Self-deleting vectors

Details Self-deleting vectors Self-deleting vectors

2000-06-12

2003-03-25

Crouch, Deborah (Department: 1632)

Chemistry: molecular biology and microbiology

Vector, per se

C536S023100, C536S023400, C536S024100

Reexamination Certificate

active

06537805

ABSTRACT:

The present application claims priority to German Application 198 34 430.9, filed Jul. 30, 1998.
The present invention relates to a recombinant vector, to a pharmaceutical composition containing the vector according to the invention, to the use of the vector according to the invention for treating tumor patients and to transduced eukaryotic cells.
So far benign and malign tumors have predominantly been treated either invasively, i.e. by surgical removal of the tumor, or conservatively, e.g. by administration of cytostatics or radiation of the organs affected, or by a combination of said methods. Great successes have already been achieved with these therapeutical possibilities because of permanently improving surgical techniques and a tremendous development in the field of cytostatics.
Nevertheless, the chances of success in the treatment of a tumor by said therapeutical methods vary considerably and are unpredictable. Moreover, each of the three methods has serious drawbacks. For instance, the surgical removal of a tumor and possibly the incision of healthy tissue greatly affects the patient because of the surgery itself. It is only in exceptional cases that the treatment with cytostatics and the radiation of tumors can be restricted to the target cells proper, so that it is almost unavoidable to subject healthy cells and healthy tissue to the treatment as well. A treatment with cytostatics means the inhibition of mitoses, which e.g. has the unpleasant side effect of alopecia. Both cytostatic treatment and radiation therapy may entail childlessness in patients of child-bearing and procreative age.
On account of the above-mentioned impairments attempts have already been made to transport therapeutics, in particular cytostatics, in a targeted manner to tumor cells and to have them internalized by said cells. An example thereof is the attempt to couple cytostatics to antibodies which bind to tumor cell-specific antigens. Despite the theoretical attractivity of such a proposal, this entails considerable difficulties, e.g. in obtaining sufficient amounts of antibodies, and also entails considerable risks, e.g. an immunological reaction to the foreign protein supplied.
A further approach is based on the knowledge about the role of transcription factors in the development of tumors. It is generally known that a few nucelar transcription factors serve to monitor the integrity of the cellular genome. When genomic DNA is damaged, said transcription factors will induce either a cell cycle arrest, which is required for repair, or apoptosis in the case of irreparable damage. Thus said transcription factors have an important tumor suppressor function. Many recent papers have been concerned with the transcription factor p53, in particular in connection with impaired p53 function in the development of cancer (Levine, 1997). The phenotype of p53
−/−
mice, produced by means of “gene targeting”, demonstrates said connection: the absence of the protein results in an increased occurrence of spontaneous tumors (Donehower et al., 1992). Moreover, it could be demonstrated with the help of said mice that p53 plays a key role in the induction of apoptosis (Lowe et al., 1994).
In conventional tumor therapies, such as radiation or chemotherapy, it is this p53-mediated apoptosis induction that plays an important role (Lowe et al., 1993). On account of the accompanying resistance to cytostatics or radiation therapy, cancer types with mutated p53 (p53
mut
) have a poor prognosis most of the time. In humans mutations of or deletions in p53 are observed in 50-80% of all cancer types (Levine et al, 1991). They always regard the DNA binding domain and entail loss in the p53 transactivator function.
The genetic difference between p53 molecules in normal and transformed cells has recently been exploited for the selective elimination of tumor cells. An adenovirus mutant which can only propagate in p53-deficient cells destroyed transplanted human p53
mut
tumors selectively and efficiently in nude mice experiments (Bischoff et al., 1996). In humans, however, an already existing immunity to adenoviruses could turn out to be a great problem. Since the majority of the population has already been immunized by preceding adenovirus infections, it could be that most of the patients intended for therapy will eliminate the therapeutical virus before it can develop its desired killing potential. Therefore, adenoviral vectors are only suited to a limited degree for use in gene therapy.
Starting from said prior art, it has been the object of the present invention to provide ways and means to fight tumor cells in a targeted manner.
According to the invention, this object is achieved by a recombinant vector comprising:
(a) at least one first transcription cassette containing a sequence coding for a recombinase, a minimal promoter MP functionally linked thereto, a transcription factor binding site and optionally a polyadenylation sequence, wherein the minimal promoter MP depends on the activation by one or several transcription factors,
(b) at least one second transcription cassette containing a suicide gene, a promoter P functionally linked thereto and optionally a polyadenylation sequence;
(c) a 5′-flanked sequence and/or a 3′-flanked sequence, wherein the 5′-and/or 3′-flanked sequence contains a recombinase target sequence.
The vector according to the invention permits the targeted and selective elimination of tumor cells in that a suicide gene is introduced by the vector into the cells, said gene being immediately eliminated in healthy cells by expression of recombinase whereas it can be activated in tumor cells with inactive transcription factors and leads to cell death (i) by expression of the suicide protein, (ii) by transcription of antisense RNA or (iii) by production of cytopathogenic virus.
In the context of the present invention the term “vector” means a linear or circular nucleic acid molecule which may consist of deoxyribonucleic acid and also of ribonucleic acid. Vectors suited for gene therapy, which may serve as starting material for the inventive vectors, are known in the prior art. Preferred vectors are vectors derived from viral or retroviral genomes because these can be packaged into viruses and can easily be introduced into cells by transduction. Vectors on a non-viral basis are also possible, but require further transfection measures. Suitable vectors would e.g. be fully synthetic vectors or vectors transduced by attenuated bacteria.
“Replication competence” means the ability of a vector to replicate in host cells. A very high replication competence with respect to mammalian cells is e.g. found in adenovirus, retroviruses, such as mouse leukemia virus MuLV, in particular Moloney mouse leukemia virus (MoMuLV). The invention generally comprises vectors with replication competence in eukaryotic cells. If the vectors are retroviruses, infectious retroviruses are preferred.
“Transcription cassettes” are nucleic acid units which apart from the sequence coding for a protein contain the necessary regulatory regions, e.g. promoter or minimal promoter with transcription factor binding site and polyadenylation sequences. The first transcription cassette may be located 5′ or 3′ relative to the second transcription cassette.
A “minimal promoter” is a natural or synthetic promoter or enhancer which can only activate the gene expression in the presence of a specific transcription factor. It contains at least one natural or synthetic transcription factor binding site.
A “transcription factor binding site” is a natural or synthetic nucleic acid sequence to which a transcription factor required for activating a minimal promoter can bind.
A “recombinase” is a natural or synthetic enzyme which recognizes and cuts specific target sequences and recombines the same with one another. Examples thereof are the Cre recombinase from the P1 coliphage (Sternberg and Hamilton, 1981) and the Flp recombinase from
S. cerevisiae
(Broach and Hicks, 1980).
“A target sequence” is a natural or

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