AAV capsid-promoter interactions determines CNS cell selective gene expression in vivo

摘要 腺相关病毒（AAV）载体在最近的CNS临床试验中占据突出的作用，特别是在 涉及AAV血清型9（AAV 9）用于单基因遗传疾病，如脊髓性肌萎缩和巨轴突的用途 神经病（Mendell等人，2018; Bailey等人，2018年）。考虑到选择性细胞转导的需要， 修饰、细胞特异性启动子和细胞特异性增强子都已证明成功实现了特异性的免疫调节。 载体性质（Asokan等人，2012; Dimidschstein等人，2016; Grimm and Buning，2017）.在最近接受的 在本论文中，我们建立了AAV 9衣壳和不同组成型之间先前未知的相互作用。 启动子，即直接影响CNS中细胞特异性基因表达的能力。使用相同的转基因 而AAV 9衣壳、CBA启动子驱动的基因表达在大鼠中表现出显性神经元基因表达 纹状体，但当基因表达由截短的Cbh启动子驱动时，基因表达显著增加， 转移到纹状体少突胶质细胞。此外，在氨基酸序列之后含有六个谷氨酸插入的AAV 9嵌合体， VP 1/2中139例显示CBA和Cbh驱动基因表达的少突胶质细胞基因表达，而VP 1/2中6例显示CBA和Cbh驱动基因表达的少突胶质细胞基因表达。 在相同位点插入丙氨酸将Cbh驱动的基因表达逆转回神经元。近日，初步 研究结果揭示了类似的AAV 9衣壳与JetI合成启动子的相互作用， 体内表达。考虑到这种captain-启动子相互作用的高度新颖性，体外研究将定义captain-启动子相互作用。 细胞基因表达中谷氨酸和丙氨酸转移的基础机制，包括衣壳构象， 细胞内运输，RNA剪接和VP 1，2，3相互作用。体内研究将评估那些启动子元件， 有助于相互作用，以及影响体内细胞基因变化的特异性AAV 9衣壳元件 表情考虑到AAV 9载体的众多应用，这些发现将大大推进我们的研究。 这是对基本的captain-启动子相互作用的理解，并证明对基于AAV 9的基因疗法的未来设计至关重要。

ABSTRACT Adeno-associated virus (AAV) vectors occupy a prominent role in recent CNS clinical trials particularly with respect to the use of AAV serotype 9 (AAV9) for single gene genetic disorders, such as spinal muscular atrophy and giant axon neuropathy (Mendell et al., 2018; Bailey et al., 2018). Given the need for selective cellular transduction, capsid modification, cell specific promoters and cell specific enhancers all have demonstrated success in achieving specific vector properties (Asokan et al., 2012; Dimidschstein et al., 2016; Grimm and Buning, 2017). In a recently accepted manuscript, we established a previously unknown interaction between the AAV9 capsid and different constitutive promoters, namely the ability to directly influence cell specific gene expression in the CNS. Using identical transgenes and the AAV9 capsid, CBA promoter driven gene expression exhibited a dominant neuronal gene expression in the rat striatum, but when gene expression was driven by the truncated Cbh promoter, gene expression was significantly shifted to striatal oligodendrocytes. Moreover, an AAV9 chimera containing six glutamate insertions after amino acid 139 in VP1/2 exhibited oligodendrocyte gene expression for both CBA and Cbh driven gene expression while a six alanine insertion in the same site reversed the Cbh driven gene expression back to neurons. Recently, preliminary findings revealed a similar AAV9 capsid interaction with the JetI synthetic promoter that influenced cellular gene expression in vivo. Given the highly novel nature of this capsid-promoter interaction, in vitro studies will define the mechanisms that underlie the glutamate and alanine shifts in cellular gene expression, including capsid conformation, intracellular trafficking, RNA splicing and VP1,2,3 interactions. In vivo studies will assess those promoter elements that contribute to the interactions, and specific AAV9 capsid elements that influence changes in in vivo cellular gene expression. Given the numerous applications of AAV9 vectors, the findings should significantly advance our understanding of basic capsid-promoter interactions and prove crucial to future design of AAV9 based gene therapies.