Method for preparing minicircle DNA and its applications

Abstract

This invention provides a method for preparing minicircle DNA and its applications. Specifically, it provides a recombinant plasmid containing a CinH resolvase for generating minicircle DNA and a specific recognition site thereof. Furthermore, it provides a method for generating minicircle DNA using the recombinant plasmid, a kit for generating minicircle DNA containing the recombinant plasmid, and applications of the minicircle DNA prepared using the above method in gene therapy and cell therapy.

Description

[Technical Field]

[0001] The present invention relates to the biomedical field, and specifically relates to a method for preparing minicircle DNA, and uses thereof in gene therapy and cell therapy. [Background Art]

[0002] DNA carriers are widely used in fields such as vaccines, gene therapy, and cell therapy. Conventional DNA carriers are plasmids comprising a prokaryotic origin of replication and an antibiotic resistance gene. However, these prokaryote-derived DNA sequences inhibit the expression of cis-linked mammalian cell expression cassettes and stimulate the DNA sensing pathway; activation of T lymphocytes, B lymphocytes, NK cells and monocytes promotes immunoinflammatory responses and induces programmed cell death. All of these responses limit the effectiveness of applications of plasmid DNA carriers. Transmission of prokaryotic origins of replication and antibiotic resistance genes is also one of the risks associated with the use of plasmid DNA carriers.

[0003] Superhelical circular DNA, which removes prokaryotic cell replication origins and antibiotic resistance genes, was first prepared by Darquet et al. in 1997 and exhibits superior protein expression capabilities compared to plasmid DNA carriers in various mammalian cells. This type of DNA is called minicircle DNA. Chen et al. further demonstrated, using mice as a model, that when the DNA carrier was transported to mouse liver, minicircle DNA expressed proteins with 200 to 560 times greater clinical potential in peripheral blood compared to plasmid DNA. Subsequently, the high stability and efficient expression capabilities of minicircle DNA have led to its widespread use in the development of gene therapies or cell therapies for various diseases. Minicircle DNA is being considered for application as a gene therapy carrier in preclinical research on rheumatoid arthritis or osteoarthritis. Rim et al. successfully used minicircle DNA to express BMP2 and TGFβ3, inducing differentiation of hiPSC-derived mesenchymal stem cells into chondrocytes and treating osteochondral damage in a rat model. Florian et al. used minicircle DNA to express the Angiopoietin 1 protein in mesenchymal stem cells, improving the effectiveness of mesenchymal stem cells in mitigating acute lung injury. Development is also underway on technologies using minicircle DNA to deliver proteins with clinical therapeutic potential to retinal epithelial cells, retinal ganglion cells, or photosensitive cells for use in treating eye-related diseases. In summary, minicircle DNA is a gene therapy and cell therapy carrier with high potential and a wide range of applications.

[0004] The preparation of minicircle DNA relies on recombinases to split a parent plasmid with two recombination sites into two smaller circular DNAs. By designing the relative positions of the recombination sites and other units on the parent plasmid, one circular DNA can carry a functional protein or functional RNA expression cassette, i.e., minicircle DNA, necessary for gene therapy or cell therapy, while the other circular DNA can carry a unit containing a prokaryotic origin of replication and antibiotic resistance genes to be removed; this is commonly called a microplasmid. Currently reported recombinases used for minicircle DNA preparation include phage λ integrase, phage ΦC31 integrase, Cre recombinase, and ParA resolverase. Minicircles prepared using phage λ integrase, ΦC31 integrase, and Cre recombinase are all contaminated with dimers and multimers. Because the sequences of these dimers and multimers match those of the target minicircle DNA, obtaining high-purity minicircle DNA subsequently becomes difficult. ParA resolver achieves nearly 100% recombination efficiency, and the recombined product is free from dimer or polymer contamination. To obtain high-purity minicircles, techniques for removing residual parental plasmids and microplasmids are also necessary. Currently, common methods include in vitro enzymatic digestion to remove parental plasmids and microplasmids, affinity chromatography to remove parental plasmids and microplasmids, or purification of minicircle DNA. Whole column chromatography is also used to further purify superhelical minicircle DNA to meet clinical use standards.

[0005] Adoptive cellular immunotherapy has made remarkable progress in recent years. The U.S. Food and Drug Administration (FDA) has approved KYMRIAH (Novartis Pharmaceuticals Corporation), YESCARTA and TECARTUS (Kite Pharma, Inc.), BREYANZI (Juno Therapeutics, Inc.), ABECMA (Celgene Corporation), CARVYKTI (Janssen Biotech, Inc.), and others for the clinical treatment of relapsed and refractory hematological malignancies, including diffuse large B-cell lymphoma, mantle cell lymphoma, and multiple myeloma, providing a new and reliable treatment strategy that extends patient survival and improves quality of life. All of these FDA-approved adoptive immunotherapies are prepared using retroviral or lentiviral carriers. In clinically used CAR-T cells prepared using retroviral and lentiviral carriers, 65% and 54% of insertion sites are located within RefSeq genes, respectively. The insertion of these exogenous genes into RefSeq genes raises concerns regarding their application in gene therapy and cell therapy using retroviral and lentiviral carriers. The Sleeping Beauty transposon system has been shown to enable nearly random insertions into the human genome and is expected to offer higher safety in gene therapy compared to carriers based on retroviral, lentiviral, and other transposon systems. However, the biggest obstacle to using non-viral carrier systems in gene therapy or cell therapy is the low efficiency of carrier delivery.

[0006] Due to its high stability and high expression levels, minicircle DNA can significantly improve the efficiency of non-viral DNA carriers. Therefore, by using minicircle DNA from the Sleeping Beauty transposon system to carry CAR genes, CAR-T can be prepared, effectively replacing high-risk retroviral or lentiviral carriers.

[0007] Therefore, it can be said that there is a very high demand for research and development of minicircle DNA drugs in this technological field. [Overview of the project] [Problems that the invention aims to solve]

[0008] The object of the present invention is to provide a recombinant plasmid for generating circular superhelical minicircle DNA that incorporates elements such as a prokaryotic origin of replication, a prokaryotic selection marker, a regulatory and controllable expression element that controls recombinase expression, a recombinase, two recombinase recognition sites, and a multicloning site into which a target DNA sequence can be inserted, and that does not contain prokaryotic DNA sequences such as a prokaryotic origin of replication and a prokaryotic selection marker, as well as a method for generating minicircle DNA using the recombinant plasmid. [Means for solving the problem]

[0009] A first aspect of the present invention provides a recombinant plasmid, the plasmid being: It includes elements such as a prokaryotic replication origin, a prokaryotic selection marker, a recombinase coding sequence, a regulatory and controllable expression element that controls recombinase expression, a multicloning site into which a target DNA sequence can be inserted, and two recombinase recognition sites located on either side of the multicloning site. Furthermore, depending on the arrangement of the elements described above, the plasmid is cleaved at two recombinase-recognition sites by the expressed recombinase and then classified into a microplasmid and a minicircle DNA, where the formed minicircle DNA includes a multicloning site into which a target DNA sequence can be inserted, the target DNA sequence inserted therein, and a remaining recombinase-recognition site. The recombinase is a CinH resolvase, and the recombinase identification site has a nucleotide sequence or a derived sequence thereof as shown in SEQ ID NO: 3.

[0010] In another preferred example, the CinH resolverase includes wild-type CinH resolverase and its mutants, wherein the mutants retain the activity of a CinH resolverase that recognizes and cleaves a recombinase recognition site as shown in SEQ ID NO: 3.

[0011] In another preferred example, the amino acid sequence of the CinH resolverase is as shown in SEQ ID NO: 1.

[0012] In another preferred example, the nucleotide sequence encoding the CinH resolver is as shown in SEQ ID NO: 2.

[0013] In another preferred example, the orientation of the two recombinase identification sites located on either side of the multicloning site is the same, i.e., →multicloning identification site→.

[0014] In another preferred example, the target DNA sequence is selected from the group consisting of a functional DNA sequence segment, a functional RNA expression cassette set, a functional protein expression cassette set, or a combination thereof.

[0015] In another preferred example, the target DNA sequence is a protein expression cassette that expresses a transposase in a eukaryotic cell, the transposase including, but not limited to, Sleepy Beauty, PiggyBac, and Tol2.

[0016] In another preferred example, the target DNA sequence is a transposon that, in cooperation with a transposase, stably inserts the carried genetic information into a prokaryotic or eukaryotic gene set.

[0017] In another preferred example, the target DNA sequence includes a chimeric antigen receptor (CAR) coding sequence.

[0018] In another preferred example, the moduloable and controllable expression element that controls the recombinase expression is selected from the group consisting of the arabinose operator group pBAD / AraC system, the lactose / IPTG operator group LacI / LacO system, the tetracycline operator group rtTA / TRE system, a heat shock induction system based on heat shock protein 70 or 90, and a photoinduction system based on the blue light-sensing protein YFI and downstream FixJ / FixK2 / cI / pR.

[0019] In another preferred example, the recombinant plasmid further comprises a microplasmid identification and removal element located within a microplasmid generated after the plasmid is cleaved by a recombinase expressed on the plasmid.

[0020] In another preferred example, the microplasmid identification and removal elements are selected from the group consisting of restriction enzyme cleavage sites, LacO sequences, specifically designed oligonucleotide or deoxynucleotide complementary sequences, specifically designed inactivated Cas ribonucleoprotein (e.g., dead Cas9, Cas12, Cas3, Cas8, Cas10, and Cas13 RNP) recognition sequences, specifically designed zinc finger DNA binding domain recognition sequences, or specifically designed transcription activator-like effector DNA binding domain recognition sequences, or combinations thereof.

[0021] In another preferred example, the microplasmid identification and removal element is at least one restriction enzyme cleavage site.

[0022] In another preferred example, the recombinant plasmid further comprises a minicircle DNA identification and separation element, which is located within the minicircle DNA generated after the plasmid is cleaved by the expressed recombinase.

[0023] In another preferred embodiment, the minicircle DNA identification and separation element is selected from the group consisting of LacO sequence, specifically designed oligonucleotide or deoxynucleotide complementary sequence, specifically designed recognition sequence for inactivated Cas ribonucleoprotein (such as dead Cas9, Cas12, Cas3, Cas8, Cas10 and Cas13 RNP, etc.), specifically designed zinc finger DNA binding domain recognition sequence or specifically designed transcription activator-like effector DNA binding domain recognition sequence, or a combination thereof.

[0024] In another preferred embodiment, the recombinant plasmid further comprises a microplasmid identification and removal element, and a minicircle DNA identification and separation element.

[0025] The second aspect of the present invention provides a reaction system for producing minicircle DNA, wherein the reaction system comprises: (Z1) the recombinant plasmid according to the first aspect of the present invention; (Z2) an inducing reagent or inducing condition that induces expression of the recombinase encoded by the recombinant plasmid; In another preferred embodiment, the inducing reagent is selected from the group consisting of arabinose, lactose or IPTG, and tetracycline.

[0026] In another preferred embodiment, the inducing condition comprises heat shock or light induction.

[0027] In another preferred embodiment, the reaction system further comprises a microplasmid identification and removal reagent, and / or a minicircle DNA identification and separation reagent.

[0028] In another preferred embodiment, the microplasmid identification and removal reagent acts on the microplasmid identification and removal element in the recombinant plasmid and the produced microplasmid, and is used for removing the microplasmid and unreacted recombinant plasmid in the removed product.

[0029] In another preferred example, the minicircle DNA identification and separation reagent is used to act on the minicircle DNA identification and separation element in the minicircle DNA and to separate the minicircle DNA in the product.

[0030] In another preferred example, the microplasmid identification and removal reagent is a restriction endonuclease, a LacI protein, an oligonucleotide or deoxynucleotide having a specific design sequence, an inactivated Cas ribonucleoprotein (e.g., dead Cas9, Cas12, Cas3, Cas8, Cas10 and Cas13 RNP, etc.), a zinc finger DNA binding domain, a transcription activator-like effector DNA binding domain, or a combination thereof.

[0031] In another preferred example, the microplasmid identification and removal reagent is a restriction endonuclease.

[0032] In another preferred example, the minicircle DNA identification and separation reagent comprises, and is selectable for, a protein capable of binding to the minicircle DNA identification and separation element in the minicircle DNA to form a stable DNA-protein complex, wherein the protein is immobilized on a solid carrier.

[0033] In another preferred example, the minicircle DNA identification and separation reagent is selected from the group consisting of LacI protein, oligonucleotides or deoxynucleotides of a specifically designed sequence, inactivated Cas ribonucleoproteins (e.g., dead Cas9, Cas12, Cas3, Cas8, Cas10 and Cas13 RNPs, etc.), zinc finger DNA-binding domains, transcription activator-like effector DNA-binding domains, or combinations thereof.

[0034] A third aspect of the present invention provides a kit for generating minicircle DNA, the kit comprising: (C1) A recombinant plasmid according to the first aspect of the present invention, and (C2) optionally includes an induction reagent for inducing recombinase expression in the recombinant plasmid.

[0035] In another preferred example, the kit is: (C3) Microplasmid identification and removal reagent, which acts on the microplasmid identification and removal elements in the recombinant plasmid and the generated microplasmid, and is used to remove the microplasmid and unreacted recombinant plasmid from the product, and / or (C4) Further comprising a minicircle DNA identification and separation reagent, the reagent is used to act on the minicircle DNA identification and separation element in the minicircle DNA and to separate the minicircle DNA in the product.

[0036] In another preferred example, the inducing reagent is selected from the group consisting of arabinose, lactose, or IPTG and tetracycline.

[0037] In another preferred example, the microplasmid identification and removal reagent is selected from restriction endonucleases, LacI proteins, oligonucleotides or deoxynucleotides of a specifically designed sequence, inactivated Cas ribonucleoproteins (e.g., dead Cas9, Cas12, Cas3, Cas8, Cas10 and Cas13 RNPs), zinc finger DNA-binding domains, transcription activator-like effector DNA-binding domains, or a combination thereof.

[0038] In another preferred example, the microplasmid identification and removal reagent is a restriction endonuclease.

[0039] In another preferred example, the minicircle DNA identification and separation reagent comprises, and is selectable for, a protein capable of binding to the minicircle DNA identification and separation element in the minicircle DNA to form a stable DNA-protein complex, wherein the protein is immobilized on a solid carrier.

[0040] In another preferred example, the minicircle DNA identification and separation reagent is selected from LacI protein, oligonucleotides or deoxynucleotides of a specifically designed sequence, inactivated Cas ribonucleoproteins (e.g., dead Cas9, Cas12, Cas3, Cas8, Cas10 and Cas13 RNPs, etc.), zinc finger DNA-binding domains, transcription activator-like effector DNA-binding domains, or a combination thereof.

[0041] In another preferred example, the kit further includes a label or instructions describing a method for generating minicircle DNA using the kit, wherein the method includes inducing recombinase expression in the recombinant plasmid, if an inducing reagent or inducing conditions are present.

[0042] In another preferred example, the induction conditions include thermal shock or photoinduction.

[0043] A fourth aspect of the present invention provides a method for generating minicircle DNA, the method comprising the following steps: (S1) The recombinant plasmid described in the first aspect of the present invention is introduced into prokaryotic cells, (S2) The prokaryotic cells are cultured and the expression of the recombinase encoded in the recombinant plasmid is induced to produce an intermediate product containing a microplasmid and minicircle DNA, and Minicircle DNA is isolated from the intermediate product of (S3)(S2).

[0044] In another preferred example, in step (S2), recombinase expression is induced by adding an induction reagent and activating a controllable expression element that controls recombinase expression in the recombinant plasmid.

[0045] In another preferred example, in step (S2), recombinase expression is induced by activating a controllable expression element that controls recombinase expression in a recombinant plasmid using induction conditions.

[0046] In another preferred example, in step (S3), minicircle DNA is separated using a minicircle DNA identification and separation element and a minicircle DNA identification and separation reagent.

[0047] In another preferred example, in step (S3), the minicircle DNA is isolated by removing the microplasmid and unreacted recombinant plasmid in the intermediate product using a microplasmid identification and removal reagent.

[0048] In another preferred example, in step (S3), first, the microplasmid and unreacted recombinant plasmid in the intermediate product are removed using a microplasmid identification and removal reagent, and then the minicircle DNA is separated using a minicircle DNA identification and separation reagent with a minicircle DNA identification and separation element in the minicircle DNA.

[0049] In another preferred example, the prokaryotic cells include (but are not limited to) Escherichia coli, Agrobacterium rhizoma, Bacillus subtilis, Lactobacillus, Salmonella, Bifidobacterium, Listeria monocytogenes, Pseudomonas, Neisseria meningitidis, and Vibrio cholerae.

[0050] A fifth aspect of the present invention provides minicircle DNA prepared by the method described in the fourth aspect of the present invention, which is, The system includes a multi-cloning site into which a target DNA sequence can be inserted, the target DNA sequence inserted therein, and a remaining recombinase identification site, wherein the recombinase identification site has a nucleotide sequence or a derived sequence thereof as shown in SEQ ID NO: 3.

[0051] In another preferred example, the target DNA sequence is selected from the group consisting of a functional DNA sequence segment, a functional RNA expression cassette set, a functional protein expression cassette set, or a combination thereof.

[0052] In another preferred example, the target DNA sequence is a protein expression cassette that expresses a transposase in eukaryotic cells, the transposase including (but not limited to) Sleepy Beauty, PiggyBac, and Tol2.

[0053] In another preferred example, the target DNA sequence is a transposon that, in cooperation with a transposase, stably inserts the genetic information it carries into a prokaryotic or eukaryotic gene set.

[0054] In another preferred example, the target DNA sequence includes a chimeric antigen receptor (CAR) coding sequence.

[0055] In another preferred example, the minicircle DNA further comprises minicircle DNA identification and separation elements.

[0056] In another preferred example, the minicircle DNA identification and separation element is selected from a LacO sequence, a specifically designed oligonucleotide or deoxynucleotide complementary sequence, a specifically designed inactivated Cas ribonucleoprotein recognition sequence, a specifically designed zinc finger DNA-binding domain recognition sequence, or a specifically designed transcription activator-like effector DNA-binding domain recognition sequence, or a combination thereof.

[0057] A sixth aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition is (a) Minicircle DNA as described in the fifth aspect of the present invention, and (b) a pharmaceutically acceptable carrier.

[0058] In another preferred example, the pharmaceutical composition is used for the treatment and / or prevention of a disease.

[0059] In another preferred example, the pharmaceutical composition is used for gene therapy or cell therapy.

[0060] In another preferred example, the pharmaceutical composition is used as a vaccine for treating and / or preventing a disease.

[0061] A seventh aspect of the present invention provides an application in the preparation of recombinant plasmid minicircle DNA drugs as described in the first aspect of the present invention.

[0062] In another preferred example, the minicircle DNA drug is used for gene therapy or cell therapy.

[0063] An eighth aspect of the present invention provides an application of the minicircle DNA described in the fifth aspect of the present invention in the preparation of drugs for treating and / or preventing diseases.

[0064] In another preferred example, the disease can be treated by gene therapy or cell therapy.

[0065] In another preferred example, the disease includes (but is not limited to) cancer or tumors, eye diseases, rheumatoid arthritis, osteoarthritis, osteochondral damage, and acute lung injury.

[0066] A ninth aspect of the present invention provides a method for treating and / or preventing a disease, comprising administering the minicircle DNA described in the fifth aspect of the present invention to a subject who requires it.

[0067] In another preferred example, the minicircle DNA serves as a delivery carrier to deliver therapeutic / preventive components into the subject's body.

[0068] In another preferred example, the subject may include a human or a non-human mammal.

[0069] In another preferred example, the disease can be treated by gene therapy or cell therapy.

[0070] In another preferred example, the disease includes (but is not limited to) cancer or tumors, eye diseases, rheumatoid arthritis, osteoarthritis, osteochondral damage, and acute lung injury. [Effects of the Invention]

[0071] It should be understood that, within the scope of the present invention, new or preferred technical solutions can be constructed by combining the above-described technical features of the present invention with the technical features specifically described below (e.g., in the examples). Due to space limitations, this will not be repeated here. [Brief explanation of the drawing]

[0072] [Figure 1] This diagram shows the schematic arrangement of the elements of the minicircle DNA parent plasmid, microplasmid, and minicircle DNA. [Figure 2a] This shows the preparation process for a transposon minicircle carrying an EGFP expression cassette. [Figure 2b] This image shows the successful preparation of transposon minicircle DNA carrying an EGFP expression cassette. [Figure 3] This shows the preparation of transposon minicircle DNA carrying a CD19-CAR expression cassette. [Figure 4] This shows the preparation of minicircle DNA with a transposase expression cassette. [Figure 5]We demonstrate that minicircle DNA is significantly superior to plasmid DNA when delivering exogenous genes to T cells as a delivery carrier. [Modes for carrying out the invention]

[0073] The inventors, through extensive and meticulous research, have developed a novel recombinant plasmid for the first time. By arranging the elements mounted on the recombinant plasmid provided in this invention, recombinase expression is induced, and then the parent plasmid is separated into two smaller circular DNAs. One is a microplasmid, containing a prokaryotic origin of replication, a prokaryotic selection marker, a regulatory and controllable expression element that controls recombinase expression, and non-target sequences such as the recombinase coding sequence. The other is a minicircle DNA, containing only the target sequence, a remaining multicloning site, and a remaining recombinase identification site. The microplasmid can be removed by methods such as a specific restricting enzyme site or a specific identification sequence on the microplasmid, thereby purifying the minicircle DNA, or the minicircle DNA can be purified by a specific identification sequence on the minicircle DNA.

[0074] Based on this, the present invention was completed.

[0075] term To facilitate understanding of the present invention, certain technical and chemical terms are defined below. Unless otherwise explicitly defined herein, all other technical and chemical terms used herein have meanings that are generally understood by those skilled in the art to which the present invention pertains. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may change. The terms used herein are intended to describe specific embodiments only, and not to limit them, and the scope of the present invention is limited only by the appended claims.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention belongs.

[0077] As used herein, when used in reference to a specific numerical value, the term "about" means that the value can only vary by 1% or less from the stated value. For example, as used herein, the term "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0078] The three-letter and one-letter codes for amino acids used in this invention are as described in J.biol.chem, 243, p3558 (1968).

[0079] As used herein, the terms “optional” or “optionally” mean that the events or circumstances described below may occur, but are not required to occur. For example, “optionally, an inducing reagent for inducing recombinase expression in the recombinant plasmid” means that such an inducing reagent may be present, but is not required to be present.

[0080] The "sequence identity" described in this invention refers to the degree of identity between two nucleic acids or two amino acid sequences when optimally compared under conditions of having appropriate mutations such as exchanges, insertions, or deletions. The sequence identity between the sequences described in this invention and sequences having that identity may be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0081] Recombinant plasmid of the present invention One aspect of the present invention provides a recombinant plasmid comprising elements of a prokaryotic origin of replication, a prokaryotic selection marker, a recombinase coding sequence, a regulatory and controllable expression element that controls recombinase expression, a multicloning site into which a target DNA sequence can be inserted, and two recombinase recognition sites located on either side of the multicloning site, wherein, by the arrangement of the above elements, the plasmid is separated into a microplasmid and minicircle DNA after being cleaved at the two recombinase recognition sites by the expressed recombinase, where the formed minicircle DNA comprises a multicloning site into which a target DNA sequence can be inserted, the target DNA sequence inserted therein, and the remaining recombinase recognition site, and the microplasmid is comprising non-target sequences such as a prokaryotic origin of replication, a prokaryotic selection marker, a regulatory and controllable expression element that controls recombinase expression, and a recombinase coding sequence. The recombinase carried by the recombinant plasmid of the present invention is a CinH resolvase, and its corresponding recombinase identification site has a nucleotide sequence or a derived sequence thereof as shown in SEQ ID NO: 3.

[0082] In the present invention, CinH resolvase includes wild-type CinH resolvase and its mutants, wherein the mutants retain the activity of CinH resolvase to identify and cleave a recombinase recognition site as shown in SEQ ID NO: 3. Preferably, the mutants have at least 85% sequence identity with respect to the amino acid sequence of wild-type CinH resolvase (SEQ ID NO: 1). In one embodiment of the present invention, the CinH resolvase coding sequence has a nucleotide sequence as shown in SEQ ID NO: 2.

[0083] Amino acid sequence of wild-type CinH resolverase (SEQ ID NO: 1) MKGQKVGYVRVSSVEQNTGRQLEGIEVDRIFVDRASGKNTDRPKFQEMLNYVREGDRVIVHSMDRFARSLKDLVTEVDKLVKRGIAIQFVKENITFTAQSTPMDNLMQLMGAFAQFEREIILERQKEGIKIAAAQGKYKGRVHKLNPDQAKALLQAWKEGKYSSKVDLAKAFGISRQAVRYLKQIN* CinHリゾルバーゼコードショード (SEQ ID NO: 2) Gtgaaaggccaaaaaagtagggtatgtgcgagtgagttcggtcgagcaaaatacagggcgtcaacttgagggaattgaagtcgaccggatttttgttgaccgtgcttcgggtaaaaaataccgaccgaccgaaatttcaagaa atgttgaactatgtccgggaaggggatagggtgattgtacattccatggatcgttttgcacgaagtctaaaagatttggtcactgaagtagataaactggtcaaaagagggatcgccatccagtttgtaaaagagaatatta cttttactgcccaatccacgccgatggataatttgatgctgcaactgatgggtgcttttgctcaattcgagcgggaaattatcttagaacgtcagaaagaagggattaagatcgcagcagctcagggcaaatataaaaggtcg tgtccataaattgaatccagatcaagctaaagcattactgcaagcctggaaagaagggaagtattcatcaaaagttgatctggcaaaagcgtttggtatcagtagacaggctgtctatcggtatttaaaacaaattaattag

[0084] As used herein, the term “recombinase coding sequence” refers to any sequence necessary for recombinase expression, such as a promoter or termination sequence. The recombinase itself may be any recombinase that can catalyze a parent plasmid into a microplasmid and separable minicircle DNA at a determined identification sequence. In the present invention, recombinase refers in particular to CinH resolverase.

[0085] As used herein, the terms “recombinase identification site” and “RS2” are interchangeable and all refer to sequences that can be identified by recombinase. The recombinant plasmid of the present invention is equipped with two recombinase identification sites, and these two recombinase identification site sequences must be positioned on the plasmid in such a manner that the recombinase can identify and cleave the sequences to produce a microplasmid and minicircle DNA. This means that the recombinase identification sites must be on both sides of the multicloning site, and the orientation of the two recombinase identification sites must be the same, i.e., →multicloning identification site→, thereby producing a minicircle DNA containing the target DNA sequence to be inserted into the multicloning site. The produced minicircle DNA may contain at least a portion of one and / or another recombinase identification site sequence. The two recombinase identification site sequences may be the same or may be derived from the same recombinase identification sequence. In the present invention, the recombinase identification site has a nucleotide sequence or a derived sequence thereof, as shown in SEQ ID NO: 3.

[0086] Recombinase identification site (SEQ ID NO: 3) CGTTACTTTGGGGTATACCCTAAAGTTACAATATAAAAGTTCTTAAAACTATGTAACATTTAAATGATTTTTAACCATATATAACATGTAACTTTGATATTTAAAGTTTATAATTTACG

[0087] The term "multicloning site" refers to a site containing at least two restriction endonuclease sites, preferably multiple sites containing various restriction endonucleases.

[0088] As used herein, the term “target DNA” refers to a DNA sequence that is inserted into the multicloning site of the recombinant plasmid, contained in the resulting minicircle DNA, and ultimately delivered to a test subject to exert a biological function. This may be a functional DNA sequence segment, a functional RNA expression cassette set, or a functional protein expression cassette set. Preferably, the target DNA is a protein gene with therapeutic effect. Since the minicircle DNA is primarily used for therapeutic purposes, it preferably contains a protein gene with therapeutic effect. For example, minicircle DNA for expressing a chimeric antigen receptor (CAR) is generated by inserting a gene encoding a CAR into the multicloning site of the recombinant plasmid of the present invention. However, it is possible to insert any gene encoding any protein, in which case the minicircle can be used in detection methods, biochemical pathway studies, etc.

[0089] The recombinant plasmid of the present invention contains a regulatory and controllable expression element that controls recombinase expression. By providing a regulatory element to control recombinase expression, recombinase expression can be inhibited or induced, thereby allowing recombinase to be expressed as needed. By using this system to generate large quantities of plasmid before inducing recombinase expression, the production efficiency of minicircle DNA can be maximized. Various regulatory elements are well known to those skilled in the art. Preferably, the regulatory element for controlling recombinase expression includes a strong promoter. This allows for sufficient expression of recombinase and catalyzes the recombination of the total amount of plasmid present in the sample, thereby achieving the maximum yield of minicircle DNA. In one embodiment of the present invention, recombinase expression is controlled using the pBAD / AraC system of arabinose operators. As long as arabinose is not present in the culture system, this regulatory system can inhibit recombinase expression. Adding arabinose, such as L-arabinose, to the culture medium induces recombinase expression. This is a very simple and efficient method for controlling recombinase expression.

[0090] As used herein, a “prokaryotic selection marker” may be an antibiotic resistance gene. This is a well-known method in the art for selecting bacteria transfected with a plasmid containing a prokaryotic selection marker and effectively generating high-copy-number plasmids.

[0091] The recombinant plasmid of the present invention further comprises a minicircle DNA and / or microplasmid identification sequence for identifying and isolating minicircle DNA. The identification sequence provides an effective method for isolating minicircle DNA from a microplasmid, thereby further improving the efficiency of minicircle DNA generation.

[0092] The identification sequence for minicircle DNA or microplasmid is any sequence that is present in the minicircle but not on the microplasmid (or vice versa), and that can identify and isolate (or remove the microplasmid). In one embodiment of the present invention, the identification sequence present on the microplasmid is referred to as the "microplasmid identification and removal element." In another embodiment of the present invention, the identification sequence present on the minicircle DNA is referred to as the "minicircle DNA identification and isolation element." The above identification sequence is present only on the microplasmid or minicircle DNA, but is also applicable when therapies for both microplasmid and minicircle DNA are present simultaneously.

[0093] Microplasmid identification and removal elements may be specific sequences that are identified by a particular reagent and then removed from the product. For example, a microplasmid identification and removal element may be a restriction endonuclease identification site that is identified by the corresponding restriction endonuclease, cleaved into linear DNA, and then digested and removed from the product.

[0094] The identification and separation elements of minicircle DNA may be specific sequences, which, upon binding to a predetermined ligand, form a complex with the ligand. This complex is then separated from microplasmids, cellular components, etc., by, for example, an immobilized ligand (the ligand is immobilized on a solid support such as magnetic beads, agarose beads, or an agarose column).

[0095] The present invention kit The present invention further provides a kit for generating minicircle DNA, comprising a recombinant plasmid as described in a first aspect of the present invention.

[0096] In one embodiment of the present invention, the kit comprises components of a recombinant plasmid according to the first aspect of the present invention, an induction reagent for inducing recombinase expression in the recombinant plasmid, and a microplasmid identification and removal reagent. In another embodiment of the present invention, the kit comprises components of a recombinant plasmid according to the first aspect of the present invention, an induction reagent for inducing recombinase expression in the recombinant plasmid, and a minicircle DNA identification and separation reagent. In yet another embodiment of the present invention, the kit comprises components of a recombinant plasmid according to the first aspect of the present invention, an induction reagent for inducing recombinase expression in the recombinant plasmid, a microplasmid identification and removal reagent, and a minicircle DNA identification and separation reagent.

[0097] Here, the microplasmid identification and removal reagent acts on the microplasmid identification and removal elements in the recombinant plasmid and the generated microplasmid, and is used to remove the microplasmid and unreacted recombinant plasmid from the product. In the present invention, the microplasmid identification and removal elements / microplasmid identification and removal reagent can be selected from a combination of a restriction endonuclease identification site / restriction endonuclease, a LacO sequence / LacI protein, a specifically designed oligonucleotide or deoxynucleotide complementary sequence / specifically designed oligonucleotide or deoxynucleotide, a specifically designed inactivated Cas ribonucleoprotein recognition sequence / inactivated Cas ribonucleoprotein, a specifically designed zinc finger DNA binding domain recognition sequence / zinc finger DNA binding domain, or a specifically designed transcription activator-like effector DNA binding domain recognition sequence / transcription activator-like effector DNA binding domain (microplasmid identification and removal elements / microplasmid identification and removal reagent). In a preferred embodiment of the present invention, the microplasmid identification and removal reagent comprises a restriction endonuclease, the identification of which is located at a restriction endonuclease identification site on the microplasmid and the unreacted recombinant plasmid, and cleaving the microplasmid and the unreacted recombinant plasmid into linear DNA. Preferably, the reagent further comprises a linear DNA digestion reagent, such as a T5 exonuclease, for digesting and removing the linear DNA.

[0098] The minicircle DNA identification and separation reagent acts on the minicircle DNA identification and separation elements in the minicircle DNA and is used to separate the minicircle DNA in the product. In a preferred embodiment, the minicircle DNA identification and separation reagent is selectable and includes a protein capable of binding to the minicircle DNA identification and separation elements in the minicircle DNA to form a stable DNA-protein complex, the protein being immobilized on a solid support (e.g., magnetic beads, agarose beads, agarose column, etc.). In the present invention, the minicircle DNA identification and separation element / minicircle DNA identification and separation reagent can be selected from a combination of LacO sequence / LacI protein, a specifically designed oligonucleotide or deoxynucleotide complementary sequence / specifically designed oligonucleotide or deoxynucleotide sequence, a specifically designed inactivated Cas ribonucleoprotein recognition sequence / inactivated Cas ribonucleoprotein, a specifically designed zinc finger DNA binding domain recognition sequence / zinc finger DNA binding domain, or a specifically designed transcription activator-like effector DNA binding domain recognition sequence / transcription activator-like effector DNA binding domain (minicircle DNA identification and separation element / minicircle DNA identification and separation reagent).

[0099] Method of the present invention The present invention provides a method for generating minicircle DNA, the method for generating minicircle DNA using a recombinant plasmid constructed according to a first aspect of the present invention.

[0100] In one embodiment of the present invention, the method includes the following steps: (S1) The recombinant plasmid described in the first aspect of the present invention is introduced into prokaryotic cells, (S2) The prokaryotic cells are cultured, and then an induction reagent (or induction conditions) is added to induce the expression of the recombinase encoded in the recombinant plasmid. The expressed recombinase cleaves two recombinase recognition sites on the recombinant plasmid to produce an intermediate product containing a microplasmid and minicircle DNA. The microplasmid identification and removal reagents are added to the intermediate product of (S3)(S2), and the microplasmids and unreacted recombinant plasmids are removed to isolate the minicircle DNA.

[0101] In another embodiment of the present invention, the method comprises the following steps: (S1) The recombinant plasmid described in the first aspect of the present invention is introduced into prokaryotic cells, (S2) The prokaryotic cells are cultured, and then an induction reagent (or induction conditions) is added to induce the expression of the recombinase encoded in the recombinant plasmid. The expressed recombinase cleaves two recombinase recognition sites on the recombinant plasmid to produce an intermediate product containing a microplasmid and minicircle DNA. Minicircle DNA is separated by adding a minicircle DNA identification and separation reagent to the intermediate product of (S3)(S2).

[0102] In yet another embodiment of the present invention, the method comprises the following steps: (S1) The recombinant plasmid described in the first aspect of the present invention is introduced into prokaryotic cells, (S2) The prokaryotic cells are cultured, and then an induction reagent (or induction conditions) is added to induce the expression of the recombinase encoded in the recombinant plasmid. The expressed recombinase cleaves two recombinase recognition sites on the recombinant plasmid to produce an intermediate product containing a microplasmid and minicircle DNA. (S3) The intermediate product of (S2) is mixed with a microplasmid identification and removal reagent to remove the microplasmid and unreacted recombinant plasmid, and then the minicircle DNA is separated by adding a minicircle DNA identification and separation reagent.

[0103] The present invention: Mini-circle DNA, pharmaceutical composition, and applications. The present invention further provides minicircle DNA prepared using the method of the present invention, comprising a multicloning site into which a target DNA sequence can be inserted, a target DNA sequence inserted therein, and a remaining recombinase identification site, wherein the recombinase identification site has a nucleotide sequence or a derived sequence thereof as shown in SEQ ID NO: 3.

[0104] Here, the target DNA sequence includes (but is not limited to) a functional DNA sequence segment, a functional RNA expression cassette set, a functional protein expression cassette set, or a combination thereof. In one embodiment, the target DNA sequence is a protein expression cassette that expresses a transposase in eukaryotic cells. In another embodiment, the target DNA sequence is a transposon that cooperates with a transposase to stably insert its carried genetic information into the genome of a prokaryotic or eukaryotic cell.

[0105] In a preferred embodiment, the minicircle DNA further comprises a minicircle DNA identification and separation element. Alternatively, it does not include a minicircle DNA identification and separation element, in which case the microplasmid produced simultaneously during the production process comprises a microplasmid identification and removal element.

[0106] The mini-circle DNA of the present invention can be used in the preparation of pharmaceutical compositions. Accordingly, the present invention further provides pharmaceutical compositions comprising the mini-circle DNA of the present invention as a safe and effective amount of active ingredient, and a pharmaceutically acceptable carrier.

[0107] As used herein, “safe and effective amount” means an amount of the active ingredient sufficient to significantly improve a medical condition or symptom without causing serious side effects. “Pharmacologically acceptable vector” means one or more compatible solid or liquid fillers or gels that are suitable for use in humans and must be of sufficient purity and sufficiently low toxicity. “Compatibility” here means that each component in the composition can be blended with and among the active ingredients of the present invention without significantly reducing the potency of the active ingredients.

[0108] The composition may be a liquid or a solid, such as a powder, gel, or paste. Preferably, the composition is a liquid, and preferably an injectable liquid.

[0109] Some examples of pharmaceutically acceptable vectors include cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., Tween®), wetting agents (e.g., sodium dodecyl sulfate), colorants, fragrances, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0110] The pharmaceutical compositions of the present invention can be used, for example, as gene therapy or cell therapy drugs, or as vaccines, for the treatment and / or prevention of diseases. This depends on the specific type and use of the target DNA sequence inserted into the minicircle DNA. Diseases that can be treated by gene therapy or cells include, but are not limited to, cancer or tumors, eye diseases, rheumatoid arthritis, osteoarthritis, osteochondral damage, and acute lung injury.

[0111] The beneficial effects of the present invention are as follows: The present invention provides a highly efficient and low-toxicity method and kit for preparing DNA carriers, which can be applied to research and development, preclinical studies, process development, and clinical studies in the fields of gene therapy and cell therapy, thereby promoting rapid development in these fields.

[0112] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are used solely for illustrative purposes and do not limit the scope of the invention. In the following examples, experimental methods that do not specify conditions are generally carried out under conventional conditions or conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are calculated as weight percentages and weight parts. Experiments in the examples or test cases of the present invention that do not specify conditions are generally carried out under conventional conditions or conditions recommended by the raw material / product manufacturer, and reagents that do not specify a particular source are commercially available conventional reagents.

[0113] Example 1. Preparation of minicircle DNA expressing EGFP The EGFP expression cassette is designed between two RS sequences. After inducing CinH resolverase expression with arabinose and promoting plasmid recombination, the microplasmid is removed by a combination of EcoR V restriction enzyme and T5 exonuclease. Following DNA purification, a minicircle DNA capable of expressing EGFP (Figure 2a) is obtained.

[0114] Monoclonal E. coli carrying the EGFP-expressing cassette parent plasmid is inoculated into LB medium and incubated overnight in a constant temperature incubator at 200 rpm and 37°C. After overnight incubation, 1% arabinose (BBI, A610071-0100) is added to induce recombination, and the cells are incubated under the same conditions for 1 hour. The recombinated intermediate product DNA (including the recombinated minicircle DNA, microplasmid DNA, and residual parent plasmid DNA) is then extracted and purified. The microplasmid and parent plasmid contain specific restriction enzyme sites for EcoR V-restrictive endonuclease, but do not contain minicircle DNA. The intermediate product DNA is treated with EcoR V-restrictive endonuclease (NEB, R3195L) for 3 hours or overnight to enzymatically cleave the microplasmid and parent plasmid into linear DNA. The T5 exonuclease (NEB, M0663L) is added and digested for 3 hours or overnight to remove linear DNA, leaving only minicircle DNA. After purification, the minicircle DNA is obtained (Figure 2b).

[0115] Example 2. Preparation of transposon minicircle DNA carrying a CD19-CAR expression cassette. The CD19-CAR expression cassette is constructed on a parent plasmid capable of preparing minicircles. The recombinant microplasmid and parent plasmid contain specific restriction enzyme sites for EcoR V-restrictive endonuclease, but lack minicircle DNA leaves.

[0116] Monoclonal E. coli carrying the CD19-CAR expression cassette parent plasmid is inoculated into LB medium and incubated overnight in a constant temperature incubator at 200 rpm and 37°C. After overnight incubation, 1% arabinose (BBI, A610071-0100) is added to induce recombination, and the cells are incubated under the same conditions for 1 hour. The recombinated intermediate product DNA (including the recombinated minicircle DNA, microplasmid DNA, and residual parent plasmid DNA) is then extracted and purified. The microplasmid and parent plasmid contain specific restriction enzyme sites for EcoR V restriction endonuclease, but do not contain minicircle DNA. The intermediate product DNA is treated with EcoR V restriction endonuclease (NEB, R3195L) for 3 hours or overnight to enzymatically cleave the microplasmid and parent plasmid into linear DNA. The T5 exonuclease (NEB, M0663L) was added and digested for 3 hours or overnight to remove linear DNA, leaving only minicircle DNA, which was then purified to obtain the minicircle DNA (Figure 3).

[0117] Example 3. Preparation of minicircle DNA with a transposase expression cassette. The transposase expression cassette is constructed on a parent plasmid capable of preparing minicircles. The recombinant microplasmid and parent plasmid contain specific restriction enzyme sites for EcoR V-restrictive endonuclease but lack minicircle DNA leaves.

[0118] Monoclonal E. coli carrying a transposase-expressing cassette parent plasmid is inoculated into LB medium and incubated overnight in a constant temperature incubator at 200 rpm and 37°C. After overnight incubation, 1% arabinose (BBI, A610071-0100) is added to induce recombination, and the cells are incubated under the same conditions for 1 hour. The recombinated intermediate product DNA (including the recombinated minicircle DNA, microplasmid DNA, and residual parent plasmid DNA) is then extracted and purified. The microplasmid and parent plasmid contain specific restriction enzyme sites for EcoR V-restrictive endonuclease, but do not contain minicircle DNA. The intermediate product DNA is treated with EcoR V-restrictive endonuclease (NEB, R3195L) for 3 hours or overnight to enzymatically cleave the microplasmid and parent plasmid into linear DNA. The T5 exonuclease (NEB, M0663L) was added and digested for 4 hours or overnight to remove linear DNA, leaving only minicircle DNA, which was then purified to obtain the minicircle DNA (Figure 4).

[0119] Example 4. Minicircle DNA that delivers exogenous genes to T cells as a delivery carrier, superior to plasmid DNA. EGFP-expressing plasmid (400 ng) and EGFP-expressing minicircle DNA (MD) (400 ng) or molar amount (176 ng) were delivered to T cells by electroporation (Lonza, AAF-1003X). After electroporation, the cells were cultured in RPMI-1640 medium (Hyclone, SH30027.01) containing 10% FBS (Hyclone, SV30208.02). EGFP positivity and cell viability were detected using flow cytometry (Agilent, NovoCyte3110) 24 hours and 5 days after culture. Cells stained positive with 7AAD (BD, 559925) were considered dead cells. Results are the mean ± SEM of three repeated trials. Statistical significance of the data was analyzed by t-tests, where ** represents P<0.01 and *** represents P<0.001.

[0120] The results show that when the same amount of DNA mass is electroporated, the minicircle DNA group exhibits a higher positive rate and MFI than the plasmid group. Furthermore, at the same molar mass, the minicircle DNA group still shows significantly higher EGFP positive rate, cell viability, and MFI than the plasmid group. Comparing the two groups of minicircles with different dosages, the low-dose minicircle group shows lower EGFP positive rate and MFI compared to the high-dose group, but the low-dose group shows significantly higher viability compared to the high-dose group. Because minicircle DNA exhibits lower cytotoxicity and higher expression levels compared to plasmid DNA, its practicality as a DNA carrier in gene therapy or cell therapy is greatly improved (Figure 5).

[0121] All documents referenced in this invention are cited as references in this application, as if each document were cited individually. Furthermore, after reading the above teachings of this invention, persons skilled in the art can make various changes or modifications to the invention, and these equivalent forms are also included within the scope defined by the claims appended to this application.

Claims

1. Recombinant plasmid, The plasmid described above is It includes elements such as a prokaryotic replication origin, a prokaryotic selection marker, a recombinase coding sequence, a regulatory and controllable expression element that controls recombinase expression, a multicloning site into which a target DNA sequence can be inserted, and two recombinase recognition sites located on either side of the multicloning site. Furthermore, depending on the arrangement of the elements described above, the plasmid is cleaved at two recombinase-recognition sites by the expressed recombinase and then classified into a microplasmid and minicircle DNA, where the formed minicircle DNA includes a multicloning site into which a target DNA sequence can be inserted, the target DNA sequence inserted therein, and a remaining recombinase-recognition site. The recombinant plasmid is characterized in that the recombinase is a CinH resolver, and the recombinase identification site has a nucleotide sequence or a derived sequence thereof as shown in SEQ ID NO:

3.

2. The CinH resolver includes wild-type CinH resolver and its mutant, wherein the mutant is characterized in that the CinH resolver retains the activity to identify and cleave the recombinase identification site as shown in SEQ ID NO:

3. The recombinant plasmid according to claim 1.

3. The target DNA sequence is selected from the group consisting of a functional DNA sequence segment, a functional RNA expression cassette set, a functional protein expression cassette set, or a combination thereof. The recombinant plasmid according to claim 1.

4. The target DNA sequence is characterized by containing a chimeric antigen receptor (CAR) coding sequence. Recombinant plasmid according to claim 1

5. The regulating and controllable expression element that controls the recombinase expression is selected from the group consisting of the pBAD / AraC system of the arabinose operator group, the LacI / LacO system of the lactose / IPTG operator group, the rtTA / TRE system of the tetracycline operator group, a heat shock induction system based on heat shock protein 70 or 90, and a photoinduction system based on blue light-sensing protein YFI and downstream FixJ / FixK2 / cI / pR. The recombinant plasmid according to claim 1.

6. The recombinant plasmid further comprises a microplasmid identification and removal element, the element being located within a microplasmid generated after the plasmid is cleaved by the expressed recombinase. The recombinant plasmid according to claim 1.

7. The microplasmid identification and removal elements are selected from the group consisting of restriction enzyme cleavage sites, LacO sequences, specifically designed oligonucleotide or deoxynucleotide complementary sequences, specifically designed inactivated Cas ribonucleoprotein (e.g., dead Cas9, Cas12, Cas3, Cas8, Cas10, and Cas13 RNP) recognition sequences, specifically designed zinc finger DNA binding domain recognition sequences, or specifically designed transcription activator-like effector DNA binding domain recognition sequences, or combinations thereof. The recombinant plasmid according to claim 6.

8. The recombinant plasmid further comprises a minicircle DNA identification and separation element, the element being located within the minicircle DNA generated after the plasmid is cleaved by the expressed recombinase. The recombinant plasmid according to claim 1.

9. The minicircle DNA recognition and separation element is characterized by being selected from the group consisting of a LacO sequence, a specifically designed oligonucleotide or deoxynucleotide complementary sequence, a specifically designed inactivated Cas ribonucleoprotein (e.g., dead Cas9, Cas12, Cas3, Cas8, Cas10 and Cas13 RNP, etc.) recognition sequence, a specifically designed zinc finger DNA binding domain recognition sequence, or a specifically designed transcription activator-like effector DNA binding domain recognition sequence, or a combination thereof. The recombinant plasmid according to claim 8.

10. A reaction system for generating minicircle DNA, The aforementioned reaction system is (Z1) A recombinant plasmid according to any one of claims 1 to 9, (Z2) comprising an induction reagent or induction conditions for inducing the expression of the encoded recombinase in the recombinant plasmid, In another preferred example, the reaction system is characterized in that the derivative reagent is selected from the group consisting of arabinose, lactose, or IPTG and tetracycline.

11. The reaction system is characterized by further comprising a microplasmid identification and removal reagent, and / or a minicircle DNA identification and separation reagent. The reaction system according to claim 10.

12. This is a kit for generating miniature DNA. The aforementioned kit is (C1) A recombinant plasmid according to any one of claims 1 to 9, and (C2) The kit comprising, optionally, an induction reagent for inducing recombinase expression in the recombinant plasmid.

13. The aforementioned kit is (C3) Microplasmid identification and removal reagents, which act on microplasmid identification and removal elements in recombinant plasmids and the generated microplasmids, and are used to remove microplasmids and unreacted recombinant plasmids from the product, and / or (C4) Further comprising a minicircle DNA identification and separation reagent, the reagent is characterized in that it acts on the minicircle DNA identification and separation elements in the minicircle DNA and is used to separate the minicircle DNA in the product. The kit according to claim 12.

14. A method for generating minicircle DNA, The above method includes the following steps: (S1) Introducing a recombinant plasmid according to any one of claims 1 to 9 into a prokaryotic cell, (S2) The prokaryotic cells are cultured and the expression of the recombinase encoded in the recombinant plasmid is induced to produce an intermediate product containing a microplasmid and minicircle DNA, and A method for producing minicircle DNA, characterized by separating the minicircle DNA from the intermediate product of (S3)(S2).

15. Minicircle DNA prepared by the method of claim 14, it is, Minicircle DNA prepared by the method of claim 14, comprising a multicloning site into which a target DNA sequence can be inserted, a target DNA sequence inserted therein, and a remaining recombinase identification site, wherein the recombinase identification site has a nucleotide sequence or a derived sequence thereof as shown in SEQ ID NO:

3.

16. A pharmaceutical composition, The aforementioned pharmaceutical composition, The pharmaceutical composition comprising (a) the minicircle DNA described in claim 15, and (b) a pharmaceutically acceptable carrier.

17. Uses of the recombinant plasmid according to any one of claims 1 to 9 in the preparation of a minicircle DNA drug.

18. Uses of the minicircle DNA according to claim 15 in the preparation of a drug for treating and / or preventing a disease.

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