Linear DNA with enhanced resistance to exonuclease and method for producing same

Abstract

(a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single, contiguous aqueous volume; and (b) incubating the single, contiguous aqueous volume to produce a linear DNA product. Further disclosed are linear deoxyribonucleic acid (DNA) products having enhanced resistance to nuclease digestion and uses thereof.

Description

[Technical field]

[0001] Technical Field The present invention relates to a method for producing a linear deoxyribonucleic acid (DNA) product (e.g., a closed linear DNA product) having enhanced resistance to nuclease digestion. The present invention relates to a method comprising the steps of: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to produce a linear DNA product (e.g., a closed linear DNA product). The present invention also relates to a linear deoxyribonucleic acid (DNA) product (e.g., a closed linear DNA product) having enhanced resistance to nuclease digestion and uses thereof. [Background technology]

[0002] background DNA is susceptible to degradation by nucleases, enzymes that occur naturally in living organisms and play an important role in the control of many cellular processes, as well as protecting DNA from foreign DNA species. Enzymatic DNA degradation can render gene therapy ineffective, and is an important consideration when developing gene therapy or DNA vaccines.

[0003] Considerable efforts have been made to increase the resistance of nucleic acid molecules to both extracellular and intracellular nucleases, thereby increasing the effective molecular life of nucleic acids.

[0004] For linear molecules, one proposed solution involves the use of phosphorothioated nucleotides (ie, 2'-deoxynucleotide-5'-(α-thio)-triphosphates).

[0005] Phosphorothioated nucleotides contain a sulfur atom instead of a non-bridging oxygen atom. These modified nucleotides exhibit the same physical and chemical properties as the corresponding unmodified nucleotides, but are resistant to exonuclease digestion. Thus, the incorporation of phosphorothioate functional groups can increase the half-life of nucleic acid molecules.

[0006] Phosphorothioate modifications are used in nucleic acid drug development programs. In therapeutic nucleic acids, phosphorothioated nucleotides are incorporated into short single-stranded polynucleotide chains. For example, the antisense oligonucleotide fomivirsen is a 21-mer phosphorothioate oligodeoxynucleotide used to treat cytomegalovirus retinitis (Stein and Castanotto, “FDA-approved oligonucleotide therapies in 2017”. Molecular Therapy 25.5 (2017):1069-1075). Similarly, pegaptanib (trade name Macugen) is a short (27 nucleotide) aptamer with a phosphorothioate 3'-3' deoxythymidine cap used to treat age-related macular degeneration of the retina.

[0007] Phosphorothioate modifications have also been used in linear double-stranded polynucleotide chains (e.g., double-stranded DNA) to cap the ends of the polynucleotide chains to increase their resistance to exonuclease digestion (Putney et al. "A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicate in vivo." Proceedings of the National Academy of Sciences 78.12 (1981):7350-7354). To cap the ends of a polynucleotide chain, the ends are digested with a restriction enzyme and treated with a mixture of DNA polymerase and deoxyribonucleotide triphosphates (dNTPs). At least one of the deoxyribonucleotide triphosphates is a phosphorothioated nucleotide complementary to a nucleotide in the overhanging strand. Because DNA polymerase adds nucleotides in a 5' to 3' direction, this process results in a blunt-ended polynucleotide fragment that has a phosphorothioated nucleotide at the 3' end of each strand (ie, within the "cap").

[0008] Resistance to nuclease digestion can also be achieved by using closed DNA molecules such as plasmids or minicircles, however, plasmids and minicircles are of limited utility in vivo due to frequent contamination with toxic substances from cellular components, fidelity issues that alter the sequence of interest, and the existence of different species (supercoiled, linear and open circular).

[0009] Alternatively, resistance to nuclease digestion can be achieved by creating closed linear DNA molecules. For example, WO2010 / 086626 A1 describes a method for producing closed linear DNA using protelomerase. However, this method has a limitation in that the action of protelomerase produces the same sequence at both ends of the closed linear DNA molecule.

[0010] Thus, there is a need for more flexible methods for producing linear DNA products that have increased resistance to nuclease (eg, exonuclease) digestion. Summary of the Invention

[0011] explanation The present invention provides a method for producing a linear deoxyribonucleic acid (DNA) product (e.g., a closed linear DNA product) with enhanced resistance to nuclease digestion. The present invention is based on the addition of an adapter molecule to a double-stranded DNA molecule. The method of the present invention includes adding an adapter molecule, an endonuclease, and a ligase to a double-stranded DNA molecule in a single reaction volume (or a single continuous aqueous volume). Thus, a method for producing a linear DNA product includes the steps of (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and a first and a second adapter molecule to form a single continuous aqueous volume; and (b) incubating the single continuous aqueous volume to produce a linear DNA product. Preferably, the linear DNA product has enhanced resistance to exonuclease (e.g., exonuclease I, exonuclease III, and / or exonuclease VIII) digestion. The linear DNA product may be a closed linear DNA product. The linear DNA product may comprise nuclease resistant nucleotides (i.e., protected nucleotides), such as phosphorothioated nucleotides. The linear DNA product may be a partially closed linear DNA product. The partially closed linear DNA product may comprise nuclease resistant nucleotides (i.e., protected nucleotides), such as phosphorothioated nucleotides. The linear DNA product may comprise a cassette. The cassette may comprise a coding sequence.

[0012] The present invention provides a method for producing a linear DNA product, the method comprising: (a) contacting a double-stranded DNA molecule with an endonuclease and first and second adaptor molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a portion of a double-stranded DNA molecule, and the first adaptor molecule is attached to a first end of the linear double-stranded region and a second adaptor molecule is attached to a second end of the linear double-stranded region; Includes.

[0013] The first and second adaptor molecules may be the same or different molecules. For example, the first and / or second adaptor molecules may comprise a hairpin. The first and / or second adaptor molecules may be double-stranded linear nucleic acid molecules comprising one or more nuclease-resistant nucleotides. The first adaptor molecule may comprise a hairpin and the second adaptor molecule may be a double-stranded linear nucleic acid molecule comprising one or more nuclease-resistant nucleotides. Thus, the linear DNA product produced by the methods described herein is resistant to nuclease (e.g., exonuclease) digestion.

[0014] The step of contacting the double-stranded DNA molecule with an endonuclease and a first and a second adaptor molecule is preferably carried out in the presence of a ligase. Thus, the present invention provides a method for producing a linear deoxyribonucleic acid (DNA) product, the method comprising: (a) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and wherein the first adaptor molecule is attached to a first end of the linear double-stranded region and a second adaptor molecule is attached to a second end of the linear double-stranded region; Includes.

[0015] The addition (or linking or closing) of the first and / or second adaptor molecule can be performed by hybridization or ligation of the adaptor molecule to the end of the linear double-stranded region. Thus, the first adaptor molecule can be hybridized to the first end of the linear double-stranded region. The second adaptor molecule can be hybridized to the second end of the linear double-stranded region. The first adaptor molecule can be ligated to the first end of the linear double-stranded region. The second adaptor molecule can be ligated to the second end of the linear double-stranded region. The addition of the first and second adaptor molecules can be performed by both hybridization and ligation of the adaptor molecule to the end of the linear double-stranded region. Thus, the first adaptor molecule can be hybridized and ligated to the first end of the linear double-stranded region. A second adapter molecule can hybridize and ligate to the second end of the linear double-stranded region. Attachment can be via a linker or spacer molecule that facilitates attachment of the adapter molecule to the first and / or second ends of the linear double-stranded region.

[0016] The method may further comprise amplifying a DNA template molecule to generate a double-stranded DNA molecule prior to step (a) (i.e., contacting the double-stranded DNA molecule with an endonuclease, a ligase, and a first and second adaptor molecule). Thus, the present invention provides a method for producing a linear DNA product, comprising: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; and (c) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, and the first adaptor molecule is attached to a first end of the linear double-stranded region and a second adaptor molecule is attached to a second end of the linear double-stranded region. The present invention provides a method comprising:

[0017] The amplification may be in vitro or in vivo amplification. Preferably, the amplification is in vitro amplification. For example, the amplification may be performed by rolling circle amplification (RCA), MALBAC, conventional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA). Preferably, the amplification is rolling circle amplification. Thus, the present invention provides a method for producing a linear deoxyribonucleic acid (DNA) product, comprising: (a) rolling circle amplifying a DNA template molecule containing at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; and (c) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and wherein the first adaptor molecule is attached to a first end of the linear double-stranded region and a second adaptor molecule is attached to a second end of the linear double-stranded region. The present invention provides a method comprising:

[0018] The method of the invention may further comprise a step of heat inactivation (after the amplification step and prior to the step of contacting the double stranded DNA molecule with an endonuclease, a ligase and a first and second adaptor molecule). Thus, the invention provides a method of producing a linear DNA product, comprising: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) heat inactivating the reaction of step (a); (c) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; and (d) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, and the first adaptor molecule is attached to a first end of the linear double-stranded region and a second adaptor molecule is attached to a second end of the linear double-stranded region. The present invention provides a method comprising:

[0019] Preferably, the amplification is rolling circle amplification.

[0020] The heat inactivation step may be performed under conditions sufficient to inactivate the reagents used during the amplification reaction. The heat inactivation step may be performed at a temperature of at least 50° C., at least 55° C., at least 60° C., at least 65° C., at least 70° C., at least 75° C., at least 80° C., at least 85° C., at least 90° C., at least 95° C., or at least 100° C. The heat inactivation step may be performed for at least 1 minute, at least 3 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, or at least 20 minutes.

[0021] The present inventors have surprisingly found that the large linear concatemeric products of rolling circle amplification reactions can be used to produce the DNA products described herein, which is surprising because the products of rolling circle amplification are highly viscous and typically require purification steps before they can be utilized in further applications.

[0022] In the method described herein, after the amplification step, the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and a first and a second adaptor molecule to form a single continuous aqueous volume can be performed without purifying the product of the amplification reaction. That is, the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and a first and a second adaptor molecule can be performed directly after the amplification step. The step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and a first and a second adaptor molecule can be performed directly after the heat inactivation step.

[0023] The inventors have found a method that requires significantly fewer steps to produce the DNA products described herein. The methods described herein are very time-efficient, since no purification steps are required after the amplification reaction. If necessary, the amplification reaction products may be heat-inactivated. Surprisingly, the methods described herein, in which the step of contacting the double-stranded DNA molecules with an endonuclease, a ligase, and a first and a second adaptor molecule is performed directly after the amplification step (i.e., without a purification step), result in a higher yield of the DNA products described herein compared to the method in which the two steps are performed separately by purifying the amplification product.

[0024] The method may further comprise the step of purifying the linear DNA product after the step of incubating the single continuous aqueous volume.

[0025] The method may further comprise a nuclease digestion step after the step of incubating the single continuous aqueous volume. The nuclease digestion may be an exonuclease digestion, such as exonuclease I and / or exonuclease III digestion. The nuclease digestion step may be before or after the purification step. The nuclease digestion step may remove double-stranded DNA molecules and / or adapter molecules that were not used to generate the linear DNA product. Thus, the method for producing a linear DNA product includes: (a) contacting double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (b) incubating a single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, and the first adaptor molecule is attached to a first end of the linear double-stranded region and a second adaptor molecule is attached to a second end of the linear double-stranded region; and (c) incubating the single contiguous aqueous volume with a nuclease (e.g., an exonuclease); may include.

[0026] The method for producing a linear DNA product comprises: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (c) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, and the first adaptor molecule is attached to a first end of the linear double-stranded region and a second adaptor molecule is attached to a second end of the linear double-stranded region; and (d) incubating the single contiguous aqueous volume with a nuclease (e.g., an exonuclease) may include.

[0027] The method for producing a linear DNA product comprises: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (c) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, and the first adaptor molecule is attached to a first end of the linear double-stranded region and a second adaptor molecule is attached to a second end of the linear double-stranded region; (d) a purification step for the linear DNA product; and (e) incubating the product of step (d) with a nuclease (e.g., an exonuclease). may include.

[0028] The method for producing a linear DNA product comprises: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (c) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, and the first adaptor molecule is attached to a first end of the linear double-stranded region and a second adaptor molecule is attached to a second end of the linear double-stranded region; (d) incubating the single contiguous aqueous volume with a nuclease (e.g., an exonuclease); and (e) Purification of the linear DNA product may include.

[0029] The endonuclease may be a restriction enzyme endonuclease. The endonuclease may be a type IIS restriction enzyme. The endonuclease may be any enzyme that recognizes a DNA sequence and cleaves outside the recognition sequence. For example, endonucleases include BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, BceAI, BcgI, B ciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, Btg ZI, BtsCI, BtsI-v2, BtsMutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, The restriction enzyme may be FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI.

[0030] Type IIS restriction endonucleases cleave double-stranded DNA molecules outside of the recognition sequence (i.e., the endonuclease target sequence), and therefore the recognition sequence (i.e., the endonuclease target sequence) is not included in the linear DNA product.

[0031] The inventors have surprisingly found a method for producing a linear DNA product with enhanced resistance to nuclease digestion. Specifically, the linear DNA product produced by the method described herein has enhanced resistance to exonuclease digestion (e.g., exonuclease III digestion). The enhanced resistance to exonuclease digestion extends the life span of the linear DNA product in cells (i.e., the linear DNA product has improved resistance to intracellular exonucleases) and in cell-free systems (i.e., the linear DNA product has improved resistance to extracellular exonucleases). The inventors have developed a method based on the addition of adapter molecules (i.e., a first adapter molecule and a second adapter molecule) to both ends of a double-stranded DNA molecule. The adapter molecules may include hairpins or loops to form a closed linear DNA product with enhanced resistance to nuclease (e.g., exonuclease) digestion. Furthermore, the inventors have found a method to efficiently introduce protective nucleotides in the form of a first and a second adapter molecule at both ends of a linear double-stranded region of a linear DNA product. The methods described herein may use a linear adapter that includes a hairpin or loop adapter at one end of the DNA product and a protective nucleotide at the other end of the DNA product. That is, any type of adapter described herein can be used as long as the final DNA product is protected from nuclease (e.g., exonuclease) digestion. The methods of the present invention provide protection from digestion by exonucleases that cleave 3'-terminal nucleotides (e.g., exonuclease III) and exonucleases that cleave 5'-terminal nucleotides (e.g., exonuclease VIII). Thus, the linear DNA product produced by the methods of the present invention has extended in vivo expression compared to linear DNA products that do not include the adapter molecules described herein.

[0032] The term "protected nucleotides" or "nuclease-resistant nucleotides" as used herein is intended to encompass any type of molecule that provides or enhances resistance to nuclease digestion, particularly exonuclease digestion. Although adapter molecules are described herein as comprising phosphorothioated nucleotides, one of skill in the art will appreciate that adapter molecules may alternatively comprise any molecule that provides resistance to nuclease digestion (e.g., exonuclease III digestion). For example, adapter molecules may comprise nuclease-resistant nucleotides, i.e., modified nucleotides that provide or enhance resistance to nucleases (e.g., exonucleases). Adapter molecules may comprise peptides, polypeptides, or proteins that provide or enhance resistance to nuclease (e.g., exonuclease) digestion. Adapter molecules may comprise 2'-O-methyl nucleotides or 2'-O-methoxyethyl (MOE) nucleotides.

[0033] As used herein, the term "phosphorothioated nucleotide" refers to a nucleotide with an altered phosphate backbone in which the sugar moieties are linked by a phosphorothioate bond. The phosphorothioate bond contains a sulfur atom in place of a non-bridging oxygen atom in the phosphate backbone of the oligonucleotide sequence. This modification makes the internucleotide bond less susceptible to digestion by nucleases.

[0034] The linear DNA products (e.g., closed linear DNA products) produced by the methods of the invention have additional advantageous properties, such as being substantially free of bacterial backbones and / or antibiotic resistance genes. The lack of these characteristics is particularly beneficial in the manufacture of cell delivery systems, such as viral vectors or nanoparticles for cell therapy. The lack of these characteristics makes the linear DNA products produced by the methods of the invention particularly suitable for use in pharmaceutical compositions.

[0035] Unexpectedly, the present inventors have discovered a method for producing a linear DNA product with enhanced resistance to nuclease digestion that allows efficient mass production of a linear DNA product with enhanced resistance to exonuclease digestion. Large-scale production of the product can be performed in a cell-free system, resulting in the production of a pure sample containing a linear DNA product that is substantially free of bacterial contaminants (e.g., remaining after cell lysis).

[0036] 1. Method for producing closed linear DNA products The methods described herein can be used to produce closed linear DNA products, for example covalently closed linear DNA products.

[0037] The present invention provides a method for producing a closed linear DNA product, comprising the steps of: (a) contacting a double-stranded DNA molecule with an endonuclease and first and second adaptor molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to produce a closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by a first adaptor molecule and at a second end by a second adaptor molecule; The present invention provides a method comprising:

[0038] The step of contacting the double-stranded DNA molecule with an endonuclease and a first and a second adaptor molecule is preferably carried out in the presence of a ligase. Thus, the present invention provides a method for producing a closed linear DNA product, comprising: (a) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adapter molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to produce a closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by a first adaptor molecule and at a second end by a second adaptor molecule; The present invention provides a method comprising:

[0039] A linear double-stranded region is a linear portion of a double-stranded DNA molecule.

[0040] The present invention provides a method for producing a closed linear DNA product, comprising the steps of: (a) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adapter molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to produce a closed linear DNA product, wherein the closed linear DNA product comprises a linear portion of a double stranded DNA molecule, and the linear portion of the double stranded DNA molecule is closed at a first end by a first adaptor molecule and at a second end by a second adaptor molecule; The present invention provides a method comprising:

[0041] Closed linear DNA products are particularly useful as therapeutics (i.e., DNA therapeutics) that can be used to express gene products in vivo because their closed structure (e.g., covalently closed structure) prevents attack by enzymes such as exonucleases, leading to increased stability and longevity of gene expression compared to "open" DNA molecules with exposed DNA ends. Linear double-stranded open-ended cassettes have been demonstrated to be inefficient for gene expression when introduced into host tissues. This was attributed to instability of the cassette due to the action of exonucleases in the extracellular space.

[0042] Segregating the DNA ends in a closed structure has other advantages: Closed linear DNA products have improved safety because the DNA ends are prevented from integrating with genomic DNA. In addition, a closed linear structure reduces ligation of the DNA product within the host cell, allowing for more sensitive regulation of gene product expression levels.

[0043] The method of the present invention can be used to produce DNA, such as a DNA vaccine, for in vitro expression in a host cell. A DNA vaccine typically encodes modified DNA of an infectious organism. The DNA vaccine is administered to a subject to express a selected protein of the infectious organism and mount an immune response against that protein. A DNA vaccine may also encode a tumor antigen in a cancer immunotherapy approach.

[0044] This method can also generate other types of therapeutic DNA molecules, such as those used in gene therapy. For example, such DNA molecules can be used to express functional genes when a subject has a genetic disorder caused by a malfunctioning version of that gene. Examples of such diseases include sickle cell anemia, cystic fibrosis, Huntington's disease, Duchenne muscular dystrophy, hemophilia A, α1-antitrypsin deficiency, primary ciliary dyskinesia or respiratory distress syndrome of prematurity. Other diseases for which gene therapy is useful include metabolic diseases, respiratory diseases, inflammatory diseases, autoimmune diseases, chronic infectious diseases (AIDS, cancer, neurological diseases, cardiovascular diseases, hypercholesterolemia, various anemias, thalassemia, blood diseases such as hemophilia, emphysema, etc.). For the treatment of solid tumors, genes encoding toxic peptides (i.e., chemotherapeutic agents such as ricin, diphtheria toxin, cobra venom factor, etc.), tumor suppressor genes such as p53, mRNA sequences that are antisense to transforming oncogenes, anti-tumor peptides such as tumor necrosis factor (TNF) and other cytokines, or genes encoding transdominant negative mutants of transforming oncogenes can be expressed.

[0045] Closure of the linear double-stranded region by the first and / or second adaptor molecules can be achieved by hybridization or ligation of the adaptor molecules to the ends of the linear double-stranded region. Thus, the first adaptor molecule can hybridize to the first end of the linear double-stranded region. The second adaptor molecule can hybridize to the second end of the linear double-stranded region. The first adaptor molecule can be ligated to the first end of the linear double-stranded region. The second adaptor molecule can be ligated to the second end of the linear double-stranded region. Closure of the linear double-stranded region by the first and second adaptor molecules can be achieved by both hybridization and ligation of the adaptor molecules to the ends of the linear double-stranded region. Thus, the first adaptor molecule can hybridize and ligate to the first end of the linear double-stranded region. A second adaptor molecule can hybridize and ligate to the second end of the linear double-stranded region.

[0046] The step of incubating a single continuous aqueous volume to create a closed linear DNA product can include creating a linear portion of the double-stranded DNA molecule by digesting the double-stranded DNA molecule with an endonuclease.

[0047] The step of incubating the single continuous aqueous volume can be performed under conditions that promote the addition (or ligation) of a first and a second adaptor molecule to the linear double-stranded region to generate a closed linear DNA product, the addition being accomplished by forming a covalent bond between the first and / or second adaptor molecule and the first and / or second terminus of the linear double-stranded region.

[0048] The step of incubating the single continuous aqueous volume can be performed under conditions that promote digestion of the double-stranded DNA molecule and generate a linear portion of the double-stranded DNA molecule. The digestion of the double-stranded DNA molecule to generate a linear portion of the double-stranded DNA molecule can be performed at a first temperature of 1° C. to 100° C., 1° C. to 80° C., 5° C. to 70° C., 10° C. to 60° C., 15° C. to 55° C., 20° C. to 50° C., 25° C. to 45° C., 30° C. to 40° C., 35° C. to 39° C., 36° C. to 38° C., or about 37° C. The digestion can be an endonuclease digestion, preferably a type IIS endonuclease digestion.

[0049] The step of incubating the single continuous aqueous volume can be performed under conditions that promote ligation of the linear double-stranded region to the first and second adaptor molecules. Ligation can be performed with an efficiency of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95%. For example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the linear double-stranded region (or portion of the double-stranded DNA molecule) can be incorporated into the closed linear DNA product. Preferably, the efficiency of ligation is at least 15%.

[0050] The ligation efficiency can be established based on the DNA quantification values ​​before and after the digestion / ligation reaction. Therefore, the ligation efficiency can be determined based on the formula: (amount of starting DNA for amplification) / (amount of final linear DNA)×100%.

[0051] Ligation efficiency can also be established based on DNA quantification before and after digestion / ligation reactions and subsequent exonuclease treatment to remove remaining open DNA constructs and excess adapter molecules.

[0052] For example, the double-stranded DNA molecules generated by rolling circle amplification are first quantified to know the amount of double-stranded DNA molecules used as starting material during the digestion / ligation reaction. After all enzymatic reactions, the linear DNA product is quantified and the ligation efficiency is calculated according to the formula above.

[0053] Methods for DNA quantification are known to those skilled in the art. For example, DNA quantification can be performed using Thermo Fisher's Qubit dsDNA BR Assay (https: / / www.thermofisher.com / order / catalog / product / Q32850# / Q32850).

[0054] The step of ligating the linear double-stranded region to the first and second adaptor molecules can be carried out at a second temperature of 1°C to 90°C, 2°C to 70°C, 5°C to 60°C, 8°C to 55°C, 9°C to 50°C, 10°C to 45°C, 11°C to 40°C, 12°C to 37°C, 13°C to 30°C, 14°C to 25°C, 15°C to 20°C, or about 16°C.

[0055] The step of incubating the single continuous aqueous volume may include incubating at a first temperature and then incubating at a second temperature. The first temperature may be 1°C to 100°C, 1°C to 80°C, 5°C to 70°C, 10°C to 60°C, 15°C to 55°C, 20°C to 50°C, 25°C to 45°C, 30°C to 40°C, 35°C to 39°C, 36°C to 38°C, or about 37°C. The second temperature may be 1°C to 90°C, 2°C to 70°C, 5°C to 60°C, 8°C to 55°C, 9°C to 50°C, 10°C to 45°C, 11°C to 40°C, 12°C to 37°C, 13°C to 30°C, 14°C to 25°C, 15°C to 20°C, or about 16°C. Preferably, the first temperature is 35°C to 39°C and the second temperature is 14°C to 18°C. Using these conditions, the endonuclease may be a type IIS restriction endonuclease (e.g., BsaI) and the ligase may be T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase or E. coli DNA ligase.

[0056] The step of incubating the single continuous aqueous volume may be performed isothermally. The step of incubating the single continuous aqueous volume may include incubating at a constant temperature. The constant temperature simultaneously promotes digestion of the double-stranded DNA molecule, creating a linear portion of the double-stranded DNA molecule, and ligating the linear double-stranded region to the first and second adaptor molecules. For example, the constant temperature can be 20°C, 21°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, 37°C, 38°C, 39°C, or 40°C. Preferably, the constant temperature is 30°C. A constant temperature means that the temperature does not change significantly during the reaction. By constant temperature is meant that the temperature change between steps of incubating a single consecutive aqueous volume is less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1°C. In a preferred embodiment, the temperature between steps of incubating a single consecutive aqueous volume does not deviate by more than 5°C, preferably less than 3°C, and more preferably less than 1°C. Thus, a constant temperature can be a temperature in the range of 20°C to 30°C, 22°C to 32°C, 24°C to 34°C, 26°C to 36°C, 28°C to 38°C, 30°C to 40°C, 22°C to 28°C, 32°C to 38°C, 25°C to 35°C, 26°C to 34°C, 27°C to 33°C, 27.5°C to 32.5°C, 28°C to 32°C, 28.5°C to 31.5°C, 29°C to 31°C, or 29.5°C to 30.5°C. Preferably, the constant temperature may be in the range of 27.5° C. to 32.5° C. Alternatively, the constant temperature may be in the range of 32° C. to 42° C., 33° C. to 41° C., 34° C. to 40° C., 35° C. to 39° C., or 36° C. to 38° C. Preferably, the constant temperature is in the range of 34.5° C. to 39.5° C.

[0057] The step of incubating a single continuous aqueous volume may include cycling between a first temperature and a second temperature. The step of incubating a single continuous aqueous volume may include cycling between a first temperature and a second temperature at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 65 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times, preferably at least 20 times. The step of incubating a single continuous aqueous volume may include cycling between a first temperature and a second temperature less than 40 times, less than 35 times, less than 30 times, less than 29 times, less than 25 times. Incubating a single continuous aqueous volume may include cycling between a first temperature and a second temperature 2-100 times, 5-80 times, 10-70 times, 20-60 times, or 30-60 times. Incubating a single continuous aqueous volume may include cycling between a first temperature and a second temperature 2-20 times, 5-29 times, 61-100 times, or 65-80 times.

[0058] The method may further comprise a step of amplifying a DNA template molecule to generate a double-stranded DNA molecule prior to step (a) (i.e., contacting the double-stranded DNA molecule with an endonuclease, a ligase, and a first and second adaptor molecule). Thus, the present invention provides a method for producing a closed linear DNA product, comprising: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adapter molecules to form a single contiguous aqueous volume; and (c) incubating the single contiguous aqueous volume to produce a closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by a first adaptor molecule and at a second end by a second adaptor molecule. The present invention provides a method comprising:

[0059] The amplifying step may be carried out by in vitro amplification or in vivo amplification. Preferably, the amplifying step is carried out by in vitro amplification. For example, the amplifying step may be carried out by rolling circle amplification (RCA), MALBAC, conventional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA). Preferably, the amplifying step is carried out by rolling circle amplification. Thus, the present invention provides a method for producing a closed linear DNA product, comprising: (a) amplifying a DNA template molecule that includes at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule, where the DNA template molecule is amplified by rolling circle amplification; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; and (c) incubating the single contiguous aqueous volume to produce a closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by a first adaptor molecule and at a second end by a second adaptor molecule. The present invention provides a method comprising:

[0060] Rolling circle amplification may be performed without primers or in the presence of a primer or primers. For example, the primers may be synthetic primers. The primers may be random primers. Rolling circle amplification may be performed in the presence of a primase. The primase may be TthPrimPol. Preferably, when rolling circle amplification is performed without primers, it is performed in the presence of a primase such as TthPrimPol. Similarly, when primers are used in the amplification reaction, no primase is used. Double-stranded DNA products may be generated by rolling circle amplification in vitro under isothermal conditions using a suitable nucleic acid polymerase such as Phi29 DNA polymerase.

[0061] In the method described herein, the DNA template molecule may comprise at least one cleavable target sequence. The cleavable target sequence may be an endonuclease target sequence. Thus, the DNA template molecule may comprise at least one endonuclease target sequence. Preferably, the DNA template molecule comprises at least two endonuclease target sequences. The endonuclease target sequences may be the same or different. Preferably, at least one endonuclease target sequence is a restriction endonuclease target sequence. Different restriction endonuclease target sequences may be known to those skilled in the art. The cleavable target sequence may be a type IIS restriction endonuclease target sequence. For example, restriction endonuclease target sequences include BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, Btg ZI, BtsCI, BtsI-v2, BtsMutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, The target sequence may be FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI.The at least one cleavable sequence (e.g., an endonuclease target sequence) may be a naturally occurring cleavable sequence (i.e., a cleavable sequence present in the template molecule), or alternatively, the at least one cleavable sequence (e.g., an endonuclease target sequence) may be introduced into the DNA template molecule prior to producing the closed linear DNA product.

[0062] The endonuclease may be a restriction enzyme endonuclease. The endonuclease may be a type IIS restriction enzyme. The endonuclease may be any enzyme that recognizes a DNA sequence and cleaves outside the recognition sequence. For example, endonucleases include BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, A juI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, Bfu AI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, B seNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, Bs pTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI-v2, Bts MutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI , HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI restriction enzymes.

[0063] The ligase may be a DNA ligase such as T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase.

[0064] The DNA template molecule used in the methods described herein may be single-stranded or double-stranded. Preferably, the DNA template molecule is double-stranded. The DNA template molecule may be a naturally occurring circular DNA molecule. For example, the DNA template molecule may be (i) a plasmid, (ii) a minicircle, (iii) a cosmid, (iv) a bacterial artificial chromosome (BAC), or (v) a molecular inversion probe (MIP). The DNA template molecule may be an enzymatically produced circular DNA molecule. For example, the DNA template molecule may be (i) a circular DNA molecule obtained from a recombinase reaction, preferably a Cre recombinase reaction, or (ii) a circular DNA molecule obtained using a ligase reaction, preferably Golden Gate assembly. The DNA template molecule may be an enzymatically produced covalently closed linear DNA molecule. For example, the DNA template molecule may be (i) a DNA molecule treated with TelN protelomerase, or (ii) a DNA molecule generated by ligation of the DNA ends with an adaptor. The DNA template molecule may include double-stranded and single-stranded elements. For example, the template DNA molecule may include a double-stranded DNA and a single-stranded hairpin loop.

[0065] The DNA template molecule may be linear. When the DNA template molecule is linear, it may be circularized prior to amplification (e.g., rolling circle amplification) to generate a DNA template molecule suitable for use in the methods described herein.

[0066] The template DNA molecule may include a cassette. The cassette may be a mammalian expression cassette. The cassette may further include a promoter. The promoter may be a CMV promoter. The cassette may further include an enhancer. The cassette may further include a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further include a homopolymer sequence. The cassette may further include a LoxP sequence, preferably two LoxP sequences. When the two LoxP sequences are in the same orientation, the DNA sequence between the two LoxP sequences is excised as a circular loop of DNA. When the two LoxP sequences are in the opposite orientation, the DNA sequence between the two LoxP sequences is inverted. Thus, preferably, the two LoxP sequences are in the same orientation (i.e., in the same direction) in the template DNA molecule.

[0067] The DNA template molecule may contain a homopolymer sequence at the 5' or 3' end, or at both the 5' and 3' ends. The homopolymer sequence may be added to the DNA template molecule prior to circularization. The homopolymer sequence may be a polyA, polyC, polyG, or polyT sequence. The homopolymer sequence may be 3-200 nucleotides in length. The homopolymer sequence may be used to facilitate purification of the linear double-stranded DNA product, in which case the homopolymer sequence may be between 4-12 nucleotides, or between 5-10 nucleotides in length. The homopolymer sequence may be used to improve expression of mRNA, in which case the homopolymer sequence may be between 10-200 nucleotides, preferably between 80-150 nucleotides in length. The length of the homopolymer sequence can be at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 110 nucleotides, at least 120 nucleotides, at least 130 nucleotides, at least 140 nucleotides, at least 150 nucleotides, at least 160 nucleotides, at least 170 nucleotides, at least 180 nucleotides, at least 190 nucleotides, or at least 200 nucleotides.Preferably, the homopolymer sequence is at least 100 nucleotides long.More preferably, the homopolymer sequence is at least 120 nucleotides long.For example, the homopolymer sequence can include a polyA sequence of at least 120 nucleotides long.

[0068] The method may further comprise a heat inactivation step (after the amplification step and prior to the step of contacting the double stranded DNA molecule with an endonuclease, a ligase and a first and second adaptor molecule). Thus, the present invention provides a method for producing a closed linear DNA product, comprising: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) thermally quenching the reaction of step (a); (c) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adapter molecules to form a single contiguous aqueous volume; and (d) incubating the single contiguous aqueous volume to produce a closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by a first adaptor molecule and at a second end by a second adaptor molecule. The present invention provides a method comprising:

[0069] Preferably, the amplification is rolling circle amplification.

[0070] The heat inactivation step can be carried out under conditions sufficient to inactivate the reagents used during the amplification reaction. The heat inactivation step can be carried out at a temperature of at least 50° C., at least 55° C., at least 60° C., at least 65° C., at least 70° C., at least 75° C., at least 80° C., at least 85° C., at least 90° C., at least 95° C., or at least 100° C. The heat inactivation step can be carried out for at least 1 minute, at least 3 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, or at least 20 minutes.

[0071] In the method described herein, after the amplification step, the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and a first and a second adaptor molecule to form a single continuous aqueous volume can be performed without purifying the amplification reaction product. That is, the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and a first and a second adaptor molecule can be performed directly after the amplification step. The step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and a first and a second adaptor molecule can be performed directly after the heat inactivation step.

[0072] The method may further comprise the step of purifying the closed linear DNA product after the step of incubating the single continuous aqueous volume.

[0073] The method may further comprise a nuclease digestion step after the step of incubating the single continuous aqueous volume. The nuclease digestion may be an exonuclease digestion, such as exonuclease I and / or exonuclease III digestion. The nuclease digestion step may be before or after the purification step. This step allows for the removal of double-stranded DNA molecules and / or adapter molecules that were not used during the performance of the method. Thus, the method comprises: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (c) incubating the single contiguous aqueous volume to produce a closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by a first adaptor molecule and at a second end by a second adaptor molecule; and (d) incubating the single contiguous aqueous volume with a nuclease (e.g., an exonuclease) may include.

[0074] In the method described herein, after the amplification step, the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and a first and a second adaptor molecule to form a single continuous aqueous volume can be performed without purifying the product of the amplification reaction. That is, the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and a first and a second adaptor molecule can be performed directly after the amplification step. The step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and a first and a second adaptor molecule can be performed directly after the heat inactivation step.

[0075] The method comprises: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (c) incubating the single contiguous aqueous volume to produce a closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by a first adaptor molecule and at a second end by a second adaptor molecule; (d) purifying the closed linear DNA product; and (e) incubating the purified product of step (d) with a nuclease (e.g., an exonuclease). Includes.

[0076] The method comprises: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) thermally quenching the reaction of step (a); (c) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (d) incubating the single contiguous aqueous volume to produce a closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by a first adaptor molecule and closed at a second end by a second adaptor molecule; (e) purifying the closed linear DNA product; and (f) incubating the purified product of step (d) with a nuclease (e.g., an exonuclease); Includes.

[0077] The method comprises: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (c) incubating the single contiguous aqueous volume to produce a closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by a first adaptor molecule and at a second end by a second adaptor molecule; (d) incubating the single contiguous aqueous volume with a nuclease (e.g., an exonuclease); and (e) purifying the closed linear DNA product; Includes.

[0078] The method comprises: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) thermally quenching the reaction of step (a); (c) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (d) incubating the single contiguous aqueous volume to produce a closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by a first adaptor molecule and at a second end by a second adaptor molecule; (e) incubating the single contiguous aqueous volume with a nuclease (e.g., an exonuclease); and (f) purifying the closed linear DNA product; Includes.

[0079] The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) with the nuclease can be carried out at a temperature of 5-90°C, 10-80°C, 15-70°C, 20-60°C, 25-50°C, 30-45°C, or 35°C-40°C. The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) with the nuclease can be carried out for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes. The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) with the nuclease may be carried out at two different temperatures. For example, the step of incubating the single contiguous aqueous volume (or the purified product of step (d)) with the nuclease can be carried out at a temperature of 15-40°C for 10-60 minutes, followed by a temperature of 60-90°C for 10-30 minutes. Higher temperatures generally inactivate nucleases (e.g., exonucleases). Thus, the method further provides a step of inactivating the nuclease (e.g., exonuclease). The step of incubating the single continuous aqueous volume (or the purified product of step (d)) with the nuclease can be performed at 37°C for 30 minutes and at 80°C for 20 minutes. Preferably, the step of inactivating the nuclease (e.g., exonuclease) is performed at a temperature of 70-80°C. The step of inactivating the nuclease (e.g., exonuclease) can be performed for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, or at least 30 minutes. Preferably, the step of inactivating the nuclease (e.g., exonuclease) is performed for at least 5 minutes.

[0080] The method may be a cell-free method.

[0081] The closed linear DNA product may be partially double-stranded or partially single-stranded. The closed linear DNA product may contain double-stranded and single-stranded portions.

[0082] The closed linear DNA product may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, such as a gene encoding a protein. The cassette may comprise a promoter and at least a portion of a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosome binding site, and a translation termination sequence. The cassette may further comprise a sequence that aids in the expression of the protein, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) can be used for CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode a CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymer sequence, such as a polyA, polyC, polyT or polyG sequence. The homopolymer sequence may be 3-200 nucleotides in length. The homopolymer sequence may be used to facilitate purification of the cassette, in which case the homopolymer sequence may be 4-12 nucleotides in length, or 5-10 nucleotides in length. The homopolymer sequence may be used to improve expression of mRNA, in which case the homopolymer sequence may be 10-200 nucleotides in length, preferably 80-150 nucleotides in length.The homopolymer sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. For example, the homopolymer sequence may include a polyA sequence of at least 120 nucleotides.

[0083] The closed linear DNA product may comprise a spacer that is at least 10 base pairs long, at least 20 base pairs long, at least 30 base pairs long, at least 40 base pairs long, at least 50 base pairs long, at least 60 base pairs long, at least 70 base pairs long, at least 80 base pairs long, at least 90 base pairs long, at least 100 base pairs long, at least 125 base pairs long, at least 150 base pairs long, at least 175 base pairs long, or at least 200 base pairs long.

[0084] The closed linear DNA product may contain inverted terminal repeat sequences.

[0085] The closed linear DNA product is at least 50 base pairs long, at least 100 base pairs long, at least 250 base pairs long, at least 500 base pairs long, at least 1000 base pairs long, at least 2000 base pairs long, at least 3000 base pairs long, at least 4000 base pairs long, at least 5000 base pairs long, at least 6000 base pairs long, at least 7000 base pairs long, at least 8000 base pairs long, at least 9000 base pairs long, at least 10000 base pairs long, at least 11000 base pairs long, at least 12000 base pairs long, at least 13000 base pairs long, at least 14000 base pairs long, or at least 15000 base pairs long. Preferably, the closed linear DNA product is at least 50 base pairs long.

[0086] The double-stranded DNA molecule can be circular or branched.

[0087] The double-stranded DNA molecule may not contain an adaptor. The double-stranded DNA molecule may not contain a hairpin, loop or stem-loop structure.

[0088] The double stranded DNA molecule may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise a promoter and at least a portion of a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosome binding site and a translation termination sequence. The cassette may further comprise a sequence that aids in the expression of the protein, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) can be used for CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode a CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymer sequence, such as a polyA, polyC, polyT or polyG sequence. The homopolymer sequence is 3-200 nucleotides in length. The homopolymer sequence may be used to facilitate purification of the cassette, in which case the length of the homopolymer sequence may be between 4-12 nucleotides, or between 5-10 nucleotides. The homopolymer sequence may be used to improve mRNA expression, in which case the length of the homopolymer sequence may be between 10-200 nucleotides, preferably between 80-150 nucleotides.The homopolymer sequence may be at least 10 nucleotides long, at least 20 nucleotides long, at least 30 nucleotides long, at least 40 nucleotides long, at least 50 nucleotides long, at least 60 nucleotides long, at least 70 nucleotides long, at least 80 nucleotides long, at least 90 nucleotides long, at least 100 nucleotides long, at least 110 nucleotides long, at least 120 nucleotides long, at least 130 nucleotides long, at least 140 nucleotides long, at least 150 nucleotides long, at least 160 nucleotides long, at least 170 nucleotides long, at least 180 nucleotides long, at least 190 nucleotides long, or at least 200 nucleotides long. Preferably, the homopolymer sequence is at least 100 nucleotides long. More preferably, the homopolymer sequence is at least 120 nucleotides long. For example, the homopolymer sequence may include a polyA sequence of at least 120 nucleotides.

[0089] The double-stranded DNA molecule may comprise a spacer. The spacer is at least 10 base pairs long, at least 20 base pairs long, at least 30 base pairs long, at least 40 base pairs long, at least 50 base pairs long, at least 60 base pairs long, at least 70 base pairs long, at least 80 base pairs long, at least 90 base pairs long, at least 100 base pairs long, at least 125 base pairs long, at least 150 base pairs long, at least 175 base pairs long, or at least 200 base pairs long. The spacer can improve the amplification rate of the double-stranded DNA molecule.

[0090] The double-stranded DNA molecule is at least 50 base pairs long, at least 100 base pairs long, at least 250 base pairs long, at least 500 base pairs long, at least 1000 base pairs long, at least 2000 base pairs long, at least 3000 base pairs long, at least 4000 base pairs long, at least 5000 base pairs long, at least 6000 base pairs long, at least 7000 base pairs long, at least 8000 base pairs long, at least 9000 base pairs long, at least 10000 base pairs long, at least 11000 base pairs long, at least 12000 base pairs long, at least 13000 base pairs long, at least 14000 base pairs long, or at least 15000 base pairs long.Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0091] The double stranded DNA molecule may comprise one or more cleavable (e.g., endonuclease) target sequences. The double stranded DNA molecule may comprise two cleavable (e.g., endonuclease) target sequences. The one or more cleavable (e.g., endonuclease) target sequences may be type IIS endonuclease target sequences. The one or more cleavable (e.g., endonuclease) target sequences include BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I , AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, BceAI, BcgI, Bci VI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseM I, BseMII, BseNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI- v2, BtsMutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, The target sequence may be GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI.

[0092] The double-stranded DNA molecule may be the product of amplification. Preferably, the amplification is rolling circle amplification.

[0093] The linear double-stranded region (e.g., the linear portion of the double-stranded molecule) can be at least 50 base pairs long, at least 100 base pairs long, at least 250 base pairs long, at least 500 base pairs long, at least 1000 base pairs long, at least 2000 base pairs long, at least 3000 base pairs long, at least 4000 base pairs long, at least 5000 base pairs long, at least 6000 base pairs long, at least 7000 base pairs long, at least 8000 base pairs long, at least 9000 base pairs long, at least 10000 base pairs long, at least 11000 base pairs long, at least 12000 base pairs long, at least 13000 base pairs long, at least 14000 base pairs long, or at least 15000 base pairs long.Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0094] A linear double-stranded region (e.g., the linear portion of a double-stranded molecule) may contain a sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a sequence of a double-stranded DNA molecule.

[0095] The first and second ends of the linear double-stranded region (e.g., the linear portion of the double-stranded molecule) may be resistant to nuclease digestion. Preferably, the first and second ends of the linear double-stranded region are resistant to exonuclease digestion, such as exonuclease III digestion and / or exonuclease I digestion.

[0096] The linear double-stranded region may include a 3'-OH group at the first end and / or the second end. The 3'-OH group facilitates ligation to a first and / or second adaptor molecule(s) (which may include a 5' phosphate). The linear double-stranded region may include a 5' phosphate at the first end and / or the second end. The 5' phosphate facilitates ligation to a first and / or second adaptor molecule(s) (which may include a 3'-OH group).

[0097] A linear double-stranded region (e.g., the linear portion of a double-stranded molecule) may comprise an overhang. For example, a linear double-stranded region may comprise a 5' overhang or a 3' overhang. A linear double-stranded region may comprise a blunt end(s). A linear double-stranded region may comprise a 5' overhang and a blunt end, two 5' overhangs, a 3' overhang and a blunt end, two 3' overhangs, or a 5' overhang and a 3' overhang. An overhang may have at least 3 nucleotides (preferably 4 to 8 nucleotides). An overhang may be present on the sense strand or the antisense strand of a linear double-stranded region.

[0098] The linear portion of the double-stranded DNA molecule (e.g., the linear portion of the double-stranded molecule) can be at least 50 base pairs long, at least 100 base pairs long, at least 250 base pairs long, at least 500 base pairs long, at least 1000 base pairs long, at least 2000 base pairs long, at least 3000 base pairs long, at least 4000 base pairs long, at least 5000 base pairs long, at least 6000 base pairs long, at least 7000 base pairs long, at least 8000 base pairs long, at least 9000 base pairs long, at least 10000 base pairs long, at least 11000 base pairs long, at least 12000 base pairs long, at least 13000 base pairs long, at least 14000 base pairs long, or at least 15000 base pairs long.Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0099] The first adaptor molecule and / or the second adaptor molecule can be a synthetic adaptor molecule.

[0100] The first adaptor molecule may be a nucleic acid adaptor molecule. The second adaptor molecule may be a nucleic acid adaptor molecule. The first adaptor molecule and / or the second adaptor molecule may comprise a self-complementary element that forms a loop, such as a hairpin loop or a stem loop. Thus, the first adaptor molecule may comprise a hairpin or a stem loop. The second adaptor molecule may comprise a hairpin or a stem loop. Both the first and second adaptor molecules may comprise a hairpin or a stem loop. Each of the adaptor molecules may comprise a double-stranded portion comprising a sense strand and an antisense strand, the sense strand and the antisense strand being linked by a hairpin such that the sense strand hybridizes to the antisense strand. The double-stranded portion of the adaptor may comprise a 3' or 5' overhang of at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, or at least 5 nucleotides. Preferably, the 3' or 5' overhang is 4 to 8 nucleotides. Each end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule) may include a 3' or 5' overhang. A portion of the first adaptor molecule (e.g., an overhang) may be complementary to a first end of the linear double-stranded region. A portion of the second adaptor molecule may be complementary to a second end of the linear double-stranded region.

[0101] A closed linear DNA product can be a covalently closed linear DNA product. Thus, in embodiments where the adapter molecule comprises a loop (e.g., a hairpin), the adapter molecule closes the ends of the linear double-stranded region to form a covalently closed linear DNA product.

[0102] The present invention provides a method for producing a covalently closed linear DNA product, comprising the steps of: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume, where the first and second adaptor molecules are each a nucleic acid adaptor molecule comprising a hairpin; and (b) incubating a single contiguous aqueous volume to produce a covalently closed linear DNA product, wherein the covalently closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, the linear double-stranded region being closed at a first end by the first adaptor molecule and closed at a second end by the second adaptor molecule, and wherein (i) the first adaptor molecule comprises an overhang that is complementary to and anneals to the overhang at the first end of the linear double-stranded region, thereby closing the first end of the linear double-stranded region, and (ii) the second adaptor molecule comprises an overhang that is complementary to and anneals to the overhang at the second end of the linear double-stranded region. The present invention provides a method comprising:

[0103] The present invention provides a method for producing a covalently closed linear DNA product, comprising the steps of: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume, where the first and second adaptor molecules are each a nucleic acid adaptor molecule comprising a hairpin; and (b) incubating the single contiguous aqueous volume to produce a covalently closed linear DNA product, wherein the covalently closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, the linear double-stranded region being closed at a first end by a first adaptor molecule and closed at a second end by a second adaptor molecule, (i) the first adaptor molecule being complementary to an overhang at the first end of the linear double-stranded region, and comprises an overhang that is complementary to and anneals to the overhang at the second end of the linear double-stranded region, thereby closing the first end of the linear double-stranded region, and (ii) the second adapter molecule comprises an overhang that is complementary to and anneals to the overhang at the second end of the linear double-stranded region, thereby closing the second end of the linear double-stranded region, and the first adapter molecule is ligated to the first end of the linear double-stranded region, and the second adapter molecule is ligated to the second end of the linear double-stranded region. The present invention provides a method comprising:

[0104] The present invention provides a method for producing a covalently closed linear DNA product, comprising the steps of: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume, where the first and second adaptor molecules are each a nucleic acid adaptor molecule comprising a hairpin; and (b) incubating the single contiguous aqueous volume to produce a covalently closed linear DNA product, wherein the covalently closed linear DNA product comprises a linear portion of a double-stranded DNA molecule, the linear portion of the double-stranded DNA molecule being closed at a first end by a first adaptor molecule and closed at a second end by a second adaptor molecule, (i) the first adaptor molecule being complementary to, and anneals to, an overhang at the first end of the linear portion of the double-stranded DNA molecule; (ii) a second adaptor molecule includes an overhang that is complementary to and anneals to the overhang at the second end of the linear portion of the double-stranded DNA molecule, thereby closing the second end of the linear portion of the double-stranded DNA molecule, and the first adaptor molecule is ligated to the first end of the linear portion of the double-stranded DNA molecule, and a second adaptor molecule is ligated to the second end of the linear portion of the double-stranded DNA molecule. The present invention provides a method comprising:

[0105] The first adaptor molecule and / or the second adaptor molecule do not have to be plasmid or vector DNA.

[0106] The first and / or second adaptor molecules may comprise a single-stranded portion. The single-stranded portion may form a hairpin or a stem loop. Thus, the first and / or second adaptor molecules may comprise a loop portion. The single-stranded portion may comprise less than 10 nucleotides, 9 nucleotides, 8 nucleotides, 7 nucleotides, 6 nucleotides, 5 nucleotides, 4 nucleotides, 3 nucleotides, 2 nucleotides. Preferably, the single-stranded portion may comprise 5 nucleotides.

[0107] The first adaptor molecule and / or the second adaptor molecule may comprise a double-stranded portion. The double-stranded portion may comprise less than 50 base pairs, less than 45 base pairs, less than 40 base pairs, less than 35 base pairs, less than 30 base pairs, less than 25 base pairs, less than 20 base pairs, less than 15 base pairs, or less than 10 base pairs. The double-stranded portion may comprise at least 5 base pairs, at least 6 base pairs, at least 7 base pairs, at least 8 base pairs, at least 9 base pairs, at least 10 base pairs, at least 11 base pairs, at least 12 base pairs, at least 13 base pairs, at least 14 base pairs, or at least 15 base pairs.

[0108] The first adaptor molecule and / or the second adaptor molecule may comprise a 5' phosphate. The 5' phosphate may facilitate ligation to a linear double-stranded region (which may comprise a 3'-OH group at the first and / or second end). The first adaptor molecule and / or the second adaptor molecule may comprise a 3'-OH. The 3'-OH may facilitate ligation to a linear double-stranded region (which may comprise a 5' phosphate at the first and / or second end).

[0109] The first and / or second adaptor molecule may comprise a sequence of SEQ ID NO:1 or a portion thereof. The first and / or second adaptor molecule may comprise at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of SEQ ID NO:1. The double-stranded portion of the first and / or second adaptor molecule may comprise a sequence of SEQ ID NO:2 or a portion thereof. The double-stranded portion of the first and / or second adaptor molecule may comprise at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 consecutive nucleotides of SEQ ID NO:2. The single-stranded portion of the first and / or second adaptor molecule may comprise a sequence of ACTCA. The single-stranded portion of the first and / or second adaptor molecule may comprise at least 1, at least 2, at least 3, at least 4, or at least 5 consecutive nucleotides of the sequence ACTCA. The first and second adaptor molecules may comprise the same nucleic acid sequence. The first and second adaptor molecules may comprise different nucleic acid sequences.

[0110] The first adaptor molecule may comprise a portion that is complementary to a first end of the linear double-stranded region (or linear portion of the double-stranded DNA molecule). The second adaptor molecule may comprise a portion that is complementary to a second end of the linear double-stranded region (or linear portion of the double-stranded DNA molecule). The first adaptor molecule may comprise a portion that anneals to a first end of the linear double-stranded region (or linear portion of the double-stranded DNA molecule). The second adaptor molecule may comprise a portion that anneals to a second end of the linear double-stranded region (or linear portion of the double-stranded DNA molecule). The first adaptor molecule may comprise a portion that anneals complementarily to a first end of the linear double-stranded region (or linear portion of the double-stranded DNA molecule). The second adaptor molecule may comprise a portion that anneals complementarily to a second end of the linear double-stranded region (or linear portion of the double-stranded DNA molecule).

[0111] The portion that is complementary or anneals to the first or second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule) may be the 5'-overhang or 3'-overhang of the first adapter molecule and / or the second adapter molecule. The overhang of the first adapter molecule may be complementary to the first end of the linear double-stranded region, and / or the overhang of the second adapter molecule may be complementary to the second end of the double-stranded region. The overhang of the first adapter molecule may be annealed to the first end of the linear double-stranded region, and / or the overhang of the second adapter molecule may be annealed to the second end of the linear double-stranded region. The overhang of the first adapter molecule may be complementary to and annealed to the first end of the linear double-stranded region, and / or the overhang of the second adapter molecule may be complementary to and annealed to the second end of the linear double-stranded region.

[0112] The first adaptor molecule and / or the second adaptor molecule may not contain a type IIS endonuclease target sequence. The first adapter molecule and / or the second adapter molecule may be BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQ I, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI-v2, Bt sMutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI, It may not contain the HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI target sequences.

[0113] The first adaptor molecule and / or the second adaptor molecule may comprise one or more locked nucleic acids (LNAs).

[0114] The first adaptor molecule and / or the second adaptor molecule may include one or more protected nucleotides (i.e., nuclease-resistant nucleotides), such as phosphorothioated nucleotides. The protected nucleotides may be located in a single-stranded portion (e.g., a hairpin portion) or a double-stranded portion. The protected nucleotides may be located in an overhang portion of the adaptor molecule.

[0115] The closed linear DNA product may contain multiple phosphorothioated nucleotides at the internal position of each strand.For example, the closed linear DNA product may contain at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g., phosphorothioated nucleotides) at the internal position of each strand.Preferably, the closed linear DNA product contains at least 2 protected nucleotides (e.g., phosphorothioated nucleotides) at the internal position of each strand.

[0116] The internal position does not have to be located between the second and the last nucleotide of the closed linear DNA product.

[0117] A linear double-stranded region (or a linear portion of a double-stranded molecule) may contain a plurality of phosphorothioated nucleotides at internal positions of each strand. For example, a linear double-stranded region (or a linear portion of a double-stranded molecule) may contain at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g., phosphorothioated nucleotides) at internal positions of each strand. Preferably, the linear double-stranded region (or the linear portion of the double-stranded molecule) contains at least two protected nucleotides (e.g., phosphorothioated nucleotides) at an internal position of each strand, which does not have to be located between the second and last nucleotide of the linear double-stranded region (or the linear portion of the double-stranded molecule).

[0118] Nucleotides resistant to exonuclease digestion (i.e., protected nucleotides) suitable for use in the methods described herein may be phosphorothioated nucleotides. For example, phosphorothioated nucleotides may be α-S-dATP (i.e., 2'-deoxyadenosine-5'-(α-thio)-triphosphate), α-S-dCTP (i.e., 2'-deoxycytidine-5'-(α-thio)-triphosphate), α-S-dGTP (i.e., 2'-deoxyguanosine-5'-(α-thio)-triphosphate), α-S-dTTP (i.e., 2'-deoxythymidine-5'-(α-thio)-triphosphate), α-S-dUTP (i.e., 2'-deoxyuridine-5'-(α-thio)-triphosphate), and / or uridine 2',3'-cyclophosphorothioate.

[0119] The phosphorothioated nucleotides may be Sp-isomers, Rp-isomers, or a mixture of both Sp- and Rp-isomers.

[0120] Nucleotides that are resistant to exonuclease digestion (i.e., protected nucleotides) may be 2'-O-methyl nucleotides or 2'-O-methoxyethyl (MOE) nucleotides. For example, MOE nucleotides may be 2'-O-methoxy-ethyl guanosine, 2'-O-methoxy-ethyl cytidine, 2'-O-methoxy-ethyl adenosine, and / or 2'-O-methoxy-ethyl thymidine.

[0121] The first end of the linear double-stranded region may be complementary to a portion of a first adaptor molecule. The second end of the linear double-stranded region may be complementary to a portion of a second adaptor molecule. The first and / or second ends of the linear double-stranded region may be generated by endonuclease digestion.

[0122] The first adaptor molecule and / or the second adaptor molecule may comprise a functional moiety. The functional moiety may be a binding molecule, a targeting sequence, or a probe.

[0123] The functional moiety may be a probe. As used herein, the term "probe" refers to a fragment of DNA, RNA or DNA / RNA chimera of variable length (e.g., 3-1000 bases long) that is used to detect the presence of a target nucleotide sequence that is complementary to the sequence in the probe. Typically, a probe hybridizes to a single-stranded nucleic acid of a base sequence that allows probe-target base pairing due to the complementarity between the probe and the target. Thus, the functional moiety may be a DNA sequence, an RNA sequence, or a DNA / RNA chimera sequence. As used herein, the term "complementary" means that the nucleotide sequences pair according to the Watson / Crick pairing rules. For example, the sequence 5'-GCGGTCCCA-3' has a complementary sequence of 5'-TGGGACCGC-3'. The complementary sequence may also be an RNA sequence that is complementary to the DNA sequence.

[0124] The functional moiety may be a binding molecule. The term "binding molecule" refers to any molecule capable of binding to a linear DNA product as described herein and / or capable of binding to a further molecule or target. The binding molecule may be a protein, a polypeptide, or a peptide. The binding molecule may be an antibody, such as a monoclonal or polyclonal antibody. The binding molecule may be an antibody fragment.

[0125] The functional moiety can facilitate detection of the DNA product by binding to a capture molecule (e.g., a capture antibody bound by a protein-protein interaction). The functional moiety can bind to a cellular target, e.g., a cellular receptor.

[0126] The functional moiety may be a label. A "label" may be any chemical entity that allows detection of a double-stranded nucleic acid molecule by physical, chemical and / or biological means. The label may be either a chromophore, a fluorophore and / or a radioactive molecule.

[0127] The functional part may be a targeting sequence. The targeting sequence is a DNA or RNA fragment, which may vary in length, and is used to target the DNA product to a specific location in the cell. The targeting sequence can be used to enhance the transfection efficiency of non-viral gene transfer by promoting the nuclear uptake of closed linear DNA products. For example, the targeting sequence may be a DNA nuclear targeting sequence (i.e., a recognition sequence for endogenous DNA binding proteins), such as the SV40 enhancer sequence (preferably downstream of the cassette).

[0128] To facilitate detection and / or quantification of the DNA product, the functional moiety may comprise a fluorophore, a radioactive compound or a barcode.

[0129] A signal corresponding to the presence, absence and / or level of a closed linear double-stranded DNA product can be measured using a barcode. The barcode may comprise at least one binding moiety linked to a barcode portion, where the barcode portion comprises at least one nucleotide (i.e., the barcode portion comprises a nucleotide sequence at least one nucleotide long), and the binding moiety can bind to a 3' overhang, a 5' overhang or a blunt end of the closed linear double-stranded DNA product. The binding site can bind to the 3' end and / or the 5' end of the closed linear double-stranded DNA product. The signal can be measured by determining the presence, absence and / or level of the barcode portion (e.g., by sequencing or PCR). The barcoded portion may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 nucleotides. The barcode may comprise at least two binding moieties (e.g., a first binding moiety and a second binding moiety). For example, a first binding moiety linked to a first barcode portion can bind to the 3' end of the closed linear double-stranded DNA product, and a second binding moiety linked to a second barcode portion can bind to the 5' end of the closed linear double-stranded DNA product. The 3' and 5' ends can include a 3' overhang, a 5' overhang, or a blunt end.

[0130] A signal corresponding to the presence, absence, and / or level of the closed linear DNA product can be measured using a fluorophore (i.e., a fluorescently labeled molecule) attached or bound to the 3' overhang, 5' overhang, or blunt end of the closed linear DNA product. The signal can be measured by flow cytometry and / or fluorescence activated cell sorting.

[0131] The functional moiety can also facilitate DNA sequencing. For example, the functional moiety can be a sequence adapter. The term "sequence adapter" refers to the Illumina (登録商標) (e.g., HiSeq (商標), MiSeq (商標) and / or Genome Analyzer (商標) Sequencing System), Oxford Nanopore (商標) Technologies (e.g., MinION sequencing system), Ion Torrent (商標) (For example, Ion PGM (商標) and / or Ion Proton (商標) sequencing systems); Pacific Biosciences (e.g., PACBIO RS II sequencing system); Life Technologies (商標) The term "sequencing platform" is intended to encompass one or more nucleic acid domains that contain at least a portion of a nucleic acid sequence (or a complement thereof) utilized by a sequencing platform of interest, such as a sequencing platform provided by Axis (e.g., the SOLiD sequencing system), Roche (e.g., the 454 GS FLX+ and / or GS Junior sequencing systems), or other sequencing platform of interest.

[0132] The first and / or second adaptor molecules may comprise inverted terminal repeat sequences. The inverted terminal repeat sequences of the first and second adaptor molecules may be symmetric (i.e., have the same symmetric three-dimensional structure relative to each other) or asymmetric (i.e., have different three-dimensional structures relative to each other). The inverted terminal repeat sequences of the first and second adaptor molecules may be from the same or different serotypes. The inverted terminal repeat sequences may comprise a terminal resolution site and a Rep binding site.

[0133] The first adaptor molecule and / or the second adaptor molecule may comprise an aptamer.

[0134] The first adaptor molecule and / or the second adaptor molecule can confer resistance to nuclease digestion, such as exonuclease digestion (e.g., exonuclease I and / or exonuclease III digestion).

[0135] Closure at a first end of the linear double-stranded region (or linear portion of the double-stranded DNA molecule) may generate a first closed end of a closed linear DNA product. Closure at a second end of the linear double-stranded region (or linear portion of the double-stranded DNA molecule) may generate a second closed end of a closed linear DNA product. The first and second closed ends of the closed linear DNA product may be resistant to nuclease digestion. The nuclease digestion may be exonuclease digestion. Preferably, the nuclease digestion is exonuclease III digestion and / or exonuclease I digestion.

[0136] 2. Method for producing linear DNA products containing nuclease-resistant nucleotides The methods described herein can be used to generate linear DNA products that contain nuclease-resistant (ie, protected nucleotides).

[0137] The present invention provides a method for producing a linear deoxyribonucleic acid (DNA) product, comprising the steps of: (a) contacting the double-stranded DNA molecule with an endonuclease and first and second adaptor molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, a first adaptor molecule is attached to a first end of the linear double-stranded region, and a second adaptor molecule is attached to a second end of the linear double-stranded region, and the first and second adaptor molecules are nucleic acid molecules that include one or more nuclease-resistant nucleotides (i.e., protected nucleotides); The present invention provides a method comprising:

[0138] The step of contacting the double-stranded DNA molecule with the endonuclease and the first and second adaptor molecules is preferably carried out in the presence of a ligase. Thus, the method for producing a linear DNA product may comprise the steps of: (a) contacting double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adapter molecule is attached to a first end of the linear double-stranded region, and the second adapter molecule is attached to a second end of the linear double-stranded region, and the first and second adapter molecules are nucleic acid molecules that include one or more nuclease-resistant nucleotides (i.e., protected nucleotides).

[0139] The linear DNA products produced by the methods described herein have enhanced resistance to nuclease (e.g., exonuclease) digestion, e.g., the linear DNA products have extended expression in vivo compared to linear DNA products that do not contain blocking nucleotides.

[0140] The addition of the first and / or second adapter molecule can be performed by hybridization or ligation of the adapter molecule to the end of the linear double-stranded region. Thus, the first adapter molecule can hybridize to the first end of the linear double-stranded region. The second adapter molecule can hybridize to the second end of the linear double-stranded region. The first adapter molecule is ligated to the first end of the linear double-stranded region. The second adapter molecule can be ligated to the second end of the linear double-stranded region. The addition of the first and second adapter molecules can be performed by both hybridization and ligation of the adapter molecule to the end of the linear double-stranded region. Thus, the first adapter molecule can hybridize and ligate to the first end of the linear double-stranded region. The second adapter molecule can hybridize and ligate to the second end of the linear double-stranded region. Hybridization is based on the complementarity of a portion of the first and / or second adapter molecule to the first and / or second ends of the linear double-stranded region.

[0141] The method for producing a linear DNA product may comprise the steps of: (a) contacting double-stranded DNA molecules with an endonuclease, a ligase, and first and second adapter molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to produce a linear DNA product, where the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adapter molecule is ligated to a first end of the linear double-stranded region, and a second adapter molecule is ligated to a second end of the linear double-stranded region, and the first and second adapter molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e., protected nucleotides).

[0142] The method for producing a linear DNA product may comprise the steps of: (a) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adapter molecules to form a single contiguous aqueous volume; and (b) incubating a single contiguous aqueous volume to produce a linear DNA product, where the linear DNA product comprises a linear portion of a double-stranded DNA molecule, a first adapter molecule is ligated to a first end of the linear portion of the double-stranded DNA molecule, and a second adapter molecule is ligated to a second end of the linear portion of the double-stranded DNA molecule, and the first and second adapter molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e., protected nucleotides).

[0143] As used herein, the term "complementary" means that the nucleotide sequences pair according to the Watson / Crick pairing rules. For example, the sequence 5'-GCGGTCCCA-3' has a complementary sequence of 5'-TGGGACCGC-3'. A complementary sequence can also be an RNA sequence that is complementary to a DNA sequence.

[0144] Preferably, the steps of contacting the double-stranded DNA molecule with the endonuclease, the ligase and the first and second adaptor molecules are performed in one reaction (ie, a single step).

[0145] The step of incubating the single continuous aqueous volume to generate a linear DNA product may include generating a linear portion of the double-stranded DNA molecule by digesting the double-stranded DNA molecule with an endonuclease.

[0146] The step of incubating the single continuous aqueous volume can be performed under conditions that promote the addition (or ligation) of a first and a second adaptor molecule to the linear double-stranded region to generate a linear DNA product. The addition can be performed by forming a covalent bond between the first and / or second adaptor molecule and the end(s) of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule).

[0147] The step of incubating the single continuous aqueous volume can be performed under conditions that promote digestion of the double-stranded DNA molecules and generate a linear portion of the double-stranded DNA molecules. The digestion of the double-stranded DNA molecules to generate a linear portion of the double-stranded DNA molecules can be performed at a first temperature of 1° C. to 100° C., 1° C. to 80° C., 5° C. to 70° C., 10° C. to 60° C., 15° C. to 55° C., 20° C. to 50° C., 25° C. to 45° C., 30° C. to 40° C., 35° C. to 39° C., 36° C. to 38° C., or about 37° C. The digestion is an endonuclease digestion, preferably a type IIS endonuclease digestion.

[0148] The step of incubating the single continuous aqueous solution can be performed under conditions that promote ligation of the linear double-stranded region to the first and second adaptor molecules, the ligation being performed with an efficiency of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95%. For example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the linear double-stranded region (or portion of the double-stranded DNA molecule) can be incorporated into the closed linear DNA product. Preferably, the efficiency of ligation is at least 15%.

[0149] The step of ligating the linear double-stranded region to the first and second adaptor molecules can be carried out at a second temperature of 1°C to 90°C, 2°C to 70°C, 5°C to 60°C, 8°C to 55°C, 9°C to 50°C, 10°C to 45°C, 11°C to 40°C, 12°C to 37°C, 13°C to 30°C, 14°C to 25°C, 15°C to 20°C, or about 16°C.

[0150] The step of incubating the single continuous aqueous volume may include incubating at a first temperature followed by incubation at a second temperature. The first temperature may be 1°C-100°C, 1°C-80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C-45°C, 30°C-40°C, 35°C-39°C, 36°C-38°C, or about 37°C. The second temperature may be 1°C-90°C, 2°C-70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 11°C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C, or about 16°C. Preferably, the first temperature is 35°C-39°C. Preferably, the second temperature is 14°C-18°C.

[0151] The step of incubating a single continuous aqueous volume may include cycling between a first temperature and a second temperature. The step of incubating a single continuous aqueous volume may include cycling between a first temperature and a second temperature at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 65 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times, preferably at least 20 times. The step of incubating a single continuous aqueous volume may include cycling between a first temperature and a second temperature less than 40 times, less than 35 times, less than 30 times, less than 29 times, less than 25 times. Incubating a single continuous aqueous volume may include cycling between a first temperature and a second temperature 2-100 times, 5-80 times, 10-70 times, 20-60 times, or 30-60 times. Incubating a single continuous aqueous volume may include cycling between a first temperature and a second temperature 2-20 times, 5-29 times, 61-100 times, or 65-80 times.

[0152] The step of incubating the single continuous aqueous volume can be performed isothermally. The step of incubating the single continuous aqueous volume can include incubating at a constant temperature. The constant temperature simultaneously promotes digestion of the double-stranded DNA molecule, generates a linear portion of the double-stranded DNA molecule, and ligates the linear double-stranded region to the first and second adaptor molecules. For example, the constant temperature can be 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, 37°C, 38°C, 39°C, or 40°C. Preferably, the constant temperature is 30°C. By constant temperature, it is intended that the temperature does not change significantly during the reaction. By constant temperature is meant that the temperature change during the step of incubating a single continuous aqueous volume is less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1°C. In a preferred embodiment, the temperature during the step of incubating a single continuous aqueous volume is 5°C or less, preferably 3°C or less, more preferably 1°C or less. Thus, the constant temperature may be a temperature in the range of 20°C to 30°C, 22°C to 32°C, 24°C to 34°C, 26°C to 36°C, 28°C to 38°C, 30°C to 40°C, 22°C to 28°C, 32°C to 38°C, 25°C to 35°C, 26°C to 34°C, 27°C to 33°C, 27.5°C to 32.5°C, 28°C to 32°C, 28.5°C to 31.5°C, 29°C to 31°C, or 29.5°C to 30.5°C. Preferably, the constant temperature is 27.5° C. to 32.5° C. Alternatively, the constant temperature may be in the range of 32° C. to 42° C., 33° C. to 41° C., 33° C. to 41° C., 34° C. to 40° C., 35° C. to 39° C., or 36° C. to 38° C. Preferably, the constant temperature is 34.5° C. to 39.5° C.

[0153] The first and second adaptor molecules may comprise one or more phosphorothioate nucleotides, and when the adaptor molecules are added (e.g., ligated) to the linear double-stranded region, the linear DNA product is resistant to nuclease digestion or has improved or enhanced resistance to nuclease digestion. The linear DNA product may be resistant to 3'-end exonuclease digestion (e.g., by exonuclease III) and / or 5'-end exonuclease digestion (e.g., by exonuclease VIII).

[0154] The adapter molecule may comprise multiple phosphorothioated nucleotides, for example, the adapter molecule may comprise 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, or at least 16 phosphorothioated nucleotides on each strand.

[0155] The adaptor molecule may be a nucleic acid adaptor molecule. The adaptor molecule may be double-stranded. The adaptor molecule may include a double-stranded portion.

[0156] The first adaptor molecule and / or the second adaptor molecule can comprise at least 3 base pairs, at least 4 base pairs, at least 5 base pairs, at least 6 base pairs, at least 7 base pairs, at least 8 base pairs, at least 9 base pairs, at least 10 base pairs, at least 11 base pairs, at least 12 base pairs, at least 13 base pairs, at least 14 base pairs, at least 15 base pairs, or at least 16 base pairs.

[0157] The adapter molecule may comprise multiple phosphorothioated nucleotides on each strand, for example, the adapter molecule may comprise 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, or at least 16 phosphorothioated nucleotides on each strand.

[0158] The adapter molecule may comprise multiple phosphorothioated nucleotides at an internal position of each strand. For example, the adapter molecule may comprise 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, or at least 16 phosphorothioated nucleotides at an internal position of each strand. Preferably, the adapter molecule comprises at least 2 phosphorothioated nucleotides at an internal position of each strand.

[0159] An internal position does not have to be located between the second and penultimate nucleotide of the adaptor molecule. An internal position can be anywhere in the adaptor molecule other than the last nucleotide at each strand end.

[0160] The adapter molecule may comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% protected nucleotides.

[0161] When an adaptor molecule is added to the linear double-stranded region, the linear DNA product may contain protected nucleotides (e.g., phosphorothioated nucleotides) at the 5'-end (or 5'-end region) of one or both strands. Preferably, the linear DNA product contains phosphorothioated nucleotides at the 5'-end (or 5'-end region) of one or both strands. The linear DNA product may contain phosphorothioated nucleotides at the 5'-end (or 5'-end region) of one or both strands. Since most exonucleases, such as exonuclease III, remove nucleotides from the 3'-end of a polynucleotide strand, the linear DNA product may contain protected nucleotides at the 3'-end (or 3'-end region) of one or both strands. Preferably, the linear DNA product contains phosphorothioated nucleotides at the 3'-end (or 3'-end region) of one or both strands. The linear DNA product may contain at least one phosphorothioated nucleotide at the 3'-end (or 3'-end region) and at least one phosphorothioated nucleotide at the 5'-end (or 5'-end region) of one or both strands. The linear DNA product may contain phosphorothioated nucleotides at the 3'-end (or 3'-end region) and 5'-end (or 5'-end region) of one or both strands.

[0162] The linear DNA product may further comprise a plurality of protected nucleotides (e.g., phosphorothioated nucleotides) at the internal position of each strand. For example, the linear DNA product may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g., phosphorothioated nucleotides) at the internal position of each strand. For example, phosphorothioated nucleotides) are present at the internal position of each strand. Preferably, the linear DNA product contains at least two protected nucleotides (eg, phosphorothioated nucleotides) at internal positions on each strand.

[0163] An internal position does not have to be located between the second and last nucleotide of the linear DNA product. An internal position can be anywhere in the adaptor molecule other than the last nucleotide at the end of each strand.

[0164] A linear double-stranded region (or a linear portion of a double-stranded molecule) may contain a plurality of phosphorothioated nucleotides at the internal positions of each strand. For example, a linear double-stranded region (or a linear portion of a double-stranded molecule) may have at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g., phosphorothioated nucleotides) at the internal positions of each strand. Preferably, the linear double-stranded region (or the linear portion of the double-stranded molecule) contains at least two protected nucleotides (e.g., phosphorothioated nucleotides) at internal positions of each strand. The internal positions do not have to be located between the second and the last nucleotide of the linear double-stranded region (or the linear portion of the double-stranded molecule).

[0165] The nucleotides resistant to exonuclease digestion (i.e., protected nucleotides) are at least one phosphorothioated nucleotide. For example, the at least one phosphorothioated nucleotide is α-S-dATP (i.e., 2'-deoxyadenosine-5'-(α-thio)-triphosphate), α-S-dCTP (i.e., 2'-deoxycytidine-5'-(α-thio)-triphosphate), α-S-dGTP (i.e., 2'-deoxyguanosine-5'-(α-thio)-triphosphate), α-S-dTTP (i.e., 2'-deoxythymidine-5'-(α-thio)-triphosphate), α-S-dUTP (i.e., 2'-deoxyuridine-5'-(α-thio)-triphosphate), and / or uridine 2',3'-cyclophosphorothioate.

[0166] The adapter molecule may comprise at least two phosphorothioated nucleotides, for example, the at least two phosphorothioated nucleotides are as follows: α-S-dATP and α-S-dCTP, α-S-dATP and α-S-dGTP, α-S-dATP and α-S-dTTP, α-S-dCTP and α-S-dGTP, α-S-dCTP and α-S-dTTP, or α-S-dGTP and α-S-dTTP.

[0167] The adapter molecule can include at least three phosphorothioated nucleotides. For example, the at least three phosphorothioated nucleotides can be (a) α-S-dATP, α-S-dCTP and α-S-dGTP; (b) α-S-dATP, α-S-dCTP and α-S-dTTP; (c) α-S-dATP, α-S-dGTP and α-S-dTTP; or (d) α-S-dCTP, α-S-dGTP and α-S-dTTP It is.

[0168] The adapter molecule can include at least four phosphorothioated nucleotides, for example, the at least four protected nucleotides are α-S-dATP, α-S-dCTP, α-S-dGTP and α-S-dTTP.

[0169] The phosphorothioated nucleotides may be Sp-isomers, Rp-isomers, or a mixture of both Sp- and Rp-isomers.

[0170] The nucleotides that are resistant to exonuclease digestion (i.e., protected nucleotides) may be at least one, or at least two, three, or four MOE nucleotides. For example, the MOE nucleotides may be 2'-O-methoxy-ethylguanosine, 2'-O-methoxy-ethylcytidine, 2'-O-methoxy-ethyladenosine, and / or 2'-O-methoxy-ethylthymidine.

[0171] The method may further comprise a step of amplifying a DNA template molecule to generate a double-stranded DNA molecule prior to step (a) (i.e., contacting the double-stranded DNA molecule with an endonuclease, a ligase, and a first and second adaptor molecule). Thus, the present invention provides a method for producing a linear DNA product, comprising: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; and (c) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is attached to a first end of the linear double-stranded region, and a second adaptor molecule is attached to a second end of the linear double-stranded region, and the first and second adaptor molecules are nucleic acid molecules that include one or more nuclease-resistant nucleotides (i.e., protected nucleotides); The present invention provides a method comprising:

[0172] The amplifying step can be carried out by in vitro or in vivo amplification. Preferably, the amplifying step is carried out by in vitro amplification. For example, the amplifying step can be carried out by rolling circle amplification (RCA), MALBAC, conventional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA). Preferably, the amplification is carried out by rolling circle amplification. Thus, the present invention provides a method for producing a linear DNA product, comprising: (a) amplifying a DNA template molecule that includes at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule, where the DNA template molecule is amplified by rolling circle amplification; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adapter molecules to form a single contiguous aqueous volume; and (c) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, wherein the first adapter molecule is attached to a first end of the linear double-stranded region and a second adapter molecule is attached to a second end of the linear double-stranded region, and wherein the first and second adapter molecules are nucleic acid molecules that include one or more nuclease-resistant nucleotides (i.e., protected nucleotides). The present invention provides a method for producing a linear DNA product, comprising:

[0173] Rolling circle amplification may be performed without primers or in the presence of a primer or primers. For example, the primers may be synthetic primers. The primers may be random primers. Rolling circle amplification may be performed in the presence of a primase. The primase may be TthPrimPol. Preferably, when rolling circle amplification is performed without primers, it is performed in the presence of a primase such as TthPrimPol. Similarly, when primers are used in the amplification reaction, no primase is used. Double-stranded DNA products may be generated by rolling circle amplification in vitro under isothermal conditions using a suitable nucleic acid polymerase such as Phi29 DNA polymerase.

[0174] In the method described herein, the DNA template molecule may comprise at least one cleavable target sequence. The cleavable target sequence may be an endonuclease target sequence. Thus, the DNA template molecule may comprise at least one endonuclease target sequence. Preferably, the DNA template molecule comprises at least two endonuclease target sequences. The endonuclease target sequences may be the same or different. Preferably, at least one endonuclease target sequence is a restriction endonuclease target sequence. Different restriction endonuclease target sequences may be known to those skilled in the art. The cleavable target sequence may be a type IIS restriction endonuclease target sequence. For example, restriction endonuclease target sequences include BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI , BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI-v2, B tsMutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, Gs The target sequence may be uI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI.The at least one cleavable sequence (e.g., endonuclease target sequence) may be a naturally occurring cleavable sequence (i.e., a cleavable sequence present in the template molecule). Alternatively, the at least one cleavable sequence (e.g., endonuclease target sequence) may be introduced into the DNA template molecule prior to producing the linear DNA product.

[0175] The endonuclease may be a restriction enzyme endonuclease. The endonuclease may be a type IIS restriction enzyme. The endonuclease may be any enzyme that recognizes a DNA sequence and cleaves outside the recognition sequence. For example, endonucleases include BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, BceAI, BcgI, B ciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, Btg ZI, BtsCI, BtsI-v2, BtsMutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, The restriction enzyme may be FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI.

[0176] The ligase may be a DNA ligase such as T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase.

[0177] The DNA template molecule used in the methods described herein may be single-stranded or double-stranded. Preferably, the DNA template molecule is double-stranded. The DNA template molecule may be a naturally occurring circular DNA molecule. For example, the DNA template molecule may be (i) a plasmid, (ii) a minicircle, (iii) a cosmid, (iv) a bacterial artificial chromosome (BAC), or (v) a molecular inversion probe (MIP). The DNA template molecule may be an enzymatically produced circular DNA molecule. For example, the DNA template molecule may be (i) a circular DNA molecule obtained from a recombinase reaction, preferably a Cre recombinase reaction, or (ii) a circular DNA molecule obtained using a ligase reaction, preferably Golden Gate assembly. The DNA template molecule may be an enzymatically produced covalently closed linear DNA molecule. For example, the DNA template molecule may be (i) a DNA molecule treated with TelN protelomerase, or (ii) a DNA molecule generated by ligation of the DNA ends with an adaptor. The DNA template molecule may include double-stranded and single-stranded elements. For example, the template DNA molecule may include a double-stranded DNA and a single-stranded hairpin loop.

[0178] The DNA template molecule may be linear. When the DNA template molecule is linear, the DNA template molecule may be circularized prior to amplification (e.g., rolling circle amplification) to generate a DNA template molecule suitable for use in the methods described herein.

[0179] The template DNA molecule may include a cassette. The cassette may be a mammalian expression cassette. The cassette may further include a promoter. The promoter may be a CMV promoter. The cassette may further include an enhancer. The cassette may further include a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further include a homopolymer sequence. The cassette may further include a LoxP sequence, preferably two LoxP sequences. When the two LoxP sequences are in the same orientation, the DNA sequence between the two LoxP sequences is excised as a circular loop of DNA. When the two LoxP sequences are in the opposite orientation, the DNA sequence between the two LoxP sequences is inverted. Thus, preferably, the two LoxP sequences are in the same orientation in the template DNA molecule.

[0180] The DNA template molecule may contain a homopolymer sequence at the 5' or 3' end, or at both the 5' and 3' ends. The homopolymer sequence may be added to the DNA template molecule prior to circularization. The homopolymer sequence may be a polyA, polyC, polyG, or polyT sequence. The homopolymer sequence may be 3-200 nucleotides in length. The homopolymer sequence may be used to facilitate purification of the linear DNA product, in which case the homopolymer sequence may be between 4-12 nucleotides, or between 5-10 nucleotides in length. The homopolymer sequence may be used to improve expression of mRNA, in which case the homopolymer sequence may be between 10-200 nucleotides, preferably between 80-150 nucleotides in length. The length of the homopolymer sequence can be at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 110 nucleotides, at least 120 nucleotides, at least 130 nucleotides, at least 140 nucleotides, at least 150 nucleotides, at least 160 nucleotides, at least 170 nucleotides, at least 180 nucleotides, at least 190 nucleotides, or at least 200 nucleotides.Preferably, the homopolymer sequence is at least 100 nucleotides long.More preferably, the homopolymer sequence is at least 120 nucleotides long.For example, the homopolymer sequence can include a polyA sequence of at least 120 nucleotides long.

[0181] The method may further comprise the step of purifying the linear DNA product after the step of incubating the single continuous aqueous volume.

[0182] The method may further comprise a nuclease digestion step after the step of incubating the single continuous aqueous volume. The nuclease digestion may be an exonuclease digestion, such as exonuclease I and / or exonuclease III digestion. The nuclease digestion step may be before or after the purification step. This step allows for the removal of double-stranded DNA molecules and / or adapter molecules that were not used during the performance of the method. Thus, the method of the present invention comprises: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (c) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is attached to a first end of the linear double-stranded region, and a second adaptor molecule is attached to a second end of the linear double-stranded region, and the first and second adaptor molecules are nucleic acid molecules that include one or more nuclease-resistant nucleotides (i.e., protected nucleotides); and (d) incubating the single continuous aqueous solution with a nuclease (e.g., an exonuclease) may include.

[0183] The method of the present invention comprises the steps of: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (c) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is attached to a first end of the linear double-stranded region, and a second adaptor molecule is attached to a second end of the linear double-stranded region, and the first and second adaptor molecules are nucleic acid molecules that include one or more nuclease-resistant nucleotides (i.e., protected nucleotides); (d) purifying the closed linear DNA product; and (e) incubating the purified product of step (d) with a nuclease (e.g., an exonuclease). may include.

[0184] The method of the present invention comprises the steps of: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (c) incubating the single contiguous aqueous volume to produce a linear DNA product, where the linear DNA product comprises a linear double-stranded region, where the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, where a first adaptor molecule is attached to a first end of the linear double-stranded region and a second adaptor molecule is attached to a second end of the linear double-stranded region, and where the first and second adaptor molecules are nucleic acid molecules that include one or more nuclease-resistant nucleotides (i.e., protected nucleotides); (d) incubating the single contiguous aqueous volume with a nuclease (e.g., an exonuclease); and (e) Purifying the closed linear DNA product may include.

[0185] The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) with the nuclease can be carried out at a temperature of 5-90°C, 10-80°C, 15-70°C, 20-60°C, 25-50°C, 30-45°C, or 35-40°C. The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) with the nuclease can be carried out for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes. The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) can be carried out at two different temperatures. For example, the step of incubating the single contiguous aqueous volume (or the purified product of step (d)) can be carried out at a temperature of 15-40°C for 10-60 minutes and then at a temperature of 60-90°C for 10-30 minutes. The higher temperature generally inactivates the nuclease (e.g., exonuclease). Thus, the method further provides a step of inactivating the nuclease (e.g., exonuclease). The step of incubating the single continuous aqueous volume (or the purified product of step (d)) can be performed at 37°C for 30 minutes and at 80°C for 20 minutes. Preferably, the step of inactivating the nuclease (e.g., exonuclease) is performed at a temperature of 70-80°C. The step of inactivating the nuclease (e.g., exonuclease) can be performed for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, or at least 30 minutes. Preferably, the step of inactivating the nuclease (e.g., exonuclease) is performed for at least 5 minutes.

[0186] The first adaptor molecule and / or the second adaptor molecule may comprise an overhang. The end of the linear double-stranded region may comprise a 3' or 5' overhang. A portion of the first adaptor molecule (e.g., the overhang) may be complementary to the first end of the linear double-stranded region. A portion of the second adaptor molecule may be complementary to the second end of the linear double-stranded region.

[0187] The first and second ends of the linear double-stranded region may be resistant to nuclease digestion. Preferably, the first and second ends of the linear double-stranded region are resistant to exonuclease digestion, such as exonuclease III digestion and / or exonuclease I digestion.

[0188] A linear DNA product may be partially double-stranded or partially single-stranded. A linear DNA product may contain double-stranded and single-stranded portions.

[0189] The linear DNA product may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, such as a gene encoding a protein. The cassette may comprise a promoter and at least a portion of a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosome binding site, and a translation termination sequence. The cassette may further comprise a sequence that aids in the expression of the protein, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) can be used for CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode a CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymer sequence, such as a polyA, polyC, polyT or polyG sequence. The homopolymer sequence may be 3-200 nucleotides in length. The homopolymer sequence may be used to facilitate purification of the cassette, in which case the homopolymer sequence may be 4-12 nucleotides in length, or 5-10 nucleotides in length. The homopolymer sequence may be used to improve expression of mRNA, in which case the homopolymer sequence may be 10-200 nucleotides in length, preferably 80-150 nucleotides in length.The homopolymer sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. For example, the homopolymer sequence may include a polyA sequence of at least 120 nucleotides.

[0190] The linear DNA product may include a spacer. The spacer is at least 10 base pairs long, at least 20 base pairs long, at least 30 base pairs long, at least 40 base pairs long, at least 50 base pairs long, at least 60 base pairs long, at least 70 base pairs long, at least 80 base pairs long, at least 90 base pairs long, at least 100 base pairs long, at least 125 base pairs long, at least 150 base pairs long, at least 175 base pairs long, or at least 200 base pairs long. The spacer may improve the ligation efficiency of the first and second adaptor molecules to the linear double-stranded region. The spacer may improve the yield of cell transfection.

[0191] The linear DNA product may contain inverted terminal repeat sequences.

[0192] The linear DNA product is at least 50 base pairs long, at least 100 base pairs long, at least 250 base pairs long, at least 500 base pairs long, at least 1000 base pairs long, at least 2000 base pairs long, at least 3000 base pairs long, at least 4000 base pairs long, at least 5000 base pairs long, at least 6000 base pairs long, at least 7000 base pairs long, at least 8000 base pairs long, at least 9000 base pairs long, at least 10000 base pairs long, at least 11000 base pairs long, at least 12000 base pairs long, at least 13000 base pairs long, at least 14000 base pairs long, or at least 15000 base pairs long. Preferably, the linear DNA product is at least 50 base pairs long.

[0193] The double-stranded DNA molecule can be circular or branched.

[0194] The double-stranded DNA molecule does not include an adaptor molecule. The double-stranded DNA molecule does not include a hairpin structure, a loop structure, or a stem-loop structure.

[0195] The double stranded DNA molecule may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise a promoter and at least a portion of a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosome binding site and a translation termination sequence. The cassette may further comprise a sequence that aids in the expression of the protein, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) can be used for CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode a CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymer sequence, such as a polyA, polyC, polyT or polyG sequence. The homopolymer sequence is 3-200 nucleotides in length. The homopolymer sequence may be used to facilitate purification of the cassette, in which case the length of the homopolymer sequence may be between 4-12 nucleotides, or between 5-10 nucleotides. The homopolymer sequence may be used to improve mRNA expression, in which case the length of the homopolymer sequence may be between 10-200 nucleotides, preferably between 80-150 nucleotides.The homopolymer sequence may be at least 10 nucleotides long, at least 20 nucleotides long, at least 30 nucleotides long, at least 40 nucleotides long, at least 50 nucleotides long, at least 60 nucleotides long, at least 70 nucleotides long, at least 80 nucleotides long, at least 90 nucleotides long, at least 100 nucleotides long, at least 110 nucleotides long, at least 120 nucleotides long, at least 130 nucleotides long, at least 140 nucleotides long, at least 150 nucleotides long, at least 160 nucleotides long, at least 170 nucleotides long, at least 180 nucleotides long, at least 190 nucleotides long, or at least 200 nucleotides long. Preferably, the homopolymer sequence is at least 100 nucleotides long. More preferably, the homopolymer sequence is at least 120 nucleotides long. For example, the homopolymer sequence may include a polyA sequence of at least 120 nucleotides.

[0196] The double-stranded DNA molecule may include a spacer. The spacer is at least 10 base pairs long, at least 20 base pairs long, at least 30 base pairs long, at least 40 base pairs long, at least 50 base pairs long, at least 60 base pairs long, at least 70 base pairs long, at least 80 base pairs long, at least 90 base pairs long, at least 100 base pairs long, at least 125 base pairs long, at least 150 base pairs long, at least 175 base pairs long, or at least 200 base pairs long. The spacer may improve the amplification rate of the double-stranded DNA molecule. The spacer may improve the ligation efficiency of the first and second adaptor molecules to the linear double-stranded region. The spacer may improve the yield of cell transfection.

[0197] The double-stranded DNA molecule is at least 50 base pairs long, at least 100 base pairs long, at least 250 base pairs long, at least 500 base pairs long, at least 1000 base pairs long, at least 2000 base pairs long, at least 3000 base pairs long, at least 4000 base pairs long, at least 5000 base pairs long, at least 6000 base pairs long, at least 7000 base pairs long, at least 8000 base pairs long, at least 9000 base pairs long, at least 10000 base pairs long, at least 11000 base pairs long, at least 12000 base pairs long, at least 13000 base pairs long, at least 14000 base pairs long, or at least 15000 base pairs long.Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0198] The double stranded DNA molecule may comprise one or more cleavable (e.g., endonuclease) target sequences. The double stranded DNA molecule may comprise two cleavable (e.g., endonuclease) target sequences. The one or more cleavable (e.g., endonuclease) target sequences may be type IIS endonuclease target sequences. The one or more cleavable (e.g. endonuclease) target sequences include BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I , AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, BceAI, BcgI, Bci VI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseM I, BseMII, BseNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI- v2, BtsMutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, The target sequence may be GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI.

[0199] The double-stranded DNA molecule may be the product of amplification. Preferably, the amplification is rolling circle amplification.

[0200] The linear double-stranded region can be at least 50 base pairs long, at least 100 base pairs long, at least 250 base pairs long, at least 500 base pairs long, at least 1000 base pairs long, at least 2000 base pairs long, at least 3000 base pairs long, at least 4000 base pairs long, at least 5000 base pairs long, at least 6000 base pairs long, at least 7000 base pairs long, at least 8000 base pairs long, at least 9000 base pairs long, at least 10000 base pairs long, at least 11000 base pairs long, at least 12000 base pairs long, at least 13000 base pairs long, at least 14000 base pairs long, or at least 15000 base pairs long.Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0201] The linear double-stranded region may include a 3'-OH group at the first end and / or the second end. The 3'-OH group facilitates ligation to a first and / or second adaptor molecule(s) (which may include a 5' phosphate). The linear double-stranded region may include a 5' phosphate at the first end and / or the second end. The 5' phosphate facilitates ligation to a first and / or second adaptor molecule(s) (which may include a 3'-OH group).

[0202] A linear double-stranded region (e.g., the linear portion of a double-stranded molecule) may comprise an overhang. For example, a linear double-stranded region may comprise a 5' overhang or a 3' overhang. A linear double-stranded region may comprise a blunt end(s). A linear double-stranded region may comprise a 5' overhang and a blunt end, two 5' overhangs, a 3' overhang and a blunt end, two 3' overhangs, or a 5' overhang and a 3' overhang. An overhang may have at least 3 nucleotides (preferably 4 to 8 nucleotides). An overhang may be present on the sense strand or the antisense strand of a linear double-stranded region.

[0203] The linear portion of the double-stranded DNA molecule (e.g., the linear portion of the double-stranded molecule) can be at least 50 base pairs long, at least 100 base pairs long, at least 250 base pairs long, at least 500 base pairs long, at least 1000 base pairs long, at least 2000 base pairs long, at least 3000 base pairs long, at least 4000 base pairs long, at least 5000 base pairs long, at least 6000 base pairs long, at least 7000 base pairs long, at least 8000 base pairs long, at least 9000 base pairs long, at least 10000 base pairs long, at least 11000 base pairs long, at least 12000 base pairs long, at least 13000 base pairs long, at least 14000 base pairs long, or at least 15000 base pairs long.Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0204] The first adaptor molecule and / or the second adaptor molecule may be a synthetic adaptor molecule.

[0205] The first adaptor molecule and / or the second adaptor molecule do not have to be plasmid or vector DNA.

[0206] The first adaptor molecule and / or the second adaptor molecule may comprise a single-stranded portion. The single-stranded portion may comprise less than 10 nucleotides, 9 nucleotides, 8 nucleotides, 7 nucleotides, 6 nucleotides, 5 nucleotides, 4 nucleotides, 3 nucleotides, or 2 nucleotides. Preferably, the single-stranded portion may comprise 5 nucleotides.

[0207] The first adaptor molecule and / or the second adaptor molecule may comprise a double-stranded portion. The double-stranded portion may comprise less than 50 base pairs, less than 45 base pairs, less than 40 base pairs, less than 35 base pairs, less than 30 base pairs, less than 25 base pairs, less than 20 base pairs, less than 15 base pairs, or less than 10 base pairs. The double-stranded portion may comprise at least 5 base pairs, at least 6 base pairs, at least 7 base pairs, at least 8 base pairs, at least 9 base pairs, at least 10 base pairs, at least 11 base pairs, at least 12 base pairs, at least 13 base pairs, at least 14 base pairs, or at least 15 base pairs.

[0208] The first adaptor molecule and / or the second adaptor molecule may include a 5' phosphate, which may facilitate ligation to a linear double-stranded region.

[0209] The first adaptor molecule may comprise a portion complementary to a first end of the linear double-stranded region. The second adaptor molecule may comprise a portion complementary to a second end of the linear double-stranded region. The first adaptor molecule may comprise a portion that anneals to a first end of the linear double-stranded region. The second adaptor molecule may comprise a portion that anneals to a second end of the linear double-stranded region.

[0210] The first and / or second adaptor molecules may include an overhang. For example, the first and / or second adaptor molecules may include a 5' overhang or a 3' overhang. The first and / or second adaptor molecules may include a blunt end. The overhang has at least 3 nucleotides (preferably 4 to 6 nucleotides). The overhang of the first and / or second adaptor molecule may be complementary to the first end and / or the second end of the linear double-stranded region. The overhang of the first and / or second adaptor molecule may anneal to the first end and / or the second end of the linear double-stranded region.

[0211] The first adaptor molecule and / or the second adaptor molecule may not contain a type IIS endonuclease target sequence. The first adapter molecule and / or the second adapter molecule may be selected from the group consisting of BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI-v2, BtsMutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, The FaqI, FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI SapI target sequence may not be included.

[0212] The first adaptor molecule and / or the second adaptor molecule may comprise a functional moiety. The functional moiety may be a binding molecule, a targeting sequence, or a probe.

[0213] The functional moiety may be a probe. As used herein, the term "probe" refers to a fragment of DNA, RNA or DNA / RNA chimera of variable length (e.g., 3-1000 bases long) that is used to detect the presence of a target nucleotide sequence that is complementary to the sequence in the probe. Typically, a probe hybridizes to a single-stranded nucleic acid of a base sequence that allows probe-target base pairing due to the complementarity between the probe and the target. Thus, the functional moiety may be a DNA sequence, an RNA sequence, or a DNA / RNA chimera sequence. As used herein, the term "complementary" means that the nucleotide sequences pair according to the Watson / Crick pairing rules. For example, the sequence 5'-GCGGTCCCA-3' has a complementary sequence of 5'-TGGGACCGC-3'. The complementary sequence may also be an RNA sequence that is complementary to the DNA sequence.

[0214] The functional moiety may be a binding molecule. The term "binding molecule" refers to any molecule capable of binding to a linear DNA product as described herein and / or capable of binding to a further molecule or target. The binding molecule may be a protein, a polypeptide, or a peptide. The binding molecule may be an antibody, such as a monoclonal or polyclonal antibody. The binding molecule may be an antibody fragment.

[0215] The functional moiety can facilitate detection of the DNA product by binding to a capture molecule (e.g., a capture antibody bound by a protein-protein interaction). The functional moiety can bind to a cellular target, e.g., a cellular receptor.

[0216] The functional moiety may be a label. A "label" may be any chemical entity that allows detection of a double-stranded nucleic acid molecule by physical, chemical and / or biological means. The label may be either a chromophore, a fluorophore and / or a radioactive molecule.

[0217] The functional part may be a targeting sequence. The targeting sequence is a DNA or RNA fragment, which may vary in length, and is used to target the DNA product to a specific location in the cell. The targeting sequence can be used to enhance the transfection efficiency of non-viral gene transfer by promoting the nuclear uptake of linear DNA product. For example, the targeting sequence may be a DNA nuclear targeting sequence (i.e., a recognition sequence for endogenous DNA binding protein), such as the SV40 enhancer sequence (preferably downstream of the cassette).

[0218] To facilitate detection and / or quantification of the DNA product, the functional moiety may comprise a fluorophore, a radioactive compound or a barcode.

[0219] A signal corresponding to the presence, absence and / or level of a linear DNA product can be measured using a barcode. The barcode may comprise at least one binding moiety linked to a barcoded moiety, where the barcoded moiety comprises at least one nucleotide (i.e., the barcoded moiety comprises a nucleotide sequence at least one nucleotide long), and the binding moiety can bind to a 3' overhang, a 5' overhang or a blunt end of the linear DNA product. The binding site can bind to the 3' end and / or the 5' end of the linear DNA product. The signal can be measured by determining the presence, absence and / or level of the barcoded moiety (e.g., by sequencing or PCR). The barcoded moiety may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 nucleotides. The barcode may comprise at least two binding moieties (e.g., a first binding moiety and a second binding moiety). For example, a first binding moiety linked to a first barcode portion can bind to the 3' end of a linear double-stranded DNA product, and a second binding moiety linked to a second barcode portion can bind to the 5' end of the linear DNA product. The 3' and 5' ends can include a 3' overhang, a 5' overhang, or a blunt end.

[0220] A signal corresponding to the presence, absence, and / or level of the linear DNA product can be measured using a fluorophore (i.e., a fluorescently labeled molecule) attached or bound to the 3' overhang, 5' overhang, or blunt end of the linear DNA product. The signal can be measured by flow cytometry and / or fluorescence activated cell sorting.

[0221] The functional moiety can also facilitate DNA sequencing. For example, the functional moiety can be a sequence adapter. The term "sequence adapter" refers to the Illumina (登録商標) (e.g., HiSeq (商標) , MiSeq (商標)and / or Genome Analyzer (商標) Sequencing System), Oxford Nanopore (商標) Technologies (e.g., MinION sequencing system), Ion Torrent (商標) (For example, Ion PGM (商標) and / or Ion Proton (商標) sequencing systems); Pacific Biosciences (e.g., PACBIO RS II sequencing system); Life Technologies (商標) The term "sequencing platform" is intended to encompass one or more nucleic acid domains that contain at least a portion of a nucleic acid sequence (or a complement thereof) utilized by a sequencing platform of interest, such as a sequencing platform provided by Axis (e.g., the SOLiD sequencing system), Roche (e.g., the 454 GS FLX+ and / or GS Junior sequencing systems), or other sequencing platform of interest.

[0222] An example of the present invention is provided in FIG. 3 , which shows a workflow starting from amplified DNA obtained by rolling circle amplification of a circular DNA template generated by the action of Cre recombinase on a substrate containing two LoxP sequences flanking the DNA of interest in the same orientation, and obtaining a linear DNA product in a single step by digestion and ligation of adapter molecules.

[0223] The method of the present invention is further described in Figure 4, which shows the sequences that facilitate ligation of an adapter molecule after BsaI digestion of the amplified double-stranded DNA molecule for each cycle of the process. BsaI digestion generates 4 nucleotide 5' overhangs (TCCC5') on both sides of the expression cassette. Adapter molecules are ligated on both sides of the expression cassette, resulting in a linear protected DNA product due to the presence of protective nucleotides (marked with asterisks) on both sides and both strands of the expression cassette, which prevents exonuclease degradation of the linear DNA product.

[0224] 3. Method for producing partially closed linear DNA products containing nuclease-resistant nucleotides The methods described herein can be used to produce partially closed linear DNA products that contain nuclease-resistant (ie, protected nucleotides).

[0225] The present invention provides a method for producing a partially closed deoxyribonucleic acid (DNA) product, comprising the steps of: (a) contacting the double-stranded DNA molecule with an endonuclease and first and second adaptor molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to produce a partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, a first adaptor molecule is attached to a first end of the linear double-stranded region, and a second adaptor molecule is attached to a second end of the linear double-stranded region, the first and second adaptor molecules are nucleic acid molecules that include one or more nuclease-resistant nucleotides (i.e., protected nucleotides), and the linear double-stranded region is closed at its second end by a second adaptor molecule. The present invention provides a method comprising:

[0226] The step of contacting the double-stranded DNA molecule with an endonuclease and a first and a second adaptor molecule is preferably carried out in the presence of a ligase. Thus, the method for producing a partially closed linear DNA product comprises: (a) contacting double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to produce a partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is attached to a first end of the linear double-stranded region, and the second adaptor molecule is attached to a second end of the linear double-stranded region, the first and second adaptor molecules are nucleic acid molecules that include one or more nuclease-resistant nucleotides (i.e., protected nucleotides), and the linear double-stranded region is closed at its second end by a second adaptor molecule; may include.

[0227] The partially closed linear DNA products produced by the methods described herein have enhanced resistance to nuclease (eg, exonuclease) digestion.

[0228] The addition of the first and / or second adapter molecule can be performed by hybridization or ligation of the adapter molecule to the end of the linear double-stranded region. Thus, the first adapter molecule can hybridize to the first end of the linear double-stranded region. The second adapter molecule can hybridize to the second end of the linear double-stranded region. The first adapter molecule is ligated to the first end of the linear double-stranded region. The second adapter molecule can be ligated to the second end of the linear double-stranded region. The addition of the first and second adapter molecules can be performed by both hybridization and ligation of the adapter molecule to the end of the linear double-stranded region. Thus, the first adapter molecule can hybridize and ligate to the first end of the linear double-stranded region. The second adapter molecule can hybridize and ligate to the second end of the linear double-stranded region. Hybridization is based on the complementarity of a portion of the first and / or second adapter molecule to the first and / or second ends of the linear double-stranded region.

[0229] The method for producing a partially closed linear DNA product may comprise the steps of: (a) contacting double-stranded DNA molecules with an endonuclease, a ligase, and first and second adapter molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to produce a partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is ligated to a first end of the linear double-stranded region, and a second adaptor molecule is ligated to a second end of the linear double-stranded region, the first and second adaptor molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e., protected nucleotides), and the linear double-stranded region is closed at the second end by the second adaptor molecule.

[0230] The method for producing a partially closed linear DNA product may comprise the steps of: (a) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adapter molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to produce a partially closed linear DNA product, where the partially closed linear DNA product comprises a linear portion of a double-stranded DNA molecule, a first adaptor molecule is ligated to a first end of the linear portion of the double-stranded DNA molecule, and a second adaptor molecule is ligated to a second end of the linear portion of the double-stranded DNA molecule, the first and second adaptor molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e., protected nucleotides), and the linear double-stranded region is closed at the second end by the second adaptor molecule.

[0231] As used herein, the term "complementary" means that the nucleotide sequences pair according to the Watson / Crick pairing rules. For example, the sequence 5'-GCGGTCCCA-3' has a complementary sequence of 5'-TGGGACCGC-3'. A complementary sequence can also be an RNA sequence that is complementary to a DNA sequence.

[0232] Preferably, the steps of contacting the double-stranded DNA molecule with the endonuclease, the ligase and the first and second adaptor molecules are performed in one reaction (ie, a single step).

[0233] The step of incubating the single continuous aqueous volume to generate a partially closed linear DNA product can include generating a linear portion of the double-stranded DNA molecule by digesting the double-stranded DNA molecule with an endonuclease.

[0234] The step of incubating the single continuous aqueous volume can be performed under conditions that promote the addition (or ligation) of a first and a second adaptor molecule to the linear double-stranded region to generate a partially closed linear DNA product. The addition can be performed by forming a covalent bond between the first and / or second adaptor molecule and the end(s) of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule).

[0235] The step of incubating the single continuous aqueous volume can be performed under conditions that promote digestion of the double-stranded DNA molecule and generate a linear portion of the double-stranded DNA molecule. The digestion of the double-stranded DNA molecule to generate a linear portion of the double-stranded DNA molecule can be performed at a first temperature of 1° C. to 100° C., 1° C. to 80° C., 5° C. to 70° C., 10° C. to 60° C., 15° C. to 55° C., 20° C. to 50° C., 25° C. to 45° C., 30° C. to 40° C., 35° C. to 39° C., 36° C. to 38° C., or about 37° C. The digestion can be an endonuclease digestion, preferably a type IIS endonuclease digestion.

[0236] The step of incubating the single continuous aqueous volume can be performed under conditions that promote ligation of the linear double-stranded region to the first and second adaptor molecules. Ligation can be performed with an efficiency of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95%. For example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the linear double-stranded region (or portion of the double-stranded DNA molecule) can be incorporated into the closed linear DNA product. Preferably, the efficiency of ligation is at least 15%.

[0237] The step of ligating the linear double-stranded region to the first and second adaptor molecules can be carried out at a second temperature of 1°C to 90°C, 2°C to 70°C, 5°C to 60°C, 8°C to 55°C, 9°C to 50°C, 10°C to 45°C, 11°C to 40°C, 12°C to 37°C, 13°C to 30°C, 14°C to 25°C, 15°C to 20°C, or about 16°C.

[0238] The step of incubating a single continuous aqueous volume may include incubating at a first temperature and then incubating at a second temperature. The first temperature may be 1°C to 100°C, 1°C to 80°C, 5°C to 70°C, 10°C to 60°C, 15°C to 55°C, 20°C to 50°C, 25°C to 45°C, 30°C to 40°C, 35°C to 39°C, 36°C to 38°C, or about 37°C. The second temperature may be 1°C to 90°C, 2°C to 70°C, 5°C to 60°C, 8°C to 55°C, 9°C to 50°C, 10°C to 45°C, 11°C to 40°C, 12°C to 37°C, 13°C to 30°C, 14°C to 25°C, 15°C to 20°C, or about 16°C. Preferably, the first temperature is 35°C to 39°C. Preferably, the second temperature is 14°C to 18°C.

[0239] The step of incubating a single continuous aqueous volume may include cycling between a first temperature and a second temperature. The step of incubating a single continuous aqueous volume may include cycling between a first temperature and a second temperature at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 65 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times, preferably at least 20 times. The step of incubating a single continuous aqueous volume may include cycling between a first temperature and a second temperature less than 40 times, less than 35 times, less than 30 times, less than 29 times, less than 25 times. Incubating a single continuous aqueous volume may include cycling between a first temperature and a second temperature 2-100 times, 5-80 times, 10-70 times, 20-60 times, or 30-60 times. Incubating a single continuous aqueous volume may include cycling between a first temperature and a second temperature 2-20 times, 5-29 times, 61-100 times, or 65-80 times.

[0240] The step of incubating the single continuous aqueous volume can be performed isothermally. The step of incubating the single continuous aqueous volume can include incubating at a constant temperature. The constant temperature simultaneously promotes digestion of the double-stranded DNA molecule, generates a linear portion of the double-stranded DNA molecule, and ligates the linear double-stranded region to the first and second adaptor molecules. For example, the constant temperature can be 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, 37°C, 38°C, 39°C, or 40°C. Preferably, the constant temperature is 30°C. By constant temperature, it is intended that the temperature does not change significantly during the reaction. By constant temperature is meant that the temperature change during the step of incubating a single continuous aqueous volume is less than 10° C., less than 9° C., less than 8° C., less than 7° C., less than 6° C., less than 5° C., less than 4° C., less than 3° C., less than 2° C., or less than 1° C. In a preferred embodiment, the temperature during the step of incubating a single continuous aqueous volume is 5° C. or less, preferably 3° C. or less, more preferably 1° C. or less. Thus, the constant temperature may be a temperature in the range of 20° C.-30° C., 22° C.-32° C., 24° C.-34° C., 26° C.-36° C., 28° C.-38° C., 30° C.-40° C., 22° C.-28° C., 32° C.-38° C., 25° C.-35° C., 26° C.-34° C., 27° C.-33° C., 27.5° C.-32.5° C., 28° C.-32° C., 28.5° C.-31.5° C., 29° C.-31° C., or 29.5° C.-30.5° C. Preferably, the constant temperature is between 27.5° C. and 32.5° C. Alternatively, the constant temperature may be in the range of 32° C.-42° C., 33° C.-41° C., 33° C.-41° C., 34° C.-40° C., 35° C.-39° C., 36° C.-38° C. Preferably, the constant temperature is between 34.5° C. and 39.5° C.

[0241] The first and second adaptor molecules may comprise one or more phosphorothioated nucleotides, and when the adaptor molecules are added (e.g., ligated) to the linear double-stranded region, the partially closed linear DNA product is resistant to nuclease digestion or has improved or enhanced resistance to nuclease digestion. The partially closed linear DNA product may be resistant to 3'-end exonuclease digestion (e.g., by exonuclease III) and / or 5'-end exonuclease digestion (e.g., by exonuclease VIII).

[0242] The adapter molecule may comprise multiple phosphorothioated nucleotides, for example, the adapter molecule may comprise 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, or at least 16 phosphorothioated nucleotides on each strand.

[0243] The adaptor molecule may be a nucleic acid adaptor molecule. The adaptor molecule may be double-stranded. The adaptor molecule may include a double-stranded portion.

[0244] The first adaptor molecule and / or the second adaptor molecule can comprise at least 3 base pairs, at least 4 base pairs, at least 5 base pairs, at least 6 base pairs, at least 7 base pairs, at least 8 base pairs, at least 9 base pairs, at least 10 base pairs, at least 11 base pairs, at least 12 base pairs, at least 13 base pairs, at least 14 base pairs, at least 15 base pairs, or at least 16 base pairs.

[0245] The adapter molecule may comprise multiple phosphorothioated nucleotides on each strand, for example, the adapter molecule may comprise 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, or at least 16 phosphorothioated nucleotides on each strand.

[0246] The adapter molecule may comprise multiple phosphorothioated nucleotides at an internal position of each strand. For example, the adapter molecule may comprise 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, or at least 16 phosphorothioated nucleotides at an internal position of each strand. Preferably, the adapter molecule comprises at least 2 phosphorothioated nucleotides at an internal position of each strand.

[0247] An internal position does not have to be located between the second and penultimate nucleotide of the adaptor molecule. An internal position can be anywhere in the adaptor molecule other than the last nucleotide at each strand end.

[0248] The adapter molecule may comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% protected nucleotides.

[0249] When an adaptor molecule is added to the linear double-stranded region, the partially closed linear DNA product may contain protected nucleotides (e.g., phosphorothioated nucleotides) at the 5'-end (or 5'-end region) of one or both strands. Preferably, the partially closed linear DNA product contains phosphorothioated nucleotides at the 5'-end (or 5'-end region) of one strand. The partially closed linear DNA product may contain phosphorothioated nucleotides at the 5'-end (or 5'-end region) of one strand. Since most exonucleases, such as exonuclease III, remove nucleotides from the 3'-end of a polynucleotide strand, the linear DNA product may contain protected nucleotides at the 3'-end (or 3'-end region) of one strand. Preferably, the partially closed linear DNA product contains phosphorothioated nucleotides at the 3'-end (or 3'-end region) of one strand. The partially closed linear DNA product may contain at least one phosphorothioated nucleotide at the 3'-end (or 3'-end region) of one strand and at least one phosphorothioated nucleotide at the 5'-end (or 5'-end region). The partially closed linear DNA product may contain a phosphorothioated nucleotide at the 3'-end (or 3'-end region) of the sense strand and at the 5'-end (or 5'-end region) of the antisense strand. The partially closed linear DNA product may contain at least one phosphorothioated nucleotide at the 5'-end (or 5'-end region) of the sense strand and at the 3'-end (or 3'-end region) of the antisense strand. Thus, both the sense and antisense strands of a double-stranded partially closed linear DNA product can be protected from nuclease digestion by using nuclease-resistant nucleotides at one end of the partially closed linear DNA product.

[0250] One of the adaptor molecules used in the methods described herein may contain a self-complementary element that forms a loop, such as a hairpin loop or a stem loop. Thus, one of the adaptor molecules may contain a hairpin or a stem loop. The adaptor molecule may contain a double-stranded portion that includes a sense strand and an antisense strand, the sense strand and the antisense strand being linked by a hairpin such that the sense strand hybridizes to the antisense strand. The double-stranded portion of the adaptor may contain a 3' or 5' overhang of at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotides. Preferably, the 3' or 5' overhang is 4-8 nucleotides. Each end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule) may contain a 3' or 5' overhang. A portion of the adaptor molecule (e.g., an overhang) may be complementary to a first end or a second end of the linear double-stranded region.

[0251] The methods described herein may use a first adaptor molecule that is a linear nucleic acid molecule that contains nuclease resistant nucleotides, and a second adaptor molecule that contains a hairpin loop or stem loop, or any other structure that can close one end of a linear DNA molecule to generate a partially closed linear DNA product.

[0252] A partially closed linear DNA product can be a partially covalently closed DNA product. Thus, in embodiments where the adapter molecule comprises a loop (e.g., a hairpin), the adapter molecule closes one end of the linear double-stranded region, forming a partially covalently closed DNA product.

[0253] The adaptor molecule may comprise a single-stranded portion. The single-stranded portion may form a hairpin or a stem loop. The single-stranded portion may comprise less than 10 nucleotides, less than 9 nucleotides, less than 8 nucleotides, less than 7 nucleotides, less than 6 nucleotides, less than 5 nucleotides, less than 4 nucleotides, less than 3 nucleotides, less than 2 nucleotides. Preferably, the single-stranded portion comprises 5 nucleotides.

[0254] The partially closed linear DNA product may further comprise a plurality of protected nucleotides (e.g., phosphorothioated nucleotides) at the internal position of each strand. For example, the linear DNA product may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g., phosphorothioated nucleotides) at the internal position of each strand. For example, phosphorothioated nucleotides) are present at the internal position of each strand. Preferably, the partially closed linear DNA product contains at least two protected nucleotides (eg, phosphorothioated nucleotides) at internal positions on each strand.

[0255] An internal position does not have to be located between the second and last nucleotide of the partially closed linear DNA product. An internal position can be anywhere in the adaptor molecule other than the last nucleotide at the end of each strand.

[0256] A linear double-stranded region (or a linear portion of a double-stranded molecule) may contain a plurality of phosphorothioated nucleotides at the internal positions of each strand. For example, a linear double-stranded region (or a linear portion of a double-stranded molecule) may have at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g., phosphorothioated nucleotides) at the internal positions of each strand. Preferably, the linear double-stranded region (or the linear portion of the double-stranded molecule) contains at least two protected nucleotides (e.g., phosphorothioated nucleotides) at internal positions of each strand. The internal positions do not have to be located between the second and the last nucleotide of the linear double-stranded region (or the linear portion of the double-stranded molecule).

[0257] The nucleotides resistant to exonuclease digestion (i.e., protected nucleotides) are at least one phosphorothioated nucleotide. For example, the at least one phosphorothioated nucleotide is α-S-dATP (i.e., 2'-deoxyadenosine-5'-(α-thio)-triphosphate), α-S-dCTP (i.e., 2'-deoxycytidine-5'-(α-thio)-triphosphate), α-S-dGTP (i.e., 2'-deoxyguanosine-5'-(α-thio)-triphosphate), α-S-dTTP (i.e., 2'-deoxythymidine-5'-(α-thio)-triphosphate), α-S-dUTP (i.e., 2'-deoxyuridine-5'-(α-thio)-triphosphate), and / or uridine 2',3'-cyclophosphorothioate.

[0258] The adapter molecule may comprise at least two phosphorothioated nucleotides, for example, the at least two phosphorothioated nucleotides are as follows: α-S-dATP and α-S-dCTP, α-S-dATP and α-S-dGTP, α-S-dATP and α-S-dTTP, α-S-dCTP and α-S-dGTP, α-S-dCTP and α-S-dTTP, or α-S-dGTP and α-S-dTTP.

[0259] The adapter molecule can include at least three phosphorothioated nucleotides. For example, the at least three phosphorothioated nucleotides can be (e) α-S-dATP, α-S-dCTP, and α-S-dGTP; (f) α-S-dATP, α-S-dCTP and α-S-dTTP; (g) α-S-dATP, α-S-dGTP and α-S-dTTP; or (h) α-S-dCTP, α-S-dGTP and α-S-dTTP It is.

[0260] The adapter molecule can include at least four phosphorothioated nucleotides, for example, the at least four protected nucleotides are α-S-dATP, α-S-dCTP, α-S-dGTP and α-S-dTTP.

[0261] The phosphorothioated nucleotides may be Sp-isomers, Rp-isomers, or a mixture of both Sp- and Rp-isomers.

[0262] The nucleotides that are resistant to exonuclease digestion (i.e., protected nucleotides) may be at least one, or at least two, three, or four MOE nucleotides. For example, the MOE nucleotides may be 2'-O-methoxy-ethylguanosine, 2'-O-methoxy-ethylcytidine, 2'-O-methoxy-ethyladenosine, and / or 2'-O-methoxy-ethylthymidine.

[0263] The method may further comprise amplifying a DNA template molecule to generate a double-stranded DNA molecule prior to step (a) (i.e., contacting the double-stranded DNA molecule with an endonuclease, a ligase, and a first and second adaptor molecule). Thus, the present invention provides a method for producing a partially closed linear DNA product, comprising: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; and (c) incubating the single contiguous aqueous volume to produce a partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is attached to a first end of the linear double-stranded region, and a second adaptor molecule is attached to a second end of the linear double-stranded region, the first adaptor molecule is a nucleic acid molecule that comprises one or more nuclease-resistant nucleotides (i.e., protected nucleotides), and the linear double-stranded region is closed at its second end by a second adaptor molecule; The present invention provides a method comprising:

[0264] The amplifying step can be carried out by in vitro or in vivo amplification. Preferably, the amplifying step is carried out by in vitro amplification. For example, the amplifying step can be carried out by rolling circle amplification (RCA), MALBAC, conventional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA). Preferably, the amplification is carried out by rolling circle amplification. Thus, the present invention provides a method for producing a partially closed linear DNA product, comprising: (a) amplifying a DNA template molecule that includes at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule, where the DNA template molecule is amplified by rolling circle amplification; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adapter molecules to form a single contiguous aqueous volume; and (c) incubating the single contiguous aqueous volume to produce a partially closed linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is attached to a first end of the linear double-stranded region, and a second adaptor molecule is attached to a second end of the linear double-stranded region, and the first and second adaptor molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e., protected nucleotides), and the linear double-stranded region is closed at its second end by the second adaptor molecule; The present invention provides a method for producing a partially closed linear DNA product, comprising:

[0265] Rolling circle amplification may be performed without primers or in the presence of a primer or primers. For example, the primers may be synthetic primers. The primers may be random primers. Rolling circle amplification may be performed in the presence of a primase. The primase may be TthPrimPol. Preferably, when rolling circle amplification is performed without primers, it is performed in the presence of a primase such as TthPrimPol. Similarly, when primers are used in the amplification reaction, no primase is used. Double-stranded DNA products may be generated by rolling circle amplification in vitro under isothermal conditions using a suitable nucleic acid polymerase such as Phi29 DNA polymerase.

[0266] In the method described herein, the DNA template molecule may comprise at least one cleavable target sequence. The cleavable target sequence may be an endonuclease target sequence. Thus, the DNA template molecule may comprise at least one endonuclease target sequence. Preferably, the DNA template molecule comprises at least two endonuclease target sequences. The endonuclease target sequences may be the same or different. Preferably, at least one endonuclease target sequence is a restriction endonuclease target sequence. Different restriction endonuclease target sequences may be known to those skilled in the art. The cleavable target sequence may be a type IIS restriction endonuclease target sequence. For example, restriction endonuclease target sequences include BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI , BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI-v2, B tsMutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, Gs The target sequence may be uI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI.The at least one cleavable sequence (e.g., endonuclease target sequence) may be a naturally occurring cleavable sequence (i.e., a cleavable sequence present in the template molecule). Alternatively, the at least one cleavable sequence (e.g., endonuclease target sequence) may be introduced into the DNA template molecule prior to producing the linear DNA product.

[0267] The endonuclease may be a restriction enzyme endonuclease. The endonuclease may be a type IIS restriction enzyme. The endonuclease may be any enzyme that recognizes a DNA sequence and cleaves outside the recognition sequence. For example, endonucleases include BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, A juI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, Bfu AI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, B seNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, Bs pTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI-v2, Bts MutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI , HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI restriction enzymes.

[0268] The ligase may be a DNA ligase such as T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase.

[0269] The DNA template molecule used in the methods described herein may be single-stranded or double-stranded. Preferably, the DNA template molecule is double-stranded. The DNA template molecule may be a naturally occurring circular DNA molecule. For example, the DNA template molecule may be (i) a plasmid, (ii) a minicircle, (iii) a cosmid, (iv) a bacterial artificial chromosome (BAC), or (v) a molecular inversion probe (MIP). The DNA template molecule may be an enzymatically produced circular DNA molecule. For example, the DNA template molecule may be (i) a circular DNA molecule obtained from a recombinase reaction, preferably a Cre recombinase reaction, or (ii) a circular DNA molecule obtained using a ligase reaction, preferably Golden Gate assembly. The DNA template molecule may be an enzymatically produced covalently closed linear DNA molecule. For example, the DNA template molecule may be (i) a DNA molecule treated with TelN protelomerase, or (ii) a DNA molecule generated by ligation of the DNA ends with an adaptor. The DNA template molecule may include double-stranded and single-stranded elements. For example, the template DNA molecule may include a double-stranded DNA and a single-stranded hairpin loop.

[0270] The DNA template molecule may be linear. When the DNA template molecule is linear, the DNA template molecule may be circularized prior to amplification (e.g., rolling circle amplification) to generate a DNA template molecule suitable for use in the methods described herein.

[0271] The template DNA molecule may include a cassette. The cassette may be a mammalian expression cassette. The cassette may further include a promoter. The promoter may be a CMV promoter. The cassette may further include an enhancer. The cassette may further include a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further include a homopolymer sequence. The cassette may further include a LoxP sequence, preferably two LoxP sequences. When the two LoxP sequences are in the same orientation, the DNA sequence between the two LoxP sequences is excised as a circular loop of DNA. When the two LoxP sequences are in the opposite orientation, the DNA sequence between the two LoxP sequences is inverted. Thus, preferably, the two LoxP sequences are in the same orientation in the template DNA molecule.

[0272] The DNA template molecule may contain a homopolymer sequence at the 5' or 3' end, or at both the 5' and 3' ends. The homopolymer sequence may be added to the DNA template molecule prior to circularization. The homopolymer sequence may be a polyA, polyC, polyG, or polyT sequence. The homopolymer sequence may be between 3 and 200 nucleotides in length. The homopolymer sequence may be used to facilitate purification of the partially closed linear DNA product, in which case the homopolymer sequence may be between 4 and 12 nucleotides, or between 5 and 10 nucleotides in length. The homopolymer sequence may be used to improve mRNA expression, in which case the homopolymer sequence may be between 10 and 200 nucleotides, preferably between 80 and 150 nucleotides in length. The length of the homopolymer sequence can be at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 110 nucleotides, at least 120 nucleotides, at least 130 nucleotides, at least 140 nucleotides, at least 150 nucleotides, at least 160 nucleotides, at least 170 nucleotides, at least 180 nucleotides, at least 190 nucleotides, or at least 200 nucleotides.Preferably, the homopolymer sequence is at least 100 nucleotides long.More preferably, the homopolymer sequence is at least 120 nucleotides long.For example, the homopolymer sequence can include a polyA sequence of at least 120 nucleotides long.

[0273] The method may further comprise the step of purifying the partially closed linear DNA product after the step of incubating the single continuous aqueous volume.

[0274] The method may further comprise a nuclease digestion step after the step of incubating the single continuous aqueous volume. The nuclease digestion may be an exonuclease digestion, such as exonuclease I and / or exonuclease III digestion. The nuclease digestion step may be before or after the purification step. This step allows for the removal of double-stranded DNA molecules and / or adapter molecules that were not used during the performance of the method. Thus, the method of the present invention comprises: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (c) incubating the single contiguous aqueous volume to produce a partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is ligated to a first end of the linear double-stranded region, and a second adaptor molecule is ligated to a second end of the linear double-stranded region, and the first adaptor molecule is a nucleic acid molecule that comprises one or more nuclease-resistant nucleotides (i.e., protected nucleotides), and the linear double-stranded region is closed at the second end by the second adaptor molecule; and (d) incubating the single continuous aqueous solution with a nuclease (e.g., an exonuclease) may include.

[0275] The method of the present invention comprises the steps of: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (c) incubating the single contiguous aqueous volume to produce a partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is ligated to a first end of the linear double-stranded region, and a second adaptor molecule is ligated to a second end of the linear double-stranded region, the first adaptor molecule is a nucleic acid molecule that comprises one or more nuclease-resistant nucleotides (i.e., protected nucleotides), and the linear double-stranded region is closed at the second end by the second adaptor molecule; (d) purifying the closed linear DNA product; and (e) incubating the purified product of step (d) with a nuclease (e.g., an exonuclease). may include.

[0276] The method of the present invention comprises the steps of: (a) amplifying a DNA template molecule that contains at least one cleavable (e.g., endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (c) incubating the single contiguous aqueous volume to produce a partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, wherein a first adaptor molecule is ligated to a first end of the linear double-stranded region and a second adaptor molecule is ligated to a second end of the linear double-stranded region, the first adaptor molecule being a nucleic acid molecule comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides), and the linear double-stranded region is closed at the second end by the second adaptor molecule; (d) incubating the single contiguous aqueous volume with a nuclease (e.g., an exonuclease); and (e) Purifying the closed linear DNA product may include.

[0277] The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) with the nuclease can be carried out at a temperature of 5-90°C, 10-80°C, 15-70°C, 20-60°C, 25-50°C, 30-45°C, or 35-40°C. The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) with the nuclease can be carried out for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes. The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) can be carried out at two different temperatures. For example, the step of incubating the single contiguous aqueous volume (or the purified product of step (d)) can be carried out at a temperature of 15-40°C for 10-60 minutes and then at a temperature of 60-90°C for 10-30 minutes. Higher temperatures generally inactivate nucleases (e.g., exonucleases). Thus, the method further provides a step of inactivating nucleases (e.g., exonucleases). The step of incubating the single continuous aqueous volume (or the purified product of step (d)) can be performed at 37°C for 30 minutes and at 80°C for 20 minutes. Preferably, the step of inactivating nucleases (e.g., exonucleases) is performed at a temperature of 70-80°C. The step of inactivating nucleases (e.g., exonucleases) can be performed for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, or at least 30 minutes. Preferably, the step of inactivating nucleases (e.g., exonucleases) is performed for at least 5 minutes.

[0278] The first adaptor molecule and / or the second adaptor molecule may comprise an overhang. The end of the linear double-stranded region may comprise a 3' or 5' overhang. A portion of the first adaptor molecule (e.g., the overhang) may be complementary to the first end of the linear double-stranded region. A portion of the second adaptor molecule may be complementary to the second end of the linear double-stranded region.

[0279] The first and second ends of the linear double-stranded region may be resistant to nuclease digestion. Preferably, the first and second ends of the linear double-stranded region are resistant to exonuclease digestion, such as exonuclease III digestion and / or exonuclease I digestion.

[0280] A linear DNA product may be partially double-stranded or partially single-stranded. A linear DNA product may contain double-stranded and single-stranded portions.

[0281] The partially closed linear DNA product may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, such as a gene encoding a protein. The cassette may comprise a promoter and at least a portion of a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosome binding site, and a translation termination sequence. The cassette may further comprise a sequence that aids in the expression of the protein, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) can be used for CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode a CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymer sequence, such as a polyA, polyC, polyT or polyG sequence. The homopolymer sequence may be 3-200 nucleotides in length. The homopolymer sequence may be used to facilitate purification of the cassette, in which case the homopolymer sequence may be 4-12 nucleotides in length, or 5-10 nucleotides in length. The homopolymer sequence may be used to improve expression of mRNA, in which case the homopolymer sequence may be 10-200 nucleotides in length, preferably 80-150 nucleotides in length.The homopolymer sequence may be at least 10 nucleotides long, at least 20 nucleotides long, at least 30 nucleotides long, at least 40 nucleotides long, at least 50 nucleotides long, at least 60 nucleotides long, at least 70 nucleotides long, at least 80 nucleotides long, at least 90 nucleotides long, at least 100 nucleotides long, at least 110 nucleotides long, at least 120 nucleotides long, at least 130 nucleotides long, at least 140 nucleotides long, at least 150 nucleotides long, at least 160 nucleotides long, at least 170 nucleotides long, at least 180 nucleotides long, at least 190 nucleotides long, or at least 200 nucleotides long. Preferably, the homopolymer sequence is at least 100 nucleotides long. More preferably, the homopolymer sequence is at least 120 nucleotides long. For example, the homopolymer sequence may include a polyA sequence of at least 120 nucleotides.

[0282] The partially closed linear DNA product may include a spacer. The length of the spacer may be at least 10 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 60 base pairs, at least 70 base pairs, at least 80 base pairs, at least 90 base pairs, at least 100 base pairs, at least 125 base pairs, at least 150 base pairs, at least 175 base pairs, or at least 200 base pairs. The spacer may improve the ligation efficiency of the first and second adaptor molecules to the linear double-stranded region. The spacer may improve the yield of cell transfection.

[0283] The partially closed linear DNA product may contain inverted terminal repeat sequences.

[0284] The partially closed linear DNA product is at least 50 base pairs long, at least 100 base pairs long, at least 250 base pairs long, at least 500 base pairs long, at least 1000 base pairs long, at least 2000 base pairs long, at least 3000 base pairs long, at least 4000 base pairs long, at least 5000 base pairs long, at least 6000 base pairs long, at least 7000 base pairs long, at least 8000 base pairs long, at least 9000 base pairs long, at least 10000 base pairs long, at least 11000 base pairs long, at least 12000 base pairs long, at least 13000 base pairs long, at least 14000 base pairs long, or at least 15000 base pairs long. Preferably, the partially closed linear DNA product is at least 50 base pairs long.

[0285] The double-stranded DNA molecule can be circular or branched.

[0286] The double-stranded DNA molecule does not include an adaptor molecule. The double-stranded DNA molecule does not include a hairpin structure, a loop structure, or a stem-loop structure.

[0287] The double stranded DNA molecule may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise a promoter and at least a portion of a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosome binding site and a translation termination sequence. The cassette may further comprise a sequence that aids in the expression of the protein, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) can be used for CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode a CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymer sequence, such as a polyA, polyC, polyT or polyG sequence. The homopolymer sequence is 3-200 nucleotides in length. The homopolymer sequence may be used to facilitate purification of the cassette, in which case the length of the homopolymer sequence may be between 4-12 nucleotides, or between 5-10 nucleotides. The homopolymer sequence may be used to improve mRNA expression, in which case the length of the homopolymer sequence may be between 10-200 nucleotides, preferably between 80-150 nucleotides.The homopolymer sequence may be at least 10 nucleotides long, at least 20 nucleotides long, at least 30 nucleotides long, at least 40 nucleotides long, at least 50 nucleotides long, at least 60 nucleotides long, at least 70 nucleotides long, at least 80 nucleotides long, at least 90 nucleotides long, at least 100 nucleotides long, at least 110 nucleotides long, at least 120 nucleotides long, at least 130 nucleotides long, at least 140 nucleotides long, at least 150 nucleotides long, at least 160 nucleotides long, at least 170 nucleotides long, at least 180 nucleotides long, at least 190 nucleotides long, or at least 200 nucleotides long. Preferably, the homopolymer sequence is at least 100 nucleotides long. More preferably, the homopolymer sequence is at least 120 nucleotides long. For example, the homopolymer sequence may include a polyA sequence of at least 120 nucleotides.

[0288] The double-stranded DNA molecule may include a spacer. The spacer is at least 10 base pairs long, at least 20 base pairs long, at least 30 base pairs long, at least 40 base pairs long, at least 50 base pairs long, at least 60 base pairs long, at least 70 base pairs long, at least 80 base pairs long, at least 90 base pairs long, at least 100 base pairs long, at least 125 base pairs long, at least 150 base pairs long, at least 175 base pairs long, or at least 200 base pairs long. The spacer may improve the amplification rate of the double-stranded DNA molecule. The spacer may improve the ligation efficiency of the first and second adaptor molecules to the linear double-stranded region. The spacer may improve the yield of cell transfection.

[0289] The double-stranded DNA molecule is at least 50 base pairs long, at least 100 base pairs long, at least 250 base pairs long, at least 500 base pairs long, at least 1000 base pairs long, at least 2000 base pairs long, at least 3000 base pairs long, at least 4000 base pairs long, at least 5000 base pairs long, at least 6000 base pairs long, at least 7000 base pairs long, at least 8000 base pairs long, at least 9000 base pairs long, at least 10000 base pairs long, at least 11000 base pairs long, at least 12000 base pairs long, at least 13000 base pairs long, at least 14000 base pairs long, or at least 15000 base pairs long.Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0290] The double stranded DNA molecule may comprise one or more cleavable (e.g., endonuclease) target sequences. The double stranded DNA molecule may comprise two cleavable (e.g., endonuclease) target sequences. The one or more cleavable (e.g., endonuclease) target sequences may be type IIS endonuclease target sequences. The one or more cleavable (e.g., endonuclease) target sequences include BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, BceAI , BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse 3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, Btg ZI, BtsCI, BtsI-v2, BtsMutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, The target sequence may be FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI.

[0291] The double-stranded DNA molecule may be the product of amplification. Preferably, the amplification is rolling circle amplification.

[0292] The linear double-stranded region can be at least 50 base pairs long, at least 100 base pairs long, at least 250 base pairs long, at least 500 base pairs long, at least 1000 base pairs long, at least 2000 base pairs long, at least 3000 base pairs long, at least 4000 base pairs long, at least 5000 base pairs long, at least 6000 base pairs long, at least 7000 base pairs long, at least 8000 base pairs long, at least 9000 base pairs long, at least 10000 base pairs long, at least 11000 base pairs long, at least 12000 base pairs long, at least 13000 base pairs long, at least 14000 base pairs long, or at least 15000 base pairs long.Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0293] The linear double-stranded region may include a 3'-OH group at the first end and / or the second end. The 3'-OH group facilitates ligation to a first and / or second adaptor molecule(s) (which may include a 5' phosphate). The linear double-stranded region may include a 5' phosphate at the first end and / or the second end. The 5' phosphate facilitates ligation to a first and / or second adaptor molecule(s) (which may include a 3'-OH group).

[0294] A linear double-stranded region (e.g., the linear portion of a double-stranded molecule) may comprise an overhang. For example, a linear double-stranded region may comprise a 5' overhang or a 3' overhang. A linear double-stranded region may comprise a blunt end(s). A linear double-stranded region may comprise a 5' overhang and a blunt end, two 5' overhangs, a 3' overhang and a blunt end, two 3' overhangs, or a 5' overhang and a 3' overhang. An overhang may have at least 3 nucleotides (preferably 4 to 8 nucleotides). An overhang may be present on the sense strand or the antisense strand of a linear double-stranded region.

[0295] The linear portion of the double-stranded DNA molecule (e.g., the linear portion of the double-stranded molecule) can be at least 50 base pairs long, at least 100 base pairs long, at least 250 base pairs long, at least 500 base pairs long, at least 1000 base pairs long, at least 2000 base pairs long, at least 3000 base pairs long, at least 4000 base pairs long, at least 5000 base pairs long, at least 6000 base pairs long, at least 7000 base pairs long, at least 8000 base pairs long, at least 9000 base pairs long, at least 10000 base pairs long, at least 11000 base pairs long, at least 12000 base pairs long, at least 13000 base pairs long, at least 14000 base pairs long, or at least 15000 base pairs long.Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0296] The first adaptor molecule and / or the second adaptor molecule may be a synthetic adaptor molecule.

[0297] The first adaptor molecule and / or the second adaptor molecule do not have to be plasmid or vector DNA.

[0298] The adaptor molecule may comprise a double-stranded portion. The double-stranded portion may comprise less than 50 base pairs, less than 45 base pairs, less than 40 base pairs, less than 35 base pairs, less than 30 base pairs, less than 25 base pairs, less than 20 base pairs, less than 15 base pairs, or less than 10 base pairs. The double-stranded portion may comprise at least 5 base pairs, at least 6 base pairs, at least 7 base pairs, at least 8 base pairs, at least 9 base pairs, at least 10 base pairs, at least 11 base pairs, at least 12 base pairs, at least 13 base pairs, at least 14 base pairs, or at least 15 base pairs.

[0299] The adaptor molecule may include a 5' phosphate, which may facilitate ligation to a linear double-stranded region.

[0300] The first adaptor molecule may comprise a portion complementary to a first end of the linear double-stranded region. The second adaptor molecule may comprise a portion complementary to a second end of the linear double-stranded region. The first adaptor molecule may comprise a portion that anneals to a first end of the linear double-stranded region. The second adaptor molecule may comprise a portion that anneals to a second end of the linear double-stranded region.

[0301] The first and / or second adaptor molecules may comprise an overhang. For example, the first and / or second adaptor molecules may comprise a 5' overhang or a 3' overhang. The first and / or second adaptor molecules may comprise a blunt end. The overhang has at least 3 nucleotides (preferably 4 to 6 nucleotides). The overhang of the first and / or second adaptor molecule may anneal to the first end and / or the second end of the linear double-stranded region.

[0302] The first adaptor molecule and / or the second adaptor molecule may not contain a type IIS endonuclease target sequence. The first adapter molecule and / or the second adapter molecule may be BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, Bs pPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI-v2, BtsMutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, Fau The polypeptides may not contain the SapI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI target sequences.

[0303] The first adaptor molecule and / or the second adaptor molecule may comprise a functional moiety. The functional moiety may be a binding molecule, a targeting sequence, or a probe.

[0304] The functional moiety may be a probe. As used herein, the term "probe" refers to a fragment of DNA, RNA or DNA / RNA chimera of variable length (e.g., 3-1000 bases long) that is used to detect the presence of a target nucleotide sequence that is complementary to the sequence in the probe. Typically, a probe hybridizes to a single-stranded nucleic acid of a base sequence that allows probe-target base pairing due to the complementarity between the probe and the target. Thus, the functional moiety may be a DNA sequence, an RNA sequence, or a DNA / RNA chimera sequence. As used herein, the term "complementary" means that the nucleotide sequences pair according to the Watson / Crick pairing rules. For example, the sequence 5'-GCGGTCCCA-3' has a complementary sequence of 5'-TGGGACCGC-3'. The complementary sequence may also be an RNA sequence that is complementary to the DNA sequence.

[0305] The functional moiety may be a binding molecule. The term "binding molecule" refers to any molecule capable of binding to a linear DNA product as described herein and / or capable of binding to a further molecule or target. The binding molecule may be a protein, a polypeptide, or a peptide. The binding molecule may be an antibody, such as a monoclonal or polyclonal antibody. The binding molecule may be an antibody fragment.

[0306] The functional moiety can facilitate detection of the DNA product by binding to a capture molecule (e.g., a capture antibody bound by a protein-protein interaction). The functional moiety can bind to a cellular target, e.g., a cellular receptor.

[0307] The functional moiety may be a label. A "label" may be any chemical entity that allows detection of a double-stranded nucleic acid molecule by physical, chemical and / or biological means. The label may be either a chromophore, a fluorophore and / or a radioactive molecule.

[0308] The functional part may be a targeting sequence. The targeting sequence is a DNA or RNA fragment, which may vary in length, and is used to target the DNA product to a specific location in the cell. The targeting sequence can be used to enhance the transfection efficiency of non-viral gene transfer by promoting the nuclear uptake of partially closed linear DNA products. For example, the targeting sequence may be a DNA nuclear targeting sequence (i.e., a recognition sequence for endogenous DNA binding proteins), such as the SV40 enhancer sequence (preferably downstream of the cassette).

[0309] To facilitate detection and / or quantification of the DNA product, the functional moiety may comprise a fluorophore, a radioactive compound or a barcode.

[0310] A signal corresponding to the presence, absence and / or level of a partially closed linear DNA product can be measured using a barcode. The barcode may comprise at least one binding moiety linked to a barcoded moiety, where the barcoded moiety comprises at least one nucleotide (i.e., the barcoded moiety comprises a nucleotide sequence at least one nucleotide long), and the binding moiety can bind to a 3' overhang, a 5' overhang or a blunt end of the partially closed linear DNA product. The binding site can bind to the 3' end and / or the 5' end of the partially closed linear DNA product. The signal can be measured by determining the presence, absence and / or level of the barcoded moiety (e.g., by sequencing or PCR). The barcoded moiety may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 nucleotides. The barcode may comprise at least two binding moieties (e.g., a first binding moiety and a second binding moiety). For example, a first binding moiety linked to a first barcode portion can bind to the 3' end of a partially closed linear double-stranded DNA product, and a second binding moiety linked to a second barcode portion can bind to the 5' end of a partially closed linear DNA product.

[0311] A signal corresponding to the presence, absence, and / or level of the partially closed linear DNA product can be measured using a fluorophore (i.e., a fluorescently labeled molecule) attached or bound to the 3' overhang, 5' overhang, or blunt end of the partially closed linear DNA product. The signal can be measured by flow cytometry and / or fluorescence activated cell sorting.

[0312] The functional moiety can also facilitate DNA sequencing. For example, the functional moiety can be a sequence adapter. The term "sequence adapter" refers to the Illumina (登録商標) (e.g., HiSeq (商標) , MiSeq (商標)and / or Genome Analyzer (商標) Sequencing System), Oxford Nanopore (商標) Technologies (e.g., MinION sequencing system), Ion Torrent (商標) (For example, Ion PGM (商標) and / or Ion Proton (商標) sequencing systems); Pacific Biosciences (e.g., PACBIO RS II sequencing system); Life Technologies (商標) The term "sequencing platform" is intended to encompass one or more nucleic acid domains that contain at least a portion of a nucleic acid sequence (or a complement thereof) utilized by a sequencing platform of interest, such as a sequencing platform provided by Axis (e.g., the SOLiD sequencing system), Roche (e.g., the 454 GS FLX+ and / or GS Junior sequencing systems), or other sequencing platform of interest.

[0313] An example of the method of the invention is shown in FIG. 17, which shows a workflow starting from amplified DNA obtained by rolling circle amplification of a circular DNA template generated by the action of Cre recombinase on a substrate containing two LoxP sequences flanking the DNA of interest in the same orientation, and obtaining a partially closed linear DNA product in a single step by digestion and ligation of adapter molecules.

[0314] The method of the present invention is further illustrated in Figure 18, which shows the sequences facilitating ligation of an adapter molecule at each cycle of the process after BsaI digestion of the amplified double-stranded DNA molecule. BsaI digestion generates a four nucleotide cohesive end at 5' on both sides of the expression cassette (upstream TCCC 5' and downstream TTTT 5'). A self-complementary adapter molecule (SEQ ID NO: 4, containing a four nucleotide cohesive end at 5' (GGGA 5')) is then ligated to the upstream side of the expression cassette. The downstream adapter is formed by hybridization of a complementary oligonucleotide (SEQ ID NO: 13 and 14) that contains a phosphorothioated internucleotide bond (indicated by an asterisk) and forms a four nucleotide cohesive end at 5' (AAAA 5'). Complementary adapter molecules are ligated to either side of the expression cassette resulting in a partially closed linear DNA product that has increased resistance to exonucleases because phosphorothioated internucleotide linkages on one side of the expression cassette prevent exonuclease degradation of the partially closed linear DNA product.

[0315] 4. Transcription and protein expression methods The present invention provides methods for in vitro transcription of a linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product), the method comprising contacting a linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product) produced by the methods described herein with a polymerase to produce a transcript encoded by the linear DNA product (e.g., the closed linear DNA product or the partially closed linear DNA product).

[0316] The present invention provides a method for the in vitro transcription of a linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product), comprising: (a) producing a linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product) by any of the methods described herein; (b) contacting the linear DNA product (e.g., the closed linear DNA product or the partially closed linear DNA product) with a polymerase; and (c) producing a transcript encoded by the linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product); The present invention provides a method comprising:

[0317] The present invention provides a method for in vitro transcription of a linear DNA product, comprising the steps of: (a) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (b) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, and wherein the first adaptor molecule is attached to a first end of the linear double-stranded region and a second adaptor molecule is attached to a second end of the linear double-stranded region; (c) contacting the linear DNA product with a polymerase; and (d) producing a transcript encoded by the linear DNA product. The present invention provides a method comprising:

[0318] The method may use an adapter molecule that generates a closed linear DNA product, such as the adapter molecules described herein. A method for in vitro transcription of a closed linear DNA product includes: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (b) incubating the single contiguous aqueous volume to produce a closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by a first adaptor molecule and closed at a second end by a second adaptor molecule; (c) contacting the closed linear DNA product with a polymerase; and (d) producing a transcript encoded by a closed linear DNA product. Includes.

[0319] The method may use an adapter molecule that includes a protected nucleotide, such as an adapter molecule described herein. The method of in vitro transcription of a linear DNA product includes: (a) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (b) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is ligated to a first end of the linear double-stranded region, and a second adaptor molecule is ligated to a second end of the linear double-stranded region, and the first and second adaptor molecules are nucleic acid molecules that include one or more nuclease-resistant nucleotides (i.e., protected nucleotides); (c) contacting the linear DNA product with a polymerase; and (d) producing a transcript encoded by the linear DNA product. Includes.

[0320] The method may use multiple adapter molecules, including adapter molecules containing protected nucleotides and hairpins or stem loops, such as the adapter molecules described herein. The method of in vitro transcription of partially closed linear DNA products includes: (a) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (b) incubating the single contiguous aqueous volume to produce a partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is ligated to a first end of the linear double-stranded region, and a second adaptor molecule is ligated to a second end of the linear double-stranded region, the first adaptor molecule is a nucleic acid molecule comprising one or more nuclease-resistant nucleotides, and the linear double-stranded region is closed at the second end by the second adaptor molecule; (c) contacting the partially closed linear DNA product with a polymerase; and (d) producing a transcript encoded by a partially closed linear DNA product. Includes.

[0321] The invention provides methods for producing a protein, the methods comprising introducing a linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product) produced by the methods described herein into a cell (e.g., a prokaryotic or eukaryotic cell) or a cell-free expression system to make the protein (e.g., the closed linear DNA product or the partially closed linear DNA product) encoded by the linear DNA product.

[0322] The present invention provides a method for producing a protein, comprising the steps of: (a) producing a linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product) by any of the methods described herein; and (b) introducing the linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product) into a cell (e.g., a prokaryotic or eukaryotic cell) or a cell-free expression system to produce a protein encoded by the linear DNA product (e.g., the closed linear DNA product or the partially closed linear DNA product). The present invention provides a method comprising:

[0323] The present invention relates to (a) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second adapter molecules to form a single contiguous aqueous volume; (b) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, and the first adaptor molecule is attached to a first end of the linear double-stranded region and a second adaptor molecule is attached to a second end of the linear double-stranded region; and (c) introducing the linear DNA product into a cell (e.g., a prokaryotic or eukaryotic cell) or into a cell-free expression system to produce a protein encoded by the linear DNA product. The present invention provides a method for producing a protein comprising the steps of:

[0324] The method can use an adapter molecule that generates a closed linear DNA product, such as the adapter molecules described herein. (a) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (b) incubating the single continuous aqueous volume to produce a closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by a first adaptor molecule and at a second end by a second adaptor molecule; and (c) introducing the closed linear DNA product into a cell (e.g., a prokaryotic or eukaryotic cell) or a cell-free expression system to produce a protein encoded by the closed linear DNA product. Includes.

[0325] The method can use an adapter molecule that includes a protected nucleotide, such as an adapter molecule described herein. (a) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (b) incubating a single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is ligated to a first end of the linear double-stranded region, and a second adaptor molecule is ligated to a second end of the linear double-stranded region, and the first and second adaptor molecules are nucleic acid molecules that include one or more nuclease-resistant nucleotides (i.e., protected nucleotides); and (c) introducing the linear DNA product into a cell (e.g., a prokaryotic or eukaryotic cell) or into a cell-free expression system to produce a protein encoded by the linear DNA product. Includes.

[0326] The method can use an adapter molecule that generates a partially closed linear DNA product, such as the adapter molecules described herein. (a) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (b) incubating the single contiguous aqueous volume to produce a partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is ligated to a first end of the linear double-stranded region, and a second adaptor molecule is ligated to a second end of the linear double-stranded region, the first adaptor molecule is a nucleic acid molecule comprising one or more nuclease-resistant nucleotides, and the linear double-stranded region is closed at the second end by the second adaptor molecule; and (c) introducing the partially closed linear DNA product into a cell (e.g., a prokaryotic or eukaryotic cell) or a cell-free expression system to produce a protein encoded by the partially closed linear DNA product. Includes.

[0327] Cell-free expression systems can be derived from (or be derived from) prokaryotic or eukaryotic cells, for example, cell-free expression systems can be derived from (or be derived from) rabbit reticulocytes, wheat germ or E. coli.

[0328] The cell may be a prokaryotic or eukaryotic cell. The cell may be an animal cell, such as a mammalian cell (e.g., a human cell), a fungal cell, a microbial cell (e.g., a prokaryotic or eukaryotic cell), or a plant cell. Preferably, the cell is a human cell.

[0329] The linear DNA product or the closed linear DNA product may comprise a cassette. The protein of interest may be encoded by the cassette.

[0330] The step of introducing the linear DNA product into a cell can be carried out in vivo or in vitro.

[0331] The nuclease resistant nucleotide (ie, the protected nucleotide) can be any protected nucleotide described herein.

[0332] 5. Cell Transfection Methods and Cell Transfection Compositions The invention provides methods of transfecting a cell with a linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product) produced by any of the methods described herein.

[0333] The present invention provides a method for transfecting a linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product) into a cell, comprising: (a) producing a linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product) by any of the methods described herein; (b) contacting the cell with a linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product); and (c) transfecting a linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product) into the cytosol of the cell. The present invention provides a method comprising:

[0334] The present invention provides a method for transfecting a cell with linear DNA, comprising the steps of: (a) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second adapter molecules to form a single contiguous aqueous volume; (b) incubating a single continuous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, and wherein the first adaptor molecule is attached to a first end of the linear double-stranded region and a second adaptor molecule is attached to a second end of the linear double-stranded region; (c) contacting the linear DNA product with the cell; and (d) transfecting the linear DNA product into the cytoplasm of the cell. The present invention provides a method comprising:

[0335] The method can use an adapter molecule that generates a closed linear DNA product, such as the adapter molecules described herein. A method for transfecting a cell with a closed linear DNA product can include: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (b) incubating the single contiguous aqueous volume to produce a closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by a first adaptor molecule and closed at a second end by a second adaptor molecule; (c) contacting the closed linear DNA product with a cell; and (d) transfecting the closed linear DNA product into the cytoplasm of the cell. may include.

[0336] The method can use an adapter molecule that includes a protected nucleotide, such as the adapter molecules described herein. The method for transfecting a linear DNA product into a cell can include: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (b) incubating a single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is ligated to a first end of the linear double-stranded region, and a second adaptor molecule is ligated to a second end of the linear double-stranded region, and the first and second adaptor molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e., protected nucleotides); (c) contacting the linear DNA product with the cell; and (d) transfecting the linear DNA product into the cytosol of the cell. may include.

[0337] The method can use an adapter molecule that includes a protected nucleotide, such as the adapter molecules described herein. The method for transfecting a partially closed linear DNA product into a cell can include: (a) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (b) incubating the single contiguous aqueous volume to produce a partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is ligated to a first end of the linear double-stranded region, and a second adaptor molecule is ligated to a second end of the linear double-stranded region, the first adaptor molecule is a nucleic acid molecule comprising one or more nuclease-resistant nucleotides, and the linear double-stranded region is closed at the second end by the second adaptor molecule; (c) contacting the cell with the partially closed linear DNA product; and (d) transfecting the partially closed linear DNA product into the cytosol of the cell. may include.

[0338] The invention provides cell transfection compositions comprising a linear DNA product (eg, a closed linear DNA product) produced (or obtained) by the methods described herein.

[0339] The cell transfection composition may or may not include a carrier, such as a drug or pharmaceutical. Preferably, the carrier promotes the accumulation of the linear DNA product at the target site and / or protects the linear DNA product from undesired interactions with components of the biological environment and / or protects the linear DNA product from metabolism and / or degradation.

[0340] The invention further provides a method of cell transfection comprising contacting (in vitro) a cell to be transfected with a linear DNA product (e.g., a closed linear DNA product) produced by the methods of the invention, wherein the linear DNA product (e.g., the closed linear DNA product) is transfected into the cytosol of the cell.

[0341] The cells to be transfected can be provided in cell culture medium (e.g., a petri dish, culture vessel, well, etc.). The linear DNA product (e.g., a closed linear DNA product) can be added directly to the cell culture medium, or the cells can be added to a solution, such as saline, buffer, cell culture medium, etc., that contains the linear DNA product (e.g., a closed linear DNA product).

[0342] The carrier can be a viral carrier or a non-viral carrier. Viral carriers include lentiviral vectors, adenoviral vectors, retroviral vectors, or adeno-associated viral vectors for delivering linear double-stranded DNA products. Non-viral carriers (or vectors) include complexing linear double-stranded DNA products with cationic agents such as cationic cell-penetrating peptides (CPPs); DNA-binding cationic moieties such as polylysine chains; cationic polymers or dendrimers, such as polyethyleneimine (PEI) and poly-D,L-lactide-co-glycolide (PLGA); and / or cationic lipids (e.g., lipofectamine).

[0343] The carrier may be a small molecule (e.g., cholesterol, bile acid, and / or lipid), polymer, protein (e.g., antibody), and / or aptamer (e.g., RNA) that binds to the linear double-stranded DNA product. The carrier may be a nanoparticle formulation used to encapsulate the linear double-stranded DNA product (e.g., closed linear DNA product).

[0344] The carrier may be a linear double-stranded DNA product modified with a targeting ligand (e.g., an antibody), a peptide, a small molecule (e.g., folic acid and / or biotin), or a polymer present in the extracellular matrix (e.g., hyaluronic acid and / or chondroitin sulfate), or a hydrophobically modified double-stranded nucleic acid molecule (e.g., with cholesterol and / or α-tocopherol).

[0345] The cell transfection composition may or may not include an agent selected from a photosensitizer and / or a radical initiator, such as a photoinitiator. Preferably, the agent improves the function of the linear double-stranded DNA product at the target site and / or protects the linear double-stranded DNA product from metabolism and / or degradation.

[0346] The present invention further provides a cell obtainable by the method of the present invention. Thus, the cell may contain a linear double-stranded DNA product (e.g., a closed linear DNA product) produced by the method of the present invention.

[0347] The invention further provides cells transfected with a linear double-stranded DNA product (eg, a closed linear DNA product) produced by the methods of the invention.

[0348] The cell may be an animal cell, such as a mammalian cell (e.g., a human cell), a fungal cell, a microbial cell (e.g., a prokaryotic or eukaryotic cell), or a plant cell. Preferably, the cell is a human cell.

[0349] The step of contacting the cell with the linear DNA product (e.g., the closed linear DNA product) may be performed in vivo. For example, the linear double-stranded DNA product (e.g., the closed linear DNA product) can be administered to an organism (e.g., a subject) that requires it. The organism (e.g., a subject) may be an animal, such as a mammal (e.g., a human), a fungus, a microorganism, or a plant.

[0350] Any or all of the linear DNA products (e.g., closed linear DNA products) described herein can be delivered to cells via delivery particles such as liposomes, nanoparticles, exosomes, macrovesicles, viral or non-viral vectors. Any or all of the linear double-stranded DNA products (e.g., closed linear DNA products) described herein can be delivered to cells using a gene gun. Any or all of the linear double-stranded DNA products (e.g., closed linear DNA products) described herein can be delivered to cells by electroporation. Any or all of the linear DNA products (e.g., closed linear DNA products) described herein can be delivered to cells by a hydrodynamic needle. Any linear DNA products (e.g., closed linear DNA products) described herein can be delivered to cells without a carrier.

[0351] The nanoparticles are preferably self-assembled nanoparticles. They may be nanoparticles produced by a process in which pre-existing components (e.g., lipid components, DNA products as described herein) form organized structures as a result of specific local interactions between the components themselves without external guidance.

[0352] The DNA product and the lipid component can reversibly interact to form a self-assembled nanoparticle. The DNA product and the lipid component can reversibly interact in the self-assembled nanoparticle by intermolecular forces. The DNA product and the lipid component can reversibly interact in the self-assembled nanoparticle by non-covalent interactions. The DNA product and the lipid component can reversibly interact in the self-assembled nanoparticle through hydrogen bonds, van der Waals interactions, hydrophobic interactions, and / or electrostatic interactions. The DNA product and the lipid component should not be bound or linked in the self-assembled nanoparticle by forces other than intermolecular forces.

[0353] 6. Pharmaceutical composition and method for producing pharmaceutical composition The invention provides pharmaceutical compositions comprising a linear DNA product described herein (eg, a closed linear DNA product) and a pharma- ceutically acceptable carrier or excipient.

[0354] The invention provides pharmaceutical compositions comprising a linear DNA product (e.g., a closed linear DNA product) produced (or obtained) by the methods described herein, and a pharma- ceutically acceptable carrier or excipient.

[0355] The present invention provides a method for producing a pharmaceutical composition comprising a linear DNA product, the method comprising carrying out a method as described herein and formulating the resulting linear DNA product with a pharma- ceutically acceptable carrier or excipient.

[0356] The present invention provides a method for producing a pharmaceutical composition, comprising the steps of: (a) producing a linear DNA product (e.g., a closed linear DNA product) by any of the methods described herein; (b) formulating the linear DNA product (e.g., the closed linear DNA product) with a pharma- ceutically acceptable carrier or excipient. The present invention provides a method comprising:

[0357] The present invention provides a method for producing a pharmaceutical composition, comprising the steps of: (a) contacting double-stranded DNA molecules with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (b) incubating the single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, and the first adaptor molecule is attached to a first end of the linear double-stranded region and a second adaptor molecule is attached to a second end of the linear double-stranded region; and (c) formulating the linear DNA product with a pharma- ceutically acceptable carrier or excipient; The present invention provides a method comprising:

[0358] The method can use an adapter molecule that generates a closed linear DNA product, such as an adapter molecule described herein. (a) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (b) incubating the single continuous aqueous volume to produce a closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprising a linear portion of a double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by a first adaptor molecule and at a second end by a second adaptor molecule; and (c) formulating the closed linear DNA product with a pharma- ceutically acceptable carrier or excipient. may include.

[0359] The method can use an adapter molecule that includes a protected nucleotide, such as an adapter molecule described herein. The method for producing a pharmaceutical composition can include: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (b) incubating a single contiguous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is ligated to a first end of the linear double-stranded region, and a second adaptor molecule is ligated to a second end of the linear double-stranded region, and the first and second adaptor molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e., protected nucleotides); (c) formulating the linear DNA product with a pharma- ceutically acceptable carrier or excipient; may include.

[0360] The method can use an adapter molecule that generates a partially closed linear DNA product, such as the adapter molecules described herein. (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second adaptor molecules to form a single contiguous aqueous volume; (b) incubating the single contiguous aqueous volume to produce a partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is ligated to a first end of the linear double-stranded region, and a second adaptor molecule is ligated to a second end of the linear double-stranded region, the first adaptor molecule is a nucleic acid molecule comprising one or more nuclease-resistant nucleotides, and the linear double-stranded region is closed at the second end by a second adaptor molecule; and (c) formulating the partially closed linear DNA product with a pharma- ceutically acceptable carrier or excipient. may include.

[0361] The pharmaceutical compositions can be formulated as pills, tablets or capsules in combination with one or more pharma- ceutically acceptable solid carriers, or as a solution in one or more pharma- ceutically acceptable solvents, or as an emulsion, suspension or dispersion in one or more pharma- ceutically acceptable solvents or carriers. The formulations may also contain other pharma- ceutically acceptable excipients, such as stabilizers, antioxidants, binders, colorants, emulsifiers or taste-modifying agents, sustained-release formulations, and the like.

[0362] The pharmaceutical composition can be administered orally, topically, parenterally, transdermally, or by inhalation.The pharmaceutical composition can be administered by injection or intravenous infusion using a suitable sterile solution.Topical dosage forms can be creams, ointments, patches, or similar vehicles suitable for transdermal and topical dosage forms.

[0363] Pharmaceutical compositions may be dissolved or suspended in a liquid vehicle or formulated as granules (small particles or granules), pellets (small sterile solid masses of highly purified composition produced by granulation or compression molding, with or without excipients), or pellet coated sustained release (a solid dosage form in which the composition itself is in the form of granules coated with varying amounts of coating, which releases the composition in a manner that allows for reduced dosing frequency compared to compositions presented as conventional dosage forms).

[0364] Other forms of pharmaceutical compositions include tablets (small round solid dosage forms containing the composition intended for oral administration), powders (intimate mixtures of dry, finely divided composition and one or more pharma- ceutically acceptable excipients, which may be intended for internal or external use), elixirs (clear, flavorful, sweetened hydroalcoholic liquids containing the dissolved composition, which are intended for oral administration), chewing gums (sweetened, flavorful, insoluble plastic materials of various shapes that release the composition in the mouth when chewed), syrups (oral solutions containing the composition and a high concentration of sucrose or other sugar; this term also applies to sweetened, viscous liquids, including oral suspensions). These include tablet (a solid dosage form containing a composition with or without a suitable diluent), chewable tablet (a solid dosage form containing a composition with or without a suitable diluent that is intended to be chewed, that leaves a pleasant tasting residual in the mouth, is easily swallowed, and does not leave a bitter or unpleasant aftertaste), tablet coated or tablet delayed release, tablet dispersible, tablet effervescent, tablet extended release, tablet film coated, or tablet film coated extended release, where the tablet is formulated to make the contained drug available for an extended period of time after ingestion.

[0365] In other forms of pharmaceutical compositions, tablets for solution, tablets for suspension, multilayer tablets, multilayer extended release tablets may be provided, where the tablets are formulated in such a way as to allow at least a reduction in the frequency of administration compared to compositions presented as conventional dosage forms. Oral disintegrating tablets, oral disintegrating delayed release tablets, soluble tablets, sugar-coated tablets, osmotic tablets, etc. are also suitable.

[0366] The oral dosage form pharmaceutical composition may contain, in addition to the composition, one or more inactive pharmaceutical ingredients such as diluents, solubilizers, alcohols, binders, release-controlling polymers, enteric polymers, disintegrants, excipients, colorants, flavoring agents, sweeteners, antioxidants, preservatives, pigments, additives, fillers, suspending agents, surfactants (e.g., anionic, cationic, amphoteric and nonionic), etc. A variety of topical inactive ingredients approved by the FDA can be found in the FDA's "The Inactive Ingredients Database."

[0367] Dosage forms of injections and infusions for use in the present invention include, but are not limited to, liposomal injections consisting of liposomes (lipid bilayer vesicles typically composed of phospholipids used to encapsulate the composition) or forming liposomes; injections comprising sterile preparations intended for parenteral use; emulsion injections comprising emulsions consisting of sterile pyrogen-free preparations intended for parenteral administration; or lipid complex injections.

[0368] For example, linear double-stranded DNA products (e.g., closed linear DNA products) can be administered by intratympanic injection (e.g., injection into the middle ear) and / or injection into the outer ear, middle ear and / or inner ear. Such methods are routinely used in the art, for example, for administering steroids and antibiotics to the human ear. For example, injection can be through the round window of the ear or through the cochlear capsule.

[0369] Other forms of pharmaceutical compositions include solution injectable powders, which are sterile preparations intended for reconstitution to form a solution for parenteral use; suspension injectable powders, which are sterile preparations intended for reconstitution to form a suspension for parenteral use; liposomal suspension injectable lyophilized powders, which are sterile lyophilized preparations intended for reconstitution for parenteral use and formulated to form liposomes (lipid bilayer vesicles usually composed of phospholipids and used to encapsulate compositions within the lipid bilayer or aqueous space) upon reconstitution; or lyophilized powders for solution injection (where lyophilization (“freeze drying”) is the process of removing water from a product in a frozen state at very low pressure).

[0370] Suspension injections include liquid formulations suitable for injection that consist of solid particles dispersed throughout a liquid phase in which the particles do not dissolve, and an oil phase dispersed throughout an aqueous phase, or vice versa. Suspension liposome injections are liquid formulations suitable for injection that consist of an oil phase dispersed throughout an aqueous phase to form liposomes (lipid bilayer vesicles, usually composed of phospholipids, used to encapsulate compositions within the lipid bilayer or aqueous space). Suspension sonicated injections include liquid formulations suitable for injection that consist of solid particles dispersed throughout a liquid phase in which the particles do not dissolve. Additionally, sonicating the product while bubbling gas through the suspension causes the solid particles to form microspheres.

[0371] In another method of administration, the pharmaceutical composition can be administered in situ via a catheter or pump, for example, which can direct the composition to the target site.

[0372] Parenteral carrier systems include one or more pharma- ceutically suitable excipients, such as solvents and cosolvents, solubilizers, wetting agents, suspending agents, thickening agents, emulsifying agents, chelating agents, buffers, pH adjusters, antioxidants, reducing agents, antimicrobial preservatives, bulking agents, protectants, tonicity adjusters, and special additives. Formulations suitable for parenteral administration conveniently include a sterile oily or aqueous preparation of the composition, preferably isotonic with the blood of the recipient, although this is not required.

[0373] Inhalation dosage forms for use in the present invention include, but are not limited to, aerosols (products containing a composition packaged under pressure and released upon actuation of a suitable valve system intended for topical application to the skin as well as to the nose (nasal aerosols), mouth (lingual and sublingual aerosols) or lungs (inhalation aerosols). Foam aerosols are dosage forms containing a composition, a surfactant, an aqueous or non-aqueous liquid, and a propellant, which delivers a stable foam when the propellant is in the internal (discontinuous) phase (i.e., oil-in-water type), or a mist or fast-breaking foam when the propellant is in the external (continuous) phase (i.e., water-in-oil type). Metered-dose aerosols are pressurized dosage forms consisting of a metered-dose valve that allows a uniform amount of spray with each actuation. Powder aerosols are products containing a composition in the form of a powder packaged under pressure and released upon actuation of a suitable valve system. Aerosol sprays are aerosol products that utilize compressed gas as a propellant to provide the force necessary to expel the product as a wet spray, and are applicable to a solution of the composition in an aqueous solvent.

[0374] Transdermal dosage forms for use in the present invention include, but are not limited to, patches, which are drug delivery systems that often include an adhesive backing that is typically applied to an external site on the body, whereby ingredients (including the composition) are passively diffused or actively transported through a portion of the patch, thereby delivering ingredients (including the composition) to the external surface of the body or internally. Various types of transdermal patches are known in the art, such as matrix, reservoir, etc.

[0375] Topical dosage forms for use in the present invention include various dosage forms known in the art, such as lotions (emulsions, liquid dosage forms, which dosage forms are generally for external application to the skin), lotion enhancers (lotion dosage forms which enhance the delivery of the composition, where enhancement does not refer to the strength of the composition in the dosage form), gels (semi-solid dosage forms that contain a gelling composition to provide rigidity to a solution or colloidal dispersion, where the gel may contain suspended particles), and ointments (semi-solid dosage forms that typically contain less than 20% water and volatile materials, and 50% or more hydrocarbons, waxes, or polyols as a vehicle, which dosage forms are generally for external application to the skin or mucous membranes). Further embodiments include ointment enhancers (ointment dosage forms that enhance the delivery of the composition, where enhancement does not refer to the strength of the composition in the dosage form), creams (emulsions, usually containing 20% ​​or more water and volatiles and / or less than 50% hydrocarbons, waxes or polyols, semi-solid dosage forms can also be used as vehicles, whereby the dosage form is typically applied externally to the skin or mucosa) and cream enhancers (cream dosage forms that enhance the delivery of the composition, where enhancement does not refer to the strength of the composition in the dosage form).As used herein, "emulsion" refers to a dosage form that consists of a two-phase system of at least two immiscible liquids, one of which is dispersed as droplets, an internal or dispersed phase, within another liquid, an external or continuous phase, and is typically stabilized with one or more emulsifiers. Further embodiments include suspensions (liquid dosage forms containing solid particles dispersed in a liquid vehicle), suspension sustained release, pastes (semi-solid dosage forms containing as much as 20-50% finely dispersed solids in a fatty vehicle, which dosage forms are typically applied topically to the skin or mucous membranes), solutions (clear, homogeneous liquid dosage forms containing one or more chemicals dissolved in a solvent or mixture of mutually miscible solvents), and powders.

[0376] Topical dosage form compositions contain the composition and one or more inactive pharmaceutical ingredients, such as excipients, colorants, pigments, additives, fillers, emollients, surfactants (e.g., anionic, cationic, amphoteric and nonionic), penetration enhancers (alcohols, fatty alcohols, fatty acids, fatty acid esters, polyols, etc.), etc. A variety of topical inactive ingredients approved by the FDA can be found in the FDA's "The Inactive Ingredients Database."

[0377] 7. Linear DNA products The present invention provides linear DNA products (eg, closed linear DNA products) as described herein.

[0378] The present invention provides a closed linear DNA product comprising a linear double-stranded region, where the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by a first adaptor molecule and closed at a second end by a second adaptor molecule.

[0379] The present invention provides a closed linear DNA product comprising a linear portion of a double-stranded DNA molecule, where the linear portion of the double-stranded DNA molecule is closed at a first end by a first adaptor molecule and at a second end by a second adaptor molecule.

[0380] The present invention provides a linear DNA product comprising a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of a double-stranded DNA molecule, the first adaptor molecule is ligated to a first end of the linear double-stranded region and a second adaptor molecule is ligated to a second end of the linear double-stranded region, and the first and second adaptor molecules are nucleic acid molecules comprising one or more nuclease resistant nucleotides.

[0381] The present invention provides a linear DNA product comprising a linear portion of a double-stranded DNA molecule, where a first adaptor molecule is ligated to a first end of the linear portion of the double-stranded DNA molecule and a second adaptor molecule is ligated to a second end of the linear portion of the double-stranded DNA molecule, and the first and second adaptor molecules are nucleic acid molecules comprising one or more nuclease resistant nucleotides.

[0382] The present invention provides a partially closed linear DNA product comprising a linear portion of a double-stranded DNA molecule, where a first adaptor molecule is ligated to a first end of the linear portion of the double-stranded DNA molecule, and a second adaptor molecule is ligated to a second end of the linear portion of the double-stranded DNA molecule, and the first adaptor molecule is a nucleic acid molecule comprising one or more nuclease resistant nucleotides, and the linear portion of the double-stranded DNA molecule is closed at the second end by the second adaptor molecule. Thus, the present invention provides a partially closed linear DNA product, the second end of which is closed (or covalently closed) and the first end is open. The partially closed linear DNA product comprises an open-ended region and / or one or more nuclease resistant nucleotides at the first end. The open-ended region is at the 3' or 5' end of the molecule.

[0383] By open-end region is meant at least 5 base pairs, at least 10 base pairs, at least 15 base pairs, or at least 20 base pairs located closest to the open end of the DNA product. The partially closed linear DNA product may contain one or more nuclease-resistant nucleotides in the sense and / or antisense strand. Thus, for example, one or more nucleotides of the 20 base pairs located closest to the open end of the partially closed linear DNA product may be nuclease-resistant nucleotides. Preferably, the partially closed linear DNA product contains at least 5 nuclease-resistant nucleotides in the open end region.

[0384] The partially closed linear DNA product may comprise a double-stranded DNA portion that is closed at a first end and open at a second end. The partially closed linear DNA product may comprise a double-stranded DNA portion that is closed at a first end by a single-stranded portion (i.e., comprises a first hairpin at the first end) and open at the first end. The partially closed linear DNA product may comprise one or more nuclease resistant nucleotides in the open-ended region adjacent to the second end. The open-ended region adjacent to the second end may be at the 3' or 5' end of the molecule. The open ended region adjacent to the second end has 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 25, at least 30, at least 35, at least 40, at least 45, or at least 50 nucleotides located at the second end of the partially closed linear DNA product. That is, the open-ended region adjacent to the second end may include any nucleotide between and including the terminal nucleotide of the second end and a nucleotide that is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 away from the terminal nucleotide of the second end.

[0385] The open-ended region adjacent to the second end may comprise a sense strand and an antisense strand. The open-ended region adjacent to the second end may comprise one or more nuclease-resistant nucleotides in the sense strand or the antisense strand. The open-ended region adjacent to the second end may comprise one or more nuclease-resistant nucleotides in both the sense strand and the antisense strand. The open-ended region adjacent to the second end may comprise two or more, three or more, four or more, or five or more nuclease-resistant nucleotides in both the sense strand and the antisense strand. Preferably, the open-ended region adjacent to the second end comprises five nuclease-resistant nucleotides in both the sense strand and the antisense strand.

[0386] The partially closed linear DNA product may comprise a hairpin loop at the 5' or 3' end. The partially closed linear DNA product may comprise a first adaptor molecule at a first end and a second adaptor molecule at a second end. The first adaptor molecule may comprise a hairpin and the second adaptor may comprise one or more protected nucleotides (i.e., nucleotides that are resistant to nuclease (e.g., exonuclease) digestion). The hairpin may confer resistance to nuclease (e.g., exonuclease) digestion. The presence of the protected nucleotides may confer resistance to nuclease (e.g., exonuclease) digestion. A partially closed linear DNA product may be a DNA molecule that has a double-stranded portion closed at a first end by ligation of a first adaptor (e.g., a hairpin adaptor) to the first end, and includes a double-stranded linear adaptor that includes protecting nucleotides (i.e., nucleotides that are resistant to nuclease (e.g., exonuclease) digestion) at the second end.

[0387] The partially closed linear DNA product may comprise (i) a cassette, where the cassette comprises a sense strand and an antisense strand; and (ii) one or more nuclease resistant nucleotides in an open-ended region of the partially closed linear DNA product, where the open-ended region is 5' of the sense strand of the cassette. The partially closed linear DNA product may comprise (i) a cassette, where the cassette comprises a sense strand and an antisense strand; and (ii) one or more nuclease resistant nucleotides in an open-ended region of the partially closed linear DNA product, where the open-ended region is 5' of the antisense strand of the cassette. The partially closed linear DNA product may comprise (i) a cassette, where the cassette comprises a sense strand and an antisense strand; and (ii) one or more nuclease resistant nucleotides in an open-ended region of the partially closed linear DNA product, where the open-ended region is 3' of the sense strand of the cassette. The partially closed linear DNA product may comprise: (i) a cassette, where the cassette comprises a sense strand and an antisense strand; and (ii) one or more nuclease resistant nucleotides in an open-ended region of the partially closed linear DNA product, where the open-ended region is 3' of the antisense strand of the cassette.

[0388] The partially closed linear DNA product may comprise: (i) a cassette, where the cassette comprises a sense strand and an antisense strand; (ii) one or more nuclease resistant nucleotides in an open ended region of the partially closed linear DNA product, where the open ended region is 5' of the sense strand of the cassette; and (iii) one or more nuclease resistant nucleotides in an open ended region of the partially closed linear DNA product, where the open ended region is 3' of the antisense strand of the cassette.

[0389] The partially closed linear DNA product may comprise: (i) a cassette, where the cassette comprises a sense strand and an antisense strand; (ii) one or more nuclease resistant nucleotides in an open ended region of the partially closed linear DNA product, where the open ended region is 3' of the sense strand of the cassette; and (iii) one or more nuclease resistant nucleotides in an open ended region of the partially closed linear DNA product, where the open ended region is 5' of the antisense strand of the cassette.

[0390] The closed linear DNA product, the linear DNA product, or the partially closed linear DNA product may comprise a cassette, optionally a single cassette. The linear portion of the double-stranded DNA molecule (of the closed linear DNA product, the linear DNA product, or the partially closed linear DNA product) may comprise a cassette, optionally a single cassette. Thus, the cassette (or the single cassette) is located between the first adaptor molecule and the second adaptor molecule.

[0391] The term "single cassette" as used herein is intended to encompass molecules that do not contain or consist of multiple cassettes. That is, a closed linear DNA product, a linear DNA product, or a partially closed linear DNA product contains only a single cassette, which may contain a single coding sequence of a gene of interest. A single cassette does not contain or consist of multiple tandem repeat sequences and / or concatenated DNA. The term "single cassette" as used herein is intended to encompass a single copy of a DNA sequence of interest, e.g., a single copy of a coding sequence. Thus, a "single cassette" does not encompass cassettes that contain multiple copies of the same DNA sequence linked in tandem. A single cassette may contain a collection of genes of interest. For example, a single cassette may contain sequences of at least two, three, four, or five genes of interest. The genes of interest are not necessarily the same in a single cassette.

[0392] The invention provides linear DNA products (eg, closed linear DNA products or partially closed linear DNA products) obtained by any of the methods described herein.

[0393] 8. Uses and Applications The present invention provides for the use of a linear DNA product (eg, a closed linear DNA product) described herein in the manufacture of a viral or non-viral delivery system.

[0394] The present invention provides the use of a linear DNA product (e.g., a closed linear DNA product) in the manufacture of a viral or non-viral delivery system, wherein the linear DNA product (e.g., a closed linear DNA product) is produced by carrying out a method described herein.

[0395] The present invention provides viral or non-viral delivery systems comprising a linear DNA product (e.g., a closed linear DNA product) as described herein.The present invention provides viral or non-viral delivery systems comprising a linear DNA product (e.g., a closed linear DNA product), wherein the linear DNA product (e.g., a closed linear DNA product) is produced by carrying out a method as described herein.

[0396] Viral Vectors Methods for producing viral vectors, such as AAV vectors, are known in the art. The most widely used method involves co-transfection of three bacterial plasmids into HEK293. The first plasmid encodes the Rep and Cap elements, the second is a helper plasmid, while the third encodes the gene payload of interest with inverted terminal repeats (ITRs). There are several problems when using plasmids for virus preparation (e.g., AAV): 1) plasmid production is time-consuming and costly; 2) propagation of ITR sequences in E. coli is difficult; 3) integration of the plasmid backbone into the viral capsid (e.g., AAV capsid) is difficult (e.g., antibiotic resistance marker problems). Methods for producing viral vectors may use other cell lines. For example, cell lines suitable for viral vector production may include Vero cells or other stable cell lines. Together, these are the main bottlenecks in viral vector production (e.g., AAV production). Thus, the methods described herein provide linear double-stranded DNA products (e.g., closed linear DNA products) suitable for use in the manufacture of viral vectors. The linear double-stranded DNA products (e.g., closed linear DNA products) overcome the above-mentioned problems of plasmid vectors.

[0397] The present invention provides a method for producing a viral vector, comprising introducing a linear DNA product (e.g., a closed linear DNA product) produced by the method described herein into a cell under conditions such that a viral vector is produced. The linear DNA product (e.g., a closed linear DNA product) may encode at least one element required for the production of a viral vector. For example, the linear DNA product (e.g., a closed linear DNA product) may encode a Rep and / or Cap element. The linear DNA product (e.g., a closed linear DNA product) may encode a helper plasmid element. The linear DNA product (e.g., a closed linear DNA product) may encode a Rep, Cap and helper plasmid element. The linear DNA product (e.g., a closed linear DNA product) may encode a transgene. The method may be an in vivo method or an in vitro method. The cell may be an animal cell, preferably a mammalian cell, such as a human cell (e.g., HEK293T, HEK293, CAP, CAP-T, CHO). The cell may be an in vitro cultured cell in a tissue culture cell line.

[0398] Preferably, the vector is an AAV vector or a lentiviral vector.

[0399] The present invention also provides a method of delivering a viral vector (e.g., a closed linear DNA product) to a cell, comprising contacting the cell with a viral vector produced by the methods described herein. The cell can be an animal cell, preferably a mammalian cell, such as a human cell.

[0400] The present invention also provides cells obtainable by the methods described herein.

[0401] Non-viral vectors Methods for producing non-viral vectors are known in the art. Non-viral vectors have several advantages over viral vectors, including the opportunity for repeated administration due to non-immunogenicity, unlimited packaging capacity, and low associated toxicity. Most methods for producing non-viral vectors use plasmid DNA. Thus, the linear DNA products (e.g., closed linear DNA products) produced by the methods described herein are suitable for use in producing non-viral vectors. The linear DNA products (e.g., closed linear DNA products) produced by the methods of the present invention overcome the problems of using plasmid vectors in non-viral vector preparation. For example, the linear DNA products (e.g., closed linear DNA products) do not constitute a bacterial backbone, unlike plasmid DNA, allowing for more transgene copies per mg of DNA. In addition, the linear DNA products (e.g., closed linear DNA products) are free of antibiotic resistance genes and bacterial contaminants. Additionally, linear DNA products (e.g., closed linear DNA products) prolong transgene expression (due to the presence of exonuclease resistant nucleotides) and provide a more cost-effective process for the production of non-viral vectors.

[0402] The present invention provides a method of delivering a non-viral vector to a cell, the method comprising contacting a cell with a non-viral vector comprising a linear DNA product (e.g., a closed linear DNA product). The cell can be an animal cell, preferably a mammalian cell, such as a human cell.

[0403] The present invention also provides cells obtainable by the methods described herein.

[0404] General therapeutic and diagnostic applications The linear DNA products (e.g., closed linear DNA products) produced by the methods described herein are particularly suitable for use in therapy. The present invention provides linear DNA products (e.g., closed linear DNA products) described herein for use in therapy. The present invention provides linear DNA products (e.g., closed linear DNA products) obtained by the methods described herein for use in therapy. The linear DNA products (e.g., closed linear DNA products) may code for sequences of therapeutic proteins, parts of vaccines, or elements of genetic engineering mechanisms, and may be used to treat disease or infection in a subject.

[0405] The present invention further provides a linear DNA product (e.g., a closed linear DNA product) as described herein for use as a medicament. The present invention further provides a linear DNA product (e.g., a closed linear DNA product) obtained by the methods described herein for use as a medicament. The present invention also provides the use of a linear DNA product (e.g., a closed linear DNA product) as described herein for the manufacture of a medicament for treating a disease. The present invention also provides the use of a linear DNA product (e.g., a closed linear DNA product) obtained by the methods described herein for the manufacture of a medicament for treating a disease.

[0406] The linear DNA product (e.g., a closed linear DNA product) may encode a therapeutic protein sequence, a part of a vaccine, or an element of a genetic engineering mechanism and may be used to treat a disease or infection in a subject.

[0407] The present invention further provides a linear DNA product produced by the methods described herein for use in the treatment of disease.

[0408] The present invention also provides a method for treating a disease in a subject, comprising administering to the subject a linear DNA product (e.g., a closed linear DNA product) described herein. The present invention also provides a method for treating a disease in a subject, comprising administering to the subject a linear DNA product (e.g., a closed linear DNA product) obtained by the method described herein. Preferably, the amount of linear DNA product administered to the subject is a therapeutically effective amount.

[0409] The linear DNA products (e.g., closed linear DNA products) described herein may be used to treat any disease or disorder. For example, the linear DNA products (e.g., closed linear DNA products) may be used to treat one or more diseases and / or disorders selected from genetic diseases (e.g., monogenic diseases), cancer, HIV, other viral infections (e.g., infections caused by coronaviruses (e.g., COVID-19), hepatitis A, hepatitis B, herpes simplex virus type 2, influenza, measles, and / or respiratory syncytial virus), neurodegenerative diseases (e.g., polyglutamine diseases such as Parkinson's disease and / or Huntington's disease), eye diseases (e.g., macular degeneration) and liver failure. Preferably, the linear DNA products (e.g., closed linear DNA products) are used to treat genetic diseases. More preferably, the linear DNA products (e.g., closed linear DNA products) are used to treat monogenic diseases. For example, linear DNA products (e.g., closed linear DNA products) may be used to treat sickle cell anemia, cystic fibrosis, Huntington's disease and Duchenne muscular dystrophy, hemophilia A, alpha-1-antitrypsin deficiency, primary ciliary dyskinesia, or respiratory distress syndrome of prematurity.

[0410] A subject to be treated with a linear DNA product (e.g., a closed linear DNA product) may receive the linear DNA product (e.g., a closed linear DNA product) in the form of any of the pharmaceutical compositions described herein.

[0411] The subject treated with linear DNA product (e.g., closed linear DNA product) can receive linear DNA product (e.g., closed linear DNA product) in combination with other forms of treatment for said disorder, including treatment with drugs commonly used to treat said disorder. The drug is administered once or multiple times. Those skilled in the art (e.g., attending physician) are fully capable of determining the appropriate dosing regimen for a subject, depending on the specific situation of the subject.

[0412] As used herein, "administering" refers to introducing the linear DNA product into the subject's body, as described in more detail above (see "Method of Manufacturing Pharmaceutical Compositions" section), including, but not limited to, oral, topical, buccal, sublingual, pulmonary, transdermal, mucosal, subcutaneous, intraperitoneal, intravenous and intramuscular injections, or in liquid or solid form via the gastrointestinal canal.

[0413] The term "therapeutically effective amount" as used herein means an amount of linear DNA product that, when administered to a subject to treat a disease, is sufficient to effectively treat the disease. A "therapeutically effective amount" will vary depending on factors such as the particular product used, the severity of the disease in the subject, the age and relative health of the subject, the route and form of administration, etc. It is routine for a person skilled in the art (e.g., a physician) to determine the appropriate therapeutically effective amount for a particular subject based on such factors. Treatment of a disease as described herein should be understood to mean the amelioration of one or more symptoms of the disease.

[0414] The present invention also provides the use of a linear DNA product (e.g., a closed linear DNA product) as described herein in a method for diagnosing a disease and / or disorder.The present invention also provides the use of a linear DNA product (e.g., a closed linear DNA product) obtained by a method as described herein in a method for diagnosing a disease and / or disorder.

[0415] The present invention provides for the use of a linear DNA product (e.g., a closed linear DNA product) in the "in vitro" diagnosis of disease. The present invention provides for the use of a linear DNA product (e.g., a closed linear DNA product) obtained by the methods described herein in the "in vitro" diagnosis of disease.

[0416] The present invention also provides linear DNA products (eg, closed linear DNA products) for use in "in vivo" methods for the diagnosis of disease.

[0417] The method can be used for the diagnosis of any disease and / or disorder. For example, the disease and / or disorder can be selected from genetic disorders (e.g., single gene disorders), cancer, HIV, other viral infections (e.g., infections with coronaviruses (e.g., COVID-19), hepatitis A, hepatitis B, herpes simplex virus type 2, influenza, measles and / or respiratory syncytial virus), neurodegenerative diseases (e.g., polyglutamine diseases such as Parkinson's disease and / or Huntington's disease), eye diseases (e.g., macular degeneration) and liver failure. Preferably, the linear DNA product is used for the diagnosis of genetic diseases. More preferably, the linear DNA product is used for the diagnosis of single gene diseases. For example, the linear DNA product can be used for the diagnosis of sickle cell anemia, cystic fibrosis, Huntington's disease, and Duchenne muscular dystrophy, hemophilia A, alpha 1-antitrypsin deficiency, primary ciliary dyskinesia or respiratory distress syndrome of prematurity.

[0418] The diagnostic and treatment methods described herein may be in vitro or in vivo methods.

[0419] Diagnostic methods can vary, depending on the detection and / or quantification of linear DNA products (eg, closed linear DNA products).

[0420] To facilitate detection and / or quantification of the linear DNA product (e.g., closed linear DNA product), the linear DNA product can be attached or bound to a functional moiety. The functional moiety can be any functional moiety described herein. For example, the functional moiety can be a probe. The functional moiety can include a fluorophore, a radioactive compound, or a barcode. The functional moiety can be a protein, such as an antibody.

[0421] Diagnosis can vary depending on the detection of a signal corresponding to the presence, absence and / or level of the linear DNA product. For example, the signal can be measured by flow cytometry and / or fluorescence activated cell sorting of the linear DNA product bound to a fluorescent probe.

[0422] The linear DNA product (e.g., a closed linear DNA product) can be detected by binding to a capture site, for example, in a lateral flow assay. In this example, the functional moiety is a protein, for example, an antibody specific for the capture moiety. Capture of the antibody bound to the linear DNA product can result in a visual signal (e.g., a band of a different color).

[0423] The present invention also provides a combined method of disease diagnosis and disease treatment.

[0424] cell therapy The products produced by the methods of the invention may be substantially less contaminated than plasmid DNA. In addition, the products produced by the methods of the invention are very simple in nature (e.g., do not have a bacterial backbone), which means that they are generally easier to work with. The pure and simple nature of the products described herein makes them particularly suitable for use in cell therapy. For example, cells containing a linear DNA product (e.g., a closed linear DNA product) can be injected or otherwise implanted into a patient to induce a desired effect. The cell (or cells) can combat cancer cells, for example, via cell-mediated immunity in the course of immunotherapy. The cell (or cells) can be implanted to regenerate diseased tissue.

[0425] The present invention provides linear DNA products (e.g., closed linear DNA products) as described herein for use in cell therapy. The present invention provides linear DNA products (e.g., closed linear DNA products) obtained by the methods described herein for use in cell therapy.

[0426] The present invention provides linear DNA products (e.g., closed linear DNA products) as described herein for use in cell therapy. The present invention provides linear DNA products (e.g., closed linear DNA products) obtained by the methods described herein for use in cell therapy.

[0427] Preferably, the cell therapy is ex vivo cell therapy. The cells are animal cells, preferably mammalian cells, such as human cells.

[0428] The invention also provides a cell obtained by any of the methods described herein, e.g., the cell is suitable for use in cell therapy.

[0429] vaccine The linear DNA product (e.g., closed linear DNA product) produced by the methods described herein is particularly suitable for use in vaccine production. The vaccine may comprise the linear DNA product (e.g., closed linear DNA product) described herein. The vaccine may comprise the linear DNA product (e.g., closed linear DNA product) obtained by the methods described herein. Alternatively, the linear DNA product (e.g., closed linear DNA product) can be used to produce a vaccine, preferably an mRNA-based vaccine, such as the BioNTech vaccine and the Moderna mRNA vaccine against COVID-19.

[0430] Thus, the present invention provides the use of a linear DNA product (e.g., a closed linear DNA product) as described herein in the manufacture of a vaccine. The present invention also provides the use of a linear DNA product (e.g., a closed linear DNA product) obtainable by a method as described herein in the manufacture of a vaccine.

[0431] The linear DNA product (e.g., the closed linear DNA product) may encode an antigen and may elicit an immune response in a subject. The subject may be a human. Preferably, the antigen is encoded by a cassette.

[0432] CAR-T cells The present invention provides for the use of a linear DNA product (e.g., a closed linear DNA product) as described herein in the production of CAR-T cells. The present invention provides for the use of a linear DNA product (e.g., a closed linear DNA product) obtained by the methods described herein in the production of CAR-T cells.

[0433] The present invention provides a method for producing a genetically modified CAR-T cell, comprising: (a) introducing into a T cell a linear DNA product (e.g., a closed linear DNA product) described herein; and (b) expressing a gene of interest encoded by the linear DNA product (e.g., the closed linear DNA product). Preferably, the gene of interest is a tumor-specific CAR.

[0434] The present invention provides a method for generating engineered CAR-T cells, comprising: (a) introducing into a T cell a linear DNA product (e.g., a closed linear DNA product) as described herein; and (b) expressing a gene of interest encoded by the linear DNA product (e.g., the closed linear DNA product). Preferably, the gene of interest is a tumor-specific CAR.

[0435] The method may further comprise the step of removing mononuclear cells from the patient prior to step (a). Preferably, the removing step is performed using leukocyte therapy. Preferably, the mononuclear cells are T cells. The method may further comprise the step of returning the genetically modified cells to the patient after step (b).

[0436] The present invention also provides modified T cells obtained by any of the methods described herein. The modified T cells may be suitable for use in CAR T cell therapy.

[0437] CRISPR delivery The products produced by the methods described herein are particularly suitable for use with the CRISPR system for delivery to cells, for example in cell therapy or in vivo therapy.

[0438] A variety of different cargoes and delivery vehicles are commonly used in CRISPR systems, including physical delivery methods (e.g., microinjection, electroporation), viral delivery methods (e.g., adeno-associated viruses (AAV); full-length adenoviruses and lentiviruses), and non-viral delivery methods (e.g., liposomes; polyplexes; gold particles).

[0439] A linear DNA product (e.g., a closed linear DNA product) may contain a gene sequence encoding any component of the CRIPSR system. A linear double-stranded DNA product may encode all components of the CRIPSR system.

[0440] The linear DNA product (e.g., closed linear DNA product) may comprise (or code for) a repair template (or editing template). The repair template (or editing template) may be for editing a genome, for example, using a CRISPR-Cas system. The repair template (or editing template) comprises or consists of a homology region (e.g., a homology arm) that is homologous to a desired DNA region (i.e., a target molecule). The repair template (or editing template) may be for use in CRISPR-Cas-mediated homology directed repair (HDR). The repair template (or editing template) may be used to repair a target molecule that has a strand break (such as a single-strand break or a double-strand break). The strand break may be generated by a nuclease (e.g., Cas9, Cpf1 or MAD7) of the CRISPR system. The repair template (or editing template) is capable of introducing at least one mutation (e.g., an insertion, deletion, and / or substitution) into a desired DNA region (i.e., a target molecule). The repair template (or editing template) is at least 10 base pairs, 20 base pairs, 30 base pairs, 40 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, 700 base pairs, 800 base pairs, 900 base pairs, 1000 base pairs, 1500 base pairs, 2000 base pairs, 2500 base pairs, 3000 base pairs, 3500 base pairs, 4000 base pairs, 4500 base pairs, 5000 base pairs, 5500 base pairs, 6000 base pairs, 6500 base pairs, 7000 base pairs, 7500 base pairs, 8000 base pairs, 8500 base pairs, 9000 base pairs, 9500 base pairs, or 10000 base pairs in length. The linear DNA product (e.g., closed linear DNA product) encoding the repair template can be delivered to cells by nanoparticles, non-viral vectors, viral vectors, or without any carrier. The linear DNA product (e.g., closed linear DNA product) encoding the repair template can be delivered to cells by electroporation.A linear DNA product (eg, a closed linear DNA product) encoding a repair template can be delivered to a cell by a hydrodynamic needle.

[0441] The linear DNA product (e.g., closed linear DNA product) may comprise (or may further comprise) a gene sequence encoding a nuclease protein (e.g., Cas9, Cpf1 or MAD7) of the CRISPR system, and / or a guide RNA. The linear DNA product (e.g., closed linear DNA product) may comprise (or may further comprise) a gene sequence encoding a nuclease protein (e.g., Cas9, Cpf1 or MAD7) of the CRISPR system. The linear DNA product (e.g., closed linear DNA product) may comprise (or may further comprise) a gene sequence encoding a guide RNA. The linear DNA product (e.g., closed linear DNA product) may comprise (or may further comprise) a gene sequence encoding a nuclease protein (e.g., Cas9, Cpf1 or MAD7) of the CRISPR system, and a guide RNA. The linear DNA product (e.g., closed linear DNA product) may contain (or may further contain) a gene sequence (e.g., a spacer) that codes for the genome target to be modified. The linear DNA product (e.g., closed linear DNA product) may also be ligated to a vector. The vector may contain the sequence of some components of the CRIPSR system. The vector may contain the sequence of the guide RNA or a part of the sequence of the guide RNA. When the vector contains a part of the base sequence of the guide RNA, the linear DNA product (e.g., closed linear DNA product) may contain the deleted base sequence part of the guide RNA, and upon ligation, the ligated vector contains the entire base sequence of the guide RNA. The nuclease and guide RNA of the CRISPR system may be encoded on a single vector or on two different vectors. The linear DNA product (e.g., closed linear DNA product) may code for the nuclease and guide RNA of the CRISPR system. One linear DNA product (e.g., one closed linear DNA product) may encode a nuclease of the CRISPR system, and the other linear double-stranded DNA product (e.g., the other closed linear DNA product) may encode a guide RNA.

[0442] The linear DNA product (e.g., a closed linear DNA product) can be used for CRISPR-Cas mediated recombination, homology directed repair, or repair by non-homologous end joining.

[0443] When the nuclease of the CRISPR system and the guide RNA are encoded by different linear DNA products (e.g., different closed linear DNA products), they may be part of different or the same delivery mechanism. For example, the linear DNA product (e.g., closed linear DNA product) encoding the nuclease of the CRISPR system can be delivered to the cell by a first nanoparticle, non-viral vector or viral vector, while the linear DNA product (e.g., closed linear DNA product) encoding the guide RNA can be delivered to the cell by a second nanoparticle, non-viral vector or viral vector. For example, the linear DNA product (e.g., closed linear DNA product) encoding the nuclease of the CRISPR system and the linear DNA product (e.g., closed linear DNA product) encoding the guide RNA can be delivered to the cell by the same nanoparticle, non-viral vector or viral vector.

[0444] When the nuclease and guide RNA of the CRISPR system are encoded by the same linear DNA product (e.g., a closed linear DNA product) (or a vector containing a linear DNA product (e.g., a closed linear DNA product)), they may be part of the same delivery mechanism. For example, the linear DNA product (e.g., a closed linear DNA product) or vector encoding the nuclease and guide RNA of the CRISPR system may be delivered to a cell by a nanoparticle, a non-viral vector, or a viral vector.

[0445] The linear DNA product (e.g., closed linear DNA product) encoding the nuclease of the CRISPR system and the linear DNA product (e.g., closed linear DNA product) encoding the guide RNA can be delivered to cells by electroporation. The linear DNA product (e.g., closed linear DNA product) encoding the nuclease of the CRISPR system and the linear DNA product (e.g., closed linear DNA product) encoding the guide RNA can be delivered to cells by hydrodynamic needle.

[0446] Thus, the present invention provides a linear DNA product (e.g., a closed linear DNA product) described herein for use in delivering a CRISPR system to a cell. The present invention provides a method of delivering a CRISPR system to a cell, comprising contacting a linear DNA product (e.g., a closed linear DNA product) described herein with the cell. The present invention also provides a linear DNA product (e.g., a closed linear DNA product) obtained by the methods described herein for use in delivering a CRISPR system to a cell. The present invention provides a method of delivering a CRISPR system to a cell, comprising contacting a linear DNA product (e.g., a closed linear DNA product) obtained by the methods described herein with the cell.

[0447] The cell is an animal cell, preferably a mammalian cell, such as a human cell.

[0448] The present invention also provides a cell obtainable by the method described herein, which is particularly suitable for cell therapy and / or in vivo therapy.

[0449] The linear DNA product produced by the methods described herein can be used for transcription in vitro or in vivo to produce RNA, preferably mRNA.

[0450] 9. Kit The present invention provides kits containing the necessary components to carry out the methods described herein. The kits include at least: (a) a first and a second adaptor molecule; (b) an endonuclease; and (c) ligase Includes.

[0451] The kit may further comprise a DNA polymerase, at least one buffer and / or a nuclease.

[0452] The first and / or second adapter molecule may comprise the sequence of SEQ ID NO:1 or a portion thereof. The first and / or second adapter molecule may comprise at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of SEQ ID NO:1. The first and second adapter molecules may comprise identical nucleic acid sequences. The first and second adapter molecules may comprise different nucleic acid sequences. The first adapter molecule may comprise one or more protected nucleotides and the second adapter molecule may comprise a hairpin or stem-loop region.

[0453] The first and second adaptor molecules may be provided together in a kit or se...

Claims

1. (a) amplifying a DNA template molecule comprising at least one endonuclease target sequence to generate a double-stranded DNA molecule, wherein the DNA template molecule is amplified by rolling circle amplification; (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second adapter molecules to form a single contiguous aqueous volume; and (c) incubating the single contiguous aqueous volume to generate a closed circular linear DNA product, wherein the closed circular linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises the linear portion of the double-stranded DNA molecule, and the linear double-stranded region is closed at a first end by the first adapter molecule and closed at a second end by the second adapter molecule, comprising, wherein the double-stranded DNA molecule generated in step (a) is not purified prior to step (b), a method for producing a closed circular linear deoxyribonucleic acid (DNA) product.

2. The method according to claim 1, wherein the closed circular linear DNA product comprises a spacer, and optionally, the spacer is at least 20 base pairs in length.

3. The method according to claim 1 or 2, wherein the endonuclease is a type IIS restriction enzyme, and optionally, the endonuclease is BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI-v2, BtsMutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnlII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI restriction enzyme.

4. The method according to claim 1 or 2, wherein the first adapter molecule and / or the second adapter molecule is a nucleic acid adapter molecule.

5. The method according to claim 1 or 2, wherein the first adapter molecule comprises a hairpin and / or the second adapter molecule comprises a hairpin.

6. The method according to claim 1 or 2, wherein the first adapter molecule and / or the second adapter molecule comprises a double-stranded region having an overhang.

7. (a) amplifying a DNA template molecule comprising at least one endonuclease target sequence to generate a double-stranded DNA molecule, wherein the DNA template molecule is amplified by rolling circle amplification; (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second adapter molecules to form a single continuous aqueous volume; and (c) incubating the single continuous aqueous volume to produce a linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, the first adapter molecule is linked to a first end of the linear double-stranded region, and the second adapter molecule is linked to a second end of the linear double-stranded region, and the first and second adapter molecules are nucleic acid molecules comprising one or more nuclease-resistant nucleotides, comprising, wherein the double-stranded DNA molecule produced in step (a) is not purified prior to step (b), a method for producing a linear deoxyribonucleic acid (DNA) product. **Claim 8** (a) amplifying a DNA template molecule comprising at least one endonuclease target sequence to produce a double-stranded DNA molecule, wherein the DNA template molecule is amplified by rolling circle amplification; (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second adapter molecules to form a single continuous aqueous volume; and (c) incubating the single continuous aqueous volume to produce a partially closed circular linear DNA product, wherein the partially closed circular linear DNA product comprises a linear double-stranded region, the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, the first adapter molecule is linked to a first end of the linear double-stranded region, and the second adapter molecule is linked to a second end of the linear double-stranded region, the first adapter molecule is a nucleic acid molecule comprising one or more nuclease-resistant nucleotides, and the linear double-stranded region is closed at the second end by the second adapter molecule, comprising, wherein the double-stranded DNA molecule produced in step (a) is not purified prior to step (b), a method for producing a partially closed circular deoxyribonucleic acid (DNA) product. **Claim 9** The method according to claim 7 or 8, wherein the one or more nuclease-resistant nucleotides are one or more phosphorothioated nucleotides. **Claim 10** (a) Producing a closed circular DNA product according to the method recited in claim 1 or 2, producing a linear DNA product according to the method recited in claim 7, or producing a partially closed circular linear DNA product according to the method recited in claim 8; (b) Contacting the closed circular DNA product, linear DNA product, or partially closed circular linear DNA product with a polymerase; and (c) Producing a transcription product encoded by the closed circular DNA product, linear DNA product, or partially closed circular linear DNA product A method for in vitro transcription of a closed circular deoxyribonucleic acid (DNA) product, linear DNA product, or partially closed circular linear DNA product, comprising:

11. (a) Producing a closed circular DNA product according to the method recited in claim 1 or 2, producing a linear DNA product according to the method recited in claim 7, or producing a partially closed circular linear DNA product according to the method recited in claim 8; and (b) Introducing the closed circular DNA product, linear DNA product, or partially closed circular linear DNA product into a cell or cell-free expression system to produce a protein encoded by the closed circular DNA product, linear DNA product, or partially closed circular linear DNA product A method for producing a protein, wherein step (b) is performed in vitro.

12. (a) Producing a closed circular DNA product according to the method recited in claim 1 or 2, producing a linear DNA product according to the method recited in claim 7, or producing a partially closed circular linear DNA product according to the method recited in claim 8; (b) Contacting a cell with the closed circular DNA product, linear DNA product, or partially closed circular linear DNA product; and (c) Transfecting the closed circular DNA product, linear DNA product, or partially closed circular linear DNA product into the cytosol of the cell A method for intracellular transfection of a closed circular deoxyribonucleic acid (DNA) product, linear DNA product, or partially closed circular linear DNA product, wherein steps (b) and (c) are performed in vitro.

13. The method according to claim 12, wherein transfection of the closed circular DNA product or linear DNA product into the cytosol of the cell is performed by electroporation. a) Producing a closed circular DNA product by the method according to claim 1 or 2, producing a linear DNA product by the method according to claim 7, or producing a partially closed circular linear DNA product by the method according to claim 8; and b) manufacturing a non-viral delivery system using the closed circular DNA product, the linear DNA product, or the partially closed circular linear DNA product A method for manufacturing a non-viral delivery system, comprising the above steps. a) Producing a closed circular DNA product by the method according to claim 1 or 2, producing a linear DNA product by the method according to claim 7, or producing a partially closed circular linear DNA product by the method according to claim 8; and b) manufacturing a viral delivery system using the closed circular DNA product, the linear DNA product, or the partially closed circular linear DNA product A method for manufacturing a viral delivery system, comprising the above steps.

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