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Compositions and methods for regulated protein expression in gut

A digestive tract and protein technology, applied in animal/human protein, fusion cells, pharmaceutical formulations, etc., can solve problems such as desensitization of the body's insulin response

Inactive Publication Date: 2008-01-09
ENGENE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these patients can hardly achieve ideal blood sugar levels by insulin injections (Turner, R.C. et al., JAMA 281:2005(1999))
Moreover, chronically elevated insulin levels may have harmful side effects such as hypoglycemic shock and desensitization of the body's insulin response

Method used

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  • Compositions and methods for regulated protein expression in gut
  • Compositions and methods for regulated protein expression in gut
  • Compositions and methods for regulated protein expression in gut

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0160] This example describes the establishment of gut endocrine cell lines for the study of regulation of insulin production and in vivo targeting of insulin expression. This example also describes the construction of a human insulin gene expression vector.

[0161] Establish GIP expressing cell line to study whether GIP promoter is effective for K cells to express insulin gene. This cell line was cloned from the mouse intestinal cell line STC-1, a mixed enteroendocrine cell population (Rindi et al., Am. J. Pathol. 136:1349 (1990)). K cells in mixed cells can be visually identified by transfection of a green fluorescent protein expression plasmid driven by the approximately 2.5 Kb rat GIP promoter. The rat GIP promoter was obtained from the rat genome lambda DASH library (Strategene) by plaque hybridization with rat GIP cDNA clone plaques as previously described (Boylan et al., J. Biol. Chem. 273: 17438 (1997)), and It was subcloned into the promoterless pEGFP-1 plasmid (Cl...

Embodiment II

[0171] This example describes transgenic mice that produce insulin in response to glucose.

[0172] Using the human insulin expression construct GIP / Ins described in Example 1, the GIP / insulin fragment (about 4.1 Kb) was digested with HindIII. Transgenic mice were generated by microinjecting approximately 4.1 Kb transgenic pronuclei into fertilized eggs and implanting the fertilized eggs into surrogate female mice. Transgenic offspring were identified by Southern blot analysis. The otic DNA (Figure 3) was digested with XhoI and PvuII, separated by electrophoresis and transferred to a nylon membrane. To detect the transgene, primers 2 and 4 were used to amplify a 416 bp human insulin gene fragment containing intron 2 (Figure 3). By using [α- 32 P]dCTP was randomly labeled to prepare PCR products as probes, and the bands were detected by autoradiography. DNA analysis results were further confirmed by PCR amplification of genomic DNA using primers 2 and 4. The positive mice ...

Embodiment III

[0179] This example shows that insulin production in transgenic mice provides normal glucose homeostasis and protects against developing diabetes. Human insulin production by gut K cells in transgenic mice was also regulated by diet. This example also describes data showing that glucose-inducible insulin production in transgenic mice provides glucose homeostasis following pancreatic β-cell destruction.

[0180] Plasma human insulin levels in transgenic mice were analyzed in response to food intake. Briefly, plasma insulin levels were measured using a human-specific insulin ELISA kit (ALPCO) following the supplier's instructions. The assay has <0.01% cross-reactivity with human proinsulin and C-peptide and cannot detect mouse insulin. Plasma C-peptide detection was performed with a rat / mouse C-peptide RIA kit (Linco). This assay showed no cross-reactivity with human C-peptide.

[0181] In pooled plasma samples collected after oral glucose administration, insulin in transgen...

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Abstract

The invention provides compositions and methods useful for treating disorders treatable by producing a protein in a regulatable manner in a mucosal cell or tissue of an animal. The treatment methods include in vivo and ex vivo methods, including transplanting in vitro transformed cells that secrete the protein into a mammalian subject.

Description

technical field [0001] The present invention relates to the regulatable production of gut proteins, and more particularly, to the nutrient-regulated production of glucose-lowering factors by gut endocrine cells. Background of the invention [0002] Peptides and proteins, by virtue of their conformational pluripotency and functional specificity, have been used to treat many diseases, including diabetes, hemophilia, cancer, cardiovascular disease, infectious disease, and arthritis (Russell C.S. & Clarke L.A. Clin Gent 55(6) : 389 (1999); Ryffel B. Biomed environ Sci 10: 65 (1997); Koths K. Curr Opin Biotechnol 6: 681 (1995); Buckel P. Trends Pharmacol Sci 17: 450 (1996)). At present, more than 2 / 3 of the approved bioengineering drugs are systemic protein drugs. With recent developments in the fields of functional genomics, proteomics, and genetic engineering, more and more protein drugs are entering the biopharmaceutical market. [0003] Initially, protein drugs were purifie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N15/63C07K14/62C07K14/605C12N5/16
Inventor T·J·基菲尔A·T·钟
Owner ENGENE INC
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