Nucleic acid constructs whose activity is affected by...

Details Nucleic acid constructs whose activity is affected by... Nucleic acid constructs whose activity is affected by...

1998-12-18

2001-07-24

Hauda, Karen M. (Department: 1632)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C536S023500

Reexamination Certificate

active

06265562

ABSTRACT:

INFORMATION ON RELATED APPLICATIONS
The present application claims the priority benefit, under 35 U.S.C. §119, of Federal Republic of Germany Application No. 19756975.7, filed Dec. 20, 1997.
BACKGROUND OF THE INVENTION
The cell cycle of eukaryotic cells is controlled by cyclin-dependent kinases (cdks); these are kinases which require a regulatory subunit (“cyclin”) in order to be active. Different processes in the cell cycle (such as replication and entry into mitosis) are controlled by different cdks (Morgan, Nature 374, 131 (1995)). In association with this, the activity of cyclin-dependent kinases is subject to a high degree of regulation. In this context, internal control mechanisms exist which, for example, prevent entry into mitosis until the DNA has completed its replication. Control by external factors, such as growth factors, only occurs before DNA replication begins; replication is initiated by active cyclin E/cdk2 complexes.
In addition to the quantity of cyclin in the kinase subunit, the activity of cyclin-dependent kinases is also regulated by small inhibitor proteins (Sherr and Roberts, Gene Dev. 9, 1149 (1995)). For example, two inhibitors, which are designated p21 and p27 in accordance with their size, are crucial for cyclin E/cdk2.
The cyclin E/cdk2 kinase is normally inactive in cells which are expressing high quantities of p27, and the entry into DNA replication is blocked.
While positive cell cycle regulators are overexpressed or at least expressed constitutively in many human tumors (Sherr, Science 274, 1672 (1996)), negative regulators are frequently mutated or only weakly expressed (Fero et al., Cell 85, 733 (1996)). Specific correlations exist: for example, the cyclin D1 gene is found to be overexpressed in many neck tumors. The hope therefore exists that cyclin-dependent kinases, and their function, might be target structures in the search for novel, selective substances which have an antiproliferative effect.
The gene for the p27 protein has been known for some years (K. Polyak et al. Cell 78, 59-66 (1994)) and is available in Genbank [murine p27; accession number K 09968; human p27: K 10906]. Despite intensive searching, mutations in the p27 gene have not so far been found in human tumors. This is all the more surprising since mice in which the gene for p27 has been inactivated exhibit a phenotype with multiple dysplasias and an increased incidence of tumors (Fero et al., Cell 85, 733 (1996), Kiyokawa et al., Cell 85, 721 (1996)). Instead of this, the function of p27 is evidently in the main regulated posttranscriptionally.
Thus, p27 is degraded by proteolysis; this also occurs at the beginning of DNA replication in normal cells. The ability to degrade p27 proteolytically is markedly increased in many tumors, as compared with the normal tissue, and this appears to correlate directly with an unfavorable prognosis (Loda et al., Nature Med. 3, 231 (1997)).
However, there must also be other mechanisms as well which are able to lead to p27 inactivation. For example, after cells have been transformed with the Myc oncogene, p27 is first of all inactivated functionally (i.e. it no longer binds to cyclin/cdk complexes) and is only degraded at a much later stage. It has not so far been possible to understand why large quantities of p27 proteins are expressed in a number of breast tumors, for example, and these tumors nevertheless grow very rapidly. The mechanisms which inactivate p27 in these tumors are so far unknown (Fredersdorf et al., Proc. Natl. Acad. Sci. USA 94, 6380 (1997)).
There is consequently a great interest among experts in finding mechanisms or substances which are responsible for inactivating p27 in tumors. The present invention has solved this problem. The protein p163, which is described in the application, can bind p27, can inhibit the function of p27 and can lead p27 to proteolysis in the cytoplasm.
SUMMARY OF THE INVENTION
The present invention relates to nucleic acid constructs comprising (a) a first activation sequence; (b) a transcription factor gene, the transcription of which is induced by activation of the first activation sequence, comprising (1) an activation domain, (2) a sequence for binding an inhibitor, and (3) a DNA-binding domain; (c) a second activation sequence that is activated by binding the expression product of the transcription factor gene; and (d) an effector gene, the transcription of which is induced by the activation of the second activation sequence. In one embodiment of the invention, the first activation sequence and the second activation sequence are the same.
The first activation sequence may be activated non-specifically, cell-specifically, metabolically specifically, virus--specifically and/or cell cycle-specifically. Examples of first activation sequence include promoters which are activated in endothelial cells, peritoneal cells, pleural cells, epithelial cells of the skin, epithelial cells of the lung, epithelial cells of the gastrointestinal tract, epithelial cells of the kidney, epithelial cells of the urinary tracts, muscle cells, connective tissue cells, hematopoietic cells, macrophages, lymphocytes, leukemia cells, tumor cells or glia cells; promoter sequences of viruses; promoter or enhancer sequences which are activated by hypoxia or cell cycle-specific activation sequences of the genes for cdc25C, cyclin A, cdc2, E2F-1, B-myb, or DHFR; and sequences for binding transcription factors which appear or are activated in a cell proliferation-dependent manner.
According to one aspect of the present invention, the activation domain of the transcriptional factor gene may be at least one of the activation domains of Oct-2, Sp1, NFY, ITF-2, VP-16, c-Myc or CTF. According to another aspect of the invention, the sequence for binding an inhibitor encodes an inhibitor of protein p27. In one embodiment, the inhibitor of protein p27 is protein p163 protein or a homologue, derivative, or part thereof. Derivatives of the include those comprising a deletion or mutation in the p27- or Ran-binding domain. In one embodiment, the inhibitor of protein p27 is the p27-binding domain of protein p163. In another embodiment, the inhibitor is a human p163 protein or a homologue, derivative or part thereof. In still another embodiment, the inhibitor is an antibody or antibody fragment that binds to p163 protein.
In another aspect of the present invention, the second activation sequence comprises at least one DNA sequence for binding the transcription factor.
In yet another aspect of the present invention, the effector gene encodes an active compound selected from the group consisting of cytokines, chemokines, growth factors, receptors for cytokines, chemokines or growth factors, proteins having an antiproliferative or cytostatic or apoptotic effect, antibodies, antibody fragments, angiogenesis inhibitors, peptide hormones, coagulation factors, coagulation inhibitors, fibrinolytic proteins, peptides or proteins having an effect on the blood circulation, blood plasma proteins and antigens of infectious pathogens or of cells or of tumors, with the selected antigen bringing about an immune reaction. In one embodiment, the effector gene encodes an enzyme which converts a precursor of a drug into a drug. In another embodiment, the effector gene encodes a ligand-active compound fusion protein or a ligand-enzyme fusion protein, wherein the ligand is selected from the group consisting of cytokines, growth factors, antibodies, antibody fragments, peptide hormones, mediators, and cell adhesion molecules.
The present invention also relates to vectors and isolated cells comprising the nucleic acid constructs
The present invention further relates to methods of treating or preventing a disease, comprising administering to a patient a nucleic acid construct or isolated cell according to present invention. The disease treated or prevented can be, for example, infections, tumors, leukemias, autoimmune diseases, allergies, arthritides, inflammations, organ rejections, graft versus host reactions, blood coagulation diseases, circulatory

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