SBIR-STTR Award

Nucleosomes are the fundamental repeating units of chromatin, comprised of four core histone proteins that are subject to a variety of post-translational modifications (PTMs, e.g. lysine methylation and acetylation). Epigenetic reader proteins coordinate the cellular response to these PTMs by influencing chromatin compaction and gene expression through domains that recognize specific PTMs. A rapidly expanding area of research has shown that reader proteins often contain combinations of domains (i.e. multivalency) in order to simultaneously translate multiple PTMs that decorate a single nucleosome. Readout of this "histone code" has profound functional outcomes for cellular functions (e.g. cell cycle regulation) as well as disease states (e.g. cancer and others). Despite increasing recognition that quantifying combinations of epigenetic marks can improve predictive validity in biomarker studies and also increase the magnitude of response to epigenetic inhibitor treatment, the dearth of available technologies to quantify co-occurring PTMs has impeded scientific discovery in this field. A major limitation for developing assays to quantify combinatorial PTMs has been the lack of control reagents that offer sufficient resolution to detect the combinations of PTMs that are read by multivalent readers. Readouts based on histone proteins (mass spectrometry, histone ELISAs) require elaborate sample processing protocols and simply cannot detect epigenetic marks in trans (e.g. PTMs on different histone tails within a nucleosome, histone-DNA interactions, etc.). Approaches capable of assaying PTMs at nucleosomal resolution are low throughput, laborious, and/or not quantitative (e.g. single nucleosome imaging, re-ChIP). Clearly, development of streamlined, high-throughput, quantitative assays using defined nucleosomal calibrants is urgently needed to decipher combinatorial histone codes and accelerate epigenetic therapies. Here, we propose to develop "QuantiNucTM", a high-throughput platform of nucleosome-calibrated assays to measure PTMs from biological samples with high precision. The innovation of this proposal is the novel application of modified recombinant nucleosome calibrants to enable minimal sample processing and reliable quantification of both single and combinatorial PTMs. In Aim 1, we will develop a combinatorially-modified nucleosome and employ it to optimize ELISAs (low-throughput but widely accessible applications) as well as AlphaLISAs (bead-based proximity assays for high-throughput applications). The optimized reagents and assays developed in Aim 1 will be used for proof-of-principle biological validation in Aim 2, where we will apply QuantiNuc to recapitulate the unique biology of combinatorial PTMs. Completion of these aims will demonstrate feasibility for developing an expanded QuantiNuc platform in Phase II to commercialize assays that will help decipher the histone code, enable biomarker discovery, and facilitate drug development.

Public Health Relevance Statement:
PROJECT NARRATIVE After deciphering the genetic code, the next major frontier is to understand the "epigenetic code", a collection of chemical changes that direct DNA function and that are emerging as highly predictive indicators of disease state and treatment response. Scientific discovery in this field is impeded by a dearth of available technologies with the appropriate controls for rapid, reliable quantification of combinations of epigenetic marks. In this proposal, EpiCypher is developing innovative, high-throughput assays using defined controls to enable unparalleled resolution of epigenetic marks in readily accessible biological samples. Commercial development of these revolutionary assays will help to decipher the epigenetic code, thereby accelerating biomarker development to diagnose and treat complex diseases.

Project Terms:
Acetylation; Acids; inhibitor/antagonist; inhibitor; Antibodies; Autoimmune Diseases; autoimmune disorder; Biological Assay; Biologic Assays; Bioassay; Assay; Biology; Calibration; Malignant Neoplasms; neoplasm/cancer; malignancy; Malignant Tumor; Cancers; cultured cell line; Strains Cell Lines; CellLine; Cell Line; Subcellular Process; Cellular Process; Cellular Physiology; Cellular Function; Cell Process; Cell Function; Cell physiology; Cell Body; Cells; Chromatin; High Speed Liquid Chromatography; High Performance Liquid Chromatography; HPLC; High Pressure Liquid Chromatography; Diagnosis; Disorder; Disease; Deoxyribonucleic Acid; DNA; Enzyme Gene; Enzymes; Fibroblasts; Gene Expression; Genes; Genetic Code; HDAC Proteins; HDAC; Histone Deacetylase; Histones; Immunoblotting; Immunoadsorbents; Immunosorbents; Language; pulmonary; Lung Respiratory System; Lung; L-Lysine; Lysine; Methylation; Nucleosomes; Peptides; Protein Modification; Posttranslational Protein Processing; Posttranslational Modifications; Post-Translational Protein Modification; Post-Translational Modifications; Post-Translational Modification Protein/Amino Acid Biochemistry; Post-Translational Protein Processing; Proteins; Quality Control; Reagent; Research; Mass Spectrum Analyses; Mass Spectrum; Mass Spectroscopy; Mass Spectrometry; Mass Photometry/Spectrum Analysis; Mass Spectrum Analysis; Standardization; Tail; Technology; Translating; Histone Acetylase; histone acetyltransferase; histone methylase; histone H3 methyltransferase; histone methyltransferase; Measures; Mediating; Transcriptional Activation; Transcription Activation; base; improved; Area; Phase; Biological; Physiological; Physiologic; Link; Chemicals; insight; Individual; Nature; Catalogs; Complex; Protocols documentation; Protocol; Neurodegenerative Disorders; neurodegenerative illness; degenerative neurological diseases; degenerative diseases of motor and sensory neurons; Neurologic Degenerative Conditions; Neurodegenerative Diseases; Neural degenerative Disorders; Neural Degenerative Diseases; Nervous System Degenerative Diseases; Degenerative Neurologic Disorders; Degenerative Neurologic Diseases; stoichiometry; novel; Disease model; disorder model; Code; Coding System; Sampling; Property; response; assay development; drug development; high throughput screening; High Throughput Assay; Cell Cycle Regulation; Cell Cycle Control; histone modification; DNA Modification; DNA Modification Process; Histone Code; Length; Detection; Reader; Recombinants; Resolution; Chromatin Structure; Collection; Epigenetic Process; Epigenetic Mechanism; Epigenetic Change; Epigenetic; Validation; Monitor; Molecular; Modification; Development; developmental; Image; imaging; functional outcomes; frontier; innovation; innovative; innovate; combinatorial; user-friendly; treatment response; therapeutic response; response to treatment; Biological Markers; biomarker; biologic marker; bio-markers; biomarker development; biomarker discovery; liquid biopsy; epigenetic therapy

Post-translational modification of histone tails (histone PTMs) and DNA methylation (DNAme) on nucleosomes form a sophisticated molecular code that regulates gene transcription. Aberrant regulation of these chromatin modifications is associated with a vast array of human pathologies. While the majority of work in the field has focused on signatures of individual modifications, combinations of histone PTMs and/or DNAme can be more specific and informative than single marks alone. For instance, although healthy cells and cancerous cells both have H3K27me3 and DNAme distributed genome-wide, the co-localization of these two modifications occurs uniquely in cancer cells. However, existing tools to measure global levels of chromatin modifications are low-throughput, display low sensitivity, and are unable to measure combinatorial modifications (e.g. immunoblot). The development of assays that overcome these limitations and are compatible with multiple sample types (including cellular samples or plasma [for detection of circulating nucleosomes, i.e. liquid biopsy]) will make the study of chromatin modifications widely accessible for academic, clinical, and pharmaceutical research. Here, EpiCypher will develop QuantiNucTM assays, a breakthrough epigenetics platform to quantify single and combinatorial chromatin modifications directly on nucleosomes from cells or plasma samples. The innovation of this proposal includes the a) application of designer nucleosomes (dNucs) to systematically identify top-performing detection reagents and to serve as quantitative assay standards, b) development of recombinant EpiSensors for unbiased detection of DNA and DNAme, and c) development of a proprietary targeted sample processing method for high-throughput cell-based assays. Overall, this platform will provide a quantitative, low- cost, and scalable approach to leverage analysis of chromatin modifications (i.e. histone PTMs and/or DNAme) for chromatin research, drug development, and novel biomarker discovery. In Phase I, we developed a QuantiNuc assay targeting combinatorial H3K4me3+H3K27ac, PTMs that are co-enriched at actively expressed genes. We validated the specificity and performance of this QuantiNuc assay by establishing key analytical parameters and applying the assay to quantify levels of H3K4me3+H3K27ac nucleosomes from human plasma samples. In Phase II, we will develop new QuantiNuc assays to measure other high-value single and combinatorial chromatin modifications and further validate these assays for use with human plasma samples (i.e. liquid biopsy). In addition, we will develop a novel targeted sample processing method for cell-based QuantiNuc assays, which will streamline the process of cell lysis and chromatin fragmentation to deliver a high- throughput, low-cost approach for clinical research. Finally, we will prepare for commercial launch of QuantiNuc assays by assembling beta-kits and performing internal and external validation testing of both liquid biopsy and cell-based assays, which will be used to develop reliable assay protocols and product literature. Market availability of these assays will transform biomarker discovery and accelerate epigenetic drug development.

Public Health Relevance Statement:
PROJECT NARRATIVE Histone post-translational modifications (PTMs) and DNA methylation (DNAme) on nucleosomes form a complex molecular language that regulates gene transcription, and these chromatin features (and specific combinations of them) are attractive targets for drug and biomarker development. However, there is a lack of simple, low-cost tools to measure global levels of these chromatin features. Here, EpiCypher will develop QuantiNucTM assays, a breakthrough epigenetics platform to quantify single and combinatorial chromatin modifications from cells and plasma samples, making chromatin research scalable and highly accessible.

Project Terms:
Posttranslational Protein Processing; Protein Modification; Post-Translational Protein Processing; Reagent; Research; Specificity; Tail; Testing; Time; Genetic Transcription; Gene Transcription; RNA Expression; Transcription; Work; Measures; Site-Directed Mutagenesis; Site-Specific Mutagenesis; Targeted DNA Modification; Targeted Modification; base; Clinical; Phase; Individual; Recovery; Malignant Cell; cancer cell; tool; DNA Methylation; Complex; Protocol; Protocols documentation; In Situ; Services; Performance; novel; Coding System; Code; Regulation; Sampling; response; assay development; drug development; Genomics; drug discovery; chromatin modification; histone modification; Molecular Interaction; Binding; Pharmaceutical Agent; Pharmaceuticals; Pharmacological Substance; Pharmacologic Substance; Lysis; Cytolysis; Dose; Data; Detection; Human Pathology; Recombinants; Epigenetic Process; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Validation; Preparation; Molecular; Modification; Development; developmental; cost; innovation; innovate; innovative; Cancerous; combinatorial; novel marker; new marker; novel biomarker; genome-wide; genome scale; genomewide; stability testing; ChIP-seq; ChIP Sequencing; chromatin immunoprecipitation-sequencing; Drug Targeting; biomarker development; biomarker discovery; epigenetic drug; epigenetic modifying drugs; liquid biopsy; genomic signature; genomic classifier; detection limit; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; Genes; Histones; Human; Modern Man; Language; Literature; Methods; Molecular Conformation; Molecular Configuration; Molecular Stereochemistry; conformation; conformational state; Nucleosomes; Blood Plasma; Plasma Serum; Reticuloendothelial System, Serum, Plasma; Plasma; Blood Plasma Cell; Plasmacytes; plasmocyte; Plasma Cells; Post-Translational Modification Protein/Amino Acid Biochemistry; Post-Translational Modifications; Post-Translational Protein Modification; Posttranslational Modifications; Antibodies; Automation; Biological Assay; Assay; Bioassay; Biologic Assays; Malignant Neoplasms; Cancers; Malignant Tumor; malignancy; neoplasm/cancer; Cell physiology; Cell Function; Cell Process; Cellular Function; Cellular Physiology; Cellular Process; Subcellular Process; Cells; Cell Body; Chromatin; Clinical Research; Clinical Study; Disease; Disorder; Pharmacotherapy; Drug Therapy; drug treatment