Reimsnider Sharon, Manfredsson Fredric P, Muzyczka Nicholas, Mandel Ronald J
Department of Neuroscience, Powell Gene Therapy Center, McKnight Brain Institute, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Mol Ther. 2007 Aug;15(8):1504-11. doi: 10.1038/sj.mt.6300227. Epub 2007 Jun 12.
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.
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，其特征是在达到稳定的长期蛋白质水平之前，基因表达起效缓慢且逐渐增加。使用新获得的rAAV衣壳血清型尚未对转基因表达的早期时间进程进行量化。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）表达的起效情况进行了量化。天然GFP荧光显示，所有假型rAAV载体的表达起效均延迟至少7天。然而，GFP免疫组织化学染色显示，所有血清型在转导后4天均有明显的转基因表达，且所有血清型介导的稳定GFP（+）神经元群体最迟在转导后14天内形成。rAAV2/1和rAAV2/2的GFP（+）纹状体神经元数量没有随时间增加；在注射后4天达到纹状体细胞GFP（+）计数的最大值。所有血清型在注射后4周时转基因表达达到峰值，此时天然GFP（+）神经元与免疫染色的纹状体GFP（+）神经元数量相等。对这些rAAV载体的炎症反应在转导后4周内一直存在，但在注射后9个月时不明显。因此，rAAV介导的转基因表达比以前认为的开始得更早。
