Department of Anesthesiology, University of California San Diego, San Diego, California, United States of America.
PLoS One. 2012;7(3):e32581. doi: 10.1371/journal.pone.0032581. Epub 2012 Mar 5.
We report here in adult rat viral vector mediate-gene knockdown in the primary sensory neurons and the associated cellular and behavior consequences. Self-complementary adeno-associated virus serotype 5 (AAV5) was constructed to express green fluorescent protein (GFP) and a small interfering RNA (siRNA) targeting mammalian target of rapamycin (mTOR). The AAV vectors were injected via an intrathecal catheter. We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection. Of those neurons, over 85% were large to medium-diameter and co-labeled with NF200, a marker for myelinated fibers. Western blotting of mTOR revealed an 80% reduction in the lumbar DRGs (L4-L6) of rats treated with the active siRNA vectors compared to the control siRNA vector. Gene knockdown became apparent as early as 7-day post-injection and lasted for at least 5 weeks. Importantly, mTOR knockdown occurred in large (NF200) and small-diameter neurons (nociceptors). The viral administration induced an increase of Iba1 immunoreactivity in the DRGs, which was likely attributed to the expression of GFP but not siRNA. Rats with mTOR knockdown in DRG neurons showed normal general behavior and unaltered responses to noxious stimuli. In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons. This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons.
我们在此报告了在成年大鼠初级感觉神经元中病毒载体介导的基因敲低作用，以及相关的细胞和行为后果。构建了自我互补的腺相关病毒血清型 5（AAV5），以表达绿色荧光蛋白（GFP）和针对哺乳动物雷帕霉素靶蛋白（mTOR）的小干扰 RNA（siRNA）。AAV 载体通过鞘内导管注射。我们观察到在注射后 2 周开始，腰椎 DRG 神经元中出现明显的 GFP 表达。在这些神经元中，超过 85%是大到中直径的，并且与 NF200 共标记，NF200 是有髓纤维的标志物。用活性 siRNA 载体处理的大鼠的腰椎 DRGs（L4-L6）中 mTOR 的 Western blot 显示其表达降低了 80%，与对照 siRNA 载体相比。基因敲低在注射后 7 天即可显现，并持续至少 5 周。重要的是，mTOR 敲低发生在大（NF200）和小直径神经元（伤害感受器）中。病毒给药导致 DRG 中 Iba1 免疫反应性增加，这可能归因于 GFP 的表达而不是 siRNA。DRG 神经元中 mTOR 敲低的大鼠表现出正常的一般行为，对有害刺激的反应没有改变。总之，鞘内 AAV5 是一种将 siRNA 递送至 DRG 神经元并产生基因敲低的高效载体。这对于涉及初级感觉神经元的疾病的基础研究和临床干预都将非常有价值。
