Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-11-06
2004-08-17
Kemmerer, Elizabeth (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069400, C435S471000, C435S325000, C435S358000, C435S360000, C435S368000, C435S365100, C435S320100, C530S350000
Reexamination Certificate
active
06777205
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates, in general, to a host cell and a vector comprising a nucleotide sequence coding for recombinant human etythropoietin (EPO). In particular, the expression vector comprises only one promoter that regulates the EPO expression. The present invention also refers to a method of producing EPO and the EPO thus produced.
2. Background Information
EPO is a glycoprotein that stimulates erythroblast differentiation in the bone marrow, thus increasing the circulating blood erythrocyte count. The mean life of erythrocytes in humans is 120 days and therefore, a human being loses 1/120 erythrocytes each day. This loss must be continuously restored to maintain an adequate level of red blood cells.
The existence of EPO was first postulated by the turn of the century and was definitely proved by Reissman and Erslev early in the '50s. See Carnot, et al.,
C.R. Acad. Sci
. (France)143:384-386 (1906); Carnot, et al.,
C.R. Acad. Sci
. (France), 143:432-435 (1906); Carnot, et al.,
C.R. Soc. Biol
., 111:344-346 (1906); Carnot,
C.R. Soc. Biol
., 111:463-465 (1906); Reissman,
Blood
, 5:372-380 (1950) and Erslev,
Blood
8:349-357 (1953). Reissman and Erslev's experiments were promptly confirmed by other researchers. See Hodgson, et al.,
Blood
, 9:299-309 (1954); Gordon, et al.,
Proc. Soc. Exp. Biol. Med
., 86:255-258 (1954) and Borsook, et al.,
Blood
, 9:734-742 (1954).
The identification of the EPO production site in the organism was an issue of debate. Successive experiments led to the identification of the kidney as the main organ and peritubular interstitial cells as the synthesis site. See Jacobson, et al.,
Nature
, 179:633-634 (1957); Kuratowska, et al.,
Blood
, 18:527-534 (1961); Fisher,
Acta Hematol
., 26:224-32 (1961); Fisher, et al.,
Nature
, 205:611-612 (1965); Frenkel, et al.,
Ann. N.Y. Acad. Sci
., 149:292-293 (1968); Busuttil, et al.,
Proc. Soc. Exp. Biol. Med
, 137:327-330 (1971); Busuttil,
Acta Haematol
., (Switzerland), 47:238-242 (1972); Erslev,
Blood
, 44:77-85 (1974); Kazal,
Ann. Clin. Lab. Sci
., 5:98-109 (1975); Sherwood, et al.,
Endocrinology
, 99:504-510 (1976); Fisher,
Ann. Rev. Pharmacol. Toxicol
., 28:101-122 (1988); Jelkmann, et al.,
Exp. Hematol
., 11:581-588 (1983); Kurtz, et al.,
Proc. Natl. Acad. Sci
. (
USA
), 80:4008-4011 (1983); Caro, et al.,
J. Lab. Clin. Med
., 103:922-931 (1984); Caro, et al.,
Exp. Hematol
., 12:357 (1984); Schuster, et al.,
Blood
, 70:316-318 (1986); Bondurant, et al.,
Mol. Cell. Biol
., 6:2731-2733 (1986); Schuster, et al.,
Blood
, 71:524-527 (1988); Koury, et al.,
Blood
, 71:524-527 (1988); Lacombe, et al.,
J. Clin. Invest
., 81:620-623 (1988); Koury, et al.,
Blood
, 74:645-651 (1989).
A smaller proportion, ranging from 10% to 15% of total EPO, is produced by the liver in adults. See Naughton, et al.,
J. Surg. Oncol
., 12:227-242 (1979); Liu, et al.,
J. Surg. Oncol
., 15:121-132 (1980); Domfest, et al.,
Ann. Clin. Lab. Sci
., 11:37-46 (1981); Dinkelaar, et al.,
Exp. Hematol
., 9:796-803 (1981); Caro, et al.,
Am. J. Physiol
., 244:5 (1983); Dornfest, et al.,
J. Lab. Clin. Med
., 102:274-285 (1983); Naughton, et al.,
Ann. Clin. Lab. Sci
., 13:432-438 (1983); Jacobs, et al.,
Nature
, 313:806-810 (1985); Erslev, et al.,
Med. Oncol. Tumor. Pharmacother
., 3:159-164 (1986). The EPO produced is directly proportional to the extent of tisular hypoxia and its expression rises by increasing the number of the EPO producing cells.
EPO has shown great efficiency in the treatment of anemia, especially anemia derived from renal failure. See Eschbach, et al.,
N. Enigland J. of Med
., 316:73-78 (1987); Krane,
Henry Ford Hosp. Med. J
., 31:177-181 (1983). Its therapeutical usefulness, however, has been limited due to the unavailability of a massive production method. The quantity and quality of the EPO obtained by the extractive systems known were insufficient. Recently, the use of recombinant DNA technology has made it possible to obtain large amounts of proteins. The application of these techniques to eukaryotic cells has allowed a large-scale production of EPO. See patents U.S. Pat. No. 5,688,679 (to Powell), U.S. Pat. No. 5,547,933 (to Lin), U.S. Pat. No. 5,756,349 (to Lin), U.S. Pat. No. 4,703,008 (to Lin) and U.S. Pat. No. 4,677,195 (to Hewick et al.).
At the present, recombinant DNA techniques are widely known and used. These techniques involve the use of genetic elements such as DNA fragments and enzymes to assemble and transfer genetic constructions for the production of recombinant proteins. The recombinant DNA techniques also facilitate the study of biological mechanisms. See Frank-Kamenetskii, “Unraveling DNA” [Samaia Glavnaia Molekula] (Addison Wesley Longman Inc., Reading, Mass., 1997); Brown, “Gene Cloning” (Chapman & Hall, London, England, 1995); Watson, et al., “Recombinant DNA”, 2nd Ed. (Scientific American Books, New York, N.Y., 1992); Alberts et al., “Molecular Biology of the Cell” (Garland Publishing Inc., New York, N.Y., 1990); Innis et al., Eds., “PCR Protocols. A Guide to Methods and Applications” (Academic Press Inc., San Diego, Calif., 1990); Ehrlich, Ed., “PCR Technology. Principles and Applications for DNA Amplification” (Stockton Press, New York, N.Y., 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, Mass., 1987); Davis et al., “Basic Methods in Molecular Biology” (Elsevier Science Publishing Co., New York, N.Y., 1986); Watson, “The Double Helix” (Penguin Books USA Inc., New York, N.Y., 1969).
SUMMARY OF THE INVENTION
The claimed invention comprises an eukaryotic cell line that produces recombinant human EPO, obtained by means of its transfection with an expression vector that comprises a gene coding for human EPO. The vector further comprises an unique promoter and terminator as expression control elements. SEQ ID NO:1 identifies the EPO amino acid sequence codified by the gene used.
The invention provides a host cell comprising a vector which comprises a nucleotide sequence encoding the erythropoietin polypeptide consisting of the amino acid sequence in SEQ ID NO:1, a viral promoter and a viral terminator.
The invention further provides a method for producing an EPO polypeptide, comprising culturing the above host cell under such conditions that said polypeptide is expressed and recovered.
One of the advantages of this invention is that the EPO coding gene utilized does not include non-coding fragments of the 5′ and 3′ regions. However, the system claimed produces an unexpectedly high amount of EPO.
An additional advantage of this invention is the use of expression vectors comprising only one promoter, which exhibit a high EPO productivity. By utilizing the claimed method, it is possible to obtain more than 50 mg of EPO per liter of cell culture per day, that is, over five times the EPO production level claimed by the best method reported so far utilizing one promoter.
The combination of the EPO coding gene claimed in this invention and a simple promoter showed, surprisingly, to operate efficiently, resulting in a stable EPO producing cell. The transfected cells yielded an amount of EPO comparable to, or even higher than, those reported using in theory more adequate, though more complex and difficult to manipulate, genetic constructions.
An additional advantage of the claimed invention is the cotransfection with two vectors that confer different resistance, thus simplifying and facilitating the selection, genetic amplification and maintenance of the cotransfected EPO producing cells.
Further objects and advantages of the present invention will be clear from the description that follows.
REFERENCES:
patent: 4667016 (1987-05-01), Lai et al.
patent: 4677195 (1987-06-01), Hewick et al.
patent: 4703008 (1987-10-01), Lin
patent: 4806524 (1989-02-01), Kawag
Carcagno Carlos Miguel
Criscuolo Marcelo Eduardo
Melo Carlos Alberto
Vidal Juan Alejandro
Hamud Fozia
Kemmerer Elizabeth
Sterne Kessler Goldstein & Fox PLLC
Sterrenbeld Biotechnologie North America, Inc.
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