Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-03-17
2001-05-08
Yucel, Remy (Department: 1635)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S091100, C435S091500, C435S325000, C435S456000, C435S458000, C536S023100, C536S024500, C536S025300, C514S04400A
Reexamination Certificate
active
06228648
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of ADAM10. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding ADAM10. Such oligonucleotides have been shown to modulate the expression of ADAM10.
BACKGROUND OF THE INVENTION
The ADAM (A Disintegrin And Metalloproteinase) family of mammalian zinc metalloproteinases is a recently characterized group of membrane bound proteins which have been implicated in cell adhesion and extracellular matrix proteolysis. These proteins have also been referred to as MDCs (metalloproteinase/disintegrin/cysteine-rich), cellular disintegrins, disintegrin-metalloproteinases and metalloproteinase-disintegrins. All ADAM family members display a common domain organization and are unique among cell surface proteins in possessing both a potential adhesion domain as well as a potential protease domain. Since cell surface proteolysis plays an important role in development and pathology and since so few cell-surface membrane-anchored proteases have been described, ADAMs are believed to play important roles in development and disease (Stone, et al.,
J. Protein Chem.,
1999, 18, 447-65).
ADAMs are expressed in a wide range of animal species, tissues and cell types and have been implicated in sperm-egg fusion, spermatogenesis, neutrophil infiltration, platelet aggregation, neurogenesis and cachexia. In addition, ADAMs have been shown to be involved in proteolysis of membrane anchored cytokines, growth factors and their receptors (Wolfsberg et al.,
J. Cell. Biol.,
1995, 131, 275-8; Yamamoto et al.,
Immunol. Today,
1999, 20, 278-84).
ADAM10 (also known as MADM, HuAD10 and kuzbanian) was originally isolated from bovine brain and shown to act as a metalloprotease involved in the degradation of myelin basic protein (Howard et al.,
Biochem. J.,
1996, 317, 45-50). This same enzyme was also shown to act as a type IV collagenase in the bovine kidney where it was shown to cleave a human placental basement membrane collagen preparation (Millichip et al.,
Biochem. Biophys. Res. Commun.,
1998, 245, 594-8). In human cells, ADAM10 was first identified in THP-1 membrane extracts. Here it was shown to cleave a peptide substrate of tumor necrosis factor (TNF) alpha (Rosendahl et al.,
J. Biol. Chem.,
1997, 272, 24588-93). Subsequently, it has been shown to act as a convertase with the ability to cleave both a recombinant pro-TNF-alpha and a synthetic peptide to the mature TNF-alpha protein (Lunn et al.,
FEBS Lett.,
1997, 400, 333-5).
In the skeletal system, ADAM10 is expressed in specific regions of articular cartilage and metaphyseal bone of the neonatal rat tibia. In the bone and cartilage, it is expressed as two different transcripts of 4.5 kb and 7.5 kb by periosteal cells, osteoblasts and osteocytes at areas of active bone formation (McKie et al.,
Biochem. Biophys. Res. Commun.,
1997, 230, 335-9; Dallas et al.,
Bone,
1999, 25, 9-15). These two isoforms are believed to be generated by the mechanisms of alternative splicing and/or alternative polyadenylation. In primary human osteoblasts and osteoblast cell lines, ADAM10 is localized in the trans-Golgi network and on the plasma membrane (Dallas et al.,
Bone,
1999, 25, 9-15).
More recently it has been demonstrated that the levels of ADAM10 mRNA are elevated in osteoarthritis tissues. These levels appear to be related to the degree of cartilage damage and/or degradation which suggests a role for ADAM10 in cartilage matrix destruction associated with osteoarthritis (Chubinskaya et al.,
J. Histochem. Cytochem.,
1998, 46, 723-9).
ADAM10 has also been identified as an autoantigen in a patient with pulmonary fibrosis associated with dermatomyositis (Fujita et al.,
Ann. Rheum. Dis.,
1999, 58, 770-2). Pulmonary fibrosis is an inflammatory disease of the lung.
In studies of cell lines from hematologic malignancies such as leukemia, erythroleukemia, lymphoma, and myeloma, ADAM10 expression was shown to be significantly elevated (Wu et al.,
Biochem. Biophys. Res. Commun.,
1997, 235, 437-42). Here again, two transcripts were observed by Northern blot analysis.
The pharmacological modulation of ADAM10 activity and/or expression is therefore believed to be an appropriate point of therapeutic intervention in pathological conditions such as connective tissue disorders, inflammation and hematologic malignancies.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of ADAM10 and to date, strategies aimed at investigating ADAM10 function have involved the use of antibodies to localize ADAM10 (Millichip et al.,
Biochem. Biophys. Res. Commun.,
1998, 245, 594-8). These antibodies were not shown to inhibit ADAM10 function and/or activity. Consequently, there remains a long felt need for agents capable of effectively inhibiting ADAM10 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of ADAM10 expression.
The present invention provides compositions and methods for modulating ADAM10 expression, including modulation of the alternatively spliced form of ADAM10.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding ADAM10, and which modulate the expression of ADAM10. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of ADAM10 in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of ADAM10 by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention employs oligomeric antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding ADAM10, ultimately modulating the amount of ADAM10 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding ADAM10. As used herein, the terms “target nucleic acid” and “nucleic acid encoding ADAM10” encompass DNA encoding ADAM10, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of ADAM10. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.
It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The
Condon Thomas P.
Flournoy Shin Cheng
ISIS Pharmaceuticals Inc.
Licata & Tyrrell P.C.
Yucel Remy
Zara Jane
LandOfFree
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