Drug Screening with a Biofrabricated 3-D Immunocompetent Skin Model for Drug Discovery in Psoriatic Disease

使用生物破碎 3D 免疫活性皮肤模型进行药物筛选，用于银屑病药物发现

• 批准号: 10249327
• 负责人: Angela M Christiano
• 金额: $ 64.12万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-28 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10249327
• 关键词: 3-Dimensional; Affect; Allogenic; Animal Model; Biological Assay; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Development; Disease; Drug Modelings; Drug Screening; Drug usage; Electrical Resistance; Endothelium; Enzyme-Linked Immunosorbent Assay; Extramural Activities; Fibroblasts; Fluorescence; Generations; Genes; Germ Cells; Goals; Histologic; IL6 gene; IL8 gene; Immunocompetent; Impairment; In Vitro; Individual; Inflammation; Inflammatory; Interleukin-17; Keratin; Lactate Dehydrogenase; Lead; Libraries; Methodology; Modeling; Molecular; Monitor; Neonatal; Organ; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Population; Procedures; Psoriasis; Reporter; Reproducibility; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Skin; Skin Tissue; Structure; System; T-Lymphocyte; TNF gene; Technology; Testing; Th1 Cells; Tissues; Toxic effect; Validation; Western Blotting; base; biofabrication; bioprinting; cytokine; disease phenotype; drug candidate; drug development; drug discovery; filaggrin; genome editing; high throughput screening; human tissue; immune function; induced pluripotent stem cell; induced pluripotent stem cell technology; innovation; keratinocyte; miniaturize; novel therapeutics; promoter; response; screening; skin disorder; small molecule; tool; transcriptome sequencing

Project Summary Psoriasis affects 2-3% of the US population. Currently, there are no high throughput screening methodologies to develop new psoriasis drugs. To meet this significant unmet pharmaceutical need, we will screen for drugs to treat this disease by using a 3D skin system established with 3D bioprinting, induced pluripotent stem cell (iPSC) reprograming and CRISPR genome-editing technologies. We successfully constructed 3D skin using exclusively iPSC-derived fibroblasts iFBs) and keratinocytes (iKCs), demonstrating the feasibility of this approach. 3D bioprinting can allow us to reproducibly construct complex structures to recapitulate human tissues/organs, and miniaturize the 3D fabricated skin for high throughput screening. Psoriasis is an inflammatory skin disorder with abnormal epidermal hyperproliferation, compromised barrier function, upregulated keratin 16 (KRT16), downregulated filaggrin (FLG) and excessive secretion of cytokines such as IL-8, which can be monitored with transepithelial/transendothelial electrical resistance (TEER), fluorescence reporters and IL-8 ELISA as readouts. We postulate that compounds will manifest their anti-psoriatic effects through the reduction in disease phenotypes that can be detected by relevant readouts. Psoriasis can be recapitulated and assessed using cytokine treatment on skin constructs derived from primary fibroblasts (FBs) and keratinocytes (KCs), however it is labor intensive and inefficient to produce large quantity of T cells and reporter-containing iPSC-derived keratinocytes (iKCs), Therefore, in this project, we will use cytokine treatment and skin constructs fabricated with primary cells for initial screening and then use iPSC-derived 3D skin containing T cells and reporters for subsequent compound validation, confirmation and the mechanism of actions (MOA) studies. Specifically, we will use FBs and KCs to bioprint 3D skin in a 96 well format, and treat with a cytokine cocktail containing TNF-a, IL-1a, IL6 and IL17A, for a pilot screen of 50 inflammation-related compounds to optimize the high throughput procedure that then will be applied to screen a library of 1000 compounds using TEER and IL-8 ELISA. Subsequently, we will validate the lead compounds using lactate dehydrogenase (LDH) toxicity assays and iPSC-derived skin constructs that are generated from healthy donor cells and containing GFP reporters for KRT16 and mCherry for FLG. Finally, bioprinted psoriasis-specific iPSC derived skin constructs containing reporters and Th17/Th1 cells will be used to confirm the lead hits and delineate the MOA of compounds using RNA-seq. Completion of this project will provide us with compounds that may be developed into drugs to treat psoriasis. Moreover, our 3D skin model-based high throughput system can be easily adapted to screen for drugs to treat other inflammatory skin diseases.

项目概要 牛皮癣影响着 2-3% 的美国人口。目前尚无高通量筛选 开发新的牛皮癣药物的方法。为了满足这一重大未满足的制药需求 如果需要的话，我们会利用建立的3D皮肤系统来筛选治疗这种疾病的药物 3D 生物打印、诱导多能干细胞 (iPSC) 重编程和 CRISPR 基因组编辑 技术。我们仅使用 iPSC 衍生的成纤维细胞成功构建了 3D 皮肤 iFBs）和角质形成细胞（iKCs），证明了这种方法的可行性。 3D生物打印可以 使我们能够可重复地构建复杂的结构来概括人体组织/器官，并且 微型化 3D 制造的皮肤以进行高通量筛选。牛皮癣是一种炎症 皮肤病伴有异常表皮过度增殖、屏障功能受损、 角蛋白 16 (KRT16) 上调、聚丝蛋白 (FLG) 下调以及角蛋白 16 过度分泌 细胞因子，例如 IL-8，可以通过跨上皮/跨内皮电监测 电阻 (TEER)、荧光报告基因和 IL-8 ELISA 作为读数。我们假设 化合物将通过减少疾病表型来发挥其抗牛皮癣作用 可以通过相关读数检测到。牛皮癣可以使用以下方法进行概括和评估： 对原代成纤维细胞 (FB) 和角质形成细胞衍生的皮肤结构进行细胞因子处理 （KC），然而，生产大量 T 细胞是劳动密集型且低效的 含有报告基因的 iPSC 衍生角质形成细胞 (iKC)，因此，在本项目中，我们将使用 细胞因子处理和用原代细胞制造的皮肤结构进行初步筛选，然后 使用含有 T 细胞和报告基因的 iPSC 衍生 3D 皮肤进行后续化合物验证， 确认和作用机制（MOA）研究。具体来说，我们将使用 FB 和 KC 以 96 孔格式生物打印 3D 皮肤，并用含有 TNF-a 的细胞因子混合物进行治疗， IL-1a、IL6 和 IL17A，用于 50 种炎症相关化合物的初步筛选，以优化 高通量程序，然后将用于筛选 1000 种化合物的库 TEER 和 IL-8 ELISA。随后，我们将使用乳酸验证先导化合物 脱氢酶 (LDH) 毒性测定和 iPSC 衍生的皮肤构建体 健康的供体细胞，含有 KRT16 的 GFP 报告基因和 FLG 的 mCherry。最后， 生物打印银屑病特异性 iPSC 衍生皮肤构建体，包含报告基因和 Th17/Th1 细胞将用于确认先导命中并使用 RNA-seq 描绘化合物的 MOA。 该项目的完成将为我们提供可开发成药物的化合物 治疗牛皮癣。此外，我们基于 3D 皮肤模型的高通量系统可以轻松 适合筛选治疗其他炎症性皮肤病的药物。