Zelleintritt und intrazelluläre Prozessierung von rAAV targeting Vektoren und ihrer Genome im lympho-hämatopoetischen System

点击翻译按钮获取中文摘要

The possibility to modify the tropism of adeno-associated virus (AAV) by genetic manipulation of the viral capsid (AAV targeting vector) has created the possibility for using this virus for a cell or tissue specific gene transfer after systemic application. This technology produces vectors with improved transduction efficiency on target cell. At the same time, lower does of vector are required (circumvention of unspecific uptake of vectors in non-target tissue). However, little is known about cell entry, endosomal processing and nuclear transport of such AAV targeting vectors. Since the inserted ligand mediates the cell entry of the targeting vectors, it is likely that the cell entry pathway and the intracellular fate of targeting vectors differ from wild-type AAV (wtAAV). In addition, targeting vectors may be used to perform gene transfer into wtAAV non-permissive cells. Utilization of different entry routes or intracellular pathways may trigger unforeseen side effects like genomic integration in proximity of proto-oncogenes or dysregulation of intracellular signalling pathways controlling cell growth or programmed cell death. For this reason, the aim of the proposed project is a detailed analysis of cell entry, intracellular trafficking and nuclear entry of AAV targeting vectors in cells of the lympho-hematopoietic system. Furthermore, the occurrence and location of viral integration sites into the cellular genome will be analysed. Finally, microarray analyses, cell survival and cell cycle analyses will be performed to measure the influence of a (retargeted) AAV infection on cell homeostasis.