Broad Institute Inc

LARGE SCALE SEQUENCING AND ANALYSIS OF GENOMES

CENTER FOR COMMON DISEASE GENETICS

INFECTIOUS DISEASE GENOMICS: PATHOGEN EVOLUTION, EMERGENCE, AND HOST INTERACTION

GENOME SEQUENCING IN SUPPORT OF THE GABRIELLA MILLER KIDS FIRST PEDIATRIC RESEARCH PROGRAM

AN ATLAS OF HUMAN BRAIN CELL VARIATION - PROJECT SUMMARY/ABSTRACT THE HUMAN BRAIN EXHIBITS PROFOUND DIVERSITY IN BIOLOGICAL FUNCTION AND VULNERABILITY TO DISEASE. DESPITE THE BIOMEDICAL AND CULTURAL IMPORTANCE OF INTER-INDIVIDUAL VARIATION, WE KNOW RELATIVELY LITTLE ABOUT ITS UNDERLYING CELLULAR AND MOLECULAR SUBSTRATES. IN THIS WORK WE WILL LEVERAGE NEW TECHNOLOGIES IN SINGLE-CELL AND SPATIAL GENOMICS TO CONSTRUCT AN ATLAS OF HUMAN BRAIN CELL VARIATION. WE WILL ANALYZE TENS OF MILLIONS OF CELLS FROM MORE THAN 200 PEOPLE BY SINGLE-NUCLEUS RNA-SEQ AND SINGLE-NUCLEUS ATAC-SEQ, AND A SUBSET OF THESE BY SPATIAL TRANSCRIPTOMICS. ANALYSIS OF THESE DATA WILL SEEK TO UNDERSTAND: THE STATIC VERSUS DYNAMIC MOLECULAR AND SPATIAL FEATURES OF EACH CELL TYPE; THE WAYS IN WHICH HUMAN GENETIC VARIATION SHAPES THE MOLECULAR REPERTOIRE OF EACH CELL TYPE; AND THE CONSTELLATIONS OF CELLULAR AND MOLECULAR FEATURES THAT CO-VARY, APPEARING TOGETHER IN THE SAME BRAINS. WE SEEK ESPECIALLY WITH THIS WORK TO UNDERSTAND THE FUNCTIONAL CONNECTIONS BETWEEN THESE PHENOTYPES AND: (I) THE GENE REGULATORY PROCESSES THAT DRIVE AND SHAPE IT; (II) GENETIC VARIATION ASSOCIATED WITH BRAIN DISEASES; AND (III) STRUCTURAL BRAIN VARIATION MEASURED IN HUNDREDS OF NEUROIMAGING STUDIES. THE PROJECT WILL DELIVER AN ESSENTIAL DATA RESOURCE FOR CELLULAR, MOLECULAR, GENETIC AND TRANSLATIONAL NEUROSCIENCE – WHILE EXPANDING OUR UNDERSTANDING OF THE WAYS THE HUMAN BRAIN VARIES ACROSS DIFFERENT PEOPLE.

THE ANVIL DATA ECOSYSTEM

INNOVATIVE TECHNOLOGIES TO TRANSFORM ANTIBIOTIC DISCOVERY.

THERAPEUTIC EDITING TO LOWER PRP IN PRION DISEASE

THE GENOME AGGREGATION DATABASE (GNOMAD) - PROJECT SUMMARY THE GENOME AGGREGATION DATABASE (GNOMAD) IS A UBIQUITOUS RESOURCE FOR BASIC RESEARCH AND CLINICAL INTERPRETATION. THE WORLD’S LARGEST GENETIC VARIATION RESOURCE, THE GNOMAD DATASET IS USED IN VIRTUALLY ALL CLINICAL GENETIC DIAGNOSTIC PIPELINES WORLDWIDE, AND THE WEBSITE HAS OVER 20 MILLION PAGE VIEWS TO DATE. HERE WE OUTLINE A PROPOSAL THAT WILL EXPAND THE GNOMAD RESOURCE TO MILLIONS OF SAMPLES ACROSS DIVERSE GLOBAL POPULATIONS. OUR PROPOSAL WILL SCALE VARIANT-CALLING AND QUALITY CONTROL TO MATCH THIS SAMPLE SIZE, INTEGRATE STATISTICAL TOOLS AND OTHER GENOMIC RESOURCES CRITICAL TO CLINICAL INTERPRETATION, AND ENSURE THAT THE DATA WE AGGREGATE WILL CONTINUE TO BE SHARED FREELY WITH THE BIOMEDICAL COMMUNITY. TO ACCOMPLISH THIS WE WILL APPLY A HIGHLY COMPUTATIONALLY EFFICIENT STRATEGY TO CALL ALL CLASSES OF VARIATION (INCLUDING SNVS, SMALL INDELS, AND THE MUTATIONAL SPECTRUM OF STRUCTURAL VARIANTS) ACROSS MILLIONS OF SEQUENCED SAMPLES ENRICHED FOR UNDER- REPRESENTED ANCESTRY GROUPS. WE WILL DEPLOY A CLOUD-BASED FRAMEWORK FOR THE EFFICIENT STORAGE AND AUTOMATED QUALITY CONTROL OF THESE VERY LARGE AND HETEROGENEOUS SEQUENCE DATA SETS USING THE MASSIVELY PARALLEL HAIL ARCHITECTURE. WE WILL LEVERAGE THE SCALE OF GNOMAD TO PROVIDE INCREASINGLY HIGH-RESOLUTION MAPS OF THE DEPLETION OF FUNCTIONAL VARIATION ACROSS REGIONS OF THE GENOME (HIGHLIGHTING GENOME REGIONS WHERE NATURAL SELECTION CONSTRAINS DNA CHANGE) AND PROVIDE STATISTICAL FRAMEWORKS FOR QUANTITATIVELY ASSESSING WHETHER THE POPULATION FREQUENCY OF A VARIANT IS CONSISTENT WITH PATHOGENICITY, LINKING THIS INFORMATION WITH EVIDENCE FROM THE CLINVAR RESOURCE. WE WILL CONTINUE TO SHARE ALL OF THIS DATA AS RAPIDLY AND OPENLY AS POSSIBLE WITH THE BIOMEDICAL COMMUNITY, LONG BEFORE PUBLICATION. WE WILL SUPPORT AND EXPAND FUNCTIONALITY IN OUR WIDELY ACCESSED DATA BROWSER AS WELL AS CREATE SCALABLE AND PUBLICLY ACCESSIBLE DATASETS THAT INTEGRATE OUR VARIATION DATA WITH CLINICAL AND FUNCTIONAL GENOMIC ANNOTATIONS, ACCESSIBLE THROUGH API FRAMEWORKS TO EMPOWER NOVEL APPLICATIONS OF THE DATASETS. WE WILL ALSO PROVIDE RESOURCES AND TRAINING TO IMPROVE THE USE OF GNOMAD RESOURCES BY THE CLINICAL GENETICS AND WIDER BIOMEDICAL COMMUNITIES.

BROAD-GEISINGER CLINICAL GENOME RESOURCE - PROJECT SUMMARY GENOMIC VARIATION UNDERLIES ALMOST ALL HUMAN DISEASE. TECHNOLOGICAL ADVANCES HAVE MADE VARIANT DETECTION ACROSS THE HUMAN GENOME COMMONPLACE IN MEDICAL CARE, SPARKING AN EXPANSION OF BASIC AND CLINICAL RESEARCH. TO ACCURATELY ASSESS THE IMPACT OF ANY GIVEN GENOMIC VARIANT ON HUMAN HEALTH, CLINICIANS AND RESEARCHERS MUST HAVE QUICK, EASY ACCESS TO HIGH-QUALITY KNOWLEDGE SOURCES. SINCE 2013, THE CLINICAL GENOME RESOURCE (CLINGEN) HAS BEEN DEDICATED TO CREATING SUCH KNOWLEDGE BASES, DEFINING THE CLINICAL RELEVANCE OF GENES AND VARIANTS FOR USE IN MEDICINE AND RESEARCH. THIS PROPOSAL BUILDS UPON CLINGEN’S SUCCESSFUL FOUNDATION, USING EXISTING TOOLS AND APPROACHES WHILE DEVELOPING NEW ONES, TO EXPAND AND SCALE OUR EFFORTS. WE WILL WORK CLOSELY WITH OUR COLLEAGUES SUBMITTING LINKED U24 GRANT PROPOSALS (BAYLOR/STANFORD; UNIVERSITY OF NORTH CAROLINA/KAISER), AS OUR WORK IS HIGHLY INTEGRATED AND SYNERGISTIC FOR ACHIEVING OUR SHARED GOALS, WHICH ARE EMBODIED BY OUR SPECIFIC AIMS: 1) DEVELOP AND IMPLEMENT STANDARDS TO SUPPORT CLINICAL ANNOTATION AND INTERPRETATION OF GENES AND VARIANTS; 2) SHARE GENOMIC AND PHENOTYPIC DATA BETWEEN CLINICIANS, RESEARCHERS, AND PATIENTS THROUGH ENHANCED KNOWLEDGE BASES FOR CLINICAL AND RESEARCH USE; 3) ENHANCE AND ACCELERATE EXPERT REVIEW OF THE CLINICAL RELEVANCE OF GENES AND VARIANTS; AND 4) DISSEMINATE AND INTEGRATE CLINGEN KNOWLEDGE AND RESOURCES TO THE BROADER COMMUNITY. CLINGEN’S APPROACH TO PROVIDING CURATED CLINICAL GENOMIC RESOURCES TO AID THE INTERPRETATION OF INDIVIDUAL GENOMES INVOLVES CURATING GENES TO UNDERSTAND WHICH HAVE BEEN VALIDLY IMPLICATED IN DISEASE (AND THROUGH WHAT MUTATIONAL MECHANISMS), AND CURATING VARIANTS (AND THE EVIDENCE SUPPORTING CLAIMS OF PATHOGENICITY) TO IDENTIFY WHICH ARE CAUSAL FOR EXISTING DISEASE OR PREDICTIVE OF RISK FOR FUTURE DISEASE. TO ADDRESS THESE NEEDS, WE HAVE ORGANIZED GENE AND VARIANT CURATION EXPERT PANELS BY CLINICAL DOMAINS TO CURATE CLAIMS OF GENE-DISEASE ASSOCIATION AND VARIANT PATHOGENICITY. IN ADDITION, WE CURATE THE DOSAGE SENSITIVITY FOR EACH GENE TO AID IN THE INTERPRETATION OF STRUCTURAL VARIANTS, AS WELL AS THE ACTIONABILITY OF GENE-DISEASE PAIRS TO GUIDE THE USE OF THIS INFORMATION FOR DETERMINING DISEASE RISK. TO SUPPORT VARIANT INTERPRETATION, WE DEVELOP STANDARDS TO ENABLE THE GLOBAL COMMUNITY TO CLASSIFY VARIANTS WITH THE SAME RIGOROUS STANDARDS AS CLINGEN. WE CONTINUE TO IMPROVE GLOBAL DATA SHARING OF GENOMIC KNOWLEDGE THROUGH OUR SUCCESSFUL PARTNERSHIP WITH CLINVAR, THE NATIONAL CENTER FOR BIOTECHNOLOGY INFORMATION’S REPOSITORY FOR GENOMIC VARIANTS AND THEIR RELATIONSHIPS TO HUMAN HEALTH, AS WELL AS THROUGH DIRECT DATA SHARING FROM PATIENTS VIA GENOME CONNECT, CLINGEN’S PATIENT REGISTRY. WE WILL CONTINUE TO SOLICIT AND SUPPORT CLINVAR SUBMISSIONS, WITH PARTICULAR FOCUS ON GLOBAL AND DIVERSE DATA SOURCES, ALLOWING TRANSPARENCY, COMPARISON OF RESULTS, AND CROWDSOURCING VARIANT CLASSIFICATION. FINALLY, WE DISSEMINATE CLINGEN’S WORK THROUGH MULTIPLE MODALITIES, ENABLING WEB-BASED AND COMPUTATIONAL DATA ACCESS, WHILE EDUCATING THE COMMUNITY ON ITS AVAILABILITY AND UTILITY. CLINGEN HAS BECOME AN INVALUABLE COMMUNITY RESOURCE FOR GENOMICS; CONTINUED EFFORTS WILL ALLOW US TO REALIZE THE FULL PROMISE FOR PRECISION MEDICINE.

THE NEXT ITERATION OF THE AMP-T2D KNOWLEDGE PORTAL

GENETIC NEUROSCIENCE: HOW HUMAN GENES AND ALLELES SHAPE NEURONAL PHENOTYPES

GENOME CHARACTERIZATION CENTER (GCC)

COUNT ME IN: PARTNERING WITH PATIENTS TO DEFINE THE CLINICAL AND GENOMIC LANDSCAPE OF RARE AGGRESSIVE SARCOMAS IN CHILDREN AND ADULTS

JOINT CENTER FOR MENDELIAN GENOMICS

PROTEO-GENOMIC DISCOVERY, PRIORITIZATION AND VERIFICATION OF CANCER BIOMARKERS

A BIOCHEMICAL ROADMAP OF EXERCISE SIGNALING

THE CANCER GENOME ATLAS DATA ANALYSIS CENTER

MULTIETHNIC STUDY OF TYPE 2 DIABETES GENES

CENTER FOR CELL CIRCUITS

PRODUCTION SEQUENCING OF REFERENCE HUMAN EPIGENOMES

CENTER FOR CELL CIRCUITS

COMPREHENSIVE SEQUENCING AND ANALYSIS OF VARIATION IN NHLBI COHORTS

BROAD INSTITUTE MENDELIAN GENOMIC RESEARCH CENTER - PROJECT SUMMARY DESPITE SIGNIFICANT PROGRESS TOWARD DECIPHERING THE GENETIC CAUSE OF MANY RARE DISEASE PHENOTYPES IN THE NHGRI CENTERS FOR MENDELIAN GENETICS (CMG), MORE THAN HALF OF THE GENES UNDERLYING MENDELIAN DISEASES REMAIN UNDISCOVERED. HOWEVER, REMARKABLE DEVELOPMENTS IN GENOMICS TECHNOLOGIES AND THE AGGREGATION OF MASSIVE REFERENCE DATASETS ARE POISED TO ADVANCE MENDELIAN GENE DISCOVERY, PROVIDED THESE TECHNOLOGIES CAN BE EXPLOITED USING SOPHISTICATED ANALYTIC TOOLS IN LARGE AND DIVERSE COHORTS. IMPORTANTLY, THESE METHODS AND DATASETS CAN ONLY CATALYZE GENE DISCOVERY IF THEY ARE RAPIDLY SHARED WITH THE COMMUNITY, AND ENABLING THIS GOAL HAS BEEN A PRIMARY FOCUS OF OUR BROAD INSTITUTE CMG. HERE, WE BRING TOGETHER AN EXTRAORDINARY TEAM OF INVESTIGATORS WITH DIVERSE EXPERTISE, COMPLEMENTARY TECHNOLOGIES, NOVEL ANALYTIC METHODS, AN ESTABLISHED RECRUITMENT NETWORK, AND PLATFORMS THAT WE HAVE DEVELOPED FOR DATA SHARING TO EXPLORE THE GENETIC UNDERPINNINGS OF MENDELIAN DISEASE, AND TO FURTHER ENABLE THERAPEUTIC DEVELOPMENT FOR RARE DISEASES. THE BROAD INSTITUTE MENDELIAN GENOMICS RESEARCH CENTER (MGRC) BUILDS UPON THE WORLD-CLASS TRACK RECORD OF MENDELIAN GENE DISCOVERY, METHODS DEVELOPMENT, AND DATA SHARING SET BY THE BROAD CMG. OUR TEAM HAS INVESTED CONSIDERABLE EFFORT TO DEVELOP WIDELY ADOPTED TOOLS AND PLATFORMS EMPOWERING VARIANT ANALYSIS, SUCH AS GATK, GNOMAD, AND SEQR, AND TO FACILITATE OPEN SHARING OF VARIANTS, DATA, AND ANALYSIS TOOLS. OVER THE LAST FOUR YEARS, WE HAVE GENERATED AND SHARED DATA FOR OVER 15,000 SAMPLES FROM 7,600 FAMILIES. IN THE PROCESS, WE HAVE UNCOVERED 256 NOVEL DISEASE-GENE RELATIONSHIPS, WITH 473 ADDITIONAL GENES UNDERGOING FOLLOW-UP. OUR MGRC ROADMAP WILL RELY ON EXOME SEQUENCING AND RAPID DATA SHARING AS THE MOST EFFICIENT FRONTLINE APPROACH, GIVEN THAT THE VAST MAJORITY OF CMG DISCOVERIES ARE DERIVED FROM CODING VARIANTS, FOLLOWED BY GENOME SEQUENCING ON UNSOLVED CASES (AIM 1). COMPLEMENTARY APPROACHES TO DISCOVER VARIATION NOT CAPTURED BY CONVENTIONAL METHODS WILL INCLUDE EMERGING SEQUENCING TECHNOLOGIES, REFERENCE-FREE ASSEMBLY, IMPROVED ANNOTATION OF EVOLUTIONARY CONSTRAINT, LARGE-SCALE DATA AGGREGATION, AND NOVEL ANALYTIC METHODS. TRANSCRIPTOME SEQUENCING, EPIGENETIC PROFILING, AND CRISPR EDITING, AS WELL AS IN VITRO AND IN VIVO FUNCTIONAL MODELING, WILL THEN INFORM FUNCTIONAL INTERPRETATION AND MECHANISTIC DISSECTION (AIM 2). FINALLY, WE WILL USE OUR PLATFORMS TO CREATE NEW TOOLS AND APPROACHES FOR TRANSFORMATIVE DATA SHARING ACROSS THE MGRCS AND THE BROADER COMMUNITY (AIM 3). AT THEIR CONCLUSION, THESE STUDIES WILL SIGNIFICANTLY CONTRIBUTE TO COMPLETING THE CATALOG OF GENES UNDERLYING MENDELIAN DISEASE, PROVIDING NEW BIOLOGICAL INSIGHTS INTO THEIR FUNCTIONAL MECHANISMS, AND OPENLY SHARING THE DATA, TOOLS, AND DISCOVERIES THAT WE PRODUCE.

A CENTER FOR GEI ASSOCIATION STUDIES

SIGNATURES OF KINASE ACTIVATION IN CANCER

BROAD INSTITUTE LINCS CENTER FOR TRANSCRIPTOMICS

PROPOSAL FOR THE AMP T2D-GENES DATA COORDINATION CENTER AND WEB PORTAL

THE PURPOSE OF THIS AGREEMENT IS TO FUND RESEARCH SUPPORTING THE DEFENSE ADVANCED RESEARCH PROJECTS AGENCY (DARPA) BIOLOGICAL TECHNOLOGIES OFFICES (BTO) HARNESSING ENZYMATIC ACTIVITY FOR LIFESAVING REMEDIES (HEALR) PROGRAM

WHOLE INDIVIDUAL COMPREHENSIVE KNOWLEDGE: SOMATIC MOSAICISM ACROSS HUMAN TISSUES (WICKED SMAHT) - ABSTRACT: (MAX 30 LINES) THERE IS AN ENORMOUS NEED TO CREATE A FOUNDATIONAL DATASET THAT WILL COMPREHENSIVELY CATALOG SOMATIC MUTATIONS ACROSS A DIVERSE RANGE OF HUMAN TISSUES, TO UNDERSTAND THEIR TYPE, FREQUENCY, THE CELLS, AND ANATOMICAL COMPARTMENTS IN WHICH THEY ARE FOUND, AND THEIR ORGANIZATIONAL, FUNCTIONAL RELATIONSHIPS AND CONSEQUENCES. SOMATIC VARIANTS CAN OCCUR WIDELY THROUGHOUT THE GENOME AND ACROSS THE LIFESPAN PRODUCING STOCHASTIC, CLONAL, AND DYNAMIC SOMATIC MOSAICISM. A GROWING NUMBER OF STUDIES INTERROGATING MUTATIONS IN NORMAL TISSUES HAVE DEMONSTRATED CAUSAL ROLES FOR SOMATIC VARIANTS IN AGE-RELATED PROCESSES AND BOTH ONCOLOGIC AND NON-ONCOLOGIC CONDITIONS, BUT THESE STUDIES HAVE GENERALLY BEEN LIMITED TO FEW TISSUES OR FEW INDIVIDUALS. THE BROADER FUNCTIONAL CONSEQUENCES OF SOMATIC VARIATION REMAIN LARGELY UNKNOWN. ADDITIONALLY, VARIANTS IN NON-CODING, REGULATORY REGIONS OF THE GENOME MAY AFFECT GENE EXPRESSION IN DIFFERENT CELLS OR TISSUES WITHIN INDIVIDUALS, BUT THEIR PATTERNS ALSO REFLECT A LIFETIME OF EXPOSURES TO CERTAIN MUTATIONAL SIGNATURES, WITH LIKELY DIFFERENCES BETWEEN TISSUES AND INDIVIDUALS. RECENT ADVANCES IN GENOMICS PROVIDE AN OPPORTUNITY TO BUILD A SOMATIC MUTATION CATALOG FOR HUMAN TISSUES AND ORGANS. A CROSS-TISSUE CATALOG OF HUMAN SOMATIC MUTATIONS AT HIGH CELLULAR AND GENETIC RESOLUTION WILL PROVIDE AN EXTRAORDINARY OPPORTUNITY TO DISCOVER THE PROCESSES UNDERLYING NORMAL TISSUE FUNCTION, AND LEADING TO DISEASE, BUT THE DEVELOPMENT OF SUCH A CATALOG REQUIRES STATE-OF-THE-ART, AND NOVEL, EXPERIMENTAL AND COMPUTATIONAL FRAMEWORKS. AS A GENOME CHARACTERIZATION CENTER (GCC) FOR THE SOMATIC MOSAICISM ACROSS HUMAN TISSUES (SMAHT) PROGRAM, OUR PROJECT WILL CONTRIBUTE TO THE CHARACTERIZATION, AND CREATION OF A RESOURCE CATALOG OF SOMATIC VARIATION ACROSS HUMAN TISSUES AT SCALE. WE WILL (1) WORK WITH THE TISSUE PROCUREMENT CENTERS AND THE NETWORK TO SHARE TISSUE SAMPLING PROTOCOLS; (2) COLLABORATE WITH OTHER GCCS TO UNDERTAKE BENCHMARKING OF SAMPLES ACROSS CENTERS TO COMPARE PRODUCTION DATA AND PIPELINES; (3) GENERATE CORE ASSAYS (SHORT AND LONG-READ DNA SEQUENCING AND RNA SEQUENCING) ACROSS 750 SAMPLES, AND AN ADDITIONAL DUPLEX SEQUENCING ASSAY ACROSS ~250 SAMPLES; (4) QC AND ANALYZE ALL DATA AND SUBMIT TO THE DATA ANALYSIS CENTER; AND (5) COLLABORATE ACROSS THE NETWORK BROADLY ON BENCHMARKING COMPARISONS, THE DEVELOPMENT OF COMMON ANALYTICAL PIPELINES AND QC METRICS, AND TO PERFORM JOINT DATA ANALYSIS. OUR GCC BRINGS TOGETHER A TEAM WITH EXPERTISE IN HUMAN GENETICS/GENOMICS, PRODUCTION SCIENCE, COMPUTATIONAL BIOLOGY, NOVEL TECHNOLOGIES, AND THE CHARACTERIZATION AND INTERPRETATION OF SOMATIC MUTATIONS. WE WILL HELP CREATE A SOMATIC DATA RESOURCE FOR THE SCIENTIFIC COMMUNITY THAT WILL CATALYZE STUDIES TO UNDERSTAND CELLULAR AND TISSUE CHANGES DURING HUMAN DEVELOPMENT, AGING, AND DISEASE VARIANT INTERPRETATION.

2/3-WHOLE GENOME SEQUENCING FOR SCHIZOPHRENIA AND BIPOLAR DISORDER IN THE GPC

DEVELOPMENTAL GTEX LABORATORY, DATA ANALYSIS AND COORDINATION CENTER - ABSTRACT UNDERSTANDING THE TRANSCRIPTIONAL PROGRAMS GOVERNING CELL IDENTITY AND FUNCTION IS KEY TO MANY OUTSTANDING QUESTIONS IN BIOLOGY AND BIOMEDICINE. MOST VARIANTS ASSOCIATED WITH COMMON DISEASES AND TRAITS LIE IN NON- CODING, REGULATORY REGIONS OF THE GENOME, AND TO INTERPRET THEIR FUNCTION, WE NEED TO IDENTIFY THE GENES THEY AFFECT AND HOW THOSE ARE EXPRESSED IN THE DIFFERENT CELLS OF A TISSUE WITHIN INDIVIDUALS, AND QUANTIFY HOW THEIR EXPRESSION VARIES ACROSS INDIVIDUALS. HOW THESE MECHANISMS ARE REGULATED DURING HUMAN DEVELOPMENT REMAINS POORLY UNDERSTOOD, AND MOST STUDIES TO DATE HAVE RELIED ON HUMAN CELL LINES OR MODEL ORGANISMS AS PROXIES. GTEX DID NOT PROFILE TISSUES FROM POSTNATAL STAGES OF HUMAN DEVELOPMENT. REGULATORY ELEMENTS AND GENE NETWORKS ARE HIGHLY CELL TYPE–SPECIFIC, AND MAY ONLY BE ACTIVE DURING PRECISE DEVELOPMENTAL STAGES, DURING WHICH THEY MAY ALSO CONTRIBUTE TO DISEASE SUSCEPTIBILITY. THUS, THERE IS A CRITICAL NEED FOR MEASURING THE TRANSCRIPTIONAL AND REGULATORY LANDSCAPE OF TISSUES AND CELL TYPES DURING HUMAN DEVELOPMENT. WE WILL SERVE AS THE LDACC FOR THE CREATION OF THE DEVELOPMENTAL GTEX (DGTEX) RESOURCE. (1) WE WILL WORK WITH THE BPC TO ESTABLISH A BIOBANK OF 30 TISSUES FROM EACH OF 120 HUMAN POSTNATAL POSTMORTEM DONORS, CONSENTED FOR BROAD RESEARCH USE AND DATA SHARING. (2) WE WILL SELECT 4 TISSUES OF IMPORTANCE FOR COMMON DISEASES, AND IN WHICH THERE IS TISSUE REMODELLING DURING DEVELOPMENT - BRAIN, GUT, LUNG AND HEART. WE WILL PROFILE THESE TISSUES WITH MULTIPLE SINGLE CELL ASSAYS INCLUDING SNRNA-SEQ, MULTIOMIC RNA/ATAC, AND SPATIAL TRANSCRIPTOMICS TO CREATE DEEP MOLECULAR MAPS FOR VARIANT CHARACTERIZATION. (3) WE WILL PERFORM BASELINE MOLECULAR PROFILING OF GENOME SEQUENCE AND BULK TRANSCRIPTOME FOR ALL SAMPLES, AND INTEGRATE THE RESOURCE WITH GTEX. WE WILL USE THESE COLLECTIVE DATA TO IDENTIFY E/SQTLS; CREATE MAPS OF ELEMENT-GENE RELATIONSHIPS ACROSS TISSUES; CHART DIFFERENTIATION HIERARCHIES WITHIN TISSUES AND ACROSS AGE RANGES; NOMINATE CELL TYPES IN WHICH A SET OF VARIANTS MANIFEST; AND MAP CELLULAR AND SPATIAL RELATIONSHIPS AMONG CELLS, CELLULAR PROGRAMS, AND TISSUE FEATURES. THIS PROJECT BRINGS TOGETHER A TEAM WITH EXPERTISE IN HUMAN GENETICS, PRODUCTION SCIENCE (GTEX, ENCODE), COMPUTATIONAL BIOLOGY, SINGLE CELL TECHNOLOGIES, AND COMMON DISEASE GENETICS. WE WILL CREATE A DATA AND BIOSPECIMEN RESOURCE FOR THE SCIENTIFIC COMMUNITY THAT WILL UNDERPIN STUDIES TO UNDERSTAND CELLULAR AND TISSUE CHANGES DURING HUMAN DEVELOPMENT, GENOME REGULATION, AND COMMON DISEASE VARIANT INTERPRETATION.

SINGLE CELL TRANSCRIPTOMIC AND EPIGENOMIC DISSECTION OF OPIOID AND COCAINE RESPONSES IN HIV - ABSTRACT SUBSTANCE USE DISORDER (SUD) IS A COMMON AND DEBILITATING CONDITION CHARACTERIZED BY COMPULSIVE USE OF AN ADDICTIVE SUBSTANCE, INABILITY TO CONTROL THE USE OF THIS SUBSTANCE, AND THE EMERGENCE OF WITHDRAWAL SYMPTOMS IN THE ABSENCE OF THE SUBSTANCE. DESPITE GREAT ADVANCES IN OUR UNDERSTANDING OF CHANGES THAT OCCUR IN VARIOUS BRAIN REGIONS IN THE CONTEXT OF ADDICTION/SUD, THERE IS A LIMITED UNDERSTANDING OF THE DIVERSITY OF CELL TYPES AND GENE EXPRESSION PROFILES OF CELLS WITHIN THESE HUMAN BRAIN REGIONS, AND HOW EXPOSURES TO ADDICTIVE SUBSTANCES SUCH AS OPIOIDS AND COCAINE INFLUENCE THE MOLECULAR PROPERTIES AND FUNCTIONS OF THESE CELLS. OVER 50% OF HIV- INFECTED PATIENTS EXPERIENCE NEUROLOGICAL DISORDERS COLLECTIVELY TERMED HIV-ASSOCIATED NEUROCOGNITIVE DISORDERS (HAND), WHICH RANGES FROM THE ASYMPTOMATIC NEUROCOGNITIVE IMPAIRMENT TO SEVERELY DISABLING DEMENTIA. THE USE OF ADDICTIVE SUBSTANCES BY HIV-INFECTED INDIVIDUALS HAS BEEN LINKED TO DIMINISHED IMMUNE FUNCTION, INCREASED NEUROINFLAMMATION AND NEURONAL INJURY, AND EXACERBATION OF HAND. HERE, WE SEEK TO DIRECTLY DISSECT THE COMMON AND DISTINCT MOLECULAR BASES OF SUD/HIV INFECTION/HAND EFFECTS ON DISTINCT CELL TYPES BY SYSTEMATIC PROFILING, DISSECTION, COMPUTATIONAL INTEGRATION, AND EXPERIMENTAL VALIDATION OF THEIR TRANSCRIPTIONAL, EPIGENOMIC, AND GENETIC SIGNATURES ACROSS INDIVIDUALS, BRAIN REGIONS, AND CELL TYPES. AIM 1: WE USE GENETIC, EPIGENOMIC, AND TRANSCRIPTIONAL PROFILES, GENERATING A TOTAL OF ~28 MILLION GENOME-WIDE MAPS AT THE SINGLE-CELL (SC) LEVEL USING 2,800 SAMPLES WITH 10,000 CELLS PER SAMPLE; THESE SPAN 7 BRAIN REGIONS (PREFRONTAL CORTEX, NUCLEUS ACCUMBENS, VENTRAL TEGMENTAL AREA, DORSAL STRIATUM, INSULA, AMYGDALA, HIPPOCAMPUS), TWO ASSAYS (SCRNA, SCATAC), FOUR PHENOTYPIC GROUPS (SUD+/HIV+, SUD+/HIV-, SUD-/HIV+, SUD-/HIV-), AND A TOTAL OF 200 SUBJECTS (50 IN EACH PHENOTYPIC GROUP). AIM 2: WE INTEGRATE THESE DATASETS TO PREDICT DRIVER GENES, REGULATORY REGIONS, VARIANTS, AND PATHWAYS, AND THE CELL TYPES AND BRAIN REGIONS WHERE THEY ACT. AIM 3: WE VALIDATE HIGH-PRIORITY FINDINGS AT THE MOLECULAR LEVEL, AND FUNCTIONALLY VALIDATE THE HIGHEST PRIORITY TARGETS’ ABILITY TO MODULATE ADDICTIVE BEHAVIORS IN MOUSE MODELS OF IN VIVO COCAINE SELF-ADMINISTRATION. THE RESULTING DATASETS WILL HELP GUIDE THE SEARCH FOR NEW THERAPEUTICS, BY PROVIDING DETAILED THERAPEUTIC TARGETS, AND THE SPECIFIC CONDITIONS WHERE THEY ARE PREDICTED TO ACT.

1/4 POWERING GENETIC DISCOVERY FOR SEVERE MENTAL ILLNESS IN LATIN AMERICAN AND AFRICAN ANCESTRIES

A FOUNDATIONAL RESOURCE OF FUNCTIONAL ELEMENTS, TF FOOTPRINTS AND GENE REGULATORY INTERACTIONS - PROJECT SUMMARY THIS PROJECT AIMS TO ASSEMBLE A FOUNDATIONAL RESOURCE OF FUNCTIONAL DNA ELEMENTS, TRANSCRIPTION FACTOR (TF) BINDING SITES AND GENE REGULATORY INTERACTIONS FOR THE IMPACT OF GENOMIC VARIATION ON FUNCTION (IGVF) CONSORTIUM. THE RESOURCE WILL FACILITATE INTERPRETATION OF NONCODING GENETIC VARIATION ASSOCIATED WITH HUMAN TRAITS AND DISEASES, ADVANCE UNDERSTANDING OF DISEASE MECHANISMS AND HASTEN PROGRESS TOWARDS GENOMIC MEDICINE. A LARGE MAJORITY OF GENETIC VARIANTS ASSOCIATED WITH HUMAN DISEASES ARE NON-CODING, WHICH HAS HINDERED THEIR INTERPRETATION AND UTILITY FOR UNDERSTANDING DISEASE. NON-CODING DISEASE VARIANTS ARE ENRICHED WITHIN PROMOTERS, ENHANCERS AND TF BINDING SITES. HENCE, A COMPELLING HYPOTHESIS IS THAT THEY MODULATE THE ACTIVITY OF FUNCTIONAL ELEMENTS, TF INTERACTIONS AND GENE TARGETS IN SPECIFIC CELLULAR CONTEXTS. TO INTERPRET THE FUNCTION OF A VARIANT, INVESTIGATORS MUST DETERMINE THE ELEMENT AND/OR TF THAT THEY IMPACT, WHICH GENE IS AFFECTED, AND THE CELL STATE IN WHICH THE EFFECT IS MANIFESTED. THIS PROCESS IS GREATLY FACILITATED BY GENOME-WIDE MAPS OF FUNCTIONAL ELEMENTS, TFS AND REGULATORY INTERACTIONS. HOWEVER, EXISTING RESOURCES UNDER-REPRESENT DISEASE-RELEVANT FUNCTIONAL ELEMENTS THAT ARE SPECIFIC TO EARLY DEVELOPMENTAL STAGES, RARE CELL STATES, PHYSIOLOGICAL RESPONSES, GENOTYPES OR DISEASE STATES. TO OVERCOME THESE LIMITATIONS, THE PROPOSED PROJECT WILL DEPLOY AN INNOVATIVE SUITE OF SINGLE-CELL ASSAYS TO PROFILE RNA TRANSCRIPTS, CHROMATIN ACCESSIBILITY, TF FOOTPRINTS AND HISTONE MODIFICATIONS AT UNPRECEDENTED SCALE. THESE ASSAYS WILL BE APPLIED TO AN EXPANSIVE COLLECTION OF PHENOTYPICALLY- AND GENOTYPICALLY-DIVERSE BIOSAMPLES SELECTED FOR THEIR RELEVANCE TO CARDIOVASCULAR, METABOLIC, AUTOIMMUNE, NEUROPSYCHIATRIC AND NEURODEGENERATIVE DISEASES. WE WILL ACQUIRE >16 MILLION SINGLE-CELL PROFILES FOR THOUSANDS OF BIOSAMPLES THAT SPAN CADAVERIC TISSUES, SURGICAL SPECIMENS, PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) COHORTS, BRAIN ORGANOIDS AND OTHER INNOVATIVE EXPERIMENTAL MODELS. INTEGRATION OF THIS VAST DATASET WILL ENABLE US TO (1) ANNOTATE MILLIONS OF REGULATORY ELEMENTS AND TF MOTIFS; (2) PREDICT GENE TARGETS FROM CO-VARIATION OF ELEMENT ACCESSIBILITY AND GENE EXPRESSION ACROSS SINGLE CELLS; AND (3) IDENTIFY QUANTITATIVE TRAIT LOCI FOR GENE EXPRESSION (EQTLS) AND CHROMATIN ACCESSIBILITY (CAQTLS) FROM THE DIVERSE GENOTYPES REPRESENTED IN OUR COHORTS. THE PROJECT WILL BRING TOGETHER A DIVERSE TEAM OF EXPERTS IN HUMAN GENETICS, DISEASE BIOLOGY, GENOMICS AND PRODUCTION RESEARCH. THE TEAM WILL COORDINATE CLOSELY WITH IGVF COLLEAGUES AND THE DACC IN THE DESIGN, ASSEMBLY AND INTEGRATION OF THIS RESOURCE. ALL DATA WILL BE MADE FREELY AVAILABLE AND MAXIMALLY ACCESSIBLE TO THE SCIENTIFIC COMMUNITY, WITH THE GOAL TO CATALYZE HUMAN GENETICS, DISEASE BIOLOGY AND GENOMIC MEDICINE.

A CATALOG OF CELL TYPES AND GENOMIC ELEMENTS IN TISSUES, ORGANOIDS AND DISEASE

ISOGENIC HUMAN PLURIPOTENT STEM CELL-BASED MODELS OF HUMAN DISEASE MUTATIONS

SYSTEMATIC IDENTIFICATION OF ENHANCERS TO TARGET THE BREADTH OF EXCITATORY AND INHIBITORY NEURONAL CELL TYPES IN THE CEREBRAL CORTEX - PROJECT SUMMARY IN THIS PROPOSAL WE AIM TO IDENTIFY GENE REGULATORY ELEMENTS THAT PERMIT THE TARGETING AND MANIPULATION OF BRAIN CIRCUIT MODELS OF HUMAN BRAIN FUNCTION. GAINING GENETIC ACCESS TO SPECIFIC NEURON POPULATIONS IN NONTRANSGENIC ANIMALS AND HUMANS WOULD ENABLE TARGETED CIRCUIT MODULATION FOR HYPOTHESIS TESTING AND PROVIDE A MEANS TO EVALUATE THE SAFETY AND EFFICACY OF CIRCUIT MODULATION FOR THE TREATMENT OF EPILEPSY AND PSYCHIATRIC DISORDERS. OUR APPROACH CAPITALIZES ON OUR COMBINED EXPERTISE IN THE DEVELOPMENT AND MATURATION OF BRAIN CELL-TYPES AND CIRCUITS (GORD FISHELL AND PAOLA ARLOTTA), IDENTIFICATION OF CIS-REGULATORY ELEMENTS THAT FUNCTION ACROSS SPECIES (YATING WANG), AAV ENGINEERING COMBINED WITH LARGE-SCALE SCREENING METHODS (BEN DEVERMAN) AND EXPERTISE AT COMBINING RNA AND CHROMATIN BIOLOGY (JASON BUENROSTRO). OUR EFFORTS WILL ALSO BENEFIT FROM AN ONGOING COLLABORATION WITH JOHN REYNOLDS AT THE SALK INSTITUTE ON OBSERVATION AND MANIPULATION OF CORTICAL CIRCUITS DURING COMPLEX VISUAL PERCEPTION TASKS. THIS PROJECT WILL BUILD UPON SUCCESS THAT WE AND OTHERS HAVE HAD IN IDENTIFYING GENE REGULATORY ELEMENTS THAT ENABLE CELL TYPE-RESTRICTED GENE EXPRESSION WHEN USED WITHIN RECOMBINANT ADENO-ASSOCIATED VIRUS (AAV) VECTORS. IN PARTICULAR, THE PIPELINE FOR ENHANCER DISCOVERY STEMMING FROM OUR RECENT UG3 GRANT PROVIDES US WITH THE SYSTEMATIC FOR IDENTIFYING ADDITIONAL ENHANCER SEQUENCES THAT FUNCTION IN THE CONTEXT OF THE LIMITED CARRYING CAPACITY OF AAV. HERE WE AIM TO APPLY THE NOVEL HIGH-THROUGHPUT SCREENING APPROACH WE DEVISED IN THE COURSE OF THIS UG3 FOR THE RAPID IDENTIFICATION OF A SUITE OF ENHANCERS THAT ENABLE THE STUDY AND MANIPULATION OF GENETICALLY DEFINED CELL TYPES AND CIRCUITS ACROSS SPECIES. OUR PRELIMINARY DATA DEMONSTRATES THAT OUR ENHANCER IDENTIFICATION STRATEGY CAN YIELD NOVEL AND HIGHLY SPECIFIC ENHANCERS THAT RESTRICT EXPRESSION TO TARGET POPULATIONS. IN ADDITION, WE HAVE DEMONSTRATED THAT IT IS POSSIBLE TO USE THE ENGINEERED AAV-PHP.EB CAPSID TO SCREEN ENHANCERS ACROSS THE BRAIN WITH A SINGLE NONINVASIVE INJECTION. THESE SUCCESSES HAVE HIGHLIGHTED THE NEED FOR MORE RAPID AND COMPREHENSIVE ASSESSMENT OF PUTATIVE ENHANCERS. IN ADDITION, WE WILL EXAMINE THE TOLERANCE TO NEURONAL ACTIVITY MANIPULATION WITHIN THE TARGET NEURONAL POPULATIONS IN SEVERAL SPECIES. THIS PROPOSAL WILL BE TRANSFORMATIVE IN DEVISING METHODS TO TARGET AND MANIPULATE THE BRAIN ACTIVITY OF SPECIFIC NEURONAL CELL POPULATIONS ACROSS SPECIES, INCLUDING HUMAN CELL-DERIVED ORGANOIDS.

DEFINING THE HUMAN MICROBIOME

AN INTEGRATED APPROACH TO DIVERSITY-ORIENTED SYNTHESIS

COMPREHENSIVE SEQUENCING AND ANALYSIS OF VARIATION IN NHLBI COHORTS

THERE AND BACK AGAIN: EPIGENETIC REINFORCEMENT OF CELLULAR SIGNALING STATES - OVERALL

GENOMESPACE: A COMMUNITY WEB ENVIRONMENT FOR INTEGRATIVE GENOMIC ANALYSIS

LARGE SCALE GENE EXPRESSION ANALYSIS OF CELLULAR STATES

SYSTEMATIC DISSECTION OF GUT CELLULAR CIRCUITS [AT SINGLE CELL RESOLUTION]

MEASURING CANCER BIOMARKER CANDIDATES BY TARGETED MS AND AB ENRICHMENT

D18AC00006

CENTER FOR GENOME INTERPRETATION

THE GLOBAL ALLIANCE FOR GENOMICS AND HEALTH: SETTING THE STANDARDS FOR GENOMICS AND HEALTH-RELATED DATA SHARING - PROJECT SUMMARY THE DECREASING COST OF GENOMIC SEQUENCING WILL YIELD MILLIONS OF SAMPLES IN THE COMING YEARS FROM BOTH RESEARCH AND HEALTHCARE. SHARING THIS DATA IS NECESSARY TO UNDERSTAND HUMAN DISEASES AND EVENTUALLY HELP PATIENTS, BUT DOING SO REQUIRES THE COMMUNITY TO AGREE ON COMMON METHODS FOR COLLECTING, STORING, TRANSFERRING, ACCESSING, AND ANALYZING DATA. THIS PROPOSAL WILL SUPPORT THE GLOBAL ALLIANCE FOR GENOMICS AND HEALTH (GA4GH; WWW.GA4GH.ORG) TO AID GENOMIC RESEARCH AND HUMAN HEALTH BY DEVELOPING STANDARDS AND POLICIES FOR EFFECTIVE AND RESPONSIBLE DATA SHARING BETWEEN INSTITUTIONS AND COUNTRIES AROUND THE WORLD. TO ADVANCE RESPONSIBLE SHARING OF GLOBAL GENOMIC AND HEALTH-RELATED DATA, GENOMICS RESEARCHERS, CLINICIANS, BIOINFORMATICIANS, SOFTWARE ENGINEERS, AND INDUSTRY EXPERTS WILL WORK TOGETHER AS A SINGLE GA4GH COMMUNITY TO DELIVER GENOMIC DATA SHARING STANDARDS AND FRAMEWORKS (E.G., ONTOLOGIES, GUIDELINES, TECHNICAL SCHEMAS). BUILDING ON OUR FIVE YEARS OF EXPERIENCE CONVENING STAKEHOLDERS AND DEVELOPING WORK PRODUCTS, WE WILL ENGAGE THE GENOMICS AND HEALTH COMMUNITY IN THE VERY EARLIEST STAGES OF DEVELOPMENT TO ENSURE OUR WORK IS USEFUL AND READY FOR ADOPTION. WE WILL LEVERAGE THE COMBINED EFFORT OF SEVERAL HUNDRED ACTIVE CONTRIBUTORS TO ADVANCE DEVELOPMENT ACTIVITIES BEYOND THE CAPACITY OF OUR SMALL STAFF TEAM. THESE CONTRIBUTORS WILL WORK WITHIN EIGHT GA4GH WORK STREAMS, EACH FOCUSED ON DEVELOPING CRITICAL STANDARDS AND FRAMEWORKS, INCLUDING CLOUD-BASED DATA FEDERATION, SCALABLE SCHEMAS AND INTERFACES, DATA MODELS, AND FILE FORMATS. WE WILL ENGAGE DEEPLY WITHIN THE BROADER HEALTHCARE, RESEARCH, AND COMMERCIAL SECTORS, INCLUDING THE LAUNCH OF THE GENOMICS IN HEALTH IMPLEMENTATION FORUM TO DRIVE UPTAKE IN THE CLINICAL DOMAIN. A FEDERATED ECOSYSTEM FOR SEARCHING, DISCOVERING, EXCHANGING, AND ANALYZING GENOMIC AND CLINICAL DATA WILL ENABLE A GLOBAL LEARNING HEALTH SYSTEM THAT ADVANCES BOTH RESEARCH AND CLINICAL CARE BEYOND THEIR INDIVIDUAL CAPACITIES AND DEPENDS ON STANDARDS AND INTEROPERABLE FRAMEWORKS EMBRACED BY THE ENTIRE COMMUNITY. WE ENVISION A FUTURE IN WHICH THE FULL SUITE OF GA4GH STANDARDS ENABLES ALL CLINICIANS, GENETICISTS, AND RESEARCHERS TO SEARCH ACROSS THE WORLD’S COLLECTIVE GENOMIC DATA TO REVEAL UNANTICIPATED GENE-DISEASE ASSOCIATIONS, MAKE OTHERWISE IMPOSSIBLE DRUG-RESPONSE PREDICTIONS, AND GENERALLY PARTICIPATE IN GENOMICS AT A COMPETITIVE PACE—REGARDLESS OF THEIR MEANS OR LOCATION. THE PROMISE OF GENOMIC MEDICINE LIES AT A CROSSROADS THAT DEPENDS ON HARMONIZATION ACROSS THE COMMUNITY AND WILL SIGNIFICANTLY ENHANCE THE HUMAN EXPERIENCE IF WE SUCCEED. WE BELIEVE THAT GA4GH IS NECESSARY TO THAT SUCCESS.

VIROME INVESTIGATION IN DIVERSE HUMAN POPULATIONS - PROJECT SUMMARY/ABSTRACT (OVERALL): THE COMPOSITION, DIVERSITY, AND DYNAMICS OF ALL VIRUSES FOUND INSIDE AND ON THE HUMAN BODY–THE HUMAN VIROME–REMAINS POORLY UNDERSTOOD. THE GOAL OF OUR HUMAN VIROME PROGRAM (HVP) VIROME CHARACTERIZATION CENTER (VCC) IS TO DISCOVER AND CURATE THE SPECTRUM OF VIRUSES PRESENT IN HUMANS, AND TO CHARACTERIZE KEY FEATURES OF THEIR BIOLOGY AND DYNAMICS, INCLUDING MAPPING VIRAL TROPISM, MONITORING CHANGES OVER TIME, IDENTIFYING DIFFERENCES, AND CATALOGING ALL PHAGES. THE VCC WILL LEVERAGE A COMBINATION OF GENOMICS, TRANSCRIPTOMICS, IMAGING, AND COMPUTATIONAL APPROACHES TO DEVELOP A SYSTEMATIC, MULTIMODAL APPROACH FOR MAPPING AND UNDERSTANDING THE HUMAN VIROME AND ITS DIVERSITY IN PRECISE DETAIL AND AT SCALE. WE WILL ESTABLISH A BESPOKE SPECIMEN COLLECTION FOR THIS PURPOSE, LEVERAGED FROM FIVE EXISTING LARGE, LONGITUDINAL AND DIVERSE COHORTS THAT SHARE A COMMON STUDY DESIGN, COLLECTIVELY ENCOMPASSING >350,000 PARTICIPANTS ACROSS ALL 55 U.S. STATES AND TERRITORIES. FROM IT, OUR BIOSPECIMEN COLLECTION CORE (BCC) WILL SELECT A DIVERSE 4,000-PARTICIPANT COHORT OF VARYING AGES, SEX/GENDER, GEOGRAPHIC ORIGIN, AND RACIAL/ETHNIC DIVERSITY, WHICH WILL BE PROCESSED BY THE BIOSPECIMEN ANALYSIS CORE (BAC) USING EXISTING CUTTING-EDGE METAGENOMIC AND OTHER OMICS APPROACHES, AND AS NEEDED DEVELOPING AND ADAPTING NEW METHODS TO IMPROVE VIRAL GENOME RECOVERY AND CHARACTERIZATION. THE DATA ANALYSIS AND SUBMISSION CORE (DASC) WILL USE STATE-OF-THE-ART COMPUTATIONAL APPROACHES TO PROCESS, STORE, ANALYZE, AND RELEASE THESE DATA. THE ETHICAL LEGAL AND SOCIETAL IMPLICATIONS (ELSI) CORE WILL MANAGE THE RESPONSIBLE ENGAGEMENT WITH PARTICIPANTS AND EXPLORE HOW FINDINGS CAN BE EFFECTIVELY COMMUNICATED TO PARTICIPANTS. THE VCC, COMPRISED OF INVESTIGATORS FROM THE BROAD INSTITUTE, BRIGHAM AND WOMEN’S HOSPITAL, AND HARVARD T.H. CHAN SCHOOL OF PUBLIC HEALTH, EMPHASIZES EFFECTIVE LEADERSHIP AND COLLABORATION AMONG ITS FIVE CORES. IT WILL BE LED BY A TEAM OF EXPERTS IN DIVERSE AREAS, INCLUDING VIRAL GENOMICS, THE HUMAN MICROBIOME, BIOINFORMATICS, TECHNOLOGY DEVELOPMENT, AND MANAGEMENT OF LARGE COHORT STUDIES. THE ADMINISTRATIVE CORE (AC) WILL ESTABLISH AND IMPLEMENT THE PROJECT PLAN WITH CLEAR MILESTONES, METRICS, AND CONTINGENCY PLANS TO ACHIEVE ITS GOALS. THE TIMELINE ENCOMPASSES PARTICIPANT ENGAGEMENT, SAMPLE RETRIEVAL, DATA PROCESSING, AND ANALYTICS, WITH ONGOING EVALUATION AND PROACTIVE MEASURES TO IDENTIFY AND ADDRESS POTENTIAL CHALLENGES. THE AC WILL ALSO IMPLEMENT A PLAN TO ENHANCE DIVERSE PERSPECTIVES (PEDP) FOR THE STUDY TEAM AND PARTICIPANTS. THE VCC IS COMMITTED TO RAPID AND OPEN SHARING OF ALL RESOURCES GENERATED, INCLUDING DATA, REAGENTS, PROTOCOLS, AND TOOLS, THROUGH PUBLIC REPOSITORIES, JOURNALS, AND COLLABORATION WITH OTHER VCCS. THROUGH THE PROJECT, WE WILL DEVELOP AN INNOVATIVE AND COMPREHENSIVE APPROACH TO CHARACTERIZING THE HUMAN VIROME, TO UNDERSTAND WHAT CONSTITUTES AND MODULATES THE VIROME AND HOW IT INFLUENCES HUMAN HEALTH, AND DISSEMINATE THE OUTCOMES BROADLY TO THE SCIENTIFIC COMMUNITY AND PUBLIC AT LARGE.

1/4 ASIAN BIPOLAR GENETICS NETWORK (A-BIG-NET) - PROJECT SUMMARY BIPOLAR DISORDER (BP) IS A SEVERE MULTIFACTORIAL NEUROPSYCHIATRIC DISORDER THAT IMPOSES A SIGNIFICANT BURDEN ON PUBLIC HEALTH. THE MOST RECENT LARGE-SCALE GENETIC STUDY OF BP IDENTIFIED 64 ASSOCIATED GENETIC LOCI, PROVIDING INITIAL INSIGHTS IN BP PATHOGENESIS. YET, GENETIC DISCOVERY IN BP LAGS BEHIND OTHER KEY PSYCHIATRIC DISORDERS. THE REPORTED GENETIC LOCI ONLY CAPTURE A SMALL PROPORTION OF THE TOTAL BP GENETIC LIABILITY, WITH MANY MORE VARIANTS ACROSS THE COMMON AND RARE ALLELE FREQUENCY SPECTRUM REMAINING TO BE DISCOVERED. IN ADDITION, THE PREVIOUS STUDIED SAMPLES WERE OF EUROPEAN ANCESTRY, LEAVING POPULATION SPECIFIC BP VARIANTS UNCOVERED AND UNCERTAINTY IN HOW THE BP GENETIC FINDINGS GENERALIZE TO OTHER POPULATIONS, EXACERBATING HEALTH DISPARITIES, AND THESE STUDIES RARELY EMPLOYED “DEEP” PHENOTYPING OR ASSESSED RELEVANT ENVIRONMENTAL RISK FACTORS. THIS PROPOSAL BRINGS TOGETHER AN INTERNATIONAL COLLABORATION OF LEADING INVESTIGATORS FROM THE U.S., TAIWAN, SOUTH KOREA, SINGAPORE, INDIA, AND PAKISTAN TO FORM THE ASIAN BIPOLAR GENETICS NETWORK (A-BIG-NET) AND CARRY OUT A LARGE-SCALE GENETIC STUDY OF BP IN EAST AND SOUTH ASIA. A-BIG-NET WILL GENERATE A BP GENETIC RESOURCE OF 27,500 CASES AND 16,000 CONTROLS WITH RICH PHENOTYPIC INFORMATION, MEASURES OF KEY ENVIRONMENTAL STRESSORS AND GENETIC DATA FROM 4X LOW-PASS WHOLE GENOME SEQUENCING (4XWGS). THIS WILL COMPLEMENT A SCHIZOPHRENIA GENETICS RESOURCE OF 22,778 CASES AND 35,362 CONTROLS OF ASIAN ANCESTRY PREVIOUSLY ASSEMBLED BY LEADERS OF THIS NETWORK THAT WILL BE AVAILABLE FOR CROSS-DISORDER COMPARISONS. STUDYING BP GENETICS IN ASIA IS IMPORTANT TO THE WORLD AND THE U.S., AS ASIA CONSTITUTES 57% OF THE WORLD POPULATION, AND ASIAN AMERICAN COMPRISES 6.6% OF THE U.S. POPULATION (21.4 MILLION). THE FIVE COUNTRIES IN A-BIG-NET COVER 47% OF ALL ASIAN POPULATIONS. THE SPECIFIC AIMS OF THE PROPOSAL ARE TO: 1) RECRUIT AND DEEPLY PHENOTYPE 17,500 BP CASES, WITH A FOCUS ON BP-I TO MAXIMIZE HOMOGENEITY, AND 14,000 CONTROLS FROM FOUR ASIAN COUNTRIES; 2) CARRY OUT 4XWGS ON ALL RECRUITED SAMPLES PLUS 10,000 BP-I CASES AND 2,000 CONTROLS COLLECTED BY A PREVIOUS STUDY USING SIMILAR PROCEDURES IN PAKISTAN; AND 3) CARRY OUT A RANGE OF ANALYSES TO DISCOVER NEW GENETIC ASSOCIATIONS WITH BP-I ACROSS THE ALLELIC SPECTRUM IN EAST AND SOUTH ASIAN POPULATIONS, EXAMINE THE COMPARATIVE GENETIC ARCHITECTURE OF BP-I ACROSS MAJOR WORLD POPULATIONS AND WITH OTHER MAJOR NEUROPSYCHIATRIC DISORDERS, AND PERFORM A NOVEL STATISTICAL FINE-MAPPING ANALYSIS THAT LEVERAGES THE MULTI-ANCESTRY GENOMIC DIVERSITY AND PLEIOTROPY ACROSS PSYCHIATRIC DISORDERS TO IDENTIFY PUTATIVE CAUSAL VARIANTS. AIM 3 WILL ALSO EXPLORE THE GENETIC “VALIDITY” OF VARIOUS BP-I SUBTYPES AND FIT MODELS WITH JOINT GENETIC AND ENVIRONMENTAL RISK FACTORS. THIS PROPOSAL WILL DRAMATICALLY INCREASE THE WORLDWIDE DIVERSITY OF GENETICS DATA ON BP, AN IMPORTANT STEP TO ACCELERATE GENE DISCOVERY IN THIS DISORDER AND ADVANCE GLOBAL MENTAL HEALTH EQUITY.

SPATIAL AND TEMPORAL RESOLUTION TO DISSECT CELLULAR CIRCUITS CONTROLLING INTESTINAL PHYSIOLOGY, IMMUNITY, AND INFLAMMATORY PATHOLOGIES - PROJECT SUMMARY AS A CRITICAL BARRIER TISSUE, THE INTESTINAL MUCOSA MUST BALANCE COMPLEX ENVIRONMENTAL STIMULI — INCLUDING DIETARY COMPONENTS, MICROBES, METABOLITES, AND XENOBIOTICS — TO FINE-TUNE IMMUNE DEVELOPMENT AND TOLERANCE. IN HEALTH, HOMEOSTASIS IS MAINTAINED VIA A CONSTELLATION OF SPECIALIZED CELL TYPES THAT CONNECT HOST PHYSIOLOGY TO THESE EXTERNAL SIGNALS. DISRUPTION OF INTESTINAL PHYSIOLOGY RESULTS IN NUMEROUS DISEASES, INCLUDING INFLAMMATORY CONDITIONS SUCH AS CROHN’S DISEASE AND ULCERATIVE COLITIS. HERE, WE PROPOSE A FRAMEWORK TO DISSECT THE CELLULAR MECHANISMS UNDERLYING THE HOMEOSTATIC AND INFLAMED STATES IN THE SMALL INTESTINE AND COLON AT UNPRECEDENTED SPATIAL AND TEMPORAL RESOLUTION. IN AIM 1, WE WILL SPATIALLY PROFILE GENE AND PROTEIN EXPRESSION ACROSS HUMAN GUT SAMPLES IN HEALTH AND DISEASE, DEVELOPING THE NECESSARY COMPUTATIONAL TOOLS TO IDENTIFY CRITICAL DISRUPTIONS IN CELL-CELL COMMUNICATION NETWORKS THAT RESULT FROM INFLAMMATION. COMPUTATIONAL TOOLS DEVELOPED IN THIS CONTEXT WILL HAVE BROAD APPLICATION IN STUDYING TISSUE BIOLOGY. ADDITIONALLY, WE WILL MAKE CROSS-DISEASE COMPARISONS (E.G., CROHN’S DISEASE, CELIAC DISEASE, AND EOSINOPHILIC GASTROENTERITIS) TO IDENTIFY COMMON INFLAMMATORY MECHANISMS INDEPENDENT OF TISSUE TYPE. IN AIM 2, WE WILL LEVERAGE EXPERIMENTAL MOUSE MODELS TO SPATIALLY PROFILE THE DYNAMICS OF INFLAMMATION (FROM HOMEOSTASIS THROUGH INDUCTION AND RESOLUTION OF INFLAMMATION) AND DIETARY STRESS IN THE MOUSE GUT — GENERATING TEMPORALLY RESOLVED MAPS OF THESE PROCESSES. IN AIM 3, WE WILL USE ORGANOID AND CELL-CELL COCULTURE MODELS TO DETERMINE HOW CHEMOSENSORY PATHWAYS ARE TRANSLATED INTO THE COORDINATED CELLULAR RESPONSES THAT MAINTAIN HOMEOSTASIS.

EXPANDING THE CATALOG OF CHROMATIN REGULATORY ELEMENTS IN THE HUMAN GENOME

DISCOVERY PIPELINE (2 OF 4)

A COMPREHENSIVE PLATFORM FOR NOVEL THERAPY DEVELOPMENT FROM THE MICROBIOME

PROGRAMMABLE RNA-TARGETING TOOLS

TRINITY: TRANSCRIPTOME ASSEMBLY FOR GENETIC AND FUNCTIONAL ANALYSIS OF CANCER

'MULTIDIMENSIONAL CHEMOGENIC CONTROL OF CRISPR- NUCLEASES IN CELLS AND ORGANISMS'

RETURN OF GENOMIC RESULTS AND ESTIMATING PENETRANCE IN POPULATION-BASED COHORTS

JOINT SNP AND CNV CALLING IN 1000 GENOMES SEQUENCE DATA

CHARACTERIZING THE GUT MICROBIAL ECOSYSTEM FOR DIAGNOSIS AND THERAPY IN IBD

STATISTICAL METHODS TO LOCALIZE DISEASE HERITABILITY AND IDENTIFY BIOLOGICAL MECHANISMS

MAKING CANCER PRECISION MEDICINE REAL: BOTTLENECKS AND OPPORTUNITIES

CENTER FOR OPEN BIOIMAGE ANALYSIS

SCALABLE MOLECULAR PIPELINES FOR FAIR AND REUSABLE BICAN MOLECULAR DATA - PROJECT SUMMARY THIS PROJECT WILL CREATE SCALABLE RESOURCES THAT WILL BE CRITICAL TO THE DEVELOPMENT OF BRAIN INITIATIVE CELL ATLAS NETWORK (BICAN) CONSORTIUM BRAIN ATLASES. THIS TEAM WILL FOCUS ON THE DEVELOPMENT OF COMMON MOLECULAR DATA PROCESSING PIPELINES THAT ARE CLOUD-NATIVE AND FAIR. STARTING WITH AN ALREADY ESTABLISHED PORTFOLIO OF BICCN PIPELINES AND COMMUNITY RELATIONSHIPS DEVELOPED IN PREVIOUS FUNDING, THIS PORTFOLIO WILL BE UPDATED AND EXTENDED IN COLLABORATION WITH THE BICAN COMMUNITY. LEVERAGING AND IMPROVING EXISTING INTEGRATION BETWEEN THE BICCN MOLECULAR (NEMO) ARCHIVE AND CLOUD-COMPUTING ENVIRONMENT (TERRA), THIS TEAM WILL PROCESS DATA SETS, PRIORITIZED BY THE BICAN COMMUNITY AND IN SUPPORT OF JOINT ANALYSIS FOR ATLAS BUILDING. THESE PIPELINES WILL PRODUCE DATA WITH A COMPREHENSIVE PANEL OF METRICS THAT OUR TEAM WILL LEVERAGE TO PERFORM QUALITY CONTROL AND ASSURE RIGOR IN DATA PROCESSING AND USE. THIS TEAM WILL WORK WITH ANALYSIS GROUPS TO CONFIRM OUTPUTS OF THE PIPELINES ARE COMPATIBLE WITH DOWNSTREAM ANALYSIS AND ARE EXTENDED TO INCORPORATE CRITICAL DOWNSTREAM STEPS AS THEY MATURE AND ARE AGREED UPON BY THE BICAN COMMUNITY. THE CLOUD COMPUTING ENVIRONMENT USED BY THIS TEAM WILL BE OPEN AND AVAILABLE TO THE BICAN COMMUNITY AND SUPPORTED BY A HELPDESK, DOCUMENTATION, FORUMS, AND AN ANNUAL WORKSHOP. AS THIS INFRASTRUCTURE SPANS INSTITUTES, TERRA IS CURRENTLY ENABLING BICCN WORKING GROUPS TO SHARE CLOUD RESOURCES AND JOINTLY ANALYZE DATA IN A COMMON SCALABLE SPACE; THIS WILL CONTINUE TO BE TRUE IN THE BICAN COMMUNITY. TO ENABLE VISUALIZING, SHARING AND PUBLISHING OF DATA, THIS TEAM BRINGS CLOUD PROGRAMMING ENVIRONMENTS (TERRA), SPECIALIZED SCALABLE RESOURCES (CIRROCUMULUS, PEGASUS), AND PUBLICATION PORTALS (NEMO ANALYTICS, AND THE SINGLE CELL PORTAL) TO BE LEVERAGED IN CONSORTIUM ACTIVITIES. THESE COMMON RESOURCES WILL BE CRITICAL TO ENABLE THE BICAN COMMUNITY TO COME TOGETHER AND PERFORM ANALYSIS THAT SCALES TO THE NEEDS OF BICAN ATLAS BUILDING EFFORTS.

DEVELOPMENT AND VALIDATION OF AAV VECTORS TO MANIPULATE SPECIFIC NEURONAL SUBTYPES AND CIRCUITS INVOLVED IN EPILEPSY AND PSYCHIATRIC DISORDERS ACROSS MAMMALIAN SPECIES.

COMPREHENSIVE FUNCTIONAL CHARACTERIZATION AND DISSECTION OF NONCODING REGULATORY ELEMENTS AND HUMAN GENETIC VARIATION

RNA BASED DIAGNOSTICS FOR RAPID PATHOGEN IDENTIFICATION AND DRUG RESISTANCE

HIGH-THROUGHPUT SEQUENCING OF CHROMATIN REGULATORY ELEMENTS

CENTER OF EXCELLENCE FOR HIGH THROUGHPUT PROTEOGENOMIC CHARACTERIZATION

INTEGRATING CHEMISTRY AND EVOLUTION TO ILLUMINATE BIOLOGY AND ENABLE NOVEL THERAPEUTICS

EXPLORATION OF DIVERSE MOBILE GENETIC ELEMENTS FOR PRECISION GENOME MANIPULATION

EXTRACTING RICH INFORMATION FROM BIOLOGICAL IMAGES

WHOLE GENOME SEQUENCING OF BIPOLAR DISORDER AND SCHIZOPHRENIA

COVID19 SLIDE-SEQ

NEW TARGETS AGAINST TUBERCULOSIS

TARGETING VULNERABILITIES OF THERAPY-RESISTANT CANCER CELL STATES WITH SMALL MOLECULES

DEVELOPMENT OF POLYGENIC RISK SCORES FOR DIABETES AND COMPLICATIONS ACROSS THE LIFE-SPAN IN POPULATIONS OF DIVERSE ANCESTRY - ABSTRACT LARGE-SCALE GENOME WIDE ASSOCIATION STUDIES (GWAS) HAVE IDENTIFIED A LARGE NUMBER OF GENETIC VARIANTS ASSOCIATED WITH COMPLEX DISEASES. THE AGGREGATION OF ALL THE VARIANTS THAT ARE KNOWN TO CONTRIBUTE TO THE DISEASE IN THE FORM OF POLYGENIC RISK SCORES (PRS) IMPROVES THE PREDICTION OF A RANGE OF COMPLEX DISEASES. MOST PRS HAVE BEEN DEVELOPED WITHIN EUROPEAN ANCESTRY STUDY SAMPLES AND HAVE SHOWN TO PERFORM POORLY IN OTHER RACE/ETHNIC GROUPS, FURTHER EXAGGERATING HEALTH DISPARITIES ACROSS ANCESTRIES. AS GENETIC APPROACHES FOR PRECISION MEDICINE BECOME MORE POPULAR, THERE IS A CRITICAL NEED TO RESPONSIVELY AND PRO-ACTIVELY EXPAND ACCESS TO ACCURATE PRS. SPECIFICALLY, DIABETES, AND ITS ASSOCIATED COMPLICATIONS ARE ONE OF THE BIGGEST GLOBAL HEALTH PROBLEMS OF THE 21ST CENTURY. IN FACT, TYPE 1 AND TYPE 2 DIABETES (T1D AND T2D), GESTATIONAL DIABETES (GDM) AND RELATED COMPLICATIONS ARE EXCELLENT DISEASE MODELS TO STUDY THE UTILITY OF PRS FOR PREDICTING HETEROGENOUS AND COMPLEX HEALTH OUTCOMES IN A SETTING WHERE DRAMATIC RACIAL/ETHNIC AND SOCIOECONOMIC DISPARITIES EXIST. NOT ONLY ARE PRS USEFUL TO PREDICT T1D AND T2D, BUT THEY CAN DISTINGUISH BETWEEN T1D AND T2D, AND BETWEEN T2D SUBTYPES. THE WEALTH OF EXISTING TRANS-ANCESTRY GWAS DATA FROM DIABETES SUBTYPES, COMPLICATIONS, AND QUANTITATIVE TRAITS RECENTLY GENERATED PROVIDES A UNIQUE OPPORTUNITY FOR CONSTRUCTING HIGHLY TRANSFERABLE PRS ACROSS POPULATIONS. TO ADDRESS THE DISPARITIES IN PRS ACROSS ANCESTRIES, WE HAVE ASSEMBLED A MULTI-DISCIPLINARY TEAM TO AGGREGATE AND ANALYZE THE LARGEST EXISTING GENETIC DATA FROM MORE THAN 1.8 M INDIVIDUALS (35% NON- EUROPEAN) WITH T1D, T2D, GDM AND GLYCEMIA-RELATED COMPLICATIONS AND QUANTITATIVE TRAITS TO IMPROVE THE PRS PREDICTION OF DIABETES AND PROGRESSION ACROSS LIFESPAN IN DIVERSE ANCESTRIES WITH THESE AIMS: (1) COLLECTION, HARMONIZATION AND INTEGRATION OF LARGE-SCALE, MULTI-ANCESTRY COHORTS WITH DIABETES TRAITS ACROSS THE LIFE-SPAN AND GENOMICS FOR DEVELOPMENT, TRAINING AND TESTING PRS FOR DIVERSE ANCESTRIES; (2) DEVELOPMENT OF METHODS TO IMPROVE PRS PREDICTION IN NON-EUROPEAN POPULATIONS BY USING BAYESIAN APPROACHES THAT ALLOW INTEGRATION OF LINKAGE DISEQUILIBRIUM AND SUMMARY STATISTICS FROM SEVERAL ANCESTRIES. (3) DEVELOPMENT, TESTING, AND COMPARING PERFORMANCE OF PRS FOR EACH TRAIT, DEVELOPMENT OF RISK PREDICTION TOOLS THAT INTEGRATE CLINICAL AND GENETIC RISK FACTORS, AND ASSESSMENT OF SCENARIOS WHERE PRS IMPROVE THE PREDICTION. ACCOMPLISHING THE AIMS OF THIS PROPOSAL WILL DEMONSTRATE HOW GENOMIC DATA CAN INFORM MORE EFFICIENT AND TARGETED PREVENTIVE STRATEGIES WITHIN HEALTHCARE SYSTEMS AND ACROSS ETHNICALLY DIVERSE POPULATIONS. FINDINGS ARE EXPECTED TO ADVANCE PRECISION CARE OF PATIENTS WITH DIABETES AND RELATED CONDITIONS IN PEOPLE OF DIVERSE ANCESTRAL BACKGROUND AND SERVE AS A PARADIGM FOR MANY OTHER COMPLEX DISEASES.

GENETICS OF PTSD IN AFRICAN ANCESTRY POPULATIONS: ENHANCING DISCOVERY BY ADDRESSING INEQUALITY - PROJECT SUMMARY / ABSTRACT THE BROAD GOAL OF THIS APPLICATION IS TO ADVANCE PTSD GENETIC DISCOVERY AND ADDRESS INEQUITY IN PTSD GENETICS RESEARCH BY LEVERAGING A PARTNERSHIP BETWEEN THE PSYCHIATRIC GENOMICS CONSORTIUM – POSTTRAUMATIC STRESS DISORDER WORKING GROUP (PGC-PTSD) AND A NETWORK OF AFRICAN INVESTIGATORS, INCLUDING THE NEUROPSYCHIATRIC GENETICS IN AFRICAN POPULATIONS (NEUROGAP) PROGRAM AND THE UGANDAN GENOME RESOURCE (UGR). BY THE END OF 2022, THE PGC-PTSD WILL ACHIEVE THE LARGEST GENETIC MAPPING OF PTSD TO DATE BASED ON MULTIETHNIC META- ANALYSIS OF > 1,280,000 SAMPLES, LEADING TO 95 RISK LOCI MEETING GENOME-WIDE SIGNIFICANCE FOR PTSD. BEYOND DISCOVERY, RECENT WORK ON POLYGENIC RISK SCORES IN PTSD SHOWS THE POTENTIAL FOR THE UTILITY OF THESE MEASURES IN RESEARCH. THIS SUCCESS IS OVERSHADOWED, HOWEVER, BY THE PERSISTENT UNDERREPRESENTATION OF AFRICAN POPULATIONS IN PTSD GENETIC RESEARCH, WHICH POSES A CHALLENGE FOR BOTH SCIENTIFIC ADVANCES IN PTSD AND GLOBAL EQUITY. AFRICAN GENOMES ARE CHARACTERIZED BY SHORTER HAPLOTYPE BLOCKS AND CONTAIN ALMOST A MILLION MORE VARIANTS PER INDIVIDUAL THAN POPULATIONS OUTSIDE AFRICA. THE LOWER CORRELATION BETWEEN GENETIC MARKERS IN AFRICAN POPULATIONS IS ALSO USEFUL FOR FINE- MAPPING DISEASE-CAUSING ALLELES. HOWEVER, DIFFERENCES IN THE AFRICAN GENOME ALSO RESULT IN LIMITED CROSS-POPULATION TRANSFERABILITY OF POLYGENIC RISK SCORES, AND, BY EXTENSION, POORER PERFORMANCE IN UNCHARACTERIZED POPULATIONS SUCH AS THOSE FROM AFRICA. THERE IS SIGNIFICANT RISK THAT THE RECENT ADVANCES IN PTSD GENETICS WILL RESULT IN A WIDENING OF THE MASSIVE RESEARCH AND TREATMENT DISPARITIES FOR AFRICAN POPULATIONS. THIS INEQUITY IS PARTICULARLY TROUBLING GIVEN THE DISPROPORTIONATELY HIGH BURDEN OF TRAUMA AND PTSD FACED BY AFRICAN POPULATIONS BOTH IN THE US AND ON THE AFRICAN CONTINENT. THUS, DATA FROM AFRICAN POPULATIONS IN GENETIC STUDIES OF PTSD ARE CRITICAL TO GENERATE A COMPLETE PICTURE OF GENETIC RISK FACTORS, IDENTIFY POTENTIALLY MISSING NOVEL THERAPEUTIC SIGNALS GARNERED BY STUDYING ALL POPULATIONS, AND ADDRESS GROWING HEALTH INEQUITY. WE PROPOSE ADDRESSING THIS INEQUITY BY ACCOMPLISHING THE FOLLOWING SPECIFIC AIMS: (1) EXPAND THE PGC–PTSD SAMPLE BANK WITH OVER 27,000 CASES AND 112,000 CONTROLS FROM THE AFRICAN CONTINENT AND DIASPORA TO ACHIEVE A ROBUST, WELL- POWERED SAMPLE SIZE OF OVER 190,000 PARTICIPANTS (~39,000 CASES, 151,000 CONTROLS) WITH AFRICAN ANCESTRY; (2) INTEGRATE DATA ACROSS AFRICA AND AFRICAN DIASPORA AND PERFORM ANALYSES TO EXPAND PTSD RISK LOCI DISCOVERY; AND (3) LEVERAGE AFRICAN ANCESTRY POPULATIONS TO IMPROVE FINE-MAPPING AND GENE PRIORITIZATION AND TO REDUCE HEALTH INEQUALITY BY IMPROVING PRS ACCURACY FOR PTSD IN THESE POPULATIONS. THESE AIMS ARE HIGHLY CONSISTENT WITH NIMH STRATEGIC PLAN GOAL 1, OBJECTIVE 1.2, STRATEGY 1.2.A “DISCOVERING GENE VARIANCES AND OTHER GENOMICS ELEMENTS THAT CONTRIBUTE TO MENTAL ILLNESSES IN DIVERSE POPULATIONS.”

STUDIES OF MATERIALS WITH PHYSIOLOGICAL PROPERTIES

MASSIVELY-PARALLEL FUNCTIONAL INTERROGATION OF PSYCHIATRIC GENETICS

SCALABLE TOOL AND COMPREHENSIVE MAPS TO INTERPRET STRUCTURAL VARIATION ACROSS THE NEUROPSYCHIATRIC SPECTRUM

CENTER OF EXCELLENCE FOR HIGH THROUGHPUT PROTEOGENOMIC CHARACTERIZATION - PROJECT SUMMARY CANCER PROTEOGENOMICS ENCOMPASSES METHODS THAT INTEGRATE MASS SPECTROMETRY (MS)-BASED MEASUREMENTS OF PROTEIN ABUNDANCE AND POST-TRANSLATIONAL MODIFICATIONS (PTMS) WITH GENOMIC, EPIGENOMIC, AND TRANSCRIPTOMIC DATA FROM PRECLINICAL CANCER MODELS AND TUMOR SAMPLES. THE MULTIDISCIPLINARY PROTEOGENOMIC CHARACTERIZATION CENTER WE PROPOSE WILL EMPLOY A RANGE OF STATE-OF-THE-ART MS-BASED PROTEOMIC AND METABOLOMIC TECHNOLOGIES TO SYSTEMATICALLY GENERATE AND INTEGRATE HIGH QUALITY, COMPREHENSIVE AND QUANTITATIVE PROTEOMIC AND METABOLOMIC DATA WITH GENOMIC DATA. OUR OVERARCHING GOALS ARE TO LEVERAGE THE INTEGRATED DATA TO IDENTIFY SIGNATURES OF CANCER DRIVERS, DETECT SIGNALING NETWORK ADAPTATIONS AND PROVIDE INFORMATION ON PTMS THAT AFFECT CELLULAR SIGNALING, MOLECULAR COMPLEX FORMATION, AND PROTEIN LOCATION, TRANSLATION AND STABILITY IN HUMAN BIOSPECIMENS AND RELEVANT MODELS OF CANCER. PEPTIDOMES OF THE CLASS I AND II HUMAN LEUKOCYTE ANTIGENS (HLA) OF THE TUMORS WILL ALSO BE ANALYZED TO SHED LIGHT ON TUMOR-IMMUNE ESCAPE MECHANISMS AND ANTIGEN PROCESSING IN CANCER, IMPROVE ALGORITHMS FOR PREDICTION OF ANTIGEN DISPLAY AND IMMUNOGENICITY AND INFORM DEVELOPMENT OF PERSONALIZED CANCER VACCINES. WE HYPOTHESIZE THAT INTEGRATING DEEP, HIGH QUALITY, QUANTITATIVE PROTEOMIC AND, ESPECIALLY, PTM-OMIC, HLA-PEPTIDOMIC AND METABOLOMIC DATA WITH GENOMIC AND TRANSCRIPTOMIC DATA WILL PROVIDE NOVEL INSIGHTS INTO THE PATHOPHYSIOLOGY OF CANCER AND HELP TO IDENTIFY NEW, ACTIONABLE TARGETS FOR DRUG DEVELOPMENT AND TREATMENT. DATA WILL BE RAPIDLY DISTRIBUTED TO THE CANCER BIOLOGY AND CLINICAL COMMUNITIES, AS WE HAVE DONE FOR THE PAST 15 YEARS IN THE NCI-CPTAC PROGRAM. THE RESULTING DATASETS WILL ENABLE A BROAD RANGE OF INVESTIGATION BY MANY TEAMS, ACCELERATING MOLECULARLY-ORIENTED CANCER RESEARCH TOWARD BIOLOGICAL AND CLINICAL IMPACT. WE WILL ALSO SYSTEMATICALLY DEVELOP AND APPLY HIGH SENSITIVITY TARGETED MS ASSAYS TO PEPTIDE/PROTEIN TARGETS IDENTIFIED IN THE DISCOVERY ARM, WITH AN EMPHASIS ON POSTTRANSLATIONALLY-MODIFIED PEPTIDES IN SIGNALING CASCADES, ONCOGENIC PATHWAY REGULATORS AND EFFECTORS, AND DRUGGABLE PROTEINS. ASSAYS WILL USE STABLE ISOTOPE- LABELED STANDARDS FOR UNAMBIGUOUS IDENTIFICATION AND QUANTIFICATION AND FOLLOW TIER 2 GUIDELINES DEVELOPED FROM THE COMMUNITY-BASED EFFORT LED BY THE BROAD PROTEOMICS TEAM. EXISTING TECHNOLOGIES WILL BE FURTHER DEVELOPED AND AUTOMATED TO ENABLE COMPREHENSIVE ANALYSIS OF RARE TUMOR CELL POPULATIONS, TO EVALUATE TUMOR HETEROGENEITY, TO INCREASE DEPTH AND BREADTH OF POST-TRANSLATIONAL MODIFICATION ANALYSIS, AND TO IMPROVE DEPTH, RELIABILITY AND REPEATABILITY OF PEPTIDE IDENTIFICATION AND QUANTIFICATION IN GENERAL BY INTELLIGENT DATA ACQUISITION.

RARE GENETIC DISORDERS IN NEURODEV: INSIGHT INTO THE GENETIC AND PHENOTYPIC HETEROGENEITY OF ID, ASD AND ADHD IN SOUTH AFRICAN POPULATIONS

A PORTAL AND INTEGRATIVE COLLABORATIVE ANALYSIS PLATFORM FOR GTEX

TARGETING CAUSAL CANCER GENES WITH SMALL MOLECULES

MOLECULAR MECHANISMS OF AT1R SIGNALING IN FSGS

ROLE OF TRPC5 CHANNEL INHIBITION IN THE TREATMENT OF GLOMERULAR DISEASE

A CELLULAR ATLAS OF THE PRIMATE AND HUMAN BASAL GANGLIA - PROJECT SUMMARY THE HUMAN BASAL GANGLIA (BG) ARE A COLLECTION OF SUBCORTICAL REGIONS WHOSE DIVERSE, SPECIALIZED CELL TYPES INFLUENCE MOTOR CONTROL, EMOTIONAL REGULATION, HABIT FORMATION, AND HIGHER COGNITION. RECENT ADVANCES IN SINGLE-CELL TRANSCRIPTOME AND EPIGENOME SEQUENCING HAVE REVOLUTIONIZED OUR ABILITY TO SYSTEMATICALLY DEFINE CELL TYPES AND STATES ACROSS COMPLEX TISSUES, REACHING SUFFICIENT LEVELS OF THROUGHPUT AND ROBUSTNESS TO BE DEPLOYABLE TO LARGE BRAIN TISSUE REGIONS LIKE THE PRIMATE BG. HOWEVER, TO DATE, DESPITE THE CENTRAL ROLE OF BG CELL TYPES IN MANY NEURODEGENERATIVE AND NEUROPSYCHIATRIC DISEASES, THEIR MOLECULAR DEFINITIONS IN THE HUMAN AND PRIMATE ARE DISTINCTLY LACKING. HERE, WE PROPOSE TO USE A COMBINATION OF HIGH-THROUGHPUT SINGLE-NUCLEUS RNASEQ, A NOVEL HIGH-RESOLUTION SPATIAL TECHNOLOGY, SLIDE-SEQ, AND A NEW APPROACH TO JOINTLY PROFILE TRANSCRIPTION AND ATAC SIGNATURES CALLED SHARE-SEQ, TO SYSTEMATICALLY IDENTIFY AND ANATOMICALLY MAP CELL TYPES ACROSS THE MACAQUE BG. WE WILL USE THESE SAME METHODS TO CHARACTERIZE CELL TYPE DIVERSITY ACROSS A SET OF 200 POSTMORTEM HUMAN BRAINS, AN UNPRECEDENTEDLY LARGE SAMPLE SIZE THAT WILL ENRICH OUR UNDERSTANDING OF INTER-INDIVIDUAL VARIATION IN THIS CLINICALLY RELEVANT SET OF BRAIN REGIONS. WE WILL THEN USE THESE DATA TO BUILD NEW VIRAL TOOLS FOR THE FUNCTIONAL INTERROGATION OF FOUR PRINCIPAL BG CELL TYPES IN THE PRIMATE. TOGETHER, THIS WORK WILL PROVIDE A COMPREHENSIVE AND HIGH-RESOLUTION MOLECULAR CHARACTERIZATION OF BG CELL TYPES, PROVIDE TOOLS FOR LINKING THESE MOLECULAR DEFINITIONS TO FUNCTIONAL ONES, AND ESTABLISH A FRAMEWORK FOR SUCH CELL TYPE CHARACTERIZATION ACROSS THE ENTIRE HUMAN BRAIN.

DNA MICROSCOPY FOR SPATIALLY RESOLVED GENOMIC ANALYSES IN INTACT TISSUE

ACHIEVING DIVERSITY IN GENOMICS: RESEARCH EDUCATION FOR URM SCIENTISTS

ARRAYED SINGLE-CELL READOUT OF POOLED GENETIC PERTURBATION LIBRARIES

DRIVING MEDICAL PROJECTS (4 OF 4)

DEVELOPMENT AND VALIDATION OF AAV VECTORS TO MANIPULATE SPECIFIC NEURONAL SUBTYPES AND CIRCUITS INVOLVED IN EPILEPSY AND PSYCHIATRIC DISORDERS ACROSS MAMMALIAN SPECIES. - PROJECT SUMMARY IN THIS PROPOSAL WE AIM TO IDENTIFY GENE REGULATORY ELEMENTS THAT PERMIT THE TARGETING AND MANIPULATION OF BRAIN CIRCUIT MODELS OF HUMAN BRAIN FUNCTION. GAINING GENETIC ACCESS TO SPECIFIC NEURON POPULATIONS IN NONTRANSGENIC ANIMALS AND HUMANS WOULD ENABLE TARGETED CIRCUIT MODULATION FOR HYPOTHESIS TESTING AND PROVIDE A MEANS TO EVALUATE THE SAFETY AND EFFICACY OF CIRCUIT MODULATION FOR THE TREATMENT OF EPILEPSY AND PSYCHIATRIC DISORDERS. OUR APPROACH CAPITALIZES ON OUR COMBINED EXPERTISE IN THE DEVELOPMENT AND MATURATION OF BRAIN CELL-TYPES AND CIRCUITS (GORD FISHELL), IDENTIFICATION OF CIS-REGULATORY ELEMENTS THAT FUNCTION ACROSS SPECIES (JORDANE DIMIDSCHSTEIN) AND AAV ENGINEERING COMBINED WITH LARGE-SCALE SCREENING METHODS (BEN DEVERMAN). OUR EFFORTS WILL BENEFIT FROM AN ONGOING COLLABORATION WITH JOHN REYNOLDS AT THE SALK INSTITUTE ON OBSERVATION AND MANIPULATION OF CORTICAL CIRCUITS DURING COMPLEX VISUAL PERCEPTION TASKS. THIS PROJECT WILL BUILD UPON SUCCESS THAT WE AND OTHERS HAVE HAD IN IDENTIFYING GENE REGULATORY ELEMENTS THAT ENABLE CELL TYPE-RESTRICTED GENE EXPRESSION WHEN USED WITHIN RECOMBINANT ADENO-ASSOCIATED VIRUS (AAV) VECTORS. IDENTIFYING ADDITIONAL ENHANCER SEQUENCES THAT FUNCTION IN THE CONTEXT OF THE LIMITED CARRYING CAPACITY OF AAV HAS BEEN SLOW DUE TO THE LIMITED SUCCESS RATE AND LOW THROUGHPUT NATURE OF THESE EFFORTS. HERE WE AIM TO APPLY A NOVEL HIGH-THROUGHPUT SCREENING APPROACH FOR THE RAPID IDENTIFICATION OF A SUITE OF ENHANCERS THAT ENABLE THE STUDY AND MANIPULATION OF GENETICALLY DEFINED CELL TYPES AND CIRCUITS ACROSS SPECIES. OUR PRELIMINARY DATA DEMONSTRATES THAT OUR ENHANCER IDENTIFICATION STRATEGY CAN YIELD NOVEL AND HIGHLY SPECIFIC ENHANCERS THAT RESTRICT EXPRESSION TO TARGET POPULATIONS. IN ADDITION, WE HAVE DEMONSTRATED THAT IT IS POSSIBLE TO USE THE ENGINEERED AAV-PHP.EB CAPSID TO SCREEN ENHANCERS ACROSS THE BRAIN WITH A SINGLE NONINVASIVE INJECTION. THESE SUCCESSES HAVE HIGHLIGHTED THE NEED FOR MORE RAPID AND COMPREHENSIVE ASSESSMENT OF PUTATIVE ENHANCERS. IN THE UH3 PORTION OF THIS PROPOSAL WE WILL EXAMINE THE TOLERANCE TO NEURONAL ACTIVITY MANIPULATION WITHIN THE TARGET NEURONAL POPULATIONS IN SEVERAL SPECIES. WE WILL ALSO APPLY THE AAV-ENHANCER VIRUSES FOR QUERYING DISEASE-RELATED CIRCUITS USING RABIES TRACING IN CONJUNCTION WITH OPTOGENETICS. THIS PROPOSAL WILL BE TRANSFORMATIVE IN DEVISING METHODS TO TARGET AND MANIPULATE THE BRAIN ACTIVITY OF SPECIFIC NEURONAL CELL POPULATIONS ACROSS SPECIES, INCLUDING HUMAN CELL-DERIVED ORGANOIDS.

TARGET ID (3 OF 4)

DETECTION OF NUCLEIC ACID SIGNATURES OF GENETIC ENGINEERING

GLOBAL INFRASTRUCTURE FOR COLLABORATIVE HIGH-THROUGHPUT CANCER GENOMICS ANALYSIS

COASTERCHASE PROGRAM - 24 MONTHS

INTEGRATED HIGH THROUGHPUT PROTEOGENOMIC DATA ANALYSIS CENTER FOR CPTAC

3/3-IDENTIFYING REGULATORY MUTATIONS THAT INFLUENCE NEUROPSYCHIATRIC DISEASE

CANCER DEPENDENCIES ASSOCIATED WITH GENOMIC ALTERATIONS AND TARGETED BY SMALL MOL

PROBING NEUROPSYCHIATRIC DISEASES USING TARGETED EPIGENOME AND GENOME ENGINEERING

2/5-ELUCIDATING THE GENETIC ARCHITECTURE OF AUTISM BY DEEP GENOMIC SEQUENCING

DISSECTING THE ROLE OF FMRP IN RNA PROCESSING USING HPSC MODELS

MITOCHONDRIAL PARTS, PATHWAYS, AND PATHOGENESIS

DEVELOPMENT AND IMPLEMENTATION OF GENOME-WIDE EXOME RE-SEQUENCING FOR MEDICAL GEN

IDENTIFICATION AND CHARACTERIZATION OF MICROBIAL METABOLITES IN IMMUNITY - PROJECT SUMMARY THE HUMAN MICROBIOME IS A KEY REGULATOR OF HOST IMMUNE FUNCTION, AND GROWING CONSENSUS SUGGESTS THIS RELATIONSHIP IS LIKELY MEDIATED BY HOST INTERACTIONS WITH MICROBIAL DERIVED SMALL MOLECULES (I.E., METABOLITES). ACCORDINGLY, MICROBIAL DERIVED METABOLITES HAVE BEEN ASSOCIATED WITH NUMEROUS AUTOIMMUNE, ALLERGIC, AND INFECTIOUS DISEASES. HOWEVER, THE HOST-MICROBIOME CIRCUITS THAT FORM THE MOLECULAR BASIS OF THESE ASSOCIATIONS HAVE NOT BEEN FULLY CHARACTERIZED, AND MANY MICROBIAL DERIVED METABOLITES HAVE NOT EVEN BEEN FORMALLY IDENTIFIED. IN THIS PROPOSAL, WE DESCRIBE A STRATEGY TO IDENTIFY AND FUNCTIONALLY CHARACTERIZE MICROBIAL DERIVED METABOLITES ISOLATED FROM PATIENTS IN HEALTH AND AUTOIMMUNITY. OUR COLLABORATIVE TEAM WILL ISOLATE AND CHARACTERIZE NOVEL METABOLITES FROM THE HUMAN MICROBIOME, MAP THESE ASSOCIATIONS TO HUMAN IMMUNE PATHWAYS, AND IDENTIFY THE SPECIFIC HOST RECEPTORS THAT ENGAGE MICROBIAL DERIVED METABOLITES. THIS PROPOSAL WILL ESTABLISH CAUSAL RELATIONSHIPS BETWEEN MICROBIAL METABOLITES AND HUMAN IMMUNE SYSTEM FUNCTION.

CARDIOVASCULAR DISEASE, METABOLIC SYNDROME, MICROBES AND METABOLITES IN FHS - CARDIOMETABOLIC DISEASES AFFECT MILLIONS OF PEOPLE WORLDWIDE AND HAVE NUMEROUS UNDERLYING RISK FACTORS. COMMENSAL MICROBES THAT COMPRISE THE INTESTINAL MICROBIOME ARE IMPLICATED IN THE PROGRESSION AND ONSET OF MANY OF THESE DISEASES, INCLUDING TYPE 2 DIABETES, OBESITY, AND ATHEROSCLEROSIS. GUT MICROBES HAVE EXTENSIVE METABOLIC CAPABILITIES, ALLOWING THEM TO PRODUCE OR MODIFY MOLECULES THAT INFLUENCE DISEASE RISK. MEMBERS OF THE GUT MICROBIOTA HAVE BEEN KNOWN TO CONVERT CHOLESTEROL INTO THE POORLY ABSORBABLE METABOLITE COPROSTANOL FOR ALMOST 100 YEARS, HOWEVER THE MICROBIAL GENES RESPONSIBLE FOR THIS METABOLISM WERE NOT KNOWN. IN RECENT WORK, WE ANALYZED PAIRED GUT METAGENOMIC AND METABOLOMIC DATA TO IDENTIFY A NOVEL GROUP OF CHOLESTEROL DEHYDROGENASES THAT METABOLIZE CHOLESTEROL WHEN EXPRESSED IN VITRO. USING CLINICAL AND METAGENOMIC DATA FROM THE FRAMINGHAM HEART STUDY (FHS), WE OBSERVED LOWER SERUM CHOLESTEROL IN SUBJECTS WHOSE MICROBIOMES ENCODE THESE CHOLESTEROL DEHYDROGENASES. THE CHOLESTEROL DEHYDROGENASES WE ORIGINALLY DESCRIBED CONVERT CHOLESTEROL TO CHOLESTENONE AND COPROSTANONE TO COPROSTANOL, AND WE HAVE SINCE IDENTIFIED THE INTERMEDIATE ENZYME THAT CONVERTS CHOLESTENONE TO COPROSTANONE. IN THIS PROPOSAL, WE WILL FUNCTIONALLY CHARACTERIZE METABOLISM OF STEROLS IN THE GUT AND DETERMINE THE IMPACT OF THIS PROCESS ON CARDIOMETABOLIC DISEASE. IN AIM 1, WE WILL IDENTIFY DETERMINANTS OF HOST-MICROBE INTERACTIONS BY COLLECTING AND ANALYZING CLINICAL VARIABLES WITH STOOL AND SERUM SAMPLES FROM THE GEN3/OMNI2 FHS COHORTS. WE WILL GENERATE COUPLED STOOL AND SERUM METABOLOMICS AND METAGENOMICS DATASETS, PERFORM CULTUROMICS TO ASSEMBLE A MICROBIAL STRAIN LIBRARY, AND IDENTIFY HOST EXPOSOMES FROM CLINICAL DATA. WE WILL THEN UTILIZE THESE DATA TO IDENTIFY AND PRIORITIZE MICROBIALLY- DERIVED OR MODIFIED CIRCULATING METABOLITES ASSOCIATED WITH CVD FOR FURTHER MECHANISTIC INVESTIGATIONS. IN AIM 2, WE WILL COUPLE BIOINFORMATICS WITH MICROBIOLOGY AND BIOCHEMISTRY FOR TARGETED IDENTIFICATION OF ENZYMES/PROTEINS THAT ALTER STEROL STRUCTURES. THIS EFFORT WILL ALLOW US TO PROBE THE DIVERSITY OF GUT MICROBIAL STEROL METABOLIZING ENZYMES AND THEIR SUBSTRATES IN ORDER TO DETERMINE SPECIFIC MICROBES AND GENES WITH THE CAPACITY TO MODIFY CHOLESTEROL. TAKING A SYSTEMS-LEVEL APPROACH, WE WILL COLONIZE MICE WITH CHOLESTEROL-METABOLIZING MICROBIAL COMMUNITIES TO DETERMINE HOW MICROBIAL METABOLISM MODULATES SERUM CHOLESTEROL AND PATHWAYS CENTRAL TO CARDIOVASCULAR DISEASE. IN AIM 3, WE WILL FUNCTIONALLY LINK MICROBIOME ENZYME ACTIVITY TO STEROL METABOLISM AND METABOLIC DISEASE. WE WILL EMPLOY CELL-BASED TRANSCRIPTIONAL AND PROTEOMIC ASSAYS TO INTERROGATE THE EFFECTS OF MICROBIALLY-MODIFIED STEROLS ON LOCAL STEROL SENSING PATHWAYS IN EPITHELIAL CELLS AND MEASURE THE EFFECTS OF CIRCULATING STEROL METABOLITES ON HUMAN IMMUNE CELLS.

INFORMATICS TOOLS FOR HIGH-THROUGHPUT SEQUENCES DATA ANALYSIS

CENTER FOR COMPREHENSIVE PROTEOGENOMIC DATA ANALYSIS - PROJECT SUMMARY PROTEOGENOMICS INVOLVES THE INTEGRATIVE MULTI-OMIC ANALYSIS OF GENOMIC, TRANSCRIPTOMIC, PROTEOMIC AND POST- TRANSLATIONAL MODIFICATION DATA PRODUCED BY NEXT-GENERATION SEQUENCING AND MASS SPECTROMETRY-BASED PROTEOMICS. SEVERAL PUBLICATIONS BY THE CLINICAL PROTEOMIC TUMOR ANALYSIS CONSORTIUM (CPTAC) AND OTHERS HAVE HIGHLIGHTED THE IMPACT OF PROTEOGENOMICS IN ENABLING DEEPER INSIGHT INTO THE BIOLOGY OF CANCER AND IDENTIFICATION OF POTENTIAL DRUG TARGETS. INTEGRATIVE ANALYSIS OF MULTI-OMIC DATA REQUIRES THE DEPLOYMENT OF COMPLEX ALGORITHMS AND DATA PROCESSING TECHNIQUES, WHICH ARE GENERALLY INACCESSIBLE TO SCIENTISTS WITHOUT A BACKGROUND IN BIOINFORMATICS AND COMPUTATIONAL BIOLOGY. OUR PROPOSED CENTER FOR COMPREHENSIVE PROTEOGENOMIC DATA ANALYSIS WILL ENCAPSULATE A COMPREHENSIVE SET OF ANALYSIS METHODS IN A PLATFORM THAT WILL BE (I) SIMPLE TO USE (II) FLEXIBLE (III) AUTOMATED, FACILITATING THE ROUTINE APPLICATION TO ALL CPTAC PROTEOGENOMIC DATASETS AS THEY BECOME AVAILABLE, AND (IV) ABLE TO INCORPORATE NEW METHODS WITH MINIMAL EFFORT. WE WILL LEVERAGE PANOPLY--A CLOUD-BASED PLATFORM FOR AUTOMATED AND REPRODUCIBLE PROTEOGENOMIC DATA ANALYSIS--AS THE FOUNDATION, WITH SPECIFIC, CAREFULLY CHOSEN ALGORITHMS TARGETED FOR ADDITION TO PROVIDE AN EXPANSIVE SET OF ANALYSIS CAPABILITIES THAT CAN BE EASILY HARNESSED TO PROVIDE A RAPID AND EXTENSIVE BASELINE ANALYSIS FOR PROTEOGENOMIC STUDIES, LEADING TO MANY DISEASE SPECIFIC HYPOTHESES THAT CAN BE EXPLORED FURTHER USING ADDITIONAL COMPUTATIONAL AND WET-LAB EXPERIMENTS. OUR COLLECTION OF TOOLS, ALGORITHMS AND INTERACTIVE REPORTS WILL ENABLE UNPRECEDENTED, AUTOMATED AND INTEGRATIVE SYSTEMS-BIOLOGY LEVEL ANALYSES OF PROTEOME, POST-TRANSLATIONAL MODIFICATION AND METABOLOMIC DATA COMBINED WITH GENOMIC DATA FOR INDIVIDUAL DISEASE COHORTS AND PAN-CANCER ANALYSIS ACROSS COHORTS, LEADING TO DEEPER UNDERSTANDING OF CANCER BIOLOGY AND ENABLING IDENTIFICATION OF THERAPEUTIC TARGETS AND DISEASE/PROGNOSTIC BIOMARKERS.

WHOLE-GENOME SHOTGUN SEQUENCING STRATEGY AND ASSEMBLY

ADVANCED DEVELOPMENT OF THE CANCER DEPENDENCY MAP PORTAL (DEPMAP.ORG)

CLINICAL, GENETIC AND CARDIOMETABOLIC RISK CORRELATES OF THE GUT MICROBIOME

A COMPREHENSIVE PROTEOMIC MAP OF MAMMALIAN MITOCHONDRIA

SYSTEMATIC IDENTIFICATION OF ONCOGENIC KRAS SYNTHETIC LETHAL INTERACTIONS

CONTINUED DEVELOPMENT OF CELLPROFILER CELL IMAGE ANALYSIS SOFTWARE

IDENTIFICATION AND VALIDATION OF INFLUENZA HOST FACTORS AS THERAPEUTIC TARGETS

ELUCIDATING GENETIC DETERMINANTS OF RESISTANCE TO LASSA HEMORRHAGIC FEVER

THE ASSOCIATION TO FUNCTION KNOWLEDGE PORTAL: A GENOMIC DATA RESOURCE FOR TRANSLATING GWAS ASSOCIATIONS TO BIOLOGICAL EFFECTS - ABSTRACT GENOME WIDE ASSOCIATION STUDIES (GWAS) HAVE PRODUCED ASSOCIATIONS BETWEEN MANY THOUSANDS OF GENETIC VARIANTS AND MANY HUNDREDS OF TRAITS. THE “FUNCTIONAL EFFECTS” OF MOST ASSOCIATIONS, HOWEVER, HAVE NOT YET BEEN ELUCIDATED – THAT IS, THE CAUSAL VARIANTS AND EFFECTOR GENES RESPONSIBLE FOR THEM, AND THE TISSUES AND PATHWAYS THROUGH WHICH THEY ACT, REMAIN LARGELY UNKNOWN. OVER THE PAST FEW YEARS, THREE CLASSES OF GENOMIC DATA HAVE ARISEN FOR INFERRING THE FUNCTIONAL EFFECTS OF GWAS ASSOCIATIONS: SUMMARY ASSOCIATION STATISTICS (EFFECT SIZES AND P-VALUES FOR ASSOCIATIONS BETWEEN SNPS AND TRAITS), GENOMIC ANNOTATIONS (ASSAYS OF REGULATORY ACTIVITY AND GENOMIC FUNCTIONAL ELEMENTS), AND BIOINFORMATIC METHODS (COMPUTATIONALLY PREDICTED FUNCTIONAL EFFECTS). WE ARGUE THAT TWO GAPS EXIST IN THE CURRENT RESOURCES THAT AGGREGATE THESE DATA: FIRST, NO CURRENT RESOURCE AIMS TO COMPREHENSIVELY CURATE AND CATALOG ALL THAT IS KNOWN, AND ALL DATA OR METHODS THAT COULD HELP PREDICT, THE FUNCTIONAL EFFECTS OF GWAS ASSOCIATIONS; SECOND, EXISTING RESOURCES ARE DEVELOPED WITH (AT BEST) LIMITED INVOLVEMENT FROM EXPERTS WHO EITHER ORIGINALLY GENERATED THE GENOMIC DATA AND/OR UNDERSTAND HOW TO BEST USE THEM. WE PROPOSE TO ADDRESS THESE GAPS BY BUILDING A NEW GENOMIC COMMUNITY RESOURCE – THE ASSOCIATION TO FUNCTION KNOWLEDGE PORTAL (A2FKP) – USING A GENERAL SOFTWARE PLATFORM WE INITIALLY DEVELOPED FOR TYPE 2 DIABETES. OUR APPROACH MAKES USE OF A KEY INNOVATION TO BUILD A RESOURCE THAT IS BOTH HIGH QUALITY AND COMPREHENSIVE: WE COLLABORATE WITH DISEASE EXPERT COMMUNITIES TO BUILD DEDICATED KNOWLEDGE PORTALS FOR THEM, MOTIVATING THEM TO CONTRIBUTE THEIR DATA AND EXPERTISE, AND WE THEN INTEGRATE THESE DATA ALONGSIDE THOSE OF OTHER COMMUNITIES, PROVIDING USERS WITH ACCESS A COMPREHENSIVE RESOURCE. SPECIFIC AIM 1 ADDRESSES GAPS IN THE COMPREHENSIVENESS AND QUALITY OF THE DATA AGGREGATED BY CURRENT RESOURCES REGARDING THE FUNCTIONAL EFFECTS OF GWAS ASSOCIATIONS. IT WILL ESTABLISH AND MANAGE COLLABORATIONS WITH A WIDE RANGE OF DISEASE, DATA, AND METHOD EXPERTS, AND THEN WORK WITH THESE COMMUNITIES TO IDENTIFY, AGGREGATE, AND CURATE DATA FOR 11 CLASSES OF DISEASE. SPECIFIC AIM 2 ADDRESSES GAPS IN CURRENT SCHEMAS AND SOFTWARE PLATFORMS FOR THE MYRIAD TYPES OF DATA USED FOR PREDICTING THE FUNCTIONAL EFFECTS OF GWAS ASSOCIATIONS. IT WILL BUILD PIPELINES FOR PROCESSING GENETIC AND GENOMIC DATASETS THROUGH BIOINFORMATIC METHODS FOR PREDICTING THE FUNCTIONAL EFFECTS OF GWAS ASSOCIATIONS, APPLY THESE PIPELINES TO DATA AGGREGATED IN AIM 1, AND TRANSFORM THEIR OUTPUTS TO RELATIONSHIPS AMONG ENTITIES IN A KNOWLEDGE GRAPH. THE GOAL OF SPECIFIC AIM 3 IS TO PROVIDE USERS WITH DIRECT AND VISUAL ACCESS TO THE RESOURCES AGGREGATED OR COMPUTED IN AIMS 1 AND 2. IT WILL DEVELOP REST APIS AND WEB PORTALS FOR QUERYING AND VISUALIZING DATA WITHIN THE A2FKP. SIGNIFICANCE: THE PROJECT WOULD PRODUCE A HIGH QUALITY AND COMPREHENSIVE GENOMIC RESOURCE OF DATA AND METHODS FOR PREDICTING THE FUNCTIONAL EFFECTS OF GWAS ASSOCIATIONS. EASY ACCESS TO SUCH A RESOURCE WILL ACCELERATE THE PACE BY WHICH GWAS ASSOCIATIONS CAN BE TRANSLATED TO INSIGHTS INTO COMPLEX DISEASE.

ADVANCING RARE DISEASE DISCOVERY THROUGH TECHNOLOGICAL AND METHODOLOGICAL APPROACHES USING AN INTUITIVE PLATFORM, SEQR - PROJECT SUMMARY / ABSTRACT OVER HALF OF ALL RARE DISEASE FAMILIES REMAIN UNDIAGNOSED FOLLOWING EXOME AND GENOME SEQUENCING ANALYSIS. REMARKABLE DEVELOPMENTS IN GENOMIC TECHNOLOGIES, ANALYTICAL TOOLS, AND INNOVATIVE DATA SHARING ARE ADVANCING MENDELIAN GENE DISCOVERY. HOWEVER, THE BENEFITS OF THESE NOVEL ADVANCES WILL ONLY BECOME REALIZED IF THEY ARE INTEGRATED INTO WIDELY ACCESSIBLE PLATFORMS FOR GENOMIC ANALYSIS. THE BROAD INSTITUTE CENTER FOR MENDELIAN GENOMICS HAS SUPPORTED THE DEPLOYMENT AND ONGOING DEVELOPMENT OF THE SEQR GENOMIC ANALYSIS PLATFORM, WHICH IS FREELY AVAILABLE TO THE COMMUNITY EITHER ON THE ANVIL PLATFORM OR AS AN OPEN-SOURCE TOOL THAT CAN BE INSTALLED LOCALLY. THIS PROPOSAL SEEKS TO ADVANCE THE DISCOVERY RATE FOR RARE DISEASE THROUGH ONGOING DEVELOPMENT OF THE SEQR PLATFORM, INCORPORATING AND EVALUATING THE IMPACT OF THREE NOVEL ADVANCES: 1) INNOVATIVE DATA SHARING THROUGH VARIANT LEVEL MATCHING BY CONNECTING SEQR TO A GROWING FEDERATED PLATFORM OF RARE DISEASE AND POPULATION GENOMIC DATA WITH ASSOCIATED PHENOTYPE; 2) DEPLOYMENT OF AUTOMATED ANALYTICAL TOOLS ON PREVIOUSLY EXAMINED AND UNSOLVED CASES TO IDENTIFY VARIATION THAT IS NEWLY IMPLICATED AS PATHOGENIC IN GENES WITH KNOWN DISEASE ASSOCIATIONS OR IN GENES THAT ARE NEWLY IMPLICATED IN DISEASE; AND 3) INCORPORATION OF NOVEL TECHNOLOGIES SUCH AS LONG READ DNA AND RNA SEQUENCING DATA THAT CAN BE EASILY ANALYZED IN A GENOME-WIDE MANNER. WE WILL STUDY THE IMPACT OF THESE NOVEL ADVANCES BY ANALYZING THE DIAGNOSTIC YIELD OF EACH APPROACH. AS SEQR IS FREELY AVAILABLE AND OPEN SOURCE, OUR PROPOSAL DEMOCRATIZES THE PROCESS OF GENETIC DISCOVERY, EMPOWERING RESEARCHERS AND CLINICIANS TO RESOLVE DIAGNOSTIC VARIANTS, AND ACCELERATE THE RATE OF DIAGNOSIS FOR THE GLOBAL POPULATION OF INDIVIDUALS WITH RARE GENETIC DISEASE.

BRAIN CONNECTS: HIGH-THROUGHPUT SINGLE CELL PROJECTION MAPPING AND SPINE QUANTIFICATION IN THE PRIMATE BRAIN BY SYNAPTIC BARCODING - SUMMARY THE PRIMATE BRAIN CONTAINS TRILLIONS OF HIGHLY SPECIFIC SYNAPTIC CONNECTIONS THAT ARE THE BUILDING BLOCKS OF NEURAL COMPUTATION. WHILE TOOLS AVAILABLE IN RODENTS HAVE BEGUN TO SHED LIGHT ON HOW PARTICULAR CELL TYPES AND CIRCUITS PARTICIPATE IN BRAIN FUNCTIONS, THESE APPROACHES ARE NOT CURRENTLY POSSIBLE TO APPLY TO PRIMATES. RECENTLY, OUR LABS HAVE DEVELOPED TECHNOLOGY THAT: 1) DELIVERS BARCODED RNAS TO THE PRESYNAPTIC AND POSTSYNAPTIC COMPARTMENTS WITH HIGH SENSITIVITY AND SPECIFICITY USING AAVS; AND 2) ALLOWS CNS-WIDE TRANSDUCTION BY AAV THROUGH SYSTEMIC INJECTION. WE WILL COMBINE THESE INNOVATIONS, TOGETHER WITH OUR PLATFORMS FOR SINGLE-CELL AND SPATIAL TRANSCRIPTOMICS, TO SYSTEMATICALLY MAP THE PROJECTIONS OF THOUSANDS OF MIDBRAIN DOPAMINERGIC NEURONS ACROSS THE MARMOSET BRAIN. IN ADDITION, WE WILL DEVELOP STRATEGIES FOR THE RAPID AND SCALABLE QUANTIFICATION OF POSTSYNAPTIC SPINES IN INDIVIDUAL CELLS, USING KNOWN ABUNDANCE IN SPINE VARIATION AMONGST THE CELLS OF THE DENTATE GYRUS AS A VALIDATION. TOGETHER, THESE TECHNOLOGIES WILL ENABLE THE ROUTINE QUANTIFICATION OF PRESYNAPTIC AND POSTSYNAPTIC PHENOTYPES AT A LEVEL OF RESOLUTION AND SCALE THAT HAS HITHERTO BEEN IMPOSSIBLE IN PRIMATE

AN NCRR CENTER FOR HIGH THROUGHPUT SNP GENOTYPING AND ANALYSIS

USING ENHANCER-DIRECTED EXPRESSION WITHIN AAVS TO TARGET SUBTYPES WITHIN THE FOUR MAJOR NEUROMODULATORY POPULATIONS - GRANT SUMMARY NEUROMODULATORY SYSTEMS—COMPRISING NORADRENERGIC, SEROTONERGIC, CHOLINERGIC, AND DOPAMINERGIC NEURONS—PLAY A CRITICAL ROLE IN REGULATING MOOD, COGNITION, MOTOR CONTROL, AND PHYSIOLOGICAL PROCESSES. THESE SYSTEMS ARE CENTRAL TO UNDERSTANDING HOW THE BRAIN ENCODES REWARD, ACTION SELECTION, AND MEMORY, AND THEY REMAIN KEY THERAPEUTIC TARGETS FOR NEUROPSYCHIATRIC AND NEURODEGENERATIVE DISORDERS. RECENT ADVANCES IN HIGH-THROUGHPUT SINGLE-CELL AND SPATIAL GENOMICS HAVE REVEALED A REMARKABLE DIVERSITY AMONG NEUROMODULATORY CELL TYPES, WITH DOZENS TO HUNDREDS OF DISTINCT SUBPOPULATIONS. DESPITE THIS, TOOLS CAPABLE OF TARGETING THESE SUBPOPULATIONS WITH PRECISION REMAIN LIMITED, HINDERING PROGRESS IN BOTH BASIC AND TRANSLATIONAL NEUROSCIENCE. THIS PROJECT SEEKS TO ADDRESS THIS GAP BY DEVELOPING A COMPREHENSIVE PIPELINE TO NOMINATE, VALIDATE, AND DISSEMINATE CELL-TYPE-SPECIFIC ENHANCERS FOR NEUROMODULATORY SYSTEMS. BUILDING UPON PRIOR SUCCESSES IN ENHANCER DISCOVERY FOR CORTICAL INTERNEURONS AND PYRAMIDAL NEURONS, WE WILL EMPLOY ADVANCED SPATIAL MULTIOMIC PROFILING, COMPUTATIONAL PREDICTION, AND HIGH-THROUGHPUT AAV-BASED ENHANCER TESTING TO GENERATE A ROBUST SET OF VALIDATED ENHANCERS. SPECIFICALLY, WE WILL: 1) NOMINATE CANDIDATE ENHANCERS BY INTEGRATING PUBLICLY AVAILABLE DATA AND PERFORMING SPATIAL MULTIOMIC PROFILING ON SORTED CHOLINERGIC, DOPAMINERGIC, SEROTONERGIC, AND NORADRENERGIC CELL TYPES. 2) QUANTITATIVELY VALIDATE ENHANCER ACTIVITY USING CUTTING-EDGE TECHNIQUES, INCLUDING SMFISH, SLIDE-TAG MOLECULAR PROFILING, AND FUNCTIONAL ASSAYS SUCH AS OPTOGENETIC AND CHEMOGENETIC MANIPULATION, NEURONAL ACTIVITY MONITORING, AND CRISPR-BASED GENE EDITING. 3) DISSEMINATE VALIDATED TOOLS THROUGH COLLABORATION WITH THE ALLEN INSTITUTE, ADDGENE, AND OTHER PLATFORMS, ENSURING WIDE ACCESSIBILITY AND STANDARDIZATION ACROSS THE NEUROSCIENCE COMMUNITY. THE PROPOSED RESEARCH WILL PRODUCE AT LEAST 60 HIGHLY SPECIFIC AND VALIDATED ENHANCERS TARGETING DISTINCT NEUROMODULATORY SUBPOPULATIONS, EACH CHARACTERIZED FOR THEIR ACTIVITY, SPECIFICITY, AND FUNCTIONAL APPLICATIONS. THESE TOOLS WILL BE INVALUABLE FOR STUDYING NEUROMODULATORY CIRCUITS IN HEALTH AND DISEASE AND FOR ADVANCING THERAPEUTIC INTERVENTIONS TARGETING THESE SYSTEMS. BY PROVIDING THE NEUROSCIENCE COMMUNITY WITH THESE TRANSFORMATIVE TOOLS, THIS PROJECT WILL FACILITATE UNPRECEDENTED INSIGHTS INTO THE MOLECULAR AND CELLULAR UNDERPINNINGS OF BRAIN FUNCTION AND DYSFUNCTION, ADDRESSING PRESSING CHALLENGES IN THE FIELDS OF PSYCHIATRY AND NEUROLOGY. THE SUCCESSFUL COMPLETION OF THIS WORK WILL NOT ONLY ENABLE BASIC SCIENTIFIC DISCOVERIES BUT ALSO LAY THE GROUNDWORK FOR THE DEVELOPMENT OF PRECISION THERAPIES FOR NEUROPSYCHIATRIC AND NEURODEGENERATIVE DISEASES.

RAPID FUNGAL IDENTIFICATION AND ANTIFUNGAL SUSCEPTIBILITY TESTING THROUGH QUANTITATIVE, MULTIPLEXED RNA DETECTION

A FUNCTIONAL GENOMICS PIPELINE FOR GENETIC DISCOVERY IN DIABETIC KIDNEY DISEASE - ABSTRACT DIABETIC KIDNEY DISEASE (DKD) IS A DEVASTATING MICROVASCULAR COMPLICATION OF BOTH TYPE 1 (T1D) AND TYPE 2 DIABETES (T2D). IT HAS BEEN SHOWN TO HAVE A HERITABLE COMPONENT, BUT PRIOR SEARCHES FOR THE GENETIC DETERMINANTS OF THIS CONDITION HAVE HAD LIMITED SUCCESS. IN THE GENETICS OF NEPHROPATHY – AN INTERNATIONAL EFFORT (GENIE) CONSORTIUM, A COLLABORATION BETWEEN QUEEN’S UNIVERSITY BELFAST, UNIVERSITY OF DUBLIN, UNIVERSITY OF HELSINKI, UNIVERSITY OF MICHIGAN, UNIVERSITY OF PENNSYLVANIA AND THE BROAD INSTITUTE, WE HAVE LEVERAGED AN ONGOING CO-FUNDING MECHANISM BETWEEN IRELAND, NORTHERN IRELAND AND THE US. THIS INTERNATIONAL GENOMICS CONSORTIUM ENUCLEATED A LARGER DIABETIC NEPHROPATHY COLLABORATIVE RESEARCH INITIATIVE, WHICH COALESCED TO ASSEMBLE NEARLY 20,000 SAMPLES FROM PARTICIPANTS WITH T1D, WITH AND WITHOUT KIDNEY DISEASE. WE PERFORMED A GENOME-WIDE ASSOCIATION STUDY (GWAS) AND DISCOVERED 16 NEW SIGNALS AT GENOME-WIDE SIGNIFICANCE. THE STRONGEST SIGNAL CENTERED ON A PROTECTIVE MISSENSE CODING VARIANT AT COL4A3, WHICH ENCODES AN INTEGRAL COMPONENT OF THE GLOMERULAR BASEMENT MEMBRANE, IMPLICATING THIS ASPECT OF KIDNEY BIOLOGY IN DKD. IN THIS AWARD, WE PROPOSE TO BUILD ON THIS ESTABLISHED INFRASTRUCTURE TO UNDERTAKE THE FOLLOWING SPECIFIC AIMS: (1) TO SIGNIFICANTLY EXPAND OUR SAMPLE SIZE BY INCLUDING ~150,000 SAMPLES WITH DKD IN THE CONTEXT OF T2D, THEREBY SUBSTANTIALLY INCREASING OUR POWER TO DISCOVER SHARED AND DISTINCT RISK FACTORS FOR DKD IN T1D AND T2D, AND TO USE COMPUTATIONAL TOOLS TO DERIVE BIOLOGICAL INSIGHTS INTO DKD PATHOGENESIS; (2) TO GENERATE A GENOME-WIDE EPIGENOMIC DATASET IN BOTH PERIPHERAL BLOOD AND HUMAN KIDNEY TO INFORM THE RELEVANCE OF GENETIC FINDINGS, ENABLE THE CONSTRUCTION OF PREDICTIVE TOOLS, AND INFER CAUSALITY VIA MENDELIAN RANDOMIZATION; AND (3) TO CREATE AN EXPERIMENTAL PIPELINE CENTERED ON ANIMAL, CELLULAR AND ORGANOID MODELS OF DKD TO PURSUE FUNCTIONAL VALIDATION OF PROMISING GENETIC FINDINGS. THIS ONGOING CLOSE COLLABORATION OF MULTIDISCIPLINARY AND SYNERGISTIC RESEARCH GROUPS SHOULD ADVANCE OUR KNOWLEDGE OF THE MOLECULAR DETERMINANTS OF DKD, IDENTIFY POTENTIAL MOLECULAR TARGETS FOR THERAPEUTICS, AND FACILITATE CLINICAL PREDICTION.

LINCS DATA COORDINATION AND INTEGRATION CENTER

MAPPING AND CONTROLLING GENE EXPRESSION IN INHIBITORY INTERNEURONS MAMMALS

PRECLINICAL DEVELOPMENT OF AN ANTIMALARIAL AGENT WITH A NEW MECHANISM OF ACTION

ONE-COMPOUND, ONE-ISLET: A HIGH-THROUGHPUT PLATFORM FOR SMALL-MOLECULE DISCOVERY

PSYCHOSIS GENETICS RESEARCH IN AFRICA: BUILDING CAPACITY BY INVESTING IN PEOPLE

CHARACTERIZING THE GUT MICROBIAL ECOSYSTEM FOR DIAGNOSIS AND THERAPY IN IBD

INTEGRATIVE GENOMIC, EPIGENETIC AND FUNCTIONAL STUDIES IN DIABETIC KIDNEY DISEASE

UNRAVELING THE GENETIC PROGRAMS ENGAGED IN ASD NEURONS THROUGH COUPLED TRANSCRIPTOMIC AND PHENOTYPIC READOUTS - AUTISM SPECTRUM DISORDERS (ASD) ARE GENETICALLY DIVERSE, CHARACTERIZED BY BOTH RARE VARIANTS OF LARGE EFFECT SIZE AND COMMON VARIANTS OF SMALL EFFECT SIZE. IDENTIFYING THE MOLECULAR MECHANISMS RESULTING FROM THESE VARIANTS PRESENTS A KEY CHALLENGE FOR THE DEVELOPMENT OF CLINICAL INTERVENTIONS. HUMAN PLURIPOTENT STEM-CELL DERIVED NEURONS (HPSC-NS) ALLOW STUDIES AGAINST A HUMAN GENETIC BACKGROUND, AND SHOW ALTERED MORPHOLOGY AND ELECTROPHYSIOLOGY IN ASD CONDITIONS. HOWEVER, IDENTIFYING MECHANISMS REMAINS DIFFICULT WITH SMALL NUMBERS OF LINES, ESPECIALLY FOR COMMON GENETIC VARIANTS. TO OVERCOME THIS CHALLENGE, WE WILL LEVERAGE MULTI-OMIC CHARACTERIZATION OF HPSC-NS PERTURBED WITH CRISPRI KNOCKDOWN OF BOTH LARGE EFFECT SIZE ASD RISK GENES AND GENES RELATED TO NEURONAL MORPHOLOGY (AIM 1) AND ELECTROPHYSIOLOGY (AIM 2). WE WILL COMPLEMENT THESE SCREENS WITH A CHARACTERIZATION (AIM 3) OF A LARGER, DIVERSE COHORT OF 46 ASD LINES AND 46 MATCHED CONTROLS WHICH DO NOT HARBOR CODING VARIANTS IN THE GENES PERTURBED IN THE PREVIOUS AIMS. AN INTEGRATIVE ANALYSIS OF THIS DATA (AIM 4) WILL GENERATE INTERPRETABLE GENETIC SIGNATURES RELATED TO EACH OF THESE PHENOTYPES AND WILL SHOW HOW THESE SIGNATURES INTERACT WITH ASD RISK GENES. THIS APPROACH IS MADE POSSIBLE BY NEW TECHNIQUES FOR POOLED STEM CELL CULTURE DEVELOPED IN DR. RALDA NEHME’S LAB, HIGH CONTENT OPTICAL PROFILING METHODS DEVELOPED BY DR. SAMOUIL FARHI’S TEAM, AND DATA INTEGRATION TOOLS DEVELOPED BY DR. ERNEST FRAENKEL’S GROUP. THE OVERALL PROJECT WILL PROVIDE A BASIC NEUROBIOLOGICAL UNDERSTANDING OF HPSC-NS; PROVIDE VALUABLE INSIGHT INTO HOW BOTH COMMON AND RARE VARIANTS INDUCE OBSERVED CELL-INTRINSIC PHENOTYPES; AND DEFINE AN ANALYTIC FRAMEWORK AND GENETIC SIGNATURES WHICH CAN BE USED TO UNDERSTAND MECHANISTIC RECRUITMENT OF NEW GENETIC RISK LOCI AND OTHER PSYCHIATRIC DISEASES.

CENTER FOR CANCER GENOME CHARACTERIZATION

A POWERFUL WEB-BASED DISCOVERY PLATFORM FOR RARE DISEASE GENETICS

THE FUNCTION OF THE PRMT5 METHYLOSOME IN MTAP DELETED CANCERS

METHODS FOR LINKING GWAS PEAKS TO FUNCTION IN PSYCHIATRIC DISEASE

DEVELOPMENT OF PLATFORMS FOR BETA CELL-SPECIFIC DELIVERY AND LIGAND DISCOVERY - PATIENTS SUFFERING FROM TYPE 1 DIABETES MUST UNDERGO BURDENSOME, OFTEN LIFELONG, EXOGENOUS INSULIN DEPENDENCE. UP TO NOW, THE ONLY AVAILABLE REPLACEMENT THERAPY IS TO TRANSPLANT ISLETS FROM CADAVERIC DONORS. HOWEVER, SUCH PROCEDURES PRESENT HURDLES SUCH AS THE SCARCITY OF AVAILABLE DONORS AND THE REJECTION OF TRANSPLANTED CELLS BY THE PATIENT’S OWN IMMUNE SYSTEM. ALSO, OVER TIME, THE TRANSPLANTED ISLETS TEND TO DIE. TO CIRCUMVENT THESE EFFECTS, WE AND OTHER HAVE IDENTIFIED SEVERAL THERAPEUTIC TARGETS TO INDUCE BETA CELL PROLIFERATION. HOWEVER, THE REALIZATION OF THE THERAPEUTIC POTENTIAL OF THESE TARGETS REQUIRES TARGETED RELEASE AS THE THERAPEUTIC WINDOW OF THESE TARGETS IS NARROW. WE PROPOSE TO APPLY CHEMICAL BIOLOGY APPROACHES TO DEVELOP METHODS FOR TARGETED RELEASE OF BIOACTIVES IN BETA CELLS IN VIVO. 1

NOVEL AAVS ENGINEERED FOR EFFICIENT AND NONINVASIVE CROSS-SPECIES GENE EDITING THROUGHOUT THE CENTRAL NERVOUS SYSTEM - PROJECT SUMMARY: MANY GENETIC DISEASES THAT AFFECT THE CENTRAL NERVOUS SYSTEM (CNS) REMAIN UNTREATABLE DUE TO A LACK EFFECTIVE SMALL MOLECULE DRUGS OR BIOLOGICS. TARGETING THE GENETIC UNDERPINNINGS OF THESE DISEASES WITH SOMATIC CELL GENE EDITING WOULD THEREFORE BE PARTICULARLY IMPACTFUL, BUT ITS SUCCESSFUL IMPLEMENTATION WILL REQUIRE METHODS TO SAFELY AND EFFICIENTLY DELIVER GENES AND GENE EDITING MACHINERY THROUGHOUT THE CNS. AAVS ARE THE STATE-OF-THE-ART VEHICLES FOR IN VIVO GENE TRANSFER BECAUSE THEY CAN PROVIDE SAFE AND LONG LASTING IN VIVO GENE EXPRESSION. AAVS ARE THE ONLY GENE THERAPY VECTORS THAT HAVE BEEN APPROVED FOR DIRECT ADMINISTRATION TO HUMANS BY REGULATORY AGENCIES IN BOTH THE US AND EUROPE. MOREOVER, IN 2017, AAVS BECAME THE FIRST VEHICLE USED AS PART OF AN EARLY PHASE CLINICAL TRIAL TO EVALUATE THE SAFETY OF IN VIVO GENE EDITING. DESPITE THEIR IMPRESSIVE PRECLINICAL AND CLINICAL SAFETY RECORD, NATURALLY OCCURRING AAVS TESTED TO DATE LACK THE EFFICIENCY REQUIRED FOR GENE DELIVERY ACROSS MOST ORGAN SYSTEMS, INCLUDING THE CNS. TO ADDRESS THE NEED FOR BETTER VEHICLES FOR CNS GENE DELIVERY, WE RECENTLY USED DIRECTED EVOLUTION AND A NEW CELL TYPE-SPECIFIC IN VIVO SELECTION METHOD TO ENGINEER SEVERAL NOVEL AAVS, MOST NOTABLY AAV-PHP.B AND AAV-PHP.EB, THAT HAVE, FOR THE FIRST TIME, MADE IT POSSIBLE TO NONINVASIVELY TRANSFER GENES TO THE MAJORITY OF NEURONS AND ASTROCYTES THROUGHOUT THE ADULT MOUSE CNS. HERE, WE AIM TO BUILD UPON THE SUCCESS OF THIS SELECTION APPROACH BY ENGINEERING AAVS THAT ENABLE EFFICIENT GENE TRANSFER THROUGHOUT THE CNS OF MULTIPLE SPECIES, INCLUDING NONHUMAN PRIMATES. THE AAVS WE DEVELOP WILL BE EVALUATED IN SEVERAL SPECIES FOR THEIR ABILITY TO PROVIDE CNS-WIDE TRANSGENE EXPRESSION AND TARGETED GENOME EDITING IN NEURONS, AND IMPROVED AAV VARIANTS WILL BE SHARED WITH THE SCIENTIFIC COMMUNITY. SUCCESSFUL COMPLETION OF THIS PROJECT, WHICH INVOLVES PAIRING THE NEW AAVS WITH NEXT-GENERATION GENE EDITING TECHNOLOGIES, WILL PROVIDE SUPPORT FOR EVALUATING THE SAFETY OF CNS GENE EDITING IN HUMAN TRIALS.

STUDIES OF MATERIALS WITH PHYSIOLOGICAL PROPERTIES

THE 200 MAMMALS PROJECT: SEQUENCING GENOMES BY A NOVEL COST-EFFECTIVE METHOD, YIELDING A HIGH RESOLUTION ANNOTATION OF THE HUMAN GENOME.

SPATIAL GENOMIC TOOLS TO INTERROGATE T CELL CLONOTYPES, TUMOR CLONES AND THE MICROENVIRONMENT - THE CONTEMPORARY CLINICAL SUCCESSES OF IMMUNOTHERAPIES HAVE HIGHLIGHTED THE KEY ROLE OF TUMOR-INFILTRATING CELLS IN MEDIATING ANTI-TUMOR IMMUNITY AND HAVE GENERALLY ASSOCIATED THE PRESENCE OF T CELLS WITHIN TUMORS WITH THERAPEUTIC RESPONSE. HOWEVER, UNTIL NOW, A SYSTEMATIC APPROACH FOR EVALUATING HOW T CELL STATE, THEIR CLONAL IDENTITY AND LOCALIZATION ARE RELATED HAS NOT BEEN POSSIBLE. IN RECENT YEARS, THE WU LAB HAS ACHIEVED SEVERAL NOTABLE TECHNICAL ADVANCES, INCLUDING GENERATION OF A BEST-IN-CLASS HLA CLASS I EPITOPE PREDICTOR (HLATHENA); A HIGHLY ROBUST TARGETED PLATE-BASED METHOD FOR SINGLE-CELL TCR SEQUENCING (RHTCRSEQ); AND A MEANS TO PARALLELIZE THE CLONING OF HUNDREDS OF TCRS SUCH THAT THEY CAN BE INTERROGATED TO DEFINITIVELY LINK A TCR WITH ITS ANTIGEN SPECIFICITY. BECAUSE DYNAMIC INTERACTIONS BETWEEN TUMOR-REACTIVE T CELLS AND MALIGNANT CLONES OCCUR WITHIN THE DEFINED SPATIAL ORDERING OF TISSUE, OUR VALUABLE NEW INSIGHTS MOTIVATE US TO INVESTIGATE HOW THE SPATIAL ORGANIZATION OF TUMOR-SPECIFIC T CELLS RELATES TO IN SITU POSITIONING OF TUMOR CLONES. WE HYPOTHESIZE THAT ANTIGEN SPECIFICITY, WHICH DRIVES THE INTERACTIONS BETWEEN T CELLS AND TUMOR CELLS, IMPACTS THE DISTINCT REGIONAL LOCALIZATION OF T CELLS WITHIN THE TUMOR MICROENVIRONMENT AT BASELINE AND IN THE CONTEXT OF THERAPY; CONVERSELY, THAT KNOWLEDGE OF SPATIAL LOCALIZATION IDENTIFIES T CELL CLONES SPECIFIC FOR DISTINCT ANTIGEN TYPES. SLIDE-SEQ TECHNOLOGY, CREATED BY THE CHEN LAB, PROVIDES A TRACTABLE AND EXCITING PATH TO INVESTIGATE THIS HYPOTHESIS BY IMPLEMENTING A SCALABLE APPROACH TO UNDERTAKE IN-DEPTH ANALYSES OF INFORMATIVE HUMAN AND MURINE TUMOR TISSUES. BY EXPANDING THE CAPABILITIES OF THIS UNBIASED CELLULAR RESOLUTION SPATIAL CAPTURE METHOD, WE AIM TO GAIN TISSUE LEVEL UNDERSTANDING OF HOW THE ABUNDANCE AND FUNCTIONAL STATE OF T CELL CLONES AND THEIR SPATIAL ORIENTATION WITHIN THE TUMOR MICROENVIRONMENT ARE LINKED. IN PARTICULAR, THE STUDY WILL ADDRESS THE SPATIAL ORGANIZATION OF T CELL CLONES AND TUMOR SUBCLONES IN HUMAN AND MOUSE TUMORS. OUR TECHNOLOGY GOALS WILL BE TO INCREASE THE EFFICIENCY OF TRANSCRIPT CAPTURE OF THE TECHNOLOGY, EXTEND THE CAPABILITY TO INCLUDE ROBUST DETECTION OF TUMOR MUTATIONS, AND TO DEVELOP A SUITE OF ANALYTIC TOOLS TO INTEGRATE IN A MULTI MODEL FASHION THE TRANSCRIPT, DNA-LEVEL AND TCR LEVELS OF INFORMATION (AIMS 1-2). FOCUSING ON RCC TUMORS, WE WILL EVALUATE THE CELL-CELL LOCALIZATION PATTERNS OF T CELL CLONES IN RELATION TO TUMOR SUBCLONES AND STROMAL CELLS THROUGH INTEGRATED SPATIAL ANALYSES OF TCR AND DNA SLIDE-SEQ DATA (AIM 2). FINALLY, WE WILL EVALUATE THE IMPACT OF ANTIGEN SPECIFICITY (DEFINITIVELY ASSESSED BY ROBUST TCR RECONSTRUCTION AND INTERROGATION METHODS ESTABLISHED IN OUR LAB) ON T CELL PHENOTYPE AND LOCALIZATION USING TCR AND DNA SLIDE-SEQ INTEGRATED SPATIAL ANALYSES AT BASELINE AND FOLLOWING IMMUNE CHECKPOINT BLOCKADE AND NEOANTIGEN VACCINE. (AIM 3) ALTOGETHER, WE WILL DEVELOP AND TEST THESE TOOLS TO UNDERSTAND SPATIALLY LOCALIZED CELLULAR NETWORKS WHICH DRIVE IMMUNE RESPONSE, AND T-CELL RECEPTOR RELATIONSHIPS WITH THE TUMOR MICROENVIRONMENT. THE COMPLETION OF OUR WORK WILL YIELD A COMPREHENSIVE TOOLSET TO ENABLE A MOLECULAR, CELLULAR AND HISTOLOGICAL UNDERSTANDING OF THE TUMOR IMMUNE RESPONSE.

LIVE CELL TRANSCRIPTOMICS

INVESTIGATING EPIGENETIC MECHANISMS IN DOWN SYNDROME USING HUMAN CELLULAR MODELS - SUMMARY DOWN SYNDROME (DS), DRIVEN BY AN EXTRA COPY OF CHROMOSOME 21 (HSA21), IS ASSOCIATED WITH A BROAD SPECTRUM OF NEUROLOGICAL AND NON-NEUROLOGICAL PHENOTYPES, PROFOUND INTERINDIVIDUAL VARIATION AND SIGNIFICANT CHANGES IN GENOME-WIDE GENE EXPRESSION AND DNA METHYLATION PATTERNS, ALTHOUGH UNDERLYING MECHANISMS REMAIN INCOMPLETELY RESOLVED. EPIGENETIC RE-WIRING IS A PROMISING CANDIDATE FOR ACHIEVING THE TYPES OF GENOME- WIDE CHANGES THAT ARE OBSERVED IN DS AS WELL AS CONTRIBUTING TO INTERINDIVIDUAL VARIATION IN DISEASE PHENOTYPES. HOWEVER, THIS AREA OF INVESTIGATION, PARTICULARLY AT THE LEVEL OF CHROMATIN STATES, REMAINS ALMOST ENTIRELY UNEXPLORED IN DS. LEVERAGING HUMAN INDUCED PLURIPOTENT STEM CELL (IPSC) MODELS, WE RECENTLY CONDUCTED A NOVEL, UNBIASED, AND COMPREHENSIVE SURVEY OF THE RELATIVE ABUNDANCE OF OVER 80 DIFFERENT HISTONE POST- TRANSLATIONAL MODIFICATIONS (PTMS) IN DS VERSUS EUPLOID CONTROLS USING HISTONE MASS SPECTROMETRY. THESE RESULTS, COUPLED WITH ADDITIONAL VALIDATION EXPERIMENTS, REVEALED A SET OF NOVEL DISRUPTIONS TO H3K36ME2, H3K4ME1 AND H3K23AC ABUNDANCE WHICH WE PREDICT CONTRIBUTE TO DS DISEASE BIOLOGY. IN THIS PROPOSAL, WE AIM TO EXPAND OUR ANALYSES TO OBTAIN, FOR THE FIRST TIME, A COMPREHENSIVE VIEW OF HOW TRISOMY 21 DISRUPTS HISTONE PTMS AND THE DOWNSTREAM IMPACTS ON MOLECULAR AND CELLULAR FUNCTION. IMPORTANTLY, THIS PROPOSAL MOVES AWAY FROM A HSA21/GENE-CENTRIC VIEW OF DS, TRADITIONALLY STUDIED USING MURINE MODELS, TO EXPLORE HOW EPIGENETIC RE-WIRING MAY INTERSECT WITH A SET OF KEY UNANSWERED QUESTIONS IN THE FIELD USING PHYSIOLOGICALLY RELEVANT HUMAN CELLULAR MODELS. CONCEPTUALLY, OUR EXPERIMENTS ARE DESIGNED TO EXPLORE QUESTIONS AROUND THE SIGNIFICANT CHANGES IN DOSAGE OF GENES ENCODED ON EUPLOID CHROMOSOMES, THE PROFOUND INTERINDIVIDUAL VARIATION IN DS, HOW DIFFERENT CELL TYPES MAY BE IMPACTED BY TRISOMY 21 IN DIVERGENT OR CONVERGENT WAYS, AND HOW DS MECHANISMS MAY OVERLAP WITH OTHER DISEASES. SPECIFICALLY, IN AIM I, WE WILL SYSTEMATICALLY DEFINE THE SCOPE OF HISTONE PTM ABUNDANCE PHENOTYPES IN DS USING A COHORT OF DIFFERENT DONOR INDIVIDUALS AND CELL TYPES TO ANALYZE INTERINDIVIDUAL AND CELL-TYPE VARIATION, AND THEN CONNECT THESE CHANGES TO CHROMATIN BINDING AND TRANSCRIPTIONAL OUTPUT FOR SELECT MODIFICATIONS TO ELUCIDATE FUNDAMENTAL MECHANISMS. IN AIM II, WE WILL TEST THE MOLECULAR REVERSIBILITY OF HISTONE PHENOTYPES IN DS USING PHARMACOLOGICAL AND CRISPRA APPROACHES TO IDENTIFY POTENTIAL CLINICALLY RELEVANT TARGETS IN DS. IN AIM III, WE WILL TEST THE HYPOTHESIS THAT DYSREGULATION OF HISTONE PTMS DRIVES CORE NEUROBIOLOGICAL PHENOTYPES IN DS TO FURTHER UNDERSTAND THEIR FUNCTIONAL RELEVANCE AND POTENTIALLY MAP KNOWN CELLULAR PHENOTYPES TO NOVEL MOLECULAR MECHANISMS. COLLECTIVELY, THE RIGOROUS AND INNOVATIVE ANALYSES IN THIS TRANSFORMATIVE RESEARCH AWARD APPLICATION WILL ILLUMINATE NEW MECHANISMS OF EPIGENETIC DYSREGULATION IN DS, EXPLORE A SET OF KEY UNANSWERED QUESTIONS IN THE FIELD AND MAY ULTIMATELY INFORM ON THERAPEUTIC STRATEGIES FOR DS PATIENTS.

DEVELOPMENT OF SMALL MOLECULE-BASED METHODS FOR TARGETED CARGO DELIVERY TO BETA CELLS

BROAD-MIT CENTER FOR HIGH-THROUGHPUT SYNTHETIC BIOLOGY

HOW SUBSTRATE DOSAGE DRIVES PRION DISEASE KINETICS - PROJECT SUMMARY PRION DISEASE IS A UNIQUELY RAPID, UNIVERSALLY FATAL PROGRESSIVE NEURODEGENERATIVE DISEASE OF HUMANS AND OTHER MAMMALS. IT ARISES FROM A SINGLE PROTEIN, THE NATIVE PRION PROTEIN (PRP), WHICH IS CAPABLE OF POST-TRANSLATIONALLY MISFOLDING INTO A SELF-TEMPLATING AND DEADLY “PRION.” GENETIC AND PHARMACOLOGICAL PROOFS OF CONCEPT INCREASINGLY IDENTIFY PRP DOSAGE AS THE KEY TO UNDERSTANDING, AND ULTIMATELY INTERCEPTING THIS PATHOGENIC CASCADE. IN MICE WITH GENETICALLY ALTERED PRP LEVELS, LOWER PRP LEVELS LEAD TO LONGER SURVIVAL FOLLOWING PRION INFECTION, WHILE EXCESS PRP HASTENS DISEASE. PRP-LOWERING ANTISENSE OLIGONUCLEOTIDES (ASOS) NOW SHOW PROMISE AS A POTENTIAL THERAPEUTIC STRATEGY. HOWEVER, EFFECTIVE IMPLEMENTATION WILL HINGE ON A DEEPER UNDERSTANDING OF HOW PRP LEVEL CONTROLS THE RATES OF PRION NUCLEATION, REPLICATION AND NEUROTOXICITY, AND HOW THIS CONTROL TRANSLATES ACROSS DISEASE TIMEPOINTS, SPECIES AND STRAINS. THOUGH ALMOST ALL HUMAN PRION DISEASE ORIGINATES IN THE BRAIN, PRP-LOWERING INTERVENTIONS HAVE NOT YET BEEN TESTED IN SPONTANEOUS, RATHER THAN INOCULATED, PRION MODELS. MEANWHILE, THE MAGNITUDE OF PROTECTION CONVEYED BY 50% GENETIC PRP REDUCTION CAN VARY BETWEEN MODEL SYSTEMS; THE RELATIVE CONTRIBUTIONS OF SLOWED PRION REPLICATION AND SLOWED NEUROTOXICITY TO OBSERVED SURVIVAL BENEFIT IN DIFFERENT SPECIES AND PRION STRAINS REMAIN TO BE DISENTANGLED. FINALLY, PRP LOWERING MUST BE STUDIED IN THE CONTEXT OF PATIENT-DERIVED HUMAN PRIONS, TO ASSESS HOW ABOVE LEARNINGS EXTRAPOLATE TO STRAINS OF PUBLIC HEALTH INTEREST. WE WILL FILL THESE GAPS BY ASSESSING THE FOLLOWING. 1) KINETICS OF SPONTANEOUS PRION FORMATION. USING A NEW MOUSE MODEL OF SPONTANEOUS PRION DISEASE, WE WILL TRACK THE SPONTANEOUSLY DISEASE PROCESS THROUGH SERIAL MOLECULAR MEASUREMENTS OF NEURONAL DAMAGE AND PRION SEEDING ACTIVITY. THROUGH PRP-LOWERING TOOL COMPOUNDS ADMINISTERED AT DIFFERENT TIMEPOINTS, WE WILL DISENTANGLE HOW PRP DOSAGE MODULATES PRION FORMATION, AMPLIFICATION, NEUROTOXICITY AND SYMPTOMATIC PROGRESSION. 2) RAPID AND SLOW PRION SUBTYPES AS A FUNCTION OF PRP DOSAGE. USING NEWLY ENGINEERED PRP KNOCKOUT HAMSTER AND RAT MODELS, WE WILL CHARACTERIZE THE KINETICS OF PATHOLOGICAL BIOMARKER RISE RELATIVE TO DISEASE ONSET AND TERMINAL ILLNESS AS A FUNCTION OF PRP EXPRESSION LEVEL IN BOTH CANONICALLY RAPID (HAMSTER) AND MORE SLOWLY PROGRESSIVE (RAT) PRION DISEASE SYSTEMS. 3) IMPACT OF PRP LOWERING ON HUMAN PRIONS. USING A SERIES OF NOVEL “HUMANIZED” MOUSE LINES EXPRESSING HUMAN PRP AT SIX DIFFERENT DOSAGE LEVELS, THAT HAVE BEEN SHOWN SUSCEPTIBLE TO MULTIPLE CLINICALLY RELEVANT HUMAN PRION STRAINS, WE WILL CHARACTERIZE TIME TO PATHOLOGY, SYMPTOM ONSET, AND TERMINAL ILLNESS. PRP LEVEL WILL BE VARIED ON A LIFELONG BASIS THROUGH GENETICALLY MANIPULATION, AS WELL AS THROUGH POSTNATAL, PRECISELY TIMED INTERVENTION WITH PRP-LOWERING TOOL COMPOUNDS. TAKEN TOGETHER, THESE STUDIES WILL ILLUMINATE PRP’S CONTROL OF DISEASE KINETICS ACROSS A SPECTRUM OF PRION DISEASE PARADIGMS, WHILE BUILDING A SCIENTIFIC FOUNDATION TO GUIDE FUTURE DEVELOPMENT OF PRP-LOWERING THERAPEUTICS.

SLIDE-SEQ: HIGH-RESOLUTION IN SITU EXPRESSION PROFILING FOR NEUROPATHOLOGY

HIGH-THROUGHPUT CELLULAR GENETICS TO CONNECT NONCODING VARIANTS TO CORONARY ARTERY DISEASE GENES - ABSTRACT DESPITE EFFECTIVE THERAPIES TO CONTROL BLOOD PRESSURE AND CHOLESTEROL, CORONARY ARTERY DISEASE (CAD) REMAINS THE LEADING CAUSE OF DEATH IN THE UNITED STATES AND THE WORLD. RECENT GENOME-WIDE ASSOCIATION STUDIES (GWAS) HAVE IDENTIFIED >200 GENETIC LOCI SIGNIFICANTLY ASSOCIATED WITH CAD. THE MAJORITY OF THESE LOCI ARE NOT ASSOCIATED WITH HYPERTENSION OR HYPERLIPIDEMIA, BUT CONTAIN GENES EXPRESSED IN VASCULAR CELLS, SUGGESTING THE PRESENCE OF UNDISCOVERED CAD DISEASE MECHANISMS OPERATING THROUGH CELLS OF THE BLOOD VESSEL WALL. IDENTIFYING THE BIOLOGIC MECHANISMS OF THESE LOCI IS DIFFICULT BECAUSE MOST OF THE COMMON VARIANTS ASSOCIATED WITH CAD ARE NON-CODING, LIKELY FUNCTIONING THROUGH ENHANCERS THAT CAN REGULATE MULTIPLE GENES AT GREAT DISTANCES, AND BE HIGHLY CELL-TYPE SPECIFIC. THIS HAS SLOWED DISCOVERY, SO THAT ONLY A FEW SUCH LOCI HAVE BEEN ‘SOLVED’, PREVENTING THE REALIZATION OF THE FULL POTENTIAL OF GENETIC STUDIES FOR THE DEVELOPMENT OF MUCH NEEDED NEW CLASSES OF THERAPIES FOR CAD. WHAT IS NEEDED ARE MUCH HIGHER-THROUGHPUT, UNBIASED METHODS TO SYSTEMATICALLY DISSECT THESE LOCI, TO IDENTIFY MOLECULAR AND CELLULAR MECHANISMS OF DISEASE. TO ACCOMPLISH THIS GOAL, WE HAVE DEVELOPED AND VALIDATED TWO NOVEL HIGH-THROUGHPUT APPROACHES WITH THE ABILITY TO QUANTIFY THE MOLECULAR AND MORPHOLOGICAL EFFECTS OF THOUSANDS OF GENES IN CAD LOCI: OPTIMIZED PERTURB-SEQ (TO LINK GENES TO TRANSCRIPTIONAL PHENOTYPES) AND POOLED PERTURBATION CELL PAINTING (TO LINK GENES TO MORPHOLOGICAL EFFECTS). WE PROPOSE TO USE THESE TECHNOLOGIES, TOGETHER WITH NEW AND NOVEL COMPUTATIONAL APPROACHES, TO: 1) ANALYZE THE TRANSCRIPTOMIC EFFECTS OF PERTURBING CAD-LOCUS GENES IN VASCULAR ENDOTHELIAL CELLS (ECS) SUBJECTED TO FOUR STIMULI ASSOCIATED WITH ATHEROSCLEROSIS, 2) QUANTIFY THE MORPHOLOGICAL EFFECTS OF PERTURBING ALL CAD-LOCUS GENES IN ECS, 3) INTEGRATE THESE DATA WITH EPIGENOMIC AND GENETIC DATA TO NOMINATE CAUSAL GENES AND BUILD HYPOTHESES CONNECTING VARIANTS TO GENES, AND GENES TO DISEASE-ASSOCIATED CELLULAR PHENOTYPES, AND THEN TEST THE TOP 5 OF EACH OF THESE HYPOTHESES USING VARIANT-EDITING AND EC-FUNCTIONAL ASSAYS. THIS INCLUDES SEVERAL PRIORITIZED CAUSAL VARIANT HYPOTHESES THAT REGULATE EC SHEAR STRESS RESPONSE UPSTREAM OF KLF2 EXPRESSION. THE COMPLETION OF THESE STUDIES WILL ACCELERATE UNDERSTANDING OF THE LINKS BETWEEN CAD GENETICS AND DISEASE, AND PROVIDE INSIGHTS INTO EC FUNCTIONS IN CAD THAT MAY INFORM THE DEVELOPMENT OF NEW CLASSES OF THERAPIES. MORE BROADLY, THIS STUDY WILL ESTABLISH NEW SCALABLE EXPERIMENTAL AND COMPUTATIONAL METHODS TO LINK NONCODING DISEASE VARIANTS TO GENES AND CELLULAR PHENOTYPES, WHICH COULD DRAMATICALLY ACCELERATE VARIANT-TO-FUNCTION STUDIES FOR CARDIOVASCULAR AND OTHER COMMON DISEASES.

DEVELOPMENT OF P300/CBP HISTONE ACETYLTRANSFERASE INHIBITORS FOR ONCOGENE-DRIVEN CANCERS - PROJECT SUMMARY: ONCOGENE MUTATIONS FREQUENTLY DRIVE MALIGNANT TRANSFORMATION THROUGH INAPPROPRIATE ACTIVATION OF REGULATORY KINASES, AND INHIBITORS THAT DISRUPT ONCOGENIC PHOSPHORYLATION HAVE TRANSFORMED CARE FOR SUBSETS OF CANCER PATIENTS. DESPITE THESE SUCCESSES, KINASES ARE A SMALL FRACTION OF KNOWN ONCOGENES. IN PARTICULAR, SEVERAL TRANSCRIPTIONAL FACTOR ONCOGENES ARE ESSENTIAL FOR TUMOR INITIATION, PROGRESSION AND MAINTENANCE. IN A FEW CASES, TARGETING TRANSCRIPTION FACTOR ONCOGENES, SUCH AS ESTROGEN, ANDROGEN AND RETINOIC ACID RECEPTORS, HAS LED TO CLINICALLY MEANINGFUL RESPONSES. HOWEVER, DIRECT TARGETING OF MOST TRANSCRIPTION FACTORS HAS PROVEN CHALLENGING. AN ALTERNATIVE APPROACH TO DIRECTLY TARGETING ONCOGENIC TRANSCRIPTION FACTORS IS TO INHIBIT UPSTREAM EPIGENETIC MECHANISMS REGULATING CHROMATIN STATE. INDEED, MANY EPIGENETIC REGULATORS ARE RECURRENTLY SOMATICALLY MUTATED, AND SEVERAL SMALL MOLECULES TARGETING CHROMATIN REGULATORS HAVE BEEN SHOWN TO ABROGATE TUMOR GROWTH. IN PRELIMINARY STUDIES, WE PERFORMED A GENE EXPRESSION-BASED SCREEN TO IDENTIFY SMALL MOLECULES THAT INHIBIT THE ACTIVITY OF TMPRSS2-ERG, A FUSION ONCOGENE LEADING TO ABERRANT TRANSCRIPTION IN PROSTATE CANCER, AND IDENTIFIED BRD4683, A HIGHLY POTENT INHIBITOR OF P300 AND CREB BINDING PROTEIN (CBP) HISTONE ACETYL TRANSFERASE (HAT) ACTIVITY. WE HAVE SOLVED THE STRUCTURE OF BRD4683 IN COMPLEX WITH P300 AND CONFIRMED THAT INHIBITION OF P300/CBP HAT ACTIVITY IS SPECIFICALLY REQUIRED FOR SURVIVAL OF TRANSCRIPTION FACTOR-DRIVEN HUMAN CANCER CELL LINES. DESPITE SERVING AS A USEFUL TOOL COMPOUND, BRD4683 HAS SEVERAL CHEMICAL LIABILITIES THAT LIMIT ITS POTENTIAL FOR DRUG DEVELOPMENT. WE THUS USED BRD4683 TO DEVELOP AND VALIDATE A HIGH-THROUGHPUT AND COST-EFFECTIVE BIOCHEMICAL SCREEN FOR DISCOVERY OF ADDITIONAL NOVEL SMALL MOLECULE P300/CBP HAT INHIBITORS AS WELL AS A CASCADE OF FOLLOW UP- ASSAYS TO ELIMINATE FALSE POSITIVES AND PRIORITIZE CHEMICALLY TRACTABLE MOLECULES. IN THIS PROJECT, WE PROPOSE TO IDENTIFY NEW P300/CBP INHIBITORS AND TO SYSTEMATICALLY IDENTIFY TUMOR SUBTYPES DEPENDENT ON COMBINED P300/CBP HAT ACTIVITY. IN AIM 1, WE WILL PERFORM A HIGH THROUGHPUT BIOCHEMICAL SCREEN OF MORE THAN 800,000 COMPOUNDS TO IDENTIFY NEW P300/CBP HAT INHIBITORS. IN AIM 2, WE WILL VALIDATE NOVEL P300/CBP CANDIDATES USING THREE COMPLEMENTARY ASSAYS TO IDENTIFY INHIBITORS AN IC50 OF AT LEAST 10 ΜM. THESE ASSAYS WILL NOT ONLY CONFIRM INHIBITOR SPECIFICITY BUT WILL ALSO VALIDATE BIOCHEMICAL ACTIVITY IN CELLS. IN AIM 3, WE WILL PERFORM ITERATIVE MEDICINAL CHEMISTRY WITH THE GOAL OF IDENTIFYING LEAD CANDIDATES. IN PARALLEL TO THESE STUDIES, WE WILL PERFORM BOTH CHEMICAL AND GENETIC EXPERIMENTS TO SYSTEMATICALLY IDENTIFY TUMOR SUBTYPES THAT DEPEND ON P300/CBP HAT ACTIVITY FOR CELL FITNESS (AIM 4). TO PERFORM THESE STUDIES, WE HAVE ASSEMBLED A MULTIDISCIPLINARY RESEARCH TEAM WITH THE NECESSARY EXPERIENCE AND EXPERTISE IN CANCER BIOLOGY, STRUCTURAL BIOLOGY, MEDICINAL CHEMISTRY AND DRUG DEVELOPMENT. WE ANTICIPATE THAT THESE STUDIES WILL ALLOW US TO IDENTIFY SMALL MOLECULES THAT WILL SERVE AS LEAD CANDIDATES THAT WILL SPUR THE DEVELOPMENT OF CLINICAL GRADE P300/CBP HAT INHIBITORS AND DEFINE WHICH PATIENT POPULATIONS ARE LIKELY TO BENEFIT FROM SUCH INHIBITORS.

NON-CODING GENETIC VARIANTS THAT IMPACT IMMUNE PHENOTYPES AND DISEASES

SMALL-MOLECULE APPROACHES TO RESTORE GLYCEMIC CONTROL IN TYPE 1 DIABETES

DEVELOPMENT OF PLATFORMS FOR SORTING, PRODUCTION, EDITING OF BETA CELLS - PROJECT SUMMARY PATIENTS SUFFERING FROM TYPE 1 DIABETES MUST UNDERGO BURDENSOME, OFTEN LIFELONG, EXOGENOUS INSULIN DEPENDENCE. UP TO NOW, THE ONLY AVAILABLE REPLACEMENT THERAPY IS TO TRANSPLANT ISLETS FROM CADAVERIC DONORS. HOWEVER, SUCH PROCEDURES PRESENT HURDLES SUCH AS THE SCARCITY OF AVAILABLE DONORS AND THE REJECTION OF TRANSPLANTED CELLS BY THE PATIENT’S IMMUNE SYSTEM. ALSO, OVER TIME, THE TRANSPLANTED ISLETS TEND TO DIE. SEVERAL TYPES OF STEM CELLS, INCLUDING EMBRYONIC STEM CELL AND INDUCED PLURIPOTENT STEM CELL, HAVE BEEN EXTENSIVELY STUDIED TO GENERATE GLUCOSE-RESPONSIVE INSULIN-SECRETING CELLS AND REPRESENTS A MORE TENABLE SOURCE OF ISLETS. HOWEVER, A SIGNIFICANT CONCERN FOR TRANSPLANTATION THERAPY IS THE NEED FOR IMMUNOSUPPRESSIVE DRUGS, WHICH EXHIBIT LONG TERM SIDE EFFECTS AND HINDERS THE FEASIBILITY OF THIS APPROACH TO THE LARGE POPULATION OF T1D PATIENTS. FURTHERMORE, COST-EFFECTIVE AND LARGE-SCALE PRODUCTION OF THESE STEM CELL-DERIVED BETA CELLS IS STILL A MAJOR CHALLENGE. WE PROPOSE TO APPLY CHEMICAL BIOLOGY AND GENOME ENGINEERING APPROACHES TO DEVELOP PLATFORMS FOR EFFICIENT SORTING, PRODUCTION, AND EDITING OF THESE BETA CELLS. PHS 398/2590 (REV. 06/09) 1 CONTINUATION FORMAT PAGE

(PQ3) THE ROLE OF DAMAGED DNA IN INTER-INDIVIDUAL VARIATION OF TUMOR IMMUNITY

CHARACTERIZING TP53 AND PPM1D MUTATIONS AS RESISTANCE DRIVERS TO RADIATION THERAPY IN DIFFUSE INTRINSIC PONTINE GLIOMAS

FUNCTIONAL GENOMICS OF NEUROPLASTICITY IN SCHIZOPHRENIA

LEVERAGING SNAKES' EXTREME PHYSIOLOGY TO MODULATE HUMAN BETA-CELL FUNCTION

CONTINUOUS EVOLUTION OF PROTEINS WITH NOVEL THERAPEUTIC POTENTIAL - PROJECT SUMMARY: CONTINUOUS EVOLUTION OF PROTEINS WITH NOVEL THERAPEUTIC POTENTIAL THE DIRECT MANIPULATION OF GENES AND GENE PRODUCTS IN VIVO HAS ENORMOUS THERAPEUTIC POTENTIAL, AND MANY STRATEGIES TO ACHIEVE THESE GOALS ARE SWIFTLY ADVANCING TOWARD CLINICAL USE. PROTEINS THAT CAN MANIPULATE DNA, RNA, AND PROTEINS IN LIVING CELLS, INCLUDING GENOME EDITING TECHNOLOGIES THAT ENABLE THE PRECISE CORRECTION OF DISEASE-CAUSING MUTATIONS IN VIVO, HAVE EXEMPLIFIED THE PROMISE OF SUCH APPROACHES BOTH FOR RESEARCH AND THERAPEUTIC APPLICATIONS. WHILE MANY OF THESE APPROACHES HAVE SHOWN PROMISE IN INITIAL RESEARCH STUDIES, PROTEINS OFTEN REQUIRE EXTENSIVE DEVELOPMENT AND TAILORING TO ACQUIRE THE ACTIVITY, SPECIFICITY, AND STABILITY NEEDED TO SERVE AS IMPACTFUL RESEARCH TOOLS OR LEADS FOR THERAPEUTIC DEVELOPMENT. AS NEW MACROMOLECULAR THERAPEUTIC MODALITIES CONTINUE TO BE DEVELOPED AT A REMARKABLE RATE, METHODS TO GENERATE PROTEINS ON A RAPID TIME SCALE WITH TAILOR-MADE FUNCTIONS ARE NEEDED. IDEALLY SUCH METHODS WILL BE VERSATILE AND CAN BE APPLIED TO MANY CLASSES OF PROBLEMS IN THE LIFE SCIENCES. OUR LAB DEVELOPED PHAGE-ASSISTED CONTINUOUS EVOLUTION (PACE), A TECHNOLOGY TO EVOLVE BIOMOLECULES =100- FOLD FASTER THAN USING CONVENTIONAL LABORATORY EVOLUTION APPROACHES, WITH MINIMAL REQUIRED RESEARCHER INTERVENTION. WE HAVE DEMONSTRATED THE ABILITY OF PACE TO EVOLVE MANY DIFFERENT CLASSES OF PROTEINS WITH NEW AND ALTERED ACTIVITIES, SPECIFICITIES, AND OTHER DESIRABLE PROPERTIES SUCH AS SOLUBLE EXPRESSION IN E. COLI. PROTEINS EVOLVED USING PACE HAVE SHOWN BROAD UTILITY IN MULTIPLE NON-BACTERIAL SETTINGS, INCLUDING GENOME EDITING AGENTS THAT HAVE BEEN APPLIED TO RESCUE HUMAN CELL AND ANIMAL MODELS OF GENETIC DISEASES, AND INSECTICIDAL PROTEINS THAT KILL AGRICULTURAL PESTS. THESE DEVELOPMENTS ESTABLISH PACE AS A BROADLY APPLICABLE AND HIGHLY ENABLING TECHNOLOGY FOR GENERATING THERAPEUTICALLY AND BIOTECHNOLOGICALLY RELEVANT PROTEINS. WE PROPOSE TO APPLY PACE TO EVOLVE NOVEL PROTEINS WITH THERAPEUTIC POTENTIAL, OR THAT ENABLE NEW TECHNOLOGIES FOR THERAPEUTICS DISCOVERY. THESE PROTEINS INCLUDE NEXT-GENERATION PRECISION GENOME EDITING AGENTS THAT CAN BE MORE EASILY DELIVERED IN VIVO OR ARE MORE EFFICIENT AND CLINICALLY RELEVANT; SELF-DELIVERING PROTEASES THAT CLEAVE ENDOGENOUS PROTEIN TARGETS IMPLICATED IN NEURODEGENERATIVE DISORDERS AND BRAIN CANCER; AND SMALL MOLECULE-BINDING PROTEINS THAT ENABLE DRUG-INDUCED TARGET PROTEIN DEGRADATION. SUCCESS WOULD PROVIDE A FOUNDATION FOR INNOVATIVE THERAPEUTIC STRATEGIES TO CORRECT MUTATIONS THAT CAUSE HUMAN GENETIC DISEASES, AND TO REPROGRAM SELF-DELIVERING PROTEASES AS CATALYTIC DRUGS TO TREAT BRAIN DISEASES. IN ADDITION, BY CREATING DRUG-SENSITIVE ALLELES THAT ALLOW A PROTEIN OF INTEREST TO BE DEGRADED IN A SMALL MOLECULE-DEPENDENT MANNER, THE PROPOSED RESEARCH WOULD ESTABLISH POWERFUL NEW FUNCTIONAL GENOMICS TOOLS TO REVEAL BIOLOGICAL FUNCTIONS AND VALIDATE THERAPEUTICS TARGETS. COLLECTIVELY, THE PROPOSED RESEARCH INTEGRATES POWERFUL PROTEIN EVOLUTION TECHNOLOGIES WITH ENZYMES THAT PRECISELY MANIPULATE GENOMES AND PROTEOMES TO ADVANCE THERAPEUTICS SCIENCE.

THE TEMPORAL RECONFIGURATION OF REGULATORY NETWORKS: FROM YEAST TO CANCER

MENTAL HEALTH GENOMICS CONSORTIUM COORDINATING CENTER - MENTAL ILLNESSES ARE AMONG THE LEADING CAUSES OF DISABILITY WORLDWIDE, AFFECTING MORE THAN 25% OF THE POPULATION IN ANY YEAR. GENETIC STUDIES FOR MENTAL ILLNESSES HAVE MADE ENCOURAGING PROGRESS IN THE PAST DECADES. THE NIMH GENOMICS CONSORTIUM COORDINATION CENTER (GC3) AIMS TO TRANSFORM GENETICS RESEARCH IN MENTAL ILLNESSES BY BRINGING TOGETHER NIMH-SUPPORTED GENOMICS DATA TO BUILD A RESOURCE FOR THE COMMUNITY AND POWER GENOMICS DISCOVERY EFFORTS FOR MENTAL ILLNESSES. GC3 WILL ENHANCE INTEROPERABILITY OF GENOMICS DATASETS AND FACILITATE COLLABORATIVE RESEARCH EFFORTS. IN AIM 1, THE GC3 WILL ESTABLISH AND MANAGE A NETWORK OF NIMH-SUPPORTED GENOMICS PROJECTS TO BRING THE COMMUNITY TOGETHER, DEVELOP WORKING GROUPS TO ENABLE CROSS-CONSORTIUM EFFORTS; ORGANIZE AN ANNUAL IN-PERSON MEETING; AND COORDINATE THE SUBMISSION OF DATA TO THE APPROPRIATE REPOSITORIES. IN AIM 2, GC3 WILL DEVELOP AND IMPLEMENT A FLEXIBLE PHENOTYPE HARMONIZATION STRATEGY ACROSS STUDY SITES. THIS INVOLVES SUPPORTING WORKING GROUPS DEDICATED TO PHENOTYPE HARMONIZATION AND CONDUCTING COMPREHENSIVE ASSESSMENTS OF PHENOTYPING PROTOCOLS USED ACROSS DIFFERENT STUDIES. BY ALIGNING DIAGNOSTIC AND SYMPTOM-LEVEL DATA, WE WILL ENSURE THAT THE PHENOTYPIC INFORMATION INCLUDED WITH GENOMICS DATASETS CAN BE ANALYZED ACROSS SITES, A CRITICAL STEP FOR ADVANCING GENOMICS DISCOVERIES IN PSYCHIATRIC DISORDERS. IN AIM 3, GC3 WILL CONDUCT COMPREHENSIVE GENOMIC INGEST, DATA HARMONIZATION AND IMPUTATION FOR ALL SAMPLES. FROM THE CALLED AND IMUTED OF GENETIC VARIATION, GC3 WILL ALSO PERFORM QUALITY CONTROL TO ENSURE THAT DATA FROM MULTIPLE SITES ARE STANDARDIZED AND INTEGRATED. THIS ALLOWS FOR MORE POWERFUL INTEGRATED ANALYSES AND ENSURES THAT GENETIC DATA IS ACCESSIBLE AND USABLE FOR A WIDE RANGE OF RESEARCHERS. IN AIM 4, WE WILL INTEGRATE AND DISTRIBUTE DATA AND RESULTS TO INVESTIGATORS. WE WILL INTEGRATE GENETIC ASSOCIATION ANALYSES ACROSS SITES, DISORDERS, AND STUDIES, THEN CREATE DATA BROWSERS TO FACILITATE BROAD AND EASY ACCESS TO THE RESULTS OF GENOMIC INVESTIGATIONS INTO MENTAL ILLNESSES INCLUDING COMMON AND RARE VARIANTS ASSOCIATION ANALYSES. WE WILL ALSO DISTRIBUTE THE RESULTS OF PHENOTYPIC PROTOCOL HARMONIZATION. IN AIM 5, WE WILL DEVELOP AND IMPLEMENT A COMPREHENSIVE TRAINING PROGRAM FOR MEMBERS OF THE CONSTITUENT NIMH-SUPPORTED GENOMICS PROJECTS. THROUGH A STRUCTURED TRAINING PROGRAM, GC3 WILL ORGANIZE ANNUAL WORKSHOPS, DEVELOP AN ONLINE LEARNING PLATFORM, AND FACILITATE A JOURNAL CLUB TO ENGAGE EARLY CAREER RESEARCHERS. THIS COMPREHENSIVE APPROACH WILL GALVANIZE PSYCHIATRIC GENETICS RESEARCH, FOSTER COLLABORATION, AND ADVANCE RESEARCH PRACTICES, TO LEARN THE BIOLOGY OF MENTAL ILLNESSES AND ULTIMATELY HELPING TO ALLEVIATE THE BURDEN THESE ILLNESSES CREATE IN THE UNITED STATES OF AMERICA.

A SCALABLE CLOUD-BASED FRAMEWORK FOR MULTI-MODAL MAPPING ACROSS SINGLE NEURON OMICS, MORPHOLOGY AND ELECTROPHYSIOLOGY - PROJECT SUMMARY CATEGORIZING INDIVIDUAL NEURONS INTO DIFFERENT GROUPS, OR CELL TYPES, IS A CLASSICAL APPROACH TO STUDYING THE NERVOUS SYSTEM. WITH INCREASINGLY MORE TOOLS BEING INVENTED TO OBSERVE THE NEURONS, NEW CRITERIA WERE CREATED TO CHARACTERIZE DIFFERENT ASPECTS, OR MODALITIES, OF THE CELLS. WHILE THESE MODALITY-SPECIFIC CATEGORIZATIONS HAVE ENABLED IN-DEPTH KNOWLEDGE IN NEUROSCIENCE, THE INCONSISTENCIES ACROSS DIFFERENT CRITERIA LEAVE DATA INTEGRATION ACROSS MODALITIES TECHNICALLY DIFFICULT. IN ADDITION, DISAGREEMENTS OF “SIMILAR CELLS” USING DIFFERENT CATEGORIZATION CRITERIA HAVE RESULTED IN DIVISION OF THE NEUROSCIENCE COMMUNITY INTO MODALITY-CENTRIC SUBGROUPS. TO SOLVE THIS PROBLEM, OBJECTIVE APPROACHES TO DEFINE CELL SIMILARITIES INCORPORATING MULTIPLE MODALITIES MUST BE ESTABLISHED AND DEVELOPED IN A WAY THAT IS OPEN, ACCESSIBLE, AND ENGAGING TO THE NEUROSCIENCE COMMUNITY AT LARGE. WE PROPOSE TO DEVELOP A BROADLY ACCESSIBLE CLOUD-BASED FRAMEWORK TOWARD AN INTEGRATIVE, MULTI-MODAL BRAIN CELL ATLAS USING NOVEL, SCALABLE ANALYTICS TOOLS, LEVERAGING FEDERATED BRAIN INITIATIVE RESOURCES AND COMMUNITY ENGAGEMENT. THE SELF-SUPERVISED LEARNING METHODOLOGY IS PURELY DATA-DRIVEN, AND ALLOWS HIGHLY ACCURATE IDENTIFICATION OF SIMILAR CELLS BASED ON SINGLE OR MULTIPLE MODALITIES OF MEASUREMENTS, WITHOUT PRESUMPTION ABOUT MODALITY-SPECIFIC CLASSES. IT ALSO ENABLES CROSS-MODALITY MAPPING, WHERE UNOBSERVED MEASUREMENTS CAN BE INFERRED FROM SINGLE-MODAL DATA, ACHIEVING COMPUTATIONAL “SCALE-UP” OF THE BRAIN CELL MAPPING EFFORTS IN LOW- THROUGHPUT MODALITIES SUCH AS ELECTROPHYSIOLOGY OR MORPHOLOGY. WITH MORE MULTIMODAL DATA BEING COLLECTED AND ADDED TO THE STUDY, THE ALGORITHM ACCURACY OF THIS INFERENCE WILL CONTINUE TO GROW IN AN AUTOMATED WAY. THE OPEN-SOURCE METHODOLOGY WILL BE PRODUCTIONIZED INTO CLOUD-NATIVE PIPELINES FOR INDIVIDUAL MODALITIES AND FOR CROSS-MODALITY MAPPING, AND INSTALLED IN A CLOUD COMPUTING WORKBENCH AS A PART OF THE ECOSYSTEM, FOR SCALABLE, OPEN DATA ANALYSES. THIS CLOUD ECOSYSTEM WILL DEMONSTRATE ACCESS TO BRAIN INITIATIVE DATA REPOSITORIES HOSTING MOLECULAR (NEMO), NEUROPHYSIOLOGY (DANDI), AND MICROSCOPY (BIL) DATA, SUCH THAT DATASETS FROM DIFFERENT SOURCES CAN BE BROUGHT INTO A COMMON WORKSPACE FOR INTEGRATIVE ANALYSES. FURTHERMORE, THE CLOUD ECOSYSTEM WILL PROVIDE A USER-FRIENDLY DATA PORTAL FOR VISUALIZATION AND NAVIGATION OF THE MULTI-MODAL SINGLE CELL DATA FROM THE REPOSITORIES, AS WELL AS EXPLORING THE CELL SIMILARITY QUERY RESULTS. THIS ECOSYSTEM WILL SUPPORT FAIR PRINCIPLES AND PROMOTE COLLABORATIVE RESEARCH AND SEEK FOR EXTENDED INTEGRATIONS WITH DATA REPOSITORIES. THROUGH BROAD ENGAGEMENT AND OUTREACH TO NEUROSCIENCE COMMUNITIES, THIS PROJECT WILL PROVIDE RESOURCES FOR BUILDING AN INTEGRATED BRAIN CELL ATLAS AND FACILITATE THE MULTIMODAL CHARACTERIZATION OF THE BRAIN.

OPTICAL FUNCTIONAL GENOMICS - PROJECT ABSTRACT: MAMMALIAN CELL BIOLOGY HAPPENS IN 3 DIMENSIONS. ADVANCES IN HIGH-THROUGHPUT GENOMICS AND CELLULAR PROFILING ‘OMICS METHODS HAVE MADE MASSIVELY-PARALLEL CELL BIOLOGY EXPERIMENTS ROUTINE FOR MANY LABS, BUT THE VAST MAJORITY OF THESE STUDIES IN HUMAN CELLS HAVE BEEN LIMITED TO HOMOGENEOUS 2-DIMENSIONAL MODELS. THIS DRASTICALLY REDUCES THE RANGE OF CELL TYPES AND INTERACTIONS THAT CAN BE STUDIED EX VIVO. AND, DESPITE ADVANCES IN THE GROWTH OF ORGANOIDS AND MICROTISSUES FOR THE STUDY OF HUMAN BIOLOGY IN 3D, NEXT GENERATION ‘OMICS APPROACHES STILL REQUIRE THE DISSOCIATION OF THESE STRUCTURES AND THE DESTRUCTION OF THEIR CELLS TO EXTRACT BIOMOLECULES FOR STUDY. RESEARCHERS NEED TOOLS THAT CAN INTERROGATE HUMAN BIOLOGY AT SCALE AND IN SITU TO GENERATE RICH PHENOTYPING DATA FROM SINGLE CELLS WHILE STILL PRESERVING COMPLEX SPATIAL RELATIONSHIPS AND INTERACTIONS BETWEEN CELLS IN 3D. THE LACK OF SUCH TOOLS IS A MAJOR LIMITATION IN BIOMEDICAL RESEARCH. HERE I PROPOSE A TECHNOLOGY THAT COMBINES THE DISCOVERY POTENTIAL OF GENOME SCALE SCREENING WITH THE INFORMATION CONTENT OF HIGH-DIMENSIONAL SINGLE-CELL PROFILING, WITH FULL INTEGRATION OF 3D SPATIAL INFORMATION TO ENABLE RICH IN SITU PHENOTYPIC READOUT OF COMPLEX CELLULAR PHENOMENA AT SCALE.

SINGLE-CELL IN SITU ANALYSIS OF RNA MODIFICATIONS IN INTACT TISSUES - PROJECT SUMMARY CELLULAR RNAS HAVE MORE THAN 100 DIFFERENT KINDS OF CHEMICAL MODIFICATIONS, WHICH TOGETHER COMPRISE THE EPITRANSCRIPTOME. PREVIOUS STUDIES HAVE REVEALED THAT RNA-MODIFYING PATHWAYS ARE VITAL TO HIGHER EUKARYOTES, AND THE ALTERATION OF THESE PATHWAYS IS ASSOCIATED WITH A NUMBER OF HUMAN DISEASES. HOWEVER, IT IS STILL UNCLEAR WHY WE NEED SUCH A COMPLEX CHEMICAL REPERTOIRE FOR RNA AND HOW RNA MODIFICATIONS IMPACT GENE EXPRESSION IN BIOLOGICAL TISSUES WITH DIVERSE CELL TYPES. THE MAJOR BOTTLENECK FOR INVESTIGATING RNA MODIFICATIONS HAS BEEN THE LACK OF MEASUREMENT TOOLS WITH SUBCELLULAR AND SINGLE-CELL RESOLUTIONS. BULK EPITRANSCRIPTOMIC SEQUENCING METHODS CAN ONLY MEASURE THE AVERAGED DISTRIBUTION OF MODIFICATION SITES FROM MILLIONS OF CELLS, OBSCURING THE INTRINSIC HETEROGENEITY OF EPITRANSCRIPTOMIC STATES IN DISTINCT CELL TYPES. MOREOVER, SPATIAL INFORMATION IS LOST DURING RNA EXTRACTION FROM TISSUES, PREVENTING FURTHER ANALYSIS OF RNA MODIFICATION PATTERNS IN THE CONTEXT OF TISSUE MORPHOLOGY AND SUBCELLULAR COMPARTMENTS. TO ADDRESS SUCH LIMITATIONS, WE PROPOSE TO CREATE CUTTING-EDGE PLATFORMS FOR THREE-DIMENSIONAL (3D) IN SITU SEQUENCING OF RNA MODIFICATIONS. THEN WE WILL UTILIZE THE NEW TOOLS TO ANALYZE GENE REGULATION MECHANISMS MEDIATED BY RNA MODIFICATIONS AT SINGLE-CELL RESOLUTION IN INTACT BIOLOGICAL TISSUES. USING THE N6-METHYLADENOSINE (M6A) PATHWAY IN MOUSE BRAIN TISSUE AS OUR MODEL SYSTEM, WE WILL (A) DEVELOP INNOVATIVE AND BROADLY APPLICABLE 3D IN SITU SEQUENCING METHODS FOR RNA MODIFICATIONS; (B) PROFILE THE M6A CODE AT SINGLE-CELL RESOLUTION WITHIN INTACT MOUSE BRAIN TISSUE; (C) STUDY HOW M6A MODIFICATIONS IN VARIOUS CELL TYPES WORK COLLECTIVELY AND INTERACTIVELY TO REGULATE BRAIN FUNCTION. IN TOTAL, THIS PROPOSAL WILL LEAD TO (1) A TRANSFORMATIVE TOOLBOX FOR SINGLE-CELL EPITRANSCRIPTOMIC PROFILING WITH BROAD APPLICATIONS IN VARIOUS BIOLOGICAL TISSUES, (2) A SPATIALLY RESOLVED SINGLE-CELL RNA MODIFICATION ATLAS IN THE MOUSE BRAIN, AND (3) NEW SCIENTIFIC UNDERSTANDING ON HOW RNA MODIFICATIONS IMPACT TISSUE FUNCTION. IN THE LONG TERM, WE AIM TO REVEAL NEW PRINCIPLES OF POST-TRANSCRIPTIONAL GENE REGULATION AT SUBCELLULAR AND SINGLE-CELL RESOLUTIONS IN COMPLEX BIOLOGICAL SYSTEMS, AS WELL AS DISCOVER NEW CHEMICAL FINGERPRINTS OF RNAS IN HEALTH AND DISEASE.

QUANTIFYING THE IMPACT OF RARE MUTATIONS ON ADHD

PHENOTYPIC PROFILING OF ASD RISK

NOVEL AAVS ENGINEERED FOR EFFICIENT AND NONINVASIVE CROSS-SPECIES GENE EDITING THROUGHOUT THE CENTRAL NERVOUS SYSTEM

ADVANCED TOOLS FOR HCMI MODEL GENETIC PERTURBATION AND METASTASIS CHARACTERIZATION

CROSS-PLATFORM STRUCTURAL VARIANT DISCOVERY WITH DEEP LEARNING - STRUCTURAL VARIANTS (SV) ARE A MAJOR DRIVER OF THE GENETIC DIVERSITY AND DISEASE IN THE HUMAN GENOME AND THEIR DISCOVERY IS IMPERATIVE TO ADVANCES IN PRECISION MEDICINE AND OUR UNDERSTANDING OF HUMAN GENETICS. DUE TO REVOLUTIONARY BREAKTHROUGHS IN WHOLE-GENOME SEQUENCING TECHNOLOGIES, WE NOW HAVE ACCESS TO GENOMIC DATA AT AN UNPRECEDENTED SCALE AND RESOLUTION. HOWEVER, DESPITE TREMENDOUS EFFORT AND PROGRESS IN SV CALLING METHODOLOGY, GENERAL SV DISCOVERY STILL REMAINS UNSOLVED. EXISTING TECHNIQUES USE HAND-ENGINEERED FEATURES AND HEURISTICS TO MODEL SV CLASSES, RELYING HEAVILY ON DEVELOPER EXPERTISE, WHICH CANNOT SCALE TO THE VAST DIVERSITY OF SV TYPES AND SEQUENCING PLATFORMS NOR FULLY HARNESS ALL THE INFORMATION AVAILABLE IN RAW SEQUENCING DATA. AS A RESULT, THESE METHODS ARE USUALLY TIGHTLY COUPLED TO THE PROPERTIES OF A PARTICULAR SEQUENCING TECHNOLOGY AND OPERATE OPTIMALLY ONLY ON CERTAIN SV TYPES AND SIZES, RENDERING US BLIND TO MANY OTHER CLASSES OF SVS AND THEIR ROLE IN DISEASE. DEEP NEURAL NETWORKS HAVE THE ABILITY TO LEARN COMPLEX ABSTRACTIONS AUTOMATICALLY FROM THE DATA AND HENCE OFFER A PROMISING AVENUE FOR GENERAL SV DISCOVERY. DEEP LEARNING HAS RECENTLY TRANSFORMED THE FIELD OF MACHINE LEARNING AND LED TO REMARKABLE ADVANCES IN SCIENCE AND MEDICINE. IN THIS PROPOSAL WE AIM TO LEVERAGE THE POTENTIAL OF DEEP LEARNING FOR THE PROBLEM OF SV DETECTION. WE LAY OUT HOW TO EFFICIENTLY FORMULATE SV DETECTION AS A DEEP LEARNING TASK, AND PROPOSE THE DEVELOPMENT OF A COMPREHENSIVE FRAMEWORK TO CALL AND GENOTYPE SVS OF DIFFERENT SIZE AND TYPE, INCLUDING COMPLEX AND SUBCLONAL SVS, GIVEN DATA FROM A RANGE OF SEQUENCING PLATFORMS. IN PARTICULAR, WE DEMONSTRATE THAT STATE-OF-THE-ART RESULTS CAN BE OBTAINED USING OUR APPROACH FOR SHORT, LINKED, AND LONG READ DATASETS. IN ORDER TO ENSURE THAT OUR MODELS GENERALIZE ACROSS DIFFERENT DATASETS, AN IMPORTANT GOAL OF OUR PROPOSAL IS ALSO TO ASSEMBLE DIVERSE AND REPRESENTATIVE TRAINING DATA AND PERFORM EXTENSIVE EVALUATION USING PUBLICLY- AVAILABLE MULTI-PLATFORM DATASETS TO ACCURATELY ASSESS MODEL PERFORMANCE. OUR SOFTWARE WILL BE BUILT WITH EXTENSIBILITY AND SCALABILITY IN MIND, AND WILL BE RELEASED, ALONG WITH PRETRAINED MODELS AND CALLSETS, FREELY TO THE COMMUNITY.

HIGH-THROUGHPUT SCREENS FOR CAV3.3 SELECTIVE T-TYPE CALCIUM CHANNEL MODULATORS

CAUSAL REPRESENTATION LEARNING FOR THE SPATIAL ANALYSIS OF TRANSCRIPTOMIC AND IMAGING DATA IN TISSUE CONTEXTS - NIH NEW INNOVATORS AWARD ABSTRACT BY MELDING IMAGING AND GENOMICS IT IS NOW POSSIBLE TO OBTAIN SPATIALLY RESOLVED TRANSCRIPTOMIC DATASETS; HOWEVER, COMPUTATIONAL METHODS FOR ANALYZING SUCH DATASETS HAVE LAGGED BEHIND EXPERIMENTAL DEVELOPMENTS. TO REALIZE THE FULL POTENTIAL OF SPATIAL TRANSCRIPTOMIC (ST) DATA, WE CANNOT RELY ON THE METHODS THAT HAVE BEEN DEVELOPED FOR ANALYZING SINGLE CELL DATA THAT DIVORCE CELLS FROM THEIR MICROENVIRONMENT. AS WITH EXPERIMENTAL DEVELOPMENTS THAT SAW ST BREAKTHROUGHS BY MELDING IMAGING AND SEQUENCING, WE ARGUE THAT THE SAME WILL HOLD TRUE IN THE COMPUTATIONAL DOMAIN, AND, THEREFORE, PROPOSE A FRAMEWORK FOR THE ANALYSIS OF THIS DATA THAT INTEGRATES IMAGING AND SEQUENCING WITH CAUSALITY TO INFER REGULATORY MECHANISMS UNDERLYING SPATIALLY DRIVEN PROCESSES. WE PROPOSE TO ACHIEVE THIS THROUGH AN INNOVATIVE UNIFICATION OF TWO VIBRANT AREAS IN MACHINE LEARNING (ML); REPRESENTATION LEARNING AND CAUSAL INFERENCE. THIS IS A MOMENTOUS TASK SINCE REPRESENTATION LEARNING, ALTHOUGH SUCCESSFUL IN PREDICTIVE TASKS LIKE RECOMMENDER SYSTEMS, DOES NOT GENERALLY ELUCIDATE CAUSAL RELATIONSHIPS. TO OVERCOME THIS, WE WILL USE REPRESENTATION LEARNING TO IDENTIFY CORRELATIONS THAT ARE PRESENT IN ALL DATA MODALITIES AVAILABLE IN ST, AND THEREBY DISCERN SPURIOUS CORRELATIONS FROM CAUSAL ONES USING THE PRINCIPLE OF INVARIANCE. IN ADDITION, WE WILL BUILD ON THREE FUNDAMENTAL CONCEPTS IN ML: - IMAGE INPAINTING: TO IDENTIFY MOTIFS IN TISSUE ARCHITECTURE AS WELL AS ANOMALOUS TISSUE PATCHES - OPTIMAL TRANSPORT: TO INFER TISSUE LINEAGES FROM SNAPSHOTS IN TIME - CAUSAL STRUCTURE DISCOVERY: TO IDENTIFY REGULATORY MODULES & PREDICT THE EFFECT OF PERTURBATIONS THIS UNIFICATION WILL RESULT IN AN ML FRAMEWORK THAT INTEGRATES SPACE, TIME, AND EXPRESSION TO IDENTIFY BIOLOGICAL MECHANISMS UNDERLYING SPATIAL PROCESSES. ALTHOUGH THIS FRAMEWORK WILL BE BROADLY APPLICABLE, IT IS CENTERED ON THREE DISEASE CONTEXTS, WHICH WILL SERVE AS THE FOREGROUND TO TEST AND REFINE OUR METHODS AND FOR WHICH ST DATA HAVE ALREADY BEEN OBTAINED: - INFLAMMATION/FIBROSIS IN THE GUT; TO STUDY CELL RECRUITMENT, MATRIX DEPOSITION, AND CLEARANCE; - ALZHEIMER'S DISEASE; TO STUDY QUESTIONS OF SECRETION AND PROTEIN AGGREGATION; AND - CLASSIC HODGKIN LYMPHOMA; TO STUDY TUMOR-IMMUNE CELL INTERACTIONS & IMMUNOLOGICAL INVASION. UNDERSTANDING THE REGULATORY MECHANISMS OF CELL-CELL COMMUNICATION IN THESE DISEASE CONTEXTS HAS THE POTENTIAL TO GIVE RISE TO NEW THERAPEUTIC TARGETS THAT COULD BE VALIDATED IN PARTNERSHIP WITH OUR EXPERIMENTAL COLLABORATORS AND BENEFIT PATIENTS' LIVES.

CENTER FOR THE COMPREHENSIVE ANALYSIS OF SOMATIC COPY-NUMBER ALTERATIONS IN CANCER

GENOME-WIDE ASSOCIATION STUDIES OF DIABETIC NEPHROPATHY

THE PURPOSE OF THIS COOPERATIVE AGREEMENT IS TO FUND RESEARCH IN SUPPORT TO BTO IN THE AMOUNT OF 750,000 ON CONTRACT HR0011-17-2-0049.

IDENTIFYING DISEASE-RELEVANT CELL TYPES BY INTEGRATING GENETIC AND FUNCTIONAL GENOMICS DATA

CHARACTERIZING GLIOMA HETEROGENEITY WITH NOVEL MULTIPLEXED NANOSCALE IMAGING TECHNOLOGIES

THE AUTISM SEQUENCING CONSORTIUM: AUTISM GENE DISCOVERY IN >50,000 EXOMES

PQB-3: DISCOVERY AND VALIDATION OF THE DRIVING MEDIATORS OF CANCER CACHEXIA

AN IMAGING REPOSITORY FOR THE CEREBROVASCULAR DISEASE KNOWLEDGE PORTAL (ICDKP) - PROJECT ABSTRACT STROKE IS THE WORLD’S SECOND-LEADING CAUSE OF DEATH. BY 2030 AN ESTIMATED 122.4 MILLION PEOPLE WORLD-WIDE WILL BE AFFECTED WHILE STROKE-RELATED MEDICAL COSTS IN THE US ALONE ARE PROJECTED TO EXCEED $180 BILLION. FURTHERMORE, STROKE ITSELF IS ONLY THE MOST ACUTE MANIFESTATION OF CEREBROVASCULAR DISEASE, WHICH ALSO CAUSES CHRONIC PROGRESSIVE DETERIORATION IN BRAIN FUNCTION, INCLUDING VASCULAR CONTRIBUTIONS TO COGNITIVE IMPAIRMENT AND DEMENTIA (VCID). DESPITE A PRESSING NEED FOR NOVEL TREATMENTS, THE PRECISE BIOLOGICAL PATHWAYS THAT LEAD TO BRAIN ISCHEMIA AND HEMORRHAGE, THE RESPONSE OF THE BRAIN TO INJURY, AND THE BRAIN’S CAPACITY FOR RECOVERY REMAIN POORLY UNDERSTOOD. IMAGING PROVIDES CRUCIAL AND QUANTIFIABLE MEASURES OF THESE PROCESSES. WITHOUT IMAGING IT IS IMPOSSIBLE TO CHARACTERIZE THE LOCATION OF THE BRAIN AFFECTED, QUANTITY OF BRAIN TISSUE INVOLVED, OR AMOUNT OF BRAIN SWELLING/EDEMA. FURTHERMORE, SERIAL IMAGES OF THE SAME PATIENT OVER TIME CAPTURE DYNAMIC PHYSIOLOGY SUCH AS INFARCT GROWTH OR HEMORRHAGE GROWTH, WHICH ARE BOTH CRUCIAL TO STUDYING INJURY AND RECOVERY. THE INTERNATIONAL STROKE GENETICS CONSORTIUM (ISGC) WAS FOUNDED TO HARNESS HUMAN GENETICS TO IDENTIFY NOVEL BIOLOGICAL PATHWAYS THAT CAN ULTIMATELY LEAD TO EFFECTIVE TREATMENTS FOR STROKE. INDEED, CANDIDATE DRUG TREATMENTS SUPPORTED BY EVIDENCE FROM HUMAN GENETICS ARE SUBSTANTIALLY MORE LIKELY TO ACHIEVE FDA APPROVAL. THE ISGC’S COMMITMENT TO LARGE HARMONIZED DATA SETS, OPEN DATA SHARING AND COLLABORATIVE CULTURE HAS LED TO THE DISCOVERY OF ALL OF THE ROBUSTLY SUPPORTED RISK GENES FOR COMMON FORMS OF STROKE TO DATE, MANY OF WHICH ARE ALREADY BEING STUDIED AS THE BASIS FOR NOVEL TREATMENTS. IN 2017 THE ISGC LAUNCHED THE CEREBROVASCULAR DISEASE KNOWLEDGE PORTAL (CDKP) (R24NS092983) TO SHARE GENETIC DATA FREELY WITH THE RESEARCH COMMUNITY IN A MANNER ADHERENT TO FAIR DATA PRINCIPLES. A MAJOR LIMITATION OF THIS RESOURCE IS THE ABSENCE OF BRAIN IMAGES. FOR THIS PROPOSED BIOMEDICAL DATA REPOSITORY COOPERATIVE AGREEMENT (U24) WE WILL AGGREGATE BRAIN IMAGING AND GENETIC DATA ACCORDING TO FAIR DATA PRINCIPLES FROM AN ESTIMATED 30,000 INDIVIDUALS WITH STROKE AND SHARE THESE DATA ACCORDING TO FAIR DATA PRINCIPLES. BUILDING ON THE ESTABLISHED STROKE NEURO-IMAGING PHENOTYPE REPOSITORY (SNIPR) AT WASHINGTON UNIVERSITY, OUR PROGRAM WILL TRANSFORM THE CDKP INTO THE IMAGING CDKP (ICDKP), PROVIDING THE GLOBAL COMMUNITY WITH A SINGLE FAIR ACCESS POINT TO GENETIC, PHENOTYPIC, AND IMAGING DATA. MILESTONE-DRIVEN AIMS WILL: 1) AGGREGATE HIGH VALUE STROKE IMAGING DATASETS FROM ACROSS THE INTERNATIONAL INVESTIGATOR COMMUNITY; 2) IMPLEMENT FAIR DATA SHARING PRACTICES AND PROCEDURES; 3) ENGAGE THE RESEARCH COMMUNITY WITH REPOSITORY. OUR TEAM, LED BY THE PIS FOR CDKP AND SNIPR IS WELL-SITUATED TO EXECUTE THIS PROJECT GIVEN THAT WE HAVE DEVELOPED THIS PROPOSAL IN CONSULTATION WITH THE INTERNATIONAL STROKE RESEARCH COMMUNITY TO ENSURE BROAD SUPPORT AND MAXIMUM RESPONSE TO THEIR ANTICIPATED NEEDS.

ACCELERATED PROTEIN SIGNALING SIGNATURES

POTENTIATE CAV3.3 TO TREAT COGNITIVE DEFICITS ASSOCIATED WITH IMPAIRED SLEEP SPINDLE - PROJECT SUMMARY/ABSTRACT SLEEP SPINDLE IS A HALLMARK BRAIN OSCILLATION OCCURRING IN NREM (NON-RAPID EYE MOVEMENT) SLEEP THAT REFLECT INTRINSIC THALAMOCORTICAL OFFLINE PROCESSING. SPINDLES ARE WAXING AND WANING BURSTS OF 12-15 HZ OSCILLATIONS THAT ORIGINATE IN THALAMIC RETICULAR NUCLEUS (TRN) WHEN ITS NEURONS UNDERGO BURSTING FIRING, AND TRN- THALAMIC RELAY NETWORK OSCILLATES AT SPINDLE FREQUENCY THROUGH THE RECIPROCAL CONNECTION. SLEEP SPINDLES ARE MARKEDLY REDUCED IN SCHIZOPHRENIA PATIENTS AS WELL AS IN ALZHEIMER PATIENTS, TWO VERY DIFFERENT CNS DISORDERS YET BOTH DEMONSTRATED SIGNIFICANT ALTERED SLEEP SPINDLE PROPERTIES ASSOCIATED WITH COGNITIVE IMPAIRMENT. WE HYPOTHESIZE THAT HYPOFUNCTIONAL TRN MAY UNDERLIE THE RISK/SYMPTOMS OF THESE TWO DEVASTING ILLNESSES. CACNA1I IS ENCODE THE Α1 FUNCTIONAL CORE OF THE CAV3.3 VOLTAGE-ˇGATED CALCIUM CHANNELS, AND ITS EXPRESSION IS ENRICHED IN TRN. WE HAVE DISCOVERED TWO NOVEL CHEMICAL SCAFFOLDS THAT POTENTIATE CAV3.3 FUNCTION, AND THIS PROPOSAL AIMS TO DELIVER IN VIVO READY CAV3.3 POSITIVE MODULATORS. THIS MULTIDISCIPLINARY EFFORT IS EXPECTED TO YIELD A PANEL OF INCISIVE PHARMACOLOGICAL REAGENTS TO ENHANCE CAV3.3 FUNCTION IN THE BRAIN AND NOMINATE CANDIDATES FOR PRECLINICAL DEVELOPMENT FOR TREATING COGNITIVE IMPAIRMENTS IN SCHIZOPHRENIA PATIENTS.

INTERROGATING THE UBIQUITIN PATHWAY TO UNDERSTAND AND TREAT CYTOKINE-INDUCED BETA-CELL DEATH IN TYPE 1 DIABETES - PROJECT SUMMARY THE LOSS OF INSULIN-PRODUCING BETA CELLS IN THE PANCREAS RESULTS IN AN ABSOLUTE REQUIREMENT FOR INJECTED INSULIN, CAUSING SIGNIFICANT RISKS OF MORTALITY FROM HYPOGLYCEMIA AND MORBIDITY FROM DIABETIC COMPLICATIONS IN PERIPHERAL NERVES, THE RETINA, THE HEART, AND THE KIDNEY. A KEY GOAL OF EFFORTS TO TREAT T1D IS TO STOP THIS CELLULAR ATTACK, EITHER BY HALTING THE IMMUNE MIS-RECOGNITION OF BETA CELLS OR BY PROTECTING BETA CELLS FROM CELL DEATH. HOWEVER, A CRITICAL BARRIER TO PROGRESS IN THE FIELD IS A LACK OF COMPLETE UNDERSTANDING OF THE CELLULAR EVENTS IN THE ISLET THAT CONTRIBUTE TO THE LOSS OF BETA-CELL MASS. USING A PHENOTYPIC SCREENING APPROACH, WE DISCOVERED BRD0476, A COMPOUND THAT IS SELECTIVELY ACTIVE AGAINST CYTOKINE-MEDIATED APOPTOSIS. FURTHER STUDY OF THIS COMPOUND REVEALED THAT IT BINDS THE DEUBIQUITINASE USP9X TO HALT JAK2 AND STAT1 SIGNALING IN RESPONSE TO IFN. WE DETERMINED THAT JAK2 CAN BE RENDERED SIGNALING INCOMPETENT BY UBIQUITINATION, AND THAT BY MODULATING USP9X, WE CAN TIP THE BALANCE TOWARD REDUCED JAK2 KINASE ACTIVITY, EVEN IN THE PRESENCE OF IFN. THESE RESULTS POINT TO AN EMERGING ROLE FOR UBIQUITINATION IN REGULATING BETA-CELL APOPTOSIS IN T1D, AND SUGGEST THAT A GREATER UNDERSTANDING OF THIS PROCESS (AND ITS POTENTIAL DYSREGULATION) IN THE EARLY STAGES OF T1D DEVELOPMENT COULD LEAD TO 1) THE ABILITY TO IDENTIFY AT-RISK INDIVIDUALS, AND 2) NOVEL THERAPEUTIC STRATEGIES TO PRESERVE BETA-CELL MASS IN EARLY-STAGE T1D. USING OUR PROBE BRD0476 AND CHEMICAL BIOLOGY TOOLS NOT PREVIOUSLY APPLIED TO ISLET BIOLOGY, WE WILL IMPROVE OUR UNDERSTANDING OF THE ROLE OF USP9X IN BETA-CELL SURVIVAL IN VITRO AND IN VIVO THROUGH THE FOLLOWING AIMS: IN AIM 1, WE WILL CHARACTERIZE THE MODE OF JAK2 INHIBITION BY USP9X IN HUMAN ISLETS. IN AIM 2, WE WILL ASSESS EFFECTS OF INHIBITING USP9X-JAK2 (WITH BRD0476) ON DEVELOPMENT AND PROGRESSION OF AUTOIMMUNE DIABETES IN A MOUSE MODEL OF TYPE 1 DIABETES. IN AIM 3, WE WILL PROFILE DEUBIQUITINASE (DUB) EXPRESSION AND ACTIVITY IN HUMAN ISLETS DURING EARLY T1D DEVELOPMENT, USING ACTIVITY-BASED PROTEIN PROFILING (ABPP) AND GLOBAL UBIQUITOME MEASUREMENTS. THE SUCCESSFUL OUTCOMES OF THIS PROPOSAL ARE 1) A GREATER UNDERSTANDING OF MECHANISMS TO PROMOTE BETA-CELL SURVIVAL IN EARLY T1D, AND 2) A CHEMICAL PROBE TO PROVIDE TRANSLATIONAL PROOF-OF-CONCEPT. THIS PROJECT WILL SET THE STAGE FOR DEVELOPING A BIOMARKER OF EARLY-STAGE T1D DEVELOPMENT, AS WELL AS ADVANCED THERAPEUTIC STRATEGIES FOR PREVENTING BETA-CELL APOPTOSIS IN EARLY-STAGE T1D, REPRESENTING A POTENTIALLY CURATIVE APPROACH.

FACTORS REGULATING STRENGTH AND DURATION OF STING SIGNALING - DNA SENSING BY THE CGAS-STING PATHWAY IS ESSENTIAL FOR RECOGNIZING PATHOGENS, TUMOR CELLS, AND MITOCHONDRIAL OR NUCLEAR DNA UNDER CERTAIN CONDITIONS, LEADING TO HOMEOSTATIC RESPONSES, SUCH AS IMMUNE CONTROL OF PATHOGENS AND CANCER. UNDER SOME CONDITIONS, SUCH AS LOSS OF THE NUCLEASE TREX1, EXCESS DNA TRIGGERS CGAS- STING AND CAUSES A CHRONIC INFLAMMATION (E.G., AICARDI GOUTIERÈS SYNDROME, OR A CONSTITUTIVELY ACTIVE STING ALLELE, CAUSES SAVI, AN INHERITED VASCULOPATHY). UPON ACTIVATION BY ITS LIGAND, CGAMP, STING TRANSLOCATES FROM ER GOLGI ENDOSOMES WHERE IT SIGNALS THROUGH TBK1 AND IRF3 TO ACTIVATE INTERFERONS, AND IS THEN DEGRADED VIA LYSOSOMES. THESE TRAFFICKING PATTERNS ARE CENTRAL TO STING’S FUNCTION, AND YET WE LACK KNOWLEDGE OF MANY OF THE GENES REGULATING THESE PROCESSES. TO IDENTIFY REGULATORS OF STING ACTIVITY, TRAFFICKING AND DEGRADATION, WE PERFORMED TWO GENOME-WIDE CRISPR KNOCKOUT SCREENS, AS WELL AS A PROXIMITY-LIGATION MEDIATED PROTEOMIC ANALYSIS, AND A FOCUSED CRISPR SCREEN. STUDYING THE DOZENS OF FACTORS FOUND, WE MADE TWO SIGNIFICANT DISCOVERIES THAT PROVIDE THE BASIS FOR THIS PROPOSAL. FIRST, WE FOUND THAT ESCRT-DEPENDENT ENDOSOMAL MICROAUTOPHAGY REQUIRES RECOGNITION OF UBIQUITINATED STING ON ENDOSOMES, AND IS CRITICAL FOR STING DEGRADATION, AUTOPHAGOSOME SEALING AND SIGNALING REGULATION. DISRUPTION OF THIS PATHWAY BY A PATHOGENIC ESCRT SUBUNIT MUTANT (FOUND IN A HUMAN DISEASE) LEADS TO CONSTITUTIVE STING SIGNALING AT STEADY STATE. SECOND, WE UNCOVERED A NOVEL INTERACTION OF ER-LOCALIZED STING WITH ENDOSOMAL PROTEIN DNAJC13, LEADING TO RESTRICTION OF STING ER EXIT AND ACTIVATION. LOSS OF DNAJC13 OR DISRUPTION OF ER-ENDOSOME CONTACT SITES DRAMATICALLY BOOSTS STING ACTIVITY. HAVING DEFINED A MODEL OF STING UBIQUITINATION CONTROLLING AUTOPHAGY RESOLUTION, AND DNAJC13 RESTRICTING STING ER EXIT, WE PROPOSE TO FURTHER OUR UNDERSTANDING OF THIS PATHWAY BY STUDYING THE MECHANISMS UNDERLYING THESE PROCESSES, INCLUDING IDENTIFYING E3 UBIQUITIN LIGASES THAT MODIFY STING AND INDUCE ESCRT-DEPENDENT AUTOPHAGY, AND DETERMINING THE IMPACT OF STING-INDUCED ENDOSOMAL MICROAUTOPHAGY ON VIRAL INFECTIONS. WE WILL ALSO DETERMINE HOW DNAJC13 BLOCKS STING ACTIVITY BY ALTERING STING TRAFFICKING. WE WILL TEST THE ROLES OF DNAJC13 AND ER-ENDOSOMAL CONTACTS IN LIMITING STING ACTIVATION BY RESTRICTING STING ER EXIT TO THE TGN. TO DEFINE THE BIOCHEMICAL MECHANISMS OF STING INTERACTIONS, WE WILL USE DEEP MUTATIONAL SCANNING TO FIND MOTIFS ON STING THAT ARE RESPONSIBLE FOR INTERACTIONS WITH DNAJC13 AND FOR THE EFFECTS OF DNAJC13 ON STING TRAFFICKING, AS WELL AS MOTIFS THAT CONTROL OTHER ASPECTS OF STING LOCALIZATION AND SIGNALING. FINALLY, WE WILL STUDY HOW MUTATIONS IN GENES INVOLVED IN HUMAN PATHOLOGIES THAT REGULATE STING TRAFFICKING, IMPACT INFLAMMATION AND DEATH OF CELLS OF THE CENTRAL NERVOUS SYSTEM. A BETTER UNDERSTANDING OF STING TRAFFICKING, DEGRADATION AND SIGNALING WILL HELP US DEVELOP THERAPEUTIC APPROACHES TO DAMPEN AUTOIMMUNITY OR BOOST PATHOGEN AND TUMOR IMMUNITY.

1/3 AKILI: PHENOTYPIC AND GENETIC CHARACTERIZATION OF ADHD IN KENYA AND SOUTH AFRICA - PROJECT SUMMARY ADHD IS A COMMON NEURODEVELOPMENTAL DISORDER THAT INCLUDES ATTENTION DIFFICULTY, IMPULSIVITY, AND HYPERACTIVITY. THE DIAGNOSIS IS ASSOCIATED WITH MANY CHALLENGES TO EDUCATIONAL, OCCUPATIONAL, AND HEALTH OUTCOMES, PARTICULARLY WHEN UNTREATED. GENETIC STUDIES OF ADHD HAVE THE POTENTIAL TO CLARIFY THE DISORDER’S BIOLOGICAL UNDERPINNINGS, ITS HETEROGENEITY, AND ITS RELATIONSHIP TO OTHER NEUROPSYCHIATRIC DIAGNOSES. HOWEVER, GENETIC RESEARCH INTO ADHD LAGS IN TERMS OF: (1) SAMPLE SIZE, (2) ANCESTRAL DIVERSITY, AND (3) CONSIDERATION OF PHENOTYPIC HETEROGENEITY. AKILI IS DESIGNED TO ADDRESS ALL THREE OF THESE CRITICAL GAPS. AKILI (THE SWAHILI TERM FOR “MIND”) WILL ENROLL 6,000 CHILDREN IN KENYA AND SOUTH AFRICA – 4,000 WITH A CONFIRMED DIAGNOSIS OF ADHD AND 2,000 AGE- AND ANCESTRY-MATCHED CONTROLS. ALL PARTICIPANTS WILL COMPLETE A DETAILED BEHAVIORAL, COGNITIVE, AND MEDICAL PHENOTYPING BATTERY, AND PROVIDE A DNA SAMPLE. WE WILL GENETICALLY CHARACTERIZE ALL 6,000 PARTICIPANTS USING EXOME SEQUENCING AND GENOME-WIDE GENOTYPING, AND MAKE ALL AKILI DATA AND MATERIALS PUBLICLY AVAILABLE THROUGH THE NIMH. AKILI DATA WILL NEARLY DOUBLE THE NUMBER OF ADHD CASES AVAILABLE FOR EXOME SEQUENCING ANALYSIS AND PROVIDE A 20% ADDITION TO THE CURRENT PGC-ADHD GWAS ACTIVITY. IT WILL BE BY FAR THE LARGEST CONTRIBUTOR OF DIVERSE ANCESTRY DATA TO EITHER ANALYSIS. AKILI WILL GENERATE A RESEARCH RESOURCE OF INTERNATIONAL VALUE, AND PROVIDE THE FIRST LARGE-SCALE CHARACTERIZATION OF ADHD IN THE AFRICAN CONTEXT.

EXPANDING THE SCOPE OF BASE EDITING

COMPREHENSIVE ANALYSIS OF POINT MUTATIONS IN CANCER - PROJECT SUMMARY PRECISION MEDICINE IN CANCER, A DISEASE OF THE GENOME, RELIES ON A DEEP AND COMPREHENSIVE UNDERSTANDING OF THE GENETIC MUTATIONS AND ABNORMALITIES THAT ACCUMULATE IN NORMAL CELLS AND DRIVE TRANSFORMATION TO CANCER. THE GETZ AND RHEINBAY LABS HAVE EXPERTISE IN THE DISCOVERY AND CHARACTERIZATION OF POINT MUTATIONS THROUGH RIGOROUS CANCER GENOME ANALYSIS. IN THIS PROPOSAL, WE AIM TO CREATE A GENOME DATA ANALYSIS CENTER (GDAC) FOCUSED ON EMPLOYING OUR EXISTING TOOLS TO ROBUSTLY AND COMPREHENSIVELY CHARACTERIZE POINT MUTATIONS (SINGLE-NUCLEOTIDE VARIATIONS AND SMALL INDELS) ACROSS THE ENTIRE CANCER GENOME TO ADDRESS SCIENTIFIC QUESTIONS RELATED TO BIOLOGICAL UNDERPINNINGS OF CANCER THAT ARISE IN EACH PROJECT WE ARE ASSIGNED. WE ALSO HAVE THE FLEXIBILITY TO ADAPT OUR TOOLS AS DEEMED NECESSARY BY THE UNIQUE NEEDS OF EACH PROJECT. SPECIFICALLY, WE PLAN TO INTEGRATE AND CHARACTERIZE MUTATIONS, MUTATIONAL SIGNATURES, AND OTHER DATA TYPES TO COMPREHENSIVELY DISCOVER CANCER DRIVERS IN CODING AND NON-CODING REGIONS OF THE GENOME, INCLUDING THE OFTEN IGNORED MORE DIFFICULT-TO-ANALYZE REGIONS OF THE GENOME. WE WILL DO THIS BY INCORPORATING METHODS TO DETERMINE DNA METHYLATION SIGNATURES AS WELL AS BY INTERROGATING THE EPIGENOME IN BOTH CODING AND NON-CODING REGIONS OF THE GENOME. WE FURTHER PLAN TO ADVANCE OUR ABILITY TO DETERMINE TRAJECTORIES OF TUMOR EVOLUTION AND HETEROGENEITY BY ADAPTING OUR PHYLOGICNDT SUITE OF TOOLS TO ANALYZE THE EVOLUTION, SUBCLONAL HETEROGENEITY, AND TIMING AND ORDER OF MUTATIONAL EVENTS FROM MULTIPLE SAMPLES (E.G., SAMPLES ACQUIRED LONGITUDINALLY OR SPATIALLY) FROM THE SAME PATIENT, OR EVEN FROM CELL-FREE DNA (CFDNA) FROM NON-INVASIVE BLOOD BIOPSY. IN THE INTEREST OF ADVANCING THE GDC’S GOAL OF IMPROVING PERSONALIZED MEDICINE, WE TEAMED WITH EXPERT CLINICIANS AND TRANSLATIONAL SCIENTISTS, DR. KEITH FLAHERTY AND DR. KIRSTEN KÜBLER, THAT WILL INTERPRET OUR FINDINGS, ASSOCIATE THEM WITH CLINICAL DATA AND DIRECT THEM TOWARDS CLINICAL IMPACT. THEY WILL ALSO ENHANCE OUR TOOLS FOR IDENTIFYING THE TISSUE- AND CELL-OF-ORIGIN OF CANCERS TO NOT ONLY BETTER UNDERSTAND THE UNDERLYING MECHANISMS OF TRANSFORMATION IN A PARTICULAR CANCER TYPE OR SUBTYPE BUT ALSO PROVIDE MORE EFFECTIVE THERAPEUTIC TARGETS. MOREOVER, OUR FINAL AIM IS TO PERFORM PATIENT-SPECIFIC ANALYSIS TO IMPROVE AND ENABLE PRECISION MEDICINE, ESPECIALLY IN PATIENTS WHOSE TUMORS DO NOT HAVE ANY IDENTIFIED ACTIONABLE DRIVER EVENTS. HERE, WE WILL EMPLOY MACHINE LEARNING TECHNIQUES TO BUILD PREDICTIVE MODELS OF THERAPEUTIC VULNERABILITIES. OVERALL, WE OFFER PRIMARY COMPETENCIES IN DNA POINT MUTATION CHARACTERIZATION, ANALYSIS OF CFDNA, AND DETERMINATION OF MUTATIONAL SIGNATURES TO THE GDAN. WE ALSO BRING ADDED VALUE WITH SECONDARY COMPETENCIES IN METHYLATION ANALYSIS (IN THE CONTEXT OF MUTATIONAL SIGNATURES), MRNA ANALYSIS, SINGLE-CELL RNA SEQUENCING, AND PATHWAY/INTEGRATIVE DATA ANALYSIS. BRINGING OUR EXTENSIVE EXPERTISE TO THE VARIOUS NEWLY ASSEMBLED ANALYSIS WORKING GROUPS AND COLLABORATING WITH OTHER GDACS WITHIN THE GDAN CAN HELP TO ANSWER OUTSTANDING QUESTIONS IN CANCER WITH THE ULTIMATE GOAL OF IMPROVING DIAGNOSIS, PROGNOSIS, AND TREATMENT FOR EVERY CANCER PATIENT.

PRECLINICAL DEVELOPMENT OF A NOVEL ANTIMALARIAL AGENT

LOW-INPUT MULTIPLEX ISOLATION AND PROFILING OF HUMAN IMMUNE CELL SUBSETS USING AN INTEGRATED MICROFLUIDICS DEVICE

CHARACTERIZATION OF STRUCTURE-FUNCTION RELATIONSHIPS IN DISTINCT THALAMIC RETICULAR NUCLEUS NETWORKS - PROJECT SUMMARY NEARLY ALL SENSORY INFORMATION DESTINED FOR THE NEOCORTEX IS RELAYED THROUGH THE THALAMUS. THE THALAMIC RETICULAR NUCLEUS (TRN), A THIN SHELL OF GABAERGIC NEURONS SURROUNDING THE DORSAL THALAMUS, RECEIVES BOTH COLLATERALS FROM CORTICO-THALAMIC AND THALAMO-CORTICAL GLUTAMATERGIC INPUTS, BUT EXERTS ITS UNIDIRECTIONAL POWERFUL INHIBITION ONLY TO THE THALAMUS. TRN THUS IS CONSIDERED AS A MASTER CONTROLLER OF THALAMO-CORTICAL CIRCUITS REGULATING THE FLOW OF THE INFORMATION BETWEEN THALAMUS AND NEOCORTEX. PERTURBED TRN FUNCTION MAY UNDERLIE BEHAVIORAL DEFICITS IN DISORDERS RANGING FROM SCHIZOPHRENIA, ADHD AND AUTISM. ALTHOUGH THE IMPORTANCE OF THE TRN HAS LONG BEEN RECOGNIZED, OUR KNOWLEDGE OF ITS MOLECULAR IDENTITY OF CELL TYPES, THEIR ORGANIZATION AND THEIR FUNCTIONAL PROPERTIES HAS LAGGED BEHIND THAT OF THE THALAMOCORTICAL CIRCUITS THEY CONTROL. THE PAUCITY OF SUCH KNOWLEDGE HAS LIMITED OUR ABILITY TO DETERMINE EXACTLY HOW TRN CIRCUITRY CONTRIBUTES TO VARIOUS BRAIN FUNCTIONS, A PREREQUISITE FOR DETERMINING HOW IT MALFUNCTIONS IN DISEASES AND HOW ITS CIRCUITRY CAN BE LEVERAGED FOR DIAGNOSTIC AND THERAPEUTIC PURPOSES. OUR MOST RECENT WORK HAS FILLED THIS CRITICAL GAP IN KNOWLEDGE. BY USING SINGLE NUCLEUS RNASEQ, FOR THE FIRST TIME WE HAVE DISCOVERED THAT TRN NEURONS CAN BE DISSOCIATED INTO TWO MAJOR SUBTYPES WITH DISTINCT TRANSCRIPTOMIC PROFILES, ANATOMICAL LOCALIZATIONS, ELECTROPHYSIOLOGICAL PROPERTIES AND THALAMIC CONNECTIVITY. ONE SUBTYPE, LOCATED IN THE “CORE” REGION OF THE TRN AND CAN BE MARKED BY THE EXPRESSION OF THE SPP1 GENE, TARGETS FIRST-ORDER SENSORY THALAMIC NUCLEI, AND THE OTHER, LOCATED IN THE “SHELL” REGION OF THE TRN AND MARKED BY THE EXPRESSION OF ECEL1 GENE, TARGETS HIGHER-ORDER ONES. WE HAVE GENERATED TRANSGENIC MICE EXPRESSING CRE RECOMBINASE IN EACH OF THESE TWO POPULATIONS INDIVIDUALLY. THIS PROPOSAL AIMS TO USE THESE NEW KNOWLEDGE AND GENETIC TOOLS TO PROVIDE A COMPREHENSIVE MAP OF TRN CELL- TYPE SPECIFIC CONNECTIVITY PATTERNS, TRN SUBCIRCUIT ELECTROPHYSIOLOGICAL PROPERTIES AND SYNAPTIC MECHANISM, AND HOW ABNORMAL FUNCTION OF DISTINCT TRN SUBCIRCUITS CONTRIBUTE TO BEHAVIORAL DEFICITS OF AUTISM SPECTRUM DISORDER (ASD) USING A NOVEL MONOGENIC FORM OF ASD, PTCHD1 DELETION MOUSE MODEL.

2/4 THE AUTISM SEQUENCING CONSORTIUM: DISCOVERING AUTISM RISK GENES AND HOW THEY IMPACT CORE FEATURES OF THE DISORDER - PROJECT SUMMARY/ABSTRACT THE PAST DECADE HAS SEEN OUTSTANDING ADVANCES IN THE GENETICS OF AUTISM SPECTRUM DISORDER (ASD). MOST OF THIS PROGRESS HAS OCCURRED BY THE STUDY OF RARE GENETIC VARIATION, ESPECIALLY DE NOVO VARIATION, WITH THE AUTISM SEQUENCING CONSORTIUM (ASC) PLAYING A CENTRAL ROLE. THE ASC REPRESENTS A COORDINATED INTERNATIONAL EFFORT TO IDENTIFY ASD RISK GENES. IN OUR MOST RECENT, UNPUBLISHED, ANALYSES OF 72,410 INDIVIDUALS FROM ASD FAMILIES, WE IDENTIFIED 185 GENES ASSOCIATED WITH RISK (FDR < 0.05). SOME OF THESE GENES HAVE BEEN LINKED TO A BROAD ARRAY OF DEVELOPMENTAL DISORDERS, WHILE OTHERS HAVE NOT. BASED ON THESE RESULTS, WE POSIT THAT SOME RISK GENES ALTER THE CORE FEATURES OF ASD, WHILE CREATING FEWER PERTURBATIONS TO OTHER FEATURES OF DEVELOPMENT: DISCOVERY OF SUCH GENES WILL PROVIDE DEEPER INSIGHTS INTO PATHWAYS DISRUPTED IN ASD. WE WILL BUILD ON THIS PROGRESS BY ANALYSIS OF SEQUENCE DATA FROM THREE RESOURCES: ASD SUBJECTS AND FAMILIES; SUBJECTS WITH OTHER DEVELOPMENTAL AND NEURO- PSYCHIATRIC DISORDERS; AND SUBJECTS FROM POPULATION SAMPLES. WE PLAN NEW RESEARCH FOCUSING ON INTERPRETATION OF RARE VARIATION, INCLUDING SINGLE NUCLEOTIDE VARIATION (SNV), INDELS, AND COPY NUMBER VARIATION (CNV). OUR KEY TARGETS ARE INHERITED VARIANTS, INCLUDING X-LINKED INHERITED VARIANTS, WHICH TO DATE HAVE SHOWN VERY LITTLE SIGNAL, AND MISSENSE VARIANTS, FOR WHICH SIGNAL HAS BEEN CONFINED TO HIGHLY CONSERVED SUBSTITUTIONS. WE ANTICIPATE DOUBLING THE NUMBER OF ASD GENES DISCOVERED, ~ 400, BY INCREASING THE NUMBER OF FAMILIES ANALYZED AND BY REFINED METHODS TO INTERPRET INHERITED AND MISSENSE VARIATION. AND, IN PARALLEL, WE EXPECT TO RESOLVE CRITICAL AS- PECTS OF ASD GENETIC ARCHITECTURE AND TO UNVEIL KEY ASPECTS OF WHAT MAKES ASD AND ITS CORE FEATURES – SOCIAL DEFICITS AND RESTRICTIVE AND REPETITIVE BEHAVIORS – DIFFERENT FROM OTHER NEURODEVELOPMENTAL DISORDERS. TO DISCOVER ASD RISK GENES WITH A DISTINCT EFFECT ON ASD, WE HAVE THE FOLLOWING SPECIFIC AIMS: 1) TO AMALGAMATE EXISTING AND EMERGING WHOLE EXOME AND WHOLE GENOME SEQUENCE DATA; 2) TO DEVELOP NEW ANALYTICAL METHODS AND ANALYZE THE ACCUMULATED SEQUENCE DATA; AND, 3) TO CONTRAST ASD AND OTHER NEURODEVELOPMENTAL DISORDER RISK GENES, EXAMINING DEVELOPMENTAL PROFILES, CELL TYPES IMPLICATED, AND WHETHER VARIANTS IN THE SAME GENE DIFFER IN HOW THEY AFFECT RISK FOR ASD AND OTHER NEURODEVELOPMENTAL AND PSYCHIATRIC DISORDERS. WITH THIS NEW RESEARCH WE WILL ACCELERATE OUR OVERALL OBJECTIVE, WHICH IS THE IDENTIFICATION OF ASD GENES, THEREBY FACILITATING OUR LONG- TERM GOAL OF BUILDING THE FOUNDATION FROM WHICH THERAPEUTIC TARGETS FOR ASD EMERGE. OUR RATIONALE IS THAT THE IDENTIFICATION OF GENES CONFERRING SIGNIFICANT RISK TO ASD AND ASSOCIATED NEURODEVELOPMENTAL DISORDERS CAN FORM THE BASIS OF STUDIES TO UNDERSTAND PATHOGENESIS, AS WELL AS THE BASIS FOR NOVEL THERAPIES. OUR CENTRAL HYPOTHESIS – FORMULATED BASED ON RESULTS OVER THE PAST DECADE – IS THAT RARE AND COMMON VARIATION CONTRIBUTES ADDITIVELY TO RISK FOR ASD, BUT ONLY CERTAIN RARE VARIANTS CONFER SUBSTANTIAL RISK. THE RESEARCH PROPOSED IS INNOVATIVE, IN OUR OPINION, BECAUSE IT USES GROUNDBREAKING AND NOVEL STATISTICAL METHODS FOR IDENTIFYING RISK VARIANTS FOR ASD.

AN INTEGRATED MULTIPLEXED GENOMIC ASSAY FOR LOW INPUT CLINICAL SAMPLES1

AUTOPHAGY GENES AND THE MICROBIOME IN CROHN'S DISEASE

CONNECTIVITY MAP 100K

CENTER FOR THE COMPREHENSIVE ANALYSIS OF CANCER SOMATIC COPY-NUMBER ALTERATIONS, REARRANGEMENTS, AND LONG-READ SEQUENCING DATA - ABSTRACT WE PROPOSE TO CONTINUE OUR ESTABLISHED GENOMICS DATA ANALYSIS CENTER FOR THE ANALYSIS OF STRUCTURAL VARIANTS IN CANCER, INCLUDING SOMATIC COPY-NUMBER ALTERATIONS (SCNAS) AND REARRANGEMENTS, ADDRESSING THE COPY NUMBER COMPETENCY REQUIRED BY GDAN. WE ALSO ADD CAPABILITIES TO ANALYZE LONG- AND LINKED-READ DATA, ADDRESSING A SECOND COMPETENCY. WE LED SCNA ANALYSES THROUGHOUT THE CANCER GENOME ATLAS (TCGA) AND THE FIRST ITERATION OF THE GENOMICS DATA ANALYSIS NETWORK (GDAN). WE ALSO CO-LED THE STRUCTURAL VARIATIONS WORKING GROUP OF THE INTERNATIONAL CANCER GENOME CONSORTIUM PAN-CANCER ANALYSIS OF WHOLE GENOMES (PCAWG). FOR THESE EFFORTS, WE HAVE DEVELOPED A LARGE SUITE OF TOOLS AND DEEP EXPERTISE COVERING ALL ASPECTS OF ANALYSIS OF SVS IN CANCER. SPECIFICALLY, WE WILL ACCOMPLISH FIVE AIMS: IN AIMS 1 AND 2, WE WILL DETERMINE SCNA AND REARRANGEMENT PROFILES FROM EITHER SHORT-READ (WHOLE EXOME OR WHOLE GENOME) OR LONG-/LINKED-READ SEQUENCING DATA, AND DETERMINE GERMLINE GENOTYPES, PARENTAL HAPLOTYPES, AND ANCESTRY GROUPS. THE HAPLOTYPE INFORMATION WILL BE USED TO IMPROVE OUR COPY-NUMBER RESOLUTION. IN AIM 3, WE RECONSTRUCT THE TUMOR GENOME AND ITS EVOLUTIONARY HISTORY. WE EVALUATE SAMPLE HETEROGENEITY INCLUDING TUMOR PURITY, PLOIDY, AND SUBCLONAL ALTERATIONS, AND PHASE REARRANGEMENTS TO HOMOLOGOUS CHROMOSOMES—DETERMINING SOMATIC DISTANCES BETWEEN ALL PAIRS OF LOCI. USING THESE DATA, WE DETERMINE MECHANISMS OF DNA DAMAGE AND REPAIR AND INFER THE EVENTS THAT OCCURRED OVER TUMOR EVOLUTION. IN AIM 4, WE INTEGRATE DATA ACROSS SAMPLES TO IDENTIFY SVS AND GENOMIC LOCI THAT IMPACT TUMOR EVOLUTION, DETECT ASSOCIATIONS WITH OTHER MOLECULAR AND CLINICAL FEATURES, AND EVALUATE POTENTIAL SCNA-DETERMINED SUBCLASSES. MOREOVER, WE PERFORM ASSOCIATION AND ADMIXTURE ANALYSES WITH THE GERMLINE GENOTYPES DETECTED IN AIM 1. IN AIM 5, WE INDICATE WAYS IN WHICH WE WILL IMMERSE OURSELVES WITHIN THE GDAN AND DISSEMINATE OUR ANALYSIS RESULTS BOTH WITHIN THE GDAN AND TO THE WIDER COMMUNITY. WITHIN THIS, WE OFFER SECONDARY COMPETENCIES IN SINGLE-CELL AND CIRCULATING DNA ANALYSIS. OUR GDAC WILL PROVIDE A COMPREHENSIVE ANALYSIS OF THE ROLES OF STRUCTURAL VARIATIONS IN CANCER DEVELOPMENT AND PROGRESSION THROUGH TREATMENT AMONG GDAN SAMPLES. WE WILL ALSO OPTIMIZE INTERACTIONS WITH THE WIDER GDAN AND SCIENTIFIC COMMUNITY TO MAKE MAXIMAL USE OF THESE DATA.

BIOMARKERS AND MECHANISMS OF PRP MISFOLDING, MUTATION, AND DEFICIENCY - PROJECT SUMMARY PRION DISEASE IS AN INCURABLE AND UNIFORMLY FATAL NEURODEGENERATIVE DISEASE OF HUMANS AND OTHER MAMMALS. PRP LOWERING IS A PHARMACOLOGICALLY VALIDATED THERAPEUTIC STRATEGY FOR PRION DISEASE, AND ANTISENSE OLIGONUCLEOTIDES (ASOS) WITH THIS MECHANISM OF ACTION ARE CURRENTLY ADVANCING TOWARD THE CLINIC. THE PROMISE OF THIS PROGRESS, AND INDEED OUR ABILITY TO ADVANCE ANY THERAPY FOR PRION DISEASE, WILL CRITICALLY DEPEND ON OUR CAPACITY TO LEVERAGE MOLECULAR BIOMARKERS AS WINDOWS INTO THE BRAIN. TO DATE THE MOLECULAR STATES OF THE BRAIN DURING LIFELONG GENETIC RISK PRIOR TO DISEASE, FIRST PRION FORMATION, AND EARLY PHASES OF PRION REPLICATION ALL REMAIN ELUSIVE, AND YET, IT IS PRECISELY THESE UPSTREAM DISEASE STAGES THAT HAVE SHOWN THEMSELVES MOST AMENABLE TO PRP-LOWERING INTERVENTION. TO BECOME A CLINICAL REALITY, EARLY TREATMENT PARADIGMS WILL REQUIRE BIOMARKERS TO REPORT ON DRUG SAFETY AND EFFICACY BUT ALSO THE EARLIEST MOLECULAR STAGES OF RISK AND DISEASE; CRITICAL DECISIONS ABOUT TRIAL ELIGIBILITY, TIMING OF TREATMENT, AND ADVANCEMENT OR TERMINATION OF WHOLE EXPERIMENTAL DRUG PROGRAMS WILL REST ON OUR ABILITY TO ACCURATELY INTERPRET AND MECHANISTICALLY CONTEXTUALIZE THESE MOLECULAR READOUTS. THE GOAL OF THE PRESENT PROPOSAL IS TO DISCOVER AND ILLUMINATE BIOMARKERS ACROSS THREE KEY CATEGORIES. 1) MARKERS OF PRP MISFOLDING. USING CSF AND PLASMA SAMPLES FROM HEALTHY AT-RISK HUMANS AND FROM PRION-INFECTED RATS AND HAMSTERS AT PRE-SYMPTOMATIC STAGES, WE WILL TRACK THE EARLIEST MOLECULAR CHANGES RESULTING FROM PRION REPLICATION. WE WILL CHARACTERIZE NEW MARKERS AS WELL AS DETERMINE THE TEMPORAL SEQUENCE AND PROGNOSTIC VALUE OF EXISTING MARKERS. 2) MARKERS OF PRP MUTATION. IN A MOUSE MODEL OF A COMMON HUMAN PRION DISEASE MUTATION, WE WILL DISSECT THE MECHANISM BY WHICH A PATHOGENIC MISSENSE VARIANT RESULTS IN UNDER-EXPRESSION OF NOT ONLY MUTANT PRP BUT ALSO WILD-TYPE PRP AND OTHER BYSTANDER PROTEINS. WE WILL DETERMINE WHETHER RESULTING CHANGES WILL CONFOUND OTHER BIOMARKER READOUTS, AND WHETHER THE UNDER-EXPRESSION REFLECTS A HOST PROTECTIVE MECHANISM OR A HARMFUL PATHOLOGICAL CONSEQUENCE OF PRP MUTATION. 3) MARKERS OF PRP DEFICIENCY. LEVERAGING NEW PRP KNOCKOUT RATS AND HAMSTERS AS WELL AS PRP KNOCKOUT MICE, WE WILL PERFORM EXPERIMENTS TO GAIN A MECHANISTIC UNDERSTANDING OF THE MOLECULAR CHANGES RESULTING FROM PRP’S REDUCTION OR ABSENCE. TAKEN TOGETHER, THESE STUDIES WILL ON ONE HAND ILLUMINATE CORE MECHANISMS OF PRION BIOLOGY, FROM THE EARLY CONSEQUENCES OF PRP MUTATION AND MISFOLDING TO THE NATIVE FUNCTION OF PRP. ON THE OTHER HAND, THESE STUDIES WILL FACILITATE INFORMED CLINICAL DEVELOPMENT OF PRP-LOWERING THERAPIES BY ALLOWING BIOMARKERS TO BE CORRECTLY INTERPRETED SO THAT HARMFUL THERAPIES ARE HALTED WHILE PROMISING THERAPIES ARE ADVANCED.

TRANSPOSABLE ELEMENT INTERACTION AND ITS IMPACT ON HUMAN DEVELOPMENT AND HEALTH - PROJECT SUMMARY ONE OF THE MOST SURPRISING DISCOVERIES FROM THE HUMAN GENOME PROJECT IS THAT ONLY ABOUT 1.5% OF THE GENOME CODES FOR PROTEINS, WHEREAS AROUND 46% COMPRISES TRANSPOSABLE ELEMENTS (TES). FUNCTIONAL ASSESSMENT OF HOW THESE UBIQUITOUS TES AFFECT HUMAN DEVELOPMENT AND HEALTH HAS POSED A MAJOR CHALLENGE. WHILE MOST TES ARE CONSIDERED NON-FUNCTIONAL, OR “JUNK” DNA, HERE I ARGUE THAT TE-INDUCED GENE REGULATION IS STRONGLY UNDERESTIMATED DUE TO THE HISTORICAL TENDENCY TO EXPLORE TE FUNCTIONALITY BY STUDYING INDIVIDUAL TES INDEPENDENTLY OF EACH OTHER. I PROPOSE TO PROVIDE A NOVEL FRAMEWORK TO STUDY HOW INTERACTIONS BETWEEN THE HITHERTO “JUNK” TE SEQUENCES CAN REGULATE PRE-MRNA SPLICING TO AFFECT GENE FUNCTION, AND INVESTIGATE WHETHER SUCH A MECHANISM COULD SUBSTANTIALLY AFFECT BOTH HUMAN DEVELOPMENT AND EVOLUTION, AND HELP EXPLAIN THE GENETIC ETIOLOGY OF HUMAN DISEASES. THIS PROPOSAL IS INSPIRED FROM MY RECENT DISCOVERY THAT THE INTERACTION BETWEEN A PAIR OF ALU RETROTRANSPOSONS MAY EXPLAIN THE LONG-SOUGHT GENETIC BASIS FOR THE EVOLUTION OF TAIL LOSS IN HUMAN AND APES. BASED ON THIS WORK AND MY PRELIMINARY DATA, I WILL FIRST USE THE ALU PAIR INTERACTION IN TBXT GENE AS A MODEL TO DEMONSTRATE THAT THE INTERACTION BETWEEN INTRONIC TES CAN PROFOUNDLY IMPACT HUMAN DEVELOPMENT AND HEALTH, AND EXPLAIN THE ETIOLOGY OF A COMMON GENETIC DISEASE (AIM 1). AIM 2 PROPOSES TO TEST THE HYPOTHESIS THAT THE ISOFORM OF TBXT INDUCED BY INTERACTION OF THE ALU PAIR PLEIOTROPICALLY CONTRIBUTES TO STRENGTHENING OF HINDLIMBS, THUS DIRECTLY TESTING THE LONG-STANDING HYPOTHESIS THAT THE TAIL-LOSS EVOLUTION IN HOMINOIDS IS ASSOCIATED WITH BIPEDAL LOCOMOTION EVOLUTION (AIM 2). BEYOND THE SPECIFIC INTERACTION OF THE ALU PAIR IN THE TBXT GENE, AIM 3 WILL DEVELOP AN ALGORITHM CALLED TEILO (TRANSPOSABLE ELEMENT INTERACTION & LOCAL ORGANIZATION) TO SYSTEMATICALLY IDENTIFY THE FUNCTIONAL TE INTERACTIONS THAT AFFECT GENE FUNCTION AND HUMAN HEALTH BY MODULATING ALTERNATIVE SPLICING. THIS WORK PROMISES TO PROVIDE A NEW PARADIGM TO STUDYING THE INTERACTION BETWEEN TES AND ITS IMPLICATION TO HUMAN HEALTH AND DISEASES.

A HIGH-RESOLUTION MOLECULAR AND LINEAGE ATLAS OF THE MOUSE BRAIN USING SLIDE-SEQ

IMPROVING GENETIC DIAGNOSIS FOR AFRICAN ANCESTRY POPULATIONS - PROJECT SUMMARY PEOPLE OF AFRICAN ANCESTRY HAVE BEEN GROSSLY UNDERREPRESENTED IN GENETIC STUDIES, ACROSS DOMAINS AND DISCIPLINES. IN AGGREGATE, THIS IS MOST VISIBLE THROUGH THE CONSTITUTION OF LARGE GENETIC DATABASES LIKE GNOMAD, IN WHICH ONLY 14% OF INDIVIDUALS HAVE SOME AFRICAN ANCESTRY. THE GREAT MAJORITY OF THOSE AFRICAN-ANCESTRY INDIVIDUALS ARE AFRICAN AMERICAN, AND MOST COMMONLY HAVE A MIXTURE OF WEST AFRICAN AND EUROPEAN ANCESTRY. THIS MEANS THAT EAST AFRICAN POPULATIONS, AND OTHER AFRICANS LIVING ON THE AFRICAN CONTINENT, ARE EVEN FURTHER UNDERREPRESENTED IN GENETIC STUDIES TO DATE. IF AFRICAN ANCESTRY INDIVIDUALS REMAIN UNDERREPRESENTED IN GENETIC RESEARCH, THEY WILL CONTINUE TO BE LESS LIKELY TO RECEIVE ACCURATE GENETIC DIAGNOSES AND LESS LIKELY TO BENEFIT FROM ADVANCES IN GENOMIC MEDICINE. HERE, WE WILL USE DATA FROM GNOMAD, NEURODEV AND NEUROGAP-PSYCHOSIS TO ADDRESS THIS REPRESENTATION GAP AND TO IMPROVE MEDICAL GENETIC AND DIAGNOSTIC PIPELINES FOR INDIVIDUALS OF ALL TYPES OF AFRICAN ANCESTRY (AIM 1). THE PIPELINE IMPROVEMENTS WILL BE MADE IMMEDIATELY AVAILABLE THROUGH SEQR, AN OPEN ACCESS ANALYSIS PLATFORM THAT IS AVAILABLE ON ANVIL FOR USE BY THE MEDICAL GENETICS COMMUNITY. WE WILL GENETICALLY CHARACTERIZE ALL PARTICIPANTS FROM THE NEURODEV KENYA PROJECT (NDK) AND USE THIS DATA TO TEST AND IMPROVE THIS PIPELINE AND IDENTIFY GENETIC DIAGNOSES FOR PARTICIPANTS (AIM 2). USING DATA FROM NDK’S 3 HOUR MEDICAL, COGNITIVE AND BEHAVIORAL BATTERY, WE WILL CONDUCT THE LARGEST PHENOTYPIC CHARACTERIZATION OF RARE GENETIC DISORDERS IN EAST AFRICAN INDIVIDUALS TO DATE (AIM 3). AS DESCRIBED BY THE NHGRI ATLAS INITIATIVE, SYNDROMIC NEURODEVELOPMENTAL DISORDERS OFTEN VARY IN THEIR PHENOTYPIC PRESENTATION BETWEEN ETHNIC GROUPS. THE PRESENTATIONS OF RELATIVELY COMMON GENETIC DISORDERS (E.G. DDX3X AND 22Q11.2 DELETION SYNDROMES) HAVE NOT BEEN WELL CHARACTERIZED IN NON-EUROPEAN POPULATIONS. IN A COLLABORATIVE ANALYSIS, WE WILL COMPARE THE PHENOTYPIC PROFILES OF NDK CASES AND CASES FROM THE DECIPHERING DEVELOPMENTAL DISORDERS AFRICA STUDY WITH COMMON GENETIC DISORDERS AGAINST THOSE OBSERVED IN EUROPEAN ANCESTRY CASES, AS DESCRIBED IN GENEREVIEWS AND THE G2MH NETWORK. LASTLY, ALL DATA (E.G. GENETIC DATA, HPO TERMS) AND ALGORITHMS GENERATED BY THIS WORK WILL BE MADE PUBLICLY AVAILABLE IN ORDER TO RAPIDLY IMPROVE MEDICAL GENETICS RESEARCH AND RESOURCES FOR AFRICAN COMMUNITIES (AIM 4).

IMPLEMENTATION OF SLIDE-SEQ FOR HIGH-RESOLUTION, WHOLE-TRANSCRIPTOME HUMAN TISSUE MAPS.

POTENTIATE CAV3.3 TO TREAT COGNITIVE DEFICITS ASSOCIATED WITH IMPAIRED SLEEP SPINDLE - PROJECT SUMMARY/ABSTRACT SLEEP SPINDLE IS A HALLMARK BRAIN OSCILLATION OCCURRING IN NREM (NON-RAPID EYE MOVEMENT) SLEEP THAT REFLECT INTRINSIC THALAMOCORTICAL OFFLINE PROCESSING. SPINDLES ARE WAXING AND WANING BURSTS OF 12-15 HZ OSCILLATIONS THAT ORIGINATE IN THALAMIC RETICULAR NUCLEUS (TRN) WHEN ITS NEURONS UNDERGO BURSTING FIRING, AND TRN- THALAMIC RELAY NETWORK OSCILLATES AT SPINDLE FREQUENCY THROUGH THE RECIPROCAL CONNECTION. SLEEP SPINDLES ARE MARKEDLY REDUCED IN SCHIZOPHRENIA PATIENTS AS WELL AS IN ALZHEIMER PATIENTS, TWO VERY DIFFERENT CNS DISORDERS YET BOTH DEMONSTRATED SIGNIFICANT ALTERED SLEEP SPINDLE PROPERTIES ASSOCIATED WITH COGNITIVE IMPAIRMENT. WE HYPOTHESIZE THAT HYPOFUNCTIONAL TRN MAY UNDERLIE THE RISK/SYMPTOMS OF THESE TWO DEVASTING ILLNESSES. CACNA1I IS ENCODE THE A1 FUNCTIONAL CORE OF THE CAV3.3 VOLTAGE-GATED CALCIUM CHANNELS, AND ITS EXPRESSION IS ENRICHED IN TRN. WE HAVE DISCOVERED TWO NOVEL CHEMICAL SCAFFOLDS THAT POTENTIATE CAV3.3 FUNCTION, AND THIS PROPOSAL AIMS TO DELIVER IN VIVO READY CAV3.3 POSITIVE MODULATORS. THIS MULTIDISCIPLINARY EFFORT IS EXPECTED TO YIELD A PANEL OF INCISIVE PHARMACOLOGICAL REAGENTS TO ENHANCE CAV3.3 FUNCTION IN THE BRAIN AND NOMINATE CANDIDATES FOR PRECLINICAL DEVELOPMENT FOR TREATING COGNITIVE IMPAIRMENTS IN SCHIZOPHRENIA PATIENTS.

A COMPREHENSIVE CANINE GENETICS RESOURCE INCLUDING GENE AND VARIATION ANNOTATION