Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
2000-04-14
2003-12-16
Paras, Peter (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S008000
Reexamination Certificate
active
06664441
ABSTRACT:
RELATED APPLICATIONS
This application claims foreign priority benefits under Title 35, U.S.C., §119(a)-(d) or §365(a),(b) of foreign patent application no. GB 9908677.9, filed Apr. 15, 1999, the entire contents of which are incorporated herein by reference.
The invention relates to the field of genetic pharmacology and in particular to the screening of compounds for potential therapeutic activity using nematodes, principally but not exclusively, the nematode
Caenorhabditis elegans
. Specifically, the invention relates to the use of nematodes modified to have characteristics suitable for compound screening and to methods of selecting suitably modified nematodes from a population of nematodes.
Caenorhabditis elegans
is a nematode worm which occurs naturally in the soil but can be grown easily in the laboratory on nutrient agar or in liquid nutrient broth inoculated with bacteria, preferably
E. coli
, on which it feeds. Each worm grows from an embryo to an adult worm of about 1 mm long in three days or so. As it is fully transparent at all stages in its life, cell divisions, migrations and differentiation can be seen in live animals. Furthermore, although its anatomy is simple its somatic cells represent most major differentiated tissue types including muscles, neurons, intestine and epidermis. Accordingly, differences in phenotype which represent a departure from that of a wild-type worm are relatively easily observed, either directly by microscopy or by using selective staining procedures. Many
C. elegans
mutants have been identified and their phenotype described, for example see
C. elegans
II Ed. Riddle, Blumenthal, Meyer and Priess, Cold Spring Harbor Laboratory Press, 1997.
Furthermore, standard methods are known for creating mutant worms with mutations in selected
C. elegans
genes, for example see J. Sutton and J. Hodgkin in “The Nematode
Caenorhabditis elegans
”, Ed. by William B. Wood and the Community of
C. elegans
Researchers CSHL, 1988 594-595; Zwaal et al, “Target—Selected Gene Inactivation in
Caenorhabditis elegans
by using a Frozen Transposon Insertion Mutant Bank” 1993, Proc. Natl. Acad. Sci. USA 90 pp 7431 -7435; Fire et al, Potent and Specific Genetic Interference by Double-Stranded RNA in
C. elegans
1998, Nature 391, 860-811.
The
C. elegans
genome is now entirely sequenced and is available in a public database of t the Wellcome Trust Sanger Institute. As a result of the
C. elegans
genome sequencing project, it has emerged that
C. elegans
comprises genes which have equivalents that are widely distributed in most or all animals including humans.
The possibility that
C. elegans
might be useful for establishing interactions between external molecules and specific genes by comparison of
C. elegans
phenotypes which are generated by exposure to particular compounds and by selected mutations is considered by Rand and Johnson in Methods of Cell Biology, Chapter 8, volume 84
, Caenorhabditis elegans
: Modern Biological analysis of an Organism Ed. Epstein and Shakes, Academic Press, 1995 and J. Ahringer in Curr. Op. in Gen. and Dev. 7, 1997, 410-415.
Rand and Johnson in particular describe compound screening assays in which varying concentrations of the compound to be tested are added to nutrient agar or broth which is subsequently seeded with bacteria and then inoculated with worms. Any phenotypic changes in the worm as a result of exposure to the compound are then observed.
Although the nematode and in particular,
C. elegans
, is proving a powerful and efficient tool in the identification or discovery of pharmacologically active molecules, the presently known techniques for compound screening are not without drawbacks. A particular problem is that
C. elegans
, like higher organisms, is endowed with physiological barriers which prevent or minimise the ingress of foreign and potentially harmful substances. Because it normally lives in the dirt, this nematode has evolved a high number of pgp and mdr genes (Table 2) and hence has a high detoxification capacity which is a disadvantages for laboratory drug discovery purposes. In the past the only way in which these barriers have been overcome is to expose the worm to a high concentration of the compound to be tested, for example in the millimolar range. This is inconvenient because many of the compounds which might be screened in
C. elegans
are either only available in micromolar quantities or it is not economically feasible to use concentrations any higher than this. Furthermore, high concentrations of compounds may lead to death of the bacteria and hence starvation of the nematodes. It has also been observed that compounds crystallize when applied in high concentration.
If a compound is to reach a particular target in
C. elegans
it must overcome barriers in the pharynx, and in the gut and/or the cuticle.
C. elegans
feeds by taking in liquid containing its food (e.g. bacteria). It then spits out the liquid, crushes the food particles and internalises them into the gut lumen. This process is performed by the muscles of the pharynx. The process of taking up liquid and subsequently spitting it out is called pharyngeal pumping. Since this pumping process is stimulated to take place mainly in the presence of food, the pharynx is not pumping all the time in wild-type
C. elegans
. As a result, if worms are placed in a liquid culture in the presence of a dye a slow uptake and large variance in uptake amongst individual worms is observed.
Once a test compound has been internalised then the gut itself is a further frontier that may prevent the test compounds reaching their target site in the worm. This may be due to the presence of modifying and detoxifying enzymes in the gut, examples of which are the multi-drug resistance proteins (P-glycoproteins) and the multi-drug resistance related proteins.
Finally,
C. elegans
possesses a natural barrier to external substances which is the cuticle. The cuticle covers the outermost surfaces of the worm and also lines the pharynx and the rectum. It comprises mainly collagens and is substantially impermeable.
The present inventors have developed methods for compound screening in which the effect of these barriers to compound uptake is removed or diminished. These methods provide compounds assays of greater sensitivity while reducing the quantity of compound which must usually be used.
Therefore, in accordance with a first aspect of the invention there is provided a method of screening a compound for pharmacological activity which comprises observing a phenotypic change in a nematode when exposed to said compound in which said nematode exhibits constitutive pharyngeal pumping.
By “constitutive pharyngeal pumping” is meant constant pumping irrespective of the external or internal stimuli which would normally induce or repress such pumping.
The nematode may exhibit constitutive pharyngeal pumping as a result of a mutation in a relevant gene or alternatively may comprise a transgene which facilitates this phenotypic effect. Furthermore, the nematode may have other mutations or transgenes which result in the manifestation of another phenotypic characteristic which could be effected by the compound to be tested.
Caenorhabditis elegans
is the most preferred nematode worm for use in the method of the invention. However, it will be appreciated that the method may also be carried out with other nematodes and in particular with other microscopic nematodes, preferably microscopic nematodes belonging to the genus
Caenorhabditis
. As used herein the term “microscopic” nematode encompasses nematodes of approximately the same size as
C. elegans
, being of the order 1 mm long in the adult stage. Microscopic nematodes of this approximate size are extremely suited for use in mid- to high-throughput screening as they can easily be grown in the wells of a multi-well plate of the type generally used in the art to perform such screening.
As aforesaid pharyngeal pumping is facilitated in
C. elegans
by the pharynx muscles and the contraction of these muscles is controlled by a set
Behgyn Myriam
Bogaert Thierry
Feichtinger Richard
Verwaerde Philippe
Devgen NV
Paras Peter
Wolf Greenfield and Sacks, P.C.
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