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Artificial site-specific RNA endonuclease as new Myotonic Dystrophy therapeutics

人工位点特异性 RNA 核酸内切酶作为强直性肌营养不良的新疗法

基本信息

批准号：
8313940
负责人：
Zefeng Wang
金额：
$ 19.58万
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：
2011
资助国家：
美国
起止时间：
2011-08-08 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8313940
关键词：
3&apos Untranslated Regions Adopted Adult Affect Affinity Alternative Splicing Animal Model Binding Binding Proteins Biological Assay Cataract Cell Nucleus Cell model Cells Cleaved cell Clinical Code Cultured Cells Development Disease Endocrine system Endoribonucleases Engineering Enzymes Eye Functional RNA Future Gene Expression Profile Gene Mutation Genes Heart Human Hybrids In Vitro Inborn Genetic Diseases Individual Introns Messenger RNA Methodology Methods Modeling Molecular Mus Muscle Muscle function Muscular Dystrophies Myocardium Myotonic Dystrophy Nuclear Nucleotides Oligonucleotides Patients Pattern Proteins RNA RNA Binding RNA Sequences RNA Splicing RNA-Binding Proteins RNA-Protein Interaction Ribonucleases SERCA1 Site Skeletal Muscle Specificity Spliced Genes Staging Symptoms Testing Therapeutic Transgenic Organisms Yeasts adeno-associated viral vector base blind design effective therapy endoribonuclease gene therapy heart function mouse model novel novel strategies novel therapeutic intervention synthetic enzyme

项目摘要

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.

描述(由申请人提供)：强直性肌营养不良(dystrophia myotonica，DM)是成人中最常见的肌肉营养不良形式，全世界每8500人中就有一人受到影响。DMPK基因3‘非编码区(DM1)的(CUG)n重复序列或ZNF9(DM2)基因内含子的(CCUG)n重复序列是导致DM的基因突变。这种非编码的RNA重复序列结合和隔离肌肉缺陷蛋白，或者改变CUG结合蛋白的表达，这些结合蛋白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。目前还没有治愈糖尿病的方法，尽管这种疾病的并发症，包括心脏问题和白内障，可以治疗和缓解。分子治疗策略，如恢复肌肉盲蛋白或针对RNA重复序列的反义寡核苷酸的传统基因治疗，已经产生了一些有希望的结果。然而，传统的基因治疗方法受到这样一个事实的限制，即RNA重复可以对多个剪接因子的表达产生积极或消极的影响，因此恢复一个基因不能有效地扭转症状。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正在扩展的RNA重复序列，这是我们实验室首次设计的人工位点特异性RNA核酸酶(Asres)。这种人工酶是由RNA结合模块(PUM1的PUF结构域)和内切核酸酶结构域(SMG6的PIN结构域)构成的，该模块专门设计用于识别任何8-NT序列，并能有效地切割RNA。我们将设计新的Asres，它可以特异性地结合和切割积累有毒RNA的细胞核中扩展的RNA重复序列。我们将重点关注DM1，这是强直性肌营养不良最常见和最严重的形式。具体地说，我们将首先识别PUF结构域的C结合代码，并设计专门与(CUG)n重复序列结合的新PUF结构域，然后使用这些PUF生成设计者Asre。我们将确定ASRE在DM1细胞中的表达是否能恢复CUG结合蛋白1(CUGBP1)和肌盲样1(MBNL1)的正常表达水平和定位模式。此外，我们还将检查HASH治疗是否可以逆转患者和转基因DM1小鼠(如CIC-1、cTnT和SERCA1)受影响的基因错误剪接。最后，我们将使用mRNA-SEQ来确定ASRE处理如何影响DM1细胞中所有基因的表达和剪接。这些信息将有助于下一阶段ASRE在DM1小鼠模型和DM1患者中的应用。总而言之，我们的研究将为糖尿病治疗的新的治疗方法奠定基础。与能够有效地将基因输送到人类肌肉和心脏的AAV载体相结合，这种独特的方法将提供一种有效的治疗方法，可以在DM1动物模型中测试，并最终在DM1患者中进行测试。