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

项目摘要 准确剪接对于活mRNA同种型的组装是至关重要的。拼接错误，导致疾病- 引起mRNA的基因突变。事实上，约50%的癌症驱动同义突变被预测为 导致拼接错误。自从引入分析软件来定量RNA的选择性剪接（AS）以来， 通过RNA测序（RNAseq）数据，已经有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 癌症免疫治疗和预防药物的发展机会。Envisagenics开发了 SpliceIO，一种仅使用RNAseq数据进行新抗原发现的软件平台。大多数其他方法 新抗原预测依赖于全外显子组测序来发现基于突变的新抗原， 不适用于具有低肿瘤突变负荷（TMB）的癌症，如乳腺癌，前列腺癌， 胰腺、小儿肿瘤和其他良性疾病。然而，这些癌症富含剪接错误， 这可以用SpliceIO检测，使得在这个目前服务不足的领域发现新抗原成为可能 医疗需求。此外，目前的方法基于其结合和呈递的能力来预测新抗原。 MHC分子在>60%的晚期肿瘤中下调。由于SpliceIO专注于拼接 它有能力预测直接结合的MHC呈递和MHC非依赖性抗原， 细胞表面。 该直接进入第二阶段提案的目标是建立在强大的初步数据基础上， 为SpliceIO做好准备。Envisagenics一直处于基于RNAseq的靶标发现的最前沿， 开发和商业化的SpliceCore®，一个软件平台，用于识别药物 剪接异构体Envisagenics将利用成熟的商业化战略，包括全面的 实验验证，以巩固强大的价值主张，并为未来的生物制药提供商业服务 伙伴在这项提案中，我们将开发一种新的策略，使用串联 质谱法（MS/MS）。此外，我们将使用乳腺类器官对新抗原进行规模鉴定， BRCA 1/2突变携带者我们目前的初步数据相当于第一阶段SBIR的结果， 展示了剪接衍生的靶发现对于癌症治疗的效用。 为了实现这些目标并使SpliceIO具备商业化的条件，我们将完成以下工作 本提案的具体目标：目标1：为SpliceIO开发一个高深度/高灵敏度的参考蛋白质组 使用MS/MS数据进行验证。目的2：使用超深杂交技术鉴定BRCA 1/2突变携带者中的新抗原 测序目的3：剪接衍生的新抗原的实验验证。这些目标的实现将带来 SpliceIO的发展水平证明，支持成功的商业化，并作出重大的 区别患者治疗和临床护理。

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.