>> THE TRACO LECTURE'S FIRST SPEAKER IS NATASHSHA CAPLEN,. SHE DID A POST DOCTORAL FELLOWSHIP ON GENE THERAPY AND THEN IN 1996 SHE CAME TO THE NATIONAL HUMAN GENOME RESEARCH INSTITUTE AND THERE SHE STARTED WORKING ON THE RNA INTERFERENCE AND SUBSEQUENTLY WE RECRUITED HER TO NCI IN 2004. HER TITLE RNA INTERFERENCE, NATASHSHA. >> THANK YOU, TERRY. CAN ANYONE HEAR ME? COME A LITTLE FORWARD? NO? ALL RIGHT. SO MY PRESENTATION TODAY IS GOING TO FOCUS ON RNA INTERFERENCE, AND I THINK THIS IS ALWAYS A NICE SUBJECT TO INSTITUTE IN THE TRACO COURSE. AND I'M NEVER QUITE SURE WHAT THE FOCUS REALLY IS AND THE CONCEPT OF TRANSLATIONAL RESEARCH. AND THIS IS REALLY, I THINK, DOES REFLECT THE STORY THAT EN COMPASSES WHAT I FEEL IS TRANSLATIONAL RESEARCH. AND THAT IS TAKING A VERY, VERY BASIC SCIENCE OBSERVATION -- IN THIS CASE ORIGINALLY IN THE EARLY 19NOOIRNTS -- 1990S IN PLANTS IN PETUNNIAS AND A FUNGI, WHICH WAS EXTENDED INTO AN ORGAN AND THE WORK THAT I WAS INVOLVED WITH, WHICH WAS MOVING THESE OBSERVATIONS INTO MAMMALIAN CELLS AND HOW I GOT INTO THIS PROCESS IN THE EARLY 2000. AND THIS KNOWLEDGE WENT INTO BE ABLE TO BUILD A CLINICAL TRIAL WITHIN JUST A FEW YEARS. SO WE ARE GOING QUICKLY FROM A BASIC OBSERVATION, SOMETHING THAT WOULD NOT GET TRADITIONALLY FUNDED AS TRANCE LAGZAL RESEARCH, WHICH HAS HAD TRULY TRANSLATIONAL IMPACT. AND I WILL TRY TO GIVE DIFFERENT EXAMPLES THROUGH THE COURSE OF THIS PRESENTATION THAT REFLECT THESE DIFFERENT FEATURES. OKAY, SO BEFORE WE START -- I KNOW THERE IS PROBABLY PEOPLE ONLINE OR WILL BE PEOPLE ONLINE, BUT I WANT TO GET A SENSE FROM THIS AUDIENCE HOW MANY PEOPLE HAVE DONE EXPERIMENTS AND ARE REASONABLY FAMILIAR WITH THE RNA PATHWAY. TWO. THREE. DON'T BE SHY. OKAY, ALL RIGHT. OKAY, SO I DON'T WANT TO SPENT TIME TALKING TO A MECHANISTIC TYPE OF TALK BUT I DO WANT TO MAKE SURE THAT EVERYBODY IS ROUGHLY ON THE SAME PAGE IN TERMS OF WHAT WE'RE TALKING ABOUT. BECAUSE WE'RE EXPLOITING THE RNA APPLICATION FOR FUNCTIONAL GENOMIC STUDIES AND WE'RE TRYING TO ACCESS AND POLITE A NATURALLY OCCURRING PATHWAY. AND HERE IS A RATHER SIMPLISTIC MODEL OF THE RNA PATHWAY, WITH SOME OF THE JUST SOME OF THE KEY PLAYERS IT IN IT. THIS HAS EXPANDED DRAMATICALLY THE NUMBER OF KEY PLAYERS IN IT. BUT I AM JUST GOING TO HIGHLIGHT SOME KEY POINTS. SO THE PRINCIPAL ELEMENTS OF THE PATHWAY. THIS IS A COMPLETELY NATURALLY OCCURRING PATHWAY THAT CONTROLS GENE SILENCING AND THE MAJOR MEDIATORS OF IT IN ITS NATURAL SETTING ARE MICRORNA'S, GENES WITHIN THE GENOME BETWEEN APPROXIMATELY 600 IN MAMMALIAN CELLS AND THE NUMBERS VARY A LITTLE BIT DEPENDING ON YOUR DEFINITION OF WHAT EXACTLY A MICRORNA LOOKS LIKE AND WHAT IT DOES. THESE ARE TRANSCRIPTS. THEY'RE VERY CONSISTENT STRUCTURE WITHIN THE PRIMARY TRANSCRIPT, SOME OF WHICH CAN BE POLY SIS TRONNICS AND ARE BURIED WITHIN PROTEIN EN CODING GENES. THOSE ARE PROCESSED BY THIS FIRST COMPLEX, WHICH PRIMARILY CONSISTS OF TWO PROTEINS. TRASHA IN MAMMALIAN CELLS. THEY PROCESS MICRO RNA AND HAVE THIS VERY UNIQUE HAIRPIN STRUCTURE. I'VE DRAWN IT HERE AS HAVING EXACT BASE PARING, JUST NOT TRUE. THEY HAVE A LITTLE BIT OF WOBBLE IN THEM. SO THIS IS AN ARTISTIC REPRESENTATION OF IT. THIS IS TRANSPORTED FROM THE NUKE LOUIS INTO THE CYTOPLASM WHERE IT EN COUNTERS AN IMPORTANT ENZYME CALLED DICA WHICH RAPIDLY TO FORM A DOUBLE STRANDED RNA THAT CAN THEN BE LOADED INTO AN AR GONAL PROTEIN. THERE ARE FOUR DIFFERENT ARGONAL PROTEINS WITHIN THE MAMMALIAN SYSTEM AND THOSE THAT CAN INDUCE A TRANSCRIPTIONAL REPRESSION THROUGH MICRONASE COMBINE TO MANY OF THESE DIFFERENT ARGONAL PROTEINS. THESE BIND OR LOAD IN THE MICRO RNA FROM ONE STAND. IT'S A SINGLE STRAND AT THIS POINT. THIS WILL FIND A SEQUENCE THAT HAS SOME DEGREE OF HOMOLOGY TO IT WITHIN A TARGET TRANSCRIPT. THIS IS NOT THE EXACT HOMOLOGY, EXCEPT AT ONE END AND THIS END OF THE MOLECULE. THIS WILL HAVE BETWEEN SIX AND SEVEN NUCLEOTIDES OF HOMOLOGY AND DIFFERENT CAN BUBBLES BECAUSE IT'S NOT A COMPLETE MATCH AT THIS POINT. THIS WILL INTERACT TO EITHER CAUSE TRANSLATIONAL EXPRESSION, TRANSCRIPT DEGRADATION OR RNA SKWEEFRGS. AND SEQUESTRATION. AND THIS IS HOW MICRO RNA MODULATE PROTEIN EN CODING GENES AND THIS IS 15 PERCENT ARE REGULATED IN THEIR EXPRESSION BY MICRO NRNA AND EVERY SINGLE PROCESS IS REGULATED TO SOME DEGREE BY MICRO RNA. IN THIS CONTEXT OF THIS AUDIENCE, WE KNOW THAT MICRO RNA'S ARE MAJOR CONTRIBUTORS TO THE CAMFO PHENOTYPE THIS WHEN PROCESS IS DEREGULATED. WELL, WHAT I AM GOING TO PREDOMINANTLY TALK ABOUT TODAY HERE IS HOW WE'VE EXPLOITED THIS PROCESS TO TRY AND ACTUALLY TARGET SPECIFIC TRANSCRIPTS TO DEGRADATION OF THE TRANSCRIPT AND THEREFORE A LOSS OF FUNCTION PHENOTYPE. AND HERE WHAT WE'RE DOING IS WE ARE MIMMICKING TWO DIFFERENT MOLECULES THAT ARE WITHIN THIS PATHWAY, USING SHORT PIN RNA'S WITH THIS TYPE OF STRUCTURE OR WE CAN JUST GO TO SYNTHETIC SRNA'S THAT EFFECTIVELY MIMIC THIS PARTICULAR MOLECULE HERE. THESE ARE DESIGNED TO SPECIFICALLY BE TARGETED IN THE IGO 2 GROUP COMPLEX AND IT IS OF THE FOUR AND THE ONLY ARGONITE PROTEIN THAT CAUSES CLEAVAGE AND IT'S AN EXACT NUKE TYPE FROM THE END HERE AND IT WILL CLEAVE AND LATER TRANSCRIPT DEGRADATION AND THEN LOSS OF FUNCTION. SO WE HAVE A NATURAL MEDIATORS, SOME OF WHICH ARE MENTIONED HERE BUT I AM GOING TO BE FOCUSED ON TODAY ON THESE ARTIFICIAL MEDIATORS THAT WE CAN USE FOR FUNCTIONAL GENOMIC STUDIES. SO JUST TO GIVE YOU A LITTLE BIT OF SENSE OF HOW PEOPLE HAVE PERCEIVED THE IMPORTANCE OF THIS PATHWAY AND HOW IT'S BEING USED. SO THE KEY THING IN THE DISCOVERY OF RNA INTERFERENCE INITIALLY AS I SAY IN PLANTS AND THEN IN OTHER ORGANISMS AND THEN MOST RECENTLY IN MAMMALIAN CELLS IS THE CENTRAL PLAYER IN GENE EXPRESSION. WE WERE VERY FAMILIAR WITH ITS ROLE IN TRANSCRIPTION, AND IN TRANSLATION, BUT HERE WE'RE SEEING NOW IT DIRECTLY REGULATES GENE EXPRESSION. AND THAT IT ALSO, THROUGH MICROASE, APPEARS TO BE KEY IN THE CELLULAR RESPONSE TO EXTERNAL STIMULI AND HOST PATHOGEN RESPONSES. I AM GOING NOT GOING TO TALK ABOUT THED THESE TODAY BUT IF YOU ARE INTERESTED IN THOSE ELEMENTS, THERE IS A GREAT NUMBER OF REVIEWS OUT THERE THAT YOU CAN LOOK AT. WE'RE NOW VERY CLEAR THAT RNA DOES HAVE A ROLE IN DISEASE INITIATION, PARTICULARLY IN CANCER BUT POTENTIALLY IN OTHERS AND INCREASING IN RESPONSE TO THERAPY. WELL, THE FOCUS OF TODAY IS GOING TO BE THOSE PREDOMINANTLY IN THE CONCEPTS OF HOW WE'VE BEEN ABLE TO APPLY WHAT WE NOW KNOW ABOUT THE RNA PATHWAY TO USE IT FOR FUNCTIONAL GENOMIC STUDIES. SO IT BECOMES A BIOLOGICAL TOOL THAT WE CAN TEST VARIOUS HYPOTHESIS PREDOMINANTLY FROM LARGE AMOUNTS OF GENTSIC DATA AND FUNCTIONAL DATA THAT'S NOW JAVENLT BUT WHERE WE NEED ADDITIONAL TOOLS TO BE ABLE TO SIDE THIS. LOOKING AT GENE PROTEIN FUNCTION DISCOVERY, PATHWAY NETWORK ANALYSIS AND TARGET IDENTIFICATION, VALIDATION AND LOOKING FOUGHTAT ENERGY SNOIRNGIC INTERACTIONS AND I'LL TALK ABOUT THAT AND THEREFORE IT'S HAD IMPLICATIONS FOR DRUG DEVELOPMENT. AND IT HAS A LITTLE BIT BEEN USED FOR DEVELOPING MODEL SYSTEMS, SO THAT HASN'T TAKEN UP AS MUCH AS MANY PEOPLE THOUGHT AT THE BEGINNING. AND THEN RECENTLY I'LL TALK ABOUT SOME OF THE APPLICATIONS FOR THERAPEUTICS DIRECTLY. ALL RIGHT, SO WHAT I AM GOING TO TALK ABOUT TODAY I'LL DIVIDE INTO TWO DIFFERENT SECTIONS LOOKING AT HOW WE CAN USE RNA TO LOOK AT THE FUNCTION OF INDIVIDUAL GENES, PARTICULARLY IN THE CONTEXT OF CANCER BIOLOGY AND THE CONTEXT OF HOW MY LAB AND OTHERS HAVE BEEN USING IT TO LOOK AT IN TERMS OF ANTI-CANCER THERAPEUTIC APPROACHES. OKAY. SO WHY DO WE ACTUALLY NEED -- WHAT IS THE GREAT ADVANTAGE OF BEING ABLE TO CONDUCT RNA INTERFERENCE ANALYSIS? ACTUALLY, I'M GOING TO SKIP THAT ONE. WHY WE'VE BEEN DOING THIS? SO THIS IS ACTUALLY DATA THAT GLO IN MEDICAL ORNGOLOGY BRANCH WAS ABLE TO GIVE ME FROM WORK THAT HE GOT FROM ADD HARLO. AND WHAT HE HAD DONE, AND I SORT OF REANALYZED, WAS THIS IS 2009 AND I DON'T THINK IT'S REALLY CHANGED THAT MUCH IN THE LAST THREE YEARS. THESE ARE THE NUMBER OF PUBLICATIONS UP TO 2009 THAT HAVE BEEN PUBLISHED ON DIFFERENT PROTEINS. SO IF YOU LOOK AT THIS, WHAT YOU ARE SEEING IS THAT THERE ARE ACTUALLY A THIRD OF PAPERS THAT HAVE BEEN PUBLISHED UP TILL THAT DATE HAVE BEEN JUST LOOKING AT 55 PROTEINS. ALL RIGHT, SO 85 PERCENT OF PROTEINS, THERE IS LESS THAN 100 PAPERS ON THEM. SO YOU STUDY UP ON MOST PROTEINS IN CERTAINLY ONE DAY. AND MANY PROTEINS YOU CAN STUDY IN ABOUT 10 MINUTES BECAUSE THERE IS ACTUALLY NOT A SINGLE PAPER ON THEM. SO THERE IS CLEARLY A NEED FOR US TO BE ABLE TO COME UP WITH FUNCTIONAL WAYS OF LOOKING AT THE GENOME IN HIGH THROUGH PUT THAT ALSO NOW MATCHES OUR ABILITY TO BE ABLE TO SEQUENCE GENES IN HIGH-THROUGH PUT, BE ABLE TO LOOK AT RNA LEVELS IN HIGH THROUGH PUT AND TO BE ABLE TO LOOK AT EPOGENETIC CHANGES IN HIGH THROUGH PUT. AND TERRY WAS JUST SAYING YOU WENT UP TO THE CONSEQUENCING FACILITY LAST WEEK AND WE CAN DO THOSE. CAN WE NOW DO THAT IN A WAY THAT WE CAN FUNCTIONALIZE IT? WHAT I AM GOING TO TALK ABOUT IS STARTING TO ONE GENE AND GETTING UP TO WHOLE GENOME FUNCTIONALITY WITH THIS APPROACH. SO WHAT ARE WE TRYING TO DO IN THE LABORATORY FOR THOSE OF YOU WHO HAVE DONE EXPERIMENTS? THIS IS USING SRNA AND THIS IS WHAT YOU ARE TRYING TO DO. YOU ARE DELIVERING EITHER AN SRNA OUR USING A HAIRPIN STRUCTURE THAT'S BEING DELIVERED TO THE CELL EITHER USING A NON-VIRAL DELIVERY SYSTEM USUALLY A LIPID OR USING A VIRAL VEHICLETRAL OR AN EXPRESSION VECTOR FOR AN SRNA. LEADING TO REDUCE PROTEIN CINT THIS, THEREFORE A LOSS OF FUNCTION THAT CAN THEN BE READ OUT EITHER AS AN MRNA LEVEL AND AT A FUNCTIONAL GENE PHENOTYPIC LEVEL. SO WHEN YOU'RE CONDUCTING EXPERIMENTS IN THE LAB WHAT IS KEY IS TO OPTIMIZE MANY OF THESE DIFFERENT STEPS TO BE ABLE TO INTERPRET THE FUNCTIONAL DATA THAT YOU WILL GET AT THE END OF THIS. THAT MEANS MAKING SURE THAT YOU HAVE THE RIGHT MODEL SYSTEM THAT'S APPROPRIATE TO THE BIOLOGY THAT YOU ARE TALKING ABOUT, THE HE HAVE CASSISE OF ALL OF THESE DIFFERENT STEPS, AND I AM ALSO GOING TO TOUCH ON ISSUES RELATED TO SPECIFICITY, BECAUSE THESE ARE BIOLOGICAL ENTITIES THAT ARE GOING INTO A NATURALLY OCCURRING PATHWAY. THE CELL DOESN'T KNOW WHAT YOU WANT TO DO. YOU HAVE TO BE ABLE TO USE RESOURCES AND THINK ABOUT YOUR TATA IN A WAY THAT UNDERSTANDS HOW THIS PATHWAY WORKS TO MINIMIZE ISSUES RELATED TO THE SEQUENCES OF THESE AFFECTED MOLECULES. DATA. SO THIS IS A REGIONAL TYPICAL EXPERIMENTS IN FROM MY LAB BUT THE CONCEPT IS EXACTLY THE SAME AND IT'S THE TYPE OF THINGS THAT MANY OF YOU THAT ARE IN THE LAB ARE LIKELY TO DO AND I WANT TO SHOW THIS SO YOU GET A SENSE OF WHAT WE'RE LOOKING AT. SO THIS IS LOOKING AT NORMALIZED PHENOCATENNIN LEVELS AND THIS IS BEING KNOCKED DOWN IN A COLORECTAL CANCER CELL AND THIS IS A NORMALIZED RNA LEVELS AND THIS WAS DONE WITH A NON-QRTPC BUT IF YOU GET THE SAME DATA. AND WHAT WE HAVE ARE TWO DIFFERENT SRNA'S THAT HAVE DIFFERENT SEQUENCES AND DIFFERENT DIFFERENT TARGET DIFFERENT PARTS OF THE GENE BUT THEY BOTH RESULT IN A SIGNIFICANT REDUCTION IN THE MRNA LEVELS OF THIS PARTICULAR GENE. IF YOU THEN LOOK AT THE WEST END OF THIS AND LOOKING LOOKING AT THE TWO DIFFERENT SRNA'S THAT WERE SHOWN PREVIOUSLY. THIS IS SRNA 1 AND 2 AND WE ARE COMPARING A CONTROLLED SRNA VERSUS THE SRNA OF THE GENE OF INTEREST IN THIS CASE BETOKETTININ AND QUANTITIESIFIED THE LEVELS WITH MULTIPLE LOTS. AND WHAT WE'RE LOOKING AT IS 48, 72 AND 96 HOURS. SO THE KEY THING I WANT TO TAKE AWAY IS TO REMEMBER THAT RNA IS A SILENCING MECHANISM AND IS NOT A KNOCKOUT. YOU WILL STILL HAVE SOME RESIDUAL PROTEINS, AND YOU ARE RELYING ON BLOCKING NEW CINT THIS OF PROTEIN. AND SO WHATEVER PROTEIN IS THERE WHEN YOU START, YOU EXPERIMENT -- YOUR EXPERIMENT HAS TO DECAY OFF. AND THAT'S WHAT YOUR DEGREE AT SILENCING IS GOING TO BE VERY DEPENDENT ON THE HALF LIFE OF THAT PROTEIN. SO IF YOU HAVE A HALF LIFE OF A PROTEIN THAT IS WITHIN A FEW HOURS, YOU ARE GOING TO START SEEING EFFECT AT 24, 48, 72, 96 HOURS. BUT FOR SOME PROTEINS THEY'RE RELATIVELY STABLE AND THEREFORE THAT IS NOT GOING TO BE AN EFFECT THAT YOU ARE GOING TO SEE. ALSO, I AM GOING TO MENTION AND REINFORCE IN A MINUTE, THIS PROCESS IS VERY DYNAMIC, AND IS VERY DEPENDENT ON FEATURES LIKE HOW MUCH PROT TEEN YOU HAVE TO START WITH IN TERMS OF INTERPRETING YOUR RESULTS. THE ONE I ALWAYS QUOTE IS THE HSP 19 SHEPRON PROTEIN. THE TRANSCRIPT IS -- THERE ARE AT LEAST 2,000 -- OVER 2,000 COPIES OF THE HSP 90 TRANSCRIPT IN EVERY SINGLE CELL. OKAY, SOME SO YOU CAN SILENCE THAT BY 95 PERCENT. AND YOU WILL ACTUALLY STILL NOW HAVE AS MANY TRANSCRIPTS LEFT AS YOU WILL HAVE FOR MORE MOOD RATLY EXPRESSED GENES. THE CHANCES OF INTRODUCING A PHENOTYPIC EFFECT, EVEN IF YOU SILENCED IT BY 95 PERCENT IS STILL REDUCED BECAUSE THERE IS STILL A QUITE A LOT OF THAT TRANSCRIPT AND YOU HAVE QUITE A LOT OF THAT PROTEIN AROUND. TRANSCRIPTS CAN BE VERY HARD TO KNOCK DOWN. SOME SUBCLASSES OF PROTEINS DEPENDING ON THE LEVELS OF THAT PROTEIN TO BEGIN WITH, THE HALF LIFE OF THAT PROTEIN TO BEGIN WITH, AND THE THRESHOLD REQUIRED OF THAT PROTEIN TO ACTUALLY INDUCE A PHENOTYPIC EFFECT. THIS IS A SIMILAR EFFECT WITH AN SHRNA WHERE WE'VE INTRODUCED AS AN INDIVIDUAL CASSETTE, AND WITH BETAKETTININ AND LOOKING FOR SILENCING AND RNA LEVEL. SO AS I WAS JUST MENTIONING, AND I DO REALLY WANT TO REITERATE THIS BECAUSE IT'S VERY IMPORTANT IN TERMS OF TRYING TO INTERPRET RNA EXPERIMENTS AND ONE OF THE BIGGEST PROBLEMS I HAVE FOR EXAMPLE IS A REVIEWER OF MANUSCRIPTS OR TRYING TO INTERPRET TO TRY AND UNDERSTAND IS SOME OF THESE FEATURES. SO I WANT TO REITERATE HERE AT MOST OF YOU AT SOME POINTS IN YOUR CAREERS WILL DO AN RNA EXPERIMENT. THIS IS A KNOCKDOWN. IT'S A SILENCING. IT'S NOT A KNOCKOUT SO THERE WILL BE SOME PROTEIN LEVEL AND LEFT AND IT WILL BE HIGHLY VARIABLE EVEN FOR AN RNA TARGETING THE SAME TRANSCRIPT OF WHAT YOU WILL INDUCE. AND THE RNA PHENOTYPE WILL BE DEPENDENT ON A LARGE NUMBER AND MOST OF THESE ARE NOT LINEAR. JUST BECAUSE YOU HAVE THE RNA DOWN DOES NOT MEAN YOU HAVE THE PROTEIN DOWN TO A LEVEL THAT WILL PRODUCE A PHENOTYPE. ONE OF THE THINGS WE TALK ABOUT IS THE TIPPING POINT. YOU WILL SEE IF YOU ARE LOOKING FOR RNA RESOURCES THAT EVERYBODY -- A LOT OF PEOPLE TALK ABOUT IT GUARANTEES 70 PERCENT KNOCKDOWN. , THAT'S GREAT AND GOOD. YOU'RE OVER THE HETEROZYGOUS STATE SO THAT MAKES SENSE. HOWEVER, IT WILL BE PROTEIN-SPECIFIC AS TO WHAT IS THAT TIPPING POINT. FOR SOME ENZYMES A 95 PERCENT KNOCKDOWN IS STILL GOING TO HAVE NO EFFECT WHATSOEVER ON A CELLULAR PHENOTYPE. FOR FOR SOME STRUCTURAL PROTEINS YOU MAY ONLY NEED 40 TO 50 PERCENT KNOCKDOWN AND YOU CAN SEE SOME EFFECTS. IF YOU TAKE OUT NUCLEAR PORIN, YOU DON'T NEED TO TAKE AWAY MUCH OF THAT TO SEE THE CELLS. ONE OF THE THINGS THAT PEOPLE HAVE YET -- THEREFORE IS A SHAME AND IT'S OFTEN FOR ME IT'S OFTEN JUST LIMITS OF TIME AND TRYING TO DO THESE EXPERIMENT IS QUITE DIFFICULT. BUT IT'S SOMETHING I DO LIKE TO REMIND PEOPLE. THIS ALLOWS YOU TO HAVE ENORMOUS FLEXIBILITY THAT YOU CAN GENERATE HYPERMORPH AND PRODUCE POTENTIALLY CELLS THAT WILL HAVE DIFFERENT LEVELS OF YOUR PROTEIN THAT HAVE A 20 PERCENT REDUCTION, A 40 PERCENT REDUCTION, 80 PERCENT AND 95 PERCENT REDUCTION IF YOU ARE CAREFUL WITH YOUR RNA MOLECULES AND THAT CAN BE USEFUL UNDER SOME CIRCUMSTANCES. THE OTHER FEATURES THAT NEED TO REMEMBER IS THAT ONE GENE MAY NOT BE ENOUGH TO GENERATE A FENE TYPE. WE ARE VERY USED TO KNOCKOUT MICE WHERE YOU TAKE AWAY A GENE PREDEVELOPMENT AND LOOKING FOR PHENOTYPES. BUT ONCE YOU HAVE AN ESTABLISHED CELL, WE HAVE A LOT OF FUNCTIONAL REDUNDANCY AND KNOCKING OUT ONE GENE MAY NOT BE ENOUGH. YOU MAY HAVE TO LOOK AT MULTIPLE LEVELS OF A COMPLEX TO ACTUALLY GET A FENE TYPIC EFFECT. THE TIMING IS CRITICAL AND IT THIS RELATES PREDOMINANTLY TO THE COMPLEX OF PROTEIN DECAY. AND SO FOR SOME SYNTHETIC SRNA'S WE TEND TO LOOK AT THEM BETWEEN 24 AND 96 HOURS. OCCASION LL LONGER BUT VERY RARELY. A LONGER TIME SILENCING BETWEEN SHRNA'S CAN BE USEFUL BUT I CAN ALSO SEE A LOT OLOSS OF SILENCING EFFECT OVER TIME THERE. AND THE SPECIFICITY OF RNA IS RELATIVE AND I'LL TALK ABOUT THIS IN A MOMENT. YOU NEED TO BE ABLE TO MAXIMIZE THE SPECIFIC EVENT OF TARGETING A TRANSCRIPT TO PERFECT SEQUENCE ALIGNMENT SO THAT THE RNA CLEAVAGE WILL MINIMIZE TARGET EFFECT AND I AM GOING TO DESCRIBE THAT IN A MOMENT IN MORE DETAIL. YOU WANT TO MINIMIZE MAMMALIAN CELLS FOR DOUBLE STRANDED RNA'S. FOR A LONG TIME THERE WAS A SIGNIFICANT DELAY BETWEEN THE DESCRIPTION OF MICRORNA'S PREDOMINANTLY BECAUSE MOST PEOPLE HAD VERY WELL CHARACTERIZED IMMUNE RESPONSES TO DOUBLE STRANDED RNA'S AND AT SUMMATION WAS THAT THIS WOULD NOT BE A MECHANISM THAT WAS -- HAD WAS MAINTAINED WITHIN MAMMALIAN CELLS. SO YOU WANT TO ALSO THEN MINIMIZE THEISTIC ON THE INDODGENUS HAD YOU ROLE OF THE PATHWAY AND I'LL COME TO THAT. SO HOW DO WE USE RNAI? SO WE CAN LOOK AT A FEW GENES AND LOOK AT SPECIFIC INTERACTIONS AND CAN BE LOOKED AT A PATHWAY LEVEL. OR WE CAN DO SCREENS WHERE WE LOOK AT LOTS AND LOTS OF GENES BUT LOOK AT ONE PARTICULAR FUNCTION END POINT. AND WE CAN ALSO CONFINE ANY OF THIS ANALYSIS WITH OTHER INTERVENTIONS. I AM GOING TO TURN TO ONE SPECIFIC EXAMPLE AND ILLUSTRATE SOME POINTS I COVERED IN THE BEGINNING. YOU DON'T NEED TO KNOW THE BIOLOGY PARTICULARLY HERE BUT JUST TO GIVE YOU SOME CONTEXT FOR IT. SO THIS IS A STUDY WHERE WE'VE BEEN INVOLVED WITH A NUMBER OF DIFFERENT GROUPS WHERE THEY'VE BEEN INTERESTED IN TARGETING REDUCTASE COMPLEX. SO THE RIBONUKETIDE REDUCTASE COMPLEX IS MADE UP OF RNA 1 AND 2. AND IT'S BEEN LONG CONSIDERED AN ANTI-CANCER ANTIVIRALS MOLECULAR TARGET BECAUSE WE KNOW THERE IS INCREASED ACTIVITY IN CANCER CELLS BECAUSE THEY ARE TENDING TO DIVIDE AND THEY'RE ALSO USUALLY SUSCEPTIBLE TO DIFFERENT DNA DAMAGE AND THEY ARE BEING CONTINUALLY TRYING TO MEND THE DNA THERE. AND SO REQUIRING DMTP'S AND THERE HAVE BEEN A COUPLE OF CHEMICAL-BASED APPROACHES TO TRY AND INHIBIT RM 1 THROUGH THE USE OF IT WHICH IS IN CLINICICAL TRIAL. AND HYDROXYHAS AN EFFECT ON RNA, TOO. MOST IMPORTANTLY TECHNOLOGY HAS BEEN USED TOO, AND THIS I WILL DISCUSS HOW RNA STARTS BEING DEVELOPED IN THIS CONTEXT. SO A NUMBER OF GROUPS CAME TO US WHERE IT CAME UP AS ONE OF THE GENES THEY WERE INTERESTED IN STUDYING AND I AM GOING TO USE SOME OF THE DATA TO ILLUSTRATE FROM THE POINTS THAT I'VE JUST MENTIONED. SO THIS IS A STUDY THAT WE CONDUCTED IN THE GROUP HERE IN THE SAME BRANCH AS ME, THE GENETICS BRANCH AND WHAT THEY HAD OBSERVED IS IF YOU LOOKED AT THE EXPRESSION OF RM 2 IN COLORECTAL TUMORS, YOU SAW A SIGNIFICANT UPREGULATION OF THE RM 2 GENE, WHICH IS TO SAY WAS IN LINE WITH PREVIOUS STUDIES IN MANY DIFFERENT CANCERS. SO WHAT THEY WANTED TO DO WAS TO LOOK TO SEE WELL, WHAT WOULD BE THE FUNCTIONAL EFFECTS OF SILENCING RM 2 IN THESE PARTICULAR CELLS IN MORE DETAIL BECAUSE THERE HAS BEEN DISCUSSION OF DEVELOPING OR TARGETING RM 2 SPECIFICALLY. SO WHAT WE NEEDED FIRST OF ALL WAS A CELL LINE MODEL THAT WAS APPROPRIATE SO WE SCREENED A WHOLE STUDY OF LINES TO FIND A CELL LINE THAT HAD OVEREXPRESSION OF RM 2 AND A WHOLE BUNCH OF OTHER GENES THAT WE WERE INTERESTED IN PART OF THAT WORK AND WE SETTLED ON S 84 CELLS AS OUR MODEL SYSTEM FOR THIS STUDY. WE OBTAINED A SIERZ OF SMRNA'S AND HERE IS THE LOCATION OF THE SEQUENCING AND THESE HAVE AN EXACT MATCH TO THE 21 NUCLEOTIDES AND THEY HAVE EXACT MATCHES IN SEQUENCE TO DIFFERENT PROPORTIONS OF THE RM 2 TRANSCRIPT. AND HERE YOU ARE SEEING THE PRIMARILY CONSENSUS SEQUENCE FOR RM 2. THESE ARE SOME ALTERNATIVES HAVE BEEN HAVE BEEN DESCRIBED AND THESE ARE THE THREE THAT WE WERE STUDYING IN THIS PARTICULAR INVESTIGATION. THIS IS 3, 5 AND 6 AND THEIR LOCATIONS. TWO OF THEM AND ONE WITH XON 4. SO IF YOU INTRODUCE THESE SRNA'S INTO THE T CELLS, WHAT YOU SEE IS IN THE DOTTED LINE IS THE DECREASE IN MRNA OVER TIME AND SPECIFICALLY WHAT YOU WOULD SEE, THEORETICALLY A DRASTIC INCREASE OVER TIME AND EVENTUALLY WILL OWE REOCCUR. HOWEVER, YOU HAVE TO TAKE INTO ACCOUNT THAT THESE CELLS ARE DYING. OHERE OVER TIME IS THE VIABILITY OF THESE CELLS. IT'S PRETTY STATIC TO BEGIN WITH. THEY HAVE TO GO AT LEAST ONE CYCLE, WHERE THEY HAVE NO -- THEY ARE REDUCING THE LEVELS OF DNC THEY'RE ABLE TO ACCESS AND THEN THEY DIE QUITE QUICKLY. WE WERE INTERESTING TO USE EXPRESSION PROFILING IN THIS CASE USING MICRO RNA ANALYSIS TO FIND OUT WHAT WAS WHAT ARE THE TRANSCRIPTIONAL CHANGES IN THESE CELLS I OFFER TIME -- ACTUALLY, A SINGLE TIME TO FIND OUT WHAT ARE THE GENES THAT CHANGES A SEQUENCE OF SILENCING RM 2. AND WHAT WE'RE SEEING HERE IS THE DATA FOR THE C SCORE TRANSFORM DATA WHERE THIS IS A DECREASE IN EXPRESSION AND INCREASE IN EXPRESSION FOR THE TWO DIFFERENT SRNA TARGETING RM 2 IN THIS CASE 5 AND 6. AND WE SEE A GOOD CORRELATION IN THOSE GENES THAT DO SHOW DIFFERENTIAL EXPRESSION VERSUS THE CONTROLS. AND THE RED CIRCLES ARE THE TWO PROBES THAT CORRESPOND TO RM 2. THAT'S ONE OF THE MOST SIGNIFICANTLILE CHANGED GENES. WE'VE GOT A LOT OF GENES THAT ARE SIGNIFICANTLY CHANGED. NOW WHY I WANTED TO SHOW YOU THIS WAS TO USE IT TO ILLUSTRATE ONE OF THE ISSUES, MAIN ISSUES IN THE CONCEPTS OF RINNI, WHICH IS THE CONTEST OF TARGET EFFECTS. SO AS I SAID EARLIER, WHAT WE'RE DOING IS WE'RE EXPLOITING THE MICRO RNA PATHWAY PREDOMINANTLY. SO THE SRNA'S ARE DESIGNED TO FORM A PERFECT ALIGNMENT WITH A TRANSCRIPT AND TO CLEAVE THAT TRANSCRIPT. BUT THE CELL DOESN'T KNOW THAT THIS IS AN SRNA AND DOESN'T HAVE A BIG PATH THAT SAYS I WAS MADE BY A COMPANY. IT JUST SAYS A SMALL RNA MOLL XUL THAT IT CAN PUT INTO ANY OF THE ORGANAL PROTEINS AND IT HAS THE POTENTIAL THAT THIS COULD BE TREATED AS IF THIS WAS A MICRO RNA. IT WILL BIND TO THAT AND INDUCE A MICRO RNA-LIKE EFFECT. A SEQUENCE-DEPENDENT TARGET EFFECT. NOW, THERE ARE VARIOUS THINGS IN PLAY TO TRY AND MINIMIZE THIS. BUT REALLY THERE ISN'T A MAGIC HUMP AND THERE IS ISN'T A MAGIC -- WHAT IT DOES MEAN IS YOU HAVE TO BE REALLY GOOD ABOUT USING THE LOWEST POSSIBLE DOSE OF YOUR SRNA OR SHRNA AND REALLY GOOD NEGATIVE CONTROLS AND MOST IMPORTANTLY USE IT TARGETING YOUR GENES TO CONFIRM THE FENE TYPE BECAUSE YOUR SRNA'S ARE GOING TO HAVE DIFFERENT SEQUENCES AND THEREFORE FU SEE OVERLAP BETWEEN THE GENES THAT THEY -- THE DOWNSTREAM EFFECT OF TARGETING A PARTICULAR GENE ARE SIMILAR BETWEEN MULTIPLE RSRNA'S, THE CHANCES ARE THAT THIS IS REAL. AND THIS IS AN EXAMPLE FOR THAT STUDY LOOKING AT RM 2. WHAT WE FOUND WAS IN THE CASE OF WHERE -- BECAUSE WE HAD 2 WITH COMPLETELY DIFFERENT SEQUENCES, THERE WERE ACTUALLY WITHIN OUR RELATIVELY LARGE EXPRESSION PROFILE THAT THERE WERE ONLY TWO GENES WHERE THERE WAS A POTENTIAL THAT THOSE COULD BE INDUCING OFF-TARGET EFFECT. SO WE FELT THAT THIS WAS A REASONABLE APPROACH TO BEING ABLE TO SAY THAT WELL, THERE MIGHT BE SOME OFF-TARGET EFFECT BUT THEY'RE PRETTY MINIMAL. SO WE WERE ABLE TO, I HOPE, ADDRESS THEM. BUT WHAT DO YOU SEE WHEN YOU SILENCE THIS GENE? WELL, YOU ACTUALLY SEE IN THIS CASE 48 HOURS, WE SAW A SIGNIFICANT CHANGE IN 325 GENES CONSISTENTLY BETWEEN THE TWO DIFFERENT SRNA'S THAT REPRESENTS THE GENES AND BLUE ARE THE DOWNREGULATED GENES. AND WHAT WE CAN DO IS TAKE THOSE AND DO PATHWAY ANALYSES AND VARIOUS OTHER APPROACHES AND SHOW WHAT RM 2'S DECREASE IN EXPRESSION IS A TRANSCRIPTIONAL LEVEL AND WHAT IS IT CHANGING? BECAUSE THIS IS CHANGING THE LEVELS OF DMPT, THERE ARE PROBABLY PATHWAYS THAT ARE BEING SWITCHED ON TO TRY AND COMPENSATE FOR THAT, AND WHERE THIS GROUP THAT WE WERE COLLABORATING WITH IS LOOKING AT SOME OF THESE FEATURES IN MORE DETAIL. BUT WHAT I WANTED TO DO IS USE THIS AS AN EXAMPLE BECAUSE IT IS A TARGET THAT HAS BEEN CONSIDERED AS A CLINICICALLY RELEVANT TARGET. CAN YOU TAKE THIS FROM THE BENCH TO THE BEDSIDE AS AN RNA THERAPEUTICS? THIS IS WHERE WORK DONE BY MARK DAVIS' GROUP AT CALTECH AND NOW PUBLISHED A COUPLE YEARS AGO WHERE WHAT THEY DID WAS THEY TOOK A NANOPARTICLE THAT THEY COMBINED WITH AN SRNA TARGETING MR 2 AND THEY WERE ABLE TO TARGET THE NANOPARTICLE USING THE TRANSFERON RECEPTOR. THEY ARE TARGETING MELANOMA AND WHAT THEY WERE ABLE TO DO WAS -- IN THIS DATA WE WERE SHOWING IT FROM JUST TWO PATIENTS AND THEY DOSED, AS I SAY, WITH SRNA CORRESPONDING TO RM 2 THAT WAS BOUND WITH THE NANOPARTICLE AND HERE WE'RE LOOKING AT RM 2 MRNA LEVELS IN TWO PATIENTS. HERE POST-ADMINISTRATION OF THE SRNA TO RM 2. THIS IS IN PATIENT 1. SO TWO PATIENTS JUST HAD RNA AND THEN THEY HAD ONE PATIENT WHERE THEY HAD RNA AND PROTEIN LEVELS. NOW IT MAY NOT LOOK STUNNINGLY DIFFERENT, THE PROTEIN LEVELS, BUT FOR AN IN VIVO EXPERIMENT, THIS WAS A REALLY GOOD START. YOU CAN ACTUALLY ALSO SEE THIS IN IMMUNOHISTOCHEMISTRY LOOKING AT RM 2 STAINING. AND THIS IS THE TRANSFERON RECEPTOR USED TO TARGET THE HMA AND HERE IS THE PATIENT WITH BEFORE BIOPSY AND HERE IS AFTER. SO WE'RE SEEING A DEFINITE DMUNGS IN THE RM 2 SIGNALING. THIS WAS DONE AS AN EXPERIMENT EXPERIMENTATION TO LOOK AT IT AND ALSO CONFIRMED THAT THEY HAD -- THAT IT WAS SRNA-INDUCED MRNA CLEAVAGE USING SEQUENCING TO CONFIRM THAT THEY ACTUALLY -- THAT THE SRNA IS TARGETING THIS AND NOT SOME OTHER MECHANISM. SO THIS IS A COUPLE OF YEARS AGO. PEOPLE ARE STILL TRYING TO FIND NOVEL WAYS TO DELIVER SRNA-TYPE MOLECULES TO INDUCE RNA IN VIVO. AND I WANTED TO HIGHLIGHT A PAPER THAT CAME OUT JUST A FEW WEEKS AGO. THIS WAS PROBABLY TWO PAPERS, ONE WHERE THEY WERE STUDYING SILENCING OF THE HUNTINGTON'S GENE FOR TREATMENT OF HUNTINGON. BUT THIS WAS BACK-TO-BACK WITH A STUDY FROM THE ISIS PHARMACEUTICAL COMPANY, WHERE THEY'VE TAKEN PREVIOUS KNOWLEDGE OF TECHNOLOGY AND RESPONSIBLE FOR ACTUALLY THE RM 2 AND HAVE NOW TRIED TO ADAPT SOME OF THEIR KNOWLEDGE TO TRYING TO DO WHAT THEY ARE CALLING ANTI-SINGLE STRANDED SRNA'S TO INDUCE RNA'S IN ANIMALS AND HERE WE'RE SEEING A REDUCTION IN MRNA IN THE LIVER. IT SHOULD SAID THAT TARGETING ALMOST ANY ACID IN THE LIVER IS RELATIVELY EASY IN COMPARISON WITH OTHERS. BUT THIS IS A VERY EN COURAGING STUDY AND AN EXAMPLE OF HOW RAPIDLY WE'RE SEEING EFFECT GO INTO THE LIVER. -- INTO THE CLINIC. SO NOW -- DOES ANYBODY HAVE QUESTIONS AT THIS POINT? SORRY. >> [INAUDIBLE] >> THOSE AREN'T MINE. THOSE WERE MARK DAVIS' AT CALTEC CALTECH. >> SURE. SO THIS IS A POLYMER AS A TARGETED TRANSFERAN LIGAN. AND AS WITH MANY THESE, IT HAS ASSOCIATED WITH IT. SO THIS IS A GROUP THAT WORKED ON THESE TYPES OF PARTICLES FOR QUITE SOMETIME. SO IT'S NOT THAT UNUSUAL NANOPARTICLE BUT YES, YOU'RE RIGHT. IN THIS CONTEXT THEY DID NOT REPORT ANY ADVERSE SIDE EFFECTS BUT THEY STUDIED THREE PATIENTS SO IT'S A RELATIVELY SMALL STUDY. I DON'T KNOW WHAT THE STATUS OF IT IS THE FOLLOWUP FROM THIS STUDY. NOW I AM GOING TO MOVE, FOR THE REMAINING PART OF MY PRESENTATION, ON TO HOW WE CAN NOW USE RNA IN THE CONTEXT OF SCREENS. COMING BACK TO THAT ORIGINAL POINT I MADE OF WHERE WE WANT TO APPLY THIS ON A LARGE SCALE TO FUNCTIONALLY AN TATE MUCH OF THE -- AS MUCH OF THE GENE GENOME AS WE CAN. ACTUALLY BEING ABLE TO DEVELOP WHOLE ORGANISMS QUICKLY. AND IN ALGANS IT WAS FOUND QUICKLY THAT ACTUALLY WHAT YOU CAN DO IS YOU CAN FEED SEA ALGANS THE BACTERIA THAT HOST PLASMIC THAT CARRY LARGE DOUBLE STRANDED RNA'S. BECAUSE IN THE INVERT BRATS YOU CAN USE LARGE-STRANDED RNA. THEY DO NOT HAVE THE SAME RESPONSE AS WE DO AND YOU CAN FEED THEM THE BACTERIA THAT HAVE THE PLASMA AND THE RNA IS EXPRESSED AND SILENCES THESE GENES. SO YOU CAN BUY THE BACTERIA IN LITTLE PLATES, IN EUROPE AND YOU CAN JUST PUT YOUR SEA ALGANS ON TOP OF THEM AND THEY EAT IT AND IT'S SO MUCH EASIER THAN DOING IT IN MAMMALIAN CELLS. YOU CAN DO IT PASSIVELY WITH DOUBLE STRANDED RNA. SOMETIMES SOME PEOPLE DO USE SOME SORT OF DELIVERY SYSTEM WITH IT, USUALLY NON-VIRAL SYSTEM TO TRY AND PROVE PARTICULARLY TO PRIMARY CELL. BUT IT'S STILL REMARKABLY EASY IN MAMMALIAN CELLS. THE MAIN PROBLEMS IN MAMMALIAN CELLS IS THAT THESE ARE INTRINSICALLY DIFFICULT TO GET SHRNA'S. IF YOU HAVE PARTICULAR VIRAL VECTORS THAT TRANSDUCTION CAN BE DIFFICULT. BUT YOU CAN DOT WHOLE WORMS AND THERE ARE RELATIVELY -- THERE ARE MUCH LESS DIFFERENT TYPES OF LINES THAT PEOPLE WANT TO STUDY, WHEREAS IN HUMANS, EVERYONE WANTS TO DO EVERYTHING FROM MELANOMA TO BLASTOMA TO IPF CELLS -- IT'S AN INCREDIBLY BROAD RANGE OF THINGS THAT PEOPLE WANT TO DO. AND SO TRYING TO STANDARDIZE EVERYTHING IS MUCH MORE DIFFICULT. MUCH MORE FUN, THOUGH, BUT IT'S MUCH MORE DIFFICULT. OKAY, SO OVER THE LAST FEW YEARS, RNA SCREENING HAS BEEN A CRITICAL FUNCTION OF GENOMICS AND I NEED TO UPDATE THIS ONE. BUT THIS IS A PUBLICATION LOOKING AT GENOME-WIDE RNA SCREEN. THERE IS ONLY A LIMITED NUMBER OF PLACE THAT'S CAN DO THIS SORT OF WORK. BUT THE NUMBER OF PUBLICATIONS IS MAINTAINING AN INCREASE OVER TIME. SO WHAT DO I MEAN BY SCREEN? SO, SCREENS IN A WAY WHAT WE'RE DOING IS WE'RE MAKING USE OF THE DECADE OF WORK THAT HAVE BEEN CONDUCTED IN THE CONTEXT OF CHEMICAL SCREENING, WHEN WE'RE SCREENING WITH SYNTHETIC SRNA'S ON OR GENETIC SCREENING. THE PRINCIPLE IS VERY MUCH SIMILAR. SO WHATEVER IS GOING TO BE INVOLVED YOU'VE GOT TO DO AN ASSAY DEVELOPMENT STEP AND THIS CAN BE ANYTHING BETWEEN ONE WEEK AND SEVERAL YEARS. IT REALLY DEPENDS COMPLETELY ON THE BIOLOGY AND WHAT WE'RE YOU'RE LOOKING AT. IF YOU ARE GOING TO CONDUCT A SYNTHETIC SRNA, THERE NEEDS TAB A STEP AND TYPICAL IN TERMS OF DOING A DRUG SCREENING. I MIGHT GO IT PILOT STUDIES AND A FULL SCALING COMPANY. THIS BIT IS ACTUALLY THE EASY PART THESE DAYS. WE'VE GOTTEN TO A POINT WHERE THIS CAN TAKE YEARS. THIS CAN TAKE A WEEK AND THIS CAN TAKE YEARS. SO IF YOU HAVE THE RIGHT INFRASTRUCTURE, THIS IS THE EASY BIT. THIS PART'S REAL REALLY DIFFICULT AND THIS PART'S REALLY DIFFICULT BUT THAT'S THE CHALLENGE. SO I WANT TO CONTRAST THIS. THIS IS WHAT I'VE JUST DESCRIBED IS IN THE CONTEXT AND THIS CAN BE DONE WITH AN SHRNA. MORE TYPICALLY WHAT THEY'RE USED TO SOMETIMES KNOWN AS A DROPOUT SCREEN, WHICH IS WHERE YOU ARE GOING TO DO A WHOLE POOL AND INFECT WITH A VIRUS THAT CONTAINS SHRNA EXPRESSION CASTETTES TO HUNDREDS, IF NOT THOUSANDS OF GENES. YOU ARE GOING TO TRANSDUCE A POPULATION AND PARTICULARLY YOU ARE GOING TO SPLIT IT INTO TWO DIFFERENT CONTEXTS. AND YOU ARE GOING TO THEN ASSAY POTENTIALLY FOR GROWTH. SO MAYBE YOU ARE GOING TO ADD A DROP AT THIS POINT OR MAYBE YOU ARE GOING INTO TWO DIFFERENT -- YOU MIGHT HAVE HAD TWO DIFFERENT TYPES OF ASOGENIC CELL LINES OR SOMETHING THAT WILL ALLOW YOU TO DO A COMPARISON OF THE NUMBER OF -- THOSE GENES -- THOSE KREZ THAT ARE CARRYING ONE SH FLS RNA THAT IS FAVORING SURVIVAL OR DEPLETING AND INDUCING A RELATIVE DECREASE IN THE VIABILITY OF THESE CELLS. AND YOU CAN DO A GENETIC ANALYSIS BY ACTUALLY DOING A BAR CODAL BY DIRECTLY SEQUENCING THE INTEGRATION OF THE SHRNA'S INTO THESE DIFFERENT POPULATIONS. SO I AM GOING TO GIVE YOU A COUPLE OF EXAMPLES SO THAT IF YOU HAVE MORE QUESTIONS, YOU CAN GO BACK AND LOOK AT SOME OF THESE STUDIES AND SEE HOW THEY'VE DONE THIS. THIS IS AN EXAMPLE OF ONE OF THE EARLY SYNTHETIC SCREENS THAT WAS DONE WHICH WAS FOCUSING ON HUMAN KINNOME AND IT WAS USED TO LOOK AT NEW REGULATED A POP TOISIS AND CHEMORESISTANCE. AND HERE WHAT WE'RE LOOKING AT IS THE FOLD INCREASE IN A POPTOSIS. YOU SEE WHEN YOU STARTED THE PARTICULAR GENE. IN THIS CASE CD K 6 AND RO1 AND OPT 6 KL. THESE WERE THE GENES AS CHOSEN FOR FOLLOWUP. SO THIS IS A FAIRLY GOOD EXAMPLE OF WHAT YOU MIGHT DO. YOU'VE GOT 650 KINASE GENES THAT A LOT OF THEM ARE DOING SOMETHING. BUT YOU ARE MOST INTERESTED IN YOUR TOP HITS IN THIS PARTICULAR ASSAY SET. SO AS AN EXAMPLE OF LOOKING AT A GENETIC SCREEN USING SHRNA'S, I THINK THIS IS DONE AS AN ARRAY SCREEN WHERE WHAT THEY -- RAY'S GROUP AT THE CANCER INSTITUTE DID THEY WERE LOOKING AT ALL OF THE ENZYMES. THEY GENERATED SHRNA'S CORRESPONDING TO EACH OF THOSE AND INTRODUCED THOSE INTO AN ASSAY WHERE THEY WERE LOOKING AT ACTIVATION OF NF KAPPA B BUT FOLD INDUCTION IN AN NT KAPPA REPORTER ASSAY AND THE HIT WAS TARGETING A GENE CALLED CILD WHICH HAS BEEN ASSOCIATED PREVIOUSLY WITH A TUMOR SYNDROME, OR SILLIDO ROMOTOSIS. SO THIS IS REALLY INTERESTING. SO WHAT THEY WERE ABLE TO SHOW WAS THAT THE PROTEIN THAT IS RESPONSIBLE FOR THIS PARTICULAR DISORDER OF MUTATIONS RESPONSIBLE AND THAT HAS ITS ADVANTAGE IN THIS CONTEXT BECAUSE WHAT THEY WERE ABLE TO SHOW IS THAT IF YOU COULD BLOCK THE KAPPA B SIGNALING THROUGH IK, THEN YOU MIGHT HAVE A CLINICAL RESPONSE AND WE HAVE A REALLY GOOD IKK INHIBITOR, WHICH IS ACID OR ASPIRIN. AND WHAT THEY DID WAS THIS WAS PUBLISHED IN 2003. THIS IS THE CLINICAL TRIAL THAT THEN WAS ACTUALLY PUBLISHED LOOKING AT FAMILIARIAL TOSIS AND HERE YOU SEE A TUMOR. THESE ARE TUMORS OF THE SWEAT GLANDS AND YOU SEE BEFORE AND AFTER TREATMENT IN A NUMBER OF DIFFERENT PATIENTS. AND SO THEY WERE ABLE TO VERY RAPIDLY TAKE THEIR OBSERVATION FROM AN RNA SCREEN AGAIN INTO THE CLINIC. SO IT'S REALLY A TRANSLATIONAL RESPONSE. JUST GOING TO MENTION A COUPLE OF OTHER SHNA STUDY. THESE WERE DROPOUT SCREENS. THESE ARE QUITE COMPLICATED SCREENS BUT WHAT I AM GOING TO DO IS JUST HIGHLIGHT HOW ONE PARTICULAR FAMILY OF -- PAPER WAS PUT TOGETHER. THREE DIFFERENT COLORECTAL AND A CONTROLLED CELL LINE. AND THEY WERE LOOKING FOR GENES THAT WHEN THEY SILENCED SPECIFICALLY, IT RESULTED IN A REDUCTION IN THE VIABILITY OF THESE COLORECTAL CANCER CELL LINES. SO I AM GOING TO HIGHLIGHT HERE A COUPLE YOU HAVE GENES. WHEN YOU SILENCE PLK 1, YOU NEARLY ALWAYS KILL EVERYTHING. BUT WHAT THEY WERE LOOKING FOR WAS WHERE THEY HAD SOME LEVEL OF SPECIFICITY AND WHAT YOU SEE HERE IS WHEN YOU LOOK AT GENES -- THIS PARTICULAR GENES AGAINST EIS 3 S 10, THEY WERE ABLE TO GET SOME SPECIFICITY FOR THE CELL LINES IN INTEREST AND SIMILAR THINGS WITH RBX 1. AND THEY WERE ABLE TO MAP THESE TO PARTICULAR PATHWAYS. HERE IS ANOTHER VERSION OF THIS TYPE OF STUDY, WHERE HERE WHAT THEY WERE LOOKING AT WAS ACTUALLY TWO VERSIONS OF ONE COLORECTAL CANCER CELL LINE BUT THIS WAS ENGINEERED TO HAVE MUTANT RATS AND YOU ARE LOOKING TO SEE CAN YOU IDENTIFY SHRNA'S THAT INDUCE KILLING THE OF ONLY THE RAT MUTANT CELLS. ONLY IN THE COMBINATION WITH RAT MUTATION DO YOU INDUCE A DEATH. AND THEY WERE ABLE TO SCREEN EN MASSE IN THIS STUDY. AND I THOUGHT I HAD ANOTHER SLIDE IN THERE -- AND IDENTIFY SPECIFIC GENES THAT DID KILL JUST THE RAT MUTANT STUDY -- MUTANT CELLS. SO THIS IS ANOTHER APPROACH THAT CAN BE DONE FOR THIS. SO I AM NOW GOING TO TURN THE LAST FEW MINUTES SO HOW YOU CAN USE RNA IN THE CONTEXT OF DRUGS. SO I AM GOING TO DESCRIBE THIS TIME FROM MY LAB WE CONDUCT AID NUMBER OF STUDIES LOOKING AT THE SUMMERASE 1 INHIBITOR OR CPT. SO THE INHIBITORS REPRESENT AN IMPORTANT BE DRUG AND THEY PREVENT DNA RELIGATION IN REPLICATION BREAKS, WHICH DOWNSTREAM INDUCES DNA DAMAGE AND THEREFORE USUALLY CELL DEATH. SO CLINICAL ANALOGUES OF CPT HAVE BEEN DEVELOPED FOR TREATMENT OF A NUMBER OF DIFFERENT CANCERS AND ARE AND. AND WHAT WE DID WAS STILL USE THE LABORATORY-BASED STUDY WHERE WE STUDY CONDUCTED SCREENS IN THE ABSENCE AND PRESENCE OF CPT. SO THIS IS A SIMILAR TYPE OF GRAPH THAT'S SHOWING YOU SOME OF THE OTHERS,. THIS IS LOOKING AT THE FUNCTIONAL RESPONSE AND VIABILITY INDUCED BY AN VILLAIN SRNA AND WE'RE LOOKING TO SEE AGAIN LIKE IN THAT STUDY THAT I MENTIONED THAT WHAT YOU'RE LOOKING FOR HERE IS THE FULL HE CAN'T IN THAT WHAT WE WANT TO SEE IS ONLY THE SILENCING OF THIS GENE CAUSE AND EFFECT WHEN IT'S COMBINED OR WITH COUNTERSECANT. SOMETHING THAT'S DIRECTLY MODULATING THIS DRUG AND THAT IS ONLY OCCURRING OR HAS MOST OF ITS EFFECT IN THE CONTEXT OF THE DRUG AND WILL NOT HAVE THE ABILITY TO TRY AND INDUCE A COM BUT THEORRIAL EFFECT. AND ONE OF THE HITS THAT WE GOT FROM THIS SCREENING AND STUDIED MORE EXTENSIVELY WITH THIS RNA MAP 3 K 7. AND WE WERE ABLE TO SHOW THAT -- WHY SILENCING IT WE WERE ABLE TO INCREASE THE SENSITIVITY OF THESE CELLS AND THIS IS ACTUALLY DONE IN A PRAES CANCER CELL LINE. WE WERE ABLE TO INCREASE THE SENSITIVITY OF CELLS BY 5 STANDARD DEVIATIONS. WE CAN SEE THIS ACROSS THE WHOLE -- RESPONSE CURVE. SO THIS IS THE RESPONSE CURVE TO CPT IN THESE CELLS. IF WE INTRODUCED THIS AGAINST THIS GENE, WE CAN SIGNIFICANTLY MOVE THE IC 50. SO THIS IS SOMETHING THAT YOU ARE GOING TO GET INTRIGUED ABOUT BECAUSE THIS IS SOMETHING THAT WE MAY PANEL TO IMPROVE THE -- APPROVE THE USE OF THIS CLASS OF DRUGS. WHAT I FAILED TO MENTION EARLIER IS THOUGH THIS HAS BEEN SUCCESSFULLY APPLIED IN A NUMBER OF CLINICAL SETTINGS, IT IS ASSOCIATED WITH A NUMBER OF DIFFERENT SIDE EFFECT AND SO IF WE CAN FIND A WAY OF REDUCING ITS DOSE BUT STILL ENHANCING ITS EFFICACY, THIS WOULD BE HIGHLY ADVANTAGEOUS. SO BECAUSE AS I SAID, WE TALKED ABOUT OUR TARGET EFFECT EARLIER, IT'S IMPORTANT TO TEST THIS WITH MULTIPLE SRNA'S AND HERE WE SEE THE SAME FUNCTIONAL EFFECT WITH A DIFFERENT SRNA AND 7.2 IS SHOWN HERE AND AGAIN WE GET REDUCTION IN THE MRNA LEVELS AND FET A FULL 20FOLDLE SHIFT IN THE IC 50 VALUE. CAN WE ALSO SEE THE SAME EFFECT WITH A CLINICAL ANALOGUE? THIS IS THE EFFECT. SO IT'S NOT JUST THE LABORATORY-BASED EFFECT. WE ALSO SEE IT WITH THE CLINICAL ANALOGUE AND WE SEE IT IN OTHER CELL LINES AND AS I SAID A MOMENT AGO, WE CAN EXTEND IT TO A COLORECTAL CANCER CELL LINE. SO WHAT IS MAP K 37? TACK ONE WAS INITIALLY IDENTIFIED AS PART OF THE NF PATHWAY. TAP ONE FROM THE COMPLEX WITH TWO OTHER PROTEINS. TAP ONE AND THEN EITHER TAP TWO OR TAP THREE. SO WE INITIALLY LOOKED TO SEE DO WE SEE A SIMILAR EFFECT WHEN WE SIGN FIND OTHER MEMBERS OF THIS COMPLEX? WE DON'T SEE IT WITH TAB ONE. BUT WHEN WE SAW IT ON TAB THREE, WE DO SEE A SIGNIFICANT LEVEL TO CPT. EXCUSE ME. AND WHAT WE'VE BEEN ABLE TO GO ON AND SHOW IS WHAT WE'RE ACTUALLY DOING IS INCREASING CPT-INDUCED A POPTOSIS. AND OUR CURRENT WORKING MODEL IS THAT WE KNOW THE PATHWAY IS ACTIVATED WHEN CPT IS ADMINISTERED BOTH IN THE LABORATORY AND IN THE IN VIVO SETTING. IN A CLINICAL SETTING AND CAVA B SERVES TO SUPPRESS A POPTOSIS. BY SILENCING MAP K 37, WHAT WE'RE ABLE TO DO IS BLOCK THAT ACTIVATION OF NF KAPPA B AND FACILITATE A POPTOSIS AND THEREFORE INCREASE THE SENSITIVITY OF THE CELL TO CPT AND INDUCE FURTHER DEATH. AND I AM GOING TO SKIP BECAUSE OF TIME THE EVIDENCE FOR THAT. AND WE CAN INDUCE HIGHLY DAMAGE AS A QUENCE OF THAT. SO I WILL GIVE YOU ONE OF MANY EXAMPLES OF HOW RNA SCREENING HAS BEEN USED IN THE CONTEXT OF DRUGS. AND IN THE LAST FEW MINUTES, I AM GOING TO TALK ABOUT HOW SCALING UP THE WORK THAT WAS DONE IN MY LAB TO A NEW YISHT INITIATIVE NAALLOWS PEOPLE TO ACCESS RNA SCREENING ON A TRANSNIH BASIS, IF YOUR SD HAS A LOT OF MONEY. SO WHAT WE ARE TRYING TO DO IS TAKE THIS TO A MUCH LARGER SCALE. WHAT WE WANT TO BE ABLE TO DO IS NOW NOT JUST HAVE 55 PROTEINS THAT HAVE A LARGE NUMBER OF PAPERS ON THEM BUT START MOVING MORE AND MORE PROTEINS UP THIS CURVE SO WE EVENTUALLY HAVE MUCH MORE OF A FLAT LINE OVER HERE. I AM NOT SAYING WE DON'T STILL NEED TO STUDY P 53 BUT THERE IS A LOT KNOWN ABOUT IT. THERE IS A LOT OF OTHER THINGS IN HERE THAT WE SHOULD ALSO KNOW ABOUT. OKAY, SO WHAT I AM GOING TO MOVE ON TO IS DISCUSSES TRANSLN LH INITIATIVE. VE VERY LABOR-INTENSIVE AND VERY EXPENSIVE. SO TO TRY AND ADDRESS THIS, ONE OF THE PARTS THAT WE'VE BEEN ABLE TO TRY AND DO IS TO COME UP WITH A TRANSRNA SCREENING FACILITY WHICH IS NOW LED BY A POST DOC OF MINE DOING WORK ON CPT THAT I MENTIONED. AND IS NOW PART OF END CAP. AND SO WHAT THE CONCEPT WAS WHEN WE WERE ASKED BY THE SCIENTIFIC DIRECTOR FOR NIH WAS TO COME UP WITH A WAY FOR NIH INVESTIGATORS TO PERFORM ON A COLLABORATIVE BASIS GENOME-WIDE RNA SCREENING STARTING WITH SRNA SCREENING AND TO HELP PEOPLE WITH THE DEVELOPMENT OF THEIR SCREENINGS AND SOME LEVEL OF VALIDATION. THE SCREENS ARE NOT ROCKET SCIENCE BUT YOU NEED TO DO THEM RIGHT. AND IT WAS A MORE EFFICIENT WAY OF DOING IT THAN HAVE EVERYBODY TRYING TO DO IT THEMSELVES. IT'S A TRANSNIGE INITIATIVE. WE NEED TO BE ABLE TO BE READY TO DO NUMEROUS AND DIVERSE PROJECTS AND TRUST NE, THEY'RE VERY DIVERSE. AND EVENTUALLY ALL OF THE DATA THAT WE'RE DEVELOPING. WE'RE INTO THE THIRD YEAR OF THIS PROGRAM, WILL BE MADE AVAILABLE ONCE THINGS HAVE BEEN PUBLISHED. SO THIS IS ACTUALLY WHAT AN RNA SCREENING SYSTEM LOOKS LIKE. THIS IS THE REPRESENTATION OF WHAT IS NOW ACTUALLY AT NC GC. WHEN YOU ARE DOING IT NOW, IF YOU COME TO MY LAB, WE HAVE A TINY SET-UP IN COMPARISON. WE CAN STILL SCREEN BETWEEN 500 AND A THOUSAND GENES EASILY, BUT IF YOU WANT TO GET UP TO 22,000 GENES, YOU REALLY NEED TO START INVESTING IN THESE TYPES OF RESOURCES. AND I THINK YOU SAW THE SEQUENCING FACILITY. YOU KNOW TO DO THINGS ON A GENOME-WIDE SCALE, YOU REALLY HAVE TO THINK BIG AND THIS IS PART OF HOW TO THINK BIG. AND THIS ALLOWS US TO INTEGRATE ALL OF THE ROBOTICS BECAUSE ROBOTS DON'T NEED COFFEE BREAKS. IT'S NICE AND YOU CAN LOAD UP AND IT'S A LARGE NUMBER OF PLATES. SO YOU CAN LOAD THEM IT ALL UP AND MOST OF THIS IS NOW AUTOMATED AND GOES REASONABLY WELL. AND WE CAN DO EVERYTHING FROM FENETIPPIC ASSAYS, PARTICULARLY AT NCI. THEY WANT CELL. WE ARE TRYING TO DO SOMEWHAT MORE SPECIFIC THINGS LOOKING AT PARTICULAR PATHWAYS AND WE CAN DO HIGH-CONTENT CELL CYCLE ANALYSIS ON PROTEIN TRANSFER INDICATION AS WELL. NOW TO DO THIS WE HAVE HAD TO DEVELOP VERY -- THE GROUPS HAD TO DEVELOP VERY SOPHISTICATED INFORMATTICS TOOLS TO DO THIS AND VISUALIZE FIRE VERY LARGE NUMBER OF PLATES. IN MY LAB WE CAN STILL USE THIS SCALE BUT YOU NEED TO HAVE MUCH MORE SOPHISTICATED SYSTEMS. AND YOU NEED TO BE ABLE TO SHOW YOU'VE GOT GOOD QC AND GOOD STATISTICAL ANALYSIS AND YOU'VE THOUGHT THROUGH SOME OF THE OFF-TARGET EFFECT AND THAT YOU CAN GET DOWN TO REALLY GOOD BIOLOGY BY THE END OF THIS. OKAY, SO WHAT DOES THE SCREEN LOOK LIKE? I TOLD BUT A SCREEN DONE WITH CPT. SO THIS ACTUALLY -- WE TOOK AS ONE OF OUR PILOT STUDIES TO TRY TO GET THE -- WHEN WE WERE TRYING TO GET THE FACILITIES SET UP. SO WHAT SCOTT DID WAS TOOK THE SAME CELL LINE THAT HE USED IN HIS PREVIOUS STUDIES IN MY LAB AND HE DID A SCREEN OF 7,000 GENES, WHICH REPRESENT APPROXIMATELY WAS KNOWN AS SOMETIMES THE DRUG OF ALL GENOMES. AND WHAT YOU ARE SEEING HERE IS EACH OF THESE RECHT ANGLES REPRESENT A 384 WELL-PLATE. THIS IS A NORMALIZED CELL VIABILITY. SO THIS IS THE VIABILITY OF THE CELLS IN THE ABSENCE OF CPT. SO THIS IS JUST SRNA. AT THE EC 10. AND FROM THIS WE WERE ABLE TO POOL 121 GENES FOR INITIAL VALIDATION AS BEING ABLE TO SHOW THAT THEY SENSITIZED THESE CELLS TO CPT. AND WE WERE REALLY PLEASED, BECAUSE ONE OF OUR TOP HITS WAS TILL TACK 1, NOT 3 K 7 AND WAS ONE OF THE MEDIATORS OF TAP TAB TWO, TRACK 6. SO NOW WE HAD AN ENTIRE SET OF DATA THAT REALLY CONFIRMS THE INVOLVEMENT IN THE PATHWAY. WE ALSO PULLED A COUPLE OF OTHER MEMBERS FROM THAT. OBVIOUSLY, WE PULLED FROM DNA DAMAGE-RELATED GENES AND AT R IS A KNOWN SENSE TIZER OF CPT. SO THAT WAS VERY IMPORTANT. AND WE STILL DPIET FEW DOWN HERE THAT WE STILL DON'T KNOW AND THE INVESTIGATOR ON THIS IS STILL PROMISING HE'S FOLLOWING UP ON IT AND STILL VERY INTERESTING STORY. OKAY, WHEN YOU WANT TO GET REALLY BIG, IT STARTS LOOKING LIKE THIS. IT MAY NOT LOOK AS BIG AS THE PREVIOUS ONE, BUT THIS IS NOW LOOKING AT 142,848LING WITHES. THIS IS A WHOLE GENOME. SO WHAT IS REPRESENTED HERE IN EACH OF THESE TINY LITTLE DOTS IS THE EFFECT OF AN SIRNA ON A PARTICULAR END POINT AND THIS IS LOOKING AT THE BIOLOGY TRANSCRIPT WHERE WE'VE ENDED UP DOING TWO SCREENS. ONE IS THE SCREEN LOOKING AT TRANSCRIPTION FACTOR AND THE CANCER SCREEN LOOKING AT A CONTROLLED SYSTEM. AND THEN WE CAN COMPARE THE TWO DATA SETS FROM THIS. SO THIS IS REPRESENTING TWO THOUSAND SCOMBREENZ THE SILENCING HE CAN'T OF EACH INDIVIDUAL OF THOSE TRANSCRIPTS. SO THIS IS NOW WHOLE A GENOME GENE ANALYSIS. SO WHAT I'LL TRY TO DO IN THE LAST 55 MINUTES IS TRY TO GIVE YOU A STORY THAT I THINK REPRESENTS TRANSLATIONAL RESEARCH. RNI WAS DESCRIBED AS AN OBSERVATION OF PETUNIAS. THE STORY HAS BAIN COMPANY IN HOLLAND WANTED TO IMPRESS -- IMPRESS THEIR INVESTORS FROM AN ACADEMIC SETTING AND THEY WANT TO IMPRESS THEIR INVESTIGATORS HOW THEY COULD MAKE PROTEINS IN PLANTS. SO THEY WERE PUTTING PLASMA EXPRESSING DIFFERENT PROTEINS INTO PLANTS AND THEY STUDIED THEY WOULD USE ONE THAT WAS SUPPOSED TO MAKE THE PETUNIAS VERY, VERY RED. AND BY DOING THIS, THIS WOULD SHOW THEIR INVESTORS HOW THEY WERE GOING TO MAKE MONEY EXPRESSING THESE PROTEINS. AND WHAT THEY FOUND IS THEY GOT WHITE ONES. AND THIS WAS KNOWN AS COSUPPRESSION BECAUSE WHAT THEY WERE DOING IS ACTUALLY PUTTING IN GOING IN AND MAKING THE HAIRPINS AS AN RNA THEMSELVES AND SUPPRESSING THE EXPRESSION. SO YOU HAD THIS ALMOST TRANSLASHL THING BECAUSE THIS WAS DONE AS AN OBSERVATION FOLLOWING SOMEBODY TO TRYING TO MAKE MONEY OUT OF SOMETHING. AND NOW WE LOOK AT WHOLE GENOME SCREENING. SO INCLUDING RNA THERAPEUTIC TRIALS. WE'VE OROUCHE GOT A COUPLE OF MINUTES OF OUR PRESENTATION SO THANK YOU. QUICK ACKNOWLEDGEMENT. LOTS OF PEOPLE HAVE WORKED ON THIS. I HIGHLIGHTED WORK FROM SCOTT MARTIN AND SOME OF THE WORK DONE ON THE EARLY STAGES WERE DONE BY CHRISTIE DELLHOUSE AND MIKE CHELTHAM AND ALL OUR COLLABORATORS. THANKS. NOW I CAN TAKE QUESTIONS. >> HI. YOU GIVE ONE EXAMPLE OF THE DELIVERY USING NANOPARTICLES. ARE THERE ANY OTHER WAYS YOU DO THE DELIVERY? >> SO THERE ARE LOTS OF DIFFERENT VERSIONS ON THAT. SO SOME PEOPLE HAVE DONE NUKELO ACID OF SRNA'S AND YOU CAN DO THAT IN ANIMALS SO YOU DON'T HAVE TO BIND IT INTO TO ANYTHING. YOU DO WANT TO HAVE THE BACKBONE OF THE NUCLEIC ACID ALTERED IN SUCH A WAY THAT IT WON'T BE DEGRADED VERY EASILY. BUT IT'S DIFFERENT VERSIONS OF NANOPARTICLES IS MAINLY WHERE PEOPLE ARE. SO YOU USE SOME SORT OF LIPID-BASE. BUT IT'S STILL VERY EARLY STAGES. >> [INAUDIBLE] >> FOR SYNTHETIC SRNA'S. AND PEOPLE ARE USING EITHER TO DELIVER MIMICS OF MICRO R ITNA'S. REALLY, IT'S TAKING EXISTING GENE THERAPY AND TWEAKING IT TO TRY AND DELIVER RNA-BASED THERAPEUTIC THERAPEUTICS. >> ARE YOU STORING DATA FOR THIS? >> SO IT'S ALL HELD BY END CATS. WE ARE TRYING TO FIGURE OUT AS AN A WAY TO VERY SHORTLY START MOVING THE DATA FOR SOME OF THE EARLY SCREENS THAT HAVE BEEN COMPLETED AND ARE ABOUT TO BE PUBLISHED INTO A CC R-BASED FIREWALL -- AN NIH-BASED FIREWALL. SO THERE IS GOING TO BE A DELAY BETWEEN MANUSCRIPTS ARE FINISHED IN THE NUMBER OF THE EARLY STAGES -- THE EARLY SCREENS. BUT THEY'RE STILL NOT PUBLISHED AND WE ARE STILL TRYING TO GET SOME OF THOSE EARLIER ONE INTO PEER REVIEW. WHAT WE WOULD REALLY LIKE TO DO IS TO BE ABLE TO MOVE A LOT OF THAT DATA INTO -- SO NIH CAN SEE IT, THE INTRAMURAL PROGRAM. SO WE'D LIKE TO BE ABLE TO HAVE IT SO THAT NOOIMG INVESTIGATORS CAN SEE IT FOR A PERIOD OF TIME BEFORE THE REST OF THE WORLD SEES IT. AND WE'RE TRYING TO MAKE THAT HAPPEN. BUT IT'S ACTUALLY MUCH MORE DIFFICULT THAN WE THOUGHT. SAYING THAT SOUNDS REALLY EASY, BUT BETWEEN THE FACT THAT WE WANT INDIVIDUAL INVESTIGATORS, THOUGH THEY'VE SIGNED ON THE BOTTOM LINE, CAN WE WAIT UNTIL THE PAPER IS REALLY ACCEPTED AND ALL THIS SORT OF THING -- WE'RE WORKING TOWARDS THAT. I AM HOPING IN THE NEXT SIX MONTHS WE'LL SOLVE THAT PROBLEM. OBVIOUSLY, YOU WANT SOMETHING PUBLISHED OUT THERE AND THAT WILL BE LOADED ON TO THE END CATS WEBSITE. WHETHER THAT WILL FORM PART OF -- WE MAY MOVE IT INTO THE NEW VERSION OF PUB CAM AND THEY'RE REVAMPING PUB CAM AT THE MOMENT AND WE MAY GO INTO THAT. BUT I'M SORT OF LEAVING THAT TO END CATS TO FIGURE OUT. I'LL BE HONEST. THANK YOU. >> OKAY, WE'LL I NEED LITTLE HELP GETTING THE NEXT PRESENTATION UP. >> OKAY, SO WE HAVE CANCER.^ THERE IS A PRIMARY EMPHASIS ON GENE MUTATIONS ABOUT 20 YEARS AGO IT WAS DISCOVERED THAT CERTAIN GENES CAN BE SUPPRESSED AND ESPECIALLY FOR TUMOR EXPRESSOR GENES. IF THEY'RE METH LATED, THEN THEIR EXPRESSION GOES DOWN AND THIS IS ANOTHER MECHANISM OF GETTING CANCER. SO OUR NEXT SPEAKER IS MUCK ESH VERMA AND HE'S AT NCI AND HE'S GOING TO TELL US ABOUT EPIGENETIC EPIGENETICS. >> HELLO, GOOD AFTERNOON. FIRST OF ALL, I WANT TO THANK YOU FOR GIVING ME THIS OPPORTUNITY TO DISCUSS ABOUT EPIGENETICS. I WORK IN EPIDEMIOIOLOGY AND GENOMICS PROGRAM WHICH IS PART OF THE DIVISION OF CANCER CONTROL AND POPULATION SCIENCES. AND MY BRANCH IS NOT CALLED METROSEN TECHNOLOGIES BRANCH. SO THE FOCUS OF OUR WHOLE PROGRAM IS TO UNDERSTAND ETIOLOGY OF CANCER, WHAT ARE THE FACTORS WHICH CONTRIBUTE TO CANCER DEVELOPMENT AND WHAT ARE THE DIFFERENT MECHANISMS? AND CANCER, AS YOU KNOW, IT IS A VERY SERIOUS DISEASE. ABOUT FOUR MILLION MEN AND 5.3 WOMEN, THEY HAVE KERNS. AMONG MEN PROSTATE CANCER IS THE NUMBER ONE. AND WOMEN BREAST CANCER IS THE ONE. SO OUR IDEA IS THAT HOW WE CAN IDENTIFY SUCH MARKERS OF DEVELOPMENT OF ASSAYS SO THAT WE CAN FIRST OF ALL, DETECT CANCER EARLY SO THAT SOMETHING CAN BE DONE. OR EVEN IF WE IDENTIFY IN LATER STAGES, WHAT KIND OF STRATEGIES WE CAN DEVELOP? SO CANCER DEVELOPMENT IS A PERIOD FOR MOST OF THE CANCER FOR A MAJORITY OF CANCERS, WHICH ARE BREAST, CERVICAL, COLON AND PROSTATE. IT'S VERY LONG. IT TAKES MANY YEARS TO DEVELOP FULL CANCER. SO FROM NORMAL TO MUTATION TO HYPERPLAYSIA ORSISES STAGE, CELLULAR STAGES IS PROCESSED THROUGH OUR EMPHASIS FIRST ON THE EARLY PART AND THEN PATIENTS ARE UNDER TREATMENT AND LATER PART ALSO BECAUSE IF WE CAN INCREASE SURVIVAL OR REDUCE PAIN AND SURROGATE THEN ALSO, WE CAN CONTRIBUTE SOMETHING ABOUT THAT. IS IT ROUCHE IN CANCER GENETICS IS THE MAJOR CONTRIBUTOR, WHETHER IT CAUSES MUTATIONS OR DELETIONS OR INSERTIONS. THOSE MECHANISMS OF DIFFERENT KIND OF CHROMOSOME ARRANGEMENT. SO WHAT INITIALLY WAS TAUGHT FIVE, SIX YEARS AGO THAT WE SHOULD DO SOME GENOME BY ASSOCIATION STUDY. AND BY DOING THOSE STUDIES, THOUSANDS AND THOUSANDS OF PATIENTS SAMPLES ARE TAKEN AND PATIENT HISTORIES ALSO RECORDED. SO WHATEVER IS THE LIFESTYLE OR HISTORY -- ALL THOSE COMBINED WITH THE GENETIC CHANGES, WHETHER WE CAN SEE SOME TARGET OR OTHER THINGS, WHETHER THOSE CAN BE USED FOR BIOMARKERS OR NOT. AND THAT WAS DONE NOT ONLY FOR CANCER BUT OTHER DISEASES ALSO. LATER ON THESE WERE SMALL NEUCLEOTYPE POLY MORPHIS. AND TECHNOLOGY IMPROVED AND WE ARE SEEING A DIFFERENT CHROMOSOMES. THOSE LOCATIONS WERE IDENTIFIED. SO THAT PROGRESSED VERY WELL. AND THIS IS THE SITUATION IN 2008 AND MANY MORE HUNDREDS ARE THERE. IN DIFFERENT DISEASES, MOSTLY IN CANCER BUT IN OTHER DISEASES OF CARDIOVASCULAR AND ALL THOSE ARE THERE. AFTER DOING ALL THOSE THINGS, WHAT WAS OBSERVED THAT MOST OF THOSE WERE NOT LOCATED IN THE REGION OF GENES. PEOPLE EXPECTED THAT WHATEVER PATHWAYS HAD BEEN CHARACTERIZED IN CANCER, THEY EXPECTED THAT MOST OF THOSE DATA SHOULD BE THERE BUT THEY WERE NOT THERE. SO THEN WE THOUGHT WHAT WOULD BE COULD BE THE ALTERNATING MECHANISM OF CANCER HAPPENING SO WE THOUGHT ABOUT EPIGENETICS AND THOUGHT ABOUT BOTH GET TOGETHER, WHETHER EPIGENETICS IS FIRST, THAT IS DIFFERENT. AND TIME TO TIME DIFFERENT ARTICLES SCIENTIFIC OR NON-SCIENTIFIC MAGAZINES YOU WILL SEE THAT YOUR DNA IS NOT YOUR DESTINY. THE CHOICE YOU MAKE CAN CHANGE JUR GENE AND THOSE OF YOUR KIDS. SO ENVIRONMENT, LIFESTYLE, AND OTHER FACTORS -- THEY CONTRIBUTE EQUALLY WHAT WE GET THOSE GENES FROM OUR PARENTS. AND TO MAKE THIS POINT FURTHER, CERTAIN STUDIES WERE DONE IN MONOZYGOTIC TWINS. INITIALLY IN CARDIOVASCULAR DISEASE AND THEN IN CANCER. SO INSTEAD OF 100 PERCENT CONCORDANCE, ONLY 25 PERCENT WERE SEEN. AND PREDISPORTION POSITION OF CERTAIN DISEASES WERE OBSERVED. AND THAT GAVE US AN IDEA THAT WE SHOULD THINK ABOUT PHARMACO GENOMICS OR PREDISPOSITION OF DISEASE, BECAUSE OF EPIGENETIC ALTERATIONS. LIKE GENOMICS, WE TALKED ABOUT BUT FUNCTIONALLY WE ARE NOT GETTING THAT MUCH. SO THE WAY NOW STUDIES. BY THAT I MEAN PROFILING CHROMATIN. IF WE CAN MAKE THOSE MAPS IN NORMAL DISEASE STATE AND COMPARE THEM MAYBE, WE CAN FIND SOMETHING. GENOME VERSUS EPIGENOME, WHY THOSE ARE IMPORTANT AND WHAT IS THE DIFFERENT? GENOME IS GENERALLY CONSTANT. EPIGENOME CHANGES WITH TIME. IN ALL THESE FACTORS ARE INVOLVED BUT EPIGENOME IN THE SAME CELL OR IN THE SAME DISEASE, IT WILL CHANGE AGAIN AND AGAIN. IF YOU ARE DOING GENOMIC ANALYSIS, YOU MAYBE YOU HAVE TO DO ONLY ONCE. BUT FOR EPIGENOME YOU HAVE TO DO MORE TIMES SO MUCH IT IS MORE COMPLEX BUT MORE IDEAS WILL ALSO COME. AND LIFESTYLE OR ENVIRONMENT -- THEY PLAY MORE IMPORTANT ROLE. AND IN MONOZYGOTIC STUDIES WHEN THEY WERE DONE THEN IN CERTAIN DISEASES LIKE CANCER OR MULTIPLE SCLEROSIS, EPIGENETICS PLAY A MAJOR ROLE AND BRAIN DISORDER AND SOME OTHER DISEASES, GENETICS IS THE MAJOR CONTRIBUTOR. ONE MORE ABOUT THING YOU HAVE TO KEEP IN MIND IS THAT ANY TYPE OF TOXIC SUBSTANCES OR OCCUPATIONAL EXPOSURE, BECAUSE OF THAT, IF THOSE ARE HAPPENING, FOR SURE, SHORT TIME, THEN ONLY YOU SEE CHANGES IN TRANSCRIPTOR FACTORS. IF THEY ARE FOR A LONG TIME, THEN ONLY YOU SEE GENETIC CHANGES. BUT IN BETWEEN CERTAIN CHANGES ARE THERE, WHICH ARE EPIGENETIC CHANGES. SO WHENEVER YOU ANALYZE OR YOU STUDY OR YOU OBSERVE SOMETHING, THEN THINK ABOUT WHAT ARE THE CHANGES AND IF YOU WANT TO DETECT EARLY, THEN YOU HAVE TO GO THROUGH THIS WAY. THEN TO THE EPIGENETIC AND THEN TO GENETIC. AND ALL OF THESE, GENE TYPING OR PHENOTYPING IN WHICH WE HAVE EPIGENEOTYPING ALSO. SO THROUGHOUT THE DEVELOPMENT OF LIFE, FROM ZYGOTE UP TO FETUS OR ADULTHOOD OR ELDERLY, ENVIRONMENT PLAYS A MAJOR ROLE. SO INITIALLY YOU SEE THAT THE GERM LINE EPIMUTATIONS ARE THERE. WE HAVE A PROGRAM AT NIH CALLED ROAD MAP EPIGENOME IN WHICH WE WANT TO SET UP THE EPIGENOME IN CLINICALLY NORMAL PEOPLE. SO THAT THAT CAN BE A REFERENCE POINT TO USE AGAINST ALL THE DISEASES. SO FROM THAT RESULT AND FROM PUBLISHED LITERATURE WE KNOW THAT AT INITIAL STAGES THERE ARE NO MUTATIONS. BY EPIGENETIC CHANGES YOU CAN SEE. THE REASON IS THAT AT THE TIME OF FETUS DEVELOPMENT, WHATEVER DIET OR ENVIRONMENT EXPOSURE FROM MOTHER, DIET INFLUENCES NOT ONLY DURING DEVELOPMENT BUT LATER ON IN ADULTHOOD ALSO. SO THAT HAS BEEN OBSERVED. IN ADULTHOOD DIET AND LIFESTYLE PLAY A MAJOR ROLE. AND AT DIFFERENT STAGES FOR ANALYSIS YOU CAN USE DIFFERENT KINDS OF SAMPLES, LIKE CARDIOVASCULAR PLACENTA,ING UMBILICAL CORD AND STOOL LOAD OR SCHEMIA FOR EPGENOME ANALYSIS. SO A LITTLE BIT OF BACKGROUND. YOU KNOW THAT UNIT OF CHROMATIN AND IN THIS DNA IS THERE AND ABOUT TWO TYPES OF DNA, ONE -- AND TO NEUTRALIZE AND SOME ARE INVOLVED. AND THESE ARE S 2 AND S 3 J AND S 4. THOSE CHANGES ARE DOES NOT CHANGE BUT STILL YOU SEE CHANGE IN GENE EXPRESSION. YOU HAVE HEARD ABOUT GENETIC CODE BUT TUMOR CODE YOU WILL BE HEARING. WHAT KIND OF INFORMATION THAT PROVIDES US? THAT TELLS US WHAT WE ARE CAPABLE OF DOING. BUT WHEN WE SHOULD DO SOMETHING AND WHEN WE SHOULD STOP. SO EPIGENETICS IS THE -- AS THE NAME IMPLIES, THAT IS ABOUT GENETICS AND THAT REGULATES. A FEW MAJOR COMPONENTS OF EPIGENETICS ARE DNA AND PROMOTO REGION IS CG 6. 40 TO 60 PERCENT CG. ANOTHER PORTION IS HISTONE, WHICH GETS DEASSET LATED AND FOR METHYLATION ALSO A NUMBER OF ENZYMES ARE INVOLVED. THEY ARE CALLED DNA TRANSFERASES. DNT 1 REQUIRES METH LATED SUBSTRAITS BUT THEY ARE NEEDED FOR DEVELOPMENT, SO THEIR FUNCTION HAS BEEN CHARACTERIZED. CHROMATIN REMODELLING DECIDE WHICH PROTEINS CAN COME. AND HISTONE GETS MODIFIED BY METHYLATION, PHOSPHORYLATION. MICRO RNA, THEY ARE ALSO A PART OF DEATH. CERTAIN SEQUENCES, AS YOU KNOW. THOSE ARE METH LATED AND WITH AGE OR WITH THE TIME THEY GET HYPERMETH LATED. SOME OTHER PROTEINS LIKE POLY GROUPS OF PROTEINS, THOSE COMPLEXES ONE AND TWO, THEY ARE ALSO INVOLVED ESPECIALLY IN SUPPRESSION OF GENES. SO FOR EPIGENETIC ALTERATION, METHYLATION OF DNA, MODIFICATION OF HIST OHMS, NON-CODING RNA, MOSTLY MICRO RNA AND OTHERS GENOME IN PRINTING, THEY ARE THE MAJOR COMPONENTS. SO HERE I HAVE SHOWN THAT IN HYPERMETHYLATION, IF IN THE REGIONS SEQUENCES ARE METH LATED, THEN THEY CAN DO INACTIVATION OF TUMOR SUPPRESSION GENES. AT THE SAME TIME IT GETS HYPERMETH LATED AND THAT GETS HYPERMETHYLATION. SO BOTH WAYS, DEPENDING ON WHERE IT IS HAPPENING AND WHICH GENES THESE TH PROCESS IS HAPPENING, BOTH WAYS CONTINUE TO CANCER DEVELOPMENT. PETER JONES AND ANDREW FINEBERG ARE THE PIONEER INVESTIGATORS, 30 YEARS AGO THEY VISUALIZED THAT THIS CAN ALSO BE A MECHANISM TO UNDERSTAND CANCER. AND THEY GIVE DIFFERENT THEORIES AND ACCORDING TO THAT, AFTER TRANSCRIPTION STARTS SOMETIMES, A SITLATION SDWRASHTHS WHICH THE GROUP AT LYSIN ON H 3 HISTOME AT 9 POSITION. SOME METHYLATION WILL BE THERE. THEN METHYLATION ALSO OCCURS AND PROM OTOH METHYLATION STARTS AND THAT WAY GENE INITIALLY IS -- PERMANENTLY SILENCED. AND IF YOU SEE ON THE TOP PART OF THIS FIGURE, NUKE SOMES HAVE SEPARATED. A SITLATION IS SHOWN IN THE YELLOW COLOR. BUT AT THE BOTTOM PART FU SEE THEY ARE QUITE TIGHT. IN THIS DIFFERENT PROTEINS TRANSCRIPTION FACTORS AND OTHER PROTEINS, THEY CAN GET IN AND YOU CAN SEE TRANSCRIPTION. BUT GRADUALLY, HYPERMETHYLATION HAPPENS AND DEASSETLATION ALSO. BECAUSE OF THAT, GENE IS NOT ACTIVATED. SO THAT'S HOW IT WORKS. SO THIS IS A MODEL OF DIFFERENT STAGES OF CANCER DEVELOPMENT ARE THERE. FU SEE EPIGENETIC CHANGES. THEN GLOBAL HYPERMETHYLATION, THUS CANCER DEVELOPMENT HAPPENS. BUT GENE-SPECIFIC OR PROM OAT OATOHYPERMETHYLATION ARE THERE AND OBSERVED. IN DIFFERENT PATHWAYS, THIS IS ONE OF THE EXAMPLES OF SIGNALING. BECAUSE OF ANGIOGENESIS, AGGRESSIVENESS OF CANCER. SO IN THAT ALSO THOSE PATHWAYS LIKE NOTCH SIGNAL ANDING ALL THAT -- THOSE HAVE BEEN CHARACTERIZED AND MORE AND MORE GENES, WHEN PEOPLE CHARACTERIZE PATHWAYS, THEY ARE FINDING THAT EPIGENETIC MECHANISM IS INVOLVED IN THAT. MICRO RNA, THIS IS ONE EXAMPLE. THIS GROUP OF MICRO RNA AND THESE TWO GROUPS -- IF THEY ARE ACTIVE, MEANS THEY ARE GENES OF MICRO RNA IS NOT METH LATED. IF THEY ARE METH LATED, THEY CANNOT. SO THAT KIND OF A STUDY ALSO HAS BEEN DONE. IN EPIDEMIOIOLOGY ESPECIALLY, MICRO RNA ARE USED IN TWO DAYS WEIGHS. FIRST OF ALL LIKE THIS YOU CHECK THE CODING SEQUENCE FOR THOSE AND SEE WHAT ARE THE CHANGES, WHETHER THEY EXIST IN DIFFERENT KINDS OF POPULATIONS BELONGING TO DIFFERENT RACES AND ETHNICITY. OR YOU FOLLOW THE PROFILING OF A GROUP OF MICRO RNA AND CORRELATE WITH CANCER, INITIATION, DEVELOPMENT, PROGRESSION OR RECURRENCE. HISTONE, AS I MENTIONED, HAS FORCE IN DUPLICATE. SO H 2 A, H 4 -- S 2 AND 3 ARE MODIFICATIONS ARE SHOWN. THOSE WHICH I MENTIONED METHYLATION AND IN ROAD MAP PROGRAM OF NIH, WHAT WE HAVE DONE THAT AGAINST THESE CHANGES OR MODIFICATIONS WE HAVE GIVEN CONTEXT SO THAT MONOCLONAL ANTIBODIES CAN BE RAISED AND THOSE WILL BE DISTRIBUTED TO DIFFERENT INVESTIGATORS. BECAUSE IF YOU WANT TO IDENTIFY WHICH EFFECT WE ARE SEEING, THEN THOSE MONOCLONALS ARE NEEDED. AND NOW THIS TECHNOLOGY EXISTS BY WHICH AT DIFFERENT LOCATION YOU CAN FOLLOW UP THE HISTONE CHANGES. SO METHYLATION HAS BEEN DONE IN MANY EXPERIMENTS AND MANY SYSTEMS. MOSTLY IN HUMAN AND HISTONE IS NOWADAYS BEING DONE.^ AND GENOME-WIDE ALSO BOTH METHYLATION AND HISTONE HAVE BEEN DONE AND MICRO RNA AS WELL. FOR ANALYSIS, FIRST IT IS LINKED WITH SOME KIND OF CROSS LINKER AND THEN AFTER THEY ARE SONICATED AND CHARACTERIZED. SO THIS PROCESS IS CALLED CHIP ON CHIP ASSAY. I HAVE WRITTEN A BOOK ON EPIGENETICS AND PREVENTION AND THAT IS IN THE NEW YORK ACADEMY OF SCIENCES AND THIS YEAR WE ARE FOCUS ON HOW WE CAN USE FOR TRANSLATION EPIDEEM LOGICAL STUDIES AND THAT IS FOR INNER CITIES CALLED METHODS AND IN MEDICINE. AND TWO YEARS AGO TWO BOOKS BUT IN ONE EMPHASIS WAS ON WHOLE SUSCEPTIBILITY FACTORS LIKE IMMUNE LOGICAL FACTORS AND ANOTHER ONE WAS MODIFIED FACTORS LIKE LIFESTYLE OR ENERGY SCOMBLNS ALL THAT, HOW THEY AFFECT CANCER EPIDEMIOIOLOGY AND EPIGENETICS IS A MAJOR PART OF THAT. LAST YEAR I WAS INTERVIEWED FOR "NATURE" AND THEN I EMPHASIZED THAT THE DIFFERENCE IN GENETIC AND EPIGENETIC AND IF YOU WANT TO CALL ANY ADVANTAGE OF EPIGENETIC IS THAT, EPIGENETIC CHANGES THEREFORE HAVE AN EDGE OVER GENETICS. AND THIS YEAR ALSO I WAS INTERVIEWED, SO IN THAT I WANTED TO EMPHASIZE ON TRANSLATIONAL EPIGENETICS SEVERAL FACTORS BEING DEVELOPED. SO I MENTIONED THAT SUCCESSFUL APPROVAL OF FIRST GENERATION OF DRUGS INTENDING TO TARGET EPIGENETICS PATHWAYS HAVE CONVINCED OTHER COMPANIES TO INVEST. FU GET A CHANCE TO ATTEND THE MEETINGS, THEN YOU WILL SEE HOW MANY DRUGS ARE COMING. FOR METHYLATION, ONE BY ONE, THESE ARE METH LATE SOD IT IS NOT ALL OR NONE. SOMETIMES THESE ARE THE CONVERTED TO THE HOMOSIFTENE. AND USUALLY WHAT HAPPENS IS THE TREATMENT IS DONE FOR THE SUBSTRAIT DNA AND 2345 REACTS WITH THE CYTOJIVENLT IT DOES NOT REACT WITH THE CYTOIN AND THAT IS CONVERTED TO YOU AREOSIN. C BINDS WITH THE G AND THIS WILL BIND WITH A. AND THAT IS THE BASIS OF METHYLATION ANALYSIS. SO DIFFERENT KINDS OF ASSAYS FOR METHYLATION ANALYSIS, THEY ARE DONE EITHER BY PC R. YOU CAN ANG LIES A PRODUCT OR METHYLATION-SPECK PC R. IF YOU ARE FOLLOWING THE GENE APPROACH INSTEAD OF GENOME-WIDE A PROP, THEN YOU CAN SELECT PRIMEERS NEAR THE CANDIDATE GENE OF TARGET AND THEN DO PC R. OR IF YOU WANT TO DO REALTIME, THEN THIS TECHNIQUE IS CALLED METHYLITE. YOU DEPENDING ON YOUR PROJECT, DIFFERENT KIND OF EXPERIMENTS -- THESE ASSAYS ARE SELECTED. WHENEVER DOING METHYLATION ANALYSIS TO REDUCE FALSE NEGATIVE OR FALSE POSITIVE, AND ANY TIME WHEN I DISCUSS I THINK ABOUT EPIDEMIOIOLOGY TRANSLATION FIRST WHERE YOU HAVE TO USE ANY ASSAY FOR A LARGE NUMBER OF SAMPLES AND HUMAN SAMPLES, SEEING THAT ALL THOSE COMPLEXITY. SO YOU HAVE TO THINK ABOUT METHYLATION, TOTAL METHYLATION CONTENT, METHYLATION LEVEL OF A SPECIFIC GENE, METHYLATION OF A GROUP OF GENES, A PROFILE OF METHYLATION OF EITHER A SPECIFIC GENE OR A NUMBER OF GENES AND METHYLATION IN WHOLE EPIGENOME. SO WHEN YOU DESIGN YOUR EXPERIMENT, IF YOU KEEP THESE THINGS IN MIND, THEN YOUR RESULTS WILL BE MUCH MORE QUANTITATIVE AND FALSE NEGATIVE AND FALSE POSITIVE WILL NOT BE THERE. JOHNS HOPKINS UNIVERSITY, HE'S VERY GOOD IN IDENTIFYING THOSE PROTEINS WHICH BIND IN THE REGION, WHETHER REGULATION INVOLVES EPIGENETIC OR NORMAL REGULATION AND HE HAS IDENTIFIED SEQUENCE OF THOSE PROTEINS WHICH BIND. SO IF YOU THINK IN TERMS OF U CHROMATIN, FEW PROTEINS ARE THERE. BUT IF IN POLY REPRESSOR COMPLEX, THAT HAS A MAJOR ROLE IN THIS REPRESSION. AND ONE EXAMPLE IS THIS. IF S 3 -- AT THE LOCATION OF LYSINE 27, IT CAN GET TRIMETH LATED. IF IT IS TRIMETH LATED, THEN IT IS INVOLVED IN DIVISION ROUCHE DIFFERENTIATION. AT THE SAME TIME, THEIR EFFECT IS DIFFERENT. ON S 3 K THE 9 IT IS METH LATED. AND CHANGE BECOMES PERMANENT IF PHOSPHORYLATED. THOSE HAVE BEEN CHARACTERIZED. EXFOLIATED CELLS ARE USEFUL FOR DNA ANALYSIS OR EPIGENETIC STUDIES, ESPECIALLY. FU WANT TO DO JUST THIS AND IDENTIFY WHICH POPULATION IS LIKELY TO DEVELOP CANCER OR WHICH HAS CANCER, THIS EARLY DETECTION OR IF CANCER IS DEVELOPED, THEN WHAT DIAGNOSIS IS DONE? WHAT KIND OF TREATMENT YOU ARE DOING, WHAT IS THE PROGNOSIS? AND WHETHER SOMEBODY IS SURVIVING OR NOT, WHETHER SECONDARY CANCER IS COMING OR RECURRENCE. FOR ALL THESE STAGES, THOSE CELLS ARE IMPORTANT AND THOSE YOU CAN COLLECT AND ANALYZE. SO HERE I HAVE SUMMARIZED. WHAT ARE GROUPS OF GENES WELCOME BACK USE FOR METHYLATION ANALYSIS AND DIFFERENT REVIEWS, I HAVE COVERED THOSE. SO AFTER SCREENING A LOT OF GENES, THEN SOME SELECTED GROUP OF GENES HAVE BEEN IDENTIFIED FOR DIFFERENT CANCERS. NOW COMING TO EPIGENETIC INHIBITORS FOR DRUGS. HE IS A LEADING INVESTIGATOR. SO DEMETHYLATION AND HISTONE DEMETHYLATION. FOR BOTH OF THOSE, DIFFERENT KINDS OF AGENTS HAVE BEEN DEVELOPED. ALTHOUGH SOME OF THEM HAVE ADVERSE EFFECT BUT SOME OF THOSE ARE COMING. AND SOME OF THOSE I'LL BE SHOWING TO YOU. AND THESE AREAS WHICH WE CALL EPIGENETIC INHIBITOR, THEY BELONG TO DIFFERENT KINDS OF CLASSES. USUALLY WHEN THESE COMPOUNDS ARE MADE, THE TOXICITY ARE BEING IMPROVED. AND ALSO, DIFFERENT KINDS HAVE BEEN IDENTIFIED DEPENDING ON WHETHER THEY REQUIRE ZINC. AND INVESTIGATORS ARE TRYING TO MAKE THOSE AS SPECIFIC AS POSSIBLE. SO THESE INHIBITORS ARE CONSIDERED AN ANTI-CANCER CLASS OF DRUGS. AND THEY INDUCE DIFFERENTIATION MIGRATION AND A POPTOSIS. SEVERAL COMPANIES LIKE JOHNSON & JOHNSON AND SUBMITTING KLEIN BEECHHAM, ALL OF THEM HAVE MAJOR PROGRAMS IN DEVELOPING INHIBITORS OR DEMETH LATING INHIBITORS. I WILL GIVE A FEW EXAMPLES. IN THIS PHASE ONE STUDY, WHERE 55 PEOPLE WERE INVOLVED, THAT ADVANCE -- AT ADVANCED CANCER STAGES -- IN THAT THAT WAS GIVEN. ONE WAS DEMETH LATING AND ONE WAS DEASSET LATING ENZYME TOGETHER. AND DAY ONE, DAY TEN, DAY 20. AND AFTER GIVING THIS, THEY GAVE GOOD RESPONSE AND THESE PATIENTS WERE SUCH AS YOU KNOW MANY TIMES PEOPLE STOP GIVING RESPONSE TO REGULAR TREATMENT. THEN WE DON'T HAVE MANY CHOICES. AND WE OBSERVE THAT IF EPIGENETIC INHIBITORS ARE ALONE OR IN COMBINATION WITH REGULAR DRUGS ARE GIVEN, THEN THEY ARE RADIO SENSE TIESORS. BY THAT I MEAN YOU HAVE TO USE LESS AMOUNT OF REGULAR DRUG AND ANY TIME YOU REDUCE THE DRUG AMOUNT, THEN TOXICITY IS LESS AND RESPONSE IS BETTER. ANOTHER EXAMPLE, WHERE IT WAS GIVEN. HERE ALSO A NUMBER OF PATIENTS WERE USED AND OVERALL RESPONSE WAS THERE. SEVERAL PROBLEMS WITH THAT. THESE INHIBITORS ARE NOT SPECIFIC. SO PEOPLE ARE TRYING TO MAKE THAT SPECIFIC OR IN COMBINATION THERAPY, THEY ARE FOCUSING THAT, IF THEY CAN IDENTIFY SOMETHING WHICH IS VERY TARGETED, SUPPOSE FOR BREAST CANCER OR HORMONAL REGULATED SOME KINDS OF PROTEINS -- THOSE IN TARGETS ARE GOOD AND INCLUDE THESE, THEN MAYBE SOMETHING CAN HAPPEN. BUT AT THE SAME TIME, AT NCI, SEVERAL CLINICAL TRIALS ARE GOING ON AND I HAVE GIVEN THIS WEBSITE AND IN THIS CASE HISTONE INHIBITORS -- I FOUND IN 84 STUDIES HISTONE INHIBITORS ARE BEING USED. AND? N SOME OF THESE STUDIES, HISTONE ENROLLMENT IS THERE AND PATIENT-RELATED HISTORY IS THERE SO THOSE ARE GOING ON. THESE ARE FOR SOLID TUMORS AS WELL AS FOR LYMPHOMA OR BLOOD CANCERS, BREAST CANCER, SO A VARIETY HE HAVE CANCERS ARE THERE, WHICH ARE COVERED IN THESE TRIALS. AT THE SAME TIME IN 51 STUDIES I FOUND THAT METHYLATION INHIBITORS ARE USED EITHER LONE OR IN EXAMININGS AND THESE ARE THE TYPES OF CANCERS IN WHICH THEY ARE BEING USED. SO IF YOU GO TO THE SITE AND CLICK ONE OF ONE OF THESE, YOU CAN SEE WHAT IS THE HISTORY, HOW THEY WERE EN ROLLED, HOW THEY ARE RESPONDING, AND THESE SITES ARE UPDATED. SO THAT IS A VERY USEFUL INFORMATION. IF YOU TYPE CLINICAL TRIALS OR METH LATING AGENT, THEN THAT WILL COME. SO THAT WAS VERY EN COURAGING. AND AS I MENTIONED, THE CELL PHARMACEUTICAL COMPANIES, THEY HAVE THEIR ONGOING PROGRAM. IT IS NOT ONLY THEORETICAL IN SOME CLINICAL TRIALS BUT FDA HAS APPROVED TWO OF THE METHYLATION INHIBITORS AND TWO HISTONE INHIBITORS. SO FDA HAS ALSO APPROVED. NO NOW I WILL GIVE A FEW EXAMPLES IN DIFFERENT CANCER -- COLORECTAL CANCER. THOSE GENES ARE STHOUN HERE WHICH ARE REGULATED EPIGENETICALLY. AND IN COLORECTAL CANCER ALSO ONE OBSERVATION WAS MADE THAT IN SOME POPULATIONS WHICH WERE AFRICAN-AMERICAN, IN THAT IT WAS DIFFICULT FOR US TO IDENTIFY WHO ARE AT DEVELOPING COLORECTAL CANCER AND WHO WERE NOT. THEN SIMPLE PHENOTYPE WAYS OBSERVED. METH LATED PHENOTYPE. SO IN A CERTAIN GROUP OF PEOPLE THIS METHYLATION REGION THAT WAS DIFFERENT. SO IT IS SHOWN HERE. SO SIMPLE PHENOTYPE WITH THAT WE WERE ABLE TO DISTINGUISH. AND IN THAT WAY AYE NUMBER OF SAMPLES WERE TAKEN TOGETHER. AND ALONG WITH THESE EPIGENETIC MARKERS, WE WANTED TO SEE WHATEVER INFORMATION WE HAVE FROM GENETICS, HOW YOU CAN INTEGRATE GENETIC INFORMATION WITH EPIGENETIC AND WHETHER IT WAS USEFUL FOR NOT. WE WANTED TO CHECK WHETHER MICRO -- AS YOU SEE, WHEN ALL THOSE DATA WERE PUT TOGETHER, THEN CERTAIN GROUPS CAME. THEN THEY GAVE BETTER RESULTS. AND AFTER THE GROUP OF GENES OR MODIFICATIONS WERE IDENTIFIED, THEN WE OBSERVED THAT NSI MIAND BF MUTATION ARE HYPERMETH LATED ALSO A GROUP OF GENES, THEN YOU CAN INCREASE THE SENSITIVITY OF YOUR PREDICTION WILL BE VERY HIGH THAT WHICH ARE THE GROUPS WHO ARE LIKELY TO DEVELOP CANCER. THEN WE VALIDATE THOSE. IN CASE OF AM L AND AL L, THIS WAS OBSERVED JUST BY METHYLATION PROFILING FROM HELL DI. YOU CAN DISTINGUISH THOSE. IF YOU COME TO LUNG GENERATION THEN PROTEIN MARKERS, MUTATIONS AS WELL AS THESE EPIGENETIC MARKERS -- THEY WERE PUT TO THE TO INCREASE SENSITIVITY AND SPECIFICITY OF THOSE BIOMARKERS N BIOMARKERS FIELD ESPECIALLY FOR SCREENING OR RISK ASSESSMENT, IT IS UNDERSTOOD THAT IF YOU USE COMBINATION OF BIOMARKERS, THEN YOU CAN INCREASE SENSITIVITY AND SPECIFICITY INSTEAD OF USING BIOONE MARKER. STEVE BALINSKI IS VERY GOOD IN LUNG CANCER IN SMOKERS AND NON-SMOKERS. HE HAS DONE ON THOUSANDS AND THOUSANDS OF PEOPLE AND THEY CAN IDENTIFY ESPECIALLY MANY NON-SMOKERS ALSO DEVELOP CANCER. SO OTHER THINGS ARE INVOLVED. SO HE HAS VERY GOOD DATA ON LUNG CANCER. MESOTHEL IOMA. ASBESTOS IS A FACTOR AND NOT MANY MUTATIONS ARE IDENTIFIED. SO NOW WE ARE COMING UP THAT PROBABLY IT IS METHYLATION CHANGES WHICH ARE DOMINATING IN THIS DISEASE. SO THOSE HAVE BEEN IDENTIFIED. NOW ISOCARCINOMA. IN THIS CANCER CARCINOMA IS INCREASING MORE NOW THAN A STROMUS. IN THIS ALSO THESE ARE THE RISK FACTORS FOR THIS CANCER. A GROUP OF GENES WERE TAKEN. IN CLINICALLY IDENTIFIED SAMPLES. THEIR METHYLATION PROFILING WAS FOLLOWED, AND AS YOU CAN SEE, IN SOME GENES METHYLATION STARTS EARLY, IN SOME LATE. SO THAT WAY YOU CAN DETERMINE PERCENT METH LATED REGIONS AND BASED ON THAT YOU CAN DO SOME PREDICTION OR AFTER TREATMENT YOU CAN FOLLOW PROGNOSIS WHO IS RESPONDING AND WHO IS NOT RESPONDING, WHICH HELPS US IN DEVELOPING PHARMACO EPGENOMICS KINDS OF APPROACHES, EPGENETIC BACKGROUND PLAYS A ROLE IN DRUG RESPONSE OR NOT. AND THIS WAS ALSO CORRELATED WITH SURVIVAL, WHETHER METHYLATION LEVEL IS RELATED WITH SURVIVAL OR NOT. PAN CREATIC CANCER. THIS CANCER AS WELL AS OVARIAN CANCER, THESE TWO ARE SUCH CANCERS THAT THEY ARE THE SAME. IT TAKES ONLY TWO TO FIEFSH YEARS THAT PEOPLE WILL DIE. BECAUSE OF OUR DETECTION TECHNOLOGY IS NOT SUCH THAT WE CAN DETECT EARLY. YOU REMEMBER THAT -- MANY TIMES PATIENTS RELATED INFORMATION IS VERY IMPORTANT TO PREDICT WHO MAY DEVELOP THIS KIND OF CANCER. SO IF SOMEONE IS ALCOHOLIC, LONG STANDING DIABETES IS THERE AS WELL AS MUTATION OR SOME MUTATIONS ARE THERE AND P 16 AND P 14 -- IF THEY ARE METH LATE I HAD, THEN A PERSON MAY BE AT HIGH RISK YOU HAVE PAN CREATIC CANCER. IN BREAST CANCER, A NUMBER OF GENES TOGETHER IN TISSUES WERE ANALYZED AND PEOPLE WERE CHARACTERIZED FOR RISK ASSESSMENT WITH IS TREATMENT. TAM OX FIN TREATMENT WAS GIVEN. BY PROFILING YOU CAN TELL WHETHER PATIENTS ARE GIVEN RESPONSE TO TREATMENT OR NOT. SIMILARLY, THIS METHYLATION PATTERN HAS BEEN FOLLOWED IN THOSE TRIPLE NEGATIVE OR DOUBLE NEGATIVE POSITIVE OR NEGATIVE AND THIS WAS DONE IN 143 SAMPLES. IN EPITHELIAL AND STROMA AND LYMPH NODE IS ALSO DETERMINED SO THOSE WERE USED IN BREAST CANCER AS WELL. NOW ABOUT 1W5 TO 20 PERCENT CANCER IN THAT INFECTION AGENTS ARE INVOLVED. WHETHER THESE ARE HYPETITIS B OR C WITH FCC OR H IS INVOLVED OR HTLV. SAR COMA ARE THERE SO ALTOGETHER THEY COMBINE AND THAT MAKE A MAJOR PORTION. AND GRADUALLY IT WAS OBSERVED THAT EPIGENETIC REGULATION HAPPENS THERE ALSO. AND IN THIS LATENCY ASSOCIATE HE HAD NUCLEAR ANTI-GEN, THEY ARE BASICALLY PREDOMINANTLY REGULATED EPIGENETICALLY. ONE PERSON FROM SPAIN HAS RECENTLY COMPLETED METHYLATION PROFILING IN ALL OF THESE VIRUSES. AND WHY WE WANT TO DO THAT? BECAUSE MANY HEALTHY PATIENTS PATH LOGICALLY WE CANNOT TELL WHETHER SOMEBODY WILL DEVELOP CANCER OR NOT. BUT IF YOU DEVELOP TOOLS, THEN YOU CAN TELL WHO IS LIKELY TO DEVELOP THESE CANCERS JOCHT BY DOING THIS ANALYSIS, THOSE THINGS ARE POSSIBLE. AND AS AN EXAMPLE, I HAVE SHOWN HERE IN EARLY GENES OR LATE GENES AT DIFFERENT TIMES HOW MUCH METHYLATION IS THERE. WHICH STARTS FIRST AND LATE. ALL THOSE CAN BE DONE AND YOU CAN DISTINGUISH BETWEEN ALL THOSE STAGES. SO IT HAS GREAT IMPLICATION. SINCE THESE VIRUSES HAVE A SMALL GENOME, SO THAT WAS POSSIBLE. BUT NOW FOR THESE THINGS ARE BEING APPLIED. IN HELPATOCELLULAR CARCINOMA. ONE WAS FOUND ONLY IN JAPANESE POPULATION. SO CERTAIN GENES IN SOME CANCERS ARE SUCH WILL NOT WILL BE ONLY IN ONE GROUP. PROBABLY THEIR LIFESTYLE AND DIET AFFECT US A AS SUCH THAT THEY ARE DISTINCT. IN ORAL CANCER, THESE ARE THE NUMBER OF GENES WHICH IN COMBINATION YOU CAN USE FOR SCREENING OR RISK ASSESSMENT. IN IMMUNOLOGY, DEVELOPMENT OF T CELLS AND B CELLS AS WELL AS SOME OF THOSE PATHWAY ALSO, EPIGENETIC REGULATION HAS BEEN OBSERVED. IN PROSTATE CANCER, WHENEVER WE DETERMINE PSA AT LOWER-LEVEL ESPECIALLY LESS THAN 4 MILLIGRAM, A DOCTOR CANNOT PREDICT WHETHER A PERSON WILL DEVELOP PROSTATE CANCER OR NOT. AND TIME TO TIME WE HEAR THAT AFTER SPSA LEVELS ARE IN THAT RANGE IRE LITTLE BIT HIGHER, WHETHER A PERSON SHOULD UNDERGO CHEMICAL TREATMENT OR NOT. SO GST METHYLATION WHEN IT WAS OBSERVED IN A NUMBER OF PATIENTS, THEN THAT GAVE VERY GOOD RESULT. AND SIDE BY SIDE, PSA LEVELS AND GST 3 P 1 METHYLATION LEVELS WERE TESTED. SO THAT CAME OUT VERY GOOD. FOR THAT AND LATER ONLE QUEST DIAGNOSTICS, THEY PURCHASED THAT. SO THIS IS AN ASSAY WHICH IS BEING USED FOR PROSTATE CANCER ANALYSIS AT ABOUT $500 AN A.^ A BUT IT HAS A VERY GOOD RESULT. NOW IN THIS SLIDE I WANT TO SHOW THAT SCREENING CAN BE DONE USING EXFOLIATED CELLS IN URINE BY POPULARITY ON A NUMBER OF GENE IN COMBINATION HAVE ALSO BEEN USED. WHETHER IT IS SURVIVAL OR IT IS JUST DETECTION FOR BOTH PURPOSES YOU CAN USE. PETER LAYER IS VERY GOOD IN TECHNOLOGY DEVELOPMENT IN EPIGENETICS. SO IN ONE SAMPLE, DIFFERENT TISSUES, WHETHER SECONDARY CANCER WILL BE DEVELOPED OR NOT OR HOW MUCH LIKELIHOOD IS THERE FOR RECURRENCE OF YOU GENERATION THOSE KINDS OF ANALYSIS ALSO CAN BE DONE. SO HIS DATA IS COMING IN THAT DIRECTION. >> WHAT'S THE COST? >> OKAY, COST COMES USUALLY $1500 TO $2,000 PER SAMPLE. STILL THEY ARE EXPENSIVE. AND MORE THAN THAT, TISSUES ARE MORE IMPORTANT. BUT SOME OF THE INVESTIGATORS LIKE WE HAVE A PROGRAM SIMILARLY, WHENEVER EPIDEEMIOLOGY PROJECTS COME, USUALLY THEY ARE FIVE TO SEVEN TIMES MORE THAN REGULAR ONE. LIKE REGULAR GRANT OR PROJECT FU SAY, THAT IS $200 TO $300,000 PER YEAR. BUT EPIDEMIOLOGICAL STUDIES ARE $2 TO $3 MILLION PER YEAR. OUR PROGRAM ARE MAXIMUM STUDIES IN THAT AREA. SO ABOUT $230 MILLION WE SPENT ON THAT. WITHOUT THAT THOSE THINGS CANNOT BE DONE. AND SINCE FOR MANY, MANY YEARS WE HAVE FOCUSED ON BASIC SCIENCE AND COLLECTED A LOT OF INFORMATION. NOW YOU WANT TO SEE HOW MUCH WE CAN TRANSLATE OR NOT. BECAUSE IF YOU DO CLEAN EXPERIMENTS IN CELL LINE AND OTHERS, MANY THINGS YOU WILL SEE VERY NICE. BUT CANCER IN DEATH CELLS ARE HETEROGENIUS AND DON'T WORK. SO THESE KINDS OF EXPERIMENTS ARE VERY, VERY USEFUL. DIET AND EPIGENETICS. IT PLAYS A MAJOR ROLE. THE DIET CAN CHANGE MANY THINGS AND WE WANT TO EMPHASIZE CANCER PREVENTION ALSO. SO ABOUT THAT, IT IS OUR UNDERSTANDING THAT 30 TO 40 PERCENT HE HAVE CANCERS, THEY ARE PREVENTIBLE IF YOU TAKE PROPER STEPS. SO UNDERSTANDING HOW DO THEY CONTRIBUTE SKWLASHGS KIND OF PROPERTIES DIFFERENT ACTIVE FEED COMPONENT OR NATURE OF FOOD CELL -- THAT IS VERY SIGNIFICANT. AND I WILL GIVE YOU SOME EXAMPLES THAT BY USING EITHER NATURAL PRODUCT OUR THEIR FOOLPROOFED COMPONENTS OF HOW YOU CAN CONTROL THAT. FOOD COMPONENTS. AND IF YOU CAN STOP THESE PREMALIGNANT CELLS TO DEVELOP TO METASTATIC, THEN IT IS GOOD. BASIC CANCER DOES NOT DEVELOP AT ALL. BUT IF YOU CAN STOP AT CERTAIN STAGES FOR MANY, MANY YEARS, THEN IT IS LS IT IS GOOD. SO HERE I AM GIVING YOU SOME EXAMPLES OF MAIN FOOD MATERIAL, LIKE PEPPERS. SOME OF THE COMPOUNDS WHICH HAVE BEEN IDENTIFIED OR ISOLATED FROM IT LIKE TURMOIL ERICK OR PEPPER OR GINGER LIKE GREEN TEA AND THESE ARE THE COMPOUNDS LIKE LYCOPENE AND EC GC OR SIS PERCENTIN AND FROM DPRAPZ. THESE, WHEN THEY WERE ISOLATED, THEN THEY HAD DIRT PRETTIERS PROPERTIES. SO THAT HAS BEEN SHOWN. AND FURTHER CHARACTERIZATION WHEN IT WAS DONE AND I HAVE REVIEWED RECENTLY THIS. YOU CAN SEE THAT. METHYLATION INHIBITORS IN TOMATO -- THESE ARE DIFFERENT KINDS OF COMPOUNDS WHICH HAVE THAT KIND OF ACTIVITY. AND THEY ARE ACTIVE FOOD COMPONENTS. SIMILARLY METHYLATION INHIBITORS -- THEY ARE PRESENT IN COFFEE OR CINNAMON AND BROCCOLI. AND ONLY HISTONE DEASSET LAYS IN THESE. THAT KIND YOU HAVE RESEARCH IS COMING AND THESE ARE THE ACTIVE COMPONENT AND IN HISTONE ESPECIALLY IN WHICH TYPE WHICH LOCATION, WHETHER IT IS INCREASING OR DECREASING OR WHAT KIND OF MODIFICATIONS ARE THERE -- THOSE ARE ALSO BEING CHARACTERIZED. THE ADVANTAGE OF FOOD SIDE EFFECT OR TOXICITY IS LESS. SO WE ARE EMPHASIZING MANY TIME PEOPLE CALL IT ALTERNATIVE MEDICINES OR NUTOSUITICALS AND SEVERAL NAMES YOU WILL SEE. BUT WE WANT TO EN COURAGE THAT WAY ALSO. JUST BY CHANGING DIET WITHOUT USING ANYTHING, SUCH KINDS OF MODELS HAVE BEEN DEVELOPED, ESPECIALLY HELPATOCARCINOMA, WHERE YOU CAN DO UNDERSTAND BASIC MECHANISM OF STRUCTURE. NOW FOR EPIGENETIC ANALYSIS, DEPENDING ON HOW MUCH RESOLUTION YOU WANT IN YOUR ASSAY, YOU HAVE TO SELECT THE TECHNOLOGY. THIS IS VERY IMPORTANT. SUPPOSE YOU WANT TO GO TO A SPECIFIC ONE-BY-ONE CHANGES, THEN YOU HAVE TO DO TO DIRECT CONSEQUENCING. SO DEPENDING ON YOUR PROJECT, THAT WAY YOU CAN PLAN. MICRO RNA OR OTHER FIELD, YOU HAVE IN THE PREVIOUS LECTURE SO I WILL NOT SPEND TIME ON THAT. WHAT I WANT TO SHOW THAT THESE ARE DIFFERENT CANCERS, IN WHICH A DIFFERENT NUMBER OF MICRO RNA'S HAVE BEEN REPORTED. AS YOU HEARD IN PREVIOUS LECTURES PREVIOUSLY, THEY WERE FOUND IN HUMAN. THEN WE WERE NOT SURE WHETHER THEY WERE SPECIFIC OR NOT BUT NOW WE KNOW THEY ARE TISSUE-SPECIFIC ALSO. AND NOW WE ARE USING MICRO RNA POLY MORPHINES TO IDENTIFY HIGH-RISK POPULATION. AND ALONG WITH THEM, SOME PROJECTS ARE SUCH WHERE PROFILING IN A GROUP, WHETHER THEY ARE DIFFERENTIALLY EXPRESSED OR NOT. AND ONE THING YOU HAVE TO KEEP IN MIND, THAT WHEN HUMAN PROTIUM PROJECT WERE DEVELOPED, THEN PROPROTIUM CAME AND EPGYIGENOME. NOW AT NIH I AM INVOLVED IN THE PROJECT ALSO. THESE ARE GRADUALLY COMING. PROTEINS ARE VERY LABILE BUT BECAUSE OF A SMALL STRUCTURE AND SPECIAL STRUCTURE, THEY ARE VERY STABLE. SO CLINICALLY THEY HAVE MORE POTENTIAL THAN PROTEIN OR OTHER KINDS OF FEATURES SO THOSE ARE THERE. SO NOW IN THE LAST FEW MINUTES I WOULD LIKE TO TELL YOU ABOUT NIH ENROLLMENT. IT IS A MAJOR BASED ON WHOLE HUMAN GENOME SEQUENCE AND THEN PARTLY BECAUSE HUMAN PROTIUM WAS ALSO INITIATED. AND BY THAT TIME ABOUT FOUR OR FIVE YEARS AGO IT WAS REALIZED THAT WE HAVE TO UNDERSTAND EPIGENOME ALSO. SO ALL INSTITUTES CONTRIBUTED IN THIS PROGRAM, BECAUSE THIS EPIGENETICS IS INVOLVED IN MANY DISEASES LIKE CELLULAR CANCERS, AUTOIMMUNE DISORDERS. NEUROLOGICAL BEHAVIORAL DISORDERS. THE IDEA WAS THAT IF YOU HAVE SOME REFERENCE OF PROFILING OF METHYLATION OR HISTONE MODIFICATION OR MICRO RNA OR DARN IS ONE MAPPING FOR CHROMATIN COMPACTATION OR TECHNOLOGIES ARE COMING FOR IN VIVO IMAGING, THEN WE CAN UTILIZE THAT INFORMATION BETTER. SO THIS IS THE ROOM ROAD MAP PROGRAM. AND HE INITIATED THAT. NOW JUST NAME AS CHANGE FROM ROAD MAP TO COMMON FUNDS. BUT STILL IT IS THE SAME. ABOUT 1.5 PERCENT OF NIH BUDGET -- AS YOU KNOW IT IS $30 MILLION. SO IT WILL GO TO THESE KINDS OF PROGRAMS. SO TIME TO TIME AFTER TWO YEARS TOPICS ARE SELECTED AND IT SHOULD BE SKIED WHAT SHOULD BE PURSUED. THE YEAR EPIGENOME WAS RECOMMENDED SAME YEAR MICRO BIOOME PROJECT ALSO CAME. THEY GO WITH THEM HOW WE CAN UTILIZE FOR DETECTION. SO WHAT THIS ROAD MAP PROVIDES? IT PROVIDES OPPORTUNITY FOR PLAN FOR FIEFSH TO TEN YEARS, NOT LIKE ONE YEAR OR SIX MONTHS SO THAT WE CAN COMMIT THAT MONEY. AND THAT MAY BE HIGH-RISK BUT HIGH-POTENTIAL RESEARCH. AND WE WANT TO ACCELERATE THIS OF DISCOVERY. IT SHOULD TRANSFORMATIVE AND MANY SHOULD BE INTERESTED IN THAT. SO THIS IS THE PLANNING WE DID FOR $219 MILLION IN 2008. WHATEVER YOU ARE SEEING IN THAT IT MEANS AT DIFFERENT TIMES WE WROTE WHATEVER OUR OBJECTIVE IS, PEOPLE SHOULD SUBMIT PROJECT AND WE ARE GOING TO FUND AND FOLLOW THE PROGRESS. SO THESE ARE CALLED REQUESTS FOR APPLICATION, OR THE ANNOUNCEMENTS. ONE OF THE CENTERS, WHAT WE WANTED TO DO. THERE ARE CERTAIN CENTERS WHICH ARE ALREADY SLENLT FACILITIES TO DO THIS KIND OF MAPPING OR ANALYSIS -- THEY SHOULD TAKE NORMAL CELLS. EMBRYONIC CELLS AND DIFFERENT KINDS OF EPIGENOMES THEY SHOULD CHARACTERIZED AND AT THE SAME TIME TRAIN NEW INVESTIGATORS WHO ARE NOT IN THE FIELD. THEN ANOTHER ONE WAS INSTITUTE A SPECIFIC PROJECT, LIKE IF CANCER INSTITUTES IS THERE, THEN PARTLY SUPPOSE $3 MILLION PROJECT IS THERE. $1.5 MILLION NCI WILL GIVE AND $1.5 MILLION COMMON FUND WILL GIVE. AS YOU KNOW IN GENOMICS, A LOT OF DATA CAME. BUT FU GO TO PROTIUM, DATA IS 100 TIMES MORE. WHERE TO KEEP THAT? THAT IS ALSO A BIG PROBLEM. WHO WILL SUPPORT THAT ALSO? SO WE SET ASIDE SOME MONEY AD MC BI IS ALSO PITCHING IN. THAT'S WHIP ASKED WHO WILL KEEP THE DATA AND WITH WHO WILL DISTRIBUTE BECAUSE A LOT OF DATA WILL COME BUT TO KEEP TRACK OF THAT AND THERE SHOULD BE SOMEBODY TO GIVE THAT CHECKING QUALITY OF DATA, ALL THOSE THINGS ARE THERE. THEN TECHNOLOGY DEVELOPMENT. THESE ARE SUCH AREA EVERY YEAR WE WANT THAT MORE DEVELOPMENT SHOULD BE THERE AND THEY ARE BEING DEVELOPED ALSO. SO THAT ALSO WE KEEP IN MIND. AND THEN DISCOVERY OF NEW MARKERS, SOME MARKERS WE ALREADY KNOW BUT SOME MORE WE WANT TO DO. AND NC BI STARTS SUBMITTING. SO THIS IS THE PROGRAM. NOW TWO YEARS AGO, A ROAD MAP RIGHT FROM THE INCEPTION TO DATE I HAVE BEEN INVOLVED THERE. AS WELL AS EVERY SIX MONTHS WE MEET WITH INVESTIGATORS AND COMMITTEE MEMBERS AND ADVISORY COMMITTEE. AND EVERY YEAR ALL PIT.^'S WILL REPORT TO US WHAT IS THEIR PROGRESS. SIMILARLY, TWO YEARS AGO, EUROPEANS, THEY GOT TOGETHER. THEY HEARD ABOUT OUR PROGRAM, AND THEY TOLD US TO PARTICIPATE IN THAT AND THIS PROGRAM IS CALLED INTERNATIONAL HUMAN EPIGENOME CONSORTIA. THEY PROPOSE THEY WANT TO DO A THOUSAND EPIGENOME AND PROXIMATE COST THEY CALCULATED BUT THEY WANTED TO SEE THAT AGE-RELATED MODIFICATION ALSO SHOULD BE THERE, BECAUSE EPIGENETICS IS NEED FOR NORMAL DEVELOPMENT. BUT IN DISEASE DIFFERENT KINDS OF PATTERN COMES. SO ALL OF US GOT TOGETHER AND THIS PROJECT IS ALSO GOING ON AND A SPECIAL ATTENTION WE PAY THAT WHATEVER WE HAVE DONE THEY DON'T REPEAT. THEY SHOULD DO NEW AND THEY ARE USING ALSO DIFFERENT POPULATIONS. WHO WERE THE PARTICIPANTS IN THAT? THESE ARE THE COUNTRIES WHO PARTICIPATED. AND LATER ON EUROPEANS, THEY MADE A CONDITION. AND THESE WERE THE FUNDINGS AGENCIES. DIFFERENT NIH WAS ALREADY THERE. BECAUSE OF ROAD MAP, BUT EUROPEAN SCIENCE FOUNDATION, AND ALL OTHERS WHICH YOU HAVE JEEVENLT WELCOME TRUST -- ALL THESE ARE THERE. AND THEN THEY OPENED FOR OTHER COUNTRIES ALSO THAT ANY COUNTRY WHO WILL BRING $25 MILLION, THEN ONE PERSON WILL BE IN THIS THAT COMMITTEE MEETING. AND THAT INTERNATIONAL PROGRAM IS GOING ON. THEN AT THE SAME TIME THE FIRST MEETING IN PARIS GENEOSCOPE ALSO PARTICIPATED SO THEY ARE GIVING THEIR EQUIPMENT FOR THIS KIND OF ANALYSIS. SO THEY ARE VERY EN COURAGELINGING. AND INITIALLY THINK IN 2011 JANUARYOFEBRUARY, WE COVERED IN "NATURE INS" WHAT IS THE PLAN AND HOW IT IS BEING DONE. AND AC R AMERICAN ASSOCIATION OF CANCER RESEARCH, THEIR SOCIETY WAS ALSO THERE. SO THOSE PROGRAMS ARE GOING ON SIDE BY SIDE. SO I REALLY IN TERMS OF TRANSLATION, IT IS ABOUT IMAGINATION OF WHAT WE WANT THAT WHEN PEOPLE COME AND THEY GIVE BLOOD OR OTHER TISSUE SARMZ, THEN ALONG WITH OTHER TESTS, THESE EPIGENOME ANALYSIS ALSO SHOULD BE DONE AND FOLLOWUP OF TREATMENT SHOULD BE DONE AND THAT WAY WE CANS UNDERSTAND MORE ABOUT PERSONALIZED MEDICINE. AND WHAT OTHER QUESTIONS, ESPECIALLY IN OUR EPIDEEM LOGIC PROGRAM WE WANT TO ANSWER THROUGH THESE KINDS OF RESEARCH, WHETHER INCLUDING EPIGENETIC BIOMARKERS, THEY CAN IDENTIFY NEW RISK FACTORS IN DIFFERENT RACE AND ETHNIC GROUPS. IN U.S. PEOPLE COME FROM OTHER COUNTRIES. SO JMENT SO ONE DRUG WILL NOT WORK IN ALL GROUPS. SO WE WANT TO KNOW HOW WE CAN UTILIZE THAT. OHOW WE CAN COORDINATE GENETIC AND EPIGENETIC. WHETHER WE CAN IDENTIFY MICRO RNA ETHNICITY-SPECIFIC, OR HOW WE CAN DEFINE SUBCATEGORIES OF CANCER. AS YOU HAVE HEARD IN BREAST CANCER AND MANY OTHERS, THAT SO MANY KINDS OF CELLS ARE THERE THAN RECEPTORS COME AND ALL THOSE -- HOW WE CAN UTILIZE THAT. AND AS WE DISCUSSED AND THESE ARE OUR MAKE INVESTIGATORS, CHRISTOPHER PLAS, CHRISTINA AND MANY, MANY OTHERS. NOW I WOULD BE DELIGHTED TO TAKE QUESTIONS ON THAT. THANKS. [APPLAUSE] >> SO DO YOU THINK METHYLATION OF GENES AND CANCER CELLS IS AN EARLY EVENT THING? YOU WERE FAEMGS THE USE OF ALL OF THESE KOOEM PREVENTIVE AGENTS. >> YES, ESPECIALLY WHEN SIDE BY SIDE WE HAVE CHAIRED AND FROM NORMAL WHEN EPIGENETIC PROFILE NOT ONLY METHYLATION BUT HISTONE CHANGES ALSO.^ AND ANY MUTATION METHYLATION OR ANYTHING. SO THOSE GENETIC CHANGES COME MUCH LATER AND THEY COME EARLIER. WHAT WE WANT TO DO NOW THAT WE HAVE A LOT OF PROSPECTIVELY COLLECTED SAMPLES AND SOME SAMPLES WHICH MANY, MANY YEARS AGO LIKE NURSES HELD A STUDY SINCE' 72 WE STARTED. SO WHATEVER MARKS WE ARE SEEING NOW IN THOSE SAMPLES WHICH WERE COLLECTED EARLY AND LATER ON PATIENTS WERE FOLLOWED EVERY TWO OR FIVE YEARS. WE WANT TO SEE WHEN THESE MARKS CAME FIRST. BUT IT IS FOR SURE THAT METHYLATION MARKS COME FIRST THEN JETTIC MARKS. >> HOW MANY DO I HAVE TO EAT EVERY DAY? >> PERSON TO PERSON IT VARIES AND NOT ONLY COMBINED WITH THOSE OTHER FOOD ALSO. AND AS WE HEAR FROM PEOPLE DIET IN GOOD FOODS AND THESE ARE USEFUL. NOW SCIENTIFIC DOCUMENTS ARE ALSO COMING THAT THESE FRUITS HAVE SOME NATURAL COMPOUNDS OF BIOACTIVE FOOD COMPONENTS WHICH AE SHOWING ACTIVITY WHIH IS CLOSE TO THOSE DRUG AND OTHERS. SO THAT IS VERY EN COURAGE. AND WE ARE ALSO PUTTING A LOT OF MONEY IN THOSE PREVENTION TRIALS. SO KEEP EATING. >> THE INGREDIENTS ARE IN THE MET -- >> SEE, WHAT HAPPENS IN THESE KINDS OF TRIALS WE HAVE DONE, FU IDENTIFY A SINGLE COMPOUND LIKE ACCORDING TO THE POLICIES OF FDA, THEY WILL NOT APPROVE ANYTHING UNLESS YOU IDENTIFY THE COMPOUND, SHOW IT IS TOXIC OR NOT, HOW MUCH EFFICACY. BUT MANY TIMES COMBINED MANY OF THE COMPOUNDS N NATURAL FORM THEY ARE MORE EFFECTIVE THAN SINGLE COMPOUND. NOW WE ARE SUPPORTING SUCH A STUDY, THE WHOLE THING IS TAKEN IN COMBINATION. IF THAT IS EFFECTIVE, THEN IT IS EN COURAGING. >> OKAY, THAT WILL DO IT.