Time course of transgene expression after intrastriatal pseudotyped rAAV2/1, rAAV2/2, rAAV2/5, and rAAV2/8 transduction in the rat
Time course of transgene expression after intrastriatal pseudotyped rAAV2/1, rAAV2/2, rAAV2/5, and rAAV2/8 transduction in the rat
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DOI：
10.1038/sj.mt.6300227
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发表时间：
2007-08-01
影响因子：
12.4
通讯作者：
Mandel, Ronald J.
中科院分区：
文献类型：
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作者：
Reimsnider, Sharon;Manfredsson, Fredric P.;Mandel, Ronald J.
体内重组腺相关病毒载体（rAAV）介导的包括脑在内的各种组织的转导的特点是基因表达缓慢起始并逐渐增加，然后才达到稳定的长期蛋白质水平。转基因表达的早期过程尚未使用新获得的 rAAV 衣壳血清型进行量化。在本实验中，对纹状体内注射基于 rAAV2 的假型载体（rAAV1、rAAV5 和 rAAV8 衣壳）后绿色荧光蛋白 (GFP) 的表达开始进行定量。所有假型 rAAV 载体的天然 GFP 荧光均显示延迟至少 7 天的表达开始。然而，GFP 免疫组织化学染色显示，所有血清型转导后 4 天都有显着的转基因表达，并且最晚在转导后 14 天内，所有血清型介导的稳定 GFP(+) 神经元群体均显示出显着的转基因表达。 rAAV2/1 和 rAAV2/2 没有表现出 GFP(+) 纹状体神经元的时间依赖性增加；注射后 4 天达到最大纹状体细胞 GFP(+) 计数。所有血清型在注射后 4 周均显示转基因表达峰值，其中天然 GFP(+) 神经元与免疫染色纹状体 GFP(+) 神经元相同。对这些 rAAV 载体的炎症反应在转导后 4 周内出现，但在注射后 9 个月并不明显。因此，rAAV 介导的转基因表达开始得比之前想象的要早。
In vivo recombinant adeno-associated viral vector (rAAV)-mediated transduction of various tissues including brain has been characterized by slow onset and gradual increase in gene expression before reaching stable long-term protein levels. The early time course of transgene expression has not been quantified using newly available rAAV capsid serotypes. In this experiment, the onset of expression of green fluorescent protein (GFP) after intrastriatal injection of rAAV2-based pseudotyped vectors (rAAV1, rAAV5, and rAAV8 capsids) was quantified. Native GFP fluorescence displayed a delayed onset of expression of at least 7 days for all the pseudotyped rAAV vectors. However, GFP immunohistochemical staining revealed significant transgene expression by 4 days after transduction for all serotypes and stable GFP(+) neuronal populations mediated by all serotypes within 14 days post transduction at the latest. rAAV2/1 and rAAV2/2 displayed no time-dependent increase of GFP(+) striatal neurons; reaching maximal striatal cell GFP(+) counts at 4 days after injection. All serotypes displayed peak transgene expression by 4 weeks post injection where native GFP(+) neurons were equal to immunostained striatal GFP(+) neurons. The inflammatory response to these rAAV vectors was present up to 4 weeks after transduction but was not apparent 9 months post injection. Thus, rAAV-mediated transgene expression begins earlier than previously thought.