Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1997-06-16
2001-10-16
McKelvey, Terry (Department: 1636)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
Reexamination Certificate
active
06303115
ABSTRACT:
BACKGROUND
This invention relates to the field of screening for compounds which affect particular microbial strains, particularly including screening for antimicrobial agents.
Traditional screening methods have generally utilized screening one or a small set of compounds at a time against a particular strain, or separately against a relatively small set of strains of some microbe or cell of interest.
SUMMARY
Screening of large numbers of compounds against many strains or potential targets can be an extraordinarily time- and labor-consuming endeavor using conventional screening methods. This invention provides a method for evaluating the effects of one or more compounds or other environmental conditions on individual microbial or cellular strains (cell lines) while avoiding the very high level of work associated with conducting individual assays of the effect on each strain. In particular this provides screening methods which dramatically reduce the work involved in such screening by providing simultaneous screening for compounds active on many different targets in a single solution, where the targets may represent many different cellular functions. In many cases, the number of targets, or variants of targets, screened can extend to hundreds or even more. Thus, the methods are particularly suited to efficient screening of multiple cellular targets against large numbers of test compounds.
The method accomplishes the reduction in the effort associated with screening by using pools of strains rather than screening against individual strains, and distinguishing the individual strains in the pools. The ability to distinguish the individual strains in a pool allows the effect of a test compound on the growth of each of the strains to be determined. The method can be carried out in a variety of different formats, for example using many different types of strains, and any of a variety of techniques for distinguishing the individual strains in a pool.
In the context of this invention, the term “strain” refers to an organism or cell line, and especially to such organisms or cell lines suitable for use in the methods described herein. The term implies that a genetic difference exists between different strains which produces a different phenotype under at least some conditions. The term is not limited to refering to accepted strain designations for particular organisms. Thus, for example, different strains can be different forms of a single species of organism, either naturally occurring or artificially created, different species, or combinations of these. In particular, the term includes, but is not limited to bacterial strains, strains of eukaryotic microorganisms, cell lines derived from complex eukaryotes or higher eukaryotes, including humans, and cells expressing heterologous genes. The prokaryote and yeast cells are examples of a variety of different microbes. The term “microbe” refers to a microscopic organism, but is preferably a unicellular organism or has a unicellular stage in the life cycle.
As indicated above, in a first aspect, the invention provides a method for determining the effect of a test compound on the growth of a strain in a pool of more than one strain. The method involves determining whether the presence of the test compound changes the representation of any of a plurality of strains in the pool of strains. Each of the plurality of strains has a different distinguishable tag. While the method can be used to focus on the effect of a test compound on a single strain, preferably the effects on the growth, and thus the representation in the pool, of a plurality of strains is determined. A change in the representation of one or a subset of the strains in the pool indicates that the test compound acts preferentially on that strain. A change in representation of a strain in response to a test compound can be observed in a number of different ways. For example, in cases where strain growth is consistent, the numbers of a strain in the pool can be directly determined, and will reflect the effects of the test compound. Likewise, the change in representation can be determined by comparing the growth of a strain against the average growth of the pool or against one or more control strains in the pool. Alternatively, the growth of a strain or strains in the presence of a test compound can be compared to the growth of the strain in the absence of the test compound. This will often be combined with comparison of the effects on other strains in the pool. Many other modes for determining strain representations will be apparent to those skilled in the art, including variations and combination of the above.
The number of strains in a pool can vary over a wide range. Preferably, however, the pool has at least 10, more preferably at least 20, still more preferably at least 50, and most preferably at least 100 strains.
The term “growth” means an increase in numbers of cells; negative growth thus refers to a decrease in the number of cells. The term can, for example, refer to an increase in the number of cells in a pool or to an increase in the numbers of cells of a particular strain in a pool. “Changing the growth rate” means altering the rate of change in the number of cells. The change can be an increase or a decrease, therefore such a change in the growth rate can be due to cell killing, cell cycle slowing or arrest, or growth enhancement.
The term “representation” refers to the relative numbers or proportion of a strain or each of a plurality of strains in a mixture or pool of strains. In general, the methods of this invention preferably utilize determination of the representation of the strains in the pools, and the changes in those representations in response to the presence of a test compound. However, absolute or approximate numbers of cells of a strain in the pool can also be used to provide useful information.
A “distinguishable tag” refers to a characteristic of a strain which allows the strain to be conveniently distinguished from others in a pool of strains. Such tags can, for example, be cell surface molecules which differ between strains and which can be readily specifically detected. Other tags which can be used include distinct spectroscopic labels attached to strains, selectable markers such as drug resistance markers and trophic markers, and nucleic acid sequence tags, including tags differing in base sequence and/or length. The tags may be natural to the cells of the strain or may be artificially inserted, such as recombinant tags. Other methods of individually tagging strains to allow the distinguishing of strains in a pool are known to those skilled in the art and can be used in this invention.
From the variety of different types of distinguishable tags, in preferred embodiments the tags are selectable markers or nucleic acid sequence tags, for example, recombinant DNA sequence tags.
A “selectable marker” refers to a specific characteristic of a strain which allows the strain to be selected from other strains not having the marker through the inhibition of growth or killing of such other cells. Common examples of such selectable markers include drug resistance markers, such as antibiotic resistance markers, and trophic markers, such as specific auxotrophic requirements.
A “nucleic acid sequence tag” refers to a nucleic acid sequence which is present in a strain but not in other strains from which it is desired to distinguish the particular strain. Such a sequence tag can be detected as a ribonucleic acid (RNA) sequence or preferably a deoxyribonucleic acid (DNA) sequence. The distinction between strains can be length and/or sequence differences.
In preferred embodiments, the tags are recombinant DNA tags which differ in sequence and which can hybridized with non-cross-hybridizing complementary probes under appropriately stringent hybridization conditions. The tags can be extrachromosomal, such as on a plasmid vector, preferably a single copy vector, or are preferably incorporated in a chromosome of the strain. Also in preferred embodiments, the representation of
Lyon & Lyon LLP
McKelvey Terry
Microcide Pharmaceuticals, Inc.
Sandals William
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