Method for preparing closed-end linear DNA

Abstract

Provided in the present application is a DNA construct which comprises: 1) a replication origin; 2) a screening element; 3) a gene of interest; 4) an auxiliary element; and 5) a telomerase recognition site. The construct can be used for preparing a linear end-free DNA. Further provided in the present application are a method for producing an end-free linear DNA, and the use of the method.

Description

A method for preparing end-closed linear DNA Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a method for preparing end-closed linear DNA. Background Art

[0002] DNA vectors are widely used in genetic engineering and gene therapy, with applications including, but not limited to, gene overexpression, non-viral gain-of-function gene therapies including gene replacement therapy and gene addition therapy, viral vector packaging and production, DNA vaccines, mRNA production templates, large gene knock-in templates based on gene editing and homologous recombination, and DNA donors for DNA transposons. Removing resistance genes and immune activation sequences such as CpG islands from traditional DNA vectors is crucial for the clinical application of DNA vectors. Problems with existing resistance gene-free DNA vector technologies include: 1) contamination with parental and backbone DNA impurities; 2) difficulty in scaling up the production system; 3) product formation of polymers; 4) involvement of complex genetic modification of production strains; and / or 5) involvement of multiple in vitro enzymatic reactions, which are limited by the supply of enzyme raw materials.

[0003] Therefore, DNA vectors still need to be continuously optimized to solve the problems existing in existing DNA vectors. Summary of the Invention

[0004] This application provides a method for amplifying resistance-gene-free DNA in Escherichia coli and obtaining end-closed linear DNA through a one-step in vitro enzymatic reaction. This method involves fewer in vitro enzymatic reaction steps and is less restricted by the supply of enzyme raw materials. It is simple to operate and the production system is easily scalable. It also produces a high-purity final product, DNA monomers, addressing the problem of product formation in existing DNA vector technologies.

[0005] On the one hand, the present application provides a DNA construct comprising: 1) a replication initiation site; 2) a screening element; 3) a target gene; 4) an auxiliary element; and 5) a telomerase recognition site, wherein the auxiliary element enables the DNA construct to form an intermediate minicircle and a backbone heteroDNA under appropriate conditions, wherein the intermediate minicircle comprises the replication initiation site, the target gene and the telomerase recognition site, and the backbone heteroDNA comprises the screening element.

[0006] In certain embodiments, the intermediate minicircle does not comprise the screening element.

[0007] In certain embodiments, the intermediate minicircle can be amplified in an environment without selection pressure.

[0008] In certain embodiments, the backbone heteroDNA does not comprise an origin of replication.

[0009] In certain embodiments, the DNA construct is introduced into a production strain, and the backbone heterologous DNA cannot stably exist in a free state outside the genome of the production strain during the amplification process of the production strain.

[0010] In certain embodiments, the production strain is Escherichia coli.

[0011] In certain embodiments, the DNA construct is a circular double-stranded DNA construct.

[0012] In certain embodiments, the replication initiation site is selected from one or more of the following groups: R6K replication initiation site, pSC101 replication initiation site, ColE1 replication initiation site, ColE2 replication initiation site, ColE3 replication initiation site, pMB1 replication initiation site, pBR322 replication initiation site, pUC replication initiation site, and P15A replication initiation site.

[0013] In certain embodiments, the screening element is a forward screening element and / or a reverse screening element.

[0014] In certain embodiments, the selection element is a resistance gene selection element and / or a suicide gene selection element.

[0015] In certain embodiments, the resistance gene selection element is selected from one or more of the following groups: Ampicillin, kanamycin, chloramphenicol, spectinomycin, tetracycline, bleomycin, streptomycin, hygromycin, gentamicin, apramycin sulfate, erythromycin, and nourseothricin.

[0016] In certain embodiments, the counter-selection element comprises the use of SacB, ccdB, rpsL, tetAR, pheS, thyA, lacY, gata-1, upp, bla I, and mazF counter-selection markers.

[0017] In certain embodiments, the gene of interest comprises an expression cassette.

[0018] In some embodiments, the target gene comprises one or more sequences selected from the group consisting of a promoter, a gene encoding an expressed protein, and a terminator. In some embodiments, the target gene may further comprise other elements, for example, the target gene may further comprise an ITR sequence.

[0019] In certain embodiments, the auxiliary elements comprise: a recombination element, a transposase element, and / or a gene editing site.

[0020] In certain embodiments, the recombination element comprises a recombination site, which is a paired recombination site. The paired recombination site can cause the DNA construct to recombine under the action of a recombinase to obtain an intermediate minicircle and a backbone heteroDNA.

[0021] In certain embodiments, the recombinase functions under inducible conditions.

[0022] In certain embodiments, the sequences of the paired recombination sites are in the same orientation.

[0023] In certain embodiments, the recombination site comprises a site-specific recombinase site.

[0024] In certain embodiments, the site-specific recombinase site is capable of being recognized by a site-specific recombinase selected from one or more of the following groups: phiC31 integrase, FLP recombinase, Cre recombinase, Dre recombinase, BxbI integrase, and phiBT1 integrase.

[0025] In certain embodiments, the auxiliary elements enable homologous recombination of the DNA construct.

[0026] In certain embodiments, the homologous recombination comprises being achieved by the lambda phage Red homologous recombination system.

[0027] In certain embodiments, the auxiliary element enables the DNA construct to form an intermediate minicircle by a transposase.

[0028] In certain embodiments, the transposase is selected from one or more of the group consisting of Tn3, Tn5, Tn7, and Tn10.

[0029] In certain embodiments, the telomerase recognition site can be recognized and cut by telomerase to obtain a linear DNA with closed ends containing the target gene.

[0030] In certain embodiments, the DNA construct comprises at least one telomerase recognition site.

[0031] In certain embodiments, the DNA construct comprises two telomerase recognition sites.

[0032] On the other hand, the present application provides a method for producing end-free linear DNA, which comprises: 1) providing a parent plasmid comprising a replication initiation site, a screening element, a target gene, and a telomerase recognition site; 2) allowing the parent plasmid to form an intermediate minicircle and a backbone heteroDNA under suitable conditions, wherein the intermediate minicircle comprises the replication initiation site, the target gene, and the telomerase recognition site, and the backbone heteroDNA comprises the screening element; 3) amplifying the intermediate minicircle; and 4) obtaining end-free linear DNA carrying the target gene by telomerase digestion.

[0033] In certain embodiments, the step of forming an intermediate minicircle and backbone heteroDNA from the parent plasmid is achieved by one or more of the following methods: recombination induced by a site-specific recombinase, homologous recombination, transposition caused by a transposase, and gene editing.

[0034] In certain embodiments, the site-specific recombinase is selected from one or more of the following groups: phiC31 integrase, FLP recombinase, Cre recombinase, Dre recombinase, BxbI integrase, and phiBT1 integrase.

[0035] In certain embodiments, the homologous recombination comprises being achieved by the lambda phage Red homologous recombination system.

[0036] In certain embodiments, the transposase is selected from one or more of the group consisting of Tn3, Tn5, Tn7, and Tn10.

[0037] In certain embodiments, the gene editing is achieved using the CRISPR / Cas system.

[0038] In certain embodiments, the intermediate minicircle does not comprise the screening element.

[0039] In certain embodiments, the intermediate minicircle can be amplified in an environment without selection pressure.

[0040] In certain embodiments, the backbone heteroDNA does not comprise an origin of replication.

[0041] In certain embodiments, step 2) is performed in a production strain.

[0042] In certain embodiments, the production strain is Escherichia coli.

[0043] In certain embodiments, the replication initiation site is selected from one or more of the following groups: R6K replication initiation site, pSC101 replication initiation site, ColE1 replication initiation site, ColE2 replication initiation site, ColE3 replication initiation site, pMB1 replication initiation site, pBR322 replication initiation site, pUC replication initiation site, and P15A replication initiation site.

[0044] In certain embodiments, the screening element is a forward screening element and / or a reverse screening element.

[0045] In certain embodiments, the selection element is a resistance gene selection element and / or a suicide gene selection element.

[0046] In certain embodiments, the resistance gene selection element is selected from one or more of the following groups: Ampicillin, kanamycin, chloramphenicol, spectinomycin, tetracycline, bleomycin, streptomycin, hygromycin, gentamicin, apramycin sulfate, erythromycin, and nourseothricin.

[0047] In certain embodiments, the counter-selection element comprises the use of SacB, ccdB, rpsL, tetAR, pheS, thyA, lacY, gata-1, upp, bla I, and mazF counter-selection markers.

[0048] In certain embodiments, the gene of interest comprises an expression cassette.

[0049] In certain embodiments, the target gene comprises one or more sequences selected from the group consisting of a promoter, a gene encoding an expressed protein, and a terminator.

[0050] On the other hand, the present application provides an intermediate product minicircle, which is obtained by the method described in the present application, and the intermediate product minicircle comprises the replication start site, the target gene and the telomerase recognition site.

[0051] In certain embodiments, the intermediate minicircle does not comprise a screening element.

[0052] In certain embodiments, the intermediate minicircle can be amplified in an environment without selection pressure.

[0053] On the other hand, the present application provides a terminal-free linear DNA obtained by the method described in the present application.

[0054] In certain embodiments, the terminal-free linear DNA comprises closed structures at both ends.

[0055] In certain embodiments, the closed structure is obtained by telomerase cleavage.

[0056] On the other hand, the present application provides a cell comprising the construct, the intermediate minicircle or the endless linear DNA described in the present application.

[0057] On the other hand, the present application provides a production bacterium comprising the DNA construct described in the present application, or the intermediate minicircle described in the present application.

[0058] In certain embodiments, the production bacteria is Escherichia coli.

[0059] On the other hand, the present application provides a kit comprising the DNA construct described in the present application, or the intermediate minicircle described in the present application.

[0060] In certain embodiments, the kit further comprises production bacteria.

[0061] In certain embodiments, the kit further comprises telomerase.

[0062] On the other hand, the present application provides a pharmaceutical composition comprising the end-free linear DNA described herein, and optionally a pharmaceutically acceptable carrier.

[0063] On the other hand, the present application provides a method for delivering a target gene to a target cell, comprising contacting the endless linear DNA described in the present application with the target cell.

[0064] On the other hand, the present application provides uses of the DNA construct, the method, the intermediate minicircle, the endless linear DNA, the cell, and the pharmaceutical composition in the production and / or treatment of diseases, wherein the uses are selected from one or more of the following groups: 1) target gene expression; 2) gene therapy for diseases and / or disorders; 3) gene knockout, and / or gene knock-in; 4) packaging and / or production of viral vectors; 5) preparation of DNA vaccines; and 6) production of mRNA.

[0065] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0067] FIG1 shows a vector map of the arabinose-inducible phiC31 recombinase expression vector pSC101(ori)-araBAD-phiC31.

[0068] Figure 2 shows the vector map of the parental vector pFluc-attB / P-kana.

[0069] FIG3 shows a vector map of the parental vector pHTM-Fluc-attB / P-kana-TelLR1.

[0070] FIG4 shows a vector map of the parental vector pHTM-Fluc-attB / P-kana-TelLR2.

[0071] Figure 5 shows the presence of the recombinase plasmid pSC101(ori)-araBAD-phiC31 and the parental plasmid pHTM-Fluc-attB / P-kana-TelLR2 in bacterial cultures before and after arabinose induction, as well as the recombination of the parental plasmids. M: DNA molecular weight marker (250-10,000 bp). 5A. Lanes 1-4: Plasmid identification in bacterial cultures from four monoclonal clones before induction; Lanes 5-8: Plasmid digestion products identification in bacterial cultures from four monoclonal clones after EcoRI digestion before induction. 5B. Lanes 1-3: DNA in bacterial cultures 2, 5, and 7 hours after arabinose induction; Lanes 5-6: EcoRI digestion products identification in bacterial cultures 2, 5, and 7 hours after arabinose induction.

[0072] Figure 6 shows that L-arabinose concentrations ranging from 0.002% to 2% can induce phiC31 recombinase expression and mediate parental plasmid recombination. M: DNA molecular weight marker (200-10,000 bp). Lanes 1-5: DNA analysis after induction with 2%, 1%, 0.2%, 0.02%, and 0.002% L-arabinose. Lanes 6-10: EcoRI digestion analysis of DNA in culture after induction with 2%, 1%, 0.2%, 0.02%, and 0.002% L-arabinose.

[0073] Figure 7 shows the identified HTM-Fluc-attL / R-TelLR2 intermediate minicircle clones lacking the resistance gene. M: DNA molecular weight marker (200-10,000 bp). Upper lanes 1-15: Supercoiled intermediate minicircle (MC) monomers and multimers; Lower lanes 1-15: EcoRI digestion of the intermediate minicircle clones for identification.

[0074] FIG8 shows a vector map of the arabinose-inducible FLP recombinase expression vector pSC101(ori)-araBAD-FLP.

[0075] Figure 9 shows the presence of the recombinase plasmid pSC101(ori)-araBAD-FLP and the parental plasmid pFluc-FRT-kana in bacterial cultures before and after arabinose induction, as well as the recombination of the parental plasmids. M: DNA molecular weight marker (200-10,000 bp). 9A. Lane 1: Plasmid identification in bacterial cultures before induction; Lane 2: Plasmid identification in bacterial cultures after EcoRI digestion before induction; 9B. Lanes 1, 3, and 5: DNA identification in bacterial cultures 2, 5, and 8 hours after arabinose induction; Lanes 2, 4, and 6: DNA identification of EcoRI digestion products in bacterial cultures 2, 5, and 8 hours after arabinose induction.

[0076] Figure 10 shows the identified Fluc-FRT minicircle strain clones lacking the resistance gene. Among them, the clones positive for the 506bp PCR product contain the target minicircle with the kana resistance gene removed. M: DNA molecular weight marker (200-10,000bp). Lanes 1-15: PCR product identification of single clones grown only on empty LB plates after 2 hours of arabinose induction. Lanes 16-32: PCR product identification of single clones grown only on empty LB plates after 5 hours of arabinose induction. Lanes 33-49: PCR product identification of single clones grown only on empty LB plates after 8 hours of arabinose induction.

[0077] Figure 11 shows the identified Fluc-FRT minicircle strain clones lacking the resistance gene. M: DNA molecular weight marker (250-10,000 bp). Upper Lanes 1-22: Supercoiled microcircle (MC) monomers and multimers; Lower Lanes 1-22: Fluc-FRT minicircle clones identified by EcoRI digestion.

[0078] Figure 12 shows the Fluc-FRT minicircle strain clones that were identified as having no resistance gene. M: DNA molecular weight marker (200-10,000 bp). Lanes 2-19: PCR products of single-clone colonies grown only on empty LB plates after induction at 43°C for 2 hours. Lanes 20-50: PCR products of single-clone colonies grown only on empty LB plates after induction at 43°C for 2.5 hours. Lanes 51-93: PCR products of single-clone colonies grown only on empty LB plates after induction at 43°C for 3 hours. Clones positive for the 506 bp PCR product contain the target minicircle with the kana resistance gene removed; clones positive for the 1728 bp PCR product contain the parental plasmid.

[0079] Figure 13 shows the Fluc-FRT minicircle strain clones identified as lacking resistance genes. M: DNA molecular weight marker (250-10,000 bp). MC Lanes 1-9 and MC+EcoRI Lanes 1-9: Screening for supercoiled microcircle (MC) monomers and multimers in correct single clones, as well as identification of EcoRI digestion products of the minicircle plasmid. Lanes 10-29: Supercoiled microcircle monomers and multimers.

[0080] FIG14 shows a vector map of the temperature-inducible Cre recombinase gene expression vector pCP20-Cre.

[0081] FIG15 shows a vector map of the temperature-inducible Cre recombinase gene expression vector pCP20-Dre.

[0082] Figure 16 shows the identified Fluc-rox resistance gene-free minicircle strain clones. M: DNA molecular weight marker (250-10,000 bp). Lane 1: Supercoiled minicircle (MC) monomers and polymers; Lane 2: Minicircle EcoRI digestion analysis.

[0083] Figure 17 shows the identified Fluc-FRT minicircle strain clones without resistance genes. M: DNA molecular weight marker (250-10,000 bp). Lane 1: Supercoiled microcircle (MC) monomers and polymers; Lane 2: EcoRI digestion of the microcircle.

[0084] Figure 18 shows colony PCR results for clones lacking the selection gene minicircle and missing the pCP20 plasmid. The 760 bp SacB PCR product-negative clones lacked the SacB backbone fragment, and the FLP PCR product-negative clones lacked the pCP20 plasmid. M: DNA molecular weight marker (200-12,000 bp). Lanes 1-5: PCR products from single colonies grown on LB plates containing 5% sucrose after incubation at 30°C for 1 hour. Lanes 6-10: PCR products from single colonies grown on LB plates containing 5% sucrose after incubation at 30°C for 3 hours. Lanes 11-15: PCR products from single colonies grown on LB plates containing 5% sucrose after incubation at 30°C for 5 hours. Lane 16: PCR product from the sacB positive control. H2O: Negative control.

[0085] Figure 19 shows the identified Fluc-FRT minicircle strain clones without the selection gene. M: DNA molecular weight marker (250-10,000 bp). Lanes 1-9: Supercoiled microcircle (MC) monomers and polymers; Lanes 10-18: EcoRI digestion of the microcircle.

[0086] Figure 20 shows the identified HA-Fluc intermediate minicircle strain clones without the selection gene. M: DNA molecular weight marker (250-12000 bp); Upper Lanes 1-20: Supercoiled minicircle (MC) monomers and multimers; Lower Lanes 1-20: Minicircle EcoRI digestion and identification.

[0087] FIG21 shows the vector map of the parental vector pHTM-Fluc-ColE1-attB / P-kana-TelLR2.

[0088] Figure 22 shows the presence and recombination of the parental plasmid pHTM-Fluc-ColE1-attB / P-kana-TelLR2 in ZYCY10P3S2T E. coli cells containing the arabinose-inducible phiC31 recombinase expression construct before and after arabinose induction. M: DNA molecular weight marker (250-12000 bp). Lane 1: Plasmid identification in the culture medium before induction; Lane 2: EcoRI digestion product identification of the plasmid in the culture medium before induction; Lane 3: DNA identification in the culture medium 5 hours after arabinose induction; Lane 4: EcoRI digestion product identification of the DNA in the culture medium 5 hours after arabinose induction; Lane 5: DNA identification in the culture medium 8 hours after arabinose induction; Lane 6: EcoRI digestion product identification of the DNA in the culture medium 8 hours after arabinose induction.

[0089] Figure 23 shows the identified HTM-Fluc-ColE1-attL / R-TelLR2 intermediate minicircle clones lacking the resistance gene. M: DNA molecular weight marker (200-12000 bp). Lanes 1-12: Supercoiled intermediate microcircle (MC) monomers and multimers; Lanes 13-24: EcoRI digestion of intermediate minicircle clones for identification.

[0090] FIG24 shows a vector map of the parental vector pHTM-Fluc-DDITR-ColE1-attB / P-kana-TelLR2.

[0091] Figure 25 shows the presence and recombination of the parental plasmid pHTM-Fluc-DDITR-ColE1-attB / P-kana-TelLR2 in ZYCY10P3S2T E. coli cells containing the arabinose-inducible phiC31 recombinase expression construct before and after arabinose induction. M: DNA molecular weight marker (250-12000 bp). Lane 1: Plasmid identification in the culture medium before induction; Lane 2: Plasmid identification in the culture medium after EcoRI digestion before induction; Lane 3: DNA identification in the culture medium 5 hours after arabinose induction; Lane 4: EcoRI digestion product identification in the culture medium 5 hours after arabinose induction; Lane 5: DNA identification in the culture medium 8 hours after arabinose induction; Lane 6: EcoRI digestion product identification in the culture medium 8 hours after arabinose induction.

[0092] Figure 26 shows the identified HTM-Fluc-DDITR-ColE1-attL / R-TelLR2 intermediate minicircle clones lacking the resistance gene. M: DNA molecular weight marker (200-12000 bp). Lanes 1-12: Supercoiled intermediate microcircle (MC) monomers and multimers; Lanes 13-24: EcoRI digestion of intermediate minicircle clones for identification.

[0093] Figure 27 shows the Sanger sequencing results of the DDITR sequence in the HTM-Fluc-DDITR-ColE1-attL / R-TelLR2 intermediate minicircle.

[0094] FIG28 shows that the yield of the intermediate minicircle DNA is stable during amplification production in E. coli liquid.

[0095] Figure 29 shows the preparation of closed-end linear DNA by telomerase digestion of an intermediate minicircle containing a telomerase recognition site. M: DNA molecular weight marker (250-12000 bp). Lane 1: Parental plasmid containing a telomerase recognition site; Lane 2: Intermediate minicircle containing a telomerase recognition site; Lane 3: Enzyme digestion of the final closed-end linear DNA product, 2690 bp in size.

[0096] Figure 30 shows the preparation of end-closed linear DNA by telomerase digestion of an intermediate minicircle containing two telomerase recognition sites. M: DNA molecular weight marker (250-12000 bp). 30A. Isolation and purification of products from the telomerase-digested intermediate minicircle. 30B. Lane 1: Sample sampled at peak 1 in 30A contains the target end-closed linear DNA final product and the end-closed linear DNA backbone. Lanes 2-3: Sample sampled at peaks 2 and 3 in 30A contain the purified target end-closed linear DNA final product, which is 2375 bp in size.

[0097] Figure 31 shows the purified closed-end linear DNA final product, containing only the target closed-end linear DNA, with a size of 2375 bp. M: DNA molecular weight marker (250-12000 bp). Lane 1: Parental plasmid containing two telomerase recognition sites. Lane 2: Intermediate minicircle plasmid containing two telomerase recognition sites. Lane 3: Enzymatically digested closed-end linear DNA final products, with sizes of 2375 bp and 371 bp. Lane 4: Ion exchange separation and purification to obtain closed-end linear DNA, with a size of 2375 bp.

[0098] FIG32 shows the results of luciferase expression in C57BL / 6 mice using lipid nanoparticles to deliver end-closed linear DNA expressing luciferase.

[0099] Figure 33 shows the use of end-closed linear DNA as a DNA donor for the piggyBac transposon in cultured cells. A shows ARPE-19 cells co-transfected with the pPB plasmid and nelPB end-closed linear DNA, respectively, carrying the same piggyBac transposon DNA donor sequence, and the transposase PBase plasmid. Single clones of cells with successful transposon integration were obtained after 21 days of blasticidin selection. B shows the number of single clones in the two groups shown in A. **** indicates P value < 0.0001, n = 4. DETAILED DESCRIPTION

[0100] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0101] Definition of terms

[0102] In this application, the term "DNA construct" generally refers to an artificially modified / synthesized DNA molecule. In some cases, the DNA construct can be a circular DNA molecule. For example, it can be a circular double-stranded DNA molecule. In some cases, the DNA construct can have a replication initiation site so that the DNA construct can have autonomous replication ability in a host cell. In some cases, the DNA construct can also include a selectable marker gene. For example, the selectable marker gene can be an antibiotic resistance gene so that the host cell containing the DNA construct can grow under specific culture conditions, while the host cell without the DNA construct cannot grow under specific culture conditions. For example, the selectable marker gene can also be some reverse screening marker genes. In some cases, the DNA construct can also include any target gene of interest, and the DNA construct can complete the amplification of the target gene under suitable conditions. In some cases, the DNA construct can also include other auxiliary elements that can split the DNA construct, for example, into the intermediate minicircle described in this application, and the backbone heteroDNA.

[0103] In this application, the term "screening element" generally refers to an element that can have a specific performance under a specific environment, thereby being able to help screen the target you want to judge. For example, the screening element can be a forward screening element or a reverse screening element. For example, the forward screening element can be a selection marker gene. For example, it can be an antibiotic resistance gene, and cells containing antibiotic resistance genes can still exist and / or maintain properties in an environment where antibiotics are present; or it can be some nutritional deficiency selection markers, such as glucosamine synthetase selection markers and mannose phosphate isomerase selection markers. For example, the reverse screening element can be some negative selection markers, and under a specific environment, cells with reverse screening elements cannot exist and / or maintain characteristics, while cells without reverse screening elements can exist and / or maintain properties, such as the SacB selection marker.

[0104] In this application, "screening pressure" or "selection pressure" can be used interchangeably. In this specification, it refers to the application of specific substances or conditions to organisms such as host cells in culture conditions (such as culture medium), so that organisms that adapt to these specific substances or conditions can survive, while organisms that adapt to these specific substances or conditions are not adapted and are eliminated. For example, in this specification, antibiotics such as kanamycin or ampicillin are added to the culture medium of the host cells, and the culture medium contains antibiotic selection stress, which can screen out host cells with corresponding antibiotic resistance.

[0105] In this application, the term "target gene" generally refers to any sequence of interest. For example, the target gene can be a double-stranded DNA fragment that can contain the minimal sequence encoding the gene of interest. For example, the target gene can also contain other sequences required for proper gene expression, such as an expression cassette. For example, the expression cassette can include, for example, a promoter and a terminator. For example, the target gene can also include regulatory elements, such as an enhancer and / or poly A.

[0106] In this application, the term "auxiliary element" generally refers to an element that can be segmented and broken under appropriate conditions. In this application, in order to separate the different parts in the DNA construct to form a backbone hetero-DNA and an intermediate microcircle, an auxiliary element is needed. Any element known in the art that can cause DNA to be cut / broken can be used in this application, for example, a recombination element, a transposase element, and / or an element in a gene editing system. In some cases, after the breakage and / or cutting of the DNA occurs, reconnection of the DNA can also occur so that the connected DNA contains the desired target sequence of the design. For example, the reconnected DNA can be circular. Through the auxiliary element, the target intermediate microcircle can be obtained from the DNA construct. The elements desired to be contained in the target intermediate microcircle can be controlled.

[0107] In this application, the term "recombination site" generally refers to a nucleotide sequence that can be recognized by a corresponding recombinase. Under the action of a recombinase, the recombination site in the DNA construct can undergo recombination to produce an intermediate minicircle and a backbone heteroDNA. For example, the recombination site is a loxP sequence, and the corresponding recombinase is a Cre recombinase. For example, the recombination site is a FRT sequence, and the corresponding recombinase is a Flp recombinase. For example, the recombination site is an attB / attP sequence, and the corresponding recombinase is a PhiC31 recombinase. In this application, the use of the recombination site is to remove the backbone heteroDNA from the DNA construct, forming an intermediate minicircle for continued amplification. The recombination site described in the present application can also include its mutants, homologues or analogs.

[0108] As used herein, the term "recombinase" generally refers to an enzyme involved in the process of gene mapping and recombination. Recombinases are responsible for recognizing and cleaving specific recombination sites and connecting the two molecules involved in the recombination. For example, the recombinase can be Cre recombinase, Flp recombinase, or phiC31 recombinase. Alternatively, the recombinase can be any recombinase known in the art.

[0109] In this application, the term "site-specific recombinase" generally refers to a family of enzymes that mediate site-specific recombination between specific DNA sequences recognized by the enzyme, known as recombinase recognition sites. Examples of site-specific recombinases include, but are not limited to, phiC31 integrase, FLP recombinase, Cre recombinase, Dre recombinase, BxbI integrase, and phiBT1 integrase.

[0110] In the present application, the term "telomerase" is any polypeptide capable of cutting and reconnecting a template containing a telomerase recognition site to produce a covalently closed linear DNA molecule. In the present application, the telomerase can have DNA cutting and connecting functions. A typical substrate of telomerase is circular double-stranded DNA. If this DNA contains a telomerase recognition site, the enzyme can cut the DNA at this site and connect the ends to produce a linear double-stranded covalently closed DNA molecule. In the present application, the telomerase that can be used in the method of the present application includes but is not limited to protelomerases from bacteriophages, such as phiHAP-1 from Halomonas aquamarina, PY54 from Yersinia enterolytica, phiKO2 from Klebsiella oxytoca, and VP882 from Vibrio sp., as well as N15 from Escherichia coli, or variants thereof.

[0111] As used herein, the term "telomerase recognition site" generally refers to any DNA sequence in a DNA construct whose presence allows it to be converted into closed linear DNA by the enzymatic activity of telomerase. Specifically, the telomerase recognition site is required for double-stranded DNA to be cleaved and rejoined by telomerase to form covalently closed linear DNA. Typically, the telomerase recognition site comprises inverted repeat sequences, which are twofold rotationally symmetrical and may also be referred to as double-stranded palindromic sequences.

[0112] In the present application, the telomerase recognition site may encompass variants or homologs of the telomerase recognition sequence. For example, the variant includes truncations, substitutions, or deletions relative to the native sequence. A variant sequence is any sequence whose presence in a DNA construct or intermediate minicircle allows it to be converted into a closed linear DNA by the enzymatic activity of telomerase. This can be easily determined using a suitable assay for measuring the formation of closed linear DNA. Any suitable assay described in the art can be used.

[0113] In this application, the term "intermediate minicircle" generally refers to the portion containing the target gene of the two parts of the DNA construct described in this application that is divided under certain conditions. In some cases, the intermediate minicircle contains a replication initiation site and the target gene, so that the intermediate minicircle can replicate under appropriate conditions to increase the number of the target gene. In this application, the intermediate minicircle can be amplified in an environment without resistance. In some cases, the intermediate does not contain the selection element described in this application. In some cases, the intermediate minicircle also contains other sites that enable the intermediate minicircle to be cut to form a linear DNA molecule.

[0114] In the present application, the term "skeleton heteroDNA" generally refers to the part that does not include the target gene in the two parts that the DNA construct is divided into under certain conditions. This part of the skeleton heteroDNA is no longer needed for the production step of the subsequent DNA, and therefore can be removed by certain steps. For example, the skeleton heteroDNA contains skeleton sequences, and these sequences need to be removed in the subsequent DNA production process. For example, the skeleton heteroDNA includes screening elements. For example, the skeleton heteroDNA does not include a replication initiation site, and therefore, after being introduced into the production strain, the skeleton heteroDNA that does not include the replication initiation site can be lost during the amplification of the production strain, thereby being removed. In certain embodiments, the skeleton heteroDNA can be removed by the auxiliary elements described in the present application, with corresponding tools / under specific environmental conditions. In certain embodiments, the skeleton heteroDNA is integrated into the production bacteria genome and no longer exists in a free state outside the production bacteria genome.

[0115] In this application, the term "terminal-free linear DNA" generally refers to a linear, closed-end DNA. The terminal-free linear DNA can be single-stranded or double-stranded. For example, the terminal-free linear DNA is a linear, double-stranded, covalently closed DNA. The two ends of the terminal-free linear DNA can be closed by inverted repeat sequences. For example, the two ends of the terminal-free linear DNA can be closed by connecting after telomerase cleavage to form a covalently closed "dumbbell" or "dog bone" shaped structure. The terminal-free linear DNA can form a "dumbbell" structure by hybridization of two complementary sequences within the same molecule, resulting in a double-stranded middle segment with two single-stranded loops on both sides. One method of forming terminal-free linear DNA is to allow telomerase to act on a telomerase recognition site, thereby generating terminal-free linear DNA. In some cases, telomerase can act on one telomerase recognition site, and in some cases, telomerase can act on two telomerase recognition sites. For example, when acting on two telomerase recognition sites, this step can also assist in removing unwanted parts / elements in the plasmid.

[0116] In the present application, the term "production bacteria" generally refers to bacteria that are capable of maintaining and / or replicating a DNA construct / plasmid. Typically, Escherichia coli can be used as the production bacteria.

[0117] In this application, the term "cell" generally refers to a cell in which a DNA construct / plasmid can be maintained and / or replicated, including prokaryotes and eukaryotes, such as bacteria (Escherichia coli), fungi (yeast), insect cells, and mammalian cells. These cells can provide the components (such as various enzymes and nucleotide monomer molecules) necessary for the replication of the target gene, and can also provide the recombinase required for reorganization. The expression of the recombinase in the cell can be controlled expression or induced expression. In some embodiments, the coding gene of the recombinase is integrated into the genome of the cell. In some other embodiments, the coding gene of the recombinase is inserted into another expression vector, which can be introduced into the front or back of the host cell together with a precursor plasmid. In some embodiments, the coding gene of the recombinase is contained in a precursor plasmid, and the recombinase can be expressed after being introduced into the cell. In this application, the term "cell" can also cover cells for DNA delivery in addition to cells for the production of target products, for example, cells from any source. The cell can help DNA reach the target location.

[0118] In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences.Can calculate " sequence homology per-cent " in the following manner: two sequences to be compared are compared in comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number in the position to obtain the number of matching positions, with the number of matching positions divided by the total number of positions (that is, window size) in the comparison window, and result is multiplied by 100, to produce sequence homology per-cent.Comparison carried out in order to determine the sequence homology per-cent, can realize by several ways known in the art, for example, use publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared or within a region of interest. Homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Earson and D. J. Lipman, "Improved tools for biological sequence comparison," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D. J. Lipman and W. R. Earson, "Rapid and sensitive protein similarity search," Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers, and D. Lipman, "A basic local alignment search tool," J. Mol. Biol., 215: 403-410, 1990.

[0119] In this application, the term "kit" generally refers to a kit comprising at least one tool that enables the production of a terminal linear DNA product from a DNA construct. For example, the kit may comprise a DNA construct and may also comprise some enzymatic tools. For example, the kit may further comprise instructions for use. For example, the kit may also comprise production bacteria. For example, the kit may also comprise an intermediate minicircle.

[0120] In this application, the term "pharmaceutical composition" generally refers to a preparation in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is to be administered. These preparations can be sterile. In this application, the pharmaceutical composition can be formulated for administration to living humans or animals. In this application, the pharmaceutical composition may also contain other active molecules.

[0121] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0122] Furthermore, as used in this specification and the appended claims, the singular forms "a / an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "a / an" and "one or more" and "at least one" may be used interchangeably herein.

[0123] Detailed Description of the Invention

[0124] In one aspect, the present application provides a DNA construct comprising: 1) a replication origin; 2) a selection element; 3) a gene of interest; 4) an auxiliary element; and 5) a telomerase recognition site.

[0125] In the construct, under the action of auxiliary elements, the DNA construct can form an intermediate minicircle and a backbone heteroDNA under appropriate conditions. The intermediate minicircle comprises the replication origin, the target gene, and the telomerase recognition site, and the backbone heteroDNA comprises the screening element. For example, the backbone heteroDNA can be in a circular form, a linear form, or can be integrated into the genome of the production bacteria.

[0126] After performing the screening function in the early stage, the screening element in the DNA construct no longer plays other functions in the amplification step and is not a necessary element in the subsequent production process. Therefore, it can be removed by certain means after the screening function is completed without affecting the subsequent DNA production and preparation process.

[0127] On the other hand, the present application provides a method for preparing end-free linear DNA, which mainly includes the following steps: 1) constructing a parent plasmid containing a replication initiation site, a screening element, a target gene, and a telomerase recognition site; 2) allowing the parent plasmid to form an intermediate minicircle and a backbone heteroDNA under appropriate conditions, wherein the intermediate minicircle contains the replication initiation site, the target gene and the telomerase recognition site, and the backbone heteroDNA contains the screening element; 3) amplifying the intermediate minicircle; and 4) obtaining end-free linear DNA carrying the target gene by telomerase digestion.

[0128] In the DNA construct, the backbone sequence can be removed under the action of an auxiliary element. The removed backbone sequence does not contain a replication initiation site and does not have replication ability. It can be lost during the amplification of the cell / production strain, or it can be integrated into the production bacteria genome, thereby no longer stably existing in a free state outside the production bacteria genome. Under the action of the auxiliary element, the DNA construct can also form an intermediate mini-ring at the same time. The intermediate mini-ring contains a replication initiation site and can be continuously amplified during the amplification of the cell / production process.

[0129] In the present application, the intermediate microcircle does not contain the screening element and can be amplified in an environment without screening pressure.

[0130] In certain embodiments, the parent plasmid contains a target gene expression cassette, a replication origin element, a resistance gene selection element, an auxiliary element, and a telomerase recognition site (Protelomerase recognition site, TelLR).

[0131] First, in Escherichia coli containing the parent plasmid, under the action of auxiliary elements, the parent plasmid undergoes recombination to form an intermediate minicircle containing the target gene expression cassette and the replication initiation element and a backbone heteroDNA containing the resistance gene. The intermediate minicircle containing the replication initiation element can be continuously replicated in bacteria, while the backbone heteroDNA without the replication initiation element will be lost during the bacterial amplification process, or in some embodiments, integrated into the genome of the producer bacteria, thereby no longer stably existing in a free state outside the genome of the producer bacteria. Subsequently, the bacteria containing the intermediate minicircle are amplified under resistance-free culture conditions, and a large amount of minicircle DNA is extracted and prepared. Finally, telomerase digestion and column purification are used in vitro to obtain a high-purity linear DNA end product with covalently closed ends.

[0132] In the present application, the auxiliary element can be selected from any element known in the prior art that can cause DNA to be split or broken. The auxiliary element can also further have the function of reconnecting specific positions of the split or broken elements.

[0133] For example, the auxiliary element can be a recombination element. For example, the recombination element can include recombination sites, wherein the recombination sites are paired recombination sites, and the paired recombination sites can cause the DNA construct to recombine under the action of a recombinase to obtain an intermediate minicircle and a backbone heteroDNA. For example, the sequences of the paired recombination sites are oriented in the same direction.

[0134] For example, the recombinase can function under inducible conditions, such as temperature induction or chemical induction by arabinose.

[0135] In the present application, the recombination site comprises a site-specific recombinase site. The site-specific recombinase site can be recognized by a site-specific recombinase.

[0136] For example, a site-specific recombinase inducible expression system can be constructed on the auxiliary plasmid of the DNA construct / parent plasmid, and then transformed into an Escherichia coli host cell for expression; or the site-specific recombinase inducible expression system can be integrated into the Escherichia coli host cell genome for expression. For example, the expression system comprises: an inducible prokaryotic transcription promoter, such as the araBAD promoter, a site-specific recombinase gene, and a transcription terminator. The site-specific recombinase gene can include: the Cre recombinase from the P1 phage Cre-loxP recombination system, a pair of specific recombination sites, i.e., the loxP sequence. For example, it can include: the Flp-FRT recombination system, comprising the Flp recombinase, and the FRT sequence. For example, it can include phiC31 integrase, and attB / attP. For example, it can include Dre and Rox. When constructing a recombinase expression system on a plasmid vector, the vector plasmid can be a conditionally induced loss-type plasmid replication element, such as the temperature-sensitive replication element pSC101 (ts) plasmid.

[0137] For example, the site-specific recombinase can be selected from one or more of the following groups: phiC31 integrase, FLP recombinase, Cre recombinase, Dre recombinase, BxbI integrase, and phiBT1 integrase.

[0138] For example, the DNA construct does not contain a gene encoding the recombinase, and the gene encoding the recombinase can be introduced into a production strain / cell for expression by another vector. Or the production strain / cell itself contains a recombinase. In the present application, the recombinase can be a Cre recombinase, which can comprise the sequence shown in SEQ ID NO:38, or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0139] In the present application, the recombinase can be a phiC31 recombinase, which can comprise the sequence shown in SEQ ID NO:35, or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0140] In the present application, the recombinase can be a Dre recombinase, which can comprise the sequence shown in SEQ ID NO: 39, or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0141] In the present application, the recombinase can be a FLP recombinase, which can comprise the sequence shown in SEQ ID NO: 36, or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0142] In the present application, the auxiliary element can also enable the DNA construct to form an intermediate minicircle via a transposase. Any transposase known in the prior art can be used in the present application. For example, the transposase can be selected from one or more of the following groups: Tn3, Tn5, Tn7, and Tn10.

[0143] In the present application, the auxiliary element can also be an editing site of a gene editing system. Any gene editing method known in the prior art can be used in the present application to remove the backbone sequence containing the screening sequence. For example, the backbone sequence can be removed using the CRISPR / Cas system, more specifically, the backbone sequence can be removed using the CRISPR / Cas9 system, or the backbone sequence can be removed using TALENs technology.

[0144] In the present application, the replication initiation site in the DNA construct can be any replication initiation site known in the art. For example, the replication initiation site can be derived from a replication initiation site of bacteria or bacteriophage. In some specific embodiments, the replication initiation site is a replication initiation site of bacteria. For example, the replication initiation site can be selected from the group consisting of R6K replication initiation site, pSC101 replication initiation site, ColE1 replication initiation site, ColE2 replication initiation site, ColE3 replication initiation site, pMB1 replication initiation site, pBR322 replication initiation site, pUC replication initiation site, and P15A replication initiation site.

[0145] For example, the replication initiation site can be an R6K replication initiation site, which can comprise the sequence shown in SEQ ID NO:37, or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0146] For example, the replication origin can be the ColE1 replication origin, which can comprise the sequence shown in SEQ ID NO: 40, or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0147] In the present application, when the sequence comprises a sequence with at least 60% or more identity, the change of nucleotides will not affect its function as a replication origin site.

[0148] In the present application, any screening element can also be used to confirm whether the target gene is successfully introduced into the destination vector / transferred into the target cell. For example, the screening element can be a forward screening element. For example, some resistance gene screening elements. For example, the resistance gene screening element can be selected from: ampicillin (Ampicillin), kanamycin (kanamycin), chloramphenicol (chloramphenicol), spectinomycin (Spectinomycin), tetracycline (tetracycline), bleomycin (Zeocin / blemycin), streptomycin (Streptomycin), hygromycin (Hygromycin), gentamicin (Gentamicin), apramycin sulfate (Apramycin sulfate), erythromycin (Erythromycin), and nourseothricin (Nourseothricin). For example, commonly used can be kanamycin.

[0149] In the present application, a counter-selection element can also be used. For example, the counter-selection element can use SacB, ccdB, rpsL, tetAR, pheS, thyA, lacY, gata-1, upp, bla I and / or mazF counter-selection markers.

[0150] In the present application, a telomerase recognition site is introduced into the DNA construct to break the amplified intermediate minicircle, forming endless linear DNA. The DNA construct may contain one or two telomerase recognition sites. The use of these telomerase recognition sites solves the problem of product multimerization in the prior art, resulting in a monomeric endless linear DNA product.

[0151] In certain embodiments, the telomerase can be derived from any known telomerase, for example, a telomerase from a bacteriophage, such as phiHAP-1 from Halomonas aquamarina, PY54 from Yersinia enterolytica, phiKO2 from Klebsiella oxytoca, VP882 from Vibrio sp., and N15 from Escherichia coli, or variants thereof.

[0152] The target sequence recognized by protelomerase TelN consists of a 56-bp palindrome flanked by telR and telL. The center of the target sequence also contains a palindrome, telO. Telomerase TelN cleaves within the telO sequence, forming a covalently closed hairpin structure between the two cleaved ends.

[0153] Telomerase recognition sites can be located on both sides of the target gene. Telomerase recognizes the telomerase recognition sites and performs cutting-ligation activity, thereby generating linear, end-covalently linked DNA (terminal-free linear DNA) containing the target gene.

[0154] In certain embodiments, the DNA construct comprises two telomerase recognition sites, and the space between the two telomerase recognition sites only contains the target gene, but does not contain a replication initiation site and / or a recombination site.

[0155] In certain embodiments, the DNA construct comprises only one telomerase recognition site, which is located on the intermediate minicircle after recombination of the DNA construct. The intermediate minicircle can be amplified to obtain linear DNA through telomerase recognizing the telomerase recognition site.

[0156] In certain embodiments, the telomerase is bacteriophage N15 TelN or a variant thereof. For example, the sequence of the telomerase can be found in NCBI Accession No. NP_046924.1. For example, the telomerase can have the amino acid sequence of SEQ ID NO:33, or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. The variant or sequence having sequence homology does not affect the telomerase's function as a telomerase to cleave and ligate DNA to form closed-end linear DNA.

[0157] In certain embodiments, the telomerase recognition site may have the nucleic acid sequence of SEQ ID NO:8, or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0158] In a specific embodiment, the DNA construct / parent plasmid comprises a target gene expression cassette, a replication origin (Ori), an antibiotic resistance gene screening element (Antibiotic resistance gene), a pair of recombinase recognition sites (RS), and a protelomerase recognition site (TelLR). The recombinase and its recognition site combination are selected from phiC31 and attB / attP, Cre and loxP, Flp and FRT, and Dre and Rox; the replication origin is the R6K replication origin; and the resistance gene screening element is the kanamycin resistance gene.

[0159] The DNA construct / parent plasmid and the inducible recombinase expression plasmid can be co-transformed into Escherichia coli, and the expression of the recombinase can be induced under chemical induction (arabinose induction) or temperature induction (37°C induction). Alternatively, an Escherichia coli strain containing the DNA construct / parent plasmid can be first established, and then the recombinase expression plasmid can be introduced into the strain to induce the expression of the recombinase. Alternatively, a strain containing an inducible recombinase expression system in the genome can be first established, and then the parent plasmid can be introduced into the strain to induce the expression of the recombinase. After recombination occurs, Escherichia coli clones containing non-resistance gene intermediate minicircles are screened on plates containing and without resistance. Telomerase is used for digestion (the telomerase recognition site can be one or two) to form a linear DNA product with closed ends.

[0160] On the other hand, the present application provides an intermediate product minicircle, which is obtained by the method described in the present application, and the intermediate product minicircle comprises the replication initiation site, the target gene and the telomerase recognition site.

[0161] In certain embodiments, the intermediate product minicircle does not comprise a selection element. In certain embodiments, the intermediate product minicircle can be amplified in an environment without selection pressure.

[0162] In another aspect, the present application also provides a terminal-free linear DNA obtained by the method described herein. In the present application, the terminal-free linear DNA comprises closed structures at both ends. The closed structures are obtained by telomerase digestion. The closed structures are shaped like a "dumbbell" or "dog bone."

[0163] In the present application, term " cell " generally refers to individual cells, cell lines or cell cultures that can be or have been recipients of subject's plasmid or vector, and it includes DNA construct of the present invention, intermediate product minicircle or no end linear DNA.Cell can include the offspring of individual cells.Due to natural, accidental or intentional mutation, offspring may not necessarily be identical with the original mother cell (in the form of total DNA complement or on genome).Cell can include the cell transfected in vitro with the parent plasmid described in the application.Cell can be bacterial cells (for example, Escherichia coli), yeast cells or other eukaryotic cells, for example COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells or stem cells (for example ES cells, iPS cells, mesenchymal stem cells) or immune cells (for example B cells, T cells, NK cells, NKT cells, macrophages, DC cells).

[0164] In another aspect, the present application provides a production bacterium. In the present application, the production bacterium can be Escherichia coli. For example, Escherichia coli JM108, TOP10, DH5α, GT115, pir1, pir2, etc., as well as other modified strains derived from related strains.

[0165] On the other hand, the present application also provides a kit. The kit may include the construct described in the present application, as well as other tools that enable the construct to produce end-free DNA. For example, the kit may include the DNA construct. For example, the kit may also include production bacteria to be introduced into the DNA construct or intermediate minicircle. For example, the kit may also include a vector encoding a recombinase. For example, the kit may also include telomerase. For example, the kit may also include an intermediate minicircle. For example, the kit may also include instructions, which indicate a method for using the DNA construct to produce the end-free linear DNA.

[0166] On the other hand, the present application also provides a pharmaceutical composition comprising the terminal-free linear DNA produced by the method of the present application, and optionally a pharmaceutically acceptable carrier. The delivery method of the terminal-free linear DNA can be physical, such as electrofection, microinjection; or chemical, including but not limited to liposomes, lipid nanoparticles, vesicles (including exosomes), cationic polymer complexes (such as PEI), dendrimers and microparticles of various polymers (such as PLGA nanoparticles, etc.), polypeptide protein delivery vectors, and delivery systems with active targeting effects using targeting molecules such as asialoglycoprotein receptors, polysaccharides, epidermal growth factor, folic acid, transferrin, steroid hormones, monoclonal antibodies, etc.

[0167] In addition, the pharmaceutical composition may also include one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or suitable formulations of preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes but is not limited to liquid, frozen and lyophilized compositions.

[0168] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, such as sodium; and / or nonionic surfactants.

[0169] On the other hand, the present application also provides a method for delivering a target gene to a target cell, comprising contacting the terminal-free linear DNA with the target cell. The delivery method of the terminal-free linear DNA can be a physical method, such as electrofection, microinjection; or a chemical method, including but not limited to liposomes, lipid nanoparticles, vesicles (including exosomes), cationic polymer complexes (such as PEI), dendrimers and various polymer microparticles (such as PLGA nanoparticles, etc.), polypeptide protein delivery vectors, and delivery systems with active targeting effects using targeting molecules such as asialoglycoprotein receptors, polysaccharides, epidermal growth factor, folic acid, transferrin, steroid hormones, monoclonal antibodies, etc. For example, the terminal-free linear DNA can be an effective amount of terminal-free linear DNA.

[0170] On the other hand, the terminal-free linear DNA produced by the present application can be used for expression in host cells in vitro and in vivo.

[0171] The end-free linear DNA produced in this application can be used for gain-of-function therapeutic gene expression, for example, for gene replacement therapy for genetic diseases and gene addition-type anti-disease gene expression.

[0172] The methods / DNA constructs and endless linear DNA described herein can also be used to knock down endogenous gene expression in host cells, for example, to encode a microRNA gene to knock down target endogenous gene expression.

[0173] The methods / DNA constructs and the end-free linear DNA of the present application can also be used for the production of DNA vaccines. DNA vaccines generally encode modified forms of the DNA of infectious organisms. DNA vaccines are administered to subjects, where they then express selected proteins of the infectious organisms, eliciting an immune response that is generally protective or therapeutic against the proteins. DNA vaccines can also encode tumor antigens in cancer immunotherapy.

[0174] The methods / DNA constructs and endless linear DNA of the present application can also be used for the production of mRNA.

[0175] The methods / DNA constructs and endless linear DNA described herein can also be used in the field of gene editing. For example, gene knockout and gene knock-in. For example, large gene knock-in DNA donor templates for gene editing and homologous recombination.

[0176] The methods / DNA constructs and endless linear DNA described in this application can also be used as DNA donors for DNA transposons, which include but are not limited to Sleeping Beauty transposons and piggyBac transposons.

[0177] The methods / DNA constructs and the endless linear DNA described herein can also be used for packaging and / or production of viral vectors, including but not limited to adeno-associated viruses, lentiviruses, herpes simplex viruses, and adenoviruses.

[0178] Without intending to be bound by any theory, the following examples are merely for illustrating the constructs, production methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.

[0179] Example

[0180] Example 1 Chemically induced expression of phiC31 recombinase to prepare intermediate minicircles

[0181] 1.1 Construction of the arabinose-inducible phiC31 recombinase expression vector pSC101(ori)-araBAD-phiC31

[0182] The phiC31-Rep101(Ts) gene fragment (shown in SEQ ID NO:1) was synthesized conventionally and homologously recombined into the pKD46 vector containing the pSC101(ori) temperature-sensitive replication origin element (shown in SEQ ID NO:2) and the araBAD arabinose chemically inducible promoter (shown in SEQ ID NO:3) via 5' BamHI and 3' SpeI sites, resulting in the pSC101(ori)-araBAD-phiC31 vector (Figure 1). This vector is used for L-arabinose chemically induced expression of the phiC31 recombinase.

[0183] 1.2 Construction of parental vector containing recombinase recognition sites attB / attP and telomerase recognition site TelLR

[0184] 1.2.1 Construction of pFluc-attB / P-kana parental vector

[0185] A Fluc fragment (as shown in SEQ ID NO: 4) was synthesized by conventional gene synthesis and cloned into the pCpGfree-Gluc vector (invivoGen, Cat. No. pcpgf-lucia, sequence shown in SEQ ID NO: 5) through the 5'NcoI and 3'NheI sites to obtain the pCpGfree-Fluc vector. An attB-kana-attP fragment (as shown in SEQ ID NO: 6) was synthesized by conventional gene synthesis and cloned into the pCpGfree-Fluc vector through homologous recombination at the 5'EcoRI and 3'EcoRI sites to obtain the pFluc-attB / P-kana parent vector (as shown in Figure 2).

[0186] 1.2.2 Construction of pHTM-Fluc-attB / P-kana-TelLR1 parent vector

[0187] A TelLR1-HTM fragment containing a liver-specific promoter (as shown in SEQ ID NO: 9) was conventionally synthesized and cloned into the plasmid pFluc-attB / P-kana via homologous recombination at the 5' AfeI and 3' SspI sites to obtain the pHTM-Fluc-attB / P-kana-TelLR1 vector (as shown in SEQ ID NO: 10) ( FIG. 3 ).

[0188] 1.2.3 Construction of pHTM-Fluc-attB / P-kana-TelLR2 parent vector

[0189] The TelLR2 fragment (as shown in SEQ ID NO: 11) was synthesized conventionally and cloned into the pHTM-Fluc-attB / P-kana-TelLR1 vector via the 5'HpaI and 3'XmaI sites to obtain the pHTM-Fluc-attB / P-kana-TelLR2 parent vector (sequence shown in SEQ ID NO: 12) ( FIG4 ).

[0190] 1.3 Arabinose-induced expression of phiC31 recombinase to prepare intermediate minicircles

[0191] 1.3.1 Transformation of the recombinase expression vector and parental vector and verification of parental plasmid recombination

[0192] Take 1 μL of pSC101(ori)-araBAD-phiC31 and pHTM-Fluc-attB / P-kana-TelLR2 plasmids (both at a concentration of 100 ng / μL) and add them to 50 μL of GT115 competent cells. Let the mixture stand on ice for 30 minutes, heat shock at 42°C for 90 seconds, and let it stand on ice again for 3 minutes. Add 500 μL of SOC medium to the mixture and incubate on a shaker at 30°C for 1 hour at 220 rpm. Take an appropriate amount of 150 μL of bacterial solution and spread it on an LB plate containing Amp (100 μg / mL), Kana (50 μg / mL) and 1% glucose, and invert it in a 30°C incubator for overnight culture.

[0193] A single clone was picked from the LB plate and inoculated into 4 mL of LB + Amp (100 μg / mL) + Kana (50 μg / mL) + 1% glucose medium, and cultured overnight at 30°C and 220 rpm. The next day, 1 mL of the bacterial solution was centrifuged, and the bacteria were washed twice with empty LB medium. The bacteria were then resuspended in LB + 2% L-arabinose culture medium and cultured at 30°C and 220 rpm to induce phiC31 recombinase expression for 2 hours, 5 hours, and 7 hours. The remaining 3 mL of bacterial solution was used to identify the presence of plasmids in the bacterial solution before induction. The plasmids in the bacterial solution before induction were extracted and digested with EcoRI (NEB, Cat. No. R3101L). The enzyme digestion system is shown in Table 1. The plasmids and enzyme digestion products were subjected to agarose gel electrophoresis.

[0194] Table 1. EcoRI-HF enzyme digestion system

[0195] Take 4 μL of the induced bacterial solution at three induction time points and transfer it to 4 mL of empty LB, culture it at 37°C, 220 rpm, and incubate it overnight. 4 ~10 6 The cells were plated on empty LB plates and cultured overnight at 37°C. The remaining bacterial suspension was used to identify the parental plasmid recombination and pSC101(ori)-araBAD-phiC31 plasmid loss in the induced bacterial suspension. The plasmids in the induced bacterial suspension were extracted and digested with EcoRI. The plasmids and digested products were subjected to agarose gel electrophoresis.

[0196] Agarose gel electrophoresis results are shown in Figure 5. Before induction, the pSC101(ts)-araBAD-phiC31 plasmid (enzyme digestion product, 6262 bp) and the pHTM-Fluc-attB / P-kana-TelLR2 parental plasmid (enzyme digestion products, 2437 bp and 1520 bp) were coexisting in E. coli. After induction, the pHTM-Fluc-attB / P-kana-TelLR2 parental plasmid underwent intramolecular recombination in the bacterial solution, forming an intermediate minicircle (enzyme digestion products, 2437 bp and 309 bp) that lacked the kana resistance gene. Simultaneously, the pSC101(ori)-araBAD-phiC31 plasmid was lost. It was also found that different induction times had no significant effect on the molecular recombination of the parental plasmids.

[0197] 1.3.2 Effects of different concentrations of L-arabinose on phiC31 recombinase expression and parental plasmid recombination

[0198] Five 1ml aliquots of the pre-induction culture were centrifuged and washed twice with empty LB medium. The cells were then resuspended in LB medium containing 2%, 1%, 0.2%, 0.02%, and 0.002% L-arabinose, respectively, and incubated at 30°C, 220 rpm, to induce phiC31 recombinase expression for 5 hours. Four μL of the post-induction culture was transferred to 4 mL of empty LB medium and incubated overnight at 37°C, 220 rpm. The next day, the culture was sampled for identification of parental plasmid recombination within the post-induction culture. The plasmid was extracted and digested with EcoRI, and the plasmid and digestion products were subjected to agarose gel electrophoresis. The agarose gel electrophoresis results are shown in Figure 6. The digestion products of the intermediate minicircle induced at each concentration range were 2437 bp and 309 bp, respectively, with the recombinase vector plasmid lost. The above results indicate that different concentrations of L-arabinose ranging from 0.002% to 2% can induce the expression of phiC31 recombinase and mediate the recombination of the parental plasmid.

[0199] 1.3.3 Screening and morphological identification of intermediate microrings

[0200] Single colonies on the empty LB plates were picked and inoculated onto LB+Amp plates (cultured at 30°C), LB+Kana plates (cultured at 37°C), and empty LB plates (cultured at 37°C) in sequence, and cultured overnight. The next day, single colonies that grew only on the empty LB plates but not on either the LB+Amp or LB+Kana plates were picked and inoculated into 5 mL of empty LB medium and cultured overnight at 37°C. The next day, the strains were preserved, the HTM-Fluc-attL / R-TelLR2 intermediate minicircle was extracted, and identified by EcoRI digestion. Table 2 shows the screening efficiency of each step of the intermediate minicircle. The agarose gel electrophoresis results are shown in Figure 7. The digestion products of the intermediate minicircle are 2437 bp and 309 bp, and the recombinase vector plasmid is lost, indicating that the vector is correctly constructed. It can also be seen from the figure that the intermediate minicircle exists as a mixture of monomeric or polymeric minicircle plasmids.

[0201] Table 2. Screening efficiency of each step of the HTM-Fluc-attL / R-TelLR2 intermediate minicircle

[0202] Example 2 Chemically induced FLP recombinase expression to prepare intermediate minicircles

[0203] 2.1 Construction of the arabinose-inducible FLP recombinase expression vector pSC101(ori)-araBAD-FLP

[0204] The FLP-Rep101(Ts) gene fragment (as shown in SEQ ID NO: 13) was synthesized by conventional gene synthesis and homologously recombined into the pKD46 vector containing the pSC101(ori) temperature-sensitive replication origin element and the araBAD arabinose chemically inducible promoter via the 5' EcoRI and 3' SpeI sites to obtain the pSC101(ori)-araBAD-FLP vector (sequence shown in SEQ ID NO: 14) ( FIG8 ). This vector is used for L-arabinose-induced FLP recombinase expression.

[0205] 2.2 Construction of the pFluc-FRT-kana parent vector containing the FRT recombinase recognition site

[0206] The FRT-kana-FRT fragment (shown in SEQ ID NO: 15) was synthesized by conventional gene synthesis and cloned into the pCpGfree-Fluc vector via homologous recombination at the 5' EcoRI and 3' EcoRI sites to obtain the pFluc-FRT-kana parent vector (sequence shown in SEQ ID NO: 16).

[0207] 2.3 Arabinose-induced FLP recombinase expression and preparation of minicircles

[0208] 2.3.1 Transformation of the recombinase expression vector and parental vector and verification of parental plasmid recombination

[0209] 1 μL of each pSC101(ori)-araBAD-FLP and pFluc-FRT-kana plasmid (both at a concentration of 100 ng / μL) was co-transformed into 50 μL of GT115 competent cells. The transformation process was the same as in Example 1.3. 150 μL of the appropriate bacterial solution was spread on an LB plate containing Amp (100 μg / mL), Kana (50 μg / mL) and 1% glucose, and the plate was inverted and cultured in a 30°C incubator overnight.

[0210] Pick a single clone from the LB plate, inoculate it into 4mL LB+Amp (100μg / mL)+Kana (50μg / mL)+1% glucose medium, and culture it at 30℃, 220rpm overnight. The next day, take 1mL of bacterial solution and centrifuge it. Wash the bacteria twice with empty LB medium, then resuspend the bacteria with LB+2% L-arabinose culture medium, and induce FLP recombinase expression at 30℃, 220rpm for 2hrs, 5hrs and 8hrs. The remaining 3mL of bacterial solution was used to identify the presence of plasmids in the bacterial solution before induction, extract the plasmids in the bacterial solution before induction and perform EcoRI enzyme digestion, and perform agarose gel electrophoresis on the plasmids and enzyme digestion products. Take 4μL of the induced bacterial solution at three induction time points and transfer it to 4mL empty LB, and culture it at 37℃, 220rpm overnight. The next day, take 5μL of the bacterial solution and dilute it 10 4 ~10 6 The cells were plated on empty LB plates and cultured overnight at 37°C. The remaining bacterial suspension was used to identify the recombinant parental plasmid and the loss of the pSC101(ori)-araBAD-FLP plasmid in the induced bacterial suspension. The plasmids in the induced bacterial suspension were extracted and digested with EcoRI. The plasmids and digested products were subjected to agarose gel electrophoresis.

[0211] Agarose gel electrophoresis results are shown in Figure 9. Before induction, the pSC101(ori)-araBAD-FLP plasmid (digestion products of 4938 bp and 778 bp) and the pFluc-FRT-kana parental plasmid (digestion products of 2745 bp and 1530 bp) were coexisting in E. coli. After induction, the pFluc-FRT-kana parental plasmid underwent intramolecular recombination in the bacterial solution, forming a minicircle without the kana resistance gene (digestion products of 2745 and 308 bp), while the pSC101(ori)-araBAD-FLP plasmid was lost. It was also found that different induction times had no significant effect on the formation of the intermediate minicircle.

[0212] 2.3.2 Screening and morphological identification of intermediate microrings

[0213] Single colonies on the empty LB plate were picked and inoculated onto LB+Amp plates (cultured at 30°C), LB+Kana plates (cultured at 37°C), and empty LB plates (cultured at 37°C) in sequence and cultured overnight. The next day, single clones that grew only on the empty LB plate but not on either the LB+Amp or LB+Kana plates were picked and subjected to colony PCR using primers kana-F (SEQ ID NO: 17) and kana-R (SEQ ID NO: 18) that span the parental plasmid kana backbone fragment (Figure 10). As shown in the colony PCR results in Figure 10, 22 of the 49 selected clones contained the target minicircle that removed the kana resistance gene.

[0214] A single clone identified by PCR was inoculated into 5 mL of empty LB medium and cultured overnight at 37°C. The next day, the strain was preserved, minicircle DNA was extracted, and digested with EcoRI for identification. Table 3 shows the screening efficiency of each step for the Fluc-FRT intermediate minicircle. Figure 11 shows the agarose gel electrophoresis results. As can be seen, 20 of the 22 single clones contained the correctly constructed intermediate minicircle, and the recombinase vector plasmid was missing. The intermediate minicircle was also found to exist as a mixture of monomeric and multimeric minicircle plasmids.

[0215] Table 3. Screening efficiency of each step of Fluc-FRT minicircle

[0216] Example 3 Temperature-induced FLP recombinase expression to prepare intermediate minicircles

[0217] 1 μL each of the temperature-inducible FLP recombinase expression plasmid pCP20 and the pFluc-FRT-kana parental plasmid (both at a concentration of 20 ng / μL) was co-transformed into 50 μL of GT115 competent cells according to the co-transformation process described in Example 1.3. 150 μL of the appropriate bacterial solution was spread on an LB plate containing Amp (100 μg / mL), Chl (25 μg / mL), and Kana (50 μg / mL) antibiotics, and the plate was inverted and cultured in a 30°C incubator overnight.

[0218] Pick a single colony from the LB plate and inoculate it into 3 mL of empty LB medium. Incubate at 43°C on a shaker for 2, 2.5, and 3 hours to induce FLP recombinase expression, causing intramolecular recombination of the parental plasmid and simultaneous loss of the pCP20 plasmid. Spread an appropriate amount of the induction solution from each of the three induction time points onto an empty LB plate and incubate at 30°C overnight.

[0219] Single colonies from the empty LB plates were inoculated onto LB+Amp plates (incubated at 30°C), LB+Kana plates (incubated at 37°C), and then empty LB plates (incubated at 37°C) overnight. The next day, single colonies that grew only on the empty LB plates but not on either the LB+Amp or LB+Kana plates were selected for colony PCR (Figure 12) using primers kana-F and kana-R that span the kana backbone fragment of the parental plasmid. As shown in Figure 12, the colony PCR results showed that 34 of the 93 selected colonies contained the target minicircle that eliminated the kana resistance gene.

[0220] A single clone identified by PCR was inoculated into 5 mL of empty LB medium and cultured overnight at 37°C. The next day, the strain was preserved, minicircle DNA was extracted, and digested with EcoRI for identification. Table 4 shows the screening efficiency of each step for the Fluc-FRT intermediate minicircle. Figure 13 shows the agarose gel electrophoresis results of the minicircle plasmid and digestion products. As can be seen, 9 of the 34 single clones contained the correctly constructed intermediate minicircle, and the recombinase vector plasmid was missing. It was also found that the minicircle existed as a mixture of monomeric and multimeric minicircle plasmids.

[0221] Table 4. Screening efficiency of each step of Fluc-FRT minicircle

[0222] Example 4 Temperature-induced Cre recombinase expression to prepare intermediate minicircles

[0223] 4.1 Construction of the Cre recombinase gene expression vector pCP20-Cre

[0224] The Cre gene fragment (as shown in SEQ ID NO: 19) was synthesized conventionally and cloned into the pCP20 vector containing a temperature-sensitive replication initiation element and a λ repressor (ts) repressor via the 5' PshAI and 3' SgrAI sites to obtain the pCP20-Cre vector (sequence shown in SEQ ID NO: 20) ( FIG. 14 ), which is used for temperature-induced Cre recombinase expression.

[0225] 4.2 Construction of the pFluc-loxP-kana parent vector containing the loxP recombinase recognition site

[0226] A loxP-kana-loxP fragment (as shown in SEQ ID NO: 21) was synthesized conventionally and cloned into the pCpGfree-Fluc vector via homologous recombination at the 5'EcoRI and 3'EcoRI sites to obtain the pFluc-loxP-kana parent vector.

[0227] 4.3 Temperature-induced Cre recombinase expression and preparation of minicircles

[0228] 1 μL of the pCP20-Cre vector and the pFluc-loxp-kana parental vector (sequence shown in SEQ ID NO: 22) (both at a concentration of 40 ng / μL) were co-transformed into 50 μL of GT115 competent cells. The transformation process was the same as in Example 1.3. 100 μL of the bacterial solution was spread on an LB plate containing Amp (100 μg / mL), Chl (25 μg / mL), and Kana (50 μg / mL) antibiotics, and the plate was inverted and cultured in a 30°C incubator overnight.

[0229] Pick a single colony from the LB plate and inoculate it into 3 mL of empty LB medium. Incubate at 43°C on a shaker for 3 hours to induce Cre recombinase expression, causing intramolecular recombination of the parental plasmid and simultaneous loss of the pCP20-Cre plasmid. Spread an appropriate amount of the induced bacterial culture onto an empty LB plate and incubate at 30°C overnight.

[0230] Single colonies from empty LB plates were inoculated onto LB+Amp plates (incubated at 30°C), LB+Kana plates (incubated at 37°C), and empty LB plates (incubated at 37°C) overnight. The next day, single colonies that grew only on the empty LB plates but not on either the LB+Amp or LB+Kana plates were selected for colony PCR using primers kana-F and kana-R that span the kana backbone fragment of the parental plasmid.

[0231] Inoculate the correct clone identified by PCR into 5 mL of empty LB medium and incubate overnight at 37°C. The next day, preserve the strain, extract the minicircle DNA, and perform EcoRI digestion for identification.

[0232] Example 5 Temperature-induced Dre recombinase expression to prepare intermediate minicircles

[0233] 5.1 Construction of the Dre recombinase gene expression vector pCP20-Dre

[0234] The Dre gene fragment (as shown in SEQ ID NO: 23) was synthesized conventionally and cloned into the pCP20 vector containing a temperature-sensitive replication initiation element and a λ repressor M (ts) repressor via the 5' PshAI and 3' SgrAI sites to obtain the pCP20-Dre vector (sequence shown in SEQ ID NO: 24) ( FIG. 15 ), which is used for temperature-induced Dre recombinase expression.

[0235] 5.2 Construction of the pFluc-rox-kana parent vector containing the rox recombinase recognition site

[0236] The rox-kana-rox fragment (as shown in SEQ ID NO: 25) was synthesized by conventional gene synthesis and cloned into the pCpGfree-Fluc vector through homologous recombination at the 5' EcoRI and 3' EcoRI sites to obtain the pFluc-rox-kana parent vector (as shown in SEQ ID NO: 26).

[0237] 5.3 Temperature-induced Dre recombinase expression and preparation of minicircles

[0238] 80 ng of the pCP20-Dre vector and 20 ng of the pFluc-rox-kana parent vector were co-transformed into 50 μL of GT115 competent cells. The transformation process was the same as in Example 1.3. 100 μL of the bacterial solution was spread on an LB plate containing Amp (100 μg / mL), Chl (25 μg / mL), and Kana (50 μg / mL) antibiotics, and the plate was inverted and cultured in a 30°C incubator overnight.

[0239] Single colonies were picked from the LB plates and inoculated into 4 mL of empty LB medium. Cultured on a shaker at 37°C for 2, 3, and 6 hours to induce Dre recombinase expression, resulting in intramolecular recombination of the parental plasmid and simultaneous loss of the pCP20-Dre plasmid. Appropriate amounts of the induced bacterial culture at each of the three induction time points were spread onto empty LB plates and cultured overnight at 30°C.

[0240] Single colonies from empty LB plates were inoculated onto LB+Amp plates (incubated at 30°C), LB+Kana plates (incubated at 37°C), and empty LB plates (incubated at 37°C) overnight. The next day, single colonies that grew only on the empty LB plates but not on either the LB+Amp or LB+Kana plates were selected for colony PCR using primers kana-F and kana-R that span the kana backbone fragment of the parental plasmid.

[0241] The correct clone identified by PCR was inoculated into 5 mL of empty LB medium and cultured overnight at 37°C. The next day, the strain was preserved, minicircle DNA was extracted, and digested with EcoRI for identification. Table 5 shows the screening efficiency of each step for the Fluc-rox intermediate minicircle. Figure 16 shows the agarose gel electrophoresis results of the correctly identified minicircle plasmid and digestion products. The minicircle was present as a mixture of monomeric and multimeric minicircle plasmids.

[0242] Table 5. Screening efficiency of each step of Fluc-rox microcircle

[0243] Example 6 Sequential transformation and introduction of recombinase expression plasmids to prepare intermediate minicircles

[0244] 6.1 Preparation of E. coli competent cells containing pFluc-FRT-kana parent plasmid

[0245] Transform GT115 competent cells with the pFluc-FRT-kana parental plasmid, plate onto LB-Kana plates, and incubate at 37°C for 12-16 hours. The next day, select a single colony and inoculate it into 5 mL of LB+Kana medium and incubate at 37°C for 12-16 hours. Inoculate the overnight bacterial suspension into 100 mL of LB+Kana medium at a ratio of 1:100 and incubate at 37°C for 2-3 hours. Harvest the suspension when the OD600 reaches 0.4-0.5. Transfer the harvested suspension into a 50 mL pre-chilled centrifuge tube, place on ice for 10 minutes, and centrifuge at 4000g for 10 minutes at 4°C. Discard the supernatant and gently resuspend the cells in 10 mL of pre-chilled 0.1 mol / L CaCl2 solution. Place on ice for 15-30 minutes and centrifuge at 4000g for 10 minutes at 4°C. Discard the supernatant, add 4 mL of pre-cooled 0.1 mol / L CaCl2 solution containing 15% glycerol, gently suspend the cells, and place on ice for 5 minutes to obtain the competent cell suspension. Aliquot 100 μL into EP tubes and store at -80°C.

[0246] 6.2 Sequential transformation into FLP recombinase expression vector

[0247] 1 μL of pCP20 plasmid (40 ng / μL) was transformed into GT115 competent cells containing the pFluc-FRT-kana parental plasmid. The transformation process was the same as in Example 6.1. An appropriate amount of the bacterial solution was spread onto LB plates containing Amp (100 μg / mL), Chl (25 μg / mL), and Kana (50 μg / mL) antibiotics, and the cells were incubated in an inverted position at 30°C overnight. Since the pCP20 plasmid expresses sufficient FLP recombinase during the overnight incubation at 30°C, intramolecular recombination of the parental plasmid occurs.

[0248] Single colonies were picked from the LB plate and inoculated into 4 mL of empty LB medium. Incubate at 43°C on a shaker for 1, 2, 3, and 4 hours to induce loss of the pCP20 plasmid. Appropriate amounts of the culture solution from the four time points of 43°C incubation were spread onto empty LB plates and incubated at 30°C overnight.

[0249] Single clones on the empty LB plate were picked and inoculated on LB+Amp plates (cultured at 30°C), LB+Kana plates (cultured at 37oC), and empty LB plates (cultured at 37°C) in sequence, and cultured overnight. The next day, single clone colonies that grew only on the empty LB plate but not on the LB+Amp and LB+kana plates were picked and inoculated into 5 mL of empty LB medium and cultured overnight at 37°C. The next day, the strains were preserved, the minicircle DNA was extracted, and EcoRI enzyme digestion was performed for identification. Table 6 shows the screening efficiency of each step of the Fluc-FRT intermediate minicircle. Figure 17 shows the results of agarose gel electrophoresis for the identification of the correct minicircle plasmid and enzyme digestion products, indicating that the sequential transformation of the recombinase vector and the parent vector can achieve molecular recombination of the parent vector and cause the loss of the recombinase vector. It was also found that the minicircle exists as a mixture of monomeric or polymeric minicircle plasmids.

[0250] Table 6. Screening efficiency of each step of Fluc-FRT minicircle

[0251] Example 7: Obtaining an intermediate minicircle using a SacB gene reverse screening marker combination

[0252] 7.1 Construction of the pFluc-FRT-kana-SacB parent vector containing the SacB reverse selection marker

[0253] The SacB fragment (shown as SEQ ID NO: 27) was synthesized by conventional gene synthesis and cloned into the pFluc-FRT-kana vector via homologous recombination between the 5' AseI and 3' BsrBI sites to obtain the pFluc-FRT-kana-SacB parent vector.

[0254] 7.2 SacB reverse screening to prepare intermediate minicircles

[0255] Prepare competent E. coli cells containing the pFluc-FRT-kana-SacB parent plasmid. The preparation experimental process is the same as that of Example 6.1. Take 1 μL of pCP20 plasmid (concentration of 40 ng / μL) and add it to the competent cells containing the parent plasmid. Let the mixture stand on ice for 30 minutes, heat shock at 42°C for 90 seconds, and let it stand on ice again for 3 minutes. Add 500 μL of SOC medium to the mixture and incubate on a shaker at 30°C for 1 hour, 3 hours, and 5 hours at 220 rpm. Take appropriate amounts of bacterial liquid from the three incubation time points and spread them on LB plates containing 5% sucrose. Incubate them upside down in a 37°C incubator overnight. The next day, colony PCR was performed using the primer pair SacB-F (shown in SEQ ID NO:28) and SacB-R (shown in SEQ ID NO:29), which amplify the SacB gene fragment in the parental plasmid, and the primer pair FLP-F (shown in SEQ ID NO:30) and FLP-R (shown in SEQ ID NO:31), which amplify the FLP gene fragment in the pCP20 plasmid (see Figure 18). As shown in Figure 18, the colony PCR results show that 10 of the 16 selected clones were negative and did not contain the SacB backbone fragment.

[0256] Colonies identified by PCR as lacking the SacB backbone fragment were inoculated into 5 mL of empty LB medium and cultured overnight at 37°C. The next day, the strain was preserved, minicircle DNA was extracted, and digested with EcoRI for identification. Table 7 shows the screening efficiency of each step for Fluc-FRT minicircles. Figure 19 shows the agarose gel electrophoresis results of the correctly identified minicircle plasmid and digestion products. The minicircles were present as a mixture of monomeric and multimeric minicircle plasmids.

[0257] Table 7. Screening efficiency of each step of Fluc-FRT minicircle

[0258] Example 8 Preparation of Intermediate Minicircles Using the λRED Homologous Recombination System

[0259] 8.1 Preparation of E. coli competent cells containing pKD46 plasmid and pFluc-FRT-kana-SacB parent plasmid

[0260] The pKD46 plasmid and the pFluc-FRT-kana-SacB parent plasmid were co-transformed into GT115 competent cells, spread on LB plates containing Amp (100 μg / mL) and kana (50 μg / mL) antibiotics, and cultured at 30°C overnight. The next day, a single clone was picked and inoculated into 5 mL LB + Amp (100 μg / mL) + Kana (50 μg / mL) medium and cultured at 30°C overnight. The next day, the overnight bacterial solution was inoculated into 100 mL at a ratio of 1:25 to 1:50. The cells were cultured in LB + Amp (100 μg / mL) + Kana (50 μg / mL) medium at 30°C; when OD600 = 0.2, 0.2% arabinose was added to the medium; when OD600 = 0.4-0.5, the bacterial solution was collected and transferred to a 50 mL pre-cooled centrifuge tube, placed on ice for 10 min, and centrifuged at 4000 g for 10 min at 4°C; the supernatant was discarded, and the cells were gently suspended with 10 mL of pre-cooled 0.1 mol / L CaCl2 solution, placed on ice for 15-30 min, and centrifuged at 4000 g for 10 min at 4°C; the supernatant was discarded, and 4 mL of pre-cooled 0.1 mol / L CaCl2 solution containing 15% glycerol was added, the cells were gently suspended, and placed on ice for 5 min to obtain a competent cell suspension, which was recorded as pKD46-SacB-GT115 competent cells; 100 μL was dispensed into 1.5 mL EP tubes and stored at -80°C.

[0261] 8.2 Preparation of intermediate minicircles by λRED homologous recombination

[0262] A single-stranded oligonucleotide, HALR-50 Oligo (SEQ ID NO: 41), containing 50 bp homology arms on either side of the Kana-SacB fragment in the pFluc-FRT-kana-SacB parent plasmid was synthesized. Primers HALR-50-F (SEQ ID NO: 42) and HALR-50-R (SEQ ID NO: 43) were designed and PCR amplified using HALR-50 Oligo as a template to obtain the double-stranded homologous targeting fragment, HALR-50.

[0263] 150 ng of homologous targeting fragment HALR-50 was transformed into 50 μL pKD46-SacB-GT115 competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and placed on ice again for 3 min; 500 μL SOC + 2% arabinose was added, and incubated at 30°C for 3 hrs and 16 hrs; 10 μL of the bacterial solution at the corresponding induction time point was taken and diluted 10 2 ~10 4times, spread on LB + 5% sucrose plates, and culture at 37 ° C overnight. The next day, 50 single clones were picked from the LB + 5% sucrose plates coated with the bacterial solution for 3 hrs and 16 hrs of induction, and inoculated in turn on LB + Amp plates (cultured at 30 ° C), LB + Kana (cultured at 37 ° C), and LB + 5% sucrose plates (cultured at 37 ° C); the next day, single clones that grew only on the LB + 5% sucrose plates were selected and inoculated into 5 mL of empty LB culture medium, the strain was preserved, the minicircle DNA was extracted, and EcoRI enzyme digestion was performed for identification. Table 8 shows the screening efficiency of each step of the HA-Fluc intermediate minicircle. Figure 20 shows the agarose gel electrophoresis results of the correct minicircle plasmid and enzyme digestion products. The enzyme digestion products of the intermediate minicircle are 2745bp and 274bp, which are identified as correctly constructed vectors. The minicircle exists as a mixture of monomeric or multimeric minicircle plasmids.

[0264] Table 8. Screening efficiency of each step of HA-Fluc intermediate minicircle

[0265] Example 9: Transformation of the parent vector containing the ColE1 replication origin element into the genome of ZYCY10P3S2T Escherichia coli containing the arabinose-induced phiC31 recombinase expression construct to prepare an intermediate

[0266] 9.1 Construction of pHTM-Fluc-ColE1-attB / P-kana-TelLR2 parent vector containing ColE1 replication origin element

[0267] The ColE1 fragment (as shown in SEQ ID NO: 32) was synthesized conventionally and cloned into the pHTM-Fluc-attB / P-kana-TelLR2 vector via the 5'HpaI and 3'SmaI sites to obtain the pHTM-Fluc-ColE1-attB / P-kana-TelLR2 parent vector (as shown in FIG21 ).

[0268] 9.2 Arabinose-induced expression of phiC31 recombinase in ZYCY10P3S2T strain to prepare intermediate minicircles

[0269] Transform 40 ng of pHTM-Fluc-ColE1-attB / P-kana-TelLR2 plasmid into 50 μL of ZYCY10P3S2T E. coli competent cells (genome containing the araBAD-phiC31 sequence). Spread 100 μL of the bacterial solution onto an LB plate containing 50 μg / mL of Kana and incubate in a 37°C incubator overnight.

[0270] A single colony was picked from the LB plate and inoculated into 2 mL of LB (containing 50 μg / mL Kana), and incubated at 37°C with a shaker at 220 rpm for 2 hrs. 5 μL of the 2 mL LB bacterial solution was inoculated into 10 mL of TB medium (containing 50 μg / mL Kana and 1x phosphate buffer), and incubated at 37°C with a shaker at 220 rpm overnight.

[0271] The next day, 5 mL of overnight bacterial culture was taken to identify the presence of the parental plasmid in the pre-induction bacterial culture. The plasmid in the pre-induction bacterial culture was extracted and digested with EcoRI. 5 mL of fresh LB medium + 100 μL of 1 M sodium hydroxide + 100 μL of 20% arabinose was added to the remaining 5 mL of bacterial culture. The pH of the bacterial culture was adjusted to 7-7.4 and cultured on a shaking platform at 32°C for 5 hours and 8 hours. 10 μL of the bacterial culture at the two induction time points was taken and diluted 10 2 ~10 4 The remaining bacterial solution was used to identify the recombinant status of the parental plasmid in the induced bacterial solution, and the plasmid in the induced bacterial solution was extracted and digested with EcoRI.

[0272] Agarose gel electrophoresis was performed on the plasmids and enzyme digestion products before and after induction. As shown in Figure 22, before induction, the pHTM-Fluc-ColE1-attB / P-kana-TelLR2 parent plasmid had enzyme digestion products of 2443 bp and 1927 bp; 5 hrs and 8 hrs after induction, the pHTM-Fluc-ColE1-attB / P-kana-TelLR2 parent plasmid underwent intramolecular recombination in the bacterial solution to form an intermediate minicircle without the kana resistance gene, and the enzyme digestion products were 2443 bp and 718 bp.

[0273] Single colonies on the empty LB plate were picked and inoculated onto LB+Kana plates and empty LB plates in turn, and cultured at 37°C overnight. The next day, single colonies that grew only on the empty LB plate but not on the LB+Kana plate were picked and inoculated into 5 mL of empty LB medium and cultured at 37°C overnight. The next day, the strain was preserved, the HTM-Fluc-ColE1-attL / R-TelLR2 intermediate minicircle was extracted, and EcoRI enzyme digestion was performed for identification. Table 9 shows the screening efficiency of each step of the intermediate minicircle. Figure 23 shows the results of agarose gel electrophoresis. The intermediate minicircle exists as a mixture of monomeric or multimeric minicircle plasmids.

[0274] Table 9. Screening efficiency of each step of HTM-Fluc-ColE1-attL / R-TelLR2 intermediate minicircle

[0275] Example 10 Preparation of an intermediate minicircle containing a secondary structure DDITR sequence and useful for adeno-associated virus packaging

[0276] 10.1 Construction of pHTM-Fluc-DDITR-ColE1-attB / P-kana-TelLR2 parental vector

[0277] Conventional gene synthesis was performed on a DD ITR sequence fragment (SEQ ID NO: 34) derived from adeno-associated virus type 2 ITR and containing D sequences at both ends with a highly stable secondary structure. The fragment was cloned into the pHTM-Fluc-ColE1-attB / P-kana-TelLR2 vector through the 5'HpaI and 3'BamHI sites to obtain the pHTM-Fluc-DD ITR-ColE1-attB / P-kana-TelLR2 parent vector (as shown in Figure 24).

[0278] 10.2 Arabinose-induced expression of phiC31 recombinase in ZYCY10P3S2T strain to prepare intermediate minicircles

[0279] Transform 40 ng of the pHTM-Fluc-DDITR-ColE1-attB / P-kana-TelLR2 plasmid into 50 μL of ZYCY10P3S2TE.coli competent cells. Follow the same transformation procedure as in Example 8.2. Spread 100 μL of the bacterial solution onto an LB+Kana (50 μg / mL) plate and invert in a 37°C incubator overnight. The next day, pick a single colony from the LB plate and inoculate it into 2 mL of LB (containing 50 μg / mL Kana) and incubate at 37°C on a shaker at 220 rpm for 2 hours. Then, inoculate 5 μL of the 2 mL LB solution into 10 mL of TB medium (containing 50 μg / mL Kana and 1x phosphate buffer) and incubate overnight at 37°C on a shaker at 220 rpm.

[0280] The next day, 5 mL of overnight culture was taken to identify the presence of the parental plasmid in the pre-induction culture. The plasmid in the pre-induction culture was extracted and digested with EcoRI. 4 mL of fresh LB medium + 100 μl of 1 M sodium hydroxide + 1 mL of 20% arabinose was added to the remaining 5 mL of culture. The pH of the culture was adjusted to 7-7.4 and cultured on a shaking platform at 32°C for 5 and 8 hours. 10 μL of the culture at the two induction time points was taken and diluted 10 2 ~10 4 The remaining bacterial solution was used to identify the recombinant status of the parental plasmid in the induced bacterial solution, and the plasmid in the induced bacterial solution was extracted and digested with EcoRI.

[0281] Agarose gel electrophoresis was performed on the plasmids and enzyme digestion products before and after induction. As shown in Figure 25, before induction, the pHTM-Fluc-DDITR-ColE1-attB / P-kana-TelLR2 parent plasmid had enzyme digestion products of 2608 bp and 1927 bp; 5 hrs and 8 hrs after induction, the pHTM-Fluc-DDITR-ColE1-attB / P-kana-TelLR2 parent plasmid underwent intramolecular recombination in the bacterial solution to form an intermediate minicircle without the kana resistance gene, and the enzyme digestion products were 2608 bp and 718 bp.

[0282] Pick the single clones on the empty LB plate and inoculate them on the LB + Kana plate and the empty LB plate in turn, and culture them at 37 ° C overnight. The next day, pick the single clone colonies that only grow on the empty LB plate but not on the LB + kana plate, inoculate them into 5 mL of empty LB medium, and culture them at 37 ° C overnight. The next day, preserve the strain, extract the HTM-Fluc-DDITR-ColE1-attL / R-TelLR2 intermediate microcircle, and perform EcoRI enzyme digestion identification. Select the correctly identified intermediate microcircle and perform Sanger sequencing on its DDITR sequence. Table 10 shows the screening efficiency of each step of the intermediate microcircle. Figure 26 shows the results of agarose gel electrophoresis. The intermediate microcircle exists as a mixture of monomeric or polymeric microcircle plasmids. Figure 27 shows the results of Sanger sequencing of the DDITR sequence. The secondary structure of DDITR remains intact and stable during the preparation of the intermediate microcircle.

[0283] Table 10. Screening efficiency of each step of the HTM-Fluc-DDITR-ColE1-attL / R-TelLR2 intermediate minicircle

[0284] Example 11 Yield stability in scale-up production of intermediate microcircles

[0285] The E. coli clone containing the intermediate minicircle obtained in Example 6 was selected and passaged five times at a 1:100 ratio. Intermediate minicircle DNA was extracted from 4 mL of bacterial culture at each passage. As shown in Figure 28, the yield of intermediate minicircle DNA in bacterial culture from passages P0 to P5 remained stable with increasing passage number, demonstrating that the intermediate minicircle constructed in this application can maintain stable yield during scale-up production.

[0286] Example 12: Telomerase Digestion of an Intermediate Minicircle Containing a Telomerase Recognition Site to Obtain End-Closed Linear DNA

[0287] An intermediate minicircle containing a telomerase recognition site was prepared using the parent vector described in Example 1.2.2. Telomerase digestion (amino acid sequence shown in SEQ ID NO:33) was performed in vitro using the digestion system shown in Table 11. Agarose gel electrophoresis results are shown in Figure 29 , which demonstrates that the end-closed linear DNA product (2690 bp) was obtained. The end-closed linear DNA product exists as a monomer.

[0288] Table 11. Telomerase digestion system

[0289] Example 13: Telomerase Digestion of the Intermediate Minicircle Containing Two Telomerase Recognition Sites to Obtain End-Closed Linear DNA

[0290] The parent vector in Example 1.2.3 was used to prepare an intermediate minicircle containing two telomerase recognition sites. Telomerase digestion was performed in vitro, and the digestion system is shown in Table 11. The digestion product was separated and purified using a Source 15Q ion exchange column (Cytiva, Cat. No. 17518101) on an AKTA purification instrument (Cytiva), equilibration solution A (0.1M sodium chloride, 40mM Tris, 10mM EDTA, pH 5.5), and equilibration solution B (1M sodium chloride, 40mM Tris, 10mM EDTA, pH 5.5) to obtain the target end-closed linear DNA final product (Figure 30). The results of agarose gel electrophoresis identification are shown in Figure 31. The experimental results show that using telomerase digestion as the last step can effectively avoid the production of polymers, and the end-closed linear DNA final product exists in the form of monomers.

[0291] Example 14 End-closed linear DNA mediated expression of luciferase in mice

[0292] Lipid nanoparticles (LNPs) were loaded with end-closed linear DNA expressing luciferase, prepared in the example. The DNA was dissolved in sodium acetate at pH 4.0 to obtain an aqueous phase solution. An ionizable cationic lipid (DLin-MC3-DMA, MC3), a helper lipid (1,2-dioleoyl sn-glycero-3-phosphocholine, DOPC), cholesterol, and DMG-PEG2000 were dissolved in 100% ethanol to prepare a 10 mM stock solution. A lipid phase solution was obtained by mixing the stock solution at a molar ratio of MC3:cholesterol:DOPC:DMG-PEG2000 of 50 / 38 / 10 / 2 (mol%). The ionizable cationic lipid to DNA was added at a nitrogen-to-phosphorus ratio (N / P) of 6. The two phases were mixed using a microfluidic chip on a Precision Nanosystems NanoAssemblr Ignite nanomedicine preparation system at a 3:1 (v / v) aqueous to lipid phase flow rate ratio and a total flow rate of 16 mL / min. The mixture was dialyzed against 2L PBS buffer at room temperature for 2 hours using a dialysis bag with a MWCO of 8–14 kDa. The mixture was then concentrated by ultrafiltration and filtered to a final concentration of 200 μg / mL of LNP-DNA. The particle size, dispersion index (PDI), and zeta potential of the sample were measured using a Malvern Zetasizer Pro particle size analyzer. TM The sample encapsulation efficiency (EE%) was detected using a dsDNA assay kit.

[0293] LNP-DNA was introduced into C57BL / 6 mice by tail vein injection at 0.5mg / kg, 1mg / kg and 2mg / kg dosages. In order to observe luciferase expression, 3mg / 300uL luciferin substrate in PBS was intraperitoneally injected into mice. PerkinElmer's IVIS lumina III in vivo imaging system was used to observe fluorescence signal, and the total fluorescence signal at the mouse liver position was quantified. Luciferase was observed to be stably expressed in the mouse liver at 24h (as shown in Figure 32).

[0294] Example 15: End-closed linear DNA for DNA donor-mediated DNA transposition in the piggyBac transposon system

[0295] A fragment containing the piggyBac transposon DNA donor sequence (e.g., SEQ ID NO: 44) containing the blasticidin resistance gene sequence was obtained by digesting the plasmid pPB, which has a pBlueScript II KS backbone (Agilent Technologies), with XhoI and NotI. Simultaneously, the R6K-FRT-SacB-KanR fragment from the pFluc-FRT-kana-SacB vector described in Example 7 was amplified using synthetic primers XhoI-F (e.g., SEQ ID NO: 45) and NotI-R (e.g., SEQ ID NO: 46) and digested with XhoI and NotI. Subsequently, the two digested fragments were ligated to obtain the pPB-FRT-kana-SacB vector. A TelLR3 fragment (e.g., SEQ ID NO: 47) was synthesized using conventional gene synthesis and cloned into the pPB-FRT-kana-SacB vector via the 5' SalI and 3' XhoI sites to obtain the pPB-FRT-kana-SacB-TelLR1 parent vector.

[0296] A single-stranded oligonucleotide, pPB-HALR-50-Oligo (e.g., SEQ ID NO: 48), containing 50 bp homology arms on either side of the KanR-SacB fragment in the pPB-FRT-kana-SacB-TelLR1 parental plasmid, was synthesized. Primers pPB-HALR-50-F (e.g., SEQ ID NO: 49) and pPB-HALR-50-R (e.g., SEQ ID NO: 50) were designed. Using pPB-HALR-50-Oligo as a template, PCR amplification was performed to obtain the double-stranded homologous targeting fragment, pPB-HALR-50. An intermediate minicircle was generated by λRED homologous recombination using the method described in Example 8. Finally, end-closed linear DNA (nelPB) containing the piggyBac transposon DNA donor sequence was obtained by telomerase digestion as described in Example 12.

[0297] 2x10 cells were plated in each well of a 24-well cell culture plate. 5ARPE-19 cells were transfected with 800 ng of pPB plasmid carrying the piggyBac transposon DNA donor sequence and nelPB end-closed linear DNA, respectively, and 200 ng of PBase plasmid (piggyBac transposase sequence is derived from the literature Yusa K, Zhou L, Li MA, Bradley A, Craig NL. A hyperactive piggyBac transposase for mammalian applications. Proc Natl Acad Sci US A. 2011 Jan 25; 108(4): 1531-6. doi: 10.1073 / pnas.1008322108. Epub 2011 Jan 4. PMID: 21205896; PMCID: PMC3029773.) using lipofectamine 2000 to co-transfect ARPE-19 cells (4 replicate wells per group). 48 hours after transfection, cells were trypsinized and harvested by centrifugation. Resuspended in 20 mL of culture medium, the cells were plated onto 10 cm cell culture plates. After 21 days of selection for resistance to blasticidin at 4 μg / ml, the cell culture medium was discarded and cells were stained with 4% PFA containing 0.1% crystal violet. Single colonies were counted (as shown in Figure 33).

Claims

1. A DNA construct comprising: 1) a replication origin; 2) a selection element; 3) a target gene; 4) an auxiliary element; and 5) a telomerase recognition site, wherein: The auxiliary elements enable the DNA construct to form an intermediate minicircle and a backbone heteroDNA under appropriate conditions, wherein the intermediate minicircle comprises the replication origin, the target gene and the telomerase recognition site, and the backbone heteroDNA comprises the screening element.

2. The DNA construct of claim 1, wherein the intermediate minicircle does not comprise the selection element.

3. The DNA construct according to any one of claims 1-2, wherein the intermediate minicircle can be amplified in an environment without selection pressure.

4. The DNA construct according to any one of claims 1 to 3, wherein the backbone heteroDNA does not comprise the replication initiation site.

5. The DNA construct according to any one of claims 1 to 4, which is introduced into a production strain, wherein the backbone hetero-DNA cannot stably exist in a free state outside the production strain genome during the amplification process of the production strain.

6. The DNA construct according to claim 5, wherein the production strain is Escherichia coli.

7. The DNA construct according to any one of claims 1 to 6, which is a circular double-stranded DNA construct.

8. The DNA construct according to any one of claims 1 to 7, wherein the replication initiation site is selected from one or more of the following groups: an R6K replication initiation site, a pSC101 replication initiation site, a ColE1 replication initiation site, a ColE2 replication initiation site, a ColE3 replication initiation site, a pMB1 replication initiation site, a pBR322 replication initiation site, a pUC replication initiation site, and a P15A replication initiation site.

9. The DNA construct according to any one of claims 1 to 8, wherein the screening element is a forward screening element and / or a reverse screening element.

10. The DNA construct according to any one of claims 1 to 9, wherein the selection element is a resistance gene selection element and / or a suicide gene selection element.

11. The DNA construct of claim 10, wherein the resistance gene selection element is selected from one or more of the group consisting of ampicillin, kanamycin, chloramphenicol, spectinomycin, tetracycline, bleomycin, streptomycin, hygromycin, gentamicin, apramycin sulfate, erythromycin, and nourseothricin.

12. The DNA construct of claim 9, wherein the counter-selection element comprises the use of SacB, ccdB, rpsL, tetAR, pheS, thyA, lacY, gata-1, upp, bla I and / or mazF counter-selection markers.

13. The DNA construct according to any one of claims 1 to 12, wherein the gene of interest comprises an expression cassette.

14. The DNA construct according to any one of claims 1 to 13, wherein the gene of interest comprises one or more sequences selected from the group consisting of a promoter, a protein-encoding gene, and a terminator.

15. The DNA construct of any one of claims 1-14, wherein the auxiliary elements comprise: a recombination element, a transposase element, and / or a gene editing site.

16. The DNA construct according to claim 15, wherein the recombination element comprises a recombination site, and the recombination site is a paired recombination site, and the paired recombination site can cause the DNA construct to recombine under the action of a recombinase to obtain an intermediate minicircle and a backbone heteroDNA.

17. The DNA construct according to claim 16, wherein the recombinase functions under conditional induction.

18. The DNA construct according to any one of claims 16-17, wherein the sequences of the paired recombination sites are in the same orientation.

19. The DNA construct of any one of claims 16-18, wherein the recombination site comprises a site-specific recombinase site.

20. The DNA construct of claim 19, wherein the site-specific recombinase site is capable of being recognized by a site-specific recombinase selected from one or more of the following groups: phiC31 integrase, FLP recombinase, Cre recombinase, Dre recombinase, BxbI integrase, and phiBT1 integrase.

21. The DNA construct according to any one of claims 16-17, wherein the auxiliary element is capable of causing homologous recombination of the DNA construct.

22. The DNA construct according to claim 21, wherein the homologous recombination is achieved by the lambda phage Red homologous recombination system.

23. The DNA construct of any one of claims 15, wherein the auxiliary element is capable of causing the DNA construct to split into intermediate minicircles by a transposase.

24. The DNA construct of claim 23, wherein the transposase is selected from one or more of the group consisting of Tn3, Tn5, Tn7, and Tn10.

25. The DNA construct according to any one of claims 1 to 24, wherein the telomerase recognition site can be recognized and cleaved by telomerase to obtain a linear DNA with closed ends containing the target gene.

26. The DNA construct according to any one of claims 1 to 25, comprising at least one telomerase recognition site.

27. The DNA construct according to any one of claims 1 to 26, comprising two telomerase recognition sites.

28. A method for producing end-free linear DNA, comprising: 1) providing a parent plasmid comprising a replication initiation site, a screening element, a target gene, and a telomerase recognition site; 2) allowing the parent plasmid to form an intermediate minicircle and backbone heteroDNA under suitable conditions, wherein the intermediate minicircle comprises the replication initiation site, the target gene, and the telomerase recognition site, and the backbone heteroDNA comprises the screening element; 3) amplifying the intermediate minicircle; and 4) obtaining end-free linear DNA carrying the target gene by telomerase digestion.

29. The method according to claim 28, wherein the step of forming an intermediate minicircle and backbone heteroDNA from the parent plasmid is achieved by one or more of the following methods: recombination induced by site-specific recombinase, homologous recombination, transposition caused by transposase, and gene editing.

30. The method of claim 29, wherein the site-specific recombinase is selected from one or more of the following groups: phiC31 integrase, FLP recombinase, Cre recombinase, Dre recombinase, BxbI integrase, and phiBT1 integrase.

31. The method according to any one of claims 29-30, wherein the homologous recombination comprises being achieved by the lambda phage Red homologous recombination system.

32. The method of any one of claims 29-31, wherein the transposase is selected from one or more of the group consisting of Tn3, Tn5, Tn7, and Tn10.

33. The method of any one of claims 29-32, wherein the gene editing is achieved using a CRISPR / Cas system.

34. The method of any one of claims 28-33, wherein the intermediate minicircle does not comprise the screening element.

35. The method of any one of claims 28-34, wherein the intermediate minicircle can be amplified in an environment without selection pressure.

36. The method of any one of claims 29-35, wherein the backbone heteroDNA does not comprise an origin of replication.

37. The method according to any one of claims 28 to 36, wherein step 2) is performed in a production strain.

38. The method of claim 37, wherein the production strain is Escherichia coli.

39. The method of any one of claims 28-38, wherein the replication initiation site is selected from one or more of the following groups: an R6K replication initiation site, a pSC101 replication initiation site, a ColE1 replication initiation site, a ColE2 replication initiation site, a ColE3 replication initiation site, a pMB1 replication initiation site, a pBR322 replication initiation site, a pUC replication initiation site, and a P15A replication initiation site.

40. The method according to any one of claims 28 to 39, wherein the screening element is a forward screening element and / or a reverse screening element.

41. The method according to any one of claims 28 to 40, wherein the selection element is a resistance gene selection element and / or a suicide gene selection element.

42. The method of claim 41, wherein the resistance gene selection element is selected from one or more of the following groups: Ampicillin, kanamycin, chloramphenicol, spectinomycin, tetracycline, bleomycin, streptomycin, hygromycin, gentamicin, apramycin sulfate, erythromycin, and nourseothricin.

43. The method of any one of claims 40-42, wherein the counter-selection element comprises the use of SacB, ccdB, rpsL, tetAR, pheS, thyA, lacY, gata-1, upp, bla I, mazF counter-selection markers.

44. The method of any one of claims 28-43, wherein the gene of interest comprises an expression cassette.

45. The method according to any one of claims 28 to 44, wherein the target gene comprises one or more sequences selected from the group consisting of a promoter, a gene encoding an expressed protein, and a terminator.

46. ​​An intermediate product minicircle obtained by the method of any one of claims 28-45, wherein the intermediate product minicircle comprises the replication initiation site, the target gene and the telomerase recognition site.

47. The intermediate minicircle of claim 46, which does not comprise a screening element.

48. The intermediate minicircle according to any one of claims 46-47, which can be amplified in an environment without selection pressure.

49. A terminal-free linear DNA obtained by the method of any one of claims 28-45. The endless linear DNA according to claim 49 , comprising closed structures at both ends.

51. The endless linear DNA according to claim 50, wherein the closed structure is obtained by telomerase digestion.

52. A cell comprising the DNA construct of any one of claims 1-27, the intermediate minicircle of any one of claims 46-48, or the terminal-free linear DNA of any one of claims 49-51.

53. Production bacteria comprising the DNA construct described in any one of claims 1-27, or the intermediate minicircle described in any one of claims 46-48. The production bacterium according to claim 53 , which is Escherichia coli.

55. A kit comprising the DNA construct of any one of claims 1-27, or the intermediate minicircle of any one of claims 46-48.

56. The kit of claim 55, further comprising production bacteria.

57. The kit according to any one of claims 55-56, further comprising telomerase.

58. A pharmaceutical composition comprising the endless linear DNA according to any one of claims 49 to 51, and optionally a pharmaceutically acceptable carrier.

59. A method for delivering a target gene to a target cell, comprising contacting the endless linear DNA according to any one of claims 49 to 51 with the target cell.

60. Use of the DNA construct of any one of claims 1-27, the method of any one of claims 28-45, the intermediate minicircle of any one of claims 46-48, the terminal-free linear DNA of any one of claims 49-51, the cell of claim 52, or the pharmaceutical composition of claim 58 in production and / or treatment of a disease, wherein the use is selected from one or more of the following groups: 1) Target gene expression; 2) gene therapy for diseases and / or conditions; 3) gene knockout and / or gene knockin; 4) Packaging and / or production of viral vectors; 5) Preparation of DNA vaccines; and 6) Production of mRNA.

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