Fate of plasmid DNA in host cells after bacteria-mediated gene transfer

细菌作为运送治疗性DNA的载体，具有迄今为止通常使用的载体无法提供的几个特性。或许，这些优势中最重要的是载体和表达载体的独立性。因此，两者都可以独立操作。基因传递是在载体细菌的细胞内裂解后实现的。到目前为止，尽管细菌裂解后大量的质粒DNA被释放到宿主细胞中，但DNA的传递效率仍然相当低。因此，在本项目中，将确定两种细菌-单核细胞增生性李斯特菌和福氏志贺氏菌-细菌输送后表达载体的命运。首先，将调查自噬小泡在细菌感染和DNA释放过程中的参与。自噬是一种宿主防御系统，在细菌入侵后被激活，可能会严重损害质粒的转移。这些实验将通过细胞生物学研究扩展到从载体释放后跟踪质粒。特别是，将研究质粒与细菌蛋白之间的剩余联系。最后，通过使用带有可诱导重组酶的新型细菌载体，在转移过程中将产生最低限度的表达盒或最低限度的附体质粒。这将为合理设计高效细菌转移载体提供知识。

Bacteria as vehicles for the delivery of therapeutic DNA have several properties that the vectors commonly used to date cannot provide. Probably, the most import of such advantages is the independence of the vehicle and the expression plasmid. Thus, both can be manipulated independently. Gene delivery is achieved after intracellular lysis of the carrier bacteria. So far, the efficiency of DNA delivery is still rather low despite of the vast amount of plasmid DNA that is released into the host cell after bacterial lysis. Therefore, in the present project the fate of the expression plasmids after bacterial delivery by two bacteria - Listeria monocytogenes and Shigella flexneri - will be determined. First, the involvement of autophagic vesicles during bacterial infection and DNA release will be investigated. Autophagy represents a host defence system that is activated after invasion by bacteria and might seriously impair plasmid transfer. These experiments will be extended by cellbiological studies to follow the plasmids after release from the carrier. Especially the remaining association of the plasmids with bacterial proteins will be investigated. Finally, minimal expression cassettes or minimal episomal plasmids will be generated during the transfer by employing novel bacterial carriers with inducible recombinases. This should provide the knowledge for a rational design of highly efficient bacterial transfer vehicles.