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.

中文翻译：

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通过全身输注最低限度纯化的AAV-PHP.eB进行有效的全脑转导。

背景

腺相关病毒（AAV）载体具有出色的基因转移载体特性。AAV血清型9的高度BBB渗透性衣壳变异体AAV-PHP.eB的最新开发为全脑转导打开了系统应用。为了获得高转导效率，已经进行了许多努力来使用梯度离心或柱色谱法纯化AAV载体。这些方法耗时，成本高并且需要昂贵的设备。

新方法

我们提出了一种简单的纯化方法，用于生产靶向大脑的全身适用的AAV-PHP.eB。新的方法，该方法我们命名为米inimal p urification（MP）的方法，只需要2个步骤：除去使用注射器过滤器和浓度使用一次性超滤装置细胞碎片。

结果

MP方法产生的AAV-PHP.eB是标准超速离心纯化（UCP）方法的2倍。使用MP方法制备的AAV-PHP.eB静脉注射引起了健壮的全脑转导，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MP病毒溶液全身治疗的小鼠之间的细胞密度和脑小胶质细胞形态几乎没有差异，表明病毒注射对脑免疫没有影响。

与现有方法的比较

新方法只需要台式离心机，只需2-4小时即可获得现成的病毒溶液，比现有的UCP方法便宜得多，并且可以避免繁琐且费时的纯化过程。

结论

这种简化的方法进一步扩大了AAV载体在神经科学界的应用。