Therapeutic strategies for microsatellite expansion diseases using RNA-targeting CRISPR/Cas

项目摘要 簇状规则间隔短回文重复序列(CRISPR)/CAS系统已得到广泛应用 用于DNA识别，使基因组编辑和招募效应器蛋白质等应用能够 DNA影响转录或使能成像。最近，我们在Nelle等人的《细胞》杂志上展示了这一点 用含有PAM识别的修饰寡核苷酸修饰催化死亡的Cas9 序列能够结合活的哺乳动物细胞中的特定mRNAs并能够跟踪它们的运动， 为Cas蛋白的多种RNA应用打开了可能性。在这项建议中，我们的目标是开发一种 基于腺相关病毒(AAV)的微卫星扩增治疗强直性肌营养不良 以扩大CTG重复为特征的疾病，使用RNA靶向CRISPR/Cas9(RCas9)。我们会 进行我们基于AAV的治疗的体内安全性和有效性研究，并评估假设 可替代的RNA加工生物标志物可用于评估RCas9治疗强直性肌强直的疗效 营养不良。如果成功，我们将在开发针对一类人的治疗方法方面向前迈出重要的一步。 微卫星扩张性疾病。！

Project Summary The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system has become widely adopted for DNA recognition, enabling applications such as genome editing and recruiting effector proteins to DNA to affect transcription or enable imaging. Recently in Nelles et al, Cell, 2016, we showed that with modifications, catalytically dead Cas9 with a modified oligonucleotide containing the PAM recognition sequence is able to bind specific mRNAs in living mammalian cells and is able to track their movement, opening up the potential for many RNA applications of Cas proteins. In this proposal, we aim at developing a adeno-associated virus (AAV)-based therapeutic strategy for myotonic dystrophy, a microsatellite expansion disease characterized by expanded CTG repeats, using RNA-targeting CRISPR/Cas9 (RCas9). We will perform in vivo safety and efficacy studies of our AAV-based therapy and evaluate the hypothesis that alternative RNA processing biomarkers are reliable for measuring treatment efficacy of RCas9 in myotonic dystrophy. If successful, we will have taken a significant step forward in developing a treatment for a class of microsatellite expansion diseases.!