Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2001-04-26
2003-04-01
Ketter, James (Department: 1633)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C530S350000, C530S387100
Reexamination Certificate
active
06541251
ABSTRACT:
BACKGROUND OF THE INVENTION
There are 15.7 million people in the United States who have diabetes, which is the seventh leading cause of death in this country. As a chronic disease that has no cure, diabetes is one of the most costly health problems in America.
Health care and other costs directly related to diabetes treatment, as well as the costs of lost productivity, run $92 billion annually.
Type I autoimmune diabetes results from the destruction of insulin producing beta cells in the pancreatic islets of Langerhans. The adult pancreas has very limited regenerative potential, and so these islets are not replaced after they are destroyed. The patient's survival then depends on exogenous administration of insulin. There are an estimated 500,000 to 1 million people with type 1 diabetes in the United States today. The risk of developing type 1 diabetes is higher than virtually all other severe chronic diseases of childhood.
The pancreas is composed of at least three types of differentiated tissue: the hormone-producing cells in islets (4 different cell types), the exocrine zymogen-containing acini, and the centroacinar cells, ductules and ducts (ductal tree). All of these cells appear to have a common origin during embryogenesis in the form of duct-like protodifferentiated cells. Later in life, the acinar and ductal cells retain a significant proliferative capacity that can ensure cell renewal and growth, whereas the islet cells become mitotically inactive.
During embryonic development, and probably later in life, pancreatic islets of Langerhans originate from differentiating epithelial stem cells. These stem cells are situated in the pancreatic ducts but are otherwise poorly characterized.
Pancreatic islets contain four islet cell types: alpha, beta, delta and pancreatic polypeptide cells that synthesize glucagon, insulin, somatostatin and pancreatic polypeptide, respectively. The early progenitor cells to the pancreatic islets are multipotential and coactivate all the islet-specific genes from the time they first appear. As development proceeds, expression of islet-specific hormones becomes restricted to the pattern of expression characteristic of mature islet cells.
The characterization of pre-islet cells is of great interest for the development of therapeutics to treat diseases of the pancreas, particularly IDDM. Model systems have been described that permit the study of these cells. For example, Gu and Sarvetnick (1993)
Development
118:33-46 identify a model system for the study of pancreatic islet development and regeneration. Transgenic mice carrying the mouse &ggr;-interferon gene linked to the human insulin promoter exhibit inflammatory-induced islet loss. Significant duct cell proliferation occurs in these mice, leading to a striking expansion of pancreatic ducts. Endocrine progenitor cells are localized in these ducts. This model provides a source of progenitor cells for further study.
The differential expression of genes by progenitor cells, as compared to their differentiated progeny, is of interest for the characterization and isolation of the progenitor cells. Where the differentially expressed genes encode a receptor for biologically active molecules, the marker may further provide information about factors that affect the growth or differentiation of the progenitor cells. Where such genes encode proteins such as transcription factors, the marker may provide information about regulated gene expression in the progenitor cells.
Relevant Literature
Kritzik et al. (1999)
J Endocrinol
163(3):523-30 found that PDX-1, a transcription factor required for insulin gene transcription as well as for pancreatic development during embryogenesis, is expressed in the duct cells of IFN&ggr; mice. Also demonstrated was elevated expression of the homeobox-containing protein Msx-2 in the pancreata of fetal mice as well as in adult IFN&ggr; mice, identifying this molecule as a marker associated with pancreatic development and regeneration.
Oberg-Welsh and Welsh (1996)
Pancreas
12:334-339 study the expression of protein tyrosine kinases in different preparations of insulin producing cells by polymerase chain reaction (PCR). Among the tyrosine kinases were the fibroblast growth factor receptor-4 (FGFR-4), c-kit, the insulin-like growth factor (IGF-1) receptor, and the cytoplasmic tyrosine kinase Jak2, which associates with the activated receptor for growth hormone (GH).
Inoue et al. (1998)
Biochem Biophys Res Commun
243(2):628-33 isolated a full-length cDNA of mouse PAX4 gene and a human homolog. Studies have suggested that PAX4, a member of the paired box (PAX) gene family, is involved in the mechanism regulating the fate of pancreatic islet endocrine progenitor cells.
Bouwens (1998) Microsc Res Tech 43(4):332-6 review the question whether islet beta-cell regeneration or neogenesis in the pancreas depends on “embryonic-like” stem cells or on transdifferentiation of “fully differentiated” cells.
St-Onge et al. (1999) Curr Opin Genet Dev 9(3):295-300 reviews the role of transcription factors such as Pdx1, p48 and Nkx2.2 pancreas development, including the role of Sonic Hedgehog.
The uPAR/CD59/Ly-6/snake toxin family is a group of proteins characterized by cysteine-rich consensus signature motifs, as well as conserved tertiary structures and genomic organization. Wang et al. (1995)
Eur J Biochem
227(1-2):116-22 compares the exon organization of the uPAR gene with that of human CD59 and murine Ly-6.
SUMMARY OF THE INVENTION
Isolated nucleotide compositions and sequences are provided for pancreatic progenitor 1 (PP1) genes. The PP1 nucleic acid compositions find use in identifying homologous or related genes; in producing compositions that modulate the expression or function of its encoded protein, PP1; for gene therapy; mapping functional regions of the protein; and in studying associated physiological pathways. In addition, modulation of the gene activity in vivo is used for prophylactic and therapeutic purposes.
In one embodiment of the invention, antibodies specific for the PP1 protein are used in the identification and isolation of cells expressing PP1, e.g. pancreatic progenitor cells. In a related embodiment, compositions of PP1 positive cells are provided.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Nucleic acid compositions encoding pancreatic progenitor 1 (PP1) are provided. They are used in identifying homologous or related genes; in producing compositions that modulate the expression or function of its encoded protein; for gene therapy; mapping functional regions of the protein; and in studying associated physiological pathways. Antibodies that recognize PP1 are useful in the identification and isolation of cells expressing PP1, particularly pancreatic progenitor cells.
Characterization of PP1
PP1 encodes 221 a amino acid protein containing two cysteine-rich domains. Sequence analysis demonstrates that PP1 is a member of the uPAR/CD59/Ly-6/snake toxin family. PP1 is expressed in the ducts of the regenerating pancreas in regions where new islets are developing. In addition, PP1 is expressed in embryonic foregut, stomach and duodenum, but not in developing pancreas or mature pancreas, demonstrating that PP1 is a marker of progenitor or stem cells. The PP1 expressing cells in the gut are localized in the endodermal pouch; and is also found in intestinal crypt cells. These results indicate that PP1 is a progenitor or stem cell marker in multiple lineages.
The nucleotide sequence of mouse PP1 is provided as SEQ ID NO:1; and the amino acid sequence of the encoded polypeptide as SEQ ID NO:2. The genomic sequence, including the promoter region, is provided as SEQ ID NO:3.
Homologs of PP1 are identified by any of a number of methods. For example, a fragment of the cDNA may be used as a hybridization probe against a cDNA library from the target organism of interest, where low stringency conditions are used. The probe may be a large fragment, or one or more short degenerate primers. Such sequences are selected from regions that are not likely to diverge over evolutionary time
Fox Howard
Sarvetnick Nora
Bozicevic Field & Francis LLP
Ketter James
Qian Celine
Sherwood Pamela J.
The Scripps Research Institute
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