Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1997-12-19
2003-09-02
Wilson, Michael C. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S014000, C800S003000, C800S021000
Reexamination Certificate
active
06613958
ABSTRACT:
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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).
One of the primary targets affected by these diseases is type II collagen, the major structural collagen found in articular cartilage. There is a balance between the production of type II collagen and 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.
Among the enzymes that degrade extracellular matrix components are matrix metalloproteinases (MMPs), a family of zinc-dependent enzymes, and aggrecanase (Table 1).
TABLE 1
Matrix-Degrading Enzymes
SUBSTRATES
Collagen
Gelatin
Proteoglycan
Fibronectin
Laminin
Elastin
Other
I. Metalloproteinases
Collagenases
MMP-1
I, II, III,
✓
(intestinal collagenase)
VII, X
MMP-8
I, II, III
(neutrophil collagenase)
MMP-13
I, II, III
✓
(collagenase 3)
Gelatinases
MMP-2
IV, V,
✓
✓
✓
✓
(gelatinase A)
VII, XI
MMP-9
IV, V
✓
✓
(gelatinase B)
Stromelysins
MMP-3
✓
✓
✓
✓
activates
(stromelysin 1)
MMP zymogens
MMP-7
IV
✓
✓
✓
✓
✓
(matrilysin)
MMP-10
IV, V,
✓
✓
✓
activates
(stromelysin 2)
IX
MMP zymogens
MMP-11
IV
✓
✓
activates serpins
(stromelysin 3)
Other
MMP-12
✓
(metalloelastase)
MMP-14
✓
proMMP-2,
proMMP-13
MMP-15
MMP-16
proMMP-2
MMP-17
II. 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. For example, 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. In some cases, MMP-13 and its cleavage products are found at higher levels than MMP-1. Billinghurst et al.,
J. Clin. Inves.
99:1534, 1997. Thus, 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). In guinea pigs, spontaneous osteoarthritis has a long course of development (six months or more), and only certain sublines of STR/ORT mice consistently develop degenerative joint disease. Thus, the duration and/or variability of these models renders them less applicable to drug discovery studies.
Other 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. Van 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. However, 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. D'Armiento et al.,
Cell
71:955, 1992, disclose transgenic mice that express human interstitial collagenase in the lung. Liu et al.,
J. Cell Biol.
130:227, 1995, disclose transgenic animals that overexpress mutated type II collagen, resulting in connective tissue defects but not osteoarthritis. None of these transgenic animal systems provides a useful animal model for osteoarthritis. Khokha et al.,
Cancer and Metastasis Rev.
14:97, 1995; Shapiro,
Matrix Biol.
15:527, 1997.
Thus, there is a need in the art for animal model systems that mimic human degenerative joint diseases such as, e.g., osteoarthritis, rheumatoid arthritis, and chondrodysplasias. 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. In particular, such 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.
SUMMARY OF THE INVENTION
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. 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. In preferred embodiments, 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.
In one aspect, 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: 1. Preferably, 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 depic
Killar Loran Marie
Neuhold Lisa Ann
Darby & Darby
Wilson Michael C.
Wyeth
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