Generation of human inner ear organoids via genetic programming

通过基因编程生成人类内耳类器官

• 批准号: 10425588
• 负责人: Jing Nie
• 金额: $ 12.54万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2022-07-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10425588
• 关键词: 3-Dimensional; Affect; Aging; Binding; Bioinformatics; Bypass; CRISPR screen; CRISPR-mediated transcriptional activation; Cell Differentiation process; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Derivation procedure; Drug Screening; Epithelial; Epithelial Cells; Equilibrium; FBXO2 gene; Functional disorder; Gene Activation; Generations; Genes; Genetic; Genetic Programming; Genetic Transcription; Genome; Genomics; Hair Cells; Human; In Vitro; Knowledge; Labyrinth; Mammals; Mediating; Methodology; Methods; Modeling; Motion; Noise; Organoids; Pathway interactions; Persons; Pharmaceutical Preparations; Proteins; Protocols documentation; Recovery; Research; Self-Direction; Sensory; Sensory Hair; Structure; Supporting Cell; System; Testing; Therapeutic; TimeLine; Treatment Step; base; cell type; clinically relevant; design; disability; equilibration disorder; gene therapy; hair cell regeneration; hearing impairment; hearing loss treatment; hearing restoration; high throughput screening; human embryonic stem cell; human pluripotent stem cell; improved; in vivo; inner ear development; insight; model design; morphogens; next generation; novel; ototoxin; permanent hearing loss; progenitor; protein degradation; scale up; screening; single-cell RNA sequencing; sound; stem cells; three dimensional cell culture; tool; transcription factor

PROJECT ABSTRACT Hearing loss and balance disorders are the two most prevalent disabilities. Over 6% of people worldwide suffer from disabling hearing loss, and over 6% suffer from balance disorders. The cells responsible for sound and motion detections are the mechanosensory hair cells residing in the inner ear. As the degeneration of the hair cells is irreversible in mammals, there is currently no approved medications for sensory recovery. In recent years, derivation methods have been developed to generate inner ear cells from non-otic cells in vitro via stepwise morphogen treatment or forced activation of hair cell transcription factors (TFs). Despite providing unprecedented research opportunities, none of these current in vitro derivation approaches are suitable for high-throughput therapeutic discoveries due to limitations such as being difficult to scale up and relatively inefficient, or the lack of a spatially organized sensory epithelial structure. To overcome these limitations, this study aims to build a novel human inner ear organoid model by genetically converting aggregated human pluripotent stem cells (PSCs) into otic progenitor cells through CRISPR-based activation of otic progenitor TFs, followed by self-organized cellular maturation in 3D culture. A CRISPR screen will be performed to identify additional TFs that can enhance the lineage conversion efficiency. As only a single treatment step is required and the derivation protocol is otic lineage-focused, this new organoid model is expected to be more scalable and efficient than current stepwise morphogen treatment organoid models. Furthermore, as otic progenitors have been shown to be capable of autonomously generating properly organized sensory epithelium cell types in vitro, this novel organoid model is expected to harbor hair cells and supporting cells in a spatially organized manner, therefore better recapitulating the native sensory epithelium structures than the existing direct hair cell conversion models. Due to these advantages, this novel organoid model could potentially serve as a therapeutic discovery platform for screening compounds and testing gene therapy treatments for hearing loss and balance dysfunctions. In addition to establishing the new model, this study will investigate the underlying mechanism of otic differentiation by identifying the direct and indirect downstream genes of a subset of otic progenitor TFs. The identification of the transcription network that regulates the cellular identity transition from the otic progenitors towards mature sensory cell types will significantly advance our understanding of human inner ear development. Collectively, the proposed research will provide novel tools and insights in basic and translational inner ear research.

项目摘要 听力损失和平衡障碍是两种最常见的残疾。全世界有超过6%的人 超过6%的人患有平衡障碍。负责声音和 运动检测是位于内耳中的机械感觉毛细胞。因为头发的退化 细胞在哺乳动物中是不可逆的，目前还没有批准的药物用于感觉恢复。近几 多年来，已经开发了衍生方法，通过体外培养从非耳细胞产生内耳细胞。 逐步形态发生剂处理或毛细胞转录因子（TF）的强制活化。尽管提供 前所未有的研究机会，这些目前的体外衍生方法都不适合 高通量治疗发现由于诸如难以扩大规模和相对 效率低下，或缺乏空间组织的感觉上皮结构。为了克服这些局限性， 一项研究旨在通过遗传转化聚集的人类来建立一种新的人类内耳类器官模型。 通过基于CRISPR的耳祖TF活化，将多能干细胞（PSC）转化为耳祖细胞， 随后在3D培养中进行自组织细胞成熟。将进行CRISPR筛选，以确定 可以增强谱系转化效率的额外TF。由于只需要一个处理步骤， 并且衍生协议是以耳谱系为重点的，这种新的类器官模型预计更具可扩展性 并且比目前的逐步形态发生剂处理类器官模型更有效。此外，作为耳祖细胞， 已经显示能够自主产生适当组织的感觉上皮细胞类型 在体外，这种新的类器官模型有望以空间组织的方式容纳毛细胞和支持细胞， 因此，与现有的直接毛细胞相比， 转换模型。由于这些优点，这种新的类器官模型可能会成为一种新的生物学模型。 用于筛选化合物和测试听力损失基因治疗的治疗发现平台 和平衡失调除了建立新的模型外，本研究还将探讨潜在的 通过鉴定耳分化的一个子集的直接和间接下游基因， 祖TF。识别调节细胞身份转变的转录网络 耳祖细胞向成熟感觉细胞类型的转化将显著地促进我们对人类感觉细胞的理解。 内耳发育总的来说，拟议的研究将提供新的工具和见解，在基础和 翻译内耳研究。