MXPA97007727A - Recombinant cellular and uses of the mi - Google Patents

Abstract

The present invention relates to a recombinant beta cell that produces insulin regulated by tetracycline or a derivative thereof. The present invention also provides a method for treating a patient with diabetes using the recombinant beta cell of the present invention.

Description

"RECOMBINANT ß CELL AND USES OF THE SAME" BACKGROUND OF THE INVENTION Diabetes mellitus is a chronic disorder of carbohydrate metabolism characterized by insufficient insulin production by pancreatic beta cells. Diabetes affects 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 usually characterized by an absolute deficiency of insulin production, while non-insulin-dependent diabetes is characterized by relatively insufficient insulin production. In normal people, the regimen of insulin secretion by beta cells is regulated by the level of glucose in the blood. When the level of glucose in the blood rises, the islet cells are stimulated to release increased amounts of insulin into the blood, accelerating the transport of glucose to the cells and the conversion of glucose into glycogen. As the level of glucose in the blood decreases, insulin is released from the islets and decreases the release of insulin from the islets. In the diabetic patient, the production of insulin is abnormally low and insufficient, resulting in abnormally high blood glucose levels, a condition known as hyperglycemia. In addition to diet and exercise programs, constant and lifelong monitoring of blood glucose levels, along with insulin injections, is central to current methods for the treatment of a diabetic patient who depends on insulin. Many diabetic patients, however, have difficulty controlling their blood glucose levels using current treatment methods, thus constantly exposing themselves to the detrimental effects of hypoglycemia (abnormally low blood glucose levels) and hyperglycemia. . The inability to accurately control the blood glucose level also presents long-term complications such as degenerative vascular changes (e.g., atherosclerosis and microangiopathy), neuropathy (e.g., peripheral nerve degeneration) and autonomic nervous system and cranial nerve lesions), ocular disturbances (eg, blurred vision, cataracts and diabetic retinopathy), kidney diseases (eg, recurrent pyelonephritis and nephropathy), and injections. Accordingly, there is a need for an alternative method to control blood glucose levels in the diabetic patient. 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 due to the limited availability of donors; Similarly, the cost and effort, as in terms of labor, associated with obtaining sufficient quantities of animal islets for transplantation also limits their use. These and other disadvantages associated with the transplantation of human and animal islets, makes the development of cell lines derived from islets the selection method to obtain sufficient quantities of cells for transplantation. In particular, a number of beta cell lines have been generated from insulinomas and hyperplastic islets that arise in mice, expressing a transgenic encoding of the antigen SV40 T (Tag) under the control of the insulin promoter (RIP-Tag) (1-6). Several of these cell lines followed insulin secretion characteristics similar to those observed in intact adult islets, in particular the response to glucose concentrations in the physiological scale (5-15 millimoles / liter).

However, a common problem encountered with all these cell lines is their phenotypic instability. After propagation in the tissue culture, these cells become responsive in subphysiological concentrations of glucose and / or manifest decreased insulin yield (4, 6-9). Similar instability has been observed in the beta cell lines derived by other methods (10-12). The present invention overcomes the problems associated with the above beta cell lines by providing a beta cell line that not only maintains blood glucose levels within the normal range, but can also be controlled to prevent unregulated proliferation.

COMPENDIUM OF THE INVENTION The present invention provides a glucose-regulated recombinant 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 the amount sufficient to maintain physiologically acceptable glucose levels in a seat to which the microcapsule has been implanted. The present invention also provides a method for treating a patient with diabetes comprising (a) implanting in the patient, recombinant beta cells whose proliferation is controlled by tetracycline or a derivative thereof, in an amount effective to establish and maintain physiologically glucose levels acceptable in the patient's blood; and (b) inhibiting the proliferation of the implanted recombinant beta cells by administering to the patient an amount of tetracycline or a derivative thereof, which is effective to inhibit the proliferation of the implanted recombinant beta cells. The present invention also provides a method for producing a glucose-regulated combinatorial insulin producing beta cell and the proliferation is controlled by tetracycline or a derivative thereof comprising the steps of: (a) introducing a first non-human animal into a first plasmid comprising a DNA coding, a tetR-VP16 fusion protein, and an insulin promoter that controls the 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 minimal tetracycline operator (Te) promoter, such that a second genetically modified non-human animal is obtained; (c) crossing the first typically modified non-human animal or offspring thereof, with a second genetically modified non-human animal or offspring thereof, to obtain offspring; (d) selecting the offspring for double transgenic non-human animals, which carry 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 insulin-producing beta cells regulated by glucose 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 that controls the expression of a fusion protein, and a second gene comprising a DNA encoding the SV40 T antigen and a minimum tetracycline operator promoter in such a manner that the stable integration of both genes is achieved; and (b) selecting 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 into a first non-human animal a first plasmid comprising a DNA encoding a fusion protein tetR-VP16 and a promoter specific for the cell that controls the expression of the fusion protein, in such a way that a first non-human animal is genetically modified; (b) introducing into a second non-human animal a second plasmid comprising the DNA encoding the SV40 T antigen and a minimal tetracycline operator promoter such that a second genetically modified non-human animal is obtained; (c) using the first genetically modified non-human animal or offspring thereof with the second genetically modified non-human animal or offspring thereof to obtain offspring thereof; (d) selecting the offspring for non-human double-transgenic anmimals bearing tumors, the proliferation of which is controlled by tetracycline or a derivative thereof; and (e) isolating said cells.

Finally, 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 for that cell that controls the expression of a fusion protein, and a second gene comprising a DNA encoding the SV40 T antigen and a minimal promoter of tetracycline operator in such a way that it is achieved the stable integration of both genes; and (b) selecting cells whose proliferation is controlled by tetracycline or a derivative thereof.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is comprised of Figures IA and IB and represents gene constructs used in the conditional transformation strategy. Figure IA illustrates the construction of tet-Tag. The SV40 Tag gene was placed under the control of a tandem formation of PC operator sequences and a minimal promoter (shaded box). Figure IB illustrates the construction of RIP-tTA. The fusion gene is encoded tetR and the activation domain in the HSV VP16 protein was placed under the control of the RIP promoter, downstream of an intron element (int) and upstream of a polyadenylation signal (An). Figure 2 is comprised of Figures 2A, 2B, 2C, 2D, 2E and 2F, which in effect represents Te in the growth of the ßTC-tet cell and Tag expression. Equal numbers of ßTC-tet cells and two series of wells were seeded. They were grown for three weeks in the absence (Figure 2A) or presence (Figure 2B) of one microgram per milliliter of Te and photographed in a phase contrast microscope. Similar cells were incubated in 16-well slide slides for 7 days in the absence (Figures 2C and 2E) or presence (Figures 2B and 2F) of one microgram per milliliter of Te. The cells were boosted for one hour with BrdU (Figures 2C and 2D) and stained with an anti-BrdU monoclonal antibody. The cells in the separated wells were stained with a Tag antiserum (Figures 2E and 2F). The ligated antibodies were visualized with second antibodies conjugated with horseradish peroxidase. The cells shown are representative of three independent experiments. The amplification is 200 times. Figure 3 depicts the arrest of the growth of ßTC-tet cells after incubation with Te and anhydrotetracycline (ATc). 2X10 ^ cells in quadrupled wells were incubated for 7 days in the presence of the indicated concentration of Te (circles) or ATc (squares). Then they were boosted with [^ H] thymidine for 6 hours, followed by quantification of the radioactivity incorporated in the DNA. The values represent the percentage of accounts in the absence of drugs, averaging 4X10 ^ cpm per ozo. Figure 4 is comprised of Figures 4A, 4B, 4C and 4D, and represents the effect of Te on Tag expression and in vivo proliferation of the ßTC-tet cell. Mice with ßTC-tet tumors received regular drinking water (Figures 4A and 4C) or water containing Te (Figures 4B and 4D) for 7 days. Then they were boosted with BrdU antisera (Figures 4A and 4B) and Tag (Figures 4C and 4D). The ligated antibodies were visualized with second antibodies conjugated with horseradish peroxidase. The amplification is 230 times. Figure 5 shows that ßTC-tet cells maintain normal blood glucose levels in diabetic recipients. Mice that became diabetic by treatment with streptozotocin were implanted intraperitoneally with 2 × 10 6 cells (circles, frames) or did not receive cell implantation (triangles). Cell implantation time is shown as day 0. Blood glucose levels were measured weekly. When the blood glucose was corrected, the mice in a group (circums) were implanted with slow release Te granules (arrow) The blood glucose levels in this group remained stable while in the group that was not treated with Te (tables) the blood glucose continued to decrease as a result of the uncontrolled proliferation of the cells that secrete insulin. The hypoglycemia in this group results in the death of a mouse with a tumor at 32 days and two mice at 50 days. The values are medium + SEM (n = 4). The difference between the two groups injected with the cells + Te at the time points of 39 and 46 days is significant by the t test (p <0.01).

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a recombinant human or animal insulin-producing beta cell regulated by glucose whose proliferation can be controlled by incubating the cell with tetracycline or with one of its derivatives. In the preferred embodiment of the present invention, the recombinant beta cell is contained within the cell line designated ßTC-tet which was deposited under the terms of the Budapest Treaty of March 31, 1995 before the American Type Culture Collection ( ATCC), of Rockville, Maryland, and the ATCC accession number CRL-11869 was assigned. The vitality of the cell line was confirmed on April 6, 1995. The present invention also provides the beta cell line deposited with ATCC under Accession No. CRL-11869. The recombinant beta cell of the present invention can be produced by crossing two lineages of transgenic non-human animals, such as cows, pigs, mice and mice are preferred. The beta cells of a lineage contain a fusion protein consisting of tetR and the activation domain of the HSV VP16 protein under the control of an insulin promoter. The combination of tetR and HSV VP16 sequence converts tetR into a transcriptional activator. The other lineage of transgenic mice contains the Tag gene under the control of a tandem formation of sequences of Te operators, a minimal promoter. The minimal promoter alone is incapable of directing the expression of the Tag gene. However, in the presence of the transcription activator tetR-VP16, called the transactivator controlled by tetracycline (tTA), the expression of the Tag gene is activated. The double transgenic mice, the Tag expresses and this expression results in beta cell tumors. Beta cell tumors, whose proliferation is inhibited by tetracycline by one of its derivatives, are then selected to obtain the beta cell of the present invention. The recombinant beta cell of the present invention can also be produced by introducing into the beta cells the tissue culture of a first gene encoding a fusion protein consisting of the tetR and the activation domain of the HSV VP16 protein under the control of an insulin promoter together with a second gene encoding the SV40 T antigen under the control of a tandem formation of the Te operator sequences, and a minimal promoter. Beta cells can be, but are not limited to beta cells of cow, pig, mouse or human origin and are preferably of human origin. The genes can be introduced by stable transfection of the beta cells by methods well known to those skilled in the art, such as calcium phosphate precipitation, cationic liposome fusion or electroporation. Alternatively, the genes can be introduced into the beta cells using viral vectors such as herpes virus-, adenovirus- or retrovirus-based vectors by techniques well known to those skilled in the art. Beta cells whose proliferation is inhibited by tetracycline or one of its derivatives, is then selected to obtain the beta cell of the present invention. Recombinant beta cells due to their retention of normal beta cell characteristics with respect to insulin secretion and blood glucose regulation offer an alternative to insulin administration in the treatment of diabetes in both animals and humans. Accordingly, the present invention also provides a method for treating a diabetic patient. The method comprises implanting the recombinant beta cells in the body of the diabetic patient, in an amount effective to establish and maintain physiologically acceptable blood glucose levels.; and then inhibit the proliferation of recombinant beta cells by administering tetracycline or one of its derivatives, in an amount sufficient to inhibit the proliferation of recombinant beta cells. Beta cells can be implanted in any feasible location within the body where they are placed in contact with the bloodstream of the recipient. Appropriate locations include but are not limited to the peritoneal cavity or the pancreas. Other locations will be apparent to a person skilled in the art. Beta cells can be implanted by methods known to those skilled in the art such as by surgical means, injection and the like. The effective amount of the beta cells is preferably from about 100 to about 300 million cells. The effective amount of beta cells, however, will depend on the method of implantation, the pharmacokinetic characteristics of the treated patient and / or the presence of other diseases or conditions. These amounts are easily determined by a person skilled in the art. The rejection of the implanted beta cells can be controlled by the administration of immunosuppressive drugs such as cyclosporin or azathioprine and the like. Alternatively, beta cells can be microencapsulated before implantation. The term "microencapsulation" as used herein, means any method that can be used to protect foreign cells introduced into the body of a recipient from destruction by the recipient's immune system. The microencapsulation methods include but are not limited to the methods described in Patent Numbers 5,389,535, 5,334,640 and the tissue implantation systems described in US Patent Nos. 5,314,471 and 5,344,454, which are incorporated herein by reference. Other means for microencapsulation of beta cells or alternative tissue implantation systems will be apparent to a person skilled in the art. The present invention also provides a microcapsule comprising an amount of the aforementioned beta cell that is sufficient to maintain physiologically acceptable glucose levels in a patient implanted with the microcapsule. Preferably, the beta cells are obtained from the cell line deposited with ATCC under Accession No. CRL-11869. As above, the amount of beta cells can be from about 100 to about 300 million cells. Again, the actual amount will depend on the method of implantation, the pharmacokinetic characteristics of the treated seat and / or the presence of other diseases or conditions. These amounts are easily determined by a person skilled in the art. The term "microcapsule" as used herein means any vehicle, polymer composition or similar means used in a microencapsulation process for implantation in the body of a patient. Tetracycline can be obtained commercially from Sigma Chemical Company, of St. Louis, Missouri. Tetracycline derivatives include but are not limited to anhydrotetracycline, 7-chloro-tetracycline, 4-Epi-7-chloro-tetracycline, oxy-tetracycline, doxycycline, 6-deoxy-6-demethyl-tetracycline, and 7-azido-6 -deoxi-6-demethyl-tetracycline. These derivatives and others can be obtained commercially. Anhydrotetracycline is preferred because it binds tetR more efficiently than tetracycline and has a lower antibiotic activity, thus allowing long-term administration. Tetracycline and its derivatives can be administered orally, by intravenous or intramuescular injection. Other modes of administration will be apparent to a person skilled in the art. The amount of tetracycline is an amount effective to inhibit the proliferation of beta cells. The current dosage will depend on the route of administration and the individual pharmacokinetic parameters of the treated patient. The current dosage is easily determined by a person skilled in the art. The present invention also provides a general method for producing recombinant cell lines from a variety of cell types in addition to beta cells. These cell lines can be produced by any of the methods described above to generate recombinant beta cells. When producing these cells, however, the insulin promoter used to control the expression of the TetR-VP16 fusion protein in the beta cell, is replaced by a - promoter specific to the interest rate cell. These cell-specific promoters are well known to those skilled in the art. For example, by producing recombinant liver cells of the present invention, a liver-specific promoter such as the albumin promoter could be used. The present invention is described in the following Experimental Details Section which is disclosed to aid the understanding of the invention, which should not be construed as in any way limiting the invention as described in the claims that will be given below.

SECTION OF EXPERIMENTAL DETAILS A. Materials and Methods Plasmid constructions. Plasmids pUHD 10-3 and pUHD 15-1 were obtained from H. Bujard, Zentrum fur Molekulare Biologie der Universitat Heidelberg, Im Neuenheimer Feld 282, W-6900 Heidelberg, Germany. To construct the tet-Tag plasmid, the Xho I-Xba I fragment of pUHD 10-3 (13), which contains a tandem formation of 7 copies of the Te operator sequence and a minimal CMV promoter, was placed ahead of the T antigen gene in pRIP-Tag (14) suppressing the Aat II-Xba I fragment that contains the RIP of pRIP-Tag and converting the sites of Aat II and Xho I into blunt ends. To generate pRIP-tTA, the EcoR I-BamH I tTa fragment from pUHD 15-1 (13) was blunt-ended with Klenow and ligated into the Sma I site of pMLSIS.CAT (15), after removal of the Pst I - Sma I CAT fragment. This placed the tTA gene downstream of a hybrid intron element and upstream of the late SV 40 polyadenylation site. The combined 1630-bp fragment was inserted between the Xba I and Sal I sites of pRIP-Tag downstream of the insulin II promoter of the rat.

Transgenic mice. The linearized DNA plasmid was microinjected into the 1-cell mouse C3HeB / FeJ elembion. The transgenic mice were generated and cultured according to the established procedures (16).

Cell culture. The tumors were removed from the pancreas, and a ß cell line called ßTC-tet was established and propagated, as described in (2). The supply of all media was from GIBCO. Cells were grown in DMEM containing 25 mM glucose and supplemented with 15 percent horse serum, 2.5 percent fetal bovine serum, 100 units per milliliter of penicillin and 100 micrograms per milliliter of streptomycin. Tetracycline (United States Biochemical Corporation) and anhydrotetracycline (ATc) (Lederle) were included at the indicated concentrations.

Implantation of cells. The cells were trypsinized, washed in PBS, and resuspended in PBS at 5x10 ^ cells per milliliter. Receiving C3H mice were injected intraperitoneally (I.P.) with 106 cells each and kept in regular drinking water or in water containing 1 milligram per milliliter of Te, and 2.5 percent sucrose. Each group included 4 to 5 mice. They were monitored weekly for blood glucose using Glucometer strips. To generate diabetic mice, 12 male C3H mice were injected I.P. with a dose of 200 milligrams of streptozotocin (Sigma) per kilogram of body weight, followed by 7 days later by 3 doses of 50 milligrams per kilogram in 3 consecutive days, which caused hyperglycemia within 6 to 9 additional days. Then eight I.P. mice were injected. with 2x10 ^ ßTC-tet cells, while 4 mice were maintained as diabetic controls. The mice were monitored weekly to determine the levels of glucose in the blood. When euglycemia was obtained in the group implanted with the cells, 4 of the mice in this group were implanted subcutaneously with a granule of slow release Te (Innovative Research of America) designed to release 3.3 milligrams per day, which were also followed by checks of blood glucose weekly. Glucose levels less than 40 milligrams per deciliter were determined using a Beckman glucose analyzer.

Immunohistochemistry The cells were plated in 16-well slide plates (Nunc) during the indicated period in the absence or presence of Te or ATc. For the BrdU incorporation assay, cells were boosted for 60 minutes with 10 μM BrdU (Sigma) and stained with an anti-BrdU monoclonal antibody (Becton-Dickinson) according to the manufacturer's recommendations. The ligated antibody was visualized with biotinylated anti-mouse IgG and avidin conjugated with horseradish peroxidase (Vector, kit ABC) and a diaminobenzidine (DAB) substrate. The cells in the separated wells were stained with a rabbit anti-Tag serum (17). The ligated antibody was visualized with a goat anti-rabbit antibody conjugated with strong horseradish peroxidase and DAB. Mice with ßTC-tet tumors were injected I.P. with 100 micrograms of BrdU / grams of body weight. One hour later they were sacrificed and the tumors were removed, fixed with 4 percent stabilized formaldehyde, processed for paraffin incrustation and sectioned. Tumor sections were stained with anti-Tag and anti-BrdU antibodies as described above.

Thymidine incorporation assay. 2X10 ^ cells were plated in 96-well plates. After the indicated incubation, they were boosted with a micro Ci of [methyl- ^ H] thymidine (Amersham, 78 Ci / millimole) for 6 hours. The cells were then subjected to lysate in water using a cell harvester and the DNA was retained on a glass fiber filter (Whittaker). The filters were dried and the radioactivity incorporated in the DNA was quantified in a scintillation counter. Each condition was tested in quadruplicates.

B. Results The tTa gene was placed under the control of RIP (Figure 1), and the construct was used to generate transgenic mice, where tTA was constitutively expressed specifically in beta cells. In a separate lineage of the transgenic mice, the Tag gene was introduced under the control of a minimal promoter and tandem formation of Te operator sequences (Figure 1). This promoter does not allow the expression of the gene per se.

Therefore, as expected, these transgenic mice do not develop tumors. The two lines of mice were crossed to generate double-transgenic mice. In these mice, the tetR part of the tTA protein is expected to bind the sequence of white Te operators in the ß cells and allow the VP16 part of the molecule to activate the transcription of the Tag gene. This resulted in the development of multiple ß-cell tumors from 5 to 6 months of age. No tumors were detected in other organs, demonstrating the need for tTA-induced expression of the Tag for the development of specific β-cell tumor. The tumor cells are cultured to derive a stable cell line, called ßTC-tet. When incubated in the presence of one microgram per milliliter of Te, the cells undergo growth arrest as demonstrated by the difference in the number of cell size colony between Figures 2A and 2B. This effect is reversible. Removal of Te after the 3-week incubation resulted in duplication of cells resumed (not shown). The effect of Te in the duplication of DNA in the ßTC-tet cells was analyzed by visualizing the incorporation of BrdU with an anti-BrdU monoclonal antibody. In the absence of Te, many duplicate cells incorporated BrdU during a 1-hour pulse (Figure 2C). After a 3-day incubation in 1 microgram per milliliter of Te, only a small number of cells incorporated BrdU (data not shown). After a 7-day incubation, none of the cells was found to incorporate BrdU (Figure 2D). The effect of Te on Tag expression was analyzed by immunohistochemistry with a Tag antiserum. After an incubation of 7 days in 1 microgram per milliliter of Te, the immuno-stained Tag disappeared from most of the cells (Figure 2F). However, a small number of cells kept Tag dye detectable. The effect of the different concentrations of Te on the growth of the cells was analyzed by incorporation of 3 H-thymidine into the duplication DNA (Figure 3). A complete elimination of duplication was achieved in the presence of 100 ng / milliliter of Te. The binding of the derivative of Te, ATc to TetR has been shown to be 35 times more intense than that of Te with respect to TetR (18). As shown in Figure 3, incubation of ßTC-tet cells in the presence of ATc resulted in complete growth arrest at 1 ng / milliliter. Cells from another ß, ßTC3 (2) cell line, which was derived from mice expressing a RIP-Tag gene that does not respond to the regulation of Te, were used as controls in these experiments. Incubation of ßTC3 cells in the presence of 1 microgram per milliliter of either Te or ATc did not affect their growth regimen, BrdU and 3 H-thymidine incorporation, and Tag dyeing (not illustrated). To test the ability of Te to regulate cell growth in vivo, the C3H syngeneic mice were injected with 10 ^ ßTC-tet cells intraperitoneally. The ßTC cell lines are tumorigenic and form benign tumors at the site of the injection (2). Tumor development leads to hypoglycemia and can be detected by monitoring blood glucose levels. The mice in a group were kept in drinking water containing Te. None of these developed tumors within 14 weeks, as judged by blood glucose measurements and a careful autopsy. No abnormalities were observed as a result of prolonged Te treatment. Mice maintained in the absence of Te developed hypoglycemia and tumors within 8 to 13 weeks. When hypoglycemia was detected, a subgroup continued to drink water regularly. The mice were then boosted with BrdU, sacrificed, and the tumors were removed and processed for immunohistochemical analysis. The tumors of the mice that were not treated with Te contained numerous cells that were stained for BrdU and Tag (Figures 4A and 4C). In contrast, tumors of mice treated for 7 days with Te did not show BrdU or Tag stain (Figures 4B and 4D). These results demonstrate that the Te effectively inhibits Tag expression and proliferation of the β cell in vivo. ßTc-tet cells demonstrate correct response to glucose in the physiological concentration scale (data not shown). To assess its ability to maintain glucose homoestasis in vivo, ßTC-tet cells were implanted in diabetic recipients (Figure 5). The implantation of the cell led to correction of hyperglycemia within two weeks, demonstrating the ability of ßTC-tet cells to function as normal β cells in vivo. As seen in the past with other ßTC lines, the implanted cells continued to proliferate in mice not treated with Te, which resulted in hypoglycemia and premature death. In contrast, in mice implanted with slow release Te granules, blood glucose levels were stabilized within the normal range. Normal blood glucose levels were maintained as long as the mice were followed, 4 months later by Te implantation. These results indicate that the cells undergo growth arrest as a result of CT-induced inhibition or Tag expression, but remain viable and capable of detecting normal glucose and insulin production and secretion.

C. Discussion These results demonstrate the ability to regulate oncogenic expression and cell proliferation by controlling the binding of tetR to its consanguineous operator sequence in the presence of Te or its derivative ATc. The concentration of Te required to complete the inhibition of cell duplication is less than 0.1 microgram per milliliter. ATc, which binds to tetR with higher affinity, can achieve this effect or concentration less than 100 times. At the same time, the ATc has a very weak antibiotic activity, since its binding to the ribosome is greatly reduced, in comparison with Te (18). These properties yield ATc, a more attractive coordinating group for prolonged in vivo treatments. A 7-day incubation of ßTC-tet cells in the presence of one microgram per milliliter of Te did not completely eliminate the Tag protein from all cells, as judged by immunohistochemical analysis. In contrast, after a 7-day Te treatment in vivo, no Tag was detected. This may represent the prolonged stability of Tag protein or mRNA in the culture, or escape or leakage of the regulatory system. However, it should be noted that Tag transformation activity requires oncoprotein threshold levels (19) that are sufficient for stoichiometric interactions, such as evaluation of tumor suppressor gene products. As demonstrated by the incorporation assays of BrdU and [-] thymidine, the treatment of Te possibly down-regulated the Tag levels to less than this functional threshold. These results reveal the dependence of transformed beta cells on the continuous expression of Tag oncoprotein for its proliferation. The development of ß-cell tumors in these mice is a rare event that occurs in 1 percent to 2 percent of the islets. This has suggested that additional genetic changes are involved in the cells. However, our results indicate that these changes probably do not include mutations in genes that regulate the cell cycle, since the cells continue to require Tag activities to remain in cycle. The ability to control the proliferation of cells in vivo, by administration of Te in drinking water or with slow-release granules, and the fact that the inhibition of gene expression by Te is reversible during the removal of the drug, provides an experimental system to study the role of Tag oncoprotein in the different stages of tumorigenesis. In addition, the tet-Tag mice will allow the derivation of conditionally transformed cell lines from other cell types, focusing the expression of the tTA fusion protein with the specific promoters of the appropriate cell. Similarly, mice expressing the tTA protein in β-cells can be used to obtain reversible expression of other genes of interest in these cells, using them with mice expressing these genes under the control of the minimal Te operator promoter. The ßTC cell lines will allow studies on the effect of cell proliferation on the expression of differentiated functions in ß cells. The results obtained with cells transplanted in diabetic mice show that the secretion of insulin from the ßTC-tet cells arrested in their growth remains regulated correctly, which allows them to maintain the levels of glucose in the blood within the physiological scale. To determine the effect of cell proliferation on the synthesis and secretion of glucose-induced insulin in these cells, cells propagated in culture and induced to undergo growth arrest in present Te, will be studied in comparison with cells that actively proliferate grown in the absence of Te. The strategy described here will contribute to the development of ß cell lines for diabetes cell therapy, as well as for the generation of conditionally transformed cell lines from other cell types with therapeutic potential.

References 1. R. D'A bra, et al., Endocrinology 126: 2815-2822 (1990). 2. S. Efrat, et al., Proc. Nati Acad. Sci. USA 85: 9037-9041 (1988). 3. J.-I. Miyazaki et al., Endocrinology 127: 126-132 (1990). 4. F. Radvanyi, and others, Mol. Cell. Biol. 13: 4223-4232 (1983). 5. K. Ha aguchi, et al., Diabetes 40: 842-849 (1991) 6. S. Efrat, et al., Diabetes 42: 901-907 (1993). 7. H. Ishihara, et al., Diabetologia 36: 1139-1145 (1993). 8. M. Sakurada, et al., Endocrinology 132: 2659-2665 (1993). 9. M. Tal, and others. Mol. Cell. Biol. 12: 422-432 (1992). 10. M. Asfari, and others Endocrinology 130: 167-178 (1992). 11. A.F. Gazdar, and others, Proc. Nati Acad. Sci. USA 77: 3519-3523 (1980). 12. D.A., Nielsen, et al. J. Biol. Chem. 260: 13585-13589 (1985). 13. M. Gossen and H. Bujard, Proc. Nati, Acad. Sci. USA 89: 5547-5551 (1992) 14. D. Hanahan, Nature 315: 115-122 (1985). 15. M.T.F. Huang, and C.M. Gorman, Nuc. Acids Res. 18: 937-947 (1990). 16. B. Hogan, et al. (1986) Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Plainview, NY). 17. S. Efrat and D. Hanahan, Mol. Cell. Biol. 7: 192-198 (1987). 18. J. Degenkold, et al., Antimicrob. Agents Chemosther, 35: 1591-1595 (1991). 9. S. Efrat, and D. Hanhan, (1989) in Transforming Proteins of DNA Tumor Viruses (R. Knippers, and A.J. Levine, editors), pages 89-95, Springer-Verlag, Berlin.

All publications mentioned above are incorporated herein in their entirety. Although the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by a person skilled in the art of reading the exhibition, that various changes in form and detail may be made without deviating from the scope true of the invention in the appended claims.

Claims (28)

CLAIMS:

1. A beta-cell producing recombinant insulin, regulated in glucose whose proliferation is controlled by tetracycline or a derivative thereof.

2. The beta cell of claim 1, contained within the cell line deposited with ATCC under Accession No. CRL-11869.

3. The beta cell line deposited with ATCC under Accession Number CRL-11869.

4. A microcapsule comprising an amount of beta cells of claim 1, sufficient to maintain physiologically acceptable glucose levels in a patient implanted with the microcapsule.

5. A microcapsule comprising an amount of the beta cell of claim 2, sufficient to maintain physiologically acceptable glucose levels in a patient implanted with the microcapsule.

6. A method for treating a patient with diabetes comprising: (a) implanting in the patient, recombinant insulin, regulated in glucose that produces beta cells whose proliferation is controlled by tetracycline or a derivative thereof, in an amount effective to establish and maintaining physiologically acceptable glucose levels in the patient's blood; and (b) inhibiting the proliferation of the implanted recombinant beta cells by administering to the patient an amount of tetracycline, or a derivative thereof, effective to inhibit the proliferation of the implanted recombinant beta cells.

The method of claim 6, wherein the implanted cells are obtained from the cell line deposited with ATCC under Accession No. CRL-11869.

The method of claim 6, wherein the implanted beta cells are microencepsulated.

The method of claim 6, wherein the beta cells are implanted in the peritoneal cavity of the patient.

The method of claim 6, wherein the beta cells are implanted in the patient's pancreas.

The method of claim 6, wherein the tetracycline derivative is selected from the group consisting of anhydrotetracycline, 7-chloro-tetracycline, 4-Epi-7-chloro-tetracycline, oxy-tetracycline, doxycycline, 6-deoxy- 6-demethyl-tetracycline and 7-azido-6-deoxy-6-demethyl-tetracycline.

12. The method of claim 11, wherein the tetracycline derivative is anhydrotetracycline.

13. A method for producing recombinant insulin-producing beta-cells regulated in glucose 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 that controls the expression of the fusion protein, such that a first genetically modified non-human animal is obtained; (b) introducing a second non-human animal, a second plasmid comprising a DNA encoding the SV40 T antigen, and a minimal tetracycline operator 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 the offspring thereof to obtain offspring thereof; (d) selecting the offspring for non-human double-transgenic animals suffering from beta-cell tumors, the proliferation of which is controlled by tetracycline, or a derivative thereof; (e) isolate the beta cells.

The method of claim 13, wherein the non-human animal is a cow, a pig or a mouse.

15. The recombinant beta cells produced by the method of claim 13.

16. A method for producing insulin-regulated beta-cells in glucose whose proliferation is controlled by tetracycline or a derivative thereof, comprising the steps of: ) introducing into a beta cell, a first gene comprising a DNA encoding a TetR-VP16 fusion protein, and an insulin promoter that controls the expression of the fusion protein, and a second plasmid comprising a DNA encoding the SV40 T antigen, and a minimal promoter tetracycline operator, in such a way that the stable integration of both genes is achieved; and (b) selecting beta cells whose proliferation is controlled by tetracycline or a derivative thereof.

17. The method of claim 16, wherein the beta cells are of cow, pig, mouse or human origin.

18. The method of claim 16, wherein the genes are introduced by transfection.

19. The method of claim 16, wherein the genes are introduced by a viral vector.

20. The recombinant betas cells produced by the method of claim 16.

21. A method for producing recombinant cells whose proliferation is controlled by tetracycline or a derivative thereof, comprising the steps of: (a) introducing a first animal , non-human, a first plasmid comprising DNA encoding a tetR-VP16 fusion protein and a promoter specific for the cell that controls the expression of the fusion protein, such that a first genetically modified non-human animal is obtained; (b) introducing a second non-human animal, a second plasmid comprising a DNA encoding the SV40 T antigen, and a minimal operator promoter of tetracycline in such a manner 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 offspring thereof; (d) selecting the offspring for non-human double-transgenic animals, which carry tumors, the proliferation of the cells is controlled by tetracycline, or a derivative thereof; and (e) isolating these cells.

22. The method of claim 21, wherein the non-human animal is a cow, a pig or a mouse.

23. The recombinant cells produced by the method of claim 21.

24. 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 cell-specific promoter that controls the expression of the fusion protein, a second gene comprising a DNA encoding the SV40 T antigen, and a promoter minimum tetracycline operator in such a way that a stable integration of both genes is achieved; and (b) selecting cells whose proliferation is controlled by tetracycline or a derivative thereof.

25. The method of claim 24, wherein the beta cells are of cow, pig, mouse or human origin.

26. The method of claim 24, wherein the genes are introduced by transfection.

27. The method of claim 24, wherein the genes are introduced by a viral vector.

28. The recombinant cells produced by the method of claim 24.