Nucleic acid constructs useful for glucose regulated production of human insulin in somatic cell lines

Abstract

Unique multifunctional nucleotide expression cassettes comprising a nucleotide sequence encoding a human protease driven by a glucose-regulatable promoter, a nucleotide sequence encoding human proinsulin having sites at its B-C and C-A junctions altered to permit cleavage by the human protease, and a CMV promoter driving the transcription of the nucleotide sequence encoding the human proinsulin, such cassettes optionally having polyadenylation sequences after each structural gene and the nucleotide sequence encoding human proinsulin optionally carrying an H10D variant, and cells transformed thereby.

Description

RELATED APPLICATIONS [0001] The present application claims priority to U.S. provisional application No. 60 / 296,936, filed Jun. 8, 2001 and U.S. provisional application No. 60 / 299,334, filed Jun. 19, 2001, the priority benefits of which are hereby claimed.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates to gene constructs useful for modifying cell lines to effectuate glucose-regulated production of human insulin, and cell lines transformed by such gene constructs. [0004] 2. Background of the Related Art [0005] Diabetes mellitus is a chronic disorder of fat, carbohydrate, and protein metabolism. It is characterized by an under-utilization of glucose and an absolute or relative insulin deficiency. Persons suffering from the disease in full expression have a tendency to fasting hyperglycemia, glycosuria, and ultimately to the development of atherosclerosis, neuropathy, nephropathy and microangiopathy. Diabetes is the seventh leading cause...

AI Technical Summary

Benefits of technology

[0042] In preferred embodiments of the present invention, the nucleic acid construct comprises a human proinsulin nucleotide sequence that is altered to encode a human proinsulin having altered protease cleavage sites from wild-type. It is further preferred that the genetic constructs of the present invention further comprise a nucleotide sequence encoding a human protease having affinity for the altered protease cleavage sites. Advantageously, the human proinsulin nucleotide sequence that is altered at its protease cleavage sites further comprises a variant, such as an H10D variant (i.e., histidine to aspartic acid at position 10 in the B chain) (described in U.S. patent Ser. No. 4,992,418), that increases the affinity of the mature insulin for insulin-receptor binding sites.

[0044] A particularly advantageous construct of the present invention employs a nucleotide sequence, which may be cDNA, encoding for the expression of human proinsulin that is altered to incorporate altered subtilisin-like serine protease-cleavable cleavage sites, a promoter of the transcription of the altered human proinsulin nucleotide sequence, a nucleotide sequence encoding a human subtilisin-like serine protease capable of cleavage at the altered cleavage sites, a glucose-regulated promoter of the transcription of the human furin nucleotide sequence, and a translation initiation nucleotide sequence, such as a consensus Kozak sequence, operatively positioned with respect to the human furin nucleotide sequence to allow proper translation upon transcription of the same. The nucleotide sequence encoding the subtilisin-like serine protease may advantageously encode furin.

[0047] And yet in another embodiment of the present invention, there is provided nucleic acid constructs as described above, further comprising a cytomegalovirus (CMV) promoter operably linked to the nucleotide sequence encoding human proinsulin that is artificially altered at its protease cleavage sites to accommodate the specificity of furin so as to drive constitutive expression of the altered human proinsulin. The CMV promoter is optionally operably-linked to a LTR promoter-enhancer such that the LTR cis-regulatory sequence can elevate transcription due to the CMV promoter. In such construct, the CMV promoter is used to drive constitutive expression of the altered human proinsulin while the LTR promoter-enhancer is used to enhance transcription from the CMV promoter. The LTR promoter-enhancer may be modified as known in the art to improve the level of expression of the altered human proinsulin (See, e.g., Hillberg, et al., PNAS USA 84: 5232 (1987); Holland, et al., PNAS USA 84: 8662 (1987); Valerio, et al., Gene 84: 419 (1989)).

Problems solved by technology

Diabetes is a chronic disease that presently has no cure.

As in normal beta-cells glucose transport capacity is in excess relative to glycolytic flux, breakdown of glucose is the true rate-limiting step.

As many diabetic patients have difficulty in meeting all these strictures, they constantly expose themselves to the adverse effects of hypoglycemia and hyperglycemia.

Unfortunately, such technology has not been developed to a point to permit tightly controlled blood glucose levels.

Major problems associated with such transplantations include: the shortage of donor tissue, the cost and expense involved in harvesting donor tissue, the need for immunosuppression to prevent tissue rejection in non-isograft transplantations, and the difficulty in maintaining viable tissue for prolonged periods of time after harvest.

Even successful transplants suffer from the inherent autoimmune mechanism responsible for destruction of the patient's original islet beta-cells.

Such artificial pancreases are expensive and time-consuming to fabricate, and have been found to exhibit limited usefulness in practice.

A common problem associated with such cells lines is their phenotypic instability.

While encapsulation reduces immunological response to the cells, because the cells themselves are undergoing rapid cell division, the increasing oxygen and nutrient demand within the encapsulation, as well as the increase in metabolic wastes, adversely impact the survivability of the cells.

Such approach suffers from the cost associated with isolating embryonic stem cells, the problem of introducing cross-species viruses along with a transplantation of tissue from one species to another, and with respect to human stem cells, the moral implications raised with respect to the use of such cells.

Numerous problems plague prior art “artificial beta-cells.” Such cells are typically very difficult to culture and are seen in practice to frequently loose their functional capabilities very quickly in culture.

The majority of such cell lines either do not secrete sufficient insulin to be therapeutic or the insulin production is unregulated.

Transplantation of genetically-altered cells of the prior art typically also require the use of immune suppressing drugs for life, or incorporation into an immunoisolation device wherein prior art cells have frequently been found to be less than effective due to overgrowth or rapid senescence in the device.

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