Enhancement of recombinant adeno-associated virus activity by improved stoichiometry and homogeneity of capsid protein assembly

Takayuki Onishi; Michika Nonaka; Takahiro Maruno; Yuki Yamaguchi; Mitsuko Fukuhara; Tetsuo Torisu; Masaharu Maeda; Susan Abbatiello; Anisha Haris; Keith Richardson; Kevin Giles; Steve Preece; Noriko Yamano-Adachi; Takeshi Omasa; Susumu Uchiyama

doi:10.1016/j.omtm.2023.101142

Enhancement of recombinant adeno-associated virus activity by improved stoichiometry and homogeneity of capsid protein assembly

Mol Ther Methods Clin Dev. 2023 Oct 24:31:101142. doi: 10.1016/j.omtm.2023.101142. eCollection 2023 Dec 14.

Authors

Takayuki Onishi¹, Michika Nonaka¹, Takahiro Maruno^{1

2}, Yuki Yamaguchi¹, Mitsuko Fukuhara^{1

2}, Tetsuo Torisu¹, Masaharu Maeda³, Susan Abbatiello⁴, Anisha Haris⁴, Keith Richardson⁵, Kevin Giles⁴, Steve Preece⁵, Noriko Yamano-Adachi¹, Takeshi Omasa¹, Susumu Uchiyama¹

Affiliations

¹ Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
² U-Medico Inc, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
³ Osaka Consolidated Laboratory, Manufacturing Technology Association of Biologics, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
⁴ Waters Corporation, Milford, MA 01757, USA.
⁵ Waters Corporation (Micromass UK Ltd), Stamford Avenue, Altrincham Road, Wilmslow SK9 4AX, UK.

Abstract

Studies of recombinant adeno-associated virus (rAAV) revealed the mixture of full particles with different densities in rAAV. There are no conclusive results because of the lack of quantitative stoichiometric viral proteins, encapsidated DNA, and particle level analyses. We report the first comprehensive characterization of low- and high-density rAAV serotype 2 particles. Capillary gel electrophoresis showed high-density particles possessing a designed DNA encapsidated in the capsid composed of (VP1 + VP2)/VP3 = 0.27, whereas low-density particles have the same DNA but with a different capsid composition of (VP1 + VP2)/VP3 = 0.31, supported by sedimentation velocity-analytical ultracentrifugation and charge detection-mass spectrometry. In vitro analysis demonstrated that the low-density particles had 8.9% higher transduction efficacy than that of the particles before fractionation. Further, based on our recent findings of VP3 clip, we created rAAV2 single amino acid variants of the transcription start methionine of VP3 (M203V) and VP3 clip (M211V). The rAAV2-M203V variant had homogeneous particles with higher (VP1+VP2)/VP3 values (0.35) and demonstrated 24.7% higher transduction efficacy compared with the wild type. This study successfully provided highly functional rAAV by the extensive fractionation from the mixture of rAAV2 full particles or by the single amino acid replacement.

Keywords: adeno-associated virus; analytical ultracentrifugation; charge detection-mass spectrometry; transduction efficiency; viral protein stoichiometry.