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Treating Insulin Secreting Cells

a technology of secreting cells and insulin, applied in the direction of transferases, drug compositions, hormone peptides, etc., can solve the problems of limited therapies, no effective medical treatment for the devastating symptoms, and significant morbidity associated with their removal

Inactive Publication Date: 2011-10-20
BRUNICARDI F CHARLES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a novel recombinant nucleic acid construct that can selectively target and ablate cells in the pancreas, such as pancreatic cancer cells. The nucleic acid sequence contains an insulin promoter that is linked to a cytotoxic gene, such as thymidine kinase or PDX-1, which can be selectively expressed in pancreatic cells. The nucleic acid sequence can be delivered to cells using an agent, such as a recombinant virus or liposome, and can be used to treat pancreatic cancer and other metabolic diseases. The invention also provides a method for increasing insulin secretion by reducing the concentration of somatostatin receptors.

Problems solved by technology

Ninety percent of β-cell tumors are benign, however morbidity associated with their removal is significant.
Furthermore, there is no effective medical treatment for the devastating symptoms associated with hyperinsulinemia as a result of either insulinoma or nesidioblastosis (idiopathic hyperinsulinemia).
Currently, only surgery offers any chance for a cure and the majority of the time the cancer has spread before it is detected.
However, these therapies have limitations due to either the weakness of the promoter or the tissue specificity of its activation.
However, thymidine kinase with a ubiquitous promoter is not cell specific, limiting its use as a cytotoxic agent.

Method used

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  • Treating Insulin Secreting Cells
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  • Treating Insulin Secreting Cells

Examples

Experimental program
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Effect test

example 1

Demonstration of β-Cell Specific Cytotoxicity Using a Rat Insulin Promoter Thymidine Kinase Construct

[0105]Materials, methods and summary of results: 0.502 kb of RIP (SEQ ID NO:1) was ligated to the reporter gene LacZ and ligated to tk. These two genes were transfected into several cell lines to ascertain β-cell specific expression and β-cell specific cytotoxicity in vitro. RT-PCR and EMSA were performed on NIT-1 cell RNA and nuclear extract, respectively, to determine the transcription factors present and responsible for RIP activation in NIT-1 cells. A mouse (3-cell adenoma model was created with NIT-1 cells. These mice were treated with the RIP-tk gene and both blood sugars and animal viability were monitored.

[0106]Only the beta (NIT-1) cells stained blue after X-gal staining (p<0.05, n=16) or had detectable levels of beta-galactosidase protein (p<0.05, n=6) in vitro. A significant decrease in cell survival was observed in NIT-1 cells transfected with RIP-tk, in vitro (p<0.05, n=...

example 2

Human Pancreatic Ductal Carcinoma Cells can be Targeted Using a RIP-tk Construct In Vitro

[0135]Materials, methods and summary of results: 0.502 kb of RIP was ligated to the reporter gene LacZ and transfected into several human cell lines: human pancreatic ductal carcinoma cell lines (PANC-1, CAPAN-1, and MIA-1), lung carcinoma (A549), and breast carcinoma (T47D). X-gal staining and the detection of beta-galactosidase using a luminometer analyzed LacZ gene expression. RIP was ligated to tk and transfected into PANC-1, CAPAN-1, MIA-1, and A549 cells. Cell viability was compared after transfection with the RIP-tk genetic construct and daily treatment with of ganciclovir (GCV). RT-PCR was performed on PANC-1, CAPAN-1, and MIA-1 total RNA with primers specific for known insulin transcription factors PDX-1 and BETA-2. EMSA was also performed on PANG-1 and CAPAN-1 nuclear extract using an antibody specific to PDX-1. A mutated RIP-LacZ construct was created with one PDX-1 binding site chang...

example 3

Human Ductal Pancreatic Adenocarcinoma Cells that Express PDX-1 can be Targeted with a RIP-tk Gene

[0170]RIP-LacZ was created and transfected into CAPAN-1 (C-1) and MIA-1 (M−1) cell lines. RIP-tk was created and transfected into C-1 and M-1 cell lines. Tk driven by a ubiquitous promoter (U-tk), a hollow vector (HV) and untransfected cells (UT) were used as controls. Cells were treated for five days with 15 μg / ml of ganciclovir and cell viability was assessed during a MTS assay. RT-PCR was performed on C-1 and M-1 RNA with primers specific for PDX-1 and BETA-2. Nuclear extract from C-1 cells was subjected to a gel shift assay with an antibody to PDX-1. A PDX-1 binding site on RIP was mutated (mRIP) with PCR and a mRIP-LacZ construct was created and transfected in C-1 cells. Results are shown in Table 5.

TABLE 5Results: LacZ expression (LU)Percent Cell DeathRIPZRSVZmRIPZRIP-tkU-tkHVUTC-14.2 × 1059.1 × 1051.4 × 105T13 ± .1*8 ± .10 ± .14 ± .1M-11.2 × 1059.5 × 1050 ± .17 ± .10 ± .10 ± .1Ta...

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Abstract

This invention relates to a recombinant nucleic acid for an RIP-tk (rat insulin promoter-thymidine kinase) construct that selectively targets insulin secreting cells, such as β-cells, PDX-1 positive human pancreatic ductal carcinomas, and other cells containing certain transcription factors. The present invention is useful in the treatment of pancreatic cancers, such as β-cell insulinomas and can also be used to target pancreatic tumors that do not express insulin, such as pancreatic adenocarcinoma.

Description

PRIOR RELATED APPLICATIONS[0001]This application claims the benefit of U.S. application Ser. No. 10 / 656,450 filed on Sep. 5, 2003 which is a continuation of U.S. application Ser. No. 09 / 686,631 filed on Oct. 11, 2000. It also claims the benefit of U.S. Provisional Patent Application No. 60 / 161,109, filed Oct. 22, 1999, and U.S. Provisional Patent Application No. 60 / 224,382, filed Aug. 9, 2000.FEDERALLY SPONSORED RESEARCH STATEMENT[0002]This invention was made in part with United States Government support under grant number 2 RO1 DK 46441-09 awarded by the National Institute of Health, and the United States Government has certain rights in the invention.REFERENCE TO MICROFICHE APPENDIX[0003]Not applicable.FIELD OF THE INVENTION[0004]This invention relates to selective targeting of cells with cytotoxic genes using fingerprinting promoter driven specific cytotoxic genetic constructs and transcription factors, and to methods for using these constructs and transcription factors to treat ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/711A61P35/00A61K31/522A61K31/70A61K31/708C07K14/62C12N9/12
CPCA61K31/522A61K31/70A61K31/708C07K14/62C07K2319/01C12N9/1211C12Y207/01021A61K2300/00A61P35/00
Inventor BRUNICARDI, F. CHARLES
Owner BRUNICARDI F CHARLES
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