Differential Transgene Expression Profiles in Rat Brain, Using rAAV2/1 Vectors with Tetracycline-Inducible and Cytomegalovirus Promoters
Differential Transgene Expression Profiles in Rat Brain, Using rAAV2/1 Vectors with Tetracycline-Inducible and Cytomegalovirus Promoters
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DOI：
10.1089/hum.2008.099
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发表时间：
2008-11-01
影响因子：
4.2
通讯作者：
Tenenbaum, Liliane
中科院分区：
文献类型：
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作者：
Bockstael, Olivier;Chtarto, Abdelwahed;Tenenbaum, Liliane
立体定向载体定位后转基因表达在中枢神经系统的生物分布是关系到神经系统疾病基因治疗安全性的重要问题。用四环素诱导表达盒(Teton)与巨细胞病毒(CMV)启动子在大鼠黑质纹状体通路中比较了rAAV2/1载体(含血清1型病毒衣壳伪分型的重组腺相关病毒载体)转基因表达的细胞特异性。注射后，虽然绿色荧光蛋白(GFP)主要在两个载体的神经元中表达，但在CMV和Teton载体中，DARPP-32阳性投射神经元和小白蛋白阳性中间神经元的相对比例分别为13：1和2：1。投射到苍白球的DARP32阳性神经元在两种载体上均呈GFP强阳性，而投射到黑质网状部的DARP32阳性神经元在CMV载体上标记有效，而在Teton载体上标记较差。两种载体在脑室下区均可见大量GFP阳性细胞。然而，在嗅球(OB)中，CMV载体可观察到GFP阳性神经元，而Teton载体则未观察到。我们的结论是，在遥远的区域(SN和OB)缺乏大量的转基因产物构成了AAV2/1-Teton载体用于纹状体基因治疗的安全优势。中脑注射可通过Teton载体在酪氨酸羟基酶阳性神经元中选择性表达GFP，而在CMV载体中，GFP阳性细胞覆盖了中脑的广泛区域。GFP蛋白的生物分布与转录本一致，而与病毒基因组不一致。我们的结论是，rAAV2/1-Teton载体构成了一种有趣的工具，可以在中脑多巴胺能神经元中特异地表达转基因。
The biodistribution of transgene expression in the CNS after localized stereotaxic vector delivery is an important issue for the safety of gene therapy for neurological diseases. The cellular specificity of transgene expression from rAAV2/1 vectors (recombinant adeno-associated viral vectors pseudotyped with viral capsids from serotype 1) using the tetracycline-inducible (TetON) expression cassette in comparison with the cytomegalovirus (CMV) promoter was investigated in the rat nigrostriatal pathway. After intrastriatal injection, although green fluorescent protein (GFP) was expressed mainly in neurons with both vectors, the relative proportions of DARPP-32-positive projection neurons and parvalbumin-positive interneurons were, respectively, 13: 1 and 2: 1 for the CMV and TetON vectors. DARP32-positive neurons projecting to the globus pallidus were strongly GFP positive with both vectors, whereas those projecting to the substantia nigra pars reticulata (SNpr) were efficiently labeled by the CMV vector but poorly by the TetON vector. Numerous GFP-positive cells were evidenced in the subventricular zone with both vectors. However, in the olfactory bulb (OB), GFP-positive neurons were observed with the CMV vector but not the TetON vector. We conclude that the absence of significant amounts of transgene product in distant regions (SN and OB) constitutes a safety advantage of the AAV2/1-TetON vector for striatal gene therapy. Midbrain injections resulted in selective GFP expression in tyrosine hydroxylase-positive neurons by the TetON vector whereas with the CMV vector, GFP-positive cells covered a widespread area of the midbrain. The biodistribution of GFP protein corresponded to that of the transcripts and not of the viral genomes. We conclude that the rAAV2/1-TetON vector constitutes an interesting tool for specific transgene expression in midbrain dopaminergic neurons.