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

项目摘要/摘要 使用CRISPR-Cas9介导的基因编辑技术的基因疗法有可能治愈广泛的 一系列遗传性疾病，包括肌萎缩侧索硬化症(ALS)。但是，识别能够进行的编辑 如何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 临床试验的治疗方法缺乏能够产生与以下因素相关的功能指标的人体模型 并提供预测性数据来指导后续的体内实验。对于ALS和 其他神经肌肉疾病，与成熟和功能能力强相关的复杂性 培养中的神经肌肉接头(NMJ)具有足够的筛选能力是一个主要障碍 为这一努力。开发能够跨并行促进NMJ发展的多路复用平台 一系列工程化的肌肉组织将对先进疗法的发展产生实质性的积极影响， 药物疗效/毒性筛选，以及ALS神经元和NMJ病理生理学的机制研究。建房 在PI作为KL2学者的工作中，这个项目试图将来自ALS的光遗传运动神经元 患者诱导多能干细胞(IPSCs)与磁感应平台的非侵入性检测 工程化肌肉收缩建立实时、连续评估NMJ功能的系统 肌萎缩侧索硬化症的下降(目标1)。胆碱能突触激动剂和拮抗剂的测试 培养的肌肉和神经元之间的突触联系，将被用来证明这一点的适用性 NMJ功能变化的体外检测模型。优化后，所描述的系统将用于 研究恢复C9orf72突变ALS功能的多种基因编辑策略；最常见的 可遗传的疾病形式(目标2)。肌萎缩侧索硬化症患者IPSC来源的运动神经元发生双等位基因之一 重复切除或等位基因特异性C9orf72基因失活将比较它们维持NMJ的能力 随着时间的推移，在与工程肌肉组织的共培养中发挥作用。我们的磁感应的非侵入性 系统能够连续评估肌肉性能，以响应光基因控制 神经元激活，从而使治疗效果的纵向研究和平行评估成为可能 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 针对周围神经性疾病的新疗法的进一步临床前验证框架 作为辅助选择哪种基因编辑技术在C9orf72 ALS中成功几率最高的数据 病人。对本提案中概述的技术的验证将代表以下项目启动工作的高潮 作为KL2计划的一部分，PI将成为未来PI独立研究的核心。结果 从R03资助的项目中收集的数据将为继续开发 这是国际和平研究所的研究计划，并将是后续联邦拨款申请成功的核心。

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.