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• W4308996775 abstract "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 × 1012 vector genome from one φ150 mm dish (mean ± SEM)." @default.
• W4308996775 created "2022-11-20" @default.
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• W4308996775 date "2022-11-14" @default.
• W4308996775 modified "2023-10-17" @default.
• W4308996775 title "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" @default.
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• W4308996775 doi "https://doi.org/10.1096/fj.202200840rr" @default.
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