GOOD AFTERNOON, LADIES AND
GENTLEMEN OF THOSE OF YOU WHO
BRAVED THE CONDITIONS TO BE IN
THE HE AUDITORIUM THOUGHT YOUR
DESK WOULD BE COZIER SPOT.
MY PLEASURE THIS AFTERNOON TO
INTRODUCE OUR SPEAKER, STUART
ORKIN.
WHO IS THE DAVID G. NATHAN
PROFESSOR OF PEDIATRICS AND
CHAIR OF THE DEPARTMENT OF
PEDIATRIC ONCOLOGY AT THE DANA
FARBER CANCER INSTITUTE.
HE IS MANY YEARS BEEN ONE OF THE
PREEMINENT FIGURES IN THE STUDY
AND HEMOGLOBIN DISORDER.
GRADUATING FROM HARVARD MEDICAL
SCHOOL DR. ORKIN BEGAN RESEARCH
CAREER HERE AT THE NIH IN THE
LABORATORY OF DR. PHILLIP LEADER
WHERE HE STUDIED PROPERTIES OF
ERYTHROCELL LINES.
THAT INTEREST EXPANDED ON HIS
RETURN TO HARVARD.
WHEN HE BEGAN TOO EXAMINE GLOBIN
GENE ORGANIZATION IN THE ALPHA
AND BETA CELL.
AND DEVISE IMPROVED ASSAY FOR
PRENATAL DIAGNOSIS.
IN THE FOLLOWING YEARS HIS GROUP
PUBLISHED MANY PAPERS DEALING
WITH THE SICKLE CELL ANEMIA
INCREASINGLY WITH WAYS TO TREAT
THESE DISORDERS.
CHEMICAL METHODS WERE
UNSATISFACTORY BECAUSE THEY HAVE
UNDESIRABLE SIDE EFFECTS.
APPROACHES THAT WERE USED IN THE
FIELD INCREASINGLY CENTERED ON
REACTIVATION OF THE FETAL GLOBIN
GENE WHICH WOULD REPLACE OR
COMPLEMENT MUTATED ADULT GLOBIN
GENE.
AS PROFESSOR ORKIN UNDERSTOOD
QUITE EARLY ON, THE BEST
SOLUTION WERE LIKELY TO ARISE
FROM UNDERSTANDING OF GLOBIN
GENE REGULATION.
THE CLONING BY HIS LABORATORY
WAS IMPORTANT STEP IN THIS
DIRECTION.
IT'S A MASTER GENE THAT
REGULATES MOST OF THE EVENT, IS
THAT PRODUCE RED CELLS FROM STEM
CELLS IN BONE MARROW.
SINCE THEN DR. OTHER KIP AND HIS
COLLABORATORS CLONED OTHER GENES
INVOLVED IN RED AND WHITE CELL
DEVELOPMENT AND STUDIED THEIR
FUNCTION IN RECENT YEARS
INCREASINGLY IN STEM CELLS.
CONSTANT PURSUIT OF THE GOAL OF
REACTIVATING HEMOGLOBIN WAS
REWARDED IN 2008 WITH HIS
DISCOVERY OF THE BCL11A INHIBITS
GLOBIN PRODUCTION.
BEGINNING WIDESPREAD EFFORT
EFFORT IN THE FIELD.
THIS IS RELATED APPROACH MAY
SOON PROVIDE EFFECTIVE METHODS
FOR TREATMENT OF SICKLE CELL
DISEASE AND BETA THALASSEMIA.
A LARGE PART HAS BEEN DEVOTED TO
THESE PROBLEMS ALSO MADE
IMPORTANT CONTRIBUTIONS TO
UNDERSTAND THE MECHANISMS THAT
MAINTAIN POTENCY IN EMBRYONIC
STEM CELLS AND ANALYSIS OF
VARIOUS CANCERS WITH BONE AND
BREAST CANCER AND CANCERS OF THE
HEMATOPOIETIC SYSTEMS.
DR. ORKIN'S INFLUENCE AND IMPACT
ARRIVED NOT ONLY FROM THESE
MAJOR ACCOMPLISHMENTS BUT FROM
THE LARGE NUMBER OF HIS STUDENTS
THAT HAVE BECOME DISTINGUISHED
INVESTIGATORS AND -- OUR
UNDERSTANDING OF HEMATOPOIESIS
WOULD BE MUCH LESS COMPLETE
WITHOUT HIS WORK WE WELCOME HIM
BACK TO THE NIH WHERE HE
STARTED.
[APPLAUSE]
>> THANKS FOR THE KIND
INTRODUCTION.
I HAVE POST VIRAL COUGH IF I
COUGH IN THE MIDDLE, JUST IGNORE
ME.
I'LL GET THROUGH THIS.
WHAT I'D LIKE TO DO IS WHAT
WE'VE BEEN DOING RECENTLY TRYING
TO UNDERSTAND FETAL GLOBINS WHAT
WE MIGHT BE ABLE TO DO ABOUT IT.
THIS REALLY TRACES THE LAST
PROBABLY SIX YEARS WORTH OF WORK
IN ONE ASPECT IN THE LAB.
IT REALLY DOES BEGIN HERE WHILE
I WAS AT THE NIH, IN BUILDING
SIX.
I BEGAN STUDYING RED BLOOD CELLS
AND DIFFERENTIATION NOT KNOWING
THAT THIS WOULD BE THREE DECADES
LONG PROCESS.
I THOUGHT IT WAS ONLY GOOD FOR
TWO YEARS AT THE NIH.
THIS IS STANDARD VIEW OF
HEMATOPOIESIS IN THE MOUSE IN
WHICH TRANSITION DELIVER
ULTIMATELY TO THE BONE MARROW
DURING DEVELOPMENT.
AND OBVIOUSLY VERY DYNAMIC
SYSTEM AND TREMENDOUS INTEREST
TO TRY TO UNDERSTAND WHAT IT IS
THAT ALLOWS STEM CELLS HE TO
RENEW AND PROGENITORS TO DEVELOP
AT EACH STAGE.
FOR THE PURPOSES OF THIS TALK
I'M GOING TO FOCUS REALLY ONLY
ON THIS PORTION OF THE PROBLEM
AND THAT IS, HOW IT IS DURING
RED CELL DEVELOPMENT WITH
RELEVANCE, WE ACTUALLY SWITCH IN
THE HUMAN FROM EMBRYONIC TO
FETAL TO ADULT.
AND WE'VE KNOWN THE STRUCTURE OF
THE LOCUS, BETA GLOBIN LOCUS FOR
MORE THAN 0 YEARS.
IN FACT THE DNA SEQUENCE FOR
MORE THAN 30 YEARS NONE OF WHICH
REALLY TREMENDOUS INSIGHT IN TO
THIS SWITCH.
THIS SWITCH IS IMPORTANT BECAUSE
THE BETA IN SICKLE CELL ANEMIA
MAKES ONE DEPENDENT IN THE BETA
CHAIN LIFE.
THE GAMMA CHAIN WHICH IS PART OF
FEELINGS HEMOGLOBIN IS SILENCED
BUT NOT TO ZERO, SILENCED TO A
COUPLE PERCENT IN THE NORMAL
ADULT INDIVIDUAL.
WE'VE BEEN INTERESTED IN HOW RED
CELLS AS WELL AS OTHER
HEMATOPOIETIC CELLS ARE MADE,
HOW THEY'RE MADE.
HOW THEY GO AWRY IN DISEASE, IN
MALIGNANT OR NONMALIGNANT
DISEASE.
IN TERMS OF THINKING ABOUT FETAL
VERSUS ADULT.
ONE CAN ASK, WHY STUDY IT?
WELL, FIRST, IT'S REALLY A
PARADIGM FOR DEVELOPMENTAL STAGE
AND TISSUE SPECIFIC GENE
EXPRESSION.
THE GENES ARE EXPRESSED.
WHY SHOULD WE REALLY CARE.
FROM DISEASE STANDPOINT, ONE IS
SICKLE CELL DISEASE WHICH IS
FIRST DESCRIBED MORE THAN A
HUNDRED YEARS AGO BY
CARDIOLOGIST.
AND WHERE HE FIRST NOTICED THE
SICKLE CELLS.
THEN IDENTIFIED THE AMINO ACID
CHANGE.
THEN THERE'S THALASSEMIA.
NOT THE STRUCTURE.
IN TERMS OF SICKLE CELL DISEASE,
KNOWN MUTATION FOR MORE THAN 50
YEARS, YET I THINK IT'S SOBERING
THAT NONE OF THIS INFORMATION
REALLY HAS ANY BEARING ON
CURRENT THERAPY.
I THINK THERE'S A STATEMENT,
CHALLENGES INVOLVED PERHAPS
WHERE WE NEED TO GO.
IN TERMS OF THINKING ABOUT THE
HEMOGLOBIN DISORDER,
PARTICULARLY SICKLE CELL
DISEASE, IN THIS COUNTRY THERE
ARE ABOUT 100,000 INDIVIDUALS
BUT WORLDWIDE MANY MORE.
IN THE AFRICAN CONTINENT THERE
ARE ABOUT 300,000 A YEAR.
PREDICTED OVER NEXT SEVERAL
YEARS THERE WILL BE UPWARDS OF
FIVE TO SIX MILLION CHILDREN
WITH SICKLE CELL DISEASE IN THE
AFRICAN CONTINENT.
IF ONE ASSUMES BETTER CONTROL
MALARIA, HIV, CHILDREN WHO DIE
YOUNG WITH SICKLE DISEASE ARE
GOING TO LIVE LONGER AND PRESENT
GREATER CHALLENGE TO THE HEALTH
SYSTEM.
THIS IS A GLOBAL PROBLEM WHICH
IS NOT GOING AWAY AT ALL.
WE'VE KNOWN FOR MANY YEARS THAT
IF ONE IS FORTUNATE ENOUGH TO
HAVE HIGHER LEVEL OF FETAL
HEMOGLOBIN, HAVE SICKLE CELL
DISSEE OR THALASSEMIA HAS LESS
OF THE DISEASE, THIS IS FROM THE
NATURAL HISTORY STUDY OF THE NIH
SHOWING HEMOGLOBIN PREDICTS A
HIGHER -- GREATER SURVIVAL.
THIS IS TRUE IN THALASSEMIA AS
WELL.
WHAT LEVEL WOULD BE CURATIVE,
WHAT LEVEL OF FETAL HEMOGLOBIN,
CUT OFF IS ABOUT 8.6%.
SOMEWHERE IN THE RANGE OF ABOUT
15 OR 20% HEMOGLOBIN IF
DISTRIBUTED IN ALL CELLS, WOULD
HAVE TREMENDOUS BENEFICIAL
EFFECT.
AGENTS OF SICKLE CELL DISEASE IS
HYPOXIA UREA.
HYDROXY UREA GETTING CHILDREN
THROUGH CHILDHOOD YEARS REALLY
PRESENTING ADULT PHYSICIANS WITH
PATIENTS SICKLE CELL DISEASE
WITH INTENDED CONSEQUENCES.
SO WHAT DO WE DO NOW?
BASICALLY WE FOCUS ON THE
CONSEQUENCES OF THE SICKLE
MUTATION, ANEMIA, PAIN, STROKE,
PULMONARY DISEASE, WHERE WE
REALLY LIKE UP HERE, FOCUS OUR
THERAPY AT THAT POINT REALLY
ONLY TWO WAYS TO THINK OF DOING
THAT.
ONE IS CORRECTING THE SICKLE
MUTATION, PERHAPS THROUGH
EDITING TECHNIQUES WHICH AT THIS
POINT, I THINK FOR THE
FORESEEABLE FUTURE NOT PRACTICAL
AS EFFICIENCY WAS NEEDED.
ALTERNATIVE TO SOMEHOW PREVENT
SICKLE HEMOGLOBIN FROM
POLYMERIZING AND SICKLING BUT
MAY BE FEASIBLE IS SMALL
MOLECULES.
OR ALTERNATIVELY INCREASING THE
LEVEL OF FETAL HEMOGLOBIN TO
REPLACE SICKLE HEMOGLOBIN IN THE
CASE OF BETA THAL SEEM YUM.
THALASSEMIA.
ALLOW FOR BETTER HEMOGLOBIN.
THIS IS AGAIN THE PROBLEM, THE
SWITCH FROM GAMMA TO BETA, HOW
DO WE UNDERSTAND IT, WHAT DO WE
KNOW ABOUT IT.
REALLY TWO ISSUES ONE HAS TO
DISTINGUISH REALLY AT THE
OUTSET.
SOMETIMES GET CONFUSED THAT IS
SWISHING FETAL VERSUS ADULT GENE
EXPRESSION IN ERYTHROID CELLS.
THEY EXPRESS ABOUT A THOUSAND
DIFFERENT TRANSCRIPTS.
SOMETHING DIFFERENT OF THE
FETALNESS OF THE CELL, WHAT KIND
OF GLOBIN, FETAL OR ADULT
GLOBIN.
MIGHT THINK IT'S THE SAME
PROBLEM.
BUT WE THINK TRANSCRIPTIONAL
LEVEL, AT LEAST AS FAR AS WE
UNDERSTAND IT, REALLY FOR THE
MOST PART DISTINGUISHABLE.
THE SORT OF FETAL PROGRAM, WHOLE
DEVELOPMENTAL PROGRAM IS REALLY
REGULATED THROUGH A SET OF
SPECIFIC ENHANCERS IF GENES THAT
EXPRESS IN SPECIFIC MANNER.
AS OUR DATA TO DATE SUGGEST
CONTROL GLOBIN CHANGE IS LARGELY
MORE LOCUS SPECIFIC AND REALLY
INDEPENDENT OF THE FETAL STAGE.
IN OTHER WORDS, WHEN ONE TALKS
ABOUT REACTIVATING FETAL
HEMOGLOBIN TALKING ABOUT
REACTIVATING THE FETAL GENE,
GAMMA GENES, NOT TALKING ABOUT
DRIVING THE CELLS BACK FOR A
MORE FETAL STATE.
SOMETIMES THEY GET CONFUSED.
ONE OF THE SYSTEMS IS VERY
CONVENIENT IT HAS IT'S FLAWS BUT
VERY CONVENIENT TO STUDY IS THE
USE OF PRIMARY CD34 CELLS WHICH
CAN BE EASILY MOBILIZED IN
ADULTS, CAN BE EXPANDED DIFFER
IN SHATED TO FORM ERYTHROCELLS,
EVEN CELLS WITH TRANSPLANTATION.
ONE CAN OBTAIN EITHER FROM
ADULTS OR FROM FETAL LIVER, ONE
CAN HAVE FETAL CELLS OR ADULT
CELLS IN THE FETAL CELLS RETAIN
THEIR FETAL GLOBIN GENE
EXPRESSION AND GENE PROGRAM,
SHOWN HERE WITH EXPRESSION OF
GAMMA, ADULT CELLS EXPRESS THE
ADULT.
WE CAN USE THIS SYSTEM BY
CHEMISTRY, PROTEOMICS, GENE
EXPRESSION, ANY SORT OF
TECHNOLOGY ALLOW TO DISTINGUISH
WHAT IS DIFFERENT 'GOUT GENE
EXPRESSION AND CONTROL.
I'LL JUST SUMMARIZE WHAT WE'VE
LEARNED ABOUT THE FETALNESS
ASPECT.
THAT IS A SEPARATE TOPIC.
THIS IS ONE WAY OF LOOKING AT
SOME OF THE WAYS CELLS ESTABLISH
THEIR FATE IN EPIGENETIC
REGULATORS AND DRIVERS.
WHICH DEFINE THE CELL IDENTITY.
WHAT WE FOUND FOR THE MOST PART
IS THAT THE FETALNESS,
SPECIFICITY, PROGRAMMED BY STATE
SELECTED COFACTORS NOT
PROGRAMMED BY THE MASTER
TRANSCRIPTION FACTORS, IT'S
PROGRAMMED BY COFACTORS THAT
DEVELOPMENTALLY REGULATED
THEMSELVES.
MAY BE USED WIDELY IN OTHER
SETTINGS.
EXAMPLES OF THAT WOULD BE THE
ADULT SPECIFIC HEMATOPOIETIC OR
THE INTERFERON REGULATORY
FACTORS.
WHAT WE'VE LEARNED IS LARGELY
ATTRIBUTABLE TO THESE ENHANCERS
AND FACT IS THAT INTER-ACTION
WITH THEM.
THESE ARE UNRECOGNIZED COFACTORS
OR PREVIOUSLY WERE UNRECOGNIZED.
I THINK THERE IS STILL MORE TO
BE DISCOVERED.
THE MECHANISM ACCOUNTS FOR
OVERALL SPECIFICITY, PROVIDE
TREMENDOUS INSIGHT IN TO THE
EXPRESSION OF THE FETAL OR ADULT
GLOBIN IS NOT COUPLED TO STATE
SPECIFICITY.
WHAT DO WE KNOW ABOUT THE
CONTROL OF FETAL VERSUS ADULT.
THIS IS ONE OF THE FIRST
RATIONAL DESCRIPTIONS IN THE
LITERATURE AGAIN FROM ENGLAND IN
1961.
WHICH HE IMAGINED ONE SWITCHED
DOWN EXPRESSION OF THE GAMMA AND
SWITCHED ON BETA CHAIN, ALPHA
CHAIN IS CONSTANT.
THIS SOUNDS SIMPLE TODAY BUT
PRIOR TO THIS LITERATURE THEY
WERE RATHER CONFUSING MODELS
BECAUSE AT THE TIME IT WAS
UNCLEAR HOW MANY GENES THAT WERE
COAT IN THESE PROTEINS EVEN WITH
THE ORGANIZATION.
LONG TIMELINE FROM UNDERSTANDING
THIS SWITCH.
GANNETT FICK STUDIES OF FAMILIES
LARGELY IN THIS PERIOD BETWEEN
'50s AND '70s WHERE FIND
THAT HEMOGLOBIN WAS INHERITED
TRAIT, LARGELY CONTROLLED AT THE
GENETIC LEVEL.
THERE WERE FAMILIES THAT HAD
INCREASED FETAL HEMOGLOBIN SOME
OF THE MUTATIONS, GLOBIN GENES
WERE FIRST AROUND 1980 WITH
SEQUENCING SOON THEREAFTER.
THAT INFORMATION NOT
PARTICULARLY HELPFUL IN TERMS OF
UNDERSTANDING THE SWITCH,
PER SE.
WE WERE STUCK ON THE PROBLEM.
WANDERING IN THE DESERT AND
DIDN'T KNOW WHERE TO GO.
THE FIELD CAME TO A HALT WITH
THE INTRODUCTION OF GENOME-WIDE
ASSOCIATION.
SITUATION HERE IS LOT SIMPLER WE
CAN TRACE THINGS DOWN AS YOU'LL
SEE ESSENTIALLY TO THE
NUCLEOTIDE LEVEL.
GENOME-WIDE ASSOCIATION IS
MEASURED OR F CELLS WHICH IS A
MEASURE OF YELLS IN THE CONTAIN
INCREASED AMOUNTS OF F.
THESE WERE DONE BOTH IN THE U.K.
AND THERE WERE THREE SNPs IN
THE MANHATTAN PLOT.
THIS THE EM PEER STATE BUILDING
WHICH IS WHAT ONLY THING I'LL
CONIS HE TRAIT ON.
THERE WERE THREE HITS.
ONE IS CHROMOSOME TWO, THE LOCUS
ON CHROMOSOME SIX AND BETA
GLOBIN LOCUS, ALREADY KNOWN THAT
GENETIC VARIATION IN THE BETA
GLOW BIN LOCUS LEADS TO
HEMOGLOBIN F LEVELS.
MANY CASES NOT UNDERSTOOD WHY
EXACTLY.
IDENTIFIED PRIOR TO THIS, AT
LEAST THAT REGIONAL GENOME.
AND WAS A NEW KID ON THE BLOCK.
SOME CONTRIBUTION OF THESE AT
LEAST 50% OF THE GENETIC
VARIATION OF HEMOGLOBIN F.
THESE CONTRIBUTE TO A LARGE
PERCENT OF THE GENETIC
VARIATION.
ALSO THE OTHER CURIOUS
OBSERVATION IS THAT THESE --
THIS VARIATION OCCURS ACROSS ALL
POPULATIONS.
IT'S NOT LIMITED TO PARTICULAR
ETHNIC OR GEOGRAPHIC GROUPS.
IT'S WORLDWIDE DISTRIBUTED.
THIS IS WHAT ORIGINAL BETA LOOKS
LIKE ON THE NIH SICKLE CELL
COHORT.
HERE THE HEMOGLOBIN F
INDIVIDUALS WITH MEAN OF 4%.
HIGHS OF ABOUT 11%.
LOT OF STRATA IN THE DATA, IT'S
NOT PREDICTED IN ANY WAY IN A
SINGLE INDIVIDUAL.
BUT YOU CAN SEE THAT THERE'S A
DIFFERENCE OF ABOUT 4 HIGHWAY
11% ALMOST TWOFOLD DIFFERENCE IN
THE BASAL HEMOGLOBIN F LEVEL
DEPENDENT ON THE GENOTYPE AT THE
GENE.
WE'LL COME BACK TO THIS LATER TO
WHAT THIS MEANS.
I THINK IT'S IMPORTANT.
EVERYTHING WE'VE LEARNED IN THE
ENSUING FIVE YEARS OR SO, SAYS
THAT BCL11A IS QUANTITATIVE
REGULATOR OF HBF.
CONVERSELY DECREASE AMOUNT OF 1A
DECREASE HEMOGLOBIN F.
I'LL JUST SUMMARIZE SOME DATA
THAT IS ALREADY PUBLISHED THAT
SUPPORTS THIS, THE GENERAL
NOTION THAT 11A ACTS DIRECTLY IN
THE LOCUS BY PRECIPITATION OF
CELLS.
INTER-ACTS WITH THE LCR.
IT'S FOUND OVER THE EMBRYONIC
GENES AND BETWEEN GAMMA AND
DELTA IN THIS REGION WHICH IS
ASSOCIATED WITH THE KNOWN YIELD
HIGHER F IN INDIVIDUALS.
KNOWN FOR MANY YEARS.
CURIOUSLY DOES NOT BIND OVER THE
GENES.
IT DOESN'T REPRESS THE PROMOTERS
DIRECTLY.
COULD ACCESS LONG DISTANCE.
IF YOU TAKE THE CD34 CELLS I
DESCRIBE, DIP IT EXPRESSION TO
THE PROTEIN OF SHRNA YOU REDUCE
GAMMA AND FETAL HEMOGLOBIN.
THIS IS NOT ENORMOUSLY SPECIFIC.
USES AMOUNT OF IT KNOCKED OUT
INCORPORATES IN TO THAT MOUSE A
HUMAN TRANSGENERAL THAT
EXPRESSIONS THE HUMAN CLUSTER IN
THE DEVELOPMENTALLY APPROPRIATE
MANNER OF THE MOUSE.
WHAT HAPPENS IS GAMMA GENE WHICH
IS NORMALLY EXPRESSED TO SHUT
OFF AROUND BIRTH IN THE HUMAN
BUT SHUT OFF IN THE FETAL LIVER
IN THE MOUSE NO LONGER SILENCE
APPROPRIATELY IN THE
HETEROZYGOTE IN BETWEEN.
I SHOULD JUST MENTION THOSE WHO
AREN'T AWARE, THE MOUSE HAS NO
FETAL EQUIVALENT.
EMBRYONIC ADULT.
THE HUMAN FETAL GENE IS SORT OF
AN UNKNOWN COMMODITY.
HAS TO BE HANDLED EITHER AS
ADULT OR EMBRYONIC GENE.
FINALLY IF ONE USES TRADITIONAL
MOUSE, THE HUMAN TRANSGENE TO BE
REPRESSED AND REPRESSED ABOUT
TWO ORDERS OF MAGNITUDE GREATER
THAN, ABOUT .1% OR LESS OF THE
TOTAL BETA LIKE RNA.
IF YOU THEN REMOVE THE CONDITION
WHERE YOU GET ABOUT 500 TO 1,000
REDUCTION OF GAMMA RNA.
FAIRLY SUBSTANTIAL LEVEL.
ALL THESE DATA SUGGEST THAT THE
SWITCH IS REVERSIBLE AND LARGELY
MEDIATED TO THE D CELL.
MANY OTHER PROTEINS THAT HAVE
BEEN DESCRIBED IN THE LITERATURE
AS AFFECTING HEMOGLOBIN.
ONE COULD ASK, WELL HOW
IMPORTANT IS ANY SINGLE PROTEIN?
PROBABLY CLOSEST WE CAN COME TO
THIS AS AN EXPERIMENT TO USE
EXISTING MOUSE SICKLE CELL MODEL
IN WHICH THE MOUSE GENES, GLOBIN
GENES, HOMOLOGOUS, HUMAN GENES
ARE INTRODUCED DEVELOPMENTALLY
REPRESSED.
AND ONE CAN GENERATE A SICKLE
MOUSE, IT'S ANEMIC HAS ALL SORT
OF CONSEQUENCES AND CHALLENGES
OF THE SICKLE CELL INDIVIDUAL.
RENAL DAMAGE, ANEMIA, ET CETERA.
ONE CAN THEN ASK, REMOVES TO
AVOID ANY OTHER CONSEQUENCES
TAKE THE ERYTHROID CELL WHAT IS
THE PHENOTYPE OF THIS MOUSE.
ACTUALLY QUITE STRIKING.
MUCH CLEANER THAN WE MIGHT HAVE
ANTICIPATED.
THIS IS A CONTROL MOUSE, HERE IS
SICKLE MOUSE, HERE IS THE CROSS
WHICH WE REMOVE THE ERYTHROID
CELLS.
THE STANDARD HEMATOLOGIST IS
ENTIRELY NORMAL.
MEASURE OF YOUNG CELLS BROUGHT
BACK ALMOST THE NORMAL AND
CONCENTRATING ABILITY OF MEASURE
OF RENAL DAMAGE IS NORMAL.
INDICATING THAT THESE MICE ARE
PHENOTYPICALLY NORMAL WE'VE HAD
THEM FOR TWO YEARS AND PERFECTLY
FINE.
WHY ARE THEY WELL.
THEY EXPRESSED A LOT OF GAMMA,
RNA NOT SURPRISINGLY.
THE FETAL HEME GLOBE IS IN
DISTRIBUTED IN MOST OF THE
CELLS.
ABOUT 85% PLUS OF THE CELLS
SUSTAIN LOTS OF HEMOGLOBIN.
ONE CURIOUS ASPECT IS THAT IF
THE RED CELL, RED CELL FORMATION
AND PRODUCTION IS ENTIRELY
NORMAL.
THIS IS TRANSCRIPTION FACTOR
BEST WE CAN TELL CONTROLS VERY
LITTLE ELSE THAT'S CRITICAL TO
RED CELL OTHER THAN THE GLOBIN.
VIRTUALLY UNHEARD OF IN OUR
EXPERIENCE.
SO THIS IS REALLY WHAT WE'VE
COME TO THEN, SITUATION THAT WE
CAN IMAGINE SICKLE RED CELL
CONTAINING SMALL AMOUNT OF
HEMOGLOBIN, TARGET, IN HUB THAT
IT TARGET GENERATING A CELL THAT
HAS MORE HEMOGLOBIN F NOT
NECESSARILY 100% BUT ENOUGH
HEMOGLOBIN F TO PREVENT SICKLING
AND GREAT INHIBITOR OF THE
SICKLING PROCESS.
ONE NICE FEATURE NO RESISTANCE,
IT'S UNLIKE A CANCER TARGET,
SOME RESISTANT MECHANISM THAT
WILL CIRCUMVENT YOUR INHIBITION.
CELL REALLY SORT OF AGNOSTIC IN
ACTUALLY EXPRESSING.
WE IMAGINE SOME THERAPEUTIC
LEVEL AS WE DECREASE
CONCENTRATION, GENERATING MORE
HEMOGLOBIN F OBVIOUSLY SOME
POINT WOULD LIKE TO BE IN THE
RANGE.
WE'LL COME BACK TO THAT.
I SKIRTED OVER ONE MAJOR ISSUE
THAT IS THE GWAS ACTUALLY
IDENTIFIES THE GENE, GENETIC
VARIATION IN THE LOCUS SOMEHOW
ULTIMATELY AFFECTS EXPRESSION OF
THE HEMOGLOBIN.
HOW COULD THIS BE?
ONE EASY SOLUTION IT COULD HAVE
BEEN A CHANGE IN THE STRUCTURE
OF THE PROTEIN, JUST SUFFICE IT
TO SAY THAT PROTEIN IS NORMAL,
ALL GENETIC VARIATIONS OCCURS
WITHIN A LARGE INTERVENING
SEQUENCE WITHIN THE GENE.
THIS IS DEPICTION OF THE GENE.
THE CHROMATIN PROFILE IN
ERYTHROID CELLS HAS HALLMARK OF
A REGULATORY ELEMENT.
MARKED WITH METHYLATION, TO
MASTER TRANSCRIPTION FACTORS.
ALL THE SNPs, HIGHLY
CORRELATED GWAS SNPs IN THIS
REGION.
IN ADDITION AS REGULATORY
ELEMENT THIS REGION IS MARKED BY
HYPERSENSITIVITY, IT'S NOT FOUND
IN OTHERS AND BASICALLY WE HAVE
THREE SITES IN THE HUMAN,
PROMOTER TO THE RIGHT.
PLUS 55, PLUS 68, PLUS 62 IN THE
GENE.
THIS IS A REGULATORY ELEMENT.
WE KNEW BEFORE THIS THAT IT'S
EXCLUSIVELY EXPRESSED IN A
SPECIFIC MANNER.
IT LOOKS AT MOUSE CELLS PRIMARY
PRIMITIVE CELLS HAVE NO
BCL11A.
YOU SEE IN THE BONE MARROW.
HUMAN IS DIFFERENT IN THAT IN
THE CELLS, EMBRYONIC CELLS
THERE'S KNOW BCL11A BUT IN THE
FETAL LIVER WITH THE SWITCH JUST
BEGIN ONE SEES SMALL AMOUNT OF
BCL11A AND MUCH GREATER AMOUNTS
IN THE BONE MARROW SHOWING THAT
THERE'S DEVELOPMENTAL PATTERN OF
EXPRESSION BUT DIFFERENCE
BETWEEN HUMAN AND MOUSE.
MOUSE REALLY ON-OFF IN THE HUMAN
IT'S OFF THEN GRADUALLY COMES
ON.
ONE OF OUR PREDICTIONS WHICH WE
HAVEN'T YET PROVED IS THAT THIS
DEVELOPMENTAL DIFFERENCE MAY BE
RESPONSIBLE IN PART FOR
DIFFERENCE OF SWITCHING BETWEEN
MOUSE AND HUMAN.
THE BCL11A GENE IS
DEVELOPMENTALLY REGULATED IN
ERYTHROID MANNER, FACT
REGULATORY REGION BETWEEN PLUS
55 AND 62, IF ONE TAKES THIS
REGION OUT AND LINKS IT TO A
MINIMAL PROMOTER PUTS IT IN TO A
TRANSGENIC MOUSE, THE EXPRESSED
IN THE FETAL LIVER, THE BCL11A
FIRST EXPRESSED.
BUT IT'S NOT EXPRESSED IN B
CELLS WHICH EXPRESS NORMALLY
HIGH LEVELS OF BCL11A.
REGULATORY ELEMENT FOR B CELLS.
AND IT'S EXPRESSED IN THE FETAL
LIVER BUT NOT IN THE PERIPHERAL
BLOOD AT THAT TIME WHICH
CIRCULATING PRIMITIVE CELLS.
IN FACT THIS IS AGAIN ENHANCER
THEN THAT ERYTHROID AND ADULT
STAGE SPECIFICALLY.
WE WENT BACK TO OUR GENETIC
COLLEAGUES TO LOOK AT THE SNPs
FROM GWAS THERE'S ONE SNP, THIS
ONE, WHICH IS ACTUALLY ROTATING
IN THE PULSE 62 ELEMENT WHICH IS
MOST HIGHLY CORRELATED IF ONE
DOES APPROPRIATE POSITIONING OF
THE DATA, IN FACT THAT WE
BELIEVE IS CAUSAL SNP.
THAT PARTICULAR SNP CHANGES
SEQUENCE WITHIN THE COMPOSITE
SITE WHICH IN THE FIELD IS KNOWN
AS COMPOSITE ONE SITE WHICH IS
KNOWN REGULATORY ELEMENT IN
HEMATOPOIETIC CELLS.
IN FACT THIS SNP IS ASSOCIATED
WITH CHANGE IN SPECIFIC
CHROMATIN BINDING.
SUCH AS LESS BINDING ON VARIOUS
ALLELES.
ACTUALLY LEFT RNA EXPRESSED FROM
THE ALLELE INDICATING THAT THIS
IS CAUSALLY ASSOCIATED SNP.
BUT THE IMPORTANT FINDING IS
DIFFERENCE OF EXPRESSION BETWEEN
THE NORMAL ALLELE AND VARIANCE
ALLELE IS ABOUT 40%.
THAT TELLS US THAT THE HIGH UP
ALLELE PUTS OUT ABOUT 40% LESS
BCL11A THAN NORMAL ALLELE.
JUST REMEMBER BACK TO THE
ORIGINAL GWAS OF THE SICKLE
GOING FROM 4% TO 11%.
WE THINK THAT THIS IS ACCOUNTS
FOR IT, ROUGHLY A DIFFERENCE.
GENETIC VARIATION OCCURS WITH
ENHANCEER, THIS REALLY EMERGING
THEME THE SAME TIME THAT MOST OF
THE GWAS DISEASE NON-CODING DNA
MOST OF THIS IS ACTUALLY
ASSOCIATED WITH PUNITIVE
ENHANCERS.
THIS IS SPECIFIC EXAMPLE OF
THAT.
HOW IMPORTANT IS ENHANCING?
GENETIC VARIATION MODEST AFFECT
ON EXPRESSION MAYBE 40%.
MANY GWAS, ONE OF THE CRITICISMS
OF GWAS THE EFFECT SIZE WAS
RATHER SMALL.
YOU HOW CAN THE GENE BE
IMPORTANT?
THE MODEST EFFECT MAY BE
RESTRICTED IN THE SENSE BY
NATURAL VARIATION.
WHAT'S THE TRUE ROLE OF THE
ENHANCER?
COULD IT BE -- IN ORDER TO GET
AT THAT ACTUALLY TURN INITIALLY
FROM THE MOUSE TO HUMAN CELLS AS
WELL.
BUT WE LOOKED AT THE HOMOLOGOUS
REGION IN THE MOUSE WHICH IS
DEPICTED HERE IN ERYTHROID
CELLS.
ARCHITECTURE IT'S OVERALL VERY
SIMILAR BUT IN DETAIL ACTUALLY
IT IS DIFFERENT, CENTRAL
HYPERTENSE SIEVE SITE LESS
APPARENT IN THE MOUSE THAN IT IS
IN THE HUMAN.
REMOVE THE ENHANCER.
NOW I'M SURE MOST OF YOU ARE
FAMILIAR WITH THE RAPIDLY MOVING
FIELD.
FIELD STARTED WITH CONVENTIONAL
GENE TARGETING WHICH IS LOW
EFFICIENCY NOT REALLY APPLICABLE
FOR CLINICAL USE.
PROGRESSED IN THE USE MODIFIED
FINGERS AND TALONS.
ACTUALLY FIRST USE PAIR OF
TALONS TO REMOVE THE REGULATORY
ELEMENT FROM THE CELLS.
WE DID THAT IN MEL CELLS.
IF YOU DO THAT THE EXPRESSION OF
BCL11A GOES ESSENTIALLY TO ZERO.
LESS THAN 1%.
THIS ENHANCER IS OBLIGATOR REFOR
GENE EXPRESSION.
NATURAL VARIATION MODIFIES
EXPRESSION A LITTLE, TAKING
ENHANCEER OUT.
DOES SAME EXPERIMENT IN B CELLS
IF YOU EXPRESS ABOUT TEN TIMES
MORE B CELLS THAN NORMAL AND
BCL11A IS ESSENTIAL FOR B CELL
DEVELOPMENT.
IN THE CASE OF NATURAL VARIATION
CAN REDUCE HEMOGLOBIN, MODESTLY
MAYBE 40% ROUGHLY.
THIS WILL ALLEVIATE EXPRESSION A
LITTLE, BUT REALLY DOESN'T HAVE
A MAJOR AFFECT ON SICKLE
DISEASE.
WHAT WE ARE SPECULATING, REALLY
PROPOSING IS IF REMOVES THE
ENHANCEER NO BCL11A, MAXIMAL
FETAL HEMOGLOBIN PRODUCTION TO
THE LEVEL THAT STILL HAVE NOT
YET DETERMINED AND WHEN RERELIEF
SICKLE.
THIS IS CLINICAL EXPERIMENT THAT
WAS DERIVED FROM, WE HOPE WILL
BE PERFORMED IN THE NOT TOO
DISTANT FUTURE TO COLLECT
HEMATOPOIETIC CELLS, DISRUPT
ENHANCER, ONE COULD ALSO TALK
ABOUT THE BCL11A GENE CAN
DISCUSS THAT.
INFUSE THESE TO BASICALLY CREATE
A GENETIC CURE OF THE DISEASE.
ONE REASON WE MAY NOT WANT TO
REMOVE THE BCL11A ITSELF IS
BECAUSE OF THE ROLES OF
HEMATOPOIESIS IT'S IMPORTANT B  
CELLS NOT SO IMPORTANT IN THE
GENE THERAPY CONTEXT BUT IN
TERMS OF STEM CELL CONTEXT MAY
BE MORE IMPORTANT.
THIS IS EXPERIMENT OF THE
CONDITIONAL MICE IN WHICH WE
BELIEVE TO BE BCL11A GENE AND
IMPORTANT POINT IS THAT OVER
TIME A LOSS OF CHIMERS FROM THE
MUTATED CELLS INDICATING THAT
BCL11A IS REQUIRED IN SOME
MANNER, YET UNKNOWN, REALLY
MAINTENANCE OF HEMATOPOIETIC
STEM CELLS.
MOVING THE CELL ITSELF MAY NOT
BE THE BEST APPROACH.
WE THINK THERE IS THERAPEUTIC
OPPORTUNITY THEN TO COMING IN
WITH TOOLS TO DISRUPT THE
ENHANCEER.
THIS WOULD OBVIOUSLY BE DONE EX
VIVO.
THIS STRATEGY WILL PROVIDE SORT
OF STABLE AND MAXIMAL REDUCTION
ONLY IN THE ERYTHROID, THINK
THAT HEMOGLOBIN F.
IN THIS CASE IN THE MOUSE,
SIMILAR WORK GOING ON IN THE
HUMAN WHERE WE BASICALLY
SATURATED THIS REGION USING IN
THIS CASE LIBRARY OF GUIDE RNA.
ONE CAN DO A COMPREHENSIVE
SATURATION OF THE ENHANCER WITH
REPORTER ASSAY.
BUT BOTTOM LINE IS, THAT IN THE
MOUSE -- SITUATION OF THE MOUSE
THERE'S ACTUALLY ACHILLE'S HEEL
WITHIN THE PLUS 62
HYPERSENSITIVE SITE.
BUT THINK BROADER THAN THIS
WHERE ESSENTIALLY INTERRUPTION
OF THIS REGION INACTIVATES THE
ENTIRE ENHANCER FUNCTION.
EQUIVALENT TO KNOCKING THE GENE
OUT.
NOT NECESSARY TO REMOVE THE
WHOLE ENHANCER, WE THINK
DISCRETE CUT AT THE ACHILLE'S
HEEL WILL RESULT IN SIMILAR --
FAVORABLE OUTCOME.
SAME IS TRUE IN THE HUMAN
ENHANCER AS WELL.
THE ACHILLE'S HEEL LOCATED IN A
DIFFERENT POSITION.
REALLY QUESTION THEN, HOW CAN WE
LEVERAGE ALL THIS WORK FOR SOME
SORT OF THERAPEUTIC BENEFIT.
WE THINK GENETICS NOW HAS COME
TO THE POINT WHERE WE BELIEVE
IT'S REASONABLE TO CONSIDER
EDITING, PARTICULARLY ENHANCER,
COULD PROVIDE REALLY GENE
THERAPY STRATEGY FOR INCREASING
FETAL HEMOGLOBIN IN THE
PERMANENT MANNER WITHIN
INDIVIDUALS.
I THINK MOST OF THE SCIENCE HAS
BEEN DONE, SAVE ACTUALLY
PRECISELY THE OPTIMUM SITE WANTS
TO MAKE THE EDITING.
I THINK THE CHALLENGE THAT LIES
BEFORE US, ONE THAT WE'RE BEGIN
TO FACE NOW IS THAT IF ONE
REALLY GOING TO IMPACT THESE, IN
THIS CASE REMOVING EFFECTIVE
FUNCTION OF THE BCL11A IN SOME
MANNER.
THIS IS OBVIOUSLY CHALLENGING
BECAUSE OF THE TRANSCRIPTION
FACTOR.
ONE COULD ANY NUMBER OF
STRATEGIES ONE COULD THINK OF
USING ATTACKING THE BCL11A.
I THINK THE DEVELOPMENT OF SMALL
MOLECULES CERTAINLY
REVOLUTIONIZE THE MANAGEMENT OF
ALL THE HEMOGLOBIN FACILITIES.
I THINK YOU WANT TO SET OUT ONE
GOAL THAT WOULD BE THE GOAL FOR
THE FUTURE.
ONE OF THE QUESTIONS THAT COMES
UP, WHAT LEVEL OF BCL11A IS
CRITICAL?
HOW MUCH DO YOU HAVE TO INHIBIT
IT IN ORDER TO HAVE ENOUGH
HEMOGLOBIN THE ACTUALLY MAKE A
DIFFERENCE.
IN GENETICS HERE HAS TOLD US
SOMETHING VERY INTERESTING.
REALLY WANT TO BE ON THIS.
ACTUALLY NEW SYNDROME THAT
RECENTLY COME TO LIGHT VERY RARE
PATIENTS HAVE BEEN DESCRIBED
WITH DELETIONS THAT ENCOMPASS
THE BCL11A OF A GENE, SOMETIMES
TRADITIONAL, SOMETIMES ONLY
BCL11A GENES ARE ONE CHROMOSOME.
THESE INDIVIDUALS ARE
UNFORTUNATE THEY ARE
ASCERTAINING BECAUSE SEVERE
AUTISM-LIKE OR MENTAL
RETARDATION SYNDROME DESCRIBED
IN FAMILIES THUS FAR.
IN RECENT GENETIC STUDIES
ACTUALLY BCL11A IS ASSOCIATED IN
SORT OF LARGE GWTS STUDIES IN
AUTISM AND SCHIZOPHRENIA AS WELL
INDICATING AN IMPORTANT ROLE IN
NERVOUS SYSTEM DEVELOPMENT.
THIS IS UNIQUE OPPORTUNITY TO
SEE WHAT IS EFFECT OF HAVING
SUFFICIENCY OF BCL11A, WHAT DOES
IT DO IN THE CONTEXT OF
HEMOGLOBIN EXPRESSION.
IT'S REALLY QUITE REMARKABLE,
THESE ARE THREE PATIENTS WE'VE
HAD AN OPPORTUNITY TO LOOK AT,
TWO OF THEM I'VE OBTAINED.
ONE CASE INVOLVE ONLY THE BCL11A
GENE, THESE TWO INVOLVE ANOTHER
GENE PROBABLY RELEVANT.
BUT WHAT'S INTERESTING NORMAL
HEMATOLOGY IN TERMS MUCH RED
CELLS, NORMAL LYMPHOID AND D
CELLS BUT HEMOGLOBIN F LEVELS
ARE QUITE REMARKABLE.
MEASURE 23%, 16%, ALMOST 30%
STANDARD CLINICAL LAB THESE
INDIVIDUALS HAVE 50% OF BCL11A.
IN FACT THIS INDIVIDUAL IS KNOWN
TO HAVE HIGH F SNP AT REMAINING
BCL11A AS COMPARED TO THIS
INDIVIDUAL WHICH I THINK ALL
GENETICS.
EITHER IN THE ORDER OF HALF, 50%
TO GET HEMOGLOBIN F
CONCENTRATION IN ROUGHLY THESE.
GOOD NEWS IN TERMS OF HOW THE
ACTUALLY TARGET THE GENE OR
PROTEIN.
WHAT ARE WE TRYING TO DO NOW.
WELL, BCL11A PROTEIN HAS ONE
MIGHT IMAGINE IS ACTUALLY PART
OF MANY PROTEIN COMPLEXES.
ERYTHROID CELLS IS FOUND IN
ASSOCIATION WITH TRANSCRIPTION
FACTORS.
SPECIFIC CHARACTERS.
ONE OF THE THINGS WE'VE BEEN
DOING TRYING TO DETERMINE WHICH
PARTS OF THE ESSENTIAL FOR
FUNCTION BECAUSE WE THINK THAT
WILL BE IMPORTANT IN THINKING
ABOUT SMALL MOLECULE
DEVELOPMENT.
ONE OF THE CURIOUS FEATURES OF
THIS PROTEIN IS IN AN ASSAY WE
HAVE, WE CAN ACTUALLY DISCARD
MUCH OF THE PROTEIN AND STILL
CAPABLE OF ACTUALLY REGULATING
THE GLOBINS APPROPRIATELY.
ONLY CRITICAL REGION THUS FAR,
THIS REGION HERE ACTUALLY ALSO
INTER-ACTS WITH GAMMA ONE AND
TRANSCRIPTION FACTORS.
MAYBE OTHER THINGS AS WELL.
DON'T KNOW IF THESE ARE ACTUALLY
CRITICAL.
BUT REALLY ONLY THREE PARTS TO
THIS LARGE PROTEIN.
WHICH IS CURIOUS ONE THINKS THAT
ONE NEEDS TO FORM SPECIFIC
PROTEIN COMPLEXES IN ORDER TO
APPROACH THE FUNCTION.
TRYING TO UNDERSTAND THE MEANING
OF THIS.
ALSO LARGER VERSIONS OF THIS
ENCODED LIE BURIES OTHER
METHODS.
USING COMBINATION OF THESE
METHODS DIFFERENT COLLABORATORS
TO TRY TO IDENTIFY SMALL
MOLECULES INTER-ACT WITH THE
PROTEIN AS AT LEAST FIRST REALLY
GET TARGET THE PROTEIN.
THIS IS OBVIOUSLY GOING TO BE
LONG ROAD, IT'S UNCERTAIN ROAD
BUT I THINK ONE THAT NEEDS TO BE
UNDERTAKEN.
WE'VE RUN SOME PILOTS ALREADY
WITH THE SMALL MOLECULE AND
MICROARRAY.
IN PILOT SCREENS WE CAN IDENTIFY
SOME SMALL MOLECULES SUCH AS TWO
HERE THAT ACTUALLY PHYSICALLY
BIND WITH MEASURABLE.
OBVIOUSLY THIS IS JUST STARTING
POINT GIVES US SOME HOPE TO THIS
KIND OF APPROACH WITH CHEMISTS
WILL GET US TO SOME MOLECULES
FOR SUBSEQUENT STUDY.
LASTLY, HAVING SMALL MOLECULES
TO GO -- TO HAVE ANY OTHER ROLE
IN THERAPY.
JUST THIS PAST WEEK OR TWO, A
PAPER APPEARED SUGGESTING BCL11A
IS ACTUALLY A CRITICAL GENE IN
TRIPLE NEGATIVE BREAST CANCER
WHERE PROPOSED THE BREAST CANCER
STEM CELLS, AT LEAST ONE WOULD
HAVE ANOTHER AVENUE, FOR
EXAMPLE, WITH SMALL MOLECULES IF
IN FACT LONG TERM USE FOR
HEMOGLOBIN.
TAKE HOME MESSAGES AND CLOSING.
WE THINK BCL11A IS VALIDATED
TARGET REACTIVATION.
THINK VALIDATED BY GENETICS
BIOCHEMISTRY, CELL BIOLOGY.
GENETIC VARIATION MODESTLY
CONTROLS THE GENE.
EMPHASIZE MODESTLY.
HAS PROFOUND AFFECT ON
EXPRESSION AND I THINK THIS IS
IMPORTANT UNDER APPRECIATED
ASPECT RELEVANT TO GWAS STUDIES
IN GENERAL.
VERY OFTEN ASSUME THAT THE GWAS
RESULTS HAVE SMALL EFFECT SIZE
IT'S NOT VERY IMPORTANT.
WE THINK THAT BCL11A IS
EXCELLENT TEST CASE FOR
THERAPEUTIC GENOME ENGINEERING
TO BENEFIT PATIENTS WITH
HEMOGLOBIN DISORDERS.
AND WE HOPE THIS COMES ONLINE IN
THE NEXT COUPLE OF YEARS.
AND WE'RE CURRENTLY TRYING TO
IDENTIFY SELECTIVE SMALL
MOLECULE INHIBITORS THAT MIGHT
BE USED IN THE FUTURE FOR
THERAPEUTICS.
IN CLOSING JUST LIKE TO THANK
THOSE WHO ACTUALLY HAVE DONE THE
WORK.
I'D LIKE TO THANK THESE PEOPLE
LISTED HERE.
THANKS VERY MUCH.
I'D BE GLAD TO TAKE ANY
QUESTIONS.
[APPLAUSE]
>> I WONDER HAVE YOU LOOKED AT
WHETHER ALSO BINDS ENHANCER OF
THE PROTEIN IN THIS GENE AND
WHETHER TRIED TO TREAT IT.
>> ASK WHETHER ENHANCEERS WOULD
BE USEFUL IN THIS CONTEXT.
WE'VE PUT JT1 ON IT DOESN'T HAVE
MUCH OF AN AFFECT.
WE DON'T THINK THIS IS GRD4
ENHANCER EPIIN THAT CONTEXT.
THOSE ARE RELATIVELY INACTIVE.
>> WHAT ARE THE PROSPECTS FOR
REACTIVATING DELTA AND THE OTHER
QUESTION IS, WHAT IS THE ROLE OF
METHYLATION PLAYS IN CONTROL OF
THE GAMMA?
>> WHAT ABOUT REACTIVATING DELTA
A MINOR DELTA GENE IS SORT --
TRYING TO BECOME A PSEUDOGENE,
IT'S ON ITS WAY OUT.
WE DON'T ACTUALLY THINK THAT IS
GOING TO BE A GOOD CHOICE.
I DON'T EVEN KNOW, SOME PEOPLE
MAY KNOW IN THE AUDIENCE WHERE
THE DELTA CHAIN, DELTA PROTEIN
PUT IN HEMOGLOBIN DOES IT
FUNCTION WELL, I'M NOT EVEN
CERTAIN IF THAT IS KNOWN.
SECOND QUESTION IS METHYLATION.
OUR VIEW, BASED ON NOT
TREMENDOUS AMOUNT IS METHYLATION
REALLY A LATE EVENT.
IT'S AFTER PROBABLY SILENCING
HAS OCCURRED.
PARTICULARLY IF ONE USES
DEMETHYLATING AGENTS IN NORMAL
CELLS ONE DOES GET A LITTLE
BOOST BUT IF YOU GET RID OF
BCL11A YOU GET LOT MORE BOOST.
>> COULD THERE BE SOME KIND OF
SYNERGISTIC AFFECT BETWEEN
HYDROXYUREA AND KNOCK DOWN AND
CCL11A?
>> POSSIBLY.
AT LEAST IN OUR WORK DON'T SEEM
TO BE OPERATING IN THE SAME WAY.
>> MIGHT BE GOOD.
>> DURING BOTH PHASES WHEN BETA
IS ACTIVE YET INTER-ACTIONS
CHANGE BETWEEN.
DO YOU HAVE ANY IDEA WHAT BCL11A
IS DOING THERE?
>> QUESTION IS, BETWEEN GAMMA
EXPRESSED AND BETA EXPRESSED
WHERE BCL11A SITS.
WE DON'T KNOW THAT.
ALL WE KNOW WHAT HAPPENS IN
ADULT BETA PRODUCING CELL YOU
DON'T ACTUALLY KNOW IF WE KNOCK
DOWN, WE DON'T NO KNOW WHERE THE
PROTEIN IS IN THE CELLS THAT
EXPRESS GAMMA.
THERE ISN'T MUCH PROTEIN WE
DON'T THINK IT'S DOING MUCH.
WE HAVE --
>> IN YOUR MOUSE EXPERIMENT
WHERE YOU HAD ACHILLE'S HEEL.
THAT'S LOOKED A LOT LIKE SITES
THAT EKLF BINDS TO.
THERE ARE EKLF MUTATIONS
ASSOCIATED WITH THE HEMOGLOBIN
OR HPFH HOW DOES THAT ALL FIT
TOGETHER.
>> IF THERE ARE NO MORE
QUESTIONS JOIN ME IN THANKING
THE DOCTOR.
I'D LIKE TO INVITE AUDIENCE TO
RECEPTION AND MORE CONVERSATION
IN THE LIBRARY WHICH IS
SPONSORED BY THE FAES DIRECTLY
AFTER THE SEMINAR.
[APPLAUSE]
