Chapter 27
Preparation of Insulin-Releasing Chinese Hamster Ovary Cell by Transfection of Human Insulin Gene Polymers of Biological and Biomedical Significance Downloaded from pubs.acs.org by COLUMBIA UNIV on 12/02/18. For personal use only.
Its Implantation into Diabetic Mice 1
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H. Iwata , N. Ogawa, T. Takagi , and J. Mizoguchi 1
National Cardiovascular Center, Suita-city, Osaka 565, Japan Falco Biosystems, Kumiyama-cho, Kuze-gun, Kyoto 613, Japan Asahi Chemical Industry Company, Ltd., Tahou-gun, Shizuoka 410-23, Japan 2
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The feasibility of gene therapy in the treatment of diabetes was examined. Insulin releasing CHO cells were prepared by transfection of a plasmid that contained human insulin gene under control of the chicken actin promoter. They were implanted into peritoneal cavities of diabetic mice in three different forms. When singly dispersed cells were implanted, only 2 of 11 recipients showed normoglycemia. In the case of implantation of microencapsulated cells, blood glucose levels of all recipients were only transitorily decreased. In the third form of the transplants, that is cells on microcarriers, all of the recipients became normoglycemic and half of them exhibited more than 50 days of normoglycemia. This study demonstrated that insulin releasing cells prepared by genetical modification could normalize glucose levels for a long period if they were implanted in an appropriate form.
If we accept that gene therapy is thought of as in vivo protein production and its delivery system is by genetically modified cells, almost all diseases that are currently treated by the administration of protein are candidates for treatment using gene therapy. There have been only a few studies which examined the feasibility of gene therapy in the treatment of diabetes(l,2). Many problems still remain to be examined in detail. For example, recipients were frequently lost by hypoglycemia during these investigations. In the treatment of disease such as diabetes, overproduction of protein caused harmful side effects. On the other hand, genetically modified cells sometimes released a sufficient amount of the gene product in in vitro culture, but could not provide therapeutic levels of it after they were implanted into animals. The function and fate of genetically modified cells in animals have not been clarified yet. In this study we used xenogeneic cells as the host cell of the insulin gene. We tried to regulate the number of implanted cells by controlling the immune reactions to the implanted cells. We also examined methods to implant cells to 0097-6156/94/0540-0306$06.00/0 © 1994 American Chemical Society
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effectively draw the functions of the cells in vivo. Figure 1 schematically shows the strategy of this research. Experimental Cell: A mutant K l cell line of Chinese hamster ovary cells (CHO cells) which lacked the enzyme dihydrofolate reductase (DHFR) was used as a host cell (3). It could not grow without supplements, such as thymidine, glycine, and purine. The expression of DHFR was used as a selective maker of genetically modified cells. A culture medium was composed of α-Minimal Essential Medium (Sigma, St. Louis, MO) supplemented with 50 U/L Penicillin and 50U/L Streptomycin (Whittaker Bioproducts, Inc. Md.) and 5% dialyzed fetal bovine serum (Whittaker Bioproducts, Inc. Md.) Plasmid: The DHFR gene was inserted into the BamHI site of a plasmid which expressed the human insulin gene under control of the chicken actin promoter (4). The resulting construct which is termed by pACT-HIN-DHFR, is shown in Figure 2. It was prepared and purified by a conventional method. Closed circular DNA was purified by equilibrium centrifugation in a CsCl-ethidium bromide density gradient. Introduction of the plasmid into cells: The plasmid pACT-HIN-DHFR was introduced into CHO cells by a highly efficient calcium phosphate method (5). Cells which were transfected with the plasmid were selected in a culture medium without supplements such as thymidine, glycine, and purine. Selected cells expressed a cloned copy of the DHFR gene and insulin gene. Plasmid DNA introduced into cells was incorporated into chromosomal DNA during culturing. They can release insulin into the culture medium. The ability of insulin release of the obtained cells was amplified by progressive selection of cells resistant to increasing concentrations of methotrexate (MTX) (Sigma, St. Louis, MO). Implantation of insulin releasing cells into diabetic mice: The ability of the insulin releasing cells to reverse diabetes was examined. Male BALB/c (Japan CLEA, Tokyo, JAPAN) mice were made diabetic by an intraperitoneal injection of streptozotocin(Sigma, St. Louis, MO). Only mice of which plasma glucose concentrations were more than 400 mg/dl were used as diabetic recipients. Three different forms of implants, single cells, microencapsulated cells and cells on microcarriers were examined. The cells cultured on the flask (Coming 25110, Iwaki Glass, Tokyo, JAPAN) were harvested as single cells. They were intraperitoneally implanted into a diabetic mouse. The second form was the cells on the microcarrier. The microcarrier was made of microporous cellulose, about 200 μηι in diameter and carrying diethylaminoethyle groups (Asahi Chemical Industry Co., Ltd., Tokyo, Japan). Cells could grow not only on the surface of the carriers, but also inside the pores of the microcarriers. The third one was the implantation of microencapsulated cells. Cells were miroencapsulated into 5% agarose mirocapsules(6). The site of implantation was a peritoneal cavity and cell number was 3x10? in spite of the implant forms. Plasma glucose levels were measured three times a week after implantation. Samples of blood were taken from the subclavian vein. Plasma glucose levels were measured with a Beckman glucose analyzer 2 (Beckman Instruments, Fullerton, CA). Immunosuppressive therapy. The CHO cell, which is derived from the Chinese hamster, is xenogeneic to a recipient mouse. It is expected that the implanted CHO cells are rejected by the host immune system. A immunosuppressive drug, 15deoxyspergualin (DSG) (Nippon Kayaku Co., LTD., Tokyo, Japan) was given (2.5 mg/kg body weight) every day. When the non-fasting plasma glucose levels became
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Figure 1. Schematic representation of this experiment.
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Figure 2. Structure of the pACT-HN-DHFR recombinant plasmid.
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less thanlOO mg/dl, DSG treatment was stopped. When the non-fasting plasma glucose levels exceeded 200 mg/dl, DSG treatment was started again. Determination of anti-CHO antibodvi levels: Antibody levels were determined using a flowcytometer (Japan Spectroscopic Co., Ltd., Tokyo, Japan). Recipient serum was mixed with CHO cells. The mixture was incubated for 30 min. on ice to form antibody-CHO cell complexes. After three washings of CHO cells with Hanks' balanced salt solution (HBSS), a second anti-mouse IgG and IgM antibody which was conjugated withfluorescenisothiocyanate (FITC) was added and left for 30 min. on ice. Cells were washed again with HBSS. FITC positive cells were measured using aflowcytometer.Anti-CHO cells antibody levels in recipient serum were expressed as a percentage of FITC positive cells. Results The cells transfected with plasmid DNA released insulin into a culture medium. However, the amount of insulin release was not sufficient enough to be used to treat a diabetic mice. The copy number of the integrated DNA was increased during culturing cells under the pressure of MTX. Figure 3 shows the abilities of insulin release by CHO cells which was obtained in the culture medium containing a certain concentration of MTX. They were increased by a sequential passage of the cells in the medium containing increasing amounts of MTX. The cell line which was obtained at the 400 nM of MTX released more than 300 μυ insulin per day by 10 cells. The stability of the amplified gene was examined by subculturing the cell in the culture medium without MTX for 5 times during 40 days. It was not observed that the cells lost their ability to release the gene product when the MTX pressure was removed. The cell line which was obtained at 400 nM of MTX was used in the implantation experiments. Singly dispersed cells of 3.3x10^ were implanted intraperitoneally. Blood glucose changes are shown in Figure 4. Before implantation of cells, blood glucose levels of recipients were much higher than the 100 mg/dl blood glucose levels of a normal mouse. Only 2 of 11 mice became normoglycemia in response to implantation of insulin releasing cells for more than 10 days. The blood glucose levels of one of these two was maintained normoglycemic for more than 50 days. However, the other 9 mice could not demonstrate the normal blood glucose levels. Effects of implantation of insulin releasing CHO cells were limited when singly dispersed cells were implanted. Microencapsulated cells in 5% concentration of agarose hydrogel were intraperitoneally implanted into each of 5 diabetic mice. Blood glucose changes are shown in Figure 5. Three of five recipients showed transitory normoglycemia, but blood glucose reverted to preoperative levels. The other two could not demonstrate normoglycemia. Cells cultured on microcarriers were also intraperitoneally implanted into each of 4 diabetic mice. On the contrary to the implantation of the singly dispersed cells and the microencapsulated cells, blood glucose levels of all of recipients were normalized within several days as shown in Figure 6. Two of these demonstrated more than 50 days normoglycemic periods. The blood glucose levels of the other mice decreased to normal levels, but it reverted to preoperative levels after 25 days of observation. The remaining one was lost by hypoglycemia. The CHO cell is xenogeneic to the recipient mice. It is anticipated that it provokes the immune reaction. The anti-CHO cell antibody formed in the recipients was monitored. Figure 7 shows changes in anti-CHO cell antibodies levels in the blood of recipient mice. Although the immunosuppressive drug,15-deoxyspergualin, 5
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Methotrexate, nM Figure 3. The abilities of insulin release by a cell line obtained at a certain concentration of M T X .
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Days After Implantation, days Figure 4. Changes in the non-fasting blood glucose levels of diabetic mice after intraperitoneal implantation of singly dispersed cells, φ ;n=9, Q ;n=l, Δ ;n=l.
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Days After Implantation, days Figure 6 Changes in the non-fasting blood glucose levels of diabetic mice after intraperitoneal implantation of cells cultured on microcarriers.
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Days after Implantation, days Figure 7. Changes in anti-Chinese hamster antibodies levels in recipient plasma. Ο ;Singly dispersed cells, Δ "Microencapsulated cells, # ; Cells on micro carriers
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was given, anti-CHO cells antibodies were formed when free cells and cells on microcarriers were implanted. On the other hand, when microencapsulated cells were implanted, antibody levels did not increase. The immune reaction was not triggered by microencapsulated C H O cells.
Discussion When singly dispersed cells were implanted, only 2 of 11 recipients showed normoglycemia. The blood glucose levels of the other 9 mice were not normalized. Although an immunosuppresive drug was administered, anti-CHO antibody levels increased after implantation of C H O cells. In the case of implantation of microencapsulated cells, anti-CHO cell antibody levels remained low. Microencapsulated C H O cells could not present antigen to recipient mice or triggerimmune reactions of recipients. Blood glucose levels, however, were only transitorily decreased. Frequently one species possesses natural antibodies for cells of distantly related species. Natural antibodies play an important role in the rejection of xenografts(7). Natural antibodies permeated through the 5% agarose microcapsule and attacked the cells in the microcapsule. In the third form of transplants, that is cells on microcarriers, all of recipients become normoglycemic and half of them demonstrated more than 50 days of normoglycemia, in spite of provocation of immune reactions against cells on microcarriers. Cells on the surface of microcarriers were killed by antibodies against C H O cells, but cells which resided inside the porous microcarriers could proliferate. Dynamic equilibrium between cell killing and cell proliferation were attained and cell numbers was almost kept constant. It resulted in long term normoglycemia. From this study, it became clear that insulin releasing cells prepared by the gene transfection could control glucose metabolism of the diabetic mouse for a long period if cells were implanted in an appropriate form. Further studies, such as how to control cell growth, how to control the immune reaction and how to remove the implants when adverse effects are observed, are needed to apply gene therapy to clinically curing diabetes. References 1. Selden, R. F., Skoskiewicz, M,J., Russell, P.,S., Goodman, H. M., Ν Eng J Med, 1987, 317, 1067 2. Kawakami, Y., Yamaoka, T., Hirochika, R., Yamashita, K., Itakura, M., Nakauchi, H., Diabetes, 1992, 41, 956-961. 3. Kingston, R.E., In Current Protocols in Molecular Biology Editors Ausubel, F.M., Brent, R., Kingston, R., Moore,D.D., Seidman, J.G., Smith, J. Α., Struhal, K.,Greene Publishing Associates and WileyInterscience: New York, NY 1987, Vol.1, pp 9.9.1-9.9.6. 4. Y.Kaneda, K.Iwai, and T.Uchida, J Biol Chem, 1989, 264, 12126. 5. Takai, T., Ohmori, H., Methods in Molecular and Cellular Biology, 1990, 2, 6. H.Iwata, T.Takagi, H.Amemiya, H.Shimizu, K.Yamashita, K.Kobayashi, Τ.Akatsu, J Biomedical Materials Res, 1992, 26. 967. RECEIVED July 30, 1993
