MXPA99010042A - Recombinant human erythropoyetine productive cellular line and the recombinant human erythropoyetine produced by this cell - Google Patents

Abstract

The present invention relates to gene encoding human erythropoietin (EPO) was obtained from human genomic DNA. The gene used does not include fragments of 5'and 3'flanking regions of the EPO gene that do not code for the protein. The gene was cloned into an expression plasmid for eukaryotic cells having as unique expression control elements the SV40 virus early promoter and its polyadenylation signal. The recombinant cells resulting from transfection with the genetic constructs used unexpectedly produces more than 50 mg of recombinant EPO per liter of culture medium and per day.

Description

LINE CELLULAR RECOMBINANT ERYTHROPOINTINETHYTHENE AND ERYTHROPOYETINEHUMAN RECOMBINANT PRODUCIDAPORESTA CÉLULA Technical Description of the Invention A cell line producing recombinant human erythropoietin (EPO), and the EPO produced by it. Isolation and construction of a gene coding for EPO, its cloning in plasmids suitable for the transfection of mammalian cells. The selection of EPO production lines, their cultivation and production of recombinant EPO.

Technical Field of the Invention The present invention relates to a recombinant EPO producing cell line that includes a coding sequence for EPO with a single promoter that regulates its expression. The present invention also relates to a method for producing EPO.

STATE OF THE ART EPO is a glycoprotein that stimulates the differentiation of erythroblasts in the bone marrow, thereby increasing the number of erythrocytes in the blood. The average life of erythrocytes in humans is 120 days, for which a human being loses 1/120 of their erythrocytes every day.

This loss must be continuously replenished to keep the number of red blood cells stable. The existence of the EPO was postulated from the beginning of the century and was definitely demonstrated by Reissman and Erslev in the early 50's. See Carnot, et al., C.R. Acad. Sci., (France), 143, 384-6 (1906); Carnot, et al., C.R. Acad. Sel, (France), 143, 432-5 (1906); Carnot, et al., C.R. Soc. Biol., 111, 344-6 (1906); Carnot, C.R. Soc. Biol., 111, 463-5 (1906); Reissman, Blood, 1950, 5, 372-80 (1950) and Erslev, Blood, 8, 349-57 (1953). The experiments of Reissman and Erslev were quickly confirmed by other researchers. See Hodgson, et al., Blood, 9, 299-309 (1954); Gordon, et al., Proc. Soc. Exp. Biol. Med., 86, 255-8 (1954) and Borsook, et al., Blood, 9, 734-42 (1954). The individualization of the production site sparked a great debate. Successive works led to identify the kidney as the main organ and peritubular interstitial cells as the synthesis site. See Jacobson, et al., Nature, 179, 633-4 (1957); Kuratowska, et al., Blood, 18, 527-34 (1961); Fisher, Acta Hematol, 26, 224-32 (1961); Fisher, et al., Nature, 205, 611-2 (1965); Frenkel, et al., Ann. N.Y. Acad. Sci., 149, 1, 292-3 (1968); Busuttil, et al., Proc. Soc. Exp. Biol. Med., 137, 1, 327-30 (1971); Busuttil, Acta Haematol, (Switzerland), 47, 4, 238-42 (1972); Erslev, Blood 44, 1, 77-85 (1974); Kazal, Ann. Clin. Lab. Sel, 5, 2, 98-109 (1975); Sherwood, et al., Endocrinology, 99, 2, 504-10 (1976); Fisher, Ann. Rev. Pharmacol. Toxicol., 28, 101-22 (1988); Jelkmann, et al., Exp. Hematol., 11, 7, 581-8 (1983); Kurtz, et al., Proc. Nati Acad. Sci., (USA), 80, 13, 4008-11 (1983); Caro, et al., J. Lab. Clin. Med., 103, 6, 922-31 (1984); Caro, et al., Exp. Hematol., 12, 357 (1984); Schuster, et al., Blood, 70, 1, 316-8 (1986); Bondurant, et al., Mol. Cell. Biol., 6, 7, 2731-3 (1986); Bondurant, et al., O /. Cell. Biol., 6, 7, 2731-3 (1986); Schuster, et al., Blood, 71, 2, 524-7 (1988); Koury, et al., Blood, 71, 2, 524-7 (1988); Lacombe, et al., J. Clin. Invest., 81, 2, 620-3 (1988); Koury, et al., Blood, 74, 2, 645-51 (1989). A smaller proportion, from 10% to 15% of the total EPO, is produced by the liver in adults. See Naughton, et al., J. Surg. Oncol, 12, 3, 227-42 (1979); Liu, et al., J. Surg. Oncol, 15, 2, 121-32 (1980); Dornfest, et al., Ann. Clin. Lab. Sci., 11, 1, 37-46 (1981); Dinkelaar, et al., Exp. Hematol., 9, 7, 796-803 (1981); Caro, et al., Am. J. Physiol., 244, 5 (1983); Dornfest, et al., J. Lab. Clin. Med, 102, 2, 274-85 (1983); Naughton, et al., Ann. Clin. Lab. Sci., 13, 5, 432-8 (1983); Jacobs, et al., Nature, 313, 6005, 806-10 (1985); Erslev, et al., Med. Oncol. Tumor. Pharmacother., 3, 3-4, 159-64 (1986). EPO is produced proportionally to the degree of tissue ipoxia, and its expression grows by increasing the number of producer cells. EPO is a protein that has shown great efficacy for the treatment of anemia caused by different factors, especially anemia of renal origin. However, its therapeutic availability was limited until recently by the lack of a mass production method, since the quantity and quality of the EPO obtained by any of the known extractive systems were insufficient. Recently, the use of recombinant DNA techniques has made it possible to obtain proteins in large quantities. The application of these techniques to eukaryotic cells has allowed the large-scale production of EPO. See US patents 5,688,679 (Powell), 5,547,933 (Lin), 5,756,349 (Lin), 4,703,008 (Lin), and 4,677,195 (Hewick et al.). Recombinant DNA techniques are widely known and used today. . They are based on the management of different genetic elements that allow, among other uses, to assemble and transfer genetic constructions, and that as a consequence make possible the production of recombinant proteins and the study of biological mechanisms. See Frank-Kamenetskii, "Unraveling DNA" [Samaia Glavnaia Molekula] (Addisonman Inc., Reading, Massachusetts, 1997); Brown, "Gene Cloning" (Chapman &Hall, London, England, 1995); Watson, et al., "Recombinant DNA", 2nd Ed. (Scientific American Books, New York, New York, 1992); Aberts et al., "Molecular Biology of the Cell" (Editions Omega, 1990); Innis et al, Eds., "PCR Protocols, A Guide to Methods and Applications" (Academic Press Inc., San Diego, California, 1990); Ehrlich, Ed., "PCR Technology, Principles and Applications for DNA Amplification" (Stockton Press, New York, New York, 1989); Sambrook et al., "Molecular Cloning, A Laboratory Manual" (Cold Spring Harbor Laboratory Press, 1989); Bishop et al., "Nucleic Acid and Protein Sequence, A Practical Approach" (IRL Press 1987); Reznikoff, Ed., "Maximizing Gene Expression" (Butterworths Publishers, Stoneham, Massachusetts, 1987); Davis et al, "Basic Methods in Molecular Biology" (Elsevier Science Publishing Co., New York, New York, 1986); Watson, "The Double Helix" (Penguin Books USA Inc., New York, New York, 1969) A rough description of the aspects of biology on which recombinant DNA techniques are based is the following: The genetic material of all living cells and some viruses are DNA (deoxyribonucleic acid) in the form of polymeric chains composed of four different nucleosides, each of which is a purine or pyrimidine linked to a deoxyribose that has, in turn, bound a phosphate group . These four nucleosides are: adenine (A); cytosine (C); guanine (G); and thymine (T). The DNA strands are formed by junctions between the nucleosides, in which the phosphate of the 5 'position of the deoxyribose of a nucleoside is attached to the 3' position of the deoxyribose of the above nucleoside. The synthesis happens in vivo from 5 'to 3', and this is the direction in which by convention the DNA sequences are described. The functional DNA is presented as a double helix of complementary bases, in which the chains are held together by the hydrogen bonds formed between the A of one chain and the T of the other, and between the C of a chain and the G of the other. There is talk of "Base pairs". The chains are also antiparallel, that is to say that the 5 'end of each propeller is complements with the 3 'end of the other, as outlined below: 5'- TACGTAG- 3' 3'- ATGCATC- 5 'The cellular synthesis of proteins begins once certain regions of the DNA are transcribed to messenger RNA, which It is then translated into protein. Each of these DNA regions coding for a protein is called a gene. Transcription consists in the synthesis of RNA strands (ribonucleic acid), by the copying by enzymes called RNA polymerases, of certain regions of the genes that are taken as a template. An antiparallel RNA strand is then obtained and complementary to the copied DNA. Each DNA A will correspond to a U in the RNA; to each C, a G; to each G, a C and to each T, an A. See Fig. 1. RNA differs from DNA in that it is much less Stable, the sugar that binds to purines and pyrimidines is ribose instead of deoxyribose and has uracil (U) instead of Thymine. Fig. 1. Template DNA 5 'ACGTAG_3_' 3 'synthesized mRNA -UGCAUC-5' If nucleated cells are involved, the synthesized mRNA can be processed in the nucleus (splicing) to result in mature mRNA. This process is not verified in bacteria because the They do not have a nucleus. The mature mRNA is then taken as a template to be translated into protein, in a a process in which transfer RNA (tRNAs, small RNA strands that transport amino acids and specifically align them to form the protein) and ribosomes participate. Each amino acid is encoded by three mRNA bases (triplet or codon). For example, the AUG sequence in the mRNA corresponds to the amino acid Methionine. The nucleotides of each mRNA are then translated to a specific amino acid sequence, i.e. to a protein. The process of transcription and translation that results in the synthesis of the protein encoded by the gene is called "protein expression." EXPRESSION The amount of protein expressed depends, among other factors, on certain regions of the DNA that are called promoters and that influence the number of times the process is carried out per unit of time. There are also DNA sequences that determine the termination of transcription (terminators), and codons that determine the end of the translation (stop codons). From the first years of the decade of the 70 'the "tools" were developed (restriction enzymes, etc.) and techniques that gave rise to recombinant DNA technology. In the present we can, among other applications of technology, isolate fragments of DNA, natural or not, and inserted into cells (bacteria, yeast, insect cells and mammals, among others) to get the cells thus modified to produce a heterologous protein, such as EPO. The proteins obtained by this process are called recombinant proteins.

The field of application is not limited to cells in culture, since the genes can also be incorporated into multicellular organisms (plants, insects, mammals and fish, among others). The expression of heterologous proteins basically requires the following elements: 1. A gene coding for the desired protein. It can be obtained by different technologies: A. isolation from genomic libraries; B. by in vitro synthesis of DNA strands. There are commercially available equipment that synthesize relatively short strands of DNA, with which a gene can be "armed"; C. amplification. It is a technology that allows a large number of times to copy a fragment of DNA that interests us, for example a gene; D. others, for example, from messenger RNA. 2. Suitable promoters to express the protein in the cell of interest and in the desired amount and time. 3. Appropriate terminators, so that the transcription is completed correctly. 4. Vectors. Genetic constructions that allow to direct the gene with its promoter and terminator to the interior of the cell that interests us, and to install the gene, either integrated in some chromosome or extra-chromosomally. According to the system used, the incorporated gene can remain indefinitely in the cell and be transmitted to its progeny, or be lost in a more or less short time. There are multiple vector systems: natural or modified viruses and plasmids, among others.

Even physical media such as microinjection can be used. Viruses and plasmids are obtained from nature and genetically modified in vitro to achieve the desired characteristics. 5. Others. Additionally, other genetic elements may be necessary to select by some characteristic the cells that received the gene (for example, another gene that confers resistance to antibiotics) or to increase the number of copies of the gene present in each cell (genetic amplification), between others. An ideal system of expression should use simple genetic constructions, so as to minimize the risks of obtaining genetically unstable systems or erroneous sequences that result in unwanted products. The use of vectors and simple genes reduces the development time of the system. A fundamental consideration is that genetic simplicity can not be to the detriment of productivity or the quality of the protein produced. To achieve the expression of the desired protein, the gene of interest is included, by transfection processes and using suitable vectors, in the genetic material of the host cell. Transfection can be performed by different techniques, for example electroporation, calcium phosphate precipitation, the use of liposomes, etc. The gene of interest can be associated with other genes that are known to confer resistance, for example, to antibiotics such as geneticin or to toxic agents such as methotrexate (MTX). This association allows the selection of stably transfected cells, that is, those that are capable of reproducing and transmitting the gene of interest to their progeny. The association also allows to select the most productive cells.

The obtained recombinant product is identified by analysis of its molecular weight, its amino acid sequence, its biological activity, etc. The genetic engineering techniques known hitherto for the production of EPO are characterized by: 1. Utilizing EPO genes that include fragments of 5 'and 3' flanking regions of the same gene that do not code for the protein. It is considered that the presence of expression control elements located in said non-coding regions is necessary and increases the production of EPO. See US patent 5,688,679. 2. Use expression vectors with several different promoters, based on the fact that the combination of promoters induces a higher production of EPO. At present, the use of a single promoter in the vector has resulted in a low level of expression of the protein. See US patents 4,703,008, 4,677,195 and 5,688,679. The average production of EPO using a single promoter is 200 μg / l / day. The maximum EPO production reported using a single promoter is 10 mg / l / day. 3. The potential instability of the genetic systems and therefore of the EPO production due to the complexity of the genetic constructions used.

Description of the Invention The claimed invention consists of a eukaryotic cell line producing recombinant human EPO, obtained through transfection with an expression vector that includes a gene coding for human EPO, a single promoter and a terminator as control elements of The expression. SEQ ID NO: 1 identifies the amino acid sequence of EPO for which it encodes the gene used.

One of the advantages of the present invention is that the gene encoding EPO used does not include fragments of 5 'and 3' flanking regions of the EPO gene that do not code for the protein. Even so, the claimed system produces a large amount of EPO. A further advantage of the present invention is the production of large amounts of EPO by the use of expression vectors with a single promoter. By the claimed method, more than 50 mg of EPO is obtained per liter of culture per day, that is more than five times the production of EPO claimed by the known methods. The combination of the gene coding for EPO used in this invention and a simple promoter showed, surprisingly, operating with great efficiency, obtaining stably transfected cells that produce EPO amounts comparable or superior to those reported using theoretically more adequate but much more complex genetic constructions. and difficult to handle in practice. A further advantage of the claimed invention is cotransfection with two vectors that confer different resistances, facilitating the selection, genetic amplification and maintenance of cotransfected producer cells. To obtain the cell line object of the claimed invention, firstly, genomic DNA extracted from human white blood cells is prepared. The gene coding for EPO is obtained from the prepared DNA. For this, the gene is amplified using primers that avoid the presence of 5 'and 3' flanking regions of the EPO gene that do not code for the protein. These primers include at their 5 'end restriction sites, which are then inserted at both ends of the isolated gene and facilitate subsequent cloning. Subsequently, the amplified gene is cloned into a bacterial vector and sequenced. Once the sequence obtained is verified, the gene is cloned in the Xho I-Hind sites or an expression vector for eukaryotic cells that uses, to control the expression, only the SV 40 early promoter and its terminator. The vector confers resistance to geneticin and ampicillin. CHO cells are then cotransfected with the expression vector obtained and a second vector conferring resistance to methotrexate. Stably transfected cells are then selected for their geneticin resistance and EPO expression is amplified by selecting cells resistant to increasing amounts of methotrexate. Finally, clones are chosen according to their productivity measured by RTE. The culture supernatants of the most productive clones are used to verify the identity of the EPO produced and its biological activity by means of SDS-PAGE tests, "Western Blot", glycanase treatment followed by SDS-PAGE, isoelectric focusing, complete sequence of the protein and biological activity in vivo in ex hypoxic polycythaemic mice compared to the international EPO standard. The processes listed are performed following molecular biology techniques that are exemplified as follows.

Example 1 Preparation of Human Genomic DNA ml of blood was extracted in 10 mM EDTA pH 8 from a male adult and clinically healthy. It was transferred in 5 ml aliquots to two 50 ml tubes and 45 ml of a solution of 0.3 M sucrose, 10 mM Tris-HCl pH 7.5, 5 mM MgCl2 and 1% Triton X 100, maintained in each, were added. at 4 ° C. It was left to stand on ice for 10 minutes and centrifuged 10 minutes at 1,000 g and 4 ° C, the supernatant was discarded and the pellet was washed several times with a 0.075 M NaCl solution and 0.025 M EDTA pH 8, centrifuging each time 10 minutes to 1,000 g and 4 ° C.

It was resuspended in 3 ml of a 10 mM Tris-HCl pH 8 solution, 400 mM NaCl, 2 mM EDTA pH 8; then 200 μl of 10% SDS (sodium dodecylsulfate) and 500 μl of proteinase K (1 mg / ml in 1% SDS and 2 mM EDTA pH 8) were added and incubated overnight at 37 ° C.

After this incubation, 1 ml of saturated NaCl solution was added and after shaking it was centrifuged at 2,500 g for 15 minutes. The supernatant was transferred to a 15 ml tube where the volume was doubled by the addition of isopropanol. It was mixed gently by inversion of the tube and left at room temperature until a DNA precipitate formed which was "fished" with a Pasteur pipette. of bent glass. The DNA was placed in a 2 mL tube and 1 mL of 70% ethanol was added, it was left for one minute, and then the supernatant was discarded, the precipitate was allowed to dry and then dissolved in 500 μL of TE (10 mM Tris). -HCl pH 8 - 1 mM EDTA). The concentration of the DNA solution was calculated by measuring at 260 nm the absorbance of a 1: 1000 dilution of this solution; for each unit of optical density it was considered to have 50 μg of genomic DNA. Once the concentration was known, a solution with 500 ng of genomic DNA / μl in TE was prepared.

Example 2 Isolation of the Coding Gene for EPO The gene coding for EPO was prepared from 500 ng of the human genomic DNA obtained in Example 1, with 400 ng of each of the primers EPO 1 and EPO 2; in a 2.5 mM aqueous solution in each deoxynucleotide (dATP, dCTP, dGTP and dTTP), with 2.5 units of Taq DNA polymerase (Perkin-Elmer) in a final volume of 100 μl, using the buffer recommended by the manufacturer. A Thermal Cycler 480 from Perkin Elmer - Cetus was used, which was programmed for 30 cycles of: 1 minute at 93 ° C, 1 minute at 55 ° C and 3 minutes at 72 ° C. A fragment of approximately 2,170 base pairs containing the EPO gene was obtained from the reaction. The sequence of the oligonucleotides used was: EPO 1: 5 'GAATTCTCGAGATGGGGGTGCACGGTGAG 3' (SEQ ID NO: 2) which corresponds to the first bases that are translated from the EPO gene with the addition at its 5 'end of a recognition site for the Xho I enzyme and one for Eco Rl, which are used in subsequent clones.

EPO 2: 5 'AAGCTTCATCTGTCCCCTGTCCTGCA 3', (SEQ ID NO: 3) which is complementary to the last translated bases and to some of the non-coding part 3 'of the EPO gene, has at its end 31 added a recognition site for the enzyme Hind JJI to be used in subsequent clones.

The sequence obtained is as follows (SEQ ID NO: 4): Aa ^ ^ ^ cfc to cggctattggccaggaggtggctgggttcaaggaccggcgacttgtcaaggaccccggaagggggaggggggtggggcagcctcca atgggggtgcacggtgagtactcgcgggctgggcgctcccgccgcccgggtccctgtttgagcggggatttagcgccc cgtgccagcggggacttgggggagtccttggggatggcaaaaacctgacctgtgaaggggacacagtttgggggttgaggggaagaa ggtttgggggttctgctgtgccagtggagaggaagctgataagctgataacctgggcgctggagccaccacttatctgccagaggggaa gcctctgtcacaccaggattgaagtttggccggagaagtggatgctggtagctgggggtggggtgtgcacacggcagcaggattgaatg aaggccagggaggcagcacctgagtgcttgcatggttggggacaggaaggacgagctggggcagagacgtggggatgaaggaagct gtccttccacagccacccttctccctccccgcctgactctcagcctggctatctgttctagaatgtcctgcctggctgtggcttctcctgtccct gctgtcgctccctctgggcctcccagtcctgggcgccccaccacgcctcatctgtgacagccgagtcctggagaggtacctcttggaggc caaggaggccgagaatatcacggtgagaccccttccccagCacattccadagaactcacgctcagggcttcagggaactcctcccagatc caggaacctggcacttggtttggggtggagttgggaagctagacactgcccccctacataagaataagtctggtggccccaaaccatacc tggaaactaggcaaggagcaaagccagcagatcctacggcctgtgggccagggccagagccttcagggacccttgactccccgggctg tgtgcatttcagacgggctgtgctgaacactgcagcttgaatgagaatatcactgtcccagacaccaaagttaatttctatgcctggaagag gatggaggtgagttcctttttttttttttttcctttcttttggagaatctcatttgcgagcctgattttggatgaaagggagaatgatcgggggaaa ggtaaaatggagcagcagagatgaggctgcctgggcgcagaggctcacgtctataatcccaggctgagatggccgagatgggagaatt gcttgagccctggagtttcagaccaacctaggcagcatagtgagatcccccatctctacaaacatttaaaaaaattagtcaggtgaagtggt gcatggtggtagtcccagatatttggaaggctgaggcgggaggatcgcttgagcccaggaatttgaggctgcagtgagctgtgatcacac cactgcactccagcctcagtgacagagtgaggccctgtctcaaaaaagaaaagaaaaaagaaaaataatgagggctgtatggaatacatt cattattcattcactcactcactcactcattcattcattcattcattcaacaagtcttattgcataccttctgtttgctcagcttggtgcttggggct gctgaggggcaggagggagagggtgacatgggtcagctgactcccagagtccactccctgtaggtcgggcagcaggccgtagaagtct ggcagggcctggccctgctgtcggaagctgtcctgcggggccaggccctgttggtcaactcttcccagccgtgggag cccctgcagctg catgtggataaagccgtcagtggccttcgcagcctcaccactctgcttcgggctctgggagcccaggtgagtaggagcggacacttctgc ttgccctttctgtaagaaggggagaagggtcttgctaaggagtacaggaactgtccgtattccttccctttctgtggcactgcagcgacctcc tgttttctccttggcagaaggaagccatctcccctccagatgcggcctcagctgctccactccgaacaatcactgctgacactttccgcaaac tcttccgagtctactccaatttcctccggggaaagctgaagctgtacacaggggaggcctgcaggacaggggacagatga? cfl1 The first codon atg translated, as well as the tga codon of "stop", are underlined. The sequences of restriction sites used for cloning appear in italic boldface.

Example 3 Cloning and Sequencing of Isolated Gene The fragment of approximately 2,170 bases corresponding to the EPO gene was purified, its blunt ends made by treatment with the Klenow fragment of the DNA polymerase and cloned in the Sma I site of an M13mpl8 vector following standard techniques applied in molecular biology. The obtained recombinant plasmids were cut with the enzymes Xho I and Hind JJI, the presence of the insert was checked by electrophoresis of the product of the restriction cuts in a 0.8% agarose gel revealed by staining with ethidium bromide. A positive clone was chosen (two bands, one of around 2,200 base pairs and another corresponding to the linearized vector) and sequenced following the Sanger technique manually using "T7 sequencing kit" (Pharmacia) and with the use of a sequencer 370 A automatic instrument from Applied Biosystems International, following the protocols recommended by the manufacturers for each sequencing system.

Example 4 Vectors for Eukaryotic Cells Construction of Vector pVex 1 It was built following the usual techniques of molecular biology. It consists of: a) fragments of the bacterial vector pBR322, which provide an origin of bacterial replication and resistance to ampicillin, for amplification and selection of the vector in E. coli. b) immediately after 3 'of a) is the early promoter of virus S V40, allows to get expression of genes cloned 5' of this element. c) immediately after 3 'of b) follow cloning sites Xho I and Hind III, to insert the genes to be expressed. d) immediately after 3 'of c) the SV40 virus polyadenylation signal is found, in order to obtain the correct polyadenylation of the specific transcripts of the gene cloned in c). e) immediately after 3 'of d) is the TK promoter and the gene encoding neomycin phosphotransferase plus its polyadenylation signal, to allow the selection of stably transfected cells by selection for resistance to neomycin and antibiotics derived from it such as geneticin. The 3 'end of e) is linked to the 5' end of a). A specimen of the vector pVex 1 is deposited in DSMZ- Deutsche Sammlung von Mikroooganismen und Zellkulturenen, no. DSM deposit 12776.

Vector pDHFR The vector pDHFR confers resistance to ampicillin for its selection in bacteria and includes DNA coding for mouse dehydrofolate reductase (DHFR), whose expression is controlled by the early SV40 virus promoter and its polyadenylation signal. The co-expression of DHFR and the gene coding for EPO allows, by selecting with methotrexate (MTX) in the culture medium, to amplify the expression of EPO achieved with the vector pVex 1-EPO a certain number of times. A specimen of the p DHFR vector is deposited in DSMZ- Deutsche Sammlung von Mikroooganismen und Zellkulturenen, no. DSM deposit 12777.

Example 5 Cloning of the Coding Gene for EPO in the Expression Vector The sequenced gene was extracted by cleaving it with the Xho I-Hind III enzymes of the vector in which it was cloned in Example 3. It was then isolated and cloned in the same restriction sites of the pVex I vector. A pVex-positive clone was isolated. EPO. All these operations were carried out following the traditional techniques of genetic engineering. See Brown, "Gene Cloning" (Chapman &Hall, London, England, 1995); Watson, et al, "Recombinant DNA", 2nd Ed. (Scientific American Books, New York, New York, 1992); Sambrook et al, "Molecular Cloning. Laboratory Laboratory" (Cold Spring Harbor Laboratory Press, 1989); Bishop et al, "Nucleic Acid and Protein Sequence: A Practical Approach" (IRL Press 1987); Davis et al, "Basic Methods in Molecular Biology" (Elsevier Science Publishing Co., New York, New York, 1986) incorporated herein in their entirety as references.

Example 6 Cotransfection and Amplification with MTX A CHO (Chimney Hamster Ovary) cell line, from hamster ovary, mutated to be deficient in the DHFR enzyme gene (CHO-DHFR "), was used to facilitate genetic amplification with MTX. cells were grown at 37 ° C in an atmosphere with 5% CO2, CHO cells were cotransfected; for this, the calcium phosphate technique was followed, which, for a Petri dish of 90 mm in diameter, consists of: a) Changing the culture medium, which is alpha-MEM added with 10% of fetal bovine serum, through fresh 4-8 hours before transfection. b) Add to a 5 ml tube, 500 μl of a 10 gr / 1 HEPES solution of pH 7.1; 16 gr / 1 NaCl and 10 μl of a 35 mM solution of Na2HPO4 and 35 mM of NaH2PO4. c) Prepare in another 1.5 ml tube a solution with 60 μl of 2 M CaCl2 and 10 μg of each DNA to be transfected (pVex-EPO and pDHFR), carrying 500 μl with H2O. The pDHFR plasmid described in example 2 is based on pBR 322, confers resistance to ampicillin, can be replicated in E. coli, has cloned the DHFR gene between the early promoter and the SV40 terminator, and allows expression of the DHFR protein in CHO cells. This protein confers resistance to methotrexate, which can then be used to select cells that have high EPO productivity. d) Add the DNA and CaCl2 solution to the tube with Hepes dropwise while air is being bubbled in order to achieve rapid mixing and that the local concentrations are as small as possible, which allows the formation of a very fine precipitate that is incorporated more efficiently by the cells. e) Let stand 30 minutes and then pour over the cells. f) Distribute well, shaking gently, and leave overnight in an incubator at 37 ° C in a 5% CO2 atmosphere. g) Wash 2 times with PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g NaH2PO4, taken to 1 liter with H2O and to pH 7.4 with HCl) and add fresh culture medium. Twenty-four hours after transfection, the selection was made with geneticin (G 418) in a final concentration of 600 μg / ml The cells that stably incorporated the plasmid pVex-EPO were able to resist the antibiotic while all the others had died after 25 days. Resistant colonies were separated and their productivity was tested. Once the clones were isolated, the three with the best productivity were chosen. Taking advantage of the genetic constructions used in the invention, a selection was made with each of the three clones with a second selective agent: MTX at different concentrations: 10"8 M, 10" 7 M, 10"6 M, 10" 5 M. For this, the culture medium was changed to alpha-MEM without nucleosides, supplemented with 10% of dialyzed fetal bovine serum. It is a critical factor that the dialysis is carried out according to the following scheme: for 100 ml of serum, the serum is placed in a dialysis bag with a porosity of less than 3,000 Da (a greater porosity would cause the loss of growth factors, causing the cells to they could not grow and reproduce), the bag is sealed and completely immersed in a container with 5 liters of bidistilled water, left at 4 ° C for 12 hours; at 12 o'clock the water is discarded and another 5 liters are added; leave another 12 hours at 4 ° C and then remove the dialysis bag and recover the serum. Dialyzing for shorter periods, or in smaller volumes, or without changing the water would not work, since a small proportion of nucleotides could remain in the serum, so that selection with MTX would not work. Dialyzing for longer periods would not work either, since some proteins necessary for the growth of the cells could be precipitated and lost.

Example 7 Isolation of High Productivity Clones Clones that grew in 10"7 M and 10" 6 M MTX were isolated, amplified in fresh medium alpha-MEM without nucleosides supplemented with 10% of dialyzed fetal bovine serum. Once grown, the culture supernatant was tested to measure the production and secretion of EPO. For this, a specific immunoassay was used. The process described above concluded with the selection of a clone of recombinant cells that produced 50,000 μg of EPO / liter of culture medium / day. A specimen of the recombinant cells used is deposited in DSMZ-Deutsche Sammlung von Mikroooganismen und Zellkulturenen, no. DSM ACC 2397 deposit. In order to verify that there were no errors in the sequence of the gene used or in its transcription, the EPO specific cellular transcripts were controlled as described in example 8. To identify the obtained protein, the procedure was as described in FIG. example 9.

Example 8 Verification of the Sequence of the Specific Messenger RNA Produced by the Recombinant Cells Preparation of RNA from Cells Total RNA was prepared from one of the production lines according to the following protocol: a) Was washed 2 times with 10 ml of PBS a box 90 mm diameter Petri dish with confluent cells. b) 2 ml of GTC buffer was added, distributing it throughout the plate. The GTC buffer consists of: 1) 50 g of guanidinium thiocyanate 2) 0.5 g of N-Lauroyl sarcosine 3) 2.5 ml of 1 M sodium citrate pH 7 4) 0.7 ml of β-mercaptoethanol 5) 0.33 ml of 30% antiespuma (SIGMA) 6) H2O csp 100 ml, pH 7.0 The cells were lysed and a highly viscous solution remained. The solution was transferred to a 15 ml tube, and the operation was repeated with another 2 ml of GTC buffer. a) The tube was shaken vigorously, for 1 minute, to break the DNA. A gradient fractionation of cesium chloride was performed. To this end, 4 ml of CsCl solution (95.97 g CsCl and 2.5 ml of 1 M Na Acetate pH 5.4, all brought to 100 ml with H2O) were placed in an ultracentrifuge tube. On this and without mixing, the suspension of the cells was added in GTC, filling the tube with GTC buffer and ultracentrifuging at 20 ° C, 20 hours at 31,000 rpm. b) Under these conditions, the RNA remained at the bottom of the tube (pellet) and the DNA formed a band that was halfway between the cesium chloride gradient. c) The supernatant was discarded, taking care to eliminate all the DNA and allowing the RNA to dry, in the pellet, for 5 minutes. d) The pellet was dissolved in 200 μl of H2O and transferred to a 1.5 ml tube. e) 200 μl of 0.4 M Na Acetate pH 4.8 and two volumes of ethanol were added, mixing well and leaving 30 minutes at -80 ° C. f) Centrifuged in a microcentrifuge at 14,000 rpm for 15 minutes, discarding the supernatant and rinsing the precipitate with 1 ml of 80% ethanol. g) The pellet was dried and redissolved in 100 μl of H2O. h) The concentration of a 1: 100 dilution of the RNA solution was measured at 260 nm (one unit of optical density equals 40 μg of RNA).

Note: All the solutions and elements used were RNAse free.

Preparation of specific cDNA The specific cDNA was prepared with a kit for that purpose (cDNA Synthesis System Plus, Amersham-cat RPN 1256) following its instructions and using the oligonucleotide EPO 2 as the first specific.

Cloning of the coding cDNA for EPO 1/20 of the obtained cDNA was amplified, using 400 ng of each of the EPO 2 and EPO 3 oligonucleotides and 2.5 mM of each deoxynucleotide, in the appropriate buffer and with 2.5 Units of Taq DNA polymerase , in 100 μl total. 35 cycles of amplification were carried out: 1 minute at 93 ° C, 1 minute at 55 ° C and 1 minute at 72 ° C.

EPO 3 was synthesized as already described for EPO 1 and EPO 2, and its sequence (5 'GAATTCCATGGGGGTGCACGAATGTCC 3') corresponds to the first 20 coding bases of the EPO cDNA, with the addition of a recognition site for the enzyme Eco Rl , to facilitate subsequent genetic manipulations.

A fragment of approximately 600 base pairs was obtained, which was cloned in vectors M13mpl8 and M13mpl9. The presence of the insert was tested on the clones with restriction cuts and sequenced in both directions to know the complete sequence, using the Sanger method. Given the very high self-complementarity of the gene regions, which causes the appearance of many and very ambiguous compressions in radioautography, it was necessary to use a sequence kit that uses Taq DNA polymerase and modified bases, resulting in lower quality results. but that eliminate the compressions. The kit used was the Gene aTaq of Pharmacia-LKB Biotechnology. Complete DNA sequencing of isolated and cloned human EPO copy showed that it encodes for EPO, so there are no errors in the gene present in the recombinant CHO cells or in their transcription.

Example 9 Analysis of the EPO Produced The EPO obtained by culturing the cells of the present example was brought to purity and then subjected to different quality and identification studies: a) In a SDS-PAGE denaturing gel, it ran as a broad band of about 30 kDa molecular weight. See Fig. 1. b) That band was recognized by a monoclonal antibody as well as by a polyclonal antibody against human EPO in a "Western Blot" assay. See Fig. 2. c) The glycanase treatment proved the existence of the glycosidic chains in quantity and molecular weight according to what was expected. See Fig. 3. d) The EPO produced showed to be composed of a series of isoelectric point species between 3.0 and 4.5. See Fig. 4. e) The complete amino acid sequencing of the protein isolated and purified from the culture supernatant of the transfected cell lines showed complete homology with natural human EPO having the following sequence of 165 amino acids (SEQ ID NO: 1). NH2 - Ala Pro Pro Arg Leu He Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Wing Lys Glu Wing Glu Asn He Thr Thr Gly Cys Wing Glu Hys Cys Ser Leu Asn Glu Asn He Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Wing Trp Lys Arg Met Glu Val Gly Gln Gln Wing Val Glu Val Trp Gln Gly Leu Wing Leu Leu Ser Glu Wing Val Leu Arg Gly Gln Wing Leu Leu Val Asn Being Ser Gln Pro Trp Glu Pro Leu Gln Leu Hys Val Asp Lys Wing Val Ser Gly Leu Arg Be Leu Thr Thr Leu Leu Arg Wing Leu Gly Wing Gln Lys Glu Ala lie Ser Pro Pro Asp Wing Wing Wing Wing Pro Leu Arg Thr He Thr Wing Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Wing Cys Arg Thr Gly Asp- COOH X glycosylation sites f) The presence of the four glycosylation sites on the 165 amino acid chain as well as the carbohydrate structure, mainly the terminal sialic acid residues, were demonstrated together with their correct biological activity in vivo, in a experimental model of the ex-hypoxic polycythemic mouse, exhibiting total parallelism against the corresponding international standard. g) The productivity achieved measured by a specific immunoassay was 50 mg / liter of culture / day.

Description of Diagrams Fig. 1 illustrates a polyacrylamide gel analysis (SDS-PAGE) of a sample of the EPO obtained according to the method described. Molecular weight markers are shown on lanes 1, 4 and 7. In streets 2, 3, 5 and 6, different masses of pure EPO obtained according to the claimed process were run. The purity of the obtained product and its apparent molecular weight of more than 30 kDa can be appreciated, which coincides with that of human urine EPO. Fig. 2 illustrates a "Western Blot" analysis of a sample of the EPO obtained according to the method described. The identity of the EPO produced is verified, since it is recognized by a human anti-EPO antibody. In Street 1, a human EPO standard was run, in the street 2 molecular weight markers and in the streets 3 to 5 EPO samples obtained according to the claimed method. Fig. 3 illustrates an SDS-PAGE analysis of a pure EPO sample obtained according to the described method, treated with glycanase. Molecular weight markers were run on streets 1, 4 and 8. On streets 2 and 7, untreated EPO is seen. In lane 3, EPO treated with O-glycanase was run, the presence of O-glycosylation is verified. In lane 5, partially degraded EPO was run with N-glycanase, the presence of 3 N-glycosylations was verified with the molecular weights corresponding to those expected for EPO. In lane 6, degraded EPO was run with O-glycanase and N-glycanase, obtaining the expected molecular weight for the fully deglycosylated protein. Fig. 4 illustrates a study of the isoelectric points of pure EPO samples produced according to the described method. The EPO samples were run in streets 2, 3 and 4, the isoelectric point markers in streets 1 and 5. The presence of the forms corresponding to EPO, with isoelectric points between 3.0 and 4.5, is verified.

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Claims (1)

Claims Having described and exemplified the nature and main object of the present invention, as well as the manner in which it can be carried out in practice. It is claimed to claim as property and exclusive rights: 1. A recombinant human erythropoietin producing cell line characterized in that: a) The gene coding for EPO is the one detailed in SEQ ID NO: 4 and consists only of the coding part of the human gene of EPO, not including sequences of 5 'and 3' flanking regions of the EPO gene that do not code for the protein, b) the genetic constructs used possess, as control elements of EPO expression, a single promoter and viral terminator, c) the method of constructing the gene encoding EPO uses human DNA as the starting material. 2. A recombinant human erythropoietin producing cell line according to claim 1, characterized in that the expression genetic systems consist of two vectors which possess as control elements of EPO expression only the SV40 virus early promoter and its terminator. 3. A recombinant human erythropoietin producing cell line according to claim 1, characterized in that the expression genetic systems consist of two vectors which possess the SV40 virus early promoter as an element for controlling the expression of EPO. 4. A recombinant human erythropoietin producing cell line according to claim 1, characterized in that it comprises a pDHFR vector. 5. A recombinant human erythropoietin producing cell line according to claim 1, characterized in that the cell lines are selected using a double system, I) resistance to geneticin and II) resistance to increasing amounts of methotrexate, 6. An erythropoietin producing cell line recombinant human according to claim 1, characterized in that the EPO obtained consists of 165 amino acids according to the sequence SEQ ID NO

1. A recombinant human erythropoietin producing cell line according to claim 1, characterized in that it produces surprisingly high amounts of EPO, greater than 50 mg / liter of culture medium / day. Recombinant human erythropoietin producing cell line according to claim 1, characterized in that the cells are mammalian cells. Recombinant human erythropoietin producing cell line according to claim 1, characterized in that the cells are CHO, COS, BHK, Namalwa, HeLa, Hep3B, HepG2 or other mammalian cells. Recombinant human erythropoietin producing cell line according to claim 1, characterized in that the cells are CHO or COS cells. Recombinant human erythropoietin producer cell line according to claim 1, characterized in that the cells are CHO cells. Recombinant human erythropoietin producing cell line according to claim 1, characterized in that the genomic DNA used is obtained from human white blood cells.