Discovery of autoimmune antigens from non-coding sequences

7.项目摘要。 该项目解决了许多神经免疫性疾病的急性临床需求， 致病抗原，以及那些变异仍然无法解释的单一定义抗原。 NMDA受体脑炎（α-NMDAR）中的抗体已经彻底改变了 副肿瘤疾病（PND），而其他PND，如眼阵挛肌阵挛综合征（OMS）， 等待这些突破性的发现几种方法，如噬菌体免疫沉淀和 测序（PhIP-seq）已经成功地用于淘选生物标志物和定义自身抗原。 然而，对于某些疾病，这些方法都失败了。一种可能性是定义抗原 因为非传统的起源而被传统的屏幕错过。我们假设 一些重要的自身抗原可能存在于“非编码”序列中。其中包括广大 小的开放阅读框架（sORF）或“微肽”的景观，其起源于细胞的区域， 基因组，如UTR和长的“非编码”RNA，可能在过去被错误地忽视了 卡位对于这项工作来说，重要的是，它们已被证明的翻译是时间和空间调节的， 可以是肿瘤特异性抗原并呈递给免疫系统。的第二源 在这些测定中未测试的蛋白质是转座因子。它们天然编码的蛋白质，以及 那些从它们转座到活性基因中产生的，也是免疫原性的潜在来源。 材料虽然很少有活性，但它们在胚胎、成人脑和肿瘤中的表达表明它们可能具有活性。 可能与副肿瘤性自身免疫有关由宿主产生的隐蔽肽 机制仍然是对依赖于确定的靶参考肽组的筛选的进一步挑战。 我们的研究旨在通过设计和构建新的库来改进以前的PhIPseq搜索 涵盖了这些非传统的缩氨酸在目标1中，我们将设计和构建两个新的T7 PhIP-seq 文库涵盖了这个定义的“非编码”肽组：一个具有以序列阵列表示的靶点的文库。 第一个是编码重叠肽的寡核苷酸，第二个是表达全长靶标的寡核苷酸， 降低了通过序列片段化破坏表位的风险。在目标2中，我们将筛选以下样本： 儿科神经免疫疾病患者（和对照）针对这些基因特异性文库，以鉴定 反应性的特征为了捕获有关隐蔽抗原肽的信息，Aim 3将 用现存的随机肽库测试相同的样品，该库报告收敛特征 来自每种疾病的患者样品的特异性。这种高风险的方法，如果成功，将导致 很快，革命性的新诊断范式的神经免疫疾病的测试，因为它没有为 NMDAR脑炎，对其他自身免疫性疾病也具有范式转移的潜力。

7. Project Summary. This project addresses an acute clinical need for the many neuroimmune diseases with no known causative antigen, and those with variation that remains unexplained by single defining antigens. Antibodies in NMDA-receptor encephalitis (α-NMDAR) have revolutionized diagnosis and treatment of that paraneoplastic disease (PND), while other PNDs, like Opsoclonus Myoclonus Syndrome (OMS), await these groundbreaking discoveries. Several methods, such as Phage immunoprecipitation and sequencing (PhIP-seq) have been used successfully to pan for biomarkers and defining autoantigens. However, for some diseases, these methods have failed. One possibility is that defining antigens have been missed by conventional screens because of unconventional origins. We hypothesize that some important autoantigens may reside within “non-coding” sequences. These include the vast landscape of small open reading frames (sORFs), or “micropeptides”, that originate in regions of the genome, like UTRs and long “non-coding” RNAs, that may have been erroneously overlooked in past screens. Importantly for this work, their proven translation is temporally and spatially regulated, they can be tumor specific antigens and are presented to the immune system. A second source of untested proteins in these assays is transposable elements. The proteins they naturally encode, and those that arise from their transposition into active genes, are also potential sources of immunogenic material. While few are active, their expression in embryos, adult brain and in tumor suggest their potential relevance for paraneoplastic autoimmunity. Cryptic peptides generated by a host of mechanisms remain a further challenge to screens that rely on defined target reference peptidomes. Our study aims to improve upon previous PhIPseq searches, by designing and building new libraries that cover this unconventional set of peptides. In Aim 1, we will design and build two new T7 PhIP-seq libraries covering this defined “non-coding” peptidome: one with targets represented as arrays of oligos encoding overlapping peptides, and a second in which full-length targets are expressed, to mitigate risk of disrupting epitopes by sequence fragmentation. In Aim 2, we will screen samples from pediatric neuroimmune disease patients (and controls) against these gene-specific libraries, to identify distinctive signatures of reactivity. To capture information about cryptic antigenic peptides, Aim 3 will test the same samples against an extant random peptide library, which reports on convergent feature specificity of patient samples from each disorder. This high-risk approach, if successful, will lead quickly to revolutionary new diagnostic paradigms for the neuroimmune diseases tested, as it did for NMDAR encephalitis, with paradigm-shifting potential for other autoimmune diseases, as well.