Brca-1 regulators and methods of use

a tumor suppressor and regulator technology, applied in biochemistry apparatus and processes, instruments, transistors, etc., can solve the problems of cell escape from normal growth regulation mechanisms and proliferate in an uncontrolled fashion, and achieve the effect of increasing the propensity for cancer, upregulating or downregulating brca-1

Inactive Publication Date: 2005-09-22
IMMUSOL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007] The invention is directed to methods of identifying genes that contribute to the formation of cancer and to the identified genes and gene products as therapeutic targets for the treatment of cancer. The methods are directed to the identification of genes encoding proteins that regulate the tumor suppressor, BRCA-1, resulting in an increased propensity for cancer. Accordingly, the present invention provides ribozymes and encoding nucleic acids having target recognition sequences that enable the ribozymes to bind and cleave BRCA-1 regulators, resulting in upregulation or downregulation of BRCA-1 in a cell.

Problems solved by technology

Cancerous tumors result when a cell escapes from its normal growth regulatory mechanisms and proliferates in an uncontrolled fashion.

Method used

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  • Brca-1 regulators and methods of use
  • Brca-1 regulators and methods of use
  • Brca-1 regulators and methods of use

Examples

Experimental program
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Effect test

example i

Preparation of the Random Retroviral Vector Ribozyme Library

[0167] This example demonstrates the construction of a random retroviral vector ribozyme gene library. The inventors have discovered that by introducing a random retroviral vector ribozyme gene library in the PA1 ovarian carcinoma cell line, certain of the ribozymes will selectively target and inactivate mRNA molecules encoding regulators of the BRCA-1 tumor suppressor gene. A ribozyme gene library with randomized target recognition sequences was introduced into mammalian cells stably expressing a selectable marker, enhanced green fluorescent protein (EGFP) under the control of the BRCA-1 promoter. Cells in which BRCA-1 expression was upregulated by particular ribozymes were selected through their concomitant increase in EGFP expression. The ribozyme genes were then rescued from the cells with enhanced EGFP expression and sequenced across their substrate binding sites. The corresponding ribozyme binding sequence, or “targ...

example ii

Isolation of Ribozymes that Target BRCA-1 Regulator Nucleic Acids

[0178] This example demonstrates the isolation of ribozyme genes that bind to and inactivate BRCA-1 regulator nucleic acid molecules, and the identification of the nucleic acid sequences they target.

[0179] A reporter gene based cellular selection system was used in order to facilitate functional selection for cells with a change in their BRCA-1 expression. The reporter construct allowed expression of EGFP under control of the BRCA-1 promoter region contained in a 2.9 kb fragment from the BRCA-1 genomic sequence. The 2.9 kb BRCA-1 promoter region was amplified using a primer with an added PstI site (5′-ATCTTTCTGCAGCTGCTGGCCCGG-3′; SEQ ID NO: 52) and an AgeI containing primer (5′-GTGTAAACCGGTAACGCGAAGAGCAGATA-3′; SEQ ID NO: 53) and the PCR Long Template System (Boehringher Mannheim) from a pGL2 vector containing a 3.8 kb genomic BRCA-1 fragment. The PCR product was gel purified, digested with PstI and AgeI and cloned ...

example iii

Isolation and Characterization of Breast Basic Conserved Protein 1 (BBC1)

[0192] This example demonstrates the isolation of a full-length transcriptional regulator of BRCA-1 nucleic acid molecule designated Breast Basic Conserved Protein 1 cDNA and its encoded polypeptide.

[0193] Since ribozymes recognize their cognate targets by sequence complementarity, the sequence of a ribozyme that causes a phenotype through its catalytic activity predicts a sequence tag that can be used to clone the target gene. This “Target Sequence Tag” or TST is about 16 bases long, consisting of the two target regions complementary to ribozyme helices 1 and 2 and the requisite GUC (see, for example FIGS. 1, 2 and 7). The TST can thus be used to BLAST search the gene and EST databases, and also can be used as a primer for 3′ and 5′ RACE. BLAST searches of the databases with the TST corresponding to RH1 yielded no complete matches, and incomplete matches only with non-human sequences.

[0194] In light of the...

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Abstract

The invention provides ribozymes and encoding nucleic acids having target recognition sequences that allow the ribozyme to target and cleave BRCA-1 regulators, resulting in upregulation of BRCA-1 in a cell. Also provided are nucleic acids encoding BRCA-1 regulators that contain the target sequences recognized by the ribozymes of the invention. Fragments of these nucleic acid and protein sequences also are provided. Further provided is a method for identifying a gene where the expression level is affected by a BRCA-1 regulator and the identity of several such affected genes. Still further provided is a method of identifying a compound that modulates the activity of a BRCA-1 regulator. Also provided is a method of treating cancer, comprising introducing a ribozyme selectively reactive with an RNA encoding a BRCA-1 regulator into a cancerous cell. The invention further comprises a method of detecting a neoplastic cell in a sample.

Description

FIELD OF THE INVENTION [0001] This invention relates generally to proliferative diseases such as cancer and, more specifically, to regulation of the tumor suppressor BRCA-1 that can be used to diagnose and treat proliferative diseases. BACKGROUND OF THE INVENTION [0002] Cancer is one of the leading causes of death in the United States. Each year, more than half a million Americans die from cancer, and more than one million are newly diagnosed with the disease. Cancerous tumors result when a cell escapes from its normal growth regulatory mechanisms and proliferates in an uncontrolled fashion. Tumor cells can metastasize to secondary sites if treatment of the primary tumor is either not complete or not initiated before substantial progression of the disease. Early diagnosis and effective treatment of tumors is, therefore, essential for survival. [0003] Cancer involves the clonal replication of populations of cells that have gained competitive advantage over normal cells through the al...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): H01L29/78C12Q1/68H01L21/28H01L21/336H01L29/423H01L29/49
CPCC12Q1/6886C12Q2600/136C12Q2600/158G01N2800/52H01L21/28088Y10S257/915H01L29/42368H01L29/42376H01L29/4966H01L29/6659H01L21/28114H01L21/28
Inventor BARBER, JACK
Owner IMMUSOL
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