Department of Microbiology and Immunology, Sol Sherry Thrombosis Research Center, Temple University, Philadelphia, PA 19140, USA.
Nucleic Acids Res. 2013 Jul;41(13):6609-17. doi: 10.1093/nar/gkt404. Epub 2013 May 15.
Scalable and efficient production of high-quality recombinant adeno-associated virus (rAAV) for gene therapy remains a challenge despite recent clinical successes. We developed a new strategy for scalable and efficient rAAV production by sequestering the AAV helper genes and the rAAV vector DNA in two different subcellular compartments, made possible by using cytoplasmic vaccinia virus as a carrier for the AAV helper genes. For the first time, the contamination of replication-competent AAV particles (rcAAV) can be completely eliminated in theory by avoiding ubiquitous nonhomologous recombination. Vector DNA can be integrated into the host genomes or delivered by a nuclear targeting vector such as adenovirus. In suspension HeLa cells, the achieved vector yield per cell is similar to that from traditional triple-plasmid transfection method. The rcAAV contamination was undetectable at the limit of our assay. Furthermore, this new concept can be used not only for production of rAAV, but also for other DNA vectors.
尽管最近在临床方面取得了成功，但对于基因治疗而言，可规模化且高效生产高质量的重组腺相关病毒（rAAV）仍然是一个挑战。我们开发了一种新策略，通过将 AAV 辅助基因和 rAAV 载体 DNA 隔离在两个不同的亚细胞隔室中，从而实现 rAAV 的可规模化且高效生产，这一策略的实现得益于使用细胞质牛痘病毒作为 AAV 辅助基因的载体。通过避免普遍存在的非同源重组，从理论上讲，第一次可以完全消除具有复制能力的腺相关病毒颗粒（rcAAV）的污染。载体 DNA 可以整合到宿主基因组中，或者通过核靶向载体（如腺病毒）进行递送。在悬浮的 HeLa 细胞中，每个细胞的载体产量与传统的三质粒转染方法相当。在我们的检测极限下，rcAAV 的污染无法检测到。此外，这一新概念不仅可用于 rAAV 的生产，还可用于其他 DNA 载体。
