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

作为用于递送治疗性DNA的载体的细菌具有迄今为止常用的载体不能提供的几种性质。这些优点中最重要的可能是载体和表达质粒的独立性。因此，两者都可以独立操作。在载体细菌的细胞内裂解后实现基因递送。到目前为止，尽管细菌裂解后大量质粒DNA被释放到宿主细胞中，但DNA递送的效率仍然相当低。因此，在本项目中，将确定两种细菌-单核细胞增生李斯特菌和福氏志贺菌-细菌递送后表达质粒的命运。首先，将研究自噬囊泡在细菌感染和DNA释放过程中的参与。自噬是一种在细菌入侵后被激活的宿主防御系统，可能严重损害质粒转移。这些实验将通过细胞生物学研究扩展，以跟踪从载体释放后的质粒。特别是将研究质粒与细菌蛋白质的剩余缔合。最后，通过采用具有诱导型重组酶的新型细菌载体，在转移期间将产生最小表达盒或最小附加型质粒。这将为合理设计高效的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.