Method for predicting gene transfer rate

Abstract

Disclosed is a method for predicting the gene transfer rate in animal cell and / or the number of gene-transferred cells in the animal cells, the method comprising (1) a step for measuring a component in a supernatant of a culture obtained by the culturing of gene-transferred animal cells and (2) a step for predicting the gene transfer rate and / or the number of gene-transferred cells on the basis of a value measured in step (1).

Description

Gene transfer rate prediction method

[0001] The present invention relates to a method for predicting the gene transfer rate and / or the number of gene-transferred cells. (Background of the invention)

[0002] In recent years, research and development of genetically modified cell medicines (cell preparations with or without gene transfer) such as chimeric antigen receptor (CAR)-T cells has been actively pursued. For genetically modified cell medicines such as CAR-T cells, the product quantity is determined by multiplying the total cell count by the percentage of the transferred gene. Therefore, for CAR-T cell products, the CAR transfer rate is the most important indicator in the manufacturing process, shipping, and administration. However, while flow cytometry has traditionally been used to measure the rate of gene transfer into cells, it has problems such as the long time required from measurement to analysis (6-7 hours) and the complex process (manual cell staining). For example, Non-Patent Document 1 reports the measurement of CAR-T cells using flow cytometry.

[0003] Cytometry Part B: Clinical Cytometry, Volume 100, Issue 2, p. 218-224

[0004] An object of the present invention is to provide a method for predicting the gene transfer rate and / or the number of gene-transferred cells, which enables measurement of the gene transfer rate and / or the number of gene-transferred cells more simply and efficiently than conventional methods.

[0005] As a result of extensive research to achieve the above-mentioned object, the present inventors have found that there are differences in the components of the culture supernatant between gene-transfected cells and non-transfected cells, and have discovered that by analyzing the medium in which cells are cultured and using the medium components (e.g., metabolites in the medium, pH, extracellular vesicles) as indicators, it is possible to simply and efficiently predict the gene transfection rate and / or the number of gene-transfected cells in animal cells.

[0006] The present invention was completed based on these findings and through further investigation, and provides the following method for predicting the gene transfer rate and / or the number of gene-transferred cells in animal cells.

[0007] [1] A method for predicting the gene transfer rate and / or the number of gene-transferred cells in animal cells, comprising: (1) measuring components of a culture supernatant in which gene-transferred animal cells have been cultured, and (2) predicting the gene transfer rate and / or the number of gene-transferred cells based on the values ​​measured in step (1). [2] The method according to [1], wherein in step (1), the pH, metabolites, or extracellular vesicles of the culture supernatant are measured. [3] The method according to [2], wherein the metabolite is at least one selected from the group consisting of organic acids, sugars, lipids, vitamins, amines, nucleic acids, amino acids, and other metabolite ions. [4] The method according to [3], wherein the organic acid is at least one selected from the group consisting of decenoic acid, dodecenoic acid, myristoleic acid, palmitic acid, palmitoleic acid, hexadecadienoic acid, stearic acid, oleic acid, linoleic acid, γ-linolenic acid, α-linolenic acid, arachidic acid, eicosenoic acid, eicosadienoic acid, mead acid, arachidonic acid, docosapentaenoic acid, docosahexaenoic acid, nervonic acid, 2-hydroxyvaleric acid, 2-oxoglutaric acid, indoxyl sulfate, 4-pyridoxy acid, 4-ethylphenyl sulfate, adipic acid, citric acid, fumaric acid, lactic acid, malic acid, phenyllactic acid, pantothenic acid, pipecolic acid, pyruvic acid, tartronic acid, p-cresol sulfate, and xanthurenic acid. [5] The method according to [3] or [4], wherein the saccharide is at least one selected from the group consisting of fructose, inositol, galactose, glucose, and sucrose. [6] The method according to any one of [3] to [5], wherein the lipid is at least one selected from the group consisting of ceramide, Hex2-ceramide, Hex-ceramide, cholesterol, cholesterol sulfate, cardiolipin, monoglycerol, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, sphingomyelin, dihydrosphingomyelin, triglyceride, glycerol-3-phosphate, and ganglioside GM3.[7] The method according to any one of [3] to [6], wherein the vitamin is at least one selected from the group consisting of α-tocopherol, nicotinamide, and choline. [8] The method according to any one of [3] to [7], wherein the amine is at least one selected from the group consisting of ethanolamine and ethanolamine phosphate. [9] The method according to any one of [3] to [8], wherein the nucleic acid is at least one selected from the group consisting of adenosine and methylthioadenosine.

[10] The method according to any one of [3] to [9], wherein the amino acid is at least one selected from the group consisting of acylcarnitine, alanine, arginine, asparagine, aspartic acid, β-alanine, cysteine, cysteine-HPE-AM, cysteine-SS-HPE-AM, dimethylglycine, glutamic acid, glutamine, glycine, isoleucine, leucine, leucine acid, lysine, methionine, homocysteine-HPE-AM, N-acetyl-β-alanine, N-acetylcysteine, N-acetylserine, N-acetyltryptophan, N-carbamoyl-aspartate, O-acetylserine, ornithine, pyroglutamic acid, phenylalanine, proline, serine, S-sulfocysteine, taurine, threonine, tryptophan, tyrosine, and valine.

[11] The method according to any one of [3] to

[10] , wherein the other metabolite ion is at least one selected from the group consisting of ammonium ion, potassium ion, calcium ion, sodium ion, pyrophosphate, sulfate, sulfite, phenol sulfate, and SS-bis-HPE-AM.

[12] The method according to any one of [1] to

[11] , wherein the components of the culture supernatant in step (1) are measured by Raman spectroscopy.

[13] The method according to any one of [1] to

[12] , wherein the gene transfer rate and / or the number of gene-transfected cells are predicted based on one component of the culture supernatant in step (2).

[14] The method according to any one of [1] to

[12] , wherein the gene transfer rate and / or the number of gene-transfected cells are predicted based on a combination of two or more components of the culture supernatant in step (2).

[15] The method according to any one of [1] to

[14] , wherein the change in the components of the culture supernatant before and after gene transfer is used to predict the gene transfer rate and / or the number of gene-transfected cells in step (2).

[16] The method according to any one of [1] to

[15] , wherein the animal cells are immune cells.

[17] The method according to

[16] , wherein the immune cells are T cells or NK cells.

[18] The method according to any one of [1] to

[15] , wherein the animal cells are epithelial cells. [18a] The method according to

[18] , wherein the animal cells are liver cancer cells. [18b] The method according to any one of [1] to [18a], wherein the animal cells are human cells.

[19] The method according to any one of [1] to [18b], wherein the gene is a nucleic acid encoding a chimeric antigen receptor.

[20] A method for producing a gene-introduced cell preparation, comprising the steps of measuring components of a culture supernatant in which animal cells have been cultured, and determining the gene introducibility and / or the number of gene-introduced cells based on the measured values.

[0008] The method for predicting the gene transfer rate and / or the number of gene-transduced cells of the present invention enables measurement of the gene transfer rate and / or the number of gene-transduced cells with a simple procedure (e.g., the only procedure required is automatic measurement using a measurement device) without requiring complex steps compared to conventional methods. Furthermore, the method for predicting the gene transfer rate and / or the number of gene-transduced cells of the present invention enables measurement of the gene transfer rate of cells efficiently (e.g., in a short time of less than 5 minutes). Furthermore, since a culture supernatant is used, cell sampling is not required, eliminating the need for an agitation step and causing less damage to the cells. For example, in the manufacturing and quality control processes for CAR-T cell products, by measuring the components of the culture supernatant before and after gene transfer, the CAR transfer rate and the number of transduced cells can be determined quickly and simply without damaging the cells, making it possible to confirm whether a product that meets standards has been manufactured.

[0009] Figure 1 shows the results of Example 1. (A) Cell count after culture, (B) viability, (C) CAR transduction efficiency, (D) photograph of the supernatant after cell culture (N=3), (E) pH of the culture supernatant (N=4, *p<0.05, p value analyzed by t-test). Values ​​in the graph are mean ± standard deviation (same below). Figure 1 shows the results of Example 1. Graph showing each component in the supernatant after culture of control T cells (UTD) without gene transduction and CAR-T cells with CAR gene transduction. (A) glutamine (Gln), (B) glutamic acid (Glu), (C) glucose (Gluc), (D) lactic acid (Lac), (E) ammonium ion (NH4 + ), (F) sodium ion (Na + ), (G) potassium ion (K + ), (H) calcium ions (Ca ++) (N=4, *p<0.05, p-value was analyzed by t-test). Figures showing the results of Example 2. (A) Schematic diagram of the experiment, graphs showing (B) cell density at each culture time point, and the amounts of (C) glucose, (D) lactate, (E) glutamine, (F) glutamate, and (G) ammonium ions in the culture supernatant (N=4, *p<0.05, **p<0.01, ***p<0.001, p-value was analyzed by t-test). Figures showing the results of Example 3. Graph showing the glucose uptake ability of UTD or CAR-T cells (N=3, *p<0.0001, p-value was analyzed by t-test). Figures showing the results of Example 4. (A) Schematic diagram of the experiment, (B) graph showing the cell density and CAR transduction efficiency of the prepared cells. Figures showing the results of Example 4. Graph showing the correlation between each component in the culture supernatant and cell density or CAR-introduced T cell (CAR-T) density on the final day of culture (C-D: lactic acid, E-F: ammonium ion). Figure 1 shows the results of Example 4. Graph showing the correlation between each component in the culture supernatant and cell density or CAR-introduced T cell (CAR-T) density on the final day of culture (G-H: pH, I-J: glutamine, K-L: glucose). Figure 1 shows the results of Example 4. Graph showing the correlation between each component in the culture supernatant and cell density or CAR-introduced T cell (CAR-T) density on the final day of culture (M-N: glutamic acid, O-P: sodium ion, Q-R: potassium ion, O-P: calcium ion). Figure 2 shows the results of Example 5. Graph showing (A) cell density and (B) CAR introduction efficiency at each date during the amplification step. Figure 3 shows the results of Example 5. Graph showing the correlation between each component in the culture supernatant and cell density or CAR-T cell density over the entire period of the expansion process (A-B: lactate, C-D: ammonium ion, E-F: pH, G-H: glutamine, I-J: glucose). Figure 1 shows the results of Example 6. Graph showing the differences in components in the supernatant between UTD and CAR-T cells using a volcano plot. The horizontal axis shows the estimated amount of change under conditions with the highest CAR transduction efficiency, obtained by performing a Williams test on the difference in the amount of each component between UTD and CAR-T cells. The left represents components that decreased in CAR-T cells, and the right represents components that increased in CAR-T cells.The vertical axis represents the -Log(p value) of the Williams test for the difference in the amount of each component at each date during the amplification process. This figure shows the results of Example 7. This figure shows graphs illustrating prediction of the number of CAR-T cells (cells / mL) using (A) PC(aa-30:0), (B) PE(aa-36:2), (C) PC(aa-32:1) on day 2 of amplification, (D) FFA(C10:1), (E) PC(aa-30:1) on day 3 of amplification, and (F) phenyllactic acid, (G) PC(aa-28:0), (H) FFA(C10:1), and (I) PC(aa-30:1) on day 4 of amplification. This figure shows the results of Example 7. This figure shows graphs illustrating prediction of the number of CAR-T cells (cells / mL) by LASSO regression analysis (A-C) from the components in the culture supernatant at each date during the amplification process. This figure shows the results of Example 7. Graphs showing the prediction of the number of CAR-T cells (cells / mL) by (D-G) OPLS regression analysis from components in the culture supernatant at each date during the expansion process.

[0023] Figure 7 shows the results of Example 7. Graphs showing the prediction of the number of CAR-T cells (cells / mL) by simple regression analysis from components in the culture supernatant over the entire expansion process. (A) Phenyllactic acid, (B) FFA (C10:1), (C) PC (aa-28:0), (D) PC (aa-30:0), (E) PC (aa-30:1), (F) PC (aa-32:1), (G) PE (aa-34:2), (H) PE (aa-36:2), (I) PE (aa-36:3).

[0024] Figure 7 shows the results of Example 7. 1 is a graph showing the prediction of the number of CAR-T cells (cells / mL) from components in the culture supernatant during the entire amplification process by (A) LASSO regression analysis and (B) OPLS regression analysis.

[0034] Fig. 1 shows the results of Example 8.

[0035] Fig. 1 is a graph showing (A) the CAR transduction efficiency and (B) the lactic acid in the supernatant after culture of non-transfected control SK-HEP-1 cells (UTD) and CAR gene-transfected SK-HEP-1 cells (N=4, *p<0.05, p-value analyzed by t-test).

[0036] Fig. 1 shows the results of Example 9.

[0037] Fig. 1 is a graph showing (A) the CAR transduction efficiency and (B) the lactic acid in the supernatant after culture of non-transfected control gdT cells (UTD) and CAR gene-transfected CAR-gdT cells.

[0038] Fig. 1 is a graph showing the results of Example 10.

[0039] Fig. 1 is a graph showing the correlation between the number of CAR-NK cells and components in the culture supernatant.Figure 11 shows the results of Example 11. (A) Graph showing particle size of exosomes in the supernatants of UTD and CAR-T cells, (B) Volcano plot showing the results of proteome analysis of exosomes (horizontal axis: ratio of expression level, vertical axis: p-value). Figure 11 shows the results of Example 11. (C) Table showing proteins whose expression changes significantly in exosomes derived from UTD and CAR-T cells. Figure 11 shows the results of Example 11. (D) Graph showing the correlation between the number of CAR-T cells and CD63-positive exosomes. Figure 12 shows the results of Example 12. (A) An example of the overall second derivative of the Raman spectroscopy spectrum obtained for the culture supernatants of UTD and CAR-T cells, and (B) an example of a peak that is different from the ...

[0010] Hereinafter, embodiments of the present invention will be described in detail.

[0011] "Comprise(s)" or "comprising" means the inclusion of, but is not limited to, the elements that follow the phrase. Thus, it implies the inclusion of the elements that follow the phrase, but does not imply the exclusion of any other elements. "Consist(s) of" or "consisting of" means inclusive of and limited to, any elements that follow the phrase. Thus, the phrase "consisting of" indicates that the listed elements are required or essential, with other elements being substantially absent. "Consist(s) essentially of" means inclusive of any elements that follow the phrase, and is limited to other elements that do not affect the activity or function of that element as specified in this disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or function of the listed elements.

[0012] As used herein, the term "pluripotent stem cell" refers to embryonic stem cells (ES cells) and cells that have the same pluripotency, i.e., the potential to differentiate into various tissues in the body (all of the endoderm, mesoderm, and ectoderm). Cells that have the same pluripotency as ES cells include "induced pluripotent stem cells" (also referred to herein as "iPS cells"). When the pluripotent stem cell is an ES cell or any cell derived from a human embryo, the cell may be a cell produced by destroying an embryo, or a cell produced without destroying an embryo, preferably a cell produced without destroying an embryo.

[0013] As used herein, a "cell population" refers to two or more cells of the same or different types. A "cell population" also refers to a mass of cells of the same or different types.

[0014] As used herein, "culturing" refers to maintaining and / or growing cells in an in vitro environment. "Culturing" refers to maintaining and / or growing cells outside a tissue or body, for example, in a cell culture dish or flask.

[0015] The present invention provides a method for predicting the gene transfer rate and / or the number of gene-transferred cells in animal cells, which comprises: (1) measuring components of a culture supernatant in which gene-transferred animal cells have been cultured; and (2) predicting the gene transfer rate and / or the number of gene-transferred cells based on the values ​​measured in step (1).

[0016] In the present invention, the "gene transfer rate" refers to the rate of transfer of a nucleic acid into cells, and is, for example, the proportion of cells into which a nucleic acid has been transferred among all cells, the amount of nucleic acid taken up in a cell population, or the expression rate of a transgene in a whole cell population. When the transgene is a CAR, it may also be referred to as the CAR-positive rate.

[0017] In the present invention, the "number of gene-transduced cells" refers to the number of cells into which a gene has been introduced in a cell population. When the transgene is a CAR, it may also be referred to as the number of CAR-positive cells.

[0018] The term "and / or" in the "method for predicting the gene transfer rate and / or the number of gene-transferred cells in animal cells" of the present invention means that the prediction method of the present invention can be a method for predicting both the gene transfer rate and the number of gene-transferred cells, or a method for measuring either one of them. More preferably, it is a method for predicting the number of gene-transferred cells.

[0019] Step (1) In step (1), components of the culture supernatant in which gene-transfected animal cells have been cultured are measured.

[0020] The type of animal cell is not particularly limited, and a wide variety of animal cells can be used, including, for example, spleen cells, nerve cells, glial cells, pancreatic beta cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibrocytes, muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, myoblasts, and muscle satellite cells), adipocytes, immune cells (e.g., macrophages, T cells, B cells, natural killer cells (NK cells), mast cells, neutrophils, basophils, eosinophils, monocytes, and megakaryocytes), synoviocytes, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocytes, interstitial cells, egg cells, and sperm cells, as well as stem cells that can be induced to differentiate into these cells (including pluripotent stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, iPS cells, and ES cells), progenitor cells, blood cells, oocytes, and fertilized eggs. T cells include αβ T cells, γδ T cells, helper T cells, cytotoxic T cells, regulatory T cells, suppressor T cells, tumor-infiltrating T cells, memory T cells, naive T cells, NKT cells, TCR-T cells, STAR receptor T cells, CAR-T cells (e.g., CAR-αβ T cells, CAR-gdT cells), and the like. Furthermore, animal cells also include the above-mentioned cells produced by inducing differentiation in vitro of primary cells, the above-mentioned stem cells (e.g., iPS cells), and the like. Animal cells also include various cancer cells. Animal cells may contain only one type of cell, or two or more types of cells.

[0021] Furthermore, the organism from which the animal cells are derived is not particularly limited, and examples of such organisms include mammals, such as humans, mice, rats, cows, horses, pigs, rabbits, dogs, cats, goats, monkeys, chimpanzees, etc. Of these, humans are preferred.

[0022] The animal cells are those into which a foreign gene has been introduced. A "foreign gene" is a gene or monomer nucleotide introduced from the outside to cause the animal cell to express a desired protein, and can be selected appropriately depending on the intended use of the animal cell. In the present invention, "animal cells into which a gene has been introduced" includes both animal cells into which a gene has actually been introduced in an attempted gene introduction, and animal cells into which no gene has been introduced. Furthermore, in the present invention, "(animal) cells into which a gene has actually been introduced" means (animal) cells into which a gene has actually been introduced. "Cells into which a gene has been introduced" and "cells into which a gene has been introduced" can be used interchangeably depending on the context.

[0023] The foreign gene can be, for example, a gene for expressing a chimeric antigen receptor (CAR). The foreign gene can be, for example, a gene for expressing a CAR and a gene for expressing a cytokine and / or a chemokine. Similar to general or known CARs, a CAR expressed by an animal cell is basically composed of peptides at each of the following sites, linked via a spacer as necessary: ​​(i) an antigen recognition site (e.g., a single-chain antibody, a ligand, a peptide, etc.) that recognizes a cell surface antigen of a cancer cell, (ii) a cell membrane-spanning domain, and (iii) a signaling domain that induces T cell activation. Other foreign genes include TCR (T cell receptor), STAR (synthetic T cell receptor and antigen receptor), chimeric TAC (T cell antigen coupler) receptor, suicide genes (iCas9, HSV-TK, etc.), cytokines (interleukins, chemokines, etc.), molecules that suppress the expression of T cell activation inhibitors (immune checkpoint factors (CTLA-4, PD-1, TIM-3, LAG-3, TGIT, BTLA, VISTA (PD-1H), etc.), CD160, Cbl-b, endogenous TCR, etc.), and T cell activation promoters (CD28, ICOS, CD137, OX40, CD27, GITR, BAFFR, TACI, BMCA, CD40L, etc.).

[0024] The means for introducing a foreign gene into an animal cell is not particularly limited, and various known or common means can be used. Typically, the foreign gene is introduced into the animal cell using an expression vector and expressed. The expression vector may be linear or circular, and may be a non-viral vector such as a plasmid, a viral vector, or a transposon-based vector.

[0025] The technique for introducing an expression vector into animal cells can be an appropriate one depending on the embodiment. For example, the expression vector can be introduced into animal cells by known methods such as viral infection, calcium phosphate, lipofection, microinjection, and electroporation. The expression vector can be prepared in a form suitable for use in each technique by known means or using a commercially available kit (according to its instructions).

[0026] The expression vector can be introduced into animal cells by viral infection. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. When using these viral vectors, a vector containing a desired foreign gene and a packaging vector (plasmid) of each virus are transfected into host cells using a corresponding commercially available kit to produce a recombinant virus, and then the resulting recombinant virus is infected into animal cells.

[0027] When there are multiple foreign genes, all of the foreign genes may be contained in a single expression vector, all of the foreign genes may be contained in separate expression vectors, or some of the multiple foreign genes may be contained in a single expression vector and the rest may be contained in separate expression vectors.When multiple foreign genes are contained in a single expression vector, the order in which the foreign genes are arranged from upstream to downstream is not particularly limited.

[0028] The foreign gene can be composed of a nucleic acid (polynucleotide) having a base sequence encoding the amino acid sequence of the desired protein or polypeptide. Those skilled in the art can design and prepare an expression vector capable of expressing the desired protein (polypeptide) in animal cells. The nucleic acid contained in the expression vector can be prepared by chemical DNA synthesis or prepared as cDNA (cloning).

[0029] In addition to a foreign gene, an expression vector may contain sequences such as a promoter, a terminator, an enhancer, an initiation codon, a stop codon, a polyadenylation signal, a nuclear localization signal (NLS), and a multiple cloning site (MCS), as necessary. The expression vector may further contain a nucleic acid (base sequence) encoding a "functional gene" such as a reporter gene (e.g., a gene encoding a fluorescent protein of each color), a drug selection gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene), or a suicide gene (e.g., a gene encoding diphtheria A toxin, herpes simplex thymidine kinase (HSV-TK), carboxypeptidase G2 (CPG2), carboxylesterase (CA), cytosine deaminase (CD), cytochrome P450 (cyt-450), deoxycytidine kinase (dCK), nitroreductase (NR), purine nucleoside phosphorylase (PNP), thymidine phosphorylase (TP), varicella-zoster virus thymidine kinase (VZV-TK), xanthine-guanine phosphoribosyltransferase (XGPRT), inducible caspase 9, etc.).

[0030] The term "nucleic acid" refers to any molecule formed by polymerizing monomeric nucleotides or nucleotides and molecules having equivalent functions to the nucleotides. Examples include RNA, which is a polymer of ribonucleotides; DNA, which is a polymer of deoxyribonucleotides; a mixed polymer of ribonucleotides and deoxyribonucleotides; and a nucleotide polymer containing a nucleotide analog. Nucleic acids may also be nucleotide polymers containing nucleic acid derivatives. Nucleic acids may be single-stranded or double-stranded. Double-stranded nucleic acids also include double-stranded nucleic acids in which one strand hybridizes to the other strand under stringent conditions.

[0031] The nucleotide analogue may be any molecule obtained by modifying ribonucleotides, deoxyribonucleotides, RNA, or DNA to improve or stabilize nuclease resistance, increase affinity with a complementary nucleic acid strand, increase cell permeability, or enable visualization, compared to RNA or DNA. The nucleotide analogue may be a naturally occurring molecule or a non-natural molecule, and examples thereof include sugar-modified nucleotide analogues (e.g., nucleotide analogues substituted with 2'-O-methylribose, nucleotide analogues substituted with 2'-O-propylribose, nucleotide analogues substituted with 2'-methoxyethoxyribose, nucleotide analogues substituted with 2'-O-methoxyethylribose, nucleotide analogues substituted with 2'-O-[2-(guanidium)ethyl]ribose, nucleotide analogues substituted with 2'-fluororibose, bridged artificial nucleic acids (BNA), locked artificial nucleic acids (LNA), ethylene bridged artificial nucleic acids (ENA), and the like. acid), peptide nucleic acid (PNA), oxypeptide nucleic acid (OPNA), peptide ribonucleic acid (PRNA)), nucleotide analogs modified with a phosphodiester bond (e.g., nucleotide analogs substituted with a phosphorothioate bond, nucleotide analogs substituted with an N3'-P5' phosphoamidate bond), etc.

[0032] The nucleic acid derivative may be any molecule in which another chemical substance is added to the nucleic acid in order to improve nuclease resistance, stabilization, affinity with a complementary nucleic acid strand, cell permeability, or visualization, compared to nucleic acids. Specific examples include 5'-polyamine-added derivatives, cholesterol-added derivatives, steroid-added derivatives, bile acid-added derivatives, vitamin-added derivatives, Cy5-added derivatives, Cy3-added derivatives, 6-FAM-added derivatives, and biotin-added derivatives.

[0033] In another embodiment of the present invention, proteins, siRNA, shRNA, dsRNA, miRNA, antisense nucleic acids, etc. may be introduced into animal cells instead of exogenous genes. Even when these substances other than exogenous genes are introduced into animal cells, the gene introduction rate of the cells can be measured by measuring the components of the culture supernatant.

[0034] The medium used in the culture of step (1) can be a basal medium used for culturing animal cells. The basal medium is not particularly limited as long as it can be used for culturing animal cells, and examples thereof include AIMV, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, improved MEM zinc option, IMDM, 199 medium, Eagle's MEM, αMEM, DMEM, Ham, RPMI-1640, Fischer's medium, and various commercially available products for T cell culture (e.g., CTS TM OpTmizer TM T-Cell Expansion Basal Medium (Thermo Fisher Scientific), CTS OpTmizer Pro Serum Free Medium (Thermo Fisher Scientific), CTS OpTmizer Pro TM (Thermo Fisher Scientific), CTS TM OpTmizer TM T-Cell Expansion Supplement (Thermo Fisher Scientific), 4Cell Nutri-T GMP Lymphocyte Medium (Sartorius), PRIME-XV T Cell Series (Fujifilm Wako Pure Chemical Corporation), ImmunoCult-XF T cell expansion medium (Veritas Corporation), RPMI 1640 Medium Series (Thermo Fisher Scientific), iMediam for T Medium (GC Lymphotec Co., Ltd.), TexMACS TM Medium (Miltenyi Biotec), Excellerate TMT Cell Expansion Media, Xeno-Free (R&D Systems), etc.), commercially available culture media for NK cells (e.g., LGM-3 (Lonza), X-VIVO TM 15 Serum-free lymphocyte medium (Lonza), Advanced RPMI 1640 Medium (Thermo Fisher Scientific), EALI TM 515-NK series (Boldo Biotechnology Co., Ltd.), ExCellerate TM Examples of suitable media include Human NK Cell Expansion Media, Xeno-Free (R&D Systems), NK Cell Initial Medium and NK Cell Subculture Medium (Japan BioTherapy Research Institute, Inc.) in the NK cell expansion kit BINKIT, and KBM NKCC-1, -2 (Kohjin Bio Co., Ltd.) in the KBN NK Kit. The media may contain serum or may be serum-free. The media may also contain serum substitutes (e.g., albumin, transferrin, Knockout Serum Replacement (KSR), CTS Immune Cell SR (Thermo Fisher Scientific), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, ITS supplement, B27™ supplement, etc.). Furthermore, the medium may contain one or more substances such as lipids, amino acids (e.g., non-essential amino acids), L-glutamine, vitamins, growth factors, cytokines, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. The pH of the medium at the start of culture is usually 7.0 to 7.8, preferably 7.2 to 7.6. After the start of culture, the pH varies depending on the type of cells to be cultured and the culture period, but is, for example, 6.8 to 7.8. Before use, the medium is preferably sterilized by a method such as filtration, ultraviolet irradiation, heat sterilization, or radiation exposure to prevent contamination. The culture conditions are not particularly limited, and the culture temperature is, for example, about 37 to 42°C, preferably about 37 to 39°C, and the medium is kept at a constant temperature for 24 hours. ２The concentration is, for example, 2% to 10%, preferably 2% to 5%, and the oxygen concentration is, for example, 1% to 20%, preferably 5% to 20%.

[0035] The "culture supernatant components" are not particularly limited as long as they can be used to predict the gene transfer rate and / or the number of gene-transfected cells. Examples of the culture supernatant components include the pH of the culture supernatant, metabolites, and extracellular vesicles. In the present invention, the culture supernatant components also include the pH of the culture supernatant. Examples of metabolites include organic acids, sugars, lipids, vitamins, amines, nucleic acids, amino acids, and other metabolite ions.

[0036] Examples of organic acids in metabolites include decenoic acid, lauric acid, dodecenoic acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, hexadecadienoic acid, stearic acid, oleic acid, vaccenic acid, linoleic acid, γ-linolenic acid, α-linolenic acid, eleostearic acid, arachidic acid, eicosenoic acid, eicosadienoic acid, mead acid, arachidonic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. , lignoceric acid, nervonic acid, 2-hydroxyvaleric acid, 2-oxoglutaric acid, indoxyl sulfate, 4-pyridoxy acid, 4-ethylphenyl sulfate, adipic acid, citric acid, cis-aconitic acid, isocitric acid, oxalosuccinic acid, oxaloacetic acid, fumaric acid, succinic acid, lactic acid, malic acid, phenyllactic acid, pantothenic acid, pipecolic acid, pyruvic acid, tartronic acid, p-cresol sulfate, xanthurenic acid, etc. Of these, preferred organic acids are lactic acid, 2-hydroxyvaleric acid, indoxyl sulfate, 4-pyridoxy acid, phenyllactic acid, and xanthurenic acid.

[0037] Examples of sugars in metabolites include fructose, inositol, galactose, glucose, sucrose, maltose, lactose, etc. Of these, glucose and fructose are preferred as sugars.

[0038] Examples of lipid metabolites include ceramide, Hex2-ceramide, Hex-ceramide, cholesterol, cholesterol sulfate, cardiolipin, monoglycerol, diglycerol, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, lysophosphatidylserine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, sphingomyelin, dihydrosphingomyelin, triglycerides, glycerol-3-phosphate, and ganglioside GM3. More specifically, the lipids include ceramide (16:0), Hex2-ceramide ((16:0) and (24:1)), Hex-ceramide ((16:0) and (24:1)), cholesterol, cholesterol sulfate, cardiolipin ((aaaa-70:5), (aaaa-70:6), (aaaa-72:6), and (aaaa-72:7)), monoglycerides, and the like. Serol ((e-16:0), (a-16:1), (a-18:1), and (a-20:0)), lysophosphatidylcholine ((a-14:0), (a-16:0), (a-16:1), (a-18:0), (a-18:1), (a-18:2), and (a-20:4)), lysophosphatidylethanolamine ((a-18:0), (a-18:1), and (aa-20:4)), phosphatidic acid ((aa-32:0), (aa-32:1), (aa-34:1), (aa-36:1), (aa-36:2), and (aa-36:3)), phosphatidylcholine ((aa-28:0), (aa-30:0), (aa-30:1), (aa-32:0), (aa-32:1), (aa-32:2), (aa-34:0) ), (aa-34:1), (aa-34:2), (aa-36:1), (aa-36:2), (aa-36:3), (aa-36:4), (aa-36:6), (aa-38:1) , (aa-38:2), (aa-38:3), (aa-38:4), (aa-38:5), (aa-38:6), (aa-38:7), (aa-40:2), (aa-40:3), (aa-40:4), (aa-40:5), (aa-40:6), (aa-40:7), (aa-42:5),(aa-42:6), (aa-42:7), (ae-26:0), (ae-28:0), (ae-30:0), (ae-32:0), (ae-32:1), (ae-34:0), (ae-34:1), (ae-34:2), (ae-36:2), (ae-36:3), (ae-36:4), (ae-38:4), and (ae-38:5)), phosphatidylethanolamine ((aa-28:0), (aa-30:0), (aa-30:1), (aa-32:0), (aa-32:1), (aa-32:2), (aa-34:0), (aa-34:1) , (aa-34:2), (aa-34:3), (aa-34:4), (aa-36:1), (aa-36:2), (aa-36:3), ( aa-36:4), (aa-36:5), (aa-38:2), (aa-38:3), (aa-38:4), (aa-38:5), (aa- 38:6), (aa-38:7), (aa-40:3), (aa-40:4), (aa-40:5), (aa-40:6), (aa-40 :7), (aa-40:8), (ae-32:1), (ae-32:2), (ae-34:1), (ae-34:2), (ae-34:3) , (ae-34:4), (ae-36:2), (ae-36:3), (ae-36:4), (ae-36:5), (ae-36:6), (ae-38:3), (ae-38:4), (ae-38:5), (ae-38:6), (ae-38:7), (ae-40:5), (ae-40:6), (ae-40:7), and (ae-40:8)), phosphatidylglycerol ((aa-32:0), (aa-32:1), (aa-34:1), (aa-34:2), (aa-36:1), (aa-36:2), and (aa-36:3)), phosphatidyl Nositol ((aa-32:1), (aa-34:1), (aa-34:2), (aa-36:1), (aa-36:2), (aa-36 :3), (aa-38:2), (aa-38:3), (aa-38:4), (aa-38:5), (aa-38:6), (aa-40:3) , (aa-40:4), (aa-40:5), and (aa-40:6)), phosphatidylserine ((aa-32:1), (aa-34: 0), (aa-34:1), (aa-34:2), (aa-36:1), (aa-36:2), (aa-36:3), (aa-38:2),(aa-38:3), (aa-38:4), (aa-38:5), (aa-40:4), and (aa-40:5)), sphingomyelin ((14:0), (16:0), (16:1), (18:0), (20:0), (22:0), (22:1), (24:0), (24:1), (24:2), (24:3), (26:1), and (26:2)), dihydrosphingomyelin Phosphorus (16:0), triglycerides ((aaa-42:0), (aaa-44:0), (aaa-44:2), (aaa-46:0), (aaa-46:1), (aaa-46:2), (aaa-48:0), (aaa-48:1), (aaa-48:3), (aaa-50:0), (aaa-50:1), (aaa-50:2), (aaa-50:3), (aaa-50:4) ), (aaa-52:1), (aaa-52:2), (aaa-52:3), (aaa-52:4), (aaa-52:5), (aaa-54:1), (aaa-54:2), (aaa- 54:3), (aaa-54:4), (aaa-54:5), (aaa-54:6), (aaa-54:7), (aaa-56:3), (aaa-56:4), (aaa-56:5), ( aaa-56:6), (aaa-56:7), (aaa-56:8), (aaa-58:4), (aaa-58:5), (aaa-58:6), (aaa-58:7), (aaa-58:8), (aaa-58:9), (aaa-60:7), and (aaa-60:8)), glycerol 3 phosphate, ganglioside GM3 ((16:0) and (24:1)), and the like. Among the lipids, cholesterol sulfate, FFA (C10:1), FFA (C16:2), FFA (C22:5), MG (a-16:1), PC (aa-28:0), PC (aa-30:0), PC (aa-30:1), PC (aa-32:1), PC (aa-34:2), PE (aa-34:2), PE (aa-36:2), PE (aa-36:3), and SM (16:0) are preferred.

[0039] Examples of vitamins in metabolites include α-tocopherol, nicotinamide, choline, retinol, ergocalciferol, cholecalciferol, tocotrienol, phylloquinone, menaquinone, thiamine, riboflavin, nicotinic acid, pantothenic acid, pyridoxal, pyridoxamine, pyridoxine, biotin, folic acid, cyanocobalamin, methylcobalamin, hydroxocobalamin, and ascorbic acid.

[0040] Examples of amines in the metabolites include ethanolamine, ethanolamine phosphate, and the like.

[0041] Examples of nucleic acids in metabolites include adenosine, methylthioadenosine, guanosine, 5-methyluridine, uridine, cytidine, etc. Of these, methylthioadenosine is preferred as a nucleic acid.

[0042] Examples of amino acids in metabolites include acylcarnitines (e.g., acylcarnitine (C5:0)), alanine, arginine, asparagine, aspartic acid, β-alanine, cysteine, cysteine-HPE-AM, cysteine-SS-HPE-AM, dimethylglycine, glutamic acid, glutamine, glycine, isoleucine, leucine, leucine acid, lysine, methionine, homocysteine-HPE-AM, N-acetyl-β-alanine, N-acetylcysteine, N-acetylserine, N-acetyltryptophan, N-carbamoyl-aspartate, O-acetylserine, ornithine, pyroglutamic acid, phenylalanine, proline, serine, S-sulfocysteine, taurine, threonine, tryptophan, tyrosine, and valine. Among these, preferred amino acids are alanine, arginine, cysteine-SS-HPE-AM, leucine acid, N-acetylcysteine, N-acetylserine, pyroglutamic acid, and S-sulfocysteine.

[0043] Examples of other metabolite ions in metabolites include ammonium ion, potassium ion, calcium ion, sodium ion, pyrophosphate, sulfate, sulfite, phenol sulfate, SS-bis-HPE-AM, etc. Among these, ammonium ion, potassium ion, calcium ion, and SS-bis-HPE-AM are preferred as other metabolite ions.

[0044] Extracellular vesicles are tiny vesicles with a membrane structure that are secreted, released, or otherwise transported by various types of cells. They are secreted from various cells and contain functional molecules such as proteins, microRNA, and mRNA, and are known to function as intermediaries for intercellular communication within the body. Examples of extracellular vesicles include exosomes, microvesicles, and apoptotic bodies. The size of exosomes is, for example, approximately 30 nm to 100 nm in diameter, the size of microvesicles is, for example, approximately 100 nm to 1.0 μm in diameter, and the size of apoptotic bodies is, for example, approximately 1.0 μm to 5.0 μm in diameter. Extracellular vesicles can be purified, separated, and concentrated from culture supernatants according to or in accordance with known methods, including, for example, ultracentrifugation (e.g., pellet-down method, sucrose cushion method, density gradient centrifugation, etc.), methods using immunoaffinity carriers, gel filtration, field-flow fractionation, FACS, and the like. In addition, purification, separation, and concentration of extracellular vesicles can also be performed using commercially available kits, etc. Exosomes are preferred as extracellular vesicles.

[0045] The method for measuring the components of the culture supernatant is not particularly limited as long as it can measure (especially quantitate) the components of the culture supernatant. Examples include methods using various commercially available measurement devices, methods using various commercially available measurement reagents and measurement kits, etc. Measurement of the components of the culture supernatant can also be performed by Raman spectroscopy. Regarding the measurement of extracellular vesicles in the culture supernatant, the basic parameters for physically evaluating particles are particle size and zeta potential. Examples of particle size analyzers include transmission electron microscopes (TEM), submerged atomic force microscopes (AFM), nanoparticle tracking analysis (NTA), particle size analyzers using the Coulter counter method, and flow cytometers. Furthermore, for proteins, microRNAs, mRNAs, and other components of extracellular vesicles, various methods used in so-called omics analysis, such as transcriptomes and proteomes, can be used (e.g., microarrays, sequencers, Western blots, mass spectrometry, etc.). In measuring extracellular vesicles, it is preferable to measure the particle size of extracellular vesicles (particularly exosomes), and it is also preferable to measure proteins contained in extracellular vesicles (particularly exosomes).

[0046] Examples of devices for measuring metabolites in culture supernatants include FLEX2 (Novo Biomedical), STAT PROFILE Prime series (Novo Biomedical), Cedex analyzer series (Roche Diagnostics), ABL800 FLEX blood gas analyzer series (RADIOMETER), REBEL cell culture component analyzer (ST Japan Co., Ltd.), Bioflow BF series (Oji Scientific Instruments Co., Ltd.), RapidPoint 500e blood gas analyzer (SIEMENS Healthineers), and Extracellular Flux Analyzer XF series (Prime Tech Co., Ltd.). Examples of devices for measuring the pH of culture supernatants include various commercially available pH meters. Examples of devices for measuring extracellular vesicles in culture supernatants include the Nanoparticle Analysis System NANOSIGHT series (Japan Quantum Design Co., Ltd.), EXOVIEW IMAGER (Quantum Design), ExoCounter (Sysmex Corporation), VIDEODROP Nanoparticle Imaging Analyzer (Meiwafosis Co., Ltd.), NanoFCM (Meiwafosis Co., Ltd.), Exoid Nanoparticle Multi-Analyzer (Meiwafosis Co., Ltd.), ZE5 Cell Analyzer series (Bio-Rad Laboratories), CytoFLEX series (Thermo Fisher Scientific), and BD FACSymphony. TMseries (BD Bioscience), nanoparticle size and concentration counter nCS1 (Spectradyne), disc centrifugal particle size distribution analyzer CPS Disc Centrifuge series (CPS Instruments), laser diffraction / scattering particle size distribution analyzer Bettersizer2600 (Betterisize), etc. Examples of measuring devices using Raman spectroscopy include the RamanRxn Systems series (Endless+Hauser), LabRAM HR Evolution, LC-Raman System, AFM Raman, LabRAM Soleil, XploRA PLUS, Standard Microscope Spectroscopy Systems (HORIBA, Ltd.), inVia series, Virsa fiber Raman device, RA816, RA802 (Renishaw), TRS100, RapID, Vaya (Agilent Technologies), ProCellics (Merck Millipore), Ramina Process Analyzer (Thermo Fisher Scientific), BioPAT Spectro (Sartorius), BRAVO, SENTERRAII, MultiRAM, RAM II (Bruker), etc. Measurement of the components of the culture supernatant using a measuring device can be carried out according to known methods, for example, according to the manual of the measuring device.

[0047] Step (2) In step (2), the gene transfer rate and / or the number of gene-transferred cells is predicted based on the values ​​measured in step (1).

[0048] In the Examples described below, it has been confirmed that there is a correlation between the measured values ​​of the components of the culture supernatant (e.g., pH, concentration) and the gene transfer rate and / or the number of gene-transferred cells, and therefore the measured values ​​of the components of the culture supernatant can be used to predict the gene transfer rate and / or the number of gene-transferred cells. In the present invention, the term "prediction" has the same meaning as "measurement" and can be used interchangeably.

[0049] A method for using the measured values ​​of the components of the culture supernatant to predict the gene transfer rate and / or the number of gene-transfected cells in a cell population includes, for example, creating a calibration curve based on the gene transfer rate and / or the number of gene-transfected cells in cells whose gene transfer rate and / or the number of gene-transfected cells are known and the measured values ​​of the components of the culture supernatant, and using the calibration curve to determine the gene transfer rate and / or the number of gene-transfected cells from parameters obtained from the values ​​measured in step (1). The calibration curve can be created using software or the like by a standard method such as the least squares method.

[0050] In step (2), the gene transfer rate and / or the number of gene-transfected cells may be predicted based on one type of component of the culture supernatant, or based on a combination of two or more types of components of the culture supernatant. When a combination of two or more types of components of the culture supernatant is used, the combination may be 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc., with no particular upper limit, and examples thereof include 30 or less, 20 or less, 10 or less, etc.

[0051] The metabolites in the culture supernatant, glutamine, glucose, potassium ions, and calcium ions, are negatively correlated with the transfection rate and the number of transfected cells, whereas lactate, ammonium ions, PC(aa-28:0), PC(aa-30:0), PC(aa-30:1), PC(aa-32:1), PC(aa-34:2), PC(aa-36:2), PC(aa-36:3), FFA(C10:1), and phenyllactic acid are positively correlated with the number of transfected cells. Furthermore, the pH of the culture supernatant is negatively correlated with the transfection rate and the number of transfected cells.

[0052] For example, glutamine, glutamic acid, glucose, potassium ions, calcium ions, lactate, ammonium ions, pH, PC(aa-28:0), PC(aa-30:0), PC(aa-30:1), PC(aa-32:1), PC(aa-36:2), FFA(C10:1), phenyllactic acid, etc. can be used individually to predict the gene transfer rate and / or the number of gene-transfected cells in a cell population for each number of days of culture. When predicting the gene transfer rate and / or the number of gene-transfected cells in a cell population for each number of days of culture, multiple components can also be combined to make the prediction. Components used for such prediction include, for example, alanine, arginine, N-acetylcysteine, S-methyl-5-thioadenosine, indoxyl sulfate, SS-bis-HPE-AM, N-acetylserine, 2-hydroxyvaleric acid, PC(aa-28:0), PC(aa-30:0), PC(aa-30:1), PC(aa-32:1), PE(aa-34:2), PE(aa-36:2), PE(aa-36:3), FFA(C10:1), FFA(C16:2), FFA(C22:5), lactic acid, leucine acid, phenyllactic acid, SM(16:0), fructose, 4-pyridoxalic acid, MG(a-16:1), xanthurenic acid, pyroglutamic acid, S-sulfocysteine, cholesterol sulfate, and the like.

[0053] When predicting the gene transfer rate and / or the number of gene-transfected cells of a cell population regardless of the number of days of culture, PC(aa-28:0), PC(aa-30:0), PC(aa-30:1), PC(aa-32:1), PC(aa-34:2), PC(aa-36:2), PC(aa-36:3), FFA(C10:1), phenyllactic acid, etc. can be used alone to predict the gene transfer rate and / or the number of gene-transfected cells of a cell population. When predicting the gene transfer rate and / or the number of gene-transfected cells of a cell population regardless of the number of days of culture, it is preferable to perform the prediction by combining multiple components. Components used for such prediction include, for example, PC(aa-28:0), PC(aa-30:0), PC(aa-30:1), PC(aa-32:1), PC(aa-34:2), PE(aa-34:2), PE(aa-36:2), PE(aa-36:3), FFA(C10:1), FFA(C16:2), lactic acid, leucine acid, phenyllactic acid, cysteine-SS-HPE-AM, etc.

[0054] When predicting the gene transfer rate and / or the number of transfected cells based on a combination of two or more components of a culture supernatant, the prediction can be performed, for example, by multivariate analysis, particularly by using a prediction model obtained by regression analysis. Such regression analysis methods can be any method capable of performing regression analysis, including lasso regression, PLS (Partial Least Squares), OPLS (Orthogonal Partial Least Squares), ridge regression, elastic net regression, principal component regression, partial least squares regression, random forest, neural network, deep learning, and support vector machine. Furthermore, components with high variable importance can be selected from the results of these regression analyses, and these selected components can be used for prediction. When performing multivariate analysis, even components with low variable importance can contribute to improved accuracy by using them in the multivariate analysis. Therefore, even components with low variable importance, or components that do not show a correlation with the gene transfer rate and / or the number of transfected cells on their own, can be used to predict the gene transfer rate and / or the number of transfected cells.

[0055] In the Examples described below, it has been confirmed that the measured values ​​of extracellular vesicles in the culture supernatant (particularly the particle size of exosomes and the profile of exosome proteins) correlate with the gene transfer rate and / or the number of gene-transferred cells. Therefore, the gene transfer rate and / or the number of gene-transferred cells can be predicted using the measured values ​​of extracellular vesicles in the culture supernatant. The particle size of extracellular vesicles is negatively correlated with the gene transfer rate and the number of gene-transferred cells. Proteins encapsulated in extracellular vesicles, such as those shown in FIG. 16C, can be used alone or in combination of two or more types to predict the gene transfer rate and / or the number of gene-transferred cells in a cell population.

[0056] In the Examples described below, it has been confirmed that there is a correlation between the gene transfer rate and / or the number of gene-transfected cells measured by Raman spectroscopy (Raman spectroscopy spectrum) of the components of the culture supernatant and the gene transfer rate and / or the number of gene-transfected cells, and therefore the gene transfer rate and / or the number of gene-transfected cells can be predicted by using the Raman spectroscopy measured values ​​of the components of the culture supernatant. For example, the height of the peak in the Raman spectroscopy spectrum can be used to predict the gene transfer rate and / or the number of gene-transfected cells of a cell population.

[0057] In step (2), the changes in the components of the culture supernatant before and after gene transfer can also be used to predict the gene transfer rate and / or the number of gene-transferred cells. In the Examples described below, it has been shown that the components of the culture supernatant change before and after gene transfer.

[0058] When the cells into which a gene (e.g., a nucleic acid encoding a chimeric antigen receptor) has been introduced are αβ T cells, the component measured in step (1) is preferably at least one selected from the group consisting of lactic acid, ammonium ions, pH, glutamine, glucose, phenyllactic acid, FFA (C10:1), PC (aa-28:0), PC (aa-30:0), PC (aa-30:1), PC (aa-32:1), PE (aa-34:2), PE (aa-36:2), and PE (aa-36:3), and more preferably at least one selected from the group consisting of lactic acid, ammonium ions, pH, glutamine, and glucose.

[0059] When the cells into which a gene (e.g., a nucleic acid encoding a chimeric antigen receptor) has been introduced are NK cells, the component measured in step (1) is preferably at least one selected from the group consisting of lactic acid, ammonium ions, glucose, glutamic acid, sodium ions, and pH.

[0060] When the gene (e.g., nucleic acid encoding a chimeric antigen receptor)-transfected cells are gdT cells, the component to be measured in step (1) is preferably lactic acid.

[0061] The method for producing a gene-introduced cell preparation of the present invention (sometimes referred to simply as the "production method of the present invention" in this specification) is characterized by comprising the steps of: (I) measuring components of a culture supernatant in which animal cells have been cultured, and determining the gene introduction rate and / or the number of gene-introduced cells based on the measured values.

[0062] The production method of the present invention may further comprise the step (II) of introducing a gene into an animal cell.

[0063] Steps (I) and (II) of the production method of the present invention can be carried out by the method described above. The cell preparation produced by the production method of the present invention (hereinafter sometimes referred to as the cell preparation of the present invention) is preferably produced as a parenteral preparation by mixing an effective amount of transfected animal cells with a pharmaceutically acceptable carrier according to known means (e.g., the method described in the Japanese Pharmacopoeia, etc.). The cell preparation of the present invention is preferably produced as a parenteral preparation such as an injection, suspension, or infusion. Parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. Pharmaceutically acceptable carriers include, for example, solvents, bases, diluents, excipients, soothing agents, buffers, preservatives, stabilizers, suspending agents, isotonic agents, surfactants, and solubilizing agents.

[0064] The dosage of the cell preparation of the present invention can be determined appropriately depending on various conditions, such as the patient's weight, age, sex, and symptoms. It may be administered once or multiple times. The cell preparation of the present invention can be in a known form suitable for parenteral administration, such as an injection or infusion. Furthermore, the cell preparation of the present invention may contain physiological saline, phosphate-buffered saline (PBS), a culture medium, etc., to stably maintain the cells. Examples of culture media include, but are not limited to, RPMI, AIM-V, and X-VIVO10. Furthermore, the cell preparation may contain a pharmaceutically acceptable carrier (e.g., human serum albumin), a preservative, etc., for stabilization purposes. The cell preparation of the present invention is intended for use in mammals, including humans.

[0065] The method for predicting the gene transfer rate and / or the number of gene-transduced cells of the present invention does not require the complicated steps required for conventional methods using flow cytometry or the like, which require manual labor. For example, the method requires only automated measurement using a measuring device, making it possible to measure the gene transfer rate and / or the number of gene-transduced cells with a simple procedure. Furthermore, the method for predicting the gene transfer rate and / or the number of gene-transduced cells of the present invention enables efficient measurement of the gene transfer rate in a short time, for example, less than 5 minutes, compared to the 6-7 hours required for conventional methods. Furthermore, since sampling is not required, there is no stirring step and damage to the cells is minimal. Furthermore, the method of the present invention is not particularly limited to the type of cell and can be applied to a variety of cells. The method of the present invention is anticipated to be applied to gene therapies such as CAR-T cell therapy.

[0066] As used in this specification and claims, singular terms include plurals and plural terms include the singular, unless the context otherwise requires. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept, unless otherwise specified.

[0067] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0068] Culture medium for CAR-αβ T cells (hereinafter also referred to simply as CAR-T cells) and CAR-gdT cells: A basal medium was prepared by adding 2.6% OpTmizer Expansion Basal Supplement (Thermo Fisher Scientific), 1% L-Glutamine (Thermo Fisher Scientific), 1% Streptomycin, and 2% CTS Immune Cell SR (Thermo Fisher Scientific) to OpTmizer CTS T-Cell Expansion basal medium (Thermo Fisher Scientific). MACS GMP IL-2 (Miltenyi Biotec) was added to the medium at a concentration of 20 IU / mL or 40 IU / mL. SK-HEP-1 cell culture medium: MEM, L-Gln(+) (Thermo Fisher Scientific) supplemented with 10% FBS (Biosera), 1% non-essential amino acids (Fujifilm Wako Pure Chemical Corporation), 1% penicillin-streptomycin solution (Fujifilm Wako Pure Chemical Corporation), and 1 mM sodium pyruvate (Fujifilm Wako Pure Chemical Corporation).

[0069] <Production of CAR-T cells and CAR-gdT cells> After thawing Leukopak (Charles River Laboratories) or gdT cells (Charles River Laboratories), the cells were diluted to 2.0 x 10 6The CAR-T cell and CAR-gdT cell culture medium was diluted to a concentration of 0.01x10 cells / mL or less (raw material before manufacture). Cell suspension: MACS GMP T-Cell TransACT (Miltenyi Biotec) was seeded into culture bags at a ratio of 17.5:1 and cultured for more than 36-48 hours. After culture, the cells were diluted with culture medium using a LOVO Cell processing system (Fresenius Kabi) or a centrifuge, and 6.07x10 cells were added to culture bags pre-coated with RetroNectin™ (Takara Bio Inc.) and a retrovirus carrying the CAR gene. 5 cells / cm 2 The following cells were seeded and cultured until the next day: 2.2x10 6 cells / cm 2 The cells were seeded in culture bottles (G-REX, Wilson Wolf) and cultured for 3-7 days to produce CAR-T cells and CAR-gdT cells (amplification step). The CAR gene used had the nucleotide sequence shown in SEQ ID NO: 1 (the same applies to SK-HEP-1 cells below).

[0070] <Production of CAR-NK cells> PBMCs (Charles River Laboratories) were thawed and then cultured using BINKIT™ for NK cells expansion from PBMCs (Japan BioTherapy Institute, Inc.), and CAR-NK cells were produced using RetroNectin™ (Takara Bio Inc.) and a retrovirus carrying a CAR gene.

[0071] <Transfection of CAR gene into SK-HEP-1 cells> SK-HEP-1 cells (ATCC) were cultured at 2.0 x 10 6 The cells were diluted with SK-HEP-1 cell culture medium to a concentration of 6.07 x 10 cells / mL or less and plated onto a culture plate pre-coated with RetroNectin (Takara Bio Inc.) and a retrovirus carrying the CAR gene. 5 cells / cm 2 The CAR gene was introduced into SK-HEP-1 cells by seeding them as follows and culturing them for 4 days.

[0072] <Measurement of gene transfer efficiency using flow cytometry> Gene transfer efficiency was measured using a BD FACSCanto II flow cytometer (BD Biosciences) using a CAR antigen.

[0073] <Measurement of Culture Supernatant Components Using a Cell Environment Analyzer> The components of the culture supernatant were quantified using Bioprofile FLEX2 (Novo Biomedical).

[0074] <Measurement of Glucose Uptake Ability> Glucose uptake ability was measured using a Glucose Uptake Detection Kit Green (Dojindo Laboratories, Inc.) according to the manual.

[0075] <Metabolome and lipidome analysis> Nine volumes of methanol were added to the culture supernatant, which was then vortexed to homogenize it, and the mixture was analyzed by hydrophilic interaction chromatography / tandem mass spectrometry (HILIC / MS / MS), lipidomics, and gas chromatography / tandem mass spectrometry (GC / MS / MS).

[0076] For HILIC / MS / MS, 100 μL of homogenate was centrifuged at 21,500 g for 5 min at 4°C. 76 μL of the resulting supernatant was mixed with 4 μL of 400 mM ammonium formate. The mixture was vortexed and centrifuged at 21,500 g for 5 min at 4°C. 2 μL of the supernatant was injected into a liquid chromatography / tandem mass spectrometry (LC / MS / MS) system consisting of a UHPLC Nexera liquid chromatography system (Shimadzu Corporation) and a 5500QTRAP mass spectrometer (AB Sciex Pte.). Chromatographic separation was performed using a ZIC-cHILIC column (2.1 × 100 mm, 3 μm, Merck Millipore). Metabolites were detected using selected reaction monitoring (SRM), and the abundance of each metabolite was assessed as the peak area on the SRM chromatogram.

[0077] For lipidomics, 2 μL of the supernatant obtained by centrifugation of the homogenate was analyzed using an LC / MS / MS system consisting of an Ultimate3000 RSLC system (Thermo Fisher Scientific) and a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). Liquid chromatographic separation was performed using a CORTECS T3 reversed-phase column (2.1 × 50 mm, 2.7 μm, 120 Å, Waters).

[0078] GC / MS / MS analysis was performed by centrifuging the homogenate, drying 10 μL of the supernatant under a nitrogen stream, and then derivatizing the compounds through a two-step reaction: oximation and trimethylsilylation. 1 μL of the reaction mixture was injected into an Agilent 7890B Series gas chromatograph using a GC Injector 80 autosampler (Agilent Technologies). Gas chromatographic separation was performed on a J&W Scientific DB-5MS-DG column (30 m × 0.25 mm id, df = 0.25 μm, Agilent Technologies). Each target molecule was detected by SRM, and its peak area was evaluated using MassHunter software (Agilent Technologies).

[0079] Statistical Analysis of Metabolomic and Lipidomic Data Using the statistical analysis software R, statistical analysis was performed on the metabolite measurement data of the culture supernatant obtained by metabolomic and lipidomic analysis. Regarding the differences in the supernatant components between untransfected T cells (UTD) and CAR-T cells, a Williams test was performed, assuming that the difference between each component increases with the CAR transduction rate when UTD was used as the control group. Furthermore, a simple regression analysis was performed for all metabolites and each metabolite with CAR cell count. Furthermore, LASSO regression and OPLS regression were performed as multivariate analyses to analyze the selection of components contributing to the regression to CAR cell count or the importance of variables. Regression analysis was performed on measurements for each day during the amplification process and for the entire day.

[0080] <Exosome particle size analysis> Exosomes in the culture supernatant were analyzed using MagCapture. TM Exosomes were isolated using Exosome Isolation Kit PS ver.2 (Fujifilm Wako Pure Chemical Industries, Ltd.), and particle size was then measured using Nanosite LM10 (Japan Quantum Design Co., Ltd.).

[0081] <Proteome analysis of exosomes> Exosomes in the culture supernatant were analyzed using MagCapture. TMExosomes were isolated using the Exosome Isolation Kit PS ver.2 (Fujifilm Wako Pure Chemical Corporation). Eight volumes of cold acetone (Fujifilm Wako Pure Chemical Corporation) were added to the exosome suspension and allowed to stand overnight at -20°C. The supernatant was removed by centrifugation (15,000 × g, 4°C, 20 minutes), and the resulting precipitate was suspended in cold acetone. The suspension was mixed by inversion and centrifuged again (15,000 × g, 4°C, 5 minutes), and the supernatant was removed. Finally, the precipitate was air-dried (left at room temperature for 30 minutes). The dried precipitate was dissolved in 10 μL of solubilization buffer [8 M urea (Fujifilm Wako Pure Chemical Corporation), 50 mM Tris-HCl, pH 8.0 (Nippon Gene Co., Ltd., 312-90061)] (protein lysate). The entire protein lysate was subjected to hydrolysis with trypsin (Promega). Specifically, 1.1 μL of 100 mM dithiothreitol (DTT) (Fujifilm Wako Pure Chemical Corporation) solution was added to the protein solution and incubated at 37°C for 30 minutes (final DTT concentration: 10 mM). Next, 1.2 μL of 250 mM iodoacetamide (IAA) (Fujifilm Wako Pure Chemical Corporation, 095-02151) solution was added and incubated in the dark for 20 minutes (final IAA concentration: 25 mM) (reductive alkylation). After reductive alkylation, 27.7 μL of 50 mM Tris-HCl (pH 8.0) was added to reduce the urea concentration to 2 M. Finally, 1 μL of 1 μg / μL trypsin solution was added, and the solution was incubated at 37°C for 16 hours for hydrolysis (final trypsin concentration: 24.4 ng / μL). The peptide solution after the reaction was desalted using a C18 + SDB STAGE Tip (Rappsilber, J. et al., Anal. Chem. 2003, 75: 663-70). The desalted sample was dried under reduced pressure. The dried peptide sample was dissolved in a solvent consisting of water (Fujifilm Wako Pure Chemical Corporation, 210-01303), acetonitrile (Fujifilm Wako Pure Chemical Corporation, 018-19853), and trifluoroacetic acid (Fujifilm Wako Pure Chemical Corporation, 204-02743) (volume ratio 98:2:0.1). The volume of the solvent added was adjusted to 0.1 μg / μL based on the total protein amount (equivalent to 2 μg), and the solution was subjected to LC-MS / MS.

[0082] The LC-MS / MS data from each sample was processed as follows. Specifically, peak alignment and sequence database searches were performed using Thermo Fisher Scientific's Proteome Discoverer (ver. 3.0) (hereafter referred to as "PD 3.0") software (https: / / www.thermofisher.com / jp / ja / home / industrial / mass-spectrometry / liquid-chromatography-mass-spectrometry-lc-ms / lc-ms-software / multi-omics-data-analysis / proteome-discoverer-software.html). Sequest HT software was used for the sequence database search. The identification information of significant peptides was linked to each detected peak, and the intensity value of the linked detected peak was used as the detected intensity of that peptide (two types: detected intensity before normalization "Abundance" and intensities after normalization "Abundances (Normalized)"). Peak detected intensities were normalized according to the PD 3.0 algorithm.

[0083] <Raman spectroscopy measurement> Raman spectroscopy of the culture supernatant was measured using the RamanRxn2 system, a Raman analyzer manufactured by Endress+Hauser. The obtained spectra were then subjected to second-order differentiation using the statistical analysis software R, and the spectra of samples with different CAR gene transfection rates were compared.

[0084] Example 1: Changes in Culture Supernatant Composition in T Cells Following CAR Gene Transduction CAR-T cells were produced, and the cell density, viability, and gene transduction efficiency after production were measured. The results are shown in Figure 1. As a result, a slight decrease in viability was observed with or without CAR gene transduction (Figure 1B), but there was no change in cell density (Figure 1A). However, in the culture supernatant containing phenol red, the pH of CAR-T cells was observed to be lower and more acidic than that of T cells not transfected with the gene (UTD) (Figures 1D-1E). Furthermore, to investigate changes in the supernatant composition, metabolites were measured. The results are shown in Figure 2. As a result, in the culture supernatant of CAR-T cells, decreases in glutamine (Gln), glucose (Gluc), potassium ions, and calcium ions were observed, and increases in lactate (Lac) and ammonium ions were observed compared to UTD. However, no changes were observed in glutamate (Glu) and sodium ions (Figures 2A-2H).

[0085] Example 2: Changes in Metabolism in T Cells Following CAR Gene Transfer Since changes were observed in the culture supernatant in Example 1, changes in metabolism in T cells following CAR gene transfer were confirmed. The results are shown in Figure 3. After production, untransfected T cells (UTD) and CAR-T cells were replaced with fresh medium and cultured. Cell counts and analysis of the culture supernatants were performed 1, 3, 6, and 24 hours later (Figure 3A). No increase in cell number was observed in UTD and CAR-T cells during the 24-hour culture period (Figure 3B). However, a decrease in glucose and an increase in lactate were observed in the culture supernatant of CAR-T cells, confirming an increase in glycolysis (Figures 3C-3D). Furthermore, a decrease in glutamine and an increase in ammonium ions were observed in the culture supernatant of CAR-T cells, confirming an increase in glutamine metabolism (Figures 3E-3G). These results confirmed that CAR gene transfer alters metabolism.

[0086] Example 3: Change in glucose uptake in T cells due to CAR gene transfer Since the results of Example 2 confirmed that CAR gene transfer changes metabolism, the actual amount of glucose uptake was quantified. The results are shown in Figure 4. As a result, it was confirmed that CAR gene transfer increases the amount of glucose uptake.

[0087] Example 4: Prediction of CAR-T Cell Count on the Final Day of Production from Components in the Culture Supernatant Based on the results of Examples 1-3, it is inferred that the CAR-T cell count correlates with the components in the culture supernatant. To confirm this, CAR gene-transduced and untransduced T cells were prepared from T cells from one donor. Subsequently, cells were mixed so that the gene-transduced T cells were 0, 25, 50, 75, and 100% and cultured for 4 days. Although there was no significant difference in cell count, cell populations containing CAR-T cells at a ratio corresponding to the mixing ratio were produced in terms of the CAR transduction rate ( Figures 5-1A and 5-1B ). Correlations between the culture supernatant and cell density and CAR-T cell density on the final day of production were confirmed, and none of the components in the culture supernatant showed a correlation with cell density. Meanwhile, CAR-T cell density showed a positive correlation with lactate and ammonium ions, and a negative correlation with pH, ​​glutamine, and glucose. However, other components showed no correlation with CAR-T cell density (Fig. 5-2C-F, Fig. 5-3G-L, Fig. 5-4M-T). Based on these results, it is possible to predict the number of CAR-T cells from the lactate, ammonium ion, pH, glutamine, and glucose in the culture supernatant on the final day of production.

[0088] Example 5: Prediction of CAR-T cell numbers over time from components in culture supernatants from multiple donors Using the same production method as in Example 4, CAR-T cells were prepared from T cells from three donors, and the cell density and CAR transduction rate were measured on all culture days from expansion days 1 to 4. The results are shown in Figures 6A-B. Subsequently, when the correlation between cell density and CAR-T cell density was confirmed from the culture components found in Example 4, a correlation was also found with CAR-T cell density, but a higher correlation was observed with cell density (Figures 7A-J).

[0089] Example 6: Comprehensive Analysis of Culture Supernatant Using Metabolomic and Lipidomic Analysis Metabolomic and lipidomic analysis was performed using the culture supernatant used in Example 5. Regarding the differences in supernatant components between non-transfected T cells (UTD) and CAR-T cells, a Williams test was performed assuming that the difference between each component increases with the CAR transduction rate when UTD was used as the control group, and components whose difference increases with the CAR transduction rate were extracted. The results are shown in Figure 8. By plotting the -log(p value) from the Williams test on the vertical axis and the estimated value of the change under the highest CAR transduction rate on the horizontal axis, components accumulated in the supernatant in a CAR transduction rate-dependent manner are plotted in the upper right, and components consumed in a CAR transduction rate-dependent manner are plotted in the upper left. The abbreviations for lipid components in the figure are as follows: PC: phosphatidylcholine; PE: phosphatidylethanolamine; SM: sphingomyelin; FFA: free fatty acid. For more detailed classification, ester bonds between glycerol and fatty acids are designated as a, while ether bonds are designated as e. The a and e values ​​indicate the number of fatty acid residues attached to the glycerol moiety. The total carbon number (x) and the number of double bonds (y) in the attached fatty acid residues are designated as x:y. For example, PC (aa-34:1) indicates that two fatty acids are attached to the glycerol moiety of phosphatidylcholine via ester bonds, with a total of 34 carbon atoms and one double bond. HPE-AM is an abbreviation for β-(4-hydroxyphenyl)ethyl iodoacetamide and is used to stabilize and detect sulfur compounds such as persulfides and polysulfides. Therefore, Cysteine-SS-HPE-AM in the figure indicates cysteine ​​persulfide, etc., and SS-bis-HPE-AM indicates sulfur metabolites such as H2S2.

[0090] It was observed that PE (aa-34:2), PC (aa-30:1), PE (aa-36:2), PC (aa-30:0), FFA (C10:1), etc. accumulated in the supernatant in a CAR introduction rate-dependent manner, and FFA (C16:2), arginine, glutamine, etc. were consumed in a CAR introduction rate-dependent manner.

[0091] Example 7 Prediction of CAR-T cell number from components in culture supernatant using metabolomic / lipidomic analysis Metabolomic / lipidomic analysis was performed using the culture supernatant used in Example 5 to confirm whether there was a component that could predict the number of CAR-T cells.

[0092] For all metabolites, the measured values ​​of each metabolite were calculated at a cell density of 10 6 After correcting for the number of CAR-T cells per cell, simple regression analysis with the number of CAR-T cells was performed. The results are shown in Figure 9. The coefficient of determination R 2 The components with high values ​​were PC(aa-30:0), PC(aa-32:1), and PE(aa-36:2) on day 2 of amplification; FFA(C10:1) and PC(aa-30:1) on day 3 of amplification; and FFA(C10:1), PC(aa-28:0), PC(aa-30:1), and phenyllactic acid on day 4 of amplification. Furthermore, LASSO regression and OPLS regression were performed as multivariate analyses using the same adjusted values ​​to analyze the selection of components contributing to the regression to CAR-T cell count or the importance of variables. The results are shown in Figure 10. For LASSO regression, the actual and predicted CAR-T cell counts on days 2, 3, and 4 of amplification during the amplification process are shown (Figures 10A-C), and the coefficients of metabolites selected during regression are also shown in the same graphs. Similarly, for OPLS regression, the actual and predicted numbers of CAR-T cells on days 1, 2, 3, and 4 of amplification during the amplification process are shown (Figures 10D-10G), and metabolites with high variable importance are also shown in the same graph.

[0093] In addition, simple regression, LASSO regression, and OPLS regression were also performed on the measurements taken over the entire period to confirm whether the number of CAR-T cells could be predicted regardless of the period. The results are shown in Figures 11-12. The coefficient of determination in simple regression, R 2The components with high variable importance were extracted as follows: phenyllactic acid, FFA (C10:1), PC (aa-28:0), PC (aa-30:0), PC (aa-30:1), PC (aa-32:1), PE (aa-34:2), PE (aa-36:2), and PE (aa-36:3) (Figure 11A-I). In addition, the predictions of the LASSO regression and the selected compounds and their coefficients (Figure 12A) and the predictions of the OPLS regression and the compounds with high variable importance (Figure 12B) were confirmed.

[0094] Example 8: Changes in metabolism in SK-Hep-1 cells due to CAR gene transfer SK-Hep-1 cells, a hepatic cancer cell line, were used to confirm whether cellular metabolism was changed by transfer of the CAR gene used in Example 1, regardless of the type of cells transferred (i.e., other than T cells). The results are shown in Figure 13. As in Example 1, an increase in lactic acid in the culture supernatant was observed due to CAR gene transfer.

[0095] Example 9: Change in metabolism in gdT cells due to CAR gene introduction Using gdT cells, it was confirmed whether cell metabolism was changed by introduction of the CAR gene used in Example 1. The results are shown in Figure 14. As a result, an increase in lactic acid in the culture supernatant was observed due to CAR gene introduction.

[0096] Example 10: Changes in metabolism in NK cells due to CAR gene introduction CAR-NK cells were produced, and the correlation between the components in the culture supernatant and the number of CAR-NK cells was confirmed. The results are shown in Figure 15. There was a positive correlation between the number of CAR-NK cells and lactate and ammonium ions, and a negative correlation between glucose, glutamate, sodium ions, and pH. The number of CAR-NK cells can be predicted from the components of the culture supernatant.

[0097] Example 11: Changes in exosomes due to CAR gene transfer We examined whether exosomes, which are intracellular vesicles in the culture supernatant, were changed by the CAR gene transfer used in Example 1. The results are shown in Figure 16. We confirmed that the particle size of exosomes was reduced compared to that of T cells by CAR gene transfer (Figure 16A). Next, to confirm the exosome profile, proteome analysis was performed on exosomes derived from CAR-T cells or T cells. Volcano plot analysis revealed that the exosome protein profiles were different between CAR-T cells and T cells (Figure 16B). A t-test was performed on exosome proteins that were significantly increased in CAR-T cells (Figure 16C, N=3). Furthermore, analysis was performed using the proteins in Figure 16C for predicting CAR-T cell count as an example, and we confirmed that the number of CAR-T cells could be predicted from CD63-positive exosomes (Figure 16D, R 2 =0.9846).

[0098] Example 12: Changes in Raman spectroscopy due to CAR gene introduction It was confirmed whether the Raman spectroscopy of the culture supernatant changes due to introduction of the CAR gene used in Example 1. The results are shown in Figure 17. Figure 17A shows the entire second derivative of the acquired Raman spectroscopy spectrum. Differences were observed in various peaks between CAR-introduced cells and non-introduced cells. Figure 17B shows an example of such peaks.

[0099] This application is based on Japanese Patent Application No. 2023-99695 filed on June 16, 2023, the contents of which are incorporated in their entirety herein.

Claims

1. A method for predicting the gene transfer rate and / or the number of gene-transferred cells in animal cells, comprising: (1) measuring components of a culture supernatant in which gene-transferred animal cells have been cultured; and (2) predicting the gene transfer rate and / or the number of gene-transferred cells based on the values ​​measured in step (1).

2. The method according to claim 1, wherein in step (1), the pH, metabolites or extracellular vesicles of the culture supernatant are measured.

3. The method according to claim 2, wherein the metabolite is at least one selected from the group consisting of organic acids, sugars, lipids, vitamins, amines, nucleic acids, amino acids and other metabolite ions.

4. The method of claim 3, wherein the organic acid is at least one selected from the group consisting of decenoic acid, dodecenoic acid, myristoleic acid, palmitic acid, palmitoleic acid, hexadecadienoic acid, stearic acid, oleic acid, linoleic acid, gamma-linolenic acid, alpha-linolenic acid, arachidic acid, eicosenoic acid, eicosadienoic acid, mead acid, arachidonic acid, docosapentaenoic acid, docosahexaenoic acid, nervonic acid, 2-hydroxyvaleric acid, 2-oxoglutaric acid, indoxyl sulfate, 4-pyridoxy acid, 4-ethylphenyl sulfate, adipic acid, citric acid, fumaric acid, lactic acid, malic acid, phenyllactic acid, pantothenic acid, pipecolic acid, pyruvic acid, tartronic acid, paracresol sulfate, and xanthurenic acid.

5. The method of claim 3, wherein the sugar is at least one selected from the group consisting of fructose, inositol, galactose, glucose, and sucrose.

6. The method of claim 3, wherein the lipid is at least one selected from the group consisting of ceramide, Hex2-ceramide, Hex-ceramide, cholesterol, cholesterol sulfate, cardiolipin, monoglycerol, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, sphingomyelin, dihydrosphingomyelin, triglyceride, glycerol 3-phosphate, and ganglioside GM3.

7. The method of claim 3, wherein the vitamin is at least one selected from the group consisting of alpha-tocopherol, nicotinamide, and choline.

8. The method according to claim 3, wherein the amine is at least one selected from the group consisting of ethanolamine and ethanolamine phosphate.

9. The method according to claim 3, wherein the nucleic acids are at least one selected from the group consisting of adenosine and methylthioadenosine.

10. The method of claim 3, wherein the amino acid is at least one selected from the group consisting of acylcarnitine, alanine, arginine, asparagine, aspartic acid, β-alanine, cysteine, cysteine-HPE-AM, cysteine-SS-HPE-AM, dimethylglycine, glutamic acid, glutamine, glycine, isoleucine, leucine, leucine acid, lysine, methionine, homocysteine-HPE-AM, N-acetyl-β-alanine, N-acetylcysteine, N-acetylserine, N-acetyltryptophan, N-carbamoyl-aspartate, O-acetylserine, ornithine, pyroglutamic acid, phenylalanine, proline, serine, S-sulfocysteine, taurine, threonine, tryptophan, tyrosine, and valine.

11. The method according to claim 3, wherein the other metabolite ion is at least one selected from the group consisting of ammonium ion, potassium ion, calcium ion, sodium ion, pyrophosphate, sulfate, sulfite, phenol sulfate, and SS-bis-HPE-AM.

12. The method according to claim 1, wherein the components of the culture supernatant in step (1) are measured by Raman spectroscopy.

13. The method according to claim 1, wherein in step (2), the gene transfer rate and / or the number of gene-transferred cells is predicted based on one type of component of the culture supernatant.

14. The method according to claim 1, wherein in step (2), the gene transfer rate and / or the number of gene-transferred cells is predicted based on a combination of two or more components of the culture supernatant.

15. The method according to claim 1, wherein in step (2), the change in the composition of the culture supernatant before and after gene transfer is used to predict the gene transfer rate and / or the number of gene-transferred cells.

16. The method of claim 1, wherein the animal cell is an immune cell.

17. The method of claim 16, wherein the immune cell is a T cell or a NK cell.

18. The method of claim 1, wherein the animal cell is an epithelial cell.

19. The method of claim 1, wherein the gene is a nucleic acid encoding a chimeric antigen receptor.

20. A method for producing a gene-introduced cell preparation, comprising the steps of measuring the components of a culture supernatant in which animal cells have been cultured, and determining the gene introducion rate and / or the number of gene-introduced cells based on the measured values.

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