High-efficiency purification of divergent AAV serotypes using AAVX affinity chromatography.
High-efficiency purification of divergent AAV serotypes using AAVX affinity chromatography.
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DOI：
10.1016/j.omtm.2022.12.009
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发表时间：
2023-03-09
期刊：
影响因子：
--
通讯作者：
Vandenberghe, Luk H.
中科院分区：
文献类型：
--
作者：
Florea, Michael;Nicolaou, Fotini;Pacouret, Simon;Zinn, Eric M.;Sanmiguel, Julio;Andres-Mateos, Eva;Unzu, Carmen;Wagers, Amy J.;Vandenberghe, Luk H.
腺相关病毒载体(AAV)提供了一个安全有效的基因治疗平台，包括几种已获批准的产品，对患者具有显著的治疗效果。然而，AAV开发和商业化的一个主要瓶颈仍然是AAV生产的效率、成本和可扩展性。层析方法有可能以更大的规模和更低的成本进行纯化，但通常需要针对每种血清型进行优化。在这里，我们展示了Poros CaptureSelect AAVX亲和树脂有效地捕获了一组15种不同的AAV血清型，包括常用的AAV2、AAV8、AAV9、PHP.B和Anc80。我们还发现，AAVX树脂可以重复再生，而不会损失效率或残留污染。AAVX纯化的AAV PreP的空衣壳比例高于超速离心法纯化的PreP，但AAVX纯化的载体在体内外的效力与Iodixano纯化的载体相当。最后，对纯化方案的优化导致了在所有规模和测试AAV血清型的总体效率为65%-80%的过程。这些数据确立了AAVX亲和层析作为一种通用和有效的方法来纯化广泛的AAV血清型。Florea等人。描述一种使用Poros CaptureSelect AAVX亲和层析纯化AAV的通用方法，适用于大量不同的AAV血清型。该方法具有成本效益，并可在实验室规模生产高质量的AAV。提供了一份详细的协议，使对AAV研究感兴趣的研究实验室能够随时采用该协议。
The adeno-associated viral vector (AAV) provides a safe and efficient gene therapy platform with several approved products that have marked therapeutic impact for patients. However, a major bottleneck in the development and commercialization of AAV remains the efficiency, cost, and scalability of AAV production. Chromatographic methods have the potential to allow purification at increased scales and lower cost but often require optimization specific to each serotype. Here, we demonstrate that the POROS CaptureSelect AAVX affinity resin efficiently captures a panel of 15 divergent AAV serotypes, including the commonly used AAV2, AAV8, AAV9, PHP.B, and Anc80. We also find that AAVX resin can be regenerated repeatedly without loss of efficiency or carry-over contamination. While AAV preps purified with AAVX showed a higher fraction of empty capsids than preps purified using iodixanol ultracentrifugation, the potency of the AAVX purified vectors was comparable with that of iodixanol purified vectors both in vitro and in vivo. Finally, optimization of the purification protocol resulted in a process with an overall efficiency of 65%–80% across all scales and AAV serotypes tested. These data establish AAVX affinity chromatography as a versatile and efficient method for purification of a broad range of AAV serotypes. Florea et al. describe a versatile AAV purification method using POROS CaptureSelect AAVX affinity chromatography applicable to a large set of divergent AAV serotypes. The method is cost effective and yields high-quality AAV at laboratory scale. A detailed protocol is provided to enable ready adoption in research laboratories interested in AAV research.
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影响因子：
14.9
作者：
Goujon M;McWilliam H;Li W;Valentin F;Squizzato S;Paern J;Lopez R
通讯作者：
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DOI：
10.1016/j.omtm.2017.12.004
发表时间：
2018-06-15
期刊：
Molecular therapy. Methods & clinical development
影响因子：
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作者：
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作者：
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DOI：
10.1073/pnas.182412299
发表时间：
2002-09-03
影响因子：
11.1
作者：
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通讯作者：
Wilson, JM
影响因子：
12.4
作者：
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