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...