Optimizing Promoters for Recombinant Adeno-Associated Virus-Mediated Gene Expression in the Peripheral and Central Nervous System Using Self-Complementary Vectors
Optimizing Promoters for Recombinant Adeno-Associated Virus-Mediated Gene Expression in the Peripheral and Central Nervous System Using Self-Complementary Vectors
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DOI：
10.1089/hum.2010.245
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发表时间：
2011-09-01
影响因子：
4.2
通讯作者：
Samulski, R. Jude
中科院分区：
文献类型：
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作者：
Gray, Steven J.;Foti, Stacey B.;Samulski, R. Jude
随着小分子自补充腺相关病毒(AAV)载体的广泛使用，设计紧凑的启动子对于在普遍存在或细胞特异性控制下包装和表达较大的转基因变得至关重要。在对常用的800bp巨细胞病毒(CMV)启动子和鸡β-肌动蛋白(CBA)启动子的比较研究中，我们报告了中枢和外周神经系统细胞特异性基因表达模式的显著差异。CMV启动子提供了随着时间推移而减弱的高初始神经表达。CBA启动子在神经细胞中的表达普遍较高，但在运动神经元(MN)中的表达较低。我们报告了一种新的CBA启动子(CBH)的混合形式的创建，它在所有用CMV或CBA观察到的细胞中提供了强大的长期表达，包括MNS。为了开发一个较短的神经元启动子来将更大的转基因包装到AAV载体中，我们还发现小鼠甲基CpG结合蛋白-2(MeCP2)启动子的229个碱基片段能够在中枢神经系统内驱动神经元特异性表达。因此，800bp的CBH启动子提供了强大的、长期的和普遍存在的中枢神经系统表达，而MeCP2启动子允许额外的570bp的包装能力，与MeCP2转录因子相似，在中枢神经系统中低水平且主要是神经元表达。
With the increased use of small self-complementary adeno-associated viral (AAV) vectors, the design of compact promoters becomes critical for packaging and expressing larger transgenes under ubiquitous or cell-specific control. In a comparative study of commonly used 800-bp cytomegalovirus (CMV) and chicken beta-actin (CBA) promoters, we report significant differences in the patterns of cell-specific gene expression in the central and peripheral nervous systems. The CMV promoter provides high initial neural expression that diminishes over time. The CBA promoter displayed mostly ubiquitous and high neural expression, but substantially lower expression in motor neurons (MNs). We report the creation of a novel hybrid form of the CBA promoter (CBh) that provides robust long-term expression in all cells observed with CMV or CBA, including MNs. To develop a short neuronal promoter to package larger transgenes into AAV vectors, we also found that a 229-bp fragment of the mouse methyl-CpG-binding protein-2 (MeCP2) promoter was able to drive neuron-specific expression within the CNS. Thus the 800-bp CBh promoter provides strong, long-term, and ubiquitous CNS expression whereas the MeCP2 promoter allows an extra 570-bp packaging capacity, with low and mostly neuronal expression within the CNS, similar to the MeCP2 transcription factor.