Agent and method for introducing nucleic acid and / or protein into cell

Abstract

The present invention addresses the problem of providing a novel electroporation introduction agent.　The problem is solved by an introduction agent for introducing a nucleic acid and / or a protein into a cell by electroporation, the introduction agent containing (1) a basal medium for cell culture, (2) a C5 or C6 monosaccharide sugar alcohol, (3) an aqueous polysaccharide solution, (4) a nonionic surfactant, (5) a pyrazoline compound, and (6) N-acetyl-L-cysteine.

Description

Agents for introducing nucleic acids and / or proteins into cells, and methods for introducing them.

[0001] This specification discloses agents and methods for introducing nucleic acids and / or proteins into cells by electroporation.

[0002] In electroporation, a method for introducing nucleic acids and proteins into cells, it is necessary to purchase and use expensive commercially available reagents whose compositions are not publicly disclosed.

[0003] For example, as a commercially available kit, Human T Cell Nucleofector TM Kit (Lonza Corporation) (Non-Patent Document 1) and Human CD34 + Cell Nucleofector TM Kit (Lonza) (Non-Patent Document 2) can be cited.

[0004] https: / / www.lonzabio.jp / catalog / 1207 / https: / / www.lonzabio.jp / catalog / 1698 /

[0005] However, the electroporation method using the commercially available reagents mentioned above suffers from poor efficiency in introducing nucleic acids and proteins into cells, as well as poor cell viability. This poses a significant obstacle when introducing nucleic acids and proteins into rare cells (such as human hematopoietic stem cells). One of the objectives of this invention is to provide a novel introduction agent for electroporation.

[0006] Item 1. An introduction agent for introducing nucleic acids and / or proteins into cells by electroporation, comprising (1) a basal culture medium for cell culture, (2) a monosaccharide alcohol having 5 or 6 carbon atoms, (3) an aqueous polysaccharide solution, (4) a nonionic surfactant, (5) a pyrazoline compound, and (6) N-acetyl-L-cysteine. Item 2. The introduction agent according to claim 1, wherein, when the introduction agent is 100% by mass, the content of the monosaccharide alcohol having 5 or 6 carbon atoms is 0.5 to 1.5% by mass, the aqueous polysaccharide solution contains 1% to 10% by mass of polysaccharide, the content of the aqueous polysaccharide solution is 0.01 to 0.5% by mass, the content of the nonionic surfactant is 0.0005 to 0.02% by mass, the content of the pyrazoline compound is 0.0005 to 0.02% by mass, and the content of the N-acetyl-L-cysteine ​​is 0.000008 to 0.00002% by mass. Claim 3. The introduction agent according to claim 1, wherein the monosaccharide alcohol is mannitol and / or sorbitol. Claim 4. The introduction agent according to item 1, wherein the polysaccharide solution is a 1% to 10% by mass aqueous solution of Ficol™ PM400 or Ficol™ 400, or Lymphoprep™. Item 5. The introduction agent according to item 1, wherein the nonionic surfactant is poloxamer 188. Item 6. The introduction agent according to item 1, wherein the pyrazoline compound is edaravone. Item 7. The introduction agent according to item 1, wherein the basal culture medium for cell culture is Opti-MEM® or Dulbecco's modified Eagle medium. Item 8. A method for introducing nucleic acids and / or proteins into cells by electroporation, comprising: preparing a first mixture by mixing the introduction agent according to item 1 with the nucleic acids and / or proteins to be introduced; preparing a second mixture by mixing the first mixture with cells to be introduced; and applying an electrical load to the second mixture.

[0007] We can provide an electroporation agent with high introduction efficiency.

[0008] This shows the transduction efficiency when a GFP expression plasmid is introduced into human hematopoietic stem cells. Figure 1A shows the conventional method using Human T Cell Nucleofector TM This section shows the flow cytometer analysis results when gene transfer was performed using Kit (Lonza). Figure 1B shows the flow cytometer analysis results when gene transfer was performed using Introducer 1. It shows the ratio of GFP-positive cells (live cells) to the number of cells seeded per well during gene transfer. It shows the transfer efficiency when the RNP complex was introduced into human hematopoietic stem cells. Figure 3A shows the results when a negative control was introduced using the conventional method. Figure 3B shows the results when an RNP complex containing gRNA targeting CD34 was introduced using the conventional method. Figure 3C shows the results when a negative control was introduced using Introducer 1. Figure 3D shows the results when an RNP complex containing gRNA targeting CD34 was introduced using Introducer 1. It shows the ratio of recovered CD34- / CD133+ cells to the number of cells seeded per well during RNP transfer. This section shows the results when a GFP expression plasmid was introduced into human iPS cell line (201B7) using Introducer 1. The results of introducing a GFP expression plasmid into a mouse fibroblast cell line (MS-5) using induction agent 1 are shown.

[0009] 1. Introducing Agents One embodiment of the present invention relates to an introducer for introducing nucleic acids and / or proteins into cells by electroporation.

[0010] The introduction agent contains component (1) a basal culture medium for cell culture (hereinafter sometimes simply referred to as "basal culture medium"), component (2) a monosaccharide alcohol having 5 or 6 carbon atoms, component (3) an aqueous polysaccharide solution, component (4) a nonionic surfactant, component (5) a pyrazoline compound, and component (6) N-acetyl-L-cysteine.

[0011] The cells are not particularly limited. For example, primary cultured cells and cultured cells can be used as examples of cells. Preferably, stem cells can be used as examples of cells. Stem cells may include fetal stem cells, pluripotent stem cells, induced pluripotent stem cells, tissue stem cells, etc.

[0012] The basal medium of component (1) is not limited as long as it is a medium that can be used for culturing cells, preferably mammalian cells. Examples of basal media include Opti-MEM®, Dulbecco's Modified Eagle Medium (DMEM), Eagle's Minimum Essential Medium (αMEM), RPMI-1640 medium, Ham's F-12 medium, and mixed media thereof. Opti-MEM and DMEM are preferred as the basal medium. The content of the basal medium is the amount obtained by subtracting the total amount of the other components from 100% by mass when the introduction agent is considered to be 100% by mass.

[0013] Examples of the monosaccharide alcohol having 5 or 6 carbon atoms in component (2) include mannitol, sorbitol, iditol, galactitol, xylitol, arabitol, and ribitol. Mannitol and sorbitol are preferred as monosaccharide alcohols. When the introducing agent is 100% by mass, the content of the monosaccharide alcohol having 5 or 6 carbon atoms is 0.5 to 1.5% by mass, preferably 0.8 to 1.2% by mass.

[0014] The polysaccharide contained in the polysaccharide aqueous solution, which is component (3), may include polysaccharide itself and / or copolymers of saccharide with other molecules. Examples of polysaccharide aqueous solutions include aqueous solutions containing polysucrose itself, which has an average molecular weight of approximately 400,000 (as stated by the manufacturer), and / or copolymers of sucrose with other molecules. Examples of polysucrose aqueous solutions include at least one selected from the group consisting of Ficol™ PM400 or Ficol™ 400 1% to 10% by mass aqueous solutions, and Lymphoprep™. Ficol™ PM400 is a copolymer of sucrose and epichlorohydrin with an average molecular weight of 400,000. It is also called Type 400 and is available for purchase from Merck KGaA GmbH (Germany).

[0015] Ficol™ PM400 can be prepared as an aqueous solution by dissolving the powder in water. However, aqueous solutions of Ficol™ PM400 are commercially available as Ficol-Paque™ PLUS (containing approximately 5.7% by mass of Ficol™ PM400), Ficol-Paque™ PREMIUM, Ficol-Paque™ PREMIUM 1.084, and Ficol-Paque™ PREMIUM 1.073 (all manufactured by Cytiva). These products may contain sodium diatrizoate (Paque) and ethylenediaminetetraacetic acid.

[0016] Ficol™ 400 is a copolymer of sucrose and epichlorohydrin. Also known as Type 400-DL, it is available for purchase from Merck KGaA GmbH (Germany).

[0017] Ficol™ PM400 or Ficol™ 400 is preferably prepared as a 1% to 10% by mass aqueous solution. More preferably, Ficol™ PM400 or Ficol™ 400 is prepared as a 4% to 8% by mass aqueous solution, and even more preferably as a 5% to 6% by mass aqueous solution.

[0018] Lymphoprep® is a solution containing polysaccharide and sodium diatrizoate. The polysaccharide may include polysaccharide itself and / or copolymers of polysaccharide with other molecules. According to the manufacturer, the polysaccharide content is 5.7% by mass (mass / volume). Lymphoprep® is a commercially available product and can be purchased, for example, from Veritas Corporation.

[0019] The aqueous polysaccharide solution preferably contains, for example, 1% to 10% by mass, preferably 4% to 8% by mass, and more preferably 5% to 6% by mass of polysaccharide.

[0020] When the amount of the introductory agent is considered to be 100% by mass, the content of component (3) is 0.01 to 0.5% by mass, preferably 0.03 to 0.01% by mass.

[0021] The nonionic surfactant as component (4) preferably has amphiphilicity. As the nonionic surfactant having amphiphilicity, for example, a compound belonging to a nonionic triblock copolymer surfactant (Pluronic (registered trademark) - type surfactant, also referred to as poloxamer) is preferred. A more preferred amphiphilic nonionic surfactant is, for example, an amphiphilic nonionic surfactant having an average molecular weight of about 8,800 (manufacturer's notation). As the amphiphilic nonionic surfactant, for example, poloxamer 188 can be mentioned. Poloxamer 188 is a commercially available product and can be purchased, for example, from Merck KGaA (Germany). When the introducing agent is 100% by mass, the content of poloxamer 188 is 0.0005 to 0.02% by mass, preferably 0.0001 to 0.01% by mass.

[0022] The pyrazoline compound as component (5) is preferably a compound having a radical scavenging action. As the pyrazoline compound having a radical scavenging action, for example, a compound represented by the following general formula (I) can be mentioned: (In the formula, R １ represents an alkyl group having 1 to 3 carbon atoms, preferably a methyl group or an ethyl group, more preferably a methyl group. R ２ represents an unsaturated or saturated ring having 5 to 7 carbon atoms, preferably an unsaturated ring having 5 to 7 carbon atoms, more preferably an unsaturated ring having 6 carbon atoms, and even more preferably a benzene ring.).

[0023] As the pyrazoline compound, edaravone is most preferred. Edaravone is a commercially available product and can be purchased, for example, from Merck KGaA (Germany).

[0024] When the introducing agent is 100% by mass, the content of the pyrazoline compound is 0.0005 to 0.02% by mass, preferably 0.0001 to 0.01% by mass.

[0025] Component (6), N-acetyl-L-cysteine, is a commercially available product and can be purchased, for example, from Merck KGaA (Germany). When the total amount of the introduction agent is 100% by mass, the content of N-acetyl-L-cysteine ​​is 0.000008 to 0.00002% by mass, preferably 0.000008 to 0.0002% by mass.

[0026] 2. Method of Introduction One embodiment of the present invention relates to a method (hereinafter simply referred to as "the method") for introducing nucleic acids and / or proteins into cells by electroporation.

[0027] The method includes preparing a first mixture by mixing the introduction agent described in 1. above with the nucleic acid and / or protein to be introduced; preparing a second mixture by mixing the first mixture with the cells to be introduced; and applying an electrical load to the second mixture.

[0028] The mixing ratio of the introduction agent and the nucleic acid and / or protein to be introduced in the first mixture is not limited. For example, 1 ng to 100 μg of nucleic acid and / or protein can be mixed per 100 μL of introduction agent.

[0029] The mixing ratio of target cells and nucleic acids and / or proteins to be introduced when preparing the second mixture is not limited. For example, 1.5 × 10⁶ cells ５ For each individual, 10 μL to 1 mL of the first mixture can be added.

[0030] The electrical load on the second mixture can be applied using a general-purpose electroporator. For example, the second mixture can be placed in a cuvette suitable for the electroporator, and an electrical load can be applied according to a predetermined program of the electroporator. The predetermined program is set appropriately depending on the target cells, the nucleic acids and / or proteins to be introduced, and the model of the electroporator.

[0031] As an electroporator, for example, Nucleofector series of Lonza such as 4D-Nucleofector, Nucleofector I, Nucleofector II, Nucleofector 2b; Neon Transfection System (Thermo Fisher Scientific); NEPA21 (Neppagen) and the like can be used. Cells that have completed the electrical load are cultured in a cell culture medium suitable for each cell and under culture conditions.

[0032] Examples are shown below to explain the present invention in more detail. However, the present invention is not construed as being limited to the examples.

[0033] 1. Example: Preparation of transfection agent for electroporation Each reagent was mixed so that the final concentrations of the transfection agent for electroporation were 90% Opti-MEM (registered trademark), 50 mM D-mannitol (about 0.911%), 0.10% Ficoll-paque (trademark), 0.005% poloxamer 188, 300 μM edaravone (about 0.0052%), and 5.5 μM N-Acetyl-L-cysteine (about 0.00009%), and a transfection agent for nucleofection (hereinafter simply referred to as "transfection agent 1") was prepared. In addition, Opti-MEM (registered trademark) was replaced with DMEM to prepare a transfection agent for electroporation (hereinafter simply referred to as "transfection agent 2"). The control group used Human T Cell Nucleofector TM Kit (Lonza) (hereinafter simply referred to as "conventional method").

[0034] 2. Example: Introduction of GFP expression plasmid The GFP expression plasmid was introduced using transfection agent 1, transfection agent 2, or the conventional method, and the introduction efficiency was evaluated by the expression of GFP. Cells used were human hematopoietic stem cells (HSC), human iPS cell line (201B7), and mouse fibroblast cell line (MS-5). (1) Cells that had been cultured in advance were collected to obtain the required number of cells (1.5 × 10 5(1) Prepare the cells / group. (2) Transfer the required number of cells into a 1.5 mL microtube and centrifuge. (3) Add 1 mL of opti-MEM and centrifuge at 300 × g for 5 min. (4) Discard the supernatant from the microtube in (3) and add 101 μl of a mixture of 1 μl of GFP expression plasmid (1 ng / μL) and 100 μl of each transdermal agent to the precipitate. (5) After thoroughly suspending the cells, transfer them to a cuvette for electroporation. (6) Perform gene transfer by running a program appropriate for each cell type. Specifically, when using transdermal agent 1 and transdermal agent 2, program Q-003 of the electroporator (Nucleofector® 2b, Lonza) was used. The conventional method is Human T Cell Nucleofector TM The program recommended by the kit was used. (7) 500 μL of culture medium was added to the cuvette. The basal medium for the culture medium was ThermoFisher Cat. # 10639011 Gibco. TM StemPro TM -34 SFM (1X) was used, and thrombopoietin (TPO) and human stem cell factor (SCF) were added to the basal medium to final concentrations of 10 ng / mL and 50 ng / mL, respectively, and used as the culture medium. (8) The entire volume of cells was transferred to a 6-well plate that had been pre-filled with 1.4 mL of culture medium. (9) The cells were incubated in a 5% CO2 incubator. (10) Two days later, the percentage of GFP-positive cells that had undergone gene transfer was analyzed using a flow cytometer (FACSCantoII (BD Biosciences)).

[0035] 3. Examples: Introducing gRNA / Cas9 protein complexes (RNP complexes) in the CRISPR / Cas9 system using introduction agent 1 or a conventional method to introduce human hematopoietic stem cells (human umbilical cord blood-derived Lineage-CD34 + / CD38 - / CD133 + / GPI-80 +The RNP complex was introduced into cells, and the knockout efficiency of the CD34 antigen was evaluated. (1) The previously cultured cells were harvested from the culture flask to obtain the required number of cells (1.5 × 10⁶). 5 (1) Prepare the cells / group. (2) Transfer the required number of cells into a 1.5 mL microtube and centrifuge. (3) Add 1 mL of opti-MEM and centrifuge at 300 × g for 5 min. (4) Add CD34 target gRNA to 5 μL of PBS to a final concentration of 48 μM and Cas9 protein to 20 μM to make the RNP complex solution. (5) Discard the supernatant in the microtube from (3) and add 105 μL of a mixture of 5 μL of RNP complex solution and 1100 μL of transdermal agent to the sediment. A mixture of Cas9 protein only and transdermal agent 1 was used as a negative control. (6) After thoroughly suspending the cells, transfer them to a cuvette for electroporation. (7) Execute a program appropriate for each cell type and perform complex transduction. Specifically, when using transdermal agent 1 and transdermal agent 2, program Q-003 of the electroporator (Nucleofector® 2b, Lonza) was used. The conventional method is Human T Cell Nucleofector TM The program recommended by the kit was used. (8) 500 μL of the same culture medium as in 2. above was added to the cuvette. (9) The entire volume of cells was transferred to a 6-well plate that had been pre-filled with 1.4 mL of culture medium. (10) The cells were incubated in a 5% CO2 incubator. (11) After 72 hours from the introduction of the complex, the editing of the target gene was confirmed using a cell sorter (FACSAria III (BD Biosciences)).

[0036] 4. Evaluation of the number of GFP-positive cells after plasmid introduction (1) Cells cultured 48 hours after introducing a GFP expression plasmid into human hematopoietic stem cells were collected and the number of viable cells was counted.

[0037] (2) To remove dead cells, the collected cells were stained with 7-Amino-Actinomycin D (7-AAD) (0.25 μg / mL) dissolved in FACS buffer (phosphate-buffered saline (PBS) + 2% fetal bovine serum (FBS)). (3) The proportion of GFP-positive cells among viable cells (7-AAD negative) in each group was analyzed using a flow cytometer (FACSCantoII). (4) The number of GFP-positive cells was calculated from the cell count and the proportion of GFP-positive cells.

[0038] 5. Evaluation of gene knockout efficiency by FACS (1) Human hematopoietic stem cells were cultured after introducing an RNP complex targeting the CD34 antigen and harvested 72 hours later. (2) The harvested cells were stained with fluorescently labeled anti-human CD34 antibody, anti-human CD133 antibody, anti-human CD45 antibody, and 7-AAD. Then, the CD45-positive and 7-AAD-negative fractions were gated using a cell sorter (FACSAriaIII), and the results were developed based on the expression levels of CD34 and CD133. (3) CD34 in each sample + / CD133 + and CD34 + / CD133 - The fractions were gated and their proportions were analyzed. (4) CD34 in each gated sample + / CD133 + and CD34 + / CD133 - The fractions were collected by cell sorting, and the proportion of cells that had undergone the desired gene editing relative to the seeded cells was evaluated.

[0039] 6. Results (1) Evaluation of gene transfer efficiency Figure 1 shows the transfer efficiency when the GFP expression plasmid was introduced into human hematopoietic stem cells. Figure 1A shows the conventional method using Human T Cell Nucleofector TMThe flow cytometer analysis results when gene transfer was performed using Kit (Lonza) are shown. Figure 1B shows the flow cytometer analysis results when gene transfer was performed using Introducer 1. With the conventional method, GFP expression was 63.4%, but when gene transfer was performed using the Introducer according to the present invention, it was 79.0%.

[0040] Figure 2 shows the percentage of GFP-positive cells (live cells) relative to the number of cells seeded per well during gene transfer. In the conventional method, the percentage of GFP-positive cells was 1.6%, but when gene transfer was performed using induction agent 1, it was 5.5%. When gene transfer was performed using induction agent 2, it was 2.6%. This demonstrates that the induction agent according to the present invention is more efficient in gene transfer compared to the conventional method.

[0041] (2) Evaluation of RNP complex introduction efficiency Figure 3 shows the introduction efficiency when the RNP complex was introduced into human hematopoietic stem cells. Surface markers were analyzed using a flow cytometer. The surface marker of human umbilical cord blood-derived hematopoietic stem cells is CD34. + / CD38 － / CD133 + / GPI-80 + Figure 3A shows the results when a negative control was introduced using the conventional method. Figure 3B shows the results when an RNP complex containing a CD34-targeting gRNA was introduced using the conventional method. Figure 3C shows the results when a negative control was introduced using induction agent 1. Figure 3D shows the results when an RNP complex containing a CD34-targeting gRNA was introduced using induction agent 1. The gates used were CD34 and CD133. When an RNP complex containing a CD34-targeting gRNA was introduced using the conventional method, CD34 ＋ / CD133 ＋ The cell percentage was 29.7%, and CD34 － / CD133 ＋ The cell percentage was 17.7%. When an RNP complex containing a gRNA targeting CD34 was introduced using induction agent 1, CD34 ＋ / CD133 ＋ The percentage of cells was 22.4%, and CD34 － / CD133 ＋The cell percentage was 27.1%. In the negative control, CD34 － / CD133 ＋ No cells were found.

[0042] Next, CD34 － / CD133 ＋ The cell recovery rate was examined. Figure 4 shows the number of CD34 cells recovered relative to the number of cells seeded per well at the time of RNP introduction. － / CD133 ＋ This shows the percentage of cells. In the conventional method, CD34 － / CD133 ＋ The cell percentage was 1.6%, but it was 3.2% when gene transfer was performed using introduction agent 1.

[0043] The results above demonstrate that the induction agent according to the present invention is more efficient in introducing nucleic acids and proteins compared to conventional methods. Since the proportion of hematopoietic stem cells in bone marrow and umbilical cord blood is remarkably low, a high recovery rate after induction is considered important.

[0044] (3) Gene transfer to human iPS cell line (201B7) Instead of human hematopoietic stem cells, a GFP expression plasmid was introduced into the human iPS cell line (201B7) using introduction agent 1. The results are shown in Figure 5. GFP expression was observed in 79.9% of the cells.

[0045] (4) Gene transfer to mouse fibroblast cell line (MS-5) Instead of human hematopoietic stem cells, a GFP expression plasmid was introduced into mouse fibroblast cell line (MS-5) using introduction agent 1. The results are shown in Figure 6. GFP expression was observed in 52.0% of the cells.

[0046] The results above suggest that the introduction agent according to the present invention can introduce nucleic acids and proteins into various cells.

Claims

1. An introduction agent for introducing nucleic acids and / or proteins into cells by electroporation, comprising (1) a basal culture medium for cell culture, (2) a monosaccharide alcohol having 5 or 6 carbon atoms, (3) an aqueous polysaccharide solution, (4) a nonionic surfactant, (5) a pyrazoline compound, and (6) N-acetyl-L-cysteine.

2. The introduction agent according to claim 1, wherein, when the introduction agent is 100% by mass, the content of the monosaccharide alcohol having 5 or 6 carbon atoms is 0.5 to 1.5% by mass, the aqueous polysaccharide solution contains 1% to 10% by mass of polysaccharide, the content of the aqueous polysaccharide solution is 0.01 to 0.5% by mass, the content of the nonionic surfactant is 0.0005 to 0.02% by mass, the content of the pyrazoline compound is 0.0005 to 0.02% by mass, and the content of the N-acetyl-L-cysteine ​​is 0.000008 to 0.00002% by mass.

3. The introduction agent according to claim 1, wherein the monosaccharide alcohol is mannitol and / or sorbitol.

4. The introduction agent according to claim 1, wherein the polysaccharide solution is a 1% to 10% by mass aqueous solution of Ficol™ PM400 or Ficol™ 400, or Lymphoprep™.

5. The introduction agent according to claim 1, wherein the nonionic surfactant is poloxamer 188.

6. The introduction agent according to claim 1, wherein the pyrazoline compound is edaravone.

7. The introduction agent according to claim 1, wherein the basal culture medium for cell culture is Opti-MEM® or Dulbecco's modified Eagle medium.

8. A method for introducing nucleic acids and / or proteins into cells by electroporation, comprising: preparing a first mixture by mixing the introduction agent described in claim 1 with the nucleic acids and / or proteins to be introduced; preparing a second mixture by mixing the first mixture with cells to be introduced; and applying an electrical load to the second mixture.

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