>> GOOD AFTERNOON. LET ME WELCOME YOU TO A TALK BY DENNIS CLEGG IN THE AUDACIOUS GOALS INITIATIVE SEMINAR SERIES. I HAVE TO THANK DENNIS FOR EVEN SHOWING UP. HE SAYS IT WAS 84 IN SANTA BARBARA WHEN HE LEFT. SO DENNIS WE ARRANGED REALLY SPECIAL WEATHER FOR YOU TODAY HERE. THE AUDACIOUS GOALS INITIATIVE, AGI IS AS YOU KNOW AN EFFORT, A CONCERTED EFFORT TOWARD NEUROREGENRATIVE MEDICINE. THE TOPIC IS TO REGENERATE NEURONS AND NEUROCONNECTIONS IN THIS THE EYE AND THE VISUAL SYSTEM. THERE ARE MANY PARTS TO THE EYE BUT ONE OF THE CENTRAL PARTS IS THE NEURAL RETINA, THE TISSUE THAT IS RECEPTIVE TO LIGHT, LIGHT SENSITIVE, CONVERTS THAT INTO APPROPRIATE PANOPLY OF NERVE SIGNALING AND GIVES US THE ABILITY TO LOOK AT EACH OTHER HERE TODAY. DENNIS IS A BIOCHEMIST BY TRAINING, RECEIVED A Ph.D. FROM UC BERKELEY. IT'S NOT 23-DEGREES IN BERKELEY TODAY EITHER. AS A POST DOCTORAL FELLOW AT UCSF, WE STUDIED NEURAL DEVELOPMENT AND REGENERATION, HE THEN JOINED THE FACULTY AT UC SAPTA BARBARA WHERE HE STUDIES EXTRA CELLULAR MATRIX AND FUNCTION IN THE DEVELOPING EYE. HIS WORK TOOK HIM INTO THE DOMAIN OF STEM CELL BIOLOGY AND HE IS NOW FOCUSED ON DEVELOPING THERAPIES FOR OCULAR DISEASE USING THE TOOLS OF IPS AND OTHER DEVELOPMENTAL CELLS. HE IS THE PRINCIPAL CO INVESTIGATOR ON A DEC TEAM, ON A GRANT FOR THE CALIFORNIA INSTITUTE OF REGENRE MEDICINE. THIS A MULTIDISCIPLINARY EFFORT TO DEVELOP STEM CELLS FOR AGE RELATED MACULAR DEGENERATION. AS YOU CAN IMAGINE C CLEGG IS A RESENTIENT OF A NUMBER OF AWARDS FROM UCSB, AND IN 2013 HE WAS ONE OF 10 WINNERS OF THE NEI AUDACIOUS GOALS INITIATIVE IDEATION CHALLENGE. AS A PRESENTER HE HAS BEEN TO THE FRONTIERS IN VISION RESEARCH LECTURE AT NEI PREVIOUSLY. A KEY NOTE LECTURE AT THE STEM CELL WORLD CONGRESS AND HAS GIVEN A TED TALK. I AM IMPRESSED THAT HE IS A MEMBER OF ADVISORY BOARDS FOR THE C. I. R. CALIFORNIA INITIATIVE FOR REGENRATIVE MEDICINE AND HAS FAMILIARITY FOR THE NIH CENTER FOR REGENRATIVE MEDICINE. HIS THOUGHT TODAY IS VISION FOR THE FUTURE, CELL THERAPY FOR OCULAR DISEASE, PLEASE JOIN ME IN WELCOMING DENNIS CLEGG, DENNIS THANK YOU. >> THANK YOU. [ APPLAUSE ] ALL RIGHT, WELL, THANK YOU PAUL FOR THAT INTRODUCTION AND I'D LIKE TO THANK THE ORGANIZERS FOR THE INVITATION TO PRESENT TO YOU TODAY AND THANK YOU ALL FOR COMING. IT WAS 84 IN SANTA BARBARA AND I DID GO TO BERKELEY BUT I GREW UP IN MINNESOTA SO DISCIPLINARY PAY MY DUES. BELIEVE ME. --I DID PAY MY DUES. BELIEVE ME. HERE'S A PICTURE OF WHERE I WORK AND BEFORE I GET STARTED, I DO HAVE ONE FINANCIAL DISCLOSURE, I'M CO-FOUNDER WITH MARK HUMAYUNAND DAVID HINTON CALLED REGENRATIVE PATCH TECHNOLOGIES AND OUR GOAL IS WHAT I WILL TELL YOU SOME OF TODAY, WHICH IS TO DEVELOP STEM CELL BASED THERAPY FOR AGE RELATED MACULAR DEGENERATION. NEW NOW WHEN YOU LOOK FOR AN IMAGE FOR CELL THERAPY THIS IS WHAT I CAME UP WITH. [LAUGHTER] , IT'S A SHAMPOO, ANYBODY USE IT OUT THERE? I WANT TO KNOW WHAT IT LOOKS LIKE. OH, OH! OH, SEE, IT WORKS. [LAUGHTER] OKAY, THAT'S NOT WHAT I'M GOING TO BE TALKING ABOUT INSTEAD I'M GOING TO BE TALKING ABOUT OF WHAT SOME HAVE DESCRIBED AS THE NEXT PILLAR OF MEDICINE, SMALL MOLECULES AND BIOLOGICS AND IN THIS REVIEW ARTICLE BY FISCHBACH, THEY COMPARE SMALL MOLECULES AND BIOLOGICS WHICH WORK ON RECOGNITION TO CELLS WHICH ARE COMPLEX RESPONSE SYSTEMS SO A WHOLE DIFFERENT BALL OF WAX TO THINK ABOUT AS YOU'RE DEVELOPING A THERAPY AND A VERY EXCITING TIME, I THINK IN REGENRATIVE MEDICINE AS WE CONTEMPLATE USING CELLS TO TREAT A VARIETY OF AILMENTS. A LOT OF PEOPLE THINK THE EYE MAY BE A GOOD PLACE TO DEVELOP CELL THERAPIES, IT HAS ADVANTAGES OVER OTHER TISSUES. IF YOU THINK ABOUT FOR EXAMPLE, FINAL CORD INJURY OR HEART DISEASE, THE EYE IS IN SOME WAYS BETTER SUITED FOR DEVELOPING A THERAPY USING CELLS, FIRST THEIR ADVANCED SURGICAL METHODS TO GET INTO THE BACK OF THE EYE, THERE ARE GOOD WAYS TO IMAGE THE TISSUE USING NONINVASIVE IMAGING AND THEY'RE GOOD END POINT PARAMETERS TO TELL IF YOU'RE THERAPY IS WORKING NOT ONLY LOOKING AT AN EYE CHART BUT DETECTING WHICH PARTS OF THE RETINA ARE ACTUALLY WORKING. ANOTHER ADVANTAGE WE THINK IS THAT WE DON'T THINK THAT WE WILL NEED THAT MANY CELLS. IF YOU THINK ABOUT SPINAL CORD INJURY, THEY WERE TALKING ABOUT AND THEY'RE DOING CLINICAL TRIALS USING BILLIONS OF CELLS INJECTED IN THE SPINAL CORD AND THEN THEY DISAPPEAR AND THEY CAN'T FOLLOW THEM. IN THE EYE, WE THINK WE CAN GET AWAY WITH A HUNDRED THOUSAND CELLINGS IN THE THERAPY I'LL TELL YOU ABOUT TODAY THAT WE'RE DEVELOPING AND WE THINK THAT'S A REAL ADVANTAGE. AND WE LACK TREATMENTS FOR OCULAR DISEASE, THINGS LIKE RETINITIS PIGMENTOSA AND GLAUCOMA AND MACULAR DEGENERATION AND YOU PROBABLY MAYBE KNOW PEOPLE THAT ARE EFFECTED BY THESE DISEASES. HERE'S A PICTURE OF ME WITH LILLY WASH WHO HAS RETINITIS PIGMENTOSA AND MEGAN DOWNING WHO HAS EARLY ONSET OF MACULAR DEGENERATION SO THERE'S A UNMET MEDICAL NEED TO DEVELOP THERAPIES AND WE MIGHT BE ABLE TO USE STEM CELL THERAPY AND APPLICATIONSYS TO TREAT THESE DISEASES, I WILL TELL BUT AGE RELATED MACULAR DEGENERATION TODAY AND I KNOW MANY OF YOU ARE FAMILIAR WITH IT SO I'LL GIVE GIVE A SHORT INTRODUCTION AND I'M SURE YOU MAY KNOW PEOPLE WITH THIS DISEASE AS WELL. HERE'S A PICTURE OF ME WITH DAM JUDY DENCH AS YOU KNOW SUFFERS FROM MACULAR DEGENERATION. OKAY, SO A LITTLE BIT ABOUT MACULAR DEGENERATION, WHEN YOU GO INTO THE OPT TOM TRYST OR THE OPHTHALMOLOGIST, THEY TAKE PICTURES OF YOUR RETINA, AND THEY ARE LOOKING AT THESE YELLOW DOTS AND HA THAT'S EARLY A. M. D. AND IT CAN PROGRESS TO THE WET OR DRY FORM, THE WET FORM, INVOLVES IMPROPER GROWTH OF BLOOD VESSELS WHICH CAN THEN LEAK AND IT'S SERIOUS AND YOU CAN LOSE VISION QUITE RAPIDLY. THAT'S ONLY ABOUT FIVE-10% OF THE CASES AND FORTUNATELY, THERE ARE SEVERAL SMALL MOLECULE DRUGS THAT ARE BIOLOGICS THAT INHIBIT THE ACTIVITY OF VEG F AND WORK FOR MOST PATIENTS. THE TRY FORM WHICH INVOLVES THE FORMATION OF WHAT THEY CALL GEOGRAPHIC ATROPHIES, BIG YELLOW AREAS WHERE YOU'VE LOST BOTH PHOTOREACCEPTEDDORS AND RPECELLS IS ABOUT 90% OF THE CASES AND WE DON'T HAVE AN EFFECTIVE THERAPY FOR THAT FORM. NOW WE KNOW SOMETHING ABOUT THESE DISEASES AND WE MAY BE TALKING ABOUT MULTIPLE DISEASES HERE AND THERE'S STILL A LOT OF ONGOING RESEARCH BUT IN MOST CASES WE THINK IT'S THE DEATH OF THESE RPE CELLS, RETINAL PIGMENTED EPITHELIAL CELLS THAT LEAD TO THE PHOTORECEPTOR DEATH. RPE CELLS ARE SUPPORT CELLS FOR THE RODS AND CONES AND ONCE YOUR RPE DIES YOUR PHOTORECEPTORS DIE SOON AFTER AND YOU LOSE VISION. SO WHY DO WE THINK WE CAN DEVELOP A CELLULAR THERAPY. WELL WE KNOW THERE'S A WINDOW WHEN THE RP CELLS GENERATE AND YOU STILL HAVE VIABLE PHOTORECEPTORS AND IF WE COULD INTERVENE AND ADD IN FRESH RPE MAYBE WE COULD RESCUE THEM AND PREVENT THEM FROM DYING. AND IN FACT, TODAYS IN RODENT MODELS AND PATIENTS USING EXPERIMENTAL SURGERIES WHERE PARTS OF THE PERIPHERAL RP E ARE TRANSPLANTED TO THE CENTRAL RETINA SHOW THAT YOU CAN RESTORE THAT RPE PHOTORECEPTOR INTERACTION AND PRESENT PHOTORECEPTOR LOSS IN SOME PATIENTS. THERE ARE MANY CHALLENGES THOUGH TO THINK ABOUT IF YOU'RE TRYING TO DEVELOP THERAPY USING STEM CELL DERIVED RPE FOR AGE RELATED MACULAR DEGENERATION AND I WILL TALK ABOUT THREE GENERAL AREAS TODAY. FIRST OF ALL, CULTIVATION HOW DO WE MAKE THE RPE, WHAT STEM CELL DO WE START WITH, IPS, OR H. E. S., BOTH CAN GIVE RISE TO RPE CELLS. HOW DO WE CHANGE THOSE CELLS FROM AN UNDIFFERENTIATE THE PLURIPOTENT CELL TO A SPECIFIC DIFFERENTIATED SOMATIC CELL, WHAT'S THE PROTOCOL? AND HOW CAN WE EXPAND AND GROW ENOUGH TO TREAT A LOT OF THESE PATIENTS. SO THAT'S CULTIVATION, NEXT IS INSTALLATION AND IN PARTICULAR CAN WE SQUIRT IN A SUSPENSION OF CELLS OR WOULD IT BE BET TORE IMPLANT A MONOLAYER GROWN ON A SCAFFOLD, A SORT OF GRAPH THAT YOU WOULD--GRAFT THAT YOU WOULD PUT IN. AND SHOULD IT BE MADE OF? SHOULD IT BE BIODEGRADABLE SO IT DISAPPEARS OVER TIME OR SOMETHING BIOSTABLE SO IT WILL STAY THERE FOR THE LIFE OF THE PATIENT. AND THEN COURSE OF TREATMENT, HOW CAN WE BRING ABOUT SURVIVAL AND FUNCTION OF THE CELLS THAT WE TRANSPLANT AND WE WANT TO AVOID UNWANTED PROLIFERATION SPORTSMANSHIP MIGRATION. WE DON'T WANT THE CELLS TO GROW OUT OF CONTROL, AS YOU KNOW PLURIPOTENT STEM CELLS LIKE TO GROW AND HAVE THE ABILITY TO FORM KERRA TELOMERES IN MOUSE MODELS, WE WOULDN'T WANT THAT TO HAPPEN IN A PATIENT. THOSE ARE OUR CHALLENGES, CULTERATION, INSTALLATION AND PRESERVATION. SO LET ME DIVE IN AND FIRST TALK ABOUT WHAT STEM CELL DO YOU THINK SHOULD BE USED TO MAKE RPE, AND PREVIOUS STUDIES HAVE SHOWN THAT IPS AND H. E. S. CAN BE COAXED TO GENERATE RPE CELLS. WE WERE INVOLVED EARLY ON IN STUDIES OF IPS CELLS FROM JAMIE THOMPSON'S LAB AND WE SHOWED THAT IF YOU JUST TAKE A CULTURE OF IPS GROWN AS THESE COLONIES IN UNDIFFERENTIATED FORM IN THE PRESENCE EVER FIBROBLAST GROWTH FACTOR AND IF YOU JUST REMOVE THE FGF, THOSE CELLS WILL DIFFERENTIATE INTO EVERYTHING UNDER THE SUN. YOU CAN GET HEART CELLS, NEURONS, ET CETERA, AND IF YOU WAIT LONG ENOUGH, SOMETIMES YOU CAN SEE PIGMENTED COLONIES THAT APPEAR. THESE CAN BE EXCISED AND EXPANDED AND WE SHOWED A FEW YEARS BACK THAT YOU CAN MAKE NICE LOOKING RPEs THAT ARE SIMILAR BUT NOT IDENTICAL TO FETAL HUMAN RPE CELLS AND THEY FORM THIS NICE COBBLESTONE MONOLAYER WITH VARIABLE PIGMENTATION IN CULTURE. AND IN FACT THE IPS RP, TRANSITION TO A CLINICAL TRIAL IN JAPAN AS MANY OF YOU ARE AWARE, MASAYO TAKAHASHI, GREW THIS LAYER ON A COLLAGEN SUBSTRATE FOR THE TREATMENT OF WET AMD IN JAPAN AND SHE GAVE A REPORT AT THE STEM CELL SUMMIT LAST YEAR. THE 50 PATIENT WAS IMPLANTED IN--THE FIRST PATIENT WAS IMPLANTED IN SEPTEMBER OF 2014, THEY STARTED MAKING 24 DIFFERENT IPS LINES FROM THE PATIENT'S SKIN OR BLOOD, I'M NOT SURE WHICH, THAT TOOK FOUR MONTHS, THEN FROM THOSE LINES THEY FOUND SIX THAT WERE ABLE TO MAKE RPE, ANOTHER FOUR MONTHS AND THEN THREE THAT WERE GROWN UP AND MADE FOR THE IMPLANT. WHAT THEY REPORTED ON THIS PATIENT WAS LOOKING AT SAFETY, THERE WERE NO TUMORS WHICH IS GOOD, NO IMMUNE REJECTION, THEY HAD DIDN'T USE IMUE O SUPPRESSION, NO DETACHMENT OR TEARS AND THEY COULD DETECT THE IPS -RPE, THERE WAS A TEMPORARY HEMORRHAGE BUT THEY DEALT WITH IT SURGICALLY. THE SECONDARY EFFECT WAS DECREASED EDEMA, NO RECURRENCE OF VASCULARIZATION, BETTER QUALITY OF LIFE BY THE PATIENT BUT NO INCREASE IN VISUAL OOH CUITY OR IMPROVEMENT OF MFERG, NOW THEY WENT IN AND LASERRED BLOOD VESSELS IN THIS PATIENT BEFORE DOING THE IMPLANT. THEY WENT ON TO A SECOND PATIENT AND IN THE SECOND PATIENT, THEY FOUND IN DOING SEQUENCING OF THE IPS LINES, THAT THEY PICKED UP A MUTATION IN AN ONCA GENE. AND SO THEY STOPPED THE TRIAL. THEY WERE WORRIED ABOUT THAT AND NOW THEY'VE DECIDED TO GO ON TO AN ALOE GENERATED AIC APPROACH WITH A PHASE ONE CLINICAL TRIAL IN 2017. SO CAN YOU SEE THAT THERE'S CERTAIN CHALLENGES WITH THE AHATOLOGIOUS APPROACH AND I THINK WE'RE MAKING PROGRESS TOWARDS SOLVING SOME OF THESE CHALLENGES AND THE GROUP HERE FOR EXAMPLE, AT NEI IS MAKING TREMENDOUS PROGRESS TOWARDS AN AHATOLOGIOUS TREATMENT. BUT ANOTHER APPROACH IS TO START WITH A BANK OF SUPER DONOR HLA-MAPPED IPS LINES AND WE'RE INVOLVED IN A COLLABORATION WITH DAVID GAMM SHOWN HERE THIS, IS TO SHOW HE'S VERY, TALL, TALL AS THE LIGHT HOUSE AND WE'RE FUNDED BY THE FOUNDATION FIGHTING BLINDNESS IN A WYNN GUND TRANSLATIONAL ACCELERATION GRANT. THE GOAL IS TO MAKE THESE IPS LINES AND GENERATE PHOTORECEPTORS AND COMBINE THEM AND TREAT PATIENTS THAT ARE LACKING BOTH CELL TYPES. THAT WORK IS UNDERWAY AND THE IDEA IS TO TAKE RPE WHICH WE CAN GROW ON A PLANAR SCAFFOLD AND ADD IN PHOTORECEPTORS AND USE THOSE FOR A COMBINATION TO TREAT LATE STAGE ATROPIC AMD FOR EXAMPLE WHERE YOU LOST BOTH PHOTORECEPTORS AND RPE, AND OTHER RETINAL DISEASES, BUT I'M NOT GOING TO TALK TOO MUCH ABOUT THAT WORK TODAY. IT'S STILL IN EARLY STAGES. YOU CAN ALSO MAKE RPE FROM EMBRYONIC STEM CELLS AND WE'RE MUCH FURTHER ALONG ON THIS PROJECT AND HERE'S A COLORED PICTURE FROM DAVID HINTON'S GROUP SHOWING STEM CELL DERIVED RPE WITH APICAL MICROVILLI, ONE CELL IS OUTLINED HERE AND YOU CAN SEE NICE POLARIZED MONOLAYERS GROWN FROM HES DERIVED--HES DERIVED RPE. WE STARTED THE CALIFORNIA PROJECT TO CURE BLINDNESS WITH FUNDING FROM THE CALIFORNIA INSTITUTE FOR REGENRATIVE MEDICINE WHICH THE COLLABORATION BETWEEN USC, UC SANTA BARBARA, CALL, TECH, CITY OF HOPE AND THE START UP COMPANY I MENTIONED, REGENRATIVE PATCH TECHNOLOGY WERE LED BY MARK HUNAYUN, AND MYSELF AND OTHERS AT THE USC KECK SCHOOL OF MEDICINE. WE WANTED TO START WITH THE STEM CELLS AND MAKE A HOMOGENIUS POPULATION OF RPE. AND WE'RE ACTUALLY INDEBTED TO THIS NEW GROUP, AMERICANS FOR CURES THAT HAVE DEVELOPED A WHITE BOARD PRESENTATION TO TELL YOU ABOUT OUR PROJECT AND I'LL SHOW YOU PART OF THIS. IT'S CLEVER, IT'S DESIGNED FOR A GENERAL ADIENCE TO SHOW WHAT THEY'VE FUNDED. >> CAN YOU IMAGINE GRADUALLY LOADING THE ABILITY TO SEE, MOST SURVEYS OF PEOPLE AROUND THE WORLD AGREE THAT VISION IS THE SENSE PEOPLE FEAR LOSING THE MOST. WE RELY ON THIS PRECIOUS SENSE TO ENGAGE WITH THE WORLD AROUND US, DEVELOP RELATIONSHIPS, REACT TO THE SITE OF A UNIQUE--SIGHT OF A UNIQUE BUILDING OR A BEAUTIFUL FLOWER. UNFORTUNATELY THERE'S 11 MILLION AMERICANS WITH A DISEASE CALLED AMD, AGE RELATED MACULAR DEGENERATION ALREADY 2 MILLION HAVE LOST VISION BECAUSE OF THE DEVASTATING FORM OF THIS DISEASE, IT'S A CURABLE DISEASE MOST COMMONLY SEEN IN THOSE OVER 50. IT BECOMES COMMON AS PEOPLE AGE. ONE IN FIVE AMERICANS OVER THE AGE OF 50 AND ONE IN FOUR AMERICANS OVER THE AGE OF 65 WILL DEVELOP AMD. WITHIN THE RAISE OF POPULATION, IT WILL RISE MAKING IT A CRITICAL HEALTH PROBLEM. AMD IS CAUSED BY THE DEGENERATION OF LIGHT SENSING CELLS IN THE EYE, SPECIFICALLY IN THE PART OF THE EYE KNOWN AS THE RETINA. NORMALLY WE CAN SEE THESE BECAUSE THESE CELLS TAKE IN THE LIGHT AROUND US AND THEN SEND IT IN ELECTRICAL IMPULSES TO OUR BRAIN. ANYWAY I THINK YOU KNOW THAT SO I'LL SKIP THAT. I CAN'T DRAW THAT FASTOT WHITE BOARD, BUT AMERICANS FOR CURE, HAVE A SUMMARY OF FUNDED PROJECT THERE IS AND IT'S A NEW EFFORT BY BOB KLEIN WHO WROTE PROP 71 TO GET THE WORD OUT TO THE PUBLIC ABOUT THE GOOD STUFF THAT SERUM HAS BEEN DOING SO I WOULD ENCOURAGE TO YOU LOOK AT THAT WEB SITE. SO ONE OF THE THINGS WE WANTED TO DO WHEN WE STARTED THIS PROJECT WAS ANSWER THIS QUESTION, CAN WE IMPROVE THE EFFICIENCY OF DIFFERENTIATION, THIS IS THE FIRST REPORT OF MAKING RPE CELLS FROM EMBRYONIC STEM CELLS FROM THE GROUP AT ACT LED BY BOB LANZA, AND IT'S A SIX FILLED WELLS AND YOU CAN SEE IT FULL OF WHITE GELAT NOWS MASS, SO IN THIS THIS PROTOCOL YOU'RE NOT GETTING MANY RPE CELLS AND CAN WE ESTABLISH A WAY TO REALLY PUSH THE CELLS TOWARDS RPE USING FACTORS THAT ARE FOUND IN NORMAL DEVELOPMENT OF RPE. WELL, ONE METHOD THAT WE USED WAS TO START WITH THAT SPONTANEOUS METHOD WE CALL SPONTANEOUS WHERE YOU'RE CONTINUOUSLY GROWING CELLS IN CULTURE, REMOVE THE FGF AND WE SCREENED A NUMBER OF CELL LINES AND CULTURE CONDITION AND THE CELL LINE FROM WISCONSIN IF YOU REMOVE FGF AND YOU CAN SEE SOME OF THE CELLS BECOMING BIG MENTORSHIP SKILLED. WE THEN WORKED OUT WAYS TO ENZYMATICALLY HARVEST THESE, ENRICH FOR THESE AND IT TURNS OUT THE RPE, HELPED US OUT HERE IN THAT THEY'RE ADHERENT AND THEY OUTCOMPETE THE CONTAMINATING UNPIGMENTED CELLS SUCH THAT AFTER SEVERAL PASSAGES YOU CAN GET CULTURES THAT ARE NOON% POSITIVE FOR RPE MARKERS LIKE PMEL SHOWN IN GREEN HERE. AND SOME CELL LINES DON'T DO THIS BUT H-NINE IS GOOD AT MAKING RPE, WHICH IS GOOD AS THE DEFAULT PATH WAYS DIFFERENTIATE AND VERY IMPORTANTLY WE DON'T SEE ANY UNDIFFERENTIATED HESC, GROW. LET SO THAT'S ONE WAY TO MAKE THEM BUT IT TAKES A LONG TIME AND SOME CELL LINES IT DOESN'T REALLY WORK VERY WELL. SO WE ALSO LOOKED AT A WAY TO REALLY FORCE THEM TO BECOME RPE BY ADDING GROWTH FACTORS. CAN WE MIMIC THE IN VIVO PROCESS WHERE CELLS FIRST URPD GO NEURAL INDUCTION AND THEN BECOME EARLY FIELD, THEN OPTIC VESICLE THEN MATURE RPE. AND A LOT IS KNOWN FROM STUDIES OF MICE AND FROGS AND ANOTHER THING WE WANTED TO KNOW IS CAN WE SPEED THIS PROCESS UP. YOU ARE STARTING WITH AN EMBRYONIC STEM CELL, HUMAN RPE PIGMENTATION APPEARS AFTER 42 DAYS, A NEW WAY TO SPEED THAT UP. AND DAVE BUCK HOLTS IN THE LAB EXPERIMENTED WHEN HE TRIED THIS PROJECT HE GAVE UP AND THEN WHEN HE WAS A FOURTH YEAR STUNTED HE CAME BACK TO ME AND I SAID I THINK I KNOW HOW TO DO THIS AND IT'S DIFFICULT TO DO BECAUSE IT'S MORE OR LESS IMPERICAL, YOU'RE ADDING GROWTH FACTORS THAT YOU KNOW ARE IMPORTANT IN DIFFERENT SEQUENCE AT DIFFERENT TIMES AND HE CAME UP WITH WHAT WE CALL THE 14 DAY DIRECTED DIFFERENTIATION PROTOCOL WHERE YOU START BY ADDING IGF AND INHIBIT BOTH THE WET AND PATHWAYS WITH THESE FACTORS UP HERE AND THEN ADD FGF AT DAY TWO THEN LET THEM GROW FOR A WHILE AND THEN INHIBIT FGF AT A LATER STAGE WITH THIS SMALL MOLECULE COMPOUND AND THEN STIMULATE WITH ACTIVEIN A. MENT AND DAVE SHOWED AFTER 14 DAYS HE COULD GET CULTURES THAT WERE 80% POSITIVE FOR EARLY RPE MARKERS. THEY WEREN'T QUITE PIGMENTED YET AND IT TAKES AN ADDITIONAL 60 DAYS TO MAKE MORE MATURE RPE, BUT AS YOU LOOK AT REALTIME PC R, YOU CAN SEE OUR CORE EXPRESSION AND NANOG GOING DOWN AND BEST AND TYROSINASE IMPROVE IN TIME. WE SHOWED WITH LINDSAY LEACH IN THE LAB WHO SHOWED IF YOU ADDED A LATE STAGE, USING AN AGONIST, CAN YOU IMPROVE THE RPE DIFFERENTIATION AND GET CULTURES THAT ARE VERY HOMOGENIUS FOR RPE. SO THERE ARE DIFFERENT WAYS TO MAKE THEM, THE FAST WAY THAT I JUST TOLD YOU ABOUT, AND THE SLOWER SPONTANEOUS METHOD, AND RECENTLY, ROXANNE CROW AND LINDSAY LEACH ASKED THIS QUESTION: HOW DO YOU COMPARE THE DIFFERENTIATED CELLS VIA THE DIRECTED PATHWAY TO THE SLOWER METHOD. HOW DO THEY COMPARE? WHICH ONE'S BETTER? AND HERE ARE THE TWO METHODS, YOU CAN SEE THE TIMELINE. THE SLOW ONE ON THE BOTTOM TAKES ABOUT 170 DAYS, A LONG TIME. THE FASTER ONE, YOU CAN GET 80% AFTER 14 DAYS BUT THEN AS I SAID, YOU NEED ABOUT 60 DAYS TO REALLY GET THESE TO A MORE MATURE STATE AND WE CAN ACTUALLY CRYOPRESERVE THESE AT AN INTERMEDIATE STATE OF PASSAGE ONE AND TWO HERE AND COMPARE THEM. WE COMPARED FIVE INTEGRATION FREE IPS LINES FROM JAMIE THOMPSON'S LAB AND WHAT WE FOUND IS THAT THIS SPONTANEOUS METHOD ONLY WORKED FOR THREE OF THE FIVE LINES WHEREAS THE DIRECTED METHOD WORKED FOR ALL FIVE LINES. SO THIS ONE, FORCING THEM WITH GROWTH FACTORS IS MORE RELIABLE AND THEN WE LOOKED BY RNA SEQ TO COMPARE THEM TO FETAL RPE AND YOU CAN SEE THIS IS ALL RED BECAUSE THEY'RE VERY CLOSE, THE SPONTANEOUS OR SP AND THE DIRECTED ARE D. I. R. AND HERE ARE THREE FETAL RPE AND THREE DIFFERENT BATCHES FROM BOTH METHODS. AND IF YOU LOOK AT THE DIFFERENTIALLY EXPRESSED GENES, COMPARING TO FETAL RPE, THEY'RE BOTH ABOUT FIVE% DIFFERENT FROM FETAL RPE WHICH WE USE AS SORT OF A GOLD STANDARD TO COMPARE TO THESE STEM CELL RPE SO THEY'RE SIMILAR BUT NOT IDENTICAL, FIVE OF THEM EXPRESS GREATER THAN TWO FOLD AND IN FACT, COMPARED TO EACH OTHER, THEY'RE VERY CLOSE, ONLY SIX% ARE DIFIENTIALLY EXPRESSED GENES AND THIS HAS BEEN SUBMITTED FOR PUBLICATION. ONE OF THE UNDERLYING MECHANISMS THAT REGULATE RPE DIFFERENTIATION, WE WOULD LIKE TO KNOW MORE ABOUT THAT PATHWAY SO WE CAN CONTROL IT AND KERRY HOO IN THE LAB HAS BEEN LOOKING AT THE MICRORNA POPULATION AND I KNOW THIS IS HARD TO SEE BUT HE'S IDENTIFIED MicroRNA IN THE LAB FROM THE DATA REPORTED BY PING'S LAB IN UCLA AND HERE ARE THE MIRAKA 19 RNAs AND ON THE X AXIS YOU'RE GOING GOING FROM IMMATURE UNDIFREPRESENTIATED PLURIPOTENT CELLS TO IMMATURE TO MATURE LEVELS AND YOU CAN SEE THE LEVELS GO DOWN AND I WILL MAKE A LONG STORY SHORT HERE AND JUST SHOW YOU SOME OF HIS WORK ON THE WORK OF MIR-19S IN DIFFERENTIATION AND HE FOUND THAT RAX IS AN IMPORTANT GENE AND HE SHOWED IF YOU TAKE THE UNTRANSLATED REGION OF RAX AND ADD IT TO A LUCIFERASE REPORTER, YOU GET DOWN REGULATION OF GENE EXPRESSION VIA INTERACTION WITH THESE THREE PRIME REGION AND THEN HE WENT ON TO SHOW IF YOU KNOCK DOWN MIR-19 YOU INCREASE RAS EXPRESSION AND YOU INCREASE GENERATION OF RPE AND THIS IS IN THE HNINE CELL LINE WHERE THIS IS THE NUMBER OF PIGMENTED COLONIESOT Y-AXIS AND DAYS IN CULTURE AND YOU CAN SEE THE KNOCK DOWN OF MIRs INCREASES THE RAX REGENERATION, HE SHOWED THAT DECREASE RAS EXPRESSION IN THIS EXPERIMENT, DECREASED IN TWO DIFFERENT LINES THE GENERATION OF RPE, SO WE THINK MIR 19 ARE ONE OF THE KEY MOLECULAR FACTORS THAT REGULATE RPE PRODUCTION AND MAYBE AN INDICATOR IF YOU'RE LOOKING FOR A MARKER THAT MIGHT PREDICT HOW THESE CELLS ARE GOING TO RESPOND. ANOTHER ISSUE, THOSE OF YOU THAT WORK ON RPE, KNOW ABOUT, IS THAT IF YOU TRY TO CULTURE THEM TOO LONG, THEY START CHANGE NOTHING CULTURE, THERE'S AN EMT OR TRANSITION THAT OCCURS BOTH IN FETAL RPE, AND IN STEM CELL DERIVED RPE, TYPICALLY AFTER FIVE PASSAGES, THEY GO FROM THIS MONOLAYER LOOKING LIKE FIBROBLAST. AND ROXANNE CROZE, DECIDED TO LOOK AT WAYS WE MIGHT AVOID THESE AND LOOK AT INHIBITORS OF THE ROCK ONE AND TWO, KINASES AND THOSE ASSOCIATED KINASES THAT ARE KNOWN TO BE MODULATORS OF THE CYTOSKELETON. AND WE KNEW THAT YOU CAN PREVENT APOPTOSIS IN BY ADDING ROCK INHIBITOR SO SHE DEVELOPED A PROTOCOL TO EXPAND THESE CELLS USING ROCK INHIBITOR Y-27 AND SHE SHOWED YOU COULD GET UP ALL THE PASSAGES BUT THEY'RE SIMILAR TO THESE PASSAGE ONE OR TWO CELLS. AFTER PASS 18 THEY START TO UNDERGO THIS TRANSITION AND START TO FINESSE, BUT BY ADDING JUST FOR THE FIRST FIVE DAYS OF CULTURE AND USING THIS PASSAGE METHOD, SHE COULD GET UP TO 13 PASSAGES: OF THESE CELLS, SO IT'S A GREAT WAY TO EXPAND YOUR NUMBERS OF RPE. WHAT'S GOING ON THERE? WELL SHE LOOKED AT TRANSCRIPTIONAL PATTERNS AND AGAIN TO MAKE A LONG STORY SHORT, SEE SAW THAT THE CELL CYCLE WAS INCREASED BY THE ROCK INHIBITION WHICH MAY ALLOW THE CELL TO DIVIDE AND REGAIN TIGHT JUNCTIONS AND CONTINUE TO GROW AND SEE SAW DECREASED EXPRESSION OF TGF BETA AND WNT SIGNALING PATHWAYS SO THAT MIGHT INHIBIT THE TGF BETA OR WNT. SO WHEN I WAS DRIVING DOWN THE STREET AND I SAW THIS SIGN, YOU'VE SEEN IT BEFORE, COVER THE EARTH. SO I CAME IN THE LAB AND I SAID DO YOU THINK YOU COULD COVER THE EARTH WITH RPE CELLS? SO WE DID THIS CALCULATION SURFACE AREA OF THE EARTH, RPE, SURFACE AREA, IT WOULD BE DIFFICULT. SO YOU COULD COVER THE EARTH IF YOU START WIDE 125 BILLION SIX-WELL PLATES AND ROXANNE'S LIKE YOU'RE CRAZY. WANT NO BUT THEN I THOUGHT, WELL, WHAT ABOUT LOOKING AT ALL THE PEOPLE WITH MACULAR DEGENERATION. COULD WE MAKE ENOUGH RPE TO COVER THE EYE WITH RPE AND TREAT LET'S SAY 20 MILLION, A ROUGH NUMBER OF PATIENTS IN THE WORLD, A HUNDRED K PERPATIENT. YOU COULD GENERATE THAT MANY RPE STARTING FROM A HUNDRED SIX WELL PLATES IN 55 DAYS. SO WE THINK WE HAVE AN ABUNDANT SOURCE OF RPE TO TREAT A LARGE NUMBER OF PATIENTS. OKAY, CAN WE CONVERT THESE RPE TO DIFFERENT CELL TYPES? WELL WE LOOKED AT WHETHER OR NOT WE COULD REPROGRAM THESE CELLS TO NEURONS USING RECOMBINANT PROTEIN, A FOX TWO FACTOR WITH A CELL MEMBRANE PENETRATING PEPTIDE THAT WAS SHOWED HERE, AND YOU COULD MAKE THESE INTO NEURONS USING THIS APPROACH AND THERE'S SOME EVIDENCE THAT RPE CAN BE CONVERTED TO PHOTORECEPTORS FROM THE WONG LAB IN ALABAMA. SALLY KEMPO UP IN NEW YORK HAS SHOWN EVIDENCE THAT THE ADULT HUMAN RPE MIGHT INCLUDE A STEM CELL THAT COULD MAKE MULTIPLE CELL TYPES. SO NOW THAT WE HAVE A WAY TO MAKE A HOMOGENIUS POPULATION OF CELLS AND MAKE LOTS OF THEM, THERE MIGHT BE A POSSIBILITY TO CONVERT THEM TO DIFFERENT CELL TYPES FOR OTHER CELL THERAPY APPLICATION. ALL RIGHT, LET'S GO ON TO INSTALLATION. HOW DO WE DELIVER THESE CELLS, INJECTION OF A SUSPENSION OR A GRAFT AND WE'RE TARGETING THE SUBRETINAL SPACE, RIGHT? BETWEEN THE RPE AND THE PHOTORECEPTORS SHOWN HERE IN THIS CARTOON. WHEN WE COLLABORATES WITH PETE COFFEE TO LOOK AT IPS RPE IN THE RCS RAT MODEL WE FOUND WHEN WE INJEBTED THE CELLS THEY FORM THESE CLUMPS AND THEY'RE STAINED FOR HUMAN SPECIFIC MARKER IN GREEN HERE AND YOU CAN SEE CLUMPS OF CELLS, NOT IDEAL, YOU HOPE THEY WOULD INTEGRATE MORE INTO THE EXISTING MODEL LAYER OF RPE AND WE ASKED WHETHER AN UPDATE COULD OUTER PH AGOCYTOSE THIS, AND OCCASIONALLY YOU COULD SEE THEM PICK UP THE RHODOPSIN, BUT NOT A LOT. IN FACT IN THIS EXPERIMENT REPORTED BY AMANDA CARR, ET, AL, WE FOUND THE CELLS DISAPPEARED AFTER A WHILE AND THERE'S A WELL KNOWN CELL BIOLOGICAL PHENOMENON CALLED ANOIKIS, WHICH IS HOMELESSNESS AND WE KNOW IF CELLS ARE NOT IN CONTACT WITH OTHER MATRIX, THEY UNDER GO APOPTOSIS. THEY HAVE TO BE BOUND TO SEND SURVIVAL SIGNALS TO THE CELL AND THIS MIGHT BE A ROAD BLOCK TO CELL TRANSPLANTATION. AND IF YOU THINK ABOUT AMD AND YOU HAVE GEOGRAPHIC ATROPHY, STUDIES HAVE SHOWN THAT THE BROOKS MEMBRANE THAT AMD, IN AMD PATIENTS AND COMPROMISED AND MAY NOT SUPPORT RPE CELLS, SUSPENSIONS THAT ARE INJECTED. THE OPHTHALMOLOGIST OUT THERE LOOKING AT THAT PHOTOAND SAYING THAT LOOKS REALLY MESSED UP, BUT THAT'S ACTUALLY MARS, THE PLANET. BUT IT MAY BE LIKE PUTTING YOURSELF ON MARS; OKAY, SO OUR APPROACH IS TO PROVIDE THE CELLS WITH A HOME. BASICALLY, PUT THEM ON A SCAFFOLD THAT'S COATED WITH AN EXTRA CELLULAR MATRIX PROTEIN SO THAT THE INTEGRINS ARE ENGAGED AND THEY HAVE A SURVIVAL ADVANTAGE. WE'RE USING A SCAFFOLD MADE OUT OF A XYLENE POLYMER CALLED PARALEAN AND SHOWN IN PURPLE HERE AND HERE'S A SCANNING E. M. PICTURE FROM DAVID HINTON'S LAB SHOWING THE CELLS WITH THE MICROVILLI SITTING ON THIS SUBSTRATE. NOW THE ENGINEERS ATICAL TECH DESIGNED THIS TO HAVE ULTRA THIN AREAS SUPPORTED BY A MESH OF THICKER [INDISCERNIBLE] SO THAT THEY WOULD WILL BE GOOD DIFFUSION PROPERTIES AND IN FACT THE DIFFUSION PROPERTIES ARE SIMILAR TO NATIVE BROOKS MEMBRANE. SO THERE ARE A LOT OF REASONS I HAVEN'T MENTIONED WHY A POLARIZED RPE ON A SCAFFOLD MIGHT BE BETTER THAN A SUSPENSION. WORK FROM DAVID HINTON'S LAB JUST AS ONE EXAMPLE IS SHOWN THE POLARIZED RPE MAKE A LOT MORE PEDF, COMPARED TO NONPOLARRIZED RPE AND THEY'RE MORE RESIST APT TO STRESS AND THEY'RE GOING TO BE ORIENTED TO CARRY OUT PHAGOCYTOSIS AND THIS IS ALREADY APPROVED FOR USE IN THE EYE, IT'S WHAT THE R-GUS TWO IS COATED WITH AS AN IMPLANT AND IT'S BEEN USED TO COAT STINTS AND ELECTRODES FOR DEEP BRAIN STIMULATION. AND WE GROW THE CELLS ON THIS FLAT PART OF THE PARALEAN, DOESN'T HAVE ANY HOLES IN IT, AND THE CELLS ARE VERY HAPPY AND MAKE A NICE MONOLAYER ON THERE AND THIS IS DATA SHOWING THAT THE DIFFUSION OF THE PARALEAN IS SIMILAR TO BROOKS MEMBRANE WHERE YOU HAVE THREES LARGE BARS BUT IN THE SAME RANGE WHEN YOU LOOK AT WAVES VERSUS FLUXES SO WE WERE FAMILIES WITH THIS AND IN OUR HANDS SOME OF THE BIODEGRADABLE SCAFFOLDS WE LOOKED AT WERE QUITE TOXIC TO THE RETINA AND BECAUSE OF THE DEED LINES WE HAD TO LOCK DOWN A SCAFFOLD. THERE MIGHT BE A GOOD BIODEGRADABLE SOLUTION BUT WE JUST DIDN'T HAVE TIME TO FIND IT AND WE LOCKED DOWN ON THE PARALEAN SCAFFOLD. WE SHOWED IN SIDE BY SIDE COMPARISONS OF SUSPENSIONS VERSES CELLS ON THEARA LEAN THAT THE CELLS SURVIVED FOR MUCH LONGER AS MONOLAYERS OF RPE COMPARED TO CELLS AS SUSPENSION AND I WON'T GO OVER THIS DATA IN DETAIL BUT WE THINK THAT'S BETTER. OKAY, WHAT ABOUT ANIMAL MODELS, CAN WE SHOW THAT THIS WORKS IN VIVO. WE USE THE RCS RAT MODEL AND I TALK ABOUT THIS WITH PEOPLE TODAY, IT'S NOT A PERFECT MODEL FOR MACULAR DEGENERATION AND IN FACT, THERE IS NO PERFECT ANIMAL MODEL FOR MACULAR DEGENERATION. THESE RATS LACK FUNCTION OF THE MERTK RECEPTOR INVOLVED IN PHAGOCYTOSIS BUT THEN THE RECEPTORS DEGENERATE AFTER ABOUT 12 WEEKS. SO THE IDEA IS TO PLANT THE PATCH AT POSTNATAL DAY 28 AND SEE IF WE CAN RESCUE THOSE PHOTORECEPTORS AND WE CAN MEASURE THE PHOTORECEPTOR SURVIVAL AND VISUAL FUNCTION OVER TIME AND I'LL SHOW YOU DATA FROM DAVID HINTON'S LAB WHERE HERE'S THE PARALEAN IMPLANT RIGHT HERE WITH THE RPE CELLS AND HERE'S THE PHOTORECEPTOR NUCLEAR LAYER AND YOU CAN SEE RESCUE IN THE AREA OF THE IMPLANT BUT NO RESCUE AWAY FROM THE IMPLANT, AREA IN THE RCS RAT, SO WE'VE SHOWN THAT WE CAN RESCUE IN THAT MODEL AND THAT'S TWO MONTHS AFTER TRANSPLANTATION AND THEN WE WONDERED HOW LONG CAN THESE SURVIVE? AND THIS IS A SECTION FROM A RAT THAT WAS TRANSPLANTED YEAR BEFORE AND YOU CAN DETECT THIS TRIAL IN 85 HUMAN SPECIFIC ANTIBODY LABELING THIS MONOLAYER A YEAR AFTER TRANSPLANTATION. NOW SOME OF YOU MIGHT BE SAYING WELL THAT'S NOT A COMPLETE PHOTORECEPTOR ON A NUCLEAR LAYER BUT WE IMPLANT IT AT DAY 28 AFTER ACTUALLY MANY OF THE FET O RECEPTORS ALREADY STARTED TO DEGENERATE AND THE REASON IS IT'S A HARD SURGERY TO DO AND THE EYE IS SO SMALL IN THE RAT THAT WE HAD TO WAIT UNTIL 28 DAYS FOR THE SUSPENSION EXPERIMENTS YOU'VE SEEN IN THE LITERATURE THEY GO IN AT DAY 21 AND THEY SEE MORE ROBUST RESCUE BECAUSE FEWER HAVE ALREADY DEGENERATED. OKAY, SO CAN THEYA FAG O SIGNIFYITOSE OUTER SEGMENTS? WELL WE LOOKED AT RHODOBSIN, AND IF YOU COMPARE TO UNTRANSPLANTED CELLS YOU DON'T SEE ANY PHAGOCYTOSIS AND IF YOU DO, YOU SEE NO PHAGOCYTOSIS, SO WE SEE FAG O STUDIES OF STUDIES OF MULTIPLE ENDOCRINES--PH AGOSOMES 60 DAYS AFTER TRANSLATION. NOW THESE CELLS IN THE RETINA, WOOING AND ARE THEY HOOKED UP TO THE BRAIN. THOMAS DID THESE STUDIES LOOKING AT A MAP IN THE RETINA AND WHAT HE FOUND IS RESPONSES TO LIGHT IN THAT AREA OF THE BRAIN THAT CORSPANNED TO WHERE THE IMPLANT IS IN THE RETINA. SO NOT ONLY ARE THESE PHOTORECEPTORS SURVIVE AG, THEY'RE STILL CONNECTED TO THE BRAIN: WELL THIS REPRESENTS A MANUFACTURING CHALLENGE AND I'M GOING TO JUMP NOW TOWARDS MOVING THIS TOWARDS THE CLINIC. WE WORKED WITH CITY OF HOPE TO TRANSFER OUR PROTOCOL TO MAKE THE RPE, TO A GMP FACILITY AND WE WORKED WITH A COMPANY TO COMMERCIALIZE PRODUCTION OF THIS PARALEAN SCAFFOLD. HERE'S A COMPARISON OF THE SCAFFOLD COVERED WITH RPE CELLS WITH A PENNY, IT'S 3.5 BY 6.2-MILLIMETERS AND IF YOU ZOOM IN, IT COMES ATTACHED TO A REMOVABLE TAB AND THE SURGEONS CUT THE IMPLANT OFF HERE AND HAS A HANDLE HERE THAT THEY GRAB WITH A TOOL TO IMPLANT IN THE SUBRETINAL SPACE. AND THIS LITTLE NOSE IS THE ORIENTATION BECAUSE IN EARLY EXPERIMENTS THEY WOULD SOMETIMES PUT IT IN UPSIDE DOWN SO NOW THEY KNOW WHICH SIDE IS UP BY WHERE THE NOSE IS POINTING. WELL, CITY OF HOPE DEVELOPED A GMP PROCESS WHERE WE GO THROUGH THE SPONTANEOUS METHOD, LONG TIME, SIX MONTHS BUT WE END UP WITH A CRYO PRESERVED INTERMEDIATE CELL BANK, THOSE ARE THOUGHT OUT, GROWN, FOR 30 DAYS, SHIPPED TO THE POINT OF CARE AND THEY'RE READY FOR IMPLANTATION. MENT AND WE WORKED WITH LARRY CO TOUR AT THE CENTER FOR APPLIED TECHNOLOGY DEVELOPMENT AND WE'VE NOW COMPLETED GMP PRODUCTION OF AN ICB THAT HAS CELLS FOR THE PHASE ONE CLINICAL TRIAL AND WE SUBMITTED OUR DATA TO THE IND AND WE WERE CLEARED TO BEGIN A PHASE ONE TRIAL. NOW THE IND ENABLING STUDIES INVOLVED A HUGE TEAM OF PEOPLE WITH MANY, MANY RATS MARKED HERE ON A WHITE BOARD WHERE PEOPLE FROM SANTA BARBARA AND USC WENT DOWN TO A GOP LAB IN SAN DIEGO TO LOOK AT TOXICITY TUMOR GENERATEDISSITY AND BIOINTRUSION AND EFFICACY AND WE USED THE GMP LOT OF CELLS TO DO THESE STUDIES. EFFICACY WAS AT LOCAL TOXICITY WAS DONE IN THE SCS RAT, TUMOR GENERATEDISSITY AND SYSTEMIC TOXICITY AND BIODISTRIBUTION WERE IN THE NUDE RAT AND THEN WE DID SOME EXPERIMENTINGS WITH THE SURGICAL TOOL IN CADAVER EYES. ONE OF THE QUESTIONS THE FDA HAD WAS WITH REGARD TO OUR TOOL WHICH I'LL SHOW YOU IN A MINUTE, ARE WE STRESSING THE CELLS BY LOADING THEM INTO THE TOOL, SO WE HAD TO DO LOAD, UNLOAD STUDIES AND SHOWED THEY SURVIVED WELL AND DID NOT GET STRESSED. OKAY, BUT WE UPON ABLE TO ESTABLISH ACTIVITY AND SAFETY AND THIS IS A PICTURE OF OUR INJECTOR TOOL FOR SUBRETINAL PLACEMENT OF THE IMPLANT. IT HAS A--A FORSEPTORS BASICALLY INSIDE A CANULA, AND AS YOU PULL THIS BACK INTO THE CANULA, THE FORCEP PINCHES CLOSED AND IT PULLS THE IMPLANT BACK INTO THE CANULA, AND THESE ARE SINGLE USE STERILE INSTRUMENTS THAT WE NOW HAVE IN PRODUCTION FOR USE IN THE CLINIC. AND THIS IS AN ACTUAL SURGICAL VIDEO FROM ROD REG O BRANDT AND MARK HUMAYUN AND THEY'RE LIGHTING UP THE VITRIOUS AND REMOVING IT, IT'S KIND OF STICKY SPHIEBEROUS STAFF AND YOU CAN SEE IT'S FAIRLY MORE DIFFICULT TO REMOVE IN THE PIG AND THEN THEY'RE INJECTING FLUID BEHIND THE RETINA TO CREATE A RETINAL LOCATION NAT DETACHMENT AND THIS LITTLE BLISTER, THEN THEY MAKE A SMALL RETINOPATHYATHY AND THIS IS AN EARLY EARLY VERSION OF THE TOOL BUT YOU CAN SEE IT GETS PULLED INSIDE THE CANULA AND FOLDS UP LIKE A TACO SHELL TO PROTECT THE CELLS ON TOP, THAT SEA THEN INSERTED THROUGH THE SIDE OF THE A, THROUGH THE HOLE IN THE RETINA AND THEN THE IMPLANT IS EXTRUDED WHERE IT UNFOLDS IN THE SUBRETINAL SPACE. NOW I DON'T KNOW ABOUT YOU BUT I GET NERVOUS WITH SHARP OBJECTS ARE HELD NEAR MY EYEBALL BUT THE SURGEONS ARE FAIRLY COMFORTABLE WITH THIS TYPE OF MANIPULATION AND THEY THINK THEY CAN DO IT IN AN OUTPATIENT PROCEDURE IN ABOUT AN HOUR. SO WE'VE DONE MANY STUDIES IN IN THE PIG. WE WEREN'T ABLE TO SHOW EFFICACY IN THE PIG BUT WE WERE ABLE TO SHOW WE COULD RELIAISONNABLY PLACE THIS IN THE PIG AND NOT DAMAGE THE RETINA. THEY'RE LASERRING THE LASERRING THE RETINOTOMY. I THINK I WENT OVER THAT IN THE VIDEO. SO RIGHT NOW WE'RE RECRUITING ON THE CLINICAL TRIAL.GOV, AND IT'S STARTED BY THE REGENRATIVE, TECHNOLOGIES AT TWO SITES IN LOS ANGELES AT USCI INSTITUTE AND RBA ASSOCIATES IN BEVERLY HILLS SO WE'RE EXCITED TO GET STARTED ON THIS CLEANICAL TRIAL. THE FIRST COHORT WILL ACTUALLY BE 2400 OR WORSE, 10 PATIENTS AND THEN A SECOND COHORT OF 2100 OR WORSE. THERE ARE A NUMBER OF EXCLUSION CRITERIA AND SURPRISED ME BUT A LOT OF PATIENTS THAT WERE EXCLUDED BECAUSE OF ALL OF THESE DIFFERENT CRITERIA, AND THE PRIMARY END POINT IS SAFETY AND THE SECONDARY END POINT IS VISUAL FUNCTION. SO WE'RE EXCITED TO GET STARTED WHICH IT SHOULD HAPPEN SOON. SO LOOKING FORWARD, AND THIS IS A PAINTING OF AN EARLY OPHTHALMOLOGIST LOOKING FORWARD, THIS IS A BAD IDEA OF ANYBODY'S' RETINAL SURGEON. YOU SHOULD BE LOOKING AT YOUR PATIENT IF YOU'RE REMOVING A CATARACT FROM A CHILD. LOOKING FORWARD AS YOU PROBABLY KNOW, THERE ARE A LOT OF PEOPLE IN THIS SPACE AND A LOT OF PEOPLE ARE USING THIS KIND OF APPROACH TO GO AFTER MACULAR DEGENERATION. BUT NOT EVERYBODY'S USING THE SAME METHODS SOME ARE USING ACT WHICH IS NOW OKADA, AND THEY'VE COMPLETED THEIR PHASE ONE CLINICAL TRIAL. SOME ARE TRYING NEURAL PROGENITORS, STEM CELLS INC. AND I HEARD TODAY CLAUS JANSSEN, AND A SUBSIDY OF JOHNSON & JANUARY, THEY HAVE REPORTED DATA. THE JAPANESE GROUP IS GROWING SMALLER LITTLE SLIVERRINGS OF RPE MONOLAYER ON A COLLAGEN BASE AND USING A DIFFERENT APPROACH SURGICALLY. THERE'S AHATOLOGIOUS, IPS RPE, APPROACH GOING ON AT UCLA AND HERE AT NEI BED LIE [INDISCERNIBLE]. MENT THE NEW YORK GROUP IS TALKING ABOUT USING ADULT RPE. THEY'RE GETTING STARTED IN PRECLINICAL STUDIES, THE LONDON PROJECT LED BY PETE COFFEE WITH SUPPORT FROM PFIZER IS USING A DIFFERENT CELL LINE FROM US, A DIFFERENT SCAFFOLD AND GOING AFTER A DIFFERENT PATIENT POPULATION. THEY'VE IMPLANTED TWO PATIENTS SO FAR AND HAVEN'T REPORTED THEIR DATA YET. IN OUR PROJECT AT THE BOTTOM. SO THERE ARE A LOT OF DIFFERENT APPROACHES, AND YOU KNOW SINCE I'M IN WASHINGTON IT KIND OF REMINDED ME OF THE PRESIDENTIAL CAMPAIGN. THERE ARE A LOT OF DIFFERENT APPROACHES. WE DON'T KNOW WHICH ONE WILL SUCCEED EVENTUALLY, WHICH CANDIDATE IS THE BEST. BUT IT'S WORTHED IT GIVING IT A TRY. SO WE HOPE THAT THIS WILL BE PART OF THAT NEXT PILLAR OF MEDICINE, USING CELLS TO TREAT DISEASE, ACCORDING TO THE ALLIANCE FOR GENRATIVE MEDICINE AND USING STEM CELLS, SERM HAS 15 TRIALS THEY'VE SPONSORED AND THEY HOPE TO HAVE 50 BY THE END OF 2020 AND THERE'S 74 OF APPROVED PRODUCTS THAT UTILIZE STEM CELLS. SO TO WRAP UP, THE THREE THINGS I TALKED ABOUT CULTERATION, INSTALLATION, AND PRESERVATION, WE THINK WE SOLVED THESE PROBLEMS TO SOME EXTENT WHERE WE CAN MAKE RPE FROM STEM CELLS, WE CAN PUT IT ON A SCAFFOLD AND DELIVER IT TO THE SUBRETINAL STAGE AND WE THINK THAT THE VITROUGH ATOM NECKIN COATED SCAFFOLD WILL GIVE THOSE CELLS A SURVIVAL ADVANTAGE. AND WE'RE MOVING FORWARD WITH OUR CELLS NOW WITH THE PHASE 12A AND WITH THE CLINICAL TRIAL. AND I WILL STOP THERE AND THANK THE PEOPLE INVOLVED IN THIS PROJECT THAT ALL THE GREAT UNIVERSITIES AND INSTITUTIONS AND ESPECIALLY MY COLLABORATORS MARK AND DAVID HINTON AT USC AND LARRY CO TOUR AND THE PEOPLE IN THE LAB THAT DID THIS WORK ESPECIALENTIALLY SHERRY, AND DAVE, AND MY COLLABORATORS, LINK JOHNSON WHO'S MY PARTNER IN CRIME CROSS THE HALL THAT HELPED ME OUT EVERY STEP OF THE WAY. AND FINALLY THE FUNDING FROM THE CALIFORNIA INSTITUTE FOR REGENRE MEDICINE, THE WILLIAM K. BOSE, AND THE U.S. ARMY RESEARCH OPERATION, THANK YOU VERY MUCH. [ APPLAUSE ] >> HAPPY TO TAKE QUESTIONS. >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> YEAH, THE TIMELINE IS WE'RE RECRUITING PATIENTS NOW. I'M A BASIC SCIENTIST, I'VE NEVER BEEN INVOLVED IN A CLINICAL TRIAL. IT'S INCREDIBLY COMPLICATED. LET IN FACT I HEARD A TALK FROM KEN CHIEN, HE'S AT HARVARD, HE'S GOING TOWARD CLINICAL TRIALS. HE SAID CLINICAL TRIALS ARE HARDER THAN GETTING A PAPER ITSELF. SO, WE'RE--WE HAVE EVERYTHING ALIGNED NOW. WE MANUFACTURE ENOUGH CELLS IN THE ICB, WE'RE SCREENING PATIENTS NOW DOWN AT USC AND WE HOPE TO START REAL SOON. THERE'S A 30 DAY STAGGER THOUGH, FDA WANTS US TO WAIT 30 DAYS AFTER THE FIRST PATIENT AND THEN WE CAN DO THE SECOND AND THEN THERE'S A 10 DAY STAGGER AND THEN THE THIRD AND THEN THE PATIENTS WILL BE FOLLOWED. THE FDA WANTS US TO FOLLOW THEM FOR 14 YEARS SO WE'RE HOPING TO GET A READ OUT AFTER ONE YEAR TO LOOK AT THE SAFETY AND EFFICACY >> [INAUDIBLE QUESTION FROM AND MAKE SOME CONCLUSIONS. >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> [INAUDIBLE QUESTION FROM >> RIGHT. >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> RIGHT THE QUESTION WAS HAVE WE LOOKED AT BIOMARKERS AND AND WE HAVE, WE HAVEN'T--FOR THE H-NINE DERIVED RPE, WE'VE DONE A BIT OF RNA SEQ, BUT MOSTLY WE'VE DONE QUANTITATIVE PC R, LOOKING AT A WHOLE BATTERY OF RPE MARKERS BOTH EARLY AND LATE AND COMPARING TO FETAL HUMAN RPE AND THEN WE DO EXPERIMENTS TO TRY TO DETECT MARKERS OF UNDIFFERENTIATED STEM CELLS WHERE WE SPIKE IN UNDIFFERENTIATED CELLS AND DIDN'T MAKE A CURVE AND I DIDN'T SHOW THAT DATA BUT WE DON'T SHOW ANYTHING FOR THAT BACKGROUND FOR UNDIFFERENTIATED MARKERS AND WE SEE LEVELS OF EXPRESSION THAT ARE SIMILAR FOR RPE AND THINGS FOR RPE 65, TYROSINASE, AND ET CETERA. I SAID 99% WHAT'S THE OTHER ONE% AND WE WANTED TO KNOW THAT TOO, WHAT WE DO SEE IS A LITTLE BIT OF NEURAL CONTISSUE AND MACROPHAGES NATION, ISSUES SOMETIMES WE WILL SEE A NEURAL CELL WITH A NEURITE AND WE'VE LOOKED AT THOSE CELLSOT--WE CAN TAKE A PARALEAN MEMBRANE AND LOOK AT EVERY SINGLE CELL WITH AUTOMATED MICROSCOPY AND WE DON'T SEE ANY OX FOUR POSITIVE CELLS FOR EXAMPLE BUT WE SEE SOME THAT ARE MAP TWO MOSSATIVE. THOSE ARE NOT DIVIDING THOUGH. WE DON'T THINK THEY'RE A PROBLEM AND THEY MAY BE IMMATURE RPE, RPE ARE DERIVED FROM THE ANTERIOR NEURAL PLATE AND MAYBE THAT'S WHAT THOSE CELLS ARE. SO YEAH, WE HAVE LOOKED AT BIOMARKERS EXTENSIVELY. WE ALSO LOOKED AT APICAL SECRETION OF PEDF AND BASAL SECRETION OF VEG F AND THEY DO WELL THERE. YES? >> [INAUDIBLE QUESTION FROM AUDIENCE ]? YEAH WE IMMUNO SURVEYS PRECIOUSES THE RATS WITH CYCLOSPORIN IN THEIR FOODS. IN HUMANS WE'RE GOING TO USE SYSTEMIC IMMUNOSUPPRESSION USING [INDISCERNIBLE] AND THEN TAPER OFF. I TALKED TO SOME OF YOU ABOUT THIS TODAY. ON PAPER THE SUBRETINAL PLACE IS AN IMMUNE PRIVILEGE BUT WE DON'T KNOW WHAT TO EXPECT IN PATIENTS AND WE DID SOME EXPERIMENTS IN BIG WHERE WE DIDN'T USE IMMUNOSUPPRESSION AND WE SAW NO REJECTION, WE DID OTHER EXPERIMENTS WHERE WE USE LOCALIZED SUPPRESSION AND USING THE DECKS WE PUT IN THE PULLET AND IT LEAKS OUT DEXAMETHA SEWN AND THERE WE SAW RP E PRESERVATION BUT IT WAS A REVIEWER FROM SIRM AND WHO USE SYSTEMIC AND EVEN THE TRANSPLANT SURGEON WHO SAID, DON'T YOU WANT TO GIVE YOUR TRANSPLANT THE BEST CHANCE IT HAS TO SURVIVE AND CONVINCED US THERE ARE COMPLICATIONS FROM IMMUNOSUPPRESSION BUT PEOPLE DO IT ALL THE TIME FOR ORGAN TRANSPLANTS. SO THAT'S THE WAY WE DECIDED TO GO AND ACT BEFORE US WAS USING A SIMILAR REGIMEN SO THAT'S WHAT WE'RE TRYING BUT LATER WE MAY TRY IN A PHASE TWO B TRIAL. COMPARING NO IMMUNOSUPPRESSION, AND SEE IF WE CAN GET AWAY WITHOUT IT. OKAY, THANK YOU [ APPLAUSE ] VERY MUCH. [ APPLAUSE ] [ APPLAUSE ]