WO1995034572A1 - THE PRODUCTION OF HEMOGLOBIN HAVING A δ-LIKE GLOBIN - Google Patents
THE PRODUCTION OF HEMOGLOBIN HAVING A δ-LIKE GLOBIN Download PDFInfo
- Publication number
- WO1995034572A1 WO1995034572A1 PCT/US1995/007286 US9507286W WO9534572A1 WO 1995034572 A1 WO1995034572 A1 WO 1995034572A1 US 9507286 W US9507286 W US 9507286W WO 9534572 A1 WO9534572 A1 WO 9534572A1
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- WIPO (PCT)
- Prior art keywords
- globin
- human
- hemoglobin
- nucleic acid
- gene
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
Definitions
- the present invention concerns the production of human hemoglobin in animals other than humans.
- the hemoglobin is to be used as a blood substitute, thus avoiding the disadvantages of obtaining human blood donors.
- the present invention concerns novel nucleic acid molecules that can be used to construct transgenic animals that will produce large amounts of human ⁇ - globin and 6"-globin. These animals are a source of Hemoglobin A 2 , which contains two and two 5-chains in place of the two ⁇ and two jS-chains found in the most common adult hemoglobin, Hemoglobin A.
- Oxygen is absorbed through the lungs and carried by hemoglobin in red blood cells (RBC) for delivery to tissues throughout the body. At high oxygen concentrations, such as those found in the proximity of the lungs, oxygen binds to hemoglobin, but is released in areas of low oxygen concentration, where it is needed.
- RBC red blood cells
- Each hemoglobin molecule in an adult consists of two ⁇ -globin subunits and two ⁇ - or ⁇ - globin subunits or chains.
- the globin chains themselves are synthesized from templates, called mRNAs which are, in turn, synthesized from a master template which is the globin gene.
- the gene contains three regions (exons) which code for the amino acids of the globin and contains other regions, called intervening sequences (IVS) , that are transcribed, but then removed from the primary transcript when the mature mRNA is formed. The mature mRNA is then translated into the globin protein.
- IVS intervening sequences
- Each globin chain contains an iron atom coordinated by the two imidazole nitrogens of two histidines.
- the iron atom is surrounded by a protoporphyrin ring which is contained within the hemoglobin molecule but not chemically linked to it.
- the protoporphyrin and the iron atom together form a heme group which binds the oxygen molecule and of course, gives hemoglobin its name.
- each hemoglobin tetramer is capable of binding four molecules of oxygen.
- Hemoglobin that contains two ⁇ - globin chains and two ⁇ -globin chains is called Hemoglobin A (HbA) ; the other form of adult hemoglobin, which has two ⁇ -globin chains is termed
- HbA 2 Both types of hemoglobins are synthesized in the same cells, called erythrocyte precursors, which, as they mature become recticulocytes, and finally become erythrocytes or red blood cells (RBC) . Normally only about 2.0-2.5% of the hemoglobin will be HbA 2 , although persons having a genetically defective /3-globin gene are known to express as much as 15% HbA 2 . Schroeder, .A. , et al., 1975, BIOCHEM. GENET. 10:135; Steinberg, M.H. , et al., 1982, J.LAB.CLIN.MED. 100:548. No hemoglobin has both a ⁇ and a ⁇ * chain.
- hemoglobin As a blood substitute rather than obtain blood from human volunteers. Because the function of hemoglobin is entirely independent of the RBC, the hemoglobin derived from transgenic animals could be used as a blood substitute with only slight modifications such as cross-linking, Ogden, J.E., et al., 1992,
- HbA 2 The preferred type of hemoglobin for this purpose is HbA 2 .
- HbA 2 is more stable than HbA, Kinderlerer, J., et al., 1970, BIOCHEM.J. 119:66P, which would allow for a longer life-span of the product and would also allow the use of harsher conditions during the manufacture of the synthetic product.
- the physico-chemical differences between HbA 2 and the HbAs of other animals are larger than between human HbA and the nonhuman HbAs. Therefore, the process of purifying the desired human Hb from other types of hemoglobins is significantly easier.
- LaFlamme et al. 1987, J.Biol.Chem. 262:4819, considered the production of human 5-globin mRNA in a transfected mouse erythroleukemia cell line, which was induced to produce quantities of globins.
- LaFlamme's studies concerned the effects on the production of 5-globin mRNA of the intervening sequences, the portions of the gene between the sequences that encode the globin protein. Each globin gene has two regions of intervening sequences (IVS) , sequences that are removed from the primary transcript also known as introns.
- IVS intervening sequences
- the present invention concerns a chimeric globin gene expression cassette wherein the control elements, i.e., the Locus Control Region, the promoter, the 5' and 3" untranslated portion of the mature message and the longer intervening sequence are all derived from a globin gene which encodes a major globin product, such as or ⁇ globin.
- the coding sequences of the chimeric gene encodes human ⁇ * globin.
- Transgenic animals having incorporated the chimeric gene of the invention and a human ⁇ -globin gene linked to it make large quantities of human Hemoglobin A 2 .
- the invention provides: the chimeric gene comprising exons encoding human ⁇ globin but having a promoter and untranslated segments from an ⁇ or ⁇ globin gene; the expression cassette comprising the above described chimeric gene and a suitable globin Locus Control Region (LCR) ; a transgenic animal expressing the cassette; and erythrocytes from the transgenic animals and methods of purifying Hemoglobin A 2 from the erythrocytes.
- LCR globin Locus Control Region
- FIGURES Figure 1 The overall organization of a construct suitable for transfection into a transgenic animal having a Locus Control Region (LCR) with promoters and structural genes for the ⁇ , e and ⁇ or ⁇ globin.
- LCR Locus Control Region
- B A schematic illustrating the placement of exons I, II and III (broader rectangles) of the human ⁇ S-globin gene into a human jS-globin gene having intervening sequences (narrower rectangles) . The nucleotide length of each region is superposed.
- IEF Isoelectric Focusing
- FIG. 3 A: Chro atogram of the proteins of a hemolysate of a transgenic mouse produced according to the invention eluted from a Mono P® ion exchange column by a linear NaCl gradient. Inset: IEF of peaks of the chromatogram. B: Chromatogram of proteins from the hemolysate of a transgenic mouse producing human HbA.
- the present invention involves the production of human Hemoglobin A 2 (HbA 2 ) in transgenic animals.
- the invention involves a chimeric gene to encode human 5-globin.
- the exons that encode ⁇ "-globin replace the exons of a gene that encodes a major component of the hemoglobin of an adult, i.e., an ⁇ or a ⁇ globin gene.
- the sequence of the human ⁇ - and ⁇ -globin genes are known. Lawn, R.M. et al., 1980, CELL 21:647-651; Spritz, R.A.
- primary transcript of the chimeric gene of the invention differs from that of the ⁇ - globin gene in that the longer intervening sequence of the ⁇ "-globin gene ( ⁇ TVS-2) is replaced by at least a portion IVS-2 of a major globin and in one embodiment by the entire IVS-2 of an ⁇ -globin gene or a 0-globin gene.
- the smaller intervening sequence ( ⁇ TVS-1) can be derived from either the 6" IVS-1 or IVS-1 from a major globin gene.
- the gene of the invention further has a promoter of an ⁇ -globin gene or a ⁇ -globin gene, and, in one embodiment, also has the 3' and 5• untranslated regions are derived from a major globin sequence.
- the IVS-2 of jS-globin but not of ⁇ * -globin contains three binding sites for the erythroid specific transcription factor NF-E1.
- the sites can have any of the sequences: 5'GATAAG3' or
- GATA-1 5' (T/A)GATA(G/A)3• or their complements, hereinafter any of these sequences are termed a "GATA-1" site.
- NF-III which can be the sequence 5 ⁇ TTTGCAT3 1 or its complement, hereinafter an "Oct-1" site. Pruijn, J.M., et al., 1987, EMBO J.
- the genes within the scope of the invention have one or two Oct-1 sites, preferably one, and between two and six, preferably three, GATA-1 sites disposed between the splice donor and splice acceptor sites that define the intron or intervening sequence.
- the chimeric genes of the invention are used to engineer transgenic animals that express the chimeric gene and a normal ⁇ -globin gene in erythroid cells so that a hemoglobin similar to human HbA 2 and useful as a blood substitute is produced. See, e.g., U.S. Patent Application, Logan et al., United States Patent Application Serial No.
- the net effect of the substitutions on the charge of the hemoglobin is the replacement of a Glu by an Arg.
- the more basic isoelectric point allows for the separation of HbA 2 from the blood of the transgenic animal more readily than the 3-globin containing HbA.
- the proteins encoded by the gene of the present invention thus, encompass globins that are homologous to either a ⁇ - or ⁇ - globin at every position except ait 22 and 116 where a different tests apply: at position 22, the residue is uncharged at physiologic pH and at position 116, an arginine is present.
- a globin meeting these conditions is termed a ⁇ -homologous globin hereinafter.
- the hemoglobin having two ⁇ "-homlogous globins and two human ⁇ -globins has a pi of between 7.3 and 7.5; a ⁇ - homologous globin that forms such a hemoglobin is referred to hereinafter as a "6 * -like globin.”
- the ⁇ S-like globin is homologous to human ⁇ or ⁇ globin at all positions except for 22 and 116 will be referred to as a human ⁇ "-like globin.
- the human 6 * -like globin is human 5-globin.
- the scope of the invention includes the placement of the chimeric gene in a vector constructed for the generation of transgenic animals.
- the vector is designed to direct expression of the chimeric globin gene in erythropoietic cells of the animal.
- the vector should further contain a globin Locus Control Region from a major globin locus, i.e. the ⁇ -globin or 0-globin locus, and a human ⁇ -globin gene operably linked to a promoter.
- LCR locus control region
- a shortened LCR which is suited for use with the present invention has been constructed and contains only the DNase super-hypersensitive regions of the human /3-globin locus control region (hereinafter " ⁇ LCR") .
- This 6.5 kb LCR consists of four fragments of between 1.1 kb and 2.1 kb, and has been shown to be active in an orientation independent manner in transfected mouse erythroleuke ia cells.
- the detailed structure of the ⁇ LCR is provided in Grosveld, F.G., 1989, WO 89/01517 and 1992, WO 92/11380 which are hereby incorporated by reference in its entirety.
- the chimeric globin gene encoding a ⁇ * -like globin and the vectors to be used in accordance with the invention can be constructed using reco binant DNA techniques well known to those skilled in the art, e.g., see Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.
- the chimeric gene of the present invention can also be synthesized in whole or in part by a combination of readily available techniques.
- Such techniques include solid-phase nucleotide synthesis to construct primers containing new restriction sites, polymerase chain reaction amplification using such primers, to readily obtain fragments containing ⁇ " globin gene sequences for insertion into an isolated major globin gene and recombinant DNA techniques for the insertion of the fragments into the major globin gene and for production of the chimeric gene and the vectors for constructing the transgenic animals.
- 5.2 GENERATION OF THE TRANSGENIC ANIMALS The invention provides the above-described chimeric nucleic acid which has been made substantially free of proteins, as by (for example) the well known chloroform/phenol extraction procedure or its equivalent. The invention also contemplates the above-described chimeric gene which has been introduced into any organism.
- Examples of such organisms would include plasmid cloning vectors and expression vectors suited for the production of the nucleic acid of the invention in bacterial hosts; eukaryotic expression systems such as erythroleukemia cell lines that can be induced to differentiate and to produce hemoglobin as well as constitutive, non- inducible eukaryotic expression systems.
- the invention further contemplates that the above-described ⁇ -globin/ ⁇ "-like globin chimeric gene expression cassette can be placed into the genome of a non-human animal.
- the invention further contemplates that the transgenic animals of the invention can be constructed by any of the available methods including pronuclear injection and transfection of embryonic stem cells followed by blastocyst fusion to create chimeric animals. The offspring of the chimeric animals are transgenic animals.
- mice Animals of any species, including but not limited to mice, rats, rabbits, guinea pigs, pigs, micro-pigs, and non-human primates, e.g.. baboons, squirrel monkeys and chimpanzees can be used to generate the transgenic animals of the invention so long as a known globin LCR is functional in that species.
- the preferred embodiments employ mice and pigs.
- Any technique known in the art can be used to introduce the transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Gordon et al., 1980, PROC NATL. ACAD. SCI.
- the present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all cells, i.e. , mosaic animals.
- the transgene can be integrated as a single transgene or in tandem, e.g.. head to head tandems, or head to tail or tail to tail.
- the human HbA 2 is superior to human HbA for use in the manufacture of a blood substitute.
- the superiority can be readily appreciated from the study of the differences in primary structures of the ⁇ and ⁇ globin genes.
- the Glu 22 and adjacent His 116,117 of 0-globin are replaced in 5-globin by Ala, Arg and Asn respectively. These substitutions lead to two significant changes. Firstly, there is an additional interaction between the ⁇ and ⁇ globin chains, specifically between ⁇ SArg 116 and ⁇ Pro 114 , which confers additional stability to the HbA 2 molecule. Perutz, M. and Raidt, H. , 1975, NATURE 255:256.
- HbA 2 is the first hemoglobin to elute from the ion exchange resin used for the preparation of HbA 2 from transgenic hemolysate.
- Mono-P®. Pulcoa
- DEAE Cellulose Whatman
- Maenprep® Biorad
- the large scale purification of the HbA 2 is accomplished by application of the hemoglobin mixture to a Mono-P® ion exchange column or its equivalent in a buffer, which is about 10 mM NaCl, which will allow only the HbA 2 to pass through the column without binding.
- the hemoglobin made and purified according to the invention can then be polymerized by means well known to those in the art, dissolved in a physiological buffer using pharmaceutically acceptable water and salts.
- the solution of between about 2.5 and 100 mg/ml of protein is then administered intravenously by means and at doses selected to restore the subjects blood volume and/or hemoglobin concentration.
- United States Patents concern part or all of these processes and are hereby incorporated by reference.
- step l a chimeric ⁇ / ⁇ globin gene was created by a 0 PCR based cloning strategy. There are only four amino-acid changes between ⁇ and ⁇ globin proteins in the region of amino acid residues 110 to 146 encoded by DNA sequences located between unique Bam HI and Eco Rl restriction enzyme sites in plasmid pSelect ⁇ (ATCC S Accession No. 75782) .
- ⁇ * A contains sequences of the ⁇ globin 2nd exon, 2nd intron and part of the 3rd exon where the sequences encoding residues 116 and 117 were converted to the ⁇ * globin sequence.
- This fragment was digested with Eco Rl and Mlu 1 to obtain ⁇ B which contains the 3rd exon of ⁇ globin with codons 125 and 126 converted to the ⁇ " globin sequence, the ⁇ globin gene 3' UTR and downstream sequences.
- the remaining portions of the ⁇ * globin geno ic coding sequences were obtained by digestion of plasmid pSelect/Jp ⁇ " with Nco 1 and Bam HI. The three fragments described above were ligated into Nco 1 and Mlu 1 digested plasmid pSelect ⁇ to create plasmid pSelect ⁇ p ⁇ * with ⁇ intron and /?3• .
- Step 2 The modified chimeric ⁇ globin gene was obtained as a Kpn 1 and Mlu 1 fragment and cloned into similarly digested pLCR ⁇ e.
- the resultant plasmid pLCR ⁇ e ⁇ "* was designated construct 534 (deposited as
- Transgenic animals were engineered using a human 5-globin gene (Construct # 487, Figure IB) or a chimeric gene consisting of sequences encoding a ⁇ - globin exons and introns and 3-globin 3• and 5•
- a Construct #534 containing plasmid was deposited with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852 on May 23, 1994 as Accession No. ATCC 75785.
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Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP8502334A JPH10505226A (en) | 1994-06-10 | 1995-06-08 | Production of hemoglobin with δ-like globin |
EP95923762A EP0765337A4 (en) | 1994-06-10 | 1995-06-08 | The production of hemoglobin having a delta-like globin |
AU28203/95A AU707436B2 (en) | 1994-06-10 | 1995-06-08 | The production of hemoglobin having a delta-like globin |
FI964917A FI964917A (en) | 1994-06-10 | 1996-12-09 | Preparation of hemoglobin with alpha-like globin |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US258,311 | 1994-06-10 | ||
US08/258,311 US5821351A (en) | 1994-06-10 | 1994-06-10 | Production of hemoglobin having a delta-like globin |
Publications (1)
Publication Number | Publication Date |
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WO1995034572A1 true WO1995034572A1 (en) | 1995-12-21 |
Family
ID=22980021
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/US1995/007286 WO1995034572A1 (en) | 1994-06-10 | 1995-06-08 | THE PRODUCTION OF HEMOGLOBIN HAVING A δ-LIKE GLOBIN |
Country Status (7)
Country | Link |
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US (2) | US5821351A (en) |
EP (1) | EP0765337A4 (en) |
JP (1) | JPH10505226A (en) |
AU (1) | AU707436B2 (en) |
CA (1) | CA2192066A1 (en) |
FI (1) | FI964917A (en) |
WO (1) | WO1995034572A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8129151B2 (en) | 1998-03-18 | 2012-03-06 | Ajinomoto Co., Inc. | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5821351A (en) * | 1994-06-10 | 1998-10-13 | Dnx Biotherapeutics | Production of hemoglobin having a delta-like globin |
JP4420539B2 (en) | 1999-09-24 | 2010-02-24 | 積水化学工業株式会社 | Method for separating hemoglobin A2 |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB8718779D0 (en) * | 1987-08-07 | 1987-09-16 | Grosveld F G | Dna sequence & expression vector |
GB9027917D0 (en) * | 1990-12-21 | 1991-02-13 | Ici Plc | Expression systems |
FR2677652B1 (en) * | 1991-06-12 | 2005-05-27 | Agronomique Inst Nat Rech | PROCESS FOR PREPARING A PROTEIN OF INTEREST IN MILK OF A TRANSGENIC ANIMAL, PRODUCT OBTAINED, AND EUCARYOTIC CELL USED |
CA2111348A1 (en) * | 1991-06-14 | 1992-12-23 | John S. Logan | Production of human hemoglobin in transgenic pigs |
US5922854A (en) * | 1991-06-14 | 1999-07-13 | Kumar; Ramesh | Purifying Human Hemogloblin from transgenic pig red cells and plasmids containing pig globin nucleic acids |
WO1993025071A1 (en) * | 1992-06-12 | 1993-12-23 | Dnx Corporation | Production of human hemoglobin in transgenic pigs |
CA2165679A1 (en) * | 1993-06-21 | 1995-01-05 | Tim M. Townes | Anti-sickling hemoglobin |
US5821351A (en) * | 1994-06-10 | 1998-10-13 | Dnx Biotherapeutics | Production of hemoglobin having a delta-like globin |
-
1994
- 1994-06-10 US US08/258,311 patent/US5821351A/en not_active Expired - Fee Related
-
1995
- 1995-06-08 CA CA002192066A patent/CA2192066A1/en not_active Abandoned
- 1995-06-08 JP JP8502334A patent/JPH10505226A/en not_active Ceased
- 1995-06-08 AU AU28203/95A patent/AU707436B2/en not_active Ceased
- 1995-06-08 EP EP95923762A patent/EP0765337A4/en not_active Withdrawn
- 1995-06-08 WO PCT/US1995/007286 patent/WO1995034572A1/en not_active Application Discontinuation
-
1996
- 1996-12-09 FI FI964917A patent/FI964917A/en unknown
-
1997
- 1997-07-09 US US08/890,328 patent/US5952482A/en not_active Expired - Fee Related
Non-Patent Citations (8)
Title |
---|
BLOOD, Volume 77, Number 10, issued 1991, MOON et al., "Functional Properties of the Beta-Globin Locus Control Region in K562 Erythroleukemia Cells", pages 2272-2284. * |
BLOOD, Volume 77, Number 6, issued 1991, CONSTANTOULAKIS et al., "Locus Control Region-A Gamma Transgenic Mice: A New Model for Studying Induction of Fetal Hemoglobin in the Adult", pages 1326-1333. * |
CELLULAR AND MOLECULAR BIOLOGY, Volume 30, Number 3, issued 1984, STAVRIDIS et al., "Expression of the Human beta-delta-Globin Genes in Rabbits", pages 209-216. * |
CURRENT OPINIONS IN STRUCTURAL BIOLOGY, Volume 4, Number 1, issued 1994, BURLEY, "DNA-Binding Motifs From Eukaryotic Transcription Factors", pages 3-11. * |
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 269, Number 52, issued 30 December 1994, PEREZ-STABLE, "Distinct Negative Regulatory Mechanisms Involved in the Repression of Human Embryonic Epsilon- and Fetal G Gamma-Globin Genes in Transgenic Mice", pages 33109-33115. * |
MOLECULAR AND CELLULAR BIOLOGY, Volume 13, Number 12, issued 1993, STAMATOYANNOPOULOS et al., "Developmental Regulation of Human Gamma-Globin Genes in Transgenic Mice", pages 7636-7634. * |
MOLECULAR AND CELLULAR NEUROSCIENCES, Volume 3, Number 2, issued 1992, RUBINSTEIN et al., "Targeted Expression of Somatostatin in Vasopressinergic Magnocellular Hypothalmic Neurons of Transgenic Mice", pages 152-161. * |
See also references of EP0765337A4 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8129151B2 (en) | 1998-03-18 | 2012-03-06 | Ajinomoto Co., Inc. | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
Also Published As
Publication number | Publication date |
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JPH10505226A (en) | 1998-05-26 |
US5952482A (en) | 1999-09-14 |
EP0765337A1 (en) | 1997-04-02 |
US5821351A (en) | 1998-10-13 |
EP0765337A4 (en) | 1999-11-24 |
CA2192066A1 (en) | 1995-12-21 |
FI964917A0 (en) | 1996-12-09 |
FI964917A (en) | 1996-12-09 |
AU2820395A (en) | 1996-01-05 |
AU707436B2 (en) | 1999-07-08 |
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