WO1999058699A2 - Facteurs de coagulation du sang humain derives de plantes transgeniques - Google Patents

Facteurs de coagulation du sang humain derives de plantes transgeniques Download PDF

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WO1999058699A2
WO1999058699A2 PCT/US1999/010732 US9910732W WO9958699A2 WO 1999058699 A2 WO1999058699 A2 WO 1999058699A2 US 9910732 W US9910732 W US 9910732W WO 9958699 A2 WO9958699 A2 WO 9958699A2
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factor
plant
human
coagulation
protein
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WO1999058699A3 (fr
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Brian S. Hooker
Jianwei Gao
Daniel B. Anderson
Ziyu Dai
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Battelle Memorial Institute
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/644—Coagulation factor IXa (3.4.21.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6429—Thrombin (3.4.21.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21005—Thrombin (3.4.21.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21022—Coagulation factor IXa (3.4.21.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to transgenic plant-derived human coagulation factors. More specifically, the invention relates to producing human-like blood coagulation factors (e.g, factors VIII, IX, XIII, thrombin) from transgenic plant cells encoded to produce human coagulation factors.
  • human-like blood coagulation factors e.g, factors VIII, IX, XIII, thrombin
  • human factor and “human coagulation factor” are interchangeable and refer to human-like proteins possessing human factor-like procoagulant or coagulant activity derived from transgenic plants.
  • the present invention relates to the introduction of genes or DNA encoding human blood coagulation factors into plants.
  • the present invention also relates to the production of active human-like blood clotting proteins from transgenic plant materials which provides a cost effective means for producing viral free, human-like blood coagulation factors.
  • the invention further relates to the use of plant-derived human factors for blood factor replacement, wound healing or other therapeutic applications contemplated for human blood coagulation factors.
  • plant shall refer to the plant itself, cells or tissues derived from the plant, seeds, cuttings, or other plant-derived structure. Both monocotyledonous and dicotyledonous angiosperm plants are included within the definition of "plant”.
  • Damage to the human vascular system leads to the participation of many physiological processes that are important in controlling blood loss.
  • the platelet adhesion process occurs, where platelets become sticky and bound to the endothelial connective tissue structures, leading to platelet plug formation.
  • the platelet release reaction gives rise to vasoactive amines, such as serotonin which causes vasorestriction ofthe injured vessel.
  • the third effect is the triggering ofthe coagulation process, involving a cascade of proteins in both the intrinsic and extrinsic systems, to arrest bleeding.
  • the fourth important effect is the activation ofthe fibrinolytic system, which leads to the degradation ofthe fibrin clot, healing and regeneration ofthe vessel wall. This particular invention is focused on production of proteins involved in the coagulation cascade.
  • the primary role ofthe coagulation cascade is to stabilize the initial platelet plug. This system consists of over a dozen interacting proteins present in plasma as well as released or activated cellular proteins. Each step ofthe cascade involves the activation of a specific inactive (zymogen) form of a protease to the catalytically active form.
  • the zymogen form of each protein is assigned a Roman numeral designation, while the activated form is designated by a Roman numeral followed by a subscript "a”.
  • the activated form ofthe protease in each step ofthe cascade catalyzes activation ofthe subsequent protease in the cascade.
  • the intrinsic coagulation pathway is initiated by contact between factor XII and an active surface (e.g., unbroken skin, articular cartilage, vascular basement membrane, sebum, long-chain fatty acids, etc.) or through fluid-phase proteolysis via kallikrein or some other enzymatic activator.
  • an active surface e.g., unbroken skin, articular cartilage, vascular basement membrane, sebum, long-chain fatty acids, etc.
  • factor XII Subsequent activation of factor XII initiates a series of reactions involving factors XI, IX, VIII, prekallikrein, high molecular weight kininogen (HWMK), and platelet factor 3 (PF- 3), which lead to the activation of factor X.
  • the extrinsic coagulation pathway is initiated by interactions between tissue factor and factor VII in the presence of Ca 2+ , leading to production of an enzyme that also activates factor X. This pathway does not require contact activation.
  • proteolytic activity that evolves early in the contact phase of coagulation greatly enhances the activity of factor VII.
  • Subsequent steps in the blood coagulation process are referred to as the common pathway of coagulation because they are common to both the intrinsic and extrinsic pathways.
  • This pathway involves factors X and V, PF-3, prothrombin, and fibrinogen and proceeds in essentially the same manner regardless of whether factor X is activated by factor IXa, PF-3, and factor Villa (intrinsic pathway) or by factor Vila and tissue factor (extrinsic pathway).
  • factor X is activated by factor IXa, PF-3, and factor Villa (intrinsic pathway) or by factor Vila and tissue factor (extrinsic pathway).
  • factor soluble polymeric fibrin is stabilized into a non-soluble clot through interactions with factor XHIa.
  • coagulation factors of current or potential pharmaceutical value include factors VIII, IX, VII, XIII as well as thrombin, fibrin, and fibrinogen.
  • factors VIII and IX are respectively used in the treatment ofthe two most common hemophilic disorders, hemophilia A (classical hemophilia) and hemophilia B (Christmas disease).
  • Hemophilia A is a sex-linked bleeding disorder, characterized by a deficiency in human coagulation factor VIII.
  • hemophilia B is a sex-linked bleeding disorder resulting from a deficiency of human coagulation factor IX.
  • Approximately 80% of all hemophilia disorders are due to a deficiency of factor VIII.
  • hemophilia Both types of hemophilia clinically result in the lack of sufficient fibrin formation required for clot stabilization. Subsequently, hemophiliacs suffer chronic bleeding episodes resulting at sites of relatively weak clot formation.
  • a single defective allele results in hemophilia in males, who only have one copy ofthe X chromosome. Since deficiency of other coagulation factors is autosomally linked (i.e., generally, two copies are needed to result in deficiency), hemophilia A and B are by far the most common hereditary blood clotting disorders, appearing almost exclusively in males.
  • Factor VIII is a highly specialized type of protein used primarily in the treatment of Hemophilia A as well as for other research and commercial applications.
  • Factor VIII is a large glycoprotein that, when activated, functions in the blood coagulation cascade as a cofactor, along with calcium ions and phospholipid, in the factor IX mediated activation of factor X in the intrinsic coagulation pathway. It can be activated proteolytically by several coagulation enzymes, including thrombin. Plasma-derived factor VIII is in chronically short supply, therefore making this type of therapy highly cost prohibitive.
  • resulting pharmaceutical products derived from plasma are highly impure, with a specific activity of 0.5 to 2 factor VIII units per milligram protein (one unit of factor VIII activity is by definition the activity present in one miililiter of normal plasma).
  • Resulting plasma-derived factor VIII purity is typically lower than 1% by weight (Wood et al. 1984. Nature 312:330).
  • the high level of impurities result in a variety of serious complications including transmission of hepatitis A, B, and C, human parvovirus and human immunodeficiency virus (HIV) pathogens.
  • HIV human immunodeficiency virus
  • recombinant factor VIII was produced in baby hamster kidney (BHK) cell lines, using the calcium-phosphate coprecipitation method (Simonsen et al. 1983. Proc Natl Acad Sci USA 80:2495, Wigler et al. 1979. Proc Natl Acad Sci USA 76:1373) for integration ofthe 7kb protein encoding region (Wood et al. supra 1984, Capon et al. 1997. U.S. Patent No. 5618788). Active recombinant human factor VIII has also been produced in Chinese hamster ovary (CHO) cells (Kaufman et al. 1988. J Biol Chem 263:6352) and monkey COS-7 cells (Toole et al.
  • Factor IX is the zymogen of a serine protease responsible for the activation of factor X in the intrinsic pathway ofthe coagulation cascade and is used primarily in the treatment of Hemophilia B. Factor IX is activated directly by factor XI a .
  • Factor IX is normally synthesized in the liver and undergoes extensive post-translational modifications including glycosylation, ⁇ -carboxy lation of specific glutamic acid residues (DiScipio et al. 1979. Biochem. 18:899), and ⁇ -hydroxylation of a single aspartic acid residue (McMullen et al. 1983 Biochem Biophys Res Commun 115:8).
  • factor VIII commercially available factor IX is produced via both plasma purification and genetically modified mammalian cell culture.
  • Factor VII is a zymogen of an enzyme that forms a complex with tissue factor for the activation of factor X in the extrinsic coagulation pathway.
  • Factor VII may be activated predominantly by kallikrein and factor Xllf under physiological conditions, although factor X a , plasmin, and factor IX a have been reported to also accomplish activation.
  • factor VII is synthesized in the liver and undergoes glycosylation as well as ⁇ -carboxy lation of specific glutamic acid residues (O'Hara et al. 1987 Proc Natl Acad Sci USA 84:5158).
  • factor VII a was used initially for treatment of severe bleeding episodes in hemophiliacs who could not receive factor VIII due to immune system response. However, it is currently contemplated for use as a replacement therapeutic for factor VIII and factor IX in the treatment of hemophilia A and B, respectively.
  • Factor VII may be more efficient in coagulation than these other factors since it travels directly to the site ofthe injury, rather than dispersing throughout the bloodstream.
  • Recombinant factor VII is currently produced in its activated form (factor VII a ) in baby hamster kidney (BHK) cells.
  • thrombin is a multifunctional protein catalyzing several key reactions in the coagulation cascade (Mann et al. 1988 Ann Rev Biochem, 57:915). In one reaction, this enzyme acts as a catalyst in the conversion of fibrinogen to fibrin, which is the main component in stable clots. In addition to this reaction, thrombin also activates platelets as well as factors V, VIII, IX and XIII. Therapeutically, thrombin is used primarily in tissue sealants, usually in conjunction with fibrinogen.
  • Prothrombin the zymogen for thrombin, requires ⁇ —carboxylation of specific glutamic acid residues for activity.
  • Prothrombin (Mr 72,000) is a vitamin K dependent glycoprotein protein that participates in the final phase of blood coagulation (Mann et al 1980 in: CRC Handbook Series in Clinical Laboratory Science, Section I: Hematology Schmidt RM Ed. Vol. 3 pp.
  • prothrombin is activated by cleavage ofthe first factor Xa site to form prethrombin-2, which is then cleaved at the second factor Xa site to form thrombin.
  • Thrombin (M r 34,000) consists of a 259 amino acid heavy chain and a 49 amino acid light chain connected by a single disulfide bond (Friezner Degen et al. 1983 Biochem
  • Factor XIII is the heterotetramer (a b ) zymogen to factor XIII a , which catalyzes the formation of intermolecular ⁇ -glutamyl-e-lysine bridges between fibrin molecules, which strengthens the clot against lysis (Lorand 1972.
  • factor XIII is not currently used in therapeutic applications, it is being contemplated for future use as a wound closure aid.
  • Transgenic plants can be used for the production of high value, medicinally important proteins, for example, monoclonal antibodies (Hiatt et al. 1989. Nature 342:76, During et al. 1990. Plant Mol Biol 15:281, Benvenuto et al. 1991. Plant Mol Biol 17:865, Firek et al. 1993. Plant Mol Biol 23:861, Magnuson et al. 1996. Prot Expr Purif 7:220), human growth hormone (Kay et al. 1987. Science 236:1299), human serum albumin (Sijmons et al. 1990. Bio/Technol 8:217), human ⁇ -interferon (DeZoeten et al. 1989.
  • monoclonal antibodies Hiatt et al. 1989. Nature 342:76, During et al. 1990. Plant Mol Biol 15:281, Benvenuto et al. 1991. Plant Mol Biol 17:865, Firek et al. 1993
  • Genetically engineered plant-based production of therapeutic proteins offers several advantages when compared to other production sources including human fluids/tissues, recombinant microbes, transfected animal cell lines or transgenic animals.
  • farming of transgenic plants can significantly reduce the direct production cost of recombinant proteins.
  • Large-scale agricultural systems may yield production costs as low as $6 to 60 per kg of raw protein product which are more than an order of magnitude less than similar direct production costs of recombinants in E.coli, currently estimated at $250 per kg (Kusnadi et al. 1997. Biotechnol Bioeng 56:473).
  • Equally important, plant systems are capable of performing complex post-translational modifications necessary for activity in many human protein therapeutics.
  • Plants may be transformed to express foreign DNA using a variety of methods including Agrobacterium transformation (An 1986. Plant Physiol 81 :86, Hoekema et al. 1985. Plant Mol Biol 5:8589), microprojectile bombardment (Klein et al., Gene Transfer by Particle Bombardment, Plant Tissue Culture Manual, Dl, p. 112, 1991, Kluwer Academic Publishers), pollen transformation (Saunders et al. 1997. U.S. Patent 5629183), chemical mediated uptake by protoplasts (Krens et al. 1982. Nature 296:72), and electroporation (Langridge et al. 1985 Plant Cell Reports 4:355).
  • Agrobacterium transformation An 1986. Plant Physiol 81 :86, Hoekema et al. 1985. Plant Mol Biol 5:8589
  • microprojectile bombardment Klein et al., Gene Transfer by Particle Bombardment, Plant Tissue Culture Manual, Dl, p
  • Agrobacterium transformation A. tumefaciens is the etiological agent of crown gall, a disease of a wide range of dicotyledons and gymnosperms (DeCleene et al. 1976. Bot Rev 42:389).
  • Virulent strains of A. tumefaciens contain large, tumor-inducing (Ti) plasmids (at about 200 kb).
  • Ti tumor-inducing
  • T-DNA a portion ofthe Ti plasmid, called T-DNA, is transferred to and integrated into the nuclear genome ofthe infected plant cells (Hemalsteens et al. 1980. Nature 287:654, Lichtenstein et al. 1987.
  • T-DNA encoded genes are transcribed and translated in the plant tissues, resulting in auxin, cytokinin and opine synthesis.
  • auxin and cytokinin levels cause rapid plant cell proliferation, resulting in gall formation (Gheysen et al. 1985. DNA flux across genetic barriers: the crown gall phenomenon, In: Genetic Flux in Plants, Hohn et al., Eds., Springer, Wein, pp. 11-49).
  • the native T-DNA sequence responsible for gall formation, can be replaced with foreign DNA, including selectable markers as well as the gene of interest.
  • the only components required for DNA transfer in the T-DNA region are the left and right border sequences (Zambryski et al. 1982. J Mol Appl Genet 1 :361).
  • Ti plasmids are difficult to manipulate directly in vitro due to their large size, simplified systems have been developed.
  • the most advanced method utilizes a binary vector system in which the binary vector contains the minimum elements required in cis (An supra 1986, An 1987. Meth Enzymol 153:292). Other functions necessary for the gene transfer mechanism are donated from a separate helper, Ti plasmid.
  • the binary vector may be directly manipulated in an E. coli host and transferred to A. tumefaciens containing the helper Ti plasmid, through biparental or triparental mating. Finally, the T-DNA region is integrated into the plant nuclear genome by cocultivation of plant material with transformed A. tumefaciens.
  • Representative tissues that have been transformed using an Agrobacterium method include tobacco (Barton et al. 1983. Cell 32:1033), tomato (Fillarti et al. 1987. Bio/Technol 5:726), sunflower (Everett et al. 1987. Bio/Technol 5:1201), cotton (Umbeck et al. 1987.
  • Bio/Technol 5:263) rapeseed (Pua et al. 1987. Bio/Technol 5:815), potato (Facciotti et al. 1985. Bio/Technol 3:241), poplar (Pythoud et al. 1987. Bio/Technol 5:1323) and soybean (Hinchee et al. 1988. Bio/Technol 6:915).
  • plant germplasm is transformed with foreign DNA by introducing the DNA into pollen grains by techniques such as electroporation (Mishra et al. 1987. Plant Sci 52:135), mating ova ofthe desired plant line with the transformed pollen, and selecting for the transformed germplasm.
  • the germinating pollen, resulting seed, and the progeny can each be screened for expression ofthe foreign gene.
  • the transformed pollen can be used as a vector for introducing the foreign DNA into plant lines of similar or dissimilar origin, including both monocots and dicots.
  • pollen collected from tobacco and corn plants has been stably transformed via electroporation (Saunders et al. supra 1997).
  • tobacco plants have been stably transformed to produce ⁇ -glucoronidase by mating ova with transformed tobacco pollen.
  • DNA uptake via chemical stimulation was developed for the direct transformation of both monocot and dicot protoplasts (Krens et al. supra 1982).
  • the plant cell wall is first degraded enzymatically to form protoplasts, using standard techniques.
  • the protoplasts and vector are then incubated in the presence of polyethylene glycol, which facilitates transformation via direct insertion.
  • Either a direct gene transfer vector or a Ti plasmid may be used in transformation. Since protoplast transformation can facilitate measurable gene expression within 24 to 48 hours, this method has been used widely by many researchers (Prols et al. 1988. Plant Cell Reports 7:221, Topfer et al. 1988. Plant Cell Reports 7:225).
  • plants After transformation of plant tissues, plants must be regenerated for characterization of stable transformants, selection, breeding and segregation analysis, and foreign protein (blood coagulation factor) production. Plants may be regenerated from a variety of tissues including callus culture, protoplasts, and A. tumefaciens tissues (i.e., explants or calli). Regeneration from callus tissue has been demonstrated in monocots, such as corn, rice, barley, wheat and rye and dicots, such as sunflower, soybean, cotton, rapeseed and tobacco.
  • monocots such as corn, rice, barley, wheat and rye
  • dicots such as sunflower, soybean, cotton, rapeseed and tobacco.
  • Regeneration of plants from protoplasts is particularly useful for tissues transformed via direct gene transfer methods including electroporation, PEG-mediated transformation, or microparticle bombardment. Regeneration of plants from protoplasts has been demonstrated for rice (Abdulah et al. 1987. Bio/Technol 4:1987), tobacco (Potrykus et al. 1985. Mol Gen Genet 199:169), rapeseed (Kansha et al. 1986. Plant Cell Reports 5:101), and potato (Tavazza et al. 1986. Plant Cell Reports 5:243), among others.
  • the present invention is directed to transgenic plants that contain DNA sequences encoding for human coagulation factors. We have surprisingly discovered that correctly processed, active blood coagulation factors can be produced inplanta.
  • the present invention is further directed to compositions in transgenic plants or plant cell culture wherein the human coagulation factor is produced either as a protein having a human coagulation like procoagulant or coagulant activity or as an amino acid sequence substantially equivalent to that of a human coagulation factor.
  • transgenic plant compositions are useful for producing viral free, active human-like blood coagulation proteins necessary for use in blood factor replacement (hemophilia), wound healing or other therapeutic applications contemplated for human blood coagulation factors.
  • Viral pathogenicity is a major problem for blood-derived therapeutics because current production methods rely on human serum fractionation or transgenic mammalian cell culture methods. Both of these mammalian based systems can harbor pathogenic viruses resulting in contamination of derived therapeutic proteins with human pathogens. Plants systems are not known to harbor or transmit any human or mammalian viruses.
  • the transgenic plants are produced by transforming plants using known plant molecular biology methods, constructing vectors containing a DNA sequence or sequences encoding for human coagulation factors.
  • a reference herein to "human factor”, “coagulation factor” or “coagulation factor N” may include a reference to any of these, or to any combination of these.
  • the scope of this specification should not be deemed limited to this list, but to any similar factor performing the equivalent function in the human body, and any derivative of such factor.
  • Transgenic plants are useful for the production of human-like coagulation factors because they can perform necessary posttranslational modifications (e.g. glycosylation, folding and peptide cleavage) of these blood factor proteins by natural processing mechanisms, through further genetic engineering modifications, and/or through in vitro processing.
  • FIG. 1 depicts schematically the blood coagulation cascade including intrinsic, extrinsic and common pathways. Figure reproduced from “Blood Coagulation,” by Bithell in The Normal Hematopoietic System (Bloom and Thomas, Eds., Churchill, Livingston, N.Y., 1981, p. 579).
  • FIG. El-1 depicts schematically the construction of pSP64-FVIIIc, a vector used to obtain the encoding sequence for pre-coagulation factor VIII for cloning into the plant expression vector pGA748.
  • Pre-coagulation factor VIII cDNA encoding the 2332 amino acid mature protein plus the 19 amino acid native signal was inserted into the pSP64 plasmid at the Sal I site using a Sal I adapter, resulting in the new 10200 bp pSP64- FVIIIc plasmid.
  • This plasmid carried the ampicillin resistance gene for positive transformant selection in E. coli.
  • FIG. ⁇ l-2 depicts schematically the construction of pZD201, the plant expression vector carrying the pre-coagulation factor VIII encoding region.
  • the full length pre- coagulation factor VIII cDNA was excised with Sal I restriction enzyme and sequentially ligated into the compatible restriction enzyme site Xho I located between the CaMV 35S promoter and T7-T5 transcript terminator of binary vector pGA748, forming the 18800 bp plasmid pZD201.
  • This plasmid carried the tetracycline resistance gene for positive transformant selection in E. coli and Agrobacterium tumefaciens as well as the kanamycin resistance gene for selection of whole plant and plant cell culture positive transformants.
  • FIG. El -3 depicts schematically the construction of pGA2020, a vector used to obtain the encoding sequence for coagulation factor XIII A-domain for cloning into the plant expression vector pGA643.
  • Coagulation factor XIII A-domain cDNA encoding the 731 amino acid protein and 29 amino acid signal peptide was inserted into pBluescript SK- at the Pst I site to adapt the Xba I site at the 5' end and the Cla I site at the 3'end, resulting in the new 5.3 kb pGA2020 plasmid.
  • This plasmid carried the ampicillin resistance gene for positive transformant selection in E. coli.
  • FIG. El -4 depicts schematically the construction of pGA2023, the plant expression vector carrying the coagulation factor XIII A-domain encoding region.
  • the full length coagulation factor XIII A-domain cDNA was excised at Xba I/Cla I restriction sites and sequentially ligated into the compatible Xba I and Cla I restriction enzyme sites located between the CaMV 35S promoter and T7-T5 transcript terminator of binary vector pGA643, forming the 14.0 kb plasmid pGA2023.
  • This plasmid carried the tetracycline resistance gene for positive transformant selection in E. coli and A. tumefaciens as well as the kanamycin resistance gene for selection of whole plant and plant cell culture positive transformants.
  • FIG. El -5 is a construction map of plasmid vector pGA2042 containing the prethrombin-2 (PT2) gene.
  • FIG. El -6 is a construction map ofthe binary vector pGA2043 containing the human prethrombin-2 gene.
  • FIG. El -7 is a construction map ofthe plasmid vector pGA2049 containing the human prethrombin-2 gene.
  • FIG. E 1 -8 is a construction map of plasmid vector pGA2029 for the expression of human factor IX in transgenic plant.
  • FIG. El -9 is a construction map of plasmid vector pGA2030 for the expression of human factor IX in transgenic plant.
  • FIG. E3-1 shows the result of dot blot immunoassay for TO Factor VIII plant transformants. Positive control plasma-derived factor VIII standards (American
  • Negative control leaf protein extracted from untransformed Nicotiana tabacum cv. SRI is shown as SR.
  • Positive plant primary transformants are shown as 1004-3, 1006-2 and 1006-3.
  • FIG. E3-2 shows protein gel bands resulting from Western blot immunoassay completed on protein extracts from several plant transformants as compared to a non- transformed control culture and plasma-derived factor VIII standard (American Diagnostica, Greenwich, CT).
  • Lane 1 shows the plasma-derived factor VIII (FVIIIc) standard;
  • lane 2 shows total protein extracts from leaf explants taken from an untransformed N tabacum cv. SRI control;
  • lanes 3 through 9 show total protein extracts from leaf explants taken from TI plant lines derived from primary transformant tobacco plants.
  • FIG. E4-1 is a reverse electrophoresis gel image of PCR products of transgenic plant genomic D ⁇ A samples and control D ⁇ A samples.
  • FIG. E4-2 is a western blot analysis image of transgenic factor XIII A-subunit protein samples and various control protein samples.
  • FIG. E4-3 is a western blot analysis image of various transgenic factor XIII A- subunit protein samples and control protein samples.
  • FIG. E4-4 is a western blot analysis image of factor XIII A-subunit expression at different leaf positions.
  • FIG. E5-1 is a reverse electrophoresis gel image ofthe PCR products of prethrombin-2 transgenic plant genomic DNA samples and control DNA.
  • FIG. E5-2 is a western blot image of transgenic prethrombin-2 plant protein samples.
  • FIG. E5-3 is a silver stain analysis of protein samples purified by metal chelating sepharose column.
  • FIG. E5-4 is a construction map of transient expression plasmid vector pGA2054a.
  • FIG. E5-5 is a construction map of plasmid vector pGA2056 containing prothrombin gene.
  • FIG. E5-6 is a construction map of plasmid vector pGA2057 containing prethrombin-2 gene.
  • FIG. E5-7 is a construction map of plasmid vector pGA2058 for prothrombin transient expression.
  • FIG. E5-8 is a construction map of plasmid vector pGA2059 for prethrombin-2 transient expression.
  • FIG. E6-1 is a reverse electrophoresis gel image ofthe PCR products of factor IX transgenic plant genomic DNA samples transformed by pGA2029 and control DNA samples.
  • FIG. E6-2 is a reverse electrophoresis gel image ofthe PCR products of factor IX transgenic plant genomic DNA samples transformed by pGA2030 and control DNA samples.
  • FIG. E6-3 is an image ofthe protein gel Coomassie blue stain analysis of purified transgenic factor IX plant protein samples and control sample.
  • FIG. E8-1 is the construction map of transient expression plasmid vector pGA2052a.
  • the present invention includes (a), plant, seeds and plant tissue capable of expressing human and human-like blood coagulation factors; (b) compositions capable of eliciting an activation response in human blood to induce clotting pathways upon administration; (c) compositions useful for blood factor replacement, wound healing or other therapeutic applications contemplated for human blood coagulation factors; (d) methods for producing virus-free, human-like coagulation factors; (e) unique vectors containing DNA sequences encoding for blood coagulation factors and (f ) methods for producing proteins having human and human-like procoagulant or coagulant activity or an amino acid sequence substantially that of a human coagulation factor in transgenic plants.
  • This invention was based on the discovery that plant cells may be genetically transformed to produce human factor VIII in sufficient quantities to conduct biological testing and prove biological functionality. Similarly, it is also possible to transform plants to produce other human coagulation factors, such as but not limited to factors IX, XIII and thrombin. After having validated appropriate biological functionality, it is possible to genetically manipulate transgenic plants to produce commercially practicable quantities of coagulation factors. This invention is directed to these associated embodiments in all respects.
  • Factor VIII is the zymogen of a large glycoprotein that functions in the blood coagulation cascade as a cofactor, functioning with calcium ions and phospholipid, in the factor IX a -mediated activation of factor X in the intrinsic coagulation pathway.
  • VIII can be activated proteolytically by several coagulation enzymes, including thrombin.
  • Factor VIII is in chronically short supply, therefore making its use as a hemophilia A therapy highly cost prohibitive.
  • resulting pharmaceutical products derived from plasma are highly impure, with a specific activity of 0.5 to 2 factor VIII units per milligram protein (one unit of factor VIII activity is by definition the activity present in one miililiter of normal plasma).
  • Resulting plasma derived factor VIII purity is typically lower than 1% by weight (Wood et al. supra 1984).
  • the high level of impurities result in a variety of serious complications including transmission of hepatitis A, B, and C, human parvovirus and human immunodeficiency virus (HIV) pathogens.
  • HIV human immunodeficiency virus
  • Factor VIII at a relative mass of 300kD, is a heterodimeric molecule consisting of heavy chain and light chain peptide segments ionically held by a divalent cation, presumed to be Ca 2+ .
  • the full-length protein coding sequence is recovered from a genomic library enriched for the human X chromosome by Wood et al. supra (1984). This sequence predicted a polypeptide of 2,351 amino acids, including a 19 amino acid signal peptide.
  • the mature protein, at 2,332 amino acids possesses a molecular weight of 330kD, which includes glycosylation, as determined by SDS polyacrylamide gel electrophoresis. Bare peptide molecular weight is calculated at approximately 265kD.
  • Factor VIII undergoes extensive post-translational processing in vivo including N- and O-linked glycosylation, cleavage ofthe primary translational protein after arginine at residue 1648 to yield light and heavy chains, and metal ion association of light and heavy chains (Kaufman et al. supra 1988).
  • factor VIII has been successfully expressed in a variety of mammalian cell culture systems. Initially, factor VIII was expressed in baby hamster kidney (BHK) cell lines, using the calcium phosphate coprecipitation method (Simonsen et al. supra 1983, Wigler et al.
  • factor VIII levels, determined through epitope screening, increased by 300-fold as compared to control T-cell hybridomas.
  • Significant activation of factor IX a determined using the Coatest assay was seen in the transfected BHK cells.
  • Active recombinant human factor VIII has also been produced in Chinese hamster ovary (CHO) cells (Kaufman et al. supra 1988) and monkey COS7 cells (Toole et al. supra 1984, Truett et al. supra 1985).
  • factor VIII is produced using these mammalian cell lines. Consequently, these cell lines will require in vivo and in vitro virus testing as well as mycoplasma detection during biosafety trials. Beyond initial mammalian cell culture production studies, several modifications of recombinant factor VIII have been made at the molecular level to increase expression levels as well as reduce undesired immune responses in patients. Deletion ofthe B- domain of cDNA encoding factor VIII has been shown to increase expression in genetically modified Chinese Hamster Ovary (CHO) cells without affecting biological activity ofthe foreign peptide (Pittman et al. 1993. Blood 81 :2925).
  • CHO Chinese Hamster Ovary
  • factor VIII secretion of factor VIII in mammalian cell culture systems was increased 10-fold by replacing a carboxyterminal 110 amino acid sequence ofthe Al domain with a homologous sequence from the factor V Al domain (Marquette et al. 1995. J Biol Chem 270:10297). The replaced region is clustered with multiple short peptide sequences that have potential to bind BiP, thus inhibiting secretion. Also, the addition of von Willebrand Factor may be necessary for correct assembly of heavy and light domains of factor VIII and subsequent biological activity ofthe construct (Wise et al. 1991. J Biol Chem 266:21948). Von Willebrand Factor is a large (220 kDa) glycoprotein of complex multimeric structure which has been shown to stabilize factor VIII in vitro in mammalian cell culture applications (Kaufman et al. supra 1988).
  • factor VIII contained the epitopes targeted by most inhibitory allo and autoantibodies (Lubin et al. 1994. J Biol Chem 269:8639). Since human inhibitors usually display limited or no reaction with porcine factor VIII, recombinant human/porcine factor VIII molecules were constructed by replacing the putative human A2 domain sequence (residues 387-604) with the homologous porcine sequence. This hybrid maintained full activity in the presence of A2 domain epitope specific antibodies but was inactivated in the presence of anti-C2 antibodies. Outside the research reported in this document, production of factor VIII in recombinant hosts other than mammalian cells has not yet been successfully completed.
  • factor VIII does not require ⁇ -carboxylation of glutamic acid residues for activity, factor VIII does possess 25 separate glycosylation sites, which may preclude the use of prokaryotic host organisms for its production, especially if glycosylation is necessary for activity.
  • the large (7.3 kb) protein coding region may preclude the use of many prokaryotic and lower eukaryotic hosts.
  • Factor IX or Christmas Factor is the zymogen of a serine protease involved in the intrinsic coagulation pathway and specifically activates Factor X in the presence of Factor VIII a , phospholipid, and calcium ion.
  • Factor IX is used therapeutically for
  • Hemophilia B specific deficiency of human factor IX
  • impurities in plasma-derived factor IX concentrates may result in a variety of serious complications including transmission of hepatitis A, B, and C, human parvovirus and human immunodeficiency virus (HIV) pathogens.
  • HAV human immunodeficiency virus
  • the mature protein at approximately 56kD, contains 416 amino acids, including 12 ⁇ -carboxyglutamic acid (Gla) residues near the amino terminus and one ⁇ - hydroxyaspartic acid (Hya) residue at position 64. Vitamin K-dependent ⁇ - carboxylation of at least a portion of these glutamic acid residues is necessary for factor IX activity.
  • the Gla residues confer metal binding properties to factor IX, enabling two sequential conformational changes that are essential for the expression of membrane binding properties and coagulant activity (Borowski et al. 1986. J Biol Chem 261 : 14969, Liebman et al. 1985. Thromb Haemost 54:226).
  • factor IX procoagulant function Activation of factor IX is catalyzed by factor XI a as well as several other serine proteases and results in an activation peptide (35 amino acids), and a light-chain (145 amino acids)/heavy-chain (236 amino acids) complex, linked by disulfide bonds (Kurachi et al. 1982. Proc Natl Acad Sci USA 79:6461).
  • CHO cell-derived recombinant Factor IX required vitamin K to achieve ⁇ -carboxy lation in an average of 6.5 of 12 glutamic acid residues. At this level of ⁇ -carboxylation, recombinant factor IX possessed about 50% ofthe specific activity of plasma-derived, fully carboxylated factor IX.
  • factor IX Some partially carboxylated forms of factor IX are likely to be partially active if only non-essential ⁇ - carboxyglutamic acid residues are missing (Kaufman et al. supra 1986). ⁇ -carboxylation of factor IX has also been achieved in vitro using a partially purified carboxylating enzyme system from bovine liver. Use of this in vitro system boosted apparent carboxylation levels from 3 to 8 residues per factor IX molecule (Soute et al. 1989. Thromb Haemost 61:238).
  • Factor XIII functions after vascular injury to catalyze the formation of ⁇ - glutamyl- ⁇ -lysyl covalent bonds between fibrin molecules, thereby strengthening the forming fibrin clot. Because of this clot strengthening ability, exogenous factor XIII has been contemplated as a wound healing therapeutic, although it is not currently approved for this type of use. Structurally, plasma-borne factor XIII is a tetramer composed of two A-chains (81kD) and two B-chains (75kD) (Lai et al. supra 1994). Factor XIII derived from platelets and placenta consists of an A-chain homodimer, only.
  • the A-chains possess the catalytic domain of factor XIII and appear to be unglycosylated and non- disulfide bonded.
  • the B-chains appear to prevent D -thrombin mediated proteolytic inactivation of factor XIII a in plasma (Mary et al. supra 1988).
  • Factor XIII activation is initiated by thrombin cleavage ofthe 4 kD activation peptide from the N-terminus of A- chains. B-chains, when present, subsequently undergo calcium-dependent dissociation from the rest ofthe molecule.
  • factor VII a tissue factor
  • prothrombin contains 10 ⁇ -carboxy glutamic acid residues within the first forty amino acids from the N-terminus.
  • Human prothrombin cDNA has been expressed in CHO cells in the presence of vitamin K, yielding a fully ⁇ -carboxylated protein with specific coagulant activity equivalent to that of plasma derived prothrombin (Jorgensen et al. supra 1987).
  • prethrombin-2 an intermediate in prothrombin activation, was cloned into CHO cells (Russo et al. supra 1997) as well as E. coli (DiBella et al. supra 1995).
  • Prethrombin-2 lacks the N-terminal pro-sequence of prothrombin, but has not yet been cleaved to form distinct thrombin light and heavy chains.
  • the resulting recombinant prethrombin-2 was proteolytically activated by ecarin, a protease derived from Echis carinatus snake venom.
  • the E. co//-derived prethrombin-2 also required refolding prior to activation.
  • Plant Transformation Vectors contain DNA coding for specific blood coagulation factors and are capable of transforming plants.
  • Foreign DNA is DNA that is exogenous to or not naturally found in the organism to be transformed.
  • Foreign DNA can be inserted into cloning vectors to transform plants and is derived from or has substantial sequence homology to DNA encoding specific blood coagulation factors.
  • Plant expression vectors in this invention are produced using standard molecular cloning procedures (Ausubel et al. 1992. Current protocols in molecular biology. Wiley, New York) and splicing PCR techniques (Marks et al. 1992. J Biol Chem 267:16007). However, the vector produced will depend on which type of transformation and which species of plant is being transformed.
  • a Ti-plasmid-derived vector is appropriate for Agrobacterium-mediated transformation of both whole plants and plant cell culture.
  • a Ti-plasmid or another appropriate direct gene transfer vector may be used to accomplish transformation. If specific plant tissues are to be transformed, an appropriate vector that is active in these tissues must be used.
  • Specific examples of plant expression vectors are given by Hoekema et al. (supra 1985), Mushegian et al. (1995. Microbiol Rev 59:548) and An (supra 1987).
  • the construction of vectors can be accomplished using an E. coli host, such as
  • MCI 000 MCI 000, among others. These vectors may be used directly for direct gene transfer techniques such as protoplast electroporation or micro-injection. If an Agrobacterium- mediated transformation technique is to be used, the vector must first be transferred to a suitable strain, such as A. tumefaciens LBA4404, among others. The vector may be transferred from E. coli to the Agrobacterium using the "freeze-thaw” technique (An et al. 1988. In: Plant Molecular Biology Manual A3, pp. 1-19, Kluwer Academic, Dordrecht), among others.
  • the vectors ofthe present invention contain DNA sequences encoding blood coagulation factors as well as blood coagulation factor-like molecules with coagulant or procoagulant activity.
  • DNA sequences may consist of full-length cDNA or genomic clones, which encode intact blood coagulation factors, such as the 7.2 kb full-length cDNA clone for coagulation factor VIII encoding for the full length, mature 2,332 amino acid protein, among others.
  • DNA sequences may consist of specific fragments of encoding sequences for blood coagulation factors, such as the fragment of 2.0 kb prothrombin cDNA that encodes prethrombin-2, the direct precursor to thrombin (Russo et al.
  • DNA sequences may consist of hybrid constructs of encoding regions of two or more different blood coagulation factors such as the factor V/factor VIII hybrid construct in which 330 bp of the Al domain of factor VIII was replaced with a homologous region of factor V (Marquette et al. supra 1995).
  • Specific genetic regulatory elements i.e., transcriptional promoters, transcriptional terminators, signal peptide encode regions, untranslated leader sequences
  • the transcriptional promoter which is necessary to initiate transcription, is placed at the upstream or 5' end ofthe ligation site ofthe human blood coagulation factor encoding sequence.
  • transcriptional promoters include the CaMV 35 S promoter which generally affords constitutive expression throughout plant tissues (Odell et al. 1985. Nature 313:810) and the ribulose bis-phosphate carboxylase small subunit gene promoter which limits expression ofthe foreign gene to tissues possessing chloroplasts (i.e., green tissues, such as the leaf and stem) (Pichersky et al. 1986. Proc Natl Acad Sci USA 83:3880), among others.
  • the transcriptional terminator which is necessary to terminate transcription, is inserted at the downstream or 3' end ofthe said ligation site.
  • Specific examples include the A. tumefaciens Ti plasmid nopaline synthase (T nos ) and the mannopine synthase (T mas ) transcription terminators, among others.
  • an additional regulatory element encoding a signal peptide may be added between the promoter and the 5' end ofthe ligation site or between the 3' end ofthe ligation site and the terminator in order to relegate the product human blood coagulation factor to a specific cellular organelle.
  • signal peptides include the ribulose bis-phosphate carboxylase small subunit signal peptide which affords chloroplast localization (Fritz et al. 1993. Gene 137: 271), the tobacco PR-1 protein signal peptide which affords apoplast localization (Cornellissen et al. 1986. Nature 321 :531) and the C-terminal KDEL coding region which affords endoplasmic reticulum localization (Pelham 1990. Trends Biochem Sci 15:483), among others.
  • untranslated leader sequences may be added either between the promoter and the additional regulatory element encoding the signal peptide or at the 3' end ofthe encoding sequence to obtain greater mRNA stability between transcription and translation events.
  • UTL untranslated leader sequences
  • Specific examples include the 36bp UTL ofthe alfalfa mosaic virus subgenomic RNA 4 (Jobling et al. 1987. Nature, 325:622) and the ribulose bis-phosphate carboxylase small subunit UTL (Fritz et al. supra 1993).
  • the cells of plants are transformed with the vectors described previously by any technique known in the art, including those described in the references discussed previously and those to follow. These techniques include but are not limited to the Agrobacterium method in which Ti-plasmid bearing A. tumefaciens is cocultivated with plant tissue as described by Hoekema et al. supra 1985. Other suitable transformation techniques include electroporation (Langridge et al. supra 1985), chemical mediated uptake by protoplasts (Krens et al. supra 1982), microinjection (Crossway et al. 1986. Mol Gen Genet 303:179), microprojectile bombardment (Klein et al. supra 1991), and pollen transformation (Saunders et al. supra 1997).
  • the transformed plant tissue is selected by conventional techniques, such as antibiotic resistance screening.
  • Positively transformed tissues may be regenerated using techniques known in the art including shoot root regeneration or suspension cell cultivation, among others. Regenerated plants and/or suspension cell cultures are screened to confirm gene insertion, transcription, foreign protein accumulation and foreign protein activity. Progeny ofthe regenerated plants are similarly screened to develop improved plant and seed lines.
  • the foreign gene can be moved into other genetic lines using a variety of techniques, including classical breeding, protoplast fusion, nuclear transfer, and chromosome transfer.
  • All blood coagulation factors derived from plant material should demonstrate immunological cross-reactivity of antibodies raised against plasma-derived blood coagulation factor with clone-derived blood coagulation factor as validated by enzyme-linked immunosorbent assay.
  • all blood coagulation factors derived from plant material should possess comparable relative protein size with respective plasma-derived coagulation factors.
  • Functional plant derived blood coagulation proteins should specifically perform proper activation function in the coagulation cascade within in vitro assays.
  • Factor VIII Activation of factor X in the presence of factor IX a , calcium ion, and phospholipid following factor VIII activation in the presence of thrombin.
  • Factor IX Activation of factor X in the presence of factor VIII a , calcium ion, and phospholipid following factor IX activation in the presence of calcium ion and factor XI a .
  • Factor XIII Transglutaminase activity (i.e., ⁇ -glutamyl- ⁇ -lysyl crosslinking of fibrin) following factor XIII activation in the presence of thrombin.
  • Thrombin Release of fibrinopeptide A during the conversion of fibrinogen to soluble fibrin in the presence of calcium ions.
  • factor VIII and IX activity of factors VIII and IX may be tested through the correction of factor Vlll-deficient and factor IX-deficient plasma samples, respectively.
  • Factor VIII can also be tested for binding and subsequent elution from immobilized von Willebrand' s factor.
  • Vectors useful for transforming plants with DNA sequences encoding for human coagulation factors are pZD201, pGA2023, pGA2049, pGA 2029 and pGA2030. Sufficient information has been provided in Example 1 in order to enable the construction of these vectors from starting materials and methods that are widely known and generally available. The information available herein will enable the construction of similar vectors from other starting materials.
  • Plasmid Vector pZD201 The E. coli plasmid pSP64-FVIIIc (ATCC No. 39812) containing the gene encoding the full-length polypeptide of factor VIII cDNA, derived from human fetal liver, was obtained from ATCC (FIG. El-1).
  • Coagulation factor VIII is the 2332 amino acid protein that contains a heavy chain, B-domain and a light chain.
  • the 7.2kb pre- coagulation Factor VIII cDNA encodes native signal peptide (the first 19 amino acid at the NH 2 -termini) as well as the mature protein.
  • the full length pre-coagulation factor VIII cDNA was excised with Sal I restriction enzyme and sequentially ligated into the compatible restriction enzyme site Xho I located between the CaMV 35 S promoter and T7-T5 transcription terminator of the binary vector pGA748, forming the 18.8 kb plasmid pZD201 (FIG. El-2).
  • Coagulation factor XIII is a tetramer composed of two A subunits linked as a dimer and two loosely associated B subunits (Schwartz et al. 1973. J Biol Chem 248:1395).
  • the A subunit consists of 731 amino acid residues with a molecular weight of 83,150.
  • the B subunit polypeptide is composed of 641 amino acid residues with a molecular weight of 73,183 and contains a carbohydrate component that adds significantly to the molecular weight ofthe circulating protein.
  • the A subunit zymogen is activated by the thrombin catalyzed cleavage of an amino terminal peptide (4 kDa).
  • the activated A subunits catalyze the formation of ⁇ -glutamyl-e-lysine peptide bonds, facilitating the crosslinking of fibrin and thereby strengthening the formed fibrin clot (Lorand et al. 1980. Prog Haemost Thromb 5:245).
  • the 3.9kb pre-coagulation factor XIII A domain cDNA encodes the mature protein preceded by the native signal peptide.
  • the full length pre-coagulation factor XIII A-domain cDNA is excised from pUCl 8-FXIII with a Pst I restriction enzyme and sequentially ligated into the compatible restriction enzyme site Pst I located on the multiple cloning site on pBluescript SK- (Strategene, La Jolla, CA) as shown in FIG. El-3.
  • the factor XIII A- domain clone is excised from pGA2020 at Xba I/Cla I restriction sites and ligated into compatible Xba I and Cla I restriction sites, respectively, located between the CaMV 35S promoter and T7-T5 transcription terminator ofthe binary vector pGA643, forming the 14.0 kb plasmid pGA2023 (FIG. El-4).
  • the vector pHII-3 containing the cDNA encoding the full-length polypeptide of prothrombin may be obtained from Earl W. Davie of University of Washington, Seattle, WA, (Friezner Degen et al. supra 1983).
  • Prothrombin (M r 72,000) is a vitamin K dependent protein that participates in the final phase of blood coagulation. During the blood coagulation process, prothrombin may be activated by cleavage at the first factor X a site to form prethrombin-2. Prethrombin-2 is subsequently cleaved at the second factor X a site to form thrombin.
  • Correctly processed thrombin (M r 34,000) consists of a 259 amino acid heavy chain and a 49 amino acid light chain connected by a single disulfide bond.
  • the cloning ofthe thrombin expression vector consisted of several steps.
  • the native thrombin signal peptide sequence (36 amino acids) was cloned using the full-length human prothrombin gene as the DNA template and the following primers.
  • the forward primer was designed to adapt a restriction enzyme site Xba I and an initiation codon (ATG) at the 5' end ofthe signal peptide sequence, and the 3' end sequence ofthe signal peptide was used for the reverse primer.
  • the PCR signal peptide sequence contained 111 bp counting from the adapted Xba I site.
  • Reverse primer (P2) 5'- TCG CCG GAC CCG CTG GAG CAG-3'
  • PCR reaction was conducted using the purified PCR product to add a 12-bp sequence ofthe prethrombin-2 gene to the signal sequence using the purified PCR product from the first step as the DNA template, the PI primer and the following reverse primer.
  • the reverse primer was designed using the P2 primer sequence flanked with a 12-bp prethrombin-2 sequence starting at the first factor Xa codon site.
  • the PCR product was designated as prothrombin SP.
  • the PCR product sequence had a length of 123 bp counting from the adapted Xba I site.
  • the prethrombin-2 gene without signal peptide sequence (PT2 w/o SP) was cloned at the first factor Xa codon using the prothrombin gene as the DNA template and the following primers.
  • the forward primer was designed directly downstream ofthe first factor Xa codon site ofthe 5' end ofthe prethrombin-2 gene and the reverse primer was designed to adapt a 6-histidine tag sequence (His) 6 followed by a stop codon (TGA) and a restriction enzyme site Cla I at the 3' end.
  • the 6-histidine peptide at the C-terminus can be used for purification of expressed prethrombin-2 protein.
  • the prothrombin SP sequence and the prethrombin-2 gene sequence were attached via PCR using the purified PCR products, 123 SP and PT2 w/o SP as the DNA templates, and the primers PI and P4.
  • the PCR product had 1062 bp and was designated as PT2, containing restriction enzymes sites, Xba I at the 5' end and Cla I at the 3' end.
  • Resulting purified PCR PT2 DNA was cut with Xba I and Cla I and consequently cloned into Xba I and Cla I restriction enzyme sites ofthe pBluescript SK- vector (Strategene, La Jolla, CA) to form vector pGA2042 (4.02 kb) as shown in FIG. El-5.
  • the constructed prethrombin-2 gene with the native signal peptide sequence was excised using Cla I and Xba I and sequentially ligated into the compatible restriction enzyme sites located between the Rubisco (RbcS-3C) promoter and T5-T7 transcription terminator of a binary expression vector pZD261 (13.4 kb), forming the 14.5 kb vector pGA2043 as shown in FIG.
  • BL is the T-DNA left border
  • BR is the T-DNA right border
  • npt is the neomycin phosphotransferase gene
  • oriT is the RK2 origin of conjugal transfer
  • oriV is the RK2 origin of replication
  • pRBC/AMV is the Rubisco (RbcS-3C) promoter with a alfalfa mosaic virus leader sequence
  • T5 is the transcription terminator 5
  • T7 is the transcription terminator 7
  • tet is the tetracycline resistance gene of
  • RK2 plasmid RK2 plasmid
  • trfA is the segment for a replication protein.
  • the prethrombin-2 gene with native signal sequence was also cloned into another binary expression vector pGA643 (11.7 kb), forming the 12.8 kb vector pGA2049 as shown in FIG. El-7.
  • the expression ofthe prethrombin-2 gene in the binary vector pGA2049 was under the control ofthe 35S promoter (P35S) and T5-T7 terminator.
  • Coagulation factor IX is a single chain, 416 amino acid glycoprotein with a molecular weight of approximately 56,000 and contains 12 ⁇ -carboxyglutamic acid (gla) residues in the amino-terminal region.
  • factor IX is converted to factor IX a by factor XI a .
  • Factor IX a then converts factor X to factor X a in the presence of factor VIII a , phospholipid, and Ca 2+ . At least a portion ofthe 12 N-terminal gla residues are necessary for factor IX activity.
  • the 1.5kb coagulation factor IX cDNA encodes the mature protein, an 18 amino acid pro- sequence and a 28 amino acid signal peptide.
  • the full length pre-pro-coagulation factor IX cDNA was cloned by PCR using the primers below.
  • the forward primer was designed to adapt the Xba I restriction enzyme site at the 5' end ofthe factor IX cDNA coding for the mature factor IX protein and the reverse primer was designed to adapt a 6-histidine tag sequence (His) 6 followed by a stop codon (TAG) and a restriction enzyme site Cla I at the 3 ' end.
  • the 6-histidine peptide at the C-terminus can be used for the purification of expressed pre-pro-factor IX after expression.
  • IX DNA (1281 bp) was ligated into compatible restriction enzyme sites located between the Rubisco (RbcS-3C) promoter (Sugita et al. 1987 Mol. Gen. Genet. 209:247) and T5- T7 transcription terminator of binary expression vectors, pZD256 and pZD261 (13.4 kb), forming the 14.8 kb vectors pGA2029 and pGA2030 as shown in FIG.'s El-8, El-9, respectively, where BL is the T-DNA left border; BR is the T-DNA right border; npt is the neomycin phosphotransferase gene; oriT is the RK2 origin of conjugal transfer; oriV is the RK2 origin of replication; pRBC/AMV is the RbcS-3C promoter with an alfalfa mosaic virus leader sequence; pRBC VSP is the RbcC-3C promoter with a vacuole signal sequence T5 is the transcription terminator 5;
  • pre-pro-factor IX is under the control ofthe RbcS-3C promoter with a vacuole signal peptide sequence in pGA2029 and with an alfalfa mosaic virus (AMV) leader sequence in pGA2030.
  • AMV alfalfa mosaic virus
  • N. tabacum plants were transformed by A. tumefaciens LBA4404 containing the expression vectors for factors VIII, IX, XIII-A domain and thrombin production prepared according to Example 1. These plasmids were directly transferred into A. tumefaciens LBA4404 using the freeze-thaw method (An supra 1988). Subsequent plant transformation was accomplished by co-cultivation of tobacco leaf disks or calli with the Agrobacterium. After 2 to 3 days, explant tissues were removed to fresh medium containing the antibiotics carbenicillin to kill the Agrobacterium and kanamycin to select positive transformants. Whole plants were regenerated on shoot and root regeneration media containing kanamycin.
  • Example 3 Factor VIII Production A. Protein Immunoblotting Vector pZD201 described in Example IA is used. The tobacco plants are transformed using the Agrobacterium method described in Example 2. After obtaining positive transformants via kanamycin resistance screening, mature tobacco plants and calli are assayed for the presence ofthe human coagulation factor VIII. Preliminary protein immunoblotting (dot blot assays-FIG. E3-1) completed using extractable leaf protein showed the presence of coagulation factor VIII antigen in the leaf tissues of TO whole plant transformants. As seen in FIG. E3-1, strong factor VIILag expression characteristics were seen in plants 1004-3, 1006-2 and 1006-3.
  • Positive control factor VIII standards (American Diagnostica, Greenwich, CT) are shown as S 1 and S2 whereas negative control leaf protein derived from untransformed N tabacum cv. SRI is shown as SR.
  • S 1 and S2 negative control leaf protein derived from untransformed N tabacum cv. SRI is shown as SR.
  • TO plants were self-pollenated, resulting in TI seedstock. TI seeds were subsequently germinated on kanamycin selective media and mature plants were grown in a controlled environment.
  • Factor VIII Activity Assay The transgenic plant leaf material was harvested and total soluble protein was extracted using standard techniques. Functionality of recombinant human factor VIII was analyzed using the Coatest method (Helena Laboratories, Beaumont, Texas). After activation by thrombin, factor VIII acts as a cofactor in the conversion of factor X to factor X a by factor IX a when calcium and phospholipid are present. In the Coatest assay, the quantity of factor X a generated was determined using a specific chromogenic substrate (MeO-CO-D-CHG-Gly-Arg-pNa) and was directly proportional to the amount of factor VIII in the sample tested. During the functional analysis, total protein samples from plants expressing factor VIII protein as well as untransformed control plants and CBHI cellulase expressing tobacco plants were tested. Results ofthe Coatest assay are shown in Table E3-1. In each individual sample,
  • Tests A, B and C were completed on separate days and each required a separate untransformed plant control. In tests A and B, the duration of incubation after factor X a addition was 5 minutes. In test C, the duration of incubation after factor X a addition was 4 minutes. Also in test C, aliquots of factor VIII reference plasma standard (Helena Laboratories, Beaumont, TX) were added to two separate tobacco plant controls. Results from tests A and B, as compared to increases in absorbance mediated by the addition of factor VIII in test C, clearly show the presence of factor VIII procoagulant activity in tobacco plant lines 1005-5, 1005-6, and 1006-3.
  • Vector pGA2023 described in Example 1 is used.
  • the tobacco plants were transformed using the Agrobacterium method described in Example 2. After obtaining positive transformants via kanamycin resistance screening, fifty tobacco plant transformants were obtained through selective media screening and used to confirm the insertion of the Factor XIII gene in the transgenic plant genome. Genomic DNA of these plants was prepared using a hexadecyltrimethyl ammonium bromide (CTAB) mini- preparation method.
  • CTAB hexadecyltrimethyl ammonium bromide
  • a small amount of plant material (25-100 mg) was ground into a fine powder with liquid nitrogen and extracted at 65°C for genomic DNA in an extraction buffer containing 3% CTAB, 1.42 M NaCl, 20 mM EDTA, 100 mM Tris-HCl, 5 mM ascorbic acid, and 2% (w/v) poly vinypyrolidone (PVP- 40; Sigma Chemical Co.), subsequently, a phenol-chloroform extraction was conducted. The upper aqueous phase was saved after centrifugation at 10,000 x g for five minutes and genomic DNA was precipitated in isopropanol at the presence of sodium acetate.
  • the resulting DNA samples were used for factor XIII gene amplification by PCR using the following primers derived from the factor XIII gene.
  • the forward primer is specific to the sequence immediately downstream the initiation codon (ATG) and the reverse primer is specific to sequence immediately upstream ofthe stop codon (TGA).
  • FIG. E4-1 5'-GGA AGG TCG TCT TTG AAT CTG CAC GTC CAG-3' PCR results are shown in FIG. E4-1, where lane S is the size marker, lane SRI is the non-transformed plant as the negative control, lane FXIII is the factor XIII A-subunit DNA as the positive control, and lanes 11, 23, 33, 35, 45, 47 are transgenic plant samples. Results show that a DNA band of 2.3 kb is present for all shown transgenic plants, matching up with the PCR band of factor XIII DNA. No PCR 2.3 kb band is observed in the non-transformed plant DNA sample. The results here confirm insertion ofthe factor XIII gene into the tobacco plant genome. A total of fifty transformed plants were screened for factor XIII gene insertion and forty plants showed factor XIII A subunit gene insertion.
  • HSA high salt buffer
  • Nonidet P-40 and 1.0 mM phenylmethyl sulfonyfluoride (PMSF) was used for total protein extraction.
  • Leaf materials ofthe transgenic plants grown in square culturing vessels were ground and extracted in the HSB in an eppendorf tube using an eppendorf tube grinder. Plant extract supematants were collected after centrifugation at 20,000 x g for 10 min. Total protein was determined using a Bradford Reagent (Bio-Rad
  • FIG. E4-2 The Western blot results of transgenic factor XIII plants are shown in FIG. E4-2 and FIG. E4-3.
  • FIG. E4-2 two transgenic plant protein samples (lanes 1 and 2) were used in the Western blot analysis while three control protein samples were used as the negative controls, Cl: non-transformed plant protein sample; C2 and C3: transgenic plant protein samples transformed with a binary expression vector containing no factor XIII gene. Results indicate that the 83 kDa factor XIII bands are expressed only in the transgenic factor XIII plants but not in the non-transformed control plant and transformed control plants.
  • FIG. E4-2 two transgenic plant protein samples (lanes 1 and 2) were used in the Western blot analysis while three control protein samples were used as the negative controls, Cl: non-transformed plant protein sample; C2 and C3: transgenic plant protein samples transformed with a binary expression vector containing no factor XIII gene. Results indicate that the 83 kDa factor XIII bands are expressed only in the transgenic factor
  • E4-3 shows the results of another Western blot analysis for various factor XIII transgenic plants, where lane FXIII is human plasma coagulation factor XIII as the positive control, lane SRI is the non-transformed plant as the negative control, and lanes 11, 23, 33, 35, 45, 47 are the transgenic plant samples.
  • the Western blots reveal at least two major proteins. One ofthe protein bands is the major 83 kDa protein and has the identical size as the human plasma factor XIII. The other protein band has a size of 210 kDa which may be a complex of unprocessed, dimerized factor XIII A subunit protein.
  • a total of forty transgenic plants were screened using Western blot analysis and thirty seven out of forty plants expressed the human factor XIII A-subunit.
  • the expression yield based on the Western blot analysis ranged approximately from 0.1 to 1.8% ofthe total extracted soluble leaf protein.
  • Plant protein samples were extracted from plant leaf materials in a buffer containing 50 mM pH 7.6 mM Tris-HCl . The extraction mixture was centrifuged at 4°C at 20,000 x g for 15 minutes and the supernatant was collected and used for both protein and factor XIII activity assay. The activity of plant derived human factor XIII was measured using a method described by Hornyak et al. (1989 Biochem 28:7326).
  • Factor XIII A subunit activity is defined as the amount of monodansylcadaverine inco ⁇ orated into casein by the transamidase activity of activated factor XIII A-subunit (Coggan et al 1984 Anal Biochem 137:402). Monodansylcadaverine incorporation into casein is measured as the fluorescence increase in the reaction mixture. Before the fluorescence incorporation reaction, factor XIII A-subunit is activated by the thrombin catalyzed cleavage of an amino terminal peptide (4 kDa).
  • 100 ⁇ l extracted plant protein sample was added to a 2.0 ml reaction tube, along with 100 ⁇ l of 50 mM pH 7.6 Tris-HCl buffer, 20 ⁇ l of 4 mM monodansylcadaverine prepared in 50 mM Tris-HCl buffer, 50 ⁇ l 0.4 M CaCl 2 prepared in 50 mM Tris-HCl buffer, 1200 ⁇ l 50 mM, pH 9.0 Bicine buffer.
  • the reaction mixture was warmed in a 37°C water bath for one minute. Reactions with non-transformed plant protein sample, factor XIII standard (3.65 ⁇ g) and blank (200 ⁇ l Tris-HCl buffer) were also set up as controls.
  • ⁇ -thrombin solution 50 ⁇ l of ⁇ -thrombin solution was added to the above reaction mixture and incubated in a 37°C water bath for 10 minutes to activate factor XIII.
  • the ⁇ -thrombin reagent 500 unit/ml, Haematologic Technologies Inc., Essex Junction, VT) was prepared in 25 mM pH 7.5 Tris-HCl buffer and 25% glycerol.
  • 50 ⁇ l 0.2 M DTT solution was added to the reaction mixture, which was incubated at 37°C for one minute.
  • 200 ⁇ l 2% N,N'- dimethylcasein was added to the reaction mixture and the time zero fluorescence was measured in a fluorometer.
  • Table E4-1 shows the activity of plant derived factor XIII A-subunit using human plasma factor XIII as the positive control and the non-transformed SRI plant protein sample as the negative. Pure human plasma factor XIII has an activity of 50.2 unit per milligram of total protein. Results show that the activity of plant recombinant factor XIII A subunit ranges from 0.012 to 0.237 unit per milligram of soluble protein, which is equivalent to 0.1 to 2.5% factor XIII A-subunit protein of total plant soluble protein. TABLE E4-1
  • Transgenic tobacco plants expressing human factor XIII A-subunit protein were cultivated to maturity in the greenhouse.
  • the expression of factor XIII A subunit was tested at different leaf positions using Western blot analysis. Protein samples were extracted using HSB buffer from leaves at positions 1, 3, 5, and 7 counted from top to bottom excluding necrotic yellow leaves. Protein samples were analyzed using 7.5 % SDS-PAGE and subsequently blotted onto a nitrocellulose membrane.
  • lane FXIII is the human factor XIII protein sample (3.65 ⁇ g) as the positive control
  • lane SRI is the non-transformed plant protein sample (100 ⁇ g)
  • lane SJ is a transgenic plant protein sample (100 ⁇ g ) from the top leaf grown in a square culturing vessel
  • lanes Gl, G3, G5, and G7 are the protein samples (100 ⁇ g ) from leaf positions 1, 3, 5, and 7, respectively from the transgenic plant. It can be seen that the expression of factor XIII A-subunit has similar expression levels at different leaf positions. In addition, the expression level ofthe factor XIII protein is the same between the transgenic plant from a square culturing vessel and the plant grown in a greenhouse.
  • the immuno- active 210 kDa protein band vanishes gradually as the leaf position changes from 1 to 3, 5, and 7. This is probably due to the completion of post-translational modification ofthe presumed factor XIII A-subunit complex, releasing single chains of factor XIII A- subunit.
  • Vectors pGA2043 and pGA2049 described in Example 1 were used.
  • the tobacco plants were transformed using the Agrobacterium method described in Example 2. After obtaining positive transformants via kanamycin resistance screening, mature tobacco seventeen tobacco plant transformants were obtained through the selective media screening process and used to confirm the insertion ofthe prethrombin-2 gene in the transgenic plant genome. Genomic DNA of these plants was prepared using a hexadecyltrimethyl ammonium bromide (CTAB) mini-preparation method.
  • CTAB hexadecyltrimethyl ammonium bromide
  • lane S is the size marker
  • lane PT is the prethrombin-2 DNA with the signal sequence as the positive control
  • lane SRI is the non- transformed plant as the negative control
  • lanes 1, 2, 3 are the transgenic plant samples transformed with the binary vector pGA2043
  • lane 4 is the transgenic plant sample transformed with the binary vector pGA2049.
  • Results show that a distinct DNA band of 1.06 kb is present for all the transgenic plants, matching up with the PCR band ofthe prethrombin-2 gene with the signal sequence. No 1.06 kb band is observed in the non- transformed plant DNA sample.
  • the results here confirm insertion ofthe prethrombin-2 gene into the tobacco plant genome.
  • Total plant leaf protein was extracted from transformed and non-transformed plants using a high salt buffer (HSB), which contains 50 mM pH 7.5 Tris-HCl, 0.5 M NaCl, 0.05% Nonidet P-40 and 1.0 mM phenylmethyl sulfonyfluoride (PMSF).
  • HLB high salt buffer
  • PMSF phenylmethyl sulfonyfluoride
  • Leaf materials ofthe transgenic plants grown in square culturing vessels were ground and extracted in the HSB in an eppendorf tube using an eppendorf tube grinder. Plant extract supernatant samples were collected after centrifugation at 20,000 x g for 10 min. Total protein was determined using a Bradford Reagent (Bio-Rad Laboratories, Hercules, CA) and bovine serum albumin as the protein standard.
  • Protein samples containing 100 ⁇ g total protein were denatured at 95°C for 5 minutes with sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and subsequently analyzed by a 4-20 % gradient SDS-PAGE (Bio-Rad Laboratories, Richmond, CA). An amount of 6.5 ⁇ g human plasma ⁇ -thrombin was used in the SDS-PAGE as the positive control (Haematologic Technologies Inc., Essex Junction, VT). The separated protein was electrophoretically blotted onto a Protran nitrocellulose membrane (Schleicher & Schuell, Keene, NH).
  • SDS-PAGE sodium dodecyl sulfate- polyacrylamide gel electrophoresis
  • lane TH is the human plasma ⁇ -thrombin served as the positive control
  • lanes 1, 2, 3, and 4 are the prethrombin-2 transgenic plant samples
  • lane C is the non-transformed plant served as the negative control.
  • the transgenic samples 3 and 4 show a 39 kDa prethrombin-2 band, which is about 5 kDa larger than human alpha-thrombin (34 kDa).
  • the 5-kDa difference is probably due to incomplete cleavage ofthe signal peptide (4.5 kDa) and the 6-histidine tag (0.66 kDa).
  • Total protein samples were extracted from the leaf materials of transgenic prethrombin-2 plants using HSB buffer supplemented with 1 mM PMSF.
  • One gram of leaf tissue was first ground into power in liquid nitrogen and extracted in 5 ml of HSB buffer using a mortar and a pestle. The sample was subsequently centrifuged at 20,000 x g for 10 min and the supernatant was saved for the following protein purification.
  • Metal chelating Sepharose Fast Flow (Pharmacia Biotech, Piscataway, NJ) was used for the prethrombin-2 protein purification.
  • a 3 -ml disposable column was filled with one miililiter of metal chelating sepharose .
  • the sepharose was prepared as follows: wash with four volumes of deionized water, activate with two volume of 0.1 M NiSO 4 solution, wash again with six volumes of deionized water, and equilibrate with four volumes ofthe HSB buffer. Five milliliters ofthe protein sample supernatant was loaded into the column, which was consequently washed with six volumes of 10 mM pH 8.0 imidazole solution. The protein bound to the sepharose was eluted with 500 mM pH 8.0 imidazole solution and the eluted sample was collected in 0.5-ml aliquots.
  • the second and the third 0.50 ml samples were combined since they contained most ofthe eluted protein based on the protein concentration analysis using the Bio-Rad Protein Assay reagent (Bio-Rad Laboratories). Protein samples containing 10 ⁇ g eluted protein were denatured at 95°C in a SDS-PAGE sample buffer and subsequently analyzed by 4- 20 % gradient SDS-PAGE. After separation, the gel was stained using a Silver Stain Plus kit (Bio-Rad Laboratories). The results are shown in FIG.
  • lane SM is the protein size marker
  • lane TH is the human alpha-thrombin protein sample
  • lane Cl is the non-transformed plant protein sample
  • lane C2 is the transgenic plant control protein sample
  • lanes 1, 2, and 3 are the prethrombin-2 transgenic plant protein samples.
  • samples 2 and 3 indicate a 39 kDa protein band, the plant derived prethrombin-2 protein, which is identical to the one shown in FIG. E5-2. This band is not shown in either the non-transformed or transformed control samples.
  • the full-length human prothrombin gene (2006 bp) in vector pHII-3 was obtained from Dr. Earl W. Davie, Biochemistry Department, University of Washington, Seattle, Washington (Friezner Degen et al., supra 1983).
  • Several steps were involved in constructing the prothrombin transient expression vector.
  • an expression cassette with only the cauliflower mosaic virus promoter CaMV 35S promoter (P 35S), the multiple cloning sites (MSC), and the transcription terminator (T7-T5) in a binary vector pGA643 (An, 1995) was cloned using PCR by Taq polymerase (Stratagene, La Jolla, C A) with the following primers.
  • the cloned expression cassette was ligated into a plasmid vector pGEM-T (Promega, Madison, WI) to form a transient plant expression vector pGA2054a, as shown in FIG. E5-4.
  • the vector pGA2054a also contains an ampicillin resistance gene (Amp) and a fl phage origin (fl ori). Foreign genes can be cloned into this transient vector pGA2054a for expression tests.
  • human prothrombin and prethrombin genes were cloned using PCR by Taq polymerase (Stratagene) to adapt a Xba I cloning site at the 5' end ofthe gene and a Cla I cloning site at the 3' end.
  • the following primers PI and P3 were used for the prothrombin PCR cloning reaction and the primers P2 and P3 were used for the prethrombin-2 cloning reaction.
  • the cloned prethrombin-2 was also adapted with the initiation codon ATG at the 5' end ofthe gene.
  • the cloned prothrombin gene was subsequently ligated into the vector pGEM-T (Promega, Madison, WI) to form the vector pGA2056 as shown in FIG. E5-5. Also, the prethrombin2 gene was ligated into pGEM-T to form the vector pGA2057 also as shown in FIG. E5-6.
  • the prothrombin gene in pGA2056 and prethrombin-2 gene pGA2057 were, respectively, excised out with Xba I and Cla I restriction enzymes.
  • a 3 -day old NT1 tobacco cell suspension was used for the preparation of protoplasts. Briefly, protoplasts were isolated by treating the suspension cells with a pH 5.8 solution containing 10 mg/1 cellulase, 500 ⁇ g/ml pectoplyase (Kanematsu-Gosho, Los Angeles, CA) and 0.4 M D-mannitol at 28°C for 20 minutes at 100 rpm. The protoplasts were then extensively washed with 0.4 M mannitol to remove cellulase and pectolyase.
  • lxlO 6 protoplasts were resuspended in 0.5 ml of pH 5.5 electroporation buffer containing 2.38 mg/ml HEPES, 8.76 mg/ml NaCl, 735 ⁇ g/ml CaC12 and 0.4 M D-mannitol.
  • the protoplasts were then electroporated using a 300 volt pulse with 210 ⁇ F capacitor. The treated protoplasts were subsequently transferred into 7 ml of protoplast culture medium in a Petri dish and cultured for 48 hours at 28°C.
  • the culture medium is a modified Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 4.3 mg/ml MS salt supplemented with 3% sucrose, 0.18 mg/ml KH2PO4, 0.1 mg/ml inositol, 1 ⁇ g/ml thiamine hydrochloride, and 0.2 ⁇ g/ml 2.4-dichlorophenoxyacetic acid (2.4-D), and 0.4 M D-mannitol.
  • MS Murashige and Skoog
  • the cultured protoplasts were collected by gentle centrifugation and suspended in 100 ⁇ l extraction buffer containing 50 mM Tris-HCl pH 8.3, 227 mM NaCl, 1 mg/ml bovine serum albumin, and 1 mg/ml sodium azide. Protein samples were extracted by sonicating the protoplasts three times for 8 seconds at 30-second intervals. The protein samples were harvested by centrifuging the sonicated mixture at 15,000 g for 5 minutes. The supernatant was saved and protein concentration was measured using the Bio-Rad Protein Assay method (Bio-Rad, Hercules, CA).
  • ⁇ -thrombin activity released by either transiently expressed prothrombin or prethrombin-2 Fifty micrograms of extracted protein was used to measure the ⁇ -thrombin activity released by either transiently expressed prothrombin or prethrombin-2.
  • Human prothrombin Human prothrombin (Haematologic Technologies Inc., Vermont) was used as a positive and the protein samples from electroporated non-transformed NT1 protoplasts were used as negative controls.
  • Ecarin Sigma was used to cleave the prothrombin and prethrombin-2 to release the active ⁇ -thrombin protein.
  • One unit of ecarin was added to each sample and the sample was incubated at 37°C for 15 minutes.
  • Vectors pGA2029 and pGA2030 described in Example 1 are used.
  • the tobacco plants are transformed using the Agrobacterium method described in Example 2.
  • mature tobacco forty and thirty seven tobacco plant transformants, respectively for the transformation of pGA2029 and pGA2030 were obtained through the selective media screening process and used to confirm the insertion ofthe factor IX gene in the transgenic plant genome.
  • Genomic DNA of these plants was prepared using a hexadecyltrimethyl ammonium bromide (CTAB) mini-preparation method.
  • CTAB hexadecyltrimethyl ammonium bromide
  • FIG. E6-1 shows lane S is the size marker
  • lane FIX is the factor IX DNA serving as a positive control
  • lane SRI is the non-transformed plant . serving as the negative control
  • lanes 1, 2, 3, and 4 are transgenic plant samples transformed with the binary vector pGA2029.
  • FIG. E6-2 shows analogous results for the transgenic plants transformed with the binary vector pGA2030. Results in both FIG. E6- 1 and FIG. E6-2 show that a distinct DNA band of 1.28 kb, corresponding to the size of the pre-pro-F actor IX gene (positive control), is present for all the transgenic plants. Factor IX gene insertion was confirmed in a total of thirty eight and thirty four transgenic plants for binary vectors pGA2029 and pGA2030, respectively.
  • HSA high salt buffer
  • PMSF phenylmethyl sulfonyfluoride
  • Total protein was determined using a Bradford Reagent (Bio-Rad Laboratories, Hercules, CA) and bovine serum albumin as protein standard.
  • the protein extract was purified for the 6- histidine-tagged factor IX protein using metal chelating sepharose (Pharmacia Biotech, Piscataway, NJ).
  • metal chelating sepharose Pharmacia Biotech, Piscataway, NJ.
  • One miililiter ofthe metal chelating sepharose was loaded into a 3-ml size column and activated by 0.1 M NiSO 4 solution. After washed thoroughly with six volumes of deionized water and equilibrated with four volumes ofthe HSB buffer, the protein sample supernatant (5 ml) was loaded into the column, which was consequently washed with six volumes of 10 mM pH 8.0 imidazole solution.
  • the protein bound to the sepharose was eluted with a 50-mM pH 6.0 imidazole solution and the eluted sample was collected in 0.5-ml aliquots. The second 0.5-ml aliquot contained the most protein and was used for the gel electrophoresis. Protein samples containing 40 ⁇ g eluted protein were denatured at 95°C in a SDS-PAGE sample buffer and subsequently analyzed by 4- 20 % gradient SDS-PAGE. After separation, the gel was stained using a Coommasie Brilliant blue solution. The results are shown in FIG.
  • lane FIX is the factor IX standard (Haematologic Technologies Inc., Essex Junction, VT)
  • lane C is a non- transformed plant protein sample
  • lanes 1 and 2 are the transformed plant protein samples.
  • Results indicate that the recombinant plant derived human factor IX has a size of about 65 kD.
  • the transformed plant sample in lane 1 shows a unique band of about 58 kD, which is not observed in the non-transformed plant sample.
  • the transformed plant sample in lane 2 shows no similar 58 kD protein band in the gel stain.
  • Acarboxyl- glutamyl residues in acarboxyl-pro-coagulation factor IX can be ⁇ -carboxylated by treatment with vitamin K-dependent carboxylase (Soute et al. supra 1989).
  • Vitamin K-dependent carboxylase is prepared from normal cow liver and partly purified as described by Soute et al. (1987. Thromb Haemostas 57:77).
  • Standard ⁇ -carboxylation reaction mixtures as reported by Van Haarlem et al. (1987. FEBS Lett 222:353), consist of 1.0 mg partially purified carboxylase, 2 ⁇ g recombinant acarboxyl-pro-factor IX, 0.15 M
  • Vitamin K hydroquinone is solubilized by mixing it with the phosphatidylcholine before preparing the mixed micelles according to the method of De Metz et al. (1981. J Biol Chem 256: 10843).
  • the presence of gla residues in pro-factor IX can be tested by completing the ⁇ -carboxylation reaction using NaH 14 CO 3 and measuring incorporation of l4 CO 2 using scintillation counting. Alternatively, the presence of gla residues can be confirmed based on colorimetric detection using 4-diazobenzenesulfonic acid staining of polyacrylamide gels as reported by Jie et al. supra (1995). Correctly ⁇ -carboxylated pro-factor IX may be separated from the reaction mixture using standard protein purification methods.
  • Western blot immunoassay can be used to determine the extent of cleavage of the N-terminal, 18 amino acid pro-peptide.
  • Factor IX coagulant activity is determined with a two-stage assay using factor IX-deficient plasma (Proctor et al. 1961. J Clin Pathol 36:212).
  • One unit of factor IX activity represents the amount of factor IX in 1 mL of normal human pooled plasma.
  • Example 8 Transient Expression of Human Coagulation Factor XIII A Subunit in Maize Protoplasts.
  • Factor XIII Transient Expression Vector Construction The expression cassette of factor XIII A subunit in the binary plasmid vector pGA2023 as shown in FIG. El-4 was used for the construction of a transient expression vector. The expression of factor XIII A subunit is under the control ofthe 35S promoter and transcription is terminated by the T7 terminator.
  • the factor XIII expression cassette (3.6 kb) was cloned by PCR with Taq polymerase (Stratagene, La Jolla, CA) using the following primers:
  • the cloned expression cassette was directly ligated into a plasmid vector pGEM- T (Promega, Madison, WI) to form a transient factor XIII expression vector pGA2052a, as shown in
  • FIG. E8-1 The vector pGA2052a also contains an ampicillin resistance gene (Amp) and a fl phage origin (fl ori).
  • Amicillin resistance gene Amp
  • fl ori a fl phage origin
  • Maize (Zea mays) protoplasts will be used for transient factor XIII expression analysis.
  • Maize cell suspension culture is grown in a modified Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 4.3 mg/ml MS salt supplemented with 3% sucrose, 0.18 mg/ml KH2PO4, 0.1 mg/ml inositol, 1 ⁇ g/ml thiamine hydrochloride, and 0.5 ⁇ g/ml 2.4-dichlorophenoxyacetic acid (2.4-D).
  • MS Murashige and Skoog
  • protoplasts are isolated by treating the suspension cells with a pH 5.8 solution containing 10 mg/1 cellulase, 500 ⁇ g/ml pectoplyase (Kanematsu-Gosho, Los Angeles, CA) and 0.4 M D- mannitol at 28°C for 2 to 4 hours with gentle shaking at 100 ⁇ m. The protoplasts are checked every 30 minutes to avoid overdigestion. Upon the completion of enzyme digestion, the protoplasts are extensively washed with 0.4 M mannitol to remove cellulase and pectolyase.
  • l-2 ⁇ l0 6 protoplasts are resuspended in 0.5 ml of pH 5.5 electroporation buffer containing 2.38 mg/ml HEPES, 8.76 mg ml NaCl, 735 ⁇ g/ml CaC12 and 0.4 M D-mannitol.
  • the protoplasts can be electroporated using a 300 volt pulse with a 210 ⁇ F capacitor.
  • the treated protoplasts are subsequently transferred in 7 ml of protoplast culture medium in a Petri dish and cultured for 48 hours at 28°C prior to protein extraction.
  • the culture medium is modified MS medium with the addition of 0.4 M D-mannitol.
  • the cultured protoplasts can be collected by gentle centrifugation and suspended in plant extraction buffer containing 50 mM pH 7.5 Tris-HCl, 0.5 M NaCl, 0.05% Nonidet P-40 and 1.0 mM phenylmethyl sulfonyfiuoride (PMSF).
  • PMSF phenylmethyl sulfonyfiuoride
  • Protein samples can be extracted by sonicating the protoplasts on ice three times for 8 seconds at 30-second intervals.
  • the protein samples can be harvested by centrifuging the sonicated mixture at 15,000 g for 5 minutes. Supernatant will retained used for protein concentration analyses, Western blot analyses and activity assays.
  • the Western blot analysis will follow the same procedure outlined in previous tobacco-based factor XIII expression examples.
  • factor XIII A subunit activity will be assayed using the method described in the previous tobacco-based factor XIII expression example, where factor XIII A subunit activity is defined as the amount of monodansylcadaverine inco ⁇ orated into casein by the transamidase activity of activated factor XIII A-subunit.

Abstract

L'invention concerne une composition pour plantes transgéniques et pour des facteurs de coagulation humains dérivés de ces plantes transgéniques et capables d'éliciter une réaction d'activation dans le processus de coagulation et par conséquent utiles pour le traitement de patients humains chez lesquels une déficience des protéines du facteur de coagulation a été diagnostiquée. Ces protéines peuvent être produites par des procédés permettant une production non virale, comprenant l'utilisation de plantes entières et de cultures de cellules de plantes. L'invention concerne également des vecteurs d'expression assurant une transformation correcte du tissu végétal pour la production de ces facteurs, ainsi que des cellules de plantes transformées et des procédés permettant de produire des facteurs de coagulation humains par des techniques de biologie moléculaire végétale.
PCT/US1999/010732 1998-05-14 1999-05-14 Facteurs de coagulation du sang humain derives de plantes transgeniques WO1999058699A2 (fr)

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WO2002008382A2 (fr) * 2000-07-24 2002-01-31 Battelle Memorial Institute Bioreacteur pour agriculture en milieu controle utilise pour la production de proteines heterologues
WO2003039584A1 (fr) * 2001-11-09 2003-05-15 Novo Nordisk Health Care Ag Composition pharmaceutique comprenant des polypeptides de facteur vii et des polypeptides de facteur v
EP1405910A1 (fr) * 2001-07-06 2004-04-07 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Procede relatif a l'elaboration de thrombine humaine par modification genique
WO2005123928A1 (fr) * 2004-06-08 2005-12-29 Battelle Memorial Institute Production du facteur viii de coagulation humain à partir de cellules végétales et de plantes entières
WO2006004675A2 (fr) 2004-06-25 2006-01-12 Altor Bioscience Corporation Production d'un facteur tissulaire chez des plantes
US7125846B2 (en) 2001-11-09 2006-10-24 Novo Nordisk Healthcare A/G Pharmaceutical composition comprising factor VII polypeptides and factor V polypeptides
EP4357450A1 (fr) * 2022-10-18 2024-04-24 Proview-Mbd Biotech Co., Ltd. Production d'a-thrombine humaine recombinante dans un système à base de plantes et son application

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EP1593389A1 (fr) * 2000-05-10 2005-11-09 Novo Nordisk Health Care AG Composition pharmaceutique comprenant un facteur VII1 et un facteur XIII
CN1304049C (zh) * 2000-05-10 2007-03-14 诺沃挪第克健康护理股份公司 包含因子VIIα和因子XIII的药物组合物
WO2001085198A1 (fr) * 2000-05-10 2001-11-15 Novo Nordisk A/S Composition pharmaceutique comprenant un facteur viia et un facteur xiii
AU2001258228B2 (en) * 2000-05-10 2006-01-12 Novo Nordisk Health Care Ag Pharmaceutical composition comprising a factor VIIA and a factor XIII
WO2002008382A2 (fr) * 2000-07-24 2002-01-31 Battelle Memorial Institute Bioreacteur pour agriculture en milieu controle utilise pour la production de proteines heterologues
WO2002008382A3 (fr) * 2000-07-24 2003-05-30 Battelle Memorial Institute Bioreacteur pour agriculture en milieu controle utilise pour la production de proteines heterologues
EP1405910A1 (fr) * 2001-07-06 2004-04-07 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Procede relatif a l'elaboration de thrombine humaine par modification genique
EP1405910A4 (fr) * 2001-07-06 2005-02-02 Juridical Foundation Procede relatif a l'elaboration de thrombine humaine par modification genique
US7635577B2 (en) 2001-07-06 2009-12-22 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Process for producing human thrombin by gene modification technique
US7125846B2 (en) 2001-11-09 2006-10-24 Novo Nordisk Healthcare A/G Pharmaceutical composition comprising factor VII polypeptides and factor V polypeptides
WO2003039584A1 (fr) * 2001-11-09 2003-05-15 Novo Nordisk Health Care Ag Composition pharmaceutique comprenant des polypeptides de facteur vii et des polypeptides de facteur v
WO2005123928A1 (fr) * 2004-06-08 2005-12-29 Battelle Memorial Institute Production du facteur viii de coagulation humain à partir de cellules végétales et de plantes entières
WO2006004675A2 (fr) 2004-06-25 2006-01-12 Altor Bioscience Corporation Production d'un facteur tissulaire chez des plantes
WO2006004675A3 (fr) * 2004-06-25 2006-05-04 Altor Bioscience Corp Production d'un facteur tissulaire chez des plantes
JP2008504033A (ja) * 2004-06-25 2008-02-14 アルター・バイオサイエンス・コーポレーション 植物に於ける組織因子の産生
EP4357450A1 (fr) * 2022-10-18 2024-04-24 Proview-Mbd Biotech Co., Ltd. Production d'a-thrombine humaine recombinante dans un système à base de plantes et son application

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CA2328493A1 (fr) 1999-11-18

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