Preparation method for minicircle DNA and use of minicircle DNA

Abstract

Provided are a preparation method for a minicircle DNA and a use of the minicircle DNA. Specifically, provided is a recombinant plasmid, which carries a CinH resolvase and a specific recognition site therefor, and is used for generating a minicircle DNA. Further provided are a method for generating a minicircle DNA by using the recombinant plasmid, a kit comprising the recombinant plasmid and used for generating a minicircle DNA, and a use of the minicircle DNA, which is prepared by using the method, in gene therapy and cell therapy.

Description

A method for preparing minicircle DNA and its application Technical Field

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

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

[0003] Supercoiled circular DNA, minus the prokaryotic replication origin and antibiotic resistance genes, was first generated by Darquet et al. in 1997 and demonstrated superior protein expression in various mammalian cell lines compared to plasmid DNA vectors. This type of DNA is known as minicircle DNA. Chen et al. further demonstrated, using a mouse model, that when DNA vectors were delivered to the liver, minicircle DNA could express 200-560 times more clinically promising proteins in peripheral blood than plasmid DNA. Since then, minicircle DNA's high stability and high expression capacity have led to its widespread application in the development of gene and cell therapies for various diseases. Minicircle DNA has been explored as a gene therapy vector in preclinical studies for rheumatoid arthritis and osteoarthritis. Rim et al. used minicircles to express BMP2 and TGFβ3, inducing the differentiation of hiPSC-derived mesenchymal stem cells into chondrocytes and successfully treating osteochondral lesions in a rat model. Florian et al. used microcircle DNA to express angiopoietin 1 in mesenchymal stem cells, enhancing their efficacy in alleviating acute lung injury. Technologies are also being developed to deliver proteins with clinical therapeutic potential to retinal epithelial cells, retinal ganglion cells, or photoreceptor cells to treat eye-related diseases. Overall, microcircle DNA is a highly promising gene therapy and cell therapy vector with a wide range of applications.

[0004] The production of minicircle DNA relies on a recombinase that splits a parent plasmid carrying two recombination sites into two smaller circular DNAs. By designing the relative positioning of the recombination sites and other units on the parent plasmid, one of the circular DNAs can carry the functional protein or RNA expression cassette required for gene therapy or cell therapy, known as the minicircle DNA, while the other carries the prokaryotic replication origin and antibiotic resistance gene to be removed, commonly known as the miniplasmid. Currently reported recombinases for minicircle DNA production include bacteriophage λ integrase, bacteriophage ΦC31 integrase, Cre recombinase, and ParA resolvase. Minicircles produced using bacteriophage λ integrase, ΦC31 integrase, and Cre recombinase all contain contaminants ranging from dimers to multimers. Because these dimers and multimers share the same sequence as the target minicircle DNA, subsequent production of high-purity minicircle DNA is difficult. However, ParA resolvase achieves near-100% recombination efficiency, and the recombined product is free of dimer or multimer contamination. Obtaining high-purity minicircles requires the use of techniques to remove residual parent and minicircle plasmids. Common methods currently include in vitro enzymatic removal of the parent and minicircle plasmids; in vivo enzymatic removal of the parent and minicircle plasmids; affinity chromatography to remove the parent and minicircle plasmids; or purification of the minicircle DNA. Monolithic column chromatography is also used to further purify supercoiled minicircle DNA to meet clinical standards.

[0005] Adoptive cellular immunotherapy (ACCT) has achieved remarkable success in recent years. With the US Food and Drug Administration's approval of KYMRIAH (Novartis Pharmaceuticals Corporation), YESCARTA and TECARTUS (Kite Pharma, Inc.), BREYANZI (Juno Therapeutics, Inc.), ABECMA (Celgene Corporation), and CARVYKTI (Janssen Biotech, Inc.), these drugs are now available for the clinical treatment of relapsed or refractory hematologic malignancies, including large B-cell lymphoma, mantle cell lymphoma, and multiple myeloma. These FDA-approved ACCs are all developed using retroviral or lentiviral vectors. Of the clinically used CAR-T cells developed using retroviral and lentiviral vectors, 65% and 54% of insertion sites are located within RefSeq genes, respectively. These exogenous gene insertions within RefSeq genes raise concerns about the application of retroviral and lentiviral vectors in gene and cell therapy. The Sleeping Beauty transposon system has been demonstrated to insert nearly randomly into the human genome, offering a higher safety profile for gene therapy compared to retroviral, lentiviral, and other transposon-based vectors. However, the primary obstacle to the application of non-viral vector systems in gene therapy or cell therapy is their low delivery efficiency.

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

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

[0008] Summary of the Invention

[0009] The present invention aims to provide a recombinant plasmid containing a prokaryotic origin of replication, a prokaryotic selection marker, a regulatable expression element for controlling recombinase expression, a recombinase, two recombinase recognition sites, and a multiple cloning site for inserting a target DNA sequence. The plasmid is used to produce circular supercoiled minicircle DNA that is free of prokaryotic DNA sequences such as the prokaryotic origin of replication and the prokaryotic selection marker. The plasmid also provides a method for producing minicircle DNA using the recombinant plasmid.

[0010] The first aspect of the present invention provides a recombinant plasmid comprising the following elements:

[0011] 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;

[0012] Furthermore, the arrangement of the above elements allows the plasmid to be divided into a miniplasmid and a minicircle DNA after being cut by the expressed recombinase at the two recombinase recognition sites, wherein the minicircle DNA formed contains a multiple cloning site for inserting a target DNA sequence, the target DNA sequence inserted therein, and a residual recombinase recognition site;

[0013] 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.

[0014] In another preferred embodiment, the CinH resolvase includes wild-type CinH resolvase and mutants thereof, wherein the mutants retain the activity of CinH resolvase in recognizing and cleaving the recombinase recognition site shown in SEQ ID NO: 3.

[0015] In another preferred embodiment, the amino acid sequence of the CinH resolvase is shown in SEQ ID NO: 1.

[0016] In another preferred embodiment, the nucleotide sequence encoding the CinH resolvase is shown in SEQ ID NO: 2.

[0017] 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→.

[0018] 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.

[0019] 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.

[0020] 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 the genome of a prokaryotic or eukaryotic cell.

[0021] In another preferred embodiment, the target DNA sequence includes a chimeric antigen receptor (CAR) coding sequence.

[0022] In another preferred embodiment, the regulatable expression element that controls 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 sensor protein YFI and downstream FixJ / FixK2 / cI / pR.

[0023] 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.

[0024] 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 recognition sequence or a specifically designed transcription activator-like effector DNA binding domain recognition sequence, or a combination thereof.

[0025] In another preferred embodiment, the miniplasmid recognition and removal element is at least one restriction enzyme site.

[0026] 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.

[0027] In another preferred embodiment, the minicircle DNA recognition and separation element is selected from the following group: LacO sequence, a specifically designed oligonucleotide or deoxynucleotide complementary sequence, a specifically designed inactivated Cas ribonucleoprotein (such as 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 DNA binding domain recognition sequence, or a combination thereof.

[0028] In another preferred embodiment, the recombinant plasmid further comprises a miniplasmid recognition and removal element, and a minicircle DNA recognition and separation element.

[0029] A second aspect of the present invention provides a reaction system for producing minicircle DNA, the reaction system comprising:

[0030] (Z1) the recombinant plasmid according to the first aspect of the present invention;

[0031] (Z2) an inducing agent or inducing conditions for inducing the expression of the recombinase encoded in the recombinant plasmid;

[0032] In another preferred embodiment, the induction agent is selected from the following group: arabinose, lactose or IPTG, tetracycline.

[0033] In another preferred embodiment, the induction conditions include heat shock or light induction.

[0034] In another preferred embodiment, the reaction system further comprises: a miniplasmid recognition and removal reagent, and / or a minicircle DNA recognition and separation reagent.

[0035] 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 unreacted recombinant plasmid in the product.

[0036] In another preferred embodiment, 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.

[0037] 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 (Zinc finger DNA binding domain), transsciption activator-like effector DNA binding domains (Transsciption activator-like effector DNA binding domain), or a combination thereof.

[0038] In another preferred embodiment, the miniplasmid recognition and removal reagent is a restriction endonuclease.

[0039] In another preferred embodiment, the minicircle DNA recognition and separation reagent includes a protein capable of binding 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 support.

[0040] 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.

[0041] A third aspect of the present invention provides a kit for producing minicircle DNA, the kit comprising:

[0042] (C1) the recombinant plasmid according to the first aspect of the present invention; and

[0043] (C2) Optionally, an inducing agent that induces the expression of the recombinase in the recombinant plasmid.

[0044] In another preferred embodiment, the kit further comprises:

[0045] (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 unreacted recombinant plasmid in the product; and / or

[0046] (C4) 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.

[0047] In another preferred embodiment, the induction agent is selected from the following group: arabinose, lactose or IPTG, tetracycline.

[0048] 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, transcription activator-like DNA binding domains, or combinations thereof.

[0049] In another preferred embodiment, the miniplasmid recognition and removal reagent is a restriction endonuclease.

[0050] In another preferred embodiment, the minicircle DNA recognition and separation reagent includes a protein capable of binding 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 support.

[0051] In another preferred embodiment, the minicircle DNA recognition and separation reagent is selected from: LacI protein, oligonucleotides or deoxynucleotides of a specific designed sequence, inactivated Cas ribonucleoprotein (such as dead Cas9, Cas12, Cas3, Cas8, Cas10 and Cas13 RNP, etc.), zinc finger DNA binding domain, transcription activator-like DNA binding domain, or a combination thereof.

[0052] 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.

[0053] In another preferred embodiment, the induction conditions include heat shock or light induction.

[0054] A fourth aspect of the present invention provides a method for producing minicircle DNA, comprising:

[0055] (S1) introducing the recombinant plasmid according to the first aspect of the present invention into a prokaryotic cell;

[0056] (S2) culturing the prokaryotic cell and inducing the expression of the recombinase encoded in the recombinant plasmid, thereby producing an intermediate product containing the miniplasmid and the minicircle DNA; and

[0057] (S3) Isolate the minicircle DNA from the intermediate product of (S2).

[0058] In another preferred embodiment, in step (S2), an induction agent is added to activate the regulatable expression element that controls the expression of the recombinase in the recombinant plasmid, thereby inducing the expression of the recombinase.

[0059] In another preferred embodiment, in step (S2), the regulatable expression element for controlling the expression of the recombinase in the recombinant plasmid is activated using induction conditions, thereby inducing the expression of the recombinase.

[0060] In another preferred embodiment, in step (S3), the minicircle DNA is separated using a minicircle DNA recognition and separation reagent utilizing the minicircle DNA recognition and separation element in the minicircle DNA.

[0061] 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.

[0062] In another preferred example, in step (S3), the microplasmid recognition and removal reagent is first used to remove the microplasmid and unreacted recombinant plasmid in the intermediate product, and then the microcircle DNA recognition and separation element in the microcircle DNA is utilized to separate the microcircle DNA using the microcircle DNA recognition and separation reagent.

[0063] 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.

[0064] 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:

[0065] A multiple cloning site for inserting a target DNA sequence, 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.

[0066] 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.

[0067] 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.

[0068] 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 the genome of a prokaryotic or eukaryotic cell.

[0069] In another preferred embodiment, the target DNA sequence includes a chimeric antigen receptor (CAR) coding sequence.

[0070] In another preferred embodiment, the minicircle DNA further comprises minicircle DNA recognition and separation elements.

[0071] 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.

[0072] A sixth aspect of the present invention provides a pharmaceutical composition comprising:

[0073] (a) the minicircle DNA according to the fifth aspect of the present invention; and (b) a pharmaceutically acceptable carrier.

[0074] In another preferred embodiment, the pharmaceutical composition is used for treating and / or preventing a disease.

[0075] In another preferred embodiment, the pharmaceutical composition is used for gene therapy or cell therapy.

[0076] In another preferred embodiment, the pharmaceutical composition is used as a vaccine for treating and / or preventing diseases.

[0077] 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.

[0078] In another preferred embodiment, the minicircle DNA drug is used for gene therapy or cell therapy.

[0079] 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 treating and / or preventing a disease.

[0080] In another preferred embodiment, the disease can be treated by gene therapy or cell therapy.

[0081] In another preferred embodiment, the disease includes (but is not limited to) cancer or tumor, eye disease, rheumatoid arthritis, osteoarthritis, osteochondral injury, and acute lung injury.

[0082] 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.

[0083] In another preferred embodiment, the minicircle DNA is used as a delivery vector to deliver therapeutic / preventive components into the subject.

[0084] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0085] In another preferred embodiment, the disease can be treated by gene therapy or cell therapy.

[0086] In another preferred embodiment, the disease includes (but is not limited to) cancer or tumor, eye disease, rheumatoid arthritis, osteoarthritis, osteochondral injury, and acute lung injury.

[0087] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 shows a schematic diagram of the positional arrangement of the minicircle DNA parent plasmid, miniplasmid, and various minicircle DNA elements.

[0089] Figure 2a shows the process for preparing transposon minicircles carrying the EGFP expression cassette.

[0090] Figure 2b shows that the transposon minicircle DNA carrying the EGFP expression cassette was successfully prepared.

[0091] FIG3 shows the preparation of transposon minicircle DNA carrying the CD19-CAR expression cassette.

[0092] FIG4 shows the preparation of minicircle DNA carrying a transposase expression cassette.

[0093] Figure 5 shows that minicircle DNA is significantly superior to plasmid DNA as a delivery vector for delivering exogenous genes to T cells. DETAILED DESCRIPTION

[0094] After extensive and in-depth research, the inventors have developed a novel recombinant plasmid. The arrangement of the components within this recombinant plasmid allows the induction of recombinase expression, splitting the parent plasmid into two smaller circular DNAs. One of these, a miniplasmid, contains non-target sequences such as a prokaryotic replication origin, a prokaryotic selection marker, regulatable expression elements for controlling recombinase expression, and the recombinase encoding sequence. The other, a minicircle DNA, contains only the target sequence, a residual multiple cloning site, and a residual recombinase recognition site. The miniplasmid can be removed using specific restriction enzyme sites or recognition sequences on the miniplasmid to purify the minicircle DNA. Alternatively, the minicircle DNA can be purified using specific recognition sequences on the minicircle DNA.

[0095] On this basis, the present invention was completed.

[0096] the term

[0097] In order to make the present invention easier to understand, certain 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 generally 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 change. 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.

[0098] 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.

[0099] As used herein, when used in reference to a specific recited 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.).

[0100] 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).

[0101] As used herein, the term "optional" or "optionally" means that the event or situation described subsequently may 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 may be present but does not have to be present.

[0102] As used herein, "sequence identity" 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 a sequence described herein and a sequence to which it is 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%, and 100%.

[0103] Recombinant plasmid of the present invention

[0104] In one aspect of the present invention, a recombinant plasmid is provided, which comprises the following elements: prokaryotic origin of replication, prokaryotic screening marker, recombinase coding sequence, regulatable expression element for controlling recombinase expression, multiple cloning site for inserting target DNA sequence and two recombinase recognition sites respectively located on both sides of the multiple cloning site; The arrangement of the above-mentioned elements makes it possible for the recombinase to be expressed in the plasmid to be divided into miniplasmid and minicircle DNA after cutting at two recombinase recognition sites; The minicircle DNA formed comprises a multiple cloning site for inserting target DNA sequence and a target DNA sequence inserted therein, and a residual recombinase recognition site; The miniplasmid comprises prokaryotic origin of replication, prokaryotic screening marker, regulatable expression element for controlling recombinase expression, a recombinase coding sequence and other non-target sequences. The recombinase carried by the recombinant plasmid of the present invention is CinH resolvase (CinH resolvase), and its corresponding recombinase recognition site has a nucleotide sequence as shown in SEQ ID NO:3 or a sequence derived therefrom.

[0105] In the present invention, CinH resolvase includes wild-type CinH resolvase and mutants thereof, wherein the mutants retain the activity of the CinH resolvase in recognizing and cleaving the recombinase recognition site as shown in SEQ ID NO: 3. Preferably, the mutants have at least 85% sequence identity with the amino acid sequence of the wild-type CinH resolvase (SEQ ID NO: 1). In one embodiment of the present invention, the CinH resolvase encoding sequence has the nucleotide sequence shown in SEQ ID NO: 2.

[0106] Amino acid sequence of wild-type CinH resolvase (SEQ ID NO: 1)

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

[0108] As used herein, the term "recombinase coding sequence" refers to any sequence required for recombinase expression, such as a promoter, termination sequence, etc. The recombinase itself can be any recombinase capable of catalyzing the recombination of the parent plasmid into a miniplasmid and an isolatable minicircle DNA at a defined recognition sequence. In the present invention, the recombinase specifically refers to a CinH resolvase.

[0109] As used herein, the terms "recombinase recognition site" and "RS2" can be used interchangeably and both refer to sequences that can be recognized by a recombinase. The recombinant plasmid of the present invention is equipped with two recombinase recognition sites, 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.

[0110] Recombinase recognition site (SEQ ID NO: 3)

[0111] 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.

[0112] 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 a biological function, 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 mini-ring DNA for expressing CAR. However, of course, any gene encoding any protein can be inserted. In this case, the mini-ring can be used for detection methods, biochemical pathway research, etc.

[0113] The recombinant plasmid of the present invention contains a controllable expression element for controlling the expression of the recombinase. 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 maximum efficiency in 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 plasmid present in the sample, thereby achieving 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 expression of the recombinase is induced. This is a very simple and effective method for regulating the expression of the recombinase.

[0114] 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.

[0115] 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 miniplasmids, which further improves the efficiency of minicircle DNA production.

[0116] The minicircle DNA or miniplasmid identification sequence is any sequence that is 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 a "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 a "minicircle DNA identification and separation element" in the present invention. The above identification sequence can be present only on the miniplasmid or minicircle DNA, but is also applicable to the presence of both on the miniplasmid and the minicircle DNA.

[0117] The miniplasmid recognition and removal element can be a specific sequence that is recognized by a specific reagent and removed from the product. For example, the miniplasmid recognition and removal element can be a restriction endonuclease recognition site that is recognized by the corresponding restriction endonuclease and cut into linear DNA, which is further digested and removed from the product.

[0118] The microcircle DNA recognition and separation element can be a specific sequence that will bind to a given ligand, thereby complexing the microcircle DNA with the ligand, and then separating the complex from the microplasmid, cellular components, etc., for example, by immobilized ligand (the ligand is immobilized on a solid phase carrier, such as magnetic beads, agarose beads, agarose columns, etc.).

[0119] Kit of the present invention

[0120] 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.

[0121] 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 expression of the recombinase in the recombinant plasmid; and a miniplasmid recognition and removal reagent. 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 expression of the recombinase in the recombinant plasmid; and a minicircle DNA recognition and separation reagent. 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 expression of the recombinase in the recombinant plasmid; and a minicircle DNA recognition and separation reagent.

[0122] Wherein, the miniplasmid recognition and removal reagent acts on the miniplasmid recognition and removal element in the recombinant plasmid and the generated miniplasmid, and is used to remove the miniplasmid and unreacted recombinant plasmid in the product. In the present invention, the miniplasmid recognition and removal element and the miniplasmid recognition and removal reagent can be selected from the following pairing combinations (miniplasmid recognition and removal element / miniplasmid 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 that recognizes restriction endonuclease recognition sites located on the miniplasmid and unreacted recombinant plasmid, thereby cutting the miniplasmid and unreacted recombinant plasmid into linear DNA. Preferably, the reagent further includes a linear DNA digestion reagent, such as T5 exonuclease, to digest and remove the linear DNA.

[0123] 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 from the product. In a preferred embodiment, the minicircle DNA recognition and separation reagent comprises a protein capable of binding 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 support (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 oligonucleotide or deoxynucleotide sequence, 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.

[0124] Method of the present invention

[0125] The present invention provides a method for producing minicircle DNA, which utilizes the recombinant plasmid constructed according to the first aspect of the present invention to produce minicircle DNA.

[0126] In one embodiment of the present invention, the method comprises the following steps:

[0127] (S1) introducing the recombinant plasmid described in the first aspect of the present invention into a prokaryotic cell;

[0128] (S2) culturing the prokaryotic cells, and then adding an inducing agent (or under inducing conditions) to induce expression of a recombinase encoded in the recombinant plasmid, wherein the expressed recombinase cleaves two recombinase recognition sites on the recombinant plasmid, thereby generating an intermediate product containing a miniplasmid and a minicircle DNA;

[0129] (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.

[0130] In another embodiment of the present invention, the method comprises the following steps:

[0131] (S1) introducing the recombinant plasmid described in the first aspect of the present invention into a prokaryotic cell;

[0132] (S2) culturing the prokaryotic cells, and then adding an inducing agent (or under inducing conditions) to induce expression of a recombinase encoded in the recombinant plasmid, wherein the expressed recombinase cleaves two recombinase recognition sites on the recombinant plasmid, thereby generating an intermediate product containing a miniplasmid and a minicircle DNA;

[0133] (S3) Adding a minicircle DNA recognition and separation reagent to the intermediate product of (S2) to separate the minicircle DNA.

[0134] In another embodiment of the present invention, the method comprises the following steps:

[0135] (S1) introducing the recombinant plasmid described in the first aspect of the present invention into a prokaryotic cell;

[0136] (S2) culturing the prokaryotic cells, and then adding an inducing agent (or under inducing conditions) to induce expression of a recombinase encoded in the recombinant plasmid, wherein the expressed recombinase cleaves two recombinase recognition sites on the recombinant plasmid, thereby generating an intermediate product containing a miniplasmid and a minicircle DNA;

[0137] (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.

[0138] Minicircle DNA, pharmaceutical composition and application of the present invention

[0139] The present invention also provides a minicircle DNA prepared using the method of the present invention, which 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 recognition site has a nucleotide sequence as shown in SEQ ID NO: 3 or a derivative sequence thereof

[0140] 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 that cooperates with a transposase to stably insert the genetic information it carries into the genome of a prokaryotic or eukaryotic cell.

[0141] 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 during its production process comprises a miniplasmid recognition and removal element.

[0142] 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.

[0143] As used herein, "safe and effective amount" means an amount of the active ingredient sufficient to significantly improve the condition or symptoms without causing serious side effects. "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gels suitable for human use, of sufficient purity, and with sufficiently low toxicity. "Compatibility" as used herein refers to the ability of the components of the composition to be compatible with the active ingredient of the present invention, and with each other, without significantly reducing the efficacy of the active ingredient.

[0144] 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.

[0145] 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 Tween ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0146] The pharmaceutical compositions of the present invention can be used for the treatment and / or prevention of diseases, for example, as gene therapy or cell therapy drugs, or as vaccines. This depends on the specific type and intended use of the target DNA sequence inserted into the minicircle DNA. Diseases amenable to 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.

[0147] The beneficial effects of the present invention include:

[0148] 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, and clinical research stages in the fields of gene therapy and cell therapy, thereby promoting the rapid development of the fields of gene therapy and cell therapy.

[0149] The present invention will be 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 stated, percentages and parts are weight percentages and parts by weight. 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.

[0150] Example 1 Preparation of minicircle DNA expressing EGFP

[0151] The EGFP expression cassette was designed between two RS sequences. After arabinose-induced CinH resolvase expression promoted plasmid recombination, the miniplasmid was removed using the EcoRV restriction enzyme and T5 exonuclease. DNA purification yielded minicircle DNA expressing EGFP (Figure 2a).

[0152] A single E. coli clone carrying the parent plasmid expressing the EGFP expression cassette was inoculated into LB medium and cultured overnight in a constant temperature incubator at 200 rpm and 37°C. After overnight incubation, 1% arabinose (BBI, A610071-0100) was added for induction. After 1 hour of incubation under the same conditions, the recombinant intermediate DNA (containing 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, while the minicircle DNA does not. The intermediate DNA was treated with EcoR V restriction endonuclease (NEB, R3195L) for 3 hours or overnight to cleave the miniplasmid and parent plasmid into linear DNA. T5 exonuclease (NEB, M0663L) was then added for digestion for 3 hours or overnight to remove the linear DNA, leaving only the minicircle DNA, which was then purified (Figure 2b).

[0153] Example 2 Preparation of transposon minicircle DNA carrying CD19-CAR expression cassette

[0154] The CD19-CAR expression cassette was constructed into a parent plasmid that can be used to produce minicircles. The recombinant miniplasmid and parent plasmid contain specific restriction endonuclease sites for EcoRV, which are not present in the minicircle DNA.

[0155] A single clone of E. 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 200 rpm and 37°C. After overnight incubation, 1% arabinose (BBI, A610071-0100) was added for induction. After 1 hour of incubation under the same conditions, the recombinant intermediate DNA (containing 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, while the minicircle DNA does not. The intermediate DNA was treated with EcoR V restriction endonuclease (NEB, R3195L) for 3 hours or overnight to cleave the miniplasmid and parent plasmid into linear DNA. T5 exonuclease (NEB, M0663L) was then added for digestion for 3 hours or overnight to remove the linear DNA, leaving only the minicircle DNA, which was then purified (Figure 3).

[0156] Example 3 Preparation of minicircle DNA carrying transposase expression cassette

[0157] The transposase expression cassette is constructed into a parent plasmid that can produce minicircles. The recombinant miniplasmid and parent plasmid contain specific restriction endonuclease sites for EcoRV, which are absent in the minicircle DNA.

[0158] A single E. coli clone carrying the parent plasmid expressing the transposase cassette was inoculated into LB medium and cultured overnight in a constant temperature incubator at 200 rpm and 37°C. After overnight incubation, 1% arabinose (BBI, A610071-0100) was added for induction. After 1 hour of incubation under the same conditions, the recombinant intermediate DNA (containing 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, while the minicircle DNA does not. The intermediate DNA was treated with EcoR V restriction endonuclease (NEB, R3195L) for 3 hours or overnight to cleave the miniplasmid and parent plasmid into linear DNA. T5 exonuclease (NEB, M0663L) was then added for digestion for 3 hours or overnight to remove the linear DNA, leaving only the minicircle DNA, which was then purified (Figure 4).

[0159] Example 4: Minicircle DNA is significantly superior to plasmid DNA as a delivery vector for delivering exogenous genes to T cells

[0160] 400 ng of EGFP-expressing plasmid and the same mass (400 ng) or molar amount (176 ng) of EGFP-expressing minicircle DNA (MD) were delivered into T cells by electroporation (Lonza, AAF-1003X). After electroporation, cells were cultured in RPMI-1640 medium (Hyclone, SH30027.01) supplemented with 10% FBS (Hyclone, SV30208.02). Flow cytometry (Agilent, NovoCyte 3110) was used to determine EGFP expression and cell viability 24 hours and 5 days after culture. Cells positive for 7AAD (BD, 559925) were considered dead. Results are the mean ± SEM of three replicates. Data were analyzed for significance by T-test, where ** indicates P < 0.01 and *** indicates P < 0.001.

[0161] The results showed that when the same DNA mass was electroporated, the EGFP-positive rate and MFI were higher in the microcircle DNA group than in the plasmid group. At the same molar mass, the EGFP-positive rate, cell viability, and MFI in the microcircle DNA group were still significantly higher than those in the plasmid group. Comparing the two groups of microcircles with different dosages, although the low-dose microcircle group was inferior to the high-dose group in terms of EGFP-positive rate and MFI, the low-dose group had a significantly better viability than the high-dose group. The low toxicity and high expression levels of microcircle DNA compared to plasmid DNA significantly increase its practicality as a DNA vector for gene or cell therapy (Figure 5).

[0162] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

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. The recombinant plasmid according to claim 1, characterized in that The target DNA sequence includes a chimeric antigen receptor (CAR) encoding sequence.

5. The recombinant plasmid according to claim 1, characterized in that The regulatable expression elements for controlling the expression of the recombinase are selected from the following groups: the pBAD / AraC system of the arabinose operator, the LacI / LacO system of the lactose / IPTG operator, the rtTA / TRE system of the tetracycline operator, the heat shock induction system based on heat shock protein 70 or 90, and the light induction system based on the blue light sensing protein YFI and downstream FixJ / FixK2 / cI / pR.

6. The recombinant plasmid according to claim 1, characterized in that The recombinant plasmid also 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.

7. The recombinant plasmid according to claim 6, characterized in that 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, Cas10and 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.

8. The recombinant plasmid according to claim 1, characterized in that The recombinant plasmid also 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.

9. The recombinant plasmid according to claim 8, characterized in that The minicircle DNA recognition and separation element is selected from the following group: 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 DNA binding domain recognition sequence, or a combination thereof.

10. A reaction system for producing minicircle DNA, characterized in that: The reaction system comprises: (Z1) The recombinant plasmid according to any one of claims 1 to 9; (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.

11. The reaction system according to claim 10, characterized in that The reaction system further comprises: a miniplasmid recognition and removal reagent, and / or a minicircle DNA recognition and separation reagent.

12. A kit for producing minicircle DNA, the kit comprising: (C1) the recombinant plasmid according to any one of claims 1 to 9; and (C2) Optionally, an inducing agent that induces the expression of the recombinase in the recombinant plasmid.

13. The kit according to claim 12, characterized in that The kit also comprises: (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 (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.

14. A method for producing minicircle DNA, the method comprising: (S1) introducing the recombinant plasmid according to any one of claims 1 to 9 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).

15. A minicircle DNA prepared by the method according to claim 14, 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.

16. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (a) the minicircle DNA as claimed in claim 15; and (b) a pharmaceutically acceptable carrier.

17. Use of the recombinant plasmid according to any one of claims 1 to 9 in the preparation of minicircle DNA drugs.

18. Use of the minicircle DNA according to claim 15 in the preparation of a drug for the treatment and / or prevention of a disease.

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