Human Cell Assay Core

人体细胞检测核心

• 批准号: 10668163
• 负责人: David M Gamm
• 金额: $ 167.98万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-16 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10668163
• 关键词: Acceleration; Adverse event; Affect; Aftercare; Alleles; Animal Model; Biological Assay; Biological Markers; Biological Models; Biomedical Engineering; Biopsy; Blood; CLCA2 gene; Calcium; Cell physiology; Cells; Cellular Assay; Chloride Channels; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; DNA Sequence; Data; Disease; Dose; Electrophysiology (science); Epigenetic Process; Epithelial Cells; Exposure to; Eye; Frequencies; Gene therapy trial; Genes; Genetic Transcription; Genome; Genomics; Genotype; Goals; Guide RNA; Human; Image; Inherited; Innate Immune Response; Investigational Drugs; Ion Channel; Knock-in; Lead; Leber' s amaurosis; Libraries; Measurement; Modification; Mutation; Optics; Organoids; Outcome; Pathogenicity; Patients; Phase; Phenotype; Photoreceptors; Phototransduction; Pluripotent Stem Cells; Preparation; Publishing; Reproducibility; Retina; Route; Safety; Skin; Source; Structure of retinal pigment epithelium; TP53 gene; Testing; Therapeutic; Time; Tissues; Translations; Universities; Vision; Vitelliform macular dystrophy; Wisconsin; Work; autosome; biological systems; biomarker identification; biomarker panel; cell type; cellular targeting; cost; first-in-human; gene regulatory network; gene therapy; genome editing; genome integrity; human pluripotent stem cell; in vivo; induced pluripotent stem cell; insight; metabolic imaging; mutant; next generation sequencing; off-target site; pre-clinical; programs; response; retinal progenitor cell; stem cell biology; stem cell model; therapeutic candidate; tool; transcriptomics

PROJECT SUMMARY/ABSTRACT – HUMAN CELL ASSAYS CORE The overarching goal of this Core is to support the preclinical and IND-enabling studies in human cells with the therapeutic leads generated in Projects 1-3. Human pluripotent stem cells (hPSCs) are a unique cell source to generate retinal cells, tissue, and organoids. The unique attributes of hPSC model systems are particularly critical for diseases that have high genotypic diversity, like channelopathies, as generating an animal model with a knocked-in human allele for every patient mutation would be time and cost prohibitive and – even if accomplished – would not provide a platform to assess potential off-target effects. Assays using hPSC-derived cells, organoids, and tissues containing pathogenic mutations can provide patient-relevant information regarding (1) the efficiency of intended on-target editing of mutant alleles, (2) the frequency of unintended on-target genomic editing outcomes (e.g., large deletions, translocations) and off-target genomic editing (in both the target cell type and other cell types exposed to the therapeutic product), and (3) the strength of functional rescue and adverse responses. Because our proposed subretinal route of nonviral editor administration will likely deliver payload to photoreceptors (PRs) as a cellular off-target, we developed an imaging and single cell transcriptional pipeline to identify biomarkers of adverse events in PR-containing hPSC-derived retinal organoids (ROs), pioneered by our team. We will apply these tools to profile potential adverse events in PRs, including changes in phototransduction, p53 response, and innate immune response. We plan to produce iPSC-RPE suitable for on- and off-target analysis of therapeutic editors, perform electrophysiology assays for functional analysis of iPSC-RPE treated with therapeutic candidates, and produce hPSC-ROs for profiling potential adverse events in human photoreceptors exposed to therapeutic candidates. The expected outcomes of HCA Core activities include continuous provision or execution of disease-relevant, rigorous, and reproducible human cell products and ion channel assays. We expect to leverage a world-class stem cell biology and bioengineering community at the University of Wisconsin-Madison to pave the way to use hPSCs in IND-enabling studies in the genome editing field. If successful, this effort could reduce the need for preclinical animal models and provide relevant safety and efficacy information to accelerate the translation of genome editing into first-in- human trials.

项目摘要/摘要 - 人类细胞分析核心 该核心的总体目标是支持人类细胞中的临床前和核对研究 项目1-3中产生的治疗铅。人多能干细胞（HPSC）是独特的细胞来源 产生残留的细胞，组织和器官。 HPSC模型系统的独特属性特别是 对于具有高基因型多样性（如通道病）的疾病至关重要，因为 每个患者突变的人类等位基因都将是时间和成本，即使 完成的 - 不会提供评估潜在脱靶效应的平台。使用HPSC衍生的测定 细胞，器官和含有致病突变的组织可以提供有关患者的信息 （1）突变等位基因的预期目标编辑的效率，（2）意外目标的频率 基因组编辑结果（例如，大删除，易位）和靶向基因组编辑（在两个目标中， 细胞类型和其他暴露于治疗产品的细胞类型），以及（3）功能救援的强度和 不良反应。因为我们提议的非病毒编辑管理下的视网膜下路线可能会交付 有效载荷对光感受器（PR）作为细胞脱离目标，我们开发了一个成像和单细胞转录 识别含PR的HPSC衍生的视网膜器官（ROS）中不良事件的生物标志物的管道， 由我们的团队开创。我们将把这些工具应用于PR中的潜在不良事件，包括更改 在光转导，p53反应和先天免疫反应中。我们计划生产适合的IPSC-RPE 治疗编辑器的靶向分析和非目标分析，对功能性进行电生理测定 用治疗候选者处理的IPSC-RPE分析，并产生HPSC-ROS以进行分析潜力 暴露于治疗候选者的人类光感受器中的不良事件。预期的结果 HCA核心活动包括连续提供或执行与疾病相关的，严格和可重复的 人类细胞产品和离子通道分析。我们希望利用世界一流的干细胞生物学和 威斯康星大学麦迪逊分校的生物工程社区，以铺平使用HPSC的方式 基因组编辑领域的研究。如果成功，这种努力可以减少对临床前动物模型的需求 并提供相关的安全性和效率信息，以加速基因组编辑为首先的转换 人类试验。