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WO1999031969A2 - Modele animal transgenique de lutte contre des maladies degeneratives du cartilage - Google Patents

Modele animal transgenique de lutte contre des maladies degeneratives du cartilage Download PDF

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Publication number
WO1999031969A2
WO1999031969A2 PCT/US1998/027056 US9827056W WO9931969A2 WO 1999031969 A2 WO1999031969 A2 WO 1999031969A2 US 9827056 W US9827056 W US 9827056W WO 9931969 A2 WO9931969 A2 WO 9931969A2
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Prior art keywords
mmp
cartilage
animal
composition
transgenic
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PCT/US1998/027056
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English (en)
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WO1999031969A3 (fr
Inventor
Lisa Ann Neuhold
Loran Marie Killar
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American Home Products Corporation
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Publication date
Priority claimed from US08/994,689 external-priority patent/US6613958B1/en
Application filed by American Home Products Corporation filed Critical American Home Products Corporation
Priority to IL13662698A priority Critical patent/IL136626A0/xx
Priority to CA002314357A priority patent/CA2314357A1/fr
Priority to JP2000524983A priority patent/JP2002533055A/ja
Priority to AU19316/99A priority patent/AU1931699A/en
Priority to KR1020007006731A priority patent/KR100558288B1/ko
Priority to EP98964125A priority patent/EP1040196A2/fr
Publication of WO1999031969A2 publication Critical patent/WO1999031969A2/fr
Publication of WO1999031969A3 publication Critical patent/WO1999031969A3/fr
Priority to AU2003257928A priority patent/AU2003257928B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
    • 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
    • A01K2217/05—Animals comprising random inserted nucleic acids (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
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • 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
    • 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
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention pertains to transgenic mammals that express recombinant matrix-degrading enzymes in a temporally and spatially regulated manner.
  • the invention further pertains to model systems incorporating such transgenic mammals for studying degenerative joint diseases, including systems for identifying therapeutic agents and treatment regimens .
  • Degenerative diseases of cartilage including joint and disc diseases such as osteoarthritis, rheumatoid arthritis, and osteochondrodysplasias, are widespread, particularly in the elderly. Early symptoms common to these diseases include progressive loss of proteoglycans in the joint (as evidenced by loss of metachromasia); collagen degradation; fibrillation of the cartilage surface; and, ultimately, loss of cartilage (which is evidenced radiologically as joint space narrowing).
  • type II collagen the major structural collagen found in articular cartilage.
  • catabolic enzymes that degrade type II collagen during normal remodeling of cartilage and bone.
  • Pathological conditions such as, e.g., degenerative joint diseases, may result when this balance is disrupted.
  • MMPs matrix metalloproteinases
  • Table 1 a family of zinc-dependent enzymes, and aggrecanase
  • MMPs are synthesized in articulating joints by chondrocytes, which, in mature articular cartilage, are terminally differentiated cells that maintain the cartilage- specific matrix phenotype. Overexpression of MMPs relative to endogenous MMP inhibitors, as occurs in degenerative joint diseases, may result in cartilage degradation.
  • Type II collagen is a substrate for MMP-13 and MMP-1 (Knauper et al., J. Biol. Chem. 271:1544, 1996) and both MMP-1 and MMP- 13 proteins can be detected immunohistochemically in human osteoarthritic tissues.
  • MMP- 13 and its cleavage products are found at higher levels than MMP-1 (Billinghurst et al., J. Clin.
  • MMP- 13 may play an important role in cartilage degradation associated with osteoarthritis and other degenerative joint diseases (Mitchell et al., J. Clin. Inves. 97:761, 1996).
  • Animal models for osteoarthritis-related syndromes have been described in guinea pigs (Watson et al., Arth. Rheum. 39; 1327, 1996) and in the inbred STR/ORT strain of mice (Das-Gupta et al, Int. J. Exp. Path. 74:627, 1993).
  • osteoarthritis-related models include surgically-induced joint destabilization, e.g., anterior cruciate ligament transection and/or partial meniscectomy in rabbits and dogs, which stimulates cartilage degradation (Hulth et al., Acta Orthop. Scand. 41:522, 1970).
  • Another model employs injection of bacterial collagenase into the joints of an animal to induce a biochemical ligament transection (Nan der Kraan et al, J. Exp. Pathol. 71:19, 1990). Because (i) surgical or other manipulation of individual animals is required; (ii) the animals are large and expensive; and/or (iii) the course of disease is not consistent, these models cannot easily be used in large-scale studies, including drug screening.
  • Transgenic animal models in principle, can provide the opportunity for a reproducible animal model system for degenerative joint diseases.
  • previous attempts to engineer transgenic animals expressing MMPs such as MMP-1 and stromelysin have not resulted in an observable joint degeneration phenotype in the transgenic animals. This could be due to embryonic lethality caused by constitutive expression of these enzymes.
  • Witty et al., Mol.Biol. Cell 6 . : 1287, 1995 have created transgenic animals that constitutively express MMP-1 and stromelysin in mammary tissue, but these animals do not exhibit symptoms of osteoarthritis.
  • Transgenic animals containing regulatable heterologous genes whose expression results in cartilage degeneration are particularly advantageous in providing reproducible experimental control over the timing and the level of expression of the transgenes and, thereby, over the pathological syndrome itself.
  • Such animals can be used to determine what level of expression of the transgene is required to cause disease and, importantly, can be used for drug discovery and optimization of treatment regimens.
  • transgenic animals can be used to further define the role of matrix- degrading enzymes in cartilage degradation and as an in vivo screen to identify compounds that modulate these enzymes or compounds that inhibit the progression of degenerative joint diseases.
  • the present invention provides transgenic non-human animals or the progeny thereof whose somatic and germline cells contain, in stably integrated form, one or more heterologous or recombinant genes encoding polypeptides comprising enzymatically active matrix-degrading enzymes (MDEs), preferably MMPs.
  • MDEs enzymatically active matrix-degrading enzymes
  • MMPs for use in the invention comprise one or more of MMP- 1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, and MMP-17; preferably one or more of MMP-1, MMP-3, MMP-8, and MMP-13; and most preferably one or more of MMP- 1 and MMP-13; and include enzymatically active variants, fragments, and combinations of these polypeptides.
  • Other matrix-degrading enzymes can also be used, including, e.g., aggrecanase.
  • the MDEs may be derived from any species, preferably human.
  • the recombinant MDE- encoding genes are selectively expressed in articular chondrocytes of the transgenic animal and expression results in pathological symptoms characteristic of degenerative joint disease.
  • the invention provides a transgenic animal or the progeny thereof whose somatic and germline cells contain a stably integrated first recombinant gene encoding an MDE or an enzymatically active derivative or variant thereof, preferably a constitutively active proMMP-13 variant (designated MMP- 13*) comprising the sequence depicted in SEQ ID NO: 4.
  • the first recombinant gene is under the control of a first regulatable promoter; most preferably, the first regulatable promoter comprises a tet07 sequence, such as, e.g., the promoter depicted in SEQ ID NO: 5.
  • the transgenic animal may further comprise a second recombinant gene encoding a polypeptide that regulates the first regulatable promoter and is preferably a tTA polypeptide.
  • the second recombinant gene is under the control of a second regulatable promoter, preferably one that comprises sequences derived from a joint-specific promoter, and most preferably a type II collagen promoter, such as, e.g., the promoter depicted in SEQ ID NO: 6.
  • a second regulatable promoter preferably one that comprises sequences derived from a joint-specific promoter, and most preferably a type II collagen promoter, such as, e.g., the promoter depicted in SEQ ID NO: 6.
  • the invention provides isolated nucleic acids encoding enzymatically active MMP variants, preferably human proMMP-13 variants, and most preferably MMP-13*.
  • the invention also encompasses recombinant cloning vectors comprising these nucleic acids; cells comprising the vectors; methods for producing
  • MMP- 13 -derived polypeptides comprising culturing the cells under conditions appropriate for MMP-13 expression; and isolated MMP- 13 -derived polypeptides.
  • the invention provides methods for producing phenotypic changes characteristic of cartilage-degenerative disease in a mammal, which comprise exposing the transgenic animals of the invention to conditions that result in expression of the MDEs encoded by the transgenes.
  • a transgenic animal comprising a first recombinant gene encoding MMP- 13* operably linked to a tet07 promoter and a second recombinant gene encoding a tTA protein operably linked to a type II collagen promoter is maintained in the presence of tetracycline or a tetracycline analogue.
  • MMP-13* When it is desired to induce expression of MMP-13*, tetracycline or the tetracycline analogue is withdrawn, MMP-13* is selectively expressed in joint tissues, and phenotypic changes characteristic of cartilage-degenerative disease result.
  • the invention provides methods for determining the potential of a composition to counteract cartilage-degenerative disease.
  • the methods are carried out by administering a known dose of the composition to the transgenic animals of the invention, either before or after phenotypic indicators of cartilage-degenerative disease have developed; monitoring the indicators for a predetermined time following administration of the composition; and comparing the extent of the indicators in the animal to which the composition was administered relative to a control transgenic animal that had not been exposed to the composition. Any difference in (i) the nature or extent of phenotypic indicators of cartilage-degenerative disease, (ii) the time required for the indicators to develop, or (iii) the need for other ameliorative treatments indicates the potential of the composition to counteract cartilage-degenerative disease.
  • FIG. 1A and IB (A) Schematic illustration of the structure of human MMP-13 (collagenase-3).
  • the first box (corresponding to the extreme aminoterminus) represents the pre domain (signal peptide) that targets nascent proMMP-13 for secretion.
  • the second box represents the pro domain, which is involved in maintaining the latency of the enzyme.
  • a conserved sequence (SEQ ID NO: 1) within the pro domain that is important for maintaining enzyme latency is shown.
  • the third box represents the 170- amino acid catalytic domain, which contains a conserved region (shown; SEQ ID NO:2) that is important for catalytic activity.
  • the fourth box represents the 200-amino acid carboxyterminal domain.
  • FIG. 3 Illustration of the nucleic acid sequence (SEQ ID NO:3) encoding a constitutively active variant of human pro MMP-13, designated MMP- 13*, and the amino acid sequence of MMP-13* (SEQ ID NO:4).
  • the residues that are mutated relative to wild-type MMP-13, which are depicted in larger type, are GTC at nucleotide positions 299-301.
  • Figures 2A and 2B Binary strategy used to obtain chondrocyte specific, doxycycline (DOX) regulated MMP13* expression.
  • DOX doxycycline
  • the first construct illustrates the rat type II collagen promoter driving expression of the tetracycline repressor - VP16 activator fusion protein (TA), followed by an SV40 splice and polyadenylation signal (this construct is referred to as CPE - TA).
  • the second construct illustrates the Tet07 promoter (Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89:5547, 1992), driving expression of a constitutively active human MMP 13 protein, followed by an SN40 splice and polyadenylation signal (construct referred to as Tet07 - MMP 13*).
  • transgene constructs were co-mi croinejected into fertilized mouse embryos to generate a double transgenic harboring both genes.
  • DOX did not affect the expression of the chromosome.
  • transgene expression is off; when DOX is removed, transgene expression is turned on.
  • An arrow denotes the transcription start site, while the asterisk indicates a constitutively active mutation in the MMP 13 transgene.
  • FIG. 3A and 3B The type II collagen gene promoter directs expression to the joints of tester transgenic mice expressing ⁇ -galactosidase under the control of the rat type II collagen promoter.
  • A Diagram of the CPE -lacZ construct. The rat type II collagen promoter (first box) drives expression of the ⁇ -galactosidase (lacZ) gene (second box), which is followed by a ⁇ -globin splice and polyadenylation signal (final boxes joined by splice symbol).
  • B Whole mount staining for ⁇ -galactosidase activity on El 6 embryos. The embryo on the left shows the staining (arrows) of the transgenic compared to a wild-type embryo.
  • C Higher magnification of a transgenic elbow and front paw; ⁇ -gal staining is visible (arrows).
  • Figures 3B and 3C more dramatically illustrate the invention when rendered in color, although black and white rendering is sufficient for understanding the invention.
  • FIG. 4A and 4B Expression profile of the TA and MMP 13 R ⁇ A by RT-PCR.
  • A Amplification of TA cD ⁇ A from total R ⁇ A.
  • B Amplification of MMP13* cDNA from total RNA.
  • Lane 1 l64 Hae III MW markers;
  • Lane 2 PCR amplification of transgenic (line 6) genomic DNA;
  • Lane 3 PCR amplification of non-trans genie genomic DNA;
  • Lane 4 a wild-type mouse maintained on DOX;
  • Lane 5 a wild-type mouse off DOX;
  • Lanes 6-7 transgenic mice (-four months) maintained on DOX;
  • Lanes 8-9 transgenic mice (Hfour months) removed from DOX at birth.
  • the arrows la and lb indicate a 648 bp MMP13* specific fragment and a 859 bp specific fragment, respectively.
  • Each reaction was run using c-fos primers as an internal control, spliced mRNA yielding 187 bp (arrow 3) and unspliced mRNA 303 bp (arrow 2). No bands were detected in corresponding lanes containing RNA for PCR that was not treated with RT (data not shown).
  • FIG. 5A and 5B Photographic illustrations of immunohistochemical localization of type II collagen cleavage products in the growth plate and articular cartilage of transgenic mice expressing the transgenes shown in Figure 2. The tissues were stained with an antibody that recognizes cleavage products of type II collagen.
  • A Tissue derived from a mouse that had been maintained on doxycycline to repress MMP- 13* expression.
  • B Tissue derived from a mouse that had been withdrawn from doxycycline, allowing expression of MMP-13*, for 30 days at 3 months of age.
  • Figures 5 A and B more dramatically illustrate the invention when rendered in color, although black and white rendering is sufficient for understanding the invention.
  • Figure 6A, 6B, and 6C Figure 6A, 6B, and 6C.
  • FIG. 6A-C A color photographic illustration of Safranin O staining of the articular cartilage and growth plate of the patella of double transgenic mice.
  • A Tissue derived from a mouse maintained on doxycycline.
  • B Tissue derived from a mouse 7 days after withdrawal from doxycycline.
  • C Tissue derived from a mouse 14 days after withdrawal from doxycycline.
  • Figures 6A-C more dramatically illustrate the invention when rendered in color, although black and white rendering is sufficient for understanding the invention.
  • Figures 7A, 7B, 7C, and 7D Longitudinal section through the hind knee joints (A and B) and synovium (C and D).
  • a and C Age match littermate control and (B and D) line 6 removed from DOX.
  • Figures 7A-D more dramatically illustrate the invention when rendered in color, although black and white rendering is sufficient for understanding the invention.
  • the present invention is based on the discovery regulated expression of matrix-degrading enzymes in cartilage in transgenic mice results in characteristic phenotypic changes associated with matrix degenerative diseases of the joints and intervertebral discs.
  • the animal models of the invention provide novel model systems for matrix degenerative disease syndromes which can be used for detailed characterization of human joint and intervertebral disc pathologies as well as for drug discovery and optimization of treatment regimens.
  • a transgenic animal according to the invention is an animal having cells that contain a transgene which was introduced into the animal or an ancestor of the animal at a prenatal (embryonic) stage.
  • a transgenic animal can be created, for example, by introducing the gene of interest into the male pronucleus of a fertilized oocyte by, e.g., microinjection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the gene of interest may include appropriate promoter sequences, as well as intronic sequences and polyadenylation signal sequences. Methods for producing transgenic animals are disclosed in, e.g., U.S. Patent Nos.
  • a transgenic founder animal can be used to breed additional animals carrying the transgene.
  • a transgenic animal carrying one transgene can also be bred to another transgenic animal carrying a second transgene to create a "double transgenic" animal carrying two transgenes.
  • two transgenes can be co-microinjected to produce a double transgenic animal. Animals carrying more than two transgenes are also possible.
  • heterozygous transgenic animals i.e., animals carrying one copy of a transgene, can be bred to a second animal heterozygous for the same transgene to produce homozygous animals carrying two copies of the transgene.
  • the present invention encompasses transgenic animals, preferably mammals, which express MDEs, particularly MMPs, and most particularly those MMPs having collagenase activity, from a recombinant gene.
  • MDEs for use in the invention include without limitation MMPs and aggrecanase.
  • Useful MMPs include without limitation the collagenases designated MMP-1, MMP-8 and MMP-13; the stromelysins designated MMP-3, MMP-10, and MMP-11; the gelatinases designated MMP -2 and MMP-9; the metalloelastase designated MMP- 12; and membrane-type MMPs designated MMP-14, MMP-15, MMP-16, and MMP-17 ( Matrisian, BioEssays, 14:455, 1992).
  • Matrix-degrading activity refers to the proteolytic degradation of matrix components, including, e.g., collagen, particularly type II collagen and most particularly the triple helical form of type II collagen. Any polypeptide exhibiting matrix-degrading activity may be used in practicing the invention, including enzymatically active fragments of the above-described enzymes. Preferably, MMP- 13 enzymatic activity is expressed. MMP- 13 enzymatic activity as used herein refers to the proteolytic degradation of type II collagen. Any MMP-13 polypeptide or fragment or derivative thereof that exhibits MMP- 13 enzymatic activity may be used.
  • the enzymes may be derived from any animal species, including without limitation human, mouse, rat, rabbit, pig, cow, or non-human primate, or combinations thereof. Preferably, the MMP- 13 or derivative thereof is of human origin.
  • MMPs are synthesized as precursors (i.e., zymogens or proenzymes) whose enzymatic activity is latent; proteolytic removal of the pro region after secretion produces the enzymatically active protein.
  • the need for proteolytic processing is circumvented by the use of enzyme or proenzyme variants that are enzymatically active even when uncleaved.
  • Such variants can be produced using conventional techniques for site-directed or random mutagenesis coupled with analysis of collagenase enzymatic activity (see below).
  • modifications may be introduced into a proenzyme sequence, particularly within the pro region or near the pro region cleavage site, to produce a constitutively active polypeptide which does not require proteolytic processing for activation.
  • the pro region may be deleted entirely.
  • recombinant genes may be used in which the sequence encoding the native signal peptide is replaced by a heterologous sequence that functions as a signal peptide, i.e., promotes secretion.
  • the use of genes encoding any such modified MMP polypeptides is encompassed by the invention.
  • a constitutively active MMP- 13 variant is used in practicing the invention.
  • the MMP- 13 variant comprises a sequence containing a mutation in the sequence encoding the PRCGVPDN region, SEQ ID ⁇ O:7, specifically a substitution of Pro" to Val; the sequence of this polypeptide is depicted in SEQ ID NO: 4 and this polypeptide is designated MMP-13*.
  • the constitutively active MMP- 13 variant comprises a substitution of Val 98 to Gly.
  • the transgenic animals of the invention preferably express MMP activity in a regulated manner. Regulated expression as used herein refers to temporal and/or spatial control.
  • Temporal control refers to the ability to repress expression of MMP activity until a predetermined time in the development of the transgenic animal, after which MMP expression may be activated and maintained for as long as desired. Preferably, MMP expression is repressed throughout embryonic development and activated in the adult animal. Spatial control refers to the ability to selectively express MMP activity in particular tissues. Preferably, MMP activity is selectively expressed in joint tissues, most preferably in articular chondrocytes.
  • Temporal control of MMP expression is achieved by use of one or more polypeptides comprising a transcriptional repressor, a transcriptional activator or enhancer, or combinations thereof, in conjunction with a promoter responsive to the transcriptional repressor/activator used to which the MMP-encoding sequence is operably linked.
  • temporal control of MMP expression is achieved by (i) expression in the transgenic animal of a repressor polypeptide operably linked to a polypeptide that directly or indirectly activates transcription in eucaryotic cells, creating a repressor- activator fusion polypeptide; and (ii) the coupled use of a target promoter operably linked to an MMP-encoding sequence whose transcriptional activity is responsive to the repressor-activator fusion polypeptide.
  • nucleotide sequences encoding the repressor polypeptide are ligated in-frame to sequences encoding the transcriptional activator polypeptide to create a chimeric gene encoding a fusion protein.
  • Useful repressor polypeptides include without limitation polypeptides comprising sequences derived from bacterial repressors, including without limitation tetracycline repressor, LacR repressor, KRAB domain, and lambda repressor (cro and cl), as well as eukaryotic repressors, including without limitation those involved in amino acid or sugar synthesis.
  • Useful direct transcriptional activator polypeptides include without limitation herpes simplex virus protein 16 (VP16); yeast GAL14; yeast STAT; steroid receptors such as, e.g., progesterone receptor and estrogen receptor; and constitutive activators such as, e.g., c-fos, c-jun, and SP-1.
  • the repressor polypeptide may be linked to a polypeptide that indirectly activates transcription by recruiting a transcriptional activator to interact with the repressor-activator fusion protein; such indirect activator polypeptides include without limitation TATA Box Binding Protein (TBP) and basic transcription factors, including, e.g., basic transcription factor D.
  • TBP TATA Box Binding Protein
  • basic transcription factors including, e.g., basic transcription factor D.
  • each repressor-activator fusion protein is used in conjunction with a target promoter that is responsive to the particular fusion protein and that regulates transcription of an MDE-encoding sequence.
  • the promoter comprises at least one operator sequence responsive to the repressor component of the repressor-activator fusion polypeptide, which is operably linked to at least a minimal promoter that supports transcription in eucaryotic cells.
  • suitable repressor- responsive operator sequences include without limitation sequences derived from the tetracycline resistance operon encoded in TnlO in E. coli, the lambda repressor operon, and the yeast GAL repressor operon.
  • CCN cytomegalovirus
  • PtK-1 thymidine kinase
  • HSP heat shock protein
  • 5,650,298 discloses a repressor-activator fusion protein comprised of sequences derived from the tetracycline repressor fused to VP16 sequences, which is designated tTA, and a tTA-responsive promoter, designated tet07, which comprises a TnlO-derived sequence linked to a portion of the CMN IE promoter.
  • temporal control is achieved by (i) expression in the transgenic animal of a heterologous or recombinant transcriptional activator polypeptide or polypeptides and (ii) the coupled use of a target promoter operably linked to an MMP- encoding sequence whose transcriptional activity is responsive to the heterologous or recombinant transcriptional activator.
  • Useful transcriptional activators include without limitation a modified ecdysone receptor, in which a NP16 transactivation domain linked to the aminoterminal transactivation domain of the glucocorticoid receptor is fused to the ligand-binding domain and carboxyterminal sequence of the ecdysone receptor (No et al., Proc. Natl. Acad. Sci.
  • a chimeric protein designated pGL-VP, comprising VP16 activator sequences, GAL4 activation sequences, and a mutated human progesterone receptor ligand-binding domain (Wang et al., Proc. Natl. Acad. Sci. USA 91:8180, 1994; Wang et al, Gene Therapy 4:432, 1997); and chimeric proteins comprising transcriptional activators fused to estrogen (or other steroid) binding domains (Mattioni et al., Meth. Cell Biol. 43:335, 1994).
  • the ecdysone receptor system utilizes retinoid X receptor (RXR) to form heterodimers with the chimeric receptor, and responds to ecdysone, muristerone (an ecdysone analogue) or dexamethasone.
  • RXR retinoid X receptor
  • the pGL-VP system is responsive to mifepristone (RU486).
  • Chimeric receptors containing an estrogen binding domain respond to hydroxytamoxifen (an estrogen analogue). Spatial control of MDE expression is achieved by the use of transcriptional promoters that direct transcription selectively in joint tissues.
  • Joint-specific expression refers to expression that is greater in joints than in other cells; typically, the level of expression in non-joint tissues is less than 10% of the level of expression in joints. Preferably, expression in non-joint tissues is undetectable.
  • Useful promoter sequences that confer joint-specific expression on a sequence to which they are operably linked include without limitation sequences derived from the collagen type II promoter. It will be understood that a joint-specific promoter according to the invention may comprise one or more copies of particular sequences or sub-sequences, and these sequences may be in direct or inverted orientation relative to each other and relative to the sequence whose expression is regulated by the promoter.
  • Coordinated spatial and temporal control of MDE expression is preferably achieved by (i) placing expression of the repressor-activator fusion polypeptide or the transcriptional activator polypeptide under the control of a joint-specific promoter; (ii) placing the expression of the MDE or a derivative thereof under the control of a promoter responsive to the repressor-activator fusion polypeptide or the transcriptional activator polypeptide; and (iii) maintaining the transgenic animal during fetal development and early life under conditions in which MDE expression is repressed.
  • transgenic animals are maintained during fetal and early post-natal development so that MDE expression is repressed will depend on the particular transgenes being expressed.
  • a repressor-activator fusion polypeptide When a repressor-activator fusion polypeptide is used, repression is achieved by providing the animal with an agent that binds to the repressor-activator fusion protein and results in repression of transcription of the target MDE gene.
  • repression is achieved by providing tetracycline or a tetracycline analogue in the food or drinking water of the mother and, following birth, of the progeny.
  • Tetracycline or an analogue may also be provided using surgically implanted subcutaneous time-release pellets (Innovative Research of America, Inc., Sarasota FL). In this case, binding of tetracycline or a tetracycline analogue to the repressor-activator fusion protein prevents the fusion protein from binding to, and activating transcription of, the cognate promoter.
  • Tetracycline analogues are compounds closely related to tetracycline which bind to the tet repressor with a Ka of at least about 10 6 M "1 , preferably with an affinity of about lO ⁇ "1 or greater.
  • Useful tetracycline analogues include without limitation doxycycline, anhdryrotetracycline, chlortetracycline, epioxytetracycline, and the like.
  • the dosage used is one that will result in substantial repression of MMP expression.
  • tetracycline or a tetracycline analogue is administered in the animal's drinking water at a dosage of about 1 mg/ml. When it is desired that MMPs be expressed, the tetracycline or analogue thereof is withheld.
  • repression is achieved by withholding from the animal an agent required for activity of the transcriptional activator polypeptide.
  • the transcriptional activator is a modified ecdysone receptor
  • the animals are maintained in the absence of ecdysone or an ecdysone analogue throughout fetal and early post-natal development.
  • Ecdysone analogues are compounds closely related to ecdysone which bind to the modified ecdysone receptor with a Ka of at least about 10 6 M _1 .
  • Useful ecdysone analogues include without limitation muristerone A.
  • the animals are given, e.g., ecdysone or muristerone A via intraperitoneal injections at dosages of between about 10 mg and about 20 mg/animal.
  • ecdysone or muristerone A via intraperitoneal injections at dosages of between about 10 mg and about 20 mg/animal.
  • pGL-VP activation is achieved by providing mifepristone.
  • a transgenic animal is constructed whose somatic and germline cells contain in stably integrated form two recombinant genes: (i) a first recombinant gene comprising a sequence encoding MMP- 13*, wherein the sequence is operably linked to atetO7 promoter; and (ii) a second recombinant gene encoding a tTA protein operatively linked to a collagen type II promoter.
  • animals are maintained in the presence of tetracycline or a tetracycline analogue throughout fetal and early post-natal development to repress the gene. Afterwards, tetracycline or the tetracycline analogue is withdrawn, and MMP-13 enzymatic activity is selectively expressed in joint tissues.
  • the present invention provides animal model systems in which phenotypic changes characteristic of cartilage-degenerative diseases, such as, e.g., joint or disc disease, are reproducibly exhibited. These diseases include without limitation osteoarthritis, rheumatoid arthritis, chondrodysplasias, and degenerative intervertebral disc diseases.
  • the model systems of the invention exhibit one or more phenotypic indicators common to these diseases, which include without limitation loss of proteoglycan (as indicated by, e.g., loss of Safranin O staining) and cleavage of type II collagen in the affected tissues.
  • the systems encompass the transgenic animals described above, in which recombinant or heterologous MDEs, particularly MMPs, are expressed in cartilage at a predetermined time in the life of the transgenic animal.
  • the timing of the appearance of cartilage-degenerative indicators is determined by activating MDE expression and monitoring the effects on cartilage (see below).
  • one or more MDEs are expressed after birth, most preferably after the animal has reached adulthood.
  • transgenes are typically monitored by extracting mRNA from different tissues and subjecting the extracted mRNA to one or more of the following: (i) reverse transcriptase-polymerase chain reaction (RT-PCR), using primers homologous to the transgene; (ii) RNAase protection; and (iii) Northern blot analysis. Alternatively, in situ hybridization may used.
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • RNAase protection RNAase protection
  • Northern blot analysis may be used.
  • the physiological effects of MDE expression on articular cartilage are monitored in test animals by sacrificing the animals and subjecting paraffin-embedded decalcified cartilage to staining with (i) hematoxylin and eosin (using conventional techniques) followed by double staining with (ii) Safranin O and fast green (Peter et al., J. Exp. Pathol. 21: 19, 1990).
  • frozen sections may be obtained and stained with antibodies that are specific for cleavage fragments derived from type II collagen (Billinghurst et al., J. Clin. Invest. 99:1534, 1997).
  • MMP transgene(s) for at least about 7 days results in detectable loss of proteoglycan and changes in growth plate morphology (see, e.g., Example 5 below).
  • Animal models in which expression of MDEs, particularly MMPs, and most particularly an enzymatically active form of MMP-13, results in proteoglycan loss and/or cleavage of type II collagen are within the scope of the invention.
  • phenotypic indicators of cartilage-degenerative disease which can be monitored in transgenic animals produced according to the invention include without limitation gross observations of changes in joint function and histological evidence of (i) fibrillation and loss of articular cartilage and (ii) osteophyte formation.
  • Syndromes for which the transgenic animals of the invention provide useful models include without limitation any pathological condition that manifests a disturbance in the composition, morphology, and/or function of cartilage, including osteoarthritis; rheumatoid arthritis; degenerative intervertebral disc diseases; chondrodysplasias, including, e.g., Kniest dysplasia, achondrogenesis, and hypophosphatasia; and proteoglycan-mediated disorders, such as occur, e.g., in brachymorphic animals (Hall et al, Cartilage: Molecular Aspects, CRC Press, 1991, pp. 201-203).
  • the transgenic animals can be subjected to additional treatments to modulate the cartilage-degenerative indicators and/or to supplement the animals' disease phenotype with additional physiological effects such as, e.g., those associated with a particular disease.
  • the transgenic animals may be further treated with inflammatory mediators to augment collagen degradation and/or induce loss of proteoglycan (see, e.g., Example 6 below).
  • the timing and extent of MDE induction, with or without additional treatments can be adapted to replicate the symptomatology of a particular disease or syndrome.
  • the present invention encompasses methods for discovery and evaluation of drugs and therapies for their efficacy against degenerative diseases of cartilage, particularly degenerative joint diseases.
  • the transgenic animals of the invention are maintained under conditions in which expression of one or more MDEs results in one or more phenotypic indicators of cartilage- degenerative disease.
  • the potential of a composition to counteract cartilage-degenerative disease can be evaluated by administering a known dose of the composition to the animal in which the symptoms have developed; monitoring the phenotypic indicators for a predetermined time following administration of the composition; and comparing the extent of the phenotypic indicators in the animal to which the composition was administered relative to a control animal.
  • Control animals comprise age- and sex-matched transgenic animals that are maintained under an identical regimen (i.e., express the transgenes) but which do not receive the composition. Any statistically significant difference in the extent or nature of the phenotypic indicators indicates the potential of the composition to counteract cartilage-degenerative disease.
  • phenotypic indicators of cartilage-degenerative disease refer to proteoglycan loss, joint space narrowing, collagen degradation, and destruction of cartilage.
  • the potential of a composition to counteract degenerative diseases of cartilage is evaluated by administering to a transgenic animal a known dose of the composition before and/or simultaneous with the induction of MDE expression in the transgenic animal; monitoring phenotypic indicators of cartilage-degenerative disease for a predetermined time following administration of the composition and MDE induction; and comparing the extent of the phenotypic indicators and/or disease in the animal to which the composition was administered relative to a control animal that had not been exposed to the composition.
  • any statistically significant difference in the extent or nature of the phenotypic indicators and/or disease, or any statistically significant delay in appearance of the phentoypic indicators or disease indicates the potential of the composition to counteract cartilage-degenerative disease.
  • a further indication of the potential of a composition to counteract cartilage-degenerative disease is the ability of the composition to cause any reduction in the extent or duration of other treatments, including, e.g., the dosage and timing of administration of other therapeutic agents used to alleviate symptoms of the disease.
  • Compounds that may be tested for anti-cartilage-degenerative disease potential may be found in, for example, natural product libraries, fermentation libraries (encompassing plants and microorganisms), combinatorial libraries, compound files, synthetic compound libraries, and compounds resulting from directed rational drug design and synthesis.
  • synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT).
  • a rare chemical library is available from Aldrich Chemical Company, Inc. (Milwaukee, WI).
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from, for example, Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily producible.
  • natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., TibTech 14_:60, 1996).
  • Transgenic animals refers to animals into which one or more heterologous and/or recombinant genes have been introduced.
  • the transgenes may be from a different species, or from the same species as the transgenic animal but are not naturally found in the animal in the configuration and/or at the chromosomal locus conferred by the transgene.
  • Transgenes may comprise foreign DNA sequences, i.e., sequences not normally found in the genome of the host animal.
  • transgenes may comprise endogenous DNA sequences that have been rearranged or mutated in vitro in order to alter the normal in vivo pattern of expression of the gene, or to alter or eliminate the biological activity of an endogenous gene product encoded by the gene.
  • DNA fragments that are introduced into a pre-existing gene to, e.g., change patterns of expression or to provide additional means of regulating the expression of the gene
  • change patterns of expression or to provide additional means of regulating the expression of the gene
  • the transgenic non-human animals of the invention are produced by introducing transgenes into the germline of the non-human animal. Embryonal target cells at various developmental stages are used to introduce the transgenes of the invention.
  • Such methods include, but are not limited to, microinjection of zygotes, viral integration, and transformation of embryonic stem cells as described below.
  • Microinjection of zygotes is the preferred method for incorporating transgenes into animal genomes.
  • a zygote which is a fertilized ovum that has not undergone pronuclei fusion or subsequent cell division, is the preferred target cell for microinjection of transgenic D ⁇ A sequences.
  • the murine male pronucleus reaches a size of approximately 20 micrometers in diameter, a feature which allows for the reproducible injection of 1-2 picoliters of a solution containing transgenic D ⁇ A sequences.
  • transgenic allele demonstrates Mendelian inheritance, i.e., half of the offspring resulting from the cross of a transgenic animal with a non-transgenic animal will inherit the transgenic allele, in accordance with Mendel's rules of random assortment.
  • Viral integration can also be used to introduce the transgenes of the invention into an animal.
  • the developing embryos are cultured in vitro to the blastocyte developmental stage.
  • the blastomeres may be infected with appropriate retroviruses
  • Transgenes are introduced via viral vectors which are typically replication-defective but which remain competent for integration of viral-associated DNA sequences, including transgenic DNA sequences linked to such viral sequences, into the host animal's genome. Transfection is easily and efficiently obtained by culture of blastomeres on a monolayer of cells producing the transgene-containing viral vector. Alternatively, infection may be performed using cells at a later developmental stage, such as blastocoeles.
  • transgenic founder animals produced by viral integration will be mosaics for the transgenic allele; that is, the transgene is incorporated into only a subset of all the cells that form the transgenic founder animals.
  • multiple viral integration events may occur in a single founder animal, generating multiple transgenic alleles which will segregate in future generations of offspring.
  • Introduction of transgenes into germline cells by this method is possible but probably occurs at a low frequency.
  • offspring may be produced in which the transgenic allele is present in all of the animal's cells, i.e., in both somatic and germline cells.
  • Embryonal stem (ES) cells can also serve as target cells for introduction of the transgenes of the invention into animals.
  • ES cells are obtained from pre-implantation embryos that are cultured in vitro (Evans et al., Nature 292: 154, 1981).
  • ES cells that have been transformed with a transgene can be combined with an animal blastocyst, after which the ES cells colonize the embryo and contribute to the germline of the resulting animal (which is a chimera, i.e., composed of cells derived from two or more animals).
  • transgenes of the invention may be stably integrated into germ line cells and transmitted to offspring of the transgenic animal as Mendelian loci.
  • Other transgenic techniques result in mosaic transgenic animals, in which some cells carry the transgenes and other cells do not. In mosaic transgenic animals in which germ line cells do not carry the transgenes, transmission of the transgenes to offspring does not occur. Nevertheless, mosaic transgenic animals are capable of demonstrating phenotypes associated with the transgenes.
  • animals of the transgenic maintenance line are crossed with animals having a genetic background in which expression of the transgene results in symptoms of cartilage-degenerative disease.
  • Offspring that have inherited the transgenes of the invention are distinguished from littermates that have not inherited transgenes by analysis of genetic material from the offspring for the presence of nucleic acid sequences derived from the transgenes of the invention.
  • biological fluids that contain polypeptides uniquely encoded by the transgenes of the invention may be immunoassayed for the presence of the polypeptides.
  • a simpler and more reliable means of identifying transgenic offspring comprises obtaining a tissue sample from an extremity of an animal, such as, for example, a tail, and analyzing the sample for the presence of nucleic acid sequences corresponding to the DNA sequence of a unique portion or portions of the transgenes of the invention.
  • the presence of such nucleic acid sequences may be determined by, e.g., hybridization ("Southern") analysis with DNA sequences corresponding to unique portions of the transgene, analysis of the products of PCR reactions using DNA sequences in a sample as substrates, oligonucleotides derived from the transgene's DNA sequence, and the like.
  • the present invention encompasses isolated nucleic acids encoding MDEs, particularly MMPs, and enzymatically active fragments derived therefrom, as well as constitutively active MMP variants and enzymatically active fragments derived therefrom.
  • the invention also encompasses complements of the above nucleic acids; vectors comprising the nucleic acids; cells comprising the vectors; and isolated polypeptides encoded by the nucleic acids.
  • Nucleic acid or “polynucleotide” as used herein refers to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides. This includes single- and double-stranded molecules, such as, for example, DNA-DNA, DNA-RNA and
  • RNA-RNA hybrids as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases.
  • a "coding sequence” or a “protein-coding sequence” is a polynucleotide sequence capable of being transcribed into mRNA and/or capable of being translated into a polypeptide. The boundaries of the coding sequence are typically determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus.
  • a "complement" of a nucleic acid sequence as used herein refers to the
  • nucleic acid or polypeptide refers to a component that is removed from its original environment (for example, its natural environment if it is naturally occurring).
  • An isolated nucleic acid or polypeptide typically contains less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components with which it was originally associated.
  • a nucleic acid or polypeptide sequence that is "derived from” a designated sequence refers to a sequence that corresponds to a region of the designated sequence.
  • sequences that are homologous or complementary to the sequence as well as “sequence-conservative variants” and “function-conservative variants.”
  • sequence-conservative variants for polypeptide sequences, this encompasses “function- conservative variants.” Sequence-conservative variants are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position.
  • Function-conservative variants are those in which a given amino acid residue in a polypeptide has been changed without altering the overall conformation and function of the native polypeptide, including, but not limited to, replacement of an amino acid with one having similar physico-chemical properties (such as, for example, acidic, basic, hydrophobic, and the like). "Function-conservative" variants also include any polypeptides that have the ability to elicit antibodies specific to a designated polypeptide.
  • Nucleic acids comprising any of the sequences disclosed herein or subsequences thereof can be prepared by conventional methods.
  • DNA can be chemically synthesized using, e.g., the phosphoramidite solid support method of Matteucci et al, 1981, J. Am. Chem. Soc. 103:3185, the method of Yoo et al, 1989, J. Biol. Chem. 764:17078, or other well known methods. This can be performed by sequentially linking a series of oligonucleotide cassettes comprising pairs of synthetic oligonucleotides.
  • nucleotide sequences can encode polypeptides having the amino acid sequences defined herein or subsequences thereof.
  • the codons can be selected for optimal expression in prokaryotic or eukaryotic systems. Such degenerate variants are also encompassed by this invention.
  • the nucleic acids may also be modified by many means known in the art.
  • Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • Nucleic acids may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators.
  • PNAs are also encompassed by the term "nucleic acid”.
  • the nucleic acid may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage.
  • nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly.
  • exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
  • the polypeptides of the invention may be expressed by using many known vectors, such as pUC plasmids, pET plasmids (Novagen, Inc., Madison, WI), or pRSET or pREP plasmids (Invitrogen, San Diego, CA), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. The particular choice of vector/host is not critical to the practice of the invention.
  • Recombinant cloning vectors will often include one or more replication systems for cloning or expression; one or more markers for selection in the host, such as, for example, antibiotic resistance; and one or more expression cassettes.
  • the inserted coding sequences may be synthesized by standard methods, isolated from natural sources, prepared as hybrids, or the like. Ligation of the coding sequences to transcriptional regulatory elements and/or to other amino acid coding sequences may be achieved by known methods. Suitable host cells may be transform ed/transfected/infected as appropriate by any suitable method including electroporation, CaCl 2 mediated DNA uptake, fungal infection, microinjection, microprojectile, or other established methods.
  • Appropriate host cells include bacteria, archebacteria, fungi, yeast, plant, and animal cells, and especially mammalian cells.
  • mammalian cells Of particular interest areE. coli, S. aureus, B. subtilis, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Schizosaccharomyces pombi, SF9 cells, C129 cells, 293 cells, Neurospora, CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloid and lymphoid cell lines.
  • Preferred replication systems include M13, Col ⁇ l, SV40, baculovirus, lambda, adenovirus, cytomegalovirus, and the like.
  • a large number of transcription initiation and termination regulatory regions have been isolated and are effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner of manipulation, etc. are known in the art (Ausubel et al., Current Protocols in Molecular Biology, John Wiley, 1997). Under appropriate expression conditions, host cells can be used as a source of recombinantly produced peptides and polypeptides.
  • the MDEs of the present invention may be isolated from native or heterologous organisms or cells (including, but not limited to, bacteria, fungi, insect, plant, and mammalian cells) into which the protein-coding sequence has been introduced and expressed.
  • these polypeptides may be produced in cell-free protein synthesis systems, which may additionally be supplemented with microsomal membranes to achieve glycosylation and signal peptide processing of preprocollagenases.
  • the polypeptides may be chemically synthesized by commercially available automated procedures, including, without limitation, exclusive solid phase synthesis, partial solid phase methods, fragment condensation, or classical solution synthesis.
  • polypeptide purification is well-known in the art, including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, and countercurrent distribution.
  • the polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix.
  • antibodies produced against the protein or against peptides derived therefrom can be used as purification reagents. Other purification methods are possible.
  • a nucleic acid encoding an MMP is modified either by site-directed or random mutagenesis, or is used in a construction scheme as one segment of a fusion gene.
  • the procedure results in a modification either contained within the sequence encoding the pro region or near the pro region cleavage site; this includes deleting the pro region entirely.
  • sequences may be constructed that encode fusion proteins either between enzymatically active MMP domains and other polypeptides, or between different MMPs.
  • the modified nucleic acid is then used to program synthesis of a variant MMP, either in a cell-free system, in intact cells (including permeabilized cells), or in a transgenic animal.
  • a cell-free system or a cell culture system is used to express the MMP variant or derivative.
  • the extent of pro region cleavage is assessed by metabolic labelling and resolution of the MMP product by SDS-PAGE.
  • MMP enzymatic activity is measured using conventional assays, such as, by quantifying the cleavage of natural substrates or model peptides, as disclosed, e.g., in Weingarten et al, Biochem. 24:6730, 1985; Woessner et al., J.
  • MMP variants and derivatives including, e.g., function-conservative variants of MMP-13*, can be created routinely and assayed for MMP enzymatic activity.
  • Example 1 Construction of a Gene Encoding a Modified, Constitutively Active
  • MMP13* The sequence of this proMMP-13 variant, designated MMP13*, is shown in SEQ ID NO:4.
  • a cDNA fragment encoding proMMP was obtained by digesting plasmid pNot3A (Freije et al., J. Biol. Chem. 269:16766, 1994; GENBANK accession number X75308) with Xbal and Hindlll and purifying the resulting -1515 bp fragment. This fragment was subcloned into the Tet-resistant/ Amp-sensitive pAlter plasmid (Promega, Madison, WI) that had been digested with Xbal and Hindi ⁇ .
  • Site-directed mutagenesis was performed using the Altered Sites II in vitro Mutagenesis System (Promega, Madison, WI). Briefly, phagemid single-stranded DNA was purified from cultures containing the helper phage R408 (Promega). In addition to the Amp repair - Tet knock-out conversion oligos (Promega), an oligonucleotide having the sequence 5'-AAGCCAAGATGCGGGGTTGTCGATGTGGGTGAATACAAT-3', SEQ ID NO:8, was phosphorylated and annealed to the single-stranded DNA, followed by mutant strand synthesis. The reaction mixture was then used to transform the repair-minus E. coli strain ES1301 mutS, and the culture was grown in ampicillin selective media. Plasmid DNA was isolated from isolated clones and transformed into JM109 cells, which were then plated on LB plates containing 120 ⁇ g/ml ampicillin.
  • the above procedure resulted in a proline-to-valine substitution at amino acid 99.
  • the modified proMMP was designated MMP13* (SEQ ID NO:4).
  • cDNA encoding MMP13* cDNA was excised from the pAlter-MMP13* vector by digestion with EcoRl and Hindlll.
  • cDNAs encoding both mutant forms of MMP 13 and wild-type MMP- 13 were subcloned into a BS(SK-) vector (Stratagene) containing the CMV promoter (Xho I - Eco RI) and the SV40 splice poly (A)n (Xba I - Nco I).
  • Duplicate cultures of Hela cells (10 cm dishes) were transfected with 50 ⁇ g of these plasmids using the CaPO 4 precipitation method (Promega). Five hours later, cells were subjected to a 1 -minute glycerol shock using a solution containing an equal volume of 2 X HBS + 30 % glycerol. This procedure is described in the Profection Mammalian Transfection Systems technical manual (Promega).
  • the culture medium (D-MEM containing 10 % fetal bovine serum) was replaced with D-MEM containing no serum and 10 ⁇ M CGS-27023A (Ciba-Geigy), an MMP inhibitor. It is believed that, in the absence of an added MMP inhibitor, MMPs produced by the culture autodigest; thus, addition of an MMP inhibitor to the culture medium resulted in a detectable MMP 13 band.
  • MMPs are synthesized as precursors (i.e., procollagenases or zymogens) whose enzymatic activity is latent; proteolytic removal of the proregion after secretion produces the enzymatically active protein.
  • the need for proteolytic processing is circumvented by the use of a procollagenase variant that is enzymatically active even when uncleaved.
  • the constitutively active MMP 13 variant used as the transgene contains a proline to valine substitution in the sequence encoding the PRCGVPDV (SEQ ID NO:7) region, which is highly conserved among MMPs and important for controlling enzyme latency.
  • MMP13 The activity of the altered MMP 13 protein, was determined on a casein zymogram (data not shown).
  • Three MMP species are secreted from control HeLa cells.
  • Cleavage of the prodomain of MMPs during enzyme activation results in a loss of approximately 10 kDa.
  • the molecular weight of a second band is consistent with the processed form of gelatinase B.
  • Stromelysin-1 (MMP3) and stromelysin-2 (MMPIO) are both 54 kDa and one or both may represent the fourth band.
  • Pro-MMP13 is expected to migrate -60 kDa, which is observed in lane 1 containing uncleaved recombinant
  • MMP13 HeLa cells were transfected with constructs expressing both parental and mutant MMP13. Only the ⁇ 60-kDa pro-MMP13 form was detected from cells expressing both constructs, indicating that autoproteolysis of the proenzyme to the mature form is not likely to occur in an exogenous system. These results showed that the proline to valine substitution did not interfere with its native MMP 13 activity or substrate specificity.
  • This method provides a rapid screen for MMP 13 variants that retain MMP 13 enzymatic activity.
  • cDNA encoding MMP-13* was operably linked to a transcriptional regulatory sequence derived from the tet07 promoter as follows:
  • the BS(SK-) vector (Stratagene) was digested with Kpnl and Notl.
  • a synthetic duplex oligonucleotide having the following sequence was digested with Kpnl and Not I and ligated to the vector:
  • the BS(SK-) vector as modified above was digested with Xbal and Ncol.
  • the resulting vector was linearized by digestion with Xhol and EcoRI and ligated to a 460 bp XhoI-EcoRI fragment containing the tetO7 promoter region from pUHD 10-3 (Gossen et al., Proc. Natl. Acad. Sci. USA 89:5547, 1992). This vector was then digested with Spel, blunt-ended with Klenow polymerase, and digested with EcoRI. pAlter-MMP13* was digested with Hindlll, blunt-ended with Klenow polymerase, and digested with EcoRI to obtain an MMP13*-encoding fragment. The MMP13* EcoRI fragment was cloned into the EcoRI digested vector obtained above.
  • the 2792 bp transgene, SEQ ID NO: 11 ( Figure 2B), was excised by digestion with Xhol and Notl and purified using CsCl gradient centrifugation prior to microinjection into fertilized eggs.
  • a gene encoding a tTA repressor-activator fusion protein was operably linked to a joint-specific (type II collagen) promoter.
  • the modified BS(SK-) vector containing the SV40 splice site and polyadenylation signal as described in Example 1 above was digested with Ndel and Sma I and ligated to a 1897 bp fragment containing the collagen II promoter and enhancer. This fragment was obtained by digesting plasmid PBS ⁇ H1 with Hindi ⁇ , after which it was blunt-ended with Klenow and digested with Ndel.
  • the plasmid was then digested with EcoRI and BamHI and ligated to a 1025 bp fragment encoding the tetracycline/VP16 repressor-activator fusion protein that had been excised from the pUHG15-l plasmid (Gossen et al, supra) using EcoRI and BamHI.
  • the plasmid was linearized by digestion with Bgi ⁇ , dephosphorylated using calf intestinal phosphatase, and ligated to a 1554 BamHI enhancer fragment obtained from plasmid PBS ⁇ H1.
  • Type II collagen promoter- ⁇ -galactosidase gene A reporter gene, suitable for assessing the tissue-specific expression conferred by the type II collagen promoter, was operably linked to the type II collagen promoter.
  • a 4179 bp BamHI-Bgi ⁇ fragment containing the ⁇ -galactosidase gene fused to the ⁇ -globin splice sequence and polyadenylation signal was excised from plasmid pUGH16-3 (Gossen et al, supra) and cloned into the BamHI site of unmodified BS(SK-) (Stratagene).
  • This plasmid was digested with EcoRI and Hindlll and ligated to a 655 bp Hind III-Eco RI fragment containing the type II collagen promoter sequence, which was excised from the plasmid described above. The plasmid was then digested with EcoRI and ligated to a 2807 bp Eco RI fragment which had been excised from the type II collagen promoter plasmid described above. Restriction mapping was used to verify the orientation of each insert.
  • the 7664 bp transgene, SEQ ID NO: 13 ( Figure 3 A), was excised by digestion with Hindlll and Notl, gel purified, purified by CsCl gradient centrifugation, dialyzed against microinjection buffer (5 mM Tris pH 7.4, 0.1 mM EDTA pH 8.0), and used for microinjection into mouse embryos.
  • Results Figure 2A and 2B shows a schematic diagram of the synthetic genes generated to achieve regulated expression of MMP 13 in chondrocytes. Inducible expression of the transgene was accomplished using the tetracycline regulatable gene expression system (Gossen et al., supra; Furth et al., Proc. Natl. Acad. Sci. USA,
  • the first construct shown in Figure 2A places expression of the tetracycline-controlled VP16 transactivator fusion protein under the control of the type II collagen gene promoter. This construct directs expression of the VP16 fusion protein to chondrocytes.
  • the second construct shown in Figure 2B places expression a cDNA encoding a modified version of MMP 13 protein (MMP 13*) under the direction of the
  • the VP16 fusion protein In the presence of doxycycline, a tetracycline analog provided in the drinking water, the VP16 fusion protein does not bind to the Tet07 promoter of the synthetic MMP13* gene and the gene is silent. Upon removal of doxycycline, the transactivator stimulates transcription of the human MMP13* cDNA and the production of the modified protein product.
  • FIG. 2A is a schematic illustration of a transgene used to assess tissue-specific regulation conferred by type II collagen promoter.
  • the nucleic acid construct comprises: (i) sequences derived from a rate type II collagen promoter; (ii) sequences encoding bacterial ⁇ -galactosidase (Lac Z); and (iii) sequences comprising an ⁇ -globin splice and polyadenylation signal.
  • telomeres were first identified by PCR as follows.
  • the tTA-encoding transgene was identified using a primer corresponding to the tTA sequence (5'- CGAGGGCCTGCTCGATCTCC-3', SEQ ID NO: 14) and a primer corresponding to a 3 ' untranslated sequence (5'-GGCATTCCACCACTGCTCCC-3', SEQ ID NO: 15).
  • the resulting PCR product was 584 bp in size.
  • the MMP13* -encoding transgene was identified using primers corresponding to sequences encoding MMP13* (5'- GAGCACCCTTCTCATGACCTC-3', SEQ ID NO:16) and the 3' untranslated region, respectively.
  • the resulting PCR product was 731 bp in size.
  • LacZ-encoding transgene was identified using primers corresponding to the nuclear localization signal of the ⁇ -galactosidase gene (5'-
  • Transgenic lines were generated by mating founder animals to FVB/N wild-type mice and the subsequent generations were identified by PCR. All mice were administered doxycycline (Sigma Chemical Co., St. Louis MO) prepared as a 100 mg/ml stock solution in 50% ethanol, and diluted to a final concentration of 1.0 mg/ml in acidic drinking water, which was changed on a daily basis (Schultze et al., Nature Biotech. 14:499, 1996).
  • doxycycline Sigma Chemical Co., St. Louis MO
  • PCR analysis of the cDNA was done using the following primers sets (5' to 3' sequences): tet activator: CGCCCAGAAGCTAGGTGTAGAG (SEQ ID NO: 19) and CGGCCATATCCAGAGCGCCG (SEQ ID NO:20); MMP13*: GCCCTCTGGCCTGCTGGCTCATG (SEQ ID NO:21) and
  • each PCR reaction also contained the following c-fos primer set: 5'-AGGAGGGAGCTGACAGATACACTCC-3' (SEQ ID NO:23) and 5'-AGGCCACAGACATCTCCTCTGG-3' (SEQ ID NO:24).
  • PCR analysis was performed on the cDNA using Taq-gold (Perkin Elmer) for 10 minutes at 95 °C, followed by 35 cycles of 60 seconds at 96°C, 90 seconds at 67°C, and 60 seconds at 72°C.
  • fibroblast were subsequently transfected via the calciumphosphate precipitation method with the desired expression plasmids. Forty-eight hours after transfection, total RNA was prepared from the transfected cells using the Trizol method (Gibco/BRL). First strand cDNA synthesis was prepared using the Superscript preamplification system (Gibco/BRL). MMP13* expression was identified using primers specific for human MMP 13 (GCCCTCTGGCCTGCTGGCTCATG) (SEQ ID NO:21) and
  • Tetracycline and their analogues are known inhibitors of MMP activity.
  • the IC 50 59.1 ⁇ M
  • the serum levels measured 2.64 ⁇ M using a zone of inhibition assay.
  • Figure 3B is a photographic illustration of whole mount staining for ⁇ -galactosidase activity of embryonic day 16 transgenic mouse embryos expressing the transgene illustrated in Figure 3 A.
  • Blue staining (arrows) is evident in the joints throughout the body of the transgenic animal, while no staining is observed in the non-transgenic, wild-type littermate.
  • joints including the ankles, knees, hips, phalanges, wrists, elbows, shoulders, and vertebrae showed expression of the transgene.
  • cartilage that has not ossified to the bone at this stage of development i.e., some of the facial, skull, and rib bones, also stained blue.
  • Figure 3C shows an enlargement of the elbow and paw.
  • transgene copy number ranged from 1-32 and 1-20 for the tet activator and MMP13*, respectively. Specifically, line 6 contained -8 copies of the tet activator and -3 copies of the MMP13* transgene.
  • TA and MMP13* transgenes were initially evaluated in the hind-knee joints of four-month old mice (line 6). Amplification of the c-fos endogenous cDNA was used as a control to verify the efficacy of each reaction.
  • Figure 4 shows amplification of an 890 bp fragment resulting from a TA-specific primer set. RT-PCR showed the TA transgene to be expressed in transgenic mice, both on and off DOX, but was not expressed in the non-transgenic controls (lanes 4-5). Constitutive expression of the TA is expected since it is driven by a constitutively active promoter.
  • Figure 4B shows amplification of a 645 bp fragment resulting from an
  • MMP13* specific primer set is specific for human MMP13 and does not react with its endogenous mouse homologue, collagenase-1.
  • RT-PCR showed that MMP13* was not expressed in the non-transgenic controls (lanes 4-5).
  • Lanes 6-7 show that there is expression of the MMP13* transgene in mice maintained on DOX. Removal of DOX from the drinking water induces a significant amount of expression (lanes 8-9). We estimate a 5-10 fold induction.
  • PCR fragments were transferred to a nylon membrane and hybridized to a TA or MMP13* specific probe to verify the identity of the PCR product (data not shown).
  • Fibroblasts from several transgenic lines were capable of expressing MMP13*, as evidenced by the appearance of a PCR product of the predicted size.
  • No MMP13* RT-PCR band was detected from cells transfected with vehicle alone.
  • mice were withdrawn from doxycycline for increasing times, after which they were sacrificed. Paraffin-embedded formaldehyde-fixed sections of decalcified cartilage were sectioned and stained with (i) hematoxylin and eosin (H&E) and (ii) Safranin O followed by fast green (American Histo Labs, Gaithersburg MD). Staining techniques in articular cartilage have been described (Peter et al., J. Exp. Pathol. 21; 19, 1990).
  • mice from line 6 were maintained or removed from DOX for 114 days, and their joints were sectioned and stained with H&E. When compared with an age matched littermate, control the transgenic removed from DOX developed a pathology reminiscent of osteoarthritis.
  • FIG. 7A Shown in Figure 7A, the control animal showed no lesions or other osteoarthritis pathologies, whereas the transgenic animal shows the formation of lesions in its articular cartilage (Figure 7B). More specifically, the H&E sections show considerable loss of cartilage, focal erosions, erosions that extend into the bone, and an inflamed synovium. Within the synovium, there is evidence of fibroid necrosis, metaplasia, and synovial cell hyperplasia. In addition to these symptoms of osteoarthritis, some changes observed are more characteristic of rheumatoid arthritis. These changes include angiogenesis as seen by an infiltration of red blood cells, monocytes, and macrophages. Figures 7C and 7D show the synovium at a higher magnification.
  • Type II collagen, as well as type I collagen are known to be substrates for proteolytic cleavage by MMP 13. Therefore, we predicted that unregulated expression of MMP13 during embryogenesis would be lethal.
  • Four transgenic lines containing both transgenes were established. In the two lines that expressed hMMP13*, one expressed at very low levels, whereas the other line expressed significant amounts of hMMP13*.
  • the line that expressed only marginal amounts of transgene displayed histological evidence of proteoglycan loss. However, after six months off DOX no lesions were observed. Line 6, which expressed significantly greater amounts of hMMP13*, showed osteoarthritis pathologies including lesion formation, cartilage degradation, and an inflamed synovium after four months off DOX.
  • hMMP13* in line 6 can be controlled, i.e., turned on and off by the investigator. This result provides additional evidence that the phenotype is due to expression of hMMP13* and not the result of the integration site in the chromatin.
  • the data presented in this paper provides direct evidence that MMP 13 is a critical player in the development of osteoarthritis.
  • the transgenics described in this paper provide an animal model to test the efficacy of therapeutics.
  • Compounds that modulate the activity of MMP 13 or inhibit progression of osteoarthritis can be monitored by determining lesion formation and other OA pathologies at various times during the progression of the disease.
  • the ability to turn on and off hMMP13* expression, thus controlling production/timing of lesion formation will be advantageous to determining the compound efficacy.
  • This OA-like transgenic model can also be used to answer a growing list of biological questions.
  • MMPl 3 interstitial collagenase (MMPl) and neutrophil collagenase (MMP8) have been shown to cleave type II collagen.
  • MMPl interstitial collagenase
  • MMP8 neutrophil collagenase
  • mice are treated to induce expression of the transgenes at 4-12 weeks of age. Two to six weeks after induction, the mice are injected intraperitonealy with an inflammatory agent, including without limitation, lipopolysaccharide (10-100 ⁇ g), zymosan (1-10 mg), the superantigen Staphylococcal
  • an inflammatory agent including without limitation, lipopolysaccharide (10-100 ⁇ g), zymosan (1-10 mg), the superantigen Staphylococcal
  • Enterotoxin B (1-100 ⁇ g), or TGF- ⁇ (1-10 ⁇ g).
  • the animals are injected intraarticularly with an inflammatory or chondrocyte function-modulating agent, including without limitation, lipopolysaccharide (1-100 ng), zymosan (50-250 ⁇ g), papain (10-100 ⁇ g), TGF- ⁇ (0.01-1 ⁇ g), Bone Morphogenic Protein -2 (2-1000 ng), IL-1 (1-100 ng), TGF- ⁇ (10-200 ng), IGF (0.01-1 ⁇ g), or FGF (0.01-1 ⁇ g).
  • Age- and sex-matched transgenic mice maintained under a regimen in which the transgenes are not expressed receive the same treatment and serve as controls.
  • the agents will induce an acute inflammatory response and/or transient loss of proteoglycan with a duration of less than one week.
  • the acute inflammatory response and/or transient cartilage changes will upregulate gene expression in the chondrocytes, enhancing the expression of the transgene and increasing the levels of MMP-13 produced.

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Abstract

L'invention concerne des systèmes modèles animaux de lutte contre une maladie dégénérative du cartilage. Ces systèmes modèles comprennent des animaux transgéniques pouvant exprimer des enzymes recombinantes d'altération matricielle (MDE), en particulier des métalloprotéases matricielles (MMP), par régulation temporelle et spatiale. L'invention concerne également des procédés de production d'indicateurs phénotypiques d'une maladie dégénérative du cartilage chez un mammifère, et des procédés pour déterminer le pouvoir d'une composition de lutter contre une maladie dégénérative du cartilage. L'invention concerne enfin des acides nucléiques isolés codant des polypeptides proMMP présentant une activité enzymatique constitutive et des polypeptides proMMP isolés.
PCT/US1998/027056 1997-12-19 1998-12-18 Modele animal transgenique de lutte contre des maladies degeneratives du cartilage WO1999031969A2 (fr)

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IL13662698A IL136626A0 (en) 1997-12-19 1998-12-18 Transgenic animal model for degenerative diseases of cartilage
CA002314357A CA2314357A1 (fr) 1997-12-19 1998-12-18 Modele animal transgenique de lutte contre des maladies degeneratives du cartilage
JP2000524983A JP2002533055A (ja) 1997-12-19 1998-12-18 軟骨の変性疾患のためのトランスジェニック動物モデル
AU19316/99A AU1931699A (en) 1997-12-19 1998-12-18 Transgenic animal model for degenerative diseases of cartilage
KR1020007006731A KR100558288B1 (ko) 1997-12-19 1998-12-18 연골의 퇴행성 질환용 트랜스제닉 동물 모델
EP98964125A EP1040196A2 (fr) 1997-12-19 1998-12-18 Modele animal transgenique de lutte contre des maladies degeneratives du cartilage
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US7005295B1 (en) 1997-04-16 2006-02-28 Wyeth β-amyloid peptide-binding proteins and polynucleotides encoding the same
US7071164B2 (en) 2001-08-16 2006-07-04 Kimberly-Clark Worldwide, Inc. Anti-cancer and wound healing compounds
US7101973B2 (en) 1997-04-16 2006-09-05 Wyeth β-amyloid peptide-binding proteins and polynucleotides encoding the same
EP1399537A4 (fr) * 2001-04-03 2006-09-06 Curagen Corp Polypeptides therapeutiques, acides nucleiques les codant, et procedes d'utilisation
US7148194B2 (en) 2002-12-30 2006-12-12 Kimberly-Clark Worldwide, Inc. Method to increase fibronectin
US7186693B2 (en) 2001-08-16 2007-03-06 Kimberly - Clark Worldwide, Inc. Metalloproteinase inhibitors for wound healing
US7189700B2 (en) 2003-06-20 2007-03-13 Kimberly-Clark Worldwide, Inc. Anti-chrondrosarcoma compounds
EP1469717A4 (fr) * 2001-12-20 2007-08-22 Univ California Modele de souris triplement transgenique de la maladie d'alzheimer
WO2007124751A3 (fr) * 2006-05-01 2008-03-06 Univ Aarhus Modèle animal et méthode d'obtention d'un tel modèle
US8137671B2 (en) 2009-05-05 2012-03-20 Genentech, Inc. Anti-IL-17F antibodies
US9650437B2 (en) 2008-05-05 2017-05-16 Novimmune S.A. Nucleic acid encoding and method of producing anti-IL-17A/IL-17F cross-reactive antibodies
EP3081081A4 (fr) * 2013-12-12 2017-05-17 Mie University Mammifère transgénique non humain exprimant la protéine humaine mmp2
WO2020196990A1 (fr) * 2019-03-25 2020-10-01 박동휘 Modèle d'ostéoarthrite in vitro et procédé de criblage d'agent de traitement de l'ostéoarthrite l'utilisant
WO2025176195A1 (fr) * 2024-02-23 2025-08-28 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Animal non humain génétiquement modifié porteur d'une mmp-7 humaine ou chimérique

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US7005295B1 (en) 1997-04-16 2006-02-28 Wyeth β-amyloid peptide-binding proteins and polynucleotides encoding the same
US7101973B2 (en) 1997-04-16 2006-09-05 Wyeth β-amyloid peptide-binding proteins and polynucleotides encoding the same
EP1399537A4 (fr) * 2001-04-03 2006-09-06 Curagen Corp Polypeptides therapeutiques, acides nucleiques les codant, et procedes d'utilisation
US7196162B2 (en) 2001-08-16 2007-03-27 Kimberly-Clark Worldwide, Inc. Anti-aging and wound healing compounds
US6906036B2 (en) 2001-08-16 2005-06-14 Kimberly-Clark Worldwide, Inc. Anti-aging and wound healing compounds
US7071164B2 (en) 2001-08-16 2006-07-04 Kimberly-Clark Worldwide, Inc. Anti-cancer and wound healing compounds
EP1513542A4 (fr) * 2001-08-16 2009-07-15 Kimberly Clark Co Composes anticancereux et de cicatrisation de plaies
US7186693B2 (en) 2001-08-16 2007-03-06 Kimberly - Clark Worldwide, Inc. Metalloproteinase inhibitors for wound healing
WO2003016520A1 (fr) * 2001-08-16 2003-02-27 Kimberly-Clark Worldwide, Inc. Composes anti-age et de cicatrisation de plaies
EP1469717A4 (fr) * 2001-12-20 2007-08-22 Univ California Modele de souris triplement transgenique de la maladie d'alzheimer
US7148194B2 (en) 2002-12-30 2006-12-12 Kimberly-Clark Worldwide, Inc. Method to increase fibronectin
US7795225B2 (en) 2003-06-20 2010-09-14 Kimberly-Clark Worldwide, Inc. Anti-chrondrosarcoma compounds
US7189700B2 (en) 2003-06-20 2007-03-13 Kimberly-Clark Worldwide, Inc. Anti-chrondrosarcoma compounds
US8173861B2 (en) 2006-05-01 2012-05-08 Aarhus Universitet Transgenic pig model for a hereditary neurodegenerative autosomal dominant disease
EP2377941A1 (fr) * 2006-05-01 2011-10-19 Aarhus Universitet Modèle animal et méthode d'obtention d'un tel modèle
WO2007124751A3 (fr) * 2006-05-01 2008-03-06 Univ Aarhus Modèle animal et méthode d'obtention d'un tel modèle
US9650437B2 (en) 2008-05-05 2017-05-16 Novimmune S.A. Nucleic acid encoding and method of producing anti-IL-17A/IL-17F cross-reactive antibodies
US8137671B2 (en) 2009-05-05 2012-03-20 Genentech, Inc. Anti-IL-17F antibodies
US8609093B2 (en) 2009-05-05 2013-12-17 Novimmune S. A. Methods of treatment using anti-IL-17F antibodies
US9475873B2 (en) 2009-05-05 2016-10-25 Novimmune Sa Nucleic acids encoding anti-IL-17F antibodies and methods of use thereof
EP3081081A4 (fr) * 2013-12-12 2017-05-17 Mie University Mammifère transgénique non humain exprimant la protéine humaine mmp2
WO2020196990A1 (fr) * 2019-03-25 2020-10-01 박동휘 Modèle d'ostéoarthrite in vitro et procédé de criblage d'agent de traitement de l'ostéoarthrite l'utilisant
WO2025176195A1 (fr) * 2024-02-23 2025-08-28 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Animal non humain génétiquement modifié porteur d'une mmp-7 humaine ou chimérique

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