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

• Published in: Molecular therapy. Methods & clinical development 2023-12, Vol.31, p.101142-101142, Article 101142
• Main Authors: Onishi, Takayuki, Nonaka, Michika, Maruno, Takahiro, Yamaguchi, Yuki, Fukuhara, Mitsuko, Torisu, Tetsuo, Maeda, Masaharu, Abbatiello, Susan, Haris, Anisha, Richardson, Keith, Giles, Kevin, Preece, Steve, Yamano-Adachi, Noriko, Omasa, Takeshi, Uchiyama, Susumu
• Format: Article
• Language: English
• Subjects: adeno-associated virus; analytical ultracentrifugation; charge detection-mass spectrometry; transduction efficiency; viral protein stoichiometry
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2023.101142

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. [Display omitted] Uchiyama and colleagues discovered protein heterogeneity in rAAV particles and enhanced them through a single amino acid substitution. This is crucial for improving the accuracy and control of VP stoichiometry in gene therapy, where rAAV vectors inherently exhibit variability in VP composition.