Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-10-27
2002-08-20
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S375000, C424S009100, C424S009200
Reexamination Certificate
active
06436629
ABSTRACT:
FIELD OF THE INVENTION
The field of the invention is modulating angiogenesis by targeting a protein known as Kuz.
BACKGROUND OF THE INVENTION
Genes of the ADAM family encode transmembrane proteins containing both metalloprotease and disintegrin domains (reviewed in Black and White, 1998 Curr.Opin.Cell Biol. 10, 654-659; Wolfsberg and White, 1996 Dev.Biol. 180, 389-401), and are involved in diverse biological processes in mammals such as fertilization (Cho et al., 1998 Science 281, 1857-1859), myoblast fusion (Yagami-Hiromasa et al., 1995 Nature 377, 652-656) and ectodomain shedding (Moss et al., 1997 Nature 385, 733-736; Black et al., 1997 Nature 385, 729-733; Peschon et al., 1998 Science 282, 1281-1284). The Drosophila kuzbanian (kuz) gene represents the first ADAM family member identified in invertebrates (Rooke et al., 1996 Science 273, 1227-1231). Previous genetic studies showed that kuz is required for lateral inhibition and axonal outgrowth during Drosophila neural development (Rooke et al., 1996; Fambrough et al., 1996 PNAS.USA 93, 13233-13238.; Pan and Rubin, 1997 Cell 90, 271-280; Sotillos et al., 1997 Development 124, 4769-4779). Specifically, during the lateral inhibition process, kuz acts upstream of Notch (Pan and Rubin, 1997; Sotillos et al., 1997), which encodes the transmembrane receptor for the lateral inhibition signal encoded by the Delta gene. More recently, a homolog of kuz was identified in
C. elegans
(SUP-17) that modulates the activity of a
C. elegans
homolog of Notch in a similar manner (Wen et al., 1997 Development 124, 4759-4767).
Vertebrate homologs of kuz have been isolated in Xenopus, bovine, mouse, rat and human. The bovine homolog of KUZ (also called MADM or ADAM 10) was initially isolated serendipitously based on its in vitro proteolytic activity on myelin basic protein, a cytolasmic protein that is unlikely the physiological substrate for the bovine KUZ protease (Howard et al., 1996 Biochem.J. 317, 45-50). In a recent study, we showed that expression of a dominant negative form of the murine kuz homolog (mkuz) in Xenopus leads to the generation of extra neurons, suggesting an evolutionarily conserved role for mkuz in regulating Notch signaling in vertebrate neurogenesis (Pan and Rubin, 1997). We have now generated mkuz-deficient mice using gene targeting in embryonic stem (ES) cells. We show that mkuz is essential for embryonic development. mkuz mutant mice die around embryonic day (E) 9.5, with severe defects in the nervous system, the paraxial mesoderm and the yolk sac vasculature. In the nervous system, mkuz mutant embryos show ectopic neuronal differentiation. In the paraxial mesoderm, mkuz mutant embryos show delayed and uncoordinated segmentation of the somites. These phenotypes are similar to those of mice lacking Notch-1 or components of the Notch pathway such as RBP-Jk (Conlon et al, 1995, Development 121, 1533-1545; Oka et al., 1995), indicating a conserved role for mkuz in modulating Notch signaling in mouse development. Furthermore, we detect no visible defect in Notch processing in our knockout animals. Besides the neurogenesis and somitogenesis defect, mkuz mutant mice also show severe defects in the yolk sac vasculature, with an enlarged and disordered capillary plexus and the absence of large vitelline vessels. Since such phenotype has not been observed in mice lacking Notch-l or RBP-Jk (Swiatek et al., 1994 Genes Dev 15, 707-719; Conlon et al, 1995; Oka et al., 1995 Development 121, 3291-3301), we determine that this phenotype reveals a novel function of mkuz that is distinct from its role in modulating Notch signaling. Taken together, our studies reveal the essential role for an ADAM family disintegrin metalloprotease in mammalian neurogenesis, somitogenesis and angiogenesis.
SUMMARY OF THE INVENTION
We disclosed that Kuz is involved in somitogenesis, neurogenesis and angiogenesis and provides a useful therapeutic target for intervention in associated pathologies. Accordingly, the invention provides methods and compositions relating to Kuz involvement in somitogenesis, neurogenesis, and particularly, angiogenesis. In one embodiment, the invention provides methods for modulating angiogenesis comprising the step of specifically modulating the activity of Kuz in a vertebrate animal predetermined to have a pathogenic angiogenesis. A wide variety of methods for specifically modulating Kuz activity are disclosed, including contacting the animal with an agent which specifically binds the Kuz or competes with the Kuz for substrate or a required cofactor.
In another embodiment, the invention provides methods for modulating angiogenesis comprising the steps of specifically modulating the activity of Kuz in a vertebrate animal not necessarily predetermined to have a pathogenic angiogenesis, but rather subsequently detecting a resultant angiogenic modulation in the animal.
The invention also provides methods for specifically detecting Kuz activity in a vertebrate animal predetermined to have a pathogenic angiogenesis; for example, using a KUZ specific protease assay or a KUZ specific immunobinding assay. The invention also provides methods for specifically detecting a pathogenic angiogenesis in a vertebrate animal having a predetermined Kuz activity; for example, by detecting a tumor associated with pathogenic angiogenesis.
The invention also provides methods for identifying a modulator of angiogenesis, comprising the steps of (a) contacting an angiogenic assay system comprising a predetermined amount of Kuz with a candidate agent, under conditions whereby but for the presence of the agent, the system provides a reference angiogenesis; and (b) detecting an agent-biased angiogenesis of the system; wherein a difference between the agent-biased angiogenesis and the reference angiogenesis indicates that the agent modulates angiogenesis in the system. Such methods may be embodied in an in vitro, cell based assay or an in vivo, animal-based assay.
The invention also provides kits and reagents adapted to the subject methods.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or. Kuz refers to an art-recognized family of natural proteins which have been extensively described, encompassing natural orthologs and variants also well known in the art. For example, several forms of human KUZ have been described including WO98/37092 and WO97/31931; Mayer et al. (U.S. Pat. No. 5,922,546); and Rubin et al. (U.S. Pat. No. 5,935,792). Though often discussed and exemplified in terms of angiogenesis, the disclosed methods and reagents are to be understood to be generally applicable to pathogenic somitogenesis and neurogenesis as well.
Several disclosed applications involve specifically modulating the activity of Kuz in a vertebrate animal. A wide variety of methods for specifically modulating Kuz activity are disclosed, including contacting the animal with an agent which specifically binds the Kuz or competes with the Kuz for substrate or a required cofactor.
Agents which specifically bind kuz include metalloprotease inhibitors, such as hydroxamate metalloprotease inhibitors and TACE (TNF-alpha converting enzyme) inhibitors (for review, see Amour A, et al. Ann N Y Acad Sci 1999 Jun 30;878:728-31). Exemplary inhibitors include IC-3 (N-{D,L-[2-(hydroxyaminocaronyl)mehyl]-4-methyl-pentanoyl}-L-alanine, 2-aminoethyl amide, Black et al., Nature, 1997, Vol 385, 729-73; Galko and Tessier-Lavigne, Science, 2000, Vol 289, 1365-1367), GM6001 (NHOHCOCH
2
CH(I-Bu)CO-Trp-NHMe); GW9471 (see structure of GW9277, a biotinylated derivative of GW9471 used during the purification of TACE as shown in Moss et al, Nature, 1997, Vol 385, 733-736); and BB-94 (batimastat), a synthetic hydroxamate peptidomimetic matrix metalloproteinase inhibitor, see Hemandez-Pando
Pan Duojia
Rubin Gerald M.
Zhang Hongbing
Caputa Anthony C.
Nickol Gary B.
Osman Richard Aron
The Regents of the University of California
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