Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-03-18
2002-07-02
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06413717
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to cancer therapy, and more particularly to the identification of agents that modulate the expression of genes encoding, for example, proteins with tumor suppressor or tumor promoting activity.
BACKGROUND OF THE INVENTION
Cancer is a significant health problem in the United States and throughout the world. Although advances have been made in cancer detection and treatment, no vaccine or other universally successful preventive or therapeutic method is currently available. Management of the disease currently relies on a combination of early diagnosis and aggressive treatment, which may include one or more of a variety of therapies such as surgery, radiotherapy, chemotherapy and hormone therapy. While such therapies provide benefit to many patients, a high mortality continues to be observed for many cancers.
The development of improved anti-tumor agents would facilitate cancer prevention and treatment. Currently, such agents may be identified through the use of cumbersome in vivo screens, in which the effect of an agent on tumor development in a test animal is evaluated. Such assays are slow and expensive, and are not suitable for screening large numbers of candidate agents. Other screens for anti-tumor agents involve the use of reporter genes linked to promoters of genes believed to play a role in cancer development. Such screens may be used to identify transcriptional regulators of gene expression, but the need to clone the relevant promoter increases the time required, as well as the likelihood of errors due to the removal of the promoter sequence from its endogenous chromosomal environment.
Accordingly, there is a need in the art for improved methods for identifying anti-cancer agents. The present invention fulfills this need and further provides other related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides methods for identifying agents that modulate the expression of a gene of interest. Within certain aspects, the present invention provides methods for screening an agent for the ability to modulate expression of a gene of interest, comprising the steps of: (a) contacting a cell comprising a gene of interest with a candidate agent under conditions and for a time sufficient to permit modulation of the level of mRNA transcribed from the gene of interest; (b) generating a cell lysate comprising mRNA; (c) stabilizing the mRNA; (d) generating amplified cDNA from the mRNA using polymerase chain reaction and two primers specific for the gene of interest; (e) separating the cDNA from free primers; (f) detecting an amount of the cDNA; and (g) comparing the amount of detected cDNA with an amount detected in the absence of candidate agent, and therefrom determining the ability of the candidate agent to modulate expression of the gene of interest. One or more of the primers may be covalently linked to a tag, such as biotin. Within certain specific embodiments, the gene of interest may be a tumor suppressor gene, such as gene is mda-7, or a tumor-promoting gene; the cell may be a human cancer cell, such as a melanoma cell; step (c) may comprise contacting the mRNA with a vanidyl ribonucleoside complex; and the candidate agent may be present within an aliquot of a small molecule combinatorial library.
Within further aspects, the present invention provides methods for screening an agent for the ability to modulate expression of a tumor suppressor gene, comprising the steps of: (a) contacting a human melanoma cell capable of expressing a tumor suppressor gene with an aliquot of a small molecule combinatorial library comprising a candidate agent, wherein the step of contacting is performed under conditions and for a time sufficient to permit modulation of the level of mRNA transcribed from the tumor suppressor gene; (b) generating a cell lysate comprising mRNA; (c) adding vanidyl ribonucleoside complex to the cell lysate in an amount ranging from 1 to 10 mM; (d) generating cDNA from the mRNA using polymerase chain reaction and two primers specific for the tumor suppressor gene, wherein at least one primer is covalently linked to a tag; (e) separating the cDNA from free primers; (f) detecting an amount of the tag; and (g) comparing the amount of detected tag with an amount detected in the absence of candidate agent, and therefrom determining the ability of the candidate agent to modulate expression of the tumor suppressor gene.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.
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patent: 5744300 (1998-04-01), Linskens et al.
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Tse and Forget, “Reverse Transcription and Direct Amplification of Cellular RNA Transcripts by Taq Polymerase,”Gene88(2):93-296, 1990.
Jiang et al., “Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression,”Oncogene 11: 2477-2486, 1995.
Fisher Paul B.
Goldstein Neil I.
Sun Yilong
Campbell Eggerton A.
Corixa Corporation
Seed Intellectual Property Law Group PLLC
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