Process for creating closed linear DNA

Abstract

The present invention provides a process for producing closed linear DNA, comprising the steps of: a) providing a template DNA containing a DNA sequence of interest; b) amplifying DNA from the template DNA of step (a), wherein amplification is stimulated with a primase / polymerase enzyme; c) producing closed linear DNA using the amplified DNA produced in step (b); and d) purifying the closed linear DNA produced in step (c). The present invention also provides closed linear DNA obtained according to the process of the present invention, pharmaceutical compositions comprising a therapeutically effective amount of the closed linear DNA of the present invention, and concatemeric DNA containing repeats of the DNA sequence of interest.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of European Patent Application No. 20382064, filed January 31, 2020.

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

[0003] Gene therapy holds great promise for the treatment of several diseases. It is based on the targeted transfer of genetic material into the nucleus of human cells. Gene delivery systems can be viral or non-viral in design. Compared to viral DNA vectors, non-viral transgene delivery systems offer a safer approach to gene transfer and vaccine design, are less likely to induce inflammatory and immune responses in the host, have a greater transgene capacity, and are easier to store.

[0004] However, the efficacy of non-viral vectors is severely limited, hindering their introduction into the clinic. For example, the use of conventional plasmid DNA vectors for gene therapy can induce harmful immune responses due to the bacterial sequences they contain, 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 dumbbell-shaped molecules containing only the DNA sequence of interest without the largely immunogenic bacterial backbone, thus ensuring greater bioavailability, higher transfection efficiency, and longer duration of gene expression. The linear nature of clDNA minimizes the possibility of insertional mutagenesis due to random genomic integration. clDNA vectors have been successfully used for a variety of therapeutic indications with promising results in vitro and in vivo.

[0006] The production of nucleotide vectors, such as cDNA, for therapeutic use presents several challenges. First, vectors must be free of any bacterial components or toxins that could cause adverse reactions in patients. Furthermore, the transfection efficiency of vectors into cells in vivo is rather low. Therefore, very high production yields are required to reach the amounts required for in vivo administration. Finally, injecting exogenous nucleotide sequences into patients carries significant risks. For example, the expression of mutant proteins or nonspecific products generated during the production process can cause significant harm to patients.

[0007] In view of the above, vectors (especially clDNA) for use in the clinic must be produced by a process that simultaneously ensures the absence of bacterial residues and antibiotic resistance sequences, is amenable to large-scale production at reduced cost, and ensures a very high degree of sequence uniformity (i.e., by having a very low percentage of altered sequences).

[0008] Therefore, despite previous efforts, there remains a need for an efficient process for the large-scale, safe production of clDNA of suitable quality for therapeutic purposes. Summary of the Invention [Problem to be solved by the invention]

[0009] The present inventors have developed a process for large-scale production of clDNA with very high sequence quality that does not involve the use of microorganisms. The process provided herein is based on the use of primase / polymerase to prime the amplification of template DNA, followed by processing of the amplified product to generate clDNA suitable for therapeutic use.

[0010] Surprisingly, as shown in the Examples below, the inventors found that performing a primase / polymerase enzyme-primed amplification step prior to clDNA formation not only resulted in good clDNA production yields, but also significantly improved the sequence fidelity of the generated clDNA compared to using other amplification processes, such as random priming (see Figure 2). Thus, the results provided herein demonstrate the importance of priming with respect to the final properties of the resulting clDNA.

[0011] The high sequence quality provided by primase / polymerase also impacts the efficiency of post-amplification steps, which require the use of enzymes that recognize specific sequences on the amplified DNA. Thus, the high sequence fidelity provided by primase / polymerase priming ensures that all target sequences on the amplified DNA are highly conserved and efficiently targeted by processing enzymes such as restriction enzymes or protelomerases.

[0012] Notably, the absence of any steps requiring the use of microorganisms makes the process of the present invention much easier and safer to scale up.

[0013] In comparison with what has been disclosed in the prior art, the present inventors have further discovered that the use of primase / polymerase to prime amplification of template clDNA does not require the presence of a primase recognition site within the single-stranded loop (i.e., adapter) of the template DNA. This greatly expands the repertoire of template DNA that can be used in the process of the present invention. For example, primase / polymerase can be used to prime template clDNA created by ligating adapters of any sequence (i.e., not containing a primase / polymerase priming site) or template clDNA generated by the action of protelomerase containing a minimal single-stranded loop (see FIG. 4).

[0014] Finally, the clDNA obtained by the process of the present invention is suitable for transfection into mammalian cells, which also allows for efficient expression of the DNA sequence of interest contained therein (see Figures 5 and 6).

[0015] Taken together, in the examples provided below, we demonstrate the utility of the process of the present invention for large-scale production of high-quality clDNA suitable for gene therapy. [Means for solving the problem]

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

[0017] As described above, the process of the present invention allows for the production of clDNA with very high sequence fidelity (i.e., very low amounts of amplification artifacts or mutant sequences), making it particularly suitable for therapeutic uses of clDNA where high sequence quality is essential.

[0018] Thus, in a second aspect, the present invention provides a closed linear DNA obtained according to the process described in the fifth aspect of the invention.

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

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

[0021] In a fifth aspect, the present invention provides a concatemer 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] 1 shows Illumina sequencing comparing TthPrimPol stimulated amplification with RP stimulated amplification, in relation to Example 1. [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 to be understood in their ordinary meaning as known in the art unless otherwise specified. Other, more specific definitions for certain terms used in this application are provided below. These definitions are also intended to be applied uniformly throughout the specification and claims unless otherwise expressly stated to provide a broader definition.

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

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

[0026] Amplification of template DNA using primase / polymerase as the priming enzyme 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 single-stranded, covalently closed DNA molecule that forms a "dumbbell" or "dogbone"-shaped structure under conditions that allow nucleotide hybridization. Thus, even though clDNA is formed by a closed single-stranded DNA molecule, hybridization of two complementary sequences within the same molecule forms a "dumbbell" structure, producing a structure consisting of a double-stranded intermediate segment flanked by two single-stranded loops. Those skilled in the art will understand how to produce clDNA from open or closed double-stranded DNA (e.g., the amplified DNA produced in step (b)) using conventional molecular biology techniques. For example, those skilled in the art will understand that clDNA can be produced by joining single-stranded hairpin adapters to both ends of open double-stranded DNA, e.g., by the action of a ligase. Another method known to those skilled in the art for generating closed linear DNA is by the action of protelomerase on double-stranded DNA containing at least two protelomerase target sequences.

[0028] A "sequence of interest" is understood to be a double-stranded DNA fragment containing the minimally necessary sequence encoding a gene of interest together with other sequences required for correct gene expression, such as an expression cassette. The sequence of interest may additionally contain other sequences flanking the expression cassette, such as inverted terminal repeats (ITRs).

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

[0030] The term "primase / polymerase enzyme" refers to a DNA-dependent primase / polymerase enzyme, such as enzymes from the archaeological-eukaryotic primase (AEP) superfamily. These enzymes exhibit the ability to prime a starting DNA strand containing dNTPs. Enzymes from this superfamily that can be used in the present invention include, for example, Thermus thermophilus primase / polymerase (TthPrimPol) or human primase / polymerase (hsPrimPol, CCDC111, FLJ33167, EukPrim2, or hPrimPol1). "Thermus thermophilus primase / polymerase" or "TthPrimPol" refers to the primase / polymerase of the bacterium Thermus thermophilus, having the sequence set forth in SEQ ID NO: 1. Nucleotide and protein sequences are available in the NCBI Entrez database, such as NC_005835 and WP_01 1173100.1.

[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 provided above or below, the process comprises the steps of: a) providing a template DNA comprising a DNA sequence of interest; b) amplifying DNA from the template DNA of step (a), wherein the amplification comprises (b1) priming the template DNA with a primase / polymerase enzyme and (b2) extending the resulting sequence with 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 provided above or below, the primase / polymerase enzyme is selected from TthPrimPol or hsPrimPol. In particular embodiments, the primase polymerase enzyme is TthPrimPol. In more particular embodiments, the primase polymerase enzyme is TthPrimPol of SEQ ID NO: 1, or a variant thereof, having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1. Those skilled in the art will appreciate that any variant of TthPrimPol that maintains its primase activity is suitable for use in the process of the invention.

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

[0035] Several mathematical algorithms for rapidly obtaining optimal alignments and calculating identity between two or more sequences are known and are incorporated into several available software programs. Examples of such programs include, inter alia, the MATCH-BOX, MULTAIN, GCG, FASTA, and ROBUST programs for amino acid sequence analysis. Preferred software analysis programs include the ALIGN, CLUSTAL W, and BLAST programs (e.g., BLAST2.1, BL2SEQ, and later versions thereof).

[0036] For amino acid sequence analysis, a weight matrix such as a BLOSUM matrix (e.g., BLOSUM45, BLOSUM50, BLOSUM62, and BLOSUM80 matrices), a Gonnet matrix, or a PAM matrix (e.g., PAM30, PAM70, PAM120, PAM160, PAM250, and PAM350 matrices) is used to determine identity.

[0037] The BLAST program provides analysis of at least two amino acid sequences, either against multiple sequences in a database (e.g., GenSeq) or by aligning a selected sequence between two selected sequences using BL2SEQ. The BLAST program is preferably modified with a low-complexity filtering program, such as the DUST or SEG programs, which are preferably integrated into the operation of the BLAST program. When using a gap existence cost (or gap score), the gap existence cost is preferably set to about -5 to -15. Similar gap parameters may be used appropriately with other programs. BLAST programs and their underlying principles are further described, for example, in Altschul et al., "Basic local alignment search tool," 1990, J. Mol. Biol., v. 215, pp. 403-410. A specific percentage of identity encompasses sequence variations due to conservative mutations of one or more amino acids that still allow the TthPrimPol enzyme to be effective, thereby priming a suitable sequence. Protein variations may also result from the insertion or deletion of one or more amino acids.

[0038] In particular embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the process is a cell-free in vitro process for generating closed linear DNA.

[0039] In a particular embodiment of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, step (b) is rolling circle amplification.

[0040] The term "rolling circle amplification" or "RCA" refers to a nucleic acid amplification method involving the amplification of covalently closed DNA molecules, such as clDNA or double-stranded circular DNA. In this case, a polymerase performs primer extension around the closed DNA molecule. The polymerase displaces hybridized copies and continues polynucleotide extension around the template, creating concatemeric DNA containing tandem units of amplified DNA. These linear, single-stranded products serve as the basis for multiple hybridization, primer extension, and strand displacement events, resulting in the formation of concatemeric double-stranded DNA products. Thus, multiple copies of each amplified single unit of DNA are present in the concatemeric double-stranded DNA product. Using general knowledge and / or manufacturer's instructions, those skilled in the art will understand how to adjust the conditions of the amplification process depending on the enzyme and characteristics of the template being amplified. Depending on how the template DNA is generated, the concatemeric DNA will contain different sequences flanking each amplified DNA sequence of interest. For example, in concatemeric DNA, the repeated DNA sequence of interest may be flanked by restriction sites, protelomerase target sequences, recombinase recognition sites, or any combination thereof.

[0041] In a specific embodiment of the process of the first aspect of the present invention, optionally in combination with any of the embodiments provided above or below, the amplification in step (b) is carried out using a strand-displacing DNA polymerase. The term "strand-displacing DNA polymerase" refers to a DNA polymerase that performs a 3'-end extension reaction while removing the double-stranded portion of the template DNA. The strand-displacing DNA polymerase that can be used in the present invention is not particularly limited as long as it has strand-displacing activity, such as phi29 DNA polymerase and Bst DNA polymerase. Depending on the polymerase type selected in this manner, those skilled in the art will understand that the reaction conditions for the 3'-end extension reaction can be appropriately set. For example, when phi29 DNA polymerase is used, the reaction can be carried out at an optimal temperature for the reaction, which is 25°C to 35°C.

[0042] Thus, in certain embodiments, the strand-displacing DNA polymerase is selected from the group consisting of phi29 DNA polymerase, Bst DNA polymerase, Bca (exo) DNA polymerase, Klenow fragment of E. coli DNA polymerase I, Vent (exo) DNA polymerase, DeepVent (exo) DNA polymerase, and KOD DNA polymerase. In more particular embodiments, the strand-displacing DNA polymerase is phi29 DNA polymerase. In even more particular embodiments, the strand-displacing DNA polymerase is a chimeric protein comprising phi29 DNA polymerase. Those skilled in the art will understand how to obtain chimeric DNA polymerases with improved properties, for example, as disclosed in WO2011000997.

[0043] In particular embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the template DNA is selected from a closed linear 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 molecule.

[0045] The process for generating closed linear DNA of the present invention may also be carried out by priming the amplification of step (b) with random primers.

[0046] In particular embodiments of the process of the first aspect of the present invention, optionally in combination with any of the embodiments provided above or below, when the template DNA is a closed linear template DNA, step (a) comprises: - contacting a plasmid vector containing at least two restriction sites flanking the DNA sequence of interest with at least one restriction enzyme, thereby generating an open double-stranded DNA containing the DNA sequence of interest, and ligating single-stranded DNA adapters to both ends of the open double-stranded DNA containing the DNA sequence of interest; or alternatively, step (a) is carried out by: - contacting a plasmid vector containing at least two protelomerase target sequences flanking a DNA sequence of interest with a protelomerase, more particularly TelN, This is done by obtaining a template DNA, which is a closed linear DNA template containing the DNA sequence of interest.

[0047] The inventors surprisingly found that, in contrast to what is disclosed in the state of the art, primase / polymerase enzymes can prime clDNA that does not contain adapters with primase recognition sites. In particular, when template clDNA is generated by the action of protelomerase, the resulting clDNA exhibits a structure with a very small single-stranded loop at its middle end that does not contain the protelomerase target sequence. Unexpectedly, primase / polymerase can prime this type of clDNA, allowing the polymerase to initiate the amplification process, even when the template clDNA is not subjected to denaturing conditions (see Figure 4).

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

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

[0050] The use of restriction enzymes and ligases (for joining purposes) is routine in the field of molecular biology, and therefore those skilled in the art know how to adjust the conditions of the reaction depending on the enzyme being used, and which restriction enzyme should be used depending on the restriction site being targeted.

[0051] Those skilled in the art also understand that some restriction enzymes generate DNA overhangs (sticky ends), while others do not (blunt ends). Both types of restriction enzymes can be used in the methods of the present invention. Those skilled in the art will understand that adapters with sticky ends can be ligated to open double-stranded DNA with sticky ends (sticky end ligation). Open double-stranded DNA with blunt ends can also be dA-tailed by a process that adds terminal 3' deoxyadenosine nucleotides, for example, using Taq polymerase, and then ligated to adapters with overhanging Ts.

[0052] In particular embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the restriction enzyme generates blunt or sticky ends. In more particular embodiments, contacting at least one restriction enzyme with a plasmid vector containing at least two restriction sites flanking the DNA sequence of interest generates an open double-stranded DNA with sticky ends or an open double-stranded DNA with blunt ends.

[0053] In particular embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the single-stranded DNA adaptor has a hairpin structure. In more particular embodiments, the single-stranded DNA adaptor is of the sequence SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. The adaptors attached to both ends of the open double-stranded DNA to form the de clDNA can be the same or different adaptors.

[0054] In certain embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the single-stranded DNA adaptor contains one or more, e.g., at least two, modified nucleotides.

[0055] A "modified nucleotide" is any nucleotide (e.g., adenosine, guanosine, cytidine, and thymidine) that has been chemically modified by modification of the base, sugar, or phosphate group, or that incorporates a non-natural moiety into its structure. Thus, a modified nucleotide may or may not be naturally occurring, depending on the modification.

[0056] As used herein, modified nucleotides are preferably 5-methyl-deoxycytidine, 2-amino-deoxyadenosine, 1-methyl-adenosine, 1-methyl-guanosine, 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´-araadenosine, 2´-aracytidine, 2´-arauridine, 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´-fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine, 2-methyl-thio-N6-isopenenyl-adenosine, 2´-O-methyl-2-aminoadenosine, 2´-O-methyl-2´-deoxyadenosine, 2´-O-methyl 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-methylpseudouridine, 2-thiocytidine, 2-thio-cytidine, 3-methyl-cytidine, 4-acetyl-cytidine, 4-thiouridine, 5-(carboxyhydroxymethyl)-uridine, 5,6-Dihydrouridine, 5-aminoallylcytidine, 5-aminoallyl-deoxyuridine, 5-bromouridine, 5-carboxymethylaminomethyl-2-thio-uracil, 5-carboxymethylamonomethyl-uracil, 5-chloro-ara-cytosine, 5-fluoro-uridine, 5-iodouridine, 5-methoxycarbonylmethyl-uridine, 5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-azacylate Thiidine, 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-azaadenosine, 8-bromo-adenosine, 8-bromo-guanosine, 8-mercapto-guanosine, 8-oxoguanosine, benzimidazole- Variants of guanosine, uridine, adenosine, thymidine, and cytidine, including, but not limited to, naturally occurring or non-naturally occurring guanosine, uridine, adenosine, thymidine, or cytidine that have been chemically modified, e.g., acetylated, methylated, hydroxylated, etc., including ribosides, β-D-mannosyl-queosine, dihydro-uridine, inosine, N1-methyladenosine, N6-([6-aminohexyl]carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine, N6-methyl-adenosine, N7-methyl-xanthosine, N-uracil-5-oxyacetic acid methyl ester, puromycin, queosine, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, wybutoxocine, xanthosine, and xylo-adenosine. The preparation of such variants is known to those skilled in the art, for example, from U.S. Pat. No. 4,373,071.

[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-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-uridine, 5-hydroxy ... Taurinomethyl-2-thio-uridine, 1-taurinomethyl-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, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-meth oxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4 Also included are, but are not limited to, 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-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-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-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydrogen purine), ... Also included are, but are not limited to, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

[0059] Modified nucleotides also include, but are not limited to, inosine, 1-methyl-inosine, wyosine, wybutosine, 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,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0060] Modified nucleotides also include 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, 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, 5-methyl-uridine, pyrrolo-cytidine, inosine, α-thioguanidine, Also included are, but are not limited to, anosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine, 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, and 7-deaza-adenosine.

[0061] The modified nucleotide may be chemically modified at the 2' position. Preferably, the modified nucleotide comprises a substituent at the 2' carbon atom. This substituent is selected from the group consisting of halogen, alkoxy, hydrogen, aryloxy, amino, and aminoalkoxy, preferably 2'-hydrogen (2'-deoxy), 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro.

[0062] Chemical modifications involving the 2' position of a nucleotide described herein are locked nucleic acid (LNA) nucleotides, ethylene bridged nucleic acid (ENA) nucleotides, and (S)-constrained ethyl cEt nucleotides. These backbone modifications lock the sugar of the modified nucleotide 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. Furthermore, modified nucleotides can include complete replacement of unmodified phosphate moieties with modified phosphates as described herein. Examples of modified phosphate groups include, but are not limited to, the group consisting of phosphorothioates (also known as thiophosphates), phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkylphosphonates, arylphosphonates, and phosphotriesters. Phosphate-containing linkers can also be modified by replacing the connecting oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene phosphonates).

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

[0065] In certain embodiments of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the at least two modified nucleotides are independently selected from the group consisting of 2-amino-deoxyadenosine, 5-methyl-deoxycytidine, thiophosphate nucleotides, LNA nucleotides, inosine, 8-oxo-deoxyadenosine and 5-fluoro-deoxyuracil and L-DNA nucleotides.

[0066] In certain embodiments of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the at least two modified nucleotides are not L-DNA nucleotides, 5-bromouridine or 5-iodouridine.

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

[0068] 5-Methyl-deoxycytidine (5-methyl-dCTP) is a derivative derived from deoxycytidine and has the IUPAC name ([[(2R,3S,5R)-5-(4-amino-5-methyl-2-oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]phosphonohydrogen phosphate and CAS number 22003-12-9.

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

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

[0071] L-DNA nucleotides refer to nucleotides that contain the L enantiomer of ribose or deoxyribose.

[0072] In more particular embodiments of the first aspect of the present invention, optionally in combination with any of the embodiments provided above or below, the c1DNA comprises at least three, at least four, or at least five modified nucleotides independently selected from the group consisting of thiophosphate, locked nucleic acid, 2,6-diaminopurine, 5-methyl-deoxycytidine, inosine, 8-oxodeoxyadenosine, and 5-fluoro-deoxyuracil, and L-DNA nucleotides.

[0073] In a more particular embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the clDNA comprises two LNA nucleotides.

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

[0075] In particular embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the single-stranded DNA adaptor does not contain a primase recognition site, hi more particular embodiments, the single-stranded DNA adaptor does not contain the sequence XTC.

[0076] In a more particular embodiment of the process of the first aspect of the present invention, optionally in combination with any of the embodiments provided above or below, when the template DNA is a closed linear template DNA, step (a) comprises: - contacting a plasmid vector containing at least two restriction sites flanking the DNA sequence of interest with at least one restriction enzyme, thereby generating an open double-stranded DNA containing the DNA sequence of interest, and ligating single-stranded DNA adaptors to both ends of the open double-stranded DNA containing the DNA sequence of interest, provided that the single-stranded DNA adaptors do not contain primase / polymerase priming sites; or alternatively, step (a) is carried out by: - contacting a plasmid vector containing at least two protelomerase target sequences flanking a DNA sequence of interest with a protelomerase, more particularly TelN, This is achieved by obtaining a template DNA that is a closed linear DNA template containing the DNA sequence of interest. In a more particular embodiment, the single-stranded DNA adapter does not contain the sequence XTC.

[0077] In more particular embodiments, optionally in combination with any of the embodiments provided above or below, the single-stranded DNA adaptor contains a protelomerase target sequence. In another particular embodiment, optionally in combination with any of the embodiments provided above or below, the single-stranded DNA adaptor does not contain a protelomerase target sequence. In another particular embodiment, the single-stranded DNA adaptor contains a portion of a protelomerase target sequence, where the portion of the protelomerase target sequence is not recognized by protelomerase.

[0078] As used herein, a "protelomerase" is any polypeptide capable of cleaving and religating a template containing a protelomerase target site to create a covalently closed linear DNA molecule. Thus, a protelomerase has DNA cleavage and ligation functions. Enzymes with protelomerase-type activity have also been described as telomere resolving bases (e.g., Borrelia burgdorferi). A typical substrate for protelomerase is circular double-stranded DNA. If this DNA contains a protelomerase target site, the enzyme can cleave the DNA at this site and ligate the ends to generate a linear, double-stranded, covalently closed DNA molecule. The ability of a given polypeptide to catalyze the creation of closed linear DNA from a template containing a protelomerase target site can be determined using any suitable assay described in the art.

[0079] Examples of suitable protelomerases for use in the process of the present invention include phiHAP-1 from Halomonas aquamarina, PY54 from Yersinia enterolytica, phiKO2 from Klebsiella oxytoca, and VP882 from Vibrio species, N15 from Escherichia coli, or any variants thereof.

[0080] In particular embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the protelomerase is bacteriophage N15 TelN of SEQ ID NO:2, or a variant thereof comprising a sequence having at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to SEQ ID NO:2.

[0081] A "protelomerase target sequence" is any DNA sequence whose presence in a template DNA allows for conversion to closed linear DNA by the enzymatic activity of protelomerase. In other words, the protelomerase target sequence is required for the cleavage and rejoining of double-stranded DNA by protelomerase to form a covalently closed linear DNA. Typically, a protelomerase target sequence is any double-stranded DNA sequence with a two-fold greater rotational symmetry, including any perfect palindromic sequence described herein as a perfect inverted repeat.

[0082] In certain embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, at least two protelomerase target sequences comprise perfect inverted repeat DNA sequences.

[0083] In particular embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the protelomerase target sequence comprises the sequence of SEQ ID NO:3, or a variant thereof, including a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:3.

[0084] The length of the perfect inverted repeat varies depending on the particular organism. In Borrelia burgdorferi, the perfect inverted repeat is 14 base pairs in length. In various mesophilic bacteriophages, the perfect inverted repeat is 22 base pairs or more in length. Also, in some cases (e.g., E. coli N15), the central perfect inverted palindrome is flanked by perfect inverted sequences, i.e., it forms part of a larger imperfect inverted palindrome.

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

[0086] The perfect inverted repeat may be flanked by additional inverted repeat sequences. The flanking inverted repeats may be perfect or imperfect repeats; i.e., they may be perfectly symmetrical or partially symmetrical. The flanking inverted repeats may be contiguous with the central palindrome or may be discontinuous. The protelomerase target sequence may comprise an imperfect inverted repeat, including a perfect inverted repeat of at least 14 base pairs in length.

[0087] A protelomerase target sequence comprising the sequence of SEQ ID NO: 3 or variants thereof is preferred for use in combination with the E. coli N15 TelN protelomerase of SEQ ID NO: 2 and variants thereof.

[0088] Variants of any of the above palindromic sequences or protelomerase target sequences include homologs or mutants thereof. Variants include truncations, substitutions, or deletions relative to the native sequence. A variant sequence is any sequence whose presence in the template DNA allows for conversion to closed linear DNA by the enzymatic activity of protelomerase. This can be easily determined by using an appropriate assay for forming closed linear DNA. Any suitable assay in the art may be used. Preferably, the variant allows protelomerase binding and activity comparable to that observed with the native sequence. Examples of preferred variants of the palindromic sequences described herein include truncated palindromic sequences that preserve the complete repeat structure and remain capable of forming closed linear DNA. However, variant protelomerase target sequences may be modified so that they no longer preserve the complete palindrome, provided they are capable of acting as a substrate for protelomerase activity.

[0089] It will be appreciated that one of skill in the art can readily identify suitable protelomerase target sequences for use in the present invention based on the structural principles summarized above. Candidate protelomerase target sequences can be screened for their ability to promote the formation of closed linear DNA using the assays described above.

[0090] In a particular embodiment of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, when the template DNA is a circular double-stranded template DNA containing the DNA sequence of interest, step (a) is carried out by contacting a plasmid vector comprising at least two recombinase recognition sites flanking the DNA sequence of interest with a site-specific recombinase, more particularly Cre recombinase.

[0091] The action of the site-specific recombinase on the plasmid vector causes the two recombinase recognition sites to recombine, thereby generating a small circular double-stranded DNA containing the DNA sequence of interest that was located between the recombinase recognition sites in the plasmid vector.

[0092] "Site-specific recombinase," as used herein, refers to a family of enzymes that mediate site-specific recombination between specific DNA sequences recognized by the enzyme, known as recombinase recognition sites. Examples of site-specific recombinases include, but are not limited to, Cre recombinase, Flp recombinase, λ integrase, γ-δ resolvase, Tn3 resolvase, Sin resolvase, Gin invertase, Hin invertase, Tn5044 resolvase, Tn3 transposase, sleeping beauty transposase, IS607 transposase, Bxb l integrase, wBeta integrase, BL3 integrase, phiR4 integrase, All l 8 integrase, TGI integrase, MRU integrase, phi370 integrase, SPBc integrase, SV1 integrase, TP901-1 integrase, phiRV integrase, FC1 integrase, K38 integrase, phiBTl integrase, and phiC31 integrase.

[0093] "Recombinase recognition site" refers to a nucleotide sequence recognized by a site-specific recombinase and can serve as a substrate for a recombination event. Non-limiting examples of recombinase recognition sites include FRT, FRT11, FRT71, attp, att, rox, and loxP, and lox sites such as lox511, lox2272, lox66, lox71, loxM2, and lox5171.

[0094] Those skilled in the art will understand, using their general knowledge, that site-specific recombinases recognize specific recombinase recognition sites, and therefore, depending on the recognition sequences contained in the plasmid vector, different recombinases must be used to generate circular double-stranded template DNA from the plasmid vector.

[0095] In particular embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the site-specific recombinase is Cre recombinase. In more particular embodiments, the recombinase recognition site is loxP. In even more particular embodiments, the site-specific recombinase is Cre recombinase and the recombinase recognition site is loxP.

[0096] Depending on the sequences flanking the DNA sequence of interest in the plasmid vector and the process used to generate the template DNA in step (a), the concatemer product generated in step (b) will contain the DNA sequence of interest flanked by different sequences. For example, if the DNA of interest is only flanked by a protelomerase target sequence in the plasmid vector, the DNA sequence of interest in the concatemer DNA will be flanked by the protelomerase target sequence. Furthermore, the DNA sequence of interest in the plasmid vector may be flanked by a combination of different sequences to allow for the generation of template DNA by one type of reaction (e.g., by TelN) and thereby the generation of clDNA from the amplified product by another reaction (e.g., restriction enzyme digestion and adapter ligation). In these particular cases, the DNA sequence of interest must be flanked by restriction sites that in turn flank the protelomerase target site.

[0097] Thus, in particular embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amplified DNA resulting from step (b) is concatemeric DNA comprising repeats of DNA sequences of interest, each one of which is flanked by restriction sites, protelomerase target sequences, and / or recombinase recognition sites.

[0098] Those skilled in the art will understand that the excision of tandem units of a DNA sequence of interest in the form of a closed linear DNA can be carried out by different conventional molecular biology techniques that are able to cleave the tandem units and close the open ends of the fragments to form a covalently closed molecule. These two steps can be carried out sequentially, for example by digestion with a restriction enzyme and ligation of an adapter, or simultaneously by the action of a protelomerase.

[0099] In a particular embodiment of the process of the first aspect of the present invention, optionally in combination with any of the embodiments provided above or below, when the concatemer DNA contains repeats of the DNA sequence of interest flanked by at least restriction sites, step (c) is carried out by (c1) contacting the concatemer DNA with at least one restriction enzyme, thereby generating a plurality of open, double-stranded DNA fragments, each containing the DNA sequence of interest, and (c2) ligating single-stranded DNA adapters to both ends of the open, double-stranded DNA fragments. All embodiments provided above regarding restriction enzymes, restriction sites, and single-stranded DNA adapters are meant to apply to this embodiment as well. Furthermore, those skilled in the art will appreciate that when a restriction enzyme is used to generate template clDNA, the same restriction enzyme can subsequently be used to generate clDNA from the amplified DNA generated in step (b). The single-stranded DNA adapters used in step (a) to generate template clDNA can be similar to or different from those used in step (c).

[0100] In a particular embodiment of the process of the first aspect of the present invention, optionally in combination with any of the embodiments provided above or below, when the concatemer DNA comprises repeats of the DNA sequence of interest flanked by at least protelomerase target sequences, step (c) is carried out by contacting the concatemer DNA with a protelomerase, more particularly TelN. All embodiments provided above regarding protelomerase and protelomerase target sites are meant to apply to this embodiment as well. Furthermore, those skilled in the art will appreciate that when a protelomerase is used to generate template clDNA in step (a), the same protelomerase can subsequently be used in step (c) to generate clDNA from the amplified DNA.

[0101] In a particular embodiment of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the process is for producing a closed linear expression cassette DNA.

[0102] In particular embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, step (a) is carried out by contacting a plasmid vector containing at least two restriction sites flanking the DNA sequence of interest with at least one restriction enzyme, thereby generating an open double-stranded DNA containing the DNA sequence of interest, and ligating single-stranded DNA adaptors to both ends of the open double-stranded DNA containing the DNA sequence of interest, and step (c) is carried out by (c1) contacting the concatemer DNA with at least one restriction enzyme, thereby generating a plurality of open double-stranded DNA fragments each containing the DNA sequence of interest, and (c2) ligating single-stranded DNA adaptors according to the first aspect of the invention to both ends of the open double-stranded DNA fragments. In more particular embodiments, the restriction enzyme generates sticky ends or blunt ends. If the restriction enzyme generates blunt ends, the resulting fragment can be ligated to an adaptor containing blunt ends, or alternatively, it can be dA-tailed and ligated to an adaptor with an overhanging T, as described above.

[0103] In particular embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, step (a) is carried out by contacting a plasmid vector comprising at least two restriction sites flanked by at least two protelomerase recognition sites flanking the DNA sequence of interest with at least one restriction enzyme, thereby generating an open double-stranded DNA containing the DNA sequence of interest flanked by the protelomerase recognition sequences, and attaching single-stranded DNA adapters to both ends of the open double-stranded DNA, and step (c) is carried out by contacting the concatemer DNA with a protelomerase, more particularly TelN.

[0104] In particular embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the template DNA is a circular double-stranded template DNA containing a DNA sequence of interest flanked by restriction sites, step (a) is carried out by contacting a plasmid vector comprising at least two recombinase recognition sites flanked by at least two restriction sites flanking the DNA sequence of interest with a site-specific recombinase, more particularly Cre recombinase, and step (c) is carried out by (c1) contacting the concatemer DNA with at least one restriction enzyme, thereby generating a plurality of open double-stranded DNA fragments each containing the DNA sequence of interest, and (c2) ligating single-stranded DNA adaptors to both ends of the double-stranded DNA fragments as described in the first aspect.

[0105] In a particular embodiment of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, when the template DNA is a circular double-stranded template DNA containing a DNA sequence of interest flanked by recognition sites, step (a) is carried out by contacting a plasmid vector comprising at least two recombinase recognition sites flanked by at least two restriction sites flanking the DNA sequence of interest with a site-specific recombinase, more particularly Cre recombinase, and step (c) is carried out by contacting the concatemer DNA with a protelomerase, more particularly TelN.

[0106] In particular embodiments of the process of the first aspect of the present invention, optionally in combination with any of the embodiments provided above or below, step (a) is carried out by contacting a plasmid vector comprising at least two protelomerase target sequences flanked by at least two restriction sites flanking the DNA sequence of interest with a protelomerase, more particularly TelN, and step (c) is carried out by (c1) contacting the concatemer DNA with at least one restriction enzyme, thereby generating a plurality of open, double-stranded DNA fragments each containing the DNA sequence of interest, and (c2) ligating single-stranded DNA adaptors to both ends of the open, double-stranded DNA fragments.

[0107] In certain embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, step (a) is carried out by contacting a plasmid vector containing two protelomerase target sequences flanked by at least two restriction sites flanking the DNA sequence of interest with a protelomerase, such as TelN; and step (c) is carried out by (c1) contacting the concatemer DNA with at least one restriction enzyme, thereby generating a plurality of open, double-stranded DNA fragments, each containing the DNA sequence of interest, and (c2) ligating single-stranded DNA adaptors to both ends of the open, double-stranded DNA fragments.

[0108] In certain embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, step (a) is carried out by contacting a plasmid vector comprising at least two protelomerase target sequences (e.g., for protelomerase A) flanked by at least two protelomerase recognition sites different from a first one (e.g., for protelomerase B) flanking the DNA sequence of interest with the corresponding protelomerase (e.g., for protelomerase A), and step (c) is carried out by contacting the concatemer DNA with the corresponding protelomerase (e.g., for protelomerase B). In certain embodiments, the protelomerase in step (a) or step (c) is TelN.

[0109] In certain embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, step (a) is carried out by contacting the protelomerase with a plasmid vector comprising two protelomerase target sites flanking the DNA sequence of interest, and step (c) is carried out by contacting the concatemer DNA with the protelomerase. In certain embodiments, the protelomerase in step (a) or 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 provided above or below, step (a) is carried out by contacting a plasmid vector comprising at least two restriction sites and a non-protelomerase target site flanking the DNA sequence of interest with at least one restriction enzyme, thereby generating an open double-stranded DNA containing the DNA sequence of interest, and attaching single-stranded DNA adaptors to both ends of the open double-stranded DNA containing the DNA sequence of interest, provided that the single-stranded DNA adaptors do not contain a protelomerase target site; and step (c) is carried out by (c1) contacting the concatemer DNA with at least one restriction enzyme, thereby generating a plurality of open double-stranded DNA fragments each containing the DNA sequence of interest, and (c2) attaching single-stranded DNA adaptors according to the first aspect of the invention to both ends of the open double-stranded DNA fragments.

[0111] In particular embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the DNA sequence of interest comprises an expression cassette. In more particular embodiments, the expression cassette consists of a eukaryotic promoter, and optionally an enhancer, and / or a eukaryotic transcription termination sequence operably linked to the sequence encoding the protein of interest.

[0112] The term "expression cassette" refers to a DNA sequence containing one or more promoter or enhancer elements and a gene or coding sequence that encodes an mRNA, miRNA, siRNA, or protein of interest. An expression cassette may further contain other elements that regulate expression of the coding sequence, such as a transcription termination site.

[0113] In certain embodiments of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the DNA sequence of interest comprises an expression cassette flanked by inverted terminal repeats (ITRs). The ITRs can be at any suitable distance from the expression cassette. For example, the ITRs can be directly attached to the expression cassette or can be at a distance of 1 to 50 nucleotides, 50 to 200 nucleotides, or 200 to 1000 nucleotides. Thus, in certain embodiments, optionally in combination with any of the embodiments provided above or below, the DNA of interest comprises an expression cassette flanked by inverted terminal repeats (ITRs) at a distance of 1 to 50 nucleotides.

[0114] As used herein, "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that contains at least one minimally required origin of replication and a region containing a palindromic hairpin structure. The Rep-binding sequence ("RBS") (also called the Rep-binding element (RBE)) and terminal resolution site (TRS) constitute the "minimally required origin of replication," and therefore contain at least one RBS and at least one TRS. TRs that are reverse-complementary sequences of each other within a given stretch of polynucleotide sequence are typically referred to as "inverted terminal repeats" or "ITRs," respectively. With respect to viruses, ITRs are involved in replication, viral packaging, integration, and proviral rescue.

[0115] Those skilled in the art will understand that in complex clDNA constructs, there may be three or more ITRs or asymmetric ITR pairs. The ITRs may be AAV or non-AAV ITRs, or may be derived from AAV or non-AAV ITRs. For example, the ITRs may be derived from the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, and human parvovirus B-19). Alternatively, the SV40 hairpin that functions as the origin of SV40 replication may be used as an ITR. This may be further modified by truncation, substitution, deletion, insertion, and / or addition. The Parvoviridae family of viruses consists of two subfamilies: the Parvoviridae family infects vertebrates, and the Densovirinae subfamily infects invertebrates. Dependoparvoviruses include the adeno-associated virus (AAV) family of viruses that are capable of replication in vertebrate hosts, including, but not limited to, humans, primates, bovine, canine, equine, and ovine species. For convenience herein, the ITR located 5' to (upstream of) the expression cassette of the clDNA vector is referred to as the "5' ITR" or "left ITR," and the ITR located 3' to (downstream of) the expression cassette of the clDNA vector is referred to as the "3' ITR" or "right ITR."

[0116] In a particular embodiment of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the DNA sequence of interest comprises an expression cassette flanked by at least one inverted terminal repeat of the sequence of SEQ ID NO:8 or SEQ ID NO:9.

[0117] In a particular embodiment of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the DNA sequence of interest comprises an expression cassette flanked by a 5' inverted terminal repeat of the sequence of SEQ ID NO: 8 and / or a 3' inverted terminal repeat of the sequence of SEQ ID NO: 9.

[0118] In a particular embodiment of the process of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the DNA sequence of interest comprises an expression cassette flanked by at least one DD-ITR. A "DD-ITR" is an ITR containing flanking D elements as disclosed in 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. 71(2), pp. 941-948.

[0119] With regard to step (d) of the method, purifying the generated clDNA, those skilled in the art will understand that any known method suitable for purifying nucleic acids, particularly clDNA, can be used.

[0120] As mentioned above, in a second aspect, the present invention provides a closed linear DNA obtained according to the process described in the first aspect.

[0121] For the purposes of the present invention, the terms "obtained", "obtained" and equivalent terms are used interchangeably, and in each case the term "obtained" encompasses the term "obtained". All embodiments provided under the first aspect of the invention are also embodiments of the closed linear DNA of the second aspect of the invention.

[0122] In a particular embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the closed linear DNA comprises one or more expression cassettes.

[0123] In particular embodiments of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the expression cassette comprises a eukaryotic promoter operably linked to a sequence encoding an mRNA, miRNA, siRNA or protein.

[0124] In particular embodiments of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the expression cassette further comprises a eukaryotic transcription termination sequence.

[0125] In a particular embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, 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, optionally in combination with any of the embodiments provided above or below, the DNA sequence of interest comprises an expression cassette flanked by inverted terminal repeats (ITRs).

[0127] As already mentioned, the present invention also provides in a third aspect a closed linear DNA according to the first aspect for use in therapy.

[0128] The clDNA of the present invention may be used for in vitro expression in host cells, particularly in DNA vaccines or gene therapy. DNA vaccines typically encode modified forms of infectious organism DNA. The DNA vaccines are administered to subjects, where they express selected proteins of the infectious organism and initiate an immune response against those proteins, which is typically protective. DNA vaccines can also encode tumor antigens in cancer immunotherapy approaches.

[0129] DNA vaccines are available for fungi, human papillomavirus (HPV), HIV, HSV2 / HSV1, influenza viruses (A, B, and C), poliovirus, RSV, rhinovirus, rotavirus, hepatitis A virus, Norwalk virus complex, enterovirus, astrovirus, measles virus, parainfluenza virus, mumps virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, adenovirus, rubella virus, human T-cell leukemia virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus, poxvirus, viruses including Marburg and Ebola; Mycobacterium tuberculosis, chlamydia, Neisseria gonorrhoeae, shigellosis, Salmonella, Vibrio cholerae, Treponema pallidum, Pseudomonas, Bordetella pertussis, Brucella The nucleic acid sequences may include nucleic acid sequences encoding antigens for treating or preventing a number of medical conditions, including, but not limited to, cancer, allergy, toxicity, and infection by pathogens such as, but not limited to, bacteria including T. typhimurium, Francisella tularensis, Helicobacter pylori, pathogenic Leptospira, Legionella pneumophila, Yersinia pestis, Streptococcus (types A and B), Pneumococcus, Neisseria meningitidis, Haemophilus influenzae (type b), Toxoplasma gondii, Campylobacter spp., Moraxella catarrhalis, Donovanosis, and Actinomycosis; fungal pathogens including candidiasis and aspergillosis; and parasitic pathogens including cestodes, trematodes, nematodes, amoebic dysentery, giardiasis, Cryptosporidium, schistosomiasis, Pneumocystis carinii, trichomoniasis, and trichinellosis.

[0130] DNA vaccines include those directed against viruses such as Adenoviridae (including, for example, human adenovirus), Herpesviridae (including, for example, HSV-1, HSV-2, EBV, CMV, and VZV), Papovaviridae (including, for example, HPV), Poxviridae (including, for example, smallpox and vaccinia), Parvoviridae (including, for example, parvovirus B19), Reoviridae (including, for example, rotavirus), Coronaviridae (including, for example, SARS), Flaviviridae (including, for example, yellow fever, West Nile virus, dengue virus, hepatitis C virus, and tick-borne encephalitis virus), Picornaviridae (including, for example, poliovirus, rhinovirus, and The nucleic acid sequences may include nucleic acid sequences encoding antigens from members of the following families: Hepatitis A virus, Togaviridae (including, for example, rubella virus), Filoviridae (e.g., Marburg virus and Ebola virus), Paramyxoviridae (e.g., parainfluenza virus, respiratory syncytial virus, mumps virus, and measles virus), Rabviridae (including, for example, rabies virus), Bunyaviridae (including, for example, hantavirus), Orthomyxoviridae (including, for example, influenza A, B, and C), Retroviridae (including, for example, HIV and HTLV), and Hepadnaviridae (including, for example, hepatitis B virus).

[0131] Antigens may be derived from pathogens causing veterinary diseases, particularly viral pathogens, including, for example, reoviruses (such as African horse sickness or bluetongue virus) and herpesviruses (including equine herpesviruses). Antigens may be derived from foot-and-mouth disease virus, tick-borne encephalitis virus, dengue virus, SARS, West Nile virus, and hantavirus. Antigens may also be derived from immunodeficiency viruses, such as SIV or feline immunodeficiency virus.

[0132] The clDNA produced by the process of the invention may also contain nucleic acid sequences encoding tumor antigens. Examples of tumor-associated antigens include, but are not limited to, cancer-testis antigens such as members of the MAGE family (e.g., MAGE 1, 2, 3), NY-ESO-1 and SSX-2, differentiation antigens such as tyrosinase, gp100, PSA, Her-2 and CEA, mutated autoantigens such as E6 and / or E7 from oncogenic HPV types, and viral tumor antigens. Further examples of specific tumor antigens include MART-1, Melan-A, p97, β-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-4, MAGE-12, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, P1A, EpCam, melanoma antigen gp75, Hker8, high molecular weight melanoma antigen, K19, Tyr1, Tyr2, pMel17 gene family, c-Met, PSM (prostate mucin antigen), PSMA (prostate specific membrane antigen), prostate secretory protein, alpha-fetoprotein, CA125, CA19.9, TAG-72, BRCA-1 and BRCA-2 antigens.

[0133] In addition, the process of the present invention can generate other types of therapeutic clDNA, such as clDNA for use in gene therapy. For example, such DNA molecules can be used to express a functional gene when a subject has a genetic disorder caused by a malfunctioning version of that gene. Examples of such diseases include Duchenne muscular dystrophy, cystic fibrosis, Gaucher disease, and adenosine deaminase (ADA) deficiency. Other diseases for which gene therapy may be useful include inflammatory diseases, autoimmune, chronic, and infectious diseases, including disorders such as AIDS, cancer, neurological diseases, cardiovascular disease, hypercholestemia, various anemias, various blood disorders including thalassemia and hemophilia, and emphysema. For the treatment of solid tumors, genes encoding toxic peptides (i.e., chemotherapeutic drugs such as ricin, diptheria toxin, and cobra venom factor), tumor suppressor genes such as p53, genes encoding mRNA sequences that are antisense to transforming oncogenes, anti-tumor peptides such as tumor necrosis factor (TNF) and other cytokines, or transdominant negative mutants of transforming oncogenes may be expressed.

[0134] Other types of therapeutic clDNA are also contemplated for production by the processes of the invention, e.g., clDNA that is transcribed into active RNA forms, such as small interfering RNA (siRNA), can be produced by the processes of the invention.

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

[0136] As used herein, phrases such as "therapeutically effective amount" refer to an amount of clDNA that, upon administration, is sufficient to prevent the onset of, or alleviate to some extent, one or more symptoms of the disease being addressed. The specific amount of an agent administered in accordance with the present invention will, of course, be determined by the particular circumstances surrounding the case, including the clDNA administered, the route of administration, the particular condition being treated, and similar considerations.

[0137] The expression "pharmaceutical composition" encompasses both compositions intended for human use and compositions intended for non-human animals (ie, veterinary compositions).

[0138] The phrase "pharmaceutically acceptable carrier or excipient" refers to a pharmaceutically acceptable substance, composition, or vehicle. Each component must be pharmaceutically acceptable in the sense of being compatible with the other components of the pharmaceutical composition. The component must also be suitable for use in contact with human or non-human animal tissues or organs without undue toxicity, irritation, allergic response, immunogenicity, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0139] Examples of suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, viscosity stabilizers or emulsifiers, preservatives, solid binders, lubricants, etc. To the extent that any conventional excipient medium is incompatible with the substance or its derivatives, for example, by producing any undesirable biological effect or by interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is contemplated within the scope of the present invention.

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

[0141] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils. Excipients such as coloring agents, coating agents, 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 processes of the present invention may be in any dosage form, e.g., solid or liquid, and may be administered by any suitable route, e.g., oral, parenteral, rectal, topical, intranasal, or sublingual, and may contain pharmaceutically acceptable excipients necessary to formulate the desired dosage form, e.g., topical formulations (e.g., ointments, creams, lipogels, hydrogels), eye drops, aerosol sprays, injectable solutions, osmotic pumps, etc.

[0143] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, 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 thereof.

[0144] Exemplary granulating and / or dispersing agents include, but are not limited to, 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 resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone crospovidone, sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and combinations thereof.

[0145] Exemplary binding excipients include, but are not limited to, starch (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, psyllium mucilage, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, polyvinylpyrrolidone, silicon aluminum magnesium (Veegum) and larch arabinogalactan, alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, water, alcohol, and combinations thereof.

[0146] Exemplary preservatives may include antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, ascorbyl stearate, ascorbyl oleate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate.

[0147] Exemplary buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, sodium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propionic acid, calcium levulinate, pentanoic acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydrogen phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, potassium phosphate dibasic, potassium phosphate monobasic, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, sodium phosphate dibasic, sodium phosphate monobasic, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and combinations thereof.

[0148] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, leucine, sodium lauryl sulfate, lauryl sulfate, and combinations thereof.

[0149] As disclosed above, the present invention provides in a fifth aspect a concatemer 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.

[0150] In a particular embodiment of the process of the fifth aspect of the invention, optionally in combination with any of the embodiments provided above or below, each one of the repeated DNA sequences of interest 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 provided above or below, the concatemer DNA comprises 10 or more repeats of the DNA sequence of interest.

[0152] In a particular embodiment of the process of the fifth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the concatemer DNA is at least 5 kb in size.

[0153] Throughout this specification and claims, the term "comprises" and variations of this term are not intended to exclude other technical features, additives, ingredients, or steps. Furthermore, the term "comprises" encompasses instances of "consisting of." Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon review of this description or may be learned by practice of the present invention. The following examples and drawings are provided by way of illustration and are not intended to limit the invention. Reference signs in parentheses in connection with the drawings and in the claims are merely an attempt to enhance the comprehension of the claims and should not be construed as limiting the scope of the claims. Furthermore, the present invention covers all possible combinations of the specific and preferred embodiments described herein. [Example]

[0154] Example 1: TthPrimPol-based amplification of template DNA provides higher sequence fidelity than random primers.

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

[0156] As shown in Figure 1, amplification stimulated with TthPrimPol did not produce any amplification products without template DNA (left column, NTC). In contrast, amplification stimulated with RP produced high DNA yields even in the absence of template DNA. This suggests that TthPrimPol priming provides a highly specific amplification reaction. Furthermore, the DNA yields produced by TthPrimPol priming were similar in magnitude to those produced by RP in the presence of template DNA (right column, plasmid).

[0157] Finally, the amplification products from either TthPrimPol or RP primers were purified and sequenced using standard protocols using Illumina technology (5 million read pairs, 2 × 150 bps). As shown in Figure 2, bioinformatic analysis of the sequencing results indicated that TthPrimPol-based amplification was able to generate 80% usable reads, while amplification using random synthetic primers generated only 66% usable reads, confirming that fewer DNA artifacts were generated when the priming method was based on the use of TthPrimPol.

[0158] In conclusion, the above results suggest that the use of TthPrimPol in the process of generating therapeutic polynucleotides may be advantageous due to the high fidelity that TthPrimPol confers in the amplification step.

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

[0160] 10 ng of the plasmid pUC57-Kan_TELO-CMVEGFP was amplified by RCA as described in the above example, and the amplified product was then purified using standard protocols for DNA purification.

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

[0162] Finally, the products from the reaction with TelN were digested with restriction enzymes and treated with exonucleases to remove unwanted DNA fragments resulting from the protelomerase reaction.

[0163] HindIII and EcoRI digestion was performed according to the manufacturer's instructions. 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. Briefly: Reaction volume: 1628 μL DNA input: 350 μg of amplified DNA digested with TelN, HindIII, and EcoRI -ExoIII input: 6 μL (600 units) -Reaction time: 45 minutes at 37°C, 20 minutes at 80°C.

[0165] As shown in Figure 3 , amplification stimulated by TthPrimPol produced amplification products that could successfully convert clDNA through the action of TelN.

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

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

[0168] To test whether TthPrimPol can prime clDNA using the minimal single-stranded loop that does not contain its target sequence, "XTC," the plasmid pUC57-Kan_TELO-CMVEGFP, which contains two protelomerase recognition sequences flanking the expression cassette, was treated with protelomerase (TelN) as described in Example 2 to generate clDNA containing the expression cassette.

[0169] One ng of the resulting clDNA or initial plasmid was subjected to primed RCA using either TthPrimPol or random primers, and the amplification product was quantified, as described in Example 1. As shown in Figure 4A, TthPrimPol priming allowed amplification of TelN-generated clDNA, although the yield was lower than that obtained with random primers.

[0170] The amplification products (DNA concatemers) were then treated with TelN to again generate clDNA, which was analyzed in an agarose gel by restriction enzyme and exonuclease treatment as described in Example 2.

[0171] Although random primer amplification resulted in high yields of DNA products (Fig. 4A), analysis of amplified DNA generated by TelN showed that TthPrimPol priming allowed for the generation of high levels of clDNA containing the target DNA sequence (Fig. 4B).

[0172] These results demonstrate that TthPrimPol can not only prime clDNA containing minimal adapter sequences (i.e., without its recognition sequence), but more importantly, it can also generate higher quality amplification products than random primers, which increases the efficiency of generating final clDNA.

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

[0174] The clDNA containing the coding sequence for eGFP (enhanced green fluorescent protein), the plasmid vector containing the coding sequence for eGFP, and the empty vector, prepared as disclosed above in Example 3, were transiently transfected into HEK293 cells as described in Heinrich, M. et al., "Linear closed mini DNA generated by the prokaryotic cleaving-joining enzyme TelN is functional in mammalian cells", J Mol Med, 2002, vol. 80, pp. 648-654.

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

[0176] As shown in Figures 4 and 5, cells transfected with clDNA synthesized according to the process of the present invention exhibited strong expression of eGFP. These results demonstrate that the process of the present invention can produce highly functional clDNA suitable for gene therapy.

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

[0178] Synthesis of customized single-stranded DNA adapters containing natural and modified nucleotides

[0179] Customized single-stranded DNA adapters containing natural and modified nucleotides were synthesized according to standard phosphoramidite chemistry (Beaucage Slet et al., 1981) with at least two of the following modified nucleotides: 8-oxo-deoxyadenosine (8-oxo-dA), 5-fluoro-deoxyuracil (5FU), inosine, thiophosphate nucleotides, or locked nucleic acid (LNA) nucleotides.

[0180] Briefly, phosphoramidite synthesis begins with the 3'-most distal nucleotide and proceeds through a series of repeated cycles consisting of four steps: deprotection (i), coupling (ii), oxidation (iii), and capping (iv) until the 5'-most distal nucleotide is attached.

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

[0182] Corresponding hairpin DNA adapters containing natural oligonucleotides were also synthesized. A list of the synthesized adapters is provided in Table 2.

[0183] At the end of synthesis, the oligonucleotides are cleaved from the support, the protecting groups are removed, and standard purification steps (e.g., PAGE, HPLC, and / or RNase-free HPLC) are then used to separate the full-length product from the cleaved sequence.

[0184] [Table 2]

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

[0186] Using some of the customized adapters in Table 2, clDNA was prepared starting from plasmid DNA (pDNA). First, pDNA, such as an eGFP plasmid with SEQ ID NO: 20 (containing a sequence of interest encoding Gfp flanked by BsaI restriction sites) and a protelomerase target sequence (see Figure 9), was treated with protelomerase to obtain clDNA containing the sequence of interest flanked by endonuclease restriction sites. This clDNA was then amplified by rolling circle amplification (RCA) using TthPrimPol and Phi29. The resulting concatemers were purified, treated with the corresponding restriction enzyme (e.g., BsaI), and ligated with customized adapters (e.g., oligo 21 and oligo 41 in Table 2). Details of an exemplary protocol are provided below.

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

[0188] 1.1 TelN digestion The eGFP plasmid was digested with TelN enzyme for 2 hours at 30° C. and inactivated for 10 minutes at 75° C. If several reactions are performed simultaneously, scale up accordingly. [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 sample was then inactivated for 15 minutes at 65° C. If several reactions are performed simultaneously, scale up accordingly. [Table 7]

[0190] 1.2.2 Exo III Digestion Exo III digestion was carried out for 1 hour at 37° C. and inactivated for 10 minutes at 75° C. If several reactions are performed simultaneously, scale up accordingly. [Table 8]

[0191] 1.3 Purifying clDNA using gel filtration chromatography and isopropanol 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 are added to the sample from the last step and mixed by vortexing. The sample is kept at 4°C for 5 minutes. Then, it is centrifuged at 12000g for 20 minutes at 25°C. After centrifugation, the supernatant is collected, and an equal volume of isopropanol is added to the supernatant and mixed thoroughly. The sample is kept at room temperature for 5 minutes. It is then centrifuged at 12000g for 20 minutes, and the supernatant is removed. Finally, the precipitate is suspended in 10mM Tris-HCl (pH 7.5). [Table 9]

[0193] After three steps of enzyme digestion, gel chromatography, Triton 114 treatment, and isopropanol precipitation, eGFP_BSaI_cl DNA was successfully produced. The DNA homogeneity (percentage) of the sample was 97% by HPLC chromatogram. The endotoxin content of the sample was less than 10EU / mg.

[0194] B. Protocol for Obtaining Customized Adapter-Containing clDNA from clDNA by RCA This experiment is designed to generate clDNA containing customized adapters from eGFP_BSaI_clDNA obtained in the above section by Trueprime-RCA kit. [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 min. Cool to 4°C. Store the amplified DNA at 4°C for short-term storage or at -20°C for long-term storage. ( * ) 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.

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

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

[0198] 1.3 Purify the RCA products (concatamers) using an Axygen kit (optional). If the sample is 100 μL or less, an Axygen kit can also be used to purify clDNA. The protocol is described below, and the bottles containing the buffers 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 from the last step to the column. Centrifuge at 12,000 x g for 1 minute. 3) Discard the filtrate from the 2 mL microfuge tube. Place the Miniprep column back into the 2 mL microfuge tube and add 500 μL of Buffer W1. Centrifuge at 12,000 x g for 30 seconds. 4) Discard the filtrate from the 2 mL microfuge tube. Place the Miniprep column back into the 2 mL microfuge tube and add 700 μL of Buffer W2. Centrifuge at 12,000 x g for 30 seconds. 5) Discard the filtrate from the 2 mL microfuge tube. Place the Miniprep column back into the 2 mL 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 into a clean 1.5 mL microfuge tube (provided). To elute the DNA, add 50 μL of 10 mM Tris-HCl (pH 7.5) to the center of the membrane. Let this sit at room temperature for 1 minute. Centrifuge at 12,000 x g for 1 minute.

[0199] 1.4 Oligo denaturation and annealing Oligos (e.g., oligo 21 or oligo 41 in Table 3) were denatured at 95°C for 10 minutes and allowed to spontaneously anneal at room temperature for 30 minutes. If several reactions are performed simultaneously, scale up accordingly. [Table 13]

[0200] 1.5 Oligophosphorylation (optional, skip this step if the oligos are already phosphorylated) 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 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 typically require several cloning steps to generate the desired construct, with each step transferring a single DNA fragment from a donor plasmid or PCR product into a recipient vector.

[0206] During Golden Gate cloning, it is possible to assemble up to 15 fragments at a time in a recipient plasmid. Cloning is performed by pipetting all the plasmid donors, recipient vectors, type IIS restriction enzymes, and ligase in a single tube and incubating the mix in a thermal cycler. Therefore, we also propose to generate oDNA using Golden Gate assembly. The system and conditions are described in Tables 14 and 15, respectively. If several reactions are performed simultaneously, scale up accordingly.

[0207] [Table 17] [Table 18]

[0208] 1.11 Unexpected DNA digestion Exo III digestion was carried out for 1 hour at 37° C. and inactivated for 10 minutes at 75° C. If several reactions are performed simultaneously, scale up accordingly. [Table 19]

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

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

[0211] A similar procedure was used to prepare Luc plasmid having SEQ ID NO: 21 (containing a luciferase-encoding sequence flanked by BsaI restriction sites and a protelomerase target sequence) and clDNA starting from oligos 15, 37, 4, 28, 29, 17, 22, 37, 28, 29, 19, and 22 in Table 2. Again, a similar procedure was used to prepare Luc-ITR plasmid having SEQ ID NO: 22 (containing an ITR sequence flanked by luciferase-encoding sequences flanked by BsaI restriction sites and additionally containing a protelomerase target sequence; see Figure 10) and clDNA starting from oligo 4. This resulted in the preparation of oDNA4. ITR (6.2 μg in total) was obtained (see FIG. 11 (agarose gel electrophoresis)).

[0212] The quality of the obtained clDNA was assessed by standard procedures (specifically agarose gel electrophoresis, grayscale analysis, anion exchange chromatography-HPLC, and Sanger sequencing). All clDNAs were found to exhibit good quality characteristics in terms of purity, peak resolution, and sequence verification. For illustrative purposes, the results for oDNA41 and oDNA21 are shown in Figures 7 and 8, respectively.

[0213] Example 6: Generation of clDNA from TelN-generated clDNA by RCA followed by TelN processing

[0214] Alternatively, clDNA of the invention may be prepared by the procedures described in Sections A (1.1) through B (1.3) of Example 5, followed by TelN processing of the resulting concatemers. This final step, processing of the concatemers resulting from RCA using protelomerase (Section B, 1.3), is described in detail below.

[0215] 1.4 TelN digestion The purified RCA product was digested with TelN enzyme for 2 hours at 30° C. and inactivated for 10 minutes at 75° C. If several reactions are performed simultaneously, scale up accordingly. [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 sample was then inactivated for 15 minutes at 65° C. If several reactions are performed simultaneously, scale up accordingly. [Table 22]

[0217] 1.4.2 Exo III Digestion Exo III digestion was carried out for 1 hour at 37° C. and inactivated for 10 minutes at 75° C. If several reactions are performed simultaneously, scale up accordingly. [Table 23]

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

[0219] 1.7 Purify the clDNA product using an Axygen kit (as above, optionally with a sample volume of 100 μL or less) Synthetic clDNA, whose sequence of interest encodes GFP (see Figure 9) and has a constant 28 base pair protelomerase sequence, was obtained after cleavage / ligation. Details regarding the synthesis performance, step by step, are described in Table 24 (below). The resulting clDNA showed 96.6% homogeneity according to agarose gel electrophoresis (AGE) (Figure 12). [Table 24]

[0220] List of cited references Heinrich, M. et al. “Linear closed mini DNA generated by the prokaryotic cleaving-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. 71(2), pp. 941-948. WO2011000997 Beaucage S. L. et al, Deoxynucleoside phosphoramidites-A new class of key intermediates 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 closed linear template DNA comprising a double-stranded intermediate segment flanked by two single-stranded loops containing a DNA sequence of interest, wherein the single-stranded loops do not contain primase / polymerase priming sites; b) amplifying DNA from the closed linear template DNA of step (a), wherein said amplification is stimulated with a primase / 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. 2. The process of claim 1, wherein the amplification performed in step (b) is rolling circle amplification.

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

1.

4. The process of any one of claims 1 to 3, wherein the amplification in step (b) is carried out using a strand-displacing DNA polymerase.

5. A process described in any one of claims 1 to 3, wherein step (a) is carried out by contacting a plasmid vector containing at least two restriction sites flanking the target DNA sequence with at least one restriction enzyme, thereby creating an open double-stranded DNA containing the target DNA sequence, and attaching single-stranded DNA adapters to both ends of the open double-stranded DNA containing the target DNA sequence.

6. A process described in any one of claims 1 to 3, wherein step (a) is carried out by contacting a protelomerase with a plasmid vector containing at least two protelomerase target sequences flanking the target DNA sequence.

7. 7. The process of any one of claims 1 to 6, 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.

8. 8. The process of claim 7, wherein when the concatemer DNA contains repeats of the DNA sequence of interest flanked by at least restriction sites, step (c) is carried out by: (c1) contacting the concatemer DNA with at least one restriction enzyme, thereby generating a plurality of open, double-stranded DNA fragments, each containing the DNA sequence of interest; and (c2) ligating single-stranded DNA adaptors to both ends of the open, double-stranded DNA fragments.

9. 8. The process of claim 7, wherein when the concatemer DNA comprises repeats of the DNA sequence of interest flanked by at least protelomerase target sequences, step (c) is carried out by contacting the concatemer DNA with protelomerase.

10. The process according to any one of claims 1 to 9, wherein the template DNA is a closed linear template DNA that does not contain a protelomerase target site.

11. 11. The process of claim 10, wherein step (a) is carried out by contacting a protelomerase with a plasmid vector comprising two protelomerase target sequences flanked by at least two restriction sites flanking the DNA sequence of interest; and step (c) is carried out by (c1) contacting the concatemer DNA with at least one restriction enzyme, thereby generating a plurality of open, double-stranded DNA fragments, each containing the DNA sequence of interest; and (c2) ligating single-stranded DNA adaptors to both ends of the open, double-stranded DNA fragments.

12. 11. The process of claim 10, wherein step (a) is carried out by contacting a plasmid vector containing at least two restriction sites and a non-protelomerase target site flanking the DNA sequence of interest with at least one restriction enzyme, thereby creating an open double-stranded DNA containing the DNA sequence of interest, and ligating single-stranded DNA adaptors to both ends of the open double-stranded DNA containing the DNA sequence of interest, provided that the single-stranded DNA adaptors do not contain a protelomerase target site; and step (c) is carried out by (c1) contacting the concatemer DNA with at least one restriction enzyme, thereby creating a plurality of open double-stranded DNA fragments each containing the DNA sequence of interest, and (c2) ligating single-stranded DNA adaptors to both ends of the open double-stranded DNA fragments.

13. Step (a) - contacting a plasmid vector containing at least two restriction sites flanking said DNA sequence of interest with at least one restriction enzyme, thereby creating an open double-stranded DNA containing said DNA sequence of interest, and ligating said single-stranded DNA adaptors to both ends of said open double-stranded DNA containing said DNA sequence of interest, provided that said single-stranded DNA adaptors do not contain primase / polymerase priming sites; or alternatively, - by contacting a protelomerase with a plasmid vector containing at least two protelomerase target sequences flanking said DNA sequence of interest, The process of claim 12, thereby obtaining a template DNA that is a closed linear template DNA containing the desired DNA sequence.

14. The process of any one of claims 1 to 13, wherein the sequence of interest comprises inverted terminal repeats (ITRs) flanking the expression cassette.

15. A process described in any one of claims 5, 8, 11 to 13, wherein the single-stranded DNA adapter comprises a modified oligonucleotide.

16. The process according to any one of claims 1 to 15, which is a cell-free in vitro process.

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