Devices comprising genetically engineered .beta.cells

• US 5,747,325 A
• Filed: 05/26/1995
• Issued: 05/05/1998
• Est. Priority Date: 06/03/1991
• Status: Expired due to Fees

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Newgard, Christopher B.
• Primary Examiner(s): Stone, Jacqueline M.
• Assistant Examiner(s): TWOMEY, PATRICK EDWARD
• Application Number:
  US08/452,667
• Time in Patent Office: 1,075 Days
• Field of Search: 210/192, 210/310, 435/240, 424/93.21, 424/425, 604/891.1
• US Class Current: 435/325
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 3/08 for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: C07K 14/62 Insulins
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12Y 207/01001 Hexokinase (2.7.1.1)
• CPC Class Codes: G01N 2800/042 Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
• CPC Class Codes: G01N 33/50 Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q
• CPC Class Codes: G01N 33/564 for pre-existing immune complex or autoimmune disease , i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• CPC Class Codes: G01N 33/567 utilising isolate of tissue or organ as binding agent
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

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.

Claims

1. An artificial β

cell device, comprising a population of engineered cells that secrete insulin, wherein cells of the population comprise a glucokinase gene encoding low affinity, high Km glucokinase (hexokinase IV), an insulin gene and a GLUT-2 glucose transporter gene, wherein at least one of said genes is a recombinant gene, wherein the cell population is positioned into a selectively permeable membrane.

2. The device of claim 1, wherein the selectively permeable membrane is further defined as a biocompatible coating.

3. The device of claim 2, wherein the cell population is encapsulated by the biocompatible coating.

4. The device of claim 2, wherein the biocompatible coating is a semipermeable capsule.

5. The device of claim 4, wherein the cells are microencapsulated.

6. The device of claim 4, wherein the cells are encapsulated in a hydrogel coating.

7. The device of claim 4, wherein the cells are encapsulated in an alginate coating.

8. The device of claim 1, wherein selectively permeable membrane is further defined as a semipermeable fiber, and the cells are seeded into the fiber.

9. The device of claim 8, wherein the cell population comprises from about 1,000 to about 10,000 engineered cells.

10. The device of claim 1, wherein the engineered cells are positioned in a tubular semipermeable membrane positioned within a protective housing.

11. The device of claim 10, wherein each end of said tubular membrane is attached to an arterial graft that extends beyond said housing and joins the device to a vascular system as a arteriovenous shunt.

12. The device of claim 1, wherein the GLUT-2 glucose transporter gene is a recombinant gene.

13. The device of claim 12, wherein the GLUT-2 glucose transporter gene is a cDNA.

14. The device of claim 1, wherein the cell population comprises cells from a cell line derived from a cell capable of forming secretory granules.

15. The device of claim 14, wherein the cell population comprises cells from a cell line derived from an endocrine cell.

16. The device of claim 14, wherein the cell population comprises cells from a cell line derived from a pituitary or thyroid cell.

17. The device of claim 14, wherein the cell population comprises cells from a cell line derived from AtT-20 cells.

18. The device of claim 14, wherein the cell population comprises cells from a cell line derived from GH-1 or GH-3 cells.

19. The device of claim 14, wherein the cell population comprises cells from a cell line derived from a β

cell.

20. The device of claim 14, wherein the cell population comprises cells from a cell line derived from β

TC, RIN or HIT cells.

21. The device of claim 1, wherein the cells secrete recombinant insulin.

22. The device of claim 1, wherein the glucokinase gene is a recombinant glucokinase gene.

23. The device of claim 22, wherein the recombinant glucokinase gene is a cDNA.

24. The device of claim 22, wherein the glucokinase gene is an islet isoform of the glucokinase gene.

25. The device of claim 1, wherein the engineered cells have a reduced hexokinase I activity relative to the cell line from which they were prepared.

26. The device of claim 25, wherein the hexokinase I activity has been reduced by an antisense RNA molecule that is complementary to and capable of binding to an RNA transcript of a hexokinase gene.

27. The device of claim 25, wherein the hexokinase I activity has been reduced through the application of a positive/negative selection protocol.

28. The device of claim 1, wherein the cells secrete human insulin.

29. The device of claim 12, wherein the GLUT-2 glucose transporter gene is an islet isoform GLUT-2 gene.