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

摘要 腺相关病毒(AAV)载体在最近的中枢神经系统临床试验中占有重要地位，特别是在 AAV9血清型用于治疗单基因遗传性疾病，如脊髓性肌萎缩症和巨大轴突 神经病(Mendell等人，2018；Bailey等人，2018)。鉴于选择性细胞转导的需要，衣壳 修饰、细胞特异性启动子和细胞特异性增强剂都证明了在获得特异性 矢量性质(Asokan等人，2012；Dimidschstein等人，2016；Grimm和Buning，2017)。在最近接受的一份 ，我们在AAV9衣壳与不同成分之间建立了一种未知的相互作用 启动子，即直接影响中枢神经系统中细胞特异性基因表达的能力。使用相同的转基因 AAV9衣壳、CBA启动子驱动的基因表达在大鼠中表现出明显的神经性基因表达 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 向纹状体少突胶质细胞转移。此外，在氨基酸后含有6个谷氨酸插入的AAV9嵌合体 VP1/2中139例同时表现为CBA和CBH驱动的少突胶质细胞基因表达，而6例 在同一位置插入丙氨酸将CBH驱动的基因表达逆转回神经元。最近，初步的 结果显示，aav9衣壳与影响细胞基因的jeti合成启动子有类似的相互作用。 在体内表达。鉴于衣壳-启动子相互作用的高度新颖性，体外研究将定义 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， 细胞内转运、RNA剪接和VP1、2、3相互作用。体内研究将评估这些启动子元件 与影响体内细胞基因变化的AAV9衣壳元件的相互作用有关 表情。鉴于AAV9载体的大量应用，这些发现应该会显著推动我们的 了解基本的衣壳-启动子相互作用，并被证明对未来基于AAV9的基因疗法的设计至关重要。

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.