AAV2/8 Vectors Purified from Culture Medium with a Simple and Rapid Protocol Transduce Murine Liver, Muscle, and Retina Efficiently
AAV2/8 Vectors Purified from Culture Medium with a Simple and Rapid Protocol Transduce Murine Liver, Muscle, and Retina Efficiently
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DOI：
10.1089/hgtb.2013.155
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发表时间：
2013-12-01
影响因子：
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通讯作者：
Auricchio, Alberto
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文献类型：
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作者：
Doria, Monica;Ferrara, Antonella;Auricchio, Alberto
在某些腺相关病毒（AAV）血清型的生产过程中，在生产细胞的培养基中发现了大量的载体。为了进行纯化，以前的方案使用介质的切向流过滤 (TFF)，然后进行碘克沙醇梯度离心。利用比细胞来源材料更高纯度的培养基作为 AAV 来源，我们测试了一种简单的方法，该方法将细胞堆中含有 AAV 的大体积培养基生产与培养基澄清 + TFF 结合起来，无需进一步耗时且不可扩展的离心。为了测试这一点，我们选择了 AAV2/8，它正在成为肝脏、肌肉和视网膜转导的首选血清型，并且在细胞外培养基中大量存在。我们表明，使用这种方法从培养基产生的 AAV2/8 载体的产量和体外感染性高于使用传统的 CsCl2 梯度超速离心方法从相同细胞裂解物中纯化的载体，尽管纯度似乎较差。此外，我们发现从培养基中纯化的 AAV2/8 的转导效率与从小鼠肝脏、肌肉和视网膜中相同细胞裂解物中纯化的 AAV2/8 的转导效率相似。考虑到我们描述的培养基纯化方案需要 3 小时，而传统的两轮 CsCl2 梯度超速离心 + 脱盐需要 63 小时，我们得出结论，含有 AAV2/8 的培养基的 TFF 代表了一种快速且可扩展的方法，用于纯化用于动物模型的研究级载体。
During the production of some adeno-associated virus (AAV) serotypes, a large amount of vectors is found in the medium of producing cells. For their purification, previous protocols used tangential flow filtration (TFF) of the medium followed by iodixanol gradient centrifugation. Taking advantage of the higher purity of the medium than the cell-derived material as the source of AAV, we tested a simple method that combines production of large culture medium volumes containing AAV from cell stacks with medium clarification + TFF without further time-consuming and nonscalable centrifugation. To test this, we selected AAV2/8, which is emerging as a favored serotype for transduction of liver, muscle, and retina and abundantly found in the extracellular medium. We show that yields and in vitro infectivity of AAV2/8 vectors produced from the culture medium using this method are higher than those of vectors purified from the same cell lysate using a conventional CsCl2 gradient ultracentrifugation-based method, although purity appears inferior. In addition, we found that the transduction efficiency of AAV2/8 purified from medium was similar to that of AAV2/8 purified from the same cell lysate in the murine liver, muscle, and retina. Considering that the purification protocol from the medium we describe requires 3 hr as opposed to the 63 hr of a conventional two-round CsCl2-gradient ultracentrifugation + desalting, we conclude that TFF of the medium containing AAV2/ 8 represents a quick and scalable method to purify research-grade vectors for use in animal models.