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

项目摘要 银屑病影响2-3%的美国人口。目前，没有高通量筛选 方法来开发新的牛皮癣药物。为了满足这一重大的未满足的药物需求， 需要，我们将筛选药物来治疗这种疾病，通过使用3D皮肤系统建立， 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.我们成功地使用专门的iPSC衍生的成纤维细胞构建了3D皮肤。 iFB）和角质形成细胞（iKC），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，以重现人体组织/器官， 将3D制造的皮肤切片用于高通量筛选。牛皮癣是一种炎症性疾病， 具有异常表皮过度增殖，屏障功能受损， 上调角蛋白16（KRT 16），下调聚丝蛋白（FLG）和过度分泌 细胞因子如IL-8，其可以用跨上皮/跨内皮电生理监测。 抗性（TEER）、荧光报告基因和IL-8 ELISA作为读数。我们推测 化合物将通过减少疾病表型 可以通过相关读数检测到。可以使用以下方法概括和评估银屑病 对源自原代成纤维细胞（FB）和角质形成细胞的皮肤构建体的细胞因子处理 (KCs)然而，生产大量的T细胞是劳动密集型和低效的， 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构建体进行初始筛选， 使用含有T细胞和报告基因的iPSC衍生的3D皮肤用于随后的化合物验证， 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-α的细胞因子混合物处理， IL-1a、IL-6和IL-17 A，用于50种炎症相关化合物的中试筛选，以优化 高通量程序，然后将应用于筛选1000种化合物库， TEER和IL-8 ELISA。随后，我们将使用乳酸盐验证先导化合物 在一些实施方案中，使用由人成纤维细胞产生的诱导多能干细胞（iPSC）脱氢酶（LDH）毒性测定和iPSC衍生的皮肤构建体，所述诱导多能干细胞衍生的皮肤构建体由人成纤维细胞产生。 健康供体细胞，并含有KRT 16的GFP报告基因和FLG的mCherry。最后， 含有报道基因和Th 17/Th 1的生物打印的银屑病特异性iPSC衍生的皮肤构建体 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，我们的基于3D皮肤模型的高通量系统可以很容易地 适用于筛选治疗其他炎症性皮肤病的药物。

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.