>> THANK YOU ALL FOR BEING HERE. WE'RE VERY EXCITED ABOUT THIS WORKSHOP. AND GETTING YOUR STRATEGY DEVELOPMENT EXPERTISE FOR WHAT NEEDS TO BE DONE IN THE FIELD OF GENE THERAPY. TO START WITH WE'RE GOING TO OPEN WITH NINA SCHOR, AND SO WE HAVE MULTIPLE LEADERSHIP, WALTER KOROSHETZ IS COMING, HE WILL BE SITTING NEXT TO NINA. WE HAVE DR. MIR TIMAS, OFFICE OF TRANSLATIONAL RESEARCH, LYNN JAKEMAN IS OUR DIRECTOR OF THE DIVISION OF NEUROSCIENCE. SO, NINA WAS -- BEFORE SHE CAME TO NINDS WAS CHAIR OF THE DEPARTMENT OF PEDIATRICS AND PEDIATRICIAN IN CHIEF AT THE UNIVERSITY OF ROCHESTER, SHE'S A PEDIATRIC NEUROLOGIST, AND ALSO HAS HER PhD in medical biochemistry, she came to NINDS in Januar of 2018, and she has just been a fantastic addition to the team. SHE'S VERY ENERGETIC, VERY INTELLIGENT, AND BRINGS A LOT OF ENERGY AND DYNAMICS TO THE GROUP. >> THANKS, JILL. I PROBABLY WILL SPEND THE REST OF THE CONFERENCE TRYING TO LIVE UP TO THE WONDERFUL INTRODUCTION. WELCOME, EVERYBODY. WE'RE DELIGHTED THAT YOU'RE HERE. WE FEEL LIKE THIS CONFERENCE IS IN SOME WAYS A LONG TIME IN COMING, AND WE'RE VERY, VERY EXCITED THAT TODAY HAS ARRIVED. THIS IS GOING TO BE A VIGOROUS DIALOGUE, AND WELCOME AS WELL TO THOSE WHO ARE WATCHING BY VIDEOCAST. WE HAVE BEEN EXTREMELY ENCOURAGED BY THE LEVEL OF INTEREST AND BY THE NUMBER OF PEOPLE WHO REALLY WANT TO PARTICIPATE IN THIS DISCUSSION. MY JOB THIS MORNING IS JUST TO SET THE STAGE FOR WHAT IS TO COME, TO REALLY CHARGE THE GROUP WITH WHAT WE HOPE WILL TRANSPIRE AND TO GIVE YOU AN ENVIRONMENTAL SCAN OF WHERE WE AT NINDS ARE IN THE MIDST OF THIS NATIONAL AND INTERNATIONAL EFFORT TO MAKE GENE THERAPY AVAILABLE, ACCESSIBLE, EFFECTIVE, SO WHAT DO WE MEAN BY GENE THERAPY IN THE CONTEXT OF THIS DISCUSSION TODAY? WELL, THIS SLIDE IS HERE REALLY TO GIVE YOU A SENSE OF THE FACT THAT WE MEAN NOT ONLY GENE REPLACEMENT OR GENE CORRECTION AS WILL BE DOWN THE ROAD, BUT ALSO siRNA THERAPY, WE PUT THIS MANIPULATION OR MODIFICATION OF GENE PRODUCTS REALLY IS WHAT THIS CONFERENCE IS ABOUT. SO WHAT IS IT THAT NINDS CURRENTLY DOES IN THIS SPACE? WE CERTAINLY INVEST IN DIFFERENT STAGES OF RESEARCH, AND I'LL SHOW YOU IN A MOMENT SOME OF THE MECHANISMS THAT WE USE TO DO THIS, BUT THIS WILL COME AS NO SURPRISE. THESE ARE VERY CONVENTIONAL MECHANISMS THAT WE HAVE TRIED TO BRING TO BEAR ON CURRENT PROBLEMS. SO, WE INVEST ACROSS THE RESEARCH SPECTRUM, AND I WOULD VENTURA GUESS THAT THIS ROOM CONTAINS PEOPLE AT EVERY PIECE IN THE CONTINUUM. WE INVEST IN BASIC RESEARCH TO LOOK AT MECHANISMS, AND TO LOOK AT TARGETS. WE INVEST IN TRANSLATIONAL RESEARCH TO SAY HOW DO WE TAKE THAT AND BRING IT TO THE CLINIC, AND THEN IN THE CLINICAL TRIALS THAT ACTUALLY TEST THOSE TECHNOLOGIES. THE PARENT R01, THIS COIN IS HERE, THE COIN OF THE REALM. TRADITIONALLY, AND I AM A TRADITIONALIST, BEING A SCIENTIST OF A CERTAIN AGE, TRADITIONALLY THE R01 HAS BEEN THE WAY IN WHICH WE HAVE DONE BUSINESS AT MUCH OF NIH. BUT I WILL SAY THAT IN THE CONTEXT OF SOMETHING AS COMPLEX AS MULTI-FACETED AS SOMETIMES INDIVIDUALISTIC AND SOMETIMES PROTOCOLISTIC AS GENE THERAPY, WE REALIZE AT NIH THAT WE ARE GOING TO HAVE TO DEVELOP MECHANISMS THAT REALLY CALL UPON EXPERTISE FROM MANY DIFFERENT SECTORS, AND BRING THEM TOGETHER IN A WAY THAT MAKES A WHOLE THAT'S GREATER THAN THE SUM OF ITS PARTS. WE HAVE BEGUN SOME OF THAT TRANSLATION AND SOME OF THAT MOVE INTO SECTORS THAT TRADITIONALLY WERE NOT INVOLVED IN NIH-FUNDED RESEARCH BY BRINGING INDUSTRY INTO THE EQUATION, BY BRINGING THINGS ALONG THE TRANSLATIONAL SPECTRUM THROUGH GRANT MECHANISMS THAT FUND SEQUENTIALLY EACH OF THE STAGES OF THE DEVELOPMENT OF SOMETHING WE HOPE TO BRING EVENTUALLY TO THE CLINIC AND ACTUALLY ESTABLISH MILESTONES SO WE CAN TRACK AND TROUBLESHOOT WITH THE INVESTIGATORS, THAT'S OUR IGNITE AND CREATE BIO PROGRAMS, TO BRING THINGS BASED ON FUNDAMENTAL SCIENTIFIC PRINCIPLES AND GET THEM TO THE CLINIC. WE CERTAINLY FUND CLINICAL RESEARCH AS WELL, AND REALIZING THAT THERE OFTEN ARE PRECURSORS TO PERFORMING A CLINICAL TRIAL THAT THEMSELVES NEED FUNDING, DEVELOPING BENCHMARKS AND METRICS AND OUTCOME MEASURES AND PROCESS MEASURES, DEVELOPING COHORTS OF PATIENTS, CHARTING THE NATURAL HISTORY OF A DISEASE BEFORE YOU ASK YOURSELF IF A THERAPY MODIFIES IT, ALL OF THESE THINGS REQUIRE FUNDING TO DEVELOP AND SO WE FUND CLINICAL TRIAL READINESS STUDIES AND BIOMARKERS PROGRAMS, IN ADDITION TO THE CLINICAL TRIALS THEMSELVES. SO, WHAT COULD WE DO THAT WE'RE NOT DOING NOW? WE ARE IN THE PROCESS, AND "WE" IS NOT JUST NINDS, IT INCLUDES A VERY BROAD SPECTRUM OF THE INSTITUTES REPRESENTED AT NIH, OF EXPLORING THE POTENTIAL TO FORM ONE OF OUR ACCELERATED MEDICINES PARTNERSHIPS, DEDICATED TO GENE THERAPY, TO REALLY SAY HOW DO WE GET ALL THE IMPORTANT STAKEHOLDERS AND PLAYERS AT THE TABLE, AT THE SAME TIME, SPEAKING THE SAME SCIENTIFIC LANGUAGE TO BRING THIS TO FRUITION. BUT FOR THE TIME THAT WE ARE TOGETHER AT THIS CONFERENCE TODAY AND TOMORROW WE HOPE WE'LL ARRIVE AT SOME FORM OF MARCHING ORDERS FOR NINDS IN PARTICULAR AND NIH IN GENERAL, WITH REGARD TO WHAT IT IS THAT WE CAN DO TO FACILITATE THE DEVELOPMENT OF THESE THERAPIES AND THE BRINGING OF THESE THERAPIES TO THE PATIENTS AND THE FAMILIES WHO NEED THEM. WHAT IS IT THAT WE COULD CREATE, FACILITATE, FUND, DEVELOP, THAT WILL HELP THE GENE THERAPY COMMUNITY IN ITS BROADEST ITERATION, BRING THIS TO BEAR IN A WAY THAT IS EFFECTIVE AND ACCESSIBLE TO OUR PATIENTS. AND SO THAT VERY SMALL ORDER FOR A COUPLE OF DAYS WORK IS WHAT I HOPE THIS CONFERENCE WILL LAUNCH AS A DIALOGUE. WHAT IS IT THAT YOU NEED FROM US AND HOW IS IT THAT WE CAN WORK WITH YOU AS PART OF THE TEAM TO BRING THIS TO FRUITION. SO, AGAIN, WELCOME TO ALL OF YOU. I KNOW FIRST HAND HOW HARD IT IS NOT ONLY TO TRAVEL BUT ALSO TO TAKE THE TIME AWAY FROM WHAT YOU DO EVERY DAY TO HELP US OUT IN THIS AND TO WORK WITH US, SO WE ARE VERY, VERY GRATEFUL. AND WE TRULY THINK OF OURSELVES AS PARTNERS ON THIS VERY LARGE, VERY DIVERSE TEAM. SO THANK YOU VERY MUCH. >> GOOD MORNING. I'M CHRIS BOSHOFF AT THE DIVISION OF TRANSLATIONAL RESEARCH, AND I WANTED TO TAKE A MINUTE OR TWO TO INTRODUCE MYSELF AND ALSO TO WELCOME AND THANK YOU FOR BEING HERE TODAY. IT SEEMS SOMETIMES THERE'S A GENE THERAPY MEETING ALMOST EVERY WEEK NOW, SOMEWHERE IN THE WORLD WHERE CAN GO AND YOU DECIDED TO BE AT THIS ONE AND WE'RE REALLY GRATEFUL FOR THAT. FIRST OF ALL, YOU KNOW, THINKING BACK HOW THIS DEVELOPED AND WHERE WE ARE NOW, I WANTED TO PUT A FEW THINGS IN PERSPECTIVE BY LOOKING BACK ON SOME PRIOR MEETINGS THAT WE WERE PART OF THAT HELPED US A GREAT DEAL IN TERMS OF DESIGN OF THIS MEETING, IN TERMS OF FORMAT OF THE MEETING AND CONTENT OF THE MEETING, SO I WANTED TO HIGHLIGHT THREE OF THOSE MEETINGS THE FIRST POINT I WANTED TO MAKE IS THAT THIS IS NOT AN ISOLATED MEETING. THEN AGAIN, NO MEETING IS REALLY ISOLATED. THERE'S ALWAYS LINES ON PAST DISCUSSION, ON ONE ON ONE DISCUSSION OR PART OF A BIG CONFERENCE. AND THIS MEETING IS NO EXCEPTION. THERE WAS A VERY CONSCIOUS AND DELIBERATE EFFORT TO TAKE POINTS FROM PREVIOUS MEETINGS AND SEE CAN WE ADVANCE THE DISCUSSION, SO WE DON'T GET INTO THE PERPETUAL RECITING OF IDEAS, THE SAME ARGUMENTS OVER AND OVER AGAIN, BUT ADVANCE THE CONVERSATION TO THE POINT WHERE WE CANNOT JUST REACH CONSENSUS ON WHAT THE GAPS ARE, WHAT THE ISSUES ARE, BUT MOVE ALONG AND SAY WHAT CAN WE DO ABOUT IT, AND WHAT WILL IT LOOK LIKE IF WE ADDRESS THESE ISSUES AND MORE IMPORTANTLY IF WE STRETCH EVEN FURTHERMORE IN WHAT KIND OF COLLABORATION MODEL, WHAT KIND OF STRUCTURE, WITH THOSE QUESTIONS BE ADDRESSED, TO MAXIMIZE IMPACT, MINIMIZE REDUNDANCIES. SO, THE FIRST OF THESE MEETINGS WAS BY OUR COLLEAGUES AT THE NATIONAL CENTER FOR ADVANCEMENT TRANSLATIONAL SCIENCES, NCATS, THEY HAD A MEETING JUST OVER A YEAR AGO ON MAIN CAMPUS WHERE THEY ADDRESED VARIOUS TOPICS, AND THEY INVITED SPEAKERS AND PANELISTS TO DISCUSS FROM ANYTHING FROM PRE-CLINICAL DEVELOPMENT, MOVING INTO THE CLINIC, MANUFACTURING ISSUES, JUST ALWAYS THERE, HOW CAN WE WORK TOGETHER IN TERMS OF PARTNERSHIPS AND PATIENT ACCESS. IT WAS FOLLOWED UP RECENTLY IN JUNE WITH A FOCUS ON SPECIFIC ISSUE, THAT WAS THE CNS IMMUNOGENICITY CONSIDERATIONS FOR AAV-MEDIATED GENE THERAPY, INCLUDING HOW DOES CNS SPECIFIC IMMUNE RESPONSE DIFFER FOR DIFFERENT ROUTES OF CNS-SPECIFIC ADMINISTRATION, THEY LOOKED AT CAN WE STANDARDIZE SOME OF THESE ASSAYS AND USE BIOMARKERS FOR IMMUNE RESPONSES AND TOLERANCE INDUCTION. LOOKING AT WHAT CAN WE DO TO IMPROVE PREDICTIVE ANIMAL MODELS, LARGE ANIMAL MODELS, THEY LOOKED AT STRATEGIES TO DEVELOP BETTER AAV AND NON-VITAL DELIVERY METHODS, AND HOW CAN WE IMPROVE DURABILITY OF THE EFFICACY. THEY WILL CONTINUE HAVING THESE MEETINGS, WHICH IS A VERY GOOD THING, THE NEXT ONE IS GOING TO BE EARLY NEXT YEAR IN JANUARY I BELIEVE, FOCUSED ON MANUFACTURING. THE SECOND MEETING I WANT TO HIGHLIGHT WAS ALSO MEETING NINDS AND NCATS WERE INVOLVED IN, BY THE UNITED STATES PHARMACOPEIA, USP, THEY LOOK AT THINGS FROM THE EARLY STAGE, FROM RAW MATERIALS COMING IN TO FINAL PRODUCT RELEASE, ACCESS TO PATIENTS, AND THEY LOOK AT ANALYTICAL AND VALIDATION CHALLENGES, HOW WE CAN APPROVE ASSURANCES TO DEVELOPERS AND REGULATORS. A FOLLOW-UP WORKSHOP IN FEBRUARY 2020, YOU CAN TAKE A LOOK AT THEIR WEBSITE. I JUST WANTED TO ALSO ADD I BELIEVE ORGANIZATIONS SUCH AS USP, STANDARDS COOPERATING BODY, THEY WILL PLAY A KEY ROLE IN TRANSLATING WHAT WE WILL BE TALKING ABOUT INTO MAINSTREAM MEDICAL PRACTICE OVER THE NEXT YEARS AND DECADES TO COME. LAST BUT NOT LEAST, THE NATIONAL ACADEMIES WORKSHOP HELD IN APRIL THIS YEAR. IT WAS AN EXCELLENT MEETING, THEY TOUCHED ON A WHOLE LOT OF SUBJECTS, VERY DISCUSSION-BASED, JUST LIKE WE PLAN TO DO TODAY AND TOMORROW. THEY LOOKED AT THE LANDSCAPE DIFFERENT APPROACHES, OPPORTUNITIES, AND WHAT CAN WE DO TO ENABLE TRANSLATION. PRE-CLINICAL MODELS, DELIVERY, DELIVERY, DELIVERY. THIS IS NOT A TYPO. I'M NOT STUTTERING EITHER. IT WAS EMPHASIZED OVER AND OVER AGAIN DELIVERY IS JUST FRONT AND CENTER TO ALL OF THIS. AND WE HAVE TO ADDRESS THIS. AND THEN MANUFACTURING ISSUES, SCALE, COST, CAPACITY, WE HOPE TO ADDRESS THAT AS WELL TODAY. PATIENT ENGAGEMENT WAS ALSO DISCUSSED. AND THEN FUTURE DIRECTIONS LIKE NEW EMERGING TECHNOLOGIES, THINKING ABOUT GENOME EDITING AND HOW THAT IS EVOLVING, CAN BENEFIT FROM WHAT WE'VE LEARNED OVER THE PAST 20 YEARS IN GENE THERAPY. AND PERSONALIZED MEDICINE OF COURSE, THINKING ABOUT PROJECTS WHERE YOU HAVE SMALL PATIENT POPULATIONS, SOMETIMES JUST AN N EQUALS 1 KIND OF SITUATION. THEY ALSO LOOKED AT POTENTIAL FOR COLLABORATIONS, PRE-COMPETITIVE COLLABORATIONS, WE REALIZE COLLABORATION CAN TAKE ON MANY SHAPES AND FORMS, THIS IS HOPEFULLY SOMETHING WE CAN ALSO ADDRESS OVER THE NEXT DAY OR TWO. THE SUMMARY OF THIS MEETING WAS PUBLISHED JUST A FEW DAYS AGO, YOU CAN TAKE A LOOK AT THAT. I WANT TO END HERE, I HOPE THIS GIVES YOU SOME PERSPECTIVE WHY THIS MEETING IS IN THIS WAY AND I'LL HAND OFF NOW TO JILL MORRIS WHO WILL BE LOOKING FORWARD AND GIVE YOU SOME CONTEXT ON WHAT WE HOPE TO ACCOMPLISH WITH THIS MEETING. >> SO, I JUST WANT TO TALK A LITTLE BIT ABOUT WHAT THE EXPECTATIONS AND WHAT WE HOPE TO GET OUT OF THIS WORKSHOP. SO FIRST I WANTED TO MENTION ORGANIZING COMMITTEE. SO, BEV DAVIDSON AND GUANGPING GAO ARE THE CO-CHAIRS, I WANT TO THANK THEM FOR THE EFFORTS THEY PUT IN HELPING US PUT TOGETHER THIS WORKSHOP. IT TAKES US CLOSES TO A YEAR TO ORGANIZE THESE EVENTS. AND THEY'VE HAD MANY, MANY PHONE CALLS WITH US. AND THEN I WANT TO RECOGNIZE THE NINDS STAFF THAT WERE ON TH ORGANIZING COMMITTEE, IN PARTICULAR WE WANT TO ACKNOWLEDGE LING WONG, WE WOULD NOT BE HEAR TODAY IF NOT FOR LING. SHE'S BEYOND FANTASTIC AT, WELL, KEEPING ME IN LINE AND THEN JUST, YOU KNOW, ON TOP OF EVERY LITTLE BIT OF ORGANIZATION AND THE SCIENCE AS WELL, SHE WAS INSTRUMENTAL IN GETTING THIS WORKSHOP UNDERWAY. SO, WHAT WAS THE BASIS FOR THIS WORKSHOP? AT NINDS, WE SAW A LOT OF ACTIVITY IN THE GENE THERAPY SPACE. AND THE QUESTION WAS ASKED ARE WE DOING EVERYTHING WE SHOULD BE DOING IN ORDER TO FACILITATE GENE THERAPY. AND WE KNOW THAT THERE HAS BEEN AN INCREASE IN GENE THERAPY INDS SUBMITTED TO THE FDA, AND THIS IS A QUOTE FROM THE FDA COMMISSIONER WHERE HE MENTIONS THAT THERE WERE -- THEY EXPECT TO RECEIVE MORE THAN 200 INDs PER YEAR IN 2020, FOR GENE THERAPY AND BY 2025 THAT THEY EXPECT APPROVING 10 TO 20 CELL AND GENE THERAPY PRODUCTS A YEAR. WE WANTED TO FOCUS THIS MEETING ON CNS, BRAIN AND SPINAL CORD, AND CNS PROVIDES VERY UNIQUE CHALLENGES AS ALL OF YOU KNOW. SO GETTING APPROPRIATE DISTRIBUTION THROUGHOUT THE BRAIN, METHOD OF DELIVERY, YOU KNOW, GETTING APPROPRIATE CELL TYPE, EVEN APPROPRIATE REGION OF THE BRAIN. WE WANT TO ASK HOW CAN WE ADDRESS THESE CHALLENGES, AND HOW CAN NIH FACILITATE THESE NEXT GENERATION STRATEGIES. AND AT THE OUTCOME OF THE WORKSHOP, ONE OF THE THINGS WE'LL BE DOING IS PUTTING TOGETHER A WHITE PAPER. WE WORKED WITH THE CHAIRS AND CAME UP WITH SESSIONS AND TALKED ABOUT WHERE THE BIGGEST GAPS WERE SO THE SESSION 1 IS ON CONTROL OF LEVEL AND LOCATION, 2 IMPROVING DELIVERY AND DISTRIBUTION, 3 ENHANCING MODELS OF MANUFACTURING, AND THEN TOMORROW'S SESSION IS ON IMPACTING PATIENTS. AND HOW WE ORGANIZED THIS, WE SELECTED CHAIRS FOR EACH OF THESE SESSIONS, AND STARTING IN AUGUST WE HAD CALLS WITH THE CHAIRS. WE CALLED THEM BY WHAT FDA DOES, CALLED THEM PRE-PRE-CALLS. WITH THE CHAIRS WE WENT OVER WHAT WE WANTED OUT OF THIS MEETING AND IT WOULD BE VERY FOCUSED ON DISCUSSION AND STRATEGY DEVELOPMENT. THEN WE HAD PRE-CALLS. WITH PEOPLE WHO ARE IN THIS ROOM, WE PUT YOU IN DIFFERENT PANELS, AND YOU WERE ON THOSE PRE-PRE-CALLS AND WHAT THE PURPOSE -- PRE-CALLS. THE PURPOSE, WE WANTED TO IDENTIFY WHAT THE GAPS WERE AHEAD OF TIME, WHAT ARE THE BIGGEST BE ON OBSTACLES SO WE WOULD BE READY TO TALK ABOUT STRATEGIES WHEN WE CAME TO THE MEETING SO WE WOULDN'T TAKE TIME ACTIVITYING GAPS AND RIGHT AWAY WE'RE WORKING ON STRATEGY DEVELOPMENT. SO THE CHAIRS PUT TOGETHER SLIDES THAT WILL GUIDE THE DISCUSSION DURING THOSE SESSIONS SO THEY WILL START WITH THE BRIEF DISCUSSION, WITH THE SLIDES. THEN WE GO RIGHT INTO A ROUNDTABLE FORMAT OF HEAVY STRATEGY DEVELOPMENT AND DISCUSSION, AND EVERYONE IN THE ROOM SHOULD BE PARTICIPATING. FOR THOSE CHAIRS, WE HAVE THE MIC FOR YOU TO GET UP. WE WELCOME ALL COMMENTS. WE WILL ALSO BE MONITORING, FOR THOSE ON THE VIDEOCAST, WE'LL BE MONITORING THE TWITTER AND THE E-MAIL THAT IS AVAILABLE SO THAT WE CAN INCORPORATE YOUR COMMENTS AS WELL. SO, FOR THE STRATEGY DEVELOP. WE WANT TO THINK ABOUT HIGH LEVEL BIG PICTURE THINKING ACROSS DISEASES SO NOT TO GET BOGGED DOWN INTO ONE SPECIFIC. COME AND AND RARE, INCLUDING ASOs AN NON-VIRAL VECTORS, HOW THE COMMUNITY CAN WORK TOGETHER SO INDUSTRY, ACADEMIA AND NON-PROFITS. THIS IS YOUR CHANCE TO TELL US WHAT WE NEED TO KNOW, AS THE NIH. SO NOT ABOUT THE PAYLINE. WHAT WE NEED TO KNOW ABOUT WHAT THE OBSTACLES ARE IN GENE THERAPY, AND -- YES. THEN WE HAVE SESSION 5. IN SESSION 5 THE CHAIRS WILL GET UP AND SUMMARIZE THE BIGGEST RESULTS FROM THEIR SESSIONS. AND THEN WHAT WE WANT TO HAVE IS THINK ABOUT THESE QUESTIONS, SO THINK ABOUT THESE QUESTIONS NOW, WHILE WE'RE HAVING DISCUSSIONS, AND THEN DURING SESSION 5 WE'LL HOPEFULLY BE ABLE TO ANSWER A LOT OF THESE QUESTIONS. WHAT COULD THE RESEARCH FIELD, NINDS, AND/OR NIH DO, WHAT ARE THE GAPS, WHAT ROAD BLOCKS ARE IMMEDIATELY FEASIBLY ADDRESSABLE, REALISTIC GOALS, HIGHEST PRIORITY AND APPROPRIATE TIMELINE. WE WANT THE DISCUSSION TO CONTINUE. SO, AFTER THIS WORKSHOP WE DON'T WANT IT TO END THERE. ON OUR END WE WILL BE WORKING TO MOVE THE FIELD FORWARD. YOU CAN CONTINUE TO CONTACT US. YOU CAN EITHER CONTACT CHRIS AND I DIRECTLY OR SEND IT TO THE GENE THERAPY E-MAIL THAT WE SET UP. AND OF COURSE I MENTIONED THE WHITE PAPER. SO, WE'RE GOING TO GET STARTED. NEXT WE'LL HAVE A TALK FROM BEV DAVIDSON. >> I TOO WANT TO THANK LING FOR HER PATIENCE AND CREATIVITY IN HERDING A BUNCH OF CATS, GETTING US ALL ON THE PHONE CALLS, REPEATEDLY, KEEPING US ON TASK. SO, I'M HERE TO ESSENTIALLY PRESENT TO YOU WHERE WE LEFT OFF FROM THESE OTHER WORKSHOPS THAT CHRIS MENTIONED, IN A BIT MORE DETAIL SO WE, AGAIN, DON'T HAVE TO REHASH THESE BUT WE CAN MOVE STRAIGHT INTO STRATEGY DEVELOPMENT AROUND THESE CHALLENGES. SO, AS CHRIS MENTIONED, THERE WAS A NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF MEDICINE WORKSHOP, SOME OF US WERE PART OF THAT WORKSHOP IN THE SPRING THAT REALLY SUMMARIZED IN BULLET POINTS HERE THESE ARE OBJECTIVES OF THE WORKSHOP, WHERE WE DREW MANY OF OUR CHALLENGES IN ADDITION TO THE MEETINGS THAT CHRIS MENTIONED. SO, WHAT IS THE CURRENT LANDSCAPE AS WE LEFT THE SPRING AND SUMMER WITH REGARDS TO ANIMAL MODELS? YOU'LL HEAR SOME OF THESE AGAIN, THE QUESTIONS AROUND DO THESE MODELS ACTUALLY MIMIC THE STATUS OF THE PATIENTS WHEN THOSE PATIENTS ENTER INTO THE TRIALS. THINK ABOUT WHERE YOU INTERCEDE WITH YOUR GENE THERAPY IN YOUR MOUSE OR YOUR DOG OR OTHER ANIMALS, TO REPRESENT DISEASE AND IS THAT THE STAGE WE GO INTO DO LARGE ANIMAL MODELS SOME OF WHICH ARE AVAILABLE TO US DO THEY HELP US BETTER DEFINE DOSING STRATEGY AND I THINK SOMETHING MANY OF US ARE AWARE IN THE FIELD ARE THAT THE ANIMAL MODELS MAY GIVE US SOME INDICATION OF WHAT'S GOING ON BUT IF WE USE NORMAL ANIMALS IT MAY NOT GIVE US AN INDICATION OF THE GENE THERAPY EFFICACIES THAT MAY EXIST IN A DISEASE STATE. NON-HUMAN PRIMATES ARE THEY BETTER PREDICTORS OF SAFE IN HUMANS, IT'S DIFFICULT WHEN YOU THINK ABOUT SOME OF THE GENE THERAPIES THAT ARE ENCOMPASSED IN THE WORKSHOP. ASOs, AGAINST HUMAN GENE, siRNAs AGAINST A HUMAN GENE, EDITING STRATEGIES TARGETING HUMAN GENES. AND HOW DOES THE ANIMAL MODEL HELP US THERE IF IT DOESN'T ITSELF HAVE THE SAME HUMAN SEQUENCES. WILL THEY ALSO PREDICT THE DURABILITY OF THE EFFECT? OUR ANIMAL MODELS HAVE DIFFERENT IMMUNE SYSTEMS THAN HUMANS. HOW CAN WE BETTER GAIN MORE KNOWLEDGE IN AGGREGATE FROM TRIALS HAPPENING IN HUMANS TO BETTER UNDERSTAND AND PREDICT WHAT WE MAY DO IN THE FUTURE. WHAT CAN WE DO IN ABSENCE OF ANIMAL MODELS MAL AND IS IT REASONABLE. WE KNOW FOR THOSE IN THE RARE DISEASE SPACE NATURAL HISTORY STUDIES ARE JUST OF CRITICAL IMPORTANCE. BUT THERE'S A LOT OF VARIABILITY IN THESE RARE AND ULTRA RARE DISORDERS, AND WE NEED TO OF COURSE BE VERY AWARE OF THAT WHEN YOU SET UP TRIAL DESIGN. AND HOW DO WE DEFINE AN ACCEPTABLE CLINICALLY MEANINGFUL CHANGE IN THESE TRIALS? AND SHOULD WE CONSIDER MOVING FROM SUBJECTIVE RATING SCALES TO TECHNOLOGY DRIVEN ENDPOINTS. WE'RE BECOMING BETTER AND DEVICES ANDS WEARABLES AND OTHER LESS SUBJECTIVE TOOLS TO RATE A SUBJECT'S -- THE IMPACT ON THE SUBJECT OF THE INTERVENTION. WILL MULTI ENDPOINT DESIGN HELP IN IN THE HISTORIES? FOR MOST OF US THIS IS COMMON SENSE, FOR CNS DISORDERS EARLY TREATMENT IS ALWAYS BETTER, IF YOU CAN INTERCEDE BEFORE THERE'S ANY NEURODEGENERATION, THE IMPACT WILL BE MORE PROFOUND. BUT HOW CAN WE ADVANCE THIS OBJECTIVE, ESPECIALLY WHEN THE TIME TO AN EFFECT IS MANY YEARS? HOW CAN WE SUPPORT GOOD SCIENCE AND GOOD TRIAL DESIGN OVER MANY, MANY YEARS TO REACH A CLINICALLY MEANINGFUL ENDPOINT? WHAT'S THE MINIMAL THRESHOLD FOR CLINICAL TRIAL READINESS AND DO WE NEED MORE SO GROUPS CAN ADVANCE INTO THERAPIES? THERE'S THE ISSUE OF COST. WHO IS GOING TO PAY FOR THESE YEARS-LONG TRIALS? AND OF COURSE SOMETHING THAT NONE OF US KNOW THE ANSWER TO BUT WE'D LIKE A STRATEGY HOW TO FIGURE THIS OUT, ARE THESE FAILURES DUE TO INADEQUATE MODELS, INADEQUATE TARGETING IN THE PATIENT, LACK OF DURABILITY OF THE EFFECT, OR EVEN MISINTERPRETATION OF CLINICAL DATA, PARTICULARLY FOR OPEN LABEL TRIALS WHERE EVERYBODY KNOWS WHAT EVERYBODY GOT AND WE DON'T REALLY HAVE ANY INFERENCE ON WHAT PLACEBO EFFECT MAY BE. I THINK THIS IS SOMETHING FOR THOSE THAT WORK IN THE RARE DISEASE SPACE THINK ABOUT ALL THE TIME, AND BRINGS UP CERTAIN ETHICAL CONSIDERATIONS AS WELL, AND HOW DO WE PRIORITIZE COMPETING TRIALS FOR RARE DISEASES WHERE THERE'S REALLY A LIMITED POOL OF SUBJECTS, AND AGAIN THE ETHICS OF PUTTING SOMEBODY IN A TRIAL THAT WE THINK MAYBE ISN'T AS GOOD AS ANOTHER TRIAL THAT'S ON THE HORIZON. s WHAT ABOUT ETHICS OF PATIENT SELECTION AND EXCLUSION FOR CLINICAL TRIALS? ESPECIALLY FOR FAMILY SUPPORTED TRIALS. HOW DO WE KEEP FAMILIES ENGAGE AND INTERESTED BUT MAKE SURE THE BES SCIENCE IS DONE MOVING FORWARD? THE FOCUS TO DATE HAS BEEN ON MONOGENIC DISORDERS, WHAT ABOUT COMPLEX DISORDERS, ARE THERE CERTAIN DEBILITATING ASPECTS IN THESE DISORDERS THAT WE SHOULD CONSIDER MOVING FORWARD ON, TACKLING FOR CLINICALLY MEANINGFUL ENDPOINT? AND FINALLY, ONCE WE SOLVED ALL CNS DISORDERS WITH GENE-BASED MEDICINES, WHAT ABOUT THE PERIPHERAL DISEASE? AND AS WE KEEP PATIENTS ALIVE LONGER AND HAVE THEM LIVE MEANINGFUL LIVES, WITH THE DISEASE, THEN WE HAVE TO WORRY ABOUT SOME OF THE PERIPHERAL MANIFESTATIONS, WOULD WE BE ABLE TO GO IN WITH A SIMILAR KIND OF THERAPY OR DO WE NEED TO THINK ABOUT TREATING THE WHOLE BODY ALL AT ONCE. SO THINKING BEYOND NOT ONLY ABOUT AAV BUT ALSO CAN REGULATORS -- THIS IS A CRITICAL QUESTION FOR THOSE OF US IN THE ACADEMIC SPACE, CAN REGULATORS HELP US ADVANCE THERAPIES THAT REQUIRE VERY LITTLE DRUG? WE MAKE ONE MANUFACTURING PRODUCT, IT'S ENOUGH UNTIL MOST OF US IN THIS ROOM HAVE LONG PASSED BUT REGULATORS WILL REQUIRE US TO MAKE ADDITIONAL LOTS, AND CAN WE COME UP WITH NOT JUST ONE-SIZE-FITS-ALL BUT SOMETHING THAT MAKES SENSE FOR THE ULTRA ORPHAN DISEASES? LENTIVIRUSES HAVE SHOWN A LOT OF PROMISE IN CNS DISEASES, SHOULD WE WORK HARDER TO CONDITIONS AND EXPAND THIS APPROACH, HOW BEST TO APPLY IT, FOR OTHER VIRAL VECTORS AS WELL. LENTIVIRUS, THIS WOULD MITIGATE REDOSING ISSUES. OF COURSE, WE'LL HAVE TO CONSIDER THE FACT THAT LENTIVIRUSES INTEGRATE AND WE NEED BETTER TOOLS FOR NON-INTEGRATIVE LENTIVIRUS VECTORS. NON-VIRAL APPROACHES HAVE BEEN ADVANCING AS siRNA SPACE HAS BEEN ADVANCING AND HOW CAN WE CAPITALIZE ON THIS FOR GENE THERAPY, LIPID NANOPARTICLES AND OTHER TROJAN HORSE MOIETIES. MAYBE THESE WOULD BE MORE APPROPRIATE FOR GENE EDITING, WE DON'T HAVE TO WORRY ABOUT LONG-TERM EXPRESSION OF EDITING MACHINERY. IT ALSO PROVIDES OPPORTUNITIES FOR REDOSING TO SITES THAT WOULD BE MISSED WITH ONE DELIVERY STRATEGY OVER ANOTHER. WE MAY ALSO BE BETTER ABLE TO TAKE ADVANTAGE OF ADVANCED DELIVERY TECHNIQUES TARGETED DELIVERY USING GUIDEDMAN, OR INFUSING CAPSIDS INTO THE BODY. AND THEN FINALLY WHEN DOES A NON-GENE THERAPY APPROACH MAKE MORE SENSE? I THINK ETHICALLY WE NEED TO CONSIDER EVERYTHING IS NOT A NAIL, EVEN THOUGH WE'RE ALL HAMMERS. AND, AGAIN, WE WANT TO THINK ABOUT WHERE DO SMALL MOLECULES MAKE SENSE AND CAN WE ADVANCE THAT EITHER IN COMBINATION WITH GENE THERAPIES OR SHOULD WE THINK ABOUT COLLABORATING WITH PHARMA WITH OUR MODELS AND THINGS LIKE THAT TO ADVANCE DRUGS WHILE WE DEVELOP THE BEST GENE THERAPIES. AND SO THESE ARE SOME OF THE CHALLENGES YOU'RE GOING TO SEE BROUGHT UP AGAIN IN THE SESSION SLIDES. AND AGAIN AS JILL MENTIONED, WE WANT TO COME OUT OF EVERY SESSION WITH FOUR TO FIVE HIGHLIGHT POINTS OF STRATEGY DEVELOPMENT THAT GUANGPING GAO AND I CAN ASSIMILATE AT THE END OF THE WORKSHOP AND PRESENT BACK FOR FURTHER DISCUSSION. SO THANK YOU ALL AGAIN FOR PARTICIPATING IN THIS VERY IMPORTANT OBJECTIVE. AND I LOOK FORWARD TO WHAT WE CAN UNVEIL IN THE NEXT COUPLE DAYS. SO WITH THAT, I'D LIKE TO INTRODUCE GUANGPING. >> THANK YOU, OUR NIH COLLEAGUES, FOR A VERY NICE INTRODUCTION FOR THE MEETING AND SET THE GOAL, OBJECTIVES FOR THE MEETING. AND WE THANK BEVERLY THAT VERY COMPREHENSIVELY LAID OUT POTENTIAL CHALLENGES TO THE FIELD. SO, I JUST WANT TO USE THE TIME KIND OF DESCRIBE SOME VIEWS AND EXPERIENCE HOW TO ADDRESS THESE CHALLENGES. SO, THIS IS MY DISCLOSURE. AND SO AAV ACTUALLY IF YOU THINK ABOUT CAREFULLY FOR THE PROCESS OF DEVELOPMENT, AAV FROM A REALLY VIRUS, ALMOST A VIRUS, TO AN AMAZING VECTOR, WE START WITH VIRUS DISCOVERED MANY YEARS AGO BY JUDE AND OTHERS PIONEERS, AND CHARACTERIZE AND SEQUENCED. AND THEN IN THE PROCESS OF THE JUDE CONVERTING THIS KIND OF VIRUSES INTO A VECTOR, THE FIRST THING WE TOOK ADVANTAGE IS CAPSID. HOST IMMUNE RESPONSE, DEALS WITH CELL TISSUE TROPISM AND SERVED, YOU KNOW, ACCOMPLISHED EFFICIENT GENE DELIVERY. SAME TIME FOR GENE THERAPY WE HAVE TO CONSIDER INSIGHT AS AAV TRANSDUCTION, REALLY IS A TEAM WORK, INSIDE-OUT, SO FROM INSIDE VIEW THIS VECTOR GENOME IS VERY CRITICAL BECAUSE IT STABILIZE GENOME BY CIRCULATION ACCOMPLISH LONG-TERM TRANSDUCTION, AND EXPRESS TRANSGENE. SO TO REALLY, YOU KNOW, I HAVE TAKE SOME TIME TO RESEARCH AND SURVEY THE FIELD, AND I WANT TO USE THE CURRENT COMMERCIAL AND TRANSLATIONAL ARENAS AS SURVEY FOR THE FIELD OF THE GENE THERAPY. SO TOTALLY ANALYZED NINE DIFFERENT THERAPEUTIC AREAS. OOPS. OKAY. SUMMARIZED THE COMPANY'S FOCUS ON THESE AREAS, GENE THERAPY, DRUGS THEY ARE DEVELOPING. AMONG THE NINE AREAS YOU CAN IMMEDIATELY SAY THAT CNS GENE THERAPY IS ONE -- SORRY. LING, AS A BACKUP CAN WE USE WHAT I GAVE TO YOU? OH, IT'S ON. YEAH, THE NEW ONE. THANK YOU. SO, VERY HOT TARGET HERE. YEAH. SO FOR CNS GENE THERAPY I STARTED MY CAREER IN CNS DISORDER, RARE DISEASE, CANAVAN DISEASE DISCOVERED IN 1931, A LETHAL DISEASE, PATIENT CANNOT SURVIVE FOR FIVE YEARS, AND SO AT THIS POINT THERE'S NO GENE THERAPY, NO ANY THERAPY. SO THIS IS A FIGURE I SHOW IN THAT 2013 I MADE KIDS, CANAVAN PATIENTS, THEN A FEW STILL AROUND, REMIND ME MY URGENCY AND MY RESPONSIBILITY TO DEVELOP GENE THERAPY AS FAST AS POSSIBLE TO SAVE THE KIDS' LIVES. THIS IS A LOOK AT MY MENTOR, THIS AMINO ACID GENERATES IN A NEURON, TRANSPORTS INTO DENDROCYTE, ACCUMULATE, CAUSE ALL PROBLEMS. MY Ph.D. IN 1993 I DISCOVERED THE GENE AND MUTATIONS, AND THIS MAKE GENE THERAPY POSSIBLE. SO OUR COLLEAGUE ACTUALLY INITIALLY GENE THERAPY, THEY TRIED BY PROTOTYPE AAV 2, INJECTION INTRACRANIALLY, GENERATED LIMITED CLINICAL IMPROVEMENT BUT CONFIRMED THE SAFETY OF THE GENE THERAPY. THIS IS PUBLISHED IN 2013. SO WHAT WE LEARN WHEN YOU HAVE WIDE DISTRIBUTION OF WHITE MATTER GENERATION YOU NEED GLOBAL GENE TRANSFER FOR GENE THERAPY. SO I JOINED AFTER MY Ph.D. JIM WILSON FOR VENTURE AT UNIVERSITY OF PENNSYLVANIA TO DISCOVER NEW AAV THAT CAN TRANSDUCE CNS USE PCR STRATEGY AND ISOLATE LIBRARY OF CAPSID. WE HIT A GOLD MINE. ONE OF THE VIRUS I WANT TO SHOW YOU, QUITE POPULAR NOWADAYS, THAT'S AAV9, THIS SHOWS YOU WE ISOLATE AAV9 FROM HUMAN LIVER SAMPLE AND THIS IS A PCR BANK, AAV9, THE CLONE I REMEMBER, 28.4, THAT IS OUR AAV9. WITH THE VIRUS AVAILABLE, SO WE START AFTER I JOIN UMass, MY FIRST GRADUATE STUDENT THERE START THE GENE THERAPY PROOF-OF-CONCEPT AAV 9. AAV GENE THERAPY IS TEAM WORK. CAPSID IS NOT ENOUGH. ANOTHER MD/PHD STUDENT DR. GESSLE EXPRESSING CASSETTE ENHANCE GENE THERAPY POTENCY FIVE-FOLD MAKE HUGE DIFFERENCE IN EFFICACY. AND WHAT I CAN SHOW YOU HERE IS BASICALLY AFTER GENE THERAPY, IF YOU START FROM ADULT MICE YOU CAN SEE DECREASE OF BIOMARKER FOR THE DISEASE, AND WHAT YOU DO HUMAN AND WHAT YOU SEE WILDTYPE BEAUTIFUL DISEASE TERRIBLE, DEMYELINATION, EDEMA, AFTER GENE THERAPY ONE WEEK YOU DON'T SEE MUCH CHANGE BUT FOUR WEEKS LATER YOU SEE KIND OF A REVERSE NEUROPATHOLOGY COMPARED WILDTYPE WITH THIS AND RESTORED MYELINATION. ANOTHER QUESTION ABOUT THE GENE THERAPY WE DISCUSSED A BEVERLY INDICATED IS THAT CELL TYPE SPECIFIC EXPRESSION MAY PLAY A ROLE IN GENE THERAPY. WE DID A SURVEY LOOKING AT PERIPHERAL TISSUE VERSUS CNS DIFFERENT CELL TYPES, WHAT IS MOSTLY INTERESTING TO US IS THAT WE FOUND IF YOU DO A LIVER SPECIFIC GENE EXPRESSION, YOU CAN RESCUE ACTUALLY 50% OF THE ANIMAL EXTEND LIFE, THAT TELLS YOU CANAVAN MAY OR MAY NOT BE JUST A CNS DISEASE. WHAT CELL TYPE PLAY A ROLE, BELIEVE OLIGODENDROCYTES IS A TARGET, WE PUT ASTROCYTE SPECIFIC PROMOTER, WE CAN SEE ACCOMPLISH SAY THE POTENCY AS UBIQUITOUS EXPRESSION. THIS TELLS US THAT THE ALTERNATIVE METABOLICAL SINK IS THERAPEUTIC. WE DO NOT JUST NEED TO FOCUS ON OLIGODENDROCYTES. I START COLLABORATION WITH DR. BARRY BYRNE, START EXPANDED TRIAL WITH SINGLE PATIENT, 2-YEAR-OLD, BASICALLY THIS IND IS IMMUNE EXPRESSION AT THE SAME TIME, SO WHAT I CAN SEE IS BY SUMMARY, BARRY PRESENTED SEVERAL TIMES ABOUT OUTCOME BUT I WANT TO TELL YOU IS THAT IMMUNE MODULATION BLOCKED THE ANTI-CAPSID IMMUNE RESPONSE MAY LEAVE THE OPPORTUNITY FOR REDOSING IF NEEDED. ALSO GENE THERAPY IMPROVED MYELINATION OF THE WHITE MATTER AND LED TO IMPROVEMENT OF THE MOTOR DEVELOPMENT AND RESTORE THE VISION, CHILD CANNOT SEE, NOW HE CAN SEE. AND ALSO NEUROIMAGING SUPPORT THOSE DEVELOPMENT FINDINGS. THIS IS A PICTURE BEFORE GENE THERAPY, HAPPY KID AFTER GENE THERAPY, ABOUT 15 MONTHS. EVERY TIME I SAW HIM HE TRY TO PLAY WITH ME, ALWAYS TRY TO GRAB MY GLASSES AND LAUGHING, EXTREMELY HAPPY. WHEN I SEE THESE KIND OF PICTURES YOU FEEL RESEARCH IS SUCH REWARDING PROCESS, PARTICULARLY WHEN YOU DEAL WITH RARE DISEASE, YOU CAN SAVE PEOPLE'S LIFE. AND SO NOW WE KNOW GENE THERAPY WORK BUT BETWEEN OUR GENE THERAPY EXPRESS TO REALLY TRANSLATIONAL OR COMMERCIALIZATION, MY PERSONAL VIEW, FOUR BLOCKS. THE FIRST ONE MAKE ENOUGH HIGH POTENCY VECTOR. SECOND IS GOING TO BE WE HAD DIMINISH CAPSID IMMUNITY AND TRANSGENE IMMUNITY. THIRD ONE IS GOING TO BE IMPROVED EFFICACY, EFFICIENCY, AND REAL TISSUE AND CELL TYPE SPECIFIC GENE THERAPY. FINALLY NOW WE GIVE A.A.V GENE THERAPY THE, THOSE ARE THE THINGS IN MY MIND AND I THINK ALL THE FIELD NOW EVERYBODY FROM DIFFERENT ANGLE TRY AND ADDRESS THOSE ISSUES. ONCE WE HAVE THIS ADDRESSED WE HAVE REAL GENE THERAPY CLINICAL APPLICATIONS. ONE WAY TO ADDRESS THIS, I STARTED THIS NATURAL DISCOVERY FOR AAV CAPSID AND CONTINUE TAKE THIS APPROACH. OUR PURPOSE IS TRYING TO ENHANCE PACKAGING EFFICIENCY WITH NEW VECTORS AND IMPROVE TISSUE TROPISM AND SEROLOGICAL PROFILE, ALL THOSE ISSUES ARE CHALLENGES. THIS IS MY LAB AND COLLABORATORS AT UMass. APPROACH, BASICALLY TAKE 840 HUMAN TISSUES, GENERATE LIBRARY AND GO THROUGH HIGH-THROUGHPUT MOLECULE SEQUENCE, CAPTURE SEQUENCES, ISOLATE A TOTAL OF 1,058 NEW CAPSID OF DIFFERENT SEROTYPES, AND THEN THE FURTHER THING WE DID, THAT'S THE FIRST SCREEN, PACKAGING. WE FOUND FOR EXAMPLE MORE THAN 25% OF AAV2 VARIANTS HAVE HIGHER PACKAGING. AND THEN WE LOOK AT -- PARTICULARLY WITH THE FIRST 50 WE SCREENED, WE IDENTIFIED A VIRUS CALLED V66, 13 FOLD HIGHER TRANSDUCTION EFFICIENCY, AND WITH ANOTHER 80 WE JUST FOUND VIRUS THAT ABOUT 30-FOLD MORE EFFICIENT THAN AAV9, WE'RE IN PROCESS OF CONFIRM THAT, HIGH-THROUGHPUT SEQUENCING. THIS IS AAV2, THIS IS OUR VIRUS ON THE SAME SITE. THE SPREAD, AND EFFICIENCY OF THE GENE TRANSFER THAT IS ABOUT 13-FOLD AS DESCRIBED. AND THEN WE FURTHER LOOK INTO THE PROFILE OF THE CELL TYPE SPECIFICITY THROUGH CONFOCAL ANALYSIS WHAT WE FOUND HERE IS NEURONAL MARKER, ASTROCYTIC MARKER, AS WELL ASKING OLIGODENDROCYTE, 30% ASTROCYTE, 22% MICROGLIA, 12% OF OLIGODENDROCYTE. AND THEN WE DID FURTHER BIOCHEMICAL -- BIOPHYSICAL ANALYSIS, YOU CAN SEE BY LOOK AT YIELD THIS VIRUS IS HIGHER, BETTER THAN AAV2, GENOME STABILITY, CAPSID STABILITY, DIFFERENT CHARACTERISTICS COMPARED TO A.A.V2 AND DID MUTATION TO FIGURE OUT WHAT POSITION CONTRIBUTE TO POVERTIES. AND THEN WE TRY TO FIGURE OUT RECEPTORS, WHAT IT TELLS US IS AGAIN IT'S HEPARIN RESISTANT, DOES NOT REQUIRE HEPARIN FOR TRANSDUCTION. WE LOOK AT CHARGES BY POTENTIAL, MUCH MORE NEGATIVELY CHARGED, WE FOUND STILL HAVE THIS CHARGE POTENCY AND CONTINUE CryoEM AND ADD 2.24 RESOLUTION, AND WHAT WE FOUND IS BY CryoEM YOU CAN CONFIRM THIS NEGATIVELY CHARGED SURFACE. SO, NOW WITH THIS AVAILABLE WHAT WE LOOK TO IS HOW THE STRUCTURE LOOK LIKE. THIS IS THE NEW AAV, UNIQUE COMPARED TO GREEN ONES, COMPARED TO AAV2, AND THIS IS TWO-FOLD AXIS AND FIVE-FOLD AND THREE-FOLD AXIS. THREE-FOLD IS MOST UNIQUE COMPARED TO OTHER POSITIONS. WE CONTINUE LOOKING TO USE CryoEM AND LOOKING TO POSSIBILITY AND WHETHER WE CAN SEE VECTOR GENOME INSIDE AND THIS IS THREE-FOLD PROTRUSION, FIVE FOLD PORE, WE NEED VECTOR GENOME START SHOWING UP. HERE YOU CAN SEE THE THREE-FOLD -- YOU SEE A BASKET IN THE FIVE-FOLD. SO THIS TELLS YOU THAT USE CryoEM YOU MAY GET SOME INFORMATION HOW GET PACKAGED, WHETHER YOU CAN ENHANCE PACKAGING EFFICIENCY AND STUFF LIKE THAT. SO THIS IS I THINK KIND OF INFORMATIVE FOR US, WHERE TO WORK. IMPORTANT ONE IS SEROLOGIC PROFILE. IS THIS VIRUS LIKE AAV2? AND THEN CAN WE DO IN VIVO NEUTRALIZATION WITH AAV2 CAN NEUTRALIZE THIS VIRUS. YOU DON'T GET MUCH NEUTRALIZATION IN VIVO, AND WE LOOK AT COMPARED TO OTHER SEROTYPES, AND YOU CAN SEE THIS VIRUS IS MANY FOLD NEUTRALIZATION POTENCY. NOW WITH THIS I ALSO WANT TO INTRODUCE A VERY RECENT ARTICLE PUBLISHED, GIVE US SOME HINT HOW YOU SCREEN LARGE LIBRARIES. IN THIS CASE THIS IS PUBLISHED IN "NATURE COMMUNICATION," CHANGE SCREEN AN ANIMAL MODELS TO CRE INDUCED TRANSDUCTION, WHAT IT SHOWS YOU CAN SEE COMPARED TO THE REGULAR AND IMPROVED WAY YOU HAVE DRAMATICALLY ENHANCED SENSITIVITY, GREAT FOR SCREENING, SO THIS IS VERY INTERESTING AND ELEGANT WAY TO SCREEN THE LARGE LIBRARIES. AND THE NEXT ISSUE IS POTENTIAL, ANTIGEN PRESENTING CELL AND IMMUNOGENICITY WE HAD TO ADDRESS, USING MICRON PROCESS TO ADDRESS FROM IN THIS CASE WANT ANTIGEN PRESENTING CELLS. AS YOU CAN SEE THROUGH THIS STUDY WE PUBLISH RECENTLY WE FOUND THAT BY MECHANISM IS REALLY REDUCED, SPECIFIC T CELL AS WELL AS B CELL, AND ALSO BY DOWNREGULATING THE CYTOKINES AND MOLECULES AND ALSO BY REALLY REDUCE FUNCTIONAL CTO AND CD8 INFILTRATION AND CLEARANCE OF TRANSDUCED MUSCLE FIBERS. UNIVERSITY OF MIAMI, WE'RE ABLE TO ACCOMPLISH THIS MIAMI MONKEY, WE USE SINGLE AAV INVECTION, CONTROL AND WE CALL MICRO MANGLED AAV VECTORED ANTI-HIV ANTIBODY. WE TEST USE OVA AS ANTIGEN. YOU CAN SEE LITTLE EXPRESSION, CD8, AND YOU HAVE THE OTHER INFLAMMATION MARKERS, BUT AFTER DETARGETTING YOU SEE DECREASE, EARLY DATA THAT WE CONTINUE TO MONITOR. GENE THERAPY, PARTICULARLY WITH THOSE NEURODEGENERATIVE, TO SILENCE TRANSGENE EXPRESSION, WE COLLABORATE WITH SOMEBODY AT UMass AND SOME P.I.s AT UMass, WERE ABLE TO ACCOMPLISH THE PHARMACOLOGICALLY INDUCED CELL TYPE AND REGIONAL SPECIFIC GENE LOCKDOWN. AND FINALLY WANT TO INDICATE THAT THE ANIMAL MODELING IS INTERESTING, IT'S A VERY IMPORTANT FOR RARE DISEASE, GENE THERAPY AND OTHER DISEASE SO WE FIRST DEVELOPED AAV INFECTION EMBRYO, GENERATE MOUSE, VERY EFFICIENT. THEN WE MOVIE DOWN, CAN WE DO THIS N MONKEYS. SEEM TO BE VERY EFFECTIVE IN MONEY. YOU CAN GET 100% EDITING. TRIED TO GENERATE CANAVAN MONKEY, IT FAILED. CURRENTLY TRYING TO ESTABLISH IN-HOUSE SIMPLE WAY MAKE MOUSE MODEL, AND KEVIN AND HEATHER, WE'RE TRYING TO MAKE LARGE ANIMAL, ONE EXAMPLE TRYING TO DO GENE THERAPY, 50 OR 40 KG ANIMALS. THANK YOU VERY MUCH. WE'RE GOING TO GO INTO SESSION 1, SPLIT INTO SESSION 1A AND SESSION 1B, BETWEEN THOSE WE'LL HAVE A COFFEE BREAK. AND OBVIOUSLY WE DON'T HAVE ANY FOOD SO YOU CAN PULL WHATEVER FROM YOUR BACKPACKS. SO, I'M BEV DAVIDSON, AS I'VE BEEN INTRODUCED BEFORE. I WANT TO INTRODUCE MY COLLEAGUE, STEVEN GRAY, IF HE CAN STAND UP. STEVEN-- I'M AT CHILDREN'S HOSPITAL OF PHILADELPHIA, UNIVERSITY OF PENNSYLVANIA. STEVEN IS AT TEXAS SOUTHWESTERN. AND SO JUST GOING TO PUT THESE UP HERE, SO SESSION 1A IS REGULATED EXPRESSION. WE WANT TO TALK ABOUT STRATEGIES TO AVOID OVEREXPRESSION. YOU SAW ONE STRATEGY BEING APPROACHED BY GUANGPING'S TEAM. ALSO REGULATION OF GENE EDITING, GENE SILENCING METHODOLOGIES, AND THEN UNIQUE CHALLENGES WITH CELL AUTONOMOUS GENE THERAPY, HOW DO WE MANAGE LEVELS ON A PER CELL BASIS. SESSION 1B WILL BE ON FOCUSED TARGETING, YOU SAW ONE STRATEGY ON FINDING NEW VECTORS THAT CAN GO EVERYWHERE AND HOW DO WE GO ABOUT FINDING NEW VECTORS THAT ARE REALLY RESTRICTED NATURALLY TO PARTICULAR CELLS AND TISSUES OF INTEREST. THEN AGAIN IN DETECTING AND MINIMIZING OFF-TARGET EFFECTS, DO WE HAVE THE MOST SENSITIVE TOOLS AVAILABLE. SO SESSION 1 REGULATED EXPRESSION, WE WERE ALL QUITE AWARE THAT TO DATE MOST OF THE GENE THERAPY APPROACHES FOR CNS DELIVERY ARE INTO THE CSF THROUGH VARIOUS ROOTS, SOME ARE FINE FOR ANIMALS, SOME ARE FINE FOR HUMANS. SOME MAY BE A LITTLE BIT MORE RISKY, PARTICULARLY IN DISEASE PATIENTS. WE INFUSE TO BLOOD, IN THE BRAIN PARENCHYMA FOR MRI-GUIDED DELIVERY, WORK THAT CHRIS PIONEERED OVER THE YEARS. LOSS OF FUNCTION DISORDERS WE KNOW TOO MUCH EXPRESSION MAY NOT BE NECESSARILY A GOOD THING AS GUANGPING ALLUDED TO. OVEREXPRESSION IN THE WRONG CELLS WITH BE DELETERIOUS, THINK ABOUT EPILEPSY AND ION CHANNELS, THE WRONG TIMES OF NEURONS COULD BE JUST AS BAD AS EXPRESSING TOO MUCH IN THE RIGHT KINDS OF CELLS. AND FOR GAIN OF FUNCTION DISORDERS HOW SHOULD WE ADVANCE DETECTION OF OFF-TARGETS AND HOW COULD WE STRATEGIZE METHODS TO REDUCE THEM? SO I'M GOING TO LEAVE THIS SLIDE UP HERE FOR SESSION -- IS THIS THE RIGHT ONE? YES, OKAY. HERE WE ARE. I REALLY WANT TO OPEN UP THE FLOOR TO BEGIN THIS DISCUSSION. I WANT -- WE DON'T WANT TO BE TALKING AT YOU. AGAIN, THIS IS NOT US SHOWING YOU OUR DATA AT THIS POINT. WE PRETTY MUCH KNOW AS A FIELD WHERE WE ARE AND SO WHAT ARE THE CHALLENGES. DO YOU ALL HAVE IDEAS AND THOUGHTS ON STRATEGIES TO OVERCOME SOME OF THESE CHALLENGES THAT WE AS A FIELD COULD PUT FORWARD TO THE NIH? >> MARK SANDS FROM WASHINGTON UNIVERSITY SCHOOL OF MEDICINE. THE TOPIC AVOIDING OVEREXPRESSION, I GUESS FROM MY PERSPECTIVE I WOULD EVEN EXPAND THAT AND INCLUDE IS OVEREXPRESSION NECESSARY. WE HAVE JUST GENERATED SOME DATA IN ONE OF OUR MOUSE MODELS WHERE WE'VE DIRECTLY COMPARED THE KAG PROMOTER TO A PROMOTER FROM STEVEN GRAY, WE GET THE SAME EFFICACY IN THE MOUSE MODEL. AND I REALLY AT LEAST FOR THE DISEASES I STUDY AND FOR NUMBER OF OTHERS I SUSPECT IT PROBABLY MATTERS MORE THE DISTRIBUTION OF THE VECTOR RATHER THAN THE LEVEL OF EXPRESSION. SO AGAIN, IS OVEREXPRESSION REALLY NECESSARY? >> (INAUDIBLE). >> TURN ON YOUR MIC PLEASE. >> OH, HERE. YEAH, DRAMATIC APPEARANCE WITH YOUR MONKEY STUDY. COULD YOU PLEASE SHARE SOME OF YOUR THOUGHTS WITH THAT STUDY? >> SURE. UMass MEDICAL SCHOOL. I AGREE WITH MARK ENTIRELY. I THINK THE NOTION OF OVEREXPRESSION BEING THE GOAL OF EVERY GENE THERAPY, I AT LEAST VIEW IT AS SORT OF A HISTORICAL LEFTOVER WHERE THE FIELD STARTED IN HUMAN APPLICATION, RIGHT? WHICH THE MANTRA HAS BEEN WE NEVER FAILED FOR HAVING TOO MUCH BUT FOR HAVING TOO LITTLE, AND FACTOR 9 AND TRYPSIN HAVE INFLUENCED THAT. I THINK MARK IS CORRECT. AT LEAST IN CNS, IF YOU THINK ABOUT LYSOSOMAL STORAGE DISEASE, NORMAL INDIVIDUALS WITH 10 TO 15% IN EVERY CELL, I THINK THIS IS WHERE THERE IS SOME CONFUSION HOW PEOPLE APPROACH, I'M TALKING ABOUT THIS IN PAPERS, OFTEN QUOTED, 10% IS ENOUGH BUT THEY FORGET TO TELL THE OTHER SIDE OF THE STORY. 10% IN EVERY CELL. I THINK THAT'S THE KEY. SO FAR WE'VE BEEN DEALING WITH VECTORS THAT ARE GREAT, ABLE TO ACHIEVE WONDERFUL THINGS, BUT WE'RE NOT THERE YET, AT THE LEVEL WE CAN SAY, OKAY, NOW WE'RE IN EVERY CELL, RIGHT? I THINK THAT'S WHERE WE WANT TO BE, THAT EVERY CELL. WHAT WE FOUND IS THAT DESPITE AN EXPERIMENT IN MICE, CATS, SHEEP, THAT EVERYTHING LOOKED FINE, WHEN WE DID INTERPARENCHYMAL INJECTION IN MONKEYS WITH ONE OF THESE VECTORS, PUT THE ENTIRE KITCHEN SINK OLD PHILOSOPHY, WE FOUND ENORMOUS TOXICITY IN THE CENTRAL NERVOUS SYSTEM. IF YOU ASK WHAT WAS THE THRESHOLD WHERE CELLS CANNOT STAND IT ANY LONGER, I HAVE ABSOLUTELY NO IDEA. BUT I AGREE THAT IN GENERAL I THINK THE CELL CAN WITH STAND DOING WITH LESS OF OF A PROGRAM THAN NECESSARILY A LOT MORE PROTEIN SO I'M NOT SURPRISED, YOU MAY THAT ABOUT THE KAG. OUR TECHNOLOGY IS ONE STEP UP IN TERMS OF DELIVERY WE CAN OBTAIN. I AGREE ENTIRELY. I THINK FOR THE CNS ESPECIALLY WE HAVE TO MOVE AWAY FROM THE NOTION OF THE CURRENT PACE OF PUTTING EVERYTHING INTO A KAG PROMOTER. WE'LL ENCOUNTER MANY DISEASES AND PROTEINS THAT'S NOT THE CASE AT ALL. WE'LL BE BETTER OFF USING WEAK PROMOTERS AS' OPPOSED TO STRONG ONES. >> XANDA BRAKEFIELD, MASS GENERAL HOSPITAL. I AGREE BUT THINK IN SOME THERAPEUTIC MODELS WHERE WE'RE TRYING TO DELIVER LET'S SAY CASPASE 1 OR SOMETHING THAT'S ACTUALLY GOING TO SHRINK A TUMOR, I ACTUALLY HIGH LEVEL EXPRESSION ALLOWS THE PRODUCT TO BE INCORPORATED INTO WHAT WE CALL EXOSOMES, THEY CAN DELIVER THE PROTEIN TO CELLS NEIGHBORING THE CELL THAT YOU TRANSDUCE, SO IF YOU HAVE LESS EFFICIENT DELIVERY SOMETIMES YOU CAN PASS YOUR GENE PRODUCT TO OTHER CELLS THROUGH THE EXOSOMES RELEASED BY THE PARENTS OF A TRANSDUCED CELL. >> YEAH, I THINK YOU'RE ABSOLUTELY RIGHT. BASICALLY IT'S GOING TO DEPEND ON THE DISEASE OR THE CONDITION YOU'RE TRYING TO TREAT. ONE OF THE REASONS WHY I BROUGHT IT UP IS IT'S HARD TO GET STUDIES LIKE THAT FUNDED. WHEN YOU WRITE A GRANT, YOU KNOW, YOU GET DINGED ON INNOVATION. IT'S NOT VERY SEXY TO JUST RUN THROUGH A BUNCH OF PROMOTERS AND SEE WHAT WORKS BEST. >> BRIAN BIGHAM, MANCHESTER UNIVERSITY. AGAIN, I AGREE WITH WHAT'S BEEN SAID ALREADY BUT SOME OF THIS COMES DOWN TO ONE'S APPROACH. SO FOR EXAMPLE IF YOU'RE TAKING AN EX VIVO APPROACH YOU GO WITH A LENTIVIRAL VECTOR EX VIVO, FOR EXAMPLE, YOUR APPROACH IS RELY ON SECRETION. PRODUCT. IN THOSE INSTANCES YOU INSTANCES NEED TO RELY ON OVEREXPRESSION BUT NEED TO BE MINDFUL OF TOXICITY. >> SO, WHAT ABOUT STRATEGIES TO THINK ABOUT OPTIMIZING EXPRESSION IN CELLS? RIGHT NOW YOU DO A TWO PROMOTER COMPARISON, YOU DO A THREE PROMOTER COMPARISON, FUNDING FOR THOSE NON-SEXY EXPERIMENTS ARE LAGGING BEHIND. DO PEOPLE HAVE SUGGESTIONS ON WAY TO RAMP UP AND HOW WOULD WE KNOW WE HAVE OPTIMAL EXPRESSION? IN VIVO EXPERIMENTS ARE ABSOLUTELY REQUIRED. DO PEOPLE WANT TO COMMENT, EVEN IN THE AUDIENCE? LUKE, THERE'S A -- >> LUKE VANDENBERG, HARVARD. NOT SPECIFICALLY ON YOUR SECOND POINT BUT COME BACK MAYBE TO SOME POINTS RAISED, I THINK OVEREXPRESSION CLEARLY ON A CASE TO CASE BASIS IS POSSIBLY TOXIC BUT I WOULD ARGUE THAT IN MOST OF OUR SETTINGS WE TRY TO DEVELOP THE MOST POTENT PARTICLES, BUT THEN USE THOSE AS A METRIC TO LIMIT OVEREXPRESSION, WHEREAS HANDICAPPING YOUR VIRUS BY MAYBE REDUCING THE EXPRESSION FROM THE PROMOTER I THINK COULD HAVE UNTOWARD CONSEQUENCES IN TERMS OF VECTOR TOXICITY THAT YOU THEN HAVE TO COMPENSATE. SO I GENERALLY AM ALWAYS A LITTLE BIT CHALLENGED BY THESE APPROACHES WHERE YOU INTENTIONALLY TRY TO REDUCE POTENCY ON PARTICLE BY PARTICLE BASIS SO I WANTED TO BRING UP THAT POINT. BUT THAT CLEARLY DOESN'T ADDRESS THE ISSUE OF DIFFERENT PROMOTERS THAT ARE ACTIVE IN DIFFERENT CELLS IN DIFFERENT WAYS AND I THINK REALLY THAT'S THE CHALLENGE THAT WE'RE OFTEN TRYING TO BALANCE. >> YEAH, I'M GOING TO ADD ON. WHAT MIGUEL SAID INITIALLY I THINK WE HAVE TO THINK, THE LEGACY HERE WITH THE HISTORY OF GENE THERAPY WE STARTED WITH EASIER THINGS, VERSUS SECRETED PROTEIN. IN THAT CASE IF OVEREXPRESSION IS TOLERATED YOU WANT TO EXPRESS AS HIGHLY AS YOU POSSIBLY CAN, AND THEN YOU CAN CONTROL THE DOSE OF THE VIRUS PARTICLE. BUT MORE AND MORE OFTEN WE'RE MOVING NOW INTO MOST OF THE SECRETED PROTEINS, QUOTE/UNQUOTE EASIER GENE THERAPIES ARE SPOKEN MORE SO WE'RE MOVING INTO DIFFICULT DISEASES WHERE I HAVEN'T SEEN ANYTHING RIGHT NOW WITH AAV VECTORS WHERE YOU CAN REALLY SATURATE AND TARGET EVERY CELL IN THE BRAIN. WE CAN'T GET CLOSE TO THAT. IT'S MOVING TO A STRATEGY OF KEEPING PARTICLE DOSE AS HIGH AS YOU POSSIBLY CAN AND THEN YOU REALLY HAVE TO REGULATE THE EXPRESSION ON THE PROMOTER LEVEL. THE LAST POINT I WANT TO MAKE THAT I THINK WE CAN LEAD INTO IS THE ANALYTICS OF HOW WE'RE ASSESSING EXPRESSION, YOU KNOW, THE TRADITIONAL WAY WOULD BE TO TAKE A WHOLE CHUNK OF BRAIN TISSUE AND INTO qPCR OR WESTERN BLOT, BULK EXPRESSION, AND THAT'S REALLY NOT GOING TO WORK ANYMORE. WE'VE GOT TO SHALL MORE SOPHISTICATED AND LOOK AT EXPRESSION ON A CELL-BY-CELL BASIS, WHICH IS A LOT HARDER, IT TAKES A LOT MORE RESOURCES, MORE TIME, BUT THIS IS KIND OF THE DIRECTION WE'RE MOVING INTO. >> I THINK I WANT TO BRING US BACK TO FIXING THE EXPRESSION PROBLEM. WE KNOW BEST VECTORS, LET'S SAY WE HAVE THE BEST VECTORS ON THE PLANET. WE'RE GOING TO GET TO SOME OF THE VECTOR TARGETING IN SESSION 1B. AND KEEPING IN MIND I AGREE WITH STEVEN EARLY STUFF WITH SECRETED PROTEINS BUT NOT PROTEINS ARE SECRETED EQUALLY AS MIGUEL AND WE FOUND OUT. YOU JUST END UP WITH EVERYTHING IN THE CELL AS OPPOSED TO ALLOWING FOR CROSS-CORRECTION BUT AGAIN I WANT TO GET BACK TO A STRATEGY, DEVELOPMENT PERSPECTIVE, HOW DO WE GO THROUGH THE THOUGHT PROCESS OF IDENTIFYING THE BEST EXPRESSION CASSETTES FOR PARTICULAR INDICATIONS. YOU KNOW, ARE THROUGH HIGH-THROUGHPUT MACHINE LEARNING APPROACHES WE CAN TAKE ADVANTAGE OF, DO WE DO IT IN CELLS, IN ANIMALS? DO WE DO THAT IN LARGE ANIMAL MODELS? AND WHAT'S THE READOUT FOR WHEN WE KNOW SOMETHING IS WORKING? HEATHER? >> HEATHER GRAY, UMass. ONE THING I WANT TO BRING UP, THE CELL KIND OF DOES THIS FOR US TO SOME DEGREE. WITH A CASE OF LYSOSOMAL STORAGE DISEASE WE SEE LYSOSOMAL DYSFUNCTION. YOU HAVE ONE ENZYME REGULATED, MOST BECOME DYSREGULATED, ALMOST A COMPENSATORY MECHANISM, UPREGULATED, AND A LOT OF GROUPS HAVE DONE THIS, LOOKED AT REGULATION OR NORMALIZATION OF COMPENSATORY INCREASES IN RESPONSE TO OVEREXPRESSION OR SAY THIS HAS COME BACK DOWN TO NORMAL, FIFTY FOLD, A HUNDRED-FOLD. IF WE COULD DO THAT ON A CELLULAR BASIS I THINK THAT WOULD BE EXTREMELY USEFUL BECAUSE IT DOES HAVE LIKE WARNING SIGNS, YOU CAN SEE THEM VISUALLY, IF YOU COULD DO THAT WILL LIVING ANIMAL. IT DOES HAVE MECHANISMS BY WHICH CAN YOU MEASURE THE SECRETION WHERE THE MICROGLIA HAVE HAVING A COME-APART, INFLAME AND UP UPSET, ON A SINGLE CELL LEVEL IF WE COULD EVALUATE THAT, I DON'T KNOW THE TECHNOLOGY IS THERE. >> CHARLES WHEAT, UNIVERSITY OF PENNSYLVANIA. LOOKING AT THE SLIDE THE WAY YOU SET UP, BEV, IT'S NOT ONLY JUST THE QUESTION OF OVEREXPRESSION BUT YOU'VE TIED THEM TOGETHER. IT IS THE QUESTION OF DELIVERY AS WELL BECAUSE WHEN YOU'RE LOOKING AT -- FORGET GENE THERAPY. YOU'RE LOOKING AT SMALL MOLECULE THERAPY, INTRATHECAL OR INTRACEREBRAL, WELLS WILL BE VERY HIGH AND WELLS WILL BE VERY LOW. I THINK THE DISTRIBUTION IS TIED IN COMPLETELY IN THE TOXICITY QUESTION, BECAUSE IF YOU'RE LOOKING AT THE PERFECT SITUATION YOU'RE LOOKING AT A SITUATION WHERE YOU WANT TO GET SOMETHING ACROSS VESSELS WHERE YOU'RE GOING TO BE RIGHT UP AGAINST EACH CELL SO THEN YOU CAN CONTROL THE DOSE, ALL OF THOSE CELLS, BECAUSE THERE'S PRESUMABLY THEY WILL BE GETTING SIMILAR. I THINK THE WAYS WE'RE LEFT WITH METHOD OF GIVING DRUGS, METHOD OF GIVING GENE THERAPY YOU'LL ALWAYS END UP WITH AREAS THAT WILL HAVE VERY HIGH EXPRESSION AND AREAS WITH VERY, VERY LOW EXPRESSION. I THINK THERE'S A LOT TO BE LEARNED FROM PHARMACOTHERAPY FIELD OF INTRATHECAL ADMINISTRATION. >> ARE THERE BETTER METHODS FOR EVALUATING BIODISTRIBUTION WE'VE NOT CONSIDERED? VIVIANA? >> I THINK THEY ARE RELATED. I WANT FOR A SECOND TO GO BACK TO HEATHER'S POINT BECAUSE I THINK THEY ARE CONNECTED. HEATHER WAS CALLING FOR METHODS TO CONTROL EXPRESSION AND WAS HINTING TO THE PROTEIN DEGRADATION PATHWAY, RIGHT? SOME REQUIREMENTS WOULD THIS NEED TO BE ORTHOGONAL SO WE MAKE SURE WE ONLY ACT UPON THE GENES WE INTRODUCE, AND THERE ARE PROTEASES FROM BACTERIA THAT DO THIS BUT THE CHALLENGES ARE LINKED TO THE IMMUNE RESPONSE AND BIODISTRIBUTION. SO IN THE SYNTHETIC BIOLOGY, BIOENGINEERING FIELD THERE'S GROUPS THAT WORK EXCLUSIVELY IN CELL CULTURE BECAUSE IT'S FASTER THROUGHPUT AND YOU CAN APPLY HOW TO PUT ASSAYS AND MACHINE LEARNING TO COME UP WITH PROTEASES THAT CAN ACT ORTHOGONAL ON TARGET OF INTEREST. IT CAN BE COMBINATORIAL AS WELL. HOWEVER, THE TRICKY PART TO TRANSLATE INTO GENE THERAPY FIELD MOST OF THESE POTENT AND/ AND THE ORTHOGONAL, THEY HAVE A SOLUTION, WELL TOLERATED IN VIVO, A BIG QUESTION, GOING TO THE BIODISTRIBUTION TO BE TOLERATED IN VIVO YOU NEED TO REDUCE AS MUCH AS POSSIBLE, PREVENT ANTIGEN EXPRESSING CELLS, ET CETERA. >> (INAUDIBLE) PARDON ME? YEAH, THERE IT IS. I WAS WONDERING ABOUT THE POSSIBILITY OF USING AN INDUCIBLE PROMOTER INSTEAD, THE ADVANTAGE THERE IS THEN YOU CAN TITRATE THE DOSAGE OF GENE EXPRESSION AND ZERO EXPRESSION, VERY HIGH EXPRESSION, TAILOR IT TO DIFFERENT INDIVIDUALS AT DIFFERENT TIMES. >> YEAH, I THINK I WROTE IT DOWN >> OH. >> THIS COMES BACK TO CHARLES' POINT THOUGH ABOUT DEPOTS. IF WE HAVE 500 COPIES OF THE AAV IN ONE DEVELOP, 5 COPIES IN ANOTHER CELL, INDUCE IT, WE'LL GET 500-FOLD EXPRESSION IN ONE CELL AND 5-FOLD IN ANOTHER CELL. AGAIN, I THINK THAT'S A GREAT WAY TO DO IT ONCE WE OPTIMIZED UNIFORMITY OF DISTRIBUTION. I DON'T KNOW IF CHRIS HAS A COMMENT. >> YEAH WHEN I THINK ABOUT TRANSLATION MOST OF THE TIME, THAT'S WHAT I NEED TO DO EVERY DAY, ONE OF THE QUESTIONS I REALLY HAVE IS ALL THE IDEAS, I COMPLETELY AGREE, WHICH IS THE MODEL WE USE FOR BECAUSE WE ALL KNOW THAT CELLS ARE NOT THE SAME, RODENTS ARE NOT THE SAME, ULTIMATELY WE DO WANT TO GET INTO HUMANS. THAT TO ME IS A VERY IMPORTANT QUESTION WE NEED TO ADDRESS. >> I THINK AS WE CONSIDER THE CNS ALSO IT'S INCREDIBLY IMPORTANT BECAUSE YOU MAKE A CHANGE IN ONE NEURON, IT'S NOT IN ISOLATION. IT'S IN A CIRCUIT. AND SO WHAT HAPPENS TO ALL OF THE CIRCUITS THAT ARE CONNECTED TO THAT EITHER IMMEDIATELY OR OVER TIME? AND ARE THERE STRATEGIES TO BEGIN TO TEST THAT? >> SO I'M GOING TO THINK THERE ARE MULTIPLE WAYS TO DO THIS, BUT EACH OF THEM IS COUCHED IN DIFFICULTY. LENTIFIELD, FOR EXAMPLE, WE HAD TO STEP BACK FROM YOUR UBIQUITOUS AND VIRAL PROMOTERS BECAUSE OF INITIAL ISSUES WITH INTEGRATION AND MUTAGENESIS. WHAT WE FOUND WAS WHEN WE SWITCHED TO MAKING PROMOTERS MORE SPECIFIC FOR MICRO GLIAL CELLS, FOR EXAMPLE, A LOW PUSHBACK ON THAT. AND WE BASICALLY HAD TO SHOW THEY WERE GOING TO BE AS EFFECTIVE AS UBIQUITOUS CELL-SPECIFIC PROMOTER. AND THE UPSHOT WAS THAT WE ENDED UP WITH A PROMOTER THAT WAS LEAKY IN FACT BUT DID PUSH SPECIFICITY TO MICROGLIA, THE UPSHOT BEING MORE EXPRESSION IN THE BRAIN, LESS IN THE PERIPHERY, WHICH IS WHAT WE WERE AIMING FOR. FOR AAV YOU'VE GOT MORE OF AN ISSUE BECAUSE OF THIS PROBLEM OF DISTRIBUTION. WHILE REGULATION IS GREAT, INDUCIBLE PROMOTERS ARE GREAT, SYSTEMS ARE NOT FIT FOR PURPOSE, USUALLY THEY REQUIRED TWO COMPONENTS, TWO-COMPONENT SYSTEMS ARE A PROBLEM TO DELIVER. UNTIL WE GET SINGLE COMPONENT INDUCIBLE PROMOTERS WORKING WELL, WHICH IS DIFFICULT WE WON'T BE THERE FOR INDUCIBILITY PROMOTERS. CELL SPECIFIC EXPRESSION SHOULD BE THE GOAL HERE. PROMOTERS ARE TRICKY. IT DEPEND HOW SPECIFIC YOU WANT THEM TO BE. AND I THINK YOU HAVE TO JUST KIND OF TAKE A TRIAL AND ERROR APPROACH WITH THEM. >> (INDISCERNIBLE), STANFORD. I WANT TO PICK UP ON MIGUEL'S AND VIVIANA'S COMMENTS REPORTING PROMOTER STRENGTH AND EXPAND OUT IN THE CONTEXT OF POTENTIAL IMMUNOTOXICITY, OTHER MANEUVERS I GUESS WE'VE DEVELOPED OVER THE YEARS, THEY ARE A BIT HISTORICAL AS WELL, USE OF MODIFICATION, THINKS THAT ELEVATE CG MOTIF WHICH MAY BE IMMUNOTOX IS AND AFFECT DURABILITY AND WPREs, MANY VECTOR DESIGNS, SOME HAVE MOVED AWAY, PERHAPS THERE'S SOME RESIDUAL, THEY HAVE THE POTENTIAL FOR UNTOWARDS TOXIC. SIMILAR MODULATIONS OF EXPRESSION CASSETTE OUTSIDE PROMOTER. >> ONE QUICK COMMENT ABOUT THE DISTRIBUTION. SORRY, ONE QUICK THING. I'VE DONE LOTS OF CS DELIVERY OF BOTH GENE THERAPY, ALSO OLIGONUCLEOTIDES, DISTRIBUTIVE PROPERTIES ARE DIFFERENT. IT CAN GO EVERYWHERE, DOES A GREAT JOB, AAV DOESN'T PENETRATE DEEPER. FOR THOSE WHO WORK ON VECTORS, IF YOU CAN GET ONE -- CORTICAL DISTRIBUTION IS FABULOUS, SUPERFICIAL CORTICAL DISTRIBUTION, IF YOU CAN MAKE THAT TRANSFER INTO DEEPER BRAIN STRUCTURES EFFICIENTLY THAT WOULD PROBABLY FIX MOST OF THE ISSUES, A LOT OF ISSUES THAT WE'RE SEEING. >> I'M GOING TO JUMP IN. IN TERMS OF REGULATED EXPRESSION, THE BIG CONSIDERATION IS SIZE. ALL VECTORS, WE ALWAYS HAVE LIMITATIONS ON SIZE. SO REALLY CONCERTED EFFORT TO MAKE SMALLER PROMOTERS THAT MIGHT HAVE LEVELS OF SPECIFICITY, THIS IS IN MY VIEW A VERY HIGH PRIORITY FOR THE FIELD. AND ALSO SOMETHING WE HAVEN'T TOUCHED ON IS -- GUANGPING TOUCHED A LITTLE BIT ON microRNA BINDING SITES. THE OTHER APPROACH MIGHT HAVE A PROMOTER THAT IS A LITTLE BIT MORE PROMISCUOUS BUT HAVE MULTIPLE microRNA BINDING SITES THAT WOULD DETARGET IT FROM CERTAIN CELLS THAT COULD BE PROBLEMATIC. THE SITES ARE SPACE EFFICIENT IN THE SCHEME OF THINGS SO THESE ARE THE COMBINATORIAL STRATEGY OF BEING VERY CONSCIOUS OF SIZE. BUT IN MY VIEW THIS IS A HIGH PRIORITY FOR THE FIELD. >> TO ADD TO THIS COMPLETELY AGREE, GIVEN REAL ESTATE IN THE AAV WE HEARD A SUGGESTION THERE ARE ALREADY MOTIFS THAT WE GET FOR HISTORICAL REASONS, WPRE DO WE NEED TO KEEP IT, MIGHT BE IMMUNOGENIC, TAU EXPRESSION IS ENHANCED FROM MY KNOWLEDGE WE KEEP IT TO ENHANCE EXPRESSION BUT IF WE DON'T NEED IT, IT'S A SMALL MOTIF BUT ANY SPACE WOULD BE HELPFUL. I WANT TO OPEN, WHAT'S PEOPLE'S EXPERIENCE WITH WPRESs, CAN WE REMOVE IT? >> CAN I COME BACK HOW DO WE BEGIN TO IDENTIFY THESE? I LIKE HEATHER'S SUGGESTION OF USING CELL-BASED SCREENING BUT YOU BROUGHT UP THE IMPORTANT POINT THAT WE NEED TO CONSIDER CELLS NOT IN ISOLATION BUT IN A NETWORK, IN A COMPLEX ENVIRONMENT LIKE THE BRAIN. AND IS THERE ROOM, DO WE HAVE EVIDENCE OF A STEP-WISE FASHION FOR DOING THINGS IN CELL ISOLATION THAT TRANSLATE WELL TO THE CNS, AND IS THIS A CHALLENGE FOR US TO TRY AND SEE IF WE CAN DO THAT? >> SO (INDISCERNIBLE) FROM USB I'M GOING TO OFFER A DIFFERENT PERSPECTIVE FROM ASSAY DEVELOPMENT STANDPOINT. I THINK THE CELL-BASED ASSAY APPROACH IS ACTUALLY IMPORTANT, TO ME, AND I THINK IT HAS TO CORRELATE MAYBE WITH THE OUTCOME IN THE ANIMAL MODEL. I THINK IF YOU'RE ABLE TO HAVE THOSE ASSAYS RUN SIDE BY SIDE, IT'S ADDITIONAL WORK BUT I THINK DOING IT AT THE FRONT WILL HELP ACTUALLY INFORM ON THE TYPE OF POTENCY ASSAY ONCE YOU ADVANCE THE PRODUCT TO THE CLINIC. AND USING CELL LINES ACTUALLY CAN PROVIDE LIKE ASSAYS THAT CAN GIVE YOU DATA THAT IS MORE PREDICTABLE, AND I THINK THERE ARE A LOT OF LESSONS TO BE LEARNED FROM PRODUCTS LIKE INSULIN, TROPIN, INSULIN WAS DISCOVERED IN THE 1920s, I THINK ANIMAL-DERIVED EXTRACT GIVEN TO PATIENTS I THINK WERE ABLE TO NORMALIZE GLYCEMIA AS PURIFICATION ADVANCED IT WAS PURER SOME OF THE EARLY PATIENTS WHO CARRIED THE INSULIN THAT WAS PURE EXPERIENCED EPISODES OF HYPOGLYCEMIA, BECAUSE THERE'S TOO MUCH INSULIN IN THE PRODUCT. I THINK OVEREXPRESSION IS MAYBE THERE IS ANALOGY HERE, IT'S NOT ABOUT OVEREXPRESSION OR INDUCE ABLE. I THINK ALL OF THOSE REGULATED EXPRESSION INDUCIBLE EXPRESSION IS IMPORTANT BUT I THINK HAVING ASSAYS EARLY ON CAN ACTUALLY HELP ADVANCE THE THINGS AS YOU GO TO THE CLINIC. >> SO I HAVE ONE COMMENT FROM ERNESTO FROM UIC, WAS SUPPOSED TO BE HERE BUT HAD A BIKING ACCIDENT OVER THE WEEKEND AND WAS UNABLE TO ATTEND. HE E-MAILED ME, AND HE MAKES A GREAT POINT THAT WE HAVE TO CONSIDER OVEREXPRESSION IN THE CONTEXT OF ESPECIALLY WITH A LOT OF PEDIATRIC DISEASES, DEVELOPMENTAL EXPRESSION LEVELS OF THE PROTEIN DURING LIKE FROM, YOU KNOW, EARLY POSTNATAL DEVELOPMENT INTO ADULTHOOD, AND SO THINKING ABOUT THE FACT THAT YOU WOULD WANT TO MIMIC WHAT ACTUALLY HAPPENS DURING DEVELOPMENT AND THAT THIS CAN BE COMPLICATED BY THE CELL AUTONOMOUS PROBLEM AND WE HAVE TO CONSIDER THE FACT NANOPROTEINS ARE SECRETED AS STEVE MENTION, DIFFERENT PROTEINS HAVE DIFFERENT HALF LIVES, WHICH WILL IN FACT HAVE IMPACTS ON EFFICACY AND EXPRESSION OF THE FUNCTIONAL PROTEIN >> YES, EXACTLY. EXACTLY. WELL SAID. THANK YOU, ERNEST. SORRY TO HEAR ABOUT YOUR BICYCLE ACCIDENT. DID EVERYONE HEAR JILL? GOOD. >> I'M LAURA FROM NINDS. TO FOLLOW UP ON JILL'S COMMENT, RHESS SYNDROME, FRAGILE X, DELETERIOUS, SOME GROUPS ARE TRYING RNA OR DNA APPROACHES. GUANGPING TALK ABOUT CRISPR/CAS9, I'VE SEEN DATA FOR FRAGILE X BUT EFFICIENCY SO LOW AND THERE'S CONCERN ABOUT TARGET EFFECTS, DIFFERENTIATED NEURONS WOULD BE A DECENT TARGET. AND THEN RNA EDITING GROUPS ARE DOING THAT, THAT HAS TO BE CONTINUOUSLY DELIVERED. I WAS WONDERING IF YOU COULD TALK WHETHER THAT IS AT ALL PROMISING, EITHER DNA OR RNA EDITING, AND IF THERE'S A PATH FORWARD OR ARE WE JUST SO FAR FROM EVEN THINKING ABOUT THAT? >> I CAN COMMENT A LITTLE BIT ABOUT THAT. THERE ARE GROUPS THAT HAVE COME OUT OF GENE YAO'S WORK ON CRISPR RX AND SMALL CAS 13As, FOR PROLONGED EXPRESSION. THINK OF RNAi 2.0, BECAUSE THE GUIDE RNAs MAKE THE MACHINERY MORE SPECIFIC THAN AN INHIBITORY RNA THAT ENGAGES RISK PATHWAY. AND SO I THINK THERE ARE GREAT ADVANCES. THAT'S FOR -- AGAIN WHY I ADDED THIS THING FOR EDITING. IT WAS ON THE EARLIER SLIDE. AND THAT WAS DO WE NEED TO DEVELOP BETTER METHODS TO SEE WHERE IT'S GOING AND HOW SAFE AND SPECIFIC IT REALLY IS? AND DO WE NEED TO WORK ON THE EFFICIENCY OF EDITING IN THE CNS? THERE'S QUITE A FEW PEOPLE IN THAT SPACE. IT IS ADVANCING, ON THE HORIZON, WE NEED TO BE READY FOR IT. MOST GROUPS HAVE MOVED AWAY FROM AN ACTIVE CAS9 BUT NOT ALL. IF YOU USE A DEAD CAS9, IT HAS BEENS TO EXPRESSED ALL THE TIME TO MAINTAIN THAT EPIGENETIC CHANGE, MAYBE THAT'S ANOTHER OPPORTUNITY FOR PULSATILE REGULATION, FIGURING OUT THE TIMING IS A HUGE UNMET NEED SCIENTIFICALLY. IF YOU AGREE I THINK THIS IS DEFINITELY A WAY WE NEED TO MOVE FOR REGULATED EXPRESSION FOR EDITING. >> THANK YOU FOR BRINGING THAT UP. >> BEV, THIS IS LUKE HERE. MAYBE INTEGRATING A FEW THINGS I'VE HEARD, THE WAY I LOOK AT THE PROBLEM OF OVEREXPRESSION AND REGULATION OF EXPRESSION SHOULD ACHIEVE ADEQUATE EXPRESSION AND ADEQUATE DISTRIBUTION AS WAS MENTIONED EARLIER. THAT THOSE BOTH CONTROL EFFICACY PARAMETERS AS WELL AS SAFETY PARAMETER. THE QUESTION THAT YOU ASKED BEFORE, BEV, HOW DO WE GET THERE, AND I THINK WE'RE ALL DEVELOPING TECHNOLOGICAL PARADIGMS TO WORK ON OUR FAVORITE CAPSIDS AND I THINK IT'S CLEAR TO ME THERE'S BEEN A LACK OF EMPHASIS OR INCENTIVES TO WORK ON PROMOTERS, I THINK I'VE HEARD HERE THAT WHETHER IT'S REGULATED OR SMALL PROMOTERS, THAT'S SOMETHING THAT CLEARLY SHOULD BE PUT OH THE MAP MORE. BUT THEN SECONDARILY ALSO WE KNOW THE SYSTEMS THAT WE WORK WITH ACTUALLY VERY POORLY. WE'VE BEEN THROWING DARTS ON A WHITE PAPER OVER THE PAST TEN YEARS, SOMEWHAT SUCCESSFULLY, AT THE SAME TIME TOUGH TO CONNECT THE DOTS WE'VE GENERATED TO LOOK AT PHARMACOLOGY OF AAV BECAUSE MAYBE THE OVEREXPRESSION PROBLEM IS REALLY A DISTRIBUTION, EXPRESSION IN CELL TYPE SCAG PROMOTER IS ACTIVE BUT NOT THE OTHER PROMOTER. SINGLE CELL ANALYSIS WHICH WE KNOW HAVE CAPABILITY OF OR THINGS WE LEARNED IN TERMS OF USING PET IMAGING AND TRACKING PARTICLES ARE HELPFUL. WE LIKE AAV9, I DON'T THINK WE KNOW WHERE OUR INPUT DOSE TRACKS. >> WHAT ABOUT PET TECHNOLOGIES OR MOW DAD, OTHER THAN GADOLINIUM, DO PEOPLE HAVE THOUGHTS ON EMPLOYING PET-BASED METHODS OR CAPSID LABELING METHODS? >> I THINK PET-BASED METHODS ARE REALLY EXCITING. THE PROBLEM WITH THEM IS TYPICALLY THE DEVELOPMENT OF TRACERS. VERY, VERY EXPENSIVE TO DEVELOP. BUT I TOTALLY AGREE WITH YOU THAT THEY ARE A POTENTIAL WAY OF TRACKING A GOOD -- AS LONG AS YOU GET A GOOD TRACER WITH A GOOD TARGET CAN BE VERY -- GIVE YOU A LOT OF INFORMATION. >> IF I COULD ADD TO THAT, VANTAK FROM HOPKINS RADIOLOGY. I DO THINK IMAGING EXPRESSION IS IMPORTANT. WE CAN REACH BEYOND PET, PET IS EXCELLENT QUANTITATIVE TECHNIQUE AND THERE ARE SOME REPORTER SYSTEMS, GOOD PROGRESS IN THAT AREA TO USE REPORTER GENES, PET REPORTER GENES, TO LOOK AT EXPRESSION IN THE TARGET AND FOR OFF-TARGET EXPRESSION. THERE ARE ALSO OTHER MODALITIES WE CAN EXPLOIT SO I THINK IT WOULD BE VERY IMPORTANT TO USE MULTIPLE MODALITY APPROACH WITH HIGH SENSITIVITY THERMO GRAPHIC FEATURES, IT'S GREAT FOR ORGANIS ASSESSMENT BUT INTRAVITAL MICROSCOPY WITH FLUORESCENT REPORTERS, WE CAN GET INTO PRETTY MUCH SINGLE-CELL LEVEL. AND LOOK AT THE HOMOGENEITY OF EXPRESSION OR THESE POCKETS THAT WE WERE DISCUSSING BEFORE. SO THE QUESTION IS WHETHER IT IS FEASIBLE TO CONSTRUCT REPORTERS, GENE DELIVERY SYSTEMS, AND INCLUDE IMAGING COMPONENT OR THERE HAVE TO BE SEPARATE FROM THERAPEUTIC COMPONENT. >> CAN YOU COMMENT A LITTLE BIT ON THE INTRAVITAL MICROSCOPY IN LARGE MODELS? >> THERE'S DEFINITELY LIMITATION OF THAT, INTRAVITAL MICROSCOPY GIVES US COUPLE HUNDRED MICRONS WINDOW. BUT THERE ARE SOME APPROACHES WITH GREEN LENS THAT YOU CAN INSERT CYLINDERS, REACH INTO DEEPER STRUCTURES BUT STILL LOOK LOCALLY AROUND THE GREEN LENS. BUT WITH THIS TOOL, WE CAN REACH UP TO EVEN A CENTIMETER INTO THE BRAIN. >> SO, THIS IS -- IN TERMS OF TRANSLATIONAL PROMOTERS, THIS IS THE POINT I WANTED TO MAKE. WHEN WE TRYING TO OPTIMIZE DEVELOP PROMOTERS, IT'S THE SAME ISSUE WITH CAPSIDS. THEY WORK IN A RODENT, DO THEY WORK IN A PRIMATE AND HUMAN? ONE OF THE BIG CHALLENGES WE FACE THAT I THINK THE PET IS TOUCHING ON IS A POSSIBLE SOLUTION SOMETIMES, HOW DO WE KNOW HOW WELL THINGS REALLY TRANSLATE? ESPECIALLY WITH CELL AUTONOMOUS PROTEINS, THERE'S NO WAY IN HUMANS ASIDE FROM BIOPSY, IN CNS WE DON'T WANT TO DO BIOPSIES OF THE CNS. IT'S FUNCTIONAL OUTCOMES BUT WE'RE GOING BLIND UNDERSTANDING HOW WELL THE EXPRESSION TRANSLATES FROM ANIMAL MODELS INTO HUMANS. >> CHRIS FROM ICU. I WANT TO GET BACK TO THE PET AND TELL YOU THAT THIS HAS BEEN PROVEN TO BE QUITE POWERFUL TOOL OVER THE YEARS IN THE CLINICAL MEDICINE BUT SPECIFICALLY GENE THERAPY USED IT FOR CLINICAL TRIALS RIGHT NOW THAT DIRECTLY SHOW LEVEL OF EXPRESSION, SO THIS HAS TO DO WITH ENZYME THAT WE ARE EXPRESSING FOR WHICH THERE'S A LIG AND THAT HAS TO BE CONVERTED BY EXPRESSING GENE TO BE VISIBLE ON PET. IN FACT IT GIVES US MULTIPLE INFORMATIONS, THE LEVEL OF EXPRESSION BECAUSE WE FOUND ACTUALLY THAT CORRELATES NICELY WITH THE AMOUNT OF ENZYME BEING PRODUCED IN BIOMARKERS BUT EVEN LOCATION IN THE BRAIN OF THE EXPRESSION AND GENE HAS DELIVERED WITH THE REGULAR ADMINISTRATION SO WE CAN CORRELATE VERY NICELY THE PLACE OF THE GENE EXPRESSION BUT ALSO LEVELS. BUT ALSO IN ADDITION TO LEVELS OF EXPRESSION WITH STUDY FOR HOW LONG THE GENE HAS BEEN EXPRESSED, ANOTHER BIG ISSUE, LONGEVITY OF GENE EXPRESSION. THINKING ALONG THOSE LINES WOULD PROBABLY BE VERY INFORMATIVE AS WE BRING THESE THERAPIES INTO THE CLINIC. FINDING THE RIGHT TRACER, DEVELOPING IT OF COURSE IS A CHALLENGE, BUT I THINK PROPER EXPERIENCE IN PARKINSON'S, ABC DEFICIENCY AS WE MOVE FORWARD, PRIMARY AND SECONDARY GENE EXPRESSION, THAT PARTICULAR ENZYME HAS BEEN TRANSPORTED IN THE BRAIN, THOSE ARE CRITICAL QUESTIONS AND VERY PRACTICAL AS WELL AND I THINK HOW YOU WELCOME I THINK IN DEVELOPING THIS APPROACH IN THE CLINIC. >> I THINK WHAT YOU JUST MENTIONED HELPS ADDRESS WHAT STEVE WAS TYING TOGETHER, HOW DO WE KNOW IF IT'S WORKING IN THE REAL MODEL, WHICH IS THE HUMAN PATIENT? SO I THINK THE CHALLENGE FOR US THAT I'M HEARING HERE IS COMING DOWN TO WE NEED BETTER WAYS TO SEE WHAT WE'RE DOING AND BRAIN INITIATIVE 2.0 MAKING BETTER TRACERS AND, YOU KNOW, I WANT TO BE ABLE TO SEE HEATHER'S LYSOSOMAL ENZYME IN THE BRAIN OF A LARGE ANIMAL MODEL AND KNOW IT'S THERE AND ACTIVE, AND WE HAVE A PENETRATABLE TRACER FOR THE SPECIFIC RECOMBINANT PROTEIN MAY BE JUST THE TICKET. I WANT TO TURN THE TABLE OVER TO JUDE. MIGUEL, IS THAT WHO YOU WERE POINTING TO? >> I WAS SCRATCHING MY HEAD, SORRY. [LAUGHTER] SINCE YOU PUT ME ON THE SPOT, LET ME WALK BACKWARDS, JUDE, UNC GENE THERAPY CENTER. I WANT TO WALK BACKWARDS AND CONNECT THE DOTS FROM HISTORICAL PERSPECTIVE. I THINK IN THE EARLY DAYS WHEN WE TOOK THE FIRST GENE THERAPY EFFORTS INTO THE CLINIC, WE WERE ASKING QUESTIONS ABOUT A VECTOR BIODISTRIBUTION AND TARGETING THE SAME AS TODAY, BUT WE WERE TRYING TO MINIMIZE THE OPEN-ENDED QUESTIONS. SO THE PROMOTERS THAT WERE CHOSEN WERE CHOSEN BECAUSE WE KNEW THEY WORKED IN HUMANS, SO CMV WAS ONE OF THE MOST POPULAR PROMOTERS, USED BECAUSE CYTOMEGALY WAS ESTABLISHED AS EFFICIENT IN HUMANS AND THEREFORE THAT PROMOTER WAS PUT INTO THE AAV CASSETTE. IN FACT THE FIRST DMD CLINICAL TRIAL WE DID WAS A CMV PROMOTER BECAUSE WE WEREN'T SURE IN A MUSCLE SPECIFIC PROMOTER WOULD WORK IN THE CONTEXT OF AAV IN A HUMAN. THAT LED THE COMMUNITY LOOKING AT WHAT WAS REFERRED TO AS ALREADY ESTABLISHED EXPRESSION PROFILES THAT WOULD WORK IN HUMANS. AND IT LED FROM THE VIRAL VECTOR, VIRAL PROMOTER SUCH AS CMV TO THE CELLULAR PROMOTERS, THOSE WERE THE DEFAULT REAGENTS TO REMOVE A QUESTION ON OF NOT ANSWERED AT THAT POINT, COULD YOU PUT A TISSUE SPECIFIC PROMOTER IN THE CONTEXT OF AAV AND GET IT TO WORK. AS A CONSEQUENCE TO THAT, WE ALSO HAD BIODISTRIBUTION PHENOMENON, WHICH WAS AAV8 IN A RODENT, 200% FACTOR 9, A MONEY WOULD GET 46%, BUT IN A HUMAN 2 TO 4%, SAME CAPSID, SAME DOSE, SAME PROMOTER. AND SO WHAT WE WERE LEFT WITH WAS THAT VECTOR TROPISM WAS NOT TRANSLATABLE FROM RODENT TO HUMAN. I WOULD PAUSE AND SAY WHAT'S PROBABLY NEEDED FOR THE FIELD IS A CAPSID ATLAS BIODISTRIBUTIONING -- - BIODISTRIBUTION THAT WOULD BE MADE AVAILABLE TO THE COMMUNITY HOPEFULLY WITH VECTORS IN THE HUMAN BRAIN, DIFFERENT TRACERS WHETHER THEY GO, SO IT ENDS UP BEING USEFUL AND NOT JUST AN EXERCISE THAT GETS PENALIZED BY VIRUS TROPISM. I HAVING SAID THAT I WANT TO PAUSE AND MAKE SURE WE UNDERSTAND TWO BOXES. TRANSCRIPTION IS CONSERVED THROUGH SPEECHES, WE HAVE A UNIQUE OPPORTUNITY AS BRIAN WAS MENTIONING TO GO AFTER CELL TYPE SPECIFIC PROMOTERS AND TISSUE CULTURE IN ANIMALS AND HOPEFULLY IN HUMANS AND SHOULD BE COMFORTABLE IT WILL TRANSLATE. VIRUS EVOLUTION IS NOT ABLE TO TRANSLATE ACROSS SPECIES, WHY CANINE PARVO VIRUS AFFECTS CANINES, FELINE AFFECTS FELINES. AAV -- IT'S PLAGUED BY THE FACT BIODISTRIBUTION WILL NOT REPRESENT WHAT HAPPENS IN HUMANS, STILL A COMPLETE UNKNOWN UNTIL WE GET INTO PATIENTS. SO WHILE WE CAN EVOLVE THE NEXT GENERATION OF CELL TYPE SPECIFIC PROMOTERS IN ANIMAL MODELS, IT'S UNLIKELY WE'RE GOING TO EVOLVE THE NEXT GENERATION OF PROMOTERS IN HUMANS SO WE NEED TO ACCEPT THAT AS PART OF OUR MOVING FORWARD. I THINK WITHOUT SPEAKING FOR THERESA CHEN AND FDA, ADDING ELEMENTS THAT COME FROM ORTHOGENIC VIRUSES I WOULD SAY WE'RE GETTING INTO A WITCH'S BREW OF MAKING MOLECULAR GYMNASTICS OF REGULATORY ELEMENTS WE DON'T KNOW HOW THEY ARE GOING TO BEHAVE, NOT JUST IN SHORT TERM BUT LONG TERM AND SOME OF THESE CELL TYPES. SO I ENCOURAGE THE COMMUNITY TO MOVE DOWN THE TISSUE SPECIFIC PROMOTERS BECAUSE WE EVOLVED AWAY FROM THE CONSTITUATIVE FIRST GENERATION, SECOND GENERATION. BUT STEVE BROUGHT UP THE POINT ON THE SIZE IS ALWAYS THE CONSTRAINT THAT WE ARE ALWAYS LOOKING FOR A WAY TO MINIMIZE THE SIZE OF THE PROMOTER. WHAT I WOULD LIKE TO ADD AS A SECOND CAVEAT AND ARGUE THERE SHOULD BE A DEDICATED EFFORT TO UNRAVEL THIS, IT'S THE CIS ELEMENT FROM VIRAL LECTORS ARE ALSO CONTRIBUTING TO THE PROMOTER ACTIVITY THAT WE END UP CALLING THERAPEUTIC PRODUCT. AND AS YOU ALL KNOW, THESE VIRUSES HAVE EFFICIENTLY PACKED THEIR SEQUENCES WITH LOTS OF ELEMENTS THAT CHROMOSOMES HAVE SPREAD OUT OVER THE GENOME, AND AS A CONSEQUENCE THE FIRST CF VECTOR THAT WENT IN THE PATIENTS USED AAV TERMINAL REPEAT AS THE PROMOTER. WE LATER SHOWED THOSE ACTIVITIES TODAY EVERY PROMOTER WE PUT IN A VIRAL VECTOR AND CONTEXT AT TERMINAL REPEAT IS EITHER HAVING CROSS-TALK OR THEY ARE WORKING INDEPENDENTLY. AND SO WE HAVE THE SWITCHBOARDS THAT MAY BE ACTIVE AT THE SAME TIME, DIVIDES IN CELL TYPE PROMOTERS WITHOUT THE CONTEXT OF VECTOR IS AN EXERCISE IN FUTILITY. IT WON'T DRIVE ANYTHING OF USE. AND I THINK A NUMBER OF US ARE BEGINNING TO SEE THAT PLAY OUT. IT'S EASILY DONE. YOU CAN TAKE A LIBRARY OF PROMOTERS AND PUT THEM IN AAV AND BARCODE SO WHEN YOU PUT IT IN THE CELL TYPE YOU CAN DERIVE THE ONE THAT'S GOING TO BE MOST EFFECTIVE IN THAT CELL TYPE, AND THEN YOU HAVE A CONTEXTUAL PROMOTER THAT'S EVOLVED IN PRESENCE OF THE TERMINAL REPEAT. IF YOU'RE NOT DOING SOMETHING LIKE THAT, AND YOU'RE MOVING DOWN THE MORE TRADITIONAL WORK IN TRANSGENIC, PUT IT IN AAV I CAUTION YOU THE FIRST EXAMPLE OF THE TISSUE SPECIFIC PROMOTER IN AAV FAILING WAS DONE BY OUR COLLEAGUES IN FLORIDA, WHERE THEY TOOK AN ASTROCYTE SPECIFIC PROMOTER PUT IT IN AN AAV VECTOR, IT WAS EXPRESSED IN NEURONS. LATER EACH LAB BLAMED THE OTHER ONE FOR SCREWING UP WITH THE CLONING. THE CONSEQUENCE WAS CORRECT, IT WAS EXPRESSING IN THE NEURONS. AND SO ONCE AGAIN I THINK WE'RE EVOLVING DOWN A PATH OF FINDING OUT WHAT THE RULES ARE, AND I STRONGLY ENCOURAGE DATABASE WITH ATLAS FOR VECTORS BIODISTRIBUTIONS BOTH PHYSICAL PROTEIN, THEN WE CAN LAY OTHER THE EXPRESSION PROFILES THAT EITHER DO OR DON'T MAKE SENSE. AFTER THAT I THINK EXPRESSION PROFILES WILL FALL OUT WHERE WHEN THEY ARE IN THE CLINIC LIKE THE JET PROMOTER THAT STEVE MADE AND TESTED, AND MUSCLE SPECIFIC PROMOTERS WILL GET PASSED ON HISTORICALLY TO THE NEXT GENERATION OF VECTORS TARGETING DISEASES. I WAS SCRATCHING MY HEAD SO. >> I THINK THESE ARE EXCEPTIONAL POINTS, JUDE. I WANT TO COMMENT THE VIRAL OR TISSUE-SPECIFIC PROMOTERS IN THE PAPER OF OURS THAT JUST CAME OUT THAT GUANGPING GAO ALLUDED TO WE TESTED A LIVE SPECIFIC PROMOTER ADVANCED TO THE CLINIC AND SHOWED VERY EFFICIENTLY TRANSDUCES LEAN CELLS, KIDNEY CELLS AND THE BRAIN. MOST LIKELY THROUGH CONTRIBUTION OF ITR AND CAPSID. HOW ARE WE DOING ON TIME? WE HAVE FIVE MINUTES. WE HAVE GOOD MARCHING ORDERS. XANDA? >> IT'S OFF TARGET BUT I WANT TO JUST BRING UP YOU CAN NOW USE HUMAN iPS CELLS AND MAKE ORGANOIDS WHERE YOU CAN GET A NUMBER OF DIFFERENT CELL TYPES, AND THEN OF COURSE DELIVERY, I'M NOT SURE HOW YOU WOULD DO DELIVERY TO MAKE IT REALISTIC BUT YOU CAN DISASSOCIATE ORGANOIDS AND EVEN DO SINGLE CELL SEQUENCING TO GET LEVELS OF PARTICULAR MESSAGES SO I THINK THERE ARE SOME NEW TECHNOLOGIES THAT COULD HELP ANSWER SOME OF THE QUESTIONS. >> THE NICE THING ABOUT ORGANOIDS YOU CAN MAKE THEM FROM PATIENTS WHERE EXPRESSION PROFILES ARE LIKELY TO BE VARIABLE FROM A NORMAL. BUT AGAIN WE MISSED THE NETWORK. THAT MIGHT BE IMPORTANT. MIGUEL? WAIT, WE HAVE ONE IN THE BACK. >> THAT'S OKAY. GO AHEAD, MIGUEL. >> ONE OF THE THINGS WE HAVEN'T TALKED ABOUT WE TOOK FOR GRANTED WE KNOW HOW TO DO CSF DELIVERY. I'M GOING TO SPEAK FOR MYSELF, NOT FOR EVERYONE ELSE. WE JUST PUT A CATHETER IN, INJECT IT. HOPE FOR THE BEST. I MEAN, I THINK SOME EFFORT TRYING TO UNDERSTAND PRESSURES, SLOW DELIVERY, FAST DELIVERY, TRY TO REALLY UNDERSTAND THE PHYSICS OF IT I THINK IS FUNDAMENTAL. I'M NOT ONE TO DO THAT BECAUSE I COULDN'T TELL YOU THE FIRST THING TO DO IN TERMS OF PHYSICS, BUT IT SOUNDS THAT SO FAR CSF WOULD HOPE FOR THE BEST. INJECT THINGS WELL, LET'S HOPE IT GETS WHERE WE WANT IT TO GET, RIGHT? THERE'S A LOT TO BE LEARNED IN TERMS OF BASIC DISTRIBUTION, WHAT REGULATES RESIDENCE TIME AND I THINK ALL THOSE THINGS ARE CRITICAL TO REALLY UNDERSTAND PROFILES. SO FAR EVERYTHING I'VE SEEN IS HOPE FOR THE BEST AND WELL, SOMETIMES IT WORKS, SOMETIMES IT DOESN'T. I SEE A LOT OF PARALLELS BETWEEN SMALL MOLECULES siRNAs, BUT THAT'S ONE PARAMETER WE HAVEN'T TOUCHED UPON, SORT OF UNDERSTANDING. CHRIS HAS DONE A GREAT JOB IN TERMS OF PARENCHYMAL INJECTION AND DELIVERY BUT WE NEED TO APPLY THE SAME TYPE OF APPROACH TO CSF DELIVERY BECAUSE THERE'S MULTIPLE THINGS WE DON'T NEED TO TALK ABOUT BUT AN IMPORTANT ASPECT TRY TO UNDERSTAND UNDERPINNINGS THAT DETERMINE DISTRIBUTION AND RESIDENCE TIME. >> I WOULD LIKE TO ADD ONE MORE POINT TO EXCELLENT POINTS MIGUEL HAS MADE. IT'S A COMPLEX QUESTION THAT REQUIRES I HOPE FROM NINDS SUPPORT BECAUSE JUST AS WE DEVELOPED INTRAPARENCHYMAL, INTRATHECAL IS MORE COMPLEX, BUT PLEASE REMEMBER AT LEAST MY GROUP FOUND PREDICTABILITY OF GENE EXPRESSION, IT'S REALLY DRIVEN BY THE CONCENTRATION OF THE VECTOR BEING GIVEN INTO SPECIFIC VOLUME OF TISSUE. ALWAYS DRIVEN BY THE NUMBER OF PARTICLES THAT YOU CAN EXPOSE THE CELLS TO. BUT CSF, WE ARE DILUTING DRAMATICALLY VERY QUICKLY SO I THINK THAT IS ANOTHER ISSUE THAT I THINK IT'S SOMETHING TO REALLY CONSIDER BECAUSE UNPREDICTABLE LEVELS OF EXPRESSION FROM OCCIPITAL OR LUMBAR INFUSIONS I THINK HAS TO DO WITH THE FACT YOU'RE DUMPING INTO PRETTY LARGE VOLUME OF CSF A NUMBER OF PARTICLES, QUICKLY GET DILUTED OUT, I THINK THE PROMOTER, TYPE OF VECTOR BEING USED, WILL DRIVE THE TYPE OF EXPRESSION WE'LL BE GETTING SPECIFIC CELLS SO I THINK THAT UNLESS WE SOLVE THAT ISSUE I THINK EVERYTHING WE'LL BE DISCUSSING IN TERMS OF HOW GREAT PROMOTERS ARE AND PARTICLES WE MAKE WILL BE USELESS IN CLINICAL ENVIRONMENT BECAUSE IT WON'T ACHIEVE THE CLINICAL TRANSDUCTION LEVELS THAT YOU'RE HOPING FOR, FOR THE THERAPEUTIC EFFECT >> CYNTHIA? >> YEAH, CYNTHIA TIF, NHGRI. IN FOLLOW-UP WE RECENTLY USED PATIENT CELLS TO MAKE CEREBRAL ORGANOIDS. THEY HAVE VENTRICULAR-LIKE CAVITIES, WE COULD SEE EXPRESSION. IT WAS SPOTTY AROUND THE ORGANOID AND IT'S A GREAT IDEA THAT YOU COULD THINK TO TAKE THOSE APART AND DISASSOCIATE AND DO SINGLE CELL ANALYSIS BUT WE DID IT FOR GM-1 VECTOR WE RECENTLY PUT IN HUMANS. >> ONE MORE POINT >> OKAY. >> IN TERMS OF THE PROMOTERS, WHAT WE'VE BEEN TALKING ABOUT IN TERMS OF DEVELOPING NEW PROMOTERS AND REALLY DEVELOPING TRANSLATIONAL PROMOTERS I THINK ANY KIND OF SCREENS THAT WE'RE DOING NEED TO BE DONE IN RODENT CELLS AS WELL AS iPSC DERIVED HUMAN CELLS. YOU REALLY CAN'T HAVE SOMETHING THAT WORKS IN A HUMAN, DOESN'T WORK IN THE ANIMAL MODEL. AND VICE VERSA. BUT THE LAST THING I'LL MAYBE CAUTION WITH THE ORGANOIDS OR WITH ANY KIND OF IN VITRO SELECTION IS WHEN WE CULTURE iPSC NEURONS, OR iPSC GLIA, THEY ARE DEVELOPMENTALLY IMMATURE, COMPARED TO WHAT WOULD BE IN A MATURE HUMAN BRAIN. AND WE REALLY GOT BURNED ON THIS WITH TRYING TO DEVELOP THE MECB 2 PROMPTER BECAUSE WHEN WE USE THAT IN CULTURED NEURONS, IT DOESN'T WORK. IT DOESN'T EXPRESS. WHEN WE PUT THAT INTO A MATURE BRAIN IN AN ANIMAL IT EXPRESSES ROBUSTLY. AND THE OPPOSITE COULD HAPPEN. WE MAY SCREEN OUT AND IDENTIFY PROMOTERS THAT WORK IN IMMATURE NEURONS AND GLIA, THAT GET SHUT DOWN IN A MATURE BRAIN. SO JUST A WORD OF CAUTION TO BE CAREFUL ABOUT WHAT WE'RE DOING THERE. >> A QUICK POINT, WE ALSO HAVE THE OPTION OF HUMAN SLICES FROM EPILEPTIC TISSUE WHICH WILL ADDRESS THE DEVELOPMENTAL CHALLENGE. >> YEAH, MORE IN TERMS OF MOVING AS QUICKLY AS POSSIBLE WITH YOUR SELECTED PROMOTER, IN VIVO SETTING, FROM OUR EXPERIENCE IT'S SO DIFFERENT. WHETHER IT DOESN'T WORK OR DOESN'T TRANSLATE, MOST OF THE CASES WHAT HAPPENED. >> I WANT TO CHALLENGE JUDE FOR A SECOND. CAN YOU INSULATE OUT THE RTRs? >> IT'S BEG TRIED NOW TO PUT ELEMENTS BETWEEN THE ITR AND PROMOTER AND SEE IF IT WILL BUFFER ACTIVITY. I DON'T THINK THERE'S ENOUGH EXPERIENCE TO UNDERSTAND ENHANCING, BINDING OVER THE SECOND PROMOTER. >> SO, WHY DON'T WE TAKE -- HOW LONG IS THE BREAK? 15-MINUTE BREAK. AND PLEASE COME BACK AND THIS IS GOING EXTREMELY WELL. SO COME BACK IN 15 AND WE'LL CONTINUE THE DISCUSSION. [ RECESS ]. SESSION 1B, WE TALKED ABOUT PROMOTERS SO WE DON'T NEED TO GO BACK THERE, BRINGING BACK SOME COMMENTS LUKE BROUGHT UP ABOUT CAPSID EVOLUTION AND I THINK GUANGPING SHOWED US BEAUTIFUL STUFF OUT OF HUMANS, DETECTING AND MINIMUM ICING OFF-TARGET EFFECTS FOR EDITS, ASOs, ET CETERA. I WANT TO SUMMARIZE THE HIGH POINTS AND IF I MISS ANYTHING BRICK -- BRING IT UP AT THE NEED. WE NEED AN ALLEN VIRUS ATLAS. FOR THOSE THAT USE THE ALAN BRAIN ATLAS A KEY BOOK MARK ON YOUR COMPUTER, YOU'LL KNOW WHAT I'M TALKING ABOUT, I LIKE THE IDEA. MAYBE WE CALL IT THE JILL AND CHRIS OR WALTER AND NINA VIRUS ATLAS BUT KNOWING WHERE THESE THINGS GO IN MODEL SYSTEMS IS PARAMOUNT. ANOTHER, IMPROVE METHODS TO DETERMINE BIODISTRIBUTION, WE HAD A LOT OF DISCUSSION ABOUT THE REAL NEED FOR NOVEL DEVELOPMENT IN THE PET TRACER, RADIO LIGAND FIELD TO BEGIN TO SEE WHERE THE ACTIVITY OF OUR GENE PRODUCT IS IN A TEMPORAL WAY. ANOTHER WAS TO USE SINGLE-CELL ANALYSIS POST GENE DELIVERY TO EVALUATE LEVELS OF EXPRESSION HOW IT MAY VARY BASED ON DIFFERENT DELIVERY STRATEGIES. THAT'S SUMMARIZE THE MORNING. NOW WE'LL OPEN IT UP FOR FOCUSED TARGETING. >> CAN I ASK A QUESTION TO GET CLARIFICATION, THE ATLAS IS A DISTRIBUTION ATLAS, THE QUESTION IS DISTRIBUTION OF THE VIRUS, AND THE SECOND LEVEL QUESTION IS HOW DOES THAT CORRELATE WITH DISTRIBUTION OF THE EXPRESSION, TWO LEVELS, OKAY. AND THEN -- >> HOW DO WE MODULATE SUCH FOR OUR USES >> AND THEN SO I THOUGHT YOU WOULD COULD POTENTIALLY AAV WITH A RADIOACTIVE TAG OR EXPRESSING A REPORTER AS OPPOSED TO YOU HAVE YOUR GENE BUT MIX IN A LITTLE BIT? >> THIS IS JUDE. YOU WANT TO PHYSICALLY LABEL THE VIRUS. WHAT YOU NEED IS THE FINGERPRINT OF THE VIRUS ITSELF'S DISTRIBUTION, AND THEN IT ALLOWS WHAT MARK WAS SAYING TO TOTALLY UNINNOVATIVE SCREENING OF PROMOTERS AND HOW THEY PERFORM IN A CERTAIN CAPSID WITH A CERTAIN ATLAS, BIODISTRIBUTION. YOU MAY FIND OUT THAT THE VIRUS IS IN MANY LAYERS OF THE BRAIN BUT THE PROMOTER IS ON CERTAIN SECTIONS, LETS THE INVESTIGATOR REALIZE IF HE WANTS ADDITIONAL CELL TYPES THE VECTOR WILL ALREADY DEPOSIT THE GENOME THERE, THE PROMOTER IS FUNCTIONAL, YOU NEED TWO SEPARATE APPROACHES, PHYSICAL DISTRIBUTION OF THE CAPSID ITSELF THAT LAYS DOWN THE BLUEPRINT AND ON TOP BEGIN TO LAYER MANY CELL TYPE SPECIFIC PROMOTERS. IF THEY DON'T SHOW UP IN THE RESPECTIVE BLUEPRINT WE HAVE AN ANOMALY. IF IT'S 100% THAT WILL MIMIC WHATEVER CAPSID DISTRIBUTION SO PERFORMING IN HUMANS. >> AFTER LUNCH WE'LL HAVE A DETAILED DISCUSSION OF OTHER METHODS. >> GREAT. AT THE CULMINATION WE WILL SUMMARIZE STRATEGIC GOALS FOR YOU. WE'LL BE ABLE TO SEE WHAT WE'RE GOING TO BE DOING ON FOCUSED TARGETING DOWN THE ROAD, HOW ARE WE GOING TO ACCOMPLISH THAT? WE HEARD ABOUT CAPSID ENGINEERING AND SOME REALLY NEW CAPSIDS YOU'RE PULLING OUT OF HUMAN CELLS, AND DO YOU THINK WE'RE DONE, MORE WORK TO BE DONE, BROADER EFFORT THAT SHOULD BE ACCOMPLISHED, WE SHOULD DO IT AND SEE HOW UNIFORM THIS IS FOR MANY HUMANS? I'M QUESTIONING MY CO-CHAIR, GUANGPING GAO. >> I DON'T THINK WE'RE DONE AS A FIELD. WE MAY STILL HAVE OPPORTUNITIES. WE START EARLY STAGE PULLING FOR MORE VECTORS FROM HUMANS AND MONKEYS. WE STOPPED FOR A WHILE. AFTER A WHILE YOU FOUND ALMOST EVERYTHING THE SAME BUT IF YOU USE DIFFERENT POPULATION, THEN SITUATION MAY BE DIFFERENT. IF YOU FOCUS ON U.S. POPULATION, VERSUS YOU GO TO EUROPEAN POPULATION, ASIA POPULATION, MAYBE DIFFERENT. WE MAY STILL HAVE SOME CHANCE. BUT OVERALL AS OUR EARLY CONSENSUS I THINK WE MAY HAVE SOME BUT YOU CAN FIND ANOTHER AAV8 OR AAV9, NOT SURE UNTIL WE HAVE FURTHER ASSESSMENT. >> CASEY MAGUIRE, OTHERS ALSO, ARE MAKING HUGE LIBRARIES WITH DIFFERENT VARIANTS OF THE PEPTIDE, AND FINDING AMAZING RESULTS. THEY SELECT A TISSUE THEY ARE GOING AFTER AND CAN GET DRAMATICALLY INCREASED DELIVERY TO PARTICULAR CELL TYPE IN THE BRAIN. MODIFIED FROM AAV9 BUT THEY HAVE DIFFERENT PEPTIDES. AND HE BASICALLY DOES IT IN MICE FIRST AND THEN WILL DO IT IN SOME KIND OF AN IMPLANTED ORGANOID IN XENOGRAFT SITUATION TO SEE IF IT WILL GO TO HUMAN. >> MANY, MANY GROUPS ARE DOING SOMETHING SIMILAR. >> I COULD COMMENT FIRST WITH THE BIG PICTURE NOTE, ENGINEERING CAPSID ITSELF FOR CELL TYPE SPECIFICITY IS VALUABLE OVERALL, IT CAN FREE UP REALLY ESTATE FOR GENE REGULATORY ELEMENTS, A WAY TO GET SPECIFICITY. EPILEPSY, YOU NEED SPECIFICITY. IN THAT CASE A WEAK PROMOTER MIGHT HAVE ADVANTAGES BECAUSE WEAK PROMOTERS CAN BE MORE SPECIFIC. SO KNOWING THE ISSUE OF ENGINEERING, CHALLENGES HAVE BEEN MENTIONED, TRANSFERABILITY ACROSS SPECIES, AND CELL TYPING. CELL TYPES IN RODENTS, IN HUMANS MIGHT HAVE DIFFERENT RECEPTOR PROFILE AT THE MEMBRANE AND IN TERMS OF EXPRESSION SOMETIMES WE FORGET ONCE THE VIRUS IS INTERNALIZED THERE'S MANY STEPS WHERE THINGS CAN BREAK DOWN. AND THESE PROCESSES MIGHT BE DIFFERENT IN, FOR EXAMPLE, DIVIDING VERSUS NON-DIVIDING CELLS, HUMAN VERSUS RODENT CELLS, A COMPLEX PLATFORM. THE DIRECT EVOLUTION APPROACH AND HIGH-THROUGHPUT SCREENING GIVES YOU WHAT YOU ARE LOOKING FOR SO IF YOU'RE SCREENING IN A RODENT YOU'RE LIKELY TO GET A SUCCESS CASE IN RODENT. THAT MIGHT OR MOST OFTEN WILL NOT TRANSLATE INTO HUMANS. WHAT'S IMPORTANT IS THINK AS A COMMUNITY THE RIGHT SCREENING APPROACHES AVAILABLE TO US AND SOME WERE MENTIONED ORGANOIDS THAT CAME FROM YOU, XANDRA, AND HUMAN SLICES TO ALLOW US TO CLOSELY MIMIC THE HUMAN ENVIRONMENT. THIS ASIDE IT'S A POWERFUL PATHWAY TO TAKE THE CAPSID AND EITHER BY MUTATION, PEPTIDE INS, THERE'S NUMEROUS METHODS TO CHANGE THE LANDSCAPE, CELL TYPE SPECIFICITY AND MIGHT BIAS ORGAN PROFILE. OF NOTE JUST AS YOU DO POSITIVE SELECTION FOR PARTICULAR FEATURES, YOU COULD WORK TOWARDS NEGATIVE SELECTIONS FOR ITEMS SUCH AS LET'S REMOVE EXPRESSION FROM THE LIVER. SO THERE'S A LOT OF POINTS THERE SO I'LL PAUSE AND SEE? WE WANT TO ELABORATE ON ANY OF THOSE. >> LUKE? >> SO YOU MAY WANT TO BRING A DIFFERENT PERSPECTIVE BUT FIRST ACKNOWLEDGE MANY OF US ARE INFATUATED WITH CAPSIDS AND SPENT A LOT OF TIME ON IT, SOME OF US MORE THAN OTHERS, FOR ALL THE RIGHT REASONS. ACTUALLY THE CURRENT MOMENT IN TIME MANY GROUPS ARE DOING UNBELIEVABLY REFINED AND INNOVATIVE THINGS IS ULTIMATELY HOPEFULLY GOING TO GET US PLACES TO THE NEXT GENERATION OF TECHNOLOGIES, PROBABLY TOUGH TO GET CONSENSUS ON THE BEST METHOD BECAUSE WE ALL HAVE OUR BETTER IDEAS BUT IT'S WORTH THE DISCUSSION. I WANT TO BRING UP THERE ARE SOME FUNDAMENTAL QUESTIONS, THAT WE HAVE NOT BEEN ABLE TO ADDRESS OVER THE PAST 40 YEARS STUDYING THE VIRUS, CLINICALLY HIGHLY RELEVANT, ACTUALLY AT SOME LEVEL EMBARRASSING IN TERMS OF WE'RE NOW GETTING CLINICAL DATA AND TRYING TO FIX THINGS BUT THE ONLY THING WE HAVE IS AN AGNOSTIC TECHNOLOGY DEVELOPMENT APPROACH TOWARDS GOING AFTER THESE TO BRING UP ONE THING, THE EPISOMAL STATE OF THE DNA IN THE CELL, AND HOW THAT IMPACTS STABILITIES, WHICH OF THE FORMS THAT ARE THERE ARE TRANSCRIPTIONALLY ACTIVE, THESE ARE I THINK INCREDIBLY RELEVANT POINTS TO STUDY BUT INCREDIBLE RETURN OF INVESTMENT IN WE FIGURE THEM OUT IN TERMS OF ADDRESSING SOMEI ISSUES IN THE CLINIC. I WANT TO MAKE SURE THEY ARE NOT OVERLOOKED WHEN WE HAVE THE CAPSID DISCUSSION. >> EXCELLENT POINTS. >> DUKE UNIVERSITY. I DIDN'T WANT TO SPEAK OUT OF TURN. THE DEBATE SORTED WITH THE QUESTION OF VIRUS DIVISION OVER EXPRESSION LEVELS IN DIFFERENT CELLS. AND THE PIECES THAT WE HAVE ARE THE CAPSID, THE PROMOTER AND DIFFERENT ANIMAL MODELS, CELLS, TISSUES, DEPENDING WHAT YOU'RE LOOKING AT. TENDENCY HAD BEEN TO LOOK AT ONE PLANE, CAPSID AND PROMOTER OR PROMOTER AND CELL TYPE OR CAPSID AND CELL TYPE. SO I THINK ANYTHING WE DISCUSS AND TRY TO DO NEEDS TO BE IN THE CONTEXT OF THIS INCREASING NUMBER OF DIMENSIONS WE'RE HAVING TO DEAL WITH AND ATLAS MAP NEEDS TO TAKE THAT INTO HE ACCOUNT. THE CAPSIDS, WHAT IS THE BIOLOGY OF THE CAPSID, WHAT DOES IT LEND ITSELF TO IN THE FIRST PLACE? MAYBE THERE'S NOT A POSSIBILITY OF GETTING ONE PERFECT COPY OR TEN COPIES IN EVERY CEREAL. CELL. MAYBE THE VIRUS CANNOT ACHIEVE THAT. WHAT ARE THE STEPS THAT PREVENT THAT FROM HAPPENING? AFTER ENTRY, SUPPOSED ENTRY, WE'RE SEEING DATA AND OTHER GROUPS PUBLISHED AS WELL THAT THE CAPSID IN THE EARLY STAGES SEEMS TO IMPACT TRANSCRIPTION, SEEMS TO BE ATTACHED TO GENOME AND PULLING TRANSCRIPTION AND HOST FACTORS TO THE MIX TO CONTROL THIS EXPRESSION PATTERN. AND THE THIRD LEVEL THE SPECIES SPECIFIC DIFFERENCES, WE'VE IDENTIFIED SPECIES-SPECIFIC HOST RESTRICTION FACTORS OR ESSENTIAL FACTORS, THAT ADDS ANOTHER LAYER TO THE WHOLE PROCESS. ON THE CAPSID AT LEAST ONE OF THE THINGS WE'VE STARTED DOING EVOLVING ACROSS SPECIES, MICE, PIGS, MONKEYS. AND THEN EVENTUALLY THE HOPE IS MAYBE IF YOU ADAPT MULTI-SYSTEM APPROACHES MAYBE YOU'RE GOING TO GET VERSIONS THAT ARE COMPATIBLE OR A BUNCH OF BEAUTIFUL MOUSE CAPSIDS AND MONKEY CAPSIDS AND PIG CAPSIDS BUT I WOULD ARGUE THE SAME RATIONALE COULD APPLY TO PROMOTER SPACE, ENHANCER SPACE, WE HAVE TO LOOK AT CAPSIDS IN CONTEXT OF PROMOTERS AND ENHANCERS BECAUSE IT SEEMS THE TRANSCRIPTION CONTROL AND OTHER HOST RESTRICTION FACTORS ALL SEEM TO KICK IN AT THAT LEVEL. SO OVERALL I THINK IT'S A MULTI-DID I -- MULTI-DIMENSIONAL PROBLEM. EVERY TIME YOU THINK ABOUT THIS, IT'S GOT TO BE A DOT IN THE 3D OR 4D MATRIX. >> GO BACK TO NINA'S SLIDE ABOUT BASIC BIOLOGY, TO THE TRANSLATIONAL AND CLINICAL, AND THINK ABOUT THE BASIC BIOLOGY THAT WE DON'T UNDERSTAND, THAT IF WE DID UNDERSTAND COULD ENABLE US TO TARGET SPECIFIC CELLS AND TISSUE TYPES AND IF WE THINK ABOUT THAT WE UNDERSTAND VERY, VERY LITTLE ABOUT HOW THE CAPSID INTERACTS WITH THE TRANSGENE, AND HOW THAT VARIES BETWEEN CELLS, SO THANK YOU, IRVING, FOR BRINGING THAT UP. NINA? >> I THINK NOT TO INCUR WRATH BY ADDING YET ANOTHER DIMENSION BUT PEOPLE HINTED AT IT ALL MORNING, THE NOTION OF SUPERIMPOSING THE SPATIAL AND CELL TYPE ATLAS, A TEMPORAL ATLAS, AND TO THINK ABOUT TIME FROM THE STANDPOINT OF LONGITUDINAL DEVELOPMENTAL TIME BUT ALSO DIURNAL TIME. I MEAN, EVEN AT ONE DEVELOPMENTAL MOMENT, OVER THE COURSE OF A DAY OR OVER THE COURSE OF CIRCUMSTANCES, ENVIRONMENTALLY CHANGING, EXPRESSION OF A PARTICULAR GENE HAS TO CHANGE AS WELL. I TEND TO THINK OF THAT AS BEING A PROMOTER REGULATED THING BUT I ACTUALLY -- AS I THINK ABOUT IT SITTING HERE I DON'T KNOW WHY ANY OF THESE OTHER THINGS COULDN'T BE USED TO REGULATE IT AS WELL. I MEAN, I THINK WE JUST KNOW MORE ABOUT IT AT THE PROMOTER LEVEL BUT YOU COULD REGULATE ENTRY TO BE THERE AT A CERTAIN TIME AND NOT AT ANOTHER. SO -- >> THINKING ABOUT THE CELL AS THE ENVIRONMENT OF THE CELL AND HOW THAT IMPACTS THE CAPSID, IRVING AND CHRIS. >> SO, AGAIN, I TOTALLY AGREE. I THINK THERE ARE THREE SORT OF MORE VAGUE DIMENSIONS THAT THE FIELD HASN'T TOUCHED ON, THERE'S SOME DATA EMERGING. ONE IS GENDER DIFFERENCES, WE'RE TALKING ABOUT DIFFERENT EXPRESSION PATTERNS BASED ON THAT. TWO IS I'M STARTING TO SEE DATA THAT CIRCADIAN RHYTHM CAN AFFECT GENE COMPRESSION PATTERNS, PROMOTERS WORK THAT WAY. SOMEONE ASKED ME DURING ONE OF MY TALKS IF THE MICROBIOME AFFECTS GENE THERAPY, AND THAT'S JUST A COMPLETE HAND WAVE THERE BECAUSE WE HAVE NO IDEA. SO I AGREE, I THINK THERE ARE ADDITIONAL DIMENSIONS TO ADD THERE. ON THE TOPIC OF SPECIFICITY TO CELLS AND TISSUES, ONE MORE POINT I'LL MAKE. AT LEAST FROM THE CAPSID PERSPECTIVE, THE UNDERSTANDING THAT SEEMS TO BE EMERGING IN THE FIELD IS IT'S ABOUT RELATIVE CONCENTRATIONS OR LEVELS OF RECEPTORS IN SEPARATE CELLS AND TISSUES, AS OPPOSED TO EXQUISITE PRESENCE OF A SINGLE RECEPTOR IN ONE CELL TYPE OR THE OTHER. SO FOR SURE I THINK AT LEAST THE PERSPECTIVE IS YOU CAN GET PREFERENTIAL ENTRY TO CERTAIN CELL TYPES. BUT I DON'T THINK IT'S ESSENTIALLY WHAT WE WOULD CALL SPECIFICITY OR TARGETING AT THAT POINT. I THINK CAPSID ALONE IS NOT GOING TO DO THAT. >> CHRIS AND THEN KIRSTEN WAS UP. CHRIS, BRIEFLY. >> SURE. SO I THINK THE POINT I WISH TO BRING UP, BECAUSE I THINK IN TERMS OF ATLAS AND HOW WE DISCUSS THE ISSUES OF ENGINEERING CAPSIDS AND GETTING THE EXPRESSION THE WAY WE LIKE TO HAVE IT, SPECIFIC CELLS THAT WE ARE TARGETING, I THINK, I HOPE MOST OF YOU WOULD AGREE AT THIS POINT WE HAVE A LOT OF HETEROGENICITY IN TERMS OF GENE EXPRESSION, A LOT OF OFF-TARGET EFFECTS. IF WE TARGET WITH SPECIFIC VECTOR MAY NOT ALWAYS GET THE EXPRESSION, THE CELL TYPE WE'RE AFTER. SO THERE'S A LOT OF I THINK EVIDENCE A WIDE RANGE OF CELL TRANSDUCTION. BEV MENTIONED IN SOME INSTANCES WE HAVE A TREMENDOUS AMOUNT OF COPIES PER CELL. THE QUESTION IS WHAT DO WE KNOW ABOUT WHAT IT MEANS TO NORMAL FUNCTION OF THE CELL BECAUSE IN THE CONTEXT OF THE ATLAS, THE PART OF THE BRAIN THAT'S NOT BEEN TRANSDUCE IT'S HAS A SPECIFIC FUNCTION WHAT HAPPENS WHEN HAVE YOU HIGHLY UNREGULATED LEVELS OF GENE EXPRESSION WILL LAST FOREVER, IS THAT GOING TO AFFECT NORMAL FUNCTION OF THE BRAIN, THAT SOMETHING WE SHOULD START THINKING ABOUT BECAUSE WE DO GET QUITE HIGH LEVELS OF EXPRESSION, ESPECIALLY WITH PARENCHYMAL ADMINISTRATION, THAT TO ME IS SOMETHING WE SHOULD EQUALLY PAY ATTENTION TO, SIMPLY BECAUSE OF THE HUGE SAFETY FACTORS AS WE MOVE FORWARD HAVE DEVELOPING TREATMENTS IN PATIENTS. >> CARSTON BONAMIN, NINDS. CAPSID TARGETING AND DETARGETTING, SAFETY AND SCALABILITY FACTORS PARTICULARLY MOVING TO SYSTEMIC ADMINISTRATION IN LARGER PEOPLE, LIMITING FACTOR OF SAFETY IS TOTAL DOSE DELIVERY. IN ANY ENGINEERING THAT ALLOWS US TO DECREASE TOTAL VIRAL LOAD THAT WE HAVE TO GIVE IN A LARGER PERSON IN SYSTEMIC DELIVERY BY DETARGETTING SINKS AND IMPROVING TARGETING OF THE TARGET TISSUE WE'LL BE IMPROVING SAFETY AS WELL AS CLINICAL EFFICACY. >> ABSOLUTELY. ABSOLUTELY. AND LUKE WAS INFERRING THAT MAKING THE BEST CAPSID TO USE LESS. I WANT TO COME BACK TO THE POINT IT'S REALLY A PRETTY BAD VECTOR, AAV. IT TAKES A TON TO GET INTO CELLS. WE REALLY DON'T UNDERSTAND THE BASIC BIOLOGY OF WHAT HAPPENS ONCE IT'S IN A CELL IN VIVO, IN THE BRAIN, IN GLIA, NEURONS, MICROGLIA, WE MAY KNOW A LITTLE BIT MORE IN THE LIVER. AND, AGAIN, STRATEGIES TO UNDERSTAND WHAT'S HAPPENING WITH THOSE GENOMES IN NORMAL ANIMALS, AND HOW THAT IMPACTS NORMAL FUNCTION, AND THEN HOW THAT MAY CHANGE IN DISEASE OR TEMPORALLY IN A DIURNAL FASHION, AS WE DEVELOP FROM A YOUNG BRAIN TO AN OLD BRAIN, STEVE WAS ALLUDING TO THAT. SO, AGAIN, PUSHING ON THAT. >> JUDE FROM UNC. BEV, WHO USED TO BE MY FRIEND ... [LAUGHTER] I THINK YOU TOUCHED ON SOMETHING, I WOULDN'T CALL IT A REALLY BAD VIRUS. I THINK WHAT I WOULD SAY IS IT'S AN ELEGANT VIRUS THAT EVOLVED DIFFERENT END GAME. ITS OBJECTIVE IS TO GO LATENT AND DORMANT AND NOT WAKE UP THE IMMUNE SYSTEM AND/OR CREATE A PROBLEM. WHAT WE'RE TRYING TO TURN IT INTO IS A VERY ROBUST AND EXPRESSION TYPE OF VIRUS AND YOU'RE FIGHTING AGAINST EVOLUTION IN THE SENSE THAT IT HASN'T EVOLVED TO DO WHAT WE'RE TRYING TO MAKE IT DO AND MAYBE THE LIBRARY APPROACHES AND PROMOTERS WILL OVERRIDE SOME OF THOSE SALIENT POINTS, BUT LONG LONG TIME AGO A PERSON IN THE LAB DID A VERY SIMPLE SET OF EXPERIMENTS, JACKY STILLWELL INFECTED CELLS WITH AD VERSUS AAV AND LOOKED AT THE PROFILE OF WHAT WAS HAPPENING, WITH RESPECT TO GENES GOING UP, IMMUNE SIGNALING, ADENOVIRUS HAD THE CLASSIC TURN I DON'T KNOW OF EVERYTHING, INTERFERON, AAV DAMPENED ALERT SIGNALS. WE'RE BORROWING ONE ASPECT OF THE VIRUS EVOLUTION TO SLIDE INTO ORGANS WITHOUT CREATING A KIND OF ENVIRON, MOVE THEATER TYPE SIGNAL, AND YET WE'RE THEN ASKING FOR IT TO DO SOMETHING THAT IT HASN'T EVOLVED AND HAS BECOME VERY ROBUST AND PRODUCED LOTS OF EXPRESSION AND WHERE IT'S TYPICALLY GOING SILENT AND LAYING DORMANT UNTIL AN ADENOVIRUS OR HELPER COMES BACK IN. THERE IS A DICHOTOMY IN WHAT WE'RE TRYING TO MAKE IT DO AND WHAT'S IT'S EVOLVED TO DO. >> VERY GOOD POINT. JOHN? AND THEN FRASIER. >> THERE WE GO. OKAY. YEAH, I THINK NOT TO FIGHT WITH JUDE BUT THE IDEA OF IT BEING A BAD VIRUS COMES FROM 10 TO THE 5th FOR AAVs, MOST VIRUSES ESPECIALLY REPLICATING VIRUSES ARE 10 OR 100, I THINK THIS CAME UP IN THE PRE-DISCUSSION ON THE THIRD SESSION ABOUT -- SOMEBODY MENTIONED EARLIER BRINGING UP REVISITING LENTIVIRUSES, BUT ALSO I THINK IT'S WORTHWHILE CONSIDERING HERPES VIRUSES WHICH CAN BE NON-PATHOGENIC IN THE BRAIN AND THERE ARE SEVERAL PEOPLE, LAST THREE DECADES, HAVE WORKED ON HERPES IN VARIOUS WAYS. AND IT ALSO HAS POTENTIAL ADVANTAGES OF BEING TRANSPORTABLE WITHIN NEURONAL PATHWAYS, WHICH CAN INCREASE THE -- POTENTIALLY THE -- REDUCE THE AMOUNT YOU ACTUALLY HAVE TO DELIVER TO GET SPECIALLY FOR GLOBAL DISEASES, CHANGES. THE OTHER THING I THINK THAT'S RELEVANT TO THAT WHEN WE START SCALING UP TO ADULT HUMANS, WE'RE BUMPING INTO INABILITY TO PRODUCE THE DRUG, AND YOU'RE PUTTING IN, YOU KNOW, HUGE AMOUNTS MORE OF THE DRUG AS IT WERE THAN YOU EVER GET EFFECTIVE TRANSDUCTION WITH. SO -- >> CAN YOU COMMENT BRIEFLY ON THE HERPES SYSTEM, FOR EXAMPLE, AND IT HAS EXQUISITE TROPISM FOR SENSORY NEURONS AND ET CETERA, AND HAS IT BEEN APPLIED IN OTHER SETTINGS AND SOME OF THE MANUFACTURING ISSUES THAT I KNEW ABOUT A DECADE AGO, HAVE BEEN SOLVED? >> YEAH, WELL, I DON'T KNOW ABOUT THE SENSORY PER SE. IF YOU INJECT IN DIFFERENT PARTS OF THE BRAIN IT WILL TRAVEL TO OTHER PARTS OF THE BRAIN BUT THROUGH CONNECTIVITY, KNOWN CONNECTED PATHWAYS. AND I DON'T -- IT'S BEEN INJECTED IN MANY DIFFERENT PLACES OF THE BRAIN. NOT SENSORY CORTEX OR MOTOR CORTEX OR THINGS LIKE THAT, CAN BE USED. THERE'S-- PROBABLY COULD BE BENEFICIAL WORK DONE ON THAT BUT THERE IS A FORM OF THE VIRUS THAT HAS PATHOGENICITY GENE COLLEAGUES. IT'S IN CLINICAL TRIALS FRANKLY. BECAUSE IT'S AN ONCOLYTIC VECTOR THAT DOES NOT AFFECT NORMAL NEURONS ADJACENT TO IT, HAS POTENTIAL PROMISE BUT ANYTIME YOU MENTION THE WORD "HERPES" IN A BRAIN OR AN EYE CONTEXT, PEOPLE FREAK OUT. THEY DON'T LISTEN TO THE DATA. A POTENTIAL BIG BARRIER. >> A QUICK SHOT ON THIS MOI, THE CONCEPT OF THE MOIs IN VITRO ASSAYS VERSUS WHAT HAPPENS IN VIVO, I THINK MIGHT DIMINISH THAT TO SOME DEGREE. >> IF YOU LOOK IN VIVO SIDE BY SEED COMPARED TO LENTI OR AAV YOU'RE TALKING ABOUT 10 TO THE 5th MORE THAN THE OTHERS TO GET THE SAME TOTAL NUMBER OF CELLS TRANSDUCED IN AN IN VIVO SETTING. >> I DON'T KNOW WHAT THE RATIO IS NOW BUT WHEN I STARTED OUT WITH AAV IT WAS SOMETHING LIKE YOU HAVE TO HIT IT WITH 500 AAVs TO GET ONE IN. WITH HERPES IN A NEURON IT'S ONE AND ONE. IT'S EXTREMELY INFECTIOUS. ACTUALLY JOE GLORIOSO HAS USED THEM FOR PAIN TO DELIVER ENDORPHINS, USED IN SOME NON-ONCOLYTIC. >> I THINK THE IMPORTANCE OF THE NON-PATHOGENIC VARIANT OF IT, IT'S BEEN USED IN THE BRAIN IN HUMAN TRIALS. WENT TO AT LEAST PHASE 3 TRIALS IN EUROPE, I DON'T KNOW WHAT'S HAPPENED. I THINK IT'S G2O7 VIRUS, ACTUAL EYE -- ACTUALLY BOB AND SAM ARE USING. >> THERE'S THEY DIFFERENT ONCOLYTIC VIRUSES NOW, HERPES, IN TRIALS. >> I THINK WE'RE COMING BACK TO A STRATEGY TO INVESTIGATE, TO REVISIT OTHER VIRUSES. >> THE PRIMARY POINT I WANTED TO BRING UP WAS KIND OF A FOLLOW-UP TO KRYS' AND JUDE'S COMMENTS, TO PUT ON THE TABLE THE CONCEPT OF TRANSDUCTION, AT LEAST SOME TYPE OF ENTRY OF AAV, NOVEL AAVS INTO ANTIGEN PRESENTING CELLS, RELATED TO IMMUNOTOXICITY RISK, DURABILITY OF EXPRESSION. IN THE MAP PROPOSED, TENDENCY EVEN FOR VIRAL PARTICLES TO ENTER ANTIGEN PRESENTING CELLS WHICH MIGHT BE ENOUGH TO TRIGGER CERTAIN SIGNALS WOULD BE I THINK IMPORTANT. >> SO CAN I MAKE A QUICK COMMENT? SO I WANTED TO FOLLOW UP ON YOUR COMMENT, JOHN, REGARDING USE OF OTHER VIRAL VECTORS. SO THERE ARE A NUMBER OF NEUROTROPIC VIRUSES THAT AFFECT HUMANS. YOU HAVE RABIES AFFECT NEURONS, TRANSSYNAPTICALLY, THE ENTEROVIRUSES, POLIO AND OTHER FAMILY OF VIRUSES THAT GO INTO THE ANTERIOR HORN CELLS, POLYOMA VISIERS SPARE THE NEURONS. YOU HAVE SOME OF THE RETROVIRUSES THAT WILL INFECT PREDOMINANTLY MICRO GLIAL CELLS AND MACROPHAGES, NATURE PRODUCED THESE VIRUSES, THEY HAVE REPLICATION CAPACITY SO SIMPLE DOG BY THE IS -- BITE, SMALL INNOCULUM, YOU CAN SPREAD THROUGH THE BRAIN BECAUSE THEY HAVE ABILITY TO REPLICATE IN CELL TYPES THEY INFECT AND SPREAD. IF ONE WERE TO MAKE THEM NON-PATHOGENIC THERE'S AN OPPORTUNITY TO MAYBE TARGET SPECIFIC CELL TYPES WITHIN THE BRAIN AND DELIVER WHAT WE NEED. I WAS WONDERING YOUR THOUGHTS ON THAT. >> ONE ISSUE, SO RABIES HAS BEEN KICKED AROUND FOR A LONG TIME, OF COURSE YOU CAN GET TRANSPORT FROM SOME OF THE PROTEINS, G-PROTEINS AND SO ON. KICKED AROUND. BUT IT'S AN RNA VIRUS. SO THE ISSUE FORTUNE AT LEAST INHERITED DISORDERS, NOW FOR TRANSIENT EXPRESSION THAT MIGHT BE A DIFFERENT STORY BUT INHERENTED DISORDERS THE IDEA IS TO GET LONG-TERM GENE EXPRESSION AND I -- UNLESS SOMEBODY HAS CLEVER WAYS TO GET RNAs TO DO THAT -- >> YOU HAVE RABIES, YOU EVENTUALLY DIE FROM IT. >> ANY OF THEM. A NUMBER OF THEM. THERE'S DIFFERENT WHETHER IT'S RNA OR DNA. >> PERSISTENT VIRUSES, MEASLES, EXPRESSED FOR YEARS AND YEARS. JUST BECAUSE IT'S AN RNA VIRUS DOESN'T MEAN IT CANNOT BE EXPRESSED FOR EXTENDED PERIODS OF TIME. >> RIGHT. >> SO I THINK -- >> SO YEAH, MY COMMENT ON THAT IS THAT MAY BE POSSIBLE BUT IT HAS TO BE ENGINEERED TO BE NON-PATHOGENIC AND A LOT OF TIMES THOSE ARE WORKING AT OPPOSITE -- >> YEAH. >> AGAIN ANOTHER GOOD COMMENT THAT MAYBE THERE'S ELEMENTS OF NEUROTROPIC VIRUSES WE CAN TAKE ADVANTAGE OF, YOU KNOW, IF OUR QUEST FOR FOCUS TARGETING. BRIAN? >> THE WAY TO GET AROUND THAT IS PSEUDOTYPING. YOU'VE GOT AN INTEGRATING VIRUS LENTI, WHICH WILL TAKE A PSEUDOVIRUS RABIES ENVELOPE. IT'S BEEN DONE BEFORE, MANY PEOPLE STILL SETTLE FOR VSVG, I KNOW A COUPLE GROUPS USE RABIES FOR CLINICAL TRIALS. >> AND TAKING ADVANTAGE OF ADVANCES IN SCREENING NEW ENVELOPES FOR LENTIVIRUSES, ENGINEERING THOSE ENVELOPES FOR BETTER MANUFACTURING MIGHT BE WORTH INVESTIGATING. BECAUSE YOU'RE RIGHT, EVERYBODY GOES BACK TO VSVG FROM A MANUFACTURING PERSPECTIVE. >> WE SCREENED EBOLA ONCE, I WOULDN'T RECOMMEND USING IT. DIDN'T WORK VERY WELL. >> STEVE? >> YEAH, I MEAN, IN CO-CHAIRING THIS ONE OF THE TASKS THAT I WAS GIVEN WAS TO TRY TO CREATE ACTION ITEMS THAT WERE PRACTICAL, WE COULD MOVE FORWARD WITH. YOU KNOW, COMING BACK TO THIS IDEA OF LIKE A DISTRIBUTION ATLAS, ONE OF THE THINGS I WANT -- WE'VE HAD A LOT OF THINGS THROWN OUT, AND SO I REALLY WANT TO HONE IN ON WHAT ARE THE CRITICAL ASPECTS OF THIS BECAUSE WE COULD INVEST A LOT IN SOMETHING LIKE AAV9, A STANDARD RIGHT NOW, AND THEN TWO YEARS FROM NOW SOMEBODY PUBLISHES SOMETHING THAT IS TEN TIMES BETTER THAN AAV9 AND TRANSLATE REALLY WELL, ALL OF A SUDDEN ALL THAT BRAIN, ATLAS DAY FOR AAV9 IS ALMOST OBSOLETE. SO IT'S LIKE I THINK WE NEED TO THINK ABOUT WHAT ARE THE CORE PIECES OF INFORMATION THAT WE NEED TO GO INTO AN ATLAS AND NOT OVERDO ANY VECTOR. >> THIS IS JUDE, UNC, BLAH, BLAH, BLAH. ONE THING THAT WE SHOULDN'T LOSE SIGHT OF IS THAT THESE CAPSIDS ARE IN HUMANS, RIGHT NOW. AND ANY DATA THAT YOU DERIVE THAT CAN CONTINUE TO BUILD A FOUNDATION OF AN UNDERSTANDING OUTSIDE OF HUMAN THAT LETS YOU INFER BACK TO ALMOST A 1:1 CORRELATE AS FAR AS WHAT COULD BE GOING ON. I AGREE WITH RESPECT TO IF WE WAIT ON THE NEXT GENERATION AND NEXT GENERATION WE'LL BE CHASING OUR TAIL OVER AND OVER AS TO WHICH CAPSID SHOULD WE FOCUS ON. BUT I WOULD SAY THAT THERE'S HUMAN DATA FROM THE GAN TRIAL WITH CARSSTON, DMD TRIAL, TO THE POINT IF WE FOCUSED AN EFFORT, THAT WOULD BE LIKE A STANDARD EVEN IF IT'S NOT AN OPTIMUM STANDARD, IT BECOMES A STANDARD FOR UNDERSTANDING WHAT DOES IT DO IN A MONKEY, INDEED WE KNOW HOW DATA IN HUMANS, WHAT IS IT DOING IN A RODENT, WE HAVE DATA IN HUMAN, AND GIVE YOU AT LEAST A REFERENCE POINT SO IF A NEW ONE COMES ALONG YOU MAY ALREADY HAVE A PRESCRIBED APPROACH WHAT YOU WOULD LIKE TO DO WITH A NEW ONE THAT MAY GO INTO THE CLINIC. >> ONE OF THE THINGS THAT WE DON'T KNOW FROM THE HUMAN DATA IS WHERE ARE ALL THE UNPACKAGED CAPSIDS. AND WE THINK THAT MANY OF THESE CAPSIDS CAN EXIST IN CELLS FOR MANY, MANY YEARS. AND CAN WE TAKE ADVANTAGE OF THAT WITH NEW LIGANDS FOR IMAGING, FOR EXAMPLE, FOR THE PATIENTS THAT ARE STILL WALKING AROUND AND DOING FINE, CAN WE ACTUALLY SEE, SEE, OR GAIN THAT INFORMATION? >> YEAH, THERE ARE LIGANDS THAT CAN BE USED. DOPAMINE 2 RECEPTOR THAT HAS A MUTATION THAT'S INACTIVE. >> NO, I'M TALKING ABOUT THE CAPSIDS. >> FOR THE CAPSIDS THEMSELVES. >> POST >> POSTMORTEM TISSUE IS A GREAT EXAMPLE, DIFFICULT TO GET HOLD OF THOUGH. THERE ETHICAL ISSUES WITH CONSENTING PATIENTS. >> ALSO YOU HAVE TO REALIZE THAT PET SCAN, FOR EXAMPLE, IT HAS PRODUCED LIMITATIONS, DETECTION LEVELS, WHAT YOU ARE PROBABLY TALKING ABOUT MAY BE VERY DIFFICULT TO IMAGE WITH THAT PARTICULAR MODALITY. ESPECIALLY IN THE BRAIN. RIGHT NOW SLICE OF 6 MILLIMETERS, IT'S AVERAGE DATA. BUT I THINK BIOPSY DISEASE OF THE BRAIN. >> SO IN TERMS OF LIKE CORE LIKE WHAT ARE THE CRITICAL THINGS, THE MOST IMPORTANT PRIORITIES, IF WE'RE TALKING ABOUT AN ATLAS JUDE RAISED WHERE DO THE PARTICLES GO, WHETHER LABELING THE PARTICLE OR USING CRE SYSTEM THAT WOULD BE INDEPENDENT OF PROMOTER. I MEAN, THAT'S KEY. THEN IF WE BUILD IN SOME PROMOTER DATA, INTO THOSE, THAT'S ALMOST -- AS LONG AS YOU INTERPRET THE DATA RIGHT, THAT INFORMATION CAN BE VECTOR INDEPENDENT. IF WE'RE GATHERING ABOUT PROMOTERS ACROSS SPECIES WITH ONE VECTOR, I MEAN, AGAIN, AS LONG AS YOU ASSESS THAT DATA PROPERLY WITH RIGHT CAVEATS CAN YOU TRANSFER ACROSS VECTORS. >> WE CAN'T LOSE SIGHT OF THE FACT THERE'S AN INTERACTION BETWEEN THE CAPSID AND TRANSGENE AND PROMOTER, AND SO I THINK WE HAVE TO DO THE EXPERIMENT. ARAVI THIS >> WE HAD A SIMILAR DECISION, GUANGPING AND MYSELF, FOLKS AT BAYLOR COLLEGE OF MEDICINE, WE'VE INITIATED THIS DUAL INDEXING APPROACH FOR TWO AAVs AT A TIME USING BARCODES YOU CO-ADMINISTER INTO MICE AND PRIMATES, AND ESSENTIALLY THE STRATEGY IS YOU'RE GOING TO GET DIFFERENT ORGANS FROM PRIMATES OR MICE AND DISSEMINATE TO PEOPLE INTERESTED IN THE SPECIFIC ORGANS TO LOOK WITH A LENS AND SINGLE CELL OR SPECIFIC DISTRIBUTION IN THE ORGANS, THE GOAL TO ESTABLISH A CORRELATION BETWEEN RNA AND DNA LEVELS. FOR STARTERS EVERYTHING IS DONE WITH YOUR -- YOUR UBIQUITOUS PROMOTERS, ALL BEING ADMINISTERED SYSTEMICALLY, THAT'S A START BUT ONE COULD ARGUE SOMETHING SIMILAR CAN BE DONE IN THE CONTEXT OF THE BRAIN, DIRECT ROUTE OR OTHER ROUTES OF ADMINISTRATION, TRYING TO GET SOME INDEXING DONE. THIS WAS DONE IN THE EARLY DAYS OF JOHN ENGELHARDT AND OTHERS, CHIMPANZEE AND RHESUS AND MACAQUE ARRAYS, WE HAVE SOME UNDERSTANDING ON AWARE EPITHELIAL MORE THAN ANY OTHER SEROTYPES FROM THE GROUPS IN IOWA AND OTHERS. THERE ARE BLUEPRINTS FOR TRYING TO BUILD SOMETHING ALONG THOSE LINES. >> WE'RE DOING SOME SIMILAR FOR THE BRAIN, BARCODED USING THREE IN-CLINIC LIBRARIES. SO A LOT OF THE PARTS ARE JUST SITTING THERE WAITING TO BE, YOU KNOW, DISBURSED, SUBJECTED TO SINGLE CELL Seq TO FIGURE OUT WHERE THE THINGS ARE GOING. AND HOPEFULLY COMPARING RNA AND DNA YOU GET A SENSE OF ESSENTIALLY THE COPIES AND WHERE THE SINKS ARE. GUANGPING? >> YES, I'M SORRY. ANOTHER COMPLICITY IS HOW YOU DELIVER THE VIRUS CELL LIBRARY, YOU COULD MAKE THREE MORE DIFFERENT STUDIES. >> YEAH, YEAH, EXACTLY. DEFINITELY DELIVERY DEPENDENT. >> SO IT SEEMS LIKE THERE'S SO MANY DIFFERENT GROUPS, I THINK THAT ARE TACKLING PIECES OF THIS, I THINK ONE OF THE -- ONE STRATEGY WOULD BE TO HAVE A CONCERTED EFFORT TO BUILD THIS. ANOTHER STRATEGY WOULD BE IF YOU CAN ESTABLISH EVEN LIKE SOME KIND OF ARCHIVAL DEPOT, SOMETHING MAYBE THE NIH CAN FACILITATE WHEN INFORMATION IS PUBLISHED IT'S A CONSOLIDATED DATABASE WHERE THEY CAN EVEN REACH OUT TO THE INVESTIGATORS, GET PRIMARY DATA, AND SOMEBODY TO REALLY HANDLE ALL THE COLLATING AND THE PRESENTATION OF THAT DATA FROM ALL KINDS OF STUDIES. >> WE DO NEED AN ALLEN VIRUS ATLAS, SOMETHING AS OPEN ACCESS, YOU KNOW, WE NEED SOMEBODY TO BUILD THE DATASET, TO BUILD THE SITE THAT ALLOWS THE IMPORTATION OF DATA FROM VARIOUS GROUPS SO THAT WE CAN BEGIN TO POPULATE WITH HUMAN, MONKEY, MOUSE. >> MAYBE NINDS SHOULD CONTACT ALLEN BRAIN ATLAS. THEY ARE GOOD, WELL ORGANIZED, THEY KNOW HOW TO DO THIS. NOW, GETTING UP DIFFERENT SPECIES, BUT THEY HAVE DONE IT IN A SYSTEMATIC WAY, PRECISION INJECTIONS, THAT THEY COULD DO. THEY ARE NOW DOING TO LOOK AT RNA EXPRESSION ALL OVER, DIFFERENT AGES, THINGS LIKE THAT. IT SEEMS LIKE THEY KNOW HOW TO DO THIS. >> ONCE YOU'VE GOT CAPSID LIBRARIES, GOT YOUR PROMOTERS SORTED OUT HOW ARE YOU GOING TO ENSURE THESE ARE GOING TO BE AVAILABLE TO PEOPLE TO USE IN CLINICAL TRIAL? THIS IS ONE OF THE PROBLEMS WE'VE SEEN, POST AAV BUT WITH NEW VECTORS ON THE BLOCK THEY ARE ALL PARCELED OUT BETWEEN DIFFERENT COMPANIES AND SOME PEOPLE DON'T HAVE ACCESS TO THEM >> THIS IS AN INCREDIBLY SALIENT POINT BUT WE'RE JUMPING AHEAD TO SESSION 4. AND IN SESSION 4 WE'RE GOING TO SOLVE THOSE ISSUES. [LAUGHTER] THAT'S THE CHALLENGE FOR SESSION 4, CO-CHAIRS. >> SO, I WOULD LIKE TO COMMENT ON THIS CONCEPT OF CREATING ATLAS. IT WOULD BE GREAT BUT THE QUESTION ABOUT THE VARIABILITY, REPRODUCIBILITY, WE HAVE A POOL OF PATIENTS, WILL THIS DISTRIBUTION BE THE SAME OR DIFFERENCES BETWEEN PATIENTS WOULD BE SO GREAT THAT IT HAS TO BE TREATED ON THE INDIVIDUAL BASIS. >> I THINK WE NEED TO KNOW THAT, RIGHT IT WOULD BE REALLY IMPORTANT TO KNOW HOW UNIFORM OR NOT THE PATIENT DATA IS, AND JUST BEGINNING TO POPULATE THAT I THINK WILL GIVE US A SENSE OF THE VARIABILITY WHICH WILL ALSO HELP INFORM FUTURE CLINICAL TRIALS BECAUSE WE'LL BE ABLE TO BUILD THAT INTO THE POWER ANALYSES. >> WE NEED VERY EFFICIENT METHODS FOR MAPPING THE DISTRIBUTION AND ADVOCATE IMAGING, THAT SEEMS TO BE REALLY EFFICIENT AND WE SEE A LOT OF VARIABILITY IN DISTRIBUTION OF VIRUS THERAPEUTICS, CELLS, MACROMOLECULES, THAT THE SAME WOULD BE FOR GENES. >> AND I DO AGREE WITH THAT BECAUSE ACTUALLY IF YOU LOOK AT CLINICAL TRIALS, EVERYONE IS CONCENTRATING ON PATIENTS THAT ARE RESPONDING, BUT IT'S GOOD NUMBER OF NON-RESPONDERS AND WE HAVE NO IDEA WHY. UNDERSTANDING VARIABILITY WOULD BE EXTREMELY IMPORTANT TO UNDERSTAND WHERE THE VIRUS GOES, WHERE IT GOES, WHAT IT DOES AND DOESN'T DO. >> LET'S LET NINA TALK. >> ACTUALLY, I'M ENJOYING LISTENING TO ALL OF YOU. I'M NOT AN EXPERT IN THIS AREA. SO IN A WAY I HAVE A QUESTION THAT I HOPE WILL RAISE SOME DISCUSSION. I WONDER IF I'M BEING NAIVE IN WORRYING THAT WE WILL DUMP DATA INTO THIS DATA BASE THAT WE PURPORT SHOWS VARIABILITY AMONG THE PATIENTS OR AMONG THE ANIMALS OR AMONG THE SPECIES OR WHATEVER IT IS, BUT IN FACT WHAT IT MIGHT SHOW IS THAT WE HAD VARIABILITY IN DOING THE EXPERIMENT OR MAKING THE MEASUREMENT OR WHAT HAVE YOU, AND I WONDER THOSE OF US WHO HAVE WORKED IN THE PEDIATRIC OR CHILD NEUROLOGY SPACE ARE AWARE OF CHILD CONSORTIUM ON CANCER THAT PUTS TOGETHER THE PROTOCOLS THAT EVERYBODY AGREES TO FOLLOW NATIONALLY AND THEN YOU ENROLL EVERYBODY, AND I SAY THIS BECAUSE I -- THIS IS PROBABLY NIH HERESY BUT I'M NEW ENOUGH I CAN SAY I DON'T YET KNOW ALL THE RULES. [LAUGHTER] BUT I FIND MYSELF CONTINUALLY WORRIED ABOUT THESE THINGS THAT WE'RE DOING WITH DATABASES AND OTHER SPHERES WHERE WE PAY SOMEBODY TO HARMONIZE DATA THAT WERE COLLECTED UNDER VERY NON-HARMONIOUS CIRCUMSTANCES, AND I WORRY IT MAKES US FEEL GOOD ABOUT SOMETHING WE OUGHT THE NEW TO FEEL SO GOOD ABOUT. I JUST WONDER WHAT PEOPLE THINK ABOUT PUTTING SOME KIND OF CONSORTIUM TOGETHER THAT SAYS HOW WE'RE GOING TO COLLECT THESE DATA. >> CAN I GET SOME COMMENTS FROM THE OTHER CLINICIAN SCIENTIST PEDIATRICIANS ON NINA'S COME WHICH IS INCREDIBLY VALUABLE. CARSTON, BECCA, YOU'RE THE ONLY TWO I KNOW. KEVIN? >> SO I THINK THIS IDEA OF CONSORTIA FOR COLLECTING IS CRITICAL ACTUALLY, WITH THE SAME CHALLENGES RELATED TO VECTORS BEING, YOU KNOW, OWNED BY COMPANIES, WILL WE FACE THE EXACT SAME QUESTION, NO MATTER HOW WE WANT TO THINK ABOUT CAPTURING DATA FROM THE COG MODEL BECAUSE WE'RE TALKING IN SESSION 4, WE'RE GOING TO FACE CHALLENGES OF INDIVIDUAL INVESTIGATORS, INDIVIDUAL INVESTIGATORS CONFLICT MANAGEMENT WITH COMPANIES THEY ARE ASSOCIATED WITH AS WELL. I THINK THERE'S CHALLENGES TO THINKING OF THE COG MODEL AS A WAYTO CAPTURE THIS. HOWEVER I THINK THERE'S OPTIONS FOR MINIMAL DATA COLLECTION, COMPONENTS OF DATA WE COLLECT AT ALL TRIALS, ASSURING DATA IS COMPARABLE BETWEEN TRIALS IN RESPONSE TO BOTH HOW WE JUDGE BIOCHEMICAL MARKERS OF EXPRESSION AS WELL AS IMMUNE RESPONSES TO IN PARTICULAR MAKING SURE WE'RE UNIFORMLY CAPTURING THOSE ACROSS GROUPS. >> I THINK THESE POINTS -- CYNTHIA? REALLY IMPORTANT AS YOU CONSIDER FUTURE APPLICATIONS TO THE NIH'S -- GROUPS COMING FORWARD AND UNIFORMITY OF THE DAY WHICH CAN BE A CHALLENGE FOR A LOT OF RARE DISORDERS. CYNTHIA? >> RIGHT. ACTUALLY A NUMBER OF US WERE SITTING IN THE BACK LAUGHING ABOUT THIS, WERE ON MONDAY AT A DAY-LONG THINK TANK WORKSHOP ON RARE DISEASE, AND WE WERE EXPLORINGED COG MODEL. IT WAS A WHOLE BUNCH OF ONCOLOGISTS TRYING TO TELL US HOW THEY DO THEIR JOB AND HOW WE MIGHT FIT RARE DISEASE IN. AND THERE'S VERY INTERESTING MODELS, ACTUALLY THE CANCER COMMUNITY IS USING, EVEN ABOUT BRINGING TOGETHER KIDS WITH SPECIFIC CANCERS, DOING GENOTYPING, AND DECIDING WHICH CHEMOTHERAPY THEY MIGHT BE MOST SUITABLE FOR, AND THEN PARCELING THEM INTO DIFFERENT PROTOCOLS, EACH ARE RUN BY DIFFERENT COMPANIES. SO IT'S NOT IMPOSSIBLE THAT COMPANIES COULD WORK TOGETHER IN A MODEL WHERE YOU HAD SORT OF A CONSOLIDATED EFFORT. AND WE'RE EXPLORING THAT AS A RARE DISEASE COMMUNITY AT THE MOMENT. SO I THINK IT'S AT LEAST DOABLE. >> IF I MAY, I THINK IT WOULD ACTUALLY TREMENDOUSLY HELP TO DE-RISK DEVELOPMENT OF THESE THERAPIES FOR COMPANIES AS WELL AS NON-COMPANY DEVELOPERS, IF THERE WAS -- THIS IS NOT DATA THAT WOULD INFRINGE ON ANY COMMERCIALIZATION PROJECT, IT WOULD BE DATA THAT WOULD HELP DE-RISK THE DEVELOPMENT IN VERY RARE DISEASES AND CREATE SOPs THAT HAVE TOKING A -- TO AGGREGATE DATA TO UNDERSTAND SAFETY EVENTS BETTER. IF A SAFETY EVENT HAPPENS, COMPANY IS AT RISK OF DROPPING LIKE 40% IN STOCK PRICE, IF IT'S AN EVENT THAT IS REALLY PART OF OUR THERAPEUTIC APPROACH, AND SOMETHING THAT'S PREDICTABLE AND MANAGEABLE, IF THAT'S KNOWN AND OUT THERE, I THINK THINGS WILL BE MUCH CALMER, AND MUCH MORE DIRECTED TOWARDS SAFE DEVELOPMENT. I THINK IT'S CRITICAL TO DO THAT. >> THE LAST FEW MINUTES, CHARLES, WE'LL GO TO YOU. AND THEN I WANT TO -- WOULD LIKE TO CHALLENGE THE GROUP TO THINK ABOUT STRATEGIES TO BETTER UNDERSTAND OFF-TARGET EFFECTS OF EDITING MACHINERY AND WHERE DO WE WANT THE FIELD TO GO, WHERE DO WE BELIEVE THE FIELD NEEDS TO GO. OR IS EVERYTHING PERFECT AND WE CAN GO HAVE LUNCH? SO CHARLES? >> I BRING THIS UP THEORETICAL FOR A MOMENT, IF YOU LOOK AT SMALL MOLECULE THERAPY AND SIMILARITIES BETWEEN GENE THERAPY AND SMALL MOLECULE THERAPY, ONE THING THAT COMES UP WHEN DEALING WITH PHARMACEUTICALS IS DEVELOPING WHAT KIND OF INFORMATION YOU WANT TO UNDERSTAND ACROSS A BROAD RANGE OF DRUGS THAT IS GOING TO THEN BE APPLICABLE TO ALL DRUGS. AND THOSE TEND TO INCLUDE QUESTIONS ON PK AND QUESTIONS ON EFFICACY. CAN GATHER THIS INFORMATIONCT WE- WHERE WE'RE SEEING PROTEIN EXPRESSED, GENE EXPRESSED AND ALL THAT AND DON'T HAVE AN OUTCOME MEASURE FOR ANYTHING THAT'S HAVING AN EFFECT ON ANYTHING. SO WE END UP BEING DATA COLLECTORS, WITHOUT TAKING THAT AND MAKING IT BROAD ACROSS -- WE BROUGHT UP DIFFERENT VECTORS, ONCE WE SHIFT WHAT DOES THAT DATA BECOME USEFUL FOR? WE'VE TALKED ABOUT USING DIFFERENT PROMOTERS, DIFFERENT ITRs, DIFFERENT EFFECTS, I WANT TO CAUTION AGAINST GOING DOWN A PATHWAY WHEREAS A GROUP WE DECIDE WE'RE GOING TO COLLECT THIS DATA AND IT BECOMES JUST A DATA COLLECTION TASK. I THINK THE TIME HAS TO BE SPENT ASKING WHAT ARE THE THINGS YOU'RE INTERESTED IN COLLECTING. WHAT ARE YOU GOING TO USE WITH THIS OUTCOME, DO IF IN FIVE YEARS IF YOU HAVE AAV17 THAT BECOMES THE ONE THAT'S USEFUL, YOU DON'T WANT TO AGAIN JUST COLLECT IT. IT'S LIKE ANYTHING ELSE, YOU'RE LOOKING FOR AN OUTCOME MEASURE. WE CAN UNDERSTAND BIOLOGY TO THE HILT BUT WE DON'T -- IF WHEN YOU GIVE THE DRUGS TO PEOPLE, YOU DON'T HAVE INFORMATION, HOW MUCH GOT INTO THE CAUDATE NUCLEUS, SO WE HAVE TO BE CONCERNED, REGARDLESS OF THE FACT HAVE YOU AN ON/OFF SWITCH USING THE DRUGS, YOU HAVE TO ASK WHY YOU'RE ASKING AND WHAT IS YOUR OUTCOME MEASURE FOR HOW YOU'RE GOING TO GENERALIZE THAT TO SOMETHING ELSE. THERE ARE A LOT OF INFORMATION IN THE PHARMA COMMUNITY THAT WE'RE REHASHING HERE, THAT'S ALREADY AVAILABLE HOW THEY UNDERSTAND AND HOW THEY ARE GOING TO QUESTION, AND DATA THEY ARE GOING TO COLLECT, THAT THEY ARE GOING TO NEED TO KEEP GOING FORWARD TO MAKE NEW COMPOUND, NEXT GENERAION AND ALL THAT. THAT'S MY ONLY COMMENT. IT'S EASY TO COLLECT DATA, IT'S HARDER TO SAY WHAT ARE WE GOING TO COLLECT, WHY DO WE CARE ABOUT IT AND WHAT ARE WE GOING TO DO WITH IT. >> ALSO, EXCELLENT POINTS, ALSO TO BE ABLE TO CAPITALIZE ON THE SURPRISES. YOU KNOW, IF WE FIND THESE THINGS ARE GOING TO TISSUES THAT WE DIDN'T EXPECT, CELLS AND NETWORKS WE DIDN'T KNOW ABOUT FROM THE ANIMAL MODELS, WE CAN BEGIN TO ASK THOSE QUESTIONS IN PATIENTS, WHETHER OR NOT WE SEE EFFECT IN THAT SYSTEM. SO, YOU KNOW, IF WE FINALLY FOUND OUT THAT OH MY GOSH, ALL THE STUFF GOES TO THE AMYGDALA MAYBE WE CAN ASK RELEVANT CLINICAL QUESTIONS THAT ENGAGE THE AMYGDALA SO AS NEUROSCIENTISTS WE NEED TO REALLY BE COGNIZANT THAT WE'RE DEALING WITH A NETWORK AND ENTIRE BRAIN, SO I AGREE TO SOME EXTENT. WE NEED TO KNOW WHAT WE'RE ASKING BUT ALSO NEED TO CAPITALIZE ON WHAT WE FIND. >> SO NOW I WOULD LIKE TO SPEND THE LAST TEN MINUTES SORT OF, AGAIN, TALKING ABOUT OFF-TARGET EFFECTS, IF THERE'S ANY BURNING QUESTIONS, AND THEN ANYTHING ELSE IN GENERAL FOR THE ENTIRETY OF SESSION 1. WE'RE GOING TO DO A HARD STOP AT 11:45 BECAUE WE HAVE TO WALK AROUND THE BLOCK AND FIND SOME FOOD. >> YEAH, I THINK TALKING ABOUT OFF-TARGET EFFECTS, WE RAISED THINGS ABOUT OVEREXPRESSION BUT THIS IS REALLY DIRECTED TO, SAY, INTERFERON RNA KNOCKDOWN OR OLIGO NUCLEOTIDE, GENE EDITING, VERY SPECIFIC TO A HUMAN SEQUENCE. SO HOW DO WE EVALUATE TOXICITY WHEN IT'S SO SPECIFIC TO HUMAN SEQUENCE? OFF-TARGET EFFECTS MAY BE SPECIFIC TO HUMAN SEQUENCE. >> SO, I'M NOT GOING TO ADDRESS THAT SPECIFICALLY BUT I THINK AT LEAST IN MY OPINION THERE'S A GAP IN OUR KNOWLEDGE. I LOSE SLEEP OVER THE TOXICITY ISSUE. AND MOST PEOPLE KNOW ME HERE. WHAT WE DISCOVERED A LONG TIME AGO BUT AS I THOUGHT ABOUT THIS OVER THE YEARS, ONE THING THAT CONTINUES TO BOTHER ME IS WHEN YOU DELIVER AN AAV VECTOR, SOME CELLS MAY HAVE 500 COPIES IN THE NUCLEUS FOR A LONG TIME. OTHERS MAY HAVE SMALLER NUMBERS. WHAT IS THE LONG-TERM EFFECT OF 500 FOREIGN EPISOMES SITTING IN A CELL? I DON'T KNOW THE ANSWER. BUT, AGAIN, THIS IS THE STUFF I LOSE SLEEP OVER. AND WE ALL LIKE TO SAY THAT AAV IS EPISOMAL, WHICH OF COURSE IT IS, BUT THERE ARE FRAGMENTS OF AAV THAT INTEGRATE AND IF YOU LOOK THROUGH THE LITERATURE THERE'S LOTS. AGAIN, THESE ARE THE SORTS OF THINGS I LOSE SLEEP OVER. WHAT IS HAPPENING IN THAT CELL? AGAIN, IF WE'RE THINKING OF DEVELOPING A THERAPY THAT WILL LAST FOR 20, 30, 40 YEARS, AND AGAIN I DON'T EVEN KNOW HOW TO DESIGN THE EXPERIMENT FRANKLY, WHAT IS CELLULAR EFFECT OF 500 EPISOMES IN THE NUCLEUS, IF SOMEBODY HAS A WAY TO STUDY -- >> RELEVANT TO ANYTHING, EDITING MACHINE OR NOT. ANYBODY? WHAT ABOUT THE FINDING RECENTLY OF THE INTEGRATION OF AAV GENOMES WITH WHEN YOU DELIVER EDITING MACHINERY WITH AAVs, IS THAT A WORRY? >> YEAH, I THINK BEFORE GOING DOWN THAT PATH TO ANSWER SOME QUESTIONS RELATED TO THE PERSISTENCE THERE, THERE IS DATA FROM THE AVIGAN TRIAL WHERE THE MUSCLE BIOPSY, I THINK IT WAS TEN YEARS, WAS SHOWN TO HAVE THE VECTOR GENOME PERSISTING, GETTING BACK TO MARK'S QUESTION OF WHAT'S HAPPENING OVER TIME, AND THAT'S IN HUMANS. SO, YOU CAN START TO DRAW SOME CORRELATES AS TO TODAY YOU WOULD SEQUENCE THOSE AND BE ABLE TO COME BACK WITH WHAT DOES IT LOOK LIKE AS FAR AS INTEGRITY AND STUFF LIKE THAT. THAT'S CONTINUE BE TO BE AVAILABLE WITH RESPECT TO BIOPSIES AND THINGS OF THAT NATURE. I'M NOT GOING TO APPROACH THE QUESTION YOU BROUGHT UP BECAUSE I THINK GETTING A BETTER FOOTPRINT WITH AAV VECTORS TODAY IS CRUCIAL. ANIMAL DATA AS EVERYONE KNOWS FOR LIFE IN THE MOUSE, NINE YEARS IN THE DOG, 11 TO 13 IN PRIMATE, PEOPLE HAVE BEEN ABLE TO TRACK THAT TYPE OF QUESTION, BUT IN HUMANS IT'S, FROM WHAT I UNDERSTAND, PRIMARILY THE MUSCLE BIOPSY AND THE AVIGEN TRIBAL FACTOR 9 HAS THE LONGEST DATA. >> YEAH, AND CONFIRMING I THINK THEY ARE POST MORTEM, LONG LIVED. >> (INAUDIBLE). >> THAT COULD BE A KATHY QUESTION I BELIEVE. >> I WANT TO SAY SOMETHING ABOUT THE CRISPR DELIVERY WITH THE AAV. I THINK IT'S THE BEST WAY TO DELIVER IT. IT DOES HANG AROUND FOR A LONG TIME. IF YOU ENCODE EVERYTHING IN IT, Cas9 AND GUIDE RNA, IT TENDS TO INTEGRATE INTO BREAK SITES, WHAT INTEGRATES MOST IS ITR ELEMENT WHICH WE KNOW HAS SOME PROMOTER ACTIVITY. SO I WOULD -- MAYBE IF YOU JUST DID -- PUT YOUR CAS ELEMENT IN THE AAV AND BROUGHT YOUR GUIDE RNA SO YOU DIDN'T HAVE THEM BOTH FOREVER, BUT MY TENDENCY, PUTTING THEM BOTH IN AAV IS NOT GOING TO BE THE WAY TO GO FOR CRISPR. >> A COUPLE QUICK THOUGHTS. THEY RECENT HEMOPHILIA FOUNDATION MEETING DATA PRESENTED WERE PBMCs FROM PATIENTS, ISOLATED, AND EPISOMAL FORMS AND LOOKING AT SINGLE ITR FUSIONS, ALL THAT DATA SEEMS TO BE COMING OUT AS WELL. SO THERE IS SOME DISCUSSION AROUND USING PBMCs AS A SURROGATED IN TERMS OF ANALYZING PERSISTENCE, AND I THOUGHT I SAW CHUCK HERE BUT I WOULD ASK CHUCK TO COMMENT BECAUSE HE'S DONE EXCELLENT WORK ON ELEMENTS THAT CAN OR CANNOT GO IN, RISK FACTORS. >> GOOD MORNING, EVERYBODY. NIH. FROM THE GENOME INSTITUTE. I'M A PEDIATRIC METABOLISM DOC, I DON'T WORK SPECIFICALLY ON NEUROLOGIC SYNDROMES, MANY PATIENTS OF COURSE HAVE CNS MANIFESTATION. PERSISTENCE AND INTEGRATION, WE'VE SPENT A LOT OF TIME WORKING ON THIS. I'VE MADE A COMMENT BEFORE FOR THOSE WHO DON'T KNOW SOME WORK WE'VE PUBLISHED, WE'VE FOUND EXACTLY THE SAME THING THAT MARK SANDS FOUND MANY YEARS AGO WHEN HE PUBLISHED THE FIRST ASSOCIATION BETWEEN AAV, RECOMBINANT AAV INTEGRATION AND DEVELOPMENT OF HCC, HEPATOCARCINOMA IN MICE. THERE'S CONTROVERSY. WE WROTE A PAPER ON THIS IN 2015, PUBLISHED IN THE JCI. AND WHAT WE FOUND WAS THAT THERE'S SOME GOOD NEWS AND BAD NEWS. I THINK FOR THE FIELD. OF COURSE, TO SAY THIS OUT LOUD, I'VE SAID IT BEFORE, I WAS HOPING WE WOULD NEVER FIND ANY TOXICITY IN OUR MICE. BECAUSE THAT'S WHAT EVERYONE BELIEVES. THAT RECOMBINANT AAV DOESN'T INTEGRATE. THAT'S NOT TRUE. WHAT WE FOUND IN THE MICE AND AGAIN SOME IS PUBLISHED, I'M END WITH A PREVIEW OF UNPUBLISHED DATA, HIGH DOSES OF A.A.V TO NEONATAL MICE WITH, MORE THAN 50% OF THE MICE, STRONG ASSOCIATION WITH HHC. WE STARTED SUBMITTING ABSTRACTS NEVER GOT PLATFORM TALKS, I'M NOT SORE BUT I'LL THROW THAT OUT THERE. WE HAD A RATHER MATURE STORY. WHEN THE POSTER WENT UP, I JUST REMEMBER THAT HUNDREDS OF PEOPLE WERE CROWDED AROUND THIS AND THE CROWD OF PEOPLE WERE SAYING I'VE SEEN CANCER IN MY MICE TOO. IT'S OUT THERE. I THINK PEOPLE ARE NOT STUDYING IT AS MUCH. TO GET BACK TO MY POINT ABOUT CAUSALITY OF AAV AND POTENTIAL GENE OH TOXICITY, THEY TENDED TO INTEGRATE ALMOST TO THE EXACT BASE CARE AS IN OTHER DISEASE MODELS, OTT, SANDOFF DISEASE, MMA MOUSE MODELS. DEPENDING ON ENHANCER STRENGTH, MORE TO SAY ABOUT AAV, ITR, WHAT THEY CARRY IN THE REGION, SOME FLOATING AROUND IN LABS, RELATED TO ENHANCER ELEMENT A COUPLE YEARS AGO, BUT THERE WAS A PROPENSITY FOR INTEGRATE INTO A SMALL microRNA IN THE MICE MISSING IN THE HUMAN GENOME. NOT EVERY INTEGRATION EVENT THAT WAS ASSOCIATED WITH HCC IN MICE WAS IN THAT microRNA. AND SO THERE'S SOMETHING ELSE TO SAY, WHICH IS THE WAY PEOPLE ARE CAPTURING THE INTEGRATION EVENTS, THIS IS GETTING INTO -- I DON'T KNOW IF WE'LL APPROACH THIS IN LATER IN ITEMS OF VIROLOGY, THERE ARE RARE EVENTS USUALLY. UNLESS IT'S A CLONE. IF IT'S A CLONE, YOU CAN DO THINGS LIKE GENOME SEQUENCING WHICH WE'RE DOING, I CAN'T DISCLOSE WHAT WE'RE FINDING BUT IT'S VERY INTERESTING. OTHERWISE YOU'RE LIMITED TO SORT OF AMPLIFICATION STEPS, CAPTURE, WHICH PRODUCES AMBIGUITY ABOUT WHERE THE AAV COULD POTENTIALLY INTEGRATE. WHAT WE FOUND, MARK AND DAVID AND RUSSELL AND OTHERS, YOU COULD ONLY ISOLATE ONE SIDE. THINK OF AAV, SIMPLE MODEL, INVERTED REPEATS, TRANSGENES, YOU FIND A FRAGMENT AND BROKEN GENOME, YOU CAN'T FIND THE OTHER SITE. SO, WITH THAT, WE'RE DIGRESSING AND GETTING HEAVY INTO THIS, BUT THE COMMENT IS I'M GOING TO MAKE ANOTHER COMMENT ABOUT THE PAPER THAT WAS PUBLISHED BY NAULT ET AL., DESCRIBING HCC ASSOCIATED AAV INTEGRATED EVENTS, VERY CONTROVERSIAL, BUT ONE OF THE EVENTS OF NAULT FOUND CAUSATIVE EVENT THAT WAS ASSOCIATED WITH ITR INSERTION INTO THE TERT CHANGE, IT HAD A NUMBER OF CHROMOSOMAL BITS ON THE END BEFORE IT REJOINED THE TERT PROMOTER. SO THE REASON I'M MAKING THESE COMMENTS, AAV INTEGRATION EFFECTS ARE REAL, STUDYING THEM IS A CHALLENGE. WE'RE WORKING ON IT IN MY LAB. THAT'S NOT MY DEDICATED FOCUS. I FEEL LIKE IT'S SUPER IMPORTANT BECAUSE I'M A BELIEVE IN AAV GENE THEY WERE BUT AN UNEXPLORED AREA IN CNS. IF YOU GAVE ENOUGH MICE HIGH ENOUGH DOSES OF AAV VECTORS AND TOOK YOUR FAVORITE CELL OUT OF THE BRAIN YOU'RE GOING TO FIND INTEGRATIONS, THAT'S MY PREDICTION. I I CAN'T TELL YOU HOW TO DO IT BUT I THINK THE TECHNOLOGY WILL BE THERE WITH ADVANCED SEQUENCE METHODS THAT ALLOW US TO USE LONGER READS WITH BETTER ALGORITHMS AND BETTER ALIGNMENT METHODS TO FIND THESE RARE EVENTS. AND THEN THE NEXT THING IS TO PATTERN THEM. FIGURE OUT IS THERE A PATTERN, WHAT IS THE PATTERN, IS IT DEPENDENT AS WE HEARD ON VECTOR TRANSGENE CAPSID COMBINATION OR IS THERE SOMETHING ELSE TO IT? SO THAT'S A COMMENT ABOUT I THINK THESE ARE THEORETICAL COMMENTS, BUT GOING BACK TO REALITY WHICH IS, YOU KNOW, I THINK THERE ARE DETERMINANTS THAT WE KNOW OF BASED ON WORK OF LOGAN, WHEN WE DESIGN CASSETTES THAT WE COULD USE TO MITIGATE POTENTIAL GENE OH TOXICITY, REMOVING THIS LITTLE ENHANCER-LIKE ELEMENT IN THE SUBITR REGION TO POLISH YOUR ITRs, GET RID OF IT. WHY WOULD YOU KEEP IT? I'M NOT THE FIRST TO DISCUSS THIS, JUST TELLING YOU THIS IS A CONSIDERATION AS WE EXPAND OUR THOUGHTS ABOUT GENOTOXICITY. I SEE THE CHAIR SAYING SHUT UP. I'LL STOP. I DO BELIEVE IN AAV. I THINK IT'S IMPORTANT. IT'S GOING TO HELP OUR PATIENTS. I WAS CALLED OUT. >> NO, THIS IS EXACTLY THE KIND OF OFF-TARGETING THAT WE NEED TO FOCUS ON. AND REALLY FABULOUS. THANK YOU FOR YOUR YOUR COMMENTS. IT'S 11:45-ISH, BE BACK AT 12:45 >> SESSION 2, I'LL HAND IT OVER TO VIVIANA. >> THANK YOU FOR COMING BACK FROM LUNCH. AS YOU'LL NOTICE SESSION 1 AND 2 HAVE COMMON TOPICS BUT I WANT TO START BY AND OVERVIEW OF THE KEY POINTS OF SESSION 1, BECAUSE WE'VE BEEN TASKED TO HAVE ACTIONABLE ITEMS. WE'VE BEEN TASKED TO CREATE FOLLOW-UP. SO THE GOAL IS NOT ONLY TO DISCUSS HERE AND GO HOME AND FORGET. THE GOAL IS TO ACT UPON IT. IF I MAY RESTATE SOME OF THE POINTS THAT WERE MADE IN SESSION 1, AND I THINK A COMMON THEME THAT EMERGED, WE NEED AN ATLAS, THAT IN WAY IS SIMILAR TO WHAT THE ALAN BRAIN INSTITUTE DID, BUT THIS SHOULD BE FOCUSED ON VECTORS, AND WHAT WE HEARD, WE HEARD A LOT OF VARIABLES, THAT SHOULD BE TAKEN INTO ACCOUNT, IN GENERATING A VECTOR ATLAS, THERE ARE SO MANY VARIABLES, DAY AND IT NIGHT, THE MAIN QUESTION OUT OF THE MANY VARIABLES HOW DO WE DISCIPLINE OURSELVES TO IDENTIFY THE KEY ONES THAT ARE ACTIONABLE. LET'S KEEP THIS IN MIND AS WE PROGRESS WITH SESSION DID, WORKING TOWARDS THE IDEA LET'S GENERATE AN ATLAS USEFUL FOR THE COMMUNITY AND TRANSLATION, AND WHAT ARE THE KEY FACTORS IN THE ATLAS AND WHAT WOULD IT TAKE TO ACTUALLY MAKE IT HAPPEN IN TERMS OF FEASIBILITY. ONE ITEM IS DIFFERENCE, PRACTICAL CHALLENGES IN GENERATING THIS. THAT'S THE OVERVIEW. THAT'S OUR TASK. LET'S MOVE TO SESSION 2. AND THESE POINTS WERE ASSEMBLED WITH INPUT FROM WORKING GROUP OF SESSION 1 AND SESSION 2, JUST GUIDANCE. I WILL START BY SAYING OUR FOCUSING ON TWO SECTIONS, THE PRESENT, CURRENT ART, AND THEN WE'LL TAKE A VERY SHORT BREAK AND GO INTO THE FUTURE. FOR THE FIRST PART WE WANT TO FOCUS ON TWO KEY ITEMS HAS TO DO WITH IMMUNE RESPONSE AND BIODISTRIBUTION, BOTH SO IMPORTANT AND RELEVANT THAT THEY CAME UP MULTIPLE TIMES DURING SESSION 1. HOWEVER FOR THE PURPOSE OF SESSION 2A, WE WANT TO FOCUS ON THESE PARTICULAR POINTS. WE CANNOT DISCONNECT GENE THERAPY FROM ADMINISTRATION, SO AGAIN WE'RE GOING TO INCORPORATE ALL OF THE DIFFERENT ROUTES YOU HEARD FROM BEV AND SESSION 1 AND VECTOR TYPES. WE'VE DISCUSSED MAYBE TOO MUCH ABOUT THE AAV, LET'S ALSO BRING IN KNOWN VIRAL VECTORS, ASOs, OTHER MODALITIES, THAT ALLOW US TO DO THESE INTERVENTIONS. LET'S OPEN BROADER THAN THE VIRAL VECTORS, AND ADMINISTRATION ROUTE, VECTOR TYPES AND TRANSGENE TYPES. FOR SOME OF THE THERAPIES WE MIGHT NEED TO INSERT CHANNELINGS AUTOLOG TO THE BRAIN AND WORRIES THEY MIGHT BE OF NON-MAMMALIAN ORIGIN, LET'S DISCUSS NOT ONLY THE CAPSID AND ROUTE AND NEUTRALIZING ANTIBODIES BUT TRANSGENE ITSELF. ANTIGEN DISPLAYING CELLS, WHAT WOULD THEY LEAD TO? IMMUNOSUPPRESSION, WHAT CAN WE DO TO HELP, CAN WE ACT AT THE DELIVERY OR LATER ON? HOW CAN WE ENABLE SECOND ADMINISTRATION AS WELL? A VERY IMPORTANT ONE THAT WAS DISTRIBUTION.S VECTOR BY- WE TALKED ABOUT IN VITRO METHODS. HOWEVER, LOOKING FORWARD, THE MAIN PART IS DEVELOP METHODS THAT ALLOW US BIODISTRIBUTION IN VIVO AT THE TRANSGENE AND CAPSID FOR PURPOSE OF INFORMING DELIVERY ROUTES, FOR EXAMPLE PRESSURE OR AMOUNT OR TIME OF WE'VE TALKED ABOUT PET AND CURRENT METHODS AND IMPROVEMENT. ONE THING I WANT US TO KEEP IN MIND IS HOW LABELING OF CAPSID MIGHT AFFECT THE BIODISTRIBUTION. SO TO TALK ABOUT THE DIFFERENCES. CROSS-TALK BETWEEN CAPSID AND TRANSGENE HAS BEEN TOUCHED UPON, I THINK IT'S IMPORTANT TO KEEP IN MIND WHEN WE DISCUSS BIODISTRIBUTION. AND ALSO WHEN WE DISCUSS HOW TO IMPROVE THESE VECTORS. AND DIFFERENCES IN BIODISTRIBUTION, DEPENDING ON GENOME YOU DELIVER, WHETHER SINGLE STRAND OR SELF COMPLEMENTARIES AND GENE THERAPY STATE OF THE ART RELIES ON SELF COMPLEMENTARY. WE'RE MISSING ON PACKAGING, WHAT ARE WE GAINING AND WHAT ARE DIFFERENCES? SO WITHOUT FIRST ADO ALL PAUSE AND OPEN TO DISCUSSION, AND THEN IN SESSION 2B WE'LL GO IN TERMS OF FUTURE DEVELOPMENTS, WHAT CAN WE DO BETTER ABOUT VECTORS, VIRAL AND NON-VIRAL, BETTER ABOUT ALLOWING SECOND DELIVERY, ET CETERA. >> THIS IS CHI CHAN, IMMUNOLOGIST BY TRAINING, TRANSITION FROM ACADEMIA TO INDUSTRY. ON THE FIRST POINT ON ADMINISTRATION ROUTES, THE REAL ADMINISTRATION REALLY MATTERS IN TERMS OF HOW VECTOR AFFECTS IMMUNE RESPONSES BY BIODISTRIBUTION, PATHOLOGY THAT YOU CAN ELICIT. I THOUGHT I WOULD ADD ADDITIONAL TWO LAYERS FOR OUR DISCUSSION. ONE LAYER IS OFTEN WHEN I DISCUSS IMMUNE RESPONSES WITH GENE THERAPIES, THERE'S A VAGUE SENSE THERE'S THIS BIG BUCKET THING CALLED IMMUNE RESPONSES, SO THE WAY I LIKE TO THINK ABOUT IT IS THAT THE IMMUNE RESPONSE CAN BE AGAINST A CAPSID, OR CAN BE AGAINST A TRANSGENE PRODUCT. ANOTHER THING THAT I FIND SOMETIMES CONFUSING IS THAT WHEN IT COMES TO ADAPTIVE IMMUNITY THERE'S TWO ARMS, CYTOTOXIC T A LOT OF PEOPLE WILL MEASURE NEUTRALIZING ANTIBODIES BUT WHEN IT COMES TO IMMUNE RESPONSES IF YOU REALLY LAYER THE TWO CELL TYPES, ANTIBODIES OR T CELLS, CAPSID OR TRANSGENE, I THINK THAT MAKES THE DISCUSSION THE MOST DETAILED AND MOST USEFUL. THE LAST THING I WOULD LIKE TO ADD AS A LAYER IS LOCATION. I'VE SEEN PEOPLE SAY WE TESTED THE BLOOD FOR CYTOKINES, NO CYTOKINES, OR WE TESTED FOR T-CELL RESPONSES AGAINST TRANSGENE, OR THAT'S IN THE BLOOD. I THINK THE LOCATION REALLY MATTERS ESPECIALLY FOR CNS BECAUSE WE'RE PUTTING THE VECTOR INTO THE SPINAL CORD OR BRAIN THAT COULD BE LOCAL IMMUNE RESPONSES SO MICROGLIA LIVE INSIDE THESE CNS TISSUES, AND IF ACTIVATED THEY CAN ALSO RECRUIT T CELLS THAT GO IN THERE. THEY MIGHT BE DIFFERENT THAN T CELLS THAT LIVE IN THE BLOOD. I THOUGHT I WOULD ADD IMMUNE RESPONSES, AS WELL AS LOCATION OF THE IMMUNE RESPONSE. >> I WONDER IF YOU COULD TAKE IT A BIT DEEPER AND TALK ABOUT CONTRIBUTIONS OF REGULATORY T CELLS FOR ONE THING, AND THE OTHER IS THE DIFFERENCE BETWEEN IMMUNE RESPONSE IN A CHILD VERSUS ADULT, IS IT THE SAME, AND THE IMMUNE RESPONSE IN A NORMAL BRAIN OR ANIMAL MODEL VERSUS DEGENERATING BRAIN WHERE THERE MAY BE PROBLEMS WITH ALREADY THE BRAIN IS IN AN IMMUNE ACTIVATED STATE. >> SO FROM OUR EXPERIENCE, WE'VE DONE EXPERIMENTS MOSTLY IN MICE THAT WE HAVE TESTED NEONATAL MICE AS WELL AS ADULT NICE. NONE WAS IN CNS. SEEMS LIKE THERE WAS A TREND THAT NEONATAL MICE HAD LESS IMMUNE RESPONSE. WHEN IT COMES TO LARGE MAMMALS, ANIMAL MODELS AND HUMANS, IT'S A GOOD QUESTION. WE DON'T KNOW IF IMMUNE RESPONSE IN PEDIATRIC POPULATION OR IN INFANTS WOULD BE LESS THAN WHAT WE'RE CURRENTLY SEEING IN ADULTS. CERTAINLY ONE THING THAT HAS BEEN ATTRACTIVE TO THE RESEARCH GROUP IS THAT IN CERTAIN DISEASES THERE'S A DEGENERATION COMPONENT, DEGENERATION OFTEN HAS AN INFLAMMATORY CONNECTION SO CELLS THAT ARE DYING, APOPTOSIS, THEY CAN BRING IN OTHER IMMUNE CELLS TO THE SITE, SO WE'VE ALSO BEEN FASCINATED THAT PERHAPS THE BASELINE INFLAMMATION IN A DISEASED SPINAL CORD OR BRAIN IS LIKELY TO BE HIGHER, THAT MAKES A LOT OF SENSE. IF YOU LOOK AT THESE PATHOLOGY, HISTOPATHOLOGIES SLIDES, THEY HAVE T CELLS TO BEGIN WITH. THAT COULD REALLY AFFECT THE EFFICACY OF THERAPY. >> I ALWAYS -- I MEAN I AGREE ESSENTIALLY WITH WHAT CHI SAID. I WILL SAY IT'S INTERESTING TO LOOK AT EXPERIENCE IN OTHER TRIALS BECAUSE IN CNS BECAUSE OF THE NATURE OF THE ORGAN THERE'S A RELATIVELY LITTLE INFORMATION, SOMETIMES WHAT HAPPENS IN THE LOCAL LEVEL AND SO ON. BUT WHEN IT COMES TO THE AGE AND THE IMMUNE RESPONSE I THINK GENERALLY SPEAKING THE YOUNGER, THE LOWER IS THE RESPONSE. WE CAN SEE THAT IN A WAY SOME OF THE DATA THAT'S EMERGING ALSO IN GENE THERAPY FOR DMD, WHERE THERE WERE SOME ADVERSE EVENTS THAT WERE DESCRIBED, BUT NOT OTHERS, AND THE DIFFERENCE IS THE AGE OF THE PATIENTS. THE YOUNGER SEEM TO BE LESS LIKELIHOOD OF HAVING SOME KIND OF ADVERSE REACTION. AND THEN THE OTHER POINT THAT I THINK IS VERY IMPORTANT IS LOCAL LEVEL, THERE'S A LOT THAT COMES INTO PLAY. AND THEN THE PROBLEM IS THAT OFTENTIMES WHEN WE MEASURE, WE MEASURE SYSTEMICALLY AND WE MISS EXACTLY WHAT HAPPENS AT THE VERY LOCAL LEVEL WHICH IS HARD TO CATCH, RIGHT? BECAUSE THE WHOLE POINT ABOUT CPG IN OUR VECTORS THAT WAS BROUGHT UP BY FRASIER, THERE'S NEVER A CYTOKINE STORM. BUT THEN THE QUESTION IS IS THERE A WAY TO DETECT SOMETHING IN CSF AFTER AN AAV ADMINISTRATION THAT MAY MAYBE IS A MORE CLOSE COME COMPARTMENT AND MAYBE WE CAN SEE IT AFTER VECTOR, SO MAYBE IS WHERE WE DO OUR ANALYSIS AND THE TIMING DEPENDING WHAT WE'RE TRYING TO LOOK FOR, THAT MAY MAKE A DIFFERENCE. >> SO AS A FOLLOW-ON TO FEDERICO'S COMMENTS, NOT A STATEMENT, MORE A QUESTION TO MAYBE THE CLINICAL AUDIENCE HERE WHO HAS BEEN BRINGING THINGS TO THE CNS, WHAT HAVE BEEN THE OBSERVATIONS IN TERMS OF INFLAMMATORY SEQUELAE BECAUSE AS WE'VE LEARNED FROM THE SYSTEMIC TRIALS, HEMOPHILIA TRIALS, THE CLINICAL BENCHMARK GUIDES US TO THEN FOCUS ON CERTAIN QUESTIONS. YEAH, I WAS WONDERING IN TERMS OF MAYBE KRYS OR PEOPLE IN THE CLINIC, WHAT THE OBSERVATIONS ARE BECAUSE ONE OF THE PROBLEMS WITH MANY PRE-CLINICAL STUDIES THERE'S A POTENTIAL CONFOUNDER, OTHER THINGS THAT MAY PLAY INTO OBSERVATIONS AT LEAST MINIMIZED AT THE CLINICAL STAGE. >> I HAVE ANOTHER POINT THAT IT'S IMMUNE RESPONSE SEROTYPE, CAPSID SEROTYPE DEPENDENT OR UNIVERSAL, THE REASON I'M ASKING THIS QUESTION I OFTEN GET ASKED ABOUT RECENT PUBLICATIONS AND DATA PRESENTATIONS FROM A FEW COMPANIES ABOUT HOW AAV 5 IS DIFFERENT FROM OTHERS. I WANT TO HEAR OPINIONS OF THE IMMUNOLOGIC PEOPLE HERE, SEE WHETHER YOU HAVE ANY COMMENTS ON THAT. >> I GUESS IF I CAN MAYBE ADDRESS THE ISSUE OF OUR EXPERIENCE WITH IMMUNE RESPONSES IN THE THREE GENE THERAPY TRIALS WE'VE INITIATED, TWO IN PARKINSON'S DISEASE, ONE IN PEDIATRIC POPULATION, THIS IS INTERPARENCHYMAL DELIVERY, VERY LOCAL. THE ISSUE WE HAVE IS THAT JUST CAN'T SEE INSIDE THE BRAIN VERY WELL, SO THAT'S A PROBLEM. IT'S HIGHLY LOCALIZED IN TERMS OF WHERE THE GENE HAS BEEN DELIVERED. BUT THAT SAID, THERE ARE SOME OBVIOUSL IMAGING PARAMETERS WE'VE BEEN FOLLOWING IN TERMS OF SEEING EDEMA AS A RESULT OF THE GENE EXPRESSION AND THAT HAS NOT BEEN THE CASE. THERE HASN'T BEEN ANYTHING DETECTED IN THE PERIPHERY EITHER. ON THE OTHER HAND, WE HAVE DATA ON THE LONG-TERM GENE EXPRESSION WITH DELIVERY TO THE PARKINSON'S DISEASE PATIENTS, OVER 15 YEARS, AND I THINK THAT IS HOLDING VERY WELL. SO I THINK IF WE HAD ANY ISSUES WITH IMMUNE RESPONSES I THINK THAT EXPRESSION AND MAYBE RESULTS CLINICAL RESPONSES WOULD DIMINISH. I THINK THE THING I'D LIKE TO POINT OUT THAT HAS TO DO WITH GUANGPING'S QUESTION, WE'VE BEEN USING AAV 2, PRODUCED BY FRASIER WRIGHT, CHARACTERIZED CAREFULLY BEFORE GOING INTO PATIENTS, THE TRANSACTION WE'RE SEEING IS NEURONAL. AND I THINK WE'VE SEEN IN MULTIPLE EXPERIMENTAL PARADIGMS WHEN WE EXPRESS THROUGH THE AAV2 VECTOR, HUMAN PROTEIN IN THE NON-HUMAN SPECIES, WE GET IMMUNE RESPONSES. SO THE ISSUE REALLY HAS TO DO WITH AAV5 YIELDS AAV5 VECTOR WITH TRANSGENE THAT ENCODES FOR HUMAN PROTEIN IN NON-HUMAN SPECIES AND WE GET IMMUNE RESPONSES. WE DON'T GET YOU THROUGH AAV 2. I BELIEVE IT'S BECAUSE OF THE ANTIGEN-PRESENTING CELLS. NOW, ALSO BEING TRANSDUCED THROUGH THE NON-AAV2 TYPE VECTORS. AND THAT ACTUALLY IN DRAMATIC WAY, WHEN WE EXPRESS GFP FOR EXAMPLE IN NON-HUMAN PRIMATES THROUGH NOT AAV 2 VECTOR DEVELOP ATAXIA WITH LOSS OF CELLS, VERY PREDICTABLE. WE EXPRESS HUMAN PROTEIN, THROUGH AAV 5 OR 9 IN RODENTS GET IMMUNE RESPONSE, WE DON'T GET IT THROUGH AAV2, IT'S A POINT I'D LIKE TO EMPHASIZE BECAUSE GOING WITH THIS APPROACH IN PATIENTS WITH NEUROINFLAMMATION AS PARKINSON'S DISEASE IT'S CRITICAL TO THINK ABOUT NOT AGGRAVATING THIS ISSUE BY HAVING POTENTIAL IMMUNE RESPONSES, SO THIS IS OUR EXPERIENCE. BY NO MEANS I CAN TELL YOU THAT WE HAVE NOT SEEN ANY IMMUNE RESPONSES, WE SIMPLY HAVE NOT GOTTEN ANY MATERIAL FROM THOSE SUBJECTS, IT'S DIFFICULT TO SAY. BUT LONG-TERM EXPRESSION SEEING IN MULTIPLE TRIALS NOW WOULD SUGGEST THAT PERHAPS WE DON'T HAVE THAT ISSUE, AT LEAST NOT AT SIGNIFICANT LEVELS THROUGH AAV2. >> I WAS GOING TO SAY IN RODENTS IN YOU MAKE INJECTION INTO THE BRAIN PARENCHYMA YOU GET VERY RAPID INVASION OF MICROGLIA. I WONDER IF ANYBODY'S LOOKED AT THAT. MICROGLIA IN GENERAL ARE NOT VER INFECTIBLE WITH MOST OF THE AAV SEROTYPES BUT IT'S INNATE IMMUNE RESPONSE, ONE FUNCTION IS RESPOND TO INJURY, ANOTHER IS TO RESPOND TO ANY KIND OF VIRUS. SO I JUST WONDER IF ANYBODY LOOKED, IN ANIMAL MODEL, AFTER INJECTION DUE SEE INVASION OF MICROGLIA? >> WE DO BUT IT'S TRANSIENT. >> US ALSO. >> SO BASICALLY I THINK IT'S GOING TO BE TRANSGENE DEPENDENT, IN OUR CASES, AS KRYS SAID IT'S A TIMING ISSUE. >> KRYS, TO CLOSE THE LOOP BETWEEN THE THREE TRIALS AND PRIMATE STUDIES TWO VERSUS FIVE WERE THERE PROMOTER DIFFERENCES OR SAME CASSETTE? >> WE'VE NOT TAKENNING AAV5 INTO THE CLINIC, THIS IS ALL AAV 2, SAME PROMOTER. >> SAME PROMOTER? >> IN PRIMATES WE HAD MUST BE PROMOTERS THROUGH AAV 5. BUT THEN AGAIN I DIDN'T SEE ANY DIFFERENCE BETWEEN PROMOTERS WITH AAV 5 WITH IMMUNE RESPONSES SO I THINK PROBABLY HAD TO DO WITH THE TRANSDUCTION OF NON-NEURONAL CELLS, SO THERE'S TREMENDOUS TRANSDUCTION OF ASTROCYTES, GLIA OF COURSE, AND OTHER IMMUNOPRESENTING CELLS. >> FRASER? >> THANKS. THE FIRST COMMENT WITH KRYS, AGAIN THIS CONCEPT OF APC, WEAN ENGULFMENT OF PARTICLE BY APCs OR TRANSDUCTION IS A KEY COMPONENT POTENTIALLY TO IMMUNE RESPONSES. I ALSO -- KAI'S COMMENT, TALKING ABOUT IMMUNE RESPONSE, CAPSID, IMMUNOGEN, AND CTLs AND IMMUNE RESPONSE BUT THE CONCEPT OF DNA IN VECTOR PRODUCTS HOW MUCH OF THAT IS A COMPONENT OF TRIGGERING? TRIGGERING IMMUNE RESPONSES THROUGH INNATE PATHWAY, PATTERNS, PATHWAYS? >> ADDING INTRONS YOU CAN GET RANSOM OF THE PROBLEM BECAUSE YOU HAVE SPLICED DNA, I'M SORRY, SPLICED RNA AT THAT POINT. BUT THAT WILL GET YOU RANSOM OF THE INITIAL RESPONSE, TLR RESPONSE PATHWAY. >> BUT THAT'S GOOD, AND I THINK THERE ARE MANY PATHWAYS THROUGH THE TLRs AS WELL. SO BE IT -- I'M NOT SURE WHICH ONES, A NUMBER OF PATHWAYS TO CONSIDER IN THE CASE. >> LIKE DIANA HAS PRESENTED IN JUNE WE'VE EXPERIENCED INTRATHECAL OF AAV9 IN THE SPACE AND SEE A DOSE DEPENDENT INFLAMMATORY RESPONSE TO AAV9 AT THREE MONTHS, INCREASED PROTEIN, CLINICALLY SILENT, RESPONSIVE TO STEROIDS. WE DO NOT -- THESE PATIENTS ALL DEVELOP ALSO NEUTRALIZES ANTIBODIES THAT SHOW UP IN THE PERIPHERY AND CSF. THE ORIGIN OF THAT INFLAMMATORY RESPONSE IS NOT CLEAR TO US. WE HAVE A CYTOKINE DIFFERENCES WE'RE ANALYZING AT THE MOMENT SO WE'LL BE ABLE TO SHOW CYTOKINE PROFILES ON THE RESPONSE. WE'RE NOT SEEING ANTI-TRANSGENE RESPONSE IN OUR DELIVERY TRIAL BUT ALL OUR PATIENTS WHO ARE TRANSGENE OR NEGATIVE HAVE NO BASELINE, PROTEIN EXPERIENCE, IMMUNOMODULATORY PROTOCOL, WE HAVEN'T SEEN ANY TRANSGENE PROBLEMS IN OUR TRIAL. >> KAI PLEASE. >> I WAS WONDERING IF YOU COULD COME WHEN YOU SAY YOU ANALYZE CYTOKINES IN THE SERUM OF THE PATIENTS OR IS THAT CSF FLUID? >> IT'S IN THE CSF, YES. >> AND I WAS WONDERING HAVING HEARD YOUR WORK IF THE CSF PLEA OH CYTOSIS IF YOU'D TRIED TO ANALYZE WHAT THE WHITE BLOOD CELLS ARE IN ITEMS OF ACTIVITY AND ANTIGEN SPECIFICITY. >> WE'RE NOT ENTIRELY SURE. THERE ARE LYMPHOCYTIC ELEMENTS, PLEA OH CYTOSIS MOSTLY LYMPHOCYTIC IN NATURE. >> KEVIN FLANAGAN, NATIONWIDE. I THOUGHT I WOULD ADD OUR EXPERIENCE WITH AAV 9 FOR SYSTEMIC DELIVERY, AND THAT NOW THE IND IS WITH THE COMPANY, THE DATA WE'VE PUBLICLY PRESENTED BEFORE I CAN SAY REALLY WE'VE NOT SEEN ANY EVIDENCE FOR SYSTEMIC -- WITH SYSTEMIC DELIVERY, NOT AT 30 DAYS OR 180 DAYS FROM CSF INFLAMMATORY CELL RESPONSES WHATSOEVER. THOSE WERE PATIENTS WHO WERE NOT NULL MUTATIONS, SO IT'S NOT TO BE UNEXPECTED. AND IT'S INTERESTING IN SOME WAYS, THERE'S ANIMAL MODELS, MPS SUGGESTED INFLAMMATIONS PLAYS A ROLE, WE'VE NOT SEEN SIGNIFICANT CNS INFLAMMATORY RESPONSES. >> KEVIN, COULD I ASK A QUICK QUESTION ON THAT. ARE YOU ABLE TO SAY, PERHAPS IN THE PUBLICATIONS, THE SYSTEMIC INFLAMMATION PERHAPS LIVER SOURCE INFLAMMATION IN SYSTEMIC ADMINISTRATION. >> WE'VE SEEN EVIDENCE, LIKE ALL SYSTEM SERUM AAV9, WE HAD TO RESTART PREDNISONE AT DIFFERENT TIME POINTS IN PATIENTS. THERE'S EVIDENCE OF LIVER RESPONSE BUT NOT IN THE TARGET -- TIME POINTS ARE ONLY 30-DAY POST INJECTION LP AND SIX-MONTH POST-INJECTION LP. WE HAVEN'T SEEN IT IN THOSE. >> I HAVE A QUESTION FOR THE GROUP. SO I AS WELL HAVE SEEN EOSINOPHIL -- PLEIOCYTOSE IN THIS LARGE ANIMAL MODELS. I'VE RUN IT BY FRIENDS IN BIOTECH FIELD THAT DO VARIOUS DELIVERY MODALITIES INCLUDING GENE THERAPY BUT NOT LIMITED TO IT, ALSO VARIOUS ENZYME. THEY KIND OF COMMENTS THAT IT'S NOT REALLY A BIG DEAL, DON'T WORRY BIT. IT'S NOT CLINICALLY SIGNIFICANT. CAN YOU COMMENT AS TO WHETHER OR NOT IF THAT'S AN AGREEMNT OR IF THAT'S MAYBE SOME INFORMATION, BAD INFORMATION THAT I GOT FROM COLLEAGUES? >> WE DON'T KNOW AND WE DON'T WANT TO FIND OUT. [LAUGHTER] SO, WHEN WE REALIZED THE INFLAMMATORY RESPONSE IS DOSE DEPENDENT AND STEROID SUPPRESSIBLE WE ADJUSTED OUR IMMUNE MODULATION PROTOCOL TO COVER THAT BECAUSE IT IS SELF LIMITED, IT GOES AWAY. I DON'T KNOW WHETHER I WANT TO FIND OUT WHAT HAPPENS IF YOU JUST LET IT RAMPANT. THAT'S A PROBLEM WITH HUMAN TRIALS. YOU HAVE TO PROTECT YOUR SUBJECT AND YOU CAN'T EASILY DO AN EXPERIMENT WHERE YOU KNOW THAT THE RISK COULD BE INCREASED. >> I DID IT BOTH WAYS. BOTH GAVE STEROIDS AND NOT GAVE STEROIDS, I DIDN'T SEE A DIFFERENCE BETWEEN THE TWO GROUPS CLINICALLY. >> THAT'S ON THE PLEIOCYTOSIS, BUT THE INFLAMMATORY RESPONSE WAS STEROID RESPONSIVE AND PREVENTIBLE. >> KAI, COULD YOU PLEASE AMPLIFY ON THE TO 2 AND TO 9 RESPONSE, YOUR EXPERT AREA. >> I WORK ON INNATE IMMUNITY. IT DETECTS GENOME OF AAV VECTORS, BINDS TO CPG MOTIFS, THAT'S SOMETHING THAT FREQUENTLY IS ENRICHED WHEN COMPANIES CODON OPTIMIZE TRANSGENES TO GET MAX MA'AM -- MAXIMAL EXPRESSION, ABUNDANT IN UBIQUITOUS PROMOTERS. KAG PROMOTER. THE SECOND DETERMINANT IS RECEPTOR 2, IMPLICATED IS SENSING CAPSID, UNDEFINED WHAT IS THE MOTIF ON THE VIRAL CAPSID THAT COULD SET THAT OFF BUT THAT ALSO SEEMS LIKE A SECOND SENSOR, IN TERMS OF TOELACK RECEPTOR 9 STUDIES IN MICE HAVE SHOWN THE MICE HAVE MUCH LESS CYTOTOXIC T CELLS AGAINST AAV CAPSID, SO IT'S NOT JUST INNATE IMMUNE RESPONSE, ALSO A DOWNSTREAM FUNCTIONAL T-CELL RESPONSE IN FACT. >> IN TERMS OF STRATEGIES TO MINIMIZE THAT WOULD USING BLOCKING OLIGOS BE HELPFUL? >> YES, THAT'S A STRATEGY THAT WE'RE PURSUING. SO THERE ARE CERTAIN DNA SEQUENCES, ONE OF THEM DERIVED FROM HUMAN TELOMERES KNOWN TO BIND AND ANTAGONIZE IT SO ACTING AN ECTOMER, TRYING TO USE THOSE SEQUENCES AND INCORPORATE THEM INTO AAV VECTORS. WE'VE NOT TESTED IN CNS CONTEXT BUT HAVE DONE IT SUBRETINAL AND INTRAVITRIOLE, THE EYE IS AN EXTENSION OF CNS. >> DO YOU PERCEIVE CHALLENGES USING THIS FOR SYSTEMIC DELIVERY? TO USE BLOCKERS, TRL9 BLOCKERS, WHAT ABOUT SYSTEMIC, SOME OF THE TRIALS NOW USING SYSTEMIC AAVs, WOULD THERE BE CHALLENGES TO SUPPRESSING AT THAT LEVEL? >> SEEMS TO ME THE CHALLENGES WITH HIGH DOSE SYSTEMIC IS NUMBER ONE THE AMOUNT OF VECTOR YOU NEED. NUMBER TWO, THE PERCEIVED POTENTIAL OF HIGHER IMMUNE RESPONSES. SO I THINK THE WAY I THINK ABOUT IT IS THAT IF YOU CAN REDUCE IMMUNE RESPONSE, AND MAYBE GET MORE EXPRESSION, THAT COULD HELP ALLEVIATE SOME OF THE HIGH AMOUNTS OF VECTOR YOU NEED TO PRODUCE. WE HAVE NOT TESTED OUR STRATEGY IN HIGH DOSE SYSTEMIC ADMINISTRATION. SO TBD ON HOW THAT LOOKS LIKE. I THINK HIGH DOSE SYSTEMIC IS A NATURAL PLACE WE WOULD WANT TO MANAGE IMMUNE RESPONSES. >> SO I'M UNCLEAR OF THE ACTIMER APPROACH, SUPPRESSED OH CO-PACKAGED SELF LIMITING SEQUENCE AND IF EXPRESSED WOULD YOU WANT IT ON ALL THE TIME? >> WE PRESENTED THIS, THERE ARE ABSTRACTS ONLINE. IT'S NOT AN EXPRESS SEQUENCE. IT'S DNA ECTOMER SEQUENCE PART OF THE VECTOR GENOME AND LOOKS TRL 9. THERE'S NO TRANSCRIBED PROTEIN. >> YOU WERE IN GEORGE'S -- >> THAT'S RIGHT >> YOU HELPED GEORGE WITH HIS PLATFORM PRESENTATION, NOW I REMEMBER. >> I KIND OF WANT TO GO BACK TO THE MECHANISM ON THIS. IS THERE ANY CLARITY OR IS THERE THOUGHT ON WHETHER WE NEED TO LOOK INTO THIS FURTHER BUT IS THIS THE VIRAL PARTICLE AND VIRAL DNA, ARE WE SURE, DOES IT HAVE THE WITH TO WITH PREP OR FREE VIRAL GENOMES? TECHNICALLY THE VIRUS IS NOT SUPPOSED TO PRESENT ITS GENOME TO TRL9 BECAUSE IT'S IN THE ENDOSOME AND GETS IN IN THE NUCLEUSS THERE ANY UNDERSTANDING OR THOUGHT PROCESS THERE? >> (INAUDIBLE). >> I'VE SEEN STUDIES WHERE THEY COMPARED PURIFIED AAV VECTORS, FILLED CAPSID WITH DNA GENOME VERSUS STUDY EMPTY CAPSIDS, A RECENT PUBLICATION THAT CAME OUT THIS WEEK FROM AGTC, INJECTEDE EMPTY OR CAPSID. EMPTY CAPSIDS DID NOT ELICIT INFLAMMATION IN FRONT OF THE EYE. IT WAS THE SAME AMOUNT OF CAPSID PROTEIN. VECTORS THAT HAD DNA IN IT DID. SO THAT SUGGESTED DNA IS AT LEAST A MAJOR COMPONENT. I THOUGHT THAT ONE THING THAT WAS BROUGHT UP ABOUT WE DON'T NECESSARILY WANT TO FIND OUT EXACTLY WHAT THESE IMMUNE RESPONSES CORRESPOND TO, I THOUGHT THAT ONE THING THAT COULD BE USEFUL IS THAT THIS CSF PLEIOCYTOSE IS OBSERVED IN NON-HUMAN PRIMATES, SO MANY OF YOU ARE PROBABLY FAMILIAR WITH THE HEMOPHILIA B STORY, YOU CANNOT MODEL THE ALT ELEVATIONS, LOSS OF TRANSGENE EXPRESSION IN MONKEYS. YOU ONLY SEE IT IN HUMANS. BUT ACTUALLY FOR CNS WE DO SEE STUDIES PUBLISHED STUDY SHOWING CSF PLEIOCYTOSIS HAPPENS WITH INTRATHECAL NON-HUMAN PRIMATE, THAT MIGHT BE THE PERFECT AVENUE TO TEST WHETHER IMMUNOSUPPRESSION MATTERS, WHETHER CPG MATTERS, WHETHER THE CAPSID MATTERS. I THINK WE HAVE ACTUALLY MODELLABLE SYSTEMS HERE THAT COULD BE USEFUL WITH A FEW BIG ANSWERS. >> CAN WE HAVE -- YEAH. SO, I AGREE WITH THE COMMENTS. I WANTED TO ADD A COUPLE THINGS. ONE OF THEM, KAI, YOU MENTIONED CPG. THE CULPRIT THAT DOES ACTIVATE TLR 9 IS UNMETHYLATED CPG. THEY TEND TO BE LARGELY METHYLATED, SO I WANT TO MAKE THAT COMMENT. IT'S GOING BACK TO HEMOPHILIA, BASED ON PUBLISH DATA, WORKSHOPS, WITH DIFFERENT LEVELS OF PURITY GOING BACK TO CONCEPT OF PURITY, EXTRA VECTOR OR ACTUALLY DNA IMMUNE SIDE, DIFFERENT PREPS FROM DIFFERENT LABS HAVE SHOWN THERE'S CORRELATION WITH MORE CPGs IN TRANSGENE LEADING TO MORE PROBLEMS AND LESS CPGs LEADING TO LESS PROBLEMS, DOES SUPPORT TLR9 LOOKING AT DNA ON THE INSIDE OF THE VECTOR. >> I'M GOING TO PLAY DEVIL'S ADVOCATE. THE WAY I'M THINKING ABOUT IT AND PROBABLY IN TERMS OF OUR OWN DOGMATIC SORT OF THINGS THAT ARE DONE, AND JUDE AND OTHERS SHOULD COMMENT ON THIS TOO, IT'S UNDERSTOOD IN GENERAL THERE'S NO SUCH THING AS A PURE EMPTY IN THE FIELD. ALL EMPTY PARTICLES AND GROUPS HAVE SHOWN 200 BASES OR SMALLER FRAGMENTS OF DNA ARE PACKAGED IN THEM, NOT TECHNICALLY EMPTY. THE PROPERTY OF DNA CONTAMINATION AND EMPTY AS OPPOSED TO FULLS RELEASING GENOMES IN FULLS IS HIGHER, AGAIN IT COMES DOWN TO I AGREE WITH CPG COMMENT AND TRIGGERS TRL 9 BUT WHERE DOES IT COME FROM, MAYBE IT'S THE PREP. SORRY. >> IT'S DOSE DEPENDENT. IF YOU PUSH THE DOSE, EVEN IF YOUR CONTAMINANTS, CAPSID LOAD WILL BE MORE, YOUR DNA, EVEN IF IT'S CPG DEPLEATED WILL INCREASE DNA LOAD. SO I THINK WHAT WE ARE LEARNING FOR EXAMPLE FROM TO TALK ABOUT HEMOPHILIA TRIALS, THERE'S ALWAYS BEEN A VERY DIRECT -- WE HAVE TRIALS, ALWAYS A DIRECT MEASURE OF THE IMMUNOGENICITY WITH LIVER ENZYME ELEVATION. EARLY GENERATION VECTORS, IMMUNOGENIC, EVEN IN LOW DOSES BECAUSE THEY HAVE HIGH CPG CONTENT, NOW EVEN THE LAST MEETING TURN OUT EVEN VECTORS THAT WERE CPG DEPLETED IF GIVEN AT HIGH DOSES WOULD ELICIT LIVER ENZYME ELEVATION BECAUSE DNA SOMEHOW ENGAGES TLR 9 EVEN IF IT'S COME WHAT CPG DEPLETED, SO IT'S NOT THAT WE MAKE OUR VECTOR INVISIBLE. I THINK AS WE PUSH THE DOSE THEN WE'LL HAVE AN ISSUE WITH IMMUNOGENICITY. I THINK IN THIS CNS WHAT PERHAPS IS MISSING IS WORK DONE IN THE CONTEXT BY ROLAND HERZOD, RESPONSE IN HEPATOCYTES IN THE LIVER, PARENCHYMAL CELLS, INTERESTING TO LOOK TO FIND SOME TARGETS, SOME IDEAS IF IT'S WORKING REALLY THE SAME WAY AND THEN FIND INTERVENTION OR INTERVENTIONS TO DECREASE THE IMMUNOGENICITY BUT MY SENSE IS THAT IF YOU PUSH THE DOSE YOU'LL SEE IMMUNOGENICITY. OF COURSE YOU NEED TO DESIGN YOUR VECTOR TO BE THE LOW -- WITH THE LOWEST IMMUNOGENICITY PROFILE BUT SECOND DOSES YOU'LL SEE IT. >> I JUST WANT TO DRAW EVERYBODY'S ATTENTION TO COMPONENT THAT WE'VE KIND OF ACCEPTED AS PART OF THE VECTOR HERE. I THINK AMET WAS EARLY IN DETERMINING STEROIDS WOULD BLUNT IMMUNE RESPONSE, AND HIS GROUP LIKE OURS THOUGHT IT WAS EMPTY PARTICLES THAT WERE RESPONSIBLE FOR CREATING THAT. AND I THINK EACH GROUP HAS GOTTEN BETTER AT REMOVING EMPTIES AND SHOWING IT'S NOT RELATED TO EMPTY PARTICLES. AND I THINK FEDERICO'S COMMENT IS ON TARGET BUT IF YOU THINK ABOUT WHAT WE DO KNOW AND HAVE ACCEPTED AS PART OF THE CLINICAL PREPS, IT'S ABOUT THREE TO FIVE PER CENT IS PACKAGING BACTERIAL BACKBONE IN THESE PARTICLES. IF YOU WERE JUST ADDING THE DOSE THAT WE ARE USING IN THE DMD TRIAL, TWO TIMES 10 TO THE 14th PER KILL OH GRAM OF BODY WAYS, 5 TIMES 10 TO THE 13 PARTICLES OF BACTERIAL SEQUENCES. I'M SURE IF I DESIGNED A TRIAL AND WENT TO THE FDA AND SAID I WANT TO DELIVER ONLY BACTERIAL SEQUENCES WITH AAV AND 10 DO THE 13 PER KILO, WE MAY ELICIT SOME TYPE OF DELAYED OR INNATE IMMUNE RESPONSE TYPE THING SO I THINK THERE'S A CONTAMINANT COMPONENT THAT WE'VE ACCEPTED IN THE FIELD, THAT IF WE WERE ABLE TO REMOVE THAT COMPLETELY THAT WOULD AT LEAST BE ONE MORE VARIABLE THAT WE COULD EITHER SAY IS CONTRIBUTING OR NOT CONTRIBUTING. >> SO AS FOLLOW-P TO THAT, IS THERE ANY DATA THAT HAS COME OUT FROM THE INSECT CELL PREPS VIRUSES THAT WENT INTO TRIALS BECAUSE THERE SHOULD BE NO BACTERIAL ELEMENTS. >> YEAH, THEY DO PACKAGE THE SEQUENCE, HERPES, EVERYBODY IS GETTING SAME AMOUNT OF CONTAMINATION. THE QUESTION IS ARE THE METHYLATED BACTERIAL SEQUENCES DIFFERENT THAN THE HERPES AND THAT -- I DON'T THINK THAT'S KNOWN, AT LEAST I'M NOT AWARE OF IT. >> SO ONE OF THE THINGS IS TO WHAT EXTENT IS THERE DNA STUCK ON THE SURFACE OF THE VIRUS? WITHOUT DNA TREATMENT, IT'S DIFFERENT. SO THAT'S ONE COMPONENT THAT WHEN YOU TALK ABOUT THEORETICALLY ENDOSOME, THERE'S A LOT OF DNA THAT IS STUCK DESPITE TREATMENTS WE DO AND PAY A LOT FOR, I DON'T THINK WE COMPLETELY ELIMINATE IT ACTUALLY. >> MAYBE. >> REALLY QUICKLY, YEAH, NO, THINK THAT'S TRUE IF YOU HAVE DNA, I DO BELIEVE THAT IS -- I DON'T WANT TO CALL IT A MORE MINOR PROBLEM BUT THERE ARE MORE OBVIOUS AVENUES TO GET RID OF THAT. THE MATERIAL I THINK, JUDE, YOU REFER TO, I AGREE WITH YOUR COMMENTS, REVERSE PACKAGING OFF IS SIGNIFICANT. HOW CAN YOU SEPARATE THOSE PARTICLES BASED ON PURIFICATION SO IT DOES COME, I WANTED TO MAKE A COMMENT ABOUT USING TRANSIENT TRANSFECTION SYSTEM, USING OVERSIZE BACKBONE IN VECTOR PLASMID HELPS A LOT, MAYBE FIVE TO TEN-FOLD, BUT STILL BRINGS IT DOWN MAYBE 5E-13 BACTERIAL PARTICLES PER KILO TO 1 OR SOMETHING LIKE THAT, STILL A PROBLEM. >> I THINK IF YOU JUST LOOK AT THE DATA IN HUMANS, AS THE DOSE GOES UP YOU SEE THIS RESPONSE, YOU CAN IMAGINE IT'S SIMPLY RELATED TO BACTERIAL SEQUENCES THAT ARE BECOMING AN ANTIGEN AS DOSE GOES UP. >> I AGREE WITH THE POINT ABOUT BACTERIAL SEQUENCES, BEING PACKAGED, ALSO AGREE WITH THE NOTION GENOMES OF THE VIRUS CAN GET EXPOSED BECAUSE OF DEFECTIVE PARTICLES BUT I DEFINITELY PUSH BACK AGAINST THE NOTION DNA BOUND TO THE SURFACE OF THE VIRAL CAPSID BECAUSE THERE'S ABSOLUTELY NO STRUCTURAL DATA TO DATE THAT HAS SHOWN ANY EVIDENCE THERE'S ANY PIECE OF DNA STUCK TO ANY AAV POST PURIFICATION, METHODS STRUCTURAL FOLKS USE SIMILAR METHODS TO WHAT'S HAPPENING IN THE PURIFICATION SPACE, ZERO EVIDENCE THERE'S DNA ON THE SURFACE OF THE VIRAL PARTICLES, EVEN IN AAV2 POSITIVE CHARGES THAT BIND HEPARAN SULFATE WE DO NOT SEE PIECES OF DNA STUCK ON THE VIRAL SURFACE. NOW, WHETHER IT'S SOME KIND OF TRANSIENT INTERACTION THAT CAN HAPPEN OR QUITE POSSIBLE, BUT I ARGUE THE GENOME BEING EXPOSED TO ARTICLES IS HIGHLY LIKELY, AND THAT SHOULD GET REMOVED BY DNA AND THAT'S WHY WE DON'T -- WE GET A HIGHER TITER. >> I'LL INTERJECT. I WOULD SAY THAT HAVING RUN A COURSE THAT SUPPORTED SEVERAL CLINICAL STUDIES FOR A LONG TIME, I THINK IF PROPERLY PURIFIED, I SPECK THE CORES ESTABLISHED HAVE ESTABLISHED THESE PURIFICATION PROCESSES, I WOULD AGREE THERE SHOULDN'T BE ANY. THAT WOULD COME UP IN THE PLUS OR MINUS DNA'S DIGESTING IN qPCR, THERE SHOULDN'T BE A DELTA THERE, WE NEED TO AIM FOR CLINICAL DEVELOPMENT. >> CLEARLY THERE ARE A LOT OF PROBLEMS ASSOCIATED WITH THIS SEE NORMOUS DOSE OF GENE THERAPEUTICS THAT HAVE TO BE INJECTED SYSTEMICALLY AND IN THIS CONTEXT I HAVE TO SAY I WAS DEVASTATED NOT TO SEE ON THE LIST OF ADMINISTRATION ROUTES MY FAVORITE DELIVERY ROUTE. AND THIS IS INTRAARTERIAL INJECTION. THIS ROUTE IS UNDERAPPRECIATED FOR SEVERAL REASONS, PEOPLE THINK IT'S COMPLICATED, IT'S DANGEROUS, EXPENSIVE TO DO. HOW EFFECTIVE MECHANICAL IS, INFRASTRUCTURE IN VASCULAR PROCEDURE AND EXPERTISE IS GROWING AND THIS ROUTE HAS A LOT OF VERY IMPORTANT ADVANTAGES, WE DON'T HAVE DATA FOR GENE THERAPEUTICS YET BUT WE HAVE DATA FOR MACROMOLECULES FOR CELLS. YOU CAN TAKE ADVANTAGE OF THE FIRST, ADVANTAGE OF ARTERIAL DELIVERY. WE CAN GET UP TO 1,000-FOLD INCREASE OF THE CEREBRAL, ESPECIALLY WHEN TARGETING THE BRAIN, OF CEREBRAL UPTAKE. THERE ARE OPPORTUNITIES OF CAPSID ENGINEERING TO IMPROVE ENDOTHELIAL CAPTURE, EVEN IN CONTEXT OF IMMUNITY IF THERE ARE NEUTRALIZING ANTIBODIES, IF YOU INJECT INTRAARTERIALLY WE DON'T KNOW FOR A FACT BUT QUITE LIKELY YOU COULD AVOID THIS INACTIVATING EFFECT, AND I WILL COME BACK THERE AND WILL TRY TO ADVOCATE FOR INTRAARTERIAL DELIVERY. WE HAVE A LOT OF DATA. WE WORKED FOR MANY YEARS AND WE CAN DO IT IN A PRECISE WAY, PREDICTABLE, AND WE CAN POST THERAPEUTICS ACROSS THE ENDOTHELIAL BARRIER. >> I'M GOING TO JUMP IN. WITH AAV9 WE DID THAT IN NON-HUMAN PRIMATE, LOW Ns, BUT DIDN'T MAKE ANY DIFFERENCE BETWEEN VENOUS AND CAROTID ARTERY. >> YEAH, THIS IS ACTUALLY -- THAT'S ALSO IN THE FIELD OF ONCOLOGY, ARTERIAL INJECTION WAS ABANDONED BECAUSE OF HIGH VARIABILITY AND EFFICACY. HAVE YOU THAT ADVANTAGE, THAT POTENTIAL ADVANTAGE, AND IT HAS TO BE DONE IN THE PRECISE WAY WITH IMAGING, PROVING THAT TARGETING IS GOOD AND THERE ARE SOME TOOLS THAT HAVE TO BE DEVELOPED. ONE OF THE THINGS THAT I SAID WOULD BE CAPSID ENGINEERING TO INCREASE ENDOTHELIAL CAPTURE AND MAKE SURE, VISUAL IDES -- VISUALIZE YOU HAVE THE ANSWER OF TARGETIG. >> WE DID IT IN CATS AND DIDN'T SEE ANY DIFFERENCE BETWEEN THE TWO ON VENOUS DELIVERY EITHER FOR AAV9. >> BEFORE WE GET INTO DISCUSSION ABOUT STRATEGIES, HOW TO FACE THOSE CHALLENGES, I HAVE ONE QUESTION. I HEARD MENTIONED BY ONE OF THE DISCUSSANTS, BECAUSE THIS TOPIC WAS VERY EXTENSIVELY DISCUSSED AT LAST IMMUNOGENICITY MEETING, I THINK STEVE AND FEDERICO BOTH THERE, CAN YOU PLEASE AMPLIFY THE DEBATE ON THE CRIME. >> CROSS REACTIVE IMMUNE MATERIAL, CRIM. WE'RE ALL CRIMINALS. WELL, YEAH, I THINK THIS IS ALSO SUCH AN IMPORTANT TOPIC BECAUSE CROSS REACTIVE IMMUNE MATERIALS BASICALLY ARE THE PATIENTS PRE-TOLERANT TO THE TRANSGENE OR WOULD THEY BE PREDICTED TO ELICIT IMMUNE RESPONSE AGAINST THE TRANSGENE. SO IF WE'RE TALKING ABOUT DELIVERING NON-SELF PROTEIN THAT WOULD BE CRIM NEGATIVE, NAIVE. SO SINCE A LOT OF THESE ARE MONOGENIC DISEASES, A LOT OF PATIENTS MAY HAVE DELETION MUTATIONS, IT'S A SIGNIFICANT PORTION OF THE PATIENT POPULATION FOR MOST DISEASES, THEY MAY BE CRIM NEGATIVE. THEIR BODY HAS NEVER SEEN THAT PROTEIN. I THINK THERE'S TWO CHALLENGES HERE. ONE IS MANAGING PROPHYLACTICALLY POTENTIAL IMMUNE RESPONSE AGAINST TRANSGENE, I THINK THE OTHER CHALLENGE IS PREDICTING WHICH PATIENTS ARE IN FACT CRIM NEGATIVE OR PREDICTED TO BE CRIM NEGATIVE, EVEN A SINGLE POINT MUTATION COULD HIT JUST THE RIGHT EPITOPE AND BE IMMUNOGENETIC. PRACTICALLY, WE HAVE ANTICIPATE MAYBE THEY WON'T MOUNT RESPONSE, IF IT'S A DELETION OR NONSENSE MUTATION MAYBE THEY WOULD MOUNT AN IMMUNE RESPONSE. >> THAT'S AN AREA THAT ESPECIALLY COMPANIES THAT ARE DOING ENZYME REPLACEMENT THERAPY FACE AND UNDERSTAND VERY WELL. THAT'S WERE SOME INFORMATION COULD -- THE KEY QUESTION WHETHER YOU DEVELOP NEUTRALIZING ANTIBODIES, OTHERWISE THE FIELD HAS A WAY TO DEAL WITH THAT. >> I WAS GOING TO ADD TO THAT, WE'VE DONE A LOT LOOKING AT BOTH STANDARD AND INHIBITORY ANTIBODIES IN MPS1, SERIOUSLY IN POMPE DISEASE, THE OUTCOME IN THE TWO DISEASES IS EXTREMELY DIFFERENT. POMPE DISEASE CRIM STATUS IS CRITICAL, IF THE PATIENT HAS ANTIBODIES THEY OFTEN DIE. IN MPS1, IF THE PATIENT HAS A NEGATIVE CRIM STATUS THEY WILL ALMOST ALWAYS SHOW ANTIBODY RESPONSE, BUT IT'S RARE THAT THEY WILL BE IN ANTIBODY RESPONSE AND THAT JUST BLOCKS EFFICACY OF THE TREATMENT RATHER THAN ACTUALLY BEING A LIFE-LIMITING EXPERIENCE FOR THEM. SO IT CAN BE VERY DIFFERENT IN DIFFERENT DISEASES, AND THAT'S WHY WE CAN TAKE A LOT OF EXPERIENCE FROM COMPANIES. >> AND I'D LIKE TO ADD TO THAT THAT I WONDER HOW DIFFERENT IT IS ALSO FROM BOLUS ADMINISTERED ERT DELIVERED, ERT THERAPIES, THE IMMUNE RESPONSES CAN BE OVERCOME BY GIVING MORE MATERIAL OR SOMEHOW MANAGING THE PLASMA RESPONSES LIKE THEY DO IN POMPE VERSUS EXPRESSION FROM ANTIGEN PRESENTING CELL, FROM A NEURON, OR EXPRESSION FROM PERI VASCULAR MACROPHAGE IN THE BRAIN TAKING UP THE RECOMBINANT PRODUCT. I'D LIKE TO ECHO AND DRIVE HOME BRIAN'S POINT THAT WE CAN'T TREAT ALL THESE PROTEINS EXACTLY THE SAME BECAUSE WE SEE AN IMMUNE RESPONSE TO ONE, MAY NOT IN ANOTHER BECAUSE WE DON'T IN ONE, WE MAY IN ANOTHER, AND THIS GETS BACK I THINK A LITTLE BIT TO THE FIRST SESSION WHERE THE MORE HUMAN DATA WE HAVE THAT'S PUBLICLY ACCESSIBLE, THE BETTER WE CAN BEGIN TO PREDICT HOW SUCCESSFUL OUR THERAPIES ARE GOING TO BE. >> AND THERE ARE TWO THINGS THERE AS WELL. HAVE YOU AN IgG -- SORRY, IgE RESPONSE TO THE INITIAL BOLUS DELIVERY OF ERT, MORE LIKE WHAT YOU'D SEE WITH AAV. AND THEN YOU'VE GOT THAT SORT OF SYSTEMIC RESPONSE WHICH WILL IgM AND IgG LATER. INTERESTINGLY WHEN YOU TRANSPORT THEY GENERATE TOLERANCE TO THE PROTEIN, WE SHOULD ALL BE TRANSPLANTING OBVIOUSLY. >> YOUR QUESTION AND COMMENT IN TERMS OF APC CELLS ONE OF THE DIFFICULTIES FROM GENE THERAPY PERSPECTIVE THEY ARE DONE WITH HUMAN cDNA, IN TERMS OF ANTIGEN PRESENTING CELLS THEY BEHAVE DIFFERENTLY WHETHER OR NOT THEY ARE EXPRESSED IN HUMANS VERSUS IMMUNE BACKGROUND. >> I THINK CRIM TOPIC WAS VERY SENSITIVE AND TIMELY WHEN HEMOPHILIA PROGRAMS WERE GOING FORWARD. ONE OF THE RESTRICTIONS PLACED ON TRIALS THE PATIENTS HAD TO TAKE 50 EXOGENOUS DOSES OF FACTOR 9 WITHOUT HAVING AN INHIBITOR RESPONSE BEFORE THEY COULD BE ENTERED INTO THE TRIAL. AND SO THEY WERE BASICALLY -- THE FDA WAS HELPING US SEGREGATE PATIENTS LESS LIKELY TO GENERATE INHIBITORS, THOSE WENT INTO ALL OF THE EARLY TRIALS. I'M NOT SURE IF THAT'S CHANGED. IF IT HAS CHANGED IT WOULD BE VALUABLE INFORMATION BECAUSE IT WOULD THEN SAY THERE'S ENOUGH HUMAN DATA WITH CRIM PLUS CRIM MINUS THEY ARE NOW LETTING OTHER PATIENTS BE TREATED BUT I DON'T KNOW IF THAT'S CHANGED, BUT IT WAS A RESTRICTION FOR THE EARLY TRIALS. >> ONE MORE QUESTION FOR THE NEUROIMMUNOLOGYISTS IN THE ROOM IF THERE ARE ANY. WHAT'S THE DIFFERENCE WITHIN A RESPONSE FROM PERIVASCULAR MACROPHAGE VERSUS MIKE LOW GLIA CELL? BRIAN, MAYBE YOU KNOW THIS A LITTLE BIT. DOES IT MATTER, IS EVERYTHING WE HAVE FROM MICE, IS THERE ANY WAY TO GET THIS INFORMATION FROM SOME OF THE ERT DATA IN HUMANS OR EVEN NON-HUMAN PRIMATES? >> I THINK THE SHORT ANSWER IS I DON'T KNOW. THEY ARE NOT THE SAME CELL, DON'T EXPRESS THE SAME THINGS, EVEN AFTER WE'VE DONE BONE MARROW TRANSPLANT THE CELLS ARE TRAFFICKED INTO THE BRAIN AND REENGRAFT, NOT MICROGLIA PER SE, THEY ARE MACROPHAGES THAT HAVE& TAKEN ON SOME SIMILAR FUNCTIONS TO MICROGLIA BUT THEY ARE NOT TRANSCRIPTIONALLY IDENTICAL. THEY ARE SIMILAR. SO I DON'T KNOW IS THE BOTTOM LINE. CONTEXT IS REALLY IMPORTANT. SO THE PLACE THAT THE IMMUNE SYSTEM FIRST ENCOUNTERS ANTIGEN I THINK IS PROBABLY QUITE IMPORTANT, KAI KNOWS MORE ABOUT THIS THAN I DO. >> FROM MY TEXT BOOK, KNOWLEDGE AND PRESENTING OF THIS MICROGLIA, TYPICALLY THEY LOOK RAM FIDE, RESTING STATE, IF THEY RECEIVED SIGNALS FROM RECEPTORS THEY CAN RECEIVE ONE AND TWO, THEY CAN BECOME LIKE AN ANTIGEN PRESENTING CELL. I THINK TEXTBOOK DIFFERENCE BETWEEN MICROGLIA AND MACROPHAGES, MACROPHAGES FROM THE OUTSIDE ARE ANTIGEN PRESENTING CELLS TO BEGIN WITH, THE THRESHOLD, THE BAR FOR ACTIVATING IS LOWER. THE LAST PIECE WHETHER THEY CROSS-TALK. AS YOU SHARED, MACROPHAGES CAN COME IN. SOMEONE IS CALLING THEM IN. MICROGLIA MIGHT BE A PROMINENT CANDIDATE FOR THAT. SO I THINK IN TERMS OF ANTIGEN PRESENTING CELLS, THE QUESTION MICROGLIA TO TARGET OR TO AVOID, I I DON'T KNOW IF IT'S A TRICK QUESTION BUT WOULD AVOID THE MICROGLIA IF POSSIBLE. >> I THINK THE BIG PROBLEM IS IN MOST CNS DISEASES, THERE'S A SIGNIFICANT NEUROINFLAMMATORY COMPONENT WHICH MEANS MICROGLIA ARE ALREADY KICKING OUT ONE ALPHA, WHICH IS A CYTOKINE FOR WHICH FUNCTION WHEN REALLY NOT QUITE SURE WHAT IT'S DOING VERSUS MCP AND YOU'LL HAVE OTHER CYTOKINES WHICH UPREGULATE AND POTENTIATE SUCH AS IL-1, TNF ALPHA AND SO ON. SO YEAH, WE NEED TO KNOW A LOT MORE ABOUT IT BUT, AGAIN, IT'S OFTEN DISEASE SPECIFIC. >> A FOLLOW-UP, ARE WE SAYING WE DON'T WANT VIRUSES TO GET INTO THE MICROGLIA OR DON'T WANT EXPRESSION FROM THE MICROGLIA? >> IN AN IDEAL WORLD WOULDN'T WANT EITHER OF THEM TO HAPPEN. BUT PROBABLY BOTH ARE HAPPENING. SO YOU COULD IMAGINE A NEURONAL SPECIFIC PROMOTER AT LEAST REDUCED EXPRESSION IN THE MICROGLIA AND MACROPHAGES, AND THAT MIGHT BE THEORETICALLY MIGHT BE BENEFICIAL FOR CRIM NEGATIVE PATIENTS. >> A COUPLE COMMENTS, IF I COULD. I PUT ON THE TABLE IS IT POSSIBLE THAT IF YOU HAD AN AAV VECTOR, TO PUT IT ON THE TABLE, I WANT TO GO BACK TO FEDERICO'S COMMENT ABOUT REFERRING BACK TO ROLAND HERZOG, TRANSGENE TOLERIZING EFFECT, WE'RE NOT SURE TO HOW THAT APPLIES IN THE BRAIN, YOU'VE DELIVERED PART OF THE VECTOR TO THE LIVER, ANY SYSTEMIC ADMINISTRATION IS PRIMARILY TO THE LIVER. >> TWO QUESTIONS ON A DIFFERENT HOPEFULLY THE QUESTIONS ARE NOT TOO INFLAMMATORY. [LAUGHTER] FIRST OF ALL I'D LOVE TO HEAR FROM THE PANEL WHAP THEY THINK OF THE OBSERVATIONS OF THE DRG TOXICITIES, PARTICULARLY IN HIGH DOSE APPLICATIONS, WHETHER OR NOT THE NEWS OUT OF RECENT STUDIES IN DND SPACE AROUND COMPLEMENT ACTIVATION HOW THEY MAY IMPACT PARTICULARLY SYSTEMIC HIGH DOSE APPLICATIONS. >> COMPLEMENT ACTIVATION IS PART OF THE INNATE IMMUNE RESPONSE. I DON'T KNOW SPECIFIC BUT MIGHT IMPLICATE MORE INNATE COMPONENTS OF AN IMMUNE RESPONSE AND THEIR IMPORTANCE AT HIGH DOSE. >> ANYBODY ON DMD? ANY COMMENTS? OKAY. TO BRIEFLY TRY TO SUM IT UP, IF POSSIBLE -- >> ONE VARIABLE WITH DMD COULD BE IN THE SCREENING PROCESS, TYPICALLY IF YOU'RE LOOKING ONLY AT NEUTRALIZING ANTIBODIES WHAT YOU WILL MISS IS ANTIBODIES THAT BIND BUT DON'T NEUTRALIZE. SO PATIENT MAY SCREEN OKAY TO GO FORWARD FOR NOT HAVING NEUTRALIZING ANTIBODIES BUT MAY HAVE BINDING ANTIBODIES AND THOSE WOULD TRIGGER COMPLEMENT SYMPTOMS SEEN IN THE ONE PATIENT, BAMBOO IN ONE PATIENT, SOLID TRIAL. WHAT ARE OUR CRITERIA OF ANALYZING THE PATIENT BEFORE THEY GO INTO A TRIAL, ONLY ANTIBODY BINDING LIKE ELISA WOULD CAPTURE EVERYTHING BUT WEREN'T DETERMINE IF THEY WERE NEUTRALIZING AT THAT LEVEL. THAT SEEMS TO BE A COMPONENT THAT PLAYED A ROLE IN THAT COMPLEMENT, FEDERICO CAN -- YOU KNOW. >> BEFORE WE MOVE TO THE NEXT PART I WANT TO HEAR ABOUT THE STRATEGIES, SO WHAT CAN WE DO TO ADDRESS THESE PROBLEMS? SO IF YOU WERE GIVEN UNLIMITED FUNDS WHAT WOULD YOU DO TO HYPOTHETICALLY -- >> ARE YOU OFFERING? >> BEV HAS ONE. >> YEAH, ONE STRATEGY THAT I HEAD, AND YOU ALL CORRECT ME IF I'M WRONG, WE DON'T HAVE THE TOOLS AT THE PRESENT TIME TO EVALUATED IMMUNE RESPONSE IN SITU IN A LIVING ANIMAL. THIS GETS TO VIVIANA'S SLIDE WHERE AGAIN THESE OTHER IMAGING MODALITIES MAY BETTER TELL US WHAT'S GOING ON IN CNS. KRYS TOLD US ABOUT EDEMA AND WATER AND THINGS LIKE THAT BUT WE NEEDED TO BE A LITTLE MORE SPECIFIC. MAYBE TOOLS TO UNDERSTAND WHAT IS THIS PLEIOCYTOSEIS, AND HOW IS IT INDUCED, COULD THAT BETTER INFORM OUR VECTOR MANUFACTURING OR OUR CAPSID ENGINEERING. >> ONE QUESTION I HAVE, THIS CAME UP AT THE NCATS WORKSHOP ABOUT IMMUNOGENICITY WHEN YOU TALK ABOUT LOOKING AT RESPONSE IN SITU WHAT'S THE BEST ANIMAL MODEL THAT WILL WOULD REFLECT WHAT'S HAPPENING IN HUMANS. >> MAYBE STEVE AND I COMMENT. OUR IMMUNE MODULATION PROTEST COLLABORATION IN THE GUNN TRIAL FOR THE CRIM NEGATIVE PATIENTS IS BASED ON STEVE'S PRE-CLINICAL NON-HUMAN PRIMATES. STEVE, DO YOU WANT TO BRIEFLY COMMENT AND I'LL EXPLAIN THE PROTOCOL. >> YEAH, THERE WAS A PAPER THAT WE PUBLISHED WITH -- NON-HUMAN PRIMATE STUDY DELIVERING GPF. IN THE CNS, MONKEYS WILL ELICIT CAN BE DELETERIOUS.PONSE THAT- WE DID A REGIMEN WHERE THEY WERE PUT ON RAPAMYCIN, AND THAT WAS A SHORT COURSE OF RAPAMYCIN BUT THAT DELAYED AND DIMINISHED BUT DIDN'T COMPLETELY PREVENT THE IMMUNE RESPONSE AGAINST GFP, BUT THAT PROMPTED ALONG WITH ANALYSIS OF REALLY THE TRANSPLANT LITERATURE TO PUT TOGETHER THIS PROTOCOL TO TRY TO ADDRESS THE CRIM NEGATIVE PATIENTS IN OUR GANN TRIAL. >> FROM THAT EXPERIENCE WE WERE CONCERNED ABOUT T CELL MEDIATED RESPONSE, TRANSGENE EXPRESSION IN THE NERVOUS SYSTEM. OUR PROTOCOL IS OF COURSE BASED ON NO CONTROLLED EXPERIMENT IN THE HUMAN AGAIN BUT FOR ACTIVELY TREATING PATIENTS THAT ARE PREDICTED TO BE CRIM NEGATIVE, NOT KNOWING WHETHER THEY WOULD MOUNT AN IMMUNE RESPONSE BECAUSE GFP IS NOT GANN BUT WE PUT THEM ON A PROTOCOL THAT INCLUDES RAPAMYCIN AND AGENTS, AND THEN KEPT THEM LONG ON RAPAMYCIN, HOPING FOR IMMUNE TOLERANCE TO DEVELOP, BUT ADMITTEDLY WE HAVE NO ASSAY. IT'S A QUESTION FOR THE ENTIRE PANEL. WE HAVE NO ASSAY TO DETERMINE WHEN IMMUNE TOLERANCE IS REACHED. SO FAR, SO GOOD. AS I SAID BEFORE WE HAVE NO T-CELL RESPONSE. T CELL RESPONSE AGAINST THE TRANSGENE. >> YEAH, SO JUST TO SUMMARIZE, I MEAN, THESE PATIENTS WILL ESSENTIALLY BE KEPT LONG TERM PERHAPS FOREVER ON A LOW DOSE OF RAPAMYCIN. SIMILAR TO WHAT WOULD BE DONE WITH AN ORGAN TRANSPLANT. BUT IT'S AN N OF 3 RIGHT NOW, NONE OF THESE PATIENTS WOULD HAVE BEEN PREDICTED TO MOUNT T-CELL RESPONSE DONE IN. WE DON'T HAVE THE CONTROL IN THE HUMAN EXPERIMENT OF PATIENTS THAT DIDN'T GET THE IMMUNOSUPPRESSION. BUT AS FAR AS MANAGING T-CELL RESPONSE SO FAR THIS IS AT LEAST LOOKING ENCOURAGING AS A REGIMEN TO MANAGE THAT. >> ONE OTHER THOUGHT. YOU WERE TALKING ABOUT -- DIANA FROM THE NIH TEAM WORKING ON THE GANN TRIAL. SO YOU'RE ASKING WHAT ANIMAL MODEL COULD YOU USE TO BETTER ASSESS RESPONSES. I THINK AS DIFFERENT CLINICAL TRIALS ARE GOING FORWARD I THINK MAYBE TRYING TO UNDERSTAND ARE THERE CERTAIN ELEMENTS IN TESTING AGAIN LOOKING AT CERTAIN RESPONSES, THE WAY WE'RE TESTING, LOOKING IN CSF OR IN THE PERIPHERY TO LOOK FOR THESE RESPONSES, MAYBE HAVING SOME KIND OF COMMON DATA ELEMENTS, RECOMMENDED ELEMENTS FOR TESTING. WE'RE DEALING WITH ULTRA RARE DISEASE, SO ARE MANY OTHER PEOPLE. COULD WE POOL THAT KNOWLEDGE FROM CLINICAL STUDIES, MAYBE TRY TO BETTER IDENTIFY TRENDS IF POSSIBLE. ANOTHER THOUGHT, FOR GANN IT'S NOT A SECRETED PROTEIN, WHETHER A LEVEL IN IS DECLINING IN RESPONSE TO T-CELL RESPONSE, WE DON'T KNOW WHEN WE SEE THE PLEIOCYTOSIS WE CAN ONLY UNDERSTAND SAFETY IMPLICATIONS, NOT SURE IF IT'S DAMPEN A SIGNAL WE NEVER SAW BECAUSE THE RESPONSE WAS THERE. SO IN SOME OF THE CNS TARGETING THERAPIES WHERE THERE IS A SECRETED ENZYME OR PROTEIN THAT CAN BE EVALUATED, THAT WOULD BE IMMENSELY HELPFUL FOR OTHERS, WE'RE NOT TARGETING SOMETHING WITH SECRETED PROTEIN WE CAN FOLLOW TO SEE IF THE LEVEL OF DYNAMICS CHANGES, AS YOU SEE, IF YOU SEE AN IMMUNE RESPONSE MOUNTED. >> JILL, ANTIBODY RESPONSES, RESPONSES TO GFP SEEN IN CATS AND SHEEP CLASSICALLY, SIMILAR TO HUMAN PATIENTS, LITERATURE SUPPORTS NON-HUMAN PRIMATE DATA AS WAS ALLUDED TO. ALL OF THOSE ARE FAIR. PLEIOCYTOSIS AND CSF AFTER 14 DAYS, I CAN ATTEST FOR THAT BEING PRESENT IN SHEEP. MAYBE CHARLES CAN TALK ABOUT SOME OTHER SPECIES YOU MIGHT HAVE EXPERIENCE WITH AS TO& WHETHER OR NOT THOSE ARE GOOD MODELS FOR THIS. >> I THINK CHANGES IN THE CSF, ONE OF THE THINGS WE COULD ADD THAT I DON'T THINK WE'VE DONE IS LOOKING TO SEE PARENCHYMAL CHANGES. MY GUESS WHAT WE'RE SEEING IS A PERIVASCULAR RESPONSE, ALMOST ALWAYS LYMPHOCYTIC, USUALLY PERIVASCULAR CUFFING LIKE WITH A VIRAL INFECTION THAT GOES AWAY BUT I DON'T HAVE THAT DATA. I DON'T KNOW >> ALL RIGHT. AS YOU NOTICED, THERE'S NO BREAK BETWEEN 2A AND 2B, SO STAND UP AND STRETCH. WE'LL GO STRAIGHT INTO 2B, HOLD YOUR THOUGHTS BECAUSE YOU'LL HAVE A CHANCE TO GET THEM IN. WHILE YOU STRETCH, SERIOUSLY IT'S BEEN A LONG TIME, I WILL TRY TO SUM UP THE KEY ITEMS FOCUSING ON ACTIONABLE. WE HEARD NEED FOR MORE HUMAN DATA AND DOSE DEPENDENCY, INTRATHECAL WAS MENTIONED, IN THAT CASE, IS IT BECAUSE OF THE EXPRESSION OR IS IT LEAK IN THE PERIPHERY, AND BOTH ITEMS POINT TO THE NEED TO DEVELOP HUMAN FRIENDLY BIODISTRIBUTION METHODS. BY THEY BEING NON-INVASIVE IDEALLY AND IN HUMANS. SO YOU MIGHT SEE THE PRESENT AND FUTURE, THEY HAVE THE BRIDGE POINT DEVELOPING TECHNOLOGIES FOR BIODISUSING, RELEVANCE FOR IMMUNE ASPECTS AND ASSESSING DELIVERY METHODS. INTRAARTERIAL WAS MENTIONED, WE NEED A GOOD ASSESSMENT METHOD. I WOULD WANT TO KEEP THAT IN MIND, HOW WE CAN ADVANCE BIODISTRIBUTION METHODS AND HOW CAN WE RECRUIT TO HELP WITH IMMUNE RESPONSE AND WITH ASSESSING DELIVERY ROUTES AND IMPROVING DELIVERY ROUTES BECAUSE IF WE HAVE FEEDBACK ON HOW CAPSID IS PERFORMING THAT COULD ENENGINEER OUT OF THE PROBLEM. 2B, VECTOR BY DISTRIBUTION AS WELL. I'LL COME BACK TO THAT. FOR 2B HAS TO DO WITH NOVEL DELIVERY METHODS. AND THIS WILL HAVE TO DO WITH BOTH DELIVERY ROUTES BUT ALSO WITH SOME FUNDAMENTAL BARRIERS, WE'VE HEARD ABOUT SOME OF THE VECTOR SYSTEMS. CONVEX OF AAV SIZE LIMITATION POINT TO CONSIDERING NON-AAV DELIVERY METHODS. OTHER VECTORS WOULDN'T HAVE THESE LIMITATIONS. CO-DELIVERY COULD BYPASS BUT IS LOW EFFICIENCY AND MIGHT NOT SOLVE ALL OF OUR PROBLEMS ESPECIALLY AS WE GO FROM MONOGENIC TO MULTI-GENIC DISORDERS. WE NEED TO BYPASS THIS PROBLEM. THERE'S CROSS-TALK AS WE DISCUSSED BETWEEN CAPSID AND TRANSGENE. AND THIS IS GOING TO BE A THEME IN THE BIODISTRIBUTION AS WELL. AND STRATEGIES TO DEVELOP BETTER DELIVERY METHODS, WITH RATIONAL DESIGN OR DIRECTED EVOLUTION, THERE'S ROOM FOR ONE OR THE OTHER, TO DISCUSS IN BROAD ASPECT, LENTIVIRUS HAS ADVANTAGES, YOU DON'T HAVE THE PROBLEM WITH SECOND ADMINISTRATION, ARE WE USING IT ENOUGH AND CAN WE ENGINEER FOR THE BETTER. HSV WAS MENTIONED, ASOs, A POINT MENTIONED IN SESSION 1, HOW GOOD IS GOOD ENOUGH. SHOULD WE AIM FOR THE MOST EXPRESSION KNOWING WE CAN DECREASE THE DOSE? AND IS THAT -- ARE THESE TWO FACTORS SEPARABLE? AND IN THE CONTEXT OF THIS SECOND ADMINISTRATION, WHICH OF COURSE IT'S MORE OF A PROBLEM WITH SOME DELIVERY METHODS THAN OTHERS. SO I WOULD WANT TO START IT, IF AGREEABLE WITH THE BIODISTRIBUTION TOPIC, BECAUSE IT WAS MENTIONED A LOT IN SESSION 1. I WANT TO POINT TO DOUG THAT HAD SOME THOUGHTS ABOUT IT. AND SINCE IT WAS THE TOPIC THAT WAS MENTIONED SO MUCH I'M BREAKING THE RULE OF THE MEETING IN SHOWING DATA. IT'S NOT MINE THOUGH. >> THANKS. DOUG BALEN, CORNELL. THERE ARE A NUMBER OF QUESTIONS THAT COULD BE ADDRESSED BY PET, EFFECT OF IMMUNE RESPONSE, OFF-TARGET VERSUS ON-TARGET AN INDIVIDUAL VARIABILITY. IT'S ONE SLIDE. THERE'S A LOT ON IT. I'LL TAKE YOU THROUGH IT. THE METHOD HERE IS LABELING AAV WITH IODINE 124, A PET TRACER, HAS A FOUR-DAY HALF-LIFE. THAT MEANS YOU CAN LOOK OUT TO ABOUT 10 TO 12 TO 14 DAYS USING I-124. IT'S CAPSID LABELING, IODINE IS DIRECTLY BOUND TO TYROSINE RESIDUES ON THE CAPSID. WE'RE BINDING TYROSINE RESIDUES SO THERE'S A CONCERN WHAT THAT MIGHT BE DOING. WITH THIS METHOD WE SEEM TO BE BINDING ABOUT 2 IODINE ATOMS PER CAPSID, FIVE OF 5 TIMES 10 TO THE 12th CAPSID ADMINISTRATIONS, HALF A MILICURIE OF EYE DON 124, NOT HUMAN DOSES BUT NOT TOO FAR, WITHIN AN ORDER OF MAGNITUDE OF MAKING HUMAN DOSES OF THIS. WE TESTED INTRAVENOUS AND INTRASISSERNAL DELIVERY, N EQUALS 1, RH TO AND AAV9. DID INJECTION AND IMMEDIATELY AFTER IV TOOK LONGER TO GET READY TO DO THIS, 20 MINUTES. EIGHT WEEKS LATER, THIS IS SOMEDAY ZERO, 1, 2 AND 3. EIGHT WEEKS LATER, WE DID THE SAME THING, SAME ANIMALS, NOW DEVELOPED IMMUNITY. ONE CONTROL, SODIUM IODIDE I.V AND INTRACISTERNAL. WE NOTICED AAV APPEARS TO GO EVERYWHERE. WE CAN DO FORMAL DOSIMETRY WITH THIS, AND IT SHOWS. YOU CAN SEE DISTRIBUTION ON DAY 3 HERE, SO THIS IS AFTER GOING OUT OF THE VASCULATURE, ALL THROUGHOUT THE BODY. I'M GOING TO PRESUME THAT AAV IS IN MUSCLE, SO 50%, IF YOU INTEGRATE OVER THE WHOLE BODY. THERE IS IN BOTH INTRAIV AND INTRACISTERNAL ADMINISTRATION A LOT GOES TO LIVER. WE WERE SURPRISED MAYBE WE HAD QUITE A BIT OF BONE MARROW SIGNAL ESPECIALLY WITH AAV9. THE BIGGEST EFFECT WE SAW WAS AFTER DEVELOPING IMMUNITY. THIS IS 8 WEEKS LATER. IT LOOKS DIFFERENT.& THERE'S CLEARANCE FROM THE BODY. THESE ARE ALL CORRECTED FOR PHYSICAL DECAY. AND THEY ARE NORMALIZED TO INJECTED DOSE SO THEY ARE ALL SCALED HERE TO BE SELF CONSISTENT. AFTER IMMUNITY, WE HAVE A VERY HIGH SIGNAL IN THE SPLEEN. IN ALL CASES. AND THESE ARE TIME DEPENDENT. WE CAN LOOK AT INFECTION DYNAMICS WITH OUR MODEL. WE HAVE A TIME COURSE OF HOW LONG THE SIGNAL STAYS IN THE ORGAN. THE BIGGEST CAVEAT TO THIS OF COURSE IS IT IS IODINE 124, NOT DIRECTLY VECTOR. WE HAVE A LOT OF WORK TO DO TO VERIFY WHAT WE'RE SEEING.& WE KNOW FOR EXAMPLE IN THE THYROID YOU SEE IMMUNITY, EVERYWHERE. THAT'S LIKELY FREE IODINE, GETTING THE THYROID AFTER INFECTION, AFTER UNCODING. AND WE STILL HAVE QUESTIONS WHAT HAPPENS IN LIVER LATER, IS THIS I 124 AAV, IS THERE CAPSID FRAGMENTS THERE? THIS IS WHAT WE'RE WORKING ON. >> THIS DATA, DOUG SAYS SOMETHING RATHER WORRYING, AAV GOES EVERYWHERE. THE OTHER QUESTION IS DOES IT STAY EVERYWHERE. AND WHICH ORGANS RETAIN HOW MUCH OF THE VECTOR WHEN DELIVERED TO VASCULATURE, CAN WE USE TO TO ESTIMATE OUT OF 100% HOW MUCH GOES TO THE BRAIN FIT CROSSES THE BBB. ARE THERE WAYS ONE COULD MONITOR IN VIVO CAPSID AND TRANSGENE TO MAKE SURE MODIFIED CAPSID BEHAVES IN A PREDICTABLE WAY AND ONE THAT'S RELEVANT FOR THERAPY. WE'LL OPEN TO QUESTIONS WITH PROMPTERS. LET'S START ON BIODISTRIBUTION. >> YEAH, I'M NOT SURPRISED AAV9 GOES WHEREVER. WE USED A MORE SENSITIVE METHOD, SCREEN THE VECTORS, YOU SEE IT IN THE SPLEEN AND BONE MARROW, IN PLACES YOU DIDN'T THINK IT WENT, EVEN WHEN YOU USED LIVER-SPECIFIC PROMOTER. I THINK THAT COMING UP WITH METHODS LIKE THIS WILL BE ABSOLUTELY CRITICAL AS WE WANT TO UNDERSTAND WHERE THESE VECTORS GO IN THE BODY, BUT THE THING I WOULD REALLY LIKE TO KNOW CAN WE MAKE LIGANDS SOMETHING LIKE WHAT KRYS DOES FOR AADC WHERE YOU LOOK AT DOPAMINE RECEPTORS BUT NOW WE WANT TO DO THIS FOR LYSOSOMAL PROTEINS. >> COULD WE MAKE A WAY TOO ASSESSING A RESPONSE? BASICALLY MEASURING MARKERS, ARE THERE MARKERS WE COULD MEASURE NON-INVASIVELY TO ASSESS THE RESPONSE? >> ONE POINT ABOUT THE PET RELATIVE TO LET'S SAY M.R. WE'RE ABLE TO SEE HERE 10 TO THE 12th CAPSIDS WITH THAT LABELING. PET IS A HIGH ENERGY TECHNIQUE. SO JUST KEEP THAT IN MIND. IF YOU GO TO SOMETHING LIKE MAGNETIC RESONANCE YOU NEED MILLIMOLAR CONCENTRATIONS BEFORE YOU CAN SEE THEM. IF YOU CAN LABEL, IF WHAT YOU'RE LOOKING FOR IS AT THAT LEVEL, 10 TO THE 12th, 10 TO THE 14th, CAN YOU POTENTIALLY SEE IT WITH PET. >> I TOTALLY AGREE PET IMAGING IS ESSENTIAL FOR VISUALIZING BIODISTRIBUTION OF GENE THERAPEUTICS, AND AS WE CAN SEE AFTER SYSTEMIC DELIVERY IT'S JUST NOT ONLY GOES EVERYWHERE, BUT UNFORTUNATELY IF WE TARGET THE BRAIN PRACTICALLY NOTHING GOES WHERE WE WANT IT TO GO. FOR EVERY CAPSID THAT WE WANT TO GET TO THE BRAIN WE HAVE TO INJECT 10,000 OR 100,000 THAT WILL JUST GO ELSEWHERE. SO WE NEED TO DO BETTER THAN THAT. AND I KNOW THIS INTRAARTERIAL INJECTIONS WERE TRIED, AND IT DID NOT WORK BUT I DON'T THINK WE SHOULD DISMISS OPPORTUNITIES WITH APPROACHING -- WITH CATHETER, BEING SUPER SELECTIVE AND BEING ABLE TO INJECT LOCALLY, IF WE ADDRESS SOME CHALLENGES. THE REASON WHY IT DIDN'T WORK, IT JUST FLEW THROUGH THE CAPILLARIES AND VECTORS DIDN'T STOP. WE HAVE TO MAKE THEM STOP AND MAKE THEM EXTRAVASATE. THERE ARE WAYS OF DOING IT, AND OUR GROUP ACTUALLY IS TRYING TO DO THE CONTROLLING THE FLOW DURING INFUSION AND ENGINEERING THE THERAPEUTICS, ARE THERE ANTIBODIES YOU COULD TARGET TO RECEPTORS OR ENGINEER CAPSIDS TO TARGET. >> I WANTED TO SAY WE DID THAT WITH HERPES VECTORS, INTERARTERIALLY, I THINK USED MANNATOL AND GOT GOOD DELIVERY INTO THE BRAIN WITH THAT. I DON'T KNOW WITH DISRUPTION. >> DONE CLINICALLY IN THOUSANDS OF PATIENTS, THAT'S ONE OF THE WAYS, ULTRASOUND IS ANOTHER ONE THAT IS BEING DEVELOPED. >> I'D LIKE TO ADD THAT FOR INTRAARTERIAL DELIVERY DOUG AND I HAVE PUBLISHED A PAPER WHERE IN MICE THAT WE DID SHOW THAT INTRAARTERIAL DELIVERY USING MANNITOL TO OPEN THE BLOOD-BRAIN BARRIER EXTREMELY SUCCESSFUL DELIVERY OF THE TRANSGENE. BUT IN FUTURE WE'VE DONE SEVERAL KIND OF - SHALL IT WRITTEN SEVERAL GRANTS, LOTS OF OTHER THINGS, ALWAYS ISSUE RAISED ABOUT USE OF MANNITOL EVEN THOUGH MANNITOL IS USED CLINICALLY BUT THERE'S BEEN A LOT OF ISSUES. I THINK THAT'S SOMETHING. ULTRASOUND FOCUSED OPENING IS REALLY A VERY GOOD OPTION. AND THE OTHER POINT I WANTED TO MAKE IS THAT THE PET IMAGING, IT REALLY OPENS UP REALLY WONDERFUL WAY OF TRYING TO FIGURE OUT, AS YOU'RE SAYING WE'RE MAKING NEW DESIGNER CAPSIDS AND MAKING ALL OF THESE CHANGES, IT'S A REALLY GOOD WAY TO VERY QUICK WAY AND VISUAL WAY DETERMINE THAT ARE THOSE CAPSID CHANGES LEADING TO CHANGES THAT YOU ACTUALLY THINK PUBLISHING EVERY DAY THAT THESE ARE VERY CNS-TROPIC VECTORS, AND WE'RE GOING TO GET HUNDRED-FOLD BETTER EXPRESSION, BUT IS THAT REALLY HAPPENING? AS POINTED OUT, WHEN YOU DELIVER AS WE SEE HERE NOT REALLY VERY MINUSCULE AMOUNT IS GETTING TO THE BRAIN WHEN DELIVERED I.V >> I WOULD LIKE TO JUST MAKE ONE COMMENT IN TERMS OF THE BLOOD-BRAIN BARRIER DISRUPTION, WE NEED TO KEEP IN MIND THAT IT'S NOT JUST A.A.V THAT'S GETTING INTO THE BRAIN. THERE'S A LOT OF THINGS FROM THE BLOODSTREAM THAT WILL ENTER AND HAVE YOU TO BE MINDFUL OF IMMUNE CONSEQUENCES, DISRUPTION DOESN'T CAUSE IMMUNITY, IT WILL LINGER. >> WE LOOKED FOR TIMING OF THE BLOOD-BRAIN BARRIER OPENING AND COMPARED OSMOTIC VERSUS FOCUSED ULTRA SOUND, ONE HOUR BEFORE IT RECEIVES, WITH FOCUSED ULTRASOUND STAYS OPEN UP TO A WEEK, ONE CAN CHOOSE TECHNIQUE THAT IS LESS INVASIVE. >> DOES ANYBODY KNOW THE BLOOD BLOOD-BRAIN BARRIER IN INFANTS IS MORE ACCESSIBLE? IN RODENT STUDIES IV INJECTION, IN INFANT RODENT YOU MAY HAVE GOTTEN MORE, DOES ANYBODY KNOW? >> I CAN ADDRESS SOME OF THAT IN THE CONTEXT OF MICE AT LEAST. AND THEN ALSO SOME QUESTIONS PERTAINING TO PET. IN THE CONTEXT OF MICE WE'VE DONE SOME STUDIES TO EVALUATE BLOOD-BRAIN BARRIER PERMEABILITY AT DIFFERENT AGES, AND THE DEVELOPMENTAL BIOLOGY AND WHEN YOU LOOK AT THE DATA CLEARLY SUGGESTS NEONATES HAVE FULLY FORMED ININTACT BLOOD-BRAIN BARRIER, OTHERWISE THEIR SURVIVAL IS IN QUESTION. DELIVERY ACROSS THE BLOOD-BRAIN BARRIER IN NEONATES SEEMS TO BE A DOSE THING MORE THAN ANYTHING ELSE. BUT OLDER MICE, WE'VE PUBLISHED ON THIS, TOP CSF FLUX, THE RESULT OF THAT BEING INTRACRANIAL PRESSURE, MIKE PUBLISHED IN THIS SPACE. WE SEE IN CHOOSING AAV INTO OLDER RODENTS THEY TEND TO STICK AROUND IN THE PARENCHYMA, IN THE BRAIN, AND DO NOT LEAK INTO THE SYSTEMIC CIRCULATION THROUGH GLYMPHATICS. THERE WAS A DISCUSSION WHERE IT WAS PROPOSED WE USE LABELING AND STRATEGIES TO UNDERSTAND CSF FLUX IN PATIENTS, THAT MIGHT HAVE DIFFERENT DISORDERS AND HOW THAT IMPACTS THE BIOLOGY OF THE VIRUS. WITH REGARD TO PET, I THINK THE ONLY CAVEAT I WOULD PUT TO THAT IS IN THE CONTEXT OF IODINE THE WAY THE CHEMISTRY IS LOADED ON TYROSINES, I WOULD BE CAUTIOUS ABOUT INTERPRETING THAT AS AAV DISTRIBUTION, IT MAY BE IODINE, AND IT WOULD BE REALLY IMPORTANT TO DO THAT ON MULTIPLE CAPSIDS, ON TRANSFERRIN, ALBUMIN, UNDERSTAND, NOT TO MENTION IODINE ALSO REACTS WITH HISTODEANS, I'D BE CURIOUS ABOUT DNA AS WELL. I THINK THERE ARE MANY PIECES TO IT THAT COULD GET LABELED, AND SO BEFORE WE INTERPRET THAT AS VIRAL DISTRIBUTION I THINK SOME MORE CONTROLS WOULD BE. >> I AGREE. WE HAVE A LOT OF WORK TO DO. >> LOOKING AT FUNCTIONAL DISTRIBUTION IS CRITICAL. GADOLINIUM, FOR EXAMPLE, IT TELLS YOU THAT THE LIQUID THAT YOU'VE PUT IN HAS SOMEONE SOMEWHERE BUT HASN'T TOLD YOU WHAT CELLS IT'S INFECTED. SO IF YOU LOOK AT FUNCTIONAL OUTPUT SUCH AS CHANGE IN ENZYME, THAT'S GREAT, OR CHANGE IN LIGAND, THE PROBLEM WITH MANY OF THE LIGANDS THAT WE WORK WITH IS THEY ARE ALL CARBOHYDRATES WHICH MAKES THEM A NIGHTMARE FOR PET LABELING. >> TRANSITIONING TO CELL TYPE AND LONG-TERM EFFECT WITH VECTOR, ONE METHOD WOULD BE 10X, HIGH-THROUGHPUT SEQUENCING AND LONG-TERM EFFECTS, HOW ARE DIFFERENT CELL TYPES CHANGING, IN RESPONSE TO INFECTION. AND 10 X IS ONE METHOD. DOWN SIDE IS THAT PREP CAN BE INTENSE AND SEQUENCING WITH ENOUGH DEPTH IS QUITE RESOURCE INTENSIVE, THAT'S ONE CHALLENGE. >> I DON'T MEAN TO DISTRACT FROM THE POINT BUT JUST ON THE POINTS ABOUT IMAGING, WHEN WE TRANSLATE THIS INTO PATIENTS, WE'RE TALKING ABOUT SCAN TIMES THAT WILL BE DIFFERENT AS WELL, RIGHT? WHAT IS AN AVERAGE SCAN TIME WHEN YOU LOOK AT PET OVER, SAY, MRI? >> SO WE ALWAYS MEASURE AS TOTAL TIME SUBJECTS IN, SUBJECTS OUT. WE DID THEM DYNAMICICALLY TO SEE THE TIME COURSE WHICH I DIDN'T SHOW. WE SCANNED FOR AN HOUR. WE HAD SIGNAL TO NOISE YOU COULD DROP BACK TO 30 MINUTES. ANOTHER POINT ABOUT THE IODINE VERSUS AAV, IF ALL THAT IODINE CAME OFF WE WOULD SEE IT, IN THE THYROID, THE CONTROLS. WE BELIEVE THIS IS A SURROGATE. >> I AGREE. I DON'T THINK THAT'S ALL IODINE. I'M SAYING TO EXTRAPOLATE THAT TO COPY NUMBER IS A CORRELATION WE HAVE TO ESTABLISH. >> IT'S JUST A SURROGATE, WE'VE MADE NO CORRELATIONS. >> IT HIGHLIGHTS THE FACT THIS IS A FUTURE AREA OF DEVELOPMENT TO FOCUS ON MULTIPLE CHEMISTRIES FOR CAPSIDS, HAVING DIFFERENT LABELS THAT CAN ALLOW FOR FASTER SCAN TIME AS WELL. SO THAT SINCE WE'RE GIVEN TASK TO LOOK FORWARD THESE ARE AREAS WHERE WE IDENTIFY QUESTIONS TO SEE HOW WE CAN ADVANCE. >> JUST LAT POINT, WE HAVE TO BE CLEAR, LOOKING IT'S A TWO, ONE IS DISTRIBUTION YOU JUST PRESENTED, IT'S A PHENOMENALLY IMPORTANT QUESTION, THE OTHER ONE IS EFFECT OF GENE THERAPY SO MORE FUNCTIONAL TYPE OF OUTPUT WHICH I THINK HAVING ANY WAY OF BEING ABLE TO DEVELOP A TRACER THAT ACTUALLY DETECTS THE EXPRESSING GENE OVER TIME I THINK THAT'S ALSO VERY HELPFUL, IN THE CONTEXT PERHAPS OF THE OUTCOME OF THE GENE THERAPY ON THE CLINICAL FOLLOW-UP OF THE PATIENTS. >> MAYBE SOMETHING THAT WE COULD DO, YOU KNOW, AS A GROUP OR AGAIN I WANT TO COME BACK TO THIS POINT THAT HEATHER RAISED, EARLIER, IN SESSION 1, WHERE IS THERE SOMETHING WE CAN FIND OUT AT THE SINGLE CELL LEVEL TO GUIDE US IN UNDERSTANDING THE IMPACT WE'RE HAVING ON CELLS IN SITU, AND IF IT THE LIGANDS WE HAVE NOW WON'T WORK CAN WE FOCUS ON DEVELOPING BETTER LIGANDS THAT MAYBE ARE NOT CARBOHYDRATE BASED, BOUND ENZYME POCKET IN TRANSIENTS WAY, SHOW IT'S EXPRESSED, HAVE A SHORT HALF-LIFE AND GO AWAY. THE OTHER, ARE THERE THINGS WE DON'T UNDERSTAND IN OUR DISEASE SYSTEMS WHERE THERE'S CHANGES IN CELL RECEPTORS WE HAVEN'T BEGUN TO THINK ABOUT, PARTICULARLY FOR SOME DISORDERS WHERE YOU'VE SCREWED UP PRESIDENT ENDOSOME LYSOSOME PATHWAY, RESIDENT TIME IS LONG OTHER CELL MEMBRANE THAN NORMAL CELLS, CAN WE USE THAT TO OUR ADVANTAGE TO SAY SOMETHING IS GETTING BETTER, STARTING AT BASELINE, BUT THAT WOULD GIVE US POSSIBLY A PROXY AS TO WHERE THE GENE THERAPY IS HAVING AN EFFECT. I GUESS I CHALLENGE US TO THINK ABOUT WAYS TO THINK OUTSIDE OF THE BOX, OUTSIDE OF THE CELL, OUTSIDE OF JUST THE CAPSID ON TRYING TO UNDERSTAND IN SITU AGAIN METHODS TO BETTER UNDERSTAND WHERE ARE WE DOING SOMETHING IN THE BRAIN, AND THEN HOW IS THAT IMPACTING THE BRAIN DOWN THE ROAD? AND I THINK THE POSSIBILITIES ARE REALLY BROAD. WE JUST NEED TO BE A BIT MORE CLEVER AND THINK MORE DEEPLY. >> THE FOLLOW YOUR POINT, YOU MENTIONED ABOUT THE BRAIN CIRCUITRY. I MEAN IF WE THINK ABOUT USE OF GENE THERAPY IN CONTEXT OF CNS DISORDERS, THERE ARE A NUMBER OF WAYS TO IMAGE CIRCUITRY USING DIFFERENT IMAGING MODALITIES, AND THEN TRY TO NOT HAVE A DIRECT EFFECT OR READOUT OF GENE EXPRESSION BUT CONSEQUENCES BECAUSE THAT'S WHAT WE'RE TRYING TO ALTER THE BRAIN CIRCUITRIES, PET IMAGING, FUNCTIONAL M.R. SCANS THAT COULD BE UTILIZED. >> IT'S NOT AS HIGH RESOLUTION BUT THERE'S PHRASEs LIKE NANOLOOP THAT GIVE YOU AMAZING SIGNAL. WE ROUTINELY DO BIOLUMINESCENCE IMAGING ON OUR MICE. EVERY WEEK AND TRACK THINGS. >> TALKING ABOUT HUMAN THERAPIES THOUGH. >> YEAH. >> IT'S PROBABLY NOT SOMETHING YOU WANT TO PUT IN A PERSON. >> ONE THOUGHT THAT COMES TO MIND IS WHETHER WE CAN CAPITALIZE ON SOME OF THE TOOLS AND TECHNIQUES DEVELOPED FOR THE BRAIN INITIATIVE. PURPOSE OF NON-INVASIVE CONTROL AND MODULATION. AND WHETHER TO WHAT KRYS MENTIONED WHERE WE COULD USE THAT AS ULTIMATE READOUT IN TERMS OF FUNCTIONALITY OF GENE THERAPY. NEURONAL EXCITABILITY, FOR EXAMPLE, ARE WE CHANGING THAT AS A RESULT OF INSERTING SOME CHANNELS OR microRNA TARGET SITES? AND ARE THERE ALREADY TOOLS THERE THAT HAVE BEEN VALIDATED IN LARGER ANIMALS THAT WE DON'T -- JUST DON'T USE IN THE CONTEXT OF GENE THERAPY? AND I KNOW WE HAVE SOME BRAIN INITIATIVE REPRESENTATIVES SO THAT'S SOMETHING THAT COMES TO MIND. ARE WE NOT USING EVERYTHING THAT WAS DEVELOPED? >> DO YOU WANT TO TALK ABOUT THAT, SOMEBODY? >> (INAUDIBLE). >> ARE THERE ANY SECRET METHODS THAT ARE NOT SO SECRET THAT COULD YOU SHARE THAT WORK IN VIVO, IDEALLY WITH MINIMUM INVASIVENESS TO MEASURE CIRCUIT, THAT WOULD ALLOW US TO MEASURE END RULED OF GENE THERAPY, SORRY FOR PUTTING YOU ON THE SPOT, IT JUST CAME TO MIND THERE'S BEEN A WONDERFUL INITIATIVE, DEVELOPING TOOLS AND METHODS, AND WITH THE PREMIUM ON NON-INVASIVENESS, COULD SERVE AT READOUT FOR SOME INTERVENTIONS IN GENE THERAPYS THERE SOMETHING WE'RE MISSING AND SHOULD READ UPON AND EMBED WITHIN THE CONTEXT OF THIS GROUP? >> I GUESS I WOULD HAVE PUT THAT QUESTION ACTUALLY TO YOU SINCE YOU DO BRIDGE THESE TWO AREAS. IS THERE SOMETHING THAT YOU'RE THINKING SORT OF THAT YOU HAVE IN MIND THAT IS SOMETHING THAT WOULD -- I MEAN WHEN I THINK ABOUT GENE THERAPY FOR DEVASTATING DISORDERS, NEUROLOGICAL DISORDERS IN CHILDHOOD, THE READOUTS FOR NEURAL ACTIVITY ARE, YOU KNOW, THE PATIENT'S BEHAVIOR, RIGHT? AND IT SEEMS LIKE THAT'S OUR PROXIMAL GOAL UNTIL WE GET SOME WINS IN THAT REGARD AND THEN WE CAN TALK ABOUT CIRCUIT CURES WHERE YOU WOULD WANT TO MERGE THIS WITH ACTIVITY INDICATORS TO MAKE SURE THAT YOU REALLY ARE INTERVENING IN THE CIRCUIT THAT YOU WANT TO. THE FOCUS OF THE BRAIN INITIATIVE I THINK HAS BEEN ON LARGELY THE FUNDAMENTAL NEUROBIOLOGY, PARTLY FOR THE REASON THAT, YOU KNOW, IT'S A LITTLE BIT EARLY TO BE THINKING ABOUT TAKING SOME OF THESE TO HUMANS. BUT IT IS, YOU KNOW, IT'S NOT THAT WE'RE NOT INTERESTED IN THAT. I CERTAINLY THINK THE TOOL SET FOR LARGE ANIMAL -- LARGE BRAINS AND FOR HUMANS IS SOMETHING THAT WE'RE INCREASINGLY LOOKING AT. PART OF IT IS BECAUSE OF ALL THE TRACTION THAT'S BEEN GAINED THROUGH THE GENE THERAPY. >> JUST TO ADD ON THAT, WE'RE FUNDING NON-INVASIVE STIMULATION AND IMAGING, IF YOU WANT DO CHAT AFTERWARDS, THE YBT IDEA WAS BROUGHT UP BEFORE, WE CAN HAVE OBJECTIVE MEASURES OF NEURAL ACTIVITY SO SOMETHING MAYBE MORE FOCAL AND PRECISE COULD BE USED TO ASSESS OVER TIME ANY CHANGES. >> VIVIANA, CAN I ADD ON TO THAT? SPECIFICALLY ADDRESSING THE CIRCUITRY QUESTION, THERE ARE TOOLS, IMAGING TOOLS OUT THERE THAT CAN DO THIS NOW. SO A COUPLE THINGS THAT COME TO MIND. EEG IS KIND OF OLD-FASHIONED TO SOME DEGREE. WE'VE SHOWN PRETTY AMAZING RESULTS IN CAT MODEL, SIMILARITIES OF EEG, DEGENERATES OVER TIME, IMPROVEMENT AFTER GENE THERAPY, IN ADDITION SOME EPILEPTI FORM. YOU CAN DO BOTH AT THE SAME TIME. STRENGTH OF THE EEG IT PROVIDES GREAT TEMPORAL INFORMATION, WHAT'S HAPPENING WEN. fMRI TELLS YOU WHERE, WHICH PART OF THE BRAINS ARE TALKING. IF YOU COMBINE THE TWO AVENUES YOU CAN REALLY START ANSWERING SOME OF THESE QUESTIONS ABOUT CIRCUITRY AND HOW THINGS ARE DIFFERENT. I WOULD LOVE TO SEE SOMETHING LIKE THAT DONE IN PATIENTS. WE DO IT IN ANIMALS BUT IN PATIENTS THAT WOULD BE AMAZING I THINK. >> I'M WONDERING IF WE'RE AT A POINTS WHERE WE OUGHT TO THINK ABOUT ALMOST CO-REGISTERING THE CELL TYPE ATLAS THAT WE'RE BUILDING FROM BRAIN, WITH THE DISTRIBUTION AND CHANGE IN EXPRESSION YOU SEE WITH GENE THERAPY. I MEAN, I WOULD THINK THAT AT MINIMUM, CELL-BASE THE INFORMATION WE GET THROUGH BRAIN -- CELL-BASED INFORMATION THROUGH BRAIN MIGHT GIVE YOU A SENSE OF WHICH CELLS WE'RE TALKING ABOUT WHEN YOU SEE ENTRY AND CHANGE IN EXPRESSION IN A CELL AFTER GENE THERAPY. I DON'T HAVE A GOOD IDEA OF HOW TO CO-REGISTER THOSE TWO AT THIS POINT. I DON'T KNOW IF THE TECHNOLOGY IS QUITE THERE YET. >> I THINK IT MAY BE CLOSE, AS WE MOVE TO SINGLE CELL METHODOLOGIES ANALYSIS AT LEAST IN MODEL SYSTEMS TO START WITH. AND YOU CAN LOOK AT THE RNA EXPRESSION PROFILES, YOU CAN LOOK AT THE DNA INTEGRITY, AAV FOOTPRINT, WHICH YOU WOULDN'T SEE IN THE BRAIN INITIATIVE CELL ATLAS BUT AT LEAST YOU WOULD BE ABLE TO GO BACK AND MAP AS TO WHAT CELLS WERE GETTING WHAT. AND THEN LOOK AT THE IMPACT ON THE TRANSCRIPTIONAL SIGNATURE IN THOSE CELLS. SO I DEFINITELY THINK WE COULD TAKE ADVANTAGE OF THAT. NOT JUST FOR WHERE THE VECTOR GOES BUT IMPACT OF THE VECTOR ONCE IT'S IN THOSE CELLS. >> I HAVE A QUESTION. IT'S RELATED TO CIRCUITRY. YOU KNOW THAT RABIES VIRUS, IT'S RETROGRADELY TRANSPORTED, THEN TRANSSYNAPTICALLY TAKEN UP BY NEURONS. DO WE KNOW THE BIOLOGY OF THE OTHER VECTORS? THAT COULD BE A WAY TO EXPLOIT IF WE UNDERSTOOD BIOLOGY TO INCREASE DISTRIBUTION IN THE BRAIN, YOU COULD TARGET IT FOR EXAMPLE IF YOU'RE GOING TO HAVE SOME VECTOR THAT COULD BE TRANSPORTED AND THEN TRANSSYNAPTICALLY TAKEN UP BY THE POST-SYNAPTIC NEURON YOU COULD TARGET IT TO RAFAY OR NEUROMERGIC NEURONS, DO WE KNOW MUCH ABOUT THE BIOLOGY OTHER THAN RABIES? >> YEAH, THAT'S MY FAVORITE TOPIC ACTUALLY. [LAUGHTER] IT'S MY FAVORITE TOPIC. YES, EXACTLY WHAT YOU SAID. I THINK WE'VE BEEN STUDYING IN ITEMS OF AAV AND SEROTYPES, YOU'RE ABSOLUTELY RIGHT. THE TRIAL WE'RE RUNNING NOW, FIRST PROSPECTIVE STUDY USING AXONAL TRANSPORT, NEURONS IN THE BRAIN, HAVING THE PATHWAY BRING THE VECTOR TO PROJECTION AREA. SO, YEAH, THERE'S A LOT OF EMERGING DATA ON DIFFERENT SEROTYPES AND ANTEROGRADE VERSUS RETROGRADE THAT SEEMS TO BE DOSE DEPEND, HOW MANY VECTORS THE& DOSE WILL JUMP THROUGH. THIS IS EFFECTIVE, SPECIFIC, AND ALSO PRECISE WAY OF TARGETING SPECIFIC PATHWAYS WITHIN THE BRAIN BY BEING ABLE TO DO THE PRIMARY TRANSDUCTION WITHIN CERTAIN PART OF THE BRAIN AND KNOWING ANATOMICAL PROJECTIONS WE CAN DEFINITELY HAVE THAT EXPRESSION EXTENTED. FOR EXAMPLE, IF YOU TRANSDUCE (INDISCERNIBLE) YOU'LL GET TREMENDOUS EXPRESSION IN THE CORTEX, WE PUBLISHED TEN YEARS, TELOMERE CORTICAL PROJECTION, TREMENDOUS WORK OF MANY OF YOU IN THE ROOM AS WELL. SO THE ADVANTAGE IN MY MIND YOU HAVE A TARGETED DELIVERY, SMALL PART OF THE BRAIN, AND THEN YOU CAN USE GREAT EFFICACY THE SPECIFIC PATHWAYS. NOW, IN THE CONTEXT OF, YOU CAN BYPASS THE PARTS NOT FUNCTIONING BY TARGETING THOSE WHICH MAKE A DETOUR AROUND THE PATHWAY THAT HAS BEEN AFFECTED BY THE DISEASE SO THERE'S TREMENDOUS POTENTIAL, I THINK, IN BOTH CORTICAL, SUBCORTICAL AND SPINAL DISEASE FOR USING THIS TYPE OF APPROACH. AGAIN, IT'S REALLY SPECIFIC TO NOT ONLY THE SEROTYPE BUT WE ALSO FOUND TO PURIFICATION METHOD, THERE'S A LOT OF UNKNOWNS THERE THAT I THINK ARE DIFFICULT TO COMPREHEND AT THIS POINT BUT I THINK THEY HAVE GREAT THERAPEUTIC POTENTIAL. SOMEONE MENTIONED HERPES. IF YOU GO BACK TO THE VERY OLD NEUROANATOMY BOOKS, THIS IS HOW IT'S BEEN STUDIED, WITH HERPES. THIS IS A CHOICE USED BY ANATOMIST, TO FIGURE OUT PATHWAYS, THEY WERE THE FIRST GREEN PATHWAY GUYS. >> THE ADVANTAGE OF HERPES, YOU THIRD AND FOURTH ORDER NEURONS PROCESS, TRACK TRACING AND ELIMINATED PATHWAYS THAT WEREN'T KNOWN FROM THE LESIONING TECHNIQUES AND SO ON. IN TERMS OF AAV, LIMITED NONE OF SEROTYPES THAT GET TRANSPORTED IN NEURONS, DATES BACK TEN YEARS, WE'VE STUDIED THAT. WE'RE LOOKING AT OTHER CANDIDATES. NINE WAS THE ONE THAT STICKS OUT AS THAT. WHAT YOU GET IS IN SENSE AMPLIFICATION EFFECT, IF YOU GET THE GENOME TRANSFERRED TO DISTAL CELL BODY WHAT CAN HAPPEN IN A LIMITED NUMBER OF THEM, THEN IT CAN GET TRANSPORTED WITHIN THAT NEURON OR IN THE CASE OF SECRETED PROTEINS, STORAGE DISEASES, YOU GET AMPLIFICATION OF YOUR EFFECTIVE DISTRIBUTION OF THERAPEUTIC PROTEIN THAT GETS TAKEN UP BY OTHER CELLS. WE'VE LOOKED AT BIOLOGY OF SEROTYPES, THEY BEHAVED THE SAME WITH RESPECT TO THE COMPARTMENTS THAT THEY ATTACH TO EITHER AT THE ANTEROGRADE OR RETROGRADE AXONAL OR CELL BODY END AND GET TRANSPORTED, COMPARTMENTS. SO IT'S PROBABLY THE DIFFERENCE BETWEEN SEROTYPES IS NOT IN CELL BIOLOGY SO IT MUST BE ENTERING RECEPTORS, A WHOLE LOT OF REASONS YOU CAN THINK ABOUT. BUT GETTING AT THOSE QUESTIONS IS DIFFICULT BECAUSE OF COURSE WE HAVE STILL A VERY LIMITED INFORMATION ON THE RECEPTORS OR BINDING AN INTERNALIZATION RECEPTOR USED IN ANY GIVEN CELL. THERE'S GOOD INFORMATION ON A FEW SEROTYPES BUT IN GENERAL IT'S NOT VERY WELL KNOWN. ANOTHER AREA THAT WOULD, IF YOU KNOW WHAT RECEPTORS WERE USED YOU COULD DO A BLOT MORE PROSPECTIVE EXPERIMENTS TESTING HYPOTHESES, IN OR NOT. THAT'S THE REALM OF CELL BIOLOGISTS AND SOME PEOPLE IN THE ROOM DO THAT KIND OF WORK. I WOULD SUGGEST THAT MIGHT BE A FRUITFUL AREA TO EXPAND. >> SO TO FOLLOW UP ON THAT, A COMMENT AND QUESTION. I THINK IT WAS KRYS AND JUDE AND FOLKS THAT WROTE A COMMENTARY ON THE PARKINSON'S, THE VERY FIRST ONE, THE TRIAL, I REMEMBER THE TITLE, SUCCESS BY DESIGN, FAILURE BY DELIVERY. >> (INAUDIBLE). >> THAT WAS YOU, YES. AND I THINK THE GIST, CORRECT ME IN I'M WRONG, GOING BACK TO BEV'S POINT, IN THE DISEASED BRAIN SOME OF THESE RETROGRADE ANTEROGRADE PATHWAYS ARE MESSED UP, NOT TRANSPORTING PROPERLY BETWEEN THE DIFFERENT NEURONS, DOWN THE DIFFERENT PATHWAYS. SO THAT'S A COMMENT. THE QUESTION IS, IS THERE ENOUGH KNOWN ABOUT THE PATHWAYS THAT WE DON'T EVER THINK ABOUT THE VIRUSES BUT THE CARGO BEING MODIFIED TO TRANSPORT ACROSS MULTIPLE CELLS? >> YOU MIGHT KNOW SOMETHING FROM HERPES. >> I WAS GOING TO MAKE THE POINT I DON'T THINK A NON-REPLICATING VIRUS CAN CROSS A SYNAPSE. HOW DOES IT DO IT? >> AAV, WE DO NOT KNOW. OR I DON'T KNOW. MAYBE SOMEBODY ELSE IN THE ROOM KNOWS. >> YEAH, WE'VE SEEN THAT TOO. >> TO SANDRA'S POINT, REPLICATING VIRUS, HERPES VERSUS AMPLICONS. AMPLICONS DON'T. >> IT KILLS THE FIRST CELL. >> WITH GAMMA 34.5 IT DOESN'T. IF YOU WANT TO USE THAT TO GET OF GET WIDESPREAD TRANSPORT YOU'RE USING A HERPES VIRUS. >> WE NEED TO KNOW MORE ABOUT SUBCELLULAR EFFECTS OF VIRUSES. ALSO TO ENCOURAGE THAT ONE POINT WE DIDN'T DISCUSS IS NON-VIRAL METHODS. THE POINT WAS MENTIONING TO SOME EXTENT THAT IF WE PACKAGE SOMETHING THAT WE CAN USE OTHER MOIETIES OR DELIVER IN A NON-VIRAL VECTOR MEDIATED WAY. TWO POINTS HERE. THE EVS OR ARC-LIKE CAPSIDS RECENTLY DISCOVERED, ONE COULD THINK YOU COULD MAKE THAT SPECIFIC TO A CARGO THAT YOU CAN TRANSPORT BETWEEN CELLS, A CELLULAR PROPERTY WE'RE ABLE TO RECRUIT. I OPEN FOR DISCUSSION NON-VIRAL VECTOR METHODS BECAUSE WE HAVEN'T TOUCHED POP THAT, AND WE SHOULD BECAUSE THEY ARE IN THE CLINIC AND THEY WORK AND WE SHOULD TALK ABOUT THEM, ASOs, ET CETERA. >> I WOULD LOVE TO SEE THE KIND OF THINGS WE'RE PROPOSING TO DO WITH AAV, BE APPLIED IN AN UNBIASED WAY TO OTHER PLATFORMS. LENTIVIRUSES, SEROTYPES, OR PSEUDOTYPES OF LENTIVIRUSES AS WELL AS SOME OF THE ENGINEERING FOR ENVELOPES THAT HAVE BEING DONE FOR A.A.V, NO REASON THAT CAN'T BE APPLIED TO ENVELOPES THAT CAN PACKAGE LENTIVIRUSES. I THINK I'D ALSO LIKE TO SEE LNPs ARE WONDERFUL, HOW DO WE GET THEM MORE MAINSTREAM? YOU HAVE TO FIND A PARTICULAR PHARMACEUTICAL COMPANY TO WORK WITH YOU TO MAKE NANOPARTICLES IN A UNIFORM WAY. IS THERE SOME SORT OF RESOURCE THAT THE NIH COULD PROVIDE FOR YOUR FAVORITE LIPID NANOPARTICLES, OR LIBRARIES OF LIPID NANOPARTICLES THAT COULD BE SCREENED IN LARGE ANIMAL MODELS, DISEASED AND NORMAL, THAT WE COULD TAKE ADVANTAGE OF. >> SO, I TRAINED AS A LIPID CHEMIST BEFORE JUDE CONVERTED ME TO VIROLOGIST. AND I THINK JUST HAVING SEEN THE FIELD GROW DEFINITELY THERE'S IMPROVED EFFICIENCY IN THE ORGANS THEY CAN STILL CONTINUE TO HIT BUT I THINK IN CONTEXT OF TOUGHER ORGANS LIKE CNS, FIELD HAS MOVED AS MUCH FROM THE LIKE AND PERSPECTIVE. I DO SEE GOLD NANOPARTICLES AND THINGS LIKE THAT COMING UP ON THE RADAR FOR CNS DELIVERY ACTUALLY BUT I DON'T KNOW ABOUT THE LNP FIELD. >> I WOULD AGREE UNTIL WE TRIED SOME NEWER LNPs, THESE ARE IN RODENTS. AND THEN PRELIMINARY WORK IN NON-HUMAN PRIMATES. IT'S PROFOUND HOW MUCH BETTER THEY DISTRIBUTE IN TODAY'S WORLD THAN THEY DID 8 YEARS AGO. SO CLEARLY THEY FIGURED SOMETHING OUT. AND WE NEED TO RECRUIT MORE CHEMISTS BACK INTO THE SPACE. THERE'S YOUR CHALLENGE. >> A COMMENT. I MEAN, THAT WOULD BE GREAT TO GET RID OF THE CAPSID COMPONENT IF WE COULD. BECAUSE THAT'S A SOURCE OF A LOT OF IMMUNOLOGY HEADACHES, IMMUNE RESPONSE HEADACHES. HAVE EXPERIENCE WHAT IS RELATIVE EFFICIENCY, AAV VECTOR WE'RE DEALING WITH VIRAL CAPSID, IT'S SO EFFICIENT AT DELIVERING PAYLOAD, WHERE IT NEEDS TO GO, IT'S HARDS TO REENGINEER THAT. NOT HAVING WORKED IN NANOPARTICLES I DON'T KNOW THE RELATIVE, IS IT 10 LOGS OR 1 LOG? >> IT'S MORE EFFICIENT, LIKE A REGULAR LIPID VIRUS ON THE ORDER OF 1 TO 10 VERSION US 10,000. >> MORE EFFICIENT THAN AAV, YEAH. >> I WOULD MAYBE WANT TO CHALLENGE THAT A LITTLE BIT AND JUMP ON THE POINT WHILE WE WANT TO MAYBE GET RID OF CAPSIDS, THERE'S ALWAYS SOMETHING THAT COMES IN, THAT'S LESS STUDIED, WE'RE SEEING ON ONE OF THE SLIDES ADMINISTRATION WITH LENTI, WOULD BE LESS OF AN ISSUE, I'M NOT SURE IF THAT'S BEEN STUDIED AS THOROUGHLY AS AAV, AGAIN AN ENVELOPE COULD BE ANTIGENIC, AND SO CAN BY THE WAY A NON-VIRAL PARTICLE. AND CAN HAVE RESPONSES AND SO FORTH, DOGMATIC CLASSIFICATION, THESE SYSTEMS ARE BETTER WE SHOULD BE CAUTIOUS. IN GENERAL I THINK AAV IS NOT GOING TO STICK AROUND BUT IF YOU STACK THINGS UP EVEN LOOKING AT THE LIVER, NON-VIRAL AGENTS, PARTICULARLY IS GOAL IS NUCLEAR, LIPID NANOPARTICLES TO GO FROM ENDOSOME TO NUCLEUS ARE ORDERS OF MAGNITUDE UP FROM WHAT A VIRUS CAN DO. HISTORICALLY WE'VE WALKED THROUGH VARIOUS CLASSES OF SYSTEMS AND LANDED ON AV AS A VERY WORKABLE STARTING POINT. DOESN'T MEAN WE SHOULD GIVE UP OBVIOUSLY. >> I'M TALKING WHERE THE CARGO IS AN RNA, OR RNP, AND NOT A GENE. SO WHERE YOU WOULD WANT SOMETHING TRANSIENT IN THE DATA, IN THE LENTIVIRUS THAT I'M AWARE OF IS ALL MOUSE DATA, AND I THINK IT SHOULD BE PUSHED INTO LARGER ANIMAL MODELS WHERE THEY HAVE GIVEN DIRECT INFUSIONS INTO ANIMALS UP TO SEVEN TIMES WITH INCREASING LEVELS OF TRANSGENE EXPRESSION, NOT INHIBITORY, AND THAT JUST NEEDS TO BE PUSHED A LITTLE BIT MORE IN CNS AND LOOKING AT THAT. >> I THINK THAT'S A CHALLENGE WITH LENTIS, ABOUT MANUFACTURING. AND BECAUSE YOU JUST CAN'T PRODUCE VERY HIGH TITERS WITH LENTI. UNTIL WE GET OVER THAT HURDLE YOU'LL ONLY IN CNS VERY SPECIFIC TARGETS LIKE SUBSTANTIA NIGRA, NOT THE SAME DISTRIBUTION, EVEN THOUGH VIRUS FOR VIRUS YOU GET A BETTER EFFECT THAN AAV, SO IT'S A PROBLEM BECAUSE IT'S A PLASMA-BASED PRODUCTION SYSTEM, ENVELOPE, EXACTLY. IT'S SOFT. IT BREAKS VERY EASILY. >> HAVING WORKED WITH BOTH AND ON THE CNC SIDE, AAV IS TOUGH, LOTS OF CMC CHALLENGES BUT IT PALES IN COMPARISON TO CHALLENGES WITH LENTICS. IT'S HETEROGENEOUS ENTITY. WE DO HAVE SOME CELL LINES. YEAH, I CONCUR. WE'VE GOT SOME REAL CHALLENGES ON THAT. >> SO, BEV, IF YOU WANT A SUGGESTION ON WHAT THE NIH COULD DO, I WOULD CONSIDER MODELING AFTER THE MICHAEL FOX PARKINSON'S PROGRAM. THEY CONTRACTED SOMEONE TO MAKE CAPSIDS WITH SPECIFIC CASSETTES AND THEN THEY MADE THEM AVAILABLE TO INVESTIGATORS, SO IT REMOVED THE RISK OF WHAT'S HAPPENING IF IT'S PRODUCED IN THIS SETTING VERSUS THAT SETTING. IF YOU WERE GOING TO DO A SURVEY ACROSS DIFFERENT VECTORS, SUCH AS HERPES, RETROS, WHATEVER, IT WOULD MAKE MORE SENSE TO HAVE THOSE CONTRACTED SO THAT YOU CAN REMOVE THAT VARIABILITY. THEN YOU WOULD GET CLOSER TO DOING THE FUNCTIONAL ASSAYS. IT WORKED REALLY WELL AND HAS CONTINUED TO BE PRODUCTIVE FOR THEM. I THINK A NUMBER OF INVESTIGATORS WHICH ENTERED INTO THE SPACE NOT KNOWING THE VECTORS ASSUMED THEY WOULD BUY RESTRICTION ENZYMES, WE'RE GETTING INTO TROUBLE GETTING RESULTS WHEN THEY MADE IT OR SOMEONE DOWN THE HALL MADE IT. ONCE THEY WENT TO CONTRACTORS, THEY GOT SOME STANDARDS SO I WOULD SAY IF YOU WERE GOING TO MAKE A PROPOSAL HOW YOU COULD ASSIST THIS COMMUNITY IS MAYBE MIMICKING SOMETHING LIKE THAT, WHETHER IT'S THE LNPs OR WHATEVER. >> JUST A COMMENT I THINK FOR ALL THESE TECHNOLOGIES, NON-VIRAL AND ALSO LENTI, IN VIVO, THERE'S STILL SOME TECHNOLOGICAL AND HAVING AN INITIATIVE WOULD BE IMPORTANT THAT ALLOWS TECHNOLOGIES TO EVOLVE A LITTLE BIT BECAUSE I THINK THE PROBLEM IS, AGAIN, WE TESTED IN ANIMAL MODELS, INITIAL CARGOES, BUT THEN I THINK OVERALL IMMUNOGENICITY WILL BE A PROBLEM, IN PARTICULAR TRANSGENE IMMUNOGENICITY FOR LNPs, PARTICLES ARE TAKING UP IMMUNE CELLS SO THEY EXPRESS, WE DON'T WANT IMMUNOGENIC BUT THEY ARE LESS IMMUNOGENIC BECAUSE THEY TARGET LESS WHICH HAS BEEN STUDIED FOR THE PROBLEM WITH LENTIVIRAL VECTORS. I THINK MY FEELING IS THAT THERE'S STILL A LOT OF WORK TO BE DONE AND THERE'S NEED FOR RESOURCES TO DEVELOP THESE TECHNOLOGIES AND MAKE THEM SUITABLE FOR IN VIVO. THEY HAVE GREAT POTENTIAL, FOR SURE. >> I'D LIKE TO MAKE ANOTHER SUGGESTION BECAUSE I THINK WE WERE POINTED IN A DIRECTION WITH microRNA TECHNOLOGY BEING ABLE TO SILENCE IN CERTAIN CELL TYPES IF AS A COMMUNITY WE GOT CONSENSUS THAT THE USE OF CERTAIN microRNAs WOULD AVOID ANTIGEN PRESENTING CELLS BECAUSE THEY HAVE BEEN TESTED AND VALIDATED BY A LAB, AND THOSE WERE MADE AVAILABLE CASSETTES, AS A STARTING CASSETTE THAT YOU WOULD POP YOUR GENE INTO, EVERYBODY WOULD START AGAIN AT THE SAME BASELINE LEVEL OF ABORTING CERTAIN CONCERNS IRRESPECTIVE OF CAPSID THEY WERE USING. IT'S A MATTER OF TIME BUT COMING OUT IN THE LITERATURE BUT COULD BE A CONCERTED EFFORT TO GENERATE THOSE AND THEN CONTRIBUTE THEM TO A DATA BASE WHERE PEOPLE COULD PULL THEM DOWN AND GET ACCESS. >> 2006 PAPER THEY PUBLISHED WITH LENTIVIRUS BACKGROUND, SO AFTERWARDS WE AND A FEW OTHER LABS GET INTO ADENO, AND A.A.V, FROM THE MECHANISTIC POINTS OF VIEW HAS BEEN QUITE EFFECTIVE. AND BUT THE QUESTION IS WILL THIS BE TRANSLATABLE TO THE LARGE ANIMALS, I THINK WE'RE STILL DEBATING. WE DID SEE SOME IMPROVEMENT IN THE MIAMI MONKEY I WAS TELLING YOU, BUT WE HAVE BEEN TRYING TO SCREEN ABOUT 30 MORE THE ANTIGEN PRESENTING CELL ASSOCIATED microRNAs, WE HAVE NOT FINISHED OUR IN VIVO SCREENING YET. BUT I THINK SOMEBODY MENTIONED THIS IN THIS DISCUSSION THAT I THINK WE NEED TO REALLY PERFORM SOME SINGLE CELL SEQUENCING TO REALLY FIGURE OUT microRNA PROFILE BUT UNFORTUNATELY I HAVE PROBLEM THERE IS BECAUSE THEY CAN DO TRANSCRIPTOME BUT CANNOT DO SMALL RNA SEQUENCING, WE TRY TO GET ISOLATED MACROPHAGE, PMCs AND OTHER CELL TYPES, AND LOOK AT microRNA SIGNATURES, AND WE STILL CANNOT DO THAT BUT I AGREE WITH JUDE, IT'S KIND OF VERY MUCH A SPACE-SAVING STRATEGY COMPARED TO TISSUE PROMOTERS, AND ALSO THE IDEAL THING WHICH I'VE BEEN TALKING TO KAI ABOUT THIS IF WE CAN COMBINE THIS, THIS TELOMERE SEQUENCE WITH INNATE AND ADAPTIVE RESPONSES WE MAY CHANGE THE LANDSCAPE QUITE SIGNIFICANTLY. >> SO, AGAIN, IF YOU TAKE THE SUMMARY OF TODAY AND BEGIN TO BREAK IT DOWN INTO SPECIFIC TO-DO LIST OR BOXES OF PACKAGES, WE TALKED ABOUT AN ATLAS TO -- REGARDLESS OF CAPSID TO GET A BASELINE FINGERPRINT, BIODISTRIBUTION, AND IT COULD BE DONE IN CONCERT WITH THE PET IMAGING OR WHATEVER, JUST TO HELP US UNDERSTAND IF YOU USE THE FALLING CAPSID IN BRAIN EXPECT THIS TYPE OF DISTRIBUTION. AND THEN BRIAN BROUGHT UP THE IDEA OF GOING AFTER CELL TYPE SPECIFIC PROMOTERS THAT IF THERE'S DIRECTED EFFORT TO BEGIN TO ISOLATE AND VALIDATE CELL TYPE SPECIFIC PROMOTERS, YOU HAVE THE OPPORTUNITY VALIDATING THOSE IN A CAPSID BACKBONE THAT HAS A SPECIFIC BIODISTRIBUTION. AND THEN IF YOU AGAIN HAVE A TARGETED EFFORT TO IDENTIFY THREE PRIME IN microRNAS WOULD AVOID ANTIGEN OR TARGET CELLS, PUTTING TAG PACKAGES ADDRESSING WHAT'S BEEN THE THEME HERE TODAY WHICH IS GETTING TARGETING, GETTING SPECIFIC RESPONSES IMMUNE AVOIDANCE AND THINGS LIKE THAT. WHETHER OR NOT IT EVER GETS USED CLINICALLY GIVES US TOOLS TO GO BACK INTO DISEASE MODELS OR INTO LARGE ANIMAL MODELS, TO SEE HOW FAITHFUL THOSE REAGENTS ARE WHEN THEY ARE BUILT INTO CASSETTES. I WOULD ASSUME THAT, AGAIN, IF EVERYONE IN HERE HAD ACCESS TO THAT AND ALL WENT BACK TO OUR MODELS AND TESTED WE WOULD COME BACK AND SAY THIS CELL SPECIFIC PROMOTER IS OR ISN'T AS GOOD AS IT WAS ORIGINALLY DESCRIBED. AND IT WOULD FALL INTO A CLASS OF IT'S ADEQUATE FOR SECRETING PROTEINS BUT NOT FOR CELL AUTONOMOUS. YOU MOVE DOWN THE GENOME LIKE THAT AND COME BACK WITH A CONSENSUS. IN THE END IF THAT WAS MADE AVAILABLE BY A THIRD PARTY CONTRACTOR, THEN THE SCIENCE GETS DONE AND WE'RE NOT SPENDING A LOT OF TIME AND RESOURCES TRYING TO DEVELOP REAGENTS. IF YOU BREAK IT INTO PACKAGES, AND HOPEFULLY THE TEAM WILL BE ABLE TO PRIORITIZE BEFORE FRIDAY'S OVER, YOU'RE LOOKING AT THINGS THAT ARE GLOBAL, IMPACTING EVERYBODY HERE, AT SOME LEVEL YOU CAN BEGIN TO CHIP AWAY AT THEM. I THINK AGAIN WHETHER IT'S THE RIGHT ONE OR NOT IS NOT AS IMPORTANT AS IT IS TO START TO FUND MENTALLY ESTABLISH SOME TYPE OF STANDARDS. >> IN ADDITION TO THAT, TO NOT LOSE SIGHT OF WHAT WE CAN GAIN FROM HUMAN CLINICAL DATA, AND TO FOCUS REALLY HARD ON TRYING TO ACCESS THOSE TISSUES AND FIGURE AGAIN MAYBE A THIRD PARTY CAN HELP, DATA WAREHOUSE, IF YOU WILL, THAT CAN BEGIN TO CO-LATE AND MAKE THAT AVAILABLE THROUGH QUERY TYPE MECHANISM SO PEOPLE CAN BEGIN TO UNDERSTAND WHAT GOES WHERE, WHY, AND HOW. VIVIANA, DO YOU WANT TO WRAP THIS UP. >> I THINK THESE WERE FINAL PARTING THOUGHTS, THE ONE FINAL THING IS TO HAVE THIS, SO WE GOT AN ORDER FOR NINDS FOR ONE SITE FOR STANDARDS, WHETHER THIS TAKES THE FORM OF MANUFACTURING THAT HAS STANDARDS CAPSIDS, CARGOES, THAT WOULD BE A GREAT ENABLER, AND ONCE WE HAVE THAT WE JUST NEED TO WORK ON TWO ATLASES, ONE FOR CELL TYPING, AND ALL OF THE BASIC KNOWLEDGE, IN SYNC WITH THE SECONDARY ATLAS FROM HUMAN DATA AND WE TALKED ABOUT GENE THERAPY, MAYBE HAVING BEHAVIORAL DATA, AND RECORDINGS, EEG, fMRI, TWO ATLASES AND ONE MAGNIFYING CENTER I THINK WE CAN MAKE A LOT OF PROGRESS. LET'S SEE HOW WE BREAK THAT DOWN. >> IT'S INTERESTING TO ME ABOUT A YEAR AND A HALF, TWO YEARS AGO, WHEN MANY OF US SAT TOGETHER AND WALTER WAS TALKING WITH US ABOUT TRYING TO SEE WHAT WE COULD DO TO MOVE THIS FURTHER, I THINK WE WERE VERY MUCH FOCUSED ON THE SORT OF BOTTLENECK IN CAPACITY FOR PRODUCTION OF THESE, AND NEED FOR SOME KIND OF PLUG AND PLAY, MAYBE WE CREATE THE VECTOR AND WE LEAVE A HOLE AND YOU PLUG IN WHATEVER CONSTRUCT YOU WANT, WE MASS MANUFACTURE. IT BECAME VERY CLEAR TO US THAT EVERYBODY IS USING SOMETHING A LITTLE BIT DIFFERENT. EVERYBODY IS INSERTING THEIR CONSTRUCT A LITTLE BIT DIFFERENTLY. NOBODY IS QUITE SURE FROM THE STANDPOINT OF EFFICACY, DELIVERY, WHATEVER, WHICH ONE OF THESE WORKS IN WHICH CIRCUMSTANCES UNDER WHICH CONDITIONS. AND IT'S ALSO CLEAR THAT IF WE WERE GOING TO DO PURELY A SCALEUP AND FACILITATE THE ACCESS TO THIS KIND OF THING, THAT ACTUALLY INDUSTRY DOES THAT MUCH BETTER THAN THE NIH EVER WOULD DO SO I THINK THE NOTION OF US HELPING WITH THE STANDARDIZATION AND DETERMINING THE PROTOCOLS THAT MIGHT GOVERN HOW AND WHAT YOU PRODUCE FOR WHAT APPLICATION OR PURPOSE IS NOT ONLY HELPFUL FOR YOU BUT IT ACTUALLY PLAYS TO WHAT WE DO UNIQUELY I THINK RATHER THAN PLAYING TO SOMETHING THAT WE DON'T DO AS WELL AS THEY DO OUTSIDE OF OUR CAMPUS. SO I THINK THAT'S VERY, VERY HELPFUL TO US. >> LET'S TAKE A BREAK UNTIL 3:05 AND WE'LL START THE SESSION 3. EVERYBODY IS DOING A GREAT JOB. KEEP UP THE ENERGY. [ RECESS ] I'M HEATHER GRAY FROM UMass. I DO LARGE ANIMAL WORK SO I'M GOING TO TRY TO KEEP AN OPEN MIND FOR MOUSE AS WE GO THROUGH THIS PRE-CLINICAL MODEL SECTION. I MAY HAVE GOTTEN CARRIED AWAY IN SLIDES, POINTS TO DISCUSS, TWO PAGES INSTEAD OF ONE. MAYBE WE CAN TALK ABOUT THE FIRST PAGE AND THEN GO TO THE SECOND PAGE, THESE WERE GOOD POINTS BROUGHT UP IN THE PRE-MEETING BY THE EXPERTS. I THINK IT'S WORTH PUTTING EVERYTHING UP THERE. THE FIRST SECTION THAT'S A GOOD SEGUE FROM THE LAST SECTION WE DISCUSSED IS TAKINGI FROM CLINICAL TRIALS BACK TO ANIMAL MODELS, CAN WE GO BACK AND LEARN THESE THINGS IN AN BECAUSE CHARLES AGREED WE HAVEN'T LOOKED IN DEPTH AND I DON'T KNOW THAT WE CLEARLY HAVE ANSWERS. THE IMMUNE RESPONSE TO GENE THERAPY, I'M SPECIFICALLY SPEAKING TO TRANSANIMASE ELEVATION, THAT'S THE CONSENSUS THAT THEY DOESN'T ACTUALLY HAPPEN IN ANIMALS. SO I'M NOT CONVINCED IT DOESN'T, I DON'T KNOW THAT MANY ANIMALS MIGHT LIVE IN TOO CLEAN OF AN ENVIRONMENT, RESEARCH ANIMALS ANYWAY, AND DON'T HAVE COMPLEX EXPERIENCES. ARE WE USING ACCURATE DOSING, FROM MICE TO PEOPLE, ARE WE UNDERDOSING PATIENTS AND THE OTHER THING IS A LOT -- MOST STUDIES IN ANIMAL MODELING DOES NOT INCLUDE IMMUNOSUPPRESSION IN PRE-CLINICAL WORK BECAUSE WE MAY OR MAY NOT NEED IT BUT WHAT ARE WE MISSING IN PRE-CLINICAL STUDIES BY NOT INCLUDING IMMUNOSUPPRESSIVE PROTOCOLS. THOSE ARE KIND OF SOME BIG POINTS THAT FIT WELL WITH THE LAST SECTION. SO I DON'T KNOW IF ANYBODY WANTS TO COMMENT ON ANY OF THAT ESPECIALLY MAYBE SOME -- RIGHT NOW A LOT OF IMMUNOLOGIST, ESPECIALLY NEUROIMMUNOLOGYISTS ARE IN MICE. WE NEED INPUT IN THE LARGE ANIMAL FIELD AND HOW MAYBE WE CAN INFORM A LITTLE BIT MORE IN THAT. I'M GOING TO SIT DOWN AND THEN WE'LL TALK AND MAYBE I'LL STAY SITTING DOWN OR COME BACK UP. >> CAN I COMMENT ON THE IMMUNE RESPONSES IN TERMS OF WHAT WE CAN MODEL IN ANIMALS AND WHAT WE CAN'T. I MEAN, I THINK INNATE RESPONSES WE PROBABLY HAVE A REASONABLE CORRELATE FOR WHAT'S GOING ON BETWEEN MICE AND HUMANS TO SOME EXTENT. FOR ADAPTIVE RESPONSES THERE ARE CLEARLY QUITE SIGNIFICANT DIFFERENCES BETWEEN RODENTS AND HUMANS. ONE IS THEY ARE IMMUNOLOGICALLY NAIVE BECAUSE WE TEND TO USE INBRED MICE. OTHER IS THERE ARE CHANGES IN TERMS OF HOW CD4 AND CD8 SPONGESES HAPPEN IN MICE AND HUMANS. THE PROBLEM IN LARGE ANIMAL MODEL YOU LOSE THE TOOLS FOR MICE AND HUMANS, IDENTIFYING APPROPRIATE IMMUNE CELLS, BECOMES MUCH MORE DIFFICULT IN LARGE ANIMAL MODELS. THERE ARE SOME ASPECTS OF IMMUNE RESPONSE YOU CAN MODEL WELL IN MICE, OTHERS YOU CAN'T. YOU JUST HAVE TO BE COGNIZANT OF THAT FACT. >> MAYBE I'LL COMMENT, HAVING BEEN INVOLVED IN THE ORIGINAL LIVER DIRECTED GENE THERAPY, PERHAPS MOST FOLKS KNEW THIS, KNOW THIS, HEMOPHILIA B AGAIN, INSTRUCTIVE THAT THE LARGE OUTBRED HEMOPHILIA B DOG MODEL I THINK WAS, YOU KNOW, SUCCESSFULLY CONVERTED LONG TERM TO EXPRESSION OF THE FACTOR 9, CANINE FACTOR 9, NOT PREDICTIVE, A BASELINE. SO EVEN IN A LARGE ANIMAL MODEL HAS ITS OWN LIMITATIONS. >> OKAY. GO AHEAD. >> I THINK FROM A CLINICAL PERSPECTIVE IT'S EXTREMELY IMPORTANT TO DEVELOP PREDICTIVE PRE-CLINICAL MODELS OF THAT BECAUSE I THINK YOU WANT TO GO INTO CLINICAL TRIALS PREPARED, INTO THE RIGHT PROTOCOL FROM THE GET-GO BECAUSE IF YOU DON'T, YOU DEVELOP ANTI-TRANSGENE IMMUNITY, YOU LOSE TRANSGENE EXPRESSION, YOU'VE BASICALLY LOST PATIENT, THE CORRECTION OF A PATIENT, IMMUNIZED THE PATIENT BUT YOU'RE NOT GIVING THEM THE RIGHT TO BE FULLY BENEFICIAL IN THAT PATIENT SO I THINK THE MORE ONE CAN PRE-CLINICALLY DETERMINE WHAT'S NEEDED IN TERMS OF IMMUNOPROTOCOLS BEFORE YOU GO INTO PATIENTS IN VARIOUS SITUATIONS, THE BETTER IT IS FOR THE CLINICAL TRIALS SO I THINK THERE'S AN URGENCY TO DEVELOP THAT PRE-CLINICALLY. >> WHAT SPECIES, THERE ARE RELATIVELY SMALL NUMBERS OF NATURALLY OCCURRING DOGS, CATS, SO ON. THEY HAVE BEEN VERY USEFUL GENETIC DISEASES BUT IT'S ON THE ORDER OF A HUNDRED MAYBE TOTAL MODELS, PROBABLY LESS, ABOUT 100. WITH CRISPR YOU CAN DO SPECIES MODELS, EVERYBODY SAYS NON-HUMAN PRIMATE, YOU GET SINGLE BABIES, LONG GESTATIONAL PERIODS AND CUMBERSOMENESS WITH HUMANS. I DON'T MEAN THAT HOW IT SOUNDS. PIGS, YOU GET LARGE LITTERS, A LOT OF ASPECTS MORE SIMILAR TO HUMAN, VASCULATURE, SKIN, THINGS LIKE THAT. GIVEN CRISPR CAN MAKE HUGE GENETIC DISEASES IN WOULD BE HUGE, WE'RE TRYING IT BUT IT'S A HUGE INVESTMENT, IS THERE AMONGST IMMUNOLOGISTS A PICK OF SPECIES IN WHICH ONE WOULD WANT TO MAKE MODELS? >> NOT ANSWERING AS AN IMMUNOLOGIST IN TERMS OF REAGENTS AVAILABLE IF YOU WANTED TO TRY AND TEST IMMUNOSUPPRESSIVE REGIMENS FOR THINGS LIKE THAT, I THINK THERE'S REAGENTS FOR HUMANS OFTEN WILL TRANSLATE TO NON-HUMAN PRIMATES BUT OUTSIDE OF THAT IF YOU'RE GOING DOO INTO DOGS, SHEEP, PIGS, OTHER ANIMAL MODELS A BIG PROBLEM IS THAT YOU DON'T NECESSARILY HAVE THE SAME REPERTOIRE OF IMMUNOSUPPRESSIVE AGENTS AS YOU WOULD FOR HUMANS. >> IN DOGS, THERE'S ACTUALLY -- I DON'T KNOW, I CAN'T SPEAK TO PIGS AND SHEEP BUT IN DOGS IT'S ACTUALLY A PRETTY EXTENSIVE SET OF REAGENTS FOR BOTH ANTIBODIES TO DETECT THINGS AND WE'RE CURRENTLY USING TRIPLE IMMUNOSUPPRESSION IN DOGS FOR RETINAL CELL TRANSPLANTS, WORKS ON HUMAN STUDIES, DONE IN DOGS. IT IS NOT THE SAME AS A HUMAN, YOU KNOW, THERE ARE MANY THINGS THAT WORK WELL THERE. CATS ARE MUCH HARDER TO WORK WITH IN THAT REGARD, CATS COME FROM A PARALLEL UNIVERSE, AND I GUESS THE QUESTION IS, IS THE DOG -- HOW MUCH IS THE DOG OFF IN TERMS OF IMMUNOLOGY? >> WELL. >> VERSUS WHAT OTHER SPECIES WOULD BE BETTER, GIVEN THAT IT WILL TAKE A LARGE INVESTMENT TO CREATE THE BONA FIDE GENETIC DEFECT. >> CAN YOU ASK YOU AND CHARLES AND HEATHER, ANYBODY ELSE WITH LARGE ANIMALS, THE DOG EXPERIENCE SPECIFICALLY, ANECDOTALLY I HEAR MAYBE DOGS HAVE ALMOST AN OVERREACTIVE IMMUNE SYSTEM. I'VE HEARD THAT BEFORE, BUT I JUST -- LET ME GET THE EXPERTS' OPINION HERE. >> I DON'T THINK I WOULD WORD IT THAT WAY. IN THE YEARS OF VETERINARY WORK IMMUNE MEDIATED DISEASE I'VE SEEN IN PEOPLE ARE SEEN IN DOGS, CANCERS SEEN IN PEOPLE ARE SEEN IN DOGS. THEY ARE PROBABLY THE CLOSEST FROM AN IMMUNE RESPONSE ASPECT, OF THE SAME KIND OF INCIDENCE, THE SAME KIND OF OCCURRENCES. THAT DOESN'T -- IT SPEAKS BACK TO YOUR REAGENT QUESTION BUT YOU HAVE THE BENEFIT THAT YOU HAVE A LARGE NUMBER OF FOR EXAMPLE VETERINARY IMMUNOLOGISTS TRYING TO DO THE SAME THINGS IN DOGS AS ARE BEING DONE IN PEOPLE. AND I'LL JUST BRING UP IT'S NOTHING I DO BUT I'LL BRING UP THIS GENE THERAPY ISSUE FOR A FIELD OF VETERINARIANS IS BECOMING THE SAME KIND OF QUESTION OF HOW WE'RE GOING TO TREAT THOSE ANIMALS WHEN THEY COME ALONG. IT'S NOT A JOKE. THAT'S REALITY. AND SO I THINK THAT I WOULD CONCUR THAT PROBABLY OF THE MODELS IN A ARE AVAILABLE, THE DOG IS PROBABLY THE CLOSEST IN TERMS OF REAGENT AVAILABILITY AND IMMUNE SYSTEM, CLOSEST TO WHAT I WOULD SAY IS YOU'RE SAYING NON-HUMAN PRIMATES, BUT THEY BRING WITH THEM THEIR OWN ISSUES. WE STILL HAVE A LOT OF UNDERSTANDING TO DO. BT NOW THERE ARE FULL FIELD OF VETERINARY IMMUNOLOGISTS ANSWERING THOSE QUESTIONS, THAT'S COME OUT OF BONE MARROW TRANSPLANT STUDIES, A LOT OF OTHER STUDIES, SO I THINK IT IS -- YOU HAVE A FIELD THAT'S RUNNING PARALLEL TO THE HUMAN FIELD WHICH IS TRYING TO DEVELOP THESE SAME KIND OF TECHNIQUES, DOGS ARE PROBABLY THE ONE THAT'S GOING TO LEAD THE WAY SO I THINK THAT IT'S USEFUL TO TALK ABOUT DOGS AS BEING A VERY ACCURATE MODEL, BASED ON REAGENTS, BASED ON DISEASE INCIDENCES, BASED ON STUFF WE CAN HAVE. IN TERMS OF THE SAME KIND OF TESTING WE CAN DO, I WOULD -- OUTSIDE OF THE PRIMATE MODEL I WOULD SAY IT'S LEADING. THAT BRINGS ITS OWN PROBLEMS WE'RE CREATING GENE EDITED DOGS IS YEARS OFF BECAUSE OF THEIR REPRODUCTIVE SYSTEM. >> I MIGHT ALSO ADD THAT IT'S ONE THING TO USE DOGS THAT WERE -- THAT NATURE PROVIDED YOU WITH A MUTATION BUT GIVING A MUTATION DOGS, PEOPLE FEEL STRONGLY ABOUT THAT. AND I KNOW IN THE FIELD OF GLIOMAS THERE WAS A VET IN ARIZONA THAT WOULD PUT TUMORS INTO BEAGLE DOGS, AND HE RECEIVED SUCH THREATS INCLUDING PEOPLE TRYING TO BURN HIS HOUSE DOWN AND EVERYTHING ELSE. HE DOESN'T DO IT ANYMORE. I'M JUST SAYING, I AGREE WITH YOU. I'M A SCIENTIST. I REALLY WANT TO HELP PEOPLE. AND IF THAT'S THE BEST MODEL I WOULD BE IN FAVOR. I'M SAYING THERE'S A LOT OF PEOPLE OUT THERE THAT WILL GET VERY UPSET AND THERE MIGHT BE REPERCUSSIONS. >> THAT'S THE LIFE WE LEAD RIGHT NOW. >> I'VE GOT TWO IMMUNOLOGISTS WITH COMMENTS. >> WHEN IT COMES TO CARCINOGENICITY THERE'S NO ANIMAL MODEL. INNATE IMMUNITY. WHEN IT COMES TO AN EASY MODEL IT'S ACTUALLY THE RAT. IN MY EXPERIENCE, THE ADVANTAGE, DISADVANTAGE OF THE RAT IS I'VE SEEN THEY EXHIBIT MORE INFLAMMATION AND TRANSGENE IMMUNOGENICITY, IT'S ALWAYS BEEN A MORE STRINGENT MODEL OF DISEASES. I'VE SEEN THAT IN DMD, IN POMPE AND OTHER DISEASES, TENDS TO BE IF YOU YOU WANT TO TEST MORE SEVERE, MORE INFLAMMATORY ANIMAL MODEL WHEN IT COMES TO TRANSGENE THE RAT HAS BEEN QUITE GOOD AND IT'S VERY EASY TO MAKE. CRISPR/CAS9, YOU CAN MAKE AN ANY MODEL YOU WASN'T, THERE'S A VARIETY OF REAGENTS. IN MY EXPERIENCE I FOUND A CHALLENGING AND STRINGENT MODEL THAT MAYBE WOULD HAVE DEVELOPED STRATEGIES ALSO WHEN IT COMES TO -- NOT ALL IMMUNOMODULATORY DRUGS WILL WORK IN RAT BECAUSE IF YOU'RE THINKING ABOUT MONOCLONALS AND SO ON, THEY DON'T USUALLY DON'T CROSS HERE. >> I WOULD SAY ONE THING VERY QUICKLY, LIKE ANYTHING ELSE WE'RE TALKING ABOUT WHAT ARE YOU MODELING, WHETHER YOU'RE MODELING BECAUSE BASED ON REAGENTS OR IMMUNE SYSTEM OR MODELING BASED ON SIZE OF BRAIN AND NERVOUS SYSTEM AND THOSE ARE ALL WHAT MAKE EACH MODEL UNIQUE. AND I THINK THAT I DO MOST OF MY WORK ON THE CAT IT'S PROBABLY EASIER TO WORK WITH. THE DOG IS -- WE HAVE OVER -- MOST OF THE LYSOSOMAL STORAGE DISEASES IDENTIFIED IN DOGS, MOST THE BRAIN TUMORS, CERTAINLY IN PEOPLE, IDENTIFIED IN DOGS, EPILEPSY IDENTIFIED IN DOGS, ALL OF THESE ARE VERY, VERY -- WHEN YOU'RE CREATING A NEW DISEASE YOU GET THE CHANCE OF CREATING SOMETHING THAT LOOKS DIFFERENT PHENOTYPEICALLY THAN THE DISEASE IN HUMANS, THE DOGS HAVE BEEN PICKED BECAUSE THEY HAVE A PHENOTYPE SIMILAR TO THE DISEASE IN HUMANS. AND THAT'S HOW THE MUTATION THEN GETS IDENTIFIED AND YOU GO -- IT'S A BACKWARDS CHALLENGE.& SO THEY ARE GOING TO BE MUCH MORE SIMILAR TO THE HUMAN DISEASES JUST BASED ON THE FACT THAT THAT'S HOW THEY WERE IDENTIFIED. I BRING THAT UP TOO AS A MODEL. >> FOR MODELING THE IMMUNE RESPONSES, IN GENERAL, I FAVOR THE LARGE ANIMALS. THEY SEEM TO HAVE AN IMMUNE RESPONSE CLOSER TO WHAT YOU MIGHT EXPECT TO SEE IN THE CLINIC. I THINK THE MODEL THAT I THINK MANY PEOPLE HAVE CAST OUT ON IS THE MOUSE MODEL. IT SEEMS TO HAVE VERY LITTLE IMMUNE RESPONSES TO THE AAV CAPSID, VERY LITTLE IMMUNE RESPONSE TO TRANSGENE. THE MOST STRIKING EXAMPLE BEING ALMOST EVERYONE CAN EXPRESS GFP, IN MICE, FOR A VERY LONG TIME. PUT THAT INTO DOGS, MONKEYS, WITHIN A WEEK THE GFP ITSELF IS INFLAMMATORY. I THINK IT'S CRITICAL AT LEAST TO MOVE PAST MICE AS THE VERY FIRST STEP, SO THAT WE CAN MODEL ACCURATELY. >> I WANT TO SAY A WORD IN FAVOR OF SHEEP. THEY ARE FLUFFY, CUDDLY, THEY DON'T BITE AS OFTEN AS DOGS. SERIOUSLY, I THINK THE BOTTOM LINE IS AN OUTBRED ANIMAL IS USEFUL BECAUSE THEY ARE NOT SO IMMUNOLOGICALLY NAIVE. AND I THINK THAT'S PROBABLY ONE OF THE REASONS WHY LARGE ANIMAL MODELS GIVE YOU BETTER MODELS THAN RODENTS. AND RODENTS IN GENERAL. WHEN WE LOOK IN SHEEP FROM THE LIMITED WORK THAT WE'VE HAD IN SHEEP FROM INTERPARENCHYMAL INJECTIONS, WE SEE PRE-EXISTING ANTIBODIES IN SOME SHEEP, AND WE SEE ANTIBODIES RAISED AS A RESULT, THIS IS TO CAPSID, AS A RESULT OF THE TREATMENT. AND IT DOESN'T SEEM TO IMPACT VERY MUCH ON GENE EXPRESSION INTERESTINGLY. THERE'S CLEARLY A RESPONSE THERE BUT WE'VE ONLY LOOKED AT AN IgG RESPONSE, HAVEN'T LOOKED IN MORE DEPTH BECAUSE IT'S NOT EASY TO DO IT IN SHEEP. >> THE DOG MODEL, MIGHT BE BEST SHORT OF NON-HUMAN PRIMATE TO MODEL THE IMMUNE SYSTEM, THE GENERAL OBSERVATION, GOING TO THE IMMUNE SYSTEM IT DOES MISS THAT KEY ELEMENT OF THE POST ADMINISTRATION CTL RESPONSE, OBSERVED IN HUMANS, HEME A AND HEME B. WE CAN'T REALLY DO GENETIC MODEL -- OR ENGINEERING I SUPPOSE ON DOGS FOR OTHER REASONS. >> WE CAN DO CATS. YOU CAN DO DOGS, ONCE YOU UNDERSTAND THEIR REPRODUCTIVE CYCLE MORE. THAT'S THE PROBLEM. BECAUSE CATS ARE INDUCIBLE OVULATORS, DOGS HAVE CYCLES, IT REQUIRES MORE WORK BUT IS NOT IMPOSSIBLE, WE'LL HAVE THAT ANSWEREDDED IN FIVE YEARS I SUSPECT. THE IMMUNE SYSTEM ONE IS A FAIR ONE, THAT'S WHERE I THINK THAT THE IMMUNE SYSTEM BETWEEN -- IT'S GREAT TO HAVE IMMUNOLOGISTS TO DISCUSS THIS WITH BECAUSE I THINK THAT'S WHERE WE WAVE OUR HANDS AND GO, IT'S DIFFERENT, YEAH. >> CAN I -- I WANT TO COME BACK TO WHAT FEDERICO WAS SAYING AND PUSH IT A LITTLE BIT. SO, YOU'RE TALKING ABOUT THE RAT AS A GOOD MODEL OR HELPFUL MODEL FOR SEEING AN IMMUNE RESPONSE. I'M WONDERING IF THIS IS WITH A CLINICAL TRIAL IN MIND, YOU SEE THAT IT'S PREDICTIVE OF A RESPONSE IN HUMANS, I WANT TO PUSH AND FIGURE OUT WHERE YOU WERE GOING AS THE RAT AS A GOOD MODEL. >> IT'S ESSENTIALLY A MODEL WHERE IT'S NOT LIKE THE MOUSE THAT YOU CAN EXPRESS HUMAN PROTEIN AND GET AWAY BECAUSE YOU NEVER SEE A RESPONSE. IT'S A MODEL WHERE YOU ARE TO EXPRESS RAT PROTEIN, IT'S MORE STRINGENT, GENERALLY SPEAKING I MEAN OF COURSE IT MAY CHANGE FROM DISEASE TO DISEASE BUT A FEW MODELS THAT WE'VE MADE IN THE PAST AND I'VE SEEN WERE CHARACTERIZED BY SOME FEATURES OF DISEASES, NEURODEGENERATIVE -- NEUROMUSCULAR DISEASE WITH SIGNIFICANT INFLAMMATION PERHAPS IN THE MOUSE DOESN'T SEEM TO BE EITHER TOO PROMINENT OR TOO -- DOES NOT SEQUENCE ON GENE TRANSFER, IN RATS SEEMS TO BE WAY MORE PROMINENT. THAT'S WHY I SAY, IF IT'S A SITUATION WHERE I'D RATHER START MY -- OR TEST MY STRATEGY IN A CONTEXT OF MORE STRINGENT THAN CONTEXT THAT TAKES -- THAT IS WORKING. >> RIGHT. >> THE ADVANTAGE OF A LARGER SIZE ANIMAL, IT GOES TO THE SIZE OF THE ORGAN WHICH POSES ANOTHER CHALLENGE AGAIN, YOU WILL NOT TRY TO SEE HOW FAR YOU CAN REACH WITH YOUR ROUTE OF ADMINISTRATION, TO SEE HOW GOOD YOU TRANSDUCE THE TARGET ORGAN. BUT THEN IN THE RAT IT'S BEEN EASY TO MAKE AND CHALLENGING AT THE SAME TIME, WHEN IT COMES TO DEVELOPING GENE TRANSFER APPROACHES. >> BUT IS IT PREDICTIVE OF WHAT YOU'RE SEEING IN HUMANS? >> WELL, I HOPE. >> THE QUESTION HERE CAN THIS BE MODELED IN HUMANS -- I MEAN ANIMALS, AND IS THE RAT THE ANIMAL BASED ON YOUR EXPERIENCE. >> I'M TRYING TO THINK IF I HAVE THE ANSWER TO THAT. I THINK I DON'T BECAUSE I HAVEN'T SEEN THE HUMAN PART YET. >> SO FEDERICO, I NEED ASK YOU A QUESTION. SO ANOTHER WAY TO MODEL IMMUNE RESPONSE IN AAV MEDIATEDDED GENE DELIVERY IS USE EXTREME IMMUNOGENIC TRANSGENE SUCH AS OVA YOU THEN YOU CAN DO IT IN MOUSE. HOW INFORMATIVE THAT WOULD BE IF YOU CAN OVERCOME THE OVA IMMUNITY IN MOUSE, EQUIVALENCE TO SOMETHING IN LARGE ANIMAL, HUGE DIFFERENCE? >> I DON'T THINK SO SIMPLY BECAUSE YOU'RE MODELING YOUR -- I MEAN THE OVA IS A GREAT MODEL TO STUDY IMMUNOLOGY BUT I THINK AT THE SAME TIME YOU ARE NOT -- YOU'RE NOT EVALUATING THE DISEASE BACKGROUND, AND THE IMMUNOGENICITY OF A PROTEIN, ALBUMIN IS NOT THE SAME AS FACTOR 9, NOT THE SAME AS MUSCULAR DYSTROPHY, A PROTEIN IN A INTRACELLULAR MEMBRANE PROTEIN, DO IT IN THE DISEASE SETTING AND OFFICIAL PROTEIN. HAVING SAID THAT I LOVE THE ALBUMIN MODEL BECAUSE REAGENTS YOU HAVE WITH ALBUMIN MODEL ARE INCREDIBLE AND ARE DIFFICULT TO EQUAL. >> ANOTHER COMMENT I'D LIKE TO MAKE SINCE THE ISSUE OF ORGAN SIZE WAS BROUGHT UP, TALKING ABOUT HEMATOLOGY, BODY WEIGHT DIFFERENCE, THE BRAIN IS A DIFFERENT STORY. ADULT HUMAN BRAIN, 3,000 TIMES BIGGER THAN ADULT MOUSE BRAIN. AND VARIOUS DOMESTIC ANIMAL MODELS ARE IN BETWEEN. DOMESTIC ANIMAL MODELS HAVE -- SORRY, DOMESTIC ANIMAL MODELS HAVE CORTICAL STRUCTURES, ALSO VERY IMPORTANT, AT LEAST THE DISEASES THAT INVOLVE MENTAL RETARDATION, THERE ISN'T MUCH DIRECT EVIDENCE BUT IT'S PRESUMED THAT THE CORTEX MULTIPLE INVOLVED, SO TO GET CORRECTION OF THAT ANOTHER OPEN QUESTION, WE HAVE NO IDEA HOW MANY CORRECTION YOU NEED TO IMPROVE THINGS. BUT THE ORGAN SIZE IN BRAIN IS A DIFFERENT THING THAN OTHER ORGANS. >> YEAH, I'D LIKE TO BUILD ON THAT AND ECHO WHAT CHARLES WAS SAYING. SO WE HAVE ACCESS TO AN INCL SHEEP MADE AT THE ROSLYN INSTITUTE. I WAS INVOLVED IN EARLY DISCUSSIONS. I'M NOT AN IMMUNOLOGIST AND CAN'T SPEAK INTELLIGENTLY AT ALL ABOUT THAT. BUT THE CHOICE WAS BASICALLY WE'VE MADE THE MODEL, MOSTLY BECAUSE OF SIZE ISSUE. CAN YOU SCALE IT UP? AGAIN, GETS TO CHARLES' ISSUE OF WHY ARE YOU MAKING THE MODEL, ARE YOU MAKING IT TO STUDY IMMUNOLOGY OR ARE YOU AGAIN LOOKING FOR SCALE? OBVIOUSLY WE CHOSE SCALE. THE SHEEP WERE GOOD, BY THE WAY. IT WAS ONE OF THE OTHER PARTS OF THE DISCUSSION. THERE WERE SEVERAL OTHER NATURALLY OCCURRING SHEEP MODELS OF BATTEN DISEASE, THEY MODELED THE HUMAN CONDITION VERY CLOSELY. AND IN FACT INCL SHEEP LOOKS JUST LIKE THE HUMAN DISEASE AS WELL. AGAIN, IMMUNOLOGY, MAY NOT BE THAT USEFUL. BUT MODELS THE HUMAN CONDITION AND, AGAIN, WE NOW HAVE SCALE >> THAT BRINGS US TO DISTANCES INVOLVED, TARGET TISSUES, TRANSSECTION IN SMALL ANIMALS VERSUS LARGE ANIMALS, MILLIMETERS TO CENTIMETERS OR MORE, DISTRIBUTION IS DIFFERENT. THANK YOU FOR BRINGING US THERE. I WENT HEAVY ON THE SLIDES SO I'LL ADVANCE ON THE NEXT -- MAYBE NOT. THANK YOU. I'M JUST GETTING THE NUDGE. KEEP THE CONVERSATION MOVING FORWARD. OKAY, CENTRAL NERVOUS SYSTEM, PERIPHERAL, AUTONOMIC, MANIFESTATION, IT'S CONTINUOUSLY OCCURRING IN MULTIPLE DISEASES, SOMETHING WE HAVE TO TALK ABOUT. NEW NEUROLOGIC PHENOTYPES, WHAT ARE WE NOT EFFECTIVELY TREATING, HAVE WE IDENTIFIED THAT? WE HAVE TOOLS TO DO THAT IN LARGE ANIMAL MODELS, SO MAYBE TALK ABOUT WHAT WE SEE IN MICE. AND THEN LIKE ANCILLARY FINDINGS, HOW WE CAN TEASE THAT APART IN LARGE ANIMALS, TRACK GENE EXPRESSION, WE TALKED ABOUT THAT ALREADY. WHAT IS HAPPENING IN CERTAIN NEURONAL POPULATIONS, ESPECIALLY MYELINATION, WE'RE SEEING CORRECTION, DEFINITELY IN PRE-CLINICAL WORK AS WELL. MAYBE TALK ABOUT THAT A LITTLE BIT. ANYBODY WANT TO COMMENT ON PERIPHERAL DISEASE IN LARGE ANIMAL MODELS? >> IT GOES BACK TO THE, FOR ME, BACK TO THE DISTRIBUTION ISSUE BECAUSE IF YOU'RE GIVING AN INTRATHECAL GENE THERAPY, JUST AS AGAIN WE SEE WITH SMALL MOLECULE THERAPIES, THE BRAIN ACTS LIKE A SPONGE WHERE THE OUTSIDE SEES IT AND THE INSIDE DOESN'T SEE IT. YOU'VE GOT HUGE AREAS THAT YOU'RE JUST NOT GETTING. THERE'S NO WAY THAT WE'RE GETTING ANY VECTOR IF IT'S NOT UTILIZING THE CIRCULATORY SYSTEM. AND SO I THINK THAT THOSE QUESTIONS ARE PRIMARILY ANSWERED IN THE LARGE ANIMAL MODELS WHERE WE'RE TRYING TO GET INTO DEEPER AREAS. THIS QUESTION OF WHETHER YOU WANT TO GIVE LUMBAR INTRATHECAL,ER CEREBELLAR, NOTHING WILL GET THE DEEP AREAS, THE CAUDATE, DEEP THYLAMIC NUCLEI, PRE-CLINICAL MODELS ARE PRIMED FOR LOOKING AT METHODS, WHETHER WE'VE TALKED ABOUT ARTERIAL, TRYING TO GET THESE VECTORS TO PARTS OF THE BRAIN THAT YOU CAN EASILY GET TO IN A MOUSE, BUT YOU JUST CAN'T COME CLOSE TO IN A DOG OR CAT. I WILL -- THIS WILL BE ONE -- THIS IS AN NIH-SPONSORED THING I WILL REMIND PEOPLE WE RUN A P 40 PROBLEM WHICH COLLECTS THESE DOGS AND CATS, NATURALLY OCCURRING DISEASE, FOR THIS AUDIENCE AND OTHERS AND MAKE THEM AVAILABLE TO PEOPLE AND DO STUFF. I THINK FOR THOSE WHO HAVE QUESTIONS ON IMMUNOLOGY, FOR THOSE WHO HAVE QUESTIONS ON NEW METHODS TO TRY TO GET TO OTHER BRAIN STRUCTURES, THOSE ANIMALS ARE WAITING FOR SOMEBODY TO DO OUTSIDE OF THE WORK THAT I DO THAT HEATHER DOES, THAT JOHN DOES, OTHERS. I WILL REMIND PEOPLE AGAINS THAT AVAILABILITY TO YOU, NIH-SPONSORED, AND I THINK THAT IT'S REAL. I BELIEVE WHAT HEATHER -- HEATHER IS POINTING AT WE'RE SEEING PERIPHERAL NERVOUS SYSTEM CHANGES, THAT ARE TOTALLY UNEXPECTED, WITH INTRATHECAL DELIVERY. AND TO TRY TO UNDERSTAND WHAT'S GOING ON, DOES FEEDBACK TO MYELINATION ISSUE IS REALLY AN IMPORTANT -- WHEN YOU'RE INJECTING CEREBELLAR MEDULLARY CISTERN SEEING TRANSDUCTION OF SCHWANN CELLS IN THE TIBIAL NERVE AS FAR FROM THE BRAIN AS YOU CAN POSSIBLY GET HOW IS IT GETTING THERE? WHAT'S IT DOING? AND I BRING THOSE UP BECAUSE I THINK THEY STILL ARE THINGS THAT WE'RE SEEING IN THE DOG AND CAT, THAT YOU CAN SIT THERE AND GO, WOW, IF WE SAW IT IN THE MOUSE THIS BIG, NOT SO WOW. HERE, THIS IS A WOW. I THINK SHARING THAT INFORMATION AND TRYING TO FIGURE OUT WITH PEOPLE WHO HAVE DIFFERENT INTERESTS MAY HELP UNDERSTAND WHAT'S GOING ON AND THEY ARE NOT BEING LOOKED AT RIGHT NOW IN PEOPLE. BUT I THINK THAT WE CAN NECROPSY EVERY SINGLE ANIMAL AND GET THAT DATA SO I THINK THAT -- I'LL LEAVE IT AT THAT. >> SO I'M HEARING A STRATEGY IS TO TAKE BETTER ADVANTAGE OF THE U54, THAT IS HOUSED AT THE PENN VET SCHOOL. AND YOU KNOW, TO UNDERSTAND BETTER, BEAUTIFUL WEBSITE, CAN YOU UNDERSTAND WHAT MODELS ARE THERE. ONE OF THE CONSIDERATIONS AS YOU THINK ABOUT GRANTS, USING THESE MODELS, HOW LONG IT TAKES TO GET ENOUGH ANIMALS TO DO AN EXPERIMENT. AND SO REALLY PRIORITIZING THE KINDS OF EXPERIMENTS THAT YOU WANT TO DO IN THESE ANIMALS IN A TIMELY WAY SO IT DOESN'T TAKE FOUR TO FIVE YEARS TO GET ENOUGH ANIMALS TO ACTUALLY DO THE STUDIES AND WHAT ARE THOSE HIGH PRIORITY EXPERIMENTS, AND I WONDER IF YOU CAN COMMENT A LITTLE ABOUT THAT, AND BECAUSE I DO THINK IT'S REALLY IMPORTANT RESOURCE AND WE OUGHT TO TAKE BETTER THANK AS A COMMUNITY. IT'S ALMOST -- A LITTLE BIT AKIN TO WHAT JUDE WAS SAYING EARLIER, WE HAVE STANDARD METHODS FOR GENERATING VIRUSES THAT CAN BE DISTRICTED TO -- DISTRIBUTED TO THE COMMUNITY TO ASK IMPORTANT QUESTIONS. IT'S CRITICALLY RELEVANT TO UNDERSTAND WHAT'S HAPPENING TO THE PHENOTYPE IN THE MODELS THAT MAY HELP US BE READY FOR THOSE KINDS OF THINGS IN KIDS AND ADULTS WITH THESE NEUROLOGIC DISEASES. SO COULD YOU JUST GIVE US A LITTLE BIT MORE INFORMATION ON THE SCOPE OF ANIMALS THAT WOULD BE AVAILABLE, IF TEN OF US CONTACTED YOU NEXT WEEK, TO SET UP STUDIES IN -- ARE THESE DOGS ON THE GROUND, OR CATS ON THE GROUND? >> VERY BRIEFLY, WE KEEP EVERYTHING -- WE KEEP SPERM AND EGGS FROZEN, IF SOMEBODY WANTS A NEW MODEL, WE HAVEN'T BEEN BREEDING FOR, WE CAN BREED THEM UP, THAT'S GOING TO TAKE APPROXIMATELY A YEAR BEFORE YOU ARE GOING TO HAVE A HETEROZYGOTE POPULATION TO BREED AND YOU'LL HAVE THE GESTATION TIME, ANOTHER TWO MONTHS, BECAUSE MOST OF THESE ANIMALS YOU'RE STUDYING TO SIX MONTHS OF AGE, STUDY IS TYPICALLY GOING TO TAKE A YEAR AND A HALF AT MINIMUM. THAT'S PROVIDED WE CAN PRODUCE ENOUGH ANIMALS FOR YOU RIGHT NOW BECAUSE FUNDING HAS BEEN THERE, THE -- WE HAVE THREE ANIMAL MODELS, NIEMANN PICK AND ANIMALSADOSIS, AND PS 1s, WHAT USUALLY HAPPENS IF SOMEBODY CALLS US AND SAYS WE HAVE FNAD MITAFUSIN DEFICIENCY, WE COULD GET THAT MODEL UP AND RUNNING IN APPROXIMATELY A YEAR AND A HALF AND WE'LL PROVIDE YOU WITHIN TWO DAYS A BUDGET AS WELL AS TIMELINE OF HOW LONG IT WILL TAKE US TO DO THAT. AND THAT BECOMES ABOUT APPROXIMATELY IF YOU'RE LOOKING AT A SIX-MONTH STUDY TO SIX-MONTH LIVING, IT WILL TAKE THREE YEARS. BUT YOU'LL GET REPEATED CSF SAMPLES EVERY WEEK IF YOU'D LIKE, BLOOD SAMPLES EVERY WEEK, URINE SAMPLES EVERY WEEK, ELECTRO DIAGNOSTICS, MRI, PET SCANNING, WE CAN DO ALL OF THE SAME THINGS THAT ARE DONE FOR PEOPLE. GENERALLY, WHAT'S SPEAKING, YOU'RE RIGHT, THERE'S ALWAYS LIMITATION OF CAGE SPACE. I HAVE 300 ANIMALS IN CAGES, AND THAT'S APPROACHING 90% CAPACITY RIGHT NOW. BUT LIKE ANYTHING ELSE WHEN THERE'S A NEED, THEN IF SUDDENLY WE HAD A BIG PUSH TO DO 700, 800 ANIMALS, IF YOU HAVE THE EVIDENCE ON PAPER AND CONTRACT, THAT CHANGES THINGS FOR HOW WE CAN GO OUT AND SPEND MONEY. I DO BRING IT UP BECAUSE I THINK THAT FOR WHAT PEOPLE HAVE TALKED ABOUT, I'VE NEVER -- IMMUNOLOGICAL ASPECTS, I'M A NEUROLOGIST, A NOVICE, NOTHING I SPENT A LOT OF TIME ON BUT FOR CONNECTIVITY ISSUES FOR LOOKING AT SLAB SECTIONS THAT ARE TAKEN AND STUDIED IN VIVO AFTERWARDS, YOU KNOW, EX VIVO, AFTERWARDS, ALL THOSE HAVE BEEN DONE AND ARE AVAILABLE AND I THINK THAT I STILL THINK, FOR ME, THE BIGGEST QUESTIONS THAT WE DEAL WITH WHEN WE LOOK AT THE LARGE ANIMALS IS HOW WHEN YOU'RE INJECTING A VECTOR IN THE CEREBELLAR SYSTEM HOW ARE YOU GET STUFF OUT OF THE CORTEX AND DEEPER AND THERE'S A LOT OF EXPERTISE IN THIS ROOM. WE HAVE ABILITY FOR SLOW INFUSIONS, FAST INFUSIONS, INTRACAROTID INFUSIONS, ALL OF THOSE. I DON'T DO THOSE BUT WE HAVE 300 ANIMALS THAT CAN BE -- THAT ARE LIVING NOW, ON THE FLOOR, DEPENDING, THEY CAN BE NORMAL ANIMALS VERSUS DISEASED ANIMALS. AND I THINK REALLY THE BENEFIT OF THIS NIH-SPONSORED CENTER IS THAT MOST OF THEM ARE DISEASED, AND I STILL THINK THAT THAT IS THE MODEL SYSTEM TO BE WORKING IN IF WE CAN PRODUCE 300 NIEMANN PICK C ANIMALS FOR STUDY IN A FIVE-YEAR PERIOD WE CAN GET A LOT MORE INFORMATION ON IMMUNOLOGY, DISTRIBUTIONAL, THAT WOULD BE MORE USEFUL THAN WE COULD GET IN NORMAL CATS. THAT'S THE STRENGTH. >> I WOULD AGREE. >> I'D LIKE TO ADD ONE COMMENT. THESE ARE MENDELIAN INHERITANCES FOR THE MOST PART. AND OVER TIME, WITH DISEASES OFTENTIMES YOU GET LESS THAN 25% AFFECTED BUT ON A GIVEN LITTER PRODUCED, IT'S HIGHLY VARIABLE. SO ACCUMULATIVELY AND AFFECTED ANIMALS OFTEN DON'T THRIVE VERY WELL IN NEONATAL PERIODS. SO TO GET ENOUGH ANIMALS TO ENTER INTO A STUDY AND HAVE FOR THE FULL STUDY TAKES ACTUALLY QUITE A BIT MORE THAN JUST 25% AND DOING YOUR CALCULATION, SOMETHING TO BE AWARE OF, CAN STILL BE DONE. VARIABILITY I DON'T THINK WE CAN EXPLAIN MOST OF THE TIME. IT'S JUST ANIMAL BIOLOGY. SOMETIMES WE GET A LITTER WITH THREE OUT OF FIVE, SOMETIMES THREE LITTERS IN A ROW WITH NOTHING. >> ONE THING WE HAVEN'T HEARD IN OUR DISCUSSION YET IS STANDARDIZATION. FOR EXAMPLE, STANDARDIZATION OF ASSAYS AND THESE DIFFERENT ANIMAL MODELS. IF YOU HAVE DIFFERENT LABS WORKING ON DIFFERENT SPECIES WHAT ASSAYS COULD THEY BE USING SUCH THAT FINDINGS COULD BE TRANSLATABLE TO INFORM RESEARCH IN OTHER SPECIES? >> SO THAT'S EXACTLY WHAT I WAS TRYING TO JUMP IN ON IS WHEREVER POSSIBLE WHAT WE TRIED TO DO IF WE'RE GOING TO DO NON-HUMAN PRIMATE, TRY TO DO A MOUSE STUDY IN PARALLEL, SAME CAPSID, PROMOTER, PREP, TRYING TO NORMALIZE THE DOSE AS MUCH AS POSSIBLE. THAT'S IN WILDTYPE ANIMALS SO WE CAN -- IF YOU CAN ANSWER THE BASIC QUESTION IS THE BIODISTRIBUTION THE SAME IN A MOUSE AS IT IS IN A DOG, AS IT IS IN A MONKEY, THEN YOU LIMIT TO THERE'S SPECIFIC QUESTIONS WHERE THE LARGE ANIMAL MODEL IS USEFUL, BUT THERE'S A LOT OF QUESTIONS YOU CAN ANSWER IN A MOUSE BECAUSE IT'S JUST NOT PRACTICAL TO DO EVERY STUDY IN A LARGE ANIMAL. SO FROM MY STANDPOINT, THE FIRST PRIORITY IS JUST BRIDGING THOSE DIFFERENT SPECIES AND GETTING THAT HARD DATA, SO WE KNOW WHEN WE ARE OKAY IN A MOUSE AND WHEN WE NEED A LARGE ANIMAL. >> AND I'M GOING TO ASK YOU WHAT POINT DO YOU MAKE THAT DECISION WHEN YOU SAY, OKAY, WE LEARNED EVERYTHING WE CAN LEARN FROM A MOUSE, BECAUSE YOU'VE ALWAYS BEEN -- I REMEMBER ONE TIME I GAVE A TALK, YOU WERE LIKE I JUST SHOWED A PRESENTATION WITH 400 MICE, YOU SHOW ONE SHEEP. WHAT POINT DO YOU DECIDE, WHEN DO YOU, IN YOUR OPINION, WHERE DOES THAT LINE GET DRAWN? >> I MEAN, CRAB A, I'M WORKING WITH CHARLES ON THAT. WE'VE DONE STUDIES, REALLY EXTENSIVE STUDIES IN THE MOUSE MODEL. AND WE'VE DONE NOW PRETTY EXTENSIVE STUDIES IN THE DOG MODEL. WE CAN SEE WHAT THE SIMILARITIES ARE AND WHAT THE DIFFERENCES ARE. IF WE'RE SEEING THE DOG AND THE MICE ARE IN AGREEMENT, OKAY, THEN IT'S FINE DOING THE STUDY IN A MOUSE. IF YOU VALIDATE THAT FOR ONE DISEASE, I MEAN, YOU CAN'T ALWAYS -- EVERYTHING ISN'T APPLES AND APPLES ALWAYS, BUT YOU MAKE THE BEST GUESS THAT YOU CAN. AND IF YOU HAVE THAT BRIDGING DATA IN ONE CONTEXT, YOU CAN EXTRAPOLATE THAT TO OTHER SITUATIONS. AGAIN, NOT 100% BUT MAKING AN EDUCATED GUESS. >> QUICK QUESTION TO STEVEN. YOU MENTIONED WILDTYPE MICE. IS THERE ROOM FOR HUMANIZED MICE AS WELL? USE OF HUMANIZED MICE? >> OF COURSE. JUST DEPENDS ON THE RESEARCH QUESTION. >> SO I THINK I'D LIKE THE LARGE ANIMAL MODELS FOR PREDICTING THE KIND OF PHENOTYPES YOU MIGHT SEE FROM A THERAPY BECAUSE THESE ANIMALS HAVE DISEASE. YOU HAVE ABILITY TO KEEP VECTOR IN THE ANIMAL FOR YEARS AS OPPOSED TO MONTHS. WHICH YOU CANNOT DO IN A MOUSE. I WONDER IF THERE'S A NEED FOR TOOLS IN THE LARGE ANIMALS. A LOT OF REASONS WE USE MICE IS NOT JUST BECAUSE OF EXPENSE BUT IT'S ALSO BECAUSE ALL OF THE TOOLS THAT YOU CAN USE TO UNDERSTAND THE IMMUNE RESPONSES, ALL THE ANTIBODIES, ET CETERA, ET CETERA, AND I DON'T KNOW WHERE THE IMMUNE FIELD STANDS IN TERMS OF DEVELOPING REAGENTS FOR OTHER SPECIES FOR STUDYING THESE SORTS OF SYSTEMS BUT I THINK IT WOULD BE REALLY IMPORTANT TO HAVE A HANDLE AND THINK ABOUT THAT BECAUSE IT COULD HELP US AS A FIELD. >> I AGREE. THAT'S THE THING I WAS ALLUDED TO EARLIER, DO YOU WE KNOW THESE RESPONSES AREN'T HAPPENING BECAUSE WE CAN'T ASK, YOU DON'T TO ASK QUESTIONS IN SHEEP, MAYBE YOU DO FOR DOGS BUT IT'S NOT THERE. IF YOU DON'T ASK, YOU DON'T KNOW. IF YOU DON'T LOOK, YOU WON'T SEE IT. IT'S POSSIBLE THAT IT'S BEEN THERE THE WHOLE TIME. I'M JUST SAYING. >> MOVING ON, SYSTEMIC ADMINISTRATION AND REPEAT ADMINISTRATION, THIS IS A BIG QUESTION IN THE FIELD. I THINK EVERYBODY SHOULD RAISE THEIR HAND. DO YOU THINK THAT WE'LL EVER BE ABLE TO ACHIEVE REPEATED ADMINISTRATION OF AAV SYSTEMICALLY IN PATIENTS? LET'S SEE WHO THINKS YES? OKAY. WHO THINKS NO? >> (INAUDIBLE). >> RIGHT. BUT SOMEBODY SHOULD TAKE OVER. YEAH, GO AHEAD. EVEN WITH IMMUNE SUPPRESSION. >> WOULD THAT INCLUDE COMBINATORIAL APPROACHES? COULD YOU EASILY IMAGINE AT THE TIME OF ADMINISTRATION TO TREAT SEVERAL COMPARTMENTS AT THE SAME TIME WHILE YOU HAVE THE IMMUNE PRIVILEGE TO DO THAT. >> THE OBVIOUS RESPONSE -- >> GO AHEAD. >> IS THAT AN EX VIVO APPROACH WITH LENTI IS CLEARLY THE WAY FORWARD FOR MULTI-SYSTEM DISEASES. IT'S TRUE. AGAIN, IT OBVIOUSLY AS ITS OWN LIMITATIONS, YOU HAVE TO GO IN VERY EARLY. AND POTENTIALLY YOU'VE GOT A MYELOABLATIVE REGIMEN, IT'S NOT GREAT EITHER. WHEN YOU LOOK AT SCENARIOS WHERE FOR EXAMPLE THAT HAS BEEN DROPPED AND REDUCED THE OUTCOME HAS BEEN MUCH LESS GOOD. EX VIVO ADMINISTRATION THE HOLY GRAIL IS TO GET RID OF CHEMOTHERAPY WHICH MIGHT BE DOABLE EVENTUALLY. THAT MAKES BIG CHALLENGES FOR GETTING INTO THE CNS BECAUSE WE RELY ON PART OF THAT CHEMOTHERAPEUTIC TREATMENT BUT THAT GIVES YOU BOTH YOUR SYSTEMIC AND CENTRAL ADMINISTRATION VERY NICELY AND YOU HAVE YOUR SCALEUP ALL IN ONE. >> ONE OF THE THINGS GENE THERAPY COMMUNITY HAS BEEN VERY GOOD AT IS CAR T CELL THERAPY. AND AT LEAST ONE OF THE STRATEGIES THAT I'M AWARE OF, NOT FROM MY TEAM BUT FROM OTHERS IS TO ESSENTIALLY DEVELOP CAR T CELLS THAT WILL DESTROY, YOU KNOW, SOME OF THE CELLS THAT ARE ESSENTIALLY EITHER CYTOTOXIC T CELLS OR NEUTRALIZING ANTIBODY CELLS THAT MAY BE CAUSING PROBLEM. WE COULD REALLY AFFORD TO DIVE DEEPER INTO THIS AND USE THE LARGE ANIMAL MODELS TO DEVELOP SOME TOOLS BUT ALSO THINK ABOUT EXPLORING THE SPACE MORE. YOU CAN EVEN MOVE AWAY FROM A VECTOR SYSTEM TO DEVELOP THE CAR T APPROACH, CRISPR EDITING EX VIVO AND EITHER AGAIN WE STILL HAVE THE TRANSPLANT ISSUES BUT IT MIGHT BE A WAY TO ALLOW FOR REIMMUNIZATION. WE CAN LEARN FROM POMPE COLLEAGUES, PATIENTS THAT DEVELOP IMMUNE RESPONSES TO RECOMBINANT PROTEIN BECAUSE THERE ARE WAYS TO MITIGATE THOSE REALLY DEBILITATING EFFECTS. >> YEAH, KRISHNANI USES A METHOTREXATE APPROACH TO INDUCE IN HER POMPE PATIENTS WHICH SHE SWEARS BY. SHE MOVED AWAY FROM RITUXIMAB, WHICH IS QUITE NICE, BUT IT MIGHT BE A DOSING ISSUE. MIGHT JUST NEED TO GO HIGH OTHER D OSING-- HIGHER ON DOSING. THEY ARE QUITE CRUDE TOOLS IF I'M HONEST. >> WE HAVE TO INCLUDE THE AAV ON THE IN VITRO SITE AS WELL. THERE ARE APPROACHES THERE. IT DOESN'T GET AROUND THE IMMUNE ABLATION AND THINGS LIKE THAT. REGARDING HEATHER'S QUESTION ABOUT READMINISTRATION, I KNOW THERE IS THE CONCEPT, DIFFERENT INDIVIDUALS HAVE DIFFERENT RESPONSES, IF YOU GO WITH ONE SEROTYPE, IF IT DOESN'T WORK WE CAN COME IN WITH ANOTHER SEROTYPE. MY GENERAL FEELING IS THAT'S PROBABLY NOT THE CASE BECAUSE OF THE CROSS-REACTIVITY BUT I'M NOT SURE IN THAT'S A BROADLY HELD VIEW. THERE ARE A COUPLE PAPERS OUT THERE, PREPARES ON BOTH SIDE. >> CARSTON, DANA, KEVIN, WE'RE ALL TALKING ABOUT AND AGREEING WITH YOU ON THIS. >> HI. JERRY LIPSHOOTS, UCLA. WE HAD DONE SOME WORK WITH ALICE TERTENTAL DELIVERING AAVs AND SAW THIS PATTERN THAT YOU DESCRIBED. I WONDER ABOUT THAT ALSO. AND THEN SOME OF YOU MAY OR MAY NOT BE AWARE OF THE WORK THAT SELECTED BIOSCIENCE HAD DONE USING LIPID NANOPARTICLE WITH RAPAMYCIN, SAME SEROTYPE COULD BE ADMINISTERED TO MICE, NOT SURE WHY THE COMPANY IS HAVING SUCH CHALLENGING STOCK PRICE IS LOW, I HAVE NO INSIDER INFORMATION BUT THERE'S TALK THEY COULD GO OUT OF BUSINESS. SEEMS LIKE THIS IS POTENTIALLY PARADIGM SHIFT IN WHAT COULD BE READMINISTRATION. >> WE PUBLISHED THE PAPER. >> RIGHT, YES. >> I THINK, OKAY, THE SETTING WHERE THE COMPANY IS IN THE CLINIC IS A CHALLENGING ONE, LIKE A CHRONIC ADMINISTRATION OF HIGHLY IMMUNOGENIC PROTEIN, I MEAN, THAT COULD BE PART OF THE TROUBLE THEY ARE HAVING. IN THE CONTEXT OF GENE THERAPY, THEY HAVEN'T TESTED YET. THEY ARE TRYING. I THINK THIS IS A POTENTIAL APPROACH TO BLOCK ANTIBODIES TO THE CAPSID ALLOWING FOR ADMINISTRATION, I THINK WHAT THIS TECHNOLOGY DOESN'T DO IS TO REVERSE PRIMING SYSTEM BECAUSE IT'S ACTING ON T CELLS, RIGHT, SO WHENEVER YOU WANT TO -- IF THERE'S PERSISTENT IMMUNITY YOU HAVE TO TARGET B CELLS. THE OTHER APPROACH CURRENTLY IN THE CLINIC IS THE ONE OF COMBINATION OF RAPAMYCIN, NOT NANOPARTICLES, AND RITUXIMAB IS BEING CONDUCTED BY THE UNIVERSITY OF FLORIDA, BARRY BYRNE, AN INTENSIVE REGIMEN SO WHEN WE THINK ABOUT THIS KIND OF APPROACH WE NEED TO THINK ABOUT HOW INTENSE THE REGIMEN, WHAT IS THE RISK/BENEFIT, RAPAMYCIN NANOPARTICLES SELECTED AS ADVANTAGE IN THEORY WORKS WITH SINGLE ADMINISTRATION AT THE TIME OF VECTOR INFUSION, SO IT'S ONE OF THE TECHNOLOGIES BEING EXPLORED. I THINK OVERALL THE CONCEPT IS PREVENTING ANTIBODIES TO AAV OR TO ANY PROTEIN REALLY WOULD BE EASIER, CONCEPTUALLY, THAN ERADICATING. THERE'S A PAPER THAT JUST CAME OUT WHERE THEY TESTED IN LARGE NUMBER OF MONKEYS, IMMUNOABSORPTION, AS A STRATEGY, A PAPER, WITH SOME SUCCESS. >> IMMUNOABSORPTION IN THE BLOOD TO REDUCE NEUTRALIZING ANTIBODIES, RIGHT, PLASMA PHORESIS. >> CASEY MAGUIRE DEVELOPED A METHOD WHERE YOU PUT AAVs INSIDE EXOSOMES, LESS MUTAGENIC AND HAVE A DIFFERENT TARGETING CAPACITY. THEY TARGET DIFFERENT CELL TYPES. HE HAS A COMPANY NOW CALLED CHAMELEON. >> WE TESTED THAT TOO, NOT CHAMELEON TECHNOLOGY. BUT THEY ARE SHOWN TO BE VERY EFFECTIVE IN TRANSDUCING LIVER AND DO OFFER SOME RESISTANCE TO NEUTRALIZING ANTIBODIES. I OUR HANDS, NOT HIGH TITERS. >> SO INTRATHECAL ADMINISTRATION IN THE FACE OF NEUTRALIZING ANTIBODIES, THAT'S A THOUGHT. THERE'S SOME DATA OUT THERE TO SUGGEST YOU CAN DO IT, SOME SAYS YOU CAN'T. WHAT DO YOU THINK ABOUT THAT STRATEGY FOR ADULT PATIENTS THAT ARE, YOU KNOW, JUST FULL OF NEUTRALIZING ANTIBODIES, AAV IS OUT THERE, WHAT ARE YOUR THOUGHTS ABOUT THAT? >> SO WE'VE BEEN DOING IT. SO THE PRESENCE OF NEUTRALIZING ANTIBODIES WAS NOT EXCLUSION CRITERIA. NONE OF THE PATIENTS ARE BASELINE NEUTRALIZING NEUTRALIZING ANTIBODIES. IN THOSE THAT DID THE REAWAKENING OF THE AAV IMMUNE RESPONSE WAS QUICKER THAN THE NEW IMMUNE RESPONSE IN THE NEGATIVE PATIENTS, BUT THE OUTCOME, LEVELS OF NEUTRALIZING ANTIBODIES WAS THE SAME. WE HAVEN'T SEEN ANY DIFFERENCE OTHER THAN THAT. >> I'M GOING TO ADD ONTO THAT. WE PRESENTED THIS, WE HAD AUTOPSY DATA FROM ONE PATIENT THAT WAS SERUM POSITIVE AT BASELINE, 1 TO 300 TITER, WE HAD GENE TRANSFER IN THAT PATIENT THAT MATCHED WHAT WE WOULD HAVE EXPECTED FROM OUR PRE-CLINICAL STUDIES. THAT WAS INTRATHECAL. NOW, THE PRIVILEGE WALL GENE TRANSFER BECAUSE A LOT OF AAV9 ESCAPES TO PERIPHERY AFTER INTRATHECAL, IT WAS MINIMAL IN THE PATIENT. >> HOW COULD YOU CIRCUMVENT, DO YOU THINK YOU COULD LOWER PLASMA PHORESIS AND MAKE AN EFFORT TO HELP WITH TRANSDUCTION OR DO YOU THINK -- >> YOU PROBABLY HAVE THE SAME ISSUES AS IF YOU'RE GOING TO PUT IN I.V I GUESS THAT'S AN OPEN QUESTION. IF IT'S SECRETD ENZYME LIKE LYSOSOMAL STORAGE DISEASE WITH SECRETED ENZYME WHETHER IF YOU'RE GOING TO PRODUCE ENOUGH ENZYME IN THE CNS THAT WOULD GET OUT AND TREAT SYSTEMIC BECAUSE WHAT WE'VE SEEN IS TO GET THE LEVELS OF TRANSGENE HIGH ENOUGH IN THE CNS A LOT OF TIMES WE'RE SEEING 100 OR 1,000 TIMES NORMAL ENZYME LEVEL IN THE BLOOD. >> OKAY. SO, WE TALKED ABOUT TOXICITY IN LARGE ANIMAL MODELS. >> THE UNANSWERED QUESTION, WHAT ABOUT READMINISTRATION INTRATHECALLY AFTER YOU ADMINISTERED ONCE INTRATHECAL. WE HAVEN'T CHECKED. I DON'T KNOW WHETHER THERE'S PRE-CLINICAL DATA TO SEE WE SEE NEUTRALIZING ANTIBODIES IN CNS AFTER TREATMENT. >> HOW HIGH? >> I CAN SHOW YOU. >> WE'RE MOVING TO MANUFACTURING. THANK YOU. >> WAIT, WAIT. LET ME ADDRESS CARSON'S QUESTION. UNFORTUNATELY THERE'S NO REAL GOOD WAY TO TEST IT BECAUSE IT'S GOING TO TAKE, I DON'T KNOW, MAYBE NINE OR TEN YEARS BEFORE YOU WOULD READMINISTER IT. AND YOU DON'T REALLY MIRROR READMINISTRATION EXPERIMENT IN A MODEL THAT YOU WOULD DO IN A PATIENT, IF YOU DO IT AT THREE MONTHS LATER OR SIX MONTHS LATER OR A YEAR LATER. IT'S NOT THE SAME >> WE'VE DONE IT TWO MONTHS LATER IN A RAT, WE HAVEN'T PUBLISHED THAT BUT IT BLOCKED A LOT OF IT. >> I THINK OUR LAST SESSION FOR THE AFTERNOON, IF I UNDERSTAND, MANUFACTURING, NUTS AND BOLTS. I'VE GOT TWO PAGES HERE ON THE ORIGINAL FONT IT WAS ON ONE SLIDE BUT IT WAS MADE VISIBLE SO WE'RE ON TWO PAGES. AND I THINK THEY ARE WELL RECOGNIZED IN THE CELL AND GENE THERAPY SPACE. I MIGHT PREFACE REMARKS BY REMARKS MADE BY THE PREVIOUS FDA COMMISSIONER, SCOTT GOTTLIEB, LAST YEAR IN 2018 AT THE AARM MEETING ABOUT HOW REALLY IN SO MANY THERAPEUTIC AREAS 80% OF CHALLENGES FOR DRUG DEVELOPMENT AND FDA REVIEW ARE CLINICAL, MAYBE 20% ON MANUFACTURING, CMC SIDE, COMPLETELY REVERSED FOR CELL AND GENE THERAPY AT THIS JUNCTURE BECAUSE THEY ARE GOING THROUGH A MATURATION OF THE TECHNOLOGIES TO USE. I'M NOT SURE, IT'S SEMI QUANTITATIVE, IT'S A BIG CHALLENGE I THINK. THESE ARE PROVIDED BY THE NINDS AS THE KEY POINTS. WE'VE GOT CAPACITY ISSUES, CAN WE MAKE ENOUGH IN A TIMELY MANNER, SO MANY PEOPLE ARE DEALING WITH 12 MONTHS FOR THIS STAGE, 12 MONTHS FOR THAT STAGE, FOR 24 MONTHS TO HAVE SOME MATERIAL AVAILABLE, IT'S TOUGH AND EXPENSIVE. THE SECOND POINT, COMPLEXITY AND DIVERSITY OF PRODUCTION SYSTEMS AVAILABLE. WE HAVE TWO OR THREE MAKE UPSTREAM PRODUCTION SYSTEMS, CELL CULTURE SYSTEMS THAT PRODUCE VECTOR BEFORE THEY ARE PURIFIED, MADE MORE STREAMLINED, PURIFICATION. TRANSGENT TRANSFECTION, I'M THINKING OF AAV AND LENTI, SIMILAR COMPLEXITY, COMPLEX COMPONENTS, USE OF HIGH-QUALITY PLASMID OR RECOMBINANT VIRUSES OR HERPES SIMPLEX VIRUSES AS STARTING MATERIALS. THERE'S COMPLEXITY, CAN WE TRIM THEN DOWN. PRODUCT QUALITY ATTRIBUTES, A SPECIAL AREA OF INTEREST FOR ME, WHICH ARE THE MOST CRITICAL FOR SAFETY, WE NEED TO MAKE THE STUFF, HAVE IT CLEAN AND MOVE ON? OR ARE THERE SOME THINGS WE DON'T UNDERSTAND YET? I THINK WE TALKED A LITTLE BIT ABOUT IT IN TERMS OF THE FIRST CHEMISTRY, VECTOR DESIGN, ARE WE PUTTING COMPONENTS WITHIN OUR EXPRESSION CASSETTES, MAKING THEM IN WAYS THAT ARE PERHAPS INADVERTENTLY POTENTIALLY PRO IMMUNOGENETIC. THE SECOND PAGE, BATCH PRODUCTION FOR ULTRA ORPHAN DISEASE, BEV MENTIONED THIS EARLY ON. 30 PATIENTS WITH A MODEST DOSE IN PRINCIPLE YOU COULD MAKE ONE GOOD BATCH AND TREAT THE WORLD AND IF THERE'S ONE COMING UP EVERY YEAR OR TWO YEARS, I DON'T KNOW, THIS IS AN EXTREME EXAMPLE. THIS COULD GO FOR A LONG TIME. SOMEBODY WILL HAVE TO MAKE ANOTHER BATCH AT SOME POINT, THEY WILL HAVE TO REMEMBER HOW YOU MADE THE FIRST BATCH, ONE OF THE CHALLENGES. HAVING TO GO THROUGH THE WHOLE RIGOROUS PROCESS OF VALIDATION, QUALIFICATION, THAT TAKES A LOT OF TIME, A LOT OF EFFORT, FOCUSED AT QUALITY BUT ARE THERE WAYS TO STREAMLINE THIS AND MAKE A PLATFORM. FEASIBILITY OF STANDARDIZED PROCESSES AND PROCEDURES, ACROSS THE BOARD THIS IS IMPORTANT. FOUR OF THE FIVE COMPONENTS, WE MIGHT HAVE 20 DISEASES, FOR CMC COMMON, MAYBE ONLY ONE COMPONENTS IS CHANGED. FOR EXAMPLE IDENTITY OF TRANSGENE CAN WE USE OTHER DATA CMC-WISE, MOVE FORWARD TO ACCELERATE AVAILABILITY OF THAT MATERIAL FOR CLINICAL TRIALS. SO I'LL SIT DOWN. >> KRYS IS GONE. SO, I WANT TO COME BACK TO THIS ONE BATCH, YOU KNOW, QUESTION THAT YOU RAISED, THAT WAS RAISED EARLIER, SORT OF OVERVIEW OF THE CHALLENGES. AND I WONDER IF THERESA, MAYBE YOU WANT TO COMMENT. I DON'T MEAN TO PUT YOU ON THE SPOT. IF YOU THINK ABOUT THIS, YOU WON'T NEED ANOTHER BATCH FOR 10, 20, 30 YEARS. EVEN IF YOU COULD REPEAT HOW THE BATCH WAS MADE, IN 2019, THE KIND OF EQUIPMENT YOU HAVE IN 2035 MAY BE COMPLETELY DIFFERENT SO YOU WOULD NOT EVEN REPEAT THAT BATCH PROCESS IN THE SAME WAY OR YOU MAY HAVE A COMPLETELY NEW SYSTEM TO MOVE FORWARD FOR. FOR ULTRA RARE DISORDERS WHERE WE CAN GET BY WITH ONE MANUFACTURING PROCESS FOR IND-ENABLING STUDIES, PATIENTS FOR THE FORESEEABLE AGAIN FUTURE, FOR DECADES, IS THERE A WAY THAT WE CAN WORK TOGETHER TO THINK ABOUT JUST MOVING THIS FORWARD SO THE PATIENTS CAN HAVE ACCESS TO THIS MATERIAL, THESE ARE NOT -- YOU KNOW, REAGENTS THAT DRUG COMPANIES ARE GOING TO WANT TO DEVELOP. THEY ARE NOT COMMERCIALLY VIABLE FROM ANYONE'S PERSPECTIVE BUT YET THIS IS AN UNMET NEED IN THE RARE DISEASE COMMUNITY. AND I THINK WE AS A FIELD ARE REALLY PASSIONATE ABOUT THIS, IN THE UNNECESSARY COST OF MOVING FORWARD. SO I SEE AS A CHALLENGE TO US AS A COMMUNITY TOGETHER WITH NIH AND TOGETHER WITH THE FDA IS HOW DO WE SOLVE THIS PROBLEM? >> SO, TO SPEAK TOWARDS THE FUTURE OF THAT PATHWAY, THE ULTRA RARE, WE'RE NOT TALKING ABOUT WHAT YOU CALL COMMERCIALLY VIABLE PRODUCT. THESE ARE PRODUCTS THAT HAVE 10 PATIENTS IN THE ENTIRE COUNTRY, HAVE THE DISEASE, ONCE YOU TREAT THEM YOU'RE WAITING FOR PEOPLE TO BE BORN. REALISTICALLY WHAT'S NEEDED IS AN NIH-FDA COLLABORATION TO GO TO A THIRD PARTY CONTRACTOR, TO GO TO INDUSTRY, AND RATTLE OFF IN THE SAME PRODUCTION SYSTEM, SAME METHODOLOGY, A WAY TO TAKE THESE DISEASES QUICKLY THROUGH THE ACADEMIC PROCESS, INTO CLINICAL TRIAL, AND YOU'RE NOT TALKING ABOUT A CLINICAL TRIAL THAT'S HEADED TOWARDS PHASE 3 OR COMMERCIAL. THIS IS FALLING ALONG YOUR ORPHAN DISEASE METHODOLOGY. YOUR COMPASSIONATE USE METHODOLOGY. WHERE WE CREATE A SYSTEM, IT'S GOING TO HAVE TO BE EITHER FUNDED FULLY BY THE GOVERNMENT OR FULLY BY A GOVERNMENT/FOUNDATIONAL APPROACH. BUT IT CAN CHEAPLY AND EASILY BE DONE. THERE ARE RARE EYE DISORDERS WHERE THERE MAY BE ONLY 500 PATIENTS, AND WITH SUSPENSION SYSTEM PROCESS YOU CAN TREAT 20 YEARS OF PATIENTS WITH ONE RUN. IT'S ECONOMICALLY FEASIBLE. AND THAT APPLICATION GOES TO A VERY LARGE PORTION OF RARE DISEASE. YOU WOULD CREATE AN ENTIRE INDUSTRY PARTNER, WE'RE PARTNERS, THE MODEL IS TO DO THAT QUICKLY AND ESSENTIALLY CREATE AN ARCHIVE OR LIBRARY OF RARE DISEASE, ON THE SHELF. >> SO IS THERE A WAY TO SCALE THIS DOWN? YOU KNOW, WE'RE ASSUMING THIS ONE BATCH IS ACTUALLY GOING TO NOT HAVE ITERATIONS THAT NEED TO FOLLOW TO MAKE IT BETTER OR THINGS WE MIGHT LEARN. IS THERE A WAY -- SCALING HAS GONE UP COMMERCIALLY, NOT DOWN. BATCH SIZES HAVE GOTTEN BIGGER, NOT SMALLER. >> METHODS STILL PRODUCE RELATVELY SMALL BATCHES, CERTAIN CLINICAL MANUFACTURING FACILITIES CAN PRODUCE SMALL BATCHES. YOU NEED HALF FOR STABILITY TESTING AND CMC BUT EVEN WITH HALF THE BATCH YOU CAN TREAT A LOT OF PATIENTS. I LIKE THE IDEA OF ESSENTIALLY A GOVERNMENT-SPONSORED THIRD PARTY GROUP THAT MANUFACTURES THESE, DOES THE CMC, GETS THE MATERIAL TO A COMMON DISTRIBUTION CENTER THAT CAN THEN DISTRIBUTE THE MATERIAL TO THE PATIENTS >> I THINK FROM AN INDUSTRY PERSPECTIVE THIS IS THE COMMENT& I'M GOING TO MAKE. IT SOUNDS THE PERFECT SOLUTION BUT I THINK IT'S ONE OF THE THINGS TO CONSIDER IS THE SHELF STABILITY OF THE PRODUCT. THAT IS A BIG, BIG, BIG CONCERN. I DON'T THINK IT HAS BEEN SOLVED YET. >> (INAUDIBLE). >> SO I MEAN, THIS IS A TOPIC THAT I'M DEEPLY PASSIONATE ABOUT, FOR A NUMBER OF YEARS. I CAN MAYBE SHARE EXPERIENCE BECAUSE TO SOME EXTENT SPECIFIC TO THE RETINA SPACE, WE HAVE SET UP SOMETHING THAT IS ALONG THE LINES PROPOSED NON-PROFIT, THERAPEUTICS, WITH NCATS THERE'S EFFORTS TO FORM CONSORTIA TO ACCOMPLISH THE MISSION, THE QUESTION TO REPHRASE FROM WHAT BEV SAID, A SAD IRONY, WE CAN DO THIS, WE ALL KNOW WE HAVE THE TOOLS, THERE'S NO DRIVER OP THE INDUSTRY SIDE WHETHER WE ULTIMATELY NEED TO GET THE PROGRAMS SOON. I THINK IT'S AN INCREDIBLY COMPLEX DISCUSSION. BUT THE MAIN THING HERE PARTICULARLY IN ITEMS OF CMC I DON'T THINK IT'S A CMC ISSUE, I DON'T THINK IT'S A MANUFACTURING ISSUE. I THINK THE COST OF GOODS AND MANUFACTURING IS SURMOUNTABLE, PATIENTS ARE WILLING TO PAY $20,000, $30,000, MAYBE $100,000 TO GET ACCESS. BUT IT REALLY IS CAN WE GET THESE THINGS TO PHASE 1/2 CLINICAL TRIAL AND THAT IS OBVIOUSLY MUCH MORE THAN JUST A CMC QUESTION, RIGHT? THAT'S A REGULATORY QUESTION. WE STILL -- I DON'T THINK WE'RE EVER GOING TO CONVINCE THE FDA THAT WE'RE GOING TO GET A BLIND EYE FOR ULTRA RARE DISEASES, WE'LL STILL HAVE TO DO FORMAL STUDIES AND THOSE ARE INHERENTLY EXPENSIVE AND THEY WILL BE NOT QUITE AS EXPENSIVE ON THE INDUSTRY SIDE BUT STILL MILLIONS OF DOLLARS, WE STILL HAVE TO GET TO THAT BOLUS TO GET EACH OF THESE DISEASES ACROSS, AND THE ECONOMIES OF SCALE WE CAN GENERATE. WE CAN GET DISCOUNTS ON THOSE. BUT THEY WILL COME OVER TIME AFTER WE HAVE INDEED ONE FRAMEWORK, MAYBE ONE VECTOR TO PLUG AND PLAY APPROACH, WHERE WE HAVE A FEW ROUNDS THAT WE CAN GO TO THE CLINIC AND MAYBE THE NEXT WILL BE SHORTER. VERY BRIEFLY, ODELLIA, CREATIVE CONSORTIA, AS WELL AS PHARMA PARTNERS, THERE'S A LOT OF THE IT REMAINS A HEAVY LIFT. >> I DO WANT TO COMMENT ON A STANDARDIZATION BUILD QUESTION FOR THE VECTOR WE MADE, KRYS, IN 2012, STILL SOILED AS CAN BE. YOU CONTINUE TO DO MONITORING AND STABILITY BUT THAT'S PART. CRO. >> JUDE? >> I HAVE A SLIGHTLY DIFFERENT PERSPECTIVE. I THINK KRYS IS A PERFECT EXAMPLE. EVERYBODY IS FOLLOWING THE SAME OF LUKE, FOUNDATIONS ARE BEING STARTED TO TRY TO SOLVE IT. KRYS IS A PERFECT EXAMPLE, HIS EFFORT WITH THE KLOMIS FOUNDATION, MAKING VECTOR, SO FORTH, THERE'S A BIGGER OPPORTUNITY HERE. THAT IS ORPHAN DISEASES GOT A NOMENCLATURE OF 200,000 PATIENTS OR LESS, NO AVAILABILITY OF THERAPY FOR ANYBODY, SO IT WAS EASY TO GROUP THEM LIKE THAT. I THINK THERE'S AN OPPORTUNITY NOW TO RECLASSIFY A NEW SUBCLASS OF ORPHAN DISEASES, ULTRA ORPHAN DISEASES, WHERE THE NUMBER MAY BE 300 OR LESS, 1,000 OR LESS OR SOMETHING LIKE THAT. THAT GROUP OF DISEASES COULD COME UP WITH A CONSENSUS BETWEEN THE NIH, FDA, RESEARCHERS, HOW BEST TO PUT IN GUIDELINES TO ASSIST THAT COMMUNITY BECAUSE WHAT YOU'RE LEFT WITH IS YOU'RE TRYING TO SQUEEZE IN THE WRONG SIZE FOOT IN A SHOE THAT DOESN'T WORK AND WE REALIZE THAT'S NOT GOING TO HAPPEN. I THINK IF YOU ARE BRAVE ENOUGH TO ENTERTAIN SOMETHING LIKE THAT YOU HAVE AN OPPORTUNITY TO REALIZE THAT IF A BETTER CAPSID COMES ALONG, VALIDATED IN ANOTHER STUDY IN HUMANS, YOU COULD TAKE THAT AND WITH A BRIEF BRIDGING STUDY BRING IT TO THE ULTRA ORPHAN DISEASE WITHOUT HAVING TO GO THROUGH THE PACKAGE IF BETTER PRODUCTION CAME ALONG AND SO FORTH. WITHOUT REINVENTING THE WHEEL, THERE'S A CHANCE TO SAY THEY ARE NOT ALL ORPHAN DISEASES. THEY FALL INTO CLASSES. AND THIS TECHNOLOGY BEING AVAILABLE BUT NOT ACCESSIBLE IS CRIMINAL FOR US AS A COMMUNITY. AND I THINK IF WE DON'T TAKE BROAD STROKES TO DO SOMETHING YOU'RE GOING TO BE RELYING ON FOUNDATIONS AND YOU'RE STILL GOING TO LOSE PEOPLE THAT ARE SAYING WE DON'T HAVE ENOUGH TO EVEN GET 10 PATIENTS' ATTENTION. >> I THINK ALL OF THE DISEASES THAT WE'RE TALKING ABOUT, ALMOST ALL WOULD FALL IN THAT, UNDER A THOUSAND. >> QUICK POINT OF FOCUS TO CLASSIFY ODELLIA, TRYING TO GET THOSE TO THE CLINIC, THE FACT THAT WE WILL UNLIKELY APPROVE LEXTERNA 2 EVEN IF IT'S TOO RARE TO BE REDEVELOPED. SO WE HAVE TO FIGURE OUT A WAY TO GET THAT. THERE'S 100+ GENES THAT ARE BASICALLY CARBON COPIES OF THAT. >> KRYS AND ANOTHER SPEAKER AT THE PODIUM. I WANTED TO COMMENT ON THE LEXTERNA. 17 OR 18 YEARS AGO, IT WAS DEEMED NOT REALLY VIABLE FOR BUSINESS REASONS. YET IT'S BEEN IMPORTANT, THE WHOLE DEVELOPMENT OF THE PROCESS IN THE U.S. YEAH, I'M NOT SURE, NUMBER 2, NUMBER 3, IF I CAN FOLLOW THE SAME PATHWAY, GREAT COMMENT. >> YEAH, HI. P.J. BROOKS FROM NCATS. WE'RE VERY MUCH INTERESTED IN THIS. TRYING TO FIND VARIOUS PLATFORM APPROACHES TO MAKE THE PROCESS FASTER, EASIER, SIMPLER. JUST IN RESPONSE TO JUDE, I APPRECIATE THE GOAL, THERE ARE IS A RISK IN TRYING TO CHANGE THAT CLASSIFICATION, WHAT'S A RARE DISEASE, BECAUSE THE ORPHAN DRUG ACT IS A LAW, THAT COULD BE CHANGED. IF YOU CHANGE THAT, YOU CAN MESS UP THE DRUG DEVELOPMENT PATHWAY FOR ALL RARE DISEASES. I THINK THAT COULD BE UNINTENDED CONSEQUENTIALS OF THAT SO I THINK THERE'S A RISK. >> WE SEE ENOUGH CHANGES TO KNOW THE LAW STANDS AND YOU CAN HAVE MANY VARIATIONS THAT FALL UNDERNEATH IT. SO I WOULD SAY THE CHALLENGE IS NOT CHANGING THE IDEA OF ORPHAN DISEASES, IT'S COMING UP WITH SOMETHING MORE COMPASSIONATE AND PROVOCATIVE ABOUT HOW TO ADDRESS AN UNMET NEED, RESOURCES IS NOT THERE FROM THE COMMERCIAL INDUSTRY. THE REST WILL TAKE CARE OF ITSELF. THINK ABOUT A POINT OF VIEW, IF YOU WERE SUCCESSFUL, SAYING WE'RE GOING TO RECLASSIFY DISEASES ABOVE OR BELOW TOTAL NUMBER IN THE WORLD, AND GIVE THEM ACCELERATED PATH, REVIEW PRODUCTION, SO FORTH AND SO ON, LENIENCY, YOU COULD GO DOWN WHAT LUKE IS PROPOSING, WHERE FOUNDATIONS WOULD COME TOGETHER TO TO MEET OBJECTIVES AND OUR EFFORT WITH FOUNDATION, WE'LL MAKE IT FREE THE FIRST TIME BUT IF YOU HAVE TO MAKE IT AGAIN WE DON'T HAVE THE RESOURCES TO DO IT AGAIN AND AGAIN AND AGAIN. EVERYBODY IS PASSIONATE ENOUGH TO. WE NEED GUIDANCE FROM MORE SENIOR INSTITUTES AND GOVERNMENTAL PEOPLE TO SPEAK UP FOR US, HELP US OUT HERE. >> UP TO THIS POINT, I THINK I HAVE -- SORRY. A COUPLE I THINK MAIN POINTS I'D LIKE TO MAKE. THE DISEASES WE'RE DEALING WITH, WITHIN MAYBE HUNDRED OR SO PATIENTS, THIS IN MY MIND CANNOT REALLY GO THROUGH TYPICAL CLINICAL TRIAL DEVELOPMENT. IT'S JUST IMPOSSIBLE. SIMPLY BECAUSE THERE'S NOT ENOUGH PATIENTS, I THINK THE WHOLE ISSUE OF CONTROL AND SO I THINK IT'S A HUGE ISSUE BUT ALSO THE MODEL THAT'S BEEN PROPOSED THAT ACTUALLY I APPLIED BECAUSE I DID GET VECTOR PRODUCED BY YOU GUYS UNDER WHAT'S CALLED XO1 AWARD, WHICH WAS PAID BY THE NIH, FOR THE CLINICAL STUDY PAID BY NIH THROUGH OUR CLINICAL R01, HAS TAKEN MANY, MANY YEARS. THAT'S THE REALITY. THE FIELD HAS MOVED DRAMATICALLY FORWARD NOW SO I THINK THOSE THINGS CAN BE GREATLY ACCELERATED, BUT I THINK THE REALITY IS STILL THERE THAT IF WE WERE TO REGISTER THIS, I MEAN, WHAT'S GOING TO HAPPEN NEXT? BECAUSE THE WHOLE DISTRIBUTION ISSUE, AND THEN HAVING TO RAISE A LOT OF MONEY TO HAVE MULTIPLE PRODUCTION CYCLES TO REALLY GET THE VECTOR RELEASED AS A COMMERCIAL PRODUCT, IT'S A TOTALLY DIFFERENT LEVEL OF COMPLEXITY WHICH I HAVE NO IDEA WHAT TO EVEN DO ABOUT IT. ON THE OTHER HAND, I FEEL STRONGLY THAT HAVING DEVELOPED I THINK SUCCESSFUL -- I'M NOT SAYING TREATMENT BUT ALLEVIATION OF CLINICAL SUFFERING I THINK YOU WANT TO ACCELERATE AND MAKE SURE THIS IS WIDELY BEING MADE AVAILABLE TO PATIENTS, AND THIS I THINK IS A HUGE CHALLENGE FOR US AS A GROUP, FDA, NIH, I THINK AS AN INVESTIGATOR, I WANT TO MAKE ONE MORE COMMENT BECAUSE THE REMARKABLE STABILITY OF THE PRODUCT WHICH I THINK BEING STABLE FOR SO LONG WILL BE STABLE FOR ANOTHER TEN YEARS I SUPPOSE. YOU THINK YOU HAVE FIVE PATIENTS TODAY, IF YOU HAVE THAT PRODUCT WHICH IS AVAILABLE TO PATIENTS IN THE FUTURE, I THINK YOU'VE ACCOMPLISHED A HUGE THING BECAUSE IT'S NOT FOR JUST VERY FEW SUBJECTS AVAILABLE TODAY BUT THERE WILL BE A NUMBER OF PATIENTS LATER ON, AS LONG AS THE PIPELINE IS THERE AND WE DO HAVE THE DRUG AVAILABLE. SO I MEAN I HOPE TO HEAR SOME SOLUTIONS TO THIS, I THINK OTHERWISE REALLY HUGE PROBLEM. >> YEAH, SO THIS WAS IN FACT EXACTLY THE CONCEPT WITH WHICH WALTER CAME TO ME AND I WOULD BE SURPRISED IF HE HADN'T GONE TO KRYS AND JILL AND P.J. EARLIER. AND BASICALLY SAID SHOULD WE BUY A BARN SOMEWHERE AND PAY SOMEONE TO SIT IN THERE ALL NIGHT AND DAY LONG AND MAKE WHATEVER CONSTRUCTS ARE NECESSARY AND, YOU KNOW, JUST PICK A VECTOR AND WE MAKE HOWEVER MANY GAZILLION KILOS OF THIS VECTOR AND PEOPLE COME AND GIVE US THEIR CONSTRUCT AND WE AMPLIFY AND STICK IT IN THE VECTORS AND GIVE IT BACK TO THEM AND WE USE GOOD MANUFACTURING PRACTICE TO DO IT AND WE GET THE FDA TO SAY IF THE VECTOR IS THE SAME EVERY TIME AND ONLY THING IS DIFFERENT IS THE INSERT, THEN THE HOOPS PEOPLE HAVE TO JUMP THROUGH FROM A REGULATORY STANDPOINT CAN BE REDUCED OR WHATEVER AND AT LEAST STANDARDIZED FROM BATCH TO BATCH TO BATCH. AND AFTER ATTENDING THIS NATIONAL ACADEMY OF MEDICINE MEETING I CAME AWAY THINKING THERE'S NO WAY THAT ANY TWO PEOPLE EVEN WORKING ON THE SAME DISEASE ARE GOING TO AGREE THAT THE SAME VECTOR IS THE RIGHT THING TO USE. I DON'T KNOW WHAT WE WOULD BE MANUFACTURING. WE'RE GOING TO MAKE TEN DIFFERENT AAVs AND INSERT IT INSIDE OUT, UPSIDE DOWN, I DON'T KNOW. BUT I THINK THE STANDARD -- I MEAN, I STILL THINK THIS IS THE WAY TO GO. AND I STILL THINK IT'S THE ONLY WAY WE'RE GOING TO GET THERAPIES TO PEOPLE WHO HAVE NOT A COMMERCIALLY VIABLE DISEASE. BUT I SUSPECT, AND NOTHING WOULD MAKE ME HAPPIER THAN FOR EVERYBODY HERE TO SAY I WAS WRONG ABOUT IT AND WE SHOULD JUST MOVE AHEAD. BUT I THINK WE HAVE TO GO THROUGH THE PROCESS OF DOING THE STUDIES THAT CONVINCE THIS COMMUNITY THAT THERE IS A STANDARD WE SHOULD USE, AND THEN WE CAN'T MANUFACTURE -- CUSTOM-MAKE HUNDREDS AND HUNDREDS. I DON'T KNOW. >> I THINK WE NEED A STANDARD PROCESS FOR HOW TO ADVANCE ACCESS TO HUMANS. WE DON'T NEED THE NIH TO MANUFACTURE AAVs THE SAME OVER AND OVER AND OVER AGAIN. WE WOULD ARGUE THAT SOME INDICATIONS WOULD REQUIRE AN AAV9, OTHERS MAY REQUIRE AN AAV66, SO THERE ARE DIFFERENCES AND THERE ARE NUANCES DEPENDING WHAT IT IS YOU WANT TO ACCOMPLISH BUT I THINK WHAT JUDE WAS GETTING AT, IF I CAN PARAPHRASE, IS JUDE AND LUKE AND KRYS AND OTHERS AND MYSELF INCLUDED IS THAT WE NEED A STANDARD PATHWAY, WE NEED A ROAD MAP ON HOW TO ADVANCE THE ULTRA ORPHAN DISEASES IN A TIMELY WAY. IN A WAY WHERE WE'RE HELD TO THE SAME SAFETY STANDARDS, BUT MAYBE NOT THE SAME REGULATORY STANDARDS IN TERMS OF THE NUMBERS OF MANUFACTURING RUNS YOU HAVE TO REPEAT OR BECAUSE IT'S NOT -- YOU CAN'T ABSORB THE COST. NOT EVERYBODY CAN ABSORB THE COST OF A MANUFACTURING RUN. IT'S ABOUT A MILLION DOLLARS A POP. AND THEN YOU HAVE -- IF THE NIH COULD TAKE THE LEAD IN MAKING SURE THAT THE DRUG WAS BOUGHT AND PAID FOR AND DISTRIBUTED AND PAID FOR THAT, IN A FEE FOR SERVICE KIND OF WAY, I THINK THAT'S WHAT WE'RE ASKING. >> I NEED -- IF I UNDERSTAND CORRECTLY, YOU NEED A FUNDING MECHANISM, FUNDING AND DISTRIBUTION MECHANISM, AND YOU NEED STANDARDIZATION OF THE CRITERIA BY WHICH WE DECIDE THIS IS SAFE ENOUGH TO MOVE AHEAD IN THIS POPULATION. >> YES. >> I'M GLAD -- I'M GLAD I ASKED. NOW I THINK I'M CLEAR ON WHAT IT IS. >> CAN I ADD SOMETHING TO THAT AS WELL? I THINK THE PHARMACOLOGICAL CHARACTERIZATION OF VIRAL VECTORS IS LARGELY BASED UPON SMALL MOLECULE DRUGS. AND SOME OF THOSE RELEASE CRITERIA ARE NOT FIT FOR PURPOSE. THAT'S WHY WE END UP USING HALF A BATCH FOR RELEASE TESTING. SO WOULD IT BE POSSIBLE TO FORM A WORKING PARTY TO WORK WITH THE FDA, FOR EXAMPLE, TO TRY AND REDUCE THE VOLUME AND BURDEN OF THOSE TESTS AND MAKE THEM MORE APPROPRIATE FOR VIRAL VECTORS WHICH MIGHT ACTUALLY MEAN CHANGING THE LAW AS WELL? >> I WOULD COMMENT TOO. I THINK THAT'S A GREAT IDEA, PART OF THE CHALLENGE WITH GENE AND CELL THERAPIES RIGHT NOW, ANALYTICS ARE RELATIVELY IMMATURE, IMPRECISE, GO BACK TO FDA SPONSORED TITERRING ASSAY, A FULL-DAY SYMPOSIUM A YEAR AND A HALF AGO SPONSORED BY FDA, WHY ARE WE SO VARIABLE IN DIFFERENT INDIVIDUALS DOING DIFFERENT TITERS, REALLY ON THE SAME MATERIAL, WE NEED TO TIGHTEN THOSE THINGS UP BECAUSE OTHERWISE IT'S HARD TO MAKE THE CONCLUSIONS, THERE'S A LOT OF WORK. THAT'S A GREAT CONCEPT. ALMOST ON THE SECOND SLIDE CAN WE MAKE A PLATFORM FOR THE PROCESS, BUT ALSO CAN WE SOMEHOW SIMPLIFY WHAT MIGHT BE THE -- I DON'T KNOW IF IT'S NIH SPONSORED, IF THAT'S THE RIGHT WORD, BUT MANUFACTURING PROCESS BECAUSE COMPLEXITY RIGHT NOW, THERE ARE COMMERCIAL REASONS TO CERTAIN GROUPS WHO MIGHT BE USING ONE UPSTREAM PROCESS, IT'S HARD TO SORT OF SAY YOU'VE GOT COMPARABLE VECTOR MADE BY PROCESS A, COMPARE ABLE TO MADE BY PROCESS, ANOTHER CHALLENGE. >> THAT'S AN AREA WHERE I THINK -- I'M SORRY. >> I DON'T THINK IT SHOULD GET LOST THE FDA HAS STEPPED UP EVERY TIME WE'VE INTERFACED WITH THEM AS INDIVIDUALS OR A COMMUNITY TO TRY TO HELP OUT, EVEN MORE RECENTLY DENISE GAVIN AND HER COLLEAGUES HAVE SAID WITH THREE TIMES REPEAT WE CAN CONSIDER YOUR G.O.P. PREPS AS MEETING STANDARDS. I DON'T THINK WE'RE LOCKED INTO SOMETHING. I THINK EVERYBODY'S TRYING TO FIND CREATIVE WAY TO MAKE THIS HAPPEN. WE SEEM TO BE BOXED IN BY WHAT'S BEEN DONE HISTORICALLY, SIMILAR TO WHAT BRIAN WAS SAYING. IT DOESN'T FIT WHO WE ARE. AND WE'RE NOT ONLY FOR THE WHOLE FIELD OVERALL SELLING GENE THERAPY BUT NOW WE'RE FRAGMENTING PATIENTS THAT WE'RE GOING TO SAY YOU DON'T HAVE ACCESS TO SOMETHING WE KNOW WORKS, BECAUSE THE COST IT TAKES TO DEVELOP IT IS NOT ACCESSIBLE TO YOU. AND THAT'S THE PART THAT I THINK BOTHERS THE MAJORITY OF US AS INVESTIGATORS. I THINK GOING FORWARD YOU'LL SEE THAT THE REGULATORY COMMUNITY IS MORE THAN HAPPY TO GIVE US GUIDANCE ON WHAT THEY THINK WE CAN DO TO MAKE THINGS GO QUICKER OR FASTER, BORROW DATA FROM OTHER INDs AND THINGS LIKE THAT. IT COMES BACK TO SOME OF THE THINGS THAT WE NEED TO BE CREATIVE ABOUT, AND I BRING UP ONE OF THE THINGS THAT WAS DONE FOR THE NATIONAL VECTOR LABS THAT ANY FDA TRIAL THAT WAS USING NIH MONEY, THE PRE-CLINICAL PACKAGES WERE DEPOSITED AT THE NATIONAL VECTOR LAB AND PEOPLE HAD ACCESS TO THAT INFORMATION LIKE YOU DIDN'T HAVE TO DO IT THREE TIMES, YOU COULD WRITE A CROSS-REFERENCE, BUT AT THE SAME TIME THAT'S AN EARLY START OF SOMETHING TO BE MORE CREATIVE GOING DOWN THE PATH OF WHAT KRYS AN OTHERS ARE SAYING WHEN YOU ONLY HAVE SO MANY PATIENTS HOW DO YOU EVEN DESIGN A TRIAL TO ANSWER A QUESTION WHEN YOU KNOW THAT ALL YOU'RE REALLY DOING IS COMPASSION, COMPASSION. >> I WANT TO TOUCH ON THE ADDITIONAL POINT THAT YOU -- JUDE MIGHT HAVE RAISED THIS EARLY, WE'D LIKE GUIDANCE ON PATH FORWARD FOR BRIDGIG STUDY THAT ALLOWS YOU TO MOVE IN WITHOUT REPEATING THE ENTIRE PHASE 1. AGAIN, THIS ISN'T A SMALL MOLECULE, IT'S NOT SOMETHING YOU CAN ADVANCE IN THAT KIND OF WAY. SO I WOULD SUGGEST THAT ONE OF THE RECOMMENDATIONS IS TO FORM A WORKING GROUP AROUND THIS SO THAT WE CAN FLESH OUT HOW TO MOVE FORWARD, HOW TO TAKE ADVANTAGE OF SOME OF THE WORK THAT'S ALREADY BEEN DONE, IN THE FOUNDATION SPACE, AND I GUESS KEVIN'S THERE. >> I JUST WANT TO ADD, A QUESTION FOR LUKE, BUT I -- OBVIOUSLY THIS IS IN SOME WAYS A POLITICAL DISCUSSION, THE STRUCTURE, TALKING ABOUT THINGS, ACCESS FOR PATIENTS AT SOME LEVEL IS NO LONGER GOING TO BE DRIVEN BY PURELY COMMERCIAL DECISIONS, BY LICENSING OF PRODUCTS TO COMPANIES, BY, YOU KNOW, INSTITUTIONS TAKING ADVANTAGE TO GET LICENSING BACK FROM IT. I THINK WE HAVE TO RECOGNIZE WHAT WE'RE TALKING ABOUT IS IN THE NON-PROFIT SPACE, IN SOME WAYS, AND I ALSO WANT TO POINT OUT JUST THE OTHER THING ABOUT ACCESS TO THIS, I THINK THE NIH HAS TO TAKE THE LEAD, I KNOW THERE'S BEEN SOME DISCUSSION, I'VE SEEN RFAs, LOOKING AT ACCESS DISPARITIES, RACIAL AND ETHNIC, NOW SOCIOECONOMIC DISPARITIES. SO I DON'T REALLY KNOW THAT WE HAVE ACCESS TO THAT DATA. I DO KNOW THAT THE FAMILIES WHO SHOW UP ON DOORSTEPS FOR STUDIES, TRAVEL GREAT DISTANCE, THERE'S ENRICHMENT FOR SOCIOECONOMIC CLASSES WE HAVE TO VIEW DIFFERENTLY IF WE'RE GOING TO PROVIDE ACCESS FOR THESE, TO EVERYBODY. SO I THINK THE NIH IS THE ONLY GROUP THAT CAN HELP FUND RESEARCH ON THAT, THE ONLY GROUP THAT CAN ENFORCE COLLECTION OF DATA IN A WAY. ONE QUESTION, THE QUESTION WAS UNDER A MODEL OF ULTRA RARE DISEASES, ACCESS TO DIFFERENT COMPANIES, HOW DO YOU HANDLE THINGS LIKE INDEMNIFICATION FOR THIS? HOW IS THAT DISTRIBUTED ACROSS MULTIPLE COMPANIES OR HOW WOULD THAT BE DISTRIBUTED IN A SYSTEM LIKE THIS, FOR THE RISKS THAT GO WITH THE NOVEL GENE THERAPY TREATMENTS? >> IT'S SOMETHING WE STRUGGLE WITH. I HAVEN'T FOUND A FULL SOLUTION TO. THE INSTITUTION ITSELF, THIS IS THE ECONOMIC INSTITUTION, MOST OF THESE PROGRAMMING COME OUT OF, YOURS, MINE, WE MUST THINK OF INDEMNIFICATION. WE HOPE WE CAN BUILD OUT ODELLIA AS AN ENTITY THAT CAN TAKE OUT THAT INDEMNIFICATION BUT WE'RE STILL TRYING TO WORK WITH SPONSORS TO OFFLOAD THAT. THAT'S ONE OF THE MANY ISSUES AND MANY OF THE OTHER ONES LIKE YOU RAISED THAT, AGAIN, I THINK TO -- I AGREE FULLY WITH JUDE, IN ADDITION TO THE FUNDING MECHANISM IDEA PROPOSED IT REQUIRES CREATIVITY AND US TRYING COLLECTIVELY TO WORK TOWARDS PARADIGM SHIFT FOR THIS NICHE OF APPLICATIONS WHERE WE HAVE ETHICAL RESPONSIBILITY TO GO AFTER THAT PARADIGM SHIFT, IT'S NOT A SIMPLE SOLUTION, NOT ONE VECTOR FOR ALL. IT'S GOES DOWN TO THESE THINGS, INDEMNIFICATION REQUIRES A CONSORTIUM TO COME TOGETHER. THAT BEING SAID I DON'T WANT TO OVERESTIMATE THE PROBLEM, TO SOME EXTENT ON THE INDUSTRY SIDE FOR THE COMMERCIALLY VIABLE PROGRAMS, THE SAME THING HAPPENS TO SOME EXTENT, ALMOST EVERY COMPANY IS TRYING TO BUILD OUT ONE MODEL THAT THEY CRANK THROUGH FOR ONE DRUG AND THE NEXT DRUG AND NEXT DRUG. WHAT THEY BENEFIT FROM IS ALL THESE DATA LAND UNDERONE ROOF AND THEY CAN CROSS REFERENCE THEIR OWN PROTOCOL. WHAT ONE OF THE CONCEPTS WE'RE TRYING TO PURSUE BUILD THAT ONE ROOF FOR COLLECTIVELY ALL ULTRA RARE DISORDERS THAT WE CAN ALL COLLECTIVELY AS A COMMUNITY TAP INTO AND DELETE THOSE BARRIERS, SUCH AS LICENSING, AND OTHERWISE THAT THAT CREATE OTHERWISE THOSE WALLS THAT WE CAN'T TAP INTO THE LEXTERNA IND, CROSS-REFERENCE FOR EXAMPLE FOR ALL THE RIGHT REASONS ON COMMERCIAL SIDE BUT NOT APPLICABLE TO THIS SPACE. THAT'S ONE LAST THOUGHT, WE'RE ACTUALLY CULPABLE AS WELL, EACH OF US BUILD TECHNOLOGIES AND FILE PATENTS AND I WANT TO THROW THIS OVER TO WALT. THAT'S PART OF THE PROBLEM. THOSE THINGS BECOME INACCESSIBLE FOR THESE DISEASES, SOME OF OUR TECHNOLOGY, PRO BONO HAVE DEPOSITED IT IN ODELLIA. >> YEAH, I JUST WANT TO MAKE A FEW COMMENTS. FIRST, ABOUT WHAT JUDE WAS TALKING ABOUT, NATIONAL GENE THERAPY VECTOR LABORATORIES BY NHLBI, IT'S A GOOD MODEL FOR EARLY STAGE DEVELOPMENT OF THE GENE THERAPY. NOW YOU NIH TRYING TO DO THIS AGAIN, I THINK WE HAVE TOTALLY DIFFERENT MINDSET, IN MANY DIFFERENT WAYS, BUT THAT'S A GOOD MODEL TO START. AND THE SECOND THING IS CROSS-REF RENNES. SIX YEARS AGO JUDE ORGANIZED SGCT CONVERSATION WITH FDA, NIH CAMPUS, THAT'S BEEN DISCUSSED EXTENSIVELY, SEROTYPES. IF YOU HAVE SPECIFIC GENE PRODUCT CROSS-REFERENCE MAY NOT WORK. WE STILL NEED THIS KIND OF SYSTEM FROM GOVERNMENT OR FROM NON-PRIVATE MONEY TO DO THIS KIND OF CHARACTERIZATIONS. >> I WAS JUST GOING TO SAY PATENTS AND ALL OF THAT FALL BY THE WAYSIDE IF YOU'RE NOT DEVELOPING A DRUG FOR COMMERCIAL SALE. AND SO I MEAN A PHASE 1 SAFETY STUDY THAT STAYS OPEN AND TREATS TEN KIDS OR 30 KIDS IS NOT AN INFRINGEMENT ON PATENT, SO IT'S AVAILABLE TO US. I THINK AGAIN IT'S BEING CREATIVE, TRYING TO GET A ROAD MAP FROM OUR COLLEAUES AT NIH, AND AGREEMENT WITH OUR FDA COLLEAGUES, SCIENCE IS POISED AND WAITING, IT'S JUST A MATTER OF PUTTING THINGS TOGETHER. >> IF WE PUT TOGETHER THIS WORKING GROUP I DO WANT TO ENCOURAGE US TO THINK ABOUT HOW THIS HAS BEEN DONE BEFORE IN THE RARE DISEASE SPACE WITH ENZYME REPLACEMENT THERAPY, (INDISCERNIBLE) A NON-PROFIT TO DEAL WITH THE ISSUES OF DEVELOPING RECOMBINANT PROTEIN THERAPIES BECAUSE CERTAIN COMPANIES DIDN'T WANT TO MOVE IT FORWARD, AND HE MAY HAVE SOME GUIDANCE ON HOW HE WENT THROUGH THIS PROCESS IN TERMS OF MAKING SURE THINGS WERE AVAILABLE AND ALLOWED, HOW DID THEY WORK WITH THE DISTRIBUTION, HOW DID THEY WORK FOR FEE FOR SERVICE STRUCTURE. >> BECCA ARONS, TWO QUICK CLINICAL ISSUES. THIS PROBLEM IS ONLY GETTING BIGGER IN THE AREA OF GENOMIC SEQUENCING, SO MANY TIMES WE SEE KIDS WHERE WE DIAGNOSE THE FIRST COUPLE PATIENTS WITH THIS DISEASE AND THEY FIND THE OTHER TEN PATIENTS IN THE WORLD ON FACEBOOK AND THEY WANT TO GO ABOUT FORMING A FOUNDATION INTO THESE TYPES OF TRIALS. AND SO A LOT OF TIMES IT GOES BACK TO THAT ETHICAL ISSUE, WHO HAS ACCESS TO THIS TYPE OF INFORMATION. IT'S THE MOST SOCIOECONOMIC EDUCATED FAMILIES THAT CAN PUSH THIS FORWARD, THEY SHOP IT AROUND AND GET OF GET DIFFERENT INFORMATION FROM EVERY PERSON. TO HAVE A FRAMEWORK TO GO TO TO START THE PROCESS OF WHAT DOES MANUFACTURING LOOK LIKE FOR WHEN A FOUNDATION IS TRYING TO FUND THIS WOULD BE A HUGE BENEFIT TO THE PATIENT COMMUNITIES. AND LIKE I SAID IT'S GOING TO KEEP GETTING BIGGER AND WE FIND MORE RARE DISEASES. >> SO I HAVE A MANUFACTURING GENERAL QUESTION THAT'S THE INVERSE OF WHAT THIS STARTED OUT WITH, A HUGE AMOUNT OF VECTOR THAT MAY LAST YEARS. >> (INAUDIBLE). >> WELL, YOU DON'T NEED VERY MUCH. BUT YOU'VE GOT -- THAT CAN LAST YEARS. THAT SEEMS TO BE COMING UP AS THE SYSTEMIC SYSTEMS COME ONLINE, IF YOU SCALE UP FROM A MOUSE TO A CAT OR DOG AND THEN TO A HUMAN, EVEN SMALL HUMAN, CHILD, YOU'RE PUSHING WHAT I UNDERSTAND IS LIMITS OF MANUFACTURABILITY FOR A BATCH NOW. I MEAN, LOOKS LIKE I'M TAKING FROM OUR DATA, NOT PUBLISHED YET BUT IT WILL BE 10 TO THE 15th OR GREATER PER PATIENT. ARE THE MANUFACTURING SYSTEMS IMPROVING IN A WAY THAT IT WILL GET LARGER, OR I MEAN WE TALKED ABOUT OTHER STRATEGIES, REDUCING THE DOSE THAT MAY BE NEEDED, IF YOU CAN GET IT TO ENTER THE CNS BETTER, THINGS LIKE THAT. A LOT OF THESE DISEASES, PREDOMINANTLY CNS, NOT ONLY CNS DISEASE. SO -- >> YEAH, I CAN COMMENT. I'LL RESPOND. I THINK, JOHN, THAT'S CORRECT. WE HAVE THE ONE SITUATION WHERE THE DOSES ARE RELATIVELY SMALL AND VERY FEW PATIENTS. AND THEN YOU GET INTO THE SITUATION WHERE, WELL, ONE REASONABLE-SIZED LOT COULD GO FOR A LONG TIME, YEARS. I AGREE WITH YOU COMPLETELY, THERE'S BEEN BIG INVESTMENT, I DON'T KNOW THE DETAILS TO TRY TO -- THAT'S STILL AN ULTRA ORPHAN DISEASE, COMING ACROSS, IT'S A HUGE MANUFACTURING CAPACITY BECAUSE I THINK THE DOSES ARE -- E 15 AND E 16, HIGH PER DOSE, THAT'S EXACTLY -- THE OTHER SIDE OF THE COIN IS THAT WE'RE PROBABLY SHORT ON OUR MANUFACTURING CAPACITY FOR THESE LARGER DOSE INDICATIONS, EVEN WHEN THEY ARE STILL ORPHAN, ULTRA ORPHAN, THE COST AND TIMELINES I THINK ARE VERY FRUSTRATING RIGHT NOW BECAUSE THE TIMELINES CAN BE TWO YEARS. I'VE HEARD THIS FROM THE GET-GO, TO GET STARTED. >> AND WE'RE LARGELY TALKING ABOUT THINGS THAT MAY NOT HAVE TO BE INFUSED, I.V. TO GO EVERYWHERE. YOU CAN GET ENOUGH PERIPHERAL EXPOSURE FROM INTRATHECAL, WORK IN PRIMATES AND HUMANS, GENE THERAPY, TINY DOSES, DIRECTED STUFF CHRIS IS DOING TAKING ADVANTAGE OF THE NETWORK. >> I DON'T THINK IT'S CLEAR WHEN YOU DOSE PER BRAIN SIZE, HUMANS THAT'S GOING TO GO WAY UP, VERSUS BODY WEIGHT FOR PERIPHERAL INJECTION, IT'S NOT ENTIRELY CLEAR TO ME BASED ON PRE-CLINICAL DATA THAT THOSE ARE GOING TO REQUIRE THAT MUCH LESS. YOU KNOW, TO DO. EVEN IF YOU GET EFFECTS OF SOME OF THE VECTOR GETTING OUT INTO THE SYSTEM. THE QUESTION IS, IS THAT ENOUGH TO DO ANYTHING TO HELP THE PERIPHERAL SYMPTOMS. >> I WANT TO ADD A FEW DATA POINTS ON THE SYSTEMIC DELIVERY, SMA EXAMPLE. MANUFACTURING CAN BE ONE WAY TO SOLVE THE PROBLEM. ANOTHER ONE COULD BE CAPSID ENGINEERING TO AVOID GOING INTO THE ORGANS WE DON'T NEED TO TOUCH BECAUSE THAT MAKES MORE CAPSID AVAILABLE TO FOR EXAMPLE CROSS. I'M GOING TO GIVE A FEW NUMBERS, A FEW EXAMPLES FROM OUR MOUSE WORK. IT'S LIMITED TO THE MOUSE BUT WE MADE THIS VECTOR THAT CAN CROSS THE BBB IN THE MOUSE AND WITH COLLABORATORS LEARNED 10% OF THE SYSTEMIC DELIVERY WE KNOW OUT OF THE VOLUME THAT WE PUT, 10% IS IN THE BRAIN. NOW, WE TOOK IT A STEP FURTHER AND USED DIRECTED EVOLUTION TO TRY TO DETARGET IT FROM OTHER ORGANS THAT MAYBE WE DON'T WANT SUCH AS THE LIVER, WE'RE ABLE TO FIND SOME CAPSIDS THAT DO NOT TRANSDUCE THE LIVER AND NOW WE'RE MEASURING BY PET TO SEE DO WE INCREASE THIS PERCENTAGE IN THE BRAIN. MANUFACTURING IS ONE. THE OTHER IS LET'S PREVENT ENTRY INTO ORGANS WE DON'T NEED. >> I THINK THE AAV 9 SYSTEMIC ADMINISTRATION, IT'S PUBLISHED, EXCESS PRODUCTS, 90% TO THE LIVER, MAYBE SOME IN THE LIVER IS BENEFICIAL YOU PROBABLY DON'T NEED THAT MUCH. >> TOO MUCH, AS IT IS NOW. IF WE COULD DECREASE THAT SOAKING FROM THE LIVER THAT WOULD JUST REDUCE MANUFACTURING PRESSURE AS WELL. WE SHOULD -- >> SORRY. PLUS AAV9 IS IN LARGE ANIMALS GOING PRIMARILY TO LOWER BRAIN SPINAL CORD, GOOD FOR SMA, NOT TREATING THE REST OF THE BRAIN. >> DEEP BRAIN AREAS. >> NOT JUST DEEP BRAIN, CORTEX. >> YES, YES. >> SMA IS A GOOD DISEASE TO APPLY IT TO BASED ON THE BIOLOGY THAT'S KNOWN NOW. >> FROM A PRODUCTION PERSPECTIVE TO SOME QUESTIONS, HERPES VIRUS COMMUNITY IS SHOWING DATA THAT INFECTIVITY IS 1 IN 5 PARTICLES, SO YOU'RE NOT USING AS MUCH. I THINK THE BACULOVIRUS SYSTEM IS BEGINNING TO GET IRONED OUT WHERE IT HAS ROBUSTNESS ATTACHED TO IT. THE TRIPLE TRANSFECTION APPROACH WE AND OTHERS HAVE USED FOR AN EXTENDED PERIOD OF TIME IS GETTING TWEAKED TO THE POINT WHERE I THINK THE LARGEST PRINTS WE MADE AT UNC VECTOR CORES THAT 10 TO THE 14th TOLD, TODAY 10 TO THE 18th,PURIFYING 10 TO THE 16th, 150-LITER BIOREACTOR, YOU'RE SEEING INCREMENTAL CHANGES BASED ON TECHNOLOGY THAT'S BECOMING AVAILABLE. AND I'M SURE WITH OUR BIG PHARMA COMING IN AND EMBRACING THIS IT'S GOING TO CONTINUE TO GET BETTER AND PRICE SHOULD COME DOWN SIGNIFICANTLY. SO THE FIELD IS PROBABLY PUTTING MORE ENERGY INTO PRODUCTION. EVERYBODY WANTED TO PUT IT IN THE BRAIN RATHER THAN MAKE A PREP SO IT WAS NOT A PRIORITY BUT I THINK IT'S BEING ADDRESSED. >> WELL, WE'RE PROBABLY COMING CLOSE TO THE END. THERE'S ONE MORE SLIDE. WE TALKED ABOUT THE PLATFORM QUITE A BIT. OH, THANKS. YEAH, THE ONE WE TALKED, BATCH PRODUCTION FOR ULTRA ORPHAN AND WE TALKED ABOUT THE OTHER SIDE OF THE COIN, REALLY LARGE, WE TALKED ABOUT FEASIBILITY OF STANDARDIZED PROCESS AND PROCEDURES, I THINK TRYING TO WORK THROUGH IS THIS KIND OF THE CMC OVERALL DEVELOPMENT PROCESS AND WHAT ARE THE MILESTONES VERSUS STANDARIZATION, THE ACTUAL WAY WE DO IT TO SUPPORT A LOT OF CASSETTE TYPE SITUATIONS. MAYBE THERE ARE TWO ELEMENTS THERE. I'D LIKE TO PUT ONE MORE ELEMENT ON THE TABLE, JUDE OUTLINED THE THREE MAIN UPSTREAM PROCESSES FOR AAV, LESS OPTIONS FOR LENTI. AND I'M INTERESTED, THERE ARE OPPORTUNITIES FOR YET BETTER WAYS BECAUSE IT IS TRUE THAT IT TAKES A LONG TIME TO GET PLASMID, ADEQUATE QUALITY, AND RECOMBINANT BACULO AND HERPES. SIMPLIFIED CELL LINES, I DO BELIEVE THERE MAY BE QUALITY ATTRIBUTES IN THE PRODUCT MAYBE NOT THOUGHT OF THAT MIGHT CONTRIBUTE TO BETTER FEATURES FOR THE IMMUNOSTEALTH APPROACHES, MAYBE THEY COULD BE CONTRIBUTED BY PRODUCTION CELL SYSTEMS SO I KNOW THIS IS AN OLD INITIATIVE PRODUCTION CELLS GOING BACK 20 YEARS AT LEAST, JUDE, I DON'T KNOW IF YOU COULD COMMENT ON FEASIBILITY OF PRODUCTION AND OTHERS WHO HAD EXPERIENCE. >> I THINK REID CLARK AND PHIL JOHNSON WERE THE ONES THAT PIONEERED THAT EFFORT EARLY WITH BARRY CARTER. >> YEAH. >> THEY TOOK IT TO TARGET GENETICS AND MADE ALL THEIR PRODUCTS THAT WAY. >> STILL HAVE THE ADENOVIRUS. >> ADENOVIRUS COMPONENT, AND I THINK WHAT THEN ABLE TO SHOW IS THEY COULD MAKE PRODUCER CELL LINES. I THINK THE FACT THAT THE FIELD WAS SO -- REQUIRED NIMBLENESS SO IF YOU MADE CHANGED IN PROMOTER OR OPTIMIZED A CODON YOU DIDN'T WANT TO MAKE A NEW CELL LINE EVERY TIME. PEOPLE GRAVITATED TOWARDS QUICKNESS OF BEING ABLE TO MODULATED THE CASSETTE AND THEN MAKE VIRUS. I THINK IN THE TRANSITION OF DOING THAT, THINKING ABOUT SUPPLY AND STUFF LIKE THAT, EVERYBODY'S LOOKING BACK TO IF I KNOW WHAT IT IS, IT'S NEVER GOING TO CHANGE, WHAT'S BEST WAY TO MAKE IT OVER AND OVER. >> I WAS GOING TO MAKE A COMMENT BACK TO THE NIH TALKING ABOUT GETTING THEIR BARN AND MAKING AAV. THERE'S A LOT OF BAD WAYS TO MAKE AAV BUT THERE'S NUMBER GOOD WAYS OF MAKING AAV. IF YOU'RE THINKING ABOUT DOING THIS, I WOULD STEER AWAY FROM TRYING TO FIND THE BEST WAY TO MAKE AAV BECAUSE YOU'LL NEVER GET A CONSENSUS. I THINK YOU'VE GOT TO PICK A METHOD THAT IS GOOD OF THE MULTIPLE AVAILABLE AND TRY TO DO IT REALLY WELL. >> YEAH, I MEAN, THAT WAS MY -- THAT'S WHAT GAVE ME THE SENSE OF FUTILITY SITTING IN THAT NATIONAL ACADEMY OF MEDICINE MEEING, THAT IN FACT BEFORE WE GO MANUFACTURING SOMETHING THAT HALF THE PEOPLE THINK IS THE WORST, THE OTHER HALF THINK IS THE BEST, WE OUGHT TO WORK TO FUEL THE STUDIES THAT COME UP WITH SOME KIND OF CONSENSUS AND THE ANSWER MAY BE DIFFERENT FOR EVERY DISEASE OR PATIENT OR WHAT HAVE YOU, BUT THE CRITERIA THAT ALLOW YOU TO DECIDE WHAT IS THE BEST INVESTMENT OUGHT TO BE DEVELOPED BY THE SCIENTIFIC COMMUNITY. THE OTHER THING WE WERE TALKING ABOUT, AND I HAVE TO SAY I HAVE TO THINK ABOUT THIS SOME MORE, IT'S NOT AN ANSWER, IT'S MORE A QUESTION, IS I THINK WE'RE ALL ACUTELY AWARE OF THE ISSUES OF DISTRIBUTION FOR CLINICAL CARE AND OF THE MAL DISTRIBUTION RELATIVE TO DISADVANTAGED COMMUNITIES AND UNDERRESOURCED COMMUNITIES AND COMMUNITIES OF COLOR AND YOU NAME IT. THERE ARE UNFAIR EXCLUSION CRITERIA FOR NOT ONLY THIS THERAPY BUT MANY OTHER THINGS. AND THE NIH IS A RESEARCH ORGANIZATION. IT CAN CERTAINLY JUSTIFY AND WE'VE JUST HIRED SOMEONE WHO WILL BE IN CHARGE OF HELPING TO FUEL THE RESEARCH THAT SAYS ARE WE REACHING THE PEOPLE THAT NEED TO BE REACHED AND THEN ACTUALLY LOOKS AT DISPARITIES, NOT ONLY WITHIN THE UNITED STATES BUT GLOBALLY, BUT WHEN WE ANSWER THAT QUESTION TO WHICH I THINK MANY OF US THINK WE ALREADY HAVE AT LEAST A PARTIAL ANSWER, THE AGENCIES AND PARTS OF THE FEDERAL GOVERNMENT THAT ACTUALLY COME UP WITH THE WAY TO DISTRIBUTE THINGS MORE FAIRLY OR REACH COMMUNITIES THAT ARE NOT CURRENTLY REACHED, THE ONES THAT DEAL WITH GETTING DISTRIBUTING HEALTH CARE, HEALTH CARE DELIVERY, THAT'S NOT THE NIH. AND SO THE QUESTION IS WITH WHOM WE NEED TO PARTNER OR TO WHOM WE NEED TO DELIVER THESE DATA THAT WE ACCRUE TO SAY THIS IS NOW YOUR MANDATE, AND WE'LL HELP YOU UNDERSTAND THE PROBLEM AND FASHION A WORKABLE SOLUTION BUT THE NIH BUDGET BY FEDERAL MANDATE CANNOT GO TO SPEND ON HEALTH CARE DELIVERY. WE CAN DO THE RESEARCH, SAY WHAT'S THE BEST PRACTICE, BUT WE CAN'T DO THE DELIVERING OF THE CARE. SO PART OF THE COMPLEXITY IS HOW THE FEDERAL PIE GETS DIVIDED UP. >> SO TO FOLLOW UP ON THAT, I THINK WHAT WE CURRENTLY DO IS PROBABLY IMPORTANT FOR YOU TO CONSIDER AS YOU SPEND TIME BETWEEN NOW AND TOMORROW. SO, CURRENTLY WE HAVE ACTUALLY A ROGRAM THAT'S LED BY KRYS, AND WE LOOK FOR PROJECTS THAT ARE AT PRE-CLINICAL STAGE AND LOOK FOR P.I.s TO BRING THOSE PROJECTS TO US FOR PEER REVIEW. AND THERE ARE TWO DIFFERENT WAYS THAT WE CAN PROVIDE THAT FUNDING TO DO THE PRE-CLINICAL WORK AND EARLY STAGE CLINICAL WORK. EITHER THE P.I. HAS A RESOURCE WITHIN THE UNIVERSITY OR WITHIN A COMPANY TO DO THE MANUFACTURE OR THEY COME UP WITH THE RIGHT PARTNER THROUGH WHICH THEY CAN DO THE MANUFACTURE, AS A COLLABORATIVE EFFORT TO DO THIS. SO, WHAT WE WANT TO KNOW IS HOW DO WE MAKE THIS PROGRAM BETTER? ARE THERE PARTS OF THE PROGRAM THAT'S NOT WORKING OUT? HOW DO WE MAKE IT SCALE? AND WE ALSO ARE ALWAYS REMINDED THAT THIS IS GOING TO GET BIGGER AS PATIENTS ARE BEING IDENTIFIED, TECHNOLOGIES ARE BEING DEVELOPED, WE'RE GOING TO GET A LOT MORE CUSTOMERS THAN WE HAVE CURRENTLY. CURRENTLY WE CAN PROVIDE UP TO ABOUT, WHAT IS IT, ABOUT $2 MILLION A YEAR TO DO THIS WORK, UP TO ABOUT 5 YEARS. KRYS CAN TELL YOU MORE DETAILS ABOUT WHAT WE DO. WE ARE OPEN FOR BUSINESS. WE ARE LOOKING FOR GOOD PROJECTS. AND WE ACTUALLY HAVE A FEW P.I.s WITHIN THIS GROUP THAT HAVE BENEFITED FROM THIS PROGRAM. SO THE QUESTION FOR US IS HOW DO WE MAKE THIS PROGRAM BIGGER, BETTER, DO WE BRING IN A SUITE OF CLINICAL -- CONTRACTORS OR CONSULTANTS TO WORK WITH P.I.s TO IDENTIFY BEST PRACTICES, SAMPLES, PROCESSES WE CAN PROVIDE OR PERHAPS BRING IN THE FDA. YOU GET AC SETS SAYS -- ACCESS TO FUNDING AND RECOLLECT LAKER TO EXPERTISE THAT'S CRITICAL. REGULATORY EXPERTISE. IT REMINDS ME OF THE VOUCHER PROJECT, THAT'S BROUGHT LOTS OF INDUSTRIES INTO THE FOLD. MAYBE THERE'S AN OPPORTUNITY FOR US TO WORK WITH THE FDA AND PERHAPS CREATE THIS ULTRA RARE SYSTEM THROUGH WHICH AN ACCELERATED OPTION IS NOW AVAILABLE FOR ALL DISEASES, NOT JUST PEDIATRICS. SO THINK ABOUT THAT. WE'RE DEFINITELY LOOKING FOR IDEAS, HOW TO MAKE THIS PROGRAM BETTER, AND THIS IS THE RIGHT COMMUNITY. AND THE IDEA OF WORKING OR CREATING A WORKING GROUP THAT CAN THINK ABOUT PROCESSES, STANDARDIZATION WOULD BE HELPFUL TO US. >> I THINK YOU SHOULD OPEN IT TO P.I.s FROM THE U.K. [LAUGHTER] >> ACTUALLY -- >> IT IS. >> IT IS DEFINITELY OPEN TO P.I.S FROM THE U.K. THERE'S A FOREIGN COMPONENT SYSTEM THAT WE CAN PROVIDE A >> OKAY. >> IF THERE ARE PATIENTS THAT ARE NOT AVAILABLE IN THE U.S. OR TECHNOLOGY THAT'S JUST NOT AVAILABLE IN THE U.S., SO THERE'S LOTS OF OPPORTUNITIES FOR INTERNATIONAL P.I.s TO PARTICIPATE IN OUR PROGRAMS. >> I WANT TO END ON ON ONE NOTE FROM NATIONAL ACADEMY MEETINGS THIS WEEK BROUGHT UP, IT WAS RAISED BY BECCA'S COMMENT, AND YOU PUT THE GLITCH IN THERE ABOUT HEALTH CARE DELIVERY FROM THE NIH. AND THAT IS -- SO ONE OF THE THINGS WE TALKED ABOUT WAS PATIENT SELECTION, EXCLUSION CRITERIA, AND HOW WE'RE ALL APPROACHED BY FOUNDATIONS ON A WEEKLY BASIS OR FAMILIES ON A WEEKLY BASIS, AND WHETHER OR NOT WE CAN DEVELOP THE GENE THERAPY PRODUCT FOR THEM AND IF IT MAKES -- IF IT'S SCIENTIFICALLY SOUND WE WORK TOWARD MOVING THAT FORWARD. IF IT'S NOT SCIENTIFICALLY SOUND WE TRY TO TELL THEM WHY AND THEY WILL MARCH DOWN THE VARIOUS INVESTIGATORS THEY CAN FIND UNTIL THEY FIND SOMEONE THAT CAN SAY YES TO THEM. AND THIS BRINGS UP A REALLY IMPORTANT CONSIDERATION THAT I THOUGHT WOULD BE SOLVED BY HAVING A ROAD MAP WHERE YOU GO, WHEN YOU HAVE THESE ULTRA ORPHAN DISEASES, TO GET GUIDANCE TO MOVE THERAPIES FORWARD OR NOT AND GO DOWN A DIFFERENT THERAPEUTIC PATHWAY AND TO SET UP THIS WORKING GROUP TO REALLY DEFINE HOW DO WE TAKE ADVANTAGE OF THAT, AND CENTRALIZE THIS AGAIN TAKING ADVANTAGE OF LUKE'S MODEL, WHAT EMA KOKUS DID FOR THE ENZYME REPLACEMENT COMMUNITY, JUDE AND KRYS AND OTHERS HAVE WORKED ON IN A ONE-OFF WAY WITH FAMILY FOUNDATIONS, AND I THINK THIS GETS BACK AND MAY SOLVE THE ISSUE THAT WE FACE, OR HELP ANYWAY, IN THE PATIENT EXCLUSION/INCLUSION CRITERIA BECAUSE IT'S NOT THOSE WITH HIGH SOCIOECONOMIC STATUS THAT CAN PAY FOR THE VECTOR FOR THEIR CHILDREN OR FAMILY MEMBER THAT NEEDS THE THERAPY. SO I WANT TO SUMMARIZEED KEY TAKEAWAYS AS I SEE THEM, STRATEGIES BROUGHT UP. ONE IS FROM THE FIRST SESSION, 3A, NEED TO USE THESE LARGE ANIMAL MODELS AND MAYBE FUNDING TO SUPPORT LARGE ANIMAL RESEARCH SO WE CAN UNDERSTAND WHAT WE'RE NOT TREATING WHEN WE DO GENE THERAPY TO THE BRAIN. AND I THINK THAT'S REALLY AN INTERESTING POINT. AND THAT WAS BROUGHT UP, SOMETHING THAT REALLY WE AREN'T THINKING ABOUT. WE CAN BE READY WHEN WE MOVE INTO THE PATIENTS PERHAPS OR DEVELOP AN ALTERNATIVE THERAPY TO GET AROUND THAT ISSUE. THE SECOND IS HOW TO SOLVE THE CHALLENGE OF REDOSING, REALLY NOT ENOUGH WORK DONE IN THAT SPACE. AND WE NEED TO THINK MORE BROADLY ABOUT HOW TO BRING IN OUR IMMUNOLOGY COLLEAGUES AND KAI I'M GLAD YOU'RE PART OF THE PROGRAM TO TALK ABOUT THIS A LITTLE BIT MORE. THE THIRD THING WAS TO SOMEHOW INITIATE THIS NIH-FDA COLLABORATIVE WORKING GROUP, TAKE ADVANTAGE OF THE FUNDING THROUGH CREATE AND UNITE PATHS AND DISTRIBUTE IT, AND WHO DO WE WORK WITH TO DISTRIBUTE IF GUIDANCE FROM THE NIH AND FDA AND HOW BEST TO ACCOMPLISH THAT. HOW TO RELEASE THE PRODUCT FOR HUMAN USE. AND THEN THE FIFTH THING AGAIN IS TO ADD ADDITIONAL WORKING GROUP TO WORK ON THE ANALYTICS SIDE OF THINGS AND SOPs THAT CAN HELP EVERYBODY IN THE CMC SPACE. AND SO I REALLY SEE TWO WORKING GROUPS FALLING OUT OF TODAY'S SESSIONS FOCUSED ON THESE VARIOUS ISSUES. >> OKAY.