Development of a high-yield, high-quality purification process for adeno-associated virus vectors that can be used in vivo without ultracentrifugation: Application to a lung endothelial cell-targeted adeno-associated virus
Development of a high-yield, high-quality purification process for adeno-associated virus vectors that can be used in vivo without ultracentrifugation: Application to a lung endothelial cell-targeted adeno-associated virus
复制标题
开发一种高产量、高质量的腺相关病毒载体纯化方法，无需超速离心即可在体内使用：在肺内皮细胞靶向腺相关病毒中的应用
DOI：
10.1096/fj.202200840rr
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发表时间：
2022
期刊：
影响因子：
4.8
通讯作者：
Ido Y.
中科院分区：
文献类型：
--
作者：
Shiraishi;Y; Adachi;T;Cacicedo;J;Ido Y.
重组腺相关病毒（rAAV）是用于在体内表达目的基因的有用载体，因为它们的低免疫原性和长期基因表达。近年来已经引入了各种突变，并且已经实现了高效、稳定和器官定向的转导。我们使用rAAV的目的是在小鼠肺中表达我们的目的基因，以研究肺动脉高压。我们构建了一种自身互补的AAV，其具有带有ESGHGYF插入物的突变衣壳，其将载体引导至肺内皮细胞。然而，当通过使用超离心的常规方法从生产细胞中纯化该突变病毒时，其导致低产率。此外，使用超离心的纯化方法是繁琐且劳动密集的。因此，我们的目标是开发一种简单，高质量的方法来获得足够的肺靶向rAAV。首先，我们修饰了衣壳的氨基酸（T491V和Y730F）以稳定rAAV免于降解，并且我们优化了培养条件。接下来，我们注意到许多rAAV从细胞释放到培养基中。因此，我们改进了我们的纯化方法，即从培养基中纯化而不进行超离心步骤。没有超浓缩的纯化存在杂质混入的问题，引起炎症。然而，通过进行PEG沉淀和氯仿提取两次，我们能够纯化rAAV，其仅引起与通过超离心法获得的一样少的炎症。获得了足够的rAAV，并且现在可以从单个培养皿中向大鼠和小鼠施用：来自一个φ150 mm培养皿的1.50 × 1013± 3.58 × 1012个载体基因组（平均值± SEM）。
Recombinant adeno‐associated viruses (rAAVs) are useful vectors for expressing genes of interest in vivo because of their low immunogenicity and long‐term gene expression. Various mutations have been introduced in recent years and have enabled high‐efficacy, stabilized, and organ‐oriented transduction. Our purpose for using rAAV is to express our target gene in the mouse lung to investigate pulmonary artery hypertension. We constructed a self‐complementary AAV having mutant capsids with the ESGHGYF insert, which directs the vectors to lung endothelial cells. However, when this mutant virus was purified from the producing cells by the conventional method using an ultracentrifuge, it resulted in a low yield. In addition, the purification method using an ultracentrifuge is tedious and labor‐intensive. Therefore, we aimed to develop a simple, high‐quality method for obtaining enough lung‐targeted rAAV. First, we modified amino acids (T491V and Y730F) of the capsid to stabilize the rAAV from degradation, and we optimized culture conditions. Next, we noticed that many rAAVs were released from the cells into the culture medium. We, therefore, improved our purification method by purifying from the culture medium without the ultracentrifugation step. Purification without ultracentrifugation had the problem that impurities were mixed in, causing inflammation. However, by performing PEG precipitation and chloroform extraction twice, we were able to purify rAAV that caused only as little inflammation as that obtained by the ultracentrifuge method. Sufficient rAAV was obtained and can now be administered to a rat as well as mice from a single dish: 1.50 × 1013± 3.58 × 1012vector genome from one φ150 mm dish (mean ± SEM).