Human Cell Assay Core

项目摘要/摘要-人体细胞分析核心 这个核心的首要目标是支持人类细胞的临床前和使能IND的研究 在项目1-3中产生的治疗线索。人类多能干细胞是一种独特的细胞来源 产生视网膜细胞、组织和器官。HPSC模型系统的独特属性尤其是 对于具有高度基因多样性的疾病，如通道病，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令人望而却步的时间和成本-即使 已完成--不会提供评估潜在偏离目标影响的平台。使用hPSC派生的分析 包含致病突变的细胞、器官和组织可以提供与患者相关的信息 (1)对突变等位基因进行有意的靶上编辑的效率，(2)非预期靶上编辑的频率 基因组编辑结果(例如，大量缺失、易位)和非目标基因组编辑(在两个目标中 细胞类型和暴露于治疗产品的其他细胞类型)，以及(3)功能抢救和 不良反应。因为我们提出的视网膜下非病毒编辑管理路线很可能会提供 光感受器的有效载荷作为细胞外靶点，我们发展了一种成像和单细胞转录 在含有PR的hPSC衍生的视网膜有机物(ROS)中识别不良事件的生物标志物的管道， 由我们的团队首创。我们将应用这些工具来描述PR中的潜在不良事件，包括变化 光转导、P53反应和先天免疫反应。我们计划生产适用于以下产品的IPSC-RPE 治疗编辑的目标上和目标外分析，执行功能的电生理学测试 分析治疗候选的IPSC-RPE，并产生用于分析潜力的hPSC-ROS 暴露在候选治疗药物中的人类光感受器的不良事件。的预期结果 HCA的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 人体细胞产物和离子通道分析。我们希望利用世界级的干细胞生物学和 威斯康星大学麦迪逊分校生物工程界为在IND中使用hPSCs铺平道路 基因组编辑领域的研究。如果成功，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，以加速基因组编辑向第一位基因组编辑的转化。 人体试验。

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.