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

Hirohata, Kiichi; Kino, Shinichiro; Yamane, Takuya; Bandoh, Karin; Bamba, Takeshi; Sternisha, Shawn M.; Torisu, Tetsuo; Fukuhara, Mitsuko; Yamaguchi, Yuki; Uchiyama, Susumu

doi:https://doi.org/10.1007/s00249-025-01751-1

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

アクセス数:10件（2025-07-27 09:27 集計）

固定URL: https://hdl.handle.net/11094/102249

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タイトル	Use of cesium chloride density gradient ultracentrifugation for the purification and characterization of recombinant adeno-associated virus
著者	著者名 Hirohata, Kiichi
	著者名 Kino, Shinichiro
	著者名 Yamane, Takuya
	著者名 Bandoh, Karin
	NRID 1000010432444 1000010432444 著者名 Bamba, Takeshi
	著者名 Sternisha, Shawn M.
	NRID 1000010730492 1000010730492 ORCID 0000-0002-8269-7803 0000-0002-8269-7803 researchmap permalink torisutetsuo torisutetsuo Scopus Author ID 57361287600 57361287600 著者名 Torisu, Tetsuo
	著者名 Fukuhara, Mitsuko
	ORCID 0000-0001-7856-4031 0000-0001-7856-4031 researchmap permalink 32902 32902 著者名 Yamaguchi, Yuki
	NRID 1000090335381 1000090335381 ORCID 0000-0002-5181-179X 0000-0002-5181-179X researchmap permalink read0090387 read0090387 Scopus Author ID 35429790100 35429790100 著者名 Uchiyama, Susumu
キーワード等	Adeno-associated virus
	Cesium chloride
	Transduction efficiency
	Ultracentrifugation
	Vertical rotor
	Viral protein stoichiometry
内容	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.
抄録	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.
出版者	Springer Science and Business Media Deutschland GmbH
収録物名	European Biophysics Journal
刊行年月	2025-05-19
言語	英語
ハンドルURL	https://hdl.handle.net/11094/102249
PISSN	01757571
eISSN	14321017
関連情報 (isIdenticalTo)	DOI (出版社版) https://doi.org/10.1007/s00249-025-01751-1
アクセス権	open access
権利情報	This article is licensed under a Creative Commons Attribution 4.0 International License.
出版タイプ	Version of Record
カテゴリ	学術雑誌論文 Journal Article

資源タイプ	学術雑誌論文
ローカル資源タイプ	学術雑誌論文
DCMI資源タイプ	text
DCTERMS.bibliographicCitation	European Biophysics Journal. 2025
DC.title	Use of cesium chloride density gradient ultracentrifugation for the purification and characterization of recombinant adeno-associated virus
DC.creator	Hirohata, Kiichi
	Kino, Shinichiro
	Yamane, Takuya
	Bandoh, Karin
	Bamba, Takeshi
	Sternisha, Shawn M.
	Torisu, Tetsuo
	Fukuhara, Mitsuko
	Yamaguchi, Yuki
	Uchiyama, Susumu
DC.publisher	Springer Science and Business Media Deutschland GmbH
DC.language' scheme='DCTERMS.RFC1766	英語
DC.type' scheme='DCTERMS.DCMIType	text
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2025-05-19
DC.identifier	https://doi.org/10.1007/s00249-025-01751-1
DC.subject	Adeno-associated virus
	Cesium chloride
	Transduction efficiency
	Ultracentrifugation
	Vertical rotor
	Viral protein stoichiometry
DC.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.
DCTERMS.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.
DC.rights	This article is licensed under a Creative Commons Attribution 4.0 International License.
DC.format	102249
citation_title	Use of cesium chloride density gradient ultracentrifugation for the purification and characterization of recombinant adeno-associated virus
citation_author	Hirohata, Kiichi
	Kino, Shinichiro
	Yamane, Takuya
	Bandoh, Karin
	Bamba, Takeshi
	Sternisha, Shawn M.
	Torisu, Tetsuo
	Fukuhara, Mitsuko
	Yamaguchi, Yuki
	Uchiyama, Susumu
citation_publisher	Springer Science and Business Media Deutschland GmbH
citation_language	英語
citation_date	2025-05-19
citation_journal_title	European Biophysics Journal
citation_doi	https://doi.org/10.1007/s00249-025-01751-1
citation_public_url	https://hdl.handle.net/11094/102249

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

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