Pignataro Diego, Sucunza Diego, Vanrell Lucia, Lopez-Franco Esperanza, Dopeso-Reyes Iria G, Vales Africa, Hommel Mirja, Rico Alberto J, Lanciego Jose L, Gonzalez-Aseguinolaza Gloria
Department of Gene Therapy and Regulation of Gene Expression, Center for Applied Medical ResearchPamplona, Spain; Department of Neurosciences, Center for Applied Medical ResearchPamplona, Spain.
Department of Gene Therapy and Regulation of Gene Expression, Center for Applied Medical ResearchPamplona, Spain; Department of Neurosciences, Center for Applied Medical ResearchPamplona, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades NeurodegenerativasSpain.
Front Neuroanat. 2017 Feb 10;11:2. doi: 10.3389/fnana.2017.00002. eCollection 2017.
Adeno-associated viruses (AAVs) have become highly promising tools for research and clinical applications in the central nervous system (CNS). However, specific delivery of genes to the cell type of interest is essential for the success of gene therapy and therefore a correct selection of the promoter plays a very important role. Here, AAV8 vectors carrying enhanced green fluorescent protein (eGFP) as reporter gene under the transcriptional control of different CNS-specific promoters were used and compared with a strong ubiquitous promoter. Since one of the main limitations of AAV-mediated gene delivery lies in its restricted cloning capacity, we focused our work on small-sized promoters. We tested the transduction efficacy and specificity of each vector after stereotactic injection into the mouse striatum. Three glia-specific AAV vectors were generated using two truncated forms of the human promoter for glial fibrillar acidic protein (GFAP) as well as a truncated form of the murine GFAP promoter. All three vectors resulted in predominantly glial expression; however we also observed eGFP expression in other cell-types such as oligodendrocytes, but never in neurons. In addition, robust and neuron-specific eGFP expression was observed using the minimal promoters for the neural protein BM88 and the neuronal nicotinic receptor β2 (CHRNB2). In summary, we developed a set of AAV vectors designed for specific expression in cells of the CNS using minimal promoters to drive gene expression when the size of the therapeutic gene matters.
腺相关病毒（AAV）已成为中枢神经系统（CNS）研究和临床应用中极具前景的工具。然而，将基因特异性递送至目标细胞类型对于基因治疗的成功至关重要，因此正确选择启动子起着非常重要的作用。在此，使用了携带增强型绿色荧光蛋白（eGFP）作为报告基因的AAV8载体，该报告基因受不同中枢神经系统特异性启动子的转录控制，并与一个强大的组成型启动子进行比较。由于AAV介导的基因递送的主要限制之一在于其有限的克隆能力，我们将工作重点放在小型启动子上。我们在立体定向注射到小鼠纹状体后测试了每种载体的转导效率和特异性。使用两种截短形式的人类胶质纤维酸性蛋白（GFAP）启动子以及截短形式的小鼠GFAP启动子生成了三种胶质细胞特异性AAV载体。所有三种载体均主要导致胶质细胞表达；然而，我们也在其他细胞类型如少突胶质细胞中观察到eGFP表达，但在神经元中从未观察到。此外，使用神经蛋白BM88和神经元烟碱受体β2（CHRNB2）的最小启动子观察到了强大且神经元特异性的eGFP表达。总之，当治疗基因的大小很重要时，我们开发了一组AAV载体，设计用于使用最小启动子在中枢神经系统细胞中进行特异性表达以驱动基因表达。
