Human Cell Assay Core

人体细胞检测核心

基本信息

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
项目类别：
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&apos 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

项目摘要

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 – 人类细胞检测核心该核心的总体目标是支持人类细胞的临床前和 IND 研究项目 1-3 中产生的治疗线索是人类多能干细胞 (hPSC) 的独特细胞来源。 hPSC 模型系统的独特属性尤其突出。对于通道病等具有高基因型多样性的疾病至关重要，因为生成具有为每个患者突变敲入人类等位基因将是时间和成本高昂的——即使 – 已完成不会提供使用 hPSC 衍生的检测来评估潜在脱靶效应的平台。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) 突变等位基因的有意定点编辑的效率，(2) 非预期定点编辑的频率基因组编辑结果（例如，大量缺失、易位）和脱靶基因组编辑（在目标基因组中）细胞类型和暴露于治疗产品的其他细胞类型），以及（3）功能救援的强度和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产生不良反应。作为细胞脱靶，我们开发了一种成像和单细胞转录鉴定含有 PR 的 hPSC 衍生视网膜类器官 (RO) 中不良事件生物标志物的管道，我们将应用这些工具来分析 PR 中的潜在不良事件，包括变更。我们计划生产适用于光转导、p53 反应和先天免疫反应的 iPSC-RPE。治疗编辑器的靶向和脱靶分析，对功能进行电生理学分析分析用候选治疗药物治疗的 iPSC-RPE，并生成 hPSC-RO 用于分析潜力暴露于候选治疗药物的人类光感受器的不良事件。 HCA 核心活动包括持续提供或执行与疾病相关的、严格的和可重复的我们期望利用世界一流的干细胞生物学和技术。威斯康星大学麦迪逊分校生物工程界为使用 hPSC 进行 IND 铺平道路如果成功，这项工作可以减少对临床前动物模型的需求。并提供相关安全性和有效性信息，加速基因组编辑向首创转化人体试验。