Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-11-06
2001-11-27
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S005000, C435S029000, C435S243000, C435S069100, C435S320100, C435S325000, C435S410000, C435S455000, C435S471000, C536S023100
Reexamination Certificate
active
06322973
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates generally to the fields of molecular biology and drug discovery. More particularly, the invention relates to identification and evaluation of compounds which modulate the activity of specific biomolecules, including the identification and evaluation of potential therapeutic agents. It also concerns elucidation of gene function(s).
Conventionally, the identification of compounds which usefully modulate the activity of a biomolecule has been performed by either of two methods.
First, biomolecules implicated as playing a critical role in a particular disease are often used as targets in biochemical assays to find specific inhibitors or other modulators of the specific biomolecules. This approach, however, generally requires a great deal of prior research to identify, characterize, and validate the target, information which is unavailable for the vast majority of human genes (or the genes of other commercially important eukaryotic organisms) despite the identification of large numbers of putative coding regions from genome sequencing efforts. As a result, a biochemical assay approach remains unavailable for the majority of potential targets.
Second, screening is often performed using whole cell assays, typically by screening compounds against a cell of interest and looking for compounds which produce a particular readout. This process is often conducted with little or no information on the specific target affected by a particular compound.
Due to the limitations inherent in these conventional approaches, there remains a need for improved techniques for identifying the function of potential target genes and gene products. Additionally, useful targets have been identified, there also remains a need for improved screening techniques to identify potential modulators of these target genes and gene products.
SUMMARY OF THE INVENTION
The present invention provides a method which combines inhibitor screening and target validation in a single process, and thereby provides a more efficient process for identifying new therapeutic compounds and gene functions.
As indicated above, conventionally the identification of compounds which usefully modulate the activity of a biomolecule has been performed using either biochemical assays with validated target molecules, or non-specific, cell-based screening to detect desired cellular responses or readouts.
In contrast, the methods of the present invention provide both a cell-based screening method utilizing convenient surrogate assays and partial target characterization, which can include indication of therapeutic relevancy, thus allowing the identification and use of previously unidentified cellular targets to screen for potential lead compounds. Advantageously, these methods can be applied to the screening and characterizing of large numbers of partially and/or completely uncharacterized genes, thereby providing both combined modulator screening and target screening methods. This combination results in the use of cellular targets which would have been ignored in conventional screening methods due to the lack of specific functional and/or structural information and the large amount of work required to obtain that information.
Thus, the invention provides a method for both determining the cellular function of a gene of unknown function and identifying modulators of that gene, preferably modulators with therapeutic potential. The method involves contacting a first cell population with a test substance, where the cell expresses a biomolecule encoded by a heterologous gene, and determining whether the test substance alters a phenotype of the cell population which is created by the presence of the heterologous biomolecule. Such alteration of the phenotype indicates that the test substance is an actual modulator. A modulator identified in this way can then optimally be used to contact one or more second cell populations which provide a model system for the function of the protein in its natural cellular environment. Such a model system can, for example, be a cell line or cells of an organism naturally expressing the specific biomolecule or a cell line, transgenic animal, or microbial strain which naturally expresses a close homolog of the specific biomolecule and which expresses a recombinant or heterologous copy of the specific biomolecule. Thus, as understood by those skilled in the art, both in vivo and in vitro model systems may be used. The cellular effects resulting from the presence of the modulator are indicative of the cellular function of the protein. Preferably the second cell population provides a disease model and the cellular effects include the effect of the modulator on the status of the disease model or the progress of the disease.
Defined Terms
Unless otherwise expressly defined, the terms used below and in the claims will be understood according to their ordinary meaning in the art, although the following terms will be understood to have the following meanings, unless otherwise indicated.
The term “biomolecule” refers to molecules produced by an organism. Examples of biomolecules include nucleic acids such as DNA and RNA, proteins, carbohydrates, lipids, terpenes, and small molecules such as nucleotides, carbohydrates, and amino acids.
In the context of this invention, “gene” refers to an inheritable unit of genetic material. Each gene is composed of a linear chain of deoxyribonucleotides which can be referred to by the sequence of nucleotides forming the chain. Thus, “sequence” is used to indicate both the ordered listing of the nucleotides which form the chain, and the chain, itself, which has that sequence of nucleotides. “Sequence” is used in the same way when referring to RNA chains, which are linear chains made of ribonucleotides, or to the amino acid sequence of a polypeptide.
In the context of this invention, the term “gene product” or “product of a gene” refers to a biomolecule encoded by a DNA sequence, and is thus an RNA molecule or a protein, but which may undergo post-transcriptional or post-translational processing. Which genes are expressed in a specific cell line or tissue will depend on factors such as tissue or cell type, stage of development of the cell, tissue, or individual, and whether the cells are normal or transformed into, for example, cancerous cells.
A DNA sequence encoding a biomolecule whose function is unknown is referred to as a “gene of unknown function.” In this context, “unknown function” may be understood to describe a gene or biomolecule with no apparent homology to any gene or biomolecule with a previously identified function. Alternatively, “unknown function” may describe a gene or biomolecule with a function predicted from sequence homology to a known gene or biomolecule, but where that function has not been confirmed by other means. Typically, homology (or lack thereof) will be determined by comparison to nucleotide or protein sequences in any of the several sequence databanks used by those skilled in the art.
Additionally, “unknown function” may also describe a gene or biomolecule with a previously known function, but which also has at least one function which has yet to be identified.
The DNA sequence encoding the biomolecule may be a complete gene containing all of the necessary information to produce an RNA or protein molecule, or may be a DNA copy of a messenger RNA molecule, known in the art as a “cDNA”. Alternatively, the DNA sequence may only potentially encode a portion of an RNA or protein molecule.
“Protein” refers to a polypeptide translated from an RNA, or to a complex which includes at least one such polypeptide in an active complex. Examples include homodimers, heterodimers, multi-subunit complexes, and complexes including prosthetic groups, one or more polypeptides and one or more RNA chains.
In the context of the present invention, a “heterologous DNA sequence ” refers to a DNA sequence present in a cell which is derived from another source, including, without limitation, another cell or a virus. Typically in this invention, the
Bostian Keith
Natsoulis Georges
Guzo David
Iconix Pharmaceuticals Inc.
Leffers, Jr. Gerald G.
Robins & Pasternak LLP
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