A construct for creating recombinant minivectors that do not contain a predetermined sequence.

Abstract

To provide a simple and readily usable method for producing recombinant minivectors that do not contain a predetermined sequence. [Solution] The present invention provides a method for producing a recombinant minivector that does not contain a predetermined sequence from a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, wherein the predetermined sequence is arranged so as to be sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence, the method comprising: 1) cleaving the construct with the first restriction enzyme and the second restriction enzyme to obtain a cleaved fragment comprising the predetermined sequence and the first restriction enzyme recognition sequence, and a cleaved fragment that does not contain the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence; and 2) subjecting the two obtained cleaved fragments to a ligation reaction to obtain a reaction product comprising two self-ligated cyclic nucleic acids of the two cleaved fragments.

Description

Technical Field

[0001] The present invention generally relates to constructs and the like for producing a recombinant minivector that does not comprise a predetermined sequence. Background Art

[0002] Plasmid DNA is produced in large quantities by proliferating Escherichia coli transformed with the plasmid DNA, and purifying the plasmid DNA from the proliferated Escherichia coli. When plasmid DNA is mass-produced using Escherichia coli in this manner, the plasmid DNA contains selection markers, the origin of replication of the plasmid DNA, and the like, but these regions are unnecessary regions when a gene is introduced into mammalian cells.

[0003] As a technique for removing such unnecessary regions, a technique is known in which after amplifying plasmid DNA in Escherichia coli, the expression of recombinase and the endonuclease I-SceI is induced to reduce the size of the plasmid DNA (Patent Document 1, Non-Patent Document 1). However, since the usable sites of Escherichia coli that have expressed recombinase and I-SceI are limited, a method that can be conveniently used is desired. Prior Art Documents Patent Documents

[0004] Patent Document 1 International Publication No. WO 2010 / 002470 Non-Patent Documents

[0005] Non-Patent Document 1 Silva-Santos, R., et al. "Minicircle Biopharmaceuticals-An Overview of Purification Strategies." Frontiers in Biotechnology, 2019. Summary of the Invention [Problems that the invention aims to solve]

[0006] The problem that this invention aims to solve is to provide a simple and readily usable method for producing recombinant minivectors that do not contain a predetermined sequence. [Means for solving the problem]

[0007] As a result of diligent research by the present inventors, it has been found that a recombinant minivector that does not contain the predetermined sequence can be produced from a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, wherein the predetermined sequence is arranged so as to be sandwiched between the first and second restriction enzyme recognition sequences, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence.

[0008] In other words, this application encompasses the following inventions. [1] A construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, A predetermined sequence is arranged so as to be sandwiched between a first restriction enzyme recognition sequence and a second restriction enzyme recognition sequence. The first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a different restriction enzyme recognition sequence from the first restriction enzyme recognition sequence. A construct for creating recombinant minivectors that do not contain a predetermined sequence. [2] The construct according to [1], wherein a predetermined sequence includes a selection marker and a replication origin. [3] The construct according to [1] or [2], wherein a portion of the other region of the two regions sandwiched between a first restriction enzyme recognition sequence and a second restriction enzyme recognition sequence, the region not containing the predetermined sequence, is included in the recombinant minivector, and the other region includes the cleavage site by the first restriction enzyme and the target gene. [4] The construct according to any one of [1] to [3], wherein the region where a predetermined sequence is located among the two regions sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence does not include a cleavage site by the second restriction enzyme. [5] A construct according to any one of [1] to [4], wherein the recombinant minivector has a superhelical structure. [6] The construct described in any one of [1] to [5], wherein the recombinant minivector is a recombinant minivector for transforming animal cells. [7] A construct according to any one of [1] to [6], wherein the recombinant minivector is introduced into cells with higher efficiency compared to the construct described above. [8] A construct described in any one of [1] to [7], wherein the recombinant minivector has a base length of 1000 to 5000. [9] A construct described in any one of [1] to [8], having a base length of 3000 or more.

[10] A recombinant minivector containing the target gene but without a replication origin.

[11] A recombinant minivector as described in

[10] , having a base length of 1000 to 5000.

[12] A method for producing a recombinant minivector that does not contain a predetermined sequence from a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, wherein the predetermined sequence is arranged so as to be sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence, 1) A step of cleaving the construct with a first restriction enzyme and a second restriction enzyme to obtain a cleaved fragment containing a predetermined sequence and a first restriction enzyme recognition sequence, and a cleaved fragment not containing the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, and 2) A method comprising the step of subjecting the two obtained cleavage fragments to a ligation reaction to obtain a reaction product containing two self-ligated cyclic nucleic acids.

[13] The method according to

[13] , further comprising the step of cleaving with a first restriction enzyme a nucleic acid ligated by the two types of cleavage fragments contained in the reaction product, or a cyclic nucleic acid self-ligated by cleavage fragments containing a predetermined sequence and a first restriction enzyme recognition sequence.

[14] The method according to

[12] or

[13] , wherein each of the steps is carried out in a solution containing a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, a first restriction enzyme, a second restriction enzyme, and a ligase.

[15] A method according to any one of

[12] to

[14] , wherein a given sequence includes a selection marker and a replication origin.

[16] The method according to any one of

[12] to

[15] , wherein the recombinant minivector is a self-ligated circular nucleic acid in which the cleavage fragments not containing a first restriction enzyme recognition sequence and a second restriction enzyme recognition sequence are self-ligated.

[17] The method according to any one of

[12] to

[16] , wherein the recombinant minivector has a superhelical structure.

[18] The method according to any one of

[12] to

[17] , wherein the recombinant minivector is a recombinant minivector for transforming animal cells.

[19] The method according to any one of

[12] to

[18] , wherein the recombinant minivector is introduced into cells with higher efficiency compared to the construct.

[20] The method according to any one of

[12] to

[19] , further comprising a step of digesting linear nucleic acid contained in the reaction product with an exonuclease.

[21] the cleavage fragment comprising a predetermined sequence and a first restriction enzyme recognition sequence further comprises a third restriction enzyme recognition sequence different from the first and second restriction enzyme recognition sequences, The method according to any one of

[12] to

[20] , further comprising a step of cleaving, with a third restriction enzyme, a nucleic acid obtained by ligation of said two types of cleavage fragments contained in said reaction product, or a circular nucleic acid obtained by self-ligation of a cleavage fragment comprising a predetermined sequence and a first restriction enzyme recognition sequence. Effects of the Invention

[0009] According to the present invention, it is possible to provide a readily usable method for producing a recombinant minivector that does not contain a predetermined sequence. Furthermore, according to the present invention, it is also possible to provide a construct for producing a recombinant minivector that does not contain a predetermined sequence. Brief Description of the Drawings

[0010] [Figure 1] An example is shown of a construct comprising a first restriction enzyme recognition sequence, a cleavage site by a first restriction enzyme, a target gene, a second restriction enzyme recognition sequence, a cleavage site by a second restriction enzyme, and a predetermined sequence, a cleavage fragment obtained when the construct is cleaved with the first and second restriction enzymes, and a ligation reaction product obtained when the cleavage fragment is ligated. [Figure 2] A simplified plasmid DNA map is shown. [Figure 3]The results of confirming restriction enzyme activity in T4 DNA ligase reaction buffer and confirming the conjugation of linear DNA by T4 DNA ligase under reaction conditions of 37°C are shown. [Figure 4] The results of the reaction product obtained from the co-existence reaction of restriction enzymes and T4 DNA ligase are shown. [Figure 5] The results of the reaction product obtained from the co-existence reaction of restriction enzymes and T4 DNA ligase are shown. [Figure 6] The results of the reaction product obtained from the co-existence reaction of restriction enzymes and T4 DNA ligase using two types of restriction enzymes are shown. [Figure 7] The results of investigating the conditions for the coexistence reaction of restriction enzymes and T4 DNA ligase are shown. [Figure 8] Figure 8 shows the results confirming that the extra band is the target reaction product. [Figure 9] The results of investigating the conditions for the coexistence reaction of restriction enzymes and T4 DNA ligase are shown. [Figure 10] The results of investigating the conditions for the coexistence reaction of restriction enzymes and T4 DNA ligase are shown. [Figure 11] The results of examining the digestion of by-products by exonuclease III are shown. [Figure 12] The results of the purification and XhoI treatment of the target reaction product after the co-reaction of restriction enzymes and T4 DNA ligase are shown. [Figure 13] The sequence analysis results of the junction in the target reaction product obtained by the co-reaction of restriction enzymes and T4 DNA ligase are shown. [Figure 14] The results of investigating the open circularization of the purified reaction product are shown. [Figure 15] The results of comparing the yields of a reaction involving the coexistence of restriction enzymes and T4 DNA ligase with a reaction in which restriction enzyme treatment is followed by purification and then ligation are shown. [Figure 16] This paper presents the results of investigating the amount of ligase and reaction time in a reaction involving restriction enzyme treatment, purification, and ligation. [Figure 17]The results of our investigation into higher-order structural changes in plasmid DNA are shown below. [Figure 18] The results of our evaluation of MiniVector's advantages are shown below. [Figure 19] The results of our evaluation of MiniVector's advantages are shown below. [Figure 20] The results of our evaluation of MiniVector's advantages are shown below. [Figure 21] The results of our evaluation of MiniVector's advantages are shown below. [Figure 22] The results of examining the amount of gene transfer using MiniVector are shown. [Figure 23] The results of our evaluation of MiniVector's advantages are shown below. [Figure 24] The results of our evaluation of MiniVector's advantages are shown below. [Figure 25] The results of our evaluation of MiniVector's advantages are shown below. [Figure 26] The results of our evaluation of MiniVector's advantages are shown below. [Figure 27] The results of our evaluation of MiniVector's advantages are shown below. [Figure 28] The results of our evaluation of MiniVector's advantages are shown below. [Figure 29] The results of our evaluation of MiniVector's advantages are shown below. [Figure 30] The results of our evaluation of MiniVector's advantages are shown below. [Modes for carrying out the invention]

[0011] The following describes embodiments of the present invention (hereinafter referred to as "these embodiments"), but the scope of the present invention is not limited to these embodiments.

[0012] In a first embodiment, a method is provided for producing a recombinant minivector that does not contain a predetermined sequence from a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, wherein the predetermined sequence is arranged so as to be sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence, the method comprising: 1) cleaving the construct with the first restriction enzyme and the second restriction enzyme to obtain a cleavage fragment comprising the predetermined sequence and the first restriction enzyme recognition sequence, and a cleavage fragment comprising the cleavage fragment that does not contain the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence; and 2) subjecting the two obtained cleavage fragments to a ligation reaction to obtain a reaction product comprising two self-ligated cyclic nucleic acids of the two cleavage fragments.

[0013] By the method of this embodiment, a recombinant minivector that does not contain a predetermined sequence can be produced by removing a predetermined sequence from a construct. For example, the method of this embodiment is preferably used to produce recombinant vectors for introduction into animal cells. When a recombinant vector is amplified using E. coli, the recombinant vector needs to contain replication origins and selection markers for growing and selecting E. coli into which the recombinant vector has been introduced, but these replication origins and selection markers are regions that are unnecessary for introduction into animal cells. Using the method of this embodiment, a recombinant minivector can be obtained by amplifying a construct using E. coli and then removing the regions that were only necessary for the amplification and selection of E. coli from the construct. Furthermore, compared to methods that use recombinase and the endonuclease I-SceI to obtain recombinant vectors from which a predetermined sequence has been removed, the method of this embodiment does not use reagents that are restricted in terms of where they can be used, making it easier to use.

[0014] (construct) As used herein, "construct" refers to a nucleic acid used to recombinate a target gene, specifically a nucleic acid that carries the target gene to the gene sequence to be recombined. The recombinant vector is not particularly limited, as long as it allows the target gene to be incorporated into the host gene and achieve stable expression. The construct can be used interchangeably with the recombinant vector.

[0015] The construct in this embodiment includes a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence. The construct in this embodiment may be one that has been amplified in E. coli.

[0016] In this specification, the "predetermined sequence" may be any sequence, but when removing sequences necessary for amplification in E. coli from the construct, the predetermined sequence may include, for example, selection markers such as antibiotic resistance genes, or origins of replication for the construct. Examples of antibiotic resistance genes that may be included in the predetermined sequence include ampicillin resistance genes, kanamycin resistance genes, and chloramphenicol resistance genes.

[0017] As used herein, "first restriction enzyme" refers to an IIS-type restriction enzyme. An IIS-type restriction enzyme generally refers to a restriction enzyme characterized by having a restriction enzyme recognition sequence and cleavage sites that are separated. Among IIS-type restriction enzymes, those having all cleavage sites on one side away from the restriction enzyme recognition sequence are preferred. Examples of IIS-type restriction enzymes include BbsI, BbvI, BbvII, BcefI, BccI, BcgI, BciVI, BinI, BmrI, BpmI, BsaI, BseRI, BsgI, BsmAI, BsmBI, BspMI, BsrDI, BstF5I, BsmI, BsrI, BsmFI, BseMII, BspQI, BtgZI, AcuI, AlwI, Al Examples include wXI, Alw26I, EarI, Eco31I, Eco57I, Esp3I, Esp3I, FauI, FokI, GsuI, HgaI, HinGUII, HphI, Ksp632I, MboII, MmeI, Mn1I, NgoVIII, PaqCI, PleI, PsrI, RleAI, SapI, SfaNI, TaqII, and Tth111II. Among these, BbsI is preferred.

[0018] In the construct of this embodiment, a predetermined sequence is positioned so as to be sandwiched between a first restriction enzyme recognition sequence and a second restriction enzyme recognition sequence. Of the two regions sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, a portion of the other region that does not contain the predetermined sequence is included in the recombinant minivector, and this other region includes a cleavage site by the first restriction enzyme. The first restriction enzyme recognition sequence, the second restriction enzyme recognition sequence, and the predetermined sequence are arranged in the construct such that when the construct is cut with the first and second restriction enzymes, a cut fragment containing a predetermined sequence and the first restriction enzyme recognition sequence is obtained, and a cut fragment that does not contain the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence. Specifically, in the construct, each sequence and cleavage site are located in the following order: first restriction enzyme recognition sequence, first restriction enzyme cleavage site, region contained in the recombinant minivector, second restriction enzyme recognition sequence, and predetermined sequence. In this case, the second restriction enzyme cleavage site is located within the second restriction enzyme recognition sequence. Alternatively, in the construct, each sequence and cleavage site are located in the following order: first restriction enzyme recognition sequence, first restriction enzyme cleavage site, region contained in the recombinant minivector, second restriction enzyme cleavage site, second restriction enzyme recognition sequence, and predetermined sequence. In this case, the second restriction enzyme cleavage site is located in the region between the first and second restriction enzyme recognition sequences that is not the region where the predetermined sequence is located.

[0019] As used herein, "second restriction enzyme" refers to a restriction enzyme having a restriction enzyme recognition sequence different from that of the first restriction enzyme, and is not particularly limited, but is preferably a restriction enzyme such that when a construct is cleaved with the first and second restriction enzymes, a cleavage fragment containing a predetermined sequence and the first restriction enzyme recognition sequence is obtained, and a cleavage fragment not containing the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence is obtained. Examples of second restriction enzymes include NotI, BssHII, and MluI.

[0020] As used herein, "recombinant minivector" refers to a recombinant vector from which a specific sequence has been removed. Examples of recombinant minivectors include non-viral vectors such as transposon vectors and plasmid vectors, and viral vectors.

[0021] The region included in the recombinant minivector in this embodiment may be any sequence, but when introducing a target gene into cells using the recombinant minivector, the region included in the recombinant minivector may include, for example, the target gene, its promoter, a poly-A tail, etc. In this case, of the two regions sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the other region that does not contain the predetermined sequence includes the cleavage site by the first restriction enzyme, the target gene, its promoter, and a poly-A tail. Preferably, of the two regions sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the other region that does not contain the predetermined sequence preferably includes at least the cleavage site by the first restriction enzyme and the target gene. If the other region that does not contain the predetermined sequence contains the target gene, the target gene does not need to include a selection marker. Preferably, the selection marker that can be included in the recombinant vector is different from the selection marker that can be included in the predetermined sequence. The selection markers included in the recombinant vector may be antibiotic resistance genes or reporter genes, and examples include puromycin resistance genes, hygromycin resistance genes, genetisin resistance genes, blastosidine resistance genes, green fluorescent protein (GFP) expression genes, mCherry expression genes, nanoluciferase expression genes, β-glucuronidase expression genes, and LacZ genes. When the selection markers included in the recombinant vector are antibiotic resistance genes, and the selection markers included in a predetermined sequence are also antibiotic resistance genes, it is preferable that these antibiotic resistance genes are resistance genes for different antibiotics.

[0022] To distinguish between selection markers that may be included in a given sequence and selection markers that may be included in a recombinant vector, these are referred to as selection marker I and selection marker II in this specification, respectively. It is preferable that different selection markers are used for selection marker I and selection marker II. As one embodiment, this embodiment also provides a method for producing a recombinant minivector from a construct comprising a selection marker I, a replication origin, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, the minivector comprising a selection marker II which does not include selection marker I and a replication origin, and is a selection marker different from selection marker I.

[0023] The length of the recombinant minivector varies depending on the length of the target gene contained in the recombinant minivector, but it can be, for example, 1000 to 5000 base pairs long. The construct length varies depending on the length of the recombinant minivector or the given sequence, but it may be, for example, 3000 base pairs or longer.

[0024] (The process of cleaving the construct with the first restriction enzyme and the second restriction enzyme) The method of this embodiment includes the step of 1) cleaving a construct with a first restriction enzyme and a second restriction enzyme to obtain a cleaved fragment containing a predetermined sequence and a first restriction enzyme recognition sequence, and a cleaved fragment not containing the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence (hereinafter also referred to as "step 1").

[0025] When using a construct in which each sequence and each cleavage site is located in the following order: first restriction enzyme recognition sequence, first restriction enzyme cleavage site, region contained in recombinant minivector, second restriction enzyme cleavage site, second restriction enzyme recognition sequence, and predetermined sequence, the cleavage fragment obtained by step 1) containing the predetermined sequence and the first restriction enzyme recognition sequence also contains the second restriction enzyme recognition sequence.

[0026] The reaction conditions, such as the reaction time and temperature for the first and second restriction enzymes and the construct, can be appropriately determined by a person skilled in the art based on the types of first and second restriction enzymes.

[0027] The cleavage of the construct with the first restriction enzyme and the cleavage of the construct with the second restriction enzyme may be carried out in separate reaction systems, but it is preferable that they be carried out in parallel. That is, it is preferable that step 1) be carried out in a solution in which the construct, the first restriction enzyme, and the second restriction enzyme are present together.

[0028] (A process of subjecting two types of cleavage fragments to a ligation reaction) This embodiment includes the step of 2) subjecting the two types of cleavage fragments obtained to a ligation reaction to obtain a reaction product containing two types of self-ligated cyclic nucleic acids (hereinafter also referred to as "step 2").

[0029] As used herein, "ligation" refers to a reaction in which nucleic acids are linked together by a phosphate diester bond using a ligase. The ligase used in the ligation reaction, the reaction time with the ligase, and the reaction temperature and other reaction conditions can be appropriately determined by those skilled in the art. Commercially available ligation reagents may also be used. Self-ligation refers to a reaction in which both ends of a single linear nucleic acid are joined. When both ends of a linear nucleic acid are joined, it becomes a cyclic nucleic acid.

[0030] Step 2) yields a reaction product containing two types of cyclic nucleic acids, each obtained by self-ligating two types of cleavage fragments: one containing a predetermined sequence and a first restriction enzyme recognition sequence, and another containing a cleavage fragment that does not contain the first restriction enzyme recognition sequence or the second restriction enzyme recognition sequence. Of these two types of cyclic nucleic acids, the cyclic nucleic acid obtained by self-ligating the cleavage fragment that does not contain the first restriction enzyme recognition sequence or the second restriction enzyme recognition sequence is a recombinant vector that does not contain the predetermined sequence.

[0031] The construct is designed and the first and second restriction enzymes are selected so that cleavage fragments containing a predetermined sequence and a first restriction enzyme recognition sequence, as well as cleavage fragments not containing the first and second restriction enzyme recognition sequences, can self-ligate. For example, if cleavage by the first restriction enzyme produces sticky ends, the construct is designed and the first and second restriction enzymes are selected so that cleavage by the second restriction enzyme produces the same sticky ends as cleavage by the first restriction enzyme. Specifically, if cleavage of the construct by the first restriction enzyme produces 5'-GCCC-3' and 5'-GGGC-3' sticky ends, the construct is designed and the first and second restriction enzymes are selected so that cleavage of the construct by the second restriction enzyme also produces the same 5'-GCCC-3' and 5'-GGGC-3' sticky ends. If cleavage by the first and second restriction enzymes produces blunt ends, the above-described construct design and restriction enzyme selection are not necessary.

[0032] Step 2) may be performed after step 1), but it is more preferable that steps 1) and 2) be performed in parallel. That is, it is preferable that steps 1) and 2) be performed in a solution in which a construct containing a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, the first restriction enzyme, the second restriction enzyme, and a ligase are present together.

[0033] The reaction products in step 2) may include, in addition to self-ligated circular nucleic acids in which cleavage fragments not containing the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence are self-ligated, and circular nucleic acids in which cleavage fragments containing a predetermined sequence and the first restriction enzyme recognition sequence are self-ligated, nucleic acids in which two types of cleavage fragments are ligated (ligated circular nucleic acids in which two types of cleavage fragments are ligated or ligated linear nucleic acids in which two types of cleavage fragments are ligated), ligated linear nucleic acids in which either of the cleavage fragments are ligated with each other, and two types of cleavage fragments that were not ligated. When steps 1) and 2) are carried out in parallel, nucleic acids from the reaction product obtained in step 2), such as nucleic acids derived from cleavage fragments containing a predetermined sequence and a first restriction enzyme recognition sequence, circular nucleic acids in which cleavage fragments containing a predetermined sequence and a first restriction enzyme recognition sequence are self-ligated, nucleic acids in which two types of cleavage fragments are ligated, and linear nucleic acids in which cleavage fragments containing a predetermined sequence and a first restriction enzyme recognition sequence are ligated, are cleaved by the first restriction enzyme present in the same solution. When using a construct in which each sequence and each cleavage site is located in the order of first restriction enzyme recognition sequence, cleavage site by the first restriction enzyme, region contained in the recombinant minivector, cleavage site by the second restriction enzyme, second restriction enzyme recognition sequence, and predetermined sequence, the cleavage fragment containing the predetermined sequence and the first restriction enzyme recognition sequence also contains the second restriction enzyme recognition sequence, so nucleic acids derived from cleavage fragments containing a predetermined sequence and a first restriction enzyme recognition sequence may also be cleaved by the second restriction enzyme. A recombinant vector, a circular nucleic acid in which cleavage fragments that do not contain the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence are self-ligated, is not cleaved by the first and second restriction enzymes. The method of this embodiment may further include a step of cleaving a nucleic acid ligated with two types of cleavage fragments contained in the reaction product, or a circular nucleic acid self-ligated with cleavage fragments containing a predetermined sequence and a first restriction enzyme recognition sequence, with a first restriction enzyme. This step is preferably achieved by carrying out steps 1) and 2) in parallel, but may also be achieved by carrying out steps 1) and 2) separately and then reacting with the first restriction enzyme.

[0034] Figure 1 shows an example of a construct containing a first restriction enzyme recognition sequence, a cleavage site by the first restriction enzyme, a target gene, a second restriction enzyme recognition sequence, a cleavage site by the second restriction enzyme, and a predetermined sequence; cleavage fragments obtained when the construct is cleaved with the first and second restriction enzymes; and ligation reaction products obtained when the cleavage fragments are ligated.

[0035] When steps 1) and 2) are carried out in parallel, that is, when steps 1) and 2) are carried out in a solution in which a construct containing a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, the first restriction enzyme, the second restriction enzyme, and a ligase are present together, for example, the following: <1> and <2> After that, the following <3> and <4> This process is repeated, and the reaction progresses. <1> The construct is cleaved by first and second restriction enzymes, yielding two types of cleavage fragments: one containing a predetermined sequence and the first restriction enzyme recognition sequence, and another containing neither the first nor the second restriction enzyme recognition sequence. <2> The two types of cleavage fragments react with the ligase to obtain a ligation reaction product. <3> Among the reaction products, nucleic acids containing the first restriction enzyme recognition sequence (such as circular nucleic acids in which cleavage fragments containing a predetermined sequence and the first restriction enzyme recognition sequence are self-ligated, circular nucleic acids in which two types of cleavage fragments are ligated, linear nucleic acids in which two types of cleavage fragments are ligated, and linear nucleic acids in which cleavage fragments containing a predetermined sequence and the first restriction enzyme recognition sequence are ligated to each other) are cleaved by the first restriction enzyme coexisting in the same solution. <4> The ligation reaction product is obtained when the two cleavage fragments produced by the cleavage of the reaction product by the first restriction enzyme react with the ligase.

[0036] Among the reaction products, the recombinant minivector, which is a cyclic nucleic acid that has self-ligated from a cleavage fragment that does not contain the first and second restriction enzyme recognition sequences, is not cleaved by the first and second restriction enzymes. Therefore, by performing steps 1) and 2) in parallel, recombinant minivectors can be accumulated.

[0037] When steps 1) and 2) are carried out in parallel, the ratio of the concentrations of the first and second restriction enzymes to the ligase can be appropriately determined by those skilled in the art depending on the concentration of the construct, etc. For example, the ratio of the concentration of the first or second restriction enzyme to the concentration of the ligase may be 1:0.1 to 10. When steps 1) and 2) are carried out in parallel, the reaction time can be appropriately determined by those skilled in the art depending on the concentrations of the first and second restriction enzymes, ligases, and constructs, but may be, for example, 2 hours or more.

[0038] (The process of cleaving with a third restriction enzyme) Furthermore, the construct may be designed such that the cleavage fragment containing the predetermined sequence and the first restriction enzyme recognition sequence further contains a third restriction enzyme recognition sequence different from the first and second restriction enzyme recognition sequences. This makes it possible to cleave the nucleic acid derived from the cleavage fragment containing the predetermined sequence and the first restriction enzyme, which is contained in the reaction product, by reacting the reaction product with the third restriction enzyme after steps 1) and 2). If steps 1) and 2) are carried out in parallel and the third restriction enzyme is reacted afterward, the nucleic acid product derived from the cleavage fragment containing the predetermined sequence and the first restriction enzyme recognition sequence that was not completely cleaved by the first restriction enzyme will be cleaved by the third restriction enzyme. The first restriction enzyme may also be reacted together with the third restriction enzyme. The method of this embodiment may further include a step of cleaving a nucleic acid ligated by two types of cleavage fragments contained in the reaction product, or a cyclic nucleic acid self-ligated by cleavage fragments containing a predetermined sequence and a first restriction enzyme recognition sequence, with a third restriction enzyme. Furthermore, the method of this embodiment may further include the step of cleaving a nucleic acid ligated by two types of cleavage fragments contained in the reaction product, or a cyclic nucleic acid self-ligated by cleavage fragments containing a predetermined sequence and a first restriction enzyme recognition sequence, with a first restriction enzyme and a third restriction enzyme.

[0039] (The process of digestion using exonuclease) After steps 1) and 2), the reaction product may be reacted with an exonuclease to decompose the linear nucleic acid contained in the reaction product. The linear nucleic acid contained in the reaction product may be, for example, a linear nucleic acid formed by the ligation of two types of cleavage fragments, a linear nucleic acid formed by the ligation of one of the cleavage fragments with another, or two types of cleavage fragments that were not ligated. The method of this embodiment may further include a step of digesting the linear nucleic acids contained in the reaction product with an exonuclease.

[0040] After steps 1) and 2), both the cleavage with a third restriction enzyme and the digestion with an exonuclease may be performed. In this case, the cleavage with the third restriction enzyme and the digestion with an exonuclease may be performed separately or in parallel. When the digestion with an exonuclease is performed after the cleavage with the third restriction enzyme, or when both steps are performed in parallel, the linear cleavage fragments produced by the cleavage with the third restriction enzyme are digested by the exonuclease, so nucleic acids other than the target recombinant minivector can be digested more effectively than when the cleavage with the third restriction enzyme is performed after the digestion with an exonuclease.

[0041] As one embodiment, the method of this embodiment includes the steps of: 1) cleaving a construct with a first restriction enzyme and a second restriction enzyme to obtain a cleaved fragment containing a predetermined sequence and a first restriction enzyme recognition sequence, and a cleaved fragment not containing the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence; 2) subjecting the two obtained cleaved fragments to a ligation reaction to obtain a reaction product containing two self-ligated cyclic nucleic acids; 3) cleaving the nucleic acid contained in the reaction product, which is either the nucleic acid contained in the reaction product that is the ligated nucleic acid of the two cleaved fragments, or the cyclic nucleic acid contained in the reaction product that is the self-ligated nucleic acid containing the predetermined sequence and the first restriction enzyme recognition sequence, with a third restriction enzyme (hereinafter also referred to as "step 3"); and 4) digesting the linear nucleic acid contained in the reaction product with an exonuclease (hereinafter also referred to as "step 4"). It is preferable that steps 1) and 2) be carried out in a solution containing a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, the first restriction enzyme, the second restriction enzyme, and a ligase, and it is also preferable that steps 3) and 4) be carried out in a solution containing a ligation reaction product, a third restriction enzyme, and an exonuclease. Compared to when steps 1) and 2) are carried out separately, more recombinant minivectors can be produced when steps 1) and 2) are carried out in a solution containing a construct with a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, the first restriction enzyme, the second restriction enzyme, and a ligase.

[0042] If steps 3) and / or 4) are performed after steps 1) and 2), the process may include a step of purifying the recombinant minivector from the reaction mixture after steps 3) and / or 4). The reagents used for purification and the reaction conditions can be appropriately determined by those skilled in the art. A commercially available purification kit may also be used.

[0043] Whether recombinant minivectors were successfully produced by the method of this embodiment can be confirmed using conventionally known methods, such as electrophoresis or sequence analysis.

[0044] The recombinant minivectors produced by the method of this embodiment preferably have a superhelical structure.

[0045] The recombinant minivectors obtained by the method of this embodiment are not particularly limited in their applications and can be used in the same applications as general recombinant vectors.

[0046] The cells into which the target gene is introduced by the recombinant minivector are not particularly limited and may be prokaryotic or eukaryotic cells, adherent or suspension cells, or primary cells. Examples include bacterial cells, fungal cells, plant cells, animal cells, and insect cells, but animal cells are preferred, and mammalian cells are more preferred. Examples of mammalian cells include hematopoietic cells, cancer cells, and ovarian cells. Specifically, examples include Jurkat cells, Ramos cells, Raji cells, THP-1 cells, Namalwa cells, HL60 cells, U266B1 cells, T cells, B cells, NK cells, dendritic cells, macrophages, monocytes, and CHO cells.

[0047] The gene introduced into cells by a recombinant minivector, i.e., the target gene in this embodiment, is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. For example, the target gene may be a gene for expressing a target protein, a reporter gene, an internal standard gene, a gene for gene therapy, hairpin RNA (shRNA), guide RNA (gRNA), etc.

[0048] The method of introducing genes using recombinant minivectors is not particularly limited and includes, for example, electroporation, heat shock, lipofection, calcium phosphate, and methods using dextran derivatives such as DEAE-dextran.

[0049] When a recombinant minivector produced by the method of this embodiment is used to introduce a gene into cells, the gene transfer efficiency, gene transfer rate, and / or cell viability may be improved compared to using the construct before the removal of a predetermined sequence by the method of this embodiment. The recombinant minivector produced by the method of this embodiment may have a superhelical structure, which may further improve the gene transfer efficiency, gene transfer rate per cell, and / or cell viability.

[0050] This embodiment also provides recombinant minivectors manufactured by the method of this embodiment.

[0051] Embodiments of methods for producing recombinant minivectors that do not contain a predetermined sequence also apply to constructs for producing recombinant minivectors that do not contain a predetermined sequence, recombinant minivectors, and kits for producing recombinant minivectors, as described below.

[0052] This embodiment also provides a construct for producing a recombinant minivector that does not include the predetermined sequence, comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, wherein the predetermined sequence is arranged so as to be sandwiched between the first and second restriction enzyme recognition sequences, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence. As one embodiment, this embodiment also provides a construct comprising a selection marker I, a replication origin, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence for producing a recombinant minivector containing a target gene, without including a selection marker I and a replication origin. Here, the target gene may include a selection marker II, which is a selection marker different from selection marker I.

[0053] This embodiment also provides a recombinant minivector that does not contain a replication origin and contains the target gene. This recombinant minivector may be manufactured by the method of this embodiment.

[0054] This embodiment also provides: 1) a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, wherein the predetermined sequence is arranged so as to be sandwiched between the first and second restriction enzyme recognition sequences, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence; 2) a first restriction enzyme; and 3) a kit for producing a recombinant minivector comprising a second restriction enzyme.

[0055] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0056] <Restriction enzyme reaction> The samples were treated at 37°C for 1 hour in a 1×rCutSmart Buffer (New England Biolabs, Cat: B6004S) reaction mixture containing one or more restriction enzymes: 10 ng / μL template plasmid DNA, 2 ng / μL purified nucleic acid, or 20 U / μg DNA. The restriction enzymes used were MluI-HF (Cat: R3198L), BssHII (Cat: R0199L), BbsI-HF (Cat: R3539L), NotI-HF (Cat: R3189L), and XhoI (Cat: R0146L), all products of New England Biolabs. Prior to the ligase co-reaction, the components necessary for each ligase reaction were added to confirm that they would not affect the restriction enzyme reaction.

[0057] <Ligases> When using DNA ligases derived from various thermophilic bacteria, use a solution containing 170 ng / μg of one of the following self-prepared DNAs: Taq LigA, Tfi LigA, or Tth LigA, with 2 ng / μL of template DNA, 1 mM dithiothreitol (DTT, Fujifilm Wako, Cat: 042-29222), 1 mM β-nicotinamide adenine dinucleotide (NAD, Nacalai Tesque, Cat: 24338-57), 0.1% Triton® X-100 (Fujifilm Wako, Cat: 160-24751) in 1×rCutSmart Buffer or Ideal Buffer (20 mM Tris-HCl (Nacalai Tesque, Cat: 35434-05, pH 8.5), 150 mM potassium chloride (Nacalai Tesque, Cat: 28538-75), 10 mM The samples were treated at 37°C for 1 hour in magnesium chloride (Nacalai Tesque, Cat: 20937-72), 10 mM DTT, 1 mM NAD, and 0.1% Triton® X-100. When using T4 DNA Ligase, use either 1X T4 DNA Ligase Buffer (the buffer provided with the enzyme) containing 8750 U / μg DNA T4 DNA Ligase (TaKaRa, Cat:2011A) and 2 ng / μL of template DNA, or 1 mM DTT, 1 mM NAD, and 0.1 mM adenosine 5'-triglycerides. The samples were treated in 1×CutSmart Buffer containing phosphate (ATP, Sigma-Aldrich, Cat: A7699-1G) at 16°C or 37°C for 1 hour.

[0058] <Restriction enzyme-ligase co-reaction> The samples were treated at 37°C for 1 to 16 hours in a reaction mixture containing 10 ng / μL template plasmid DNA, 1–1750 U / μL T4 DNA ligase, and one or more restriction enzymes of 2.5–20 U / μg DNA (a combination of MluI-HF and BssHII, or a combination of BbsI-HF and NotI-HF), 1 mM DTT, and 0.1 mM ATP in 1×rCutSmart Buffer (New England Biolabs, Cat: B6004S).

[0059] <Exonuclease III reaction> The samples were treated with a 1×Exonuclease III Buffer (the buffer provided with the enzyme) containing 2 ng / μL nucleic acid and 5000 U / μg DNA exonuclease III (RaKaRa, Cat: 2170A) at 37°C for 1 hour, followed by inactivation at 65°C for 5 minutes. To perform co-reaction with restriction enzymes, 1mM DTT-containing 1X rCutSmart Buffer was used instead of the attached buffer. The restriction enzymes used in the co-reaction were BssHII, PstI-HF (New England Biolabs, Cat:R3140L), HindIII-HF (New England Biolabs, Cat:R3104L), XhoI, PvuI-HF (New England Biolabs, Cat:R3150L), and EcoRI-HF (Cat:R3101L). DNA was added at enzyme concentrations of 10 U or 20 U / μg, and the mixture was treated at 37°C for 1 hour, followed by inactivation at 65°C for 10 minutes.

[0060] <Suspension Cell Line Culture> Jurkat beta-del (JCRB Cell Bank, Cat: JCRB0147, hereinafter referred to as Jurkat), Raji (JCRB Cell Bank, Cat: JCRB1647), and Ramos (JCRB Cell Bank, Cat: JCRB9119) were cultured in RPMI-1640 (FUJIFILM Wako Pure Chemical Corporation, Cat: 189-02025) supplemented with 10% (v / v) inactivated (56°C, 30 minutes) fetal bovine serum (Nichirei Bioscience, Cat: 175012-500ML) (hereinafter referred to as complete medium) in a CO₂ incubator at 37°C under 5% CO₂ conditions.

[0061] <Primary T Cell Culture> Cryopreserved PBMCs were seeded at 200,000 cells / well in a 24-well plate pre-coated with 0.5 μg / mL anti-CD3 antibody (BioLegend, Cat: BL317302). In this step, the culture volume per well was 1.0 mL, and the culture medium used was AIM-V (gibco, Cat: 12055-091) supplemented with 0.5 μg / mL anti-CD28 antibody (BioLegend, Cat: BL302902), 40 U / mL IL-2 (Peprotech, Cat: AF-200-02), and 5% inactivated human type AB serum (GeminiBio, Cat: 100-512). Three days after the start of culture, 1 mL / well of AIM-V containing 80 U / mL IL-2 and 5% inactivated human type AB serum was added; subculture was repeated as necessary, and culture was performed for a total of 5 days.

[0062] <Gene Transfection by DEAE / Dextran Method> Cells in the logarithmic growth phase are collected, washed with D-PBS(-) (Fujifilm Wako Pure Chemical, Cat: 049-29793), 2 million cells are aliquoted into a 1.5 mL microcentrifuge tube, pelleted after centrifugation (400×g, 5 minutes, room temperature). 1.25 μg of GFP expression vector (if tested at different doses, the results are described in the results) is diluted in 62.5 μL of STBS buffer (25 mM Tris (Nacalai Tesque, Cat: 35434-21)-HCl (Nacalai Tesque, Cat: 18321-05), pH 7.5, 137 mM NaCl (Nacalai Tesque, Cat: 31333-45), 5 mM KCl (Nacalai Tesque, Cat: 28538-75), 0.6 mM Na₂HPO₄ (Nacalai Tesque, Cat: 31738-55), 0.7 mM CaCl₂ (Fujifilm Wako Pure Chemical, Cat: 3819735), 0.5 mM MgCl₂ (Nacalai Tesque, Cat: 20937-72)) (tube 1). DEAE / Dextran with an average molecular weight of 500 kDa (Fujifilm Wako Pure Chemical, Cat: 591-03161) is diluted with STBS buffer to a concentration of 250 μg / mL (tube 2). An equal volume of the solution from tube 2 is mixed with tube 1, and the cell pellet is suspended in the mixed solution (DEAE / Dextran-DNA complex). After treating the suspended cell solution at 29°C for 30 minutes, 1 mL of ice-cold STBS buffer is added, and the mixture is immediately centrifuged. The obtained cell pellet is washed once with ice-cold STBS and once with RPMI-1640 medium, suspended in 2 mL of complete medium, the entire volume is seeded into a 6-well multi-well plate, and cultured in a CO₂ incubator for 48 hours. The above is the basic condition, and conditions modified during investigation are described in each test example and example.

[0063] <Gene Transfer Using Lipofectamine 3000> Cells in logarithmic growth phase were collected, and 0.5 mL aliquots of a cell suspension adjusted to 2×10⁵ cells / mL in complete medium were seeded into a 24-well multi-well plate. 0.125 to 0.5 µg of EGFP expression vector (promoter: PGK1) was diluted in 50 µL of OptiMEM (Thermo Fisher Scientific, Cat: 31985-062) (Tube 1). On the other hand, 3 µL of Lipofectamine 3000 (Thermo Fisher Scientific, Cat: L3000-01), 0.5 to 4 µL of P3000 reagent (included with the product), and 50 µL of OptiMEM were placed in a separate tube and mixed (Tube 2). The entire volume from Tube 1 was transferred to Tube 2, mixed well, and then allowed to stand at room temperature for 15 minutes. The mixed solution was added dropwise onto the wells seeded with cells, and the cells were cultured for 48 hours in a CO₂ incubator.

[0064] <Gene Transfer Using Neon Electroporation System> For gene transfer, the Neon NxT Electroporation System (Thermo Fisher Scientific), Neon NxT Buffer Kit, 10 µL (Thermo Fisher Scientific, Cat: N1096) or Neon NxT Buffer Kit, 100 µL (Thermo Fisher Scientific, Cat: N10096) were used. Jurkat cells in logarithmic growth phase were collected, and a cell suspension at 2×10⁷ cells / mL was prepared in Buffer R containing plasmid DNA. 10 µL of the cell suspension was subjected to electroporation treatment (Voltage: 1325, Width: 10, Pulse: 3), and the cells were quickly transferred to a 24-well multi-well plate containing 1 mL of pre-warmed complete medium per well. Thereafter, the cells were cultured for 48 hours in a CO₂ incubator. Regarding the amount of nucleic acid used for gene transfer, the basic amount was 500 ng / reaction for the original vector and 100 ng / reaction for MiniVector. Raji cells in the logarithmic growth phase were collected, and a cell suspension at 7,000,000 cells / mL was prepared in Buffer R containing plasmid DNA. 100 µL of the cell suspension was subjected to electroporation (Voltage: 1,300, Width: 30, Pulse: 1), and the cells were quickly transferred to a 6-well multiplate containing 2 mL of pre-warmed complete medium per well. Thereafter, the cells were cultured for 48 hours in a CO₂ incubator. The basic amounts of nucleic acid used for gene transfer were 5000 ng / reaction for the original vector and 250 ng / reaction for MiniVector. Ramos cells in the logarithmic growth phase were collected, and a cell suspension at 7,600,000 cells / mL was prepared in Buffer R containing plasmid DNA. 100 µL of the cell suspension was subjected to electroporation (Voltage: 1,550, Width: 20, Pulse: 1), and the cells were quickly transferred to a 6-well multiplate containing 2 mL of pre-warmed complete medium per well. Thereafter, the cells were cultured for 48 hours in a CO₂ incubator. The basic amounts of nucleic acid used for gene transfer were 10 µg / reaction for the original vector and 250 ng / reaction for MiniVector

[0065] <Evaluation of GFP-introduced cells> 48 hours after gene transfer, the survival rate was calculated by trypan blue (Gibco, Cat: 15250-061) exclusion test. After collecting the cells, the GFP introduction rate was evaluated using a flow cytometer (KSRFortessa X-20, Beckton Dickenson or CytoFLEX LX 4, Beckman Coulter).

[0066] <Evaluation of NanoLuciferase-introduced cells> 48 hours after gene transfer, the cell survival rate was calculated by trypan blue exclusion test (Gibco, Cat: 15250-061). Cells were harvested, 5,000 viable cells / 100 μL were transferred to a 96-well white well plate, and luminescence from NanoLuciferase was measured with a plate reader using the Nano-Glo Luciferase Assay (Promega, Cat: N1120) according to the attached protocol.

[0067] <Evaluation of CAR-introduced cells> 48 hours after gene transfer, the cell survival rate was calculated by trypan blue exclusion test (Gibco, Cat: 15250-061). Cells were harvested, 500,000 viable cells were reacted with a biotin-labeled anti-ScFv antibody cocktail (GeneScript, Cat: A02283-200), and then stained with PE-labeled streptavidin (BioLegend, Cat: 405245). PE-positive CAR-expressing cells were evaluated using a flow cytometer (CytoFLEX LX 4, Beckman Coulter).

[0068] [Test Example 1] Confirmation of restriction enzyme activity in T4 DNA ligase reaction buffer and confirmation of conjugation of linear DNA by T4 DNA ligase under 37°C reaction conditions The plasmid DNA shown in the simplified plasmid DNA map in Figure 2 was digested with MluI, BssHII, or both restriction enzymes. In addition to the basic reaction buffer 1X rCutSmart Buffer, 0.1 mM ATP or additionally 1 mM DTT was added (ALL). It was confirmed that regardless of the effect of the additives, the plasmid DNA was digested and linearized after 1 hour of reaction at 37°C with any of the restriction enzymes. Each restriction enzyme product (MluI / BssHII) processed under conditions without the above-mentioned additives was purified using the QIAquick PCR Purification Kit. The purified products were reacted in 1X T4 DNA Ligase Buffer at 16°C or 37°C, as shown in Figure 3. In addition to T4 DNA Ligase Buffer (T4), rCutSmart Buffer conditions (CB) with the above-mentioned ATP and DTT added were used as the reaction buffer. Since MluI produces a 5' overhang (5'-CGCG-3') and BssHII also produces the same overhang, conjugation is possible even between cut surfaces derived from different restriction enzymes. In both conditions, the linear DNA disappeared and a shift to the high-molecular-weight side was observed, suggesting that the conjugation reaction occurred.

[0069] [Test Example 2] Co-existence reaction of restriction enzyme and T4 DNA ligase The restriction enzyme reaction was performed under the same conditions as in Test Example 4 (ALL). During the restriction enzyme reaction, 1 / 5 the amount of T4 DNA Ligase (1750 U / μg DNA) as in Test Example 4 was co-administered ("T4 co-administration," hereinafter referred to as the co-administration reaction). The "T4 separate treatment" in Figure 4 was under the same conditions as in Test Example 4 (37°C, CB) (hereinafter referred to as the continuous reaction). However, as with Test Example 4, no linear nucleic acids were detected, suggesting that ligase activity was sufficient even with the enzyme amount reduced to 1 / 5. The co-administration reaction resulted in a band pattern different from both the pre-restriction enzyme treatment (Non-cut) and the T4 separate treatment conditions. It was decided to confirm that the product obtained from the co-administration reaction was conjugated and cyclic.

[0070] [Test Example 3] Confirmation of Cyclization of Coexisting Reaction Products The reaction was carried out under the same conditions as in Test Example 5, either as a continuous reaction (RE treatment followed by purification and T4 treatment) or a co-existence reaction (T4 co-existence with RE treatment). Each reaction product was purified and treated with exonuclease III or MluI / BssHII (Figure 5). Almost no unconjugated linear nucleic acids were observed in the continuous reaction product, and its resistance to exonuclease III indicated that it was cyclized. Furthermore, it was re-cleaved by restriction enzyme treatment, and the two target fragments were detected. The co-existence reaction product showed a supershift towards the high molecular weight side under unpurified conditions, but the band was detected at the same position as the continuous reaction product after purification. Some unconjugated linear nucleic acids were also confirmed, and since only this band was digested by exonuclease III, the main band is a cyclized product due to self-ligation. Similar to the continuous reaction product, restriction enzyme treatment produced two fragments. From these results, it is suggested that in the co-existence reaction, the restriction enzyme reaction and the ligase reaction antagonize each other, forming a cyclized product and creating some kind of higher-order structure. We decided to investigate whether self-ligation products of different nucleic acid fragments, which are the target product, are produced by combining two types of restriction enzymes that produce two nucleic acid fragments with T4 DNA Ligase.

[0071] [Test Example 4] Confirmation of cyclization of coexisting reaction products (using two restriction enzymes) The reaction was carried out under the same conditions as in Test Example 5, either as a continuous reaction (MluI / BssHII, purified → T4lig) or as a co-reaction reaction (MluI / BssHII + T4lig) (Figure 6). However, the restriction enzyme concentration was set to 10 U / μg DNA, and T4 DNA Ligase was set to 875 U / μg DNA. Each reaction product, either unpurified or purified, was treated in Exonuclease III Buffer (EB) or DTT-containing rCutSmart Buffer (CB) with either ExoIII alone or in the co-reaction of MluI / BssHII (MB). It was confirmed that Exonuclease III had the same activity in CB as in EB. As with Test Example 4, the original vector band was not detected by enzyme treatment alone, suggesting that digestion was sufficient with half the amount of restriction enzyme. Furthermore, as with Test Example 5, linear nucleic acids were not detected in the continuous reaction, suggesting that ligase activity was sufficient even when the amount of enzyme was reduced by half. When the purified product after MluI / BssHII treatment was treated with exonuclease III, two bands (fragment 1 and fragment 2) completely disappeared, indicating that the amount of nuclease in the reaction solution was sufficient to digest the linear double-stranded DNA, and no differences between buffers were observed. When the continuous reaction product was treated with exonuclease III, three bands were detected. However, when the uppermost band was treated with exonuclease III in the presence of MB, it was thought that the uppermost band contained a product in which fragments 1 and 2 were joined in the forward direction to regenerate the original vector, and a product in which they were joined in the reverse direction, appearing to be the same size as the original vector, but no longer being re-cleaved by restriction enzymes. These are all unintended by-products. The two lower bands are thought to be products of self-ligation of fragments 1 and 2, respectively, and are the products we intend to obtain. On the other hand, many of the coexisting reaction products were re-cleaved by restriction enzymes (purified, ExoIII(-), CB / MB), and since most of the bands disappeared with ExoIII treatment alone, it was thought that the product contained many linear fragments or cyclized products formed by the joining of the same restriction enzyme fragments. However, the number of cyclized products formed by the ligation of fragment 1 and fragment 2 in the forward or reverse direction was small compared to the continuous reaction, suggesting that the target product could be selectively obtained by optimizing the reaction conditions, and we decided to proceed with further investigations.

[0072] [Test Example 5] Investigation of coexistence reaction conditions (restriction enzyme amount, T4 DNA ligase amount) A co-existence reaction was performed using the same buffer and reaction conditions as in Test Example 7. The restriction enzymes and T4 DNA ligase amounts used are shown in Figure 7. After the co-existence reaction, exonuclease III was added to digest the linear nucleic acids. Compared to the conditions in Test Example 7 (restriction enzyme: 10 U / μg DNA, T4 DNA ligase: 875 U / μg DNA), reducing the amount of T4 ligase significantly increased the amount of the target product detected after exonuclease III treatment (the group of multiple bands detected on the lower side). Almost the same results were obtained in the concentration range of 1 to 25 U / μg DNA for T4 DNA ligase. On the other hand, there was no significant change in the amount of the target product when the restriction enzyme amount was halved, but it decreased dramatically when it was halved again. Interestingly, when the amount of T4 DNA ligase was reduced, multiple extra bands were detected below the two main bands that were detected under the high-concentration T4 DNA ligase conditions. In the next investigation, we decided to confirm that the extra bands were the target product.

[0073] [Test Example 6] Confirmation that the extra band is the target product Eight samples from the specimens prepared in Test Example 8 were used in a co-reaction of exonuclease III with either PstI or HindIII (Figure 8). The lower series of multiple bands detected during the reaction with exonuclease III alone disappeared in the presence of PstI, suggesting that both are self-ligating products of 3282 bp fragments and adopt different higher-order structures. All bands disappeared in the presence of HindIII. The restriction enzyme concentration range was narrowed to 2.5–10 U / μg DNA, and the T4 DNA Ligase concentration range to 50 U / μg DNA or less, and the reaction time was investigated.

[0074] [Test Example 7] Investigation of coexistence reaction conditions (restriction enzyme amount, T4 DNA ligase amount, reaction time) A coexistence reaction was carried out in the same manner as in Test Example 8. The restriction enzymes and T4 DNA ligase amounts used are shown in Figure 9. After the coexistence reaction, linear nucleic acid digestion was performed with exonuclease III. A tendency was observed for both the amount of the target product and the number of multiple bands to increase with increasing reaction time. Since there was almost no difference between conditions at the enzyme concentrations investigated, the enzyme concentration was set to 5 U / μg DNA for all tests, and further condition optimization will be pursued.

[0075] [Example 11] Investigation of coexistence reaction conditions using a new plasmid DNA (reaction time) As shown in the results of the studies up to Test Example 10, by mixing restriction enzymes and T4 DNA Ligase in appropriate ratios and causing competition between cleavage and conjugation reactions, we were able to obtain self-ligated products of the target nucleic acid fragment. To obtain the target product even more efficiently, we constructed the plasmid DNA shown in Figure 10. This plasmid DNA has recognition sequences for BbsI and NotI on both sides of the target gene product (the 3252 bp fragment shown in Figure 10). BbsI is an IIS-type restriction enzyme that cleaves sites distant from the recognition site, but this plasmid DNA was designed so that BbsI digestion produces sticky ends identical to those of NotI. By coexisting with ligase, a reaction occurs in which the BbsI or NotI cleavage sites rejoin, and the two fragments resulting from the digestion of both restriction enzymes each undergo self-ligation. The self-ligated product of the 2008 bp fragment has an intact BbsI recognition site, so it is re-cleaved by BbsI. On the other hand, the self-ligating product (target product) of the 3252bp fragment lacks a BbsI recognition site, and the NotI recognition site also disappears, so it is not digested by any coexisting restriction enzymes. We hypothesized that we could utilize this property of plasmid DNA to accumulate the self-ligating product of the 3252bp fragment, which is the target product. When this plasmid was reacted with restriction enzymes at 5 U / μg DNA concentrations and T4 DNA Ligase, the results of Test Example 10 were reproduced, and multiple bands were detected at the bottom in a time-dependent manner. Furthermore, the bands above 7000 kDa, which originated from the original vector, were significantly reduced compared to the results of Test Example 10, suggesting that the target self-ligation product had accumulated as expected. The bands that disappeared after exonuclease III treatment were the remaining linear nucleic acids, and the bands above 7000 bp that disappeared with the presence of XhoI are thought to be the original plasmid DNA, while the bands around 3000-5000 bp are thought to be the target product (self-ligation product of a 3252 bp fragment). The remaining bands detected around 1500-2000 bp at this time are the self-ligation product of a 2008 bp fragment (by-product). Comparing the amounts of the target product and by-products when digested with exonuclease III alone, it can be seen that the amount of by-products is smaller than the target product, especially at reaction times of 2-6 hours. As expected, the by-product underwent redigestion by BbsI, remaining partially in a linear form, and was likely digested by exonuclease III. Increasing the coexistence reaction time clearly reduced the amount of bands derived from the original plasmid DNA, while increasing the amounts of the target product and by-products. Furthermore, the ratio of multiple bands tended to converge to a certain ratio. Based on these results, a reaction time of 2 hours or more was considered sufficient; however, future studies will use a coexistence reaction time of 16 hours or more.

[0076] [Example 12] Investigation of by-product digestion by exonuclease III In Example 11, since most of the byproducts remained as cyclized products without being re-cleaved by BbsI after a reaction time of 12 hours, it was considered that the activity of BbsI was being lost. Therefore, in order to completely digest all products other than the target product with exonuclease III, BbsI or PvuI (10 U / μg DNA) was added during the exonuclease III digestion following the coexistence reaction. As shown in Figure 11, most of the byproducts were digested, but some were still detected. Therefore, the amount of PvuI, which was digested more efficiently, was doubled and digestion was performed, followed by purification using the QIAprep Spin Miniprep Kit (QIAGEN, Cat:27106) or the QIAquick PCR Purification Kit (QIAGEN, Cat:28106).

[0077] [Example 13] Purification study of the target product after coexistence reaction and cleavage by intrastructural restriction enzymes The same method as in Example 12 was used, but the additional PvuI concentration during exonuclease III treatment was set to 20 U / μg DNA. After exonuclease III treatment, purification was performed using two different purification kits, and the QIAprep Spin Miniprep Kit yielded a higher yield than the QIAquick PCR Purification Kit. Multiple bands were still detected after purification, suggesting that the higher-order structure was preserved. These multiple bands converged into a single linear nucleic acid of 3252 bp after XhoI treatment (Figure 12). From these results, it was considered that the multiple bands obtained by the coexistence reaction were the same structure with multiple higher-order structures.

[0078] [Example 14] Junction sequencing analysis of the target product after coexistence reaction The target product purified in Example 13 was sequenced using Eurofins Genomics. It was confirmed that the NotI and BbsI sections of the original vector were joined (Figure 13).

[0079] [Example 15] Investigation of open circularization of the target purified product after coexistence reaction Two lots of the target product purified using the method of Example 13 were treated with a restriction enzyme (Nb.BtsI) that cleaves only one strand of the recognition site. As a result, an open circular band (white arrowhead in Figure 14) was detected as the main band. A small amount of linear bands, in which both strands were cleaved, were also detected, as shown by the black arrowhead. From these results, it was considered that the multiple bands detected in NC represent multiple superhelical structures. Hereafter, this target product will be referred to as MiniVector.

[0080] [Example 16] Comparison of yields between coexistence reaction and normal reaction In previous studies, byproducts have been digested by co-administering PvuI and exonuclease III after the co-administration reaction. To minivectorize various gene products, vectors were created with BamHI, HindIII, and ExoRI recognition sequences inserted as restriction enzyme sites to be co-administered during byproduct digestion (see Figure 15, simplified map). It was previously confirmed that changing the co-administered restriction enzymes during digestion does not affect yield (data omitted). Normally, when obtaining self-ligation products of restriction enzyme fragments, nucleic acid fragments are purified after restriction enzyme treatment and then ligated (see Figure 15, right side of the flow chart). In this study, we confirmed whether the co-administration reaction is superior in terms of yield. First, a T4 DNA Ligase (5U / μg DNA, 16hr) reaction was attempted on the restriction enzyme-treated purified product under the same conditions as the coexistence reaction. However, the target 2230bp fragment was fragmented into two by XhoI digestion after the reaction, suggesting that no conjugation occurred at all (Figure 16, left four lanes). Therefore, when the amount of T4 DNA Ligase was increased by more than 200 times, the original fragment could no longer be detected even after a 30-minute reaction. XhoI treatment then allowed the detection of the 2230bp linear target fragment, suggesting that a sufficient conjugation reaction occurred and the fragments were circularized (Figure 16, right four lanes). In a yield comparison test with the coexistence reaction under these conditions, the yield of MiniVector was 55% in the coexistence reaction, while the yield was less than 2% in the method shown on the right side of the flowchart in Figure 15.

[0081] [Example 17] Structural changes of plasmid DNA by ligation reaction of restriction enzyme fragments In the previous study, it was found that MiniVectors could be produced even if the restriction enzyme digestion and ligation processes were separated, although the yield decreased. Therefore, the same treatment was applied to plasmid DNA purified from E. coli to confirm that a change in higher-order structure occurred. The plasmid DNA used in the previous study was digested with NotI alone, subjected to heat inactivation, and then ligated with T4 ligase (Figure 17, flow chart). As a result of the study, MiniVector-like multiple bands were detected, which were not seen in the electrophoresis of the original plasmid DNA, as shown in lanes 3-4 of the right panel of Figure 17.

[0082] [Example 18] Evaluation of MiniVector's superiority (Jurkat cells, Nano Luciferase, DEAE / Dextran method) Purified Nano Luciferase-expressing MiniVectors (MiniVectors) after co-existence reaction and their original vectors (Nluc MV-1, -2) were introduced into Jurkat cells using the DEAE / Dextran method. Two days after introduction, the cells were lysed, and the luciferase activity in the lysate was compared (Figure 18). Compared to when the original vector was used, approximately six times higher luciferase activity was detected when the MiniVector was used, suggesting that the gene transfer efficiency and the amount introduced per cell increased due to the MiniVectorization. The Nluc MV vector used in this study had a total length of 5159 bp, which became 3160 bp after MiniVectorization. When the same amount of nucleic acid was introduced, the molar amount of MiniVector was approximately 1.6 times greater. Therefore, we decided to investigate the luciferase activity of MiniVectors that is dependent on the amount of nucleic acid introduced.

[0083] [Example 19] MiniVector dose-dependent effect (Jurkat cells, Nano Luciferase, DEAE / Dextran method) Purified Nano Luciferase-expressing MiniVectors (MiniVectors) after co-existence reaction and their original vectors were introduced into Jurkat cells using the DEAE / Dextran method. Two days after introduction, the cells were lysed, and the luciferase activity in the lysates was compared (Figure 19). However, the amount of MiniVector introduced was also evaluated at four levels of reduction from 1.25 μg using a common ratio of 2. When using normal pDNA, the introduction efficiency showed a gradual bell-shaped curve with a peak at 1.25 μg in the range of introduced nucleic acid amounts from 0.5 to 2.0 μg (data omitted). Surprisingly, it was found that the introduction rate of MiniVector reached a plateau even at approximately 0.3 μg, which is below the equivalent molar amount of the original vector of 0.78 μg. Hereafter, unless otherwise noted, the amount of MiniVector introduced was 0.3 μg.

[0084] [Example 20] Evaluation of MiniVector's superiority (Jurkat cells, GFP, DEAE / Dextran method) Purified GFP-expressing MiniVectors (mvEGFP) after co-existence reaction, their original vector (EGFP / pMV), and a control GFP-expressing vector (EGFP / pRP) were introduced into Jurkat cells using the DEAE / Dextran method. The percentage of GFP-positive cells and the average fluorescence intensity of GFP-positive cells were compared two days after introduction (Figure 20; the promoters and sizes of each vector are shown in the table on the right). Compared to using both EGFP expression vectors, the gene transfer efficiency was more than 1.5 times higher when using MiniVectors, and the MFI, an indicator of expression level per cell, was more than 6 times higher. These results suggest that MiniVectors are useful not only for increasing the transfer efficiency but also for increasing the expression level of the target gene per cell.

[0085] [Example 21] Evaluation of MiniVector's superiority (Jurkat cells, GFP, DEAE / Dextran method) Using three lots of MiniVector (mvEGFP) prepared on separate test days, independent studies were conducted with the original vector (EGFP / pMV) in n=2 and n=3 trials each, following the example of Test Example 9 (Figure 21). The results showed that using MiniVector compared to the original vector resulted in a statistically significant improvement in transduction efficiency of approximately 1.6 times and an increase in transgene expression per cell of approximately 4 times.

[0086] [Example 22] MiniVector dose-dependent effect (Jurkat cells, GFP, DEAE / Dextran method) Purified EGFP-expressing MiniVector, obtained after co-reaction, was used to introduce genes into Jurkat cells using the DEAE / Dextran method with amounts reduced in three steps from 600 ng at a common ratio of 2. The percentage of GFP-positive cells and the average fluorescence intensity of GFP-positive cells were analyzed two days after introduction. The results showed that gene transfer efficiency (Figure 22, line graph) plateaued at 150 ng, but the expression level per cell (Figure 22, bar graph) increased in a nucleic acid-dependent manner and did not reach a plateau within the scope of this study. At least in terms of gene transfer efficiency, 150 ng / reaction was sufficient, and the required amount was found to be less than 1 / 8 of the original vector. Based on the results up to Example 10, the amount of nucleic acid used for MiniVector introduction should be approximately 1 / 4 of the original vector as a guideline. [Example 23] Evaluation of MiniVector's superiority (Jurkat cells, GFP, Lipofection method) To evaluate the usefulness of MiniVectors in other gene transfer methods, we attempted gene transfer into Jurkat cells using Lipofectamine 3000. The amount of nucleic acid introduced was 500 ng for the original vector and 125 ng for the MiniVector, according to the package insert. The results showed that Jurkat cells had very low transfer efficiency using the Lipofection method, with the original vector having an efficiency of only a few percent. However, applying MiniVectors improved this efficiency by approximately three times, and the MFI (Metabolic Factor Intake), an indicator of the amount of introduced per cell, also improved by approximately three times (Figure 23). Since it was thought that MiniVectorization would also improve other gene transfer methods, we decided to investigate this using electroporation.

[0087] [Example 24] Evaluation of MiniVector's superiority (Jurkat cells, GFP, electroporation method) To evaluate the usefulness of MiniVector in electroporation, gene transfer into Jurkat cells was attempted using the Neon NxT Electroporation system (Figure 24). The amount of introduced nucleic acid was adjusted according to the package insert, with 500 ng as the base for the original vector, and 100 ng and 30 ng for MiniVector. Under the 500 ng original vector condition, the transfer efficiency was high (over 90%), but the survival rate was low (approximately 60%). Reducing the amount of introduced nucleic acid to 30 ng restored the survival rate to over 90%, but the transfer efficiency fell below 90%. On the other hand, with MiniVector, the survival rate was over 90% even with a 100 ng introduction of nucleic acid, and the transfer efficiency was almost 100%. Cell-level expression levels followed the same trend as the GFP positivity rate, with the 100 ng MiniVector condition showing the highest expression. Even when the amount of introduced nucleic acid was reduced to 30 ng, the GFP positivity rate was almost equivalent to that under the 500 ng original vector condition. These results demonstrate that MiniVector is an effective tool for improving survival rates and delivery efficiency in electroporation.

[0088] [Example 25] Evaluation of the superiority of MiniVector (Raji cells, GFP, electroporation method) Next, to evaluate the superiority of MiniVector over suspension cell lines other than Jurkat, we attempted to introduce MiniVector via Neon NxT electroporation into Burkitt lymphoma cell lines, Raji cells and Ramos cells. For Raji cells, the amount of introduced nucleic acid was adjusted according to the package insert, using 5000 ng as the base for the original vector, then 250 ng and 100 ng, and 250 ng and 100 ng for MiniVector (Figure 25). Under the 5000 ng original vector introduction condition, the introduction efficiency was high at over 80%, but the survival rate was less than 20%. Reducing the amount of introduced nucleic acid to 250 ng restored the survival rate to over 90%, but the introduction efficiency fell below 80%. On the other hand, with MiniVector, the survival rate was over 90% even with an introduced nucleic acid amount of 250 ng, and the introduction efficiency was almost 100%. Even when the amount of introduced nucleic acid was reduced to 100 ng, the GFP positivity rate was equivalent to or higher than that under the 5000 ng original vector introduction condition.

[0089] [Example 26] Evaluation of MiniVector's superiority (Ramos cells, GFP, electroporation method) For Ramos cells, the amount of introduced nucleic acid was adjusted according to the package insert, using 10 μg as the base for the original vector, then 250 ng and 100 ng, and 250 ng and 100 ng for MiniVector (Figure 26). Under the condition of introducing 10 μg of original vector, the introduction efficiency was high at over 50%, but the survival rate was approximately 20%. When the amount of introduced nucleic acid was reduced to 250 ng, the survival rate recovered to about 80%, similar to that of the decoy gene-introduced sample, but the introduction efficiency fell below 10%. On the other hand, even with an introduced nucleic acid amount of 250 ng, the survival rate of MiniVector was as high as that of the control sample, and the introduction efficiency was also high at over 40%. These results demonstrate the usefulness of MiniVector for other suspension cell lines as well, using the electroporation method.

[0090] [Example 27] Evaluation of MiniVector's superiority (Raji / Ramos cells, GFP, DEAE / Dextran method) Next, the superiority of MiniVector (MV) over Raji and Ramos cells using the DEAE / Dextran method was evaluated (Figure 27). While the gene transfer efficiency using the original vector was approximately 5% in both cell lines, using MiniVector improved it to approximately 14% in Raji cells and approximately 10% in Ramos cells. Cell-specific expression levels also increased by approximately 25% in Raji cells and approximately doubled in Ramos cells. However, no change was observed in cell viability. These results demonstrate the usefulness of MiniVector for other suspension cell lines using the DEAE / Dextran method.

[0091] [Example 28] Evaluation of MiniVector's superiority (primary T cells, GFP, DEAE / Dextran method) Next, we evaluated the superiority of MiniVector (MV) over primary T cells using the DEAE / Dextran method. The gene transfer efficiency using the original vector was 2.44% (Figure 28, Δ value in the second quadrant), but using MiniVector improved it to 16.5%, an improvement of approximately 10 times. The expression level per cell also clearly improved, and the survival rate improved from approximately 50% when using the original vector to over 70% when using MiniVector (data omitted). These results demonstrate the usefulness of MiniVector even for primary T cells in the DEAE / Dextran method.

[0092] [Example 29] Production of CAR-T cells using MiniVector (primary T cells, CAR, DEAE / Dextran method) We decided to confirm that other genes could also be introduced into primary T cells using MiniVector. We attempted to express CD19 CAR (chimeric antigen receptor), which is attracting attention as a new treatment for diffuse large B-cell lymphoma and other diseases. As shown in Figure 29, the Δ value in the second quadrant, which indicates CAR-positive cells, was approximately 30% compared to non-genetically modified cells. This suggests that CAR-T cells can be prepared by chemical methods using MiniVector.

[0093] [Example 30] Establishment of stable expression cells using the Tol2 Transposon system (Jurkat cells, NanoLuciferase, DEAE / Dextran method) Traditionally, viral vector methods, such as lentiviruses and retroviruses, have been used to obtain suspension cell lines that stably express a target gene. Viral vector production is technically difficult, time-consuming, and labor-intensive, and requires equipment that meets biosafety requirements. While methods exist for inserting the target gene into genomic DNA using transposon systems like PiggyBac, SleepingBeauty, and Tol2, chemical methods often fail to achieve sufficient gene transfer efficiency in suspension cells, necessitating electroporation. Electroporation, however, lacks versatility due to the need for specialized equipment and devices. We evaluated whether stable expression cells could be efficiently obtained using the transposon method by leveraging the characteristics of MiniVector, which demonstrated high gene transfer efficiency even with chemical gene transfer methods. We mini-vectorized both a Puromycin-selective marker-carrying Nano Luciferase-expressing Tol2 vector and a transposase (TPase) vector. This reduced the vector size from 5180 bp to 3160 bp for the former and from 6042 bp to 4025 bp for the latter. Jurkat cells were introduced into various Jurkat cells using the DEAE / Dextran method, with Tol2 source vectors and transposase source vectors, or Tol2 MiniVectors and TPase MiniVectors in different ratios. Luciferase activity was measured the day after introduction, puromycin selection was performed for 3 days, and then the luciferase activity of the proliferating cells was evaluated over the following 2 weeks. When the original vector was introduced, stable Jurkat cells were obtained in only one case for each of the three conditions. In contrast, when MiniVector was used, stable expression cells were obtained in both cases for all two conditions (Table 1). This suggests that applying MiniVector to the transposon method allows for stable expression of Jurkat cells even using chemical methods.

[0094] [Table 1]

[0095] [Example 31] Evaluation of the superiority of MiniVector-like plasmid DNA (Jurkat cells, GFP, DEAE / Dextran method) We decided to evaluate the superiority of the gene transfer efficiency of the plasmid DNA that underwent a MiniVector-like structural change, prepared in Example 17, compared to the original plasmid DNA, when using the DEAE / Dextran method. As shown in Figure 30, the gene transfer efficiency was significantly improved compared to the original pDNA by introducing a MiniVector-like structure.

Claims

1. A construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, A predetermined sequence is arranged so as to be sandwiched between a first restriction enzyme recognition sequence and a second restriction enzyme recognition sequence. The first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a different restriction enzyme recognition sequence from the first restriction enzyme recognition sequence. A construct for creating recombinant minivectors that do not contain a predetermined sequence.

2. The construct according to claim 1, wherein a predetermined sequence includes a selection marker and a replication origin.

3. The construct according to claim 1 or 2, wherein a portion of the other region of the two regions sandwiched between a first restriction enzyme recognition sequence and a second restriction enzyme recognition sequence, the region not containing a predetermined sequence, is included in the recombinant minivector, and the other region contains the cleavage site by the first restriction enzyme and the target gene.

4. The construct according to claim 3, wherein, of the two regions sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the region in which a predetermined sequence is located does not include a cleavage site by the second restriction enzyme.

5. The construct according to claim 1 or 2, wherein the recombinant minivector has a superhelical structure.

6. The construct according to claim 1 or 2, wherein the recombinant minivector is a recombinant minivector for transforming animal cells.

7. The construct according to claim 1 or 2, wherein the recombinant minivector is introduced into cells with higher efficiency compared to the construct.

8. The construct according to claim 1 or 2, wherein the recombinant minivector has a base length of 1,000 to 5,000.

9. The construct according to claim 1 or 2, having a base length of 3000 or more.

10. A recombinant minivector containing the target gene but without a replication origin.

11. A recombinant minivector according to claim 10, having a base length of 1,000 to 5,000.

12. A method for producing a recombinant minivector that does not contain the predetermined sequence from a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, wherein the predetermined sequence is arranged so as to be sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence, 1) A step of cleaving the construct with a first restriction enzyme and a second restriction enzyme to obtain a cleaved fragment containing a predetermined sequence and a first restriction enzyme recognition sequence, and a cleaved fragment not containing the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, and 2) A method comprising the step of subjecting the two obtained cleavage fragments to a ligation reaction to obtain a reaction product containing two self-ligated cyclic nucleic acids.

13. The method according to claim 12, further comprising the step of cleaving with a first restriction enzyme a nucleic acid ligated by the two types of cleavage fragments contained in the reaction product, or a cyclic nucleic acid self-ligated by cleavage fragments containing a predetermined sequence and a first restriction enzyme recognition sequence.

14. The method according to claim 12 or 13, wherein each of the steps is carried out in a solution containing a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, a first restriction enzyme, a second restriction enzyme, and a ligase.

15. The method according to claim 12 or 13, wherein the predetermined sequence includes a selection marker and a replication origin.

16. The method according to claim 12 or 13, wherein the recombinant minivector is a self-ligated cyclic nucleic acid in which the cleavage fragments not containing a first restriction enzyme recognition sequence and a second restriction enzyme recognition sequence are self-ligated.

17. The method according to claim 12 or 13, wherein the recombinant minivector has a superhelical structure.

18. The method according to claim 12 or 13, wherein the recombinant minivector is a recombinant minivector for transforming animal cells.

19. The method according to claim 12 or 13, wherein the recombinant minivector is introduced into cells with higher efficiency compared to the construct.

20. The method according to claim 12 or 13, further comprising the step of digesting the linear nucleic acid contained in the reaction product with an exonuclease.

21. A cleavage fragment containing a predetermined sequence and a first restriction enzyme recognition sequence further contains a third restriction enzyme recognition sequence that is different from the first and second restriction enzyme recognition sequences. The method according to claim 12 or 13, further comprising the step of cleaving a nucleic acid ligated by the two types of cleavage fragments contained in the reaction product, or a circular nucleic acid self-ligated by a cleavage fragment containing a predetermined sequence and a first restriction enzyme recognition sequence, with a third restriction enzyme.

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