|dc.description.abstracteng||Gene delivery tools play a key role in research and gene therapy. Since 1989, when the first trial in humans took place, the improvement of transduction effciency and safety gained enormous attention to increase the therapy success.
In this context, the biotechnological aim of this thesis is to engineer three viral- and non-viral delivery tools.
I developed a production and purification pipeline for in vivo grade recombinant adeno-associated viruses (rAAVs) and established in vitro and ex vivo quality tests. By examination of the expression profiles of five rAAV serotypes on human cell lines and primary cells of human, rodent and non-human primate origin, I can additionally provide a source which enables the selection of the best-performing serotype for future applications. Additionally, I generated a genetic toolbox for a straightforward expression construct generation, useful for all three provided delivery tools.
Exosomes cannot only be used as biomarkers of diseases or as delivery tools for therapy, but in combination with rAAVs, they are a powerful tool to enhance the AAVs‘ transduction efficiency, and immunological safety. Here, we used the expertise in boosting the exosome release by over-expression of the tetraspanin CD9, an exosomal marker-protein, to engineer the production of exosome-associated AAVs (exo-AAVs), the second part of my thesis. For the first time, we demonstrated that with this modification of producer cells, exo-AAV production yields up to 32% more e cient viruses.
Lentiviruses, another well-established vector system in gene therapy, were introduced and investigated to complete our gene delivery platform. Which is useful to unravel the mechanism behind the CD9‘s boosting e ect.
The third tool is represented by JC polyoma virus-derived virus-like particles (VLPs). This quasi non-viral system is a promising tool for both research and clinical applications. In order to increase the immunological safety of linear DNA delivery with the VLPs, we adopted and investigated the utilisation of so-called MIDGE vectors, a monomolecular linear DNA cassette, as cargo.
By attachment of targeting molecules to the outer surface of the VLPs, we could alter the VLPs‘ tropism towards new target cells. Using this strategy, I aimed to develop a retargeting towards nervous tissue which can by interchanging the targeting molecule also be applied for lentiviruses and exo-AAVs.
In summary, these developments provide a handy way to improve the rAAVs‘ and exo- AAVs‘ production, purification and quality assessment. In addition, we provide a platform for viral (rAAV, Lentivirus) and non-viral (VLP) gene delivery systems with interchangeable expression features and cell-/tissue targeting opportunities.||de