Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2002-08-06
2004-11-30
Chan, Christina (Department: 1644)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S455000, C435S252300, C435S320100
Reexamination Certificate
active
06825007
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to therapeutic and diagnostic methods and compositions based on Jagged/Notch proteins and nucleic acids, and on the role of their signaling pathway in endothelial cell migration and/or differentiation.
2. Background of the Invention
The functional integrity of the human vascular system is maintained by the endothelial cell which monitors the non-thrombogenic interface between blood and tissue in vivo. Thus, factors that influence human endothelial cell function may contribute significantly to the regulation and maintenance of homeostasis (see, Maciag, in
Progress in Hemostasis and Thrombosis
, T. Spaet, ed. (New York: A. R. Liss), pp.167-182 (1984); Folkman and Klagsbum,
Science
235:442-447 (1987); Burgess and Maciag,
Annu. Rev. Biochen
. 58:575-606 (1989)). Likewise, events that perturb this complex equilibrium are relevant to the pathophysiology of human disease states in which cellular components of the vascular tree are active participants including, e.g., atherogenesis, coronary insufficiency, hypertension, rheumatoid arthritis, solid tumor growth and metastasis, and wound repair.
Since the endothelium is present in all organs and tissues, endothelial cell function is also fundamental to the physiology and integration of these multicellular systems. This includes the ability to monitor and interface with repair systems that employ the tightly regulated inflammatory, angiogenic and neurotropic responses. Indeed, biochemical signals that are responsible for the modification of these responses have been well characterized as polypeptide growth factors and cytokines; however, their mechanisms of operation have, prior to the present invention, been poorly understood, impeding their acceptance as valuable tools in clinical management.
A major accomplishment of modern biology has been the recognition that structural elements responsible for physiologic functions are conserved throughout the animal kingdom. Genetic analysis of yeast,
C. elegans
, Xenopus, Zebra fish, and
Drosophila
, among others, have provided new insight into the regulation of the cell cycle, organelle biosynthesis and trafficking, cell fate and lineage decisions during development, as well as providing the fundamental principles for transcriptional/translational/post-translational regulation. Indeed, the conservation of structure-function principles exhibited by such systems has generated new insight into these and other regulatory systems utilized by mammalian cells. Moreover, a resolution of the genetic structure of the mammalian homologs for such genes in non-mammalian species has often led to a discernment of their function in mammals, even though the delineation of the function of a particular, homologous mammalian gene or gene fragment may well be serendipitous. In many cases, it is the result produced by expression and differential cDNA cloning strategies that manifest mammalian DNA sequences with homology to genes previously identified in more primitive species.
During the past decade, differential cDNA cloning methods, including e.g., conventional subtractive hybridization (Hla and Maciag,
Biochem. Biophys. Res. Commun
. 167:637-643 (1990a)), differential polymerase chain reaction (PCR)-oriented hybridization (Hla and Maciag,
J. Biol. Chem
. 265:9308-9313 (1990b)), and more recently, a modification of the differential display (Zimrin et al.,
Biochem. Biophys. Res. Commun
. 213:630-638 (1995)) were used to identify genes induced during the process of human umbilical vein endothelial cell (HUVEC) differentiation in vitro. Very early studies disclosed that HUVEC populations are able to generate capillary-like, lumen-containing structures when introduced into a growth-limited environment in vitro (Maciag et al.,
J. Cell Biol
. 94:511-520 (1982)). These studies permitted the identification and characterization of protein components of the extracellular matrix as inducers of this differentiation process, while at the same time defining the capillary-like structures as non-terminally differentiated (Maciag, 1984). Additional experiments have elucidated the importance of polypeptide cytokines, such as IL1 (Maier et al.,
J. Biol. Chem
. 265:10805-10808 (1990a)), and IFN&ggr; (Friesel et al.,
J. Cell Biol
. 104:689-696 (1987)) as inducers of HUVEC differentiation in vitro, and ultimately lead to an understanding that the precursor form of IL-1&agr; was responsible for the induction of HUVEC senescence in vitro (Maciag et al.,
J. Cell Biol
. 91:420-426 (1981); Maier et al.,
Science
249:1570-1574 (1990b))—the only truly terminal HUVEC phenotype identified to date. Summarized in FIG.
1
.
Recent research has employed differential cDNA cloning methods, which permits the identification of new and very interesting genes. However, until very recently, establishing their identity did not provide insight into the mechanism of HUVEC differentiation. Current research has focused upon the fibroblast growth factor (FGF) and interleukin (IL)-1 gene families as regulators of the angiogenesis process, both in vitro and in vivo (Friesel et al., FASEB J9:919-925 (1995); Zimrin et al.,
J. Clin. Invest
. 97:1359 (1996)). The human umbilical vein endothelial cell (HUVEC) has proven to be an effective model for studying the signal pathways utilized by FGF-1 to initiate HUVEC migration and growth, the role of IL-1&agr; as an intracellular inhibitor of FGF-1 function and modifier of HUVEC senescence, and the interplay between the FGF and the IL-1 gene families as key effectors of HUVEC differentiation in vitro. Such insight has enabled the present inventors to use modern molecular methods to identify a key regulatory ligand-receptor signaling system, which is able to both induce capillary endothelial cell migration and repress large vessel endothelial cell migration.
The Jagged/Serrate/Delta-Notch/Lin/Glp signaling system, originally described during the development of
C. elegans
and
Drosophila
as an essential system instrumental in cell fate decisions, has been found to be highly conserved in mammalian cells (Nye and Kopan,
Curr. Biol
. 5:966-969 (1995)). Notch proteins comprise a family of closely-related transmembrane receptors initially identified in embryologic studies in Drosophila (Fortini and Artavanis-Tsakonas,
Cell
75:1245-1247 (1993)). The genes encoding the Notch receptor show a high degree of structural conservation, and contain multiple EGF repeats in their extracellular domains (Coffinan et al.,
Science
249:1438-1441 (1990); Ellisen et al.,
Cell
66:649-661 (1991); Weinmaster et al.,
Development
113:199-205 (1991); Weinmaster et al.,
Development
116:931-941 (1992); Franco del Amo et al.,
Development
115:73 7-744 (1992); Reaume et al.,
Dev. Biol
. 154:377-387 (1992); Lardelli and Lendahi,
Mech. Dev
. 46:123-136 (1993); Bierkamp and Campos-Ortega,
Mech. Dev
. 43:87-100 (1993); Lardelli et al.,
Exp. Cell Res
. 204:364-372 (1994)). In addition to the 36 EGF repeats within the extracellular domain of Notch 1, there is a cys-rich domain composed of three
N
otch
L
in
G
lp (NLG) repeats, which is important for ligand function, followed by a cys-poor region between the transmembrane and NLG domain.
The intracellular domain of Notch 1 contains six ankyrin/Cdc10 repeats positioned between two nuclear localization sequences (NLS) (Artavanis-Tsakonas et al.,
Science
268:225-232 (1995)). This motif is found in many functionally diverse proteins (see e.g., Bork,
Proteins
17:363-374 (1993)), including members of the rel/NF-kB family (Blank et al., TIBS 17:135-140 (1992)), and is thought to be responsible for protein-protein interactions. Notch has been shown to interact with a novel ubiquitously distributed cytoplasmic protein deltex through its ankyrin repeats, a domain shown by deletion analysis to be necessary for activity (Matsuno et al.,
Development
121:2633-2644 (1995)).
Carboxy terminal to this region is a polyglutamine-rich domain (OPA) and a pro-glu-ser-thr (PEST) domain which may be involved in sign
Maciag Thomas
Montesano Roberto
Pepper Michael S.
Wong Michael
Zimrin Ann B.
Chan Christina
Haddad Maher
Maine Medical Center Research Institute
Morgan & Lewis & Bockius, LLP
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