Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-02-27
2001-03-06
Zitomer, Stephanie W. (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C536S024100, C435S252300, C435S320100, C435S325000, C530S350000
Reexamination Certificate
active
06197945
ABSTRACT:
BACKGROUND OF THE INVENTION
Each year, over 728,000 new cases of diabetes are diagnosed and 150,000 Americans die from the disease and its complications; the total yearly cost in the United States is over 20 billion dollars (Langer et al. (1993)
Science
260:920-926). For instance, diabetes is characterized by pancreatic islet destruction or dysfunction leading to loss of glucose control. Diabetes mellitus is a metabolic disorder defined by the presence of chronically elevated levels of blood glucose (hyperglycemia). Insulin-dependent (Type 1) diabetes mellitus (“IDDM”) results from an autoimmune-mediated destruction of the pancreatic &bgr;-cells with consequent loss of insulin production, which results in hyperglycemia. Type 1 diabetics require insulin replacement therapy to ensure survival. Non-insulin-dependent (Type 2) diabetes mellitus (“NIDDM”) is initially characterized by hyperglycemia in the presence of higher-than-normal levels of plasma insulin (hyperinsulinemia). In Type 2 diabetes, tissue processes which control carbohydrate metabolism are believed to have decreased sensitivity to insulin. Progression of the Type 2 diabetic state is associated with increasing concentrations of blood glucose, and coupled with a relative decrease in the rate of glucose-induced insulin secretion.
The primary aim of treatment in both forms of diabetes mellitus is the same, namely, the reduction of blood glucose levels to as near normal as possible. Treatment of Type 1 diabetes involves administration of replacement doses of insulin. In contrast, treatment of Type 2 diabetes frequently does not require administration of insulin. For example, initial therapy of Type 2 diabetes may be based on diet and lifestyle changes augmented by therapy with oral hypoglycemic agents such as sulfonylurea. Insulin therapy may be required, however, especially in the later stages of the disease, to produce control of hyperglycemia in an attempt to minimize complications of the disease.
More recently, tissue-engineering approaches to treatment have focused on transplanting healthy pancreatic islets, usually encapsulated in a membrane to avoid immune rejection. Three general approaches have been tested in animal models. In the first, a tubular membrane is coiled in a housing that contained islets. The membrane is connected to a polymer graph that in turn connects the device to blood vessels. By manipulation of the membrane permeability, so as to allow freediffusion of glucose and insulin back and forth through the membrane, yet block passage of antibodies and lymphocytes, normoglycemia was maintained in pancreatectomized animals treated with this device (Sullivan et al. (1991)
Science
252:718).
In a second approach, hollow fibers containing islet cells were immobilized in the polysaccharide alginate. When the device was place intraperitoneally in diabetic animals, blood glucose levels were lowered and good tissue compatibility was observed (Lacey et al. (1991)
Science
254:1782).
Finally, islets have been placed in microcapsules composed of alginate or polyacrylates. In some cases, animals treated with these microcapsules maintained normoglycemia for over two years (Lim et al. (1980)
Science
210:908; O'Shea et al. (1984)
Biochim. Biochys. Acta
. 840:133; Sugamori et al. (1989)
Trans. Am. Soc. Artif. Intern. Organs
35:791; Levesque et al. (1992)
Endocrinology
130:644; and Lim et al. (1992)
Transplantation
53:1180).
However, all of these transplantation strategies require a large, reliable source of donor islets.
SUMMARY OF THE INVENTION
The present invention relates to the discovery in eukaryotic cells, particularly mammalian cells, of novel a transcriptional regulatory factor, referred to hereinafter as “Insulin Promoter Factor 1” or “Ipf1”.
In general, the invention features an Ipf1 polypeptide, preferably a substantially pure preparation of the polypeptide, or a recombinant Ipf1 polypeptide. In preferred embodiments the polypeptide has a biological activity associated with its binding to Ipf1-responsive elements, such as the P1 insulin promoter site, and with its binding to other transcriptional regulatory proteins. The polypeptide can be identical to the polypeptide shown in SEQ ID No: 2, or it can merely be homologous to that sequence. For instance, the polypeptide preferably has an amino acid sequence at least 60% homologous to the amino acid sequence in SEQ ID No: 2, though higher sequence homologies of, for example, 80%, 90% or 95% are also contemplated. The polypeptide of the present invention can comprise the full length protein represented in SEQ ID No: 2, or it can comprise a fragment of that protein, which fragment may be, for instance, at least 5, 10, 20, 50 or 100 amino acids in length. The fragment can be derived to include, for example, regions of the protein which are likely to be involved in protein—protein interactions with other transcriptional regulatory proteins or which may influence the DNA-binding specficity of the homeodomain of Ipf1 (Glu146-Ser211) relative to other heterologous homeodomains. For instance, the fragment can include at least 4 amino acid residues between Metl to Glu145 and/or Ser212 to Arg284, though more preferably includes portions of at least 10, 20, 30 or 50 residues from one or both of those regions. Exemplary fragments include N-terminal fragments within or including Met1 to Glu145, or C-terminal fragments within or including Glu146 to Arg284.
Moreover, as described below, the Ipf1 polypeptide of the present invention can be either an agonist (e.g. mimics), or alternatively, an antagonist of a biological activity of a naturally occurring form of Ipf1. That is, the polypeptide is an Ipf1 homolog which is able to modulate Ipf1-mediated gene expression (e.g., a gene containing an Ipf1-responsive element) in at least one tissue in which wild-type Ipf1 is expressed, such as in pancreatic tissue, particularly &bgr;-islet cells.
In a preferred embodiment, a peptide having at least one biological activity of the subject polypeptide may differ in amino acid sequence from the sequence in SEQ ID No: 2, but such differences result in a modified protein which functions in the same or similar manner as the native Ipf1 or which has the same or similar characteristics of the native Ipf1. Moreover, homologs of the naturally occurring protein are contemplated which are antagonistic of the normal cellular role of the naturally occurring form of Ipf1. For example, the homolog may be capable of interfering with the ability of wild-type Ipf1 to modulate gene expression, e.g. of developmentally or growth regulated genes. Preferred antagonistic forms of an Ipf1 polypeptide either (i) retains the DNA binding ability of authentic Ipf1 but lack the ability to assemble transcriptionally-competent protein complexes, or (ii) lacks DNA binding ability (e.g. to Ipf1-RE2) yet retains the ability to bind to other transcription regulatory complexes normally involving authentic Ipf1.
In yet other preferred embodiments, the Ipf1 polypeptide is a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated to Ipf1, e.g. the second polypeptide portion is glutathione-S-transferase, e.g. the second polypeptide portion is a DNA binding domain of a heterologous transcriptional regulatory protein, or the second polypeptide portion is an RNA polymerase activating domain, e.g. the fusion protein is functional in a two-hybrid assay.
Yet another aspect of the present invention concerns an immunogen comprising an Ipf1 peptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for said Ipf1 polypeptide. The response can be in the form of a humoral response, e.g. an antibody response or a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g. a unique determinant, from a protein represented by SEQ ID No: 2.
A still further aspect of the present invention features an antibody preparation specifically reactive with an epit
Foley Hoag & Eliot LLP
Halstead David P.
Ontogeny, Inc.
Vincent Matthew P.
Wilder Cynthia
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