Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
2000-04-06
2003-01-07
Clark, Deborah J. R. (Department: 1633)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C536S023100, C536S023500, C435S320100, C435S325000, C435S455000
Reexamination Certificate
active
06503502
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the demonstration of genes which are involved in the molecular pathways of tumor suppression and to the use of the genes which have thus been demonstrated for treating certain genetic malfunctions, in particular cancers.
BACKGROUND OF THE INVENTION
The present invention was made possible by isolating cDNA which corresponded to the messenger RNAs which are expressed or repressed during the process of apoptosis which is induced by the p53 suppressor gene.
A global analysis of the molecular events which take place during the cell cycle at the time of development and cell apoptosis is required in order to better understand the importance of the p53 gene in the process of tumor suppression or, on the contrary, of canceration.
The transformation of a normal cell into a tumor cell is a process which takes place in several stages and which requires a sequence of molecular events. At the physiological level, these events find expression in the tumor cell becoming independent of external signals and in an internal deregulation which leads to uncontrolled growth.
Two groups of genes are responsible for this so-called “malignant” transformation; on the one hand oncogenes and, on the other hand, suppressor genes or anti-oncogenes. Because of their deregulation in cancer (resulting most frequently from a mutation or a translocation), oncogenes induce a positive signal which promotes neoplastic growth. By contrast, the suppressor genes are unable, either because they have been deleted, because they are not being expressed due to mutation of the promoter, for example, or because of mutations which modify the structure and function of the protein, to supply, in the cancer, the signal which would normally retard this abnormal growth. As a consequence, malfunction of the suppressor genes contributes to neoplastic transformation.
SUMMARY OF THE INVENTION
The object of the present invention is to isolate genes which normally play a part in tumor suppression and any possible malfunctions of which can then be monitored and treated.
In particular, isolation of these genes makes it possible to carry out a gene replacement therapy or else to synthesize protein or non-protein pharmacological agents which, directly or indirectly, induce activation and expression of these genes by acting on the promoters, or else to synthesize pharmacological agents which mimic the physiological effect of these suppressor genes.
The final objective is either to inhibit tumor growth or, even better, induce the apoptotic process in these tumor cells, that is to cause the tumor cells to “commit suicide”.
Thus, in one embodiment the invention relates to an isolated DNA molecule comprising a sequence selected from the group consisting of:
(a) a nucleotide sequence of one of SEQ ID Nos. 4 to 11,
(b) a nucleotide sequence that hybridizes with one of the sequences of
(a), and
(c) a nucleotide sequence that is at least 80% homologous with a
sequence of either (a) or (b). Expression of this DNA molecule may activate cell apoptosis and/or tumor suppression.
In another embodiment, the invention relates to an isolated DNA molecule comprising a sequence selected from the group consisting of:
(a) a nucleotide sequence of one of SEQ ID Nos. 1 or 3;
(b) a nucleotide sequence that hybridizes with one of the sequences of
(a), and
(c) a sequence that is at least 80% homologous with a sequence of either
(a) or (b). Tumor suppression induces expression of this DNA molecule.
In another embodiment, the invention relates to an isolated DNA molecule comprising a sequence selected from the group consisting of:
(a) SEQ ID No. 2,
(b) a nucleotide sequence that hybridizes with SEQ ID No. 2, and
(c) a nucleotide sequence that is at least 80% homologous with either (a) or (b); wherein cell apoptosis induces expression of this DNA molecule.
In a preferred embodiment, the DNA molecule of this invention is SEQ ID No. 11 or fragment thereof.
In yet another embodiment, the invention relates to a biologically functional vector comprising one of the above described DNA molecules. Another embodiment relates to a host cell stably transformed with this vector. In yet another embodiment, the invention relates to a protein obtained by culturing the host cell under appropriate nutrient conditions so as to allow the cell to express the protein.
In another embodiment, the invention relates to a pharmaceutical composition comprising the above described vector and a pharmaceutically acceptable carrier. Another embodiment relates to a method of preventing tumorigenesis, the method comprising contacting cells with a tumorigenesis inhibiting amount of the pharmaceutical composition. In a preferred embodiment, the pharmaceutical composition comprises the DNA SEQ ID NO. 11 or TSAP 3.
In other embodiments, the invention relates to a DNA probe or a PCR amplification primer comprising a nucleotide sequence selected from the group consisting of SEQ ID No. 1-11, or a fragment thereof.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to demonstrating genes which are involved in this apoptosis, particularly TSAP 3, which has been discovered to directly activate apoptosis. Thus, each cell contains within itself a program of physiological death. This is also a physiological process which is involved in development for the purpose of maintaining homeostasis of the body and of preventing abnormal cell proliferations from becoming established even if, for all that, they are not malignant in nature.
One of the most important suppressor genes involved in apoptosis is the p53 gene. In its normal function, this gene controls cell growth and the apoptotic process; in particular, it is this gene which blocks cell growth and which is responsible for inducing the apoptotic process in order to avoid the development of a cancer. Thus, it has been demonstrated that mice which are nullizygous for p53 are much more sensitive to the formation of tumors. The fact has also been demonstrated that, in cancers, the p53 gene is very often altered and leads to the production of proteins which are unable to serve as a vehicle for the apoptotic message.
It is this distinctive feature which has been employed within the context of the present invention.
Thus, the present invention is based on the observation that it is not possible, or that it at least appears very difficult, to institute a direct replacement therapy when the p53 gene is malfunctioning. Thus, when p53 is mutated as it is in cancer, it nullifies the physiological effect of the normal p53.
It was therefore necessary, at least initially, to abandon a replacement therapy which acted directly at the level of p53.
The present invention is therefore linked to studying the genes which are situated downstream of p53 in order to bypass the abovementioned difficulty.
In order to isolate the genes which are activated or inhibited by normal p53 (wild-type p53), a global screening was carried out of gene expression in a cell in which apoptosis had been induced and in the same malignant cell, more specifically in a cell which was expressing a p53 whose function was normal and in a cell which was expressing a p53 which was mutated and whose function was oncogenic. Comparison of the expressed genes (messenger RNAs expressed in the two types of cell) made it possible to identify genes which were expressed differentially, that is which were expressed in one of the cells but not the other (the genes can be activated or inhibited).
It was readily deduced that these genes are involved in the process of canceration, in the one case by their absence, and in the other case by their presence.
The method used for carrying out this differential study is the method described in 1992 by Liang and Pardee (Differential display of eucaryotic mRNA by means of a polymerase chain reaction)
Science
257: 967-971 (1992), which is herewith incorporated by reference.
Until now, genes involved in suppression have been isolated either by positional cloning or by using double hybrids. The first method has made
Amson Robert
Cohen Daniel
Telerman Adam
Chen Shin-Lin
Clark Deborah J. R.
Société Molecular Engines Laboratories a French Société anonyme
LandOfFree
Nucleotide sequences, proteins, drugs and diagnostic agents... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nucleotide sequences, proteins, drugs and diagnostic agents..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleotide sequences, proteins, drugs and diagnostic agents... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3016511