embodiment 45) The DNA molecule according to Embodiment 42 or 44, wherein the parvovirus is dependent parvovirus, boca parvovirus, erythroparvovirus, protoparvovirus, or tetraparvovirus.

[0184] (Embodiment 46) The DNA molecule according to Embodiment 45, wherein the nucleotide sequence of the modified ITR is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% identical to the ITR of the parvovirus.

[0185] (Embodiment 47) The DNA molecule according to any one of embodiments 37 to 46, wherein the expression cassette includes a promoter operatively linked to a transcription unit.

[0186] (Embodiment 48) The DNA molecule according to Embodiment 47, wherein the transcription unit includes an open reading frame.

[0187] (Embodiment 49) The DNA molecule according to embodiment 47 or 48, wherein the expression cassette further comprises a post-transcriptional regulatory factor.

[0188] (Embodiment 50) The DNA molecule according to embodiment 47 or 48, wherein the expression cassette further comprises a polyadenylation and termination sequence.

[0189] (Embodiment 51) The DNA molecule according to any one of embodiments 37 to 50, wherein the size of the expression cassette is at least 4 kb, at least 4.5 kb, at least 5 kb, at least 5.5 kb, at least 6 kb, at least 6.5 kb, at least 7 kb, at least 7.5 kb, at least 8 kb, at least 8.5 kb, at least 9 kb, at least 9.5 kb, or at least 10 kb.

[0190] (Embodiment 52) The DNA molecule according to any one of embodiments 37 to 51, which is protected from exonuclease.

[0191] (Embodiment 53) The DNA molecule according to embodiment 52, wherein the exonuclease is RecBCD exonuclease.

[0192] (Embodiment 54) The DNA molecule according to any one of embodiments 1 to 53, which lacks a replication (Rep) protein binding site.

[0193] (Embodiment 55) A delivery vehicle comprising the DNA molecule according to any one of embodiments 37 to 51.

[0194] (Embodiment 56) The delivery vehicle according to embodiment 55, comprising a hydridosome, a lysosome, or a lipid nanoparticle.

[0195] (Embodiment 57) A method for producing a covalently closed-ended DNA molecule, comprising: a. culturing a host cell comprising the DNA molecule according to any one of embodiments 1 to 35 under conditions that result in amplification of the DNA molecule; b. To cause the DNA molecule to be released from the host cell; c. Incubating the DNA molecule with one or more nicking endonucleases that recognize the four restriction sites and produce four nicks; d. To perform denaturation, thereby generating a DNA fragment that includes the expression cassette and is adjacent to the two single-stranded DNA overhangs; e. To perform intramolecular annealing on the single-stranded DNA overhang, thereby generating hairpinned reverse repeats at both ends of the DNA fragment obtained as a result of step d. The method comprising the above.

[0196] (Embodiment 58) A method for preparing hairpin-end DNA: a. Culturing host cells containing the plasmid described in Embodiment 20 under conditions that result in amplification of the plasmid; b. To cause the plasmid to be released from the host cell; c. Incubating the DNA molecule with one or more nicking endonucleases that recognize the four restriction sites and produce four nicks; d. To perform denaturation, thereby generating a DNA fragment that includes the expression cassette and is adjacent to the two single-stranded DNA overhangs; e. To perform intramolecular annealing on the single-stranded DNA overhang, thereby generating hairpinned reverse repeats at both ends of the DNA fragment obtained as a result of step d; f. Incubating the plasmid or the fragment obtained as a result of step d with the restriction enzyme, and thereby cleaving the plasmid or the fragment of the plasmid; and g. Incubate the fragment of the plasmid with an exonuclease, thereby digesting the fragment of the plasmid, excluding the fragment obtained as a result of step e. The method, including the method described above.

[0197] (Embodiment 59) A method for preparing hairpin-end DNA: a. Culturing host cells containing the plasmid described in Embodiment 24 under conditions that result in amplification of the plasmid; b. To cause the plasmid to be released from the host cell; c. Incubating the DNA molecule with one or more nicking endonucleases that recognize the first, second, third, and fourth restriction sites and produce four nicks; d. To perform denaturation, thereby generating a DNA fragment that includes the expression cassette and is adjacent to the two single-stranded DNA overhangs; e. To perform intramolecular annealing on the single-stranded DNA overhang, thereby generating hairpinned reverse repeats at both ends of the DNA fragment obtained as a result of step d; f. Incubating the plasmid or the fragment obtained as a result of step d with one or more nickel endonucleases that recognize the fifth and sixth restriction sites resulting in cleavage in the double-stranded DNA molecule; and g. Incubate the fragment of the plasmid with an exonuclease, thereby digesting the fragment of the plasmid, excluding the fragment obtained as a result of step e. The method, including the method described above.

[0198] (Embodiment 60) The method according to embodiment 57, 58, or 59, further comprising repairing the nick using h. ligase to generate circular DNA.

[0199] (Embodiment 61) The method according to any one of embodiments 57 to 60, wherein the steps are performed in the order in which they appear in the embodiment.

[0200] (Embodiment 62) The method according to any one of embodiments 57 to 61, wherein the hairpin-terminal DNA consists of two hairpin terminals.

[0201] (Embodiment 63) The method according to any one of embodiments 57 to 62, wherein the hairpin terminal DNA is a viral genome.

[0202] (Embodiment 64) The method according to embodiment 63, wherein the viral genome is a parvovirus genome.

[0203] (Embodiment 65) The method according to Embodiment 64, wherein the parvovirus is dependent parvovirus, bocaparvovirus, erythroparvovirus, protoparvovirus, or tetraparvovirus. [Brief explanation of the drawing]

[0204] (4. Brief explanation of the drawing) [Figure 1] Figure 1 shows various exemplary hairpin structures and their structural elements.

[0205] [Figure 2] Figures 2A and 2B show exemplary chain conformations and linear interaction plots illustrating intramolecular forces within overhangs, as well as intermolecular forces between chains, while Figure 2C shows the expected annealed structures of Figures 2A and 2B.

[0206] [Figure 3] Figure 3 shows various exemplary arrangements of hairpins, the locations of various restriction sites, and the restriction site for type II nicking endonucleases in the primary stem of the hairpin.

[0207] [Figure 4] Figure 4 shows the structures of various exemplary hairpins, as well as the structural elements of human mitochondrial DNA OriL and OriL-derived ITR.

[0208] [Figure 5] Figure 5 shows the hairpin structure of exemplary aptamers and aptamer ITRs.

[0209] [Figure 6] Figure 6 shows a visualization of the DNA product derived from construct 1 after performing the steps of the method described in construct 1 and Example 1.

[0210] [Figure 7] Figure 7 shows a visualization of the DNA product derived from construct 1 after performing the steps of the method described in construct 2 and Example 1.

[0211] [Figure 8] Figures 8A to 8C illustrate the multiple regeneration / modification cycles described in Example 2.

[0212] [Figure 9] Figures 9A and 9B illustrate the isothermal denaturation of construct 1 as described in Example 3.

[0213] [Figure 10] Figure 10 shows the expression levels of luciferase from various DNA vector concentrations. Cells were transfected with DNA vectors of varying concentrations, either hybridosomes or lipid nanoparticles. Luciferase activity was determined 48 hours after transfection.

[0214] [Figure 11]Figures 11A to 11D show luciferase expression in dividing and non-dividing cells as described in Section 6.5 (Example 5 Expression in dividing and non-dividing cells). Figures 11A and 11B show the expression of non-secreted Turboluc (constructor 1) in dividing cells (11A) and non-dividing cells (11B). In the case of non-secreted Turboluc (constructor 1), luciferase activity in dividing cells peaks on day 2, and expression continues to increase in non-dividing cells. Figures 11C and 11D show the expression of secreted Turboluc (constructor 2) in non-dividing cells (11C) and dividing cells (11D). In the case of secreted Turboluc (constructor 2), luciferase activity peaks on day 2 in dividing cells, expression increases in non-dividing cells, and then remains stable for 9 days. For direct comparison, equimolar amounts of a complete circular plasmid encoding constructor 2 were also transfected. As shown in Figures 11C and 11D, generally, lower luciferase activity is recorded, indicating improved nuclear delivery of purified construct 2 with folded ITR.

[0215] [Figure 12] Figure 12 illustrates the vector construction strategy for the novel synthesis of hairpin-encoding plasmids.

[0216] [Figure 13] Figure 13 shows the sequence alignment of the ITR derived from AAV1, highlighting the sequence modifications that create recognition sites for various nickel endonucleases.

[0217] [Figure 14] Figure 14 shows the sequence alignment of the ITR derived from AAV2, highlighting the sequence modifications that create recognition sites for various nickel endonuclease recognition sites.

[0218] [Figure 15] Figure 15 shows the sequence alignment of the ITR derived from AAV3, highlighting the sequence modifications that create recognition sites for various nickel endonuclease recognition sites.

[0219] [Figure 16] Figure 16 shows the sequence alignment of the ITR derived from AAV4 left, highlighting the sequence modifications that create recognition sites for various nickel endonuclease recognition sites.

[0220] [Figure 17] Figure 17 shows the sequence alignment of the ITR derived from AAV4 right, highlighting the sequence modifications that create recognition sites for various nickel endonuclease recognition sites.

[0221] [Figure 18] Figure 18 shows the sequence alignment of the ITR derived from AAV5, highlighting the sequence modifications that create recognition sites for various nickel endonuclease recognition sites.

[0222] [Figure 19] Figure 19 shows the sequence alignment of the ITR derived from AAV7 left, highlighting the sequence modifications that create recognition sites for various nickel endonuclease recognition sites.

[0223] [Figure 20] Figures 20A and 20B show agarose gels demonstrating successful ligation of DNA constructs and corresponding luciferase expression in non-dividing hepatocytes transfected using hybridosomes that encapsulate ligated and unligated constructs, as well as the original plasmid, respectively.

[0224] [Figure 21] Figure 21 shows the time course of luciferase expression in non-dividing cells transfected with equimolar amounts of hairpin-terminated DNA molecules encoding secreted luciferase encapsulated within LNPs or hybridosomes.

[0225] [Figure 22] Figure 22 shows the percentage of RFP-positive color-switched HEK293 cells after 72 hours of transfection with lipid nanoparticles, hybridosomes, and hairpin-terminal DNA encoding Cre recombinase delivered by jetprime, as described in Example 9.

[0226] [Figure 23] Figures 23A and 23B show agarose gels demonstrating the successful formation of hairpin-terminal DNA from plasmids containing right and left ITRs with wild-type AAV RBE, compared to mutants in which the RBE was replaced with the corresponding sequence shown in the figure. Figure 23B shows luciferase expression in non-dividing hepatocytes transfected with the corresponding ITR sequence.

[0227] [Figure 24] Figures 24A and 24B show a further exemplary cloning method (Figure 24A) and a map of the obtained plasmid (Figure 24B). From this plasmid, a hairpinned reverse repeat DNA molecule as disclosed herein can be prepared by following the steps of the method described in Example 11. In this example, six restriction sites for the nicking endonuclease are located in a region that is 5' relative to the left ITR and 3' relative to the right ITR.

[0228] [Figure 25] Figures 25A and 25B visualize the luminescence readings of the products obtained by nicking, denaturation / annealing, and exonuclease digestion, starting from the plasmid shown in Figure 24B on an agarose gel, as well as the transfection of the products.

[0229] [Figure 26]Figures 26A and 26B visualize the nicking, denaturation / annealing, and exonuclease digestion products of the ITR-containing OriL-derived construct described in Example 13 on an agarose gel, as well as the luminescence readings of secreted TurboLuc in the supernatant of Hek293 cells. [Modes for carrying out the invention]

[0230] (5. Detailed explanation) This specification provides methods for constructing hairpin-terminated DNA molecules. Also provided are methods for using hairpin-terminated DNA molecules, such as using them in gene therapy. Various methods for constructing hairpin-terminated DNA molecules are further described in Section 5.2 below. Various methods for using hairpin-terminated DNA molecules are described in Section 5.7 below. The hairpin-terminated DNA produced by these methods is shown in Section 5.4 below, each comprising reverse hairpinned repeats at both ends and an expression cassette, each further described below. In some embodiments, the hairpin-terminated DNA also includes one or two nicks, as further described in Section 5.4 below. Hairpins, reverse hairpinned repeats, and hairpinned ends are described in Section 5.4 below; reverse repeats forming hairpinned ends are described in Section 5.3.1 below; nicks, nicking endonucleases, and restriction sites for nicking endonucleases are described in Sections 5.3.2 and 5.4 below; expression cassettes are described in Sections 5.3.3 and 5.4 below; and the functional properties of hairpin-terminated DNA molecules are described in Section 5.5 below. Accordingly, this disclosure provides hairpin-terminated DNA molecules, methods for producing them, and methods for using them, along with any combination or permutation of the elements provided herein.

[0231] Furthermore, provided herein are parent DNA molecules used in a method for producing the hairpin-terminated DNA molecules, each comprising two reverse repeats, two or more restriction sites for nickeling endonucleases, and an expression cassette, as further described below. The restriction sites for nickeling endonucleases are positioned such that, upon nickeling and denaturation by the nickeling endonuclease, a single-stranded overhang having a reverse repeat sequence is generated, which then folds upon annealing to form a hairpin (each step being as described in Section 5.2). The reverse repeats are described in Section 5.3.1 below; the nickels, nickeling endonucleases, and restriction sites for nickeling endonucleases are described in Section 5.3.2 below; and the expression cassette is described in Section 5.3.3 below. Accordingly, this disclosure provides parent DNA molecules used in a method for production, along with any combination or permutation of the elements provided herein.

[0232] (5.1 definition) As used herein, the term “isolated” in relation to DNA molecules is intended to mean that the DNA molecule referred to does not contain at least one component found in its natural, natural, or synthetic environment. This term includes DNA molecules isolated from some or all other components found in its natural, natural, or synthetic environment. Components of the natural, natural, or synthetic environment of a DNA molecule include anything found in that environment that is necessary, used, or otherwise plays a role in the replication and maintenance of the DNA molecule in that environment. Other components of the natural, natural, or synthetic environment of a DNA molecule include, for example, cells, necrotic tissue fragments, organelles, proteins, peptides, amino acids, lipids, polysaccharides, nucleic acids other than the DNA molecule referred to, salts, nutrients for cell culture, and / or chemicals used in DNA synthesis. The DNA molecules of this disclosure may be partially, completely, or substantially free of all of these components or any other components of the natural, natural, or synthetic environment from which the DNA molecule was isolated, synthetically produced, naturally produced, or recombinantly produced. Specific examples of isolated DNA molecules include partially pure DNA molecules and substantially pure DNA molecules.

[0233] As used herein, the term “delivery vehicle” means a substance that can be used to administer or deliver one or more drugs to cells, tissues, or subjects containing or not containing the drugs to be delivered, in particular to human subjects. A delivery vehicle can preferentially deliver drugs to a particular subset or type of cells. The selective or preferential delivery achieved by a delivery vehicle can be achieved by the properties of the vehicle, or by a site conjugated to the delivery vehicle, a site bound to the delivery vehicle, or a site contained in the delivery vehicle. This site specifically or preferentially binds to a particular subset of cells. A delivery vehicle can also increase the in vivo half-life of the drug to be delivered, the efficiency of drug delivery compared to delivery without the delivery vehicle, and / or the bioavailability of the drug to be delivered. Non-limiting examples of delivery vehicles include hybridosomes, liposomes, lipid nanoparticles, polymerosomes, mixtures of natural / synthetic lipids, membranes or lipid extracts, exosomes, viral particles, proteins or protein complexes, peptides, and / or polysaccharides.

[0234] In this specification, the term "and / or" as used in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0235] (5.2 Method for producing hairpin-ended DNA molecules) In one embodiment, the foregoing provides a method for preparing a hairpin-terminated DNA molecule, comprising: a. culturing a host cell containing the DNA molecule described in Section 5.3 under conditions that result in amplification of the DNA molecule; b. releasing the DNA molecule from the host cell; c. incubating the DNA molecule with one or more nicking endonucleases that recognize the four restriction sites and result in four nicks; d. denaturing the DNA molecule, thereby generating a DNA fragment containing the expression cassette and adjacent to the two single-stranded DNA overhangs; e. intramolecular annealing the single-stranded DNA overhangs, thereby generating hairpin-terminated reverse repeats at both ends of the DNA fragment resulting from step d.

[0236] In another embodiment, provided herein is a method for preparing hairpin-terminated DNA, comprising: a. culturing a host cell containing the plasmid described in Section 5.3.5 under conditions that result in amplification of the plasmid; b. releasing the plasmid from the host cell; c. incubating the DNA molecule with one or more nicking endonucleases that recognize the four restriction sites and result in four nicks; d. denaturing, thereby generating a DNA fragment containing the expression cassette and adjacent to the two single-stranded DNA overhangs; e. intramolecular annealing of the single-stranded DNA overhangs, thereby generating hairpin-shaped reverse repeats at both ends of the DNA fragment resulting from step d; f. incubating the plasmid or the fragment resulting from step d with the restriction enzyme, thereby cleaving the plasmid or the fragment of the plasmid; and g. incubating the fragment of the plasmid with an exonuclease, thereby digesting the fragment of the plasmid, except for the fragment resulting from step e.

[0237] In a further embodiment, provided herein is a method for preparing hairpin-terminated DNA, comprising: a. culturing host cells containing the DNA molecule described in Embodiment 24 of Section 3.1 under conditions that result in amplification of the plasmid; b. releasing the plasmid from the host cells; c. incubating the DNA molecule with one or more nicking endonucleases that recognize the first, second, third, and fourth restriction sites and result in four nicks; d. denaturing and thereby denaturing the DNA fragments containing the expression cassette and adjacent to the two single-stranded DNA overhangs. The method comprises: e. generating a fragment; f. intramolecular annealing of the single-stranded DNA overhang and thereby generating hairpinned reverse repeats at both ends of the DNA fragment obtained as a result of step d; f. incubation the plasmid or the fragment obtained as a result of step d with one or more nickeling endonucleases that recognize the fifth and sixth restriction sites resulting in cleavage in the double-stranded DNA molecule; and g. incubation the fragment of the plasmid with an exonuclease to digest the fragment of the plasmid, excluding the fragment obtained as a result of step e.

[0238] In one embodiment, the foregoing provides a method for preparing a hairpin-terminated DNA molecule, comprising: a. culturing a host cell containing the DNA molecule described in Section 5.3 under conditions that result in amplification of the DNA molecule; b. releasing the DNA molecule from the host cell; c. incubating the DNA molecule with one or more programmable nicking enzymes that recognize the four target sites for the guide nucleic acid and result in four nicks; d. denaturing the DNA, thereby generating a DNA fragment containing the expression cassette and adjacent to the two single-stranded DNA overhangs; e. intramolecular annealing the single-stranded DNA overhangs, thereby generating hairpin-terminated reverse repeats at both ends of the DNA fragment resulting from step d.

[0239] In another embodiment, provided herein is a method for preparing hairpin-terminated DNA, comprising: a. culturing a host cell containing the plasmid described in 5.3.5 under conditions that result in amplification of the plasmid; b. releasing the plasmid from the host cell; c. incubating the DNA molecule with one or more programmable nicking enzymes that recognize the four target sites for guide nucleic acids and result in four nicks; d. denaturing, thereby generating a DNA fragment containing the expression cassette and adjacent to the two single-stranded DNA overhangs; e. intramolecular annealing of the single-stranded DNA overhangs, thereby generating hairpin-terminated reverse repeats at both ends of the DNA fragment resulting from step d; f. incubating the plasmid or the fragment resulting from step d with the restriction enzyme, thereby cleaving the plasmid or the fragment of the plasmid; and g. incubating the fragment of the plasmid with an exonuclease, thereby digesting the fragment of the plasmid, except for the fragment resulting from step e.

[0240] In a further embodiment, provided herein is a method for preparing hairpin-terminated DNA, comprising: a. culturing a host cell containing the DNA molecule described in Embodiment 24 of Section 3.1 under conditions that result in amplification of the plasmid; b. releasing the plasmid from the host cell; c. incubating the DNA molecule with one or more programmable nicking enzymes that recognize first, second, third, and fourth target sites for guide nucleic acids to result in four nicks; d. denaturing the DNA containing the expression cassette and adjacent to the two single-stranded DNA overhangs. The method comprises: generating a fragment; e. intramolecular annealing of the single-stranded DNA overhang and thereby generating hairpinned reverse repeats at both ends of the DNA fragment obtained as a result of step d; f. incubation the plasmid or the fragment obtained as a result of step d with a programmable nickeling enzyme that recognizes fifth and sixth target sites for the guide nucleic acid and causes a cleavage in the double-stranded DNA molecule; and g. incubation the fragment of the plasmid with an exonuclease and thereby digesting the fragment of the plasmid, except for the fragment obtained as a result of step e. In another embodiment, step f of this paragraph may be replaced with step f: incubation the plasmid or the fragment obtained as a result of step d with one or more nickeling endonucleases that recognize the two restriction sites that cause a cleavage in the double-stranded DNA molecule.

[0241] In one embodiment, a DNA molecule containing an expression cassette adjacent to a reverse repeat (as described in Section 5.3) can be provided by culturing a host cell containing the DNA molecule or plasmid, and releasing the DNA molecule or plasmid from the host cell, as provided in steps a and b of the previous paragraph. Alternatively, such DNA molecules can be synthesized in a cell-free system or in a combination of a cell-free system and a host cell-based system. For example, the chemical synthesis of DNA fragments and plasmids of various sizes and sequences is known and widely used in the art; fragments can be chemically synthesized and then ligated by any means known in the art or recombined in host cells. In another embodiment, the DNA molecule or plasmid can be provided by in vitro replication. Various methods, including amplification by polymerase chain reaction (PCR), can be used for in vitro replication. For example, PCR methods for replicating DNA fragments or plasmids of various sizes are well known and widely used in the art, as described in Michael Green and Joseph Sambrook's "Molecular Cloning: A Laboratory Manual," 4th edition, ISBN 978-1-936113-42-2 (2012), which is incorporated herein by whole citation. In some embodiments, the in vitro replication method can be isothermal DNA amplification. In some embodiments, steps a and b can be replaced with steps for preparing the DNA molecule by chemical synthesis or PCR. In another embodiment, steps a, b, c, and d can be replaced by preparing the DNA molecule by chemical synthesis.

[0242] In one embodiment, the method provided herein can be used to prepare a hairpin-ended DNA molecule containing an expression cassette, thereby comprising: a. preparing a double-stranded DNA molecule as described in Section 5.3; b. incubating the DNA molecule with at least one nicking enzyme under conditions that result in nicking of the double-stranded DNA molecule, thereby producing at least two stoichiometric DNA fragments; c. denaturing the DNA fragments; d. annealing the DNA fragments, thereby annealing such that at least one DNA fragment contains a single-stranded DNA overhang that can be annealed within the expression cassette and molecule, and thereby generating hairpin-reverse repeats at both ends of the DNA; e. incubating the DNA fragments with at least one exonuclease, thereby digesting the stoichiometric DNA fragments of step b, excluding the hairpin-ended fragment containing the expression cassette obtained as a result of step d. In specific embodiments, step b of the method in this paragraph produces at least two, at least three, at least four, at least five, at least six, or more stoichiometric fragments. In further embodiments, step b of the method in this paragraph produces at least two stoichiometric DNA fragments, thereby making the DNA fragment containing the expression cassette stoichiometrically equivalent to the DNA molecule prepared in step a. In some embodiments, the digestion-resistant hairpin-end fragment containing the expression cassette obtained as a result of step e in this paragraph can be made approximately stochiometrically equivalent to the DNA molecule prepared in step a.

[0243] In some embodiments, the method provided herein can be used to prepare a hairpin-ended DNA molecule containing an expression cassette, thereby comprising: a. preparing a double-stranded DNA molecule as described in Section 5.3; b. incubating the DNA molecule with at least one nicking enzyme under conditions that result in nicking of the double-stranded DNA molecule, thereby producing at least two stoichiometric DNA fragments; c. denaturing the DNA fragments into single-stranded DNA; d. annealing the sense and antisense strands of the DNA fragment containing the expression cassette, thereby annealing the sense and / or antisense strands, including a single-stranded DNA overhang that can be annealed intramolecularly, thereby generating hairpin-shaped reverse repeats at both ends of the DNA fragment containing the expression cassette; e. incubating the DNA fragment with at least one exonuclease, thereby digesting the stoichiometric DNA fragments of step b, excluding the hairpin-ended DNA fragment containing the expression cassette obtained as a result of step d.

[0244] In some embodiments, the method provided herein can be used to prepare a hairpin-terminated DNA molecule containing an expression cassette, thereby comprising: a. preparing a double-stranded DNA molecule as described in Section 5.3; b. incubating the DNA molecule with at least one nicking enzyme under conditions that result in nicking of the double-stranded DNA molecule; c. denaturing the double-stranded DNA to produce at least two stoichiometric DNA fragments; d. annealing the DNA fragments, thereby annealing such that at least one DNA fragment includes a single-stranded DNA overhang that can be annealed within the expression cassette and molecule, and thereby generating hairpin-terminated reverse repeats at both ends of the DNA fragments.

[0245] In a further embodiment, the method provided herein can be used to prepare hairpin-terminated DNA molecules, the method comprising at least one pot (e.g., a container, vessel, well, tube, plate, or other receptacle) containing a double-stranded DNA molecule as described in Section 5.3 in an aqueous buffer to which (i) a nicking enzyme, (ii) a denaturant (e.g., a base), (iii) an annealing agent (e.g., an acid), and (iv) an exonuclease are sequentially added. To enable the method provided herein to be carried out as a one-pot reaction, the method for producing hairpin-terminated DNA molecules can be completed without requiring the purification of intermediates, contaminants (e.g., enzymes), or DNA digestion by-products (i.e., nucleotides, oligonucleotides, or single-stranded DNA fragments) between steps (i) to (iv) of the method, thereby providing a preferred method in terms of cost and risk of manufacturing failure (e.g., by minimizing losses due to purification, reducing the required starting materials, and having stricter control over process variables).

[0246] In a further embodiment, the method provided herein may be used to produce a hairpin-terminated DNA molecule, the method comprising: a. preparing a pot (e.g., a container, bowl, well, tube, plate, or other container) containing a double-stranded DNA molecule as described in Section 5.3 and at least one nicking enzyme under conditions that result in nicking of the double-stranded DNA molecule; b. denaturing and annealing the DNA (e.g., by changing temperature, pH, or buffer composition); and c. adding an exonuclease without requiring purification of the intermediate (e.g., between steps a and c). In a specific embodiment, the pot of the method of this paragraph comprises: step a, at least one double-stranded DNA molecule (e.g., a plasmid or a derivative thereof), at least one nicking enzyme, and an aqueous buffer; and step c, at least one hairpin-terminated DNA, at least one nicking enzyme, at least one exonuclease, and an aqueous buffer containing a DNA digest product (e.g., dNMP, dinucleotide, and / or short oligonucleotide).

[0247] In further embodiments, the method provided herein can be used to produce a hairpin-terminated DNA molecule, thereby comprising: a. preparing a double-stranded DNA molecule and at least one nicking enzyme in at least one pot (e.g., a container, bowl, well, tube, plate, or other container) under conditions that result in nicking of the double-stranded DNA molecule; b. denaturing the DNA molecule and thereby producing a DNA fragment containing the expression cassette and adjacent to the two single-stranded DNA overhangs; c. intramolecularly annealing the single-stranded DNA overhangs and thereby producing hairpin-terminated reverse repeats at both ends of the DNA fragment obtained as a result of step b; and d. adding an exonuclease to the pot and thereby digesting the DNA fragment of the DNA molecule in step b, except for the fragment obtained as a result of step c.

[0248] In some embodiments, the method provided herein can be used to prepare a hairpin-ended DNA molecule containing an expression cassette, thereby comprising: a. preparing a double-stranded DNA molecule and at least one nicking enzyme as described in Section 5.3 in at least one pot (e.g., a container, bowl, well, tube, plate, or other container) under conditions that result in nicking of the double-stranded DNA molecule, thereby producing at least two stoichiometric DNA fragments; b. denaturing the DNA fragments; c. annealing the DNA fragments, thereby annealing such that at least one DNA fragment contains a single-stranded DNA overhang that can be annealed intramolecularly, and thereby generating hairpin-reverse repeats at both ends of the DNA fragment containing the expression cassette; d. adding at least one exonuclease to the pot, thereby digesting the DNA fragments of step a, excluding the hairpin-ended DNA fragment containing the expression cassette obtained as a result of step c. In a further embodiment, step a of the method of this paragraph produces at least two, at least three, at least four, at least five, at least six, or more stoichiometric fragments.

[0249] In some embodiments, the method provided herein can be used to prepare a hairpin-ended DNA molecule containing an expression cassette, thereby comprising: a. preparing a double-stranded DNA molecule as described in Section 5.3 in at least one pot (e.g., a container, bowl, well, tube, plate, or other container); b. adding at least one nicking enzyme to the pot under conditions that result in nicking of the double-stranded DNA molecule, thereby producing at least two stoichiometric DNA fragments; c. denaturing the DNA fragments; d. annealing the DNA fragments, thereby annealing such that at least one DNA fragment contains a single-stranded DNA overhang that can be annealed intramolecularly, and thereby generating hairpin-reverse repeats at both ends of the DNA fragment containing the expression cassette; e. adding at least one exonuclease to the pot, thereby digesting the stoichiometric DNA fragments of step b, excluding the hairpin-ended fragment containing the expression cassette obtained as a result of step d. In a specific embodiment, the pot of the method of this paragraph comprises, in step a, one DNA molecule, and in step b, at least two, at least three, at least four, at least five, at least six, or more stoichiometric fragments compared to the DNA molecule in step a, thereby the fragment containing the expression cassette is stoichiometrically equivalent to the DNA molecule prepared in step a. In a specific embodiment, the pot in step b of the method of this paragraph comprises at least two, at least three, at least four, at least five, at least six, or more stoichiometric fragments compared to the DNA molecule in step a, thereby the fragment containing the expression cassette is stoichiometrically equivalent to the DNA molecule prepared in step a. In a non-limiting example, the pot in step b of the method of this paragraph comprises three stoichiometric DNA fragments; (i) two fragments lacking an expression cassette and one fragment containing said expression cassette, thereby the fragment containing said expression cassette is stoichiometrically equivalent to the DNA molecule prepared in step a.In a more specific embodiment, the pot in step e of the method described in this paragraph contains a stoichiometrically equivalent amount of digestion-resistant hairpin-terminated DNA molecules compared to the DNA molecule prepared in step a. In a more specific embodiment, the pot in step e of the method described in this paragraph contains at most a stoichiometrically equivalent amount of digestion-resistant hairpin-terminated DNA molecules compared to the DNA molecule prepared in step a. In some embodiments, the pot in step e of the method described in this paragraph contains at most a stoichiometrically equivalent amount of digestion-resistant hairpin-terminated DNA molecules compared to the DNA molecule prepared in step a, thereby reducing the total mass of the DNA molecule by approximately the ratio of nucleotides present in the hairpin-terminated DNA molecule containing the expression cassette to the nuclotides present in the DNA molecule prepared in step a. In a more specific embodiment, the pot in step e of the method described in this paragraph contains a stoichiometrically equivalent amount of digestion-resistant hairpin-terminated DNA molecules compared to the expression cassette of the DNA molecule prepared in step a.

[0250] In some embodiments, the method provided herein can be used to prepare hairpin-terminated DNA molecules containing an expression cassette, thereby: a. culturing host cells containing the DNA molecule described in Section 5.3 under conditions that result in amplification of the DNA molecule; b. releasing the DNA molecule from the host cells; c. adding the DNA molecule to at least one pot (e.g., a container, vessel, well, tube, plate, or other container); d. adding at least one nicking enzyme to the pot under conditions that result in nicking of the double-stranded DNA molecule, thereby... e. to produce at least two stoichiometric DNA fragments; f. to denature the DNA fragments; f. to anneal the DNA fragments, thereby annealing such that at least one DNA fragment includes a single-stranded DNA overhang that can be annealed within the expression cassette and molecule, and thereby generating hairpinned reverse repeats at both ends of the DNA fragment; g. to add at least one exonuclease to the pot, thereby digesting the stoichiometric DNA fragments of step f, excluding the hairpin-ended fragments containing the expression cassette obtained as a result of step f. In a specific embodiment, the pot in step g of the method of this paragraph contains a stoichiometrically equivalent amount of digestion-resistant hairpin-ended DNA molecules compared to the DNA molecules prepared in step c.

[0251] In some embodiments, the method provided herein can be used to prepare a hairpin-terminated DNA molecule containing an expression cassette, thereby: a. culturing host cells containing the plasmid described in Section 5.3.5 under conditions that result in amplification of the plasmid; b. releasing the plasmid from the host cells; c. adding the plasmid to at least one pot (e.g., a container, bowl, well, tube, plate, or other container); d. adding at least one nicking enzyme to the pot under conditions that result in nicking of the plasmid, thereby less The process comprises: e. producing two stoichiometric DNA fragments; f. denaturing the DNA fragments; annealing the DNA fragments such that at least one DNA fragment includes a single-stranded DNA overhang that can be annealed within the expression cassette and molecule, and thereby generating hairpinned reverse repeats at both ends of the DNA fragments; g. adding at least one exonuclease to the pot to digest the stoichiometric DNA fragments of step f, excluding the hairpin-ended fragments containing the expression cassette resulting from step f. In specific embodiments, the pot in step g of the method of this paragraph contains a stoichiometrically equivalent amount of digestion-resistant hairpin-ended DNA molecules compared to the plasmid prepared in step c.

[0252] The order of steps of the method is listed in the exemplary method of the objective. In some embodiments, the steps of the method are performed in the order in which they appear as described herein. In some embodiments, the steps of the method can be performed in a different order than in which they appear as described herein. Specifically, in some embodiments, the steps of the method for producing hairpin-terminated DNA molecules can be performed in the order in which they appear, or in the order listed alphabetically as described herein from a to e or a to g. Alternatively, the steps of the method for producing hairpin-terminated DNA molecules can be performed in a different order than in which they appear as described herein. In one embodiment, if the host cells naturally express one or more nickeling endonucleases, or are manipulated to express them, or otherwise contain them, step c (incubating the DNA molecule with one or more nickeling endonucleases that recognize the four restriction sites and result in four nicks) can be performed before step b (releasing the plasmid from the host cells). In another embodiment, step f (incubating the plasmid or the fragment obtained as a result of step d with the restriction enzyme, or incubating the plasmid or the fragment obtained as a result of step d with one or more nickel endonucleases) can be performed before step d (denaturation, thereby generating a DNA fragment containing the expression cassette and adjacent to the two single-stranded DNA overhangs) or before step c (incubating the DNA molecule with one or more nickel endonucleases). Furthermore, one or more steps can be combined into a single step that performs all the actions of separate steps. In one embodiment, step a (culturing host cells) can be combined with step c (incubating the DNA molecule with one or more nickel endonucleases) if the host cells naturally express one or more nickel endonucleases, are engineered to express them, or otherwise contain them.In another embodiment, step f (incubating a plasmid or a fragment obtained as a result of step d with a restriction enzyme, or incubating a plasmid or a fragment obtained as a result of step d with one or more nickel endonucleases) can be incubated with the nickel endonucleases or restriction enzymes described in steps f and c, thereby being combined with step c (incubating a DNA molecule with one or more nickel endonucleases). Accordingly, this disclosure specifies that the steps can be carried out in various combinations and permutations depending on the latest technology.

[0253] Additional steps may be added to the method provided herein, either before all steps of the method, after all steps of the method, or during any of the steps of the method. In one embodiment, the method provided herein further includes step h, repairing the nick using a ligase to generate circular DNA. In another embodiment, step h, repairing the nick using a ligase to generate circular DNA, is performed after all other steps of the method described herein.

[0254] As further described below in Sections 5.3.1 and 5.4, the hairpins formed at the ends of a DNA molecule are determined by the properties of the overhangs between the restriction sites for the nickel endonuclease. Thus, by designing properties including the sequence and structural properties of the overhangs between the restriction sites for the nickel endonuclease in accordance with Sections 5.3.1 and 5.4, one, two, or more hairpinned ends can be produced using this method. In one embodiment, the method produces hairpin-ended DNA containing one hairpin end. In another embodiment, the method produces hairpin-ended DNA consisting of one hairpin end. In yet another embodiment, the method produces hairpin-ended DNA containing two hairpin ends. In yet another embodiment, the method produces hairpin-ended DNA consisting of two hairpin ends.

[0255] The methods provided herein can be used to produce DNA molecules containing artificial sequences, natural DNA sequences, or sequences containing both natural and artificial DNA sequences. In one embodiment, the method produces a hairpin-ended DNA molecule containing an artificial sequence. In another embodiment, the method produces a hairpin-ended DNA molecule containing a natural sequence. In yet another embodiment, the method produces a hairpin-ended DNA molecule containing both natural and artificial sequences. In one embodiment, the method produces a hairpin-ended DNA molecule containing a viral reverse-end repeat (ITR). In yet another embodiment, the method produces a hairpin-ended DNA molecule containing a hairpin-ended reverse repeat lacking RABS. In yet another embodiment, the method produces a hairpin-ended DNA molecule containing two hairpin-ended reverse repeats, wherein both hairpin-ended reverse repeats lack RABS. In yet another embodiment, the method produces a hairpin-ended DNA molecule containing two hairpin-ended reverse repeats, wherein both hairpin-ended reverse repeats lack TRS. In a further embodiment, the method produces a hairpin-terminal DNA molecule comprising two hairpin-reverse repeats, wherein both hairpin-reverse repeats lack RABS and TRS. In another embodiment, the method produces a hairpin-terminal DNA molecule comprising two hairpin-reverse repeats, wherein both hairpin-reverse repeats lack promoter activity (e.g., P5 promoter activity) and transcriptional activity (e.g., transcription start site [TSS]). In yet another embodiment, the method produces a hairpin-terminal DNA molecule comprising two hairpin-reverse repeats, wherein both hairpin-reverse repeats lack RABS, promoter activity (e.g., P5 promoter activity), transcriptional activity (e.g., transcription start site [TSS]), and TRS. In yet another embodiment, the method produces a hairpin-terminal DNA molecule comprising two hairpin-reverse repeats, wherein both hairpin-reverse repeats lack RABS.In a further embodiment, the method produces a hairpin-terminated DNA molecule containing a viral genome. In some embodiments, the viral genome is an engineered viral genome containing one or more nonviral genes within an expression cassette. In some embodiments, the viral genome is an engineered viral genome in which one or more viral genes are knocked out. In some specific embodiments, the viral genome is an engineered viral genome in which the replication-associated protein ("RAP", i.e., Rep or NS1) gene, the capsid (Cap) gene, or both the RAP gene and the Cap gene are knocked out. In another embodiment, the viral genome is a parvovirus genome. In yet another embodiment, the parvovirus is dependent parvovirus, bocaparvovirus, erythroparvovirus, protoparvovirus, or tetraparvovirus.

[0256] The various methods of performing the steps described herein are described in further detail below. Embodiments of host cells and the culture of said host cells are described in Section 5.2.1; embodiments of the step of releasing DNA molecules from host cells are described in Section 5.2.2; embodiments of the step of denaturing DNA molecules are described in Section 5.2.3; embodiments of the step of annealing are described in Section 5.2.5; embodiments of the step of incubating DNA molecules with a nickel endonuclease or restriction enzyme are described in Section 5.2.4; embodiments of the step of incubating with an exonuclease are described in Section 5.2.6; and embodiments of the step of ligation are described in Section 5.2.7. Accordingly, this disclosure provides methods including permutations and combinations of various embodiments of the steps described herein.

[0257] (5.2.1 Host cells and culture of said host cells) This disclosure stipulates that various host cells can be cultured to amplify the DNA molecule. The host cells for use in the methods provided herein may be eukaryotic host cells, prokaryotic host cells, or any transformable organism capable of replicating or amplifying recombinant DNA molecules. In some embodiments, the host cells may be microbial host cells. In further embodiments, the host cells may be host microbial cells selected from bacteria, yeasts, fungi, or various other microbial cells applicable to replicating or amplifying DNA molecules. The bacterial host cells include Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, and Lactococcus lactis. It may be any of the following species selected from Lactobacillus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.The yeast or fungal host cell can be any of the following species selected from among Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, and Rhizobus oryzae. Escherichia coli is a particularly useful host cell because it is a well-characterized microbial cell and is widely used in molecular cloning. Other particularly useful host cells include yeasts such as Saccharomyces cerevisiae. It is understood that DNA molecules can be amplified using any suitable microbial host cell known in the art.

[0258] Similarly, eukaryotic host cells for use in the methods provided herein can be any eukaryotic cell capable of replicating or amplifying recombinant DNA molecules known and used in the art. In some embodiments, the host cells for use in the methods provided herein can be mammalian host cells. In further embodiments, the host cells can be human or non-human mammalian host cells. In another embodiment, the host cells can be insect host cells. Some widely used non-human mammalian host cells include CHO, mouse myeloma cell lines (e.g., NS0, SP2 / 0), rat myeloma cell lines (e.g., YB2 / 0), and BHK. Some widely used human host cells include HEK293 and its derivatives, HT-1080, PER.C6, and Huh-7. In one embodiment, the host cells are selected from the group consisting of HeLa, NIH3T3, Jurkat, HEK293, COS, CHO, Saos, SF9, SF21, High 5, NS0, SP2 / 0, PC12, YB2 / 0, BHK, HT-1080, PER.C6, and Huh-7.

[0259] Host cells are culturable because each host cell is known and cultured in the art. Culture conditions and culture media for different host cells may differ, as is known and practiced in the art. For example, bacterial or other microbial host cells can be cultured at 37°C with a maximum stirring speed of 300 rpm and with or without forced aeration. Some insect host cells can generally be optimally cultured at 25-30°C with or without stirring and a maximum stirring speed of 150 rpm and with or without forced aeration. Some mammalian host cells can be optimally cultured at 37°C with or without stirring and a maximum stirring speed of 150 rpm and with or without forced aeration. Furthermore, conditions for culturing various host cells can be determined by investigating the growth curves of the host cells under various conditions, as is known and practiced in the art. Several widely used host cell culture media and conditions are described in Michael Green and Joseph Sambrook's "Molecular Cloning: A Laboratory Manual," 4th edition, ISBN 978-1-936113-42-2 (2012), which is incorporated herein by reference in its entirety.

[0260] (5.2.2 Releasing DNA molecules from host cells) DNA molecules can be released from host cells in a variety of ways known and practiced in the art. For example, DNA molecules can be released by physically, mechanically, enzymatically, chemically, or by a combination of physical, mechanical, enzymatic, and chemical actions of the host cell. In some embodiments, DNA molecules can be released from host cells by exposing the cell to a solution of a cell lysis reagent. The cell lysis reagent includes surfactants such as Triton, SDS, Tween, NP-40, and / or CHAPS. In another embodiment, DNA molecules can be released from host cells by exposing the host cell to a difference in molar osmotic concentration, for example, by exposing the host cell to a hypotonic solution. In yet another embodiment, DNA molecules can be released from host cells by exposing the host cell to a high-pH or low-pH solution. In one embodiment, DNA molecules can be released from host cells by enzymatic treatment of the host cell, for example, treatment with lysozyme. In some further embodiments, DNA molecules can be released from host cells by exposing the host cells to any combination of a surfactant, molar osmotic pressure, high or low pH, and / or an enzyme (e.g., lysozyme).

[0261] Alternatively, DNA molecules can be released from host cells by applying physical force to them. In one embodiment, DNA molecules can be released from host cells by directly applying force to them, for example, using a Waring blender and a Polytron. A Waring blender uses rapidly rotating blades to break down cells, while a Polytron pulls in tissue within a shaft containing rotating blades. In another embodiment, DNA molecules can be released from host cells by applying shear stress or shear force to them. Various homogenizers can be used to force host cells into a narrow space, thereby shearing the cell membrane. In some embodiments, DNA molecules can be released from host cells by liquid-based homogenization. In one particular embodiment, DNA molecules can be released from host cells using a Dounce homogenizer. In another specific embodiment, DNA molecules can be released from host cells using a Potter-Elvehjem homogenizer. In yet another specific embodiment, DNA molecules can be released from host cells using a French press. Other physical forces that release DNA molecules from host cells include manual grinding, for example, using a mortar and pestle. In manual grinding, host cells are often frozen, for example, in liquid nitrogen, and then ground using a mortar and pestle, during which the host cells are destroyed by the tensile strength of the cellulose and other polysaccharides in the cell wall.

[0262] Furthermore, DNA molecules can be released from host cells by performing freeze-thaw cycles on the cells. In some embodiments, the suspension in the host cells is frozen and then thawed over several such freeze-thaw cycles. In some embodiments, DNA molecules can be released from host cells by performing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 freeze-thaw cycles on the host cells.

[0263] The methods described above for releasing DNA molecules from host cells are not mutually exclusive. Therefore, this disclosure stipulates that DNA molecules can be released from host cells by any combination of the DNA release methods provided in this Section 5.2.2.

[0264] (5.2.3 Denaturing DNA molecules) DNA molecules can be denatured in various ways known and practiced in this art. The process of denaturing DNA molecules can separate double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA). To separate the two DNA strands, the DNA is unwound, the hydrogen bonds holding the two strands together weaken, and finally, the temperature can be increased until they separate. The process of separating double-stranded DNA into single strands is known as DNA denaturation or DNA denaturing.

[0265] In some embodiments, the DNA denaturation step can separate two DNA strands of one or more segments of a dsDNA molecule while retaining other segments of the DNA molecule as dsDNA. In some embodiments, the DNA denaturation step can separate all DNA strands of one or more segments of the dsDNA molecule into ssDNA strands. In some further embodiments, the DNA denaturation step can separate the dsDNA in the segment between the first and second restriction sites for the nickel endonuclease on the top and bottom strands of the DNA (e.g., the DNA molecule described in Section 5.3) into ssDNA while retaining the rest of the DNA molecule as dsDNA, thereby creating an overhang between the first and second restriction sites. In one embodiment, the DNA denaturation step can separate the dsDNA into ssDNA in the segment between the third and fourth restriction sites for the nickeling endonuclease on the top and bottom strands of the DNA, while retaining the rest of the DNA molecule (e.g., the DNA molecule described in Section 5.3) as dsDNA, thereby creating an overhang between the third and fourth restriction sites. In another embodiment, the DNA denaturation step can separate the dsDNA into ssDNA in the segment between the first and second restriction sites for the nickeling endonuclease on the top and bottom strands of the DNA and in the segment between the third and fourth restriction sites, while retaining the rest of the DNA molecule (e.g., the DNA molecule described in Section 5.3) as dsDNA, thereby (1) decomposing the DNA molecule into two daughter DNA molecules, and (2) creating one overhang between the first and second restriction sites and one overhang between the third and fourth restriction sites. In one embodiment, the overhang between the first and second restriction sites for the nicking end nuclease can be a top chain 5' overhang.In another embodiment, the overhang between the first and second restriction sites for the nickeling endonuclease can be a bottom strand 3' overhang. In yet another embodiment, the overhang between the third and fourth restriction sites for the nickeling endonuclease can be a top strand 3' overhang. In yet another embodiment, the overhang between the third and fourth restriction sites for the nickeling endonuclease can be a bottom strand 5' overhang. In some embodiments, the step of denaturing a DNA molecule can separate the DNA molecule in any combination of the embodiments provided herein.

[0266] The overhangs may vary in length depending on the distance between each restriction site for the nickel end nuclease. In one embodiment, the overhangs may be identical in length and / or arrangement. In another embodiment, the overhangs may vary in length and / or arrangement. In some embodiments, the top chain 5' overhangs may vary in length to at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least The number of nucleotides can be 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, 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 at least 100.In another embodiment, the top chain 5' overhang is approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, The number of nucleotides can be approximately 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more.In one embodiment, the bottom chain 3' overhang has a length of at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 5 9, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, 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 at least 100 nucleotides.In a further embodiment, the bottom chain 3' overhang is approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 The number of nucleotides can be approximately 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more.In yet another embodiment, the top chain 3' overhang has a length of at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least The number of nucleotides can be 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, 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 at least 100.In another embodiment, the top chain 3' overhang is approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, The number of nucleotides can be approximately 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more.In some embodiments, the bottom chain 5' overhang has a length of at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least The number of nucleotides can be 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, 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 at least 100.In another embodiment, the bottom chain 5' overhang is approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, The number of nucleotides can be approximately 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more.

[0267] As is known and practiced in the art, DNA molecules can be denatured by heat, by changing the pH in the environment surrounding the DNA molecule, by increasing the salt concentration, or by any combination of these and other known means. This disclosure specifies that the DNA molecule can be denatured in the manner described above by using denaturing conditions that selectively separate the dsDNA into ssDNA in the segments between first and second restriction sites and / or between third and fourth restriction sites on the top and bottom strands of the DNA, while retaining the rest of the DNA molecule as dsDNA. In some embodiments, the denaturation completely separates the dsDNA into ssDNA. Such selective separation of dsDNA into ssDNA can be achieved by controlling the denaturing conditions and / or the time the DNA molecule is exposed to the denaturing conditions. In one embodiment, DNA molecules denature at temperatures of at least 70°C, at least 71°C, at least 72°C, at least 73°C, at least 74°C, at least 75°C, at least 76°C, at least 77°C, at least 78°C, at least 79°C, at least 80°C, at least 81°C, at least 82°C, at least 83°C, at least 84°C, at least 85°C, at least 86°C, at least 87°C, at least 88°C, at least 89°C, at least 90°C, at least 91°C, at least 92°C, at least 93°C, at least 94°C, or at least 95°C. In another embodiment, DNA molecules denature at temperatures of approximately 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In a particular embodiment, DNA molecules denature at a temperature of approximately 90°C.

[0268] In addition to thermal denaturation, some or all of the DNA molecules provided herein can undergo denaturation processes by adding various chemicals such as guanidine, formamide, sodium salicylate, dimethyl sulfoxide, propylene glycol, and urea. These chemical denaturants lower the melting temperature by competing with existing nitrogen base pairs for hydrogen bond donors and acceptors, thereby enabling isothermal denaturation. In some embodiments, the chemicals can induce denaturation at room temperature. In some specific embodiments, an alkaline agent (e.g., NaOH) can be used to denaturate DNA by changing the pH and removing protons contributing to hydrogen bonding. In another embodiment, chemical denaturation of the DNA molecules provided herein can be a milder treatment in terms of DNA stability compared to thermally induced denaturation. In another embodiment, chemical denaturation and regeneration of the DNA molecules provided herein (e.g., by changing the pH) can be faster than by heating. In some embodiments, the DNA of this disclosure can be replicated and nicked in bacteria and simultaneously denatured during the period of release from bacteria (e.g., alkaline dissolution step).

[0269] In one embodiment, DNA molecules denature at pH levels of at least 10, at least 10.1, at least 10.2, at least 10.3, at least 10.4, at least 10.5, at least 10.6, at least 10.7, at least 10.8, at least 10.9, at least 11, at least 11.1, at least 11.2, at least 11.3, at least 11.4, at least 11.5, at least 11.6, at least 11.7, at least 11.8, at least 11.9, at least 12, at least 12.1, at least 12.2, at least 12.3, at least 12.4, at least 12.5, at least 13, at least 13.5, or at least 14. In another embodiment, DNA molecules denature at pH values ​​of approximately 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 13, 13.5, or 14. In yet another embodiment, DNA molecules denature at salt concentrations of at least 1 M, at least 1.5 M, at least 2 M, at least 2.5 M, at least 3 M, at least 3.5 M, or at least 4 M. In a further embodiment, the DNA molecule is denatured at salt concentrations of approximately 1 M, 1.5 M, 2 M, 2.5 M, 3 M, 3.5 M, or 4 M. In one embodiment, the DNA molecule is exposed to denaturing conditions for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes. In another embodiment, the DNA molecule is exposed to denaturing conditions for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes.In some embodiments, DNA molecules can be denatured by any combination of denaturation conditions and denaturation periods provided herein.

[0270] For a method of selectively denaturing a DNA molecule while preserving the rest of the DNA molecule as dsDNA, denaturation conditions can be determined for the steps of a method for selectively denaturing segments between first and second restriction sites on the top and bottom strands of the DNA and segments between third and fourth restriction sites. Such selective denaturation conditions can be determined depending on the properties of the DNA segment to be selectively denatured. The stability of the DNA double helix correlates with the length of the DNA segment and the percentage of G / C content. This disclosure specifies that selective denaturation conditions can be determined by the sequence of the DNA segment to be selectively denatured or the sequence of the resulting overhang. For example, the temperature for selective denaturation can be roughly determined for the DNA sequence to be selectively denatured as Tm = 2°C × number of AT pairs + 4°C × number of GC pairs. Furthermore, other more rigorous Tm calculations are known and used in the art, such as those described in the literature by Freier SM et al., Proc Natl Acad Sci, 83, 9373-9377 (1986), which is incorporated herein by collective citation; the literature by Breslauer KJ et al., Proc Natl Acad Sci, 83, 3746-3750 (1986); the literature by Panjkovich, A. and Melo, F., Bioinformatics 21:711-722 (2005); and the literature by Panjkovich, A. et al., Nucleic Acids Res 33:W570-W572 (2005).

[0271] The overhang can include a variety of DNA sequences. In one embodiment, the overhang includes a reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of a reverse repeat). In another embodiment, the overhang includes a reverse repeat or a fragment thereof of a virus (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of a reverse repeat of a virus). In yet another embodiment, the overhang includes or is derived from any embodiment of the sequences described in sections 5.3.1, 5.3.2, 5.3.3, and 5.4. In yet another embodiment, the overhang includes or is derived from any one of the sequences described in sections 5.3.1 and 5.4. In some embodiments, the overhang does not include one or more viral replication-related sequences (e.g., RABS, RBE, or TRS) as described in Section 5.3.4. In some embodiments, the overhang does not include one or more transcription activity-related sequences (e.g., TSS or CpG motifs) as described in Section 5.3.4.

[0272] (5.2.4 Incubating DNA molecules with one or more nickel end nucleases or restriction enzymes) This disclosure provides steps of one or more methods for incubating a DNA molecule with one or more nicking endonucleases or restriction enzymes, as described in Sections 3 and 5.2. While not theoretical, a nicking endonuclease recognizes a restriction site for the nicking endonuclease within the DNA molecule and cleaves either inside or outside the restriction site on only one strand of the dsDNA (e.g., by hydrolyzing a phosphodiester bond on one DNA strand), thereby creating a nick within the dsDNA. A restriction enzyme, on the other hand, recognizes a restriction site for the restriction enzyme and cleaves both strands of the dsDNA, thereby cleaving the DNA molecule at or near a specific restriction site.

[0273] In various embodiments of the compositions and methods provided herein, the nickeling endonuclease may be methylation-dependent, methylation-sensitive, or methylation-insensitive. Various nickeling endonucleases known and practiced in the art are presented herein. In some embodiments, the nickeling endonuclease for the compositions and methods provided herein may be naturally occurring nickeling endonucleases that are not 5-methylcytosine-dependent, such as Nb.Bsml, Nb.BbvCI, Nb.BsrDI, Nb.Btsl, Nt.BbvCI, Nt.Alwl, Nt.CviPII, Nt.BsmAI, Nt.Alwl, and Nt.BstNBI. Nicking endonucleases for compositions and methods provided herein can also be engineered from type IIs restriction enzymes (e.g., Alwl, BpulOI, BbvCI, Bsal, BsmBI, BsmAI, Bsml, BspOJ, mlyl, Mval269l, and Sapl), and methods for producing nickeling endonucleases are described in reference, for example, US 7,081,358; US 7,011,966; US 7,943,303; US 7,820,424, WO201804514, all of which are incorporated herein by reference in whole.

[0274] Alternatively, a programmable nickeling enzyme can be used in place of a nickeling endonuclease in the compositions and methods provided herein. Such programmable nickeling enzymes include, for example, Cas9 or its functional equivalent (such as Pyrococcus furiosus Argonaut (PfAgo) or Cpfl). Cas9 comprises two catalytic domains, RuvC and HNH. Inactivation of one of these domains yields a programmable nickeling enzyme that can replace the nickeling endonuclease for the methods and compositions provided herein. In Cas9, the RuvC domain can be inactivated by an amino acid substitution at position D10 (e.g., D10A), the HNH domain can be inactivated by an amino acid substitution at H840 (e.g., H840A), or by amino acid substitutions at the corresponding positions in other Cas9-equivalent proteins. Such a programmable nicking enzyme may also be an Argonaut or type II CRISPR / Cas endonuclease containing two components: a nicking enzyme that cleaves target DNA (e.g., D10A Cas9 nicking enzyme or its variant or ortholog), and a guide nucleic acid, e.g., guide DNA or RNA (gDNA or gRNA), that targets or programs the nicking enzyme to a specific site within the target DNA (see, for example, Hsu et al., whose entire work is incorporated herein by reference: Nature Biotechnology 2013 31: 827-832). A programmable nicking enzyme can also be created by fusing a site-specific DNA-binding domain (targeting domain), such as the DNA-binding domain of a DNA-binding protein (e.g., restriction endonuclease, transcription factor, zinc finger, or another domain that binds to DNA at a non-random site), with the nicking endonuclease so that it acts on a specific non-random site.As is evident from the foregoing, programmable cleavage by programmable nicking enzymes is due to a targeting domain within or fused to the nicking enzyme, or a guide molecule (gDNA or gRNA) that directs the nicking enzyme to a specific, non-random site that is a programmable site by altering the targeting domain or guide molecule. Such programmable nicking enzymes are described in reference, e.g., US7,081,358 and WO2010021692A, which are incorporated herein by reference in whole.

[0275] Suitable guide nucleic acid (e.g., gDNA or gRNA) sequences and target sites suitable for guide nucleic acids are known and widely used in the art. A guide nucleic acid (e.g., gDNA or gRNA) is a specific nucleic acid (e.g., gDNA or gRNA) sequence that recognizes a target DNA region and directs a programmable nicking enzyme (e.g., Cas nuclease) to it for editing. A guide nucleic acid (e.g., gDNA or gRNA) often consists of two parts: a targeting nucleic acid, which is a 15-20 nucleotide sequence complementary to the target DNA, and a scaffold nucleic acid that acts as a binding scaffold for the programmable nicking enzyme (e.g., Cas nuclease). A target site suitable for a guide nucleic acid must have two elements: a sequence complementary to the targeting nucleic acid within the programmable nicking enzyme and an adjacent protospacer adjacency motif (PAM). The PAM acts as a binding signal for the programmable nicking enzyme (e.g., Cas nuclease). Various PAMs are known, well-defined, and utilized in the art, as described, for example, in the literature by Daniel Gleditzsch et al., RNA Biol. 16(4): 504-517 (April 2019); and the literature by Ryan T. Leenay et al., Mol Cell. 62(1): 137-147 (April 7, 2016), both of which are incorporated herein by collective citation. Exemplary gRNA and gDNA sequences that target the main stem sequence of the AAV2 ITR include those listed in Table 1. (Table 1: Exemplary nicking endonucleases and their corresponding restriction sites) [Table 1]

[0276] Various nickel endonucleases known and used in the art can be used in the methods provided herein. An exemplary list of nickel endonucleases provided as embodiments for use in the methods, and corresponding restriction sites for some of these nickel endonucleases, is available in the Restriction Enzyme Database (known in the art as REBASE), which is available at www.rebase.neb.com / cgi-bin / azlist?nick and incorporated herein by reference in its entirety. In one embodiment, nickel endonucleases that recognize the first, second, third, and / or fourth restriction sites are all for the same nickel endonuclease target sequence. In another embodiment, the first, second, third, and fourth restriction sites for the nickel endonuclease are target sequences for two different nickel endonucleases, including all possible combinations of assigning the four sites to two different nickel endonuclease target sequences (e.g., the first restriction site for the first nickel endonuclease and the remaining restriction sites for the second nickel endonuclease, the first and second restriction sites for the first nickel endonuclease and the remaining restriction sites for the second nickel endonuclease, etc.). In yet another embodiment, the first, second, third, and fourth restriction sites for the nickel endonuclease are target sequences for three different nickel endonucleases, including all possible combinations of assigning the four sites to three different endonuclease target sequences. In yet another embodiment, the first, second, third, and fourth restriction sites for the nickel endonuclease are target sequences for four different nickel endonucleases. In some embodiments, the nicking end nuclease can be any one selected from those listed in Table 2. (Table 2: Exemplary nicking endonucleases and their corresponding restriction sites) [Table 2]

[0277] Conditions for various nickeling endonucleases to cleave one strand of dsDNA are known for the various nickeling endonucleases presented herein, including temperature, salt concentration, pH, buffering reagents, presence or absence of certain surfactants, and incubation period to achieve a desired percentage of nickeled DNA molecules. These conditions are readily available from the websites or catalogs of various suppliers of nickeling endonucleases, for example, from New England BioLabs. This disclosure specifies that the step of incubating the DNA molecules with one or more nickeling endonucleases is carried out according to incubation conditions known and practiced in the art. In some embodiments, the step of incubating the DNA molecules with one or more nickeling endonucleases is carried out according to incubation conditions optimized by methods known in the art.

[0278] Various restriction enzymes known and used in the art can be used in the methods provided herein. An exemplary list of restriction enzymes provided as embodiments for use in the methods and the corresponding restriction sites for said restriction enzymes is available at neb.com / products / restriction-endonucleases and is described in the New England Biolabs catalog, which is incorporated herein by reference in its entirety. Conditions under which various restriction enzymes cleave dsDNA, including temperature, salt concentration, pH, buffer reagents, certain surfactants, and incubation period to achieve a desired percentage of nicked DNA molecules, are known for the various restriction enzymes provided herein. These conditions are readily available from various restriction enzyme suppliers, for example, the website or catalog of New England BioLabs. This disclosure specifies that the step of incubating the DNA molecules with the restriction enzyme is carried out according to incubation conditions known and practiced in the art.

[0279] (5.2.5 Annealing) The annealing step in the method provided herein is performed to selectively anneal the ssDNA overhang within the molecule, thereby generating a hairpinned reverse repeat at one end of the DNA fragment obtained in the denaturing step (sections 5.2.3) (e.g., those in sections 5.3 and 5.4). In one embodiment, the annealing step in the method provided herein is performed to selectively anneal the ssDNA overhang within the molecule, thereby generating hairpinned reverse repeats at both ends of the DNA fragment obtained in the denaturing step (sections 5.2.3) (e.g., those in sections 5.3 and 5.4). Without being constrained by or otherwise limited by theory, such selective intramolecular annealing of ssDNA overhangs is achieved because the intramolecular complementary sequences within the ssDNA overhang make the intramolecular annealing of the ssDNA overhang thermodynamically and / or kinetically favorable compared to the intermolecular annealing of the ssDNA overhang.

[0280] While not being confined to or otherwise limited by theory, it is recognized that certain lengths and / or arrangements of overhangs can make intramolecular annealing of ssDNA overhangs thermodynamically and / or kinetically more favorable than intermolecular annealing of the same ssDNA overhangs. For example, linear interaction plots showing intramolecular forces within the overhang, intermolecular forces between chains, and the resulting structure are shown in Figures 2A to C. The thermodynamics and dynamics of ssDNA overhang annealing are determined by various factors, particularly enthalpy (ΔH) and entropy (ΔS). The inventors recognize that the entropy decrease in intramolecular annealing is smaller than that in intramolecular annealing because the decrease in degrees of freedom of motion from a free ssDNA overhang to an intramolecularly annealed overhang is less than the decrease in degrees of freedom of motion from a free ssDNA overhang to an intermolecularly annealed overhang. On the other hand, because the number of complementary nucleotide pairs in an intramolecularly annealed overhang is less than the number of complementary nucleotide pairs in an intermolecularly annealed overhang (and therefore fewer Watson-Crick and Hoogsteen-type hydrogen bonds), the enthalpy increase in intramolecular annealing may be less than that in intermolecular annealing. This disclosure specifies that ssDNA overhangs can be designed to have a specific length, number of complementary nucleotide pairs, and percentages of GC pairs and AT pairs such that the free energy increase for intramolecular annealing of the overhang (ΔG=ΔH-TΔS) is greater than that for intermolecular annealing, thereby making intramolecular annealing thermodynamically more favorable than intermolecular annealing. The inventors further recognize that the reaction rate of intramolecular annealing of ssDNA overhangs may be higher than that of intermolecular annealing because nucleotides within an ssDNA overhang have a higher probability of mutual contact than the probability of contact with nucleotides in another ssDNA overhang during molecular motion.This disclosure stipulates that even when intramolecular annealing is thermodynamically unfavorable compared to intermolecular annealing, the superior reaction rate of intramolecular annealing of ssDNA overhangs can preferentially lead to the formation of intramolecularly annealed overhangs than intermolecularly annealed overhangs.

[0281] The annealing process can be carried out at a variety of temperatures that are more favorable to intramolecular annealing than to intermolecular annealing. In one embodiment, the ssDNA overhang can be performed at a minimum of 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, and at least It is annealed at a temperature of 37°C, at least 38°C, at least 39°C, at least 40°C, at least 41°C, at least 42°C, at least 43°C, at least 44°C, at least 45°C, at least 46°C, at least 47°C, at least 48°C, at least 49°C, at least 50°C, at least 51°C, at least 52°C, at least 53°C, at least 54°C, at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, or at least 60°C. In another example, the ssDNA overhang is approximately 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, and 36°C. Annealing takes place at temperatures of approximately 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C. In one particular embodiment, the ssDNA overhang is annealed at a temperature of at least 25°C. In another particular embodiment, the ssDNA overhang is annealed at a temperature of approximately 25°C. In yet another particular embodiment, the ssDNA overhang is annealed at room temperature.

[0282] Furthermore, the annealing process can be performed for a variety of times that are more favorable to intramolecular annealing than to intermolecular annealing. In one embodiment, the ssDNA overhang is annealed for 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 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 minutes. In another embodiment, the ssDNA overhang is annealed for approximately 1, 2, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 minutes. In a particular embodiment, the ssDNA overhang is annealed for at least 20 minutes. In another specific embodiment, the ssDNA overhang is annealed for approximately 20 minutes.

[0283] In some embodiments, annealing can be achieved by lowering the temperature to a temperature below the melting temperature of the calculated sense and antisense sequence pair. The melting temperature depends on the specific nucleotide base content and the properties of the solution used, e.g., the salt concentration. The melting temperature of any sequence-solution combination can be readily calculated, as is known and practiced in the art.

[0284] In some embodiments, annealing can be achieved isothermally by reducing the amount of denaturing chemicals to allow interaction between pairs of sense and antisense sequences. The minimum concentration of denaturing chemicals required to denature a DNA sequence may depend on the specific nucleotide base content and the properties of the solution used, such as temperature or salt concentration. The concentration of a chemical denaturing agent that does not cause denaturation for any given sequence-solution combination can be readily determined, as is known and practiced in the art. The concentration of the chemical denaturing agent can also be readily modified, as is known and practiced in the art. For example, the amount of urea can be reduced by dialysis or tangential flow filtration, or the pH can be changed by adding an acid or base.

[0285] The annealing temperature and annealing time for intramolecular annealing correlate with the length of the ssDNA overhang, the number of complementary nucleotide pairs, the percentages of GC and AT pairs, and the sequence of the ssDNA overhang (arrangement of complementary nucleotide pairs). In one embodiment, the ssDNA overhang provided in the method provided herein contains any number of nucleotides of any length, as described in Section 5.2.3. In one embodiment, the ssDNA overhang provided in the method provided herein includes at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 pairs of intramolecular complementary nucleotide pairs. In some embodiments, the ssDNA overhang provided in the method provided herein comprises about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 pairs of intramolecular complementary nucleotide pairs.In some embodiments, the ssDNA overhang provided in the methods provided herein includes at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 90% of intramolecular complementary nucleotide pairs. In one embodiment, the ssDNA overhang provided in the method provided herein comprises approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, or approximately 90% of the intramolecular complementary nucleotide pairs.

[0286] Furthermore, the inventors recognize that the concentration of DNA molecules, which correlates with the concentration of overhangs, can affect the equilibrium and dynamics of intramolecular and intermolecular annealing of the overhangs. Without being confined to or otherwise limited to theory, if the concentration of overhangs is too high, the probability of intermolecular contact between overhangs increases, and in that case, the kinetic superiority of intramolecular contact over intermolecular contact observed at the low concentrations described above decreases.

[0287] As described above, in some embodiments, intramolecular interactions may occur at a rapid rate, while intermolecular interactions occur at a slower rate. In some embodiments, base-pair interactions involving three or more molecules (e.g., three different strands) occur at the slowest rate. In some embodiments, the kinetic rate of intramolecular interactions to intermolecular interactions is governed by the concentration of each molecule. In some embodiments, the lower the concentration of DNA strands, the faster the kinetic rate of intramolecular interactions or the stronger the intramolecular forces.

[0288] Viewed individually, the absolute free energies forming each complementary domain of an IR or ITR may differ, resulting in regions of the IR or ITR that may fold locally earlier when the chain transitions from a denatured state to an annealed state. The presence of locally folded domains (e.g., a central hairpin or branched hairpin, as in the AAV2 ITR, as described elsewhere in this section (Section 5.3.1) and in Section 5.4) can reduce the amount of bases available for pairing with other chains, thus reducing the likelihood of intermolecular annealing or hybridization and shifting the equilibrium from intermolecular annealing to intramolecular annealing or ITR formation.

[0289] Accordingly, this disclosure specifies that the annealing process can be carried out at various concentrations that are more favorable to intramolecular annealing than to intermolecular annealing. In some embodiments, the ssDNA overhang is 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, 20 or less, 21 or less, 22 or less, 23 or less, 24 or less, 25 or less, 26 or less, 27 or less, 28 or less, 29 or less, 30 or less, 31 or less, 32 or less, 33 or less, 34 or less, 35 or less, 36 or less, 37 or less, 38 or less, 39 or less, 40 or less, 41 or less, 42 or less, 43 or less, 44 or less, 45 or less, 46 or less, 47 or less, 48 ​​or less, 49 or less, 50 or less, 55 or less, 60 or less , 65 or less, 70 or less, 75 or less, 80 or less, 85 or less, 90 or less, 95 or less, 100 or less, 110 or less, 120 or less, 130 or less, 140 or less, 150 or less, 160 or less, 170 or less, 180 or less, 190 or less, 200 or less, 210 or less, 220 or less, 230 or less, 240 or less, 250 or less, 260 or less, 270 or less, 280 or less, 290 or less, 300 or less, 325 or less, 350 or less, 375 or less, 400 or less, 425 or less, 450 or less, 475 or less, 500 or less, 550 or less, Annealed at ng / μl concentrations of 600 or less, 650 or less, 700 or less, 750 or less, 800 or less, 850 or less, 900 or less, 950 or less, 1000 or less.In one embodiment, the ssDNA overhang is approximately 1, 2, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65 over the DNA molecule. Annealing occurs at concentrations of approximately 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000 ng / μl.

[0290] Similarly, this disclosure specifies that the annealing process can be carried out at various molar concentrations that are more favorable to intramolecular annealing than to intermolecular annealing. In some embodiments, the ssDNA overhang is 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, 20 or less, 21 or less, 22 or less, 23 or less, 24 or less, 25 or less, 26 or less, 27 or less, 28 or less, 29 or less, 30 or less, 31 or less, 32 or less, 33 or less, 34 or less, 35 or less, 36 or less, 37 or less, 38 or less, 39 or less, 40 or less, 41 or less, 42 or less, 43 or less, 44 or less, 45 or less, 46 or less, 47 or less, 48 ​​or less, 49 or less, 50 or less, 55 or less, 60 or more Below, 65 or less, 70 or less, 75 or less, 80 or less, 85 or less, 90 or less, 95 or less, 100 or less, 110 or less, 120 or less, 130 or less, 140 or less, 150 or less Lower, 160 or less, 170 or less, 180 or less, 190 or less, 200 or less, 210 or less, 220 or less, 230 or less, 240 or less, 250 or less, 260 or less, 270 or less Lower, 280 or less, 290 or less, 300 or less, 325 or less, 350 or less, 375 or less, 400 or less, 425 or less, 450 or less, 475 or less, 500 or less, 550 or less Bottom, annealed at nM concentrations below 600, below 650, below 700, below 750, below 800, below 850, below 900, below 950, and below 1000.In one embodiment, the ssDNA overhang is approximately 1, 2, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, and 60 over the DNA molecule. Annealing is performed at concentrations of approximately 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000 nM. In some further embodiments, the ssDNA overhang is annealed at a concentration of 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, and 20 or less μM. In yet another embodiment, the ssDNA overhang is annealed at a concentration of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, and about 20 μM. In a particular embodiment, the ssDNA overhang is annealed at a concentration of about 10 nM with respect to the DNA molecule. In another particular embodiment, the ssDNA overhang is annealed at a concentration of about 20 nM with respect to the DNA molecule. In yet another specific embodiment, the ssDNA overhang is annealed at a concentration of approximately 30 nM with respect to the DNA molecule. In yet another specific embodiment, the ssDNA overhang is annealed at a concentration of approximately 40 nM with respect to the DNA molecule.In yet another specific embodiment, the ssDNA overhang is annealed at a concentration of about 50 nM with respect to the DNA molecule. In yet another specific embodiment, the ssDNA overhang is annealed at a concentration of about 60 nM with respect to the DNA molecule. In one specific embodiment, the ssDNA overhang is annealed at a concentration of about 10 ng / μl with respect to the DNA molecule. In yet another specific embodiment, the ssDNA overhang is annealed at a concentration of about 20 ng / μl with respect to the DNA molecule. In yet another specific embodiment, the ssDNA overhang is annealed at a concentration of about 30 ng / μl with respect to the DNA molecule. In yet another specific embodiment, the ssDNA overhang is annealed at a concentration of about 40 ng / μl with respect to the DNA molecule. In one specific embodiment, the ssDNA overhang is annealed at a concentration of about 50 ng / μl with respect to the DNA molecule. In another specific embodiment, the ssDNA overhang is annealed at a concentration of approximately 60 ng / μl with respect to the DNA molecule. In yet another specific embodiment, the ssDNA overhang is annealed at a concentration of approximately 70 ng / μl with respect to the DNA molecule. In one particular embodiment, the ssDNA overhang is annealed at a concentration of approximately 80 ng / μl with respect to the DNA molecule. In yet another specific embodiment, the ssDNA overhang is annealed at a concentration of approximately 90 ng / μl with respect to the DNA molecule. In yet another specific embodiment, the ssDNA overhang is annealed at a concentration of approximately 100 ng / μl with respect to the DNA molecule.

[0291] In some embodiments, the ssDNA overhangs provided in the methods provided herein include any sequence listed in Table 3. (Table 3: Sequence of ssDNA overhang and corresponding structure after annealing) [Table 3]

[0292] In some embodiments, the structure of the DNA molecule provided herein is identical after 2, 3, 4, 5, 10, or 20 cycles of denaturation / regeneration (e.g., denaturation as described in Section 5.2.3 and re-annealing as described in this Section (Section 5.2.5)). The DNA structure can be described by an ensemble of structures with minimum or near minimum energy. In some embodiments, the ensemble DNA structure is identical after 2, 3, 4, 5, 10, or 20 cycles of denaturation / regeneration. In one embodiment, the folded hairpin structure formed from ITR or IR provided herein is identical after 2, 3, 4, 5, 10, or 20 cycles of denaturation / regeneration. In another embodiment, the ensemble structure of the folded hairpin is identical after 2, 3, 4, 5, 10, or 20 cycles of denaturation / regeneration.

[0293] (5.2.6 Incubate with exonuclease) This disclosure provides a step of incubation with an exonuclease as described in Section 3. An exonuclease cleaves nucleotides from the ends (exo) of a DNA molecule. An exonuclease can cleave nucleotides in the 5' to 3' direction, the 3' to 5' direction, or bidirectionally. In some embodiments, the exonuclease for use in the methods provided herein cleaves nucleotides without exhibiting sequence specificity. In some embodiments, the exonuclease for use in the methods provided herein digests a DNA fragment containing ends produced by one or more nickeling endonucleases that recognize and cleave the fifth and sixth restriction sites, or by restriction enzymes that cleave a plasmid or fragment of a plasmid provided in Section 3.

[0294] Various exonucleases known and used in the art can be used in the methods provided herein. An exemplary list of exonucleases provided as embodiments of restriction enzymes for use in the methods is available at neb.com / products / dna-modifying-enzymes-and-cloning-technologies / nucleases and is included in the catalog of New England Biolabs, which is incorporated herein by reference in its entirety. Conditions under which various exonucleases digest DNA molecules, including temperature, salt concentration, pH, buffering reagents, presence or absence of certain surfactants, and incubation period to achieve the desired percentage of digestion, are known for the various exonucleases provided herein. These conditions are readily available from various restriction enzyme suppliers, for example, the website or catalog of New England BioLabs. This disclosure specifies that the step of incubating the DNA molecule with the restriction enzyme is carried out according to incubation conditions known and practiced in the art.

[0295] The step of incubating the exonuclease selectively digests DNA molecules having one or more ends while leaving hairpin-terminated DNA molecules intact. As is evident from the descriptions in Sections 5.2.5 and 5.4, hairpin-terminated DNA molecules contain zero, one, two, or more nicks. In some embodiments, the exonuclease for use in the methods provided herein may be an exonuclease that selectively digests DNA molecules having one or more ends while leaving circular ssDNA / dsDNA molecules or DNA molecules containing one or more nicks but no ends intact. In one embodiment, the exonuclease for use in the methods provided herein may be exonuclease V (RecBCD). In one embodiment, the exonuclease for use in the methods provided herein may be exonuclease VIII or cleavage-type exonuclease VIII. Exonuclease V (RecBCD), exonuclease VIII, and cleavage-type exonuclease VIII include the selectivity described in this paragraph. In some embodiments, the exonuclease for use in the methods provided herein may be an exonuclease that starts at one or more nicks and selectively digests linear segments of a DNA molecule, but cannot proceed through folded hairpins, terminating the digestion at the hairpins and leaving ssDNA behind. In some embodiments, the exonuclease used to initiate one or more nicks and / or double-strand breaks may be a T7 exonuclease. Other suitable exonucleases are also known and used in the art and presented herein, as described on the websites and catalogs of various exonuclease suppliers, including, for example, New England BioLabs.

[0296] In some embodiments, after exonuclease treatment, the DNA molecules of this disclosure substantially contain no prokaryotic skeletal sequence. In some embodiments, the skeletal sequence means a plasmid sequence that is not part of the sequence containing the expression cassette between two ITRs. In some embodiments, the skeletal sequence means a vector sequence that is not part of the sequence containing the expression cassette between two ITRs. In some embodiments, the isolated DNA molecules of this disclosure contain 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% of the prokaryotic skeletal sequence of the original plasmid.

[0297] (5.2.7 Repairing the nick using ligauze) This disclosure provides an optional step of repairing the nick using a ligase as described in Section 3. DNA ligases catalyze the linking of two ends of a DNA molecule by forming one or more new covalent bonds. For example, a commonly used T4 DNA ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl ends in DNA. The formation of a new covalent bond linking two DNA molecules, catalyzed by a ligase, is called "ligation." In some embodiments, the DNA ligase for use in the methods provided herein ligates nucleotides without exhibiting sequence specificity. In some embodiments, the DNA ligase for use in the methods provided herein ligates two ends at one nick of a DNA molecule as described in Section 5.4, thereby repairing the one nick. In some embodiments, the DNA ligase for use in the methods provided herein ligates pairs of ends at two nicks of a DNA molecule as described in Section 5.4, thereby repairing the two nicks. In some embodiments, the DNA ligase for use in the methods provided herein ligates each pair of ends at all nicks of the DNA molecule described in Section 5.4, thereby repairing all nicks of the DNA molecule, if the DNA molecule described in Section 5.4 forms a circular DNA after all nicks of the DNA molecule described in Section 5.4 have been repaired. As described in Section 5.4, in some embodiments, the DNA molecule described in Section 5.4 consists of two nicks. In one embodiment, the DNA molecule described in Section 5.4 contains two nicks. In another embodiment, the DNA molecule described in Section 5.4 consists of one nick. In yet another embodiment, the DNA molecule described in Section 5.4 contains one nick.

[0298] In some embodiments, the step of repairing the nicks using ligase can be carried out by incubation conditions known and practiced in the art.

[0299] Various ligases known and used in the art can be used in the methods provided herein. An exemplary list of ligases provided as embodiments for use in the methods is available at neb.com / products / dna-modifying-enzymes-and-cloning-technologies / dna-ligases / dna-ligases and is included in its entirety in the New England Biolabs catalog, which is incorporated herein by reference. Conditions under which various ligases digest DNA molecules, including temperature, salt concentration, pH, buffer reagents, the presence or absence of certain surfactants, and incubation period to achieve the desired percentage of digestion, are known for the various ligases presented herein. These conditions are readily available from various restriction enzyme suppliers, e.g., the New England BioLabs website or catalog. Ligation conditions also correlate with the degrees of freedom of movement of the two DNA ends to be ligated. For example, annealing both ends to a common DNA strand can bring two DNA ends closer to each other, or enhance ligation if there is a high probability that the two DNA ends will be close to each other. In one embodiment, the steps of the method provided in this section (Section 5.2.7) involve repairing a nick using a ligase to produce circular DNA, where the two DNA ends at any nick of the DNA molecule described in Section 5.4 are annealed to a common DNA strand. In some embodiments, the step of repairing the nick using a ligase is carried out according to incubation conditions known and practiced in the art.

[0300] In some embodiments, the hairpin-terminated DNA molecules described herein can be produced on a large scale, in high yield, and / or in high purity using the method provided in this Section 5.2. In some embodiments, the large scale, high yield, and / or high purity can be achieved in a single reaction vessel. In some embodiments, the large scale is at least 1 mg, 10 mg, 100 mg, 1 g, 10 g, 100 g, 1 kg, or at least 10 kg. In some embodiments, the high yield is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% (comparing the number of plasmid copies used as input to the number of hairpin-terminated DNA molecules as product). In some embodiments, the high purity is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% purity of the hairpin-terminated DNA molecules as product resulting from the method provided herein.

[0301] (5.3 DNA molecules used in this method) This disclosure provides various aspects and embodiments of DNA molecules for use in the methods provided herein as described in Section 3 above. In one aspect, provided herein is a DNA molecule in the 5'-3' direction of the top strand: i) a first reverse repeat (e.g., described in Section 5.3.1), wherein when the top strand separates from the bottom strand of the first reverse repeat, the nicking results in a top strand 5' overhang containing the first reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), with first and second restriction sites for nicking endonucleases located on the opposing strand near the first reverse repeat (e.g., described in Sections 5.2.3, 5.2.4, and 5.3.2), the first reverse repeat; ii) expression iii) a cassette (e.g., described in Section 5.3.3); and iii) a second reverse repeat (e.g., described in Section 5.3.1), wherein when the top strand separates from the bottom strand of the second reverse repeat, the nicking results in a top strand 3' overhang containing the second reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), the third and fourth restriction sites for the nicking endonuclease are located on the opposing strand near the second reverse repeat (e.g., described in Sections 5.2.3, 5.2.4, and 5.3.2), and the double-stranded DNA molecule containing the second reverse repeat. In one embodiment, the top strand 5' overhang contains the first reverse repeat. In one embodiment, the top strand 3' overhang contains the second reverse repeat. In one embodiment, the top chain 5' overhang includes the first reverse repeat, and the top chain 3' overhang includes the second reverse repeat.

[0302] In another embodiment, provided herein are first and second restriction sites for nicking endonucleases located on opposing strands near the first reverse repeat (e.g., described in sections 5.2.3, 5.2.4, and 5.3.2), such that when the top strand separates from the bottom strand of the first reverse repeat, nicking results in a bottom strand 3' overhang containing the first reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), the first reverse repeat; ii) an expression cassette (e.g. (i) a double-stranded DNA molecule containing the second reverse repeat, wherein, when the top strand separates from the bottom strand of the second reverse repeat, the nicking results in a bottom strand 5' overhang containing the second reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), the third and fourth restriction sites for the nicking endonuclease are located on the opposing strand near the second reverse repeat (e.g., as described in sections 5.2.3, 5.2.4, and 5.3.2 or shown in Figures 2A and 2C). In one embodiment, the bottom strand 3' overhang contains the first reverse repeat. In one embodiment, the bottom strand 5' overhang contains the second reverse repeat. In one embodiment, the bottom chain 3' overhang includes the first reverse repeat, and the bottom chain 5' overhang includes the second reverse repeat.

[0303] In yet another embodiment, provided herein are first and second limiting sites for nicking endonucleases located on a chain opposite to the first reverse repeat (e.g., described in sections 5.3.1) such that when the top chain separates from the bottom chain of the first reverse repeat, nicking results in a top chain 5' overhang containing the first reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) of the first reverse repeat (e.g., described in sections 5.2.3, 5.2.4, and 5.3.2); ii) iii) a current cassette (e.g., described in Section 5.3.3); and iii) a second reverse repeat (e.g., described in Section 5.3.1), wherein when the top strand separates from the bottom strand of the second reverse repeat, the nicking results in a bottom strand 5' overhang containing the second reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), the third and fourth restriction sites for the nicking endonuclease are located on the opposing strand near the second reverse repeat (e.g., described in Sections 5.2.3, 5.2.4, and 5.3.2), the second reverse repeat, a double-stranded DNA molecule comprising the second reverse repeat. In one embodiment, the top strand 5' overhang contains the first reverse repeat. In one embodiment, the bottom strand 5' overhang contains the second reverse repeat. In one embodiment, the top chain 5' overhang includes the first reverse repeat, and the bottom chain 5' overhang includes the second reverse repeat.

[0304] In a further embodiment, provided herein are first and second limiting sites for the nicking endonuclease located on the opposing strand near the first reverse repeat (e.g., described in sections 5.2.3, 5.2.4, and 5.3.2), such that when the top strand separates from the bottom strand of the first reverse repeat, the nicking results in a bottom strand 3' overhang containing the first reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), the first reverse repeat; and ii) an expression cassette (e.g., , as described in Section 5.3.3); and iii) a double-stranded DNA molecule containing the second reverse repeat, wherein, when the top strand separates from the bottom strand of the second reverse repeat, the nickeling provides a top strand 3' overhang containing the second reverse repeat, the third and fourth restriction sites for the nickeling endonuclease are located on the opposing strand near the second reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the second reverse repeat) (e.g., as described in Sections 5.2.3, 5.2.4, and 5.3.2 or shown in Figures 2B and 2C). In one embodiment, the bottom strand 3' overhang contains the first reverse repeat. In one embodiment, the top strand 3' overhang contains the second reverse repeat. In one embodiment, the bottom chain 3' overhang includes the first reverse repeat, and the top chain 3' overhang includes the second reverse repeat.

[0305] In one embodiment, provided herein are: i) a first reverse repeat (e.g., described in Section 5.3.1) in the 5'-3' direction of the top strand, wherein when the top strand separates from the bottom strand of the first reverse repeat, nicking by a programmable nicking enzyme results in a top strand 5' overhang containing the first reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), wherein first and second target sites for guide nucleic acids for a programmable nicking enzyme are located on the opposite strand near the first reverse repeat (e.g., described in Sections 5.2.3, 5.2.4, and 5.3.2); ii) expression iii) a set (e.g., described in Section 5.3.3); and iii) a second reverse repeat (e.g., described in Section 5.3.1), wherein when the top strand separates from the bottom strand of the second reverse repeat, the nickeling by a programmable nickeling enzyme yields a top strand 3' overhang containing the second reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), the third and fourth target sites for guide nucleic acids for the programmable nickeling enzyme are located on the opposing strand near the second reverse repeat (e.g., described in Sections 5.2.3, 5.2.4, and 5.3.2), the second reverse repeat, a double-stranded DNA molecule containing the second reverse repeat. In one embodiment, the top strand 5' overhang contains the first reverse repeat. In one embodiment, the top strand 3' overhang contains the second reverse repeat. In one embodiment, the top chain 5' overhang includes the first reverse repeat, and the top chain 3' overhang includes the second reverse repeat.

[0306] In another embodiment, provided herein are: i) a first reverse repeat (e.g., as described in Section 5.3.1) in the 5' to 3' direction of the top strand, wherein when the top strand separates from the bottom strand of the first reverse repeat, the nicking by the programmable nicking enzyme results in a bottom strand 3' overhang containing the first reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), wherein first and second target sites for guide nucleic acids for a programmable nicking enzyme are located on the opposite strand near the first reverse repeat (e.g., as described in Sections 5.2.3, 5.2.4, and 5.3.2), the first reverse repeat; ii) an expression cassette (e.g. (i) a double-stranded DNA molecule containing the second reverse repeat, wherein, when the top strand separates from the bottom strand of the second reverse repeat, the nicking by the programmable nicking enzyme results in a bottom strand 5' overhang containing the second reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), the third and fourth target sites for guide nucleic acids for the programmable nicking enzyme are located on the opposing strand near the second reverse repeat (e.g., as described in sections 5.2.3, 5.2.4, and 5.3.2 or shown in Figures 2A and 2C). In one embodiment, the bottom strand 3' overhang contains the first reverse repeat. In one embodiment, the bottom chain 5' overhang includes the second reverse repeat. In another embodiment, the bottom chain 3' overhang includes the first reverse repeat, and the bottom chain 5' overhang includes the second reverse repeat.

[0307] In yet another embodiment, provided herein are: i) a first reverse repeat (e.g., described in Section 5.3.1) in the 5' to 3' direction of the top strand, wherein when the top strand separates from the bottom strand of the first reverse repeat, nicking by a programmable nicking enzyme results in a top strand 5' overhang containing the first reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), wherein first and second target sites for guide nucleic acids for a programmable nicking enzyme are located on the opposite strand near the first reverse repeat (e.g., described in Sections 5.2.3, 5.2.4, and 5.3.2); ii) iii) an expression cassette (e.g., described in Section 5.3.3); and iii) a second reverse repeat (e.g., described in Section 5.3.1), wherein third and fourth target sites for guide nucleic acids for a programmable nickeling enzyme are located on the opposing strand near the second reverse repeat (e.g., described in Sections 5.2.3, 5.2.4, and 5.3.2), such that when the top strand separates from the bottom strand of the second reverse repeat, nickeling by a programmable nickeling enzyme results in a bottom strand 5' overhang containing the second reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), and the second reverse repeat is a double-stranded DNA molecule containing the second reverse repeat. In one embodiment, the top strand 5' overhang contains the first reverse repeat. In one embodiment, the bottom strand 5' overhang contains the second reverse repeat. In one embodiment, the top chain 5' overhang includes the first reverse repeat, and the bottom chain 5' overhang includes the second reverse repeat.

[0308] In a further embodiment, provided herein are: i) a first reverse repeat (e.g., as described in Section 5.3.1) in the 5' to 3' direction of the top strand, wherein when the top strand separates from the bottom strand of the first reverse repeat, nicking by a programmable nicking enzyme results in a bottom strand 3' overhang containing the first reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), wherein first and second target sites for guide nucleic acids for a programmable nicking enzyme are located on opposite strands near the first reverse repeat (e.g., as described in Sections 5.2.3, 5.2.4, and 5.3.2), the first reverse repeat; ii) an expression cassette (e.g., iii) a double-stranded DNA molecule comprising the second reverse repeat, wherein, when the top strand separates from the bottom strand of the second reverse repeat, nicking by a programmable nicking enzyme yields a top strand 3' overhang containing the second reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), the third and fourth target sites for guide nucleic acids for a programmable nicking enzyme are located on the opposing strand near the second reverse repeat (e.g., as described in sections 5.2.3, 5.2.4, and 5.3.2 or shown in Figures 2B and 2C). In one embodiment, the bottom strand 3' overhang contains the first reverse repeat. In one embodiment, the top chain 3' overhang includes the second reverse repeat. In another embodiment, the bottom chain 3' overhang includes the first reverse repeat, and the top chain 3' overhang includes the second reverse repeat. In one embodiment, the first, second, third, and fourth target sites for the programmable nicking enzyme in this paragraph and the three preceding paragraphs are all identical.In another embodiment, three of the first, second, third, and fourth target sites for the programmable nicking enzyme in this paragraph and the three preceding paragraphs are identical. In yet another embodiment, two of the first, second, third, and fourth target sites for the programmable nicking enzyme in this paragraph and the three preceding paragraphs are identical. In yet another embodiment, all of the first, second, third, and fourth target sites for the programmable nicking enzyme in this paragraph and the three preceding paragraphs are different.

[0309] The DNA molecules provided herein include a variety of features and have a variety of embodiments, as described in Section 3 and the paragraphs above in this section (Section 5.3), and these features and embodiments are further described in the following various subsections: an embodiment for reverse repeats including a first reverse repeat and / or a second reverse repeat is described in Section 5.3.1; an embodiment for restriction enzymes, nickeling endonucleases, and their respective restriction sites is described in Sections 5.3.2 and 5.2.4; an embodiment for programmable nickeling enzymes and their target sites is described in Section 5.2.4; an embodiment for expression cassettes is described in Section 5.3.3; an embodiment for plasmids and vectors is described in Section 5.3.5; and an embodiment for DNA molecules containing fewer than four restriction sites for nickeling endonucleases is described in Section 5.3.6. Accordingly, this disclosure provides DNA molecules including any permutations and combinations of the various embodiments of the DNA molecules, as well as embodiments of the features of the DNA molecules described herein. In further embodiments, the arrangement of the ITR, expression cassette, restriction sites for nickel endonucleases or restriction enzymes, and programmable nickel enzymes and their target sites can be any arrangement as described in sections 5.2.3, 5.2.4, 5.2.5, 5.3.1, 5.3.2, 5.3.3, 5.3.6, and 5.4.

[0310] In one embodiment, provided herein are first and second limiting sites for nicking endonucleases located on opposing strands near the first reverse repeat (as described in sections 5.2.3, 5.2.4, 5.3.2, and 5.3.4), wherein, in the 5' to 3' direction of the top strand: i) a first viral replication-deficient reverse repeat (e.g., as described in sections 5.3.1 and 5.3.4), wherein when the top strand separates from the bottom strand of the first reverse repeat, nicking results in a top strand 5' overhang containing the first reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%); ii iii) an expression cassette (e.g., described in Section 5.3.3); and iii) a second viral replication-deficient reverse repeat (e.g., described in Sections 5.3.1 and 5.3.4), wherein when the top strand separates from the bottom strand of the second reverse repeat, the nicking yields a top strand 3' overhang containing the second reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), and third and fourth restriction sites for nicking endonucleases are located on the opposing strand near the second reverse repeat (e.g., described in Sections 5.2.3, 5.2.4, and 5.3.2), and the double-stranded DNA molecule containing the second viral replication-deficient reverse repeat. In one embodiment, the top strand 5' overhang contains the first viral replication-deficient reverse repeat. In one embodiment, the top strand 3' overhang includes the second viral replication-deficient reverse repeat. In another embodiment, the top strand 5' overhang includes the first viral replication-deficient reverse repeat, and the top strand 3' overhang includes the second viral replication-deficient reverse repeat.

[0311] In another embodiment, provided herein are: i) a first viral replication-deficient reverse repeat (e.g., described in sections 5.3.1 and 5.3.4) in the 5'-to-3' direction of the top strand, wherein the first viral replication-deficient reverse repeat (e.g., described in sections 5.2.3, 5.2.4, and 5.3.2) has first and second restriction sites for a nicking endonuclease located on the opposite strand near the first reverse repeat so that nicking results in: ii) an expression cassette (e.g., described in section 5.3.3); and iii) a second viral replication-deficient reverse repeat (e.g., described in sections 5.3.1 and 5.3.4) in which, when the top strand separates from the bottom strand of the second reverse repeat, nicking results in the second reverse repeat A double-stranded DNA molecule containing the second viral replication-deficient reverse repeat, wherein third and fourth restriction sites for the nickeling endonuclease are located on the opposing strand near the second reverse repeat (for example, as described in sections 5.2.3, 5.2.4, and 5.3.2 or shown in Figures 2A and 2C), resulting in a bottom strand 5' overhang containing the reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the second reverse repeat). In one embodiment, the bottom strand 3' overhang contains the first viral replication-deficient reverse repeat. In one embodiment, the bottom strand 5' overhang contains the second viral replication-deficient reverse repeat. In one embodiment, the bottom strand 3' overhang contains the first viral replication-deficient reverse repeat, and the bottom strand 5' overhang contains the second viral replication-deficient reverse repeat.

[0312] In yet another embodiment, provided herein are: i) a first viral replication-deficient reverse repeat (e.g., described in sections 5.3.1 and 5.3.4) in the 5' to 3' direction of the top strand, wherein first and second limiting sites for nicking endonucleases are located on the opposing strand near the first viral replication-deficient reverse repeat (e.g., described in sections 5.2.3, 5.2.4, and 5.3.2), such that when the top strand separates from the bottom strand of the first reverse repeat, nicking results in a top strand 5' overhang containing the first reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%); ii) iii) an expression cassette (e.g., described in Section 5.3.3); and iii) a second viral replication-deficient reverse repeat (e.g., described in Sections 5.3.1 and 5.3.4), wherein third and fourth restriction sites for the nicking endonuclease are located on the opposing strand near the second reverse repeat (e.g., described in Sections 5.2.3, 5.2.4, and 5.3.2), such that when the top strand separates from the bottom strand of the second reverse repeat, the nicking results in a bottom strand 5' overhang containing the second reverse repeat or a fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%), and the double-stranded DNA molecule containing the second viral replication-deficient reverse repeat. In one embodiment, the top strand 5' overhang contains the first viral replication-deficient reverse repeat. In one embodiment, the bottom strand 5' overhang includes the second viral replication-deficient reverse repeat. In another embodiment, the top strand 5' overhang includes the first viral replication-deficient reverse repeat, and the bottom strand 5' overhang includes the second viral replication-deficient reverse repeat.

[0313] In further embodiments, provided herein are: i) a first viral replication-deficient reverse repeat (e.g., described in sections 5.3.1 and 5.3.4) in the 5' to 3' direction of the top strand, wherein first and second restriction sites for nicking endonucleases are located on opposite strands near the first viral replication-deficient reverse repeat, such that nicking results in the bottom strand (as described in sections 5.2.3, 5.2.4, and 5.3.2); ii) an expression cassette (e.g., described in section 5.3.3); and iii) a second viral replication-deficient reverse repeat (e.g., described in sections 5.3.1 and 5.3.2). A double-stranded DNA molecule containing the second viral replication-deficient reverse repeat, wherein, when the top strand separates from the bottom strand of the second reverse repeat, the nickeling results in a top strand 3' overhang containing the second reverse repeat, such that the nickeling has third and fourth restriction sites for the nickeling endonuclease located on the opposing strand near the second reverse repeat or fragment thereof (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the second reverse repeat) (e.g., as described in sections 5.2.3, 5.2.4, and 5.3.2 or shown in Figures 2B and 2C). In one embodiment, the bottom strand 3' overhang contains the first viral replication-deficient reverse repeat. In one embodiment, the top strand 3' overhang contains the second viral replication-deficient reverse repeat. In one embodiment, the bottom strand 3' overhang includes the first viral replication-deficient reverse repeat, and the top strand 3' overhang includes the second viral replication-deficient reverse repeat.

[0314] The DNA molecules provided herein may be DNA molecules in their natural environment or isolated DNA molecules. In some embodiments, the DNA molecule is a DNA molecule in its natural environment. In some embodiments, the DNA molecule is an isolated DNA molecule. In one instance, isolated DNA molecules were found to be at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, The DNA molecules can be at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% pure.In another instance, isolated DNA molecules were approximately 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, and 54%. This can result in DNA molecules with a purity of approximately 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or approximately 99%. Other embodiments of the isolated DNA molecules provided herein in terms of purity are further described in Section 5.3.7, which can be combined in any suitable combination with the embodiments provided in this paragraph.

[0315] Because DNA molecules can be entirely engineered (e.g., manufactured by synthesis or recombinant production), the DNA molecules provided herein, including those in Section 3 and this Section 5.3, may lack certain sequences or features, as further described in Section 5.3.4.

[0316] (5.3.1 Reverse repetition) The ITRs or IRs provided in Section 3 and this Section (Section 5.3.1) can be formed, for example, by performing the steps of the methods described in Sections 3, 5.2.3, 5.2.4, and 5.2.5, to form hairpinned ITRs within the hairpin-terminated DNA molecules provided in Section 5.4. Thus, in some embodiments, the ITRs or IRs provided in Section 3 and this Section (Section 5.3.1) may include any combination of embodiments of any IR or ITR provided in Sections 3 and 5.4 and additional embodiments provided in this Section (Section 5.3.1).

[0317] Most DNA in cells consists of two strands joined by Watson-Crick base pairing, which sequesters most functional groups and limits structural and functional diversity. While this is a desirable property for molecules whose function is to store genetic information, single-stranded viruses have evolved to form secondary structures that add functional complexity to another layer by utilizing intramolecular interactions of linear single-stranded DNA (ssDNA). One of the major contributing factors is that these ssDNA viral genomes consist of only a single strand that folds over itself to form a hairpin.

[0318] The secondary structure of a single-stranded DNA molecule can be considered to represent the complementary base pairing patterns formed between constituent nucleotides based on the initial DNA sequence. The sequence, represented as a sequence of four different letters (one for each nucleotide species), is generally a single strand composed of nucleotides that are thought to form different secondary structures with the lowest free energy governed by thermodynamic interactions.

[0319] A “reverse repeat” or “IR” refers to a single-stranded nucleic acid sequence containing a palindromic sequence region. This palindromic structure region contains a sequence of nucleotides and its reverse complement, i.e., a “palindromic sequence” as further described below, on the same strand as further described below. In a denatured state, meaning that the hydrophobic stacking attraction between bases is neutralized, the IR nucleic acid sequence exists in a random coil state (e.g., at high temperatures, in the presence of chemicals, at high pH, ​​etc.). As the conditions become more physiological, the IR can fold to form a secondary structure in which the outermost regions are held together non-covalently by base pairing. In some embodiments, the IR can be an ITR. In some embodiments, the IR includes an ITR. In some embodiments, the IR can be a hairpinned reverse repeat. In some embodiments, when a reverse repeat folds on itself, it can form a hairpin loop (also known as a stem loop) in which a pairless loop of single-stranded DNA is formed when the DNA strand folds to form base pairs with another section of the same strand. When folded, the reverse repeat can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 such hairpin loop structures.

[0320] "Reverse terminal repeat," "terminal repeat," "TR," or "ITR" refers to a reverse repeat region at or near the end of a single-stranded DNA molecule, or a reverse repeat in or within a single-stranded overhang of a dsDNA molecule. An ITR can fold on itself as a result of a palindromic sequence in the ITR. In one embodiment, the ITR is at or near one end of the ssDNA. In another embodiment, the ITR is at or near one end of the dsDNA. In yet another embodiment, two ITRs are each at or near the two respective ends of the ssDNA. In yet another embodiment, two ITRs are each at or near the two respective ends of the dsDNA. In some embodiments, a non-ITR portion of the ssDNA or dsDNA is heterogeneous with respect to the ITR. In some embodiments, a non-ITR portion of the ssDNA or dsDNA is homologous with respect to the ITR. In a denatured state, meaning that the hydrophobic stacking attraction between bases is neutralized, nucleic acid sequences containing ITRs exist in a random coil state (e.g., at high temperatures, in the presence of chemicals, at high pH, ​​etc.). In some embodiments, as the conditions become more suitable for annealing as described in Section 5.2.5, the ITRs can fold on themselves to form a structure that is held together non-covalently by base pairing, while the heterogeneous non-ITR portion of the dsDNA remains intact, or the heterogeneous non-ITR portion of the ssDNA molecule can hybridize with a second ssDNA molecule containing the inverted complementary sequence of the heterogeneous DNA molecule. The resulting complex of two hybridized DNA strands contains three distinct regions, namely, a first folded single-stranded ITR covalently linked to a double-stranded DNA region covalently linked to a second folded single-stranded ITR, in that order. In one embodiment, the ITR sequence may begin at one of the restriction sites for nickel end nucleases described in Sections 3, 5.2.4, and 5.3.2, and end at the last base preceding the dsDNA.In one embodiment, in contrast to linear double-stranded DNA molecules, ITRs located at the 5' and 3' ends of the top and bottom strands at both ends of a DNA molecule can fold inward and face each other (e.g., 3' to 5', 5' to 3', or vice versa), thus not exposing the free 5' or 3' ends on either side of the nucleic acid double strand. When ITRs fold on themselves, in some embodiments, the dsDNA within the folded ITR can be placed immediately next to the dsDNA in the non-ITR portion of the DNA molecule, creating a nick adjacent to the dsDNA; or in other embodiments, the dsDNA within the folded ITR can be placed one or more nucleotides away from the dsDNA in the non-ITR portion of the DNA molecule, creating an "ssDNA gap" adjacent to the dsDNA. Two ITRs located on either side of a non-ITR DNA sequence are referred to as an "ITR pair." In some embodiments, when the ITR is in a folded state, it becomes resistant to exonuclease digestion (e.g., exonuclease V) for a period of time exceeding 1 hour at 37°C.

[0321] The boundary between the terminal bases of the ITR, which folds to form its secondary structure, and the terminal bases of the DNA hybridized double helix can be further stabilized by stacking interactions (e.g., coaxial stacking) between base pairs located on either side of the nick or ssDNA gap, and these interactions are sequence-dependent. In the case of nick-like structures, an equilibrium may exist between two conformations, where the first conformation is very close to that of an intact double helix, where stacking between base pairs located on either side of the nick is conserved, and the other conformation corresponds to a complete loss of stacking at the nick site, thus inducing twisting in the DNA. Nicked molecules are known to migrate somewhat slower than intact molecules of the same size during polyacrylamide and agarose gel electrophoresis. In some cases, this delay is enhanced at higher temperatures. The rapid equilibration between the stacked / linear conformation and the unstacked / bent conformation of the nick is thought to result in a unique delay characteristic of nick-containing DNA molecules, directly affecting the mobility of the DNA molecule during gel electrophoresis.

[0322] While not strictly theoretical, it is thought that cellular proteins can recognize parallel 5' and 3' ends as double-strand breaks, bind to them, and process them, which can have detrimental effects on the fate of DNA within the cell. Therefore, ITRs can prevent premature and unwanted degradation of expression cassettes having ITRs, such as those provided in Sections 3 and 5.4 and in this section (Section 5.3.1), at one or both of their two ends.

[0323] By positioning the first and second restriction sites for the nicking endonuclease on opposing strands and near the reverse repeat, and by subsequently separating the top strand from the bottom strand of the reverse repeat, the resulting overhang can fold back over itself to form a double-stranded end containing at least one restriction site for the nicking endonuclease. In some embodiments, the folded ITR is similar in secondary structural conformation to that of a viral ITR. In one embodiment, the ITR is located at both the 5' and 3' ends of the bottom strand (e.g., a left ITR and a right ITR). In another embodiment, the ITR is located at both the 5' and 3' ends of the top strand. In yet another embodiment, one ITR is located at the 5' end of the top strand, and the other ITR is located at the opposite end of the bottom strand (e.g., the left ITR is at the 5' end of the top strand, and the right ITR is at the 5' end of the bottom strand). In yet another embodiment, one ITR is located at the 3' end of the top chain, and the other ITR is located at the 3' end of the bottom chain.

[0324] In some embodiments, the Disclosure provides DNA molecules comprising palindromic sequences. A “palindromic sequence” or “palindrome” is a self-complementary DNA sequence that can be folded back to form a segment of dsDNA in a self-complementary region under conditions favorable for intramolecular annealing. In some embodiments, a palindromic sequence comprises a contiguous segment of polynucleotides that, when read forward, is identical to a segment of polynucleotides that, when read backward, is identical to a segment of polynucleotides that, when read forward, is identical to a segment of polynucleotides that, when read backward, is interrupted by one or more segments of polynucleotides of a non-palindromic structure. In another embodiment, the palindromic sequence includes a polynucleotide segment that, when read forward, is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical when read forward on the complementary strand. In yet another embodiment, the palindrome sequence is such that when read forward, the percentages when read backward on the complementary chain are 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%. , including a polynucleotide segment that is 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and which is interrupted by one or more non-palindral polynucleotide segments.ssDNA encoding one or more palindromic sequences can fold over itself to form double-stranded base pairs with secondary structures (e.g., hairpin loops or three-way junctions).

[0325] For example, under suitable conditions as described in Sections 5.2.3, 5.2.4, and 5.2.5, the IR or ITR provided in this Section (Section 5.3.1) can be folded to form a hairpin structure as described in this Section (Section 5.3.1) and Section 5.4, including any combination or permutation of the stem, main stem, loop, turning point, bulge, branch, branch loop, internal loop, and / or structural features described in Section 5.4.

[0326] In one embodiment, the IR or ITR for methods and compositions provided herein comprises one or more palindromic sequences. In some embodiments, the IR or ITR described herein comprises a palindromic sequence or domain that, in addition to forming a main stem domain, can form a branched hairpin structure. In some embodiments, the IR or ITR comprises a palindromic sequence that can form any number of branched hairpins. In a specific embodiment, the IR or ITR comprises a palindromic sequence that can form 1 to 30 branched hairpins, or any sub-range number of branched hairpins from 1 to 30. In some specific embodiments, the IR or ITR comprises a palindromic sequence that can form 1, 2, 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, or 30 branched hairpins. In some embodiments, the IR or ITR includes sequences that can form two branched hairpin structures resulting in a tridirectional junction domain (T-shaped). In some embodiments, the IR or ITR includes sequences that can form three branched hairpin structures resulting in a quaternary junction domain (or cruciform structure). In some embodiments, the IR or ITR includes sequences that can form a non-T-shaped hairpin structure, e.g., a U-shaped hairpin structure. In some embodiments, the IR or ITR includes sequences that can form a broken U-shaped hairpin structure including a series of bulges and base pair mismatches. In some embodiments, all branched hairpins have stems and / or loops of the same length. In some embodiments, one branched hairpin is smaller than the other (e.g., cut). Several exemplary embodiments of the hairpin structure and structural elements of the hairpin structure are shown in Figure 1.

[0327] A "hairpin closing base pair" refers to the first base pair following a pairless loop sequence. Certain stem-loop sequences have a preferred closing base pair (e.g., GC in AAV2 ITR). In one embodiment, the stem-loop sequence includes a GC pair as the closing base pair. In another embodiment, the stem-loop sequence includes a CG pair as the closing base pair.

[0328] An "ITR closed base pair" refers to the first and last nucleotides that form a base pair within a folded ITR. Terminal base pairs are typically the pair of nucleotides in the main stem domain closest to the non-ITR sequence of the DNA molecule (e.g., the expression cassette). ITR closed base pairs can be of any type (e.g., CG, AT, GC, or TA). In one embodiment, the ITR closed base pair is a GC base pair. In another embodiment, the ITR closed base pair is an AT base pair. In yet another embodiment, the ITR closed base pair is a CG base pair. In yet another embodiment, the ITR closed base pair is a TA base pair.

[0329] This disclosure stipulates that DNA secondary structure can be computationally predicted, as is known and practiced in the art. DNA secondary structure can be represented in several ways: squiggle plot, graph representation, dot-bracket notation, circular plot, arc diagram, mountain plot, dot plot, etc. In a circular plot, the skeleton is represented by circles, and base pairs are represented by arcs inside the circles. In an arc diagram, the DNA skeleton is drawn as straight lines, and the nucleotides of each base pair are connected by arcs. Both circular and arc plots allow for the identification of similarities and differences in secondary structure.

[0330] One of the many methods for predicting DNA secondary structure uses the nearest neighbor model, which minimizes the total free energy associated with the DNA structure. The minimum free energy is estimated by summing the individual energy contributions from base pair stacking, hairpins, bulges, internal loops, and multi-branched loops. The energy contributions of these elements are sequence and length-dependent and have been determined experimentally. The separation of a sequence into stem-loops and substems can be illustrated, for example, by showing the structure as a graph plot. In a linear interaction plot, each residue is represented on the horizontal axis, and semi-elliptical lines connect pairs of bases (e.g., Figures 2A and B).

[0331] In some embodiments, ITR promotes the long-term persistence of nucleic acid molecules in the cell nucleus. In some embodiments, ITR promotes the permanent persistence of nucleic acid molecules in the cell nucleus (e.g., throughout the entire lifespan of the cell). In some embodiments, ITR enhances the stability of nucleic acid molecules in the cell nucleus. In some embodiments, ITR inhibits or prevents the degradation of nucleic acid molecules in the cell nucleus.

[0332] In one embodiment, the IR or ITR may include any viral ITR. In another embodiment, the IR or ITR may include a synthetic palindrome sequence that can form a palindrome hairpin structure in which the 5' or 3' ends are not exposed at the outermost vertices or transition points of the repeats.

[0333] In some embodiments, a single-stranded ITR sequence extending from one nucleotide of an ITR closed base pair to the other nucleotide of an ITR closed base pair has an unfolding Gibbs free energy (ΔG) in the range of -10 kcal / mol to -100 kcal / mol under physiological conditions. In one embodiment, the Gibbs free energy (ΔG) (kcal / mol) of unfolding as described in the preceding text is -10 or less (meaning ≤ -10, including, for example, -20, -30, etc.), -11 or less, -12 or less, -13 or less, -14 or less, -15 or less, -16 or less, -17 or less, -18 or less, -19 or less, -20 or less, -21 or less, -22 or less, -23 or less, -24 or less, -25 or less, -26 or less, -27 or less, -28 or less, -29 or less, -30 or less, -31 or less, -32 or less, -33 or less, -34 or less, -35 or less, -36 or less, -37 or less, -38 or less, -39 or less, -40 or less, -41 or less, -42 or less, -43 or less, -44 or less, -45 or less, -46 or less, -47 or less, -48 or less, - 49 or less, -50 or less, -51 or less, -52 or less, -53 or less, -54 or less, -55 or less, -56 or less, -57 or less, -58 or less, -59 or less, -60 or less, -61 or less, -6 2 or less, -63 or less, -64 or less, -65 or less, -66 or less, -67 or less, -68 or less, -69 or less, -70 or less, -71 or less, -72 or less, -73 or less, -74 or less, -75 The following are the cases: -76 or less, -77 or less, -78 or less, -79 or less, -80 or less, -81 or less, -82 or less, -83 or less, -84 or less, -85 or less, -86 or less, -87 or less, -88 or less, -89 or less, -90 or less, -91 or less, -92 or less, -93 or less, -94 or less, -95 or less, -96 or less, -97 or less, -98 or less, -99 or less, or -100 or less.In another embodiment, the Gibbs free energy (ΔG) (kcal / mol) of unfolding as referred to in the preceding sentence is approximately -10 (meaning ≤ -10, including, for example, -20, -30, etc.), approximately -11, approximately -12, approximately -13, approximately -14, approximately -15, approximately -16, approximately -17, approximately -18, approximately -19, approximately -20, approximately -21, approximately -22, approximately -23, approximately -24, approximately -25, approximately -26, approximately -27, approximately -28, approximately -29, approximately -30, approximately -31, approximately -32, approximately -33, approximately -34, approximately -35, approximately -36, approximately -37, approximately -38, approximately -39, approximately -40, approximately -41, approximately -42, approximately -43, approximately -44, approximately -45, approximately -46, approximately -47 , approximately -48, approximately -49, approximately -50, approximately -51, approximately -52, approximately -53, approximately -54, approximately -55, approximately -56, approximately -57, approximately -58, approximately -59, approximately -60, approximately -61, approximately -62, approximately -63, approximately -64, approximately -65, approximately -66, approximately -67, approximately -68, approximately -69, approximately -70, approximately -71, approximately -72, approximately -73, approximately -74, These values ​​are approximately -75, -76, -77, -78, -79, -80, -81, -82, -83, -84, -85, -86, -87, -88, -89, -90, -91, -92, -93, -94, -95, -96, -97, -98, -99, or approximately -100. In some embodiments, the ITR sequence extending from one nucleotide of an ITR closed base pair to the other nucleotide of the ITR closed base pair has an unfolding Gibbs free energy (ΔG) in the range of -26 kcal / mol to -95 kcal / mol under physiological conditions. In some embodiments, an ITR sequence extending from one nucleotide of an ITR closed base pair to the other nucleotide of the ITR closed base pair contributes to the entire Gibbs free energy (ΔG) of unfolding for that ITR sequence under physiological conditions.

[0334] In some embodiments, in the folded state, single-stranded IR or ITR has approximately 50% to 98% overall Watson-Crick self-complementarity. In one embodiment, in the folded state, single-stranded IR or ITR has approximately 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, and 7 It has a total Watson-Crick self-complementarity of 3%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99%. In another embodiment, in the folded state, single-stranded IR or ITR is at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, and less It has a combined Watson-Crick self-complementarity of at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99%.In some embodiments, in the folded state, the IR or ITR has a total Watson-Crick complementarity of about 60% to 98%. In some embodiments, the single-stranded IR or ITR has a total GC content of about 60% to 95%. In one embodiment, a single-stranded IR or ITR has a total GC content of at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%. In another embodiment, the single-stranded IR or ITR has a total GC content of approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, or approximately 95%. In some embodiments, the single-stranded IR has a total GC content of approximately 60-91%.

[0335] Table 4 lists the folding free energy, GC content, complementarity percentage, and length of exemplary ITRs, and Table 5 lists the sequences of the ITRs in Table 4. (Table 4: Exemplary ITR folding free energy, GC content, complementarity percentage, and length) [Table 4] (Table 5: ITR sequence from Table 4) Table 5

[0336] DNA molecules for methods and compositions provided herein may include IRs or ITRs of various origins. In one embodiment, the IR or ITR within the DNA molecule is a viral ITR. A “viral ITR” includes a synthetic sequence comprising a terminal repeat or at least one minimum required origin of replication and a region containing a palindromic hairpin structure of any virus. In one embodiment, the viral ITR is derived from the family Parvoviridae. In another embodiment, a viral ITR derived from Parvoviridae includes a “minimum required origin of replication” comprising at least one viral replication-associated protein binding sequence (“RABS”). RABS means a DNA sequence to which a viral DNA replication-associated protein (“RAP”) or its isoform, encoded by the gene Rep and / or NS1 of the family Parvoviridae, can bind. In some embodiments, the RABS is a Rep-binding sequence (“RBS”). In some embodiments, the RABS comprises a Rep-binding sequence (“RBS”) to which Rep can bind to two elements within the ITR. This can bind to a nucleotide sequence within the stem structure of the ITR (i.e., the nucleotide sequence recognized by the Rep protein for replication of the viral nucleic acid molecule). Such an RBS is also referred to as an RBE (Rep-binding element). Rep can also bind to a nucleotide sequence within the ITR that forms a small palindrome containing a single tip of an internal hairpin, thereby stabilizing the binding between Rep and the ITR. Such an RBS is also referred to as an RBE'. In another embodiment, a viral ITR derived from the Parvoviridae family includes an RABS containing an NS1-binding element ("NSBE") to which the replication-associated viral protein NS1 can bind. In another embodiment, the RABS is an NS1-binding element ("NSBE") to which the replication-associated viral protein NS1 can bind. In some embodiments, the viral ITR is derived from the Parvoviridae family and includes terminal separation sites ("TRS") in which the viral DNA replication-associated proteins NS1 and / or Rep can make endonucleolytic cleavage within the sequence.In yet another embodiment, the viral ITR comprises at least one RBS or NSBE and at least one TRS. In connection with the production of viral or recombinant RAP (i.e., Rep or NS1) based viral genomes, the ITR mediates replication and viral packaging. As unexpectedly discovered by the inventors and provided herein, a double-stranded linear DNA vector having an ITR similar to the viral ITR can be produced without requiring the Rep or NS1 protein, and consequently, without relying on the RABS or TRS sequence for DNA replication. Thus, RABS and TRS can optionally be encoded within the nucleotide sequences disclosed herein, but are not required, providing flexibility in the design of the ITR. In one embodiment, the ITR for the methods and compositions provided herein does not contain at least one RABS (e.g., one RABS, two RABS, or three or more RABS). In another embodiment, the ITR for the methods and compositions provided herein does not contain any RABS. In another embodiment, the ITR for the methods and compositions provided herein does not contain at least one RBS. In another embodiment, the ITR for the methods and compositions provided herein does not contain any RBS. In another embodiment, the ITR for the methods and compositions provided herein does not contain RBE. In another embodiment, the ITR for the methods and compositions provided herein does not contain RBE'. In another embodiment, the ITR for the methods and compositions provided herein does not contain RBE and RBE'. In another embodiment, the ITR for the methods and compositions provided herein does not contain NSBE. In yet another embodiment, the ITR for the methods and compositions provided herein does not contain TRS. In a further embodiment, the ITR for the methods and compositions provided herein does not contain at least one RABS (e.g., one RABS, two RABS, or three or more RABS) and does not contain TRS. In a further embodiment, the ITR for the methods and compositions provided herein does not contain any RABS and does not contain TRS.In further embodiments, the ITR for the methods and compositions provided herein includes RBS (i.e., RBE and / or RBE'), TRS, or both RBS (i.e., RBE and / or RBE') and TRS. In further embodiments, the ITR for the methods and compositions provided herein includes NBSE, TRS, or both NBSE and TRS.

[0337] An "ITR pair" refers to two ITRs within a single DNA molecule. In some embodiments, both ITRs in an ITR pair originate from a wild-type viral ITR (e.g., an AAV2 ITR) that has a reverse complementary sequence over its entire length. An ITR can be considered a wild-type sequence even if it has one or more nucleotides that deviate from a standard naturally occurring sequence, as long as the change does not affect the nature of the sequence and its overall three-dimensional structure. In some embodiments, this disclosure specifies that one or more nucleotide insertions, deletions, or substitutions can create a restriction site for a nicking endonuclease without altering the overall three-dimensional structure of the viral ITR. In some embodiments, the deviating nucleotides refer to a conserved sequence change. In one embodiment, the ITR sequences provided herein can have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to a standard sequence (for example, measured using BLAST with default settings), and have restriction sites for a nicking endonuclease such that the 3D structure has the same shape in geometric space. In another embodiment, the ITR sequences provided herein can have about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with respect to a standard sequence (for example, measured using BLAST with default settings), and have restriction sites for a nicking endonuclease such that the 3D structure has the same shape in geometric space.

[0338] In some embodiments, the DNA molecules for the methods and compositions provided herein include a set of wt-ITRs. In a specific embodiment, the DNA molecules for the methods and compositions provided herein include a set of wt-ITRs selected from the group shown in Table 6. Table 6 shows the genomes of AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12); AAVrh8, AAVrhlO, AAV-DJ, and AAV-DJ8 (e.g., NCBI: NC 002077; NC 001401; NC001729; NC001829; NC006152; NC 006260; NC This section shows exemplary ITRs from the same or different serotypes, or from other parvoviruses, including ITRs from warm-blooded animals (avian AAV (AAAV), bovine AAV (BAAV), dog, horse, and sheep AAV), B19 parvovirus (GenBank accession No. NC 000883), mouse microvirus (MVM) (GenBank accession No. NC 001510); goose: goose parvovirus (GenBank accession No. NC 001701); snake: snake parvovirus 1 (GenBank accession No. NC 006148). (Table 6: Exemplary ITR sequences) [Table 6] TIFF0007927684000007.tif77170

[0339] In some embodiments, the DNA molecules for the methods and compositions provided herein comprise all or part of the genome of a parvovirus. The genome of a parvovirus is linear, 3.9–6.3 kb in size, and the coding region is flanked by terminal repeats that can fold into a hairpin-like structure, either different (heterotelomeres, e.g., HBoV) or identical (homotelomeres, e.g., AAV2). In one embodiment, the DNA molecule for the methods and compositions provided herein comprises two different ITRs at two ends of the DNA molecule. In another embodiment, the DNA molecule for the methods and compositions provided herein comprises two identical ITRs at two ends of the DNA molecule. In yet another embodiment, the DNA molecule for the methods and compositions provided herein comprises two different ITRs corresponding to two HBoV ITRs at two ends of the DNA molecule. In yet another embodiment, the DNA molecule for the methods and compositions provided herein comprises two identical ITRs corresponding to AAV2 ITRs at two ends of the DNA molecule.

[0340] In one embodiment, the ITR in the DNA molecule provided herein may be an AAV ITR. In another embodiment, the ITR may be a non-AAV ITR. In one embodiment, the ITR in the DNA molecule provided herein may be derived from an AAV ITR or a non-AAV ITR. In some specific embodiments, the ITR may be derived from any one of the Parvoviridae family, which includes parvoviruses and dependent viruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19). In another specific embodiment, the ITR may be derived from an SV40 hairpin that acts as the origin of SV40 replication. Viruses of the Parvoviridae family consist of two subfamilies: the Parvovirinae, which infects vertebrates, and the Densovirinae, which infects invertebrates. Therefore, in one embodiment, the ITR may be derived from any one of the Parvovirinae subfamilies. In another embodiment, the ITR may be derived from any one of the Densovirinae subfamilies.

[0341] Compared to the T-type AAV ITR, human erythrovirus B19 has an ITR that folds into a long linear double-stranded structure with a small number of unpaired nucleotides, ending in an incomplete palindrome that can produce a series of small but highly conserved mismatch bulges. In some embodiments, any parvovirus ITR can be used as an ITR for the DNA molecules provided herein (e.g., wild-type or modified ITR), or any parvovirus ITR can act as a template ITR for modification and its incorporation into the DNA molecules provided herein. In some specific embodiments, the parvovirus from which the ITR of the DNA molecule is derived is dependvirus, erythroparvovirus, or bocaparvovirus. In another specific embodiment, the ITR of the DNA molecule provided herein is derived from AAV, B19, or HBoV. In some embodiments, the serotype of the AAV ITR selected for the DNA molecule provided herein may be based on the histotropy of the serotype. AAV2 has broad tissue tropism; AAV1 preferentially targets neurons and skeletal muscle; AAV5 preferentially targets neurons, retinal pigment epithelial cells, and photoreceptor cells; AAV6 preferentially targets skeletal muscle and lungs; AAV8 preferentially targets liver, skeletal muscle, heart, and pancreatic tissue; AAV9 preferentially targets liver, skeletal, and lung tissue. In one embodiment, the ITR or modified ITR of the DNA molecule provided herein is based on the AAV2 ITR. In one embodiment, the ITR or modified ITR of the DNA molecule provided herein is based on the AAV1 ITR. In one embodiment, the ITR or modified ITR of the DNA molecule provided herein is based on the AAV5 ITR. In one embodiment, the ITR or modified ITR of the DNA molecule provided herein is based on the AAV6 ITR. In one embodiment, the ITR or modified ITR of the DNA molecule provided herein is based on the AAV8 ITR. In one embodiment, the ITR or modified ITR of the DNA molecule provided herein is based on the AAV9 ITR.

[0342] In one embodiment, the DNA molecules for the methods and compositions provided herein comprise one or more non-AAV ITRs. In a further embodiment, such non-AAV ITRs can be derived from hairpin sequences found in mammalian genomes. In a particular embodiment, such non-AAV ITRs are OriL hairpin sequences that adopt a stem-loop structure and are involved in initiating mitochondrial DNA synthesis. [ka] (See Fuste et al., Molecular Cell, 37, 67-78, January 15, 2010, the entire work of which is incorporated herein by reference.) These can be derived from hairpin sequences found in the mitochondrial genome. In another specific embodiment, the DNA molecule for the methods and compositions provided herein comprises an ITR derived from an OriL sequence, mirrored to form a T-junction having two self-complementary palindromic structural regions and 12-nucleotide loops at both vertices of the hairpin. In one embodiment, the DNA molecule for the methods and compositions provided herein comprises an ITR derived from an OriL sequence that maintains the OriL hairpin loop and the subsequent paired bulge and GC-rich stem. Several exemplary embodiments of ITRs derived from mitochondrial OriL are shown in Figure 2.

[0343] In one embodiment, the DNA molecule for the methods and compositions provided herein comprises one or more non-AAV ITRs derived from an aptamer. Similar to viral ITRs, the aptamer is composed of ssDNA that folds to form a three-dimensional structure and has the ability to recognize a biological target with high affinity and specificity. DNA aptamers can be produced by phylogenetic evolution of ligands by exponential enrichment (SELEX). For example, some aptamers have already been shown to be able to target the nucleus of human cells (see Shen et al., ACS Sens. 2019, 4, 6, 1612-1618, which is incorporated herein by reference in its entirety). In one embodiment, the DNA molecule for the methods and compositions provided herein comprises a nuclear targeting aptamer ITR or a derivative thereof, wherein the aptamer specifically binds to a nuclear protein. In some embodiments, the aptamer ITR folds to form a secondary structure which may include hairpins and internal loops, as well as bulge and stem regions. Figure 3 shows some exemplary embodiments of aptamers or ITRs derived from this.

[0344] In some specific embodiments, the DNA molecules for the methods and compositions provided herein comprise one or more AAV2 ITRs, human erythrovirus B19 ITRs, goose parvovirus ITRs, and / or derivatives thereof in any combination. In another specific embodiment, the DNA molecules for the methods and compositions provided herein comprise two ITRs selected from AAV2 ITRs, human erythrovirus B19 ITRs, goose parvovirus ITRs, and derivatives thereof in any combination. In some specific embodiments, the DNA molecules for the methods and compositions provided herein comprise one or more AAV2 ITRs, human erythrovirus B19 ITRs, goose parvovirus ITRs, and / or derivatives thereof in any combination, wherein the ITRs remain functional regardless of whether the palindromic structural regions of those ITRs are direct, opposite, or any possible combination of 5' and 3' ITR orientations relative to the expression cassette (described in whole in WO2019143885, which is incorporated herein by reference).

[0345] In some embodiments, the modified IR or ITR within the DNA molecule provided herein is a synthetic IR sequence that includes a restriction site for endonucleases, such as 5'-GAGTC-3', in addition to various palindromic sequences that enable hairpin secondary structure formation as described in this section (Section 5.3.1).

[0346] In one embodiment, the IR or ITR within a DNA molecule provided herein may be an IR or ITR having varying sequence homology to the IR or ITR sequences described in this section (Section 5.3.1). In another embodiment, the IR or ITR within a DNA molecule provided herein may be an IR or ITR having varying sequence homology to known IR or ITR sequences of various ITR origins described in this section (Section 5.3.1) (e.g., viral ITRs, mitochondrial ITRs, artificial or synthetic ITRs such as aptamers). In one embodiment, such homology provided in this paragraph may be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% homology. In another embodiment, such homology provided in this paragraph may be about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homology.

[0347] In some embodiments, the IR or ITR within a DNA molecule provided herein may include one or more of the features described in this section (Section 5.3.1) in various permutations and combinations.

[0348] (5.3.2 Restriction enzymes, nickeling end nucleases, and their respective restriction sites; programmable nickeling enzymes and their target sites) Various embodiments of the nickeling endonucleases, restriction enzymes, and / or their restriction sites described in Section 5.2.4 are provided herein for DNA molecules. In some embodiments, the first, second, third, and fourth restriction sites for nickeling endonucleases provided for DNA molecules such as those described in Section 3 and this Section (Section 5.3) can all be the same target sequence for nickeling endonuclease. In some embodiments, the first, second, third, and fourth restriction sites for nickeling endonucleases provided for DNA molecules such as those described in Section 3 and this Section (Section 5.3) can be four different target sequences for nickeling endonucleases. In another embodiment, the first, second, third, and fourth restriction sites for the nickel endonuclease are target sequences for two different nickel endonucleases, comprising all possible combinations of assigning the four sites to two different nickel endonuclease target sequences (e.g., the first restriction site for the first nickel endonuclease and the remaining restriction sites for the second nickel endonuclease, the first and second restriction sites for the first nickel endonuclease and the remaining restriction sites for the second nickel endonuclease, etc.). In one embodiment, the first, second, third, and fourth restriction sites for the nickel endonuclease are target sequences for three different nickel endonucleases, comprising all possible combinations of assigning the four sites to three different nickel endonuclease target sequences. In some embodiments, the nicking endonuclease and the restriction site for the nicking endonuclease may be any one selected from those listed in Section 5.2.4, including Table 2. In further embodiments, each of the first, second, third, and fourth restriction sites for the nicking endonuclease may be any site for the nicking endonuclease selected from those listed in Section 5.2.4, including Table 2.

[0349] Tables 7 to 16 show exemplary modified AAV ITR sequences that contain two antiparallel recognition sites for the same nickeling endonuclease species, grouped by nickeling endonuclease species. The modified ITR sequences and their corresponding alignments with wild-type AAV1, AAV2, AAV3, AAV4 left, AAV4 right, AAV5, and AAV7 are shown in Figures 13 to 19. (Table 7: Exemplary AAV-derived ITRs containing antiparallel recognition sites for the nickel endonuclease Nb.BvCI) [Table 7] TIFF0007927684000010.tif244170TIFF0007927684000011.tif23170 (Table 8: Exemplary AAV-derived ITRs containing an antiparallel recognition region for the nickel endonuclease Nb.BsmI) [Table 8] TIFF0007927684000013.tif156170 (Table 9: Exemplary AAV-derived ITRs containing an antiparallel recognition region for the nickel endonuclease Nb.BsrDI) [Table 9] TIFF0007927684000015.tif233170 (Table 10: Exemplary AAV-derived ITRs containing an antiparallel recognition region for the nickel endonuclease Nb.BssSi) [Table 10] TIFF0007927684000017.tif82170 (Table 11: Exemplary AAV-derived ITR with an internal antiparallel recognition region for the nickel endonuclease Nb.BtsI) [Table 11] TIFF0007927684000019.tif154170 (Table 12: Exemplary AAV-derived ITR containing an antiparallel recognition region for the nickel endonuclease Nt.AlwI) [Table 12] TIFF0007927684000021.tif223170 (Table 13: Exemplary AAV-derived ITR containing an antiparallel recognition region for the nickel endonuclease Nt.BbvCI) [Table 13] TIFF0007927684000023.tif81170 (Table 14: Exemplary AAV-derived ITR containing an antiparallel recognition region for the nickel endonuclease Nt.BsmAI) [Table 14] TIFF0007927684000025.tif155170 (Table 15: Exemplary AAV-derived ITR containing an antiparallel recognition region for the nickel endonuclease Nt.BspQI) [Table 15] TIFF0007927684000027.tif233170 (Table 16: Exemplary AAV-derived ITR with an internal antiparallel recognition region for the nickel endonuclease Nt.BstNBI) [Table 16] TIFF0007927684000029.tif110170 (Table 17: Inverted complementary sequences of Nicking enzyme targets) [Table 17] TIFF0007927684000031.tif244170TIFF0007927684000032.tif244170TIFF0007927684000033.tif243170TIFF0007927684000034.tif244170TIFF0007927684000035.tif245170TIFF0007927684000036.tif244170TIFF0007927684000037.tif244170TIFF0007927684000038.tif244170TIFF0007927684000039.tif248170TIFF0007927684000040.tif244170TIFF0007927684000041.tif244170TIFF0007927684000042.tif244170TIFF0007927684000043.tif56170

[0350] The first, second, third, and fourth restriction sites for the nicking end nuclease can be arranged in various configurations.In some embodiments, the first and second restriction sites for the nickel end nuclease are at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64 , at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, They are separated by at least 95, at least 96, at least 97, at least 98, at least 99, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, or at least 200 nucleotides.In another embodiment, the first and second restriction sites for the nicking end nuclease are approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 , approximately 65, approximately 66, approximately 67, approximately 68, approximately 69, approximately 70, approximately 71, approximately 72, approximately 73, approximately 74, approximately 75, approximately 76, approximately 77, approximately 78, approximately 79, approximately 80, approximately 81, approximately 82, approximately 83, approximately 84, approximately 85, approximately 86, approximately 87, approximately 88, approximately 89, approximately 90, approximately 91, approximately 92, approximately 93, approximately 94, approximately 95, approximately 96, They are separated by approximately 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or approximately 200 nucleotides.

[0351] Similarly, in one embodiment, the third and fourth restriction sites for the nickel end nuclease are at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64 , at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, They are separated by at least 95, at least 96, at least 97, at least 98, at least 99, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, or at least 200 nucleotides.In a further embodiment, the third and fourth restriction sites for the nicking end nuclease are approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 6 4, approximately 65, approximately 66, approximately 67, approximately 68, approximately 69, approximately 70, approximately 71, approximately 72, approximately 73, approximately 74, approximately 75, approximately 76, approximately 77, approximately 78, approximately 79, approximately 80, approximately 81, approximately 82, approximately 83, approximately 84, approximately 85, approximately 86, approximately 87, approximately 88, approximately 89, approximately 90, approximately 91, approximately 92, approximately 93, approximately 94, approximately 95, approximately 96, They are separated by approximately 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or approximately 200 nucleotides.

[0352] This disclosure specifies that the overhangs described in Section 3, 5.2 (including 5.2.3), and 5.3 (including 5.3.1) may be the result of nickeling at the first and second restriction sites by nickeling endonucleases and the denaturation described in Section 3 and 5.2 (including 5.2.3). Accordingly, in some embodiments, the overhangs resulting from nickeling at the first and second restriction sites may be the same length (in terms of the number of nucleotides) as the distance between the first and second restriction sites as described in the preceding paragraph of this section (Section 5.3.2). Because a nickeling endonuclease can cleave DNA inside or outside the restriction site for the nickeling endonuclease, in one embodiment, the overhang resulting from nickeling at the first and second restriction sites may be at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 nucleotides longer or shorter than the length at which the first and second restriction sites are separated. In another embodiment, the overhang resulting from nicking at the first and second restriction sites may be about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides longer or shorter than the length at which the first and second restriction sites are separated.

[0353] Similarly, the disclosure specifies that the overhangs described in Sections 3, 5.2 (including 5.2.3), and 5.3 (including 5.3.1) may be the result of nickeling at the third and fourth restriction sites by nickeling endonucleases and the denaturation described in Sections 3 and 5.2 (including 5.2.3). Accordingly, in some embodiments, the overhangs resulting from nickeling at the third and fourth restriction sites may be the same length (in terms of nucleotides) as the separation of the third and fourth restriction sites as described in the preceding paragraph of this section (Section 5.3.2). Because a nickeling endonuclease can cleave DNA inside or outside the restriction site for the nickeling endonuclease, in one embodiment, the overhang resulting from nickeling at the third and fourth restriction sites may be at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 nucleotides longer or shorter than the length at which the third and fourth restriction sites are separated. In another embodiment, the overhang resulting from nicking at the third and fourth restriction sites may be about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides longer or shorter than the length by which the third and fourth restriction sites are separated.

[0354] As is evident from Sections 3 and 5.4 and this Section (Section 5.3), the DNA molecules provided herein include an expression cassette. In some embodiments, the expression cassette is located between first and second restriction sites for one nickel end nuclease(s) and third and fourth restriction sites for the other nickel end nuclease(s). In another embodiment, the expression cassette is located inside the dsDNA segment of a DNA molecule produced by performing steps a-d of the methods described in Sections 3 and 5.2, which include a denaturation step that provides two DNA overhangs as described in Section 5.2.3. In one embodiment, the first, second, third, and fourth restriction sites for the nickel endonuclease are arranged such that the length of the dsDNA segment described in this paragraph is at least 0.2kb, at least 0.3kb, at least 0.4kb, at least 0.5kb, at least 0.6kb, at least 0.7kb, at least 0.8kb, at least 0.9kb, at least 1kb, at least 1.5kb, at least 2kb, at least 2.5kb, at least 3kb, at least 3.5kb, at least 4kb, at least 4.5kb, at least 5kb, at least 5.5kb, at least 6kb, at least 6.5kb, at least 7kb, at least 7.5kb, at least 8kb, at least 8.5kb, at least 9kb, at least 9.5kb, or at least 10kb. In another embodiment, the first, second, third, and fourth restriction sites for the nickel endonuclease are arranged such that the length of the dsDNA segment described in this paragraph is approximately 0.2kb, approximately 0.3kb, approximately 0.4kb, approximately 0.5kb, approximately 0.6kb, approximately 0.7kb, approximately 0.8kb, approximately 0.9kb, approximately 1kb, approximately 1.5kb, approximately 2kb, approximately 2.5kb, approximately 3kb, approximately 3.5kb, approximately 4kb, approximately 4.5kb, approximately 5kb, approximately 5.5kb, approximately 6kb, approximately 6.5kb, approximately 7kb, approximately 7.5kb, approximately 8kb, approximately 8.5kb, approximately 9kb, approximately 9.5kb, or approximately 10kb.

[0355] As described in Section 5.2.4, incubation with a nicking endonuclease yields a first nick corresponding to a first restriction site for the nicking endonuclease, a second nick corresponding to a second restriction site for the nicking endonuclease, a third nick corresponding to a third restriction site for the nicking endonuclease, and / or a fourth nick corresponding to a fourth restriction site for the nicking endonuclease. The disclosure specifies that the first, second, third, and / or fourth nicks may be located at various positions relative to the reverse repeat. In one embodiment, the first nick is located within 1, 2, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 5' nucleotide of the ITR closed base pair of the first reverse repeat. In another embodiment, the first nick is within 1, 2, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 3' nucleotide of the ITR closed base pair of the first reverse repeat. In yet another embodiment, the second nick is within 1, 2, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 5' nucleotide of the ITR closed base pair of the first reverse repeat.In a further embodiment, the second nick is located within 1, 2, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 3' nucleotide of the ITR closed base pair of the first reverse repeat. In one embodiment, the third nick is located within 1, 2, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 5' nucleotide of the ITR closed base pair of the second reverse repeat. In another embodiment, the third nick is within 1, 2, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 3' nucleotide of the ITR closed base pair of the second reverse repeat. In yet another embodiment, the fourth nick is within 1, 2, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 5' nucleotide of the ITR closed base pair of the second reverse repeat.In a further embodiment, the fourth nick is within 1, 2, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides from the 3' nucleotide of the ITR closed base pair of the second reverse repeat. In some additional embodiments, the first, second, third, and fourth nicks may have any relative positions in any combination or permutation between themselves, between any of them and the reverse repeat, and / or between any of them and the expression cassette described in this section (Section 5.3.2).

[0356] (5.3.3 Expression Cassette) As is evident from Sections 3 and 5.4 and this Section (Section 5.3), the DNA molecules provided herein include expression cassettes. An expression cassette is a nucleic acid molecule or a portion of a nucleic acid molecule containing a sequence or other information that instructs the cell machinery to produce RNA or proteins. In some embodiments, the expression cassette includes a promoter sequence. In some embodiments, the expression cassette includes a transcription unit. In some yet another embodiment, the expression cassette includes a promoter operatively linked to the transcription unit. In one embodiment, the transcription unit includes an open reading frame (ORF). Embodiments of ORFs used with the methods and compositions provided herein are further described in the final paragraph of this Section (Section 5.3.3). The expression cassette may further include features that instruct the cell machinery to produce RNA and proteins. In one embodiment, the expression cassette includes a post-transcriptional regulator. In another embodiment, the expression cassette further includes a polyadenylation and / or termination signal. In yet another embodiment, the expression cassette includes regulatory elements known and used in the art to perform modulation (promote, inhibit, and / or turn on / off ORF expression). Such regulatory elements include, for example, the 5' untranslated region (UTR), the 3'-UTR, or both the 5'UTR and the 3'UTR. In some further embodiments, the expression cassette includes any combination or permutation of one or more features provided in this section (Section 5.3.3).

[0357] An expression cassette may contain a protein-coding sequence in its ORF (sense strand). Alternatively, an expression cassette may contain a complementary sequence (antisense strand) to the protein-coding ORF, as well as regulatory components and / or other signals to cause the cell machinery to produce sense strand DNA / RNA and the corresponding protein. In some embodiments, the expression cassette contains a protein sequence without introns. In another embodiment, the expression cassette contains a protein sequence with introns that are removed when transcribed and spliced. An expression cassette may also contain a variety of ORFs or transcription units. In one embodiment, the expression cassette contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ORFs. In another embodiment, the expression cassette contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 transcription units.

[0358] An expression cassette may include one or more transcription factors, one or more post-transcriptional factors, or both one or more transcription factors and one or more post-transcriptional factors. Such regulatory elements are any sequences that enable, contribute to, or regulate the functional regulation of nucleic acid molecules, including replication, duplication, transcription, splicing, translation, stability, and / or transport of one of the nucleic acids or its derivatives (e.g., mRNA) into a host cell or organism. Examples of such regulatory elements include, but are not limited to, promoters, enhancers, polyadenylation signals, translation termination codons, ribosome-binding elements, transcriptional terminators, selection markers, and origins of replication.

[0359] In some embodiments, the expression cassette includes an enhancer. Any enhancer sequence known to those skilled in the art may be used in consideration of this disclosure. In some embodiments, the enhancer sequence may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as one of CMV, HA, RSV, or EBV. In some specific embodiments, the enhancer may be a woodchuck HBV post-transcriptional regulator (WPRE), an intron / exon sequence derived from human apolipoprotein A1 precursor (ApoAI), the untranslated R-U5 domain of a human T-cell leukemia virus type 1 (HTLV-1) terminal repeat sequence (LTR), a splicing enhancer, a synthetic rabbit β-globin intron, the P5 promoter of AAV, or any combination thereof.

[0360] As described above, an expression cassette may include a promoter that controls the expression of the target protein. The promoter includes any nucleotide sequence that initiates the transcription of an operablely linked nucleotide sequence. The promoter can be constitutive, inducible, or repressible. The promoter can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. The promoter can be homologous (e.g., derived from the same genetic source) or heterologous (e.g., derived from different genetic sources). In some embodiments, the promoter can be a monkey virus 40 (SV40) promoter, a mouse mammary cancer virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, such as a bovine immunodeficiency virus (BIV) terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukemia virus (ALV) promoter, a cytomegalovirus (CMV) promoter, such as a CMV very early promoter (CMV-IE), an Epstein-Barr virus (EBV) promoter, or a Roussarcoma virus (RSV) promoter. In another embodiment, the promoter may be a human gene-derived promoter such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein. In yet another embodiment, the promoter may be a tissue-specific promoter, such as a natural or synthetic muscle or skin-specific promoter.

[0361] As described above, the expression cassette may include polyadenylation, a termination signal, or both polyadenylation and a termination signal. Any polyadenylation signal known to those skilled in the art in consideration of this disclosure may be used. In some embodiments, the polyadenylation signal may be an SV40 polyadenylation signal, an AAV2 polyadenylation signal (bp 4411-4466, NC_001401), a polyadenylation signal derived from the herpes simplex virus thymidine kinase gene, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal.

[0362] The expression cassette can have various sizes to accommodate one or more ORFs of varying lengths. In one embodiment, the size of the expression cassette is at least 0.2kb, at least 0.3kb, at least 0.4kb, at least 0.5kb, at least 0.6kb, at least 0.7kb, at least 0.8kb, at least 0.9kb, at least 1kb, at least 1.5kb, at least 2kb, at least 2.5kb, at least 3kb, at least 3.5kb, at least 4kb, at least 4.5kb, at least 5kb, at least 5.5kb, at least 6kb, At least 6.5kb, at least 7kb, at least 7.5kb, at least 8kb, at least 8.5kb, at least 9kb, at least 9.5kb, at least 10kb, at least 15kb, at least 20kb, at least 25kb, at least 30kb, at least 35kb, at least 40kb, at least 45kb, at least 50kb, at least 55kb, at least 60kb, at least 65kb, at least 70kb, at least 75kb, or at least 80kb. In one particular embodiment, the expression cassette is at least 4.5kb. In another particular embodiment, the expression cassette is at least 4.6kb. In yet another particular embodiment, the expression cassette is at least 4.7kb. In yet another particular embodiment, the expression cassette is at least 4.8kb. In one particular embodiment, the expression cassette is at least 4.9kb. In yet another particular embodiment, the expression cassette is at least 5kb.In another embodiment, the size of the expression cassette is approximately 0.2kb, approximately 0.3kb, approximately 0.4kb, approximately 0.5kb, approximately 0.6kb, approximately 0.7kb, approximately 0.8kb, approximately 0.9kb, approximately 1kb, approximately 1.5kb, approximately 2kb, approximately 2.5kb, approximately 3kb, approximately 3.5kb, approximately 4kb, approximately 4.5kb, approximately 5kb, approximately 5.5kb, The expression cassette is approximately 6kb, 6.5kb, 7kb, 7.5kb, 8kb, 8.5kb, 9kb, 9.5kb, 10kb, 15kb, 20kb, 25kb, 30kb, 35kb, 40kb, 45kb, 50kb, 55kb, 60kb, 65kb, 70kb, 75kb, or 80kb. In one particular embodiment, the expression cassette is approximately 4.5kb. In another particular embodiment, the expression cassette is approximately 4.6kb. In yet another particular embodiment, the expression cassette is approximately 4.7kb. In yet another particular embodiment, the expression cassette is approximately 4.8kb. In one particular embodiment, the expression cassette is approximately 4.9kb. In yet another particular embodiment, the expression cassette is approximately 5kb. An expression cassette may also include a variety of target genes ("transgenes"). In one embodiment, the expression cassette includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 transgenes. In some specific embodiments, the expression cassette includes one transgene. In some embodiments, the transgene is a recombinant gene. In some further embodiments, the transgene includes a cDNA sequence (e.g., no introns in the transgene).

[0363] In some embodiments, the DNA molecules provided herein do not have size limitations on the capsid-forming AAV vector, and therefore enable the delivery of large expression cassettes, providing efficient transgenes. In some embodiments, the DNA molecules provided herein include an expression cassette that is the same size as or larger than the size of any natural AAV genome.

[0364] Expression cassettes can occupy various positions relative to the reverse repeat. In some embodiments, expression cassettes can occupy 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 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, 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 at least 100 nucleotides apart.In one embodiment, the expression cassette is located at least 0.2kb, at least 0.3kb, at least 0.4kb, at least 0.5kb, at least 0.6kb, at least 0.7kb, at least 0.8kb, at least 0.9kb, at least 1kb, at least 1.5kb, or at least 2kb away from the reverse repeat. In another embodiment, the expression cassette is approximately 1, 2, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 5 from the reverse repeat. They are separated by 0, approximately 51, approximately 52, approximately 53, approximately 54, approximately 55, approximately 56, approximately 57, approximately 58, approximately 59, approximately 60, approximately 61, approximately 62, approximately 63, approximately 64, approximately 65, approximately 66, approximately 67, approximately 68, approximately 69, approximately 70, approximately 71, approximately 72, approximately 73, approximately 74, approximately 75, approximately 76, approximately 77, approximately 78, approximately 79, approximately 80, approximately 81, approximately 82, approximately 83, approximately 84, approximately 85, approximately 86, approximately 87, approximately 88, approximately 89, approximately 90, approximately 91, approximately 92, approximately 93, approximately 94, approximately 95, approximately 96, approximately 97, approximately 98, approximately 99, or approximately 100 nucleotides. In further embodiments, the expression cassette is located approximately 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb, 1kb, 1.5kb, or 2kb away from the reverse repeat. In one embodiment, the reverse repeat of this paragraph is the first reverse repeat described in sections 3 and 5.3 (including 5.3.1). In another embodiment, the reverse repeat of this paragraph is the second reverse repeat described in sections 3 and 5.3 (including 5.3.1). In yet another embodiment, the reverse repeat of this paragraph is both the first and second reverse repeats described in sections 3 and 5.3 (including 5.3.1).

[0365] In one embodiment, provided herein is a first reverse repeat (e.g., as described in Section 5.3.1) in the 5'-3' direction of the sense strand, wherein when the sense strand separates from the antisense strand of the first reverse repeat, the first and second limiting regions for the nicking endonuclease are located on the opposing strand near the first reverse repeat (e.g., as described in Sections 5.2.3, 5.2.4, and 5.3.2), such that nicking results in a sense strand 5' overhang containing the first reverse repeat. A double-stranded DNA molecule comprising a second reverse repeat, wherein third and fourth restriction sites for a nickeling endonuclease are located on the opposite strand near the second reverse repeat (e.g., as described in sections 5.2.3, 5.2.4, and 5.3.2), such that when the top strand separates from the antisense strand of the second reverse repeat, nickeling results in a sense strand 3' overhang containing the second reverse repeat.

[0366] In another embodiment, provided herein are first and second restricting regions for a nicking endonuclease located on the opposing chain near the first reverse repeat, such that when the sense chain separates from the antisense chain of the first reverse repeat, the nicking results in an antisense chain 3' overhang containing the first reverse repeat (e.g., sections 5.2.3, 5.2.4, ii) the first reverse repeat (as described in Section 5.3.2); ii) a sense expression cassette; and iii) a second reverse repeat (e.g., as described in Section 5.3.1), wherein third and fourth restriction sites for a nickeling endonuclease are located on the opposite strand near the second reverse repeat, such that when sense is separated from antisense of the second reverse repeat, nickeling results in a 5' overhang of the antisense strand containing the second reverse repeat.

[0367] In yet another embodiment, provided herein are a first and second restricting regions for a nicking endonuclease located on an opposing chain near the first reverse repeat (as described in Sections 5.2.3, 5.2.4, and 5.3.2), such that when the sense chain separates from the antisense chain of the first reverse repeat, the nicking results in a sense chain 5' overhang containing the first reverse repeat. A double-stranded DNA molecule comprising a second reverse repeat, wherein third and fourth restriction sites for a nicking endonuclease are located on the opposing strand near the second reverse repeat (as described in, for example, sections 5.2.3, 5.2.4, and 5.3.2), such that when the sense strand of the second reverse repeat separates from the antisense strand, the nicking results in an antisense strand 5' overhang containing the second reverse repeat.

[0368] In a further embodiment, provided herein are: i) a first reverse repeat (e.g., as described in Section 5.3.1) in the 5'-3' direction of the sense strand, wherein first and second limiting sites for a nicking endonuclease are located on a nearby opposing strand (e.g., as described in Sections 5.2.3, 5.2.4, and 5.3.2) such that when the sense strand separates from the antisense strand of the first reverse repeat, the nicking results in an antisense strand 3' overhang containing the first reverse repeat; ii iii) a sense expression cassette; and iii) a double-stranded DNA molecule comprising a second reverse repeat (e.g., as described in Section 5.3.1), wherein, when the sense strand of the second reverse repeat separates from the antisense strand, the nickeling results in a sense strand 3' overhang containing the second reverse repeat, with third and fourth restriction sites for nickeling endonucleases located on the opposing strand near the second reverse repeat (e.g., as described in Sections 5.2.3, 5.2.4, and 5.3.2 or shown in Figures 2B and 2C).

[0369] In one embodiment, provided herein is a first reverse repeat (e.g., as described in Section 5.3.1) in the 5' to 3' direction of the sense strand, wherein when the sense strand separates from the antisense strand of the first reverse repeat, the first and second target sites for a guide nucleic acid for a programmable nicking enzyme are located on the opposite strand near the first reverse repeat (e.g., as described in Sections 5.2.3, 5.2.4, and 5.3.2), such that nicking by a programmable nicking enzyme results in a sense strand 5' overhang containing the first reverse repeat. ii) a sense expression cassette; and iii) a second reverse repeat (e.g., as described in Section 5.3.1), wherein third and fourth target sites for guide nucleic acids for a programmable nicking enzyme are located on opposite strands near the second reverse repeat (e.g., as described in Sections 5.2.3, 5.2.4, and 5.3.2), such that when sense is separated from antisense of the second reverse repeat, nicking by a programmable nicking enzyme results in a sense strand 3' overhang containing the second reverse repeat.

[0370] In another embodiment, provided herein is a first reverse repeat (e.g., as described in Section 5.3.1) in the 5' to 3' direction of the sense strand, wherein when the sense strand separates from the antisense strand of the first reverse repeat, the first and second target sites for a guide nucleic acid for a programmable nicking enzyme are located on the opposite strand near the first reverse repeat (e.g., as described in Sections 5.2.3, 5.2.4, and 5.3.2), such that nicking by a programmable nicking enzyme results in an antisense strand 3' overhang containing the first reverse repeat. ii) a sense expression cassette; and iii) a second reverse repeat (e.g., as described in Section 5.3.1), wherein third and fourth target sites for guide nucleic acids for a programmable nicking enzyme are located on the opposite strand near the second reverse repeat (e.g., as described in Sections 5.2.3, 5.2.4, and 5.3.2), such that when the sense strand of the second reverse repeat separates from the antisense strand, nicking by a programmable nicking enzyme results in an antisense strand 5' overhang containing the second reverse repeat.

[0371] In yet another embodiment, provided herein is a first reverse repeat (e.g., as described in Section 5.3.1) in the 5'-3' direction of the sense strand, wherein when the sense strand separates from the antisense strand of the first reverse repeat, the first and second target sites for a guide nucleic acid for a programmable nicking enzyme are located on the opposite strand near the first reverse repeat (e.g., as described in Sections 5.2.3, 5.2.4, and 5.3.2), such that nicking by a programmable nicking enzyme results in a sense strand 5' overhang containing the first reverse repeat. ii) a sense expression cassette; and iii) a second reverse repeat (e.g., as described in Section 5.3.1), wherein third and fourth target sites for guide nucleic acids for a programmable nicking enzyme are located on the opposite strand near the second reverse repeat (e.g., as described in Sections 5.2.3, 5.2.4, and 5.3.2), such that when the sense strand of the second reverse repeat separates from the antisense strand, nicking by a programmable nicking enzyme results in an antisense strand 5' overhang containing the second reverse repeat.

[0372] In a further embodiment, provided herein are: i) a first reverse repeat (e.g., as described in Section 5.3.1) in the 5' to 3' direction of the sense strand, wherein first and second target sites for a guide nucleic acid for a programmable nicking enzyme are located on a contralateral strand near the first reverse repeat (e.g., as described in Sections 5.2.3, 5.2.4, and 5.3.2), such that when the sense strand separates from the antisense strand of the first reverse repeat, nicking by a programmable nicking enzyme results in an antisense strand 3' overhang containing the first reverse repeat; ii iii) a sense expression cassette; and iii) a double-stranded DNA molecule comprising a second reverse repeat (e.g., as described in Section 5.3.1), wherein third and fourth target sites for guide nucleic acids for a programmable nicking enzyme are located on the opposite strand near the second reverse repeat (e.g., as described in Sections 5.2.3, 5.2.4, and 5.3.2 or shown in Figures 2B and 2C), such that when the sense strand of the second reverse repeat separates from the antisense strand, nicking by a programmable nicking enzyme results in a sense strand 3' overhang containing the second reverse repeat.

[0373] An expression cassette may include one or more transcription factors, one or more post-transcriptional factors, or both one or more transcription factors and one or more post-transcriptional factors. Such regulatory elements are any sequences that enable, contribute to, or regulate the functional regulation of nucleic acid molecules, including replication, duplication, transcription, splicing, translation, stability, and / or transport of one of the nucleic acids or its derivatives (e.g., mRNA) into a host cell or organism. Such regulatory elements include, but are not limited to, promoters, enhancers, polyadenylation signals, translation termination codons, ribosome-binding elements, transcriptional terminators, selection markers, and / or origins of replication.

[0374] The expression cassette can have various sizes to accommodate one or more ORFs of varying lengths. In one embodiment, the size of the expression cassette is at least 2kb, at least 2.5kb, at least 3kb, at least 3.5kb, at least 4kb, at least 4.5kb, at least 5kb, at least 5.5kb, at least 6kb, at least 6.5kb, at least 7kb, at least 7.5kb, at least 8kb, at least 8.5kb, at least 9kb, at least 9.5kb, at least 10kb, at least 15kb, at least 20kb, at least 25kb, at least 30kb, at least 35kb, at least 40kb, at least 45kb, at least 50kb, at least 55kb, at least 60kb, at least 65kb, at least 70kb, at least 75kb, or at least 80kb. In one particular embodiment, the expression cassette is at least 7.5kb. In another specific embodiment, the expression cassette is at least 7.6kb. In yet another specific embodiment, the expression cassette is at least 7.7kb. In yet another specific embodiment, the expression cassette is at least 7.8kb. In one particular embodiment, the expression cassette is at least 7.9kb. In yet another specific embodiment, the expression cassette is at least 8kb. In another embodiment, the size of the expression cassette is approximately 2kb, 2.5kb, 3kb, 3.5kb, 4kb, 4.5kb, 5kb, 5.5kb, 6kb, 6.5kb, 7kb, 7.5kb, 8kb, 8.5kb, 9kb, 9.5kb, 10kb, 15kb, 20kb, 25kb, 30kb, 35kb, 40kb, 45kb, 50kb, 55kb, 60kb, 65kb, 70kb, 75kb, or 80kb. In one particular embodiment, the expression cassette is approximately 7.5kb. In another specific embodiment, the expression cassette is approximately 7.6kb. In yet another specific embodiment, the expression cassette is approximately 7.7kb. In yet another specific embodiment, the expression cassette is approximately 7.8kb. In one particular embodiment, the expression cassette is approximately 7.9kb.In another specific embodiment, the expression cassette is approximately 8 kb. The expression cassette may also contain a variety of target genes ("transgenes"). In one embodiment, the expression cassette contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 transgenes. In some specific embodiments, the expression cassette contains one transgene. In some embodiments, the transgene is a recombinant gene. In some further embodiments, the transgene contains a cDNA sequence (e.g., no introns in the transgene).

[0375] Furthermore, the expression cassette may contain at least 4,000 nucleotides, at least 5,000 nucleotides, at least 10,000 nucleotides, at least 20,000 nucleotides, at least 30,000 nucleotides, at least 40,000 nucleotides, or at least 50,000 nucleotides. In some embodiments, the expression cassette may contain any range of nucleotides between approximately 4,000 and approximately 10,000 nucleotides, approximately 10,000 and approximately 50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, the expression cassette may contain transgenes in the range of approximately 500 to approximately 50,000 nucleotides in length. In some embodiments, the expression cassette may contain transgenes in the range of approximately 500 to approximately 75,000 nucleotides in length. In some embodiments, the expression cassette may contain transgenes in the range of approximately 500 to approximately 10,000 nucleotides in length. In some embodiments, the expression cassette may contain transgenes in the range of approximately 1,000 to approximately 10,000 nucleotides in length. In some embodiments, the expression cassette may include a transgene ranging in length from approximately 500 to approximately 5,000 nucleotides. In some embodiments, the DNA molecules provided herein do not have size limitations on the capsid-forming AAV vector, thus enabling the delivery of large expression cassettes and providing efficient transgenes. In some embodiments, the DNA molecules provided herein include an expression cassette that is the same size as or larger than the size of any natural AAV genome.

[0376] Expression cassettes can occupy various positions relative to the reverse repeat. In some embodiments, expression cassettes can occupy 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 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, 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 at least 100 nucleotides apart.In one embodiment, the expression cassette is located at least 0.2kb, at least 0.3kb, at least 0.4kb, at least 0.5kb, at least 0.6kb, at least 0.7kb, at least 0.8kb, at least 0.9kb, at least 1kb, at least 1.5kb, or at least 2kb away from the reverse repeat. In another embodiment, the expression cassette is approximately 1, 2, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 5 from the reverse repeat. They are separated by 0, approximately 51, approximately 52, approximately 53, approximately 54, approximately 55, approximately 56, approximately 57, approximately 58, approximately 59, approximately 60, approximately 61, approximately 62, approximately 63, approximately 64, approximately 65, approximately 66, approximately 67, approximately 68, approximately 69, approximately 70, approximately 71, approximately 72, approximately 73, approximately 74, approximately 75, approximately 76, approximately 77, approximately 78, approximately 79, approximately 80, approximately 81, approximately 82, approximately 83, approximately 84, approximately 85, approximately 86, approximately 87, approximately 88, approximately 89, approximately 90, approximately 91, approximately 92, approximately 93, approximately 94, approximately 95, approximately 96, approximately 97, approximately 98, approximately 99, or approximately 100 nucleotides. In further embodiments, the expression cassette is located approximately 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb, 1kb, 1.5kb, or 2kb away from the reverse repeat. In one embodiment, the reverse repeat of this paragraph is the first reverse repeat described in sections 3 and 5.3 (including 5.3.1). In another embodiment, the reverse repeat of this paragraph is the second reverse repeat described in sections 3 and 5.3 (including 5.3.1). In yet another embodiment, the reverse repeat of this paragraph is both the first and second reverse repeats described in sections 3 and 5.3 (including 5.3.1).

[0377] As described above in this section (Section 5.3.3), an expression cassette may contain one or more ORFs. In one embodiment, the ORF is an ORF of a human gene, wherein a gene mutation in the human gene is known to cause a disease. In another embodiment, the ORF is an ORF of a human gene, wherein a gene mutation in the human gene is known to cause a hereditary disease. In yet another embodiment, the ORF encodes a therapeutic protein. In yet another embodiment, the ORF encodes an enzyme. In one embodiment, the ORF encodes a metabolic enzyme. In several embodiments, the ORF encodes an enzyme that replaces or complements the function of a defective enzyme in humans. In one embodiment, the ORF encodes an antibody. In another embodiment, the ORF encodes a therapeutic antibody. In yet another embodiment, the ORF encodes a cytokine. In yet another embodiment, the ORF encodes RNA. In one embodiment, the ORF encodes regulatory RNA. In another embodiment, the ORF encodes antisense RNA. In yet another embodiment, the ORF encodes siRNA. In further embodiments, the ORF encodes shRNA. In one embodiment, the ORF encodes miRNA. In another embodiment, the ORF encodes piRNA (PIWI-interacting RNA). In some embodiments, the expression cassette includes one or more of the features described in this section (Section 5.3.3) in various permutations and combinations.

[0378] The various embodiments described in this section (Section 5.3.3) using a nickeling endonuclease and / or a restriction site for a nickeling endonuclease are further provided by replacing the nickeling endonuclease with a programmable nickeling enzyme and replacing the restriction site with a target site for a programmable nickeling enzyme. Programmable nickeling enzymes and their target sites for this paragraph and this section (Section 5.3.3) are provided in Section 5.2.4.

[0379] (5.3.4 Characteristics of viral DNA sequences not present in the DNA mo...

Claims

1. A method for preparing hairpin-terminated DNA molecules, a. Preparing a double-stranded DNA molecule, wherein the double-stranded DNA molecule is positioned in the 5' to 3' direction of the top strand, i. A first reverse repeat, wherein the first and second restriction sites for the nicking endonuclease are located on opposing chains, and the nicking is (1) When the top strand separates from the bottom strand of the first reverse repeat, it results in a top strand 5' single-stranded DNA overhang containing the first reverse repeat or a fragment thereof, or (2) When the top strand separates from the bottom strand of the first reverse repeat, the first reverse repeat and the bottom strand 3' single-stranded DNA overhang containing the first reverse repeat are obtained. ii. Expression cassette, and iii. A second reverse repeat, in which the third and fourth restriction sites for the nicking endonuclease are located on opposing chains, and the nicking is (1) When the top strand separates from the bottom strand of the second reverse repeat, it results in a top strand 3' single-stranded DNA overhang containing the second reverse repeat or a fragment thereof, or (2) When the top strand separates from the bottom strand of the second reverse repeat, the second reverse repeat results in a bottom strand 5' single-stranded DNA overhang containing the second reverse repeat. Including the preparations mentioned above, b. Incubating the double-stranded DNA molecule with one or more nickel end nucleases that recognize the four restriction sites, thereby generating the two single-stranded DNA overhangs specified in step a when separating the top strand from the bottom strand, wherein the length of each single-stranded DNA overhang is independently at least 20 nucleotides. c. To perform denaturation, and thereby generate a DNA fragment that includes a single-stranded expression cassette and is adjacent to the two single-stranded DNA overhangs, and d. To perform intramolecular annealing on the single-stranded DNA overhang, thereby generating hairpinned reverse repeats at both ends of the DNA fragment obtained as a result of step c. Includes, The method wherein the denaturation is performed by exposing the DNA to heat, a change in pH, an increase in salt concentration, or a chemical denaturing agent.

2. The double-stranded DNA molecule in step a is a. Culturing host cells containing the double-stranded DNA molecule under conditions that result in amplification of the double-stranded DNA molecule, and b. To release the double-stranded DNA molecule from the host cell. Prepared by, or The method according to claim 1, wherein the double-stranded DNA molecule in step a is prepared by in vitro replication.

3. The method according to claim 1 or 2, wherein the first, second, third, and fourth restricting sites for the nickel endonuclease are all the same restricting site for the nickel endonuclease.

4. The double-stranded DNA molecule in step a is a. The first and / or second reverse repeats described above have nucleotide sequences that are at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% identical to the parvovirus ITR, and / or b. The first and / or second reverse repeats described above are modified parvovirus ITRs, or c. The first and / or second reverse repeats described above originate from adeno-associated virus (AAV). A method according to any one of claims 1 to 3, characterized by the above.

5. The double-stranded DNA molecule in step a is a plasmid, and the plasmid is a. It is ring-shaped, or b. The method according to any one of claims 1 to 4, wherein the chain is linear.

6. The method according to any one of claims 1 to 5, wherein the expression cassette of the double-stranded DNA molecule in step a includes one or more open reading frames (ORFs).

7. The ORF encodes a therapeutic protein, or The method according to claim 6, wherein the ORF encodes RNA.

8. The size of the expression cassette of the double-stranded DNA molecule in step a is at least 4kb, at least 4.5kb, at least 5kb, at least 5.5kb, at least 6kb, at least 6.5kb, at least 7kb, at least 7.5kb, at least 8kb, at least 8.5kb, at least 9kb, at least 9.5kb, or at least 10kb, and / or The method according to any one of claims 1 to 7, wherein the expression cassette of the DNA fragment generated in step c includes a transgene having a length in the range of about 500 to about 50,000 nucleotides, about 500 to about 75,000 nucleotides, about 500 to about 10,000 nucleotides, about 1,000 to about 10,000 nucleotides, or about 500 to about 5,000 nucleotides.

9. The method according to any one of claims 1 to 8, wherein the first and second reverse repeats of the double-stranded DNA molecule in step a each lack functional RABS and functional TRS, respectively.

10. e. Incubating the double-stranded DNA molecule from step a or step b or the fragment obtained as a result of step d with one or more restriction enzymes that cause cleavage in the double-stranded DNA molecule, and f. Incubating the fragment of the double-stranded DNA molecule with an exonuclease, thereby digesting the fragment of the double-stranded DNA molecule, excluding the fragment obtained as a result of step d. The method according to any one of claims 1 to 9, further comprising:

11. The method according to any one of claims 1 to 10, further comprising repairing the nicks in the fragment obtained as a result of step d using a ligase to generate circular DNA.

12. a. The bottom chain is antisense-oriented with respect to the open reading frame in the expression cassette, or b. The method according to any one of claims 1 to 11, wherein the top strand is antisense-oriented with respect to the open reading frame in the expression cassette.

13. The double-stranded DNA molecule of step a is characterized by containing a palindromic sequence in which the first reverse repeat and the second reverse repeat are 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical when read forward and reversed on the complementary strand, and a. The first and second restriction sites for the nickel endonuclease are separated by at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, or at least 200 nucleotides, and / or b. The method according to any one of claims 1 to 12, wherein the third and fourth limiting sites for the nickel endonuclease are separated by at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, or at least 200 nucleotides.

14. The method according to any one of claims 1 to 13, wherein either or both of the reverse hairpinning iterations of step d include a main stem and a vertex.

15. The first and second reverse repeats of the double-stranded DNA molecule of step a each further include at least one nucleotide modification to replace or deplete the presence of a CpG motif, and / or The method according to any one of claims 1 to 14, wherein the first and second reverse repeats of the double-stranded DNA molecule in step d include the following: The sequence from one nucleotide of an ITR closed base pair to the other nucleotide of the ITR closed base pair has an unfolding Gibbs free energy of -10 kcal / mol or less under physiological conditions.

16. The length of each single-stranded DNA overhang is independently at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, and less The method according to any one of claims 1 to 15, wherein the length of each nucleotide is at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, 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 at least 100 nucleotides.

17. The double-stranded DNA molecule further includes fifth and sixth restriction sites for a nickeling endonuclease in a region that is 5' relative to the first reverse repeat of the top strand and 3' relative to the second reverse repeat of the top strand, wherein the fifth and sixth restriction sites for a nickeling endonuclease are a. Located on opposing chains, b. The method according to any one of claims 1 to 16, wherein the cleavage occurs within the double-stranded DNA molecule such that the single-stranded DNA overhang of the cleavage does not undergo intermolecular or intramolecular annealing at a level detectable under conditions suitable for annealing of the first reverse repeat and / or the second reverse repeat.

18. a. The fifth and sixth nicks are separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, and / or b. The first, second, third, fourth, fifth, and sixth restriction sites for nickel endonucleases are all the same nickel endonuclease target sequence and / or c. The double-stranded DNA molecule further comprises an open reading frame encoding a nickeling endonuclease that recognizes the first, second, third, fourth, fifth, and / or sixth restriction sites for the nickeling endonuclease, and / or d. The method according to claim 17, wherein the nickeling endonuclease that recognizes the fifth and sixth restriction sites for nickeling endonuclease is Nt. BsmAI; Nt. BtsCI; N. Alwl; N. BstNBI; N. BspD6I; Nb. Mva1269I; Nb. BsrDI; Nt. BtsI; Nt. BsaI; Nt. Bpu10I; Nt. BsmBI; Nb. BbvCI; Nt. BbvCI; or Nt. BspQI.

19. e. Incubating the double-stranded DNA molecule from step a or step b or the fragment obtained as a result of step c with one or more nickeling endonucleases that recognize the fifth and sixth restriction sites and cause cleavage in the double-stranded DNA molecule, and f. Incubating the fragment of the double-stranded DNA molecule with an exonuclease, thereby digesting the fragment of the double-stranded DNA molecule, excluding the fragment obtained as a result of step d. The method according to claim 17 or 18, further comprising:

20. The method according to claim 2, wherein the host cell is a bacterial host cell.

21. The method according to claim 4, wherein the parvovirus is dependent parvovirus, bocaparvovirus, erythroparvovirus, protoparvovirus, or tetraparvovirus.

22. The method according to claim 4, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrhlO, AAV-DJ, or AAV-DJ8.

23. (i) The plasmid further comprises a restriction enzyme site in a region of the top strand that is 5' with respect to the first reverse repeat and 3' with respect to the second reverse repeat, wherein the restriction enzyme site is not located in the first reverse repeat, the second reverse repeat, and / or in the region between the first reverse repeat and the second reverse repeat, and (ii) The method according to claim 5, wherein the restriction enzyme cleavage results in a single-stranded DNA overhang that does not anneal under the annealing conditions of the first reverse repeat and / or the second reverse repeat.

24. The method according to claim 7, wherein the therapeutic protein is an enzyme, an antibody, or a cytokine.

25. The method according to claim 7, wherein the RNA is regulatory RNA.

26. The method according to claim 7, wherein the RNA is antisense RNA, siRNA, shRNA, miRNA, or piRNA.

27. One or both of the reverse hairpinning iterations of step d are one or more a. Branching hairpin, b. Bulge and / or c. The method according to any one of claims 1 to 26, further comprising an internal loop.

28. The method according to claim 15, wherein the modification is a deletion, substitution, or addition.

Images