Glycosylation of recombinant adeno-associated virus serotype 6

Yuki Yamaguchi; Kentaro Ishii; Sachiko Koizumi; Hiroaki Sakaue; Takahiro Maruno; Mitsuko Fukuhara; Risa Shibuya; Yasuo Tsunaka; Aoba Matsushita; Karin Bandoh; Tetsuo Torisu; Chie Murata-Kishimoto; Azusa Tomioka; Saho Mizukado; Hiroyuki Kaji; Yuji Kashiwakura; Tsukasa Ohmori; Atsushi Kuno; Susumu Uchiyama

Molecular Therapy: Methods & Clinical Development (Jun 2024)

Glycosylation of recombinant adeno-associated virus serotype 6

Yuki Yamaguchi,
Kentaro Ishii,
Sachiko Koizumi,
Hiroaki Sakaue,
Takahiro Maruno,
Mitsuko Fukuhara,
Risa Shibuya,
Yasuo Tsunaka,
Aoba Matsushita,
Karin Bandoh,
Tetsuo Torisu,
Chie Murata-Kishimoto,
Azusa Tomioka,
Saho Mizukado,
Hiroyuki Kaji,
Yuji Kashiwakura,
Tsukasa Ohmori,
Atsushi Kuno,
Susumu Uchiyama

Affiliations

Yuki Yamaguchi: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Kentaro Ishii: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Sachiko Koizumi: GlycoTechnica Ltd., Yokohama, Japan; Precision System Science Co. Ltd., 88 Kamihongo, Matsudo, Chiba 271-0064, Japan
Hiroaki Sakaue: Molecular and Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8565, Japan
Takahiro Maruno: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; U-Medico Inc., 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Mitsuko Fukuhara: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; U-Medico Inc., 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Risa Shibuya: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Yasuo Tsunaka: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Aoba Matsushita: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Karin Bandoh: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Tetsuo Torisu: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Chie Murata-Kishimoto: GlycoTechnica Ltd., Yokohama, Japan
Azusa Tomioka: Molecular and Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8565, Japan
Saho Mizukado: Molecular and Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8565, Japan
Hiroyuki Kaji: Institute for Glyco-core Research (iGCORE), Nagoya University, Furo-cho, Chikusa, Nagoya, Aichi 464-8601, Japan
Yuji Kashiwakura: Department of Biochemistry, Jichi Medical University School of Medicine, 3111-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan; Center for Gene Therapy Research, Jichi Medical University, 3111-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan
Tsukasa Ohmori: Department of Biochemistry, Jichi Medical University School of Medicine, 3111-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan; Center for Gene Therapy Research, Jichi Medical University, 3111-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan
Atsushi Kuno: Molecular and Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8565, Japan
Susumu Uchiyama: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Corresponding author: Susumu Uchiyama, PhD, Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

Journal volume & issue: Vol. 32, no. 2
p. 101256

Abstract

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Glycosylation of biopharmaceuticals can affect their safety and efficacy. Glycans can occur on recombinant adeno-associated viruses (rAAVs) that are used for gene therapy; however, the types of glycans that attach to rAAVs are controversial. Here, we conducted lectin microarray analyses on six rAAV serotype 6 (rAAV6) preparations that were produced differently. We demonstrate that O-glycans considered to be attached to rAAV6 were recognized by Agaricus bisporus agglutinin (ABA) and that N-glycans were detected in rAAV6 purified without affinity chromatography. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the N-glycans detected in rAAV6 were derived from host cell proteins. A combination of ABA-based fractionation and LC-MS/MS revealed that rAAV6 was O-glycosylated with the mucin-type glycans, O-GalNAc (Tn antigen), and mono- and di-sialylated Galβ1-3GalNAc (T antigen) at S156, T162, T194, and T201 in viral protein (VP) 2 and with O-GlcNAc at T242 in VP3. The mucin-type O-glycosylated rAAV6 particles were 0.1%–1% of total particles. Further physicochemical and biological analyses revealed that mucin-type O-glycosylated rAAV6 had a lower ratio of VP1 to VP2/VP3, resulting in a lower transduction efficiency both in vitro and in vivo compared with rAAV6 without mucin-type O-glycans. This report details conclusive evidence of rAAV glycosylation and its impact on rAAV-based therapeutics.

Published in Molecular Therapy: Methods & Clinical Development

ISSN: 2329-0501 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Biology (General): Genetics; Science: Biology (General): Cytology
Website: http://www.cell.com/molecular-therapy-family/methods/latest-content

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