Project Details
Projekt
Therapeutic potential of DARPins for treatment of HPV induced tumors
Subject Area
Virology
Biochemistry
Biochemistry
Term
since 2025
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 569665351
High-risk human papillomaviruses (HR-HPV, especially HPV16) cause anogenital and oropharyngeal cancers. The molecular mechanism of HR-HPV-induced tumorigenesis is based on the inactivation of key cellular factors by viral oncoproteins. Mechanistic investigations have revealed that the viral E6 protein binds to the cellular E3 ligase E6AP and to the DNA binding domain (DBD) of p53. The recruitment of E6AP to the vicinity of p53 results in the ubiquitination and degradation of p53. Recently, the Dötsch lab developed a Designed Ankyrin Repeat Protein (DARPins) that binds to the p53 DBD (DARPin C10). DARPins are very stable and do not contain disulfide bonds which makes them ideally suited for intracellular applications. In competition assays we showed that DARPin C10 prevents binding of E6 to p53. Furthermore, in Hela (HPV18 positive) and SiHa (HPV16 positive) cells expression of DARPin C10 increased endogenous p53 levels, p53 target gene expression and induced apoptosis. In addition, crystal structure of DARPin C10 in complex with the p53 DBD confirmed that neither the DNA-binding nor crucial protein-protein binding are affected upon DARPin binding. Taken together, we have shown that our novel DARPin C10 can block the interaction of E6 with p53, stabilize p53 and drive HPV positive cells into apoptosis. The Akgül lab, which is part of the German reference center for papilloma and polyomaviruses, has longstanding expertise in HPV biology and HPV mouse models. We now propose to combine the expertise of the Dötsch lab for the development and characterization of DARPins and the expertise of the Akgül lab in HPV mouse models to investigate the potential of DARPins as therapeutic strategy for HPV related malignancies. Since the affinity of DARPin C10 to mouse p53 is significantly lower to human p53, we will use immunodeficient NRG mice (NOD-Rag1null IL2rgnull), as preclinical animal model to induce xenograft tumors from transplanted human HPV positive cancer cell lines. We will intratumorally inject mRNA/lipid-nanoparticles coding for the DARPin C10 and determine their ability to restore p53 and induce an anti-tumor effect after treatment. We will then treat mice systemically with DARPin C10, determine the therapeutic dose and test for the anti-tumor effect either alone or as a combination therapy with cisplatin chemotherapy. The toxicity of the therapeutic dose of DARPin C10 will be valuated in the HUPKI mouse model, genetically modified to encode human p53. Since blocking the oncogenic functions of E6 represents a promising therapeutic approach for HPV-positive cancers we will develop novel E6 specific DARPins and characterize their anti-tumorigenic activity.
DFG Programme
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