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WO2025226122A1 - Procédé d'inactivation de gène de glutamine synthétase et procédé de production de protéine cible l'utilisant - Google Patents

Procédé d'inactivation de gène de glutamine synthétase et procédé de production de protéine cible l'utilisant

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Publication number
WO2025226122A1
WO2025226122A1 PCT/KR2025/095295 KR2025095295W WO2025226122A1 WO 2025226122 A1 WO2025226122 A1 WO 2025226122A1 KR 2025095295 W KR2025095295 W KR 2025095295W WO 2025226122 A1 WO2025226122 A1 WO 2025226122A1
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zinc finger
gene
seq
cells
cell
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English (en)
Korean (ko)
Inventor
이현주
김형미
김동준
김만수
김민수
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Celltrion Inc
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Celltrion Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present invention relates to a method for inactivating a glutamine synthetase (GS) gene involved in the synthesis of glutamine, a major energy source for cell growth, using a zinc finger nuclease, a cell line in which the glutamine synthetase gene is inactivated by the method, and a method for producing a target protein using the method.
  • GS glutamine synthetase
  • CHO Chinese hamster ovary
  • CHO cell lines exhibit similar posttranslational protein modifications, such as glycosylation and phosphorylation, to human cells. They are also capable of suspension culture, allowing for relatively high-density cultivation in serum-free media. Their safety has also been proven. For these reasons, they are the most widely used cell line for industrial mass production of target proteins.
  • DHFR dihydrofolate reductase
  • MTX methotrexate
  • GS glutamine synthetase
  • MSX methionine sulfoximine
  • a vector containing a selectable marker gene (DHFR or GS) of the system along with a gene that induces expression of the target recombinant protein is transfected into a host cell line, and the cells are cultured in a medium containing an inhibitor appropriate for the selectable marker protein expressed by each selectable marker gene.
  • the selectable marker gene In order for the cells to survive in a medium containing an inhibitor of DHFR or GS, which is necessary for cell growth, the selectable marker gene must be introduced into an active region of the genome or sufficiently expressed through gene amplification. Therefore, if the selectable marker gene is sufficiently expressed to allow survival in a medium containing an inhibitor, the expression of the target recombinant protein contained in the vector will also increase, allowing the selection of a cell line with high productivity per unit cell.
  • the selection marker gene In order to effectively apply the above genetic selection system, it is necessary to suppress the influence of the selection marker gene inherent in the host cell. If the selection marker gene is already present at a high concentration in the host cell, the selection effect of the selection marker gene in the vector will be inhibited by the selection marker gene in the host cell even if an inhibitor is treated during the selection process. As a result, cells that have not introduced the target recombinant gene into their genome can survive in the medium containing the inhibitor, making it impossible to select cell lines with high productivity per unit cell. Therefore, in the case of the DHFR/MTX system, a CHO dhfr(-) cell line deficient in the selection marker gene dhfr has been developed to produce cell lines that effectively express the target protein. In the case of the GS/MSX system, a CHO cell line in which the selection marker gene endogenous glutamine synthetase has been inactivated is also required to effectively select cell lines that express the target protein.
  • Zinc-finger nuclease is an enzyme that can recognize and modify a specific DNA sequence. It is completed through the fusion of a zinc finger DNA-binding protein that can bind to a specific DNA sequence and Fok1, a restriction endonuclease derived from Flavobacterium okeanokoites . When the zinc finger DNA-binding domains that can bind to the target DNA sequence bind to the target DNA sequence, FoK1 is activated, causing double-stranded breaks (DSBs). In the presence of donor DNA, the broken sequence is repaired through homology-directed repair (HDR) or non-homologous end joining (NHEJ), and this process completes the incomplete correction of the sequence.
  • HDR homology-directed repair
  • NHEJ non-homologous end joining
  • the problem to be solved by the present invention is to provide a zinc finger DNA-binding domain that binds to a target site within exon 5 of the glutamine synthetase gene for use in partial or complete inactivation of endogenous glutamine synthetase (GS) in mammalian cells.
  • Another problem to be solved by the present invention is to provide a fusion protein comprising the zinc finger DNA-binding domain and one or more cleavage domains.
  • Another problem to be solved by the present invention is to provide a polynucleotide encoding the zinc finger DNA-binding domain.
  • Another problem to be solved by the present invention is to provide a polynucleotide encoding the fusion protein.
  • Another problem to be solved by the present invention is to provide an isolated cell comprising a polynucleotide encoding the zinc finger DNA-binding domain.
  • Another problem to be solved by the present invention is to provide an isolated cell comprising a polynucleotide encoding the fusion protein.
  • Another problem to be solved by the present invention is to provide a cell line in which glutamine synthetase (GS) is partially or completely inactivated by the fusion protein.
  • GS glutamine synthetase
  • Another problem to be solved by the present invention is to provide a method for inactivating an endogenous cellular GS gene in a cell using the fusion protein.
  • Another problem to be solved by the present invention is to provide a method for producing a target recombinant protein in a host cell using a method for inactivating the endogenous GS gene.
  • Another problem to be solved by the present invention is to provide a cell line in which the GS gene is inactivated, produced using a method for inactivating the endogenous GS gene.
  • the present invention provides a zinc finger DNA-binding domain that binds to a target site in exon 5 of a glutamine synthetase gene for use in partial or complete inactivation of endogenous glutamine synthetase (GS) in mammalian cells,
  • the target sequence of the zinc finger DNA-binding domain is SEQ ID NO: 3,
  • Zinc finger 1 comprising a recognition helix region consisting of SEQ ID NO: 4, zinc finger 2 comprising a recognition helix region consisting of SEQ ID NO: 5, zinc finger 3 comprising a recognition helix region consisting of SEQ ID NO: 6, and zinc finger 4 comprising a recognition helix region consisting of SEQ ID NO: 7, zinc finger 5 comprising a recognition helix region consisting of SEQ ID NO: 8; or
  • the target sequence of the zinc finger DNA-binding domain is SEQ ID NO: 9
  • a zinc finger DNA-binding domain comprising zinc finger 1 comprising a recognition helix region consisting of SEQ ID NO: 10, zinc finger 2 comprising a recognition helix region consisting of SEQ ID NO: 11, zinc finger 3 comprising a recognition helix region consisting of SEQ ID NO: 12, zinc finger 4 comprising a recognition helix region consisting of SEQ ID NO: 13, and zinc finger 5 comprising a recognition helix region consisting of SEQ ID NO: 14.
  • the present invention also provides a fusion protein comprising the zinc finger DNA-binding domain and one or more cleavage domains, wherein the cleavage domains may be wild-type or engineered Fok1 cleavage domains.
  • the present invention provides a polynucleotide encoding the zinc finger DNA-binding domain.
  • the present invention provides a polynucleotide encoding the fusion protein.
  • the present invention also provides an isolated cell comprising a polynucleotide encoding the zinc finger DNA-binding domain.
  • the present invention also provides an isolated cell comprising a polynucleotide encoding the fusion protein.
  • the present invention provides a cell line in which glutamine synthetase (GS) is partially or completely inactivated by the fusion protein.
  • GS glutamine synthetase
  • a first polynucleotide encoding a first polypeptide comprising (i) a zinc finger DNA-binding domain engineered to bind to a first target site in an endogenous glutamine synthetase (GS) gene, and (ii) a cleavage domain; and
  • a method for inactivating an endogenous glutamine synthetase (GS) gene in a cell comprising introducing into the cell a second polynucleotide encoding a second polypeptide comprising (i) a zinc finger DNA-binding domain engineered to bind to a second target site in an endogenous glutamine synthetase (GS) gene, and (ii) a cleavage domain, thereby causing the first polypeptide and the second polypeptide to be expressed in the cell, such that the first and second polypeptides bind to their respective target sites and cleave the GS gene;
  • the target sequence of the zinc finger DNA-binding domain is SEQ ID NO: 3,
  • a zinc finger DNA-binding domain comprising a zinc finger 1 comprising a recognition helix region consisting of SEQ ID NO: 4, a zinc finger 2 comprising a recognition helix region consisting of SEQ ID NO: 5, a zinc finger 3 comprising a recognition helix region consisting of SEQ ID NO: 6, a zinc finger 4 comprising a recognition helix region consisting of SEQ ID NO: 7, and a zinc finger 5 comprising a recognition helix region consisting of SEQ ID NO: 8,
  • the target sequence of the zinc finger DNA-binding domain is SEQ ID NO: 9
  • a method comprising a zinc finger DNA-binding domain comprising zinc finger 1 comprising a recognition helix region consisting of SEQ ID NO: 10, zinc finger 2 comprising a recognition helix region consisting of SEQ ID NO: 11, zinc finger 3 comprising a recognition helix region consisting of SEQ ID NO: 12, zinc finger 4 comprising a recognition helix region consisting of SEQ ID NO: 13, and zinc finger 5 comprising a recognition helix region consisting of SEQ ID NO: 14.
  • the present invention also provides a first polynucleotide encoding a first polypeptide comprising (i) a zinc finger DNA-binding domain engineered to bind to a first target site in an endogenous glutamine synthetase (GS) gene, and (ii) a cleavage domain.
  • GS glutamine synthetase
  • a method for inactivating an endogenous GS gene in a cell by introducing into the cell a second polynucleotide encoding a second polypeptide comprising (i) a zinc finger DNA-binding domain engineered to bind to a second target site in a GS gene, and (ii) a cleavage domain, thereby causing the first polypeptide and the second polypeptide to be expressed in the cell, thereby causing the first and second polypeptides to bind to their respective target sites.
  • a method for producing a target recombinant protein within a host cell is provided.
  • the target protein may be an antibody, but is not limited thereto.
  • a cell line in which the GS gene is partially or completely inactivated produced by a step of culturing cells under conditions suitable for producing a cell line in which the GS gene is partially or completely inactivated.
  • the cell may be a cell selected from the group consisting of CHO cells, SP2/0-Ag14 cells, HEK293 cells, COS cells, VERO cells, MDCK cells, WI38 cells, V79 cells, B14AF28-G3 cells, BHK cells, HaK cells, NS0 cells, HeLa cells, and perC6 cells, but is not limited thereto.
  • the GS gene can be effectively inactivated by inducing deletion or addition of the GS gene in a host cell through a zinc finger nuclease that selectively binds to the GS gene.
  • a zinc finger nuclease that selectively binds to the GS gene.
  • a cell line in which the GS gene is inactivated has lost the ability to synthesize glutamine, and thus, using a vector containing the GS gene as a selection marker, a cell line that highly expresses a target recombinant protein can be effectively selected using only a glutamine-deficient culture medium without the addition of an inhibitor, and a cell line in which the expression of the target protein is stably maintained can be effectively selected. Furthermore, a high-quality target protein can be produced using a GS gene-deficient cell line according to the present invention.
  • Figure 1 shows the structure of glutamine synthetase of CHO cells and the location within the 5th exon of the GS gene to which the zinc finger nuclease used in the present invention selectively binds.
  • Figures 2a to 2d are schematic diagrams of zinc finger nuclease expression vectors that selectively bind to a sequence within the 5th exon of the GS gene.
  • Figure 3 shows the results of transient transfection of CHO-K1 cells with a zinc finger nuclease expression vector that selectively binds to a sequence within the 5th exon of the GS gene, followed by T7 nuclease treatment, confirming cleavage of the 5th exon of the GS gene (see the “cleaved” symbol in Figure 3). This indicates that a sequence change was induced in the 5th exon of the GS gene.
  • Figure 4 shows the results of confirming the growth curve in a culture medium containing glutamine using cell lines deficient in the GS gene.
  • Figures 5a to 5c are schematic diagrams of an expression vector for Ixekizumab, an IL-17A inhibitor, an expression vector for Dupilumab, an IL-4 and IL-13 inhibitor, and an expression vector for Daratumumab, a CD38 inhibitor, respectively.
  • Figure 6 shows the results of confirming the short-term expression level in GS gene-deficient cell lines through transient transduction of ixekizumab.
  • Figures 7a and 7b show the results of confirming cell viability (Figure 7a) and cell growth concentration (Figure 7b) in a glutamine-free culture medium using GS gene-deficient cell lines, respectively.
  • Figure 8 shows the results of confirming the deleted or inserted gene sequence of the GS gene in GS gene-deficient cell line #9, GS gene-deficient cell line #10, and GS gene-deficient cell line #11.
  • Figures 9a to 9e show the results of confirming the difference in expression levels of genes related to lipid metabolism, lysosomal pathway, DNA repair process, cell cycle process, and oxidation-reduction reaction in GS gene-deficient cell line #9 compared to CHO-K1 host cells.
  • Figures 10a to 10e show the results of comparing the expression levels of genes related to lipid metabolism, lysosomal pathway, DNA repair process, cell cycle process, and oxidation-reduction reaction in GS gene-deficient cell line #10 with those in CHO-K1 host cells.
  • Figures 11a to 11d show the results of comparing the expression levels of genes related to lipid metabolism, lysosomal pathway, DNA repair process, and cell cycle process in GS gene-deficient cell line #11 with those in CHO-K1 host cells.
  • Figure 12 shows the results comparing the dupilumab or daratumumab productivity of dupilumab or daratumumab expressing cell lines using GS gene deficient cell line #9, GS gene deficient cell line #10, and GS gene deficient cell line #11 with CHO-K1 derived dupilumab or daratumumab expressing cell lines.
  • Figures 13a and 13b show the results of confirming the intact IgG ratio of dupilumab or daratumumab produced in the expression cell line derived from GS gene deficient cell line #9, GS gene deficient cell line #10, GS gene deficient cell line #11, and CHO-K1-derived expression cell line through the ratio of high molecular weight (HMW) and low molecular weight (LMW) separated by size exclusion chromatography.
  • HMW high molecular weight
  • LMW low molecular weight
  • Figure 14 shows the results of confirming the impurity ratio of dupilumab or daratumumab produced in expression cell lines derived from GS gene-deficient cell line #9, GS gene-deficient cell line #10, and GS gene-deficient cell line #11 and dupilumab or daratumumab produced in expression cell lines derived from CHO-K1 using high-performance capillary electrophoresis-sodium dodecyl sulfate (CE-SDS).
  • CE-SDS capillary electrophoresis-sodium dodecyl sulfate
  • Figure 15 shows the results of comparing the change in production (stability) during the passage process of dupilumab or daratumumab-expressing cell lines derived from GS gene-deficient cell line #9, GS gene-deficient cell line #10, and GS gene-deficient cell line #11 and dupilumab or daratumumab-expressing cell lines derived from CHO-K1.
  • gene inactivation refers to a specific reduction in gene expression compared to cells that have not introduced zinc finger nucleases as described herein. Thus, gene inactivation may be complete (knock-out) or partial.
  • zinc finger DNA-binding protein or binding domain refers to a domain within a larger protein or a protein that binds DNA in a sequence-specific manner via one or more zinc fingers, the structure of which is a region of amino acid sequence within the binding domain stabilized by coordination of a zinc ion.
  • the term “zinc finger DNA-binding protein” is often abbreviated as “zinc finger protein” or "ZFP”.
  • zinc finger nuclease refers to a fusion of a zinc finger DNA-binding protein that selectively binds to a target gene and a nuclease that cleaves the gene, which, when transduced into a cell, induces a sequence change in the target gene through target gene binding and cleavage, thereby inducing inactivation of a protein derived from the target gene.
  • ZFN zinc finger nuclease
  • an endogenous molecule refers to a molecule that is typically present within a specific cell at a specific developmental stage under specific environmental conditions.
  • an endogenous polynucleotide may include the genome of a chromosome, mitochondrion, chloroplast, or other organelle, or a naturally occurring episomal polynucleotide. Additional endogenous molecules may include proteins, such as transcription factors and enzymes.
  • fusion refers to a molecule having two or more moieties, preferably covalently bonded to two or more subunit molecules.
  • the subunit molecules may be of the same chemical type or may be of different chemical types.
  • Examples of fusion molecules include, but are not limited to, fusion proteins (e.g., a fusion between a ZFP DNA binding domain and a cleavage domain) and fusion polynucleotides (e.g., a polynucleotide encoding a fusion protein as described above).
  • Fusion protein in a cell can be achieved by delivery of the fusion protein into the cell or by delivery of a polynucleotide encoding the fusion protein into the cell, wherein the polynucleotide is transcribed and the transcript is translated to produce the fusion protein.
  • Trans-splicing, polypeptide cleavage, and polypeptide ligation can also be involved in expression of the protein in the cell.
  • mammal as used herein may include, but is not limited to, a human, monkey, dog, cat, rabbit, horse, pig, cow, goat, sheep, mouse, rat, or hamster.
  • the mammal may be a mammal having endogenous glutamine synthetase.
  • the zinc finger binding domain can be engineered to bind to a selected target sequence.
  • the engineered zinc finger binding domain can have novel binding specificities compared to naturally occurring zinc finger DNA-binding proteins.
  • the present invention relates to a zinc finger DNA-binding domain that binds to a target site within exon 5 of a glutamine synthetase gene for use in partial or complete inactivation of endogenous glutamine synthetase (GS) in mammalian cells,
  • the target sequence of the zinc finger DNA-binding domain is SEQ ID NO: 3,
  • Zinc finger 1 comprising a recognition helix region consisting of SEQ ID NO: 4, zinc finger 2 comprising a recognition helix region consisting of SEQ ID NO: 5, zinc finger 3 comprising a recognition helix region consisting of SEQ ID NO: 6, zinc finger 4 comprising a recognition helix region consisting of SEQ ID NO: 7, and zinc finger 5 comprising a recognition helix region consisting of SEQ ID NO: 8; or
  • the target sequence of the zinc finger DNA-binding domain is SEQ ID NO: 9
  • Zinc finger 1 comprising a recognition helix region consisting of SEQ ID NO: 10
  • zinc finger 2 comprising a recognition helix region consisting of SEQ ID NO: 11
  • zinc finger 3 comprising a recognition helix region consisting of SEQ ID NO: 12
  • zinc finger 4 comprising a recognition helix region consisting of SEQ ID NO: 13
  • zinc finger 5 comprising a recognition helix region consisting of SEQ ID NO: 14
  • a zinc finger DNA-binding domain comprising:
  • Table 1 describes two or more zinc finger DNA-binding domains engineered to bind to a target sequence within exon 5 of the GS gene.
  • 'target sequence' represents the DNA target sequence within exon 5 of the GS gene to which each zinc finger DNA-binding domain binds
  • columns 'F1 to F5' represent the amino acid sequence of the recognition region of the zinc finger within the zinc finger DNA-binding domain.
  • RSDELVR (Sequence number: 4)
  • QSSSLVR (Sequence number: 5)
  • RSDDLVR (Sequence number: 6)
  • DPGHLVR (Sequence number: 7)
  • QSGDLRR (Sequence number: 8) GCTGGGGTCAAGATT (SEQ ID NO: 9)
  • HKNALQN Sequence number: 10
  • RKDNLKN Sequence number: 11
  • DPGALVR (Sequence number: 12)
  • RSDKLVR (Sequence number: 13)
  • TSGELVR (Sequence number: 14)
  • the present invention provides a zinc finger DNA-binding protein (Exon5 L1) comprising a sequence of SEQ ID NO: 15 that selectively binds to a target sequence in the 5th exon of the GS gene of a CHO cell.
  • a zinc finger DNA-binding protein (Exon5 L1) comprising a sequence of SEQ ID NO: 15 that selectively binds to a target sequence in the 5th exon of the GS gene of a CHO cell.
  • the present invention provides a zinc finger DNA-binding protein (Exon5 R1) comprising a sequence of SEQ ID NO: 16 that selectively binds to a target sequence in the 5th exon of the GS gene of a CHO cell.
  • a zinc finger DNA-binding protein (Exon5 R1) comprising a sequence of SEQ ID NO: 16 that selectively binds to a target sequence in the 5th exon of the GS gene of a CHO cell.
  • the present invention also provides a fusion protein comprising the zinc finger DNA-binding domain and one or more cleavage domains.
  • the cleavage domains may be wild-type or engineered Fok1 cleavage domains.
  • the fusion proteins may be interchangeably referred to as zinc finger nucleases (ZFNs).
  • zinc finger nucleases SEQ ID NO: 17 (Exon5 L1-Fok1), SEQ ID NO: 18 (Exon5 R1-Fok1) are doubly bound to SEQ ID NO: 1 (cactaccgcgcctgcttgtatgctggggtcaagatt) in exon 5 of the GS gene, and then induce cleavage (double-stranded breaks, DSB) of the target sequence by the activity of FoK1.
  • the sequence of the 5th exon in the GS gene cut by the zinc finger nucleases provided as a specific example of the present invention is repaired by Non-Homologous End Joining (NHEJ), which is one of the DNA repair mechanisms in a cell.
  • NHEJ Non-Homologous End Joining
  • the zinc finger nuclease provided as a specific example of the present invention has the characteristic of selectively binding to sequence number: 1 of the 5th exon of the GS gene, thereby efficiently inducing a deficiency of the GS gene in CHO cells.
  • the present invention provides a polynucleotide encoding the zinc finger DNA-binding domain.
  • the present invention provides a polynucleotide encoding the fusion protein.
  • the present invention also provides an isolated cell comprising a polynucleotide encoding the zinc finger DNA-binding domain.
  • the present invention also provides an isolated cell comprising a polynucleotide encoding the fusion protein.
  • the present invention also provides a cell line in which glutamine synthetase (GS) is partially or completely inactivated by the fusion protein.
  • the present invention provides a CHO (Chinese hamster ovary) cell line in which GS is inactivated.
  • the cell line is characterized by a mutation in the base sequence of the 5th exon of the GS gene, and the mutation may occur due to a deletion or insertion of a base in the sequence of the 5th exon.
  • the CHO cell line in which GS is inactivated according to the present invention has high expression of Fabp4 (fatty acid binding protein 4) genes involved in lipid metabolism, high expression of Elovl7 (ELOVL fatty acid elongase 7), low expression of Ugcg (UDP-glucose ceramide glucosyltransferase), high expression of Gja1 (gap junction protein alpha 1) involved in the lysosomal pathway, high expression of Fuca1 (alpha-L-fucosidase 1), low expression of Ctsa (cathepsin A), high expression of Blm (BLM RecQ like helicase) involved in the DNA repair process, high expression of Myc (MYC proto-oncogene) involved in the DNA repair process and the cell cycle process, high expression of Aurkb (aurora kinase B) involved in the cell cycle process, and Nusap1 (nucleolar and spindle associated protein 1) It has the characteristics of
  • the CHO cell line in which GS is inactivated according to the present invention has high expression of Fabp4 (fatty acid binding protein 4) gene involved in lipid metabolism, low expression of Ugcg (UDP-glucose ceramide glucosyltransferase), high expression of Gja1 (gap junction protein alpha 1) involved in lysosomal pathway, low expression of Ctsa (cathepsin A), high expression of Blm (BLM RecQ like helicase) involved in DNA repair process, high expression of Myc (MYC proto-oncogene) involved in DNA repair process and cell cycle process, high expression of Aurkb (aurora kinase B) involved in cell cycle process, high expression of Qsox2 (quiescin sulfhydryl oxidase 2) involved in oxidation-reduction reaction, low expression of P3h3 (prolyl 3-hydroxylase 3), and/or It is characterized by, but not limited to, low expression of Hsd
  • the CHO cell line in which GS is inactivated according to the present invention has low expression of Elovl7 (Elongation of Very Long Chain Fatty Acids Protein 7), a gene involved in lipid metabolism, low expression of Ugcg (UDP-glucose ceramide glucosyltransferase), high expression of Gja1 (gap junction protein alpha 1) involved in the lysosomal pathway, low expression of Sgsh (N-sulfoglucosamine sulfohydrolase), low expression of Lamp2 (Lysosomal Associated Membrane Protein 2), low expression of Top2a (Topoisomerase II) involved in the DNA repair process, low expression of Gnl1 (Guanine nucleotide-binding protein-like 1), low expression of Myc (MYC proto-oncogene) involved in the DNA repair process and the cell cycle process, and high expression of Mcm5 (Minichromosome maintenance complex component 5) involved in the cell cycle process.
  • a first polynucleotide encoding a first polypeptide comprising (i) a zinc finger DNA-binding domain engineered to bind to a first target site in an endogenous glutamine synthetase (GS) gene, and (ii) a cleavage domain; and
  • a method for inactivating an endogenous glutamine synthetase (GS) gene in a cell comprising introducing into the cell a second polynucleotide encoding a second polypeptide comprising (i) a zinc finger DNA-binding domain engineered to bind to a second target site in an endogenous glutamine synthetase (GS) gene, and (ii) a cleavage domain, thereby causing the first polypeptide and the second polypeptide to be expressed in the cell, such that the first and second polypeptides bind to their respective target sites and cleave the GS gene;
  • the target sequence of the zinc finger DNA-binding domain is SEQ ID NO: 3,
  • a zinc finger DNA-binding domain comprising a zinc finger 1 comprising a recognition helix region consisting of SEQ ID NO: 4, a zinc finger 2 comprising a recognition helix region consisting of SEQ ID NO: 5, a zinc finger 3 comprising a recognition helix region consisting of SEQ ID NO: 6, a zinc finger 4 comprising a recognition helix region consisting of SEQ ID NO: 7, and a zinc finger 5 comprising a recognition helix region consisting of SEQ ID NO: 8,
  • a method comprising a zinc finger DNA-binding domain comprising a zinc finger 1 comprising a recognition helix region consisting of SEQ ID NO: 9, SEQ ID NO: 10, zinc finger 2 comprising a recognition helix region consisting of SEQ ID NO: 11, zinc finger 3 comprising a recognition helix region consisting of SEQ ID NO: 12, zinc finger 4 comprising a recognition helix region consisting of SEQ ID NO: 13, and zinc finger 5 comprising a recognition helix region consisting of SEQ ID NO: 14, wherein the target sequence of the zinc finger DNA-binding domain is SEQ ID NO: 9;
  • a method for producing a target recombinant protein within a host cell is provided.
  • the target protein may be an antibody, and the antibody may be, but is not limited to, Ixekizumab, Dupilumab, or Daratumumab.
  • a cell line in which the GS gene is inactivated has glutamine dependence, and thus, selection of a cell line expressing a target protein can be facilitated using a target protein expression vector including a glutamine selection marker in a glutamine-deficient culture medium.
  • a cell line in which the GS gene is partially or completely inactivated produced by a step of culturing cells under conditions suitable for producing a cell line in which the GS gene is partially or completely inactivated.
  • the cell may be a cell selected from the group consisting of CHO cells, SP2/0-Ag14 cells, HEK293 cells, COS cells, VERO cells, MDCK cells, WI38 cells, V79 cells, B14AF28-G3 cells, BHK cells, HaK cells, NS0 cells, HeLa cells, and perC6 cells, but is not limited thereto.
  • Example 1 Design of zinc finger nuclease sequences that induce GS gene deficiency.
  • CHO-K1 cell line ATCC, CCL-62 was first cultured in suspension in SFM4CHO medium (Hyclone, SH30549.02).
  • the sequences were designed based on the binding affinity of each zinc finger provided by Barbas Lab (http:/zincfingertools.org) to selectively bind to the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 located within the 5th exon of the GS gene.
  • Gene synthesis was performed using GeneArt® to fuse NLS (nuclear localization signal) and nuclease (Fok1) to the four designed zinc finger DNA-binding proteins Exon5 L1, Exon5 R1, Exon5 L2, and Exon5 R2, and the DNA sequences were also optimized to induce high expression in CHO cells.
  • Expression vectors for Exon5 L1 zinc finger nuclease designed to bind to SEQ ID NO: 1 expression vectors for Exon5 R1 zinc finger nuclease, and expression vectors for Exon5 L2 zinc finger nuclease designed to bind to SEQ ID NO: 2, and expression vectors for Exon5 R2 zinc finger nuclease were mixed respectively and transfected into CHO-K1 cells in suspension culture using the cationic polymer Lipofectamine TM LTX (Invitrogen, 15338-100) according to the manufacturer's instructions.
  • cationic polymer Lipofectamine TM LTX Invitrogen, 15338-100
  • Zinc finger nucleases expressed by the transfected vectors selectively bound to the 5th exon, then cleavage of the target sequence by the duplexed nuclease, and subsequent induced sequence changes in the 5th exon of the GS gene during the repair process were confirmed through T7 endonuclease assay (Fig. 3).
  • the sequences SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, which have excellent sequence change induction rates within the GS gene, were selected as the final zinc finger sequences (Table 1).
  • Table 2 shows the sequences of zinc finger DNA-binding proteins that selectively bind to the target sequence within the 5th exon of the GS gene of CHO cells selected in the present invention. In Table 2, the zinc finger sequences that bind to the target sequence within the 5th exon of the GS gene are underlined.
  • Table 3 shows the target sequences in the 5th exon of the GS gene to which the zinc finger pair Exon5 L1 and Exon5 R1 described in Table 2 above bind, and the recognition region sequences of the zinc finger DNA-binding domains that bind to each triplet subsite of the target sequence.
  • Zinc fingers Exon5 L1 and Exon5 R1 each contain five zinc finger DNA-binding domains.
  • T7 endonuclease cleaves DNA strands when they are not complementary. Therefore, when a zinc finger nuclease induces a sequence change within a DNA strand, cleavage occurs. By confirming this result, the function of the zinc finger nuclease can be assessed.
  • Example 2 Establishment of a CHO-K1 cell line deficient in the GS gene.
  • CHO-K1 cell line (ATCC, CCL-62) was first suspended and cultured in EX-CELL Fusion medium (SIGMA, 14365C).
  • the vectors expressing the zinc finger nuclease of SEQ ID NO: 17 and the zinc finger nuclease of SEQ ID NO: 18 were transduced into CHO-K1 cells in suspension culture using the cationic polymer Lipofectamine TM LTX (Invitrogen, 15338-100) according to the manufacturer's instructions.
  • CHO-K1 cells were cultured in 96-well plates using EX-CELL Fusion medium (SIGMA, 14365C) supplemented with 6 mM glutamine for 3 weeks to obtain growing cell lines.
  • EX-CELL Fusion medium SIGMA, 14365C
  • the secured cell lines were cultured in 96-well plates using glutamine-supplemented and glutamine-deficient EX-CELL Fusion media, respectively. Cell growth was observed to initially select cell lines that did not grow in the glutamine-deficient EX-CELL Fusion media. Afterwards, the initially selected cell lines were gradually expanded to 24-well plates, 12-well plates, and 6-well plates and cultured. Each well was cultured additionally in the glutamine-deficient EX-CELL Fusion media to continuously select cell lines that did not grow in the glutamine-deficient media. The cell lines selected up to the 6-well plate stage were finally selected through cell growth rate and T7 endonuclease assay, and these cell lines were cultured by isolating 1 cell at a time into 96-well plates using a single cell dispenser.
  • Example 3 Confirmation of growth potential of CHO-K1 cell line deficient in GS gene
  • Example 2 14 cell lines confirmed to have GS gene deficiency were cultured in suspension in EX-CELL CD CHO Fusion medium supplemented with 6 mM glutamine in a 125 mL shaker flask at 120 rpm, with passage every 3-4 days.
  • Doubling time indicates the growth rate of cells, and when creating cell lines for production, doubling time and maximum cell number can be used to predict the ease of selection and productivity.
  • the results in Figure 4 confirmed the ability to create GS gene-deficient CHO host cells with various doubling times and maximum cell numbers.
  • Example 4 Ixekizumab, dupilumab, and Construction of a daratumumab expression vector
  • the heavy and light chain genes of ixekizumab, dupilumab, and daratumumab were cloned into the MarEx vector (Korean Patent No. 10-1076602, hereinafter referred to as pCT) containing glutamine synthetase as a selection marker into the Nhe/PmeI and HpaI/ClaI sites, respectively, to complete the ixekizumab expression vector pCT562 (Fig. 5a), the dupilumab expression vector pCT586 (Fig.
  • Example 5 Short-term production of target protein using a CHO cell line deficient in the GS gene.
  • Ixekizumab was produced by transient transfection of GS gene-deficient CHO cell lines using polyethylenimine (PEI) with the pCT562 vector that induces the expression of ixekizumab, and the amount of ixekizumab produced in each cell line was compared.
  • GS gene-deficient CHO cell lines transfected with the ixekizumab expression vector were cultured in an incubator at 30–34°C and 5–8% CO2 for 1–3 weeks to induce ixekizumab production, and the amount of ixekizumab production was confirmed using the protein A assay of Octet Qke (Forte Biosciences, Inc).
  • Example 6 Measurement of glutamine dependence in CHO-K1 cell lines deficient in the GS gene.
  • CHO-K1 cell lines were used as a control, and 0.2 x 10 6 cells/mL were inoculated into medium environments containing 6 mM glutamine and medium environments without glutamine, and suspension cultures were performed. On the 4th day, the concentration and viability of living cells were determined.
  • Example 7 Confirmation of useful gene expression in a GS gene-deficient CHO cell line.
  • FIG. 8 shows the results of confirming the deleted or inserted gene sequence of the GS gene in GS gene-deficient cell line #9, GS gene-deficient cell line #10, and GS gene-deficient CHO cell line #11.
  • RNA was isolated from CHO-K1 and GS gene-deficient CHO cell lines #9, #10, and #11, and residual DNA was removed using DNase. Afterwards, mRNA was purified using an mRNA purification kit for library construction and randomly fragmented for sequence confirmation. The finely fragmented RNA fragments were reverse-transcribed to produce cDNA, and different adapters were ligated to both ends. After PCR amplification to an amount that could be sequenced, the sequence information of all mRNAs of 200-400 bp was obtained through a size selection process using an Illumina Sequencer.
  • transcript assembly was performed using the StringTie program, and the expression profile was extracted using the TPM (Transcripts Per Kilobase Million) value, which is a normalization value considering the read count, transcript length, and depth of coverage, obtained through transcript quantification of each sample. Then, the expression levels of useful genes (Fabp4, Elovl7, Ugcg, Gja1, Sgsh, Lamp2, Myc, Top2a, Gnl1, Mcm5, Fuca1, Ctsa, Blm, Aurkb, Nusap1, Qsox2, P3h3, Hsd3b7, and Sc5d) were confirmed.
  • TPM Transcripts Per Kilobase Million
  • Example 8 Production of dupilumab or daratumumab-expressing cell lines using GS gene-deficient CHO cell lines
  • the expression level of dupilumab or daratumumab antibodies was confirmed, and among these, the cell lines with high antibody expression were cultured stepwise from 24-well plate, 12-well plate, 6-well plate, and 125 ml shake flask to secure a high-expression preliminary cell line (p-clone). After that, the secured high-expression preliminary cell line with antibody was cultured by isolating 1 cell per 96-well plate using a single cell dispenser, and the image results of each well were confirmed to select a single-cell-derived cell line.
  • the cells were sequentially cultured in 24-well plates, 12-well plates, 6-well plates, and 125 ml shake flasks, and high-expressing cell lines were selected based on the antibody expression levels at each stage, thereby producing CHO-K1 cell lines expressing dupilumab or daratumumab derived from CHO-K1 host cells, CHO cell line #9 expressing dupilumab or daratumumab derived from GS gene-deficient CHO cell line #9, CHO cell line #10 expressing dupilumab or daratumumab derived from GS gene-deficient CHO cell line #11 expressing dupilumab or daratumumab.
  • Example 9 Confirmation of dupilumab or daratumumab productivity in GS gene-deficient CHO cell lines.
  • a culture solution was produced using the CHO-K1-derived cell line expressing dupilumab or daratumumab antibody produced in Example 8 above, the cell line derived from GS gene-deficient CHO cell line #9 expressing dupilumab or daratumumab antibody, the cell line derived from GS gene-deficient CHO cell line #10, and the cell line derived from GS gene-deficient CHO cell line #11.
  • cultures containing antibodies were produced by culturing for 7 to 9 days in an incubator at 37°C and 5% CO2 using SFM4CHO medium (HyClone, SH30549.02) or EX-CELL® CD CHO Fusion medium (MERCK, 14365C), or SFM4CHO medium
  • the cells were cultured in an incubator at 37°C, 5% CO2 using EX-CELL® Advanced TM CHO Fed-batch medium (MERCK, 24366C), and cell boost 7a/b (HyClone TM , SH31026/SH31120) or EX-CELL® Advanced TM Feed 1 (Sigma, 24368) and glucose were added on days 3, 5, 7, 9, and 11 of culture, respectively, to produce a culture solution containing antibodies for 14 days.
  • SFM4CHO medium HyClone, SH30549.02
  • EX-CELL® CD CHO Fusion medium MERCK, 14365C
  • SFM4CHO medium the cells were cultured in an incubator at 37°C, 5% CO2
  • dupilumab or daratumumab antibody-expressing cell lines derived from CHO-K1 cell lines and GS gene-deficient CHO cell line #9, GS gene-deficient CHO cell line #10, and GS gene-deficient CHO cell line #11 was confirmed using protein A analysis using Octet Qke (Forte Biosciences, Inc) equipment.
  • Example 10 Dupilumab produced in GS gene-deficient CHO cell lines or confirmation of the physical properties of daratumumab
  • the culture solutions containing the dupilumab or daratumumab antibodies produced in Example 9 were purified by protein A chromatography to obtain dupilumab or daratumumab antibodies. Thereafter, the purified antibodies were separated by size using a size exclusion chromatography analysis method using a porous gel, and the ratio of high and low molecular weight antibodies was confirmed.
  • dupilumab or daratumumab produced from expression cell lines derived from GS gene-deficient CHO cell lines had a higher proportion of intact antibodies than dupilumab or daratumumab produced from expression cell lines derived from CHO-K1.
  • dupilumab or daratumumab was confirmed by electrophoresis of the antibody purified by protein A chromatography onto microtubules containing SDS using CE-SDS (Capillary Electrophoresis Sodium Dodecyl Sulfate) analysis, and then size-separated dupilumab or daratumumab was confirmed.
  • CE-SDS Capillary Electrophoresis Sodium Dodecyl Sulfate
  • dupilumab or daratumumab produced from expression cell lines derived from GS gene-deficient CHO cell lines had a purity level equivalent to that of dupilumab or daratumumab derived from CHO-K1. Based on these results, it was confirmed that a high-quality target protein can be obtained when producing a target protein using the GS gene-deficient CHO cell line provided in this patent.
  • Example 11 Expression stability test using a GS gene-deficient CHO cell line
  • SFM4CHO medium HyClone, SH30549.02
  • EX-CELL Fusion medium MERCK, 14365C
  • dupilumab or daratumumab was confirmed using cell lines from the 5th, 15th, and 25th passages during a total of 30 passages.
  • the productivity of dupilumab or daratumumab antibody-expressing cell lines derived from CHO-K1-derived cell lines and GS gene-deficient CHO cell line #9, GS gene-deficient CHO cell line #10, and GS gene-deficient CHO cell line #11 at the 15th and 25th passages was compared with the productivity at the 5th passage, thereby measuring the maintenance ratio of productivity according to passage and confirming production stability.
  • dupilumab or daratumumab expressing cell lines derived from GS gene-deficient CHO cell lines had higher production stability than the dupilumab or daratumumab producing cell lines derived from CHO-K1.

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Abstract

La présente invention concerne : un procédé d'inactivation d'un gène de glutamine synthétase (GS) impliqué dans la synthèse de glutamine, qui est une source d'énergie majeure de croissance cellulaire, au moyen d'une nucléase à doigt de zinc; une lignée cellulaire dans laquelle un gène GS est inactivé par le procédé; et un procédé de production d'une protéine cible au moyen du procédé. Selon la présente invention, la délétion ou l'addition d'un gène GS d'une cellule hôte est induite par l'intermédiaire d'une nucléase à doigt de zinc, qui se lie sélectivement au gène GS, ce qui permet au gène GS d'être efficacement inactivé. Selon la présente invention, étant donné qu'une lignée cellulaire ayant un gène GS inactivé perd la capacité de synthèse de glutamine, une lignée cellulaire dans laquelle une protéine recombinante cible est hautement exprimée peut être efficacement sélectionnée, simplement par l'intermédiaire d'un milieu de culture déficient en glutamine, sans ajout d'un inhibiteur, au moyen d'un vecteur comprenant un gène GS comme marqueur de sélection, et une lignée cellulaire dans laquelle l'expression d'une protéine cible est maintenue de manière stable peut être efficacement sélectionnée. De plus, selon la présente invention, une protéine cible de haute qualité peut être produite au moyen de la lignée cellulaire déficiente en gène GS.
PCT/KR2025/095295 2024-04-25 2025-04-25 Procédé d'inactivation de gène de glutamine synthétase et procédé de production de protéine cible l'utilisant Pending WO2025226122A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6140081A (en) * 1998-10-16 2000-10-31 The Scripps Research Institute Zinc finger binding domains for GNN
KR101673566B1 (ko) * 2008-10-29 2016-11-07 상가모 바이오사이언스 인코포레이티드 글루타민 신테타제 유전자 발현을 불활성화시키기 위한 방법 및 조성물
US20190352631A1 (en) * 2016-11-16 2019-11-21 Agency For Science Technology And Research Attenuated Glutamine Synthetase as a Selection Marker
WO2023180398A1 (fr) * 2022-03-23 2023-09-28 Boehringer Ingelheim International Gmbh Glutamine synthétase bactérienne en tant que marqueur de sélection dans des cellules de mammifère
WO2023180374A1 (fr) * 2022-03-23 2023-09-28 Boehringer Ingelheim International Gmbh Nouveaux variants de glutamine synthétase en tant que marqueur de sélection
KR20230147644A (ko) * 2021-02-04 2023-10-23 뉴욕 유니버시티 천연 기능을 징발하기 위한 내인성 전사 인자에 조작된 징크 핑거의 원활한 통합

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6140081A (en) * 1998-10-16 2000-10-31 The Scripps Research Institute Zinc finger binding domains for GNN
KR101673566B1 (ko) * 2008-10-29 2016-11-07 상가모 바이오사이언스 인코포레이티드 글루타민 신테타제 유전자 발현을 불활성화시키기 위한 방법 및 조성물
US20190352631A1 (en) * 2016-11-16 2019-11-21 Agency For Science Technology And Research Attenuated Glutamine Synthetase as a Selection Marker
KR20230147644A (ko) * 2021-02-04 2023-10-23 뉴욕 유니버시티 천연 기능을 징발하기 위한 내인성 전사 인자에 조작된 징크 핑거의 원활한 통합
WO2023180398A1 (fr) * 2022-03-23 2023-09-28 Boehringer Ingelheim International Gmbh Glutamine synthétase bactérienne en tant que marqueur de sélection dans des cellules de mammifère
WO2023180374A1 (fr) * 2022-03-23 2023-09-28 Boehringer Ingelheim International Gmbh Nouveaux variants de glutamine synthétase en tant que marqueur de sélection

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