Ablation of the pathogenic RNA transcript associated with myotonic dystrophy, DM1

与强直性肌营养不良 (DM1) 相关的致病性 RNA 转录物的消融

基本信息

批准号：
9919285
负责人：
JOSEPH C. RUIZ
金额：
$ 22.4万
依托单位：
ENZERNA BIOSCIENCES, LLC
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9919285
关键词：
Ablation Address Adult Affect Animal Model Antisense Oligonucleotides Base Sequence Binding Biological Assay Cell Line Cell Nucleus Cells Clinical DNA Degenerative Disorder Disease Endoribonucleases Engineering Fibroblasts Functional disorder Genes Genetic Genetic Diseases Institutes Lead Legal patent Mediating Messenger RNA Molecular Muscle Muscle Weakness Muscular Dystrophies Mutation Myotonic Dystrophy Myotonic Muscular Dystrophy Nuclear Nucleotides Pathogenicity Patients Phenotype Production Protein Kinase Proteins RNA RNA Recognition Motif RNA Sequences RNA Splicing Research Ribonucleotides Route Site Specificity Technology Testing Therapeutic Transcript Untranslated Regions Viral adverse outcome base curative treatments design efficacy study genetic regulatory protein innovation lead candidate molecular phenotype muscle degeneration novel therapeutics off-patent palliative phase 2 study protein complex restoration safety study small molecule success symptom management targeted treatment therapeutic gene transcriptome transcriptome sequencing translation factor vector

项目摘要

ABSTRACT Myotonic muscular dystrophy (DMD) is a genetic disorder characterized by muscle degeneration and weakness. It is a common form of muscular dystrophy that generally begins in adulthood. Unfortunately, there are no approved curative therapies for DM1; treatments are largely palliative. The more severe form, myotonic dystrophy type 1 (DM1), is caused by the (CTG)n expansion in the 3 UTR of the dystrophia myotonica-protein kinase gene (DMPK). In DM1-affected cells, (CUG)n repeats in the DMPK mRNA specifically bind to splicing regulatory proteins, forming RNA-protein complexes that accumulate within nucleus as foci that disrupt RNA splicing and ultimately lead to cellular dysfunction. Therapeutic strategies directly targeting expanded repeats in DMPK mRNA, such as antisense oligonucleotides (ASO) or CUG-array specific small molecules, that “release” the bound splicing factors have produced promising results. However, difficulties in ASO delivery and need for lifelong administration of the ASO therapeutic remain limiting factors for ASO-based therapies. In addition, it appears that interfering the ability of CUG repeats to bind factors mitigates only a subset of the adverse consequences of the pathogenic DM1 expanded mRNA. In this proposal, we propose to our Artificial Site- Specific RNA Endonuclease (ASRE) technology to finalize the design of CUG repeat specific RNA endonuclease that may selectively eliminate the pathogenic transcript. The distinguishing feature of ASRE technology is the presence of PUF ribonucleotide binding domains that can be arranged in an array to recognize any 8- to 16-ribonucleotide sequence. In Aim 1, we will finalize the design of 10-base ASRE that specifically recognizes the (CUG)n repeat, clone the ASRE, with or without a nuclear localization sequence into a piggyBac cumate-inducible transposon vector, and transduce the constructs into fibroblasts derived from patients affected by DM1 (GM04602; Coriell Institute). This aim will seek to identify a lead ASRE candidate that can reverse the molecular phenotypes associated DM1 (e.g., accumulation of nuclear foci and aberrant splicing of muscle specific genes) and validate that nuclear expression of the ASRE gene therapeutic is required for activity. In Aim 2, we will generate AAV vectors that express the lead ASRE candidate and validate that viral transduction can rescue the phenotypic anomalies associated with DM1. Once feasibility is demonstrated, Phase II studies will focus on the use of research grade AAV ASRE stocks for efficacy and safety studies in animal models of DM1 before progressing to production of clinical grade AAV for IND enabling safety and efficacy studies of this innovative curative gene therapeutic for DM1.

抽象的强直性肌营养不良症（DMD）是一种以肌肉退化和无力为特征的遗传性疾病。是一种常见的肌营养不良症，通常始于成年期，不幸的是，目前尚无批准的治疗方法。 DM1 的治疗主要是姑息性治疗，引起更严重的 1 型强直性肌营养不良 (DM1)。在受 DM1 影响的细胞中，肌强直性营养不良蛋白激酶基因 (DMPK) 的 3 UTR 中 (CTG)n 扩展。 DMPK mRNA 中的 (CUG)n 重复序列特异性结合剪接调节蛋白，形成 RNA-蛋白复合物它们在细胞核内积累，作为破坏 RNA 剪接的焦点，最终导致细胞功能障碍。直接靶向 DMPK mRNA 中扩展重复序列的策略，例如反义寡核苷酸 (ASO) 或 CUG 阵列 “释放”结合剪接因子的特定小分子具有良好的结果，但也存在困难。 ASO 递送和终生施用 ASO 治疗的需要仍然是基于 ASO 的限制因素此外，干扰 CUG 重复结合因子的能力似乎只能减轻一部分。致病性 DM1 扩增 mRNA 的不良后果在本提案中，我们向我们的人工站点提出建议- 特异性 RNA 核酸内切酶 (ASRE) 技术最终确定了 CUG 重复特异性 RNA 核酸内切酶的设计， ASRE技术的显着特征是PUF的存在。可以排列成阵列以识别任何8至16个核糖核苷酸序列的核糖核苷酸结合域。在目标 1 中，我们将最终确定专门识别 (CUG) 重复的 10 碱基 ASRE 的设计，克隆 ASRE，带有或不带有核定位序列到piggyBac cumate诱导转座子载体中，并转导该构建体进入来自受 DM1 影响的患者的成纤维细胞（GM04602；Coriell Institute）。确定一个主要的 ASRE 候选者，它可以逆转 DM1 相关的分子表型（例如，细胞核的积累）病灶和肌肉特异性基因的异常剪接）并验证 ASRE 基因治疗剂的核表达是在目标 2 中，我们将生成表达主要 ASRE 候选者的 AAV 载体并验证该病毒。一旦可行性得到证实，转导可以挽救与 DM1 相关的表型异常。之前将重点使用研究级 AAV ASRE 库存在 DM1 动物模型中进行功效和安全性研究进展到用于 IND 的临床级 AAV 的生产，从而能够对这种创新治疗药物进行安全性和有效性研究 DM1 的基因治疗。