Lentiviral gene transfer into the dorsal root ganglion of adult rats.
Lentiviral gene transfer into the dorsal root ganglion of adult rats.
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DOI：
10.1186/1744-8069-7-63
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发表时间：
2011-08-23
期刊：
影响因子：
3.3
通讯作者：
Hogan QH
中科院分区：
文献类型：
--
作者：
Yu H;Fischer G;Jia G;Reiser J;Park F;Hogan QH
慢载体介导的基因传递到背根神经节（DRG）是一种很有前途的方法来探索疼痛病理生理和遗传治疗慢性神经性疼痛。本研究制备了一系列具有不同细胞启动子、包膜糖蛋白和病毒辅助蛋白的修饰慢载体颗粒，以评估其在体外和体内高效转导到神经细胞和成年大鼠DRG的需求。在体外实验中，在人延伸因子1α (EF1α)启动子控制下，用常规水泡性口炎病毒G蛋白（svv -G）包膜表达增强绿色荧光蛋白（EGFP）的慢载体在低感染倍数（MOIs）下向永活DRG感觉神经元细胞系转移的效果最好，在高感染倍数（MOIs）下向原代培养DRG神经元转移的效果最好。在体内，将第一代或第二代EF1α-EGFP慢载体直接注射到成年大鼠DRG中，在注射后4周检测到egfp阳性DRG神经元的转导率分别为19±9%和20±8%。转导细胞包括全范围的神经元表型，包括髓鞘神经元以及非肽能性和肽能性伤害性无髓鞘神经元。含有人延伸因子1α (EF1α)-EGFP表达盒的VSV-G假型慢载体在直接注射到DRG后显示出相对有效的向感觉神经元的转导。这些结果清楚地表明，慢载体作为一种可行的系统，可以将靶基因传递到DRGs中，以探索神经性疼痛的基本机制，并有可能在未来的临床应用中治疗慢性疼痛。
Lentivector-mediated gene delivery into the dorsal root ganglion (DRG) is a promising method for exploring pain pathophysiology and for genetic treatment of chronic neuropathic pain. In this study, a series of modified lentivector particles with different cellular promoters, envelope glycoproteins, and viral accessory proteins were generated to evaluate the requirements for efficient transduction into neuronal cells in vitro and adult rat DRG in vivo. In vitro, lentivectors expressing enhanced green fluorescent protein (EGFP) under control of the human elongation factor 1α (EF1α) promoter and pseudotyped with the conventional vesicular stomatitis virus G protein (VSV-G) envelope exhibited the best performance in the transfer of EGFP into an immortalized DRG sensory neuron cell line at low multiplicities of infection (MOIs), and into primary cultured DRG neurons at higher MOIs. In vivo, injection of either first or second-generation EF1α-EGFP lentivectors directly into adult rat DRGs led to transduction rates of 19 ± 9% and 20 ± 8% EGFP-positive DRG neurons, respectively, detected at 4 weeks post injection. Transduced cells included a full range of neuronal phenotypes, including myelinated neurons as well as both non-peptidergic and peptidergic nociceptive unmyelinated neurons. VSV-G pseudotyped lentivectors containing the human elongation factor 1α (EF1α)-EGFP expression cassette demonstrated relatively efficient transduction to sensory neurons following direct injection into the DRG. These results clearly show the potential of lentivectors as a viable system for delivering target genes into DRGs to explore basic mechanisms of neuropathic pain, with the potential for future clinical use in treating chronic pain.
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