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
2000-03-24
2004-03-09
Kunz, Gary (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S325000, C435S320100, C435S252300, C536S023500, C536S023100
Reexamination Certificate
active
06703220
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to the field of nucleotide sequences encoding transcription factors involved in growth and differentiation, particularly development of pancreatic islet cells.
BACKGROUND OF THE INVENTION
Diabetes mellitus is the third leading cause of death in the U.S. and the leading cause of blindness, renal failure, and amputation. Diabetes is also a major cause of premature heart attacks and stroke and accounts for 15% of U.S. health care costs. Approximately 5% of Americans, and as many as 20% of those over the age of 65, have diabetes.
Diabetes results from the failure of the &bgr;-cells in the islets of Langerhans in the endocrine pancreas to produce adequate insulin to meet metabolic needs. Diabetes is categorized into two clinical forms: Type 1 diabetes (or insulin-dependent diabetes) and Type 2 diabetes (or non-insulin-dependent diabetes). Type 1 diabetes is caused by the loss of the insulin-producing &bgr;-cells. Type 2 diabetes is a more strongly genetic disease than Type 1 (Zonana & Rimoin, 1976 N. Engl. J. Med. 295:603), usually has its onset alter in life, and accounts for approximately 90% of diabetes in the U.S. Affected individuals usually have both a decrease in the capacity of the pancreas to produce insulin and a defect in the ability to utilize the insulin (insulin resistance). Obesity causes insulin resistance, and approximately 80% of individuals with Type 2 diabetes are clinically obese (greater than 20% above ideal body weight). Unfortunately, about one-half of the people in the U.S. affected by Type 2 diabetes are unaware that they have the disease. Clinical symptoms associated with Type 2 diabetes may not become obvious until late in the disease, and the early signs are often misdiagnosed, causing a delay in treatment and increased complications. While the role of genetics in the etiology of type 2 diabetes is clear, the precise genes involved are largely unknown.
Insulin is made exclusively by the &bgr;-cells in the islets of Langerhans in the pancreas. During development, the islet cells, including the &bgr;-cells, develop from an undifferentiated precursor within the growing pancreatic bud. As the bud grows, the undifferentiated cells form into ducts, and it is these cells that function as precursors. Duct cells appear to retain the capacity to differentiate into islet cells throughout life, and when the pancreas is damaged, new islet cells form from the duct cells.
This developmental process is clinically relevant for several reasons. First, the formation of islet cells and especially &bgr;-cells is necessary in order to make insulin and control energy metabolism. If the process of &bgr;-cell development is in anyway impaired, it predisposes that individual to the later development of diabetes. Therefore genes involved in this process are candidate genes for neonatal diabetes, maturity onset diabetes of the young (MODY) or type 2 diabetes. The sequence of these genes could be used to identify individuals at risk for the development of diabetes, or to develop new pharmacological agents to prevent and treat diabetes.
Second, as discussed above, insulin production is impaired in individuals with diabetes. In type 1 diabetes the impairment is caused by the destruction of the beta-cells, while in type 2 diabetes, insulin production is intact, but inadequate. Treatment of type 1 diabetes, as well as many cases of type 2 diabetes, may involve replacement of the &bgr;-cells. While replacement of &bgr;-cells may be accomplished in several ways, the development of new &bgr;-cells from precursor cells, either in culture or in vivo in the patient, would be the most physiologic. To do this, the molecules that control beta-cell differentiation are needed.
For these reasons, the diabetes field has spent considerable effort in attempts to identify islet precursor cells, and to develop methods for differentiating beta-cells in vitro. To date this has been largely unsuccessful. The present invention addresses this problem.
Relevant Literature
A cloned fragment of mouse Ngn3 is described in Sommer et al. 1996 Mol. Cell. Neurosci. 8:221.
cDNA and amino acid sequences of murine Ngn3 and murine mammalian atonal homology 4B (MATH4B) are described at GenBank Accession Nos. U76208 and Y09167, respectively.
cDNA and amino acid sequences of the rat relax transcriptional regulator are described at GenBank Accession No. Y10619.
SUMMARY OF THE INVENTION
The present invention features a human neurogenin3 (Ngn3) polypeptide and nucleotide sequences encoding Ngn3 polypeptides. In a particular aspect, the polynucleotide is the nucleotide sequence of SEQ ID NO:1. In addition, the invention features isolated nucleic acid sequence comprising an Ngn3 promoter, as well as a polynucleotide sequences that hybridize under stringent conditions to SEQ ID NO:1. In related aspects the invention features expression vectors and host cells comprising polynucleotides that encode a human Ngn3 polypeptide. The present invention also relates to antibodies that bind specifically to a human Ngn3 polypeptide, methods for producing human Ngn3 polypeptides, methods for identifying &bgr;-cell precursor cells expressing Ngn3, methods for using the Ngn3 gene and the Ngn3 polypeptide to alter cellular differentiation in culture or in vivo to produce new &bgr;-cells to treat patients with diabetes mellitus, and identification of individuals at risk for diabetes by detecting alteration in Ngn3 coding and regulatory sequences and Ngn3 expression levels.
A primary object of the invention is to provide an isolated human Ngn3 polypeptide-encoding polynucleotide for use in expression of human Ngn3 (e.g, in a recombinant host cell) and for use in, for example, identification of human Ngn3 polypeptide binding compounds (especially those compounds that affect human Ngn3 polypeptide-mediated activity, which compounds can be used to modulate Ngn3 activity).
Another object of the invention is to provide an isolated human Ngn3 polypeptide-encoding polynucleotide for use in generation of non-human transgenic animal models for Ngn3 gene function, particularly “knock-in” Ngn3 non-human transgenic animals characterized by excess or ectopic expression of the Ngn3 gene.
These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the invention more fully set forth below.
The invention will now be described in further detail.
DETAILED DESCRIPTION OF THE INVENTION
Before the present nucleotide and polypeptide sequences are described, it is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, vectors and reagents described as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a host cell” includes a plurality of such host cells and reference to “the antibody” includes reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.
All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the cell lines, vectors, and methodologies which are described in the publications which might be used in connection with the presen
German Michael S.
Lin Joseph
Bozicevic Field & Francis LLP
Francis Carol
Hayes Robert C.
Keddie James S.
Kunz Gary
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