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2009
Doctoral Thesis
Titel
Development of novel RNA- and protein-based cancer therapeutics by either silencing of potential oncogenes or active restoration of a tumor suppressor gene
Alternative
Entwicklung neuartiger RNA- und Protein-basierter Krebstherapeutika durch Inhibition potenter Onkogene oder durch aktive Rückführung von Tumorsuppressorgenen
Abstract
Cancer has become more and more prevalent over the last few years, reaching its highest levels per capita in the Western world in 2008. Mutations that produce dominant gain of function oncogenes and loss of function tumor suppressor genes have been discovered due to their alteration in human and animal cancer cells, and by their ability to elicit cancer phenotypes in experimental models. Typical treatments for cancer include surgery, radiation therapy and chemotherapy. However, chemotherapy also kills normal cells because tumor cells are not specifically targeted, resulting in significant side effects. In the last 10 years, the problem of resistance against chemotherapeutic agents has become more common, and further investigation revealed that the resistant tumor cells contained mutations that allowed them to regulate drug cytotoxicity. Novel approaches have therefore been tested. These strategies are more specific to cancer cells and their metabolism. In clinic, they achieve promising levels of cytotoxicity towards cancer cells and reduce unwanted side effects. Targeted therapy is based on the specific elimination of cancer cells without damaging the surrounding, healthy tissues. For this approach to work, two major components are needed: first, a specific binding moiety that only targets cancer cells, and second, a toxic moiety that is taken up by endocytosis and induces cell death. These two components are often functionally combined to achieve a drug which selectively targets and kills the diseased cell.APTAMER-SIRNA-TRANSCRIPTSA prerequisite for successful RNAi-based cancer therapy is the identification of potent siRNA sequences that selectively silence genes crucial for tumor cell survival. Here the challenge was addressed by silencing key components of the translational machinery that are sensitive to apoptosis. This thesis has shown that the transfection of cytotoxic siRNAs into tumor-derived cell lines efficiently induces apoptosis after specific mRNA degradation. One of the major challenges in the development of siRNA-based drugs is the incorporation of effective strategies for cell type-specific siRNA delivery. We chose the well-established PSMA binding aptamer xPSM-A10-3 as a cell surface-specific ligand for the development of aptamer based siRNA-delivery vehicles. Furthermore, we also used A30, a novel RNA aptamer that binds to HER3 expressed on MCF-7 cells. This is the first study demonstrating the specific binding of A30 to HER3-expressing MCF-7, cells as well as its subsequent internalization. In agreement with the results obtained by McNamara et al., this thesis demonstrates that it is possible to deliver functional siRNAs into PSMA and HER3 expressing cells. The monovalent aptamer-siRNA transcripts induced the siRNA sequence-specific knockdown of mRNA and protein expression. The administration of aptamer-siRNA transcripts specifically induced apoptosis and cell death only in antigen-expressing cells without activating the nonspecific type I Interferon response. As a global parameter for the overall efficacy of the aptamer siRNA transcripts, cell viability was evaluated in a dose-dependent manner.IMMUNOKINASESThe unique expression of surface markers on tumor cells makes it possible to explore rational strategies for molecular targeting cancer cells without harming the surrounding normal cells. We developed a strategy to deliver a tumor suppressor protein specifically to Hodgkin lymphoma cells carrying the tumor-associated CD30 receptor. This was achieved by genetically fusing a constitutively active version of DAPK2 to the CD30 ligand, thus decreasing the likelihood of systemic toxicity and providing proof-of-principle that compounds ineffective or unsuitable as systemic drugs can be developed as targeted therapeutic reagents. We have shown that DAPK2 protein is downregulated in different CD30+ Hodgkin lymphoma-derived tumor cells by DNA promoter hypermethylation and that receptor-specific targeting of those cells with a constitutively-active version of DAPK2 fused to the CD30 ligand can restore this kinase activity and induce cell death in vitro and in vivo. For this purpose a fusion proteins was designed, expressed, purified and characterized concerning affinity, internalization activity, apoptosis induction and serum stability. These experiments were essential for the following in vivo experiments. In a disseminated mice model we could prove that the purified immunokinase was able to prevent tumor development in SCID mice. Even after more than 100 days no cancer development could be observed in any tissue analyzed. In conclusion, our data support the therapeutic potential of restoring the catalytic activity of kinases such as DAPK2 is an attractive alternative to commonly used toxins in combination with a specific ligand targeting diseased tumor cells in patients.
ThesisNote
Aachen, TH, Diss., 2009
Verlagsort
Aachen