Papé Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science University, Portland, OR 97239, USA.
San Raffaele Telethon Institute for Gene Therapy, IRCCS Ospedale San Raffaele Scientific Institute, Milan, Italy.
Mol Ther. 2021 Oct 6;29(10):2898-2909. doi: 10.1016/j.ymthe.2021.08.031. Epub 2021 Aug 28.
Recombinant adeno-associated viral (rAAV) vectors are considered promising tools for gene therapy directed at the liver. Whereas rAAV is thought to be an episomal vector, its single-stranded DNA genome is prone to intra- and inter-molecular recombination leading to rearrangements and integration into the host cell genome. Here, we ascertained the integration frequency of rAAV in human hepatocytes transduced either ex vivo or in vivo and subsequently expanded in a mouse model of xenogeneic liver regeneration. Chromosomal rAAV integration events and vector integrity were determined using the capture-PacBio sequencing approach, a long-read next-generation sequencing method that has not previously been used for this purpose. Chromosomal integrations were found at a surprisingly high frequency of 1%-3% both in vitro and in vivo. Importantly, most of the inserted rAAV sequences were heavily rearranged and were accompanied by deletions of the host genomic sequence at the integration site.
重组腺相关病毒 (rAAV) 载体被认为是针对肝脏的基因治疗的有前途的工具。虽然 rAAV 被认为是一种附加体载体，但它的单链 DNA 基因组容易发生内和分子间重组，导致重排和整合到宿主细胞基因组中。在这里，我们确定了 rAAV 在体外转导或体内转导随后在异种肝再生小鼠模型中扩增的人肝细胞中的整合频率。使用捕获 PacBio 测序方法确定了染色体 rAAV 整合事件和载体完整性，这是一种长读长下一代测序方法，以前未用于此目的。在体外和体内都发现了令人惊讶的高频率 1%-3%的染色体整合。重要的是，大多数插入的 rAAV 序列都发生了严重的重排，并伴有整合部位宿主基因组序列的缺失。
