KR20230136325A - Recombinant human cell having GSTP1 gene knockout and use thereof - Google Patents

Abstract

The present invention relates to a recombinant human cell resistant to hyperosmotic stress conditions and a method for producing target products using the same. Specifically, the present invention relates to a recombinant human cell in which a GSPT1 gene has been knocked out and a method for producing target products such as proteins like antibodies, genes, and vectors using the same.

Description

Recombinant human cell having GSTP1 gene knockout and use thereof {Recombinant human cell having GSTP1 gene knockout and use thereof}

Recombinant human cells resistant to high osmotic stress conditions and a method for producing a target product using the same, specifically, a recombinant human cell with the GSPT1 gene knocked out and using the same to produce a target product such as proteins such as antibodies, genes, vectors, etc. It's about how to do it.

The human embryonic kidney (HEK) 293 cell line is widely used as a production host for a variety of purposes, such as the production of recombinant proteins for therapeutic use or structural studies, virus-like particles (VLPs) for vaccine production, and viral vectors for gene therapy. It is becoming. As a production host, the HEK293 cell line has several advantages, such as ease of transfection, growth in suspension culture, and human-like post-translational modification. To enhance the production of recombinant proteins by HEK 293 cells, genetic manipulation of target genes involved in culture performance, such as cell proliferation and death, metabolism, glycosylation, protein folding and secretion, has been attempted, with limited success. It was seen (Abaandou et al., 2021, Cells 10, 1667). Research is actively underway to utilize human cells, such as HEK 293 cells, as an expression platform for the production of biological drugs such as recombinant proteins and vectors.

In order to be developed as a cell line for producing target products, including therapeutic proteins, resistance to stress occurring in culture conditions is required. During mass cultivation of recombinant cell lines that produce therapeutic proteins, various stresses occur and inhibit cell proliferation and productivity. Osmotic stress is caused by the difference between extracellular and intracellular osmolarity due to the supply of nutrients and base for pH adjustment in fed-batch culture of cells for production of the target product, and reduces cell activity for survival. brings about change Hyperosmotic stress activates signaling cascades mediated by mitogen-activated protein kinases (MAPKs), such as p38 and c-Jun N-terminal kinase (JNK), which phosphorylate transcription factors and downstream kinases. , thereby inducing apoptosis (de Nadal et al., 2002, EMBO Rep. 3, 735-740; Wada and Penninger, 2004, Oncogene 23, 2838-2849). To overcome the effects of hyperosmotic stress on cell viability and productivity, various genetic manipulations have been attempted in mammalian cells. Overexpression of anti-apoptotic regulators, such as Bcl-2 and 30Kc6 , inhibited apoptosis under hyperosmotic stress and increased cell viability and productivity of recombinant proteins in recombinant CHO cells (Kim and Lee, 2002, J. Biotechnol 95, 237-248; Wang et al., 2012, Process Biochem. 47, 735-741). However, these knowledge-based approaches were often limited or insufficiently effective on cellular phenotypes. Under hyperosmotic stress in human cells, inhibition of caspase activity increased cell viability, but inhibition of MAPKs, including p38 and JNK, had a minor effect on cell viability (Thiemicke and Neuert, 2021, Sci. Adv. 7, eabe1122 ). The improvement of mammalian cells through genetic engineering has been widely attempted to efficiently produce therapeutic proteins of appropriate quality, but identification of new target genes for cell engineering is labor-intensive and has not yet been fully proven.

Recently, CRISPR/Cas9 library screening has been used to discover essential genes and therapeutic targets for diseases in various types of human cells, mainly using virus-mediated integration of gRNA cassettes. The recombinase-mediated cassette exchange (RMCE) system is an attractive option for the delivery of gRNAs that require single-copy integration for unbiased and sustained expression. Development of recombinant cell lines using RMCE makes it possible to sort out non-fluorescent cells carrying donor gene cassettes and avoid random integration of gene cassettes using promoters or poly(A) traps positioned beyond the landing pad, thereby allowing the gene cassette to reach a predetermined site. enables single-copy integration of the transgene (Hamaker and Lee, 2018, Curr. Opin. Chem. Eng. 22, 152-160). This strategy of cell line development has gained great prominence in the production of therapeutic proteins due to uniform and stable expression of the transgene (Srirangan et al., 2020, Crit. Rev. Biotechnol. 40, 833-851).

Accordingly, the present inventors developed an RMCE-based CRISPR/Cas9 screening system and used it to discover genes related to resistance to osmotic stress in the culture of recombinant human cell lines and completed the present invention.

The purpose of the present invention is to provide target genes associated with response to osmotic stress in human cells.

The purpose of the present invention is to provide recombinant human cells with resistance to osmotic stress.

The purpose of the present invention is to provide a method for producing a target product using recombinant human cells that have resistance to osmotic stress.

One aspect of the present invention provides recombinant human cells in which the GSPT1 gene has been knocked out.

As used herein, the term “recombinant human cell” refers to a cell in which genetic modification has occurred in an original human cell through transduction or gene editing. Recombinant human cells provide human-like post-translational modifications and are therefore being developed and utilized as a useful expression platform for the production of desired products such as therapeutic proteins. As a platform for the production of target products, attempts are being made to improve cell viability, growth rate, and productivity of the target product.

When producing a target product by culturing recombinant human cells, various factors have an effect limiting the growth rate, viability, and productivity of cells. One of these factors is a gene related to resistance to osmotic pressure, such as osmotic pressure stress. It was confirmed that the viability, growth rate, and productivity of cells were improved compared to the parent cells by knocking out the gene encoding GSPT1 under these conditions.

GSPT1 (G1 to S phase transition 1) protein functions as an antagonist to inhibitors of apoptosis proteins in human cells. GSPT1 protein is transported from the endoplasmic reticulum to the cytoplasm, enhances caspase activation, and regulates signal transduction under various stresses. , is known to promote apoptosis by regulating the activity of apoptosis signal-regulating kinase 1 (Lee et al., 2008, Oncogene 27, 1297-1305).

GSPT1 was discovered as a gene that increases cell growth rate, viability, and productivity of the target product when knocked out under hyperosmotic conditions through CRISPR/Cas9 library screening using transfer of a gRNA cassette by RMCE (recombinase-mediated cassette exchange). . Attempts have been made to increase cell viability and productivity by knocking out genes that induce apoptosis or overexpressing genes that inhibit apoptosis, but inhibition of apoptosis does not necessarily lead to increased cell viability and productivity. The GSPT1 gene, discovered by the present inventors, confers resistance to osmotic pressure, which is one of the factors that most limits the viability and productivity of cells in cell culture, especially fed-batch culture, and increases cell viability and productivity. is useful as a new target in the development or improvement of human cell lines for production of the desired product.

As used herein, the term “knockout” refers to physical removal from the genome so that the relevant gene does not function, and includes, but is limited to, removal of the gene by homologous recombination using CRISPR/Cas9. Gene knockout can be achieved using methods that do not work.

In one embodiment of the present invention, the recombinant human cell may be more resistant to hyperosmotic stress than the parent cell before the GSPT1 gene is knocked out.

As used herein, the term “parental cell” refers to a cell before the desired modification, such as gene knockout, occurs. For example, the parent cell of a human recombinant cell in which the GSPT1 gene has been knocked out is identical to the human recombinant cell in everything except the knockout of the GSPT1 gene.

As used herein, the term "osmotic stress" or "hyperosmotic stress" refers to the proliferation and proliferation of cells by an increase in osmotic pressure due to the production of target products or metabolic by-products according to the composition of the medium or the progress of the culture under cell culture conditions. It refers to a condition that negatively affects viability. It is a stress that accompanies industrially used fed-batch culture, and although the level that can be recognized as osmotic stress varies depending on the cell or culture conditions, it can be defined as the osmolarity at which significant inhibition of cell proliferation occurs. For example, in the culture of HEK293 cells, osmotic stress can be defined as an osmolarity of 450 mOsm/kg or more.

In one embodiment of the present invention, the recombinant human cells may have higher viability and growth rate under hyperosmotic stress conditions than the parental cells before the GSPT1 gene is knocked out.

In one embodiment of the present invention, the recombinant human cells may have a longer culture life than the parental cells before the GSPT1 gene is knocked out.

As used herein, the term “culture life” refers to the period during which cells proliferate and maintain viability in the culture of cells for the production of the target product and significantly produce the target product, depending on the target product, culture conditions, and cells. It may vary depending on the As the culture life is prolonged, the total number of cells in the culture increases, thereby increasing the total production of the desired product by the culture.

In one embodiment of the present invention, the hyperosmotic stress condition may result from fed-batch culture.

In one embodiment of the present invention, the recombinant human cell may contain a gene encoding the desired product.

As used herein, the term "target product" refers to a substance to be obtained by culturing a recombinant cell containing a gene encoding the same or a gene related to a metabolic pathway including its biosynthesis or a modification thereof, such as a protein, nucleic acid, virus, etc. Including, but not limited to.

In one embodiment of the present invention, the target product may be a therapeutic protein, gene therapy, cell therapy, vector, or vaccine, but is not limited thereto.

In one embodiment of the present invention, the gene encoding the target product may be inserted into the genome of the recombinant human cell, or may exist as a vector.

In one embodiment of the present invention, the recombinant human cell may include two or more landing pads on the genome.

In one embodiment of the present invention, the recombinant human cell may contain landing pads at each of the AAVS1 locus and the ROSA26 locus, and a gene encoding the same desired product is present in each landing pad, thereby comprising two copies of the gene. can do.

In one embodiment of the present invention, the recombinant human cell may include landing pads at each of the AAVS1 locus and the ROSA26 locus, one landing pad contains a gene encoding the desired product, and the remaining landing pad contains the gene's An effector that regulates expression may exist to promote the expression of a gene encoding a desired product.

In one embodiment of the invention, the recombinant human cells can be generated by targeted integration of a gene expression cassette encoding the desired product into a landing pad.

As used herein, the term "landing pad" refers to a site-specific recognition sequence or site-specific recombination site, such as an attP site, that is stably integrated into the genome of a host cell, It is used for site-specific or targeted integration of exogenous sequences, such as genetic sequences encoding the desired product.

In one embodiment of the present invention, the recombinant human cells may be HEK293 cells, HT-1080 cells, or PER.C6 cells.

In one embodiment of the present invention, the recombinant human cells are HEK293 cells, and the target product may be an antibody.

Another aspect of the present invention is

A method of producing GSPT1 knockout recombinant human cells, comprising:

It includes the step of transducing human cells using a donor containing a gRNA targeting the gene encoding GSPT1 and a recombinant enzyme,

The method provides a method in which the recombinant enzyme is tagged with an NLS (Nuclear Localization Sequence) at its end.

In one embodiment of the present invention, NLS may be tagged at the N-terminus, C-terminus, or both ends of the recombinant enzyme to increase recombination efficiency by RMCE.

In one embodiment of the present invention, the recombinant enzyme may be Cre recombinase, Bxb1 integrase, FLP recombinase, or R4 integrase, but is not limited to site-specific recombinase.

In one embodiment of the present invention, the NLS may be derived from nucleoplasmin (NP).

In one embodiment of the present invention, the recombinant enzyme may be in the form of a Bxb1 integrase expression plasmid having an NP NLS at the C-terminus.

In one embodiment of the present invention, the donor is attB-gRNA(GSPT1)-attB ^mut , The recombinant enzyme is Bxb1 integrase, and the donor and recombinant enzyme can be used in a ratio of 4:1 (w/w).

In one embodiment of the present invention, the gRNA targeting the gene encoding GSPT1 may have the sequence of SEQ ID NO: 1.

In one embodiment of the present invention, the donor further includes a gene encoding Cas9, or the method may further include transducing a vector containing a gene encoding Cas9 into the recombinant human cell. .

In one embodiment of the present invention, the donor comprising a gRNA targeting the GSPT1 gene and a gene encoding Cas9 is an all-in-one CRISPR/Cas9 vector, for example, gRNA(GSPT1)-Cas9 -2A-HygR.

In one embodiment of the present invention, the vector containing the gene encoding Cas9 may be a GFP-2A-Cas9-HygR plasmid.

In one embodiment of the present invention, the human cells may be HEK293 cells.

Another aspect of the present invention is

As a method for producing a target product,

cultivating GSPT1 knockout recombinant human cells containing a gene encoding the desired product, and

A method comprising the step of recovering the desired product from the culture is provided.

In one embodiment of the present invention, the target product may be a therapeutic protein, gene therapy, cell therapy, vector, or vaccine, but is not limited thereto.

In one embodiment of the present invention, the recombinant human cells can be cultured for a longer time and produce a greater amount of the target product than parent cells in which the gene encoding GSPT1 has not been knocked out.

In one embodiment of the present invention, the recombinant human cells are HEK293 cells, and the target product may be an antibody.

In one embodiment of the present invention, the step of culturing the recombinant human cells may be performed by adherent culture or suspension culture, and may be performed by batch culture, fed-batch culture, or continuous culture.

The step of culturing the recombinant human cells may be performed in an appropriate medium, temperature, humidity, etc. for production of the target product, and may be appropriately selected by a person skilled in the art to which the present invention pertains.

Recombinant human cells in which the GSPT1 gene has been knocked out according to one embodiment of the present invention have resistance to osmotic stress and can overcome cell proliferation inhibition and apoptosis caused by osmotic stress, and therefore, therapeutic proteins such as antibodies It can be useful in the production of viral vectors or vaccines.

Figure 1 is a schematic diagram of RMCE (recombinase-mediated cassette exchange)-based CRISPR/Cas9 library generation in HEK293 cells according to one embodiment of the present invention. Using Bxb1-mediated RMCE, a gRNA library containing a gRNA flanked by the attB and attB ^mut recombination sites and a blasticidin resistance gene (BSD) was inserted into the ROSA26 locus with the attP-EF1α-TagBFP-attP ^mut landing pad. were integrated into the master cell line. Library cells were enriched by blasticidin (20 μg/mL) selection for 12 days, and TagBFP-negative cells were isolated using FACS. Knockout library cells were generated by transient transfection of a master cell line expressing Cas9, and cells expressing Cas9 were enriched by hygromycin (50 μg/mL) selection for 3 days.
Figure 2 shows coverage of the kinase and cell cycle gene pools of plasmids and cells in a knockout library constructed for screening of genes related to osmotic resistance according to an embodiment of the present invention. (A) and (B) represent normalized counts per million (CPM) per gRNA and gene, respectively. Lines and boxes represent median and interquartile range (IQR), respectively, and whiskers were 1.5X IQR.
Figure 3 shows the profile of cell proliferation during osmotic stress screening of kinase- and cell cycle-knockout library cells according to one embodiment of the present invention. (A) Viable cell concentration (kinase-knockout library cells under conditions without osmotic stress (black circles), kinase-knockout library cells under osmotic stress conditions (white circles), cells under conditions without osmotic stress. Cycle-knockout library cells (black cells), and cell cycle-knockout library cells (white triangles) under osmotic stress conditions, (B) first passage (P1), second passage (P2), third passage The specific proliferation rate ( μ ) of knockout library cells in standard medium or osmotic medium at passage (P3), and fourth passage (P4) is shown. The knockout cell library was inoculated in standard medium or high-osmolar medium and then passaged every 4 days. The osmolarity of the high osmotic pressure medium was increased by 150 mOsm/kg compared to the control group by adding NaCl.
Figure 4 shows the results of batch culture of attachment-adapted recombinant human cells according to one embodiment of the present invention. The effects of osmotic stress were compared in batch cultures of cells with knockouts of target genes and control cells without knockouts. (A), (B), and (C) represent cell proliferation, viability, and mAb productivity following culture in conditions without osmotic stress, respectively, and (D), (E), and (F) represent cell proliferation, (E), and (F) from the start of culture. Cell proliferation, viability, and mAb productivity are shown during culture under osmotic stress conditions created by adding NaCl to increase the osmolarity of the medium to 150 mOsm/kg. Non-targeting control (black circles), CDK8 (open circles), TRIM28 (black triangles), DBF4 (white triangles), GAS2L1 (black squares), GSPT1 (white squares), and ING1 (black diamonds) ). Error bars represent the standard deviation of three independent experiments.
Figure 5 shows the results of batch culture of suspension-adapted recombinant human cells according to one embodiment of the present invention. The effects of osmotic stress were compared in batch cultures of cells with knockouts of target genes and control cells without knockouts. (A), (B), and (C) show cell proliferation, viability, and mAb productivity according to culture under conditions without osmotic stress, respectively, and (D), (E), and (F) show cell proliferation, (E), and (F) under osmotic stress conditions. Cell proliferation, viability, and mAb productivity according to culture are shown. On the third day of osmotic stress conditions, the osmolarity of the medium was increased to 150 mOsm/kg by adding NaCl, as indicated by the arrow. Non-targeting control (black circles), CDK8 (open circles), GAS2L1 (black triangles), and GSPT1 (white triangles). Error bars represent the standard deviation of three independent experiments.
Figure 6 shows osmotic stress-induced apoptosis resistance in a knockout (KO) cell pool according to one embodiment of the present invention. Western blot results of apoptosis markers cleaved PARP, cleaved caspase-3, and cleaved caspase-7 are shown during batch culture of suspension-adapted cells. Cells were harvested on days 4, 5, and 6 of culture, and β-actin was used as a loading control.
Figure 7 shows the results of fed-batch culture of the knockout cell pool according to one embodiment of the present invention: (A) osmolality, (B) cell proliferation, (C) cell viability, and (D) mAb concentration. Feed medium (feed) was added at a rate of 5% v/v every day from the 3rd day of culture, as indicated by the arrow. Non-targeting control (NT-Control) (black circles), CDK8 (open circles), GAS2L1 (black triangles), and GSPT1 (white triangles). Error bars represent the standard deviation of three independent experiments.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate one or more embodiments and the scope of the present invention is not limited to these examples.

Example 1. Screening of target genes related to osmotic stress resistance by CRISPR/Cas9 library screening platform

To identify novel targets associated with osmotic stress, RMCE-based CRISPR/Cas9 library screening was applied to HEK293 cells. A schematic diagram of RMCE-based CRISPR/Cas9 library screening is shown in Figure 1. Because osmotic pressure has been reported to induce growth inhibition, cell cycle arrest, and apoptosis through a kinase-mediated signaling cascade (Burg et al., 2007, Physiol. Rev. 87, 1441-1474), osmotic stress To screen for novel targets that may confer resistance, we used two gRNA sub-pools targeting kinase and cell cycle-related genes, consisting of 507 and 983 genes, respectively. Osmotic screening was performed under hyperosmotic conditions by adding NaCl to the culture medium.

1-1. Construction of a gRNA library targeting kinase and cell cycle-related genes

gRNA sub-pools for kinases and cell cycle were provided by David Sabatini & Eric Lander (Addgene plasmid # 51044 and 51046). The gRNA sequence was amplified using Phusion High-Fidelity PCR Master Mix (Thermo Fisher Scientific) and purified from a 2% agarose gel using NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). The gRNA sequence was cloned into the attB-Esp3I-BSD plasmid (Xiong et al., 2021) treated with FastDigest Esp3I enzyme (Thermo Fisher Scientific) using Gibson Assembly Master Mix (New England Biolabs) according to the manufacturer's instructions. , attB-gRNA plasmid was constructed. The assembled gRNA library plasmids were transferred by electroporation into Endura ElectroCompetent cells (Lucigen, Middleton, WI) using a MicroPulser Electroporator (Bio-Rad, Hercules, CA) at a yield of 500 copies per gRNA for each library, and the resulting The cells obtained were incubated overnight at 37°C in LB-ampicillin medium. Afterwards, plasmid DNA was purified using NucleoBond Xtra Maxi EF Kit (Macherey-Nagel).

Analysis of gRNA library representativeness

To assess the representativeness of the library, samples were prepared by amplifying gRNA sequences from the library plasmid or genomic DNA of library cells and sequencing using NGS. Coverage of the gRNA library for the kinase and cell cycle in the library plasmid was 99.9% and 99.4%, respectively, and in library cells was 100% and 99.3%, respectively. Read counts were normalized to counts per million (CPM), and the median CPM for gRNA was 172 and 82 for kinase and cell cycle, respectively. Coverage of genes in the library plasmids and cells was 100% for both kinases and cell cycle. The median CPM for genes was 1913 and 983 for kinase and cell cycle, respectively, indicating that each library contained 10 gRNAs per gene. Therefore, the two sub-pool libraries generated using RMCE were confirmed to represent most gRNAs and genes and were used for screening. Data confirming the representativeness of gRNA are shown in Figure 2.

1-2. Construction of knockout cell libraries

A knockout cell library was constructed using the gRNA library constructed in 1-1.

To overcome the shortcomings of virus-mediated construction of CRISPR/Cas9 libraries, recombinase-mediated cassette exchange (RMCE) was used as a gRNA delivery method. It has a loxP-mCherry (loxP-EF1α-mCherry-lox2272-BGH poly(A)) landing pad at the AAVS1 locus and an attP-TagBFP (attP-EF1α-TagBFP-attP ^mut -BGH poly(A)) landing pad at the ROSA26 locus. An RMCE-based knockout platform was constructed in the dual-landing pad master cell line, HEK293 cells (Shin et al., 2021, ACS Synth. Biol. 10, 1715-1727).

Cultivation of dual-landing pad master cell lines

The dual-landing pad master cell line was grown in DMEM supplemented with 10% fetal bovine serum (HyClone, Logan, UT), non-essential amino acid solution (Sigma-Aldrich, St. Louis, MO), and GlutaMAX supplement (Thermo Fisher Scientific). (Gibco, Grand Island, NY) medium. Cells were maintained in 6-well plates (Thermo Fisher Scientific) at 37°C under 5% CO ₂ and inoculated with 0.2 x 10 ⁶ cells/mL medium every 3 days. Viable cell concentration and viability were measured on a CountessII FL automated cell counter (Invitrogen, Carlsbad, CA) using a trypan blue exclusion assay.

Additionally, the dual-landing pad master cell line was adapted to grow in serum-free EX-CELL 293 medium (Sigma-Aldrich) supplemented with 2 mM glutamine (HyClone). Afterwards, the cells were placed in a 125 mL Erlenmeyer flask (Corning, Corning, NY) containing the medium in a climo-shaking CO ₂ incubator (ISF1-X, Adolf Kuhner AG, Birsfelden, Switzerland) at 37°C, 85°C. % humidity, 5% CO ₂ , and 110 rpm conditions were maintained, and inoculated at 0.3 x 10 ⁶ cells/mL every 3 days.

Transduction by RMCE

A dual-landing pad master cell line with attP-EF1α-TagBFP-attP ^mut -BGH poly(A) at the ROSA26 locus was used for gRNA library transfection. To achieve coverage of ⁵⁰⁰ copies ^per gRNA for the kinase and cell cycle sub-pools, a total of ^1.8 Inoculated at the concentration of medium, ACS Synth. Biol. Transfection was performed using the gRNA donor plasmid (attB-Esp3I-BSD) and Bxb1 integrase at a ratio of 4:1 (w/w) calculated based on the RMCE efficiency measured as described in 10, 1715-1727 ( Figure 1). After 48 hours, cells were treated with 20 μg/mL blasticidin (Sigma-Aldrich) for 12 days. The concentration of blasticidin was determined using the death curve of the dual-landing pad master cell line. For enrichment of cells with inserted gRNA libraries, TagBFP-negative cells with gRNA cassettes were sorted by MoFlo Astrios EQ (Beckman Coulter, Brea, CA) using a 405 nm violet laser and 448/59 filter. To ensure sufficient coverage of the gRNA library, for the kinase and cell cycle sub-pools, 2.5 x 10 ⁶ and 5.0 x 10 ⁶ cells were sorted and recovered for Cas9 transfection. Cells into which the gRNA library was inserted were transfected with the GFP-2A-Cas9-HygR plasmid, and 48 hours later, they were treated with 50 μg/mL hygromycin for enrichment of cells expressing Cas9. After 3 days of selection, cells were cultured in hygromycin-free medium for 9 days. The recovered cells were used as a knockout library.

1-3. Screening under osmotic pressure conditions

Osmotic pressure affects cellular function and structure through kinase-mediated signaling pathways, resulting in cell cycle arrest and apoptosis (Burg et al., 2007, Physiol. Rev. 87, 1441-1474). To identify target genes that confer resistance to hyperosmotic stress in human cells, knockout libraries for kinases and cell cycle obtained in 1-2 were cultured in standard or hyperosmolar media. The osmotic pressure of the hyperosmolar medium was determined to be 450 mOsm/kg, where significant inhibition of cell proliferation was observed based on the growth profile of host cells in media containing various concentrations of NaCl.

Standard media (DMEM (Gibco, Grand Island) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), non-essential amino acid solution (Sigma-Aldrich, St. Louis, MO), and GlutaMAX supplement (Thermo Fisher Scientific) , NY) medium: hyperosmolar medium (455 ± 3 mOsm/kg) was prepared by adding 5 M NaCl (Sigma-Aldrich) to 304 ± 1 mOsm/kg). Specifically, for the kinase and cell cycle knockout library cells obtained in 1-2, a total of 7.5 x 10 ⁶ and 1.5 x 10 ⁷ cells, respectively, were placed in a T75 flask (Thermo Fisher Scientific) containing 15 ml standard or hyperosmolar medium. Inoculated at 0.5 x 10 ⁶ cells/mL and passaged every 4 days. The osmotic pressure of the culture medium was measured using a Fiske Micro-Osmometer (Thermo Fisher Scientific).

The growth rate of cells was measured during subculture. The results are shown in Figure 3. During passage every 4 days, at the fourth passage, the specific growth rates μ of the kinase- and cell cycle-knockout library cells in standard medium were respectively 0.493 ± 0.008 and 0.414 ± 0.005 day^-OneIt was. On the other hand, in hyperosmolar medium μ was 0.366 ± 0.019 and 0.366 ± 0.016 day, respectively.^-OneSubsequently, it was shown that cell proliferation of cells from both libraries was significantly reduced at 450 mOsm/kg. On day 16 of culture, for NGS analysis, 2.0 x 10 cells were aliquoted using Exgene Blood SV (GeneAll Biotechnology) according to the manufacturer's instructions.⁷canine kinase knockout library cells and 4.0 x 10⁷Genomic DNA samples were obtained from canine cell cycle knockout library cells.

NGS analysis

Analysis of NGS data using the adjusted robust rank aggregation (aRRA) method (Li et al., 2014, Genome Biol. 15, 554) between samples from standard or hyperosmotic media identified genes that were significantly enriched or depleted by osmotic pressure. revealed. Candidate genes for validation were selected by ranking enriched or depleted gRNAs using the aRRA method and comparing significantly enriched gRNAs in all three replicates using the SUMLFC method (Spahn et al. , 2017, Sci. Rep. 7, 15854). In the kinase knockout library, 31 genes were significantly enriched and 6 genes were significantly depleted in all three replicates. In the cell cycle knockout library, 34 genes were significantly enriched and 15 genes were significantly depleted in all three replicates. The physical and functional interactions of proteins encoded by enriched or depleted genes were analyzed using the STRING database (Szklarczyk et al., 2021, Nucleic Acids Res. 49, D605-D612). The network of candidate genes from each sub-pool library showed significantly clustered interactions and functional enrichment.

For the identification of target genes for resistance to osmotic stress, NGS data were analyzed using the SUMLFC ranking scale and identified two genes in the kinase sub-pool ( CDK8 and TRIM28 ) and four genes in the cell cycle sub-pool ( DBF4 , GAS2L1 , GSPT1 , and ING1 ) were identified as significant genes in all three replicates. Table 1 below shows the gene score for each target found to be enriched in osmotic screening using the SUMLFC method.

Gene (GenBank Accession No.) SigmaFC CDK8 (Cyclin dependent kinase 8: CH471075) 9.19 ± 2.18 TRIM28 (Tripartite motif containing 28: BC004978) 8.56 ± 2.61 DBF4 (DBF4 zinc finger: AF160876) 1.72 ± 0.65 GAS2L1 (Growth arrest specific 2 like 1: AC002059) 1.12 ± 0.05 GSPT1 (G1 to S phase transition 1: BC009503) 1.21 ± 0.10 ING1 (Inhibitor of growth family member 1: AF044076) 1.42 ± 0.48

Example 2. Effect of target genes related to osmotic stress resistance on cell proliferation and production of target products

In order to confirm the function of the six genes selected in Example 1, CDK8 , TRIM28 , DBF4 , GAS2L1 , GSPT1 , and ING1 , as target genes related to osmotic stress resistance, knockout cells of these genes were produced, and the growth rate and The effect of osmotic pressure on the productivity of the target product was investigated.

2-1. Construction of mAb-producing recombinant HEK293 (rHEK293) cells

It has a loxP-mCherry (loxP-EF1α-mCherry-lox2272-BGH poly(A)) landing pad at the AAVS1 locus used in Example 1, and attP-TagBFP (attP-EF1α-TagBFP-attP ^mut -BGH poly) at the ROSA26 locus. (A)) Recombinant HEK293 cells for mAb production were constructed from the dual-landing pad master cell line, which is HEK293 cells with landing pads (Shin et al., 2021, ACS Synth. Biol. 10, 1715-1727). Specifically, the loxP-mCherry landing pad at the AAVS1 locus of the attached dual-landing pad master cell line was used to site-specifically insert the gene encoding the mAb, rituximab (DrugBank Accession Number: DB00073) through RMCE. A cell line expressing the mAb was created by insertion using the method, and a recombinant cell line adapted to adherent culture and a recombinant cell line adapted to suspension culture were created by adapting it to suspension culture in EX-CELL 293 medium, a medium for suspension culture.

2-2. Construction of knockout cells

Attachment- and suspension-adapted, mAb-producing recombinant HEK 293 (rHEK293) cells were transfected with Lipofectamine 3000 (Invitrogen) or 293fectin (Thermo Fisher Scientific) according to the manufacturer's instructions, targeting the genes identified in 1-3. All-in-one CRISPR/Cas9 plasmid, gene-targeting gRNA-Cas9-2A-HygR plasmid and non-targeting control plasmid were transfected. Knockout efficiency was estimated by measuring the percentage of mCherry-negative cells using mCherry-M3 gRNA in the dual-landing master cell line and found to be increased to 96.8% by hygromycin selection.

Knockout cell pools were cultured in 12-well plates using standard or hyperosmolar media. After 48 h, cells were harvested by culturing in the presence of 200 μg/mL hygromycin for 3 days and for 9 days without selection pressure. The knockout efficiency of each target was measured using Tracking of Indels by Decomposition (TIDE) analysis (Brinkman et al., 2014).

2-3. Adherent batch culture

The obtained knockout cells of each gene were subjected to adherent batch culture to examine the effect on the growth rate due to osmotic pressure and the productivity of the mAb, the target product. For adherent cultures, knockout cell pools were seeded at 0.3 x 10 ⁶ cells/mL in 12-well plates containing 1 mL of standard or hyperosmolar medium and incubated at 37°C and 5% CO ₂ . Using a single well at each time point, viable cell concentration and viability were measured every two days. For titer determination, culture supernatants were harvested every 2 days and stored at -70°C.

mAb concentrations were measured using ELISA, as previously described (Hwang and Lee, 2008, Biotechnol. Bioeng. 99, 678-685). The specific productivity of mAb ( q _mAb ) was calculated as integrated viable cell concentration (IVCC) and titer.

The results are shown in Figure 4. osmotic stress (450 mOsm/kg) reduced cell growth and increased mAb production in all knockout cell pools. In standard medium, knockout of the gene did not improve cell growth or mAb production (Figure 4, A, B, and C). Knockout of TRIM28 , DBF4 , or ING1 decreased maximum viable cell concentration (MVCC) (* p < 0.05) (Figure 4A). In contrast, knockout of CDK8 , GAS2L1 , or GSPT1 in cells grown in hyperosmolar medium resulted in significant increases in MVCC, day 12 cell viability, and maximum mAb concentration compared to control cells (* p < 0.05) ( Figure 4D, E, and F). Likewise, knockout of CDK8 , GAS2L1 , or GSPT1 significantly increased μ in hyperosmolar medium. Table 2 below shows the specific proliferation rate ( μ ) and specific productivity of mAb ( q _mAb ) of each gene knockout cell in adherent batch culture.

Therefore, these three target genes ( CDK8 , GAS2L1 , and GSPT1 ) were selected for the generation of osmo-resistant, suspension-adapted rHEK293 cells to further study the effect of knocking out these genes on mAb production in suspension culture. .

2-4. Suspension batch culture

Knockout cells for each of the three target genes, CDK8 , GAS2L1 , and GSPT1 , selected based on the adherent culture results in 2-3, were produced as described in 2-2 using suspension-adapted, mAb producing rHEK293 cells.

Knockout of each target gene was confirmed by tracking of indels by decomposition (TIDE) analysis and Western blot analysis. According to TIDE analysis, the total editing efficiency of CDK8 , GAS2L1 , and GSPT1 was 82.0%, 80.2%, and 80.9%, respectively, and Western blot analysis of each target gene showed reduced protein expression in the knockout cell pool compared to control cells. showed it

To evaluate the effect of target gene knockout on mAb production in suspension culture, pools of knockout cells of the constructed target gene were cultured in 125 mL flasks in the presence or absence of osmotic stress. _Specifically , ^0.5 Cultured. To induce osmotic stress, the osmolality of the culture medium was increased to 450 mOsm/kg by adding 5M NaCl to the culture on day 3. The results are shown in Figure 5.

In cultures without osmotic stress, all three knockout cell pools maintained higher viable cell concentrations during the attenuation phase of growth compared to control cells and resulted in increased mAb production (* p < 0.05) (Figure 5) A, B, and C). The maximum mAb concentration was 37.4 - 40.6 mg/L in the knockout cell pool and 33.2 ± 0.7 mg/L in control cells (Figure 5C). Osmotic stress inhibited cell proliferation and reduced cell viability in all cell pools, including control cells, but to a lesser extent in the knockout cell pool compared to control cells (Figure 5D and E). As a result, as observed in adherent cell cultures, the maximum mAb concentration in the knockout cell pool (43.1 - 59.5 mg/L) was significantly higher than the maximum mAb concentration in control cells (36.6 ± 2.4 mg/L) (* p < 0.05) (Figure 5F).

Additionally, to confirm the anti-apoptotic effect of CDK8 -, GAS2L1 -, and GSPT1 -knockout, cells cultured under osmotic stress (Figure 5D) were collected on days 4, 5, and 6, and sectioned. The expression levels of apoptosis markers such as cleaved poly (ADP-ribose) polymerase (PARP), cleaved caspase-3, and cleaved caspase-7 were measured using anti-CDK8 rabbit antibody (Abcam, Cambridge, UK), anti- GAS2L1 rabbit antibody (Abcam), anti-GSPT1 rabbit antibody (Abcam), anti-cleaved PARP rabbit antibody (Cell Signaling Technology, Danvers, MA), anti-cleaved caspase-3 rabbit antibody (Cell Signaling Technology), anti-cleaved Analyzed by Western blot analysis using caspase-7 rabbit antibody (Cell Signaling Technology) and anti-β-actin mouse antibody (Sigma-Aldrich). The results are shown in Figure 6. In cultures exposed to osmotic stress, the expression levels of PARP, caspase-3, and cleaved forms of caspase-7 in control cells gradually increased, indicating hyperosmotic stress-induced apoptosis. However, the expression levels of apoptotic markers were reduced in all three knockout cell pools compared to control cells, confirming that CDK8 -, GAS2L1 -, or GSPT1 -knockout improved resistance to osmotic stress-induced apoptosis. .

2-5. fed-batch culture

Fed-batch culture of recombinant cells to produce a target product such as an antibody involves adding a medium containing nutrients at regular intervals, adding a base to adjust pH, and increasing the osmolarity of the culture medium due to by-products as the culture progresses. As the concentration increases, it causes hyperosmotic stress in cells, thereby slowing down the growth rate and viability of cells and causing apoptosis. In 2-3 and 2-4, respectively, knockout cells confirmed to have effects on osmotic stress-induced cell growth rate, mAb productivity, and apoptosis in adherent culture and suspension culture were applied to fed-batch culture to demonstrate resistance to osmotic stress. Confirmed.

To evaluate the effect of target gene knockout on mAb production in fed-batch culture, knockout cells were seeded in 30 mL of culture medium (serum-free EX-CELL 293 medium (Sigma-Aldrich) supplemented with 2 mM glutamine (HyClone)). ) were inoculated into a 125 _mL Erlenmeyer flask containing ^0.5 From the third day after culture, a mixture (1:1:1) of Cell Boost 2, 5, and 6 supplements (HyClone) was added to the culture medium at a ratio of 5% v/v every day. The results are shown in Figure 7.

The osmolarity of the culture medium continued to increase with daily supply of nutrients during fed-batch culture. Osmolarity at the end of fed-batch culture of CDK8 -, GAS2L1 -, and GSPT1 -knockout cell pools were 522 ± 12, 526 ± 10, and 561 ± 10 mOsm/kg, respectively (Figure 7A).

All three knockout cell pools showed increased cell growth and extended culture life compared to control cells (Figure 7B). On day 8, when the osmolarity was increased to 457 ± 5.0 mOsm/kg, the cell viability of control cells was reduced to less than 50%, but the cell viability of the knockout cell pool remained above 70% (Figure 7) C). In particular, the GSPT1 -knockout cell pool showed the highest MVCC (6.5 ± 0.2 x 10 ⁶ cells/mL) and IVCC (5.1 ± 0.2 x 10 ⁷ cells/day/mL), compared to control cells, 1.4- and 2.1-fold, respectively. -It was twice higher. As a result, the GSPT1 -knockout cell pool showed the highest mAb concentration (103.7 ± 7.2 mg/L), which was 2.3 times higher than that of the control cells (Figure 7D).

Accordingly, CDK8 -, GAS2L1 -, or GSPT1 -knockout in mAb-producing rHEK293 cells improved cell growth and culture longevity, resulting in increased mAb production in fed-batch culture, which improved against hyperosmolar stress-induced apoptosis. It was confirmed that this was due to resistance.

Among the three candidate genes, GSPT1 -knockout showed the best performance in terms of cell growth, culture life, and mAb production in fed-batch culture (Figure 7) and showed reduced caspase-3 activation in the stationary phase of culture. The GSPT1 protein binds IAPs through a conserved N-terminal IAP binding motif, thereby promoting caspase-3 activation (J. Biol. Chem. 278, 38699-38706). Therefore, the GSPT1 gene is a promising target for improving cell proliferation and productivity of rHEK293 cells in fed-batch culture, where increased osmolarity can induce apoptotic stress during mass production of therapeutic proteins.

SEQUENCE LISTING <110> Korea Advanced Institute of Science and Technology <120> Recombinant human cell having GSTP1 gene knockout human cell and use that <130> PN143322 <160> 7 <170> PatentIn version 3.2 <210> 1 <211> 23 <212> DNA <213> Artificial <220> <223> gRNA target sequence for GSPT1 <400> 1 gggagatgga aggccgccag agg 23 <210> 2 <211> 20 <212> DNA <213> Artificial <220> <223> gRNA target sequence for NT control <400> 2 gtagcgaacg tgtccggcgt 20 <210> 3 <211> 23 <212> DNA <213> Artificial <220> <223> gRNA target sequence for CDK8 <400> 3 cgaggacctg tttgaatacg agg 23 <210> 4 <211> 23 <212> DNA <213> Artificial <220> <223> gRNA target sequence for TRIM28 <400> 4 cgtgtgaatg gcggcctccg cgg 23 <210> 5 <211> 23 <212> DNA <213> Artificial <220> <223> gRNA target sequence for DBF4 <400> 5 ttataggtgt gtttaagcag agg 23 <210> 6 <211> 23 <212> DNA <213> Artificial <220> <223> gRNA target sequence for GAS2L1 <400> 6 agtggcgggc atcgcgggct cgg 23 <210> 7 <211> 23 <212> DNA <213> Artificial <220> <223> gRNA target sequence for ING1 <400> 7 tcttctcgtc gcccagctcc tgg 23

Claims (15)

Recombinant human cells in which the GSPT1 (G1 to S phase transition 1) gene has been knocked out. The recombinant human cell according to claim 1, wherein the recombinant human cell has a higher viability and growth rate under osmotic stress conditions compared to the parent cell before the GSPT1 gene is knocked out. The recombinant human cell according to claim 1, wherein the recombinant human cell has a longer culture life than the parent cell before the GSPT1 gene is knocked out. The recombinant human cell according to claim 1, wherein the recombinant human cell contains a gene encoding the desired product. The recombinant human cell of claim 4, wherein the desired product is a therapeutic protein, gene therapy, cell therapy, viral vector, or viral antigen. The recombinant human cell of claim 5, wherein the desired product is an antibody. The recombinant human cell according to claim 6, wherein the gene encoding the target product is inserted into the genome of the recombinant human cell or exists as a vector. The recombinant human cell according to claim 1, wherein the recombinant human cell is a HEK293 cell. The recombinant human cell according to claim 4, wherein the recombinant human cell is a HEK293 cell, and the target product is an antibody. A method of producing GSPT1 knockout recombinant human cells, comprising:
It includes the step of transducing human cells using a donor containing a gRNA targeting the gene encoding GSPT1 and a recombinant enzyme,
The method wherein the recombinant enzyme is tagged with an NLS (Nuclear Localization Sequence) at its end. The method of claim 10, wherein the donor further includes a gene encoding Cas9, or
The method further comprises the step of transducing a vector containing a gene encoding Cas9 into the human cell. As a method for producing a target product,
cultivating GSPT1 knockout recombinant human cells containing a gene encoding the desired product, and
A method comprising recovering the desired product from the culture. The method of claim 12, wherein the target product is a therapeutic protein, gene therapy, cell therapy, vector, or vaccine. The method of claim 12, wherein the recombinant human cells are cultured for a longer time and can produce a greater amount of the desired product than parent cells in which the gene encoding GSPT1 is not knocked out. The method of claim 12, wherein the recombinant human cells are HEK293 cells, and the target product is an antibody.