Bockstael Olivier, Chtarto Abdelwahed, Wakkinen Janica, Yang Xin, Melas Catherine, Levivier Marc, Brotchi Jacques, Tenenbaum Liliane
Multidisciplinary Research Institute (IRIBHM), Université Libre de Bruxelles, Hôpital Erasme, Brussels, Belgium.
Hum Gene Ther. 2008 Nov;19(11):1293-305. doi: 10.1089/hum.2008.099.
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.
在局部立体定向载体递送后，转基因在中枢神经系统中的生物分布是神经疾病基因治疗安全性的一个重要问题。我们在大鼠黑质纹状体通路中研究了使用四环素诱导型（TetON）表达盒的rAAV2/1载体（用1型病毒衣壳假型化的重组腺相关病毒载体）与巨细胞病毒（CMV）启动子相比转基因表达的细胞特异性。纹状体内注射后，尽管两种载体的绿色荧光蛋白（GFP）主要在神经元中表达，但对于CMV和TetON载体，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。两种载体中，投射到苍白球的DARP32阳性神经元均强烈表达GFP，而投射到黑质网状部（SNpr）的神经元被CMV载体有效标记，但被TetON载体标记较差。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，用CMV载体可观察到GFP阳性神经元，而用TetON载体则未观察到。我们得出结论，在远处区域（SN和OB）缺乏大量转基因产物是AAV2/1 - TetON载体用于纹状体基因治疗的一个安全优势。中脑注射导致TetON载体在酪氨酸羟化酶阳性神经元中选择性表达GFP，而CMV载体则使GFP阳性细胞覆盖中脑的广泛区域。GFP蛋白的生物分布与转录本一致，而非病毒基因组。我们得出结论，rAAV2/1 - TetON载体是中脑多巴胺能神经元特异性转基因表达的一个有趣工具。
