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Poster Presentations
Day 3, June 24（Tue.）
Room P (Maesato East, Foyer, Ocean Wing)
- 3P-AM-28
LC-MS/MS-based approach for glycosylation of recombinant adeno-associated virus
(1Osaka Univ., 2GlycoTechnica, 3Precision System Science, 4AIST, 5U-Medico, 6Nagoya Univ., 7Jichi Med. Univ. Sch. of Med., 8Jichi Med. Univ.)
oYuki Yamaguchi1, Kentaro Ishii1, Sachiko Koizumi2,3, Hiroaki Sakaue4, Takahiro Maruno1,5, Mitsuko Fukuhara1,5, Risa Shibuya1, Yasuo Tsunaka1, Aoba Matsushita1, Karin Bandoh1, Tetsuo Torisu1, Chie Murata-Kishimoto2, Azusa Tomioka4, Saho Mizukado4, Hiroyuki Kaji6, Yuji Kashiwakura7,8, Tsukasa Ohmori7,8, Atsushi Kuno4, Susumu Uchiyama1
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 using 42 lectins on six rAAV serotype 6 (rAAV6) preparations. As a result, Agaricus bisporus agglutinin (ABA) potentially recognized O-glycans attached to rAAV6. 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 based on western blot and polymerase-chain reaction analyses. 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.