Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-08-10
2002-10-29
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S325000, C435S320100, C435S440000, C435S455000, C435S471000, C435S476000, C435S004000, C435S029000, C536S023100
Reexamination Certificate
active
06472151
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides a method for functionally expressing and isolating molecular targets for drug discovery without utilizing information about the nucleic acid or amino acid sequence of the target under study. This procedure for the first time allows one to develop fast high throughput screens against molecular targets whose specific nucleic acid and amino acid sequences are not available. Screening tools such as cell lines or Xenopus oocytes are generated by expression of cDNA libraries and validated based on the pharmacological and physiological properties of the desired protein. No specific sequence information (e.g., purified or isolated nucleic acid sequences) is necessary to practice the screening tool of the present invention.
BACKGROUND OF THE INVENTION
Obtaining isolated cDNA clones of potential molecular targets for drug discovery is a standard method used throughout the pharmaceutical industry. Such clones often allow more facile screening against libraries of potential therapeutic compounds. For example, if one wishes to screen for compounds that modulate the activity of inosine 5′-monophosphate dehydrogenase (“IMPDH”), the nucleic acid sequence is useful to manufacture recombinant protein and run a screen. (See, e.g., U.S. Pat. No. 5,665,583).
At present, molecular targets are cloned using available nucleic acid or protein sequence information, and polymerase chain reaction (“PCR”) or homology screening. Verification is accomplished by characterizing the functional or physical properties of the potential target. It is important to note that the functional properties of the protein are of greatest value to drug discovery. In many cases the nucleic acid or amino acid sequences are of lessor interest and value.
One common approach to identifying the nucleic acid sequence of a molecular target is referred to as expression cloning. A single cDNA is isolated and cloned based on its ability to direct the synthesis of a functional protein. The properties conferred upon host cells are used as the endpoint in the search for an appropriate cDNA clone. Normally, this individual cDNA is then sequenced. However, availability or knowledge of the sequence (or lack thereof) does not alter the ability to functionally characterize the expressed protein or to use it in a screen. A major drawback to this historic method of screening molecular targets is that expression cloning is laborious and time consuming. Newer technology for detecting the actions of receptors, enzymes and ion channels has made this task markedly easier.
The present invention for the first time presents a new method for combining classical expression cloning with high sensitivity methods for detecting functional proteins in host cells to produce molecular and cellular tools for high throughput screens and other drug discovery efforts. The novelty of this method is that desired endpoint of DNA manipulation is not the isolation and sequencing of a single DNA molecule, but rather the generation of cells and cell lines that express a particular desired phenotype. This approach provides significant advantages over current screening methods that rely upon expression cloning and sequencing, and facilitates the efforts of those skilled in the art to find molecules that interact with nearly any human protein and develop drugs that target these proteins.
SUMMARY OF THE INVENTION
The present invention provides a novel method for generation of cells and cell lines and to screen for compounds that interact with, and potentially modulate the activity of, a given molecular target. The present invention eliminates the need to isolate and clone a specific nucleic acid sequence encoding the molecular target. Rather, the method relies upon techniques such as retroviral activation of cellular gene transcription and detecting the functional or physical properties of the potential target. The present method is quicker than the screening methods of the prior art, and is adaptable to test compounds against any molecular target protein.
DETAILED DESCRIPTION OF THE INVENTION
Functional or expression cloning provides a method of expressing, isolating, and maintaining clones of potential targets of therapeutic intervention. Messenger RNA (“mRNA”) is isolated by methods known in the art. Preferably, the MRNA is isolated from tissue known to express a certain molecular target of interest. In one embodiment, using known methods (Chirgwin et al., (1979)
Biochem.
18:5294-5299), the isolated mRNA is reverse transcribed to produce a “library” of cDNAs, which ideally will contain full length copies of all mRNAs expressed in that tissue. The library of cDNAs is then subcloned into commercially available vectors that are optimized to express proteins encoded by the cDNAs in a host cell, for example in mammalian cells, Xenopus oocytes,
E. coli,
or yeast.
The library is then subdivided into pools containing any number of individual clones, for example 1000-5000 individual clones, per pool. Expression is accomplished by introducing individual pools of the library into the appropriate host cells and testing for functional activity with a variety of assays. The exact host cell and measured endpoint will depend on the nature of the molecular target one wishes to test.
Once a pool containing the cDNA of the molecular target is identified, that cDNA pool is preferably further subdivided until a single cDNA that confers the appropriate function is found. Reduction to a single plasmid DNA is not necessary because permanent cell lines could be made from a mixture of cDNAs based solely on the fact that the final clonal line responded in a fashion expected of the molecular target.
If further molecular biological manipulations are necessary, such as for example high level expression in baculovirus-infected Sf9 cells, a plasmid comprising the recombinant DNA will be “rescued” from the transfected cells. Chromosomal DNA may then be isolated from positive cells and amplified by PCR with primers that recognize the flanking sequences from the original expression vector. The resulting PCR fragment is subcloned into a baculovirus shuttle vector for development of the appropriate recombinant virus.
Once a pool, or single cDNA, that confers an appropriate function is found, test chemical compounds can be screened to determine if they have an affect on the function of the specific molecular target. The term “chemical compound” encompasses proteins, peptides, and other molecules. This method never requires the use of an isolated nucleic acid sequence that encodes the actual target molecule.
In another embodiment, this invention utilizes the propensity of certain RNA-based viruses, known as retroviruses or tumor viruses, to efficiently infect and then integrate DNA copies of their genome (or proviruses) into the chromosomal DNA of a host cell. The mechanism of integration varies among retroviruses, but is fairly non-specific in terms of chromosomal integration sites. Significantly, proviral integration often results in the physical linkage of strong viral expression elements (long terminal repeat enhancers) proximal to inactive or weakly active cellular genes. At some frequency, this results in the aberrant over-expression of the cellular gene, a phenomenon known as transcriptional activation. Retroviral activation of oncogenes has been very well documented. If the over-expressed RNA is translated to produce a target protein that has sufficient activity in the host cell, such a cell can be detected in a screening assay based on a functional or physical property of the protein. It follows that in a sufficiently large pool of retrovirally infected cells, a panel of cellular genes potentially including every one in the genome will be activated by different integration events, and from this population, a cell over-producing any particular molecular target can be identified, isolated and propagated indefinitely. The method of this invention thus comprises the following sequential steps: 1) infection of host cells with an integrating ret
Robinson Gordon W.
Verdoorn Todd A.
Weaver C. David
Bristol--Myers Squibb Company
Guzo David
Klein Christopher A.
Leffers, Jr. Gerald G.
Sher Audrey F.
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