Method for producing molecularly introduced cells

Abstract

This invention provides a method for producing cells into which a nucleic acid molecule encoding a target protein is introduced, thereby increasing the expression level of the target protein within those target cells. [Solution] A method for producing molecularly introduced cells in which a target protein is expressed by introducing a nucleic acid molecule encoding the target protein into the target cells, comprising a molecular introduction step of introducing the nucleic acid molecule and at least one of the first siRNA and the second siRNA into the target cells by contacting the target cells with a liquid containing at least one of a first siRNA that reduces the expression level of Dom34 which may be endogenous in the target cells and a second siRNA that reduces the expression level of SAMHD1 which may be endogenous in the target cells, and the nucleic acid molecule, and then irradiating the target cells with plasma.

Description

Technical Field

[0001] The present invention relates to a method for producing a molecule-introduced cell using plasma irradiation treatment.

Background Art

[0002] In recent years, in the research and development of pharmaceuticals, there has been an increasing need to introduce various molecules into target cells for testing. The molecules to be introduced are, for example, nucleic acid molecules encoding genes, proteins involved in signal transduction and transcriptional regulation, physiologically active substances, drug candidates, and the like.

[0003] Examples of methods for introducing molecules into target cells include chemical methods, physical methods, and biological methods. The chemical method is a method of causing endocytosis to take up molecules into target cells using transfection reagents such as cationic polymers, cationic lipids, or calcium phosphate. The physical method is a method of directly introducing molecules into target cells by physical operations such as sonoporation, laser irradiation, or electroporation. The biological method is a method of using viral vectors such as retroviruses, lentiviruses, adeno-associated viruses (AAVs), or adenoviruses to transfer the nucleic acid in the virus particles into target cells by the infectivity of the virus.

[0004] By the way, naturally occurring pathogenic viruses have a mechanism for introducing a nucleic acid molecule encoding a gene required for virus replication into a host cell. On the other hand, host cells have the SAMHD1 protein, which is an endogenous host restriction factor, to counter virus infection. In Non-Patent Document 1, it has been reported that Vpx possessed by human immunodeficiency virus (HIV) is phosphorylated and activated by host PIM kinase and degrades SAMHD1.

[0005] When a pathogenic virus introduces single-stranded RNA into a host cell, the 2',5'-oligoadenylate synthetase (OAS) / RNaseL pathway, one of the host cell's innate immune systems, acts as an immune mechanism. Non-patent document 2 reports that the Dom34 protein forms a complex with RNaseL, recognizing and eliminating exogenous RNA. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Dom34 mediates targeting of exogenous RNA in the antiviral OAS / RNase L pathway, Nucleic Acids Research, Volume 47, Issue 1, 2019, Pages 432-449 [Non-Patent Document 2] PIM kinases facilitate lentiviral evasion from SAMHD1 restriction via Vpx phosphorylation. Nat Commun 10, 1844 (2019). [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The inventors hypothesized that when producing target cells expressing the target protein encoded by a nucleic acid molecule by introducing a nucleic acid molecule into target cells, the expression level of the target protein may be reduced due to the influence of SAMHD1 or Dom34, which are endogenous in the target cells. Therefore, the inventors conceived the idea that it may be possible to increase the expression level of the target protein expressed in target cells by eliminating the influence of SAMHD1 or Dom34, and thus completed the present invention.

[0008] The present invention provides a method for producing molecularly introduced cells, which allows for the introduction of nucleic acid molecules encoding a target protein into target cells and the increase in the expression level of the target protein within those target cells. [Means for solving the problem]

[0009] The present invention encompasses the following embodiments. [1] A method for producing molecularly introduced cells in which a target protein is expressed in target cells by introducing a nucleic acid molecule encoding the target protein into the target cells, comprising a molecular introduction step of introducing the nucleic acid molecule and at least one of the first siRNA and the second siRNA into the target cells by contacting the target cells with a liquid containing at least one of a first siRNA that reduces the expression level of Dom34 which may be endogenous in the target cells and a second siRNA that reduces the expression level of SAMHD1 which may be endogenous in the target cells, and the nucleic acid molecule, into the target cells by irradiating the target cells with plasma. [2] A method for producing molecularly introduced cells in which a target protein is expressed in target cells by introducing a nucleic acid molecule encoding the target protein into the target cells, comprising: a first molecular introduction step in which the target cells are brought into contact with a pretreatment solution containing at least one of a first siRNA that reduces the expression level of Dom34 which may be endogenous in the target cells, and a second siRNA that reduces the expression level of SAMHD1 which may be endogenous in the target cells, and then the target cells are irradiated with plasma to introduce at least one of the first siRNA and the second siRNA into the target cells; and a second molecular introduction step in which the target cells that have undergone the first molecular introduction step are brought into contact with a liquid containing the nucleic acid molecule, and then the target cells are irradiated with plasma to introduce the nucleic acid molecule into the target cells. [3] The method for producing a molecule-transformed cell according to [1] or [2], wherein the nucleic acid molecule is mRNA encoding the target protein. [4] A method for producing molecularly introduced cells according to any one of [1] to [3], wherein the target cell is at least one selected from the group consisting of blood-derived cells, adherent cells, and iPS cells. [5] A method for producing molecularly introduced cells according to any one of [1] to [4], wherein the target cells are blood-derived cells including CD3-positive cells. [6] The method for producing molecularly introduced cells according to [5], wherein the CD3-positive cells include T cells. [7] A method for producing molecularly introduced cells according to any one of [1] to [6], wherein the target cells are adherent cells, and the adherent cells include HEK293F cells, mesenchymal stem cells, adipose-derived stem cells, or hematopoietic stem cells. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a method for producing molecularly introduced cells in which a nucleic acid molecule encoding a target protein is introduced into target cells, thereby increasing the expression level of the target protein within those target cells. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a cross-sectional view of an example of a molecular introduction device that can be used in the present invention. [Figure 2] Figure 2 is an end view of the irradiating body 14 shown in Figure 1. [Modes for carrying out the invention]

[0012] In this invention, the "~" indicating a numerical range means that the numerical values ​​written before and after it are included as the lower limit and upper limit, respectively.

[0013] ≪Method for producing molecularly introduced cells≫ <First aspect> A first aspect of the present invention is a method for producing molecule-transformed cells in which a target protein is expressed by introducing a nucleic acid molecule encoding the target protein into the target cells. This aspect preferably comprises the following molecule introduction steps.

[0014] In the molecular introduction step of this embodiment, after contacting a liquid containing at least one of a first siRNA that reduces the expression level of Dom34 that may be endogenous to the target cell and a second siRNA that reduces the expression level of SAMHD1 that may be endogenous to the target cell, and the nucleic acid molecule, with the target cell, plasma is irradiated to the target cell, thereby introducing the nucleic acid molecule and at least one of the first siRNA and the second siRNA into the target cell.

[0015] The nucleic acid molecule encoding the target protein may be in a form in which the target protein is expressed in the cell, and may be any of DNA, RNA, and artificial nucleic acid molecules derived therefrom. The nucleic acid molecule is a polynucleotide formed by linking a plurality of nucleotides. From the viewpoint of rapidly expressing the target protein, RNA is preferred, and mRNA is more preferred.

[0016] When using mRNA, in addition to the region encoding the target protein, from the viewpoint of enhancing the expression efficiency, it preferably has a cap structure and a polyA tail. The nucleotides constituting the mRNA may be subject to known chemical modifications.

[0017] When using DNA, from the viewpoint of high efficiency of conversion to mRNA in the cell, cDNA is preferred. In addition to the region encoding the target protein, from the viewpoint of enhancing the expression efficiency, cDNA preferably has a promoter that operates in the target cell to be introduced and preferably has a polyA signal.

[0018] The target protein encoded by the nucleic acid molecule introduced into the target cell is expressed by a known protein expression system including ribosomes in the target cell. In this embodiment, the target cell in which the target protein is expressed is referred to as a molecular introduction cell.

[0019] The target protein is not particularly limited as long as it can be expressed in the target cells, and it may be a protein that functions in the cytoplasm, a membrane protein that moves to the cell membrane and functions, or a secreted protein that is secreted outside the cell. Specifically, for example, fluorescent proteins such as green fluorescent protein (GFP), yellow fluorescent protein, red fluorescent protein, and blue fluorescent protein, proteins containing protein tags such as His tag and epitope tag, and CAR (chimeric antigen receptor) can be mentioned.

[0020] In this embodiment, from the viewpoint of increasing the expression level of the target protein, at least one of the first siRNA that reduces the expression level of Dom34 that can be endogenous in the target cell and the second siRNA that reduces the expression level of SAMHD1 that can be endogenous in the target cell is introduced into the target cell.

[0021] The first or second siRNA used in this embodiment is preferably a double-stranded RNA (dsRNA) of 21 to 25 bp with a dinucleotide 3' overhang. The first or second siRNA used in this embodiment assumes that long dsRNA is cleaved by Dicer in the RNA interference pathway in the target cell, and may be, for example, long dsRNA of about 20 to 1000 bp.

[0022] Dom34 is a protein known to form a Dom34-Hbs1 complex in the cell, and this complex shows homology with the translation termination factor eRF1:eRF3 complex. The Dom34 targeted by this embodiment is a Dom34 that can be endogenously expressed in the target cell. When the target cell is a cell derived from a human, the amino acid sequence of Dom34 preferably has at least 80%, preferably 90% or more, more preferably 95% or more, and still more preferably 99% or more sequence identity to the sequence number 1 registered in a known database (database name: NCBI, accession number: NP_057030.3), or preferably contains a fragment in which a part thereof is deleted or missing. The Dom34 targeted by this embodiment preferably exhibits the above-described known functions.

[0023] The base sequence of the first siRNA used in this embodiment is any base sequence that has the function of reducing the expression level of Dom34 which may be endogenous in target cells by RNA interference (RNAi). The base sequence can be designed by a method well known to those skilled in the art based on the amino acid sequence of Dom34. As an example, the first siRNA has the base sequence of SEQ ID NO: 2. As an example of the base sequence of the first siRNA used in this embodiment, for example, one that has sequence identity of at least 80%, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more with respect to SEQ ID NO: 2.

[0024] SAMHD1 is a type of enzyme produced by human cells and is known to inhibit HIV (human immunodeficiency virus) infection of macrophages and CD4-positive T cells. The SAMHD1 targeted in this embodiment is SAMHD1 that can be endogenously expressed in target cells. When the target cells are derived from humans, the amino acid sequence of SAMHD1 is preferably a sequence that has at least 80%, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more sequence identity with SEQ ID NO: 3 (database name: HGNC, accession number: 15925) registered in a known database, or contains a fragment in which a part of it has been deleted or removed. The SAMHD1 targeted in this embodiment preferably exhibits the known functions described above.

[0025] The base sequence of the second siRNA used in this embodiment is any base sequence that has the function of reducing the expression level of SAMHD1 which may be endogenous in target cells by RNA interference. The base sequence can be designed by a method well known to those skilled in the art based on the amino acid sequence of SAMHD1. As an example, a second siRNA having the base sequence of SEQ ID NO: 4 can be cited. Examples of the base sequence of the second siRNA used in this embodiment include one having at least 80%, preferably 90% or more, more preferably 95% or more, sequence identity with SEQ ID NO: 4.

[0026] In this embodiment, the method for contacting target cells with a liquid containing at least one of the first and second siRNAs and a nucleic acid molecule encoding the target protein (hereinafter sometimes referred to as "nucleic acid solution") is not particularly limited. By increasing the contact efficiency between the nucleic acid solution and the target cells, the efficiency of molecule introduction can be increased. From this viewpoint, it is preferable that the concentration of siRNA and nucleic acid molecules in the nucleic acid solution is sufficiently high when it is in contact with the target cells. For example, if the target cells have been pre-cultured before contacting the nucleic acid solution with the target cells, a method can be used in which the medium used for pre-culture is removed and then the nucleic acid solution is dropped onto the surface of the target cells. By dropping the nucleic acid solution into the medium in the presence of as little medium as possible so as not to dry out the target cells, it is possible to contact the target cells with a sufficiently high concentration of nucleic acid solution while preventing the target cells from drying out.

[0027] The components contained in the nucleic acid solution include at least one of the first and second siRNAs, a nucleic acid molecule encoding the target protein, a solvent, and optional components. Examples of solvents include water and DMSO. Examples of optional components include pH buffers and salts. The solvent and optional components in the nucleic acid solution are preferably selected considering good solubility of the nucleic acid molecule and low toxicity to target cells.

[0028] The molar ratio of the first and second siRNAs to the nucleic acid molecule encoding the target protein that is brought into contact with the target cells is not particularly limited and can be, for example, in the range of (total moles of siRNA / moles of nucleic acid molecule) = 0.1 to 10.

[0029] (target cell) In this embodiment, target cells refer to cells that are targeted to express the target protein. Specific examples of 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. A single molecular introduction treatment may target one type of target cell or two or more types.

[0030] Cells taken from human organisms 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. The aforementioned cells taken from human organisms also include cells cultured from cells taken from human organisms.

[0031] 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.

[0032] In this embodiment, the target cells are preferably at least one selected from the group consisting of blood-derived cells, adherent cells, and iPS cells.

[0033] Blood-derived cells include, for example, T cells. Adherent cells include, for example, HEK293 cells, mesenchymal stem cells, adipose-derived stem cells, and hematopoietic stem cells.

[0034] T cells are a type of lymphocyte that differentiates and matures from progenitor cells produced in the bone marrow through selection in the thymus, and they make up 70-80% of lymphocytes in peripheral blood. T cells have a T cell receptor (TCR) on their cell surface. When using T cells as target cells, not only T cells collected from peripheral blood but also cultured T cells may be used. T cells are included in CD3-positive cells. In addition to T cells, Jurkat cells are another example of CD3-positive cells.

[0035] 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 as a host for recombinant protein production, recombinant adenovirus creation, and amplification. HEK293F cells are a commercially available product.

[0036] Mesenchymal stem cells (MSCs) are a type of stem cell found in the body that have the ability to differentiate into mesoderm-derived tissues such as bone, cartilage, blood vessels, and cardiomyocytes. They have also been reported to be able to differentiate into ectoderm-derived nerve cells and glial cells, and endoderm-derived liver cells.

[0037] Adipose-derived stem cells (MSCs) are a type of mesenchymal stem cell and can be harvested from adipose tissue. Adipose-derived MSCs are present in higher concentrations in the body compared to bone marrow-derived MSCs. Therefore, large quantities of MSCs can be harvested from adipose tissue throughout the body. MSCs have several advantageous characteristics: 1) they produce many growth factors such as hepatocyte growth factor and vascular endothelial growth factor, which contribute to organ repair; 2) they have high immunosuppressive capacity; and 3) they can proliferate without problems even when obtained from the adipose tissue of elderly individuals.

[0038] Hematopoietic stem cells are stem cells that have the ability to differentiate into red blood cells, white blood cells, and platelets.

[0039] iPS cells (induced pluripotent stem cells) are cells that have been given pluripotency, the ability to differentiate into a variety of cell types like ES cells (embryonic stem cells), and the ability to self-renew, which allows them to maintain this differentiation even after cell division and proliferation, by introducing four types of genes into somatic cells.

[0040] The target cells used in this embodiment may be artificially formed cells having a lipid bilayer structure, such as erythrocyte ghosts or liposomes, but it is preferable that the target cells have a protein expression system and that they may contain Dom34 or SAMHD1.

[0041] According to the manufacturing method of this embodiment, siRNA and nucleic acid molecules can be introduced into multiple target cells simultaneously. The multiple target cells may be cells of the same species or cells of different species.

[0042] The target cells used in this embodiment may be contained in tissue. The tissue containing the target cells is not particularly limited and includes, for example, organs for organ transplantation, tissues such as skin and tooth roots reconstructed in regenerative medicine, and pre-differentiation plant tissues constructed by callus culture.

[0043] (Plasma irradiation) Target cells are irradiated with plasma while in contact with a nucleic acid solution. When the target cells are irradiated with plasma, they are activated, and siRNA and nucleic acid molecules are introduced into the target cells. Endocytosis is thought to occur at this time. The first and second siRNAs introduced into the target cells independently reduce the expression levels of Dom34 and SAMHD1, which may be endogenous within the target cells, through RNA interference within the target cells. Dom34 and SAMHD1 are thought to have the function of degrading foreign RNA that has entered the target cells. By reducing the expression levels of these proteins, the efficiency of the nucleic acid encoding the target protein introduced by plasma irradiation in expressing the target protein via ribosomes in the target cells can be increased.

[0044] In this embodiment, irradiating target cells with plasma means generating plasma in the vicinity of the nucleic acid solution and the target cells while the nucleic acid solution is in contact with the target cells. From the viewpoint of ensuring that the plasma sufficiently activates the target cells, it is preferable that the amount of liquid present between the plasma generation space and the target cells be small enough to prevent the target cells from drying out. The plasma irradiation conditions will be described in detail in the description of the molecular introduction device, which will be discussed later.

[0045] In this embodiment, the nucleic acid solution brought into contact with target cells contains at least one of the following siRNAs: a first siRNA that reduces the expression level of Dom34, a second siRNA that reduces the expression level of SAMHD1, and a nucleic acid molecule encoding the target protein. It is preferable that these are simultaneously introduced into the target cells via endocytosis through a single plasma irradiation treatment. Completing molecular introduction in a single plasma irradiation treatment reduces the burden on target cells compared to two or more plasma irradiation treatments, thereby increasing the expression level of the target protein within the target cells.

[0046] The molecular introduction method by plasma irradiation in this embodiment is a technique that introduces nucleic acid molecules into target cells by irradiating them with discharge plasma. This method has the advantage 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 an electric current through the cells, it causes less damage to the cells, and the survival rate of cells into which nucleic acid molecules have been introduced is high.

[0047] 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.

[0048] While "plasma" in a narrow sense sometimes refers to ionized gas, the plasma in this invention 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.

[0049] [Molecular introduction device] Below, one embodiment of a molecular introduction apparatus that can be used in the method for producing molecularly introduced cells according to this embodiment will be described. The molecular introduction 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 molecular delivery device can introduce desired nucleic acid molecules (nucleic acid molecules in a general sense, including siRNA used in this embodiment) into target cells. The molecular delivery device generates plasma from the first electrode and irradiates the irradiated object where the target cells and nucleic acid molecules coexist, thereby introducing the nucleic acid molecules into the target cells.

[0050] The molecular introduction device 1 shown in Figure 1 comprises a first electrode 10, a second electrode 20, a power supply unit 30, and a container 40.

[0051] 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.

[0052] 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.

[0053] The first electrode 10 comprises an electrode body 12 and an irradiating element 14 provided at the tip of the electrode body 12. In contrast to the embodiment illustrated in Figure 1, the first electrode 10 may consist only of the irradiating element 14. In this embodiment, the first electrode 10 is a high-voltage electrode.

[0054] 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.

[0055] 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.

[0056] 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. The shape of the wall portion 16 is not limited to the cylindrical shape exemplified in Figure 1, but may also 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.

[0057] 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.

[0058] 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.

[0059] 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 nucleic acid molecules into 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.

[0060] The outer diameter R16 is preferably larger than the outer diameter R12. A larger outer diameter R16 allows for plasma irradiation over a wider area, further increasing the introduction efficiency. If 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. If 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.

[0061] 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.

[0062] 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 less than or equal to the above upper limit, the electric field concentrates at the edge, making it easier to generate plasma between the electrodes.

[0063] The angle between the lower end surface of the wall portion 16 and the inner surface of the wall portion 16 can be 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.

[0064] 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.

[0065] The height h16 of the wall portion 16 is not particularly limited, but is preferably 1 to 10 mm.

[0066] 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.

[0067] In a plan view, it is preferable that the area of ​​the second electrode 20 is larger than the area of ​​the first electrode 10. A larger area of ​​the second electrode 20 allows the plasma to be irradiated over a wider area from the first electrode 10 onto the target object.

[0068] The power supply unit 30 only needs to be able to generate plasma between the first electrode 10 and the second electrode 20 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.

[0069] The container 40 functions as an irradiation field. In the method for producing molecularly introduced cells according to the present invention, the irradiation field can be any location that holds the irradiated object in a desired position. For this reason, if the container 40 is not part of the configuration of the molecular introduction device 1, the second electrode 20 on which the container 40 is placed may be used as the irradiation field.

[0070] The container 40 only needs to be able to accommodate the object to be irradiated, and can be exemplified by the multiple 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).

[0071] 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.

[0072] The molecular introduction device 1 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 area, and input / output (I / O) circuits. It reads programs recorded in the storage and uses the memory as a working area to perform processing by the CPU.

[0073] The control unit 50 controls the liquid injection step, which causes the liquid addition unit to inject nucleic acid solution 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.

[0074] The liquid addition unit injects the nucleic acid solution into the container 40 based on a command from the control unit 50.

[0075] The molecular introduction device 1 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 liquid injection step, which causes the liquid addition unit to inject nucleic acid solution 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.

[0076] 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, desired nucleic acid molecules 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.

[0077] The distance L40 from the lower end of the first electrode 10 (the lower end surface of the wall portion 16) to the irradiated object (the nucleic acid solution 42 and target cells 44) is preferably 0.1 to 5 mm. Plasma can be generated more stably when the distance L40 is within the above range.

[0078] 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.

[0079] 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.

[0080] 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 nucleic acid molecules introduced into the target cells 44 can be increased. If the irradiation time is below the upper limit, damage to the target cells 44 can be reduced.

[0081] During 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.

[0082] [Effects and Effects] The present invention's method for producing molecules-introduced cells involves introducing not only a nucleic acid molecule encoding the target protein, but also at least one of two siRNAs into the target cells: a first siRNA that reduces the expression level of Dom34, which may be endogenous in the target cells, and a second siRNA that reduces the expression level of SAMHD1, which may be endogenous in the target cells. This is thought to suppress the degradation of the introduced mRNA or mRNA obtained by transcription of the introduced DNA within the target cells by the action of Dom34 or SAMHD1. As a result, the expression level of the target protein within the target cells is improved. However, the mechanism by which the effects of the present invention are obtained is not limited to the above description.

[0083] <Second aspect> A second aspect of the present invention is a method for producing molecules that express the target protein in target cells by introducing a nucleic acid molecule encoding the target protein into the target cells. This aspect differs from the first aspect in that the introduction of the first and second siRNAs and the introduction of the nucleic acid molecule encoding the target protein are carried out in separate steps. In other words, this embodiment comprises the following first molecule introduction step and second molecule introduction step.

[0084] The first molecule introduction step involves contacting the target cells with a pretreatment solution containing at least one of the following siRNAs: a first siRNA that reduces the expression level of Dom34 which may be endogenous in the target cells, and a second siRNA that reduces the expression level of SAMHD1 which may be endogenous in the target cells. The process then involves irradiating the target cells with plasma to introduce at least one of the first and second siRNAs into the target cells.

[0085] The second molecule introduction step involves bringing a liquid containing the nucleic acid molecule into contact with the target cells that have undergone the first molecule introduction step, and then irradiating the target cells with plasma to introduce the nucleic acid molecule into the target cells. This step may be performed immediately after the completion of the first molecule introduction step, or after a culture period of several minutes to several hours.

[0086] The pretreatment solution used in the first molecule introduction step and the liquid containing the nucleic acid molecule used in the second molecule introduction step can each be prepared in the same manner as the nucleic acid solution of the first embodiment.

[0087] The plasma irradiation treatment performed in the first molecule introduction step and the plasma irradiation treatment performed in the second molecule introduction step can be carried out in the same manner as the plasma irradiation treatment performed in the first embodiment.

[0088] In this embodiment, since siRNA is introduced before the introduction of the nucleic acid molecule, RNA interference by Dom34 or SAMHD1 can be performed first within the target cells. As a result, the nucleic acid molecule can be introduced into target cells in which the expression level of Dom34 or SAMHD1 has been sufficiently reduced, which may lead to a higher expression level of the target protein in the target cells. In this embodiment, plasma irradiation is performed in both the first molecule introduction step and the second molecule introduction step. From the viewpoint of reducing the burden on target cells, the first embodiment, in which plasma irradiation is performed only once, is preferable. [Examples]

[0089] The present invention will be explained in more detail below with experimental examples, but these examples are not intended to limit the present invention.

[0090] <Molecular introduction device> Using a molecular delivery system outlined in Figure 1, target cells pre-cultured in a 48-well plate were subjected to plasma irradiation. 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: 40ms Discharge points per well: 1 point in the center Distance between the end of the first electrode and the top surface of the container (the surface on which target cells adhere): 1.0 mm

[0091] <material> The following materials will be used. Target cells: PMBC cells (human peripheral blood mononuclear cells, manufactured by Precision MEDICINE) mRNA: mRNA containing the base sequence (SEQ ID NO: 5) encoding the fluorescent protein mCherry (manufactured by VectorBuilder, Inc.) First siRNA: Double-stranded RNA (21 bp) containing a base sequence (SEQ ID NO: 2) encoding a portion of Dom34. Second siRNA: Double-stranded RNA (21 bp) containing a base sequence (SEQ ID NO: 4) encoding a portion of SAMHD1. Container: 48-well plate (Corning)

[0092] <Experimental Procedure> [Experimental Example 1-1: Suppression of SAMHD1] Each well of a 48-well plate contains 2 × 10⁶ PMBC cells. 6 0.4 mL of cells per well were seeded, StemFit medium (Ajinomoto Co., Ltd.) was added, and the cells were incubated in a CO2 incubator (37°C, 5 vol% CO2 concentration) for 48 hours until plasma irradiation. Before plasma irradiation, the medium was removed, and 4 μL of nucleic acid solution, in which the above mRNA and the second siRNA were dissolved in physiological salt solution, was added to each well. The cells were then allowed to stand for 3 minutes with the nucleic acid solution in contact with them. The mRNA concentration in the nucleic acid solution was 1 μg / μL, and the concentration of the second siRNA was 2.5 nM.

[0093] Subsequently, plasma irradiation was performed using a molecular delivery device under the above conditions, and after standing for 3 minutes, the above mRNA and the second siRNA were introduced into the PMBC cells. Next, 0.4 mL of TexMACS medium was added to each well to restore the PMBC cells, and the PMBC cells were continued to be cultured in a CO2 incubator.

[0094] On days 2 and 5 after molecular transduction, the expression level of the fluorescent protein mCherry expressed in PMBC cells was quantified using a fluorescence microscope (Keyence Corporation).

[0095] [Experimental Example 1-2; Suppression of Dom34] The test was carried out in the same manner as in Experimental Example 1-1, except that the second siRNA in the nucleic acid solution was replaced with the first siRNA, and the expression level of the fluorescent protein mCherry was quantified. The mRNA concentration in the nucleic acid solution used in this example was 1 μg / μL, and the concentration of the first siRNA was 25 nM.

[0096] [Experimental Examples 1-3; Negative Controls] The test was conducted in the same manner as in Experimental Example 1-1, except that a physiological salt solution was used instead of the nucleic acid solution, and the expression level of the fluorescent protein mCherry was quantified.

[0097] [Experimental Examples 1-4; Positive Control] The test was carried out in the same manner as in Experimental Example 1-1, except that a nucleic acid solution containing only the mRNA and not the first siRNA and the second siRNA was used, and the expression level of the fluorescent protein mCherry was quantified.

[0098] Table 1 shows the measurement results for the group in Experimental Example 1 described above. The measured values ​​are relative values ​​obtained by quantifying fluorescence intensity using analysis software attached to the fluorescence microscope.

[0099] [Table 1]

[0100] The results from the group in Experimental Example 1 confirmed that the expression level of the target protein can be increased by introducing not only mRNA encoding the target protein (e.g., mCherry) but also siRNA that suppresses SAMHD1 expression or siRNA that suppresses Dom34 expression into target cells (e.g., PMBC cells). Furthermore, it was found that the expression level of the target protein remained sufficient for several days (at least 5 days) after mRNA introduction. [Explanation of Symbols]

[0101] 1...Molecular introduction device, 10...First electrode, 12...Electrode body, 14...Irradiator, 16...Wall, 20...Second electrode, 30...Power supply unit, 40...Container, 42...Nucleic acid molecule, 44...Target cell, O1...Tube axis, θ1...Angle

Claims

1. A method for producing molecules that express the target protein within target cells by introducing a nucleic acid molecule encoding the target protein into the target cells, After bringing a liquid containing at least one of a first siRNA that reduces the expression level of Dom34 which may be endogenous in the target cells, and a second siRNA that reduces the expression level of SAMHD1 which may be endogenous in the target cells, and the nucleic acid molecule, into contact with the target cells, A method for producing molecularly introduced cells, comprising a molecular introduction step of introducing the nucleic acid molecule and at least one of the first siRNA and the second siRNA into the target cells by irradiating the target cells with plasma.

2. A method for producing molecules that express the target protein within target cells by introducing a nucleic acid molecule encoding the target protein into the target cells, After bringing the target cells into contact with a pretreatment solution containing at least one of the following siRNAs: a first siRNA that reduces the expression level of Dom34 which may be endogenous in the target cells, and a second siRNA that reduces the expression level of SAMHD1 which may be endogenous in the target cells, A first molecular introduction step involves irradiating the target cells with plasma to introduce at least one of the first siRNA and the second siRNA into the target cells. After bringing the liquid containing the nucleic acid molecule into contact with the target cells that have undergone the first molecule introduction step, A second molecule introduction step involves introducing the nucleic acid molecule into the target cell by irradiating the target cell with plasma, A method for producing molecularly introduced cells.

3. The method for producing molecularly introduced cells according to claim 1 or 2, wherein the nucleic acid molecule is mRNA encoding the target protein.

4. The method for producing molecularly introduced cells according to claim 1 or 2, wherein the target cell is at least one selected from the group consisting of blood-derived cells, adherent cells, and iPS cells.

5. A method for producing molecularly introduced cells according to claim 1 or 2, wherein the target cells are blood-derived cells including CD3-positive cells.

6. The method for producing molecularly introduced cells according to claim 5, wherein the CD3-positive cells include T cells.

7. A method for producing molecularly introduced cells according to claim 1 or 2, wherein the target cells are adherent cells, and the adherent cells include HEK293F cells, mesenchymal stem cells, adipose-derived stem cells, or hematopoietic stem cells.

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