Analyzing Genes for Autoimmune Thyroiditis and Diabetes: Translation to Therapy

项目摘要 自身免疫性甲状腺疾病（AITD）经常与1型糖尿病（T1 D）一起发生， 被认为是自身免疫多腺体综合征3型（APS 3v）的变体。我们的目标是确定 AITD+ T1 D（APS 3v）的联合易感基因，以剖析它们引起疾病的机制， 将这些知识转化为新的疗法。在上一个赠款周期，我们取得了实质性进展， 这些目标：（1）我们利用全基因组方法定位了APS 3v的新易感基因/位点;（2）我们 发现了一个独特的HLA-DR-pocket签名，对APS 3v的发展至关重要;（3）我们确定了4个 与APS 3v特异性HLA-DR结合并激活T细胞的致病性甲状腺和胰岛肽;（4）我们鉴定了 遗传-表观遗传相互作用，触发AITD和T1 D;（5）我们发现了一种化合物，千金藤素， 阻断甲状腺球蛋白肽呈递，预防小鼠自身免疫性甲状腺炎。根据这些调查结果， 我们提出：（1）我们定位的序列3 APS 3v基因座，以鉴定易患APS 3v的罕见变体;和（2） 通过阻断这个口袋，将我们对APS 3v-DR口袋的发现转化为新的疗法。我们的假设 除了常见的变体，罕见的非HLA变体对AITD+ T1 D的关节病因也至关重要， 识别它们将揭示新的机制和治疗靶点;此外，我们假设， 致病肽与APS 3v-DR口袋的结合及其对T细胞的呈递触发APS 3v，并且 阻断致病肽与HLA-DR的结合可用于治疗或预防APS 3v。我们的具体目标是： 具体目标1：使用靶向next鉴定与APS 3v连锁的3个基因座（2 q，8 p，Xp）中的罕见变异 在我们独特的AITD+ T1 D聚类的家庭数据集中，我们使用了第二代测序（NGS）。鉴定的变体 将在2个大的APS 3v患者和家族队列中复制。 具体目标2：筛选阻断致病肽的小分子抑制剂（SMI）的大型文库 与APS 3v-DR口袋结合以鉴定命中。将在体外、离体和体内确认命中SMI。铅 SMI将通过药物化学进行优化，以提高效力，选择性和药代动力学特性。 具体目标3：设计与APS 3v HLA-DR结合的致病肽的D-氨基酸肽阻断剂 口袋我们将在计算机上设计D-氨基酸肽（D-肽），预测阻断结合和呈递 致病性甲状腺/胰岛肽。将在体外、离体和体内证实预测的D肽阻断剂。 总之，我们的多学科翻译项目建立在上一个资助期获得的知识基础上。 我们的目标是继续我们成功的基因发现，使用定位的APS 3v基因座的靶向NGS来鉴定 新的治疗靶点，并寻求通过阻断 我们确定的APS 3v-DR囊袋。我们的治疗方法的优势在于它既有选择性， 识别致病肽的细胞被靶向和个性化，因为只有携带APS 3v的患者， DR将接受治疗。我们的研究有望为APS 3v以及AITD和T1 D带来新的治疗方法。

PROJECT SUMMARY Autoimmune thyroid diseases (AITD) frequently develop together with type 1 diabetes (T1D), a combination considered a variant of the Autoimmune Polyglandular Syndrome type 3 (APS3v). Our goals are to identify the joint susceptibility genes for AITD+T1D (APS3v), to dissect the mechanisms by which they cause disease, and to translate this knowledge into new therapies. In the last grant cycle we made substantial progress toward these goals: (1) We mapped new susceptibility genes/loci for APS3v using whole genome approaches; (2) We discovered a unique HLA-DR-pocket signature critical for the development of APS3v; (3) We identified 4 pathogenic thyroid & islet peptides that bind to the APS3v-specific HLA-DR & activate T-cells; (4) We identified genetic-epigenetic interactions that trigger AITD & T1D; (5) We discovered a compound, Cepharanthine, that blocks thyroglobulin peptide presentation & prevents autoimmune thyroiditis in mice. Building on these findings we propose to: (1) Sequence 3 APS3v loci we mapped to identify rare variants predisposing to APS3v; and (2) Translate our discovery of the APS3v-DR pocket into new therapies by blocking this pocket. Our hypothesis is that in addition to common variants, rare non-HLA variants are also critical to the joint etiology of AITD+T1D, and that identifying them will reveal new mechanisms & therapeutic targets; furthermore, we hypothesize that binding of pathogenic peptides to the APS3v-DR pocket & their presentation to T-cells triggers APS3v, and that blocking pathogenic peptide binding to HLA-DR can be used to treat or prevent APS3v. Our specific aims are: Specific Aim 1: Identifying rare variants in 3 loci (2q, 8p, Xp) that are linked with APS3v using targeted next generation sequencing (NGS) in our unique dataset of families in which AITD+T1D cluster. Identified variants that are predicted to alter gene function will be replicated in 2 large cohorts of APS3v patients and families. Specific Aim 2: Screening of a large library for small molecule inhibitors (SMI's) that block pathogenic peptide binding to the APS3v-DR pocket to identify hits. Hit SMI's will be confirmed in vitro, ex vivo and in vivo. Lead SMI's will be optimized by medicinal chemistry to improve potency, selectivity, & pharmacokinetic properties. Specific Aim 3: Designing D-amino acid peptide blockers of pathogenic peptide binding to the APS3v HLA-DR pocket. We will design in silico D-amino acid peptides (D-peptides) predicted to block binding and presentation of pathogenic thyroid/islet peptides. Predicted D-peptide blockers will be confirmed in vitro, ex vivo, and in vivo. In summary, our multidisciplinary translational project builds on the knowledge gained in the last grant period. Our goals are to continue our successful gene discovery using targeted NGS of mapped APS3v loci to identify new therapeutic targets, and to pursue pre-clinical development of novel therapies for APS3v by blocking the APS3v-DR pocket we identified. The strength of our therapeutic approach is that it is both selective as only T- cells recognizing pathogenic peptides are targeted and personalized since only patients carrying the APS3v- DR will be treated. Our studies will hopefully lead to new therapies for APS3v, as well as for AITD and T1D.