New York Genome Center Inc

NEW YORK CENTER FOR COLLABORATIVE RESEARCH IN COMMON DISEASE GENOMICS

CENTER FOR INTEGRATED CELLULAR ANALYSIS

NEW YORK GENOME CHARACTERIZATION CENTER: SOMATIC MOSAICISM ACROSS HUMAN TISSUES - PROJECT SUMMARY/ABSTRACT - NEW YORK GENOME CHARACTERIZATION CENTER LARGE-SCALE SEQUENCING EFFORTS OVER THE LAST TWO DECADES HAVE BEEN FOCUSED ON GENERATING DNA SEQUENCE DATASETS FROM READILY AVAILABLE TISSUES SUCH AS BLOOD OR SALIVA TO IDENTIFY GERMLINE VARIANTS ASSOCIATED WITH DISEASE PHENOTYPES. HOWEVER, LIMITED PROGRESS HAS BEEN MADE IN CHARACTERIZING SOMATIC VARIANTS IN HEALTHY TISSUES AND THEIR CONTRIBUTION TO HEALTH AND DISEASE OVER THE COURSE OF THE HUMAN LIFESPAN. SOMATIC VARIATION HAS HISTORICALLY BEEN STUDIED IN THE CONTEXT OF TUMOR BIOLOGY; HOWEVER, THERE IS MOUNTING EVIDENCE THAT SOMATIC VARIATION PLAYS AN IMPORTANT ROLE IN THE AGING PROCESS, AS WELL AS IN CARDIOVASCULAR, NEURODEGENERATIVE, IMMUNOLOGIC, AND NEURODEVELOPMENTAL DISEASES. THERE IS THEREFORE A CRITICAL NEED TO CHARACTERIZE THE SOMATIC VARIANT LANDSCAPE IN HEALTHY HUMAN TISSUES IN INDIVIDUALS OF DIVERSE RACE AND ETHNICITY ACROSS THE HUMAN LIFESPAN. THE SOMATIC MOSAICISM ACROSS HUMAN TISSUES (SMAHT) PROGRAM WILL ADDRESS THIS GAP BY ESTABLISHING A COHESIVE NETWORK THAT WILL WORK TOGETHER TO CREATE HIGH-QUALITY SOMATIC VARIANT CATALOG; A CATALOG THAT IS BROADLY SHAREABLE ACROSS THE SCIENTIFIC COMMUNITY AND THAT ENABLES STUDIES INVESTIGATING THE RATES AND PATTERNS OF SOMATIC MOSAICISM ACROSS CELL POPULATIONS AND TISSUES, THAT CAN ELUCIDATE THE MECHANISMS UNDERLYING CLONAL DEVELOPMENT, EVOLUTION, AND EXPANSION, AND THAT ENABLES STUDIES OF THE ROLE OF SOMATIC MUTATION IN DISEASE PATHOGENESIS AND PROGRESSION. THE NEW YORK GENOME CHARACTERIZATION CENTER (NYGCC) WILL WORK COLLABORATIVELY WITH OTHER SMAHT NETWORK CENTERS TO GENERATE A HIGH-QUALITY SOMATIC VARIANT CATALOG USING THREE CORE HIGH-DEPTH SEQUENCING ASSAYS: DUPLEX WHOLE GENOME SEQUENCING (WGS), MRNA SEQUENCING, AND LONG- READ OXFORD NANOPORE WGS. THESE THREE CORE ASSAYS WILL PROVIDE AN UNPRECEDENTED AND COMPREHENSIVE VIEW OF SOMATIC MUTATIONS ACROSS A VARIETY OF HEALTHY TISSUES. THE DATA FROM DEEP WGS WILL ENABLE DISCOVERY OF SOMATIC SNVS, INDELS, MOBILE ELEMENTS, COPY NUMBER CHANGES, AND STRUCTURAL VARIANTS. THE RNA SEQUENCING DATA WILL BE USED TO CONFIRM THE PRESENCE OF THOSE VARIANTS THAT FALL IN EXPRESSED GENES, AND FURTHER EVALUATE THEIR EFFECT ON SPLICING. THE LONG READ WGS SEQUENCING WILL BE USED AS A COROLLARY TO SHORT READ WGS TO CONFIRM AND ENHANCE DISCOVERY OF MOBILE ELEMENTS, COPY NUMBER CHANGES AND STRUCTURAL VARIANTS. TO THESE CORE ASSAYS WE PROPOSE ADDING SINGLE CELL WGS SEQUENCING USING DIRECT LIBRARY PREPARATION PLUS (DLP+) AND GENOTYPING OF TRANSCRIPTOMES (GOT). DLP+ IS AN AMPLIFICATION-FREE SINGLE CELL WGS ASSAY THAT ALLOWS HIGH SENSITIVITY DETECTION OF COPY NUMBER CHANGES, LOSS OF HETEROZYGOSITY, AND STRUCTURAL VARIATION. IT FURTHER ENABLES THE STUDY OF REPLICATION TIMING, CLONAL EXPANSION AND FITNESS AND IS COMPATIBLE WITH POOLED PSEUDO-BULK ANALYSIS TO COMPARE AGAINST DEEP BULK WGS. THE GENOTYPING OF TRANSCRIPTOMES ASSAY WILL ALLOW US TO EXPLORE, FOR EXPRESSED SOMATIC VARIANTS, THE CELL TYPE OR LINEAGE IN WHICH THEY OCCURRED AND BY PAIRING WITH SINGLE CELL EXPRESSION DATA (AND CELL SURFACE MARKER DETECTION AND LONG READ TRANSCRIPT SEQUENCING) THE FUNCTIONAL EFFECTS OF THESE MUTATIONS.

INTEGRATING SPATIAL MULTI-OMICS AND CLINICAL COVARIATES TO IDENTIFY MECHANISMS OF DISEASE IN ALS-FTD

IN SITU FUNCTIONAL GENOMICS TO UNDERSTAND TRANSCRIPTIONAL REGULATION

DELINEATING THE NETWORK EFFECTS OF MENTAL DISORDER-ASSOCIATED VARIANTS USING CONVEX OPTIMIZATION METHODS - PROJECT SUMMARY/ABSTRACT DRIVEN BY INTERNATIONAL OPEN SCIENTIFIC COLLABORATION THROUGH GROUPS SUCH AS THE PSYCHIATRIC GENOMICS CONSORTIUM (PGC, IN WHICH CO-I MULLINS IS A LEADING ANALYST) BOTH GENOME-WIDE ASSOCIATION STUDIES (GWAS) AND WHOLE EXOME AND GENOME SEQUENCING STUDIES OF NEUROPSYCHIATRIC DISORDERS (NPDS) ARE RAPIDLY INCREASING IN SAMPLE SIZE. WITH THIS INCREASED SAMPLE SIZE COMES INCREASED STATISTICAL POWER TO DETECT MANY MORE, SMALLER GENETIC EFFECTS ON DISEASE RISK, KNOWN AS THE POLYGENIC COMPONENT. THE CHALLENGE NOW IS TO UNDERSTAND WHAT THESE FINDINGS TELL US ABOUT NPD RISK, ETIOLOGY AND BIOLOGY. HERE WE PROPOSE A SUITE OF METHODS FOR MULTI-TRAIT ANALYSIS TO DETERMINE UNDERLYING LATENT STRUCTURE, CAUSAL NETWORKS OF GENES AND TRAITS, AND ENRICHED DATA-DERIVED REGULATORY PATHWAYS. WE MAKE EXTENSIVE USE OF CONVEX OPTIMIZATION METHODS THAT ALLOW BOTH COMPUTATIONAL EFFICIENCY AND GUARANTEES ON REPRODUCIBILITY. IN AIM 1 WE WILL DECOMPOSE A WIDE RANGE OF NPDS AND THEIR SUBPHENOTYPES INTO SHARED AND UNIQUE GENETIC COMPONENTS USING A NOVEL CONVEX FORMULATION OF OBSERVED-WEIGHTED PRINCIPAL COMPONENTS ANALYSIS (PCA) AND DEVELOP EXTENSIONS TO HANDLE SAMPLE OVERLAP, LINKAGE DISEQUILIBRIUM (LD), AND DIFFERENT ANCESTRIES. IN AIM 2 WE WILL EXTEND AND CUSTOMIZE OUR EXISTING WORK ON CAUSAL NETWORK INFERENCE USING BICONVEX OPTIMIZATION TO ESTIMATE BOTH CIS AND TRANS GENE REGULATORY NETWORKS IN THE BRAIN USING LARGE-SCALE UNIFORMLY PROCESSED CHROMATIN ACCESSIBILITY AND EXPRESSION QUANTITATIVE TRAIT LOCI (QTLS). WE WILL REGULARIZE ESTIMATES OF CIS INTERACTIONS USING CHROMATIN CONFORMATION DATA, MODEL LATENT GENETIC CONFOUNDERS IN THESE NETWORKS USING AN EXPECTATION-MAXIMIZATION (EM) APPROACH AND ESTIMATE NETWORKS OVER BOTH GENES AND NPDS IN ORDER TO DETERMINE THE MOST DIRECT CAUSES (“CORE” GENES IN THE OMNIGENIC MODEL). IN AIM 3 WE WILL ANALYZE BOTH RARE AND COMMON GENETIC ASSOCIATIONS IN THEIR GENE REGULATORY NETWORK CONTEXT. BORROWING FROM CANCER GENOMICS, WE WILL USE HEAT DIFFUSION MODELS TO PROPAGATE STATISTICAL INFORMATION ON THE LOCAL NETWORK OVER BOTH GENES AND REGULATORY ELEMENTS (RES) AND THEN USE GRAPH CLUSTERING ALGORITHMS TO EXTRACT “HOT” SUBNETWORKS, CORRESPONDING TO PATHWAYS IMPLICATED IN THE NPD UNDER STUDY. THE METHODS WE DEVELOP FOR THESE ANALYSES WILL BE MADE PUBLICLY AVAILABLE UNDER SOURCE LICENSES WITH EXTENSIVE SUPPORT IN TERMS OF DOCUMENTATION, TUTORIALS, AND VIGNETTES. THROUGH THIS WE HOPE TO EMPOWER FUTURE “POST-GWAS” ANALYSES THAT CAN LEVERAGE THE GENETIC, SUBPHENOTYPE AND TRAIT NETWORKS UNDERLYING HUMAN NEUROPSYCHIATRIC HEALTH, AND EVENTUALLY POINT THE WAY TO THERAPEUTIC INTERVENTIONS.

SPATIALLY RESOLVED DYNAMICS OF MOLECULAR PATHOLOGY AND INTERCELLULAR INTERACTIONS IN AMYTROPHIC LATERAL SCLEROSIS

MULTISCALE GENOME ENGINEERING TO MAP CIS-REGULATORY VARIANTS IN HUMAN AND MOUSE - PROJECT SUMMARY GENOME-WIDE ASSOCIATION STUDIES (GWAS) HAVE LINKED 1000S OF GENOMIC LOCI WITH HUMAN TRAITS AND DISEASES. HOWEVER, THE MECHANISTIC INNER WORKINGS OF THESE LOCI ARE LARGELY UNKNOWN, LEAVING THE PRINCIPAL GOAL OF GWAS – ILLUMINATING THE CAUSAL BIOLOGICAL ETIOLOGY OF HERITABLE PHENOTYPES – UNFULFILLED. MOST GWAS LOCI OCCUR WITHIN NONCODING REGIONS OF THE GENOME WHOSE FUNCTIONAL IMPACT ON GENE REGULATION IS DIFFICULT TO UNRAVEL. HERE, WE PROPOSE TO DEVELOP A HIGH-THROUGHPUT, INTEGRATED GENOME ENGINEERING TOOLBOX TO BUILD CONTEXT-SPECIFIC MAPS OF ENHANCERS AND VARIANTS FOR IMMUNE TRAITS AND AUTOIMMUNE DISORDERS. OUR MULTI-PI TEAM CONSISTS OF EXPERTS IN COMPLEMENTARY FIELDS: MOLECULAR GENOMICS AND CRISPR SCREENS, LARGE-SCALE HUMAN GENETICS AND FUNCTIONAL GENOMICS DATA ANALYSIS, IMMUNOLOGY, STATISTICAL MODELING, AND SINGLE-CELL MULTIOMICS. SPECIFICALLY, WE PROPOSE TO: 1) IDENTIFY GENES AND CIS-REGULATORY ELEMENTS (CRES) RELEVANT FOR T CELL FUNCTION. T CELLS ARE A CENTRAL CELL TYPE IMPLICATED IN MULTIPLE AUTOIMMUNE DISEASES. WE WILL FIRST PERFORM GENOME-WIDE LOSS- AND GAIN-OF-FUNCTION SCREENS FOR 9 PHENOTYPES REFLECTING T-CELL DIFFERENTIATION AND ACTIVATION USING PRIMARY HUMAN T CELLS. FOR TOP- RANKED GENES, WE WILL INTERROGATE CRES NEAR EACH GENE AND EXPLORE THEIR MECHANISMS VIA SINGLE-CELL PROFILING AND SATURATION MUTAGENESIS. 2) BUILD A CONTEXT-SPECIFIC ENHANCER MAP OF GWAS LOCI IN T CELLS. WE WILL TEST 1,000 CANDIDATE CRES THAT OVERLAP GWAS LOCI USING CRISPRI/A SCREENS IN THE SAME PRIMARY T-CELL SYSTEM, COMPLEMENTED BY SINGLE-CELL ECCITE-SEQ TO MEASURE EFFECTS ON THE TRANSCRIPTOME AND SURFACE PROTEOME. THIS WILL PRODUCE A COMPREHENSIVE MAP OF REGULATORY ELEMENTS FOR A LARGE NUMBER OF LOCI, AND THEIR CONTEXT-SPECIFIC IMPACT ON TRANSCRIPTOMIC AND CELLULAR PHENOTYPES. THEN, WE WILL CONSTRUCT A CONTEXT-SPECIFIC VARIANT MAP OF REGULATORY ELEMENTS IN T CELLS BY INSERTING SPECIFIC ALLELES VIA BASE EDITING AT 100 VALIDATED CRES. THIS WILL PRODUCE A FINE-RESOLUTION MAP OF REGULATORY SITES WITHIN CRES. 3) TEST 100 SYNTENIC CRES FROM IN MOUSE MODELS OF GUT HOMEOSTASIS AND INFLAMMATION IN VIVO. WE WILL FOCUS ON T-CELL TISSUE ACCUMULATION (REFLECTING ACTIVATION AND MIGRATION) AND ALTERATIONS IN TRANSCRIPTIONAL AND CELL SURFACE PHENOTYPES OF THE MIGRATING CELLS. BY DOING SO, WE WILL DETERMINE IF THE RELEVANT VARIANTS HAVE SIMILAR ROLES IN HUMAN DISEASE AND PROVIDE PATHWAYS TOWARDS TARGETING THE PATHOGENIC FUNCTIONS OF THOSE GENES. THROUGH THESE AIMS, WE WILL BUILD A HIGHLY-GENERALIZABLE TOOLKIT FOR MULTI-SCALE INTERROGATION OF NONCODING ELEMENTS AND AN ACCESSIBLE, OPEN, AND REUSABLE RESOURCE OF ENHANCER AND VARIANT EFFECTS ON MOLECULAR, CELLULAR, AND PHYSIOLOGICAL TRAITS. ALTOGETHER, WE WILL ANALYZE THE REGULATORY ARCHITECTURE OF THE GENOME, LEVERAGING OUR DIVERSE PERTURBATIONS AND PHENOTYPIC LAYERS, AND CHARACTERIZE FUNCTIONAL MECHANISMS OF LOCI ASSOCIATED WITH AUTOIMMUNE DISORDERS.

LTBR CARS AS NEXT-GENERATION THERAPIES FOR R/R LYMPHOMA - PROJECT SUMMARY UP TO 50% OF PATIENTS WITH DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL) RELAPSE AFTER FIRST-LINE TREATMENT. CHIMERIC ANTIGEN RECEPTOR (CAR) T-CELLS HAVE RECENTLY EMERGED AS A CURATIVE THERAPY FOR RELAPSED OR REFRACTORY (R/R) DLBCL. HOWEVER, ONLY 35% OF R/R DLBCL PATIENTS TREATED WITH CAR T-CELLS HAVE A DURABLE RESPONSE, AND SURVIVAL IS MEASURED IN MONTHS FOR PATIENTS WHO FAIL TO BENEFIT. IMPROVEMENTS IN CAR T-CELLS ARE URGENTLY REQUIRED TO IMPROVE OUTCOMES. RECENTLY, WE IDENTIFIED THE CELL SURFACE LYMPHOTOXIN BETA RECEPTOR (LTBR) AS A POSITIVE T- CELL REGULATOR THAT ENHANCES CD19 CAR T-CELL EFFICACY IN VITRO AND IN VIVO. LTBR IS TYPICALLY EXPRESSED IN A SUBSET OF MYELOID CELLS BUT ABSENT IN LYMPHOCYTES; HOWEVER WHEN EXPRESSED IN T-CELLS, LTBR INDUCES PROINFLAMMATORY CYTOKINE RELEASE, AND IMPROVES ANTIGEN-SPECIFIC CAR T- AND D T-CELL RESPONSES WITH NO APPRECIABLE OFF-TARGET TOXICITY. BASED ON THESE OBSERVATIONS, WE HYPOTHESIZE THAT LTBR CAN EFFECTIVELY POTENTIATE ANTI-TUMOR ACTIVITY IN R/R LYMPHOMA T-CELLS, REDUCING MARKERS OF T CELL EXHAUSTION AND OUTPERFORMING CURRENT FDA-APPROVED CAR-TS ACROSS R/R DLBCL SUBTYPES. IN AIM 1, WE CHARACTERIZE DIFFERENCES IN EXPRESSION OF T-CELL DIFFERENTIATION, ACTIVATION, AND EXHAUSTION MARKERS AND MYELOID POPULATIONS IN 25 TREATMENT-NAIVE AND 25 R/R DLBCL PATIENT SAMPLES. TO UNDERSTAND IF LTBR CAN SIMILARLY IMPROVE CAR-T RESPONSE IN THE R/R CONTEXT, WE WILL USE SINGLE- CELL PROFILING AND FUNCTIONAL ASSAYS TO TEST AUTOLOGOUS CD19+ CELL KILLING, WITH AND WITHOUT LTBR. IN AIM 2 WE WILL EVALUATE THE IMPACT OF DLBCL SUBTYPE ON CAR T-CELL ACTIVITY BY INTRODUCING LTBR AND CAR T-CELLS INTO MICE XENOTRANSPLANTED WITH MULTIPLE GERMINAL CENTER B-CELL (GCB) AND ACTIVATED B-CELL (ABC) CELL LINES. SINCE T-CELL ACTIVATION AND KINETICS ARE FURTHER INFLUENCED BY PATIENT TUMOR BURDEN, WE WILL ALSO INVESTIGATE THE EFFICACY OF LTBR-CAR T-CELL THERAPY IN A HIGH TUMOR BURDEN CONTEXT AND TEST FOR DURABLE IMMUNE MEMORY AFTER COMPLETE TUMOR REGRESSION. RECENTLY, BY FUSING THE INTRACELLULAR SIGNALING DOMAIN OF LBTR DIRECTLY TO EXISTING (CD28 AND 4-1BB) CARS, WE HAVE DEVELOPED A NOVEL CAR CONSTRUCT WITH MORE POTENT ANTITUMOR RESPONSE. IN AIM 3 USING COMPREHENSIVE SCANNING MUTAGENESIS OF THE LTBR DOMAIN, WE WILL CREATE A LIBRARY OF CAR VARIANTS AND TEST THEIR ABILITY TO IMPROVE TUMOR KILLING, RESISTANCE TO EXHAUSTION AND CYTOKINE SECTION. WE WILL ALSO MEASURE CHANGES IN T-TO-B CELL IMMUNE SYNAPSES AND RESISTANCE TO IMMUNOSUPPRESSION BY MYELOID-DERIVED SUPPRESSOR CELLS (MDSCS) IN THE MOST PROMISING LTBR-CARS. THIS PROJECT IS THE FIRST TO COMPREHENSIVELY CHARACTERIZE T CELLS STATES IN TREATMENT-NAIVE AND R/R DLBCL AND EVALUATE LTBR AS A T-CELL ACTIVATING STRATEGY TO MAXIMIZE INTRINSIC ANTI-TUMOR ACTIVITY IN R/R DLBCL. THERE IS SUBSTANTIAL POTENTIAL FOR OUR WORK TO SERVE AS A BRIDGE FROM LABORATORY STUDIES TO CLINICAL TRIALS AND TO HELP THE 40,000 PATIENTS PER YEAR WITH R/R DLBCL AND OTHER B-CELL NHLS.

LEARNING THE METADATA OF THE CELL WITH SINGLE CELL GENOMICS

NEXTGEN SPATIAL MULTI-OMICS TECHNOLOGIES - PROJECT SUMMARY TISSUE ORGANIZATION AND FUNCTION REQUIRES AN INTRICATE AND DYNAMIC INTERPLAY OF MULTIPLE CELL TYPES AND CELL STATES. SUCH LOCAL AND TISSUE SCALE CELLULAR RELATIONSHIPS ARE THE PRODUCT OF INTERACTING GENE REGULATORY NETWORKS WITHIN CELLS AND SIGNAL TRANSDUCTION BETWEEN CELLS. THIS COMPLEXITY MUST BE BOTH FINELY TUNED TO MAINTAIN HOMEOSTASIS AND RECONFIGURABLE IN RESPONSE TO CHANGING ENVIRONMENTAL OR PHYSIOLOGICAL NEEDS. EPIGENETIC MARKS, TRANSCRIPTIONAL ACTIVITY, RNA PROCESSING, TRANSLATION, POST-TRANSLATIONAL MODIFICATION, AND REGULATED DEGRADATION ARE ALL REQUIRED TO ACHIEVE THIS. ALTHOUGH SUCH MULTIMODAL PROFILING CAN BE CONDUCTED AT A SINGLE CELL LEVEL, CURRENT METHODS ARE NOT ABLE TO MAP THESE MODALITIES SIMULTANEOUSLY IN TISSUES WITH SPATIAL RESOLUTION. ON THE OTHER HAND, THE METHODS THAT DO PROVIDE SPATIAL INFORMATION, LIKE SPATIAL TRANSCRIPTOMICS, ARE NOT ONLY UNIMODAL, BUT ALSO PROHIBITIVELY EXPENSIVE, REQUIRING COSTLY CONSUMABLES OR SPECIALIZED CAPITAL EQUIPMENT. THEREFORE, WE CURRENTLY LACK SPATIAL INFORMATION NEEDED FOR UNDERSTANDING THE CELLULAR MICROENVIRONMENT AND TISSUE ARCHITECTURE. AS SUCH, OUR UNDERSTANDING OF HOW CHROMATIN STATES RELATE TO GENE EXPRESSION IS LIMITED, AND WE HAVE YET TO MEASURE HOW TISSUE-SPECIFIC AND -INDEPENDENT FACTORS INFLUENCE THESE PROCESSES. HERE, WE PROPOSE TO OVERCOME THESE TECHNICAL CHALLENGES, AS WELL AS FINANCIAL BARRIERS, BY DEVISING THE FIRST COST-EFFECTIVE, USER-FRIENDLY PLATFORM FOR SPATIAL MULTI-OMIC PROFILING. WE WILL ACHIEVE THIS BY IMPLEMENTING A TECHNOLOGY DEVELOPMENT STRATEGY THAT IS CAREFULLY DESIGNED SUCH THAT EACH NEW CAPABILITY WE BUILD CAN BE EMPLOYED INDEPENDENTLY TO ENHANCE CAPABILITIES OF SPATIAL TECHNOLOGIES ALREADY IN WIDESPREAD USE, AS WELL AS JOINTLY AS AN INTEGRATED PLATFORM. TOWARDS THIS OVERARCHING GOAL WE PLAN TO INTRODUCE SEVERAL SIGNIFICANT INNOVATIONS BY: (1) DEVELOPING COST- EFFECTIVE AND EASY-TO-USE FABRICATION TECHNIQUES FOR NUCLEIC ACID CAPTURE SURFACES THAT ENCODE SPATIAL INFORMATION AT 1ΜM RESOLUTION ACROSS A LARGE FIELD OF VIEW AND CAN EASILY AND CHEAPLY BE TAILORED TO THE TISSUE AND BIOLOGICAL QUESTION UNDER STUDY; (2) ESTABLISHING A NOVEL ALL-SEQUENCING BASED APPROACH FOR SPATIALLY RESOLVED MULTI-OMICS PROFILING AND VALIDATING ITS PERFORMANCE BY PROFILING THE AGING BRAIN; AND (3) BUILDING A NEW SPATIAL PCR-FREE SEQUENCING METHOD WE TERMED SPATIAL INFERENCE VIA LOCALIZED CONCATEMERIZATION (SILC). SILC WILL OBTAIN SPATIAL INFORMATION BY SEQUENCING ALONE, THEREBY DRAMATICALLY LOWERING BARRIERS TO ENTRY FOR USE OF SPATIAL TECHNOLOGIES. OVERALL, OUR EFFORTS WILL DEMOCRATIZE RESEARCH INVOLVING SPATIAL PROFILING, AND CATALYZE ADVANCES ACROSS A BROAD SWATH OF BIOLOGICAL INVESTIGATION.

SCALABLE MULTI-ANCESTRY FUNCTIONAL GENOMICS OF BLOOD TRAITS AND CARDIOVASCULAR DISEASE - PROJECT SUMMARY CARDIOVASCULAR DISEASE (CVD) IS THE MOST COMMON CAUSE OF MORTALITY IN THE WORLD. GENOME-WIDE ASSOCIATION STUDIES (GWAS) HAVE SUCCESSFULLY IDENTIFIED NUMEROUS GENETIC VARIANTS ASSOCIATED WITH CVD. HOWEVER, MOST VARIANTS DO NOT NECESSARILY CAUSE THE OBSERVED PHENOTYPES, BUT RATHER ARE IN LINKAGE DISEQUILIBRIUM WITH THE TRULY CAUSAL VARIANTS THAT INFLUENCE DISEASE PATHOPHYSIOLOGY VIA LARGELY UNKNOWN MOLECULAR AND CELLULAR MECHANISMS. THUS, THREE CENTRAL CHALLENGES FOR CVD GWAS ARE: 1) IDENTIFYING CAUSAL VARIANTS, 2) UNDERSTANDING WHICH CELL TYPES ARE MOST RELEVANT FOR SPECIFIC VARIANTS, AND 3) IDENTIFYING THE CIS- AND TRANS-REGULATORY TARGET GENES WHOSE EXPRESSION IS MODULATED BY CVD VARIANTS. CURRENTLY, THERE IS NO CONSENSUS ON HOW BEST TO IDENTIFY RELEVANT CELL TYPES AND TARGET GENES. FURTHERMORE, EFFORTS TO CONNECT SPECIFIC NONCODING VARIANTS TO TARGET GENES, SUCH AS CRISPR-BASED INSERTION OF SPECIFIC VARIANTS COUPLED WITH PHENOTYPIC ASSAYS HAVE BEEN HAMPERED BY LOW THROUGHPUT. HERE, WE SEEK TO DEVELOP SCALABLE METHODS TO ADDRESS ALL THREE CENTRAL CHALLENGES FOR CVD DISEASE GWAS, WHILE LEVERAGING THE CONSIDERABLE BENEFITS OF POPULATION-SCALE, MULTI-ANCESTRY GWAS — NAMELY, IMPROVED DISCOVERY OF NOVEL GWAS LOCI AND INCREASED RESOLUTION FOR CAUSAL VARIANTS AT ALREADY KNOWN LOCI. OF THE MANY CELL TYPES AND ORGAN SYSTEMS UNDERLYING CVD PATHOPHYSIOLOGY, WE WILL FOCUS ON BLOOD-RELATED MECHANISMS, WHICH ARE CURRENTLY UNDERSTUDIED DESPITE THE IMPORTANCE OF E.G., IMMUNE RESPONSE AND COAGULATION IN CVD, DEMONSTRATED BY PRIOR WORK AND OUR PRELIMINARY DATA. IN AIM 1, WE WILL PERFORM COLOCALIZATION OF CVD AND BLOOD CELL TRAIT GWASES (AS BLOOD CELLS TRAITS ARE NATURALLY CELL-TYPE-SPECIFIC) AND PRODUCE A REFERENCE ATLAS OF 3D ENHANCER MAPS FOR 13 BLOOD CELL TYPES AND 2 ENDOTHELIAL CELL TYPES IN DONORS OF DIVERSE ANCESTRIES. IN AIM 2, WE WILL COMBINE CRISPR SCREENS AND SINGLE-CELL MULTIOMICS (STING-SEQ) IN BLOOD AND ENDOTHELIAL CELLS TO IDENTIFY CAUSAL VARIANTS AND TARGET GENES FOR CVD. WE WILL FURTHER EXTEND IT BY DEVELOPING BEESTING-SEQ, WHICH COMBINES CYTOSINE AND ADENINE BASE EDITORS WITH A MORE FLEXIBLE CRISPR ENZYME TO INSERT PRECISE SNPS. FOR BOTH STING METHODS, WE WILL USE A THOROUGHLY-VALIDATED COMPUTATIONAL APPROACH TO IDENTIFY CIS AND TRANS TARGET GENES AND REGULATORY NETWORKS. FURTHER, WE WILL DEEPLY VALIDATE TOP VARIANTS USING KEY FUNCTIONAL ASSAYS (ELECTRICAL IMPEDANCE, MIGRATION AND STRESS RESPONSE). THIS PROPOSAL TAKES AN INTERDISCIPLINARY APPROACH WITH A TEAM OF EXPERTS IN NONCODING BIOLOGY AND HIGH-THROUGHPUT SINGLE-CELL CRISPR SCREENS (SANJANA), IN GENETICS AND SYSTEMS BIOLOGY (LAPPALAINEN) AND IN CVD GWAS, CARDIOLOGY AND ENDOTHELIAL CELL FUNCTION (GUPTA). OUR INTEGRATED EXPERIMENTAL AND COMPUTATIONAL APPROACH WILL NOT ONLY REVEAL HOW GENOMIC VARIATION SHAPES CVD RISK, BUT ALSO DEVELOP A GENERALIZABLE TOOLKIT THAT LEVERAGES CUTTING-EDGE 3D GENOME MAPPING, GENE EDITING AND SINGLE-CELL PROFILING TO MAP GENE REGULATORY ELEMENTS, SPECIFIC VARIANTS AND TARGET GENES TO INFORM FUTURE CVD THERAPEUTICS.

ROLES OF SMALL RNAS IN GUARDING GERM CELL GENOMES

STATISTICAL METHODS FOR CHARACTERIZING MOLECULAR MECHANISMS OF HUMAN TISSUE DEVELOPMENT AND DISEASE - PROJECT SUMMARY THE UNIQUE BIOSPECIMENS AND DATA FROM THE DEVELOPMENTAL GTEX (DGTEX) PROJECT CREATE AN EXCITING OPPORTUNITY AND NEED FOR NOVEL METHODS DEVELOPMENT. IN THIS PROJECT, WE PROPOSE TO DEVELOP A SET OF STATISTICAL METHODS AND ANALYTICAL APPROACHES THAT ARE DESIGNED TO EXTRACT INSIGHTS INTO HUMAN AND NON-HUMAN PRIMATE DEVELOPMENT FROM THE MULTI-MODAL AND MULTI-TISSUE DATA FROM DGTEX POST-MORTEM DONORS. ANALYSIS OF THE DGTEX DATA REQUIRES STATISTICAL MODELS THAT CAN TAKE ADVANTAGE OF THE RICH STRUCTURE AND DIVERSITY OF THE DATA ACROSS AGES AND MODALITIES, WHILE ADDRESSING SOME OF THE INHERENT CHALLENGES. MODELS EXPLICITLY INFORMED BY AGE CAN CAPTURE DEVELOPMENTAL TRAJECTORIES OF GENE REGULATION, GENETIC EFFECTS, AND TISSUE STRUCTURE. THE RANGE OF DATA MODALITIES ALSO CREATES AN OPPORTUNITY FOR NOVEL METHODS TO CAPTURE ADDITIONAL EFFECTS AND IMPROVED RESOLUTION AT THE CELL-TYPE, ISOFORM, AND STRUCTURAL LEVELS. HOWEVER, THESE DATA ARE ALSO INHERENTLY COMPLEX, REPRESENTING A MIXTURE OF CELL TYPES ALONG WITH BIOLOGICAL AND TECHNICAL NOISE. THE AMBITIOUS STUDY DESIGN OF DGTEX ALSO COMES WITH CHALLENGES OF DONOR RECRUITMENT THAT LIMIT SAMPLE SIZE. THE METHODS PROPOSED HERE ARE DESIGNED TO LEVERAGE THE BENEFITS OF TEMPORAL MULTI-MODAL DATA, WHILE ADDRESSING DATA COMPLEXITY AND LIMITED SAMPLE SIZE. IN OUR FIRST AIM, WE WILL ANALYZE TRANSCRIPTOME VARIATION ACROSS THE HUMAN LIFESPAN, WITH IMPROVED TRANSCRIPT ANNOTATION AND NEW METHODS TO CHARACTERIZE HOW GENE EXPRESSION, ALTERNATIVE SPLICING AND CELL TYPE COMPOSITION CHANGE DURING DEVELOPMENT AND HOW THEY CONTRIBUTE IN DRIVING PHENOTYPIC CHANGE. SECONDLY, WE WILL USE MULTI-MODAL DATA FROM GTEX TO CAPTURE CHANGES IN GENE REGULATORY NETWORKS DURING DEVELOPMENT. FROM THE DGTEX HISTOLOGY IMAGES AND SPATIAL TRANSCRIPTOMICS DATA WE WILL MODEL DEVELOPMENTAL TRAJECTORIES OF TISSUE STRUCTURES, AND DESCRIBE THEIR MOLECULAR CHARACTERISTICS AS WELL AS ROLE IN DISEASE. IN OUR THIRD AIM WE WILL MAP AND CHARACTERIZE GENETIC REGULATORY VARIATION IN DGTEX AND APPLY PREDICTIVE MODELS FOR IMPROVED PREDICTIONS OF REGULATORY VARIANTS IN PEDIATRIC TISSUES. IN ADDITION TO EMPOWERING BIOLOGICAL DISCOVERY, THIS WORK HAS THE POTENTIAL TO UNCOVER DISEASE RISK FACTORS AND MECHANISMS THAT ORIGINATE OR MANIFEST DURING EARLY LIFE.

STUDYING THE REGULATORY DYNAMICS WITH SINGLE-CELL MULTIOMICS - PROJECT SUMMARY/ABSTRACT A MULTITUDE OF EPIGENOMIC VARIABLES AND MECHANISMS CONTRIBUTE TO CELL-TYPE-SPECIFIC GENE EXPRESSION PROGRAMS, AND THE SPATIOTEMPORAL DYNAMICS OF THESE COMPLEX GENE REGULATORY MACHINERY LAID THE FOUNDATIONS FOR DIVERSE BIOLOGICAL PROCESSES, PARTICULARLY IN DEVELOPMENT, DISEASE, AND AGING. SINGLE-CELL GENOMICS TECHNOLOGIES ALLOWED CAPTURING THE STATIC SNAPSHOTS, SUCH AS TRANSCRIPTOMIC OR EPIGENOMIC STATES OF THE CELLS; WHILE IT REMAINED CHALLENGING TO STUDY THE TEMPORAL DYNAMICS OF THE CELL’S STATE TRANSITION PROCESSES. WE HYPOTHESIZED THAT THE REGULATORY DYNAMICS ARE SHAPED BY THE BALANCE BETWEEN “WRITING” AND “ERASING” OF EPIGENOMIC VARIABLES, AND THUS CAN BE INFERRED FROM MEASURING THE LINKED MOLECULAR LAYERS THAT MAINTAIN REGULATORY EQUILIBRIUMS BY THE DEVELOPMENT OF SINGLE-CELL MULTIOMICS TECHNOLOGIES. STUDYING THE REGULATORY DYNAMICS OF CELL STATE TRANSITION IS PARTICULARLY CHALLENGING IN AGING BRAINS: AGING OF THE BRAIN INVOLVES COMPLEX CELLULAR AND MOLECULAR CHANGES, INCLUDING VARIATIONS IN MOLECULAR SIGNATURES OF CERTAIN CELL TYPES, CHANGES IN CELL POPULATION COMPOSITIONS, AND DECLINED COMMUNICATIONS BETWEEN NEURON CELLS IN THIS TISSUE WITH THE MOST SOPHISTICATED CELLULAR COMPOSITION AND SPATIAL ORGANIZATIONS. AGING CONTRIBUTES TO MANY DISEASES THAT AFFECT ALL ORGAN SYSTEMS AND IS THE GREATEST RISK FACTOR FOR MULTIPLE DISEASES, INCLUDING NEURODEGENERATION AND CANCERS. UNDERSTANDING THE FUNDAMENTAL BIOLOGY OF AGING IS ESSENTIAL FOR THE DEVELOPMENT OF CLINICAL INTERVENTIONS. BUT CURRENT OMICS ANALYSIS OF AGING CAN ONLY CAPTURE THE STATIC PICTURES OF INDIVIDUAL MODALITIES, WHICH CANNOT DIFFERENTIATE WELL-MAINTAINED COMPONENTS (YOUNG) FROM THOSE WHO ARE ABOUT TO LOSE FIDELITY (PRE-DECAY) NOR RECORD THE COMPLEX RELATIONSHIPS BETWEEN DIFFERENT MOLECULE TYPES. IN THIS PROPOSAL, WE AIM TO FUNDAMENTALLY TRANSFORM OUR APPROACHES TO STUDYING THE PRINCIPLES OF CELL STATE TRANSITION, FOCUSING ON THE MOUSE AGING BRAIN AS A MODEL SYSTEM, BY DEVELOPING INNOVATIVE SINGLE-CELL GENOMICS TECHNOLOGIES FOR JOINT ANALYSIS OF THE CELL’S REGULATORY DYNAMICS AND TRANSCRIPTIONAL STATES. FIRSTLY, WE WILL DEVELOP A SET OF SINGLE-CELL MULTIOMICS TOOLS FOR INTEGRATED ANALYSIS OF THE RATES OF FORWARD AND REVERSE REACTIONS IN MAINTAINING THE CELL’S REGULATORY STATES, INCLUDING EPIGENOME (DNA METHYLATION AND ACTIVE DEMETHYLATION) AND DNA DAMAGES (OXIDATIVE DAMAGES AND BASE EXCISION REPAIR) WITH THE TRANSCRIPTIONAL STATES. NEXT, WE WILL DEVELOP A TECHNOLOGY FOR THE DETECTION OF COLOCALIZED REGULATORY ELEMENTS AND THEIR EPIGENETIC STATES JOINTLY WITH TRANSCRIPTOMES FROM SINGLE CELLS, TO EVALUATE THE CELL’S REGULATORY FUNCTIONALITY. FINALLY, WE WILL DEVELOP A MODULARIZED PLATFORM FOR TISSUE-SCALE HIGH-DEFINITION 3-D SPATIAL REGISTRATION OF SINGLE CELLS (AMBER) AND THEN COMBINE IT WITH THESE SINGLE-CELL MULTIOMICS TOOLS TO RECONSTRUCT THE WHOLE TISSUE STRUCTURE WITH MULTIMODAL MOLECULAR PROFILES. WE WILL APPLY OUR METHODS TO INVESTIGATE THE MOLECULAR CHANGES OF AGING IN NERVOUS SYSTEMS WITH 3-D SPATIAL INFORMATION FROM MOUSE MODELS, AND BELIEVE OUR APPROACH IS BROADLY APPLICABLE TO STUDYING REGULATORY DYNAMICS ACROSS VARIOUS BIOLOGICAL SYSTEMS BOTH IN HEALTH AND DISEASES.

REGULATORY MODIFIERS OF CODING VARIANT PENETRANCE VIA HAPLOTYPE EPISTASIS IN HUMAN POPULATIONS AND DISEASES

FINE-MAPPING PSYCHIATRIC DISEASE VARIANTS THAT AFFECT POST-TRANSCRIPTIONAL GENE REGULATION - PROJECT SUMMARY NEUROPSYCHIATRIC DISORDERS (NPD) SUCH AS SCHIZOPHRENIA (SZ), AUTISM SPECTRUM DISORDERS (ASD) AND BIPOLAR DISORDERS (BD) ARE REMARKABLY COMMON, WITH SZ ALONE AFFECTING NEARLY THREE MILLION AMERICANS. DESPITE MORE THAN FIFTY YEARS OF RESEARCH, NO CURES EXIST FOR THESE CONDITIONS AND THE STANDARD OF TREATMENT REMAINS UNSATISFACTORY. GENOME-WIDE ASSOCIATION STUDIES (GWAS) INDICATE THAT, IN ADDITION TO HIGHLY PENETRANT RARE MUTATIONS, NPD RISK ALSO REFLECTS THE IMPACT OF HUNDREDS OF COMMON SINGLE NUCLEOTIDE POLYMORPHISMS WITH SMALL EFFECT SIZES. A MAJOR CHALLENGE IN THE FIELD HAS BEEN ILLUMINATING THE PATHWAYS CONNECTING THESE GENETIC VARIANTS (THE VAST MAJORITY OF WHICH FALL IN NON-CODING SEQUENCES) TO TARGET GENES AND CAUSAL CELLULAR PHENOTYPES. TO UNDERSTAND HOW THESE MYRIAD RISK LOCI CAUSALLY CONTRIBUTE TO DISEASE RISK, IT IS ESSENTIAL TO SCREEN FOR PUTATIVELY CAUSAL VARIANT(S) AND DETERMINE HOW THEY INFLUENCE GENE EXPRESSION, WHICH HAS BEEN SHOWN TO BE CELL-TYPE SPECIFIC, AS WELL AS CELLULAR FUNCTION. RECENT EVIDENCE HAS EMERGED INDICATING A SUBSTANTIAL CONTRIBUTION OF RNA SPLICING VARIATION TO HERITABILITY ACROSS MANY COMPLEX GENETIC DISEASES, INCLUDING SZ. BASED ON OUR PRELIMINARY ANALYSES AND THE WORK OF OTHERS, WE HYPOTHESIZE THAT A SUBSTANTIAL PROPORTION OF NPD GWAS LOCI EXERT THEIR PATHOGENIC EFFECTS ON NEURONAL FUNCTION BY IMPACTING RNA: ITS STRUCTURE, MODIFICATIONS, PROTEIN INTERACTIONS AND SPLICING. TO TEST THIS, WE WILL APPLY NOVEL TOOLS AND MACHINE LEARNING METHODS TO PREDICT AND QUANTIFY RNA SPLICING IN THE LARGEST SZ, ASD AND BD GWAS, IN ORDER TO PREDICT SPLICING QUANTITATIVE TRAIT LOCI (SQTLS, AIM 1). TO CONFIRM TRUE EFFECTS ON EXON INCLUSION INDEPENDENTLY IN GLUTAMATERGIC AND GABAERGIC NEURONS (I.E., THE MAJOR CELL-TYPES IMPACTED IN NPD), UP TO SEVERAL THOUSAND OF THE PREDICTED SPLICE VARIANTS WILL BE TESTED BY A MASSIVELY PARALLEL REPORTER ASSAY, MAPSY (AIM 2). FINALLY, IN ORDER TO EVALUATE THE CELL-TYPE-SPECIFIC IMPACT OF PUTATIVE CAUSAL SQTLS IDENTIFIED IN AIMS 1 AND 2 ON NEURONAL MATURATION AND SYNAPTIC FUNCTION, WE WILL USE CRISPR GENE EDITING TO ENGINEER THESE MUTATIONS WITHIN HUMAN INDUCED PLURIPOTENT STEM CELL (HIPSC)-BASED MODELS OF BOTH NEURAL CELL TYPES (AIM 3). OUR OVERARCHING GOAL IS TO MAP AND FUNCTIONALLY EVALUATE THE NPD-GWAS LOCI THAT IMPACT ALTERNATIVE SPLICING AND NEURONAL FUNCTION. OUR WORK MAY IMPACT THE FIELD BY DELIVERING NEW INSIGHTS INTO THE ROLE OF COMMON VARIANTS IN NPD PATHOPHYSIOLOGY, WHICH COULD INFORM WAYS OF IMPROVING DIAGNOSTICS, PREDICTING CLINICAL TRAJECTORIES, AND DEVELOPING NOVEL THERAPEUTIC INTERVENTIONS.

ADVANCED DEVELOPMENT OF LANCET, AN EMERGING TOOL FOR COMPLEX VARIANT CALLING IN CANCER GENOMICS - ABSTRACT ONE OF THE CENTRAL CHALLENGES IN CANCER GENOMICS IS THE ABILITY TO ACCURATELY DETECT SOMATIC MUTATIONS IN HETEROGENEOUS TUMORS, AND PRECISELY DETERMINE THEIR CLONAL ORIGIN AND EVOLUTION. THIS FUNDAMENTAL KNOWLEDGE IS CENTRAL TO THE DISCOVERY OF NEW CANCER THERAPIES. IN RECENT YEARS, REDUCTIONS IN THE COST OF WHOLE-GENOME AND WHOLE-EXOME SEQUENCING HAVE ENABLED RESEARCHERS TO ADDRESS THESE QUESTIONS IN UNPRECEDENTED DETAIL. HOWEVER, A MAJOR LIMITATION IN THE FIELD HAS BEEN A PAUCITY OF METHODS FOR VARIANT CALLING THAT EXTEND BEYOND IDENTIFYING SIMPLE SINGLE-NUCLEOTIDE VARIANTS (SNVS) AND SMALL INDELS TO ALLOW THE CHARACTERIZATION OF COMPLEX STRUCTURAL CHANGES THAT ALSO PLAY A SIGNIFICANT ROLE IN TUMORIGENESIS AND CANCER PROGRESSION. INDELS OF MORE THAN A FEW BASES ARE CHALLENGING TO DISCOVER WITH TYPICALLY USED ALIGNMENT-BASED METHODS. IN ADDITION, MOST VARIANT CALLERS ANALYZE TUMOR AND NORMAL DATA SEPARATELY, WHICH CAN INTRODUCE FALSE POSITIVES SUCH AS WHEN A MUTATION SHOWS PARTIAL SUPPORT IN THE NORMAL SAMPLE. TOWARDS ADDRESSING THESE SHORTCOMINGS, WE RECENTLY INTRODUCED LANCET, A NEW SOMATIC VARIANT CALLER DEVELOPED UNDER THE AUSPICES OF THE ITCR R21. LANCET LEVERAGES LOCAL ASSEMBLY AND JOINT ANALYSIS OF TUMOR-NORMAL PAIRED DATA USING REGION-FOCUSED COLORED DE BRUIJN GRAPHS, WITH ON- THE-FLY REPEAT COMPOSITION ANALYSIS AND A SELF-TUNING K-MER STRATEGY. THIS RESULTS IN RELATIVELY REDUCED REFERENCE BIAS; AN IMPROVED ABILITY TO DETECT VARIATIONS THAT SIGNIFICANTLY DIVERGE FROM THE REFERENCE CHROMOSOME REPRESENTATIONS; A REDUCTION IN THE SCALE OF THE ANALYSIS, LEADING TO INCREASED POWER AND SENSITIVITY TO DETECT VARIANTS THROUGH LOCALIZED, COMPREHENSIVE GRAPH EXPLORATION; AND DYNAMIC ADJUSTMENT OF CALLING BEHAVIOR ACCORDING TO THE SEQUENCE CONDITIONS OF EACH GENOMIC REGION. IN TESTING, LANCET SHOWS SUPERIOR PERFORMANCE TO ALL MAJOR ALIGNMENT-BASED METHODS IN TERMS OF ACCURACY, PARTICULARLY IN THE DETECTION OF ‘TWILIGHT ZONE’ INDELS (30- 250 BP). GIVEN ITS CONTINUED ADOPTION AND SUCCESSFUL APPLICATION IN OVER A DOZEN HIGH-IMPACT PUBLICATIONS, LANCET IS POISED FOR MORE ADVANCED DEVELOPMENT TO ENABLE CONTINUED IMPROVEMENTS IN ITS VARIANT CALLING POWER, PRECISION, AND ANALYTICAL CAPABILITIES. SPECIFICALLY, LANCET IS CURRENTLY LIMITED BY LONGER RUNTIMES THAN ALIGNMENT- BASED METHODS, REDUCED SENSITIVITY FOR LONGER INSERTIONS, LACK OF INTERACTIVE VISUALIZATION OF THE COLORED DE BRUIJN GRAPH, AND THE INABILITY TO JOINTLY ANALYZE LONGITUDINAL DATA. TO ADDRESS THESE LIMITATIONS, WE PROPOSE THE FOLLOWING SPECIFIC AIMS: 1) INCREASE COMPUTATIONAL PERFORMANCE AND FACILITATE USER ADOPTION AND THIRD-PARTY DEVELOPMENT; 2) ADD NEW FEATURES AND ENHANCEMENTS TO IMPROVE VARIANT DETECTION, PHASING, AND DATA VISUALIZATION; AND 3) ENABLE JOINT ASSEMBLY AND ANALYSIS OF LONGITUDINAL DATA. IMPACT: WITH ADDITIONAL DEVELOPMENT, THE NEXT ITERATION OF LANCET WILL FEATURE ADVANCED ALGORITHMS FOR FAST, EFFICIENT, ACCURATE, LOCALIZED, USER-FRIENDLY, AND APPLICATION- MODIFIABLE VARIANT ANALYSIS OF PHASED GENOME-WIDE TIMESERIES DATA, ESTABLISHING IT AS ONE OF THE LEADING METHODS FOR VARIANT CALLING IN CANCER RESEARCH.

2/5 CLINICAL OUTCOME PREDICTION OF PSYCHOSIS FROM ELECTRONIC HEALTH RECORDS (COPPER) - PROJECT SUMMARY CLINICAL PREDICTORS ARE NOW FIRMLY INCORPORATED INTO ROUTINE STANDARD-OF-CARE IN MANY FIELDS OF MEDICINE, IN CONTRAST WITH PSYCHIATRY WHERE QUANTITATIVE PREDICTORS THAT GUIDE CLINICAL DECISION-MAKING REMAIN EXTREMELY LIMITED. PSYCHOSIS-RELATED DISORDERS ARE RESPONSIBLE FOR A SUBSTANTIAL PUBLIC HEALTH BURDEN, FOR WHICH THERE ARE SIGNIFICANT UNMET NEEDS THAT WOULD BE SUBSERVED BY CLINICAL PREDICTORS. FOR EXAMPLE, LONG-TERM OUTCOMES VARY WIDELY AND IDENTIFYING INDIVIDUALS WITH POOR OR ADVANTAGEOUS FUTURE OUTCOMES WOULD HELP TO OPTIMIZE TREATMENT PLANNING AND RESOURCE ALLOCATION. FURTHERMORE, ANTIPSYCHOTICS ARE ASSOCIATED WITH ADVERSE SIDE EFFECTS, SUCH AS INCREASED RISK OF DIABETES. IN THIS APPLICATION, WE PROPOSE TO USE MACHINE LEARNING APPROACHES TO BUILD PREDICTORS AND IDENTIFY SUBTYPES OF CLINICAL OUTCOMES AMONG INDIVIDUALS WITH SCHIZOPHRENIA, THROUGH INTEGRATION OF LONGITUDINAL ELECTRONIC HEALTH RECORDS (EHRS), DIMENSIONAL PHENOTYPING, AND GENETIC ANALYSES. WE WILL ALSO EXPLORE THE PSYCHOSOCIAL AND ETHICAL IMPLICATIONS OF PSYCHIATRIC CLINICAL PREDICTORS. OUR LONG-TERM OBJECTIVE IS TO ADVANCE THE GOALS OF PRECISION PSYCHIATRY TO ACHIEVE INDIVIDUALIZED TREATMENT PLANNING, OUTCOME MONITORING, AND PREVENTIVE INTERVENTIONS. WE PROPOSE THE FOLLOWING SPECIFIC AIMS: AIM 1: LEVERAGE TWO INDEPENDENT EHR DATABASES FOR OUTCOME PREDICTION AND SUB-CLASSIFICATION OF PSYCHOSIS-RELATED DISORDERS. (A) WE WILL USE THE LONGITUDINAL PSYCKES AND MARKETSCAN DATABASES TO BUILD MACHINE LEARNING-BASED INDIVIDUAL-LEVEL PREDICTION MODELS TO FORECAST THE ONSET OF FOUR MAJOR PROGNOSTIC OUTCOMES: TREATMENT RESPONSE (ANTIPSYCHOTIC RESISTANCE), ILLNESS SEVERITY (LONG-TERM HOSPITALIZATION), MEDICAL COMORBIDITY (DIABETES), AND DIAGNOSTIC TRANSITION FROM A PSYCHOSIS-RELATED DISORDER TO SCHIZOPHRENIA. (B) WE WILL PERFORM COHORT-LEVEL ANALYSES USING UNSUPERVISED METHODS TO DISCOVER NOVEL PSYCHOSIS-RELATED DIAGNOSIS AND PROGNOSIS SUBTYPES. AIM 2: ENHANCE PREDICTIVE MODELING THROUGH DIMENSIONAL PHENOTYPING AND WHOLE GENOME SEQUENCING. (A) WE WILL RECRUIT N = 10,000 PATIENTS WITH SCHIZOPHRENIA FROM THE PSYCKES DATABASE POPULATION FOR ENRICHED DATA COLLECTION: 1) DIMENSIONAL PHENOTYPING (COGNITION, EXPOSOME, AND SOCIAL DETERMINANTS OF HEALTH), AND 2) WHOLE GENOME SEQUENCING TO ENABLE CALLING OF RARE VARIANTS, STRUCTURAL VARIANTS, AND COMMON VARIANTS (POLYGENIC RISK). (B) WE WILL INVESTIGATE THE EXTENT TO WHICH DIMENSIONAL PHENOTYPES AND GENOMIC DATA CAN IMPROVE THE MODELS DEVELOPED IN AIM 1. AIM 3: EXPLORE THE PSYCHOSOCIAL AND ETHICAL IMPLICATIONS OF PSYCHIATRIC CLINICAL PREDICTORS. (A) WE WILL SURVEY A SUBSET OF PATIENTS AND THEIR CLINICIANS REGARDING THEIR ATTITUDES TOWARDS IMPLEMENTATION OF CLINICAL OUTCOME PREDICTORS. (B) WE WILL RETURN PATHOGENIC FINDINGS TO PATIENTS THROUGH GENETIC COUNSELING AND SURVEY THE EXPERIENCE OF PATIENTS AND THEIR CLINICIANS ON THEIR EMOTIONAL REACTIONS AND PERCEPTIONS OF IMPAIRMENT, TREATABILITY, AND LIFE-PLANNING.

SINGLE NUCLEUS TRANSCRIPTOMIC PROFILING IN POSTMORTEM SPINAL CORD OF ALS PATIENTS

PRIVACY PRESERVATION IN TRANSCRIPTOMIC DATA ANALYSIS - PROJECT SUMMARY UNDERSTANDING THE MECHANISM BEHIND CELLULAR ACTIVITIES REQUIRES LARGE-SCALE MINING OF GENETIC AND TRANSCRIPTOMIC OBSERVATIONS FROM LARGE AMOUNTS OF HUMAN DATA. WIDESPREAD AND EASY ACCESS TO SUCH DATA IS IMPERATIVE TO MAKE BIOLOGICAL CONNECTIONS BETWEEN GENES AND DISEASES. HOWEVER, THERE IS A DIRECT CONFLICT BETWEEN PROTECTING THE PRIVACY OF PATIENTS AND RESEARCH PARTICIPANTS AND BROAD SHARING OF GENETIC AND TRANSCRIPTOMIC DATA FOR BIOMEDICAL ADVANCES. IN ORDER TO ADDRESS THESE PRIVACY CONCERNS DURING TRANSCRIPTOMIC ANALYSIS, WE PROPOSE TO TAKE ADVANTAGE OF CRYPTOGRAPHIC APPROACHES THAT ENABLE DIRECT COMPUTATIONS ON ENCRYPTED DATA WITHOUT REVEALING THE SENSITIVE INFORMATION IN THEM. WE WILL CREATE AN EVOLVING AND MODULAR TOOL SUITE TO PRESERVE PRIVACY; THIS SUITE WILL HAVE THE ABILITY TO BE ADOPTED TO NEW DATA MODALITIES AND ANALYSIS NEEDS AS THEY ARISE. IN PARTICULAR, WE PROPOSE TO DEVELOP A SERIES OF TOOLS THAT CAN QUANTIFY THE BULK TRANSCRIPT AND SINGLE-CELL GENE EXPRESSION AND PERFORM EQTL MAPPING ON THE ENCRYPTED GENOTYPES IN A SHARED SERVER AND CLOUD SETTING. THE PROPOSED TOOLS WILL HELP PREVENT FUTURE CATASTROPHIC PRIVACY LEAKS, WHICH MAY RESULT IN A LOSS OF ACCESS TO ALL MEDICALLY ACTIONABLE DATA. OUR LONG-TERM GOAL IS TO DEMOCRATIZE DATA ACCESS FOR ALL RESEARCHERS AND CREATE TRUST BETWEEN PATIENTS AND RESEARCHERS, THUS INCREASING PARTICIPATION IN STUDIES.

MASSIVELY PARALLEL MULTI-MODAL SINGLE-CELL PHENOTYPING USING A PORTABLE DEVICE

STUDYING THE MAMMALIAN REGULATORY CIRCUITS BY DEVELOPING SINGLE-CELL MULTI-OMICS TECHNOLOGIES - PROJECT SUMMARY/ABSTRACT HOW THE IDENTICAL GENOME SEQUENCE PRODUCES DIVERSE CELL TYPES DURING DEVELOPMENT REMAINS A FUNDAMENTAL QUESTION IN BIOLOGY. RECENT TECHNOLOGY ADVANCEMENTS IN SINGLE-CELL GENOMICS PROVIDED EXCELLENT OPPORTUNITIES TO STUDY THE MOLECULAR PROFILES DURING DEVELOPMENT AND IN DISEASE AT UNPRECEDENTED RESOLUTION. HOWEVER, MONITORING INDIVIDUAL MODALITIES FROM SINGLE CELLS AT A TIME RUNS THE RISK OF OBTAINING ONLY PARTIAL PICTURES FROM THE COMPLEX REGULATORY NETWORK. MULTI-MODAL SINGLE-CELL GENOMICS TOOLS WOULD BE DESIRED TO OVERCOME THIS LIMITATION. I RECENTLY INVENTED A METHOD FOR ULTRA-HIGH-THROUGHPUT JOINT ANALYSIS OF OPEN CHROMATIN AND TRANSCRIPTOME FROM THE SAME SINGLE CELLS (PAIRED-SEQ) AND DEMONSTRATED ITS POTENTIAL FOR COMPREHENSIVE INVESTIGATIONS OF THE CELL- TYPE-SPECIFIC REGULATORY PROGRAMS FROM HETEROGENOUS BRAIN TISSUES. IN THIS K99/R00 APPLICATION, I PROPOSE TO FURTHER DEVELOP A SET OF NEW SINGLE-CELL MULTI-OMICS TOOLS TO STUDY THE DYNAMIC AND CELL-TYPE-SPECIFIC REGULATORY CIRCUITS DURING MAMMALIAN DEVELOPMENT. I WILL IMPROVE THE SENSITIVITIES AND COVERAGES OF PAIRED-SEQ AND DEVELOP A COMPUTATIONAL METHOD FOR SINGLE-CELL MULTI-OMICS ANALYSIS FROM THE PHENOTYPIC LEVEL (AIM1). SUBSEQUENTLY, I WILL FURTHER DEVELOP A METHOD FOR HIGH-THROUGHPUT SINGLE-CELL JOINT ANALYSIS OF HISTONE MODIFICATIONS/TRANSCRIPTION FACTORS BINDING WITH GENE EXPRESSION (PAIRED-TAG) FOR ANALYSIS OF MOLECULAR PROGRAMS FROM THE MECHANISTIC LEVEL (AIM2). FINALLY, I WILL APPLY THESE TECHNOLOGIES TO STUDY THE DYNAMIC AND CELL-TYPE-SPECIFIC MOLECULAR PROGRAMS IN MAMMALIAN DEVELOPING GERM CELLS, AND TO IDENTIFY AND VALIDATE NOVEL REGULATORS DURING THIS PROCESS (AIM3). OVERALL, THE RESULTS FROM THIS PROPOSAL WILL PROVIDE NEW TECHNOLOGIES FOR THE STUDY OF EPIGENETIC PROGRAMS IN COMPLEX TISSUES AND DURING DEVELOPMENT AT SINGLE-CELL RESOLUTION, AND PROVIDING MORE COMPLETE VIEWS OF THE GENE REGULATORY CIRCUITS DURING MAMMALIAN GERM CELL DEVELOPMENT. MY CAREER GOAL IS TO LEAD AN INDEPENDENT RESEARCH GROUP FOCUSING ON INTEGRATING NOVEL EXPERIMENTAL AND COMPUTATIONAL TECHNOLOGIES TO UNDERSTAND THE UNDERLYING PRINCIPLES CONTROLLING MAMMALIAN DEVELOPMENT. DURING THE K99 PHASE, I WILL CONTINUE TO RECEIVE EXPERIMENTAL AND COMPUTATIONAL TRAINING FROM MY POSTDOCTORAL MENTOR DR. REN AND COLLABORATORS/ADVISORY COMMITTEE AT UC SAN DIEGO AND THE SALK INSTITUTE. THE RIGOROUS MENTORED SUPPORT AND RESULTS OBTAINED IN THE K99 PHASE WILL FACILITATE MY TRANSITION TO AN INDEPENDENT INVESTIGATOR IN THE R00 PHASE AND LAY THE FOUNDATION FOR MY FUTURE CAREER.

GENOME ENGINEERING TOOLS FOR FUNCTIONAL SCREENING OF NON-CODING ELEMENTS

A CRISPR/CAS13 APPROACH FOR IDENTIFYING INDIVIDUAL TRANSCRIPT ISOFORM FUNCTION IN CANCER - ABSTRACT ONE OF THE MAJOR CHALLENGES IN CHARACTERIZING TRANSCRIPT ISOFORM CELLULAR FUNCTION IN HEALTH AND DISEASE IS THE LACK OF METHODS TO SPECIFICALLY AND EFFICIENTLY DOWNREGULATE THEIR EXPRESSION. RNA-TARGETING RNA-GUIDED TYPE VI CRISPR/CAS13 SYSTEMS CONSTITUTE A RECENTLY DEVELOPED TOOL TO KNOCKDOWN TRANSCRIPTS IN MAMMALIAN CELLS. THEIR RNASE ACTIVITY IS ACTIVATED BY BINDING OF A CRISPR RNA GUIDE (GRNA) COMPLEMENTARY TO A SINGLE-STRANDED RNA TARGET. THE CRISPR/CAS13 SYSTEMS TESTED TO DATE HAVE BEEN SHOWN TO ACHIEVE HIGHLY SPECIFIC KNOCKDOWN OF ENDOGENOUS TRANSCRIPTS, WITH MINIMAL OFF-TARGET EFFECTS, OUTPERFORMING CURRENT METHODOLOGIES SUCH AS RNAI. WHILE CRISPR/CAS13 HAS BEEN SHOWN TO WORK EFFICIENTLY WHEN TARGETED TO THE PRE-MRNA, TRANSCRIPTOME-WIDE STRATEGIES THAT WOULD ALLOW KNOCKDOWN OF TRANSCRIPT ISOFORMS BY TARGETING UNIQUE JUNCTIONS IN THE MATURE MRNA MOLECULE HAVE NOT BEEN EXPLORED. SELECTING THE BEST GRNA SEQUENCES IS CRUCIAL FOR SUCCESSFUL AND SPECIFIC RNA KNOCKDOWN MEDIATED BY CAS13. PRELIMINARY DATA FROM OUR LAB SHOWS THAT USING GRNAS TARGETING SEQUENCES SPANNING EXON-EXON JUNCTIONS IN MATURE MRNA MOLECULES WITH CRISPR/CAS13D (THE SMALLEST CAS13 EFFECTOR) EFFICIENTLY REDUCES TRANSCRIPT EXPRESSION UP TO 80%. MOREOVER, TARGETING ISOFORM-SPECIFIC JUNCTIONS ALLOWS FOR THEIR INDIVIDUAL KNOCKDOWN WITHOUT AFFECTING NON-TARGETED ISOFORMS. THESE RESULTS SUGGEST THAT THERE IS NO STERIC CONSTRAINT IN TARGETING JUNCTIONS IN MATURE MRNA MOLECULES. TRANSCRIPTOME ANALYSES OF TUMORS AND CANCER CELL LINES HAVE COMPREHENSIVELY DESCRIBED THE EXPRESSION LEVELS AND IDENTITIES OF ALTERNATIVE TRANSCRIPT ISOFORMS. IN PARALLEL, A NUMBER OF STUDIES HAVE SHOWN THAT SPLICING DYSREGULATION IS A HALLMARK OF CANCER. THE CAUSAL LINK BETWEEN TRANSCRIPT ISOFORM EXPRESSION AND CANCER RELATED PHENOTYPES HAS NOT BEEN THOROUGHLY STUDIED. HERE, WE WILL EXPAND THE APPLICABILITY OF CRISPR/CAS13 SYSTEMS TO THE SYSTEMATIC STUDY OF TRANSCRIPT ISOFORMS IN CANCER BY 1) COMBINING LARGE-SCALE EXPERIMENTAL DATA WITH MACHINE LEARNING APPROACHES TO DEFINE RULES FOR GRNA DESIGN WHEN TARGETING SPECIFIC TRANSCRIPT JUNCTIONS 2) OPTIMIZING A PIPELINE AND THE COMPUTATIONAL ANALYSIS REQUIRED FOR USING CRISPR/CAS13 SYSTEMS IN FORWARD TRANSCRIPTOMIC POOLED SCREENS TO INTERROGATE THE CELLULAR FUNCTION OF TRANSCRIPT ISOFORMS. BY BROADENING THE APPLICATION OF THE CRISPR/CAS13 SYSTEM, OUR PIPELINE WILL PROVIDE THE MOLECULAR TOOLS AND COMPUTATIONAL ANALYSIS REQUIRED FOR INTERROGATING TRANSCRIPT ISOFORM FUNCTION IN A ROBUST, UNBIASED AND HIGHLY EXPANDABLE MANNER. WE EXPECT THAT OUR CRISPR/CAS13 APPROACH WILL BE ABLE TO OVERCOME THE LIMITATIONS OF CURRENT METHODS IN IDENTIFYING CELL-SPECIFIC ISOFORM EXPRESSION AND/OR RATIOS UNDERLYING TUMORIGENESIS AND DRUG RESISTANCE. LASTLY, THE CRISPR/CAS13 APPROACH COULD BE FURTHER ADAPTED TO PERFORM TARGETING OF TRANSCRIPT ISOFORMS IN VIVO AS AN RNA-BASED THERAPEUTIC.

ROBUST STR CALLING FROM HIGH-THROUGHPUT SEQUENCING TECHNOLOGIES

SOMATIC VARIANT CALLING AND PHASING USING COLORED DE BRUIJN GRAPHS IN HETEROGENEOUS TUMORS

COLLABORATIVE RESEARCH: ABI DEVELOPMENT: A NEW PLATFORM FOR HIGHLY-OPTIMIZED, LOW-LATENCY PIPELINES FOR GENOMIC DATA ANALYSIS

POSE: PHASE I: CREATION OF AN OPEN SOURCE ECOSYSTEM TO REPURPOSE DNA SEQUENCERS AS AN AUTOMATION PLATFORM -AS DNA SEQUENCING TECHNOLOGY RAPIDLY EVOLVES, ONCE STATE-OF-THE-ART EQUIPMENT, ORIGINALLY PRICED IN THE HUNDREDS OF THOUSANDS OF DOLLARS, OFTEN BECOMES OBSOLETE IN LESS THAN A DECADE. CONSEQUENTLY, THOUSANDS OF DNA SEQUENCERS ARE BEING DECOMMISSIONED AND, WITHOUT A CLEAR USE, ARE EITHER DISCARDED OR SOLD ON SECONDARY MARKETS AT A FRACTION OF THEIR ORIGINAL COST. DESPITE BEING OUTDATED FOR SEQUENCING, THESE INSTRUMENTS STILL CONTAIN HIGHLY VALUABLE COMPONENTS INCLUDING PRECISION ROBOTICS, ADVANCED OPTICS, TEMPERATURE CONTROL, AND MICROFLUIDIC SYSTEMS. WITH THE RIGHT SOFTWARE, ENGINEERING SUPPORT, AND EXPERIMENTAL FRAMEWORK, THESE COMPONENTS COULD BE REPURPOSED INTO POWERFUL PLATFORMS FOR AUTOMATING A WIDE RANGE OF MODERN BIOLOGICAL ASSAYS, THEREBY ACCELERATING RESEARCH AND DISCOVERY. IN PARTICULAR, THE EMERGING FIELD OF SPATIAL OMICS OFFERS TRANSFORMATIVE INSIGHTS INTO IMPORTANT BIOLOGICAL PROCESSES SUCH AS CANCER, NEURODEGENERATION, AND IMMUNE RESPONSE, BUT IS CURRENTLY HINDERED BY ITS RELIANCE ON COMMERCIAL AUTOMATION PLATFORMS THAT REMAIN PROHIBITIVELY EXPENSIVE TO MANY LABORATORIES. THIS PROJECT ADDRESSES THIS CHALLENGE BY ESTABLISHING AN OPEN-SOURCE ECOSYSTEM THAT UNITES RESEARCHERS, ENGINEERS, AND DEVELOPERS WORKING TOWARDS THE COMMON GOAL OF TRANSFORMING DECOMMISSIONED DNA SEQUENCERS INTO FLEXIBLE AUTOMATION PLATFORMS. THIS INITIATIVE WILL IMPROVE THROUGHPUT, INCREASE ACCESSIBILITY, REDUCE ELECTRONIC WASTE, AND PROMOTE GREATER STANDARDIZATION IN BIOLOGICAL RESEARCH. THIS PROJECT BUILDS UPON PYSEQ, AN OPEN-SOURCE TOOLKIT DEVELOPED TO CONTROL INDIVIDUAL COMPONENTS WITHIN DNA SEQUENCERS, ENABLING THEIR USE IN SPATIAL OMICS AND OTHER BIOMEDICAL RESEARCH APPLICATIONS. THE PRIMARY GOAL IS TO UNLOCK THE FULL POTENTIAL OF DECOMMISSIONED DNA SEQUENCERS BY CREATING AN OPEN-SOURCE ECOSYSTEM CENTERED AROUND PYSEQ, TRANSFORMING THESE POWERFUL BUT UNUSED INSTRUMENTS INTO FLEXIBLE PLATFORMS FOR LABORATORY AUTOMATION. THE PROJECT IS REALIZED THROUGH THREE MAIN AIMS: (I) ENGAGE DIRECTLY WITH CURRENT PYSEQ USERS TO ESTABLISH A CORE COMMUNITY OF CONTRIBUTORS FOCUSED ON DEVELOPING CONTROL TOOLS, PROTOCOLS, AND DOCUMENTATION; (II) COLLABORATE WITH USERS AND DEVELOPERS TO ESTABLISH SUSTAINABLE GOVERNANCE STRUCTURES THAT SUPPORT LONG-TERM COMMUNITY DEVELOPMENT AND OPEN-SOURCE PHILOSOPHY; AND (III) EXPAND THE USER BASE BY DISSEMINATING TECHNOLOGICAL KNOWLEDGE, PROCEDURES, BEST PRACTICES, AND NEW PERSPECTIVES TO RESEARCHERS UTILIZING LABORATORY AUTOMATION. THROUGH THE EXCHANGE OF PROTOCOLS, HARDWARE MODIFICATIONS, MAINTENANCE PROCEDURES, AND SOFTWARE TOOLS, THE OVERARCHING GOAL IS TO BUILD A SELF-SUSTAINED COMMUNITY THAT WILL LAUNCH PYSEQ OSE AS A ROBUST PLATFORM THAT TRANSFORMS ONCE-OBSOLETE INSTRUMENTS INTO POWERFUL, ACCESSIBLE TOOLS FOR MODERN SCIENCE. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.- SUBAWARDS ARE NOT PLANNED FOR THIS AWARD.

SINGLE-CELL JOINT PROFILING OF SINGLE- AND DOUBLE-STRANDED DNA BREAKS WITH TRANSCRIPTOME - PROJECT SUMMARY/ABSTRACT BOTH GENOME INSTABILITY AND EPIGENETIC DYSREGULATION ARE HALLMARKS OF CANCER, AND THE INTERACT WITH EACH OTHER IN COMPLEX WAYS. DEFECTS IN DNA REPAIR PATHWAYS IS A NEAR-UNIVERSAL FEATURE OF CANCER AND DRIVES A CASCADE OF DETRIMENTAL EFFECTS. THIS RELIANCE ALSO PRESENTS AS AN OPPORTUNITY FOR CANCER THERAPY: BY DELIBERATELY INTRODUCE DNA DAMAGE, E.G., SINGLE- AND DOUBLE- STRAND DNA BREAKS (SSBS AND DSBS), TO PROMOTE TUMOR CELL DEATH. GENOME-WIDE DNA DAMAGE SEQUENCING METHODS WERE DEVELOPED FOR BULK CELL POPULATIONS ANALYSIS AND SIGNIFICANTLY ADVANCED OUR KNOWLEDGE. HOWEVER, THE INTRINSIC HETEROGENEITY OF CANCER ENVIRONMENTS REPRESENTS A FORMIDABLE CHALLENGE. UNDERSTANDING THE INTERPLAY BETWEEN DIFFERENT TYPES OF DNA STRAND BREAKS AND THEIR IMPACTS ON GENE REGULATION IS CRITICAL FOR DEVELOPING EFFECTIVE AND PERSONALIZED CANCER THERAPIES; HOWEVER, WHICH IS HINDERED BY THE LACK OF PROPER SINGLE-CELL TECHNOLOGIES. SINGLE-CELL MULTIOMICS METHODS, BY MEASURING MULTIPLE MOLECULAR LAYERS SIMULTANEOUSLY FROM SINGLE CELLS, ENABLED THE CHARACTERIZATION OF COMPLEX RELATIONSHIPS BETWEEN BIOLOGICAL DISTINCT MODALITIES IN HETEROGENEOUS CELLULAR POPULATIONS. RECENTLY, WE DEVELOPED PAIRED-DAMAGE-SEQ FOR SIMULTANEOUS ANALYSIS OF OXIDATIVE AND SINGLE-STRAND DNA BREAKS WITH GENE EXPRESSION. THIS WORK REVEALED THAT HOTSPOTS OF DNA DAMAGE ARE ALSO VULNERABLE REGIONS FOR EPIGENETIC MEMORY LOSS, EXHIBITING CELL-TYPE SPECIFIC DISTRIBUTION PATTERNS AND LINKING DYSREGULATED GENE PROGRAMS TO DISEASE RISKS. TO ELUCIDATE THEIR COMBINATORIAL ROLES OF MULTIPLE TYPES OF DNA DAMAGE IN COMPLEX CELLULAR PROCESSES, NEW METHODS ARE STILL DESIRED. TO FILL THIS TECHNICAL GAP, IN THIS PROPOSAL WE WILL DEVELOP AND VALIDATE PAIRED-BREAK-SEQ, A NOVEL HIGH-THROUGHPUT SINGLE- CELL MULTIOMICS SEQUENCING TECHNIQUE FOR JOINT ANALYSIS OF DNA DSBS, SSBS, AND TRANSCRIPTOME. FIRSTLY, WE WILL PARALLELLY TEST AND COMPARE TWO APPROACHES FOR ORTHOGONAL LABELING OF DNA DSBS AND SSBS ON BULK CULTURED CELLS (AIM 1). NEXT, WE WILL COUPLE THE OPTIMAL ONE WITH TRANSCRIPTOME PROFILING AND COMBINATORIAL INDEXING AND VALIDATE ITS PERFORMANCES IN SINGLE-CELL ANALYSIS (AIM 2). FINALLY, TO JUSTIFY THE FEASIBILITY FOR R33 TRANSITION, WE WILL APPLY PAIRED-BREAK-SEQ TO THREE CANCER CELL LINES TREATED WITH DNA DAMAGE-INDUCING DRUGS (AIM 3). PAIRED- BREAK-SEQ CAN BE WIDELY ADOPTED TO STUDY DRUG RESPONSES, TO REVEAL HOW CANCER CELLS’ EPIGENOME REMODELED FOR DRUG RESISTANCE. THE INTEGRATED SINGLE-CELL TOOLKIT CREATES OPPORTUNITIES FOR IDENTIFYING NOVEL BIOMARKERS TO PREDICT TREATMENT RESPONSES AND MAXIMIZE EFFICACY AND MINIMIZE TOXICITY FOR INDIVIDUAL PATIENTS. THE OUTCOME OF THIS PROJECT WILL ALSO BE COMPLEMENTARY TO OTHER IMAT INITIATIVES FOCUSING ON DIFFERENT ASPECTS OF CANCER GENOMICS.

COMPREHENSIVE MAPPING OF MULTIMODAL CHROMATIN STATE IN SINGLE CELLS - PROJECT SUMMARY MANY NON-MENDELIAN (POLYGENIC) HUMAN GENETIC DISEASES INVOLVE MULTIPLE CAUSAL LOCI IN NONCODING REGIONS OF THE GENOME, IMPLICATING MUTATIONS IN REGULATORY ELEMENTS RATHER THAN PROTEIN-CODING GENES AS THE CAUSE OF THESE DISEASES. DECIPHERING THE ETIOLOGY OF THESE HUMAN GENETIC DISEASES REQUIRES AN UNDERSTANDING OF HOW GENES ARE REGULATED DURING DEVELOPMENT AND HOMEOSTASIS TO PRODUCE FUNCTIONAL CELL STATES. THIS REGULATION IS ENCODED BOTH THROUGH EPIGENETIC CHROMATIN STATE, AND GENETICALLY ENCODED IN THE DNA SEQUENCE OF REGULATORY ELEMENTS SUCH AS ENHANCERS, PROMOTERS, SILENCERS, AND INSULATORS. HOWEVER, THESE REGULATORY ELEMENTS AND THEIR ACTIVITY STATES IN HUMAN CELLS ARE NOT RESOLVABLE WITH CURRENT TECHNOLOGIES. AS ABERRANT GENE REGULATION LIKELY UNDERLIES MANY HUMAN DISEASES, UNDERSTANDING (1) THE FUNCTION OF REGULATORY DNA ELEMENTS AND (2) THEIR ACTIVITY DYNAMICS DURING HEALTHY HUMAN DEVELOPMENT ARE ESSENTIAL. TO ADDRESS THESE TWO PROBLEMS, I PROPOSE TO: (I) DEVELOP NEW EXPERIMENTAL METHODS TO PROFILE MULTIMODAL CHROMATIN STATE IN SINGLE CELLS; (II) IDENTIFY ALTERATIONS IN REGULATORY ELEMENT ACTIVATION STATES THAT GUIDE CELL FATE CHOICE DURING HUMAN HEMATOPOIESIS; (III) IDENTIFY THE DNA SEQUENCE FEATURES IMPORTANT FOR REGULATORY ELEMENT FUNCTION; (IV) BUILD COMMUNITY TOOLS AND RESOURCES FOR THE ANALYSIS OF SINGLE-CELL CHROMATIN DATA. TOGETHER THESE AIMS WILL PROVIDE METHODS AND RESOURCES FOR THE INTERROGATION OF THE HUMAN FUNCTIONAL GENOME, AND THE IDENTIFICATION OF REGULATORY STATE DYNAMICS THAT GENERATE HUMAN CELL TYPES. TO SUCCEED IN ACHIEVING THESE AIMS, I WILL PURSUE ADDITIONAL TRAINING SUPPORTED BY CO-MENTORS DR. RAHUL SATIJA (SINGLE-CELL BIOLOGY), DR. VIJAY SANKARAN (HEMATOPOIESIS), DR. DANNY REINBERG (GENE REGULATION), AND DR. DAVID KNOWLES (MACHINE LEARNING). I HAVE DEVELOPED A 5-YEAR CAREER DEVELOPMENT PLAN THAT INTEGRATES SCIENTIFIC TRAINING IN HEMATOPOIESIS AND GENE REGULATION, PRACTICAL TRAINING AND MENTORSHIP IN DEEP LEARNING, EXTENSIVE LEADERSHIP TRAINING THROUGH COURSES AND MENTORSHIP, AND SEMINARS AND WORKSHOPS ON ACADEMIC WRITING. MY TEAM OF SCIENTIFIC MENTORS WILL PROVIDE FURTHER GUIDANCE AND MENTORSHIP IN ACADEMIC JOB SEARCHES. THE NEW YORK GENOME CENTER IS AN IDEAL ENVIRONMENT FOR RESEARCH AND FURTHER CAREER DEVELOPMENT, PROVIDING THE CUTTING-EDGE RESEARCH FACILITIES AND OPPORTUNITIES FOR FURTHER CAREER DEVELOPMENT IN A RICH INTERDISCIPLINARY ENVIRONMENT. COMPLETION OF THE PROPOSED RESEARCH PROGRAM AND CAREER DEVELOPMENT PLAN WILL LAUNCH MY INDEPENDENT SCIENTIFIC CAREER AS A LEADER IN THE FIELD OF SINGLE-CELL EPIGENOMICS.

PERTURBATION-RESPONSE APPROACHES TO DETERMINING THE REGULATORY NETWORKS UNDERLYING HUMAN COMPLEX TRAITS - NO ABSTRACT AVAILABLE

INTEGRATED ANALYSES OF THE EPIGENOME TO UNDERSTAND THE MOLECULAR BASIS OF HEMATOPOIETIC MALIGNANCIES - PROJECT SUMMARY RESEARCH PLAN: AN IMPAIRED HEMATOPOIETIC DIFFERENTIATION PROCESS UNDERLIES BONE MARROW MALIGNANCIES LIKE LEUKEMIA, BUT WE STILL LACK THE MECHANISTIC UNDERSTANDING OF THE SEQUENCE OF REGULATORY EVENTS THAT MISLEADS THE DIFFERENTIATION PROCESS. SINCE EPIGENOMIC REGULATORY PATTERNS ARE MAJOR FEATURES OF LEUKEMIC DEVELOPMENT, UNDERSTANDING THE CHROMATIN DYNAMICS OF A FAILED (MALIGNANT) HEMATOPOIETIC DIFFERENTIATION PROCESS CAN HELP DEFINE THE MOLECULAR BASIS OF LEUKEMIA. A PREREQUISITE TO SUCH AN UNDERSTANDING IS A FRAMEWORK THAT ALLOWS INVESTIGATION OF THE PROGRESSIVE CHANGES IN THE ACTIVITY OF THE REGULATORY ELEMENTS (RE) DURING HEMATOPOIETIC DIFFERENTIATION. SINGLE-CELL CUT&TAG (SCCUT&TAG) TECHNOLOGY IS WELL-SUITED FOR SUCH STUDIES AS RE ACTIVITY THROUGH HISTONE MODIFICATION PROFILES CAN BE INVESTIGATED IN A LINEAGE-SPECIFIC MANNER. HOWEVER, POOR UNDERSTANDING OF THE CELL-TYPE-SPECIFIC HISTONE MODIFICATION PATTERNS MAKES THE TASK CHALLENGING. TO OVERCOME THIS CHALLENGE, WE DESIGNED SCCUT&TAG-PRO WHICH ALLOWS SIMULTANEOUS MEASUREMENT OF CELL-SURFACE PROTEIN AND IN-SILICO INTEGRATION OF GENE-EXPRESSION AND CHROMATIN ACCESSIBILITY. I WILL LEVERAGE THIS NOVEL MULTIMODAL FRAMEWORK TO INVESTIGATE THE RE AND PROGRESSIVE CHANGES IN THEIR ACTIVITY DURING HEMATOPOIESIS. FIRST, I WILL DEFINE A MULTIMODAL REFERENCE MAPPING FRAMEWORK FOR MOUSE HEMATOPOIESIS. THIS FRAMEWORK WILL ALLOW ME TO INTEGRATE MULTIPLE HISTONE MODIFICATION PROFILES ONTO ONE REFERENCE AND COMPARE THE CHROMATIN STATES OF THE RE BETWEEN A WILD TYPE (WT) AND MOUSE MODEL WITH LOSS OF FUNCTION IN HISTONE METHYL TRANSFERASE (HMT) (AIM 1). SECOND, SINCE HMTS REGULATE TRANSCRIPTION THROUGH THE INTERACTION NETWORK OF RE. I WILL DEFINE A CHROMATIN STATE AWARE MAP THAT DYNAMICALLY LINKS RES ACROSS DEVELOPMENTAL TRAJECTORIES. I WILL USE THIS FRAMEWORK TO INVESTIGATE THE CHANGES IN THE INTERACTION OF RES DUE TO HMT LOSS (AIM 2). THIRD, SINCE THE TRANSCRIPTIONAL STATE OF A CELL EMERGES FROM THE UNDERLYING GENE REGULATORY NETWORK (GRN), I WILL INTEGRATE SINGLE-CELL GENE EXPRESSION DATA WITH HISTONE MODIFICATION PROFILES AND EXTEND IT TO DEFINE A CHROMATIN STATE AWARE MODEL OF GRN. I WILL COMPARE THE WT AND HMT LOSS EXPERIMENTS AND DEFINE THE DIFFERENTIAL GRN (AIM 3). ALTOGETHER, THIS RESEARCH PROPOSAL SEEKS TO PIONEER THE COMPUTATIONAL METHODS FOR THE INTEGRATED ANALYSES OF MULTIMODAL SINGLE-CELL HISTONE MODIFICATIONS AND SYSTEMATICALLY DISSECT PROGRESSIVE CHANGES IN THE SYSTEM-LEVEL FUNCTION OF THE REGULATORY CIRCUITS THAT MISLEADS HEMATOPOIETIC DIFFERENTIATION USING MOUSE MODELS WITH CONDITIONAL HMT LOSS OF FUNCTION IN THE HEMATOPOIETIC COMPARTMENT. I HAVE DEVELOPED A 5-YEAR CAREER DEVELOPMENT PLAN TO MEET MY GOAL OF BECOMING AN INDEPENDENT INVESTIGATOR IN THE MULTI-DISCIPLINARY FIELD OF COMPUTATIONAL CANCER BIOLOGY. THE MENTORSHIP COMMITTEE WILL ALSO PROVIDE ME THE GUIDANCE IN MY RESEARCH AND ACADEMIC JOB SEARCH. GIVEN THE EXCELLENT THE OUTSTANDING RECORD OF TRAINING MULTIPLE INDEPENDENT SCIENTISTS, NEW YORK GENOME CENTER PROVIDES ME AN IDEAL ENVIRONMENT TO ATTAIN MY SCIENTIFIC CAREER GOALS.

MEASURING ALLELE AND ISOFORM-SPECIFIC RBP BINDING TO IMPROVE PREDICTIVE MODELS OF RNA SPLICING - PROJECT SUMMARY ALTERNATIVE SPLICING (AS) IS A FUNDAMENTAL CELLULAR PROCESS THAT REGULATES 95% OF MULTI-EXON GENES TO DIVERSIFY PROTEIN OUTPUT AND DEFINE CELL-TYPE SPECIFIC FUNCTIONS. BOTH CONSTITUTIVE SPLICING AND AS ARE CONTROLLED BY COMBINATIONS OF CIS-ACTING PRE-MESSENGER RNA SEQUENCES (PRE-MRNA) AND TRANS-ACTING RNA-BINDING PROTEINS (RBPS). THEREFORE, DEFECTS IN SPLICING REGULATORY RNA SEQUENCE OR RBPS CAN BE HIGHLY DISRUPTIVE TO BASIC CELLULAR ACTIVITIES AND OFTEN LEAD TO DISEASE, ESPECIALLY NEUROLOGICAL AND MUSCULAR DISORDERS AND CANCER. WHILE THE CONSTITUTIVE SPLICING CODE IS WELL ESTABLISHED, THE AS CODE IS MORE COMPLICATED AND, THUS, POORLY UNDERSTOOD. THIS PROPOSAL INTEGRATES MULTIPLE CUTTING-EDGE APPROACHES TO TAKE AN RBP-CENTRIC VIEW OF AS TO STUDY BOTH CIS GENETIC VARIANTS AND TRANS RBP EXPRESSION EFFECTS ON RBP BINDING AND SPLICING OUTCOME. WHILE THOUSANDS OF NON-CODING GENETIC VARIANTS ARE ASSOCIATED WITH SPLICING VARIATION, AND ARE THUS TERMED PUTATIVE SPLICING QUANTITATIVE TRAIT LOCI (SQTLS), THE CAUSAL VARIANTS AND THEIR MOLECULAR EFFECTS, SUCH AS RBP BINDING, ARE LARGELY UNCLEAR. AIM 1 WILL ADDRESS THIS GAP BY INTEGRATING RBP-FOCUSED EXPERIMENTS, ALLELE-SPECIFIC GENOMICS, AND STATE- OF-THE-ART MACHINE LEARNING PREDICTIVE MODELS TO CHARACTERIZE AN IMPORTANT CATEGORY OF FUNCTIONAL, CIS NON-CODING VARIANTS THAT ALTER RBP BINDING. IMPORTANTLY, I WILL TAKE A UNIQUE APPROACH TO INCLUDE THESE ALLELE-SPECIFIC RBP BINDING DATA AS ADDITIONAL TRAINING DATA FOR OUR CONVOLUTIONAL NEURAL NET MODEL. MODEL OUTPUT IS EXPECTED TO MUCH MORE ACCURATELY PREDICT FUNCTIONAL RBP BINDING EFFECTS OF EVEN A SINGLE NUCLEOTIDE CHANGE IN SEQUENCE, ENABLING IMPROVED INTERPRETATION OF SQTLS. IN ADDITION TO GENETIC VARIANT EFFECTS, CHANGES IN RBP EXPRESSION CAN HAVE AMPLIFIED DOWNSTREAM EFFECTS ON RNA SPLICING. INTERESTINGLY, ~86% OF RBP GENES CAN BE EXPRESSED AS MORE THAN ONE SPLICE ISOFORM, BUT MOST STUDIES TO DATE HAVE IGNORED RBP ISOFORM-SPECIFIC ABUNDANCE AND FUNCTION. AIM 2 WILL PROVIDE FOUNDATIONAL EXPERIMENTS TO UNDERSTAND DIFFERENTIAL RBP ISOFORM EFFECTS BY USING A NOVEL APPROACH TO KNOCKDOWN RBP ISOFORMS BY TARGETING CAS13 TO UNIQUE EXON JUNCTIONS. DATA FROM DOWNSTREAM ASSAYS THAT ASSESS CHANGES IN RBP BINDING, SPLICING, AND RNA LOCALIZATION WILL BE INTEGRATED TO CONSTRUCT THE MOST COMPREHENSIVE RBP REGULATORY NETWORKS TO DATE. RESULTS FROM BOTH AIMS ARE ESSENTIAL TO MECHANISTICALLY LINK RNA SEQUENCE AND RBP BINDING TO SPLICING OUTCOME AND, ULTIMATELY, TO PHENOTYPE AND DISEASE. MY LONG-TERM GOAL IS TO BECOME A PRINCIPAL INVESTIGATOR, WHERE I WILL CONTINUE TO LEVERAGE MOLECULAR BIOLOGY, MACHINE LEARNING, AND STATISTICAL GENETICS TO ANSWER UNIQUE QUESTIONS ABOUT RNA-MEDIATED ASSOCIATIONS BETWEEN NON-CODING SEQUENCE AND CELLULAR AND DISEASE PHENOTYPE. THE RESEARCH AND TRAINING PLANS PROPOSED HERE ARE STRATEGICALLY TAILORED TO PROVIDE AMPLE OPPORTUNITIES TO LEARN AND APPLY MACHINE LEARNING AND STATISTICAL GENETICS METHODS THAT COMPLEMENT MY FORMER PHD TRAINING IN MOLECULAR BIOLOGY AND GENOMICS. MY SPONSOR, CO- SPONSOR, AND COLLABORATORS AT THE NYGC ARE COMMITTED TO PROVIDING THE SCIENTIFIC EXPERTISE, COMPUTATIONAL TRAINING, AND CAREER DEVELOPMENT MENTORING TO ENSURE THE SUCCESSFUL ACHIEVEMENT OF MY GOALS.

LINKING GWAS VARIANTS TO FUNCTION WITH SINGLE-CELL POOLED CRISPR SCREENS - PROJECT SUMMARY/ABSTRACT GENOME-WIDE ASSOCIATION STUDIES (GWAS) HAVE IDENTIFIED THOUSANDS OF COMMON AND RARE GENETIC VARIANTS ASSOCIATED WITH COMPLEX TRAITS AND COMMON DISEASES. MOST VARIANTS MAP TO THE 98% OF THE GENOME THAT IS NONCODING, WITH THEIR TARGET GENES OR FUNCTION LARGELY UNKNOWN. THIS IS THE VARIANT-TO-FUNCTION PROBLEM (V2F), AND SOLVING IT REMAINS A MAJOR HURDLE IN HUMAN GENETICS RESEARCH. TO HELP SOLVE V2F, I PROPOSE TO DEVELOP MODULAR WORKFLOWS COMBINING GWAS VARIANT PRIORITIZATION METHODS AND POOLED SINGLE-CELL CRISPR SCREENS FOR TARGET GENE IDENTIFICATION. I HAVE DEVELOPED AN INTEGRATIVE APPROACH COMBINING HIGHLY POLYGENIC BLOOD TRAIT GWASS AND POOLED SINGLE-CELL CRISPR INHIBITION (CRISPRI) SCREENS IN A HUMAN ERYTHROID PROGENITOR CELL MODEL (K562), TO IDENTIFY TARGET GENES: SYSTEMATIC TARGETING AND INHIBITION OF NONCODING GWAS LOCI WITH SINGLE-CELL SEQUENCING (STING-SEQ). STING-SEQ CAN FUNCTIONALLY DISSECT MULTIPLE GWAS LOCI IN A MASSIVELY PARALLEL FASHION, IDENTIFYING TARGET GENES IN CIS AS WELL AS TRANS-REGULATORY NETWORKS. HERE, I WILL DEVELOP STING-SEQ FURTHER AND EXAMINE ITS GENERALIZABILITY FOR OTHER GWAS TRAITS AND THEIR CELL MODELS. FIRST, I WILL EXPAND STING-SEQ WITH PRECISE VARIANT INSERTION, DEVELOPING BASE EDITING STING-SEQ (BEE-STING) FOR HIGH-THROUGHPUT MEASUREMENTS OF GWAS VARIANT EFFECTS ON TARGET GENES AND REGULATORY NETWORKS. SECOND, I WILL DEVELOP MODULAR WORKFLOWS FOR GWAS VARIANT PRIORITIZATION FOR STING-SEQ, TARGETING SETS OF VARIANTS WITH DISTINCT SELECTION CRITERIA TO INCREASE STING-SEQ’S TARGET GENE AND REGULATORY NETWORK DISCOVERY RATE. THIRD, I WILL FOCUS STING-SEQ ON NEW GWAS TRAITS AND CELL MODELS TO EXAMINE ITS GENERALIZABILITY, FIRST PILOTING STING-SEQ FOR ANOTHER HIGHLY POLYGENIC COMPLEX TRAIT, BONE MINERAL DENSITY, WITH A HUMAN OSTEOBLAST CELL MODEL (HFOB). IN THE LONG-TERM, THESE AIMS WILL HELP SOLVE V2F FOR HUMAN GENETICS RESEARCH, AS THEIR CONTINUED DEVELOPMENT AND APPLICATION WILL IMPROVE OUR UNDERSTANDING OF HOW GWAS VARIANTS CAUSALLY INFLUENCE COMPLEX TRAITS AND COMMON DISEASES. I HAVE A COMPREHENSIVE TRAINING PLAN IN PLACE WITH MY PRIMARY MENTORS, DR. NEVILLE SANJANA (GENOME ENGINEERING) AND DR. TUULI LAPPALAINEN (GENE REGULATION), MY MENTORSHIP COMMITTEE MEMBERS, DR. DAVID KNOWLES (MACHINE LEARNING), DR. ARAVINDA CHAKRAVARTI (HUMAN GENETICS), DR. CHARLES FARBER (BONE BIOLOGY), AND MY COLLABORATOR DR. EUGENE KATSEVICH (STATISTICAL METHODS). THIS PLAN WILL CONTINUE MY TRAINING IN DISSECTING GWAS VARIANT FUNCTION WITH MULTIPLE COMPUTATIONAL AND EXPERIMENTAL APPROACHES, ALONG WITH ADDITIONAL TRAINING IN GRANT WRITING, MENTORING STUDENTS, TEACHING COURSES, AND PRESENTING AT RESEARCH CONFERENCES. THE FULL MENTORSHIP COMMITTEE WILL DIRECT ME TO PERTINENT LITERATURE, OFFER ADVICE ON MY RESEARCH PROGRAM, AND PROVIDE GUIDANCE AS I NAVIGATE THE ACADEMIC JOB MARKET. THE NEW YORK GENOME CENTER IS THE IDEAL TRAINING LOCATION FOR ME, GIVEN ITS CUTTING-EDGE FACILITIES, PLENTIFUL OPPORTUNITIES FOR CAREER AND INTELLECTUAL DEVELOPMENT, AND COLLABORATIVE RESEARCH ENVIRONMENT. UPON COMPLETION OF THIS TRAINING PROGRAM, I WILL BE WELL-POSITIONED TO LEAD MY OWN INTERDISCIPLINARY RESEARCH LAB AND BECOME A LEADER IN THE FIELDS OF HUMAN COMPLEX TRAITS GENETICS AND GENOME ENGINEERING.

DISENTANGLING TISSUE AND OME SHARED AND PRIVATE EFFECTS OF EXERCISE - PROJECT SUMMARY/ABSTRACT EXERCISE IS A POWERFUL INTERVENTION AGAINST MANY CHRONIC DISEASES BUT THE MOLECULAR MECHANISMS OF THIS PROTECTION REMAIN UNCLEAR. WE PROPOSE AN INNOVATIVE, INTEGRATIVE APPROACH TO ANALYZE THE COMPLEX EFFECTS OF EXERCISE ON MULTIPLE BIOLOGICAL LEVELS USING THE EXTENSIVE DATA COLLECTED BY THE MOLECULAR TRANSDUCERS OF PHYSICAL ACTIVITY CONSORTIUM (MOTRPAC). THIS DATASET INCLUDES MULTIOMIC, MULTI-TISSUE, AND MULTI-TIME POINT PROFILING AFTER ENDURANCE EXERCISE TRAINING IN BOTH RATS AND HUMANS, OFFERING AN UNPRECEDENTED OPPORTUNITY TO EXPLORE THE MECHANISMS UNDERLYING THE HEALTH BENEFITS OF EXERCISE. EXISTING STUDIES ANALYZE INDIVIDUAL OMES AND TISSUES SEPARATELY, WHICH RISKS MISSING SUBTLE SHARED SIGNALS AND CONFLATING DIFFERENCES IN STATISTICAL POWER WITH TRUE BIOLOGICAL VARIATION. TO OVERCOME THESE LIMITATIONS, WE PROPOSE AN INTEGRATIVE ANALYSIS BASED ON OUR RECENTLY PUBLISHED MULTISET CORRELATION AND FACTOR ANALYSIS (MCFA). OUR FIRST AIM IS TO EXTEND MCFA TO HANDLE TENSOR-VALUED DATA, CREATING A NEW MODEL WE CALL TENSOR-MCFA (TMCFA). THIS MODEL WILL EXPLICITLY ACCOUNT FOR MULTIMODAL, MULTI-TISSUE DATA BY DIVIDING VARIATION INTO FOUR CATEGORIES: (TISSUE, OME)-TUPLE PRIVATE, TISSUE-PRIVATE, OME-PRIVATE, AND SHARED. WE WILL DESIGN TMCFA TO HANDLE AND IMPUTE MISSING DATA PAIRS AND EXPLORE LINKING RELATED FEATURES ACROSS OMES USING A GRAPH-GUIDANCE STRATEGY. IN OUR SECOND AIM, WE WILL APPLY TMCFA TO THE PRECLINICAL ANIMAL STUDY SITE (PASS) DATASET FROM MOTRPAC. WE WILL INCORPORATE ALTERNATIVE SPLICING AS AN ADDITIONAL MODALITY FOR ANALYSIS AND ASSESS THE PROPORTION OF VARIANCE EXPLAINED BY EACH OF THE FOUR CATEGORIES. TO INTERPRET THESE COMPONENTS, WE WILL USE GENE SET ENRICHMENT ANALYSIS AND PROTEIN-PROTEIN INTERACTION ENRICHMENT. BY RELATING OUR FINDINGS TO HUMAN EXPRESSION DATA AND VARIOUS METABOLIC DISEASES, WE AIM TO IDENTIFY HOW EXERCISE-RELATED CHANGES OBSERVED IN RATS CORRESPOND TO PROTECTIVE EFFECTS AGAINST HUMAN DISEASES. THIS COMPREHENSIVE ANALYSIS ACROSS TISSUES AND OMES WILL ALLOW US TO PINPOINT THE KEY DRIVERS OF EXERCISE'S PROTECTIVE EFFECTS. THROUGH THIS WORK, WE HOPE TO PROVIDE NOVEL INSIGHTS INTO THE MOLECULAR MECHANISMS UNDERLYING THE HEALTH BENEFITS OF PHYSICAL ACTIVITY. OUR FINDINGS COULD PAVE THE WAY FOR NEW THERAPEUTIC STRATEGIES AND PERSONALIZED EXERCISE INTERVENTIONS THAT MAXIMIZE THESE PROTECTIVE EFFECTS.

CHARACTERIZING GENETIC, LONGITUDINAL, AND DEVELOPMENTAL EFFECTS ON THE TRANSCRIPTOME USING A NOVEL NON-INVASIVE RNA-SEQUENCING METHOD

MYELOID CELL-EXPRESSED PTPN22 AND ANTI-TUMOR IMMUNITY - PROJECT SUMMARY IMMUNOTHERAPIES THAT BLOCK THE CHECKPOINT MOLECULES PROGRAMMED CELL DEATH 1 (PD-1) AND ITS LIGAND PD-L1 HAVE REVOLUTIONIZED CANCER TREATMENT; HOWEVER, A SIGNIFICANT NUMBER OF PATIENTS DISPLAY RESISTANCE TO IMMUNOTHERAPY, EITHER DE NOVO OR WITH DISEASE RELAPSE AFTER INITIAL RESPONSE [1-3]. IMMUNOTHERAPY-REFRACTORY CASES HAVE PROMPTED INSIGHT INTO MECHANISMS OF RESISTANCE, WHICH SHOULD ULTIMATELY LEAD TO NEW STRATEGIES TO EXPAND CLINICAL EFFICACY. PRIMARY RESISTANCE IS LINKED TO LACK OF IMMUNE CELL INFILTRATION WITHIN THE TUMOR MICROENVIRONMENT (TME), AN OBSERVATION WHICH HAS PROMPTED DEEPER INVESTIGATION INTO THE TUMOR AND HOST FACTORS THAT REGULATE THE DEGREE OF SPONTANEOUS T CELL ACTIVATION AND INFILTRATION INTO TUMOR SITES. ONE RELEVANT SOURCE OF INTER-PATIENT HETEROGENEITY IS THE VARIABLE PRESENCE OF POLYMORPHISMS (SNPS) IN IMMUNE-REGULATORY GENES, MANY OF WHICH HAVE BEEN LINKED PREVIOUSLY TO THE PROPENSITY TOWARDS AUTOIMMUNITY. IN THE F99 PHASE OF THIS PROPOSED PLAN, I WILL EVALUATE THE UTILITY OF TARGETING AN AUTOIMMUNE-ASSOCIATED GENE TO INCREASE IMMUNOTHERAPY EFFICACY. A SNP IN THE TYROSINE-PROTEIN PHOSPHATASE NON-RECEPTOR TYPE 22 (PTPN22) GENE IS ATTRIBUTED WITH THE GREATEST RISK FOR AUTOIMMUNE DISEASE OUTSIDE MUTATIONS IN THE HUMAN LEUKOCYTE ANTIGEN LOCUS [4-6]. PTPN22 NEGATIVELY REGULATES THE ACTIVATION OF MULTIPLE IMMUNE COMPARTMENTS, WITH LOSS-OF-FUNCTION VARIANTS LEADING TO HEIGHTENED IMMUNE CELL ACTIVATION IN MICE AND HUMANS [7-8]. THIS INCREASE IN IMMUNE ACTIVITY IS ATTRIBUTED TO THE EXPANSION OF ACTIVATED CD8+ T CELLS, HOWEVER, WORK REPORTED TO DATE HAVE UTILIZED GLOBAL KNOCKOUT MICE (KO), CONFOUNDING THE SPECIFIC ROLE OF PTPN22 IN OTHER CELL LINEAGES RELEVANT FOR ANTI-TUMOR IMMUNITY, IN PARTICULAR MYELOID CELLS. WE THUS DEVELOPED A PTPN22FL/FL MOUSE TO STUDY ITS EFFECT IN DIFFERENT IMMUNE CELL TYPES VIA CONDITIONAL KO MOUSE MODELS. I HYPOTHESIZE THAT LOSS OF PTPN22 AUGMENTS THE ABILITY OF DCS TO ACTIVATE ANTIGEN SPECIFIC CD8+ T CELLS THROUGH 1) IMPROVED PRIMING IN THE TDLN OR RECRUITMENT TO AND/OR SURVIVAL SIGNALING IN THE TME AND THAT 2) DELETION OF PTPN22 IN MACROPHAGES ALSO MAY POTENTIATE ANTI-TUMOR IMMUNITY. IN THE K00 PHASE OF THE PROPOSED PLAN, I AIM TO IDENTIFY NOVEL TARGETS GOVERNING ANTI-TUMOR IMMUNITY AND IMMUNOTHERAPY EFFICACY BY 1) IDENTIFYING AUTOIMMUNE RELATED SNPS WHOSE LOSS OF FUNCTION VARIANTS CORRELATE WITH INCREASED TUMOR IMMUNE INFILTRATION AND 2) CHARACTERIZING THE EFFECT OF THESE TARGETS ON TUMOR PROGRESSION AND IMMUNOTHERAPY RESPONSE THROUGH FUNCTIONAL STUDIES USING CONDITIONAL KO MICE. THIS WORK HOLDS THE POTENTIAL TO ELUCIDATE NOVEL THERAPEUTIC TARGETS TO POTENTIATE ANTI-TUMOR IMMUNITY. MY ULTIMATE GOAL IS TO BECOME A TENURE- TRACK FACULTY MEMBER AT A LEADING ACADEMIC RESEARCH INSTITUTION AND CONDUCT NIH FUNDED WORK CONTRIBUTING TO THE FIELD OF TUMOR IMMUNOLOGY BY ELUCIDATING ANTI-TUMOR IMMUNITY AND DEVELOPING NOVEL IMMUNOTHERAPIES.

THE ROLE OF EPIGENETIC TUMOR SUPPRESSORS IN REGULATING TUMORIGENESIS AND TUMOR-IMMUNE CROSSTALK