Cell containing exogenous insertion sequence comprising plurality of homologous sequences arranged in tandem, and method for producing same

Abstract

The present disclosure relates to: a cell that contains an exogenous insertion sequence comprising a plurality of homologous sequences arranged in tandem; and a method for producing the cell. The plurality of homologous sequences arranged in tandem may have sequences that are different from each other to the extent that homologous recombination is not induced. The present disclosure also relates to: a cell that contains an exogenous insertion sequence comprising an EF1α promoter and a ubiquitin promoter; and a method for producing the cell.

Description

Cells containing an exogenous insertion sequence comprising a plurality of homologous sequences arranged in tandem and a method for producing the same

[0001] The present disclosure relates to cells containing an exogenous insertion sequence comprising a plurality of homologous sequences arranged in tandem and a method for producing the same.

[0002] Techniques for introducing foreign genes into mammalian cells, particularly for simultaneously introducing multiple genes, are important basic techniques in modern biological research. The introduction of multiple genes is essential for modifying cell functions and reconstructing complex biological systems.

[0003] Conventionally, a method using a plasmid vector has been known as a method for introducing multiple genes. For example, Kriz et al. developed a multiple gene expression system for mammalian cells and succeeded in simultaneously expressing multiple genes. However, in the method using a plasmid vector, the introduced genes are not integrated into the genome.

[0004] On the other hand, as a method for stably introducing genes into the genome, a method using a retroviral vector has been known. Szymczak et al. succeeded in integrating the expression sequences of four types of CD3 proteins (CD3ε, γ, δ, ζ) into the genome using a single retroviral vector utilizing a 2A peptide. In this method, multiple proteins were expressed from a single promoter via multiple 2A peptide sequences. However, in this method, the expression of the protein located on the C-terminal side decreases.

[0005] Kriz, A., Schmid, K., Baumgartner, N. et al., Nat Commun 1, 120 (2010). https: / / doi.org / 10.1038 / ncomms1120Szymczak, A., Workman, C., Wang, Y. et al, Nat Biotechnol 22, 589-594 (2004). https: / / doi.org / 10.1038 / nbt957

[0006] The present disclosure provides cells containing an exogenous insertion sequence comprising a plurality of homologous sequences arranged in tandem and a method for producing the same.

[0007] The inventors have found that when an exogenous insertion sequence containing multiple homologous sequences (including identical sequences) arranged in tandem is inserted by genome editing using homologous recombination, the expression of some genes contained in the exogenous insertion sequence decreases. This suggests that homologous recombination occurs between multiple homologous sequences during insertion, potentially causing sequences to be lost between them. When the exogenous insertion sequence contains multiple gene expression cassettes, the promoter portion can become a homologous sequence. To resolve this issue, the inventors have found that the decrease in gene expression can be suppressed by introducing mutations into the promoter portion. The inventors have also found that the decrease in gene expression can be suppressed by using different promoters with different sequences.

[0008] According to this disclosure, for example, the following inventions may be provided: (1) A cell having an exogenous insertion sequence, wherein the exogenous insertion sequence comprises a first sequence and a second sequence, each having a first common homologous sequence and a second common homologous sequence, the common homologous sequences having 90% or more sequence identity with each other, and having sequences that are different to the extent that homologous recombination does not occur between the common homologous sequences (especially during gene insertion formation). (2) A cell having an exogenous insertion sequence, wherein the exogenous insertion sequence comprises a first sequence and a second sequence, each having a first promoter and a second promoter that drive gene expression, the first promoter and the second promoter are operably linked to a first exogenous gene and a second exogenous gene, respectively, and the first promoter and the second promoter have sequences that are different to the extent that homologous recombination does not occur between the promoter sequences. (3) The cell according to (1) or (2) above, wherein the first sequence comprises a promoter or a first modified promoter, the second sequence comprises a second modified promoter, and the first modified promoter and the second modified promoter each maintain all or part of the promoter activity. (4) The cell according to (2) or (3) above, wherein the promoter is an EF1α promoter. (5) The cell according to (2) or (3) above, wherein the promoter is a ubiquitin promoter. (6) The cell according to (4) or (5) above, wherein each modified promoter maintains all or part of the promoter activity and exhibits 90% sequence identity with respect to the promoter sequence. (7) The exogenous insertion sequence comprises n sequences from the first sequence to the nth sequence {where n is a natural number of 2 or more}. A cell in which the first to nth sequences each contain first to nth promoters, each of which contains a selected promoter consisting of the group comprising the EF1α promoter, a modified EF1α promoter, a ubiquitin promoter, and a modified ubiquitin promoter, and each of the first to nth promoters has sequences that are different to the extent that homologous recombination does not occur between them.(8) The cell according to (7) above, wherein the first sequence comprises an EF1α promoter or a modified EF1α promoter, the second to nth sequences comprises a modified EF1α promoter, and each modified EF1p promoter sequence maintains all or part of the promoter activity and exhibits 90% or more sequence identity with the EF1α promoter sequence of SEQ ID NO: 1, or the first sequence comprises a ubiquitin promoter or a modified ubiquitin promoter, the second to nth sequences comprises a modified ubiquitin promoter, and the modified ubiquitin promoter sequence maintains all or part of the promoter activity and exhibits 90% sequence identity with the ubiquitin promoter sequence of SEQ ID NO: 5 or 6. (9) The cell as described in (7) above, wherein the first sequence comprises an EF1α promoter or a modified EF1α promoter, the second sequence comprises a ubiquitin promoter or a modified ubiquitin promoter, and if n is 3 or more, the third to n sequences comprise one promoter selected from the group consisting of an EF1α promoter or a modified EF1α promoter and a ubiquitin promoter or a modified ubiquitin promoter, each modified EF1p promoter sequence maintains all or part of the promoter activity and exhibits 90% or more sequence identity with the EF1α promoter sequence of SEQ ID NO: 1, and each modified ubiquitin promoter sequence maintains all or part of the promoter activity and exhibits 90% sequence identity with the ubiquitin promoter sequence of SEQ ID NO: 5 or 6. (10) The cell according to (1) above, wherein the first common homologous sequence and the second common homologous sequence (preferably comprising homologous sequences, wherein the homologous sequences comprise one selected from the group consisting of nucleic acids encoding a chimeric antigen receptor, nucleic acids encoding a T cell receptor, and nucleic acids encoding an antibody or an antigen-binding fragment thereof. (11) The cell according to any one of (1) to (10) above, which is a pluripotent cell. (12) A composition comprising the cell according to any one of (1) to (11) above.

[0009] The scheme for an experiment in which a genomic region of a cell is cut and homologous recombination is induced with a donor plasmid containing four genes, each operably linked to the EF1α promoter, is shown. The results of cloning cells after homologous recombination induction using the scheme in Figure 1A and confirming the integration of each gene into the genome by PCR are shown. In clone 6, all four genes were detected, but the integration of some genes into the genome could not be confirmed in the other clones. The scheme for an experiment in which a genomic region of a cell is cut and homologous recombination is induced with a donor plasmid containing the SctHLA-E and GFP-PuroR genes, and the presence of SctHLA-E is detected with an anti-ELA-E antibody are shown. The results of experiments in which the expression of SctHLA-E and GFP in cells after homologous recombination were confirmed when SctHLA-E was operably linked to the EF1α promoter (EF1p) and the GFP-PuroR gene was linked to the ubiquitin promoter (UBCp), EF1p, or modified EF1p, respectively, are shown. This section shows the results of experiments confirming the expression of SctHLA-E and GFP in cells after homologous recombination when the GFP-PuroR gene was linked to various modified EF1p genes. It also shows the results of experiments confirming the expression of SctHLA-E and GFP in cells after homologous recombination when SctHLA-E was linked to UBCp and the GFP-PuroR gene was linked to UBCp. The results demonstrate that the expression of tandem-linked genes is enhanced by introducing mutations into the promoter. In Figure 6, four genes were functionally linked to both wild-type and modified EF1α promoters. It is also shown that promoter activity is maintained even when a single mutation is introduced between 30 and 50 nucleotides.

[0010] In this specification, "exogenous insertion sequence" refers to an insertion sequence added to a cell, which is artificially created through genetic recombination. The sequence itself is included in the definition of an exogenous insertion sequence, whether it is an endogenous sequence or a sequence different from an endogenous sequence. This term is used specifically to distinguish it from other sequences on the genome.

[0011] In this specification, "homologous recombination" refers to recombination that occurs specifically between sequences with high homology. Homologous recombination is considered an important process for gene repair and maintaining genome stability. It is thought that double-strand breaks in DNA activate homologous recombination. A certain level of high homology and the length of a sequence-matching region play an important role in homologous recombination, and it is generally considered preferable to have a sequence-matching region that extends for 200 bases or more. Therefore, it is expected that the frequency of homologous recombination can be reduced by making the length of the sequence-matching region, for example, about 200 bases or less. "About" may mean a numerical range of ±25%, ±20%, ±15%, ±10%, or ±5% of the following number.

[0012] In this specification, "sequence identity" can be calculated by aligning the sequences and determining the number of positions where the sequences match, with the total length of the aligned sequences as the denominator. Examples of alignment methods that can be used include Crystal Omega, EMBOSS Needle (https: / / www.ebii.ac.uk / jdispatcher / psa / emboss_needle), etc.

[0013] In this specification, “cell” may be a eukaryotic cell. Eukaryotic cells may be cells selected from the group consisting of pluripotent cells and pluripotent stem cells (such as embryonic stem cells and induced pluripotent stem cells), tissue stem cells, somatic cells, germline cells (e.g., germ cells), primary cells, cell lines, immortalized cells, cancer cells, non-cancer cells, cells of diseased patients, cells of healthy individuals, animal cells (e.g., mammalian cells, e.g., human cells), insect cells (e.g., silkworm cells), HeLa cells, MCF7 cells, HCT116 cells, HEK293 cells, HEK293T cells, Expi293F® cells, FreeStyle® 293F cells, Chinese hamster ovary cells (CHO cells), CHO-S cells, CHO-K1 cells, and ExpiCHO cells, as well as cells derived from these cells.

[0014] In this specification, a "promoter" is a sequence capable of driving gene expression, which can operably link a gene to the promoter, thereby inducing the expression of RNA such as mRNA from the gene. The promoter is not particularly limited, and various types of pol II promoters can be used, for example. Examples of pol II promoters are not particularly limited, but include the CMV promoter, EF1 promoter (EF1α promoter), ubiquitin promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, CBh promoter, etc. Inducible promoters are also included as promoters. An inducible promoter is a promoter that can induce the expression of a polynucleotide functionally linked to the promoter only in the presence of an inducing factor that drives the promoter. Examples of inducible promoters include promoters that induce gene expression by heating, such as heat shock promoters. Inducible promoters also include promoters in which the inducing factor that drives the promoter is a drug. Examples of such drug-inducible promoters include Cumate operator sequences, λ operator sequences (e.g., 12×λOp), and tetracycline-based inducible promoters. Examples of tetracycline-based inducible promoters include promoters that drive gene expression in the presence of tetracycline or its derivatives (e.g., doxycycline), or reverse tetracycline-regulating transactivators (rtTA). An example of a tetracycline-based inducible promoter is the TRE3G promoter. Genes typically have an open reading frame. Promoters from different animal species can also be used in combination.

[0015] In this specification, “antibody” means immunoglobulin. Antibodies can be various isotypes, for example, IgG. Antibodies are preferably monoclonal antibodies. Antibodies can be human chimeric antibodies, humanized antibodies, or human antibodies. Human chimeric antibodies can be produced by replacing the constant region of a non-human antibody with the constant region of a human antibody. Humanized antibodies can be produced by replacing the six CDRs of a human antibody with the six corresponding CDRs of a non-human antibody. Human antibodies can be produced using animals (e.g., mice) in which at least the heavy chain variable region of the immunoglobulin has been replaced with the corresponding region of a human gene locus. If the constant region is non-human, a human antibody can be obtained by replacing the constant region with the amino acid sequence of a human antibody. In this specification, antibodies are preferably humanized antibodies. In this specification, antibodies are preferably human antibodies. Antibodies have a signal peptide when produced intracellularly, but this signal peptide is excised when secreted extracellularly. Therefore, when administered as a pharmaceutical, the signal peptide is not necessary for the antibody.

[0016] In this specification, "CDR" refers to complementarity-determining regions located in the heavy chain variable region and the light chain variable region of an antibody. There are three CDRs in both the heavy chain and light chain variable regions, and they are referred to as CDR1, CDR2, and CDR3 from the N-terminus. CDRs can be determined, for example, based on the numbering by Kabat et al. (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, Bethesda: US Dept. of Health and Human Services, PHS, NIH.).

[0017] In this specification, "antigen-binding fragment of an antibody" means a fragment of an antibody that maintains its ability to bind to an antigen. Examples of antigen-binding fragments include Fab, Fab', and F(ab'). ２ Fv, scFv (single-chain Fv), diabody, bsDb, scBsDb, scBs TaFv, BiTE, sc(Fv) ２ (Single chain (Fv)２ For example, digesting an antibody with papain can yield Fab. Alternatively, digesting an antibody with pepsin yields F(ab'). ２ This can be obtained, and further reduction can yield Fab'. Other antigen-binding fragments of antibodies can also be prepared by methods well known to those skilled in the art. In this disclosure, such antigen-binding fragments of antibodies, in particular fragments having multiple heavy chain variable regions and / or light chain variable regions, can be used.

[0018] In this specification, a “chimeric antigen receptor” (CAR) is a chimeric molecule having an antigen-binding fragment of an antibody (particularly scFv) and an immune cell activation domain. A CAR is generally a molecule consisting of a linked antigen-binding domain (e.g., scFv), an extracellular hinge domain, a transmembrane domain (e.g., CD8α or CD28), and an activation signaling domain (e.g., CD3ζ). CARs can be introduced into cells and expressed on the cell surface. Cells expressing CARs can be targeted against specific antigens. CARs can be introduced into immune cells, such as T cells or NK cells, to target these immune cells against cancer. While first-generation CARs consisted of a linked scFv, an extracellular hinge domain, a transmembrane domain (e.g., CD8α or CD28), and an activation signaling domain (e.g., CD3ζ), second-generation CARs further include a co-stimulatory molecule signaling domain for activation of the immune cell into which the CAR is introduced. Co-stimulatory factors such as CD28, 4-1BB, OX40, CD27, and ICOS are used as the co-stimulatory molecular signaling domain. In third-generation CARs, multiple co-stimulatory factors are incorporated. Thus, improvements have been made to CARs to enable the sustained proliferation of CAR-transformed immune cells in vivo. It is preferable that all domains other than the scFv portion are derived from human proteins.

[0019] In this specification, “T cell receptor” (TCR) is a protein complex presented on the cell surface by T cells, which are immune cells, for the purpose of recognizing antigens. A TCR is a heterodimer composed of two polypeptide chains: an α chain (TCRα) and a β chain (TCRβ). A TCR may include a variable region (V region), a constant region (C region), a transmembrane region, and a signaling region. A TCR typically recognizes antigen peptides presented on MHC (or HLA). A TCR may recognize both MHC (or HLA) and antigen peptides. Heterodimers of TCRs, composed of two polypeptide chains: a γ chain (TCRγ) and a δ chain (TCRδ), are also known and may be used in this disclosure. TCRs can be cloned from T cells that possess antigen specificity.

[0020] In this specification, when referring to the first through nth arrays, for convenience, the elements contained in the tth array (where t is a natural number satisfying 1 < t ≤ n) are referred to as the tth element. For example, if the tth array contains elements A, B, and C, then elements A, B, and C are referred to as the tth element A, the tth element B, and the tth element C, respectively. Conversely, when the tth element D is mentioned, it means that the tth array contains element D, and that element D is identified as the tth element D.

[0021] <Cells of this Disclosure> This disclosure provides cells having exogenous insertion sequences. The exogenous insertion sequence is, for example, loaded onto the genome of the cell. The exogenous insertion sequence may also be loaded onto an extragenomic gene element. For example, the exogenous insertion sequence may be loaded onto an artificial chromosome. Alternatively, the exogenous insertion sequence may be loaded onto an extrachromosomal vector such as a cosmid or plasmid.

[0022] Examples of artificial chromosomes include episomal artificial chromosomes (e.g., circular DNA without centromeres or telomeres), centromere artificial chromosomes (e.g., having a centromere sequence derived from a host animal), mini artificial chromosomes (e.g., composed of the minimum necessary regions such as centromeres, telomeres, and origin regions), telomere artificial chromosomes (e.g., having telomeres derived from a host animal), and reconstructed artificial chromosomes (e.g., created by modifying natural chromosomes derived from a host animal).

[0023] In one embodiment, the foreign insertion sequence includes n sequences, namely the first to the nth sequence {where n is a natural number greater than or equal to 2}, i.e., n sequences in total, where the first to the nth sequences have homologous sequences (also simply called "common homologous sequences"), and these homologous sequences are different to the extent that they do not undergo homologous recombination. Therefore, although the term "common" is used for common homologous sequences, this is to clarify that they can be consistently included in the first to the nth sequences, and the common homologous sequences in the t and k sequences are not identical {where t and k are any natural numbers satisfying 1 ≤ t < k ≤ n}. Preferably, all common homologous sequences in the first to nth sequences are sequences with a length of 200 bp or more. n can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or a natural number greater than or equal to 11. n can be, for example, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, or 2-4; 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, or 3-4; or 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5.

[0024] In one embodiment, in an inserted foreign sequence, the first to nth sequences all have the same orientation. Same orientation means that each common homologous sequence faces the same direction.

[0025] However, in some embodiments, some or all of the first to n sequences may have different orientations. Since homologous recombination between sequences with different orientations only results in inversion, the common homologous sequence may be a sequence that undergoes homologous recombination, and for example, it may contain identical sequences in whole or in part. This can be applied in all embodiments of the present disclosure. In some preferred embodiments, each of the first to n sequences comprises a gene expression cassette, all of which have the same orientation.

[0026] The length of the foreign insertion sequence containing the first to n sequences is not particularly limited, but may be, for example, 5kb or more, 6kb or more, 7kb or more, 8kb or more, 9kb or more, 9.5kb or more, or 10kb or more, and is not particularly limited, but may be, for example, 1Mb or less, 500kb or less, 400kb or less, 300kb or less, 200kb or less, 100kb or less, 50kb or less, 45kb or less, 40kb or less, 35kb or less, 30kb or less, 25kb or less, 20kb or less, 15kb or less, 14kb or less, 13kb or less, 12kb or less, 11kb or less, 10kb or less, 9kb or less, 8kb or less, 7kb or less, or 6kb or less. Since the knock-in of an exogenous insertion sequence does not necessarily need to be achieved in a single knock-in, and can be achieved through multiple knock-ins, there is no particular limit to the maximum length of the exogenous insertion sequence, as long as cell proliferation and / or survival are not significantly inhibited.

[0027] The length of the first to nth common homologous sequences is not particularly limited, but could be, for example, approximately 300 bp or more, approximately 400 bp or more, approximately 500 bp or more, approximately 600 bp or more, approximately 700 bp or more, approximately 800 bp or more, approximately 900 bp or more, approximately 1000 bp or more, approximately 1100 bp or more, approximately 1200 bp or more, approximately 1300 bp or more, approximately 1400 bp or more, approximately 1500 bp or more, approximately 1600 bp or more, approximately 1700 bp or more, approximately 1800 bp or more, approximately 1900 bp or more, or approximately 2000 bp or more. The length of the common homologous sequences is not particularly limited, but could be, for example, approximately 5 kb or less, approximately 4 kb or less, approximately 3 kb or less, or approximately 2 kb or less. The length of the common homologous sequences is not particularly limited, but could be, for example, approximately 300 bp to approximately 5 kb.

[0028] Common homologous sequences in the t-th sequence (where t is a natural number satisfying 1 < t ≤ n) may have sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, or 99.5% or more with respect to the common homologous sequences in the first sequence (or the t-th sequence). The first sequence may be, for example, a reference sequence, and sequence identity may be determined with respect to the first sequence. Common homologous sequences in the first sequence (or the t-th sequence) may be, for example, wild-type sequences. In one embodiment, the first to n-th sequences are arranged in order from upstream, but in another embodiment, the order of the first to n sequences does not coincide with the order from upstream.

[0029] The first to nth common homologous sequences have sequences that are different from each other to the extent that they do not undergo homologous recombination. The frequency of homologous recombination can be reduced as the sequence identity decreases, but for example, even if there is one sequence difference at approximately 20 bp to 300 bp, approximately 30 bp to 300 bp, approximately 35 bp to 300 bp, approximately 40 bp to 300 bp, approximately 45 bp to 300 bp, and approximately 50 bp to 300 bp, the frequency of homologous recombination can be sufficiently reduced. For this purpose, the frequency of recombination between promoters can be reduced by introducing multiple mutations (especially point mutations, etc.) into a single promoter. Alternatively, the frequency of recombination between promoters can be reduced by using promoters from different animal species or by introducing one or more mutations (especially point mutations, etc.) into them. A person skilled in the art can introduce mutations that appropriately reduce sequence identity to achieve this objective.

[0030] In one embodiment, the first to nth common homologous sequences are such that there is at least one every approximately 20 bp to approximately 300 bp, at least one every approximately 30 bp to approximately 300 bp, at least one every approximately 35 bp to approximately 300 bp, at least one every approximately 40 bp to approximately 300 bp, at least one every approximately 45 bp to approximately 300 bp, at least one every approximately 50 bp to approximately 300 bp, at least one every approximately 60 bp to approximately 300 bp, and approximately The sequences may contain different bases at the following rates: one or more every 70 bp to approximately 300 bp, one or more every approximately 80 bp to approximately 300 bp, one or more every approximately 90 bp to approximately 300 bp, one or more every approximately 100 bp to approximately 300 bp, one or more every approximately 150 bp to approximately 250 bp, one or more every approximately 175 bp to approximately 225 bp, one or more every approximately 180 bp to approximately 220 bp, and one or more every approximately 200 bp. This reduces the possibility of inducing homologous recombination between common homologous sequences, making it easier to maintain each gene on the cellular genome or extragenomic gene element with a higher probability, while not significantly impairing the expected activity of the sequence, and maintaining some or all of that activity {without excluding cases where the activity is improved}. In the above, "one or more" includes one, two, three, four, or five, but is acceptable as long as it does not significantly impair the expected activity of the sequence.

[0031] In some embodiments, it is desirable that the modified common homologous sequence has high sequence identity with the original common homologous sequence in order to avoid significantly impairing its expected activity. High sequence identity increases the likelihood that all or part of the sequences necessary for the activity are maintained, thus eliminating the need for excessive trial and error to select a sequence that maintains all or part of the activity from a group of sequences with high sequence identity. In this sense, it is preferable that the modified common homologous sequence exhibits sequence identity of 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, or 99.7% or more with the original common homologous sequence that has activity. It is self-evident that the higher the sequence identity, the greater the likelihood that the sequences necessary for maintaining all or part of the activity are preserved. However, in the examples described later, even when a single mutation was introduced between 40 bp and 50 bp, no particular effect on promoter activity was observed. Furthermore, although promoters with various modifications were designed in the examples described later, all of them maintained the original promoter activity.

[0032] In one embodiment, the common homologous sequences of the first to nth are approximately 300 bp or more, approximately 290 bp or more, approximately 280 bp or more, approximately 270 bp or more, approximately 260 bp or more, approximately 250 bp or more, approximately 240 bp or more, approximately 230 bp or more, approximately 220 bp or more, approximately 210 bp or more, approximately 200 bp or more, approximately 190 bp or more, approximately 180 bp or more, approximately 170 bp or more, or approximately 150 bp It does not have a sequence that is completely identical over a continuous sequence of p or more, approximately 140 bp or more, approximately 130 bp or more, approximately 120 bp or more, approximately 110 bp or more, approximately 100 bp or more, approximately 90 bp or more, approximately 80 bp or more, approximately 70 bp or more, approximately 60 bp or more, approximately 50 bp or more, 45 bp or more, 40 bp or more, 35 bp or more, 30 bp or more, 25 bp or more, or 20 bp or more. In this embodiment, it is further preferable that the first to n common homologous sequences have different bases at a rate of one or more every approximately 20 bp to approximately 300 bp, one or more every approximately 30 bp to approximately 300 bp, one or more every approximately 35 bp to approximately 300 bp, one or more every approximately 40 bp to approximately 300 bp, one or more every approximately 45 bp to approximately 300 bp, one or more every approximately 50 bp to approximately 300 bp, one or more every approximately 100 bp to approximately 300 bp, one or more every approximately 150 bp to approximately 250 bp, one or more every approximately 175 bp to approximately 225 bp, one or more every approximately 180 bp to approximately 220 bp, and one or more every approximately 200 bp. This configuration reduces the frequency of homologous recombination between common homologous sequences during and after the insertion of exogenous insertion sequences, making it easier to maintain each gene on the cell's genome or extragenomic gene elements with a higher probability, while not significantly impairing the expected activity of the sequence, and maintaining some or all of that activity (without excluding cases where the activity is improved).

[0033] This configuration suppresses homologous recombination between the first to nth common homologous sequences, making it easier to maintain each gene on the cell's genome or extragenomic gene elements with a higher probability, without significantly impairing the expected activity of the sequence, and maintaining some or all of that activity {without excluding cases where the activity is improved}. In particular, if only one mutation is introduced at intervals of approximately 20 bp to 300 bp, 30 bp to 300 bp, 35 bp to 300 bp, 40 bp to 300 bp, 45 bp to 300 bp, 50 bp to 300 bp, 60 bp to 300 bp, 70 bp to 300 bp, 80 bp to 300 bp, 90 bp to 300 bp, 100 bp to 300 bp, or 100 bp to 300 bp, the activity can be appropriately maintained.

[0034] In some embodiments, the first to nth sequences include multiple gene expression cassettes, and for example, the first to nth common homologous sequences may, without particular limitation, include a chimeric antigen receptor (CAR), a T cell receptor (TCR), an antibody, or an antigen-binding fragment thereof. For example, this disclosure can be utilized when maintaining in tandem genes on DNA that encode multiple chimeric antigen receptors having different antigen-binding properties, when maintaining in tandem genes on DNA that encode multiple TCRs having different antigen-binding properties, or when maintaining in tandem genes on DNA that encode multiple antibodies or antigen-binding fragments thereof having different antigen-binding properties. In particular, these molecules may have a common backbone within the molecule (i.e., the backbone includes a common homologous sequence). When protein units containing the same amino acid sequence are linked in tandem, the incidence of homologous recombination between each unit can be reduced by introducing mutations in the nucleotide sequence encoding them. Nucleotide sequence mutations can be introduced by changing codons without changing the amino acids, thereby changing the amino acids but not affecting protein function. To introduce mutations that alter amino acids without affecting protein function, the mutations are preferably conservative substitutions of amino acids. Conservative substitutions of amino acids are substitutions to amino acids with similar properties. Examples include substitutions between hydrophobic amino acids (e.g., alanine (Ala), leucine (Leu), isoleucine (Ile), valine (Val), methionine (Met)), mutations between polar uncharged amino acids (e.g., serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln)), mutations between acidic amino acids (e.g., aspartic acid (Asp), glutamic acid (Glu)), mutations between basic amino acids (e.g., lysine (Lys), arginine (Arg), histidine (His)), and mutations between aromatic amino acids (e.g., phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp)). The range of permissible conservative substitutions depends on the structure and function of the protein; therefore, molecular dynamics simulations and experimental verification are sometimes used to predict the effects of substitutions.In this embodiment, the promoter may also contain common homologous sequences, as described below.

[0035] In one embodiment, the first to nth sequences comprise a plurality of gene expression cassettes, for example, the first to nth common homologous sequences each comprise a promoter sequence that drives gene expression. The exogenous insertion sequence comprises, for example, a first sequence and a second sequence, each comprising a promoter that drives gene expression. If the first to nth sequences each comprise a promoter that drives gene expression, the first to nth promoters may each be a wild-type promoter sequence or an active modified sequence thereof. In this embodiment, the gene itself may or may not contain another common homologous sequence.

[0036] Wild-type promoters are not particularly limited, but examples include the CMV promoter, EF1 promoter (EF1α promoter), ubiquitin promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, and CBh promoter. Inducible promoters can also be used as promoters. The EF1α promoter and its modified promoters may have, for example, any of the sequences of SEQ ID NOs: 1 to 4. The ubiquitin promoter and its modified promoters may have, for example, any of the sequences of SEQ ID NOs: 5 and 6. Promoters derived from other animal species may also be used.

[0037] In cases where the first to nth common homologous sequences each contain a promoter, homologous recombination between the common homologous sequences can be suppressed by changing the type of promoter for each sequence. However, even when using the same type of promoter, homologous recombination between the common homologous sequences can also be suppressed by designing (i.e., modifying) the first to nth sequences to have sequences that are different enough not to cause homologous recombination, as described above. This invention may be useful when there are few types of promoters suitable for target cells into which exogenous insertion sequences are introduced, or when it is desired to make the gene expression levels from the first to nth sequences equivalent.

[0038] In the context of expression level, "equivalent" means that the expression levels do not differ by more than 10-fold. When desiring to make the expression levels equivalent, it is preferable that the expression levels of each gene do not differ by more than 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, or 2-fold. The expression level is the expression level of mRNA.

[0039] In the case where the first to nth sequences, particularly the common homologous sequences, each contain a promoter, the first to nth sequences may further contain a foreign gene, and the foreign gene may be operably linked to each promoter. When a promoter is operably linked to a foreign gene, the combination of the promoter and the foreign gene operably linked thereto is referred to as a gene expression cassette.

[0040] In one aspect, the exogenous insertion sequence includes a plurality of first to nth gene expression cassettes, and each gene expression cassette includes the first to nth promoters and the first to nth foreign genes operably linked thereto, respectively. A spacer may be interposed between each gene expression cassette. The spacer may have a length of, for example, ~1 kb, ~2 kb, or ~3 kb.

[0041] The first to nth common homologous sequences may include common homologous sequences belonging to a plurality of clusters. For example, the first to nth common homologous sequences may have a certain type of common homologous sequence and one or more common homologous sequences of another species. At this time, the certain type of common homologous sequence and the one or more common homologous sequences of the other species may have a low sequence identity with each other (for example, a sequence identity of 60% or less, 50% or less, 40% or less, or 30% or less). When the common homologous sequences are aligned and clustered based on sequence identity, they may be clustered into a plurality. For example, when the common homologous sequence is a promoter, in addition to the EF1α promoter and its modified promoter, it may also include the ubiquitin promoter and its modified promoter.

[0042] <Method for Driving Multiple Genes by Multiple Promoters> In one aspect, the foreign inserted sequence includes the first to nth sequences {where n is a natural number of 2 or more}, that is, n sequences, and the first to nth sequences each have a different promoter. The sequences of the promoters are different to such an extent that they do not undergo homologous recombination with each other.

[0043] In one aspect, the cell can be a pluripotent cell such as a pluripotent stem cell (which means "pluripotent cell" in this specification). Examples of pluripotent stem cells include embryonic stem cells and induced pluripotent stem cells, which can be preferably used in the present disclosure.

[0044] When n is 2, the different promoters can have sequence identities with each other of, for example, 60% or less, 50% or less, 40% or less, or 30% or less. As the promoters, for example, the EF1α promoter and the ubiquitin promoter can be preferably used. In another aspect, when n is 2, the different promoters have sequence identity and have sequences that are different to such an extent that homologous recombination does not occur between the promoter sequences. Since the conditions therefor are as described above, reference is made thereto and duplicate description is avoided here.

[0045] When the cell is a pluripotent cell, as the promoters, for example, the EF1α promoter and the ubiquitin promoter can be preferably used. The EF1α promoter can be used as the first promoter, and the ubiquitin promoter can be used as the t-th promoter. Other promoters can be variants or modified forms of the EF1α promoter or variants or modified forms of the ubiquitin promoter. In this specification, variants are assumed to include promoters having different sequences of different animals. Different promoters have sequence identity and have sequences that are different to such an extent that homologous recombination does not occur between the promoter sequences. Since the conditions therefor are as described above, reference is made thereto and duplicate description is avoided here.

[0046] In one embodiment, the cells are pluripotent cells such as embryonic stem cells or induced pluripotent stem cells, and the exogenous insertion sequence comprises multiple gene expression cassettes, one of which may contain a gene operably linked to the EF1α promoter, and another gene expression cassette may contain a gene operably linked to the ubiquitin promoter.

[0047] In one embodiment, the cells are pluripotent cells such as embryonic stem cells or induced pluripotent stem cells, the exogenous insertion sequence contains multiple, i.e., first to nth gene expression cassettes, the first to nth common homologous sequences each have a different promoter, and the first to nth common homologous sequences have relative sizes of approximately 300 bp or more, approximately 290 bp or more, approximately 280 bp or more, approximately 270 bp or more, approximately 260 bp or more, approximately 250 bp or more, approximately 240 bp or more, approximately 230 bp or more, approximately 220 bp or more, and approximately 210 bp or more. , does not have a completely identical sequence over a continuous sequence of approximately 200 bp or more, approximately 190 bp or more, approximately 180 bp or more, approximately 170 bp or more, or approximately 150 bp or more, approximately 140 bp or more, approximately 130 bp or more, approximately 120 bp or more, approximately 110 bp or more, approximately 100 bp or more, approximately 90 bp or more, approximately 80 bp or more, approximately 70 bp or more, approximately 60 bp or more, approximately 50 bp or more, approximately 45 bp or more, approximately 40 bp or more, approximately 35 bp or more, approximately 30 bp or more, approximately 25 bp or more, or approximately 20 bp or more. Promoters with various mutations can be used as long as they do not significantly adversely affect promoter activity and prevent the achievement of the objective, but point mutations of this frequency are unlikely to have a significant impact on promoter activity.

[0048] Although not bound by theory, the phenomenon in which the expression of one gene decreases when a foreign sequence containing the same promoter sequence in tandem is introduced into the genome is thought to be because homologous recombination occurs between the same promoter sequences when the foreign sequence is introduced into the genome, and the region sandwiched between the same promoter sequences is lost before the foreign sequence is inserted into the genome. In terms of preventing this loss, the cells and method of this disclosure are particularly effective when the foreign sequence contains sequences 1 to m (where m is a natural number between 2 and n).

[0049] Examples of foreign genes, though not limited to them, include transcription factors, differentiation-inducing factors, and growth factors that determine the differentiation fate of cells; therapeutic genes; selection marker genes (e.g., drug resistance genes, genes encoding visualization markers, etc.); and suicide genes.

[0050] Examples of drug resistance genes include, but are not limited to, puromycin resistance genes, blastisidin resistance genes, genetisin resistance genes, neomycin resistance genes, tetracycline resistance genes, kanamycin resistance genes, zeosin resistance genes, hygromycin resistance genes, and chloramphenicol resistance genes. Examples of genes encoding visualization markers include fluorescent protein genes and chromogenic enzyme genes. Examples of fluorescent protein genes include, but are not limited to, green fluorescent protein (GFP) genes, yellow fluorescent protein (YFP) genes, and red fluorescent protein (RFP) genes. Examples of luminescent enzyme genes include, but are not limited to, luciferase genes. Examples of chromogenic enzyme genes include, but are not limited to, β-galactosidase genes, β-glucuronidase genes, and alkaline phosphatase genes. Examples of suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase 9. However, suicide genes can be incorporated in a way that allows for control over their expression timing so that they are induced at a desired time.

[0051] <Method for producing the cells of this disclosure> The cells of this disclosure can be produced by inducing double-strand breaks near regions on gene elements such as the genome that induce homologous recombination with the donor DNA in the presence of donor DNA containing foreign sequences.

[0052] The donor DNA has an upstream homology arm, the foreign sequence, and a downstream homology arm, the foreign sequence being located between the upstream and downstream homology arms.

[0053] The upstream homology arm has a sequence that can homologously recombine with the sequence upstream of the target region in the genome to be modified, for example, a sequence homologous to the sequence adjacent to the upstream side of the target sequence. The downstream homology arm has a sequence that can homologously recombine with the sequence upstream of the target region in the genome to be modified, for example, a sequence homologous to the sequence adjacent to the downstream side of the target sequence. The length and sequence of the upstream and downstream homology arms are not particularly limited, as long as they can homologously recombine with the surrounding region of the target region. The upstream and downstream homology arms do not necessarily have to be perfectly identical to the upstream or downstream sequence of the target region, as long as homologous recombination is possible. For example, the upstream homology arm can be a sequence that has 90% or more sequence identity (homology) with the sequence adjacent to the upstream side of the target region, and it is preferable that it has 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity. For example, the downstream homology arm can be a sequence having 90% or more sequence identity (homology) with a nucleotide sequence adjacent to the downstream side of the target region, and preferably has 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity. Furthermore, the efficiency of allele modification can be further increased if at least one of the upstream homology arm and the downstream homology arm is closer to the cleavage site in or near the target region. Here, "close" may mean that the distance between the two sequences is 100 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less. In some embodiments, there is one cleavage site. In other embodiments, there can be two or more cleavage sites. When introducing multiple cleavage sites into the genome, one cleavage site can be set upstream of the target region and the other can be set downstream of the target region.

[0054] The upstream homology arm and the downstream homology arm may each have a length of, for example, 500 bp to 3 kbp.

[0055] Donor DNA may contain an insulator sequence. An "insulator" is a sequence that blocks or mitigates the influence of the adjacent chromosomal environment, ensuring or enhancing the independence of transcriptional regulation of the DNA sandwiched within that region. Insulators are defined by their enhancer blocking effect (the effect of blocking the influence of an enhancer on promoter activity by inserting it between an enhancer and a promoter) and their positional effect suppression effect (the effect of preventing the expression of a transgene from being affected by its position on the genome where it is inserted by sandwiching both sides of the transgene with insulators). Donor DNA may contain an insulator sequence between the upstream arm and the selection marker gene (or between the upstream arm and the promoter that controls the selection marker gene). Donor DNA may contain an insulator sequence between the downstream arm and the selection marker gene.

[0056] The invention of this disclosure will be described below based on the following examples, but the invention of this disclosure is not limited to the description of the examples.

[0057] • Method for seeding iPS cells: Add 1.2 mL of a coating solution prepared by mixing 1.2 mL of DPBS and 8 μL of iMatrix-511 silk to a 6-well plate and incubate at 25-37°C for at least 1 hour to coat the plate. After coating, remove the coating solution from the plate and add 1.5 mL of iPS cell culture medium (AHS, Cat No. AK02N) to which Y-27632 solution has been added to a final concentration of 10 μM. Add the iPS cell suspension to the plate with the culture medium and incubate at 37°C under 5% CO2 conditions.

[0058] • Transfection method (introducing gRNA / Cas9 expression plasmid and donor plasmid into iPS cells) 5×10 5Individual iPS cells were seeded in a 6-well plate and incubated overnight at 37°C under 5% CO2 conditions. The following day, the medium was changed with 2 mL of iPS cell culture medium to which Y-27632 solution had been added to achieve a final concentration of 10 μM. Immediately afterward, a total of 2000 ng of gRNA / Cas9 expression plasmid and donor plasmid were mixed with lipofection reagent in a 1.5 mL tube, incubated at room temperature for 10 minutes, and then added to the 6-well plate in which the cells had been seeded. After 24 hours, the medium was changed with 2 mL of iPS cell culture medium to which Y-27632 solution had been added to achieve a final concentration of 10 μM. The sequence of the gRNA protospacer region was as shown in Sequence ID No. 7.

[0059] Drug selection method: Starting two days after transfection, replace the iPS cell culture medium with 2 mL of puromycin added to achieve a final concentration of 1 μg / mL daily. Perform drug selection for approximately 7-10 days, and then perform GFP and SctHLA-E expression analysis on the surviving cells using immunostaining and flow cytometry.

[0060] - Expression analysis of GFP and HL-E using immunohistochemistry and flow cytometry (1 × 10⁻¹⁶) 5 Each iPS cell was collected in a 1.5 mL tube and centrifuged at 300 × g for 10 minutes at room temperature to form a pellet. To the resulting pellet, 50 μL of antibody reaction solution prepared by mixing 0.25 μL of anti-HLA-E antibody (BioLegend, Cat. No. 342608) with 50 μL of iPS cell culture medium was added, and the cells were completely suspended. This suspension was incubated at 4°C in the dark for 30 minutes. After incubation, the cells were suspended in 500 μL of iPS cell culture medium and centrifuged at 300 × g for 10 minutes at room temperature, after which the supernatant was removed. The remaining pellet was resuspended in 300 μL of iPS cell culture medium and collected in a 5 mL tube through a 40 μm cell strainer. The obtained samples were analyzed for GFP fluorescence and HLA-E expression using flow cytometry.

[0061] - Modification of the EF1α promoter (EF1p) EF1p had the sequence described in Sequence ID No. 1 and was approximately 2kb in length. Modified EF1p (SynEF1p #1 to #3) were created by introducing point mutations at a rate of one per 200bp. The sequences of the wild-type (WT) EF1p and each modified EF1p were as described in Sequence IDs No. 2 to 4, respectively. SynEF1p#1 contains nine mutations, with the 85th T in a total length of 2,203 base pairs being replaced with G, the 96th G being replaced with A, the 100th A being replaced with G, the 501st G being replaced with C, the 736th C being replaced with T, the 827th C being replaced with A, the 1100th G being replaced with A, the 1431st T being replaced with C, and the 1824th C being replaced with T. SynEF1p#2 contains nine mutations, with the 41st G in a total length of 2,203 base pairs being replaced with A, and the 140th base Replace the G in the first position with A, delete the TAC at positions 176-178, change the A at position 370 to G, change the C at position 442 to G, change the G at position 584 to A, change the T at position 860 to C, change the C at position 971 to T, change the C at position 1050 to T, change the C at position 1297 to T, change the G at position 1340 to T, change the C at position 1399 to T, change the T at position 1519 to C, change the C in the G at position 1633, change the G at position 1699 to C, change the G at position 2026 SynEF1p#3 contains 16 mutations in which a chromosome is replaced with C. The 2,203 base pairs in total length have the following mutations: the 41st G is replaced with A, the 85th T with G, the 96th G with A, the 100th A with G, and the 140th G with A. The 176-178th TAC is deleted, the 370th A with G, the 442nd C with G, the 501st G with C, the 584th G with A, and the 736th C with T. It included 25 mutations, in which the C at the first base was replaced with A, the T at the 860th base was replaced with C, the C at the 971st base was replaced with T, the C at the 1050th base was replaced with T, the G at the 1100th base was replaced with A, the C at the 1297th base was replaced with T, the G at the 1340th base was replaced with T, the C at the 1399th base was replaced with T, the T at the 1431st base was replaced with C, the T at the 1519th base was replaced with C, the C at the 1633rd base was replaced with G at the 1699th base was replaced with C, the C at the 1824th base was replaced with T, and the G at the 2026th base was replaced with C.

[0062] In the figure, EF1p represents the EF1α promoter, SctHLA-E represents a fusion protein of β2-microglobulin (B2M) and HLA-E, SV40pA represents the polyadenylation signal of SV40, spacer represents a spacer of approximately 1 kb, UBCp represents the ubiquitin promoter, GFP-PuroR represents a fusion protein of green fluorescent protein (GFP) and the puromycin resistance gene, and bGHpA represents the polyadenylation signal of bovine growth hormone. Also, Syn. EF1p#1-3 in the figure are modified versions of EF1p that maintain the transcriptional inducing ability of EF1p.

[0063] As shown in Figure 1A, homologous recombination was induced in the iPS cell genome in the presence of plasmids containing gene expression cassette clusters, each ligated to the EF1α promoter with HLA-E, CD155, rapaCASP, and HLA-G, respectively. Amplification of each gene was attempted by PCR. As shown in Figure 1B, all four genes were detected in only one out of six cases; in the others, one or more genes were missing. All gene expression cassettes possessed the EF1α promoter, suggesting that homologous recombination was induced between the EF1α promoters, potentially leading to gene loss.

[0064] Foreign genes, each linked to a promoter, were introduced upstream of the HLA-E gene in iPS cells, and the expression levels of each foreign gene were monitored (see Figure 2). Flow cytometry was used to evaluate the expression levels of GFP encoded by the foreign genes and the expression levels of endogenous HLA-E. sctHLA-E is a modified form of HLA-E, and sctHLA-E was detected using an anti-HLA-E antibody.

[0065] Wild-type iPS cells showed no expression of sctHLA-E or GFP. In iPS cells into which the construct shown in Figure 3(1) was introduced, the promoters EF1p and ubiquitin promoter (UBCp) were operably linked to the exogenous genes. The iPS cells into which the construct shown in Figure 3(1) was introduced showed almost complete expression levels for both sctHLA-E and GFP (see Figure 3).

[0066] In contrast, the construct in (2) of Figure 3 has EF1p as the promoter for both foreign genes, but iPS cells into which this construct was introduced produced a large number of cells that were negative for sctHLA-E (see Figure 3).

[0067] In the construct (3) in Figure 3, one of the exogenous genes was linked to wild-type EF1p, and the other to SynEF1p#1. iPS cells into which this construct (3) in Figure 3 was introduced showed almost complete expression levels for sctHLA-E and GFP (see Figure 3).

[0068] The results above indicate that when the same promoter is inserted in tandem into the genome, gene expression is lost in some cells. However, this loss of gene expression does not occur when the promoters are different or when the promoter sequences are not completely identical (in this example, there are 9 mutations in 2kb). The loss of gene expression was thought to be due to the loss of the gene from the genome caused by the two homologous tandem sequences. Since no such loss of gene expression was observed between EF1p and Syn. EF1p#1, it is suggested that gene loss can be prevented by slight differences in the tandem sequences.

[0069] Next, we investigated the relationship between the number of mutations introduced into EF1p and the effect of restoring reduced gene expression. Figure 4 shows Syn. EF1p#1, which contains nine mutations, restored complete expression of SctHLA-E, and SctHLA-E expression was not strongly related to the increase in the number of mutation sites.

[0070] The above describes the case where EF1p was incorporated in tandem. Below, we show the results for genomes in which UBCp was incorporated in tandem instead of EF1p. Figure 5 shows that when the two promoters are different, almost complete expression levels are obtained for sctHLA-E and GFP. In contrast, when UBCp (1.2kb: SEQ ID NO: 6) or UBCp (2.0kb: SEQ ID NO: 5) was tandem-linked, SctHLA-E expression disappeared in some cells, and the expression level was greatly reduced in most cells.

[0071] As described above, in the above embodiment, it is thought that tandem linking of identical sequences destabilizes the genome, inducing homologous recombination between the tandem sequences, for example, and causing the intermediate sequence to be lost. This gene loss could be prevented even by slightly modifying the sequences arranged in tandem. Therefore, when tandem linking sequences, it is possible to suppress recombination between tandem sequences by introducing mutations while maintaining function, thereby ensuring higher gene expression.

[0072] Next, we examined the number of EF1p mutations introduced into the genome and the deletion rate from the genome, as well as the relationship between the number of mutations introduced into EF1p and the effect of restoring reduced gene expression.

[0073] Constructs expressing four genes—sctHLA-E, CD155, CD47, and CD55—from four wild-type EF1p cells were incorporated into the genome of human iPSCs. Immunostaining was used to stain the proteins derived from the four membrane-expressed transgenes, and flow cytometry was used to analyze the expression of each gene.

[0074] Assuming that cells expressing CD55, which in principle does not undergo deletion due to recombination between homologous sequences, were cells whose genomes had been modified as expected, we checked the expression levels of the other three genes in CD55-expressing cells. As a result, the expression rate of none of the three genes was 100% (see Figure 6 (1)). When the three genes other than sctHLA-E were expressed from three different mutant EF1p cells, each containing approximately 10 to 20 mutations within its 2.2 kb length, the expression rate of all three genes improved (see Figure 6 (2)). Furthermore, when the genes were expressed from three different mutant EF1p cells containing approximately 40 to 50 mutations, a further improvement in expression rate was observed (see Figure 6 (3)).

[0075] As described above, in the above embodiment, the more identical sequences are arranged in tandem, the more unstable the genome becomes, and the higher the frequency of homologous recombination being induced between tandem sequences (i.e., between promoter sequences), resulting in the loss of intermediate sequences. In contrast, it was suggested that the frequency of intermediate sequence loss can be reduced by using a promoter sequence with a higher mutation frequency. Furthermore, it was shown that it is possible to appropriately create a promoter that maintains its function even when approximately 50 mutations are added to the total length of 2.2 kb.

[0076]

Claims

1. A cell having an exogenous insertion sequence, wherein the exogenous insertion sequence comprises a first sequence and a second sequence, the first sequence and the second sequence each having a first common homologous sequence and a second common homologous sequence, the common homologous sequences having 90% or more sequence identity with each other, and having sequences that are different to the extent that homologous recombination does not occur between the common homologous sequences.

2. A cell having an exogenous insertion sequence, wherein the exogenous insertion sequence comprises a first sequence and a second sequence, the first sequence and the second sequence each comprising a first promoter and a second promoter that drive gene expression, the first promoter and the second promoter each operably ligated to a first exogenous gene and a second exogenous gene, and the first promoter and the second promoter have sequences that are different to the extent that homologous recombination does not occur between the promoter sequences.

3. The cell according to claim 1 or 2, wherein the first sequence comprises a promoter or a first modified promoter, and the second sequence comprises a second modified promoter, and the first modified promoter and the second modified promoter each maintain all or part of the promoter activity.

4. The cell according to claim 2 or 3, wherein the promoter is the EF1α promoter.

5. The cell according to claim 2 or 3, wherein the promoter is a ubiquitin promoter.

6. The cell according to claim 4 or 5, wherein each modified promoter maintains all or part of the promoter activity and exhibits 90% sequence identity with respect to the promoter sequence.

7. A cell in which the exogenous insertion sequence comprises n sequences, from the first sequence to the nth sequence {where n is a natural number greater than or equal to 2}, each of the first to nth sequences comprises a first to nth promoter, each of the first to nth promoters comprises a selected promoter from the group consisting of the EF1α promoter, a modified EF1α promoter, a ubiquitin promoter, and a modified ubiquitin promoter, and each of the first to nth promoters has sequences that are different to the extent that homologous recombination does not occur between them.

8. The cell according to claim 7, wherein the first sequence comprises an EF1α promoter or a modified EF1α promoter, the second to nth sequences comprises a modified EF1α promoter, and each modified EF1p promoter sequence maintains all or part of the promoter activity and exhibits 90% or more sequence identity with the EF1α promoter sequence of SEQ ID NO: 1, or the first sequence comprises a ubiquitin promoter or a modified ubiquitin promoter, the second to nth sequences comprises a modified ubiquitin promoter, and the modified ubiquitin promoter sequence maintains all or part of the promoter activity and exhibits 90% sequence identity with the ubiquitin promoter sequence of SEQ ID NO: 5 or 6.

9. The cell according to claim 7, wherein the first sequence comprises an EF1α promoter or a modified EF1α promoter, the second sequence comprises a ubiquitin promoter or a modified ubiquitin promoter, and if n is 3 or more, the third to n sequences comprise one promoter selected from the group consisting of an EF1α promoter or a modified EF1α promoter and a ubiquitin promoter or a modified ubiquitin promoter, each modified EF1p promoter sequence maintains all or part of the promoter activity and exhibits 90% or more sequence identity with the EF1α promoter sequence of SEQ ID NO: 1, and each modified ubiquitin promoter sequence maintains all or part of the promoter activity and exhibits 90% sequence identity with the ubiquitin promoter sequence of SEQ ID NO: 5 or 6.

10. The cell according to claim 1, wherein the first common homologous sequence and the second common homologous sequence include one selected from the group consisting of nucleic acids encoding a chimeric antigen receptor, nucleic acids encoding a T cell receptor, and nucleic acids encoding an antibody or an antigen-binding fragment thereof.

11. A cell according to any one of claims 1 to 10, which is a pluripotent cell.

12. A composition comprising the cells described in any one of claims 1 to 11.

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