RU2820830C2 - Cell line containing novel selection marker and use thereof for production of proteins - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a mammalian cell line containing an endogenous dehydroorotate dehydrogenase (DHODH) gene which is partially or completely inactivated and an expression vector suitable for producing a recombinant protein, where the expression vector comprises a sequence coding DHODH, as well as an expression system comprising a mammalian cell line comprising an endogenous dehydroorotate dehydrogenase (DHODH) gene which is partially or completely inactivated, and an expression vector containing a nucleotide sequence coding DHODH of a mammal, and at least one expression cassette. Also disclosed is a kit containing said cell line or expression system, and a culture medium without uridine.

EFFECT: invention is effective for production of recombinant protein in vitro.

17 cl, 8 dwg, 6 ex

Description

Technical field

The present invention relates to cell lines and selection markers (selectable markers) for the production of proteins.

Prerequisites for creating the invention

The production of recombinant proteins on an industrial scale requires the isolation of clones that produce large quantities of recombinant proteins. Introducing heterologous genes into animal host cells and screening for expression of the added genes is a lengthy and complex process. The process involves transfection and selection of clones with stable long-term expression, as well as screening for clones with high levels of expression of the corresponding recombinant protein.

When generating clones expressing recombinant protein from expression vectors, host cells are typically transfected with a DNA vector encoding both the protein of interest and the selectable marker in the same vector. Thus, such an expression vector contains a selectable marker allowing the selection of clones in which the expression vector is present. Such a selectable marker can also lead to coamplification of transfected DNA, which allows the isolation of recombinant protein producing clones at a high level.

Most selectable markers are either a protein that confers resistance to an antibiotic or other toxic substance, or a protein essential for cell survival. Several such selectable markers are known in the art, including, for example, G418, hygromycin, puromycin, zeomycin, dihydrofolate reductase (DHFR), glutamine synthetase (GS), and hypoxanthine guanine phosphoribosyltransferase (HPRT). In particular, GS is widely used as a selectable marker in the field of industrial production of recombinant proteins in eukaryotic cells. The GS gene allows the synthesis of glutamine, necessary for cell growth, and is inhibited by MSX (L-methionine sulfoximine). In the presence of MSX, only cells expressing more GS survive. After appropriate screening, cells that produce exogenous proteins can be selected.

In the previous application WO2016/062837, the present inventors developed an expression system based on the use of dehydroorotate dehydrogenase (DHODH) as a selectable marker. DHODH is an enzyme required for the synthesis of pyrimidine. Therefore, compounds that inhibit DHODH inhibit DNA synthesis and therefore cell proliferation. Thus, this selection marker includes an expression vector encoding DHODH used in combination with a DHODH inhibitor such as leflunomide and teriflunomide.

However, most inhibitors used with the above selection markers are toxic. In the case of the selection marker DHODH, teriflunomide is, for example, a potent immunosuppressant, and treatment with it, especially on a large scale, can be problematic for safety reasons. In the case of the GS selection marker, MSX causes seizures at high doses and thus may also cause problems when treated with it. In the case of the DHFR selection marker, methotrexate is known to exhibit hematopoietic and digestive toxicity, which also causes problems when treated with it.

Accordingly, there is a need for expression systems in which selection of a clone producing a protein of interest can be accomplished without the addition of a compound that is difficult to process.

The present invention satisfies this need.

Summary of the Present Invention

The present invention results from the development by the present inventors of a cell line in which cells producing a protein of interest can be selected in a uridine-depleted environment due to partial or complete inactivation of the DHODH gene in the cell line. This cell line, in which the DHODH gene is partially or completely inactivated, is grown typically in medium supplemented with uridine but transfected with an expression vector containing a nucleotide sequence encoding mammalian DHODH, in particular encoding mutated mammalian DHODH, and an expression cassette for expression of the protein of interest, the culture medium is typically replaced with culture medium lacking uridine, thereby selecting for cells producing the protein of interest.

This expression system is particularly advantageous because, by avoiding the use of inhibitors as selection pressure, it increases the viability of the producing cells. The present inventors have further demonstrated that this reduction in toxicity is associated with high productivity.

Thus, the present invention relates to a cell line containing an endogenous dehydroorotate dehydrogenase (DHODH) gene that is partially or completely inactivated.

In a specific embodiment, said cell line is a Chinese hamster ovary (CHO) cell line.

In a more specific embodiment, the cell line is obtained by

a) inactivating the endogenous DHODH gene in the cell, in particular using a gene editing method such as a method using the CRISPR-Cas9 system, and

b) culturing the cell in a culture medium containing uridine under conditions suitable for creating a cell line in which the endogenous DHODH gene is partially or completely inactivated.

In a specific embodiment, all alleles of the endogenous DHODH gene of the specified cell line are partially or completely inactivated.

In a further embodiment, said cell line further comprises an expression vector comprising a nucleotide sequence encoding exogenous mammalian DHODH and at least one expression cassette for expressing a recombinant protein, wherein said exogenous DHODH comprises a sequence that is at least 60% identical. sequence SEQ ID NO:2 or sequence SEQ ID NO:4.

In a particular embodiment, said nucleotide sequence includes the sequence of SEQ ID NO: 1 or the sequence of SEQ ID NO:3.

In another specific embodiment, said recombinant protein is a monoclonal antibody.

In yet another specific embodiment, the vector comprises a first expression cassette suitable for cloning an antibody light chain and a second expression cassette suitable for cloning an antibody heavy chain.

Another object of the present invention is an expression system comprising:

(i) a cell line containing an endogenous dehydroorotate dehydrogenase (DHODH) gene that is partially or completely inactivated as defined above, and

(ii) an expression vector comprising a nucleotide sequence encoding exogenous mammalian DHODH and at least one expression cassette for expressing a recombinant protein, wherein said exogenous DHODH comprises a sequence that is at least 60% identical to the sequence of SEQ ID NO:2 or the sequence of SEQ ID NO:2 ID NO:4.

In a specific embodiment, said nucleotide sequence includes the sequence of SEQ ID NO:1 or the sequence of SEQ ID NO:3.

In another specific embodiment, said recombinant protein is a monoclonal antibody.

In yet another specific embodiment, the vector comprises a first expression cassette suitable for cloning an antibody light chain and a second expression cassette suitable for cloning an antibody heavy chain.

The present invention further relates to (i) a cell line as defined above or an expression system as defined above, and (ii) a culture medium lacking uridine.

Another aspect of the present invention relates to a method for producing a recombinant protein in vitro, comprising the steps of:

A a1) providing a cell line as defined above, further comprising an expression vector containing a nucleotide sequence encoding exogenous mammalian DHODH, and at least one expression cassette for expression of a recombinant protein, wherein said exogenous DHODH includes a sequence identical to at least at least 60% of the sequence of SEQ ID NO:2 or the sequence of SEQ ID NO:4;

or

a2) providing the cell line defined above, and

a2') introducing the expression vector defined above into the cell line provided in step a2);

or

a3) providing a cell line containing the endogenous DHODH gene,

a3') partial or complete inactivation of the endogenous DHODH gene in the cell line provided in step a3), and

a3") introducing the expression vector defined above into a cell line containing partially or completely inactivated endogenous DHODH gene obtained in step a3');

B) culturing said cell line under conditions suitable for production of the recombinant protein; And

C) isolating and/or purifying said recombinant protein.

In a specific embodiment, step B) of said method is carried out in a culture medium lacking uridine.

In another specific embodiment, said method further includes step D) of forming said recombinant protein into a pharmaceutical composition.

The present invention further relates to the use of a cell line as defined above, an expression system as defined above, or a kit as defined above for the production of a recombinant protein.

In a specific embodiment, the cell line, expression system or kit is used in combination with a culture medium lacking uridine.

Brief description of drawings

In fig. Figure 1 shows the genomic structure of the human DHODH gene, registered under Gene ID: 100756632, available December 21, 2018 in NCBI Genbank.

In fig. Figure 2 shows the alignment of DHODH exon 2 sequence #1. PAM: protospacer-adjacent motif (TGG) sequence.

In fig. Figure 3 shows the screening of various DHODH KO clones for antibody production in the presence of various concentrations of teriflunomide as a selection agent.

In fig. Figure 4 shows the amount of protein produced in mg/ml using different DHODH variants as selection markers.

In fig. 5 shows lipase production at day 14 using human DHODH G202A or human GS as the selectable marker and DHODH KO or wild type CHO cells.

In fig. 6 shows the production of monoclonal antibodies, mAb-B, on day 14 using human DHODH G202A or human GS as the selectable marker and DHODH KO or wild-type CHO cells.

In fig. 7 shows bispecific antibody production at day 14 using human DHODH G202A and/or human GS as a selectable marker and DHODH KO or wild type CHO cells.

In fig. 8 shows trispecific antibody production at day 14 using human DHODH G202A and human GS as selectable markers and DHODH KO or wild type CHO cells.

Detailed Description of the Present Invention

Dihydroorotate dehydrogenase

As used herein, the term "dihydroorotate dehydrogenase" or "DHODH" refers to a polypeptide capable of catalyzing the conversion of dihydroorotate (4,5-dihydroorotic acid or 2,6-dioxo-1,3-diazinan-4-carboxylic acid) to orotate (orotic acid or 1 ,2,3,6-tetrahydro-2,6-dioxo-4-pyrimidinecarboxylic acid), as represented by the following reaction:

(S)-dihydroorotate+O ₂ ↔ orotate+H ₂ O ₂

This polypeptide is classified under Enzyme Commission (EC) number 1.3.3.1. Polypeptides capable of catalyzing the above reaction exhibit "DHODH activity".

The above reaction is the fourth step in the de novo synthesis of uridine monophosphate (rUMP), required for DNA and RNA synthesis. Thus, inhibition or inactivation of DHODH has the effect of inhibiting DNA and RNA synthesis and therefore inhibiting cell proliferation.

Cell line

The present invention relates to a cell line containing an endogenous dehydroorotate dehydrogenase (DHODH) gene that is partially or completely inactivated.

The cell line is a eukaryotic cell line, for example a mammalian cell line such as a Chinese hamster ovary (CHO) cell line, a monkey cell line or a human cell line.

In a specific embodiment, the cell line is a CHO cell line.

CHO cell lines are commonly used for industrial protein production, and many CHO cell lines are known to those skilled in the art. For example, such CHO cell lines include those that are publicly available from the American Type Culture Collection, such as the CHO-K1 cell line (ATCC number: CCL-61), the CHO-S cell line (sold, for example, by Invitrogen and Gibco), the CHO DP-12 cells (ATCC number: CRL-12444 and 12445) and CHO cell line 1-15 (ATCC number: CRL-9606). Another cell line suitable for industrial protein production is the CHO 9E4 cell line. The 9E4 cell line was derived from a clone of the CHO-K1 cell line through a single cell progeny cloning process. The development of the 9E4 cell line is presented in more detail in Example 1. The CHO-K1 cell line was obtained by Puck in 1957 and deposited in the ATCC under the number CCL-61.

Human cells such as HEK293 cells (ATCC number: CRL-1573), HKB11 (ATCC number: CRL-12568), PER-C6 (Crucell), HT1080 (ATCC number: CRL-121), Jurkat , Daudi, Raji and CAP (ATCC number: CRL-1098), can also be used for protein production to obtain a naturally occurring glycosylation profile of recombinant human proteins.

In one embodiment, the cell line is capable of growing in a serum-free medium (eg, a chemically defined medium) and/or in suspension. Such a cell line can be readily generated by those skilled in the art by adapting the parent cell line to grow in serum-free medium and/or suspension (e.g., by cloning single cell progeny, by gradual adaptation, and/or by a starvation and salvation").

The cell line of the present invention is a cell line containing an endogenous dehydroorotate dehydrogenase (DHODH) gene that is partially or completely inactivated.

By “endogenous DHODH gene” herein is meant a DHODH gene typically present in a specified specific cell at a specific stage of development under certain environmental conditions.

An "endogenous DHODH gene" differs from an "exogenous DHODH gene" defined below in that said exogenous DHODH gene is provided by an expression vector defined below, which may be present in a cell line of the present invention if said expression vector has been introduced into said cell line .

As will be appreciated by one skilled in the art, the endogenous DHODH gene will depend on the cell line. For example, in the CHO cell line, the endogenous DHODH gene is the Chinese hamster DHODH gene; in a human cell line, the endogenous DHODH gene is a human DHODH gene.

In general, wild-type Chinese hamster DHODH refers to the sequence comprising or consisting of SEQ ID NO:2, as well as variants thereof exhibiting DHODH activity. Such variants may, for example, correspond to variants that occur naturally in hamster species (such as allelic variants or splice variants).

Generally, wild-type human DHODH refers to the sequence comprising or consisting of SEQ ID NO:4, as well as variants thereof exhibiting DHODH activity. Such variants may, for example, correspond to variants that occur naturally in the human species (eg, allelic variants or splice variants).

As used herein, the term “gene” includes the region of DNA encoding the gene product, as well as all regions of DNA that regulate the production of the gene product, whether such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, the gene includes promoter sequences, terminators, translation-regulating sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites and locus control regions (long-range regulatory regions) .

"Inactivation" of a gene refers to any decrease in gene expression compared to the corresponding wild-type cell. Inactivation of a gene can be complete (complete inactivation or knockout) or partial (for example, a hypomorphic gene (mutant gene) whose expression level is lower than normal, or the product of a mutant gene that shows a partial reduction in the activity it affects).

In a specific embodiment, all alleles of the endogenous DHODH gene are partially or completely inactivated.

In a specific embodiment, said endogenous DHODH gene is completely inactivated.

In a more specific embodiment, all alleles of the endogenous DHODH gene are completely inactivated.

In a specific embodiment, the endogenous DHODH gene is inactivated by a method using the CRISPR-Cas9 system described in Aga et al. (2015) BMC Proceedings 9(suppl 9):P2.

As is well known in the art, the CRISPR-Cas9 system is a prokaryotic adaptive immune response system that uses non-coding RNAs to direct the Cas9 nuclease to cause site-specific cleavage of DNA. Repair of this DNA damage is accomplished by cellular DNA repair mechanisms, either through the non-homologous end joining (NHEJ) DNA repair pathway or the homology-directed repair (HDR) pathway. To generate gene disruptions, a single guide RNA (gRNA), consisting of a crRNA sequence specific to the DNA target and a tracrRNA sequence that interacts with the Cas9 protein, binds to a recombinant form of the Cas9 protein that has DNA endonuclease activity. The resulting complex will cause target-specific cleavage of double-stranded DNA. Repair of the cleavage site will be accomplished by DNA repair by non-homologous end joining (NHEJ), an error-prone process that can result in insertions/deletions (INDELs) that can disrupt gene function.

In a specific embodiment, at least one exon of the DHODH gene is targeted for inactivation, in particular, using a gene editing method, such as a method using the CRIPR-Cas9 system. In a more specific embodiment, a portion of the DHODH gene encoding the N-terminal portion of the DHODH protein is targeted for inactivation, in particular, using a gene editing method, such as a method using the CRISPR-Cas9 system. In yet another embodiment, the second exon of the DHODH gene is targeted for inactivation, in particular, using a gene editing method, such as a method using the CRISPR-Cas9 system.

In one embodiment, a 20-nucleotide sequence with the sequence CAAGGATGATGGCTGCATCC (SEQ ID NO:23) or the sequence GGATGCAGCCATCATCCTTG (SEQ ID NO:5) or any sequence compatible with knockout of the DHODH gene without reducing the lifespan of CHO is used as the corresponding DNA fragment to create a gRNA that targets the second exon of the DHODH gene. This gRNA is typically prepared using oligonucleotides with the sequence CACCGCACCGGGATGCAGCCATCATCCTTG (SEQ ID NO:6) and AAAACCAAGGATGATGGCTGCATCC (SEQ ID NO:7) or using oligonucleotides with the sequence GGATGCAGCCATCATCCTTGGTTTT (SEQ ID NO:24) and CAAGGATGATGGCTGCATCCCGGTG (SEQ ID NO:25) ), usually cloned into a unique plasmid restriction site, such as the BaeI site of plasmid pCM3561 (turned into Invitrogen's moneymaker), so that the cloned DNA sequence is under the control of the U6 promoter, and once said plasmid is introduced into the cell, it is transcribed into one a transcription unit containing crRNA fused to tracrRNA, wherein part of the crRNA is specific for the second exon of the DHODH gene, and part of tracrRNA is recognized by the Cas9 enzyme.

To identify a DHODH gene inactivated cell line, individual cells are typically isolated by limiting dilution in well plates, and after achieving adequate confluence, such as 90% confluence, the cells are divided into at least 2 conditions, such as one in culture medium supplemented with uridine, and the other in culture medium lacking uridine. The clones of interest are generally those that are sensitive to uridine deficiency.

Once isolated, these cells of interest can be cultured in a culture medium containing a pyrimidine base, in particular a culture medium containing uridine.

By "pyrimidine base" is meant herein pyrimidine itself and various pyrimidine derivatives having a pyrimidine core as a backbone. Examples of such pyrimidine bases include substances related to uracil-containing components of nucleic acids, such as uracil, uridine, uridine phosphates, in particular uridine monophosphate (UMP), uridine diphosphate (UDP) and uridine triphosphate (UTP), deoxyuridine, deoxyuridine phosphates, in particular deoxyuridine monophosphate (dUMP ), deoxyuridine diphosphate (dUDP) and deoxyuridine triphosphate (dUTP); substances related to the cytosine-containing components of nucleic acids, such as cytosine, cytidine, cytidine phosphates, in particular cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), deoxycytidine, 2'-deoxycytidine, deoxycytidine phosphates, in particular deoxycytidine monophosphate ( dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP); thymine, thymidine, thymidine phosphates, in particular thymidine monophosphate (TMP), thymidine diphosphate (TDP) and thymidine triphosphate (TTP), deoxythymidine, deoxythymidine phosphates, in particular deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTTP) and deoxythymidine triphosphate (dTTP) and orotate.

In a specific embodiment, said pyrimidine base is uridine.

By "uridine" is meant a nucleoside of the following formula:

By "culture medium lacking uridine" is meant any basic culture medium suitable for growing a particular cell line, wherein the medium contains less than 1 mM uridine, in particular the medium does not contain any uridine.

By “culture medium containing uridine” is meant any basic culture medium suitable for growing a particular cell line, said medium further containing from 1 to 25 mM uridine, in particular from 5 to 10 mM uridine.

By "basic culture medium" is meant a medium without additives that is suitable for treating cells, such as CHO cells. As will be appreciated by one skilled in the art, the basic culture medium used will depend on the type of cells used. Examples of basic culture media include CDCHO medium, OPTiCHO ^™ medium, Fecto CHO ^™ medium, FortiCHO ^™ medium, ExpiCHO ^™ medium, Ex-Cell ^™ medium, ActiPRO ^™ medium, MAM PF77 ^™ medium and PowerCHO ^™ medium.

In a specific embodiment, additional glutamine is added to the basic culture medium, typically 4 to 6 mM glutamine.

Accordingly, in a specific embodiment, the cell line of the present invention is created by

a) inactivating the endogenous DHODH gene in the cell, in particular using a gene editing method such as the CRISPR-Cas9 system, and

b) culturing the cell in a culture medium containing uridine under conditions suitable for creating a cell line in which the endogenous DHODH gene is partially or completely inactivated.

The generation of a CHO cell line containing the endogenous DHODH gene, which is completely or partially inactivated using a CRISPR-Cas9 approach, is illustrated in more detail in Examples 2 and 3.

The creation of a cell line, such as a CHO cell line, containing an endogenous DHODH gene that is completely or partially inactivated can be accomplished using a variety of other molecular biology techniques known in the art. For example, other gene editing techniques useful for creating a cell line containing an endogenous DHODH gene that is completely or partially inactivated include the use of zinc finger nucleases (ZFNs) or transcription factor type effector nucleases (TALENs). The Cre/Lox combination system method can also be used to knock out one or more or all alleles of the DHODH gene.

In a specific embodiment, the cell line of the present invention further comprises an expression vector as defined below in the "Expression Vector" section.

Said expression vector can be introduced into a cell line by any suitable method well known to the person skilled in the art, for example by transfection, in particular by electroporation or chemical transfection or transduction.

In a specific embodiment, said cell line of the present invention may further comprise an additional expression vector containing a selection marker different from the expression vector of the present invention, typically an additional expression vector containing a glutamine synthetase coding sequence.

Exogenous DHODH

The DHODH encoded by the expression vector used in the present invention (hereinafter referred to as "exogenous DHODH") may include or consist of a sequence identical to at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91%; 92%; 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100% SEQ ID NO:2 or SEQ ID NO:4. It may also include or consist of a fragment of at least 100, 150, 200, 250, 300 or 350 consecutive amino acids of SEQ ID NO:2 or SEQ ID NO:4, provided that the protein retains DHODH activity.

In some embodiments, exogenous DHODH in accordance with the present invention includes or consists of a sequence that is at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91% identical; 92%; 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100% as sequences SEQ ID NO:2 and SEQ ID NO:4.

In some embodiments, the exogenous DHODH of the present invention is human DHODH, i.e. DHODH of human origin.

As used herein, the term “human DHODH” refers to a protein with the sequence comprising or consisting of SEQ ID NO:4, as well as variants thereof that exhibit DHODH activity. Such variants may, for example, correspond to variants that occur naturally in the human species (eg, allelic variants or splice variants). Alternatively, such variants may correspond to genetically engineered variants. In one embodiment, such variants differ from the sequence of SEQ ID NO:4 only by having at most 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 25, 24, 23. 22, 21, 20, 19, 18,17,16,15,14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid change from SEQ ID NO:4 (where these changes include substitutions, insertions and deletions).

In a specific embodiment, said human DHODH is a variant containing the G202A mutation compared to the wild type sequence, typically a protein comprising or consisting of the amino acid sequence of SEQ ID NO:26.

In some embodiments, the exogenous DHODH is hamster DHODH, i.e. DHODH is of hamster origin. Hamster DHODH may be, for example, Chinese hamster DHODH (Cetulus griseus).

As used herein, the term “Chinese hamster DHODH” refers to the sequence comprising or consisting of SEQ ID NO:2, as well as variants thereof exhibiting DHODH activity. Such variants may, for example, correspond to variants that occur naturally in the hamster species (eg, allelic variants or splice variants). Alternatively, such variants may correspond to genetically engineered variants. In one embodiment, such variants differ from the sequence of SEQ ID NO:2 only by having at most 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid change compared to SEQ ID NO:2 (where these changes include substitutions, insertions and deletions).

In another embodiment, the DHODH variant will have DHODH activity, optionally the same level of activity as the wild type protein, or 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%. 130%, 140% or more of the wild-type protein activity level.

By a polypeptide having an amino acid sequence identical to at least, for example, 95% identical to the query amino acid sequence of the present invention, it is meant that the amino acid sequence of the subject polypeptide is identical to the query sequence, except that the subject polypeptide sequence may include up to five amino acid changes for every 100 amino acids of the requested amino acid sequence. In other words, to produce a polypeptide having an amino acid sequence that is at least 95% identical to the target amino acid sequence, up to 5% (5 out of 100) of the amino acid residues in the subject sequence may be inserted, deleted, or replaced by another amino acid.

Sequence identity can be determined over the entire length of the variant sequence, over the entire length of the reference sequence, or both. For example, percent identity can be calculated using global alignment (i.e., two sequences are compared along their entire length). Methods for comparing the identity and homology of two or more sequences are well known in the art. The "Needle" program, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453) to find the optimal alignment (including breaks) of two sequences, taking into account their entire length , can, for example, be used when performing global alignment. This "Needle" program, for example, is available on the global website ebi.ac.uk. The percent identity according to the present invention is preferably calculated using the EMBOSS::needle (global) program using a "Gap Open" parameter of 10.0, a "Gap Extend" parameter, equal to 0.5, and the substitution matrix Blosum62.

Variants of the reference sequence may contain mutations, such as deletions, insertions and/or substitutions, compared to the reference sequence. In case of substitutions, the substitution preferably corresponds to a conservative substitution as indicated in the table below.

Conservative substitutions Amino acid type Ala, Val, Leu, Ile, Met, Pro, Phe, Trp Amino acids with aliphatic hydrophobic side chains Ser, Tyr, Asn, Gln, Cys Amino acids with uncharged but polar side chains Asp, Glu Amino acids with acidic side chains Lys, Arg, His Amino acids with basic side chains Gly Neutral side chain

Expression vector

The expression vector used in the context of the present invention is suitable for the production of recombinant protein and contains a sequence encoding dihydroorotate dehydrogenase (DHODH).

The expression vector is preferably a DNA vector.

The expression vector used in the context of the present invention contains a sequence encoding exogenous DHODH, as defined above in the section "Exogenous DHODH".

In a specific embodiment, the cell line into which the expression vector is to be introduced is a CHO cell line, and the exogenous DHODH is of heterologous origin (ie, the exogenous DHODH is not hamster DHODH).

The sequence encoding such exogenous DHODH may be a naturally occurring nucleotide sequence. Alternatively, the triplet codons of the sequence encoding such DHODH may be shifted for expression in CHO cells. Software and algorithms for shifting codons in a sequence to obtain optimal expression are known in the art and include, for example, the algorithm described in Raab et al. (2010) Syst Synth Biol. 4:215-225. This algorithm not only provides the best available codons for expression, but also takes into account GC content and the absence of unwanted DNA motifs.

For example, the sequence encoding exogenous DHODH may include or consist of a sequence identical to at least 60%, 62%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% SEQ ID sequences NO:3 (i.e., the human DHODH coding sequence with SEQ ID NO:4, which was designed for optimal expression in CHO cells) and/or the SEQ ID NO:1 sequence (i.e., the hamster DHODH coding sequence with SEQ ID NO:2, which was designed for optimal expression in CHO cells).

In one embodiment, the exogenous DHODH coding sequence includes or consists of the sequence SEQ ID NO:1 or SEQ ID NO:3.

In the expression vector used in the context of the present invention, the sequence encoding exogenous DHODH as defined above may be placed under the control of any promoter known to those skilled in the art.

For example, the sequence encoding exogenous DHODH as defined above may, for example, be placed under the control of a promoter suitable for driving DHODH expression, e.g., simian vacuolating virus 40 (SV40) promoter (e.g., SV40 late or early promoter), CMV promoter, elongation factor 1, GAPDH promoter, RPL37 promoter, actin promoter. The SV40 early promoter is described, for example, in Benoist and Chambon (1981) Nature 290:304-310 and Moreau et al. (1981) Nucleic Acids Res. 9:6047–6068. In particular, said SV40 promoter is a full-length promoter. The SV40 promoter may also have an origin of replication containing a 72 base pair repeat.

In some embodiments, said SV40 promoter is not an SV40 promoter from which positions 128 to 270 have been deleted, i.e. the said SV40 promoter is not the SV40 promoter described in Korean Patent No. 10-0267720 and the E. coli transformant deposited in Gene Bank, Institute of Bioengineering, KIST, December 17, 1997 under deposit number: KCTC 8860 P.

In other embodiments, the exogenous DHODH coding sequence defined above is not under the control of the SV40 promoter.

Expression vectors suitable for the production of recombinant proteins are known to those skilled in the art. Such vectors typically correspond to expression vectors that contain an origin of replication and at least one expression cassette allowing cloning and expression of the recombinant protein whose production is desired. An expression cassette typically contains a 5' untranslated region (including or consisting of a promoter and optionally an enhancer sequence), one or more restriction sites allowing cloning of a recombinant protein coding sequence, a 3' untranslated region (e.g., a signal ( poly)A) and optionally one or more introns. The promoter sequence may correspond to any strong promoter well known in the art, such as, for example, the human CMV promoter. Optionally, expression vectors used in the context of the present invention contain a prokaryotic origin of replication (for example, a prokaryotic replicon such as ColE1 in E. coli ) and at least a prokaryotic selection marker gene, also known as a prokaryotic selection marker, such that these vectors do possible replication in prokaryotic cells. Cells that replicate the vectors also express the prokaryotic selection marker gene and can therefore be identified and selected. Genes for prokaryotic-selective markers are well known to one skilled in the art. Examples of prokaryotic selection marker genes are, for example, nucleic acid sequences encoding a protein conferring resistance to antibiotics (eg, a sequence encoding a protein conferring resistance to ampicillin, chloramphenicol, blasticidin or kanamycin).

The recombinant protein can correspond to any protein that is of interest to those skilled in the art.

As used herein, the term “protein” is intended to include peptides (i.e., amino acid chains of less than 50 amino acids), polypeptides (i.e., amino acid chains of at least 50 amino acids), monomeric proteins (i.e., proteins consisting of of one amino acid chain) and multimeric proteins (i.e. proteins consisting of two or more amino acid chains, such as, for example, monoclonal antibodies).

An expression vector used in the context of the present invention typically includes a number of expression cassettes that are identical to a number of different amino acid chains constituting the protein (for example, one expression cassette in the case of a monomeric protein or homodimeric protein, two in the case of a heterodimeric protein or monoclonal antibody, and etc.).

Alternatively, the expression vector used in the context of the present invention may contain only one expression cassette, even when production of a heterodimeric protein or monoclonal antibody is desired. In such a case, the sequence(s) encoding the other amino acid chain(s) of the protein is present in a separate expression vector, which is cotransfected together with the expression vector in accordance with the present invention into a host cell line, in particular a CHO cell line .

In this case, additional separate expression vectors may contain selection markers other than the DHODH selection marker described herein, such as DHFR, GS or HPRT.

In one embodiment, the expression vector used in the context of the present invention may lack an expression cassette. In such a case, the expression cassette(s) suitable for expression of the recombinant protein are present in a separate vector, which is cotransfected together with the expression vector in accordance with the present invention into a host cell line, in particular into a DHODH-inactivated cell line. the present invention, more specifically, the DHODH-inactivated CHO cell line of the present invention.

Thus, in some embodiments, an expression vector used in the context of the present invention comprises:

- a sequence encoding exogenous DHODH as defined above, placed under the control of the SV40 early promoter;

- a first expression cassette in which the sequence encoding the light chain of the antibody is placed under the control of the CMV promoter;

- a second expression cassette in which the sequence encoding the heavy chain of the antibody is placed under the control of the CMV promoter;

- prokaryotic origin of replication; And

- a selectable marker for use in prokaryotic cells, namely a sequence encoding a protein conferring resistance to ampicillin, placed under the control of its natural promoter.

Throughout the present description, the term “recombinant protein” refers to any recombinant protein, the production of which is desired. It may, for example, correspond to a therapeutic and/or prophylactic protein, i.e. protein intended for use as a medicine (including a vaccine). In a specific embodiment, the recombinant protein whose production is desired is not DHODH. In another specific embodiment, the recombinant protein whose production is desired is an antibody, such as a monoclonal antibody. In yet another specific embodiment, the recombinant protein whose production is desired is an antigenic protein.

The term "antibody" is used herein in its broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies) of any isotype, such as IgG, IgM, IgA, IgD and IgE, polyclonal antibodies, multispecific antibodies (including bispecific and trispecific antibodies), antibody fragments (such as, for example, Fv, scFv, dsFab, Fab' or F(ab) fragments, single domain antibodies and fragments thereof, as well as fusion proteins containing an antibody fragment. Antibody that reacts with a specific antigen, can be created using recombinant methods, such as selection from libraries of recombinant antibodies in phage or similar vectors, or by immunizing an animal with an antigen or antigen-encoding nucleic acid.

A "monoclonal antibody" as used herein is an antibody derived from a population of substantially homogeneous antibodies, i.e. the antibodies making up this population are essentially identical, except for possible naturally occurring mutations that may be present in minute quantities. These antibodies are directed against one epitope (or one group of epitopes in the case of polyspecific monoclonal antibodies) and are therefore highly specific.

A typical monoclonal antibody consists of two identical heavy chains and two identical light chains linked by disulfide bonds. Each heavy and light chain contains a constant region and a variable region. Each variable region contains three segments called “complementarity determining regions” (“CDRs”) or “hypervariable regions”, which are primarily responsible for binding to an epitope of the antigen. They are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus (see Kabat et al. , Sequences of Proteins of Immunological Interest, 5th edition, National Institute of Health, Bethesda, MD, 1991). The more highly conserved parts of the variable regions are called "framework regions".

The monoclonal antibody may, for example, be a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

The monoclonal antibody may be a monospecific, bispecific or trispecific antibody.

When the recombinant protein whose production is desired is a monoclonal antibody, the expression vector in accordance with the present invention may contain a first expression cassette suitable for cloning an antibody light chain and a second expression cassette suitable for cloning an antibody heavy chain.

In a specific embodiment, each of the first and second expression cassettes comprises a cytomegalovirus (CMV) promoter, for example a CMV promoter from human or mouse CMV. More specifically, said first and second expression cassettes may comprise:

- CMV immediate early promoter enhancer (eg, one that has the sequence described in Teschendorf et al. (2002) Anticancer Res. 22:3325-3330); or

- the IE2 promoter/enhancer region from mouse CMV (eg, having the sequence described in Chatellard et al. (2007) Biotechnol Bioeng. 96:106-117); or

- hCMV-MIE regulatory element (for example, having the sequence described in WO 89/01036).

The term "antigenic protein" is used here in its broadest sense and covers any protein capable of inducing an immune response, either alone or in combination with an adjuvant. It may be intended for use in either a prophylactic vaccine or a therapeutic vaccine. In a specific embodiment, the antigenic protein is a vaccine protein, i.e. a protein intended for use in a prophylactic vaccine.

The expression vector may either contain at least one sequence encoding the recombinant protein of interest (e.g., one sequence encoding a monomeric protein, one sequence encoding an antibody chain, or two sequences encoding an antibody light chain and an antibody heavy chain, respectively), or it may be empty (ie, lacking such sequence encoding the recombinant protein of interest).

Expression system, kits, methods and applications

The present invention provides an expression system comprising:

(i) a cell line, as defined in the "Cell Line" section above, containing an endogenous DHODH gene that is partially or completely inactivated, as defined in the "Cell Line" section above, and

(ii) an expression vector as defined in the “Expression Vector” section above.

The expression system of the present invention may further include additional separate expression vectors, each of which contains a nucleotide sequence encoding a selection marker other than DHODH, such as DHFR, GS or HPRT, and at least one expression cassette for expressing a recombinant protein .

Alternatively, the expression system of the present invention may further include additional expression vectors as defined in the "Expression Vector" section above.

The present invention provides a kit comprising (i) a cell line in accordance with the present invention containing an expression vector as defined in the "Expression Vector" section above or an expression system in accordance with the present invention and (ii) a culture medium lacking uridine as defined above .

The kit may include an expression vector encoding exogenous DHODH (in an expression system) as described above. In such a kit, the vector is preferably empty, since this allows the cloning of a protein of interest to those skilled in the art. Moreover, the expression vector is preferably isolated from a cell line in such a set.

The kit also includes culture medium lacking uridine as defined in the Cell Line section above.

The kit may further include a medium suitable for culturing the cell line, a medium suitable for transfecting the vector into the cell line, packaging material and/or instructions for use of the expression system.

In a specific embodiment, the kit does not contain a DHODH inhibitor.

Examples of DHODH inhibitors include bicinchoninic acid, brequinar (6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinolinecarboxylic acid), naphthoquinone derivatives such as dichloroalli- lawson, isoxazole derivatives such as leflunomide (5-methyl- N- [4-(trifluoromethyl)phenyl]isoxazole-4-carboxamide) and its active metabolite teriflunomide ((2Z)-2-cyano-3-hydroxy-N-[ 4-(trifluoromethyl)phenyl]but-2-enamide), quinolonecarboxylic acids, naphthoquinones, isoxazoles, phenoxyquinolines, redoxal and derivatives, lawson, lapachol, atovaquone and (8-chloro-4-(2-chloro-4-fluorophenoxy)quinoline ). A DHODH inhibitor can inhibit DHODH activity by at least 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100%.

In a specific embodiment, the kit does not contain teriflunomide.

The present invention further provides for the use of a cell line in accordance with the present invention containing an expression vector as defined in the section "Expression Vector" above, an expression system in accordance with the present invention or a kit in accordance with the present invention for the production of recombinant protein in vitro .

In a specific embodiment, said cell line, expression system or kit is used in combination with a culture medium lacking uridine as defined above, more specifically in the absence of a DHODH inhibitor.

In addition, the present invention also provides for the use of an expression system in accordance with the present invention, a cell line in accordance with the present invention containing an expression vector as defined in the "Expression Vector" section above, or a kit in accordance with the present invention for isolating a cell clone, which produces high-level recombinant protein (“high-producing clones”) in vitro , particularly in the absence of a DHODH inhibitor.

In the context of the present invention, the term “high level of recombinant protein” is intended to mean that in the culture medium the concentration of recombinant protein is at least 0.05 g/L, preferably at least 0.1 g/L, even more preferably at at least 0.2 g/l, more preferably 0.3 to 1 g/l. The concentration of the recombinant protein can be determined using methods that are well known to one skilled in the art, including, but not limited to, enzyme-linked immunosorbent assay (ELISA), Western blotting, caliper method, and a purified protein concentration range corresponding to the recombinant protein.

The present invention further provides a method for producing recombinant protein in vitro, comprising the steps of:

A a1) providing a cell line in accordance with the present invention containing the expression vector defined in the "Expression Vector" section above;

or

a2) providing a cell line in accordance with the present invention; and

a2') introducing the expression vector defined in the "Expression Vector" section above into the cell line provided in step a2);

or

a3) providing a cell line containing the endogenous DHODH gene,

a3') partial or complete inactivation of the endogenous DHODH gene in the cell line provided in step a3), and

a3") introducing the expression vector defined in the "Expression Vector" section above into the cell line containing the partially or completely inactivated endogenous DHODH gene obtained in step a3');

B) culturing said cell line under conditions suitable for the production of recombinant protein; And

C) isolating and/or purifying said recombinant protein.

In a specific embodiment, step B) of the above method is carried out in a culture medium devoid of uridine, more specifically also devoid of a DHODH inhibitor, and in particular includes the sub-step of selecting transfected cells that grow despite the absence of uridine, in particular additionally in no DHODH inhibitor.

The present invention further provides an in vitro method for isolating a cell clone that produces a recombinant protein at a high level, the method comprising or consisting of the following steps:

A a1) providing a cell line in accordance with the present invention containing the expression vector defined in the "Expression Vector" section above;

or

a2) providing a cell line in accordance with the present invention; and

a2') introducing the expression vector defined in the "Expression Vector" section above into the cell line provided in step a2);

or

a3) providing a cell line containing the endogenous DHODH gene,

a3') partial or complete inactivation of the endogenous DHODH gene in the cell line provided in step a3), and

a3") introducing the expression vector defined in the "Expression Vector" section above into the cell line containing the partially or completely inactivated endogenous DHODH gene obtained in step a3');

B) culturing said cell line under conditions suitable for the production of recombinant protein; And

C) isolating a clone that produces recombinant protein at a high level.

In a specific embodiment, step B) of the above method is carried out in a culture medium devoid of uridine, more specifically also devoid of a DHODH inhibitor, and in particular includes the sub-step of selecting transfected cells that grow despite the absence of uridine, in particular additionally in no DHODH inhibitor.

Said expression vector can be introduced into said cell line at stages a2') or a3") by any method well known to the person skilled in the art, for example by transfection, in particular by electroporation, or chemical transfection, or transduction.

Conditions suitable for the production of recombinant proteins are well known to those skilled in the art. For example, you can use the protocols described in the Examples section.

In a specific embodiment, the culture medium used in step B) contains decreasing concentrations of uridine. This allows the selection of clones in which amplification of the exogenous vector-derived DHODH gene (and thus the recombinant protein coding sequence) has occurred.

The above methods may further include the step of forming the recombinant protein into a pharmaceutical composition.

Throughout the specification, terms such as “comprises”, “comprising” and “comprising” have the meaning ascribed to them in most patent jurisdictions, preferably the jurisdiction in question; for example, they can mean "includes", "included", "including", etc. Terms such as "consisting of", "substantially consisting of" and "essentially consisting of" have the meaning ascribed to them in most patent jurisdictions, preferably in the jurisdiction in question; for example, they purport to exclude all, most, or all but a small number of other elements; they admit elements not expressly listed, but exclude elements that appear in the prior art or that affect the essential or novel features of the present invention.

Several documents are cited throughout this description. Each of the documents contained herein (including any journal article or abstract, published or unpublished patent application, issued patent, manufacturer's specifications, instructions, etc.) is hereby incorporated by reference. However, it cannot be assumed that any document given herein is actually prior art regarding the present invention.

The present invention will now be described with reference to the following drawings and examples, which are illustrative only and not intended to limit the present invention.

The present invention is defined by the claims, which are to be interpreted with the help of the description and drawings.

Brief description of sequences

SEQ ID NO: Description 1 cDNA sequence encoding DHODH with origins in the Chinese hamster ( Cricetulus griseus ) 2 Amino acid sequence of DHODH with origins in the Chinese hamster 3 cDNA sequence encoding DHODH of human origin 4 Amino acid sequence of DHODH of human origin 5 The appropriate DNA fragment to create gRNA 6 Oligonucleotide used to produce gRNA (sequence 1) 7 Oligonucleotide used to produce gRNA (sequence 1) 8 Oligonucleotide used to produce gRNA (sequence 2) 9 Oligonucleotide used to produce gRNA (sequence 2) 10 Oligonucleotide used to produce gRNA (sequence 3) eleven Oligonucleotide used to produce gRNA (sequence 3) 12 Oligonucleotide used to produce gRNA (sequence 4) 13 Oligonucleotide used to produce gRNA (sequence 4) 14 Oligonucleotide used to produce gRNA (sequence 5) 15 Oligonucleotide used to produce gRNA (sequence 5) 16 Oligonucleotide used to produce gRNA (sequence 6) 17 Oligonucleotide used to produce gRNA (sequence 6) 18 Oligonucleotide used to produce gRNA (sequence 7) 19 Oligonucleotide used to produce gRNA (sequence 7) 20 Oligonucleotide used to produce gRNA (sequence 8) 21 Oligonucleotide used to produce gRNA (sequence 8) 22 DHODH CHO gene sequence 23 The appropriate DNA fragment to create gRNA 24 Oligonucleotide used to produce gRNA (sequence 1’) 25 Oligonucleotide used to produce gRNA (sequence 1') 26 Amino acid sequence of human DHODH G202A 27 Oligonucleotide used to produce gRNA (sequence 2') 28 Oligonucleotide used to produce gRNA (sequence 2’) 29 Oligonucleotide used to produce gRNA (sequence 3’) thirty Oligonucleotide used to produce gRNA (sequence 3’) 31 Oligonucleotide used to produce gRNA (sequence 4’) 32 Oligonucleotide used to produce gRNA (sequence 4’) 33 Oligonucleotide used to produce gRNA (sequence 5’) 34 Oligonucleotide used to produce gRNA (sequence 5’) 35 Oligonucleotide used to produce gRNA (sequence 6’) 36 Oligonucleotide used to produce gRNA (sequence 6’) 37 Oligonucleotide used to produce gRNA (sequence 7’) 38 Oligonucleotide used to produce gRNA (sequence 7’) 39 Oligonucleotide used to produce gRNA (sequence 8’) 40 Oligonucleotide used to produce gRNA (sequence 8’) 41 Sense oligonucleotide 603 42 Antisense oligonucleotide 503 43 A sequence including a target sequence and a PAM. 44 DHODH exon2 sense sequence region including sequence n°1 for CrispR. 45 DHODH exon2 antisense sequence region including sequence n°1 for CrispR.

Examples

Example 1: Generation of CHO 9E4 Cell Line

This example describes the generation of the CHO 9E4 cell line from the CHO-K1 cell line commercially available from ATCC under ATCC accession number: CCL-61.

1. CHO-K1 cell line

A vial of CHO-K1 cells (ATCC: CCL-61) frozen in the presence of calf serum in 1969 was obtained from ATCC.

2. Thaw the vial in Ex-Cell™ 302 and prepare the CHO-LG-APF jar.

CHO-K1 in a vial was thawed directly in Ex-Cell™ 302 (SAFC) medium supplemented with 4 mM glutamine and propagated on a stationary support, then in a rotator. The resulting CHO-LG-APF bank was frozen in Ex-Cell™ 302 medium after 12 passages and 17.3 generations.

3. Thaw the CHO-LG-APF jar in Ex-Cell™ 302 and prepare the ABC-024 P22 jar.

The CHO-LG-APF vial was thawed in Ex-Cell™ 302 medium and propagated. The resulting bank ABC-024 P22 was thawed after 18.5 generations.

4. Adaptation of bank CMV07-024 to the CDCHO Fusion environment and preparation of bank ABC-003.

The CMV07-024 bank was thawed and directly adapted to Ex-cell™ CDCHO Fusion (SAFC) medium supplemented with 4 mM glutamine and shaker-adapted for over 12.5 generations before freezing the ABC-003 bank in Ex-cell™ CDCHO Fusion medium .

5. Thaw jar ABC-003 in CDCHO Fusion and prepare jar ABC-053 in CDCHO Fusion

ABC-003 in the vial was thawed in CDCHO Fusion medium, and after dilution, the ABC-53 jar was frozen after 4.2 generations.

6. Thaw bank ABC-053 in CDCHO Fusion, select, clone and prepare bank P15A11 in CDCHO Fusion

Bank ABC-053 was thawed in Ex-cell™ CDCHO Fusion (SAFC) medium supplemented with 4 mM glutamine. After propagation of the culture, it was cloned by limiting dilution in plates and then propagated in CDCHO Fusion medium. The P15A11 clone bank resulting from this cloning was frozen. This cloning and propagation corresponds to approximately 94 generations.

7. Thaw jar P15A11 in CDCHO Fusion, adapt the jar by direct passage in CDCHO, and prepare jar CHOSP10-002 in CDCHO.

The P15A11 bank was thawed in Ex-cell™ CDCHO Fusion (SAFC) medium supplemented with 4 mM glutamine, and after 2 passages in CDCHO Fusion medium, the cells were diluted in CDCHO medium. After 3 passages in CDCHO medium, the CHOSP10-002 bank was frozen after only 15.9 generations.

8. Thaw the CHOSP10-002 bank in CDCHO medium, multiply, remove masses by centrifugation and select by subcultivation without masses in 96-well plates, multiply in 6-well plates and on a shaker to prepare the CHOSP10-012 bank in CDCHO medium.

The CHOSP10-002 bank was thawed in CDCHO medium (Invitrogen) supplemented with 6 mM glutamine and then propagated. The culture was centrifuged to remove cell masses and culture continued using only cells isolated from the supernatant. At this stage, 11.3 generations have passed since thawing.

This culture was divided into 96-well plates with 10 cells per well. Wells with cells that multiply in isolation in suspension were propagated in 6-well plates, then on a shaker. 23.2 additional generations passed before the CHOSP10-012 bank was frozen.

9. Thawing the CHOSP10-012 jar, multiplying and preparing the CHOSP11-008 jar (9E4 jar)

A bank of CHOSP10-012 was thawed in CDCHO medium (Invitrogen) supplemented with 6 mM glutamine, then propagated from the Erlenmeyer flask stage to a 17-L bioreactor.

Bank 9E4 was frozen after a total of 10 generations.

Example 2: Generation of a CHO cell line in which the DHODH gene is inactivated.

A - Design and construction of guide RNA (gRNA) for the CRISPR-CAS9 system.

To knock down the DHDOH gene in CHO cells, we began by obtaining the sequence of the hamster DHODH gene and using the publicly available Tefor software to design various guide RNAs (gRNAs) for transfection along with CRISPR-Cas9 into the CHO genome. The full-length sequence of DHODH CHO, with introns and exons, is shown in FIG. 1.

The program identified 8 sequences that can target the DHODH gene:

Sequence 1

CACCGGGATGCAGCCATCATCCTTG (SEQ ID NO:6)

AAACCAAGGATGATGGCTGCATCC (SEQ ID NO:7)

Sequence 2

CACCGGATGCAGCCATCATCCTTGG (SEQ ID NO:8)

AAAACCCAAGGATGATGGCTGCATC (SEQ ID NO:9)

Sequence 3

CACCGGCAGCCATCATCCTTGGGGG (SEQ ID NO:10)

AAAACCCCCCAAGGATGATGGCTGC (SEQ ID NO:11)

Sequence 4

CACCGGCCATCATCCTTGGGGGAGG (SEQ ID NO:12)

AAAACCCTCCCCCAAGGATGATGGC (SEQ ID NO:13)

Sequence 5

CACCGGCTATTCGCTTCACGTCCCT (SEQ ID NO:14)

AAACAGGGACGTGAAGCGAATAGC (SEQ ID NO:15)

Sequence 6

CACCGGCCTCTACAAACTGGGCTTT (SEQ ID NO:16)

AAAACAAAGCCCAGTTTGTAGAGGC (SEQ ID NO:17)

Sequence 7

CACCGGGCTTTGGGTTTGTCGAGGT (SEQ ID NO:18)

AAAACACCTCGACAAACCCAAAGCC (SEQ ID NO:19)

Sequence 8

CACCGGCTGGTCTGAGGAGCCTACA (SEQ ID NO:20)

AAAACTGTAGGCTCCTCAGACCAGC (SEQ ID NO:21)

Although 8 sequences were tested and cloned, of the 8 sequences, only four cloned sequences were transfected, and only one was successful in causing DHODH gene knockout. The following 20 nucleotide sequence GGATGCAGCCATCATCCTTG (SEQ ID NO:5) was used as the corresponding DNA fragment to generate the gRNA, as shown in FIG. 2. It targets the second exon of the DHODH gene. To achieve transcription of the proper gRNA, two oligonucleotides CACCGGGATGCAGCCATCATCCTTG (Oligonucleotide 1, SEQ ID NO:6) and AAAACCAAGGATGATGGCTGCATCC (Oligonucleotide 2, SEQ ID NO:7) were synthesized, annealed and cloned into the unique BaeI site of pCM3561 (turned into a profit center for Invitrogen ).

Thus, the cloned DNA sequence was under the control of the U6 promoter, and after DNA transfection into CHO cells, it was transcribed into a single transcription unit containing crRNA fused to tracrRNA. The crRNA portion was specific for the second exon of the DHODH gene, while the tracrRNA was recognized by the Cas9 enzyme itself.

B-Preparation of material for gene editing using the CRISPR-Cas9 system

CHO 9E4 cells were isolated and selected from CHO K-1 cells purchased from ATCC as described in Example 1, and grown and maintained as suspension cultures in serum-free, chemically defined CDCHO medium optimized for the culture of Chinese hamster ovary (CHO) cells. ), with the addition of 6 mM L-glutamine at 37°C in an incubator with 8% CO ₂ and 80% humidity.

10 μg of sgRNA expression vector (pCM3561) was digested with 1 μl of BaeI enzyme by adding 5 units/μl with 20 μM S-adenosylmethionine (SAM) at 25°C for 1 hour, then the digested plasmid was separated by electrophoresis using 1% agarose gel. The resulting sgRNA cloning vector was then extracted using a gel extraction kit (Qiagen Kit).

The sgRNA cloning vector and annealed guide oligonucleotides were ligated using T4 DNA ligase enzyme (Biolabs) and incubated for 10 min at room temperature.

5 μl of ligation products was added to 50 μl of competent E. coli DH5a cells (Invitrogen).

Cells and DNA were incubated for 30 min on ice and then heat shocked at 42°C for 45 s. After adding 500 ml of SOC medium, a 1-hour incubation at 37°C (at 800 rpm) allowed the bacteria time to develop the antibiotic resistance proteins encoded in the plasmid backbone. After incubation, each tube was distributed into one LB layer plate supplemented with 100 pg/ml ampicillin. The dishes were incubated overnight at 37°C. Negative controls (with water instead of DNA insert) were used to evaluate the success of transformation.

For the amplification step, two colonies for each construct were selected and inoculated in 2 ml of LB medium supplemented with 100 μg/ml ampicillin in a tube placed in an incubator overnight (at 37°C, 700 rpm). The overnight incubated culture was harvested by centrifugation. The QIAprep Miniprep Kit™ (QIAGEN) was used to extract the amplified DNA (elute into EB buffer). The sequence of the guide oligonucleotides of interest was then verified by Sanger sequencing (sense and antisense sequencing, GATC Company). After checking by alignment in Vector NTI software (Thermofisher Scientific), appropriate colonies were used to inoculate 200 ml of LB medium supplemented with 100 μg/ml ampicillin. After incubation for 24 hours, bacteria were collected by centrifugation at 6000 x g for 15 minutes at 4°C. The EndoFree Plasmid Maxi Kit™ (QIAGEN) was used to prepare MaxiPrep. DNA was precipitated by adding isopropanol at room temperature. After centrifugation for 1 hour (at 4°C, 8000 rpm), the DNA pellet was washed with 70% endotoxin-free ethanol at room temperature. After a short further centrifugation, the pellet was air-dried for 1 h and redissolved in an appropriate volume of sterile endotoxin-free water to obtain a DNA concentration of 5 mg/ml. A NanoDrop device was used to measure DNA concentration.

Four different plasmids were produced, namely plasmid pBH6840 (KO DHODH SEQ1), plasmid pBH6841 (KO DHODH SEQ4), plasmid pBH6842 (KO DHODH SEQ5) and plasmid pBH6843 (KO DHODH SEQ7). The target of these plasmids in the DHODH CHO gene is shown in the sequence SEQ ID NO:22.

DNA sequencing was performed by GATC subcontractor Eurofins Genomics Company.

C-Gene editing using the CRISPR-Cas9 system

Transfections were performed by electroporation using MaxCyte STX and its CHO-specific protocol. They were performed in OC-100 process configurations (20 million cells per transfection).

The day before transfection, cells were seeded at 1.5 x 10 ⁶ cells/ml in CDCHO medium supplemented with 6 mM L-glutamine.

On the day of transfection, cells were counted using a ViCell instrument (Beckman & Coulter). The required number of cells was centrifuged at 250 x g for 10 min, and the supernatant was discarded.

For each transfection condition, 20 × 10 ⁶ cells were centrifuged for 10 min at 250 × g. The pellet was resuspended in 70 μl Maxcyte buffer. 30 µg DNA was added and the mixture (cells, buffer and DNA) was transferred to a 100 µl Maxcyte electroporation cassette. The process assembly used was an OC-100 process assembly configured for a 100 μL cassette, and a CHO optimized program was selected.

The following transfections were carried out.

T1 pBH6840 KO DHODH SEQ1 T2 pBH6841 KO DHODH SEQ4 T3 pBH6842 KO DHODH SEQ5 T4 pBH6843 KO DHODH SEQ7 T5 W/O ADN H2O

After electroporation, the cells were transferred to Erlenmeyer flasks with a working volume of 25 ml. They were placed in a static incubator at 37°C, 5% CO ₂ for 45 minutes. 25 ml of CDCHO medium supplemented with 6 mM L-glutamine was then added to resuspend the cells, and the Erlenmeyer flasks were placed on shakers at 110 rpm at 37°C, 5% CO ₂ , 70% humidity.

The day after electroporation, one cell per well was seeded by limiting dilution from the CH09E4-transfected pools described above. After approximately 20 days, when the cells were approximately 90% confluent and appeared healthy when examined under a microscope, the cells were divided into 2 new 96-well plates with or without uridine.

Several clones were selected for their sensitivity to uridine deficiency. These clones were adapted for cultivation in CDCHO medium supplemented with 6 mM glutamine and 5 mM uridine.

To confirm that gene editing was successful, genomic DNA was extracted from CRISPR-Cas9 clone cells using the Qiagen DNeasy™ kit (Qiagen). The target locus was amplified by PCR using appropriate primers for the region of the DHODH locus targeted by the CRISPR-Cas9 system, and PCR products were sequenced by NGS using PCR fragments covering the potentially deleted regions.

Example 3: Alternative generation of a CHO cell line in which the DHODH gene is deleted.

A - Design and construction of guide RNA (gRNA) for the CRISPR-CAS9 system.

To knock down the DHDOH gene in CHO cells, we began by obtaining the sequence of the hamster DHODH gene and using the publicly available Tefor software to design various guide RNAs (gRNAs) for transfection along with CRISPR-Cas9 into the CHO genome.

The program identified 8 sequences that can target the DHODH gene:

Sequence 1'

GGATGCAGCCATCATCCTTGGTTTT (SEQ ID NO:24)

CAAGGATGATGGCTGCATCCCGGTG (SEQ ID NO:25)

Sequence 2'

GATGCAGCCATCATCCTTGGGTTTT (SEQ ID NO:27)

CCAAGGATGATGGCTGCATCCGGTG (SEQ ID NO:28)

Sequence 3'

GCAGCCATCATCCTTGGGGGGTTTT (SEQ ID NO:29)

CCCCCAAGGATGATGGCTGCCGGTG (SEQ ID NO:30)

Sequence 4'

GCCATCATCCTTGGGGGAGGTTTT (SEQ ID NO:31)

CCTCCCCCAAGGATGATGGCCGGTG (SEQ ID NO:32)

Sequence 5'

GCTATTCGCTTCACGTCCCTGTTTT (SEQ ID NO:33)

AGGGACGTGAAGCGAATAGCCGGTG (SEQ ID NO:34)

Sequence 6'

GCCTCTACAAACTGGGCTTTGTTTT (SEQ ID NO:35)

AAAGCCCAGTTTGTAGAGGCCGGTG (SEQ ID NO:36)

Sequence 7'

GGCTTTGGGTTTGTCGAGGTGTTTT (SEQ ID NO:37)

ACCTCGACAAACCCAAAGCCCGGTG (SEQ ID NO:38)

Sequence 8'

GCTGGTCTGAGGAGCCTACAGTTTT (SEQ ID NO:39)

TGTAGGCTCTCAGACCAGCCGGTG (SEQ ID NO:40)

Although 8 sequences were tested and cloned, of the 8 sequences, only four cloned sequences were transfected, and only one was successful in causing DHODH gene knockout. The following 20 nucleotide sequence GGATGCAGCCATCATCCTTG (SEQ ID NO: 5) was used as the corresponding DNA fragment to create gRNA. It targets the second exon of the DHODH gene. To achieve transcription of the proper gRNA, two oligonucleotides GGATGCAGCCATCATCCTTGGTTTT (oligonucleotide 1', SEQ ID NO:24) and CAAGGATGATGGCTGCATCCCGGTG (oligonucleotide 2', SEQ ID NO:25) were synthesized, annealed and cloned into the unique BaeI site of plasmid pCM3561 (converted to source of Invitrogen's profit).

Thus, the cloned DNA sequence was under the control of the U6 promoter, and after DNA transfection into CHO cells, it was transcribed into a single transcription unit containing crRNA fused to tracrRNA. The crRNA portion was specific for the second exon of the DHODH gene, while the tracrRNA was recognized by the Cas9 enzyme itself.

B-Preparation of material for gene editing using the CRISPR-Cas9 system

CHO 9E4 cells were isolated and selected from CHO K-1 cells purchased from ATCC as described in Example 1, and grown and maintained as suspension cultures in serum-free, chemically defined CDCHO medium optimized for the culture of Chinese hamster ovary (CHO) cells. ), with the addition of 6 mM L-glutamine at 37°C in an incubator with 8% CO ₂ and 80% humidity.

10 μg of sgRNA expression vector (pCM3561) was digested with 1 μl of BaeI enzyme by adding 5 units/μl with 20 μM S-adenosylmethionine (SAM) at 25°C for 1 hour, then the digested plasmid was separated by electrophoresis using 1% agarose gel. The resulting sgRNA cloning vector was then extracted using a gel extraction kit (Qiagen Kit).

The sgRNA cloning vector and annealed guide oligonucleotides were ligated using T4 DNA ligase enzyme (Biolabs) and incubated for 10 min at room temperature.

5 μl of ligation products was added to 50 μl of competent E. coli DH5a cells (Invitrogen).

Cells and DNA were incubated for 30 min on ice and then heat shocked at 42°C for 45 s. After adding 500 ml of SOC medium, a 1-hour incubation at 37°C (at 800 rpm) allowed the bacteria time to develop the antibiotic resistance proteins encoded in the plasmid backbone. After incubation, each tube was distributed into one LB layer plate supplemented with 100 pg/ml ampicillin. The dishes were incubated overnight at 37°C. Negative controls (with water instead of DNA insert) were used to evaluate the success of transformation.

For the amplification step, two colonies for each construct were selected and inoculated in 2 ml of LB medium supplemented with 100 μg/ml ampicillin in a tube placed in an incubator overnight (at 37°C, 700 rpm). The overnight incubated culture was harvested by centrifugation. The QIAprep Miniprep Kit™ (QIAGEN) was used to extract the amplified DNA (elute into EB buffer). The sequence of the guide oligonucleotides of interest was then verified by Sanger sequencing (sense and antisense sequencing, GATC Company). After checking by alignment in Vector NTI software (Thermofisher Scientific), appropriate colonies were used to inoculate 200 ml of LB medium supplemented with 100 μg/ml ampicillin. After incubation for 24 hours, bacteria were collected by centrifugation at 6000 x g for 15 minutes at 4°C. The EndoFree Plasmid Maxi Kit™ (QIAGEN) was used to prepare MaxiPrep. DNA was precipitated by adding isopropanol at room temperature. After centrifugation for 1 hour (at 4°C, 8000 rpm), the DNA pellet was washed with 70% endotoxin-free ethanol at room temperature. After a short further centrifugation, the pellet was air-dried for 1 h and redissolved in an appropriate volume of sterile endotoxin-free water to obtain a DNA concentration of 5 mg/ml. A NanoDrop device was used to measure DNA concentration.

Four different plasmids were produced, namely plasmid pBH6840 (KO DHODH SEQ1), plasmid pBH6841 (KO DHODH SEQ4), plasmid pBH6842 (KO DHODH SEQ5) and plasmid pBH6843 (KO DHODH SEQ7).

DNA sequencing was performed by GATC subcontractor Eurofins Genomics Company.

C-Gene editing using the CRISPR-Cas9 system

Transfections were performed by electroporation using MaxCyte STX and its CHO-specific protocol. They were performed in OC-100 process configurations (20 million cells per transfection).

The day before transfection, cells were seeded at 1.5 x 10 ⁶ cells/ml in CDCHO medium supplemented with 6 mM L-glutamine.

On the day of transfection, cells were counted using a ViCell instrument (Beckman & Coulter). The required number of cells was centrifuged at 250 x g for 10 min, and the supernatant was discarded.

For each transfection condition, 20 × 10 ⁶ cells were centrifuged for 10 min at 250 × g. The pellet was resuspended in 70 μl Maxcyte buffer. 30 µg DNA was added and the mixture (cells, buffer and DNA) was transferred to a 100 µl Maxcyte electroporation cassette. The process assembly used was an OC-100 process assembly configured for a 100 μL cassette, and a CHO optimized program was selected.

The following transfections were carried out.

T1 pBH6840 KO DHODH SEQ1 T2 pBH6841 KO DHODH SEQ4 T3 pBH6842 KO DHODH SEQ5 T4 pBH6843 KO DHODH SEQ7 T5 W/O ADN H2O

After electroporation, the cells were transferred to Erlenmeyer flasks with a working volume of 25 ml. They were placed in a static incubator at 37°C, 5% CO ₂ for 45 minutes. 25 ml of CDCHO medium supplemented with 6 mM L-glutamine was then added to resuspend the cells, and the Erlenmeyer flasks were placed on shakers at 110 rpm at 37°C, 5% CO ₂ , 70% humidity.

The day after electroporation, one cell per well was seeded by limiting dilution from the CH09E4-transfected pools described above. After approximately 20 days, when the cells were approximately 90% confluent and appeared healthy when examined under a microscope, the cells were divided into 2 new 96-well plates with or without uridine.

Several clones were selected for their sensitivity to uridine deficiency. These clones were adapted for cultivation in CDCHO medium supplemented with 6 mM glutamine and 5 mM uridine.

To confirm that gene editing was successful, genomic DNA was extracted from CRISPR-Cas9 clone cells using the Qiagen DNeasy™ kit (Qiagen). The target locus was amplified by PCR using appropriate primers for the region of the DHODH locus targeted by the CRISPR-Cas9 system, and PCR products were sequenced by NGS using PCR fragments covering the potentially deleted regions.

Example 4: Use of DHODH Deficient CHO Cell Line to Produce Recombinant Proteins

Antibody production was tested on confirmed DHODH-deficient CHO clones obtained in Example 2 or 3 to test whether these clones could express antibodies without teriflunomide.

The developed vectors were obtained and prepared at a concentration of 5 mg/ml. All of them contain ITRs that allow the use of a transposon system to integrate plasmids into the genome of producing cells, except pBH6209, which is a plasmid encoding a transposase.

The cell lines used were wild-type CHO 9E4_SP11 and DHODH knockout KO2 and KO19.

CHO 9E4_SP11 was cultured in CDCHO medium supplemented with 6 mM L-glutamine.

KO2 and KO19 were cultured in CDCHO medium supplemented with 6 mM L-glutamine and 5 mM uridine.

Initially, they were cultured in Erlenmeyer flasks with a working volume of 25 ml and multiplied until the required number of viable cells was reached.

Various proteins were produced using a high-efficiency electroporation protocol developed by Maxcyte on a Maxcyte STX machine.

Cells were divided into 1.5 x 10 ⁶ portions the day before transfection.

On the day of transfection, cells were cotransfected with two vectors: a plasmid DNA expression vector containing anti-human CD38 heavy chain (HC) and light chain (LC) expression cassettes and the DHODH selection marker (as described in WO2016/062837), which were flanked PiggyBac recognition sites (inverted terminal repeats, ITRs), and a transposase-encoding vector from Transposagen that catalyzes the mobilization of transposons into the CHO genome into TTAA sites.

For each transfection condition, 80 x 10 ⁶ cells were centrifuged for 10 min at 250 x g. The pellet was resuspended in 250 μl Maxcyte buffer. 120 µg DNA was added and the mixture (cells, buffer and DNA) was transferred to a 400 µl Maxcyte electroporation cassette. The process assembly used was an OC-400 process assembly configured for a 400 μL cassette, and a CHO optimized program was selected.

For the recovery phase, transfected cells were immediately transferred to a 125 ml flask at 37°C for 40 min without stirring. 25 ml of prewarmed CDCHO medium supplemented with 6 mM glutamine (+5 mM uridine for KO cells) was added and transfected cultures were maintained at 37°C in an incubator with 8% CO ₂ and 80% humidity. 1 day after transfection, cells were centrifuged and resuspended in CD OPTiCHO™ selective medium (Gibco) supplemented with 6 mM glutamine, 30% FeedB (Gibco) and various amounts of teriflunomide (0, 5, 15 and 25 μM), up to 1x10 ⁶ cells/ ml.

On day 14 after transfection, cells were centrifuged at 200 × g for 10 min at 25°C. The supernatant was filtered through a 0.22-μm PES filter, and the antibody titer was measured using an Octet instrument.

As shown in FIG. 3, two clones (KO2 and KO19) showed good production without teriflunomide, and genomic NGS showed that these two clones contained a knockout mutation in two alleles of the DHODH locus.

Notably, all KO clones produced antibodies even in the absence of teriflunomide as a selective agent. Two clones KO2 and KO19 were selected for further studies. Moreover, these referenced clones were the only clones exhibiting homozygous knockout of the DHODH gene.

Thus, this example shows that the present invention provides a number of cell lines and vectors that allow the production of antibodies without the use of any selective pressure.

Example 5: Evaluation of three human DHODH variants.

To test whether the use of disrupted forms of human DHODH increases the number of copies of integration into the CHO genome and thereby provides greater productivity, the DHODH cell lines KO2, KO19, and WT CHO 9E4 were transfected with three cDNA variants for human DHODH described in patients with Miller syndrome ( R135C, G202A and R346W, see especially Fang et al. (2012) Biosci 32:631-639). As a control vector, a plasmid containing cDNA encoding wild-type human DHODH was transfected.

50 ng of a plasmid vector encoding a monoclonal antibody against human CD38, mAb-A (pBH6204), digested with Sal I- Bgl II restriction enzymes, was mixed with 37.5 ng of purified Sal I- Bgl II DNA fragments corresponding to each variant (R135C, G202A and R346W). After adding 1 μl of T4 DNA ligase (BioLabs) and concentrated ligation buffer, the ligation reaction (in a final volume of 10 μl) was carried out for 10 minutes at room temperature.

Aliquots of this DNA pool were then used to transform competent E. coli cells (Stellar™, Takara).

Small scale plasmid preparation was performed using a commercially available Min-Prep kit (Qiagen) according to the manufacturer's recommendations.

DNA sequencing using 603 sense (sequence GTTGGCCTTCCAATGGCTT, SEQ ID NO:41) and 503 antisense (sequence GTTCCTTCACAAAGAT, SEQ ID NO:42) oligonucleotides was performed by GATC subcontractor Eurofins Genomics Company.

Transfection of DHODH variants was performed by electroporation using MaxCyte STX. They were performed in OC-100 process assemblies as described above.

One day after transfection, cells were centrifuged and resuspended in CD CHO selective medium supplemented with 6 mM glutamine and 25 μM teriflunomide for 9E4 CHO cells, and without teriflunomide for clones K02 and K019 at a concentration of 1x10 ⁶ cells/ml.

After two passages, mAb-A production began in CD OPTiCHO medium supplemented with 30% FeedB and 6 mM glutamine and 25 μM teriflunomide for 9E4 CHO cells, and without teriflunomide for clones K02 and K019 at a concentration of 0.3x10 ⁶ cells/ml.

On day 14 after transfection, cells were centrifuged at 200 x g for 10 min at 25°C. The supernatant was filtered through a 0.22-μm PES filter, and the antibody titer was measured using an Octet instrument.

As shown in FIG. 4, no significant effect of the R138C and R346W mutations in DHODH was observed on WT CHO 9E4 and DHODH KO clones. On the other hand, the G202A mutation allowed the production of antibodies on a gram scale using the WT CHO 9E4 line and increased the productivity of the DHODH clones KO2 and KO19.

Example 6: Protein production using the expression system of the present invention.

Various types of proteins were produced using the expression system of the present invention, in particular using cDNA for human DHODH, including the G202A mutation described above, in the confirmed CHO 9E4 KO2 and KO19 clones described in example 3 above, to confirm that the final productivity is at least in the same range as in the case of a prior art expression system that uses glutamine synthetase (GS) as a selectable marker.

The following proteins were produced:

- Lipase:

using the monocistronic PLBL2-His cDNA, and

using a plasmid encoding hDHODH G202A or a plasmid encoding hGS as selectable markers.

- Monoclonal antibody (mAb-B):

using a bicistronic cDNA encoding the VH and VL chains of the antibody, and

using a plasmid encoding hDHODH G202A or a plasmid encoding hGS as a selectable marker.

- Bispecific antibody:

using (i) a bicistronic cDNA encoding the VH and VL chains of an antibody, or (ii) two monocistronic cDNAs encoding the VH and VL chains of an antibody, respectively, and

using (i) a plasmid encoding hDHODH G202A (in the case of bicistronic cDNAs) or (ii) two plasmids encoding hDHODH G202A, or a plasmid encoding hDHODH G202A and a plasmid encoding hGS (in the case of monocistronic cDNAs) as selectable markers .

- Trispecific antibodies:

using two bicistronic cDNAs and

using a plasmid encoding hDHODH G202A or a plasmid encoding hGS as selectable markers.

The day before FectoPRO® transfection, cells were diluted to 1.5 x 10 ⁶ cells/ml in CD CHO medium supplemented with 6 mM glutamine and 5 mM uridine.

On the day of transfection, the cell suspension was diluted to 1.1x10 ⁶ cells/ml in CD CHO medium supplemented with 6 mM L-glutamine and 5 mM uridine. FectoPRO® reagent was vortexed for 5 s and stopped spinning before adding 25 µl/tube to an empty 50 ml tube. In a second 50-ml tube, 12.5 μg of cDNA was diluted in CD CHO medium, and the diluted DNA was immediately poured into pure FectoPRO® reagent. The solution was immediately homogenized and incubated for 10 min. The FectoPRO®/DNA transfection mixture was poured onto the cells and the culture was incubated at 37°C, 190 rpm and CO ₂ levels of 8%.

24 hours after transfection, cells were counted using an Invitrogen™ Countess™ instrument. The whole cell culture was centrifuged at 200 x g for 10 min. The pellet was resuspended in 2.5 ml of prewarmed production medium under the conditions described below.

For complex proteins containing 3 or more subunits, perform a second transfection, again using the transfection protocol described above.

- For lipase, monocistronic vector (protein with His tag)

Expression vectors used for transfection Cells Selection marker Wednesday on the day of transfection Selective medium after transfection AVEC-30778 KO2 DHODH_G202A CD CHO+Uri+Gln CD OPTiCHO+FeedB+Gln pBH6450 G.S. CD CHO+Gln CD OPTiCHO+FeedB+MSX AVEC-30778 KO19 DHODH_G202A CD CHO+Uri+Gln CD OPTiCHO+FeedB+Gln pBH6450 G.S. CD CHO+Gln CD OPTiCHO+FeedB+MSX AVEC-30778 9E4WT DHODH G202A CD CHO+Gln CD OPTiCHO+FeedB+Gln+TNF pBH6450 G.S. CD CHO+Gln CD OPTiCHO+FeedB+MSX

MSX: L-methionine sulfoximine; Gln: glutamine; Uri: uridine; TNF: teriflunomide

- For the monoclonal antibody mAb-B, the bicistronic, VH and VL encoding vector:

Expression vectors used for transfection Cells Selection marker Wednesday on the day of transfection Selective medium after transfection pVA4-00072 KO2 DHODH G202A CD CHO+Uri+Gln CD OPTiCHO+FeedB+Gln pVA4-00070 G.S. CD CHO+Gln CD OPTiCHO+FeedB+MSX pVA4-00072 KO19 DHODH G202A CD CHO+Uri+Gln CD OPTiCHO+FeedB+Gln pVA4-00070 G.S. CD CHO+Gln CD OPTiCHO+FeedB+MSX pVA4-00072 9E4WT DHODH G202A CD CHO+Gln CD OPTiCHO+FeedB+Gln+TNF pVA4-00070 G.S. CD CHO+Gln CD OPTiCHO+FeedB+MSX

MSX: L-methionine sulfoximine; Gln: glutamine; Uri: uridine; TNF: teriflunomide; FeedB: product feed.

- For bispecific antibodies, bicistronic or two monocistronic, encoding VH and VL vectors:

Expression vectors used for transfection. Cells Selection marker Wednesday on the day of transfection Selective medium after transfection pVA4-00073/pVA4-00076 KO2 GS/DHODH G202A CD CHO+Uri+Gln CD OPTiCHO+FeedB+MSX pVA4-00073/pVA4-00076 KO19 GS/DHODH G202A CD CHO+Uri+Gln CD OPTiCHO+FeedB+MSX pVA4-00073/pVA4-00076 9E4WT GS/DHODH G202A CD CHO+Gln CD OPTiCHO+FeedB+MSX/TNF pVA4-00074/pVA4-00076 KO2 DHODH G202A/ DHODH G202A CD CHO+Uri+Gln CD OPTiCHO+FeedB pVA4-00074/pVA4-00076 KO19 DHODH G202A/ DHODH G202A CD CHO+Uri+Gln CD OPTiCHO+FeedB pVA4-00074/pVA4-00076 9E4WT DHODH G202A/ DHODH G202A CD CHO+Gln CD OPTiCHO+FeedB+TNF pVA4-00077 KO2 DHODH G202A CD CHO+Uri+Gln CD OPTiCHO+FeedB pVA4-00077 KO19 DHODH G202A CD CHO+Uri+Gln CD OPTiCHO+FeedB pVA4-00077 9E4WT DHODH G202A CD CHO+Gln CD OPToCHO+FeedB+TNF

MSX: L-methionine sulfoximine; Gln: glutamine; Uri: uridine; TNF: teriflunomide; FeedB: product feed.

- For trispecific antibodies, bicistronic, VH and VL encoding vectors

Transfection Cells Selection marker Wednesday on the day of transfection Selective medium after transfection pVA4-00080/pVA4-00081 KO2 GS/DHODH CD CHO+Uri+Gln CD OPTiCHO+FeedB+MSX pVA4-00080/pVA4-00081 KO19 GS/DHODH CD CHO+Uri+Gln CD OPTiCHO+FeedB+MSX pVA4-00080/pVA4-00081 9E4WT GS/DHODH CD CHO+Gln CD OPTiCHO+FeedB+MSX/TNF

MSX: L-methionine sulfoximine; Gln: glutamine; Uri: uridine; TNF: teriflunomide; FeedB: product feed.

72 hours after transfection, cells were counted using an Invitrogen™ Countess™ instrument, and protein production was increased with another media change. In addition, the viability of transfected cells was measured 3 and 7 days after transfection.

On day 14 after transfection, cells were centrifuged at 200 xg, 10 min and 25°C. The supernatant was filtered through a 0.22-μm PES filter, and the protein titer was measured using an Octet instrument.

The following results were obtained.

a) Lipase

CHO cell viability at day 3 CHO cell viability at day 7 Living cells (10 ⁶ ) Viability (%) Living cells (10 ⁶ ) Viability (%) LIPASE DHODH_G202A KO2 4.2 90 5.7 94 LIPASE GS KO2 2.5 80 5.9 80 LIPASE DHODH_G202A KO19 4.4 92 5.9 80 LIPASE GS KO19 2.3 77 5.3 85 LIPASE DHODH_G202A 9E4 2.8 78 5.4 90 LIPASE GS 9E4 3.5 76 5.9 93

Lipase production on day 14 is shown in FIG. 5.

These results indicate that both DHODH KO cell lines have the ability to produce lipase in the absence of selective pressure from teriflunomide, thereby reducing toxicity to the producing cells.

In addition, the DHODH KO cell lines were able to produce lipase in the same range as the prior art GS system (in wild-type CHO 9E4 cells), i.e. 1 g/l on a 25 ml scale.

b) Monoclonal antibody mAb-B

CHO cell viability at day 3 CHO cell viability at day 7 Living cells (10 ⁶ ) Viability (%) Living cells (10 ⁶ ) Viability (%) mAb-B DHODH_G202A KO2 3.2 94 6.1 96 mAb-B GS KO2 1.7 74 4.7 88 mAb-B
DHODH_G202A KO19 4.2 95 6.5 95 mAb-B GS KO19 1.9 73 4 85 mAb-B
DHODH_G202A 9E4 1.5 72 4.5 90 mAb-B GS 9E4 2.3 76 5.3 96

mAb-B monoclonal antibody production at day 14 is shown in FIG. 6.

These results indicate that the DHODH KO cell lines behaved differently during the production of this particular antibody. Indeed, although both clones showed greater productivity than prior art teriflunomide, clone KO19 showed significantly greater productivity than clone KO2.

In the best KO cell line (K019), antibody production was in the same range as the prior art GS system (in wild-type CHO 9E4 cells), approximately 0.67 g/L on a 25-ml scale.

Additionally, increased viability was observed when using KO cell lines.

c) Bispecific antibody

CHO cell viability at day 3 CHO cell viability at day 7 Living cells (10 ⁶ ) Viability (%) Living cells (10 ⁶ ) Viability (%) Bispe GS/DHODH_G202A KO2 2.4 80 5.3 91 Bispe GS/DHODH_G202A KO19 2.5 84 5.4 92 Bispe GS/DHODH_G202A 9E4 1.9 69 4.1 90 Bispe DHODH_G202A/DHODH_G202A KO2 1.5 84 4.5 94 Bispe DHODH_G202A/DHODH_G202A KO19 1.6 86 4.8 95 Bispe DHODH_G202A/DHODH_G202A 9E4 1.6 72 5.1 91 Bispe DHODH_G202A KO2 1.7 85 4.8 94 Bispe DHODH_G202A KO19 1.8 90 4.9 95 Bispe DHODH_G202A 9E4 1.5 68 4.5 91

Bispecific antibody production at day 14 is shown in FIG. 7.

Bispecific antibodies are not produced as efficiently as monospecific antibodies in all cases tested. However, despite the difficulties in producing bispecific antibodies of this type, the productivity of the best KO cell line was in the same range as the prior art GS system (in wild-type 9E4 CHO cells), approximately 0.145 g/L on a 25-ml scale .

Additionally, increased viability was observed when using KO cell lines.

d) Trispecific antibody

CHO cell viability at day 3 CHO cell viability at day 7 Living cells (10 ⁶ ) Viability (%) Living cells (10 ⁶ ) Viability (%) Trispe GS/DHODH_G202A KO2 2.8 78 5.7 90 Trispe GS/DHODH_G202A KO19 3.7 86 5.9 95 Trispe GS/DHODH_G202A 9E4 2.2 67 4.6 90

Trispecific antibody production at day 14 is shown in FIG. 8.

Under all conditions, the two KO cell lines performed in the same range as the prior art selection system, i.e. 0.5 g/l.

Thus, this example confirms that KO DHODH CHO clones are suitable for expressing various forms of proteins (proteins, monoclonal antibodies, bispecific antibodies, trispecific antibodies). These clones can even be used for double transfection to produce complex proteins.

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LIST OF SEQUENCES

<110> SANOFI

<120> Cell line containing a new selection marker and its application

for the production of proteins

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130 135 140

Arg Tyr Gly Phe Asn Ser His Gly Leu Ser Val Val Glu His Arg Leu

145 150 155 160

Arg Ala Arg Gln Gln Lys Gln Ala Lys Leu Thr Glu Asp Gly Leu Pro

165 170 175

Leu Gly Val Asn Leu Gly Lys Asn Lys Thr Ser Val Asp Ala Ala Glu

180 185 190

Asp Tyr Ala Glu Gly Val Arg Val Leu Gly Pro Leu Ala Asp Tyr Leu

195 200 205

Val Val Asn Val Ser Ser Pro Asn Thr Ala Gly Leu Arg Ser Leu Gln

210 215 220

Gly Lys Ala Glu Leu Arg Arg Leu Leu Thr Lys Val Leu Gln Glu Arg

225 230 235 240

Asp Gly Leu Arg Arg Val His Arg Pro Ala Val Leu Val Lys Ile Ala

245 250 255

Pro Asp Leu Thr Ser Gln Asp Lys Glu Asp Ile Ala Ser Val Val Lys

260 265 270

Glu Leu Gly Ile Asp Gly Leu Ile Val Thr Asn Thr Thr Val Ser Arg

275 280 285

Pro Ala Gly Leu Gln Gly Ala Leu Arg Ser Glu Thr Gly Gly Leu Ser

290 295 300

Gly Lys Pro Leu Arg Asp Leu Ser Thr Gln Thr Ile Arg Glu Met Tyr

305 310 315 320

Ala Leu Thr Gln Gly Arg Val Pro Ile Ile Gly Val Gly Gly Val Ser

325 330 335

Ser Gly Gln Asp Ala Leu Glu Lys Ile Arg Ala Gly Ala Ser Leu Val

340 345 350

Gln Leu Tyr Thr Ala Leu Thr Phe Trp Gly Pro Pro Val Val Gly Lys

355 360 365

Val Lys Arg Glu Leu Glu Ala Leu Leu Lys Glu Gln Gly Phe Gly Gly

370 375 380

Val Thr Asp Ala Ile Gly Ala Asp His Arg Arg

385 390 395

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> appropriate DNA fragment to create gRNA

<400> 5

ggatgcagcc atcatccttg 20

<210> 6

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence1)

<400> 6

caccgggatg cagccatcat ccttg 25

<210> 7

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence1)

<400> 7

aaaaccaagg atgatggctg catcc 25

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence2)

<400> 8

caccggatgc agccatcatc cttgg 25

<210> 9

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence2)

<400> 9

aaaacccaag gatgatggct gcatc 25

<210> 10

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence3)

<400> 10

caccggcagc catcatcctt ggggg 25

<210> 11

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence3)

<400> 11

aaaacccccc aaggatgatg gctgc 25

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence4)

<400> 12

caccggccat catccttggg ggagg 25

<210> 13

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence4)

<400> 13

aaaaccctcc cccaaggatg atggc 25

<210> 14

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence5)

<400> 14

caccggctat tcgcttcacg tccct 25

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence5)

<400> 15

aaaacaggga cgtgaagcga atagc 25

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence6)

<400> 16

caccggcctc tacaaactgg gcttt 25

<210> 17

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence6)

<400> 17

aaaacaaagc ccagtttgta gaggc 25

<210> 18

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence7)

<400> 18

caccgggctt tgggtttgtc gaggt 25

<210> 19

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence7)

<400> 19

aaaacacctc gacaaaccca aagcc 25

<210> 20

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence8)

<400> 20

caccggctgg tctgaggagc ctaca 25

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence8)

<400> 21

aaaactgtag gctcctcaga ccagc 25

<210> 22

<211> 14437

<212> DNA

<213> Cricetulus griseus

<220>

<221> miscellaneous sign

<222> (1992)..(2015)

<223> target of plasmids pBH6840 KO DHODH SEQ1 and pBH6841 KO DHODH

SEQ4

<220>

<221> miscellaneous sign

<222> (2129)..(2147)

<223> target of plasmid pBH6842 KO DHODH SEQ5

<220>

<221> miscellaneous sign

<222> (4015)..(4047)

<223> target of plasmid pBH6843 KO DHODH SEQ7

<400> 22

ccgagcccac attctccaat ggagtggagg cagaggcggg cccagtggca gaaggagcat 60

ggcgtggaga cagatgagag tgagtctgtt gcgggtgctt acgcttgttc ggaaaacagg 120

ttgggagtgg acgaggctgg gagattgaaa ggctggggcc cttgcagtgt gggctgcatg 180

tgtgctcagc taggggcatg caaaaagcaa gcgtgctggg cagaggctga gttgattgtg 240

agagatcgcg ggcttttttt tttttttttt tcttaaactg agctagtatc gggcccctgt 300

gagcacatgg tggctagcat tatggaaagg atccagggcg gaatgaaata agacacgcag 360

ccacccttcc agataaagtg tggttagaat gggagtcccg tgcgaagagt ccgtagattg 420

tgtccctggc ttgggaaaga gtgcgcagat attgtttctt gacacgtgag aatttctccc 480

tccagaggag ggagaatgaa tacttggttt gctgtacagg caaatctgat cacatcctct 540

acttccttat ttagtggaca aatcccatta aaaatataat cactgattgt ggtgaggggt 600

tgtgctgtga catgggagct ggaaccgaac tctgcactta accactgaac catctccagc 660

ccccaaatct gtattttggc tgacaccccc acatttctcc taccttactt taagaaatgc 720

tttcctttta gttccaaaca gacctgtctt cctctacccc attctctggc ccttctcatt 780

ggcttcagcc acctagagat tccttctcca gctgcttcct cctctttagc ttttgtgtct 840

ccttggaaag tcaaccttag cctggaagga gcctcaagat ccttccatta cttatctctt 900

cgtgtcagtt ttcttgggtt tgtgtttaca tggccattaa tttctctctt cattagatag 960

attacacact tctgaaggac agggacctgg tgtggtttgc ttgcctttgg tgaaggattg

1020

aatgaatgca ttagccactt ggcatctggc agtgataatt atcaaaaagc attgtgagcc

1080

tcctttgaac tagggtagtg attctctacc cagagtgtga agctatgttt gtattacaac

1140

cacttttata ttatacacat aggacatttc aggccagtaa cttttctagg tataatttta

1200

aaatatagac accatgtcct aactacagta taaaggagaa atgaaaacat gtatttcagt

1260

atgtagaagt cagaacacag ttcacctaca tagatggcca ggtgtttaag cctctattta

1320

gaaggtgtgc gttggtatac aaacagcact gtataccctt tctggtgggg ttttaggaac

1380

agtgaccaag tcttagtaat gttttgtgca aactgtacaa ttgtccttaa attacaaggt

1440

atctttattt ctgtaaaagt caccctgtac atgtaaacca tgtggttgtg tggtgaacta

1500

tagcttgctt cttgcctctc ctattatcct cagataatag gaagttttcc tcacataaat

1560

agcaatagca agttcaaaca tcatacagta catagggaaa ctagtgtgtt agctttaatt

1620

aaagaagaat attgacagga ctgatttctc cacaccaaat gtattagcat cattccctca

1680

ttaactagta aaaattttcc catgtgaccc ctagggggca gtactgcctg tcttgaaaat

1740

tccctgcctg gtccttagga aaatattcag ggctagaacc catgtaagca gtgtaggtac

1800

tgcatctacc tttgtagatg tgtaagtggg caggatatga agggggacag tgtgagcagg

1860

gaaggactgt cactttgtgt agttgttatg tcctctgggc aggtgtgaag ggtcaggaag

1920

ggtactgcag gctggtctga tggggttccc catggggcct tttctctccc ccactagaag

1980

cgggccctgg atgcagccat catccttggg gggaggaggac ttctcttcac ctcttacctg

2040

acagccacgg gcgatgacca tttctatgct gaatatctga tgccggccct gcagaggctg

2100

ctagacccag aatcagccca ccggctagct attcgcttca cgtccctggg gctccttcct

2160

cgagctacat ttcaggactc tgacatgctg gtaggtcccc cggtcaccct gtgttacatt

2220

tgtgtttgta ggggaggtca catccttcag cacttcggta ttcctctgct gccactgggc

2280

ttttgaaaac cagagctgca ggttcacaag cagctgggac ctggagccca gcccgaggaa

2340

gggacttttc ttctgtgtta caccctgtac ttacagaaca gcactcagca tatagccggt

2400

gcttacagct ttaatcctca cagataccct ttgagggagg tcctatctca tctccatcta

2460

caaatcagga aactgaggta cagactaagt aagtgacttg cccaaggtca cacagctggg

2520

aggtggcagg accaggattc aaacctatag agtttgactc cagtgtccaa ccttggtcac

2580

tggcctgcct tgctgagatc cctctgctga acaggaacat cttctggagt cattaagcag

2640

ctactggatg caggggacag ggtcagcagg gaagggctgt cacttgtaga tactgtgtcc

2700

tctgtctgga taggtgtgaa gtctcagagt gggtgctgca gcctggcctg atgggtccgt

2760

cctttccccc catcagaagc agaccctggc tgtagtcatc accctgaggg gtggaggact

2820

taagtcctag gcaacctcct gccccagcca cattcagcca caactcactc ctgtggccca

2880

cctggcacca tttataacag agccagtgtg gcccttctac tgtctctctg aatgctcccc

2940

ttttgagatg ggcctaggtt gtccaggttg gtctcaaact tggggtccca aatgatttca

3000

gaatctcagc ttcctgagta agtgggatca cacagtatgc accactgtgc gcagtggtcc

3060

ctgttccccg atagccaggc cggttgatac tcttagcccg attcaccttt ctcggttttc

3120

taagtcttca gttttctatc atgacgagac tctcctgggg gagggggatg gctgggtgct

3180

tctggaatgc ttggctgacc cggtctcctg attgaccggt tttaccataa tctcaaagaa

3240

gcctgacatc aacaccccag gctctgtcac acagccagg atgagaactg gcttgaggt

3300

agcctcaagg aaggcctgtt ctagctgaac ttctcgcctc atggttatga agtgctgcgt

3360

gcacattttt accttccttg tgctcattct cgtctcctgt ggattttgag agcatgtagc

3420

tttccccttg gggactggcc tctaagagct cactggcagc accattgcct gccagtgtcc

3480

acccttcagc tctccatggt gcccatgggc tggcttcttg acagttatgg aggaagggcc

3540

agctagggct tgtatgctag gctggctaca ggctacctca aggctggtga caccaaggac

3600

tccttctcct gcccaggttg agtagatagg aagatacaca gtaattgtgc aagacagcac

3660

ctgtctcctc ttcacttgtg gtgggacact atctttctct ccccactgcc ttgagacccc

3720

ctggtgacag actccagcag acatcagaag cacctgcaga atgctgatgc agatagatac

3780

cccagggtgt ccttcctccc aaagatccct attttctggg cccagtgatg cttagcaagc

3840

tccaggggaa ttcttataca gtatgctggg atccctgctg tgagaaccac ttggtatagc

3900

tgcttatacc tctgacctgt ctccttccca ggaagtgaga gtcctgggcc ataaattccg

3960

aaatccggta gggattgctg cgggatttga caagcacggg gaagctgtag atggcctcta

4020

caaactgggc tttgggtttg tcgaggtagg aagtgtgact ccccagcctc aggaaggaaa

4080

ccccagaccc agagtgttcc gcctcccgga ggaccaagct gtcattaaca ggtaggtggt

4140

ggctcaatgt catagggaca tctcctcccc tgctgggatc tctgagatgg aacctgtttt

4200

gtgcttttct catatgatca gtggacagtc tgtcctttgg gatagtcagg agcacctttg

4260

aacacctgtt gtgtactagg cagctctcgc cccaaccttg ggaaacagct gttaggattt

4320

taccaggtat tcccactgag gctctgagag ctggtgctgg ttgcaaagaa cagcttggga

4380

aggcttgggc tgttgggctt cagtgtgagc ttttttacat tgttgctcta ttgcctttgt

4440

ccaatgttgg ctaacaggaa tataatgatc tcacatgtaa tttgaaatat tctaatgatg

4500

cattaaaaac tagtgaactt gataatacat ttataattat tatattttta attattatga

4560

tttaattata aacagtgtgt tctaaagtca ttttaacctg cagtcaatgt aaaaatcatt

4620

ggttaactat cgtcctcttt ttattggttt ccggtccccc aatgccttta cttctggttc

4680

ctctcacttg gccagggctg gtggccacaa gcttcagcag cgttggtact gtacaggtga

4740

cccttgcttt caatgaacag ggacatggta ttcaaatgta cagctgctct taggaggaga

4800

atccttcgac tgtatgaagc cttgcggttt gttgttctgt atagttagta taactgcaaa

4860

gggaagacct tcgtactttt tgctaggaga tttttggaga acctccttgt ggggattttg

4920

gcaggcctgt ggcagtgctt cctactgctt gtttcaggaa actgatttac aagtcataga

4980

tctttctgtt acattacagg cagggactga tgtctttagg actggccaga caaaatactt

5040

gtgtttctga tttggctgtt acaagtgact tcgggtctta ggatgatggt acttgggaca

5100

gggagtggtg agggacaggc acctgtgctc ttgggcatgt gcgaacttag ccttcttgct

5160

catatacaca gtgtgtgttc agtataggca tgctaacgga tgggtattta cacctgcaga

5220

cccacatgcc acaatccatg cagtgttgct ctggctgcaa gcaacaaagt gatgtaacca

5280

gatctcattt gttatcattc ttgtcactag cacaaaataa ctgacagcag gcagcttaag

5340

gaatggggtc aggtattttg gctcacagtt tgaggggaca cgccctcccg gtggcaggag

5400

cctgaggcag agggtcactc tgtacctaca gtgaggaagc agcaagggaa ggatgctggt

5460

gcttgggtct ctttctccgt cttgctcagc tggggtgtga atccatggtg ctgcccatgt

5520

ttgcggtggg tcttccctgc tccattcaac cttcccagaa acagtcttcc agacacacaa

5580

caggtgtgtt tccatggtga ctctaagtca catctagttg acaatgaagc ataaccagtg

5640

gtgagagcat ggaaatggtc tcaggtacgt tttccaaact gccacctttt aattccaccc

5700

cttacctgtg ggtttatgtg ctccaggtac ggattcaaca gccacgggct ctcagtggtg

5760

gaacacaggc tacggggccag acagcagaag cagaacaagc tcactgcagg taaactcagg

5820

tgttgtggga gggacttact aactctatta caaagaaaca tggggagggc atgggattca

5880

gcaataagaa acaaatgagc aaacaattaa aaccccacag aacaactcag gggattgtcc

5940

agcttctacc acagcctcaa aggtctcaac cagatggcat aattcagcaa aaccttgctg

6000

gtctctggca tccatggaac ctgaattgtg tcttaactta ggggcttgaa gatcagcaga

6060

gagcatgtgt ggagctaaga tgcgccccga gttcatcttc ccacatcccg ccctgctgct

6120

gtccttctgt tcactgtgta aatagtgtgt attagttctg gaagagctac agaagagaat

6180

agagttgtca cactcctgtc cttccactca ggggaccagc tgtcctcaaa tctcacttcg

6240

gaaacaacct ttgcttttgg tggattctca tggagacgct ccagtttgtc ctctgtggtc

6300

tgcatggctc tctgaagtgc attttggggt caaagccctg ggtggtggcc cccctcccca

6360

tgtctccctg tgtggcttct gcagtccatg gagattttc tcctttgcct ctggaatgtc

6420

tttcaggagg tcttagtctc atttgatgtc cccatagtgt gacaattaag ccaaggactt

6480

ctgcatttat ttgaggagct aaggaagaga gggctgaagc atttgacgag tttagagggt

6540

gggaacagga aatgacaatg tttaaaaaaa cccctggttt tgacggaacc gggtctgcca

6600

gccctgtaga accttgaaca caatgtttgt ggtttgtggc gttctcctgc ctcctgcctt

6660

ccgtttcttc gttttaatgc cttctgtggg ctaggtttat tgccattgtc ctgtgtccac

6720

ccttcatcct cattctgtgc cctcacagtc actggagtga ggccaagttc tgtgtctttg

6780

ccctgcctac atgtgtctgc tgacagctag cagcctcata gaaacttttc atcagcacca

6840

tgcctaggaa ccctagtgga tagaagagac tgtgctttgt gtaaagtgta gttcagcctt

6900

cctgactcca gaactgtgac tgtgggcagg tgaccactaa tgaaaggctc agttgcctct

6960

tggaagccat actctcaatc cagtttactt gtgctaaagc ttacagtgat cctggcttgc

7020

tgggtgctag acttgaagct ttgagacctt gttctgtata tagtctaatc cctggctcca

7080

cccagcccac ttaggaaatt agttacacaa atcttacttt taagttgatg acccagaaga

7140

tgacgggttg tgagagatgg actgtgagaa gggggctagc aatacagtgg gggatgttgc

7200

ccgtgtcctc cctggcctgg tatgggtgag ctgacttggt gttctaggtc tggctggatt

7260

ggcagatgga cagcagcctg ggccactgac atactaggag ttgtaagaaa gttgagttgt

7320

aataaagatg aatccaggaa actcctttct gccaaagaga acatgagtct gaaagagcat

7380

ttttctagag gcttggcctt ttgagagtga caggggggcag gtgtgagtaa ttacacccag

7440

gccccaggag tgtcttgagg ctacagagat gtatcatcac cctaattata agttcttttc

7500

tcagagaaca agaaagattt attacacact tccatctact ttttaggcat gatagttgaa

7560

aacatttcct agtgttttcc tgtgtagctc aggctggcct cagacttaca gcaatcctcc

7620

tgcagactaa atttccatgt gtggcctcca gtgttttata cttttgtttt taatcatttc

7680

acatacagtc ccttgaccaa ggctcatgag attcaggttg ttcttgctga catgcccatc

7740

aatacctgcc tactctctct tttgttatct ctcaaaccac tgatctgtga atatagcaag

7800

tgtcattggg agtcatttta ttgctacata cttcaagaag agcagtagta tttgtttttt

7860

tccccatgtc cctgatttgt ctggactcag atccttggcc acccatgcag tcagtgttgg

7920

ggatgggttc catctcatgg attgggcctt aaatacaatc agattctggt tggttactcc

7980

cgcagctttg tgctactgtt tcactagtgc atccagggca ggtcatcatt gtagatcaaa

8040

agatgtgtag cggggttggt gtttaccttt ctcctttggt aacatgcaga atacctccca

8100

gtaccatgga caccaccaga gggatgaagg ctgtaggtag gcaccagctc gacttctccc

8160

tatttaataa cttgtgtagg tgttgtcttt agcaatagag ccttgtcagt tttcagagag

8220

caaccaatat ccttgacaat agcctgggtt gtttgggtat tctcatgggg cccctttggc

8280

caacaacttc tttgaaagtc tggtcaaatt cttcactaaa accatctggc actgggcatt

8340

ttttggttgg gaggctttta tgactgcttc tattttacta ggggttataa gtttgtttac

8400

attgcttatc cgatcttgat ttaactttag tagatggtat gtatcaagaa aattattcat

8460

ttattttcaa ttttccatt aggtagagta taggttttta aaacacatcc ctatgattct

8520

ttggatttcc ttggtgtctg ttgttatgtc actcttttca tctctaattt tgttaatttg

8580

gatctttcct ctttgccttt taattaattt ggctaagggt ttgtcaatct tactggtttt

8640

ctcaaagaac caactcttcg tttcattgat tctttgcatt atttttgtct gtttctattt

8700

cattgatttc agccctgagt ttgattattt cctgccatct actcctcctg ggtgtgactt

8760

cttgttgttc tagagctttc aggtgtgcta gtatgaaatc tctctaattt atgtaggcac

8820

ttagtgctac ataaagtttt gggtttttgt tttgttttga cctatttttc attcagtagt

8880

gagttgttca gtttcagttg ttcagtttcc atgagtttgt aagctttctg ttgtttttgt

8940

aggtgttgat attcagcttt aatctgtggt ggtatgatag ggtattattt cagttttctt

9000

gtatcttttg agacttgctt tatggcctag tatatggtca gttttggaga aagttccatg

9060

aggtgctgag aagaaacgtt ttgtgtttgg gtgaaatgtt ctataaatta ggtccattg

9120

gtttaacaca tcatttagct ccatcatttc tttgttcagt ttttgtctgg atgacctgtc

9180

tattgtcgag agttggggag tatcaaagta tcccactatg tgtgtgtgtg tgcggagggg

9240

tcaatatgtg atttaagcta tagtagagtt tcttttatga acttggatgc ctttgtgctt

9300

ggtgcataga tgtttagaat ttcaatatcc tcttggtggg tttttccttt gatgagtatg

9360

tagtttcctt ccctatctat ctcttgatta gatttgattt gaaatctatt ctgtcatata

9420

ttaaaatggc ttcacccatt tgctttcttg tccatttgct tggaatatct ttttccatcc

9480

ttttactctg aggtgatatc tatccttgat gttaaagtat gtttcttgga ttcagttaaa

9540

gaatgaatcc tatttttgaa cccaattttt tagtctgtgt cttatttatt gggaaattga

9600

ggccactgat atcagtgttt gttgattcct gttatattat tgttatggcg taggcccctt

9660

cccttttgat ttgctggtct gagattattt attcaggctg aagttttcct tgtagtacct

9720

tctatcaagc tggatttgta gacagaaacc acttaagttt cattttattg tagattgtca

9780

ttctttcttc atctctgtga ttaaatgttt tgctggctat agtagtctgg gctggcatct

9840

gtggtagaat gtctgcccag gacctttgac ttttagagtc tcgattgaag tcaggggtta

9900

ttctaatagg tttgtctgcc gttatatgtt atctggtctt tctctattgt ggcttttggt

9960

attctttctt tgttctgtac attctgtttt ttgattatta tgtgttgtgg ggaatttatt

10020

tttggcccag tccgtttggt gttctatttg tttcttatac catgataggc acttccttct

10080

ttaggttagg taacttttca tctgtgattt tgttgaaaat attttctgtg cctttgacct

10140

gggtttcttc tccttttgct atccctgtta cttatagatt tttggtgttt tcatagtatc

10200

ctagatttcc tggatgtttt gtgcctggat tttgttttct ttcttttttt gtagatttaa

10260

cattttcttt gactgcagta tccttgtctc ctatcttgtc ctcagtgctt gagactctgt

10320

cttccattc ttgtatgatt ttggtgaggc ttgcttctaa gatttctgtt ctagttccta

10380

aatttttcat ttccagcttt acctcaattt gggttttctt tagcaattcc atttcctctt

10440

tcatgtgttg aacaattttc ttcatttcat tgcactgttt gtgttttcat taatgggttt

10500

attcatatct tctttaggga ccttgaacat attcataata gctattctga gagccttgtc

10560

ttatgcttcc ctgtattgca tttctcggag cctgctgtgg tagggttgct gggctctagt

10620

ggagacatgt cgttctggct gttactgggt ttttacactg gtgtctaggc gactgggttt

10680

gagaagattg taattctagg tgctgatatc tggtcttgtt tttgttgggg tgatgttcag

10740

ttccttggtt tctgttgccc ccccccctca gggggggtgt ggtaactgga ttggttgcct

10800

ggtagggaat gcttctgaga tcctgccaga accctgccac tggcagtctt gggtagaaag

10860

tgtttctagg tgttgggagc tgacactaat gattgaggat gggttagaag cagtggtggc

10920

ggggagagtcc acaggaggag gagagctggg tgtgccacca gggtctgcac agaggcctgg

10980

gaatgagaac agagatgaag gtgaggcctc agcaggtagt ctgctacaag gttgggataa

11040

ggctggggaga ttggaacttg gggacaggag ggagagtgaa gatcgcagac ccatcccctg

11100

gccaggtggg ggaagcctgg aggagaagat ctgtgtgatc tgctggaaat gggtccctta

11160

gtaacttctt gaagtttctt tgattttgag gctgctgttg ggggtcccct gtatgccaag

11220

catgtggtat atcactgaga tagaatctcc atagccctgc ttcttttttg agactgggtc

11280

tcatatcggc caggttggcc ttgaactagc tatgttgacc ttgaactttt ccttacctat

11340

acctgagaac tgggattaca agtgcccatc acacccagct tcctattgtt tttgcttttt

11400

tcctagtatt taagcttctg gcttccccat taaaatttaa aagatgaaag gcttagtaca

11460

cgggaagcat ccttgaaggt gcacacactt gccatataga gaggatgaag tggttctaag

11520

tcatcaggca gcagccccag gatcagacag ttcacattct ccatcatctg gttcagggga

11580

gccccacctg tactctgaat gttgcctggg aaactgtggg gacacactct tgatatttac

11640

agatgggctg cctctgggaa taaacctggg gaagaataag acttcggagg atgctgctgc

11700

agactatgta gagggtgttc gtgtcctggg ccccttggct gactacctgg tggtgaatgt

11760

gtccagtccc aacactgctg gtctgaggag cctacaggga aaggctgagc tgcgccgcct

11820

gctggccaag gtgtgtcatt acaccatcac atgcctgttg tccttactcc ttttcattct

11880

tcaggtgaag attcaggaca actgaggaga aactgttctt cacagctggc ctaggagccc

11940

tcatcacaca ttttccaagt accctctcat gtctcacagc cccggtcata cacaatagac

12000

agttcactgc tttaaaacca caggcaagca gagcagagca ggcggggctc ccatgtatca

12060

ctctgtcccc agaactgtga atgctcagca cttgtgacac acctccttgt ttgttttcag

12120

gaacaaaatc aaaacccttg agcagttatc tcggcggggg gtgcgggggt gggggaattg

12180

ctggtgcttt agttgacctt aggtgaaaat gctggcctgc actgcggggc tatgggctgg

12240

gtccccatct ctggcttgtc aacctaggtg ctgcaggaga gggacgcttt gaagggagcg

12300

cagaagccag cagtgctggt gaagatcgcc cccgacctca cggcccagga caaggaggac

12360

attgccagtg tggcgagaga ggtttgagtt ggggtggtcc agggcagggt gggggtagcc

12420

ttcatcgtcc actgctgctg ggataacaca gaagggcatg attggtgact tcctctgtgt

12480

gaagtggaca gctggttgat tgtctgtcat tgtatagttc atctggtagc aaacagtgag

12540

tttgaaaatg tgtttggtga gtctttttct tgcttctgat ctgtcttcat ccagaaagtc

12600

tgaggcctgt gctagtctct gccagtctca cctgggactt agaatggtgt ctgtcccttt

12660

ccagcttgtg taaacaccag gcttctctgg ctaaaaaggt agtaggaaca cagtctgctg

12720

ttctggactt cagtcttgtt ctactgtgta ctccctgaga tactcacaga cagcttgttc

12780

taagggtcaa actctgtgta ggcactgtga tgggttcagc aggagatgct gctgtgtctt

12840

cagcattgag aggctaattc tgatggcttc tccaatcaag atgtaggtga ggcctgtgag

12900

ggtcttgctc tgcagagctg gcccctggcc tggtggctgc ttcaatcctc aaaagacagt

12960

ttccttgagt acttcagatc catggcttaa ggctcttttt ctgtcttgtg ctgcagctgg

13020

gcattgatgg attaattgtc acaaacacca cagtgagtcg cccgactggc ctccaaggtg

13080

ctctgcgttc tgagatggga ggactgagcg ggaagccact ccgagatctg tcgacccaga

13140

ccatccggga gatgtacacc ctcactcaag gtaaggcttt tttgtttgtt tgtttgaggc

13200

aggatttctc tgtttaacac ctctggctgt cctggaactc acttggtaga ccaggctggc

13260

ctggaactca cagcttccct agtgctggga ttaaaggcat gtgccaccat tgcctggcta

13320

aggcaagact tctttggaca aatgtgaggt cctggatctt ccctcattca ctgtgtttgc

13380

tgagaactct ggttctgccc tcatcggctg tgtttgctgg gactgaggcc tgatggagcc

13440

ctgggtcttt agccctccct tcccggcctt gctatgtgct tctctccagg caggattccc

13500

attatcgggg ttggtggtgt gagcagtggg caagatgcgc tggagaagat ccaggcaggg

13560

gcctccctgg tgcagctgta cacggccctc accttcctgg ggccacccgt cgtggtcagg

13620

gtcaagcgtg agctggaggc acttctaaag tgagtagggt tcgatgcagc tgagacgtag

13680

aaagtgacac ttgtcatcag tctattgtgt actcccagag ggccggaggg aacactgagg

13740

gcatggtggg acgatttctc tgctgcagct ttggccaagg acaaacagtg ccagaggaat

13800

tcagaatgct tctgaggcag ggctcattac aaggcaggac ttgccacttg cccaggggct

13860

gggcatggag gatgaagtag taatttacat tgactcagtg tctggaagct gcaggttata

13920

aagtcacttc ccttcctgca cagaaggcct ggctgtacat atgtgcaggg gcctgggtga

13980

gggcagagta gcagtggttg taaggtgtgt tgggtgggac ggtgtggtaa gcggtgtgct

14040

catggtgagt gtgggcttcc ttaggacagg ctatttgttt tctgtccaga gagcggggtt

14100

ttaacacagt cacagaagcc attggagcag atcatcggag gtgacggttc ctgccagatg

14160

ccccatccag aacgtgccca ccaactcaag caagccttgt ggctgcatca taagaggaag

14220

atctgtctca agctatgtcc cttgactgtg tgacctggct ggactgcata agccagtcac

14280

ggttatcact agacagtaaa ggctttctct aatgagacca tgaactctac agtcactttc

14340

tggatctaag tcctgggatc cctcagtatt ataaggacat tggctctttg ggaggaaaaa

14400

tcatggagaa aataaagcca tttcaatctg ttttcaa 14437

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> appropriate DNA fragment to create gRNA

<400> 23

caaggatgat ggctgcatcc 20

<210> 24

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence1')

<400> 24

ggatgcagcc atcatccttg gtttt 25

<210> 25

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence1')

<400> 25

caaggatgat ggctgcatcc cggtg 25

<210> 26

<211> 395

<212> Protein

<213> Artificial sequence

<220>

<223> amino acid sequence of human DHODH G202A

<400> 26

Met Ala Trp Arg His Leu Lys Lys Arg Ala Gln Asp Ala Val Ile Ile

1 5 10 15

Leu Gly Gly Gly Gly Leu Leu Phe Ala Ser Tyr Leu Met Ala Thr Gly

20 25 30

Asp Glu Arg Phe Tyr Ala Glu His Leu Met Pro Thr Leu Gln Gly Leu

35 40 45

Leu Asp Pro Glu Ser Ala His Arg Leu Ala Val Arg Phe Thr Ser Leu

50 55 60

Gly Leu Leu Pro Arg Ala Arg Phe Gln Asp Ser Asp Met Leu Glu Val

65 70 75 80

Arg Val Leu Gly His Lys Phe Arg Asn Pro Val Gly Ile Ala Ala Gly

85 90 95

Phe Asp Lys His Gly Glu Ala Val Asp Gly Leu Tyr Lys Met Gly Phe

100 105 110

Gly Phe Val Glu Ile Gly Ser Val Thr Pro Lys Pro Gln Glu Gly Asn

115 120 125

Pro Arg Pro Arg Val Phe Arg Leu Pro Glu Asp Gln Ala Val Ile Asn

130 135 140

Arg Tyr Gly Phe Asn Ser His Gly Leu Ser Val Val Glu His Arg Leu

145 150 155 160

Arg Ala Arg Gln Gln Lys Gln Ala Lys Leu Thr Glu Asp Gly Leu Pro

165 170 175

Leu Gly Val Asn Leu Gly Lys Asn Lys Thr Ser Val Asp Ala Ala Glu

180 185 190

Asp Tyr Ala Glu Gly Val Arg Val Leu Ala Pro Leu Ala Asp Tyr Leu

195 200 205

Val Val Asn Val Ser Ser Pro Asn Thr Ala Gly Leu Arg Ser Leu Gln

210 215 220

Gly Lys Ala Glu Leu Arg Arg Leu Leu Thr Lys Val Leu Gln Glu Arg

225 230 235 240

Asp Gly Leu Arg Arg Val His Arg Pro Ala Val Leu Val Lys Ile Ala

245 250 255

Pro Asp Leu Thr Ser Gln Asp Lys Glu Asp Ile Ala Ser Val Val Lys

260 265 270

Glu Leu Gly Ile Asp Gly Leu Ile Val Thr Asn Thr Thr Val Ser Arg

275 280 285

Pro Ala Gly Leu Gln Gly Ala Leu Arg Ser Glu Thr Gly Gly Leu Ser

290 295 300

Gly Lys Pro Leu Arg Asp Leu Ser Thr Gln Thr Ile Arg Glu Met Tyr

305 310 315 320

Ala Leu Thr Gln Gly Arg Val Pro Ile Ile Gly Val Gly Gly Val Ser

325 330 335

Ser Gly Gln Asp Ala Leu Glu Lys Ile Arg Ala Gly Ala Ser Leu Val

340 345 350

Gln Leu Tyr Thr Ala Leu Thr Phe Trp Gly Pro Pro Val Val Gly Lys

355 360 365

Val Lys Arg Glu Leu Glu Ala Leu Leu Lys Glu Gln Gly Phe Gly Gly

370 375 380

Val Thr Asp Ala Ile Gly Ala Asp His Arg Arg

385 390 395

<210> 27

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence2')

<400> 27

gatgcagcca tcatccttgg gtttt 25

<210> 28

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence2')

<400> 28

ccaaggatga tggctgcatc cggtg 25

<210> 29

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence3')

<400> 29

gcagccatca tccttggggg gtttt 25

<210> 30

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence3')

<400> 30

cccccaagga tgatggctgc cggtg 25

<210> 31

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence4')

<400> 31

gccatcatcc ttgggggagg gtttt 25

<210> 32

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence4')

<400> 32

cctcccccaa ggatgatggc cggtg 25

<210> 33

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence5')

<400> 33

gctattcgct tcacgtccct gtttt 25

<210> 34

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence5')

<400> 34

agggacgtga agcgaatagc cggtg 25

<210> 35

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence6')

<400> 35

gcctctacaa actgggcttt gtttt 25

<210> 36

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence6')

<400> 36

aaagcccagt ttgtagaggc cggtg 25

<210> 37

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence7')

<400> 37

ggctttgggt ttgtcgaggt gtttt 25

<210> 38

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence7')

<400> 38

acctcgacaa acccaaagcc cggtg 25

<210> 39

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence8')

<400> 39

gctggtctga ggagcctaca gtttt 25

<210> 40

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide used to produce gRNA,

(sequence8')

<400> 40

tgtaggctcc tcagaccagc cggtg 25

<210> 41

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> 603 sense oligonucleotide

<400> 41

gttggccttc caatggctt 19

<210> 42

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> 503 antisense oligonucleotide

<400> 42

gttccttcac aaagat 16

<210> 43

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> A sequence including the target sequence and

PAM.

<400> 43

ggatgcagcc atcatccttg g 21

<210> 44

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> DHODH exon2 sense sequence region including

sequence n°1 for CrispR.

<400> 44

gacgaaacac cgggatgcag ccatcatcct tggttttaga 40

<210> 45

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> DHODH exon2 antisense sequence region,

including sequence n°1 for CrispR.

n°1

<400> 45

tctaaaacca aggatgatgg ctgcatcccg gtgtttcgtc 40

<---

Claims (32)

1. A mammalian cell line for producing a recombinant protein, comprising an endogenous dehydroorotate dehydrogenase (DHODH) gene that is partially or completely inactivated and an expression vector suitable for producing the recombinant protein, wherein the expression vector includes a sequence encoding DHODH, and wherein the cell line is selected from Chinese hamster ovary (CHO) cell lines, HEK293, HKB11, PER-C6, HT1080, Jurkat, Daudi, Raji and CAP cell lines. 2. The cell line according to claim 1, which is a Chinese hamster ovary (CHO) cell line. 3. The cell line according to claim 1 or 2, which is obtained by a) inactivation of the endogenous DHODH gene in the cell and b) culturing the cell in a culture medium containing uridine under conditions suitable for creating a cell line in which the endogenous DHODH gene is partially or completely inactivated. 4. The cell line according to claim 3, in which the endogenous DHODH gene is inactivated using a gene editing method. 5. The cell line according to claim 4, in which the endogenous DHODH gene is inactivated using a method using the CRISPR-Cas9 system. 6. Cell line according to any one of paragraphs. 1-5, in which one or more or all alleles of the endogenous DHODH gene are partially or completely inactivated. 7. Cell line according to any one of paragraphs. 1-6, wherein the expression vector contains a nucleotide sequence encoding exogenous mammalian DHODH and at least one expression cassette for expressing a recombinant protein, wherein said exogenous DHODH includes a sequence that is at least 60% identical to the sequence of SEQ ID NO: 2 or sequence SEQ ID NO: 4. 8. Expression system, including: (i) a mammalian cell line comprising an endogenous dehydroorotate dehydrogenase (DHODH) gene that is partially or completely inactivated, where the cell line is selected from Chinese hamster ovary (CHO) cell lines, HEK293, HKB11, PER-C6, HT1080, Jurkat, Daudi cell lines , Raji and CAP, and (ii) an expression vector comprising a nucleotide sequence encoding mammalian DHODH and at least one expression cassette for expressing a recombinant protein, wherein said DHODH comprises a sequence that is at least 60% identical to the sequence of SEQ ID NO: 2 or the sequence of SEQ ID NO: : 4. 9. Cell line according to any one of paragraphs. 1-7 or the expression system of claim 8, wherein said nucleotide sequence encoding DHODH comprises the sequence SEQ ID NO: 1 or the sequence SEQ ID NO: 3. 10. Cell line according to any one of paragraphs. 1-7 or 9 or the expression system according to claim 8 or 9, in which the specified recombinant protein is a monoclonal antibody. 11. Cell line according to any one of paragraphs. 1-7, 9 and 10 or the expression system according to any one of paragraphs. 8-10, wherein said vector contains a first expression cassette suitable for cloning an antibody light chain and a second expression cassette suitable for cloning an antibody heavy chain. 12. A kit for the production of recombinant protein, comprising (i) a cell line according to any one of paragraphs. 1-7 and 9-11, or the expression system according to any one of paragraphs. 8-11, and (ii) a culture medium lacking uridine, in particular further lacking a DHODH inhibitor. 13. A method for producing a recombinant protein in vitro, including the stages: And a1) providing a cell line according to any one of claims. 1-7 and 9-11; or a2) providing a mammalian cell line comprising an endogenous dehydroorotate dehydrogenase (DHODH) gene that is partially or completely inactivated, where the cell line is selected from Chinese hamster ovary (CHO) cell lines, HEK293, HKB11, PER-C6, HT1080, Jurkat, Daudi cell lines , Raji and CAP, and a2') introducing an expression vector comprising a nucleotide sequence encoding mammalian DHODH and at least one expression cassette for expression of a recombinant protein, wherein said DHODH includes a sequence at least 60% identical to the sequence of SEQ ID NO: 2 or the sequence of SEQ ID NO : 4, into the cell line provided in step a2); or a3) providing a mammalian cell line containing the endogenous DHODH gene, where the cell line is selected from Chinese hamster ovary (CHO) cell lines, HEK293, HKB11, PER-C6, HT1080, Jurkat, Daudi, Raji and CAP cell lines, a3') partial or complete inactivation of the endogenous DHODH gene in the cell line provided in step a3), and a3'') introducing an expression vector comprising a nucleotide sequence encoding mammalian DHODH and at least one expression cassette for expression of a recombinant protein, wherein said DHODH includes a sequence at least 60% identical to the sequence of SEQ ID NO: 2 or the sequence of SEQ ID NO: 4, into a cell line containing partially or completely inactivated endogenous DHODH gene obtained at stage a3'); B) culturing said cell line under conditions suitable for production of the recombinant protein; And C) isolating and/or purifying said recombinant protein. 14. The method according to claim 13, in which step B) is carried out in a culture medium devoid of uridine, in particular devoid, in addition, of a DHODH inhibitor. 15. The method according to claim 13 or 14, further comprising step D) of forming said recombinant protein into a pharmaceutical composition. 16. Use of a cell line according to any one of paragraphs. 1-7 and 9-11, the expression system according to any one of paragraphs. 9-11 or kit according to claim 12 for the production of recombinant protein. 17. Use according to claim 16, wherein the cell line, expression system or kit is used in combination with a culture medium lacking uridine, in particular in the absence of a DHODH inhibitor.

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