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：
10573146
负责人：
Ryan Hon Hean Hsu
金额：
$ 4.62万
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/10573146
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 Goals Immune response In Vitro Introns Length Libraries Limb structure Mammals Measures Mechanics Mediating Molecular Monitor Morphology Mus Muscle Muscle function Mutation Myocardium Nonsense Mutation Paralysed Patients Physiological 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 improved in vivo insertion/deletion mutation liver function mRNA Expression mRNA Translation male mdx mouse 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 technology validation 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.

项目摘要和摘要 Duchenne肌肉营养不良症（DMD）是一种遗传疾病，是由胡说八道引起的肌营养不良基因，DMD。肌营养不良蛋白在保护肌肉免受机械损伤和基因的突变导致肌肉变性。疾病的自然发展涉及肌肉肢体变性导致迁移率丧失和心脏和隔膜肌肉的最终变性，导致死亡。 Duchenne肌肉营养不良会影响5,000个男性的1分，使大多数患者瘫痪到12岁，平均而到26岁时死亡。没有已知的治愈方法，当前治疗只延迟疾病的进展或针对生活质量的改善。基因治疗是一种旨在治愈遗传疾病的新兴技术。目前，两种主要方法正在探索。尽管CRISPR/CAS9的开发已大大提高了基因工程近年来，许多障碍仍在临床治疗DMD等疾病的临床治疗中，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希望的替代和潜在的治愈策略。基因- 腺相关病毒（AAV）提供的替代疗法通过更换而替代，而不是而不是编辑，有缺陷的基因。使用AAVS，将外源性DNA传递到靶细胞，稳定居住外染色体。然后可以表达肌营养不良蛋白基因，从功能上代替内源性副本有缺陷。但是，由于AAV的货物容量限制，当前的临床测试具有仅限于高度截短的肌营养不良蛋白，导致有限的功效。最近开发的技术克服了AAV的货物容量限制，为递送全长基因替代品。首先，通过将完整基因分为碎片，侧翼是包含内含子和基生成结合序列的区域，其中每个片段通过AAV鸡尾酒的子种群交付。在细胞内，表达的RNA片段在局部通过结合结构域的碱基对稳定，然后经历剪接体介导的连接反应，因此，将片段连接到全长mRNA中，以翻译和恢复功能蛋白。初步数据证明了这种方法在小鼠肌肉。拟议的研究旨在通过通过可肌营养不良蛋白基因递送的稳健构建体的工程，优化靶向和脱靶的递送体外和体内，以及在杜钦（Duchenne）中的治疗和意外宿主反应的表征肌肉营养不良小鼠模型。该项目将展示新型类别的治疗潜力基因替代疗法。此外，该项目为在上级哺乳动物中的未来研究奠定了基础，为了使未来的治疗和治愈患者的杜钦肌营养不良。