Single Cell Profiling To Define Biomarkers Of Photoreceptor Dysfunction After Gene Editing Within PSC-Derived Organoids

在 PSC 衍生类器官中进行基因编辑后，通过单细胞分析来定义光感受器功能障碍的生物标志物

• 批准号: 10254334
• 负责人: David M Gamm
• 金额: $ 61.2万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-05 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10254334
• 关键词: 3-Dimensional; Advanced Development; Adverse effects; Adverse event; All-Trans-Retinol; Basic Science; Biological Markers; Biomedical Engineering; CRISPR/Cas technology; Cell Membrane Permeability; Cell Separation; Cell physiology; Cells; Chemicals; Coculture Techniques; Complex; Computing Methodologies; Cone; Cyclic GMP; Data; Data Set; Development; Disease; Dose; Electrophysiology (science); Functional disorder; Genes; Genome; Genomic DNA; Harvest; Human; Image; Immune; Immune response; Immunology procedure; Label; Lead; Lentivirus Vector; Light; Maps; Measures; Metabolic; Metabolism; Methods; Minority; Morphology; NADH; Network-based; Optic vesicle; Optics; Organoids; Photoreceptors; Phototransduction; Physiological; Pluripotent Stem Cells; Population; Regulator Genes; Reporter Genes; Retina; Retinal Cone; Rod; Rod Outer Segments; Second Messenger Systems; Structure; Technology; Testing; Time; Tissues; Translational Research; Tretinoin; Validation; Vertebrate Photoreceptors; Vision; Work; biomarker panel; gene discovery; genome editing; genotoxicity; human pluripotent stem cell; immunocytochemistry; in situ imaging; metabolic imaging; novel; response; retinal rods; screening; single-cell RNA sequencing; therapeutic genome editing; transcriptomics; two-photon

PROJECT SUMMARY Genome editors make targeted changes in the genome and hold great promise in both basic and translational research. Unfortunately, they often produce unwanted adverse effects, including genotoxicity, immune response, and reductions in cellular function. Therefore, screening for adverse events is essential for the development of safe genome editing therapies. Here we propose to develop a generalizable and scalable approach to define biomarkers for adverse events after delivery of a genome editor. Our strategy combines state-of-the-art, label-free optical metabolic imaging (OMI) to measure the physiological, functional, and high-content morphological status, with single cell transcriptomic profiling (scRNA-seq) and regulatory network-based methods to analyze single cell data. The inferred gene regulatory networks can be used to develop a small (~50) set of biomarkers for adverse events within functional cells. Proof-of-concept studies will focus on the retina, specifically on rod and cone photoreceptors (PR) within 3D optic vesicle (OV) organoids derived from human pluripotent stem cells (PSCs). Creation of this dataset and validation of this approach will leverage these bioengineering technologies toward the development of safer genome editing therapeutics. In Aim 1, we will adapt an existing imaging and culture platform to administer Cas9 genome editors into OVs. Cells will be edited with important PR master regulators and challenged with light and chemical perturbations to test functional phototransduction post genome editing. In Aim 2, we will discover gene regulatory networks and biomarkers associated with abnormal metabolism within normal and dysfunctional gene-edited OVs. We will perform scRNA-seq and OMI on metabolically-distinct, gene-edited OVs, and then map the gene regulatory network associated with adverse events within PRs. We plan to validate the biomarker panel with qPCR/immunocytochemistry (ICC) and electrophysiology. In Aim 3, we will test and refine the platform with novel sgRNAs and genome editors within the SCGE toolkit. And finally, in Aim 4, we will expand the platform to detect adverse events that occur only in cone PRs, which constitute a minority of PRs within the retina, yet are critical for human vision. By tackling a 3D, heterogeneous organoid culture, our approach will extend to more complex cultures. Thus, the impact of this work could be broad, with the potential to advance the development of genome editors administered to any tissue.

项目摘要 基因组编辑器在基因组中进行有针对性的改变，在基础和翻译方面都有很大的希望。 research.不幸的是，它们经常产生不想要的副作用，包括遗传毒性、免疫反应， 以及细胞功能下降。因此，筛查不良事件对于开发 安全的基因组编辑疗法。 在这里，我们建议开发一种可推广和可扩展的方法来定义不良事件的生物标志物 基因组编辑器交付后。我们的策略结合了最先进的无标记光学代谢成像技术 (OMI)用单细胞测量生理、功能和高含量形态学状态， 转录组学分析（scRNA-seq）和基于调控网络的方法来分析单细胞数据。的 推断出的基因调控网络可用于开发一小组（约50个）不良事件的生物标志物 在功能细胞中。概念验证的研究将集中在视网膜上，特别是在杆和锥 来源于人多能干细胞的3D视泡（OV）类器官内的光感受器（PR） （私营保安公司）。创建该数据集和验证该方法将利用这些生物工程技术 开发更安全的基因组编辑疗法。 在目标1中，我们将调整现有的成像和培养平台，以将Cas9基因组编辑器管理到OV中。 细胞将用重要的PR主调节剂进行编辑，并用光和化学扰动进行挑战， 测试基因组编辑后的功能性光转导。在目标2中，我们将发现基因调控网络， 与正常和功能失调的基因编辑的OV内的异常代谢相关的生物标志物。我们将 对代谢上不同的基因编辑的OV进行scRNA-seq和OMI，然后绘制基因调控序列。 与PR内不良事件相关的网络。我们计划验证生物标志物面板， qPCR/免疫细胞化学（ICC）和电生理学。在目标3中，我们将使用新颖的方法测试和改进该平台 SCGE工具包中的sgRNA和基因组编辑器。最后，在目标4中，我们将扩展平台以检测 仅发生在视锥PR中的不良事件，视锥PR构成视网膜内的少数PR，但仍是关键的 for human人的vision视力. 通过处理3D异质类器官培养，我们的方法将扩展到更复杂的培养。因此 这项工作的影响可能是广泛的，有可能推动基因组编辑器的发展 施用于任何组织。