Nucleic acid encoding a transcription factor, IDX-1

Details Nucleic acid encoding a transcription factor, IDX-1 Nucleic acid encoding a transcription factor, IDX-1

1996-11-19

2001-04-03

Pak, Michael (Department: 1646)

Chemistry: molecular biology and microbiology

Vector, per se

C435S325000, C536S023500

Reexamination Certificate

active

06210960

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to proteins or polypeptides useful for treatment of the disease Diabetes mellitus. The invention was made with Government funding and, therefore, the U.S. Government has rights in the invention.
BACKGROUND OF THE INVENTION
Diabetes mellitus type I, or insulin-dependent diabetes, results from a genetically conferred vulnerability that causes a primary deficiency of insulin. This deficiency of insulin is believed to be the consequence of destruction of a specialized population of cells that produce insulin in the body, i.e., pancreatic &bgr;-cells. An autoimmune process may also contribute to &bgr;-cell damage. As a consequence of insulin lack (and glucagon excess) , glucose production is augmented, and the efficiency of peripheral glucose use is reduced until a new equilibrium between these processes is reached at a very high plasma glucose level. Because of the high plasma glucose levels, the filtered load of glucose exceeds the renal tubular capacity for reabsorption. Glucose therefore is excreted in the urine in large quantities, causing, by its osmotic effect, increased excretion of water and salts and frequent urination. The goal of insulin treatment is to systemically lower plasma levels of glucose, free fatty acids, and ketoacids to normal and reduce urine nitrogen losses. This result is achieved by direct actions of insulin and also by diminishing the secretion of the insulin antagonist glucagon.
Another more common form of diabetes mellitus, non insulin-dependent or type II, often is associated with obesity. In this disease, there appears to be both a deficit of insulin production (Weir et al., 1982, Amer. Jour. Med. 73:461) in combination with a resistance to the action of insulin on major target tissues. The locus of resistance is distal to the insulin receptor binding site, but defects in receptor tyrosine kinase activity, glucose transport, and activities of insulin-sensitive enzymes have been found. In addition, there is a derangement in &bgr;-cell recognition of glucose as a stimulus, so that first phase insulin secretion is lost, though a delayed release does occur. Treatment of type II diabetes does not normally require insulin administration. Caloric regulation, weight reduction if obesity is present, and use of sulfonylurea drugs simultaneously improve tissue responsiveness to endogenous insulin and &bgr;-cell responsiveness to glucose. In late stages, insulin administration is usually required.
Insulin excess is usually caused by tumors of the &bgr;-cells. The cardinal manifestation is a low plasma glucose level in the fasting state. With chronic insulin excess and persistent hypoglycemia, disturbed central nervous system function results in bizarre behavior, defects in cerebration, loss of consciousness, or convulsions. Removal of the tumor may cure the condition. Alternatively, drugs that inhibit insulin secretion may ameliorate the condition.
It is an object of the invention to provide a nucleic acid sequence encoding a novel transcriptional activator that is present in certain pancreatic cell populations, and the encoded transcriptional activator.
Another object of the invention is to provide an in vitro method for producing a desired protein using the novel transcriptional activator to activate transcription of the gene encoding the desired protein.
Yet another object of the invention is to provide methods of treating Diabetes mellitus type I, type II, or diseases in which insulin is produced in excess.
Another object of the invention is to provide a transgenic mouse model for diabetes, in which expression of the novel transcriptional activator is altered.
These and further objects of the invention will be apparent for one skilled in the art.
SUMMARY OF THE INVENTION
The invention features a novel recombinant polypeptide, i.e., IDX-1 or a variant thereof, that transactivates the somatostatin promoter, the polypeptide being present in pancreatic duct cells and not present in pancreatic &agr;-cells. The polypeptide is encoded by a gene which encodes a protein. As used herein, “transactivates” means to activate or aid in activating transcription of a gene associated with the promoter: “present” means expressed from a gene that is active in such cells.
The invention also encompasses a nucleic acid sequence encoding IDX-1 or its variant, replicable expression vectors comprising and capable of expressing the nucleic acid sequence in a transformant host cell, and microorganisms and cell cultures transformed with the vector.
As used herein, an IDX-1 variant is a protein sequence that is substantially similar to (i.e., having at least 70% homology and preferably 80-90% homology) the IDX-1 sequence presented in
FIG. 1
[SEQ ID NO: 2]. The sequence of an IDX-1 variant will be sufficiently duplicative of the IDX-1 sequence such that the variant retains the tissue expression pattern of IDX-1 and also retains the ability to transactivate the somatostatin promoter. An IDX-1 variant will include deletion, insertion, or point mutants of IDX-1.
The invention also encompasses IDX-1 variants, including agonists or antagonists of IDX-1, having enhanced or reduced transactivating activity for the somatostatin promoter, and methods for making such variants, comprising: (a) introducing an amino acid alteration into IDX-1 at a site or sites recognized as conferring transactivating activity or binding activity with respect to the somatostatin promoter; and (b) screening the resultant IDX-1 variant for enhanced or reduced transactivating or binding activity in comparison to native IDX-1. Thus, an IDX-1 variant having enhanced binding activity but reduced transactivating activity will include competitive inhibitors of IDX-1, i.e., that competitively inhibit IDX-1 from binding to a given promoter, while providing reduced promoter activation. Alternatively, an IDX-1 variant may have enhanced transactivating activity and enhanced binding activity, and therefore may be considered an agonist of IDX-1. An antagonist or agonist of IDX-1 may contain an insertion, deletion or point mutation of the native IDX-1 sequence.
IDX-1 variants of the invention with enhanced promoter transactivating activity can be used to obtain enhanced expression of a desired gene that is under control of the somatostatin promoter. IDX-1 variants having reduced promoter transactivating activity can be used to obtain reduced expression of a desired gene that is under control of the somatostatin promoter.
Accordingly, the present invention also provides methods for the treatment or prevention of a symptom or condition associated with diabetes comprising administering to a patient having or at risk of developing such symptom or condition a therapeutically effective amount of IDX-1. As used herein, a “therapeutically effective amount” of IDX-1 means the amount of IDX-1 protein that is necessary to restore the level of insulin in the body to a normal level. Symptoms or conditions associated with diabetes include but are not limited to excretion of abnormally high levels of glucose and salts in the urine, frequent urination, and abnormally high levels of glucose in the blood.
IDX-1 as a therapeutic protein can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the protein is combined in admixture with a pharmaceutically acceptable carrier. Such pharmaceutical compositions are within the scope of the present invention, although the nature of the carrier itself is not an essential aspect of the invention.
The invention features in another aspect, methods of treating diabetes mellitus type I or type II. Thus, according to the invention, the method includes administering to a patient in need thereof a recombinant polypeptide that transactivates sequences found in the somatostatin promoter, the polypeptide being expressed in pancreatic duct cells and not expressed in pancreatic &agr;-cells, the polypeptide being encoded by a gene which encodes a protein, the polypeptide being administered in an amount and for a time suffi

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