Process for creating closed linear DNA

Abstract

A process for producing DNA is provided. [Solution] A process for producing DNA is provided, the process comprising: a) providing a closed linear template DNA containing a DNA sequence of interest; and b) amplifying DNA from the closed linear template DNA of step (a), wherein the closed linear template DNA does not contain single-stranded loops containing primase / polymerase priming sites, and the amplification is stimulated with a primase / polymerase enzyme.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of European Patent Application No. 20382064 filed on January 31, 2020. Claim the grace of the book.

[0002] The present invention is in the field of nucleic acids and therapeutics. In particular, the present invention relates to closed linear DNA and its The present invention also relates to a process for the preparation and a pharmaceutical composition comprising the closed linear DNA. The closed linear DNA obtained by this process is particularly useful for therapeutic purposes. [Background technology]

[0003] Gene therapy holds great promise for the treatment of several diseases. The gene delivery system is based on the desired transfer to the nucleus of targeted human cells. In comparison to viral DNA vectors, Non-viral transgene delivery systems offer a safe approach to gene transfer and vaccine design. Larger transgenes are less likely to induce inflammatory and immune responses in the host It is potent and easy to store.

[0004] However, the efficacy of non-viral vectors is very limited, which has limited their translation into the clinic. For example, the use of conventional plasmid DNA vectors for gene therapy has hindered the introduction of Therefore, the bacterial sequences contained in this vector may induce harmful immune responses. and their large molecular size impairs bioavailability. Therefore, new types of non-viral DNA constructs have been developed in recent years.

[0005] Closed linear DNA vectors (clDNA) are intended to be used without the immunogenic, mostly bacterial backbone. It is a dumbbell-shaped molecule that contains only the DNA sequence of the Higher transfection efficiency and longer duration of gene expression The linear nature of cDNA ensures that it is integrated into the genome at random. The clDNA vectors are suitable for in vitro and in vivo applications. It has been successfully used for a variety of therapeutic indications with promising results in vivo.

[0006] The production of nucleotide vectors, such as cDNA, for therapeutic use involves various challenges. First, the vector may contain toxins or other bacterial compounds that may cause adverse reactions in patients. Furthermore, the vector should be free of any chemicals or toxins. Therefore, the injection efficiency is rather low. Finally, delivery of the exogenous nucleotide sequence to the patient requires a very high production yield. Injecting a protein is nothing but risky. For example, the mutations generated during the manufacturing process can lead to serious complications. Expression of proteins or non-specific products can result in significant harm to the patient.

[0007] From the above perspective, vectors used in clinics (especially clDNA) are prone to bacterial residues. The absence of residues and antibiotic resistance sequences is simultaneously confirmed, reducing costs. It is possible to produce large-scale DNA fragments in a homogeneous state, and it is possible to ensure a very high degree of sequence uniformity. (i.e., due to a very low percentage of altered sequences) It must be made from

[0008] Therefore, despite previous efforts, no clDNA of suitable quality for therapeutic purposes has been identified. There is a need for an efficient process for the large-scale and safe production of Summary of the Invention [Problem to be solved by the invention]

[0009] The present inventors have generated large amounts of clDNA with very high sequence quality without using microorganisms. The process provided herein uses template DNA. Use primase / polymerase to stimulate amplification of the gene, followed by a therapeutically suitable clone It is based on the processing of the amplification product to produce DNA.

[0010] Surprisingly, as shown in the following examples, the present inventors found that prior to clDNA formation A primase / polymerase enzyme-driven amplification step is performed to obtain good quality cDNA. Not only did we obtain good production yields, but we also demonstrated the use of other amplification processes such as random priming. We found that the sequence fidelity of the generated clDNA was much improved compared to that of the conventional method. (See Figure 2). Therefore, the results provided herein are based on the obtained clDN This shows that priming is important for the final property of A.

[0011] The high sequence quality provided by the primase / polymerase also contributes to the post-amplification process. These steps also affect the efficiency of the enzymes that recognize specific sequences on the amplified DNA. Therefore, the use of primase / polymerase priming The high sequence fidelity ensures high preservation of all target sequences on the amplified DNA, while It can be efficiently targeted by processing enzymes such as restriction enzymes or protelomerases. It becomes certain.

[0012] Notably, there are no processes that require the use of microorganisms. Scaling up the invention process also becomes much easier and safer.

[0013] Compared with what has been disclosed in the prior art, the present inventors have The use of primase / polymerase to prime the template DNA creates a single-stranded loop (i.e., We further found that the presence of a primase recognition site within the DNA (i.e., the adapter) is not required. This greatly expands the repertoire of template DNAs that can be used in the process of the present invention. For example, a primase / polymerase can be used to amplify any sequence (i.e., a primer) Ligate the adapter (containing no enzyme / polymerase priming site) template cDNA generated by the cleavage of a protelomerase containing a minimal single-stranded loop can be used to stimulate template c1DNA generated by the action of sea ​​bream).

[0014] Finally, the cDNA obtained by the process of the present invention can be transferred into mammalian cells. This allows for efficient transfer of the target DNA sequence contained therein. It is also possible to express the protein in a cellular context (see Figures 5 and 6).

[0015] In summary, in the examples provided below, the inventors have demonstrated that high-quality This demonstrates the utility of the process of the present invention for large-scale production of quality clDNA. [Means for solving the problem]

[0016] Thus, in a first aspect, the present invention provides a process for producing closed linear DNA. a) providing a template DNA containing a DNA sequence of interest; b) reacting the template of step (a) with the template DNA; A process for amplifying DNA from template DNA, wherein the amplification is performed with a primase / polymerase enzyme. step (c) generating a closed linear DNA using the amplified DNA prepared in step (b); and d) purifying the closed linear DNA produced in step (c). Provide a process for

[0017] As described above, the process of the present invention allows for the production of sequences with very high sequence fidelity (i.e. This allows for the production of cDNA with very low amounts of amplification artifacts or mutated sequences. This makes clDNA particularly suitable for therapeutic use, where high sequence quality is essential. .

[0018] Thus, in a second aspect, the present invention provides a method for producing a medicament for the treatment of ... The resulting closed linear DNA is provided.

[0019] In a third aspect, the present invention relates to a closed linear DNA molecule according to the second aspect for use in therapy. Provide A.

[0020] In a fourth aspect, the present invention provides a therapeutically effective amount of closed linear DNA according to the second aspect, and a pharmaceutical and a physiologically acceptable carrier or excipient.

[0021] In a fifth aspect, the present invention provides a concatemeric DNA comprising repeats of a DNA sequence of interest, wherein each one of the repeated DNA sequences of interest is flanked by at least a recombinase recognition site, or alternatively by at least a restriction site and a protelomerase target sequence. [Brief explanation of the drawings]

[0022] [Figure 1]

[0039] Figure 1 shows the DNA yields obtained from RCA amplification of template DNA when amplification is primed by either TthPrimPol or random primers (RP), relative to Example 1. The y-axis represents DNA yield in μg. NTC refers to the control reaction without template DNA, and Plasmid refers to the amplification reaction using plasmid template DNA. [Figure 2]

[0033] Referring to Example 1, Illumina sequencing comparing TthPrimPol stimulated amplification with RP stimulated amplification is shown. [Figure 3] Photographs of agarose gels showing various product reactions performed in Example 2 are shown, including: 1) DNA ladder, 2) TthPrimPol-stimulated amplification of pUC57-Kan_TELO-CMV-EGFP, 3) TthPrimPol-stimulated amplification of pUC57-Kan_TELO-CMV-EGFP digested with TelN, 4) DNA ladder, 5) TthPrimPol-stimulated amplification of pUC57-Kan_TELO-CMV-EGFP digested with TelN, EcoRI, and HindIII, and 6) TthPrimPol-stimulated amplification of pUC57-Kan_TELO-CMV-EGFP digested with TelN, EcoRI, HindIII, and ExoIII. Arrows indicate the band sizes corresponding to the cassettes (target molecules). [Figure 4A]For Example 3, RCA-amplified clDNA produced in TelN is shown, and the DNA yield obtained from RCA amplification of TelN-produced template plasmid or template clDNA is shown when amplification is stimulated by either TthPrimPol or random primers (RP). The y-axis represents DNA yield in μg. NTC refers to a control reaction without template DNA. [Figure 4B] Photographs of agarose gels loaded with DNA amplification products obtained under the indicated conditions with or without treatment with TelN. Prior to loading, the DNA products were digested with EcoRI, HindIII, and ExoIII. Arrows indicate the band sizes corresponding to the cassette (target molecule). [Figure 5] Quantification of fluorescence intensity of HEK293 cells 24 and 48 hours after transfection with the indicated constructs is shown for Example 4. NT denotes untreated cells. The y-axis represents arbitrary units of fluorescence intensity. [Figure 6] With reference to Example 4, representative images of HEK293 cells 24 and 48 hours after transfection with the indicated constructs are shown. [Figure 7A] Quality control parameters for oDNA41 are shown. Agarose gel electrophoresis (M1, supercoiled DNA ladder marker: TAKARA:3585A; M2, 1 kb DNA ladder TIAGEN MD111; lane 5, oDNA41) is shown. [Figure 7B] Quality control parameters of oDNA41 are shown. Grayscale analysis is shown. [Figure 7D] Quality control parameters for oDNA41 are shown. Sanger sequencing is shown. [Figure 8A] Quality control parameters for oDNA21 are shown. Agarose gel electrophoresis (M1, supercoiled DNA ladder marker: TAKARA:3585A; M2, 1 kb DNA ladder TIAGEN MD111; lane 5, oDNA41) is shown. [Figure 8B] Quality control parameters of oDNA41 are shown. Grayscale analysis is shown. [Figure 8D]Quality control parameters for oDNA41 are shown. Sanger sequencing is shown. [Figure 9]

[0023] Figure 1 shows a representation of a fragment of the eGFP plasmid (a plasmid having SEQ ID NO: 20) containing a sequence of interest for preparing a clDNA of the invention. The represented fragment contains the sequence of interest (in this case, the sequence encoding GFP) along with additional sequences such as the corresponding promoter and enhancer. The sequence of interest is flanked by a BsaI restriction site and a protelomerase target sequence. [Figure 10]

[0033] Figure 2 shows a representation of a fragment of Luc-ITR (a plasmid having SEQ ID NO: 22) containing a sequence of interest for preparing a clDNA of the invention. The represented fragment contains the sequence of interest (in this case, a sequence encoding luciferase) along with additional sequences such as the corresponding promoter and enhancer, as well as the AVV2-ITR. The sequence of interest is flanked by a BsaI restriction site and a protelomerase target sequence. [Figure 11] Agarose gel electrophoresis of oDNA4ITR (M, DL3000 ladder, lane 12, oDNA4ITR) is shown. [Figure 12] Agarose gel electrophoresis of clDNA obtained from eGFP plasmid (plasmid having SEQ ID NO: 20) as in Example 6 (RCA followed by protelomerase treatment) is shown (M1, supercoiled DNA ladder marker: TAKARA:3585A; M2, 1 kb DNA ladder TIAGEN MD111; lane 2, clDNA from Example 6). DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description of the Invention All terms used herein in this application are understood to be of the art unless otherwise specified. The term "a" is to be understood in its general meaning as known in the art. Other more specific definitions of terms are provided below. Definitions may also be used in conjunction with broader definitions. This definition applies throughout the specification and claims unless expressly stated otherwise. It is intended to be applied uniformly throughout.

[0024] As used herein, the indefinite articles "a" and "an" mean "at least one" or Unless otherwise indicated, "the" as used herein is synonymous with "one or more." Definite articles such as these also include the plural of the noun.

[0025] In a first aspect, the present invention provides a process for producing closed linear DNA, comprising: b) providing a template DNA containing a desired DNA sequence; and c) cleaving the template DNA from step (a). amplifying DNA, wherein the amplification is stimulated with a primase / polymerase enzyme; Step (c) generating closed linear DNA using the amplified DNA produced in step (b). and d) purifying the closed linear DNA produced in step (c). provide.

[0026] Amplify template DNA using primase / polymerase as the priming enzyme This produces amplified DNA with very high efficiency and fidelity. , which can then be processed to produce closed linear DNA suitable for therapeutic applications.

[0027] As used herein, the term "closed linear DNA" or "clDNA" refers to a nucleotide sequence. "Dumbbell" or "dogbone" under conditions that allow hybridization of the octides. It refers to a single-stranded, covalently closed DNA molecule that forms a structure of the shape Although the cDNA is formed by a closed single-stranded DNA molecule, the two phases within the same molecule The hybridization of complementary sequences forms a "dumbbell" structure, allowing two identical This produces a structure consisting of a double-stranded intermediate segment flanked by single-stranded loops. Using molecular biology techniques, open or closed double-stranded DNA (e.g., the DNA produced in step (b)) can be isolated. Those skilled in the art will understand how to generate cDNA from amplified DNA (e.g., amplified DNA). Single-stranded hairpin adapters are ligated to both ends of the double-stranded DNA opened by the action of bar ligase. We understand that clDNA can be produced by doing this. Another method known to those skilled in the art for cleaving at least two protelomerase targets is This is due to the action of protelomerase on double-stranded DNA, including the sequence

[0028] A "sequence of interest" is any sequence required for correct gene expression, such as an expression cassette. A double-stranded DNA fragment containing the minimal sequence encoding the gene of interest, along with the sequence of The sequence of interest is understood as an inverted terminal repeat. It may additionally contain other sequences flanking the expression cassette, such as interleukin (ITR) repeats. That's fine.

[0029] As used herein, the term "priming" refers to the process of priming a polynucleotide by an enzyme. Refers to the generation of an oligonucleotide primer on a template.

[0030] The term "primase / polymerase enzyme" refers to the archaeological-eukaryotic primase (AE) DNA-dependent primase / polymerase enzymes, such as enzymes from the P) superfamily These enzymes exhibit the ability to degrade a starting DNA strand containing dNTPs. Enzymes from this superfamily that can be used include, for example, Thermus thermophilus primers Human primase / polymerase (TthPrimPol) or human primase / polymerase (h sPrimPol, CCDC111, FLJ33167, EukPrim2 or hPri mPol1) is a primase / polymerase of Thermus thermophilus. "TthPrimPol" is a primer for the bacterium Thermus thermophilus, sequence of which is shown in SEQ ID NO: 1. The nucleotide and protein sequences are listed under NC_005835 and available in the NCBI Entrez database, such as WP_01 1173100.1 It is possible.

[0031] [Table 1]

[0032] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described herein, the process may include: a) obtaining a desired D b) providing a template DNA containing a DNA sequence; and c) amplifying DNA from the template DNA of step (a). (b1) amplifying a template DNA fragment using a primase / polymerase enzyme; (b2) priming A; and (b3) extending the resulting sequence using a polymerase. (c) generating closed linear DNA using the amplified DNA produced in step (b). and (d) purifying the closed linear DNA produced in step (c).

[0033] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the primase / polymerase The enzyme is selected from TthPrimPol or hsPrimPol. In a more specific embodiment, the primase polymerase enzyme is TthPrimPol. In this embodiment, the primase polymerase enzyme is at least 70% specific to SEQ ID NO: 1, at least at least 75%, at least 80%, at least 85%, at least 90%, or at least TthPrimPol of SEQ ID NO: 1 having 95% sequence identity, or a variant thereof Those skilled in the art will recognize that any of the TthPrimPols that maintain their primase activity. It will be understood that either variant may be suitable for use in the process of the present invention.

[0034] In the present invention, the term "identity" refers to the degree of identity between two sequences when the sequences are optimally aligned. In an optimal alignment, the first sequence position is If the corresponding positions in two sequences are occupied by the same amino acid residue, then the sequences are The level of identity (or "percent sequence identity") between two sequences is expressed as a percentage of the sequence identity. ") is the ratio of the number of identical positions shared by the sequences to the size of the sequences (i.e., The percent sequence identity is measured as: percent sequence identity = (number of identical positions / total number of positions) × 100).

[0035] To quickly obtain optimal alignments and calculate identity between two or more sequences, Several mathematical algorithms for this are known and several software programs are available. Examples of such programs include, among others, amino acid sequence MATCH-BOX, MULTAIN, GCG, FASTA and ROB for sequence analysis A preferred software analysis program is the ALI GN, CLUSTAL W and BLAST programs (e.g., BLAST2.1, BL 2SEQ and their later versions).

[0036] For amino acid sequence analysis, a BLOSUM matrix (e.g., BLOSUM45, BLOSUM50, BLOSUM62 and BLOSUM80 matrices), Gonne t matrix, or PAM matrix (e.g., PAM30, PAM70, PAM1 20, PAM160, PAM250 and PAM350 matrices) Used to determine identity.

[0037] The BLAST program searches for multiple sequences in a database (e.g., GenSeq). or use BL2SEQ to align the selected sequences between two selected sequences. BLAST provides an analysis of at least two amino acid sequences by either The program is preferably integrated into the operation of the BLAST program DUST or SEG The modification is preferably performed by a low-complexity filtering program such as a When using gap existence costs (or gap scores), the gap existence costs are preferably The gap parameter is set to approximately -5 to -15. BLAST programs and their underlying principles can be suitably used in Altschul et al.,´´Basic local alignment search tool´´,1990,J.Mol.Biol,v.215,403~ Further explained on page 410. A specific percentage of identity is One or more mPol enzymes that are still effective and can thereby stimulate the desired sequence. Conservative changes in the sequence of one or more amino acids are also included. is due to the insertion or deletion of multiple amino acids.

[0038] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the process A cell-free in vitro process for producing A.

[0039] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, step (b) may include rolling stamping. It is circle amplification.

[0040] The term "rolling circle amplification" or "RCA" refers to the amplification of clDNA or double-stranded circular DNA. refers to a nucleic acid amplification method involving the amplification reaction of covalently closed DNA molecules such as In this case, the polymerase extends the primer around the closed DNA molecule. The polymerase displaces the hybridized copy and creates polynucleotides around the template. Continue nucleotide extension to obtain concatemer DNA containing tandem units of amplified DNA. These linear single-stranded products are then subjected to multiple hybridizations, primer serves as a basis for extension and strand displacement events, resulting in the formation of concatemeric double-stranded DNA products Thus, the concatemeric double-stranded DNA product contains multiple copies of each amplified single DNA unit. A person skilled in the art can easily identify the source of the virus by using general knowledge and / or the manufacturer's instructions. and how to adjust the conditions of the amplification process depending on the enzyme and the properties of the template being amplified. Depending on how the template DNA is generated, the concatemer DNA can be The amplified DNA sequence contains different sequences flanking it. For example, in concatemeric DNA, The repeated DNA sequences are restriction sites, protelomerase target sequences, and recombinase recognition sites. The nucleotide sequence may be adjacent to a site, a region, a nucleotide sequence ...

[0041] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the amplification in step (b) may be performed by strand displacement. It is carried out using a DNA polymerase. The term "strand-displacing DNA polymerase" refers to a strand-displacing DNA polymerase that displaces the template D A DNA polymerase is used to carry out a 3'-end extension reaction while removing the double-stranded portion of the DNA. The strand-displacing DNA polymerase that can be used in the present invention is phi29 DNA polymerase. There is no particular limitation on the DNA polymerase as long as it has strand displacement activity. Depending on the type of polymerase selected in this way, the skilled artisan may It is understood that the reaction conditions for the reaction can be appropriately set. For example, phi29D If NA polymerase is used, the reaction temperature is 25°C to 35°C. It can be implemented in

[0042] Thus, in certain embodiments, the strand-displacing DNA polymerase is DNA polymerase, Bst DNA polymerase, Bca (exo) DNA polymerase, Escherichia coli Klenow fragment of DNA polymerase I, Vent (exo) DNA polymerase, D Consists of eepVent (exo) DNA polymerase and KOD DNA polymerase In a more particular embodiment, the strand-displacing DNA polymerase is selected from the group consisting of phi29D In an even more particular embodiment, the strand-displacing DNA polymerase is and phi29 DNA polymerase. As disclosed in US Pat. No. 1,000,997, those skilled in the art can develop textures with improved properties. Understand how to obtain DNA polymerase.

[0043] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the template DNA is a closed linear The template is selected from a DNA template or a circular double-stranded DNA template.

[0044] As used herein, the term "circular double-stranded DNA" refers to a covalently closed double stranded DNA. Refers to a single-stranded DNA molecule.

[0045] The process for producing closed linear DNA of the present invention can also be performed with random primers. This may be accomplished by priming the amplification of step (b).

[0046] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the template DNA is a closed linear template. In the case of the DNA, step (a) is - a plasmid vector containing at least two restriction sites flanking the DNA sequence of interest and at least one restriction enzyme, thereby obtaining a fragment containing the desired DNA sequence. The open double-stranded DNA containing the DNA sequence of interest is then inserted into the single-stranded DNA adapter. Alternatively, step (a) may be carried out by attaching a nucleotide to both ends of the double-stranded DNA. , - a protelomerase containing at least two protelomerase target sequences flanking a DNA sequence of interest; contacting the plasmid vector with protelomerase, more specifically TelN, This results in the formation of a closed linear DNA template containing the desired DNA sequence. This is done by obtaining the template DNA.

[0047] The inventors have surprisingly found that the primers The primase / polymerase enzyme does not contain an adapter with a primase recognition site. In particular, we found that template cDNA is able to stimulate the action of protelomerase. When generated by this method, the resulting cDNA has a very small single-stranded loop at the middle end. The single-stranded loop exhibits a structure that does not contain the protelomerase target sequence. However, the primase / polymerase did not react properly unless the template c1DNA was subjected to denaturing conditions. Even if the DNA fragment is not fragmented, it stimulates this type of clDNA, thereby allowing the polymerase can initiate the amplification process (see Figure 4).

[0048] Thus, in one embodiment of the process of the first aspect of the invention, optionally as described above or as described below In combination with any of the embodiments provided in In certain embodiments, the template DNA does not contain a priming site. A is template clDNA that does not contain a primase / polymerase priming site.

[0049] As used herein, a "plasmid vector" refers to a vector that transports another nucleic acid to which it is linked. A circular double-stranded nucleic acid that can be delivered and autonomously replicate within a cell independently of chromosomal DNA Thus, a plasmid vector is a molecule that contains all the components necessary for replication in a cell, especially a bacterial cell. Contains all the required elements.

[0050] The use of restriction enzymes and ligases (for joining purposes) is routine in molecular biology. Therefore, the skilled artisan will know how to adjust the reaction conditions depending on the enzyme being used and the target enzyme. Know which restriction enzymes should be used depending on the restriction site being used.

[0051] Those skilled in the art will also appreciate that some restriction enzymes generate DNA overhangs (sticky ends), It is understood that the others do not produce this (blunt ends). Both types The following restriction enzymes can be used in the methods of the present invention. Those skilled in the art will recognize that adapters with sticky ends include: Able to ligate to open double-stranded DNA with sticky ends (sticky end ligation) It is understood that open double-stranded DNA with blunt ends can also be synthesized using, for example, Taq polymerase. dA tein is synthesized by the process of adding a terminal 3' deoxyadenosine nucleotide using enzymes. The DNA fragment can be attached to an adapter with an overhanging T and then ligated to an adapter with an overhanging T.

[0052] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the restriction enzyme may be blunt-ended or In a more particular embodiment, at least two nucleotides flanking the DNA sequence of interest are generated. contacting a plasmid vector containing at least two restriction sites with at least one restriction enzyme; This results in open double-stranded DNA with sticky ends or open double-stranded DNA with blunt ends. Produces strand DNA.

[0053] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described herein, the single-stranded DNA adapter In a more particular embodiment, the single-stranded DNA adapter has the structure of SEQ ID NO: 4, The sequence is SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. Therefore, the adapters ligated to both ends of the open double-stranded DNA may be the same or different. It can be an adapter.

[0054] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the single-stranded DNA adapter comprises: It contains one or more, for example at least two, modified nucleotides.

[0055] "Modified nucleotide" refers to a nucleotide that has been chemically modified by modification of the base, sugar, or phosphate group, or Any nucleotide (e.g., adenosine, guanylate, thiamin ... Modified nucleotides are nucleotides that react to the modification. It may or may not be naturally occurring.

[0056] As used herein, modified nucleotides preferably include 5-methyl-deoxyribonucleotides. Adenosine, 2-amino-deoxyadenosine, 1-methyl-adenosine, 1-methyl-guanidine Inosine, 1-methyl-inosine, 2,2-dimethyl-guanosine, 2,6-diaminopurine 2'-amino-2'-deoxyadenosine, 2'-amino-2'-deoxycytidine , 2'-amino-2'-deoxyguanosine, 2'-amino-2'-deoxyuridine, 2-Amino-6-chloropurine riboside, 2-aminopurine riboside, 2´-aladecyl Nosine, 2'-aracytidine, 2'-arauidine, 2'-azido-2'-deoxyadenosine 2-azido-2'-deoxycytidine, 2'-azido-2'-deoxyguanosine 2'-azido-2'-deoxyuridine, 2-chloroadenosine, 2'-fluoro- 2´-Deoxyadenosine, 2´-fluoro-2´-deoxycytidine, 2´-fluoro -2´-deoxyguanosine, 2´-fluoro-2´-deoxyuridine, 2´-fluoro Rotimidine, 2-methyl-adenosine, 2-methyl-guanosine, 2-methyl-thio-N 6-Isopenenyl-adenosine, 2´-O-methyl-2-aminoadenosine, 2´-O- Methyl-2´-deoxyadenosine, 2´-O-methyl-2´-deoxycytidine, 2´ -O-methyl-2´-deoxyguanosine, 2´-O-methyl-2´-deoxyuridine , 2´-O-methyl-5-methyluridine, 2´-O-methylinosine, 2´-O-methyl Luseudolidine, 2-thiocytidine, 2-thio-cytidine, 3-methyl-cytidine, 4-acetyl-cytidine, 4-thiouridine, 5-(carboxyhydroxymethyl)-uridine Lysine, 5,6-dihydrouridine, 5-aminoallylcytidine, 5-aminoallyl-de Oxyuridine, 5-bromouridine, 5-carboxymethylaminomethyl-2-thio- Uracil, 5-carboxymethylammonomethyl-uracil, 5-chloro-ara-cytosine , 5-fluorouridine, 5-iodouridine, 5-methoxycarbonylmethyluridine uridine, 5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-azacytidine, 6-Azauridine, 6-chloro-7-deaza-guanosine, 6-chloropurine riboside, 6-Mercapto-guanosine, 6-methyl-mercaptopurine-riboside, 7-deaza- 2´-deoxy-guanosine, 7-deazaadenosine, 7-methyl-guanosine, 8- Zaadenosine, 8-bromo-adenosine, 8-bromo-guanosine, 8-mercapto-guanosine Guanosine, 8-oxoguanosine, benzimidazole riboside, β-D-mannosyl -queosine, dihydrouridine, inosine, N1-methylaridine Adenosine, N6-([6-aminohexyl]carbamoylmethyl)-adenosine, N6- Isopentenyl-adenosine, N6-methyl-adenosine, N7-methyl-xanthosine , N-uracil-5-hydroxyacetic acid methyl ester, puromycin, queosin (que osine), uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, including acetylated hydroxybenzoates, including hydroxybenzoates, ... Chemically modified, such as methylated or hydroxylated, naturally occurring or non-naturally occurring Any of guanosine, uridine, adenosine, thymidine, or cytidine, but Without limitation, variances of guanosine, uridine, adenosine, thymidine and cytidine are given. The preparation of such variants is described, for example, in U.S. Pat. No. 4,373,071. more familiar to those skilled in the art.

[0057] Modified nucleotides also include pyridin-4-one ribonucleosides, 5-aza-uridine , 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2 -thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carbo 1-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine Lysine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurine 5-Taurinomethyl-2-thiouridine, 1-Taurinomethyl-pseudouridine Methyl-4-thio-uridine, 5-methyluridine, 1-methyl-pseudouridine, 4 -thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1 -methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine Douridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine uridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4- Thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio- Douridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N 4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxycytidine Methylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- Isocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- Pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1- Methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine Zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2- Thio-Zebularine, 2-Thio-Zebularine, 2-Methoxy-Cytidine, 2-Methoxy-5 4-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl This also includes, but is not limited to, til-pseudoisocytidine.

[0058] Modified nucleotides also include 2-aminopurine, 2,6-diaminopurine, 7-deaza- Adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza 7-Deaza-2,6-diaminopurine, 7-Deaza- 8-Aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-Isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6- Lysinylcarbamoyl adenosine, N6-threonylcarbamoyl adenosine, 2-methyl Ruthio-N6-threonylcarbamoyl adenosine, N6,N6-dimethyl adenosine, Also includes 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine , but not limited to these.

[0059] Modified nucleotides also include inosine, 1-methyl-inosine, yiosine, ybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine , 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-Methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N 2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N This also includes, but is not limited to, 2,N2-dimethyl-6-thio-guanosine.

[0060] Modified nucleotides also include 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, Uridine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine , N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine uridine, 5-methyl-uridine, pyrrolo-cytidine, inosine, α-thio-guanosine, 6 -methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine sine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro- Purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro- Purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, This also includes, but is not limited to, 7-deaza-adenosine.

[0061] The modified nucleotide may be chemically modified at the 2' position. The tide contains a substituent at the 2' carbon atom. This substituent can be a halogen, an alkoxy group, hydrogen, From the group consisting of aryloxy groups, amino groups and aminoalkoxy groups, preferably 2'- Hydrogen (2'-deoxy), 2'-O-methyl, 2'-O-methoxyethyl and 2'-fluoro Selected from Oro.

[0062] The chemical modifications involving the 2' position of the nucleotide described herein are referred to as locked nucleic acids (lo ethylene-bridged nucleic acid (ENA) nucleotides, Ethylene bridged nucleic acid (ENA) nucleotides and (S)-constrained ethyl cEt nucleotides. These backbone modifications are The sugars of the nucleotides are locked into a preferred northern conformation.

[0063] The backbone phosphate groups can be modified, for example, by replacing one or more of the oxygen atoms with different substituents. Additionally, modified nucleotides may have an unmodified phosphate moiety modified with any of the nucleotides described herein. Examples of modified phosphate groups include phospholipids. thioates (also known as thiophosphates), phosphoroselenates, boranophosphatides phosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkylphosphonate esters, aryl phosphonates and phosphotriesters. Phosphate-containing linkers also can be used to convert the bonded oxygen to a nitrogen (bridging phosphonate). thiamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene phosphonates ) can be modified by substituting

[0064] The modified nucleotide may be an abasic site. A "site" is a nucleotide lacking an organic base. In a preferred embodiment, an abasic nucleotide The nucleotide further comprises a chemical modification described herein at the 2' position of the ribose. Preferably, the 2'C atom of ribose is selected from the group consisting of halogen, alkoxy, hydrogen, and aryloxy. , the group consisting of amino groups and aminoalkoxy groups, preferably 2'-hydrogen (2'-deoxy) ), 2'-O-methyl, 2'-O-methoxyethyl and 2'-fluoro It is substituted with a substituent.

[0065] In particular embodiments of the first aspect of the invention, the compound of formula (I) is optionally as provided above or below. In combination with any of the embodiments, at least two modified nucleotides , 2-amino-deoxyadenosine, 5-methyl-deoxycytidine, thiophosphate Nucleotides, LNA nucleotides, inosine, 8-oxo-deoxyadenosine and 5 - independently selected from the group consisting of fluoro-deoxyuracil and L-DNA nucleotides will be done.

[0066] In particular embodiments of the first aspect of the invention, the compound of formula (I) is optionally as provided above or below. In combination with any of the embodiments, at least two modified nucleotides Not an L-DNA nucleotide, 5-bromouridine or 5-iodouridine.

[0067] 2-amino-deoxyadenosine (2-amino-2'-deoxyadenosine or 2-amino Amino-dA (also known as dA) is a derivative derived from deoxyadenosine. -Amino-deoxyadenosine is (2R,3S,5R)-5-(2,6-diaminopurine) IUPAC name: (benzo-9-yl)-2-(hydroxymethyl)oxolan-3-ol , and has CAS number 4546-70-7.

[0068] 5-methyl-deoxycytidine (5-methyl-dCTP) is derived from deoxycytidine It is a derivative of ([[(2R,3S,5R)-5-(4-amino-5-methyl-2- Oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-hydr It has the IUPAC name [hydroxyphosphoryl]phosphonohydrogen phosphate and CAS number 22 It has 003-12-9.

[0069] Thiophosphate nucleotides contain thiophosphate (phosphorothioate) as the phosphate group. A thiophosphate is any nucleotide containing a thiophosphate (also known as a thiophosphate). It has CAS number 15181-41-6.

[0070] LNA nucleotides are modified RNA nucleotides in which the ribose moiety is substituted with the 2' oxygen atom. and modified with an extra bridging moiety connecting the 4' carbon.

[0071] L-DNA nucleotides contain the L enantiomer of ribose or deoxyribose It refers to a nucleotide that

[0072] In a further particular embodiment of the first aspect of the invention, In combination with any of the embodiments described above, thiophosphates, locked nucleic acids, , 2,6-diaminopurine, 5-methyl-deoxycytidine, inosine, 8-oxodeoxy independently selected from the group consisting of oxyadenosine and 5-fluoro-deoxyuracil The cDNA contains at least three, at least four, or at least five modified nucleotides. It includes L-nucleotides as well as L-DNA nucleotides.

[0073] In a further particular embodiment of the first aspect of the invention, In combination with any of the embodiments described above, the cDNA contains two LNA nucleotides. Contains leotide.

[0074] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the single-stranded DNA adapter comprises: It contains at least one restriction site. In a more particular embodiment, the restriction site is a BsaI restriction site. restriction site, AfIII restriction site, HindIII restriction site, Nhel restriction site, and EcoRV In an even more particular embodiment, the restriction site is selected from the group consisting of Bsa I restriction site.

[0075] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the single-stranded DNA adapter comprises: In a more particular embodiment, the single-stranded DNA adapter does not contain a primase recognition site. It does not contain the sequence XTC.

[0076] In a more particular embodiment of the process of the first aspect of the present invention, optionally as described above or below In combination with any of the provided embodiments, the template DNA is When the template DNA is a strand, step (a) is - a plasmid vector containing at least two restriction sites flanking the DNA sequence of interest and at least one restriction enzyme, thereby obtaining a fragment containing the desired DNA sequence. The single-stranded DNA adapter creates an open double-stranded DNA that can be used for primase / polymerase protease reactions. The single-stranded DNA adapter was then inserted into the target DNA under the condition that it did not contain a priming site. This can be done by attaching the sequence to both ends of an open double-stranded DNA containing the In step (a), - a protelomerase containing at least two protelomerase target sequences flanking a DNA sequence of interest; contacting the plasmid vector with protelomerase, more specifically TelN, This results in the formation of a closed linear DNA template containing the desired DNA sequence. In a more particular embodiment, the method is carried out by obtaining single-stranded DNA. The data does not contain the sequence XTC.

[0077] In more particular embodiments, any of the embodiments provided above or below may be used. In combination with either the single-stranded DNA adapter, the protelomerase target sequence In another particular embodiment, optionally any of the embodiments provided above or below. When combined with either of these, the single-stranded DNA adapter acts as a protelomerase target. In another specific embodiment, the single-stranded DNA adapter does not contain a target sequence. In this case, the portion of the protelomerase target sequence is Not recognized by telomerase.

[0078] As used herein, "protelomerase" refers to a covalently closed linear D A template containing a protelomerase target site can be cleaved and recombined to generate NA molecules. Therefore, protelomerase is a polypeptide that acts as a catalyst for DNA cleavage and ligation. Enzymes with protelomerase-type activity also have a gating function. They have also been described as resolvase (e.g., Borrelia burgdorferi). The typical substrate for protelomerase is circular double-stranded DNA. If it contains the target site, the enzyme cuts the DNA at this site and ligates the ends to form It is possible to generate linear double-stranded covalently closed DNA molecules. A given polymerase catalyzes the creation of a closed linear DNA from a template containing a target site. The potency of the polypeptide can be assessed using any suitable assay described in the art. It can be determined as follows.

[0079] Examples of suitable protelomerases for use in the processes of the present invention include Halomonas phiHAP-1 from Aquamarina, PY54 from Yersinia enterolytica , phiKO2 from Klebsiella oxytoca and VP882 from Vibrio species, large intestine N15 derived from fungi, or any variant thereof.

[0080] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described herein, the protelomerase may comprise a sequence bacteriophage N15 TelN of number 2, or at least 80 for SEQ ID NO: 2 % identity, at least 85% identity, at least 90% identity, or at least 9 These variants contain sequences with 5% identity.

[0081] A "protelomerase target sequence" is any DNA sequence that exists in the template DNA. This allows the enzyme activity of protelomerase to convert it into closed linear DNA. In other words, the protelomerase target sequence forms a covalently closed linear DNA strand. It is required for the breakage and rejoining of double-stranded DNA by protelomerase in order to Typically, the protelomerase target sequence is any double-stranded DNA sequence with a two-fold greater rotational symmetry. and any perfect palindromic sequence described herein as a perfect inverted repeat. include.

[0082] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, at least two prothrombins The nucleotide sequence of the target DNA fragment contains a perfect inverted repeat DNA sequence.

[0083] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, a protelomerase target sequence The sequence is SEQ ID NO: 3, or at least 80%, at least 85%, or at least These barriers include sequences with at least 90%, or at least 95% sequence identity. Includes ent.

[0084] The length of the perfect inverted repeat varies depending on the specific organism. In various mesophilic bacteriophages, the perfect inverted repeat is 14 base pairs in length. In most cases, perfect inverted repeats are 22 base pairs or more in length. In E. coli N15, the central perfect inverted palindrome is flanked by perfect inverted sequences. , i.e. it forms part of a larger incomplete inverted palindrome.

[0085] The protelomerase target sequence used in the present invention is at least 14 base pairs in length. It preferably contains a double-stranded palindrome (perfect inverted repeat) sequence.

[0086] The perfect inverted repeat may be flanked by additional inverted repeat sequences. can be perfect or imperfectly repeated, i.e. it is perfectly symmetrical or partially symmetrical. The flanking inverted repeats may be contiguous with the central palindrome, or The protelomerase target sequence may be at least 14 bases in length. It may contain imperfect inverted repeats, including pairs of perfect inverted repeats.

[0087] The protelomerase target sequence comprising the sequence of SEQ ID NO: 3 or a variant thereof is SEQ ID NO: 2 and the E. coli N15 TelN protelomerase and its variants. I wish.

[0088] A variant of either the palindromic sequence or the protelomerase target sequence may be Variants include truncations, substitutions, or deletions to the native sequence. A variant sequence is any sequence whose presence in the template DNA results in a proterologous sequence. The enzymatic activity of the cleavage enzyme allows the conversion of the closed linear DNA into a closed linear DNA. This can be readily determined by using an appropriate assay for forming DNA. Any suitable assay in the art may be used. The activity of the protelomerase binding assay was comparable to that observed with the native sequence. Examples of preferred variants of the palindromic sequences described herein As for the structure, it preserves the perfect repeat structure and maintains the state where the closed linear DNA can be formed. However, variant protelomerase targets The target sequence is a complete palindromic sequence if it can act as a substrate for protelomerase activity. The program may be modified so that it no longer stores the program.

[0089] Based on the structural principles summarized above, one skilled in the art can easily design suitable protelomers for use in the present invention. It should be appreciated that enzyme target sequences can be readily identified. Candidate protelomerase targets The sequences were tested for their ability to promote the formation of closed linear DNA using the assay described above. The antibodies can be screened for potency.

[0090] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the template DNA is a DNA of interest. In the case where the template DNA is a circular double-stranded DNA containing a sequence, step (a) comprises a plasmid vector containing at least two adjacent recombinase recognition sites; by contacting the gene with a specific recombinase, more particularly Cre recombinase. It will be carried out.

[0091] The action of a site-specific recombinase on the plasmid vector results in the fusion of two recombinases. This causes recombination of the recombinase recognition sites in the plasmid vector. A small circular double-stranded DNA is generated containing the desired DNA sequence located between the recognition sites. can be.

[0092] "Site-specific recombinase," as used herein, refers to a recombinase that recognizes a site-specific recombination between specific DNA sequences recognized by enzymes known as It refers to a family of enzymes that mediate site-specific recombination. Examples of site-specific recombinases are Cre recombinase and Recombinase, Flp recombinase, λ integrase, γ-δ resolvase, Tn3 Resolvase, Sin resolvase, Gin invertase, Hin invertase, T n5044 resolvase, Tn3 transposase, sleeping beauty transposase, IS607 transposase, Bxb l integrase, wBeta Integrase, BL3 integrase, phiR4 integrase, All l 8 in Tegulases, TGI integrases, MRU integrases, phi370 integrases ze, SPBc integrase, SV1 integrase, TP901-1 integrase, phiRV integrase, FC1 integrase, K38 integrase, phiBT integrase and phiC31 integrase. stomach.

[0093] A "recombinase recognition site" is a nucleoside that is recognized by a site-specific recombinase. Recombinase recognition sites are non-recombinase recognition sites that can serve as substrates for recombination events. Non-limiting examples include FRT, FRT11, FRT71, attp, att, rox, and loxP, lox511, 1οχ2272, 1οχ66, 1οχ71, loxM2 and l Examples of lox sites include ox5171.

[0094] Those skilled in the art will understand, using their general knowledge, that a site-specific recombinase is a specific recombinase. The enzyme recognizes the enzyme recognition site, thereby responding to the recognition sequence contained within the plasmid vector. Therefore, different recombinases are required to generate circular double-stranded template DNA from the plasmid vector. I understand that I have to use enzymes.

[0095] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, a site-specific recombinase is a Cre recombinase. In a more particular embodiment, the recombinase recognition site is In an even more particular embodiment, the site-specific recombinase is Cre recombinase. The recombinase recognition site is loxP.

[0096] The sequences flanking the DNA sequence of interest in the plasmid vector and the template D in step (a) Depending on the process used to produce NAs, the concatemer products produced in step (b) may be The product contains a DNA sequence of interest flanked by different sequences. For example, the DNA of interest is In the case of a DNA fragment containing only the protelomerase target sequence in the vector, The DNA sequence of interest is adjacent to the protelomerase target sequence. The DNA sequence of interest in the vector can be introduced by some type of reaction (e.g., by TelN). Template DNA can be generated, which can then be used in other reactions (e.g., restriction enzyme digestion and adapter ligation) to generate cDNA from the amplified product, In these particular cases, the DNA sequence of interest may then be proteolytically The restriction site must be adjacent to the target site for the nucleotide sequence.

[0097] Thus, in certain embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments provided below, steps (b) to The amplified DNA obtained from the PCR reaction is a concatemer DNA containing repeats of the target DNA sequence. Each of the repeated DNA sequences contains a restriction site, a protelomerase target sequence, and and / or flanked by recombinase recognition sites.

[0098] Those skilled in the art will appreciate that tandem excision of a DNA sequence of interest in the form of a closed linear DNA sequence can be achieved by: The tandem units are cleaved and the open ends of the fragments are closed to form a covalently closed molecule. It will be understood that this can be achieved by different conventional molecular biology techniques. These two steps can be accomplished, for example, by restriction enzyme digestion and adapter ligation. This can be done sequentially or simultaneously by the action of protelomerase.

[0099] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the concatemer DNA may comprise at least If the DNA sequence contains repeats of the desired DNA sequence flanked by restriction sites, step (c) is contacting the chromatin DNA with at least one restriction enzyme, thereby isolating the desired DNA sequence; (c2) creating a plurality of open double-stranded DNA fragments each containing a single-stranded DNA fragment; This is done by ligating an adapter to both ends of an open double-stranded DNA fragment. All of the embodiments provided above regarding the restriction sites and single-stranded DNA adapters are Furthermore, those skilled in the art can easily create template cDNA. If restriction enzymes are used to prepare the cDNA from the amplified DNA produced in step (b), Similar restriction enzymes can later be used to generate A. Generate template c1DNA. The single-stranded DNA adapter used in step (a) for the purpose of It may be similar to or different from.

[0100] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the concatemer DNA may comprise at least If the DNA sequence contains repeats of the desired DNA sequence flanked by protelomerase target sequences, step (c ) is produced by contacting concatemeric DNA with protelomerase, more specifically TelN. The method is carried out by the method provided above with respect to protelomerase and protelomerase target sites. All embodiments mentioned above are meant to apply to this embodiment as well. When a manufacturer uses protelomerase to generate template clDNA in step (a), In step (c), a similar protelomerase is used to generate clDNA from the amplified DNA. It can be used later.

[0101] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the process is a closed linear expression This is a process for creating cassette DNA.

[0102] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, step (a) may further comprise: A plasmid vector containing at least two restriction sites flanking the A sequence and at least A restriction enzyme is then applied to the target DNA fragment, thereby creating an open double strand containing the target DNA sequence. Create an open double-stranded DNA containing the DNA sequence of interest by ligating a single-stranded DNA adapter to the open double-stranded DNA. and step (c) is carried out by binding to both ends of NA, and (c1) concatemer D The DNA is contacted with at least one restriction enzyme, thereby cleaving the target DNA sequence. (c2) generating a plurality of open double-stranded DNA fragments containing the DNA fragments according to the first aspect of the present invention; The single-stranded DNA adapters described above are then attached to both ends of the open double-stranded DNA fragments. In more particular embodiments, the restriction enzyme generates sticky or blunt ends. If the restriction enzyme generates blunt ends, the resulting fragments will be fused to adapters containing blunt ends. This can be coupled to a DA tail as explained above, or alternatively, it can be DA tailed and An overhanging T can be used to connect to the adapter.

[0103] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, step (a) may further comprise: At least two protelomerase recognition sites flanking the A sequence A plasmid vector containing two restriction sites is contacted with at least one restriction enzyme. thereby generating a gene encoding a DNA fragment containing the DNA sequence of interest flanked by protelomerase recognition sequences. Double-stranded DNA was created by binding single-stranded DNA adapters to both ends of the open double-stranded DNA. and step (c) is carried out by reacting the concatemer DNA with protelomerase, more particularly Typically, this is accomplished by contacting the substrate with TelN.

[0104] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the template DNA may be a circular double-stranded template DNA containing adjacent DNA sequences of interest, and step (a) At least two restriction sites flanking the DNA sequence of interest A plasmid vector containing a recombinase recognition site and a site-specific recombinase is more particularly carried out by contacting with Cre recombinase, c) (c1) contacting the concatemer DNA with at least one restriction enzyme, thereby generating a plurality of open double-stranded DNA fragments each containing a DNA sequence of interest; 2) Attaching a single-stranded DNA adapter to both ends of a double-stranded DNA fragment as described in the first embodiment This is done by attaching to the termini.

[0105] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the template DNA is In the case where the template DNA is a circular double-stranded DNA containing a target DNA sequence adjacent to the target DNA, step (a) At least two restriction sites flanking the target DNA sequence A plasmid vector containing a recombinase recognition site for a site-specific recombinase, More particularly, step (c) is carried out by contacting the gene with Cre recombinase. By contacting the concatemer DNA with protelomerase, more specifically TelN, It will be carried out.

[0106] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, step (a) may further comprise: At least two promoter sequences flanked by at least two restriction sites flanked by A sequences A plasmid vector containing a telomerase target sequence and a protelomerase, more particularly and step (c) is carried out by contacting (c1) concatemer DNA with TelN. and at least one restriction enzyme, thereby forming fragments each containing the desired DNA sequence. (c2) creating multiple open double-stranded DNA fragments having single-stranded DNA adapters; This is carried out by ligating to both ends of a double-stranded DNA fragment.

[0107] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, step (a) may further comprise: Two protelomeres flanked by at least two restriction sites flanked by A sequences A plasmid vector containing a protelomerase target sequence is contacted with a protelomerase, such as TelN. The step (c) is carried out by: (c1) reacting a concatemer DNA with at least one a restriction enzyme, thereby creating multiple open fragments each containing the desired DNA sequence. (c2) creating double-stranded DNA fragments, and (c3) connecting single-stranded DNA adapters to the open double-stranded DNA fragments; This is carried out by binding to both ends of the

[0108] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, step (a) may further comprise: At least one of the sequences (e.g., of protelomerase B) that is different from the first one adjacent to the A sequence At least two protelomerase recognition sites (e.g., protelomerase A) are adjacent to each other. a plasmid vector containing at least two protelomerase target sequences (e.g., a protelomerase target sequence); The step ( c) The concatemer DNA and the corresponding protelomerase (e.g., protelomerase B) In certain embodiments, step (a) or step (c) is carried out by contacting The protelomerase is TelN.

[0109] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, step (a) comprises: A plasmid vector containing two protelomerase target sites flanking the sequence and a protein and step (c) is carried out by contacting the concatemer DNA with protease. In certain embodiments, step (a) or (b) is carried out by contacting the enzyme with a ferrocene. The protelomerase in step (c) is TelN.

[0110] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, step (a) may further comprise: Contains at least two restriction sites and a non-protelomerase target site flanking the A sequence A plasmid vector is contacted with at least one restriction enzyme, thereby generating a DNA fragment of interest. A sequence containing double-stranded open DNA is generated, and a single-stranded DNA adapter is inserted between the protelomeres. The single-stranded DNA adapter was then ligated to the target DNA, provided that it did not contain a target site for the enzyme. This is done by attaching the sequence to both ends of an open double-stranded DNA containing the and step (c) comprises (c1) contacting the concatemer DNA with at least one restriction enzyme; This generates multiple open double-stranded DNA fragments, each containing the desired DNA sequence. and (c2) ligating the single-stranded DNA adaptor according to the first aspect of the present invention to an open double-stranded DNA. This is done by ligating both ends of the fragment.

[0111] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described herein, the DNA sequence of interest is In a more particular embodiment, the expression cassette comprises a set of a eukaryotic promoter, and optionally an enhancer, operably linked to a sequence comprising consists of a eukaryotic transcription termination sequence.

[0112] The term "expression cassette" refers to a set of one or more promoter or enhancer elements and a gene encoding a desired gene. a gene or coding sequence encoding an RNA, miRNA, siRNA, or protein; An expression cassette refers to a DNA sequence that contains a sequence of genes that regulate the expression of a coding sequence, such as a transcription termination site. It may further include other elements.

[0113] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the DNA sequence of interest may be an inverted The expression cassette is flanked by inter-terminal repeats (ITRs). The ITRs are preferably For example, the ITRs can be directly attached to the expression cassette, or are 1 to 50 nucleotides, 50 to 200 nucleotides, and 200 to 1000 nucleotides. Therefore, in certain embodiments, the method of the present invention may be at a distance from the In combination with any of the embodiments described above, the DNA of interest may be 1 to 50 nucleotides long. It contains an expression cassette flanked at a distance of 100 kJ by inverted terminal repeats (ITRs).

[0114] As used herein, a "terminal repeat" or "TR" refers to at least one minimally required Any virus containing the region containing the required replication origin and palindromic hairpin structure. Contains a Rep-binding sequence. uence: "RBS") (also called RBE (Rep-binding element) and terminal resolution sites ( TRS) constitutes the "minimal required origin of replication" and therefore requires at least one A given stretch of a polynucleotide sequence contains an RBS and at least one TRS. Internal reverse-complementary sequences of the TRs are typically called "inverted terminal repeats" or "ITRs." In viruses, the ITRs are involved in replication, viral packaging, and , involved in integration and proviral rescue.

[0115] In complex clDNA configurations, there may be more than two ITRs or asymmetric ITR pairs. Those skilled in the art will understand that an ITR may be an AAV ITR or a non-AAV ITR. or may be derived from AAV ITRs or non-AAV ITRs. For example, this ITR Parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus) Viruses, mouse parvovirus, porcine parvovirus, human parvovirus B-19) or may function as an origin of SV40 replication. SV40 hairpins can be used as ITRs, allowing for truncations, substitutions, deletions, insertions, and Parvoviridae viruses can be further modified by addition of the following: The Parvoviridae family infects vertebrates, while the Densovirinae subfamily infects invertebrates. Dependoparvoviruses affect humans, primates, bovine, canine, equine, and ovine species. Adeno-associated viruses (AAVs) capable of replicating in vertebrate hosts, including but not limited to: For convenience, the term "expression vector of a cDNA" is used herein to refer to the virus family AAV. The ITR located 5' to the cassette (upstream) is referred to as the "5' ITR" or "left ITR." TR” and is located 3′ to the expression cassette (downstream) of the clDNA vector. The ITR is called the "3' ITR" or "right ITR."

[0116] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the DNA sequence of interest may be An expression cassette flanked by at least one inverted terminal repeat of the sequence of SEQ ID NO: 8 or SEQ ID NO: 9 Includes

[0117] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the DNA sequence of interest may be adjacent to the 5' inverted terminal repeat of sequence number 8 and / or the 3' inverted terminal repeat of sequence number 9 It contains a contiguous expression cassette.

[0118] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described herein, the DNA sequence of interest may be at least The expression cassette is flanked by at least one DD-ITR. iao X.et al.,´´A novel 165-base-pair ter minal repeat sequence is the sole cis re requirement for the adeno-associated virus life cycle´´,1997,J Virol.,vol.71(2),pp 941-948.

[0119] With regard to step (d) of the method, which purifies the generated clDNA, those skilled in the art will be able to It is understood that any known method suitable for purifying c1DNA may be used. are.

[0120] As mentioned above, in a second aspect, the present invention relates to a method for producing a soluble polymer obtained according to the process described in the first aspect. This provides a closed linear DNA that can be used for

[0121] For purposes of this invention, the terms "obtained," "obtained," and equivalents are used interchangeably. are used interchangeably and in either case the expression "obtained" is used interchangeably with the expression "obtained" All embodiments provided under the first aspect of the invention may be combined with the second aspect of the invention. It is also an embodiment of a closed linear DNA such as

[0122] In particular embodiments of the second aspect of the invention, In combination with any of the embodiments, the closed linear DNA may contain one or more expression Set includes.

[0123] In particular embodiments of the second aspect of the invention, In combination with any of the embodiments, the expression cassette may be an mRNA, miRNA, A, a eukaryotic siRNA or a protein-encoding sequence operably linked to the eukaryotic siRNA or protein-encoding sequence Contains a promoter.

[0124] In particular embodiments of the second aspect of the invention, In combination with any of the embodiments, the expression cassette may comprise a eukaryotic transcription terminator. It further comprises the sequence:

[0125] In particular embodiments of the second aspect of the invention, In combination with any of the embodiments, the expression cassette comprises: (i) bacterial origin of replication; (ii) bacterial selectable markers; (iii) unmethylated CpG motifs; The vector lacks one or more bacterial or vector sequences selected from the group consisting of:

[0126] In particular embodiments of the second aspect of the invention, In combination with any of the embodiments, the DNA sequence of interest may contain inverted terminal repeats ( It contains an expression cassette flanked by ITRs.

[0127] As already mentioned, the present invention also relates to a closed straight-through device according to the first aspect for use in medical treatment. Stranded DNA is provided in a third aspect.

[0128] The clDNA of the present invention can be used in host cells, particularly in DNA vaccines or gene therapy. DNA vaccines may be used to express infectious organism DNA in vitro. The DNA vaccine typically encodes a modified form of the These vaccines express selected proteins of the infectious organism and are usually protective. DNA vaccines are also being used as an immunotherapy approach for cancer. The vector may encode a tumor antigen in a locus.

[0129] DNA vaccines are available for fungi, human papillomavirus (HPV), HIV, HSV2 / HSV1, influenza virus (types A, B and C), poliovirus, RSV Virus, rhinovirus, rotavirus, hepatitis A virus, Norwalk virus group, Enterovirus, astrovirus, measles virus, parainfluenza virus, Chronic parotitis virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus Barr virus, adenovirus, rubella virus, human T-cell leukemia virus type 1 (HT LV-1), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus Viruses including rhesus, poxvirus, Marburg and Ebola; Mycobacterium tuberculosis, Chlamydia A, gonorrhea, bacillary dysentery, salmonella, cholera, Treponema pallidum, Pseudomonas, Bordetella pertussis, Brucella, Francisella tularensis, Helicobacter pylori, pathogenic Leptospira, Dionella pneumophila, Yersinia pestis, Streptococcus (types A and B), Pneumococcus, meningeal Haemophilus influenzae, Haemophilus influenzae type b, Toxoplasma gondii, Campylobacter Campylobacter spp. (Campylobacteriosis), Moraxella catarrhalis, Bacteria, including novanopsis and actinomycosis; candidiasis and aspergillosis Fungal pathogens including cestodes, trematodes, nematodes, amoebic dysentery, giardiasis, cryptosporidium Poridium, schistosomiasis, pneumocystis carinii, trichomoniasis and trichinellosis Cancer and allergies caused by pathogens, including but not limited to parasitic pathogens Antigens for treating or preventing a number of conditions, including but not limited to, toxicity and infection It may comprise a nucleic acid sequence encoding:

[0130] DNA vaccines are directed against viruses such as Adenoviridae (including, for example, human adenoviruses), herpes Sviridae (including, for example, HSV-1, HSV-2, EBV, CMV, and VZV); Papovaviridae (including, for example, HPV), Poxviridae (including, for example, smallpox and vaccinia) Seniors), Parvoviridae (including parvovirus B19), Reovirus Family: Mycobacterium (including, for example, rotavirus), Family: Coronaviridae (including, for example, SARS), Family: Viviridae (e.g., yellow fever, West Nile virus, dengue virus, hepatitis C virus) and tick-borne encephalitis viruses), Picornaviridae (poliovirus, rhinovirus, viruses and hepatitis A virus), Togaviridae (including, for example, rubella virus) , Filoviridae (e.g., Marburg virus and Ebola virus), Paramyxoviridae Influenza virus (e.g., parainfluenza virus, respiratory syncytial virus, mumps virus) and measles virus), Rabaviridae (including, for example, rabies virus), Bunyavirus family (including, for example, Hantaviruses), Orthomyxoviridae (including, for example, types A, B, and C) influenza), Retroviridae (including, for example, HIV and HTLV), and Encoding antigens derived from members of the Hepadnaviridae family (including, for example, Hepatitis B virus) The nucleic acid sequence may include a nucleic acid sequence

[0131] Antigens may be derived from pathogens that cause veterinary disease, particularly viruses such as reoviruses (AfV). Horse sickness or bluetongue virus) and herpesviruses (equine herpesvirus) Antigens can be derived from viral pathogens, including foot and mouth disease virus, tick-borne Encephalitis virus, dengue virus, SARS, West Nile virus and hantavirus The antigen may be derived from an immunodeficiency virus. V or feline immunodeficiency virus.

[0132] The clDNA produced by the process of the present invention also contains a nucleic acid sequence encoding a tumor antigen. Examples of tumor-associated antigens include the MAGE family (MAGE1, 2, 3 ), cancer testis antigens such as members of NY-ESO-1 and SSX-2, tyrosinase, g Differentiation antigens such as p100, PSA, Her-2 and CEA, E6 from oncogenic HPV types and / or mutated autoantigens such as E7 and viral tumor antigens. Further examples of specific tumor antigens include MART-1, Melan-A, p 97, β-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-4, MAG E-12, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, P 1A, EpCam, melanoma antigen gp75, Hker8, high molecular weight melanoma antigen, K 19, Tyr1, Tyr2, pMel17 gene family, c-Met, PSM (prostate Prostate gland mucin antigen), PSMA (prostate-specific membrane antigen), prostate secretory protein, α-fetoprotein Protein, CA125, CA19.9, TAG-72, BRCA-1 and BRCA-2 antibodies Hara is one example.

[0133] In addition, the process of the present invention can be used to generate other target genes, such as clDNA for use in gene therapy. For example, a patient may have a dysfunctional version of that gene. In cases where a person has a genetic disorder caused by DNA molecules can be used. Examples of such diseases include Duchenne muscular dystrophy. trophy, cystic fibrosis, Gaucher disease, and adenosine deaminase (ADA) deficiency Other diseases for which gene therapy may be useful include inflammatory diseases, AIDS, etc. Any disorder, including autoimmune, chronic and infectious diseases, cancer, neurological disorders, cardiovascular diseases (car Diabetic disease, hypercholesterolemia estemia), various blood disorders, including various anemias, thalassemia, and hemophilia; For the treatment of solid tumors, toxic peptides (i.e., ricin, diphtheria, genes encoding chemotherapeutic agents such as diptheria toxins and cobra venom factors Antisense to genes, tumor suppressor genes such as p53, and transforming oncogenes The gene encoding the mRNA sequence that is the tumor necrosis factor (TNF) Anti-cancer peptides and other cytokines, such as tumor necrosis factor (TNF), or tumor suppressor Transdominant-negative mutants of transforming oncogenes can be expressed.

[0134] Other types of therapeutic cDNA are also contemplated for production by the process of the present invention. They are transcribed into active RNA forms, such as small interfering RNA (siRNA). The clDNA can be produced, for example, by the process of the present invention.

[0135] As mentioned above, the present invention also provides a therapeutically effective amount of a closed linear peptide according to the second aspect of the invention. A pharmaceutical composition is provided that includes DNA and a pharmaceutically acceptable carrier or excipient.

[0136] As used herein, the phrase "therapeutically effective amount" refers to a dose that, when administered, is effective to treat the disease being treated. sufficient to prevent or alleviate to some extent one or more of the symptoms of the disease The specific amount of agent administered according to the present invention refers to the amount of clDNA administered. A. Surrounding the case, including route of administration, specific condition being treated, and similar considerations This will of course depend on the particular circumstances.

[0137] The expression "pharmaceutical composition" includes both human and non-human animal compositions (i.e. The term "veterinary composition" encompasses both compositions intended as pharmaceutical compositions and pharmaceutical preparations.

[0138] The expression "pharmaceutically acceptable carrier or excipient" means a pharmaceutically acceptable substance, composition, or Each component is meant to be compatible with the other components of the pharmaceutical composition. The ingredients must also be free of adverse toxicity, irritation, allergic reaction, immunogenicity, or other adverse events commensurate with a reasonable benefit / risk ratio contact with human or non-human animal tissues or organs without other significant problems or complications It must be suitable for use.

[0139] Examples of suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents or other liquid vehicles. , dispersing or suspending aids, surfactants, isotonicity agents, thickening stabilizers or emulsifiers, preservatives, solid binders, lubricants, etc. Any conventional excipient vehicle may be used, e.g., any desirable produce no biological effect or interact adversely with any other component of the pharmaceutical composition. Unless the substance or its derivatives are compatible with the invention by interacting in a manner that It is contemplated that the invention is within the scope of the invention.

[0140] The closed linear DNA, the pharmaceutically acceptable excipient, any additional components in the pharmaceutical composition of the present invention The relative amounts of each component will depend on the identity, size and / or condition of the subject being treated, and will vary depending on the route by which the composition is to be administered.

[0141] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include inert diluents, dispersants, Dispersing and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers These include, but are not limited to, colorants, coating agents, lubricants and / or oils. Excipients, such as sweetening agents and flavoring agents, may be present in the composition, according to the judgment of the formulator.

[0142] Pharmaceutical compositions containing closed linear DNA produced by the process of the present invention can be prepared, for example, by The pharmaceutical composition may be in any dosage form, such as solid or liquid. Administered by any suitable route, such as orally, parenterally, rectally, topically, intranasally, or sublingually. These can be used, for example, in topical formulations (ointments, creams, lipogels, hydrogels, etc.), eye drops, necessary to formulate the desired dosage form, such as aerosol sprays, injectable solutions, or osmotic pumps. The formulation contains essential pharmaceutically acceptable excipients.

[0143] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, phosphorus Dicalcium carbonate, calcium sulfate, sodium hydrogen phosphate, sodium phosphate, lactose Sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol Inositol, sodium chloride, dry starch, corn starch, powdered sugar and Combinations include, but are not limited to:

[0144] Exemplary granulating and / or dispersing agents include potato starch, corn starch, Tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum , citrus pulp, agar, bentonite, cellulose and wood products, sponge, cation exchange resin Fat, calcium carbonate, silicate, sodium carbonate, cross-linked polyvinylpyrrolidone cross-linked Vidon, sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (cross-linked sodium carboxymethylcellulose Roasted rice), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch Ingredients: water-insoluble starch, carboxymethylcellulose calcium, magnesium silicate Aluminum (Veegum), sodium lauryl sulfate, quaternary ammonium compounds and This includes, but is not limited to, combinations thereof.

[0145] Exemplary binding excipients include starches (e.g., cornstarch and starch paste). ), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), natural and synthetic gums (e.g. , acacia, sodium alginate, Irish moss extract, panwar gum (pan war gum, ghatti gum, psyllium mucilage, carbo Hydroxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose Cellulose, cellulose acetate, polyvinylpyrrolidone, silicon aluminum magnesium ( Veegum) and larch arabinogalactan, alginate, oxidized polyethylene, Polyethylene glycol, inorganic calcium salt, silicic acid, polymethacrylate, water, alco Examples of suitable ion exchange media include, but are not limited to, ion exchange media ...

[0146] Exemplary preservatives include antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohols, and the like. Included may be choline preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include: , alpha tocopherol, ascorbic acid, ascorbyl palmitate, stearic acid Ascorbyl, Ascorbyl Oleate, Butyl Hydroxyanisole, Butylated Hydroxyanisole Dihydroxytoluene, monothioglycerol, potassium pyrosulfite, propionic acid, gallic acid propionate propyl, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and Exemplary chelating agents include, but are not limited to, sodium sulfite and sodium bisulfite. Contains ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, edetate disodium, Dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, alcohol Examples include tartaric acid and edetate trisodium.

[0147] Exemplary buffers include citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, Um, calcium carbonate, calcium chloride, calcium citrate, sodium glubionate , calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate Calcium, calcium lactate, propionic acid, calcium levulinate, pentanoic acid, phosphate Calcium hydrogen, phosphate, tricalcium phosphate, calcium hydrogen phosphate, potassium acetate, Potassium chloride, potassium gluconate, potassium mixture, potassium phosphate dibasic, phosphoric acid Potassium monobasic, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride Sodium citrate, sodium lactate, sodium phosphate dibasic, sodium phosphate Thorium monobasic, sodium phosphate mixture, tromethamine, magnesium hydroxide, hydroxide Aluminum chloride, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohols, and combinations thereof.

[0148] Exemplary lubricants include magnesium stearate, calcium stearate, stearate, Allyl acetate, silica, talc, malt, glyceryl behenate ate), hydrogenated vegetable oil, polyethylene glycol, leucine, sodium lauryl sulfate These include, but are not limited to, hydroxybenzoates ...

[0149] As disclosed above, the present invention provides a concatemer DNA containing repeats of a DNA sequence of interest. Thus, each of the repeated DNA sequences of interest is cleaved by at least one recombinase. adjacent to the recognition site, or alternatively at least a restriction site and a protelomerase Concatemer DNA flanking the target sequence is provided in a fifth aspect.

[0150] In particular embodiments of the process of the fifth aspect of the invention, optionally In combination with any of the embodiments described above, the repeated DNA of interest Each one of the sequences is additionally flanked by ITRs.

[0151] In particular embodiments of the process of the fifth aspect of the invention, optionally In combination with any of the embodiments described above, the concatemer DNA is a DNA of interest. It contains 10 or more repeats of the A sequence.

[0152] In particular embodiments of the process of the fifth aspect of the invention, optionally In combination with any of the embodiments described above, the concatemer DNA is and at least 5kb.

[0153] Throughout this specification and claims, the term "comprises" and variations of this term refer to other technologies. It is not intended to exclude technical features, additives, ingredients, or steps. Further objects, advantages and features of the present invention are set forth in this description. These and other features will become apparent to those skilled in the art upon examination of the present invention or may be learned by practice of the invention. The drawings are provided by way of example only and are not intended to limit the invention. Related reference signs in parentheses in the claims are intended to aid in the understanding of the claims. is merely an attempt to increase the scope of the claims and should be interpreted as limiting the scope of the claims. Furthermore, the present invention does not encompass the specific and preferred embodiments described herein. Cover all possible combinations. [Example]

[0154] Example 1: TthPrimPol-based template DNA amplification is more efficient than random primers provides higher sequence fidelity than conventional methods.

[0155] Primed by either TthPrimPol or random primers (RP) RCA of 10 ng of plasmid vector containing the DNA sequence of interest using phi29 Amplification (pUC57-Kan_TELO-CMVEGFP with SEQ ID NO: 23) was performed The reaction conditions were 30°C for 6 hours and 65°C for 10 minutes, and the total reaction volume was 100 μL. .

[0156] As shown in Figure 1, amplification stimulated with TthPrimPol resulted in a significant increase in the number of copies of the template DNA. On the other hand, stimulation with RP did not produce any amplification products containing RP (left column, NTC). The amplification produced high DNA yields even in the absence of template DNA. , suggesting that TthPrimPol priming provides a highly specific amplification reaction. Furthermore, the DNA yield produced by TthPrimPol priming was , which was of similar magnitude to the DNA yield produced by RP in the presence of template DNA. (Right column, plasmid).

[0157] Finally, purify the primers used for amplification by either TthPrimPol or RP. and standard protocols were used, with Illumina technology (5 million reads per pair, 2 × 1 As shown in Figure 2, the raw sequencing results were Bioinformatic analysis showed that TthPrimPol-based amplification produced 80% usable reads. Although it is possible to produce a gene encoding a nucleotide sequence, amplification using randomly synthesized primers yields only 66% of the available sequences. The priming method is based on the use of TthPrimPol. It was confirmed that when this was the case, the amount of DNA artifacts produced was small.

[0158] In conclusion, the above results suggest that TthP is a promising candidate for the process of generating therapeutic polynucleotides. The use of PrimPol is due to the high fidelity of TthPrimPol in the amplification process. This suggests that this may be advantageous.

[0159] Example 2: Generation of clDNA based on TthPrimPol-stimulated amplification

[0160] As described in the examples above, 10 ng of plasmid pUC57-Ka was isolated by RCA. n_TELO-CMVEGFP was amplified and then purified using standard protocols. The amplified product was purified using

[0161] Next, in particular the following conditions: Reaction volume: 1008 μL -DNA input: 350 μg of amplified DNA -TelN Input: 125 μL (625 units) Reaction time: 30 minutes at 30°C, followed by 5 minutes at 75°C according to the manufacturer's protocol The amplification products (DNA concatemers) were treated with protelomerase (TelN). .

[0162] Finally, the product from the reaction with TelN was digested with a restriction enzyme to produce exonuclease. Treat with lyase to remove unwanted DNA fragments resulting from the protelomerase reaction did.

[0163] HindIII and EcoRI digestion was performed according to the manufacturer's instructions. This results in: Reaction volume: 1453 μL DNA input: 350 μg of amplified DNA digested with TelN -EcoRI input: 150 μL (1500 units) -HindIII input: 150 μL (1500 units) -Reaction time: 60 minutes at 37°C, 15 minutes at 65°C.

[0164] Exonuclease III digestion was performed according to the manufacturer's instructions. Then: Reaction volume: 1628 μL DNA input: 350 μg digested with TelN, HindIII and EcoRI Amplified DNA -ExoIII input: 6 μL (600 units) -Reaction time: 45 minutes at 37°C, 20 minutes at 80°C.

[0165] As shown in Figure 3, TthPrimPol-stimulated amplification was dependent on the action of TelN. This produced amplification products that could be successfully converted from c1 DNA.

[0166] Finally, the obtained DNA was purified according to standard procedures, with a purified yield of 80.5 μg ( The cl obtained by Sanger sequencing was 106ng / μL (760μL). The quality of the DNA was analyzed.

[0167] Example 3: clDNA production using TelN-generated template clDNA

[0168] TthPrimPol is the smallest single-stranded loop that does not contain its target sequence, "XTC." To test whether the cDNA could be primed using the expression cassette, Plasmid pUC57-K contains two protelomerase recognition sequences flanking the an_TELO-CMVEGFP was transfected with protelomerase (T) as described in Example 2. e1N) to generate clDNA containing the expression cassette.

[0169] As described in Example 1, either TthPrimPol or random primers 1 ng of the resulting cDNA or initial plasmid was used for RCA stimulation. As shown in Figure 4A, the amplification products were quantified using TthPrimPol priming. Although amplification of TelN-generated clDNA was possible using the primers, amplification using random primers was not possible. The yield was lower than the width.

[0170] The amplified products (DNA concatemers) were then treated with TelN and reassembled into clDNA. This was purified by restriction enzyme and exonuclease treatment as described in Example 2. The clDNA was analyzed in an agarose gel.

[0171] Although random primer amplification resulted in a high yield of DNA product (Fig. 4A), amplification D Assays generated by TelN from NA were primed with TthPrimPol. We demonstrated that high levels of clDNA containing the target DNA sequence can be produced (Figure 4 B).

[0172] These results indicate that TthPrimPol contains the minimal adapter sequence (i.e., Not only can it stimulate c1 DNA (which does not contain the recognition sequence for c1), but more importantly, In this case, higher quality amplification primers than random primers are used, which increases the efficiency of the final clDNA production. It has also been demonstrated that a wide range of products can be produced.

[0173] Example 4: Functional verification of clDNA produced by the process of the present invention

[0174] eGFP (enhanced green fluorescent protein), prepared as disclosed above in Example 3. a plasmid vector containing the coding sequence for eGFP; an empty vector; The cDNA containing the - was prepared by Heinrich, M. et al. "Linear c lost mini DNA generated by the prokaryo tic cleaving-joining enzyme TelN is func tional in mammalian cells´´,J Mol Med,20 02, vol.80, pp.648-654, into HEK293 cells. Transiently transfected.

[0175] Cells were analyzed by microscopy at 24 and 48 hours using standard microscopy protocols. The fluorescence intensity of the cells was measured according to the rule.

[0176] As shown in Figures 4 and 5, the cloned DNA was synthesized according to the process of the present invention. The transfected cells showed strong expression of eGFP. These results support the use of the present invention. This process enables the generation of highly functional cDNA suitable for gene therapy. It proves this.

[0177] Example 5: Customized single-stranded DNA from TelN-generated clDNA by RCA Preparation of clDNA containing adapters

[0178] Customized single-stranded DNA adapters containing natural and modified nucleotides Puta Synthesis

[0179] The following modified nucleotides: 8-oxo-deoxyadenosine (8-oxo-dA), 5 -fluoro-deoxyuracil (5FU), inosine, thiophosphate nucleotides, or a standard phosphoramidate containing at least two locked nucleic acid (LNA) nucleotides. Natural nucleosides were synthesized according to Daito chemistry (Beaucage, Lett. et al., 1981). Synthesize customized single-stranded DNA adapters containing nucleotides and modified nucleotides. Ta.

[0180] Briefly, phosphoramidite synthesis begins at the most distant 3' nucleotide and continues to the most distant 4' nucleotide. A series of four steps is repeated until the selected 5' nucleotide is attached. The reaction proceeds through a cycle consisting of (i) deprotection, (ii) coupling, (iii) oxidation, and (iv) (iii) and capping (iv).

[0181] This cycle is repeated for each nucleotide in the sequence. The oligonucleotide may, for example, have a 3' end still attached to the CPG and a trityl group It exists as a 25-mer with a 5'-terminal protected by . In addition, the ring structure of the bases is Protecting groups remain on three of the four bases to maintain compatibility. Protecting groups are on A and C. The benzoyl on G and the N-2 isobutyryl on G. Thymidine does not require any protecting groups. The complete synthesis is detritylated, the controlled pore glass is cleaved, and the 3' and 5' ends are ´ Leaving a hydroxyl at the end. At this point, hot ammonium hydroxide is used to base The oligo (base and phosphate) is deprotected by hydrolysis. The final product is a functional single-stranded D It is an NA molecule.

[0182] The corresponding hairpin DNA adapter containing the natural oligonucleotide was also synthesized. A list of the synthesized adapters is provided in Table 2.

[0183] At the end of the synthesis, the oligonucleotides were cleaved from the support and the protecting groups were removed. Purification steps (e.g., PAGE, HPLC, and / or RNase-free HPLC) was used to separate the full-length product from the truncated sequences.

[0184] [Table 2]

[0185] Preparation of clDNA with customized adapters from plasmid DNA

[0186] Using some of the customized adapters in Table 2, plasmid DNA (pDNA ) to prepare clDNA. First, eGFP with SEQ ID NO: 20 was used. A plasmid (containing the sequence of interest encoding Gfp flanked by BsaI restriction sites) and a pDNA, such as a protelomerase target sequence (see Figure 9), is then added to the protelomerase The clDNA containing the sequence of interest flanked by endonuclease restriction sites is treated with Then, rolling circle amplification was performed using TthPrimPol and Phi29. This cDNA was amplified by PCR (reverse catalytic amplified). The resulting concatemers were purified and the corresponding Treat with a suitable restriction enzyme (e.g., BSaI) and insert a customized adapter (e.g., , and oligo 21 and oligo 41 in Table 2. Exemplary protocol details are given below. Provided to.

[0187] A. Protocol for obtaining clDNA from plasmid DNA [Table 4] [Table 5]

[0188] 1.1 TelN digestion Digest the eGFP plasmid with TelN enzyme for 2 hours at 30°C and 10 minutes at 75°C. If several reactions are performed simultaneously, scale up accordingly. do. [Table 6]

[0189] 1.2 Removal of the skeleton 1.2.1 Kpn I and Hind III Digestion The product of the final step was digested with Kpn I and Hind III for 1 hour at 37°C. The samples were then inactivated at 65°C for 15 minutes. Several reactions were carried out simultaneously. If so, scale up accordingly. [Table 7]

[0190] 1.2.2 Exo III Digestion The fragments were digested with Exo III at 37°C for 1 hour and inactivated at 75°C for 10 minutes. If reactions are performed simultaneously, scale up accordingly. [Table 8]

[0191] 1.3 Purifying cDNA using gel filtration chromatography and isopropanol R 1.3.1 Gel filtration chromatography Buffer A: 10 mM Tris-HCl, pH 7.5 Column: Vestarose 6 FF 153mL Sample 28ml Flow: 60cm / h Recovered fraction 20mAU-20mAU, 40mL CIP: 1M ​​NaOH + deionized water Storage: Pure water

[0192] 1.3.2 Endotoxin removal and isopropanol precipitation As shown in Table 6, 3M sodium acetate and 15% Triton-114 were added to the final Add to the sample from step 1 and mix by vortexing. Incubate the sample at 4°C for 5 minutes. The mixture is then centrifuged at 12000 g for 20 minutes at 25°C. Add an equal volume of isopropanol to the supernatant and mix thoroughly. Incubate at room temperature for 5 minutes. The sample was then centrifuged at 12000g for 20 minutes and the supernatant was removed. Finally, the precipitate is suspended in 10 mM Tris-HCl (pH 7.5). [Table 9]

[0193] Enzyme digestion, gel chromatography, Triton 114 treatment, and isopropanol precipitation After three steps, eGFP_BSaI_clDNA was successfully produced. The percent DNA homogeneity of the samples was 97%. The toxin level is less than 10EU / mg.

[0194] B. Retrieval of clDNA containing customized adapters from clDNA by RCA Protocol for obtaining This experiment was carried out using the Trueprime-RCA kit, clDNA containing customized adapter derived from GFP_BSaI_clDNA Designed to produce. [Table 10] [Table 11]

[0195] 1.1 RCA Mix constantly by pipetting, do not vortex. Transfer 10 μL of clDNA (≥1 ng / μL) to a clean tube. Add 10 μL of Buffer D and incubate at room temperature for 3 minutes. Neutralize the reaction by adding 10 μL of Buffer N to each tube. Keep samples at room temperature until use. * . Prepare the amplification mix by adding the components in the order listed in the table below. Incubate at 30°C for 3 hours ** Inactivate the reaction at 65°C for 10 minutes. do. ◇ Cool to 4°C. For short term storage, keep at 4°C, or for long term storage, keep at -2°C. Store the amplified DNA at 0°C. ( * ) It is highly recommended to perform the amplification reaction immediately after denaturing the sample. ( ** If higher amplification yields are required, the incubation time can be increased to 6 hours. It can be added.

[0196] If several reactions are carried out simultaneously, scale up accordingly. [Table 12]

[0197] 1.2 Purify the RCA products (concatamers) with isopropanol (as above) R

[0198] 1.3 Purify the RCA products (concatamers) using an Axygen kit (optional). If your sample is less than 100 μL, you can also use an Axygen kit to purify clDNA. The protocol is described below and the bottle containing the buffer is They are labeled as described below: 1) Add 2x the sample volume of Buffer DE-B and mix. 2) Place the Miniprep column into a 2 mL microfuge tube. Transfer the sample to the column. Centrifuge at 12,000 x g for 1 minute. 3) Discard the filtrate from the 2 mL microfuge tube. Place the tube back into the microfuge and add 500 μL of Buffer W1. Centrifuge for 30 seconds. 4) Discard the filtrate from the 2 mL microfuge tube. Place the tube back into the microfuge and add 700 μL of Buffer W2. Centrifuge for 30 seconds. 5) Discard the filtrate from the 2 mL microfuge tube. Place back into the microfuge tube. Add a second 700 µL aliquot of Buffer W2 and centrifuge at 12,000 x g for 1 minute. 6) Place the Miniprep column in a clean 1.5 mL microfuge tube (provided). To elute the DNA, add 50 μL of 10 mM Tris-HCl (pH 7.5 Add the 100ml of PBS to the center of the membrane. Let it sit at room temperature for 1 minute. Centrifuge at 12,000 x g for 1 minute. Separating the heart.

[0199] 1.4 Oligo denaturation and annealing Denature the oligo (e.g., oligo 21 or oligo 41 in Table 3) at 95°C for 10 minutes and The reaction mixture was allowed to anneal naturally at room temperature for 30 minutes. Scale up accordingly. [Table 13]

[0200] 1.5 Oligophosphorylation (optional, if the oligo is already phosphorylated, skip this step) (omitted) Oligophosphorylation at 37°C for 1 hour [Table 14]

[0201] 1.6 BsaI digestion BsaI digestion for 2 hours at 37°C and inactivation at 75°C for 10 minutes [Table 15]

[0202] 1.7 Purify the BsaI-digested RCA product with isopropanol (as above)

[0203] 1.8 Purify the BsaI-digested RCA product using the Axygen kit (as described above) , optionally with a sample of 100 μL or less).

[0204] 1.9 T4 ligation T4 ligation is carried out overnight at 16°C and inactivated at 75°C for 10 minutes. [Table 16]

[0205] 1.10 Advanced Golden Gate Assembly (Optional) Traditional ligation methods require several cloning steps to generate the desired construct. Each step involves inserting a single DNA fragment into a donor plasmid or PC Transfer the R product into the recipient vector.

[0206] During Golden Gate cloning, up to 15 It is possible to assemble fragments in a single tube, all plasmid donors, recipe Pipette the ent vector, type IIS restriction enzyme, and ligase together and then thermal cycle. Cloning is performed by incubating the mix in a They also propose to use Golden Gate assembly to generate oDNA. The system and conditions are explained in Tables 14 and 15, respectively. If implemented, scale up accordingly.

[0207] [Table 17] [Table 18]

[0208] 1.11 Unexpected DNA digestion The fragments were digested with Exo III at 37°C for 1 hour and inactivated at 75°C for 10 minutes. If reactions are performed simultaneously, scale up accordingly. [Table 19]

[0209] 1.12 Purify oDNA using isopropanol (as above)

[0210] 1.13 Purify DNA using an Axygen kit (as above, optionally with a sample) is less than 100 μL) Oligos 21 and 41 were used to successfully generate eGFP_BSaI_oDNA: [Table 20]

[0211] A similar procedure was used to generate the Luc plasmid containing SEQ ID NO: 21 (flanked by BsaI restriction sites). a luciferase-encoding sequence adjacent to the luciferase-encoding sequence, and a protelomerase target sequence; Oligos 15, 37, 4, 28, 29, 17, 22, 37, 28, 29, 19 and 2 in Table 2 2. Again using the same procedure, a clDNA was prepared starting from 2. Luc-ITR plasmid (containing luciferase with the sequence of interest flanked by BsaI restriction enzyme) The coding sequence is flanked by ITRs, and additionally includes a protelomerase target sequence. (See Figure 10) as well as clDNA starting from oligo 4. So, oDNA4ITR A total of 6.2 μg was obtained (Figure 11 (agarose gel electrophoresis) See electrophoresis).

[0212] Standard procedures (especially agarose gel electrophoresis, grayscale analysis, anion exchange chromatography) The obtained cDNA was analyzed by chromatography-HPLC and Sanger sequencing. All clDNAs were evaluated for quality in terms of purity, peak resolution, and sequence verification. It was found that the oDNA41 and oDNA21 exhibited good quality characteristics. The results are shown in Figures 7 and 8, respectively.

[0213] Example 6: cDNA synthesis from TelN-generated cDNA by RCA followed by TelN processing lDNA preparation

[0214] Alternatively, see Section A (1.1) through Section B (1.3) of Example 5. by the procedure described, followed by TelN processing of the resulting concatemers. The cDNA of the present invention may be prepared by this final step, i.e., by using protelomerase. The processing of the concatemers obtained from the RCA (Section B, 1.3) is detailed below. I will explain in detail.

[0215] 1.4 TelN digestion Digest the purified RCA product with TelN enzyme at 30 °C for 2 h and at 75 °C for 10 min. If several reactions are performed simultaneously, scale up accordingly. do. [Table 21]

[0216] 1.5 Skeleton Removal 1.4.1 Kpn I and Hind III Digestion The product of the final step was digested with Kpn I and Hind III for 1 hour at 37°C. The samples were then inactivated at 65°C for 15 minutes. Several reactions were carried out simultaneously. If so, scale up accordingly. [Table 22]

[0217] 1.4.2 Exo III Digestion The fragments were digested with Exo III at 37°C for 1 hour and inactivated at 75°C for 10 minutes. If reactions are performed simultaneously, scale up accordingly. [Table 23]

[0218] 1.6 Purify clDNA using isopropanol (as above)

[0219] 1.7 Purify the cDNA product using an Axygen kit (optionally sampled as above). (The volume is less than 100 μL) The sequence of interest encodes GFP (see Figure 9) and contains a constant 28 base pair protelomerase. Synthetic cDNA containing the enzyme sequence was obtained after cleavage / ligation. Each step is described in Table 24 (below). The resulting clDNA was analyzed by agarose gel electrophoresis. According to the AGE (Figure 12), the homogeneity was 96.6%. [Table 24]

[0220] References list Heinrich, M. et al. “Linear closed mini DNA generated by the prokaryotic cleavi ng-joining enzyme TelN is functional in mammalian cells”, J Mol Med, 2002, vol. 80, pp. 648-654 Altschul et al., “Basic local alignment search tool”, 1990, J. Mol. Biol, vol. 215, pp. 403-410 Xiao X. et al., “A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle”, 1997, J Virol., vol. 7 1(2), pp. 941-948. WO2011000997 Beaucage S. L. et al, Deoxynucleoside phosphoramidites-A new class of key interm ediates for deoxypolynucleotide synthesis. Tetrahedron Letters, Volume 22, Issue 20, 1981, Pages 1859-1862

Claims

1. 1. A process for making closed linear DNA, said process comprising: a) providing a template DNA containing a DNA sequence of interest; b) amplifying DNA from the template DNA of step (a), wherein said amplification is performed using primers priming with a ribozyme / polymerase enzyme; c) generating closed linear DNA using the amplified DNA produced in step (b). and d) purifying the closed linear DNA produced in step (c). 。

2. The process according to claim 1, wherein the amplification carried out in step (a) is rolling circle amplification. Seth.

3. the primase / polymerase enzyme is TthPrimPol of SEQ ID NO: 1, or At least 80% sequence identity to SEQ ID NO: 1, particularly at least 85% to SEQ ID NO: 1 3. The process of claim 1 or 2, wherein the sequence of the sequence of the nucleic acid sequence of the present invention is a variant thereof having a sequence identity of 1% or more.

4. The amplification in step (b) is carried out using a strand-displacing DNA polymerase, more particularly phi29 polymerase. The process according to any one of claims 1 to 3, which is carried out using an enzyme.

5. The template DNA is selected from a closed linear template DNA or a circular double-stranded template DNA.

10. The process according to claim 1 ,

6. When the template DNA is a closed linear template DNA, step (a) a plasmid vector containing at least two restriction sites flanking the sequence and at least one and contacting the fragment with two restriction enzymes, thereby forming open fragments containing the desired DNA sequence. A double-stranded DNA is prepared, and a single-stranded DNA adapter is attached to the DNA containing the target DNA sequence. Any of claims 1 to 5, which is carried out by binding to both ends of an open double-stranded DNA. The process of any one of claims 1 to 4.

7. When the template DNA is a closed linear template DNA, step (a) Plasmid vector containing at least two protelomerase target sequences flanked by sequences This is carried out by contacting the nucleotide sequence with a protelomerase, more particularly TelN. The process according to any one of claims 1 to 5.

8. 8. The process of any one of claims 1 to 7, wherein the amplified DNA obtained from step (b) is a concatemeric DNA comprising repeats of the DNA sequence of interest, each one of the repeated DNA sequences of interest being flanked by restriction sites and / or protelomerase target sequences.

9. The concatemer DNA contains repeats of the DNA sequence of interest flanked by at least restriction sites. In the case where the step (c) comprises the steps of: (c1) reacting the concatemer DNA with at least one restriction enzyme; contacting each other, thereby forming a plurality of open strands each containing the DNA sequence of interest. (c2) attaching a single-stranded DNA adapter to the open double-stranded DNA fragment.

9. The process of claim 8, carried out by binding at both ends.

10. The concatemer DNA is flanked by at least protelomerase target sequences. When the DNA sequence contains repeats, step (c) is carried out by combining the concatemer DNA with protelomerase, 9. The process of claim 8, more particularly carried out by contacting the vinegar.

11. The template DNA is a closed linear template DNA that does not contain a protelomerase target site. The process according to any one of claims 1 to 10.

12. Step (a) comprises flanking at least two restriction sites flanking the DNA sequence of interest. A plasmid vector containing two protelomerase target sequences and a protelomerase , for example, TelN, and step (c) is carried out by: (c1) contacting said chain with contacting the target DNA sequence with at least one restriction enzyme; (c2) creating a plurality of open double-stranded DNA fragments each containing a single-stranded DNA sequence; A adapter is attached to both ends of the open double-stranded DNA fragment. The process of claim 11.

13. Step (a) comprises providing at least two restriction sites and a non- A plasmid vector containing a protelomerase target site is ligated to at least one restriction enzyme. contacting the DNA with the target DNA sequence, thereby creating an open double-stranded DNA containing the target DNA sequence. , provided that the single-stranded DNA adapter does not contain a protelomerase target site, The single-stranded DNA adapter is attached to the open double-stranded DNA containing the DNA sequence of interest. and step (c) is carried out by binding to both ends of the concatemer DNA. Each of the fragments is contacted with one restriction enzyme, thereby forming fragments each containing the target DNA sequence. (c2) creating a plurality of open double-stranded DNA fragments each having a single-stranded DNA adapter; The method according to claim 11, wherein the method is carried out by binding to both ends of a double-stranded DNA fragment. process.

14. The template DNA is a closed linear template DNA, and the closed linear template DNA is 1 to 3, provided that the nucleic acid sequence does not contain a primase / polymerase priming site.

14. The process according to any one of claims 13.

15. Step (a) - a plasmid vector containing at least two restriction sites flanking the DNA sequence of interest; and at least one restriction enzyme, thereby obtaining a fragment containing the desired DNA sequence. The single-stranded DNA adapter is then used to generate an open double-stranded DNA having a primase / polymerase chain reaction. the single-stranded DNA adapter, provided that it does not contain an enzyme priming site; by binding to both ends of the open double-stranded DNA containing the target DNA sequence. or alternatively, - containing at least two protelomerase target sequences flanking said DNA sequence of interest; contacting a plasmid vector containing the vector with protelomerase, more particularly TelN; Conducted by Thereby, a closed linear template DNA containing the target DNA sequence is obtained. The process of claim 14, wherein A is obtained.

16. The sequence of interest comprises inverted terminal repeats (ITRs) flanking the expression cassette.

16. The process of any one of paragraphs 1 to 15.

17. The closed linear DNA is separated from both the open double-stranded DNA containing the DNA sequence of interest. When a single-stranded DNA adaptor is included at the end, the adaptor may include a modified oligonucleotide. The process of any one of claims 1 to 16.

18. The process according to any one of claims 1 to 17, which is a cell-free in vitro process. Process.

19. A closed linear DNA obtained according to the process of any one of claims 1 to 18.

20. 20. The method of claim 19 for use in therapy, more particularly in DNA-based therapy. Closed linear DNA.

21. The DNA-based therapy may be a gene therapy, a gene editing, a cell therapy (CAR-T), a vaccine, or the like.

21. The method for use according to claim 20, wherein the method is selected from the expression of a gene and a monoclonal antibody. Closed linear DNA.

22. 20. A therapeutically effective amount of the closed linear DNA of claim 19 and a pharmaceutically acceptable carrier or excipient. A pharmaceutical composition comprising an excipient.

23. A concatemer DNA containing repeats of a DNA sequence of interest, Each of the sequences is flanked by at least a restriction site and a protelomerase target sequence. Concatenated DNA.

24. Each of the repeated DNA sequences of interest is additionally flanked by an ITR. The concatemer DNA of claim 23 .

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