Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-01-16
2003-02-04
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S810000, C530S388100, C530S388800, C530S387100
Reexamination Certificate
active
06514712
ABSTRACT:
The present invention relates to a novel therapeutic or diagnostic agent containing as an active ingredient at least one nucleic acid and which is particularly useful for the early diagnosis of dysplasias of the stratified squamous epithelium and the cartilage as well as for tumour diagnosis and tumour therapy.
Squamous Epithelial Carcinomas Precancerous Stages and Dysplasias
The squamous epithelial carcinoma is the type of oesophageal tumour having the highest incidence. In tumour diagnostics, this tumour type has been classified on the basis of histological criteria into four categories (G1, G2, G3, and G4) wherein the occurrence of histopathological abnormalities is least in G1 tumours and highest in G4 tumours.
Great efforts have been made in medical research to develop suitable methods for tumour therapy. This research has been based on a comprehensive morphological, histological, and molecular tumour diagnosis as well as on the investigation of the generation of tumours. The molecular etiology of tumour generation is not homogenous—however, in a high number of cases mutations (deletions, gene amplifications, translocations of chromosomal portions, etc.) can be detected in the respective tissues which gradually manifest themselves in abnormal differentiation and eventually in uncontrolled cell divisions. In the squamous epithelial carcinomas of the oesophagus, a variety of mutations of oncogenes, tumour suppressor genes, and growth factor genes has been reported (see review in Stemmermann et al., 1994). Interestingly, such mutations were also detected in the tissues adjacent to the tumour which appeared to be histologically normal. It has been assumed that for a tumour to develop the entirety of intracellular damages must exceed a critical threshold value. Therefore, a successful tumour therapy will also depend on its ability to diagnose early stages of tumour development.
From a histopathological point of view, dysplasias of the stratified squamous epithelium are precancerogenic lesions and, thus, may represent potential early stages of tumour development. One of the reasons for the development of dysplasias is the degeneration of the transcriptional control of differentiation-specific genes. Normally, this control is ensured by an orderly co-operation of transcription factors which jointly regulate the activity of these target genes. If it would be possible to show an alteration of already the expression, subcellular localization, or activity, respectively, of these transcription factors, then, these findings might be used as diagnostic markers for an early diagnosis of dysplasias. Regarding squamous epithelial tumours, a useful marker should also be able to indicate an alteration of the expression in malignant tissue.
Pax Genes
Pax genes are members of a multigene family containing a conserved DNA sequence called the “paired” box. To date, 9 different Pax genes (Pax1-Pax9) have been isolated from the genomes of humans and mouse (see review in Walther et al., 1991; Stapleton et al., 1993; Wallin et al., 1993). In addition, the “paired” box has also been detected in members of lower classes of animals such as nematodes, drosophila, zebrafish, turtles and chicken (see review in Noll, 1993). The “paired” box codes for the DNA-binding “paired” domain; thus, the proteins encoded by the Pax genes may be assigned to the class of transcription factors (Treisman et al., 1991; Chalepakis et al., 1991; Xu et al., 1995).
Pax genes play an important role in the development of embryonic structures. During the development of the mouse embryo, Pax genes are expressed spatially as well as temporally in specific patterns which partly overlap each other (Gruss & Walther, 1992). The strong instructive effect of the Pax genes during embryogenesis could be demonstrated i.a. by ectopic expression of Pax6 in the imaginal discs of drosophila wings and legs, respectively, using genetic engineering (normally, the Pax6 gene is expressed in the eye primordium). By ectopic Pax6 expression in the imaginal discs of wings or legs, respectively, a nearly complete eye develops in a wrong site (Halder et al., 1995).
By the finding that mutations in Pax genes cause congenital defects in mice but also in men (Pax1: undulated; Pax3: splotch and Waardenburg syndrome; Pax6: small eye and aniridia) the studies performed on this group of genes gained further interest (Balling et al., 1988; Epstein et al., 1991; Tassabehji et al., 1992; Hill et al., 1991; Ton et al., 1991).
The function of the Pax genes is not restricted to embryogenesis. For example, it could be shown that Pax5 protein activates the CD19 gene (CD19 codes for a protein specific for B lymphocytes) (Kozmik et al., 1992), and therefore has also functions in the adult organism. Pax8 is expressed in the thyroid of the adult organism and is involved in the activation of the thyroglobulin and thyroperoxidase genes (Zannini et al., 1992).
Pax Genes and Tumour Development
Some of the members of the family of Pax genes (Pax1, Pax2, Pax3, Pax6, Pax8) have been identified as proto-oncogenes due to their tumourigenic properties. This identification has been based on transformation tests in which the above-mentioned Pax genes are overexpressed in NIH3T3 cells or 208 cells, respectively, under the control of the cytomegalovirus promoter. Injection of the transformed 208 cells into nude mice caused sarcomas to occur in almost all of the cases (Maulbecker & Gruss, 1993).
The molecular basis for the development of tumours by activation of Pax genes is unknown. It could be shown for a number of rhabdomyosarcomas that as a result of chromosomal translocations Pax3 or Pax7 become fused to the FKHR gene, a transcription factor of the family of forkhead genes. It has been demonstrated that chimeric transcripts of Pax3-FKHR or Pax7-FKHR, respectively, are expressed in rhabdomyosarcomas (Shapiro et al., 1993; Davis et al., 1994).
Evidence for Pax2 expression has been achieved for Wilm's tumours of the kidney (Dressler & Douglass, 1992). Pax2 is necessary for kidney development—its expression, however, is down-regulated as early as during embryogenesis and is no longer detected in normal adult kidney (Dressler et al., 1990).
A therapeutic or diagnostic means is known from DE-A-42 25 569 which contains as an active ingredient at least one nucleic acid hybridizing to a Pax gene. Pax1 through Pax8 are mentioned as Pax genes. Furthermore, the use of such means as a molecular probe in tumour diagnosis and as antisense nucleic acid for the inhibition of gene expression are described. DE-A-42 25 569 does not mention a therapeutic or diagnostic means using a nucleic acid hybridizing to the Pax9 gene nor the uses of such means. This may be due to the fact that at the filing date of DE-A-42 25 569 (03/08/1992), the existence of the Pax9 gene was yet unknown. Further, this German Published Application does only disclose in general the use of such probes in tumour diagnosis or tumour therapy. There are no specific fields of tumour diagnosis or tumour therapy disclosed in which the agent might actually be useful, and the disclosure of which would be of importance considering the variety and heterogeneity of tumours. In particular, this Published Application does not disclose a means which may also be used in the early diagnosis of precancerogenic lesions, particularly of dysplasias of the stratified squamous epithelium.
It is an object of the present invention to provide a novel probe comprising as an active ingredient at least one nucleic acid which is useful for tumour diagnosis and tumour therapy of dysplasias, metaplasias and tumours of epithelial cells and cartilage cells.
According to the invention, this object has been solved by the therapeutic or diagnostic means characterized in more detail in claim
1
. Preferred embodiments become clear from the dependent claims and the alternative independent claims.
According to the invention, it has been surprisingly found that a nucleic acid coding for the amino acids corresponding to 30 to 337 of SEQ ID No:2 and hybridizing to th
Balling Rudolf
Höfler Heinz
Peters Heiko
Richter Thomas
Caputa Anthony C.
GSF-Forschungszentrum fur Umwelt und Gesundheit GmbH
Nickol Gary B.
Nixon & Vanderhye P.C.
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