Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2001-05-17
2004-04-13
Nguyen, Dave T. (Department: 1635)
Chemistry: molecular biology and microbiology
Vector, per se
C435S091400, C536S023500, C424S450000
Reexamination Certificate
active
06720180
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of wound healing. More particularly, it concerns the cloning and characterization of the epsin 3 gene, a wounded epithelium-specific transcript.
2. Description of Related Art
The epidermis consists of a multilayered epithelial sheet that provides a physical barrier against the outside environment and heals in response to injury. In unwounded skin, basal keratinocytes reside on a basement membrane (BM) that physically separates these cells from the underlying dermal connective tissue rich in type I collagen. While in contact with this extracellular matrix (ECM), keratinocytes express a programmed subset of genes that promotes proliferation and differentiation. Following injury, however, keratinocytes from the surrounding tissue are activated by exposure to ligands released into the wound site and by contact with extracellular matrix macromolecules (Grinnell, 1990; Clark, 1996; Coulombe, 1997).
Normal cutaneous wound healing requires an orderly progression of events to re-establish the integrity of the injured tissue. The initial injury sets forth a programmed series of responses that include re-epithelialization, inflammation, angiogenesis, fibroplasia, matrix accumulation, and resolution (Clark 1992). In wounds that disrupt the BM, keratinocytes from the surrounding uninjured tissue are “activated” by exposure to soluble ligands released into the wound site and by contact with dermal and provisional extracellular matrix. This activation, which typically occurs 18-24 hours prior to the onset of migration and hyperproliferation, occurs as wound edge keratinocytes switch from a gene expression program of proliferation and differentiation to one that supports sustained and directed migration leading to re-epithelialization (Coulombe, 1997).
Migration of keratinocytes from the wound edge occurs as the cells dissect under a provisional matrix of fibrin and fibronectin (Clark et al., 1982) and over or through a viable dermis, which includes structural molecules distinct from those in the basement membrane. Loss of contact with the basement membrane and subsequent exposure to the underlying dermal matrix may be a critical determinant that alters gene expression and induces the activated keratinocyte phenotype. Recent evidence supports the idea that the enzyme, matrix metalloproteinase collagenase-1, is invariantly expressed in keratinocytes not in contact with a basement membrane, but rather in keratinocytes migrating over the dermal matrix and in close apposition to collagen fibers (Inoue et al., 1995; Saarialho-Kere et al., 1992; Saarialho-Kere et al., 1993; Pilcher et al., 1997). Moreover, expression of this enzyme is selectively induced in keratinocytes following contact with fibrillar type I collagen in vitro and its proteolytic activity is essential for cell migration across this matrix (Pilcher et al., 1997; Sudbeck et al, 1997). Together, these data suggest that keratinocyte contact with dermal extracellular matrix, and in particular fibrillar type I collagen, profoundly influence keratinocyte activation following injury by inducing the expression of transcripts required for efficient repair.
Many of the genes upregulated in keratinocytes during healing, including secreted proteinases and integrin receptors, enable a fundamental shift in cell behavior that supports sustained and directed migration to re-establish the normal cytoarchitecture of the skin (Coulombe, 1997). The preponderance of studies to date attempting to identify signals that stimulate keratinocyte activation during wound healing have focused on soluble mediators, whereas the role that alterations in cell:extracellular matrix interactions play in this process has received relatively little attention.
The epsins are a family of recently characterized genes involved in clathrin-mediated endocytosis. Epsins 1 and 2 function as molecular bridges to bind epidermal growth factor pathway substrate 15 (Eps15) and clathrin adaptor protein-2 (AP-2), allowing the appropriate molecular conformation required for assembly of the coated pit. Indeed, perturbation of epsin function in fibroblasts potently inhibits assembly and internalization of clathrin-coated pits (Chen et al., 1998; Rosenthal et al., 1999). Conserved domains common to all epsins include a COOH-terminal consensus sequence of three NPF repeats that bind Eps15, multiple central region DPW motifs that bind AP-2, and a 150 amino acid NH
2
-terminal protein module, the epsin NH
2
-terminal homology domain (ENTH domain).
SUMMARY OF THE INVENTION
The instant invention encompasses methods and compositions comprising the mammalian epsin 3 protein and the gene encoding said protein. An envisioned embodiment of the invention therefore comprises an isolated and purified polynucleotide comprising a nucleic acid sequence encoding a mammalian epsin 3 gene. This polynucleotide may be further defined as a nucleic acid encoding 10, 20, 40, 50, 60, 100 contiguous amino acid residues or the full-length protein of SEQ ID NO:2.
A further embodiment of the invention encompasses a polynucleotide comprising a nucleic acid sequence encoding SEQ ID NO:1 or at least 10, 20, 30, 50, 80 or 100 contiguous bases of SEQ ID NO:1. An additional embodiment of the invention as setforth herein is considered to comprise an epsin 3 protein, peptide or polypeptide. In an envisioned embodiment, such a protein, peptide or polypeptide may be further characterized as comprising at least 10, 20, 30, 50, 80 or 100 contiguous amino acids as set forth in SEQ ID NO:2 or the full length sequence set forth in SEQ ID NO. 2.
An expression vector comprising a nucleic acid sequence encoding a mammalian epsin 3 polypeptide is also considered within the scope of the invention as claimed. In a specific embodiment of the invention, such an expression vector may be characterized as comprising a nucleic acid sequence that encodes SEQ ID NO:2 or an expression vector comprising a nucleic acid sequence encoding at least 10, 20, 30, 40, 50, 80 or 100 contiguous amino acids of SEQ ID NO:2. An alternate embodiment may encompass an expression vector comprising the nucleic acid sequence as set forth in SEQ ID NO:1 or an expression vector comprising a nucleic acid sequence encoding at least 10, 20, 30, 40, 50, 80 or 100 contiguous bases of SEQ ID NO:1. It is further envisioned that the expression vector may comprise a virus and that the expression construct may include a promoter operably linked to the epsin 3-encoding nucleic acid sequence.
Another embodiment of the invention encompasses the use of epsin 3 protein or nucleic acid sequence encoding the epsin 3 protein to detect tissue damage, particularly epithelial damage. In an envisioned embodiment, this method entails detecting epithelial damage by screening for epsin 3. More particularly, the epithelial damage may be caused by cancer, or other pathologies exhibiting altered cell:extracellular matrix interactions. Such pathologies include, but are not limited to pyogenic granuloma, pyoderma gangrenosum, decubitus ulcers, venous-stasis ulcers, diabetic ulcers, poorly-healing wounds, burns, normal surgical invasions, oral lesions, muscosal lesions, airway/lung lesions, gastric ulcerations, intestinal ulcerations, ulcerative colitis, Chrohn's disease or opthalmic ulcerations.
Yet further, another embodiment encompasses a method of enhancing re-epithelialization comprising administering an epsin 3 protein or a functional equivalent thereof to the wound of an organism. An organism may include, but is not limited to humans, rats, mice, monkeys, dogs, cats or pigs. It is envisioned that overexpression or increased abundance of epsin 3 protein may enhance the migration of keratinocytes in pathologies that fail to heal or exhibit altered cell:extracelluar matrix interactions. Such pathologies include, but are not limited to pyogenic granuloma, pyoderma gangrenosum, decubitus ulcers, venous-stasis ulcers, diabetic ulcers, poorly-healing wounds, burns, normal surgical invas
Angell J. Eric
Board of Regents , The University of Texas System
Fulbright & Jaworski L.L.P.
Nguyen Dave T.
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