Recombinant cells regulated by tetracycline

Details Recombinant cells regulated by tetracycline Recombinant cells regulated by tetracycline

2000-01-27

2001-06-05

Hauda, Karen M. (Department: 1632)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S455000

Reexamination Certificate

active

06242254

ABSTRACT:

BACKGROUND OF THE INVENTION
Diabetes mellitus is a chronic disorder of carbohydrate metabolism characterized by insufficient production of insulin by the pancreatic beta cells. Diabetes effects approximately 10 million people in the United States, with more than 250,000 new cases diagnosed each year. There are two common types of diabetes mellitus: insulin-dependent (Type-I diabetes) and non-insulin-dependent (Type-II diabetes). Insulin-dependent diabetes is generally characterized by an absolute deficiency of insulin production, whereas non-insulin-dependent diabetes is characterized by a relatively insufficient production of insulin.
In normal individuals, the rate of insulin secretion by beta cells is regulated by the level of glucose in the blood. When the blood glucose level rises, the islet cells are stimulated to release increased amounts of insulin into the blood, accelerating glucose transport into the cells and glucose conversion into glycogen. As the blood glucose level falls, insulin release from the islets is decreased. In the diabetic subject, insulin production is abnormally low or insufficient, resulting in abnormally high blood glucose levels, a condition known as hyperglycemia.
In addition to diet and exercise programs, the constant and life long monitoring of blood glucose levels in conjunction with injections of insulin is central to the current methods for the treatment of the insulin-dependent diabetic subject. Many diabetic subjects, however, have difficulty in controlling their blood glucose levels using the current treatment methods, thus constantly exposing themselves to the adverse effects of hypoglycemia (abnormally low blood glucose levels) and hyperglycemia.
The inability to precisely control the level of blood glucose also poses long term complications such as degenerative vascular changes (e.g. atherosclerosis and microangiopathy), neuropathy (e.g. peripheral nerve degeneration, autonomic nervous system, and cranial nerve lesions), ocular disturbances (e.g. blurred vision, cataracts, and diabetic retinopathy), kidney diseases (e.g. recurrent pyelonephritis and nephropathy), and infections. Accordingly, there exists a need for an alternative method for controlling blood glucose levels in the diabetic patient. The transplantation of beta cells has been proposed as an alternative therapy in the treatment of diabetes. However, large scale transplantation of human beta cells is not feasible because of the limited availability of donors; similarly, the cost and effort, in terms of labor, associated with obtaining sufficient amounts of animal islets for transplantation also limits their use. These and other disadvantages associated with transplanting human and animal islets, makes the development of islet-derived cell lines the method of choice in obtaining sufficient quantities of cells for transplantation. In particular, a number of beta cell lines have been generated from insulinomas and hyperplastic islets arising in mice expressing a transgene encoding the SV40 T antigen (Tag) oncogene under the control of the insulin promoter (RIP-Tag)(
1
-
6
). Several of these cell lines displayed insulin secretion characteristics similar to those observed in intact adult islets, in particular the response to glucose concentrations in the physiological range (5-15 mmol/l).
However, a common problem encountered with all of these cell lines is their phenotypic instability. After propagation in tissue culture, these cells become responsive to subphysiological concentrations of glucose and/or manifest diminished insulin output (
4
,
6
-
9
). A similar instability has been observed with beta cell lines derived by other methods (
10
-
12
).
The present invention overcomes the problems associated with the previous beta cell lines by providing a beta cell line which not only maintains blood glucose levels in the normal range, but also may be controlled to prevent unregulated proliferation.
SUMMARY OF THE INVENTION
The present invention provides a recombinant, glucose-regulated insulin producing beta cell whose proliferation is controlled by tetracycline or a derivative thereof.
The present invention also provides a microcapsule comprising an amount of the recombinant beta cell above sufficient to maintain physiologically acceptable levels of glucose in a subject implanted with the microcapsule.
The present invention also provides a method for treating a subject with diabetes which comprises (a) implanting in the subject recombinant beta cells whose proliferation is controlled by tetracycline or a derivative thereof, in an amount effective to establish and maintain physiologically acceptable levels of glucose in the blood of the subject; and (b) inhibiting proliferation of the implanted recombinant beta cells by administering to the subject an amount of tetracycline or a derivative thereof, effective to inhibit proliferation of the implanted recombinant beta cells.
The present invention also provides a method for producing a recombinant, glucose-regulated insulin producing beta cell whose proliferation is controlled by tetracycline or a derivative thereof, comprising the steps of: (a) introducing to a first, non-human animal a first plasmid comprising a DNA encoding a tetR-VP16 fusion protein, and an insulin promoter which controls expression of the fusion protein, such that a first, genetically controlled, non-human animal is obtained; (b) introducing to a second, non-human animal a second plasmid comprising a DNA encoding the SV40 T antigen, and a tetracycline (Tc) operator minimal promoter, such that a second genetically-modified, non-human animal is obtained; (c) crossing the first genetically-modified, non-human animal, or offspring thereof, with the second, genetically-modified, non-human animal, or offspring thereof, to obtain progeny; (d) screening the progeny for double transgenic, non-human animals which bear beta cell tumors, the proliferation of which can be controlled by tetracycline or a derivative thereof; and (e) then isolating the beta cells.
The present invention also provides a method for producing recombinant, glucose-regulated insulin producing beta cells whose proliferation is controlled by tetracycline or a derivative thereof, comprising the steps of: (a) introducing into a beta cell, a first gene comprising a DNA encoding a TetR-VP16 fusion protein, and an insulin promoter which controls expression of the fusion protein, and a second gene comprising a DNA encoding SV40 T antigen, and a tetracycline operator minimal promoter, such that stable integration of both genes is acheived; and (b) screening for cells whose proliferation is controlled by tetracycline or a derivative thereof.
The present invention further provides a method for producing recombinant cells whose proliferation is controlled by tetracycline or a derivative thereof, comprising the steps of: (a) introducing to a first, nonhuman animal a first plasmid comprising a DNA encoding a tetR-VP16 fusion protein, and a promoter specific to said cell which controls expression of said fusion protein, such that a first, genetically-modified, non-human animal is obtained; (b) introducing to a second, non-human-animal a second plasmid comprising a DNA encoding SV40 T antigen, and a tetracycline operator minimal promoter, such that a second, genetically-modified, non-human animal is obtained; (c) crossing said first, genetically-modified, non-human animal, or offspring thereof, with said second, genetically-modified, non-human animal, or offspring thereof, to obtain progeny thereof; (d) screening said progeny for double-transgenic, non-human animals which bear tumors, the proliferation of which is controlled by tetracycline, or a derivative thereof; and (e) isolating said cells.
Lastly, the present invention provides a method for producing recombinant cells whose proliferation is controlled by tetracycline or a derivative thereof, comprising the steps of: (a) introducing into a cell, a first gene comprising a DNA encoding a TetR-VP16 fusion protein, and a promoter specific to said cell which co

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