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

项目摘要 牛皮癣影响2-3%的美国人口。目前，还没有高通量的筛查 开发牛皮癣新药的方法学。以满足这一重要的未获满足的药物 需要，我们将使用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。我们成功地使用IPSC来源的成纤维细胞构建了3D皮肤 IFBs)和角质形成细胞(IKCs)，证明了这种方法的可行性。3D生物打印罐 使我们能够重复地构建复杂的结构来概括人类组织/器官，以及 将3D制作的皮肤微型化，以实现高通量筛选。牛皮癣是一种炎症性 皮肤病伴随着异常的表皮过度增殖，屏障功能受损， 上调角蛋白16(KRT16)、下调微丝蛋白(Flg)和过度分泌 细胞因子，如IL-8，可通过跨内皮/跨内皮电监测 以耐药(TEER)、荧光报告和IL-8酶标法为读数。我们假定 化合物将通过减少疾病表型来显示其抗牛皮癣的作用。 这可以通过相关读数检测到。银屑病可以用以下方法概括和评估 细胞因子对原代成纤维细胞和角质形成细胞来源皮肤结构的影响 (KCS)，但是生产大量的T细胞和 含有IPSC来源的角质形成细胞(IKCs)，因此，在这个项目中，我们将使用 细胞因子治疗和用原代细胞构建的皮肤结构进行初步筛选，然后 使用IPSC衍生的含有T细胞的3D皮肤和记者进行后续的化合物验证， 确认和作用机制(MOA)研究。具体地说，我们将使用FBS和 KCS将以96孔的格式生物打印3D皮肤，并使用含有肿瘤坏死因子-α的细胞因子鸡尾酒进行治疗， IL-1a，IL6和IL17A，用于50种与炎症相关的化合物的试点筛选，以优化 高通量程序，然后将应用于筛选包含1000个化合物的库 TERE和IL-8的酶联免疫吸附试验。随后，我们将使用乳酸盐验证先导化合物 脱氢酶(LDH)毒性分析和IPSC衍生的皮肤构建物 健康的供体细胞，包含KRT16的GFP记者和FLG的mCherry。最后， 含有Reports和Th17/Th1的Biopprint银屑病特异性IPSC来源的皮肤构建物 单元格将用于确认铅的命中，并使用RNA-SEQ描述化合物的MOA。 该项目的完成将为我们提供可能被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.