Targeting central serotonergic neurons with lentiviral vectors based on a transcriptional amplification strategy
Targeting central serotonergic neurons with lentiviral vectors based on a transcriptional amplification strategy
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DOI：
10.1038/gt.2009.7
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发表时间：
2009-05-01
期刊：
影响因子：
5.1
通讯作者：
Kasparov, S.
中科院分区：
文献类型：
--
作者：
Benzekhroufa, K.;Liu, B-H;Kasparov, S.
许多病理过程与中枢5-羟色胺(5-羟色胺)系统紊乱有关，但由于缺乏特异性靶向载体，通过调节5-羟色胺系统进行基因治疗的可能性尚未被探索。我们探索了色氨酸羟基酶-2(TPH-2)基因上游的序列，并产生了慢病毒载体，然后用显微注射到大鼠中缝进行了体内测试。所有大于1kb的片段(称为2TPH、3.6TPH和6.7TPH)在5HT神经元中具有高度的特异性表达，但由于太弱而无法在没有免疫染色的情况下被检测到。为了提高表达水平，采用了两步转录扩增策略，通过嵌合增强子GAL4/P65增强TPH-2启动子片段的活性。令人惊讶的是，以前发表的这一策略的实现损害了表达式的特殊性。因此，我们使用了一种新的方法，其中转基因和GAL4/p65通过内部核糖体进入位点共同表达，GAL4/p65以正反馈的方式放大表达。因此，我们产生了新的载体，这两者都是高度特异的5HT神经元和足够强大的。它们为研究5-羟色胺相关疾病的基因纠正提供了一系列新的机会。基因治疗(2009年)16681-688；DOI：10.1038/gt.2009.7；在线发布
Numerous pathological processes have been linked to disorders of the central 5-hydroxytryptamine (5HT) system but the possibility of gene therapy through modulation of 5HT system remained unexplored due to the lack of the specific targeting vectors. We explored the sequences upstream of the tryptophan hydroxylase-2 (TPH-2) gene, the key enzyme in neuronal 5HT synthesis and generated lentiviral vectors, which were then tested in vivo using microinjections into the rat raphe. All fragments longer than 1 kb (called 2TPH, 3.6TPH and 6.7TPH) drove highly specific expression in 5HT neurons which was, however too weak to be detectable without immunostaining. To enhance the level of expression, a two-step transcriptional amplification strategy was employed whereby the activity of a TPH-2 promoter fragment was potentiated by a chimeric enhancer GAL4/p65. Surprisingly, previously published implementations of this strategy compromised the specificity of expression. We therefore used a new approach where both, the transgene and GAL4/p65 are co-expressed using an internal ribosomal entry site and GAL4/p65 operates in a positive feedback manner to amplify the expression. Thus, we have generated new vectors, which are both, highly specific for 5HT neurons and sufficiently powerful. They open a range of new opportunities for enquiries into genetic correction of 5HT-related disorders. Gene Therapy (2009) 16, 681-688; doi: 10.1038/gt.2009.7; published online 12 February 2009