Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-05
2004-03-09
Spector, Lorraine (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C530S350000, C530S300000, C536S023100, C536S023500
Reexamination Certificate
active
06703198
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to polypeptides and peptides for regulating stem cell differentiation and renewal and to the molecular defects involved in Alagille Syndrome.
Hematopoiesis involves a delicate balance between progenitor cell self-renewal and differentiation. Self-renewal generates additional progenitor cells through cell division, and differentiation produces specialized cell types such as red blood cells or lymphocytes. The ability to reliably reproduce hematopoietic differentiation and expansion in vitro would greatly facilitate the development of clinical therapeutic treatments based on blood products and cell transplantation. For example, the ability to modulate hematopoietic differentiation and expansion would promote the production of mature blood cells for transfusion therapies and the production of mature dendritic cells for immunotherapy. In addition, the ability to manipulate a hematopoietic cell population to maintain a large number of progenitor cells would greatly improve ex vivo retroviral gene therapy since cell proliferation is required for retroviral gene transduction.
The ability to maintain the survival and proliferation of hematopoietic progenitor cells and to inhibit their differentiation would also improve cell transplantation following tumor purging. In high-dose chemotherapy, doses of toxic drugs are escalated to destroy aggressive malignancies such as hematologic, breast, testicular and ovarian cancers. These high doses also destroy many of the rapidly cycling cells of the hematopoietic system, rendering a patient vulnerable to infection. The ability to promote the survival and expansion of a limited number of remaining hematopoietic progenitor cells would increase neutrophil and platelet recovery times and reduce the danger associated with tumor purging and hematopoietic cell transplantation. However, current technology cannot effectively regulate the balance of hematopoietic progenitor cell survival and differentiation.
During embryogenesis in Drosophila, the Notch receptor plays a central role in cell fate specification during development of the central and peripheral nervous systems, eye, mesoderm, wing, bristles and ovaries. The Notch family of cell-cell signaling receptors is highly conserved in fly, worm, frog as well as higher vertebrates, and functions to determine cell fate through the transduction of signals between cells in direct contact with each other.
In higher vertebrates, the process of cell-fate determination is integral to hematopoiesis, where the balance between stem cell or progenitor cell self-renewal and differentiation is carefully regulated. Notch homologues can play a role in determining cell fate in hematopoietic cells, as evidenced by the expression of Notch1 RNA in immature hematopoietic precursor cells from adult human bone marrow. Notch homologues are implicated in T lymphocyte development since the human Notch homologue, TAN-1 (hNotch1), was isolated from a T-cell leukemia containing a translocation between Notch and the T cell receptor (TCR)-&bgr; gene. In addition, Notch1 can influence the CD4/CD8 cell-fate decision. Because an activated form of Notch1 can inhibit G-CSF-induced granulocytic differentiation of 32D myeloid progenitors, Notch also can play a role in mediating cell-fate decisions in the myeloid lineage.
The evolutionary conservation of Notch is reflected in the corresponding conservation of Notch ligands. Several Notch ligands have been identified thus far, including Delta and Serrate in Drosophila; LAG-2 and APX-1 in
C. elegans
; X-Delta-1 in Xenopus; C-Delta-1 and C-Serrate-1 in the chick; Delta-like-1 (Dll1) in the mouse; and Jagged1 and Jagged2 in the rat. Each of these Notch ligands share two important extracellular features: the DSL domain, defined by a conserved region among Delta Serrate, and LAG-2, and tandem epidermal growth factor (EGF) repeats. Delta and Serrate have been shown to interact with Notch in Drosophila, and fibroblasts expressing rat Jagged1 inhibit muscle cell differentiation of Notch1-expressing C2C12 cells. These results indicate that DSL family polypeptides including Drosophila Delta and Serrate and rat Jagged can function as Notch ligands.
However, a human Notch ligand, which would be useful in manipulating the balance of hematopoietic progenitor cell renewal and differentiation, has not yet been identified. Thus, there is a need for a human Notch ligand and for methods of using the ligand to maintain and expand hematopoietic progenitor cells to make clinical blood products and progenitor cells for transplantation. The present invention satisfies this need by providing human JAGGED1 polypeptides and provides related advantages as well.
The invention also relates to Alagille Syndrome, which is an autosomal dominant, developmental disorder affecting the liver, heart, skeleton, eye, face and kidneys. The course and prognosis of Alagille Syndrome, which occurs at a minimum estimated frequency of 1 in 70,000 live births, varies widely. This multi-system disorder traditionally has been defined by a paucity of intrahepatic bile ducts in association with several of the main clinical abnormalities, which are cholestasis, cardiac disease, skeletal abnormalities, ocular abnormalities and a characteristic facial phenotype. Fifteen percent of Alagille Syndrome patients will require liver transplantation, and seven to ten percent of patients will have severe congenital heart disease.
Unfortunately, the available therapies for Alagille Syndrome are few, and both diagnosis and treatment have been hampered by a lack of knowledge regarding the molecular defect underlying the disease. In a relatively small number of patients, gross chromosomal deletions of chromosome 20 appear to be inherited with the disorder. However, for the large majority of patients lacking such gross chromosomal abnormalities, the genetic defect responsible for Alagille Syndrome has eluded discovery. Identification of the molecular defect responsible for Alagille Syndrome would be useful in the early diagnosis and prenatal testing of individuals at risk for the disorder. In addition, knowledge of mutations resulting in Alagille Syndrome would facilitate the development of new therapies for treating the disorder. Thus, there is a need for identifying the mutations responsible for Alagille Syndrome and for methods of diagnosing the disorder by analyzing the genetic defect responsible for the disorder. The present invention satisfies this need and also provides related advantages.
SUMMARY OF THE INVENTION
The present invention provides an isolated polypeptide exhibiting substantially the same amino acid sequence as JAGGED, or an active fragment thereof, provided that the polypeptide does not have the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. The invention further provides an isolated nucleic acid molecule containing a nucleotide sequence encoding substantially the same amino acid sequence as JAGGED, or an active fragment thereof, provided that the nucleotide sequence does not encode the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. Also provided herein is a method of inhibiting differentiation of hematopoietic progenitor cells by contacting the progenitor cells with an isolated JAGGED polypeptide, or active fragment thereof. The invention additionally provides a method of diagnosing Alagille Syndrome in an individual. The method consists of detecting an Alagille Syndrome disease-associated mutation linked to a JAGGED locus.
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Corbin et al., “A Role for the Drosophila Neurogenic Genes in Mesoderm Differentiation,”Cell67:311-323
Hood Leroy
Krantz Ian D.
Li Linheng
Spinner Nancy B.
McDermott & Will & Emery
Seharaseyon Jegatheesan
Spector Lorraine
The University of Washington
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