Methods of preparing genetically engineered cells that produce insulin in response to glucose
First Claim
1. A method for preparing engineered cells having glucose-responsive insulin secretory capability, comprising the steps of:
- (a) selecting starting cells that are capable of secreting insulin and that express a glucokinase gene to produce a functional glucokinase (hexokinase IV) protein;
(b) introducing into the cells a recombinant gene encoding a GLUT-2 glucose transporter protein; and
(c) selecting cells that express the recombinant gene to produce a functional GLUT-2 glucose transporter protein.
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Abstract
The present disclosure relates to the application of genetic engineering to provide artificial β cells, i.e. cells which can secrete insulin in response to glucose. This is achieved preferably through the introduction of one or more genes selected from the insulin gene, glucokinase gene, and glucose transporter gene, so as to provide an engineered cell having all three of these genes in a biologically functional and responsive configuration. Assays for detecting the presence of diabetes-associated antibodies in biological samples using these and other engineered cells expressing diabetes-associated epitopes are described. Also disclosed are methods for the large-scale production of insulin by perfusing artificial β cells, grown in liquid culture, with glucose-containing buffers.
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Citations
50 Claims
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1. A method for preparing engineered cells having glucose-responsive insulin secretory capability, comprising the steps of:
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(a) selecting starting cells that are capable of secreting insulin and that express a glucokinase gene to produce a functional glucokinase (hexokinase IV) protein; (b) introducing into the cells a recombinant gene encoding a GLUT-2 glucose transporter protein; and (c) selecting cells that express the recombinant gene to produce a functional GLUT-2 glucose transporter protein. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 15, 16, 17, 18, 19, 23, 24, 25, 26)
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9. A method for preparing engineered cells having glucose-responsive insulin secretory capability, comprising the steps of:
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(a) selecting starting cells that are capable of forming secretory granules; (b) introducing into the starting cells one or more recombinant genes selected from the group consisting of a GLUT-2 glucose transporter gene, a glucokinase (hexokinase IV) gene, and an insulin gene;
such that the cells comprise a competent GLUT-2 glucose transporter gene, glucokinase gene and insulin gene, wherein at least one of said genes is a recombinantly introduced gene; and(c) selecting cells having glucose-responsive insulin secretory capability. - View Dependent Claims (10, 11, 12, 13, 14, 20, 21, 22, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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36. A method for preparing engineered cells having glucose-responsive insulin secretory capability, comprising the steps of:
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(a) selecting starting cells that are capable of secreting insulin and that express a GLUT-2 glucose transporter gene to produce a functional GLUT-2 glucose transporter protein; (b) introducing into the cells a recombinant glucokinase (hexokinase IV) gene; and (c) selecting cells that express the recombinant glucokinase gene to produce a functional glucokinase protein. - View Dependent Claims (37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50)
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Specification