Title:  Method for restoring glucose responsiveness to insulin secretion

United States Patent:  6,319,495

Assignee:  The Regents of the University of California (Oakland, CA)

Filed:  April 17, 1998

PCT NO:  PCT/US96/16736

102(e) Date:  November 30, 1998

PCT PUB. Date:  April 24, 1997

This invention comprises engineered cells that express a nucleic acid that encodes a calbindin molecule, and exhibit the ability to secrete insulin in a glucose-sensitive fashion. This invention also comprises an artificial tissue that, when implanted into recipients that do not secrete insulin in a glucose-sensitive fashion, imparts to the recipients the ability to secrete insulin in a glucose-sensitive fashion. The artificial tissue comprises engineered cells that express a nucleic acid that encodes a calbindin molecule and secrete insulin in a glucose-sensitive fashion, enclosed by a semipermeable porous matrix.

SUMMARY OF THE INVENTION

The present invention comprises an engineered cell that secretes insulin in a glucose-sensitive fashion and that further expresses a drug sensitivity. This engineered cell expresses a first exogenous nucleic acid that encodes a calbindin molecule and a second exogenous nucleic acid that encodes a drug sensitivity, with the proviso that in the absence of said first exogenous nucleic acid that encodes a calbindin molecule, the host cell does not secrete insulin in glucose-sensitive fashion. The engineered cell may be a primary isolate, a continuous non-transformed cell line, or an insulinoma, preferably of human origin. The engineered cell may optionally be negatively selected by exposure to metabolites or drugs that are lethal to cells that express the drug sensitivity gene.

The invention further encompasses a method for imparting glucose-sensitive insulin secretion to a host cell that does not exhibit glucose-sensitive insulin secretion, comprising the steps of a) providing a cell that is capable of producing and secreting insulin, but has an impaired ability to secrete insulin in a glucose sensitive fashion; and b) stably introducing into said cell a nucleic acid that encodes a calbindin molecule, wherein the expression of said nucleic acid that encodes a calbindin molecule imparts to the engineered host cell the ability to secrete insulin in a glucose-sensitive fashion. In different embodiments of this method, the mammal is a human patient, the engineered cell is either con-specific or syngeneic with the mammal into which it is stably introduced, and the cell is selected from the group consisting of a primary isolate, a continuous non-transformed cell line or an insulinoma.

Other embodiments of this invention are artificial implantable tissues that secrete insulin in response to humoral signals, in particular glucose. One such embodiment is an engineered cell that expresses calbindin and secretes insulin in a glucose-sensitive fashion, wherein the cell is enclosed in a semipermeable matrix or membrane that permits the diffusion of glucose and nutrients into the matrix, and the diffusion of insulin and waste products out of the matrix. The matrix may be formed from plasma, fibrinogen, casein, fibrin, limulus lysate, milk protein, collagen, agarose, carrageenan, agar, alginate, guar gum, gum arabic, pectin, tragacanth gum, xanthan gum, and mixtures thereof. These matrix-enclosed engineered glucose-sensitive insulin-secreting calbindin-expressing cells are implanted or injected into an animal that is unable to secrete insulin in a glucose-sensitive fashion.

In one embodiment, a cell-containing matrix of this invention is prepared by: (1) polymerizing matrix components or precursors in the presence of viable engineered cells that express an exogenous calbindin and secrete insulin in a glucose-sensitive fashion, and (2) recovering a porous matrix containing viable cells. Depending upon which components are used to make the matrix, the polymerization can be achieved by exposing the respective precursor to a polymerization promoting reagent and/or a polymerization promoting condition.

In another embodiment, the current invention provides methods for growing artificial .beta. cells in liquid culture and for the increased production of human insulin by perfusion of such recombinant cells with glucose-containing buffers.

Claim 1 of 19 Claims

We claim:

1. An engineered cell that secretes insulin in a glucose-sensitive fashion and that expresses a drug sensitivity, wherein said cell expresses a first exogenous nucleic acid that encodes a calbindin molecule and a second exogenous nucleic acid that encodes a drug sensitivity, with the proviso that in the absence of said first exogenous nucleic acid that encodes a calbindin molecule, the host cell does not secrete insulin in glucose-sensitive fashion.

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