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.

项目总结/摘要-人细胞试验核心 该核心的总体目标是支持人类细胞的临床前和IND使能研究， 项目1-3中产生的治疗线索。人多能干细胞（hPSC）是一种独特的细胞来源， 生成视网膜细胞、组织和类器官。hPSC模型系统的独特属性特别是 对于具有高基因型多样性的疾病，如通道病，至关重要，因为产生具有以下特征的动物模型： 对于每一个患者突变，敲入人类等位基因将是时间和成本高昂的， 完成-不会提供一个平台来评估潜在的脱靶效应。使用hPSC衍生的测定 含有致病性突变的细胞、类器官和组织可以提供与患者相关的信息， (1)突变等位基因的预期中靶编辑的效率，（2）突变等位基因的非预期中靶编辑的频率， 基因组编辑结果（例如，大的缺失、易位）和脱靶基因组编辑（在靶基因组编辑和非靶基因组编辑中）。 细胞类型和暴露于治疗产品的其他细胞类型），和（3）功能拯救的强度， 不良反应。因为我们提出的非病毒编辑器管理的视网膜下途径可能会提供 作为细胞脱靶，我们开发了成像和单细胞转录 鉴定含有PR的hPSC衍生的视网膜类器官（RO）中不良事件的生物标志物的管道， 由我们的团队开创。我们将应用这些工具描述PR中的潜在不良事件，包括变更 在光传导、p53应答和先天免疫应答中。我们计划生产适用于 治疗编辑器的靶点和脱靶分析，执行功能性电生理学测定， 分析用治疗候选物处理的iPSC-RPE，并产生hPSC-RO用于分析潜力 暴露于治疗候选物的人光感受器的不良事件。的预期成果 HCA核心活动包括持续提供或执行与疾病相关的、严格的和可复制的 人细胞产品和离子通道测定。我们希望利用世界一流的干细胞生物学， 威斯康星大学麦迪逊分校的生物工程社区，为在IND中使用hPSC铺平道路， 基因组编辑领域的研究。如果成功的话，这一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有效性信息，以加速将基因组编辑转化为先进技术。 人体试验