Closed linear DNA using modified nucleotides

Abstract

To provide a closed linear DNA containing modified nucleotides which is particularly useful for a therapeutic purpose.SOLUTION: Provided is a closed linear DNA (clDNA) comprising stem regions including a target double-stranded DNA sequence which is covalently closed at both ends by a hairpin loop, and includes at least two modified nucleotides.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of European Patent Application No. 20382063.4 filed on January 31, 2020. Claim the benefit of specification.

[0002] The present invention is in the field of nucleic acids. In particular, the present invention relates to closed linear oligonucleotides containing modified nucleotides. The closed linear DNA of the present invention is particularly useful for therapeutic purposes. [Background technology]

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

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

[0005] In this regard, it is important to note that the nucleotide sequences do not contain the majority of the immunogenic bacterial backbone and only carry the DNA sequence of interest. The use of small linear oligodeoxynucleotides (ODNs) to However, ODNs are susceptible to endonucleases and cleavage, which severely limits their therapeutic potential. It is prone to degradation by exonucleases.

[0006] To improve the stability of ODNs, several strategies have been followed in the prior art. Open linear ODNs are chemically modified to ensure their persistence in vivo. For example, L-DNA nucleotides are contained at open ends, which protect against nucleolytic degradation. However, test results have so far been modest. and the addition of modified nucleotides within these open DNA structures can be used to target off-target molecules. It often causes side effects.

[0007] Another strategy for ODN stabilization is to protect the double-stranded region by flanking it with two single-stranded loops. This results in the formation of a closed linear DNA (clDNA) molecule, which forms a dumbbell-shaped molecule. The absence of any open ends in the clDNA makes it a nucleic acid molecule. This results in a state that is highly tolerant to solutions (Non-Patent Document 2).

[0008] By modifying the stem length and loop size, or by adding sequences within the stem-loop region There have also been attempts to further improve the stability of cDNA by introducing motifs. For example, the presence of a cytosine-guanine pair in closing this loop is associated with increased loop stability, although CG motifs are highly effective. Genes known to be immunostimulatory sequences and whose activation of the immune system should be avoided This significantly hinders the use of vectors for child therapy.

[0009] Therefore, any specific target can be achieved without causing unwanted immune-related side effects. The need for stable c1DNA suitable for expressing any gene in vivo still exists. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Kapp K et al., “EnanDIM-a novel family of L-nucleotide-protected TLR9 agonists for cancer immunotherapy”2019, J Immunother Cancer., vol7(1), pp.5 [Non-patent document 2] Heinrich J. et al., “Linear closed mini DNA generated by the prokaryotic cleaving-joining enzyme TelN is functional in mammalian cells” 2002, J Mol Med, vol. 80(10), pp. 648-54 Summary of the Invention [Problem to be solved by the invention]

[0011] The present inventors have discovered novel closed loop DNA fragments suitable for use in DNA-based therapies, such as gene therapy. We have developed linear DNA (clDNA). In particular, the clDNA of the present invention has a closed molecular structure. and a small number of other molecules that improve the efficiency of clDNA when used in DNA-based therapies. It contains at least two modified nucleotides.

[0012] Surprisingly, the inventors have found that when two or more nucleotide modifications are incorporated, We found that stable clDNA can be produced even in very small molecules whose stability and functionality depend largely on their unique dumbbell-like shape. This was completely unexpected, since the prior art Increase the stability of cDNA without disrupting the fragile intermolecular interactions that maintain it. Most attempts to do so have focused on small modifications of nucleotide sequence identity or on the modification of only one nucleotide. This indicates that the effect is based on the addition of leutidine modifications.

[0013] The clDNA of the present invention can be used to treat diseases through DNA-based therapies, such as gene therapy. It constitutes a very useful alternative to the arrangements disclosed in the prior art. [Means for solving the problem]

[0014] Thus, in a first aspect, the present invention provides a method for producing a nucleotide sequence covalently linked at both ends by hairpin loops. It consists of a stem region containing a closed double-stranded DNA sequence of interest and contains at least two modified nucleic acids. A closed linear DNA ("clDNA") containing nucleotides is provided.

[0015] Advantageously, at least two modified nucleotides modulate the properties of the clDNA to be synthesized. In order to achieve this, the nucleotides may be introduced into various regions of the molecule, such as the single-stranded loop or specific regions of the stem. good.

[0016] The clDNA of the present invention, which comprises at least two modified oligonucleotides, is a naturally occurring They have several advantageous properties that give them an advantage over their counterparts. For example, increased transfection efficiency, increased expression efficiency, good stability, bioassay Increased availability, functional survival, and resistance to degradation, and the purpose of the compounds contained therein The following examples are provided to illustrate the functional properties of sequences of interest. Some clDNAs containing modified nucleotides show a surprising improvement in performance. In this case, this sequence had luciferase activity to provide proof of concept. This proves that

[0017] The clDNAs of the present invention are useful for multiple indications, for example, for therapeutic or diagnostic indications. In a second aspect, the present invention relates to a closed linear DNA according to the first aspect for use in therapy. In another aspect of the invention, a clDN according to the first aspect is provided for use in diagnosis. Provide A.

[0018] In a third aspect, the present invention provides a therapeutically effective amount of closed linear DNA according to the first aspect, and a pharmaceutical and a physiologically acceptable carrier or excipient.

[0019] In a fourth aspect, the present invention provides a method for the preparation of a nucleic acid comprising at least two modified nucleotides according to this first aspect. The present invention provides a process for producing a closed linear DNA comprising: a) a target b) providing a template DNA containing the DNA sequence of step a); amplifying the target template DNA to produce a concatemer DNA containing repeats of the target template DNA; each of the repeated DNA sequences is flanked by a restriction site; The target DNA is contacted with at least one restriction enzyme, thereby isolating the target DNA sequence. (c2) generating a plurality of open double-stranded DNA fragments each containing Attaching hairpin DNA adaptors to each end of the double-stranded DNA fragment. wherein each one of the adaptors has at least one modified nucleotide; or Alternatively, only one of the adapters bound to the DNA fragment contains at least two modified nucleotides. and (b) forming a closed linear DNA fragment using the amplified DNA fragment prepared in step (b) containing a nucleotide. d) producing the closed linear DNA produced in step c); Includes:

[0020] In a fifth aspect, the present invention relates to a closed linear D-type olefin obtainable by a process according to the fourth aspect. The present invention also provides a method for treating or diagnosing a NA according to the fourth aspect. Provide cDNA.

[0021] In a sixth aspect, the present invention provides a method for the preparation of a nucleotide sequence comprising at least one modified nucleotide, a ligase, and optionally for generating clDNA containing hairpin DNA adapters containing instructions therefor Provide a kit.

[0022] The cDNA may be used alone, in conjunction with a gene vector or carrier, or in any other suitable medium as desired. It may be provided together with other DNA molecules that contribute to the therapeutic effect of the virus. combination with a viral or non-viral vector, nanoparticle, or any other carrier. This may be advantageous, for example, for targeting desired cells or tissues. However, certain non-viral vectors and clDNA containing modified nucleotides The complexes formed by the above-mentioned methods (also referred to herein as polyplexes) are then directed to the desired cells. The transfection efficiency of the clDNA or the release process of the clDNA under physiological conditions The cDNA of the present invention can be used to further improve certain properties, such as the polynucleotide profile. Non-limiting non-viral vectors suitable for forming the complex include polycationic polymers. It's Rimmer.

[0023] Thus, in a seventh aspect, the present invention provides a method for producing a composition according to the first or fourth aspect of the present invention. A composition is provided that includes a cDNA and a carrier.

[0024] In an eighth aspect, the present invention provides a polymer, e.g., a polycationic polymer, and a method for producing the same. The present invention provides a polyplex comprising the clDNA according to the first or fourth aspect of the present invention. [Brief explanation of the drawings]

[0025] [Figure 1A] 1 shows the structure of a closed linear DNA according to the present invention, consisting of two stem-loop adaptors flanking a DNA sequence of interest. [Figure 1B] The structure of the adaptor forming the clDNA of the present invention is shown in more detail, where the stem of the adaptor represents a proximal region at the end of the stem (1) that is ligated to the DNA sequence of interest, and a distal region at the end of the stem (2) that is closed by a single-stranded loop. [Figure 2] 1 shows a scheme for preparing clDNA prepared with customized hairpin adapters. A DNA fragment (A) containing a sequence of interest (e.g., luciferase or Gfp) flanked on each side by endonuclease restriction sites (e.g., BsaI restriction sites) is treated with a specific restriction endonuclease (B) and ligated with a desired hairpin adapter (e.g., oligo 37 of SEQ ID NO: 7, containing 5 phosphothioated nucleotides shown in italics) and an exonuclease to obtain clDNA containing modified nucleotides (C). [Figure 3A]Quality control parameters of oDNA17 are shown by agarose gel electrophoresis (M1, supercoiled DNA ladder marker (TAKARA): 3585A; M2, 1 kB DNA ladder TIAGEN MD111; lane 11, oDNA17). [Figure 3B] Quality control parameters of oDNA17, showing a grey scale analysis. [Figure 3C] FIG. 1 shows quality control parameters for oDNA17, anion exchange chromatography-HPLC. [Figure 3D] FIG. 1 shows quality control parameters for oDNA17 Sanger sequencing. [Figure 4A] Quality control parameters of oDNA19 are shown by agarose gel electrophoresis (M1, supercoiled DNA ladder marker (TAKARA): 3585A; M2, 1 kB DNA ladder TIAGEN MD111; lane 2, oDNA19). [Figure 4B] oDNA19 quality control parameters, grayscale analysis. [Figure 4C] FIG. 1 shows quality control parameters for oDNA19, anion exchange chromatography-HPLC. [Figure 4D] FIG. 1 shows quality control parameters for oDNA19 Sanger sequencing. [Figure 5A] Quality control parameters of oDNA41 are shown by agarose gel electrophoresis (M1, supercoiled DNA ladder marker (TAKARA): 3585A; M2, 1 kB DNA ladder TIAGEN MD111; lane 5, oDNA41). [Figure 5B] Quality control parameters of oDNA41, showing a grey scale analysis. [Figure 5D] FIG. 1 shows quality control parameters for oDNA41 Sanger sequencing. [Figure 6A]Luciferase activity 24 hours after transfection in HaCaT cells transfected with clDNA containing natural oligonucleotides (oDNA15, oDNA4, or oDNA17) or modified oligonucleotides (oDNA37, oDNA28, oDNA19, or oDNA22) using PEI is shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, natural oDNA vs. the corresponding modified oDNA (15 vs. 37, 4 vs. 28, and 17 vs. 19 or 22), Student's t-test (n=3). [Figure 6B] Luciferase activity in HaCaT cells transfected with natural oligonucleotides (oDNA15, oDNA4, or oDNA17) or modified oligonucleotides (oDNA37, oDNA28, oDNA19, or oDNA22) using PEI is shown 48 hours after transfection. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, natural oDNA vs. the corresponding modified oDNA (15 vs. 37, 4 vs. 28, and 17 vs. 19 or 22), Student's t-test (n=3). [Figure 7A] Time evolution of luciferase activity levels versus time for HaCaT cells transfected with clDNA containing natural or modified oligonucleotides using PEI. Pairwise comparison of natural vs. modified indicates oDNA15 vs. oDNA37. *p<0.05, **p<0.01, ***p<0.001, **p<0.0001, day 2 values ​​vs. day 1 values, Student's t-test (n=3). [Figure 7B] Time evolution of luciferase activity levels versus time for HaCaT cells transfected with clDNA containing natural or modified oligonucleotides using PEI. Pairwise comparison of natural vs. modified indicates oDNA4 vs. oDNA28. *p<0.05, **p<0.01, ***p<0.001, *** *p<0.0001, day 2 values ​​vs. day 1 values, Student's t-test (n=3). [Figure 7C]Time evolution of luciferase activity levels versus time for HaCaT cells transfected with clDNA containing natural or modified oligonucleotides using PEI. Pairwise comparisons of natural versus modified indicate oDNA17 versus oDNA19 or oDNA22. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, day 2 values ​​versus day 1 values, Student's t-test (n=3). [Figure 8] Figure 1 shows the release of clDNA cargo from polyplexes formed by polymer CXP-37 and clDNA containing natural oligonucleotides (oDNA15, oDNA4) or modified oligonucleotides (oDNA37, oDNA28, oDNA29) after 12 hours of incubation with 8 U / mL heparin, which mimics physiological conditions for the release of complexed clDNA (competing with the polymer). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, native oDNA vs. the corresponding modified oDNA (15 vs. 37, 4 vs. 28 or 29), Student's t-test (n=3). [Figure 9A] The synthetic route of PAspDET / DIDPA is shown. [Figure 9B] The synthesis of poly(β-benzyl L-aspartate) (PBLA) is shown. [Figure 9C] 1 shows the synthesis of PAsp(DET / DIIPA)-compound CXP037A. [Figure 10] The 1H NMR spectrum of PBLA is shown below. 1H NMR (DMSO-d6): δ = 0.79 (t, J = 7.58 Hz, 3H), 1.13-1.37 (m, 4H), 2.96-2.52 (m, 2H, CH2), 4.61 (s, 1H, CH), 5.01 (s, 2H, benzyl CH2), 7.27 (s, 5H, aryl CH), 8.15 (s, 1H, NH). [Figure 11] The 1H NMR spectrum of CXP037 is shown below. 1H NMR (D2O): δ = 0.84 (t, J = 7.68 Hz, 3H), 1.32 (m, 3H, CH3), 2.82 (brs, 2H, CH2), 3.08-3.79 (m, 2H, CH2). [Figure 12] SEC-MALS-RI of CXP037A analysis for MW determination: MW = 14,000 Da (1.03). [Figure 13] Potentiometric titration curve for pKa determination of CXP037. Calculated pKA: 5,370 / 8,952. [Figure 14]

[0023] Figure 1 shows a representation of a fragment of the eGFP plasmid (a plasmid having SEQ ID NO: 16) 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 15]

[0033] Figure 1 shows a representation of a fragment of Luc-ITR (a plasmid having SEQ ID NO: 18) 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 16] Agarose gel electrophoresis of oDNA37ITR (M, DL3000 ladder, lane 14, oDNA37ITR) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description of the Invention All terms used herein in this application are understood to be of the art unless otherwise specified. The term "common" is to be understood in its general meaning as known in the art. Other more specific definitions of terms are provided below. Definitions may also be used in conjunction with broader definitions. This definition applies throughout the specification and claims unless expressly stated otherwise. It is intended to be applied uniformly throughout.

[0027] As used herein, the indefinite articles "a" and "an" mean "at least one" or Unless otherwise indicated, "the" as used herein is synonymous with "one or more." Definite articles such as these also include the plural of the noun.

[0028] In a first aspect, the present invention provides a method for producing a nucleic acid molecule covalently closed at both ends by a hairpin loop. The stem region contains the desired double-stranded DNA sequence and is bound to at least two modified nucleotides. The present invention provides closed linear DNA ("clDNA") containing the nucleotide.

[0029] As used herein, the term "closed linear DNA" or "clDNA" refers to a nucleotide sequence. "Dumbbell" or "dogbone" under conditions that allow hybridization of the octides. It refers to a single-stranded, covalently closed DNA molecule that forms a structure of the shape Although a single-stranded DNA molecule forms a clDNA, two complementary sequences within the same molecule Hybridization of the two single-stranded loops forms a "dumbbell" structure. The method produces a structure consisting of a double-stranded intermediate segment flanked by loops. Use biological techniques to understand how to generate clDNA from open or closed double-stranded DNA. For example, those skilled in the art can recognize the ends of double-stranded DNA that have been opened, for example, by the action of a ligase. We found that clDNA can be produced by ligating a hairpin DNA adapter to "Hairpin DNA adapters" are DNA fragments that are formed by hybridization of two complementary sequences. This refers to a single-stranded DNA that forms a stem-loop structure. It is closed at one end by a chain loop and open at the other end.

[0030] A "sequence of interest" is any sequence required for correct gene expression, such as an expression cassette. A double-stranded DNA fragment containing the minimal sequence encoding the gene of interest, along with the sequence of The sequence of interest is understood as an inverted terminal repeat. It may additionally contain other sequences flanking the expression cassette, such as interleukin (ITR) repeats. That's fine.

[0031] The term "nucleoside" refers to a nucleoside that consists of a nucleotide at the C-1' carbon of a sugar, e.g., ribose or deoxyribose. The term "nucleotide" refers to a monomeric unit or polymer. Refers to the phosphate ester of a nucleoside within a nucleotide.

[0032] "Modified nucleotide" refers to a nucleotide that has been chemically modified by modification of the base, sugar, or phosphate group, or Any nucleotide (e.g., adenosine, guanylate, thiamin ... Modified nucleotides are nucleotides that react to the modification. It may or may not be naturally occurring.

[0033] As used herein, modified nucleotides preferably include 5-methyl-deoxyribonucleotides. Adenosine, 2-amino-deoxyadenosine, 1-methyl-adenosine, 1-methyl-guanidine Inosine, 1-methyl-inosine, 2,2-dimethyl-guanosine, 2,6-diaminopurine 2'-amino-2'-deoxyadenosine, 2'-amino-2'-deoxycytidine , 2'-amino-2'-deoxyguanosine, 2'-amino-2'-deoxyuridine, 2-Amino-6-chloropurine riboside, 2-aminopurine riboside, 2´-aladecyl Nosine, 2'-aracytidine, 2'-arauidine, 2'-azido-2'-deoxyadenosine 2-azido-2'-deoxycytidine, 2'-azido-2'-deoxyguanosine 2'-azido-2'-deoxyuridine, 2-chloroadenosine, 2'-fluoro- 2´-Deoxyadenosine, 2´-fluoro-2´-deoxycytidine, 2´-fluoro -2´-deoxyguanosine, 2´-fluoro-2´-deoxyuridine, 2´-fluoro Rotimidine, 2-methyl-adenosine, 2-methyl-guanosine, 2-methyl-thio-N 6-Isopenenyl-adenosine, 2´-O-methyl-2-aminoadenosine, 2´-O- Methyl-2´-deoxyadenosine, 2´-O-methyl-2´-deoxycytidine, 2´ -O-methyl-2´-deoxyguanosine, 2´-O-methyl-2´-deoxyuridine , 2´-O-methyl-5-methyluridine, 2´-O-methylinosine, 2´-O-methyl Luseudolidine, 2-thiocytidine, 2-thio-cytidine, 3-methyl-cytidine, 4-acetyl-cytidine, 4-thiouridine, 5-(carboxyhydroxymethyl)-uridine Lysine, 5,6-dihydrouridine, 5-aminoallylcytidine, 5-aminoallyl-de Oxyuridine, 5-bromouridine, 5-carboxymethylaminomethyl-2-thio- Uracil, 5-carboxymethylammonomethyl-uracil, 5-chloro-ara-cytosine , 5-fluorouridine, 5-iodouridine, 5-methoxycarbonylmethyluridine uridine, 5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-azacytidine, 6-Azauridine, 6-chloro-7-deaza-guanosine, 6-chloropurine riboside, 6-Mercapto-guanosine, 6-methyl-mercaptopurine-riboside, 7-deaza- 2´-deoxy-guanosine, 7-deazaadenosine, 7-methyl-guanosine, 8- Zaadenosine, 8-bromo-adenosine, 8-bromo-guanosine, 8-mercapto-guanosine Guanosine, 8-oxoguanosine, benzimidazole riboside, β-D-mannosyl -queosine, dihydrouridine, inosine, N1-methylaridine Adenosine, N6-([6-aminohexyl]carbamoylmethyl)-adenosine, N6- Isopentenyl-adenosine, N6-methyl-adenosine, N7-methyl-xanthosine , N-uracil-5-hydroxyacetic acid methyl ester, puromycin, queosin (que osine), uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, including acetylated cyclobutanosine, xanthosine, and xylo-adenosine Chemically modified, such as methylated or hydroxylated, naturally occurring or non-naturally occurring Any of guanosine, uridine, adenosine, thymidine, or cytidine, but Without limitation, guanosine, uridine, adenosine, thymidine, and cytidine barriers The preparation of such variants is described, for example, in U.S. Pat. No. 4,373,071. and are known to those skilled in the art.

[0034] Modified nucleotides also include pyridin-4-one ribonucleosides, 5-aza-uridine , 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2 -thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carbo 1-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine Lysine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurine 5-Taurinomethyl-2-thiouridine, 1-Taurinomethyl-pseudouridine Methyl-4-thio-uridine, 5-methyluridine, 1-methyl-pseudouridine, 4 -thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1 -methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine Douridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine uridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4- Thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio- Douridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N 4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxycytidine Methylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- Isocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- Pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1- Methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine Zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2- Thio-Zebularine, 2-Thio-Zebularine, 2-Methoxy-Cytidine, 2-Methoxy-5 4-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl This also includes, but is not limited to, til-pseudoisocytidine.

[0035] Modified nucleotides also include 2-aminopurine, 2,6-diaminopurine, 7-deaza- Adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza 7-Deaza-2,6-diaminopurine, 7-Deaza- 8-Aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-Isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6- Lysinylcarbamoyl adenosine, N6-threonylcarbamoyl adenosine, 2-methyl Ruthio-N6-threonylcarbamoyl adenosine, N6,N6-dimethyl adenosine, Also includes 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine , but not limited to these.

[0036] Modified nucleotides also include inosine, 1-methyl-inosine, yiosine, ybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine , 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-Methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N 2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N This also includes, but is not limited to, 2,N2-dimethyl-6-thio-guanosine.

[0037] Modified nucleotides also include 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, Uridine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine , N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine uridine, 5-methyl-uridine, pyrrolo-cytidine, inosine, α-thio-guanosine, 6 -methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine sine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro- Purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro- Purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, This also includes, but is not limited to, 7-deaza-adenosine.

[0038] The modified nucleotide may be chemically modified at the 2' position. The tide contains a substituent at the 2' carbon atom. This substituent can be a halogen, an alkoxy group, hydrogen, From the group consisting of aryloxy groups, amino groups and aminoalkoxy groups, preferably 2'- Hydrogen (2'-deoxy), 2'-O-methyl, 2'-O-methoxyethyl and 2'-fluoro Selected from Oro.

[0039] The chemical modifications involving the 2' position of the nucleotide described herein are referred to as locked nucleic acids ( locked nucleic acid (LNA) nucleotides, ethylene-bridged nucleic acids ( ethylene bridged nucleic acid (ENA) nucleotide and (S)-constrained ethyl cEt nucleotides. These backbone modifications are The sugars of the tides are locked into a preferred northern conformation.

[0040] The backbone phosphate group can be modified by, for example, substituting one or more of the oxygen atoms with different substituents. Additionally, modified nucleotides may be modified with modified phosphate groups as described herein. Examples of modified phosphate groups include phosphorothioates, phosphate groups, and phosphate groups. ates (also known as thiophosphates), phosphoroselenates, boranophosphates, Boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkyl phosphonate The group consisting of, but not limited to, aryl phosphonates and phosphotriesters. A phosphate-containing linker may also convert the bonded oxygen to a nitrogen (bridging phosphoroamine). thioate), sulfur (bridged phosphorothioate) and carbon (bridged methylene phosphonate) The amino acid sequence can be modified by substituting the amino acid sequence.

[0041] The modified nucleotide may be an abasic site. A "site" is a nucleotide lacking an organic base. In a preferred embodiment, an abasic nucleotide The nucleotide further comprises a chemical modification described herein at the 2' position of the ribose. Preferably, the 2'C atom of ribose is selected from the group consisting of halogen, alkoxy, hydrogen, and aryloxy. , the group consisting of amino groups and aminoalkoxy groups, preferably 2'-hydrogen (2'-deoxy) ), 2'-O-methyl, 2'-O-methoxyethyl and 2'-fluoro It is substituted with a substituent.

[0042] In particular embodiments of the first aspect of the invention, the compound of formula (I) is optionally as provided above or below. In combination with any of the embodiments, at least two modified nucleotides , 2-amino-deoxyadenosine, 5-methyl-deoxycytidine, thiophosphate Nucleotides, LNA nucleotides, inosine, 8-oxo-deoxyadenosine and 5 - independently selected from the group consisting of fluoro-deoxyuracil and L-DNA nucleotides will be done.

[0043] In particular embodiments of the first aspect of the invention, the compound of formula (I) is optionally as provided above or below. In combination with any of the embodiments, at least two modified nucleotides Not an L-DNA nucleotide, 5-bromouridine or 5-iodouridine.

[0044] 2-amino-deoxyadenosine (2-amino-2'-deoxyadenosine or 2-amino Amino-dA (also known as dA) is a derivative derived from deoxyadenosine. -Amino-deoxyadenosine is (2R,3S,5R)-5-(2,6-diaminopurine) IUPAC name: (benzo-9-yl)-2-(hydroxymethyl)oxolan-3-ol , and has CAS number 4546-70-7.

[0045] 5-methyl-deoxycytidine (5-methyl-dCTP) is derived from deoxycytidine It is a derivative of ([[(2R,3S,5R)-5-(4-amino-5-methyl-2- Oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-hydr Its IUPAC name is [hydroxyphosphoryl]phosphonohydrogenphosphate, and its CAS number is 2 It has 2003-12-9.

[0046] Thiophosphate nucleotides contain thiophosphate (phosphorothioate) as the phosphate group. A thiophosphate is any nucleotide containing a thiophosphate (also known as a thiophosphate). It has CAS number 15181-41-6.

[0047] LNA nucleotides are modified RNA nucleotides in which the ribose moiety is substituted with the 2' oxygen atom. and modified with an extra bridging moiety connecting the 4' carbon.

[0048] L-DNA nucleotides contain the L enantiomer of ribose or deoxyribose It refers to a nucleotide that

[0049] In a further particular embodiment of the first aspect of the invention, In combination with any of the embodiments described above, thiophosphates, locked nucleic acids, , 2,6-diaminopurine, 5-methyl-deoxycytidine, inosine, 8-oxodeoxy independently selected from the group consisting of oxyadenosine and 5-fluoro-deoxyuracil The resulting cDNA contains at least three, at least four, or at least five modified nucleotides. It includes nucleotides, nucleotides, and L-DNA nucleotides.

[0050] In a further particular embodiment of the first aspect of the invention, In combination with any of the embodiments described above, the cDNA contains two LNA nucleotides. Contains leotide.

[0051] In a further particular embodiment of the first aspect of the invention, In combination with any of the embodiments described above, at least two modified nucleosides The tides are located in one or both single-stranded terminal loops of the cDNA. In certain embodiments, at least one modified nucleotide is located in one single-stranded terminal loop. and at least another modified nucleotide is located in another single-stranded terminal loop. do.

[0052] In a further particular embodiment of the first aspect of the invention, In combination with any of the embodiments described above, at least one modified nucleoside The nucleotide is located in one of the single-stranded terminal loops and contains at least another modified nucleotide. is located on one of the strands that form the stem region of the clDNA.

[0053] In a further particular embodiment of the first aspect of the invention, In combination with any of the embodiments described above, at least two modified nucleosides The tides are present on one or both strands that form the stem region of the c1 DNA.

[0054] In a further particular embodiment of the first aspect of the invention, In combination with any of the embodiments described above, at least one modified nucleoside If the modified nucleotide is present in one of the strands forming the stem region, the modified nucleotide The sequence is determined by the nucleotides 1 to 5 relative to the last nucleotide forming the loop. It is located within the chain region.

[0055] of the strand forming the stem region relative to the last nucleotide forming the loop The nucleotide at position 1 is the first nucleotide immediately after the last nucleotide of the single-stranded loop. nucleotides forming the stem region with the last nucleotide forming the loop The second nucleotide of one of the strands is immediately after the last nucleotide of the single-stranded loop. The same reasoning can be applied to the last nucleotide forming the loop. This applies to the nucleotides at positions 3, 4 and 5 relative to the base.

[0056] In a further particular embodiment of the first aspect of the invention, In combination with any of the embodiments described above, at least one modified nucleoside If the modified nucleotide is present in one of the strands forming the stem region, the modified nucleotide Nucleotides 1-10 relative to the last nucleotide that forms part of the target DNA sequence The strands are located within the region defined by

[0057] Form a stem region to the last nucleotide that forms part of the DNA sequence of interest The nucleotide at position 1 on one of the strands is the last nucleotide in this DNA sequence. It is the first nucleotide immediately following the first nucleotide. It is the last nucleotide that forms part of the desired DNA sequence. The nucleotide at position 2 on one of the strands forming the stem region for the nucleotide is the second nucleotide immediately following the last nucleotide of this DNA sequence. The theory is based on the 3rd to 10th nucleotides of the last nucleotide that forms part of the desired DNA sequence. This applies to nucleotides.

[0058] In particular embodiments of the first aspect of the invention, the compound of formula (I) is optionally as provided above or below. In combination with any of the embodiments, the stem region flanks the DNA sequence of interest. In a more particular embodiment, the restriction sites are a BsaI restriction site. , AfIII restriction site, HindIII restriction site, Nhel restriction site, and EcoRV restriction site In an even more particular embodiment, the restriction site is selected from the group consisting of BsaI Those skilled in the art will appreciate that the restriction site can be located at any distance between the loop and the DNA sequence of interest. It can be positioned.

[0059] In particular embodiments of the first aspect of the invention, the compound of formula (I) is optionally as provided above or below. In combination with any of the embodiments, the clDNA is primase / polymerase The primase recognition site may be present, for example, within the stem. In certain embodiments, the primase recognition site is contained within at least one loop. In particular embodiments of the first aspect of the invention, the method further comprises the steps of: In combination with any of the embodiments described above, the clDNA is It does not contain a polymerase priming site.

[0060] To stimulate amplification of the c1DNA of the present invention by including a primase recognition site, The use of primase is facilitated.

[0061] In particular embodiments of the first aspect of the invention, the compound of formula (I) is optionally as provided above or below. In combination with any of the embodiments, the clDNA is flanked by the gene of interest. In certain embodiments, the ITRs flank the expression cassette. In another specific embodiment, the ITRs are contained within the sequence of interest of the adapter. The ITRs can be at any suitable distance from the expression cassette. For example, the ITRs can be directly linked to the expression cassette, or can be 1 to 50 nucleotides, 5 The distance can be 0 to 200 nucleotides, or 200 to 1000 nucleotides. In certain embodiments, any of the embodiments provided above or below may be used. In combination with the target DNA sequence, the target DNA sequence is separated by inverted ends at a distance of 1–50 nucleotides. It contains an expression cassette flanked by inter-terminal repeats (ITRs).

[0062] As used herein, a "terminal repeat" or "TR" refers to at least one minimally required Any virus containing the region containing the required replication origin and palindromic hairpin structure. Contains a Rep-binding sequence. uence: "RBS") (also called RBE (Rep-binding element) and terminal resolution sites ( TRS) constitutes the "minimal required origin of replication" and therefore requires at least one A given stretch of a polynucleotide sequence contains an RBS and at least one TRS. The internal reverse complementary sequences, Tr, are typically called "inverted terminal repeats" or "ITRs." In viruses, the ITRs are involved in replication, viral packaging, and , involved in integration and proviral rescue.

[0063] In complex cDNA configurations, there may be three or more ITRs or asymmetric ITR pairs. Those skilled in the art will appreciate that the ITRs may be AAV ITRs or non-AAV ITRs. ITRs, or may be derived from AAV ITRs or non-AAV ITRs. ITR is a virus that can infect parvoviruses and depend viruses (e.g., canine parvovirus, bovine parvovirus, Parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B 19) or may function as an origin of SV40 replication. Functional SV40 hairpins can be used as ITRs. This allows for shortening, substitution, deletion, They can be further modified by insertions and / or additions. Parvoviridae viruses are divided into two subgroups: The Parvoviridae family infects vertebrates, while the Densovirinae subfamily infects invertebrates. Dependoparvovirus infects humans, primates, cattle, dogs, horses, and sheep. Adeno-associated viruses capable of replication in vertebrate hosts, including but not limited to For convenience, the term "clDNA vector" is used herein to refer to the virus family of AAVs. The ITR located 5' to (upstream of) the expression cassette is referred to as the "5'ITR" or It is called the "left ITR" and is located 3' to the expression cassette (downstream) of the clDNA vector. The positioned ITR is called the "3' ITR" or "right ITR."

[0064] In particular embodiments of the first aspect of the invention, the compound of formula (I) is optionally as provided above or below. In combination with any of the embodiments, the inverted terminal repeat is SEQ ID NO: 4 or the sequence This is the array number 5.

[0065] In particular embodiments of the first aspect of the invention, the compound of formula (I) is optionally as provided above or below. In combination with any of the embodiments, the closed linear DNA has the sequence of SEQ ID NO: 4. and / or a 5' inverted terminal repeat of the sequence of SEQ ID NO:5. In particular embodiments of the first aspect of the invention, the compound of formula (I) is optionally as provided above or below. In combination with any of the embodiments, the closed linear DNA comprises at least one "DD-ITR" includes DD-ITR. "DD-ITR" is a term used in the literature as described in Xiao X. et al., "A nov el 165-base-pair terminal repeat sequence e is the sole cis requirement for the ad eno-associated virus life cycle”,1997,J Virol., vol.71(2), pp.941-948. It is an ITR that contains a D element.

[0066] In particular embodiments of the first aspect of the invention, the compound of formula (I) is optionally as provided above or below. In combination with any of the embodiments, the DNA sequence of interest comprises an expression cassette. nothing.

[0067] The term "expression cassette" refers to a set of one or more promoter or enhancer elements and a gene encoding a desired gene. a gene or coding sequence encoding an RNA, miRNA, siRNA, or protein; An expression cassette refers to a DNA sequence that contains a sequence of genes that regulate the expression of a coding sequence, such as a transcription termination site. It may further include other elements.

[0068] In particular embodiments of the first aspect of the invention, the compound of formula (I) is optionally as provided above or below. In combination with any of the embodiments, the expression cassette may be an mRNA, miRNA, A, a eukaryotic siRNA or a protein-encoding sequence operably linked to the eukaryotic siRNA or protein-encoding sequence Contains a promoter.

[0069] In particular embodiments of the first aspect of the invention, the compound of formula (I) is optionally as provided above or below. In combination with any of the embodiments, the expression cassette comprises a eukaryotic transcription termination sequence. It also contains columns.

[0070] In particular embodiments of the first aspect of the invention, the compound of formula (I) is optionally as provided above or below. In combination with any of the embodiments, 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:

[0071] In particular embodiments of the first aspect of the invention, the compound of formula (I) is optionally as provided above or below. In combination with any of the embodiments, the cDNA may be present in a cell in vitro. It is a clDNA that does not exist.

[0072] As indicated above, in a second aspect, the present invention provides a method for treating a pulmonary arthritis, comprising administering to a subject a therapeutically effective amount of a compound of formula (I) or (II) ... This provides closed linear DNA.

[0073] The clDNA of the present invention can be used in host cells, particularly in DNA vaccines or gene therapy. DNA vaccines may be used to express infectious organism DNA in vitro. The DNA vaccine typically encodes a modified form of the These vaccines express selected proteins of the infectious organism and are usually protective. DNA vaccines are also being used as an immunotherapy approach for cancer. The vector may encode a tumor antigen in a locus.

[0074] DNA vaccines are available for fungi, human papillomavirus (HPV), HIV, HSV2 / HSV1, influenza virus (types A, B and C), poliovirus, RSV Virus, rhinovirus, rotavirus, hepatitis A virus, Norwalk virus group, Enterovirus, astrovirus, measles virus, parainfluenza virus, Chronic parotitis virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus Barr virus, adenovirus, rubella virus, human T-cell leukemia virus type 1 (HT LV-1), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus Rums, poxvirus, Marburg and Ebola, SARS-CoV-1, SARS -Viruses including CoV-2; Mycobacterium tuberculosis, chlamydia, gonorrhea, shigellosis, salmonella, Vibrio cholerae, Treponema pallidum, Pseudomonas, Bordetella pertussis, Brucella, Francisella tularensis, Helicobacter pylori, Helicobacter pylori, pathogenic Leptospira, Legionella pneumophila, Yersinia pestis, Streptococcus (types A and B), Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae ( b), Toxoplasma gondii, Campylobacter spp., Moraxella catarrhalis Bacteria, including Streptococcus aureus, Streptococcus donovanoides, and Actinomycosis; Candidiasis and Aspergillus fungal pathogens including cerebrospinal fluid disease; cestodes, trematodes, nematodes, amoebic dysentery, giardiasis, cryptococcosis Tosporidium, Schistosomiasis, Pneumocystis carinii, Trichomoniasis and Trichinella spiralis Cancer caused by pathogens, including but not limited to parasitic pathogens, including HIV-1, HIV-2, and allelic for treating or preventing a number of medical conditions, including but not limited to gas, toxicity, and infection. It may comprise a nucleic acid sequence encoding an antigen.

[0075] DNA vaccines are directed against viruses such as Adenoviridae (including, for example, human adenoviruses), herpes Sviridae (including, for example, HSV-1, HSV-2, EBV, CMV, and VZV); Papovaviridae (including, for example, HPV), Poxviridae (including, for example, smallpox and vaccinia) Seniors), Parvoviridae (including parvovirus B19), Reovirus family (including, for example, rotavirus), family Coronaviridae (including, for example, SARS-CoV-1 and and SARS, including SARS-CoV-2), Flaviviridae (e.g., yellow fever, Stone virus, dengue virus, hepatitis C virus and tick-borne encephalitis virus Picornaviridae (including poliovirus, rhinovirus, and hepatitis A virus) Togaviridae (including, for example, rubella virus), Filoviridae (including, for example, Marsh virus), Rubeolivirus and Ebola virus), Paramyxoviridae (e.g., Parainfluenza virus), measles virus, respiratory syncytial virus, mumps virus, and rabies virus), Ruminaceae (including, for example, rabies virus), Bunyaviridae (including, for example, hantaviruses) Orthomyxoviridae (including influenza A, B, and C), Toroviridae (including, for example, HIV and HTLV) and Hepadnaviridae (for example, B The virus may include nucleic acid sequences encoding antigens derived from members of the human immune system (including hepatitis viruses).

[0076] Antigens may be derived from pathogens that cause veterinary disease, particularly viruses such as reoviruses (AfV). Horse sickness or bluetongue virus) and herpesviruses (equine herpesvirus) Antigens can be derived from viral pathogens, including foot and mouth disease virus, tick-borne Encephalitis virus, dengue virus, SARS, West Nile virus and hantavirus The antigen may be derived from an immunodeficiency virus. V or feline immunodeficiency virus.

[0077] The clDNA produced by the process of the present invention also contains a nucleic acid sequence encoding a tumor antigen. Examples of tumor-associated antigens include the MAGE family (MAGE1, 2, 3 ), cancer testis antigens such as members of NY-ESO-1 and SSX-2, tyrosinase, g Differentiation antigens such as p100, PSA, Her-2 and CEA, E6 from oncogenic HPV types and / or mutated autoantigens such as E7 and viral tumor antigens. Further examples of specific tumor antigens include MART-1, Melan-A, p 97, β-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-4, MAG E-12, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, P 1A, EpCam, melanoma antigen gp75, Hker8, high molecular weight melanoma antigen, K 19, Tyr1, Tyr2, pMel17 gene family, c-Met, PSM (prostate Prostate gland mucin antigen), PSMA (prostate-specific membrane antigen), prostate secretory protein, α-fetoprotein Protein, CA125, CA19.9, TAG-72, BRCA-1 and BRCA-2 antibodies Hara is one example.

[0078] In addition, the process of the present invention can be used to generate other target genes, such as clDNA for use in gene therapy. For example, a patient may have a dysfunctional version of that gene. In cases where a person has a genetic disorder caused by DNA molecules can be used. Examples of such diseases include Duchenne muscular dystrophy. trophy, cystic fibrosis, Gaucher disease, and adenosine deaminase (ADA) deficiency Other diseases for which gene therapy may be useful include inflammatory diseases, AIDS, etc. Any disorder, including autoimmune, chronic and infectious diseases, cancer, neurological disorders, cardiovascular diseases (car Diabetic disease, hypercholesterolemia estemia), various blood disorders, including various anemias, thalassemia, and hemophilia; For the treatment of solid tumors, toxic peptides (i.e., ricin, diphtheria, genes encoding chemotherapeutic agents such as diptheria toxins and cobra venom factors Antisense to genes, tumor suppressor genes such as p53, and transforming oncogenes The gene encoding the mRNA sequence that is the tumor necrosis factor (TNF) Anti-cancer peptides and other cytokines, such as tumor necrosis factor (TNF), or tumor suppressor Transdominant-negative mutants of transforming oncogenes can be expressed.

[0079] Other types of therapeutic cDNA are also contemplated for production by the process of the present invention. They are transcribed into active RNA forms, such as small interfering RNA (siRNA). The clDNA can be produced, for example, by the process of the present invention.

[0080] In particular embodiments of the second aspect of the invention, In combination with any of the embodiments, the clDNA may be used in a DNA vaccine or a genetic It is intended for use in child treatment.

[0081] As described above, in a third aspect, the present invention provides a therapeutically effective amount of the closed linear D A pharmaceutical composition is provided that includes the NA and a pharmaceutically acceptable carrier or excipient.

[0082] As used herein, the phrase "therapeutically effective amount" refers to a dose that, when administered, is effective to treat the disease being treated. sufficient to prevent or alleviate to some extent one or more of the symptoms of the disease The specific amount of agent administered according to the present invention refers to the amount of clDNA administered. A. Surrounding the case, including route of administration, specific conditions being treated, and similar considerations This will of course depend on the particular circumstances.

[0083] The expression "pharmaceutical composition" includes both human and non-human animal compositions (i.e. The term "veterinary compositions" encompasses both compositions intended as pharmaceutical compositions and pharmaceutical preparations.

[0084] The expression "pharmaceutically acceptable carrier or excipient" means a pharmaceutically acceptable substance, composition, or Each component is meant to be compatible with the other components of the pharmaceutical composition. The ingredients must also be free of adverse toxicity, irritation, allergic reaction, immunogenicity, or other adverse events commensurate with a reasonable benefit / risk ratio contact with human or non-human animal tissues or organs without other significant problems or complications It must be suitable for use.

[0085] Examples of suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents or other liquid vehicles. , dispersing or suspending aids, surfactants, isotonicity agents, thickening stabilizers or emulsifiers, preservatives, solids binders, lubricants, etc. Any conventional excipient vehicle may be used, e.g., any desirable produce no biological effect or interact adversely with any other component of the pharmaceutical composition. Unless the substance or its derivatives are compatible with the invention by interacting in a manner that It is contemplated that the invention is within the scope of the invention.

[0086] The closed linear DNA, the pharmaceutically acceptable excipient, any additional components in the pharmaceutical composition of the present invention The relative amounts of each component will depend on the identity, size and / or condition of the subject being treated, and will vary depending on the route by which the composition is to be administered.

[0087] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include inert diluents, dispersants, Dispersing and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers These include, but are not limited to, colorants, coating agents, lubricants and / or oils. Excipients, such as sweetening agents and flavoring agents, may be present in the composition, according to the judgment of the formulator.

[0088] Pharmaceutical compositions containing closed linear DNA produced by the process of the present invention can be prepared, for example, by The pharmaceutical composition may be in any dosage form, such as solid or liquid. Administered by any suitable route, such as orally, parenterally, rectally, topically, intranasally, or sublingually. These can be used, for example, in topical formulations (ointments, creams, lipogels, hydrogels, etc.), eye drops, necessary to formulate the desired dosage form, such as aerosol sprays, injectable solutions, or osmotic pumps. The formulation contains essential pharmaceutically acceptable excipients.

[0089] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, phosphorus Dicalcium carbonate, calcium sulfate, sodium hydrogen phosphate, sodium phosphate, lactose Sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol Inositol, sodium chloride, dry starch, corn starch, powdered sugar and Combinations include, but are not limited to:

[0090] Exemplary granulating and / or dispersing agents include potato starch, corn starch, Tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum , citrus pulp, agar, bentonite, cellulose and wood products, sponge, cation exchange resin Fat, calcium carbonate, silicate, sodium carbonate, cross-linked polyvinylpyrrolidone cross-linked Vidon, sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (cross-linked sodium carboxymethylcellulose Roasted rice), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch Ingredients: water-insoluble starch, carboxymethylcellulose calcium, magnesium silicate Aluminum (Veegum), sodium lauryl sulfate, quaternary ammonium compounds and Combinations of these include, but are not limited to:

[0091] Exemplary binding excipients include starches (e.g., cornstarch and starch paste). ), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), natural and synthetic gums (e.g. , acacia, sodium alginate, Irish moss extract, panwar gum (pan war gum, ghatti gum, psyllium mucilage, carbo Hydroxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose Cellulose, cellulose acetate, polyvinylpyrrolidone, silicon aluminum magnesium ( Veegum) and larch arabinogalactan, alginate, oxidized polyethylene, Polyethylene glycol, inorganic calcium salt, silicic acid, polymethacrylate, water, alco Examples of suitable ion exchange media include, but are not limited to, ion exchange media ...

[0092] Exemplary preservatives include antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohols, and the like. Included may be choline preservatives, acid preservatives, and other preservatives. Exemplary antioxidants include: , alpha tocopherol, ascorbic acid, ascorbyl palmitate, stearic acid Ascorbyl, Ascorbyl Oleate, Butyl Hydroxyanisole, Butylated Hydroxyanisole Dihydroxytoluene, monothioglycerol, potassium pyrosulfite, propionic acid, gallic acid propionate propyl, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and Exemplary chelating agents include, but are not limited to, sodium sulfite and sodium bisulfite. Contains ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, edetate disodium, Dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, alcohol Examples include tartaric acid and edetate trisodium.

[0093] Exemplary buffers include citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, Um, calcium carbonate, calcium chloride, calcium citrate, sodium glubionate , calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate Calcium, calcium lactate, propionic acid, calcium levulinate, pentanoic acid, phosphate Calcium hydrogen, phosphate, tricalcium phosphate, calcium hydrogen phosphate, potassium acetate, Potassium chloride, potassium gluconate, potassium mixture, potassium phosphate dibasic, phosphoric acid Potassium monobasic, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride Sodium citrate, sodium lactate, sodium phosphate dibasic, sodium phosphate Thorium monobasic, sodium phosphate mixture, tromethamine, magnesium hydroxide, hydroxide Aluminum chloride, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohols, and combinations thereof.

[0094] Exemplary lubricants include magnesium stearate, calcium stearate, stearate, Allyl acetate, silica, talc, malt, glyceryl behenate ate), hydrogenated vegetable oil, polyethylene glycol, leucine, sodium lauryl sulfate These include, but are not limited to, hydroxybenzoates ...

[0095] In a fourth aspect, the present invention provides a nucleic acid sequence comprising at least two modified nucleotides according to the first aspect. The present invention provides a process for producing a cloned DNA containing a desired DNA sequence, the process comprising: a) generating a cloned DNA containing a desired DNA sequence; providing a template DNA comprising a sequence; b) amplifying DNA from the template DNA of step a) to obtain a strand containing repeats of the desired template DNA; a step of generating a repeat DNA fragment, wherein each of the repeat DNA sequences of interest is a restriction site; and step c) (c1) ligating the concatemer DNA with at least one restriction enzyme. contacting the DNA fragments, thereby forming multiple open double-stranded DNA fragments, each containing the desired DNA sequence. (c2) a step of generating NA fragments at each end of the open double-stranded DNA fragment. and attaching hairpin DNA adaptors, each of which is at least one of the adaptors. each having one modified nucleotide, or alternatively, an adapter bound to the DNA fragment. and (b) by which only one of the two modified nucleotides is obtained. d) producing closed linear DNA using the amplified DNA produced in step c); and and purifying the closed linear DNA produced in step (a).

[0096] In particular embodiments of the fourth aspect of the invention, In combination with any of the embodiments, the hairpin DNA adaptor may be In a more particular embodiment, the hairpin DNA adapter is In a more particular embodiment, the adaptor is 6 to 200 nucleotides in length. In another particular embodiment, the adaptor is at most 6 to 60 nucleotides in length. In another specific embodiment, the adapter is 10 to 60 nucleotides in length. It is 0 to 40 nucleotides.

[0097] In certain embodiments of the method of the fourth aspect, optionally the method provided above or below In combination with any of the methods, amplification is performed using random primers or primase. / primed using polymerase enzyme.

[0098] Amplify template DNA using primase / polymerase as the priming enzyme This produces amplified DNA with very high efficiency and fidelity. , which can then be processed to produce closed linear DNA suitable for therapeutic applications.

[0099] As used herein, the term "priming" refers to the act of priming an oligonucleotide on a polynucleotide template. This refers to the generation of a mononucleotide primer.

[0100] The term "primase / polymerase enzyme" refers to the archaeological-eukaryotic primase (AE) DNA-dependent primase / polymerase enzymes, such as enzymes from the P) superfamily These enzymes exhibit the ability to degrade a starting DNA strand containing dNTPs. Enzymes from this superfamily that can be used include, for example, Thermus thermophilus primers Human primase / polymerase (TthPrimPol) or human primase / polymerase (h sPrimPol, CCDC111, FLJ33167, EukPrim2 or hPri mPol1) is a primase / polymerase of Thermus thermophilus. "TthPrimPol" is a primer for the bacterium Thermus thermophilus, sequence of which is shown in SEQ ID NO: 1. The nucleotide and protein sequences are listed under NC_005835 and available in the NCBI Entrez database, such as WP_01 1173100.1 It is possible.

[0101] [Table 1]

[0102] In a particular embodiment of the fourth process of the invention, optionally In combination with any of the embodiments described above, the amplification in step (b) is A primase / polymerase enzyme selected from imPol or hsPrimPol is used. In certain embodiments, the primase polymerase enzyme is TthPrim In a more particular embodiment, the primase polymerase enzyme is SEQ ID NO: 1 at least 80%, at least 85%, at least 90%, or at least 95% % sequence identity to TthPrimPol of SEQ ID NO: 1, or a variant thereof Those skilled in the art will recognize any of the TthPrimPol constructs that maintain their primase activity. It will be understood that variants are suitable for use in the processes of the present invention.

[0103] In the present invention, the term "identity" refers to the degree of identity between two sequences when the sequences are optimally aligned. In an optimal alignment, the first sequence position is If the corresponding positions in two sequences are occupied by the same amino acid residue, then the sequences are The level of identity (or "percent sequence identity") between two sequences is expressed as a percentage of the sequence identity. ") is the ratio of the number of identical positions shared by the sequences to the size of the sequences (i.e., The percent sequence identity is measured as: percent sequence identity = (number of identical positions / total number of positions) × 100).

[0104] Quickly obtain optimal alignments and calculate identity between two or more sequences. Several mathematical algorithms for this are known and several software programs are available. Examples of such programs include Amino MATCH-BOX, MULTAIN, GCG, FASTA, and R for nucleic acid sequence analysis A preferred software analysis program is the OBUST program. LIGN, CLUSTAL W, and BLAST programs (e.g., BLAST 2.1, BL2SEQ and their later versions).

[0105] For amino acid sequence analysis, a BLOSUM matrix (e.g., BLOSUM45, BLOSUM50, BLOSUM62 and BLOSUM80 matrices), Gonne t matrix, or PAM matrix (e.g., PAM30, PAM70, PAM1 20, PAM160, PAM250 and PAM350 matrices) Used to determine identity.

[0106] The BLAST program searches for multiple sequences in a database (e.g., GenSeq). or use BL2SEQ to align the selected sequences between two selected sequences. BLAST provides an analysis of at least two amino acid sequences by either The program is preferably integrated into the operation of the BLAST program DUST or SEG The modification is preferably performed by a low-complexity filtering program such as a When using gap existence costs (or gap scores), the gap existence costs are preferably The gap parameter is set to approximately -5 to -15. BLAST programs and their underlying principles can be suitably used in Altschul et al., “Basic local alignment s search tool”, 1990, J.Mol.Biol, v.215, 403~41 Further explained on page 0. The specific percentage of identity is One or more ol enzymes that are still effective and thereby capable of priming the desired sequence. Conservative amino acid mutations are included in the sequence. Protein mutations also include those that involve one or more It is due to the insertion or deletion of several amino acids.

[0107] In particular embodiments of the process of the fourth aspect of the invention, optionally In combination with any of the embodiments described above, the process may be It is a cell-free in vitro process for producing NAs.

[0108] In particular embodiments of the process of the fourth aspect of the invention, optionally In combination with any of the embodiments described above, step (b) may include rolling stamping. It is circle amplification.

[0109] The term "rolling circle amplification" or "RCA" refers to the amplification of clDNA or double-stranded circular DNA. refers to a nucleic acid amplification method involving the amplification reaction of covalently closed DNA molecules such as In this case, the polymerase extends the primer around the closed DNA molecule. The polymerase displaces the hybridized copy and creates polynucleotides around the template. Continue nucleotide extension to obtain concatemer DNA containing tandem units of amplified DNA. These linear single-stranded products are then subjected to multiple hybridizations, primer serves as a basis for extension and strand displacement events, resulting in the formation of concatemeric double-stranded DNA products Thus, the concatemeric double-stranded DNA product contains multiple copies of each amplified single DNA unit. A person skilled in the art can easily identify the source of the virus by using general knowledge and / or the manufacturer's instructions. and how to adjust the conditions of the amplification process depending on the enzyme and the properties of the template being amplified. Depending on how the template DNA is generated, the concatemer DNA can be The amplified DNA sequence contains different sequences flanking it. For example, in concatemeric DNA, The repeated DNA sequences may be restriction sites, protelomerase target sequences, recombinase recognition sites, or may be adjacent to any combination of these.

[0110] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the amplification in step (b) may be performed by strand displacement. It is carried out using a DNA polymerase. The term "strand-displacing DNA polymerase" refers to a strand-displacing DNA polymerase that displaces the template D A DNA polymerase is used to carry out a 3'-end extension reaction while removing the double-stranded portion of the DNA. The strand-displacing DNA polymerase that can be used in the present invention is phi29 DNA polymerase. There is no particular limitation on the DNA polymerase as long as it has strand displacement activity. Depending on the type of polymerase selected in this way, the skilled artisan may It is understood that the reaction conditions for the reaction can be appropriately set. For example, phi29D If NA polymerase is used, the reaction temperature is 25°C to 35°C. It can be implemented in

[0111] Thus, in certain embodiments, the strand-displacing DNA polymerase is DNA polymerase, Bst DNA polymerase, Bca (exo) DNA polymerase, Escherichia coli Klenow fragment of DNA polymerase I, Vent (exo) DNA polymerase, D Consists of eepVent (exo) DNA polymerase and KOD DNA polymerase In a more particular embodiment, the strand-displacing DNA polymerase is selected from the group consisting of phi29D In an even more particular embodiment, the strand-displacing DNA polymerase is and phi29 DNA polymerase. As disclosed in US Pat. No. 1,000,997, those skilled in the art can develop textures with improved properties. Understand how to obtain DNA polymerase.

[0112] In particular embodiments of the process of the fourth aspect of the invention, optionally In combination with any of the embodiments described above, the template DNA is a closed linear The template is selected from a template DNA or a circular double-stranded template DNA.

[0113] As used herein, the term "circular double-stranded DNA" refers to a covalently closed double stranded DNA. Refers to a single-stranded DNA molecule.

[0114] In particular embodiments of the process of the fourth aspect of the invention, optionally In combination with any of the preceding embodiments, step (a) comprises: - a plasmid vector containing at least two restriction sites flanking the DNA sequence of interest and at least one restriction enzyme, thereby forming an open fragment containing the desired DNA sequence. Create double-stranded DNA and insert a hairpin DNA adapter into the open double strand containing the DNA sequence of interest. Alternatively, step (a) may be carried out by: - a protelomerase containing at least two protelomerase target sequences flanking a DNA sequence of interest; contacting the plasmid vector with protelomerase (particularly TelN); This produces a closed, linear template DNA containing the desired DNA sequence, called template DNA. This is done by obtaining

[0115] As used herein, a "plasmid vector" refers to a vector that transports another nucleic acid to which it is linked. A circular double-stranded nucleic acid that can be delivered and autonomously replicate within a cell independently of chromosomal DNA Thus, a plasmid vector is a molecule that contains all the components necessary for replication in a cell, especially a bacterial cell. Contains all the required elements.

[0116] The use of restriction enzymes and ligases (for joining purposes) is routine in molecular biology. Therefore, the skilled artisan will know how to adjust the reaction conditions depending on the enzyme being used and the target enzyme. Know which restriction enzymes should be used depending on the restriction site being used.

[0117] Those skilled in the art will also appreciate that some restriction enzymes generate DNA overhangs (sticky ends), It is understood that the others do not produce this (blunt ends). Both types The following restriction enzymes can be used in the methods of the present invention. that it can be ligated to open double-stranded DNA with cohesive ends (cohesive end ligation) It is understood that open double-stranded DNA with blunt ends can also be synthesized using, for example, Taq polymerase. dA tail by the process of adding a terminal 3' deoxyadenosine nucleotide using The nucleotide sequence can be ligated to an adapter with an overhanging T.

[0118] In particular embodiments of the process of the fourth aspect of the invention, optionally In combination with any of the embodiments described above, the restriction enzyme may be blunt-ended or In a more particular embodiment, at least two nucleotides flanking the DNA sequence of interest are generated. contacting a plasmid vector containing at least two restriction sites with at least one restriction enzyme; This allows for the creation of open double-stranded DNA with sticky ends or open double-stranded DNA with blunt ends. Generate A.

[0119] Adapters ligated to both ends of the open double-stranded DNA to form de clDNA may be the same adapter or different adapters.

[0120] In particular embodiments of the process of the fourth aspect of the invention, optionally In combination with any of the embodiments described above, the hairpin DNA adapter , and at least one restriction site. In a more particular embodiment, the restriction site is a BsaI restriction site. site, AfIII restriction site, HindIII restriction site, Nhel restriction site, and EcoR In an even more particular embodiment, the restriction site is selected from the group consisting of Bs V restriction sites. In a particular embodiment, the restriction sites are Bbsl and BseRI restriction sites. is selected from.

[0121] In particular embodiments of the process of the fourth aspect of the invention, optionally In combination with any of the embodiments described above, the hairpin DNA adapter In a more particular embodiment, the hairpin DNA adapter does not contain a primase recognition site. The data does not contain the sequence XTC.

[0122] In more particular embodiments, any of the embodiments provided above or below may be used. When combined with either the nucleotide sequence, the hairpin DNA adapter binds to the protelomerase target sequence. In an even more particular embodiment, the hairpin DNA adapter comprises a protelomeric Contains a portion of the target sequence.

[0123] As used herein, "protelomerase" refers to a covalently closed linear D A template containing a protelomerase target site can be cleaved and recombined to generate NA molecules. Therefore, protelomerase is a polypeptide that acts as a catalyst for DNA cleavage and ligation. Enzymes with protelomerase-type activity also have a gating function. They have also been described as resolvase (e.g., Borrelia burgdorferi). The typical substrate for protelomerase is circular double-stranded DNA. If it contains the target site, the enzyme cuts the DNA at this site and ligates the ends to form It is possible to generate linear double-stranded covalently closed DNA molecules. A given polymerase catalyzes the creation of a closed linear DNA from a template containing a target site. The potency of the polypeptide can be assessed using any suitable assay described in the art. It can be determined as follows.

[0124] Examples of suitable protelomerases for use in the processes of the present invention include Halomonas phiHAP-1 from Aquamarina, PY54 from Yersinia enterolytica , phiKO2 from Klebsiella oxytoca and VP882 from Vibrio species, large intestine N15 derived from fungi, or any variant thereof.

[0125] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described herein, the protelomerase may comprise a sequence bacteriophage N15 TelN of number 2, or at least 80 for SEQ ID NO: 2 These variants include sequences with % identity.

[0126] A "protelomerase target sequence" is any DNA sequence that exists in the template DNA. This allows the enzyme activity of protelomerase to convert it into closed linear DNA. In other words, the protelomerase target sequence forms a covalently closed linear DNA strand. It is required for the breakage and rejoining of double-stranded DNA by protelomerase in order to Typically, the protelomerase target sequence is any double-stranded DNA sequence with a two-fold greater rotational symmetry. and any perfect palindrome sequence described herein as a perfect inverted repeat. Contains columns.

[0127] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, at least two prothrombins The nucleotide sequence of the target DNA fragment contains a perfect inverted repeat DNA sequence.

[0128] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, a protelomerase target sequence The sequence is SEQ ID NO: 3, or at least 80%, at least 85%, or at least and sequences with at least 90%, or at least 95% sequence identity. Includes these variants.

[0129] The length of the perfect inverted repeat varies depending on the specific organism. In various mesophilic bacteriophages, the perfect inverted repeat is 14 base pairs in length. In most cases, perfect inverted repeats are 22 base pairs or more in length. In E. coli N15, the central perfect inverted palindrome is flanked by perfect inverted sequences. , i.e. it forms part of a larger incomplete inverted palindrome.

[0130] The protelomerase target sequence used in the present invention is at least 14 base pairs in length. It preferably contains a double-stranded palindrome (perfect inverted repeat) sequence.

[0131] The perfect inverted repeat may be flanked by additional inverted repeat sequences. can be perfect or imperfectly repeated, i.e. it is perfectly symmetrical or partially symmetrical. The flanking inverted repeats may be contiguous with the central palindrome, or The protelomerase target sequence may be at least 14 bases in length. It may contain imperfect inverted repeats, including pairs of perfect inverted repeats.

[0132] The protelomerase target sequence comprising the sequence of SEQ ID NO: 3 or a variant thereof is SEQ ID NO: 2 and the E. coli N15 TelN protelomerase and its variants. I wish.

[0133] A variant of either the palindromic sequence or the protelomerase target sequence may be Variants include truncations, substitutions, or deletions to the native sequence. A variant sequence is an arbitrary sequence whose presence in the template DNA can lead to protelomere formation. The enzymatic activity of the enzyme allows the conversion of the closed linear DNA. This can be easily determined by using an appropriate assay for the formation of NA. Any suitable assay in the art may be used. The assay demonstrated protelomerase binding and its activity comparable to that observed with the native sequence. Examples of preferred variants of the palindromic sequences described herein are: The DNA fragments must maintain a perfect repeat structure and maintain a state in which the closed linear DNA can be formed. The cleaved palindromic sequence is included. However, variant protelomerase targets A sequence is a perfect palindrome if it can act as a substrate for protelomerase activity. The system may be modified so that it no longer stores the data.

[0134] Based on the structural principles summarized above, one skilled in the art can easily design suitable protelomers for use in the present invention. It should be appreciated that enzyme target sequences can be readily identified. Candidate protelomerase targets The sequences were tested for their ability to promote the formation of closed linear DNA using the assay described above. The antibodies can be screened for potency.

[0135] In particular embodiments of the process of the fourth aspect of the invention, optionally In combination with any of the embodiments described above, the template DNA is a DNA of interest. In the case where the template DNA is a circular double-stranded DNA containing a sequence, step (a) comprises A plasmid vector containing at least two adjacent recombinase recognition sites and a site specific This is carried out by contacting the gene with a heterologous recombinase (more specifically, Cre recombinase). will be done.

[0136] The action of a site-specific recombinase on the plasmid vector results in the fusion of two recombinases. This causes recombination of the recombinase recognition sites in the plasmid vector. A small circular double-stranded DNA is generated containing the desired DNA sequence located between the recognition sites. can be.

[0137] "Site-specific recombinase," as used herein, refers to a recombinase that recognizes a site-specific recombination between specific DNA sequences recognized by enzymes known as It refers to a family of enzymes that mediate site-specific recombination. Examples of site-specific recombinases are Cre recombinase and Recombinase, Flp recombinase, λ integrase, γ-δ resolvase, Tn3 Resolvase, Sin resolvase, Gin invertase, Hin invertase, T n5044 resolvase, Tn3 transposase, sleeping beauty transposase, IS607 transposase, Bxb l integrase, wBeta Integrase, BL3 integrase, phiR4 integrase, All l 8 in Tegulases, TGI integrases, MRU integrases, phi370 integrases ze, SPBc integrase, SV1 integrase, TP901-1 integrase, phiRV integrase, FC1 integrase, K38 integrase, phiBT integrase and phiC31 integrase. stomach.

[0138] A "recombinase recognition site" is a nucleoside that is recognized by a site-specific recombinase. Recombinase recognition sites are non-recombinase recognition sites that can serve as substrates for recombination events. Non-limiting examples include FRT, FRT11, FRT71, attp, att, rox, and loxP, lox511, 1oχ2272, 1oχ66, 1oχ71, loxM2 and l Examples of lox sites include ox5171.

[0139] Those skilled in the art will understand, using their general knowledge, that a site-specific recombinase is a specific recombinase. The enzyme recognizes the enzyme recognition site, thereby responding to the recognition sequence contained within the plasmid vector. Therefore, different recombinases are required to generate circular double-stranded template DNA from the plasmid vector. I understand that I have to use enzymes.

[0140] In particular embodiments of the process of the fourth aspect of the invention, optionally In combination with any of the embodiments described above, a site-specific recombinase is a Cre recombinase. In a more particular embodiment, the recombinase recognition site is In an even more particular embodiment, the site-specific recombinase is Cre recombinase. The recombinase recognition site is loxP.

[0141] Thus, in certain embodiments of the process of the fourth aspect of the invention, optionally In combination with any of the embodiments provided below, steps (b) to The amplified DNA obtained from the PCR reaction is a concatemer DNA containing repeats of the target DNA sequence. Each of the repeated DNA sequences contains a restriction site, a protelomerase target sequence, and and / or flanked by recombinase recognition sites.

[0142] Those skilled in the art will appreciate that when a restriction enzyme is used to generate the template c1DNA, the c1DNA generated in step (b) Subsequent use of the same restriction enzymes to generate cDNA from the amplified DNA The hairpin DNA adapter used in step (a) to generate the template c1DNA can be The adapter may be similar to or different from the one used in step (c). The adapter is preferably different. It is.

[0143] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, the process is a closed linear expression This is a process for creating cassette DNA.

[0144] In particular embodiments of the process of the fourth aspect of the invention, optionally In combination with any of the embodiments described above, step (a) may further comprise: A plasmid vector containing at least two restriction sites flanking the A sequence and at least A restriction enzyme is then contacted with the target DNA sequence to form an open double-stranded DNA fragment. Prepare a hairpin DNA adapter and attach it to both ends of an open double-stranded DNA containing the DNA sequence of interest. and step (c) is carried out by binding to one end of the concatemer DNA and at least one Each fragment contains a target DNA sequence. (c2) generating a plurality of open double-stranded DNA fragments comprising the DNA fragments according to the first aspect of the present invention; ligating the hairpin DNA adapters to both ends of the open double-stranded DNA fragments; In more particular embodiments, the restriction enzyme generates sticky or blunt ends. If the restriction enzyme generates blunt ends, the resulting fragments will be fused to adapters containing blunt ends. This can be coupled to a DA tail as explained above, or alternatively, it can be DA tailed and An overhanging T can be used to connect to the adapter.

[0145] In particular embodiments of the process of the fourth aspect of the invention, optionally In combination with any of the embodiments described above, the template DNA may be a circular double-stranded template DNA containing adjacent DNA sequences of interest, and step (a) At least two restriction sites flanking the DNA sequence of interest Plasmid vectors containing two recombinase recognition sites and site-specific recombinases (e.g., and more particularly Cre recombinase), and step (c) is carried out by contacting the c1) contacting the concatemer DNA with at least one restriction enzyme, thereby obtaining the desired DNA fragment; (c2) generating a plurality of open double-stranded DNA fragments each containing the A sequence; ligating a hairpin DNA adaptor according to an embodiment to both ends of the open double-stranded DNA fragment; This is carried out by:

[0146] In particular embodiments of the process of the first aspect of the invention, optionally In combination with any of the embodiments described above, step (a) may further comprise: At least two promoter sequences flanked by at least two restriction sites flanked by A sequences A plasmid vector containing a telomerase target sequence and protelomerase (more specifically, T e1N), and step (c) is carried out by contacting (c1) concatemer DNA with at least Each fragment contains a desired DNA sequence. (c2) generating a plurality of open double-stranded DNA fragments; and (c3) forming a hairpin DNA adapter according to the first embodiment. This is done by ligating a primer to both ends of an open double-stranded DNA fragment.

[0147] In a fifth aspect, the present invention relates to a closed linear D-type olefin obtainable by a process according to the fourth aspect. Provide NA.

[0148] The seventh and eighth aspects of the present invention comprise at least two modified oligonucleotides and a carrier. The term "carrier" refers to a composition comprising a clDNA of the present invention. It may also be a viral vector. A "viral vector" is a vector that delivers exogenous genetic material to a cell. The present invention relates to a modified virus that functions as a vehicle for the introduction of the virus into the nucleus of a host. In the present paper, a "non-viral vector" is defined as a vector that functions as a carrier for delivering the cDNA. Any substance other than that derived from a virus. Non-viral vectors include nanoparticles, liposomes, and Examples include vesicles, vesicles, and polymers.

[0149] If the non-viral vector is a polymer (e.g., a polycationic polymer), The complexes and polymers formed by lDNA are called "polyplexes." The "plex" is formed by electrostatic interactions between DNA and cationic polymers (cationomers). These vectors have attracted much attention as a safe and versatile alternative to viral vectors. are.

[0150] Particularly suitable polymers in the sense of the present invention are those disclosed in EP 1 859 812. Some of these polymers are polycationic polymers such as polyethylene glycols. In certain embodiments, the present invention provides a polycationic polymer based on ethylene glycol. The polyplex of eight embodiments contains a polymer with formula I: [ka]

[0151] The present invention also relates to the above-described clDNAs and clones of the present invention for use in therapy or diagnosis. A carrier or composition comprising the polyplex is provided.

[0152] For purposes of this invention, the terms "obtained," "obtained," and equivalents are used interchangeably. are used interchangeably and in either case the expression "obtained" is used interchangeably with the expression "obtained" All embodiments provided under the first and fourth aspects of the present invention include It is also an embodiment of the closed linear DNA of the fifth aspect of the invention.

[0153] In a sixth aspect, the present invention provides a method for the preparation of a nucleotide sequence comprising at least one modified nucleotide, a ligase, and optionally for generating clDNA containing hairpin DNA adapters containing instructions therefor Provide a kit.

[0154] This kit provides a method for ligating any given DNA sequence of interest through the action of a ligase enzyme. The cDNA of the present invention is produced by ligating the adapters present in the cDNA. All of the embodiments relating to the adapter of the fourth aspect of the present invention may also be used for This is also meant to apply to the adapter of the kit according to the sixth aspect of the invention.

[0155] Throughout this specification and claims, the term "comprises" and variations of this term refer to other technologies. It is not intended to exclude technical features, additives, ingredients, or steps. Further objects, advantages and features of the present invention are set forth in this description. These and other features will become apparent to those skilled in the art upon examination of the present invention or may be learned by practice of the invention. The drawings are provided by way of example only and are not intended to limit the invention. Related reference signs in parentheses in the claims are intended to aid in the understanding of the claims. is merely an attempt to increase the scope of the claims and should be interpreted as limiting the scope of the claims. Furthermore, the present invention does not encompass the specific and preferred embodiments described herein. Cover all possible combinations. [Example]

[0156] Example 1: Synthesis of clDNA with at least two modified nucleotides

[0157] Synthesis of hairpin DNA adapters Standard phosphoramidite chemistry (Beaucage SLet et al., 1981) According to the method described above, 8-oxo-deoxyadenosine (8-oxo-dA), 5-fluoro-deoxy 5-FLU, inosine, thiophosphate nucleotides, or locked nucleic acids (L Hairpin DNA containing at least two modified nucleotides, such as NA nucleotides A adapter was synthesized.

[0158] Briefly, phosphoramidite synthesis begins at the most distant 3' nucleotide and continues to the most distant 4' nucleotide. A series of four steps is repeated until the selected 5' nucleotide is attached. The reaction proceeds through a cycle consisting of (i) deprotection, (ii) coupling, (iii) oxidation, and (iv) (iii) and capping (iv).

[0159] This cycle is repeated for each nucleotide in the sequence. The oligonucleotide may, for example, have a 3' end still attached to the CPG and a trityl group It exists as a 25-mer with a 5'-terminal protected by . In addition, the ring structure of the bases is Protecting groups remain on three of the four bases to maintain compatibility. Protecting groups are on A and C. The benzoyl on G and the N-2 isobutyryl on G. Thymidine does not require any protecting groups. The complete synthesis is detritylated, the controlled pore glass is cleaved, and the 3' and 5' ends are ´ Leaving a hydroxyl at the end. At this point, hot ammonium hydroxide is used to base The oligo (base and phosphate) is deprotected by hydrolysis. The final product is a functional single-stranded D It is an NA molecule.

[0160] [Table 2]

[0161] The corresponding hairpin DNA adapters containing natural oligonucleotides were also used for comparison. A list of the synthesized adapters is provided in Table 3. At the end of the synthesis, the oligonucleotides were cleaved from the support and the protecting groups were removed. Purification steps (e.g., PAGE, HPLC, and / or RNase-free HPLC) was used to separate the full-length product from the truncated sequences.

[0162] [Table 3]

[0163] Preparation of clDNA containing modified oligonucleotides Next, the hairpin DNA adapters of SEQ ID NOs: 6 to 15 obtained in the above section were ligated. cDNA is prepared by binding to a double-stranded DNA fragment containing the target sequence through the action of enzymes. (See Figure 1.) Figure 2 shows the cl prepared using the hairpin adapter. The preparation scheme for DNA is shown. As shown in the figure, endonuclease restriction sites (A ) to cleave a DNA fragment containing the desired sequence flanked on both sides with specific restriction endonucleases. (B) and ligated with the desired hairpin adapter (C).

[0164] The sequences of interest in these specific examples were ligated using adapters of SEQ ID NOS: 6-13. a sequence encoding the luciferase enzyme (for ligation), or a restriction site, In this example, adapters flanking the BsaI restriction site (SEQ ID NOs: 14 and 15) were used to clone the green fluorescent protein (green fluorescent protein for irrigation) Therefore, all constructs prepared in this example contained Regarding the DNA fragment to which the hairpin adapter is ligated, it is flanked on both sides by BsaI overhangs. Luciferase (with additional sequences such as the corresponding promoter and enhancer) In Figure 2, exemplary hairpin adapters on both sides were Oligo 37 (SEQ ID NO: 7), which contains 5 phosphorothioate nucleotides. (shown in italics in Figure 2). After adapter ligation, exonuclease The samples were treated with lyase and Triton-114 to remove endotoxins. , and purified.

[0165] This scheme involves the use of different hairpin adapters (oligos of SEQ ID NOs: 6 to 15) as shown in Figure 2. It is flanked on both sides by BsaI overhangs so that it Two sequences containing sequences encoding luciferase or Gfp (along with additional sequences such as enhancers) This applies to all the cDNAs in the examples obtained by ligating double-stranded DNA fragments. Each cDNA contained identical hairpin adaptors on both sides of the double-stranded DNA fragment. The resulting clDNAs were designated oDNAs and were connected to the hairpin adaptors used in their preparation. These were numbered (see Table 3), i.e., oDNA15, oDNA3 7, oDNA4, oDNA28, oDNA29, oDNA17, oDNA19, oDNA 22, oDNA21 and oDNA41. oDNA37, oDNA28, oDNA2 9, oDNA19, oDNA22, oDNA21 and oDNA41 are modified nucleotides oDNA15 is the naturally occurring counterpart of oDNA37. NA4 is the naturally occurring counterpart of oDNA28 and oDNA29. NA17 is the naturally occurring counterpart of oDNA19 and oDNA22.

[0166] Protocol for preparing clDNA using customized adapters An example of a specific protocol that can be followed to obtain the oDNA is provided below. This protocol uses modified nucleotides ( Briefly, pDNA can be prepared by adding a protein to a DNA fragment. Using ribosomal enzyme, the corresponding promoter and enhancer A sequence containing the sequence of interest (which also contains a sequence encoding GFP along with additional sequences, such as a nucleic acid sequence encoding a GFP vector) is prepared. The eGFP plasmid in column number 16 and the protelomerase target sequence (see Figure 14) (b) to obtain a cDNA fragment containing the desired sequence flanked by endonuclease restriction sites. Rolling circle amplification (RC) using TthPrimPol and Phi29 was performed. This cDNA was amplified by the method described in A). The resulting concatemers were purified and the corresponding recombinant DNA fragments were amplified. Treatment with a restriction enzyme (e.g., BSal) yields hairpin adapters containing modified nucleotides. It was combined with puta.

[0167] A. Protocol for obtaining clDNA from plasmid DNA [Table 4] [Table 5]

[0168] 1.1 TelN digestion Digest the eGFP plasmid with TelN enzyme for 2 hours at 30°C and 10 minutes at 75°C. If several reactions are performed simultaneously, scale up accordingly. do. [Table 6]

[0169] 1.2 Removal of the skeleton 1.2.1 Kpn I and Hind III digestion The product of the final step was digested with Kpn I and Hind III for 1 hour at 37°C. The samples were then inactivated at 65°C for 15 minutes. Several reactions were carried out simultaneously. If so, scale up accordingly. [Table 7]

[0170] 1.2.2 Exo III Digestion The fragments were digested with Exo III at 37°C for 1 hour and inactivated at 75°C for 10 minutes. If reactions are performed simultaneously, scale up accordingly. [Table 8]

[0171] 1.3 Purifying cDNA using gel filtration chromatography and isopropanol R 1.3.1 Gel filtration chromatography Buffer A: 10 mM Tris-HCl, pH 7.5 Column: Vestarose 6 FF 153mL Sample: 28ml Flow: 60cm / h Recovered fraction 20mAU-20mAU, 40mL CIP: 1M ​​NaOH + deionized water Storage: Pure water

[0172] 1.3.2 Endotoxin removal and isopropanol precipitation As shown in Table 6, 3M sodium acetate and 15% Triton-114 were added to the final Add to the sample from step 1 and vortex to mix. Incubate the sample at 4°C for 5 minutes. Then, the mixture is centrifuged at 12000 g for 20 minutes at 25°C. The supernatant is collected and an equal volume of isopropanol is added to the supernatant and mixed thoroughly. The sample was then centrifuged at 12000g for 20 minutes and the supernatant was collected. Finally, the precipitate is suspended in 10 mM Tris-HCl (pH 7.5). [Table 9]

[0173] Enzyme digestion, gel chromatography, Triton 114 treatment, and isopropanol precipitation After three steps, eGFP_BSaI_clDNA was successfully produced. The percent DNA homogeneity of the samples was 97%. The toxin level is less than 10EU / mg.

[0174] B. Extraction of oDNA (clDNA containing modified nucleotides) from clDNA by PCR profit This experiment was performed using the Trueprime-RCA kit (TthP as DNA primase). based on two enzymes, rimPol and Phi29 DNA polymerase) and T eGFP_BSaI_clDNA derived from the above section was synthesized by eIN. The cloned adaptor is designed to generate a clDNA containing the cloned adaptor. [Table 10] [Table 11]

[0175] 1.1 RCA Mix constantly by pipetting, do not vortex. Transfer 10 μL of clDNA (≥1 ng / μL) to a clean tube. Add 10 μL of Buffer D and incubate at room temperature for 3 minutes. Neutralize the reaction by adding 10 μL of Buffer N to each tube. Keep samples at room temperature until use. * . Prepare the amplification mix by adding the components in the order listed in the table below. Incubate at 30°C for 3 hours ** Inactivate the reaction at 65°C for 10 minutes. do. ◇ Cool to 4°C. For short term storage, keep at 4°C, or for long term storage, keep at -2°C. Store the amplified DNA at 0°C. ( * ) It is highly recommended to perform the amplification reaction immediately after denaturing the sample. ( ** If higher amplification yields are required, the incubation time can be increased to 6 hours. It can be added.

[0176] If several reactions are carried out simultaneously, scale up accordingly. [Table 12]

[0177] 1.2 Purify the RCA products (concatamers) with isopropanol (as above) do

[0178] 1.3 Purify the RCA products (concatamers) using an Axygen kit (optional).

[0179] If your sample is less than 100 μL, you can also use an Axygen kit to purify clDNA. The protocol is described below and the bottle containing the buffer is They are classified as explained below: 1) Add 2x the sample volume of Buffer DE-B and mix. 2) Place the Miniprep column into a 2 mL microfuge tube. Transfer the sample to the column. Centrifuge at 12,000 x g for 1 minute. 3) Discard the filtrate from the 2 mL microfuge tube. Place the tube back into the microfuge and add 500 μL of Buffer W1. Centrifuge for 30 seconds. 4) Discard the filtrate from the 2 mL microfuge tube. Place the tube back into the microfuge and add 700 μL of Buffer W2. Centrifuge for 30 seconds. 5) Discard the filtrate from the 2 mL microfuge tube. Place back into the microfuge tube. Add a second 700 µL aliquot of Buffer W2 and centrifuge at 12,000 x g for 1 minute. 6) Place the Miniprep column in a clean 1.5 mL microfuge tube (provided). To elute the DNA, add 50 μL of 10 mM Tris-HCl (pH 7.5 Add the 100ml of PBS to the center of the membrane. Let it sit at room temperature for 1 minute. Centrifuge at 12,000 x g for 1 minute. Separating the heart.

[0180] 1.4 Oligo denaturation and annealing Denature the oligo (e.g., oligo 21 or oligo 41 in Table 3) at 95°C for 10 minutes and The reaction mixture was allowed to anneal naturally at room temperature for 30 minutes. Scale up accordingly. [Table 13]

[0181] 1.5 Oligophosphorylation (optional, if the oligo is already phosphorylated, skip this step) (omitted) Oligophosphorylation at 37°C for 1 hour [Table 14]

[0182] 1.6 BsaI digestion BsaI digestion for 2 hours at 37°C and inactivation at 75°C for 10 minutes [Table 15]

[0183] 1.7 Purify the BsaI-digested RCA product with isopropanol (as above)

[0184] 1.8 Purify the BsaI-digested RCA product using the Axygen kit (as described above) , optionally with a sample of 100 μL or less)

[0185] 1.9 T4 ligation T4 ligation is carried out overnight at 16°C and inactivated at 75°C for 10 minutes. [Table 16]

[0186] 1.10 Advanced Golden Gate Assembly (Optional) Traditional ligation methods require several cloning steps to generate the desired construct. Each step involves inserting a single DNA fragment into a donor plasmid or PC Transfer the R product into the recipient vector.

[0187] During Golden Gate cloning, up to 15 It is possible to assemble all the plasmid donors, the recombinant vectors, and the recombinant vectors in a single tube. Pipette the spirient vector, type IIS restriction enzyme, and ligase together, then perform thermal cycling. Cloning is performed by incubating the mix in a cooler. We also propose to generate oDNA using Golden Gate assembly. The system and conditions are explained in Table 14 and Table 15, respectively. If reactions are performed simultaneously, scale up accordingly.

[0188] [Table 17] [Table 18]

[0189] 1.11 Unexpected DNA digestion The fragments were digested with Exo III at 37°C for 1 hour and inactivated at 75°C for 10 minutes. If reactions are performed simultaneously, scale up accordingly. [Table 19]

[0190] 1.12 Purify oDNA using isopropanol (as above)

[0191] 1.13 Purify DNA using an Axygen kit (as above, optionally with a sample) (The volume is less than 100 μL.) Oligos 21 and 41 were used to successfully generate eGFP_BSaI_oDNA: [Table 20]

[0192] A similar procedure was used to generate the Luc plasmid with SEQ ID NO: 17 (flanked by BsaI restriction sites). (containing the luciferase-encoding sequence adjacent to the oligos 15, 37, 4, 28, and 29) , 17, 22, 37, 28, 29, 19 and oligo 22 (see Table 3) Again using the same procedure, a Luc-I cDNA having SEQ ID NO: 18 was prepared. TR plasmid (the sequence of interest encodes luciferase flanked by BsaI restriction enzymes) See Figure 15, which additionally contains ITRs flanking the sequence, and a protelomerase target sequence. (See references below) as well as c1DNA starting from oligo 37. oDNA37ITR (total 5.6 μg) was obtained (Figure 16 (agarose gel electrophoresis) (See

[0193] The stability of the generated clDNA was confirmed by the International Conference on Harmonisation (ICHarmonisation) of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICP). In accordance with the International Conference on Harmonization (ICH), All synthetic cDNAs containing modified nucleotides were tested at -20°C for 6 months. The results showed stable values ​​suitable for use in gene therapy.

[0194] Standard procedures (especially agarose gel electrophoresis, grayscale analysis, anion exchange chromatography) The obtained cDNA was analyzed by chromatography-HPLC and Sanger sequencing. All clDNAs were evaluated for quality in terms of purity, peak resolution, and sequence verification. It was found that they exhibited good quality characteristics. The quality control results for oDNA41 and oDNA42 are shown in Figures 3, 4, and 5, respectively.

[0195] Example 2: Functional performance of clDNA containing at least two modified nucleotides

[0196] oDNA15, oDNA37, oDNA4, oDNA28, and oDNA obtained in Example 1 A29, oDNA17, oDNA22, oDNA37, oDNA28, oDNA29, o The clDNAs designated as DNA19 and oDNA22 were transfected onto HaCaT cells. and luciferase activity was determined.

[0197] Transfection: 10% fetal bovine serum (Hyclone, no. SV30160 DMEM High Glucose (Gibco No. 61965-059) containing 0.03HI Use HaCaT cells (#EP-CL-0090, Elabscience, batch The cells were trypsinized the day before transfection. The cells were then treated with PBS and placed in a 96-well plate (Greiner Bio-one number 655090). 6,000 cells were plated per well in a final volume of 100 μL per well. The plate was incubated at 5% CO2 and 37°C. The medium was aspirated immediately before transfection. The solution was removed from the well and added at 90 μL / well. EI (jetPEI® Polyplus No. 101-10N) or CXP03 7 (see Example 4 below) were used at N / P ratios of 5 to 30, respectively, and 100n Transfection was performed at gDNA / well. According to the instructions, DPBS (Hyclone, No. SH30028.02, ThermoFi A transfection mixture was prepared. More specifically, 100 ng of DNA was added. A is defined as the N / P ratio (equation: N / P ratio = 7.5 × μg of jetPET / 3 × μg of DNA). The number of nitrogen residues (N) in jetPET per phosphate (P) in DNA according to the formula )5. Transfection with CXP037 In this case, an NP ratio of 30 was used (15.8 μg of CXP037 / μg of DNA).

[0198] Luciferase activity: 100 μL / well of 100 μL ... BrightGlo (Promega #E2620), a commercially available reagent, was added directly to the wells. Luciferase activity was measured by adding 100 ml of ... Afterwards, luminescence was measured using a VictorNivo (PerkinElmer) plate reader. The luminescence of individual wells was quantified using the control pDNA(Luc) at day 1. It was normalized.

[0199] The results are shown in Figures 6 and 7. Figure 6 shows the results of cDNAs containing at least two modified nucleotides. Cells transfected with lDNA express the corresponding clDNA containing natural nucleotides. This indicates that the luciferase activity was significantly higher than that of the control. The functional performance of the sequence (in this case luciferase) is determined by the presence of at least two modified nucleotides. significantly higher (statistically significant) than when transfected in clDNA containing Figure 7 demonstrates that the cDNA assayed showed a significant change over time. The results show that the level of luciferase activity increased gradually with the addition of modified nucleotides. Only those clDNAs with luciferase activity were found to be significantly higher at 48 hours than at 24 hours. A statistically significant increase in the level of enzyme activity was observed.

[0200] Example 3: clDNs containing at least two modified nucleotides from polyplexes Emission of A

[0201] This assay measures the picogreen fluorescence produced when the molecule binds to free double-stranded DNA. The polyplexes formed were diluted 10-fold with PBS. and added 10 μL / well to a 384-well plate for a final volume of 40 μL / well. At physiological conditions, each diluted polyplex was added with 8 U / mL of heparin. After adding PicoGreen, the fluorescent signal was acquired 12 hours later. The fluorescent signal was converted to DNA quantity using a standard DNA curve included in the kit. The assay demonstrated that heparin was effective in the absence and presence of the maximum concentration (8 U / mL). It is possible to determine the amount of released DNA, defined as the difference in DNA concentration between the two conditions. do.

[0202] The results are shown in Figure 8. Polyplexes formed by polymer CXP037 and Different DNA containing natural and modified nucleotides were incubated under physiological conditions (8 U / ml Incubated with 1 L of heparin (Engelberg et al., 1961) for 12 hours. The DNA released was quantified by monitoring the time of the reaction. observed different behavior. Therefore, as cargo DNA with modified nucleotides The polyplexes containing the ATP showed a statistically significant effect on the amount of "released DNA," This results in greater DNA availability than polyplexes with native DNA.

[0203] Therefore, after incubating these polyplexes for 12 hours in a physiological environment, The results show that the use of these oDNAs containing modified nucleotides improves DNA bioactivity. The irritability is higher.

[0204] Example 4: Synthesis and characterization of polymer CXP037

[0205] CXP037 is a polyplex mix with clDNA for cell transfection. It is a cell-forming polycationic polymer vehicle.

[0206] General Reactions were carried out under a nitrogen atmosphere unless otherwise stated. Solvents containing 2-pyrrolidinone (>99%), anhydrous CH2Cl2, and anhydrous DMF were used in Aldri All reagents were obtained from commercial manufacturers and used as described. The product was used without further purification. The polymerization reaction was monitored by IR (CARY 630 ATR-FTIR spectroscopy). The measurement was performed using a photometer. Vivaspin 3000 MWCO PES was used. The aminolysis reaction was purified by centrifugal ultrafiltration.

[0207] NMR spectroscopy: on a 300 MHz Bruker Advance AC-300 spectrometer 1 H spectrum was recorded.

[0208] SEC-MALS: TDA MALVER equipped with UV-RI-RALS-MALS Multi-angle light scattering spectrophotometer was used with a MALVERN GPC MAX equipped with an N 305 detector. Size exclusion chromatography coupled to SEC-MALS measurements was performed. A precolumn of 0.1 M NaNO3 solution containing 0.005% NaN3 was used at a flow rate of 1 L / min. Both were performed using a TSKgel G3000PWXL-CP cationic column in series. The mass of the sample injected onto the column was typically 2-5 mg. The concentration of the solution was 10-20 mg / mL. OMNISEC was used for data acquisition and evaluation. 5.12 software.

[0209] pKa determination procedure: Determine the pKa of the cationic polymer by acid-base titration and process The pH of the solution is measured throughout. The pKa is then obtained from the titration graph. To carry out this experiment, a 1 mg / mL cationic polymer solution was prepared in Milli-Q water and dissolved. Add HCl (0.1M) of known quality until the pH of the solution is approximately 2. At this point Methrom 916 potentiometric autotitrator with Dosino 800 dispenser The titration was performed with NaOH (0.2 M) using a 50 mV / min signal drift. Set the titration rate to 0.1 mL / min using the . The titration is completed when the pH reaches 12. The instrument will measure the pKa of the species present and generate a .txt report. When identifying many equivalence points that do not correspond to the chemical properties of a compound, a graphical method can be used. Use the HPLC method to manually determine the pKa.

[0210] Synthetic Route of PAspDET / DIIPA-Compound CXP037A (See Figure 9A) Synthesis of poly(β-benzyl L-aspartate) (PBLA) (see Figure 9B) Following the general procedure for ring-opening polymerization of NCA and PB, using ethylamine as the initiator, LA was synthesized. The synthesis reaction was carried out in a heated and dried Schlenk flask under a nitrogen atmosphere. First, BLA NCA (3 g, 12 mmol) was dissolved in dry dichloromethane (1 The compound was dissolved in a mixture of 20 mL of HCl and 10 mL of DMF. The initiator solution (n-butylamine, 11.89 μL, 0.12 mmol) was added to the reaction mixture. The mixture was allowed to stir at 50° C. for 16 hours. Upon completion, the reaction mixture became clear. Complete conversion of the monomer could be detected by IR. The reaction mixture was diluted with diethyl ether The precipitate was isolated by centrifugation (3750 rpm, 4 min). , and dried under vacuum. PBLA was isolated as a white solid (1.5 g, η = 60%). The 1H NMR spectrum of BLA is shown in Figure 10A.

[0211] Synthesis of PAsp(DET / DIIPA)-compound CXP037A (see Figure 9C) Aminolysis of PBLA with DET and DIIPA yielded PAsp(DET / DIIPA). PBLA (DP = 67.60 mg) was dissolved in NMP (3 mL) and 4 The solution was cooled to ° C. and diluted with DET (50 equivalents of DET per Asp unit, 1.58 mL). and DIIPA (100 equivalents of DIIPA per aspartic acid unit, 5.18 mL) The mixture was added dropwise to a mixing vessel, which was cooled to 4°C, and the mixture was stirred at the same temperature for 4 hours. After filtration, the reaction mixture was added dropwise to cold HCl (6M) for neutralization (pH: 3.5). The polymer product was purified by centrifugal ultrafiltration. After filtration, the resulting aqueous polymer solution was frozen. The final product was obtained by lysing and drying (42 mg, η = 64%). SEC-MALS-RI of P037A analysis. MW=14,000 Da (1.03). Figure 11 shows the 1H NMR spectrum of CXP037.

[0212] References list Kapp K et al., “EnanDIM - a novel family of L-nucleotide-protected TLR9 agonists s for cancer immunotherapy” 2019, J Immunother Cancer., vol 7(1), pp. 5 Heinrich J. et al., “Linear closed mini DNA generated by the prokaryotic cleavi ng-joining enzyme TelN is functional in mammalian cells” 2002, J Mol Med, vol. 80(10), pp. 648-54 Xiao X. et al., “A novel 165-base-pair terminal repeat sequence is the sole cis “requirement for the adeno-associated virus life cycle”, 1997, J Virol., vol. 7 1(2), pp. 941-948. Altschul et al., “Basic local alignment search tool”, 1990, J. Mol. Biol, vol. 215, pp. 403-410. WO2011000997 US4373071 EP1859812 Engelberg H. Plasma heparin levels in normal man. Circulation. 1961;23:578-581. doi: 10.1161 / 01.CIR.23.4.578. Beaucage S. L. et al, Deoxynucleoside phosphoramidites-A new class of key interm ediates for deoxypolynucleotide synthesis. Tetrahedron Letters, Volume 22, Issue 20, 1981, Pages 1859-1862

Claims

1. containing a double-stranded DNA sequence of interest covalently closed at both ends by hairpin loops. A closed linear DNA ( clDNA).

2. - the at least two modified nucleotides are present in one or both of the clDNAs Located in a single-stranded terminal loop, - at least one modified nucleotide is located in one of said single-stranded terminal loops, At least another modified nucleotide in said strand forms the stem region of said clDNA adapter. or alternatively, - the at least two modified nucleotides form the stem region of the adapter of the clDNA; present in one or both chains forming The clDNA of claim 1.

3. one of said strands wherein said at least one modified nucleotide forms said stem region; When present in - defined by nucleotides 1 to 5 relative to the last nucleotide forming the loop located within the defined strand region, or alternatively, - nucleotides in positions 1 to 10 relative to the last nucleotide forming part of said DNA sequence of interest The clDNA of claim 2, located within a strand region defined by a nucleotide.

4. The at least two modified nucleotides are 2-amino-deoxyadenosine, 5-amino- Thiol-deoxycytidine, thiophosphate nucleotides, inosine nucleotides, LNA nucleotides, L-DNA nucleotides, 8-oxo-deoxyadenosine 5-fluoro-deoxyuracil nucleotides, and 5-fluoro-deoxyuracil nucleotides. The clDNA of any one of claims 1 to 3, independently selected.

5. For example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 3, such as 4, 15, 16, 17, 18, 19 or 20 modified nucleotides The clDNA of any one of claims 1 to 4, comprising up to 20 modified nucleotides.

6. c according to any one of claims 1 to 5, comprising at least two LNA nucleotides lDNA.

7. The clDNA of any one of claims 1 to 6, comprising two LNA nucleotides.

8. Any one of claims 1 to 7, comprising at least two thiophosphate nucleotides. The clDNA described in

9. the stem region comprises two restriction sites flanking the DNA sequence of interest. Item 9. The clDNA according to any one of Items 1 to 8.

10. The clDNA of any one of claims 1 to 9, wherein the clDNA comprises a primase recognition site. clDNA.

11. 10. The method according to claim 1, wherein at least one of the loops comprises a primase recognition site. The clDNA according to any one of claims 1 to 4.

12. 12. The method according to claim 1, wherein the sequence of interest comprises an inverted terminal repeat (ITR). The clDNA shown above.

13. 13. The method of claim 1, wherein the DNA sequence of interest comprises an expression cassette. clDNA.

14. In treatment, e.g., gene therapy, gene editing, cell therapy (e.g., CAR-T), vaccines and expression of monoclonal antibodies, particularly for use in DNA-based therapy. The clDNA according to any one of claims 1 to 13.

15. A therapeutically effective amount of the clDNA according to any one of claims 1 to 13 and a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprising a carrier or excipient.

16. A c1 comprising at least two modified nucleotides according to any one of claims 1 to 13. A process for making DNA, comprising: a) providing a template DNA containing a DNA sequence of interest; b) amplifying DNA from the template DNA of step (a) to obtain a repeat of the desired DNA sequence; a step of preparing a concatemer DNA comprising: one flanked by restriction sites; c) (c1) contacting the concatemer DNA with at least one restriction enzyme, thereby and generating a plurality of open double-stranded DNA fragments each containing the target DNA sequence. (c2) forming hairpin DNA fragments at each end of the open double-stranded DNA fragments. coupling adapters, each one of said adapters having at least one modification; or alternatively, one of the adaptors attached to the DNA fragment. only one of the nucleic acids contains the at least two modified nucleotides; generating closed linear DNA using the amplified DNA produced in step (a); and d) purifying the closed linear DNA produced in step (c).

17. 17. The method of claim 16, wherein the template DNA containing the DNA sequence of interest is clDNA. Seth.

18. 18. The process of claim 17, wherein the template clDNA does not contain a primase recognition site. 。

19. 17. The method of claim 16, wherein the amplification in step (b) is performed by random cycle amplification (RCA).

19. The process according to any one of claims 1 to 18.

20. 10. The method of claim 1, wherein the hairpin DNA adaptor is 6 to 600 nucleotides in length.

20. A process for producing the clDNA of any one of claims 6 to 19.

21. 10. The method of claim 1, wherein the hairpin DNA adaptor is 6 to 200 nucleotides in length.

21. A process for producing the clDNA of any one of 6 to 20.

22. A closed linear DNA obtained by the process according to any one of claims 16 to 21. A.

23. A composition comprising a carrier and the clDNA of any one of claims 1 to 14 or 22.

24. The composition of claim 23 , wherein the carrier is a gene vector.

25. The composition of claim 24 , wherein the genetic vector is a viral vector.

26. The composition of claim 25 , wherein the genetic vector is a non-viral vector.

27. 27. The composition of claim 26, wherein the non-viral vector is a polycationic polymer. thing.

28. A polyp comprising a polymer and a clDNA according to any one of claims 1 to 14 or 22. Rex.

29. 29. The polyplex of claim 28, wherein the polymer is a polycationic polymer.

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