>> WELCOME TO THE THIRD SESSION OF THE VIRTUAL DEMYSTIFYING MEDICINE COURSE. USING THE BROOKLYN BRIDGE AS OUR LOCAL O, THIS COURSE IS INTENDED TO BRIDGE EXCITING ADVANCES IN BIOLOGICAL AND ENGINEERING SCIENCES WITH HUMAN HEALTH. THE NEXT SLIDE GIVES SOME GENERAL INFORMATION FOR THOSE ATTENDING. ALL SESSIONS WILL BE VIRTUAL ON TUESDAYS FROM 4-530 JANUARY THROUGH MAY. YOU CAN ACCESS THE FULL PROGRAM ON OUR WEBSITE OR BY GOOGLING DEMYSTIFYING MEDICINE. THE WEBSITE INCLUDES SELECTED PAPERS, BIOSKETCHES AND WHEN AVAILABLE POWER POINTS. THOSE WHO ATTEND AT LEAST 50% OF THE SESSIONS AND PASS A FINAL EXAM CAN QUALIFY FOR CERTIFICATE. THE IDEA OF TRANSPLANTING AN ORGAN FROM 1 HUMAN BEING TO ANOTHER IS ANCIENT, BUT IT COULD NOT BE ACCOMPLISHED UNTIL RELATIVELY RECENT TIMES. THIS CARTOON BY THE DANISH CARTOONIST PIET HEIN SUMMARIZES THE SITUATION IN HIS FAMOUS CARTOON PROBLEMS WORTHY OF ATTACK PROVE THEIR WORTH BY FIGHTING BACK. THE NEXT SLIDE PROVIDES A BRIEF SUMMARY OF TRANSPLANTATION AS REFLECTED IN NOBEL PRIZES. IN 1912 ALEXIS CAROH LL, WHO WON THE NOBEL PRIZE FOR DEVELOPING SURGICAL TECHNIQUES FOR THE VASCULATURE USING TRANSPLANTED ORGANS AND LIMBS CAME TO A CONCLUSION THAT A BIOLOGIC--WHEN PETER METAWAR WON THE NOBEL PRIZE--ON IMMUNE O LOGIC TOLERANCE. HE ALSO CAME TO THE CONCLUSION THAT THERE WAS A FORCE WHICH HE STATES WOULD FOREVER PREVENT TRANSPLANTATION. HOWEVER, NOT EVERYONE ACCEPTED THIS INEVALUATION PROCESSITIABILITY, AND IN 1950, SPECIFIC TRANSPLANTATION ANTIGENS ON CELL SURFACES WERE DISCOVERED BY THE NOBELIST JOHN DOSSIER THEY'RE CALLED HLA FOR HUMAN LEUCOCYTE ANTIGENS, AND THIS DISCOVERY, FOLLOWING AMAZING ADVANCES IN IMMUNOLOGY, OPENED THE DOOR TO ORGAN AND CELL TRANSPLANTATION WHICH HAS NOW BEEN PERFORMED USING ALMOST EVERY SOLID ORGAN ACCEPT THE BRAIN. WHEN BONE MARROW TRANSPLANTATION WAS FIRST PROPOSED AND TESTED, IT IS UNCERTAIN. HOWEVER, A HALF A CENTURY AFTER EXPERIMENTS IN INBRED MICE REVEALED THE POSSIBILITY OF HEMATOPOIETIC CELL TRANSPLANTATION, THE 1990 NOBEL PRIZE WAS GIVEN TO JOSEPH MURRAY AND DONALD THOMAS FOR DISCOVERIES CONCERNING ORGAN THAT WAS MURRAY'S WORK, THE FIRST KIDNEY TRANSPLANT AND BONE MARROW STEM CELL TRANSPLANTATION WHICH ARE THE STUDIES OF THOMAS AND COLLEAGUES AT THE UNIVERSITY OF WASHINGTON, PRIMARILY USED IN THE TREATMENT OF HEMEAT O LOGIC DISEASES, LEUKEMIA IN PARTICULAR AND APLASTIC ANEMIA. THE FOLLOWING 50 YEARS SHOWED AMAZING BASIC SCIENCE DISCOVERIES IN BONE MARROW LINEAGE, HEMATOPOIETIC STEM CELLS, INDUCED PLURIPOTENT CELLS, IMMUNE TOLERANCE, AND PHARMACOLOGICAL CONTROL OF THE IMMUNE SYSTEM AND THESE HAVE PERMITTED TRUE BRIDGING INTO SUCCESSFUL TREATMENT OF AN INCREASING NUMBER OF NEOPLASTIC AND GENETIC [INDISCERNIBLE]. THIS IS 1 OF THE MOST DRAMATIC ADVANCES IN MODERN MEDICINE AND THE FUTURE PROMISES MORE DISCOVERIES IN THIS RAPIDLY CHANGING FIELD. IN TODAY'S DEMYSTIFYING MEDICINE SESSION, WE'RE FORTUNATE TO BE TAUGHT BY 2 NIH COLLEAGUES WHO ARE LEADERS IN THIS FIELD. CYNTHIA DUNBAR OF THE NATIONAL, HEART, LUNG AND BLOOD INSTITUTE AND HARRY MALECH, OF THE NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES. THE TITLE OF BOTH OF THEIR TALKS IS BONE MARROW-BASED THERAPIES, TODAY AND TOMORROW. CYNTHIA DUNBAR GRADUATED IN MEDICINE AT HARVARD AND TOOK MEDICAL TRAINING IN HEMEAT O LOGIC TRAINING, CAME TO THE HEART INSTITUTE AS A POST DOC IN 1987. AND SINCE 2000 THESE BEEN THE HEAD OF THE MOLECULAR HEMAT O POETICESIS SECTION, AND AMONG HER MANY, MANY AWARDS AND ACCOMPLISHMENTS, HER EDITORSHIP WITH THE JOURNAL OF BLOOD AND PRESIDENT OF THE AMERICAN SOCIETY FOR CELL AND GENE THERAPY. AS AN NIH DISTINGUISHED INVESTIGATOR HER RESEARCH FOCUSES ON UNDERSTANDING DIFFERENTIATION OF STEM CELLS INTO MULTIPLE TYPES OF BLOOD CELLS INVIVO. WORKING WITH NONHUMAN PRIMATES SHE HAS BECOME AN INTERNATIONAL LEADER IN IMPROVING THE SAFETY OF GENE TRANSFER AND TRANSPLANTATION OF PRIMARY HEMATOPOIETIC CELLS FOR THERAPEUTIC PURPOSES IN HUMAN TRIALS. OUR SECOND SPEAKER WILL BE DR. HARRY MALEC, WHO RECEIVED HIS MEDICAL DEGREE AT YALE, RESIDENCY IN PENNSYLVANIA FOLLOWED BY BASIC RESEARCH TRAINING AT THE NIH AFTER WHICH HE RETURNED TO YALE, THEN BECAME AN ASSOCIATE PROFESSOR AND IN 1986 RETURNED TO NIH AS A SENIOR INVESTIGATOR. HE'S CURRENTLY CHIEF OF THE NIAID GENETIC IMMUNOTHERAPY SECTION WITH COLLEAGUES HE APPLIES GENE THERAPY AND HEMATOPOIETIC STEM CELL TREATMENTS WITH THE TREATMENT OF PRIMARY IMMUNE DEFICIENCIES, THIS THERAPY PROGRAM FOCUSES ON BENCH AND CLINICAL DEVELOPMENTS OF GENE TRANSFER TREATMENTS FOR A VARIETY OF IMMUNE DEFICIENCY DISEASES. WE WILL ENTERTAIN QUESTIONS THROUGHOUT THE PRESENTATION AND EACH SPEAKER WILL RESPOND AFTER THEY HAVE FINISHED THEIR PRESENTATION. SO OUR FIRST SPEAKER IS DR. DUNBAR. >> THANKS WIN, AND IT'S A REAL PLEASURE TO SPEAK TODAY, PARTICULARLY WITH HARRY MALECH, WHO I ADMIRED MY WHOLE CAREER AT NIH AND BEEN AT THE NIH LONGER THAN ME AND SO HAS WIN, DESPITE THE FACT THAT I'VE BEEN HERE 30 YEARS AND THANK YOU FOR BEING HERE AS THE VICE PRESIDENT AND SECOND GENTLEMAN GETTING THEIR COVID SHOT AT THE NIH CENTER, SO HERE ARE MY DISCLOSURES, I WILL TALK ABOUT CLINICAL TRIALS WE'VE DONE WITH STEM CELL STIMULATING DRUGS AND A TRAUMA PACK WHICH WE RECEIVED RESEARCH FUNDING FROM THE MANUFACTURES AND THE LEARNING OBJECTIVE FOR THOSE OF YOU GETTING CME CREDITS, I WILL FLASH THIS UP QUICKLY TO GET ON TO MY TALK. SO WHAT I WILL TALK ABOUT TODAY FOR THE FIRST 2/3RDS OF THE TALK ARE APPROACHES TO INTERROGATING AND UNDERSTANDING HEMATOPOIESIS AT A CLONAL LEVEL AND I MEAN HIERARCHIES AND FAMILY LINEAGE LEADERSHIP WITHIN DIFFERENT PARTS OF THE HEMATOPOIESIS, PEOPLE HAVE MAPPED OUT THESE PRECURSOR CELLS CAN PRODUCE DIFFERENT DAUGHTER CELLS IN THAT EVERY HEMATOLOGYST GIVES AT THE BEGINNING OF THEIR TALK, OVER THE YEARS THESE HAVE BEEN REFINED WITH SOME OF THE CLONE TRACKING I WILL TALK ABOUT TODAY TO MODELS THAT SHOW QUITE EARLY COMMITMENTS TO EARLY LINEAGES WITH THE POTENT STATANT PROGENITORS THAT THE CLASSICAL SYSTEM SHOWS AND IT'S IMPORTANT TO LOOK AT THIS TO TRY TO UNDERSTAND LAWN MOWER'S GOING ON. BUT THE ABILITY TO STUDY HEMATOPOIESIS IS REALLY DIFFICULT WHEN YOU APT WANT TO LOOK AT A POLEY CLONAL PHYSIOLOGIC SETTING. IF YOU DO LIMIT DILUTION TRANSPLANTS WHERE YOU PUT A SINGLE CELL INTO A MOUSE TO PROVE THAT THE CELL CAN RECONSITUTE THE HEMATPOIETIC SYSTEM IN THE MOUSE, AND FOR A CLINICIAN WHO DOES TRANSPLANT WE DON'T WANT TO DO LIMIT DILUTION TRANSPLANTS ON OUR PATIENTS, WE WANT THEM TO RECOVER QUICKLY WITH PLENTY OF LEEWAY IN THE NUMBER OF STEM CELLS. SOPHISTICATEDY WE WANTED TO DERIVE APPROACHES TO LOOK AT THE OUTPUT IN A PHYSIOLOGIC SETTING OF THOUSANDS, HUNDREDS, MILLIONS OF INDIVIDUAL CELLS AND THE WAY WE'VE DONE THAT IS WE AND MANY OTHER VS THRIE TRIED TO COME UP WITH CLONAL IDENTIFIERS FOR AN ORIGINAL PROGENITOR CELL THAT IS PASSED ON TO THE DAUGHTER CELLS. SO CAN YOU LOOK AT THE PRESENCE OF PARTICULAR MARKERS AND DIFFERENT LINEAGES AND DIFFERENT TIME POINTS, EVEN DIFFERENT TISSUES AND YOU KNOW THIS IS A SCHEMA OF THE DIFFERENT APPROACHES THAT WE PUBLISHED IN A RECENT REVIEW, THE THINGS I WILL CONCENTRATE ON TODAY ARE TRACKING HEMATOPOIESIS POST TRANSPLABTATION, EARLY ON WE USE PROVIRAL INTEGRATION SITES WHEN WE LOOK AT THE TWEPMENT OF GENE THERAPY VECTORS, AND WE WILL HEAR MORE ABOUT THAT FROM HARRY IN OUR SECOND TALK. AND YOU CAN USE THAT AS I CLONAL TAG BUT IT'S NOT QUANTITATIVE AND IS QUITE CHALLENGING TECHNICALLY. MORE RECENTLY WHAT WOVE DONE DISPI WILL TALK MORE ABOUT TODAY IS PUTTING IN GENETIC BAR CODES WHICH ARE BASICALLY OLIGIO NUCLEOTIDES OF RANDOM SEQUENCE THAT ARE INSERTED INTO AN INTEGRATING VIRAL VECTOR AND USE THOSE BAR CODESsA CLONAL TAGS, THERE ARE A NUMBER OF OTHER APPROACHES THAT ARE DEVELOPED FOR INSTANCE AND GENETICY ENTERED YOUR MOUSE MODELS, YOU CAN USE TRANSPOSONS THAT CAN BE ACTIVATED TO MOVE,A ROUND THE GENOME AND THEN SIT DOWN AND STAY PUT AND YOU CAN USE TRANSPOSON INTEGRATION SITES TO STUDY HEMATOPOIESIS WITHOUT DOING TRANSPLANTATION IN MICE AND MORE RECENTLY WITH THE AVAILABILITY OF HITHROUGHOUT PUT GENETIC SEQUENCING FOR REASONABLE AMOUNTS OF MONEY, YOU CAN TAKE ADVANTAGE OF THE FACT THAT EVERY TIME A CELL DIVIDES, IT CIBOLS UP ENDOGENOUS MUTATIONS AND USE THOSE CLONAL TAGS, ENDOGENOUSLY AGAIN WITHOUT TRANSPLANTATION AND WE CAN ALSO USE ENDOGENOUS CLONAL TAGS INVOLVING THE T-CELL RECEPTOR OR THE IMMUNE O GLOBUE LYNN RECEPTOR THAT OCCUR TO STUDY THE CLONAL DYNAMICS OF B-CELLS AND T-CELLS, BUT THE PROGENITOR CELLS CAN ADD OTHER CONTRIBUTIONS TO OTHER LINEAGES CANNOT BE STUDIED THAT WAY AND YOU NEED THESE OTHER APPROACHES. AND THEN MORE RECEIPTLY YOU CAN IMPUGN--TRAJECTORY INFERENCE OR PSEUDOTEMPORAL ORDERING BASED ON GENE EXPRESSION OR EPIGENETIC MARKS TO IMPUTE RELATIONSHIPS BETWEEN DIFFERENT LINEAGES. SO WHAT I'M GOING TO TALK ABOUT TODAY IS USING RESUSC MACAQUES TO STUDY THESE AND THE REASON WE FOCUSED ON RHESUS MACAQUES, IS WE FOUND EARLIER WHICH I WAS INVOLVED INLET 1990S I DIDN'T HAVE THE PERSISTENCE THAT HARRY'S SHOWN, I FOUND WHAT WE DID IN MURINE MODELS DIDN'T WORK SO WELL BACK THEN SO I SWITCHED TO WOSH, ON MACAQUES. NOW THEY HAVE A VERY PROLONGED LIFE SPAN WHICH IS RELEVANT TO STUDIES I WILL SHOW LATER ABOUT AGING WITH HEMATOPOIESIS AND THEIR SIZE IS CLOSER TO HUMANS AND MICE, AND THEY HAVE AN IMMUNE SYSTEM THAT ARE VERY HOMOLOGOUS IN TERMS OF HUMANS IN TERMS OF TYPES OF CELLS AND BONE MARROW AND IMMUNE ORGANS, TELOMERE LENGTHS AND MUCH LONGER TELOMERES AND THE MARROW STRUCTURE AND IMMUNE TISSUE AND ARCHITECTURE AND FUNCTION ARE VERY, VERY SIMILAR AND ALSO EXTREMELY WELL UNDERSTOOD WHICH OCCURRED IN PART BECAUSE OF THE HIV EPIDEMIC AND MORE RECENTLY EBOLA, ZIKA AND COVID WHERE THESE ANIMALS HAVE SHOWN TO BE EXTREMELY GOOD MODELS FOR VACCINE STUDIES AND INFECTION STUDIES WITH THESE VERY IMPORTANT VIRUSES. SO 30 YEARS OF RESUSC MACAQUE AND HEMATOPOIESIS AND GENE TRANSFER SIDES HAVE CLOSELY MIRRORED CLOSE HUMAN OUTCOMES AND I HAVE BEEN VERY LUCKY IN THE INTRAMURAL PROGRAM TO HAVE ACCESS TO THIS VERY EXPENSIVE AND LONG-TERM FOCUS MODEL. SO FAR BAR CODING STUDIES THAT ARE LED BY JOY WU AND NOW A STAFF SCIENTIST IN MY LABORATORY WE CONSTRUCT THESE VERY HIGH DIVERSITY PLAZ MIDS MIDBAR CODED LIBRARIES IN THEULENTY VIRAL BACKBONE AND WE ACTUALLY COLLECT HEMATOPOIESIS AND PROGENITOR CELLS FROM RESUSC MACAQUES, WE USE MOBILIZATION OF THESE CELLS IN THE BLOOD WITH AN AGENT BECAUSE IT MIRRORS WHAT WE DO CLINICALLY PER MOST HUMANS WITH TRANSPLANTS THESE DAYS. WE CAN PURIFY THE CELLS AWAY FROM MATURE CELLS USING CD34 SELECTION AND WE BRIEFLY EXPOSE THESE HEMATOPOIESIS CELLS IN CULTURE TO THEULENTY VIRAL LIBRARY. WHILE THE ANIMAL IS GETTING EXPOSED TO RADIATION TO WIPE OUT MOST OF THE ENDOGENOUS REMAINING HEMATOPOIESIS PROGENITOR CELLS AND OPENED UP NICHES FOR OURSELVES TO HONE BACK TO THE MARROW AND GROF THE.--GRAFT. THERE THERE'S NO HLA DISPARITIES SO THESE CELLS YOU DON'T HAVE TO WORRY ABOUT REJECTION AND WE REALLY WANT TO STUDY THE INTRINSIC PROPERTY IN THE HEMATOPOIESIS IN THE CELLS THEMSELVES WITHOUT THE ISSUE OF IMMUNE REJECTION OR OTHER ISSUES THAT COME UP WITH ALOE GENERATED AIC TRANSPLANTATION, WE CAN THEN SAMPLE THE BLOOD, MARROW, OF THE ANIMALS OVERTIME, LOW CYCLE PC R TO AMPLIFY THE BAR CODE, WE CAN DO HIGH THROUGHOUT PUT SEQUENCING, PROCESSING TO BASICALLY COME UP WITH A LIST OF BAR CODES PRESENT IN THIS ANIMAL, EACH BAR CODE CORRESPONDS TO 1 TRANSDUCED PROGENITOR OR STEM CELL, WE HAVE TO MAKE SURE WE HAVE A HIGH DIVERSITY LIEBIARY SO EVERY TIE YOU HAVE A BAR CODE IT HAS TO HAVE ORIGINATED 1 INDIVIDUAL ORIGINALLY TRANSDUCED HEMATOPOIESIS OR STEM CELL. AND WE'VE SHOWN RIGOROUSLY AND THE NUMBER OF STUDIES LISTED HERE IN THE BOTTOM, THAT THE FRACTIONAL CONTRIBUTION OF EACH BAR CODE, TO A SAMPLE REFLECTS THE UNDERLYING CONTRIBUTION OF THAT CELLULAR CLONE TO THE SAMPLE, TO THE PERIPHERAL BLOOD, TO THE LYMPHNODE OR WHATEVER TISSUE OR SAMPLING. SO I WILL GO THROUGH A COUPLE OF QUESTIONS, WE'VE BEEN ABLE TO LOOK AT THIS WITH THIS MODEL. ONE IS AN IMPORTANT ISSUE OF WHETHER OR NOT STEM CELLS REALLY CAN PERSIST VR LONG-TERM STABLY IN TERMS OF CONTRIBUTIONS OVER YOUR ENTIRE LIFE SPAN WHETHER EVERY STEM CELL IN YOUR BODY AND BONE MARROW IS ALWAYS CONTRIBUTING OR WHETHER YOU HAVE A PICTURE OF CLONAL SUCCESS OR YOU HAVE GROUPS THAT CONTRIBUTE FOR MONTHS TO YEARS TO WEEKS AND EXHAUST AND DISAPPEAR AND REPLACE BY WAVES OF DIFFERENT CLONES, VERSUS THIS CLONAL STABILITY MODEL SHOWN HERE ON THE RIGHT. VERSUS A STOIKASTIC MODEL CASTIC PROCESS WHERE THEY MAY WAX AND WANE AND A COUPLE OTHER MODELS THAT PEOPLE HAVE LOOKED AT AND WHY ARE THESE CLINICALLY RELEVANT QUESTIONS, WELL IF YOU WILL GENETICALLY MODIFY A POOL OF CELLS, YOU HOPE THEY WILL LAST FOR THE REST OF YOUR LIFE AND YOU WILL NOT HAVE WAXING AND WANING LEVELS OF CORRECTION. AND THINKING ABOUT HOW AGING AFFECTS THE STEM CELL POOL AND WHAT HAPPENS WITH--IN A PROGRESSION TOWARDS MILD DISPLACIA OR FULL BLOWN LUNG CANCER KEEPIC TRANSFORMATION DEPENDS A LOT ON WHICH OF THESE 2 MODELS IS TRUE AND WHAT THE IMPACT IS OF HEMATOPOIETIC STEM CELLS TO DEPLETING THERAPIES SO IT MAY ACT ON A POOL OF CELLS AND THERE WERE STUDIES BACK IN THE 1980S THAT SUGGESTS PRIMARY REVIEWER TYPH,A PROACHS AND PROBABLY USED CONDITIONS THAT KILLED MOST OF THE STEM CELLS WHEN THEY WERE TRANSDUCED OUTSIDE THE ANIMAL. SO IT SEEMS LIKE THE STUDIES WERE DUE TO STOIKASTIC MODEL CASTIC PROCESSES BASED ON FEW STEM CELLS, BUT MORE RECENTLY THERE WAS VERY HIGH PROFILE PAPERS USING THE TRANSPOSON SYSTEM I MENTIONED EARLIER IN MICE, SUGGESTING THAT INDIVIDUAL CLONES CONTRIBUTED FOR VERY SHORT PERIODS OF TIME AND WERE REPLACED BY NEW WAVES EVERY 3 OR 4 WEEKS, IT WAS QUITE PUZZLING AND THERE'S ALSO MOLECULAR CLOCK STUDIES AND COMPLICATED GENETICALLY ENGINEERED MICE THAT CAN TRACK CELL DIVISION AND SUGGESTED PERHAPS EACH HEMATOPOIESIS IN THESE MURINE MODELS HAD VERY LIMITED NUMBERS OF MITOSIS THEY COULD UNDERGO. IT'S OPINION ANTER QUESTION TO ASK CLINICALLY. SO WE HAD A NUMBER OF ANIMALS WE CAN DONE BAR CODED TRANSPLANTATION STUDIES ON AND THIS IS AIAN VERY TALENTED POST BACKIN MY LABORATORY AND ALSO A COMPUTER SCIENTIST AND THERE'S LOTS AND LOTS OF DATA TO WORK WITH FOR THESE STUDIES AND THIS SHOWS A HEAT MAP FOR EACH ROW AS AN INDIVIDUAL BAR CODED CLONE IN TERMS OF THE PARTICULAR BAR CODE, AND EACH COLUMN IS A DIFFERENT CELL LINEAGE AND A DIFFERENT TIME POINT POST TRANSPLANTATION AND THERE'S A HEAT MAP SCALE HERE SHOWING LARGE CONAL CONTRIBUTIONS IN RED DUNE THROUGH ABSENT CLONAL CONTRIBUTIONS IN BLUE. WHAT WE FOUND IS GROUPS OF VERY SHORT-TERM CONTRIBUTING CLONES THAT WERE UNILINEAGE IN SAY T-CELLS, B-CELLS AND MYELOID CELLS THAT DISAPPEARED AFTER A MONTH OR 2 AND WERE REPLACED BY MULTIPOTENT LONG-TERM HEMATOPOIESIS STEM AND PROGENITOR CELLS WHOSE CONTRIBUTIONS AT THIS POINT ARE FOLLOWED AND STABLE UP TO AS MANY AS 7 OR 8 YEARS. WE COULD ALSO LOOK AT THE FREQUENCY OF THESE CELL AND CALCULATE THE NUMBERS, WE COME UP WITH 10S OF THOUSANDS OF INDIVIDUAL CONTRIBUTING CLONES OVER TIME. WE ALSO SAW T-CELLS THAT WERE EXPANDING AND GROWING IN SIZE, YOU KNOW PERIPHERALLY AS WE KNOW THE T-EFFECTOR MEMORY CELLS CAN RESPOND TO ANTIGEN SUCH AS VACCINE OR VIRUS AND EXPAND INDEPENDENT OF THEIR LEVEL OF CONTRIBUTION FROM ONGOING HEMATOPOIESIS. THIS IS A MODEL OF BASICALLY STABLE CONTRIBUTIONS WITHOUT [INDISCERNIBLE] DATA MENTIONED EARLIER THIS APPROACH OF DOING LARGE AMOUNTS OF SEQUENCING TO LOOK AT SOMATIC MUTATIONS THAT ACCUMULATE OVER TIME IN A PAPER BY LEE 6 THAT WAS PUBLISHED IN NATURE A COUPLE YEARS AGO, SO AGAIN, ALL THE DATA WE HAVE FROM ALL STUDIES AND EVEN FROM WITHOUT TRANSPLANTATION IN THE SOMATIC MUTATION STUDY SUGGESTS THAT ALL OF OUR HEMATOPOIESIS CONTRIBUTE STABLY OVER TIME. AND I LIST A NUMBER OF OTHER STUDIES WE FOCUSED ON USING THESE CLONAL TRACKING APPROACHES WHICH I WILL NOT HAVE TIME TO GO INTO GREAT DETAIL TODAY. ONE IS LOOKING AT WHERE HEMATOPOIESIS OCCURS IN THE MARROW AND DOES IT [INDISCERNIBLE] SPACE VERY RAPIDLY AFTER TRANSPLANTATION OR DO YOU HAVE GEOGRAPHIC RESTRICTION OF INDIVIDUAL HEMATOPOIESIS PROGENITOR CELL CLONES THAT WHEN STEM CELLS THEMSELVES RENEW, AND OUR STUDIES DO SHOW THIS CLONAL--CLONAL GEOGRAPHIC RESTRICTION OVER THE BODY THAT PERSISTSEXPOSURE. --AND THEN--THE GRADUATE STUDENT IN MY GRUP SO QUANTITATIVE TO TRY TO DETECT THE PRECOMPARE DESIGNS OF THEIR EVENTUAL SAFETY WHEN THEY WENT INTO CLINICAL TRIALS. SO THE NEXT THING I WILL TALK ABOUT AND SPEND SOME TIME ON BECAUSE IT'S BEEN MORE OF OUR RECENT WORK AND TO ME IT'S INTERESTING AND STEM CELL AGING WE'RE ALL AGING, SOME OF US ARE FURTHER ALONG THAT PATH THAN OTHERS BUT THERE'S BEEN A LOT OF INTEREST IN HEMATOPOIESIS AGING OVER THE PAST 5 OR 6 YEARS AND I WILL TALK ABOUT WHY 1 IS INFORMATION FOR MURINE MODELS SUGGESTING THAT OVERTIME YOU LOSE THE PROGENITOR CELLS THAT ARE ABLE TO MAKE LYMPHOID PROGENY SUFFICIENTLY AND GAIN INSTEADY A MYELOID BIASED HEMATOPOIESIS ROUGH ATOM GENITOR CELLS AND THIS BEING AN EXPLANATION FOR 1 OF THE REASONS AT LEAST THAT OUR IMMUNE FUNCTION DECREASES WITH AGE BECAUSE WE'RE UNABLE TO MAKE NEW BNT CELLS AS EFFICIENTLY BUT IT'S BEEN HARD TO STUDY IN MICE BECAUSE THEIR AGING PROCESS SAYS ARE QUITE DIFFERENT. AND THE SECOND FINDING THAT WAS QUITE INTERESTING WAS THE CLONAL HEMATOPOIESIS THAT I WILL TALK ABOUT IN A BIT THAT WAS DISCOVERED IN HUMANS IN 2014. SO WE HAD THIS MODEL AND WE HAPPEN TO HAVE A LOT OF OLD MACAQUES IN OUR COLONY THAT WERE USED AS BLOOD DONORS OVER TIME AND A POST DOC IN MY GROUP DECIDED TO STUDY THE CLONAL RECONSTITUTION IN OUR YOUNG ADULT MACAQUES THAT I ALREADY TALKED ABOUT AND PRESENTED ALREADY AND A GROUP OF AGE MACAQUES, 18-25 YEARS, THESE ARE KIND OF, YOU KNOW 60-70 YEAR-OLDS IN TERMS OF HUMAN AGE EQUIVALENTS. SO WHAT WE FOUND WAS A DIFFERENT CLONAL RECONSTITUTION PATTERN BOTH OVERALL AND KINETICALLY AND WE SHOWED DELAYED EMERGENCE OF POTENT CLONES IN THESE MACAQUES AND THESE ARE PEARSON PLOTS LOOKING AT ALL THE CLONAL CONTRIBUTIONS OVERTIME HERE FROM 1 MONTH TO 6 AND A HALF MONTHS IN YOUNG VERSUS OLD MACAQUES AND DIFFERENT LINEAGES, AND YOU SEE EARLY YOU HAVE CLONES THAT DON'T FORALATE WITH LINEAGES OR TIME POINTS REPLACED IN THE YOUNG MACAQUE BY MULTIPOTENT STABLE CLONES BOO I 2-3 MONTHS AND IN THE AGE MACAQUES WE SAW VERY DELAYED EMERGENCE IN MULTIPOTENT CLONE ANDS AND IN SOME CASES WE HAVE SKEWED AND BIASED CLONES PERSISTING LONG-TERM. WE FOCUSED ON LARGE CLONES AND WE FOUND IN THE AGE MACAQUES THERE WAS A NUMBER OF LARGE CLONES THAT WERE EXPANDING OVER TIME, UP TO LEVELS OF INDIVIDUAL CLONES CONSITUTING YOU KNOW 10% OF HEMATOPOIESIS, BOTH IN MYELOID CELLS AS WELL AS IN LYMPHOID CELLS LIKE THESE CELLS AND IN YOUNG MACAQUES WE NEVER SAW, EVEN IF WE LOOK THEA THE VERY LARGEST CLONES OF 1 OR 2% CONTRIBUTORS, THEY WERE STABLE OVERTIME, THEY DIDN'T EXPAND AND WE BELIEVE THIS WAS A REALLY INTERESTING ENDOGENOUS MODEL FOR WHAT'S CALLED CLONAL HEMATOPOIESIS OF AGING ARCH FOR SHORT OR CHIP AS I'LL SHOW IN THE NEXT SLIDE. SO AGE RELATED CLONAL HEMATOPOIESIS OR CLONAL HEMATOPOIESIS OF INDETERMINANT PROGNOSIS AS CHP BECAUSE YOU'LL SEE IT DOESN'T HAVE INDETERMINANT PROGNOSIS, WE KNOW MORE AND MORE ABOUT THE PROGMOWSIS SORE I WILL CALL IT ARCH. THIS WAS FOUND ON LARP SCALE POPULATION BASED SEQUENCING STUDIES THAT WERE REPORTED IN TWEBT 14 FROM 3 DIFFERENT GROUPS. AND THEY WERE LOOKING AT THOUSANDS OF SAMPLES IN THE FREEZER FROM THE NURSES TRIAL AND FRAMINGHAM HEART STUDY FROM A MILITARY DATABASE AND WHAT THEY FOUND IS THAT THEY REQUIRED SOMATIC MUTATIONS WHEN YOU LOOK IN HUMAN BLOOD CELLS THAT WERE NOT PRESENT IN THE GERMLINE, OTHER TISSUES AND THEY FOUND THESE INCREASED WITH FREQUENCY AND INCIDENTS WITH AGE SO LET'S SAY UP TO 15% OR HIGHER IN 80 YEAR-OLDS AND AS HIGH AS 30% OF INDIVIDUALS WHO WERE OVER A HUNDRED AND THEY ALSO FOUND THAT THE MUTANT ALLELE FRACTION INCREASED OVER TIME WITH AGING AS WELL, USING AIR COLLECTED SEQUENSZING IF YOU LOOK AT INDIVIDUALS THAT ARE 80 IN THIS MORE RECENT STUDY UP TO 30% OF THEM COULD BE FOUND TO HAVE THESE ISSUE ACQUIRED SOMATIC MUTATIONS AND SO FOR CLINICIANS OR STUDIES OF THIS PHENOMENON, YOU KNOW THEY HAD TO HAVE NO EVIDENCE OF A HEMEAT O LOGICNY O PLASM, THEY HAVE TO HAVE NORMAL BLOOD COUNTS AND MANY OF THESE CLINICAL RESEARCH STUDIES ARE DEFINED THAT YOU HAVE TO HAVE AT LEAST [INDISCERNIBLE] NONERROR CORRECTED SEQUENCING BY THE MORE GENERALLY AVAILABLEY SEQUENCING APPROACHES AND THE OTHER INTERESTING THING IS THAT THERE ARE A FEW GENES THAT ARE EXTREMELY COMMONLY MUTATED IN NEEZ INDIVIDUALS, DMTM3A AND TAT2 AND XLSA 1 MADE UP TO 3040% OF ALL THE MUTATIONS THAT WERE UNCOVERED AND THE MUTATIONS IN THESE 3 GENES WERE PREVIOUSLY BEEN LINKED TONY O PLASSIA AND EPIGENETIC MASTER REGULATORS OF DNA METHYLATION, THEY'RE ALL HETEROGENEOUS ROWING ZYGOUS LOSS OF FUNCTION MUTATIONS AND WHEN YOU LOOKED AT THE SURVIVAL OF THESE INDIVIDUALS, THAT HAVE BEEN FOUND TO HAVE THESE MUTATIONS IN THE POPULATION BASED STUDIES, THEY HAD LOWER SURVIVAL AND THEY WERE DYING OF HEMEAT O LOGIC MALIGNANCIES WHICH ISN'T A SURPRISE THAT THIS WAS KIND OF THE FIRST STEP - THE WAY TO DEVELOPING FULL BLOWN LEUKEMIA BUT THEY WERE ALSO DYING OF CARDIOVASCULAR DISEASE, STROKES, COB JESTIVE HEART FAIL AND YOU ARE CORONARY ARTERY DISEASE AND THAT WAS A BIG SURPRISE. AND SO, IT'S BEEN DIFFICULT TO INVESTIGATE HUMAN ARCH BECAUSE THESE ARE NOT PATIENTS, THESE ARE INDIVIDUALS THAT DON'T HAVE ABNORMAL BLOOD COUNTS, THEY'RE NOT SEEING THEIR DOCTOR FOR ANYTHING IN PARTICULAR, MAYBE THEY HAVE EARLY ONSET CORONARY DISEASE AND PEOPLE ARE STARTING TO INVESTIGATE THAT MORE NOW, BUT YOU KNOW THERE'S LIMITED SAMPLING AVAILABILITY, THERE'S LIMITED LONGITUDINAL FOLLOW UP IN THESE INDIVIDUALS AND MOST PEOPLE DON'T WANT TO HAVE A BONE MARROW EXAM DONE IF THEY DON'T HAVE ANY PROBLEMS. NOW MICE HAVE NOT BEEN VERY GOOD MODELS FOR CLONAL HEMATOPOIESIS BECAUSE IF YOU LOOK WITH AGED MOUSE POPULATION, THEY DON'T HAVE THE SAME SPECTRUM OF THESE ARCHETYPE MUTATIONS, THEY DON'T HAVE MUTATIONS OF THE [INDISCERNIBLE] THEY DON'T HAVE ANY RECURRENT MUTATIONS OF THE SAME TYPE WITH AGING. IF YOU ENGINEER THEM TO HAVE THESE MUTATIONS THEY PROGRESS TO LEUKEMIA PRETTY QUICKLY IN SOME CASES, YOU CAN'T FOLLOW THEM VERY LONG, LIFE SPANS ARE QUITE SHORT AND REALLY THEIR PROGENITOR CELL PROPERTIES, BONE MARROW ENVIRONMENT INTERACTIONS ARE QUITE DIFFERENT FROM HUMANS. SO INSTEAD WE LOOKED AT RESUSC MACAQUES AND ASKED WHETHER THEY COULD A MODEL FOR HEMATOPOIESIS OF AGING. AND SO [INDISCERNIBLE] WHO'S A GRAD UTSTUDENT IN MY GROUP, IN THE PROGRAM PUT TOGETHER A COHORT OF AGE MACAQUES TO STUDY AND THIS HAS BEEN CHALLENGING BECAUSE THERE WERE VERY HUGH COHORTS OF THE MACAQUES THAT EXIST ANYWHERE IN THE WORLD. ANIMALS ARE USED FOR VACCINE STUDIES AND OTHER STUDIES QUITE RAPIDLY AND IT'S VERY EXPENSIVE TO HOLD HAN MALS TO HOLD THEM FOR 20 OR 30 YEARS TO STUDY AGING ABOUT YOU WE MANAGED TO DO IT WITH SOME HELP FROM OUR FRIENDSA THE NATIONAL RAGING INSTITUTE, HE RAN A PANEL OF SEQUENCING ON EXOMES OF 56 GENES THAT HAVE PREVIOUSLY BEEN ASSOCIATED WITH ARCH, MDS AND OTHER BLOOD CANCERS IN HUMANS AND HE FOUND THAT TWIVEN% HAVE SOMATIC MUTATIONS WITH A MEDIAN AGE AND THE COHORT THAT HE LOOKED AT WITH MUTATIONS WAS 28. AND JUST LIKE IN HUMANS THE MOST COMMONLY MUTATED GENES ARE IDENTICAL TO WHAT WE SEE IN HUMANS, THE MOST COMMON AND THE FEW ANIMALS WERE ABLE TO FOLLOW OVERTIME, THEY DID EXPAND SO THIS DOES SUGGEST THAT THERE'S SIMILAR HEMATOPOIESIS PROGENITOR CELL DYNAMICS RESULTS IN AGE RELATED HEMATOPOIESIS IN BOTH SPECIES SO THESE MUTATIONS IN SOME WAY, ESPECIALLY IN AN AGING MICROENVIRONMENT PERHAPS HAVE A COMPETITIVE ADVANTAGE AND EXPAND AND GROW OUT OVER TIME. NOW THE PROBLEM IS, AS I MENTIONED, IT'S QUITE HARD TO FIND COHORTS OF AGE MACAQUES TO WORK WITH AND TO USE THIS AS A MODEL TO STUDY ARCH. SO WE ASKED COULD WE GENERATE AN ENGINEERED MACAQUE MODEL TARTING WITH YOUNG MACAQUES THAT ARE MUCH EASIER TO ACQUIRE AND WORK WITH, USE CRSPR CAS 9 GENE EDIT 8 HOURS TO CREATE THIS MUTATION IN THE MACAQUES AND SEE IF WE GET EXPANSION OVER TIME AND THIS IS WORK STARTED BY [INDISCERNIBLE] FOR AGE MACAQUE STUDIES AND [INDISCERNIBLE] A POST DOC IN MY LABORATORY RIGHT NOW, AND BASICALLY WE HAVE GUIDE RNAs TARGETING THESE TOP 3 GENES ASSOCIATE WIDE HUMAN ARCH, WE DON'T HAVE ANY HOMOLOGOUS [INDISCERNIBLE] WE ARE JUST USING THE GUIDE RNA IN CAS9 VIA INDELATWALS AND JOINING, AND ENDONUCLEACE TARGETING AND YOU GET HEALING OF THE SCAR WITH MUTATIONS THAT GENERALLY RELATE IN FRAME SHIFTS ON PREMATURE STOP CO DONS. SO IN THE FIRST 3 ANIMALS WE DID, LOOKING AT LOCI, WE FOUND THAT ALL 3 LOOKING AT GRANUE LOW SIGHTS, HOST TRANSPLANT OF THE EDIT ITSELF, WE GOT RAPID EXPANSION IN THESE ANIMALS OF GRANUE LOW CITES THAT CONTAINED MUTATIONS IN TET2, AND THESE REFLECT ONGOING PRODUCTION BY HEMATOPOIESIS--1 ANIMAL, ON DMT3A CLONES BUT MUCH SLOWER EXPANSION OVERALL OF DMT3A AND ASXL1 COMPARED TO TET2. IT WAS INTERESTING BUT GOES ALONG WITH COMPARATIVE DATA SUGGESTING THAT THE TET2 PHENOTYPE IS MORE PROFOUND. BUT IF WE LOOKED AT A HEAT MAP HERE SHOWING THE DIFFERENT SPECIFIC INDELATWALS THAT WE PULLED UP BY SEQUENCING THIS IS THE INFUSION PRODUCT WHERE WE HAD 99.5% IN THE WILD TYPE AND ABOUT .5% WERE MUTANT IN THE INFUSION PRODUCT BUT AFTER TRANSPLANTATION, OVER TIME, THOSE CLONES THAT HAD LOSS OF FUNCTION MUTATION, SO FRAME SHIFT MUTATIONS AND LOSS OF PREDICTED LOSS OF TET2 ACTIVITY, EXPANDED OVER TIME UP TO AT THIS POINT ABOUT 20% OF THE ALLELES IN THIS ANIMAL. AND WE REALLY, THIS DOES SUGGEST BECAUSE IT WAS MULTIPLE DIFFERENT TYPES OF INDELs RESULTING IN EXPANSION THAT NO HITS WERE NECESSARY BUT WE CARRIED OUT SEQUENCING OF THE WHAT I MENTIONED EARLIER AND THE 56 GENES, PREVIOUSLY ASSOCIATED AND WE DIDN'T FIND ANY ADDITIONAL MUTATIONS IN THESE EXPANDING CLONES. AND THIS PUTS ALL 3 OF THE ANIMALS ON THE SAMEAXIS AND IT DOES SHOW THAT THE RATE OF EXPANSION BETWEEN THE 3 MACAQUES VARIED MARKETEDLY, 2 OF THEM WERE FEMALE, 1 FERS MALE. THE AGE RANGE WAS FROM 3-10 YEARS OR SO AT THE TIME OF TRANSPLANT. SO THIS DOES SUGGEST THAT SPECIFIC HOST INTRINSIC FACTORS, YOU KNOW ARE IMPACTING ON THE RATE OF EXPANSION BECAUSE AGAIN THE SAME GUIDE RNAs, THE STARTING LEVEL OF EDITING WAS SIMILAR IN ALL 3 ANIMALS SO POTENTIALLY THERE'S OTHER INTRINSIC GENETIC FACTORS IMPACT ON THIS AND AGE OF THE MICROENVIRONMENT ITSELF MAY IMPACT ON THE RATE OF EXPANSION OR THE PRESENCE OF INFLAMMATION, COULD IMPACT ON EXPANSION. AND IT'S INTERESTING BECAUSE IN HUMAN POPULATION, BASED STUDIES IT DOES APPEAR THAT FACTORS SUCH AS SMOKING, OBESITY, DIABETES, ET CETERA, MAY IMPACT ON THE PREVALENCE OF CHP AND THE RATE OF EXPANSION ONCE THESE MUTATIONS OCCUR AND WE WILL TALK ABOUT WHY THAT COULD BE IN A MINUTE. ABOUT YOU THIS IS ANOTHER WAY IN WHICH THIS MODEL SEEMS TO BE VERY SIMILAR TO WHAT SEE IN HUMAN POPULATIONS WITH CLONAL HEMATOPOIESIS. AND WE LOOKED THEA THE BONE MARROW OF THESE ANIMALS SHOWN HERE ON THE LEFT 2 COLUMNS ARE CONTROL ANIMALS, ALSO TRANSPLANTED BUT WITHOUT EDITING AND YOU SEE FAT CELLS AND RELATIVELY HYPOCELLULAR MARROWS AT THIS TIME POINT POST TRANSPLANT ABOUT 1-2 YEARS POST TRANSPLANT AND WE SEE IN THESE CHP ANIMALS THAT WE CREATED OR ARCH ANIMALS WE CREATED WE SEE RELATIVE HYPER CELLULARITY COMPARED TO CONTROLS MEANING MORE CELLS, LESS FAT AND THIS IS SHOWN HERE GRAPHICALLY IN TERMS OF INCREASED CELLULARITY AND IN THE LOWER ROW WE SHOW MYELOPEROX DACE STAINING DEVELOPING YIEWNG NUTRIFILLS AND WE SEE A SHIFT TOWARDS A MYELOID BIAS IN THE MARROW BUT WE DID NOT SEE ANY MILD DISPLACIA AND ANY ABNORMAL DIFFERENTIATION, WE DIDN'T SEE ANY INCREASE IN BLAST CELLS SO THERE WAS NO EVIDENCE OF LEUKEMIA AND THESE ANIMALS NOW FAMILIARED UP TO 3 YEARS, HAVE NORMAL BLOOD COUNTS SO AGAIN THIS ALSO REFLECT WHAT IS YOU SEE IN HUMANS, WITH CLONAL HEMATOPOIESIS. SO IT TAKES A VERY LONG TIME AND PROBABLY SECOND EVENTS TO GO FROM CLONAL HEMATOPOIESIS TO CHANGES IN BLOOD COUNTS OR OVER TO LEUKEMIC TRANSFORMATION. THE OTHER THING WE WANT TO LOOK AT IS WHAT WAS GOING ON WITH THE INCREASE IN CARDIOVASCULAR DISEASE, AND SO WE DID RNA SEQ ON THE PROGENITORS FROM THE MACAQUES, WE GENO TYPES INDIVIDUAL COLONIES AND WE LOOK AT CONTROL VERSUS TET2 MUTANT, COLONIES WE GREW OUT OF THE MARROW AND WE FOUND INFLAMMATION CYTOKINE EXPRESSION AND SPECIFICALLY IN THE TET2 MYELOID PROGENITOR CELLS, WHEN WE SORTED OUT MATURE MACROPHAGES WHICH ARE APPLICATIONS IN ABNORMAL GLUCOSE TOLERANCE CLER O SCLEROSIS AND CARDIOVASCULAR DISEASE, WE FOUND THAT THE MA ROUGH ATOM PHAGE FROM THE EDITED ANIMALS HYPER SECRETED INTERLUKEIN 1 BETA AND 6 TO HYPER INFLAMMATORY CYTOKINES AND IF WE LOOK IN THE SERUM FROM THE BOARN MAR OR IN THE PERIPHERAL BLOOD WE FOUND ELEVATED LEVELS OF INTERLUKEIN 6 AND RECEPTOR ALPHA, WHICH HAS ALSO BEEN REPORT INDEED SOME POPULATION BASED STUDIES OF HUMANS AND THIS WAS SEEN EVEN IN THE MACAQUE CXL39 THAT HAD A LOW MUTANT FRACTION, IT WAS THE ANIMAL THAT HAD THE SLOWEST EXPANSION ONLY ABOUT 2 PNTD 5% AND THAT WAS INTERESTING, THAT LED US TO MODEL FOR INFLAMMATION AND CLENNAL EXPANSION AND TET 2 SO HERE'S THE PROGENITOR CELLS, AND MYELOID PROGENITOR CELLS AND SOME OF THEM HAVE A MUTATION AND--AND THIS IS A RECEPTOR FOR INTERLUKEIN 6. WE KNOW THESE ARE SECRETING IL6, IL8 AND THEY ACT ON VARIOUS COMPONENTS OF THE MICROENVIRONMENT IN THE MARROW SUCH AS STROAMAL CELLS AND MACROPHAGES TO INCREASE IL6 SECRETION WHICH ACTS BACK O THESE PROGENITOR CELLS AND MYELOID PROGENITORS TO FURTHER EXPAND THE ABNORMAL CLONE WHICH ARE HYPER SENSITIVE TO INTERLUKEIN 6, SO HAVE YOU THE VICIOUS CYCLE CONTINUING TO DRIVE CLONAL EXPANSION AND INFLAMMATION, SO WE BLOCKED THE CYTOKINE SUCH AS IL6 COULD HALT THIS PROCESS SO WE LOOKED AT USING [INDISCERNIBLE] A MONOCLONAL ANTIBODY THAT INHIBITS IL6 RECEPTOR AND MEMBRANE IL6 RECEPTOR BINDING TO IL6 AND WE FOUND WHEN WE TREATED FOR 4 MONTHS WITH [INDISCERNIBLE] THAT WE DEREESED IN THE PERCENT OF MUTANT TETALLELES IN GRANUE LOW SIGHTS WE DIDN'T SEE ANY CHANGE IN DMT3A OR ASXL1 AND WHEN WE STOPPED THEKS PANGZ BEGAN AGAIN, IN SUMMARY, I THINK I HAVE GONE OVER MOST OF THE THESE POINTS BUT THESE MACAQUE ARCH MODELS CAN BE QUITE USEFUL TO INVESTIGATE PATHOPHYSIOLOGY, IT'S CLEAR THAT AN AGE MICROENVIRONMENT IS NOT NECESSARY FOR CLONAL EXPANSION, AT LEAST WITH TET2, HOWEVER THESE VARIABLE RATES OF EXPANSION BETWEEN INDIVIDUAL ANIMALS DOES SUGGEST A ROLE FOR EXTRINSIC FACTORS AND THIS MODEL ALLOWS PRECLINICAL TESTS TO STOP CLONAL EXPANSION AND DEAL WITH DOWN STREAM CONSEQUENCES. THIS COMES UP WITH SOMETHING WE THOUGHT ABOUT AND OTHERS HAVE THOUGHT ABOUT ABOUT THE RELATIONSHIP WEAN THE PRESENCE OF CLONAL HEMATOPOIESIS AND COVID. IF YOU LOOK AT THE COVID FATALT RATE VERSUS AGE IT'S VERY SIMILAR TO THE CLONAL HEMATOPOIESIS VERSUS AGE. IN COVID PATIENTS DIE PRIMARILY FROM LATE HYPER INFLAMMATORY TISSUE AND ORGAN DAMAGE, NOT FROM THE ORIGINAL INFECTION DURING THE FIRST WEEK BUT DURING THE SECOND WEEK WHEN YOU THINK THEY'RE GETTING BETTER THEY GET THE HYPER INFLAMMATORY SYNDROME, IT'S NOT THAT WELL UNDERSTOOD AND IL1 BETA AND IL6 HAVE BEEN IMPLICATED. AND SO IT'S KNOWN THAT IT'S HIGHLY HETEROGENEOUS ROW GENIUS IN TERMS OF WHICH ELDERLY PATIENTS TO THIS OUTCOME AND KNOWN RISK FACTORS SUCH AS DIABETES, UNDERLYING CORONARY ARTERY DISEASE, UNDERLYING COPD, PREDICT FATALITY ONLY IN 2/3RDS OF THE CASES OF AGE PATIENTS SO WE WONDERED WHETHER ARCH MUTATIONS PREDISPOSE TO THESE POOR COVID OUTCOMES AND WE HAVE FUNDS FROM NAID ITACK TO DO A COLLABORATIVE PROJECT TO SEQUENCE PATIENTS WITH DIFFERENT SEVERITYS OF COVID FOR THE PRESENCE OF ARCH MUTATIONS, I DON'T HAVE ANY OF THAT DATA TO PRESENT TO YOU YET BUT WE'RE INTERESTED IN LOOKING AT THAT RELATIONSHIP. WE'RE ALSO INTERESTED IN BEING LOAMACYING AT WHETHER OUR ARCH MACAQUES CAN BE A MODEL FOR HYPER INFLAMMATORY COVID. THERE IS NOT A MODEL FOR HYPER INFLAMMATORY COVID. MACAQUES ARE A GREAT MODEL FOR INFECTION AND VACCINE TRIALS AND THAT'S WHERE THE MODEL CITIZEN DERNA VACCINE WAS AT THE NIESHES H INVESTIGATED FOR EFFICACY BUT THEY ONLY GET VERY MILD DISEASE AT LEAST IN YOUNG MACAQUES, THEY DON'T DIE, THEY DON'T GET HYPER INFLAMMATION SO WE'RE LOOKING AT WHETHER OUR ENGINEERED ARCH MACAQUES COULD POSSIBLY SHOW MANIFESTATIONS MORE OF THIS LATER INFLAMMATORY DISEASE TO UPON ALLOW US TO LOOK AT INTERVENTION PATHOPHYSIOLOGY. SO FINALLY I WANT TO TALK ABOUT SOMETHING SLIGHTLY MORE CLINICAL, NOTHING TO DO WITH TRANSPLANTATION BUT IT DOES HAVE TO DO WITH LESSONS WE LEARNED ABOUT STEM CELLS FROM LABORATORY STUDIES, IT'S THE PROTOTYPE, IF YOU LOOK AT THE BONE PARMARROW, THIS IS A MARROW BIOPSY FROM THE ILIAC CREST OF THE PATIENT WITH THE ANEMIA AND BASICALLY THERE'S NO HEMATOPOIESIS, THERE'S BONE, NO FAT, VERY FEW DEVELOPING HEMATOPOIESIS CELLS COMPARED TO NORMAL BONE MARROW, SO SEVERE CYTOPENIA, PLEADING AND INFECTIONS AND SHOWN IN ACQUIRED APLASTIC ANEMIA THAT YOU HAVE AUTOIMMUNE T-CELL ATTACK ON THE PROGENITOR CELLS GREATLY DECREASING THE SEMESTER CELL TOM COMFORTMENT AS WELL AS CIRCULATING BLOOD CELLS AND HOW DO YOU TREAT THIS, YOU DO AN ALOE GENERATED AIC TRANSPLANT IF YOU HAVE A GOOD DONOR AND IF YOU'RE YOUNG ENOUGH TO WITHSTAND THE RIGORS OF TRANSPLANT AS WYNN SAID HOW IMPORTANT IT WAS, IN APLASTIC ANEMIA, IF NOT YOU CAN TRY TO SHUT IT OFF BUT ONLY 60% OF THE PATIENTS RESPOND, THEY CAN RELAPSE, GET CLONAL PROGRESSION, SO UNTIL THERE REALLY WAS VERY FEW THERAPIES OR NO THERAPIES BESIDES GIVING TRANSFUSION AND HOPING PEOPLE DIDN'T GUY OF SERIOUS INFECTIONS OR BLOODING IF PEOPLE DID NOT GET TRANSPLANTED OR RESPOND TO IMMUNE O SURVEYS EXPRESSION. SO IN THE LABORATORY, IT WAS KNOWN INITIALLY WHEN THE HORMONE THROMO POTENT STATEIN WAS CLONED IT WAS IDENTIFIED AS A CRITICAL CYTOKINE NECESSARY FOR THE PRODUCTION OF THE NUMBER OF DIFFERENT STUDIES BOTH STUDYING HEMATOPOIESIS AND STEM CELLS INVITRO BY OUR LAB AND A NUMBER OF OTHERS AS WELL AS GENETIC INFORMATION IN HUMANS, SUGGESTING THAT IF YOU HAVE MUTATIONS TPO OR RECEPTOR MIPL, THAT YOU END UP WITH THE HEMATOPOIESIS FAILURE. SO IT APPEARS THAT IT'S ABLE TO STIMULATE AND NECESSARY FOR MAINTENANCE AND SELF-RENEWAL OF HEMATPOIETIC STEM CELLS. SO THE DEVELOPMENT OF THROMBO POETIN IN ITSELF HAD COMPLICATIONS AND PROBLEMS SO INSTEAD A SMALL MOLECULE ORAL AGANIST OF A THROMBOWETIN WAS DEVELOPED, AND 15 YEARS AGO CALLED TRAUMA PACK AND AS I SAID SIGNALS THROUGH THE MIPL RECEPTOR AND CAUSES PROLIFERATION, AND THIS BINDS TO THE SAME RECEPTOR AND ACTIVATES IT SEEMING 3 THROUGH THE SAME PATHWAYS. SO WE TOOK A--WE TOOK A COHORT OR REFRACTORY, SOME OF WHOM WHO HAVE BEEN EXTREMELY ILL IN REQUIRES PLATELET AND RED TRANSFUSIONS FOR YEARS, WE TREATED THEM WITH A PATH WHICH AT THAT POINT WAS ABOUT TO RECEIVE FDA APPROVAL FOR USE IN IDIO PATHIC THROMO CYTOPENIC PERIPH RA, AUTOIMMUNE DESTRUCTION OF PLATELETS AT WHICH POINT VERY LOW DOSES WERE EFFECTIVE BUT WE DOSE ESCALATED UP TO HIGHER DOSES AND LOOKED AT WHETHER INDIVIDUALS RESPONDED 3-4 MONTHS LATER, I MUST SAY WHEN WE STARTED THIS TRIAL I WASN'T PARTICULARLY OPTIMISTIC BUT IT SEEMED REASONABLE TO TRY GIVEN THE DRUG HAD VERY LITTLE TOXICITY IN THE ORIGINAL ITP TRIALS BECAUSE YOU KNOW THE IDEA OF WHETHER OR NOT YOU COULD ACTUALLY STIMULATE STEM CELLS WHETHER THEY'RE EVEN STILL PRESENT IN THE ANEMIA WAS UNCLEAR BUT WE'RE VERY EXCITED TO SEE THAT THESE INDIVIDUALS, REASONABLE PERCENTAGE OF THEM, I WILL SHOW YOU IN THE NEXT SLIDE, 40-50% ACTUALLY HAD TRIED LINEAGE RESPONSES, THESE RED ARROWS ARE TRANSFUSIONS THIS, INDIVIDUAL, 19 YEAR-OLD PATIENT WAS RECEIVING EVERY WEEK OR SO, PLATELETS AND RED BLOOD CELLS BEGONE A TRAUMA PACK AND PLATELETS BEGAN TO GO TOWARDS REMEMBER NORMAL WHEN OFF TRANSFUSIONS, NORMALIZED AND THE NUTRIFILL ACCOUNT CAME INTO A RANGE HE WAS SAFE FROM SERIOUS INFECTIONS. WE LOOK AT THE MARROW AND CELLULARITY IMPROVED AND NORMALIZED AND MARKEDLY IN RESPONDING PATIENTS AND THIS LED TO FDA APPROVAL OF A TRAUMA PAD FOR PATIENTS WITH SEVERE APLASTIC ANEMIA, THE FIRST NEW DRUG FOR APLAOF THEY ANEMIA IN OVER 30 YEARS. AS I SAID 40-50% OF THESE REFRACTORY PATIENTS RESPONDED. THEY WERE CLINICALLY SIGNIFICANT AND STABLE RESPONSES UP TO 11 YEARS AND SOME PATIENTS WE'VE BEEN TABLE TO STOP THE DRUG AND SEE A RESPONSE, KIANSWER QUESTIONS ABOUT THAT LATER AND VERY MINIMAL TOXICITY FOR A ONCE A DAY ORAL DRUG. DANIEL TOWNSLY WORKING WITH NEAL YOUNG IN THE HEMATOLOGY BRANCH, LOOK ADDADDING A TRAUMA PACK TO STANDARD UP FRONT ANEMIA WONDERING WHETHER YOU COULD APPROVE THE ORIGINAL RESPONSE RATE AND HAVING PATIENTS TO GO TO TO BEING REFRACTORY AND TRYING A TRAUMA PACK LATER ANDISK BASICALLY HE SHOWED THAT THE OVERALL RESPONSE RATE INCREASED MARKEDLY FROM YOU KNOW 60% AS I MENTIONED EARLIER TO BETWEEN 80 AND 95% DEPENDING ON ON HOW THE DRUG WAS GIVEN IN TERMS OF SCHEDULE AND COMPLETE RESPONSES SO NORMALIZATION OF BLOOD COUNTS OCCURRED IN A MUCH HIGHER PERCENTAGE ALMOST 40% COMPARED TO 12% WITHSTAND ARD ATG AND PSYCHOSPORIN SO THIS ALSO RECEIVED APPROVA AND BECOME REALLY THE STANDARD OF CARE FOR ACQUIRED APLASTIC ANEMIA. THE 1 THING THAT WAS QUITE INTERESTING ABOUT ALL THIS IS THAT YOU KNOW IF YOU LOOK AT THE TPOL LEVELS WHICH IS A PAPER I DID IN MY LAB QUITE A LONG TIME AGO, THE LEVELS WERE ALREADY REALLY HIGH IN THE SERUM OF PATIENTS WITH SEVERE APLASTIC ANNEALIA IN CONTRAST TO PATIENTS WITH ITP. SO IT MADE A LOT MORE SENSE TO TRY GIVING A PACK AN AGANIST TO PATIENTS WITH ITP THAN THOSE WITH SEVERE APLASTIC ANEMIA YOU MIGHT THINK ALREADY HAD A LOT OF T PO AROUND AND WASN'T WORKING AND ULS MUCH HIGHER LEVELS AND NORMAL HEALTHY CONTROLS, SO THERE'S A FELLOW IN MY LABORATORY AND TENURED TRACK INVESTIGATOR, HE DID THIS WORK ONCE AGAIN BECOME INDEPENDENT, WONDERED HOW IN THE WORLD IT COULD BE IMPROVING HEMATOPOIESIS IN PATIENCES WITH SEVERE APLASTIC ANEMIA WHO ALREADY HAD TPO LEVELS, SO WHY WASN'T IT WORKING? IT'S KNOWN THAT LEVELS OF INTERFERON ARE EXTREMELY HIGH IN THE SERUM AND BONE MARROW PATIENTS WITH APLASTIC ANEMIA AND ALSO FROM THESE T-CELLS THAT ARE HURTING STEM CELLS AND IT'S KNOWN THAT INTERFERON IS DPREEMLY INHIBITORY TO HEMATOPOIESIS AND PROGENITORS AND ADD INTERFERON THE NUMBER OF COLONIES DROPS WHEN YOU ADD A TRAUMA PACK, YOU ARE ABLE TO PREVENT THE IMPACT OF INTERFERON. AND THIS IS THE SAME WHEN YOU LOOK AT ENGRAPHMENT OF HUMAN PRIMITIVE CELLS IN IMMUNE O DEFICIENT MICE. SO ANDRE WONDERED HOW COULD THAT BE HAPPENING AND THROUGH THE WORK OF RECEPTOR INVITRO AND BIOLOGY HE CONCLUDED THAT INTERFERON DIRECTLY BINDS TO THROAM BO POEETIN AND PREVENT ITS FROM THE LOW AFFINITY SITE AND IT COULD BYPASS THIS ISSUE BECAUSE IT CAN DIRECTLY ACTIVATE THE RECEPTOR BY BINDING TO THE MEMBRANE DOMAIN, GET DOWN STREAM SIGNALING AND GET SURVIVAL AND PROLIFERATION. SO IT CAN BASICALLY BYPASS THIS INHIBITORY EFFECT OF INTERFERON ON CLINICAL GENETICS DODGE NOWS TPO. SO THAT WAS GREAT BECAUSE THEN WE HAD A MECHANISM AND UNDERSTANDING FOR WHY THIS DRUG WAS WORKING IN A WAY THAT YOU WOULDN'T NECESSARILY HAVE PREDICTED GIVEN THE ENDOGENOUS LEVELS OF TPO. SO I WILL FINISH HERE NTHERE A NICE ANIMATION WITH WHAT HAPPENS WITH THE BINDING OF TPO AND I WILL FINISH HERE BY THANKING THE MEMBERS OF MY LAB RAARE TOY. THIS IS ACTUALLY THE MEMBERS OF MY LABORATORY 3 OR 2 YEARS AGO WE TOOK A PICTURE AT THE PICNIC AT THE CHERRY BLOSSOM GROVE IN BETHESDA, BUT WE DON'T HAVE A NEW LAB PICTURE BECAUSE OF THE PAN DEPRIVATIONIC BUT I'M LISTING THE PEOPLE HERE IN MY LAB, I THINK I SHOWED THE PEOPLE RESPONSIBILITY FOR THE WORK THROUGHOUT MY TALK, I WOULD THANK NEAL, DANIEL AND ALL THE CLINICIANS THAT WORK WITH ME ON THE TRAUMA PACK STUDIES, PATIENTS, RESEARCH NURSES, OUR COLLABORATORS ON THE CHIP STUDIES AT CAME BRIDGE AND SANGER AND A NUMBER OF OTHER COLLABORATORS AND OUR PRIMATE PROGRAM STAFF WHO TAKE SUCH GREAT CARE OF OUR ANIMALS ANDITARY COULD AND--AND CARRY OUT THE RESEARCH WITH US AT THE FACILITY. SO THANK YOU VERY MUCH. I CAN TAKE QUESTIONS. I THINK YOU ARE MUTED. >> OKAY, THANK YOU VERY MUCH SYND WE HAVE TIME FOR SEVERAL QUESTIONS. JEFFREY INQUIRES THAT THE FIRST--WELL, HE POINTS OUT, YOU MENTION THAT THE ARCH MUTATIONS ARE RELATED TO COMPETITIVE ADVANTAGE AND THE FIRST RESPONSE TO MUTATIONS IS TYPICALLY--THIS IS BAD. HE WONDER FIST YOUR TODAYS MAY SUGGEST THAT MUTATIONS AND DECREASE IN PARTICULAR HEMATPOIETIC MIGHT BE PERINENT AS WE ENGAGE. >> THAT'S A PERINENT QUESTION AND IT MAY BE RIGHT IT MAY BE THAT IN THE CELLING WITH ANY OF US AS WE AGE, I MEKS THAD INTERFERON AND OTHER INFLAMMATORY CYTOKINES CAN BE QUITE INHIBITORY AND DELETE ORIOUS TO NORMAL AMOUNT OF PROGENITOR CELLS SO IT'S VERY POSSIBLE THESE MUTATIONS ALLOW CELLS TO SURVIVE IN THAT KIND OF MICROENVIRONMENT AND YOU KNOW IT MAY BE GOOD, I MEAN IF YOU LOSE A LOT OF YOUR NORMAL STEM CELLS WITH AGING THROUGH ENVIRONMENTAL, YOU KNOW STRESS OF VARIOUS KINDS OR EVEN EXHAUSTION OR CELLS THAT CAN BE RESISTANT TO THAT MAY HAVE AN ADVANTAGE BUT BOTH THEY HAVE A CLONAL ADVANTAGE BUT THEY MAY ALSO HAVE AN ADVABTAGE IN TERMS OF IMPROVING YOUR HEMEAT ON POEISIS OVER ALL BUT THE PROBLEM IS THEY DO SEEM TO HAVE THIS ADVERSE SIDE EFFECT OF, YOU KNOW CAUSING MORE INFLAMMATION AND ALSO HAVING A PROPENSITY TO EVENTUALLY THRANS FORM TO BE OVERLY LEUKEMIC AND LEUKEMIA, MANY OF US THINK OF LEUKEMIA AS SOMETHING THAT KIDS GET BUT ACTUALLY ACUTE MYELOID LEUKEMIA IS ABSOLUTELY A DISEASE OF AGING AND FAR MORE COMMON IN INDIVIDUALS OVER 60 AND YOU KNOW WE PROBABLY EVOLVED TO SURVIVE TO FOART AND HAVE KIDS NOT NECESSARILY TO EVOLVE TO BE 80 WITH NORMAL HEMATOPOIESIS, ANYWAY, VERY GOOD QUESTION. THANK YOU. OR A TRAUMA PACK AND AML, OKAY, SO, I DEPARTMENT GO INTO A LOT OF THAT, THERE'S BEEN CONCERN THAT IF YOU ARE STIMULATING SOME STEM CELLS WITH A DRUG SUCH AS A TRAUMA PACK THAT HYPER STIMUMENTS THEM THAT THAT COULD HASTEN PROGRESSION TO MILD DISPLASSIA OR LEUKEMIA WHICH ARE ALREADY MORE COMMON IN INDIVIDUAL PATIENTS WITH SEVERE APLASTIC ANEMIA, ESPECIALLY IF THE REFRACTORY BUT EVEN IF THEY RESPOND TO IMMUNE O SUPPRESSION AND IT'S THE REASON YOU WOULD DO A TRANSPLANT IF YOU HAVE A GOOD DONOR IN A YOUNG PERSON WITH APLASTIC ANEMIA TO REPLACE THE STEM CELLS COMPLETELY. YOU KNOW WE HAVE LOOKEDDA THE THAT PRETTY CAREFULLY AND--PROGRESSION TO MILD DISPLASSIA OR AML, WE LOOKED AT SOMATIC MUTATIONS THAT ARE PRESENT IN THESE INDIVIDUALS AND WE DON'T SEE THAT THE MUTATED CELLS ARE YOU KNOW SPECIFICALLY OR YOU KNOW STIMULATED MORE BY A TRAUMA PACK THAN NORMAL CELLS THAT ARE PRESENT IN THE MARROW OF THESE INDIVIDUALS AND THEN RANDOMIZED CONTROL TRIAL IN MILD DISPLASSIA INDIVIDUALS HAVE QUITE A HIGH RISK OF AML, THERE WAS NO RISK INCREASE IN PROGRESSION TO AML IN THE PATIENTS THAT WERE TREATED WITH THE TRAUMA PACK VERSUS PLACEBO AND OUR IDEA IS THAT MAYBE YOU COULD BE KIND OF--IF YOU EARLY ON PRESERVE STEMLES BYADAING A TRAUMA PACK EARLY TO ATP AND PSYCHE LOW SPORIN, ATG AND CYCLOSPORIN THAT THEN YOU WOULD NOT RESULT WITH SUCH AN INCREDIBLE AMOUNT OF CELLS AND THEREFORE MAYBE THE STEM CELLS WOULD BE UNDERLESS STRESS LONG-TERM BECAUSE WE KNOW THAT ANY CONDITION THAT YOU END UP LOSING STEM CELLS PREMATURELY, YOU DO HAVE AN INCREASED RISK OF LEUKEMIA AND A NUMBER OF DIFFERENT CONGENITAL STEM CELL DEFICIENCIES, THERE'S A WHOLE SERIES OF THEM, THEY ALL HAVE RISK OF PROGRESSION TO AML. SO IT'S A GOOD QUESTION AND 1 THAT YOU KNOW I'M PROBABLY BIASED ABOUT BECAUSE I DEVELOPED THIS DRUG FOR THIS DISEASE AND I AM EXCITED ABOUT HOW WELL IT WORKS, BUT THERE ARE OTHERS WHO ARE MORE CONCERNED ABOUT THIS PARTICULAR ISSUE, BUT THERE'S NO DATA YET TO SUGGEST IT REALLY IS A PROBLEM. >> THANK YOU VERY MUCH. BECAUSE OF MANY PEOPLE WHO ATTEND DEMYSTIFYING ARE NOT EXPERTS IN A PARTICULAR FIELD, BUT THEY MAY HAVE QUESTIONS ABOUT CLEANING UP THE BONE MARROW AND INJECTING IT IS STILL USED OR IS WHAT WE CALL BONE MARROW TRANSPLANTATION TODAY REALLY CELL TRANSPLANTATION BASED UPON OBTAINING STEM CELLS FROM THE PERIPHERAL BLOOD. DO YOU WANT TO BRIEFLY COMMENT ABOUT THAT? >> YEAH, IT'S INTERESTING BECAUSE SOME OF MY PATIENTS, YOU KNOW WHEN YOU TALK ABOUT DOING A BONE MARROW TRABZ PLANT OR STEM CELL TRANSPLANT, FIRST OF ALL THEY THINK YOU ARE ACTUALLY GOING TO TAKE WHATEVER THE SOURCE OF STEM CELLS IS AND MAKE A HOLE IN THE BONE AND YOU KNOW SHOOT IT DIRECTLY INTO THE BONE. IN FACT, YOU GIVE THEM INTERRAVINOUSLY AND MIRACULOUSLY THEY KNOW HOW TO HOME BACK TO THE MARROW AND SET UP SHOP. THAT'S THE FIRST REASON THAT HEMATOPOIESIS TRANSPLANTATION WAS DEVELOPED SO EARLY AND SO SUCCESSFULLY IS BECAUSE YOU DON'T ACTUALLY, YOU KNOW THE CELLS KNOW WHERE TO GO AND WHAT TO DO. BUT THE FIRST QUESTION ASKED, WAS SOCIETY SAID HAVE ALL CHANGED THEIR NAMES THERE'S NO MORE SOCIETY OF BONE MARROW TRANSPLANTATION, NO MORE BONE MARROW TRANSPLANTATION JOURNAL BECAUSE I THINK ABOUT 90% OF THE TRANSPLANTS THESE DAYS WHETHER IT'SOUS OR ALOE GENERATED AIC ARE DONE USING MOBILIZED CORE BLOOD CELLS AND BASICALLY IT WAS DISCOVERED THAT BY GIVING CYTOKINES OR CHEMO THERAPY FOLLOWED BY SIEM CYTOKINES AND SHIFT PRIMITIVE CELLS FROM THE BONE MARROW INTO THE PERIPHERAL BLOOD AND YOU CAN COLLECT THEM BY APHORESIS BASICALLY BY BEING ON A MACHINE KIND OF LIKE WHEN YOU DONATE BLOOD BUT IT'S A CONTINUOUS PROCESS, TAKES A COUPLE HOURS AND FOR MOST PEOPLE IT'S A LOT EASIER, LESS PAINFUL DON'T HAVE TO TO SLEEP IN THE O. R. AND HAVE 200 HOLES IN YOUR HIP BOAB TO GET MARROW SO THE RECIPIENTS RECOVER MORE EASILY AND WE UNDERSTAND THE PROCESS OF MOBILIZATION AND PROCESS OF HOMING AND IT GOES BACK TO DURING EMBRYO GENESIS THAT YOUR CELLS MOVE FROM PLACE TO PLACE, YOUR HEMATOPOIESIS STARTS AT THE AORTA REGION DURING DEVELOPMENT, MOVES TO THE YOLK SACK, THEN THE LIVER AND THEN ONLY AROUND THE TIME OF DELIVERY DOES IT MOVE TO THE BONE MARROW, SO THESE CELLS ARE KIND OF, YOU KNOW PRIMED TO MOVE AROUND AND WE TAKE ADVANTAGE OF THAT BY OUR ABILITY TO COLLECT THEM FOR TRANSPLANT. >> OKAY. WELL, LISTEN, THANK YOU VERY MUCH CINDY AND SO NOW DR. MALECH, WILL YOU PLEASE CONTINUE? >> I WOULD LIKE TO THANK THE ORGANIZERS FOR INVITING ME, THIS IS A LECTURE SERIES THAT I'VE ADMIRED THE SERIES AND THE MANY PEOPLE THAT YOU'VE INVITED TO SPEAK HERE. AND IT'S A SPECIAL HONOR TO SHARE THIS SESSION WITH MY ESTEEMED COLLEAGUE DR. DUNBAR. SO WHAT I AM GOING TO DO TODAY IS THAT I WOULD LIKE TO TALK TO YOU ABOUT GENE THERAPY FOR INHERITED IMMUNE DEFICIENCIES, AND I'M--OBVIOUSLY, I CAN'T COVER EVERYTHING BUT WHAT I WANT TO DO IS I WILL START OUT BY TRYING TO BREAK THIS DOWN INTO A DISCUSSION OF HOW THIS IS DONE IN A WAY THAT YOU DON'T HAVE TO BE AN EXPERT TO UNDERSTAND WHAT IT'S ABOUT. AND ONCE I DO THAT, THEN I WILL SORT OF TIP TOW AT FIRST AND THEN RACE A LITTLE FASTER INTO MORE COMPLEX DATA BUT THE IDEA IS NOT TO LEAVE ANYBODY BEHIND IF WE CAN. SO FIRST OF ALL, I HAVE NO FINANCIAL DISCLOSURES AND I'M GOING TO BE TALKING ABOUT GENETICALLY ENGINEERED HEMATPOIETIC, THE PATIENTS THEMSELVES THAT GO BACK INTO THE PATIENTS THAT'S CALLED AUTOLOGOUS AND THESE ARE NOT STANDARDS OF CARE FOR THERAPY BUT WE HOPE THEY WILL BE SO OVER THE NEXT FEW YEARS. THESE ARE THE LEARNING OBJECTIVES WHICH I WILL NOT READ IN DETAIL BUT ARE LIAISONSED ELSEWHEREOT SITE. SO I WILL START OFF BY SHOWING THIS GENERAL SCHEME FORULENTY VECTOR GENE THERAPY AND I'M--I'M FIRST GOING TO QUICKLY GO THROUGH IT AND THEN I WILL TAKE INDIVIDUAL SLIDES AND EXPLAIN EACH STEP IN WHAT IT'S ABOUT, SO IF YOU START OUT WITH A PATIENT, WITH A BLOOD DISORDER, IN THE CASE OF MY INTERESTS, THE BLOOD CELLS I'M INTERESTED IN ARE THE IMMUNE CELLS, THAT CIRCULATE IN THE BLOOD OR IN THE SPLEEN OR IN THE LYMPHNODES AND THYMUS AND SO ON, BUT ALL OF THEM DERIVE FROM THE BONE MARROW. AND WHAT 1 DOES FIRST IS TO COLLECT STEM CELLS AND THE LAST DISCUSSION THAT DR. DUNBAR HAD BEFORE TURNING OVER TO MY TALK SHE WAS ASKED ABOUT THE SOURCE OF THESE CELLS AND 1 WAY IS TO GO DIRECTLY TO THE BONE MARROW AND STICK A NEEDLE IN THE BONE AND PULL CELLS OUT, THE OTHER WAY IS TO MOBILIZE THEM FROM THE MARROW INTO THE BLOOD WHERE 1 CAN COLLECT THEM. ONCE 1 HAS THOSE, THE NEXT THING 1 DOES IS TO PURIFY THOSE CELLS TO WORK SPECIFICALLY WITH THE STEM CELLS. HAD 1 THEN TAKES A VIRUS VECTOR AND EXPOSES THOSE CELLS TO THE VECTOR IN CULTURE WHERE THE VECTOR INSERTS A NEW GENE THAT INSERTS INTO THE GENOME OF THE CELLS. ONE HARVESTS THOSE CELLS, 1 FIEWRIFYS--EITHER USES THEM FRESH OR 1 CAN CRYOPRESERVE THEM, 1 THEN NEEDS TO MAKE ROOM IN THE PATIENT'S BONE MARROW SO THAT WHEN YOU INJECT THOSE CELLS BACK IN, THEY HAVE A PLACE TO SETTLE. ONE WOULD THINK THAT JUST PUTTING THE PATIENTS OWN BEAN MARROW CELLS BACK IN, THEY FIND THEIR WAY HOME BUT IN FACT IT'S A BIT LIKE A GAME OF MUSICAL CHAIRS WHERE YOU HAVE TO EMPTY SOME OF THE CHAIRS FOR THESE CELLS TO HAVE A PLACE TO SIT DOWN. AND I WILL GO THROUGH EACH OF THESE A LITTLE MORE DETAIL. JUST TO REMIND YOU AND I WON'T GO THROUGH THIS IN DETAIL, EXCEPT TO POINT OUT THAT BLOOD STEM CELLS RIGHT HERE AT THE TON THE HEMATPOIETIC GIVES RISE TO ALL THE DIFFERENT MATURE CELLS THAT MAKE UP THE BLOOD AND THE IMMUNE SYSTEM AND SO IF 1 CAN COLLECT PURIFY AND CORRECT THESE STEM CELLS AND PUT THEM BACK, THEN THEY CAN GIVE RISE TO CORRECTED MORE MATURE CELLS. SO HEMATPOIETIC OCCUPY SPECIFIC SITES IN THE BONE MARROW, IT USED TO BE THOUGHT THAT THIS WAS AN ANALOGY BUT IN FACT INVESTIGATORS LIKE DAVID SCADEN THAT THE BONE MARROW IS A REAL PLACE, THERE'S ACTUAL SPECIFIC PLACES IN THE MARROW WHERE THE TRUE STEM CELLS SIT AND ARE NURTURED BY SURROUNDING CELLS BUT THERE ARE LOTS OF DIFFERENT WAYS IN WHICH THE--WAY IN WHICH THEY'RE HELD IN IS BY TETHERING BETWEEN STROAMAL DERIVED FACTOR WHICH IS ON THE STROAMAL CELLS THAT CODDLE THE STEM CELLS AND THE STEM CELLS THEMSELVES WHICH HAVE THE RECEPTOR CXCR4, AND WHEN THEY BIND, THIS SERVES AS A TETHERING, A HOLDING IN, LIKE A LEASH IN THE MARROW AND TREATMENT WITH GRANUE LOW CITE STIMULATING FACTOR OR THE SMALL MOLECULE DRUG, WHICH IS A DIRECT CXCR 4 INHIBITOR DISRUPTS THIS TETHER AND ALLOWS THE CELLS TO, IF YOU INHIBIT IT, THEY COME OUT OF THE MARROW INTO THE CIRCULATION AND IF 1 STOPS THAT, THEY AND GO BACK IN THE MARROW, OR IF 1 PUTS NEW CELLS IN, 1 WOULD THINK THEY COULD EASILY GET IN BUT IN FACT, AT ANY POINT IN TIME, THERE'S ONLY A FEW EMPTY SPACES IN THE MARROW, SO IF YOU PUT NEW CELLS IN, YOU NEED TO DO MYELOID CONDITIONING WHICH I WILL EXPLAIN IN MORE DETAIL BUT ESSENTIALLY IT'S A WAY TO MAKE THE RESIDENT STEM CELLS GO AWAY SO WHEN YOU PUT NEW 1S IN, THEY CAN SETTLE IN THERE, SO HOW DOES 1--SO NOW YOU'VE MOBILIZED THE CELLS TO THE PERIPHERAL BLOOD, HOW DO YOU COLLECT THAT, AND THIS IS DONE BY A PROCEDURE CALLED APHORESIS, AND THE FIRST THING YOU DO IS PUT AN . CATHETER IN THE NECK OF THE PATIENT WITH 2 BASICALLY 2 LINES, 1 FOR THE BLOOD TO FLOW OUT AND INTO THIS MACHINE THAT'S SHOWN IN AN OPEN FASHIONOT RIGHT AND THE BLOOD IS ANTICO AGULATED WITH CITRATE AS IT COMES OUT AND THEN MAGNESIUM AND CALCIUM IS ADDED GOING BACK SO THAT THE PATIENT ISN'T OVERLY ANTICO AGULATED AND THE APHORESIS SOMEONE SPINS THE BLOOD SO IT'S LIKE A BLOOD CENTRIFUGE AND IT CREATES A THIN LAYER AND THERE'S A LITTLE TINY ZIPPER THAT CAN BE JUST RIGHT TO PULL OFF THE HEMATPOIETIC STEM CELLS. THE FOLKS IN THE BLOOD BANK KNOW HOW TO ADJUST THE MACHINE SO IF YOU WANT TO GET OTHER KINDS OF CELLS YOU CAN GET PLASMA, YOU CAN GET PLATELETS, CAN YOU GET THE GRANUE LOW SIGHTS OR LYMPHOCYTES OR MONOCYTES AND SOMETHING ENRICHED AND SORT OF STEM CELLS SO THAT'S HOW THAT WORKS. SO WHAT DO DO YOU WITH THOSE, YOU HAVE A BAG FULL OF BLOOD CELLS, WHITE BLOOD CELLS AND YOU WANT TO SEPARATE OUT THE STEM CELLS, SO THERE ARE MACHINES THAT 1 CAN USE THAT ESSENTIALLY ARE AUTOMATED INSTRUMENTS THAT FIRST USE ANTIBODIES THAT BIND TO A CELL SURFACE MOLECULE CALLED CD34, THAT'S PARTICULARLY ENRICHED ON THE SURFACE OF PRIMITIVE BLOOD CELLS. AND SO THE ANTIBODY BINDS, THEY THEN PUT--AND THEN THE ANTIBODY LINKS TO THINGS IN THE COLUMN THAT COLLECT THE CELLS WHILE THE REST OF THE CELLS FLOW THROUGH AND 1 CAN RELEASE THOSE CELLS AND NOW YOU HAVE PURIFIED PATIENT CD34 CELLS THAT CAN EITHER BE KRIST O PRESERVED OR USED FRESH AND GROWN IN CULTURE IN FLASKS OR THIN GAS PERMEABLE MEMBRANES AND THIS IS A SORT OF--I'M ACTUALLY GOING TO GO DOWN HERE AND SHOW YOU THAT THESE ARE THE KINDS OF BAGS 1 COULD USE TO GROW THEM BUT 1 COULD USE JUST STANDARD TISSUE CULTURE FLASKS. SO THE QUESTION IS WHAT IS THISULENTY VECTOR THAT WE WORK WITH? WELL, WHAT THIS S&P IT'S A SELF-ENACTIVATING VECTOR THAT'S ENGINEERED FROM THE HUMAN IMMUNE O DEFICIENCY VIRUS, THERE ARE--SO YOU START OUT WITH THE HUMAN IMMUNE O DEFICIENCY VIRUS WHICH IS OF COURSE IS A DISEASE VIRUS AND IT'S HIGHLY ENGINEERED TO REMOVE ALL SORTS OF PARTS OF THIS GENE THAT MAKE THE VIRUS BAD AND YOU HOPEFULLY END UP WITH SOMETHING THAT YOU CAN USE THAT JUST HAS THE PIECES THAT ALLOW THE VIRUS TO GET INTO THE CELL AND TO DEPOSIT NOT HIV GENES BUT NOW THERAPEUTIC GENE THAT YOU PUT IN THERE, A CORRECTIVE GENE TO FIX THE PATIENT'S PROBLEM. AND I WON'T GO INTO GREAT DETAIL ABOUT THIS, BUT 1 HAS TO PACKAGE THIS IN SPECIAL CELLS, THAT CONTAIN THE PACKAGES ELEMENTS THAT I'VE SORT HAVE HAD LISTED HERE, ONCE THE VIRUS BINDS TO STEM CELLS AND GOES IN, AND AND INSERTS IN THE GENOME, 1 OF THE IMPORTANT THINGS THAT 1 DOES IN A LENTIVECTOR IS TO DELETE A PART OF THIS 3-PRIME. THAT IS THE--ONCE IT GOES INTO THE CELL IT'S RNA WHICH GETS CONVERTED TO DNA BUT IT'S LIKE A SNAKE BITING ITS TAIL. SO IT MAKES A CIRCLE. AND THE TAIL END BECOMES THE FRONT END AND SO, YOU END UP WITH THIS IMPORTANT DELETION THAT MAKES IT NO LONGER ABLE TO REPLICATE AND IT NO LONGER USING THIS AS THE PROMOTER TO PRODUCE THERAPEUTIC GENE. INSTEAD YOU HAVE A NEW PROMOTER THERE THAT THAT'S NOT THE ACTUAL VIRUS PROMOTER, PROMOTERS ARE USED TO REGULATE THE PRODUCTION OF THE THERAPEUTIC GENE. SO IT'S A VERY COMPLEX SLIDE BUT MY POINT WAS TO JUST SAY THAT THIS WAY YOU AT HAVE SOME IDEA OF WHAT A LENTIVECTOR IS AND HOW IT'S USED. I TALKED ABOUT THE BAGS, SO HOW IS THE PATIENT TREATED TO ENHANCE THE LONG-TERM INGRAFTMENT. SO NOW YOU CORRECT THESE STEM CELLS AND NOW YOU'VE GOT A SYRINGE FULL OF GENE CORRECTED STEM CELLS, AND IF YOU JUST--INJECT THEM INTO THE PATIENT, MOST OF THOSE CELLS WON'T FIND THEIR WAY INTO THE MARROW BECAUSE THOSE STEM CELL NICHES NEED TO BE EMPTY AND 1 WAY TO DO THAT IS TO USE CHEMOTHERAPY OR RADIATION, SO 1 OF THE PREFERRED AGENTS IS AN AGENT CALLED BUSULFIN WHICH IS A PARTICULAR CHEMO THERAPEUTIC AGENT THAT IS VERY, VERY EFFECTIVE AT EMPTYING THE BONE MARROW NICHES BUT DOESN'T TARGET REDUCED T OR B-CELLS VERY MUCH. NOW THERE MAY BE SOME KINDS OF TREATMENTS WHERE YOU WANT TO REDUCE TOR B CELLS AND YOU WOULD USE OTHER AGENTS IF ARE THAT BUT FOR MOST OF THE GENE THERAPY I WILL TALK ABOUT TODAY, WE JUST USE THE BUSULF AN TO MAKE ROOM IN THE MARROW SO THAT CORRECTED CELL CANS GET IN. NOW I DON'T HAVE TIME TO TALK ABOUT IT TODAY, BUT I DO WANT TO MENTION IT, THAT THERE ARE AS YOU THINK ABOUT THIS, RADIATION, CHEMO THERAPY AND ALL THAT, WOULDN'T IT BE BETTER IF WE HAD BETTER WAY TOTS EMPTY THE MARROW THAN USING CHEMO THERAPY? WELL, THERE ARE NEW APPROACHES TO MYELOID CONDITION THAGOREAN ARE IN PRECLINICAL AND EARLY CLINICAL DEVELOPMENT WHICH USING EABT BODIES THEMSELVES RATHER THAN CHEMO THERAPY TO EMPTY THE MARROW OF THESE STEM CELLS AND I WON'T--I WON'T GO INTO THIS HERE, BUT IT'S WELL WORTH LOOKING UP THE LITERATURE ON THAT WHICH IS A VERY EXCITING NEW AREA OF TRANSPLANT FOR HEMATPOIETIC STEM CELLS. SO AS A SORT OF OVERVIEW, THIS KIND OF APPROACH OF LEAPTY VECTOR GENE THERAPY HAS RESULTED IN SUSTAINED CLINICAL BENEFIT TO TREAT THESE--THIS GROUP OF IMMUNE DEFICIENCIES AND FOR OTHER PRIMARY IMMUNE DEFICIENCIES THAT ARE PLANNING OR EARLY CLINICAL THRIELS. IF I HAVE THE TIME, I WILL TALK FIRST ABOUT GENE THERAPY FOR X-LINKED SEVERE COMBINED IMMUNE DEFICIENCY AND THEN I WILL TALK ABOUT GENE THERAPY FOR X-LINKED CHRONIC GRANUE LOAMATTOUS DISEASE, BOTH OF THESE DONE AT THE NEY HEAR OR PART OF A MULTICENTER STUDY IN COLLABORATION WITH OTHER CENTERS. SO WHAT IS X-LINKED COMBINED IMMUNE DEFICIENCY, IT'S THE MOST SEVERE FORM. THERE ARE MANY DIFFERENT FORMS OF SKIN BUT ABOUT 25% OF THE PATIENTS HAVE THIS FORM, THE X-LINK SKID, SO THE FREQUENCY OF ABOUT 1 IN 200,000 BIRTHS. THE DEFECT ARE MUTATIONS IN THE INTERLUKEIN 2 RECEPTOR GAMMA CHAIN AND EVEN THOUGH IT'S CALLED--IT LOOKS LIKE IT HAS SOMETHING TO DO INTERLUKEIN 2 RECEPTOR, IT REALLY IS A COMMON GAMMA CHAIN FOR MULTIPLE RECEPTORS FOR INTERLUKEIN 2, 47, 9, 15 AND 21. SO IT'S A PROFOUND DEFICIENCY, IF YOU'RE MISS THANKSGIVING COMMON GAMMA CHAIN NONE OF THE RECEPTORS FOR THESE IMPORTANT IMPORTANT IMMUNE HORMONES DO THEIR JOB AND SO YOU END UP WITH A PROIF YOU UNDERSTAND DEFECT IN TB AND NK CELL IMMUNITY AND THERE ARE FEW T AND NK CELLS AND ALTHOUGH IMMATURE B-CELLS MAY BE MADE IN REDUCED NUMBERS, THEY DON'T MAKE IMMUNE O GLOBUE LYNN SO THEY DON'T MATURE. SO THIS IS A LIFE THREATENING DISEASE AND IN INFANTS IT REQUIRES URGENT INTERVENTION, EITHER A TRANSPLANT OR GENE THERAPY. --WHICH WAS MOST OF THE PARENTS, 1 OF THE GREAT INVENTIONS OF THE LAST 30 YEARS WAS THAT IT WAS REALIZED THAT YOU COULD TAKE CELLS FROM A PARENT, USUALLY THE MOTHER, PURIFY THE STEM CELL, AND JUST INJECT IT IN THE PATIENTS AND IN BABIES AND INFANTS ISSUES THE TURNOVER THAT IS THE IN AND OUT OF THE MARROW IS SO ACCELERATED THAT YOU REALLY DON'T NEED CHEMO THERAPY TO GET A BIT OF INGRAFTMENT IN THE MARROW AND THE PRODUCTION OF CELLS. AND SO, YOU COULD USE HAPLOIDENTICAL LYMPHOCYTE DEPLETED BONE GRAF FROM A PARENT TO SAVE THEIR LIVES. SO EVEN THOUGH THEIR LIVES ARE SAVED IF YOU DON'T USE MYELOID CONDITIONING TO GREATLY INCREASE THE ROOM IN THE MARROW FOR THE CELLS, THE CORRECTION IS SORT OF LIMITED TO ONLY T-CELL REPONCE TUITION, THAT'S ENOUGH TO SAVE THE LIVES OF THESE INFANTS BUT THEY DON'T GO ON TO GENERATE NORMAL BCELLS, THEY DON'T HAVE NORMAL NK CELLS THEY NEED LIFE LONG IMMUNE O GLOBUE LYNN SUPPLEMENTATION AND SOME OF THEM MAY EXPERIENCE DECREASING DONOR T-CELL REPERTOIRE IN NUMBER AND THEY MAY GO ON TO HAVE CHRONIC MEDICAL PROBLEMS, NOT ALL OF THEM BUT PROBABLY ABOUT A THIRD TO A HALF OF THE PATIENTS WILL HAVE PROBLEMS THAT ARE SIGNIFICANT ENOUGH THAT THAT I HAVE COME TO OUR ATTENTION AS NEEDING SOME BETTER THERAPY TO FURTHER FIX THE IMMUNE SYSTEM. SO AT THE NIH WHERE WE CAN'T TREAT INFANTS, WE'VE SORT OF MADE--WE DON'T HAVE THE CAPACITY TO TREAT INFANTS, AS YOU KNOW MANY GENE THERAPY TRIALS TREAT INFANTS SO WE'VE SORT OF MADE LEMONADE OUT OF LEMONS BY SAYING OKAY, THESE OLDER PATIENTS ARE LEFT BEHIND, THEY'RE PATIENT WHO IS STILL NEED SOMETHING, THEY NEED SOME KIND OF RESCUE TO MAKE THEIR IMMUNE FUNCTION BETTER THAN THEY ARE, THEY COME TO US WITH A PARTIAL TRANSPLANT OF PARENTAL T-CELLS AND YOU KNOW THEY GET BY, BUT THEY HAVE LOTS AND LOTS LOTS OF PROBE DEVELOPMENTS, AND WE DEVELOPED THESE TO TREAT THESE OLDER CHILDREN IN YOUNG ADULTS. WE'VE PUBLISHED THE FIRST 5 PATIENTS AND I WILL TELL YOU A BIT MORE ABOUT WHAT WE'VE DONE WITH THE OTHER PATIENTS IN OUR STUDY. THERE ARE ALSO INFORMED BY THE NIH STUDY OF GENE THERAPY OF OLDER CHILDREN FOR YOUNG ADULTS THAT BEGAN IN 2012 OF CLINICAL TRIAL USING THE SAME LENTIVECTOR BLUES BUSULF AN, A MYELOID CONDITIONING WAS USED TO TREAT NEWLY DIAGNOSED INFANTS AT OUR COLLABORATORS HOSPITAL ST. JUDE AND [INDISCERNIBLE] CHILDREN'S HOSPITAL IN SAN FRANCISCO. THIS GROUP IS NOW EXPANDED THEIR TRIAL TO CHILDREN BEING TREATED IN SEALGT, CHILDREN'S HOSPITAL AND THIS IS THE REPORT OF THE FIRST STATE PATIENTS CAME OUT LAST YEAR AND--BUT RIGHT NOW I WILL NOT TALK ABOUT THE INFANT TRIAL, I WILL JUST TALK ABOUT THE NIH TRIAL TODAY. SO THIS IS--WE COLLECT THE TARGET STEM CELLS WE TREAT THE PARENT CELLS WITH THE LEIN, TIVECTOR AND WE TREAT THE PATIENTS WITH THE LITTLE BIT OF BUSULF AN AND WE INCREASE THE CELLS AND WE WAIT. ELEVEN THING WE'VE DONE IS WE WERE USING A STANDARD REGIMEN OF CULTURE OF THE CELLS AND EXPOSURE TO THE LENTIVECTOR BUT WHAT WE FOUND WAS THAT WE WERE USING AN AWFUL LOT OF VECTOR AND WE WERE BT GETTING REALLY, REALLY HIGH LEVELS OF INSERTION INTO THE CELL, SO MORE RECENTLY, WE'VE INCORPORATED USE OF SOME NEW KINDS OF SUBSTANCES THAT ENHANCE THE TRANSDUCTION, THAT IS IT INCREASES THE EFFICIENCY WITH WHICH THE LENTI VECTOR INSERTS IN THE GENOME SO I WILL TALK ABOUT THE COMPARISON OF THE OLD GROUP AND THE NEW GROUP. SO THIS IS JUST A SLIDE SHOWING THAT IF YOU USE 20% VECTOR AND GET ABOUT 1 COPY IN ABOUT HALF OF THE CELLS WHICH IS WHAT THIS MEANS SO VECTOR COPY NUMBER MEANS OVER THE WHOLE GROUP, ONLY ABOUT 50% OF THE CELLS OR LESS HAVE A SINGLE TO MAYBE 2 COPIES BUT IF WE USE THESE TRANSDUCTION--AND SO, WE NOT ONLY SAVE VECTOR BUT WE HAVE A WHOLE LOT MORE BANG FOR OUR BUCK USING THESE TRANSDUCTION ENHANCERS. SO THIS IS SORT OF A SIMPLE LIST OF THE--THESE ARE THE PATIENTS IN THE FIRST GROUP TREATED SINCE 2012 TO 16. WE TREATED 8 PATIENTS, THERE ARE VARIOUS AGES HERE, FAIR NUMBER OF CELLS INFUSED, AND YOU CAN SEE THAT THIS IS CALLED VECTOR COPY NUMBER. SO WHEN YOU GET A LOW VECTOR COPY NUMBER, LIKE .27, IT MEANS THAT ABOUT 27% OF THE CELL GOT A SINGLE COPY, AS YOU GET HIGHER AND HIGHER THIS .57 DOESN'T MEAN THAT FEIVE% GOT 1 COPY, YOU START TO GET 2 COPIES PER CELL IN SOME OF THE CELL, SO IT REALLY MEANS ONLY PROBABLY ABOUT 40% OF THE CELLS GOT TRANSDUCED BUT WITH SOME OF THEM HAVING 1 COPY AND SOME OF THEM HAVING 2 COPIES, AND SO ON. WHEN WE USE THE TRANSDUCTION ENHANCER, YOU CAN SEE 1 GETS VERY, VERY HIGH COPY NUMBERS OF CELLS. SO 1 OF THE THINGS WE DON'T KNOW S&P THIS BETTER FOR PATIENTS, IS THIS A PROBLEM WITH THAT, APPROXIMATE SO THE STUDY AS WE LOOK AT THE SECOND GROUP ASKED THE QUESTION, DO WE GET A BETTER OUTCOME WITH THESE HIGHER COPY NUMBERS AND IS THAT SAFE. SO FIRST I WILL JUST SORT OF WITHOUT BURYING YOU IN GRAPHS AND SLIDES, I WILL SORT OF DO THIS IN TEXT. MANY OF THESE PATIENTS CAN AM TO US WITH CHRONIC NORA VIRUS, IF THEY EVER GET A NORA VIRUS, IT NEVER GOES AWAY, YOU KNOW YOU READ ABOUT NORA VIRUS AND PEOPLE GETTING SICK ON CRUISE SHIPS AND SO ON, THEY EVENTUALLY GET BETTER BUT MANY OF THESE PATIENTS IF THEY GET A NEARA VIRUS,--NORA VIRUS, IT CAN MAKE THEIR DAILY LIFE PRETTY UNCOMFORTABLE SO 6 OF 7 PATIENTS TREATED IN THIS GROUP, CURED THE NORA VIRUS AFTER THE GENE THERAPY, THEY ALSO TEND TO HAVE DIARRHEA AND OTHER PROBLEMS, THEY LOSE PROTEIN INTO THEIR GET AND THEY RESOLVE THEIR PROTEIN UPON AFTER EBTER OPERATING GLOBALLYATHY, THEY MAKE LITTLE OR NO IGM AND WE FOUND 7 OF 8 PATIENTS NORMALIZE THEIR LEVELS OF IGM OTHER HALF OF THE AND AND WERE ABLE TO SEE SOMETHING. SO 1 EVER OUR PATIENT WHO IS WAS LIKE, YOU KNOW 22 YEARS OLD AND THEY TREATED EVERY 4 WEEKS OR SO OF IMMUNE O GLOBUE LYNN, HE DOESN'T NEED IT ANYMORE AND HE'S THE 1 OUT ALMOST 8 YEARS NOW AND HASN'T NEEDED IMMUNE O GLOBUE LYNN FOR ALMOST 8 YEARS NOW, AND I WON'T READ EACH OF THESE BUT EACH OF THESE ARE IMPORTANT MILESTONES SO WE'VE DONE PRETTY WELL WITH THIS GROUP OF 8 BUT NUMBER 1, THE ORIGE NAWILL TRANSDUCTION REGIMEN, THESE WERE PRETTY LOW TRANSDUCTIONS. PATIENT 8 ACTUALLY FAILED TO ACHIEVE ANY OF THE LABORATORY OF CLINICAL LANDMARKS AND THE PACE TO ACHIEVE LAB RAARE TOY CLINICAL LANDMARKS WAS SLOW AND MOST OF THE PATIENTS THIS WAS TAKING 2-3 YEARS TO OCCUR. SO, THERE WAS A SLOW INCREASE IN PERIPHERAL BLOOD NK CELLS AND ONLY IN PATIENT 1 AT YEAR 7 DID HE EVEN THEN FINALLY ACHIEVE NORMAL LEVELS OF THESE IMPORTANT NATURAL KILLER CELLS THAT ARE IMPORTANT FOR PROTECTION AGAINST VIRUSES. AND THE VECTOR MAT USED FOR THE PATIENT WAS AN ISSUE PARTICULARLY FOR ADULTS, SO WE USE THE ENHANCERS TO SEE--TO ASSURE THAT ALL PATIENTS RECEIVED PRODUCT WITH HIGH COPY NUMBER, AS I HAD SHOWN ABOVE AND REDUCE THE AMOUNT OF VECTOR USED AND TRY TO ENHANCE BOTH THE PACE OF NK CELL GENERATION AND PERHAPS EVEN THE PACE OF B-CELL CORRECTION AND IMPROVEMENT. SO THIS IS JUST AN EXAMPLE, I'M ONLY SHOWING THE FIRST 3 MONTHS SO MANY OF THESE LADDER PATIENTS ARE FURLGTER OUT AND AS YOU KNOW THE FIRST GROUP OF PATIENTS DOWN HERE IN THE LOWER ENDER FURLGTER UP BUT WHAT I WANTED TO SHOW WAS THERE AN ACCELERATED EARLY INCREASE IN THESE NK CELLS. THIS IS SORT OF BEEN SUSTAIN AS WE WENT OUT FURTHER BUT I THINK IT PROBABLY RELATES TO WHAT I'M GOING TO TALK ABOUT FURTHER DOWN WHICH IS THAT THESE PATIENTS ALSO SEEM TO IMPROVE THEIR GI SYMPTOMS A LOT SOONER. THIS IS--THIS IS JUST ANOTHER WAY TO LOOK AT THESE PERIPHERAL BLOOD NK CELLS AND THEY ALSO INCREASE THEIR NK CELLS MUCH QUICKER, SO, YOU CAN'T SEELET LOWER END OF THIS, BUT THIS IS ONLY 6 MONTHS. PATIENT 1 WAS WAY DOWN HERE BUT AT 6 MONTHS, PATIENT 9, 10, 11, 12, WAS UP HERE. WE ALSO SAW AN ACCELERATED INCREASE IN B-CELLS, SO THE B-CELLS WHICH START OUT LOW IN MANY OF THE PATIENTS COME UP VERY SLOWLY AND MAY NOT REACH REALLY NORMAL LEVELS, SOMETIMES FOR COUPLE OF YEARS IN SOME PATIENTS BUT WE HAVE QUITE A BIT MORE RAPIDLY IN USING THESE TRANSDUCTION ENHANCERS AND THIS IS THE I GM WHICH YOU CAN SEE IGM COME UP FAIRLY EARLY IN A LOT OF THESE PATIENTS AS WELL. THIS JUST SHOWS THAT PATIENT 8 WHO UNFORTUNATELY HAD HAD A FAIRLY POOR SET OF COPY--VECTOR COPY NUMBERS HAD MUCH BETTER COPY NUMBERS AFTER THE--AT 6 MONTHS AFTER THE--HE WAS RETREATED. AND I'M JUST GOING TO ACCELERATE THIS A LITTLE BIT, AND JUST SAY, THAT OF THE 7 PATIENTS WITH NORA VIRUS IN THE EARLIER REGIMEN, THE 6 WHO EVENTUALLY CLEARED THE INFECTION REQUIRED FROM 1 TO 3 YEARS POST GENE THERAPY TO ACHIEVE THAT SYMPTOMATIC IMPROVEMENT AND CURE THE NORA VIRUS AND PATIENT 8 UNFORTUNATELY NEVER CURED HIS NORA VIRUS BUT NOW WITH THE NEW REGIMEN PATIENT 9 AND 10 CURED THE MULTIYEAR CHRONIC NORO VIRUS POST GENE THERAPY AND PATIENT 11 AND 12, RETREATED PATIENT 8 WERE SYMPTOM FREE BY 3-6 MONTHS AND SOME OF THEM HAVE NOW--DON'T HAVE IT ON THIS SLIDE HAVE FIXED THEIR GENE THERAPY. SO I'M JUST GOING TO SAY WITH THIS NEW TRANSDUCTION REGIMEN, IT REQUIRES MUCH LESS VECTOR ACHIEVESUNE FIRMLY HIGHER TRANSDUCTION, HIGHER VECTOR COPY NUMBER, NOW YOU KNOW WE--WE'RE PUSHING THESE COPY NUMBERS HIGH IN ORDER TO ACCELERATE THINGS, BUT YOU KNOW WE ALSO HAVE TO REALIZE THAT THERE MAY BE SAFETY ISSUES AND WE'RE WATCHING THESE PATIENTS VERY CLOSELY. THERE'S ACCELERATED PRODUCTION OF NK CELLS, ACCELERATED INCREASED VECTOR MARKING B-CELLS, ACCELERATED SPIKE AND IGM AND ACCELERATED CLEARANCE OF NORO VIRUS AND GI SYMPTOMS AND OF COURSE LONGER FOLLOW UP WILL BE REQUIRED TO KNOW IF THIS INCREASED VECTOR MARKET USING THIS ENHANCED REGIMEN WILL PROVE TO BE STABLE AND SAFE LONG-TERM. SAFETY OF COURSE IS THE CRITICAL ISSUE HERE. I DIDN'T HAVE TIME TO TALK ABOUT A LOT OF THE JEEP THERAPY THINGS WE'RE DOING WITH OTHER SCID BUT INCLUDED ALL THE PEOPLE NOT JUST INVOLVED IN THIS, BUT IN SOME OF THE OTHER IMMUNE DEFICIENCIES THAT ARE WE'RE WORKING ON HERE. I SEE WE'RE GETTING--SINCE I HAD A LITTLE LESS TIME THAN CINDY, I DIDN'T KNOW WHETHER I COULD GO ON TO THE NEXT SECTION OF THIS OR WHETHER WE SHOULD--BECAUSE THIS IS A BREAK POINT I COULD USE HERE IF--IT DEPENDS ON THE-- >> PLEASE GO AHEAD, HARRY. >> OKAY. SO I WOULD LIKE TO TALK ABOUT OUR OTHER GENE THERAPY TRIAL WHICH IS X-LINKED SEVERE COMBINED IMMUNE DEFICIENCY, THIS IS A DISEASE WHERE PHAGOCYTES MEANING NUTRIFILLS AND MONOCYTES AND THE ACENOFILLS ARE LACKING ENZYME REQUIRED TO GENERATE SUPEROXIDE DERIVED MICROBICIDAL HYDROGEN AND BACTERIAIAL AND FUNGAL INFECTION AND THEY GET GRANUE LOAM US INFLAMMATION, THE INCIDENCE OF THIS DISEASE IS ALSO ABOUT 1 IN 200,000 BIRTHS AND ABOUT 70, THERE ARE SOME AUTOSTUDIES OF MULTIPLE ENDOCRINAL FORMS OF CGD BUT I'M ONLY GOING TO TALK ABOUT THE XOF LINKED FORM TODAY WHICH IS WHAT WE'VE BEEN DOING THE GENE THERAPY FOR, SO THERE WAS A LENTI VECTOR DEVELOPED BY OUR COLLEAGUES AT UNIVERSITY HOSPITAL LONDON IN ADRIAN THRASHERS LAB WITH OTHERRINGS AND WE WERE FORTUNATE TO INTERACT WITH THEM AND HELP THEM TO QUALIFY THIS VECTOR. AND THIS WAS A BIG TRIAL. THIS WAS--I'M NOT GOING TO READ ALL THE GROUPS EXCEPT TO SAY, THAT THERE WERE MANY PEOPLE INVOLVED FROM A NUMBER OF DIFFERENT SECTORS AND THE PLACES WHERE PATIENTS WERE TREATED, WERE UNIVERSITY COLLEGE LONDON, THE GREAT [INDISCERNIBLE] STREET HOSPITAL AT UNIVERSITY OF CALIFORNIA SAN FRANCISCO, UCLA, LED BY DR. --LED BY DON CONE AND OURSELVES AT THIS CENTER. AND AND BASICALLY,--WE PURIFIED THEM, WE TRANSDUCED THEM IN CULTURE WITH THIS LENTIVECTOR, WE TREATED PATIENTS WITH BUT SULF AN TO MAKE ROOM IN THE MARROW, WE TRANSPLANTED THESE CELLS AND THEN WE WATCHED TO SEE WHETHER WE--WHETHER PERIPHERAL BLOOD NUTRIFILLS THAT NOW COULD MAKE THE SUPEROXIDE WERE GENERATED AND WE COULD FOLLOW THAT THROUGH AN ASSAY OF FLOW CYTOMETRY ASSAY CALLED THE DIHYDRORHODAMINE, DETECTS THE YOU HYDROGEN OXIDE AND SUPEROXIDE AND BECOMES MORE FLUORESCENT SO I WILL KIND OF GIVE A QUICK STUDY OF THE SUMMARY OF THE FIRST PATIENTS, 5 WERE TREATED IN THE U.S. TRIAL, 4 IN THE UK TRIAL, 2 PATIENTS WHO WERE QUITE SICK WITH INFECTIONS AND OTHER PROBLEMS BEFORE THE GENE THERAPY PASSED AWAY WITHIN 3 MONTHS POST GENE THERAPY, 7 PATIENTS COULD BE FOLLOWED FOR GREATER THAN 24 MONTHS AND THE RESULTS OF THESE PATIENTS WERE PUBLISHED IN THE PUBLICATION I SHOW ABOVE IN FEBRUARY 2020. ONE OF THE 7 PATIENTS UNFORTUNATELY ALSO DIED AT 2.5 YEARS AFTER THE GENE THERAPY AFTER SEVERE EBOLUS PULL MONITORARY DISEASE THAT EXISTED BEFORE HAND. ONE THING YOU SEE IS THAT THE GENE THERAPY DIDN'T KILL THESE PATIENTS, THEY CAME TO STUDY WITH LOTS AND LOTS OF PROBLEMS. THIS IS A VERY SICK GROUP OF PATIENTS THAT WERE TREATED HERE. MORE RECENTLY FOR ADDITIONAL PATIENTS WERE TREATED AND I'LL DISCUSS THEM A LITTLE BIT SEPARATELY. I'M ONLY USING THIS SLIDE TO SHOW YOU THAT THERE WERE 1, 2, 3, PATIENTS, 4 PATIENTS, TREATED IN THE UK, 1 OF THE PATIENTS IN THIS SERIES WAS TREATED AT UCLA, 1 IN BOSTON, AND 1, 2, 3 IN THIS GROUP AT THE NIH IN THIS PUBLISHED SERIES. THEY ACHIEVED A PRETTY GOOD COPY NUMBER, THIS WAS A HIGH TITER VECTOR, WE DID NOT USE TRANSDUCTION ENHANCERS HERE, THIS WAS A VERY POTENT VECTOR TO BEGIN WITH AND SO YOU CAN SEE THE VARIOUS COPY NUMBERS AND THE AMOUNT OF BUSULF AN AS MEASURED BY THIS KIND OF ASSAY WAS FAIRLY SIMILAR IN ALL THE PATIENTS AND I WON'T GO INTO IT WHAT IT MEANS AN AREA UNDER THE CURVE MEANS BUT THIS IS A FAIRLY STIFF DOSE OF BUSALVATIONARMYUSA AN TO GET GOOD INGRAFTMENT OF THE MARROW IN THE PATIENTS, THIS IS THE DSHRA IN THE VARIOUS PATIENTS AND YOU CAN SEE THAT 1 PATIENT FROM THE UK WHO ACHIEVED 12% DHR POSITIVE NUTRIFILLS AT MONTH 3 IS 1 OF THE 1S THAT DIED SOON AFTERWARDS FROM PROGRESSION OF SEVERE LUNG INFLAMMATION. NUMBER 8, NIH ACHIEVED 38% AT 1 MONTH BUT HAD A VERY, VERY SEVERE FUNGAL INFECTION AND HAD INTRA CRANIAL BLOOD AT THE SITE OF 1 OF THESE FUNGAL INFECTIONS SECONDARY TO AUTOIMMUNE THROMBOCYTE PENIA, A UK PATIENT 5 ACHIEVED 35% AT 1 MONTH BUT HAD SIGNIFICANT LOSS EVER MARKING THROUGH MONTH 12. SO CAN YOU SEE THAT'S THIS PATIENT HERE. THE UCLA PATIENT, OF COURSE I MENTIONED RETAINED HIGH LEVEL, THIS IS NUMBER 6 OF MARKING BUT UNFORTUNATELY DIED OF HIS SEVERE PROGRESSIVE BOLUS FIEB ROTTIC PULL MONITORARY DEC BUT THE 5 OTHER PATES ARE ALIVE AND WELL MAINTAINING A STEADY LEVEL OF DHR POSITIVE NUTRIFILLS AND COPY NUMBER FROM THE LAST DATA POINT SHOWN THAT YOU SEE HERE ALL THE WAY TO THE PRESENT. ALL OF THESE PATIENTS ARE NOW WELL PAST 2 YEARS. SO THE SUMMARY OF THE FIRST 9 PATIENTS WERE ALIVE AND WELL, AT 24 MONTHS, THE 1 DIED FROM THE CHRONIC PULL MONITORARY DISEASE AT 2.5 YEARS, NO NEW CGD RELATED INFECS TO THE PRESENT IN THESE PATIENTS, NO VECTOR RELATED COMPLICATIONS, NO MOLECULAR CLONAL DISREGULATION, AT 24 MONTHS 6 OF 7 SURVIVING PATIENTS DEMONSTRATED PERSISTENCE OF 16-46%OX DACE POSITIVE NUTRIFILLS AND IN OTHER STUDIES WE KNOW THAT 20% POSITIVE NUTRIFILLS IS POSITIVELY CURATIVE FOR CGD AND ANYWHERE BETWEEN 10 AND TWENTIES% IS ENOUGH TO PROVIDE THE--WITH CURATIVE LEVELS OFOX DACE ACTIVITY NO LONGER REQUIRED CGD PROPHYLACTIC ANTIBIOTICS AND NONE HAVE HAD CGD INFECTIONS AND MOST OF THOSE PATIENTS ARE OUT MORE THAN 3 YEARS NOW. SO I JUST WANT TO GO OVER 1 EXAMPLE OF PATIENT NUMBER 7 OF WHAT HE CAME IN WITH, HE HAD AN ONGOING INFECTION WITH THE FUNGUS IN HIS LUNG FOR ALMOST A YEAR AND THIS JUST SHOWS PREGENE THERAPY, THIS IS THIS DHR ASSAY, AND WHAT YOU ARE LOOKING AT WHERE IT SAYS PREGENE THERAPY IS WHERE THE CELLS SHOULD BE IF YOU HAD ANYOX DACE POSITIVE CELLS, AFTER THE GENE THERAPY, ABOUT 44% OF THE CELLS NOW GENERATED SUPEROXIDE AND THIS HAS BEEN MAINTAINED, I SHOW THIS AT TWEIVE MONTHS BUT HE'S NOW WELL OUT PASSED 3 YEARS AND STILL MAINTAINING THIS LEVEL OF CORRECTION. NOW HE HAD THIS PNEUMONIA WHICH KEPT PROGRESSING DESPITE THERAPY, AND BACK IN MAY 2017, HE STILL HAD THIS AND HAD A BIOPSY JUN. 15th THAT WAS STILL POSITIVE THAT COULD STILL GROW THE FUNGUS. ON JUNE 29th, HE HAD THE GENE THERAPY AND YOU CAN SEE THAT EVEN BY AUGUST, 2 MONTHS LATE THERAPY AND WAS ALREADY STARTING TO GET BETTER AND BY 6 MONTHS OUT THIS, WAS CURED. AND JUST TO SHOW YOU SOME OF THE OTHER PATIENTS THAT HAVE BEEN TREATED SINCE, WE HAD A PATIENT 31 YEAR-OLD PATIENT WHO HAD AN ONGOING BONE-LUNG-ASPER GILLIS INFECTION AS WELL, VERY SIMILAR AND HAD A BACTERIAL INFECTION AND HE'S BEEN MAINTAINING OUT NOW ALMOST A YEAR AND A HALF, 76% DHR POSITIVE. NOW HERE'S WHERE THINGS GET A LITTLE BIT STICKY. THERE WAS A CHILD TREATED AT UCLA AND ANOTHER CHILD TREATED AT UCLA AND EYE CHILD TREATED AT BOSTON WHERE EARLY MARKING JUST PLAIN DISAPPEARED AND WE DIDN'T KNOW WHAT WAS GOING ON IN THESE CHILDREN AND TO TELL YOU THE TRUTH, WE STILL DON'T BUT WE THINK MAYBE CHILDREN HAVE A HIGHER PERHAPS BONE MARROW INFLAMMATION OR OTHER PROBLEM AND WHAT WE'RE DOING HERE IS THAT THE PROTOCOLS BEEN ON HOLD WITH RESPECT TO TREATMENT OF ADDITIONAL PATIENTS BUT WILL REOPEN SOON WITH A PLAN TO ADD ANTIINFLAMMATORY TREATMENT IN THE FORM OF THIS AGENT, PRETREATMENT AND USING SEROLOMOUS, BUT AND PERHAPS IMMUNE RESPONSE AND WE DON'T KNOW IF AND INTERESTINGLY ALL THE ADULTS SEEM TO HAVE DONE WELL, IT'S THE LITTLE KIDS WE'RE NOT DOG AS WELL WITH SO WHEN THE PROTOCOL OPENS, WE WILL FOCUS ON LITTLE KIDS FIRST AND SEE WHETHER THIS NEW ANTIINFLAMMATORY REGIMEN MAY MAKE THIS WORK AS WELL IN CHILDREN. I THOUGHT IT VERY IMPORTANT TO SHOW YOU NOT JUST THE STUFF THAT WORKS, BUT WHEN WE HAVE PROBLEMS, WHAT WE DO, HOW WE THINK ABOUT IT, WHAT WE TRY TO DO TO FIX IT, SO NOT EVERYTHING WORKS PERFECTLY, OR IT WORKS REALLY WELL IN SOME PATIENTS AND NOT IN OTHERS. AND THAT'S WHERE WE ARE WITH THIS TRIAL WE--WE HOPE TO OPEN SOON AND WE HOPE TO MAKE THIS NOT JUST WORK IN ADULTS WITH BUT WORK IN CHILDREN WHERE THIS WOULD BE VERY IMPORTANT AND I AM GOING TO STOP THERE, I DON'T HAVE TIME TO TALK ABOUT GENE EDITING AND I KNOW THE SLIDES ARE GOING TO BE SHARED AND I WILL SHARE MY WHOLE SLIDE SET, AND AS YOU CAN SEE FROM MY SLIDES MUCH OF THIS IS SELF-EXPLANATORY SO THOSE OF YOU WHO HAVE INTEREST AND MAYBE I WILL GET INVITED BACK SOMETIME TO JUST TALK ABOUT GENE EDITING WHICH IS A VERY EXCITING AREA AND I WILL STOP HERE. THANK YOU VERY MUCH. >> THANK YOU VERY MUCH. WE HAVE SEVERAL QUESTIONS THAT HAVE BEEN ASKED. [INDISCERNIBLE] WANTS TO ENFIRE ABOUT THE SAFETY FROM THE STANDPOINT OF INSERTIONAL MUTE O GENESIS WITH LENTI VIRUS, DO YOU WANT TO COMMENT BRIEFLY ABOUT THAT? >> SO I DIDN'T TALK ABOUT THAT TODAY IN GREAT DETAIL, NOT BECAUSE IT'S NOT IMPORTANT, IT IS VERY, VERY IMPORTANT, IT'S A CRITICAL ISSUE AND AND MOST SITES INCLUDING OUR OWN ARE DOING DETAILED ANALYSIS OF THE INSERT SITES TO LOOK AT WHETHER THERE ARE EXEXPANSION CLONAL OR SO IS ON. SO DETAILED, QUITE A BIT LESS DETAIL THAN WE'VE BEEN DOING ON OUR XSCID STUDY, THERE HASN'T BEEN ANY EVIDENCE OF ANY CLONAL ISSUES IN THE XSCID STUDY HOWEVER, WE HAD SEEN SOME EVIDENCE SO WE SEE FOR EXAMPLE EXCESS CLONES AND THIS WAS PUBLISHED BY DR. RAVEN SHE REPORTED THAT IN THE REPORT OF 2016 SCIENCE TRANSLATIONAL MEDICINE THAT THERE WAS A MODEST TO MODERATE INCREASE IN INSERTS IN THE GENE CALLED HMGA 2. WE'VE BEEN TRYING TO TRACK THAT DOWN AND IN A FEW PATIENTS, CLONES THAT HAVE INSERTS IN THE HMGA2 ARE A BIT MORE PROMINENT AND ARE GROWING OUT AND WE'VE PUT A HUGE AMOUNT OF EFFORT INTO UNDERSTANDING THAT AND WE FOUND IS THAT IT APPEARS THERE MAY BE A KRIOF THETIC SPLICE SITE FOR THE SECTOR THAT ESSENTIALLY CAPTURES THE PRODUCTION OF GENES, LENTI VECTORS ALREADY INSERT ALL OF THE GENOME SO IN THEORY THEY LAND IN ALL SORTS OF GENES AND CAUSE DISRUPTIONS, FORTUNATELY WE'VE SENIOR NO ABNORMALITIES IN THE HEMATOPOIESIS OF ANY OF THESE PATIENTS AND ANY AND WE ARE PUTTING A LOT OF ATTENTION INTO THAT PARTICULAR VECTOR WHICH MAY HAVE A CRYPTIC SPLICE SITE AND MAY IN SOME GENES CAPTURE THE RNA THAT'S BEING PRODUCED BY THE GENE OHM BE AN ISSUE HAD THE NMG2 BUT NOTHING ELSE. >> THERE'S ALSO A QUESTION IF SNAWN STEM CELL BASED REQUIRE GRAFTING FOR PERSISTENCE. >> SO I THINK THE QUESTION BEING ASKED HAS TO DO WITH ARE THERE OTHER CELLS WE CAN USE? FOR EXAMPLE, COULD YOU--IN SOME DISEASES COULD WE JUST CORRECT THE T-CELLS AND PUT THEM BACK, IN A WAY THIS IS WHAT CAR T-CELL TREATMENTS FOR CANCERS ARE ALL ABOUT. YOU ARE PUTTING IN A GENE JUST IF THE T-CELLS AND INJECTING THOSE T-CELLS WITH A--WITH SOMETHING THAT WILL BIND TO, TO CAUSE T-CELLS TO BIND TO AND KILL CANCER. THE 1 THAT'S BEEN MOST SUCCESSFUL HAS BEEN FOR CANCERS AFFECTING B-CELLS. AND THERE HAVE BEEN A NUMBER OF APPROACHES TO TREATING CANCERS AND B-CELLS OR T-CELLS THAT EXPRESS CERTAIN B-CELL MARKERS AND IN THOSE STUDIES, WHAT IT'S BEEN SHOWN IS THAT IT DOESN'T WORK AS WELL AS IT SHOULD UNLESS YOU USE SOME CHEMO THERAPY TO REDUCE THE PATIENTS OWN RESIDENT T-CELLS SO IT'S A SIMILAR ISSUE WHERE YOU KIND OF NEED TO MAKE ROOM AND GIVE THE CELLS YOU'RE PUTTING IN A LITTLE BIT OF A AN ADVANTAGE TO GROW. AND SO, THAT WAS A VERY GOOD QUESTION AND THAT'S WHAT CAR-T-CELL THERAPY IS ALL ABOUT, IT'S NOT TREATING HEMATPOIETIC STEM CELLS IT'S TREATING LYMPHOCYTES. >> THERE'S ALSO AN ASSOCIATED WEB CONNECTED AS TO WHETHER THE AMOUNT OF BISULFURE AN IN ORDER TO ACCOMPLISH THE GOAL THAT YOU WANT IS DEPENDENT UPON THE AGE OF THE PATIENT? >> THE QUICK ANSWER TO THAT IS ABSOLUTELY YES. SO--AND 1 NEEDS TO ADJUST THE AMOUNT OF CONDITIONING, 1 WANTS TO USE THE MINIMUM AMOUNT OF BUT SULF AN 1 NEEDS TO GET THE JOB DONE SO WITH THE XSCID TRIAL, WE ARE USING QUITE MODEST DOSES OF BUSULF AN AND WE THINK WE NEED HAD--WE NEED AT LEAST THE MODEST DOSES WE'RE USING BUT IT'S A LOT LESS THAN WE NEEDED FOR THE CHEMO THERAPY TO MAKE THE GENE THERAPY WITH CGD WORK SO I'M DPLAD I HAD A CHANCE TO TALK ABOUT BOTH OF THEM BECAUSE I DIDN'T FOCUS ON IT BUT THE AMOUNT OF CONDITIONING IS ALMOST TWICE--TO MAKE THIS WORK WITH THE XSCID AND IN THE INFANT STUDY WHICH I HAVEN'T SPOKEN ABOUT, THEY'RE USING MUCH, MUCH, LESS MAYBE HALF OF THE CONDITIONING THAT WE'RE USING IN THE OLDER CHILDREN AND YOUNG ADULTS, SO IT'S SOMETHING THAT NEEDS TO BE TAILORED PROPERLY. AND THE GENERAL PRINCIPLE IS TRY TO USE THE LEAST AMOUNT YOU NEED TO GET THE JOB DONE. >> SO IS THERE A DIFFERENCE BETWEEN THE XCIDs WHO ARE TREATED AND WHEN THEY'RE INFANTS AND THOSE WHO SURVIVE TO ADOLESCENCE OR THE LATER 1S THAT YOU ARE DEALING WITH, IS THAT SOME SORT OF SELECTION WITHIN THE DISEASE ITSELF OR IS THAT THE EFFECTIVE GOOD THERAPY WHEN THEY'RE BABIES? >> I PROBABLY--I PROBABLY DIDN'T MAKE MYSELF--I PROBABLY DIDN'T MAKE IT ABSOLUTELY CLEAR. RIGHT NOW, THE TREATMENT OF SCID PARTICULARLY XSCID IS UNDERGOINGA A REVOLUTION IN THINKING SO THAT PEOPLE ARE TRYING TO COME UP WITH A MUCH BETTER THERAPY DURING INFANCY SO THAT THEY DON'T END UP BEING OLDER CHILDREN AND YOUNG ADULTS WHO NEED SOMETHING MORE LIKE WE'RE TREATING THEM WITH, AND SO FOR EXAMPLE, OUR PATIENTS IN THEIR 20S, THERE WAS NO OTHER OPTION. THEY EITHER DIED OR GOT THE KIND OF HAPLO-TRANSPLANT WITHOUT CONDITIONING THAT THEY GOT 20, 25 YEARS AGO AND NOW THEY'RE INCOMPLETELY IMMUNE RECONSITUTED AND SOMETIMES IN SOME CASES LOSING SOME OF THAT AND HAVE CHRONIC MEDICAL PROBLEMS AND WE'RE TRYING TO SALVAGE THEIR IMMUNE SYSTEM USING THE GENE THERAPY BUT THE IDEA WOULD BE TO TREAT EVERYBODY AS INFANTS WITH A CURATIVE APPROACH, WHETHER IT BE GENE THERAPY OR TRANSPLANT WITH CONDITIONING SO THAT THEY DON'T END UP ONLY PARTIALLY IMMUNE RESPONSE CONSTITUTED. >> JEFF ASKS, WHETHER--OH, YOUR INSIGHT A QUESTION REGARDING PROTOCOLS AND DEVELOPMENT OF STABLE IPS CELLS. THE SOURCES ARE ELDERLY PATIENTS CD34 CELLS, WHAT'S YOUR INSIGHT ON LENTIVECTORS, TRANSDUCTION ENHANCERS VERSUS THE USE OF PLASMIDS? >> LET ME TRY TO PARSE THE QUESTION. USE THE WORD INDUCE PLURIPOTENT STEM CELLS WHICH IS AN ENTIRELY DIFFERENT KIND OF STEM CELL ISSUE. ALTHOUGH I COULD HAVE TALKED ABOUT IPS CELLS TODAY, THAT WASN'T THE TOPIC OF MY CONVERSATION AND THOSE ARE A DIFFERENT KIND OF STEM CELL THAT CAN GIVE RISE TO--THEY'RE THE MOST PRIMITIVE STEM CELLS AND RIGHT NOW, THERE'S A LOT OF HOPE THAT THEY MAY BE USED FOR THERAPY BUT THEY'RE NOT REALLY THE MAIN 1S. AND THAT ISN'T WHAT I'M TALKING ABOUT TODAY. BUT, I'M SORRY COULD YOU REPEAT THE LATTER PART OF THAT QUESTION? >> THE LATTER PART WAS YOUR INSIGHT ON LENTI VECTOR TRANSDUCTION ENHANCERS, VERSUS THE USE OF PLASMIDS? >> SO THE REASON WE USE LENTI VECTORS IS THAT WE'RE ESSENTIALLY HARNESSING AN EXTRAORDINARILY EFFICIENT SET OF MACHINERY THAT IF WE TRY OURSELVES TO INVENT IT FROM SCRATCH, WE'RE AT A LOSS. MAYBE SOMEDAY WE CAN INVENT THINGS THAT CARRY PLASMIDS INTO THE--INTO THE CELLS AND THOSE PLASMIDS HAVE THE ELEMENTS THAT ALLOW IT TO EFFICIENTLY INSERT INTO CELLS BUT RIGHT NOW MOST PLAZ MIDS MIDS IF YOU JUST USE PLASMIDS AND USE CALCIUM PHOSPHATE OR ELECTROPRORATION OR WHATEVER, TO GET THAT IN, IT ISN'T THAT YOU DID CAN'T GET THE PLASMIDS TO INSERT, IT'S JUST THAT THE INSERTION RATE INTO THE GENOME IS A THOUSAND FOLD LESS EFFICIENT THAN WITH A LENTIVECTOR. SO, WE USE THESE BECAUSE THEENTIOUS FICIENCYS OF USING JUST PLAIN D NA PLASMIDS IS EXTRAORDINARY LOW. >> LET'S SEE. NTHERE'S A QUESTION, CINDY WITH TET2 ACTIVITY AND APLASTIC ANEMIA IS IT INCREASED OR IN OGLI-BONE MARROW FAILURE PATIENTS? >> YOU KNOW IT'S ACTUALLY THE TET 2 STORY WAS IN REGARD TO AGED HEMATOPOIESIS. SO IN A LARGE SEQUENCING STUDY THAT NEAL YOUNG'S GROUP DID COLLABORATIVELY WITH A GROUP IN JAPAN ON LOOKING FOR ACQUIRED SOMATIC MUTATIONS AND IN APLASTIC ANEMIA PATIENTS THEY DID FIND QUITE A HIGH RATE OF DMT 3A, BSXL1 AND FEW OR CORE JEERNS BUT THEY DID NOT FIND TET2. AND IT'S INTERESTING, IT GOES ALONG WITH CERTAIN MUTATIONS SEEM TO HELP, YOU KNOW TO GROW OUT OR TO DOMINATE IN THE SETTING OF CERTAIN MICROENVIRONMENTAL OR INFLAMMATORY SITUATIONS AND OTHER 1S, YOU KNOW MAY BE FAVORED IN A DIFFERENT SITUATION THAT WE DON'T YET UNDERSTAND BUT IT'S INTERESTING THAT YOU KNOW THE APLASTIC ANEMIA PATIENTS HAVE THE MUTATIONS AT A MUCH YOUNGER AGE IF THEY HAVE BONE MARROW FAILURE AND I THINK IT'S ABOUT LAWN MOWER I SAID EARLIER ABOUT CERTAIN MUTATIONS ALLOWING STEM CELLS TO SURVIVE IN A KIND OF VERY UNFORGIVING ENVIRONMENT. AND DMT3A REALLY SEEMS TO DO THAT. WHY TET2 IS NOT PRESENT IN THESE PATIENTS, YOU KNOW I DON'T KNOW. BUT IT IS INTERESTING AND A LOT MORE TO LEARN. THE OTHER CONFUSING THING ABOUT TET2 AND DMT3A IS THAT THEIR ACTIONS ARE OPPOSITE ON METHYLATION, IT'S REQUIRED FOR DNA METHYLATION AND TET2 IS REQUIRED FOR DEMETHYLATION AND WHY THEY HAVE SOME CHARACTER IEOF THETIC THATION SEEM TO BE SHARED WHEN YOU HAVE GET LOSS OF FUNCTION IS QUITE CONFUSING AS WELL, AND THE ACTIVITY OF TET2 ACTIVITY MAY HAVE NOTHING TO DO WITH THE DEMETHALATING CHARACTERISTICS. BUT ANYWAY. >> WELL, WE HAVE RUN OUT OF TIME. BUT I WISH TO THANK BOTH OF YOU ON BEHALF OF EVERYONE LISTENING FOR REALLY GIVING US A GLIMPSE INTO AN INCREDIBLY EXCITING AREA OF SCIENCE. THANK YOU AGAIN.