Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-03-28
2002-03-05
Schwartzman, Robert A. (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C536S024100, C536S023500
Reexamination Certificate
active
06352857
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to treatment of diabetes with synthetic beta cells, and specifically to a method of utilizing non-islet cells comprising a genetic construct that has a coding sequence for a proinsulin expressible in the cells in response to glucose levels. The proinsulin synthesized in the cells is further processed into a secretable, active insulin.
Insulin is normally produced in and secreted by the beta cells of the islets of Langerhans in the pancreas. Mature insulin is a protein having two polypeptide chains, A and B, held together by disulfide bonds. The glucose responsive release of insulin from the beta cells is a complex event including gene expression, posttranslational modification and secretion. The initial protein product and insulin precursor is preproinsulin, a single polypeptide chain having an N-terminal signal sequence and an intervening sequence, the C-peptide, between the A and B chains. The signal sequence is cleaved during transport from the rough endoplasmic reticulum to form proinsulin. The proinsulin is packaged into secretory granules along with specific enzymes required for its processing. Proinsulin folds into a specific three-dimensional structure, forming disulfide bonds. Mature insulin results from removal of the C-peptide. In beta cells, this function is catalyzed by endopeptidases that recognize the specific amino acid sequences at the junction of the A chain and the C peptide (C-A junction) and at the junction of the B chain and the C peptide (B-C junction). Mature insulin, stored in secretory granules, is released in response to elevated blood glucose levels. The detailed mechanism of insulin release is not completely understood, but the process involves migration and fusion of the secretory granules with the plasma membrane prior to release.
In normally functioning beta cells, insulin production and release is affected by the glycolytic flux. Glucokinase and glucose transporter 2 (GLUT-2) are two proteins that are believed to be involved in sensing changes in glucose concentration in beta cells. A reduction in GLUT-2, which is involved in glucose transport, is correlated with decreased expression of insulin; loss of glucokinase activity causes a rapid inhibition of insulin expression.
Autoimmune destruction of pancreatic beta cells causes insulin-dependent diabetes mellitus or Type I diabetes. As a consequence of partial or complete loss of beta cells, little or no insulin is secreted by the pancreas. Most cells, with the exception of brain cells, require insulin for the uptake of glucose. Inadequate insulin production causes reduced glucose uptake and elevated blood glucose levels. Both reduced glucose uptake and high blood glucose levels are associated with a number of very serious health problems. In fact, without proper treatment, diabetes can be fatal.
One conventional treatment for diabetes involves periodic administration of injectable exogenous insulin. This method has extended the life expectancy of millions of people with the disease. However, blood glucose levels must be carefully monitored to ensure that the individual receives an appropriate amount of insulin. Too much insulin, can cause blood glucose levels to drop to dangerously low levels. Too little insulin will result in elevated blood glucose levels. Even with careful monitoring of blood glucose levels, control of diet, and insulin injections, the health of the vast majority of individuals with diabetes is adversely impacted in some way.
Replacement of beta cell function is a treatment modality that may have certain advantages over insulin administration, because insulin would be secreted by cells in response to glucose levels in the microenvironment. One way of replacing beta cell function is by pancreas transplantation, which has met with some success. However, the supply of donors is quite limited, and pancreas transplantation is very costly and too problematic to be made widely available to those in need of beta cell function.
There have been many other proposed alternatives for beta cell replacement, including replacing beta cell function with actual beta cells or other insulin-secreting, pancreas-derived cell lines (Lacy, et al.,
Ann. Rev. Med
., 37:33, 1986). Because the immune system recognizes heterologous cells as foreign, the cells have to be protected from immunoactive cells (e.g., T-cells and macrophages mediating cytolytic processes). One approach to protect heterologous cells is physical immunoisolation; however, immunoisolation itself poses significant problems.
U.S. Pat. No. 5,427,940 issued to Newgard discloses another approach to beta cell replacement. This patent describes an artificial beta cell produced by engineering endocrine cells of the At-T-20 ACTH secreting cells. A stably transfected cell, At-T-20, is obtained by introducing cDNA encoding human insulin and the glucose transporter gene, i.e. the GLUT-2 gene, driven by the constitutive CMV promoter. The cell line already expresses the correct isoform of glucokinase required for glucose responsive expression of the proinsulin gene. Although the cell line is responsive to glucose, it is secretagogue-regulated at concentrations below the normal physiological range. Therefore, use of these cells in an animal would likely cause chronic hypoglycemia; furthermore, these cells are derived from a heterologous source and bear antigens foreign to the recipient host.
U.S. Pat. No. 5,534,404 issued to Laurance et al. discloses another approach to obtaining a cell line in which insulin production is secretagogue-regulated. Subpopulations of beta-TC-6 cells having an increased internal calcium concentration, a property associated with insulin secretion, were selected using a cell sorter. After successive passages, a subpopulation of cells that produce insulin in response to glucose in the physiological range (4-10 mM) was selected, and the cells were encapsulated for therapeutic use in alginate bounded by a PAN/PVC permselective hollow fiber membrane according to the method of Dionne (International Patent application No. PCT/US92/03327).
Valera et al.,
FASEB Journal
, 8: 440 (1994) describes transgenic mouse hepatocytes expressing insulin under the control of the PEPCK promoter driven by P-enolpyruvate. The PEPCK promoter is sensitive to the glucagon/insulin ratio and is activated at elevated glucose levels. The PEPCK/insulin chimeric gene was introduced into fertilized mouse eggs. Under conditions of severe islet destruction by streptozotocin (SZ), the production and secretion of intact insulin by the liver compensated for loss of islet function.
Despite these prior art attempts, there is a continuing need for alternative methods to conventional insulin therapy for the treatment of diabetes.
SUMMARY OF THE INVENTION
The present invention relates to a genetic construct that can be used to obtain glucose-inducible expression of active insulin in a host cell. The genetic construct permits glucose-regulated expression of proinsulin in the presence of higher than physiologic concentrations of glucose, but not at lower than physiologic glucose concentrations. The present invention further provides that the proinsulin encoded by the coding sequence of the genetic construct comprises an amino acid sequences that allows the proinsulin to be converted into a secretable, active insulin.
In one aspect, the invention provides a genetic construct for glucose-inducible expression of active insulin in host cells. The construct comprises (i) a coding sequence for a human proinsulin, the coding sequence operably connected a promoter functional in the host cell, wherein the proinsulin amino acid sequences corresponding to the B-C and C-A junctions of native human proinsulin are cleavable in the host cell; (ii) a glucose responsive regulatory module located 5′ of the promoter, the module having at least one glucose inducible regulatory element (GIRE) comprising a pair of CACGTG motifs linked by a five base nucleotide sequence; and (iii) a 5′ untranslated region (UTR), located 5&pr
Alam Tausif
Hullett Debra A.
Sollinger Hans W.
Dhruva Bharati
Quarles & Brady LLP
Schwartzman Robert A.
Wisconsin Alumni Research Foundation
LandOfFree
Treatment of diabetes with synthetic beta cells does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Treatment of diabetes with synthetic beta cells, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Treatment of diabetes with synthetic beta cells will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2889231