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.