2024-03-28T13:20:24Z
https://tsukuba.repo.nii.ac.jp/oai
oai:tsukuba.repo.nii.ac.jp:00049313
2022-04-27T09:21:49Z
2780:335
2780:922
3:62:5595:7193
Highly Efficient Ultracentrifugation-free Chromatographic Purification of Recombinant AAV Serotype 9
石井, 亜紀子
イシイ, アキコ
ISHII, Akiko
玉岡, 晃
タマオカ, アキラ
TAMAOKA, Akira
Tomono, Taro
Hirai, Yukihiko
Okada, Hironori
Miyagawa, Yoshitaka
Adachi, Kumi
Sakamoto, Shuhei
Kawano, Yasuhiro
Chono, Hideto
Mineno, Junichi
Shimada, Takashi
Onodera, Masafumi
Okada, Takashi
© 2018 The Authors.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Recombinant adeno-associated virus serotype 9 (rAAV9) can specifically transduce muscle and neuronal tissues; thus, rAAV9 can potentially be used in gene therapy. However, rAAV9 is the most challenging rAAV serotype to purify. Traditionally, rAAV9 has been purified by ultracentrifugation, which is not scalable. We recently described a chromatographic purification protocol for rAAV1; this protocol can achieve scalable purifications. In this study, we attempted to optimize this protocol for purifying rAAV9 preparations, and we developed a novel, effective method for high-yield purification of rAAV9 using quaternary ammonium anion exchangers and size-exclusion chromatography. The final purified rAAV9 contained mainly three capsid proteins, as observed by SDS-PAGE. Furthermore, negative-stain electron microscopy demonstrated that 96.1% ± 1.1% of rAAV9 particles carried the viral genome containing the EGFP transgene, indicating that impurities and empty capsids can be eliminated with our purification protocol. The final rAAV9 titer obtained by our protocol totaled 2.5 ± 0.4 × 1015 viral genomes produced from ∼3.2 × 109 HEK293EB cells. We confirmed that our protocol can also be applied to purify other varied AAV genome constructs. Our protocol can scale up production of pure rAAV9, in compliance with current good manufacturing practice, for clinical applications in human gene therapy.
American Society of Gene & Cell Therapy
Elsevier
2018-12
eng
journal article
http://hdl.handle.net/2241/00154784
https://tsukuba.repo.nii.ac.jp/records/49313
30533449
10.1016/j.omtm.2018.10.015
23290501
Molecular Therapy - Methods & Clinical Development
11
180
190
https://tsukuba.repo.nii.ac.jp/record/49313/files/MTMCD_11.pdf
application/pdf
1.7 MB
2019-03-06