SBIR-STTR Award

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.

Public Health Relevance Statement:
PROJECT NARRATIVE Immuno-oncology (IO) therapies offer great promise in effectively treating cancers, however, current tools for IO target identification are not amenable to tumors with low tumor mutational burden (TMB) and/or are skewed towards the identification of tumor neoantigens that are MHC bound. Envisagenics has developed SpliceIO, a software platform for neoantigen discovery that detects splicing errors using only RNAseq data, which can effectively identify both MHC-presented and MHC-independent tumor neoantigens, and do so in low TMB tumors. This project will validate the SpliceIO platform in BRCA1/2+ mammary tissue derived organoids, and upon completion, Envisagenics will achieve commercial readiness for SpliceIO, thus providing a valuable tool for IO target identification in numerous cancers.

Project Terms:
Alternative Splicing; Alternate Splicing; Alternative RNA Splicing; Antigens; immunogen; Surface Antigens; Cell Surface Antigens; Immunologic Surface Markers; Immunological Surface Markers; Archives; Automobile Driving; driving; Breast; malignant breast neoplasm; Breast Cancer; malignant breast tumor; Malignant Neoplasms; Cancers; Malignant Tumor; malignancy; neoplasm/cancer; Disease; Disorder; Gene Expression Regulation; Gene Action Regulation; Gene Regulation; Gene Regulation Process; Goals; Laboratories; Lead; Pb element; heavy metal Pb; heavy metal lead; Medically Underserved Area; medically under served area; Methods; Mutation; Genetic Alteration; Genetic Change; Genetic defect; genome mutation; Organoids; Pancreatic; Pancreas; Patients; Production; Prostate Gland; Prostatic Gland; Prostate; Proteins; Research; Risk; Non-Polyadenylated RNA; RNA Gene Products; Ribonucleic Acid; RNA; Splicing; RNA Splicing; mRNA; Messenger RNA; Software; Computer software; Time; Tissues; Body Tissues; Genetic Transcription; Gene Transcription; RNA Expression; Transcription; Translations; Universities; Woman; Work; Generations; Case Study; case report; base; Benign; Phase; Medical; Licensing; Collaborations; Therapeutic; tool; Event; Source; Best Practice Analysis; Benchmarking; BRCA 1/2; BRCA1/2; brca gene; Performance; Isoforms; Protein Isoforms; tandem mass spectrometry; novel; Cell surface; Proteome; Regulation; Sampling; Proteomics; immune drugs; immune-based therapeutics; immunologic preparation; immunologic therapeutics; immunotherapeutics; immunotherapy agent; Immunotherapeutic agent; cancer therapy; Cancer Treatment; Malignant Neoplasm Therapy; Malignant Neoplasm Treatment; anti-cancer therapy; anticancer therapy; cancer-directed therapy; Malignant Ovarian Neoplasm; Malignant Ovarian Tumor; Malignant Tumor of the Ovary; Ovary Cancer; ovarian cancer; Malignant neoplasm of ovary; Molecular Interaction; Binding; T-Stage; Tumor stage; Preparedness; Readiness; Childhood Neoplasm; Childhood Tumor; Pediatric Tumor; tumors in children; Pediatric Neoplasm; Tumor Load; Tumor Burden; Preventive; Breast Neoplasms; Breast Tumors; Mammary Cancer; mammary tumor; Mammary Neoplasms; Data; High Prevalence; immune-oncology; immuno oncology; immunology oncology; oncoimmunology; Immunooncology; Non-Malignant; nonmalignant; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; Validation; Mammary Gland Parenchyma; Breast Tissue; Mammary Gland Tissue; Process; Development; developmental; deep sequencing; novel strategies; new approaches; novel approaches; novel strategy; Outcome; Population; prospective; Coupled; commercialization; tumor; mutation carrier; clinical care; patient population; candidate validation; transcriptome sequencing; RNA Seq; RNA sequencing; RNAseq; exome sequencing; exome-seq; clinically actionable; transcriptome; global gene expression; global transcription profile; proteogenomics; neoantigens; neo-antigen; neo-epitopes; neoepitopes; computational pipelines; cancer immunotherapeutics; cancer immune therapeutics; BRCA mutations; BRCA 1/2 mutations; BRCA1/2 mutations; mammary

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.

Public Health Relevance Statement:
PROJECT NARRATIVE Immuno-oncology (IO) therapies offer great promise in effectively treating cancers, however, current tools for IO target identification are not amenable to tumors with low tumor mutational burden (TMB) and/or are skewed towards the identification of tumor neoantigens that are MHC bound. Envisagenics has developed SpliceIO, a software platform for neoantigen discovery that detects splicing errors using only RNAseq data, which can effectively identify both MHC-presented and MHC-independent tumor neoantigens, and do so in low TMB tumors. This project will validate the SpliceIO platform in BRCA1/2+ mammary tissue derived organoids, and upon completion, Envisagenics will achieve commercial readiness for SpliceIO, thus providing a valuable tool for IO target identification in numerous cancers.

Project Terms:
Alternate Splicing; Alternative RNA Splicing; Alternative Splicing; immunogen; Antigens; Cell Surface Antigens; Immunologic Surface Markers; Immunological Surface Markers; Surface Antigens; Automobile Driving; driving; Breast; malignant breast neoplasm; Breast Cancer; malignant breast tumor; Calibration; Malignant Neoplasms; Cancers; Malignant Tumor; malignancy; neoplasm/cancer; Disease; Disorder; Fishes; Gene Expression Regulation; Gene Action Regulation; Gene Regulation; Gene Regulation Process; Goals; Laboratories; Medically Underserved Area; medically under served area; Methods; Mutation; Genetic Alteration; Genetic Change; Genetic defect; genome mutation; Organoids; Pancreas; Pancreatic; Patients; Production; Prostate; Prostate Gland; Prostatic Gland; Proteins; Research; Risk; RNA; Non-Polyadenylated RNA; RNA Gene Products; Ribonucleic Acid; RNA Splicing; Splicing; Messenger RNA; mRNA; Computer software; Software; Tissues; Body Tissues; Genetic Transcription; Gene Transcription; RNA Expression; Transcription; Translations; translation; Universities; Woman; Work; Generations; case report; Case Study; Benign; Phase; Medical; Licensing; Collaborations; Therapeutic; tool; Event; Source; Benchmarking; Best Practice Analysis; benchmark; brca gene; BRCA 1/2; BRCA1/2; Performance; Protein Isoforms; Isoforms; tandem mass spectrometry; MHC antigen; novel; Cell surface; Proteome; Regulation; Sampling; Proteomics; Immunotherapeutic agent; immune drugs; immune-based therapeutics; immunologic therapeutics; immunotherapeutics; immunotherapy agent; cancer therapy; Cancer Treatment; Malignant Neoplasm Therapy; Malignant Neoplasm Treatment; anti-cancer therapy; anticancer therapy; cancer-directed therapy; Malignant Ovarian Neoplasm; Malignant Ovarian Tumor; Malignant Tumor of the Ovary; Ovary Cancer; ovarian cancer; Malignant neoplasm of ovary; Molecular Interaction; Binding; T-Stage; Tumor stage; Preparedness; Readiness; Pediatric Neoplasm; Childhood Neoplasm; Childhood Tumor; Pediatric Tumor; tumors in children; Tumor Burden; Tumor Load; Preventive; Mammary Neoplasms; Breast Neoplasms; Breast Tumors; Mammary Cancer; mammary tumor; Data; High Prevalence; Immunooncology; immune-oncology; immuno oncology; immunology oncology; oncoimmunology; Non-Malignant; nonmalignant; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; Validation; validations; Mammary Gland Parenchyma; Breast Tissue; Mammary Gland Tissue; Process; Development; developmental; deep sequencing; new approaches; novel approaches; novel strategy; novel strategies; Outcome; Population; prospective; Coupled; commercialization; tumor; mutation carrier; clinical care; patient population; candidate identification; candidate validation; RNA Seq; RNA sequencing; RNAseq; transcriptomic sequencing; transcriptome sequencing; exome-seq; exome sequencing; clinically actionable; global gene expression; global transcription profile; transcriptome; proteogenomics; neo-antigen; neo-epitopes; neoepitopes; neoantigens; computational pipelines; cancer immune therapeutics; cancer immunotherapeutics; BRCA 1/2 mutations; BRCA1/2 mutations; BRCA1/2mut; BRCAmut; BRCA mutations; mammary