Use of cesium chloride density gradient ultracentrifugation for the purification and characterization of recombinant adeno-associated virus

Hirohata, Kiichi; Kino, Shinichiro; Yamane, Takuya et al.

European Biophysics Journal, 2025

Number of Access:19（2025-08-14 06:53 Counts）

Identifier to cite or link to this item: https://hdl.handle.net/11094/102249

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Title
Use of cesium chloride density gradient ultracentrifugation for the purification and characterization of recombinant adeno-associated virus
Creator
CreatorName
Hirohata, Kiichi
CreatorName
Kino, Shinichiro
CreatorName
Yamane, Takuya
CreatorName
Bandoh, Karin
NRID
1000010432444 1000010432444
CreatorName
Bamba, Takeshi
CreatorName
Sternisha, Shawn M.
NRID
1000010730492 1000010730492
ORCID
0000-0002-8269-7803 0000-0002-8269-7803
researchmap permalink
torisutetsuo torisutetsuo
Scopus Author ID
57361287600 57361287600
CreatorName
Torisu, Tetsuo
CreatorName
Fukuhara, Mitsuko
ORCID
0000-0001-7856-4031 0000-0001-7856-4031
researchmap permalink
32902 32902
CreatorName
Yamaguchi, Yuki
NRID
1000090335381 1000090335381
ORCID
0000-0002-5181-179X 0000-0002-5181-179X
researchmap permalink
read0090387 read0090387
Scopus Author ID
35429790100 35429790100
CreatorName
Uchiyama, Susumu
Subject
Adeno-associated virus
Cesium chloride
Transduction efficiency
Ultracentrifugation
Vertical rotor
Viral protein stoichiometry
Description
The version of record of this article, first published in European Biophysics Journal, is available online at Publisher’s website: https://doi.org/10.1007/s00249-025-01751-1.
Abstract
Recombinant adeno-associated virus (rAAV) has been widely used as an effective delivery tool in gene therapy. One of the challenges facing the production of high-quality rAAV is optimization of the production and purification methodologies. Cesium chloride density gradient ultracentrifugation (CsCl-DGUC) has been traditionally utilized for rAAV purification; however, few studies have focused on CsCl-DGUC for rAAV purification despite this technique having a great potential for clinical-grade or large-scale rAAV production. In this study, we aimed to explore the unaddressed challenges associated with rAAV purification using CsCl-DGUC. We clarified that AAV capsids assembled by the different stoichiometries of three viral proteins (VP1, VP2, and VP3) showed heterogeneous population in the CsCl density gradient and encapsidated DNA increased the buoyant density differences of these capsids, resulting in the wider distribution. We implemented CsCl-DGUC using a vertical rotor which improved throughput and enhanced the separation of desired AAV particles from impurities. Furthermore, we examined the effect of CsCl exposure during purification, and the presence of residual CsCl in the purified rAAV. This study provides valuable insights into the application of CsCl-DGUC in the manufacturing of rAAV while ensuring adequate efficacy and safety for gene therapy.
Publisher
Springer Science and Business Media Deutschland GmbH
Source Title
European Biophysics Journal
Date of Issued
2025-05-19
Language
English
Handle URL
https://hdl.handle.net/11094/102249
PISSN
01757571
eISSN
14321017
Relation.isIdenticalTo
DOI (Publisher Version)
https://doi.org/10.1007/s00249-025-01751-1
Access Rights
open access
CopyRight
This article is licensed under a Creative Commons Attribution 4.0 International License.
oaire:version
Version of Record
Category
学術雑誌論文 Journal Article