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

• 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
• 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

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.

摘要 强直性肌营养不良症（DMD）是一种遗传性疾病，其特征是肌肉变性和无力。它 是一种常见的肌肉萎缩症，通常开始于成年期。不幸的是目前还没有 DM1的治疗;治疗主要是姑息性的。更严重的形式，强直性肌营养不良1型（DM1），是由于 通过在肌强直性肌营养不良症蛋白激酶基因（DMPK）的3个UTR中的（CTG）n扩增。在DM1受影响的细胞中， 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 其在细胞核内积累，作为破坏RNA剪接并最终导致细胞功能障碍的病灶。治疗 直接靶向DMPK mRNA中扩增重复序列的策略，如反义寡核苷酸（ASO）或CUG阵列 特异性小分子“释放”结合的剪接因子已经产生了有希望的结果。然而，困难 在ASO递送和需要终身施用ASO治疗仍然是基于ASO的治疗的限制因素。 治疗此外，似乎干扰CUG重复序列结合因子的能力仅减轻了一个亚组的免疫应答。 致病性DM1扩增mRNA的不良后果。在这份提案中，我们建议我们的人工场地- 特异性RNA内切酶（ASRE）技术，以完成CUG重复特异性RNA内切酶的设计， 可以选择性地消除致病转录物。ASRE技术的显著特点是存在PUF 核糖核苷酸结合结构域可以排列成阵列以识别任何8-至16-核糖核苷酸序列。 在目标1中，我们将完成特异性识别（CUG）n重复序列的10碱基ASRE的设计，克隆 ASRE，具有或不具有核定位序列到piggyBac表皮诱导型转座子载体中，和 将构建体导入来源于受DM 1影响的患者的成纤维细胞（GM 04602; Coriell Institute）中。这一目标将力求 鉴定可以逆转与DM1相关的分子表型的前导ASRE候选物（例如，核积累 病灶和肌肉特异性基因的异常剪接），并证实ASRE基因治疗剂的核表达是 活动所需。在目标2中，我们将产生表达前导ASRE候选物的AAV载体，并验证病毒表达。 转导可以挽救与DM1相关的表型异常。一旦证明可行性，第二阶段研究 将专注于使用研究级AAV ASRE储备用于DM 1动物模型的有效性和安全性研究， 进展到生产临床级AAV用于IND，从而能够进行这种创新治疗药物的安全性和有效性研究。 DM1的基因治疗