WELCOME TO THE COVID-19
SCIENTIFIC INTEREST GROUP
LECTURE SERIES.
THIS IS THE NO GROW LECTURE FOR
THE NEW YEAR, 2022.
WE ARE DELIGHTED TO HAVE DR.
MELANIE OTT KICKING IT OFF FOR
US.
DR. OTT IS THE DIRECTOR OF THE
GLADSTONE INSTITUTE OF VIROLOGY
AND PROFESSOR OF MEDICINE AT
UCSF.
SHE'S A WORLDWIDE LEADER IN
INVESTIGATIONS INTO THE HOST
VIRUS INTERFACE.
STUDYING BOTH VIRAL LIFE CYCLE
AND USING VIRUSES AS TOOLS TO
MAKE DISCOVERIES ABOUT THE INNER
WORKING CELL.
SHE'S MADE IMPORTANT DISCOVERIES
ABOUT HOW VIRUSES INCLUDING
HEPATITIS C ZIKA, HIV AND TODAY
YOU WILL HEAR COVID, HIJACK
HUMAN CELLS.
HER STUDIES ON HIV IN PARTICULAR
ABOUT LATENCY PHASE LED TO
SIMILAR KNOLL DISCOVERIES HOW
PROTEINS MANIPULATE THE
EPIGENETIC CODE CHANGE CHROMATIN
ORGANIZATION AND CONTRIBUTE TO
PREMATURE IMMUNE CELL AGING BY
MANIPULATING CIRCUMSTANCE
PROTEINS.
HER WORK ON HCV LED TO DEEPER
UNDERSTANDING HOW VIRUSES
REGULAR RATE THE LIPID
METABOLISM OF CELLS AND HER WORK
ON ZIKA AND HCV REVEAL
MECHANISMS WHICH VIRUSES
MODULATE RNA METABOLISM OF CELL
PERTURBING NON-SENSE MEDIATED
mRNA PATHWAYS AND RNA IMPORT
EXPORT PATHWAYS.
SHE ALSO IS TOGETHER WITH
JENNIFER AND DAN FLETCHER HAS
BEEN DEVELOPING CRISPER BASED
VIRUS TESTING PLATFORMS THAT DO
NOT NEED TO BE CARRIED OUT IN A
LAB USE MOBILE PHONE
TECHNOLOGIES, AND DELIVER
QUANTITATIVE RESULTS IN SHORT
AMOUNTS OF TIME.
MOST RECENTLY LIKE THE REST OF
THE WORLD HER FOCUSES TURN TO
SARS COV-2 AND TODAY YOU WILL BE
HEARING ABOUT SOME RECENT WORK
THAT JUST CAME OUT IN SCIENCE
WHERE SHE AND DR. CONNER
DEVELOPED COV-2 VIRUS LIKE
PARTICLES TO LOOK AT IMPACT OF
MUTATIONS ON N AND SPIKE
PROTEINS ON NOT ONLY VIRUS ENTRY
BUT INTERESTINGLY RNA RELEASE
AND RNA PACKAGING.
SO WE HOPE TO HEAR ABOUT SOME OF
THAT TODAY.
WITHOUT FURTHER ADIEU, I WOULD
LIKE TO WELCOME DR. OTT TO NIH,
WE LOOK FORWARD TO HEARING HER
TALK.
THANK YOU.
>> THANK YOU SO MUCH NIHAL FOR
THE KIND INTRODUCTION AND
INVITATION TO THIS SEMINAR.
I'M COMPUTE EXCITED TO BE HERE I
THINK WE ARE A LITTLE ZOOMED
OUT, THOUGH IT SEEMS IT WILL BE
WITH US FOR QUITE SOME TIME.
AS NIHAL SAID I'M A VIROLOGIST
FROM SAN FRANCISCO, HAVING
WORKED ON MANY VIRUSES IN THE
PAST AND HIV HEPATITIS C AND
ZIKA.
WE HAVE TURNED OUR ATTENTION TO
SARS COV-2 EXACTLY TWO YEARS AGO
WHEN IT BECAME CLEAR THAT THIS
IS GOING TO BE A WORLDWIDE
PROBLEM.
ONE OF THE FIRST THINGS WE HAVE
TO DO WAS THAT WE HAVE TO
PROVIDE THE ENVIRONMENT FOR US
TO STUDY IT AND THAT IS OF
COURSE BSL 3 LABORATORY THAT
ALLOWS RESPIRATORY PATHOGEN
WORK.
WE HAVE HAD -- WE HAVE A LARGE
BSL 3 THAT ALLOWS US TO WORK
WITH BLOOD BORNE PATHOGENS BUT
WE HAVE TO PIVOT AND BUILD A NEW
BSL 3 LABORATORY FOR RESPIRATORY
VIRUSES WHICH WE DID AND
FINISHED ACTUALLY IN EARLY
SUMMER LAST YEAR AND SINCE THEN
WORKING BASICALLY 24/7 IN THAT
LABORATORY.
THIS IS ACTUALLY TAKEN HERE AT
THE UCSF AT ONE OF THE BSL 3
THERE MY COLLEAGUE ANITA, THIS
IS THE FIRST VIRUS THAT WE
CULTURED AND LOOKING FOR
CYTOPATHIC EFFECTS IN CELL
CULTURE HERE.
THE REASON WE BECAME INTERESTED
IN OR WHY WE THOUGHT WE COULD
CONTRIBUTE TO SARS COV-2
RESEARCH WAS BECAUSE WE HAD TWO
ONGOING PROJECTS IN THE LAB.
ONE IS A TESTING PROJECT THAT
NIHAL MENTIONED TOGETHER WITH MY
COLLEAGUE JENNIFER AND DAN
FLETCHER, BOTH FROM BERKELEY BUT
BOTH ALSO HAVE AFFILIATION WITH
GLADSTONE INSTITUTES.
WHERE WE HAD BEEN LOOKING AT
RAPID CRISPER BASED TESTING FOR
HIV.
AND TODAY MORE THAN EVER IT IS
CLEAR THESE TESTS ARE ACTUALLY
EXTREMELY USEFUL FOR SARS COV-2
POTENTIALLY OTHER VIRUSES, WE
REALLY NEED FREQUENT FAST BUT
ALSO IMPORTANTLY ACCURATE TESTS
THAT WE CAN RELY ON.
SO WE PIVOTED PART OF THIS TEST
TO SARS COV-2 AND I WILL TELL
YOU MORE LATER ABOUT THIS.
THE OTHER THING THAT WE HAVE
GOING IN THE LAB THAT WE THOUGHT
COULD BE HELPFUL FOR THE
UNDERSTANDING OF SARS COV-2 IS
WE HAVE BEEN LOOKING TO WORKING
WITH PRIMARY CELL ORGANOID
MODELS ACTUALLY A FEW YEARS BACK
ALREADY, STARTING WITH HEPATITIS
C AND LIVER ORGANOIDS BUT MOVING
TO INTESTINAL ORGANOIDS AND ALSO
INTO HUMAN AIRWAY ORGANOIDS WE
ESTABLISHED FOR SPECIFICALLY
SOME RESEARCH WE WERE DOING ON
INFLUENZA A AND B TOGETHER WITH
VR BY TECHNOLOGY HERE IN SAN
FRANCISCO.
THESE ARE JUST TO MAKE SURE,
THESE ARE ORGANOIDS THAT ARE
ADULT STEM CELL DERIVED SO
COMING DIRECTLY OUT OF BIOPSY OR
RESECTION MATERIAL FROM THE
RESPECTIVE ORGAN.
IT IS METHOD LANCE HAS PIONEER
AD FEW YEARS BACK TO PUT CELLS
IN CULTURE PROLIFERATE THE
PROGENITOR CELLS AND THEN
DIFFERENTIATE THEM INTO THE
INDIVIDUAL ORGANS.
SO HAVING THESE HUMAN AIRWAY
ORGANOIDS AND OTHERS WE THOUGHT
THAT THIS COULD BE IMPORTANT TO
CONTRIBUTE TO SARS COV-2
RESEARCH AT THE TIME.
INDEED, HAVING THE BSL 3, HAVING
THE VIRUSES AND THEN HAVING ALSO
THE INDIVIDUAL ORGAN SYSTEMS
THAT ALLOWED US REALLY TO LOOK
AT SARS COV-2 INFECTION IN
VARIOUS ORGANS, HERE THE LUNG,
INTESTINE BUT ALSO IN THE HEART
CELLS WHERE YOU CAN SEE GREEN OR
HERE IN RED DOUBLE STRANDED RNA
COMPLEX SYSTEM STAINED IN CELLS
AND YOU SEE HOW DRAMATICALLY
BASICALLY THE VIRUS TAKES OVER
THE CELLS AND REPLY CASE IN
THEM, ESPECIALLY THE HEART
CELLS, WE NOTICE TOGETHER WITH
OUR COLLEAGUE COLLEAGUES HERE AT GLADSTONE
THESE CELLS ARE QUIZZICALLY
SENSITIVE TO OXIC EFFECTS OF THE
VIRUS, EVEN ON THROUGH BINDING
BUT ESPECIALLY REPLICATION CELLS
START TO BE EXTREMELY DESTROYED
AND UNDERGOING SOME FORMS OF
CELL DEATH IF THE VIRUS IS
TOUCHING THEM.
SO WE ARE STILL VERY INTERESTED
IN THAT.
WE HAVE COLLABORATED WITH MANY
PEOPLE HERE AT UCSF AND
ELSEWHERE TO LOOK AT THE
PATHOGENESIS OF THE VIRUS IN
THESE DIFFERENT ORGANS.
TODAY I WANT TO TALK ABOUT THREE
SUBJECTS THAT I POINTED OUT IN
MY TITLE.
I THINK WE WANT TO LOOK ABOUT
DETECTION, STUDY AND TREATMENT
AND I WILL DO THIS IN A LITTLE
BIT DIFFERENT ORDER THAN IS
LOGICAL, BUT I WANT TO START
WITH THE HOST DIRECTED THERAPIES
JUST BECAUSE IT IS SOMETHING
THAT WE HAVE A LONG STANDING
INTEREST IN AND WAS ALSO THE
FIRST THINGS WE CONTRIBUTED TO
SARS COV-2.
LET ME START WITH OUR INTEREST
IN HOST DIRECTED THERAPIES.
I THINK IT DEPENDS -- IT REALLY
STEMS FROM THE FACT THAT VIRUSES
ARE UNIQUELY DEPENDENT ON THE
HOST AND THIS DEPENDENCY IS A
ACHILLES HEEL AND OPPORTUNITY
FOR THERAPEUTIC INTERVENTION.
WE HAVE FANTASTIC ANTIVIRALS FOR
HIV AND FOR HEPATITIS C AND
OTHERS BUT ONE OF THE ISSUES
HERE IS THAT THE VIRUS CAN
DEVELOP RAPIDLY DRUG RESISTANCE
AND WE USUALLY HAVE A VERY
HIGHLY SPECIFIC DRUG THAT WORKS
AGAINST MOSTLY ONE PATHOGEN.
IF WE GO INTO THE HOST SIDE THE
IDEA IS THAT WE HAVE A LESS
PROBLEMS WITH DRUG RESISTANCE
BECAUSE WE CANNOT EVOLVE AS FAST
AS THE VIRUS.
AND BECAUSE OF OUR OWN RESEARCH
AND OTHERS WE KNOW THAT MANY
VIRUSES EVEN UNRELATED VIRUSES,
FROM THE CLASS STAND POINT HAVE
DEPENDENCIES ON SIMILAR HOST
PATHWAYS.
SO THE IDEA IS TO FIND THESE
PATHWAYS, TO DEVELOP DRUGS
AGAINST AND WITH THIS TO
ACTUALLY HIT NOT ONLY ONE
PATHOGEN BUT MULTIPLE PATHOGEN,
THE IDEA OF A PAN ANTIVIRAL AND
ALSO THE IDEA OF BEING BETTER
PREPARED FOR SOMETHING THAT SARS
COV-2 THAT IS GOING TO BE
POTENTIALLY EVOLVING IN THE
FUTURE EMERGING VIRUSES THAT WE
HAVE NOT TIED YET TO STUDY IN
DETAIL TO DEVELOP THESE VERY
DETAILED LASER SHARP FOCUS
ANTIVIRALS DIRECT AFFECTING
ANTIVIRALS.
WITH THIS WE HAVE A LONG
STANDING COLLABORATION WITH MY
COLLEAGUE NEVIN KROGAN UCSF AND
SEEN YOUR INVESTIGATOR AT
GLADSTONE AND DIRECTOR OF
QUANTITATIVE BIOLOGY INSTITUTE
HERE AT UCSF WHERE WE HAVE BEEN
LOOKING AT MANY OF THESE MAPS OF
HOST VIRAL INTERACTOMES.
PROTEOMICS BASED MASS
SPECTROMETRY BASED LOOKING AT
EACH INDIVIDUAL VIRAL PROTEINS
AND IDENTIFY BY MASS
SPECTROMETRY THE PROTEINS THAT
CONTACT THESE PROTEINS ARE
INTERACTING WITH VIRAL PROTEINS
IN CELLS.
SO NEVIN AND A LARGE GROUP OF
INVESTIGATORS HERE AT UCSF HAVE
MANAGED TO BUILD UP THE SARS
COV-2 INTERACTOME BY APRIL 2020
WHERE WE COULD IDENTIFY 332 SARS
COV-2 INTERACTERS AND BY JUST
LOOKING INTO THESE INTERACTERS,
69 OF THEM WERE DRUGGABLE HOST
CHARACTERS WHERE FDA APPROVED
DRUGS WERE AVAILABLE AND SO THIS
WAS THE FIRST START TO LOOK INTO
THE POTENTIAL OF HOST DIRECTED
THERAPIES AGAINST SARS COV-2 AND
THIS WORK IS VERY MUCH ONGOING
WITH MANY BASIC BIOLOGISTS HERE
AT UCSF LOOKING INTO
STRUCTURALLY OR FUNCTIONALLY
INTO THESE INTERACTIONS AND US
LIKE OTHER VIROLOGISTS AROUND
THE WORLD TESTING WHETHER ANY OF
THESE DRUGS HAVE AN EFFECT ON
SARS COV-2 IN VITRO OR IN MOUSE
OR HAMSTER MODELS.
THE OTHER WAY HOW YOU CAN
APPROACH HOST DIRECTED THERAPIES
IS NOT JUST LOOKING AT THIS
PROTEINS THAT ARE INTERACTING
WITH VIRAL PROTEINS, YOU CAN
ACTUALLY LOOK FUNCTIONALLY AT
THE WHOLE -- AT ALL HUMAN
PROTEINS AND SEE WHICH ONES OF
THEM ARE ACTUALLY DEPENDENCY
FACTORS OF THE VIRUS, THIS IS A
CLASSICAL CRISPR SCREEN WHERE
YOU KNOCK OUT INDIVIDUAL HOST
PROTEINS, INFECT THE CELL AND
THEN IN THIS CASE IT IS A LIVE
DEATH SELECTION AND LOOK FOR
SURVIVING CELLS THAT ARE NOT
KILLED BY THE VIRAL REPLICATION.
SEQUENCE THE GENE THAT IS
EXPRESSED OR THAT IS KNOCKED
DOWN IN THE CELLS AND THEN
ASSEMBLE THE LIST OF THE
DEPENDENCY FACTORS FOR AN
INDIVIDUAL VIRUS.
SO WE DID THIS ALSO VERY EARLY
IN 2020 TOGETHER WITH MY
COLLEAGUE ANDREAIAS PUSHNIK AT
THE ZUCKERBERG BIOHUB AT SAN
FRANCISCO AND WE ARE NOT ONLY
FOCUSED ON SARS COV-2 BUT
PARALLEL INCLUDE TWO COMMON COLD
CORONA VIRUSES 229E AND OC 43
JUST TO GO PRECISELY AT THE
QUESTION ARE THEY PAN VIRAL
FACTORS THAT WE CAN TARGET IN
THE FUTURE TO NOT ONLY TARGET OR
HIT SARS COV-2 BUT ALSO RELATED
CORONA VIRUSES.
THIS IS THE WORK THAT WAS DONE
BY PETER WONG AND ANDREIAS'S LAB
AND MY LAB, CAMILLE HAS SINCE
GRADUATED AND IS POST-DOC IN THE
LAB READY TO TAKE HER NEXT STEP.
SO WHEN WE DID THIS AS I
EXPLAINED BEFORE, WE IDENTIFIED
SEVERAL HOST FACTORS FOR SARS
COV-2 ENRICHED IN THIS SCREEN,
VERY IMPORTANTLY WE IDENTIFIED H
2 AS THE ENTRY FACTOR BUT
NOTICED THERE WERE QUITE A FEW
PROTEINS THAT ARE IMPORTANT IN
CHOLESTEROL HOMEOSTASIS AS WELL
AS LYSOSOME AUTOPHAGESOME
RELATED FUNCTIONS.
229E HAD MORE PROTEINS THAN WE
IDENTIFIED.
AGAIN, THE RECEPTOR WAS AMONG
THE HIGHEST ENRICHED GENES BUT
WE ALSO FOUND QUITE A FEW GOLGI
RELATED FUNCTION PROTEINS THAT
WERE HIGHLY ENRICHED.
WHILE THE OC 43 IS A GLYCAN
BASED RECEPTOR AND ENTERING
VIRUS SO THE GLYCOSAMINOGLYCAN
BIOSYNTHESIS PATHWAY WAS HIGHLY
ENRICHED IN THESE AMONG OTHERS
THAT WERE SHARED WITH THE OTHER
TWO CORONA VIRUSES.
WHEN WE LOOK AT THE OVERLAP
BETWEEN THESE THREE VIRUSES IN
TERMS OF INDIVIDUAL GENES,
COMPARING HERE SARS COV-2 AND
229E WE DIDN'T SEE MUCH OVERLAP,
JUST THE LDL RECEPTOR HERE.
LITTLE BIT MORE BETWEEN SARS
COV-2 AND OC 43.
BUT THEN QUITE A BIT BETWEEN THE
TWO COMMON COLD CORONA VIRUSES.
SO THIS EITHER INDICATED THAT
SARS COV-2 WAS UNIQUE, DIFFERENT
FROM THEM, BUT WE ALSO THOUGHT
IT MIGHT BE THAT AT -- THAT THE
OVERLAP MIGHT NOT BE HAPPENING
AT THE GENE LEVEL BUT MIGHT BE
HAPPENING AT THE PATHWAY LEVEL.
SO WE WORKED WITH NEVIN'S GROUP
AND DID A NETWORK PROPAGATION
ANALYSIS WHERE WE REALLY LOOKED
AT THE NETWORKS THAT WERE
ENRICHED BETWEEN THE THREE VIRAL
INFECTIONS.
WE COULD IDENTIFY QUITE A FEW,
ONE WAS CHOLESTEROL METABOLIC
PATHWAY, ALSO IT WAS HIGHLY
ENRICHED WITH SARS COV-2, THE
OTHER PROTEINS, THE OTHER
VIRUSES ALSO HAD CHOLESTEROL
RELATED CO-FACTORS THAT WERE
ABSOLUTELY CRITICAL FOR THEIR
REPLICATION.
THE SAME, ANOTHER PATHWAY WE
IDENTIFIED WAS MACRO AUTOPHAGY
AND PHOSPHOMETABOLISM TYROSINE
KINASE, HIGHLY ENRICHED BETWEEN
THE THREE PROTEIN WITH DIFFERENT
INDIVIDUAL FACTORS.
TO START TO LOOK INTO VALIDATION
HERE, WE DID INDIVIDUAL KNOCK
OUT EXPERIMENTS WITH CRISPR
HERE, FOR EXAMPLE WE KNOCKED
DOWN THE SARS COV-2 HITS,
SPECIFICALLY THE HITS WE
IDENTIFIED IN THE CHOLESTEROL
PATHWAY BUT ALSO OTHER RELATED
FACTORS AND YOU CAN SEE NICELY
THAT IF YOU KNOCK IT DOWN AND
YOU INFECT YOU SEE THAT YOU KILL
INFECTION, IF YOU REPLACE THE
FACTOR CLASSICAL
RESUPPLEMENTATION EXPERIMENTS
YOU CAN SEE THAT THIS IS
SPECIFIC BECAUSE YOU ENABLE
INFECTION AGAIN.
THIS WAS SHOWN WITH MULTIPLE
FACTORS AND YOU CAN SEE THAT'S
SPECIALLY ALSO THESE CHOLESTEROL
PATHWAY FACTORS WERE HIGHLY
IMPORTANT FOR THE REPLICATION OF
THE VIRUS.
WHEN WE DID THE SAME THING WITH
THE TWO OTHER COMMON COLD CORONA
VIRUSES WE SAW THAT ALSO NOT ALL
BUT MANY ESPECIALLY THE
CHOLESTEROL PATHWAY, FACTORS
WERE ALSO IMPORTANT FOR ENABLING
VIRAL REPLICATION FOR THE COMMON
COLD CORONA VIRUSES, REALLY
UNDERSCORING THE VALIDITY OF
THIS NETWORK PROPAGATION
ANALYSIS AND THE IDENTIFICATION
OF THE CHOLESTEROL PATHWAY AS
POTENTIAL PAN VIRAL PATHWAY FOR
CORONA VIRUSES.
WE DID THIS ALSO WITH OTHER
VIRUSES HERE WITH OC 43, KNOCK
DOWN IMPORTANT FACTORS AND
ENDOSOME MATURATION AND PI 3
KINASE COMPLEX HIGHLY ENRICHED,
YOU CAN SEE REALLY IMPORTANT FOR
REPLICATION.
THEY WERE ALSO IMPORTANT FOR
REPLICATION FOR 229E ALSO TO
DIFFERENT DEGREE AND WHEN WE DID
THIS WITH SARS COV-2 YOU CAN SEE
THAT ALL BUT TWO WERE IMPORTANT
FOR SARS COV-2 REPLICATION
ESPECIALLY ALSO IN THE PI 3
KINASE COMPLEX.
SO THESE ARE -- THIS SHOWS YOU
THE POWER OF THE METHOD BUT ALSO
THE OPPORTUNITIES AND REALLY
DIGGING DEEPER AND UNDERSTANDING
THE INDIVIDUAL LIFE CYCLE OF
THESE VIRUSES, BUT ALSO
IDENTIFYING POTENTIALLY COMMON
TARGETS THAT MIGHT BE IMPORTANT
FOR THERAPEUTICS.
SO THE QUESTION NOW WAS IS THIS
REALLY SOMETHING THAT WE CAN
DERIVE FROM THIS DATA.
CAN WE IDENTIFY PAN CORONA VIRUS
PHARMACOLOGICAL INHIBITORS.
SO WE FOCUSED ON PI 3 KINASE
INHIBITORS HERE.
WHERE WE LOOK FOR INFECTION IN
SARS COV-2, 229E AND OC 43 YOU
CAN SEE THIS INHIBITOR ALSO
REALLY HAS VERY PRONOUNCED
INHIBITORY ACTIVITY FOR ALL
THREE FACTORS, WHILE NOT BEING
TOXIC.
THE SAME IS FOR CHOLESTEROL
INTRACELLULAR CHOLESTEROL
PRODUCTION INHIBITOR HERE, WHERE
YOU CAN SEE ESPECIALLY THE LOWER
DOSE YOU SEE ALSO AN INTERESTING
INHIBITION OF THESE TWO VIRUSES,
A LITTLE LESS WITH OC 43 AND
HIGHER CONCENTRATION HERE WE
HAVE MORE TOXICITY BUT THIS IS
TYPE OF STUDIES WE HAVE BEEN
DOING WITH OTHER VIRUSES BEFORE
AND WE CONTINUE TO DO WITH THIS
VIRUS HERE AND THAT WE BRING NOW
INTO IN VIVO STUDIES TO SEE
REALLY POTENTIAL AND ALSO THE
PRECISE MOLECULAR FUNCTION OF
THESE IN THE LIFE CYCLE.
IF I SUMMARIZE THIS FIRST PART I
THINK THE CRISPR KNOCK OUT
SCREENS HAVE BEEN BY US AND
OTHERS PERFORMED MANY OTHERS
HAVE DONE SIMILAR SCREENS WITH
HOST FACTORS CRITICAL FOR SARS
COV-2 BUT FOR US ALSO WE
COMPARED WITH OC 43 AND 229E
INFECTION AND THAT HELPED US TO
IDENTIFY SHARED ESSENTIAL
PATHWAYS BETWEEN THE VIRUSES
INCLUDING CHOLESTEROL SYNTHESIS
AND PHOSPHOKINASE, KINASE
PATHWAYS WERE IDENTIFIED AND WE
ARE CONTINUING TO WORK ON IT
NIHAL MENTIONED WE HAVE A LONG
STANDING INTEREST IN LIPIDS AND
VIRAL INFECTION STEMMING FROM
OUR WORK IN HEPATITIS C AND WE
ARE VERY INTERESTED IN FOLLOWING
UP WITH CHOLESTEROL SYNTHESIS
PATHWAY, I THINK IT IS CLEAR
THAT ENVELOPE VIRUSES HAVE A
UNIQUE AFFINITY TO CHOLESTEROL
SYNTHESIS BUT WE THINK THERE
MIGHT BE MORE TO THE CHOLESTEROL
CONNECTION WITH SARS COV-2 ALSO
GIVEN OBVIOUS RISK OF OBESITY AS
A RISK FACTOR FOR SEVERE
DISEASE.
SO WE ARE OVERALL INTERESTING TO
EITHER USE EXISTING
PHARMACOLOGICAL INHIBITORS OR
DERIVE NEW INHIBITORS OF
IDENTIFIED PATHWAYS TO RESTRICT
PAN CORONA VIRUS REPLICATION TO
GET AHEAD OF VARIANTS BUT ALSO
NEW AND EMERGING VIRUSES.
SO I THINK THIS IS A WHOLE NEW
FIELD, I THINK THE PHARMA
COMPANIES ARE MUCH MORE OPEN TO
THIS IDEA BEFORE THEY WERE NOT
BUT I THINK SARS COV-2 HAS
CHANGED MANY PERSPECTIVES HERE
AND I THINK THAT HOST DIRECTED
THERAPY COULD BE A VERY GOOD
ADDITION TO COMBINATION THERAPY
OF ANTIVIRALS FOR SARS COV-2 IN
THE FUTURE GIVEN IT WILL REDUCE
RESISTANCE DEVELOPMENT.
NOW I SHIFT TO THE SECOND PART
OF MY TALK WHICH IS THE
DETECTION PART.
AND THE QUESTION HOW CAN WE GET
TO BETTER SARS COV-2 TESTS.
WE ALL PROBABLY HAVE ANTIGEN
TESTS NOW AT HOME TESTING OUR
KIDS, TESTING OURSELVES WHEN WE
WANT TO GET INTO THE COUNTRY,
HAVING SENTINEL TESTING IN
INSTITUTES, BUT THE QUESTION IS
ALWAYS THEY ARE FREQUENT AND
FAST AND CHEAP BUT ARE THEY ALSO
ACCURATE.
SO AS I SAID WE HAVE BEEN
THINKING ABOUT THESE TYPES OF
TESTS FOR QUITE A WHILE.
THIS IS MY -- MY COLLEAGUE DAN
FLETCHER AND JENNIFER DOUDNA
FROM BERKELEY BUT HAVE
AFFILIATION AT GLADSTONE.
AND WE REALLY SET OUT TO COMBINE
OUR RESPECTIVE EXPERTISE IN
VIROLOGY CRISPR TECHNOLOGY AND
DAN WHO IS A BIOENGINEER AND HAS
PIONEERED THE WORK IN USING
MOBILE PHONES AS DIAGNOSTICS.
AT THE CENTER OF OUR TECHNOLOGY
IS THE ENZYME CALLED CAS 13A, IT
IS A -- ONE OF THE CRISPR
ENZYMES, IT IS AN RNA BINDING
AND EDITING CRISPR ENZYME.
SO IF YOU COMBINE IT WITH
APPROPRIATE GUIDE RNA HERE FOR
SARS COV-2 YOU TARGET BASICALLY
THE ENZYME TO THE TARGET, IT
BECOMES ACTIVE AND IT STARTS
EDITING BECAUSE THAT'S WHAT IT
DOES, US BASICALLY IS A NUCLEUS
AND THE INTERESTING PART ABOUT
CAS 13 IS IT IS NOT A DIRECTED
EDITING BUT IT ALSO EDITS ANY
RNA THAT IS COLLATERALLY
SUPPLIED TO THE REACTION, SO YOU
CAN USE THIS BY JUST GIVING IT A
REPORTER RNA WITH A QUENCHER AND
FLOR FORE, IF THIS RNA IS
CLEAVED THE QUENCHER IS RELEASED
AND FLUORESCENCE IS STARTING TO
GLOW AND IT CAN BE CAPTURED WITH
FOR EXAMPLE A MOBILE PHONE OR
WITH A CLASSICAL PLATE READER IN
THE LAB.
SO INITIALLY -- SO THIS IS A
VERY SIMPLE REACTION.
IT IS BASICALLY AN ANTIGEN TEST
FOR RNA, NOT OF COURSE WITH
ANTIBODIES USUALLY BUT IT IS A
DIRECT TARGETING OF THE -- OF
DETECTING AGENT TO VIRAL RNA.
THIS ALLOWS US TO POTENTIALLY BE
AS CONVENIENT AS ANTIGEN TEST
BUT MORE PRECISE OR AS PRECISE
AS MOLECULAR TEST, THAT WAS THE
GOAL WHEN WE STARTED OUT TO MAKE
IT PORTABLE WE SWITCHED TO THE
MOBILE PHONE AS DETECTOR.
BUT MANY PEOPLE DOUBTED THIS
SIMPLE REACTION WOULD BE
SENSITIVE ENOUGH BECAUSE IT
DOESN'T HAVE -- THE INITIAL DATA
WERE NOT PROMISING WHEN YOU DID
NOT TWEAK THE ESSAY AS I'M
SHOWING WE HAVE DONE SINCE BUT
ALSO MOST OF THE CRISPR
REACTIONS YOU SEE TODAY ARE ANY
COMMERCIAL CRISPR OR
COMMERCIALLY DEVELOPED CRISPR
REACTIONS INCLUDING
AMPLIFICATION STEP IN THEIR
REACTION.
WHERE REVERSE TRANSCRIBING THE
INITIAL RNA AMPLIFYING AND FOR
TRANSCRIBING BEGIN TO MAKE
CRISPR TEST.
WHEN WE SET OUT TO DEVELOP THIS
TEST WE REALLY PURPOSEFULLY
DECIDEDECIDED THAT WE WEREN'T REALLY
PUSH THE ENVELOPE ON THIS DIRECT
DETECTION ESSAY WHERE WE DO NOT
INCLUDE ANY AMPLIFICATION STEP.
WHEN WE STARTED TO SHIFT ESSAY
FROM HIV TO SARS COV-2 IT WAS
LAST YEAR WHEN SEQUENCE OF SARS
TWO YEARS AGO WHEN THE -- WHEN
SARS -- WHEN THE SARS COV-2
SEQUENCE BECAME AVAILABLE WE
JUST ENGINEERED SOME OF THESE
GUIDE RNAs ACCORDING TO
ORIGINAL STRAIN.
THIS IS A CLASSICAL REACTION
WHERE YOU BASICALLY PUT ALL THE
COMPONENTS TOGETHER AND YOU CAN
SEE THAT IF YOU ADD THE SARS
COV-2 VIRAL RNA YOU CAN SEE
FLUORESCENCE IS TAKING OFF.
IF YOU DON'T ADD IT YOU HAVE
CERTAIN BACKGROUND OF JUST BY
THE REPORTER AND THE ENZYME BY
ITSELF.
YOU SEE THAT THERE IS -- THE
GUIDES THAT WORK VERY WELL, AND
THERE ARE GUIDES THAT WORK NOT
VERY WELL.
THIS IS LITERALLY THE FIRST 15
GUIDE RNAS WE DEVELOPED TWO
YEARS AGO.
WHAT WE FOUND IS IT WAS TRUE
WHAT OTHERS FOUND BEFORE IF WE
USE JUST ONE GUIDE AND THE
ENZYME, THE SENSITIVITY IS NOT
VERY HIGH AND WE CANNOT GET INTO
A RANGE THAT IS ATTRACTIVE FOR
ANY DIAGNOSTIC.
HOWEVER WHEN WE START COMBINE
GUIDES AND BUILD GUIDES ALONG
THE VIRAL GENOME WE CAN VERY
MUCH ENHANCE OUR SENSITIVITY
HERE IN SHORTER AND SHORTER TIME
FRAMES.
I THINK ONE OF THE THINGS WE
ALSO DID WHICH IS DIFFERENT FROM
OTHERS IS WE DON'T LOOK AT END
POINT, WE ARE NOT WAITING 30
MINUTES OR TWO HOURS AND
MEASURING THE END POINT, WE
DEVELOPED ALGORITHMS WHERE WE
DEVELOPED -- WE MEASURE THE
SLOPE OF THE REACTION AND THAT
LETS US VERY EARLY ON DETERMINE
THIS IS A POSITIVE REACTION AND
THIS IS BASICALLY A NEGATIVE
REACTION HERE.
SO WITH THIS WE HAVE BEEN
COMBINING NOW MANY GUIDES, WE
HAVE COME UP WITH AN EIGHT GUIDE
COMBINATION IN OUR PAPER END OF
2020, WE TALKED ABOUT FREE
GUIDES BUT WE HAVE SINCE SETTLED
ON AN EIGHT GUIDE COMBINATION
THAT WE HAVE TESTED AGAINST ALL
RISING VARIANTS AND THAT DETECTS
IT INCLUDING OMICRON AND DELTA
BUT NO CROSS REACTIVITY WITH
NON-SARS COV-2 VIRAL RNA AND
IMPORTANTLY ALSO NO CROSS
REACTIVITY WITH HUMAN RNA AND
ANY PREDICTABLE MICROBIOME RNA
THAT WE MIGHT HAVE IN OUR NOSE.
WITH THIS EIGHT GUIDE
COMBINATION AND SOME OTHER
TWEAKS THAT I WILL PARTIALLY
ELUDE TO IN MY TALK, WE HAVE NOW
PUSHED THE LIMIT OF DETECTION
DOWN TO TEN COPIES PER
MICROLITER, YOU CAN SEE HERE
AFTER 20 MINUTES WE CAN CALL
THIS REACTION POSITIVE WITHOUT
ANY DOUBT.
OVER THE GREEN LINE OF THE
NEGATIVE AND ONE OF THE MOST
IMPORTANT THINGS WE HAVE TO DO
IS REALLY REDUCE THE BACKGROUND
HERE IN ORDER TO HAVE THE
SENSITIVITY TO DIFFERENTIATE
BETWEEN THESE SMALL DIFFERENCES
IN SIGNAL.
ANOTHER THING THAT TURNED OUT TO
BE VERY IMPORTANT FOR THE
SUCCESS OF THIS ESSAY WAS THE
OPTICS.
INITIALLY WE THOUGHT WHILE IS
GOAL IS MOBILE PHONE WILL BE AS
SENSITIVE AS THE PLATE READER IN
THE LAB, IT TURNS OUT THAT THE
MOBILE PHONE CAMERAS THAT WE
HAVE MOST OF OUR PHONES TODAY
ARE ACTUALLY TEN TIMES BETTER
THAN THE PLATE READER WE HAVE IN
THE LAB.
THIS IS MAINLY BECAUSE OF THE
NOISINESS OF THE SIGNAL, THE
PLATE READER HAS A CONSIDERABLE
NOISE IN READS WHILE HERE THE
PIXEL 4 PHONE CAMERA IS STEADY
IN ACQUIRING THE SIGNALS AND
GIVES US OVERALL TENFOLD
INCREASE IN IF SENSITIVITY WHICH
IS REMARKABLE.
THIS IS NOT DUE ONLY TO
EXPENSIVE PHONES THAT WE HAVE
CARRYING IN OUR POCKETS.
WE HAVE TESTED $20 PHONES FROM
INDIA AND THEY ALSO HAVE PHONE
CAMERAS IN THE RANGE OF
SENSITIVITY THAT IS IMPORTANT
FOR US.
SO FIRST MODEL WE BUILT THAT DAN
BUILT WAS BASICALLY THE PHONE
HERE, ALIGN, THE CAMERA ALIGNED
TO THIS BOX WHICH CARRIES THE
REACTION.
THERE IS A DRAWER HERE YOU CAN
OPEN AND ENTER THE REACTION AND
BASICALLY THIS IS A PROGRAM, AN
APP THAT HAS BEEN WRITTEN FOR
THE PHONE THAT IMMEDIATELY
ACQUIRES FLUORESCENCE THAT IS IN
THESE THREE CHANNELS HERE AND
YOU CAN SEE THIS IS A VERY
HIGHLY POSITIVE PATIENT SAMPLE,
LESS POSITIVE PATIENT SAMPLE IN
NEGATIVE ONE AND YOU CAN SEE
THAT THE PHONE IS BASICALLY
RUNNING THE REACTION, THE
ACQUISITION AND ANALYSIS AND I
THINK THE PHONE HAS THE ADDED
BENEFIT IT ALSO IS CONNECTED TO
LOCATION, GPS SYSTEMS SO IT CAN
BE USED TO POTENTIALLY IN THE
FUTURE FOR GEOLOCATION AND O
CONTACT TRACING IF WE GO FORWARD
WITH THIS TECHNOLOGY.
NOW, THIS -- ADDING MULTIPLE
GUIDES, IMPROVING THE OPTICS,
HAS REALLY LED TO EXQUISITE
SENSITIVITY OF THAT'S SAY WE ARE
IN OUR INITIAL PAPER WE COULD
SHOW WE HAVE HAD FIVE PATIENT
SAMPLES FROM THE BIOHUB HERE IN
SAN FRANCISCO AND WE CAN DETECT
ALL FIVE PATIENT SAMPLES WITHIN
FIVE MINUTES AS POSITIVE AND
DISTINGUISH IT FROM THE
NEGATIVES.
SO THIS IS VERY PROMISING.
AND WE CONTINUING TO WORK ON
THIS SO-CALLED BULK ASSAY AS WE
USE IT WITH THE EIGHT GUIDES
THAT I HAVE INTRODUCED TO YOU.
WE ARE LOOKING BUILDING DEVICES
LIKE THIS, WHERE WE PUT THE SWAB
INTO A PEN LIKE STRUCTURE, SCREW
IT INTO THIS DETECTION BLOCK,
THE IMPORTANT PART IS THAT WE
HAVE TO HEAT, STILL HEAT AND
THAT IS ONE THING WE WOULD LIKE
TO GET RID OF BUT WE HAVE TO
BRIEFLY HEAT THE REACTION,
MAINLY TO INACTIVATE RNA,
BECAUSE THAT IS OUR MAIN ENANY
IN THIS ESSAY AND THEN WE CAN
TURN AND PUSH REACTIONS TO THE
THREE CHANNELS, IT IS A POSITIVE
AND NEGATIVE AND THE TESTING
CHANNEL.
THEN YOU CAN USE YOUR MOBILE
PHONE BASICALLY ALIGNED WITH A
CAMERA HERE, TO READ THIS WITH
APPROPRIATE APP.
SO THAT IS THE SORT OF THE BROAD
STROKE ROUTE WE ARE CURRENTLY
GOING.
WE HAVE BEEN VERY FRUSTRATED, WE
THOUGHT WE COULD DO THIS MUCH
FASTER AND COULD BE READY FOR
SOMETHING LIKE THE OMICRON SURGE
HERE BUT BUILDING SOMETHING FROM
SCRATCH AND HAVING FDA APPROVAL
IS NOT EASY BUT WE THINK THAT
THIS IS A TECHNOLOGY THAT IS NOT
ONLY IMPORTANT NOW FOR SARS
COV-2 BUT ALSO FOR OTHER
VIRUSES, RNA VIRUSES IN THE
FUTURE AND ALSO I THINK IS A
METHOD THAT IS EXTREMELY
INTERESTING FOR RNA
QUANTIFICATION ALSO IN THE LAB.
SO THIS WORK WAS MAINLY DRIVEN
BY VERY TALENTED MSTP STUDENT IN
THE LAB PARINAZ FOZOUNI, SHE IS
BACK IN MEDICAL SCHOOL, SHE
WORKED WITH SUNGMIN SON WHO HAS
HELPED WITH OPTICS AND THE
ACQUISITION OF -- WITH THE
MOBILE PHONE.
SUNGMIN HAS TAKEN THE NEXT STEP
AND PUT IT INTO A DROPLET BASED
WORK FLOW.
BECAUSE OF THE -- I TOLD YOU WE
HAVE WITH THE BULK ESSAY
ACHIEVED A UNIQUE SENSITIVITY
BUT WE REALLY WOULD LIKE TO GO
INTO PCR SENSITIVITY WHICH IS
ONE COPY FROM MICROLITER BUT
ALSO LIKE TO DECREASE NUMBER OF
GUIDES IN ORDER TO BE ABLE TO
DIFFERENTIATE BETWEEN INDIVIDUAL
VARIANTS OR VIRUSES.
SO WHAT HE DID IS VERY SIMPLE,
HE EMULSIFIED THE REACTION BY
PIPETTING UP AND DOWN AND
BUILDING THESE DROPLETS AND
IMAGING AND QUANTIFYING THE
FLUORESCENCE AND THE INDIVIDUAL
DROPLETS BY MICROSCOPY.
YOU CAN SEE WE HAVE DIFFERENT
SIZES OF DROPLETS BUT BECAUSE WE
ARE USING MICROSCOPY NORMALIZED
FOR THIS, YOU CAN SEE WITHIN A
FEW MINUTES, YOU SEE THE
DROPLETS POPPING UP AND NUMBER
OF DROPLETS IS BASICALLY THE
QUANTITATION OF THIS ASSAY.
WHILE YOU CAN SEE OVER 30
MINUTES WE HAVE AN INCREASE,
CONTINUED INCREASE IN THE SIGNAL
PER DROP, THE NUMBER OF DROPS
REMAINS STEADY AFTER FIVE
MINUTES SO WE CAN BASICALLY CALL
THIS REACTION AFTER FIVE MINUTES
WHICH IS A HUGE BENEFIT.
AND WE HAVE A HUGE BENEFIT IN
SENSITIVITY BECAUSE WE NOW PULL
FROM A 300-MICROLITER REACTION
TO A 30 PICO LITTER REACTION IN
A DROPLET.
YOU CAN SEE THAT WITH ONE GUIDE
WE CAN NOW DETECT 20 OR 10 COP
PIPS PER MICROLITER WHICH IS THE
SENSITIVITY WITH EIGHT GUIDES IN
THE BULK REACTIONND AND IF WE
COMBINE THE GUIDES HERE IN
DIFFERENT VARIATIONS WE CAN GO
TO ONE COPY PER MICROLITER OR
BELOW, EASILY IN FIVE MINUTES.
SO THIS IS REALLY A UNIQUELY
SENSITIVE METHOD THAT PUSHES
THIS DIRECT DETECTION METHOD
INTO A PCR SENSITIVITY.
BUT THE INTERESTING PART, OR THE
PART WITH THE MOST INTERESTING
IS THAT THE DROPLETS OFFER US
NOW A WAY TO DIFFERENTIATE
BETWEEN INDIVIDUAL GUIDES AND
THERE BY GIVES US A WAY TO
POTENTIALLY MEASURE DIFFERENT
VIRUSES OR DIFFERENT VARIANTS
JUST IN ONE SINGLE REACTION A.
INSTEAD OF PUTTING THEM INTO THE
THREE CHANNELS OR IN MORE
CHANNELS AND DIVIDING OUR INPUT
MATERIAL, I THINK WE CAN NOW USE
ONE SINGLE REACTION AND CAN
POTENTIALLY COMBINE MULTIPLE
GUIDES.
HOW -- WHY CAN WE DO THAT?
THE REASON IS SIMPLE.
IF YOU REMEMBER MY VERY FIRST
SLIDE I SHOWED YOU ALL THE
DIFFERENT GUIDE KINETICS, EACH
OF THEM HAS -- WE CALL THEM THE
GOOD GUIDES OR THE BAD GUIDES.
VERY STEEP SLOPE WHERE THEY HAVE
A VERY SORT OF SHALLOW SLOPE.
BUT THIS SLOPE CHARACTERISTIC IN
THE DROPLETS IS FIXED BECAUSE WE
ALWAYS HAVE ONE COPY OF THE
TARGET.
SO WE CAN USE THIS KINETIC SLOPE
BEHAVIOR OF THE INDIVIDUAL GUIDE
TO BAR CODE AND DIFFERENTIATE
BETWEEN INDIVIDUAL GUIDES.
I AM SHOWING AN EXAMPLE HERE, IF
WE HAVE A GUIDE THAT IS SPECIFIC
FOR VARIANT, THIS IS THE
CALIFORNIA VARIANT HERE WITH THE
SIGNAL POINT MUTATION.
THIS IF THAT GUIDE YIELDS TO THE
VARIANT RNA IT HAS A STEEPER
SLOPE THAN TO THE WILD TYPE
WHERE IT IS INCOMPLETE.
THIS DIFFERENCE IN SLOPE CAN NOW
BE USED BY ALLIES IN PATIENT
SAMPLES WHERE WE CAN
DIFFERENTIATE WHAT THIS ONE
GUIDE WITH ALMOST 100% ACCURACY
BETWEEN WILD TYPE SAMPLES THAT
WE HAVE IN PATIENTS, OR BETWEEN
CALIFORNIA VARIANT THAT WAS
WIDESPREAD HERE IN SAN
FRANCISCO.
IN OUR SAMPLES.
WE ARE VERY EXCITED BY THIS
KINETIC BAR CODING HERE BECAUSE
THAT ALLOWS US NOW TO MULTIPLEX
REACTION, TRULY MULTIPLEX THE
REACTION IN ONE REACTION WHERE
WE CAN NOW BASED ON KINETICS ON
THE INDIVIDUAL OF THE INDIVIDUAL
GUIDES DIFFERENTIATE BETWEEN
VARIANTS BUT ALSO POTENTIALLY
LOOK FOR MULTIPLE VIRUSES IN ONE
REACTION.
SO AS A SUMMARY I HAVE SHOWN YOU
DIRECT DETECTION WITH CRISPR
WITH CAS 13A IN BULK OR DROPLET
REACTIONS IS POSSIBLE AND I
EMPHASIZE HERE THIS IS THE
DIRECT ROUTE TO DETECTION ESSAY,
NOT AMPLIFIED REACTION AS IN
OTHER CRISPR ASSAYS.
THE SENSITIVITY IS CURRENTLY TEN
COPIES PER MICROLITER AND 30
MINUTES BULK REACTION OR ONE
COPY PER MICROLITER IN FIVE
MINUTES FOR THE DROPLET
REACTION.
THE DROPLETS OFFER UNIQUE
OPPORTUNITIES BECAUSE NOW WE
HAVE THE SINGLE MOLECULE
RESOLUTION IN THE DROPLETS THAT
ALLOWS US TO STUDY THE BASIC
BIOLOGY OF CAS 13A BUT ALSO
ALLOWS KINETIC BAR CODING BY
USING THE UNIQUE INTERACTION OR
THE UNIQUE KINETICS THAT RESULTS
FROM THE GUIDE TARGET
INTERACTION.
THIS ALLOWS US TO MULTIPLEX
DIFFERENT TARGETS WITH SINGLE
REPORTSER WE DON'T HAVE TO
CHANGE THE ENZYME, THE REPORTER
OR ANYTHING.
AND IT GIVES UNIQUE, THAT I
HAVEN'T EMPHASIZED BUT THIS IS A
UNIQUE QUANTITATIVE ASSAY
BECAUSE OUR SLOPE IS DIRECTLY
LINEARLY RELATED TO THE VIRAL
LOAD IN OUR ASSAY.
SO WE CAN DIRECTLY FROM THE
SLOPE DERIVE QUANTITATION OF
VIRAL LOADS IN OUR ASSAY SO WE
CAN HAVE ACCURATE FAST MOBILE
RNA DETECTION THAT WE THINK IS
IMPORTANT FOR SARS COV-2 IN THIS
PANDEMIC BUT POTENTIALLY OTHER
RNAS NOT ONLY VIRAL RNAs IN
THE FUTURE.
THIS LEADS ME TO MY LAST PART OF
THE TALK WHICH IS THE STUDY
PART.
AND THIS IS CONTINUED
COLLABORATION BETWEEN JENNIFER'S
LAB AND MY LAB.
JENNIFER MOVED 25% OF HER
EFFORTS A FEW YEARS BACK TO
GLADSTONE MAINLY ALSO TO HAVE AT
THE UCSF CAMPUS AND HAVE A MORE
CLINICALLY AFFILIATED LAB HERE
IN SAN FRANCISCO.
ONE OF HER POST-DOCS IS
EXTREMELY TALENTED AND HER LAB
IS NEXT TO MINE SO WE HAD
ENORMOUS INTERACTIONS DURING OUR
TIME FOR THE DEVICE AND THE TEST
BUT WE HAVE ALSO PEOPLE IN HER
LAB AND MY LAB HAVE CONTINUED TO
WORK CLOSELY TOGETHER AND
AMAZING SYNERGIES HAVE BEEN
ARISING.
SO SYED IS WORKING CLOSE WITH
VERY TALENTED POST DOC TAHA AND
MY LAB AND TAKAKO, A SENIOR
SCIENTIST WHO IS DOING ALL THE
BSL 3 WORK FOR THEM.
THIS LED IN NOVEMBER LAST YEAR
TO THE SCIENCE PAPER WHERE WE
DEVELOP NEW VIRUS LIKE PARTICLES
TO ASSESS SARS COV-2 IN BOTH
VARIANTS AND WE CONTINUED TO DO
THIS WITH DELTA AND NOW WITH
OMICRON.
WHAT IS A VIRUS LIKE PARTICLE
AND HOW IS IT DIFFERENT FROM A
PSEUDOTYPE VIRUS?
I THINK A VIRUS LIKE PARTICLE
USES ALL FOUR STRUCTURAL
PROTEINS OF SARS COV-2, NOT JUST
THE SPIKE PROTEIN THAT IS
BASICALLY ARTIFICIALLY MOUNTED
ON TO ANOTHER VIRUS PARTICLE.
WE ARE GENERATING AUTHENTIC SARS
COV-2 VIRUS PARTICLES INCLUDING
THE S PROTEIN, THE N PROTEIN,
THE NUCLEAR CAPSID, WHICH IS
CRITICAL FOR THE PARTICLE
FORMATION AND RNA INCORPORATION
BUT ALSO THE SMALLER ADDITIONAL
STRUCTURAL PROTEINS LIKE THE M
PROTEIN AND TINY LITTLE E
PROTEIN THAT CONSTITUENTS SARS
COV-2 PARTICLE.
IN ORDER TO USE A VIRAL LIKE
PARTICLE INSTEAD OF PSEUDOVIRUS,
SYED NEEDED TO IDENTIFY THE
PACKAGING SIGNAL FOR THIS
PARTICLE IN ORDER TO INCORPORATE
A REPORTER THAT WE COULD USE TO
MEASURE PRODUCTION AND
INFECTIVITY OF THIS PARTICLES.
HE DID THIS BY USING EXISTING
LITERATURE ABOUT CORONA VIRUS
PACKAGING SIGNALS, AND EXTENSIVE
MUTAGENESIS AND IDENTIFIED A
SMALL T 20 MOTIF AT THE END OF 1
AB AS PACKAGING SIGNAL AND IF HE
HOOKS UP TO GFP OR LUCIFERASE
GENE HE CAN SHOW THAT THIS
CONSTRUCT IS PACKAGED AND ALSO
DELIVERED INTO THE NEXT CELLS
WHERE EITHER GLOWS OR PRODUCES
LUCIFERASE.
SO WITH THIS AT HAND HE
DEVELOPED A VLP SYSTEM, SIMPLE
CO-TRANSFECTION SYSTEM WHERE YOU
USE THE SPIKE PROTEIN, THE N
PROTEIN EXPRESSING CONSTRUCT BUT
ALSO CISTRONIC AND EXPRESSING
REPORTER TO CO-TRANSFECT TO 2293
CELLS, YOU ISOLATE THEM PUT ON E
RECEPTOR CELLS AND THEN YOU CAN
MEASURE LUCIFERASE OR IF YOU USE
GFP YOU CAN DO SINGLE CELL
ANALYSIS BY FLOW.
THE IMPORTANT PART IS THAT THESE
VLPs ONLY FUNCTION IF YOU PUT
ALL FOUR TOGETHER IN THE RIGHT
RATIO.
IF YOU MISS OUT ON ANY OF THIS
YOU HAVE NO SIGNAL CONFIRMING
THAT YOU HAVE REALLY INTACT VLP
HERE IN SUPERRER NATANT.
WITH THIS WE CAN GO BEYOND
SPIKE.
EVERYBODY IS FOCUSING ON SPIKE.
LOOKING INTO WHAT IS THE
CONTRIBUTION OF THE SPIKE TO FOR
EXAMPLE VARIANT INFECTIVITY.
AND IMMUNE EVASION.
WE HAVE BEEN STARTING NOW TO
LOOK AT THE N M AND E PROTEINS,
TALKING ABOUT OUR FINDINGS IN
THE N PROTEIN TODAY, AS I SAID
THE N PROTEIN VERY IMPORTANT
PROTEIN, WE HAVE A LONG STANDING
INTEREST IN CAPSID NUCLEAR
CAPSID OR CORE PROTEINS IN OTHER
VIRUSES, THEY HAVE ENORMOUS HOST
BUT ALSO VIRALLY IMPORTANT
FUNCTIONS AND HERE THE NUCLEAR
CAPSID IS REALLY THE ONE THAT IS
MEDIATING RNA BINDING AND RNA
INCORPORATION TO ITS END
TERMINAL AND C TERMINAL DOMAIN
BUT ALSO OLIGOMERRIZES TO FORM
THE CAPSID AND THE PARTICLE OF
THE VIRUS.
IMPORTANTLY THE TWO N AND C
TERMINAL DOMAINS IS SEPARATED BY
A LINKER DOMAIN IN THE MIDDLE.
AND THIS LINKER DOMAIN CAUGHT
OUR ATTENTION BECAUSE MANY OF
THE MUTATIONS THAT HAVE BEEN
REPORTED FOR N PROTEINS IN
DIFFERENT VARIANTS HAVE ACTUALLY
CONCENTRATED IN THIS LINKER
REGION.
SO WHAT WE DID IN THIS PAPER IS
BASICALLY GENERATE N PROTEINS
WITH SIGNAL POINT MUTATIONS AND
ANY OF THESE THAT HAVE BEEN
REPORTED IN NUCLEAR CAPSID
PROTEINS AND WE FOUND THE ONES
MANY THE LINKER REGION
SPECIFICALLY HAVE HUGE INCREASE
IN INFECTIVITY WHEN YOU CAN DO
THE ASSAY I SHOWED YOU BEFORE.
THREE OF THEM, THE 9 -- 199L TO
WHICH WE ARE AND THE TWO OR
THREE M WHICH IS THE MUTATION
THAT IS FOUND IN DELTA VIRUS,
DELTA VARIANT, ACTUALLY VERY
MARKEDLY INCREASE INFECTIVITY UP
TO TENFOLD.
SO SYED LOOKED INTO WHAT EXACTLY
IS HAPPENING, WHY IS THERE MORE
LUCIFERASE IN THE TARGET CELLS
WHEN USES THESE MUTANTS.
HE LOOKED, PURIFIED THE
PARTICLES OF THE PRODUCTION, HE
LOOKED WHETHER THEY HAD MORE
SPIKE OR MORE PROCESS SPIKE.
THERE WAS NO CLEAR SIGNAL FOR N,
WE SAW TWO VARIANTS INCREASING
IN LEVELS BUT THE -- NOT FOR THE
THIRD WHICH WAS THE DELTA
VARIANT.
BUT THE MOST CONSISTENT SIGNAL
CAME FROM THE NORTHERN BLOT
WHERE HE COULD SHOW RNA
INCORPORATION IS ENHANCED IN ALL
THREE OF THEM.
THAT REALLY RESEMBLES THE
INCREASE IN INFECTIVITY THAT WE
SEE IN THE FUNCTIONAL ASSAY.
THE MODEL IS THESE M MUTATIONS
ARE INCREASING RNA
INCORPORATION, AND THEREFORE
INCREASE OR MAKING MORE
FUNCTIONAL VIRAL LIKE PARTICLES
IN THE SUPERNATANT, THAT LEADS
TO MORE INFECTION IN THE TARGET
CELL.
IN ORDER TO PROVE THIS IS REALLY
TRUE, WE NOW PUT THESE MUTATIONS
IN TO THE FULL LENGTH VIRUS.
WE USE INFECTIOUS CLONE
GENERATED BY PEI YONG SHI'S LAB
IN TEXAS, WE FOCUS ON THE S 2R
AND THE R 2 OR 3M MUTATION AND
YOU CAN SEE HERE INFECTIOUS
TITERS FROM THE SUPERNATANT OF
THESE TRANSFECTIONS THAT BOTH
MUTATIONS ARE REALLY INCREASING
INFECTIVITY OF THE VIRUSES TO A
SIMILAR DEGREE AS WE HAVE SEEN
IN THE BLPs.
THIS WAS ALSO TRUE FOR CLASSICAL
COMPETITION EXPERIMENTS WHERE WE
MIX THE MUTANT VIRUS WITH THE
WILD TYPE VIRUS AND WE CAN SEE
ALL THE DILUTIONS EVEN THE
HIGHEST DILUTION CAUGHT UP OR
WAS BETTER IN REPLICATING OR
CAUGHT UP WITH THE WILD TYPE IN
-- WITHIN TWO PASSAGES OF THE
VIRUSES.
INDICATING THESE HAVE INCREASE
INFECTIVITY AND REPLICATION
ADVANTAGES.
AS I SAID, THE MODEL IS THAT
THESE N MUTATIONS AND VARIANTS
ARE VERY CRITICAL.
FOR INCREASING INFECTIVITY AND
POTENTIAL TRANSMISSION OF THESE
VARIANTS.
BY ALLOWING THE VIRUS TO BE MORE
EFFECTIVE AND BUILD MORE
INFECTIOUS MORE EFFECTIVE MORE
EFFECTIVELY INFECTIOUS PARTICLES
THAT CAN MORE EFFECTIVELY INFECT
THE NEXT CELLS.
WE ARE CURRENTLY WORKING ON
THIS.
WE HAVE OF COURSE LIKE EVERYBODY
ELSE, ALSO FOCUSED OUR ATTENTION
ON THE NEW OMICRON VARIANT,
WHICH ALSO CONTAINS OF COURSE A
WHOLE RANGE OF SPIKE MUTATIONS
BUT ALSO CONTAINS A WHOLE SERIES
OF N MUTATIONS.
INCLUDING SOME IN THE LINKER
REGION I JUST POINTED OUT.
SO WHEN WE MADE A NUCLEAR CAPSID
THAT HAD ALL THE OMICRON
MUTATIONS AND COMPARED IT TO THE
NUCLEAR CAPSID, FROM DELTA, PUT
IT INTO VLPs, WE COULD SEE THE
OMICRON N PROTEIN IS MORE
EFFICIENT THAN THE DELTA TO
ENHANCE INFECTIVITY.
IF YOU JUST FOCUS ON TWO
MUTATIONS THAT ARE IN THE LINKER
REGION HERE WE SEE THAT THERE IS
ABOUT SIX FOLD INCREASE IN
INFECTIVITY BUT IT REALLY
REQUIRES THE WHOLE SLEW OF
OMICRON MUTATIONS HERE IN ORDER
TO ENHANCE DRAMATICALLY
INFECTIVITY OF PARTICLES THAT
CARRY OMICRON MY CLEAR CAPSID.
-- KNEW CLEAR CAP NUCLEAR
CAPSID.
SPIKE ALSO INCREASES
INFECTIVITY, IT IS A LITTLE BIT
DIFFERENT FROM WHAT WE OBSERVE
WITH DELTA.
BUT WE THINK THAT SPIKE AND
NUCLEAR CAPSID ARE SYNERGIZING
HERE TO MAKE THIS OMICRON VIRUS
VARIANT EXTREMELY INFECTIOUS.
THIS IS NOT THE CASE FOR THE M
AND E PROTEINS BECAUSE WHEN WE
PUT -- VERY RANDOM, WE HAVE M
AND E MUTATIONS IN VARIANTS BUT
OMICRON DOES, DELTA ALSO HAD N
MUTATION BUT YOU CAN SEE THAT
NEITHER OF THESE MUTATIONS IN
THE VARIANTS ACTUALLY ENHANCE
INFECTIVITY IN CONTRAST THEY
ACTUALLY DIMINISH THEM BUT
OBVIOUSLY THESE AFFECTS ARE
OVERRIDDEN BY THE POSITIVE
EFFECTS WE HAVE SEEN WITH
NUCLEAR CAPSID AND SPIKE BECAUSE
WHEN YOU LOOK AT THE FULL
PARTICLE THAT CONTAINS ALL OF
THE FOUR PROTEINS FROM OMICRON,
YOU CAN SEE THAT IT IS MORE
INFECTIOUS THAN DELTA VARIANT
HERE.
AND REALLY SHOWS THE SYNERGY OF
THE DIFFERENT MUTATIONS AT LEAST
IN THE STRUCTURAL PROTEINS FOR
THE VIRUS TO BECOME MORE
INFECTIOUS.
AS I SAID WE ARE VERY INTERESTED
IN THE N MUTATIONS.
WE ARE LOOKING VERY HARD INTO
HOW EXACTLY THESE N MUTATIONS
ARE ENHANCING RNA UPTAKE BECAUSE
THEY ARE IN THE LINKER REGION,
NOT NECESSARILY IN THE DOMAIN
NAYS ARE ATTACHING THE RNA
DIRECTLY.
IT IS VERY INTERESTING BECAUSE M
IS A HIGHLY PHOSPHORYLATED
PROTEIN AND PHOSPHORYLATION
SITES ARE IN THE LINKER REGIONS.
AND WE ARE TESTING DIFFERENT
ENZYMES THAT ARE KNOWN TO
PHOSPHORYLATE THIS REGION AND
SEEING HOW POTENTIALLY THE
PHOSPHORYLATION OF THIS PROTEIN
IS CHANGED.
IN THE VARIANTS AND HOW THAT
COULD EFFECT FUNCTION OF THE
NUCLEAR CAPSID.
OF COURSE THE VIRAL LIKE
PARTICLES ARE ALSO EXQUISITE,
SUBSTRATES TO TEST IMMUNE
FUNCTION.
SIMILAR TO PSEUDOTYPE VIRUS THAT
HAVE BEEN DONE QUITE A BIT FOR
OMICRON AND OTHER VIRUSES, WE
HAVE BEEN DEVELOPING VLP
NEUTRALIZATION ASSAYS.
WHERE WE JUST MEASURE THE
LUMINESCENCE AND THE ACCEPTOR
CELL AND DEVELOPING BASICALLY A
READ OF 50% INHIBITION HERE
NEUTRALIZATION, THAT WE USE AS
AN OUTREAD.
SO WITH THIS WE TESTED OMICRON
CARRYING BLPs.
AGAINST SERA OBTAINED FROM
PEOPLE WHO HAD OBTAINED TWO OF
THE PFIZER BIOTECH VACCINES, THE
MODERNA VACCINE OR J AND J
VACCINE OR RECOVERING FROM
PREVIOUS COVID INFECTIONS.
LIKE OTHERS WE HAVE OBSERVED
THAT OMICRON IS REALLY UNIQUELY
EVADING THE NEUTRALIZING
ACTIVITY IN THESE SERA BOTH FOR
THE mRNA VACCINES WITH SORT OF
SIMILAR REDUCTION WE OBSERVE
BETWEEN PFIZER AND MODERNA.
BUT WE REALLY SEE VERY LITTLE
NEUTRALIZING ACTIVITY WITH J AND
J VACCINE, THEY ALSO HAVE VERY
LITTLE TOTAL ANTI-SARS COV-2
ANTIBODIES IN THESE SERA AND IT
SPEAKS FOR POTENTIALLY THE P
CELL ACTIVATING ACTIVITY OF THIS
VACCINE TO EXPLAIN ITS
PROTECTION HERE.
WITH COVID, POST COVID SERA WE
INTERESTINGLY SAW DIFFERENT
RESULTS.
OMICRON VERY EFFECTIVE IN
EVADING THIS IMMUNITY WHILE FOR
DELTA WE SEE A WIDE RANGE OF
PROTECTIVE OR INHIBITORY RESULTS
HERE.
WE THINK THAT MOST PATIENTS HAVE
BEEN INFECTED WITH DELTA THAT
ARE INCLUDED HERE.
SO THE INFECTION WITH THE SAME
STRAIN MIGHT CONFER IN SOME
CASES QUITE EFFECTIVE IMMUNITY
HERE.
HOWEVER, THE BOOSTER WORKS IF WE
USE AND WE TEST SERA 16 DAYS OR
21 DAYS AFTER THE BOOST WE CAN
SEE THAT THE ANCESTRAL VIRUS
DELTA AND OMICRON SIGNIFICANTLY
REACTIONS ARE NEUTRALIZATION IS
SIGNIFICANTLY INCREASED.
AND BUT IF WE COMPARE THE
INCREASE BETWEEN ANCESTRAL DELTA
AND OMICRON WE SEE THE OMICRON
IS STILL ABLE TO ALSO SOMEHOW
EVADE THE NEUTRALIZING ACTIVITY
OF THE BOOST EVEN AFTER THREE
WEEKS AFTER THE BOOSTING.
SO THE BOOSTERS REALLY IS
WORKING BUT OMICRON IS STILL THE
ONE THAT CAN DEAL WITH IT THE
BEST.
WE ALSO TESTED THE AVAILABLE
MONOCLONAL ANTIBODIES FROM
REGENERON.
AS YOU KNOW THIS IS AN IC 50
EXPERIMENT SO HIGHER THE NUMBER
THE WORSE.
WE SEE THAT OMICRON REALLY HAS
ABSOLUTELY NO ACTIVITY AGAINST
THE TWO ANTIBODIES HERE.
WE ALSO GENERATED SOME SPIKE
PROTEINS THAT HAD MUTATIONS ONLY
IN THE CLASS 1 OR CLASS 3 SITES
AND THESE ANTIBODIES ARE AGAINST
CLASS 1 OR CLASS 3, YOU CAN SEE
THAT THEY ARE VERY EFFICIENT BUT
THAT THE COMBINATION OF THESE
MUTATIONS IS REALLY COMPLETELY
ABROGATING THE ACTIVITY OF THESE
IMPORTANT THERAPEUTICS.
SO AS A SUMMARY, I THINK THIS
SCURRY OF THE RNA PACKAGING
SIGNAL ALLOWS SARS COV-2 VLP
GENERAL RATION AS RAPID TOOL TO
ANALYZE IMMUNE EVASION AND
SPREAD OF THE BARS INFECTIVITY.
WE IDENTIFY SEVERAL N MUTATIONS
IN LINKER REGIONS THAT ENHANCE
VIRAL INFECTION BY ENHANCING RNA
PACKAGING.
AND THE RECOMBINANT VIRUS
CONFIRMED THIS, WE THINK VLPs
ARE RAPID TOOLS TO ANALYZE VIRUS
NEUTRALIZATION.
SORRY I REALIZE I'M A LITTLE
LATE BUT I WANT TO THANK
EVERYBODY I MENTIONED EVERYBODY
REALLY WANT TO THANK DAN,
JENNIFER, ANDREAS AND ALL OUR
COLLABORATORS AND FUNDERS HERE
FOR SUPPORTING US AND I'M HAPPY
TO TAKE -- THIS IS MY LAB, MY
WEB PAGE, IF YOU HAVE ANY MORE
QUESTIONS, HAPPY TO TAKE ANY
QUESTIONS.
>> THANK YOU, MELANIE, FOR
WONDERFUL TALK.
BEFORE I READ SOME OF THE
QUESTIONS, THE CME CODE FOR
TODAY IS 37961.
37961.
SO GOING BACK TO THE VERY
BEGINNING OF YOUR TALK, THERE
WERE A FEW QUESTIONS THAT I'M
GOING TO CONDENSE.
ONE IS IS THE RELIANCE OF THE
VIRUSES ON THE CHOLESTEROL
PATHWAY RELATED TO THE ENHANCED
VIRULENCE IN HUMANS WITH
OBESITY?
AND RELATED TO THIS, IS IT KNOWN
WHETHER BLOOD CHOLESTEROL LEVELS
ARE CORRELATED WITH COVID
INCIDENCE OR SEVERITY AND WHAT
ABOUT USE OF CHOLESTEROL
REDUCING DRUGS?
>> VERY GOOD QUESTIONS.
SO HERE, A FEW THINGS TO
CONSIDER.
IS THAT OBVIOUSLY OBESITY IS A
VERY STRONG RISK FACTOR.
WE HAVE STARTED TO MAKE FOR
SEVERE DISEASE AND WE ARE
LOOKING CAREFULLY INTO THIS.
I THINK WE HAVE TO DIFFERENTIATE
BETWEEN INFLAMMATORY EFFECTS OF
OBESITY AND ANTI-OBESE OR
ANTI-CHOLESTEROL MEDICATION AND
WE HAVE TO DIFFERENTIATE BETWEEN
DIRECT ANTIVIRAL OR ANTIVIRAL
EFFECTS.
SO WE THINK THAT THERE IS A
DIRECT LINK AND FOR EXAMPLE IF
WE FEED CELLS LBL IN VITRO WE
CAN INCREASE TITERS OF THE VIRUS
BY TWO LOG.
DEFINITELY THIS IS SOMETHING THE
VIRUS LIKES AS A PURE VIRAL
REPLICATION MACHINERY --
MACHINE.
SO THAT IS SOMETHING THAT WE ARE
VERY MUCH INTERESTED IN.
IN TERMS OF ANTI-CHOLESTEROL
DRUGS, MANY PEOPLE HAVE LOOKED
INTO STATINS AND HAVE SHOWN THAT
STATINS HAVE SOME PROTECTIVE
EFFECT AGAINST SEVERE COVID.
BUT AGAIN, THE ANTI-INFLAMMATORY
EFFECTS HERE MIGHT BE VERY
IMPORTANT.
WHAT WE ARE INTERESTED IN IS
REALLY LOOKING AT DRUGS THAT
BLOCK INTRACELLULAR CHOLESTEROL
LEVELS BECAUSE WE THINK THAT
THAT IS WHAT THE VIRUS NEEDS AND
LIKES.
THE STATINS AND OTHER
ANTI-CHOLESTEROL DRUGS ARE
REALLY SHUFFLING THE CHOLESTEROL
INTO THE CELL.
WHICH MIGHT BE A DOUBLE EDGE
SWORD BECAUSE YOU MIGHT REDUCE
IT IN THE CIRCULATION BUT
INCREASE IN THE CELL.
SO THE DRUGS WE ARE USING
CURRENTLY ARE THOSE THAT ARE
GOING AGAINST INTRACELLULAR
PRODUCTION OF CHOLESTEROL AND
ARE HOPEFULLY LOWERING
INTRACELLULAR CHOLESTEROL LEVELS
AND DECREASING VIRAL REPLICATION
REPLICATION.
THIS IS SORT OF A LARGE
OVERVIEW, THERE IS AN
INTERESTING PAPER OUT FROM MY
COLLEAGUE KATHERINE BLISS AT
STANFORD WHO SHOWS THE VIRUS
DIRECTLY INFECTS ADIPOCYTES
WHICH IS SOMETHING THAT WE ARE
VERY INTERESTED IN AND WE ARE
TALKING CLOSELY WITH KATHERINE
TO SEE HOW WE CAN VERIFY THIS
AND POTENTIALLY USE THIS AS A
SPECIFIC STRATEGY.
>> HAVE YOUR STUDIES IDENTIFIED
THE MECHANISM OF WHY SARS COV-2
BUT NOT SEASONAL CORONA VIRUSES
LIKE LC 43 CAUSE CARDIAC
PATHOLOGY OR NEURAL PATHOLOGY?
>> YES, NO WE HAVEN'T BUT WE
WOULD LOVE, I THINK AS I SAID WE
ARE VERY INTERESTED IN THE
CARDIAC PATHOLOGY BECAUSE WE
HAVE BEEN WORKING VERY CLOSELY
THERE AND IS VERY FEW VIRAL
PARTICLES ARE NECESSARY TO
DAMAGE THE HEART.
SO THAT IS SOMETHING THAT WE ARE
DEFINITELY FOLLOWING UP WITH.
WE HAVE NOT DONE THE STUDIES
WITH THE COMMON COLD CORONA
VIRUSES HERE BUT I THINK WE
SHOULD.
THE OTHER THING IS THAT WE WORK
VERY CLOSELY WITH ARNOLD
CRICKSTEEN HERE AND ALSO WARNER
GREEN UCSF GLADSTONE LOOKING
WITH BRAIN ORGANOIDS INTO THE
BRAIN TRUMPISM AND ALSO
POTENTIAL DAMAGE.
I THINK THERE IS A HUGE AMOUNT
OF WORK TO BE DONE.
I THINK THE VIRUS IN OUR HANDS
REPLY CASE MOSTLY IN ASTROCYTES.
AND NOT IN NEURONS.
I THINK THERE IS NOW PAPERS OR
PRE-PRINT OUT SHOWING THAT
PEOPLE ACTUALLY RECOVER IN LONG
COVID INFECTIOUS PARTICLE FROM
THE BRAIN SO THERE MIGHT BE A
RESERVOIR FUNCTION HERE WHICH IS
SOMETHING WE DEFINITELY NEED TO
LOOK INTO.
ONE LAST THING, I THINK THE KEY
WITH COMMON COLD CORONA VIRUS IS
THEY REPLICATE TO MUCH LOWER
LEVELS THAN SARS COV-2, EITHER
WE HAVEN'T MADE IT INTO THE
RIGHT SYSTEM BUT DIRECT
COMPARISONS HERE ARE A LITTLE
BIT HAMPERED BY THE DIFFERENT
REPLICATION KINETICS OF THE
VIRUSES.
>> WE ARE GOING TO SWITCH TO
YOUR CRISPR BASED TESTING
APPARATUS.
CAN YOU KINETIC APPROACH
DISTINGUISH VARIANTS WITH ONE OR
TWO MISMATCHES IN DIFFERENT
POSITION?
>> YES, YES WE CAN.
I THINK IT IS STILL -- I MEAN,
WHAT MAKES A GUIDE WORK AND WHAT
MAKES A GUIDE NOT WORK IS STILL
SOME -- LITTLE BIT OF A MYSTERY.
I THINK THERE IS MANY ALGORITHMS
BEING WRITTEN ABOUT IT AND MANY
PEOPLE CLAIMED THEY KNOW HOW TO
DESIGN A GOOD GUIDE VERSUS A BAD
ONE.
IF THE MUTATION IS IN A CERTAIN
PLACE AND WE HAVE TO USE THAT
AREA BASICALLY IN ORDER TO
DESIGN THE GUIDE, I THINK WE ARE
STILL DEPENDENT ON CERTAIN
ASPECTS OF THE ACCESSIBILITY,
3-D FORMATION OF LOOPS AND STUFF
LIKE THIS WHICH WE HAVEN'T
REALLY FULLY MASTERED.
BUT I THINK IF WE HAVE IT IN AN
AREA THAT WORKS AS A GUIDE AND
WE CAN MOVE THE GUIDE UP AND
DOWN AND MOVE THE MUTATION IN
THE GUIDE UP AND DOWN, I THINK
WE HAVE VERY GOOD -- WE HAVE
VERY GOOD SENSIBILITY,
OPPORTUNITIES TO MAKE GUIDES
THAT CAN DIFFERENTIATE BETWEEN
SINGLE MUTANTS.
>> AND REGARDING THE LAST TOPIC
YOU DISCUSSED, HOW MUCH DO THE
MUTATIONS IN THE END CONTRIBUTE
TO MAKING THE OMICRON VIRUS MORE
VIRULENT?
CAN YOU USE YOUR SYSTEM TO LOOK
AT THE DIFFERENT CONTRIBUTIONS
OF THE SPIKE MUTATIONS VERSUS N
MUTATIONS?
>> WE FIND THE N MUTATIONS ARE
MORE IMPORTANT IN MAKING THE
VIRUS INFECTIOUS.
SO IF WE PUT AN OMICRON N INTO
AN VLP WE SEE A HIGHER
INFECTIVITY THAN IF WE PUT THE
SPIKE FROM OMICRON SPIKE ON VLP.
BUT WE THINK THAT BOTH TOGETHER
ARE REALLY MAKING THAT VIRUS
UNIQUELY MORE INFECTIOUS.
EVEN OVERRIDING THE NEGATIVE
EFFECTS OF THE M AND E MUTATIONS
THAT WE IDENTIFIED.
>> THE FINAL QUESTION, DO YOU
THINK THAT MUTATIONS IN THE N
PROTEIN VARIANTS ARE FOR
COMPETING OVER THE RNA BINDING
PROTEINS LIKE HOST SUPPRESSION
OF THE IMMUNE RESPONSE TO
SUPPRESS IMMUNE RESPONSE?
>> POSSIBLE.
VERY POSSIBLE AND WE DON'T KNOW
YET.
I THINK EVERYTHING IS ON THE
TABLE AT THIS POINT.
>> ALL RIGHT.
THANK YOU SO MUCH, MELANIE.
THIS ENDS TODAY'S LECTURE.
THANK YOU, EVERYONE.
>> THANK YOU, BYE-BYE.