WO2015194704A1

WO2015194704A1 - Method for preparing recombinant glycoprotein having high sialic acid content, through glycosphingolipid synthesis cycle control

Info

Publication number: WO2015194704A1
Application number: PCT/KR2014/008896
Authority: WO
Inventors: 김정회; 곽찬영; 박승열; 심우용
Original assignee: 한국과학기술원
Priority date: 2014-06-18
Filing date: 2014-09-24
Publication date: 2015-12-23

Abstract

The present invention relates to a method for preparing a recombinant cell line producing recombinant glycoproteins having high sialic acid content by inhibiting glycosphingolipid (GSL) biosynthesis pathway in a cell line producing recombinant glycoproteins. Particularly, the present invention produces erythropoietin (EPO), containing a high content of sialic acid in a cell, from a cell line, which induces CGT inhibition by using siRNA and miRNA, specifically binding to ceramide glucosyltransferase (CGT), thereby increasing the in vivo half-life of a recombinant therapeutic protein, and thus can be useful in treatment using the same.

Description

[Specification】

[Name of invention]

High sialic acid content and recombinant glycoprotein production method by controlling glycolipid synthesis circuit

Technical Field

The present invention relates to a method for producing glycoproteins that increase sialic acid content by inhibiting ceramide glucosyltransferase (CGT) to regulate glycolipid biosynthesis pathways.

Background Art

Glycosylation is a major post-translational formula in mammalian cells and is divided into two classes, N— and ^ -linked glycosylation. ^ Linked glycosylation is attached to Asn (Asn-X-Ser / Thr motif) in many glycoproteins. Glycation affects enzyme activity, protein stability, and immunogenicity. N-acetylneuraminic acid O acetylneuraminic acid, sialic acid are charged sugar and attached to terminal galactose by 2-3 / 6 glycosidic linkage. This is known to affect the circulating half-life of glycoproteins released in order to prevent the recognition of a second galactose at the end from the asialoglycoprotein receptor (ASGPR) for degradation in hepatocytes. Since sialic acid regulates the stability of glycoproteins in vivo, enhanced sialylation of recombinant therapeutic proteins has been an issue in the last decade.

Sialic acid (sialic acid) is an essential component of the composition of sugar precursors. So far, about 50 kinds of sialic acid added sugar chain precursors have been found in nature. Sialic acid is important for biological phenomena in cells, such as intercellular interactions in mammals, the role of precursors in determining intracellular signals, and stabilizing glycoproteins. Play a role. In particular, sialic acid in vivo is known as one of the first recognized sugar chains during pathogen infection (Sasi sekharan and Myette, Am. Sci. 91: 432-441 (2003); Vimr and Li chtensteiger, Trends Microbiol. 10: 254-257) Sialic acid, which is present on the surface of cells, is also known as a component of polysaccharide oligosaccharides in the form of capsules for immune evasion reactions or cell protection even in the pathogenic microorganisms (Vimr et al., Microbiol. Mol. Biol. Rev. 68: 132-153 (2004)). When sialic acid is synthesized among pathogenic microorganisms itself, it is synthesized through sialic acid metabolic circuit in the cell, or sialic acid outside the cell is transferred into the cell through sialic acid transporter on the surface of the cell Sialic acid is synthesized via metabolic pathways (Vimr and Lichtensteiger, Trends Microbiol. 10: 254-257 (2002)). If not, however, sialic acid may be cleaved from the host or extracellular sialic acid sugar chain precursors by trans-sial idase, a sialic acid transferase on the cell surface, and sialic to the sugar chains on its cell surface. Acid is known to be added (Scudder et al., Patent 10-1083065 J. Biol. Chem. 268: 9886-9891 (1993)). Trans-sialidase has a trans-glycosidase activity and is sialic to various monosaccharides, disaccharides and oligosaccharides, including galactose from sialyl lactose. It is an enzyme that transfers acids.

CMP-Neu5Ac acts as a substrate in sialic acid biosynthesis. Sialylation on glycosylation carriers is carried to the luminal side of Golgi by the CMP—Neu5Ac carrier (CSAT) and attached to terminal galactose by sialyl transferase (ST). Enhancement of sialylation of recombinant therapeutic proteins is promoted as the potential linkage glycosylation sites increase. In addition, sialylation of recombinant therapeutic proteins is increased by overexpression of key enzymes for sialic acid biosynthesis such as galactosyltransferase (GT) or sialyl transferase. Since CMP-Neu5Ac acts as a limiting factor in sialylation, the increase in CMP-Neu5Ac in the Golgi lumen may be due to supplementation of ManNAc or CST. Made with overexpression

Glycosphingolipid (GSL) is another form of glycosylation carrier expressed on the cell surface and plays an important role in cell growth, adhesion, and signaling. Glycolipids are ganglioseries (GlcNAcp4Galp4GkpCer), G1 oboser i es (Ga 1 cx4Ga 1 β4 (1 c (3Cer)), lactoseries (GlcNAc) according to glycan structure (¾ ³ Gal (3 ⁴ Glc (3Cer) and neo-lactoseries) (Galp4GlcNAcP3Galp4GIcpCer). Glycolipid synthesis transfers ceramides to c / golgi by the ceramide carrier protein (CERT) via a non-follicular carrier. Ceramide glucosyltransferase (CGT) produces GlcCer (glucosyl cerimide), a precursor of GSL, to a ceramide glucosyltransferase by a chemical inhibitor, EtD0-P4, or RNAi, which inhibits glycolipid synthesis. Inhibition results in the growth and migration of cells, while neutral glycolipids are transported to Ra ;? Golgi by FAPP2, while sialylated glycolipids are synthesized in the cisternae CMP-Neu5Ac is a glycoprotein It is used in allylation as well as glycolipids such as gangliosides, and the present inventors have endeavored to develop recombinant glycoproteins that produce high amounts of sialic acid, resulting in ceramide glucosyltransferase (CGT) inhibitors involved in glycolipid biosynthesis pathways or The present invention was completed by constructing a cell line inducing the CGT inhibition through siRNA and miRNA that bind to specific ^, and confirming that erythropoietin (EP0) containing a high content of sialic acid is produced therefrom. 【Detailed Description of the Invention】

[Technical problem] An object of the present invention is to produce a recombinant glycoprotein with high sialic acid content, comprising the step of inhibiting the glycospingol ipid (GSL) biosynthetic pathway in a cell line producing recombinant glycoprotein. It is to provide a method for producing a cell line.

It is also an object of the present invention to provide a recombinant cell line that produces a recombinant glycoprotein of high sialic acid content, which inhibits the glycolipid biosynthetic pathway in a recombinant glycoprotein producing cell line.

In addition, the object of the present invention

1) culturing the recombinant cell line of the present invention; And

2) to provide a method for producing a high sialic acid recombinant glycoprotein comprising the step of separating the high sialic acid content glycoprotein from the culture of step 1).

It is also an object of the present invention to provide a use of a recombinant cell line for the production of a recombinant glycoprotein of high sialic acid content, wherein the glycolipid biosynthetic pathway is inhibited in a cell line producing glycoprotein ¾.

Technical Solution

In order to achieve the above object, the present invention comprises a step of inhibiting glycospingol ipid (GSL) biosynthetic pathway in a cell line producing a recombinant glycoprotein, a high sugar ic acid (repair sugar) content It provides a method for producing a recombinant cell line for producing a protein.

The present invention also provides a recombinant cell line that produces a recombinant glycoprotein with high sialic acid content, which inhibits the glycolipid biosynthetic pathway in the recombinant glycoprotein producing cell line.

In addition, the present invention

1) culturing the additive recombinant cell line; And

2) provides a method for producing a high sialic acid recombinant glycoprotein comprising the step of separating the high sialic acid content glycoprotein from the culture of step 1). The present invention also provides the use of a recombinant cell line for the production of a recombinant glycoprotein of high sialic acid content, wherein the glycolipid biosynthetic pathway is inhibited in the cell line producing the glycoprotein. [Beneficial] effect ^'

Cell lines that induce the CGT inhibition via siRNA and miRNA that specifically bind to ceramide glucosyltransferase (CGT) of the present invention, e.g., EC2-lH9-miRl and EC2-lH9_miR2, are the By producing erythropoietin (EP0) containing sialic acid (sial ic acid), the stability of the recombinant therapeutic protein can be increased, which can be usefully used for the prevention and treatment of diseases.

[Brief Description of Drawings]

1 is a diagram showing the change of glycolipid (GSL) and sialic acid (sial ic acid) content when EtIXH treatment in EC2-1H9 cells, a cell line producing recombinant human EP0.

Figure 2 is a diagram showing the change in the content of glycolipid and sialic acid when treated with siRNA that inhibits ceramide glucosyltransferase (CGT) in EC2-1H9 cells.

Figure 3 is a diagram showing the change in the content of glycolipids and sialic acid in stable cells transformed with pcCGT-miRNA (stable cel l).

4 is a diagram showing the concentration of CMP-NeuNAc and sialic acid content of erythropoietin (EP0) in stable cells transformed with pcCGT-miRNA.

5 is a diagram showing cell growth and production of EP0 in stable cells transformed with pcCGT-miRNA.

[Best form for implementation of the invention]

Hereinafter, the present invention will be described in detail. The present invention provides a method for preparing a recombinant cell line for producing a recombinant glycoprotein having a high sialic acid content, comprising the step of inhibiting a glycospingolipid (GSL) biosynthetic pathway in a cell line producing a recombinant glycoprotein. To provide.

The glycoprotein may be sialic acid, erythropoietin, thrombopoietin, thrombopoiet, alpha-ant i trypsin, cholinesterase, chorionic gonadodot. Pin (chorionic gonadotropin), CTLA4Ig, factor VDKFactor VI, gamma-glutamyltransf erase, granulocyte colony-stimulating factor (G-CSF) and luteinizing hormone (LH) It is preferably any one selected from the group consisting of, more preferably erythropoietin or thrombopoietin, but is not limited thereto.

Inhibition of the glycolipid biosynthetic pathway preferably inhibits ceramide glucosyltransferase (CGT), and consists of the amino acid sequence set forth in SEQ ID NO: 3.

Inhibition of the ceramide glucosyltransferase may be performed by treating a ceramide glucosyltransferase inhibitor or transforming any one selected from the group consisting of antisense nucleotides, siRNAs, shRNAs and miRNAs that bind to ceramide glucosyltransferase mRNAs. desirable.

The ceramide glucosyltransferase inhibitor

P4 (D-t hr eo-l-phen 1 2-pa Imitoylami ηο-3-pyr rol idi no- 1-pr opano 1) ，

E t D0-P4 (D-thr eo-l- (3 ', 4'-ethy 1 ened i oxy) pheny 1-2-palmitoylami ηο-3-pyrrol idi no- 1-pr opano 1) and pOH- P4 (D-thr e 4 'one hydr oxy- 1 -pheny 1-2-palmitoylami ^η ο-3-pyrrol idi no- 1-propa no 1) is preferably, but not limited to Do not. . The siRNA is preferably composed of any one of SEQ ID NO: 4 to SEQ ID NO: 9 is not limited to this, the miRNA is preferably composed of any one of SEQ ID NO: 10 or 11, but is not limited thereto. .

The siRNA may comprise a single RNA strand of stem-loop structure comprising an independent sense RNA strand and a complementary antisense RNA strand homologous to the target sequence, or wherein the sense RNA strand and antisense RNA strand are connected by a loop. The siRNA is not limited to completely paired double-chain RNA portion paired with RNA, and may have a hairpin structure forming a stem-loop (st em-loop) structure, in particular shRNA (short hairpin RNA) It is referred to as. On the other hand, the double-chain or stem region may include a portion not paired by mismatch (base is not complementary), bulge (the base does not stand in one chain) and the like. The total length is 10 to 80 _. Base is preferably 15 to 60 bases, more preferably 20 to 40 bases. In addition, the loop region has no special meaning in the sequence, and only 3-10 bases are required to connect the sense sequence and the antisense sequence at appropriate intervals. Examples that have been widely used as loop regions of siRNAs are as follows: AUG (Sui et al., Proc. Nat l. Acad. Sci. USA 99 (8): 5515-5520, 2002), CCC, CCACC or CCACACCCPaul et al. , Nature Biotechnology 20: 505—508, 2002), UUCG (Lee et al., Nature Biotechnology 20: 500—505), CTCGAG, AAGCUUC Edi tors of Nature Cel l Biology Whither RNAi, Nat Cel l Biol. 5: 489-490, 2003), UUCAAGAGA (Yu et al., Proc. Nat l. Acad. Sci. USA 99 (9): 6047-6052, 2002) and TTGATATCCGCwww. genscr ipt. com≤] default spacer). The siRNA terminal structure is possible at both blunt and black cohesive ends. The cohesive end structure can be both a 3 'protruding structure and a 5' end protruding structure, and the number of protruding bases is not limited. For example, the number of bases may be 1 to 8 bases, preferably 2 to 6 bases. In addition, siRNA For example, low molecular RNA (eg, natural RNA molecules such as tRNA, rRNA, viral RNA, or artificial RNA molecules) may be included in the protruding portion of one end. Can be. The siRNA terminal structure does not need to have a cleavage structure at both sides, and may be a stem loop structure in which one terminal site of the double-chain RNA is connected by a linker RNA. The length of the linker is not particularly limited as long as it does not interfere with pairing of stem portions.

In addition, in the siRNA according to the present invention, it is possible that the sense RNA strand and / or antisense RNA strand contain at least one chemical modification in its sugar moiety, nucleobase moiety or ¾ ternucleotide structure. Such modifications may make it possible to inhibit the destruction of siRNA by nucleases in vivo. All chemical modifications that can enhance the stability and biocompatibility of siRNA according to the present invention in vivo are included in the scope of the present invention. Among the preferred modifications to the sugar moiety, mention may be made of the position 2 'of ribose, such as 2'-deoxy, 2'-fluoro, 2'-amino, 2'-thio, or 2'-0-alkyl. And preferably is a modification that takes place in the presence of a methylene bridge between positions 2 'and 4' of 2'-0-methyl or LNA replacing the normal 2'-0H group on the ribonucleotide. For nucleobases, 5-bromo-uridine, 5-iodo-uridine, N3-methyl-uridine, 2, 6-diaminopurine (DAP, 5-methyl-2'-deoxycytidine ，

5— (1-propynyl) -2'-deoxy-uridine (pdU),

It is possible to use modified bases such as 5- (1-propynyl) -2'-teoxycytidine (pdC) or bases incorporating cholester. Finally, preferred modifications of the internucleotide backbone include substituting phosphodiester groups in the backbone by phosphorothioate, methylphosphonate, phosphorodiamidate groups, or linked by peptide bonds. A backbone consisting of N- (2-aminoethyl) -glycine (PNA, peptide nucleic acid) is used. Various modifications (bases, sugars, backbones) are modified nucleic acids of the morpholino type (bases immobilized on morpholin ring and linked by phosphorodiamidate groups) or PNA (N- (2 linked by peptide bonds) Aminoethyl) to glycine units Immobilized base).

The expression vector for expressing the miRNA is a pcDNA ™ 6.2-GW / EmGFP-mir vector from Invitrogen, miRNASelect ™ pEGP-miR cloning & expression vector from CellBiolabs, or miRNASelect ™ pEP-miR cloning & expression vector, pmR-ZsGreenl vector from Ckmtech Or pmR-mCherry vector, a pCMV-MIR vector from OriGene, and a pLV-miRNA vector from BiOSETTIA, or any vector for expressing miRNA.

The transformation is any one commercially available transfection reagent selected from the group consisting of Lipofectamine (Lipofectamine), Dojindo's Hilymax, Fugene, jetPEI, Effectene and DreamFect; Calcium-phosphate, positively charged polymers, liposomes, nanoparticles, nucleofection (∞ (; 160 £ ^^, electroporation, heat shock, magnetofection) It can be carried out using any one selected from the group consisting of a method using the method.

The transformed cells are characterized in that glycolipids (GSL) are reduced. In the method, the cell line may use mammalian cells, yeast cells or insect cells (insect eel Is), the mammalian cells are Chinese hamster ovary cells (Chinese hamster ovary cells, CHO), HT-1080, human lymphoblastoid, SP2 / 0 (mouse myeloma), NS0 (mouse myeloma), baby hamster kidney cells (BHK), human embryonic kidney cells, HEK), PERC.6 (human retinal cells) and EC2-1H9 cells, preferably selected from the group consisting of, Chinese hamster ovary cells (Chinese hamster ovary cells (CHO) is most preferred, but not limited thereto. It doesn't work. EC2-1H9 cells that mass-produce erythropoietin of the present invention Plasmid vector pCMV-dhfr was digested with restriction enzymes Hindm and Apal and creno

The expression vector pEpoG-dhfr was obtained by modification to (klenow) and addition of the genomic gene EpoG of EP0. In addition, the expression vector pEpoC-dhfr was obtained by linking the plasmid vector pCMV-dhfr with the cDNA gene EpoC of EP0. Dhfr mutant CH0 cell line (ATCC CRL 9096) cells were transformed with expression vectors pEpoG-dhfr or pEpoC-dhfr to obtain transformant EC2 with human EP0 production capacity. These cells were cultured in IA-MEM medium containing MTX. Hypoxanthin 10 in DMEM / F12 medium (Gibco Inc.) with 10% calf serum

, Thymidine 10, glycine 50 // g / ml, glutamine 587 / g / ml, glucose 4.5 mg / ml, penicillin-straptomycin

(PIC) was added to the culture medium containing penicillin-streptomycin lOOIU / ml (Sigma).

Dhfr mutated CH0 cells passaged at 37 ° C. were maintained using a centrifuge ᅳ inoculated to 5χ10 ⁵ cells / ml in a dish of 6 cm in diameter, and washed twice with 0 MEMMEM MEM KGibco the next day).

In the dhfr mutated CH0 cell line, plasmid 5 / g was diluted in 1.5 ml of 0 ΡΉ MEM I, and Lipofectin (Gibco) 20 diluted in the same amount;

After leaving for 10 minutes, they were evenly put on the prepared CH0 cells. Incubated for 6 hours at 37 ° C, 5% CO ₂ incubator, 3ml of DMEM / F12 medium containing 20% calf serum was added, and further incubated for 24 hours. Cells obtained by treatment with 0.25% trypsin and centrifuged were aliquoted into 96 well plates to be 2 ^× 10 ⁴ cells per well. IA-MEM medium (Gibco) containing 10% of dialyzed serum (Gibco 26300-020), 550 g / ml of G418 was added thereto, and cultured for 2 weeks while changing medium at 4 day intervals. Dozens of surviving clones were obtained and indirect Elisa method (Engvall & Co., Ltd.) using goat anti-mouse IgG antibody (manufactured by Sigma) in combination with mouse anti-human EP0 antibody and peroxidase to measure EP0 production capacity of each clone. Perlman, J. Immunol. 109, 129, 1972), the expression vector pEpoG-dhfr or Among the transformants containing pEpoC-dhfr, the cell line EC2 producing the most EP0 was selected. The present invention also provides a recombinant cell line that produces a recombinant glycoprotein of high sialic acid content that inhibits the glycolipid biosynthetic pathway in the recombinant glycoprotein producing cell line.

The present invention also provides the use of a recombinant cell line for the production of a recombinant glycoprotein of high sialic acid content, in which a glycolipid biosynthetic pathway is inhibited in a cell line producing a glycoprotein.

The glycoprotein is erythropoietin, thrombopoietin, alpha-ant i trypsin, chol inesterase, chorionic gonadodot that sialic acid can bind to. Chorionic gonadotropin, CTLA4Ig, factor VHKFactor VI, gamma-glutamyl transferase, granulocyte colony-stimulating factor (G-CSF) and luteinizing hormone (LH) It is preferably any one selected from the group consisting of, more preferably erythropoietin or thrombopoietin, but is not limited thereto.

Inhibition of the glycolipid biosynthesis pathway is characterized by inhibition of ceramide glucosyltransferase (CGT).

The transformed cells are characterized in that glycolipids (GSL) are reduced. The cell line can use mammalian cells (ma 瞧 alian cells), yeast cells (yeast cells) or nasal cells (insect eel Is), the mammalian cells are Chinese hamster ovary cells (CHO), HT-1080, human lymphoblastoid, SP2 / 0 mouse myeloma, NS0 (mouse myeloma), baby hamster kidney cells (BHK), human embryos It is preferably one selected from the group consisting of human embryonic kidney cel ls (HEK), PERC.6 (human retinal cells) and EC2-1H9, and Chinese hamster ovary cel ls (CHO) Most preferably, but not limited to.

As used herein, "cel ll ine" refers to one of the specific cell types that can grow in a laboratory. Cell lines can generally be grown in a permanently established cell culture and can spread indefinitely in the appropriate fresh medium and space provided. Methods of establishing cell lines from isolated cells are well known in the art. In a preferred embodiment, the cell line is a stable cell line, the cell line can be selected according to the selection marker of a specific plasmid containing miRNA that inhibits CGT, in the present invention was selected using blastidine of the present invention In a specific embodiment, as a result of confirming the effect on the glycolipid and sialic acid by treatment with EtD0-P4 in the cell line producing recombinant human EP0 ᅳ glycolipid (GSL) of erythropoietin (recombinant human erythropoiet in (rhEpo)) was reduced and sialic acid content This increase was confirmed (see FIG. 1). In addition, the effects of CGT-siRNA treatment on glycolipid and sialic acid content in EC2-1H9 cells were not detected in siRNA-treated cells, but the glycolipids were decreased and sialic acid content in purified rhEPO. Was found to increase (see Figure 2). In addition, it was confirmed that glycolipids (GSL) were reduced in cell lines stably inhibiting CGT (EC2-lH9-miR 1 and EC2-lH9-miR 2) (see FIG. 3), and compared to EC2-1H9 control cell lines. It was confirmed that the acid content was increased (see FIG. 4), and cell growth patterns and rhEPO production were similar to those in control cells (see FIG. 5). Accordingly, the present invention provides a cell line that induces the CGT inhibition through siRNA and miRNA specifically binding to ceramide glucosyltransferase (CGT), eg For example, EC2-lH9-miRl and EC2-lH9-miR2 increase the stability of recombinant therapeutic proteins by producing erythropoietin (EP0) containing high levels of sialic acid in cells, thereby improving the stability of the disease in need thereof. It can be usefully used for prevention and treatment. Furthermore, the present invention

1) culturing the recombinant cell line; And

2) provides a method for producing a high sialic acid recombinant glycoprotein comprising the step of separating the high sialic acid content glycoprotein from the culture of step 1). The glycoprotein may be sialic acid-binding, erythropoietin, trambopoietin, alpha-antitrypsin, cholinesterase, chorionic gonadotropin,

It is preferably any one selected from the group consisting of CTLA4Ig, factor VIE, gamma-glutamyltransferase, granulocyte colony stimulating factor, and luteinizing hormone, and more preferably, but not limited to erythropoietin or trvopoietin.

The erythropoietin of the present invention is mainly used to treat or prevent CNS or peripheral nervous system diseases, eye diseases, cardiovascular diseases, cardiopulmonary diseases, respiratory diseases, kidneys, urogenital diseases, gastrointestinal diseases, and endocrine and metabolic abnormalities in humans with neurological or psychiatric symptoms. useful.

The glycoprotein separation method may be a known method, but preferably immunoaffinity chromatography is used. Accordingly, the present invention relates to cell lines that induce such CGT inhibition via siRNA and miRNA that specifically bind to ceramide glucosyltransferase (CGT), e.g., EC2-lH9-miRl and EC2—lH9_miR2, which contain high amounts of cells. Erythropoietin (EP0) containing sial ic acid to increase the stability of the recombinant therapeutic protein to produce neuronal or psychiatric symptoms It can be usefully used for the prevention and treatment of peripheral nervous system diseases and metabolic disorders. Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

Example 1 Cell Line Culture and EtD0 ~ P4 Treatment

EC2-1H9, a CH0 cell line producing recombinant human erythropoietin (EP0), was provided from Kangwon National University (Korea). The cells were treated with 10% dFBS (fetal calf dialysis; SAFC, US), 3.5 g / L glucose, 20 nM MTX (methotrexate, Sigma), and 1% antibiotic in a humidified atmosphere containing 37 ° C., 5% CO 2. Antifungals' solution (Gibco) is replenished

Kept at ΜΕΜ-α. Inhibitors of the synthesis of glycosphingolipid (GSL)

EtD0-P4 (D-thr e 1 ᅳ (3 ', 4' -ethyl ened ioxy) -pheny 1-2-palmitoylami no— 3_pyr roli dino— 1 ᅳ propanol) was added to James A. Seyman (Dr. James A Shayman, University of Michigan, USA).

Example 2 Cloning of Ceramide Glucosyl Transferase

Chinese hamster (Cr / ce / us griseus) UDP—glucose ceramide glucosyl transferase (UGCG or CGT) was cloned from Chinese hamster ovary cells (GenBank® accession numbers 丽 001246692). Total RNA from Chinese hamster ovary cells was extracted using Trizol® reagent (Invitrogen, Carlsbad, Calif.), And the CGT cDNA strand was extracted with RT-PCR (AccuPower RT-PCR PreMix; Bioeer). Synthesized. Forward primer (5'-CTC GAG ATG GCG CTG CT-3 '; SEQ ID NO: 1) and A reverse primer (5'-TCT AGA TTA TAC ATC TAG GAT TTC CTC TGC-3 '; SEQ ID NO: 2) was used. The cloned CGT was inserted into a pGEM-T easy vector (PROMEGA, Madison, Wis.), Resulting in pCGT. Gene sequences were verified by di-deoxy sequencing.

Example 3 Preparation of siRNA and miRNA Expression Vectors

In the coding region, the CGT cDNA sequence of the Chinese hamster was 95.86% and 93.92, respectively, with the mouse (GenBank acession no.116_011673), rat (GenBank accession no. NM_031795) and human (GenBank acession no. 匪 _003358), respectively. % And 92.32% match, and CGT mRNACGenBank accession no. SiRNA sequences (siRNA-675, SEQ ID NOs: 4 and 5; siRNA-926, SEQ ID NOs: 6 and 7; and siRNA-1098, SEQ ID NOs: 8 and 9) for inhibiting the candidate group It was synthesized by request. Additive oligonucleotides are listed in Table 1 below. EC2-1H9 cells were transiently transformed with siRNA for CGT or negative control using liposutamine 2000 transformation reagent (Invi trogen) according to the manufacturer's protocol.

For continuous downregulation, miRNA expression vectors were used using oligonucleotides CGT-1098_F (SEQ ID NO: 10) and CGT-1098_R (SEQ ID NO: 11) listed in Table 1 below. The resulting miR-1098 was inserted into a pcDNA ™ 6.2-GW / EmGFP-miR vector (Invitrogen) to obtain pcCGT-miR. The vector contains a blastidin selection gene and the resulting transcript produces functional miRNAs in the cell.

Experimental Example 1 Confirmation of EtD0 ~ P4 Treatment Effect in Recombinant Human EP0 Cell Line

In order to investigate the effects on the glycolipid and sialic acid after treatment with EtD0-P4 in cell lines EC2-1H9 cells in which recombinant human EPO (rhEPO) is produced, the following experiment was performed. Specifically, for EtD0-P4 treatment, three cells (5xl0 ⁶ ) were incubated in 100-mm plates with ΜΕΜ-α supplemented with 10% dFBS for 24 hours, and with ΜΕΜ-α without serum.

Wash twice and incubate for 48 hours with the addition of 1 μΜ EtD0-P4 / MEM-a.

As a result, as shown in FIG. 1, glycolipid (glycosphingolipid (GSL)) based on the synthesis of glucosyl ceramide was inhibited by a ceramide glycosyltransferase (CGT) inhibitor, EtD0-P4 (Lee et al. 1999). EtD0-P4 treatment was found to completely reduce the expression of major glycolipids (GSL), GMl, GM2 and GM3 in EC2-1H9 cell line (FIG. 1A). In contrast, EtD0-P4 treatment increased the sialic acid content of the recombinant erythropoietin (erythropoietin (EPO)) for EC2-1H9. In the relative case of the Wheat Germ Agglutinin (WGA) lectin blot, it was found that the sialic acid content of purified recombinant human erythropoietin (rhEpo) increased by 22% (FIG. 1B). And 1C). This indicates that GSL biosynthetic components such as sialic acid were used for glycosylation of glycoproteins for co-substrate (FIG. 1).

Experimental Example 2 Confirmation of Temporary Downregulation of CGT by siRNA Treatment

In order to investigate the effect on the content of glycolipid and sialic acid by CGT-siRNA treatment in EC2-1H9 cells, siRNA_675 (siRNAl), siRNA_926 (siRNA2) and siRNA_1098 (siRNA3) of <Example 3> and Table 1 below. Transformation was performed using.

Specifically, EC2-1H9 cells were transformed with three different 5 nM and 10 nM CGT-siRNAs (siRNAl, siRNA2 and siRNA3) using Lipofectamine ™ LTX and PLUS ™ reagents (Invitrogen). Transformed cells were dispensed at 96 cells in a 96-well tissue culture plate: Nunc at 10 cells / well, and then a stable cell line was placed at 10% dFBS (bovine) in a humidified atmosphere containing 37 ⁰ C, 5% CO2. Dialysis of fetal serum; SAFC, US), 3.5 g / L glucose, 20 nM MTXOnethotrexate, Sigma), and 1% Modified Eagle's medium -α (ΜΕΜ-α; supplemented with antibiotic-antifungal solution (Gibco);

Gibco) for 2 weeks. 24 hours after transformation, total RNA was isolated using Trizol ^® reagent and 1.0 / g reverse transcription with cDNA

RT-PCR was performed using PreMix (Bioneer). CDNA was used as a template to identify mRNA transcripts of Chinese hamster CGT by PCR. To amplify Chinese hamster CGT, the same primers used for CGT cloning were used. Amplified genes were separated on 0.8% agarose gel and observed by ethidium bromide staining.

Glycosphingol ipid (GSL) was subjected to chloroform / methanol (C / M; 2: 1, v / v) and isopropanol / nucleic acid / water (IPA / H / W; 55:25: 20) under sonication for 30 minutes. , V / v / v). GSL extracts were dissolved in 0.1 M NaOH / MeOH and incubated at 40 ° C. for 2 hours, neutralized with 1 N HC1, added nucleic acid and left for 5 minutes. The lower layer was evaporated under N ₂ air stream, dissolved in distilled water and applied to SepPak C ₁₈ cartridge (Varian, Palo Al to, CA). After washing with distilled water, the total GSL was eluted with C / M and evaporated. The residue, dissolved in C / M, was spotted on TLC folate (Si ica Gel 60 F-254, Merck), C / M / 0.2% CaCl ₂ (55: 40:10 v / v / v) And stained by spraying with 2% orcinol in 2 MH ₂ SO ₄ .

As a result, as shown in FIG. 2, the pCGT vector prepared in Example 2 was used as a positive control (FIG. 2A, lane 1), and transcription of Chinese hamster CGT was detected in EC2-1H9 control cells (FIG. 2). 2A, lane 2). siRNA treated cells did not detect expression of CGT. Glycolipids (GSL) were reduced in siRNA treated cells (FIG. 2B). Maackia Amur ens is Lect in KMAL I) In the case of the relative strength of the lectin blot, the sialic acid content of the purified rhEPO increased by 22% (FIG. 2C). Based on RT-PCR and lectin blot analysis, one siRNA candidate group was selected to prepare miRNA expression vectors.

Table 1

<Experiment 3> Confirmation of stable cell construction that CGT is continuously down-regulated

In order to investigate the effects on glycolipid and sialylation as glycan analysis in stable cells in which CGT is continuously down-regulated, the experiment was performed by the same method described in Experimental Example 2 above. Specifically, pcCGT-miRNA prepared in Example 3 was prepared using Lipofectamine ™ LTX and PLUS ™ reagent (Invitrogen) on EC2-1H9 cells. Transformed. Stable cell lines overexpressed miRNAs for CGT were selected using blastisidine. To detect expression of the transformed genes, total RA of each transcript was isolated and RT-PCR was used to detect transcripts of Chinese hamster CGT.

As a result, as shown in Figure 3 Chinese hamster CGT transcript

Slight detection was found in EC2-1H9 control cells (FIG. 3A, lane 3) and pcCGT-miR vector was used as a positive control (FIG. 3A, lane 1). Two clones (EC2-lH9-miR 1 and EC2-lH9-miR 2) did not show CGT gene expression (FIGS. 3A, lanes 4 and 5). In addition, glycolipids were shown to decrease in siRNA treated cells (EC2-lH9-miR 1 and EC2-lH9-miR 2) (FIG. 3B). It showed an increase in sialic acid in siRNA treated cells (FIG. X).

Experimental Example 4 Confirmation of Quantitative Analysis of CMP-sialic Acid Concentration in Stable Cells

In order to investigate the effect of sialuria-mutated rat GNE / MNK expression from the stable cells, intracellular CMP-sialic acid concentration was performed as follows.

Specifically, 1.0 X 10 ⁷ cells were treated with 75% (v / v) ethanol in ice-cold using a sonic cel l disruptor (Vibra cel l 130; Sonics & Mater ials). Dissolved. The soluble portion was centrifuged at 13,000 rpm, 4 ⁰ C for 10 minutes and lyophilized. To stabilize CMP-sialic acid, samples were resuspended in 120 ^ 40 mM phosphorylation buffer pH 9.2 and centrifuged. The supernatant was filtered through a membrane that passes only Dublin than ^{10,000丽(Microcon ®; Mi ll} ipore). Sugar nucleotides were separated on a CarboPac PA-1 column (Dionex) and detected at Abs ₂₆₀ with an absorbance detector (model 486 tunable UV vi sible absorbance detector). Intracellular CMP-sialic acid levels were normalized with respect to cell number.

As a result, the concentration of CMP-NeuNAc in the cells as shown in Figure 4A It was confirmed that there was no difference between the control cell line and the engineered cell line (Fig.

4A).

Experimental Example 5 Analysis of Sialic Acid Content of rhEPO in Stable Cells

In order to analyze the sialic acid content of purified rhEPO in the stable cells,

The following experiment was performed using the 0PD-labeling method.

Specifically, EC2-1H9 cells and selected cell lines contain ΜΕΜ-α supplemented with 10% (v / v) dFBS, 3.5 g / L glucose, 1% (v / v) Ab-Am solution and 20 nM ΜΤΧ. there is

In a T-175 cm ² culture flask (Nunc) was dispensed at a concentration of 5.0 x 10 ⁶ cells. After 3 days, Seyang medium was replaced with serum-free medium (CH (^ S-SFM II; Gibco) and cells were further incubated for 2 days. Culture supernatants containing recombinant human EPO (rhEPO) were obtained and 0.45 m Filtration through pore-sized membrin To isolate rhEPO, the filtered culture medium was concentrated and buffer changed to PBS using ultrafiltration (AmiconUltra; Millipore) rhEPO was converted to mouse anti-human EP0 monoantibody. Purification by immunoaffinity chromatography made with CNBr-act ivated Sepharose 4B (Amersham Biosciences) combined with R & D systems After loading the sample and washing with PBS, the bound rhEPO was purified by 0.1 M glycine / 0.5 M NaCl,. Eluted to pH 2.8 and eluted immediately with 1.0 MTris / HCl, pH 9.0. Purified rhEPO was concentrated and dialyzed with distilled water by ultrafiltration (AmiconUltra; Millipore) .The concentration of rhEPO was Quant-iT ^™. Protein minutes The sialic acid content of rhEPO was determined using the 0PD-marker method (Anumula 1995) .The sialic acid content was determined using a 0PD—signal method for quantitative analysis of the sialic acid content of rhEPO. Isolate from purified rhEPO in 0.5 M NaHS0 ₄ at 80 ° C for min and for 40 min

Induced with 0PD ((广 phenylenediamine-2HCl; Sigma) at 80 ° C. 0PD-labeled Sialic acid was separated on a C18 reversed-phase C ₁₈ column (Shim-pack CLC-ODS; Shimadzu) using HPLC and detected at 230 nm emission and 425 nm excitation wavelength of fluorescence detector (Model 474; Waters). Human EP0 has ^ linked glycosylation sites (two sialic acid residues) and three ^ linked glycosylation sites (four sialic acid residues) so that up to 14 moles of sialic acid can be attached with 1 mole rhEPO.

As a result, as shown in FIG. 4B, the sialic acid content of rhEPO from EC2-lH9-miRl and miR2 cell lines was measured as 7.8 and 8.7 mol sialic acid / mol and rhEPO, respectively, which was compared with EC2-1H9 control cell line. The contents showed about 26. and 40. W increase, respectively (FIG. 4B).

Experimental Example 6 Confirmation of Cell Growth and rhEPO Production Analysis

Cell growth and rhEPO production assays were performed to determine gene overexpression effects on cell growth patterns and rhEPO production.

Specifically, exponentially growing cells were dispensed at a concentration of 0.5 × 10 ⁶ in 6-well culture plates (Nunc). After incubation for 3 days, the culture medium was replaced with serum-free medium (CH0-S-SFM II; Gibco) to produce rhEPO (Figures 5A and B, indicated by arrows). After trypsinization to isolate cells and staining with trypan blue, the number of viable cells was determined by counting with a hematocometer. The concentration of rhEPO excreted was measured using a sandwich ELISA assay. 96-well immune-plates (Nunc) were coated with rabbit anti-human EP0 polyclonal antibody (Sigma) at 4 ⁰ C for 18 hours and TBS-T (150 mM NaCl, 0.05¾> Tween 20 at room temperature for 1 hour). 10 mM Tris-HCb containing 7.4) was blocked with 2% BSA. After three washes with TBS-T, serially diluted rhEPO normalized and cultured media supernatants were loaded and incubated at 37 ° C for 2 hours. After washing three times with TBS-T, the plate was diluted 1: 2,000 at 37 ° C for 30 minutes.

Incubated with HRP-bound Gout anti-mouse IgG secondary antibody (Santa (： 12 ^ 016 (± 11010)) and washed three times with TBS-T. ELISA (3,3 ', 5, 5'tetramethylbenzidine; A 100 ^ TMB liquid substrate system for Sigma) was added to each well and the absorbance was measured at 370 nm using a microplate reader (Infinite M200 Megel lan, Tecan).

As a result, it was shown in FIG. 5 (FIG. 5).

Claims

[Range of request]

[Claim 1]

A method of making a recombinant cell line producing a recombinant glycoprotein of high sialic acid content, comprising the step of inhibiting a glycospingolipid (GSL) biosynthetic pathway in a cell line producing a recombinant glycoprotein.

[Claim 2]

The method of claim 1, wherein the glycoprotein is erythropoietin (EP0), thrombopoietin (thrombopoietin), alpha-antitrypsin, cholinesterase (cholinesterase), chorionic gonadotropin ( chorionic gonadotropin, CTLA4Ig, Factor VM, gamma-glutamyl transferase, granulocyte colony-stimulating factor (G—CSF) and luteinizing hormone (LH) Method for producing a recombinant cell line producing a recombinant glycoprotein of high sialic acid content, characterized in that any one selected from the group.

[Claim 3]

The method of claim 1, wherein the inhibition of the glycolipid biosynthesis pathway inhibits ceramide glucosyl transferase (CGT). [Claim 4]

The method of claim 3 wherein the ceramide glucosyltransferase of high sialic acid content, characterized in that consisting of the amino acid sequence of SEQ ID NO: 3 Method for producing recombinant cell line producing recombinant glycoprotein

[Claim 5]

The method of claim 3, wherein the inhibition of ceramide glucosaltransferase is selected from the group consisting of antisense nucleotides, siRNAs, shRNAs, and miRNAs that process a ceramide glucosyltransferase inhibitor or bind to ceramide glucosyltransferase mRNA. A method for producing a recombinant cell line for producing a recombinant glycoprotein having a high sialic acid content, characterized by transforming one.

[Claim 6]

According to claim 5, wherein the siRNA is a method for producing a recombinant cell line producing a recombinant glycoprotein of high sialic acid content, characterized in that consisting of any one of SEQ ID NO: 4 to SEQ ID NO: 9

[Claim 7]

The method of claim 5, wherein the miRNA comprises a nucleotide sequence of any one of SEQ ID NO: 10 or 11. 7.

[Claim 8]

The method of claim 5, wherein the transformed cell produces a high sialic acid recombinant glycoprotein, characterized in that the glycolipid (GSL) is reduced.

[Claim 9]

The method of claim 1, wherein the cell line is selected from the group consisting of mammalian cells (mammal ian cel ls), yeast cells (yeast cel ls) and insect cells (insect cel ls) Method for producing a recombinant cell line producing a recombinant glycoprotein of high sialic acid content, characterized in that any one. rclaim 10]

The method of claim 9, wherein the cells are Chinese hamster ovary cells (CHO), HT-1080, human lymphoblastoid, SP2 / 0 (mouse myeloma), NS0 (mouse myeloma), baby Baby hamster kidney cells (BHK), human embryonic kidney eel Is (HEK), PERC.6 (human retinal cells) and EC2-1H9, characterized in that any one selected from the group consisting of Method for producing a recombinant cell line to produce a recombinant glycoprotein of high sialic acid content.

[Claim 11]

A recombinant cell line producing a recombinant glycoprotein with high sialic acid content, which inhibits glycolipid biosynthesis pathways in a recombinant glycoprotein producing cell line.

[Claim 12]

The method of claim 11, wherein the glycoprotein is erythropoietin, thrombopoietin, alpha-antitrypsin, cholinesterase, chorionic gonadotropin, CTLA4Ig, Factor VI, gamma-glutamyltransferase, granulocyte colony stimulation A recombinant cell line producing a recombinant glycoprotein of high sialic acid content, characterized in that it is any one selected from the group consisting of factor and luteinizing hormone.

[Claim 13]

12. The recombinant cell line of claim 11, wherein the inhibition of the glycolipid biosynthetic pathway inhibits ceramide glucosyltransferase.

[Claim 14]

1) culturing the recombinant cell line of claim 11; And 2) A method for producing a recombinant high glycoprotein having a high sialic acid content, comprising separating the high sialic acid glycoprotein from the culture medium of step 1).

[Claim 15]

15. The method of claim 14, wherein the glycoprotein is erythropoietin, trambopoietin, alpha-antitrypsin, cholinesterase, chorionic gonadotropin, CTLA4Ig, Factor Vffl, gamma-glutamyltransferase, granulocyte colony stimulation Method for producing a high sialic acid recombinant glycoprotein, characterized in that any one selected from the group consisting of factor and luteinizing hormone.

[Claim 16]

Use of a recombinant cell line for producing a recombinant glycoprotein of high sialic acid content, wherein the glycolipid biosynthetic pathway is inhibited in a cell line producing a recombinant glycoprotein.