AbstractsBiology & Animal Science

Gene therapy based on adeno-associated virus (AAV) vectors becomes a very promising option for the treatment of monogenetic disorders. For broad clinical application of AAV vectors efficient and economical production methods are required to keep pace with the rapidly increasing demand. In this thesis, a scalable rAAV production system was developed with individual stable insect Sf9 cell lines harboring the AAV1–12 rep and cap genes. Upon infection with a single baculovirus carrying the rAAV vector genome stable, high-titer rAAV production is induced for an AAV serotype of choice. AAV vectors generated by these cell lines reached higher per cell burst sizes with infectivities similar to vectors produced by the current gold standard, plasmid transfection of HEK 293 cells. For transgene delivery of AAV vectors of variant serotypes to the target cells binding of the capsids to a specific receptor on the host cell is required. The capsids of different AAV serotypes have been shown to use variable cell surface glycans as their primary receptors. In this thesis the analysis of highly-purified, fluorescence-labeled AAV capsids to hundreds of synthetic glycan structures or heparins on microarrays led to the identification of specific glycans which bound to AAV capsids in a serotype-dependent manner. Furthermore, the natural heparin binding AAV serotypes 2, 3, 6, and 13 were shown for the first time to bind to different chemical-defined, synthetic heparins. Through comparative analysis of binding and non-binding glycans the minimal binding structure for various AAV serotypes could be determined thereby extending the existing definition of the host cell receptors. For a neurobiological collaboration project AAV vectors for the expression of neuropeptide Y (NPY) have been generated in this thesis. These vectors were used in genetic studies investigating the role of NPY for the development of obesity in mice. Stereotactic injections into the hypothalamus of wild type and NPY knock-out mice confirmed not only the function of NPY as an appetite stimulant but also gave evidence for a role of NPY in the reduction of energy expenditure. In addition to baculovirus-infected insect cells AAV vectors can also be produced in mammalian cells by infection of herpesviruses (HSV). In this process the HSV-encoded replication machinery is required for AAV DNA replication. The AAV replication is initiated by the formation of a ternary complex of AAV Rep78 and HSV-ICP8 at the single-stranded AAV DNA genome. Studies with different AAV Rep mutants demonstrated that an intact DNA binding domain of Rep78 is necessary for the formation of the ternary complex. With the results a model was developed, suggesting that HSV-dependent AAV replication is most probably initiated by Rep binding to the AAV ITR followed by cooperative binding of ICP8 on the single-stranded AAV DNA. Rekombinante Adeno-assoziierte Virus Vektoren (rAAV) werden mit zunehmendem Erfolg in gentherapeutischen Studien für die Behandlung von monogenetischen Krankheiten eingesetzt. Für…