Subgenomic particles in rAAV vectors result from DNA lesion/break and non-homologous end joining of vector genomes.
Subgenomic particles in rAAV vectors result from DNA lesion/break and non-homologous end joining of vector genomes.
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RAAV矢量中的亚基因组颗粒是由DNA病变/断裂和载体基因组的非同源末端连接而产生的。
DOI：
10.1016/j.omtn.2022.08.027
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发表时间：
2022-09-13
期刊：
影响因子：
--
通讯作者：
Xiao, Weidong
中科院分区：
文献类型：
--
作者：
Zhang, Junping;Guo, Ping;Yu, Xiangping;Frabutt, Dylan A.;Lam, Anh K.;Mulcrone, Patrick L.;Chrzanowski, Matthew;Firrman, Jenni;Pouchnik, Derek;Sang, Nianli;Diao, Yong;Herzog, Roland W.;Xiao, Weidong
重组腺相关病毒（rAAV）载体已被开发用于遗传疾病的治疗性治疗。目前给予受影响个体的rAAV载体通常含有载体DNA相关污染物。在这里，我们提出了一个彻底的分子分析的非标准的AAV基因组的配置过程中产生的rAAV生产使用单分子测序。除了含有不完整AAV基因组的亚载体基因组大小颗粒外，我们的结果还表明rAAV制剂被具有快速恢复基因组（SBG）构型或具有缺失的载体基因组的多种亚基因组颗粒污染。通过组织培养细胞中的CRISPR和基于核酸酶的建模，我们确定了导致非规范基因组颗粒形成的潜在机制，该机制通过宿主细胞中存在的基因组损伤或DNA断裂引起的片段化载体基因组的非同源末端连接而发生。本研究的结果推进了我们对AAV载体的理解，并为提高用于人类基因治疗的载体效率和安全性提供了新的线索。Zhang等人揭示了载体DNA损伤和NHEJ是在rAAV载体生产过程中导致亚基因组AAV颗粒产生的潜在机制。这一发现将为发现新的方法以提高AAV载体的安全性和有效性提供替代途径。
Recombinant adeno-associated virus (rAAV) vectors have been developed for therapeutic treatment of genetic diseases. Current rAAV vectors administered to affected individuals often contain vector DNA-related contaminants. Here we present a thorough molecular analysis of the configuration of non-standard AAV genomes generated during rAAV production using single-molecule sequencing. In addition to the sub-vector genomic-size particles containing incomplete AAV genomes, our results showed that rAAV preparations were contaminated with multiple categories of subgenomic particles with a snapback genome (SBG) configuration or a vector genome with deletions. Through CRISPR and nuclease-based modeling in tissue culture cells, we identified that a potential mechanism leading to formation of non-canonical genome particles occurred through non-homologous end joining of fragmented vector genomes caused by genome lesions or DNA breaks present in the host cells. The results of this study advance our understanding of AAV vectors and provide new clues for improving vector efficiency and safety profiles for use in human gene therapy. Zhang et al. revealed that vector DNA lesion and NHEJ are a potential mechanism leading to subgenomic AAV particle generation in the rAAV vector production process. This finding will provide an alternate pathway for discovering novel methodology to improve the safety and efficacy of AAV vectors.
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影响因子：
12.4
作者：
Wu, Zhijian;Yang, Hongyan;Colosi, Peter
通讯作者：
Colosi, Peter
影响因子：
82.9
作者：
Ferrari, FK;Xiao, X;Samulski, RJ
通讯作者：
Samulski, RJ
DOI：
10.3390/v13061185
发表时间：
2021-06-21
期刊：
Viruses
影响因子：
--
作者：
Zhang J;Yu X;Guo P;Firrman J;Pouchnik D;Diao Y;Samulski RJ;Xiao W
通讯作者：
Xiao W
影响因子：
5.4
作者：
DELAMAZA, LM;CARTER, BJ
通讯作者：
CARTER, BJ
影响因子：
3.7
作者：
LAUGHLIN, CA;MYERS, MW;CARTER, BJ
通讯作者：
CARTER, BJ