Modular Recombinant Virus for Tumor Targeting and Prodrug Activation

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Kristian Müller, Sven Hagen, Tobias Baumann, Hanna Wagner, Adrian Fischer, Beate Kaufmann, Volker Morath, Stefan Bergmann, Katja M. Arndt

Bielefeld University, Germany

Targeting gene expression cassettes to specific cells is a prerequisite for gene therapy, regenerative medicine and, in our case, for gene dependent prodrug activation therapy (GDEPT). Viruses provide an evolutionary optimized source for gene ferries, yet they need to be tamed and tailored. Since a recombinant Adeno Associated Virus (rAAV) recently became the first gene therapy treatment recommended for approval, we foresee a broader application of such viruses for Synthetic Biology. We established a set of BioBricks enabling the modular assembly of recombinant Adeno-Associated viruses based on serotype 2. We demonstrated that fusion of binding molecules (DARPin, Affibody) to the viral capsid enabled selective targeting to tumor cells expressing surface markers (e.g. EGF-R). As genes of interest we delivered enzymes (CD, TK) activating prodrugs (5FC, ganciclovir). The natural tropism of the viral particle was reduced by capsid mutations. Introducing specific restriction sites flanking two capsid loop coding sequences permitted additional loop insertions, which we used for further modifications or the coding of purification tags. In various cell assays we analyzed and demonstrated the selective binding and killing of tumor cells. Precise gene delivery tools provide the opportunity to apply Synthetic Biology to living organisms and are particular valuable for healthcare.