Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-02
2002-10-22
Chan, Christina (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S252300, C435S252330, C435S320100, C435S810000, C530S350000, C536S023100, C536S023500
Reexamination Certificate
active
06468750
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the cell regulation factor TTO 20 and DNA therefor, and its preparation and use for screening purposes for the discovery of modulators for TTO 20 activity.
BACKGROUND
The many biological effects of interleukin-1 (IL-1) include the action of IL-1 on the metabolism of many types of cells from the connective tissue. An example of cells of this type is articular chondrocytes. IL-1 inhibits the synthesis of proteoglycans (PG) by chondrocytes and stimulates the production of prostaglandin E
2
and metalloproteinases, which are capable of degrading molecules of the extracellular matrix.
On the basis of experimental results, and the discovery of IL-1, PG fragments, and proteolytic enzymes in inflammation-modified joints, it was concluded that IL-1 plays a part in cartilage degradation in osteoarthrosis and rheumatoid arthritis (Beuton H P & Tyler J A, 1988
, Biochem. Biophys. Res. Comm
. 154:421-428; Aydelotte M B et al.,
Comm. Tiss. Res
. 28:143-159; Wood D D et al.,
Arthritis Rheum
. 26:975-983; Lohmander L S et al.,
Trans. Orthop. Res. Soc
. 17:273). Matrix metalloproteases are potential candidates for starting points for a therapy with active compounds that interact with these enzymes. Until now, no actual molecular starting points have been identified that relate to early steps in the complex process that leads to cartilage degradation. For this reason, various approaches have been chosen in order to obtain molecular starting points of this type for a medicinal therapy of osteoarthrosis and rheumatoid arthritis.
Such an approach is described in European Patent Application EP 0 705 842 A2. The question was whether it would be possible to obtain potential molecular starting points for a medicinal therapy of IL-1&bgr;-induced cartilage degradation on the RNA plane in human, articular chondrocytes from osteoarthritic cartilage.
For this purpose, genes were identified that are expressed differentially in diseased cartilage. Total RNA from IL-1&bgr; stimulated and nonstimulated human chondrocytes was subjected to a differential display of mRNA by reverse transcription and the polymerase chain reaction (DDRT-PCR). This method can be used for the identification and isolation of genes that are expressed differentially in two cell populations (Liang P & Gardee A B (1992),
Science
257: 967-971; Liang P et al., A B (1993),
Nucl. Acids Res
. 21: 3269-3275; Bauer D et al. (1993),
Nucl. Acids Res
. 21:4272-4280). The key element of this technology is the use of a set of oligonucleotide primers, one of which binds to the polyadenylated tail of the mRNA, and the others are random decamers that bind to various other sites of the mRNA. Such mRNA subpopulations, which are defined by a specific set of primers, are amplified after reverse transcription and separated on DNA sequencing gels. Band patterns are seen that are characteristic for one of each of the cell lines studied. Thus, for example, 100 different primer combinations should afford 10,000 different PCR products, which represent at least approximately half of all the genes expressed in a cell line. A comparison of the band patterns of two different cell lines indicates those bands that correspond to differentially expressed genes. On the basis of this information, it is now possible to extract, to reamplify, to subclone, and to sequence bands of differentially expressed gene products from the gel.
However, this is to be qualified by saying that this method has a number of difficulties:
1. As a result of the high sensitivity of the DDRT-PCR, slightly artificial bands can result.
2. The analysis of complex gene expression patterns is difficult.
3. Only tiny amounts of RNA are available as starting material.
These difficulties cause uncertainty in the results obtained.
In European Patent Application EP 0 705 842 A2, a number of short DNA sequences are disclosed that have been identified in the manner described above. An analysis of these sequences showed that some are complete or have very great identity with the sequences of already known genes. Thus, a cDNA fragment having 100% identity with human osteopontin, another cDNA fragment having 97.2% identity with human calnexin, and a further fragment having 99.5% identity with human TNF-30 stimulated gene 6 (TSG-6) were found. Most of the fragments found, however, could not be assigned to any known gene based on the sequence corresponding to the fragment. This group of cDNA fragments also included the 400 bp-long clone TTO 20/2(2), 152 bp of which has been deciphered.
In the context of the present invention, the clone TTO 20/2 has now been investigated more closely. An antisense experiment is one method for investigating the functional meaning of the corresponding gene or gene product.
The expression of antisense RNA in human chondrosarcoma cells, which are regarded as model cells for cartilage differentiation, yielded indications of a role of TTO 20/2. The antisense approach is based on transforming the cultured cells with a vector that expresses antisense mRNA to TTO 20, but at the same time, the vector also expresses an indicator protein whose activity indicates whether antisense RNA was formed. Vectors of this type are called bicistronic or dicistronic vectors. The starting vector for the present constructs was pED4, whose construction has been described by Kaufmann et al. (Kaufmann et al., (1991),
Nucl. Acids Res
. 19: 4485-4490).
In antisense technology, the formation of a functional protein is restricted or even prevented via the expression of a complementary RNA (antisense RNA) that binds to the protein-encoding mRNA (sense RNA). In particular, as the present examples confirm, antisense RNA can be employed for subregions of the encoding mRNA, and for the 3′ or 5′ untranslated region, in order to prevent the formation of the target protein. With the aid of the vector, EST fragments that have no defined open reading frame can thus be used in order to work out the action of the protein that is finally encoded by the associated gene because the “antisense-expressed” EST switches off or decreases the reading of the encoding sense mRNA. If the synthesis of the target protein that is blocked in this way plays an important role in the cell, this has direct or indirect effects on cell division, cell growth, synthesis of regulated and expressed proteins etc. If the antisense expression prevents, for example, the formation of a factor that plays a role in signal cascades, then that cascade is disturbed.
If the factor is a transcription factor, the expression of a number of genes is disturbed. This can be recognized, for example, from morphologically visible changes that can be attributed to altered expression of secreted proteins, particularly proteases.
The genes, or products thereof, identified in this way can be employed as therapeutic targets for the search for pharmacologically active substances. Likewise, cells transformed in this way can be used in screening systems attempting to block the action of the antisense RNA.
DNA chip technology allows the direct analysis of such changes because the transcript profiles of transformed cells can be compared with untransformed or mock-transformed cells. The comparison then allows conclusions to be drawn as to whether an EST plays a crucial role in the context of a clinical picture and is thus suitable as a screening target. The use of the vector is thus also suitable for the discovery of novel targets and for the profiling of novel medicaments. The vector is particularly suitable for the synthesis of HTS systems for target validation. Expediently, EST clones are cultured in HTS formats, such as 96-well microtiter plates, and the insert DNA is amplified by means of PCR using suitable PCR primers that, for example, generate a PST cleavage site on the 3′ side and an Eco RI cleavage site on the 5′ side for cloning in one of the pED4 derivatives described. In a second step, the PCR fragments generated in this way are cleaved using Pstl and Eco RI and ligated into the di
Aventis Pharma Deutschland GmbH
Chan Christina
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Huynh Phoung N.
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