Method for producing genetically modified cells

Abstract

To provide a method for producing gene-modified cells that offers superior efficiency in introducing the target gene into target cells, excellent survival rates of target cells into which the target gene has been introduced, and long-term expression of the target gene. [Solution] A method for producing genetically modified cells, comprising: contacting target cells with a vector containing a target gene sequence; contacting target cells with mRNA containing a sequence encoding a transposase for introducing the target gene sequence into the genomic DNA of the target cells; irradiating the target cells with plasma; and obtaining genetically modified cells into which the vector and mRNA have been introduced.

Description

Technical Field

[0001] The present invention relates to a method for producing gene-introduced cells.

Background Art

[0002] With the decoding of the human genome and the mouse genome, when conducting research and development related to molecular biology or pharmaceutical development in fields such as medicine or pharmacy, polynucleotides such as DNA or RNA or their derivatives, proteins such as signal transduction proteins or transcriptional regulators or their derivatives, high molecular compounds, low molecular bioactive substances, or molecules such as drug candidates are introduced into target cells, and it is increasingly necessary to test the function of genes or the physiological activities of bioactive molecules in target cells.

[0003] Methods for introducing molecules into target cells include chemical methods, physical methods, and biological methods. The chemical method is a method of using a transfection reagent such as a cationic polymer, cationic lipid, or calcium phosphate to allow the molecule to be taken up into the target cell by endocytosis. The physical method is a method of directly introducing the molecule into the target cell by a physical operation such as sonoporation, laser irradiation, or electroporation. The biological method is a method of using a viral vector such as a retrovirus, lentivirus, adeno-associated virus (AAV), or adenovirus, and transferring the nucleic acid in the virus particle into the target cell by the infectivity of the virus.

[0004] As a method for introducing a target gene into a cell without using a virus, there is a transposon method. Representative examples of the transposon method include the piggyBac transposon method and the Sleeping Beauty transposon method.

[0005] Non-Patent Document 1 describes that the piggyBac transposon method is useful for long-term expression of a target gene.

[0006] To introduce a gene using the piggyBac transposon method, two types of DNA plasmids are first prepared: a target gene expression vector (transposon vector) and a piggyBac transposase vector that expresses a gene transposition enzyme. The incorporated transposase vector causes piggyBac transposase to be expressed in the host cell, and specific sequences at both ends of the target gene expression vector are excised. The excised sequences are then incorporated into the TTAA sequence (nucleotide sequence) in the host cell's genome, thereby introducing the target gene into the cell. By integrating the target gene into the host cell's genome, long-term expression of the target gene in the host cell is achieved.

[0007] Non-patent document 2 reports the initial results of a Phase I / II clinical trial using donor-derived CD19 CAR T cells differentiated into cytokine-induced killer (CIK) cells produced with Sleeping Beauty (SB) transposons in patients with B-cell acute lymphoblastic leukemia (B-ALL) who had relapsed after allogeneic hematopoietic stem cell transplantation. The Sleeping Beauty transposon method is a method for introducing target genes using jumping DNA fragments called "Sleeping Beauty," which can efficiently integrate designed foreign DNA into the genome of mammalian cells. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Nakazawa, Y. et al., “Optimization of the PiggyBac Transposon System for the Sustained Genetic Modification of Human T-Lymphocytes,” Journal of Immunotherapy, October 2009, Vol. 32, No. 8, pp. 826-836. [Non-Patent Document 2] Magnani, Chiara F. et al., “Sleeping Beauty-engineered CAR T cells achieve antileukemic activity without severe toxicities,” Journal of Clinical Investigation, November 2020, Vol. 130, No. 11, pp. 6021-6033. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] As mentioned above, the piggyBac method is an example of the transposon method. However, the transposon method has low efficiency in introducing the target gene, making it difficult to improve the efficiency of introducing the target gene into target cells and the survival rate of host cells after gene introduction.

[0010] The present invention aims to provide a method for producing gene-modified cells that exhibits excellent efficiency in introducing the target gene into target cells, high survival rates of target cells into which the target gene has been introduced, and long-term expression of the target gene. [Means for solving the problem]

[0011] The present invention encompasses the following embodiments. [1] A method for producing genetically modified cells, comprising: contacting target cells with a vector containing a target gene sequence; contacting target cells with mRNA containing a sequence encoding a transposase for introducing the target gene sequence into the genomic DNA of the target cells; irradiating the target cells with plasma; and obtaining genetically modified cells into which the vector and mRNA have been introduced. [2] A method for producing gene-transformed cells according to [1], wherein the target cells are irradiated with plasma between contacting the vector with the target cells and contacting the mRNA with the target cells. [3] A method for producing gene-transformed cells according to [1], wherein the vector is brought into contact with the target cells at the same time as the mRNA is brought into contact with the target cells. [4] A method for producing gene-transformed cells according to [1], wherein the vector is introduced into the target cells, the mRNA is introduced into the target cells, and the target cells are irradiated with plasma simultaneously. [5] The method for producing gene-transformed cells according to any one of [1] to [4], wherein the vector is a modified transposon vector into which the nucleic acid fragment of (1) or (2) below is inserted. (1) The target gene sequence is located between the 5' reversed repeat sequence and the 3' reversed repeat sequence of the transposon gene. (2) The transposon gene has a 5' inverted repeat sequence / series repeat sequence and a 3' inverted repeat sequence / series repeat sequence, and the target gene sequence is located between these sequences. [6] The method for producing gene-transformed cells according to [5], wherein the vector is a modified transposon vector in which the nucleic acid fragment of (3) below is further inserted in addition to (1) or (2) above. (3) A restriction enzyme recognition site or exogenous gene expression cassette located between the 5' reversed repeat sequence and the 3' reversed repeat sequence of a transposon gene, or between the 5' reversed repeat sequence / series repeat sequence and the 3' reversed repeat sequence / series repeat sequence. [7] A method for producing gene-transformed cells according to any one of [1] to [6], wherein the molar ratio of the amount of vector to the amount of mRNA to be brought into contact with the target cells is vector:mRNA = 1:0.1 to 1:10. [8] A method for producing genetically modified cells according to any one of [1] to [7], wherein the target cell is at least one selected from the group consisting of blood-derived cells, adherent cells, and iPS cells. [9] The method for producing gene-transformed cells according to [8], wherein the target cells are blood-derived cells including CD3-positive cells.

[10] A method for producing a gene-transformed cell according to [9], comprising the target gene sequence encoding a chimeric antigen receptor.

[11] The method for producing a gene-introduced cell according to [9] or

[10] , wherein the CD3-positive cells contain T cells.

[12] The method for producing a gene-introduced cell according to any one of [8] to

[11] , wherein the adherent cells contain HEK293F cells, mesenchymal stem cells, and adipose-derived stem cells. [Advantages of the Invention]

[0012] According to the present invention, there can be provided a method for producing a gene-introduced cell, which has excellent efficiency in introducing a target gene into a target cell and excellent survival rate of the target cell into which the target gene has been introduced, and can ensure long-term expression of the target gene. [Brief Description of the Drawings]

[0013] [Figure 1] FIG. 1 is a cross-sectional view of a gene introduction device according to an embodiment of the present invention. [Figure 2] FIG. 2 is an end view of the irradiator 14 in FIG. 1. [Figure 3] FIG. 3 is a graph showing the measurement results of the total cell amount (All Events) of each gene-introduced cell in Experimental Examples 1-1 to 1-3 by flow cytometry. [Figure 4] FIG. 4 is a graph showing the measurement results of the total cell amount (All Events) of each gene-introduced cell in Experimental Examples 1-4 to 1-6 by flow cytometry. [Modes for Carrying Out the Invention]

[0014] In the present invention, "~" indicating a numerical range means that the numerical values described before and after it are included as the lower limit value and the upper limit value.

[0015] [Method for Producing Gene-Introduced Cell] The method for producing a gene-introduced cell of the present invention includes contacting a vector containing a target gene sequence with a target cell, contacting the target cell with mRNA containing a sequence encoding a transposase for introducing the target gene sequence into the genomic DNA of the target cell, irradiating the target cell with plasma, introducing the vector into the target cell, and introducing the mRNA into the target cell.

[0016] (Target gene) In the present embodiment, the target gene is not particularly limited and may be any gene to be expressed in the target cell. Examples include DNA encoding cDNA (complementary DNA), miRNA (microRNA), or shRNA (small hairpin RNA).

[0017] The cDNA contains, in sequence, the sequence of the region of the gene that is translated into protein from the start codon to the stop codon. In the target cell into which the gene has been introduced, it is transcribed into mRNA and, through translation in liposomes, can express the target protein such as an enzyme and a fluorescent protein. Examples of the target gene include gene sequences encoding fluorescent proteins such as green fluorescent protein (GFP), yellow fluorescent protein, red fluorescent protein, and blue fluorescent protein, gene sequences encoding protein tags such as His tag and epitope tag, or gene sequences encoding CAR (chimeric antigen receptor).

[0018] miRNA is a functional nucleic acid that finally becomes a microRNA of 20 to 25 bases in length through a multi-step generation process from transcription. miRNA is classified as a functional ncRNA (non-coding RNA) and plays an important role in life phenomena by regulating the expression of other genes.

[0019] shRNA is a hairpin-type RNA sequence used for gene silencing by RNA interference.

[0020] The size (number of base pairs) of the target gene is not particularly limited, but is preferably 10 bp to 10 kbp, more preferably 500 bp to 6 kbp, and even more preferably 1 kbp to 3 kbp.

[0021] (vector) The vector is a DNA plasmid containing the target gene sequence and enabling long-term expression of the target gene. Preferably, the vector is a modified transposon vector into which a nucleic acid fragment having the characteristics of (1) or (2) below is inserted, and more preferably, a modified transposon vector into which a nucleic acid fragment having the characteristics of (3) below in addition to (1) or (2) below is inserted.

[0022] (1) The transposon gene has a 5' inverted repeat (TIR) ​​sequence and a 3' inverted repeat (TIR) ​​sequence, and the target gene sequence is inserted between these sequences.

[0023] (2) The transposon gene has a 5' inverted repeat sequence / serial repeat (IR / DR, IR: Inverted Repeat, DR: Direct Repeat) sequence and a 3' inverted repeat sequence / serial repeat (IR / DR) sequence, and the target gene sequence is inserted between these sequences.

[0024] (3) A restriction enzyme recognition site or exogenous gene expression cassette between the 5' reversed repeat (TIR) ​​sequence and the 3' reversed repeat (TIR) ​​sequence or the reversed repeat / series repeat (IR / DR) sequence of the transposon gene.

[0025] A transposon is a nucleotide sequence that can transposition its position on the genome within a cell; it is also called a mobile gene or transposable element. Transposons include DNA transposons, which are DNA fragments that transpose directly, and RNA transposons, which undergo transcription and reverse transcription. However, usually only DNA transposons are referred to as transposons. RNA transposons are usually distinguished as retrotransposons or retroposons. One example of a transposon method is the piggyBac method. For DNA transposons to transpose, an enzyme called transposase is required, and this enzyme is encoded by the transposon itself. Transposons have a reversed repeat sequence (reverse repeat sequence, TIR sequence) or a reverse repeat sequence / series repeat sequence (IR / DR sequence) at their ends. The transposase recognizes this sequence and excises the transposon from the genome sequence. Then, it reinserts the excised sequence into the appropriate genome sequence.

[0026] An exogenous gene expression cassette is defined as a nucleic acid fragment to which an exogenous gene has been modified with an appropriate promoter, stop codon, poly(A) addition signal sequence, Kozak sequence, and secretory signal. By introducing this into target cells, the exogenous gene, i.e., the target gene, can be expressed within the target cells.

[0027] One method for bringing a vector containing the target gene sequence into contact with target cells is to bring a liquid containing the vector into contact with the target cells. The target cells may be pre-cultured before contacting the vector. After removing the culture medium used for pre-culture, the liquid containing the vector may be brought into contact with the surface of the target cells. For example, the liquid containing the vector may be poured into a container containing the target cells so that it comes into contact with the target cells.

[0028] Liquids containing vectors include those in which the vector is dissolved or dispersed in a buffer solution. The buffer solution is not particularly limited, but it is preferable that it has little to no effect on the efficiency of gene transfer to target cells and has little to no cytotoxicity to target cells.

[0029] (mRNA) mRNA contains a sequence that encodes a transposase for introducing a target gene sequence into the genomic DNA of a target cell. An example of mRNA is the mRNA (SEQ ID NO: 1) derived from the cabbage looper (Trichoplusia ni) containing a transposase-encoding sequence. Because the mRNA introduced into the target cell is subsequently degraded, the transposase is expressed only for a short period.

[0030] A transposase recognizes a TIR sequence or IR / DR sequence located at the end of the target gene sequence and excises the target gene sequence from the vector. The transposase is not particularly limited as long as it functions as a transposase for the vector within the target cell after the mRNA has been taken up into the target cell, but examples include transposases derived from cabbage loopers (Trichoplusia ni), transposases derived from salmonid fish (e.g., those registered as accession number: 5CR4_A), and transposases containing sequences that have at least 80%, preferably 90% or more, more preferably 95% or more sequence identity with respect to these transposases, or fragments in which a part of such sequences has been deleted or removed.

[0031] One method for bringing mRNA into contact with target cells is to bring a liquid containing mRNA into contact with the target cells. The target cells may be pre-cultured before contacting the mRNA. After removing the culture medium used for pre-culture, the liquid containing mRNA may be brought into contact with the surface of the target cells. For example, the liquid containing mRNA may be added to a container containing the target cells so that it comes into contact with the surface of the target cells.

[0032] A liquid containing mRNA may be a vector dissolved or dispersed in a buffer solution. The buffer solution is not particularly limited, but it is preferable that it has little to no effect on the efficiency of gene transfer to target cells and has little to no cytotoxicity to target cells.

[0033] The vector and mRNA may be contained in the same solution; that is, the vector and mRNA may be brought into contact with the target cells simultaneously. The liquid containing the vector and mRNA may also contain the buffer solution mentioned above. For example, the liquid containing mRNA may be added to a container containing the liquid containing the target cells and the vector so that it comes into contact with the target cells. Alternatively, the mRNA may be brought into contact with the target cells after the vector and target cells have been brought into contact.

[0034] The molar ratio of the amount of vector to mRNA brought into contact with the target cells is preferably vector:mRNA = 1:0.1 to 1:10, and more preferably 1:2 to 1:5. For example, when bringing the target cells into contact with the vector and mRNA using a liquid containing the vector and a liquid containing mRNA, the liquids containing the vector and mRNA are prepared so that the molar ratio of the amount of vector to mRNA added to the container containing the target cells is vector:mRNA = 1:0.1 to 1:10. Alternatively, when using a liquid containing both the vector and mRNA, the molar ratio of the amount of vector to mRNA contained in the liquid is adjusted to vector:mRNA = 1:0.1 to 1:10.

[0035] (target cell) Target cells refer to cells into which a target gene is introduced, and are not limited to any particular type of cell. Specific examples of such target cells include animal cells, including human cells, non-human animal individuals, cells collected from human individuals, cells within human individuals, human individuals, plant cells, and microbial cells. These target cells may be of a single type or a mixture of two or more types.

[0036] The cells collected from human organisms mentioned above include cells used in pharmaceutical research and development, which are not intended to be returned to human organisms, and cells used in regenerative medicine, which are intended to be returned to human organisms. Furthermore, the cells collected from human organisms mentioned above also include cells cultured from cells collected from human organisms.

[0037] Furthermore, the non-human animal cells and plant cells mentioned above include cells present within an individual, cells present within a tissue, cells collected from an individual or tissue, and cells cultured from cells collected from an individual or tissue. From another perspective, the target cells used in the present invention include prokaryotic cells such as Escherichia coli, actinomycetes, and Bacillus subtilis; as well as eukaryotic cells such as yeast, non-human animal cells, cells collected from a human individual, cells contained in a human individual, and plant cells.

[0038] The target cells are preferably at least one selected from the group consisting of blood-derived cells, adherent cells, and iPS cells.

[0039] Blood-derived cells include, for example, CD3-positive cells. CD3-positive cells include T cells and Jurkat cells, etc. Adherent cells include, for example, HEK293 cells, mesenchymal stem cells, and adipose-derived stem cells, etc.

[0040] T cells are a type of lymphocyte that differentiates and matures from progenitor cells produced in the bone marrow through selection in the thymus. T cells have a characteristic T cell receptor (TCR) on their cell surface. They make up 70-80% of lymphocytes in peripheral blood. T cells can be collected from peripheral blood or cultured. T cells are classified as CD3-positive cells. In addition to T cells, Jurkat cells are another example of CD3-positive cells. T cell culture can be performed using commercially available T cell culture media according to known protocols.

[0041] HEK293 cells are a cell line established by transforming human fetal kidney cells with the adenovirus E1 gene. Due to their ease of culture and gene transfer, HEK293 cells are used for various purposes, such as substitution protein production, recombinant adenovirus creation, and as a host for amplification. They are available commercially. HEK293F cells are one example of a commercially available product.

[0042] Mesenchymal stem cells (MSCs) are a type of stem cell found in the body that has the ability to differentiate into mesoderm-derived tissues such as bone, cartilage, blood vessels, and cardiomyocytes. They were first discovered in bone marrow in the 1960s, and then in adipose tissue in the 2000s, both of which are called "mesenchymal stem cells." In recent years, it has been reported that they can also differentiate into ectoderm-derived nerve cells and glial cells (which have functions such as supporting nerve cells), and endoderm-derived liver cells.

[0043] Adipose-derived stem cells (MSCs) are a type of mesenchymal stem cell (MSC) that can be harvested from adipose tissue. Compared to bone marrow-derived MSCs, adipose-derived MSCs have several advantages: they are present in larger quantities in the body, can be harvested in large quantities from adipose tissue throughout the body, produce more growth factors (regeneration-promoting factors) such as HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor) that contribute to organ repair, have high immunosuppressive capacity, and can proliferate without problems even when obtained from the adipose tissue of elderly individuals.

[0044] iPS cells (induced pluripotent stem cells) are cells that have been given pluripotency, which allows them to differentiate into a wide variety of cell types like ES cells (embryonic stem cells), and self-renewal ability, which allows them to maintain this pluripotency even after undergoing cell division and proliferation, by introducing four types of genes into somatic cells.

[0045] The target cells used in this invention include those with lipid bilayer structures, such as red blood cell ghosts and liposomes.

[0046] The target cells used in this invention may be untreated, but in order to improve the efficiency of gene transfer, they may be treated as compliant cells, which are commonly used in gene transfer. Specific examples include compliant cells of E. coli that have been treated with calcium chloride, which alters the structure of the cell membrane and makes it easier to dialyze DNA molecules.

[0047] In the present invention, genes can be introduced into multiple target cells simultaneously. In this case, the multiple target cells may be of the same species or different species. Alternatively, it is preferable that these multiple target cells include different species. Furthermore, it is desirable that these target cells be one or more selected from the group consisting of non-human animal cells, non-human animal individuals, cells collected from human individuals, cells within human individuals, human individuals, plant cells, and microbial cells.

[0048] Target cells may be contained within tissue. In this invention, tissue containing target cells may be referred to as target tissue. Target tissue is not limited to any particular type of tissue. Specific examples of such target tissues include organs from donors used for transplantation, tissues such as skin and tooth roots reconstructed using regenerative medicine methods, and pre-differentiation plant tissues constructed by callus culture. These target tissues may be of a single type or a mixture of two or more types.

[0049] (Plasma irradiation) The target cells are irradiated with plasma. Plasma irradiation activates the target cells' functions. This makes them more receptive to the introduction of the vector and mRNA into the target cells. More specifically, irradiating the target cells with plasma induces endocytosis. As a result, the vector and mRNA are introduced into the target cells, and target cells containing the vector and mRNA are obtained.

[0050] The process of irradiating target cells with plasma means generating plasma in the vicinity of the liquid and the target cells while the vector is in contact with the target cells. The plasma irradiation conditions will be described in detail in the description of the gene transfer apparatus, which will be discussed later.

[0051] The step of irradiating the target cells with plasma may be performed between the step of bringing the vector into contact with the target cells and the step of bringing the mRNA into contact with the target cells.

[0052] Furthermore, the steps of contacting the target cells with the vector, contacting the target cells with mRNA, and irradiating the target cells with plasma may be performed simultaneously. For example, the vector and mRNA may be contacted with the target cells simultaneously, and then the target cells may be irradiated with plasma. Plasma irradiation induces endocytosis of the target cells, and the vector and mRNA are introduced into the target cells. Alternatively, the vector and mRNA may be introduced into the target cells simultaneously by endocytosis of the target cells after the step of irradiating the target cells with plasma is completed.

[0053] The plasma irradiation gene transfer method involves irradiating target cells with discharge plasma to introduce genes into those cells. This method has the advantages of having a low risk of cancer because it does not use viruses, and a low risk of cell dysfunction because it does not use chemicals. Furthermore, because it does not directly pass electric current through the cells, it causes less damage to the cells, resulting in a high survival rate for the cells into which the genes have been introduced.

[0054] In this specification, plasma is defined as a substance that satisfies the following conditions (1), (2), and (3). (1) A collection of charged particles with opposite signs that are nearly electrically neutral as a whole. (2) At least one of the charged particles described in (1) above is undergoing irregular thermal motion. (3) The charged particles described in (1) and (2) above have dimensions greater than the Debye length.

[0055] In a narrow sense, plasma can refer to ionized gas, but in this invention, plasma is not limited to plasma generated by discharge in the gas phase, but also includes plasma generated by discharge in a liquid. Furthermore, "irradiating with plasma" is not limited to directly bringing plasma into contact with the target, but also includes acting on the target with active species generated by the reaction of plasma with the surrounding gas or liquid, or electromagnetic waves such as ultraviolet rays, electrons, or heat generated by the plasma.

[0056] The present invention relates to a method for producing gene-modified cells, which involves using mRNA containing a transposase-encoding sequence and irradiating target cells with plasma. This improves gene transfer efficiency and the survival rate of target cells after gene transfer compared to using a DNA plasmid containing a transposase-encoding gene. More specifically, irradiating target cells with plasma to induce endocytosis makes it easier for target cells to take up extracellular mRNA. Furthermore, mRNA does not require introduction into the nucleus, transcription, or translocation of the mRNA transcript outside the nucleus, as is the case with DNA plasmids. Therefore, it is believed that when mRNA containing a transposase-encoding sequence is taken up into cells, the transposase translation efficiency improves. As a result, it is believed that the efficiency of gene transfer and the survival rate of target cells after gene transfer improve.

[0057] [Gene transfer device] Hereinafter, an embodiment of a gene transfer apparatus that can be suitably used in the method for producing gene-transformed cells of the present invention will be described. The gene transfer apparatus of this embodiment includes a first electrode, a second electrode, and an irradiation field located between the first electrode and the second electrode. The gene transfer device introduces the target gene into target cells. The gene transfer device generates plasma from the first electrode and irradiates the target area where the target cells and the target gene coexist, thereby introducing the target gene into the target cells.

[0058] The gene transfer device 1 shown in Figure 1 comprises a first electrode 10, a second electrode 20, a power supply unit 30, and a container 40.

[0059] The second electrode 20 is spaced apart from the first electrode 10 and is located below the lower end (tip) of the first electrode 10. In other words, the second electrode 20 is located ahead of the tip of the first electrode 10, and the first electrode 10 and the second electrode 20 face each other.

[0060] The power supply unit 30 is connected to the first electrode 10 and the second electrode 20. The container 40 is located between the first electrode 10 and the second electrode 20. The container 40 is positioned opposite the second electrode 20 and spaced apart from the first electrode 10.

[0061] The first electrode 10 comprises an electrode body 12 and an irradiating element 14 provided at the tip of the electrode body 12. In this invention, the first electrode 10 may consist only of the irradiating element 14. In this embodiment, the first electrode 10 is a high-voltage electrode.

[0062] The electrode body 12 is a rod-shaped body extending in one direction. The electrode body 12 may have a hollow structure such as a cylindrical or polygonal tube, or a solid structure such as a cylindrical or polygonal prism. In particular, a solid structure for the electrode body 12 increases durability and makes manufacturing easier. Examples of materials for the electrode body 12 include metals such as stainless steel, copper, and tungsten, and carbon.

[0063] The outer diameter (R12) of the electrode body 12 is, for example, 1 to 50 mm. If the electrode body 12 is polygonal cylindrical or polygonal prism-shaped, the outer diameter R12 is the diameter of the circumscribed circle of the cross-section of the electrode body 12.

[0064] The irradiating body 14 has a flat substrate portion 15 and a wall portion 16 that hangs down from the periphery of the substrate portion 15. The substrate portion 15 and the wall portion 16 are connected. Figure 2 shows the end face of the irradiating body 14 as seen from the lower end (i.e., in the direction of the second electrode 20) of the first electrode 10. As shown in Figure 2, the wall portion 16 is hollow inside and cylindrical with an opening at the lower end (tip). The wall portion 16 is a single cylinder with the tube axis O1 in the direction from the first electrode 10 to the second electrode 20. Note that the wall portion 16 is not limited to a cylindrical shape and may be polygonal. Reference numeral 17 indicates the inner surface of the wall portion 16. Reference numeral 18 indicates the outer surface of the wall portion.

[0065] Examples of materials for the substrate portion 15 include metals such as stainless steel, copper, and tungsten, as well as carbon. Examples of materials for the wall portion 16 include metals such as stainless steel, copper, and tungsten, as well as carbon. The substrate portion 15 and the wall portion 16 may be a single molded product, or they may be individually molded and then joined together.

[0066] If the electrode body 12 has a hollow structure, the substrate portion 15 may or may not have through holes that connect the inside of the electrode body 12 and the inside of the wall portion 16.

[0067] The thickness (outer diameter) R16 of the wall portion 16 can be appropriately determined considering the size of the container 40, etc. The outer diameter R16 is preferably more than 1 mm, more preferably 3 to 50 mm, and even more preferably 5 to 10 mm. If the outer diameter R16 is above the lower limit, the plasma can be irradiated over a wider area, further increasing the efficiency of introducing the target gene into the target cells (hereinafter sometimes simply referred to as "introduction efficiency"). If the outer diameter R16 is below the upper limit, it becomes easier to generate a stable discharge.

[0068] Furthermore, it is preferable that the outer diameter R16 is larger than the outer diameter R12. A larger outer diameter R16 allows for plasma irradiation over a wider area, further increasing the introduction efficiency. When the wall portion 16 is polygonal cylindrical, the outer diameter R16 is the diameter of the circumscribed circle of the cross-section of the wall portion 16. When the irradiating body 14 has two or more wall portions 16, the outer diameter R16 of the wall portion 16 is the outer diameter of the outermost wall portion 16.

[0069] The inner diameter r16 of the wall portion 16 is preferably 0.1 mm or more. If the wall portion 16 is polygonal cylindrical, the inner diameter r16 is the diameter of the inscribed circle of the cross-section of the wall portion 16. If the irradiating body 14 has two or more wall portions 16, the inner diameter r16 of the wall portion 16 is the inner diameter of the innermost wall portion 16.

[0070] The angle θ1 between the lower end surface of the wall portion 16 and the outer surface of the wall portion 16 is preferably 60° or less, more preferably 30° or less, and even more preferably 10° or less. The intersection point between the lower end surface of the wall portion 16 and the outer surface of the wall portion 16 is a so-called edge. When the angle θ1 is below the above upper limit, the electric field concentrates at the edge, making it easier to generate plasma between the electrodes.

[0071] The angle between the lower end surface of the wall portion 16 and the inner surface of the wall portion 16 is the same as angle θ1. The angle between the lower end surface of the wall portion 16 and the inner surface of the wall portion 16 may be the same as angle θ1 or may be different.

[0072] The thickness t16 of the wall portion 16 is preferably 1 mm or less, and more preferably 0.1 mm or less. When the thickness t16 is below the above upper limit, the strength of the electric field at the end can be further increased.

[0073] The height h16 of the wall portion 16 is not particularly limited, but is preferably 1 to 10 mm.

[0074] The second electrode 20 can function as a ground electrode, for example, a flat plate-shaped electrode. Examples of materials for the second electrode 20 include metals such as stainless steel, copper, and tungsten, and carbon.

[0075] In a plan view, the area of ​​the second electrode 20 is larger than the area of ​​the first electrode 10. A larger area for the second electrode 20 allows the plasma to be irradiated over a wider area from the first electrode 10 onto the target object.

[0076] The power supply unit 30 only needs to be able to generate plasma between the first electrode 10 and the second electrode by applying a voltage between the two electrodes. The power supply unit 30 switches the start or stop of the supply of electricity to the electrodes. The power supply unit 30 also adjusts the voltage and frequency applied to the electrodes. Examples of the power supply unit 30 include a circuit that is connected to an external power source and includes an inverter. Another example of the power supply unit 30 is a secondary battery.

[0077] In this embodiment, the container 40 functions as an irradiation field. The irradiation field can be any location where the irradiated object is positioned in the gene transfer cell manufacturing method of the present invention. Therefore, if the container 40 is not part of the configuration of the gene transfer device 1, the second electrode 20 on which the container 40 is placed may be used as the irradiation field.

[0078] The container 40 only needs to be able to accommodate the object to be irradiated, and examples include the wells of a microplate. The inner bottom surface of the container 40 is spaced apart from the tip of the first electrode 10 (i.e., the irradiating body 14).

[0079] The material of the container 40 may be a conductive material or an insulating material. However, an insulating material is preferred from the viewpoint of preventing localized discharge due to high voltage. Examples of insulating materials include resins, ceramics, and glass. Examples of resins include polystyrene, polyolefins, and polyesters.

[0080] The gene transfer device may have a control unit 50. The control unit 50 controls the power supply unit 30 and a liquid addition unit (not shown) by computer. The computer of the control unit 50 includes an arithmetic unit (CPU), main memory, storage, and input / output (I / O) circuits. It reads programs recorded in storage and uses memory as a working area to perform processing by the CPU.

[0081] The control unit 50 controls the liquid injection step, which causes the liquid addition unit to inject a liquid containing a vector or a liquid containing mRNA into the container 40; the voltage application step, which causes the power supply unit 30 to apply a voltage between the first electrode 10 and the second electrode 20; and the timing of the execution of the liquid injection step and the voltage application step.

[0082] The liquid addition unit injects a liquid containing a vector or a liquid containing mRNA into the container 40 based on a command from the control unit 50.

[0083] The gene transfer apparatus 1 of this embodiment may further include an image acquisition unit (not shown) that acquires images of target cells in the container 40. In this case, the control unit 50 analyzes the image data from the image acquisition unit and controls the execution timing of a liquid injection step in which the liquid addition unit injects a liquid containing a vector or a liquid containing mRNA into the container 40, a voltage application step in which the power supply unit 30 applies a voltage between the first electrode 10 and the second electrode 20, and the execution timing of the liquid injection step and the voltage application step.

[0084] In the method for producing gene-transformed cells of the present invention, plasma is irradiated into a container 40 while the target cells 44 and the liquid 42 containing the vector are in contact. The vector is introduced into the target cells 44 through plasma irradiation. Alternatively, in the introduction step, the plasma may be irradiated into the container 40 while the target cells 44, the vector, and the liquid 42 containing mRNA are in contact, thereby introducing the vector and mRNA into the target cells 44.

[0085] During plasma irradiation, power is supplied from the power supply unit 30 to the first electrode 10, and a voltage is applied between the first electrode 10 and the second electrode 20. When a voltage is applied between the electrodes, an electric field concentration occurs at the lower edge of the wall portion 16, and plasma is generated from the electric field concentration point toward the second electrode 20. At this time, because the wall portion 16 is cylindrical, the plasma can be irradiated over a wide area of ​​the target. By irradiating a wide area with plasma, the vector and mRNA can be introduced into target cells over a wide area of ​​the target with a single plasma irradiation. Therefore, the amount introduced per unit time can be increased, and the introduction efficiency can be further improved.

[0086] The distance L40 from the lower end of the first electrode 10 (the lower end surface of the wall portion 16) to the irradiation target (the liquid in which the vector or the vector and mRNA and target cells etc. 44 coexist) is preferably 0.1 to 5 mm. Plasma can be generated more stably when the distance L40 is within the above range.

[0087] The AC voltage applied between the first electrode 10 and the second electrode 20 is preferably 1 to 30 kVpp, and more preferably 5 to 20 kVpp. Setting the AC voltage above the lower limit further increases the introduction efficiency. Setting the AC voltage below the upper limit further reduces damage to target cells, etc. The unit "Vpp (Volt peak to peak)" representing AC voltage is the potential difference between the highest and lowest values ​​of the AC voltage waveform.

[0088] The frequency of the alternating current applied between the first electrode 10 and the second electrode 20 is preferably 1 to 500 kHz, and more preferably 10 to 200 kHz. Setting the frequency above the lower limit further increases the introduction efficiency. Setting the frequency below the upper limit further reduces damage to target cells, etc.

[0089] In the process of irradiating target cells with plasma, the plasma irradiation time is preferably, for example, 10 nanoseconds to 1 second, and more preferably 100 nanoseconds to 0.1 seconds. If the irradiation time is above the lower limit, the amount of vector (or vector and mRNA) introduced into the target cells, etc. 44 can be increased. If the irradiation time is below the upper limit, damage to the target cells, etc. can be reduced.

[0090] In the irradiation process, the environment inside the container 40 is not particularly limited, but may be, for example, in the presence of CO2. The CO2 concentration is, for example, 1 to 10 volume percent.

[0091] [Effects and Effects] The present invention relates to a method for producing gene-modified cells, which involves using mRNA containing a transposase-coding sequence and irradiating target cells with plasma. This improves gene transfer efficiency and the survival rate of target cells after gene transfer compared to using a DNA plasmid containing a transposase-coding gene. More specifically, irradiating target cells with plasma to induce endocytosis makes it easier for target cells to take up extracellular mRNA. Furthermore, mRNA does not require introduction into the nucleus, transcription, or translocation of the transcript outside the nucleus, as is the case with DNA plasmids. Therefore, it is believed that when mRNA containing a transposase-coding sequence is taken up into cells, the transposase translation efficiency improves. As a result, it is believed that the efficiency of gene transfer and the survival rate of target cells after gene transfer improve. [Examples]

[0092] The present invention will be explained in more detail below with experimental examples. However, the examples described below are not intended to limit the present invention.

[0093] [Experimental Example 1] Experimental Example 1-1 is an example, and Experimental Examples 1-2 to 1-6 are comparative examples.

[0094] <Molecular introduction device> Plasma irradiation was performed using a molecular introduction apparatus, the overview of which is shown in Figure 1. First electrode: One end of a 3mm diameter stainless steel cylinder was covered with a 0.5mm thick alumina cap. A 0.1m diameter through-hole was created on the surface of the alumina cap facing the second electrode by laser processing to create the first electrode. Second electrode: 3mm thick, copper plate. Power frequency: 50kHz Discharge voltage: 16kVp-p Discharge time: 80ms Discharge points per well: 1 point in the center Distance between the end of the first electrode and the top surface of the container: 1.0 mm

[0095] <material> The following materials will be used. Target cells: Jurkat cells (obtained from ATCC) Transposon vector: A vector pEF1α (created by VectorBuilder) in which a fragment (SEQ ID NO: 2) containing the target gene, the eGFP gene, is inserted between ITR sequences. (pEF1α-ITR-eGFP) mRNA: mRNA containing a transposase-coding sequence (SEQ ID NO: 1, Vector Builder Inc., hyPBase mRNA) Transposase expression plasmid: Transposase expression helper plasmid (VectorBuilder, product name pCMV-PBase 5957bp) Culture medium: RPMI1640 (Gibco) Container: 48-well plate (manufactured by Corning, product name Falcon®)

[0096] <Experimental Procedure> [Experimental Example 1-1] Each well of a 48-well plate contains 2 × 10⁶ Jurkat cells. 6 Cells were seeded at a rate of 0.4 mL per well and cultured in RPMI1640 medium in a CO2 incubator for 48 hours until plasma irradiation (37°C, 5 vol% CO2 concentration). Before plasma irradiation, the RPMI1640 medium and supernatant were removed, and 4 μL of nucleic acid solution containing the transposon vector pEF1α-ITR-eGFP and mRNA containing the transposase-encoding sequence was added to each well (each well contained 2 μg of the transposon vector pEF1α-ITR-eGFP and 2 μg of mRNA). The Jurkat cells were then brought into contact with the transposon vector pEF1α-ITR-eGFP and mRNA containing the transposase-encoding sequence, and allowed to stand for 3 minutes.

[0097] Subsequently, plasma irradiation was performed using a molecular delivery device (discharge time 80 ms) to introduce the transposon vector pEF1α-ITR-eGFP and mRNA containing the transposase-encoding sequence into Jurkat cells. 0.4 mL of recovery medium was added to each well, and the cells were cultured in a CO2 incubator (37°C, 5 vol% CO2 concentration).

[0098] On days 1 and 12 after molecular transduction, the total cell volume (All Events) was measured using a flow cytometer (Thermofisher). Cells were cultured on the same day, and from day 5 onward, the cells were diluted to 1 / 8 and cultured continuously.

[0099] [Experimental Example 1-2] The procedure was the same as in Experimental Example 1-1, except that the transposase expression helper plasmid pCMV-PBase was used instead of mRNA containing the transposase-coding sequence.

[0100] [Experimental Examples 1-3] The procedure was the same as in Experimental Example 1-1, except that mRNA containing a transposase-coding sequence was not used.

[0101] [Experimental Examples 1-4] The procedure was the same as in Experimental Example 1-1, except that a circular vector (pCMV-eGFP, SEQ ID NO: 3) containing a fragment of the eGFP gene sequence but without the ITR sequence was used instead of the transposon vector pEF1α-ITR-eGFP.

[0102] [Experimental Examples 1-5] The procedure was the same as in Experimental Examples 1-4, except that the transposase expression helper plasmid pCMV-PBase was used instead of mRNA containing the transposase-coding sequence.

[0103] [Experimental Examples 1-6] The procedure was the same as in Experimental Examples 1-4, except that mRNA containing a transposase-coding sequence was not used.

[0104] Figure 3 shows the results of flow cytometry measurements of total cell volume (All Events) for each gene-transformed cell in Experimental Examples 1-1 to 1-3 on day 1 and day 12 after molecular transduction. Figure 4 shows the results of flow cytometry measurements of total cell volume (All Events) for each gene-transformed cell in Experimental Examples 1-4 to 1-6 on day 1 and day 12 after molecular transduction. In Figures 3 and 4, dots in the area enclosed by the dashed line + / +(1) indicate living cells. On day 1 after molecular transduction, living cells were confirmed in all of Experimental Examples 1-1 to 1-6, but on day 12 after molecular transduction, Experimental Example 1-1 showed a larger number of living cells compared to Experimental Examples 1-2 to 1-6. In other words, it was shown that gene transduction into target cells using the transposon method with mRNA containing a transposase-coding sequence improves the efficiency of target gene transduction. [Explanation of Symbols]

[0105] 1. Molecular introduction device, 10. First electrode, 12. Electrode body, 14. Irradiator, 16. Wall section, 20. Second electrode, 30. Power supply section, 40. Container, 42. Molecules, 44. Target cells, O1. Tube axis, θ1. Angle

Claims

1. The process involves contacting target cells with a vector containing the target gene sequence, The process involves contacting the target cells with mRNA containing a sequence encoding a transposase for introducing the target gene sequence into the genomic DNA of the target cells, Irradiating the target cells with plasma, A method for producing genetically modified cells, comprising obtaining genetically modified cells into which the vector and the mRNA have been introduced.

2. A method for producing gene-transformed cells according to claim 1, wherein the target cells are irradiated with plasma between contacting the vector with the target cells and contacting the mRNA with the target cells.

3. The method for producing gene-transformed cells according to claim 1, wherein contact with the target cells of the vector is performed simultaneously with contact with the target cells of the mRNA.

4. A method for producing gene-transformed cells according to claim 1, wherein the vector is introduced into the target cells, the mRNA is introduced into the target cells, and the target cells are irradiated with plasma, all of which are performed simultaneously.

5. The method for producing gene-transformed cells according to any one of claims 1 to 4, wherein the vector is a modified transposon vector into which the nucleic acid fragment of (1) or (2) below is inserted. (1) The target gene sequence is located between the 5' reversed repeat sequence and the 3' reversed repeat sequence of the transposon gene. (2) The transposon gene has a 5' inverted repeat sequence / series repeat sequence and a 3' inverted repeat sequence / series repeat sequence, and the target gene sequence is located between these sequences.

6. The method for producing gene-transformed cells according to claim 5, wherein the vector is a modified transposon vector in which, in addition to (1) or (2), the nucleic acid fragment described in (3) below is further inserted. (3) A restriction enzyme recognition site or exogenous gene expression cassette located between the 5' reversed repeat sequence and the 3' reversed repeat sequence of the transposon gene, or between the 5' reversed repeat sequence / series repeat sequence and the 3' reversed repeat sequence / series repeat sequence.

7. A method for producing gene-transformed cells according to any one of claims 1 to 4, wherein the molar ratio of the amount of vector to the amount of mRNA to be brought into contact with the target cells is vector:mRNA = 1:0.1 to 1:

10.

8. A method for producing gene-transformed cells according to any one of claims 1 to 4, wherein the target cell is at least one selected from the group consisting of blood-derived cells, adherent cells, and iPS cells.

9. A method for producing gene-transformed cells according to any one of claims 1 to 4, wherein the target cells are blood-derived cells including CD3-positive cells.

10. A method for producing genetically modified cells according to claim 9, wherein the target gene sequence includes a gene sequence encoding a chimeric antigen receptor.

11. The method for producing genetically modified cells according to claim 9, wherein the CD3-positive cells include T cells.

12. The method for producing gene-transformed cells according to claim 8, wherein the adherent cells include HEK293F cells, mesenchymal stem cells, and adipose-derived stem cells.

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