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

通过对病毒衣壳（AAV靶向载体）的遗传操作来改变腺相关病毒（AAV）的趋向性的可能性，创造了在系统应用后使用该病毒进行细胞或组织特异性基因转移的可能性。该技术能提高载体在靶细胞上的转导效率。同时，需要较低的载体剂量（避免载体在非靶组织中的非特异性摄取）。然而，这种AAV靶向载体的细胞进入、内体加工和核运输机制尚不清楚。由于插入的配体介导靶向载体的细胞进入，因此靶向载体的细胞进入途径和细胞内命运可能与野生型AAV （wtAAV）不同。此外，靶向载体可用于将基因转移到wtAAV非允许细胞中。利用不同的进入途径或细胞内途径可能引发不可预见的副作用，如原癌基因附近的基因组整合或控制细胞生长或程序性细胞死亡的细胞内信号通路失调。因此，本项目的目的是详细分析AAV靶向载体在淋巴造血系统细胞中的细胞进入、细胞内运输和核进入。此外，将分析病毒整合位点进入细胞基因组的发生和位置。最后，将进行微阵列分析、细胞存活和细胞周期分析，以测量（重靶向）AAV感染对细胞稳态的影响。

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.