Selective and rapid uptake of adeno-associated virus type 2 in brain
Selective and rapid uptake of adeno-associated virus type 2 in brain
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DOI：
10.1089/hum.1998.9.8-1181
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发表时间：
1998-05-20
影响因子：
4.2
通讯作者：
McCown, TJ
中科院分区：
文献类型：
--
作者：
Bartlett, JS;Samulski, RJ;McCown, TJ
重组腺相关病毒（AAV）载体在脑中有效地转移和表达外源基因。然而，转移的基因在神经元中选择性表达，这种特异性的原因尚不清楚。为了解决这个问题，野生型AAV-2衣壳共价标记的荧光团，Cy 3，并注入下丘或海马。使用抗体鉴定神经元（NeuN）、星形胶质细胞（GFAP）或少突胶质细胞（OX-42），早在输注开始后6 min就观察到病毒的明确神经元特异性摄取。到输注后30分钟，AAV颗粒存在于神经元的细胞核中，但在下丘和海马中，一个神经元亚组没有摄取病毒颗粒。病毒注入后1.5分钟或24小时，在星形胶质细胞中未发现AAV颗粒。有趣的是，输注后1小时，在小胶质细胞中没有发现AAV颗粒，而到输注后24小时，在小胶质细胞中发现了AAV颗粒的点状图案。为了测试病毒摄取是否与载体转导的细胞相关，输注rAAV-CMV-GFP病毒。通过3天后输注，GFP定位于神经元群体，在星形胶质细胞或小胶质细胞中没有表达，类似于荧光病毒摄取。这些发现表明，在脑中，AAV颗粒以与体内载体转导相似的模式快速结合并主要进入神经元。此外，这些研究表明，病毒结合和摄取，独立于启动子向性，可以解释AAV脑转导的特异性。因此，这第一次描述的AAV在体内的动力学处置应有助于有针对性地应用该载体的人脑基因治疗。
Recombinant adeno-associated virus (AAV) vectors effectively transfer and express foreign genes in the brain. The transferred genes, however, are selectively expressed in neurons, and the cause of this specificity is not understood. To address this question, wild-type AAV-2 capsids were covalently labeled with the fluorophore, Cy3, and infused into the inferior colliculus or the hippocampus. Using antibodies to identify neurons (NeuN), astrocytes (GFAP), or oligodendrocytes (OX-42), clear neuron-specific uptake of the virus was observed as early as 6 min after the start of the infusion. By 30 min postinfusion, AAV particles were present in the nucleus of neurons, yet in both the inferior colliculus and hippocampus, a subset of neurons did not take up the virus particles. No AAV particles were found in astrocytes 1.5 min or 24 hr after virus infusion. Interestingly, 1 hr postinfusion, no AAV particles were found in microglia, yet by 24 hr postinfusion, a punctate pattern of AAV particles was found in microglia. To test whether virus uptake correlated with vector-transduced cells, an rAAV-CMV-GFP virus was infused. By 3 days postinfusion, GFP was localized to neuronal populations with no expression in astrocytes or microglia, similar to that of fluorescent virus uptake. These findings demonstrate that in brain, AAV particles rapidly bind and enter primarily neurons with a pattern similar to that of in vivo vector transduction. In addition, these studies indicate that viral binding and uptake, independent of promoter tropism, can explain the specificity of AAV brain transduction. Thus, this first description of AAV kinetic disposition in vivo should facilitate targeted application of this vector for human brain gene therapy.