>> GOOD AFTERNOON AND WELCOME TO
THE WEDNESDAY AFTERNOON LECTURE
SERIES.
I'M A LAB CHIEF IN NEUROGENETICS
FROM THE NATIONAL INSTITUTE ON
AGING AND HERE AND CO-CHAIR OF
THE SINGLE CELL GENOMICS GROUP
WHO NOMINATED TODAY'S SPEAKER,
DR. JONATHAN WEISSMAN AND I'LL
READ THESE BECAUSE I CAN'T
REMEMBER THIS STUFF.
IF YOU'RE WATCHING REMOTELY AND
I BELIEVE THERE'S A COUPLE
HUNDRED PEOPLE ONLINE WE
ENCOURAGE YOU TO PARTICIPATE IN
THE Q&A AND CLICK ON THE BUTTON
BELOW YOUR VIDEOCAST THAT SAYS
SEND LIVE SPEFEEDBACK AND WE'LL
RELY IT TO HIM AND YOU CAN
SUBMIT AT ANY TIME AND HOPE YOU
DO SO.
FOR THOSE HERE IN PERSON AND WE
HAVE A GREAT CROWD, THANK YOU
ALL FOR BEING HERE.
THERE'LL BE A COUPLE MICROPHONES
LEFT AND RIGHT TO ASK IN PERSON.
THIS IS CRITICAL, WE'RE OFFERING
CONTINUING MEDICAL EDUCATION
CREDIT AND THE CME CODE IS
50128.
PUT THAT AND YOU'RE GET YOUR CME
CREDIT.
SO OUR SPEAKER TODAY, DR
DR. WISEMAN IS A HEAD OF
WHITEHEAD INSTITUTE AND STUDIES
HOW CELLS ENSURE PROTEINS ARE
FOLDED AND HOW IT IMPACTS
DISEASE.
IT'S FAIR TO SAY HE'S FAMOUS FOR
A LOT OF TECHNIQUES DEVELOPED
OVER THE YEARS INCLUDING
RIBOSOME PROFILING AND GENOME
SCALE HIGH CONTENT SCREENS,
CRISPR INTERFERENCE AND
ACTIVATION TO CONTROL EXPRESSION
AND REWIRE THE EPI GENGENOME AN
RECORDING THE HISTORY OF CELLS.
VERY IMPRESSIVE TOOL SET AND
THANK YOU.
THESE ARE THINGS MANY US USE IN
OUR LABS.
HE RECEIVED AN AB IN PHYSICS
FROM HARVARD AND PURSUED AN AN
Ph.D. IN PHYSICS AT M.I.T.
HE STUDIED PROTEIN FOLDING BY
PANCREATIC INHIBITOR AND DID A
POSTDOC AT YALE AND HELD
POSITIONS IN DEPENDS OF CELL
MOLECULAR PHARMACOLOGY AT
UNIVERSITY SAN FRANCISCO AND
CO-LEADS THE LABORATORY FOR
GENOMICS RESEARCH TO DRIVE
DEVELOPMENT OF CRISPR-BASED
THERAPEUTICS.
HE CO-FOUNDED THE INNOVATIVE
GENOMICS INSTITUTE IN 2015 AND
REMAINED ON THE ADVISORY BOARD.
HE HAS RECEIVED NUMEROUS AWARDS
TO CITE A FEW ELECTED TO THE
NATIONAL ACADEMIES SCIENCES
ENGINEERING MEDICINE AND WON THE
AWARD FOR SCIENTIFIC DISCOVERY
IN 2015.
SO WITH ALL THAT SAID AND THAT
IS VERY IMPRESSIVE AND THANK YOU
FOR BEING HERE, THE TITLE OF HIS
TALK IS EPIGENOME EDIT BEING AS
A TOOL FOR BASIC DISCOVERY AND
DELIVERY OF CHARM INTERVENTION.
JOIN MIN WELCOMING DR. WEISSMAN.
>> THANK YOU FOR THE KIND
INTRODUCTION AND INVITATION TO
BE HERE.
IT'S BEEN A WONDERFUL DAY SO FAR
AND HAVE VISITED NIH MANY TIMES
AND IT'S WONDERFUL TO BE BACK.
I WISH I HAD MORE TIME BECAUSE
THERE'S SO MANY PEOPLE I DIDN'T
HAVE A CHANCE TO VISIT BUT
HOPEFULLY THERE'LL BE AN
OPPORTUNITY SOME TIME IN THE NOT
TOO DISTANT FUTURE.
I'D LIKE TO TELL YOU TODAY ABOUT
THE UNIFYING THEME OF OUR WORK
IS A TOOL OF PROGRAMMABLE
EPIGENETIC EDITORS.
I'D LIKE TO TALK HOW WE USE THIS
AS DISCOVERY TOOL AND PRESENT
FLU WORK ABOUT THE POTENTIAL OF
USING EPIGENETIC SILENCING AS A
THERAPEUTIC.
FIRST, THESE ARE MY DISCLOSURES.
I THINK THE PRESENTATION WILL BE
AVAILABLE FOR SOME TIME TO SEE
THEM IN MORE DETAIL.
SO MY INTRODUCTION TO CRISPR
STARTED THROUGH THREE VERY
FORTUITOUS ENCOUNTERS IN MY
OFFICE IN 2012 AND HAD THE GOOD
FORTUNE OF SITTING IN MY OFFICE
AND SUCCESSIVELY THESE THREE
PEOPLE CAME IN.
THE FIRST WAS JENNIFER DOWDNA I
HAD KNOWN MANY YEARS AND WITH
GREAT EXCITEMENT LIKE OTHER
PEOPLE READ HER PAPER SHOWING
THAT KAS9 AND THE CHIMERIC
POTENTIAL OF CRISPR AND SAID I
HAVE THIS GREAT TOOL, WHAT ARE
THINGS WE CAN DO WITH IT.
THEN STANLEYY WHO WAS THEN A
GRADUATE STUDENT JUST FINISHING
AT BERKELEY AND COMING TO UCSF
AS A FELLOW WAS TALKING ABOUT
WHAT TO DO WITH NEXT STEP AND
DESCRIBED REMARKABLE THING HE
COULD TAKE A DEAD VERSION OF KAS
9 AND EXPRESS SINCE A
PROGRAMMABLE WAY IN BACTERIA AND
USE IT TO BLOCK TRANSCRIPTION.
THEN FINALLY LUKE GILBERT JUST
JOINED MY LAB AS A POSTD-DOCTORL
FELLOW AND HAD EXPERIENCE IN
VECTOROLOGY AND MAMMALIAN STUDY
AND WE HIT ON WHAT WE COULD DO
WITH JENNIFER'S PROGRAMMABLE
NUCLE
NUCLEASE.
WE WERE GOING TO BREAK IT SO IT
COULDN'T CUT ANYTHING.
THE REASON WE DID THIS IS
BECAUSE NOW INSTEAD OF HAVING A
PROGRAMMABLE NUCLEASE YOU HAD A
READILY PROGRAMMABLE DNA BINDING
PROTEIN.
WE KNEW FROM SEMINOLE WORK THIS
COULD BE USED AS A PLATFORM WHEN
FUSED TO VECTOR DOMAINS AND THE
FIRST WERE TRANSCRIPTIONAL
REPRESSOR TO TURN OFF THE
NEIGHBORING GENE OR
TRANSCRIPTIONAL ACTIVATOR TO
TURN ON.
IN ESSENCE WE NOW HAD A DIAL TO
TUNE ANY GENE EXPRESSION DOWN OR
UP OF ANY GENE OR SET OF GENES
IN THIS PROGRAMMABLE WAY.
SINCE THIS TIME WE AND OTHER
LABS HAVE PUT DIFFERENT EFFECTER
DOMAINS.
PERHAPS THE MOST SPECTACULAR OF
WHICH ARE THE MANY REVERSE
TRANSCRIPTASES TO GENERATE THE
BASE EDITORS AND PRIME EDITOR
STRATEGIES.
THANKS TO THE THE ADVANCES YOU
CAN MAKE GUIDE RNAs TO TARGET
THE FULL SET OF GENES IN THE
GENOME.
NOW YOU CAN TRANSDUCE IN
INDIVIDUAL CELLS THE LIBRARIES
SO THAT EACH CELL GOT ONE OR A
COMBINATION OF DIFFERENT RNA AND
IT WOULD PROGRAM THE CHANGING
EXPRESSION OF THE TARGETED GENE
AND ACT AS A BAR CODE THAT TOLD
YOU UP THROUGH SEQUENCING WHICH
GENE HAD BEEN MODULATED.
THIS COULD THEN BE DONE FOR
EXAMPLE IN SCREENS TO ALLOW AND
IDENTIFY GENES THAT CONFER
RESISTANCE TO PRESSURE.
FOR EXAMPLE ANANTI-CANCER DRUG
YOU CAN LOOK FOR GENES WHEN YOU
KNOCKED OUT CONFERRED RESISTANCE
TO THAT THERAPEUTIC, RESISTANCE
MECHANISMS OR ACT
SYNERGISTICALLY AND LOOK AT THE
NEXT DRUG TO ADD ON TO THAT.
AND YOU MAY BE INVOLVED IN THE
BIOLOGICAL PROFESSOR.
WE WERE INTERESTED IN STRESS AND
THEN BUILD A FLUORESCENCE
REPORTER TO REPORT ON THE LEVEL
OF STRESS OF AND THEN DO A FACT
SCREEN FOR AND IDENTIFY ALL THE
GENES WHEN YOU TURN ON OR OFF
CAUSE DISRUPT FOLDING IN THE ER
AND THIS COLLECTIVELY THESE AND
PARALLEL EFFORTS WITH CRISPR
CUTTING OPENED UP MAMMALIAN
BOARD GENETICS IN A WAY THAT HAS
PROVEN TO BE QUITE
TRANSFORMATIVE.
THERE WAS SHRNA APPROACH BEFORE
AND THOSE WERE GROUNDBREAKING
FOR THE DESIGN MUCH THESE.
BUT THE SPECIFICITY AND EFFICACY
OF THE TOOLS HAD NOW ROOM
COMMODITIZED THE CRISPR SCREEN
AND NOW IT'S FIGURE 1A THE
STARTING POINT TO LAUNCH INTO
BIOLOGY.
YOU HAD TO DECIDE WHAT PART OF
BIOLOGY YOU'RE INTERESTED IN AND
IF I'M LOOKING AT THE MUTATED
CANCER CELLS I THEN DO A CRISPR
SCREEN FOR THINGS THAT MODULATE
THE TOXICITY OR IF I'M
INTERESTED AS I TALKED ABOUT IN
THE PROTEIN FOLDING IN THE ER I
BUILD A CELL LINE TO REPORT ON
PROTEIN FOLDING AND HAD TO
DEFINE AHEAD OF TIME WHAT THE
BIOLOGY WAS AND THE BIGGER
PROBLEM WAS AT THE END YOU WOULD
GET A LIST OF SCREENS AND WHEN
DONE WE WILL HAVE A HIGH
CONFIDENCE LIST OF GENES AND
DECONVOLUTING HOW THEY'RE WORK
IS AN ENORMOUS CHALLENGE AND
CELLS LIVE IN A HIGH DIMENSIONAL
MANI FOLD OF TRANSCRIPTIONAL
STATES AND WHEN YOU DO SINGLE
READ OUT ONE DIMENSIONAL SCREEN
IS DOING A DIMENSIONALITY
REDUCTION PREDICTING THE TYPES
ON THE SINGLE DIMENSION AND
DOING THIS -- IT'S NOT A U MAP
OR ANY UNDERSTOOD WAY.
IT'S A HORRIBLY NON LINEAR AND
IRREVERSIBLE APPROACH.
TO GO BACK FROM WHERE YOU STAND
HERE IN YOUR ONE DIMENSIONAL
PHENOTYPE TO WHAT IT'S DOING
UNDERSTANDING WHAT'S IT'S DOING
TO THE CELL ENDS UP BEING OFTEN
A Ph.D. THESIS.
SO IT DIDN'T SCALE WITH THE
NUMBER OF INTERESTING GENES THAT
WERE COMING OUT OF THESE TYPES
OF SCREENS.
THIS MOTIVATE THE US AND OTHERS
TO MAP THE EFFECTED PHENOTYPE IN
A RICHER PHENOTYPIC SPACE AND
APPROACH THAT WE AND WONDERFUL
COLLABORATION AND MY LAB WITH
THOSE WHO ARE NOW FACULTY
MEMBERS AT PRINCETON AND
MEMORIAL KETTERING RESPECTIVELY
AND WONDERFUL COLLABORATION WITH
A LAB TO DEVELOP THE PERTURBED
SYC APPROACH WITH READOUT.
AND THE FRAMEWORK OF THESE
EXPERIMENTS WERE WE'D START WITH
THE POOLED SCREEN AND NOW YOU
WOULD DO A DROPLET BASED SINGLE
CELL RNA SEQ AND THE GUIDE RNA
PRESENT IN THE CELL WOULD TELL
YOU WHAT THE PERTURBATION IN THE
CELL WAS AND READ OUT THE
TRANSCRIPTIONAL RESPONSE.
IN ESSENCE THIS LET YOU MAP OUT
FOR EACH OF THE PERTURBATIONS
HOW YOU WERE PUSHING THE CELL IN
THE TRANSCRIPTIONAL LANDSCAPE OR
PHENOTYPIC LANDSCAPE OF THE
CELL.
AND STEPPING BACK IT'S PART OF A
BROADER EFFORT WE AND OTHERS
HAVE DONE TO TAKE SINGLE CELL
RNA SEQ AND SEE THE STATE OF THE
CELL AND MAKE IT ESSENTIALLY AN
EXPERIMENTAL TOOL IN WHICH EACH
CELL BECOMES A TEST TUBE AND YOU
HAVE THE NATURE OF THE
EXPERIMENT IN THIS CASE THE
PERTURBATION THE GENE YOU'RE
TURNING ON AND OFF AND THE
RESULT READ OUT BY THE SINGLE
CELL RNA SEC FROM HUNDREDS TO
THOUSANDS AND NOW ROUTINELY A
MILLION CELLS WE COULD DO THESE
RICH EXPERIMENTS IN A MASSIVELY
PARALLEL WAY.
SO WHEN WE STARTED THE SCALE WAS
A FEW HUNDRED GENES AND THE
ORIGINAL VISION OF WHAT WE DO IS
DO THE POOLED SCREEN AND GET
FAVORITE HITS AND PUT IT INTO A
PERTURBED SEQ TO UNDERSTAND THE
FUNCTIONAL RELATIONSHIP BETWEEN
THE TWO.
THAT DIDN'T SOLVE THE FIRST
PROBLEM I TALKED ABOUT WHICH IS
YOU HAD TO STILL PREDEFINE WHAT
THE FIRST POOLED SCREEN WAS.
THAT MOTIVATED US AND US BEING
BRIT ADAMSON AND A TERRIFIC
STUDENT NOW HAS GONE ON TO DO
MEDICAL TRAINING M.D. Ph.D.
STUDENT TO ALLOW THEM TO BE DONE
ACROSS GENOME PERTURBATIONS.
SO IN THE FIRST EXPERIMENT HE
DID ALL THE EXPRESSED GENES IN
ONE CANCER CELL LINE K562 AND
ACROSS FOUR CELL LINES INCLUDING
BOTH CANCER AND NON-TRANSFORMED
IMMORTALIZED CELL LINES.
THIS IS ABOUT 6 MILLION CELL
EXPERIMENT.
AT THE TIME NOW A FEW YEARS AGO,
WAS FAIRLY HEROIC BUT AGAIN
BECAUSE TECHNOLOGY IS ADVANCING
SO RAPIDLY THIS WOULD BE STILL A
LARGE SCALE EXPERIMENT BUT
NOWHERE NEAR AS HEROIC AS IT WAS
JUST A COUPLE YEARS AGO.
WHAT YOU GOT OUT OF THIS WAS A
LIST OF THE GENETIC
PERTURBATIONS, KNOCKING DOWN
IRE1 OR PROTEIN RESPONSE SENSOR
AND THE TRANSCRIPTIONAL RESPONSE
FOR EACH OF THESE PERTURBATIONS.
WHAT YOU COULD DO THEN IS EITHER
CLUSTER ON THE GENES THE
TRANSCRIPTIONAL RESPONSES AND IN
THIS WAY IN A SYSTEMATIC MANNER
LOOK AT THE TRANSCRIPTIONAL
RESPONSES IN THE CELLS.
FOR EXAMPLE, INTEGRATE STRESS
RESPONSE AND THE PROTEIN
RESPONSE AND FALLOUT IN AN
AUTOMATIC WAY AND DO OTHER
STRESS RESPONSE AND OTHER
TRANSCRIPTIONAL RESPONSES THAT
HAVEN'T BEEN AS WELL STUDIED OR
HAVE A NAME ON THEM.
THE SECOND APPROACH IS TO ORDER
THE GENETIC PERTURBATIONS AND
ORDER GENES BASED ON THE
SIMILARITY OF HOW THE CELL
RESPONDS TO THE LOSS OF THAT
GENE.
SO GENES THAT ARE HAVING SIMILAR
FUNCTIONS WILL THEN CLUSTER NEXT
TO EACH OTHER.
THIS IS REALLY POWERFUL APPROACH
IN AN OBJECTIVE SYSTEMATIC WAY
OF ORDERING THE GENES AND GROUPS
AND DIDN'T PRESUPPOSE YOU HAVE
TO DEFINE WHAT THE GROUPS ARE.
IT ALLOWS A FORM OF UNBIASSED
HYPOTHESIS GENERATING
EXPERIMENTS ON A DIFFERENT SCALE
THAN PREVIOUSLY POSSIBLE.
LET'S JUST GIVE YOU A BIT OF
EXAMPLES OF THIS.
HERE'S JUST CLUSTERING
PERTURBATION CORRELATION AND
MATRIX WITH EVERY PERTURBATION
ORDERED WITH ONES MOST SIMILAR
TO IT.
THEN BLOWUP AND YOU CAN SEE HOW
MUCH BIND STRUCTURE THERE IS
HERE AND WHEN YOU GET TO THE
POINT YOU SEE GENE NAMES YOU CAN
SEE THIS IS ABLE TO PUT TOGETHER
A WELL STUDIED PROCESSES AND
METALATION AND ELONGATER AND
INTEGRATOR COMPLEX AND A TINY
CORNER OF THIS ENTIRE MAP.
SO BASICALLY WHAT WE'RE ABLE TO
DO IS RECAPITULATE THOUSANDS OF
KNOWN RELATIONSHIPS AND BECAUSE
AS YOU'LL SEE IN A VERY SPECIFIC
EXAMPLE BECAUSE THERE'S NEWER OR
POORLY CHARACTERIZED GENES THAT
OFTEN CLUSTER WITHIN THESE KNOWN
FUNCTIONAL CLUSTERS IT ALLOWS
YOU TO DISCOVER OR GENERATE
HYPOTHESES ABOUT NEW ELEMENTS.
IN ADDITION TO THIS GROUPING
GENES INTO FUNCTION THOUGH IT
CAN BE USED TO DISSECT A LARGE
MULTI-FUNCTIONAL COMPLEX INTO
FUNCTIONAL MODULES.
I'LL SPEND A COUPLE SLIDES
SHOWING A NICE EXAMPLE CENTERED
AROUND THIS INTEGRATOR COMPLEX.
THE INTEGRATOR COMPLEX THIS IS A
PIECE.
IT'S A METAZONE TRANSCRIPTIONAL
COMPLEX STUDIED OVER THE LAST
DECADE OR SO THE SUBJECT OF
INTENSE STRUCTURE AND
MECHANISTIC STUDIES.
WHAT WE FOUND IS THAT EVEN
THOUGH THIS IS A SINGLE THEN
PROTEIN AND REALLY A 15 PROTEIN
COMPLEX, THE FUNCTIONAL MAP
DISSECTED THIS INTO AT LEAST
THREE DISTINCT FUNCTIONAL
MODULES.
AND WE HAD A STRUCTURE OF MOST
THE COMPONENTS OF THE INTEGRATOR
AND THE FUNCTIONAL MODULES AS
DEFINED BY OUR PERTURB SEQ WENT
TO THE COMPLEX AND THIS IS PART
OF THE BACKBONE AND SHOULDER AND
THE ENDONUCLEASE SUB COMPLEX
RESPONSIBLE FOR CLEAVING NASCENT
TRANSCRIPTS.
WE FOUND THREE SUB UNITS AND ONE
THEN COMPLETELY UNCHARACTERIZED
PROTEIN AS A DISTINCT FUNCTIONAL
MODULE.
AND SO WE THEN AS YOU'LL SEE
WHY, WE RENAMED THIS UNNAME D AD
RUBIN SAUNDERS SHOWED THROUGH A
PULL DOWN THIS FORMED THE
SUBCOMPLEX THE SUB UNITS.
IT ALSO SHOWED THE SUB COMPLEX
WAS NOT REQUIRED FOR THE
ENDNUCLEASE ACTIVITY.
AND THIS LED RUBIN TO
HYPOTHESIZE THE REASON THE
SUBCOMPLEX WAS MISSING FROM THE
OVER ALL STRUCTURE WAS BECAUSE
THEY WERE MISSING THE SUB UNIT.
AND GRATIFYINGLY A MONTH OR SO
AGO PATRICK KRAMER PUBLISHED THE
FULL INTEGRATOR COMPLEX
EXPLICITLY PURIFYING AND
GRACEFULLY NOTING RUBIN'S
OBSERVATIONS AND ADDED IT TO THE
REST AND FOUND IT FORMED WHAT IS
CALLED THE TAIL DOMAIN AND IT
RESEMBLED A SCORPION TAIL AND
ALSO SHOWED THE SUB UNIT THAT
WITH US MISSING FROM OTHER
COMPLEXES WAS CRITICAL FOR
STAPLING THE TAIL ON TO THE REST
OF THE COMPLEX AND THEN PATRICK
SHOWED THIS TAIL COMPLEX WAS NOT
REQUIRED FOR THE ENDONUCLEUS
ACTIVITY BUT FOR REMOVAL TO
AFTER THE CLEAVAGE EVENT.
I GO THROUGH THIS EXAMPLE AND A
LITTLE BIT OF DETAIL BECAUSE IT
SHOWS HOW FROM A GENERIC
EXPERIMENT TO BE HONEST WE
BARELY KNEW WHAT THE INTEGRATOR
WAS WHEN WE CONDUCTED THE
STUDIES WE WERE ABLE IN A FAIRLY
SYSTEMATIC AND SCALABLE WAY GET
TO A SPECIFIC ENOUGH HYPOTHESIS
THAT IT COULD HAND OFF DIRECTLY
AND BE OF DIRECT UTILITY TO
PEOPLE WITH MORE FOCUSSED
INTEREST IN THIS APPROACH.
I'VE TOLD YOU FROM THE GENOTYPE,
PHENOTYPE MAPS YOU CAN DISCOVER
TRANSCRIPTIONAL PROGRAMS AND
FUNCTIONALLY CHARACTERIZE AND
DISSECT PROTEIN COMPLEXES.
THE FINAL AND I FEEL IN MANY
WAYS THE MOST EXCITING ASPECT OF
THE POTENTIAL FOR THE ANALYSIS
IS THAT YOU CAN THEN TAKE THESE
GENOTYPE/PHENOTYPE MAPS AND
ESSENTIALLY CREATE A SYNTHETIC
PHENOTYPE IN SILICO AND QUERY
THAT TO DO THE SCREEN AND IN A
NICE EXAMPLE OF THIS JOSEPH AND
RUBIN ESSENTIALLY CREATED FOR
EACH PERTURBATION A SCORE OF HOW
THEY AFFECTED CHROMOSOME
STABILITY AND HOW THEY AFFECT
WILL THE POTENTIAL FOR
ANEUPLOIDITY AND DID A GENOME
WIDE SCREEN FOR FACTORS THAT
MODULATED THE INPUT PROCESS.
AND AGAIN IT WAS THE FACT TH%T
YOU COULD DO THESE COMPLEX IN
WHAT WOULD OTHERWISE BE A
DIFFICULT AND HAVING THE FULL
DATA SET AND DO AND ILLUSTRATES
THE POTENTIAL FOR THE MAPS AS A
DISCOVERY TOOL.
I SAY THIS AND IT SOUNDS LIKE
WE'VE SOLVED ALL THE QUESTION
WHICH IS BY FAR NOT THE CASE.
THERE'S TWO BIG LIMITATIONS.
ONE IS MANY GENES -- IN FACT
MOST GENES WHEN WORKING AT LEAST
IN CANCER CELLS JUST DON'T GIVE
YOU A GOOD TRANSCRIPTIONAL
PHENOTYPE.
IT'S NOT THE RIGHT WAY TO LOOK
AT THE FUNCTION.
WE'RE NOT ASSESSING THE RIGHT
PHYSIOLOGY AND THERE'S FEATURES
NOT WELL READ OUT BY
TRANSCRIPTION AND WE HAVE
MULTIPLE EXAMPLES BUT ONE
CONCRETE ONE THE TOR PATHWAY IS
CRITICAL FOR THE SENSOR OF
CELLULAR STRESS AND AS YOU CAN
TURN IT UP AND DOWN SECONDARY
SIGNATURES FROM TRANSCRIPTION
REALLY ISN'T READ OUT WELL AND
READING THE LEVELS YOU HAVE ROOM
TO MONITOR THE TOR PATHWAY.
IT'S NOT JUST ENOUGH TO HAVE A
LOT OF DIMENSIONS WE HAVE TO
WATCH ALL THE RIGHT FUNCTIONS OF
THE CELLS.
WE HAVE TO GO TO MORAL
MULTI-MODAL READOUTS THE SECOND
IS EVERYTHING DEPENDS ON YOU
HAVING THE RIGHT MODEL SYSTEM.
THIS IS NOT MY QUOTE AND WE
DON'T WANT FUNCTIONAL GENOMICS
TO GET US TO THE WRONG ANSWER
FASTER.
CELLS LIVE IN COMMUNITIES.
WE NEED TO MOVE AWAY FROM
STUDYING MONOCULTURES OF CANCER
CELLS.
THERE'S GREAT OPTIONS OF
STUDYING ORGANOIDS AND OTHER
ASSEMBLIES OR IPS DERIVED CELLS
AND THEY'RE POWERFUL APPROACHES.
WHAT WE'VE BEEN FOCUSSING ON
THOUGH, RUBIN WHO WORKED WITH
JOSEPH ON THE PERTURB SEQ AND A
JUNIOR FELLOW WHO WILL SOON BE
AN ASSISTANT PROFESSOR AT
STANFORD RAN A WONDERFUL
COLLABORATION WITH A POSTDOC AND
HAVE BEEN DEVELOPING APPROACHES
THAT ABUT US TO DELIVER VERY
COMPLEX GENOME WIDE
PERTURBATIONS IN VIVO TO AN
INTACT LIVE ANIMAL.
FOR EXAMPLE TO LIVER OR TO THE
BRAIN OR TO THE HEART.
YOU ESSENTIALLY CREATE A GENETIC
MOSAIC.
FOR EXAMPLE, WE'VE BEEN
FOCUSSING MOST OF OUR PRIMARY
WORK BUILDING ON EARLY
APPROACHES FOR DELIVERY
INCLUDING SEMINOLE WORK AND IN
EACH HEPATOCYTE YOU GET
DIFFERENT PERTURBATIONS OR SET
OF PERTURBATIONS AND IN THE
ANIMAL YOU CAN PROFUSE THE
ANIMAL TO LOCK IN THE PHENOTYPIC
STATE AND DISASSOCIATE THE CELLS
AND DO PERTURB SEQ FROM THE IN
VIVO SAMPLES OR TAKE SECTIONS OF
TISSUES AND THEN USING THE
APPROACH.
WE'RE ABLE TO READ OUT WHAT THE
PERTURBATION IS AND WHAT THE
EFFECTS ON TRANSCRIPTS ARE AND
ALSO BY COMING WITH ANTIBODIES
AND LOOK AT A HIGH RESOLUTION
VIEW OF HOW THE FUNCTION OF THE
CELL IS MODULATED.
SO THESE ARE JUST A FEW OF THE
DIFFERENT EXAMPLES WHERE YOU GET
RICH PHENOTYPES TO MONITOR NOT
JUST TRANSCRIPTIONALLY BUT IN
OVER ABUNDANCE OR MORPHOLOGY OF
ORGANELLES AND COMBINE THESE SO
FOR EACH CELL WE BUILD IN A
MATRIX OF FEATURES WHAT THE
PERTURBATION IS AND WHAT AFFECTS
ON THE RNA AND PROTEINS AND WHAT
NEIGHBORS ARE DOING.
IN THIS WAY START TO UNDERSTAND
THINGS LIKE CELLULAR STRESS
RESPONSES NOT JUST AS A CELL
AUTONOMOUS EFFECT BUT AS AFFECT
ON CELL COMMUNITY.
MORE GENERALLY WE THINK THIS
WILL BE A GREAT TOOL FOR
DISCOVERY AND REALLY COMES AT A
VERY PROPITIOUS TIME IN BIOLOGY
WHERE THERE'S BEEN A GREAT DEAL
OF EXCITEMENT ABOUT MARRYING
MACHINE LEARNING APPROACHES WITH
DATA RICH BIOLOGICAL DATA SETS.
AND THIS IS NICELY ENCAPSULATED
BY A WONDERFUL BUT BOLD QUOTE IF
HE'D NOT JUST SOLVED THE PROTEIN
FOLDING PROBLEM MAY SEEM LIKE A
GRANDIOSE STATEMENT BUT WITH A
NOD TO GALILEO'S COMMENT THE
LANGUAGE OF THE UNIVERSITY IS
MATHEMATICS HE PROPOSED THAT IT
MAY TURN OUT TO PLAY A SIMILAR
ROLE FOR BIOLOGY AND THE
COMPLEMENT TO THIS IS A QUOTE
FROM DAPHNE KOLLOR FOUNDER AND
CEO OF INSITRO A COMPANY
DEDICATED TO APPLYING MACHINE
LEARNING FOR DRUG DISCOVERY AND
A LEADER IN MACHINE LEARNING
THAT REALLY WHAT THEY'RE
LIMITATION IS DOES NOT COMPUTE
BUT THOUGH THE COMPUTE IS
IMPORTANT BUT THEY NEED A
THOUSAND TIMES THE AMOUNT OF
DATA AND I THINK IN JUST THE
QUANTITY BUT A QUALITATIVE
DIFFERENT TYPE OF DETE -- DATA
AND HOPE THIS WILL HELP GENERATE
THIS TYPE OF DATA AND MORE
BROADLY THAT THEY'LL HELP TO
CREATE FOUNDATION MODELS OF
CELLS AND WITH SOME OF THE
FOLLOWING FEATURES THAT WE HAVE
MULTI-MODAL TO CREATE EMBEDDINGS
AND ENCOMPASS DNA AND
TRANSCRIPTS AND CELL-CELL
INTERACTIONS AND THEY'LL BE
GENERATIVE CAPABILITIES.
PREDICTING MUTATIONS OR DRUG
TREATMENTS, IDENTIFYING TARGETS,
MODELLING DISEASES AND EVEN
ALLOWING ACROSS SPECIES
PREDICTION BY COLLECTING THESE
TYPES OF DATA SETS IN DIFFERENT
ANIMAL MODELS AND CELL MODELS.
AND THEN PERHAPS MOST
AMBITIOUSLY TRY TO HAVE
INTERPRETTABLE INTERPRETABLE
APPROACHES TO PREDICT FUNCTION.
SO THAT'S THIS PART OF THE TALK
AND I'LL SWITCH GEARS.
THIS IS REALLY USING HOW WE USE
EPIGENETIC MODULATIONS AND
PERTURBATIONS AS A DISCOVERY
TOOL AND IDENTIFYING POTENTIAL
DRUG TARGETS.
WHAT ABOUT USING THESE AS
EFFECTERS FOR THERAPEUTIC?
SO THE ANSWER IS CRISPR A AS WE
ORIGINALLY DEVELOPED THEM WERE
NOT PARTICULARLY GOOD.
THAT'S BECAUSE THEY WERE
INHERENTLY REVERSIBLE.
YOU COULD TURN DOWN OR UP THE
EXPRESSION BUT ONLY FOR AS LONG
AS YOU EXPRESSED THE DEAD CAS 9
TARGETED TO THE RIGHT GENE.
FOR THIS REASON IT MOTIVATED
JAMES NUNEZ BUILDING OFF OTHER
WORK TO DEVELOP THE PROGRAMMABLE
APPROACH NOT FOR DOING TRANSIENT
REPRESSION OR ACTIVATION OF A
GENE BUT FOR METHYLATING CPG
ISLANDS IN THE DNA AND THIS WAY
PERMANENTLY IN A HIT-AND-RUN
MANNER TURNING OFF EXPRESSION OF
THE GENE.
AND THE REASON THIS WORKS IS
BECAUSE IT BUILDS OFF OF THE
CELL'S NATURAL SYSTEM FOR
SILENCING GENES IN PARTICULAR.
THIS DNA METHYLATION PROCESS AND
THE SPECIFIC BUILDING OFF
MACHINERY THE CELL USES FOR
SILENCING ENDOGENOUS
RETROVIRUSES AND FOUND FOREIGN
ELEMENTS AND MAKE SURE FOR THE
LIFE OF THE ANIMAL THEY DON'T
GET EXPRESSED.
THEY PROPAGATE RELYING ON
ENDOGENOUS MACHINERY AND
BRINGING IN A PROGRAMMABLE
TRANSFERASE IN THE ELEMENT AND
IT BUILDS OFF THE MAINTENANCE
METHYL TRANSFERASE.
WHENEVER IT SEES CPG METHYLATED
IT REMETHYLATED THE SECOND
STRAND.
NOW AS THE CELL DIVIDES YOU GET
A NEW STRAND THAT IS NOT
METHYLATED AND IT COMES IN AND
FILLS THIS IN ALLOWING THIS TO
PROPAGATE INDEFINITELY.
AND INDEED JAMES WAS ABLE TO
SHOW THAT YOU'D GET TRANSIENT
EXPRESSION OF THE CRISPR PROGRAM
TO DEFINE LOCUS THROUGH
ITERATIONS HE WAS ABLE TO FIND
ONES THAT WE'RE NOW ABLE TO ONCE
METHYLATED TO PROPAGATE
INDEFINITELY AND ONE OF THE
EARLY EXPERIMENTS HE CARRIED OUT
A CELL DIVIDING FOR 15 MONTHS
AND SHOWED EVEN 15 MONTHS THE
VAST MAJORITY OF THE PROGENY OF
THE CELL WERE METHYLATED AND
SILENCED.
EVEN MORE RECENTLY ANGELO USING
A CRISPR OFF TARGETED TO SHOW IN
THE MOUSE OVER A YEAR YOU COULD
MAINTAIN THE MENLATION AND
TARGETING OF THE GENE AND REMOVE
THE FRACTION OF THE LIVER AND
THE LIVER BEING ONE FEW ORGANS
THAT CAN REGENERATE AND
REGENERATED LIVER WOULD MAINTAIN
THE SILENCING FROM WHAT WAS
IMPARTED PRIOR TO THE REMOVAL OF
THE PART.
SO FINALLY, THIS WAS REALLY
HIGHLY SPECIFIC.
HERE WE'RE TARGETING THREE
DIFFERENT GENES AND SHOWING YOU
THE RNA SEQ DATA ANALYSIS AND
THIS IS NOT THE FIGURE LEGEND
BUT THE TARGETED GENE IN THE
GENOME.
THE REASON THIS WORKS IS BECAUSE
THIS SHOWS WHEN WE TARGET WE GET
NICE METHYLATION AT THIS POINT
AND NO METHYLATION ON THE STRAND
FOR THE REMAINDER OF THE DNA.
THE REASON IT WORK AND THE
PICTURE TO HAVE IN MIND IS MOST
CPGs IN THE GENOME ARE
METHYLATED.
IT'S ONLY THESE CPG ISLANDS AND
RICH REGIONS IN GENES EXPRESSED
THAT HAVE THIS SMALL WINDOW OF
UNMETHYLATED CPGs.
AND ESSENTIALLY WHAT THE CRISPR
IS DOING IS COMING IN AND FILL
IN THE METHYLATION IN THE CPG
ISLAND BUT IT'S NOT TARGETING
THE CPGs IN THE PROXIMAL GENE.
IN THIS WAY WE'RE ABLE TO
SILENCE IN A HERITABLE AND
STABLE MANNER WITHOUT AFFECTING
THE OTHER GENES.
THAT'S WHERE WE STOOD IN THE
FAIRLY RECENTLY.
WE THOUGHT THIS WOULD BE A GREAT
TOOL FOR UNDERSTANDING
METHYLATION AND SILENCING AND
HAD THERAPEUTIC POTENTIAL BUT
THAT WAS IN MY DISCLOSURES LUKE
GILBERT AND I TOGETHER WITH
ANGELO AND STARTED A COMPANY
THAT WOULD TRY TO USE THIS TO
THERAPEUTIC SILENCING AND
THOUGHT THERE'D BE A SEPARATION
OF CHURCH AND STATE AND IT WOULD
BE GREAT TO DEVELOP THE
THERAPEUTICS AND WE'D TWO OTHER
SCIENCE WE'RE BETTER SUITED TO
DO.
THAT WAS THE CASE UNTIL ALMOST A
LITTLE UNDER TWO YEARS AGO WHEN
ALMOST 10 YEARS TO THE DAY
JENNIFER WALKED IN MY OFFICE,
THEY WALKED IN MY OFFICE AND
SAID DO YOU THINK YOU CAN DO
EPIGENETIC SILENCING TO SILENCE
THE PRION PROTEIN?
SO SONIA AND ERIC WON A LAB IN
THE BROAD INSTITUTE NEXT TO US
DEDICATED DEVELOPING
THERAPEUTICS FOR THE PRION
DISEASE AND AS SONIA SAYS AND I
URGE YOU TO WATCH HER RECENT TED
TALK SHE SAYS THIS PROBLEM WAS
PERSONAL TO THEM.
SONIA WAS A HARVARD TRAINED
LAWYER AND ERIC WAS A CITY
PLANNER AND SONIA'S MOTHER DIED
OF FATAL FAMILIAL PION DISEASE
AND THEY STOPPED WHAT THEY WERE
DOING AND WENT TO GRADUATE
SCHOOL AND 12 YEARS OR SO LATER
HAD THIS LAB AT THE BROAD
DEVELOPING THERAPEUTICS.
SO IT WAS ALSO A COMPELLING
REASON TO WORK WITH THEM AND
IT'S BEEN WONDERFUL.
THEY'RE TRUE SCIENTIFIC PARTNERS
ON THIS AND IT'S BEEN TERRIFIC
COLLABORATION THEY'VE PROPELLED
FORWARD.
ALSO THERE'S A VERY FROM A WELL
VALIDATED TARGET AND PRIONS ARE
AN INFECTIOUS PROTEIN.
THE CAUSE OF THE SLOW VIRUS THAT
WAS DISCOVERED AND THEN IT WAS
SHOWN IT WAS NOT A VARIETY BUT A
MISFOLDED FORM OF AN ENDOGENOUS
NATURAL PRION PROTEIN.
THE BASIS OF THE PATHOLOGY IS IT
EITHER THROUGH SPORADICALLY,
THROUGH INFECTION FOR EXAMPLE
CONSUMPTION OF INFECTED MEAT IN
THAT CASE MAD COW OR THROUGH
MUTATIONS IN THE CASE OF THINGS
LIKE TSS OR FATAL FAMILIAL
STRAINS AND THE AGGREGATED FORM
FORMED A SEED AND COULD USE
ENDOGENOUS PROTEIN AS A
SUBSTRATE TO CONVERT TO MAKE
MORE OF AND IN THIS WAY YOU GET
AN EXPERIENTIAL PROCESS.
MORE OVER, WE KNEW FROM WORK
STARTED ABOUT 20 YEARS AGO FIRST
MAMMALS CAN SURVIVE WELL WITHOUT
THE PRION PROTEIN.
IT'S NOT EXACTLY CLEAR WHAT IT'S
DOING.
THE LOSS OF FUNCTION PHENOTYPES
ARE NONE OR MILD NEUROPATHY
AFTER EXTENDED PERIOD OF TIME
AND SECOND IS IF YOU EVEN IN THE
ACTIVELY INFECTED MOUSE
SYSTEMATIC FOR THE PRION
DISEASE, IF YOU SHUT OFF IN
NEURONS THE CONTINUED EXPRESSION
OF THE NATURAL PRION PROTEIN YOU
NOW DEPRIVE THE PROCESS OF A NEW
SUBSTRATE TO MAKE MORE
INFECTIOUS PROTEIN AND THE
EXISTING INFECTED PROTEIN
INSTEAD OF GROWING EXPONENTIALLY
DECAYS AND THIS IS ENOUGH TO
INTERFERE AND PREVENT THE
FURTHER DISEASE PROCESS.
SO WE HAD CLEAR THERAPEUTIC
HYPOTHESIS IF WE SILENCE THE
PROTEIN IT WILL HAVE A
THERAPEUTIC AFFECT IN A PREV
PREVENTATIVE FORM FOR PEOPLE
LIKE SONIA WHO INHERIT THE
BETWEEN -- GENETIC MUTATION AND
SOMEONE WHO THE ACTIVE INFECTION
GOING ON.
FIRST THING WE DID AND I SAY WE,
EDWIN NEWMAN A TERRIFIC STUDENT
AND A WONDERFUL POSTDOC IN THE
LAB WORKED CLOSELY ON THE
PROJECT.
FIRST THING THEY DID WAS SEE IN
CELLS CAN WE SILENCE THE PRION
PROTEIN?
THE ANSWER IS YES.
YOU COULD TRANSIENTLY EXPRESS
THE CRISPR OFF TARGETING THE
FWEEN AND THIS WOULD CAUSE
HERITABLE SILENCING OF THE PRION
PROTEIN AND ACCOMPANIED BY
METHYLATION OF THE PRION GENE.
WE LAD A POSSIBILITY OF
GENETICALLY SILENCING THE GENE.
THE CHALLENGE IS NOW HOW DO YOU
DELIVER?
THIS IS THE CHALLENGE OF ALL
GENETS IC -- GENETIC MEDICINE
THE RIGHT CELLS AT THE RIGHT
TIME AND ESSENTIALLY AT THIS
POINT THE ONLY THING VIABLE AND
APPROVED METHOD THAT HAD THE
HELP OF GETTING ACROSS THE CNS
WAS A VIRAL VECTOR.
THE PROBLEM WAS CAS 9 IS A BIG
PROTEIN AND THE GENOME SIZE IS
LIMITED.
THE CRISPR AS WE INITIALLY
DESIGN IT HAD NO CHANCE OF
THINNING IN THE AAD AND YOU MAY
BE ABLE TO BY WORKING WITH
DIFFERENT CASTS YOU MAY GET IT
IN.
IT WAS CLEARLY NOT THE BEST DNA
BINDING DOMAIN TO DELIVER THE
SILENCING.
IN FACT A MORE ATTRACTIVE ONE
WAS AN OLDER TECHNOLOGY WHICH
WAS MUCH SMALLER AND BASED ON
HUMAN PROTAIN.
YOU DON'T HAVE CONCERN ABOUT
ANTIGENISTIC AND WHEN YOU NEED A
BUBBLE TO GET THE AMYLASE IN OR
A NICK FOR THE REVERSE
TRANSCRIPTASE WE'RE LOOKING AT
THE PROTEIN AND IT WAS
INCUMBRANCE TO THE PROCESS WHERE
THE FINGERS SEEM LIKE A MORE
NATURAL DELIVERY FOR OUR
EPIGENETIC SILENCERS.
THIS IS THE SIZE OF THE AAG
GENOME AND WHAT THE FINGER
VERSION WOULD LOOK LIKE.
WHEN YOU WORK CLOSELY WITH THE
SPECIALIST AND WHEN WE MADE OUR
FIRST FING VERSION AND HOE WOULD
MAKE THE AA V FOR US WE GOT AN
E-MAIL BACK SAYING IT LOOKS
GREAT BUT CAN YOU ADD STUFF
BECAUSE IT'S SMALL AND ARAID
OTHER DNA WILL GET IN SO YOU CAN
APPRECIATE THE IRONY OF THAT.
THAT SEEMED SIMPLE WE'D GET OUR
ZINC FINGER AND TARGET THE
PROMOTER AND DELIVER AND THEN WE
GOT A RUDE UNFORTUNATE RESULT
AND THAT YOU WERE HIGHLY TOXIC
EVEN TO CANCER CELL LINES WHEN
WE TRANSIENTLY DELIVERED THEM
DUE TO THE METHYL TRANSFERASE
DOMAIN AND WE MADE ANALOGOUS
VERSIONS WHERE WE REMOVED THE
DOMAIN AND GOT RID OF THIS
TOXICITY.
WHY WAS IT TOXICITY?
I'LL HAVE TO TELL YOU A LITTLE
BIT OF A DETAIL ABOUT AND HOW WE
WERE ABLE TO ENGINEER AROUND THE
TOXICITY AND THERE'S A METHYL
TRANSFERASE AND IT EXISTS IN THE
CELL EN AN AUTO INHIBITED FORM
AND WE'VE REMOVED ALL THE
INHIBITORY DOMAINS AND TAKE THE
TRANS TERRASE AND FUSE IT WITH
THE 3L AND THIS THEN GAVE US A
COMPLETELY UNREGULATED METHYL
TRANSFERASE ONCE YOU METHYLATE
YOU HAVE THE METHYL TRANSFERASE
TO PROPAGATE AND THERE WAS A
MUTIGEN WHERE ANY GENE IT HIT IT
COULD POTENTIALLY TURN OF.
WE ALSO KNEW THANKS TO DECADES
OF FUNCTIONAL WORK THAT HOW THIS
WAS RELEASED.
AN IT'S SNIFFING AROUND AT THE
CHROMOSOME STATES ON THE
HISTONES AND THE TAIL IN THE H3
IS THE UNMETHYLATED FORM AND
IT'S AN INDICATION OF AN
INACTIVE PROMOTER AND THE KEY
THAT RELEASES THIS AUTO
INHIBITION.
WE KNEW FROM A RECENT PAPER
ABOUT THE SAME TIME THAT SHOWED
IN VITRO PEPTIDE FROM AGE 33
TAIL AND WE KNEW 3L WAS
SUFFICIENT TO RECRUIT AND
RELYING ON THE ENDOGENOUS DNMT3L
AROUND HYPOTHESIZED IF WE COULD
PUT IN THE HISTONE TAIL WE CAN
RECRUIT THE TRANSFERASE ONLY
WHERE WE HAD PROGRAMMED TO PLACE
THE 3L.
SO HE MADE VARIOUS VERSIONS.
THE ORIGINAL CHRIS PER AN
RECRUIT THE 3L AND WINNER WITH
THE HOSE HIS TONE TAIL.
REMARKABLY IT WORK AND TOOK
ITERATIONS AND WAS ABLE TO GET
TO WORK ROBUSTLY.
CRISPR I IS TRANSIENT AND
PERMANENT AND INHERITABLE AND
THE ONE WITH THE HISTONE TAIL
WORKS IN THIS CASE ALMOST AS
WELL AND I'LL SHOW YOU AFTER
SOME ITERATIONS IF NOT BETTER
THAN THE ORIGINAL CRISPR.
THIS WAS TERRIFIC BUT YOU GET A
NICE RESULT AND HAVE THIS
ELEGANT MODEL AND THINK WHAT
COULD BE WRONG.
YOU HAVE MOMENTS OF ANXIETY.
IS THIS WORKING THE WAY WE THINK
AND MAYBE ITION AND IT ENTAILS
SOMETHING AND KNEW THE LYSIN 4
WAS REQUIRED AND EDWARD MADE A
VARIANT AND CHANGED ONLY THE
SINGLE LYSIN AND THAT'S AB ROW
FA
GAITED AND GAVE HIM CONFIDENCE
AND THOUGHT HE'D OFFER THE
TRANSFERASE AND THAT'S A
MOUNLFUL BUT CHARM IS MEMORABLE
AND MADE MULTIPLE ITERATIONS OF
CHARM WITH TETHERS AND A KEY
EVENT WAS TO GO THROUGH THIS AND
THE EUROPEAN WOOD MOUSE WAS THE
WINNER.
THROUGH THE VARIOUS ITERATIONS
HE WAS ABLE TO GET CRISPR BASED
CHARM THAT WORKED AS WELL AS THE
CRISPR ALL IN THE MAGNITUDE OF
THE EFFECT.
HE PUT THIS ON A ZINC FINGER AND
ELIMINATE THE TOXICITY IN CELL
LINES AND WAS ABLE TO PROMOTE
AND NICELY INHERITABLY SILENCE
THE PRION PROTEIN AND
METHYLATING THE CPG ISLANDS AND
THE PRION.
NOW HE WAS FINALLY ABLE TO TO
THE EXPERIMENT.
HE HAD A NON-TOXIC CFP CHARM
THAT READILY FITS INTO AN AAV.
WE GAVE THE DESIGN TO BEN TO PUT
IN HIS CNS TARGETING PHP AND
THIS IS GREAT AT TARGETING THE
MOUSE FRAME BUT DOESN'T TARGET
OTHER STRAINS OF MICE AND
CERTAINLY NOT HUMANS BUT
RECENTLY BEN HAS A BEAUTIFUL
PAPER ON THE HUMAN TARGETING AAV
BASED ON THE TRANSPARENT
RECEPTOR AND OTHER COMPANIES
INCLUDING REGENERON AMONG OTHERS
HAVE TARGETING TO HUMAN BRAIN.
IT'S STILL A WORK IN PROGRESS
BUT THINK THERE'S POTENTIAL TO
BE TRANSLATED TO HUMANS.
WE USED THE PHB AND THE EMPTY
AAV OR CF CHARM TARGETING THE
PRION PROTEIN AND GET 80% TO 90%
OF THE PRION PROTEIN EXPRESSION
AND FROM THE MOUSE FRAMEWWORK W
THROUGH THE FRAMEWORK AND SEE
THIS ACROSS THE CNS AND SHOWED
WHEN THE VAST MAJORITY OF
NEURONS WE ARE SWITCHING ON AND
OFF THE PROTEIN.
IT'S NOT A FOREGONE CONCLUSION.
THERE'S A CRITICAL ROLE IN
PROPAGATION BUT THAT'S ONLY TRUE
IF THE CELLS ARE DIVIDING AND
DNA IS BEING REPLICATE AND
TARGETING THE POSTMITOTIC GENE.
AND I TOLD YOU THIS IN THE
HIT-AND-RUN MANNER.
WE DON'T WANT THE CHARM TO BE
EXPRESSED AS LONG AS AD IN THE
NEURONS AND THEY'RE NOT DIVIDING
AND WOULDN'T GET DILUTED OUT AND
THERE WAS A SELF-SILENCING
STRATEGY IN WHICHD IN THE
NEURONS AND THEY'RE NOT DIVIDING
AND WOULDN'T GET DILUTED OUT AND
THERE WAS A SELF-SILENCING
STRATEGY IN WHICH INTHE
NEURONS AND THEY'RE NOT DIVIDING
AND WOULDN'T GET DILUTED OUT AND
THERE WAS A SELF-SILENCING
STRATEGY IN IN THE
NEURONS AND THEY'RE NOT DIVIDING
AND WOULDN'T GET DILUTED OUT AND
THERE WAS A SELF-SILENCING
STRATEGY IN A IN THE
NEURONS AND THEY'RE NOT DIVIDING
AND WOULDN'T GET DILUTED OUT AND
THERE WAS A SELF-SILENCING
STRATEGY IN WHICHV IN THE
NEURONS AND THEY'RE NOT DIVIDING
AND WOULDN'T GET DILUTED OUT AND
THERE WAS A SELF-SILENCING
STRATEGY IN WHICH AND PUTTING
TWO COPIES, A PERFECT MATCH OR
SCRAMBLED KINETICS TO MAKE SURE
IT OCCURRED RAPIDLY PUT NOT
ENOUGH EXPRESSION TO SILENCE THE
PRION GENE ITSELF.
SHE WAS ABLE TO TUNE THIS AND
THIS IS THE GOLDILOCKS
EXPERIMENT WITH THE PAPA BEAR
AND MAMA BEAR AND THE BABY BEAR
THAT WAS RIGHT AND SILENCED THE
PROTEIN AND THE PROMOTER ITSELF
AND SHOWED THIS IN CELL LINES AT
GOT THIS ALLELIC SERIES OF
SELF-SILENCING EITHER TOO SLOW
OR TOO FAST OR JUST RIGHT IN THE
MOUSE AND THIS WAS ALSO WORKING
BIG METHYLATING THE AAV ITSELF
SO IT WOULD BE WE THINK IN A
PERMANENTLY SHUT OFF STATE.
SO IN THE END WHAT WOULD BE LEFT
IN THE MOUSE WOULD TRANSIENTLY
EXPRESS THE CFP CHARM AND THEN
GO IN AND METHYLATE THE
ENDOGENOUS GENE AND LEAVE THE
ANIMAL WITH THE TURNED OFF
PROTEIN AND UNEXPRESSIBLE PIECE
OF DNA AS CLOSE TO THE PERFECT
REPAIR AS WE COULD GET.
NOW WE'RE ABLE TO DO THE
EXPERIMENT.
THESE ARE LONG EXPERIMENTS SO
IT'S A WORK IN PROGRESS.
THEY INFECTED A MOUSE AND AFTER
FOUR MONTHS WHEN IT'S BECOMING
SYMPTOMATIC THEY EXPRESSED MOCK
VIRUS OR THE CHARMS BOTH CELL
SILENCING OR NON-FORM AND BY 190
DAYS AS WE EXPECT ALL THE
UNTREATED ANIMALS ARE DEAD AND
NOW A FEW MONTHS OUT THE LARGE
MAJORITY BUT NOT ALL THE TREATED
ANIMALS ARE SURVIVING AND IN
SOME MEASURE STARTING TO
IMPROVE.
THIS IS OUR FIRST HINT IT COULD
BE A VIABLE THERAPY AND THE
CHALLENGE OF DELIVERY TO THE
HUMAN BRAIN SHOULD NOT IN ANY
WAY BE UNDERESTIMATED BUT WE NOW
HAVE TAKEN THE HUGE BIOLOGICAL
PROBLEM OF THIS HERITABLE
MUTATION, MYSTERIOUS DISEASE AND
COME TO THIS DISCREET
ENGINEERING PROBLEM WE HOPE
WE'LL BE ABLE TO ADDRESS IN THE
NEAR FUTURE.
AND MORE GENERALLY WE THINK THE
CHARMS WILL BE A POWERFUL NEXT
GENERATION OF EPIDEMIOLOGIC
SILENCERS AND DON'T REQUIRE
EXPRESSION OF ACTIVITY AND
RECRUIT ENDOGENOUS AND NOT
EXPECTED TO BE PARTICULARLY
MUTIGENIC AND EFFICACIOUS AND
FOR ANY MODALITY WHETHER AAV OR
LNP BECAUSE THEY'RE A SIMPLE
SINGLE PROTEIN AND DON'T REQUIRE
GUIDE RNA THEY'LL BE MORE
READILY DELIVERABLE.
AND STEPPING BACK THIS IS PART
OF MUCH BROADER EFFORT OF WHERE
WE CAN USE CRISPR-BASED
FUNCTIONAL GENOMICS AS A WAY OF
IDENTIFYING TARGETS THAT ARE
DISEASE MODULATING AND
UNDERSTANDING THEIR MECHANISM ON
ONE HAND AND ON THE OTHER HAND
WE CAN THEN COME IN AND USE
CRISPR OR FINGER-BASED TOOLS TO
DIRECT A PROGRAM TO ADDRESS AND
TO AFFECT THE CHANGE THAT WE NOW
UNDERSTAND IS GENE MODULATORY
AND GAVE US THE TOOLS TO
MODULATE.
WITH THAT I'LL FINISH AND
IMPORTANTLY I THANK THE PEOPLE
WHO DID THE WORK.
I'VE TRIED TO MENTION THEM ALONG
THE WAY BUT IF IT BEARS
REPEATING, EDWIN AND TESSA AND
MANY ELAINE WU WORKING WITH
SONIA AND BEN LED THE WORK AND
GOING IN TRE FIRST CONCEPTION
AND FULL SPEED AHEAD FOCUSSING
ON THE HUMAN PRION PROTEIN.
AND RUBEN AND JOSEPH WORKED IN
COLLABORATION AND REUBEN WITH
WILL AND OUR COLLABORATION LEAD
THE NEXT GENERATION OF IN VIVO
FUNCTIONAL GENOMICS.
WIN THAT I'LL END AND I'LL TAKE
ANY QUESTIONS.
>> FIRST THE CME CODE IS 50128
AND SONIA AND ERIC GAVE A WALs
TALK AND THE ARCHIVE IS
AVAILABLE IF ANYONE WANTS TO
WATCH.
>> BEAUTIFUL AS ALWAYS.
ONE GENERAL QUESTION IS NOW THAT
YOU HAVE SPECIFIC EDITORS LIKE
BASE EDITING AND YOU CAN
TARGET -- WHAT ARE THE
ADVANTAGES OF THE PERMANENT
SILENCING OPPOSED TO INTRODUCING
POINT MUTATION THAT WILL
INTRODUCE A STOP.
>> WHY WOULD YOU PUT IN A STOP
CODE THAT CREATES A DAMAGED
PROTEIN AND MESSAGE THAT WOULD
EXPRESSED FOR THE REST OF THE
LIFE OF THE ORGANISM THAN
PREVENTING EXPRESSION OF THE
GENE?
SO IF YOU HAVE A GENETIC
MUTATION LIKE SICKLE CELL
TRAITOR MUTATION IN CFTR AND
NEED TO CORRECT THAT, FOR SURE
THE BASE EDITING AND PRIME
EDITING APPROACHES ARE POWERFUL
APPROACHES BUT IF YOU WANT TO
TURN OFF OR TURN UP THE FUNCTION
OF GENE, THE EPIGENETIC
MODULATORS ARE A MORE NAH
NATURAL THING.
WE HAVE A TOOL KIT OF PRECISION
MROJ LATERS AND WE'RE NOW
GETTING TOOLS THAT ALLOW US TO
PUT IN LARGER PIECES OF DNA AS
WELL.
I THINK IT'S BETTER TO SEE THE
CHANGES WANT TO MAKE AND WHAT
ARROWS DO WE PULL OUT OF THE
QUIVER TO DO THIS?
THAT SAID GENETIC DISEASES ARE
VERY IMPORTANT BUT THE MECHANISM
OF DRUG IS TO MODULATE THE
FUNCTION.
THE GENETIC MODULATORS ARE AN
IMPORTANT PART BUT NOT THE WHOLE
PART.
>> IS THIS NOW MORE EFFICIENCY?
SHOULD WE USE THESE TOOLS IN THE
RAB WHEN WE DO KNOCKOUTS INSTEAD
OF THE USUAL CAS 9?
>> SO WE DID DO GENOME WIDE
CRISPR OFFERS AND I AND IT WAS
GENERALLY MORE EFFECTIVE.
WE DON'T HAVE THE SAME -- IT'S
SOMETHING WE WANT TO DO
ESPECIALLY WITH THE CHARM FORMAT
WHERE YOU DON'T HAVE TO EXPRESS
THE METHYL TRANSFERASE.
WE SAW THE EXPRESSION AND
TOXICITY AND THAT WAS HESITATION
TO GO CRISPR OFF SCREEN AND NOW
WITH THE CHARM IT COULD BE A
MORE EFFICACIOUS FORM AND MORE
SWITCH LIKE AND IF YOU WANT TO
GO AFTER CENTRAL GENES AND LOSS
OF FUNCTION BUT IF YOU WANT A
TRUE NUL THE CRISPR WILL BE
BETTER AND IT'S PRETTY MUCH THE
SAME LIBRARY WHICH IS A NICE
VITRO OF THIS.
>> I'M EXCITED ABOUT YOUR
VIRTUAL CELL PROJECT OR YOU
MENTIONED PART OF THAT.
WHEN IT FINISHES IT WILL MAKE
HALF OF BIOLOGY OBSOLETE SO
BASED ON MY CAREER AND ANXIETY
SO ARE IN THE MULTI-DIMENSIONAL
SPACE ONE AT A TIME.
ARE THERE ANY PUBLICALLY
AVAILABLE INFORMATION ABOUT THAT
MAPPING AND DID YOU ACHIEVE FIND
PROPERTIES TO SEE HOW CLOSE THEY
ARE?
>> SO FIRST, THE VIRTUAL CELL
LINE THERE'S A LOT OF PEOPLE OF
INTEREST IN THIS AND I THINK
IT'S GOING TO BE A LONGER TERM
PROJECT SO I THINK WE ALL HAVE A
JOB FOR AT LEAST A LITTLE WHILE
AND BY THEN THE A.I. OVERLORDS
WILL BE FEEDING US ANYWAY.
IT WILL BE IRRELEVANT.
I THINK MORE SERIOUSLY THAT THE
AN ASPIRATIONAL THING BUT THE
MUCH MORE SPECIFIC THINGS LIKE
PREDICTING THE SPOKE AND THE
IMAGES OF CELL FROM TRANSCRIPT
OR FROM PERTURBATION AND THE
IMAGES ARE THE MORE DIRECT
THINGS AND WHETHER WE CAN BUILD
A FOUNDATION MODEL TO CAPTURE
ALL THESE I THINK IS AN OPEN
QUESTION AND OUR STRATEGY THE
DATA WE HAVE WILL BE USEFUL AND
MOTIVATIONAL FOR THESE EFFORTS
BUT HOW FAR WILL THEY GO?
I THINK WE HAVE A LONG WAY
BEFORE WE'RE ALL OUT OF A JOB.
WE PUBLISHED AND IT'S ALL
AVAILABLE THE MAP WE HAVE.
I THINK WHAT WE HOPE IS WE'LL BE
AND WE'RE SURE WE'LL BE
GENERATING RICHER AND LARGER
SCALE MAPS FROM THE IN VIVO
PHENOTYPES.
I THINK IN MANY WAYS AS WE GO TO
MORE PHYSIOLOGIC CELLS IPS
DERIVED CELLS OR IN VIVO, THE
LOGIC OF THE SYSTEM WILL BE
CLEARER AND MORE INTERPRETABLE.
I GUESS I WOULD SAY WE'RE AT A
PLACE WHERE THE ABILITY TO
COLLECT DATA HAS INCREASED SO
RAPIDLY UP SOME WAYS WE SHOULD
WORK ON THE METHODS BUT ALSO
MOTIVATING WHAT THE ARE NEXT
DATA SETS WE'D WANT.
>> I'D LIKE TO SLIP IN ONE
QUESTION ONLINE.
>> SURE.
>> IT'S A DOOZY.
SO POOLED IN VIVO.
PERTURBATION SCREENS ARE
ATTRACTIVE BUT ALSO SEEMS TO
PRESENT DIFFICULTY IN DATA
INTERPRETATION IT'S CONFOUNDING
THE EFFECT OF THE RNA DEPENDING
ON THE STATE.
IN VIVO CULTURES AT LEAST
PROVIDE REDUNDANT REPLICATES
WITHIN THE SAMPLE.
HOW DO YOU ADDRESS THE
CHALLENGE?
>> I GUESS I WOULD SAY THAT
CELLS MOSTLY FUNCTION AND THIS
IS HOW THEY'VE EVOLVED IN THE
NORMAL FUNCTION.
WE HAVE TO DEAL WITH THE
COMPLEXITY.
THAT SAID IT'S COMPLEX BUT NOT
SO COMPLEX.
YOU HAVE THREE ZONES FROM THE
PERIPORTAL TO THE PERICENTRAL
AND WELL DEFINED.
WE'RE ABLE TO GET GOOD COVERAGE
MUCH EACH OF THE ZONES AND YOU
CAN TELL WHICH ZONE WE'RE IN.
IT HAS A LEVEL OF COMPLEXITY
WHEREAS THE BRAIN THOUSANDS OF
CELL TYPES IT'S GOING TO BE A
REAL CHALLENGE AND I THINK MORE
CELLS, FEWER PERTURBATIONS IS
PROBABLY GOING TO BE THE ROUTE.
YOU NEED A NUMBER OF -- FOR
EVERY CELL STATE AND
NEIGHBORHOOD YOU WANT TO HAVE
EXAMPLES TO REALLY GET A ROBUST
SIGNAL OUT OF IT.
>> OKAY.
MAYBE ONE MORE QUESTION.
I DON'T WANT TO CUT THIS OFF
WE'LL HAVE A RECEPTION.
THIS WILL BE THE LAST QUESTION.
>> THAT WAS BASICALLY MY
QUESTION.
THERE'S A VARIETY OF DIFFERENT
TRANSCRIPTION STATES THAT MAY OR
MAY NOT REPRESENT DIFFERENT
FUNCTIONAL STATES WITHIN VARIOUS
TISSUES AND THE AFFECT OF A
SINGLE GENE PERTURBATION MAY
HAVE DRASTICALLY DIFFERENT
EFFECTS DEPENDING ON THE STATE
OF THE CELL IS THE ANSWER TO
JUST INCREASE THE NUMBER OF
CELLS YOU DO?
>> IT'S ABOUT EXPERIMENTAL
DESIGN AND TO MAKE COMPLEXITY
WORSE, THE LIVER CHANGES ITS
PHYSIOLOGY AND FUNCTION
DRAMATICALLY BASED ON RAPID
CHANGES LIKE FASTING AND HIGH
FAT DIETS AND SYNDROMES AND
FOLLOWING THE EFFECTS OF
PERTURBATION AND PHYSIOLOGY WILL
BE COMPLEX.
YOU HAVE THE COMPLEXITY.
ON THE OTHER HAND YOU GET 300
MILLION HEPATOCYTES FROM THE
LIVER.
YOU DON'T HAVE TO SPLIT YOUR
CELLS.
YOU FEED THE MOUSE CHOW AND KEEP
THEM IN A PHYSIOLOGIC STATE.
IN MANY WAYS THE SCALE YOU'RE
ABLE TO DO AT LEAST FROM
PRODUCING THE CELLS IS SIMPLER
FROM THE IN VIVO SETTING.
AND IT CAN GENERATE THE IMAGE
BUT IN THE END WE'RE NOT GOING
TO BE ABLE TO COLLECT ALL THE
DATA.
AND THERE'S MORE SOPHISTICATED
FOUNDATION MODELS AND HOW WE
FILL OUT THE SPACE THE REST CAN
BE IMPUTED ARE BIGGER, BROADER
CHALLENGE.
>> THERE'S A RECEPTION AND WE
MIGHT RUN OUT OF FOOD THIS TIME
BECAUSE OF THE SIZE OF THE
AUDIENCE SO THAT'S GREAT AND
THANK YOU ONCE AGAIN.
>> THANK YOU.