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

• 批准号: 8174186
• 负责人: Zefeng Wang
• 金额: $ 16.26万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-08 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8174186
• 关键词: 3' 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

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. PUBLIC HEALTH RELEVANCE: This proposal is focused on the development of a new therapeutic approach for myotonic dystrophy treatments. Myotonic dystrophy (DM) is a dominantly inherited disorder with multisystemic clinical features affecting skeletal muscle, heart, eyes, and endocrine system. It is the most common form of muscular dystrophy in adults (affecting 1 in ~8500 people worldwide), and is caused by non-coding RNA expansion that affects the level of several important splicing factors. Currently there is no cure for DM, although complications of the disease, including heart problems and cataracts, can be treated and alleviated.

描述（由申请人提供）：强直性肌营养不良（强直性肌营养不良，DM）是成人中最常见的肌营养不良形式，全球每8500例患者中就有1例受影响。DM 1和DMPKmRNA 3' UTR的（CUG）n重复序列扩增，DM 2的（CCUG）n重复序列扩增。这种非编码RNA重复序列结合并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，包括心脏问题和白内障，可以治疗和缓解，但目前还没有治愈糖尿病的方法。分子治疗策略如常规的肌盲蛋白修复基因治疗或针对RNA重复序列的反义寡核苷酸治疗已取得了一些有希望的结果。然而，传统的基因治疗方法受到RNA重复序列可以积极或消极地影响多个剪接因子表达的事实的限制，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递送到肌肉或心脏中的困难的限制。 在这里，我们提出了一种新的方法来靶向和切割的扩增RNA重复与人工位点特异性RNA核酸酶（ASRE），首先在我们的实验室工程。这种人工酶是用RNA结合模块（PUF 1的PUF结构域）和内切核糖核酸酶结构域（SMG 6的PIN结构域）构建的，所述RNA结合模块被专门设计为识别任何8-nt序列，所述内切核糖核酸酶结构域有效切割RNA。我们将设计新的ASRE，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。我们将集中在DM 1，这是最常见和最严重的形式强直性肌营养不良症。具体来说，我们将首先识别PUF域的C绑定代码，并设计专门绑定到（CUG）n重复序列的新PUF域，并进一步使用这些PUF生成设计者ASRE。我们将确定ASRE在DM 1细胞中的表达是否可以恢复CUG结合蛋白1（CUGBP 1）和肌盲样蛋白1（MBNL 1）的正常表达水平和定位模式。此外，我们将研究ARSE治疗是否可以逆转患者和转基因DM 1小鼠中受影响基因（如CIC-1，cTnT和SERCA 1）的错误剪接。最后，我们将使用mRNA-seq来确定ASRE治疗如何影响DM 1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DM 1小鼠模型和DM 1患者中的应用。累积起来，我们的研究将为糖尿病治疗的新治疗方法奠定基础。结合可以有效地将基因递送到人类肌肉和心脏的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DM 1动物模型中进行测试，并最终在DM 1患者中进行测试。 公共卫生相关性：该提案侧重于强直性肌营养不良治疗的新治疗方法的开发。强直性肌营养不良症（DM）是一种显性遗传性疾病，具有多系统的临床特征，影响骨骼肌、心脏、眼睛和内分泌系统。它是成人肌营养不良症中最常见的形式（影响全世界约8500人中的1人），由影响几个重要剪接因子水平的非编码RNA扩增引起。虽然糖尿病的并发症，包括心脏问题和白内障，可以治疗和缓解，但目前还没有治愈糖尿病的方法。