Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1998-04-17
2001-11-20
Clark, Deborah J. R. (Department: 1633)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C424S093200, C435S069100, C435S320100, C435S325000, C435S455000, C536S023500
Reexamination Certificate
active
06319495
ABSTRACT:
BACKGROUND OF THE INVENTION
A. Field of the Invention
The present invention relates generally to the field of genetic engineering and gene expression. More specifically, it relates to artificial tissues, particularly to artificial tissues comprising engineered cells having the ability to secrete insulin in a glucose-sensitive fashion. It also relates to the use of engineered cells in the production of human insulin for use in, for example, the treatment of insulin deficient conditions such as type I diabetes mellitus.
B. Description of the Related Art
The &bgr; cells of the pancreatic islets of Langerhans synthesize insulin. A portion of this insulin is slowly and continually released into the bloodstream (basal secretion). However, the bulk of the insulin is stored in secretory vesicles and released only in response to certain physiological stimuli (stimulated secretion).
In humans and other mammals, the principal physiological stimulus for the secretion of insulin is increased blood levels of glucose (e.g., following ingestion of a carbohydrate meal). The capacity of normal islet &bgr; cells to sense a rise in blood glucose concentration and to respond by secreting insulin is critical to control of blood glucose levels.
In certain insulin-deficient conditions such as diabetes mellitus, the pancreas loses its ability to manufacture and secrete insulin in response to rising blood glucose concentrations. The result is a metabolic imbalance, which causes blindness, kidney-related diseases, neurological disorders, cardiovascular diseases, non-accidental amputation of limbs, and death.
The current preferred treatment for insulin deficiency is injection of insulin once or twice daily. The objective of this regimen is to maintain glucose levels close to normal. However, daily insulin injections can not reproduce the rapid insulin secretory responses of normal islets to physiological demand. Most insulin deficient patients never achieve the finely-tuned glucose homeostasis needed to avoid long-term complications.
Consequently, a major goal of research into insulin deficiencies is to develop a system for sensing changes in blood glucose and quickly adjusting insulin output to maintain normal blood glucose levels at all times. One approach has been replacement of the malfunctioning organ by transplantation of normal pancreatic tissue. Lacy et al. (1986),
Ann. Rev. Med
., 37: 33-40; Lacey (July, 1995),
Scientif. Amer
. 273: 50-55. However, transplanted islets are recognized and destroyed by the same autoimmune mechanism responsible for destruction of the patient's original &bgr; cells. For this reason artificial pancreatic devices containing live islets have often been designed to avoid immune rejection, often by enclosing islets in a semipermeable pouch or matrix which separates the transplanted islets from immunoreactive cells and molecules.
The treatment of diabetes with peritoneal implants of encapsulated islets in vivo diabetic models has been reported by several research groups. Lum et al. (1991), “Prolonged reversal of diabetic state in NOD Mice by xenografts of microencapsulated rat islets,”
Diabetes
, 40: 1511; and Maki et al. (1991), “Successful treatment of diabetes with the biohybrid artificial pancreas in dogs,”
Transplantation
, 51: 43; Scharp etal. (1990), “Insulin independence after islet transplantation into type I diabetic patient,”
Diabetes
, 39: 515; Robertson (1991), “Pancreas Transplantation in humans with diabetes mellitus,” Diabetes, 40: 1085; Colton et al. (1991), “Bioengineering in development of the hybrid artificial pancreas,”
J. Biomech. Eng
., 113: 152; Reach (1990), “Bioartificial pancreas: Status and bottlenecks,”
Intern. J. Art. Organs
, 13: 329; and Warnock et al. (1988), “Critical mass of purified islets that induce normoglycemia after implantation into dogs,”
Diabetes
, 37: 467. See also, T. Matsumura (U.S. Pat. No. 3,827,565); Sun et al. (U.S. Pat. No. 4,323,457 (1982)); Chick etal. (U.S. Pat. Nos. 4,242,459 and 4,242,460 (1980)); Lim, U.S. Pat. No. 4,391,909; Loeb, U.S. Pat. No. 4,378,016; Newgard, U.S. Pat. No. 5,427,940 (1992); Bae, et. al., U.S. Pat. No. 5,262,055 (1993).
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 &bgr; cells in liquid culture and for the increased production of human insulin by perfusion of such recombinant cells with glucose-containing buffers.
REFERENCES:
patent: 5811266 (1998-09-01), Newgard
Mcdonald et al, Mol. Cell. Endocrin. 123(2):199-204, 1996.*
Efrat. Diabetologia. 41:1401-1409, 1998.
Christakos Sylvia
Pollock Allan S.
Reddy Daphne
Clark Deborah J. R.
Kaushal Sumesh
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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