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.

项目总结/摘要 采用CRISPR-Cas9介导的基因编辑技术的基因疗法有可能治愈广泛的癌症。 一系列遗传性疾病，包括肌萎缩侧索硬化症（ALS）。然而，识别能够 中和致病突变是一个紧迫的问题。新基因翻译中的一个关键瓶颈 临床试验的一个重要问题是缺乏能够产生与以下相关的功能指标的人类模型： 患者的结果，并提供预测数据，以指导随后的体内实验。对于ALS和 其他神经肌肉疾病，与产生成熟和功能能力相关的复杂性， 培养的神经肌肉接头（NMJ）具有足够的通量用于筛选目的是一个主要障碍 这一努力。开发一个能够促进NMJ开发的多路复用平台， 一系列工程化肌肉组织将对先进疗法的发展产生实质性的积极影响， 药物功效/毒性筛选，以及ALS中神经元和NMJ病理生理学的机制研究。建筑 在PI作为KL 2学者的工作上，该项目寻求将来自ALS的光遗传运动神经元联合收割机 患者诱导的多能干细胞（iPSC）与用于非侵入性检测的基于磁体的感测平台 设计肌肉收缩，以建立实时，连续评估NMJ功能的系统 ALS下降（目标1）。胆碱能突触激动剂和拮抗剂的试验，就其改变 培养的肌肉和神经元之间的突触通讯，将被用来证明这一适用性。 用于测定体外NMJ功能变化的模型。一旦优化，所描述的系统将用于 研究用于恢复C9 orf 72突变型ALS功能的多种基因编辑策略;最常见的 疾病的遗传形式（目的2）。ALS患者iPSC衍生的运动神经元经受双等位基因或非双等位基因。 将比较重复切除或等位基因特异性C9 orf 72基因失活对维持NMJ的能力 在与工程化肌肉组织的共培养中随时间的推移发挥作用。我们的磁感应的非侵入性 系统使得能够响应于光遗传学控制的肌肉性能的连续评估 神经元激活，从而能够纵向研究治疗效果和平行评估 多个组织接受不同的治疗方案。这些实验的结果将提供一个 进一步临床前验证靶向周围神经病性疾病的新疗法的框架 作为帮助选择哪种基因编辑技术在C9 orf 72 ALS中具有最佳成功机会的数据 患者本提案中概述的技术的验证将代表以下工作的高潮： PI作为KL 2计划的一部分，将成为PI未来独立研究的核心。结果 从R 03资助的项目中收集的数据将为继续开发 PI的研究计划，并将是中央的成功，随后的联邦拨款申请。