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.

项目总结