Deamidation of Amino Acids on the Surface of Adeno-Associated Virus Capsids Leads to Charge Heterogeneity and Altered Vector Function

• Published in: Molecular therapy 2018-12, Vol.26 (12), p.2848-2862
• Main Authors: Giles, April R., Sims, Joshua J., Turner, Kevin B., Govindasamy, Lakshmanan, Alvira, Mauricio R., Lock, Martin, Wilson, James M.
• Format: Article
• Language: English
• Subjects: adeno-associated virus; Agreements; Amides; Amino acids; bioengineering; Capsids; Expression vectors; Flexibility; Gene therapy; Mass spectrometry; Mass spectroscopy; Original; Peptides; Post-translation; post-translational modification; Proteins; Purification; Scientific imaging; Serotypes; structural biology; virus structure
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2018.09.013

Post-translational modification of the adeno-associated virus capsids is a poorly understood factor in the development of these viral vectors into pharmaceutical products. Here we report the extensive capsid deamidation of adeno-associated virus serotype 8 and seven other diverse adeno-associated virus serotypes, with supporting evidence from structural, biochemical, and mass spectrometry approaches. The extent of deamidation at each site depended on the vector’s age and multiple primary-sequence and three-dimensional structural factors. However, the extent of deamidation was largely independent of the vector recovery and purification conditions. We demonstrate the potential for deamidation to impact transduction activity and, moreover, correlate an early time point loss in vector activity to rapidly progressing spontaneous deamidation at several adeno-associated virus 8 asparagines. We explore mutational strategies that stabilize side-chain amides, improving vector transduction and reducing the lot-to-lot molecular variability that presents a key concern in biologics manufacturing. This study illuminates a previously unknown aspect of adeno-associated virus capsid heterogeneity and highlights its importance in the development of these vectors for gene therapy. Understanding and managing protein heterogeneity, often due to post-translational modifications, is critical for biologics development. Here we demonstrate widespread asparagine deamidation of adeno-associated virus gene therapy vectors and correlate its progress to a loss of transduction activity. We present mutagenic strategies that prevent deamidation, increase transduction, and improve manufacturing consistency.