TODAY'S SPEAKER IS BEN PHILPOT COMING FROM THE DEPARTMENT OF CELL BIOLOGY PHYSIOLOGY FROM THE UNIVERSITY OF NORTH CAROLINA CHAPEL HILL. BEN GOT HIS Ph.D. IN 1997 IN PSYCHOBIOLOGY UNIVERSITY OF VIRGINIA CHARLOTSVILLE AND WORKED FOR FOUR YEARS AS POST-DOCTORAL FELLOW WITH MARK BEAR AT BROWN UNIVERSITY WHERE HE DID VERY NICE GROUND BREAKING WORK ON BIDIRECTIONAL REGULATION ACTIVITY DEPENDENT REGULATION OF SYNAPSES INDIVIDUAL CORTEX. HE THEN STAYED AT BROWN LONGER, WENT TO MIT WITH MARK WHERE HE WAS ASSISTANT PROFESSOR, UNTIL THEN HE WENT TO NORTH CAROLINA WHERE HE PRESENTLY IS KING PROFESSOR IN CELL BIOLOGY AND PHYSIOLOGY AND ASSOCIATE DIRECTOR OF NEUROSCIENCE CERTAINTY. LOOKING THROUGH BEN'S CV IT'S CLEAR THAT IS/INTERESTED) MENTORING I DON'T KNOW SCIENTIST. HE'S BASIC SCIENTIST BUT ALSO AT THE SAME TIME INTERESTED IN TRANSLATING THE BASIC SCIENCE IN TO PRE-CLINICAL STUDIES IN HIS LABORATORY IDENTIFYING NOVEL THERAPEUTIC STRATEGIESES TO TREAT DEVELOPMENTAL DISORDERS AND TEST THEM IN THE PRE-CLINICAL TESTING. AN EXAMPLE OF THIS HOW BEN HAS BEEN ABLE TO BLEND HIS INTEREST IN BASIC SCIENCE AND TRANSLATIONAL RESEARCH IS HIS WORK ON UB 38 WHICH IS A UBIQUITIN LIGASE WHICH IS MUTATE ED OR DELETED IN PERSONS WITH ANGELMAN SYNDROME. BEN'S GROUP HAS DONE GROUND BREAKING WORK USING THESE GENETIC MOUSE MODELS OF ANGELMAN OF BASICALLY MONOGENETIC DELETION USUALLY IN MATERNAL DELETION, TO LOOK AT EXPERIENCE DEPENDENT CORTICAL DEVELOPMENT, HE WAS ABLE TO SHOW THAT THE LACK OF THIS GENE ALSO CAUSES LESIONS IN SYNAPSES THAT AFFECTS EXCITATORY INHIBITORY BALANCE. AFFECTS BEHAVIORS, BEHAVIORS& THAT COULD BE ASSOCIATED WITH ANGELMAN'S. HIS GROUP WENT TO SHOW THEN THAT TOPO EYE SOME RACE INHIBITORS CAN UNSILENCE THE DORMANT PARENTAL UB 3A GENE, VERY IMPORTANT BECAUSE IT POTENTIAL THERAPEUTIC APPROACH TO BE ABLE TO TREAT THE DISORDER. SO A HALLMARK OF HIS RESEARCH IS TO INTEGRATE GENETIC ELECTROPHYSIOLOGICAL, NEUROCHEMICAL, AND BEHAVIORAL EXPERIMENTS TO TRY TO GET A HOLISTIC VIEW OF THESE ANIMAL MODELS AND THEN THEY ARE ALSO THEIR TRANSLATIONAL POTENTIAL. NEEDLESS TO SAY, BEN PUBLISHED TOP TIER NEUROSCIENCE JOURNALS AND HE'S RECEIVED A LOT OF AWARDS INCLUDING THE NARSET YOUNG INVESTIGATOR AWARD, THE SIMONS FOUNDATION, AND THE CLAUDIA BENTON AWARD BY ANGELMAN SYNDROME FOUNDATION. SO I WOULD LIKE TO WELCOME BEN TO THE NIH AND HE'S GOING TO BE GIVING US A TALK ON THE ANGELMAN SYNDROME BIOMARKERS AND TREATMENT OPPORTUNITIES. WELCOME. [APPLAUSE] EYE SOME RACE >> THANKS FOR INVITING ME, IT'S A PLEASURE TO BE HERE. I ALSO WANTED TO SAY I HOPE YOU INTERRUPT ME EARLY AND OFTEN. I MUCH PREFER TO ANSWER QUESTIONS DURING MY TALK RATHER THAN THE END, OF COURSE YOU'LL ANSWER AT THE END AS WELL SO FEEL FREE TO BLURT OUR A QUESTION ANY TIME YOU WANT SO I WANT TO MAKE SURE EVERYONE IS FOLLOWING AND ENGAGED. BEFORE I START I WANT TO GIVE DISCLOSURESSH I HAVE BEEN DOING CONSULTING AND GOTTEN SPONSORED RESEARCH AGREEMENTS AND HONOR AREA FOR SPEAKING ON THESE TOPICS AT VARIOUS PHARMACEUTICAL COMPANIES AND IT'S IMPORTNT TO DISCLOSE THOSE INTERACTIONS. SO WHAT I WILL BE DOING TODAY IS FIRST TALKING TO YOU ABOUT A GENERAL OVERVIEW ABOUT ANGELMAN SYNDROME. WHAT IS ANGELMAN SYNDROME AND WHY SHOULD YOU BE INTERESTED IN IT? TALK ABOUT A COUPLE OF TREATMENT OPPORTUNITIES THAT WE NOW HAVE FOR THIS NEURODEVELOPMENTAL DISORDER. THEN IN THINKING ABOUT HOW TO MOVE THOSE TREATMENTS EVENTUALLY TO THE CLINIC, I WILL TALK ABOUT BIOMARKERS THAT WE ARE USING TO IDENTIFY IN THIS PATIENT POPULATION WHEN THEN USE IN CLINICAL TRIALS. STARTING OFF ABOUT ANGELMAN SYNDROME, THIS IS A PRETTY TYPICAL FACE OF ANGELMAN SYNDROME, YOU CAN SEE VERY DEFINING FEATURES OF ANGELMAN SYNDROME. IS THESE INDIVIDUALS HAVE APPARENTLY HAPPY DISPOSITIONS. BUT THEY ARE ALSO FACE WITH A LOT OF CHALLENGES IN LIFE. SO THESE INDIVIDUALS HAVE A LOT OF MOVEMENT PROBLEMS, IN FACT THEY ARE OFTEN MISTAKEN FOR INDIVIDUALS WITH CEREBRAL PALSY. THEY HAVE A SEVERE INTELLECTUAL DISABILITY AND VERY DEFINING FEATURE IS A LACK OF SPEECH. SO MOST INDIVIDUALS DON'T SPEAK A SINGLE WORD THEIR ENTIRE LIFETIME YET CAN LIVE A FULL LIFETIME. THEY HAVE SEIZURES, ALMOST ALL THESE INDIVIDUALS. THEY HAVE ARHYTHMIC EEG PATTERNS AND EARLY IN LIFE HAVE SLEEP PROBLEMS SO THEY NEED LESS SLEEP AND SLEEP IN A MORE INCONGRUENT MANNER, SO MORE INTERMITTENT SLEEP. EXTREMELY CHALLENGING FOR CAREGIVERS. SO WHY ARE WE INTERESTED IN ANGELMAN SYNDROME? WE ARE INTERESTED IN FIRST AND FOREMOST DUE TO THE PERSONAL& TRAGEDY OF THIS DISORDER, TRYING TO HELP THESE INDIVIDUALS WHO ARE SO WONDERFUL YET SO CHALLENGEDDED IN LIFE AND HELP THEIR CAREGIVERS. WE ARE ALSO INTERESTED BECAUSE THERE'S INCREDIBLE OPPORTUNITY FOR THERAPEUTIC. IT'S A SINGLE GENE DISORDER. WE KNOW WHAT THAT GENE IS, IT'S UB 3A. SO UB 3A IS E 3 UBIQUITIN LIGASE THAT TARGETS SUBSTRATE PROTEINS FOR PROTEOSOMAL DEGRADATION. IF YOU HAVE A DELETION OR MUTATION UB 3A YOU WILL GET ANGELMAN SYNDROME. SO WE KNOW THIS IS A GENE THAT CAUSES ANGELMAN SYNDROME. THE PREVALENCE OF ANGELMAN SYNDROME ESTIMATED AROUND 1 IN 15,000 OR 1 IN 20,000 INDIVIDUALS AND HAS A HIGH CO-MORBIDITY WITH AUTISM. INTERESTINGLY, IF YOU HAVE TOO MANY GENE COPIES OF UB 3A AND OTHER GENES AROUND, THIS IS ONE OF THE MOST GENETICALLY IDENTIFIABLE FORMS OF AUTISM. SO AUTISM NOW AFFECTS AROUND 1 IN 59 INDIVIDUALS, SOMEWHERE IN INDIVIDUALS CAUSED BY COPY NUMBER VARIATIONSES IN THE CHROMOSOME 15 Q 1 TO Q 3 REGION WHERE IT RESIDES. UB 3A IS OVEREXPRESSED IN CONJUNCTION WITH OTHER GENES AROUND IT, IT'S A VERY CLASSIC FORM OF AUTISM. HAVING TOO LITTLE OR TOO MUCH UB 3A LEADS TO NEURODEVELOP MENTAL DYSFUNCTION. UB 3A IS ALSO INTERESTING IN ANOTHER WAY, IT'S ONLY EXPRESSED OFF ALLELE INHERITED FROM MATERNAL ALLELE. WHEREAS PATERNAL ALLELE IS EPIGENETICALLY SCIENCED IN NEURONS. SILENCED TO LONG CODING ANTI-SENSE THAT TRANSCRIPTIONALLY COMPETES WITH EXPRESSION OF UB 3A OFF PATERNAL ALLELE. NOW, VERY INTERESTINGLY, WE KNOW THAT UB 3A CAN BE EXPRESSED OFF PATERNAL ALLELE BECAUSE NON-NEURONAL CELLS THEY ARE BIALEE LIKELY EXPRESSED IN MATERNAL AND PATERNAL ALLELE SO UNIQUE ARRANGEMENT THAT ONLY NEURONS IS IT EPIGENETICALLY REPRESSED. EVERYONE IN THE AUDIENCE TODAY, NEUROEXPRESSINGS UB 3A PROTEIN IN NEURONS OFTEN MATERNAL ALLELE, WHEREAS PATERNAL ALLELE IS EPIGENETICALLY SILENCED DUE TOLUENE NON-CODING ANTI-SENSE. IS THERE IS A MUTATION OR DELETION OF JUST MATERNAL ALLELE THERE'S NO PROTEIN MADE BECAUSE PATERNAL ALLELE IS SILENT. WE TO STUDIES BACK AND FORTH BETWEEN PATIENT POPULATION AND USING A ANGELMAN SYNDROME MODEL ANALYSIS. SO A MOUSE THAT GENETICALLY MODELS ANGELMAN SYNDROME IN THAT UB 3A ALLELE IS TARGETED FOR DELETION. THESE MICE ALSO FEE KNOW COPY MANY ASPECT OF THE PATIENTS. THEY HAVE LEARNNDING AND MEMORY IMPAIRMENT, MOTOR DYSFUNCTION, THEY HAVE INCREASE SEIZURE SUSCEPTIBILITY, THEY HAVE ENHANCED EEG DELTA WAVE ACTIVITY AND EXHIBIT MICRORECEIVELY AS WITH INDIVIDUALS IN ANGELMAN SYNDROME. AT LEAST IN THIS EXAMPLE OF GENETIC MOUSE MODEL, UB 3A IS EXPRESSED IN THE ENTIRE BRAIN FROM THIS EXAMPLE OF THE MOUSE BRAIN. WHAT YOU SEE IN GREEN IS LABELING FOR UB 3A, IN THE MOUSE BRAIN YOU CAN SEE THROUGHOUT THE BRAIN YOU HAVE UB 3A LABELING, VERY HIGH LEVELSES IN BOTH EXCITATORY AND INHIBITORY NEURONS. IF YOU DELETE MATERNAL ALLELE THERE'S A LOSE OF EXPRESSION OF UB 3A EVERYBODY IN THE BRAIN. IF YOU LOOK CLOSE AND LIGHTS ARE DIM ENOUGH YOU CAN SEE THE EXPRESSION IN DEN DAY EYE US SO CELLS CAN BIALEE LIKELY EXPRESS UB 3A. IF YOU ARE TO ZOOM IN FAR ON GLIA YOU SEE A LITTLE BIT OF EXPRESSION IN GLIA. SO GLIA ALSO BIALEE LIKELY EXPRESSED IN UB 3A. I WILL GIVE A COUPLE OF VIGNETTES HOW WE ARE DEVELOPING TREATMENTS FOR ANGELMAN SYNDROME. ONE TO TREAT SYMPTOMS OF SYNDROME BUT ANOTHER APPROACH IS TO TRY TO REACTIVATE THE DORMANT ALLELE AS A TREATMENT OPPORTUNITY. LIMITED ON TIME SO I WON'T TELL YOU A THIRD TRADITIONAL GENE THERAPY APPROACH, WE ARE TRYING TO REINTRODUCES UB 3A USING VIRAL VECTORS. THERE'S MANY SYMPTOMS OF ANGELMAN SYNDROME. THERE'S SEIZURES H MOVEMENT DISORDERS, INTELLECTUAL DISABILITY, YOU CAN TRY TO TREAT THESE ONE AT A TIME AND I WILL GIVE ONE EXAMPLE, THAT OF SEIZURES. SO A DEBILITATING FEATURES THAT AFFECTS 90% OF INDIVIDUALS WITH ANGELMAN SYNDROME, THAT AGE ONSET IS EARLY, AROUND 2 TO 3 YEARS OF AGE. IT'S A GENERALIZED EPILEPSY AND BASICALLY ALMOST ANY SEIZURE TYPE YOU CAN IMAGINE YOU CAN FOUND INDIVIDUALS WITH ANGELMAN SYNDROME. GENERALIZED SEIZURES H TYPICAL SEIZURES, ET CETERA. AND BOTH THESE VICTIMS ARE EXHIBITING MULTIPLE SEIZURE TYPES. SO INCREDIBLE BURDEN ON THIS PATIENT POPULATION. THESE CAN BE TREATED WITH SOME EFFICACY USING TRADITIONAL ANTI-EPILEK -- EPILEPTICS. DIETARY SUCH AS QUITO GENEIC DIET OR LOW GLEE SEEMIC INDEX DIET AND RARELY SURGICAL INTERVENTIONS. FOR THE ANTI-EPILEPTICS THE MOST COMMON ONES ARE, -- THERE'S OTHER ANTI-EPILEPTICS THAT CAN BE USED BUT EVEN WITH ALL THESE ANTI-EPILEPTICS AT OUR DISPOSAL, MANY OF THESE INDIVIDUALS ARE REFRACTORY TO THESE TRADITIONAL ANTI-EPILEPTICS SO WE NEED ANOTHER LINE OF TREATMENT FOR SEIZURES INDIVIDUALS WITH ANGELMAN SYNDROME. ONE OF THE THINGS WE HAVE BEEN LOOKING AT RECENTLY, IS THIS IDEA THAT DA NAB DIE CONTROL MIGHT BE USED TO TO ATTENUATE SEIZURES IN VICTIMS WITH ANGELMAN SYNDROME. WHEN PEOPLE THINK OF CANNABADIOL YOU THINK MARIJUANA PLANT OF COURSE BUT LAY PEOPLE THINK OF IS THE EFFECTS OF THC COME FROM THE MARIJUANA PLANT SO THE PSYCHOACTIVE COMPONENT FROM CANNABIS PLANT IS DRIVEN BY THC. SIMILAR BUT IT DOES NOT HAVE A PSYCHOACTIVE COMPONENT. INSTEAD IT'S USED FOR MANY MEDICINAL BENEFITS. IN FACT PEOPLE REFER TO CBD THE HIPPIES DISAPPOINTMENT,SINCE THERE'S NO PSYCHOACTIVE COMPONENT THAT COMES WITH THAT. SO CBD HAS A LOT OF ATTENTION RECENTLY, BECAUSE IT'S FDA APPROVED FOR THE TREATMENT OF DRAVA SYNDROME SO EPIDIALECTS APPROVED FOR THE TREATMENT OF SEIZURES IN INDIVIDUALS WITH JERVAIS SYNDROME, THERE'S MANY PRODUCTS THAT CONTAIN CBD ON THE MARKET, MANY OF THESE ARE USED FOR DIETARY SUPPLEMENTS AND NAY ARE NOT WELL REGULATED. I KNOW EVEN MY MOTHER IS RUBBING CBD OIL ON HER SKIN TO TRY TO REDUCE PAIN. SO REALLY, YOU CAN'T ESCAPE A C SEEING CBD AROUND. BUT THESE PUBLICLY AVAILABLE SOURCES OF CBD ARE REALLY NOT VERY WELL CONTROLLED AND OFTEN HAVE VERY LITTLE CBD IN THEM. WHAT WE ARE USING IN STUDIES HERE IS SYNTHETIC CANNABADIOL WHICH IS 99% PURE. THE FIRST TEST WE DID WAS TO TRY TO SEE WHAT WOULD HAPPEN IF THE ANGELMAN SYNDROME MODEL MICE, IF WE WERE TO TRY TO INDUCE SEIZURES AND TREAT AT VARIOUS LEVELS OF CANNABADIOL. TO DO THIS WE TREAT MICE WITH CANNABADIOL, WAIT AN HOUR, THEN EVENTUALLY WILL GIVE THEM A VERY LOUD ACOUSTIC STIMULUS TO -- THAT MIGHT INDUCE AN AUDIO GENIC SEIZURE. WILD TYPE MICE DON'T EXHIBIT SEIZURES TYPICALLY BUT ANGELMAN SYNDROME MODEL MICE IN THE ABSENCE OF CANNABADIOL HAVE A STRONG SEIZURE RESPONSE TO THE LOUD AUDIO GENIC STIMULUS. WITH INCREASING CONCENTRATIONS OF CANNABADIOL WE ARE STRONGLY ABLE TO ATTENUATE SEIZURES IN THESE ANGELMAN SYNDROME MODEL MICE. SO THIS IS A FIRST HINT THAT CANNABADIOL MIGHT LEAD TO THERAPEUTIC BENEFIT FOR SEIZURES. IN THIS PATIENT POPULATION A COMMON WAY TO GET SEIZURES IS NOT IOGENICALLY BUT DUE TO INCREASE IN BODY TEMPERATURE SO FEBRILE SEIZURES. SO WE CAN STUDY THIS IN THE ANGELMAN MODEL MICE BY RACING THE TEMPERATURE -- RAISING THE BODY TEMPERATURE OF THE MICE AND SEEING POINT THEY HAVE SEIZURES AND WHETHER OR NOT SEIZURE DURATION SEVERITY WERE ADJUSTED BY CANNABADIOL TREATMENT. THOUGH THE TRIGGER TEMPERATURE FOR INDUCING SEIZURES DIDN'T VARY IN ANGELMAN SYNDROME MODEL MICE WITH TREATMENT OF CANNABADIOL WE GREATLY REDUCE SEIZURE DURATION AND HOW SEVERE THE SEIZURES WERE. SO THE MICE ARE HAVING LESS SEVERE SEIZURES, AND NOT LASTING AS LONG. SO THESE TWO PIECES OF EVIDENCE WERE PART OF MANY STUDIES WE DID TO SHOW CANNABADIOLL IN ANGELMAN MICE, MODEL MICE HAVE THERAPEUTIC EFFECT IN SEIZURES. IT LEADS US TO THINK MAYBE IT ALSO HAVE A ROLE IN TREATING INDIVIDUAL WITH ANGELMAN SYNDROME. I'LL JUST PUT A WORD OF CAUTION, CLINICAL TRIALS HAVE NOT BEEN DONE YET, I SUSPECT ON THE HORIZON BUT THEY NEED TO BE PERFORMED JUST TO TEST SAFETY AND EFFICACY IN ANGELMAN SIB CHROME PATIENT POPULATION. SO JUST WANT TO GIVE THAT ONE VIGNETTE ABOUT HOW SO RESEARCH WE ARE DOING IS TRYING TO THINK ABOUT THERAPEUTICS FOR DIFFERENT SYMPTOMATIC ASPECTS AND MANIFESTATIONS OF ANGELMAN SYNDROME. BUT OF COURSE, THESE CAN BE IMPACTFUL ON AN INDIVIDUAL'S LIFE BY TREATING SOMETHING AS SEVERE AS SEIZURES, IT'S STILL ONLY AFFECTING ONE DOMAIN OF THE MANIFESTATION OF ANGELMAN SYNDROME, WE HAVE TO WORRY ABOUT EVERYTHING ELSE, ALL MOTOR AND COMMUNICATION DEFECT, EVERYTHING ELSE THAT GOES WITH ANGELMAN SYNDROME. SO A MORE TRANSFORMATIVE TREATMENT WOULD BE TO TREAT THE DISORDER AT ITS GENETIC CORE. SO TO DO THIS, YOU CAN IMAGINE USING A SMALL MOLECULE ANTI-SENSE OLIGONUCLEOTIDE OR ANOTHER TYPE OF TREATMENT THAT CAN DOWN REGULATE THE UB 3A ANTI-SENSE THAT REPRESSES EXPRESSION OF UB 3A OFF THE PATERNAL ALLELE THE ALLOW PROTEIN MADE OFF THAT PATERNAL ALLELE. THE GREAT ADVANTAGE OF THIS APPROACH IS THAT REALLY TACKLES THIS DISORDER AT ITS CORE. SO WITH THE IDEA OF INTERVENING EARLY ENOUGH YOU CAN HAVE A TRANSFORMATIVE TREATMENT. SO WE HAVE TOOLS TO STUDY THIS AND LOOK AT REACTIVATION OF SILENT ALLELE. SO THIS IS A FLUORESCENT REPORTER MOUSE MADE BY SCOTT DINDOE IN ART'S LAB THAT HAS YELLOW FLUORESCENT PROTEIN FUSED TO UB 3A KNOCKED TO ENDOGENOUS LOCUST. SO WHEN YOU INHERIT THE REPORTER FROM THE MOTHER AND YOU SLICE UP THE BRAIN LOOK AT IT THE NEURONS ARE GLOWING BECAUSE THAT GENE COPY IS ON. IF THE FLUORESCENT REPORTER IS INHERITED PATERNALLY AND THEN LOOK AT BRAIN, THE NEURONS AREN'T GLOWING BECAUSE THAT GENE COPY IS SILENT. SO WE HAVE A REALLY SIMPLE ASSAY. NEURONS GLOWING, GENE COPY ON, NEURONS NOT GLOWING, GENE COPY OFF. SO WHAT WE DID IS WE TOOK NEURONS FROM THESE MICE THAT HAD THE FLUORESCENT REPORTER ON PATERNAL ALLELE WHERE GENE COPY SILENCED. WE PLATED THOSE NEURONS IN A 384 WELL FORMAT. THIS ALLOWED US TO DO A HIGH THROUGH PUT SCREEN WHERE WE CAN ADD SMALL MOLECULES AND LOOK FOR A SIGNATURE OF NEURONS THAT WERE GLOWING. THIS MEANS THAT WE HAVE ACTIVATED THAT SILENT COPY OF PATERNAL UB 3A. IS THAT CLEAR TO EVERYONE? THIS YOU GUYS AREN'T ASKING QUESTIONS LIKE I ASKED YOU TO SO YOU HAVE TO START ASKING QUESTIONS. USING THIS APPROACH AND TAKING ADVANTAGE OF THINGS LIKE FLUID HANDLING ROBOTICS WE HAVE AT THE UNIVERSITY OF NORTH CAROLINA, THIS SLIDE IS NOW CONDENSING YEARS OF HARD WORK TO ONE SLIDE SO ALWAYS A HARD SLIDE TO SHOW. BUT WE ARE ABLE TO SHOW THAT THE DRUG KNOWN AS TOPOTEKIN CAN POWERFULLY TURN ON THAT PATERNAL ALLELE OF UB 3A. FROM MS.THIS IS REALLY EXCITING. BECAUSE THIS COMPOUND IS AN FDA APPROVED COMPOUND. SO IT WAS ALREADY CLEARED FOR HAS VERY EXCITED, IN DOING DOSE RESPONSE CURVES WE CAN SEE THAT TOPOTEKIN CAN WORK IN THE LOW NANOMOLAR RANGE, OTHER DRUGS CLASSIC TO IT CAN WORK, INACTIVE ANALOGS THAT DIDN'T SHOW TOPO ISOMERASE ACTIVITY DIDN'T SHOW ABILITY TO UNSILENCE PATERNAL UB 3A. SO THIS GOT US EXCITED. GOES US EVEN MORE EXCITED WHEN WE FOUND THAT WE CAN INJECT TOPOTEKIN INTRATHREE KALLEY INTO MICE. AND AT LEAST IT WAS GETTING DELIVERINGED IN THE SPINAL CORD WE CAN SEE UNSILENCING OF PATERNAL UB 3A. SO NOW ONLY DID IT WORK IN VITRO, BUT ALSO WORKED IN VIVO. DRUGS MIGHT HAVE TOXICITY ASSOCIATED WITH THEM SO WE INJECTED THE DRUGS INTRATHECALLY AND LOOKED AT VARIOUS TIMES IMMEDATELY AFTER WE GAVE THE LAST DRUG DELIVERY OR UP TO A YEAR LATER. AND THEN WE ARE LOOKING NOW IN THE SPINAL CORD, WHERE WE CAN EASILY DELIVER THESE DRUGS. AND WHAT WE SAW WAS SOMETHING REMARKABLE, EVEN A YEAR AFTER OUR LAST DELIVERY OF TOPOTEKIN WE STILL SAW EVIDENCE FOR UNSILENCING OF PATERNAL UB 3A. SO THIS IS REMARKABLE IN THAT A TWO WEEK DELIVERY OF TOPOTEKIN DELIVERED INTRATHECALLY COULD HAVE A LASTING EFFECT A YEAR LATER WHERE YOU HAD MAINTAINED UNSILENCING OF PATERNAL UB 3A SO EVEN IF THERE WERE TRANSIENT TOXICITIES ASSOCIATED WITH DELIVERING THESE DRUGS, THERE'S AN EXCITEMENT THAT MAYBE CAN BE A LONG LASTING BENEFIT. NOW, THIS IS A BLOWUP TO SHOW YOU THAT LONG LASTING UNSILENCING A YEAR LATER. SO WHY AREN'T WE TAKING THESE COMPOUNDS TO THE CLINIC? THERE ARE MANY, MANY REASONS WHY WE ARE NOT. I'M GO BEING TO GO THROUGH A FEW OF THOSE. ONE IS THAT FIRST IF YOU HAVE HAD THE UNPLEASANT EXPERIENCE OF WATCHING A LOVED ONE GOING THROUGH CHEMOTHERAPY YOU KNOW MOST CHEMOTHERAPEUTICS HAVE ASSOCIATED TOXICITIES. WITH TOPOTEKIN ONE IS NEUTROPENIA. OTHER PROBLEMS WITH THIS DRUG IS ITS DELIVERY TO THE BRAIN ISN'T VERY GOOD SO IF YOU DELIVER PERIPHERALLY VERY LITTLE GETS INTO THE BRAIN, IF YOU DELIVER IT INTRATHECALLY, VERY LITTLE GETS INTO THE BRAIN. SO WE THOUGHT WE WOULD BYPASS THIS PROBLEM AND BEDELIVERRED TOPOTEKI NEXT INTO THE BRAIN. TURNS OUT WHEN YOU DELIVER TOPOTEKIN INTO THE BRAIN YOU GET INCREDIBLE UNSILENCING BUT IT'S ACTIVELY PUMPED BACK OUT OF THE BRAIN SO YOU HAVE A SMALL REGION WITH UNSILENTING OF UB 3A. WE DON'T KNOW HOW LONG THIS AFFECT LASTS IN THE CORTEX, THIS LONG LASTING AFFECT IN THE SPINAL CORD WE HAVEN'T LOOKED YET IN THE CORTEX. SO WE HAVE A LOT OF NEEDS. COMPOUNDS THAT CAN BE DELIVERED PERIPHERALLY, IDEALLY AND CAN GET BIDISTRIBUTION THROUGHOUT THE BRAIN -- BIODISTRIBUTION WITHIN THE BRAIN AND DEEP MECHANISTIC INSIGHT TO OPTIMIZE THERAPEUTIC BENEFIT WHILE OFF SIZING -- MINIMIZING OFF TARGET AFFECTS SO THE STORY DIDN'T DONE THERE. WE CONTINUE TO SCREEN SMALL MOLECULE LIBRARIES IN SEARCH FOR OTHER COMPOUNDS. AND WE FOUND OTHER COMPOUNDS THAT ARE NOT TOPO ISOMERASE INHIBITORS THAN PRODUCE UNSILENCING OF PATERNAL UB 3A AND I'M GOING TO GIVE YOU A TEASER, BY TELLING YOU ONE OF THE COMPOUNDS WE IDENTIIED RECENTLY WE CAN DELIVER PERIPHERALLY, JUST WITH AN IP INJECTION, AND WE CAN GET UNSILENCING OF PATERNAL UB 3A THROUGHOUT THE ENTIRE BRAIN. THIS IS INCREDIBLY EXCITING FOR US BECAUSE WHAT THIS SAYS IS THAT WE NOW HAVE A SMALL MOLECULE COMPOUND THAT WE CAN DELIVER PERIPHERALLY AND CAN UNSILENCE PATERNAL UB 3A THROUGHOUT THE ENTIRE BRAIN, THIS IS GIVING US A REALLY REMARKABLE OPPORTUNITY TO TRY TO DEVELOP A THERAPEUTIC THAT CAN HAVE A BROAD AFFECT IN THE BRAIN AND THAT HOPEFULLY WE CAN DEVELOP SO THAT IT'S SAFE AND EFFECTIVE AND CAN EITHER PRODUCE LONG LASTING UNSILENCING OR THAT WOULD BE SAFE ENOUGH TO DELIVER VERY FREQUENTLY. SO THIS IS SOMETHING WE ARE EXCITE AND ARE PURSUING. (OFF MIC) >> SO THE PREVIOUS STUDIES FIRST OF ALL IS HERE, THIS IS IN SPINAL CORD. THIS IS USING TOPOTEKIN, WHEN WE DELIVER THOSE CONCENTRATIONS, WE ARE DOING LOWER DOSES THAT COULD BE EASILY TOLERATED. WE ARE ALSO USING, GOING TO LOWER THIS A BIT. WE ARE ALSO USING LOW DOSES BECAUSE IT ALLOWED US TO USE AUTOMATED METHODS TO DETECT NUMBER OF NEURONS. SO HERE THIS SPINAL CORD IT IS A SPARSE DISTRIBUTION OF UNSILENCING THAT WE ARE GETTING. WE DON'T KNOW WHY CERTAIN NEURONS HAD LASTING UNSILENCING AND WHETHER WE CAN GET A CUMULATIVE EFFECT. SO ONE STUDY WE WANTED TO DO IS CONTINUALLY TO GIVE TREATMENTS AND THEN WAIT A COUPLE OF MONTHS, GIVE ANOTHER TREATMENT WAIT COUPLE OF MONTHS GIVE ANOTHER TREATMENT AND SEE IF'S ADDITIVE EFFECT OR IF IT'S THE SAME NEURONS THAT HAVE PROPENSITY TO TAKE UP AND HAVE A LASTING EFFECT. SO THEN WHAT I SHOWED YOU HERE IS WITH DIFFERENT COMPOUND, AND THIS IS OBVIOUSLY BRAIN. THIS IS A COMPOUND THAT WE CAN DELIVER IP AND VERY EFFECTIVELY UNSILENCE THROUGHOUT THE BRAIN. THAT ANSWER YOUR QUESTION? (OFF MIC) >> AT THE COUPLE OF AGES WE TRIED, THE 11 TO 19 RANGE WE ARE GETTING GOOD UNSILENCING. WE HAVEN'T TRIED MUCH YOUNGER OR MUCH OLDER YET. BUT IT IS ALSO LOST TO THE IDEA THERE IS A CRITICAL PERIOD FOR INTERVENTION. SO THERE'S -- A REALLY ELEGANT WORK OUT OF THE LAB OF (INDISCERNIBLE) IN THE NETHERLANDS, WHERE HE'S GENETICALLY REINSTATED UB 3A DIFFERENT STAGES OF DEVELOPMENT AND WE KNOW THE EARLIER YOU GO THE GREATER THE BENEFIT. FROMIF YOU REINSTATE UB 3A IN ADULTHOOD YOU CAN RECOVER SYNAPTIC PLASTICITY PHENOTYPE BUT NOT A SEIZURE PHENOTYPE OR MOTOR PHENOTYPE. IF YOU TREAT IN JUVENILE MICE UX GET RECOVERY PARTIAL RECOVERY OF REMOTE PHENOTYPE, IN ADDITION IF YOU TREAT YOUNGER YOU CAN GET PRETTY MUCH FULL RECOVERY SO THERE IS A CRITICAL PERIOD FOR INTERVENTION. THIS IS GOING TO BE VERY IMPORTANT FOR TREATMENTER TO THIS DISORDER. -- TREATMENT FOR THIS DISORDER. (OFF MIC) TOPO TEE KIN >> SO THE QUESTION IS WHAT CAN ACCOUNT FOR THE LONG LASTING UNSILENCING? WHAT WE ARE SHOWING YOU IS A KNOCKDOWN OF UB 3A ANTI-SNSE YET YOU THEN SEE AFFECTS THAT CAN LAST A YEAR. SO I THINK THERE ARE COUPLE OF THINGS TO -- THAT HIT ON THAT POINT. SO ONE IS THAT THERE IS A METHYLATION ON MATERNAL ALLELE THAT SUPPRESS IT IS ANTI-SENSE SO MAYBE WE ARE INCREASING METHYLATION OF THE PATERNAL ALLELE LONG LASTING AFFECT. THAT'S ONE SUGGESTION. ANOTHER SUGGESTION, SOMETHING WE NEED TO BE CAREFUL OF, IS THERE'S ALSO A SLIGHT CHANCE THERE COULD BE SOMETHING NEUROTOXIC THAT IS CAUSING THE LONG LASTING UNSILENCING. SO MAYBE THERE'S SOME TYPE OF DNA BREAK OR SOMETHING ALONG THOSE LINES, THAT'S CAUSING A LONG LASTING UNSILENTING. WE WOULD BE MINDFUL TO MAKE SURE THAT'S NOT OCCURRING. IN APPROACHES SUCH AS THOSE THAT USE ANTI-SENSE OLIGONUCLEOTIDES TO KNOCK DOWN THE ANTI-SENSE, THOSE AFFECTS LAST AS LONG AS ASOs ARE STANDING AROUND SO GENERALLY THREE MONTHS. SO THOSE ARE TRANSIENT. (OFF MIC) >> THE REQUEST IS WHY ONLY EXPRESSED IN MATERNAL ALLELE AND IS IT JUST AS GOOD TO EXPRESS OFF PATERNAL ALLELE. IF I CAN REPHRASE, WHY IS IT MONOALLELICLY EXPRESSED TO BEGIN WITH. ONE PATIENT FAMILY WITH A MICRODUPLICATION OF UB 3A SO YOU HAVE TWO COPIES EXPRESSED, THERE'S DEPRESSION AND SKITS SEW EFFECTIVE BEHAVIOR IN THOSE INDIVIDUALS WITH OVEREXPRESSION OF UB 3A. THOSE INDIVIDUALS CAN HAVE GONE ON SO WE KNOW ITs A MODEST EFFECT ON INTELLECTUAL DISABILTY BUT IT STILL DOES HAVE A CONSEQUENCE. SO WILL IS A CONSEQUENCE OF OVEREXPRESSING UB 3A. NOW, WE THINK THAT EXPRESSION OF PATERNAL ALLELE WILL BE JUST AS GOOD. THE REASON WE THINK AS FAR AS WE CAN TELL ALL OF THE SAME PROMOTERS AND REGULATORY ELEMENTS ARE IN PLACE. SO GET EXPRESSION OF PATERNAL ALLELE WE NEVER OBSERVEOVER EXPRESSION. OVEREXPRESS. THAT IS A HUGE ADVANTAGE OF THIS APPROACH IF YOU UNSILENCE PATERNAL ALLELE OVEREXPRESSION IS NOT A CONCERN. I'LL ATTACH A LITTLE OF A CAVEAT TO THAT, THERE'S ONE SMALL SET SUBSET OF INDIVIDUALS WITH ANGELMAN SYNDROME THAT ARISE TWO PATERNAL COPIES. OF THAT CHROMOSOMAL REGION. SO IN THAT INSTANCE IF YOU UNSILENCE PATERNAL UB 3A UNSILENCING TWO GENE COPIES. OTHER THAN THAT SMALL PERCENT OF INDIVIDUALS WHO HAVE PARENTAL OTHER INDIVIDUALS WITH ANGELMAN SYNDROME ONLY HAVE ONE COPY EXPRESSED AND YOU DON'T HAVE TO WORRY ABOUT OVEREXPRESSION. (OFF MIC) >> QUESTION IS WHAT IS THE SPECIFICITY OF DRUGS ON UB 3A VERSUS OTHER GENES? GREAT QUESTION. SO FOR TOPOTECAN, FIRST I SAY TOPO TEKAN IS FDA APPROVED. WHAT OTHER GENES ARE YOU AFFECTING WHEN YOU TREAT WITH TOPOTECAN? IN BROADWAY WE START OFF DOING RNA SEQ. AT LEAST WHAT IS AFFECTED DIRECTLY AND INDIRECTLY. AND WHAT WE SAW IS THAT THERE ARE RELATIVELY SMALL NUMBER OF GENES GETTING DOWN REGULATED, VERY POWERFULLY. AND WE ARE TRYING TO FIGURE OUT WHAT THE -- KIND OF GENETIC OR MOLECULAR FINGERPRINT OF THOSE GENES MIGHT BE AND AID BID THE FACT MY COLLABORATOR MARTIN WHO I DESCRIBE AS A WELL GENE MAN IS THE GENES DOWN REGULATED ARE LONG GENES. WE THEN REORDERED OUR RNA SEQ DATA BY GENE LINK, SHORT GENE TO LONG GENE AND YOU CAN SEE FAITHFULLY THE LONGER THE GENE THE GREATER THE DROP OFF IN EXPRESSION WITH TOPO TECAN YOU CAN SEE SIGNIFICANT CHANGES OF ANY GENES OVER 100 KB WE SAW DROP OFF IN LONGER THE GENE THE GREATER THE DROP OFF IN EXPRESSION. TURNS OUT THE ANTI-SENSE IS ONE OF THE LONGEST GENES IN THE GENOME SO AROUND A THOUSAND KB IN MICE. SO IT'S VERY POTENTLY DOWN-REGULATED SO TOPO ISOMERASE TURNS OUT TO BE IMPORTANT FOR TRANSCRIPTIONAL ELONGATION. WHEN YOU DISRUPT TRANSCRIPTIONAL ELONGATION THE LONG ARE THE MOST SENSITIVE. WE TOOK OTHER DATA SETS OUT THERE IN THE CANCER FIELD AND REORDERED THEIR DATA SETS BY GENE LENGTH AND SAW THAT IN EVERY CELL TYPE, THERE'S A CONTROL OFF IN GENE LENGTH. I THINK (INDISCERNIBLE) HAD PAPER COME OUT SAME TIME AS OURS SHOWING GENE LENGTH AFFECT OF THESE DRUGS. THE NEWER DRUG WE ARE STILL LOOKING INTO WHAT OTHER GENES MIGHT BE AFFECTED. YEAH. (OFF MIC) >> I SHOULD BETTER DIRECT THAT TO BEN GU A POST DOC IN MY LAB WHO DID THOSE STUDIES AND IS NOW LOOKING FOR A JOB. HE'S FANTASTIC. SO HE DID A LOT OF EEG STUDIES AS WELL. ONE OF THE THINGS THAT HE SAW, TRYING TO REMEMBER THE PAPER JUST PUBLISHED, I SHOULD KNOW IT ALL, WAS THAT THERE'S A NORMALIZATION OF THE DELTA PHENOTYPE. SO THERE'S ENHANCED DELTA ACTIVITY IN THE ANGELMAN SYNDROME MODEL MICE ATTENUATED AND CORRECTED ACTUALLY. SO IT SEEMED TO HAVE A BOTH BEHAVIORAL AND EEG CORRECTION. DOES THAT ANSWER YOUR QUESTION? OKAY. WHILE WE ARE REALLY EXCITED ABOUT OUR SMALL MOLECULE APPROACHES, HACKED PROVIDE A TREATMENT FOR ANGELMAN SYNDROME, MY CAUTION IS SAFETY AND EFFICACY STUDIES NEED TO BE DONE OF COURSE BEFORE MOVED INTO THE PATIENT POPULATION. BUT EXCITINGLY, SINCE OUR WORK CAME OUT SHOWING THAT YOU CAN UNSILENCE PATERNAL UB 3A USING DRUGS, SEVERAL PHARMACEUTICAL COMPANIES HAVE COME UP WITH APPROACHES USING ANTI-SENSE AL GO NUCLEOTIDES TO KNOCK DOWN PATERNAL UB 3A. I THINK THERE'S AN ANTICIPATED THREE DIFFERENT TRIALS NEED TO BE RUN THIS YEAR USING ASOs TO UNSILENCE PATERNAL UB 3A SO REALLY EXCITING TIME IN THE ANGELMAN SYNDROME COMMUNITY. NOW, OUR ULTIMATE GOAL IS TO IMPROVE LIVES OF INDIVIDUALS WITH ANGELMAN SYNDROME. AND WE ARE NOW AT A STAGE WHERE WE HAVE INCREDIBLE OPPORTUNITIES TO DO SO. SO WE NOW HAVE DRUGS TO TREAT SYMPTOMS, THERE'S A CLINICAL TRIAL FOR DRUG CALLED OB 101 TO TREAT THINGS LIKE SLEEP ISSUES IN INDIVIDUAL WITH ANGELMAN SYNDROME. I THINK THERE'S A PROMISE OF THINGS LIKE CANNABIDIOL WHICH I TALKED ABOUT. WE HAVE TRANSFORMATIVE APPROACHES THAT ARE ON THE HORIZON TOO USING GENE REACTIVATION STRATEGIES SUCH AS SMALL MOLECULES OR ANTI-SENSE OLIGONUCLEOTIDES, MORE TRADITIONAL GENE THERAPY WHICH MY GROUP AND OTHERS ARE WORKING ON AS WELL AND MANY OTHER APPROACHES SO WE ARE REALLY GETTING CLOSE, IN FACT THERE'S A NUMBER OF CLINICAL TRIALS THAT ARE STARTING THIS YEAR. I WOULD ARGUE FOR A LOT OF NEURODEVELOPMENTAL DISORDERS, PEOPLE HAVE HAD IDEAS OF WHAT TO USE FOR THERAPIES BEFORE THEY ACTUALLY READY TO GO TO CLINIC. BEFORE THEY WERE GOOD OUTCOME MEASUREMENTS FOR THOSE CLINICAL TRIALS. SO I HAVE BEEN TRYING TO BE VERY MINDFUL OF THIS AND FOR SOMETIME NOW I HAVE BEEN TRYING TO THINK OF BIOMARKERS THAT WE MIGHT BE ABLE TO USE IN UPCOMING CLINICAL TRIALS BY OUR GROUP OR OTHER GROUPS. THERE ARE MANY FEATURES YOU WOULD WANT FOR BIOMARKER, I'M GOING TO LIST A FEW THAT I THINK ARE PARTICULARLY IMPORTANT, ONE, THEY SHOULD BE CLINICALLY RELEVANT, RELEVANT TO THE PATIENT POPULATION. IF IT'S OBJECTIVELY QUANTIFIABLE INSTEAD OF SOMETHING SUBJECTIVE, THERE'S A HUGE IN THE PARENT REPORTING AND DOCTOR REPORTING. SO IF YOU CAN -- MAKE OBJECTIVELY QUANTIFIABLE IT'S AN ADVANTAGE. IF IT'S MINIMALLY OR NON-INVASIVE, THAT IS IDEAL. YOU WANT SOMETHING HIGHLY PENETRANT, UNLESS USING AS A BIOMARKER FOR STRATIFICATION OF A POPULATION. AND I WOULD ARGUE THAT IF YOU OBSERVE A FEATURE IN THE PATIENT POPULATION AND IF YOU OBSERVE THE SAME FEATURE IN THE MOUSE MODEL, AND IF YOU CAN REVERSE THAT WITH THE TREATMENT IN THE MOUSE MODEL, IT GIVES YOU AN IDEA THAT YOU MIGHT BE ABLE TO REVERSE IN THE PATIENT POPULATION. SO WITH ALL THESE THINGS IN MIND FORTH BETWEEN STUDIES IN MAN AND MOUSE TO TRY TO DEVELOP BIOMARKERS. THAT HAVE THESE CHARACTERISTICS. ONE OF THEM IS JUST EEG BECAUSE EEG IS A RELATIVELY NON-INVASIVE PROCEDURE, WHERE YOU CAN COLLECT EEG ACTIVITY FROM PATIENT POPULATIONS AND YOU CAN DO IT REPEATEDLY. HERE IS AN EXAMPLE OF EEG FROM NEUROTYPICAL INDIVIDUAL AND INDIVIDUAL WITH ANGELMAN SYNDROME. I THINK YOU DON'T HAVE TO BE A VIROLOGIST TO TELL THERE'S A HUGE DIFFERENCE IN THE EEG PATTERNS FROM AN INDIVIDUAL WITH ANGELMAN SYNDROME FROM A NEUROTYPICAL INDIVIDUAL. THESE ARE REALLY HYPERARHYTHMIC. AND ONE OF THE THINGS WE DID IS WE HAVE BEEN COLLECTING DATA, I SAY WE, I SHOULD SAY MIKE SEROF (PHONETIC) I SHOULD CALL OUT WHO JUST GOT A FACULTY POSITION AT CHILDREN'S NATIONAL UP THE ROAD. HE STARTED COLLECTING E EG DATA FROM CLINICIANS DOING THESE STUDIES ON ANGELMAN SYNDROME PATIENT POPULATIONS. BECAUSE THE CLINICIANS ARE AMAZING IN THAT THEY CAN QUICKLY ASSESS EEG CAN PICK UP DIFFERENT TYPES OF SEIZURE ACTIVITY. THEY ARE REALLY GREAT PRESCRIBING ANTI-EPILEPTICS BUT THEY OVERBURDENED AND DON'T HAVE TIME TO DO A DEEPER ANALYSIS OF THE EEG ACTIVITY. SO ALL THESE CLINICIANS WERE SITTING ON TONS AND TONS OF DATA THAT WAS ACCESSIBLE ONCE WE FIGURED HOW TO -- HOW WE CAN TRANSFER THEM WITH ALL HI PA RULES OF COURSE. BUT ONCE THAT WAS FIGURED OUT, THERE WAS A HUGE AMOUNT OF DATA THAT WAS AT OUR DISPOSAL. SO MIKE SEROF DID A LOT OF DIFFERENT TYPES OF ANALYSES OF EEG DATA AND ONE WAS A POWER SPECTRA AND WHAT YOU CAN CONFIRM IS WHAT HAD BEEN POINTED OUT IN LITERATURE FOR A LONG TIME, THAT THESE INDIVIDUALS HAVE HIGHLY ENHANCED DELTA WAVE ACTIVITY. SO THIS IS ACTIVITY IN THE TWO TWO TO FOUR HERTZ RANGE. HE ALSO FOUND OTHER THINGS THAT HADN'T BEEN BROADLY REPORTED, IN THE LITERATURE TRADITIONS REPORTED OCCIPITAL BIAS FOR ENHANCED DELTA POWER BUT WE SAW THAT IN ALL AREAS OF THE BRAIN SO A GENERALIZED FEATURE OF THIS ENHANCED DELTA ACTIVITY. HE ALSO PLOTTED THIS ACROSS TIME. AND SO PLOT HERE IS IN THE AGE OF INDIVIDUAL AND X AXIS AND THE PERCENT OF THE EEG-DRIVEN BY OCCIPITAL DELTA POWER AND YOU CAN SEE HERE IN RED INDIVIDUALS WITH ANGELMAN SYNDROME HAVE MUCH HIGHER DELTA ACTIVITY THAN NEUROTYPICAL INDIVIDUALS. THIS IS MAINTAINED LARGELY THROUGHOUT DEVELOPMENT THOUGH THERE'S A SLIGHT DEVELOPMENTAL DECLINE IN TOTAL DELTA IN NEUROTYPICAL INDIVIDUALS. SO THIS SUGGESTS ONE OF MANY FEATURES SUGGESTING IT'S A GOOD-BYE MARKER. NOW, -- GOOD, BIOMARKER. WE OBSERVED ENHANCED DELTA ACTIVITY IN ANGELMAN SYNDROME MODEL MICE SO UNDER THE RIGHT CONDITIONS, IN DOING LOCAL FIELD POTENTIAL RECORDINGS, IN PRIMARY VISUAL CORTEX, MIKE OBSERVED ENHANCE DELTA WAVE ACTIVITY IN ANGELMAN MODEL MICE COMPARED TO WILD TYPE MICE. IN THE MICE WE HAVE THE POWER TO GENETICALLY REINSTATE UB 3A BOTH TO TRY TO FIGURE OUT TIME COURSE, FOR WHICH YOU CAN NORMALIZE EEG ACTIVITY AND ALSO UNDERSTAND THE RELEVANT CELL TYPES FOR DRIVING THIS TYPE OF ACTIVITY. SO JUST GIVING ONE PIECE OF DATA ON THIS, THESE WERE MICE IN WHICH WE GENETICALLY DELETED UB 3A JUST FROM INHIBITORY NEURONS. THAT RECAPITULATED THE ENHANCE DELTA PHENOTYPE OR IN MICE WE GENETICALLY REINSTATED UB 3A IN INHIBITORY NEURONS THAT WOULD NORMALIZE THE DELTA PHENOTYPE. SO WE NO THE PHENOTYPE IS DRIVEN BY LOSS OF UB 3A IN INHIBITOR NEURONS. ARE THERE OTHER BIOMARKERS FOR EEG? WE ARE REALLY JUST BEGINNING TO SCRATCH SURFACE WHAT TYPES OF INFORMATION WE CAN GET FOR THE EEG DATA BUT I WANT TO SHOW YOU JUST ONE MORE SLIDE ON THE EEG ACTIVITY. THAT WE HAVE BEEN LOOKING AT SLEEP SPINDLE ACTIVITY. SO SLEEP SPINDLES ARE IN 11 TO 16 HERTZ RANGE, THEY CAN BE MEASURED IN AN OBJECTIVE WAY, LOOK AT NUMBER AND DURATION OF SPINDLES THEY ARE RA DRAMATICALLY REDUCED IN INDIVIDUALS WITH ANGELMAN SYNDROME. IN SOME WAYS THIS IS A MUCH BETTER BIOMARKER THAN DELTA WAVE ACTIVITY BECAUSE WE KNOW MORE ABOUT THE CLINICAL RELEVANCE AND THE CIRCUITRY OF SLEEP SPINDLES THAN WE DO ABOUT DELTA WAVE ACTIVITY. SO SLEEP SPINDLES ARE ASSOCIATED WITH QUALITY AND WE KNOW A LOT ABOUT THE CORTICAL CIRCUITS OBSERVE SLEEP SPINDLES. SO WHILE DELTA WAVE ACTIVITY COULD BE A GOOD PROXY READ OUT FOR UB 3A TARGET ENGAGEMENT WE DON'T KNOW MUCH ABOUT CLINICAL RELEVANCETOR THE SLEEP SPINDLES WE DO. SO EXAMPLES OF THE TYPES OF DATA WE CAN GET FROM ANALYZING EXISTING EEG DATA. OTHER TYPES? YEAH. (OFF MIC) >> SO THESE ARE INDIVIDUALS WHO ARE MOST CASES PRESENTING WITH SEIZURES, GOING IN CLINIC FOR BASELINE STUDIES. IT SHOULD BE POINTED OUT A LOT OF STUDIES ARE RELATIVELY BRIEF DURATIONS OF TIME. SO THE RECORDINGS ARE LASTING ANYWHERE FROM THREE MINUTES OR SOME CASES UP TO AN HOUR BUT THEY ARE RELATIVELY BRIEF. SO I THINK IF WE HAD LONGER PERIODS TO ANALYZE WE CAN GET PROBABLY EVEN TIGHTER DATA. OTHER QUESTIONS? ALL RIGHT. SO THERE ARE OTHER TYPES OF DATA WE CAN GET TO LOOK AT BIOMARKERS AND IN THIS CASE THIS IS MRI DTI DATA. I SHOULD POINT OUT, THIS IS WORK DONE BY HEATHER AND MARK CHEN AT UNC AND MATT JOHNSON IN MY LAB DID MOUSE WORK I WILL SHOW YOU. YOU LEARN A LOT ABOUT PRESENTING DATA IN FRONT OF PARENTS. THIS IS AN EXAMPLE WHERE I WAS REMINDED BECAUSE THE FIRST TIME I PRESENTED THIS IN FRONT OF PARENTS I SAID THIS IS A GREAT AND A PARENT RAISED HER HAND WITH CHILD WITH ANGELMAN SYNDROME AND HE SAID EXCEPT THE FACT OUR KIDS HAVE TO BE ANESTHETIZED TO GO INTO MRI MACHINE UNLIKE NEUROTYPICAL INDIVIDUAL. SO IT'S A REMINDER TO MYSELF AND EVERYONE WORKING ON ANY TYPE OF DISORDER, STAY ENGAGED WITH THE PATIENT POPULATION. AND THE CAREGIVERS BECAUSE YOU ALWAYS LEARN A LOT AND REMINDED A LOT AND ARE ALWAYS HUMBLED IN GREAT WAYS. SO OTHER THAN THE FACT THESE INDIVIDUALS NEED TO BE ANESTHETIZED TO GO INTO AN MRI MACHINE, IT'S A NON-INVASIVE PROCEDURE. AND WE CAN GET A LOT OF DATA. SO ONE OF THE SIMPLEST PIECES OF DATA FROM MRI IS LOOKING AT GRAY MATTER AND WHITE MATTER VOLUME AND IF YOU LOOK AT A PLOT WHITE MATTER ON THE Y AXIS AND ANGELMAN SYNDROME INDIVIDUALS IN RED, TYPICALLY DEVELOPING INDIVIDUALS IN GRAY YOU CAN SEE& HUGE DIFFERENCES, INCREDIBLE LOSS OF WHITE MATTER VOLUMES IN INDIVIDUALS WITH ANGELMAN SYNDROME. THERE'S A DISPROPORTIONATE LOSS OF WHITE MATTER VOLUME THAN GRAY MATTER VOLUME WHERE LOSS IS MORE MODEST. SO THIS IS REALLY INTERESTING. WE ARE WONDERING HOW EARLY DURING DEVELOPMENT WAS THIS OBSERVED? SO HERE IS A STUDY LOOKING AT WHITE MATTER VOLUME PLOTTED BY AGE AND YOU CAN SEE INDIVIDUALS IN ANGELMAN SYNDROME IN RED START TO FALL OFF TYPICAL DEVELOPING TRAJECTORIES EARLY AROUND THREE TO FOUR YEARS OF AGE SO DOWNWARD EXTENDING THIS STUDY AND SCANNING BABIES EVEN. INTERESTINGLY, THE DEGREE OF WHITE MATTER VOLUME LOSS IS CORRELATED TO A LOSS OF MOTOR ABILITY. SO THE GREATER YOUR WHITE MATTER VOLUME LOSS, THE GREATER YOUR LOSS OF MOTOR ABILITIES. JUST TO GIVE YOU AN IDEA OF THE SIZE OF THE AFFECTS FOR DIFFERENT TYPES OF MEASUREMENTS, THESE ARE FUSION SEN TENSOR MEASURING WHICH CAPTURES MOTOR INFORMATION, MEASUREMENTS OF FACTUAL, GENERALIZE HAS BEEN SAY WHITE MATTER INTEGRITY. BUT YOU CAN SEE THAT IN ANGELMAN SYNDROME INDIVIDUALS, THERE'S A HUGE CHANGE IN WHITE MATTER INTEGRITY COMPARED TO INDIVIDUALS WITH AUTISM OR FRAGILE X SYNDROME. SO THERE IS A REALLY LARGE AFFECT SIZE. THE CHALLENGE FROM THE HUMAN STUDIES IS THAT WE KNOW THAT WHITE MATTER IS ALTERED. WE KNOW WHITE MATTER INTEGRITY IS DISRUPTED. BUT WE DON'T KNOW HOW IT'S DISRUPT SOD THERE'S MANY THINGS THAT ALTER WHITE MATTER FUNCTION SO YOU CAN HAVE CHANGE IN MYELINATION, YOU CAN HAVE A CHANGE IN THE WHITE MATTER BUNDLES. THERE COULD BE A NUMBER OF FEATURES THAT COULD CAUSE IT. BUT WHEN WE GO TO MOUTHS WE CAN ACTUALLY LOOK AT THESE IN DETAIL. SO THE FIRST THING WE DID IN GOING TO MOUSE STUDIES IS SEE IF WE CAN RECAPITULATE THE PHENOTYPES IN THE PATIENT POPULATION. SO THIS IS SHOWING THAT EARLY IN DEVELOPMENT AROUND 14 DAYS OF AGE, THERE IS VERY LITTLE CHANGE IN VOLUME IN NEOCORTEX, SO GRAY MATTER OR CORPUS CALLOSUM. BUT FOUR YEARS OF AGE THERE IS LOSS OF VOLUME IN WHITE MATTER -- SORRY GRAY MATTER NEOCORTEX AND GREATER LOSS IN WHITE MATTER IN THE CORPUS CASH FLOW SUM. SO -- CORPUS CALLOSUM. SO THEY EXHIBIT AD DISPROPORTIONATE LOSS OF WHITE MATTER VOLUME THE GRAY MATTER VOLUME. IN THE MODEL MICE WE CAN LOOK WHAT IS CAUSING THAT. SO ONCE AGAIN MAKING A LONG STORY VERY SHORT, WE ARE SURPRISED AT THE THINGS THAT WEREN'T CHANGED AND IN FACT, WHEN WE LOOK AT WHAT WAS DIFFERENT WE CAN ONLY FIND ONE DIFFERENCE, SHOWN HERE LOOKING AT LEK -- ELECTROMICROSCOPY FROM WILD TYPE MOUSE OR ANGELMAN SYNDROME MODEL MOUSE ON THE RIGHT. YOU CAN SEE THE BLACK AROUND AXONS ON FUSS IS MYELINATION AND IF YOU LOOK AT THE G RATIOS IT'S A MEASURE OF MYLYNN COMPACTION VERSUS AXON DIAMETER THAT IS UNCHANGED. BUT THE BIG DIFFERENCE WAS CHANGE IN AXON DIAMETER. IN ANGELMAN SYNDROME MODEL MICE THERE IS CONSISTENT REDUCTION IN DIAMETER SO AXONS WERE BETTER. SO WE WANT TO SEE IF THIS HAS A FUNCTIONAL IMPACT. IT'S HARD TO IN THE MOUSE BRAIN TO DO VERY ACCURATE STUDIES OF CONDUCTION VELOCITY SO WE LOOKED TO SEE IF WE SAW THE SAME THING IN THE SCIATIC NERVE, CENTRAL NEURONS ARE PROJECTING OUT OVER A MUCH LONGER LENGTH. WE SAW REDUCTION IN AXON DIAMETER, IT WAS MODEST BUT WE SAW IT THEN WHEN WE DID NERVE CONDUCTION VELOCITY STUDIES THE PEAK VELOCITY WAS NOT CHANGED BUT THE AVERAGE VELOCITY REDUCED. AND PEAK DIDN'T CHANGE BECAUSE THERE WAS LINGERING LARGE DIAMETER AXONS BUT ON AVERAGE, AXON CONDUCTION VELOCITY REDUCE SOD THIS SUGGESTED A FUNCTIONAL IMPACT FROM THIS REDUCED AXON DIAMETER SIZE. IT SUGGESTS THAT MAYBE THIS IS ONE OF THE CONTRIBUTING FACTORS TO THE MOTOR DYSFUNCTION OF INDIVIDUALS WITH ANGELMAN SYNDROME. SO JUST TO SUMMARIZE THESE DIFFERENT VIGNETTES I HAVE SHOWN YOU TODAY, I'M SHOWING YOU CANNABIDIOL IS A TREATMENT OPPORTUNITY TO TREAT SEIZURES FOR INDIVIDUALS WITH ANGELMAN SYNDROME AND THERE ARE MANY OTHER APPROACHES THAT ARE BEING DEVELOPED TO TREAT OTHER SYMPTOMS OF ANGELMAN SYNDROME. UB 3A UNSILENCING APPROACHES AND GENETHER I PI APPROACH I DIDN'T HAVE TIME -- GENE THERAPY APPROACH I DIDN'T TALK ABOUT ARE OTHER OPPORTUNITIES TO TREAT ANGELMAN SYNDROME THAT CAN CORRECT MANY ASPECTS OF ANGELMAN SYNDROME ATTACKING ATTACKING THE CORE GENETIC DEFECT OF THE DISORDER. AS ALL THESE CLINICAL TRIALS ARE COMING UP THIS YEAR, IT'S NICE TO HAVE MEASUREMENTS SUCH AS MRI DTI MEASUREMENTS AN EEG MEASUREMENTS TO FACILITATE THESE CLINICAL TRIALS AND PROVIDE BOTH RAPID OR LONGITUDINAL BIOMARKERS FOR TREATMENT. THIS IS THE LAST SLIDE I WILL SHOW YOU, THE I HAVE A DREAM -- OTHER THAN ACKNOWLEDGMENTS THE LAST SLIDE. I HAD A DREAM PORTION OF THE TALK. AND YOU CAN IMAGINE ZOOMENING TO AN ANGELMAN SYNDROME BRAIN AND LOOKING AT THE NEURONS AND THEY ARE KIND OF UNHEALTHY DUE TO LOSS OF UB 3A, WE DEPICTED THAT KIND OF GRAYISH AND THERE ARE UNHEALTHY BECAUSE THEY HAVE THIS MUTATED COPY OF UB 3A, THE OTHER COPY IS SILENT SO NOT EXPRESSING UB 3A. BUT THE DREAM IS WE ARE GOING TO TREAT WITH SMALL MOLECULE COMPOUND OR SOME OTHER TYPE OF TREATMENT THAT CAN COME IN LIKE A FLURRY OF SNOWFLAKES AND REACTIVATE DORMANT ALLELE UB 3A AND MAKE NEURONS HAPPY HEALTHY AND PINK AND TRY TO HELP THESE INDIVIDUALS WITH ANGELMAN SYNDROME. THAT'S REALLY WHAT OUR ULTIMATE GOAL IS. OF COURSE I DIDN'T GET HERE ALONE. THIS WORK WAS REALLY DRIVEN BY MEMBERS OF THE LAB INCLUDING SOME WHO LEFT LABS SUCH AS HM LEE, MIKE -- (INDISCERNIBLE) VERY MANY COLLABORATORS I ALWAYS LIKE TO GIVE A SHOUT OUT TO. WE WERE FORTUNATE TO HAVE THE FIRST ANGELMAN CLINIC IN THE UNITED STATES AT THE UNIVERSITY OF NORTH CAROLINA AND HEATHER AND MARK ARE MY MAIN COLLABORATORS THERE. MY OPINION -- THANKS TO MY FUNDING SOURCES SUCH AS ANGELMAN SYNDROME FOUNDATION, ANGELMAN SYNDROME ALLIANCE PARTICULARLY RELEVANT HERE, AND OF COURSE MY NIH FUNDING SOURCES. SO THANK YOU FOR YOUR TIME AND ATTENTION AND HAPPY TO ANSWER ANY QUESTIONS YOU MAY HAVE. [APPLAUSE] >> IF THERE'S MORE QUESTIONS YOU CAN USE THIS. >> THANK YOU FOR THAT TALK. I HAVE TWO QUESTIONS. ONE IS DO YOU HAPPEN TO KNOW THE LIKELIHOOD OF INDIVIDUALS WITH ANGELMAN SYNDROME TO DEVELOP NEURODEGENERATIVE DISEASES? ESPECIALLY THOSE THAT ARE RELATED TO ALLEGATION OF PROTEIN, THE OTHER END, HOW MANY OF THE SYMPTOMS OF ANGELMAN SYNDROME ARE RELATED TO DEVELOPMENTAL PROBLEMS? MEANING THE INABILITY TO RETRACT NEURONS AND OTHERWISE PROJECTIONS DURING DEVELOPMENT OPPOSED TO ONGOING PROCESS IN ADULT AND HAVE YOU ADDRESSED THIS IN YOUR BIOMARKERS? >> TWO GREAT QUESTIONS. THE FIRST QUESTION IS, BECAUSE THE GENETIC DIAGNOSIS ANGELMAN SYNDROME IS RELATIVELY RECENT, THE OLDEST INDIVIDUAL WITH ANGELMAN SYNDROME TYPICALLY DON'T HAVE GENETIC DIAGNOSIS. SO THERE ISN'T A LARGE POPULATION OLDER POPULATION STUDIED FOR DIFFERENCES IN NEURODEGENERATION FOR EXAMPLE. AS A LITTLE BIT OF SIDE NOTE, THERE'S RECENT STUDIES FROM JOE'S GROUP AND OTHERS THAT INDIVIDUALS WITH AUTISM ARE AT INCREASE RISK FOR GETTING PARKINSON'S. SO I THINK THIS IS SOMETHING TO BE MIND OF, ARE THESE INDIVIDUALS WITH -- MINDFUL OF, PARTICULARLY ONES THAT MIGHT AFFECT PROTEOSTAYSIS ARE YOU AT INCREASE RISK FOR NEURODEGENERATIVE DISORDER. WE DON'T KNOW THE ANSWER YET BUT IT'S SOMETHING THAT PARTICULARLY THE CLINICS AND PEOPLE STUDYING THIS DISORDER SHOULD KEEP AN EYE ON. NOW, THE NEXT QUESTION WAS WHAT ASPECT OF IT IS NEURODEVELOPMENTAL AND CIRCUIT-BASED. AND WHAT ASPECT OF IT IS DUE TO ONGOING DEFECT OF UB 3A. ONCE AGAIN I POINT TO THE WORK GENETICALLY REINSTATED UB 3A AT DIFFERENT POINTS OF DEVELOPMENT TO GET AT THIS QUESTION. SO WE KNOW INDIVIDUALS WITH -- SORRY I SHOULDN'T SAY INDIVIDUALS, MICE MODELING ANGELMAN SYNDROME HAVE DEFECTS IN SYNAPTIC PLASTICITY. THAT CAN BE RENNE -- CORRECTED BY REINSTATED UB 3A IN ATOLT HOOD SO THAT IS SOMETHING THAT'S PRETTY EXCITING BUT WE KNOW THAT OTHER ASPECTS SUCH AS THE CIRCUITRY CIRCUIT DEFECT UNDERLYING SEIZURES CAN NOT BE CORRECT BID REINSTATING UB 3A IN ADULTHOOD SO THERE SEEMS TO BE ONGOING ROLE FOR UB 3A AND NEURODEVELOPMENTAL CIRCUIT BASED ROLE FOR UB 3A SO ALL OUR EFFORTS WOULD BE TO TRY TO GET IN AS EARLY AS POSSIBLE BECAUSE THAT WOULD MAXIMIZE THE BENEFIT OF REINSTATEMENT OF UB 3A BUT IT IS STILL OUR BELIEF REINSTATEMENT IN HAY ADULTHOOD COULD HAVE SOME BENEFIT PARTICULARLY PERHAPS IN COGNITIVE DOMAIN GIVEN THESE RESTORATIONS DUE TO SYNAPTIC PLASTICITY IN THE MOUSE MODEL. WHOEVER GETS THE -- -- THE MAGIC BALL. >> SO WHAT IS THE MECHANISM OF CANNABIDIOL AND COULD IT BE SUB CORTICAL GABANERGIC NEURONS? >> IT'S A GREATS QUESTION. SO HOW IS CANNABIDIOL WORKING? WHAT IS ITS MECHANISM OF ACTION? THE SHORT ANSWER IS WE DON'T KNOW. WE REALLY WOULD LOVE TO START LOOKING AT THE CHANGES THAT ARE UNDERLYING THE AFFECTS. FROM A SLIGHTLY LONGER ANSWER WOULD BE GW IS A COMPANY THAT LICENSED CANNABIDIOL IN THE EPIDIALECTS OF TREATMENT OF GERVAIS SYNDROME, THEY HAVE A NICE DISCUSSION ON A WEB PAGE ABOUT THE SUSPECTED MECHANISM OF ACTION OF CANNABIDIOL. SO I WOULD REDIRECT YOU TO GW'S WEB PAGE, TALKING ABOUT POSSIBLE MECHANISMS OF ACTION OF CANNABIDIOL. BUT I THINK IT'S REALLY STILL REALLY UNKNOWN EXACTLY HOW IT'S ACTING. THAT'S GREAT QUESTION. LIKE MANY DRUGS WE JUST DON'T KNOW HOW IT'S WORKING. >> MANY OF THEM IN THIS PICTURE ACTUALLY GABAERGIC. >> YES. IN THIS PICTURE THEY ARE ALL GABAERGIC. THIS IS GENETICALLY DELETED UB 3A JUST FROM EXCITATORY NEURONS. SO THIS IS A PICTURE WHERE YOU ARE TRYING TO HIGHLIGHT THE INTERNEURONS EXPRESSING UB 3A. SO IN THE OTHER PICTURE UB 3A EXPRESSED IN EXCITATORY NEURONS AND INHIBITORY NEURONS. SO I CAN TELL YOU THIS MIGHT BE MORE INFORMATION THAN YOU WANT TO KNOW BUT IF YOU GENETICALLY DELETE UB 3A FROM EXCITATORY INHIBITORY NEURONS, IT ESTRAYSICALLY DIFFERENT EFFECTS. SO -- DRASTICALLY DIFFERENT EFFECTS SO YOU THINK AGE NEURONS EXCITATORY YOU DELETE THERE YOU'RE GOING TO DRIVE SEIZURE, NO, YOU'RE NOT DRIVING SEIZURE PHENOTYPE AT ALL FROM EXCITATORY NEURONS. DELETED FROM INHIBITOR NEURONS YOU ARE GETTING WORSE SEIZURE PHENOTYPE THAN IF DELETED EVERYWHERE SO YOU THROW THE EXCITATORY AND INHIBITOR NEURONS OUT OF WHACK IF YOU DELETE JUST INHIBITORY NEURONS YOU HAVE A WAY WORSE SEIZURE PHENOTYPE THAN IF YOU DELETED EVERYWHERE. SO WITHOUT DOUBT THERE'S IMPORTANT FOR UB 3A INHIBITORY NEURONS. >> JUST WONDERING ABOUT YOUR THOUGHTS ALONG THE GENE THERAPY APPROACH, BECAUSE AS YOU POINTED OUT THERE'S DOSE SENSITIVITY AND SIMILAR TREATIES SYNDROME, YOU HAVE TO GET IT , TO THE LET'S SYNDROME, YOU HAVE TO GET IT RIGHT. >> IN RHETT SYNDROME, THERE'S A PARTICULAR DANGER OF OVEREXPRESSION. PARTICULARLY IN THE MOUSE MODEL WHERE THEY ARE REINSTATING MECT OVEREXPRESSION CAN OOH EASILY LEAD TO DEATH OF THE MICE WITH A LITTLE OVEREXPRESSION OF MECP 2. THERE IS GREATER TOLERANCE OF THAT IN FOR UB 3A. WE HAVE BEEN REALLY INTERESTED IN THIS BECAUSE WE ARE TRYING TO DEVELOP GENE THERAPIES AND OBVIOUSLY OUR BIGGEST CONCERN IS WHETHER WE GET OVEREXPRESSION OF TOXICITY ASSOCIATED WITH THAT. SO TO LOOK AT THAT WE GENETICALLY ENGINEERED MICE THAT OVEREXPRESS DIFFERENT COPY NUMBERS OF UB 3A. SO THEY OVEREXPRESS ONE COPY, TWO COPY, NOW SIX COPIES OF UB 3A. WE ARE LOOKING AT THE CONSEQUENCES OF OVEREXPRESSION. AND WHAT THAT DOES. THE LONG AND SHORT, IT'S A SURPRISE HOW TOLERANT THEY ARE TO OVEREXPRESSION OF UB 3A. AND SO I THINK MANY OF THE PHENOTYPES THAT ARISE FROM OVEREXPRESSION ARE DRIVEN BY THE OTHER GENES THAT ARE OVEREXPRESSED AT THE SAME TIME. THE ONE HIT WE HAVE THAT THERE'S OVEREXPRESSION OF UB 3A ITSELF DAMAGING IS THIS ONE FAMILY, ONE ON FAMILY ABOUT FIVE INDIVIDUALS HAVE THIS MICRODUPLICATION OF UB 3A AND WE CERTAINLY HAVE SOME ISSUES WITH DEPRESSION, INTELLECT AND PSYCHOEFFECTIVE PHENOTYPES. BUT NOT NEARLY TO DEGREE WHICH THESE INDIVIDUALS HAVE A LARGE CHROMOSOME DUPLICATIONS. SO IT'S SOMETHING TO BE VERY MINDFUL OF. (OFF MIC) >> FOR THE TALK. FOR THE SMALL MOLECULES THERAPIES -- INTRATHREECAL INJECTIONS WERE YOU ABLE TO SEE AMELIORATION OR RESCUE OF LIKE MOTOR AND NEUROCOGNITIVE DEFICITS? >> SO GREAT QUESTION. AND YOU MIGHT BE SHOCKED AT THE ANSWER BUT WE HAVEN'T LOOKED AT BEHAVIORAL RECOVERY YET. THE REASON WE HAVEN'T LOOKED AT IT YET IS WE WANTED TO FIRST OPTIMIZE THE DELIVERY THAT WILL GET A BROAD BIODISTRIBUTION OF UNSILENCING AND THAT WE CAN INHIBIT EARLY ENOUGH. WE ARE THERE NOW WITH THIS NEW COMPOUND SO WE WANT TO THE A FEW -- WE STILL HAVE MORE TESTS TO DO LONGEVITY HOW LONG EFFECT LASTS BUT THEN AFTER THAT WE CAN GO WITH A REALLY NICE OPTIMIZE DOSING SCHEDULE AND DO THOSE BEHAVIORAL RECOVERY STUDIES. >> ALL RIGHT. THANK YOU. [APPLAUSE]