Functional Validation of Gene Modifications that Protect Beta Cells against Autoimmunity Identified by Genome-Wide CRISPR Cas9 Screening

项目摘要 1型糖尿病（T1D）是由胰腺中胰岛素产生细胞的免疫破坏引起的。的 疾病无法预防，唯一可用的治疗方法是终身使用多次注射胰岛素。 一天三次。现在可以在实验室中从多能干细胞中产生产生胰岛素的β细胞。 这一最新的成就提高了通过β细胞恢复患者胰岛素产生的前景。 移植然而，T1D潜在的自身免疫性是持久的，并且如果 它们在没有免疫抑制的情况下被移植。迫切需要一种保护β细胞的新疗法 不仅能够在患者中进行治疗性β细胞替代，而且还可以预防有糖尿病风险的个体的糖尿病。 发展疾病。为了发现这种疗法的遗传靶点，我们进行了全基因组搜索， 在T1D小鼠模型中使用CRIPSR-Cas9基因组编辑。这一广泛的无偏搜索确定了 肾酶基因（RNLS）。值得注意的是，RNLS以前与总体风险和年龄相关- 通过人类全基因组关联研究（GWAS）发现T1D的发病率，表明RNLS在糖尿病中的作用 发病机制我们继续证实，删除RNLS保护小鼠β细胞免受细胞应激 和自身免疫我们证实了RNLS缺失在人类干细胞衍生的β-核受体中的保护作用。 细胞基于这些发现，我们假设RNLS的小分子抑制剂也可以保护。 使用基于结构的分子建模，我们确定了FDA批准的药物帕吉林作为潜在的RNLS 抑制剂.我们发现，帕吉林治疗复制了RNLS缺失的保护作用，使其成为一种新的治疗方法。 药物是T1D预防性治疗的潜在候选药物。现在，我们的目标是更好地了解RNLS如何 缺乏保护β细胞免受细胞应激和自身免疫。此外，我们的目标是更广泛地 测试帕吉林的治疗潜力为此，我们将追求三个具体目标。一是 评估代谢可塑性作为保护RNLS缺陷细胞的可能机制基础。第二、 我们将在体外和小鼠模型中定量和描述Rnls缺陷β细胞的免疫原性， T1D第三，我们将评估帕吉林作为Rnls缺失的模拟物的治疗效用，无论是在细胞内还是在细胞外。 水平和小鼠自身免疫性糖尿病的背景下。完成前两个目标将有助于解释 RNLS改变β细胞脆弱性和1型糖尿病风险的机制。第三个目标将 通过测试药物的治疗价值，支持我们的发现转化为临床应用。 被鉴定为RNLS抑制剂。我们的研究团队由免疫学、β细胞生物学和 干细胞分化我们处于理想的位置，可以从以下角度探索RNLS在T1D中的作用： 从细胞生物学到系统性自身免疫，前景是提高我们对 疾病病理学和开发预防药物。我们的研究将利用一种特征良好的小鼠， T1D和人类β细胞的模型，以确定我们的发现与人类疾病的相关性。

PROJECT SUMMARY Type 1 diabetes (T1D) is caused by the immune destruction of insulin producing cells in the pancreas. The disease cannot be prevented, and the only treatment available is a life-long use of insulin injected multiple times a day. Insulin-producing beta cells can now be generated from pluripotent stem cells in the laboratory. This recent achievement has raised the prospect of restoring insulin production in patients by beta cell transplantation. However, the autoimmunity underlying T1D is long-lasting and will destroy new beta cells if they are transplanted without immunosuppression. A new therapy that protects beta cells is urgently needed not only enable curative beta cell replacement in patients but also to prevent diabetes in individuals at risk of developing the disease. To discover genetic targets for such a therapy, we performed a genome-wide search using CRIPSR-Cas9 genome editing in a mouse model for T1D. This broad unbiased search identified the gene Renalase (RNLS). Significantly, RNLS had previously been associated with the overall risk and the age- of-onset of T1D by human genome-wide association studies (GWAS), suggesting a role for RNLS in diabetes pathogenesis. We went on to confirm that deleting RNLS protected mouse beta cells against cellular stress and autoimmunity. We corroborated the protective effect of RNLS deletion in human stem cell-derived beta cells. Based on these findings, we hypothesized that a small molecule inhibitor of RNLS would also protect. Using structure-based molecular modeling, we identified the FDA-approved drug pargyline as a potential RNLS inhibitor. We showed that pargyline treatment replicated the protective effects of RNLS deletion, making this drug a potential candidate for a preventive treatment for T1D. Now, we aim to better understand how RNLS deficiency protects beta cells against cellular stress and autoimmunity. Further, we aim to more extensively test the therapeutic potential of pargyline. To these ends, we will pursue three Specific Aims. First, we will evaluate metabolic plasticity as a possible mechanistic basis for the protection of RNLS deficient cells. Second, we will quantify and delineate the immunogenicity of Rnls deficient beta cells in vitro and in mouse models for T1D. Third, we will evaluate the therapeutic utility of pargyline as a mimic of Rnls deletion, both at the cellular level and in the context of autoimmune diabetes in mice. Completing the first two Aims will help explain the mechanism by which RNLS modifies beta cell vulnerability and the risk of type 1 diabetes. The third Aim will support the translation of our discoveries into a clinical application by testing the therapeutic value of drug we identified as a RNLS inhibitor. Our research team is composed of experts in immunology, beta cell biology and stem cell differentiation. We are ideally positioned to explore the role of RNLS in T1D from perspectives that range from cellular biology to systemic autoimmunity, with the prospect of improving our understanding of disease pathology and developing a preventive drug. Our research will utilize both a well characterized mouse model for T1D and human beta cells to ascertain the relevance of our findings for human disease.