Zheng Qingyun, Wang Tianyi, Zhu Xiangying, Tian Xiao, Zhong Chen, Chang Guolin, Ran Gai, Xie Yilin, Zhao Bing, Zhu Liqing, Ling Chen
State Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China.
Zhejiang Hengyu Biological Technology Co., Ltd., Jiaxing, Zhejiang Province 314113, China.
Mol Ther Methods Clin Dev. 2021 Jun 24;22:293-303. doi: 10.1016/j.omtm.2021.06.009. eCollection 2021 Sep 10.
The major challenge of recombinant adeno-associated virus (rAAV) vectors is host immunological barriers. Compared to the neutralizing antibody and the cytotoxic T lymphocyte response, the host immune responses induced by unsatisfactory rAAV manufacturing were largely ignored previously. rAAV vector production usually requires large amounts of plasmid DNAs. The DNA are commonly isolated from the DH5α bacterial strain, which contains lipopolysaccharide (LPS) contamination. LPS, also named endotoxin, in plasmid DNA is intractable, and residual endotoxin in the subsequent rAAV vectors may result in substantial host immune response. Recently, a ClearColi K12 bacterial strain is commercially available, with genetically modified LPS that does not trigger endotoxic response in mammalian cells. Here, we produced rAAV-DJ vectors by plasmids yielded from either DH5α or ClearColi K12 bacterial strains. Our data indicated that the ClearColi K12 strain had satisfactory protection for the rAAV inverted terminal repeat (ITR) sequence. As expected, the ClearColi K12-derived rAAV-DJ vectors had lower endotoxin levels. The physical and biological equivalency of the purified viral stocks were confirmed by electron micrographs, Coomassie blue staining, and transduction assays. Most importantly, the ClearColi K12-derived rAAV-DJ vectors triggered reduced nuclear factor-kappa B (NF-κB) signaling pathway both in cell cultures and in C57BL/6 mice retinas . We believe that the use of the ClearColi K12 bacterial strain could eliminate the LPS in the purified vector stock at the source. Our data indicate its promising use in future clinical development.
重组腺相关病毒（rAAV）载体面临的主要挑战是宿主免疫屏障。与中和抗体和细胞毒性T淋巴细胞反应相比，先前人们很大程度上忽略了由质量欠佳的rAAV生产所诱导的宿主免疫反应。rAAV载体生产通常需要大量质粒DNA。这些DNA通常从含有脂多糖（LPS）污染的DH5α菌株中分离得到。质粒DNA中的LPS（也称为内毒素）难以处理，后续rAAV载体中的残留内毒素可能会引发大量宿主免疫反应。最近，一种ClearColi K12菌株已上市，其经过基因改造的LPS不会在哺乳动物细胞中引发内毒素反应。在此，我们用从DH5α或ClearColi K12菌株产生的质粒制备了rAAV-DJ载体。我们的数据表明，ClearColi K12菌株对rAAV反向末端重复序列（ITR）有良好的保护作用。正如预期的那样，源自ClearColi K12的rAAV-DJ载体内毒素水平较低。通过电子显微镜、考马斯亮蓝染色和转导分析证实了纯化病毒原液的物理和生物学等效性。最重要的是，源自ClearColi K12的rAAV-DJ载体在细胞培养物和C57BL/6小鼠视网膜中均触发了降低的核因子-κB（NF-κB）信号通路。我们认为，使用ClearColi K12菌株可以从源头上消除纯化载体原液中的LPS。我们的数据表明其在未来临床开发中具有广阔的应用前景。
