Deamidation of Amino Acids on the Surface of Adeno-Associated Virus Capsids Leads to Charge Heterogeneity and Altered Vector Function
Deamidation of Amino Acids on the Surface of Adeno-Associated Virus Capsids Leads to Charge Heterogeneity and Altered Vector Function
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DOI：
10.1016/j.ymthe.2018.09.013
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发表时间：
2018-12-05
影响因子：
12.4
通讯作者：
Wilson, James M.
中科院分区：
文献类型：
--
作者：
Giles, April R.;Sims, Joshua J.;Wilson, James M.
腺相关病毒衣壳的翻译后修饰是这些病毒载体发展成药物产品的一个鲜为人知的因素。在这里，我们报告了8型腺相关病毒和其他7种不同的腺相关病毒血清型的广泛衣壳脱酰胺，并从结构、生化和质谱方法中获得了支持证据。每个位点的脱酰胺程度取决于载体的年龄和多个主要序列和三维结构因素。然而，脱酰胺程度在很大程度上与载体回收和纯化条件无关。我们证明了脱酰胺对转导活性的潜在影响，此外，在几种腺相关病毒8天酰胺中，载体活性的早期时间点损失与快速进展的自发脱酰胺有关。我们探索了稳定侧链酰胺的突变策略，改善了载体转导，减少了生物制剂制造中一个关键问题的批次间分子变异性。这项研究阐明了腺相关病毒衣壳异质性的一个以前未知的方面，并强调了其在这些基因治疗载体发展中的重要性。
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.