Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-09-23
2001-03-27
Hauda, Karen (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S008000, C800S013000, C800S021000
Reexamination Certificate
active
06207877
ABSTRACT:
This invention relates to a transgenic non-human mammalian animal which is heterozygous or homozygous for an at least partially defective coagulation factor XIII gene.
Factor XIII is an enzyme of the coagulation cascade which cross-links fibrin and thus promotes the stability of the hemostatic plug.
The coagulation is a cascade system of proteolytic enzymes with subsequent activation by limited proteolysis. After wounding the intrinsic and the extrinsic pathways of coagulation are activated. The intrinsic activation includes coagulation factors XII, XI and IX which are proteolytic enzymes (proteases) and an enzyme cofactor (factor VIII). The extrinsic system includes tissue factor and factor VII. Both activation pathways result in the common pathway containing the protease factor Xa and its cofactor (factor Va) which convert the inactive prothrombin (factor II) to the enymatically active thrombin (IIa, n.b.: activated factors are marked with an “a” after the roman number, the roman number alone marks the inactive enzyme). Thrombin is considered to be the central protease of the coagulation system. It cleaves fibrinogen (factor I) resulting in fibrin which is the substrate for the wound closure. Together with blood platelets fibrin forms the hemostatic plug. Thrombin moreover activates the clotting factor XIII by limited proteolysis. In contrast to the other proteases of the coagulation cascade, factor XIII is a transglutaminase. Factor XIII cross-links the fibrin molecule via lysine and glutamine residues, rendering soluble fibrin into insoluble fibrin.
FXIII as found in plasma is composed to two nonidentical subunits, the a-chain and b-chain, with molecular weights of approximately 83.000 and 76.500 Da respectively (McDonagh J., in: Hemostasis and Thrombosis, Colman R W, Hirsh J., March V. J. and Salzmann E. W. (eds), Lippincoft, Philadelphia, 1994).
The tetrameric complex in plasma has the composition a
2
b
2
with a Mr of 320 000 Da. The concentration of the tetramer in plasma is about 0.07 &mgr;mol/l. During the activation step, thrombin cleaves an Arg-Gly bond at the positions 37 and 38 of the a-chain releasing a Mr 4500 activation peptide from the amino terminus. The b-chain dimer dissociates from the complex to release the active a-chain dimer (a
2
*, FXIIIa). FXIIIa is a glutamine-lysine transferase. The reaction, catalyzed is the formation of an isopeptide bond between the &ggr;-carbonyl group of glutamine and the &egr;-amino group of lysine between fibrin molecules, stabilizes the fibrin network. Four to six lysyl-glutamyl crosslinks are formed per mole of fibrin.
The lack of factor XIII in patients might lead to hemorrhagic diathesis and impairment of wound healing (Duckert F., Ann NY Acad Sci, vol. 202, 190-199, 1972). Patients with a congenital FXIII deficiency suffer from soft tissue and joint hemorrhage after trauma. The most serious form of bleeding is in the central nervous system, FXIII deficient patients have a high incidence of intracranial hemorrhage. Women with a FXIII deficiency often undergo abortion during pregnancy. FXIII stimulates the proliferation of fibroblasts, which is essential for wound healing. Thus, patients with FXIII deficiency were reported to have abnormal wound healing.
In this invention the generation of a transgenic coagulation factor XIII defective mouse with disturbance of the wound healing process and bleeding disorders is described. Furthermore, its use is described to develop wound healing accelerating drugs or drugs normalizing coagulation.
A transgenic animal which carries an at least partially defective coagulation factor XIII gene may be prepared by known gene manipulations, in which the factor XIII gene will be inactivated by an insertion, deletion, substitution or inversion or other suitable genetic manipulations. In the performance of the present invention a transgenic animal with a defective coagulation factor XIII gene has been prepared by a targeted disruption of the gene for the factor XIIIa subunit with deletion of the active site encoding the exon 7 sequence.
Thus, according to another aspect of the present invention there is provided a method of producing a transgenic non-human mammalian animal with an at least partially defective coagulation factor XIII gene comprising:
1. preparing a DNA construct encoding an at least partially defective coagulation factor XIII gene;
2. introducing such DNA construct into suitable carrier cells;
3. identifying cells in which the DNA construct has integrated by homologous recombination;
4. inserting those targeted cells into an embryo;
5. placing the embryo into a mother animal, and allowing the embryo to develop to full term;
6. deriving offsprings heterozygous for the disrupted factor XIIIa gene, and
7. interbreeding heterozygous offsprings to obtain animals homozygous for the defective factor XIIIa gene.
REFERENCES:
patent: 69896/87 (1987-09-01), None
patent: 0 236 978 A2 (1987-09-01), None
patent: WO 91/16931 (1991-11-01), None
patent: WO 94/04669 (1994-03-01), None
patent: WO 97/46669 (1997-12-01), None
Monsonego et al. FASEB 12:1163-1171, Sep. 1998.*
Ebert et al. Molecular Endocrinology 2(3):277-283, Feb. 1988.*
Hammer et al. Journal of ANimal Science 63:269-278, Jul. 1988.*
Schwaab et al. Thrombosis Research 61:225-234, Jan. 1991.*
Mullins et al. Journal of Clinical Investigations 98(11S):S37-S40, Sep. 1998.*
Moreadith et al. Journal of Molecular Medicine 75:208-216, Sep. 1998.*
Mustoe, T.A. et al., “Accelerated Healing of Incisional Wounds in Rats Induced by Transforming Growth Factor-&bgr;”, Science, vol. 237, pp. 1333-1336, (1987).
Duckert, F., “Documentation of the Plasma factor XIII Deficiency in Man”, Ann. NY Acad of Sci, vol. 202, pp. 190-199, (1972).
McDonagh, J., “Structure and Function of Factor XIII”,Hemostasis and Thrombosis: Basic Principles and Clinical Practice, Third Edition, Lippincott Company, Philadelphia, pp. 301-313, (1994).
Karges, H. E., “Blood Coagulation Facto III: Determination by Clot Stability Assays”, Bergmeyer: Methods of Enzymatic Analyses, Third Edition, vol. V, Verlag Chemie, Weinheim, pp. 400-410, (1984).
Anwar, Rashida et al., “Molecular Basis of Inherited Factor XIII Deficiency: Identification of Multiple Mutations Provides Insights Into Protein Function”, British Journal of Haematology, vol. 91, pp. 728-735 (1995).
Ichinose, Akitada et al., “Arg260-Cys Mutation in Severe Factor XIII Deficiency: Conformational Change of the A Subunit is Predicted by Molecular Modelling and Mechanics”, British Journal of Haematology, vol. 101, pp. 264-271 (1998).
Mikkola, Hanna et al., “Deficiency in the A-Subunit of Coagulation Factor XIII: Two Novel Point Mutations Demonstrate Different Effects on Transcript Levels”, The American Society of Hematology, pp. 517-525 (1994).
Izumi, Tomonori et al., “Novel Deletion and Insertion Mutations Cause Splicing Defects, Leading to Severe Reduction in mRNA Levels of the A Subunit in Severe Factor XIII Deficiency,” Thromb Haemost, 79, pp. 479-485 (1998).
Rosier, Florence, “Les Souris<<Knockout>> Ont La Pê ;che”, Genetique, pp. 48-50 (1996
Dickneite Gerhard
Grundmann Ulrich
Lathe Richard
Li Meng
Metzner Hubert
Aventis Behring GmbH
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Hauda Karen
Woitach Joseph T.
LandOfFree
Transgenic coagulation factor XIII defective animal and its... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Transgenic coagulation factor XIII defective animal and its..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Transgenic coagulation factor XIII defective animal and its... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2504176