Exploring method, cell preparation method, cell sorting method, cell, and cell product manufacturing method

Abstract

One approach to the exploration method includes calculating a TAD score representing transcriptional activity for each TAD present in the genome and selecting TADs based on the TAD scores. Another approach includes analyzing a genome obtained from cells expressing a foreign gene and selecting the boundaries of regions containing the foreign gene. Cell preparation involves inserting the target gene into the regions identified by the exploration method. Cell sorting involves calculating a TAD score representing transcriptional activity for each TAD present in the genome, selecting TADs based on the TAD scores, and selecting cells containing the target gene in the selected TADs. The cells are derived from Chinese hamsters and are cells in which the target gene has been inserted into at least one specified region. The method for manufacturing the cell product includes culturing the cells to express the target gene.

Description

Technical Field

[0001] This invention relates to an exploration method, a cell preparation method, a cell sorting method, and a method for manufacturing cells and cell products. Background Technology

[0002] Patent document 1 discloses a host cell that is a site-specific integrated host cell containing an endogenous Fer1L4 gene, wherein an exogenous nucleotide sequence is integrated into the Fer1L4 gene. Patent document 2 discloses a cell that possesses exogenous nucleic acid integrated at a specific site within an expression enhancement site, wherein the exogenous nucleic acid sequence encodes a bispecific antigen-binding protein. Patent document 3 discloses a cell having a first exogenous nucleic acid integrated into a first expression enhancement site and a second exogenous nucleic acid integrated into a second expression enhancement site, wherein both the first and second exogenous nucleic acids encode antigen-binding proteins. Patent document 4 discloses a cell that is a mammalian cell containing a first recombination target site (RTS) integrated into the chromosome at a first high integration (HI) site, wherein the first HI site is located within approximately 30,000 base pairs of the active genomic region chamber of the accessible chromatin and the boundary of the TAD, and the first HI site overlaps with a region of the proliferating genomic genome that interacts with at least one enhancer element. Existing technical documents Patent documents

[0003] Patent Document 1: European Patent Application Publication No. 2711428 Patent Document 2: International Publication No. 2017 / 184831 Patent Document 3: International Publication No. 2017 / 184832 Patent Document 4: International Publication No. 2020 / 072480 Summary of the Invention The technical problem to be solved by the invention

[0004] One technology aims to prepare cells that stably produce medical proteins such as humanized monoclonal antibodies by integrating the target gene into the host cell's genome. If the regions on the genome where a gene is highly or stably expressed are known in advance, cell lines that highly or stably express the target gene can be prepared with a high probability by inserting the target gene into those regions.

[0005] The present invention was made based on the above circumstances. The objective of this invention is to provide a method for identifying target regions on the genome where the target gene is inserted and where the target gene is highly expressed. The subject of this invention is to provide a method for preparing cells that highly express a target gene. The subject of this invention is to provide a method for sorting cells that highly express a target gene. The subject of this invention is to provide a cell that highly expresses a target gene. The objective of this invention is to provide a method for manufacturing a cell product with excellent productivity of a substance encoded by a target gene. means for solving technical problems

[0006] The specific methods used to solve the problem include the following. <1> An exploratory method for identifying target regions on the genome where a gene intended for insertion may be found. The exploration methods include the following (1). Identify the TAD as the target region. (1) For each TAD present in the genome, calculate the TAD score representing transcriptional activity and select TADs based on the TAD score. <2> According to the exploration method described in <1>, it also includes the following (2). (2) For at least one of the known safe harbors within the genome, determine the TAD score, i.e., the TAD score-SH, of the corresponding TAD. Determine a threshold based on at least one TAD score-SH. Select TADs based on thresholds and TAD scores. <3> An exploratory method for identifying target regions on the genome where a gene intended for insertion may be found. The exploration methods include the following (a) to (d). Find the region defined by the following boundary pair P as the target region. (a) Prepare cells with genomic F that integrates the foreign gene into the genome and expresses the foreign gene. (b) Obtain the genome F from the cell, analyze the genome F, and determine the region containing the foreign gene, i.e., region F, and the boundary between region F and the genome, i.e., boundary F. (c) For each TAD present in the genome, calculate the TAD score representing transcriptional activity. For each boundary F, determine the TAD score of the associated TAD, i.e., TAD score-F, and select the boundary F based on the TAD score-F. (d) Find the boundary pair P of the clamping region F from the selected boundary F. <4> According to the exploration method described in <3>, it also includes (p) performing (c) on the boundary F selected by (p). (p) Determine the methylation rate of exogenous genes present in region F, and select the boundary F of region F with a low methylation rate. <5> According to the exploration method described in <3> or <4>, it also includes (q) performing (d) on the boundary F selected through (c) and (q). (q) For at least one of the known safe harbors within the genome, determine the TAD score, i.e., the TAD score-SH, of the associated TAD. Determine a threshold based on at least one TAD score-SH. The boundary F is selected based on the threshold and the TAD score-F. <6> The exploration method according to any one of <3> to <5> further includes the following (r). (r) Select the region F in the genome F located between the boundary pairs P, which is not a region formed by chromosomal translocation. <7> According to any one of <1> to <6>, wherein, The TAD score is a value obtained by multiplying the density of genes present in the TAD by the average expression level. <8> According to any one of <1> to <7>, wherein, The genome is the genome of mammalian cells. <9> According to any one of <1> to <7>, wherein, The genome is the genome of CHO cells.

[0007] <10> A method for preparing cells, comprising: The TAD was located using the exploration methods described in <1> or <2>; and Insert the target gene into the TAD within the genome containing the TAD. <11> A method for preparing cells, comprising: The region defined by boundary pair P is found using any one of the exploration methods in <3> to <9>; and Insert the target gene within ±10 kbp before and after the region of the genome containing that region. <12> According to the cell preparation method described in <10> or <11>, wherein, The target gene is a gene encoding at least one of the following groups: enzymes, antibodies, interleukins, cytokines, chemokines, hormones, growth factors, transcription factors, receptors, transcriptionally regulated nucleic acids, non-coding RNAs, viral agents, vaccines, medical proteins, their subunits and their fragments.

[0008] <13> A cell sorting method, which is a method for selecting cells expressing a target gene, the sorting method includes the following (11). (11) For each TAD present in the cell genome, calculate the TAD score representing transcriptional activity, select TADs based on the TAD score, and select cells in which the target gene is present in the selected TADs. <14> The cell sorting method described in <13> also includes the following (12). (12) For at least one of the known safe harbors within the genome, determine the TAD score, i.e., the TAD score-SH, of the associated TAD. Determine a threshold based on at least one TAD score-SH. The TAD is selected based on the threshold and TAD score, and cells containing the target gene are selected from the selected TADs. <15> The cell sorting method according to <13> or <14> also includes the following (13). (13) Determine the methylation rate of the target gene present in the selected TAD and select cells with low methylation rates. <16> The cell sorting method according to any one of <13> to <15>, wherein, The TAD score is a value obtained by multiplying the density of genes present in the TAD by the average expression level. <17> The cell sorting method according to any one of <13> to <16>, wherein, The cells are mammalian cells. <18> The cell sorting method according to any one of <13> to <16>, wherein, The cells were CHO cells. <19> The cell sorting method according to any one of <13> to <18>, wherein, The target gene is a gene encoding at least one of the following groups: enzymes, antibodies, interleukins, cytokines, chemokines, hormones, growth factors, transcription factors, receptors, transcriptionally regulated nucleic acids, non-coding RNAs, viral agents, vaccines, medical proteins, their subunits and their fragments.

[0009] <20> A type of cell, derived from Chinese hamsters. Insert the target gene into at least one region selected from the 40 regions shown in Table 1. <21> A type of cell, derived from Chinese hamsters. Insert the target gene into at least one region selected from the 40 regions shown in Table 2. <22> A type of cell, derived from Chinese hamsters. Insert the target gene into at least one region selected from the 40 regions shown in Table 3. <23> The cell according to any one of <20> to <22>, wherein, The cells derived from Chinese hamsters are CHO cells. <24> The cell according to any one of <20> to <23>, wherein, The target gene is a gene encoding at least one of the following groups: enzymes, antibodies, interleukins, cytokines, chemokines, hormones, growth factors, transcription factors, receptors, transcriptionally regulated nucleic acids, non-coding RNAs, viral agents, vaccines, medical proteins, their subunits and their fragments.

[0010] <25> A method for preparing cells, comprising: The target gene was inserted into at least one region selected from the 40 regions shown in Table 1 of the genome derived from cells of Chinese hamsters. <26> A method for preparing cells, comprising: The target gene was inserted into at least one region selected from the 40 regions shown in Table 2 of the genome derived from cells of Chinese hamsters. <27> A method for preparing cells, comprising: The target gene was inserted into at least one region selected from the 40 regions shown in Table 3 of the genome derived from cells of Chinese hamsters. <28> The method for preparing cells according to any one of <25> to <27>, wherein, The cells derived from Chinese hamsters are CHO cells. <29> The method for preparing cells according to any one of <25> to <28>, wherein, The target gene is a gene encoding at least one of the following groups: enzymes, antibodies, interleukins, cytokines, chemokines, hormones, growth factors, transcription factors, receptors, transcriptionally regulated nucleic acids, non-coding RNAs, viral agents, vaccines, medical proteins, their subunits and their fragments.

[0011] <30> A method for manufacturing a cell product, comprising: Cells prepared by any one of the cell preparation methods described in <10> to <12> and <25> to <29> are cultured to express the target gene. <31> A method for manufacturing a cell product, comprising: Cells sorted by any one of the cell sorting methods in <13> to <19> are cultured to express the target gene. <32> A method for manufacturing a cell product, comprising: Culture the cells described in any one of <20> to <24> to express the target gene. <33> The method for manufacturing cell products according to any one of <30> to <32>, wherein, The target gene is a gene encoding at least one of the following groups: enzymes, antibodies, interleukins, cytokines, chemokines, hormones, growth factors, transcription factors, receptors, transcriptionally regulated nucleic acids, non-coding RNAs, viral agents, vaccines, medical proteins, their subunits and their fragments. Invention Effects

[0012] According to the present invention, a method is provided for identifying target regions on the genome where the target gene is inserted and where the target gene is highly expressed. According to the present invention, a method for preparing cells that highly express a target gene is provided. According to the present invention, a method for sorting cells that highly express a target gene is provided. According to the present invention, a cell that highly expresses a target gene is provided. According to the present invention, a method for manufacturing a cell product with excellent productivity of a substance encoded by a target gene is provided. Attached Figure Description

[0013] Figure 1 This is a concept diagram of the second exploration method. Figure 2 This is a scatter plot showing the antibody production performance of the 60 antibody production strains prepared in the examples. Figure 3 It is a histogram of copy numbers of foreign genes present within paired boundaries, obtained from genomic analysis of 31 antibody-producing strains. Figure 4 This is a histogram of the average methylation rate of promoter and antibody subunit regions, obtained from genomic analysis of 31 antibody-producing strains. Figure 5 This is an example of a contact diagram of CHO cells. Figure 6 This is a distribution map of the TAD scores involved in 2502 TADs detected in the genome of CHO cells. Figure 7 This is a scatter plot representing the performance of the high-performance region found through this embodiment. Figure 8 This is a graph representing the performance of the high-performance region found through this implementation method. Figure 9 This is a graph that shows how multiple copies of the encoded sequence can be inserted in the high-performance region found through this embodiment. Figure 10 This is a graph showing that the high-performance region found through this embodiment can stably represent multiple copies of the coding sequence. Detailed Implementation

[0014] The embodiments of the present invention will be described below. These descriptions and examples are illustrative of the embodiments and do not limit the scope of the embodiments. The mechanisms of action described in the present invention include speculation, and their correctness does not limit the scope of the embodiments.

[0015] When describing embodiments of the present invention with reference to the accompanying drawings, the structure of the embodiments of the present invention is not limited to the structure shown in the drawings. The sizes of the elements in the drawings are conceptual, and the relative sizes of the elements are not limited thereto.

[0016] In this invention, the term "process" not only includes independent processes, but also includes any process that can achieve its purpose, even if it cannot be clearly distinguished from other processes.

[0017] In this invention, the numerical range represented by “~” indicates the range included by taking the values ​​recorded before and after “~” as the minimum and maximum values, respectively. In the numerical ranges described in stages in this invention, the upper or lower limit value described in one numerical range can be replaced with the upper or lower limit value of other numerical ranges described in stages. Furthermore, in the numerical ranges described in this invention, the upper or lower limit value of that numerical range can also be replaced with the values ​​shown in the embodiments.

[0018] In this invention, each component may contain multiple corresponding substances. In this invention, when referring to the amount of each component in the composition, unless otherwise specified, the amount refers to the total amount of the multiple substances present in the composition, where multiple substances corresponding to each component are present in the composition.

[0019] In this invention, nucleic acid is a term encompassing any nucleic acid (e.g., deoxyribonucleic acid (DNA), ribonucleic acid (RNA), its analogues, natural products, and artificial products), nucleic acids having low-molecular-weight compounds, groups (e.g., methyl groups), molecules or structures other than nucleic acids linked to them. Nucleic acids can be single-stranded or double-stranded.

[0020] In this invention, the donor carrier is a substance that carries exogenous nucleic acids into cells and the cell's genome, and it is itself a nucleic acid. There are no restrictions on the source, form, or base sequence of the donor carrier. The donor carrier can be a circular nucleic acid or a linear nucleic acid. The donor carrier can be a single-stranded nucleic acid or a double-stranded nucleic acid. Double-stranded DNA is preferred as the donor carrier.

[0021] In this invention, the number of amino acid residues in the protein is not limited. The protein includes proteins with post-translational modifications of amino acids. Examples of post-translational modifications of amino acids include phosphorylation, methylation, acetylation, glycan addition, and lipid addition. In this invention, amino acids are labeled using the 3-character and 1-character labels specified by IUPAC-IUBMB JCBN (IUPAC-IUBMB Joint Commission on Biochemical Nomenclature). Unless otherwise stated, the amino acids mentioned in this invention are L-amino acids.

[0022] In this invention, the identity of the base sequence and the identity of the amino acid sequence are calculated using BLAST (Basic Local Alignment Search Tool) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0023] A TAD (topologically associating domain) is a structural unit within the genome detected through three-dimensional genome structure analysis; it represents regions with a relatively high probability of spatial contact. The size of a TAD typically ranges from hundreds of kbp to several Mbp. The algorithm for three-dimensional genome structure analysis can be a publicly available algorithm, an improved version of a publicly available algorithm, or a newly developed algorithm. Genome structure data used for three-dimensional genome structure analysis can be obtained from publicly available databases, academic papers, technical literature, etc., or from actual analysis of the genome within cells. Analytical methods for three-dimensional genome structure analysis include: Hi-C method; insitu Hi-C method, low Hi-C method, SAFE Hi-C method, Hi-CO method based on the Hi-C method; Micro-C method; etc.

[0024] Hi-C is one of the analytical methods for three-dimensional genome structure analysis. It is an analytical method that comprehensively detects spatially close regions within the genome. Hi-C is an abbreviation for high-throughput chromosome conformation capture. Based on the calculation results of Hi-C analysis, regions with a relatively high probability of spatial contact are identified as TADs, and the genome is divided into multiple TADs. The size of TADs detected by Hi-C analysis is typically from hundreds of kbp to several Mbp. Through Hi-C analysis, the mammalian genome is divided into thousands of TADs. The Hi-C analysis algorithm used for TAD detection can be a publicly available algorithm, an improved version of a publicly available algorithm, or a newly developed algorithm. The genomic structure data available for Hi-C analysis can be obtained from publicly available databases, academic papers, technical literature, etc., or it can be obtained from actual analysis of the intracellular genome.

[0025] A safe harbor within the genome refers to a region where the host cell can survive even after a gene is inserted, and where the inserted gene is expressed. Safe harbors within the genome are identified by chromosome number or accession number and base number from publicly available base sequence databases. Examples of publicly available base sequence databases include INSD (the International Nucleotide Sequence Databases) and RefSeq (NCBI Reference Sequence Database). Safe havens within the genome are sometimes referred to by the well-known gene names that exist within or near their regions.

[0026] <Exploration Methods> This invention provides a method for identifying target regions on the genome where a gene to be inserted is located. This invention provides a first exploration method and a second exploration method.

[0027] <First Exploration Method> The target region found by the first exploration method is a structural unit within the genome, namely a TAD, which is a TAD with relatively high transcriptional activity.

[0028] Among TADs, there are TADs with relatively high transcriptional activity and TADs with relatively low transcriptional activity. The first exploratory method aims to find TADs with relatively high transcriptional activity, including the following (1).

[0029] (1) For each TAD present in the genome, calculate the TAD score representing transcriptional activity and select TADs based on the TAD score.

[0030] In this invention, the TAD score is an evaluation criterion indicating the level of transcriptional activity. Generally, a higher TAD score is preferred. Selecting a TAD based on its rating means, for example, choosing a TAD with a relatively high rating or a TAD with a rating that exceeds a pre-set standard.

[0031] The TAD score is simply an indicator that identifies a TAD with relatively high transcriptional activity among multiple TADs found on the genome (e.g., the mammalian genome is divided into thousands of TADs). TAD scores can be, for example, the total, average, or median expression levels of genes present in a TAD, the density of genes present in a TAD, the reciprocal of the methylation rate or the demethylation rate of CpG sites within a TAD, the value obtained by multiplying two or more of these values, or an indicator based on two or more of these values.

[0032] Genes present in TAD can be identified from publicly available databases, academic papers, and technical literature. At least one gene must be present in TAD and relevant to the calculation of the TAD score; preferably two or more, with more being preferable. Ideally, the gene representation should comprehensively cover all genes present in TAD. Gene expression levels can be obtained from publicly available databases, academic papers, technical literature, etc., or from actual quantitative measurements of gene expression. Gene expression levels can be quantified using well-known mRNA quantification methods.

[0033] One implementation of the TAD score is to multiply the density of genes present in the TAD by the average expression level. A higher value is preferred. The density of genes present in the TAD is calculated by dividing the number of endogenous genes present in the TAD by the number of base pairs in the TAD (genes / Mb).

[0034] An example of an implementation of the first exploration method also includes the following (2). (2) For at least one of the known safe harbors within the genome, determine the TAD score, i.e., the TAD score-SH, of the corresponding TAD. Determine a threshold based on at least one TAD score-SH. Select TADs based on thresholds and TAD scores.

[0035] Well-known safe harbors within the genome can be identified from publicly available databases, academic papers, and technical literature. The TAD score-SH is determined to have at least one safe harbor, preferably two or more, but for example, no more than eight.

[0036] When multiple well-known safe harbors exist, at least one can be selected. Methods for selecting a safe harbor include, for example, selecting a safe harbor where the expression level (pg / cell / copy) of the protein encoded by the inserted gene is relatively high; or selecting a safe harbor where the expression level (pg / cell / copy) of the protein encoded by the inserted gene exceeds a pre-defined standard. The protein expression level of the safe harbor can be data obtained from publicly available databases, academic papers, technical literature, etc., or it can be data obtained by actually inserting the gene into the safe harbor and measuring the protein expression level.

[0037] The coordinates of the safe harbor (i.e., chromosome number or accession number and base number of a publicly available base sequence database) are applied to the partition of the TAD (output of a three-dimensional genome structure analysis (e.g., Hi-C analysis) to determine the TAD to which the safe harbor belongs, and then the TAD score of that TAD is determined. Here, the TAD score is the TAD score calculated for each TAD in (1). In this invention, the TAD score of the TAD to which the safe harbor belongs is referred to as "TAD score-SH". At least one TAD score-SH is determined.

[0038] The threshold based on the TAD score-SH is, for example, the minimum, maximum, average, or median value of at least one TAD score-SH.

[0039] Regarding the evaluation criteria for TAD scores, generally, higher scores are preferred. Selecting a TAD based on a threshold and its score means choosing a TAD with a score exceeding the threshold.

[0040] TADs selected based on the TAD score-SH threshold are expected to show gene expression levels that are the same as or exceed those of known safe harbors.

[0041] Even if (1) and (2) cannot be clearly distinguished from each other, the first exploration method includes (1) and (2) as long as the purpose of (1) and (2) is achieved.

[0042] <Second Exploration Method> The target regions identified by the second exploration method are safe havens within the genome and regions with relatively high transcriptional activity.

[0043] The second exploratory method aims to identify safe havens within the genome that exhibit relatively high transcriptional activity, including (a) to (d) below, identifying regions defined by boundary pairs P. Regions defined by boundary pairs P refer to regions that begin and end with boundary pairs P.

[0044] (a) Prepare cells with genome F that integrates foreign genes into the genome and expresses foreign genes. (b) Obtain the genome F from the cell, analyze the genome F, and determine the region containing the foreign gene, i.e. region F, and the boundary between region F and the genome, i.e. boundary F. (c) For each TAD in the genome, calculate the TAD score representing transcriptional activity, determine the TAD score of the TAD to which each boundary F belongs, i.e., TAD score-F, and select the boundary F based on the TAD score-F. (d) Find the boundary pair P of the clamping region F from the selected boundary F.

[0045] Figure 1This is a concept diagram of the second exploration method. Figure 1 This represents the relationship between the genome, exogenous gene, cell, genome F, region F, boundary F, TAD, and boundary-to-P as the object of the second exploration method.

[0046] In (a) and (b), exogenous genes refer to genes that are not originally present in the genome of the target genome, and are genes of interest during genome analysis with the aim of identifying safe harbors. The source, type, size, and base sequence of exogenous genes are not limited. One example of an implementation of exogenous genes is a structural gene (i.e., a gene encoding a protein).

[0047] In this invention, a foreign gene refers to a foreign gene capable of being expressed within a cell. A foreign gene contains all the sequences required for its expression. When the foreign gene is a structural gene, it contains all the sequences required for the expression of the protein encoded by the foreign gene, and includes the protein's coding sequence and all nucleic acids (e.g., promoters, transcription terminators, polyadenylated sequences) required for transcription and translation of that coding sequence within the cell. A foreign gene may contain one copy of the protein's coding sequence, or it may contain two or more copies. For example, to express all subunits of a heteropolymeric protein, the foreign gene may contain at least one copy of the coding sequence for each subunit. For example, a foreign gene may contain at least one copy each of the sequence encoding the H chain and the sequence encoding the L chain of an antibody.

[0048] The cells in (a) can be established cell lines or newly prepared cells. The cells can be polyclonal cell populations or monoclonal cells. The cell in (a) is a cell that does not die even when the foreign gene is integrated into the genome and expresses the foreign gene. Therefore, by analyzing the genome F obtained from this cell, a safe harbor can be found.

[0049] A preferred example of the cell in (a) is a monoclonal cell that stably and highly expresses a foreign gene. It is hypothesized that the region of the genome F of this cell containing the foreign gene is a high-performance safe harbor (i.e., a region where the host cell will survive even if the gene is inserted and the inserted gene is stably and highly expressed). By analyzing the genome F obtained from this cell, high-performance safe harbors can be efficiently identified.

[0050] Specifically, (b) includes, for example, extracting the genome from cells, constructing a sequencing library, sequencing the library, mapping reads onto the genome, mapping foreign genes on reads, determining the boundary between the genome and foreign genes, extracting reads containing foreign genes, determining the copy number of foreign genes contained in reads, and determining the boundary pairs holding foreign genes. From the perspective of obtaining long reads and DNA methylation data, library sequencing is preferably single-molecule real-time sequencing or nanopore sequencing.

[0051] Region F is a region within the genome F, which is distinct from the genome and contains at least one copy of a foreign gene. Boundary F is the boundary between region F within genome F and the genome itself. The coordinates of boundary F are determined by the chromosome number of the genome being explored or by the accession number and base number of a publicly available base sequence database. Examples of publicly available base sequence databases include INSD (the International Nucleotide Sequence Databases) and RefSeq (NCBI Reference Sequence Database).

[0052] One example of the implementation of (b) involves defining region F as a region containing multiple copies (i.e., more than 2 copies) of the foreign gene. In this case, the boundary F of region F containing multiple copies of the foreign gene becomes the object of (c). The number of copies of the foreign gene contained in region F can be, for example, 2 to 6 copies, 2 to 5 copies, or 2 to 4 copies. Region F, containing multiple copies of a foreign gene, exhibits multicopy tolerance, thus ensuring stable production of the target substance even after the insertion of multiple copies of the target gene with relatively long base lengths. That is, high yields of the target substance are maintained even after long-term culture. Previously, when multiple copies of a foreign gene were inserted in one location, the yield of the substance encoded by that foreign gene tended to be unstable; however, this problem is improved according to embodiments of the present invention. Examples of target genes with relatively long base lengths and multiple copies include nucleic acids consisting of two or more copies of the coding sequence and nucleic acids consisting of at least one copy of the coding sequences of each subunit of a heteropolymeric protein. Regions capable of inserting into nucleic acids consisting of two or more copies of the coding sequence are regions capable of producing the substance encoded by the target gene in high yields. Regions capable of inserting into nucleic acids consisting of at least one copy of the coding sequences of each subunit of a heteropolymeric protein are regions capable of stably producing the heteropolymeric protein.

[0053] The TAD and TAD score in (c) have the same meaning as the TAD and TAD score in (1) of the first exploration method, and the specific methods and calculation methods are also the same. One implementation of the TAD score is to multiply the density of genes present in the TAD by the average expression level. Regarding the evaluation criteria for this value, a higher value is preferred.

[0054] The coordinates of boundary F (i.e., chromosome number or accession number and base number from a publicly available base sequence database) are applied to the partition of the TAD (output of three-dimensional genome structure analysis, such as Hi-C analysis) to determine the TAD to which boundary F belongs, and then the TAD score for that TAD is determined. Here, the TAD score is the TAD score calculated for each TAD. In this invention, the TAD score of the TAD to which boundary F belongs is called "TAD score-F".

[0055] Regarding the evaluation criteria for TAD scores, higher scores are generally preferred. Selecting a boundary F based on the TAD score -F means, for example: selecting a boundary F with a relatively high TAD score -F; or selecting a boundary F where the TAD score -F exceeds a pre-defined standard.

[0056] In (d), find the boundary pair P that holds the region F from the boundary F selected by (c). The boundary pair P is one end and the other end of the same region F in the genome F.

[0057] Boundary pair P is the pair of boundaries F selected by (c), and is therefore a coordinate pair present in TADs with relatively high transcriptional activity. Therefore, the region defined by boundary pair P (i.e., the region starting and ending with boundary pair P) is expected to be a region with relatively high transcriptional activity. The coordinates of the boundary pair P are determined by the chromosome number of the genome being explored or the accession number and base number of a publicly available base sequence database. Examples of publicly available base sequence databases include INSD (the International Nucleotide Sequence Databases) and RefSeq (NCBI Reference Sequence Database).

[0058] In (b), when region F is confined to a region containing multiple copies of the foreign gene, even when a relatively long target gene (e.g., a nucleic acid consisting of two or more copies of the coding sequence linked together, or a nucleic acid consisting of at least one copy of the coding sequences of each subunit of a heteropolymeric protein linked together) is inserted into the region defined by boundary pair P, the target substance can still be stably produced. That is, a high yield of the target substance is maintained even after long-term culture.

[0059] An example of the implementation of the second exploration method also includes (p) the following (c) of performing (c) on the boundary F selected by (p). (p) Determine the methylation rate of exogenous genes present in region F, and select the boundary F of region F with a low methylation rate.

[0060] DNA methylation typically suppresses gene expression. Therefore, (p) is the region F where gene expression is not suppressed and its boundary F. The boundary F selected by (p) is then subjected to (c), followed by (d), thereby finding the boundary pair P associated with the region where high gene expression can be expected.

[0061] The methylation rate of exogenous genes can be obtained from data obtained by single-molecule real-time sequencing or nanopore sequencing, as in (b). The methylation rate is, for example, the methylated cytosine at the CpG site / all cytosine at the CpG site × 100. The methylation rate value can be the value of the entire exogenous gene, the value of a portion of the exogenous gene (e.g., the value of a protein-coding sequence), or the value of each range of the exogenous gene divided according to function (e.g., the values ​​of the promoter region and the protein-coding sequence, respectively). The minimum, maximum, average, or median value of at least one methylation rate obtained from the target range is used as a representative value for selecting region F and its boundary F.

[0062] Selecting a region F with a low methylation rate means, for example, selecting a region F with a relatively low methylation rate; or selecting a region F with a methylation rate lower than a pre-set standard (e.g., 30%, 20%, 10%).

[0063] An example of the implementation of the second exploration method also includes (q) the boundary F selected by (c) and (q) being (d). (q) For at least one of the known safe harbors within the genome, determine the TAD score, i.e., the TAD score-SH, of the associated TAD. Determine a threshold based on at least one TAD score-SH. The boundary F is selected based on the threshold and the TAD score-F.

[0064] The known safe harbors, TAD score-SH and thresholds within the genome in (q) have the same meaning as the known safe harbors, TAD score-SH and thresholds within the genome in (2) of the first exploration method, and the specific methods and determination methods are also the same.

[0065] Regarding the evaluation criteria for TAD scores, higher scores are generally preferred. Selecting the boundary F based on the threshold and TAD score -F means selecting the boundary F with TAD scores -F exceeding the threshold.

[0066] Boundary F is selected by performing (c) and (q), and then boundary pairs P are identified by performing (d) that are associated with regions that are expected to show gene expression levels that are the same as or exceed those of known safe harbors.

[0067] Even when (c) and (q) cannot be clearly distinguished from each other, the second exploration method includes (c) and (q) as long as the objectives of (c) and (q) are achieved.

[0068] An example of an implementation of the second exploration method also includes the following (r). (r) Select the region F in the genome F located between the boundary pairs P, which is not a region formed by chromosomal translocation.

[0069] To confirm that region F is not formed through chromosomal translocation, one method is to ensure that the coordinates of the boundary pair P containing region F belong to the same chromosome and that the base length of region F (in other words, the distance between boundary pairs P on genome F) is not too long. For example, if the base length of region F is less than 100 kbp, it is determined that region F is not formed through chromosomal translocation, and the boundary pair P containing region F is selected.

[0070] Even when (d) and (r) cannot be clearly distinguished from each other, the second exploration method includes (d) and (r) as long as the purpose of (d) and (r) is achieved.

[0071] The genome that serves as the object of the first exploration method can be the genome of any cell, as long as it has TADs, which are structural units within the genome. Examples of cells include fungi, yeast, insect cells, mammalian cells, and plant cells.

[0072] The genome that can be used as the object of the second exploration method can be any cell genome as long as it meets the following conditions: it has a TAD (Transformation and Adaptation) that serves as a structural unit within the genome, and it is possible to prepare cells that express the gene by integrating the gene into the genome. Examples of cells include fungi, yeast, insect cells, mammalian cells, and plant cells.

[0073] As an example of fungi, Aspergillus oryzae can be cited.

[0074] Examples of yeasts include Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha.

[0075] Examples of insect cells include BmN cells from the silkworm (Bombyx mori), Sf9 and Sf21 cells from the fall armyworm (Spodoptera frugiperda), S2 cells from the Drosophila melanogaster, and Pv11 cells from the midge (Polypedilum vanderplanki).

[0076] Examples of mammalian cells include Chinese hamster ovary cells (CHO cells), young hamster kidney cells (BHK cells), human embryonic kidney cell lines (e.g., HEK293 cells), cell lines derived from human retinoblastoma cells (e.g., PER.C6 cells), mouse myeloma cell lines (e.g., NS0 cells and SP2 / 0 cells), and cell lines derived from these cells.

[0077] Examples of CHO cells include CHO-DG44 cells, CHO-K1 cells, CHO-DXB11 cells, and CHOpro3 cells. - Cells and lineages derived from these cells.

[0078] Examples of mammalian cells include cells capable of differentiating into other cell types. Examples include pluripotent stem cells such as ES cells (embryonic stem cells) and iPS cells (induced pluripotent stem cells); multipotent stem cells such as mesenchymal stem cells, tissue stem cells, and somatic stem cells; and so on.

[0079] <Cell Preparation Methods> This invention provides a method for preparing cells that highly express a target gene. The present invention provides a cell preparation method including a first exploration method and a cell preparation method including a second exploration method.

[0080] The cell preparation method including the first exploration method includes: identifying the TAD by the first exploration method; and inserting the target gene into the TAD containing the genome of the TAD.

[0081] In cell preparation methods that include the first exploratory method, the insertion region of the target gene is within a TAD selected based on a TAD score representing transcriptional activity, specifically within a TAD with relatively high transcriptional activity. Therefore, the prepared cells have a high probability of highly expressing the target gene.

[0082] The cell preparation method including the second exploration method includes: identifying the region defined by the boundary pair P by the second exploration method; and inserting the target gene within ±10 kbp before and after the region in the genome containing the region.

[0083] In cell preparation methods that include the second exploration method, the insertion region of the target gene can be within ±10 kbp of the region defined by boundary pair P (i.e., the region with boundary pair P as the starting and ending point). This can be either within the region with boundary pair P as the starting and ending point, outside the region with boundary pair P as the starting and ending point, or spanning both the inside and outside of the region with boundary pair P as the starting and ending point. Since the insertion region of the target gene is within or near the safe harbor found through the second exploration method, the prepared cells are highly likely to not die and express the target gene at a high rate.

[0084] Cell preparation methods, including the second exploration method, can limit the insertion region of the target gene to a narrower range. Examples of the insertion region of the target gene include: the region within ±8 kbp before and after the boundary relative to P; the region within ±5 kbp before and after the boundary relative to P; the region within ±3 kbp before and after the boundary relative to P; the region within ±1 kbp before and after the boundary relative to P; and the region within the boundary relative to P.

[0085] The genome to which the target gene is inserted can be the genome of any cell, as long as it has a TAD (Transmission Adaptation Array) which serves as a structural unit within the genome. Examples of cells include fungi, yeast, insect cells, mammalian cells, and plant cells. Specific examples of cells are the same as those listed in the description of the exploration method of this invention.

[0086] In one embodiment of the cell preparation method of the present invention, the target gene is inserted into the genome of a mammalian cell. Examples of mammalian cells include Chinese hamster ovary cells (CHO cells), young hamster kidney cells (BHK cells), human embryonic kidney cell lines (e.g., HEK293 cells), cell lines derived from human retinoblastoma cells (e.g., PER.C6 cells), mouse myeloma cell lines (e.g., NSO cells and SP2 / 0 cells), and cell lines derived from these cells.

[0087] In one embodiment of the cell preparation method of the present invention, a target gene is inserted into the genome of CHO cells. Examples of CHO cells include CHO-DG44 cells, CHO-K1 cells, CHO-DXB11 cells, and CHOpro3 cells. - Cells and lineages derived from these cells.

[0088] In one embodiment of the cell preparation method of the present invention, a target gene is inserted into the genome of a cell capable of differentiating into other cells. Examples of such cells include pluripotent stem cells such as ES cells and iPS cells; and multi-differentiated stem cells such as mesenchymal stem cells, tissue stem cells, and adult stem cells.

[0089] Using well-known genome editing technologies, it is possible to insert a target gene into a target region of the genome.

[0090] The source, size, and base sequence of the target gene are not limited. The target gene contains both nucleic acids that encode proteins and nucleic acids that do not encode proteins. Examples of target genes may include genes encoding at least one of the following groups: enzymes, antibodies, interleukins, cytokines, chemokines, hormones, growth factors, transcription factors, receptors, transcriptionally regulated nucleic acids, non-coding RNAs, viral agents, vaccines, medical proteins, their subunits and their fragments.

[0091] When the target gene is a nucleic acid encoding a protein, examples of the protein encoded by the target gene (referred to as the "target protein" in this invention) may be selected from at least one of the following groups: enzymes, antibodies, interleukins, cytokines, chemokines, hormones, growth factors, transcription factors, receptors, proteins constituting viral preparations, vaccines, medical proteins, their subunits and fragments.

[0092] In this invention, antibodies are not limited to immunoglobulins; any molecule that binds to an antigen is acceptable. In this invention, the term "antibody" encompasses both antibody fragments and antigen-binding molecules. In this invention, the heavy chain of an antibody is also referred to as the H chain, and the light chain of an antibody is also referred to as the L chain.

[0093] Nucleic acids that do not encode proteins include transcriptional regulatory nucleic acids, non-coding RNAs, and nucleic acids that constitute viral agents. Examples of non-coding RNAs (ncRNAs) include miRNA (microRNA), shRNA (short hairpin RNA), siRNA (small interfering RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), and tRNA (transfer RNA).

[0094] In this invention, a target gene refers to a gene capable of being expressed within a cell. The target gene contains all the sequences required for its expression. When the target gene is a structural gene, it contains all the sequences required for the expression of the protein encoded by the target gene, and includes the protein's coding sequence and all nucleic acids (e.g., promoters, transcription terminators, polyadenylated sequences) required for transcription and translation of that coding sequence within the cell. The target gene may contain one copy of the protein's coding sequence, or it may contain two or more copies. For example, to express all subunits of a heteropolymeric protein, the target gene may contain at least one copy of the coding sequence for each subunit. For example, the target gene may contain at least one copy each of the sequence encoding the H chain and the sequence encoding the L chain of an antibody.

[0095] In one embodiment of the cell preparation method of the present invention, a nucleic acid consisting of two or more copies of a coding sequence is inserted as a target gene into a target region of the genome. Through this embodiment, cells capable of producing the substance encoded by the target gene in high yields can be prepared. From the perspective of long-term cell passaging stability, the number of copies of the coding sequence contained in the target gene is preferably not too high, preferably 2 to 6 copies, more preferably 2 to 5 copies, and even more preferably 2 to 4 copies.

[0096] In one embodiment of the cell preparation method of the present invention, a nucleic acid consisting of at least one copy of the coding sequences of all subunits of a heteropolymeric protein is inserted as a target gene into a target region of the genome. Through this embodiment, cells capable of stably producing heteropolymeric proteins can be prepared. From the viewpoint of increasing the expression level of heteropolymeric proteins, the target gene in this embodiment is preferably a nucleic acid consisting of two or more copies of the coding sequences of all subunits linked together. From the viewpoint of long-term cell passage stability, the copy number of this set contained in the target gene is preferably not more than 2 to 6 copies, more preferably 2 to 5 copies, and even more preferably 2 to 4 copies.

[0097] <Cell sorting methods> This invention provides a method for sorting cells that highly express a target gene.

[0098] The source, size, and base sequence of the target gene are not limited. The target gene contains both nucleic acids that encode proteins and nucleic acids that do not encode proteins. Examples of target genes may include genes encoding at least one of the following groups: enzymes, antibodies, interleukins, cytokines, chemokines, hormones, growth factors, transcription factors, receptors, transcriptionally regulated nucleic acids, non-coding RNAs, viral agents, vaccines, medical proteins, their subunits and their fragments. The meaning and specific method of the target gene are the same as those described in the description of the cell preparation method of the present invention.

[0099] The cell sorting method of the present invention includes the following (11). (11) For each TAD present in the cell genome, calculate the TAD score representing transcriptional activity, select TADs based on the TAD score, and select cells in which the target gene is present in the selected TADs.

[0100] The TAD and TAD score in (11) have the same meaning as the TAD and TAD score in (1) of the first exploration method, and the specific methods and calculation methods are also the same. One implementation of the TAD score is to multiply the density of genes present in the TAD by the average expression level. Regarding the evaluation criteria for this value, a higher value is preferred.

[0101] Specifically, the presence of the target gene in the TAD is confirmed by means of methods such as extracting the genome from the cell, constructing a sequencing library, sequencing the library, mapping reads onto the genome, mapping the target gene onto the reads, extracting reads containing the target gene, and determining the copy number of the target gene contained in the reads. From the perspective of obtaining long reads and DNA methylation data, library sequencing is preferably single-molecule real-time sequencing or nanopore sequencing.

[0102] Cells selected by (11) contain the target gene in TADs with relatively high transcriptional activity, and can therefore be predicted to be cells that highly express the target gene.

[0103] One example of the implementation of (11) includes selecting cells in which multiple copies (i.e., two or more copies) of the target gene are present in the selected TAD. These cells are highly likely to express the target gene. From the viewpoint of long-term cell passaging stability, the number of copies of the target gene present in the selected TAD is preferably not excessive, preferably 2 to 6 copies, more preferably 2 to 5 copies, and even more preferably 2 to 4 copies.

[0104] An example of an embodiment of the cell sorting method of the present invention also includes the following (12). (12) For at least one of the known safe harbors within the genome, determine the TAD score, i.e., the TAD score-SH, of the associated TAD. Determine a threshold based on at least one TAD score-SH. The TAD is selected based on the threshold and TAD score, and cells containing the target gene are selected from the selected TADs.

[0105] The known safe harbor, TAD score-SH and threshold in the genome in (12) have the same meaning as the known safe harbor, TAD score-SH and threshold in the genome in (2) of the first exploration method, and the specific methods and determination methods are also the same.

[0106] Cells selected by (12) are expected to show gene expression levels equivalent to or exceeding those of cells in a known safe harbor where the target gene has been inserted.

[0107] Even when (11) and (12) cannot be clearly distinguished from each other, the cell sorting method of the present invention includes (11) and (12) as long as the purpose of (11) and (12) is achieved.

[0108] An example of an embodiment of the cell sorting method of the present invention also includes the following (13). (13) Determine the methylation rate of the target gene present in the selected TAD and select cells with low methylation rates.

[0109] DNA methylation typically suppresses gene expression. Therefore, by performing (13), cells that further overexpress the target gene are selected.

[0110] The methylation rate of the target gene can be obtained from data obtained by single-molecule real-time sequencing or nanopore sequencing as a library sequencing in (11). The methylation rate is, for example, the methylated cytosine at the CpG site / all cytosine at the CpG site × 100. The methylation rate value can be the value of the entire target gene, the value of a portion of the target gene (e.g., the value of the protein-coding sequence), or the value of each range of the target gene divided according to function (e.g., the values ​​of the promoter region and the protein-coding sequence). At least one minimum, maximum, average, or median value of the methylation rate obtained from the target range is used as a representative value for selecting cells.

[0111] Selecting cells with low methylation rates of the target gene means, for example, selecting cells with relatively low methylation rates; or selecting cells with methylation rates lower than a pre-set standard (e.g., 30%, 20%, 10%).

[0112] The cells targeted by the cell sorting method of the present invention can be any cells as long as they have a genome containing the structural unit TAD. Examples of cells include fungi, yeast, insect cells, mammalian cells, and plant cells. Specific examples of cells are the same as those listed in the description of the exploration method of the present invention.

[0113] In one embodiment of the cell sorting method of the present invention, mammalian cells are used as the target. Examples of mammalian cells include Chinese hamster ovary cells (CHO cells), young hamster kidney cells (BHK cells), human embryonic kidney cell lines (e.g., HEK293 cells), cell lines derived from human retinoblastoma cells (e.g., PER.C6 cells), mouse myeloma cell lines (e.g., NSO cells and SP2 / 0 cells), and cell lines derived from these cells.

[0114] In one embodiment of the cell sorting method of the present invention, CHO cells are used as the target. Examples of CHO cells include CHO-DG44 cells, CHO-K1 cells, CHO-DXB11 cells, and CHOpro3 cells. - Cells and lineages derived from these cells.

[0115] In one embodiment of the cell sorting method of the present invention, cells differentiated from mammalian cells capable of differentiation are used as the target. For example, cells obtained by introducing a target gene into pluripotent stem cells (ES cells, iPS cells, etc.) or pluripotent stem cells (mesenchymal stem cells, tissue stem cells, adult stem cells, etc.) and causing them to differentiate are used as the target.

[0116] <Cell> The present invention provides a cell obtained by integrating an exogenous target gene into the genome, and a cell that highly expresses the target gene.

[0117] The cells used in this invention are derived from Chinese hamsters. Examples of cells derived from Chinese hamsters include fibroblasts, adipocytes, adipose-derived stem cells, bone marrow-derived stem cells, ovarian cells, and lineages derived from these cells.

[0118] One example of an implementation method using cells derived from Chinese hamsters is Chinese hamster ovary cells (CHO cells). Examples of CHO cells include CHO-DG44 cells, CHO-K1 cells, CHO-DXB11 cells, and CHOpro3 cells. -Cells and lineages derived from these cells.

[0119] The cells of the present invention are cells in which the target gene is inserted into at least one region of the genome of a cell derived from a Chinese hamster, selected from 40 regions shown in Table 1 below. The 40 regions shown in Table 1 are determined by the RefSeq accession number and base number of Chinese hamsters, namely “RefSeq ID”, “start” and “end” in Table 1.

[0120] [Table 1]

[0121] Table 1 also shows the TADs belonging to each region. The TADs here are those detected in the embodiments described later. "TAD start" and "TAD end" in Table 1 are the boundary coordinates of the TADs obtained in the embodiments described later. The meanings of "TAD start" and "TAD end" are the same in Tables 2 and 3.

[0122] The 40 regions shown in Table 1 are regions formed by the regions found by the second exploration method of the present invention and their vicinity, and are regions where genes can be highly expressed. Therefore, cells with a genome in which the target gene is inserted in at least one of the 40 regions shown in Table 1 are cells capable of high expression of the target gene.

[0123] A preferred example of the cell of the present invention is a cell in which the target gene is inserted into at least one region of the genome of a cell derived from a Chinese hamster, which is composed of a group consisting of 40 regions selected from Table 2 below and a region having more than 90% sequence homology with any one of the 40 regions. The 40 regions shown in Table 2 are determined by the RefSeq accession number and base number of Chinese hamsters, namely “RefSeq ID”, “start” and “end” in Table 2.

[0124] [Table 2]

[0125] The 40 regions shown in Table 2 are the internal regions of the 40 regions shown in Table 1. The 40 regions shown in Table 2 are regions formed by the regions found by the second exploration method of the present invention and their vicinity, and are regions where genes can be highly expressed. Therefore, cells with a genome containing the target gene inserted in at least one of the 40 regions selected from Table 2 are cells capable of high expression of the target gene.

[0126] A more preferred example of the cell of the present invention is a cell in which the target gene is inserted into at least one region of the genome of a cell derived from a Chinese hamster, selected from 40 regions shown in Table 3 below and regions having more than 90% sequence homology with any one of the 40 regions. The 40 regions shown in Table 3 are determined by the RefSeq accession number and base number of Chinese hamsters, namely “RefSeq ID”, “start” and “end” in Table 3.

[0127] [Table 3]

[0128] The 40 regions shown in Table 3 are the internal regions of the 40 regions shown in Table 2. The 40 regions shown in Table 3 are regions found using the second exploration method of this invention; they are safe harbors and regions with high gene expression. Therefore, cells with a genome containing the target gene inserted in at least one of the 40 regions selected from Table 3 are cells capable of stably and highly expressing the target gene.

[0129] The source, size, and base sequence of the target gene are not limited. The target gene contains both nucleic acids that encode proteins and nucleic acids that do not encode proteins. Examples of target genes may include genes encoding at least one of the following groups: enzymes, antibodies, interleukins, cytokines, chemokines, hormones, growth factors, transcription factors, receptors, transcriptionally regulated nucleic acids, non-coding RNAs, viral agents, vaccines, medical proteins, their subunits and their fragments.

[0130] When the target gene is a nucleic acid that encodes a protein, examples of the target protein may be selected from at least one of the following groups: enzymes, antibodies, interleukins, cytokines, chemokines, hormones, growth factors, transcription factors, receptors, proteins constituting viral preparations, vaccines, medical proteins, their subunits and their fragments.

[0131] Nucleic acids that do not encode proteins include transcriptional regulatory nucleic acids, non-coding RNAs, and nucleic acids that constitute viral agents. Examples of non-coding RNAs (ncRNAs) include miRNA (microRNA), shRNA (short hairpin RNA), siRNA (small interfering RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), and tRNA (transfer RNA).

[0132] In this invention, a target gene refers to a gene capable of being expressed within a cell. The target gene contains all the sequences required for its expression. When the target gene is a structural gene, it contains all the sequences required for the expression of the protein encoded by the target gene, and includes the protein's coding sequence and all nucleic acids (e.g., promoters, transcription terminators, polyadenylated sequences) required for transcription and translation of that coding sequence within the cell. The target gene may contain one copy of the protein's coding sequence, or it may contain two or more copies. For example, to express all subunits of a heteropolymeric protein, the target gene may contain at least one copy of the coding sequence for each subunit. For example, the target gene may contain at least one copy each of the sequence encoding the H chain and the sequence encoding the L chain of an antibody.

[0133] One embodiment of the cell of the present invention has a genome containing a target gene consisting of two or more copies of a coding sequence linked together. The cell of this embodiment is capable of producing the substance encoded by the target gene in high yield. From the perspective of long-term cell passaging stability, the number of copies of the coding sequence contained in the target gene is preferably not too high, preferably 2 to 6 copies, more preferably 2 to 5 copies, and even more preferably 2 to 4 copies.

[0134] One embodiment of the cell of the present invention has a genome containing a target gene consisting of nucleic acid containing at least one copy of the coding sequence of each subunit of a heteropolymeric protein linked together. The cell of this embodiment is capable of stably producing heteropolymeric proteins. From the viewpoint of increasing the expression level of heteropolymeric proteins, the target gene in this embodiment is preferably a nucleic acid consisting of two or more copies of the coding sequences of all subunits linked together. From the viewpoint of long-term cell passage stability, the copy number of this set contained in the target gene is preferably not more than 2 to 6 copies, more preferably 2 to 5 copies, and even more preferably 2 to 4 copies.

[0135] This invention provides a method for preparing cells derived from Chinese hamsters, which highly express the target gene. The method for preparing the cell involves inserting the target gene into at least one region selected from 40 regions shown in Table 1 of the genome of a cell derived from a Chinese hamster. A preferred example of the cell preparation method involves inserting the target gene into at least one region selected from the 40 regions shown in Table 2 of the genome of a cell derived from a Chinese hamster. A more preferred example of the cell preparation method involves inserting the target gene into at least one region selected from the 40 regions shown in Table 3 of the genome of a cell derived from a Chinese hamster. Using well-known genome editing technologies, it is possible to insert a target gene into a target region of the genome.

[0136] <Methods for Manufacturing Cell Products> This invention provides a method for manufacturing cell products with excellent productivity. The method uses cells that highly express a target gene, thereby achieving excellent productivity of the substance encoded by the target gene (referred to as the "target substance" in this invention). The cells that highly express the target gene are selected from at least one group consisting of cells prepared by the cell preparation method of this invention, cells sorted by the cell sorting method of this invention, and cells of this invention.

[0137] In the method for manufacturing cell products of the present invention, cells are cultured to express a target gene and produce a target substance. The target substance is produced within the cells through cell culture, and the target substance accumulates in the culture medium and / or cells.

[0138] The cell culture method and culture medium composition can be selected according to the cell type. Culture conditions (e.g., culture scale, cell density, temperature, and CO2 concentration) can also be selected according to the cell type.

[0139] One embodiment of the method for manufacturing cell products of the present invention includes recovering a target substance from the culture medium. Examples of methods for recovering the target substance from the culture medium include centrifugation, filtration, dialysis filtration, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and high-performance liquid chromatography (HPLC). The recovered target substance may be used, for example, in the manufacture of pharmaceutical compositions.

[0140] One embodiment of the method for manufacturing the cell product of the present invention includes recovering cells containing a target substance from a culture medium. Methods for recovering cells from a culture medium include, for example, centrifugation and filtration. The target substance accumulates inside or on the surface of the cells depending on its properties. The recovered cells can be administered, infused, or transplanted into mammals, for example. Example

[0141] The following specific examples will provide a more detailed description of the exploration method of the present invention. The materials, processing steps, etc., shown in the following specific examples can be appropriately modified without departing from the spirit of the present invention. The scope of the exploration method of the present invention should not be limited by the specific examples shown below.

[0142] <Preparation of Antibody Production Strains> [method] Using an artificial plasmid containing the dihydrofolate reductase (DHFR) gene as a backbone, a plasmid was constructed carrying genes encoding known IgG (one H-chain gene and one L-chain gene, totaling two antibody subunit genes). The H-chain and L-chain genes contain the hEF-1α promoter, the coding sequence for the fibronectin secretion signal peptide, the coding sequence for the antibody subunit, and the PolyA sequence, respectively. The fibronectin secretion signal peptide is a signal peptide that induces the extracellular secretion of polypeptides. The two antibody subunit genes on the plasmid are arranged in the L-H chain sequence. Hereinafter, this genome (L-H chain) will be referred to as "GoI".

[0143] The plasmid was linearized and introduced into CHO-DG44 cells via electroporation. MTX-resistant cell pools were established by static culture in a medium containing methotrexate (MTX) for 14–21 days. One cell was seeded in each well of a 96-well plate and cultured statically at 37°C and 10% (v / v) CO2. On day 14 of culture, the culture supernatant was recovered, and antibody concentrations were determined using the Octet Qke (Sartorius) intermolecular interaction analysis device. Clones with high antibody concentrations were selected and cultured in 24-well plates, followed by culture in bioreactor tubes for scale-up production. Sixty cell lines with high antibody concentrations were selected. To confirm the long-term passage stability of cells, cells were suspended in passage medium and passaged every 3 days. Passaging was terminated when the total number of cell divisions exceeded 60.

[0144] <Feeded Batch Culture Experiment> [method] Sixty antibody production strains, before and after long-term passage, were suspended in 40 mL of basal medium and transferred to 125 mL flasks for shaking culture. The culture was carried out at 140 rpm under an atmosphere of 37 °C and 5% (v / v) CO2 concentration. From day 3 to day 13 of culture, the prescribed amount of culture medium was added daily. Samples were taken every 1 to 3 days to measure cell density, culture medium composition, and antibody concentration. The assays used included the Vi-CELL XR cell counter (Beckman Coulter Life Sciences), the FLEX2 cell culture environment analyzer (Nova Biomedical), and the CedexBio product assay device (Roche Diagnostics). The culture medium was recovered on day 14 of culture, and cells and cell debris were removed using a depth filter (0.22 μm pore size) to obtain the culture supernatant. The antibody concentration in the culture supernatant was determined by liquid chromatography using a protein A column.

[0145] [result] Figure 2 The antibody production performance of 60 antibody production strains is shown in the figure. Figure 2 In the scatter plot, the horizontal axis represents the degree of change in relative production rate over a long period of generations, and the vertical axis represents relative production rate. From 60 antibody-producing strains, 31 strains with a horizontal axis of -20% or higher were selected.

[0146] <Determination of the insertion position of GoI> [method] Thirty-one antibody-producing strains were cultured with shaking for two weeks. The culture medium used was a liquid medium supplemented with L-glutamate and methotrexate in serum-free basal medium (CDOptiCHO Medium, model 12681-011, Thermo Fisher Scientific Inc.). 4 × 10⁴ cells were recovered from the culture medium of each cell. 6 The genome of the cells was extracted using a long-chain genome extraction kit. The genome length was adjusted and concentrated. A library was constructed from a condensed genome using a Ligation Sequencing Kit (model SQK-LSK109, Oxford Nanopore Technologies). The DNA sequences in the library were read using a nanopore sequencer (model M1CCapEx, Oxford Nanopore Technologies). Base identification was performed on the fast5 file output from the sequencer to obtain the fastq file and the corresponding fasta file. From all reads, reads containing H-chain and L-chain genes were extracted using BLAST (Basic Local Alignment Search Tool) and mapped to the CHO genome (GCF_003668045.3), obtaining the mapping data (bam file). The mapping tool used was minimap2. The BAM file was visualized using the Integrative Genomics Viewer (IGV) (Broad Institute), which comprehensively defined the boundaries between the GoI and the genome. Hereinafter, the boundary between GoI and the genome will be referred to as the "connection point", and its coordinates will be referred to as the "connection point coordinates".

[0147] [result] A total of 226 linkage sites were found in 31 antibody production strains.

[0148] <Determination of GoI's Insertion Mode> [method] Base identification was performed on the fast5 file output from the sequencer to obtain the fastq file. Reads were then mapped to the CHO genome (CriGri-PICRH). Minimap2 was used for mapping. Only reads mapped within 6500 bp of the ligation point were extracted. Using the self-developed software "TaulVis", homologous regions with the genome and homologous regions with GoI in the extracted reads were visualized. Reads that allow for the continuous reading of two junction points and GoI (i.e., "junction point-GoI-junction point") were extracted.

[0149] [result] The above continuous reading segment contains a total of 176 connection points.

[0150] <Determining the connection point for clamping GoI> [method] The H-strand and L-strand genes were mapped onto the aforementioned continuous read segment. The mapping tool used was minimap2. When the full length of the H-strand gene could be mapped, the H-strand copy number was counted. Similarly, when the full length of the L-strand gene could be mapped, the L-strand copy number was counted. Reads with a count of 1 copy or more in the H-chain and L-chain genes were extracted, and the connection points contained in the extracted reads were identified.

[0151] [result] exist Figure 3 The image shows a histogram of copy numbers for the H-chain and L-chain genes. Figure 3 In the histogram, the horizontal axis represents the number of copies, and the vertical axis represents the number of connection points. The H-chain and L-chain genes contain a total of 165 linker sites in reads with a count of 1 copy or more.

[0152] <Detection of hypomethylated regions> [method] In reads of the H-chain and L-chain genes with more than one copy count, methylated cytosine at CpG sites were detected from the aforementioned fast5 files using a base modification analysis tool (Megalodon, Oxford Nanopore Technologies). Methylation rates (methylated cytosine at CpG sites / all cytosine at CpG sites × 100) were determined for each promoter region, H-chain coding sequence, and L-chain coding sequence, and the arithmetic mean of the methylation rates was calculated.

[0153] [result] Figure 4 The figure shows the average methylation rate of the promoter region, H chain coding sequence, and L chain coding sequence. Figure 4 In the histogram, the horizontal axis represents the average methylation rate, and the vertical axis represents the number of connection points. The total number of linkage sites in reads with an average methylation rate of less than 10% in both the promoter region and antibody subunit region is 140.

[0154] <Obtaining the Boundary Coordinates of TAD> [method] The genomic structure data (fastq file, SRRID: SRR12194154) for Hi-C analysis were obtained from the paper analyzing the genomic structure of CHO cells (William Hilliard, Kelvin H. Lee. Systematic identification of safe harbor regions in the CHO genome through a comprehensive epigenome analysis. Biotechnology and Bioengineering, 2020. https: / / doi.org / 10.1002 / bit.27599). Using the mapping program bwa, the fastq file was mapped to the CHO genome (CriGri-PICRH, https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_003668045.3 / ) (alignment parameters: -A1, -B4, -E50, -L0), and the mapping data (bam file) was obtained. Next, the contact diagram was obtained from the mapping data (bam file) using the hicBuildMatrix command (binSize: 100000) of HiCExplorer, a HiC data parsing and visualization tool. Next, the hiCFindTADs command (minDepth: 300000, maxDepth: 600000, Step: 100000, minBoundaryDistance 400000) was used to obtain the boundary coordinates of the TAD (a set of pairs of start and end points) from the contact graph.

[0155] [result] The genomic structure data obtained from the aforementioned paper consisted of 281, 721, and 369 reads with paired ends. Mapping all reads to the CHO genome yielded 150 million paired reads, which were used to obtain contact maps for each chromosome. Figure 5 The image shows a contact diagram (represented as a heatmap) for RefSeq accession number NW_023276806.1. 2502 TADs were detected in the CHO genome.

[0156] <Calculation of TAD Score> [method] Based on the coordinate information of endogenous genes in CHO cells (GCF_003668045.3_CriGri-PICRH-1.0_genomic.gtf), the endogenous genes contained in each of the 2502 TADs were detected. CHO-DG44 cells were used as samples to perform RNA-Seq (Takara Bio Inc.) to quantify the amount of mRNA in endogenous genes. The mRNA amount was then corrected to TPM (transcripts per million) as the expression level of the endogenous gene. For each of the 2502 TADs, the value obtained by multiplying the density of endogenous genes by the average expression level of endogenous genes (usually the average of log-transformed TPM values) is calculated, and this value is used as the TAD score.

[0157] [result] Figure 6 The distribution of TAD scores is shown in the figure. Figure 6 The “×” in the table represents the 2502 TAD ratings displayed in descending order.

[0158] <Determination of the TAD score for the well-known safe harbor> [method] As a safe harbor for CHO cells, the Fer1l4, Hprt, and C12orf35 loci are known. The TADs associated with these three loci were identified, and their TAD scores were determined. The minimum TAD score among the three was used as the threshold.

[0159] [result] The TAD scores for the three loci's TADs are as follows. Figure 6 The distribution is shown in the diagram with three horizontal lines. Fer1l4 locus = 34.3 Hprt locus = 12.3 C12orf35 locus = 9.0 The threshold was set at 9.0.

[0160] <Selection of Connection Points with High TAD Scores> [method] The TADs and their scores for 140 connection points were determined. Connection points with TAD scores exceeding the threshold of 9.0 (i.e., TAD score of the C12orf35 locus) were selected from 140 connection points.

[0161] [result] Figure 6 The “○” in the figure represents the 140 connection points selected based on the methylation rate. Of the 140 connection points, 28 had a TAD score of over 9.0.

[0162] <Detection of junction pairs without chromosomal translocation> [method] In join pairs containing regions of the H and L genes containing more than one copy, if the join point coordinates indicate that the join pairs are located on the same chromosome, and the distance between join pairs on the read is less than 100 kbp, then no chromosomal translocation is considered to exist between the join pairs. Join pairs without chromosomal translocation were detected based on this criterion.

[0163] [result] Of the 28 junctions with a TAD score above 9.0, 14 were junction pairs with no chromosomal translocations between them. Through this series of processes, it is inferred that the seven regions held by the seven connection point pairs are safe havens for the inserted gene and regions where the inserted gene is stably highly expressed. Table 4 shows the coordinates of 7 regions and 14 connection points (the “start” and “end” of the 7 regions).

[0164] [Table 4]

[0165] <Validation of gene expression levels in 7 regions> [method] For each of the seven regions shown in Table 4, plasmids with homologous arms for the seven regions attached to both ends of the insert gene were constructed. The insert gene consisted of the two antibody subunit genes (L-chain and H-chain) mentioned above.

[0166] The constructed plasmid was introduced into CHO cells via electroporation (4D-Nucleofector X unit system, Lonza). By using PCR and digital PCR systems (QX-200, Bio-Rad Laboratories) to amplify the boundary between the genome and the inserted gene, strains with only one copy of the inserted gene introduced into a predetermined region were selected. As a comparison, strains with only one copy of the insert gene were prepared in the well-known safe harbor of CHO cells, namely the Fer1l4 locus, in the same manner as described above. As a comparison, linearized insert genes were introduced into CHO cells via electroporation and randomly integrated into the genome. Using a digital PCR system, strains with only one copy of the insert gene introduced into each whole genome were selected. Cell lines were cultured, and the mRNA levels of the H and L chains were quantified using real-time PCR (CFX96, Bio-Rad Laboratories, Inc.).

[0167] [result] exist Figure 7 The figure shows the amount of mRNA in the H chain. The vertical axis represents the ratio of mRNA amount prepared by random integration to the average mRNA amount of 47 strains with only 1 copy of the insert gene. “RI” refers to the 47 strains prepared through random integration. The "TI" numbers, from top to bottom, represent the strain inserted into the Fer1l4 locus, the strain inserted into ID029, and the strain inserted into ID040. from Figure 7 The results show that, compared with random integration, inserting genes into the regions found through this embodiment results in a higher probability of producing strains that highly express the target gene.

[0168] exist Figure 8 The diagram shows the relative production rates of the H strand for the strain inserted into ID040, ID029, ID038, ID032, and ID024. Here, relative production rate refers to the ratio of the production rate to that of the strain inserted into the Fer1l4 locus. The relative production rate of the above 5 strains exceeded 0.8, which is not inferior to that of the strain inserted into the Fer1l4 locus.

[0169] <Preparation of multi-copy inserts> [method] Multiple copy insertions of the coding sequences were attempted in the seven regions shown in Table 4. Plasmids with homologous arms for each of the seven regions were constructed for each region, with the inserted gene having homologous arms attached to both ends of the respective region. The inserted gene was a nucleic acid consisting of two or four of the aforementioned two antibody subunit genes (L-chain-H-chain).

[0170] The constructed plasmid was introduced into CHO cells via electroporation (4D-Nucleofector X unit system, Lonza). By using PCR and digital PCR systems (QX-200, Bio-Rad Laboratories, Inc.) to amplify the boundary between the genome and the inserted gene, strains with only 2 or 4 copies of the inserted gene introduced into a predetermined region were selected. To confirm the long-term passage stability of cells, cells were suspended in passage medium and passaged every 3 days. Passaging was terminated when the total number of cell divisions exceeded 60.

[0171] [result] Figure 9 The graph shows the copy numbers of the L and H strands as determined by ddPCR. The horizontal axis represents the strain name prepared, with the strain identification number following the region ID. In strains ID040-1 and ID040-2, a nucleic acid consisting of two L-strands linked together was inserted. ID040 is a region capable of inserting at least two copies of the L-strand-H strand. In strains ID029-1 and ID029-2, a nucleic acid consisting of four L-H strands linked together was inserted. ID029 is a region capable of inserting at least four copies of the L-H strand. The ID032-1 and ID032-2 strains contain an inserted nucleic acid consisting of four L-H strands linked together. ID032 is a region capable of inserting at least four copies of the L-H strand.

[0172] <Feeded Batch Culture Experiment> [method] The strains ID040-1, ID040-2, ID029-1, ID029-2, ID032-1, and ID032-2 were used for the culture experiment.

[0173] Six antibody production strains, before and after long-term passage, were suspended in 40 mL of basal medium and transferred to 125 mL flasks for shaking culture. The culture was carried out at 140 rpm under an atmosphere of 37 °C and 5% (v / v) CO2 concentration. From day 3 to day 13 of culture, the prescribed amount of culture medium was added daily. Samples were taken every 1 to 3 days to measure cell density, culture medium composition, and antibody concentration. The assays used included the Vi-CELL XR cell counter (Beckman Coulter Life Sciences), the FLEX2 cell culture environment analyzer (Nova Biomedical), and the CedexBio product assay device (Roche Diagnostics). The culture medium was recovered on day 14 of culture, and cells and cell debris were removed using a depth filter (0.22 μm pore size) to obtain the culture supernatant. The antibody concentration in the culture supernatant was determined by liquid chromatography using a protein A column.

[0174] [result] exist Figure 10 The figure shows the antibody production performance of six antibody-producing strains before and after long-term culture. "pre" represents the antibody-producing strain before long-term culture, and "post" represents the antibody-producing strain after long-term culture. The vertical axis represents the relative production rate. according to Figure 10 The results show that antibody-producing strains with 2 or 4 copies of the L-H chain inserted in ID040, ID029, or ID032 maintained stable antibody production even after long-term culture. It is evident that strains with 4 copies of the L-H chain inserted in ID029 or ID032 have higher antibody production rates compared to strains with 2 copies of the L-H chain inserted in ID040, and antibody production increases with increasing copy number of the coding sequence.

[0175] All documents, patent applications and technical standards described in this specification are incorporated herein by reference to the same extent as the specific and individually described documents, patent applications and technical standards which are incorporated herein by reference.

[0176] The invention, which was filed on November 29, 2023, under Japanese Patent Application No. 2023-202228, is incorporated herein by reference in its entirety.

Claims

1. An exploratory method for identifying target regions on the genome where a target gene is inserted. The exploration methods include the following (1). Identify the TAD as the target region. (1) Calculate a TAD score representing transcriptional activity for each TAD in the genome, and select a TAD based on the TAD score.

2. The exploration method according to claim 1, further comprising the following (2). (2) For at least one of the known safe harbors within the genome, determine the TAD score, i.e., the TAD score-SH, of the associated TAD. Determine a threshold based on at least one of the TAD scores-SH. The TAD is selected based on the threshold and the TAD score.

3. An exploratory method for identifying target regions on the genome where a target gene will be inserted. The exploration methods include the following (a) to (d). The region defined by the following boundary pair P is identified as the target region. (a) Prepare cells having a genome F that integrates a foreign gene into its genome and expresses the foreign gene. (b) Obtain the genome F from the cell, analyze the genome F, and determine the region containing the foreign gene, i.e., region F, and the boundary F between region F and the genome. (c) Calculate a TAD score representing transcriptional activity for each TAD possessed by the genome; for each boundary F, determine the TAD score, i.e., TAD score-F, belonging to the TAD; and select the boundary F based on the TAD score-F. (d) Find the boundary pair P that clamps the region F from the selected boundary F.

4. The exploration method according to claim 3, further comprising (p) performing (c) on the boundary F selected by (p). (p) Determine the methylation rate of the exogenous gene present in the region F, and select the boundary F of the region F with a low methylation rate.

5. The exploration method according to claim 3 or 4, further comprising (q) performing (d) on the boundary F selected by (c) and (q). (q) For at least one of the known safe harbors within the genome, determine the TAD score, i.e., the TAD score-SH, of the associated TAD. Determine a threshold based on at least one of the TAD scores-SH. The boundary F is selected based on the threshold and the TAD score-F.

6. The exploration method according to claim 3 or 4, further comprising the following (r). (r) Select the boundary pair P of the region F located between the boundary pairs P in the genome F that is not formed by chromosomal translocation.

7. The exploration method according to any one of claims 1 to 4, wherein, The TAD score is a value obtained by multiplying the density of genes present in the TAD by the average expression level.

8. The exploration method according to claim 1 or 3, wherein, The genome is the genome of a mammalian cell.

9. The exploration method according to claim 1 or 3, wherein, The genome is the genome of CHO cells.

10. A method for preparing cells, comprising: The TAD is found using the exploration method described in claim 1; and Insert the target gene into the TAD within the genome containing the TAD.

11. A method for preparing cells, comprising: The region determined by the boundary pair P is found using the exploration method described in claim 3; and Insert the target gene within ±10 kbp before and after the region of the genome containing the region.

12. The method for preparing cells according to claim 10 or 11, wherein, The target gene is a gene encoding at least one of the following groups: enzymes, antibodies, interleukins, cytokines, chemokines, hormones, growth factors, transcription factors, receptors, transcriptionally regulated nucleic acids, non-coding RNAs, viral agents, vaccines, medical proteins, their subunits and their fragments.

13. A cell sorting method, which is a method for selecting cells expressing a target gene, said sorting method includes the following (11). (11) Calculate a TAD score representing transcriptional activity for each TAD in the cell’s genome, select a TAD based on the TAD score, and select cells in which the target gene is present in the selected TAD.

14. The cell sorting method according to claim 13, further comprising the following (12). (12) For at least one of the known safe harbors within the genome, determine the TAD score, i.e., the TAD score-SH, of the associated TAD. Determine a threshold based on at least one of the TAD scores-SH. The TAD is selected based on the threshold and the TAD score, and cells in which the target gene is present in the selected TAD are selected.

15. The cell sorting method according to claim 13 or 14, further comprising the following (13). (13) Determine the methylation rate of the target gene present in the selected TAD and select cells with a low methylation rate.

16. The cell sorting method according to claim 13 or 14, wherein, The TAD score is a value obtained by multiplying the density of genes present in the TAD by the average expression level.

17. The cell sorting method according to claim 13, wherein, The cells in question are mammalian cells.

18. The cell sorting method according to claim 13, wherein, The cells in question are CHO cells.

19. The cell sorting method according to claim 13, wherein, The target gene is a gene encoding at least one of the following groups: enzymes, antibodies, interleukins, cytokines, chemokines, hormones, growth factors, transcription factors, receptors, transcriptionally regulated nucleic acids, non-coding RNAs, viral agents, vaccines, medical proteins, their subunits and their fragments.

20. A type of cell derived from Chinese hamsters. Insert the target gene into at least one region selected from the 40 regions shown in Table 1 below. [Table 1] 。 21. The cell according to claim 20, wherein, The cells derived from Chinese hamsters are CHO cells.

22. The cell according to claim 20 or 21, wherein, The target gene is a gene encoding at least one of the following groups: enzymes, antibodies, interleukins, cytokines, chemokines, hormones, growth factors, transcription factors, receptors, transcriptionally regulated nucleic acids, non-coding RNAs, viral agents, vaccines, medical proteins, their subunits and their fragments.

23. A method for manufacturing a cell product, comprising: Cells prepared by the cell preparation method according to claim 10 or 11 are cultured to express the target gene.

24. A method for manufacturing a cell product, comprising: Cells sorted by the cell sorting method of claim 13 or 14 are cultured to express the target gene.

25. A method for manufacturing a cell product, comprising: The cells of claim 20 or 21 are cultured to express the target gene.

Images