Jackson Laboratory

THE JACKSON LABORATORY KNOCKOUT MOUSE PRODUCTION AND PHENOTYPING PROJECT (JAX KOMP2)

MOUSE GENOME DATABASE

CANCER CENTER SUPPORT (CORE) GRANT

GENE EXPRESSION DATABASE FOR MOUSE DEVELOPMENT

INTERVENTIONS THAT RETARD MAMMALIAN AGING

GENOME DYNAMICS: EVOLUTION, ORGANIZATION AND FUNCTION

CENTER FOR SYSTEMS NEUROGENETICS OF ADDICTION

MOUSE GENOME DATABASE (MGD): A CORE KNOWLEDGE RESOURCE FOR FUNCTIONAL CHARACTERIZATION OF THE HUMAN GENOME - PROJECT SUMMARY AS THE COST OF GENOME-SCALE SEQUENCING CONTINUES TO DECREASE AND NEW TECHNOLOGIES FOR GENOME EDITING BECOME WIDELY ADOPTED, THE LABORATORY MOUSE IS MORE IMPORTANT THAN EVER AS A MODEL SYSTEM FOR UNDERSTANDING THE BIOLOGICAL SIGNIFICANCE OF HUMAN GENETIC VARIATION AND FOR ADVANCING THE EMERGENCE OF GENOMIC MEDICINE. THE MOUSE GENOME DATABASE (MGD) HAS A UNIQUE AND STRATEGIC ROLE AS A COMMUNITY RESOURCE FOR FACILITATING THE USE OF THE LABORATORY MOUSE FOR UNDERSTANDING GENOMICS UNDERLYING HUMAN BIOLOGY AND DISEASE. MGD SERVES THREE MAJOR USER COMMUNITIES: (I) BIOMEDICAL RESEARCHERS WHO USE MOUSE EXPERIMENTATION TO INVESTIGATE GENETIC AND MOLECULAR PRINCIPLES OF BIOLOGY AND DISEASE PROCESSES, (II) TRANSLATIONAL SCIENTISTS WHO USE THE LABORATORY MOUSE TO MODEL HUMAN DISEASE, AND (III) BIOINFORMATICIANS/ COMPUTATIONAL BIOLOGISTS WHO USE THE RICH INTEGRATED DATA MGD PROVIDES TO DEVELOP ALGORITHMS AND BIOINFORMATICS TOOLS FOR DATA ANALYSIS AND INTERPRETATION. DURING THE PROJECT PERIOD, WE WILL CONTINUE TO CURATE AND INTEGRATE NEW GENETIC, GENOMIC, VARIANT, FUNCTIONAL, PHENOTYPIC, AND HUMAN DISEASE MODEL DATA ESSENTIAL TO RESEARCHERS USING THE LABORATORY MOUSE IN BIOMEDICAL RESEARCH. WE WILL MAKE THESE DATA FREELY AVAILABLE THROUGH A VARIETY OF WEB-BASED AND PROGRAMMATIC USER INTERFACES. OUR CORE AIMS INCLUDE: (I) MAINTAINING THE CANONICAL CATALOG OF MOUSE GENOME FEATURES, (II) SERVING AS THE AUTHORITATIVE DATA FOR MOUSE FUNCTIONAL ANNOTATIONS, AND (III) MAINTAINING A COMPREHENSIVE CATALOG OF MOUSE MUTATIONS AND STRAINS AND THEIR PHENOTYPE AND DISEASE MODEL ASSOCIATIONS. TO SUPPORT OUR AIMS, WE WILL MAINTAIN COST-EFFECTIVE SOFTWARE, DATABASE, AND HARDWARE USING INDUSTRY BEST PRACTICES. WE WILL MAINTAIN MGD'S SECURE INFRASTRUCTURE THROUGH REGULAR MAINTENANCE, UPGRADES, AND PLANNED EVOLUTION. WE WILL LEVERAGE EXISTING SOFTWARE COMPONENTS FROM THE ALLIANCE OF GENOME RESOURCES AND OTHER RESOURCES WHERE POSSIBLE AND FOCUS OUR SOFTWARE DEVELOPMENT ACTIVITIES ON UNIQUE INFRASTRUCTURE NEEDED TO SUPPORT OUR CORE AIMS. TO ENSURE THE GREATEST IMPACT OF MGD IN THE BROADER SCIENTIFIC COMMUNITY, WE WILL PROVIDE ROBUST USER SUPPORT AND OUTREACH THROUGH ONLINE USER DOCUMENTATION, TUTORIALS, TRAINING WORKSHOPS, AND ONE-ON-ONE ASSISTANCE USING A VARIETY OF COMMUNICATION MODALITIES AND MAJOR SOCIAL MEDIA TOOLS. WE WILL ACTIVELY SOLICIT COMMUNITY INPUT, DATA SUBMISSIONS, AND COLLABORATIONS.

MODULATION OF LUNG IMMUNE RESPONSES TO VIRAL INFECTION

ESTABLISHING A MUTANT MOUSE REGIONAL RESOURCE CENTER AT THE JACKSON LABORATORY

MOUSE GENOME INFORMATICS

PRECLINICAL GENOME EDITING FOR RARE NEUROLOGICAL DISEASES - PROJECT SUMMARY OVERALL WHILE THE DEVELOPMENT OF HIGH-THROUGHPUT SEQUENCING TECHNOLOGY AND ITS APPLICATION TO CLINICAL DIAGNOSTICS HAS YIELDED THE GENETIC BASIS FOR MANY RARE GENETIC DISEASES, THE DEVELOPMENT OF EFFECTIVE TREATMENTS HAS NOT KEPT PACE. ALTHOUGH GENE REPLACEMENT AND MODULATION THERAPIES CAN BE POWERFUL, SOMETIMES EVEN LIFESAVING TREATMENT OPTIONS, THEY COME WITH MANY RISKS, SUCH AS IMMUNOGENICITY AND ONCOGENICITY. PROGRAMMABLE NUCLEASES SUCH AS CRISPR/CAS9 HAVE REVOLUTIONIZED OUR ABILITY TO MANIPULATE THE GENOME, AND PROVIDE THE POTENTIAL TO ACHIEVE LASTING, PRECISE GENOME MODIFICATION FOR THERAPEUTIC BENEFIT. THE PROPOSED U19 PROGRAM SEEKS TO ADDRESS THESE CHALLENGES THROUGH THE DEVELOPMENT, VALIDATION AND TRANSLATION OF GENE EDITING– BASED THERAPEUTIC SOLUTIONS FOR RARE NEUROLOGICAL GENETIC DISEASES. WE PROPOSE TO FOCUS ON FOUR NEUROLOGICAL CONDITIONS THAT EACH REPRESENT A SIGNIFICANT UNMET CLINICAL NEED: SPINAL MUSCULAR ATROPHY, FRIEDRICH'S ATAXIA, HUNTINGTON'S DISEASE, AND RETT SYNDROME. MEMBERS OF OUR TEAM HAVE DEVELOPED A SUITE OF BASE AND PRIME GENOME EDITING TOOLS THAT CAN INSTALL PRECISE ALTERATIONS WITHOUT CREATING A DSB OR REQUIRING A DONOR TEMPLATE. WE ALSO HAVE DEVELOPED VALIDATED IN VIVO MOUSE MODELS FOR EACH OF THESE DISEASES AND BRING DEEP EXPERTISE IN THE IND-ENABLING PRECLINICAL EVALUATION OF GENE-EDITING THERAPEUTICS. WE PROPOSE TO MERGE THESE CONSIDERABLE ASSETS WITH DISEASE-SPECIFIC EXPERTISE IN EACH OF THE FOUR NEUROLOGICAL CONDITIONS, SUPPORTED BY EXPERTISE AND RESOURCES FOR SCALED PRODUCTION OF AAV-BASED DELIVERY VECTORS FOR DELIVERY OF PRECISION GENE- EDITING THERAPIES TO TISSUES, AND FOR NAVIGATING THE REGULATORY PATH TO IND SUBMISSION. THE PROPOSED U19 TEAM HAS A TRACK RECORD OF INDIVIDUAL AND COLLABORATIVE SUCCESS AT EVERY STEP OF THE PRECLINICAL PIPELINE PATHWAY AND IS THUS WELL POSITIONED TO ACHIEVE OUR MILESTONES, WHICH INCLUDE AN IND PACKAGE SUBMITTED TO FDA FOR AT LEAST ONE THERAPY AND NEUROLOGICAL CONDITION. OUR OVERALL AIMS ARE TO: 1) ASSEMBLE A MULTI-DISCIPLINARY TEAM WITH UNIQUE STRENGTHS AND EXPERTISE TO DEVELOP AND IMPLEMENT INNOVATIVE GENOME EDITING STRATEGIES TO ADDRESS IMPORTANT DISEASE OF THE CNS, INCLUDING SPINAL MUSCULAR ATROPHY, FRIEDREICH'S ATAXIA, HUNTINGTON'S DISEASE, AND RETT SYNDROME; 2) OPTIMIZE LEAD BASE EDITOR AND PRIME EDITOR CANDIDATES FOR EACH DISEASE AREA, UTILIZING IN VITRO PLATFORMS AND VALIDATED ANIMAL MODELS; 3) EXECUTE DEFINITIVE PRECLINICAL IN VIVO PHARMACOLOGY STUDIES ON OPTIMIZED LEADS TO DEVELOP REPRODUCIBLE EFFICACY DATA, WHILE MONITORING BIODISTRIBUTION, PK/PD, TOLERABILITY, AND TOXICOLOGY; AND 4) ADVANCE ONE LEAD CANDIDATE TO AN ALLOWABLE INVESTIGATIONAL NEW DRUG (IND) APPLICATION THROUGH COORDINATED COMMUNICATION WITH THE FDA INTERACT PROGRAM, THE RESEARCH PROJECT TEAM, AND THE PROJECT CORES.

SHOCK CENTER FOR AGING RESEARCH AT THE JACKSON LABORATORY

HIGH THROUGHPUT PRODUCTION AND CRYOPRESERVATION OF KNOCKOUT MICE

IDENTIFYING AND CHARACTERIZING THE FULL SPECTRUM OF HAPLOTYPE-RESOLVED STRUCTURAL VARIATION IN HUMAN GENOMES

GENE ONTOLOGY CONSORTIUM

SPECIAL MOUSE STRAINS RESOURCE

ELECTRONIC ACCESS TO MOUSE TUMOR DATA

AN INTEGRATIVE ANALYSIS OF STRUCTURAL VARIATION FOR THE 1000 GENOMES PROJECT

THE JACKSON LABORATORY KOMP2 PHENOTYPING CENTER

THE JACKSON LABORATORY CENTER FOR PRECISION GENETICS

THE JACKSON LABORATORY SENESCENCE TISSUE MAPPING CENTER (JAX-SEN TMC) - PROJECT SUMMARY/ABSTRACT OVERALL THE JACKSON LABORATORY (JAX) CELLULAR SENESCENCE NETWORK MOUSE TISSUE MAPPING CENTER (JAX-SEN) REPRESENTS A MULTI-MODAL INTERDISCIPLINARY EFFORT THAT DRAWS UPON EXISTING, LONGSTANDING AGING RESEARCH PROGRAMS AT JAX, OUR ROLE AS A CONSORTIUM SITE OF KAPP-SEN, A FUNDED SENNET HUMAN TISSUE MAPPING CENTER, AND JAX’S CLOSE COLLABORATION WITH UCONN HEALTH. JAX-SEN IS ORGANIZED AROUND THREE CORES NESTED IN THE WELL-RESOURCED ENVIRONMENT FOR AGING RESEARCH AND MULTI-OMICS PROFILING AT JAX. JAX-SEN WILL PROFILE SENESCENT CELLS IN KIDNEY, ADIPOSE TISSUE, PLACENTA, PANCREAS, HEART, AND HYPOTHALAMUS, SELECTED FOR THEIR CLINICAL RELEVANCE TO CHRONIC DISEASES OF AGING, THE DOMAIN EXPERTISE OF THE JAX-SEN LEADERSHIP TEAM IN THE BIOLOGY OF THESE TISSUES, AND THE OVERLAP OF FOUR OF THE TISSUES WITH KAPP-SEN. WE WILL LEVERAGE CUTTING-EDGE MOUSE RESOURCES INCLUDING THE DIVERSITY OUTBRED, WHICH OFFERS UNPARALLELED GENETIC DIVERSITY FOR MODELLING A RANGE OF MOLECULAR SENESCENCE PHENOTYPES, AND NEW INBRED (C57BL/6J) TRANSGENIC MICE THAT EXPRESS P16 AND P21 DRIVEN FLUORESCENT TAGS TO ALLOW VISUALIZATION ENRICHMENT FOR SPECIFIC SENESCENT CELL SUBSETS. THE BIOLOGICAL ANALYSIS CORE WILL APPLY A MULTITUDE OF ANALYTICAL MODALITIES TO SELECTED TISSUES (BULK AND SINGLE CELL AND SINGLE NUCLEUS RNASEQ, VISIUM SPATIAL TRANSCRIPTOMICS, MULTIPLEXED ANTIBODY-BASED IMAGING, MULTIPLEXFISH, IMAGING MASS SPECTROMETRY INCLUDING LIPIDOMICS, METABOLOMICS, PROTEOMICS, 3D TISSUE RECONSTRUCTION FROM SERIAL SECTIONS), AND WILL RE- EVALUATE THESE TECHNOLOGIES THROUGHOUT THE PROJECT PERIOD TO ENSURE OUR APPROACHES ALIGN WITH THOSE OF OTHER CENTERS AND THAT WE MAXIMALLY BENEFIT FROM THE TECHNOLOGY DEVELOPMENT EFFORTS IN SENNET. THESE PROFILING ACTIVITIES WILL YIELD SUBCELLULAR RESOLUTION OF BIOMOLECULAR CONTENT IN SENESCENT CELLS NOT POSSIBLE WITH HUMAN SAMPLES, ALONG WITH AN EXPANDED SET OF SENESCENT CELL BIOMARKERS. THE DATA ANALYSIS CORE WILL COMBINE ROBUST COMPUTATIONAL CAPABILITY WITH SCALABLE AND REPRODUCIBLE SCIENTIFIC WORKFLOWS TO DEVELOP AND IMPLEMENT NETWORK- WIDE OPEN DATA AND METADATA STANDARDS. THE DATA ANALYSIS CORE WILL BENEFIT FROM JAX’S UNIQUE MOUSE RESEARCH RESOURCES TO ESTABLISH THE DIVERSITY OF SENESCENCE BY INTEGRATING WITH EXPRESSION QUANTITATIVE TRAIT LOCUS AND PROTEIN QUANTITATIVE TRAIT LOCUS DATA IN DO MICE AND JAX BIOINFORMATICS RESOURCES, E.G., MOUSE GENOME INFORMATICS, MOUSE PHENOME DATABASE, MONARCH INITIATIVE. THE INTEGRATIVE MINING OF JAX-SEN DATA WILL LEAD TO NOVEL METHODS FOR MULTI-MODAL 3D MAP RECONSTRUCTION FROM IMAGING DATA AND WILL YIELD AN IMPORTANT RESOURCE FOR SENNET INTERPRETATION AND ENRICHMENT OF HUMAN SENESCENT CELL DATA. FINALLY, THE ADMINISTRATIVE CORE WILL PROVIDE STRONG OVERSIGHT OF PROJECT PROGRESS AND NURTURE A CULTURE OF COLLABORATIVE, DYNAMIC SCIENTIFIC EXCHANGE WITHIN JAX-SEN. IT WILL ALSO MAXIMIZE JAX-SEN’S ENGAGEMENT IN THE CONSORTIUM THROUGH COLLABORATIVE ACTIVITIES THAT WILL HELP OPTIMIZE DATA GENERATION ACTIVITIES AND CREATE NEW RESEARCH SYNERGIES. IN SO DOING, JAX-SEN WILL HELP ACHIEVE THE GOAL OF SENNET TO GENERATE A UNPRECEDENTED ATLAS OF MOUSE SENESCENT CELLS AND NEW BIOMARKERS OF SENESCENCE FOR HUMAN ANALYSIS AND THERAPEUTIC APPLICATION.

TOPOLOGICAL MAPPING OF IMMUNE, MICROBIOTA, METABOLOMIC AND CLINICAL PHENOTYPES TO REVEAL ME/CFS DISEASE MECHANISMS

THE JACKSON LABORATORY CENTER FOR PRECISION GENETICS: FROM NEW MODELS TO NOVEL THERAPEUTICS

COMPREHENSIVE MAPPING OF LONG-RANGE CHROMATIN INTERACTIONS IN HUMAN AND MOUSE GENOMES

RETINAL DISEASE: MOLECULAR BASIS AND PATHOPHYSIOLOGY

JAX MORPHIC DATA PRODUCTION CENTER - PROJECT SUMMARY THIS PROPOSAL WILL ESTABLISH A MOLECULAR PHENOTYPES OF NULL ALLELES IN CELLS (MORPHIC) PHASE 1: DATA PRODUCTION RESEARCH AND DEVELOPMENT CENTER BASED ENTIRELY AT THE JACKSON LABORATORY FOR GENOMIC MEDICINE – THE JAX MORPHIC CENTER. JAX MORPHIC WILL COMPRISE, UNDER ONE ROOF, A TEAM OF HIGHLY COLLABORATIVE SCIENTISTS WITH COMPLEMENTARY SKILL SETS AND DECADES OF CUMULATIVE EXPERIENCE IN MAMMALIAN GENE KNOCKOUTS (KO), STEM CELL AND DEVELOPMENTAL BIOLOGY, MOLECULAR PHENOTYPING, SINGLE CELL ANALYSIS, AND ADVANCED METABOLOMICS AND LIPIDOMICS CAPABILITIES. WE WILL KO 250 PROTEIN-CODING GENES OVER THE PHASE 1 PERIOD OF THIS INITIATIVE AND WILL ENGINEER THESE KOS IN HUMAN INDUCED PLURIPOTENT STEM CELLS (IPSCS) THAT WILL SUBSEQUENTLY BE DIFFERENTIATED INTO TWO CELL LINEAGES, THE EXTRA-EMBRYONIC AND THE NEUROECTODERMAL, WHERE CELLS WILL THEN BE COMPREHENSIVELY PHENOTYPED. OUR RATIONALE FOR SELECTING THESE LINEAGES IS THAT THEY ARE TWO OF THE MOST EVOLUTIONARY DIVERGENT BETWEEN SPECIES, AND PRIMATE-SPECIFICITY IS ONE OF OUR CRITERIA FOR GENE PRIORITIZATION. IN ADDITION, THE EXTRA- EMBRYONIC LINEAGE RAPIDLY DEVELOPS INTO A CELLULAR FUNCTIONAL ENDPOINT RESPONSIBLE FOR MANY BIOLOGICAL PROCESSES, THUS PERMITTING INTERPRETATION OF FUNCTION OF DIVERSE GENES, WHEREAS THE EARLY NEUROECTODERMAL LINEAGE IS RELEVANT TO NEURODEVELOPMENTAL DISORDERS. IN AIM 1, WE WILL PRIORITIZE GENES FOR KNOCKOUT; SELECTION CRITERIA INCLUDE EXPRESSION IN EXTRA-EMBRYONIC OR NEUROECTODERMAL LINEAGES, PRIMATE-SPECIFIC FEATURES, BROAD CLASSES OF FUNCTIONS WHILE ENRICHING FOR TRANSCRIPTION FACTORS, AND GENES IMPLICATED IN HUMAN DISEASE. IN AIM 2, WE WILL GENERATE IPSC KO CLONES IN HIGH-THROUGHPUT USING HIGH-EFFICIENCY PROTOCOLS AND WORKFLOWS ESTABLISHED IN JAX'S CELLULAR ENGINEERING CORE. WE WILL ENGINEER KO CLONES FOR 250 GENES IN THE WELL-CHARACTERIZED, STABLE HUMAN IPSC LINE, KOLF2.1, WHILE CONSIDERING THE EFFECTS OF SEX, GENETIC BACKGROUND, POSSIBLE ADAPTIVE/COMPENSATORY RESPONSES, AND DIFFERENT KO STRATEGIES INCLUDING INCORPORATION OF CONDITIONAL/REVERSIBLE AND SCALABLE APPROACHES. IN AIM 3, WILL CARRY OUT COMPREHENSIVE PHENOTYPING OF DERIVATIVES DIFFERENTIATED FROM KO IPSCS. WE HAVE SELECTED A COMBINATION OF ASSAYS TO MAXIMALLY INTEGRATE CONSISTENCY, SCALABILITY, AND FUNCTIONAL INFORMATIVENESS TO HELP ACHIEVE THE OVERALL OBJECTIVES OF THE MORPHIC CONSORTIUM. THESE INCLUDE IMAGING, SINGLE CELL TRANSCRIPTOMICS, SINGLE NUCLEUS EPIGENOMICS, AND METABOLOMICS/LIPIDOMICS. OUR RESEARCH AIMS WILL BE COORDINATED IN ADMINISTRATIVE AIM 4, WHICH WILL ENSURE EFFICIENT PROJECT MANAGEMENT AND OVERSIGHT, INTERNAL AND EXTERNAL COMMUNICATIONS, AND DATA DISSEMINATION TO THE MORPHIC DATA RESOURCE AND ADMINISTRATIVE COORDINATION CENTER (DRACC). SUCCESSFUL COMPLETION OF THE PROPOSED WORK WILL ADDRESS SEVERAL MAIN BARRIERS HAMPERING THE ULTIMATE GOAL OF THE MORPHIC CONSORTIUM TO FUNCTIONALLY CHARACTERIZE ALL HUMAN GENES: IDENTIFYING THE MOST EFFECTIVE KO STRATEGIES AND OPTIMAL PHENOTYPING TECHNOLOGIES, AS WELL AS SCALING EFFICIENCIES TO EXPAND THIS PROGRAM TOWARDS FUTURE MORPHIC PHASES. IN THE PROCESS, WE WILL GENERATE A VALUABLE RESOURCE OF 250 HUMAN IPSC KO LINES THAT WILL BE DISTRIBUTED TO THE SCIENTIFIC COMMUNITY VIA EXISTING OPERATIONAL INFRASTRUCTURE AT JAX.

MOUSE PHENOME PROJECT

DATA COORDINATION CENTER FOR PDX NET

A DEEP LONGITUDINAL ANALYSIS OF NEXT GENERATION INFLUENZA VACCINES IN OLDER ADULTS - PROJECT SUMMARY THE WHO ESTIMATES THAT ANNUAL EPIDEMICS OF INFLUENZA RESULT IN 3-5 MILLION CASES OF SEVERE ILLNESS AND 300,000- 500,000 DEATHS. 90% OF INFLUENZA-RELATED DEATHS OCCUR IN OLDER ADULTS DESPITE WIDESPREAD VACCINATION PROGRAMS WITH VACCINES TAILORED FOR THIS HIGH-RISK GROUP. THE ESTIMATED EFFECTIVENESS OF THE INFLUENZA VACCINE IN THE U.S. FOR THE 2018-2019 INFLUENZA SEASON OVERALL WAS 47%, BUT ONLY 12-13% IN OLDER ADULTS. THERE IS THEREFORE AN URGENT NEED TO UNDERSTAND THE MECHANISMS THAT ARE TURNED ON/OFF IN OLDER ADULTS THAT RESULT IN THEIR LIMITED RESPONSE RATE TO THE MOST COMMONLY USED INFLUENZA VACCINE, FLUZONE® HIGH-DOSE. THERE IS ALSO A NEED TO UNDERSTAND WHETHER AND WHY NEXT-GENERATION INFLUENZA VACCINES MIGHT BE MORE EFFICACIOUS. IMMUNOSENESCENCE IS KNOWN TO BE ASSOCIATED WITH DECLINES IN OPTIMAL B CELL AND T CELL ADAPTIVE IMMUNITY, HOWEVER, OUR OVERALL UNDERSTANDING OF THE MECHANISMS OF IMMUNOSENESCENCE IS INCOMPLETE. THE CENTRAL GOAL OF THIS PROPOSAL IS TO UNDERSTAND THE MECHANISMS THAT LEAD TO A LOSS OF RESPONSE TO INFLUENZA VACCINE IN OLDER ADULTS THROUGH ESTABLISHMENT OF THE 3FLUAGING COHORT OF HEALTHY OLDER ADULTS WHO WILL BE VACCINATED WITH THREE DIFFERENT INFLUENZA VACCINES THREE YEARS IN A ROW. WE HYPOTHESIZE THAT AGING IMPACTS SPECIFIC REGULATORY MECHANISMS OF HUMORAL IMMUNITY TO REDUCE VACCINE EFFECTIVENESS. IN AIM 1, WE WILL ESTABLISH A COHORT OF 60 HEALTHY OLDER ADULTS (=65YRS) WHO WILL SEQUENTIALLY RECEIVE THREE DIFFERENT ANNUAL INFLUENZA VACCINES, WITH SERIAL BLOOD AND MICROBIOME SAMPLE COLLECTION DURING THREE YEARS OF FOLLOW-UP. PARTICIPANTS WILL UNDERGO REGULAR CLINICAL ASSESSMENTS. IN AIM 2, WE WILL DECIPHER THE MAGNITUDE AND IMMUNODOMINANCE PATTERN OF THE HUMORAL RESPONSE TO INFLUENZA VIRUS IN HEALTHY OLDER INDIVIDUALS UPON VACCINATION. FOR EACH VACCINE, WE WILL CHARACTERIZE ANTIBODY TITER AND QUALITY AND WILL DEFINE RESPONDERS AND NON-RESPONDERS. IN AIM 3, WE WILL CHARACTERIZE THE EPIGENOME, TRANSCRIPTOME, CYTOKINE PRODUCTION, AND CELL PROPORTIONS OF BLOOD LEUKOCYTES IN VACCINATED HEALTHY OLDER PARTICIPANTS. WE WILL IDENTIFY SPECIFIC (EPI)GENOMIC AND FUNCTIONAL SIGNATURES, AND THEIR LONGEVITY, ASSOCIATED WITH VACCINE RESPONSE. WE WILL ALSO SEQUENCE ALL PARTICIPANTS TO UNCOVER THE ROLE OF GENETIC VARIATION ON INFLUENZA VACCINE RESPONSES. IN AIM 4, WE WILL ASSESS THE FUNCTION OF T HELPER CELLS AND ANTIGEN PRESENTING CELLS, SPECIFICALLY DENDRITIC CELLS, IN INFLUENZA VACCINE RESPONDERS AND NON-RESPONDERS. BY IDENTIFYING RESPONDERS AND NON-RESPONDERS FOR EACH VACCINE AND INTEGRATING THESE DATA WITH BASELINE IMMUNE STATUS MULTI-OMIC SIGNATURES, WE WILL DETERMINE WHICH IMMUNE FEATURES CAN PREDICT VACCINE RESPONSIVENESS. WE EXPECT TO IDENTIFY HUMORAL IMMUNITY PATHWAYS THAT ARE ALTERED IN AGING THAT CAN BE USED AS THE BASIS FOR DESIGNING NOVEL APPROACHES TO BOOST EFFICACY OF THE MOST COMMONLY USED, AS WELL AS EMERGING, INFLUENZA VACCINES.

MOLECULAR REGULATION OF MAMMALIAN MEIOSIS

DISCOVERY OF ADDICTION-RELATED GENES WITH ADVANCED MOUSE RESOURCES

GENOME TECHNOLOGIES COORDINATING CENTER - PROJECT SUMMARY THE GENOME TECHNOLOGY PORTFOLIO AT NHGRI ENCOMPASSES A SUBSTANTIAL DIVERSITY OF SCIENTIFIC APPROACHES AND OBJECTIVES, FROM INSTRUMENTATION DEVELOPMENT TO HIGH-THROUGHPUT APPLICATION OF TECHNOLOGIES TO CHARACTERIZING THE GENOMIC BASIS FOR PHENOTYPIC TRAITS. THESE RESEARCH EFFORTS HAVE PRODUCED SIGNIFICANT INSIGHT INTO BIOLOGY AND DISEASE, AND YET THERE ARE MANY OPPORTUNITIES TO ACCELERATE INNOVATION, DEVELOPMENT, AND EARLY DISSEMINATION OF GENOMIC TECHNOLOGIES. THE GOAL OF THE GENOME TECHNOLOGY COORDINATING CENTER (GTCC) IS TO MAXIMIZE THESE OPPORTUNITIES BY LIAISING GRANTEES AND NHGRI PROGRAM STAFF, MANAGING RESEARCH FUNDS, AND DEVELOPING EDUCATIONAL RESOURCES TO SUPPORT AND TOUT THE EXCELLENCE OF THE GENOME TECHNOLOGY PROGRAM. WE PROPOSE THE FOLLOWING AIMS TO MEET THESE GOALS: 1) FACILITATE COLLABORATIONS ACROSS THE GENOME TECHNOLOGY PROGRAM AND PROMOTE STANDARDS THAT AID IN ACHIEVING PROGRAM GOALS. WE WILL HOST AND SUPPORT ANNUAL MEETINGS FOR THE GENOME TECHNOLOGY AND CEGS PROGRAMS. WE WILL ALSO DEVELOP WORKING GROUPS AMONG GRANTEES TO PROMOTE COLLABORATION-BUILDING AND THE DEVELOPMENT OF METRICS AND STANDARDS FOR GENOMIC TECHNOLOGIES. WE WILL USE THESE WORKING GROUPS TO IDENTIFY GAPS IN THE FIELD THAT WILL PROVIDE THE FOUNDATION FOR SOLICITATION OF OPPORTUNITY FUNDS. 2) ESTABLISH AND MANAGE AN OPPORTUNITY FUNDS PROGRAM TO SUPPORT INITIAL DEVELOPMENT OF PROMISING NEW TECHNOLOGIES AND IDEAS. THESE OPPORTUNITY FUNDS WILL BE DISTRIBUTED AS PILOT PROJECTS (EARLY STAGE OR PROOF-OF- PRINCIPLE STUDIES) AND DEVELOPMENT PROJECTS (COMPARATIVELY ADVANCED STUDIES WITH SUPPORTIVE PRELIMINARY DATA) IN YEARS 2-4. PEER REVIEW WILL BE CONDUCTED BY AN EXTERNAL REVIEW PANEL OF EXPERTS IN GENOMIC TECHNOLOGY. 3) IMPLEMENT COMMUNITY OUTREACH ACTIVITIES TO ENCOURAGE AWARENESS OF GENOME TECHNOLOGY PROGRAM SUCCESSES AND FACILITATE ACCESS BY THE SCIENTIFIC COMMUNITY. WE WILL DEVELOP AND MANAGE AN EDUCATION AND OUTREACH WORKING GROUP COMPOSED OF GRANTEES AND EDUCATIONAL EXPERTS TO IDENTIFY AND PRIORITIZE AREAS OF DEVELOPMENT FOR EDUCATIONAL MATERIALS, AND HOST AND ADVERTISE A WEBSITE TO DISTRIBUTE THOSE MATERIALS. A TARGETED EFFORT WILL BE MADE TO EXPOSE UNDERREPRESENTED AND UNDERSERVED POPULATIONS IN SCIENCE TO THE ADVANCES AND IMPORTANCE OF THE GENOME TECHNOLOGY PROGRAM. INFORMATIONAL RESOURCES RELATED TO TECHNOLOGY TRANSFER AND INTELLECTUAL PROPERTY WILL BE DEVELOPED FOR GRANTEES. THE JACKSON LABORATORY (JAX) IS UNIQUELY QUALIFIED TO SERVE AS THE GTCC FOR THE GENOME TECHNOLOGY PROGRAM. THE LABORATORY HAS OVER NINETY YEARS OF HISTORY IN SUPPORTING MAMMALIAN GENETICS RESEARCH INCLUDING THROUGH THE PROVISION OF WIDELY USED INFORMATION RESOURCES (E.G. MOUSE GENOME INFORMATICS) AND EDUCATIONAL PROGRAMS INCLUDING THE ANNUAL SHORT COURSE ON EXPERIMENTAL MODELS OF HUMAN CANCER (29 YEARS), AND THE MCKUSICK SHORT COURSE ON HUMAN AND MAMMALIAN GENETICS AND GENOMICS (NEARLY 60 YEARS). THE JACKSON LABORATORY FOR GENOMIC MEDICINE WAS ESTABLISHED IN 2012 TO APPLY THE LAB’S EXPERTISE IN GENOMICS AND TECHNOLOGY DEVELOPMENT TOWARD IMPROVEMENTS IN GENOME-BASED MEDICINE.

COMMUNITY PROJECT FUNDING/CONGRESSIONALLY DIRECTED SPENDING - CONSTRUCTION - THE RARE DISEASE TRANSLATIONAL RESEARCH CENTER (RDTC) IS A PIONEERING PROJECT THAT AIMS TO CREATE A WORLD-CLASS RESEARCH FACILITY DEDICATED SOLELY TO THE STUDY OF RARE DISEASES. THIS PROJECT IS ABOUT BUILDING A STATE-OF-THE-ART INFRASTRUCTURE THAT WILL SERVE AS A CATALYST FOR SCIENTIFIC BREAKTHROUGHS AND INNOVATIONS IN THE FIELD OF RARE DISEASES. THE FACILITY, TO BE LOCATED IN BAR HARBOR, MAINE, WILL HOUSE ADVANCED LABORATORIES EQUIPPED WITH THE LATEST TECHNOLOGIES AND EQUIPMENT. THESE LABORATORIES WILL PROVIDE A CONDUCIVE ENVIRONMENT FOR SCIENTISTS TO CONDUCT CUTTING-EDGE RESEARCH, FROM BASIC SCIENCE TO TRANSLATIONAL STUDIES, ALL AIMED AT UNDERSTANDING THE UNDERLYING MECHANISMS OF RARE DISEASES AND DEVELOPING EFFECTIVE TREATMENTS. THE RESEARCH CONDUCTED HERE WILL CONTRIBUTE TO THE GLOBAL BODY OF KNOWLEDGE ON RARE DISEASES, ACCELERATING THE PACE OF DISCOVERY AND BRINGING US CLOSER TO FINDING CURES. IT WILL ALSO FOSTER COLLABORATIONS AMONG SCIENTISTS, ENHANCING THE EXCHANGE OF IDEAS AND FACILITATING THE SHARING OF RESEARCH FINDINGS. THE PROJECT WILL INCORPORATE SUSTAINABLE DESIGN PRINCIPLES, WITH A FOCUS ON ENERGY EFFICIENCY AND ENVIRONMENTAL STEWARDSHIP. THE FACILITY WILL BE DESIGNED TO FOSTER CREATIVITY AND COLLABORATION, WITH OPEN SPACES, NATURAL LIGHT, AND A LAYOUT THAT ENCOURAGES INTERACTION AMONG RESEARCHERS. CONSTRUCTION IS EXPECTED TO BEGIN IN LATE 2024AND WILL TAKE APPROXIMATELY TWO YEARS. WE ARE COMMITTED TO KEEPING OUR COMMUNITY INFORMED OF OUR PROGRESS AND WILL MAKE EVERY EFFORT TO MINIMIZE DISRUPTION DURING THE CONSTRUCTION PHASE. THE RARE DISEASE TRANSLATIONAL RESEARCH CENTER (RDTC) IS A TESTAMENT TO OUR COMMITMENT TO ADVANCING RARE DISEASE RESEARCH. IT IS ABOUT CREATING A SPACE WHERE SCIENCE THRIVES, AND WHERE EVERY DISCOVERY BRINGS US ONE STEP CLOSER TO MAKING A DIFFERENCE IN THE LIVES OF THOSE AFFECTED BY RARE DISEASES. WE ARE EXCITED ABOUT THE JOURNEY AHEAD AND LOOK FORWARD TO SHARING MORE DETAILS AS THE PROJECT PROG RESSES. THANK YOU FOR YOUR SUPPORT AS WE EMBARK ON THIS TRANSFORMATIVE PROJECT. TOGETHER, WE CAN CHANGE THE FUTURE OF RARE DISEASE RESEARCH.

CRE DRIVER STRAIN RESOURCES

MODELS FOR VISION RESEARCH

CAT-TAILS: A NOVEL TYPE OF PROTEIN MODIFICATION IMPLICATED IN NEURODEGENERATION

SYSTEMS GENETICS ANALYSIS OF RESILIENCE TO ALZHEIMER?S DISEASE

RESOURCES FOR COMPARATIVE MENDELIAN DISEASE GENOMICS

B-LYMPHOCYTE TARGETING THERAPIES FOR AUTOIMMUNE DIABETES

MOUSE MUTANT RESOURCE

EYE MUTANT RESOURCE

VCID CWOW: IDENTIFYING NOVEL TARGETS TO TREAT CEREBRAL AMYLOID ANGIOPATHY - ALZHEIMER’S DISEASE (AD), MOST COMMON FORM OF DEMENTIA, AFFECTS OVER 55 MILLION PEOPLE WORLDWIDE. NUMBERS ARE EXPECTED TO DOUBLE EVERY 20 YEARS UNLESS IMPROVED TREATMENTS ARE DEVELOPED. AD IS CHARACTERIZED BY AMYLOID PLAQUES AND NEUROFIBRILLARY TANGLES OF TAU, BUT RECENTLY APPROVED TREATMENTS TARGETING AMYLOID DEPOSITION SHOW LIMITED EFFECTIVENESS. THEREFORE, IT IS CRITICAL THAT TREATMENTS ARE DEVELOPED THAT TARGET MULTIPLE ASPECTS OF AD AND RELATED DEMENTIAS (ADRD). SMALL VESSEL CEREBROVASCULAR DISEASE IS NOW THOUGHT TO CONTRIBUTE TO MANY ADRD CASES. HOWEVER, NO TREATMENTS THAT TARGET CEREBROVASCULAR HEALTH IN ADRD ARE AVAILABLE. THEREFORE, IN RESPONSE TO RFA-NS-24-027 (VCID CENTER WITHOUT WALLS [CWOW] FOR UNDERSTANDING AND LEVERAGING SMALL VESSEL CEREBROVASCULAR DISEASE MECHANISMS IN ADRD), WE PROPOSE TO ESTABLISH THE JACKSON LABORATORY (JAX)/EMORY UNIVERSITY/COLUMBIA UNIVERSITY/RUSH UNIVERSITY VCID CWOW TO PRIORITIZE NOVEL THERAPEUTIC TARGETS FOR VASCULAR CONTRIBUTIONS TO COGNITIVE IMPAIRMENT AND DEMENTIA (VCID). WE WILL FOCUS ON UNDERSTANDING FACTORS MEDIATING CEREBRAL AMYLOID ANGIOPATHY (CAA: THE DEPOSITION OF AMYLOID IN SMALL VESSELS), INCLUDING INTERACTIONS WITH TAU PATHOLOGY AND APOE GENOTYPE. APOE4 IS THE GREATEST GENETIC RISK FACTOR FOR ADRD AND ACCELERATES CAA. DESPITE THE PREVALENCE OF CAA, THE MOLECULES THAT MEDIATE ITS DEVELOPMENT REMAIN TO BE ELUCIDATED. WE HYPOTHESIZE THAT TARGETING CAA, AND THE UNDERLYING CEREBROVASCULAR DYSFUNCTION AND BBB BREAKDOWN, WILL PREVENT OR SLOW NEURODEGENERATION AND COGNITIVE DECLINE IN ADRD. TO TEST OUR HYPOTHESIS, WE WILL LEVERAGE EXPERTISE AND RESOURCES AVAILABLE TO THE JAX/EMORY/COLUMBIA/RUSH VCID CWOW. THESE INCLUDE HUMAN SAMPLES AND ASSOCIATED DEEP CLINICAL DATA FROM THE RELIGIOUS ORDERS STUDY/MEMORY AGING PROJECT (ROSMAP), A WEALTH OF MULTI-OMIC DATA GENERATED AS PART OF THE ACCELERATED MEDICINES PARTNERSHIPS (AMP)- AD, AND NOVEL ADRD MOUSE MODELS GENERATED AS PART OF MODEL ORGANISM DEVELOPMENT AND EVALUATION FOR LATE-ONSET AD (MODEL-AD). OUR PRELIMINARY ANALYSES HAVE IDENTIFIED PUTATIVE CAA-ASSOCIATED TARGETS THAT ARE DEPENDENT ON OR INDEPENDENT OF APOE4. HOWEVER, SMALL VESSEL-SPECIFIC MULTI-OMIC DATASETS ARE LACKING, HINDERING OUR SENSITIVITY TO IDENTIFY CEREBROVASCULAR-SPECIFIC MEDIATORS OF CAA. THEREFORE, WE WILL FIRST GENERATE AND ANALYZE VESSEL-ENRICHED PROTEOMIC AND SINGLE-NUCLEUS RNA-SEQ DATASETS FROM ROSMAP SAMPLES SHOWING VARIATION IN COGNITIVE STATUS, AMYLOID AND TAU PATHOLOGY, CAA, AND APOE GENOTYPE (AIM 1). WE WILL THEN DEEPLY PHENOTYPE OUR NOVEL ADRD MOUSE MODEL PANEL THAT SHOWS A SIMILAR VARIATION IN CAA, APOE GENOTYPE, AND TAU PATHOLOGY TO THAT IN THE ROSMAP HUMAN SAMPLES. WE WILL PERFORM COGNITIVE EXAMS, MAGNETIC RESONANCE IMAGING, FLUID BIOMARKERS, WHOLE-BRAIN CAA MAPPING, AND BULK AND VESSEL-ENRICHED PROTEOMICS (AIM 2). FINALLY, WE WILL PERFORM SPATIAL TRANSCRIPTOMICS AND PROTEOMICS IN HUMAN AND MOUSE SAMPLES AND INTEGRATE ALL DATA TO PRIORITIZE NOVEL VASCULAR-SPECIFIC CANDIDATES. THE MOST PROMISING OF THESE WILL BE VALIDATED IN OUR MOUSE MODELS (AIM 3). ALL DATA AND RESOURCES WILL BE MADE AVAILABLE THROUGH THE AD KNOWLEDGE PORTAL AND THE JAX REPOSITORY.

THE GENETICS OF THE NEUROMUSCULAR JUNCTION: MECHANISMS AND DISEASE MODELS

QTL ANALYSIS IN COMBINED INBRED LINE CROSSES

CLONING & FUNCTIONAL STUDIES OF MOUSE DEAFNESS MUTATIONS

GENETICS OF THE NEUROMUSCULAR JUNCTION: MECHANISMS AND DISEASE MODELS

HAIR CELL POLARIZATION AND SENSORY BUNDLE DEVELOPMENT

REPRODUCTIVE GENOMICS: MUTANT MODELS FOR INFERTILITY

GENETIC MODIFIERS OF RETINAL DISEASE

GENETIC PROGRAMMING OF HUMAN ISLET METABOLIC AND ENDOPLASMIC RETICULUM (ER) STRESS RESPONSES IN DIABETES - PROJECT SUMMARY TYPE 2 DIABETES (T2D) RESULTS FROM FAILURE OF PANCREATIC ISLETS TO SECRETE SUFFICIENT INSULIN TO COMPENSATE FOR INCREASED INSULIN RESISTANCE IN PERIPHERAL TISSUES. PRECISE UNDERSTANDING OF THE MOLECULAR MECHANISMS UNDERLYING GENETIC AND ENVIRONMENTAL CONTRIBUTIONS TO ISLET FAILURE IS ESSENTIAL TO DEVELOP NEW, TARGETED APPROACHES TO PREVENT AND TREAT T2D. ENDOPLASMIC RETICULUM (ER) AND (GLUCO)LIPOTOXIC STRESS RESPONSES ARE CENTRAL (PATHO)PHYSIOLOGIC PROCESSES THAT CONTRIBUTE TO ISLET DYSFUNCTION AND FAILURE. OUR OVERALL OBJECTIVE IN THIS PROPOSAL IS TO ELUCIDATE THE GENETIC REGULATION OF ISLET STRESS RESPONSES AND TO DETERMINE HOW GENETIC VARIANTS, INCLUDING SNPS ASSOCIATED WITH T2D AND OTHER METABOLIC TRAITS (T2D SNPS), MODULATE THESE RESPONSES TO CONTRIBUTE TO ISLET DYSFUNCTION AND T2D PATHOGENESIS. BASED ON PREVIOUS STUDIES AND PRELIMINARY DATA, WE HYPOTHESIZE THAT T2D SNPS ALTER HUMAN ISLET STRESS RESPONSES BY CHANGING ISLET REGULATORY ELEMENT (RE) USE/FUNCTION AND EXPRESSION OF THEIR TARGET GENES TO CONTRIBUTE TO ISLET DYSFUNCTION AND T2D. IN AIM 1, WE WILL TEST THIS HYPOTHESIS BY CHARACTERIZING ISLET ER AND (GLUCO)LIPOTOXIC STRESS RESPONSES AT THE LEVEL OF GENE EXPRESSION AND IDENTIFYING GENETIC VARIANTS ALTERING HUMAN ISLET STRESS RESPONSES. FOR THIS, WE WILL USE BULK AND SINGLE-CELL RNA-SEQ AS WELL AS COMPUTATIONAL ANALYSES TO DISCOVER RESPONSE EXPRESSION QUANTITATIVE TRAIT LOCI (REQTL). IN AIM 2, WE WILL TAKE A COMPLEMENTARY EPIGENOMIC APPROACH TO ELUCIDATE GENETIC EFFECTS ON STRESS RESPONSE REGULATORY ELEMENT (RE) USE IN HUMAN ISLETS. FROM THE SAME ISLETS AS IN AIM 1, WE WILL DETERMINE THE GENOME-WIDE LOCATION OF RES AT STEADY STATE AND AFTER EXPOSURE TO STRESSORS USING THE ASSAY FOR TRANSPOSASE ACCESSIBLE CHROMATIN-SEQUENCING (ATAC-SEQ). USING THESE DATA, WE WILL COMPUTATIONALLY IDENTIFY TRANSCRIPTION FACTORS BINDING TO THESE RES, IDENTIFY GENETIC VARIANTS ALTERING STRESS-RESPONSIVE RE USE BY CHROMATIN ACCESSIBILITY QUANTITATIVE TRAIT LOCUS (CAQTL) ANALYSES, AND TEST ALLELIC EFFECTS ON STRESS-RESPONSIVE RE ACTIVITY USING MASSIVELY PARALLEL REPORTER ASSAYS (MPRA) IN BETA CELL LINES. FINALLY, WE WILL EXPERIMENTALLY MANIPULATE THESE ISLET STRESS-RESPONSIVE GENES IN HUMAN ENDOC-SSH3 BETA CELLS USING CRISPR/CAS9 (EPI)GENOME EDITING TO DETERMINE THEIR FUNCTIONS IN BETA CELL PROLIFERATION, FUNCTION, AND SURVIVAL (AIM 3). THIS STUDY WILL PROVIDE MECHANISTIC INSIGHT INTO HOW HUMAN GENETIC VARIATION MODULATES THESE T2D-RELEVANT STRESS RESPONSES IN HUMAN ISLETS. BY DELINEATING THE GENES AND PATHWAYS THAT MODULATE ISLET (GLUCO)LIPOTOXIC AND ER STRESS RESPONSES AND EXPERIMENTALLY VALIDATING THEIR EFFECTS ON ISLET/BETA CELL RESILIENCE, THIS STUDY WILL REVEAL NOVEL THERAPEUTIC TARGETS AND GUIDE STRATEGIES FOR SUBSEQUENT STUDIES MANIPULATING THESE RESPONSES TO PREVENT OR TREAT ISLET FAILURE AND T2D.

CPDM: CLONING A GENE THAT REGULATES EOSINOPHIL FUNCTION

THE JACKSON LABORATORY GENE EDITING TESTING CENTER (JAX-GETC)

DIABETOGENIC ROLE OF H-2G7 CLASS I ALLELES IN NOD MICE

SPECIAL MOUSE STRAINS RESOURCE

INCREASING THE YIELD AND UTILITY OF PEDIATRIC GENOMIC MEDICINE WITH EXOMISER - PROJECT SUMMARY AS MUCH AS 10% OF THE POPULATION SUFFERS FROM A RARE DISEASE (RD); 80% OF THESE DISEASES ARE CAUSED BY GENE MUTATIONS AND UP TO 75% ARE PRESENT AT BIRTH OR BEGIN IN CHILDHOOD. DIAGNOSIS OF GENETIC DISEASES IS OFTEN PROBLEMATIC: ROUGHLY 25% OF RD PATIENTS MUST WAIT BETWEEN 5 AND 30 YEARS FOR A DIAGNOSIS, AND ABOUT HALF OF THE INITIAL DIAGNOSES ARE WRONG. FOR MANY AFFECTED CHILDREN, DEFINITIVE DIAGNOSIS COMES ONLY AFTER A PROTRACTED AND FRUSTRATING ODYSSEY OF VISITS TO DIFFERENT SPECIALISTS. EMERGING GENETIC SEQUENCING TECHNIQUES OFFER THE POSSIBILITY OF SHORTENING THIS LONG AND COSTLY PATH TO DIAGNOSIS. METHODS FOR DETERMINING THE CHANGES IN GENE SEQUENCES ACROSS ALL GENES (EXOME SEQUENCING) OR ALL GENETIC MATERIAL (GENOME SEQUENCING), COLLECTIVELY REFERRED TO AS NEXT-GENERATION SEQUENCING (NGS), AND WHICH WERE FIRST USED TO IDENTIFY THE GENETIC CAUSE OF A DISEASE IN 2010, ARE NOW BECOMING ROUTINE IN THE CLINIC. THE ABILITY TO MAKE A DIAGNOSIS WITH NGS HAS MORE THAN DOUBLED SINCE 2010 FOR CHILDREN WITH SUSPECTED GENETIC DISEASES. THE DIAGNOSTIC ANALYSIS OF NGS DATA INVOLVES THE ASSESSMENT OF TENS OF THOUSANDS (EXOME) OR EVEN MILLIONS (GENOME) OF CHANGES IN THE DNA (VARIANTS), WHICH REQUIRES SOPHISTICATED COMPUTER ALGORITHMS THAT CAN SIFT THROUGH THESE/THIS DATA TO FIND THE CAUSE. OUR GROUP HAS DEVELOPED THE HUMAN PHENOTYPE ONTOLOGY (HPO), A RESOURCE WIDELY USED AROUND THE WORLD FOR THE COMPUTATIONAL ANALYSIS OF CLINICAL DATA IN HUMAN GENETICS AND PEDIATRICS, ALLOWING ALGORITHMS TO MATCH THE SYMPTOMS OF A PATIENT WITH DATABASE RECORDS OF OVER 7,000 GENETIC DISEASES. OUR EXOMISER SOFTWARE COMPARES THE CLINICAL PHENOTYPES OF PATIENTS WITH KNOWN HUMAN DISEASES AND GENETICALLY MODIFIED ANIMAL MODELS, AND COUPLES THIS WITH AN ANALYSIS OF THE DISEASE-CAUSING POTENTIAL OF DNA VARIANTS, GREATLY REDUCING THE SEARCH SPACE TO IDENTIFY THE CAUSAL VARIANT. EXOMISER EFFICIENTLY PROCESSES BOTH EXOME AND GENOME DATA. IN THIS PROPOSAL, WE PLAN TO EXTEND EXOMISER TO UTILIZE NEW GENOMIC DATA TYPES INCLUDING LONG-READ GENOME SEQUENCING AND NGS-BASED ANALYSIS OF RNA DATA, WHICH WILL IMPROVE PATHOGENICITY PREDICTION FOR STRUCTURAL VARIANTS (SVS) AND FOR VARIANTS AFFECTING GENE EXPRESSION OR SPLICING. WE WILL ALSO PREDICT NOVEL DISEASE GENES THROUGH CHARACTERIZATION OF NETWORKS OF CLINICAL PHENOTYPES AND THE MOLECULAR FUNCTIONS (PATHWAYS) OF AFFECTED GENES. WE PLAN TO USE THESE ALGORITHMS TO ASSESS COLLECTIONS (COHORTS) OF UNSOLVED CASES IN PROJECTS SUCH AS THE 100,000 GENOMES PROJECT. OUR ALGORITHMIC APPROACH WILL BE APPLIED TO INTELLIGENTLY REANALYZE UNSOLVED CASES PERIODICALLY AS NEW INFORMATION IS ADDED TO THE MEDICAL LITERATURE. AND FINALLY, WE WILL DEVELOP TOOLS TO INTEGRATE EXOMISER INTO A LARGE RANGE OF SETTINGS BY ADDING SUPPORT FOR STANDARDS GENERATED BY THE GLOBAL ALLIANCE FOR GENOMICS AND HEALTH (GA4GH). THE PROPOSED ADVANCES WILL MAKE EXOMISER MORE EFFICIENT, MORE ACCURATE, AND EASIER FOR NON-SPECIALIST PEDIATRICIANS TO USE, BRINGING GENOMIC DIAGNOSTICS TO ROUTINE PEDIATRIC CLINICAL CARE.

PEDIATRIC ONCOLOGY IN VIVO TESTING PROGRAM COORDINATING CENTER - PROJECT SUMMARY THE OVERARCHING GOAL OF NCI'S PEDIATRIC IN VIVO TESTING PROGRAM (PED-IN VIVO-TP) IS TO IMPROVE OUTCOMES OF PEDIATRIC CANCER PATIENTS AND TO SATISFY THE REQUIREMENTS OF THE RESEARCH TO ACCELERATE CURES AND EQUITY (RACE) FOR CHILDREN ACT TO ASSESS THE EFFICACY OF TARGETED ANTI-CANCER AGENTS DEVELOPED FOR ADULTS IN PEDIATRIC CONTEXTS. TO COORDINATE THE ACTIVITIES OF THE NEW PED-IN VIVO-TP INITIATIVE, WE HAVE ASSEMBLED A TEAM OF INVESTIGATORS FROM THE JACKSON LABORATORY (JAX) AND SEVEN BRIDGES GENOMICS (SB) WITH UNIQUE COMBINED EXPERTISE AND EXPERIENCE WITH IN VIVO CANCER MODELS, SCALABLE CLOUD-BASED DATA MANAGEMENT AND ANALYSIS SYSTEMS, INFORMATICS RESOURCE DEVELOPMENT, AND MULTI-SITE PROJECT COORDINATION. COMBINING THE COMPLEMENTARY STRENGTHS OF JAX AND SB PROVIDES THE IDEAL FOUNDATION FOR A COORDINATING CENTER TO MAXIMIZE THE SHORT- AND LONG-TERM IMPACTS OF THE PEDIATRIC IN VIVO TESTING PROGRAM FOR ADVANCING THE APPLICATION OF PRECISION MEDICINE IN PEDIATRIC ONCOLOGY. WE WILL MANAGE A COMPREHENSIVE AND COHESIVE TESTING PROGRAM TO ADVANCE PRECISION MEDICINE IN PEDIATRIC ONCOLOGY THROUGH EFFECTIVE PUBLIC-PRIVATE PARTNERSHIPS AMONG PHARMACEUTICAL COMPANIES, REGULATORY AGENCIES, FUNDERS, AND RESEARCH ORGANIZATIONS. WE WILL ACHIEVE THIS GOAL THROUGH THE FOLLOWING AIMS: AIM 1: ESTABLISH AND MAINTAIN THE PEDIATRIC IN VIVO TESTING COORDINATING CENTER (PIVOT CC) TO PROVIDE ADMINISTRATIVE AND LOGISTICAL SUPPORT FOR DIVERSE STAKEHOLDERS IN THE PEDIATRIC IN VIVO TESTING PROGRAM CONSORTIUM. WE WILL DRAW ON OUR TEAM'S COMBINED DECADES OF EXPERIENCE WITH MULTI-SITE PROGRAM MANAGEMENT, CANCER MODEL DEVELOPMENT, STANDARDIZED TESTING OF IN VIVO CANCER MODELS, DATA MANAGEMENT AND ANALYSIS, AND INFORMATICS RESOURCE DEVELOPMENT TO ENSURE TIMELY DECISION MAKING, CONFORMANCE TO STANDARD PROTOCOLS, RESOURCE TRACKING, AND EFFECTIVE COMMUNICATIONS WITHIN THE PED-IN VIVO-TP. AIM 2: PROVIDE DATA MANAGEMENT, STATISTICAL, AND BIOINFORMATICS SUPPORT TO ENSURE DATA SECURITY AND INTEGRITY. WE WILL LEVERAGE EXISTING PROTOCOLS AND SOFTWARE SYSTEMS AT JAX AND SB TO IDENTIFY RELEVANT IN VIVO CANCER MODELS AND TO COLLECT, ANALYZE, AND SECURELY MANAGE DATA GENERATED FROM TESTING CENTERS WITHIN THE CONSORTIUM. WE WILL PERFORM STATISTICAL AND BIOINFORMATIC ANALYSES ON CONSORTIUM DATA TO RELIABLY INFORM THE EVALUATION OF THE EFFICACY OF NOVEL THERAPEUTIC AGENTS IN A PEDIATRIC ONCOLOGY SETTING. WE WILL DEVELOP A PUBLIC- FACING DATA PORTAL FOR SHARING OF DATA AND METHODS WITH THE BROADER SCIENTIFIC COMMUNITY. AIM 3: PROVIDE SCIENTIFIC COORDINATION TO MAINTAIN AN EFFICIENT AND EFFECTIVE PRECLINICAL TESTING PIPELINE. WE WILL DRAW FROM THE EXTENSIVE EXPERIENCE OF OUR TEAM WITH IN VIVO PHARMACOLOGY AND COORDINATION OF SIMILAR CONSORTIA TO MANAGE AND COORDINATE THE TESTING OF AGENTS BY THE PED-IN VIVO-TP CENTERS AT ALL STAGES OF THE PROCESS, FROM THE IDENTIFICATION OF RELEVANT IN VIVO MODELS TO THE GENERATION OF FINAL TECHNICAL REPORTS AND PUBLICATION OF RESULTS. WE WILL DEVELOP, RECORD, AND TRACK PERFORMANCE METRICS FOR THE CONSORTIUM TO INFORM THE EVALUATION OF THE PROGRAM'S SUCCESS AND IMPACT.

GENE X ENVIRONMENT INTERACTIONS IN HYPOTHALAMIC DYSFUNCTION IN ALZHEIMER'S DISEASE

DEVELOPING EFFECTIVE APPROACHES TO EXTEND HEMATOPOIETIC HEALTHSPAN BY TARGETING CELL-EXTRINSIC AND CELL-INTRINSIC ALTERATIONS AT MIDDLE AGE

ESTABLISHMENT AND CHARACTERIZATION OF NOVEL MUTANT MOUSE MODELS FOR THE ADDICTION RESEARCH COMMUNITY - PROJECT SUMMARY/ABSTRACT ADDICTION IS AN ENORMOUS ECONOMIC, PERSONAL, AND SOCIAL BURDEN, COSTING OVER $600 BILLION PER YEAR IN THE U.S. UNDERSTANDING VULNERABILITY TO ADDICTION, AND DEVELOPING EFFECTIVE THERAPIES, REQUIRES IDENTIFYING THE GENES AND PATHWAYS THAT MEDIATE THE ADDICTION PROCESS. OUR LONG-TERM GOAL IS TO DEVELOP NOVEL GENETIC MODELS FOR ADDICTION-RELEVANT PHENOTYPES, AND USE THESE MODELS TO CHARACTERIZE THE GENETIC MECHANISMS OF ADDICTION. WE PROPOSE TO LEVERAGE THE JACKSON LABORATORY KNOCKOUT MOUSE PROJECT 2 (JAX KOMP2) PIPELINE TO PRIORITIZE ADDICTION GENE CANDIDATES, AND THEN CHARACTERIZE THE EFFECTS OF CANDIDATE GENE KNOCKOUTS ON ADDICTION-RELATED BEHAVIORS AND ON ADDICTION-RELEVANT TISSUES. THE JAX KOMP2 PHENOTYPING CENTER PERFORMS HIGH-THROUGHPUT PHENOTYPING OF KNOCKOUT MICE ACROSS ORGAN SYSTEMS USING AN EFFICIENT, BROAD-BASED TESTING PIPELINE INCLUDING BEHAVIORAL ASSAYS FOR EMOTIONALITY AND SLEEP, BOTH PREDICTIVE OF ADDICTION PHENOTYPES. HERE WE PROPOSE TO EXPLOIT THIS RICH KOMP2 DATASET TO SELECT A SUBSET OF LINES WITH EMOTIONALITY AND NEURONAL PHENOTYPES (E.G. DEVIANT OPEN FIELD, LIGHT DARK, HOLE BOARD, TAIL SUSPENSION, PREPULSE INHIBITION, ROTAROD, ELECTROCONVULSIVE SEIZURE THRESHOLD, OR SLEEP PHENOTYPES) AND LACKING METABOLISM AND PHYSIOLOGY PHENOTYPES. OUR PRELIMINARY DATA PROVIDE COMPELLING EVIDENCE THAT GENE DELETIONS LEADING TO EMOTIONALITY PHENOTYPES IN THE KOMP PIPELINE HAVE ADDICTION PHENOTYPES. WE WILL SUBJECT THESE LINES TO DEEP DRUG ABUSE–RELEVANT PHENOTYPING, INCLUDING DRUG SELF-ADMINISTRATION, TRANSCRIPTIONAL PROFILING FROM KEY NEURONAL TISSUES, AND WHOLE BRAIN IMAGING. THE DATA FROM THESE WILL BE INTEGRATED USING SYSTEMS ANALYSIS. THE SUCCESSFUL COMPLETION OF THIS PROJECT WILL YIELD DOZENS OF NOVEL MOUSE MODELS WITH DETAILED TRANSCRIPTOME, AND NEUROANATOMICAL PROFILE TO ESTABLISH MECHANISTIC INSIGHT INTO THIS BEHAVIORAL ABNORMALITY. THESE CAN SERVE ARE A RESOURCE FOR THE RESEARCH COMMUNITY FOR THERAPEUTICS DEVELOPMENT.

HOW ANTIGEN EXPOSURE SHAPES THE SUBSEQUENT NK CELL RESPONSE TO HIV - THE GOAL OF THIS PROPOSAL IS TO DETERMINE HOW ANTIGEN EXPOSURE SHAPES SUBSEQUENT NATURAL KILLER CELL RESPONSES TO HIV. WE PROPOSE TO IDENTIFY NK FUNCTIONAL SUBSETS IN NAÏVE AND ANTIGEN-PRIMED HUMAN NK CELLS USING SINGLE-CELL SEQUENCING AND MULTI-PARAMETRIC FLOW CYTOMETRY. WE PROPOSE TO VERIFY THE RELEVANCE OF SUCH FUNCTIONAL SUBSETS TO NK CELL-MEDIATED HOST PROTECTION FROM HIV DISEASE USING OUR ESTABLISHED IN VITRO AND IN VIVO FUNCTIONAL ASSAYS. NK CELLS ARE INNATE LYMPHOCYTES THAT LIVE UP TO THEIR NAME BY THEIR ABILITY TO KILL INFECTED OR TUMOR CELLS WITHIN MINUTES OF EXPOSURE. HOWEVER, THE TARGETING OF NK CELL EFFECTOR FUNCTIONS HAS NOT BEEN A SIGNIFICANT FOCUS IN VACCINE DEVELOPMENT, WHICH HAS MAINLY FOCUSED ON THEIR T AND B CELL COUNTERPARTS IN THE ADAPTIVE IMMUNE SYSTEM. RECENT FINDINGS FROM THE PI OF THIS APPLICATION INDICATE THAT NK CELLS DESERVE MORE ATTENTION. WE RECENTLY PUBLISHED EXCITING NEW DATA THAT HUMAN NK CELLS REMEMBER PRIOR ANTIGEN- ENCOUNTERS AND MEDIATE ENHANCED RECALL RESPONSES TO HIV-ENVELOPE IN HUMANIZED MICE. HERE, WE PRESENT UNPUBLISHED NEW DATA THAT HIV-ENV-PRIMED MEMORY NK CELLS SUPPRESS HIV VIRAL TITERS UPON EXPERIMENTAL VIRAL CHALLENGE. THESE FINDINGS HAVE OPENED THE OPPORTUNITY TO HARNESS NK MEMORY FUNCTIONS FOR VACCINE DESIGN. HOWEVER, HIGH NK CELL RECEPTOR REPERTOIRE DIVERSITY IS ASSOCIATED WITH AN INCREASED RISK OF HIV ACQUISITION, AND NK CELL RECEPTOR REPERTOIRES DIVERSIFY THROUGHOUT LIFE, PRESUMABLY IN RESPONSE TO ANTIGEN EXPOSURE. THESE DATA PRESENT A CHALLENGE FOR VACCINE DESIGN, AS BOTH PROTECTIVE NK MEMORY RESPONSES AND POTENTIALLY RISKY NK REPERTOIRE DIVERSIFICATIONS ARE CONSEQUENCES OF ANTIGEN EXPOSURE. THE IDENTIFICATION OF SPECIFIC FUNCTIONAL SUBSETS OF HIV-RESPONSIVE, HOST-PROTECTIVE NK CELLS AND THEIR MECHANISMS OF HOST PROTECTION IS THEREFORE CRITICALLY NEEDED. THEIR DISCOVERY WILL OPEN THE DOOR FOR REVISED VACCINE DESIGNS THAT ENDURE THE INCORPORATION OF NK MEMORY, RATHER THAN HARMFUL RECEPTOR REPERTOIRE DIVERSITY, AS A HOST PROTECTIVE OUTCOME. OUR DATA WILL ENABLE THE PRE-SCREENING OF VACCINES FOR THE INDUCTION OF PROTECTIVE NK FUNCTIONAL SUBSETS IN PRE- CLINICAL MODELS AND ALLOW FOR IMPROVED VACCINE EFFICACY EVALUATIONS IN HUMANS. THEREBY, OUR STUDIES WILL PROVIDE THE RATIONALE TO DEVELOP NOVEL VACCINES THAT EXPLOIT THE ANTIVIRAL ACTIVITY OF NK CELLS TO PROTECT HUMANS FROM HIV INFECTION WHILE AVOIDING HARMFUL ACTIVITY.

HIGH THROUGHPUT PRODUCTION AND CRYOPRESERVATION OF KNOCKOUT MICE

SYSTEMS CONTROL OF NORMAL AGING AND ALZHEIMER'S DISEASE

GENETIC CONTROL OF PHOSPHOLIPID BIOSYNTHESIS AND MUSCULAR DYSTROPHY

SYSTEMS GENETICS APPROACH TO DETERMINE INTERACTORS OF APOLIPOPROTEIN E IN ALZHEIMER?S DISEASE

TRANSGENIC AND TARGETED MUTANT PRESERVATION

GENETIC CONTROL OF ADDICTION BY HOST AND MICROBIOME

MODELING THE GENETIC INTERACTION BETWEEN KLOTHO AND APOE ALLELES IN ALZHEIMER'S DISEASE - PROJECT SUMMARY UNDERSTANDING HOW COMBINATIONS OF GENETIC RISK FACTORS INFLUENCE RISK FOR LATE-ONSET ALZHEIMER’S DISEASE (LOAD) CAN LEAD TO TARGETED STRATEGIES FOR THERAPEUTIC INTERVENTION. APOLIPOPROTEIN E4 (APOE4), A COMMON VARIANT OF APOE, IS THE SINGLE LARGEST GENETIC RISK FACTOR FOR DEVELOPING LOAD.APOE4 STATUS IS LINKED TO INCREASED INFLAMMATION AND HIGHER SS-AMYLOID BURDEN IN LOAD PATIENTS. DESPITE THIS INCREASED GENETIC RISK PROFILE, APOE4 CARRIERS DO NOT ALWAYS DEVELOP LOAD IN THE COURSE OF THEIR LIFETIME. SEVERAL LARGE-SCALE GENETIC STUDIES HAVE IDENTIFIED A COMMON HAPLOTYPE OF THE AGING FACTOR KLOTHO THAT MODIFY AGE OF ONSET AND REDUCE AMYLOID PLAQUE DEPOSITION SPECIFICALLY IN APOE4 CARRIERS, SUGGESTING THAT KLOTHO VARIANTS CAN PROVIDE A PROTECTIVE EFFECT AGAINST THE DEVELOPMENT OF LOAD BY COUNTERACTING THE NEGATIVE EFFECTS OF APOE4. IN HUMANS, KLOTHO HARBORS TWO COMMON MISSENSE VARIANTS (RS9536314, P.F352V; RS9527025, P.C370S). THE COMBINATION OF THESE TWO CODING VARIANTS DEFINE THE KLOTHO V/S (KL-V/S) HAPLOTYPE, WHICH IS PROTECTIVE AGAINST LOAD IN APOE4 CARRIERS, AND THE KLOTHO F/C (KL-F/C) HAPLOTYPE, WHICH IS NOT PROTECTIVE AGAINST LOAD. THE OVERALL OBJECTIVE OF THIS PROPOSAL IS TO DETERMINE THE PHYSIOLOGICAL PROCESSES ALTERED BY KLOTHO AS AN APOE4-SPECIFIC PROTECTIVE FACTOR IN LOAD USING A SET OF RECENTLY-CREATED MOUSE MODELS HARBORING COMBINATIONS OF RELEVANT HUMAN VARIANTS IN BOTH KLOTHO AND APOE. OUR CENTRAL HYPOTHESIS IS THAT THE PROTECTIVE KL-V/S HAPLOTYPE WILL SIGNIFICANTLY DELAY AGE-DEPENDENT INFLAMMATION AND AMYLOID DEPOSITION WHILE THE REFERENCE KL-F/C HAPLOTYPE WILL FAIL TO ATTENUATE THESE HALLMARK LOAD PATHOLOGIES. WE WILL ASSESS MULTIPLE LOAD-RELEVANT OUTCOMES TO VALIDATE AND CHARACTERIZE THIS KLOTHO- APOE GENETIC INTERACTION WITH THREE SPECIFIC AIMS: (1) DETERMINE THE EFFECTS OF KLOTHO HAPLOTYPES ON AGE-RELATED FRAILTY AND KLOTHO ISOFORM LEVELS IN BLOOD AND CSF IN MICE; (2) DETERMINE CHANGES IN LOAD HALLMARK PATHOLOGIES DRIVEN BY THE INTERACTION BETWEEN KLOTHO AND APOE ALLELES IN MICE; AND (3) IDENTIFY MOLECULAR SIGNATURES SHARED IN HUMAN LOAD STRATIFIED BY KLOTHO HAPLOTYPE AND THE NOVEL KLOTHO MOUSE MODELS. THE OUTCOME OF THIS WORK WILL RESULT IN THE CHARACTERIZATION OF NEW MOUSE MODELS OF HUMAN KLOTHO HAPLOTYPES AND IDENTIFY THE PATHWAYS WHICH ARE DIFFERENTIALLY AFFECTED BY KLOTHO VARIANTS IN AN APOE-DEPENDENT MANNER. THIS INFORMATION WILL PROVIDE A BIOLOGICAL BASIS FOR THE EPISTATIC INTERACTION OBSERVED IN HUMAN GENETIC STUDIES, THEREBY PROVIDING THE NECESSARY FUNCTIONAL INFORMATION TO GUIDE POTENTIAL TREATMENTS BASED ON KL-V/S PROTECTION FOR APOE4 CARRIERS.

DATA STRUCTURES ALGORITHMS AND TOOLS FOR ONTOLOGICAL DISCOVERY

FROM QTL TO GENE FOR HDL CHOLESTEROL

PREDICTING TISSUE SPECIFIC GLI3 REGULATORY ACTIVITY USING HAND2 - PROJECT SUMMARY THE HEDGEHOG (HH) SIGNALING PATHWAY IS ESSENTIAL FOR NORMAL EMBRYONIC DEVELOPMENT AND WHEN PERTURBED, FREQUENTLY RESULTS IN HUMAN DISEASE, INCLUDING THOSE THAT IMPACT DEVELOPMENT OF THE CRANIOFACIAL COMPLEX. THE GLI TRANSCRIPTION FACTORS ARE THE DOWNSTREAM EFFECTORS OF THE PATHWAY AND HAVE BEEN THE SUBJECT OF MUCH RESEARCH AS THEY ARE ASSOCIATED WITH A NUMBER OF CRANIOFACIAL SYNDROMES (E.G., GRIEG CEPHALOPOLYSYNDACTYLY) AND CAN FUNCTION AS BOTH TRANSCRIPTIONAL ACTIVATORS AND REPRESSORS OF THE HH PATHWAY. GLI3 IS MOST STABLE AND ABUNDANT AS A REPRESSOR. DESPITE THIS, OUR RECENT WORK IDENTIFIED A SPECIFIC AND NECESSARY ROLE FOR GLI3 ACTIVATOR (GLI3A) FUNCTION DURING NORMAL DEVELOPMENT OF THE MANDIBLE THAT REQUIRES ADDITIONAL REGULATORY INPUTS TO CONVEY A ROBUST GLI3A RESPONSE. LITTLE IS KNOWN REGARDING WHAT IS REQUIRED FOR GLI3, AS A BIMODAL TRANSCRIPTION FACTOR, TO FUNCTION AS A POTENT ACTIVATOR DURING DEVELOPMENT. TO ADDRESS EXISTING KNOWLEDGE GAPS IN HOW FULL-LENGTH GLI3 IS CONVERTED INTO AN ACTIVATOR, WE ENGINEERED A SET OF ENDOGENOUSLY EPITOPE TAGGED ALLELES FOR GLI3. WITH THESE NOVEL TOOLS WE PROPOSE TO: (AIM1) DETERMINE IF/HOW CHROMATIN ACCESSIBILITY MODULATES GLI3A FUNCTION; (AIM2) INVESTIGATE THE ROLE OF CO-FACTORS AND REGULATORY GRAMMAR IN REGULATING ENHANCER OUTPUT; AND (AIM3) UNBIASEDLY IDENTIFY PROTEIN INTERACTORS OF GLI3A WITHIN THE NUCLEUS. WE WILL FOCUS ON CRANIOFACIAL DEVELOPMENT, SPECIFICALLY DEVELOPMENT OF THE MANDIBLE, AS A RELEVANT MODEL FOR TESTING THESE PRINCIPLES GIVEN THE REQUIREMENT FOR GLI ACTIVITY DURING GLOSSOGENESIS AND MANDIBULAR SKELETOGENESIS. THIS DEVELOPMENTAL SYSTEM WILL ALLOW US TO DETERMINE THE REQUIREMENT FOR CHROMATIN ACCESSIBILITY, EXHAUSTIVELY INTERROGATE THE REGULATORY GRAMMAR, AND IDENTIFY THE CONSTITUENTS OF THE GLI3 ACTIVATOR COMPLEX. RECENT TECHNOLOGICAL ADVANCES WILL ENABLE HYPOTHESIS TESTING THROUGH SINGLE-CELL ANALYSIS OF CHROMATIN ACCESSIBILITY AND TRANSCRIPTION PROFILES FROM MUTANTS PREDICTED TO HAVE PIONEERING ACTIVITY. TO VALIDATE OUR FINDINGS, WE WILL PERFORM IN VIVO EXPERIMENTS TO TEST ENHANCER ACTIVITY AND APPLY CRISPR/CAS9 MUTAGENESIS TO FUNCTIONALLY ASSESS NATIVE BINDING SITE REQUIREMENTS. COLLECTIVELY, OUR STUDIES WILL SHED LIGHT ON THE REGULATORY PRINCIPLES GOVERNING GLI-DIRECTED CELLULAR PROGRAMS THAT WHEN DISRUPTED CAN RESULT IN RANGE OF HUMAN DISORDERS RANGING FROM STRUCTURAL BIRTH DEFECTS TO CANCER. UNDERSTANDING HOW THESE PROGRAMS ARE DEPLOYED AND INTERPRETED DURING NORMAL DEVELOPMENT HAS THE POTENTIAL TO IMPROVE HUMAN HEALTH THROUGH THE EXPANSION OF THERAPEUTIC INTERVENTIONS THAT CAN HELP MITIGATE PATHWAY DYSREGULATION.

THE HUMAN PHENOTYPE ONTOLOGY: ACCELERATING COMPUTATIONAL INTEGRATION OF CLINICAL DATA FOR GENOMICS - PROJECT SUMMARY/ABSTRACT TO IMPROVE DIAGNOSTIC YIELD FOR RARE DISEASES, WE DEVELOPED THE HUMAN PHENOTYPE ONTOLOGY (HPO) IN 2008 AS A COMPREHENSIVE BIOINFORMATIC RESOURCE THAT PROVIDES A STANDARDIZED TERMINOLOGY OF PHENOTYPIC ABNORMALITIES FOR THE ANALYSIS OF HUMAN DISEASES. HPO REDUCES AMBIGUITY IN DISEASE DESCRIPTIONS—THUS ENABLING MORE ROBUST DIFFERENTIAL DIAGNOSIS AND CLINICAL CARE—AND ENABLES PHENOTYPIC CONTEXTUALIZATION OF GENOMIC DATA FOR DIAGNOSTICS AND PRECISION MEDICINE. THE PERFORMANCE OF COMPUTATIONAL ALGORITHMS FOR DIFFERENTIAL DIAGNOSTICS WITH HPO TERMS DEPENDS CRITICALLY ON THE COMPREHENSIVENESS AND DEPTH OF HPO ANNOTATIONS FOR DISEASES. HOWEVER, THE CURRENT MANUAL NATURE OF OUR BIOCURATION PROCESS HAS LIMITED THE QUALITY, DEPTH, AND COVERAGE OF THESE ANNOTATIONS. THEREFORE, THIS PROPOSAL'S OBJECTIVES ARE TO GREATLY EXPAND THE CORPUS OF DISEASE-PHENOTYPE ANNOTATIONS BY AUTOMATING PORTIONS OF THE CURATION AND EXPANDING THE COMPUTATIONAL DISEASE MODEL. THIS PROJECT, HPO: ACCELERATING COMPUTATIONAL INTEGRATION OF CLINICAL DATA FOR GENOMICS, WILL MAINTAIN AND ADVANCE HPO RESOURCES TO ADDRESS THE NEEDS OF A GROWING NUMBER OF MEDICAL DISCIPLINES THAT HAVE ADOPTED THE HPO. WE WILL ACHIEVE THIS GOAL BY 1) AUTOMATING HPO DEVELOPMENT, MAINTENANCE, AND RELEASE PROCESSES, 2) DEVELOPING REPRESENTATIONS OF RARE DISEASE TREATMENTS AND INTERVENTIONS, AND 3) EXTENDING OUR CURRENT COMPUTATIONAL DISEASE MODELS TO REPRESENT TIME COURSE, SEX BIASES, AND FREQUENCY OF EVENTS, AND TO INCORPORATE CASE REPORT DATA. WE ALSO PROVIDE A SUSTAINABLE SOLUTION TO COMMUNITY CONTRIBUTION WITH A USER-FRIENDLY, WEB- BASED PORTAL TO ENABLE CONTRIBUTORS TO VET AND SUGGEST IMPROVEMENTS TO THE ONTOLOGY AND THE ANNOTATIONS AND GROW THE HPO CONTRIBUTOR COMMUNITY. IN SUMMARY, OUR PROJECT ADDRESSES THE MOST PRESSING NEEDS FOR ADVANCEMENTS OF THE HPO TO ENSURE SUSTAINABLE, ROBUST, AND RIGOROUS DEVELOPMENT, TO ENABLE HPO RESOURCES TO SUPPORT NEW COMMUNITIES, NEW APPLICATIONS, AND MORE MEDICAL DISCIPLINES.

COMBINATION ADJUVANTS TO ACTIVATE HUMAN DENDRITIC CELL SUBSETS AND B CELLS

SYNAPTIC REFINEMENT IN THE THALAMUS

INVESTIGATING THE ROLE OF COMPLEMENT-EXPRESSING MYELOID CELLS IN SYNAPSE LOSS AND VASCULAR COMPROMISE IN ALZHEIMERS DISEASE

ETHICAL, LEGAL, SOCIAL, AND POLICY IMPLICATIONS OF WORKPLACE GENOMIC TESTING

MODULAR PLATFORM FOR COMBINATORIAL EPIGENOME MANIPULATION

NUCLEOME POSITIONING SYSTEM FOR SPATIOTEMPORAL GENOME ORGANIZATION AND REGULATION

GENETIC FACTORS THAT INFLUENCE ARSENIC TOXICITY

NOVEL MECHANISMS REGULATING IMMUNITY TO RESPIRATORY VIRUS INFECTION - INFLUENZA VIRUSES ARE RAPIDLY MUTATING RNA VIRUSES AND ARE THE CAUSATIVE AGENT OF ABOUT ONE BILLION ANNUAL RESPIRATORY VIRUS INFECTIONS AND 500,000 DEATHS WORLDWIDE. INFLUENZA-RELATED DEATHS ARE GENERALLY ATTRIBUTABLE TO VIRAL OR BACTERIAL PNEUMONIA (FROM SECONDARY BACTERIAL INFECTIONS); EXCESSIVE INFLAMMATION RESULTING IN ACUTE RESPIRATORY DISTRESS SYNDROME; AND SEVERE LUNG IMMUNOPATHOLOGY, LEADING TO HYPOXIA AND MULTI-ORGAN FAILURE. INFLUENZA VIRUSES HAVE SIGNIFICANT PANDEMIC POTENTIAL, SEASONAL EPIDEMICS BURDEN THE HUMAN POPULATION, AND VIRAL RESISTANCE HAS DEVELOPED TO ALL AVAILABLE TREATMENT OPTIONS. MUCH EMPHASIS IS PLACED ON THE HUMORAL IMMUNE RESPONSE TO INFLUENZA, AS NEUTRALIZING ANTIBODIES ARE THE DESIRED VACCINE OUTCOME. HOWEVER, B CELL- DEFICIENT MICE AND HUMANS WITH HYPER-IGM SYNDROME CLEAR INFLUENZA VIRUS INFECTIONS, WHILE T CELL-DEFICIENT MICE DO NOT. THUS, B CELL-INDEPENDENT MECHANISMS PROTECT AGAINST INFLUENZA VIRUS-RELATED MORTALITY. HOWEVER, THE IMMUNE RESPONSE TO INFLUENZA VIRUS INFECTION REMAINS POORLY UNDERSTOOD, AND MUCH-NEEDED THERAPEUTICS AUGMENTING THE ANTIVIRAL IMMUNE RESPONSE WHILE PREVENTING HARMFUL IMMUNOPATHOLOGY REMAIN TO BE DEVELOPED. TO ADDRESS THIS KNOWLEDGE GAP, WE RECENTLY GENERATED NOVEL AND COMPELLING EVIDENCE THAT INFLUENZA A VIRUS (IAV) INFECTION TRIGGERS LUNG MAST CELLS (MCS) TO PRODUCE THE ANTI-INFLAMMATORY CYTOKINE IL-10 (MC-IL-10). IN WILD- TYPE (WT) AND T- AND B-CELL DEFICIENT (RAG1-KO) MICE, IAV/MC-IL-10 INDUCES THE EXPRESSION OF THE IL-10 RECEPTOR (IL-10R) AND PROGRAMMED CELL DEATH LIGAND 1 (PD-L1) ON NATURAL KILLER (NK) CELLS. NOTABLY, IN RAG1-KO MICE, WHERE NK CELLS ARE THE SOLE VIRUS-FIGHTING LYMPHOCYTES, PD-L1 BLOCKADE, BUT NOT PD-1, PD-L2, OR CD80 BLOCKADE, SIGNIFICANTLY REDUCES IAV-RELATED LETHALITY. THE IAV/MC-IL10/NK-PD-L1 PATHWAY IS ALSO CONSERVED IN HUMANS, AT LEAST IN VITRO: IAV INFECTION OF HUMAN-LUNG TISSUE-DERIVED SINGLE-PRIMARY-CELL SUSPENSIONS OR INTACT HUMAN LUNG TISSUE SLICES ELICIT MC-IL-10 AND NK CELL-EXPRESSED IL-10R AND PD-L1. IN MICE AND HUMANS, T CELLS ALSO UPREGULATE THE IL-10R, PD-1, AND PD-L1 UPON IAV INFECTION. FURTHER, IAV-INFECTED IL-10-KO/RAG-WT MICE, WHOSE NK AND T CELLS DO NOT UPREGULATE IL-10R, PD-1, PD-L1, OR PD-L2, AND IAV-INFECTED WT MICE IN WHICH PD-L1 IS BLOCKED, DEVELOP PROLONGED IMMUNE INFILTRATION AND IMMUNOPATHOLOGY AFTER IAV CLEARANCE. OUR FINDINGS ARE NOVEL AND SURPRISING. THE INDUCTION OF THE PD/PD-L PATHWAY IS GENERALLY ASSOCIATED WITH LYMPHOCYTE EXHAUSTION (VIA T CELL-EXPRESSED PD-1) IN CANCER OR CHRONIC INFECTION RATHER THAN THE MODULATION OF LYMPHOCYTE FUNCTION IN RESPONSE TO AN ACUTE VIRAL ILLNESS. WE HYPOTHESIZE THAT INFLUENZA VIRUS-INDUCED MC-IL-10 BALANCES HELPFUL ANTIVIRAL RESPONSES WITH HARMFUL IMMUNOPATHOLOGY THROUGH PDL1 SIGNALING IN NK CELLS, AND PD-1 AND/OR PD-L1 SIGNALING IN T CELLS. WE PROPOSE IDENTIFYING THE MECHANISMS OF IAV/MC/IL-10/PD-L1-MEDIATED NK CELL AND IAV/MC/IL-10/PD-1 AND/OR PD-L1-MEDIATED T CELL REGULATION AND EACH PATHWAY'S CONTRIBUTION TO VIRAL CLEARANCE VS. LUNG TISSUE DAMAGE. OUR PROPOSAL IS HIGHLY SIGNIFICANT TO HUMAN HEALTH, AS IT HAS GREAT POTENTIAL TO IDENTIFY THERAPEUTIC TARGETS FOR ALLEVIATING IAV IMMUNOPATHOLOGY-ASSOCIATED MORTALITY AND MORBIDITY.

MECHANISM AND FUNCTIONAL SIGNIFICANCE OF POLARITY REVERSAL IN MECHANOSENSORY ORGANS

COMMUNITY PROJECT FUNDING/CONGRESSIONALLY DIRECTED SPENDING - CONSTRUCTION

GENOMIC BIOLOGY OF THE TANDEM DUPLICATOR PHENOTYPE IN MOUSE AND HUMAN CANCERS - PROJECT SUMMARY WE HAVE IDENTIFIED A GROUP OF GENOME INSTABILITY CONFIGURATIONS CALLED THE TANDEM DUPLICATOR PHENOTYPES (TDPS) THAT ARE FOUND IN ~50% OF TRIPLE NEGATIVE BREAST, OVARIAN AND ENDOMETRIAL CANCERS AND ARE CHARACTERIZED BY THE MASSIVE GENOME-WIDE DISTRIBUTION OF SOMATIC TANDEM DUPLICATIONS (TDS) OF SPECIFIC SPAN SIZES. WE HAVE IDENTIFIED THE BONA FIDE GENETIC DRIVERS OF THESE CONFIGURATIONS, DEMONSTRATED THAT LOSS OF TRP53 AND BRCA1 IN THE MOUSE MAMMARY GLAND IS SUFFICIENT TO INDUCE TUMORS WITH THE SHORT-SPAN TDP CONFIGURATION FOUND IN TP53- AND BRCA1-DEFICIENT HUMAN CANCERS, AND SHOWN THAT UPON LOSS OF BRCA1, TDS ARE FORMED THROUGH THE ABERRANT REPAIR OF STALLED REPLICATION FORKS. HERE, WE PROPOSE TO DEPLOY A COMBINATION OF COMPUTATIONAL ANALYSES, IN VIVO MODELLING AND IN VITRO EXPERIMENTATION TO ACHIEVE A DEEP MECHANISTIC UNDERSTANDING OF HOW THE DISTINCT TDP GENOMIC CONFIGURATIONS EMERGE AND IMPACT THE COURSE OF BREAST TUMORIGENESIS. SPECIFICALLY, WE WILL INVESTIGATE THE MOLECULAR MECHANISMS LEADING TO DE NOVO TD FORMATION ACROSS THE DIFFERENT TDP GROUPS BY EXPLORING HOW LOCAL DNA FEATURES ASSOCIATED WITH DNA REPLICATION AND FORK STALLING CONTRIBUTE TO THE GENERATION OF NEW TDS ACROSS A LARGE PAN-CANCER DATASET REPRESENTING ALL TDP GROUPS AND ALL TDP GENETIC DRIVERS (AIM 1A) AND HOW LOSS OF BRCA1 ACTIVITY MAY MODULATE THE SPREAD AND LOCATION OF THE DE NOVO TDS FORMED IN THE CONTEXT OF THE SHORT-SPAN TDP (AIM 1B). WE WILL ESTABLISH NEW GENETICALLY ENGINEERED MOUSE MODELS (GEMMS) OF BREAST CANCER TO VALIDATE THAT ACTIVATION OF THE CCNE1 PATHWAY OR LOSS OF CDK12 ACTIVITY, BOTH IN CONJUNCTION WITH TRP53 LOSS OF FUNCTION, INDUCES MEDIUM- AND LONG-SPAN TDP CONFIGURATIONS THAT MIMIC THEIR HUMAN COUNTERPARTS BOTH IN TERMS OF TD SPAN SIZE AND DISTRIBUTION (AIM 2A) AND OF THE GENOMIC FEATURES AND GENETIC ELEMENTS THAT ARE ASSOCIATED WITH AND AFFECTED BY TD FORMATION (AIM 2B). WE WILL ALSO ASSESS THE TUMOR NEO-ANTIGEN LOAD OF THE TDP TUMORS EMERGING FROM THE NEWLY DEVELOPED GEMMS AND TEST WHETHER IMMUNO-ONCOLOGY AGENTS ARE EFFECTIVE AGAINST MAMMARY TUMORS WITH THE TDP CONFIGURATION, AS SUGGESTED BY RECENTLY EMERGING CLINICAL OBSERVATIONS (AIM 2A). WE WILL THEN USE ISOGENIC HUMAN CANCER CELL LINES THAT ARE EITHER PROFICIENT OR DEFICIENT FOR BRCA1 ACTIVITY, TO DETERMINE THE DYNAMICS OF DE NOVO TD FORMATION UNDER DIFFERENT MODES OF CELLULAR PERTURBATION AND AS A FUNCTION OF BRCA1 STATUS (AIM 3A). FINALLY, WE WILL USE THE NEWLY DEVELOPED GEMMS TO UNDERSTAND THE EVOLUTIONARY PATH TO GENOME-WIDE TD DISTRIBUTION IN THE MAMMARY GLAND, AND TO DISCERN THE DYNAMICS OF TDP EMERGENCE, BOTH IN TERMS OF THE RATE OF DE NOVO TD FORMATION AND WITH RESPECT TO THE TIMELINE OF BREAST TUMORIGENESIS (AIM 3B). IF SUCCESSFUL, THIS PROPOSAL WILL UNCOVER THE ROOT CAUSES OF A SIGNIFICANT FORM OF GENOMIC INSTABILITY IN HUMAN CANCER, THE TDP, DEFINE THE MUTATIONAL DYNAMICS LEADING TO CANCER FORMATION IN THIS CONDITION, AND GENERATE MODEL SYSTEMS THAT CAN LEAD TO THE DEVELOPMENT OF NEW AND DIRECTED THERAPEUTICS AGAINST CANCER GROWTH.

GENETICS OF AGE-RELATED HEARING LOSS

MOUSE MUTANT RESOURCE

GENETIC VARIATION IN OPIOID INDUCED RESPIRATORY DEPRESSION IN MICE

(PQ3) CELLULAR AND MOLECULAR MECHANISMS DRIVING MYELOID COMPARTMENT VARIATION IN HUMAN TRIPLE NEGATIVE BREAST CANCER

DECODING IMMUNOLOGICAL PERTURBATIONS DURING CHRONIC FATIGUE SYNDROME

GENETICALLY DIVERSE MOUSE EMBRYONIC STEM CELLS: A PLATFORM FOR CELLULAR SYSTEMS GENETICS - PROJECT SUMMARY THE OBJECTIVE OF THIS APPLICATION IS TO GENERATE A THOROUGHLY-VALIDATED PANEL OF GENETICALLY DIVERSE MOUSE EMBRYONIC STEM CELLS (MESC) THAT WILL ENABLE WIDESPREAD ADOPTION OF CELLULAR SYSTEMS GENETICS. PHENOTYPIC VARIATION, MANIFESTING AS HETEROGENEITY IN CELL STATE, REPRESENTS A SIGNIFICANT CHALLENGE FOR REALIZING THE FULL PROMISE OF INDIVIDUALIZED, CELL-BASED THERAPIES, REGENERATIVE MEDICINE. BUT PHENOTYPIC VARIATION IN GENETICALLY DIVERSE STEM CELLS ALSO PRESENTS AN OPPORTUNITY FOR THE ADVANCEMENT OF LARGE SCALE, CELLULAR SCREENS OF GENE BY ENVIRONMENT INTERACTIONS (E.G. PHARMACOGENOMICS, TOXICOGENOMICS). A VARIETY OF APPROACHES ARE BEGINNING TO IDENTIFY THE NETWORKS THAT DRIVE CELL STATE TRANSITIONS, BUT THESE EFFORTS HAVE LARGELY FOCUSED ON BULK ASSAYS, WHICH DO NOT PROVIDE SUFFICIENT RESOLUTION OF CELL STATE HETEROGENEITY, AND MASK THE CONTRIBUTION OF UNDERLYING GENETIC VARIATION ON RARE CELL TYPES. MOREOVER, GENETIC STUDIES USING HUMAN PLURIPOTENT STEM CELLS ARE LARGELY LIMITED TO TESTING COMMON VARIANTS DUE TO LOW ALLELE FREQUENCIES AND IMBALANCED POPULATION STRUCTURE REQUIRING PROHIBITIVELY LARGE SAMPLES AND IMPEDING IDENTIFICATION OF CORE REGULATORY NETWORKS WITH HIGH POWER AND RESOLUTION. THEREFORE, WE CURRENTLY LACK A THOROUGH UNDERSTANDING OF THE GENES AND MECHANISMS THAT UNDERLIE PHENOTYPIC VARIATION IN PLURIPOTENT STEM CELLS. THE DIVERSITY OUTBRED (DO) MOUSE POPULATION AT THE JACKSON LABORATORY IS GENETICALLY DEFINED, DIVERSE, AND PRESENTS A SINGULAR, COST-EFFECTIVE OPPORTUNITY TO SYSTEMATICALLY INVESTIGATE HETEROGENEITY IN MAMMALIAN PLURIPOTENCY. OUR PILOT STUDIES USING DO MESCS ESTABLISH THE FEASIBILITY OF IDENTIFYING REGULATORY LOCI AT HIGH POWER AND RESOLUTION, AS WELL AS NETWORKS CONSERVED IN MICE AND HUMANS THAT REGULATE CELL STATE TRANSITIONS. IN AIM 1, WE WILL CREATE A REFERENCE MAPPING PANEL OF 300 DO MESC LINES THAT WILL SERVE AS A GOLD STANDARD RESOURCE FOR CELLULAR SYSTEMS GENETICS. THIS PANEL WILL BE FULLY CREDENTIALED AND BANKED FOR BROAD AVAILABILITY THROUGH THE JACKSON LABORATORY / MUTANT MOUSE RESOURCE AND RESEARCH CENTERS (MMRRC). IN AIM 2, WE WILL DETERMINE AT THE SINGLE CELL LEVEL THE TRANSCRIPTIONAL NETWORKS THAT REGULATE CELL STATE TRANSITIONS IN VITRO THROUGH THE EARLY STAGES OF DIFFERENTIATION TO MESODERM IN A REPRESENTATIVE SUBSET OF 144 LINES. IN AIM 3, WE WILL MAP QUANTITATIVE TRAIT LOCI (QTL) THAT UNDERLIE VARIATION IN CELL STATE-SPECIFIC GENE EXPRESSION AND IN THE DISTRIBUTION OF CELL STATES IN A POPULATION. IN ADDITION, WE WILL BUILD AND TEST MODELS BASED ON POLYGENIC SCORES THAT CAN PREDICT DIFFERENTIATION PROPENSITY FROM GENOTYPE. FINALLY, A WEB-BASED SEARCHABLE DATABASE OF EXPRESSION PHENOTYPES AND INTERACTIVE TOOLS FOR VISUALIZATION OF CELL COMPOSITION AND EQTL WILL BE MADE PUBLICLY AVAILABLE TO SUPPORT COMMUNITY QUERIES AND HYPOTHESIS GENERATION. IN SUM, WE WILL PRODUCE A RESOURCE OF CELL LINES AND GENE EXPRESSION DATA FOR THE RESEARCH COMMUNITY THAT WILL SPUR NEW DISCOVERIES IN REGENERATIVE MEDICINE, PHARMACOGENOMICS, AND TOXICOGENOMICS.

TECHNOLOGY PROJECT: GENETIC TOOLS AND RESOURCES FOR OROFACIAL CLEFTING RESEARCH

IMMUNOBIOLOGY OF REGULATORY T CELLS IN HIV INFECTION

CELLULAR SYSTEMS GENETIC APPROACHES TO UNDERSTANDING REGULATORY VARIATION

EVOLUTIONARY GENOMICS OF FUNCTIONAL CHROMATIN DOMAINS

DECONSTRUCTION OF A HYPOTHALAMIC EXERCISE-RESPONSIVE CIRCUIT FOR NEUROPROTECTION - PROJECT SUMMARY EXERCISE SLOWS THE COGNITIVE DECLINES ASSOCIATED WITH AGING AND PROTECTS AGAINST THE DEVELOPMENT AND PROGRESSION OF NEURODEGENERATIVE DISEASES SUCH AS ALZHEIMER'S DISEASE (AD). AT THE CELLULAR LEVEL, EXERCISE ENHANCES SYNAPTIC CONNECTIVITY AND REDUCES MARKERS OF NEUROINFLAMMATION IN AGING CORTICAL CIRCUITS. EXACTLY HOW EXERCISE SIGNALS IN THE BRAIN GENERATE THESE NEUROPROTECTIVE EFFECTS REMAINS UNKNOWN. OUR PRELIMINARY EXPERIMENTS HAVE IDENTIFIED A SET OF NEURONS IN THE MOUSE VENTROMEDIAL HYPOTHALAMUS (VMH) EXPRESSING STEROIDOGENIC FACTOR 1 (SF-1) THAT ROBUSTLY INCREASE THEIR ACTIVITY IN RESPONSE TO EXERCISE. WE HAVE FOUND THAT THE VMH SF-1 NEURAL ACTIVITY SIGNAL IS POTENTIATED SEVERALFOLD FOLLOWING REPEATED EXERCISE, SUGGESTING THAT THE EXERCISE SIGNALS GENERATED BY VMH SF-1 NEURONS ARE PLASTIC AND SHAPED BY EXPERIENCE. FURTHERMORE, WE HAVE FOUND THAT DIRECT STIMULATION OF SF-1 NEURONS SUBSTANTIALLY INCREASES SUBSEQUENT ENDURANCE CAPACITY, SUGGESTING VMH SF-1 NEURONS ARE AN IMPORTANT NEURAL NODE CONTROLLING THE PHYSIOLOGICAL BENEFITS OF EXERCISE. HOWEVER, SEVERAL IMPORTANT QUESTIONS REMAIN UNKNOWN. FIRST, WHICH FEATURES OF VMH SF-1 NEURONS ENABLES PLASTICITY OF ACTIVITY SIGNALS FOLLOWING REPEATED EXERCISE? SECOND, WHICH SPECIFIC SETS OF VMH SF-1 OUTPUT NEURONS TRANSMIT EXERCISE-RELEVANT SIGNALS? LAST, IS IT POSSIBLE TO STIMULATE VMH SF-1 NEURONS AND GENERATE THE NEUROPROTECTIVE EFFECTS OF EXERCISE ON COGNITION AND NEURAL CIRCUITRY IN THE AGING BRAIN OR IN AD-LIKE STATES? THE PROPOSED EXPERIMENTS WILL LEVERAGE ADVANCED NEUROANATOMICAL AND NEUROPHYSIOLOGICAL TOOLS WITH PRECLINICAL GENETIC MODELS TO GAIN INSIGHTS INTO THESE QUESTIONS. IN AIM 1, WE WILL PAIR LARGE-VOLUME, HIGH-RESOLUTION, AND CELL-TYPE SPECIFIC ARRAY TOMOGRAPHIC NEUROANATOMICAL RECONSTRUCTIONS WITH IN VIVO CALCIUM IMAGING AND NEURONAL ACTIVITY PERTURBATIONS TO DETERMINE HOW EXERCISE SHAPES THE SYNAPTIC ARCHITECTURE OF VMH SF-1 NEURONS. THESE EXPERIMENTS WILL DEFINE HOW CHANGES IN THE SYNAPTIC INPUTS TO THESE NEURONS MIGHT PHYSICALLY `STORE' EXERCISE HISTORY WITHIN VMH CIRCUITRY. IN AIM 2, WE WILL USE ADVANCED VIRAL MAPPING AND IN VIVO SINGLE-CELL FUNCTIONAL IMAGING TECHNIQUES TO IDENTIFY WHICH NEURONS ARE ACTIVATED BY EXERCISE AND UNDERSTAND HOW THESE EXERCISE SIGNALS ARE TRANSMITTED TO SPECIFIC CIRCUITS DOWNSTREAM OF THE VMH. THESE EXPERIMENTS WILL DEFINE THE ORGANIZATION AND LOGIC BY WHICH EXERCISE-RELATED ACTIVITY IN VMH NEURONS DRIVES FUNCTIONAL CHANGES IN THE BRAIN. IN AIM 3, WE WILL TAKE ADVANTAGE OF ADVANCED PRECLINICAL GENETIC MOUSE MODELS OF EARLY- AND LATE-ONSET AD TO DETERMINE WHETHER STIMULATING ACTIVITY IN VMH NEURONS MIGHT RECAPITULATE THE NEUROPROTECTIVE EFFECTS OF EXERCISE OBSERVED IN CORTICAL CIRCUITS. THESE EXPERIMENTS WILL INCREASE OUR UNDERSTANDING OF HOW SIGNALS IN THE VMH COULD BE HARNESSED FOR THERAPEUTIC MANIPULATION IN DISEASE STATES. BY LEVERAGING THE SYNERGISTIC EXPERTISE OF THE TEAM OF INVESTIGATORS ASSEMBLED TO ADDRESS THIS PROBLEM, INSIGHTS FROM THESE EXPERIMENTS WILL ADVANCE OUR FUNDAMENTAL UNDERSTANDING OF HOW THE BENEFICIAL EFFECTS OF EXERCISE ARE MEDIATED BY SPECIFIC SYNAPSES, CELL- TYPES, AND CIRCUITS, AND WHETHER THESE FEATURES ARE POTENTIAL THERAPEUTIC TARGETS FOR INTERVENTION IN DISEASE STATES.

TYPE 2 DIABETES RISK VARIANT EFFECTS ON MITOCHONDRIAL (PATHO)PHYSIOLOGY - PROJECT SUMMARY / ABSTRACT TYPE 2 DIABETES (T2D) RESULTS WHEN PANCREATIC ISLET SS-CELLS FAIL TO SECRETE SUFFICIENT INSULIN TO MEET PERIPHERAL INSULIN DEMAND. MITOCHONDRIAL BIOENERGETICS IS CENTRAL TO THE (PATHO)PHYSIOLOGY OF SS-CELL (DYS)FUNCTION, AND RECENT WORK SUGGESTS THAT SS-CELL MITOCHONDRIAL DYSFUNCTION PRECEDES THE DEVELOPMENT OF T2D IN SS-CELLS FROM DONORS WITH IMPAIRED GLUCOSE TOLERANCE (OR PRE-DIABETES). MITOCHONDRIAL DEFECTS HAVE BEEN REPORTED IN THE SS-CELLS OF HUMAN T2D PATIENTS, BUT THE ETIOLOGY OF MITOCHONDRIAL DYSFUNCTION IN T2D IS UNKNOWN. SUCH MECHANISTIC KNOWLEDGE IS NECESSARY TO GUIDE STRATEGIES TO PREVENT OR TREAT ISLET FAILURE AND T2D. IMPORTANTLY, GENOME-WIDE ASSOCIATION STUDIES (GWAS) LINK SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS) IN >500 GENETIC LOCI TO T2D AND ISLET DYSFUNCTION-RELATED METABOLIC TRAITS. THE MAJORITY OF THESE SNPS ARE NON-CODING AND OVERLAP REGULATORY ELEMENTS (RES) WITH BROAD TRANSCRIPTIONAL IMPLICATIONS FOR AFFECTED CELLS. IN THIS STUDY, WE COMBINE OUR EXPERTISE IN THE GENOMICS OF T2D, (EPI)GENOMIC MODIFICATION, AND MITOCHONDRIAL FUNCTION IN SS-CELLS TO BRIDGE THE GAP FROM GENOMIC ASSOCIATION TO MECHANISTIC UNDERSTANDING. WE HYPOTHESIZE THAT NON-CODING T2D SNPS CAUSE SS-CELL DYSFUNCTION BY ALTERING RE USE OR ACTIVITY, THEREBY CHANGING EXPRESSION OF EFFECTOR GENES THAT DIRECTLY IMPAIR MITOCHONDRIAL HEALTH. TO TEST THIS, WE PROPOSE TO USE SOPHISTICATED (EPI)GENOMIC EDITING TOOLS IN HUMAN ISLETS AND SS-CELL SPECIFIC MOUSE MODELS FOR PHYSIOLOGICAL RELEVANCE AND VALIDATION IN TWO COMPLEMENTARY AIMS. IN AIM 1, WE WILL TEST RE– EFFECTOR GENE LINKS IN HUMAN ISLETS USING CRISPR-QTL. IN PARALLEL, WE WILL ASSESS T2D RISK ALLELE EFFECTS ON RE CHROMATIN ACCESSIBILITY, ACTIVITY, TRANSCRIPTION FACTOR BINDING, AND SS-CELL EXPRESSION OF PUTATIVE MITOCHONDRIAL T2D EFFECTOR GENES USING COMPLEMENTARY IN VIVO (SINGLE CELL CHROMATIN ACCESSIBILITY, HISTONE ACETYLATION, AND EXPRESSION QUANTITATIVE TRAIT LOCUS ANALYSIS OF PRIMARY HUMAN ISLETS) AND IN VITRO (REPORTER GENE, DNA-BINDING ASSAY) APPROACHES. FINALLY, WE WILL DETERMINE THE CONSEQUENCES OF EFFECTOR GENE PERTURBATION ON MITOCHONDRIAL PHENOTYPES, SS-CELL VIABILITY, AND INSULIN CONTENT AND SECRETION IN HUMAN ISLETS AND ENDOC-SSH3 CELLS. IN AIM 2, WE HARNESS SS-CELL-SPECIFIC KNOCKOUT MOUSE MODELS TO ASSIGN FUNCTION TO TWO HIGH-PRIORITY MITOCHONDRIAL T2D EFFECTOR GENES IN GLYCEMIC CONTROL, SS-CELL MASS/FUNCTION, AND MITOCHONDRIAL METABOLISM. FURTHER, WE WILL ADDRESS THE IMPORTANCE OF THESE MITOCHONDRIAL T2D EFFECTOR GENES FOR SS-CELL COMPENSATION TO PERIPHERAL INSULIN RESISTANCE FOLLOWING DIET-INDUCED OBESITY. FINALLY, WE WILL USE (EPI)GENOMIC EDITING TOOLS IN HUMAN ISLETS TO DETERMINE IF MITOCHONDRIAL T2D EFFECTOR GENES IMPAIR SS-CELL FUNCTION AND GLYCEMIC CONTROL IN EX VIVO ASSAYS AS WELL AS AFTER ISLET TRANSPLANTATION INTO IMMUNODEFICIENT MICE. COMPLETION OF THIS STUDY WILL GENERATE NEW VARIANT-TO-FUNCTION CONNECTIONS THAT ASSIGN MOLECULAR AND CELLULAR FUNCTIONS TO T2D RISK ALLELES, IDENTIFY NOVEL THERAPEUTIC TARGETS, AND PROVIDE IMPORTANT KNOWLEDGE TO GUIDE SUBSEQUENT STRATEGIES TO PREVENT OR TREAT SS-CELL FAILURE AND T2D.

CHROMOSOME-WIDE MAPPING OF RECOMBINATIONAL ACTIVITY

LUNG RESIDENT MESENCHYMAL CELLS IN THE PRE-METASTATIC NICHE FORMATION - PROJECT SUMMARY/ABSTRACT METASTATIC DISEASE REMAINS THE MAJOR CAUSE OF CANCER-RELATED DEATH. AMONG THE VITAL ORGANS TO WHICH SOLID TUMORS METASTASIZE, THE LUNG IS ONE OF THE MOST COMMON. LUNG METASTASES FREQUENTLY OCCUR IN VARIOUS LATE STAGE SOLID CANCERS INCLUDING BREAST CANCER. IN THE PAST 20 YEARS, SIGNIFICANT ADVANCES IN LUNG METASTASIS RESEARCH HAVE REVEALED INTRICATE INTERACTIONS BETWEEN THE DISSEMINATED TUMOR CELLS (DTCS) AND THE LUNG MICROENVIRONMENT THAT ARE ESSENTIAL FOR THE DEVELOPMENT OF METASTATIC LUNG LESIONS. IN PARTICULAR, FORMATION OF THE IMMUNOSUPPRESSIVE LUNG PRE-METASTATIC MICROENVIRONMENT (NICHE), WHICH IS DRIVEN BY FACTORS SECRETED BY PRIMARY TUMORS, IS REGARDED AS A KEY PREPARATION STAGE PRIOR TO THE ARRIVAL OF DTCS. THE FOCUS OF THIS PROPOSAL WILL BE ON THE LUNG RESIDENT MESENCHYMAL CELLS (MCS), AN UNDER-CHARACTERIZED STROMAL CELL POPULATION WITHIN THE LUNG PRE-METASTATIC NICHE. PRELIMINARY STUDIES IN MOUSE MODELS OF BREAST CANCER SUGGEST THAT LUNG RESIDENT MCS SERVE AS A DRIVER OF IMMUNOSUPPRESSIVE PRE-METASTATIC NICHE FORMATION BY REPROGRAMMING DIVERSE TYPES OF MYELOID CELLS TO BECOME HIGHLY IMMUNOSUPPRESSIVE OR TOLEROGENIC. THE CENTRAL HYPOTHESIS OF THIS PROPOSAL IS THAT LUNG RESIDENT MCS DRIVE FORMATION OF THE LUNG PRE-METASTATIC NICHE BY ENDOWING DIVERSE INFILTRATING MYELOID CELLS WITH AN IMMUNOSUPPRESSIVE CAPACITY. TO TEST THIS HYPOTHESIS AND UNCOVER MECHANISMS BY WHICH MCS MODULATE MYELOID CELLS, WE PROPOSE THREE SPECIFIC AIMS. AIM 1: DETERMINE THE LUNG RESIDENT MC CHANGES IN THE PRE-METASTATIC NICHE USING GENETIC MANIPULATION OF ENDOGENOUS MCS IN MICE. WE WILL USE ORTHOTOPIC BREAST TUMOR CELL IMPLANTATION AND GENETICALLY ENGINEERED MOUSE (GEM) TUMOR MODELS TO MEASURE THE ENDOGENOUS MC CHANGES AT THE PRE-METASTATIC STAGE. MCS WILL ALSO BE ABLATED IN VIVO TO DETERMINE THEIR NECESSITY FOR PRE-METASTATIC NICHE FORMATION. AIM 2: CHARACTERIZE THE KEY LUNG MESENCHYMAL FACTORS THAT RECRUIT AND MODULATE MYELOID CELLS. WE WILL USE RNA SEQUENCING TO PROFILE TRANSCRIPTOME CHANGES BETWEEN ENDOGENOUS MCS RESIDING IN PRE-METASTATIC VS. NORMAL LUNGS TO IDENTIFY UPREGULATED GENES THAT MIGHT PLAY A ROLE IN MODULATING MYELOID CELLS. THE CONTRIBUTION OF CANDIDATE MC GENES TO PRE-METASTATIC NICHE FORMATION WILL BE EVALUATED THROUGH MC-SPECIFIC GENE KNOCKOUT. AIM 3: TARGET LUNG MCS AS A STRATEGY FOR ABOLISHING THE IMMUNOSUPPRESSIVE LUNG PRE-METASTATIC NICHE IN PRECLINICAL MODELS. USING ORTHOTOPIC TUMOR IMPLANTATION MODELS, WE WILL TEST THE ABILITY OF LUNG MC BLOCKADE AGENTS TO BOOST LUNG ANTI-TUMOR IMMUNITY AND PREVENT LUNG METASTASIS. THESE STUDIES WILL BE A SIGNIFICANT STEP TOWARD OUR LONG-TERM GOAL TO FULLY DISSECT THE ROLES OF ORGAN-SPECIFIC STROMAL CELLS IN ORGAN-TROPIC METASTASES OF BREAST CANCER. WE ANTICIPATE THAT OUR FINDINGS WILL ESTABLISH A STRONG FOUNDATION FOR THE DEVELOPMENT OF NOVEL THERAPEUTIC STRATEGIES THAT TARGET STROMAL FACTORS FOR THE PREVENTION AND TREATMENT OF LUNG METASTASES OF BREAST CANCER AND OTHER SOLID TUMORS.

ASSESSING THE INTERPLAY BETWEEN INFLAMMATORY SIGNALING AND EPIGENETIC DYSREGULATION IN AGE-ASSOCIATED CLONAL HEMATOPOIESIS AND LEUKEMIA INITIATION - PROJECT SUMMARY/ABSTRACT AGE IS ONE OF THE MOST CLEARLY DEFINED RISK FACTORS FOR CANCER. AS THE INCIDENCE OF CANCER INCREASES WITH AGE, RISING MORE RAPIDLY BEGINNING IN MID-LIFE (AGES 45-64 YEARS), CANCER CAN BE CONSIDERED AN AGE-RELATED DISEASE. RECENT STUDIES HAVE IDENTIFIED AGE-DEPENDENT SOMATIC MUTATIONS, INCLUDING ALLELES WITH A ROLE IN CANCER INITIATION, IN A SPECTRUM OF HUMAN TISSUES. IN THE HEMATOPOIETIC SYSTEM THIS IS TERMED CLONAL HEMATOPOIESIS (CH) AND IS MOST COMMONLY CAUSED BY MUTATIONS IN THE EPIGENETIC REGULATORS DNMT3A, TET2, AND ASXL1 WITHIN THE HEMATOPOIETIC STEM AND PROGENITOR CELL (HSPC) COMPARTMENT. HOW THE AGING PROCESS PROMOTES CLONAL SELECTION, EXPANSION, AND TRANSFORMATION FROM CH TO ACUTE MYELOID LEUKEMIA (AML) IS POORLY UNDERSTOOD. THE LONG-TERM GOAL OF THIS RESEARCH IS TO UNDERSTAND THE MECHANISMS BY WHICH AGING PROMOTES TRANSFORMATION CAUSING HEMATOLOGIC MALIGNANCIES. THE OVERALL OBJECTIVE OF THIS PROPOSAL IS TO ELUCIDATE THE MECHANISMS BY WHICH INCREASED INFLAMMATION OBSERVED DURING AGING PROMOTES EXPANSION OF CH-MUTANT HSPCS, AND THE EXTENT TO WHICH THIS FITNESS ADVANTAGE IS PROVIDED BY ALTERED EPIGENETIC REGULATION OCCURRING AS A DIRECT CONSEQUENCE OF CH MUTATIONS. PRELIMINARY DATA SHOW THAT CH-MUTANT HSPCS HAVE A MORE POTENT SELECTIVE ADVANTAGE AND UNDERGO MORE RAPID MALIGNANT TRANSFORMATION IN AGED COMPARED TO YOUNG MICE. MECHANISTICALLY, INCREASED INFLAMMATION IN AGED MICE IS A DRIVER OF THIS PHENOTYPE AND EPIGENETIC ALTERATIONS ARE FOUND TO ACCUMULATE IN CH-MUTANT HSPCS WITH AGING. THESE DATA SUPPORT THE CENTRAL HYPOTHESIS THAT AGING-ASSOCIATED INFLAMMATION IS A SELECTIVE PRESSURE FAVORING CH-MUTANT HSPC EXPANSION AND MALIGNANT TRANSFORMATION, AND THAT CLONAL EXPANSION, EPIGENETIC ALTERATIONS, AND RISK OF TRANSFORMATION CAUSED BY CH MUTATIONS ARE DEPENDENT UPON SUSTAINED CH-MUTANT ALLELE EXPRESSION. THIS PROJECT WILL USE CELLULAR AND MOLECULAR BIOLOGICAL APPROACHES IN AGED MICE INTEGRATED WITH STUDIES USING PRIMARY HUMAN CH SAMPLES TO ACHIEVE THE FOLLOWING SPECIFIC AIMS: AIM 1. DETERMINE THE EXTENT TO WHICH CLONAL HEMATOPOIETIC EXPANSION AND LEUKEMIC TRANSFORMATION IN THE AGING CONTEXT IS DUE TO INFLAMMATION; AND AIM 2. DETERMINE THE MECHANISMS BY WHICH, AND EXTENT TO WHICH, REVERSION OF A CH MUTATION DURING AGING ALTERS CLONAL EVOLUTION AND RISK OF LEUKEMIA INITIATION. THE PROPOSED RESEARCH IS CONCEPTUALLY INNOVATIVE BECAUSE IT IS THE FIRST TO DETERMINE HOW INFLAMMATION AND EPIGENETIC DYSREGULATION CONSPIRE DURING THE AGING PROCESS TO EXPAND, EVOLVE AND TRANSFORM CH-MUTANT CLONES. THE PROPOSED RESEARCH IS TECHNICALLY INNOVATIVE AS IT INCORPORATES NOVEL CO-CULTURE SYSTEMS AND THERAPEUTIC STUDIES TO ASSESS KEY INFLAMMATORY DRIVERS, AS WELL AS A NOVEL MURINE MODEL WITH CH-MUTANT INDUCTION AND REVERSION CAPABILITIES TO INVESTIGATE CH MUTANT ALLELE DEPENDENCIES IN CLONAL EXPANSION, EPIGENETIC ALTERATIONS, AND LEUKEMIC TRANSFORMATION. THIS STUDY IS SIGNIFICANT BECAUSE UNDERSTANDING THE MECHANISMS BY WHICH AGING CONTRIBUTES TO CLONAL EXPANSION AND TRANSFORMATION WILL PROVIDE INSIGHTS INTO EFFECTIVE THERAPEUTIC STRATEGIES TARGETING CLONAL EVOLUTION, ATTENUATE PATHOPHYSIOLOGY PROMOTED BY CLONAL EXPANSION, AND INTERCEPT MALIGNANT TRANSFORMATION.

CELL CYCLE REGULATION DURING SPERMATOGENESIS

HIGH-RESOLUTION SINGLE CELL PROFILING OF VACCINE RESPONSIVENESS IN THE ELDERLY

METAGENOMES TO THERAPEUTICS: DEFINING THE RULES FOR ENGINEERING THE SKIN MICROBIOME

SEQUENCING MUTANT MICE WITH ALTERED COCAINE RESPONSES

TYPE I INTERFERON RESPONSES IN THE PATHOBIOLOGY OF ANTHRACYCLINE-INDUCED CARDIOTOXICITY

DEFINING TANDEM DUPLICATOR PHENOTYPES IN TRIPLE-NEGATIVE BREAST CANCER TO GUIDE THERAPEUTIC INTERVENTIONS

THE ROLE OF LUNG RESIDENT MESENCHYMAL STEM CELLS IN POST-CHEMOTHERAPY LUNG METASTASES OF BREAST CANCER

ROLE OF A NOVEL STRESS RESPONSE MECHANISM-GENETIC BUFFERING-IN TUMOR EVOLUTION

ESTABLISHING A MUTANT MOUSE REGIONAL RESOURCE CENTER AT THE JACKSON LABORATORY

MYC-REGULATED RNA BINDING PROTEIN NETWORKS AND SPLICED ISOFORMS DRIVING CANCER - SUMMARY ALTERNATIVE RNA SPLICING IS A KEY STEP IN GENE EXPRESSION REGULATION AND CONTRIBUTES TO TRANSCRIPTIONAL DIVERSITY BY SELECTING WHICH TRANSCRIPT ISOFORMS ARE PRODUCED IN A SPECIFIC CELL AT A SPECIFIC TIME POINT. ABERRANTLY SPLICED ISOFORMS CAN IMPACT EVERY ONE OF THE HALLMARKS OF CANCER, INCLUDING INCREASED CELL PROLIFERATION, MIGRATION, OR RESISTANCE TO APOPTOSIS. REGULATORY SPLICING FACTORS (SFS) HAVE RECENTLY EMERGED AS A NEW CLASS OF ONCOPROTEINS AND TUMOR SUPPRESSORS. IN PARTICULAR, THE TUMORIGENIC CAPACITY OF THE ONCOGENIC TRANSCRIPTION FACTOR MYC, WHICH IS DYSREGULATED IN >50% OF HUMAN TUMORS, HAS BEEN SHOWN TO BE DEPENDENT ON THE SPLICING MACHINERY AND ON AT LEAST 3 SFS DIRECTLY REGULATED BY MYC. HOWEVER, WE CURRENTLY DO NOT HAVE A COMPREHENSIVE UNDERSTANDING OF WHICH COMPONENT(S) OF THE SPLICING MACHINERY ARE REGULATED BY MYC, OR OF THE FUNCTIONS OF MYC-INDUCED SPLICED ISOFORMS. THE GOAL OF THIS PROPOSAL IS TO SYSTEMATICALLY CHARACTERIZE THE MECHANISMS BY WHICH MYC-REGULATED SFS AND SPLICED ISOFORMS DRIVE TUMOR GROWTH AND MAINTENANCE. TO BEGIN TO ADDRESS THIS GAP IN KNOWLEDGE, IN OUR PRELIMINARY STUDIES WE USED A MAMMARY CELL LINE HARBORING AN INDUCIBLE FORM OF MYC TO GREATLY EXPAND THE NUMBER OF KNOWN SFS REGULATED BY MYC. WE UNCOVERED THAT MYC ACTIVATION PROMOTES ALTERNATIVE SPLICING OF >4,000 ISOFORMS AND EXPRESSION OF 125 SFS. THESE SFS ARE ALSO UPREGULATED IN MYC-ACTIVE BREAST TUMORS AND CAN BE GROUPED, BASED ON CO-EXPRESSION, INTO GROUPS OR MODULES. SIX SF-MODULES HIGHLY CORRELATE WITH MYC ACTIVITY IN BREAST TUMORS AND CELL LINES, AND ARE ENRICHED IN TRIPLE NEGATIVE BREAST CANCER (TNBC). WHICH OF THESE SFS PLAY A ROLE IN MYC-DRIVEN TRANSFORMATION, AND WHETHER CO-EXPRESSION OF MULTIPLE MYC-INDUCED SFS HAS A STRONGER TUMORIGENIC EFFECT THAN INDIVIDUAL SFS, IS NOT KNOWN. FURTHER, CO-EXPRESSION ANALYSIS IN 33 TCGA TUMORS OF DIFFERENT TISSUE ORIGIN IDENTIFIED AN SF-MODULE SHARED ACROSS ALL MYC-ACTIVE TUMORS, SUGGESTING A PAN-CANCER VULNERABILITY. WE HYPOTHESIZE THAT MYC REGULATES A NETWORK OF SFS WHICH COOPERATE IN TUMOR PATHOGENESIS AND THAT DISRUPTING THIS NETWORK COULD PROVIDE A NOVEL STRATEGY TO SLOW GROWTH OF MYC-DRIVEN TUMORS. HERE, WE WILL LEVERAGE OUR EXPERTISE IN RNA SPLICING AND CANCER BIOLOGY AND APPLY A FUNCTIONAL GENOMICS APPROACH TO GAIN NOVEL INSIGHTS INTO MYC'S ONCOGENICITY. AIM 1 WILL CHARACTERIZE THE FUNCTION OF 6 MYC-INDUCED SF MODULES AND THEIR SPLICING TARGETS IN TNBC TUMOR GROWTH IN VITRO AND IN VIVO. SINCE IT IS UNKNOWN WHETHER MYC REGULATES A SHARED SET OF ISOFORMS IN DISTINCT TISSUES, AIM 2 WILL IDENTIFY PAN-CANCER SPLICING SIGNATURES PREDICTIVE OF MYC ACTIVITY AND CLINICAL OUTCOMES, WHICH MAY SERVE AS CLINICAL BIOMARKERS, AND WILL DELIVER PUTATIVE NEO-ANTIGENS GENERATED FROM MYC-INDUCED ISOFORMS. FINALLY, AIM 3 WILL IMPLEMENT GENOMIC APPROACHES TO DETERMINE WHICH MYC-INDUCED ISOFORMS ARE ESSENTIAL FOR THE GROWTH OF MYC-DRIVEN CANCER CELLS AND PATIENT-DERIVED ORGANOIDS. THIS PROJECT WILL REVEAL FUNDAMENTAL MECHANISMS BY WHICH ONCOGENIC SFS AND THEIR TARGET SPLICED ISOFORMS DRIVE TUMORIGENESIS DOWNSTREAM OF MYC. THESE RESULTS COULD HELP INFORM DEVELOPMENT OF THERAPEUTIC STRATEGIES FOR TUMORS DRIVEN BY MYC, WHICH REMAINS AN UNDRUGGABLE TARGET.

TBD

QUANTITATIVE COMPUTATIONAL METHODS TO ACCURATELY MEASURE TUMOR HETEROGENEITY IN SOLID TUMORS TO INFORM DEVELOPMENT OF EVOLUTION-BASED TREATMENT STRATEGIES

IDENTIFYING MECHANISTIC PATHWAYS UNDERLYING RPE PATHOGENESIS IN MODELS OF PATTERN DYSTROPHY - PROJECT SUMMARY/ABSTRACT THE RETINAL PIGMENT EPITHELIUM (RPE) PLAYS AN IMPORTANT ROLE IN THE EYE TRANSPORTING NUTRIENTS, IONS, AND WATER; AND SERVING IN ABSORPTION OF LIGHT AND PROTECTION AGAINST PHOTOOXIDATION, RECYCLING OF VISUAL CYCLE COMPONENTS, AND PROVIDING ESSENTIAL FACTORS FOR THE STRUCTURAL INTEGRITY OF THE RETINA. COLLECTIVELY THESE SUPPORT METABOLIC HOMEOSTASIS AND BARRIER FUNCTION OF THE RETINA. MANY RPE DISEASE PHENOTYPES ARE SHARED BETWEEN HUMANS AND MOUSE MODELS CARRYING MUTATIONS IN CELL- ADHESION AND EXTRACELLULAR MATRIX (ECM) MOLECULES. DEFINING CAUSATIVE PATHWAYS AND NETWORKS THAT ARE INDUCED BY MUTATIONS OR PATHOGENIC VARIANTS MAY PROVIDE THERAPEUTIC TARGETS. IDENTIFYING DRUGGABLE TARGETS THAT ACT DURING THE PRE-SYMPTOMATIC STAGE OF THE DISEASE IS PARTICULARLY IMPORTANT TO ENABLE DEVELOPMENT OF THERAPIES THAT CAN PREVENT, DELAY ONSET, OR DECREASE THE SEVERITY OF RPE-ASSOCIATED DISEASES, IRRESPECTIVE OF THE INITIAL CAUSE OF THE DISEASE, BEFORE THE PATHOLOGIC CHANGES BECOME IRREVERSIBLE. THE GOAL OF THIS APPLICATION IS TO IDENTIFY SHARED PRE-CLINICAL AND END-STAGE PHENOTYPIC AND CELLULAR EFFECTS OF MUTATIONS IN TWO GENES, CTNNA1 AND LRATD2, WHICH ARE HIGHLY EXPRESSED IN THE RPE AND LEAD TO SIMILAR RETINAL DEFECTS. MUTATIONS IN CTNNA1 HAVE BEEN REPORTED IN PATIENTS WITH PATTERN DYSTROPHY AND LEBERS CONGENITAL AMAUROSIS (LCA), AND LRATD2 HAS BEEN ASSOCIATED WITH EARLY AMD IN A GENOME WIDE ASSOCIATION STUDY. OUR APPROACH IS TO USE CLINICAL, FUNCTIONAL AND BIOCHEMICAL TESTS TO PROVIDE A DEEP CHARACTERIZATION OF THE MODELS AND TO ANALYZE ASSOCIATED CELLULAR CHANGES USING SINGLE-NUCLEAR TRANSCRIPTOMICS AS WELL AS SPATIAL METABOLOMIC/PROTEOMIC MEASUREMENTS. THESE PHENOTYPIC AND GENOMICS DATA WILL BE JOINTLY ANALYZED USING COMPUTATIONAL METHODS TO IDENTIFY THE SHARED PATHWAYS PERTURBED IN THESE MODELS. OUR USE OF TWO MODELS WITH SHARED PATHOLOGIES WILL LET US FILTER OUT MUTATION SPECIFIC ALTERATIONS AND FOCUS ON THE SHARED DISEASE-CAUSING PATHWAYS. USING OUR MOUSE RESOURCES, WE ALSO PLAN TO ENHANCE CURRENT MODELS. SUCCESSFUL COMPLETION OF OUR STUDIES WILL IDENTIFY NOVEL PATHOGENIC PATHWAYS UNDERLYING RPE-RELATED DISORDERS, REVEALING POTENTIAL THERAPEUTIC TARGETS THAT COULD BE EFFECTIVE IN A BROAD RANGE OF THESE DISEASES, REGARDLESS OF THE CAUSE OF THE DISEASE. THESE WELL-CHARACTERIZED MODELS WILL BE MADE AVAILABLE TO THE RESEARCH COMMUNITY FOR FURTHER MECHANISTIC INQUIRIES AS WELL AS FOR TESTING THERAPEUTIC STRATEGIES.

FUNCTIONAL MAPPING OF ENHANCER CONSERVATION BETWEEN SPECIES TO ENABLE MECHANISTIC INSIGHTS INTO POLYGENIC DISEASE - PROJECT SUMMARY RECENT ADVANCES TO CHARACTERIZE CIS-REGULATORY ELEMENTS (CRE), INCLUDING MASSIVELY PARALLEL REPORTER ASSAYS AND CRISPR-BASED SCREENS OF NON-CODING ELEMENTS, HAVE TRANSFORMED OUR ABILITY TO COMPREHENSIVELY CHARACTERIZE THE NON-CODING GENOME AT SCALE. LARGE SCALE EFFORTS BY US AND OTHERS THROUGH THE ENCYCLOPEDIA OF DNA ELEMENTS (ENCODE) CONSORTIUM ARE NOW UNDERWAY TO APPLY THESE METHODS GENOME-WIDE ACROSS MANY CELLULAR STATES. THE RESULTS OF THESE SCREENS WILL HAVE A TRANSFORMATIVE IMPACT ON OUR ABILITY TO READ AND WRITE THE REGULATORY GRAMMAR OF THE CELL. ONE DIRECT APPLICATION WILL BE IN THE INTERPRETATION OF CAUSAL ALLELES FOR HUMAN DISEASE RISK AND OTHER PHENOTYPIC TRAITS IDENTIFIED THROUGH GENOME-WIDE ASSOCIATION STUDIES. FROM THESE STUDIES WE NOW KNOW THE MAJORITY OF HERITABILITY FOR COMPLEX TRAITS RESIDES IN NON-CODING REGIONS OF THE GENOME. UNTIL RECENTLY IT HAS BEEN DIFFICULT TO PINPOINT INDIVIDUAL CAUSAL ALLELES BUT PROGRESS IS NOW BEING MADE TO IDENTIFY AND ELUCIDATE THEIR MOLECULAR FUNCTION. DESPITE OUR BURGEONING SUCCESS IN UNDERSTANDING HOW A VARIANT IMPACTS MOLECULAR PHENOTYPES (E.G. GENE TRANSCRIPTION), WE LACK THE ABILITY TO SYSTEMATICALLY EVALUATE ALLELE(S) WITHIN MODEL ORGANISMS TO UNDERSTAND THEIR IMPACT ON PHYSIOLOGICAL FUNCTION. THIS DISCONNECT IS PARTIALLY DUE TO OUR INABILITY TO IDENTIFY THE HOMOLOGOUS NON-CODING REGION TO TARGET WITHIN MODEL ORGANISMS. TO AID IN MODELING HUMAN REGULATORY VARIATION IN THE MOUSE, IN THIS PROJECT WE WILL DEVELOP IMPROVED MAPS OF HOMOLOGOUS CRES BETWEEN HUMAN AND MOUSE. CURRENT COMPARATIVE APPROACHES RELY ON SEQUENCE HOMOLOGY AND CORRELATIVE MEASURES OF GENE EXPRESSION SUCH AS REGIONS OF DNASE HYPERSENSITIVITY AND CHROMATIN MODIFICATIONS. WHILE THESE METHODS HAVE PROVIDED VALUABLE INSIGHT, THEY LACK DIRECT QUANTITATIVE MEASUREMENTS OF A CRE'S IMPACT ON INDIVIDUAL GENES AND THE LOCATION OF THE CIS-REGULATORY MODULES (CRMS) WITHIN THE CRES RESPONSIBLE FOR ACTIVITY. TO OVERCOME THESE SHORTCOMINGS, IN THIS STUDY WE WILL DEVELOP MAPS OF CRE CONSERVATION BASED DIRECTLY ON FUNCTION. TO ACCOMPLISH THIS, WE WILL DIFFERENTIATE INDUCED PLURIPOTENT STEM CELLS (IPSCS) FROM HUMAN AND MOUSE TO EARLY DEVELOPMENTAL STATES AS THE STARTING MATERIAL FOR SCREENS OF CRE ACTIVITY. WE WILL USE (I) A CRISPR-BASED SCREEN TO ENDOGENOUSLY PERTURB PUTATIVE CRES IMPORTANT FOR NEURONAL AND EPITHELIAL FUNCTION; AND (II) CRES WITH CONCORDANT AND DISCORDANT ACTIVITY ACROSS THE TWO SPECIES WILL THEN UNDERGO SATURATION MUTAGENESIS USING A MASSIVELY PARALLEL REPORTER ASSAY (MPRA). RESULTS FROM THE MPRA WILL IDENTIFY CRMS (E.G. TF BINDING MOTIFS) WITHIN EACH CRE DRIVING REGULATORY ACTIVITY OF THE ELEMENT. WE WILL USE THE RESULTS FROM BOTH SCREENS TO CONSTRUCT IMPROVED MAPS OF CRE CONSERVATION THAT WILL INFORM HOW TO COPY THE EFFECTS OF GENETIC VARIATION RESIDING AT THESE REGIONS ACROSS SPECIES. DOING SO WILL ACCELERATE OUR PROGRESS IN MOVING HUMAN DISEASE VARIANTS INTO ANIMAL MODELS, THEREBY ALLOWING US TO BETTER UNDERSTAND THE PATHOPHYSIOLOGY OF COMPLEX DISEASES IN THE HUMAN POPULATION.

SITE-SPECIFIC INTEGRATION OF LARGE (10-100 KB) DNA CONSTRUCTS INTO THE MOUSE GENOME AND HUMAN INDUCED PLURIPOTENT STEM CELLS USING THE CAS9-BXB1 INTEGRASE TOOLBOX - PROJECT SUMMARY/ABSTRACT THE LONG-TERM GOAL OF THIS PROJECT IS TO INCREASE THE ABILITY OF RESEARCHERS TO CREATE FAITHFUL MOUSE AND STEM-CELL MODELS OF HUMAN CANCERS AND OTHER DISEASES. CURRENTLY AVAILABLE GENETIC-ENGINEERING APPROACHES, INCLUDING THE CRISPR-CAS9 SYSTEM, WHICH HAS REVOLUTIONIZED GENOME EDITING, LACK THE CAPACITY FOR EFFICIENT INTEGRATION OF LARGE DNA CONSTRUCTS (> 10 KILOBASES; KB) IN MOUSE ZYGOTES AND MOUSE AND HUMAN STEM CELLS. THIS LIMITATION SIGNIFICANTLY HINDERS THE MODELING OF HUMAN DISEASES, INCLUDING CANCER. FOR EXAMPLE, TANDEM DUPLICATIONS (TDS), SUPER-ENHANCERS (SES; LARGE CLUSTERS OF TRANSCRIPTIONAL ENHANCERS), AND LARGE NON-CODING STRUCTURAL VARIANTS HAVE BEEN LINKED TO HUMAN DISEASES, INCLUDING CANCERS, BUT AVAILABLE TECHNOLOGIES DO NOT PERMIT MODELING SUCH LARGE VARIANTS IN WHOLE ANIMALS OR CELL LINES. TO FILL THIS GAP, WE WILL DEVELOP A GENE-EDITING TOOLBOX THAT COUPLES THE PRECISION OF THE CRISPR-CAS9 SYSTEM WITH THE FIDELITY AND EFFICIENCY OF THE SERINE INTEGRASE BXB1 TO ENABLE RAPID, EFFICIENT INSERTION OF LARGE DNA CONSTRUCTS IN MICE, MOUSE EMBRYONIC STEM CELLS, AND HUMAN INDUCED PLURIPOTENT STEM CELLS (HIPSCS). BXB1 INTEGRASE USES DNA ATTACHMENT SITES (ATTP IN THE GENOME, ATTB IN THE DONOR DNA) AS SUBSTRATES FOR CATALYZING EFFICIENT TRANSGENESIS. WE SHOW THAT OUR INNOVATIVE CAS9-BXB1 TOOLBOX CAN PRECISELY INTEGRATE DNA CONSTRUCTS UP TO ~43 KB IN LENGTH IN MICE. HERE, IN THREE AIMS WE WILL FURTHER DEVELOP AND VALIDATE THE TOOLBOX TO ENABLE PRECISE TRANSGENESIS OF LARGE DNA CONSTRUCTS (~100 KB) AND TO FACILITATE GENERATION OF DNA REARRANGEMENTS. AIM 1: OPTIMIZE THE CAS9-BXB1 TOOLBOX FOR INSERTION OF LARGE DNA (10 TO 100 KB) CONSTRUCTS INTO MOUSE ZYGOTES. WE WILL USE REPORTER CONSTRUCTS WITH DIFFERING LENGTHS TO DETERMINE THE MAXIMUM LENGTH OF DNA CONSTRUCT THAT CAN BE INSERTED EFFICIENTLY, AND WILL VALIDATE A ONE-STEP PROTOCOL FOR RAPID GENERATION OF TRANSGENIC MICE WITHOUT THE NEED TO FIRST GENERATE AND CHARACTERIZE MICE WITH ATTACHMENT SITES. AIM 2: GENERATE MOUSE AND HIPSC MODELS OF HUMAN DISEASES, INCLUDING CANCER, USING THE CAS9-BXB1 TOOLBOX. USING OUR TOOLBOX TO INSERT LARGE GENOMIC VARIANTS, WE WILL GENERATE A MOUSE MODEL OF BREAST CANCER (INSERTION OF A 23.7-KB TD), HIPSC MODEL OF TRIPLE NEGATIVE BREAST CANCER (27.2-KB SE), AND MOUSE MODEL OF HIRSCHSPRUNG DISEASE (~80-KB HUMAN RISK ALLELE). AIM 3: ENABLE USE OF THE CAS9-BXB1 TOOLBOX FOR GENERATION OF DNA REARRANGEMENTS. IN CRE-LOX RECOMBINATION SYSTEMS, CRE CATALYZES RECOMBINATION BETWEEN TWO LOXP SITES FLANKING A TARGET LOCUS, ENABLING DIVERSE DNA REARRANGEMENTS. STUDIES SUGGEST THAT CRE-RECOMBINATION EFFICACY IS LIMITED BY THE INTER-LOXP-SITE DISTANCE AND THE PARTICULAR GENOMIC SITE TARGETED. WE WILL DETERMINE WHETHER THE CAS9- BXB1 TOOLBOX IS MORE EFFICIENT THAN CRE-LOX FOR GENERATION OF DNA REARRANGEMENTS, BY DETERMINING BXB1-MEDIATED RECOMBINATION EFFICACY AT DIFFERENT INTER-ATTP/ATTB DISTANCES. SUCCESSFUL COMPLETION OF THIS PROJECT WILL PROVIDE THE COMMUNITY WITH THREE NEW MODELS FOR FUTURE STUDIES, AND A VERSATILE TOOL FOR DEVELOPMENT OF NOVEL AND IMPROVED MOUSE AND HIPSC MODELS OF CANCER AND OTHER DISEASES.

CRITICAL REGULATORY CIRCUITS IN BLOOD FORMATION

GENOMICS AND EPIGENOMICS OF THE ELDERLY RESPONSE TO PNEUMOCOCCAL VACCINES

CLONING QTL GENES FOR PLASMA HDL CHOLESTEROL

TYPE 1 DIABETES MOUSE RESOURCE (T1DR)

INVESTIGATING CELL SPECIFIC ROLES OF THE COMPLEMENT CASCADE IN GLAUCOMA

ALZHEIMER'S DISEASE-RELATED DEMENTIA MODELS BY PRECISION EDITING AND RELEVANT GENETIC X ENVIRONMENTAL EXPOSURES

IDENTIFYING SHARED PATHOGENIC NETWORKS AND MOLECULAR TARGETS UNDERLYING RETINAL PIGMENTED EPITHELIAL ASSOCIATED DISEASE

MAPPING GENE MUTATIONS THAT ALTER HDL CHOLESTEROL LEVELS IN MICE

DISCOVERY OF AGING-ASSOCIATED MECHANISMS CAUSING EXPANSION AND PROGRESSION OF CLONAL HEMATOPOIESIS OF INDETERMINANT POTENTIAL (CHIP)

CRYOPRESERVATION OF MURINE GERMPLASM

AN INTEGRATIVE COMPUTATIONAL FRAMEWORK FOR DNA HYDROXYMETHYLATION DATA MINING AND INTERPRETATION

IDENTIFICATION AND INTERPRETATION OF CHROMATIN CHANGES ASSOCIATED WITH THE AGING OF HUMAN IMMUNE CELLS

DETERMINING HOW LYMPHATIC MOLECULES CONTROL CONVENTIONAL OUTFLOW

DEVELOPMENT OF SINGLE MOLECULE CHROMATIN INTERACTION ASSAYS (SMCHIA) IN SINGLE NUCLEI

MAPPING THE RNA SPLICING LANDSCAPE OF NF1 AND ITS IMPACT ON NEUROFIBROMIN FUNCTION ACROSS TISSUES

GENETIC REGULATION OF COMPLEX NEUROLOGICAL DISEASE

ROLE OF ARMS2 MUTATIONS IN AGE-RELATED MACULAR DEGENERATION - PROJECT SUMMARY/ABSTRACT GENOME-WIDE ASSOCIATION (GWA) AND LINKAGE STUDIES HAVE IDENTIFIED >50 GENES SIGNIFICANTLY ASSOCIATED WITH AGE RELATED MACULAR DEGENERATION (AMD). HOWEVER, MANY QUESTIONS REMAIN. NAMELY, ARE THE IDENTIFIED GWA HITS DISEASE-CAUSING VARIANTS OR ARE THEY SIMPLY CLOSELY LINKED MARKERS? AND IF THE GWA HITS ARE DISEASE-CAUSING VARIANTS, HOW DO DISRUPTIONS IN THESE MOLECULES LEAD TO THE OBSERVED PATHOLOGIES? FURTHER, SINCE AMD IS MULTIFACTORIAL, TO WHAT EXTENT DO PARTICULAR COMBINATIONS OF FACTORS PRECIPITATE AMD DISEASE PHENOTYPES OR INCREASE DISEASE SEVERITY, AS HAS BEEN SUGGESTED TO BE THE CASE IN LARGE SCALE GWA STUDIES, WHERE CFH AND ARMS2/HTRA1 (LOCUS ON CHR. 10Q26) APPEAR TO HAVE SYNERGISTIC EFFECTS ON DISEASE RISK. TOGETHER THEY EXPLAIN >50% OF THE GENETIC VARIABILITY OBSERVED IN AMD. FURTHERMORE, SINCE ARMS2 AND HTRA1 ARE IN STRONG LINKAGE DISEQUILIBRIUM, IT HAS BEEN DIFFICULT TO DECIPHER WHETHER EITHER OR BOTH GENES CONTRIBUTE TO AMD-PATHOLOGIES. THE ISSUES RAISED ABOVE CAN BE ADDRESSED, IN PART, WITH APPROPRIATE ANIMAL MODELS. ALTHOUGH MICE DO NOT HAVE A MACULA PER SE, THEY FAITHFULLY RECAPITULATE MANY ASPECTS OF RETINAL DEGENERATIVE DISEASES AND HAVE BEEN USED TO LEARN HOW DISRUPTION OF CERTAIN MOLECULES LEAD TO AMD-LIKE PATHOLOGIES. IN THIS APPLICATION, WE WILL SEEK IN VIVO CONFIRMATION OF THE CELL-TYPE AND SUBCELLULAR LOCALIZATION OF ARMS2, AND ESTABLISH WHETHER MICE BEARING THE ARMS2A69S ALLELE INDEPENDENTLY DEVELOP AMD-LIKE SUB-PHENOTYPES AND EXPLORE POTENTIAL MOLECULAR MECHANISMS UNDERLYING THE CHANGES. FINALLY, BECAUSE AMD IS A MULTIFACTORIAL DISEASE, WE WILL EXAMINE IF AMD- ASSOCIATED RISK FACTORS SUCH AS SUCH AS DIET OR GENETIC VARIANTS, SUCH AS CFH RISK ALLELES, CAN POTENTIATE AMD- LIKE DISEASE PHENOTYPES. IDENTIFYING THE PATHOGENIC PATHWAYS AND MECHANISMS UNDERLYING THE DISEASE SUB-PHENOTYPES, THE GOAL OF THIS PROPOSAL, IS CRITICAL FOR DEVELOPING EFFECTIVE THERAPIES THAT CAN TARGET THE PRE-SYMPTOMATIC STAGE TO PREVENT, DELAY ONSET OR DECREASE SEVERITY OF THE DISEASE. ANIMAL MODELS SERVE AN IMPORTANT AND UNIQUE ROLE FOR FURTHERING OUR UNDERSTANDING OF THE GENETIC UNDERPINNINGS OF DISEASE, AND AS A RESOURCE TO EXAMINE TISSUE PATHOLOGY AND TO TEST THERAPEUTICS THAT CANNOT BE READILY DONE IN HUMANS.

CHARACTERIZATION OF RNA-CHROMATIN INTERACTOME BY RNA-DNA LIGATION AND SEQUENCING

GENETIC MODIFIERS OF ENHANCED S-CONE SYNDROME ?ROLE OF THE EXTERNAL LIMITING MEMBRANE

TRANSACTING GENES REGULATING RECOMBINATION HOTSPOT ACTIVITIES

MECHANISMS OF POST-TRANSCRIPTIONAL REGULATION OF SPLICING FACTORS

BIOINFORMATICS EXPANSION PHASE III

REGULATION AND FUNCTION OF THE TYPE 2 DIABETES-ASSOCIATED C2CD4A/B LOCUS

GENETICS AND GENOMICS OF THE OVARIAN RESERVE AND FEMALE FERTILITY

GENOMIC ORGANIZATION OF RECOMBINATION HOT SPOTS

SPERMATOGONIAL STEM CELL MAINTENANCE

METHODS AND TOOLS TO ANALYZE GENETIC COMPLEXITY

LEVERAGING NATURAL GENETIC DIVERSITY AND SYSTEMS GENETICS TO ELUCIDATE THE COMPLEX HIERARCHY OF GENE REGULATION UNDERLYING GROUND STATE PLURIPOTENCY, CELL FATE DECISIONS AND TISSUE HOMEOSTASIS

DISARMING THE IMMUNOLOGICAL BARRIERS TO REGENERATION IN MAMMALS - PROJECT SUMMARY/ABSTRACT THE LONG-TERM GOAL OF THIS WORK IS TO FACILITATE THE FAITHFUL REGENERATION OF DAMAGED HUMAN TISSUES. REGENERATION IN ADULT MAMMALS IS EXTREMELY LIMITED; DAMAGED TISSUE IN MOST MAJOR ORGANS FAILS TO REGENERATE, AND INSTEAD UNDERGOES SCAR-BASED REPAIR. THE LACK OF ADULT REGENERATIVE CAPACITY IS AN ENORMOUS BURDEN ON THE HEALTHCARE SYSTEM AND SOCIETY AS A WHOLE. ALTHOUGH BOTH HUMAN AND MOUSE DIGIT TIPS CAN UNDERGO A TRUE REGENERATIVE RESPONSE, THIS REGENERATION IS POSITIONALLY RESTRICTED TO THE TERMINAL DISTAL PHALANX BONE. IMPORTANTLY, AMPUTATIONS WITH AN AXIS POINT BELOW THE TERMINAL DISTAL PHALANX BONE OR TOO CLOSE TO THE NAIL BED RESULT IN REGENERATION FAILURE. NOTABLY, SALAMANDER LIMBS HAVE AN ANATOMY SIMILAR TO THAT OF HUMAN LIMBS, BUT UNIQUELY REGENERATE AFTER AMPUTATION FROM ANY POSITION THROUGHOUT ADULT LIFE. THE BIOLOGICAL MECHANISMS LIMITING REGENERATION IN ADULT MAMMALS IS POORLY UNDERSTOOD. ALTHOUGH THE IMMUNE SYSTEM IS A POWERFUL REGULATOR OF WOUND REPAIR, THE EXACT ROLE OF IMMUNE-CELL NETWORKS AS A DETERMINANT OF REGENERATIVE SUCCESS HAS BEEN GROSSLY UNDERSTUDIED. IN OUR REGENERATION STUDIES, INCLUDING THOSE PROPOSED HERE, WE USE THE MOUSE DIGIT-TIP MODEL, EXAMINING REGENERATION FOLLOWING TISSUE REMOVAL AT DIFFERENT DIGIT LOCATIONS. THIS IS A POWERFUL MODEL, AS REGENERATION CAN BE MEASURED NON-INVASIVELY WITH HIGH-RESOLUTION MICRO-COMPUTED TOMOGRAPHY 3D-IMAGING (BONE/SOFT TISSUE VOLUME), AND ANALYZED COMPREHENSIVELY USING HISTOLOGY AND MOLECULAR ANALYSIS. WE IDENTIFIED SEVERAL LYMPHOID IMMUNE-CELL TYPES THAT INHIBIT MOUSE DIGIT-TIP REGENERATION VIA CYTOTOXIC ACTIVITY AGAINST PROGENITOR CELLS AND SHOWED THAT T- REGULATORY CELLS (TREGS) PLAY A CRITICAL ROLE IN PROTECTING PROGENITORS FROM THESE CELLS. WE ALSO FOUND THAT IN MICE LACKING LYMPHOID IMMUNITY, NOVEL REGENERATION IS INDUCED, PROVIDING NEW MODELS TO IDENTIFY PRO-REGENERATIVE CELLS AND MOLECULAR PATHWAYS THAT CAN BE EXPLOITED THERAPEUTICALLY. IMPORTANTLY, WE ALSO IDENTIFIED SEVERAL LYMPHOID-CELL TYPES THAT SUPPORT REGENERATION, SUGGESTING THE POTENTIAL TO THERAPEUTICALLY ENHANCE HUMAN REPAIR THROUGH TARGETED IMMUNOMODULATION. THIS PROJECT AIMS TO IDENTIFY AND CHARACTERIZE THE MECHANISMS BY WHICH LYMPHOID CELLS REGULATE ADULT REGENERATION. SPECIFICALLY, WE WILL: AIM 1: DISSECT AND CHARACTERIZE LYMPHOID-CELL MECHANISMS INHIBITING REGENERATION. WE WILL USE A RANGE OF MOUSE STRAINS WITH MUTATIONS IN CYTOTOXIC FUNCTION IN EX VIVO AND IN VIVO ANALYSES. AIM 2: DEFINE MECHANISMS OF PRO-REGENERATIVE TREG SUPPRESSION OF LYMPHOID-CELL CYTOTOXICITY USING TREG-SPECIFIC DELETION OF FUNCTIONAL GENES IN VIVO. AIM 3: TEST THE HYPOTHESIS THAT TARGETED DISARMING OF LYMPHOID CELLS COULD ENHANCE REGENERATION IN VIVO. WE WILL TEST TOLEROGENIC MOLECULES IN EX VIVO CYTOTOXICITY ASSAYS AND THEN EVALUATE TOLEROGENIC ANTIGEN OVEREXPRESSION IN VIVO BOTH DIRECT TRANSGENIC AND VIRAL APPROACHES AND THEN VIA A MODIFIED TREG DELIVERY STRATEGY. THIS PROJECT WILL FUSE DEVELOPMENTAL BIOLOGY AND IMMUNOLOGICAL METHODS TO IDENTIFY THE CRITICAL BIOLOGICAL PATHWAYS AND GENETIC MODIFIERS REQUIRED FOR TRANSIENT IMMUNOMODULATION STRATEGIES DIRECTED AT INDUCING LATENT REGENERATIVE POTENTIAL IN ADULT TISSUES IN MAMMALS. THIS WORK WILL LAY THE GROUNDWORK FOR TRANSLATION STUDIES AIMED AT ENHANCING TISSUE REPAIR IN HUMAN PATIENTS.

MUMMICHOG 3, ALIGNING MASS SPECTROMETRY DATA TO BIOLOGICAL NETWORKS

DISSECTING THE HETEROGENEITY OF HUMAN ISLET STRESS RESPONSES IN TYPE 2 DIABETES

MODULATION OF AGRP NEURONAL EXCITABILITY: ROLE OF DIET AND BODY WEIGHT

BUILDING MOUSE MODELS OF METABOLIC SYNDROME

GENETICS OF ALOPECIA AREATA IN THE C3H/HEJ MOUSE

MOLECULAR AND PHYSIOLOGICAL FUNCTION OF THE TUBBY GENE FAMILY

SHORT COURSE ON EXPERIMENTAL MODELS OF HUMAN CANCER

REVEALING SPATIAL TRANSCRIPTOMIC AND PROTEOMIC SHIFTS IN TUMOR-INFILTRATING IMMUNOCYTES: CHARTING A PATH TO ENHANCED MELANOMA THERAPEUTICS

AN EXPLAINABLE UNIFIED AI STRATEGY FOR EFFICIENT AND ROBUST INTEGRATIVE ANALYSIS OF MULTI-OMICS DATA FROM HIGHLY HETEROGENEOUS MULTIPLE STUDIES - HEALTHY CENTENARIANS CARRY PROTECTIVE VARIANTS THAT COUNTERACT AGE-RELATED DISEASE RISK VARIANTS, THE FORMER OF WHICH ARE MOSTLY RARE. THEREFORE, MARKERS ASSOCIATED WITH EXCEPTIONAL LONGEVITY (EL) NEED BE DISCOVERED THROUGH INTEGRATIVE MULTI-OMICS DATA ANALYSIS TO IMPROVE DETECTION POWER. HOWEVER, EXISTING INTEGRATIVE ANALYSIS METHOD FOR MULTI-OMICS DATA DO NOT MODEL THE RELATIONSHIPS AMONG MARKERS IN A MODALITY AND AMONG STUDIES, MUDDYING THE EFFICIENT USE OF PERTINENT INFORMATION PROVIDED BY MULTI-OMICS DATA FROM HETEROGENEOUS STUDIES. WE PROPOSE A UNIFIED AI STRATEGY THAT MODELS THE RELATIONSHIPS AMONG MARKERS, MODALITIES, AND STUDIES, AND LEARNS NONLINEAR LOW-DIMENSIONAL REPRESENTATIONS OF DATA IN A COMMON SPACE VIA GRAPH NEURAL NETWORKS (GNN). WE ACHIEVE DEEP INTEGRATION BY ENFORCING THE MAXIMIZATION OF SIMILARITIES BETWEEN STUDY REPRESENTATIONS AND THE PHENOTYPE PREDICTION ACCURACY IN A SINGLE GNN. THE PROPOSAL HAS THREE SPECIFIC AIMS: 1) DEVELOP AN EXPLAINABLE UNIFIED AI STRATEGY AND SOFTWARE FOR EFFICIENT AND ROBUST INTEGRATIVE ANALYSIS OF MULTI-OMICS DATA FROM HIGHLY HETEROGENEOUS MULTIPLE STUDIES. 2) APPLY THE METHODS DEVELOPED IN AIM 1 TO LONG-LIFE FAMILY STUDY (LLFS) AND INTEGRATIVE LONGEVITY OMICS (ILO) DATA PROVIDED BY THE EL CONSORTIUM TO IDENTIFY EL-ASSOCIATED PATHWAYS AND BIOMARKERS. 3) APPLY THE METHODS DEVELOPED IN AIM 1 TO OMICS DATA FROM HUMAN AND 100 SPECIES OF DIVERSE LIFESPAN PROVIDED BY THE EL CONSORTIUM TO IDENTIFY CONSERVED AND SPECIES-SPECIFIC EL-ASSOCIATED PATHWAYS AND MARKERS. THE OUTCOME OF THIS WORK WILL RESULT IN A PUBLICLY AVAILABLE INTEGRATIVE OMICS DATA ANALYSIS SOFTWARE WHICH NOT ONLY IS ABLE TO IDENTIFY ROBUST LONGEVITY-ASSOCIATED PATHWAYS AND BIOMARKERS, BUT WILL ALSO BE APPLICABLE TO ANY COMPLEX DISEASE STUDY WITH SIMILAR OMICS DATA ANALYSIS DEMANDS. OUR WORK WILL CONTRIBUTE SIGNIFICANTLY TO IDENTIFY THERAPEUTIC INTERVENTIONS FOR IMPROVING HUMAN HEALTH.

MOLECULAR GENETIC CHARACTERIZATION OF ALSTROM SYNDROME

ADHESION AND MATRIX MOLECULES IN THE NEURONAL DEVELOPMENT OF THE RETINA

GENETIC MODIFIERS OF BETA-LIKE GLOBIN GENE SWITCHING

RESEARCH VIVARIUM

JACKSON LABORATORY (THE)

GENETIC DEFINITION OF MECHANISMS BY WHICH RAPAMYCIN RETARDS MAMMALIAN AGING

COMPUTATIONAL BIOLOGY RESOURCES

A NOVEL WILD-DERIVED DIVERSITY MOUSE POPULATION FOR PRECISION MAPPING AND SYSTEMS GENETICS - PROJECT SUMMARY INBRED MOUSE STRAINS ARE INTEGRAL TOOLS FOR BOTH PRECLINICAL AND BASIC RESEARCH. WHILE THE ABSENCE OF GENETIC DIVERSITY IS A WIDELY PERCEIVED STRENGTH OF INBRED STRAINS, IT IS ALSO A CRITICAL LIMITATION, AS A SINGLE INBRED GENETIC BACKGROUND CANNOT CAPTURE THE FULL SPECTRUM OF PHENOTYPIC RESPONSES OBSERVED IN GENETICALLY DIVERSE HUMAN POPULATIONS. THIS LIMITS THE TRANSLATION SUCCESS FROM INBRED MOUSE MODELS TO HUMAN CLINICAL OUTCOMES AND UNDERSCORES THE NEED FOR MOUSE PLATFORMS THAT MORE ACCURATELY MODEL HUMAN GENETIC VARIATION. THE DIVERSITY OUTBRED (DO) MOUSE POPULATION WAS FOUNDED IN 2012 TO MEET THESE CRUCIAL RESEARCH NEEDS. THE DO WAS INITIATED FROM EIGHT GENETICALLY DIVERSE INBRED MOUSE STRAINS, INCLUDING WILD-DERIVED REPRESENTATIVES FROM EACH OF THREE CARDINAL HOUSE MOUSE SUBSPECIES, AND HAS BEEN MAINTAINED BY PSEUDO-RANDOM MATING FOR OVER 50 GENERATIONS. HOWEVER, THE INCLUSION OF FOUNDER STRAINS DERIVED FROM REPRODUCTIVELY ISOLATED SUBSPECIES HAS, OVER TIME, EXPOSED GENETIC INCOMPATIBILITIES AND UNCHECKED SELFISH ELEMENTS, RESULTING IN ALLELE FREQUENCY DISTORTIONS THAT HAVE COMPROMISED THE GENETIC INTEGRITY OF THE DO. HERE, WE PROPOSE TO GENERATE A NEW, HIGH DIVERSITY OUTBRED MOUSE POPULATION, THE WILD MOUSE DIVERSITY PANEL (WMDP), FOUNDED FROM NOVEL WILD-DERIVED INBRED STRAINS OF SINGLE SUBSPECIES ORIGIN (MUS MUSCULUS DOMESTICUS). OUR RECENT WORK HAS SHOWN THAT A LARGE PROPORTION OF VARIANTS IN THE GENOMES OF THESE NEWLY DEVELOPED M. M. DOMESTICUS STRAINS ARE ABSENT FROM EXISTING INBRED MOUSE STRAINS, AND THAT THESE STRAINS CAPTURE NOVEL NEUROBEHAVIORAL, METABOLIC, PHYSIOLOGICAL, AND BIOCHEMICAL PHENOTYPES NOT OBSERVED IN COMMON LABORATORY MOUSE MODELS. THUS, THE WMDP STANDS TO MINIMIZE POTENTIAL HAPLOTYPE DISTORTION DUE TO GENETIC INCOMPATIBILITIES BETWEEN SUBSPECIES, ALLOW FUNCTIONAL ASSESSMENT OF MILLIONS OF VARIANTS THAT HAVE NEVER BEEN TESTED IN THE LABORATORY, AND EMPOWER INVESTIGATIONS OF NATURAL GENOME COMPLEXITY. IN AIM 1, WE WILL INITIATE AN OUTBRED POPULATION FROM FOUR PHENOTYPICALLY AND GENETICALLY DIVERSE WILD- DERIVED M. M. DOMESTICUS INBRED STRAINS, IMPLEMENTING ROUTINE GENOMIC MONITORING OF ALLELE FREQUENCIES TO ENSURE THE GENETIC INTEGRITY AND LONGEVITY OF THE WMDP RESOURCE. TO AID DISCOVERY EFFORTS IN THIS NEW POPULATION, WE WILL GENERATE KEY GENOMIC RESOURCES FOR THE FOUNDER STRAINS IN AIM 2, INCLUDING HIGH QUALITY DE NOVO GENOME ASSEMBLIES, COMPREHENSIVE VARIANT CALL SETS, AND A GENE EXPRESSION ATLAS OF SEVERAL TISSUES. IN AIM 3, WE WILL PROVIDE A PROOF-OF-PRINCIPLE STUDY TO UNDERSCORE THE POWER AND UTILITY OF THE WMDP. WE WILL PROFILE PHENOTYPIC DIVERSITY FOR SEVERAL MORPHOLOGICAL, METABOLIC, BEHAVIORAL, AND CLINICAL TRAITS IN EARLY OUTBREEDING GENERATIONS OF THE WMDP, MAP CAUSAL LOCI, AND INTEGRATE OUR FINDINGS WITH HUMAN DATA TO IDENTIFY HUMAN-MOUSE TRANSLATIONAL PARALLELS AT THE LEVELS OF BOTH GENES AND GENE NETWORKS. THIS PROJECT WILL YIELD A NEW OUTBRED MOUSE POPULATION POISED FOR NEAR LIMITLESS DISCOVERY IN ALL AREAS OF BASIC AND PRECLINICAL RESEARCH. THIS RESOURCE WILL BECOME PART OF THE SPECIAL MOUSE STRAIN RESOURCE AT THE JACKSON LABORATORY, WHICH WILL PROVIDE THE FRAMEWORK AND SETTING FOR ITS LONG-TERM MAINTENANCE AND DISTRIBUTION TO THE GLOBAL RESEARCH COMMUNITY.

DEVELOPMENT OF MAMMALIAN OOCYTE-GRANULOSA CELL COMPLEX

TEACHING THE GENOME GENERATION: CULTIVATING HIGH SCHOOL GENOMICS THROUGH TEACHER EDUCATION - PROJECT SUMMARY/ABSTRACT THE JACKSON LABORATORY (JAX) PROPOSES A TEACHER EDUCATION INITIATIVE, 'TEACHING THE GENOME GENERATION' (TTGG), TO PROVIDE PRE-SERVICE HIGH SCHOOL TEACHERS THE CONTENT KNOWLEDGE, TEACHING STRATEGIES, AND RESOURCES NEEDED TO ENHANCE STUDENT LEARNING IN GENOMICS, BIOETHICS, AND BIOINFORMATICS, WITH AN EMPHASIS ON MATH AND DATA LITERACY. OUR PRE-PROFESSIONAL DEVELOPMENT PROGRAM WILL PROVIDE INSTRUCTION IN THE MOLECULAR GENETICS OF PERSONALIZED MEDICINE, USE OF BIOINFORMATICS TOOLS, INCORPORATING STATISTICS AND DATA ANALYSIS, AND DISCUSSION OF THE ETHICAL, LEGAL, AND SOCIAL IMPLICATIONS (ELSI) SURROUNDING GENETICS RESEARCH. IN COLLABORATION WITH PARTNERS IN HIGHER EDUCATION, UP TO 32 PRE-SERVICE TEACHERS PER YEAR WILL PARTICIPATE IN A HANDS-ON SHORT COURSE. PRE-SERVICE TEACHERS WILL ALSO HAVE THE OPPORTUNITY TO ENGAGE WITH AND PRACTICE TEACHING OUR CURRICULUM THROUGH INSTRUCTIONAL METHODS COURSEWORK AND STUDENT TEACHING/INTERNSHIPS. OUR INNOVATIVE APPROACH WEAVES THREE LEARNING STRANDS—MOLECULAR GENETICS, BIOINFORMATICS AND BIOETHICS—TOGETHER WITHIN THE CONTEXT OF THE NEXT GENERATION SCIENCE STANDARDS AND COMMON CORE MATH STANDARDS. THE TTGG TEAM WILL EXPAND DISSEMINATION OF OUR CONTENT AND SHORT COURSE THROUGH SIGNIFICANT ENHANCEMENT OF OUR ONLINE RESOURCES. A PILOT ONLINE COURSE WILL BE LAUNCHED IN LATE 2020, AND A FULL ONLINE PROGRAM WITH ADDITIONAL RESOURCES ACCESSIBLE THROUGH A NEWLY DESIGNED TTGG PUBLIC FACING WEBSITE WILL BE COMPLETED AND RELEASED BY THE FIRST QUARTER OF 2022. TO EVALUATE THE IMPACT OF THE SHORT COURSE, EVALUATORS WILL ADMINISTER PRE AND POST SURVEYS TO PRE-SERVICE TEACHER PARTICIPANTS ON GENETICS, GENOMICS AND BIOINFORMATICS CONTENT KNOWLEDGE, TEACHER SELF-EFFICACY, AND CONFIDENCE. EVALUATORS WILL ALSO CONDUCT SMALL SCALE EFFICACY STUDIES TO EXAMINE THE IMPACT OF THE TTGG MATERIALS ON STUDENTS': A) CONTENT KNOWLEDGE OF GENETICS, GENOMICS, AND APPLICATIONS OF MATHEMATICS; B) CONFIDENCE ENGAGING IN GENOMICS CONCEPTS, LAB ACTIVITIES, AND MATH PROBLEMS; C) INTEREST IN ENGAGING IN ADDITIONAL GENOMICS-RELATED BEHAVIOR, INCLUDING ACADEMIC AND CAREER PURSUITS; AND D) ABILITY TO EXPLAIN HOW AND WHY MATH SKILLS ARE REQUIRED FOR PRACTICING LIFE SCIENCES. BY TRAINING PRE-SERVICE EDUCATORS, TTGG WILL EXECUTE ON NIH'S GOAL OF STRENGTHENING THE FUTURE STEM WORKFORCE THROUGH INCREASING GENOMIC AND HEALTH LITERACY.

LIFESPAN EXTENSION DESPITE GREATLY ELEVATED INSULIN AND BODY FAT

SHOCK CENTER FOR AGING RESEARCH AT THE JACKSON LABORATO*

MOUSE PHENOME DATABASE: NIA INTERVENTIONS TESTING PROGRAM DATA COORDINATING CENTER

COMPLEX TRAIT ANALYSIS OF ERYTHROPOIESIS IN NORMAL POPULATIONS

GENETIC IDENTIFICATION OF NOVEL GENES CRITICAL IN ERYTHROPOIESIS

ALZHEIMER'S DISEASE-RELATED DEMENTIA MODELS BY PRECISION EDITING AND RELEVANT GENETIC X ENVIRONMENTAL EXPOSURES - PROJECT SUMMARY ALZHEIMER’S DISEASE IS THE MOST COMMON CAUSE OF DEMENTIA IN THE ELDERLY, BUT THERE ARE A NUMBER OF OTHER RELATED DEMENTIAS THAT EXHIBIT SUBSTANTIAL OVERLAP IN THE BEHAVIORAL, COGNITIVE, AND NEUROPATHOLOGICAL MANIFESTATIONS OF THE DISEASE. IN FACT, THE MAJORITY OF DEMENTIA CASES LIKELY ARISE FROM THE CO-OCCURRENCE OF ONE OR MORE OF THESE AD AND AD-RELATED PATHOLOGIES, WITH VERY FEW INDIVIDUALS EXHIBITING ‘PURE’ ALZHEIMER’S PATHOLOGY (E.G., ONLY AMYLOID PLAQUES). THIS COMPLEXITY MAKES DIAGNOSIS AND THERAPEUTIC DEVELOPMENT CHALLENGING, A PROBLEM EXACERBATED BY A PAUCITY OF ACCURATE ANIMAL MODELS FOR ADRD THAT FAITHFULLY RECAPITULATE THE FULL SPECTRUM OF THE MOLECULAR, CELLULAR, COGNITIVE, AND BEHAVIORAL PATHOLOGIES OF THESE DEMENTIAS. IN RESPONSE TO PAR-19-167, WE WILL CREATE A PANEL OF GENETICALLY DIVERSE KNOCK-IN MICE HARBORING KNOWN MUTATIONS ASSOCIATED WITH AD AND SEVERAL RELATED DEMENTIAS USING PRECISE GENOMIC EDITING TO ENSURE BIOLOGICALLY-RELEVANT GENE EXPRESSION PATTERNS AND LEVELS. IN AIM 1, WE WILL USE CRISPR/CAS9 TO CREATE MICE CARRYING COMBINATIONS OF DISEASE-CAUSING MUTATIONS IN APP, PSEN1, MAPT, TARDBP, AND SNCA TO PRODUCE A SET OF ‘CORE’ STRAINS WE EXPECT TO BETTER CAPTURE THE COMPLEXITY OF ADRD. TO CAPTURE THE ROLE OF GENETIC BACKGROUND IN DISEASE RISK, WE WILL THEN CROSS THESE ‘CORE’ MICE TO FOUR GENETIC BACKGROUNDS KNOWN TO PROMOTE SUSCEPTIBILITY OR RESILIENCE OF ADRD (DBA/2J, FVB/NJ, WSB/EIJ, AND C57BL/6J). WE WILL THEN LEVERAGE OUR EXPERTISE IN HIGH-THROUGHPUT MOUSE NEUROBEHAVIORAL PHENOTYPING TO SCREEN 16 NEW ADRD STRAINS TO IDENTIFY THE LINES THAT BEST MODEL ADRD. IN AIM 2, WE WILL USE OUR DEEP PHENOTYPING PIPELINE TO FULLY CHARACTERIZE OUR TOP STRAINS ACROSS THE ENTIRE SPECTRUM OF ADRD-RELATED SYMPTOMS, INCLUDING BOTH COGNITIVE AND NON-COGNITIVE DOMAINS. WE WILL ALSO USE HIGH-FIELD MRI, HISTOPATHOLOGICAL MEASUREMENTS, AND MOLECULAR PHENOTYPES TO ASSESS EFFECTS ON BRAIN STRUCTURE, EXTENT OF NEUROPATHOLOGIES, AND IMPACT ON GENE NETWORKS AND PATHWAYS ASSOCIATED WITH DISEASE. FINALLY, IN AIM 3, WE WILL VALIDATE OUR NEW MODELS FOR USE IN BASIC SCIENCE AND PRECLINICAL STUDIES BY DETERMINING CONCORDANCE BETWEEN MOUSE AND HUMAN DATA AND USE NETWORK MODELING APPROACHES TO IDENTIFY EARLY DRIVERS OF DISEASE THAT PREDICT LATE-STAGE OUTCOMES IN HUMANS. THIS PROJECT WILL PRODUCE MUCH-NEEDED NEW MODELS FOR AD AND RELATED DEMENTIAS THAT WILL GREATLY ENHANCE OUR UNDERSTANDING OF THE PATHOLOGICAL MECHANISMS UNDERLYING THESE DISEASES. FINALLY, ALL OF THE MODELS PRODUCED HERE WILL BE DISTRIBUTED TO THE COMMUNITY VIA THE JAX REPOSITORY. WE WILL ALSO MAKE ALL OF THE PHENOTYPING DATA PUBLICLY AVAILABLE USING RESOURCES SUCH AS MOUSE PHENOME DATABASE, GENEWEAVER, AND SYNAPSE.

GENETIC CONTROL OF PURKINJE CELL DEGENERATION

MULTI-OMIC PHENOTYPING OF HUMAN TRANSCRIPTIONAL REGULATORS - PROJECT SUMMARY THE MOLECULAR PHENOTYPES OF NULL ALLELES IN CELLS (MORPHIC) PROGRAM IS USING MULTIPLE PERTURBATION STRATEGIES TO REALIZE THE NHGRI'S VISION OF ASSIGNING FUNCTION TO EVERY HUMAN GENE. STRATEGIES INCLUDE POOLED AND INDIVIDUAL GENE KNOCKOUTS AND KNOCKDOWNS (KDS), GENERATED USING CRISPR TECHNOLOGIES AND AUXIN-INDUCIBLE DEGRONS. FOLLOWING APPLICATION OF SUCH ASSAYS, MOLECULAR PHENOTYPES OF THE CELLS ARE PROFILED LONGITUDINALLY AND AT INDIVIDUAL TIME POINTS USING BULK AND SINGLE-CELL (SC)RNA-SEQ. PERTURBATION STRATEGIES HAVE INTRINSIC SOURCES OF VARIABILITY, E.G., KD PENETRANCE, WHILE THE SINGLE-CELL SEQUENCING APPROACHES CONTRIBUTE TECHNICAL NOISE, E.G., `DROP OUT.' QUANTIFYING AND CONTROLLING THIS VARIABILITY ARE CRUCIAL TO ENSURE RELIABLE PHENOTYPIC ASSESSMENT AND FULFILL MORPHIC'S GOAL TO ACCURATELY CATALOG GENE FUNCTION. GIVEN THE CRITICAL ROLE OF TRANSCRIPTION FACTORS (TFS) IN REGULATING CELL STATE, ALL FOUR MORPHIC DATA PRODUCTION CENTERS (DPCS) WILL PERTURB TFS AND THEN PROFILE CELLS USING BULK OR SC-RNA-SEQ. A WIDE RANGE OF OTHER `REGULATORY PHENOTYPING' DATA, INCLUDING (BULK OR SINGLE-CELL) ATAC-SEQ, ARE BEING GENERATED WITHIN MORPHIC AND TF CHIP-SEQ, HIC, AND MASSIVELY PARALLEL REPORTER ASSAY (MPRA) DATA ARE AVAILABLE IN THE ENCODE AND IMPACT OF GENOMIC VARIATION ON FUNCTION (IGVF) CONSORTIA. TO ROBUSTLY DEFINE THE REGULATORY IMPACT OF TF PERTURBATION, WE PROPOSE A JAX MORPHIC DATA ANALYSIS AND VALIDATION CENTER (DAV) TO ANALYZE THESE MULTI-MODAL DATA. OUR TEAM IS UNIQUELY POSITIONED TO ESTABLISH THIS TF-FOCUSED DAV: WE ARE CO-LOCATED WITH THE JAX MORPHIC DPC AND HAVE CONSORTIUM-LEVEL COLLABORATIONS WITH ITS PI, WHILE OUR OWN WORK FOCUSES ON ELUCIDATING TRANSCRIPTIONAL REGULATION OF GENES AND ON DEVELOPING ROBUST COMPUTATIONAL METHODS THROUGH COMMUNITY EFFORTS. IN AIM 1, WE WILL QUANTIFY AND CONTROL VARIABILITY IN PERTURBATION-BASED REGULATORY PHENOTYPING BY USING HETEROGENEOUS DATA GENERATED WITHIN MORPHIC TO ISOLATE THEIR TECHNICAL NOISE CHARACTERISTICS AND TO DERIVE A SET OF TF-GENE TARGET PAIRS (TF-GTS). WE WILL THEN COMPUTATIONALLY SIMULATE LARGE- SCALE PERTURBATION SCREENS, THROUGH WHICH WE WILL PERFORM POWER ANALYSIS TO QUANTIFY DATA VARIABILITY AND MAKE RECOMMENDATIONS THAT AMELIORATE IT. IN AIM 2, WE WILL EVALUATE PUBLISHED GENE REGULATORY NETWORK (GRN) INFERENCE METHODS. WE WILL ALSO CONDUCT TWO “CROWD-SOURCED” DREAM CHALLENGES, IN WHICH COMMUNITY PARTICIPANTS WILL DEVELOP GRN INFERENCE METHODS THAT WE WILL OBJECTIVELY EVALUATE WITH MORPHIC DATA. USING TOP- PERFORMING METHODS, AS WELL AS A NOVEL APPROACH WE ARE DEVELOPING BASED ON DYNAMICAL SYSTEMS, WE WILL PERFORM IN SILICO TF PERTURBATION WITHIN THE GRNS TO PRIORITIZE TFS FOR EXPERIMENTAL VALIDATION IN MORPHIC. IN AIM 3, WE WILL FURTHER IMPROVE ROBUSTNESS OF INFERRED TF-GTS BY INTEGRATING THEM WITH TF CHIP-SEQ, HIC, AND MPRA DATA, KNOCKOUT MOUSE PHENOTYPING DATA (KOMP2), AND SPATIAL TRANSCRIPTOMICS DATA FROM JAX AND MORPHIC. WE WILL VALIDATE PUBLISHED METHODS FOR DEFINING TISSUE-SPECIFIC GRNS BY OVERLAPPING THEM WITH RELEVANT MORPHIC MODEL SYSTEMS AND WILL THEN USE THEM TO PREDICT TF-GTS IN SYSTEMS YET TO BE PROFILED BY MORPHIC. OUR AIMS WILL BOLSTER THE FIELD'S ABILITY TO DECIPHER THE REGULATORY FUNCTION OF THE ~ 1600 TF GENES WITHIN THE HUMAN GENOME.

ILLUMINATING THE DRUGGABLE GENOME BY KNOWLEDGE GRAPHS

BUILDING A SPATIAL TRANSCRIPTOMICS INFRASTRUCTURE FOR ISOFORM PROFILING IN AGING PRE-NEOPLASTIC TISSUES - PROJECT SUMMARY/ABSTRACT AGING IS THE GREATEST RISK FACTOR FOR CANCER, BUT IT IS NOT KNOWN WHICH AGE-DEPENDENT CELLULAR AND MOLECULAR EVENTS DRIVE CANCER INITIATION. SPATIAL TRANSCRIPTOMIC APPROACHES ARE REVOLUTIONIZING OUR UNDERSTANDING OF CANCER INITIATION, PROGRESSION, AND DRUG RESISTANCE BY REVEALING EXPRESSION PATTERNS WITH TISSUE MORPHOLOGICAL CONTEXT. HOWEVER, THESE APPROACHES HAVE NOT YET BEEN APPLIED TO THE INTERDISCIPLINARY BIOLOGY OF AGING-DRIVEN CANCERS, DESPITE THE LIKELIHOOD THAT INTRATISSUE AND MICROENVIRONMENTAL EVOLUTION MEDIATE THE AGING PHENOTYPE. MOREOVER, THE MAJORITY OF CURRENT SINGLE CELL SPATIAL PROJECTS ARE BASED ON 3' SHORT READ RNA-SEQUENCING (RNA-SEQ) AND THEREFORE LACK THE ABILITY TO DETECT FULL-LENGTH SPLICED ISOFORMS, WHICH ARE FREQUENTLY OBSERVED IN TUMORS AND ARE KNOWN TO IMPACT TUMOR INITIATION AND TREATMENT RESPONSE. WORK FROM US AND OTHERS HAS REVEALED WIDESPREAD ALTERATIONS IN ALTERNATIVE RNA SPLICING IN HUMAN TUMORS, INCLUDING IN BREAST CANCER, AND THAT HALF OF ALL SPLICED ISOFORMS DETECTED IN HUMAN BREAST TUMORS USING LONG-READ SEQUENCING (LR-SEQ) ARE MISSED BY RNA-SEQ AND ABSENT FROM REFERENCE TRANSCRIPTOMES. IN ADDITION, WE HAVE CAUSALLY LINKED THE UPREGULATION OF SPECIFIC SPLICING FACTORS WITH BREAST TUMOR INITIATION BOTH IN VITRO AND IN VIVO AND IDENTIFIED AGE-DEPENDENT CHANGES IN SPLICED ISOFORMS IN CANCER-ASSOCIATED GENES IN MAMMARY EPITHELIAL CELLS. TOGETHER, THESE RESULTS SUGGEST THAT ALTERNATIVE SPLICING IS A CRITICAL MECHANISM UNDERLYING TUMOR INITIATION WITH AGE, AND THAT SPATIAL LR-SEQ APPROACHES ARE REQUIRED TO RESOLVE THESE MECHANISMS. HOWEVER, STANDARDIZED APPROACHES AND RESOURCES TO MEASURE, QUANTIFY, AND VISUALIZE EXPRESSION OF FULL-LENGTH ISOFORMS WITHIN TISSUES ARE LACKING. THIS GAP IN INFRASTRUCTURE IMPEDES THE FIELD'S ABILITY TO IDENTIFY CELL POPULATIONS THAT EXPRESS AGE-DEPENDENT ISOFORMS, HOW SUCH ISOFORMS IMPACT CANCER INITIATION FOR EXAMPLE THROUGH CHANGES IN RECEPTOR-LIGAND INTERACTIONS. TO ADDRESS THESE INFRASTRUCTURE AND KNOWLEDGE GAPS, WE WILL FIRST DEVELOP APPROACHES TO MAP AND ANALYZE FULL-LENGTH RNA ISOFORMS SPATIALLY WITHIN TISSUE SECTIONS (R21 PHASE, AIM 1). THESE TOOLS, WHICH MERGE LR-SEQ AND SPATIAL TRANSCRIPTOMICS, WILL BE APPLICABLE ACROSS SAMPLE TYPES AND WILL THEREFORE BE OF BROAD, SUSTAINABLE UTILITY TO THE RESEARCH COMMUNITY. WE WILL THEN APPLY THESE TECHNOLOGIES TO GENERATE A SPATIAL MAP OF FULL-LENGTH RNA ISOFORMS IN HEALTHY BREAST TISSUES AND TUMORS DURING AGING (R33 PHASE, AIMS 2 AND 3). FINALLY, WE WILL DEVELOP DATA SHARING AND VISUALIZATION TOOLS FOR SPATIAL ISOFORM EXPRESSION TO ENABLE OTHERS TO MINE OUR DATA VIA A WEB RESOURCE (R33 PHASE, AIM 4). TO ACHIEVE THESE GOALS, THIS PROJECT WILL LEVERAGE THE COMPLEMENTARY AND INTERDISCIPLINARY EXPERTISE OF THE ANCZUKOW LAB IN ALTERNATIVE SPLICING AND BREAST CANCER, AND OF THE CHUANG LAB IN SYSTEMS BIOLOGY AND SPATIAL TRANSCRIPTOMICS ANALYSIS. IN RESPONSE TO NOT-CA-22-002, NOTICE OF NCI'S PARTICIPATION IN PAR-20- 070, THIS PROJECT WILL DELIVER A SPATIAL TRANSCRIPTOMIC INFRASTRUCTURE FOR ISOFORM PROFILING AND A CRITICAL INTERDISCIPLINARY DATA RESOURCE FOR AGING AND CANCER RESEARCHERS TO UNDERSTAND THE ROLE OF SPLICING IN TISSUE AGING AND ONCOGENESIS, THEREBY ADVANCING APPROACHES FOR CANCER EARLY DETECTION, INTERVENTION, AND PREVENTION.

SHORT COURSE ON MEDICAL AND EXPERIMENTAL MAMMALIAN GENETICS

BRUKER SINGLE CELL PROTEOMICS MASS SPECTROMETER SYSTEM - PROJECT SUMMARY THE LONG-TERM GOAL OF THIS PROPOSAL IS TO ADVANCE THE RESEARCH OF INVESTIGATORS AT THE JACKSON LABORATORY (JAX) AND IN THE MAINE RESEARCH COMMUNITY BY BRINGING A NEW BRUKER TIMSTOF SCP MASS SPECTROMETER PACKAGE FOR HIGH-RESOLUTION, SINGLE-CELL PROTEOMIC ANALYSIS TO JAX’S MASS SPECTROMETRY AND PROTEIN CHEMISTRY SERVICE (MSPC). JAX’S RESEARCH MISSION FOCUSES ON DISCOVERING PRECISE GENOMIC SOLUTIONS FOR DISEASE TO IMPROVE HUMAN HEALTH IN THE GLOBAL COMMUNITY. AS PART OF THIS MISSION, THERE HAS BEEN AN INCREASING EMPHASIS ON THE ANALYSIS OF SINGLE CELLS, AS BIOMEDICAL RESEARCH HAS DEMONSTRATED THAT EXAMINATION OF BIOLOGY AT THIS LEVEL IS ESSENTIAL FOR UNDERSTANDING THE BIOLOGICAL PROCESSES UNDERLYING DEVELOPMENT AND DISEASE. ACCORDINGLY, JAX HAS TECHNOLOGIES FOR CHARACTERIZATION AND QUANTIFICATION OF GENE EXPRESSION, CHROMATIN ACCESSIBILITY, AND INDIVIDUAL CELL-SURFACE PROTEIN MOLECULES PRESENT, ALL AT THE SINGLE-CELL LEVEL. IN ADDITION, RECENTLY JAX INVESTIGATORS HAVE HAD ACCESS TO INCREASINGLY ADVANCED CAPABILITIES IN THE MSPC IN MASS SPECTROMETRY-BASED PROTEOMICS AND METABOLOMICS FOR BIOLOGICAL DISCOVERIES LINKING GENOMICS WITH PHENOTYPE. HOWEVER, DESPITE OUR NUMEROUS MASS SPECTROMETRY CAPABILITIES, THERE IS CURRENTLY NO INSTRUMENTATION HERE WITH THE CAPABILITY TO PERFORM UNBIASED, HIGH-THROUGHPUT LABEL-FREE SINGLE-CELL PROTEOMICS ANALYSIS. THIS GAP LIMITS OUR INVESTIGATORS’ ABILITY TO UNDERSTAND BIOLOGY AT THE SINGLE-CELL LEVEL, INCLUDING NEW INSIGHTS THAT WOULD BE GAINED BY INTEGRATING SINGLE-CELL PROTEOME DATA WITH THE ARRAY OF OTHER SINGLE-CELL DATA GENERATED HERE, AND BY ADDRESSING THE ISSUE OF A GENERALLY WEAK TRANSCRIPTOME-PROTEOME CORRELATION IN ORGANISMS. THE BRUKER TIMSTOF SCP MASS SPECTROMETER IS THE IDEAL SYSTEM TO ADDRESS THIS GAP. THIS INSTRUMENT OFFERS INNOVATIVE TECHNOLOGIES, INCLUDING A DRAMATICALLY IMPROVED ION-SOURCE CONCEPT, AN ENHANCED TRAPPED ION MOBILITY, AND MASS ANALYZER THAT PROVIDE ULTRA-HIGH SENSITIVITY, ENABLING UNBIASED SINGLE-CELL PROTEOMICS WITH HIGH REPRODUCIBILITY. THE TIMSTOF SCP CAN ACHIEVE COVERAGE OF ABOUT 1,500 PROTEINS PER CELL, SUFFICIENTLY IDENTIFY POST-TRANSLATIONAL MODIFICATIONS IN A VERY SMALL NUMBER OF MORPHOLOGICALLY OR FUNCTIONALLY SIMILAR CELLS, AND EFFICIENTLY ANALYZE LOW-ABUNDANT SAMPLES, SUCH AS THE RARE CELL POPULATIONS THAT MANY OF OUR INVESTIGATORS STUDY. THE TIMSTOF SCP AND ITS ACCESSORIES, INCLUDING THE CELLENONE FOR SINGLE-CELL SORTING AND PROTEOMIC SAMPLE PROCESSING, WILL BE TRANSFORMATIVE FOR RESEARCH AT JAX AND OTHER MAINE RESEARCH INSTITUTIONS. VARIOUS STUDIES AT JAX FOCUSED ON RARE IMMUNE-CELL POPULATIONS, STEM-CELL DIFFERENTIATION, SUB-POPULATIONS OF CANCER CELLS, THE IDENTIFICATION OF NEW THERAPEUTIC TARGETS FOR CANCER, AND CHARACTERIZING MECHANISMS UNDERLYING COGNITIVE AGING WILL ALL BENEFIT FROM THIS INSTRUMENTATION. PROGRESS IN THESE RESEARCH AREAS ARE PARTICULARLY IMPORTANT GIVEN JAX’S BROADENING EMPHASIS ON TRANSLATING OUR BASIC RESEARCH DISCOVERIES INTO CLINICAL ADVANCES THAT WILL DIRECTLY BENEFIT HUMAN HEALTH. IN SUM, THE BRUKER TIMSTOF SCP PLATFORM WILL SUBSTANTIALLY ENHANCE JAX’S ABILITY TO FULFILL ITS RESEARCH MISSION AND WILL ALSO BRING THIS POTENTIAL TO THE GREATER MAINE COMMUNITY.

CELLULAR AND GENETIC MECHANISMS OF AUTOIMMUNE DIABETES ASSOCIATED NEURITIS - PROJECT SUMMARY TYPE 1 DIABETES (T1D) IS OFTEN ACCOMPANIED BY OTHER AUTOIMMUNE DISORDERS, INCLUDING AUTOIMMUNE NEUROPATHIES. FINDINGS IN BOTH NOD MICE AND PATIENTS HAVE REVEALED POTENTIAL OVERLAP BETWEEN IMMUNE RESPONSES TARGETING PANCREATIC B-CELLS AND NERVES. WE HYPOTHESIZED THAT LYMPHOCYTE POPULATIONS INVOLVED IN T1D PATHOGENESIS TARGETING PROTEINS CO-EXPRESSED IN THE NERVOUS SYSTEM MAY BE THE EARLIEST RESPONDERS CAUSING INITIAL DAMAGE TO PERIPHERAL NERVES. THESE FIRST RESPONDERS PROVIDE THE NECESSARY TRIGGER TO EXPAND IMMUNE RESPONSES AGAINST MYELIN AND OTHER NERVOUS SYSTEM COMPONENTS TRADITIONALLY STUDIED IN EXISTING MOUSE MODELS OF AUTOIMMUNE NEURITIS. A VAST MAJORITY OF ISLET-INFILTRATING B-LYMPHOCYTES IN NOD MICE RESPOND TO THE NERVOUS-SYSTEM PROTEIN PERIPHERIN. ANTIBODIES AGAINST PHOSPHORYLATED PERIPHERIN HAVE BEEN IDENTIFIED IN T1D PATIENTS. WE RECENTLY CREATED A NEW MOUSE MODEL (NOD-PERIG) IN WHICH B-LYMPHOCYTES TRANSGENICALLY EXPRESS THE IMMUNOGLOBULIN MOLECULE FROM THE PERIPHERIN-REACTIVE B-CELL CLONE H280 ISOLATED FROM ISLETS. T1D IS ACCELERATED IN NOD-PERIG MICE. T-CELLS FROM NOD-PERIG, BUT NOT NOD MICE, TRANSFER AN AUTOIMMUNE NEURITIS SIMILAR TO CHRONIC DEMYELINATING POLYNEUROPATHY (CIDP) TO NOD.SCID RECIPIENTS. THIS NEW NOD-PERIG A NOD.SCID MODEL OF T1D-ASSOCIATED AUTOIMMUNE NEURITIS PROVIDES AN EXPERIMENTAL SYSTEM TO DIRECTLY DISSECT THE DISCREET STAGES OF NERVE CELL INFILTRATION AND DAMAGE. WE ORIGINALLY HYPOTHESIZED THAT INSULITIS EXPANDED T- CELLS CAPABLE OF CAUSING NEURITIS. HOWEVER, WE HAVE RECENTLY DETERMINED THAT T-CELLS DERIVED FROM ISLETS OR SCIATIC NERVES IN PRIMARY NOD.SCID RECIPIENTS ARE ONLY CAPABLE OF INFILTRATING THE ORGAN FROM WHICH THEY WERE DERIVED. THEREFORE, EXPERIMENTS IN AIM 1 WILL ADDRESS THE CURRENT UNKNOWNS REGARDING THE DISCRETE TEMPORAL AND SPATIAL STEPS LEADING TO CELLULAR RECRUITMENT INTO PERIPHERAL NERVES. WE HAVE ALSO FOUND THAT IN ADDITION TO THE PRESENCE OF IFNG AND TNFA PRODUCING CD4+ T-CELLS, THERE IS PARADOXICALLY AN EXPANSION OF T-CELLS NEGATIVE FOR TRADITIONAL TH1, TH2, AND TH17 CYTOKINES WITHIN SCIATIC NERVES. AN ADDITIONAL OPEN QUESTION IS WHETHER PERIPHERIN REMAINS THE ANTIGEN TOWARDS WHICH T-CELLS ARE RESPONDING OR WHETHER THERE HAS BEEN AN EXPANSION OF RESPONSES AGAINST OTHER NEURONAL ANTIGENS. THEREFORE, STUDIES IN AIM 2 WILL DISSECT THE MECHANISMS BY WHICH T-CELLS DESTROY PERIPHERAL NERVES AND HOW AN IMMUNE RESPONSE AGAINST ONE SHARED B- CELL/NEURONAL ANTIGEN CAN SPILL OVER TO A WIDER NEURONAL RESPONSE. FINALLY, IN LARGE NOD COLONIES, SPONTANEOUS CLINICAL NEURITIS CAN BE OCCASIONALLY OBSERVED. WE HAVE FOUND THAT AT BASELINE, NOD MICE (AROUND THE AGE OF T1D ONSET) ALREADY HAVE SCIATIC NERVE INFILTRATING T-CELLS. THESE T-CELLS ARE NOT OBSERVED IN NON-AUTOIMMUNE PRONE C57BL/6 MICE. THIS INDICATES THE LIKELIHOOD THAT NOD HARBORS GENETIC LOCI CONTRIBUTING TO SPONTANEOUS NERVE INFILTRATION THAT CAN LEAD TO SPONTANEOUS NEURITIS. WHETHER THESE LOCI ARE DISTINCT OR OVERLAPPING WITH LOCI CONTRIBUTING TO T1D IS UNKNOWN. THEREFORE, STUDIES IN AIM 3 WILL MAP GENES ASSOCIATED WITH SPONTANEOUS NERVE INFILTRATION ALLOWING FUTURE GENETIC SCREENING OF PATIENTS AT RISK FOR DEVELOPING AUTOIMMUNE NEUROPATHY.

GENE EXPRESSION DATABASE FOR MOUSE DEVELOPMENT

SELECTIVE TRANSLATIONAL REGULATION OF MALE FERTILITY

IMPACT OF AGING AND CLONAL HEMATOPOIESIS ON EPIGENETIC HETEROGENEITY, EVOLVABILITY, AND LEUKEMOGENESIS - PROJECT SUMMARY ACUTE MYELOID LEUKEMIA (AML) OCCURS MOSTLY IN ADULTS 65 YEARS AND OLDER AND IS ASSOCIATED WITH AGE-RELATED CLONAL HEMATOPOIESIS (CH). THIS CONDITION THAT RESULTS FROM THE CLONAL EXPANSION OF MUTATIONALLY-MARKED HEMATOPOIETIC STEM AND PROGENITOR CELLS (HSPC). THESE HSPC EXPANSIONS OFTEN FEATURE SOMATIC MUTATIONS IN LEUKEMIA-ASSOCIATED GENES SUCH AS THE EPIGENETIC REGULATOR TET2. TET2-MUTANT CH IS A RISK FACTOR FOR AML, AND CH PROGRESSION TO AML IS PROMOTED BY ACQUIRING COOPERATING MUTATIONS SUCH AS CONSTITUTIVELY ACTIVE FLT3ITD. YET, IT IS UNCLEAR HOW AGING AND TET2 MUTATION INTERACT TO DRIVE THE EVOLUTION OF CH TO LEUKEMIA. WE SEEK TO REVEAL THE EPIGENETIC MECHANISMS FOR HOW CH EVOLVES TO LEUKEMIA IN AN AGING MICROENVIRONMENT, LAYING THE FOUNDATION FOR THERAPEUTIC STRATEGIES TO BLOCK THIS EVOLUTION AND PREVENT LEUKEMIA IN THE AGING US POPULATION. TO THIS END, WE WILL LEVERAGE OUR TEAM'S COMPLEMENTARY EXPERTISE IN INTRA-TUMOR HETEROGENEITY AND COMPUTATIONAL EPIGENOMICS; AGING, CANCER EVOLUTION, AND LEUKEMOGENESIS; INFLAMMATION AND HEMATOPOIETIC STEM CELL (HSC) BIOLOGY; AND MOUSE HSC PROLIFERATION AND FUNCTIONAL HETEROGENEITY. OUR PRELIMINARY RESULTS IN MICE SHOW THAT HSPC EPIGENETIC HETEROGENEITY INCREASES IN OLD AGE. THIS PROCESS OCCURS IN AN AGED BONE MARROW (BM) MICROENVIRONMENT CHARACTERIZED BY INFLAMMATION AND ALTERED HSPC SUPPORT. THE RISE OF EPIGENETIC AND TRANSCRIPTOMIC HETEROGENEITY IN HSC WITH TET2 MUTATION (TET2MT) AND FLT3ITD PRECEDES LEUKEMIC TRANSFORMATION. WE HYPOTHESIZE THAT AGING AND TET2 MUTATION COOPERATIVELY ENHANCE EPIGENETIC HETEROGENEITY AND EVOLVABILITY OF HSPC, THUS CONTRIBUTING TO CLONAL EXPANSION AND LEUKEMOGENESIS. IN AIM 1, WE WILL DETERMINE THE COMBINED IMPACT OF AGING AND TET2MT ON EPIGENETIC HETEROGENEITY AND GENE REGULATION IN HSPC BY EXAMINING THE SOMATIC EPIGENOMIC LANDSCAPE OF TET2MT HSPC FROM YOUNG (2-3 MONTHS) AND OLD (22 MONTHS) MICE, INCLUDING SINGLE-CELL (SC) TRANSCRIPTOMES BY SCRNA-SEQ, OPEN-CHROMATIN PROFILES BY SNATAC-SEQ, AND DNA METHYLOMES BY RRBS. WE EXPECT TO DEFINE EPIGENETIC CONFIGURATIONS IN OLD TET2MT HSPC ASSOCIATED WITH GENES IN SELF-RENEWAL, QUIESCENCE, AND RESPONSES TO INFLAMMATION AND STRESS. IN AIM 2, WE WILL DEFINE EPIGENETIC CONFIGURATIONS OF TET2MT HSPC THAT ARE ADAPTIVE IN THE AGED CONTEXT. WE WILL TRANSPLANT GENETICALLY BARCODED HSPC INTO YOUNG OR OLD MICE AND ASSESS SUBCLONE EXPANSION/CONTRACTION TO DETERMINE IF AN AGE-DEPENDENT SELECTION IS FOR ALL TET2MT HSPC OR ONLY A SUBSET. WE WILL USE SCRNA-SEQ AND SNATAC-SEQ TO DEFINE MOLECULAR SIGNATURES AND EPIGENETIC HETEROGENEITY OF BARCODED HSPC THAT ARE POSITIVELY SELECTED IN THE AGED CONTEXT. IN AIM 3, WE WILL DEFINE THE EPIGENETIC CONFIGURATIONS OF TET2MT HSPC THAT ARE PERMISSIVE TO FLT3ITD-INDUCED TRANSFORMATION IN THE AGED CONTEXT. WE WILL TRANSDUCE THE FLT3ITD GENE INTO YOUNG OR AGED TET2MT OR WILD-TYPE HSC AND THEN TRANSPLANT THESE GENETICALLY BARCODED CELLS INTO YOUNG OR OLD HOST MICE. WE WILL ASSESS THE EPIGENETIC PROFILES OF THE SELECTED HSPC BY SCRNA-SEQ AND SCATAC-SEQ OF THEIR IDENTIFIED PROGENY. WE EXPECT OUR FINDINGS WILL SHIFT THE PARADIGM FOR HOW EPIGENETIC PLASTICITY PROMOTES SOMATIC EVOLUTION AND LEADS TO CANCER INITIATION IN AGING INDIVIDUALS.

IDENTIFICATION OF KIDNEY DISEASE MODIFIER GENES IN MOUSE AND HUMAN ALPORT SYNDROME - PROJECT SUMMARY/ABSTRACT ALPORT SYNDROME IS A HUMAN HEREDITARY GLOMERULONEPHRITIS, WHICH IN MOST CASES, RESULTS IN END-STAGE RENAL DISEASE. IT IS THE MOST COMMON INHERITED GLOMERULAR DISEASE LEADING TO RENAL FAILURE AND IS CAUSED BY MUTATIONS IN ANY ONE OF THE GENES ENCODING A3, A4, OR A5 CHAINS OF TYPE IV COLLAGEN (COL4A3, COL4A4, AND COL4A5, RESPECTIVELY). THERE IS LARGE VARIATION IN THE AGE OF ONSET AND SEVERITY OF THE DISEASE, EVEN BETWEEN PATIENTS WITH SIMILAR MUTATIONS. STUDIES IN MICE HAVE SHOWN THAT THE RENAL PHENOTYPE IS HIGHLY DEPENDENT ON THE GENETIC BACKGROUND. IT IS WIDELY ACCEPTED THAT MODIFIER GENES CONTRIBUTE TO THIS VARIATION, WHICH COULD REPRESENT A SOURCE OF NOVEL THERAPEUTIC TARGETS IN ALPORT SYNDROME AND OTHER RENAL DISEASES. WE IDENTIFIED HUMAN-RELEVANT MODIFIER GENES IN A SMALL COHORT OF GENETICALLY DIVERSE MICE WITH A COL4A5 MUTATION (LEADING TO X-LINKED ALPORT SYNDROME (XLAS)) AND VALIDATED THAT DECREASED EXPRESSION OF ONE OF THESE GENES, FMN1, LEADS TO A LESS SEVERE RENAL PHENOTYPE. WE FURTHER FOUND THAT TWO OF THE CANDIDATE MODIFIER GENES (PIK3R1 AND DGKE) MODULATE OTHER FORMS OF KIDNEY DISEASE, INCLUDING DIABETIC NEPHROPATHY AND HEMATOLYTIC UREA SYNDROME. IN THIS APPLICATION WE WILL DISCOVER NOVEL CANDIDATE MODIFIER GENES OF XLAS BY HIGH-RESOLUTION GENETIC MAPPING IN A LARGE GENETICALLY DIVERSE XLAS MOUSE COHORT AND CONFIRM THE TRANSLATIONAL RELEVANCE OF THE MODIFIERS IN HUMANS. THE FUNCTIONAL IMPACT AND CAUSALITY OF THE MODIFIER GENES WILL BE ASSESSED IN PRECLINICAL MOUSE MODELS OF XLAS AND OTHER FORMS OF KIDNEY DISEASE. WE WILL GENERATE A LARGE, GENETICALLY DIVERSE XLAS MOUSE POPULATION THAT, COMBINED WITH OUR PREVIOUS POPULATION, WILL ALLOW US GENE-RESOLUTION MAPPING OF MODIFIER LOCI (AIM 1). WHOLE EXOME SEQUENCING AND TARGETED TESTING FOR THE DETECTION OF THE MOST LIKELY CANDIDATE MODIFIER GENES IN HUMAN XLAS PEDIGREES WILL BE CONDUCTED TO CONFIRM THE TRANSLATIONAL RELEVANCE OF THE CANDIDATE MODIFIER GENES FOUND IN OUR MOUSE STUDIES (AIM 2). WE WILL USE AVAILABLE KNOCKOUT RESOURCES AND/OR CRISPR- CAS9 GENE EDITING TO TEST CAUSALITY OF AS MANY AS FIVE CANDIDATE GENES IN THE XLAS MOUSE MODEL (AIM 3A). WE WILL FURTHER TEST THESE MODIFIER GENES FOR CAUSALITY IN MOUSE MODELS OF TWO COMMON FORMS OF KIDNEY DISEASE: DIABETIC NEPHROPATHY AND FOCAL SEGMENTAL GLOMERULOSCLEROSIS SYNDROME (AIM 3B). IDENTIFICATION OF THE GENES RESPONSIBLE FOR THE ONSET AND SEVERITY OF DISEASE WILL PROVIDE MEANINGFUL INSIGHTS INTO UNDERSTANDING THE MOLECULAR EVENTS UNDERLYING THE PATHOGENESIS OF KIDNEY DISEASE AND PROVIDE THE BASIS FOR DEVELOPING NOVEL THERAPEUTIC STRATEGIES.

FUNCTIONAL VALIDATION OF NOVEL VARIANTS ASSOCIATED WITH CONGENITAL DIAPHRAGMATIC HERNIA - PROJECT SUMMARY/ABSTRACT CONGENITAL DIAPHRAGMATIC HERNIA (CDH) IS A COMMON AND SEVERE STRUCTURAL BIRTH DEFECT ARISING IN 1 OUT OF 3000 LIVE BIRTHS, ACCOUNTING FOR 8% OF ALL CONGENITAL ANOMALIES AND 1-2% OF INFANT MORTALITY. THE HIGH MORTALITY RATES ARE DUE TO THE ABNORMAL LUNG HYPOPLASIA AND PULMONARY HYPERTENSION THAT ACCOMPANIES THE HERNIATION OF THE DIAPHRAGM MUSCLE. THE GENETIC ETIOLOGY OF CDH IS HIGHLY HETEROGENEOUS AND OUR UNDERSTANDING OF THE ANOMALY IS INCOMPLETE. ALTHOUGH WHOLE EXOME AND WHOLE GENOME SEQUENCING HAS IDENTIFIED MORE THAN 100 CANDIDATE GENES, ONLY ~25 HAVE BEEN VALIDATED WITH REPRODUCIBILITY, AND WE ESTIMATE THAT MANY ADDITIONAL CDH GENES AND ALLELES ARE YET TO BE DISCOVERED. ANIMAL MODEL VALIDATION OF THESE DISCOVERIES ARE OFTEN LACKING, WHICH IS DUE, IN PART, TO THE FACT THAT THE DIAPHRAGM IS A MAMMALIAN-SPECIFIC STRUCTURE. THE GENERATION AND CHARACTERIZATION OF GERMLINE MOUSE MODELS IS COSTLY AND TIME-CONSUMING, AND THEREFORE TYPICALLY INCOMPATIBLE WITH THE NEED TO SCREEN MANY CANDIDATE GENES AND VARIANTS. THE HIGH EFFICIENCY OF CRISPR/CAS9 GENOME EDITING, AND THE APPEARANCE OF PHENOTYPES IN FOUNDER ANIMALS HAS ALLOWED US TO DEVELOP A PLATFORM THAT CAN REDUCE THE TIME FOR VALIDATION FROM MORE THAN ONE YEAR, TO 2-3 MONTHS. THE OVERARCHING GOAL OF THIS PROPOSAL IS TO IDENTIFY NOVEL GENES AND VARIANTS THAT ARE ASSOCIATED WITH HUMAN CDH AND TO VALIDATE THESE DISCOVERIES IN THE MOUSE. WE WILL USE GENOME DATA FROM OUR LARGE, WELL-CHARACTERIZED AND LONGITUDINALLY STUDIED CDH COHORT TO IDENTIFY NOVEL GENES AND VARIANTS IN PATIENTS WITH CDH, AND PRIORITIZE THESE GENES AND VARIANTS FOR VALIDATION IN MOUSE MODELS. WE WILL SCREEN PRIORITIZED VARIANTS USING OUR HIGH-THROUGHPUT MOUSE F0 PLATFORM AND SELECT A SUBSET OF HITS FOR GERMLINE MODELING AND MORE EXTENSIVE CHARACTERIZATION. TOGETHER, THESE STUDIES WILL ADVANCE OUR UNDERSTANDING OF THE GENETICS AND MECHANISMS UNDERLYING CDH, IMPROVE DIAGNOSTIC TOOLS FOR PATIENTS, AND PAVE THE WAY FOR THE FUTURE DEVELOPMENT OF THERAPEUTICS AIMED AT IMPROVING LUNG MATURATION AND FUNCTION.

DISCOVERY OF HOST GENETIC ELEMENTS AFFECTING RESPONSE TO IMMUNE CHECKPOINT INHIBITORS - PROJECT SUMMARY ABROGATING IMMUNE CHECKPOINT CONTROL HAS PROVEN TO BE A POWERFUL THERAPEUTIC STRATEGY AGAINST CANCER WITH DOCUMENTED LONG-TERM REMISSIONS IN REFRACTORY METASTATIC DISEASE. HOWEVER, ONLY A SUBSET OF PATIENTS (10-40%) HAVE MEANINGFUL RESPONSES. WHILE INTRINSIC TUMOR FACTORS PLAY A ROLE IN PREDICTING IMMUNE CHECKPOINT INHIBITOR (ICI) OUTCOMES, GERMLINE VARIATIONS IN HOST IMMUNE GENES ARE ALSO LIKELY TO HAVE A SIGNIFICANT IMPACT GIVEN THEIR IMPORTANCE IN DEFINING IMMUNE RESPONSE AND AUTOIMMUNITY. THAT BOTH THE TUMOR AND THE HOST GENOMES VARY MAKES THE IDENTIFICATION OF GERMLINE FACTORS MODIFYING ICI RESPONSE HIGHLY CHALLENGING. WE HAVE SOLVED THIS PROBLEM BY DEVISING A MOUSE EXPERIMENTAL PLATFORM THAT “FIXES” THE GENOMIC CONFIGURATION OF THE TUMOR AS A TRANSPLANTABLE CELL LINE (MC38) WHILE VARYING GERMLINE HOST GENETICS, THUS PERMITTING THE UNBIASED MAPPING OF QUANTITATIVE TRAIT LOCI (QTL) FOR ANTI-TUMOR RESPONSE AFTER ANTI-PD1 (APD1) THERAPEUTICS. BY CROSSING COLLABORATIVE CROSS (CC) MULTIPARENT RECOMBINANT INBRED LINES WITH THE C57BL/6 STRAIN, THE RESULTANT F1S HAVE GENETIC VARIABILITY BUT WILL ACCEPT THE MC38 TRANSPLANT. IN PRELIMINARY DATA, WE SHOW THAT HOST GENETICS ACCOUNTS FOR 42% OF THE VARIATION IN APD1 RESPONSE (HERITABILITY, H2) AND HAVE MAPPED APD1 RESPONSE TO FOUR QTLS ON MOUSE (M)CHR 5, 9, 15, AND 17 ENRICHED FOR IMMUNE GENES. BY SELECTIVE INTERCROSSES BETWEEN RESPONDER AND NON- RESPONDER CC LINES, WE FOUND SIGNIFICANT EPISTATIC INTERACTIONS BETWEEN CHROMOSOMES 5 AND 17 AND BETWEEN 15 AND 17. WE DEVISED A CROSS-SPECIES TUMOR MICROENVIRONMENT (CST) ALGORITHM THAT REDUCED THE ~1,500 GENES IN THE QTLS TO 48 TOP CANDIDATE GENES THAT COULD PREDICT ICI RESPONSE IN HUMAN TRIALS, OUTPERFORMING CURRENT THERANOSTIC BIOMARKERS. BASED ON THESE DATA, WE PROPOSE TO EXPAND AND REFINE THE QTL MAP FOR ICI RESPONSE IN THE MC38 MODEL WITH A FOCUS ON DEFINING THE GENETIC MECHANISMS FOR THE EPISTATIC INTERACTIONS (AIM 1). IN AIM 2, WE WILL CHALLENGE THE THREE TOP CANDIDATE GENES, CSF2RB AND IL2RB (MCHR15) AND IL10RA (MCHR9), WITH AVAILABLE RECOMBINANT CYTOKINES AND BLOCKING ANTIBODIES TOWARDS PREDICTED CHANGES IN APD1 RESPONSE. THESE PERTURBATIONS WILL ALLOW US TO FURTHER MAP THE GENETIC NETWORK THAT ESTABLISHES THE OPTIMAL TUMOR IMMUNE MICROENVIRONMENT. WE WILL THEN EXPAND OUR STUDIES BY MAPPING THE QTLS ASSOCIATED IN THE MURINE MAMMARY CANCER MODEL AT3 (AIM 3). WE ANTICIPATE THAT NEW QTLS FOR RESPONSE WILL BE UNCOVERED. FINALLY, IN AIM 4, WE WILL USE THE CCF1 TUMORS TO OPTIMIZE AN INNOVATIVE IN VITRO MICRO-ORGANOSPHERE (MOS) PLATFORM THAT ENCAPSULATES CELLS OF THE TUMOR MICROENVIRONMENT IN A PERMEABLE MICROCAPSULE AMENABLE TO PHARMACOLOGICAL INTERVENTION AND CELLULAR INTERROGATION. ONCE OPTIMIZED, WE WILL TEST A PANEL OF ICI DRUGS ALONE AND IN COMBINATION ON BOTH MURINE AND HUMAN TUMORS. THE SUCCESS OF THE PROPOSED WORK WILL NOT ONLY IDENTIFY POTENTIAL NEW BIOMARKERS AND NEW TARGETS FOR AUGMENTING CLINICAL ICI RESPONSE, BUT ALSO WILL ESTABLISH A VALIDATED PLATFORM FOR THE DISCOVERY OF HOST GENES INVOLVED IN THE RESPONSE TO ANY IMMUNE CHECKPOINT INHIBITOR.

TRAINING PROGRAM IN PRECISION GENETICS OF AGING, ALZHEIMER'S DISEASE AND RELATED DEMENTIAS

INTEGRATING HIGH-THROUGHPUT HISTOLOGY WITH SYSTEMS GENETICS THROUGH CAUSAL GRAPHICAL MODELS - PROJECT SUMMARY THE OVERALL OBJECTIVE OF THIS PROPOSAL IS TO DEVELOP AND VALIDATE A DEEP LEARNING ANALYTICAL FRAMEWORK TO INTEGRATE HISTOLOGICAL TRAITS INTO SYSTEMS GENETICS ANALYSIS OF COMPLEX DISEASES. MAPPING THE RISK GENES FOR POOR HEALTH OUTCOMES IS A MAJOR FOCUS OF BIOMEDICAL RESEARCH, AND NEW APPROACHES TO IMPROVE GENETIC MAPPING POWER CAN HAVE A TRANSFORMATIVE IMPACT ON PUBLIC HEALTH. GENETIC DISEASE RISK MANIFESTS THROUGH COMPLEX INTERACTIONS BETWEEN GENE REGULATION AND TISSUE STRUCTURE, ULTIMATELY INFLUENCING ORGAN FUNCTION. HOWEVER, QUANTIFYING TISSUE STRUCTURE FOR QUANTITATIVE GENETIC MAPPING HAS NOT BEEN WIDELY ADOPTED. THIS IS PARTLY BECAUSE HISTOLOGICAL SCORING HAS TRADITIONALLY BEEN LABOR INTENSIVE AND ERROR PRONE, AND LIMITED TO COARSE MEASURES (E.G., DISCRETE CATEGORIES) THAT ARE SUBOPTIMAL FOR ASSOCIATION TESTING. IN CONTRAST, DEEP NEURAL NETWORKS (DNNS) NOW ROUTINELY AUTOMATE LABORIOUS IMAGE QUANTIFICATION TASKS FOR HISTOPATHOLOGY, MAKING THEM AN IDEAL PLATFORM FOR INTEGRATING HISTOLOGY INTO GENETIC ANALYSIS. FURTHERMORE, UNLIKE HUMAN-DEFINED HISTOLOGICAL SCORES, DNN READOUTS ENABLE OBJECTIVE HISTOLOGICAL TRAIT DISCOVERY AS A FUNCTION OF GENETIC, MOLECULAR, AND PHYSIOLOGICAL VARIATION. IN THIS PROJECT, HISTOLOGICAL FEATURES WILL BE RIGOROUSLY AND ROBUSTLY QUANTIFIED USING DNNS AND THESE DATA WILL BE INTEGRATED INTO NOVEL MULTISCALE STATISTICAL MODELS THAT WILL CONNECT GENETIC, MOLECULAR, AND HISTOLOGICAL VARIATION TO PHYSIOLOGICAL OUTCOMES. IN PARTICULAR, NOVEL METHODS WILL BE DEVELOPED TO INTEGRATE HISTOLOGY INTO THREE MAJOR CLASSES OF SYSTEMS GENETIC ANALYSIS, I.E., HERITABLE TRAIT INFERENCE, CAUSAL MEDIATION ANALYSIS, AND MOLECULAR QUANTITATIVE TRAIT LOCUS (MQTL) MAPPING. THESE METHODS WILL BE DEVELOPED AND VALIDATED USING A DATA SET OF GENETIC, HISTOLOGICAL, TRANSCRIPTOMIC, PROTEOMIC, AND PHYSIOLOGICAL DATA FROM A COHORT OF DIVERSITY OUTBRED MICE USED FOR THE STUDY OF AGE-RELATED KIDNEY DISEASE. BY USING A MODEL SYSTEM, COMPLEX GENETIC EFFECTS AND CAUSAL MEDIATION HYPOTHESES CAN BE DIRECTLY TESTED TO VALIDATE AND REFINE THE ANALYTICAL FRAMEWORK. THE SPECIFIC AIMS OF THIS PROPOSAL INCLUDE: AIM 1: IDENTIFY MAXIMALLY HERITABLE HISTOLOGICAL TRAITS THROUGH DEEP LEARNING ON PAIRED GENOTYPES AND HISTOLOGICAL IMAGES. AIM 2: GENETICALLY MAP HISTOLOGICAL MEDIATORS OF COMPLEX PHYSIOLOGICAL TRAITS USING DEEP LEARNING ON HISTOLOGICAL IMAGES. AIM 3: IDENTIFY CAUSAL PATHS CONNECTING MOLECULAR QTLS (MQTLS) TO OUTCOMES THROUGH HISTOLOGICAL MEDIATORS. THE OUTCOME OF THIS STUDY WILL BE A VALIDATED METHODOLOGICAL FRAMEWORK FOR HISTOLOGICAL SYSTEMS GENETICS THAT IS MODULAR, ENABLING A WIDE RANGE OF USERS TO INCORPORATE APPROPRIATE COMPUTER VISION TOOLS INTO STATE-OF-THE-ART SYSTEMS GENETICS WORKFLOWS FOR ANY COMPLEX DISEASE.

DNA BINDING PROPERTIES OF ZINC FINGER PROTEINS

TRANSLATIONAL RESEARCH FOR MUSCULAR DYSTROPHY

HOMOLOGOUS RECOMBINATION IN GENOME STABILITY AND TUMOR SUPPRESSION

"DEVELOPMENTAL GENETICS"

MOLECULAR MECHANISMS OF HUMAN TNBC METASTASIS IN VIVO

ADVANCING ULTRA LONG-READ SEQUENCING AND CHROMATIN INTERACTION ANALYSES FOR CHROMOSOMAL AND EXTRACHROMOSOMAL STRUCTURAL VARIATION CHARACTERIZATION IN CANCER

HOW INSULIN/IGF-1 DEFICIENCIES RETARD MAMMALIAN AGING

(PQ #4) SINGLE CELL BARCODING FOR STUDIES OF STUDY CLONAL EVOLUTION IN GLIOBLASTO

HUMANIZED MOUSE MODELS TO DISSECT IN VIVO THE INTERPLAY BETWEEN MELANOMA AND THE IMMUNE SYSTEM

GENETIC VARIATION OF ULTRA-POTENT SYNTHETIC OPIOID SENSITIVITY IN MICE - PROJECT SUMMARY/ABSTRACT THE WEAPONIZATION OF ULTRAPOTENT SYNTHETIC OPIOIDS (UPS) HAS MADE FINDING A NOVEL REVERSAL AGENT A PRIORITY. THE CURRENT OPIOID RESPONSE AGENT, NALOXONE, IS NOT AS EFFECTIVE AGAINST UPS OPIOIDS AND DOES NOT REVERSE ONE OF ITS KNOWN EFFECTS, WOODEN CHEST SYNDROME. OUR LONG-TERM GOAL IS TO DEFINE THE BIOLOGICAL BASIS OF OPIOID OVERDOSE RISK AND PROMOTE THE DISCOVERY OF SAFE AND EFFECTIVE AGENTS THAT REVERSE FENTANYL LETHALITY. A KEY OBJECTIVE IS DETERMINING THE MOLECULAR MECHANISMS UNDERLYING INDIVIDUAL VARIABILITY TO FENTANYL TOXICITY USING GENETICALLY DIVERSE MICE. IN AIM 1 WE PLAN TO IDENTIFY GENES AND VARIANTS THAT MODIFY THE INFLUENCE OF MCOLN1 ON ACUTE UPS OPIOID TOXICITY. MCOLN1 WAS IDENTIFIED IN A GWAS STUDY OF OVERDOSE RISK, AND PRELIMINARY DATA SUPPORT A GENETIC KNOCKOUT OF MCOLN1 RESULTING IN DEATH MORE RAPIDLY FROM MORPHINE OR FENTANYL. WE WILL CREATE AN ADDITIONAL CRISPR KNOCKOUT OF MCOLN1 ON A MORE SENSITIVE GENETIC BACKGROUND, NOD/SHILTJ AND COMPARE IT WITH THE C57BL/6 KNOCKOUT WE HAVE FOR RESPONSE TO THE UPS OPIOID FENTANYL. THE LD50 WILL BE DETERMINED FOR THESE STRAINS USING OUR PIEZOELECTRIC RESPIRATORY DEPRESSION DETECTION SYSTEM. WE WILL ALSO STUDY RESPIRATORY MECHANICS AND PULMONARY AND CHEST WALL IMPEDANCE IN RESPONSE TO THREE DOSES OF FENTANYL. IN ANOTHER COHORT OF MICE, THEY WILL BE TESTED BY PLETHYSMOGRAPHY TO ACQUIRE RESPIRATORY METRICS SUCH AS TIDAL VOLUME AND MINUTE VENTILATION. WE WILL ALSO COLLECT ARTERIAL BLOOD TO MEASURE BLOOD GASES OF OXYGEN AND CARBON DIOXIDE DURING THE PLETHYSMOGRAPHY SESSION TO MONITOR THE RESPONSE TO FENTANYL AT THAT LEVEL. FINALLY, ANOTHER COHORT OF NAÏVE AND FENTANYL-TREATED MICE WILL BE DISSECTED FOR BRAIN STEMS. THE PRE-BÖTZINGER COMPLEX WILL BE IDENTIFIED AND ANALYZED BY SINGLE-NUCLEUS RNA-SEQ, COMPARING THE CELLULAR POPULATIONS AND DIFFERENTIAL GENE EXPRESSION ACROSS GENOTYPES, SEXES AND TREATMENTS. IN AIM 2 WE PLAN TO IDENTIFY THE PHYSIOLOGICAL, NEURAL, AND MOLECULAR MECHANISMS OF VARIABLE FENTANYL-INDUCED TOXICITY AND LETHALITY AMONG EIGHT INBRED MOUSE STRAINS. THESE EIGHT STRAINS, WHICH SERVED AS THE FOUNDATION FOR THE ADVANCED MOUSE POPULATIONS OF THE COLLABORATIVE CROSS AND DIVERSITY OUTBRED MICE, CONTAIN APPROXIMATELY 45 MILLION SNPS SEGREGATING BETWEEN THEM. WE HAVE DETERMINED THAT THE LD50 FOR FENTANYL VARIES > 150-FOLD ACROSS BOTH SEXES OF THE EIGHT STRAINS. AS IN AIM 1, IN AIM 2 WE WILL PHENOTYPE COHORTS OF MICE TO DETECT THE DIVERSE PHENOMENA ASSOCIATED WITH UPS OPIOIDS, INCLUDING OPIOID- INDUCED RESPIRATORY DEPRESSION (OIRD), OPIOID-INDUCED PERSISTENT APNEA (OIPA), WOODEN CHEST SYNDROME (WCS), CLOSURE/COLLAPSE OF THE UPPER AND CARDIOVASCULAR/HEMODYNAMIC DISTURBANCES. THIS PHENOTYPING WILL BE COUPLED TO IDENTIFYING THE CELLULAR POPULATIONS, THROUGH SINGLE NUCLEUS RNA-SEQ, WITHIN THE BRAINSTEM PRE- BÖTZINGER REGION THAT VARIES ACROSS NAÏVE AND FENTANYL-TREATED STRAINS OF BOTH SEXES OF MICE. THE DIFFERENTIALLY EXPRESSED GENES THAT DEFINE THESE POPULATIONS WILL HELP US IDENTIFY TARGETS FOR THERAPEUTIC DEVELOPMENT ASSOCIATED WITH THE DIFFERENT FENTANYL LETHALITY PHENOTYPES.

MACHINE LEARNING BASED FRAILTY INDEX FOR THE GENETICALLY DIVERSE MICE - PROJECT SUMMARY AGING IS A TERMINAL PROCESS THAT AFFECTS ALL BIOLOGICAL SYSTEMS. BIOLOGICAL AGING—IN CONTRAST TO CHRONOLOGICAL AGING—OCCURS AT DIFFERENT RATES FOR DIFFERENT INDIVIDUALS. IN HUMANS, GROWING OLD COMES WITH INCREASED HEALTH ISSUES AND MORTALITY RATES, YET SOME INDIVIDUALS LIVE LONG AND HEALTHY LIVES, AND OTHERS SUCCUMB EARLIER TO DISEASES AND DISORDERS. THE CONCEPT OF FRAILTY IS USED TO QUANTIFY THIS HETEROGENEITY AND IS DEFINED AS THE STATE OF INCREASED VULNERABILITY TO ADVERSE HEALTH OUTCOMES. THE FRAILTY INDEX (FI) IS AN INVALUABLE AND WIDELY USED TOOL WHICH OUTPERFORMS OTHER METHODS TO QUANTIFY FRAILTY. FIS HAVE BEEN ADAPTED FOR USE IN MICE USING A VARIETY OF BOTH BEHAVIORAL AND PHYSIOLOGICAL MEASURES AS INDEX ITEMS. HOWEVER, BECAUSE CONDUCTING MOUSE FI REQUIRES TRAINED INDIVIDUALS FOR MANUAL SCORING, IT OFTEN LIMITS THE SCALABILITY OF THE TOOL. THUS, ALTHOUGH THE FI IS AN EXTREMELY USEFUL TOOL FOR AGING RESEARCH, AN INCREASE IN ITS SCALABILITY, RELIABILITY, AND REPRODUCIBILITY THROUGH AUTOMATION WOULD ENHANCE ITS UTILITY. WE USED MACHINE LEARNING APPLIED TO VIDEO DATA TO CREATE AN AUTOMATED VISUAL FI (VFI). THE IS EASY TO IMPLEMENT, UNBIASED, AND SCALABLE. HERE WE PROPOSE TO IMPROVE OUR TOOL AND CARRY OUT AN INTERVENTIONAL STUDY. WE WILL ADOPT THE VFI TO FUNCTION WITH GENETICALLY DIVERSE MICE (R61: AIM 1). WE WILL ALSO CREATE FEATURES FROM LONG-TERM MONITORING TO INCREASE ACCURACY AND BREADTH OF SYSTEMS MEASURED IN THE VFI (R61: AIM 2). FINALLY, WE WILL APPLY THE VFI TO A DIET INTERVENTION STUDY TO SHOW ITS UTILITY FOR LARGE SCALE STUDIES (R33: AIM 3). WE WILL TEST A HIGH FAT HIGH SUGAR DIET (INCREASED FRAILTY) AND CALORIC RESTRICTION GROUP (DECREASED FRAILTY) WITH NORMAL CHOW (CONTROL) IN A DIVERSITY OUTBRED POPULATION OF MICE. THE RESULT OF THIS PROJECT WILL BE A FULLY VALIDATED AND AUTOMATED VFI THAT CAN BE USED FOR HIGH-THROUGHPUT INTERVENTIONAL STUDIES, ENABLING THERAPEUTICS FOR HEALTHY AGING.

CAREER: EVOLUTION AND GENETIC CONTROL OF VARIATION IN MEIOTIC RECOMBINATION

IMPACT OF GENETIC DIVERSITY ON HUMAN XENOGRAFT TUMOR GROWTH

THE GENETICS AND CELL BIOLOGY OF THE EPILEPTIC MOUSE MUTANT FITFUL

OOCYTE CULTURE CONDITIONS THAT PROMOTE EMBRYOGENESIS

DISSECTION OF ADDICTION RELEVANT SIGNAL INTEGRATION BY CYFIP2 THROUGH PRECISE GENOME ENGINEERING - PROJECT SUMMARY ADDICTION IS AN ENORMOUS ECONOMIC, PERSONAL, AND SOCIAL BURDEN, COSTING OVER $600 BILLION PER YEAR IN THE U.S. UNDERSTANDING VULNERABILITY TO ADDICTION, AND DEVELOPING EFFECTIVE THERAPIES, REQUIRES IDENTIFYING THE GENES AND PATHWAYS THAT MEDIATE THE ADDICTION PROCESS. OUR LONG-TERM GOAL IS TO DEVELOP NOVEL GENETIC MODELS FOR ADDICTION-RELEVANT PHENOTYPES, AND USE THESE MODELS TO CHARACTERIZE THE GENETIC MECHANISMS OF ADDICTION. HERE, WE PROPOSE TO EXTEND OUR PREVIOUS WORK THAT LED TO THE CLONING OF A QTL THAT REGULATES ADDICTION AND THE SUBSEQUENT IDENTIFICATION OF CYFIP2 IN MOUSE SUBSTRAINS AS A REGULATOR OF COCAINE ACUTE AND SENSITIZED RESPONSES. WE AND OTHERS HAVE SINCE SHOWN THAT THIS MUTATION REGULATES FOOD REWARD, NICOTINE PREFERENCE, AND ALCOHOL PREFERENCE (PRELIMINARY DATA). IN ADDITION, WE HAVE SHOWN THAT CYFIP2 REGULATES VOLUNTARY SELF- ADMINISTRATION OF COCAINE IN THE IVSA ASSAY, THE GOLD STANDARD IN THE ADDICTION FIELD. CYFIP2 IS A HUB FOR SIGNAL INTEGRATION FROM MULTIPLE PATHWAYS, INCLUDING THE SMALL GTPASE RAC1, WIRS DOMAIN RECEPTORS, AND FRAGILE X FAMILY SIGNALING. WE HYPOTHESIZE THAT THIS SIGNAL INTEGRATION BY CYFIP2 IS CRITICAL FOR REWARD BEHAVIORS. IN RESPONSE TO PAR-19-278, WE NOW PROPOSE TO USE PRECISE GENOME ENGINEERING IN MICE TO GENERATE AND FUNCTIONALLY VALIDATE 5 VARIANTS IN CYFIP2 (1-2 AMINO ACID SUBSTITUTIONS EACH) THAT SPECIFICALLY PERTURB EACH OF THESE SIGNAL INTEGRATION EVENTS. THESE MUTATIONS ARE DESIGNED USING PUBLISHED BIOCHEMICAL DATA AND IN CONSULTATION WITH OUR CO- INVESTIGATOR DR. CHEN, WHO IS A LEADER IN CYFIP BIOPHYSICS AND STRUCTURE. IN THE R21 PHASE (AIM 1), WE WILL LEVERAGE THE MOUSE GENETICS EXPERTISE OF THE JACKSON LABORATORY TO GENERATE BY CRISPR/CAS9 A SET OF 5 CYFIP2 SIGNALING MUTANTS. SPECIFIC MILESTONES FOR PROGRESSION TO THE R33 PHASE ARE (I) VIABILITY OF THE MUTANTS, SINCE THE KNOCKOUT OF CYFIP2 IS POSTNATAL LETHAL, AND (II) THE LACK OF FUNCTIONAL OFF-TARGET EDITS. WE WILL THEN CHARACTERIZE THESE MUTANTS COMPREHENSIVELY FOR COCAINE AND NATURAL REWARD BEHAVIOR (R33, AIM 2). TO GAIN INSIGHT INTO MECHANISMS UNDERLYING THESE BEHAVIORS, WE WILL DETERMINE THE BIOCHEMICAL INTERACTOME OF EACH MUTANT IN MOUSE BRAIN REGIONS USING A COMPREHENSIVE MASS SPECTROMETRY-BASED STUDY (R33, AIM 3). THE SUCCESSFUL COMPLETION OF THIS PROJECT WILL YIELD 5 PRECLINICAL MOUSE MODELS OF ADDICTION TRANSITION FOR THE SCIENTIFIC COMMUNITY, AS WELL AS INFORMATION ABOUT SPECIFIC SIGNALING PATHWAYS THAT ARE CRITICAL FOR TRANSITION TO ADDICTION AND THAT CAN BE TARGETED FOR THERAPY.

A RESOURCE FOR MOUSE MODELS OF PERIPHERAL NEUROPATHY

GENETIC AND DYNAMIC ANALYSES OF PEAK BONE DENSITY IN MICE