A Novel Technology for Full-Length Gene Replacement Therapy of Duchenne Muscular Dystrophy

杜氏肌营养不良症全长基因替代治疗新技术

基本信息

批准号：
10390188
负责人：
Ryan Hon Hean Hsu
金额：
$ 4.46万
依托单位：
SALK INSTITUTE FOR BIOLOGICAL STUDIES
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2026-01-13
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10390188
关键词：
Affect Age Base Pairing Behavioral Binding Biochemical Bioinformatics Biological Assay Biological Markers Birth CRISPR/Cas technology Capsid Cardiac Catalytic RNA Cells Cessation of life Clinical Treatment Clinical Trials DNA Data Dependovirus Development Disease Disease Progression Dose Duchenne muscular dystrophy Dystrophin Electrophysiology (science)Elements Emerging Technologies Engineering Enzymes Exons Foundations Future Gene Delivery Generations Genes Genetic Diseases Genetic Engineering Genetic Transcription Genetic Translation Goals Immune response In Vitro Introns Length Libraries Limb structure Mammals Measures Mechanics Mediating Molecular Monitor Morphology Mus Muscle Muscle function Mutation Nonsense Mutation Paralysed Patients Physiological Population Protein Fragment Proteins Quality of life RNA RNA Splicing Reaction Reporter Research Respiratory Diaphragm Sequence Analysis Serum Site Spliceosomes Split Genes System Technology Testing Therapeutic Therapeutic Effect Tissues Toxic effect Transcript Translations Variant Western Blotting Work cohort combinatorial design design and construction experimental study functional restoration gene replacement gene replacement therapy gene therapy immunogenicity in vivo insertion/deletion mutation liver function mRNA Expression male micro-dystrophin mini-dystrophin mouse model muscle degeneration muscular dystrophy mouse model new technology novel novel strategies preclinical trial promoter protein complex protein expression reconstitution research clinical testing skeletal systemic toxicity targeted delivery transcriptome transcriptome sequencing

项目摘要

Project Summary and Abstract Duchenne muscular dystrophy (DMD) is a genetic disease which arises from a nonsense mutation of the dystrophin gene, dmd. Dystrophin has important functions in protecting muscle from mechanical damage and mutations in the gene results in muscle degeneration. The natural progression of the disease involves muscle degeneration in the limbs leading to loss of mobility and eventual degeneration of cardiac and diaphragm muscle, leading to death. Duchenne muscular dystrophy affects 1 in 5,000 male births, renders most patients paralyzed by age 12, and on average results in death by age 26. There is no known cure and current treatments only delay disease progression or are aimed at quality-of-life improvements. Gene therapy is an emerging technology that aims to cure genetic diseases. Currently, two main approaches are being explored. Although CRISPR/Cas9 developments have advanced genetic engineering significantly in recent years, many barriers still remain to clinical treatment of diseases such as DMD, including off-target editing, insertion/deletion mutations, delivery of the large protein complex, and immunogenicity of expressing the bacterial enzyme. Gene-replacement therapy is a promising alternate and potentially curative strategy. Gene- replacement therapy delivered by Adeno-Associated Virus (AAV) offers an elegant solution by replacing, rather than editing, the defective gene. Using AAVs, exogenous DNA is delivered to target cells and stably resides extra-chromosomally. The dystrophin gene can then be subsequently expressed, functionally replacing the defective endogenous copy. Due to cargo-capacity limitations of AAVs however, current clinical testing have been restricted to highly truncated versions of dystrophin with resulting limited efficacy. Recently developed technology overcomes the cargo capacity limitation of AAVs, providing an avenue for delivery of full-length gene replacements. First, split-gene constructs are designed by dividing the full gene into fragments, flanked by regions containing both intronic and base-pairing binding sequences, where each fragment is delivered by a sub-population of an AAV cocktail. Within the cell, the expressed RNA fragments are locally stabilized by the base-pairing of the binding domains, then undergo a spliceosome-mediated joining reaction, thus concatenating the fragments into full-length mRNA for translation and restoration of functional protein. Preliminary data demonstrates the ability of this approach to deliver three-fragment fluorescent reporters in mouse muscle. The proposed research aims to develop this technology as a gene-replacement therapy through the engineering of robust constructs for dystrophin gene delivery, optimization of on- and off-target delivery both in vitro and in vivo, and characterization of both therapeutic and unintended host responses in a Duchenne Muscular Dystrophy mouse model. This project will demonstrate the therapeutic potential of a novel class of gene-replacement therapies. Further, this project lays the groundwork for future studies in higher mammals, and for a potential future treatment and cure of Duchenne Muscular Dystrophy in patients.

项目概要和摘要杜氏肌营养不良症 (DMD) 是一种由基因无义突变引起的遗传性疾病。抗肌营养不良蛋白基因，dmd。抗肌营养不良蛋白在保护肌肉免受机械损伤和基因突变会导致肌肉退化。该疾病的自然进展涉及肌肉四肢退化导致活动能力丧失并最终导致心肌和膈肌退化，导致死亡。杜氏肌营养不良症影响五千分之一的男性新生儿，导致大多数患者瘫痪到 12 岁时就会死亡，平均到 26 岁时就会死亡。目前尚无已知的治愈方法，目前的治疗方法只能延迟疾病进展或旨在改善生活质量。基因治疗是一种旨在治愈遗传性疾病的新兴技术。目前主要有两种方法正在探索中。尽管 CRISPR/Cas9 的发展显着推进了基因工程近年来，DMD等疾病的临床治疗仍存在诸多障碍，包括脱靶编辑、插入/缺失突变、大蛋白复合物的递送以及表达的免疫原性细菌酶。基因替代疗法是一种有前途的替代疗法，并且具有潜在的治愈策略。基因- 腺相关病毒（AAV）提供的替代疗法提供了一种优雅的解决方案，通过替代而不是而不是编辑有缺陷的基因。使用 AAV，将外源 DNA 递送至靶细胞并稳定驻留染色体外。抗肌营养不良蛋白基因随后可以表达，在功能上取代有缺陷的内源复制。然而，由于 AAV 的载货能力有限，目前的临床测试已经仅限于高度截短的肌营养不良蛋白，从而导致功效有限。最近开发的技术克服了 AAV 的载货能力限制，为提供全长基因替换。首先，通过将完整基因分成片段，两侧是包含内含子和碱基配对结合序列的区域，其中每个片段由 AAV 鸡尾酒的一个亚群提供。在细胞内，表达的RNA片段在局部通过结合域的碱基配对稳定，然后进行剪接体介导的连接反应，从而将片段连接成全长 mRNA，用于翻译和恢复功能蛋白。初步数据表明该方法能够在以下情况下提供三片段荧光报告基因：老鼠的肌肉。拟议的研究旨在通过以下方式开发这项技术作为基因替代疗法抗肌营养不良蛋白基因递送的稳健构建体的工程设计、靶向和脱靶递送的优化体外和体内，以及 Duchenne 中治疗性和非预期宿主反应的表征肌营养不良症小鼠模型。该项目将展示一类新型药物的治疗潜力基因替代疗法。此外，该项目为未来高等哺乳动物的研究奠定了基础，未来可能治疗和治愈杜氏肌营养不良症患者。