Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA.
Avista Therapeutics, Pittsburgh, PA, USA.
Methods Mol Biol. 2025;2848:249-257. doi: 10.1007/978-1-0716-4087-6_15.
The production of Adeno-associated virus (AAV) vectors in the lab setting has typically involved expression in adherent cells followed by purification through ultracentrifugation in density gradients. This production method is, however, not easily scalable, presents high levels of cellular impurities that co-purify with the virus, and results in a mixture of empty and full capsids. Here we describe a detailed AAV production protocol that overcomes these limitations through AAV expression in suspension cells followed by AAV affinity purification and AAV polishing to separate empty and full capsids, resulting in high yields of ultra-pure AAV that is highly enriched in full capsids.
腺相关病毒 (AAV) 载体在实验室环境中的生产通常涉及在贴壁细胞中进行表达，然后通过密度梯度超速离心进行纯化。然而，这种生产方法不易扩展，存在与病毒一起共纯化的高水平细胞杂质，并且导致空壳和完整衣壳的混合物。在这里，我们描述了一种详细的 AAV 生产方案，该方案通过悬浮细胞中的 AAV 表达克服了这些限制，然后通过 AAV 亲和纯化和 AAV 抛光分离空壳和完整衣壳，从而获得高产率的超纯 AAV，其中富含完整衣壳。
