Using functional readouts from engineering models of innervated skeletal muscle to assess the efficacy of CRISPR-based c9orf72 ALS gene therapies

使用受神经支配的骨骼肌工程模型的功能读数来评估基于 CRISPR 的 c9orf72 ALS 基因疗法的功效

• 批准号: 10653223
• 负责人: Alec Simon Tulloch Smith
• 金额: $ 8.83万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10653223
• 关键词: ALS patients; Acceleration; Activities of Daily Living; Address; Agonist; Alleles; Amyotrophic Lateral Sclerosis; Animal Model; Applications Grants; Basic Science; Behavior; Biological Assay; C9ORF72; CRISPR/Cas technology; Cells; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Collaborations; Communication; Complex; Coupled; DNA; Data; Defect; Development; Disease; Engineering; Ensure; Etiology; Excision; Functional disorder; Funding; Future; Gene Silencing; Genes; Genetic; Genome; Hereditary Disease; Human; In Vitro; Inherited; Longitudinal Studies; Magnetism; Measures; Mediating; Modality; Modeling; Monitor; Motor Neurons; Movement; Muscle; Muscle Contraction; Mutation; Nature; Neurodegenerative Disorders; Neuromuscular Diseases; Neuromuscular Junction; Neuronal Differentiation; Neurons; Neuropathy; Non-Invasive Detection; Patient-Focused Outcomes; Patients; Performance; Peripheral; Phenotype; Population; Real-Time Systems; Recovery of Function; Regulation; Research; Research Personnel; Skeletal Muscle; Synapses; System; Techniques; Technology; Testing; Therapeutic; Therapeutic Intervention; Time; Tissue Engineering; Tissues; Toxic effect; Transgenes; Translations; Treatment Efficacy; Treatment Protocols; Untranslated RNA; Validation; Work; antagonist; base editing; bench to bedside; cholinergic; clinical development; comparative; disease-causing mutation; drug efficacy; efficacy evaluation; efficacy testing; experimental study; first-in-human; flexibility; functional decline; functional restoration; gene correction; gene therapy; human disease; human model; human tissue; in vivo; induced pluripotent stem cell; insight; loss of function; muscle engineering; mutant; neuromuscular; neuromuscular function; new therapeutic target; novel; novel therapeutics; optogenetics; organ on a chip; pre-clinical; preclinical evaluation; predictive modeling; preservation; programs; response; risk variant; screening; sensor; success; technology validation; theories; therapeutic genome editing; therapy development; translational potential

PROJECT SUMMARY/ABSTRACT Gene therapies employing CRISPR-Cas9-mediated genetic editing techniques have the potential to cure a wide range of inheritable disorders, including amyotrophic lateral sclerosis (ALS). However, identifying edits capable of neutralizing disease-causing mutations is a pressing issue. A critical bottleneck in the translation of novel gene therapies to clinical trials is a lack of human models capable of producing functional metrics that correlate with patient outcomes and provide predictive data with which to guide subsequent in vivo experiments. For ALS and other neuromuscular disorders, the complexity associated with generating mature and functionally competent neuromuscular junctions (NMJs) in culture with sufficient throughput for screening purposes is a major hindrance to this effort. The development of a multiplexed platform capable of promoting NMJ development across a parallel array of engineered muscle tissues will have a substantial positive impact on advanced therapy development, drug efficacy/toxicity screening, and mechanistic studies of neuronal and NMJ pathophysiology in ALS. Building on the PI’s work as a KL2 scholar, this project seeks to combine optogenetic motor neurons derived from ALS patient induced pluripotent stem cells (iPSCs) with a magnet-based sensing platform for non-invasively detecting engineered muscle contractions to establish a system for real-time, continuous assessment of NMJ functional decline in ALS (Aim 1). Tests with cholinergic synaptic agonists and antagonists, in terms of their ability to alter synaptic communication between cultured muscle and neurons, will be used to demonstrate the suitability of this model for assaying changes in NMJ function in vitro. Once optimized, the described system will be used to investigate multiple gene editing strategies for restoring function in C9orf72-mutant ALS; the most common inheritable form of the disorder (Aim 2). ALS patient iPSC-derived motor neurons subjected to either bi-allelic repeat excision or allele-specific C9orf72 gene inactivation will be compared for their ability to maintain NMJ function over time in co-culture with engineered muscle tissues. The non-invasive nature of our magnetic sensing system enables continuous assessment of muscle performance in response to optogenetically-controlled neuronal activation, thereby enabling longitudinal study of therapeutic efficacy and parallel assessment of multiple tissues subjected to different treatment regimens. Results from these experiments will provide a framework for further preclinical validation of novel therapies targeting peripheral neuropathic diseases as well as data to aid in the selection of which gene editing technique has the best chance of success in C9orf72 ALS patients. Validation of the technologies outlined in this proposal will represent the culmination of work started by the PI as part of the KL2 program and will form the core of the PI’s independent research going forward. Results collected from this R03 funded program will provide valuable preliminary data for the continued development of the PI’s research program and will be central to the success of subsequent federal grant applications.

项目摘要/摘要 采用CRISPR-CAS9介导的遗传编辑技术的基因疗法有可能治愈广泛的 遗传性疾病范围，包括肌萎缩性侧索硬化症（ALS）。但是，确定有能力的编辑 中和引起疾病的突变是一个紧迫的问题。新基因翻译中的关键瓶颈 临床试验的疗法是缺乏能够产生与与之相关的功能指标的人类模型 患者的结果并提供预测性数据，以指导随后的体内实验。对于ALS和 其他神经肌肉疾病，与产生成熟和功能胜任相关的复杂性 具有足够吞吐量以筛查的培养物中的神经肌肉连接（NMJ）是一个主要的障碍 为此努力。能够促进NMJ开发的多路复用平台的开发 一系列工程的肌肉时间将对先进的治疗开发产生重大积极影响， 药物效率/毒性筛查以及ALS中神经元和NMJ病理生理学的机理研究。建筑 在PI作为KL2科学的工作中，该项目试图结合源自ALS的光遗传学神经元 患者诱导多能干细胞（IPSC），具有基于磁铁的感应平台，用于非侵入性检测 设计的肌肉收缩，建立一个实时，连续评估NMJ功能的系统 ALS的下降（AIM 1）。用胆碱能突触激动剂和拮抗剂进行的测试，以改变它们的能力 培养的肌肉和神经元之间的突触交流将用于证明这一点的适合性 用于在体外测定NMJ功能变化的模型。一旦优化，描述的系统将用于 研究多种基因编辑策略，以恢复C9orf72突变的ALS中的功能；最常见的 该疾病的遗传形式（AIM 2）。 ALS患者IPSC衍生的运动神经元均受到双链接 将比较其维持NMJ的能力 随着时间的流逝，与工程肌肉组织共同培养。我们磁感应的非侵入性质 系统可以持续评估肌肉性能，以响应于胶合控制 神经元激活，从而实现了治疗效率的纵向研究和平行评估 多次接受不同的治疗方案。这些实验的结果将提供 还针对靶向周围神经性疾病的新疗法的临床前验证的框架 作为帮助选择哪种基因编辑技术在C9orf72 ALS中取得成功的最佳机会 患者。对本提案中概述的技术的验证将代表工作的高潮 PI作为KL2计划的一部分，将构成PI未来独立研究的核心。结果 从该R03资助计划中收集的将为持续开发的有价值的初步数据提供 PI的研究计划将是随后的联邦赠款申请成功的核心。