Highly Efficient Ultracentrifugation-free Chromatographic Purification of Recombinant AAV Serotype 9
Highly Efficient Ultracentrifugation-free Chromatographic Purification of Recombinant AAV Serotype 9
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DOI：
10.1016/j.omtm.2018.10.015
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发表时间：
2018-12-01
影响因子：
4.7
通讯作者：
Okada, Takashi
中科院分区：
文献类型：
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作者：
Tomono, Taro;Hirai, Yukihiko;Okada, Takashi
重组腺相关病毒血清型9（rAAV 9）可以特异性地感染肌肉和神经组织，因此，rAAV 9有可能用于基因治疗。然而，rAAV 9是纯化最具挑战性的rAAV血清型。传统上，rAAV 9已经通过超离心纯化，这是不可扩展的。我们最近描述了rAAV 1的色谱纯化方案;该方案可以实现可扩展的纯化。在这项研究中，我们试图优化这个协议纯化rAAV 9制剂，我们开发了一种新的，有效的方法，高产量纯化rAAV 9使用季铵阴离子交换剂和尺寸排阻色谱。通过SDS-PAGE观察，最终纯化的rAAV 9主要含有三种衣壳蛋白。此外，负染色电子显微镜显示96.1% +/-1.1%的rAAV 9颗粒携带含有EGFP转基因的病毒基因组，表明杂质和空衣壳可以用我们的纯化方案消除。通过我们的方案获得的最终rAAV 9滴度总计为2.5 +/-0.4 X 10（15）个病毒基因组，其由类似于3.2 X 10（9）个HEK 293 EB细胞产生。我们证实，我们的方案也可以应用于纯化其他不同的AAV基因组构建体。我们的方案可以按照目前的良好生产规范扩大纯rAAV 9的生产，用于人类基因治疗的临床应用。
Recombinant adeno-associated virus serotype 9 (rAAV9) can specifically transduce muscle and neuronal tissues; thus, rAAV9 can potentially be used in gene therapy. However, rAAV9 is the most challenging rAAV serotype to purify. Traditionally, rAAV9 has been purified by ultracentrifugation, which is not scalable. We recently described a chromatographic purification protocol for rAAV1; this protocol can achieve scalable purifications. In this study, we attempted to optimize this protocol for purifying rAAV9 preparations, and we developed a novel, effective method for high-yield purification of rAAV9 using quaternary ammonium anion exchangers and size-exclusion chromatography. The final purified rAAV9 contained mainly three capsid proteins, as observed by SDS-PAGE. Furthermore, negative-stain electron microscopy demonstrated that 96.1% +/- 1.1% of rAAV9 particles carried the viral genome containing the EGFP transgene, indicating that impurities and empty capsids can be eliminated with our purification protocol. The final rAAV9 titer obtained by our protocol totaled 2.5 +/- 0.4 x 10(15) viral genomes produced from similar to 3.2 x 10(9) HEK293EB cells. We confirmed that our protocol can also be applied to purify other varied AAV genome constructs. Our protocol can scale up production of pure rAAV9, in compliance with current good manufacturing practice, for clinical applications in human gene therapy.