Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Patent
1996-06-03
2000-10-24
Chambers, Jasemine C.
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
800 9, 435455, 435456, 4353201, 435325, 514 44, C12N 1509, C12N 1563, C12N 1500, C12N 500
Patent
active
061370290
DESCRIPTION:
BRIEF SUMMARY
This 371 application claims the benefit of PCT/ES94/00027, filed Mar. 14, 1994.
The present invention relates firstly to a chimeric gene using the gene or cDNA (complementary DNA) of insulin driven by a promoter or fusion of promoters.
More specifically, it relates to the design of a chimeric gene formed by the fusion of the promoter of P-enolpgruvate carboxyquinase to the structural gene of human insulin, which allows the production of human insulin, physiologically regulated, in a tissue different from the pancreas.
The invention further relates to others objects which are described below.
BACKGROUND OF THE INVENTION
Patients suffering from insulin dependent diabetes mellitus (IDDM) (type I) depend dramatically on the administration of the hormone. The interruption of the insulin administration results first in hyperglycemia and ketoacidosis, then coma and finally death if the hormone is not injected. Therefore, the life and the quality of life of these patients depend completely on the fluctuations of the insulin levels in their blood.
Gene therapy consists in the transfer of genetic material into cells of a patient with the purpose of treating an illness. At present, different approximations of gene therapy are being developed, based on the introduction of genes directly into animals or cells which are then transplanted.
However, the most important goal is not to be able to transplant successfully cells expressing the gene in an animal, but to make it possible for the gene to express in a regulated and physiologic way. The choice of a good promoter which drives the expression of the suitable gene is crucial in order to obtain suitable plasmatic levels of the corresponding protein.
In the case of diabetes, the question is to chose the promoter which drives the expression of the gene in order to obtain suitable insulin plasmatic levels for every condition of the individual. The overexpression of the insulin gene would result in hypoglucemia and a low expression of said gene would not modify the high glucose levels in the diabetic process.
BRIEF DESCRIPTION OF THE DRAWINGS
In order to better understand the disclosure herein, we enclose some drawings concerning examples of embodiments.
In said drawings:
FIG. 1 shows the construction of the PEPCK/insulin chimeric gene from the plasmid pB7.0 and the human insulin gene.
FIG. 2 shows the structure of PEPCK/insulin chimeric gene.
FIG. 3 shows the process for the obtention of the vPCK/Ins retroviral vector from the pPCK/Ins plasmid.
DESCRIPTION OF THE INVENTION
One object of the invention is a chimeric gene using the gene or cDNA (complementary DNA) of insulin driven by a promoter or fusion of promoters, preferably adjustable and activated by the diabetic process.
Preferably, the object of the invention is a chimeric gene which is obtained by fusion of the human insulin gene to the promoter of the P-enolpiruvate carboxyquinase (PEPCK).
P-enolpyruvate carboxyquinase is a key enzyme for the control of the gluconeogenic via, and it is found mainly in the liver, kidney, jejunum and adipose tissue. The activity of this enzyme is regulated as regards the expression of its gene. (Hanson, R. W., et al. (1976) Gluconeogenesis: Its Regulation in mammalian Species. John Wiley & Sons, Inc., New York). The expression of the gene of PEPCK is finely regulated by hormones. Within the fragment of the promoter of PEPCK used (-550 bp to +73 bp) sequences responding to AMPc, glucocorticoids and insulin have been described (Wynshaw-Boris, A. et al. (1984) J. Biol. Chem. 259, 12161-12169; Wynshaw-Boris, A., et al. (1986) J. Chem. 261, 9714-9720; Short, J. M., et al. (1986) J. Biol. Chem. 261, 9721-9726; O'Brien, R. M., et al. (1990) Science 249, 533-537). The glucagon, acting via AMPC, and the glucocorticoids activate the gene expression, while insulin inhibits said expression. The gene expression of the PEPCK is increased in diabetic animals due to the rise in the plasmatic levels of glucagon and the drop in the insulin levels (Tilghman, S. M., et al. (1974) Proc. Natl. Aca
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"Regulated Expression of Human Insulin in the Liver of Transgenic Mice Corrects Diabetic Alterations" X, Gregori et al, Journal of Cellular Biochemistry, Supplement 18A, Jan. 4-23, 1994, p. 145.
"Characterization of the Phosphoenolpyruvate Carboxykinase (GTP) Promoter-regulatory Region", Jul. 25, 1986 Jay M Short, et al., The Journal of Biological Chemistry, vol. 261, pp. 9721-9726.
Abril Alfons Valera
Tubert Fatima Bosch
Chambers Jasemine C.
Martin Jill D.
Universitat Autonoma De Barcelona
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