The present invention relates to compounds which are capable of introducing associated compounds into a cell. In particular, the present invention relates to a transfer compound which comprises the carboxy-terminal fragment of the Ki-67 protein. Furthermore, this application encompasses vectors which contain the sequence coding for the transfer compound, transfer compounds and pharmaceutical compositions containing said transfer compounds and/or vectors. Claimed are also methods for the production thereof as well as the use of said transfer compounds. Corresponding methods for the treatment or prevention of diseases by gene therapy with the aid of said transfer compounds are within the scope of the invention.
Protein targeting is a biological process of fundamental importance which is controlled by highly coordinated mechanisms.
Thus protein export or secretion occurs via specific reaction routes for which characterized signal sequences are used for directing the proteins into the subcellular compartments involved, e.g. endoplasmic reticulum, Golgi complex and vesicles. Signal sequences are also used for the intracellular transfer. For instance, nuclear localization sequences (NLS) have been described for the transfer of proteins into the cell nucleus; these nuclear localization sequences direct large proteins, which cannot pass into the nucleus by diffusion, through the nuclear pores into the cell nucleus.
The uptake of proteins in a cell is also regulated in a complex manner. One example to be given here is the receptor-mediated endocytosis which serves the import of specific proteins by binding to receptors on the cell membrane and subsequent inclusion in vesicles. This process serves, on the one hand, to supply cells with metabolites required for metabolism and, on the other hand, to degrade proteins. Furthermore, the receptor-mediated endocytosis mediates the cellular responses to many mediators, such as peptide hormones or growth factors. Finally, said process is used by viruses and toxins to pass into cells.
It is desired for many applications in biomedical research, diagnostics and therapy to introduce substances, preferably proteins, nucleic acids, non-peptide molecules, such as oligosaccharides, lipids or drugs or marker molecules, into cells. Since many of the aforementioned substances are not able to pass through the cell membrane, different methods are employed for the introduction of said substances and their intracellular production, respectively.
Apart from mechanical methods, such as e.g. microinjection, the person skilled in the art is well aware of e.g. expression techniques used in molecular biology for this purpose. The last-mentioned methods, however, are not very efficient; as a rule, an expression is only successful in 2–20% of the cells, which renders e.g. an in-vivo application very difficult. This drawback was recently overcome by using a structural protein (VP22) of the herpes simplex virus type 1 (HSV-1). After classic transfection with expression vectors it was found that, in contrast to another HSV-1 protein which (as expected) could only be detected in 2–5% of the cells, the VP22 could be detected in 100% of the cells (PCT application no. WO 97/05265). Furthermore it was found that said viral protein as a fusion protein can introduce various polypeptides into target-cell populations (WO 97/05265). However, it is well known to the person skilled in the art that viral proteins can initiate pleiotropic effects preferably in mammalian cells, cell assemblies and the whole organism, respectively.
For instance, the E1A protein of adenoviruses as well as the T antigen of the simian virus 40 (SV40) start a multitude of processes in the cells. These include, for instance, initiation of the DNA synthesis as well as the activation of various enzymes, such as dihydrofolate reductase, thymidine kinase and DNA polymerase (Nevins, J. R. Adenovirus E1A: Transcription regulation and alteration of cell growth control, in Doerfler, W. and Böhm, P., The molecular repertoire of Adenovirus III: Biology and pathogenesis, Springer Verlag Berlin, Heidelberg, N.Y., 1995). A further example are the pleiotropic properties of the structural proteins of reoviruses (Yue, Z. and Shatkin, A. J., Enzymatic and control functions of Reovirus structural proteins, in Tyler, K. L. and Oldstone, M. B. A., Reoviruses I: Structure, Proteins, and Genetics, Springer Verlag Berlin, Heidelberg, N.Y., 1998).
It is therefore the object of the present invention to provide a transfer vehicle for compounds to overcome such drawbacks. The transfer compounds can be used in gene therapy.
The transfer vehicle according to the invention is from a mammal, preferably of human origin.