Preparation method and application of minicircle DNA

Abstract

The invention provides a preparation method and application of minicircle DNA (deoxyribonucleic acid). Specifically, the invention provides a recombinant plasmid which carries a CinH dissociation enzyme (CinH resolvase) and a specific recognition site of the CinH resolvase and is used for generating the minicircle DNA. The invention also provides a method for producing the minicircle DNA by using the recombinant plasmid, a kit which contains the recombinant plasmid and is used for producing the minicircle DNA, and application of the minicircle DNA prepared by using the method disclosed by the invention in gene therapy and cell therapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for preparing minicircle DNA and its application in gene therapy and cell therapy. Background Art

[0002] DNA vectors are widely used in the fields of vaccines, gene therapy, cell therapy, etc. Traditional DNA vectors are plasmids with prokaryotic cell replication origins and antibiotic resistance genes. However, these DNA sequences derived from prokaryotes will inhibit the expression of cis-linked mammalian cell expression cassettes, stimulate the DNA sensing pathway, activate T, B lymphocytes, NK cells and monocytes to promote immune inflammatory responses, and induce programmed cell death. These reactions limit the effectiveness of the application of plasmid DNA vectors. The spread of prokaryotic cell replication origins and antibiotic resistance genes is also one of the risks of using plasmid DNA vectors.

[0003] Supercoiled circular DNA with the prokaryotic cell replication origin and antibiotic resistance gene removed was first prepared by Darquet et al. in 1997, and showed superior protein expression ability to plasmid DNA vectors in a variety of mammalian cells. This type of DNA is called minicircle DNA. Chen et al. further used mice as a model to prove that when the DNA vector was delivered to the mouse liver, minicircle DNA could express 200-560 times more proteins with clinical medical potential in peripheral blood than plasmid DNA. After that, the high stability and high expression ability of minicircle DNA were widely used in the development of gene therapy or cell therapy for various diseases. Minicircle DNA was tried as a gene therapy vector in preclinical studies of rheumatoid arthritis or osteoarthritis. Rim et al. expressed BMP2 and TGFβ3 in minicircles, induced mesenchymal stem cells derived from hiPSCs to differentiate into chondrocytes, and successfully treated osteochondral injury in a rat model. Florian et al. used microcircular DNA to express Angiopoietin 1 protein in mesenchymal stem cells, which improved the efficacy of mesenchymal stem cells in relieving acute lung injury. Technology is also being developed to use microcircular DNA to deliver proteins with clinical therapeutic potential to retinal epithelial cells, retinal ganglion cells or photoreceptor cells to treat eye-related diseases. In general, microcircular DNA is a highly potential and widely used gene therapy and cell therapy vector.

[0004] The preparation of minicircle DNA relies on the recombinase to split the parent plasmid carrying two recombination sites into two smaller circular DNAs. By designing the relative positions of the recombination sites on the parent plasmid and other units, one of the circular DNAs can carry the functional protein or functional RNA expression cassette required for gene therapy or cell therapy, i.e., minicircle DNA, while the other circular DNA carries the unit of the prokaryotic replication origin and antibiotic resistance gene to be removed, generally called miniplasmid. Currently, the recombinases reported to be used for the preparation of minicircle DNA include bacteriophage λ integrase, bacteriophage ΦC31 integrase, Cre recombinase, and ParA resolvase. The minicircles prepared by bacteriophage λ integrase, ΦC31 integrase, and Cre recombinase all carry dimer to multimer contamination. Since the sequences of these dimers and multimers are consistent with the target minicircle DNA, it increases the difficulty of obtaining high-purity minicircle DNA in the future. The recombination efficiency of ParA resolvase is close to 100%, and there is no dimer or multimer contamination in the recombined product. To obtain high-purity minicircles, the technology of removing residual mother plasmids and miniplasmids must also be used. Common practices include in vitro enzyme digestion to remove mother plasmids and miniplasmids; in vivo enzyme digestion to remove mother plasmids and miniplasmids, affinity chromatography to remove mother plasmids and miniplasmids or purify minicircle DNA, etc. Monolithic column chromatography is also used to further purify supercoiled minicircle DNA to meet clinical use standards.

[0005] Adoptive cellular immunotherapy has achieved great results in recent years. With the approval of KYMRIAH (Novartis Pharmaceuticals Corporation), YESCARTA and TECARTUS (Kite Pharma, Inc.), BREYANZI (Juno Therapeutics, Inc.), ABECMA (Celgene Corporation), CARVYKTI (Janssen Biotech, Inc.) by the U.S. Food and Drug Administration, it is used in the clinical treatment of relapsed and refractory hematological tumors, including large B lymphoma, mantle cell lymphoma and multiple myeloma and other malignant hematological tumors, providing a new and reliable treatment strategy that can prolong the patient's survival and quality of life. These adoptive immunotherapies approved by the FDA are all prepared with retroviral or lentiviral vectors. 65% and 54% of the insertion sites of clinically used CAR-T prepared by retroviral vectors and lentiviral vectors are located in RefSeq genes, respectively. The insertion of exogenous genes in these RefSeq genes has caused doubts about the application of retroviral and lentiviral vectors in gene therapy and cell therapy. The Sleeping Beauty transposon system has been proven to be able to insert into human genomes almost randomly, and is safer for gene therapy than retrovirus, lentivirus and other transposon-based vectors. However, the biggest obstacle to the use of non-viral vector systems in gene therapy or cell therapy is the low delivery efficiency of the vector.

[0006] Minicircle DNA can greatly improve the efficiency of non-viral DNA vectors due to its high stability and high expression level. Therefore, using minicircle DNA of the Sleeping Beauty transposon system to carry the CAR gene to prepare CAR-T can effectively replace high-risk retroviral or lentiviral vectors.

[0007] Therefore, there is a great need in this field for the research and development of minicircle DNA drugs. Summary of the invention

[0008] The purpose of the present invention is to provide a recombinant plasmid, which carries a prokaryotic replication origin, a prokaryotic screening marker, a regulatable expression element for controlling the expression of a recombinase, a recombinase, two recombinase recognition sites, and a multiple cloning site for inserting a target DNA sequence, and is used to produce a circular supercoiled minicircle DNA that does not contain a prokaryotic DNA sequence such as a prokaryotic replication origin and a prokaryotic screening marker. And a method for producing minicircle DNA using the recombinant plasmid.

[0009] The first aspect of the present invention provides a recombinant plasmid, wherein the plasmid comprises the following elements:

[0010] Prokaryotic replication origin, prokaryotic selection marker, recombinase coding sequence, regulatable expression element for controlling recombinase expression, multiple cloning site for inserting target DNA sequence and two recombinase recognition sites located on both sides of the multiple cloning site;

[0011] Furthermore, the arrangement of the above elements allows the plasmid to be divided into a mini-plasmid and a mini-circle DNA after being cut by the expressed recombinase at two recombinase recognition sites, wherein the formed mini-circle DNA comprises a multiple cloning site for inserting a target DNA sequence and the target DNA sequence inserted therein, and a residual recombinase recognition site;

[0012] The recombinase is CinH resolvase, and the recombinase recognition site has a nucleotide sequence as shown in SEQ ID NO: 3 or a derivative sequence thereof.

[0013] In another preferred embodiment, the CinH resolvase includes a wild-type CinH resolvase and a mutant thereof, wherein the mutant retains the activity of the CinH resolvase in recognizing and cleaving the recombinase recognition site as shown in SEQ ID NO:3.

[0014] In another preferred embodiment, the amino acid sequence of the CinH resolvase is shown in SEQ ID NO:1.

[0015] In another preferred embodiment, the nucleotide sequence encoding the CinH resolvase is shown as SEQ ID NO:2.

[0016] In another preferred embodiment, the two recombinase recognition sites located on both sides of the multiple cloning site are oriented in the same direction, ie →multiple cloning recognition site→.

[0017] In another preferred embodiment, the target DNA sequence is selected from the following group: a functional DNA sequence, a group of functional RNA expression cassettes, a group of functional protein expression cassettes, or a combination thereof.

[0018] In another preferred embodiment, the target DNA sequence is a protein expression cassette for expressing a transposase in eukaryotic cells; the transposase includes (but is not limited to) Sleepy Beauty, PiggyBac, and Tol2.

[0019] In another preferred embodiment, the target DNA sequence is a transposon, which cooperates with a transposase to stably insert the genetic information it carries into a prokaryotic or eukaryotic cell genome.

[0020] In another preferred example, the target DNA sequence includes a chimeric antigen receptor (CAR) coding sequence.

[0021] In another preferred embodiment, the regulatable expression element for controlling the expression of the recombinase is selected from the following groups: 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, the heat shock inducible system based on heat shock protein 70 or 90, and the light inducible system based on the blue light sensing protein YFI and downstream FixJ / FixK2 / cI / pR.

[0022] In another preferred embodiment, the recombinant plasmid further comprises: a mini-plasmid recognition and removal element, which is located in the mini-plasmid generated after the plasmid is cut by the expressed recombinase.

[0023] In another preferred embodiment, the miniplasmid recognition and removal element is selected from the following group: a restriction enzyme site, 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 (Zinc finger DNA binding domain) recognition sequence or a specifically designed transcription activator-like effector DNA binding domain (Transsciption activator-like effector DNA binding domain) recognition sequence, or a combination thereof.

[0024] In another preferred embodiment, the miniplasmid recognition and removal element is at least one restriction enzyme cutting site.

[0025] In another preferred embodiment, the recombinant plasmid further comprises: a minicircle DNA recognition and separation element, which is located in the minicircle DNA generated after the plasmid is cut by the expressed recombinase.

[0026] In another preferred embodiment, the minicircle DNA recognition and separation element is selected from the following group: LacO sequence, specifically designed oligonucleotide or deoxynucleotide complementary sequence, specifically designed inactivated Cas ribonucleoprotein (such as dead Cas9, Cas12, Cas3, Cas8, Cas10 and Cas13 RNP, etc.) recognition sequence, specifically designed zinc finger DNA binding domain recognition sequence or specifically designed transcription activator-like DNA binding domain recognition sequence, or a combination thereof.

[0027] In another preferred embodiment, the recombinant plasmid further comprises a miniplasmid recognition and removal element, and a minicircle DNA recognition and separation element.

[0028] A second aspect of the present invention provides a reaction system for producing minicircle DNA, the reaction system comprising:

[0029] (Z1) the recombinant plasmid as described in the first aspect of the present invention;

[0030] (Z2) an inducing agent or inducing condition for inducing the expression of the encoded recombinase in the recombinant plasmid;

[0031] In another preferred embodiment, the inducing agent is selected from the following group: arabinose, lactose or IPTG, tetracycline.

[0032] In another preferred embodiment, the induction conditions include heat shock or light induction.

[0033] In another preferred embodiment, the reaction system further comprises: a miniplasmid recognition and removal reagent, and / or a minicircle DNA recognition and separation reagent.

[0034] In another preferred embodiment, the miniplasmid recognition and removal reagent acts on the miniplasmid recognition and removal elements in the recombinant plasmid and the generated miniplasmid to remove the miniplasmid and the unreacted recombinant plasmid in the product.

[0035] In another preferred example, the minicircle DNA recognition and separation reagent acts on the minicircle DNA recognition and separation element in the minicircle DNA to separate the minicircle DNA in the product.

[0036] In another preferred embodiment, the miniplasmid recognition and removal reagent is selected from: restriction endonucleases, LacI proteins, oligonucleotides or deoxynucleotides of specific designed sequences, inactivated Cas ribonucleoproteins (such as dead Cas9, Cas12, Cas3, Cas8, Cas10 and Cas13 RNP, etc.), zinc finger DNA binding domains, transsciption activator-like effector DNA binding domains, or combinations thereof.

[0037] In another preferred embodiment, the miniplasmid recognition and removal reagent is a restriction endonuclease.

[0038] In another preferred embodiment, the minicircle DNA recognition and separation reagent includes a protein that can bind to the minicircle DNA recognition and separation element in the minicircle DNA to form a stable DNA-protein complex; optionally, the protein is immobilized on a solid phase carrier.

[0039] In another preferred embodiment, the minicircle DNA recognition and separation reagent is selected from the following group: LacI protein, oligonucleotides or deoxynucleotides of specific designed sequences, inactivated Cas ribonucleoproteins (such as dead Cas9, Cas12, Cas3, Cas8, Cas10 and Cas13 RNP, etc.), zinc finger DNA binding domains, transcription activator-like DNA binding domains, or a combination thereof.

[0040] In a third aspect of the present invention, a kit for producing minicircle DNA is provided, the kit comprising:

[0041] (C1) the recombinant plasmid as described in the first aspect of the present invention; and

[0042] (C2) Optionally, an inducing agent that induces the expression of the recombinase in the recombinant plasmid.

[0043] In another preferred embodiment, the kit further comprises:

[0044] (C3) a miniplasmid recognition and removal reagent, which acts on the miniplasmid recognition and removal elements in the recombinant plasmid and the generated miniplasmid to remove the miniplasmid and the unreacted recombinant plasmid in the product; and / or

[0045] (C4) A minicircle DNA recognition and separation reagent, which acts on the minicircle DNA recognition and separation element in the minicircle DNA to separate the minicircle DNA in the product.

[0046] In another preferred embodiment, the inducing agent is selected from the following group: arabinose, lactose or IPTG, tetracycline.

[0047] In another preferred embodiment, the miniplasmid recognition and removal reagent is selected from: restriction endonucleases, LacI proteins, oligonucleotides or deoxynucleotides of specific designed sequences, inactivated Cas ribonucleoproteins (e.g., dead Cas9, Cas12, Cas3, Cas8, Cas 10 and Cas13 RNPs, etc.), zinc finger DNA binding domains, transcription activator-like DNA binding domains, or a combination thereof.

[0048] In another preferred embodiment, the miniplasmid recognition and removal reagent is a restriction endonuclease.

[0049] In another preferred embodiment, the minicircle DNA recognition and separation reagent includes a protein that can bind to the minicircle DNA recognition and separation element in the minicircle DNA to form a stable DNA-protein complex; optionally, the protein is immobilized on a solid phase carrier.

[0050] In another preferred embodiment, the minicircle DNA recognition and separation reagent is selected from: LacI protein, oligonucleotides or deoxynucleotides of specific designed sequences, inactivated Cas ribonucleoproteins (such as dead Cas9, Cas12, Cas3, Cas8, Cas10and Cas13 RNP, etc.), zinc finger DNA binding domains, transcription activator-like DNA binding domains, or a combination thereof.

[0051] In another preferred embodiment, the kit further comprises a label or instructions describing a method for producing minicircle DNA using the kit, wherein the method comprises inducing the expression of the recombinase in the recombinant plasmid in the presence of an inducing agent or inducing conditions.

[0052] In another preferred embodiment, the induction conditions include heat shock or light induction.

[0053] A fourth aspect of the present invention provides a method for producing minicircle DNA, the method comprising:

[0054] (S1) introducing the recombinant plasmid described in the first aspect of the present invention into a prokaryotic cell;

[0055] (S2) culturing the prokaryotic cell and inducing the expression of the recombinase encoded in the recombinant plasmid, thereby producing an intermediate product containing a miniplasmid and a minicircle DNA; and

[0056] (S3) Separating the minicircle DNA from the intermediate product of (S2).

[0057] In another preferred embodiment, in step (S2), the expression of the recombinase is induced by adding an inducing agent to activate the regulatable expression element controlling the expression of the recombinase in the recombinant plasmid.

[0058] In another preferred embodiment, in step (S2), the regulatable expression element controlling the expression of the recombinase in the recombinant plasmid is activated by induction conditions, thereby inducing the expression of the recombinase.

[0059] In another preferred embodiment, in step (S3), the minicircle DNA is separated by using the minicircle DNA recognition and separation element in the minicircle DNA and a minicircle DNA recognition and separation reagent.

[0060] In another preferred embodiment, in step (S3), a miniplasmid recognition and removal reagent is used to remove the miniplasmid and unreacted recombinant plasmid in the intermediate product, thereby isolating the minicircle DNA.

[0061] In another preferred embodiment, in step (S3), the miniplasmid recognition and removal reagent is first used to remove the miniplasmid and unreacted recombinant plasmid in the intermediate product, and then the minicircle DNA recognition and separation element in the minicircle DNA is utilized to separate the minicircle DNA using the minicircle DNA recognition and separation reagent.

[0062] In another preferred embodiment, the prokaryotic cells include (but are not limited to): Escherichia coli, Agrobacterium tumefaciens, Bacillus subtilis, Lactobacillus, Salmonella, Bifidobacterium, Listeria monocytogenes, Pseudomonas, Neisseria meningitidis, and Vibrio cholerae.

[0063] In a fifth aspect, the present invention provides a minicircle DNA prepared by the method according to the fourth aspect of the present invention, comprising:

[0064] A multiple cloning site for inserting a target DNA sequence and the target DNA sequence inserted therein, and a residual recombinase recognition site; the recombinase recognition site has a nucleotide sequence as shown in SEQ ID NO: 3 or a derivative sequence thereof.

[0065] In another preferred embodiment, the target DNA sequence is selected from the following group: a functional DNA sequence, a group of functional RNA expression cassettes, a group of functional protein expression cassettes, or a combination thereof.

[0066] In another preferred embodiment, the target DNA sequence is a protein expression cassette for expressing a transposase in eukaryotic cells; the transposase includes (but is not limited to) Sleepy Beauty, PiggyBac, and Tol2.

[0067] In another preferred embodiment, the target DNA sequence is a transposon, which cooperates with a transposase to stably insert the genetic information it carries into a prokaryotic or eukaryotic cell genome.

[0068] In another preferred example, the target DNA sequence includes a chimeric antigen receptor (CAR) coding sequence.

[0069] In another preferred example, the minicircle DNA further comprises minicircle DNA recognition and separation elements.

[0070] In another preferred embodiment, the minicircle DNA recognition and separation element is selected from: 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 DNA binding domain recognition sequence, or a combination thereof.

[0071] The sixth aspect of the present invention provides a pharmaceutical composition, comprising:

[0072] (a) the minicircle DNA as described in the fifth aspect of the present invention; and (b) a pharmaceutically acceptable carrier.

[0073] In another preferred embodiment, the pharmaceutical composition is used for treating and / or preventing a disease.

[0074] In another preferred embodiment, the pharmaceutical composition is used for gene therapy or cell therapy.

[0075] In another preferred embodiment, the pharmaceutical composition is used as a vaccine for treating and / or preventing diseases.

[0076] The seventh aspect of the present invention provides use of the recombinant plasmid described in the first aspect of the present invention in the preparation of minicircle DNA drugs.

[0077] In another preferred embodiment, the minicircle DNA drug is used for gene therapy or cell therapy.

[0078] The eighth aspect of the present invention provides use of the minicircle DNA as described in the fifth aspect of the present invention in the preparation of a drug for the treatment and / or prevention of a disease.

[0079] In another preferred embodiment, the disease can be treated by gene therapy or cell therapy.

[0080] In another preferred embodiment, the disease includes (but is not limited to) cancer or tumor, eye disease, rheumatoid arthritis, osteoarthritis, osteochondral injury, acute lung injury.

[0081] The ninth aspect of the present invention provides a method for treating and / or preventing a disease, comprising administering the minicircle DNA as described in the fifth aspect of the present invention to a subject in need thereof.

[0082] In another preferred embodiment, the minicircle DNA is used as a delivery vector to deliver therapeutic / preventive components into a subject.

[0083] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0084] In another preferred embodiment, the disease can be treated by gene therapy or cell therapy.

[0085] In another preferred embodiment, the disease includes (but is not limited to) cancer or tumor, eye disease, rheumatoid arthritis, osteoarthritis, osteochondral injury, acute lung injury.

[0086] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 A schematic diagram showing the positional configuration of the minicircle DNA parent plasmid, miniplasmid, and various minicircle DNA elements is shown.

[0088] Figure 2a The process of preparing transposon minicircles carrying the EGFP expression cassette is shown.

[0089] Figure 2b It shows that the transposon minicircle DNA carrying the EGFP expression cassette was successfully prepared.

[0090] Figure 3 The transposon minicircle DNA preparation carrying the CD19-CAR expression cassette is shown.

[0091] Figure 4 The preparation of minicircle DNA carrying the transposase expression cassette is shown.

[0092] Figure 5 It was shown that minicircle DNA is significantly superior to plasmid DNA as a delivery vector for delivering foreign genes to T cells. DETAILED DESCRIPTION

[0093] After extensive and in-depth research, the inventors have developed a new type of recombinant plasmid for the first time. The arrangement of the various elements carried by the recombinant plasmid provided by the present invention allows the parent plasmid to be separated into two smaller circular DNAs after the recombinase is induced to express; one of them becomes a miniplasmid, containing non-target sequences such as prokaryotic replication origin, prokaryotic screening marker, regulatable expression elements for controlling recombinase expression, and recombinase coding sequence; the other is a minicircle DNA containing only the target sequence, residual multiple cloning sites, and residual recombinase recognition sites. The miniplasmid can be removed by means of a specific restriction enzyme site or a specific recognition sequence on the miniplasmid to purify the minicircle DNA; or the minicircle DNA can be purified by a specific recognition sequence on the minicircle DNA.

[0094] On this basis, the present invention has been completed.

[0095] the term

[0096] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0097] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0098] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0099] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

[0100] As used herein, the term "optional" or "optionally" means that the event or situation described later can occur but does not have to occur. For example, "optionally, an inducing agent that induces the expression of the recombinase in the recombinant plasmid" means that the inducing agent can be present but does not have to be present.

[0101] "Sequence identity" as used herein refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions. The sequence identity between the sequences described herein and the sequences with which they are identical 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%, 100%.

[0102] Recombinant plasmid of the present invention

[0103] In one aspect of the present invention, a recombinant plasmid is provided, comprising the following elements: a prokaryotic origin of replication, a prokaryotic screening marker, a recombinase coding sequence, a regulatable expression element for controlling the expression of the recombinase, a multiple cloning site for inserting a target DNA sequence, and two recombinase recognition sites located on both sides of the multiple cloning site; the arrangement of the above-mentioned elements allows the plasmid to be divided into a miniplasmid and a minicircle DNA after being cut by the recombinase that can be expressed at two recombinase recognition sites; wherein the minicircle DNA formed comprises a multiple cloning site for inserting a target DNA sequence and a target DNA sequence inserted therein, and a residual recombinase recognition site; the miniplasmid comprises a prokaryotic origin of replication, a prokaryotic screening marker, a regulatable expression element for controlling the expression of the recombinase, a recombinase coding sequence and other non-target sequences. The recombinase carried by the recombinant plasmid of the present invention is a CinH resolvase, and its corresponding recombinase recognition site has a nucleotide sequence or a derivative sequence thereof as shown in SEQ ID NO:3.

[0104] In the present invention, CinH resolvase includes wild-type CinH resolvase and mutants thereof, wherein the mutant retains the activity of CinH resolvase in recognizing and cleaving the recombinase recognition site as shown in SEQ ID NO: 3. Preferably, the mutant has at least 85% sequence identity compared to the amino acid sequence of wild-type CinH resolvase (SEQ ID NO: 1). In one embodiment of the present invention, the CinH resolvase encoding sequence has a nucleotide sequence as shown in SEQ ID NO: 2.

[0105] Amino acid sequence of wild-type CinH resolvase (SEQ ID NO: 1)

[0106] MKGQKVGYVRVSSVEQNTGRQLEGIEVDRIFVDRASGKNTDRPKFQEMLNYVREGDRVIVHSMDRFARSLKDLVTEVDKLVKRGIAIQFVKENITFTAQSTPMDNLMLQLMGAFAQFEREIILERQKEGIKIAAAQGKYKGRVHKLNPDQAKALLQAWKEGKYSSKVDLAKAFGISRQAVYRYLKQIN*

[0107] CinH resolvase coding sequence (SEQ ID NO: 2)

[0108] Gtgaaaggccaaaaagtagggtatgtgcgagtgagttcggtcgagcaaaatacagggcgtcaacttgagggaattgaagtcgaccggatttttgttgaccgtgcttcgggtaaaaataccgaccgaccgaaatttcaagaa atgttgaactatgtccgggaaggggatagggtgattgtacattccatggatcgttttgcacgaagtctaaaagatttggtcactgaagtagataaactggtcaaaagagggatcgccatccagtttgtaaaagagaatatta cttttactgcccaatccacgccgatggataatttgatgctgcaactgatgggtgcttttgctcaattcgagcgggaaattatcttagaacgtcagaaagaagggattaagatcgcagcagctcagggcaaatataaaggtcg tgtccataaattgaatccagatcaagctaaagcattactgcaagcctggaaagaagggaagtattcatcaaaagttgatctggcaaaagcgtttggtatcagtagacaggctgtctatcggtatttaaaacaaattaattag

[0109] As used herein, the term "recombinase coding sequence" refers to any sequence required for the expression of the recombinase, such as a promoter, a termination sequence, etc. The recombinase itself can be any recombinase capable of catalyzing the recombination of the parent plasmid into a miniplasmid and a separable minicircle DNA on a defined recognition sequence. In the present invention, the recombinase specifically refers to the CinH resolvase.

[0110] As used herein, the terms "recombinase recognition site" and "RS2" can be used interchangeably and refer to sequences that can be recognized by a recombinase. Two recombinase recognition sites are carried on the recombinant plasmid of the present invention, and the two recombinase recognition site sequences must be arranged on the plasmid in such a way that the recombinase can produce miniplasmids and minicircle DNA by recognizing and cutting the sequence. This means that the recombinase recognition site should be located on both sides of the multiple cloning site, and the directions of the two recombinase recognition sites are in the same direction, i.e. →multiple cloning recognition site→, to produce minicircle DNA containing the target DNA sequence inserted into the multiple cloning site. The minicircle DNA produced may contain at least a portion of one and / or another recombinase recognition site sequence. The two recombinase recognition site sequences may be identical, or derived from the same recombinase recognition sequence. In the present invention, the recombinase recognition site has a nucleotide sequence as shown in SEQ ID NO:3 or a sequence derived therefrom.

[0111] Recombinase recognition site (SEQ ID NO: 3)

[0112] CGTTACTTTGGGGTATACCCTAAAGTTACAATATAAAAGTTCTTAAAACTATGTAACATTTAAATGATTTTTAACCATATATAACATGTAACTTTGATATTTAAAGTTTATAATTTACG

[0113] The term "multiple cloning site" refers to a site comprising at least two restriction endonuclease sites, preferably, it comprises multiple sites for multiple restriction endonucleases.

[0114] As used herein, the term "target DNA" refers to a DNA sequence that can be inserted into the multiple cloning site of the above-mentioned recombinant plasmid, is contained in the generated mini-ring DNA, and is ultimately delivered to the subject to perform biological functions, which can be a functional DNA sequence, a set of functional RNA expression cassettes, and a set of functional protein expression cassettes. Preferably, the target DNA is a protein gene with a therapeutic effect. Since mini-ring DNA is mainly used for treatment, it preferably contains a protein gene with a therapeutic effect. For example, a gene encoding a chimeric antigen receptor (CAR) is inserted into the multiple cloning site of the recombinant plasmid of the present invention to produce a mini-ring DNA for expressing CAR. However, of course, any gene encoding any protein can be inserted, in which case the mini-ring can be used for detection methods, biochemical pathway research, and the like.

[0115] The recombinant plasmid of the present invention comprises a controllable expression element for controlling the expression of the recombinase, and by providing a regulatory element for controlling the expression of the recombinase, the expression of the recombinase can be inhibited or induced, which allows the recombinase to be expressed when necessary. Using this system, plasmids can be produced in large quantities before inducing the expression of the recombinase, thereby achieving the maximum efficiency of minicircle DNA production. Various regulatory elements are well known to those skilled in the art. Preferably, the regulatory element for controlling the expression of the recombinase includes a strong promoter. This allows the recombinase to be fully expressed to catalyze the recombination of the total amount of plasmids present in the sample, thereby achieving the maximum yield of minicircle DNA. In one embodiment of the present invention, the pBAD / AraC system of the arabinose operator group is used to control the expression of the recombinase. As long as arabinose is not present in the culture system, the control system will inhibit the expression of the recombinase. When arabinose, such as L-arabinose, is added to the culture medium, the recombinase expression is induced. This is a very simple and effective method for regulating the expression of the recombinase.

[0116] As used herein, a "prokaryotic selection marker" may be an antibiotic resistance gene. This is a method well known in the art for selecting bacteria transfected with a plasmid containing a prokaryotic selection marker in order to efficiently produce a high copy number of the plasmid.

[0117] The recombinant plasmid of the present invention also includes a minicircle DNA and / or miniplasmid identification sequence for identifying and isolating minicircle DNA. The identification sequence provides an effective method for isolating minicircle DNA from miniplasmid, which further improves the efficiency of minicircle DNA production.

[0118] The minicircle DNA or miniplasmid identification sequence is any sequence that will be present on the minicircle but not on the miniplasmid (or vice versa) and is capable of identifying and isolating the minicircle DNA (or removing the miniplasmid). In one embodiment of the present invention, the identification sequence is present on the miniplasmid, which is referred to as the "miniplasmid identification and removal element" in the present invention. In another embodiment of the present invention, the identification sequence is present on the minicircle DNA, which is referred to as the "minicircle DNA identification and separation element" in the present invention. The above identification sequence may only be present on the miniplasmid or minicircle DNA, but is also applicable to the simultaneous presence of both on the miniplasmid and the minicircle DNA.

[0119] The miniplasmid recognition and removal element can be a specific sequence that is removed from the product after being recognized by a specific reagent. For example, the miniplasmid recognition and removal element can be a restriction endonuclease recognition site that is cut into linear DNA after being recognized by the corresponding restriction endonuclease and further digested and removed from the product.

[0120] The minicircle DNA recognition and separation element can be a specific sequence that will bind to a given ligand, thereby complexing the minicircle DNA with the ligand, and then separating the complex from the miniplasmid, cellular components, etc., for example, by immobilizing the ligand (the ligand is immobilized on a solid phase carrier, such as magnetic beads, agarose beads, agarose columns, etc.).

[0121] Kit of the present invention

[0122] The present invention also provides a kit for producing minicircle DNA, which comprises the recombinant plasmid described in the first aspect of the present invention.

[0123] In one embodiment of the present invention, the kit comprises the following components: the recombinant plasmid of the first aspect of the present invention; an inducing agent for inducing the expression of the recombinase in the recombinant plasmid; a miniplasmid recognition and removal agent. In another embodiment of the present invention, the kit comprises the following components: the recombinant plasmid of the first aspect of the present invention; an inducing agent for inducing the expression of the recombinase in the recombinant plasmid; a minicircle DNA recognition and separation agent. In yet another embodiment of the present invention, the kit comprises the following components: the recombinant plasmid of the first aspect of the present invention; an inducing agent for inducing the expression of the recombinase in the recombinant plasmid; a miniplasmid recognition and removal agent and a minicircle DNA recognition and separation agent.

[0124] Wherein, the microplasmid recognition and removal reagent acts on the microplasmid recognition and removal element in the recombinant plasmid and the generated microplasmid, and is used to remove the microplasmid and the unreacted recombinant plasmid in the product. In the present invention, the microplasmid recognition and removal element and the microplasmid recognition and removal reagent can be selected from the following pairing combinations (microplasmid recognition and removal element / microplasmid recognition and removal reagent): restriction endonuclease recognition site / restriction endonuclease, LacO sequence / LacI protein, specifically designed oligonucleotide or deoxynucleotide complementary sequence / specifically designed sequence oligonucleotide or deoxynucleotide, specifically designed inactivated Cas ribonucleoprotein recognition sequence / inactivated Cas ribonucleoprotein, specifically designed zinc finger DNA binding domain recognition sequence / zinc finger DNA binding domain, or specifically designed transcription activator-like DNA binding domain recognition sequence / transcription activator-like DNA binding domain. In a preferred embodiment of the present invention, the miniplasmid recognition and removal reagent includes a restriction endonuclease, which recognizes the restriction endonuclease recognition site on the miniplasmid and the unreacted recombinant plasmid, and cuts the miniplasmid and the unreacted recombinant plasmid into linear DNA. Preferably, the reagent further comprises a linear DNA digestion reagent, such as T5 exonuclease, to digest and remove the linear DNA.

[0125] The minicircle DNA recognition and separation reagent acts on the minicircle DNA recognition and separation element in the minicircle DNA to separate the minicircle DNA in the product. In a preferred embodiment, the minicircle DNA recognition and separation reagent includes a protein that can bind to the minicircle DNA recognition and separation element in the minicircle DNA to form a stable DNA-protein complex; optionally, the protein is solidified on a solid phase carrier (e.g., magnetic beads, agarose beads, agarose columns, etc.). In the present invention, the minicircle DNA recognition and separation element and the minicircle DNA recognition and separation reagent can be selected from the following pairing combinations (minicircle DNA recognition and separation element / minicircle DNA recognition and separation reagent): LacO sequence / LacI protein, specifically designed oligonucleotide or deoxynucleotide complementary sequence / specifically designed sequence oligonucleotide or deoxynucleotide, specifically designed inactivated Cas ribonucleoprotein recognition sequence / inactivated Cas ribonucleoprotein, specifically designed zinc finger DNA binding domain recognition sequence / zinc finger DNA binding domain, or specifically designed transcription activator-like DNA binding domain recognition sequence / transcription activator-like DNA binding domain.

[0126] Method of the present invention

[0127] The present invention provides a method for producing minicircle DNA, wherein the method produces minicircle DNA using the recombinant plasmid constructed according to the first aspect of the present invention.

[0128] In one embodiment of the present invention, the method comprises the following steps:

[0129] (S1) introducing the recombinant plasmid described in the first aspect of the present invention into a prokaryotic cell;

[0130] (S2) culturing the prokaryotic cells, and then adding an inducing agent (or under inducing conditions) to induce the expression of a recombinase encoded in the recombinant plasmid, wherein the expressed recombinase cuts two recombinase recognition sites on the recombinant plasmid, thereby generating an intermediate product containing a miniplasmid and a minicircle DNA;

[0131] (S3) adding a miniplasmid recognition and removal reagent to the intermediate product of (S2) to remove the miniplasmid and unreacted recombinant plasmid therein, thereby isolating the minicircle DNA.

[0132] In another embodiment of the present invention, the method comprises the following steps:

[0133] (S1) introducing the recombinant plasmid described in the first aspect of the present invention into a prokaryotic cell;

[0134] (S2) culturing the prokaryotic cells, and then adding an inducing agent (or under inducing conditions) to induce the expression of a recombinase encoded in the recombinant plasmid, wherein the expressed recombinase cuts two recombinase recognition sites on the recombinant plasmid, thereby generating an intermediate product containing a miniplasmid and a minicircle DNA;

[0135] (S3) Adding a minicircle DNA recognition and separation reagent to the intermediate product of (S2) to separate the minicircle DNA.

[0136] In another embodiment of the present invention, the method comprises the following steps:

[0137] (S1) introducing the recombinant plasmid described in the first aspect of the present invention into a prokaryotic cell;

[0138] (S2) culturing the prokaryotic cells, and then adding an inducing agent (or under inducing conditions) to induce the expression of a recombinase encoded in the recombinant plasmid, wherein the expressed recombinase cuts two recombinase recognition sites on the recombinant plasmid, thereby generating an intermediate product containing a miniplasmid and a minicircle DNA;

[0139] (S3) adding a microplasmid recognition and removal reagent to the intermediate product of (S2) to remove the microplasmid and unreacted recombinant plasmid therein, and then adding a microcircle DNA recognition and separation reagent to separate and obtain the microcircle DNA.

[0140] Minicircle DNA, pharmaceutical composition and application of the present invention

[0141] The present invention also provides a minicircle DNA prepared by the method of the present invention, which comprises a multiple cloning site for inserting a target DNA sequence and a target DNA sequence inserted therein, and a residual recombinase recognition site; the recombinase recognition site has a nucleotide sequence as shown in SEQ ID NO: 3 or a derivative sequence thereof

[0142] Wherein, the target DNA sequence includes (but is not limited to) a functional DNA sequence, a set of functional RNA expression cassettes, a set of functional protein expression cassettes, 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, which cooperates with a transposase to stably insert the genetic information it carries into a prokaryotic or eukaryotic cell genome.

[0143] In a preferred embodiment, the minicircle DNA further comprises a minicircle DNA recognition and separation element. Alternatively, it does not comprise a minicircle DNA recognition and separation element, in which case the miniplasmid produced with it during its production process comprises a miniplasmid recognition and removal element.

[0144] The minicircle DNA of the present invention can be used to prepare a pharmaceutical composition. Therefore, the present invention also provides a pharmaceutical composition comprising a safe and effective amount of the minicircle DNA of the present invention as an active ingredient and a pharmaceutically acceptable carrier.

[0145] As used herein, "safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects. "Pharmaceutically acceptable carrier" means: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients.

[0146] The composition may be a liquid or a solid, such as a powder, gel or paste. Preferably, the composition is a liquid, preferably an injectable liquid.

[0147] Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0148] The pharmaceutical composition of the present invention can be used for disease treatment and / or prevention, for example, as a gene therapy or cell therapy drug, or as a vaccine. This depends on the specific type and purpose of the target DNA sequence inserted into the minicircle DNA. Diseases that can be treated by gene therapy or cell therapy include (but are not limited to) cancer or tumors, eye diseases, rheumatoid arthritis, osteoarthritis, osteochondral damage, and acute lung injury.

[0149] The beneficial effects of the present invention include:

[0150] The present invention provides a method and kit for preparing a highly efficient and low-toxic DNA vector, which can be applied to the research and development, preclinical research, process development, clinical research and other stages in the fields of gene therapy and cell therapy, and promote the rapid development of the fields of gene therapy and cell therapy.

[0151] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. Experiments for which specific conditions are not specified in the embodiments or test examples of the present invention are generally carried out under conventional conditions or under conditions recommended by the raw material / commodity manufacturer; reagents for which specific sources are not specified are conventional reagents purchased on the market.

[0152] Example 1 Preparation of minicircle DNA expressing EGFP

[0153] The EGFP expression cassette was designed between two RS sequences. When arabinose was used to induce the expression of CinH resolvase and promote plasmid recombination, the miniplasmid could be removed by EcoR V restriction enzyme and T5 exonuclease. After DNA purification, minicircle DNA expressing EGFP was obtained ( Figure 2a ).

[0154] The monoclonal E. coli carrying the parent plasmid of the EGFP expression cassette was inoculated into LB medium and cultured overnight in a constant temperature incubator at 200rpm and 37°C. After overnight culture, 1% arabinose (BBI, A610071-0100) was added for induction. After 1 hour of culture under the same conditions, the recombinant intermediate DNA (including recombinant minicircle DNA, miniplasmid DNA and residual parent plasmid DNA) was extracted and purified. The miniplasmid and parent plasmid contain specific restriction sites for the EcoR V restriction endonuclease, but the minicircle DNA does not. The intermediate DNA was treated with EcoR V restriction endonuclease (NEB, R3195L) for 3 hours or overnight to cut the miniplasmid and parent plasmid into linear DNA. Then T5 exonuclease (NEB, M0663L) was added for digestion for 3 hours or overnight to remove the linear DNA. Finally, only the minicircle DNA remained. After purification, the minicircle DNA ( Figure 2b ).

[0155] Example 2 Preparation of transposon minicircle DNA carrying CD19-CAR expression cassette

[0156] The CD19-CAR expression cassette is constructed into the parent plasmid that can prepare the minicircle. The recombinant miniplasmid and the parent plasmid contain specific restriction endonuclease sites for EcoR V restriction endonuclease, but the minicircle DNA does not.

[0157] The monoclonal Escherichia coli carrying the parent plasmid of the CD19-CAR expression cassette was inoculated into LB medium and cultured overnight in a constant temperature incubator at 200rpm and 37°C. After overnight culture, 1% arabinose (BBI, A610071-0100) was added for induction. After 1 hour of culture under the same conditions, the recombinant intermediate DNA (including recombinant minicircle DNA, miniplasmid DNA and residual parent plasmid DNA) was extracted and purified. The miniplasmid and parent plasmid contain specific restriction sites for EcoR V restriction endonuclease, but not in minicircle DNA. The intermediate DNA was treated with EcoR V restriction endonuclease (NEB, R3195L) for 3 hours or overnight to cut the miniplasmid and parent plasmid into linear DNA. Then T5 exonuclease (NEB, M0663L) was added for digestion for 3 hours or overnight to remove the linear DNA, leaving only minicircle DNA. After purification, the minicircle DNA ( Figure 3 ).

[0158] Example 3 Preparation of minicircle DNA carrying transposase expression cassette

[0159] The transposase expression cassette is constructed into the parent plasmid for preparing minicircles. The recombinant miniplasmid and the parent plasmid contain specific restriction endonuclease sites for EcoR V, but the minicircle DNA does not.

[0160] The monoclonal E. coli carrying the parent plasmid of the transposase expression cassette was inoculated into LB medium and cultured overnight in a constant temperature incubator at 200rpm and 37°C. After overnight culture, 1% arabinose (BBI, A610071-0100) was added for induction. After 1 hour of culture under the same conditions, the recombinant intermediate DNA (including recombinant minicircle DNA, miniplasmid DNA and residual parent plasmid DNA) was extracted and purified. The miniplasmid and parent plasmid contain specific restriction sites for EcoR V restriction endonuclease, but not in minicircle DNA. The intermediate DNA was treated with EcoR V restriction endonuclease (NEB, R3195L) for 3 hours or overnight to cut the miniplasmid and parent plasmid into linear DNA. Then T5 exonuclease (NEB, M0663L) was added for digestion for 3 hours or overnight to remove the linear DNA. Finally, only minicircle DNA remained. After purification, the minicircle DNA ( Figure 4 ).

[0161] Example 4: Minicircle DNA is significantly superior to plasmid DNA as a delivery vector for delivering foreign genes to T cells

[0162] 400 ng of plasmid expressing EGFP and the same mass (400 ng) or the same molar amount (176 ng) of minicircle DNA (MD) expressing EGFP were delivered into 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). The positive rate of EGFP and the viability of cells were detected by flow cytometry (Agilent, NovoCyte3110) after 24 hours and 5 days of culture, respectively. The positive cells stained with 7AAD (BD, 559925) were dead cells. The results are the mean ± SEM of 3 repetitions. The T test analyzed the significance of the difference in the data, where ** indicates P < 0.01 and *** indicates P < 0.001.

[0163] The results showed that when the same DNA mass was electroporated, the EGFP positivity rate and MFI of the microcircle DNA group were higher than those of the plasmid group; at the same molar mass, the EGFP positivity rate, cell viability and MFI of the microcircle DNA group were still significantly higher than those of the plasmid group. Comparison between the two groups of microcircles with different dosages showed that although the low-dose microcircle was not as good as the high-dose group in terms of EGFP positivity rate and MFI, the viability of the low-dose group was significantly better than that of the high-dose group. Compared with plasmid DNA, the low toxicity and high expression level of microcircle DNA to cells have greatly increased the practicality of microcircle DNA as a DNA vector for gene therapy or cell therapy ( Figure 5 ).

[0164] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A recombinant plasmid, characterized in that: The plasmid contains the following elements: Prokaryotic replication origin, prokaryotic selection marker, recombinase coding sequence, regulatable expression element for controlling recombinase expression, multiple cloning site for inserting target DNA sequence and two recombinase recognition sites located on both sides of the multiple cloning site; Furthermore, the arrangement of the above elements allows the plasmid to be divided into a mini-plasmid and a mini-circle DNA after being cut by the expressed recombinase at two recombinase recognition sites, wherein the formed mini-circle DNA comprises a multiple cloning site for inserting a target DNA sequence and the target DNA sequence inserted therein, and a residual recombinase recognition site; The recombinase is CinH resolvase, and the recombinase recognition site has a nucleotide sequence as shown in SEQ ID NO: 3 or a derivative sequence thereof.

2. The recombinant plasmid according to claim 1, characterized in that The CinH resolvase includes a wild-type CinH resolvase and a mutant thereof, wherein the mutant retains the activity of the CinH resolvase in recognizing and cleaving the recombinase recognition site shown in SEQ ID NO:

3.

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

4. A reaction system for producing minicircle DNA, characterized in that: The reaction system comprises: (Z1) the recombinant plasmid according to claim 1; (Z2) an inducing agent or inducing condition for inducing the expression of the encoded recombinase in the recombinant plasmid; In another preferred embodiment, the inducing agent is selected from the following group: arabinose, lactose or IPTG, tetracycline.

5. A kit for producing minicircle DNA, the kit comprising: (C1) the recombinant plasmid according to claim 1; and (C2) Optionally, an inducing agent that induces the expression of the recombinase in the recombinant plasmid.

6. A method for producing minicircle DNA, the method comprising: (S1) introducing the recombinant plasmid as claimed in claim 1 into a prokaryotic cell; (S2) culturing the prokaryotic cell and inducing the expression of the recombinase encoded in the recombinant plasmid, thereby producing an intermediate product containing a miniplasmid and a minicircle DNA; and (S3) Separating the minicircle DNA from the intermediate product of (S2).

7. A minicircle DNA prepared by the method according to claim 6, comprising: A multiple cloning site for inserting a target DNA sequence and the target DNA sequence inserted therein, and a residual recombinase recognition site; the recombinase recognition site has a nucleotide sequence as shown in SEQ ID NO: 3 or a derivative sequence thereof.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (a) the minicircle DNA as described in claim 7; and (b) a pharmaceutically acceptable carrier.

9. Use of the recombinant plasmid according to claim 1 in the preparation of minicircle DNA drugs.

10. Use of the minicircle DNA according to claim 7 in the preparation of a drug for the treatment and / or prevention of a disease.

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