Artificial site-specific RNA endonuclease as new Myotonic Dystrophy therapeutics

描述（由申请人提供）：肌强直性营养不良（肌强直性营养不良，DM）是成人中最常见的肌肉营养不良形式，全球每8500人中就有1人受到影响。DM的基因突变被确定为DMPK mRNA 3' UTR中的扩展（CUG）n重复序列（DM1）或ZNF9内含子中的（CCUG）n扩展（DM2）。这些非编码RNA重复序列结合和隔离肌盲蛋白，或改变调节对肌肉和心脏功能至关重要的内源性基因的选择性剪接的CUG结合蛋白的表达。虽然糖尿病的并发症，包括心脏病和白内障，可以治疗和减轻，但目前还没有治愈糖尿病的方法。分子治疗策略，如传统的基因治疗，恢复肌肉失明蛋白或反义寡核苷酸对RNA重复产生了一些有希望的结果。然而，传统的基因治疗方法受到RNA重复可以积极或消极地影响多个剪接因子的表达这一事实的限制，因此恢复一个基因不能有效地逆转症状。另一方面，反义方法受到将寡核苷酸输送到肌肉或心脏的困难的限制。在这里，我们提出了一种新的方法来靶向和切割扩大的RNA重复与人工位点特异性RNA核酸酶（ASREs），首先在我们的实验室设计。这些人工酶由一个RNA结合模块（PUM1的PUF结构域）和一个有效切割RNA的核糖核酸内切酶结构域（SMG6的PIN结构域）组成，PUM1的PUF结构域专门用于识别任何8-nt序列。我们将设计新的ASREs，它可以特异性地结合和切割细胞核中积累有毒RNA的扩增RNA重复序列。我们将重点关注DM1，这是肌强直性营养不良最常见和最严重的形式。具体来说，我们将首先确定PUF结构域的C结合代码，并设计新的PUF结构域，专门结合（CUG）n重复序列，并进一步使用这些PUF生成设计ASREs。我们将确定ASRE在DM1细胞中的表达是否可以恢复正常的cug结合蛋白1 （CUGBP1）和肌肉盲样蛋白1 （MBNL1）的表达水平和定位模式。此外，我们将研究在患者和转基因DM1小鼠（如CIC-1、cTnT和SERCA1）中，是否可以通过ARSE治疗逆转受影响基因的错误剪接。最后，我们将使用mRNA-seq来确定ASRE治疗如何影响DM1细胞中所有基因的表达和剪接。这些信息将有助于本项目下一阶段ASRE在DM1小鼠模型和DM1患者中的应用。总之，我们的研究将为糖尿病治疗的新方法奠定基础。结合能够有效地将基因传递到人体肌肉和心脏的AAV载体，这种独特的方法将提供一种有效的治疗方法，可以在DM1动物模型中进行测试，并最终在DM1患者中进行测试。

DESCRIPTION (provided by applicant): Myotonic dystrophy (dystrophia myotonica, DM) is the most common form of muscular dystrophy in adults that affects 1 in 8500 individuals worldwide. The genetic mutations responsible for DM were identified as the expanding (CUG)n repeats in the 3' UTR of DMPK mRNA (for DM1) or the (CCUG)n expansion in the intron of ZNF9 (for DM2). Such non-coding RNA repeats bind and sequester muscleblind proteins or change the expression of the CUG binding proteins that regulate alternative splicing of endogenous genes critical to muscle and heart function. Currently there is no cure for DM, although complications of the disease, including heart problems and cataracts, can be treated and alleviated. The molecular therapeutic strategy such as conventional gene therapy that restore muscleblind protein or antisense oligos against RNA repeats had produced some promising results. However, conventional gene therapy approaches are limited by the fact that the RNA repeats can positively or negatively affect expressions of multiple splicing factors, therefore restoring one gene cannot efficiently reverse the symptoms. The antisense method, on the other hand, is limited by the difficulties of delivering oligonucleotides into muscle or heart. Here we propose a novel approach to target and cleave the expanding RNA repeat with the artificial site- specific RNA nucleases (ASREs) that were firstly engineered in our lab. Such artificial enzymes were constructed with an RNA binding module (PUF domain of PUM1) that is specifically designed to recognize any 8-nt sequence and an endoribonuclease domain (PIN domain of SMG6) that efficiently cleave RNA. We will design novel ASREs that can specifically bind and cleave expanding RNA repeats in the cell nucleus where the toxic RNAs are accumulated. We will focus on the DM1 that is the most common and severe form of myotonic dystrophy. Specifically, we will first identify the C binding code for PUF domain and engineer new PUF domains that specifically bind to the (CUG)n repeats, and further generate designer ASREs using these PUFs. We will determine if expression of ASRE in DM1 cells can restore the normal expression level and localization pattern of CUG-binding protein 1 (CUGBP1) and muscle blind-like 1 (MBNL1). In addition, we will examine if the ARSE treatment can reverse the mis-splicing of genes affected in patients and transgenic DM1 mouse (such as CIC-1, cTnT and SERCA1). Finally, we will use mRNA-seq to determine how ASRE treatment affects the expression and splicing of all genes in DM1 cells. Such information will help the future application of ASRE in DM1 mouse model and DM1 patients in the next stage of this project. Cumulatively, our studies will establish basis for a novel therapeutic approach for DM treatment. Combined with the AAV vectors that can efficiently deliver genes to human muscle and heart, this unique method will provide an effective treatment that can be tested in DM1 animal model and eventually in DM1 patients.