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

AAV衣壳启动子相互作用决定CNS细胞体内选择性基因表达

基本信息

批准号：
10317110
负责人：
Thomas J. McCown
金额：
$ 38.88万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-15 至 2025-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10317110
关键词：
Alanine Amino Acids Back Capsid Cell Nucleus Cells Cellular Tropism Central Nervous System Diseases Chimera organism Clinic Clinical Clinical Trials Complement Confocal Microscopy Corpus striatum structure Dependovirus Elements Enhancers Exhibits Future Gene Expression Genes Genetic Diseases Genome Glutamates Immunohistochemistry In Vitro Introns Length Location Manuscripts Minor Modification Molecular Conformation Mutagenesis Nature Neurons Oligodendroglia Outcome Pattern Property Publishing RNA Splicing Rattus Role Serotyping Site Spinal Muscular Atrophy Techniques Therapeutic Transgenes Translating Tropism Virus Diseases adeno-associated viral vector base cellular transduction clinical translation design gene therapy giant axonal neuropathy in vivo mutant nonhuman primate novel promoter receptor binding success trafficking vector

项目摘要

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.

抽象的腺相关病毒（AAV）载体在最近的中枢神经系统临床试验中发挥着重要作用，特别是在以下方面使用 AAV 血清型 9 (AAV9) 治疗单基因遗传性疾病，例如脊髓性肌萎缩症和巨轴突神经病（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子都已证明在实现特异性方面取得了成功向量属性（Asokan 等人，2012；Dimidschstein 等人，2016；Grimm 和 Buning，2017）。在最近接受的手稿中，我们建立了 AAV9 衣壳和不同组成型之间以前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，基因表达显着降低转移到纹状体少突胶质细胞。此外，AAV9嵌合体在氨基酸后含有六个谷氨酸插入物 VP1/2 中的 139 个细胞表现出 CBA 和 Cbh 驱动基因表达的少突胶质细胞基因表达，而 6 个细胞则表现出少突胶质细胞基因表达。在同一位点插入丙氨酸可逆转 Cbh 驱动的基因表达回到神经元。近日，初步研究结果揭示了类似的 AAV9 衣壳与影响细胞基因的 JetI 合成启动子的相互作用体内表达。鉴于这种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变化的机制，包括衣壳构象，细胞内运输、RNA 剪接和 VP1,2,3 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定 AAV9 衣壳元件表达。鉴于 AAV9 载体的众多应用，这些发现将显着推进我们的研究了解基本的衣壳启动子相互作用，并被证明对基于 AAV9 的基因疗法的未来设计至关重要。