Pich Dagmar, Humme Sibille, Spindler Mark-Peter, Schepers Aloys, Hammerschmidt Wolfgang
Department of Gene Vectors, Helmholtz Center Munich, German Research Center for Environmental Health, Marchioninistr. 25, 81377 Munich, Germany.
Nucleic Acids Res. 2008 Aug;36(13):e83. doi: 10.1093/nar/gkn273. Epub 2008 Jun 19.
Non-integrating gene vectors, which are stably and extrachromosomally maintained in transduced cells would be perfect tools to support long-term expression of therapeutic genes but preserve the genomic integrity of the cellular host. Small extrachromosomal plasmids share some of these ideal characteristics but are primarily based on virus blueprints. These plasmids are dependent on viral trans-acting factors but they can replicate their DNA molecules in synchrony with the chromosome of the cellular host and segregate to daughter cells in an autonomous fashion. On the basis of the concept of the latent origin of DNA replication of Epstein-Barr virus, oriP, we devised novel derivatives, which exclusively rely on an artificial replication factor for both nuclear retention and replication of plasmid DNA. In addition, an allosteric switch regulates the fate of the plasmid molecules, which are rapidly lost upon addition of doxycycline. Conditional maintenance of these novel plasmid vectors allows the reversible transfer of genetic information into target cells for the first time.
非整合基因载体可在转导细胞中稳定且以染色体外的形式维持，对于支持治疗性基因的长期表达并保持细胞宿主的基因组完整性而言，它将是理想的工具。小型染色体外质粒具备其中一些理想特性，但主要基于病毒蓝图构建。这些质粒依赖病毒反式作用因子，不过它们能够使其DNA分子与细胞宿主的染色体同步复制，并以自主方式分离至子代细胞。基于爱泼斯坦-巴尔病毒DNA复制潜在起始位点oriP的概念，我们设计出了新型衍生物，其质粒DNA的核内保留及复制完全依赖一种人工复制因子。此外，一种变构开关调控着质粒分子的命运，加入强力霉素后，质粒分子会迅速丢失。这些新型质粒载体的条件性维持首次实现了将遗传信息可逆地转入靶细胞。
