A method for modifying cells

Abstract

Provided is a method for modifying cells, which comprises the following steps: non-virally transfecting a target molecule and a screening marker molecule into cells, such that the cells express the target molecule and the screening marker molecule. Also provided are modified cells obtained by using the method. The method can effectively enrich the desired cells and increase the positive rate of the cells for the target molecule.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and specifically relates to a method for modifying cells. Background Art

[0002] Non-viral transfection has great potential advantages in the field of cell therapy. When preparing cells required for engineering modified cell therapy products (such as CAR-T cells) using it, it is necessary to quickly obtain characteristics such as high cell viability, high positive rate, high cell function, and large cell quantity that are essential for cell therapy. To achieve this goal, various methods have been tried. For example, increasing the initial cell quantity or increasing the cultivation time is used to make up for the problem that the cell amplification ability is reduced when transfecting cells by non-viral transfection methods, resulting in insufficient cell quantity in the final product. However, increasing the initial cell quantity has problems such as high cost, and extending the cultivation time will change cell characteristics, thus greatly reducing the in vivo function of cells. Another example is that the electrotransfection intensity can be increased in physical transfection, or the concentration of transfection auxiliary molecule liposome (lipid) and DNA can be increased in chemical transfection methods, etc., to increase the transfection positive rate. However, these methods are often accompanied by a decrease in cell viability, continuous amplification ability, and cell function, so the treatment purpose still cannot be achieved. In addition, although the positive rate of the selected cells can be increased by using commercially available GMP-grade kits, this still cannot solve the problem of simultaneously ensuring that the selected cells have good cell viability and good cell function. Therefore, how to produce cell therapy products that meet the requirements of cell therapy in a short time using non-viral methods has long been a major problem faced by the industry. This series of problems has made non-viral transfection unable to be effectively applied to cell therapy. Summary of the Invention

[0003] The present application provides a special method for modifying non-viral cells. Using this method, T cells transfected by non-viral transfection methods can have higher transfection efficiency, higher cell viability and cell amplification rate. On this basis, an additional method of screening and enrichment is used to obtain cell products with a higher positive rate to meet the requirements of cell therapy. The method described in the present application defines and can process plasmids to meet the quality requirements of plasmids, and uses electroporation to enable cells to express target molecules that can exert biological functions (such as tumor killing effects) and screening molecules that can participate in screening to be knocked into the cell group DNA at a fixed point, so that the modified cells can co-express the target molecule and the screening marker molecule. Screening and enrichment through the screening molecule can further significantly increase the proportion of cells expressing the target molecule, and use this comprehensive step to prepare a method for cell therapy products. The method described in the present application can obtain cells with high cell viability, high continuous amplification ability, high cell function, high target molecule positive rate and unchanged above characteristics after cryopreservation within a short time (such as about 3-9 days). The method can be used to prepare CART cells. For example, the method can be used to prepare CART cells that highly express proteins (CCR7 + or CD62L + ) with characteristics of TCM memory cells.

[0004] On the one hand, the present application provides a method for modifying cells, which includes the following steps: transfecting a target molecule and / or a screening marker molecule into cells by non-viral transfection, so that the cells express the target molecule and the screening marker molecule.

[0005] On the other hand, the present application provides a method for increasing the transfection positive rate of a target molecule, which includes the following steps: transfecting the target molecule and / or a screening marker molecule into cells by non-viral transfection, so that the cells express the target molecule and the screening marker molecule.

[0006] In some embodiments, the cells co-express the target molecule and the screening marker molecule.

[0007] In some embodiments, the transfection includes transient transfection.

[0008] In some embodiments, the transfection includes electroporation.

[0009] In some embodiments, the transfection includes stable transfection.

[0010] In some embodiments, the target molecule and / or the screening marker molecule are integrated into the genome of the cells.

[0011] In some embodiments, the transfection includes using a transposon system and / or using gene editing knock-in.

[0012] In certain embodiments, the gene editing method is selected from one or more of the following groups: CRISPR / Cas systems, RNA editing system ADAR, RNA-guided endonucleases, zinc finger proteases, Mega-TAL nucleases, TALENs, and Meganucleases.

[0013] In certain embodiments, the non-viral transfection uses linear or circular nucleic acid molecules with encoding.

[0014] In certain embodiments, the circular nucleic acid molecule includes a circular plasmid or a supercoiled plasmid.

[0015] In certain embodiments, the plasmid contains a nucleic acid molecule encoding the target molecule and / or a nucleic acid molecule encoding the selection marker molecule.

[0016] In certain embodiments, the plasmid serves as a donor plasmid in the gene editing knock-in.

[0017] In certain embodiments, the nucleic acid molecule includes ssDNA and / or dsDNA.

[0018] In certain embodiments, the nucleic acid molecule includes homology arms.

[0019] In certain embodiments, the plasmid includes a plasmid produced by bacteria.

[0020] In certain embodiments, the plasmid includes a Nanoplasmid and / or a minicircle DNA vector.

[0021] In certain embodiments, in the transfection mixture containing the plasmid, the content of the nucleic acid molecule or its fragment derived from the genome of the microorganism accounts for about 10% (w / w) or less of the total nucleic acid molecule content of the transfection mixture.

[0022] In certain embodiments, in the transfection mixture containing the plasmid, the content of the nucleic acid molecule or its fragment derived from the genome of the microorganism accounts for about 2% (w / w) or less of the total nucleic acid molecule content of the transfection mixture.

[0023] In certain embodiments, in the transfection mixture containing the plasmid, the content of the nucleic acid molecule or its fragment derived from the genome of the microorganism accounts for about 5‰ (w / w) or less of the total nucleic acid molecule content of the transfection mixture.

[0024] In some embodiments, in the transfection mixture containing the plasmid, the content of the nucleic acid molecule or its fragment derived from the genome of a microorganism accounts for about 1‰ (w / w) or less of the total content of nucleic acid molecules in the transfection mixture.

[0025] In some embodiments, in the transfection mixture containing the plasmid, the content of the nucleic acid molecule or its fragment with a size of at least about 48 kb accounts for about 10% (w / w) or less of the total content of nucleic acid molecules in the transfection mixture.

[0026] In some embodiments, in the transfection mixture containing the plasmid, the content of the nucleic acid molecule or its fragment with a size of at least about 48 kb accounts for about 2% (w / w) or less of the total content of nucleic acid molecules in the transfection mixture.

[0027] In some embodiments, in the transfection mixture containing the plasmid, the content of the nucleic acid molecule or its fragment with a size of at least about 48 kb accounts for about 5‰ (w / w) or less of the total content of nucleic acid molecules in the transfection mixture.

[0028] In some embodiments, in the transfection mixture containing the plasmid, the content of the nucleic acid molecule or its fragment with a size of at least about 48 kb accounts for about 1‰ (w / w) or less of the total content of nucleic acid molecules in the transfection mixture.

[0029] In some embodiments, the nucleic acid molecule or its fragment with a size of at least about 48 kb has a size of at least about 1 Mb.

[0030] In some embodiments, the nucleic acid molecule or its fragment with a size of at least about 48 kb has a size of at least about 10 Mb.

[0031] In some embodiments, the nucleic acid molecule or its fragment with a size of at least about 48 kb is derived from a microorganism.

[0032] In some embodiments, the microorganism is selected from one or more of the following groups: bacteria, fungi, actinomycetes, mycoplasmas, chlamydias, rickettsias, and spirochetes.

[0033] In some embodiments, the microorganism includes Gram-negative bacteria.

[0034] In some embodiments, the microorganism includes Escherichia coli.

[0035] In some embodiments, the method includes the following step: treating the plasmid with deoxyribonuclease (DNase).

[0036] In some embodiments, the DNase can non-specifically cleave linear DNA.

[0037] In certain embodiments, the DNase is an exonuclease.

[0038] In certain embodiments, the treatment comprises contacting the transfection mixture with the DNase in the presence of Mg 2+ and Ca 2+ .

[0039] In certain embodiments, after treatment with the DNase, in the transfection mixture containing the plasmid, the content of nucleic acid molecules or fragments thereof derived from the genome of a microorganism accounts for about 10% (w / w) or less of the total content of nucleic acid molecules in the transfection mixture.

[0040] In certain embodiments, after treatment with the DNase, in the transfection mixture containing the plasmid, the content of nucleic acid molecules or fragments thereof having a size of at least about 48 kb accounts for about 10% (w / w) or less of the total content of nucleic acid molecules in the transfection mixture.

[0041] In certain embodiments, the cell includes a eukaryotic cell.

[0042] In certain embodiments, the cell includes an immune cell, a stem cell, a fibroblast, and / or a muscle cell.

[0043] In certain embodiments, the stem cell includes a pluripotent stem cell.

[0044] In certain embodiments, the stem cell includes a hematopoietic stem cell and / or a mesenchymal stem cell.

[0045] In certain embodiments, the immune cell is selected from the group consisting of: an unactivated or activated T lymphocyte, a B lymphocyte, an NK cell, a macrophage, a dendritic cell, a monocyte, a granulocyte, and a mast cell.

[0046] In certain embodiments, the immune cell includes a primary T cell.

[0047] In certain embodiments, the target molecule includes an antibody or an antigen-binding fragment, a chimeric antigen receptor (CAR), a cytokine, and / or a chemokine.

[0048] In certain embodiments, the chimeric antigen receptor targets one or more targets.

[0049] In certain embodiments, the chimeric antigen receptor targets CD19 and CD20.

[0050] In certain embodiments, the chimeric antigen receptor targets CD19 and CD22.

[0051] In certain embodiments, the antibody or antigen-binding fragment includes a bispecific antibody or antigen-binding fragment.

[0052] In certain embodiments, the antibody or antigen-binding fragment comprises a bispecific T cell engager (BiTE).

[0053] In certain embodiments, the selection marker molecule comprises a domain capable of binding to a selection agent.

[0054] In certain embodiments, the selection agent comprises magnetic beads.

[0055] In certain embodiments, the selection marker molecule comprises CD34 and / or an EGFRT truncation.

[0056] In certain embodiments, the type of the selection marker molecule corresponds to the type of the cell and / or the target molecule.

[0057] In certain embodiments, the cell is an immune cell and the selection marker molecule is CD34 and / or an EGFRT truncation.

[0058] In certain embodiments, the method comprises the following steps: contacting the cell expressing the target molecule and the selection marker molecule with the selection agent.

[0059] In certain embodiments, the method further comprises the following step: collecting the cell bound to the selection agent.

[0060] In certain embodiments, the method further comprises the following step: separating the cell bound to the selection agent from the selection agent.

[0061] On the other hand, the present application provides a cell and / or cell line prepared by the method described in the present application.

[0062] On the other hand, the present application provides a modified immune cell that simultaneously expresses a target molecule and a selection marker molecule.

[0063] In certain embodiments, the immune cell is selected from the group consisting of: unactivated or activated T lymphocytes, B lymphocytes, NK cells, macrophages, dendritic cells, monocytes, granulocytes, and mast cells.

[0064] In certain embodiments, the target molecule comprises an antibody or antigen-binding fragment, a chimeric antigen receptor (CAR), a cytokine, and / or a chemokine.

[0065] In certain embodiments, the chimeric antigen receptor targets one or more target sites.

[0066] In certain embodiments, the chimeric antigen receptor targets CD19 and CD20.

[0067] In certain embodiments, the chimeric antigen receptor targets CD19 and CD22.

[0068] In certain embodiments, the antibody or antigen-binding fragment comprises a bispecific antibody or antigen-binding fragment.

[0069] In certain embodiments, the antibody or antigen-binding fragment comprises a bispecific T cell engager (BiTE).

[0070] In certain embodiments, the selection marker molecule comprises a domain capable of binding to a selection agent.

[0071] In certain embodiments, the selection agent comprises magnetic beads.

[0072] In certain embodiments, the selection marker molecule comprises CD34 and / or an EGFRT truncation.

[0073] In certain embodiments, the type of the selection marker molecule is selected according to the type of the cell and / or the target molecule.

[0074] In certain embodiments, the cell is an immune cell, and the selection marker molecule is CD34 and / or an EGFRT truncation.

[0075] Those skilled in the art can readily appreciate other aspects and advantages of the present application from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the content of the present application enables those skilled in the art to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention involved in the present application. Accordingly, the descriptions in the drawings and the specification of the present application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The specific features of the invention involved in the present application are shown in the appended claims. The features and advantages of the invention involved in the present application can be better understood by referring to the exemplary embodiments and the drawings described in detail below. A brief description of the drawings is as follows:

[0077] Figure 1 Shows the cell viability of the modified cells described in the present application.

[0078] Figure 2 Shows the cell proliferation results of the modified cells described in the present application.

[0079] Figure 3 Shows the protein expression of the modified cells described in the present application.

[0080] Figure 4Shown is the situation of the protein expression after enrichment of the modified cells described in the present application.

[0081] Figure 5 Shown is the cell proliferation result after enrichment of the modified cells described in the present application.

[0082] Figure 6 A-6B shows the situation of the positive rate of protein expression after enrichment of the modified cells described in the present application.

[0083] Figure 7 Shown is the cell viability after cryopreservation and resuscitation of the enriched modified cells described in the present application.

[0084] Figure 8 Shown is the number of cells after cryopreservation and resuscitation of the enriched modified cells described in the present application.

[0085] Figure 9 Shown is the proliferation result activated by target cells after cryopreservation and resuscitation of the enriched modified cells described in the present application.

[0086] Figure 10 Shown is the killing effect on in vitro target cells after enrichment of the modified cells described in the present application.

[0087] Figure 11 Shown is the killing effect on in vivo tumors after enrichment of the modified cells described in the present application.

[0088] Figure 12 Shown is the situation of the protein expression after enrichment of the modified cells described in the present application.

[0089] Figure 13 Shown is the killing effect on in vitro target cells after enrichment of the modified cells described in the present application.

[0090] Figure 14 Shown is the killing effect on in vivo tumors after enrichment of the modified cells described in the present application.

[0091] Figure 15 Shown are the properties of plasmids with nucleic acid molecules having different contents of genomes derived from microorganisms. Detailed implementation manners

[0092] The following specific embodiments illustrate the implementation manners of the invention of the present application, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

[0093] Term definitions

[0094] In the present application, the term "stable transfection" generally refers to introducing and integrating an exogenous nucleic acid molecule into the genome of the transfected cell. For example, integrating the exogenous gene into the genome of the transfected cell.

[0095] In the present application, the term "transient transfection" generally refers to a transfection method in which the exogenous gene transfected into a cell is not integrated into the genome of the cell itself. The transient transfection can achieve rapid expression of the exogenous gene in the short term (for example, at least about 1 day, at least about 2 days). The exogenous gene transfected into the cell by the transient transfection may be gradually lost as the cell grows and / or divides. The transient transfection may have advantages selected from the group consisting of: simple operation, short experimental period, high expression efficiency, safety, and no need to screen genes. The operation steps of the transient transfection may be known to those skilled in the art. For example, the transient transfection may be carried out by liposome-mediated transfection. For example, the transient transfection may include electroporation transfection (such as electroporation). The transient transfection may use transfection reagents, for example, FuGENE6 may be used.

[0096] In the present application, the term "deoxyribonuclease (DNase)" generally refers to an enzyme capable of cleaving the phosphodiester bond on the DNA backbone. The DNase may be a type of nuclease. The DNase can digest double-stranded DNA into deoxynucleotides (for example, under weakly alkaline conditions). For example, the DNase may be inactive (for example, unable to cleave it) against circular or supercoiled nucleic acid molecules or fragments thereof (such as closed circular double-stranded DNA and / or supercoiled DNA). The DNase may also have certain activity against circular or linear single-stranded DNA nucleic acid molecules or fragments thereof, but the activity may be low (for example, the efficiency of cleaving it is low). The DNase may be an exonuclease. The DNase may be an ATP-Dependent DNase, for example, it may be Plasmid-Safe TM ATP-Dependent DNase.

[0097] In the present application, the term "transfection efficiency" generally refers to the relative amount of the material introduced into and / or expressed by the transfected cells among the cells that have undergone transfection. In the present application, the transfection may refer to introducing one or more materials (such as polynucleotides) into cells. The introduced material may be stably or transiently retained in the transfected cells. In the present application, the transfection efficiency may be measured by the amount of the introduced material.

[0098] In the present application, the term "nucleic acid molecule or fragment thereof" generally refers to nucleotides (such as ribonucleotides, deoxyribonucleic acids, and / or modified forms of the foregoing two) or fragments thereof. The nucleic acid molecule or fragment thereof may include a polymeric form of nucleotides or a fragment thereof. The nucleic acid molecule or fragment thereof may be interchangeable with "polynucleotide" in some cases. The nucleic acid molecule may include DNA, RNA, cDNA, sense and antisense strands of genomic DNA, as well as synthetic forms, mixtures, and / or polymers thereof. The nucleic acid molecule or fragment thereof may include any topological conformation, for example, may include single-stranded, double-stranded, partially double-stranded, triple-stranded, hairpin structure, circular, and / or padlock conformation. The nucleotides in the nucleic acid molecule or fragment thereof may be natural or modified. The nucleotides in the nucleic acid molecule or fragment thereof may be linked together by naturally occurring and / or non-naturally occurring nucleotide bonds.

[0099] In the present application, the term "transfection mixture" generally refers to the mixture required for transfection. In the present application, the transfection mixture may include one or more materials (such as polynucleotides) to be introduced. For example, the transfection mixture may contain a nucleic acid molecule encoding an exogenous gene to be transfected. For example, the transfection mixture may include a vector containing the nucleic acid molecule. For example, the transfection mixture may further include impurities (in some cases, the impurities may include nucleic acid molecules or fragments thereof). In the present application, the impurities may include nucleic acid molecules or fragments thereof of microbial origin. The impurities carried by the vector may include the impurities carried by the vector (for example, nucleic acid molecules or fragments thereof homologous or heterologous to the backbone of the vector).

[0100] In the present application, the term "total nucleic acid molecule content" generally refers to the total mass of all substances having nucleotides in the transfection mixture. For example, the substances having nucleotides may include the nucleic acid molecule or fragment thereof and the material.

[0101] In the present application, the term "microorganism" generally refers to eukaryotic and prokaryotic microbial species from the domains Archaea, Bacteria, and / or Eucarya. For example, the microorganisms may include bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasmas, chlamydiae, and / or spirochetes. In the present application, the term bacteria generally refers to any type of prokaryote, including prokaryotes in all phyla within the kingdom Prokaryota. The bacteria may include cocci, bacilli, spirilla, spheroplasts, and protoplasts. The bacteria may include Gram-positive bacteria and Gram-negative bacteria. "Gram-negative" and "Gram-positive" refer to the staining patterns using the Gram staining method, which are well-known in the art (see, for example, Finegold and Martin, Diagnostic Microbiology, 6th edition, C V Mosby St. Louis, pp. 13-15 (1982)).

[0102] In the present application, the term "nucleic acid molecule or fragment thereof derived from the genome of a microorganism" generally refers to a nucleic acid molecule or a fragment of a nucleic acid molecule that is derived from the genome of the microorganism. Information on the genomes of microorganisms can be found in A genomic catalog of Earth’s microbiomes, Nature Biotechnology (2020).

[0103] In the present application, the term "Gram-negative bacteria" generally refers to bacteria that do not retain the primary dye used in the Gram staining but are stained by the counterstain. Thus, Gram-negative bacteria generally appear red in the Gram staining method. The cell walls of Gram-negative bacteria have a lower content of peptidoglycan and a higher content of lipids. For example, the cell walls of Gram-negative bacteria may have a lipopolysaccharide layer. For example, the Gram-negative bacteria may include Escherichia coli, Pseudomonas aeruginosa, Proteus spp., Shigella spp., Klebsiella pneumoniae, Brucella spp., Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Acinetobacter spp., Yersinia spp., Legionella pneumophila, Bordetella pertussis, Bordetella parapertussis, Shigella spp., Pasteurella spp., Vibrio cholerae, Vibrio parahaemolyticus, and / or Plesiomonas shigelloides.

[0104] In the present application, the term "Escherichia coli" generally refers to Escherichia coli. Escherichia coli belongs to the genus Escherichia in the family Enterobacteriaceae.

[0105] In the present application, the term "immune cell" generally refers to cells that play a role in the immune response. The immune cells may include lymphocytes, monocytes, and / or granulocytes, as well as their precursors and / or mature derivatives. The immune cells may include T cells, B cells, Th cells, natural killer cells, monocytes, macrophages, eosinophils, basophils, mast cells, dendritic cells, and / or granulocytes. The immune cells may include immune effector cells. The immune effector cells may participate in immune reactions, such as promoting immune effector responses. The immune effector cells may include T cells, such as, α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NTK) cells, mast cells, and phagocytes derived from bone marrow.

[0106] In the present application, the term "stem cell" generally refers to a class of undifferentiated cells that have the ability to self-renew and at the same time retain varying degrees of potential to form differentiated cells and tissues. The stem cells may be nullipotent stem cells, pluripotent stem cells, or totipotent stem cells. Among them, the nullipotent stem cells are derivative stem cells that have lost the ability to differentiate. The totipotent stem cells can form all the cells and tissues found in a complete organism. For example, the totipotent stem cells can form a complete organism.

[0107] In the present application, the term "pluripotent stem cell" generally refers to a stem cell that has the ability to form the cells and tissues ultimately found in a complete organism but cannot form a complete organism. For example, the pluripotent stem cells can proliferate extensively in vitro or actually proliferate without limitation while maintaining their undifferentiated state and showing a normal karyotype (chromosomes). The pluripotent stem cells can have the ability to differentiate into all three germ layers (ectoderm, mesoderm, and endoderm) under appropriate conditions. For example, the pluripotent stem cells may include ES cells isolated from early embryos and / or isogenic EG cells isolated from fetal primordial germ cells.

[0108] In the present application, the term "mesenchymal stem cell" generally refers to cells that can give rise to mesenchymal lineages. The mesenchymal stem cells may be considered to belong to the pluripotent stem cells. The mesenchymal stem cells can produce one or more types of cells of the mesenchymal lineages. The cells of the mesenchymal lineages can be derived from different tissues. For example, they can be derived from bone marrow tissue, adipose tissue, muscle tissue, reproductive tissues (such as amnion, amniotic fluid, or umbilical cord tissue), skin tissue, bone tissue, and / or dental tissue.

[0109] In the present application, the term "immune cell" generally refers to a cell that plays a role in the immune response. The immune cells may include lymphocytes, monocytes, and / or granulocytes, as well as their precursors and / or mature derivatives. The immune cells may include T cells, B cells, Th cells, natural killer cells, monocytes, macrophages, eosinophils, basophils, mast cells, dendritic cells, and / or granulocytes. The immune cells may include immune effector cells. The immune effector cells may participate in immune reactions, such as promoting immune effector responses. The immune effector cells may include T cells, such as, α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NTK) cells, mast cells, and phagocytes derived from the bone marrow.

[0110] In the present application, the term "fibrocyte" generally refers to a fibroblast with inactive functions. The fibrocyte can be transformed into a fibroblast (for example, it can participate in the repair process when the tissue is damaged). The fibroblast (which can also be called a fibroblast) can secrete structural proteins that make up the extracellular matrix. The fibroblast can be closely related to physiological or pathological processes such as wound healing and fibrosis.

[0111] In the present application, the term "muscle cell" generally refers to a single cell or a group of cells derived from muscle. The muscle cells can be derived from cells and tissues of skeletal muscle, smooth muscle (for example, from the digestive tract, bladder, and blood vessels), and cardiac muscle. The muscle cells can include myocytes in vitro and in vivo. The muscle cells can also include myocytes and muscle tissues derived from differentiated and undifferentiated muscle cells such as myocytes (such as myotubes), dividing and differentiating myoblasts, cardiomyocytes, and cardiac myoblasts.

[0112] In the present application, the term "plasmid" generally refers to a construct containing genetic material. The plasmid can be designed to be capable of delivering genetic material (such as one or more nucleic acid sequences) into cells. The plasmid can contain an autonomous replication sequence of single-stranded or double-stranded nucleic acid (such as DNA or RNA) derived from any source. The plasmid can be used interchangeably with the term "vector" in the present application. The plasmid can have different configurations, for example, it can be a linear plasmid, a circular plasmid, or a supercoiled plasmid. The linear plasmid can be a linear DNA molecule. The supercoiled plasmid can contain two nucleic acid strands that maintain an intact structure (for example, it can contain covalently closed circular DNA, cccDNA), and has a supercoiled configuration. The circular plasmid can be a circular structure in which at least one nucleic acid strand remains intact. The plasmid can be not integrated into the genome of the cell.

[0113] In the present application, the term "multiple cloning site" or "multiple cloning site, MCS" generally refers to a nucleic acid sequence containing at least one restriction site. The multiple cloning site can ligate a nucleic acid molecule into the vector described in the present application. For example, the insertion of a nucleic acid molecule can be achieved at a specified site through the restriction site. The restriction site can be a recognition site for a restriction endonuclease. For example, the restriction endonuclease can be AclU HindIII, SspI, MluCI, Tsp509I, PciI, AgeI BspMI, BfuAI, SexAI, MluI, BceAI, HpyCH4IV, HpyCH4III, BaeI, BsaXI, SpeI, BsrI, BmrI, BglII, AfeI, AluI, StuI, SacI, ClaI, BspDI, PI-SceI, NsiI, Asel, SwaI, CspCI, MfeI, BssSI, BmgBI, PmlI, DraIII, AleI, EcoP15I, PvuII, AlwNI or BtsMutI.

[0114] In the present application, the term "antibody" generally refers to an immunoglobulin that is reactive with a specified protein or peptide or a fragment thereof. Antibodies can be antibodies from any class, including but not limited to IgG, IgA, IgM, IgD, and IgE, and antibodies from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). An antibody can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. An antibody can also have a light chain selected from, for example, kappa (κ) or lambda (λ). The antibodies of the present application can be derived from any species.

[0115] In the present application, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule that contains amino acid residues that interact with an antigen and confer specificity and affinity of the antibody for the antigen. Examples of antigen-binding fragments can include but are not limited to Fab, Fab’, F(ab) 2 , Fv fragments, F(ab’) 2 , scFv, di-scFv, and / or dAb. In the present application, the term "Fab" generally refers to a fragment containing a heavy chain variable domain and a light chain variable domain, and also containing the constant domain of the light chain and the first constant domain (CH1) of the heavy chain; the term "Fab’" generally refers to a fragment that is different from Fab by adding a small number of residues (including one or more cysteines from the antibody hinge region) at the carboxyl terminus of the heavy chain CH1 domain; the term "F(ab′) 2”Generally refers to a dimer of Fab’, an antibody fragment containing two Fab fragments linked by a disulfide bridge in the hinge region. The term “Fv” generally refers to the smallest antibody fragment containing a complete antigen recognition and binding site. In some cases, this fragment can consist of a dimer of a heavy chain variable region and a light chain variable region in a tight non-covalent association; the term “dsFv” generally refers to a disulfide bond-stabilized Fv fragment, where the bond between a single light chain variable region and a single heavy chain variable region is a disulfide bond. The term “dAb fragment” generally refers to an antibody fragment consisting of a VH domain. In the present application, the term “scFv” generally refers to a monovalent molecule formed by covalently linking and pairing a heavy chain variable domain and a light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules can have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.

[0116] In the present application, the term "chimeric antigen receptor" generally refers to a fusion protein comprising an extracellular domain capable of binding an antigen and at least one intracellular domain. The CAR is the core component of chimeric antigen receptor T cells (CAR-T), which may include an antigen (e.g., tumor-specific antigen and / or tumor-associated antigen) binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. In the present application, the CAR can be combined with the intracellular domain of T cell receptor activation based on the antigen (e.g., CD19) specificity of an antibody. T cells genetically modified to express the CAR can specifically recognize and eliminate malignant cells expressing the target antigen. For descriptions of CAR and CAR-T cells, see, for example, Sadelain M, Brentjens R, Rivi`ere I. The basic principles of chimeric antigen receptor design. Cancer Discov. 2013;3(4):388-398; Turtle CJ, Hudecek M, Jensen MC, Riddell SR. Engineered T cells for anti-cancer therapy. Curr Opin Immunol. 2012;24(5):633-639; Dotti G, Gottschalk S, Savoldo B, Brenner MK. Design and development of therapies using chimeric antigen receptor-expressing T cells. Immunol Rev. 2014;257(1):107-126; and WO2013154760, WO2016014789.

[0117] In the present application, the term "bispecific antibody" generally refers to an antibody having variable regions that recognize more than one epitope on one or more antigens. Bispecific antibodies include, but are not limited to, full-length antibodies, antibodies having two or more VL and VH domains, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, antibody fragments that have been covalently or non-covalently linked. In some cases, the bispecific antibody can recognize two different epitopes on the same or different antigens. In some cases, the bispecific antibody can recognize two different antigens.

[0118] In the present application, the term "antigen-binding domain" generally refers to a domain capable of binding to a target antigen. The antigen-binding domain may include a heavy chain and antigen receptor and fragments thereof that can specifically bind to an antigen, an antibody or an antigen-binding fragment thereof. The antigen-binding domain may be a domain capable of binding to a tumor-associated antigen, and the tumor-associated antigen may include, but is not limited to: CD19, CD20, CD22, CD123, CD33 / IL3Ra, CD138, CD33, BCMA, CS1, C-Met, EGFRvIII, CEA, Her2, GD2, MAG3, GPC3, and NY-ESO-1.

[0119] In the present application, the term "BiTE" generally refers to a bispecific T cell engager. The BiTE may be a single polypeptide chain molecule having two antigen-binding domains, one of the two antigen-binding domains binding to a T cell antigen and the second binding to an antigen present on the surface of a target cell (see W005 / 061547; Baeuerle, P et al. (2008) " : A New Class Of Antibodies That Recruit T Cells Drugs of the Future 33:137-147; or Bargou et al. (2008) "Tumor Regression in Cancer Patients by Very Low Doses of a T Cell-Engaging Antibody / ' Science 321:974-977).

[0120] In the present application, the term "homologous arm" generally refers to a polynucleotide suitable for targeting an exogenous gene to be knocked in the donor plasmid to the genome by homologous recombination. The homologous recombination may refer to the recombination occurring between sister chromatids or between or within DNA molecules on the same chromosome containing homologous sequences. There may be two homologous arms (for example, there may be a 5' homologous arm and / or a 3' homologous arm). The homologous arms may be located upstream and downstream of the exogenous gene to be knocked in the donor plasmid. In some cases, a break may be generated at the targeting position of the exogenous gene to be knocked in the genome due to the action of a nuclease. The homologous arms may be identical to the DNA sequences at both ends (i.e., the 5' and / or 3' ends) of the break at the targeting position or have at least 80% identity. For example, the homologous arms may have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identity with the DNA sequences at both ends of the break at the targeting position. In the present application, the donor plasmid may contain a 5' homologous arm and / or a 3' homologous arm.

[0121] In the present application, the term "cell line" generally refers to a clonal population of cells capable of continuous division. For example, the cell line may have acquired the ability to proliferate infinitely in vitro.

[0122] In the present application, the term "gene editing" generally refers to an operation of nucleic acid insertion, deletion and / or replacement into the genome. The gene editing can be achieved by homologous directed repair (HDR), non-homologous end joining (NHEJ) or single-base alteration. The gene editing can use gene editing tools familiar to those skilled in the art, for example, the zinc finger nuclease system (ZFN), TALEN system and / or CRISPR technology can be utilized.

[0123] In the present application, the term "transposon system" generally refers to a system that includes a polynucleotide capable of excising from a donor polynucleotide (such as a plasmid) and integrating into a target site (such as the genomic DNA of a cell). The polynucleotide can be integrated into the target site by a transposase. The transposon system may include a transposase. The transposase can transpose and mediate the functional nucleic acid-protein complex for transposition. The transposon system may include transposon ends. The transposon ends may be nucleotide sequences that only exhibit the necessity for forming a complex with the transposase or integrase. The transposon ends may be double-stranded nucleic acid DNA. In some cases, the transposon ends may form a functional nucleic acid-protein complex with the transposon in the transposition reaction.

[0124] In the present application, the term "nanoplasmid" generally refers to a nanoplasmid, i.e., a nanoparticle produced by one or more lipid-based nanocarriers, polymeric nanoparticles, metal nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles, peptide- or protein-based particles (such as albumin nanoparticles), and / or compositions using nanomaterials (e.g., lipid-polymer nanoparticles). In some cases, the size of the nanoplasmid may be equal to or less than about 100 nanometers.

[0125] In the present application, the term "minicircle DNA vector" generally refers to a product of site-specific recombination of a parental plasmid (PP). The minicircle DNA vector is a supercoiled DNA molecule. Under the action of a recombinase, the parental plasmid is transformed into two circular DNAs, one containing a large amount of bacterial backbone sequences, called a miniplasmid (MP); the other is a eukaryotic expression cassette containing only the target gene, i.e., a minicircle DNA (MC). The minicircle DNA vector may consist of a eukaryotic expression cassette.

[0126] In the present application, the term "gene editing knock-in" generally refers to a genetic engineering process (e.g., which can be called knock-in), which involves a one-to-one replacement or insertion of DNA sequence information at a genetic locus of sequence information not found within the endogenous locus. The gene editing knock-in can utilize gene homologous recombination. For example, by using gene homologous recombination, a foreign functional gene (a gene that did not originally exist or has been inactivated in the genome) is transferred into a cell and undergoes homologous recombination with a homologous sequence in the genome, so that it is inserted into the genome and expressed in the cell. For example, the gene editing knock-in can enable a foreign gene to at least partially replace the cell genome. The gene editing knock-in can use CRISPR technology to achieve site-directed "targeted knock-in".

[0127] In the present application, the term "about" generally refers to within 30%, within 25%, within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, within 0.5%, or within 0.05% of the specified value or numerical range involved in the present application. In the present application, when the "about" is before the first of more than two numerical values, it applies to each value in the series. Detailed Description of the Invention

[0129] On the one hand, the present application provides a method for modifying cells, which includes the following steps: non-virally transfecting a target molecule and / or a screening marker molecule into cells, such that the cells express the target molecule and the screening marker molecule.

[0130] On the other hand, the present application provides a method for increasing the transfection positive rate of a target molecule, which includes the following steps: non-virally transfecting the target molecule and / or a screening marker molecule into cells, such that the cells express the target molecule and the screening marker molecule.

[0131] In the present application, the cells can express the target molecule and the screening marker molecule simultaneously.

[0132] In the present application, the transfection can include transient transfection. For example, the transfection can include electroporation. The electroporation can refer to cell electroporation or cell electropermeabilization. The electroporation can utilize the action of a powerful instantaneous electric field to enable charged substances to enter cells through a cell membrane with certain permeability. The electroporation can produce very little cytotoxicity. Compared with chemical transfection methods and / or viral transfection methods, the cytotoxicity produced by the electroporation is significantly reduced. The electroporation can be applied to almost all types of eukaryotic cells. The electroporation can be used for transient or stable expression of foreign proteins.

[0133] In the present application, the transfection can include stable transfection.

[0134] In the present application, the transfection (such as stable transfection) can include transfection using a transposon system (such as the Sleeping beauty transposon system).

[0135] In the present application, the target molecule and / or the screening marker molecule can be integrated into the genome of the cells.

[0136] In the present application, the transfection can include gene editing knock-in.

[0137] In the present application, the gene editing method can be selected from one or more of the following groups: CRISPR / Cas system, RNA editing system ADAR, RNA-guided endonuclease, zinc finger protease, Mega-TAL nuclease, TALENs, and Meganucleases. In the present application, the gene editing method can use a variety of nucleases as long as the nuclease can help integrate the target molecule and / or the screening marker molecule into the genome of the cells.

[0138] In this application, the gene editing method can be known to those skilled in the art, as long as it can achieve the purpose of gene editing, and is not limited to a specific method. In this application, the gene editing method can be selected from one or more of the following groups: CRISPR / Cas system, RNA editing system ADAR, RNA-guided endonuclease, zinc finger protease, Mega-TAL nuclease, TALENs, and Meganucleases. For example, the gene editing method can be the use of the CRISPR / Cas system.

[0139] In this application, the gene editing can include gene editing knockout and / or gene editing knock-in. For example, the gene editing can include gene editing knock-in. For example, the CRISPR / Cas system can be used for the gene editing knock-in.

[0140] For example, the CRISPR-CAS system can include a class of clustered regularly interspaced short palindromic repeats (CRISPRs) and some functionally related proteins (CRISPR-associated, Cas). Among them, the Cas protein-encoding genes can include Cas9, Cas1, Cas2, and Csn2. For example, it can be Cas9. The CRISPR-CAS system (such as the CRISPR / Cas9 system) can have targeted cleavage specificity for DNA molecules.

[0141] For example, the gene editing knock-in can be a process of targeting a Cas protein to a specific DNA sequence in a cell for DNA sequence insertion. The gene editing knock-in can be a process mediated by a Cas protein (such as Cas9 protein) for homologous recombination of the exogenous gene to be knocked in in the donor plasmid with the specific DNA sequence in the targeted cell.

[0142] In this application, the non-viral transfection can use linear or circular nucleic acid molecules with encoding.

[0143] For example, the circular nucleic acid molecule can include a circular plasmid or a supercoiled plasmid.

[0144] In this application, the plasmid can be a DNA plasmid. For example, the DNA plasmid can be a double-stranded, closed-loop DNA molecule. For example, the DNA plasmid can be a double-stranded, linear DNA molecule. In this application, the plasmid can be artificially synthesized.

[0145] In this application, the plasmid can include plasmids produced by bacteria. In this application, the plasmid can include nanoplasmid and / or minicircle DNA vectors.

[0146] In the present application, the plasmid may contain a multiple cloning site. The plasmid may encode at least one target molecule. The target molecule may encode one or more antigen-binding fragments (such as antibodies), chimeric antigen receptors, cytokines, and / or chemokines. The target molecule may also encode any one or more functional proteins. For example, the functional protein may be used to treat diseases related to gene defects. For example, the functional protein may be a protein that is absent and / or mutated in the host of the transfected cell. For example, the exogenous gene expressing the functional protein may be transfected into the fibroblast (such as primary fibroblast), muscle cell, and / or stem cell (such as iPs cell).

[0147] In the present application, the nucleic acid molecule encoding the target molecule and / or the nucleic acid molecule encoding the selection marker molecule may be expressed by means of an endogenous promoter or an exogenous promoter. For example, the plasmid may contain the exogenous promoter.

[0148] In the present application, the plasmid may contain a nucleic acid molecule encoding the target molecule and / or a nucleic acid molecule encoding the selection marker molecule. For example, the nucleic acid molecule encoding the target molecule and the nucleic acid molecule encoding the selection marker molecule are located on the same plasmid. For example, the nucleic acid molecule encoding the target molecule and / or the nucleic acid molecule encoding the selection marker molecule may be connected by a linker sequence. The linker sequence does not affect the expression and / or function of the nucleic acid molecule encoding the target molecule and / or the nucleic acid molecule encoding the selection marker molecule. For example, the linker sequence may include a sequence encoding a self-cleaving peptide. For example, the linker sequence may include a nucleic acid molecule encoding a 2A sequence.

[0149] In the present application, the plasmid may be used as a donor plasmid in the gene editing knock-in. For example, the donor plasmid may be double-stranded DNA or single-stranded DNA. The donor plasmid may serve as a donor template for the HDR repair mechanism.

[0150] In the present application, the nucleic acid molecule may include ssDNA and / or dsDNA. In the present application, the nucleic acid molecule may be artificially synthesized.

[0151] In the present application, the plasmid may include ssDNA and / or dsDNA. In the present application, the plasmid (such as the donor plasmid) may include homologous arms. The plasmid may be artificially synthesized.

[0152] In the present application, in the transfection mixture containing the plasmid, the content of the nucleic acid molecule or its fragment derived from the genome of a microorganism may be about 10% (w / w) or less of the total content of the nucleic acid molecules in the transfection mixture (for example, it may be about 10% (w / w) or less, about 9% (w / w) or less, about 8% (w / w) or less, about 7% (w / w) or less, about 6% (w / w) or less, about 5% (w / w) or less, about 4% (w / w) or less, about 3% (w / w) or less, about 2% (w / w) or less, about 1% (w / w) or less, about 9‰ (w / w) or less, about 8‰ (w / w) or less, about 7‰ (w / w) or less, about 6‰ (w / w) or less, about 5‰ (w / w) or less, about 4‰ (w / w) or less, about 3‰ (w / w) or less, about 2‰ (w / w) or less, about 1‰ (w / w) or less, about 0.1‰ (w / w) or less, about 0.01‰ (w / w) or less, about 0.001‰ (w / w) or less, or lower).

[0153] In the present application, the size of the fragment of the nucleic acid molecule derived from the genome of a microorganism may be at least about 48 kb in size (for example, it may be at least about 50 kb, at least about 100 kb, at least about 150 kb, at least about 200 kb, at least about 250 kb, at least about 300 kb, at least about 350 kb, at least about 400 kb, at least about 450 kb, at least about 500 kb, at least about 600 kb, at least about 700 kb, at least about 800 kb, at least about 900 kb, at least about 1 Mb, at least about 2 Mb, at least about 3 Mb, at least about 4 Mb, at least about 5 Mb, at least about 6 Mb, at least about 7 Mb, at least about 8 Mb, at least about 9 Mb, at least about 10 Mb, at least about 20 Mb, at least about 50 Mb, at least about 100 Mb, at least about 200 Mb, or larger).

[0154] In the present application, in the transfection mixture containing the plasmid, the content of the nucleic acid molecule or its fragment with a size of at least about 48 kb (for example, it can be at least about 50 kb, at least about 100 kb, at least about 150 kb, at least about 200 kb, at least about 250 kb, at least about 300 kb, at least about 350 kb, at least about 400 kb, at least about 450 kb, at least about 500 kb, at least about 600 kb, at least about 700 kb, at least about 800 kb, at least about 900 kb, at least about 1 Mb, at least about 2 Mb, at least about 3 Mb, at least about 4 Mb, at least about 5 Mb, at least about 6 Mb, at least about 7 Mb, at least about 8 Mb, at least about 9 Mb, at least about 10 Mb, at least about 20 Mb, at least about 50 Mb, at least about 100 Mb, at least about 200 Mb or larger) can be less than about 10% (w / w) of the total content of nucleic acid molecules in the transfection mixture (for example, it can be less than about 10% (w / w), less than about 9% (w / w), less than about 8% (w / w), less than about 7% (w / w), less than about 6% (w / w), less than about 5% (w / w), less than about 4% (w / w), less than about 3% (w / w), less than about 2% (w / w), less than about 1% (w / w), less than about 9‰ (w / w), less than about 8‰ (w / w), less than about 7‰ (w / w), less than about 6‰ (w / w), less than about 5‰ (w / w), less than about 4‰ (w / w), less than about 3‰ (w / w), less than about 2‰ (w / w), less than about 1‰ (w / w), less than about 0.1‰ (w / w), less than about 0.01‰ (w / w), less than about 0.001‰ (w / w) or lower).

[0155] In the present application, the content of the nucleic acid molecule or its fragment with a size of at least about 48 kb may be about 10% (w / w) or less, about 9% (w / w) or less, about 8% (w / w) or less, about 7% (w / w) or less, about 6% (w / w) or less, about 5% (w / w) or less, about 4% (w / w) or less, about 3% (w / w) or less, about 2% (w / w) or less, about 1% (w / w) or less, about 9‰ (w / w) or less, about 8‰ (w / w) or less, about 7‰ (w / w) or less, about 6‰ (w / w) or less, about 5‰ (w / w) or less, about 4‰ (w / w) or less, about 3‰ (w / w) or less, about 2‰ (w / w) or less, about 1‰ (w / w) or less, about 0.1‰ (w / w) or less, about 0.01‰ (w / w) or less, about 0.001‰ (w / w) or less or lower of the total content of nucleic acid molecules in the transfection mixture. For example, the content of the nucleic acid molecule or its fragment with a size of at least 48 kb may be about 2% or less of the total content of nucleic acid molecules in the transfection mixture; it may be about 5‰ (w / w) or less of the total content of nucleic acid molecules in the transfection mixture; it may be about 1‰ (w / w) or less of the total content of nucleic acid molecules in the transfection mixture.

[0156] In the present application, the nucleic acid molecule or its fragment with a size of at least about 48 kb (for example, it may be at least about 50 kb, at least about 100 kb, at least about 150 kb, at least about 200 kb, at least about 250 kb, at least about 300 kb, at least about 350 kb, at least about 400 kb, at least about 450 kb, at least about 500 kb, at least about 600 kb, at least about 700 kb, at least about 800 kb, at least about 900 kb, at least about 1 Mb, at least about 2 Mb, at least about 3 Mb, at least about 4 Mb, at least about 5 Mb, at least about 6 Mb, at least about 7 Mb, at least about 8 Mb, at least about 9 Mb, at least about 10 Mb, at least about 20 Mb, at least about 50 Mb, at least about 100 Mb, at least about 200 Mb or larger) and the plasmid are present in different nucleic acid molecules. For example, the nucleic acid molecule or its fragment with a size of at least about 48 kb is free in the transfection mixture (the transfection mixture may contain a plasmid). For example, the nucleic acid molecule or its fragment with a size of at least about 48 kb is free from the plasmid. The free form may be in a separated state, existing in a form not attached to the plasmid.

[0157] In the present application, the plasmid (such as a linear plasmid) can be treated with (such as deoxyribonuclease, such as exonuclease, such as Exonuclease V) so that the content of the nucleic acid molecule or its fragment with a size of at least about 48 kb described in the present application (such as a linear nucleic acid molecule or its fragment) and / or the content of the nucleic acid molecule or its fragment derived from the genome of a microorganism described in the present application (such as a linear nucleic acid molecule or its fragment) meet the conditions described in the present application. In the present application, the treatment may not affect the nucleic acid molecule having a circular structure.

[0158] In the present application, the nucleic acid molecule or its fragment with a size of at least about 48 kb may be derived from a microorganism. For example, the microorganism may be selected from one or more of the following groups: bacteria, fungi, actinomycetes, mycoplasmas, chlamydias, rickettsias, and spirochetes.

[0159] In the present application, the microorganism may include Gram-negative bacteria. For example, the microorganism may be a microorganism suitable for preparing the backbone vector of a carrier. For example, the microorganism may include Escherichia coli.

[0160] In the present application, the cell may include eukaryotic cells. The eukaryotic cells may include plant cells, fungal cells, and / or animal cells. Among them, the animal cells may include mammalian cells.

[0161] In the present application, the cell may include stem cells, immune cells, fibroblast cells, and / or muscle cells. For example, the stem cells may include pluripotent stem cells. For example, the pluripotent stem cells may include embryonic stem cells (ESCs). The embryonic stem cells can differentiate into three primary germ layers (such as the ectoderm, mesoderm, and endoderm), and these three germ layers can ultimately form organs and tissues. The pluripotent stem cells may include epiblast stem cells (EpiSCs). The pluripotent stem cells may also include induced pluripotent stem cells (such as iPS), which can be formed by the dedifferentiation of mammalian somatic cells (such as skin cells from the tail of a mouse) after the transfer of transcription factors (such as Oct4, Sox2, c-Myc, and Klf4).

[0162] In the present application, the stem cells may include hematopoietic stem cells and / or mesenchymal stem cells. The hematopoietic stem cells can differentiate into blood cells (such as blood cells of the myeloid lineage and blood cells of the lymphoid lineage). The hematopoietic stem cells can have the characteristics of pluripotency and self-renewal. The hematopoietic stem cells can differentiate into cells selected from the following group: monocytes, macrophages, neutrophils, basophils, eosinophils, red blood cells, megakaryocytes, platelets, T cells, B cells, and NK cells.

[0163] The mesenchymal stem cells may be adult stem cells derived from the mesoderm in the early stage of embryonic development, with the ability of self-renewal, multi-directional differentiation potential, and still maintaining their biological characteristics after large-scale in vitro expansion. The mesenchymal stem cells may express HLA class I antigens. The mesenchymal stem cells may not express or express HLA class II antigens at a low level. The mesenchymal stem cells may have the ability to differentiate into adipocytes, osteoblasts, and chondrocytes; they may also support the differentiation of hematopoietic stem cells into granulocytes, macrophages, and megakaryocytes. The mesenchymal stem cells may secrete cytokines, such as CSF-1, GM-CSF, G-CSF, IL-6, c-kit ligand, and / or IL-3.

[0164] In the present application, the immune cells may include immune effector cells. For example, the immune effector cells may include lymphocytes (such as cytotoxic T cells, memory T cells), macrophages, dendritic cells, and NK cells. For example, the immune cells may be selected from the group consisting of: T lymphocytes (for example, they may be activated T lymphocytes or may be non-activated T lymphocytes), B lymphocytes, NK cells, macrophages, dendritic cells, monocytes, granulocytes, and mast cells. For example, the immune cells may include primary T cells. The primary T cells may include T cells obtained from an individual (such as peripheral blood T cells obtained from an individual). The primary T cells may be different from T cells maintained in culture for a long time.

[0165] In the present application, the target molecule may include an antibody or antigen-binding fragment, a chimeric antigen receptor (CAR), a cytokine, and / or a chemokine.

[0166] In the present application, the chimeric antigen receptor may target one or more targets.

[0167] In the present application, the chimeric antigen receptor may target CD19 and CD20.

[0168] In the present application, the chimeric antigen receptor may target CD19 and CD22.

[0169] In the present application, the antibody or antigen-binding fragment may include a bispecific antibody or antigen-binding fragment.

[0170] In the present application, the antibody or antigen-binding fragment may include a bispecific T cell engager (BiTE).

[0171] In the present application, the target molecule may be selected from the group consisting of: antibodies (such as monoclonal antibodies, bispecific antibodies, and / or multispecific antibodies; Ig-type intact antibodies (such as IgG antibodies); antibody fragments (such as VHH, Fab, Fab’, (Fab) 2, scFv), cytokines, chemokines, chimeric antigen receptors, and MHC complexes (e.g., HLA-A, HLA-G).

[0172] For example, the target molecule may include CART. For example, the target molecule may include CART and a cytokine (e.g., IL-15). For example, the target molecule may include CART-BiTE.

[0173] In the present application, the nucleic acid molecule encoding the target molecule and / or the nucleic acid molecule encoding the selection marker molecule may be natural or a gene obtained by engineering modification.

[0174] In the present application, the plasmid may contain homology arms homologous to the ends of the nucleic acid molecule encoding the target molecule and / or the nucleic acid molecule encoding the selection marker molecule or a fragment thereof. For example, the 5'-end and / or 3'-end of the nucleic acid molecule or a fragment thereof may have a nucleic acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the nucleic acid sequence at the 5'-end and / or 3'-end of the position to be knocked in in the genomic DNA of the cell to be transfected. For example, the homology arm may be in the size range of 100 - 1000 bp. For example, the homology arm may be about 150 bp - 1000 bp, may be about 200 bp - 1000 bp, may be about 300 bp - 1000 bp, may be about 400 bp - 1000 bp, may be about 800 bp - 1000 bp.

[0175] In the present application, the selection marker molecule may include CD34 and / or an EGFRT truncation. In the present application, the selection marker molecule may also include an antibiotic.

[0176] In the present application, the expression and / or function of the selection marker molecule do not affect the expression and / or function of the target molecule.

[0177] In the present application, the type of the selection marker molecule may correspond to the type of the cell and / or the target molecule.

[0178] For example, the cell may be an immune cell, and the selection marker molecule may be CD34 and / or an EGFRT truncation. For example, the cell may be a CART cell, and the selection marker molecule may be CD34.

[0179] In the present application, the method may include the following steps: contacting the cell expressing the target molecule and the selection marker molecule with the screening agent.

[0180] In the present application, the method may further include the following steps: collecting the cells that bind to the screening agent.

[0181] In the present application, the method may further include the following steps: separating the cells that bind to the screening agent from the screening agent.

[0182] In the present application, the screening marker molecule may include a domain capable of binding to a screening agent. In the present application, through the binding of the screening molecule to the screening agent, the cells transfected with the desired target molecule and the screening marker molecule can be collected (for example, can be separated from the screening agent), and / or can be enriched. For example, the screening marker molecule may be CD34, and the cells expressed by the method of the present application (such as CART cells) express CD34. Contact the cells expressing CD34 with magnetic beads containing anti-CD34 antibodies. Due to the binding of CD34 and anti-CD34 antibodies to each other, the cells expressing CD34 bind to the magnetic beads. Collect the cells that bind to the magnetic beads expressing CD34. Further, the collected cells can be separated from the magnetic beads. In this way, the cells expressing the target molecule and the screening marker molecule (i.e., positive) can be obtained. Therefore, the method described in the present application can increase the positive rate of cell transfection (such as non-viral transfection).

[0183] In the present application, the screening agent may include magnetic beads. For example, the screening agent may include micro magnetic beads.

[0184] In the present application, the method may further include the following steps: treating the plasmid with deoxyribonuclease (DNase).

[0185] For example, after the treatment with DNase, in the transfection mixture containing the plasmid, the content of nucleic acid molecules or fragments thereof derived from the genome of microorganisms may account for about 10% (w / w) or less of the total content of nucleic acid molecules in the transfection mixture. For another example, after the treatment with DNase, in the transfection mixture containing the plasmid, the content of nucleic acid molecules or fragments thereof with a size of at least about 48 kb may account for about 10% (w / w) or less of the total content of nucleic acid molecules in the transfection mixture.

[0186] In the present application, the method described in the present application may include the following steps: reducing the content of the nucleic acid molecules or fragments thereof in the transfection mixture. In the present application, the reduction may mean reducing the content of the nucleic acid molecules or fragments thereof in the transfection mixture to meet the requirements of the method described in the present application.

[0187] In the present application, the reduction may include purifying the transfection mixture. That is, through the purification, the content of the nucleic acid molecule or its fragment in the transfection mixture can be reduced to meet the requirements of the method described in the present application. In the present application, through the purification, the nucleic acid molecule or its fragment with a size of at least about 48 kb can be removed; and / or, the nucleic acid molecule or its fragment derived from the genome of a microorganism can be removed. For example, the purification may include using methods well-known to those skilled in the art for purifying DNA to reduce the content of the nucleic acid molecule or its fragment in the transfection mixture. In the present application, the purification may not affect the integrity and / or activity of the plasmid DNA. In the present application, the reduction may include purifying the transfection mixture using a reagent selected from the group consisting of DNase, SDS, TX-100, CTAB, and cesium chloride-ethidium bromide. For example, DNase may be used for the reduction. For example, DNase can be used to reduce the content of the nucleic acid molecule or its fragment with a size of at least about 48 kb in the transfection mixture to meet the requirements of the method described in the present application; and / or, to reduce the content of the nucleic acid molecule or its fragment derived from the genome of a microorganism to meet the requirements of the method described in the present application. For example, the DNase may not affect the integrity and / or activity of the circular plasmid DNA.

[0188] In the present application, the reduction may include using a method selected from the group consisting of DNA artificial synthesis of the plasmid described in the present application and HPLC detection of the transfection mixture described in the present application. For example, DNA artificial synthesis can be directly carried out according to the nucleotide sequence of the plasmid, so that the obtained plasmid only contains the nucleotide sequence of the plasmid. For example, according to the HPLC detection of the transfection mixture described in the present application, based on the HPLC detection result, the characteristic peak corresponding to the plasmid can be selected, and the component corresponding to this characteristic peak can be collected, so that the obtained plasmid only contains the nucleotide sequence of the plasmid.

[0189] In the present application, using the method described in the present application can significantly improve the transfection efficiency and / or the positive rate of cells, and achieve the preparation of modified cells for cell therapy using a non-viral transfection method. The modified cells prepared by the method described in the present application can have a relatively high cell viability and / or a high and continuous amplification ability (for example, can continuously maintain a high amplification ability within the detection time range after transfection (such as electroporation) (for example, at least 9 days)). The modified cells prepared by the method described in the present application can have relatively high cell functions (for example, can have the ability to effectively kill tumor cells). The method described in the present application can be used to prepare modified cells required for cell therapy (such as CART cells). The method described in the present application can be used to prepare CART cells and / or CART-BiTE cells on the basis of primary T cells using a non-viral transfection method (such as plasmid transfection).

[0190] On the other hand, the present application provides cells and / or cell lines prepared by the method described in the present application.

[0191] On the other hand, the present application provides a modified immune cell that simultaneously expresses a target molecule and a screening marker molecule.

[0192] In the present application, the immune cells may be selected from the group consisting of: unactivated or activated T lymphocytes, B lymphocytes, NK cells, macrophages, dendritic cells, monocytes, granulocytes, and mast cells.

[0193] In the present application, the target molecule may include an antibody or antigen-binding fragment, a chimeric antigen receptor (CAR), a cytokine, and / or a chemokine.

[0194] In the present application, the chimeric antigen receptor may target one or more target points.

[0195] In the present application, the chimeric antigen receptor may target CD19 and CD20.

[0196] In the present application, the chimeric antigen receptor may target CD19 and CD22.

[0197] In the present application, the antibody or antigen-binding fragment may include a bispecific antibody or antigen-binding fragment.

[0198] In the present application, the antibody or antigen-binding fragment may include a bispecific T cell engager (BiTE).

[0199] In the present application, the screening marker molecule may include a domain capable of binding to a screening agent.

[0200] In the present application, the screening agent may include magnetic beads.

[0201] In the present application, the screening marker molecule may include CD34 and / or an EGFRT truncation body.

[0202] In the present application, the type of the screening marker molecule may be selected according to the type of the cells and / or the target molecule.

[0203] In the present application, the cells may be immune cells, and the screening marker molecule may be CD34 and / or an EGFRT truncation body.

[0204] Without being limited by any theory, the examples below are only for explaining the various technical solutions of the invention of the present application, and are not used to limit the scope of the invention of the present application.

[0205] Examples

[0206] Properties of Modified Cells Obtained by Non-Viral Transfection in Example 1

[0207] (1.1) Detect the content of nucleic acid molecules of microbial-derived genomes in plasmids

[0208] Using an E. coli DNA quantification detection kit (Cat#4458435, Thermal Fisher), qPCR quantitative determination was performed on the nucleic acid molecules or their fragments (mainly E. coli genomic DNA) of microbial-derived genomes contained in commercially available plasmids (where plasmids are referred to as plasmid A from GenScript Biotech and plasmid B from Sino Biological according to different purchase sources) according to the method described in the kit instructions. qPCR was performed using a CF96 Tough qPCR instrument (purchased from Bio-Rad).

[0209] Results of the determination Figure 15 are shown as follows Figure 15 The results show that there are significant differences in the content of nucleic acid molecules or their fragments of microbial-derived genomes in these plasmids. Among them, the results of plasmid A are shown in column 1; the results of plasmid B are shown in column 2. In plasmid A, the content of nucleic acid molecules of microbial-derived genomes accounts for 12 μg / mg of the total nucleic acid molecule content; in plasmid B, the content of nucleic acid molecules of microbial-derived genomes accounts for less than 1 μg / mg of the total nucleic acid molecule content.

[0210] Let plasmid A and plasmid B contain nucleic acid molecules that can express CD19 CAR and CD34 respectively. Among them, the nucleotide sequence of the nucleic acid molecule encoding CD34 is shown in SEQ ID NO.4. The nucleotide sequence of the nucleic acid molecule encoding CD19 CAR is shown in SEQ ID NO.5.

[0211] These plasmids are respectively called CD19 CAR-CD34-A and CD19 CAR-CD34-B. These plasmids were electroporated into human T cells that had been activated for two days with Dynal beads (activated using Dynal beads and collected human T cells in a 1:1 ratio in X-vivo15 + 20 U / ml culture medium), and the cell viability, relative cell number obtained by cell proliferation, and CAR expression ability on the 1st day or 4th day after electroporation were detected respectively. The results are respectively shown as follows Figure 15 where the results of plasmid A are shown in column 1; the results of plasmid B are shown in column 2.

[0212] Figure 15 The results show that when the content of nucleic acid molecules of microbial-derived genomes in the plasmid decreases, the cell viability, cell proliferation, and / or expression ability (such as expressing CD19 CAR) of the plasmid-transfected cells can be significantly improved.

[0213] (1.2) Prepare plasmids capable of expressing GPC3 CAR and CD34. Among them, the nucleotide sequence of the nucleic acid molecule encoding CD34 is as shown in SEQ ID NO.4. The nucleotide sequence of the nucleic acid molecule encoding GPC3 CAR is as shown in SEQ ID NO.3.

[0214] Activate the collected human T cells with Dynal beads at a ratio of 1:1 in X-vivo15 + 20U / ml culture medium.

[0215] Electroporate the prepared plasmids into human T cells that have been activated with Dynal beads for two days. The nucleic acid molecules encoding GPC3 CAR and CD34 are knocked into the genome of human T cells and can be regulated and expressed by the endogenous promoter. The obtained cells are named GPC3 CAR-CD34 T cells.

[0216] (1) Cell viability

[0217] Figure 1 It shows the cell viability of human T cells for different samples on the 1st - 4th day after electroporation of the said plasmids. The cell viability is obtained by counting with NC-200. Among them, the results 1 - 4 corresponding to each sample from left to right represent the results on the 1st day, 2nd day, 3rd day, and 4th day after electroporation respectively. Therefore, modifying the cells does not affect their own cell viability.

[0218] (2) Cell proliferation

[0219] Figure 2 It shows the cell numbers of human T cells for different samples on the 0th - 4th day after electroporation of the said plasmids. The cell viability is obtained by counting with NC-200. Among them, the results 1 - 5 corresponding to each sample from left to right represent the results on the 0th day, 1st day, 2nd day, 3rd day, and 4th day after electroporation respectively. Therefore, modifying the cells does not affect their own cell proliferation.

[0220] (3) Expression ability

[0221] Respectively count the expression rates of GPC3 CAR and CD34 in GPC3 CAR-CD34 T cells. The results are as Figure 3 shown. It can be seen that there is a linearly correlated relationship between the expressions of GPC3 CAR and CD34 in GPC3 CAR-CD34 T cells.

[0222] Characteristics of the enriched modified cells in Example 2

[0223] Based on the specific binding of cells expressing CD34 to micro magnetic beads containing anti-CD34 antibodies (CD34 MicroBeads Kit UltraPure human, Order NO. 130 - 100 - 453, Miltenyi Biotech), the GPC3 CAR-CD34 T cells prepared in Example 1 were enriched using the enrichment steps provided by the kit. Enriched GPC3 CAR-CD34 T cells were obtained.

[0224] (1) Expression ability and cell viability

[0225] The expression rates of GPC3 CAR and CD34 were respectively counted in the enriched GPC3 CAR-CD34 T cells. The results are as Figure 4 shown. Among them, the results 1 - 3 from left to right corresponding to each sample represent the expression rates of GPC3 CAR and CD34, and the cell viability of the enriched GPC3 CAR-CD34 T cells respectively. It can be seen that in the enriched GPC3 CAR-CD34 T cells, the enrichment operation does not affect the expression of GPC3 CAR and CD34. At the same time, the enrichment operation does not affect the cell viability of GPC3 CAR-CD34 T cells.

[0226] (2) Cell proliferation

[0227] The doubling rate of the enriched GPC3 CAR-CD34 T cells of each sample was calculated on the 4th day after electroporating the plasmid described in Example 1. Among them, the doubling rate = (the number of enriched GPC3 CAR-CD34 T cells on the 4th day after electroporation) / (the number of T cells to be electroporated on the 0th day of electroporation), and the results are as Figure 5 shown.

[0228] Figure 5 The results of... illustrate that the enriched GPC3 CAR-CD34 T cells still have a high cell proliferation ability after electroporation.

[0229] (3) Positive rate

[0230] The positive rates of GPC3 CAR expression in the GPC3 CAR-CD34 T cells prepared in Example 1 and the enriched GPC3 CAR-CD34 T cells prepared in Example 2 were respectively counted by flow cytometry. The results are respectively as Figure 6 shown in A - 6B. The results show that the positive rate can be increased from about 20% before enrichment to 83% after enrichment. It can be seen that the enriched modified cells can effectively improve the positive rate of expression.

[0231] Recovery ability of the modified cells in Example 3

[0232] The GPC3 CAR-CD34 T cells prepared in Example 1 derived from 5 samples were enriched with microbeads (MicroBead) containing CD34 antibody on the 4th day after electroporation, and cryopreserved after enrichment. The specific steps of cryopreservation are as follows: Centrifuge the cells (1000 RPM, 5 min), aspirate the supernatant. Centrifuge again to remove the culture supernatant adhering to the tube wall. Add CryoStor CS10 cryoprotectant (Stemcell Technologies, Canada) at 4°C, place it in a CoolCell cryopreservation box, overnight at -80°C, and then place it in liquid nitrogen.

[0233] (1) Viability

[0234] The cryopreserved cells were thawed, and the viability of GPC3 CAR-CD34 T cells at different days after thawing was detected. The results are as Figure 7 shown. Figure 7 The results indicate that the modified cells described in this application have good recovery ability after cryopreservation.

[0235] (2) Cell proliferation

[0236] The cryopreserved cells were thawed, and the cell number of GPC3 CAR-CD34 T cells at different days after thawing was detected. The results are as Figure 8 shown. Among them, the results 1-4 corresponding to each sample from left to right represent the cell numbers on the 0th day, 1st day, 2nd day, and 3rd day after thawing, respectively. Figure 8 The results indicate that the modified cells described in this application still have good proliferation ability after cryopreservation and thawing.

[0237] (3) Activation ability of target cells

[0238] The cryopreserved cells were thawed. On the 3rd day after thawing, the thawed GPC3 CAR-CD34 T cells were mixed and incubated with Huh-7 target cells at an effector-to-target ratio of 1:1, and the cell number of GPC3 CAR-CD34 T cells after incubation was detected. The results are as Figure 9 shown. Figure 9 The results indicate that the modified cells described in this application still have the amplification ability to be activated by target cells after cryopreservation and thawing.

[0239] Example 4 Killing ability of modified cells against tumors

[0240] (1) In vitro killing ability

[0241] The enriched GPC3 CAR-CD34 T cells prepared in Example 2 were mixed with Huh-7 target cells at different effector-to-target ratios in vitro, and the killing ratio (%) of Huh-7 was detected. The results are asFigure 10 As shown in the figure. Among them, the control refers to T cells that have not been transfected with the plasmid described in Example 1. Among them, 1-3 represent the cases of enriched GPC3 CAR-CD34 T cells from different sample sources. Figure 11 The results show that the modified cells described in the present application still have the ability to kill target cells after cryopreservation and resuscitation.

[0242] (2) In vivo killing ability

[0243] The GPC3 CAR-CD34 T cells prepared in Example 1 were enriched with MicroBeads containing CD34 antibody 4 days after electroporation and cryopreserved after enrichment.

[0244] HepG2 cells were injected into mice (NCG mice, purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.) to obtain a mouse model with subcutaneous HepG2 tumors.

[0245] When the tumor volume reached approximately 100 3 mm, the cryopreserved and resuscitated cells were injected into the obtained mouse model via the tail vein at a dose of 1e7 (4 mice were treated for each treatment). The average value of the tumor volume in the mice after treatment was detected. The results are as Figure 11 shown. Among them, the control refers to T cells transfected with CD19 CAR (the nucleotide sequence of the nucleic acid molecule encoding CD19 CAR is as shown in SEQ ID NO.5).

[0246] Figure 11 The results show that the modified cells described in the present application still have the ability to specifically kill tumors in vivo after cryopreservation and resuscitation.

[0247] Killing ability of the modified cells in Example 5 against tumors

[0248] Prepare plasmids that can express CD19 CAR and CD34. Among them, the nucleotide sequence of the nucleic acid molecule encoding CD19 CAR is as shown in SEQ ID NO.5. The nucleotide sequence of the nucleic acid molecule encoding CD34 is as shown in SEQ ID NO.4. The nucleotide sequence of the nucleic acid molecule encoding GPC3 CAR-CD34 knocked into TRAC is as shown in SEQ ID NO.1.

[0249] Use Dynal beads to activate the collected human T cells in X-vivo15 + 20U / ml culture medium at a ratio of 1:1.

[0250] The prepared plasmid was electroporated into human T cells that had been activated with Dynal beads for two days. The nucleic acid molecules encoding CD19 CAR and CD34 were knocked into the genome of human T cells and could be regulated by the endogenous promoter for expression. The resulting cells were named CD19 CAR-CD34 T cells.

[0251] The CD19 CAR-CD34 T cells were enriched with MicroBeads containing CD34 antibodies on the 4th day after electroporation. The expression rates of CD19 CAR and CD34 were respectively counted in the enriched CD19 CAR-CD34 T cells. The results are as Figure 12 shown. The results indicate that the enriched CD19 CAR-CD34 T cells can normally express CD19 CAR and CD34.

[0252] (1) In vitro killing ability

[0253] The enriched CD19 CAR-CD34 T cells were mixed with Nalm6 target cells in vitro at different effector-to-target ratios, and the killing ratio (%) of Nalm6 was detected. The results are as Figure 13 shown. Figure 13 The results show that the enriched CD19 CAR-CD34 T cells have the ability to kill target cells in vitro.

[0254] (2) In vivo killing ability

[0255] Fluorescent Nalm6 cells (Nalm-6-Luc, purchased from PharmaLegacy, Shanghai) were intravenously injected into mice (NCG mice, purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.) to obtain a mouse model of hematological tumor Nalm6.

[0256] The enriched CD19 CAR-CD34 T cells were intravenously injected into the obtained mouse model at a dose of 1e7 (4 mice were treated for each treatment). The growth and killing of the tumor were detected by the fluorescence value of Nalm-6-Luc (Lumina XRMS, PerkinElmer). The results are as Figure 14 shown. The control refers to T cells transfected with GPC3 CAR (the nucleotide sequence of the nucleic acid molecule encoding GPC3 CAR is as shown in SEQ ID NO.3).

[0257] Figure 14 The results show that the modified cells described in this application still have the ability to kill tumors in vivo after cryopreservation and resuscitation.

[0258] The foregoing detailed description is provided by way of explanation and illustration and is not intended to limit the scope of the appended claims. Various changes to the presently recited embodiments of the present application will be apparent to those of ordinary skill in the art and are within the scope of the appended claims and their equivalents.

Claims

1. A method for modifying a cell, comprising the following steps: non-virally transfecting a nucleic acid molecule encoding a target molecule and a selection marker molecule into the cell by electroporation in vitro, such that the cell expresses the target molecule and the selection marker molecule; wherein the non-viral transfection is transfection using a circular nucleic acid molecule having a coding sequence, and the circular nucleic acid molecule comprises a circular plasmid or a supercoiled plasmid; wherein in the transfection mixture containing the plasmid, the content of the nucleic acid molecule or its fragment derived from the genome of a microorganism accounts for 1% or less of the total content of the nucleic acid molecules in the transfection mixture; the cell is a primary T cell.

2. The method according to claim 1, wherein, the method further comprises: collecting and / or enriching the cells transfected with the target molecule and the selection marker molecule by the binding of the selection marker molecule to a selection agent.

3. The method according to claim 1, wherein the nucleic acid molecule encoding the target molecule and the nucleic acid molecule encoding the selection marker molecule are integrated into the genome of the cell.

4. The method according to claim 3, wherein the transfection comprises: using a transposon system and / or using gene editing knock-in; wherein the gene editing method is selected from one or more of the following groups: CRISPR / Cas system, RNA editing system ADAR, RNA-guided endonuclease, zinc finger protease, Mega-TAL nuclease, TALENs, and Meganucleases.

5. The method according to claim 4, wherein the plasmid serves as a donor plasmid in the gene editing knock-in.

6. The method according to claim 5, wherein the donor plasmid comprises homologous arms.

7. The method according to claim 1, wherein in the transfection mixture containing the plasmid, the content of the nucleic acid molecule or its fragment derived from the genome of a microorganism accounts for 5‰ or less of the total content of the nucleic acid molecules in the transfection mixture.

8. The method according to claim 1, wherein in the transfection mixture containing the plasmid, the content of the nucleic acid molecule or its fragment derived from the genome of a microorganism accounts for 1‰ or less of the total content of the nucleic acid molecules in the transfection mixture.

9. The method according to claim 1, wherein in the transfection mixture containing the plasmid, the nucleic acid molecule derived from the genome of a microorganism is a nucleic acid molecule with a size of at least 48 kb, and the content of the nucleic acid molecule with a size of at least 48 kb or its fragment accounts for 1% or less of the total content of the nucleic acid molecules in the transfection mixture; the nucleic acid molecule with a size of at least 48 kb or its fragment is derived from a microorganism.

10. The method according to claim 1, wherein the microorganism comprises Escherichia coli.

11. The method according to claim 1, comprising the following step: treating the plasmid with deoxyribonuclease (DNase); the DNase can non-specifically cleave linear DNA.

12. The method according to claim 1, wherein the target molecule comprises an antibody or antigen-binding fragment, a chimeric antigen receptor (CAR), a cytokine, and / or a chemokine.

13. The method according to claim 12, wherein the chimeric antigen receptor targets CD19 and CD20; or the chimeric antigen receptor targets CD19 and CD22.

14. The method according to claim 1, wherein the screening marker molecule comprises a domain capable of binding to a screening agent; wherein the screening agent comprises magnetic beads.

15. The method according to claim 14, wherein the screening marker molecule comprises CD34 and / or an EGFRT (epidermal growth factor receptor truncated) truncation body.

16. The method according to claim 14, comprising the following steps: contacting the cells expressing the target molecule and the screening marker molecule with the screening agent; collecting the cells bound to the screening agent; and separating the cells bound to the screening agent from the screening agent.

17. A modified immune cell that simultaneously expresses a target molecule and a screening marker molecule, wherein, the nucleic acid molecules encoding the target molecule and the screening marker molecule are transfected into the cells by non-viral transfection, so that the cells express the target molecule and the screening marker molecule; wherein the non-viral transfection is transfection using a circular nucleic acid molecule having a coding sequence, and the circular nucleic acid molecule comprises a circular plasmid or a supercoiled plasmid; wherein in the transfection mixture containing the plasmid, the content of the nucleic acid molecule or its fragment derived from the genome of the microorganism accounts for less than 1% of the total nucleic acid content of the transfection mixture; the immune cell is a primary T cell.

18. The immune cell according to claim 17, wherein the target molecule comprises an antibody or an antigen-binding fragment, a chimeric antigen receptor (CAR), a cytokine, and / or a chemokine; and / or the screening marker molecule comprises CD34 and / or an EGFRT truncation body.

19. The immune cell according to claim 18, wherein the chimeric antigen receptor targets CD19 and CD20; or the chimeric antigen receptor targets CD19 and CD22.

20. The immune cell according to claim 17, wherein the immune cell is a CAR-T cell, and the screening marker molecule comprises CD34.

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