Mück-Häusl Martin, Solanki Manish, Zhang Wenli, Ruzsics Zsolt, Ehrhardt Anja
Max von Pettenkofer-Institute, Department of Virology, Ludwig-Maximilians-University Munich, Munich, Germany.
Institute of Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, University of Witten/Herdecke, Witten, Germany.
Nucleic Acids Res. 2015 Apr 30;43(8):e50. doi: 10.1093/nar/gkv031. Epub 2015 Jan 21.
Recombinant adenoviruses containing a double-stranded DNA genome of 26-45 kb were broadly explored in basic virology, for vaccination purposes, for treatment of tumors based on oncolytic virotherapy, or simply as a tool for efficient gene transfer. However, the majority of recombinant adenoviral vectors (AdVs) is based on a small fraction of adenovirus types and their genetic modification. Recombineering techniques provide powerful tools for arbitrary engineering of recombinant DNA. Here, we adopted a seamless recombineering technology for high-throughput and arbitrary genetic engineering of recombinant adenoviral DNA molecules. Our cloning platform which also includes a novel recombination pipeline is based on bacterial artificial chromosomes (BACs). It enables generation of novel recombinant adenoviruses from different sources and switching between commonly used early generation AdVs and the last generation high-capacity AdVs lacking all viral coding sequences making them attractive candidates for clinical use. In combination with a novel recombination pipeline allowing cloning of AdVs containing large and complex transgenes and the possibility to generate arbitrary chimeric capsid-modified adenoviruses, these techniques allow generation of tailored AdVs with distinct features. Our technologies will pave the way toward broader applications of AdVs in molecular medicine including gene therapy and vaccination studies.
含有26 - 45 kb双链DNA基因组的重组腺病毒在基础病毒学、疫苗接种、基于溶瘤病毒疗法的肿瘤治疗或仅仅作为一种高效基因转移工具方面得到了广泛研究。然而，大多数重组腺病毒载体（AdV）是基于一小部分腺病毒类型及其基因改造。重组工程技术为重组DNA的任意工程改造提供了强大工具。在此，我们采用了一种无缝重组工程技术，用于重组腺病毒DNA分子的高通量和任意基因工程改造。我们的克隆平台（其中还包括一条新型重组流程）基于细菌人工染色体（BAC）。它能够从不同来源产生新型重组腺病毒，并在常用的早期代次AdV和缺乏所有病毒编码序列的最后一代高容量AdV之间进行转换，使其成为有吸引力的临床应用候选者。结合一条允许克隆包含大而复杂转基因的AdV以及产生任意嵌合衣壳修饰腺病毒的新型重组流程，这些技术能够产生具有独特特征的定制AdV。我们的技术将为AdV在包括基因治疗和疫苗接种研究在内的分子医学中的更广泛应用铺平道路。
