Ebberink Eduard H T M, Ruisinger Alisa, Nuebel Markus, Thomann Marco, Heck Albert J R
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Mol Ther Methods Clin Dev. 2022 Nov 15;27:491-501. doi: 10.1016/j.omtm.2022.11.003. eCollection 2022 Dec 8.
Adeno-associated viruses (AAVs) are useful vehicles for gene therapy because of their stability, low immunogenicity. and non-pathogenicity. However, disparity in AAV sample preparations (e.g., in capsid composition, DNA packaging, and impurities) gives rise to product heterogeneity, with possibly undesired effects on gene delivery. Ideally, AAV production should be with full control of AAV structure and genetic payload. Therefore, robust, efficient, and low material consuming methods are essential to characterize AAVs. Here, we use two emerging single-molecule techniques, mass photometry and Orbitrap-based charge-detection mass spectrometry, and show how they may efficiently and accurately characterize AAVs. We were able to resolve heterogeneous pools of particles, evaluating AAVs from two different serotypes (AAV8 and AAV2), produced by three independent production platforms, either lacking a genome or packed with a transgene. Together our data confirm that the different AAV production methods result in rather different and diverse AAV particle distributions. Especially for the packed AAVs, frequently additional subspecies were observed, next to the expected packed genome, mostly resulting from under- or overpackaging of genome material and/or residual empty particles. This work further establishes that both these single-particle techniques may become valuable tools in characterizing AAVs before they are used in gene therapy.
腺相关病毒（AAV）因其稳定性、低免疫原性和非致病性，是基因治疗的有用载体。然而，AAV样品制备的差异（例如衣壳组成、DNA包装和杂质）会导致产品异质性，可能对基因传递产生不良影响。理想情况下，AAV的生产应该能够完全控制AAV的结构和基因载荷。因此，强大、高效且低耗材的方法对于表征AAV至关重要。在这里，我们使用两种新兴的单分子技术，即质量光度法和基于轨道阱的电荷检测质谱法，并展示它们如何高效、准确地表征AAV。我们能够解析异质颗粒池，评估由三个独立生产平台产生的两种不同血清型（AAV8和AAV2）的AAV，这些AAV要么缺乏基因组，要么包装有转基因。我们的数据共同证实，不同的AAV生产方法会导致相当不同且多样的AAV颗粒分布。特别是对于包装好的AAV，除了预期的包装基因组外，经常会观察到额外的亚群，这主要是由于基因组材料包装不足或过度包装和/或残留空颗粒所致。这项工作进一步证实，在将这些单颗粒技术用于基因治疗之前，它们都可能成为表征AAV的有价值工具。
