Efficient whole brain transduction by systemic infusion of minimally purified AAV-PHP.eB
Efficient whole brain transduction by systemic infusion of minimally purified AAV-PHP.eB
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DOI：
10.1016/j.jneumeth.2020.108914
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发表时间：
2020-12-01
影响因子：
3
通讯作者：
Hirai, Hirokazu
中科院分区：
文献类型：
--
作者：
Konno, Ayumu;Hirai, Hirokazu
背景：腺相关病毒（AAV）载体作为基因转移载体具有优良的特性。AAV-PHP.eB，AAV血清型9的高BBB渗透性衣壳变体的最新发展已经开辟了全脑转导的系统应用。为了获得高的转导效率，已经付出了很多努力来使用梯度离心或柱层析纯化AAV载体。这些方法都是耗时，成本高，需要昂贵的设备。新方法：我们提出了一种简单的纯化方法，用于生产靶向脑的全身适用的AAV-PHP.eB。结果：与传统的超滤纯化（UCP）方法相比，MP方法获得的AAV-PHP.eB产量提高了2倍。静脉内注射使用MP方法制备的AAV-PHP.eB引起稳健的全脑转导，而对肝脏和肾脏没有明显的毒性。此外，我们发现对照小鼠和用MP病毒溶液全身治疗的小鼠之间脑小胶质细胞的细胞密度和形态几乎没有差异，表明病毒注射对脑免疫没有影响。新方法只需要台式离心机，仅需2-4小时即可获得即用型病毒溶液，比现有的UCP方法便宜得多，并且可以避免繁琐和耗时的纯化processs.Conclusions：这种简化的方法进一步扩展了AAV载体在神经科学界的使用。
Background: Adeno-associated virus (AAV) vectors have excellent properties as gene transfer vehicles. The recent development of AAV-PHP.eB, highly BBB-permeable capsid variant of AAV serotype 9, has opened up systemic application for whole brain transduction. To attain high transduction efficacy, much efforts have been paid to purify AAV vectors using gradient centrifugation or column chromatography. These methods are time-consuming, cost substantially and require expensive equipment.New method: We propose a simple purification method for the production of systemically applicable AAV-PHP.eB targeting the brain. The new method, which we named minimal purification (MP) method, requires only 2 steps: removal of cell debris using a syringe filter and concentration using a disposable ultrafiltration device.Results: The MP method yielded 2 times more AAV-PHP.eB than the standard ultracentrifuge purification (UCP) method. Intravenous injection of AAV-PHP.eB prepared using the MP method caused robust whole brain transduction without overt toxicity on the liver and kidney. Moreover, we found almost no difference in cellular density and morphology of brain microglia between control mice and mice treated systemically with the MP viral solution, suggesting no influence of the viral injection on brain immunity.Comparison with existing methods: The new method, which requires only a benchtop centrifuge and takes only 2-4 h to obtain a ready-to-use viral solution, is much less expensive than the existing UCP method, and can avoid cumbersome and time-consuming purification processes.Conclusions: This simplified method further expands the use of AAV vectors in the neuroscience community.