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WO1999053032A1 - mammifères transgéniques exprimant un GP IIIa mutant - Google Patents

mammifères transgéniques exprimant un GP IIIa mutant Download PDF

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Publication number
WO1999053032A1
WO1999053032A1 PCT/US1999/008285 US9908285W WO9953032A1 WO 1999053032 A1 WO1999053032 A1 WO 1999053032A1 US 9908285 W US9908285 W US 9908285W WO 9953032 A1 WO9953032 A1 WO 9953032A1
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human mammal
tyrosine residues
mouse
mammal
gene
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PCT/US1999/008285
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English (en)
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Deborah Ann Law
David R. Phillips
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Cor Therapeutics, Inc.
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Priority to US09/673,302 priority Critical patent/US6995298B1/en
Application filed by Cor Therapeutics, Inc. filed Critical Cor Therapeutics, Inc.
Priority to EP99918586A priority patent/EP1071751A1/fr
Priority to AU36462/99A priority patent/AU3646299A/en
Priority to JP2000543580A priority patent/JP2002511249A/ja
Priority to CA002325817A priority patent/CA2325817A1/fr
Priority to IL13887399A priority patent/IL138873A0/xx
Publication of WO1999053032A1 publication Critical patent/WO1999053032A1/fr

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    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C07K14/70557—Integrin beta3-subunit-containing molecules, e.g. CD41, CD51, CD61
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • the present invention relates to mammals into which foreign DNA has been introduced or in which certain codons of integrin-encoding genes have been replaced, thereby generating transgenic or genetically-engineered mammals.
  • the present invention provides a transgenic mammal in which the endogenous GP Ilia gene (also known as ⁇ 3) has been replaced in whole or in part with a mutant GP Ilia gene in which one or both of the two phosphorylatable cytoplasmic tyrosine residues have been replaced with non-tyrosine residues, for example, such as phenylalanine.
  • the cells, platelets in particular, that are found in the blood of the resultant transgenic mammals that express an altered GP Ilia gene cannot undergo tyrosine phosphorylation, either in whole or in part, as occurs in their wild type counte ⁇ arts, these animals provide a useful tool for assessing the importance of the phosphorylation of these tyrosine residues for platelet function.
  • the present invention is also useful for studying the effect of the mutant GP Hla integrin subunit on biological processes other than platelet formation.
  • Integrins are a family of ⁇ heterodimers that mediate adhesion of cells to extracellular matrix proteins and to other cells (Clark et al., Science (1995) 268:233-239). Integrins also bind to the actin cytoskeleton through a series of intermediate proteins, and thus provide a link between the extracellular matrix and the intracellular cytoskeleton and its associated motile machinery. Such transmembrane linkages are required for cell migration.
  • integrin family also participate in signal transduction. This is evidenced by an alteration in the adhesive affinity of cell surface integrins in response to cellular activation, termed inside-out signal transduction. Additionally, effects on intracellular signaling pathways following integrin-mediated adhesion have been observed, termed outside-in signal transduction.
  • the integrin family consists of 15 related known ⁇ subunits ( ⁇ l, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, cc7, ⁇ 8, ⁇ 9, ⁇ E, ⁇ V, ⁇ llb, ⁇ L, ocM, and ⁇ X) and 8 related known ⁇ subunits ( ⁇ l, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 7, and ⁇ 8). Luscinskas et al, FASEB J., 8: 929-938 (1994). Integrin ⁇ and ⁇ subunits are known to exist in a variety of pairings as indicated in Figure 1.
  • Integrin ligand specificity is determined by the specific pairing of the ⁇ and ⁇ subunits, although some redundancy exists as several of the integrins are known to bind the same ligand. Two known parings of the ⁇ 3 subunit have been observed: with ⁇ V to make ⁇ V ⁇ 3, the Vitronectin Receptor; and with GP lib to make GP Ilb-IIIa, the Fibrinogen Receptor. ⁇ V ⁇ 3 is widely distributed, is the most promiscuous member of the integrin family and mediates cellular attachment to a wide spectrum of adhesive proteins, mostly at the R-G-D sequence on the adhesive protein.
  • ⁇ V ⁇ 3 The biological processes mediated by ⁇ V ⁇ 3 are diverse and include bone reso ⁇ tion, angiogenesis, tumor metastasis and restenosis.
  • ⁇ V ⁇ 3 is known to signal upon adhesive protein ligation (P.I. Leavesley, et al., J. Cell Biol. 121:163-170 (1993)).
  • adhesive protein ligation P.I. Leavesley, et al., J. Cell Biol. 121:163-170 (1993)
  • endothelial cells undergo apoptosis when relieved of ligation (P.C. Brooks, Cell 79:1157-1164 (1994)).
  • talin a 235 kD vinculin and actin binding protein
  • the binding of ⁇ actinin, a 100 kD vinculin binding protein and actin cross-linking protein, to the cytoplasmic domain of ⁇ l and ⁇ 3 in solid phase binding assays has also been observed.
  • CA. Otey et al J. Biol. Chem. 268(28);21193-21197 (1993); and C. A. Otey et al, J. CellBiol 111:721-729 (1990).
  • Binding studies have demonstrated an interaction between the cytoplasmic domain of ⁇ 1 and tensin, a 215 kD SH2 domain containing vinculin and actin binding protein. S. Lin et al. Mol. Biol. Cell 7 Supp. 389a, Abstract 2259 (1996).
  • Skelemin a 195 kD myosin and intermediate filament binding protein, binds to the membrane proximal regions of ⁇ l and ⁇ 3 cytoplasmic domains.
  • Paxillin a vinculin binding signaling protein also binds to the cytoplasmic domain of the ⁇ l integrin. M.D. Schaller et ⁇ /., J. CellBiol. 130:1181-1187 (1995). It is not yet known whether the ⁇ 1 -paxillin association is direct or indirect, however paxillin was postulated as being the substrate for and tyrosine phosphorylated by tyrosine kinase ppl25 FAK .
  • the actin binding protein filamin has been shown to bind to the cytoplasmic tail of the ⁇ 2 integrin subunit in vitro and co-immunoprecipitated and co-localized with ⁇ 2 integrins in vivo.
  • cytoskeletal proteins listed above have been shown to interact with cytoplasmic domains of integrin subunits with purified proteins or peptides, it is not known how these interactions occur within cells or how these interactions are regulated. Furthermore, the integrin/cytoskeletal interactions described thus far do not occur in a phosphotyrosine-dependent manner.
  • Platelet aggregation induced by a number of agonists results in the phosphorylation of tyrosine residues in the ⁇ 3 cytoplasmic tail. Law et al, J. Biol. Chem 271:10811-10815 (1996). In some respects, the phosphorylation of both tyrosine residues was necessary for binding to certain signaling proteins, whereas other signaling proteins bound following monophosphorylation. Furthermore, adhesion to vitronectin by cells transfected with ⁇ v ⁇ 3 induces a robust tyrosine phosphorylation of the ⁇ 3 subunit. Blystone et al, J. Biol. Chem 271 :31458-31462 (1996).
  • Point mutations in homologous domains in ⁇ l- and ⁇ 2 -containing integrins also modulate function, as these mutations affect integrin-cytoskeletal interactions by reducting focal adhesions, A. A. Reszka et. al., J. Cell Biol. 117: 1321-1330 (1992), and integrin activation, M. L. Hibbs et. al., J Exp. Med. 174:1227-1238 (1991), respectively.
  • Tyrosine kinases similarly were found to be essential in regulating the cytoskeletal attachment of ⁇ llb ⁇ 3. Schoenwaelder et al, J. Biol. Chem. 269(51):32479-32487 (1994).
  • the platelet plasma membrane is coated by a lattice-like structure, known as the membrane skeleton, that is composed of short actin filaments, actin-binding protein, spectrin, vinculin and various other proteins, not all yet identified. Fox et al, J. Biol.
  • the skeleton appears to be associated with a network of cytoplasmic actin filaments.
  • the membrane skeleton coats the lipid bilayer and is associated with both extracellular glycoproteins and intracellular cytoskeletal elements. Fox et al suggested that GPIIb-IIIa induces redistribution of components of the membrane skeleton and associated signaling molecules as a step in regulating integrin-induced motile events in platelets.
  • Myosin is a contractile protein that interacts with actin to produce contraction or movement.
  • the term "myosin” broadly refers to a diverse superfamily, comprised of at least 11 classes, of molecular motors capable of translocating actin filaments or of translocating vesicles or other cargo on fixed actin filaments by.
  • One characteristic of all myosins is their ability to reversibly bind to actin and to hydrolyze MgATP. See Figure 5 and J. R. Sellers and H.V. Goodson, Protein Profile 2:1323-1339 (1995).
  • the head or motor domain contains nucleotide and actin binding sites and is the most conserved region of the myosin superfamily.
  • the neck domain consists of a long single alpha helical strand from the heavy chain which is stabilized by the binding of light chain subunits.
  • the tail region which serves to anchor myosin so that it can translocate actin, is the most diverse primary sequence of all the regions and may serve to anchor certain myosin isoforms to cell or organelle membranes. It has been suggested that myosin clustering within a cell may occur on membranes or on actin filaments themselves. Titus, Trends in Cell Biology 7:119 (1997). However, the precise biochemical mechanism of interaction between the myosin tail and cytocellular structures has not heretofore been described.
  • GP Ilb-IIIa The involvement of the cytoplasmic domain of GP Ilb-IIIa in integrin signal transduction is inferred from mutagenesis experiments. Deletion of the cytoplasmic domain of GP lib results in a constitutively active receptor that binds fibrinogen with an affinity equivalent to the wild-type complex, implying that the cytoplasmic tail of GP lib has a regulatory role (T. E. O'Toole, et al, Cell Regul 1 :883-893, (1990)). Point mutations, deletions and other truncations of GP Ilb-IIIa affects the ligand binding activity of GP Ilb-IIIa and its signaling response (P. E. Hughes, et al, J. Biol. Chem. 270:12411-12417, (1995); J. Ylanne, et ⁇ /., J. Biol. Chem. 270:9550-9557, (1995)).
  • ⁇ 3-endonexin a protein identified through two hybrid screening using the cytoplasmic domain of GP Ilia as the "bait", has been found to interact directly and selectively with the cytoplasmic tail of GP Ilia (S. Shattil et al, J. Cell. Biol. 131 :807-816, (1995)).
  • ⁇ 3-endonexin shows decreased binding to the GP Ilia cytoplasmic domain containing the thrombasthenic S752-P mutation. It is not yet known whether either of these GP Ilia-binding proteins are involved in signal transduction.
  • Cytoplasmic proteins that bind to ⁇ V ⁇ 3 have also been described which may be interacting with the integrin at the GP Ilia cytoplasmic domain sequence.
  • IRS-1 was found to bind to the ⁇ V ⁇ 3 integrin following insulin stimulation of Rat- 1 cells stably transfected with DNA encoding the human insulin receptor (K. Vuori and E. Ruoslahti, Sci.
  • Cytohesin-1 specifically binds to the intracellular portion of the integrin ⁇ 2 chain, and overexpression of cytohesin-1 induces ⁇ 2 integrin-dependent binding of Jurkat cells to ICAM-1.
  • a novel serine/threonine kinase, ILK-1 was found to associate with the ⁇ l cytoplasmic domain (Hannigan et al, Nature 379:91-96, (1996)). Overexpression of ILK-1 inhibits adhesion to the integrin ligands fibronectin, laminin, and vitronectin.
  • Integrin binding to adhesive proteins and integrin signal transduction have a wide variety of physiological roles, as identified above. Enhanced signaling through integrins allows for increased cell adhesion and activation of intracellular signaling molecules which causes enhanced cell mobility and growth, enhanced cell responsiveness, and modulations in mo ⁇ hological transformations. Although integrins responsible for cellular function have been described and signaling events are beginning to be elucidated, the mechanism by which integrins transduce signals remains to be determined. To understand the molecular mechanisms of the inside-out and outside-in signaling roles mediated by the cytoplasmic tails of ⁇ 3 integrin requires the identification of the intracellular molecules that interact with the intracellular tails of integrin.
  • ILK-1 does not bind to ⁇ 3 (Hannigan et al, Nature 379:91-96 (1996)).
  • Homologous Recombination Genes can be introduced in a site directed fashion using homologous recombination. This can be used in the creation of a transgenic animal, wherein the animal would be mutated, and the phenotype of the mutation could be studied for pu ⁇ oses of drug screening, investigating physiologic processes, developing new products and the like. Papers discussing homologous recombination are discussed in R. Kucherlapati et al, (1995) U.S. Patent No. 5,413,923.
  • homologous recombination permits site-specific modifications in endogenous genes and thus inherited or acquired mutations may be corrected, and/or novel alterations may be engineered into the genome.
  • the application of homologous recombination to gene therapy depends on the ability to carry out homologous recombination or gene targeting in normal, somatic cells for transplantation.
  • embryonic stem cells or a stem cell line may be obtained.
  • Cells other than embryonic stem cells can be utilized (e.g. hematopoietic stem cells etc.) (See for more examples, J.G. Seidman et al, (1994) U.S. Patent No. 5,589,369).
  • the cells may be grown on an appropriate fibroblast fetal layer or grown in the presence of leukemia inhibiting factor (LIF) and then used.
  • LIF leukemia inhibiting factor
  • the embryonic stem cells may be injected into a blastocyst, that has been previously obtained, to provide a chimeric animal.
  • the main advantage of the embryonic stem cell technique is that the cells transfected with the "transgene" can be tested prior to reimplantation into a female animal for gestation for integration and the effect of the transgenes.
  • the homologous respective endogenous gene can be removed from a chromosome by homologous recombination with the transgene.
  • animals can be bred which carry the transgene on both chromosomes. If mutations are inco ⁇ orated into the transgenes which block expression of the normal gene production, the endogenous genes can be eliminated by this technique and functional studies can thus be performed.
  • Transgenic animals are genetically modified animals into which cloned genetic material has been experimentally transferred.
  • the cloned genetic material is often referred to as a transgene.
  • the nucleic acid sequence of the transgene is integrated at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found.
  • the transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.
  • transgenic technology allows investigators to create mammals of virtually any genotype and to assess the consequences of introducing specific foreign nucleic acid sequences on the physiological and mo ⁇ hological characteristics of the transformed animals.
  • the availability of transgenic animals permits cellular processes to be influenced and examined in a systematic and specific manner not achievable with most other test systems.
  • the development of transgenic animals provides biological and medical scientists with models that are useful in the study of disease. Such animals are also useful for the testing and development of new pharmaceutically active substances.
  • Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Patent No. 4,736,866; U.S. Patent No. 5,602,307; Mullins et al., Hypertension 22(4):630-633 (1993); Brenin et al., Surg.
  • knock-out generally refers to mutant organisms, usually mice, which contain a null allele of a specific gene.
  • knock-in generally refers to mutant organisms, also usually mice, into which a gene has been inserted through homologous recombination.
  • the knock-in gene may be a mutant form of a gene which replaces the endogenous, wild-type gene.
  • a number of recombinant murines have been produced, including those which express an activated oncogene sequence (U.S. Patent No. 4,736,866); express simian SV 40 T-antigen (U.S. Patent No. 5,728,915); lack the expression of interferon regulatory factor 1 (IRF-1) (U.S. Patent No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Patent No. 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Patent No. 5,731,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Patent No. 5,720,936); have a reduced capacity to mediate cellular adhesion (U.S.
  • Patent No. 5,602,307 possess an bovine growth hormone gene (Clutter et al., Genetics 143(4):1753-1760 (1996)); and, are capable of generating a fully human antibody response (McCarthy, The Lancet 349(9049):405 (1997)).
  • the method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules.
  • Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the recitations in U.S. Patent No. 5,489,743 and U.S. Patent No. 5,602,307.
  • the present invention provides non-human mammals comprising altered, substituted or mutant integrin cytoplasmic ⁇ subunit genes (and their expression products) in which at least one of the two (or more) cytoplasmic tyrosine residues of the expression product are replaced with a non-tyrosine residue such as phenylalanine.
  • •10- mammals encompassed by the present invention include sheep, goat, mouse, pig, dog, cat, monkey, chimpanzee, hamster, rat, rabbit, cow and guinea pig.
  • the present invention provides non-human mammals comprising a mutant GP Ilia gene and expression product wherein one or both of the cytoplasmic tyrosine residues that are capable of phosphorylation have been replaced with a non-phosphorylatable residue such as phenylalanine.
  • the present invention provides transgenic mice comprising a mutant GP Ilia gene wherein the two cytoplasmic tyrosine residues 747 and 759 have each been replaced with phenylalanine.
  • the present invention also provides platelets isolated from the blood plasma of the transgenic non-human mammals of the present invention.
  • the present invention also provides methods of preparing a transformed non- human mammal with a mutant integrin cytoplasmic ⁇ subunit gene, such as the GP Ilia gene, wherein, for example, at least one of the two tyrosine residues of the endogenous GP Ilia gene has been replaced with a non-tyrosine residue to prepare the mutant GP Ilia, wherein the methods comprise: a) introducing into embryonic stem cells a nucleic acid molecule encoding the mutant integrin cytoplasmic ⁇ subunit gene, such as a mutant or altered GP Ilia gene, and b) regenerating a transformed non-human mammal from the cells resulting from step a).
  • a mutant integrin cytoplasmic ⁇ subunit gene such as the GP Ilia gene
  • the present invention provides methods of preparing a transformed non-human mammal with, for example, a mutant GP Ilia gene wherein at least one of the two tyrosine residues of the endogenous GP Ilia gene has been replaced with a non-tyrosine residue to prepare the mutant GP Ilia, said method comprising: a) introducing into embryonic stem cells a nucleic acid molecule encoding the mutant GP Ilia gene and a selectable marker flanked by FRT sites; b) identifying and selecting transformed cells; c) removing the selectable marker from the transformed cells selected in step b) by transient transformation with FLP recombinase; d) injecting the transformed cells from step c) into blastocysts;
  • the present invention also provides methods of comparing a characteristic between two mammals of the same species, or strain, wherein one mammal has, for example, a wild-type GP Ilia gene and the other mammal has an altered or mutant GP Ilia gene, wherein at least one of the two tyrosine residues of the wild-type GP Ilia gene has been replaced with a non-tyrosine residue in the mutant GP Ilia gene. More specifically, the present invention includes such methods using transgenic mammals wherein both cytoplasmic tyrosine residues of GP Ilia have been replaced with a non-tyrosine residue such as phenylalanine. Even more specifically, the present invention includes such methods using transgenic mammals wherein the cytoplasmic tyrosine residues 747 and 759 have both been replaced with a non-tyrosine residue such as phenylalanine.
  • Examples of characteristics which can be compared use in the transgenic mammals of the present invention include comparing various physiological or biological functions and responses that are mediated, in whole or in part, by the cytoplasmic ⁇ subunit gene of various integrin molecules.
  • a comparison of such functions would include observations and comparisons of the bleeding or clotting time between the two mammal types (i.e., between wild-type and genetically engineered animals); comparing various thrombotic responses between the two mammal types; comparing the level, extent and rate of angiogenesis between the two mammal types in response to various angiogenic stimulii;
  • the present invention also provides methods of determining the effect of various agents on selected biological characteristics of a genetically engineered mammal expressing an altered or mutant integrin ⁇ subunit gene that is attributable to the expression of the GP Ilia gene, wherein the methods comprise: a) administering said agent to the transgenic mammal; b) maintaining said mammal for a desired period of time after said administration; and, c) determining whether a characteristic of said mammal that is attributable to the expression of the mutant GP Ilia gene has been affected by the administration of said agent.
  • Figure 1 shows the pairing of ⁇ and ⁇ integrin subunits.
  • Figure 2 shows the cytoplasmic domain of various integrin subunits.
  • GP Ilia tyrosine phosphorylation was dependent upon platelet aggregation, and the fact that signaling proteins as well as cytoskeletal proteins became associated with the phosphorylated GP Ilia, suggested to us that GP Ilia phosphorylation was important in outside-in GP Ilb-IIIa signaling in platelets.
  • Successful GP Ilb-IIIa-mediated outside-in signaling is required for platelet functions such as the formation of stable platelet aggregates, an important process for normal hemostasis and one which, under aberrant conditions, can lead to the formation of occlusive thrombi.
  • tyrosine phosphorylation of GP Ilia is indeed a critical step in outside-in GP Ilb-IIIa signaling, then it could be hypothesized that compounds capable of inhibiting such a phosphorylation event would have anti-thrombotic properties.
  • a mutant mouse has been generated in which the endogenous GP Ilia gene was replaced with one in which the two cytoplasmic tyrosine residues were mutated to phenylalanines (GP Ilia (Y747F, Y759F)).
  • the platelets from such animals express GP Ilia that can not undergo tyrosine phosphorylation and therefore provide a critical tool for assessing the importance of the phosphorylation reaction for platelet function.
  • assessing the effect of GP Ilia mutations in transfected cell lines i.e., in vitro
  • the importance of the GP Ilia cytoplasmic tyrosine residues in platelet function can best be determined in vivo.
  • a gene replacement project in which the endogenous murine GP Ilia gene was replaced by a mutant genes with at least one non- tyrosine substitution at one of the two cytoplasmic tyrosine residues. More specifically, the endogenous murine GP Ilia gene was replaced by a mutant gene with double tyrosine
  • PI clones containing murine genomic GP Ilia were obtained using PCR-based technology with PCR primers based on the sequence of the human GP Ilia gene. DNA corresponding to the area of interest, i.e,. the two exons encoding the GP Ilia cytoplasmic domain, was mapped using common molecular biology techniques.
  • the two tyrosines in the cytoplasmic domain (Y747 and Y759) were mutated to phenylalanines and a targeting vector was constructed which included the mutated GP Ilia genomic DNA as well as a selectable drug marker (neomycin resistance) flanked by FRT sites, the recognition sequences for the FLP recombinase.
  • This vector was transfected into embryonic stem cells and cells which had undergone a homologous recombination event were identified by Southern blotting and by PCR using specific primers.
  • ES cells that contained one normal GP Ilia allele and one GP Ilia (Y747F, Y759F) mutant allele as well as the neomycin resistance DNA were then transiently transfected with the FLP recombinase to remove the drug resistance DNA. Again, ES cells that had undergone the desired event were identified by both Southern blotting and PCR. The now standard protocols for the generation of mutant mice were employed. Basically, the desired mutant ES cells being injected into blastocysts in order to generate chimeric mice which were bred to wild-type mice to produce heterozygote animals expressing one normal and one mutant GP Ilia allele (as assessed by Southern blotting of tail genomic DNA and PCR).
  • transgenic mice provide a critical tool for assessing the importance of the GP Ilia cytoplasmic tyrosine residues on platelet function.
  • the platelets from the mutant mice express a GP Ilb-IIIa in which the GP IHa can not be tyrosine phosphorylated.
  • platelets from these mice can be used in a number of assays to assess the role of outside-in GP Ilb-IIIa signaling on platelet function. For example, bleeding time in mutant mice will assess whether tyrosine phosphorylation of GP Ilia is critical for normal hemostasis. Also if GP Ilia tyrosine phosphorylation is an
  • mutant mice will predict the mutant mice to be defective in thrombotic responses, where the formation of very large platelet aggregates is required.
  • murine thrombotic models with these mutant mice to directly assess this issue. Indeed, this mutant mouse will provide information on the utility of generating therapeutics designed to interfere either directly with GP Ilia tyrosine phosphorylation, or with an event dependent on this phosphorylation e.g. interaction of phospho-GP Ilia with an adaptor protein.
  • GP Ilia ( ⁇ 3 ) integrin subunit is not restricted to platelets. In endothelial cells this protein pairs with the ⁇ v subunit to form the ⁇ v ⁇ 3 integrin. This integrin appears to play an important role in angiogenesis and tumor metastasis.
  • ⁇ 3 is a subunit of the LRI (leucocyte responsive integrin), and is thought to be involved in inflammatory responses. Since all of the ⁇ 3 in the mutant mouse contains the tyrosine to phenylalanine mutations in the cytoplasmic domain, this mouse can be used to examine the effect of these mutations on ⁇ v ⁇ 3 and LRI function. Again, such analyses will provide information on whether or not inhibition of ⁇ 3 phosphorylation will have effects on angiogenesis, tumor metastasis and/or inflammation, and thus will be of significant clinical import.
  • FIG. 1 -16- phosphotyrosine-binding signaling proteins She and Grb2 to the cell membrane. See Philips et al. (1996).
  • the present inventors have discovered that tyrosine phosphorylation and dephosphorylation of the ⁇ 3 integrin tail may, in an analogous manner, regulate integrin association with the cytoskeleton (see, e.g., U.S. Patent Application No. 08/975,653, filed November 21, 1997, which is inco ⁇ orated by reference herein).
  • Figure 2 provides the amino acid sequences of various human integrin ⁇ subunits, showing the locations of the tyrosine residues.
  • cytoplasmic domains of ⁇ l, ⁇ 2 and ⁇ 3 which contain tyrosines are important for normal functioning of GPIIb-IIIa and of other integrins.
  • transgenic animals that express altered and mutant cytoplasmic domains of those or other integrin ⁇ subunits that natively contain phosphorylatable tyrosine residues are expressly included in the scope of this invention.
  • a conservative amino acid substitution refers to alterations of particular residues (specifically the phosphorylatable tyrosine residues) or other alterations in the amino acid sequence that, except for the inability to undergo phosphorylation at the site of the one or more relevant cytoplasmic tyrosine residues, do not otherwise adversely affect the ability of the integrin to participate in signaling activities or to bind to one or more particular binding partners, such as myosin, for example.
  • a substitution, insertion or deletion is said to adversely affect the normal functioning of the corresponding wild-type integrin ⁇ subunit peptide when the altered sequence significantly inhibits the peptide from participating in signalling activities or from associating with its native binding partners, such as myosin.
  • the overall charge, structure or hydrophobic/hydrophilic properties of the ⁇ subunit peptide can be altered without adversely affecting activity of the peptide.
  • the amino acid sequence of the ⁇ subunit peptide can be altered, for example to render the peptide more hydrophobic or hydrophilic, to the extent that this does not adversely affecting the ability of the peptide to otherwise participate in its normal signalling
  • mutant GPIIIa subunits which are
  • altered ⁇ subunit peptides can be generated using standard knock-out procedures to modify any one or all of the phosphorylatable tyrosine residues. This can be accomplished using a variety of art-known procedures such as targeted recombination. Once generated, the genetically-engineered animal can be used to
  • the present invention provides transgenic animals that contain non-tyrosine residues at sites where phosphorylatable tyrosine residues occur normally in endogenous ⁇ subunit genes. Since the non-tyrosine substitutions will not be phosphorylated as in the case where the residues are tyrosine and since normal platelet aggregation is dependent on phosphorylation occurring, the transgenic mammals of the present invention will display non-normal platelet aggregation. By comparing the physiological and mo ⁇ hological characteristics between the transformed and non- transformed animals one skilled in the art can thereby determine the effect of the presence or absence of cytoplasmic tyrosine phosphorylation in the GP Ilia gene on the corresponding animal.
  • the transgenic animals of the present invention can be used to identify agents that modulate (i.e., either promote or further inhibit) platelet aggregation or other effects that are mediated by integrin signaling pathways.
  • the evaluation of such agents can be conducted either in vitro, in situ, or in vivo by techniques known to those skilled in the art.
  • the cells, platelets, tissues and whole organisms of the disclosed transgenic animals have utility in testing the effect of various agents for their ability to reduce or increase the processes involved with integrin-mediated cytoskeletal association.
  • Agents which can be tested include various anticoagulant, thrombolytic and antiplatelet therapeutics and drugs. Examples of such agents include glycosaminoglycans such as
  • oral anticoagulants such as dicumarol, anisindione, and bromadkiolone
  • tissue plasminogen activator (t-PA) tissue plasminogen activator
  • urokinase aspirin; dipyridamole
  • ticlopidine tissue plasminogen activator
  • the cells and whole organisms of the transgenic animals of the present invention may also be used to investigate gene regulation, expression and organization in animals.
  • transgenic mammals especially transgenic mice.
  • -19- murine genomic DNA and one set generated a specific band of approximately 1.5kB in the PCR (polymerase chain reaction). This band was close in size to the predicted human fragment ( ⁇ 1.2kB) which spanned the 3' part of exon M, the 5' part of exon N, and the intron between these two exons.
  • a .3kB Pstl/SacI fragment containing exon N (which encodes the two tyrosine residues) was subcloned into pBSKS II and the two tyrosine residues were mutated to phenylalanines using standard site-directed mutagenesis techniques. Mutations were confirmed by sequencing and were also designed to introduce two new enzyme sites, a Dral site (at Y747F) and a TaqI site (at Y759F) which could be used in later identification of the mutant DNA. This mutant fragment was used to replace the wild-type Pstl/SacI fragment in the murine genomic GP Ula clone.
  • -20- can be used to excise the neo r DNA at a later stage (Dymecki S. M. (1996) PNAS USA 93: 6191-6196).
  • a ⁇ 4kB HindTII fragment of GP Ula containing exon N with the tyrosine to phenylalanine mutations was subcloned into a HindTLI site on this vector to give a construct basically consisting of the mutated GP Ula with a neo r cassette in the intron between exons M andN.
  • the above targeting construct was transfected into ES cells which were then selected for neo r using the drug G418 as described in Johnson, R and Killeen, N. P (In Gene Probes 2: a practical approach, Eds. B. D. Hanes and S. J. Higgins. Oxford University Press p. 313-327 (1995)).
  • ES cell which had undergone a successful homologous recombination event were identified by Southern blotting and PCR techniques. These cells now had one wild type GP Ula allele and one containing the neo r cassette and the tyrosine to phenylalanine mutations.
  • the drug cassette is 1.9kB in size and although it was present in an intron we wished to remove it to make sure that its presence would have no detrimental effect in our experiments. To this effect the selected ES cells were transiently transfected with the Flp recombinase which recognized the FRT sites and led to the excision of the DNA encoding the drug resistance. Thus, instead of a 1.9kB piece of extraneous DNA being present in the intron a small piece encoding the 34bp FRT recognition site (and a new Xbal site) is all that remains (Dymecki, S. M., PNAS 93: 6191-6196 (1996)). Again, ES cells that had undergone a successful event were selected for using Southern blotting and PCR techniques.
  • mutant mice To produce mutant mice a mutant ES cell clone (originally from a strain 129 mouse) was injected into blastocysts from a C57B16 mouse and these blastocysts were implanted into a pseudo-pregnant foster mother (as in Ramirez-Solis, R., Davis, A. C, and Bradley, A. Guide to techniques in mouse development. Methods in Enzymology 225. Eds. P. M. Wassarman, M. L. DePanphihs. Academic Press (1993) in particular p 855-878). Male chimeric animals identified by their mixed coat color (black from the C57B16 blastocyst and agouti from the 129
  • -21- ES cells were then mated with C57B16 wild-type females.
  • the offspring were genotyped for the presence of wildtype and mutant GP Ilia alleles by Southern blotting (making use of the new Xbal site that was present only in mutant GP Ula DNA).
  • the offspring were a mix of wild-type and heterozygote animals and the heterozygote animals were further crossed to produce litters containing wildtype animals, heterozygote animals (ie. one wildtype and one mutant GP Ula allele) and homozygotes (ie. both GP Ula alleles containing the tyrosine to phenylalanine mutations).
  • mutant animals are viable and express GP Ub-UIa on their platelets at similar levels to that seen in normal ariimals expressing non-mutant GP Ula when the platelets are stained with an anti-GP Ub-UIa antibody and examined on the FACS (fluorescent activated cell sorter).

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Abstract

La présente invention concerne des mammifères dans lesquels un ADN étranger a été introduit, ou dans lesquels diverses modifications ou substitutions ont été pratiquées sur une sous-unité de l'intégrine β, permettant ainsi de produire des mammifères non humains transgéniques ou génétiquement manipulés. La présente invention concerne en particulier un mammifère transgénique dans lequel un gène GP IIIa endogène a été remplacé par un gène GP IIIa modifié ou mutant, à l'intérieur duquel l'un des restes de tyrosine cytoplasmiques pouvant être phosphorylés, ou la totalité d'entre eux, ont été remplacés par des restes d'autres substances, par exemple la phénylalanine. Les plaquettes sanguines des mammifères transgéniques ainsi obtenus exprimant ledit gène GP IIIa modifié ou mutant ne pouvant être soumises à une phosphorylation de tyrosine identique à celle des mammifères de phénotype sauvage, ces animaux génétiquement manipulés constituent un outil permettant d'évaluer de manière critique l'importance de la réaction de phosphorylation de la fonction plaquettaire. Cette invention permet également d'étudier l'effet de la sous-unité de l'intégrine β dudit gène GP IIIa modifié ou mutant sur des processus biologiques autres que la formation plaquettaire.
PCT/US1999/008285 1998-04-15 1999-04-15 mammifères transgéniques exprimant un GP IIIa mutant WO1999053032A1 (fr)

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US09/673,302 US6995298B1 (en) 1998-04-15 1998-04-15 Transgenic mammals expressing mutant GP IIIa
EP99918586A EP1071751A1 (fr) 1998-04-15 1999-04-15 MAMMIFERES TRANSGENIQUES EXPRIMANT UN GP IIIa MUTANT
AU36462/99A AU3646299A (en) 1998-04-15 1999-04-15 Transgenic mammals expressing mutant gp iiia
JP2000543580A JP2002511249A (ja) 1998-04-15 1999-04-15 変異型ＧＰＩＩＩａを発現するトランスジェニック哺乳動物
CA002325817A CA2325817A1 (fr) 1998-04-15 1999-04-15 Mammiferes transgeniques exprimant un gp iiia mutant
IL13887399A IL138873A0 (en) 1998-04-15 1999-04-15 Transgenic mammals expressing mutant gp iiia

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Publication number Priority date Publication date Assignee Title
WO2000008137A3 (fr) * 1998-08-04 2000-03-23 Cor Therapeutics Inc Animaux transgeniques avec un gene de la glycoproteine v modifie
US8618062B2 (en) * 1999-06-26 2013-12-31 Inter-K Pty Limited Method of modulating integrin mediated cellular activity and agents useful for same

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WO1995025173A1 (fr) * 1994-03-14 1995-09-21 The Scripps Research Institute Procede d'identification d'inhibiteurs d'activation de l'integrine
WO1997008316A1 (fr) * 1995-08-31 1997-03-06 Merck & Co., Inc. Sous-unites d'integrine de souris

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WO1995025173A1 (fr) * 1994-03-14 1995-09-21 The Scripps Research Institute Procede d'identification d'inhibiteurs d'activation de l'integrine
WO1997008316A1 (fr) * 1995-08-31 1997-03-06 Merck & Co., Inc. Sous-unites d'integrine de souris

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BLYSTONE S ET AL: "Requirement of integrin beta3 tyrosine 747 for beta3 tyrosine phosphorylation and regulation of alphavbeta3 avidity", JOURNAL OF BIOLOGICAL CHEMISTRY., vol. 272, no. 45, 7 November 1997 (1997-11-07), pages 28757 - 28761, XP002112506 *
HANKS M ET AL: "Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2", SCIENCE., vol. 269, no. 5224, 4 August 1995 (1995-08-04), pages 679 - 682, XP002112508 *
LIU K ET AL: "Identification of a functionally important sequence in the cytoplasmic tail of integrin beta 3 by using cell-permeable peptide analogs", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA., vol. 93, no. 21, 15 October 1996 (1996-10-15), pages 11819 - 11824, XP002112507 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000008137A3 (fr) * 1998-08-04 2000-03-23 Cor Therapeutics Inc Animaux transgeniques avec un gene de la glycoproteine v modifie
US6953874B2 (en) 1998-08-04 2005-10-11 Millennium Pharmaceuticals, Inc. Transgenic animals having a modified glycoprotein V gene
US8618062B2 (en) * 1999-06-26 2013-12-31 Inter-K Pty Limited Method of modulating integrin mediated cellular activity and agents useful for same

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