Comprehensive validation and commercial readiness of SpliceIO, a software platform for neoantigen discovery using RNA-seq data

项目总结 准确的剪接对于组装有活力的信使核糖核酸亚型至关重要。拼接错误，导致疾病- 导致mRNAs，频繁发生。事实上，大约50%的致癌同义突变被预测为 导致拼接错误。自从引入分析软件来量化来自RNA的选择性剪接(AS)以来 在测序(RNAseq)数据中，越来越多的证据表明剪接调控在癌症中的重要性。 剪接错误是肿瘤特异性新抗原的主要来源，因此剪接研究打开了巨大的大门 癌症免疫疗法和预防的发展机遇。环境学已经发展起来了 SpliceIO，一个仅使用RNAseq数据发现新抗原的软件平台。大多数其他方法用于 新抗原的预测依赖于全外显子组测序来发现基于突变的新抗原。 对肿瘤突变负荷(TMB)低的癌症，如乳腺癌、前列腺癌， 胰腺、儿科肿瘤等良性疾病。然而，这些癌症富含剪接错误 它可以用SpliceIO检测到，使在这个目前服务不足的地区发现新抗原成为可能 出于医疗需要。此外，目前的方法根据新抗原的结合和呈现能力来预测它们 通过MHC分子，这种分子在60%的晚期肿瘤中下调。由于SpliceIO专注于拼接 它有能力预测与MHC直接结合的MHC提呈抗原和非MHC抗原 细胞表面。 这一直接到第二阶段提案的目标是建立在强大的初步数据基础上，并实现商业 为SpliceIO做好准备。Envisagenics一直处于基于RNAseq的目标发现的前沿 药品鉴定软件平台SpliceCore®的开发与商业化 剪接异构体。Envisagenics将利用成熟的商业化战略，包括全面的 巩固未来生物制药的强大价值主张和商业产品的实验验证 合伙人。在这项提案中，我们将开发一种新的策略，使用Tandem进行大规模的新抗原验证 质谱仪(MS/MS)。此外，我们还将使用乳腺器官对新抗原进行规模化鉴定 来自BRCA1/2突变携带者。我们提供的初步数据相当于第一阶段SBIR的结果 展示剪接衍生的靶点发现在癌症治疗中的效用。 为了实现这些目标并为SpliceIO做好商业准备，我们将完成以下工作 本提案的具体目标：目标1：为SpliceIO开发高深度/高灵敏度的参考蛋白质组 使用MS/MS数据进行验证。目的2：应用超深层技术鉴定BRCA1/2突变携带者的新抗原 测序。目的3：剪接衍生新抗原的实验验证。这些目标的实现将带来 SpliceIO的开发水平被证明支持成功的商业化，并取得了显著的 区别对待患者和临床护理。

PROJECT SUMMARY Accurate splicing is critical for the assembly of viable mRNA isoforms. Splicing errors, resulting in disease- causing mRNAs, occur frequently. In fact, ~50% of all cancer-driving synonymous mutations are predicted to cause splicing errors. Since the introduction of analytic software to quantify alternative splicing (AS) from RNA sequencing (RNAseq) data, evidence pointing to the importance of splicing regulation in cancer has mounted. Splicing errors are a major source of tumor-specific neoantigens, thus splicing research opens tremendous opportunities for the development of cancer immunotherapeutics and preventives. Envisagenics has developed SpliceIO, a software platform for neoantigen discovery using only RNAseq data. Most other methods for neoantigen prediction rely on whole-exome sequencing for the discovery of mutation-based neoantigens, a method that is not amenable to cancers with low tumor mutational burden (TMB), such as breast, prostate, pancreatic, pediatric tumors and other benign conditions. These cancers, however, are rich in splicing errors which can be detected with SpliceIO, making neoantigen discovery possible in this currently underserved area of medical need. Moreover, current methods predict neoantigens based on their ability to bind and be presented by MHC molecules which are downregulated in >60% of late-stage tumors. Since SpliceIO focuses on splicing errors it has the ability to predict both MHC-presented and MHC-independent antigens that are directly bound to the cell surface. The goal of this Direct-to-Phase II proposal is to build upon strong preliminary data and attain commercial readiness for SpliceIO. Envisagenics has been at the forefront of RNAseq-based target discovery since the development and commercialization of SpliceCore®, a software platform for the identification of druggable splicing isoforms. Envisagenics will utilize a proven commercialization strategy, which consists of comprehensive experimental validation to solidify a strong value proposition and commercial offering to prospective biopharma partners. In this proposal, we will develop a novel strategy for large-scale neoantigen validation using tandem mass spectrometry (MS/MS). In addition, we will scale identification of neoantigens using mammary organoids from BRCA1/2 mutation carriers. We present preliminary data equivalent to results from a Phase I SBIR and demonstrate the utility of splicing-derived target discovery for cancer therapeutics. To accomplish these goals and obtain commercial readiness for SpliceIO we will complete the following specific aims in this proposal: Aim 1: Develop a high-depth/high-sensitivity reference proteome for SpliceIO validation using MS/MS data. Aim 2: Neoantigen identification in BRCA1/2 mutation carriers using ultra-deep sequencing. Aim 3: Experimental validation of splicing-derived neoantigens. Completion of these aims will bring SpliceIO to a level of development proven to support successful commercialization and make a significant difference patient treatment and clinical care.