P53 protein variants and therapeutic uses thereof

Details P53 protein variants and therapeutic uses thereof P53 protein variants and therapeutic uses thereof

1998-02-20

2001-12-04

Ungar, Susan (Department: 1642)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

25 or more amino acid residues in defined sequence

C530S350000, C530S828000

Reexamination Certificate

active

06326464

ABSTRACT:

The present invention relates to proteins derived from the product of the tumour suppressor gene p53, having improved functions for therapeutic use. It relates advantageously to proteins having tumour suppressor and programmed cell death inducing functions superior to that of the wild-type p53 protein, more particularly in pathological situations of proliferation in which the wild-type p53 protein is inactivated. It also relates to the nucleic acids encoding these molecules, the vectors containing them and their therapeutic use, especially in gene therapy. The products of the invention are particularly adapted to the restoration of the functions of p53 in pathological situations such as especially cancers.
The wild-type p53 protein is involved in regulating the cell cycle and in maintaining the integrity of the cell genome. This protein, whose main function is to be an activator of the transcription of some genes, is capable, by a process not yet well defined, of blocking the cell in the G1 phase of the cell cycle during the appearance of mutations during the replication of the genome, and of triggering a number of DNA repair processes. Furthermore, in the event of a malfunctioning of these repair processes or in the event of the appearance of mutation events which are too many to be corrected, this protein is capable of inducing the phenomenon of programmed cell death called apoptosis.
In this manner, the p53 protein acts as a tumour suppressor, by eliminating abnormally differentiated cells or cells whose genome has been damaged.
This principal function of p53 depends on its function as transcription factor, that is to say, in other words, on its double capacity to recognize specific sequences at the level of the genomic DNA and to recruit the general transcription machinery.
The p53 protein comprises 393 amino acids which define 5 functional domains (see FIG.
1
):
the transcription activating domain consisting of amino acids 1 to 73 and capable of binding some factors of the general transcription machinery such as the TBP protein. This domain is also the seat for a number of post-translational modifications. It is also the seat of numerous interactions of the p53 protein with numerous other proteins and especially with the cellular protein mdm2 or the protein EBNA5 of the Epstein-Barr virus (EBV), which are capable of blocking the function of the wild-type protein. Furthermore, this domain has amino acid sequences termed PEST for susceptibility to proteolytic degradation;
the DNA-binding domain located between amino acids 73 and 315. The conformation of this central domain of p53 regulates the recognition of DNA sequences specific for the p53 protein. This domain is the seat of two types of alterations which affect the function of the wild-type protein:
(i) the interaction with proteins blocking the function of p53, such as the “large T” antigen of the SV40 virus or the E6 viral proteins of the HPV16 and HPV18 viruses which are capable of causing its degradation by the ubiquitin system. The latter interaction can only occur in the presence of the cellular protein E6ap (enzyme E3 of the ubiquitinilation cascade);
(ii) the point mutations which affect the function of p53, practically all of which are located in this region;
the nuclear localization signal consisting of amino acids 315 to 325 and essential for the correct directing of the protein in the compartment where it will exert its principal function;
the oligomerization domain consisting of amino acids 325 to 355. This 325 to 355 region forms a structure of the type: &bgr; sheet (326-334)-elbow (335-336)-&agr; helix (337-355). The alterations of functions located in this region are essentially due to the interaction of the wild-type protein with the various mutant forms which may lead to variable effects on the function of the wild-type protein;
the regulatory domain, consisting of amino acids 365 to 393, which is the seat of a number of post-translational modifications (glycosylations, phosphorylations, attachment of RNA and the like) which modulate the function of the p53 protein in a positive or negative manner. This domain plays an extremely important role in the modulation of the activity of the wild-type protein.
The function of the p53 protein can be disrupted in various ways:
blocking of its function by a number of factors such as for example the “large T” antigen of the SV40 virus, the EBNA5 protein of the Epstein-Barr virus, or the cellular protein mdm2;
destabilization of the protein by increasing its susceptibility to proteolysis, especially by interaction with the E6 protein of the human papilloma viruses HPV16 and HPV18, which promotes the entry of p53 into the ubiguitinilation cycle. In this case, the interaction between these two proteins can only occur through the prior attachment of a cellular protein, the E6ap protein whose site of attachment is poorly known;
point mutations at the level of the p53 gene;
deletion of one or both p53 alleles.
The latter two types of modifications are found in about 50% of the various types of cancer. In this regard, the mutations of the p53 gene recorded in cancer cells affect a very large portion of the gene encoding this protein, and they result in varying modifications of the function of this protein. It can, however, be noted that the great majority of these mutations are located in the central part of the p53 protein which is known to be the region of contact with the genomic sequences specific for the p53 protein.
This explains why most of the mutants of the p53 protein have the principal characteristic of no longer being able to attach to the DNA sequences recognized by the wild-type protein and thus of no longer being able to exert their role as transcription factor. Moreover, some mutants appear to have acquired new functions such as the activation of some genes at the transcriptional level.
The range of these modifications is currently classified into three categories:
the so-called weak mutants, of which the product is a nonfunctional protein which, in the case of a mutation on only one of the two alleles, does not affect the function of the wild-type protein encoded by the other allele. The principal representatives of this category are the H273 and W248 mutants, the latter being specific for the familial Li-Fraumeni syndrome for hypersensitivity to cancerous conditions.
the dominant-negative mutants, of which the product is a nonfunctional protein which, in the case of a mutation on only one of the two alleles and through interaction with the wild-type protein, is capable of blocking the function of the latter by forming nonactive mixed oligomers which can no longer attach to the DNA sequences specific for the wild-type protein. The main representative of this category is the G281 mutant.
the dominant-oncogenic mutants, of which the product is a protein which is capable, on the one hand, of blocking the function of the wild-type protein like the mutants of the previous category and, on the other hand, of promoting tumour development through poorly known mechanisms, thereby offering a gain in function. The principal representative of this category is the H175 mutant.
Taking into account its antitumour and apoptotic properties and its involvement in numerous pathologies of the hyperproliferative type, the wild-type p53 gene has been used in gene and cell therapy procedures. It has in particular been proposed to treat certain hyperproliferative pathologies, and especially cancers, by in vivo administration of the wild-type p53 gene and by restoring the functions of p53. The administration may be preferably carried out by viral and especially adenoviral (WO 94/24297) or retroviral (WO 94/06910) vectors.
It has thus been shown that the introduction of a nucleic acid encoding the wild-type p53 protein made it possible partially to restore a normal regulation of cell growth. However, while these results are encouraging, the effectiveness of these procedures is limited by the therapeutic efficacy of the p53 protein after transfer and expression in vivo in hyperp

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