Adeno-Associated Virus Serotypes 1, 8, and 9 Share Conserved Mechanisms for Anterograde and Retrograde Axonal Transport
Adeno-Associated Virus Serotypes 1, 8, and 9 Share Conserved Mechanisms for Anterograde and Retrograde Axonal Transport
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DOI：
10.1089/hum.2013.189
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发表时间：
2014-08-01
影响因子：
4.2
通讯作者：
Wolfe, John H.
中科院分区：
文献类型：
--
作者：
Castle, Michael J.;Gershenson, Zachary T.;Wolfe, John H.
腺相关病毒（AAV）载体在脑内注射后经常经历长距离轴突运输。这导致注射部位远端的脑区域的转导，尽管轴突运输和远端转导的程度在AAV血清型之间变化很大。驱动这种变化的机制知之甚少。这对于需要在特定脑区域内集中基因表达的应用来说是一个关键问题，并且还阻碍了将载体转运用于需要将转基因广泛递送至脑的应用。在这里，我们比较了AAV血清型1和9，其经常表现出远端转导，与血清型8，其很少传播超过注射部位。为了研究AAV在体外的定向转运，我们使用微流控室将染料标记的AAV应用于原代大鼠胚胎皮层神经元的轴突末端或细胞体。所有三种血清型都是沿着轴突主动运输的，运输的特点是在顺行和逆行方向上的高速和长时间运行。与血清型对的共感染表明AAV 1、8和9共享用于轴突运输的相同细胞内隔室。在体内，AAV8和9在注射到成年小鼠脑中后在非可逆回路内表现出顺行和逆行转运，具有高度相似的远端转导分布。然而，在大量培养的神经元中，我们发现AAV1比AAV8或9更频繁地转运，并且AAV9转运的频率可以通过增加受体可用性来增强。因此，虽然这些血清型在体外和体内都具有轴突运输的保守机制，但运输的频率在血清型之间可以变化，并且轴突运输可以通过增强载体摄取而显著增加。这表明体内远端转导的变异性可能是由质膜上的差异性摄取引起的，而不是AAV血清型之间转运机制的根本差异。
Adeno-associated virus (AAV) vectors often undergo long-distance axonal transport after brain injection. This leads to transduction of brain regions distal to the injection site, although the extent of axonal transport and distal transduction varies widely among AAV serotypes. The mechanisms driving this variability are poorly understood. This is a critical problem for applications that require focal gene expression within a specific brain region, and also impedes the utilization of vector transport for applications requiring widespread delivery of transgene to the brain. Here, we compared AAV serotypes 1 and 9, which frequently demonstrate distal transduction, with serotype 8, which rarely spreads beyond the injection site. To examine directional AAV transport in vitro, we used a microfluidic chamber to apply dye-labeled AAV to the axon termini or to the cell bodies of primary rat embryonic cortical neurons. All three serotypes were actively transported along axons, with transport characterized by high velocities and prolonged runs in both the anterograde and retrograde directions. Coinfection with pairs of serotypes indicated that AAV1, 8, and 9 share the same intracellular compartments for axonal transport. In vivo, both AAV8 and 9 demonstrated anterograde and retrograde transport within a nonreciprocal circuit after injection into adult mouse brain, with highly similar distributions of distal transduction. However, in mass-cultured neurons, we found that AAV1 was more frequently transported than AAV8 or 9, and that the frequency of AAV9 transport could be enhanced by increasing receptor availability. Thus, while these serotypes share conserved mechanisms for axonal transport both in vitro and in vivo, the frequency of transport can vary among serotypes, and axonal transport can be markedly increased by enhancing vector uptake. This suggests that variability in distal transduction in vivo likely results from differential uptake at the plasma membrane, rather than fundamental differences in transport mechanisms among AAV serotypes.