Abstract
Purpose. Recent progress in genetic engineering presents the possibility of providing physiologically regulated glucose metabolism in individuals with diabetes. The objective of this study is to explore the feasibility of obtaining glucose dependent gene expression in the pancreatic β-cell lines via recombinant adeno-associated virus type 2 (rAAV) mediated gene transfer.
Methods. Two transcription cassettes containing the luciferase gene under the control of the rat insulin I gene promoter and the enhanced green fluorescent protein (EGFP) open reading frame under the control of the immediate early gene promoter of human cytomegalovirus (CMV) were placed in series between the inverted terminal repeats (ITRs) of AAV. The rAAV vectors produced were used to transduce pancreatic β-cell line grown in the absence or presence of various concentrations of glucose. Luciferase activity assays were performed at 72 hr post-transduction.
Results. Glucose-responsive reporter gene expression was obtained in both calcium phosphate transfected HIT-T15 and βHC-9 cells, demonstrating regulated luciferase gene expression under control of the insulin gene promoter. At MOI of 100, rAAV-transduced βHC-9 cells exhibited glucose-dependent luciferase activities, which were approximately 4.3 fold higher than those transfected by the calcium phosphate coprecipitation method at 20 mM glucose.
Conclusions. Delivery of the insulin gene promoter via rAAV was shown in this study to result in glucose-dependent control of the reporter gene expression. The results suggest that rAAV is an efficient viral vector for gene transfer into the pancreatic islet cells.
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REFERENCES
K. Docherty. Gene therapy for diabetes mellitus. Clin. Sci. 92: 321–330 (1997).
M. S. German. Glucose sensing in pancreatic islet beta cells: The key role of glucokinase and the glycolytic intermediates. Proc. Natl. Acad. Sci. USA 90:1781–1785 (1993).
C. B. Newgard. Cellular engineering and gene therapy strategies for insulin replacement in diabetes. Diabetes 43:341–350 (1994).
C. B. Newgard, S. Clark, H. BeltrandelRio, H. E. Hohmeier, C. Quaade, and K. Nomington. Engineered cell lines for insulin replacement in diabetes: Current status and future prospects. Diabetologia 40(Suppl. 2):S42–27 (1997).
S. D. Hughes, J. H. Johnson, C. Quaade, and C. B. Newgard. Engineering of glucose-stimulated insulin secretion and biosynthesis in non-islet cells. Proc. Natl. Acad. Sci. USA 89:688–692 (1992).
D. Mitanchez, B. Doiron, R. Chen, and A. Kahn. Glucosestimulated genes and prospects of gene therapy for type I diabetes. Endocrin. Rev. 18:520–540 (1997).
P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen. Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA 84:7413–7417 (1987).
Q.-L. Hao, P. Malik, R. Salazar, H. Tang, E. M. Gordon, and D. B. Kohn. Expression of biologically active human factor IX in human hematopoietic cells after retroviral vector-mediated gene transduction. Hum. Gene Ther. 6:873–880 (1995).
M. E. Csete, P. Y. Benhamou, K. E. Drazan, L. Wu, D. F. McIntee, R. Afra, Y. Mullen, R. W. Busuttil, and A. Shaked. Efficient gene transfer to pancreatic islets mediated by adenoviral vectors. Transplantation 59:263–268 (1995).
R. R. Ali, M. B. Reichel, A. J. Thrasher, R. J. Levinsky, C. Kinnon, N. Kanuga, D. M. Hunt, and S. S. Bhattacharya. Gene transfer into the mouse retina mediated by an adeno-associated viral vector. Hum. Mol. Genet. 5:591–594 (1996).
P. D. Kessler, G. M. Podsakoff, X. Chen, S. A. McQuiston, P. C. Colosi, L. A. Matelis, G. J. Kurtzman, and B. J. Byrne. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Proc. Natl. Acad. Sci. USA 93:14082–14087 (1996).
T. J. Sferra, G. Qu, D. McNeely, R. Rennard, K. R. Clark, W. D. Lo, and P. R. Johnson. Recombinant adeno-associated virusmediated correction of lysosomal storage within the central nervous system of the adult mucopolysaccharidosis type VII mouse. Hum. Gene Ther. 11:507–519 (2000).
R. O. Snyder, C. H. Miao, G. A. Patijn, S. K. Spratt, O. Danos, D. Nagy, A. M. Gown, B. Winther, L. Meuse, L. K. Cohen, A. R. Thompson, and M. A. Kay. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors. Nat. Genet. 16:270–276 (1997).
T. A. Smith, B. D. White, J. M. Gardner, M. Kaleko, and A. McClelland. Transient immunosuppression permits successful repetitive intravenous administration of an adenovirus vector. Gene Ther. 3:496–502 (1996).
R. J. Samulski, L. S. Chang, and T. Shenk. Helper-free stocks of recombinant adeno-associated viruses: Normal integration does not require viral gene expression. J. Virol. 63:3822–3828 (1989).
G. Christofori, P. Naik, and D. Hanahan. A second signal supplied by insulin-like growth factor II in oncogne-induced tumorigenesis. Nature 369:414–418 (1994).
R. M. Kotin, M. Siniscalco, R. J. Samulski, X. D. Zhu, L. Hunter, C. A. Laughlin, S. McLaughlin, N. Muzyczka, M. Rocchi, and K. I. Berns. Site-specific integration by adeno-associated virus. Proc. Natl. Acad. Sci. USA 87:2211–2215 (1990).
R. F. Santerre, R. A. Cook, R. M. Crisel, J. D. Sharp, R. J. Schmidt, D. C. Williams, and C. P. Wilson. Insulin synthesis in a clonal cell line of simian virus 40-transformed hamster pancreatic beta cells. Proc. Natl. Acad. Sci. USA: 78:4339–4343 (1981).
F. Radvanyi, S. Christgau, S. Baekkeskov, C. Jolicoeur, and D. Hanahan. Pancreatic β-cell cultured from individual preneoplastic foci in a tumorigenesis pathway: A potentially general technique for isolating physiologically representative cell lines. Mol. Cell Biol. 13:4223–4232 (1993).
V. R. Braddon, J. A. Chiorini, S. Wang, R. M. Kotin, and B. J. Baum. Adeno-associated virus-mediated transfer of a functional water channel into salivary epithelial cells in vitro and in vivo. Hum. Gene Ther. 9:2777–2785 (1998).
J. A. Chiorini, C. M. Wendtner, E. Urcelay, B. Safer, M. Hallek, and R. and R. M. Kotin. High-efficiency transfer of the T-cell co-stimulatory molecule B7-2 lymphoid cells using high-titer recombinant adeno-associated virus vectors. Hum. Gene Ther. 6: 1531–1541 (1995).
H. E. Hohmeier, H. BeltrandelRio, S. A. Clark, R. Henkel-Rieger, K. Normington, and C. B. Newgard. Regulation of insulin secretion from novel engineered insulinoma cell lines. Diabetes 46:968–977 (1997).
J. Saldeen, D. T. Curiel, D. L. Eizirik, A. Andersson, E. Strandell, K. Buschard, and N. Welsh. Efficient gene transfer to dispersed human pancreatic islet cells in vitro using adenovirus-polylysine/ DNA complexes or polycationic liposomes. Diabetes 45:1197–1203 (1996).
G. Leibowitz, G. M. Beattie, T. Kafri, V. Cirulli, A. D. Lopez, A. Hayek, and F. Levine. Gene transfer to human pancreatic endocrine cells using viral vectors. Diabetes 48:745–753 (1999).
S. A. Clark, C. Quaade, H. Constandy, P. Hansen, P. Halban, S. Ferber, C. B. Newgard, and K. Normington. Novel Insulinoma cell lines produced by iterative engineering of GLUT2, glucokinase, and human insulin expression. Diabetes 46:958–967 (1997).
G. M. Beattie, T. Otonkoski, A. D. Lopez, and A. Hayek. Functional β-cell mass after transplantation of human fetal pancreatic cells: Differentiation or proliferation? Diabetes 46:244–248 (1997).
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Yang, YW., Kotin, R.M. Glucose-responsive Gene Delivery In Pancreatic Islet Cells Via Recombinant Adeno-associated Viral Vectors. Pharm Res 17, 1056–1061 (2000). https://doi.org/10.1023/A:1026445426982
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DOI: https://doi.org/10.1023/A:1026445426982