SO WELCOME. I'M ANNE PARISER FROM THE OFFICE OF RARE DISEASES RESEARCH AT THE NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES OR NCATS AT NIH, AND I'M HERE WITH DR. WILSON BRYAN, WHO IS THE DIRECTOR OF THE OFFICE OF TISSUES AND ADVANCED THERAPIES AT THE CENTER FOR BIOLOGICS EVALUATION AND RESEARCH AT FDA, SO WE'D LIKE TO WELCOME EVERYBODY HERE TODAY AND THANK YOU SO MUCH FOR COMING, SO WELCOME TO EVERYBODY WHO'S HERE IN THE ROOM AND ALSO TO EVERYONE WHO'S JOINING US BY THE WEBCAST. SO BEFORE WE GET STARTED, JUST WOULD LIKE TO GO OVER JUST A FEW LOGISTICAL ITEMS. THE GOALS FOR TODAY'S MEETING WAS TO BRING TOGETHER STAKEHOLDERS TO ASSESS THE CURRENT STATE OF GENE THERAPY AND HOW CAN WE WORK TOGETHER GOING FORWARD TO BRING PROMISING GENE THERAPIES TO BENEFIT PATIENTS WITH RARE DISEASES. SO AGAIN, THANK YOU ALL FOR BEING HERE. HERE'S JUST A FEW HOUSEKEEPING ITEMS. THIS IS THE WI-FI IF YOU CHECK YOUR WI-FI, IT SHOULD SAY NIH GUEST NETWORK. THERE'S NO PASSWORD. OR HERE'S THE LINK IF YOU NEED IT. ANYBODY WHO'S LIVE TWEETING OR POSTING ON SOCIAL MEDIA, WE'RE SUGGESTING HASHTAG GENETX2019 ALTHOUGH YOU CAN MAKE UP SOMETHING ELSE IF YOU'D LIKE. WE UNDERSTAND THE CLINICAL CENTER IS A LITTLE HARD TO GET AROUND SO THERE IS A NAVIGATION APP FOR YOUR CELL PHONE EITHER AT THE APPLE STORE OR GOOGLE PLAY AND I PUT THE I CONS THERE, BUT IT'S NIHCC TAKE ME THERE, AND WE ARE IN MASUR, ALSO KNOWN AS CLINICAL CENTER BUILDING 10. ALSO THE FACILITIES, IF YOU GO JUST PAST THE REGISTRATION DESK TO THE RIGHT OR THE LEFT, MOVING TO THE RIGHT AND THEN THE LEFT, AND THERE IS A SNACK SHOP A LITTLE FARTHER DOWN THE HALL AND THERE ARE MORE FOOD CHOICES IF YOU JUST KEEP GOING TO THE MAIN ENTRYWAY. AND WE'VE ALSO RESERVED THE TERRACE IF ANYBODY WANTS TO TAKE A BREAK, SO PAST THE REGISTRATION DESK, PAST THE BOOK STORE TO YOUR RIGHT THERE'S A PLACE YOU CAN SIT IF YOU'D LIKE. HERE'S AN OVERVIEW OF THE NEXT TWO DAYS. SO THERE ARE SIX SESSIONS, SPANNING FROM PRE-CLINICAL DEVELOPMENT TO CLINICAL QUALITY AND MANUFACTURING AND FINALLY OPERATIONS TYPE ISSUES, BUSINESS MODELS AND APPROACHING VERY LOW PREVALENCE DISORDERS. AND JUST LIKE TO TAKE ONE MINUTE TO THANK OUR PLANNING COMMITTEE, WE ACTUALLY HAD A VERY BIG PLANNING COMMITTEE AND THANK YOU SO MUCH FOR EVERYBODY A MULTIDISCIPLINARY EFFORT, LOTS OF STAKEHOLDERS, REALLY APPRECIATE THE WORK EVERYBODY PUT INTO THIS, AND WE'D LIKE TO MAKE SPECIAL THANKS TO LIZ OTTINGER WHO'S RIGHT THERE, NONE OF THIS WOULD HAVE HAPPENED WITHOUT LIZ, SO THANK YOU VERY MUCH, LIZ, FOR ALL OF YOUR HARD WORK. SO I'D LIKE TO PUT SOME CONTACT INFORMATION, THIS IS HOW YOU CAN CONNECT WITH NCATS. YOU CAN EMAIL YOU, WE ACTUALLY SET UP AN EMAIL BOX FOR THIS MEETING, GENETHERAPY2018@NIH.GOV, AND HERE'S OUR WEBSITE AND SOME OTHER POSTINGS. >> THE PURPOSE OF THIS CONFERENCE IS TO FACILITATE COMMUNICATION AMONGST STAKEHOLDERS TO HE SEE HOW WE CAN MOVE THE FIELD FORWARD AND COMMUNICATION OBVIOUSLY IS VERY IMPORTANT. THOSE OF US AT THE FDA, IF YOU'VE GOT QUESTIONS FOR THE FDA, YOU CAN TRY AND BUTTON HOLE ONE OF THE MANY FDA FOLKS WHO ARE HERE, BUT REALLY, REGULATORY QUESTIONS SHOULD GO THROUGH THIS FIRST BULLET, IT GIVES AN EMAIL ADDRESS AND PHONE NUMBER, AND THEN THE SECOND BULLET HAS OUR WEBINAR SERIES WITH A SERIES OF PRESENTATIONS ON CLINICAL, PHARM TOX AND OTHER ISSUES PERTINENT TO PEOPLE WHO ARE TRYING TO BRING THESE PRODUCTS FORWARD TO PATIENTS. SO I THINK I'LL TURN IT OVER NOW TO DR. PARISER TO INTRODUCE OUR FIRST SPEAKER. >> THANK YOU, WILSON. SO WE'LL JUST GO AHEAD AND GET STARTED. I'D LIKE TO INTRODUCE OUR FIRST SPEAKER, DR. CHRISTOPHER AUSTIN. DR. AUSTIN AS MANY OF YOU KNOW IS THE DIRECTOR OF NCATS. HE HAS ACTUALLY BEEN WITH NIH SINCE 2002, ORIGINALLY AT THE NATIONAL HUMAN GENOME RESEARCH INSTITUTE OR NHGRI, AND IS NOW THE INAUGURAL DIRECTOR OF NCATS, WHICH WAS FOUNDED IN 2012. PRIOR TO THAT, HE WORKED IN THE PHARMACEUTICAL INDUSTRY. HE'S TRAINED AS CLINICIAN AND GENETICIST AT HARVARD AND A RESEARCH FELLOWSHIP IN DEVELOPMENTAL AND NEUROGENETICS. SO WE HAVE MANY DISTINGUISHED SPEAKERS TODAY AND I CAN'T POSSIBLY GO THROUGH ALL THEIR MANY ACCOMPLISHMENTS, SO JUST BRIEFLY LEAVE IT THERE, SO THANK YOU, CHRIS. GLL ALL RIGHT. SO MY TASK TODAY IS PUT WHAT WE'RE GOING TO DO IN THIS NEXT COUPLE DAYS IN A LITTLE BIT OF A HISTORICAL PERSPECTIVE. THERE'S SO MUCH EXCITEMENT IN THE FIELD THAT THERE ARE MANY NEW PEOPLE IN THE FIELD, AND I THOUGHT IT WAS IMPORTANT FOR ALL OF US TO UNDERSTAND THE HISTORY OF HOW WE GOT HERE VERY BRIEFLY BECAUSE THAT HISTORY IS VERY IMPORTANT TO UNDERSTANDING THE REALLY REMARKABLE POINT THAT WE ARE AT NOW, I THINK, IN TRANSLATING THE POTENTIAL OF GENE THERAPY WHICH HAS BEEN AROUND FOR A WHILE INTO REALITY FOR PATIENTS. THIS IS REALLY WHERE ALL THIS BEGAN IN MANY WAYS. THIS IS A PAPER THAT ALL OF YOU SHOULD READ IF YOU HAVEN'T, IT'S BY TED FRIEDMAN, TALKING ABOUT THE PROSPECTS OF HUMAN GENE THERAPY OR GENE THERAPY FOR HUMAN GENETIC DISEASE, AND I WAS REFLECTING WITH SOME OF THE FOLKS BEFORE THE MEETING STARTED TODAY THAT I WAS IN CARTIF LAST WEEK, THE LAST TIME I WAS THERE WAS IN 1992 GIVING A TALK ABOUT HOW GENE THERAPY FOR NEUROLOGICAL DISORDERS WAS RIGHT AROUND THE CORNER. AND I REMEMBER HAVING THIS CONVERSATION WITH TED FRIEDMAN AND WE WERE SO EXCITED THAT WE WERE FINALLY GETTING TO THIS POINT, AT THE TIME WE WERE TALKING ABOUT USING GUTTED HERPES VIRUSES AND NEURONOTROPIC VIRUSES FOR THE CNS. THIS POTENTIAL HAS BEEN ONE THAT HAS CAPTIVATED MANY OF US, MYSELF INCLUDED, REALLY FOR OUR WHOLE CAREERS CAREER. THIS WAS REALLY THE FIRST SUCCESS DONE IN ADASKD, STEVE ROSENBERG AND HARVEY KLEIN ARE STILL HERE, DONE BY MICHAEL BLAZE AND FRENCH ANDERSON FOR ADASKD. THE GIRL THEN, WOMAN NOW, WHO WAS TREATED IS STILL ALIVE, AND DOING WELL WITH THIS DISORDER. THIS REALLY EXCITED PEOPLE FOR OBVIOUS REASONS, AND SO THE MOMENTUM BUILT THROUGH THE 90s UNTIL THIS HAPPENED, I DON'T HAVE TIME TO GO THROUGH THE WHOLE STORY BUT IN A TRIAL AT PENN, THERE WAS A YOUNG MAN WHO DIED OF WHAT TURNED OUT TO BE AN OVERWHELMING IMMUNE RESPONSE TO THE VECTOR, AND A LOT WAS LEARNED FROM THIS. JIM WILSON HIMSELF, WHO WAS THE P.I. ON THE PROTOCOL, HAS WRITTEN EXTENSIVELY FOR THIS. THIS IS A REALLY NICE PAPER FROM JIM 10 YEARS ON ABOUT LESSONS LEARNED FROM THIS TRIAL AND THIS REALLY UNFORTUNATE, UNFORTUNATE OUTCOME. WHAT HAPPENED AT THAT POINT WAS THAT THERE WAS REALLY -- PROTOCOLS WERE PULLED BACK AND STOPPED AND PROGRESS WAS, AT LEAST IN THE CLINICAL WORLD, WAS SLOW, BUT FOR A GOOD REASON. THE GOOD REASON WAS THAT WE APPRECIATED THAT IN OUR EAGERNESS TO TREAT PATIENTS WITH LIFE-THREATENING DISEASES, WE DIDN'T UNDERSTAND THE SCIENCE AS WELL AS WE NEEDED TO. AND SO IN THE SUCCEEDING COUPLE OF DECADES, A REMARKABLE AMOUNT OF SCIENCE, BOTH VECTORROLOGY AND IMMUNOLOGY HAS BEEN DONE SINCE THEN, SO I JUST WANT TO SHOW YOU, THIS IS ONE OF MY FAVORITE LAWS, THIS COMES FROM RON AMARA, WE TEND TO OVERESTIMATE THE EFFECT OF A TECH NOTHING IN THE SHORT RUN AND UNDERESTIMATE IT IN THE LONG RUN. THIS IS QUITE WELL-KNOWN HERE, TECHNOLOGY TRIGGER MEANS EVERYBODY GOES NUTS, THE SILVER BULLET HAS BEEN FOUND, THE END OF ALL OF OUR PROBLEMS HAVE COME AND YOU CAN FILL IN WHATEVER YOU WOULD LIKE, DNA MICRO ARRAYS OR THE HUMAN GEE GENOME, STEM CELLS, IPSLs, WHATEVER YOU WANT TO PUT IN THERE, THIS PEAK OF INFLATED EXPECTATIONS, AND THEN INEVITABLY, THERE IS A TROUGH OF DISAPPOINTMENT AND THEN MY FAVORITE, THE SLOPE OF ENLIGHTENMENT HAPPENS, AND THIS HAS VARIABLE SLOPE AND THEN THE PLATEAU OF PRODUCTIVITY, AND I WOULD JUST SUGGEST TO YOU THAT THIS IS REALLY WHAT'S HAPPENED, DURING THIS PERIOD OF TIME, '72 TO 1990, 1999, AND YOU NOW SINCE THEN, REALLY SINCE ABOUT 2010, WE'VE SEEN INCREASING PRODUCTIVITY, AND I ARGUE THAT IT'S REALLY NOT IN THIS CASE A PLATEAU OF PRODUCTIVITY, IT'S A SLOPE OF PRODUCTIVITY AND YOU'LL SEE THAT TODAY. SO GENE THERAPY IS NOW MAKING STRIDES. THE FIRST GENE THERAPY WAS APPROVED IN EUROPE A NUMBER OF YEARS AGO, CLIBERA OR STRIMBELIS AS WELL. THE PROBLEM WITH CLIBERA IS IT DID NOT HAVE ENOUGH OF A BUSINESS CASE BILTD AROUND IT TO STAY ON THE MARKET SO IT WAS ACTUALLY WITHDRAWN A COUPLE YEARS LATER BECAUSE THE COMPANY COULDN'T MAKE A COMMERCIAL CASE TO KEEP PRODUCING IT. BUT OF COURSE JUST IN THE LAST YEAR OR TWO, CAR T THERAPY HAS BEEN APPROVED FOR THE FIRST TIME BY THE FDA TARGETING RARE CANCERS, THERE ARE SEVERAL OF THESE DONE NOW. I PUT THE WORDS EX VIVO DOWN HERE BECAUSE IT'S IMPORTANT TO KEEP IN YOUR MIND THE DISTINCTION IN TYPES OF GENE THERAPY, IN ONE CASE ONE IS TAKING CELLS OUT OF THE PATIENT, TREATING THOSE CELLS WITH SOME SORT OF GENE THERAPY VECTOR AND CONSTRUCT AND THEN DELIVERING THE CELLS BACK INTO THE PATIENT, IN ANOTHER CASE, ONE IS DELIVERING THE VECTOR OR THE GENETIC CONSTRUCT DIRECTLY TO THE PATIENT, AND THAT CONSTRUCT HAS TO FIND THE TARGET CELLS IN THE PATIENT, SO CALLED IN VIVO. SO THIS WAS JUST LAST YEAR, IF I COULD FIND THE DATE HERE ON IT, PETER COULD PROBABLY GET ME THE DADE OF DATE OF IT, PROBABLY A RED LETTER DATE ON HIS CALENDAR. AND THEN LATE LAST YEAR, DECEMBER 19TH, THIS FIRST GENE THERAPY WAS APPROVED FOR AN INHERITED GENETIC DISEASE. I THINK YOU'RE GOING TO BE HEARING ABOUT THIS FROM SOMEONE FROM SPARC THERAPEUTICS LATER TODAY. SO THESE THINGS ARE GETTING OUT OF THE REALM OF THE POSSIBLE TO THE THEORETICAL TO THE RESEARCH TO THE REGULATORY TO THE APPROVED TO THE CLINICL. AND WE REALLY CELEBRATE THAT. THERE'S REALLY PRELIMINARY EVIDENCE OF DURABLE EFFECTS, SINGLE TREATMENT OF IN THIS CASE SPINAL MUSCULAR ATROA FEEF REALLY HAVING REMARKABLE EFFECTS IN CHILDREN WHO WOULD HAVE OTHERWISE SUCCUMB TO THEIR DISEASE, LIVING IN SOME CASES CLOSE TO A NORMAL LIFE OR EVEN A NORMAL LIFE. WHEN I WAS A NEUROLOGIST IN TRAINING 35 YEARS AGO, I NEVER COULD HAVE ENVISIONED. AND THAT'S THE REALITY NOW. AND ALL OF US AT THIS MEETING NEED TO THINK DEEPLY ABOUT THE FACT THAT THAT IS POSSIBLE NOW, SO WHAT SHOULD THAT DO TO THE WAY WE DO OUR RESEARCH? WE DON'T WANT TO REACH THAT HEIGHT OF HYPE THAT I SHOWED YOU BEFORE, BUT ON THE OTHER HAND, THIS POTENTIALLY IS A REALITY FOR PATIENTS NOW IN A WAY THAT WAS NEVER BEFORE, AND WE HAVE TO THINK DEEPLY ABOUT WHAT THAT AND I HOPE WE'LL DO THAT TODAY. BUT OF COURSE WHOLE GENES OR PARTIAL GENES PUT INTO A VECTOR AND DELIVERED TO A PATIENT IS NOT THE ONLY WAY ONE CAN DELIVER GENETIC MATERIAL TO A PATIENT FOR THERAPEUTIC PURPOSES. ANTISENSE OLIGONUCLEOTIDE THAT WAS APPROVED LAST YEAR, JANUARY OF 2017, THERE WAS -- FDA WAS GOOD ENOUGH AND I WANT TO THANK PETER FOR THIS FOR APPROVING THIS LAST WEEK SO THAT I COULD SHOW YOU THIS IN CONCERT WITH THIS MEETING, I KNOW THAT WAS THE REASON FOR THE TIMING. THIS IS ONE OF THE FIRST SRNA COMPANIES THAT ANNOUNCED THE APPROVAL OF THE FIRST RNAI PAIR THERAPEUTIC. CRISPR IS, OF COURSE, THE NEW KID ON THE BLOCK. VERY PROMISING, NEW. I THINK WE HAVE TO BE CAREFUL ABOUT THIS HYPE CYCLE, BUT I THINK PEOPLE ARE BEING MORE CAREFUL THAN PERHAPS THEY'D BEEN IN THE PAST, THAT WE REALLY LOOK CAREFULLY FOR SIDE EFFECTS, ADVERSE EFFECTS, AND PEOPLE ARE DOING IT ONGOINGLY. THAT SAID, CRISPR WILL PROBABLY COME TO THE CLINIC THIS YEAR FOR EX VIVO APPLICATIONS OF THALASSEMIAS AND SICKLE CELL DISEASE, WHICH FOR A VARIETY OF REASONS ARE PARTICULARLY AMENABLE TO THIS KIND OF THERAPY. SO AGAIN, THE POTENTIAL OF CURATIVE THERAPIES FOR THESE DISORDERS POTENTIALLY EVEN WITH A SINGLE APPLICATION. BUT WE CAN'T FORGET OTHER TECHNOLOGIES FOR GENE THERAPY. CRISPR HAS KIND OF SUCKED ALL THE OXYGEN OUT OF THE ROOM WHEN IT COMES TO GENE EDITING BUT OF COURSE ZINC FINGERS AND OTHER TECHNOLOGIES HAVE BEEN AROUND FOR A WHILE. JUST LAST YEAR IN A PROGRAM THAT NCATS AND ANNE HAD THE PRIVILEGE OF BEING INVOLVED WITH, THE FIRST PATIENTS WERE TREATED FOR AN INHERITED DISEASE, HUNTER'S SYNDROME, JUST LAST YEAR. SO CERTAINLY REGULATORY REVIEW HAS EVOLVED WITH THE SCIENCE. THIS IS A HEADLINE FROM BACK IN 2014 THAT NIH WILL NO LONGER REQUIRE SPECIAL REVIEW FOR U.S. GENE THERAPY TRIALS. THIS REFERS TO THE REVIEW BY THE RECOMBINANT DNA ADVISORY COMMITTEE OR THE RAC, SO THE ROLE OF THE RAC WAS CHANGED ABOUT FOUR YEARS AGO IN RECOGNITION OF THE EVOLUTION OF THIS TECHNOLOGY, AND COMING MORE IN THE MAINSTREAM. AND THEN JUST LAST WEEK, AGAIN, A REMARKABLE ADVANCE OF LANDMARK CHANGES TO THE RAC IN THIS FEDERAL REGISTER NOTICE, IN CASE YOU DON'T READ THE FEDERAL REGISTER ALONG WITH YOUR "WASHINGTON POST" EVERY MORNING, HERE IT IS, ON THE LEFT. THIS IS THE OFFICIAL ANNOUNCEMENT OF THE THE NIH AND FDA INTENDING TO CHANGE THE REVIEW OF GENE THERAPY PROTOCOLS, AND THIS REALLY NICE EDITORIAL BY SCOTT GOTTLIEB, WHO'S THE COMMISSIONER OF THE FDA AND FRANCIS COLLINS WHO'S THE HEAD OF THE NIH TALKING ABOUT THE FACT THAT GENE THERAPY WILL NOW -- GENE THERAPY PROTOCOLS WILL NOW BE CONSIDERED LIKE ANY OTHER PROTOCOL, AND REVIEWED BY THE FDA BECAUSE THIS IS THEIR ROLE, AND SHOULD BE THEIR ROLE AND APPROPRIATELY SO, NOW YOU THAT THIS THERAPY, THIS TYPE OF THERAPY HAS EVOLVED AND MATURED AS MUCH AS IT HAS. THE RAC WILL REMAIN AS AN ADVISORY BODY ON NEW TECHNOLOGIES. SO ALL OF THAT IS VERY EXCITING AND RARE DISEASE DRUG APPROVALS ARE ACCELERATING, DOING EXACTLY WHAT WE WOULD HOPE, AND ALL THIS IS VERY EXCITING, BUT I HAVE TO LEAVE YOU WITH A CHALLENGE. I HAVE THAT IN MY TITLE SLIDE. AND THE CHALLENGE IS THIS: THAT IS, THE NUMBER OF DISEASES THAT HAVE ANY REGULATORY APPROVED THERAPY, NUMBER OF RARE DISEASES THAT HAVE ANY REGULATORILY APPROVED THERAPY REMAINS AROUND 300, OUT OF THE 7,000 OR SO THAT ARE SAID TO EXIST. BUT I WOULD ASK YOU TO DO AN EVEN MORE THOUGHT-PROVOKING THOUGHT EXPERIMENT WHICH I ASKED ANNE PARISER TO DO A MONTH OR TWO AGO, THAT IS WHEN ONE IS THINKING ABOUT GENE THERAPIES AT TIMES, DEPENDING ON THE TYPE OF THERAPY, ONE IS INVOLVED WITH, SOMETIMES ONE NEEDS TO BE ALLELE SPECIFIC, AND IF ONE IS ALLELE-SPECIFIC, THEN WE'RE NOT TALKING ABOUT 7,000 DISEASES, WE'RE TALKING ABOUT 7,000 TIMES ALL THE DIFFERENT MUTATIONS THAT CAN LEAD TO THAT DISEASE, WHICH IS GREATER THAN 7,000 BY A FACTOR OF A HUNDRED IF NOT A THOUSAND. AND THAT MAKES THE WORLD OF PERSONALIZED MEDICINE AND PERSONALIZED THERAPY DEVELOPMENT BECOME EVEN MORE REAL THAN IT ALREADY WAS AND THESE NUMBERS EVEN MORE DAUNTING. SO THE QUESTION FOR US IN THE NEXT TWO DAYS IS, HOW DO WE TAKE THESE PROOF OF PRINCIPLES, AND THEY REALLY ARE THAT, PROOFS OF PRINCIPLE, GOING ALL THE WAY FROM AN IDEA TO REGULATORY APPROVAL, AND DEMONSTRATION OF SAFETY AND EFFICACY IN PEOPLE, AND RAMP THAT UP TO THE MILLIONS OF PATIENTS WHO ARE WAITING AND HOPING THAT THIS TECHNOLOGY WILL BE FOR THEM. FOR THOSE OF YOU WHO DON'T THINK ABOUT RARE DISEASES EVERY DAY, THESE ARE THE NUMBERS, THERE ARE THOUGHT TO YOU ABOUT 7,000 DISEASES, ABOUT 50% IF NOT MORE HAVE THEIR ONSET IN CHILDHOOD. A NUMBER WHICH MANY PEOPLE, MYSELF INCLUDED WHEN I FIRST HEARD IT, IS A BIT SHOCKING IS THAT THERE ARE ABOUT 250 NEW RARES BEING DEFINED EVERY SINGLE YEAR NOW. IF ONE LOOKS AT THE PLATEAU -- SORRY -- THE SLOPE OF RARE DISEASES, THE NUMBER OF RARE DISEASES BEING -- NEW RARE DISEASES BEING DEFINED EVERY YEAR, THAT SLOPE IS NOT DECREASING. THAT IS, IT'S NOT BECOME ASIM TO TICK YET BY ANY SO I KNOW FDA APPROVES A THERAPY FOR A DISEASE WHICH PREVIOUSLY HAS NO REGULATORY APPROVED TREATMENT AT A RATE OF ABOUT FIVE A YEAR, BUT WE'RE GETTING ABOUT 250 NEW DISEASES A YEAR, SO THERE'S A REAL CHALLENGE HERE FOR US WHO WORK IN THIS ARENA. THE POPULATION PREVALENCE IS IMPORTANT FOR US TO REALIZE THAT AS A GROUP, AND WE REALLY THINK ABOUT THIS VERY CLEARLY AS A GROUP OF DISORDERS, ALL OF WHICH ARE UNDER THIS COMMON BANNER OF RARE DISEASES, MUCH LIKE WE THINK ABOUT CANCER IS A BANNER UNDER WHICH THERE ARE THOUSANDS OF INDIVIDUAL DISEASES, RARE DISEASES ARE THE SAME WAY. IF ONE THINGS ABOUT THINKS ABOUT RARE DISEASES IN THAT HOLISTIC WAY, THAT POPULATION OF PREVALENCE OF RARE DISEASES IS ABOUT 8%, THAT'S THE SAME POPULATION PREVALENCE AS TYPE 2 DIABETES. AND THE EFFECT ON INDIVIDUALS, FAMILIES AND SOCIETY IN BOTH LOSS OF PRODUCTIVITY AND FINANCIAL CONSIDERATIONS IS OUTSIZED, AND SO THE OPPORTUNITY HERE IS ENORMOUS, THE NEED IS ENORMOUS, AND THE NUMBERS, I MENTIONED TO YOU BEFORE. SO I HOPE WHAT I'VE BEEN ABLE TO DO IS GIVE YOU A SENSE THAT PEOPLE HAVE BEEN AT THIS A WHILE, THERE'S A LOT OF EXCITEMENT IN THE FIELD, THERE'S GOOD REASON FOR THE EXCITEMENT IN THE FIELD. WE DON'T WANT TO GET TOO EXCITED BUT WE WANT TO STAY JUST EXCITED ENOUGH TO TAKE ACTION. HOW DO WE MOVE THIS FIELD FORWARD IN A QUANTUM LEVEL, TO DELIVER ON WHAT THIS PROMISE IS THAT MANY OF US HAVE THOUGHT ABOUT FOR MANY DECADES, BUT NOW REALLY SEEMS TO BE COMING TRUE. SO LET GET STARTED. [APPLAUSE] >> THANK YOU, DR. AUSTIN. OUR NEXT SPEAKER IS DR. PETER MARKS, WHO IS THE DIRECTOR OF THE CENTER FOR BIOLOGICS EVALUATION AND RESEARCH AT THE FDA. DR. MARKS RECEIVED HIS PH.D. IN CELL AND MOLECULAR BIOLOGY ALONG WITH HIS M.D. DEGREE IN NEW YORK UNIVERSITY, THEN TRAINED AT THE BRIGHAM IN INTERNAL MEDICINE AND HEMATOLOGY AND ONCOLOGY. HE IS A HEMATOLOGIST AND HAS HAD OUTSTANDING CAREERS IN BOTH ACADEMIA AND IN PHARMACEUTICAL INDUSTRY. HE CAME TO THE FDA IN 2012 AND HAS BEEN THE DIRECTOR OF CBER FOR THE PAST THREE YEARS. DR. MARKS IS A OUTSTANDING VOCAL ADVOCATE FOR GENE THERAPY AND MOVING THE FIELD FORWARD, AND ALSO WORKING WITH HIM ON A DAILY BASIS, I CAN TELL YOU THAT HE IS ALWAYS THINKING ABOUT HOW TO GET PRODUCTS AVAILABLE FOR PATIENTS AND I THINK WE ALL APPRECIATE THAT. DR. MARKS? >> FIRST I WANT TO THANK DR. AUSTIN FOR MAKING MY LIFE SO EASY BECAUSE I'M JUST GOING TO SUMMARIZE, FOR THOSE WHO GOT STUCK IN THE AREA WHERE YOU'RE GETTING THROUGH SECURITY, IT'S OKAY IF YOU MISSED A FEW MINUTES OF DR. AUSTIN'S REMARKS BECAUSE I'M GOING TO SUMMARIZE THEM AGAIN. FOR THOSE OF YOU WHO ARE ONLINE, SORRY ABOUT THAT. BUT FIRST THANK YOU TO THE NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES AND TO NIH AS WELL AS MY COLLEAGUES AT FDA FOR PLANNING WHAT IS, I BELIEVE WILL BE AN EXCELLENT WORKSHOP HERE THAT WILL HELP MOVE THIS FIELD FORWARD. I ALSO WANT TO THANK YOU ALL FOR TAKING THE TIME TO ATTEND BECAUSE SOME OF THE ISSUES WE NEED TO THINK ABOUT HEERYLY DESERVE SOME ATTENTION AS WE MOVE FORWARD TO TRY TO MAKE GENE THERAPIES A REALITY. IT REALLY DOES SEEM LIKE EXACTLY THE RIGHT TIME TO HAVE THIS CONVERSATION NOW BECAUSE WE'RE AT A PLACE NOW WHERE I COULDN'T AGREE MORE WITH DR. AUSTIN, WE WERE GOING TO CURE HEMOPHILIA IN THE MID 1990s, THE CURE WAS RIGHT AROUND THE CORNER. BY 2000, WE WEREN'T GOING TO NEED FACTOR CONCENTRATES ANYMORE AND WE'RE NOT QUITE THERE YET, BUT I DO THINK THAT WITH WHAT'S HAPPENED OVER THE PAST YEAR WITH THE APPROVAL OF THE COMMERCIALLY VIABLE GENE THERAPIES IN THE UNITED STATES, BOTH FOR CANCER INDICATION IN TERMS OF EX VIVO CAR T-CELL GENE THERAPIES AND FOR THE FIRST GENE THERAPY FOR A RARE GENETIC CONDITION, RPE65 MUTATIONS THAT CAUSE RETINAL DYSTROPHY, THAT'S AN IN VIVO, THAT IS A DIRECTLY ADMINISTERED GENE THERAPY, I THINK WE'RE NOW AT A PLACE WHERE -- WE ARE ON THAT PLACE WHERE THAT SLOPE OF PRODUCTIVITY AND THE S LOPE OF NEW THERAPIES WILL START TO INCREASE AND JUST THIS WEEKEND THERE WAS A PIECE THAT WAS PUBLISHED BY SOME PEOPLE OUTSIDE OF GOVERNMENT THAT SAID THEY'RE EXPECTING LITERALLY DOZENS OF GENE THERAPIES TO BE APPROVED IN THE NEXT SEVERAL YEARS. SO WE REALLY ARE AT THE RIGHT TIME HERE, AND THAT INTENSE INTEREST IS EVIDENCED, WE SEE THAT AT FDA AS EVIDENCED BY LIT THELY HUNDREDS OF ACTIVE INVESTIGATIONAL NEW DRUG APPLICATIONS IN THE AREA OF GENE THERAPY, WHETHER THEY BE GENE THERAPIES THAT ARE PERFORMED EX VIVO OR DIRECTLY ADMINISTERED GENE THERAPIES, WHETHER THEY USE OLDER GENE EDITING TECHNOLOGIES OR NEWER GENE EDITING TECHNOLOGIES OR MORE CONVENTIONAL VECTORS OR NOVEL VECTORS. WHAT I THINK IS REALLY SPECIAL ABOUT THE NEXT TWO DAYS IN TERMS OF PLANNING THIS WORKSHOP WAS THAT IT'S TAKING A HOLISTIC APPROACH OF EVERYTHING FROM THE PRE-CLINICAL ASPECT OF THIS TO THE COMMERCIAL ASPECT, AND AS WE CAN SEE FROM SOME OF THE FAILURES HERE, YOU CAN HAVE A GREAT PRE-CLINICAL PROGRAM AND GREAT CLINICAL PROGRAM BUT IF IT'S NOT COMMERCIALLY VIABLE, THE GENE THERAPY WILL WILT AND GO AWAY. SO THIS KIND OF OVERVIEW OF THE ENTIRE PROCESS IS REALLY CRITICAL. A HOLISTIC APPROACH TOWARDS GENE THERAPY MAY REALLY BE IMPORTANT AS WE THINK ABOUT THE PROCESS OF BRINGING THINGS FOR RARE DISEASES TO MARKET, WHICH IS THAT -- HERE'S A CASE WHERE THE PRE-CLINICAL DEVELOPMENT WILL NEED TO SUPPORT CLINICAL DEVELOPMENT. THE MANUFACTURING PROCESS WILL NEED TO BE SUFFICIENTLY THOUGHT UP IN ADVANCE SO IT COULD POTENTIALLY SUSTAIN A PRODUCT THAT WILL GO VERY RAPIDLY FROM THE CLINIC INTO COMMERCIALIZATION. AND THAT'S SOMETHING WHICH I THINK WE'RE GOING TO HAVE TO THINK ABOUT DIFFERENTLY, AND I WOULD SAY THAT OUR PREVIOUS PHARMACEUTICAL MODEL OF PILOT PHASE DEVELOPMENT FOR MANUFACTUREING PROCESSES FOLLOWED BY COMMERCIAL SCALE MANUFACTURING, I DON'T THINK THAT'S GOING TO WORK. THAT MODEL, I THINK WE NEED TO RE-THINK THAT PARTICULARLY FOR RARE DISEASES AND GENE THERAPIES. WE MAY HAVE TO THINK ABOUT HOW WE GET IT RIGHT THE FIRST TIME WHEN WE PUT THINGS INTO HUMANS TO BEGIN WITH SO THAT WHEN THINKS MAKE IT THROUGH A CLINICAL TRIAL THAT MIKE TAKE ONLY 20 OR 40 PATIENTS, THE PRODUCT CAN BE COMMERCIALIZED WITHOUT RE-ENGINEERING THINGS ALL OVER AGAIN BECAUSE THAT HAS TWO PROBLEMS, ONE IS I WOULD SAY PROBABLY A SOMEWHAT TRIVIAL ONE WHICH IS IT COSTS A HECK OF A LOT. IT'S TRIVIAL, IT'S JUST MONEY. BUT THE REAL PROBLEM IS, IT PREVENTS THESE THERAPIES FROM GETTING TO THE PEOPLE WHO NEED THEM, AND IF YOU'RE A PARENT OF A CHILD WHO HAS A DISEASE THAT'S GOING TO TAKE THEIR LIFE, YOU REALLY FEEL THAT. SO ALTHOUGH GENE THERAPY MIGHT NOT BE THE OPTIMAL SOLUTION FOR EVERY RARE DISEASE, IT DOES OFFER TREMENDOUS HOPE FOR MANY RARE DISEASES THAT HAVEN'T BEEN APPROACHED BY CONVENTIONAL METHODS, AND I THINK WE REALLY HAVE THE OPPORTUNITY HERE TO SEE WHETHER WE'LL APPROACH OR GET TO THE SPEED THAT I THINK DR. AUSTIN WOULD LIKE TO SEE US GET TO, I DON'T KNOW, BUT I THINK WE CERTAINLY HAVE THE OPPORTUNITY IF WE THINK ABOUT IT AND WE THINK ABOUT NOVEL DEVELOPMENT PARADIGMS TO ACCELERATE THE PROGRESS THAT WE'VE HAD. IN ADDITION, GENE THERAPY MAY ULTIMATELY TURN OUT TO BE NOT ONLY THE MOST SAFE AND EFFECTIVE METHOD FOR TREATING SOME RARE DISEASES, BUT IT ALSO MIGHT TURN OUT TO BE THE MOST COST-EFFECTIVE AND EFFICIENT WAY OF TREATING THESE DISEASES, PARTICULARLY WHEN YOU THINK ABOUT AREAS OUTSIDE OF CONVENTIONAL HIGH RESOURCE SETTINGS. BECAUSE IT MAY BE THAT FOR THINGS LIKE THALASSEMIA, SICKLE CELL DISEASE, AND SOME OTHER RARE GENETIC DISORDERS PRESENT AREAS OF THE WORLD THAT HAVE LOW AND MIDDLE INCOMES, WHAT WE CAN DO RIGHT NOW TO TREAT THOSE DISORDERS WITH SUPPORTIVE CARE IS SIMPLY NOT AVAILABLE. SO A SIMPLE OR RELATIVELY SIMPLE SAFE AND EFFECTIVE ONE-TIME TREATMENT OR FEW TIMES TREATMENT MAY BE TRANSFORMATIVE. SO WITH THAT, I JUST THINK WE HAVE A TREMENDOUS POSSIBILITY HERE. I THINK THIS IS ONE OF THOSE PLACES WHERE REALLY THE LIMIT IS WHAT WE CAN CONCEIVE OF, OF THE POSSIBILITIES OF HOW WE CAN DRIVE DEVELOPMENT FORWARD. SO I'D ENCOURAGE US TO THINK CREATIVELY OVER THE NEXT COUPLE DAYS AND THANK EVERYONE FOR ATTENDING TODAY. SO THANKS AGAIN. [APPLAUSE] >> THANK YOU, DR. MARKS. OUR NEXT SPEAKER IS DR. BEAR EE DR. BEAR E E BYRNE, DR. BEAR DR. BARRY BYRNE, UNIVERSITY OF FLORIDA. DR. BYRNE HASES HAS A PARTICULAR INTEREST IN DEVELOPING GENE THERAPIES FOR TREATMENT FOR MUSCLE DISEASES, AND TO BORROW A PHRASE FROM DR. AUSTIN, DR. BYRNE CERTAINLY IS SOMEONE WHO THINKS ABOUT RARE DISEASES EVERY DAY. HE'S HERE TODAY SPEAKING ON BEHALF OF THE AMERICAN SOCIETY OF GENE AND CELL THERAPY. DR. BYRNE. >> THANKS VERY MUCH, WILSON, AND ON BEHALF OF THE AMERICAN SOCIETY OF CELL AND GENE THERAPY, I WAS JUST ASKED TO WELCOME YOU ALL, MAKE A FEW COMMENTS ABOUT THE EFFORTS IN SOCIETY THAT HAS EVOLVED IN A MAJOR WAY OVER THE LAST 22 YEARS SINCE IT WAS FOUNDED AND THE FIRST MEETING 20 YEARS AGO IN SEATTLE, I WAS LUCKY ENOUGH TO ATTEND JUST STARTING MY OWN LAB. THERE WERE 1600 PEOPLE AT THE MEETING, WHICH WAS NOT A BAD SHOWING FOR A NEW SOCIETY. 700 ABSTRACTS AND PROBABLY ONLY A HANDFUL OF THEM ABOUT AAV, WHICH WAS THE TOPIC WE WERE INTERESTED IN, AND JUDE CAN PROBABLY ATTEST TO THIS, WE HAD -- OUR TWO LABS HAD A PAPER IN 1997 WHICH WAS A BIT OF A TIPPING POINT FOR THE AAV FIELD DEMONSTRATING LONG TERM EXPRESSION IN NON-DIVIDING CELLS. THERE WERE ONLY A THOUSAND MEMBERS THEN, THAT'S WHEN THEY FIRST STARTED TRACKING MEMBERSHIP. AT THE LAST MEETING IN CHICAGO, THE NUMBER OF ATTENDEES HAD TRIPLED AND GOOD NEWS FOR THE SOCIETY THAT NOW BELIEVES THEY THINK IT CAN SUSTAIN ITSELF BECAUSE THE MEMBERSHIP HAS NOW ALSO TRIPLED TO 2900 MEMBERS, AND THERE ARE A THOUSAND ABSTRACTS AT THE LAST MEETING, AND ANY WHO ATTENDED REALIZED THAT THE INCREASE IN INTEREST REALLY LED TO VERY CROWDED SPACE THAT WAS SO POPULAR. THE MAJORITY OF THE AB STRACTSZ ARE ABOUT AAV, UNLIKE THE EARLY DAYS WHERE RETROVIRUSES AND ADENORUSS WERE THE TOPIC. SO THE AAC HAS CONTINUED TO FOCUS ON EARLY CAREER TRAINEES WHICH HELP BUILD INTEREST IN THE FIELD AND TRAIN THE NEXT GENERATION OF SCIENTISTS. I THINK ONE OF THE INITIATIVES THAT HAS TO COME FORWARD IS THE IMPORTANCE OF TRAINING ALSO CLINICIAN SCIENTISTS BECAUSE IF THESE DRUGS ARE TO BECOME A REALITY IN CLINICAL PRACTICE AND, IN FACT, CHANGE CLINICAL PRACTICE, WE NEED PRACTITIONERS WHO ARE GOING TO BE FAMILIAR WITH THEIR SAFE USE AND HOW TO ESTABLISH TREATMENT CENTERS FOR PATIENTS WITH RARE DISEASE BECAUSE IT'S UNLIKELY AS I THINK REALLY AN EXCELLENT ASPECT OF THE APPROVAL OF THE LCA TREATMENT BY SPARC WAS THE FOCUS ON DEVELOPING CENTERS OF EXCELLENCE TO PROPERLY TREAT PATIENTS WITH LCA. SO THAT'S REALLY THE ECK TEND OF MY COMMENTS. WE HAVE REALLY AN IMPORTANT TIME IN THE FIELD AND THIS MEETING IS A GREAT TESTAMENT TO THE PARTNERSHIP THAT ASGCT, THE NIH AND THE FDA HAVE ESTABLISHED, BOTH FOR ESTABLISHING BEST PRACTICES AND CONTINUING TO SUPPORT THE FIELD OF GENE THERAPY. SO WITH THAT, I SAY WELCOME AND LET'S BEGIN THE MEETING. [APPLAUSE] >> OKAY. SO THANK YOU VERY MUCH TO ALL OUR INTRODUCTORY SPEAKERS, AND NOW WE'RE READY TO MOVE ON TO SESSION ONE, WHICH IS PRE-CLINICAL DEVELOPMENT. SO I'D JUST LIKE TO START BY INTRODUCING OUR CHAIR AND CO-CHAIR FOR THIS SESSION, DR. STEPHEN KALER, AND DR. CHARLES VENDITTI. DR. KALER IS A SENIOR INVESTIGATOR AT THE EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT OR NICHD. HE IS A WORLD LEADER IN THE MOLECULAR BASIS FOR COPPER TRANSPORT DISORDERS INCLUDING PIONEERING EARLY IDENTIFICATION OF INFANTS AT RISK FOR MENKES DISEASE. DR. KALER LEADS A CLINICAL SERVICE FOR PATIENTS WITH MENKES AND RELATED DISORDER HERE AT THE NIH CLINICAL CENTER, AND OF NOTE, DR. KALER'S LAB RECENTLY RESCUED A LETHAL -- BUT THE AAV MEDIATED GENE. THE CO-CHAIR IS DR. VENDITTI, WHO IS ALSO A SENIOR INVESTIGATOR. HE'SIN THE GENETICS MOLECULAR BIOLOGY BRANCH AT NHGRI, BOARD CERTIFIED PEDIATRICIAN, CLINICAL GENETICIST AND BIOCHEMICAL GENETICS AND IS AN ATTENDING PHYSICIAN AT THE MARK O. HATFIELD CLINICAL CENTER HERE AT NIH. HE'S INITIATED A TRANSLATIONAL RESEARCH PROGRAM IN AS METABOLIC DISORDERS AND ALONG WITH MANY OTHER ACCOMPLISHMENTS. SO WELCOME, DRS. KALER AND VENDITTI. >> GOOD MORNING, EVERYONE. IT'S A PLEASURE TO HELP CHAIR THIS SESSION OF PRE-CLINICAL DEVELOPMENT, THE FIRST SESSION OF THIS MEETING. IN TERMS OF THE FORMAT, WE'RE GOING TO ASK EACH OF OUR SPEAKERS TO DELIVERY MARKS OVER A PERIOD OF 25 MINUTES EACH. WE'D LIKE TO RESERVE QUESTIONS UNTIL THE COMPLETION OF ALL THE TALKS. WE'LL HAVE THREE TOPICAL EXPERTS DELIVER COMMENTS ON THE VARIOUS GENE THERAPY TECHNOLOGIES THAT WE ARE ENGAGED IN THESE DAYS AND WE WILL WRAP IT UP WITH COMMENTS FROM THE FOOD AND DRUG ADMINISTRATION, DR. IWEN WU, WHO WILL REACT TO THE TALKS, AND ALSO ENGENDER THE -- AND INITIATE THE DISCUSSION OF ALL OF THE TALKS, IN WHICH WE HOPE THE AUDIENCE WILL BE FULLY ENGAGED. SO WITHOUT FURTHER ADO, I'D LIKE FOR THE PANEL, DR. JAY CHIORINI FROM THE NATIONAL INSTITUTE OF DENTAL AND CRANIOFACIAL RESEARCH RIGHT HERE IN THE INTRAMURAL RESEARCH PROGRAM OF NIH. AND JAY IS GOING TO SPEAK ABOUT AV APPROACHES TO RADIATION-INDUCED XEROSTOMIA, AN IMPORTANT PROBLEM IN WHICH HE HAS LED EFFORTS TO BEGINNING FROM THE PRE-CLINICAL PHASE ALL THE WAY TO THE CLINICAL TRIAL PHASE. SO DR. CHIORINI. >> THANKS, STEVE. GOOD MORNING, EVERYONE. I'D LIKE TO ADD SOMETHING TO THE LIST OF GENES THAT DR. AUSTIN WAS TALKING ABOUT, OUR LIST OF RARE CONDITIONS, AND THAT WOULD BE THINGS THAT ARE NOT SINGLE GENE DISORDERS BUT STILL FALL IN THAT CATEGORY OF RARE DISEASES. RADIATION INDUCED XEROSTOMIA FITS IN THAT CATEGORY. THERE'S NOT ONE GENE BUT IT'S A PHYSIOLOGIC RESPONSE TO AN INSULT. AND THEY'VE BEEN TRYING TO DEVELOP THERAPIES THAT WE CAN USE TO OVERCOME THIS LOSS OF FUNCTION. SO THIS YEAR IN THE UNITED STATES, ABOUT 50,000 AMERICANS WILL BE DIAGNOSED WITH HEAD AND NECK CANCER, AND A MAJORITY OF THEM WILL ELECT TO UNDERGO IONIZING RADIATION, EXTERNAL BEAM, TO TREAT THAT TUMOR, AND TO CURE THEIR CANCER. THE PROBLEM IS ABOUT 85 PERSONS OF THEM WILL EXPERIENCE A CONDITION CALLED RADIATION INDUCED XEROSTOMIA, AND THAT'S BASICALLY THE LOSS OF THEIR SALIVARY GLAND FUNCTION. SO SOME OF THOSE INDIVIDUALS, ABOUT HALF OF THEM WILL HAVE A NATURAL RECOVERY IN THEIR GLAND ACTIVITY OVER ABOUT A TWO-YEAR PERIOD, BUT THE OTHER 40%, ABOUT 10 TO 12,000 AMERICANS A YEAR, WILL HAVE THIS CHRONIC CONDITION WHERE THEY CAN'T CHEW, THEY CAN'T SWALLOW, THEY HAVE SIGNIFICANT ORAL PAIN BECAUSE OF THEIR LOSS OF THEIR SALIVARY GLANDS. AS YOU CAN IMAGINE, WITHOUT SALIVA TO BATHE THE ORAL CAVITY, THERE'S SIGNIFICANT DENTAL CARIES AND ESSENTIALLY PEOPLE LOSE ALL THEIR TEETH OVER ABOUT A FIVE-YEAR PERIOD OF TIME. ANY TIME YOU AFFECT THE ORAL CAVITY, THERE'S PROBLEMS WITH WEIGHT LOSS, MALNUTRITION. THIS IS A SIGNIFICANT QUALITY OF LIFE ISSUE FOR THESE INDIVIDUALS. AND YOU THINK THAT WE'VE CURED THEIR CANCER, THEY'RE FIVE YEARS OUT, BUT YOU TALK TO THEM AND THIS IS PROBABLY WORSE THAN HAVING THE CANCER. THEY HAVE A DRAMATIC DIMINISHED QUALITY OF LIFE OVERALL WE'VE BEEN TRYING TO DEVELOP A THERAPY FOR THIS BECAUSE THERE IS NO TREATMENT FOR THESE INDIVIDUALS. THERE'S NO WAY TO REALLY REPLACE THE VA LIE VA THAT THEY'RE NATURALLY PRODUCING. THERE ARE FORMS OF ARTIFICIAL SALIVA BUT THEY'RE GENERALLY NOT ACCEPTED BY THE PATIENTS. HOW BIG OF A CONDITION IS THIS? AS I SAID, IT'S ABOUT 12,000 AMERICANS A YEAR, BUT MOST PEOPLE ARE DIAGNOSED IN THEIR 50s AND GETTING EVEN YOUNGER NOW, WITH OROPHARYNGEAL CANCER, NOW THERE'S A HIGHER RATE OF HPV IN THIS DISEASE. SO THIS IS REPRESENTING ABOUT 170,000 AMERICANS WHO HAVE THIS CHRONIC CONDITION AND ARE WALKING AROUND TODAY WITH NO THERAPY FOR IT. SO WE'VE SPENT ABOUT 15 YEARS TRYING TO UNDERSTAND WHAT IS THE DRIVER FOR THAT LOSS OF SALIVARY GLAND FUNCTION? AND THE SHORT ANSWER IS, WITHIN THE SALIVARY GLAND, THERE ARE TWO CELL TYPES. THERE'S THE -- THEY INITIATE FLUID MOVEMENT WHICH THEN GOES INTO WATER IMPERMEABLE DUCT WHERE IT'S MODIFIED AND THEN CONDUCTED INTO THE ORAL CAVITY. THE PROBLEM IS THESE CELLS ARE VERY SUSCEPTIBLE TO IONIZING RADIATION AND EE IS SENGSLY ESSENTIALLY AR E FALLING DOWN ON THE JOB. YOU GET VERY LITTLE SALIVA COMING OUT. SO WE HAD A PROBLEM HERE. WE UNDERSTOOD THE PHYSIOLOGY THAT WAS RESULTING IN THIS LOSS OF FUNCTION, WE NEEDED A WAY TO DEVELOP -- TO TEACH THE REMAINING CELLS WITHIN THE GLAND A NEW TRICK. HOW COULD THEY REGAIN THAT FUNCTION? SO OUR PRE-CLINICAL DEVELOPMENT HAD THREE STAGES IN IT, FIRST TO IDENTIFY A CANDIDATE GENE THAT WE COULD USE TO RE-ENGNEER THE REMAINING EPITHELIA TO HAVE THAT GAIN OF FUNCTION, DEVELOP GENE TRANSFER VECTORS THAT WE COULD USE INITIALLY IN A PROOF OF CONCEPT STUDY TO SHOW THAT THE PHYSIOLOGY WAS THE SAME IN HUMANS AS WHAT WE WERE PREDICTING BASED ON WORK WE DID IN ANIMAL MODELS AND ALSO TO DEVELOP A DURABLE VECTOR SO WE HAD TWO VECTOR SYSTEMS WE NEEDED TO DEVELOP. FINALLY, WE NEEDED AN ANIMAL MODEL THAT WOULD PHYSIOLOGICALLY REPLICATE WHAT'S GOING ON IN THE HUMAN CONDITION. SO THE GENE THAT WE FOCUSED ON OR THE CLASS OF GENES IS BASED ON A MOLECULE THAT WAS DISCOVERED UP THE ROAD AT JOHNS HOPKINS BY PETER ARGRAVES GROUP CALLED AQUA POREPORINS. THIS IS CALLED AQUAPORIN5. THIS IS VERY SPECIFIC, TIGHTLY REGULATED. IF WE'RE GOING TO RE-ENGINEER THE EPITHELIA, WE NEEDED A DIFFERENT APPROACH, WE NEEDED SOMETHING THAT WASN'T REGULATED AND WOULD GO TO BOTH CHANNELS AND WOULD RESPOND TO AN OSMOTIC GRADIENT WHICH THE DUCTAL CELLS WERE STILL ABLE TO PRODUCE. SO WE FOCUSED ON A DIFFERENT MOLECULE. IT'S CALLED AQUAPORIN1. IT FREELY ALLOWS WATER TO MOVE, IT WILL SORT TO EITHER THE BASAL OR APICAL SURFACE OF THE CELL, AND IT'S CONSTITUTIVELY ACTIVE. SO IN RESPONSE TO AN OSMOTIC GRADIENT, WATER WILL FLOW IN THAT DIRECTION, AND THE DUCTAL CELLS AS I SAID ARE STILL FUNCTIONAL, THEY CAN SECRETE POTASSIUM INTO THE SALIVA, CREATING THAT OSMOTIC GRADIENT THAT WOULD ALLOW FLUID TO MOVE. VECTOR SYSTEMS. REALLY THE THE GENE THERAPY. HOW TO EFFICIENTLY DELIVER GENES INTO A CELL. AND IN OUR PRE-CLINICAL DEVELOPMENT, WE FOCUSED ON DEVELOPING TWO VECTOR SYSTEMS. ONE WE USED AN ADENOVIRUS AS A PROOF OF CONCEPT STUDY TO SHOW IN A HUMAN GLAND, THAT WE COULD SEE RECOVERY IN FUNCTION WITH AQUA PORIN. THE SECOND WAS TO DEVELOP A DURABLE VECTOR. A NUMBER OF VECTOR SYSTEMS CAN WORK IN THE SALIVARY GLAND BUT MY BACKGROUND WAS IN AAV BIOLOGY AND THERE'S A LOT OF GREAT ATTRIBUTES TO USING AAV AS A VECTOR SYSTEM, I'M SURE MANY OF YOU FAMILIAR WITH SO I WON'T GO INTO THAT AT THIS POINT, BUT AS I SAID, OUR PRE-CLINICAL DEVELOPMENT AND OUR INITIAL CLINICAL TRIAL THAT WE DID WAS USING A SHORT-TERM VECTOR, THEN TO PROVE THE CONCEPT AND THEN COME BACK WITH A LONG TERM VECTOR USING THE AAV. HOW COULD WE TARGET A SALIVARY GLAPPED? SAL SALIVARY GLAND? THERE'S A SCAN USED FOR IMAGING STONES, THIS IS WHERE YOU CAN INSERT A PLASTIC CANNULA INTO THE LUMINA OF THE GLAND, I'M SHOWING YOU THE PAROTID THERE, THEN INFUSE A CONTRAST DYE INTO THE GLAND. THIS MADE A VERY EASY WAY FOR US TO INFUSE OUR VECTOR AND EXPOSE VECTOR TO ALL THE EPITHELIA WITHIN THE GLAND. YOU ESSENTIALLY CAN TARGET ALL THE DUCTAL AND ANY REMAINING ACINER CELLS WITH THIS KIND OF APPROACH AND THIS IS AN EXAMPLE OF A PAROTID DUCT BEING CAN LATED. IT'S VERY EASY TO DO IF YOU KNOW WHAT YOU'RE LOOKING FOR. ANIMAL MODEL, THE THIRD PART, HOW CAN WE MODEL THIS IN SOMETHING THAT WOULD BE PHYSIOLOGICALLY RELEVANT IN HUMANS. WE WORKED WITH MONKEYS, WE'VE WORKED WITH MICE, AND ONE OF OUR FAVORITE MODELS IS THE MINI PIG. THE SALIVARY GLANDS IN A MINI PIG ARE ABOUT TWICE THE SIZE AS A HUMAN AND THEY RESPOND VERY SIMILAR TO THE HUMAN CONDITION WITH IONIZING RADIATION. YOU SEE INFLAMMATION WITHIN THE GLAND, YOU SEE ATROPHY AND LOSS OF FUNCTION OVER TIME. SO AS A LARGE ANIMAL MODEL, PIGS WERE VERY USEFUL FOR OUR CLINICAL -- PRE-CLINICAL DEVELOPMENT PLANS. THIS IS AN EXAMPLE FROM ONE OF THE BIG STUDIES WE DID WHERE WE EE RADIATE THE PIG AT DAY ZERO, AND WITH ABOUT 15 GRADE, WE CAN DO A SINGLE DOSE OR FRACTION ATED SIMILAR TO WHAT A PATIENT WOULD RECEIVE. YOU CAN SEE OVER ABOUT A 16 WEEK PERIOD OF TIME, THERE'S A SLOW BUT STEADY DECREASE IN SALIVARY GLAND OUTPUT FROM THESE PIGS. AT 16 WEEKS HERE, WE INFUSED EITHER AN ADENOVIRUS VECTOR ENCODING OR ONE ENCODING THAT AQUAPORIN1 GENE. YOU CAN SEE A SIGNIFICANT INCREASE IN FLUID OUTPUT, SUGGESTING THAT AQUA PORIN IS ABLE TO RECOVER IN THIS PHYSIOLOGICALLY RELEVANT ANIMAL MODEL. BUT IN KEEPING WITH ADENOVIRUS' TRANSIENT NATURE, WE CAN SEE A SLOW BUT STEADY DECREASE IN GLAND ACTIVITY OVER ABOUT TWO WEEKS, BACK DOWN TO OUR BASELINE. SO AS EFFICACY WOULD SUGGEST THAT IN THIS MODEL, AQUAPORIN WOULD WORK. SO IN 2009, WE APPROACHED THE FDA ABOUT MOVING THIS PRO CLINICAL DATA INTO A CLINICAL APPLICATION IN THIS PROOF OF CONCEPT STUDY. THEY WERE VERY HELPFUL IN GETTING US THROUGH THESE PROCESSES. THE TRIAL WAS DONE HERE IN THE CLINICAL CENTER AND WRAPPEDUP IN 2012 AND I'LL TELL YOU SOME OF THE DATA FROM THAT. THIS IS A PHASE ONE SAFETY STUDY. OUR PRIMARY OUTCOME STUDY WAS SAFETY BUT WE BUILT IN SOME SECONDARY EFFICACY MEASURES WHERE WE SEE THE INCREASE IN ORAL PAIN AND INCREASED WETNESS AND OBJECTIVE RESPONSES SUCH AS COULD WE MEASURE AN INCREASE IN SALIVA PRODUCED BY THESE INDIVIDUALS. THERE WAS A STANDARD 3 BY 3 DESIGN, THE STUDY WAS ORIGINALLY DESIGNED TO GO FOR ONE YEAR, BUT BECAUSE OF POSITIVE RESULTS, THE FDA ASKED US TO CONTINUE IT AND FOLLOW THE PATIENTS FOR ANOTHER TWO YEARS. IN THE END, WE DOSED 11 PATIENTS WITHIN THIS TRIAL THE FIRST FOUR DOSE COHORTS. ALL THE PATIENTS TOLERATED THE VECTOR AND ASSOCIATED PROCEDURES WELL AND IMPORTANT FOR US, WE HAD SUBJECTIVE AND OBJECTIVE IMPROVEMENTS IN GLAND OUTPUT WITHIN THESE INDIVIDUALS. ABOUT HALF OF THEM HAD IMPROVED RECOVERY. IT WAS IMPORTANT TO UNDERSTAND WHO THOSE WERE AND ALSO WHY SOME DIDN'T RESPOND, AND I'M NOT GOING TO GO THROUGH ALL THE DATA BUT JUST TO SUMMARIZE IT, THE PROBLEM WAS THE VECTOR SYSTEM THAT WE WERE USING INITIALLY WITH THE ADENOVIRUS. YES, IT WAS DESIGNED AS A PROOF OF CONCEPT TO SHOW THAT AQUAPORI WOULD WORK, WE SAW INCREASE IN THOSE INDIVIDUAL, SOME HAD MORE DURABLE EXPRESSION THAN WE EXPECTED BUT WE DID SEEING EVEN AT VERY LOW DOSES OF ADENOVIRUS AN INFLAMMATORY RESPONSE. AS YOU CAN SEE BY THE DOSE ESCALATION. SO BROADLY IF YOU LOOK AT PRELEVELS OF SALIVA VERSUS POST YOU CAN SEE THERE'S A STATISTICALLY SIGNIFICANT INCREASE BUT ONLY ABOUT HALF OF THE INDIVIDUALS ARE RECOVERING, AND THAT'S DEPENDENT ON THE DOSE CORE HORT, AS WE WENT HIGHER IN THE VECTORS, WE STARTED TO SEE A PEAK AND THEN HIT WHAT WE CALLED A MAXIMUM TOLERATED DOSE. WHERE WE STARTED SEEING INFLAMMATION IN ALL THE PATIENTS WE WERE DOSING IN DOSE COHORT 4. WE WERE USING A GALLIUM UPTAKE ASSAY, SO NONE OF THESE PATIENTS EXPERIENCED SWELLING WITHIN THE GLAND, OROVERT PAIN, BUT WE COULD SEE CONCENTRATIONS OF GALLIUM, INDICATING THAT LYMPHOCYTES WERE MOVING IN TO THE AREA, AND NONE OF THESE INDIVIDUALS HAD INCREASED SALIVA FLOW. SO HAD WE ACHIEVED OUR GOAL OF DEMONSTRATING THAT AQUAPORIN WOULD WORK? YES. AND WE COULD THEN MOVE ON TO OUR DURABLE VECTOR, WHICH OVERALL IS A LOT LESS IMMUNOGENIC THAN THE ADENOVIRUS. IMPORTANTLY FOR US, AAV ALSO IS ABLE TO BE DURABLE IN THE SALIVARY GLAND AND PROMOTE LONG TERM STABLE TRANSDUCTION. SAL ARE VARY GLANDS ARE A SLOWLY DIVIDING EP PEEL YA AND IN OUR PRE-CLINICAL STUDIES, WE COULD SEE -- EFFECTIVELY FOR THE LIFE OF THE ANIMAL. ADENOVIRUS IN A MOUSE SIMILAR TO A PIG IS EXPRESSED AND THEN SHUT OF OFF BY ABOUT TWO WEEKS. WHERE AAV TAKES ABOUT 12 WEEKS BEFORE YOU START SEEING PEAK EXPRESSION BUT THEN IS DURABLE IN MICE FOR ESSENTIALLY THE LIFE OF THE MOUSE. IN PIGS WE WENT OUT AS FAR AS 32 WEEKS AND WE COULD SEE LONG TERM SUSTAINED EXPRESSION WITHIN THE PIGS. HOW DOES AAV COMPARE TO ADENOVIRUS IN TERMS OF EXPRESSION? AND RECOVERY OF FUNCTION? THIS IS A WATER MOVEMENT ASSAY, AND THIS IS SUPPOSED TO BE A DELTA, I'M SORRY IT GOT LOST IF THE SLIDE, BUT YOU CAN SEE IN TERMS OF FLUID MOVEMENT WITH AN ADD OR AN AAV THAT ENCODES AQUAPORIN 1 AND OUR CONTROL, NO TREATMENT OR -- YOU CAN SEE THAT THE ADD IS VERY SIMILAR IN TERMS OF RECOVERY TO THE AAV. BUT IF YOU LOOK AT THE AMOUNT OF PROTEIN THAT BOTH OF THOSE ARE PRODUCING, OVER HERE THE PERCENT TRANSDUCTION SET THE ADENOVIRUS AT 100. THE AAV IS REALLY ONLY PRODUCING ABOUT 10% OF THE AMOUNT OF MATERIAL THAT THE AD WAS, BUT YET WE GET THE SAME AMOUNT OF RECOVERY, SUGGESTING THAT THIS INTRACELLULAR PROTEIN, WE REALLY DON'T NEED A LOT OF IT TO GET IT TO GO, JUST A FEW MOLECULES OF AQUAPORIN TO CREATE THAT FACILITATED CHANNEL WOULD BE SUFFICIENT TO RECOVER FUNCTION AND MOVE MAXIMUM AMOUNTS OF FLUID. HOW DOES THIS WORK IN AN EFFICACY MODEL GOING BACK TO OUR PIGS, THE RADIATION SIDE IS BROKEN UP A LITTLE DIFFERENTLY HERE. WE HAVE NON-TREATED AND THEN THE TREATED SIDE. AND YOU CAN SEE IN OUR NON-IRRADIATED OVER 16 WEEK, WE SEE VERY LITTLE LOSS IN GLAND ACTIVITY. RETAINED ABOUT 80% OF IT. WHEREAS IN THE IRRADIATED SIDE, WE LOSE ALL OF THAT AGAIN AS I WAS SHOWING YOU BEFORE IN THE FIRST STUDIES. 16 WEEKS IS OUR ZERO TIME POINT HERE, WHERE WE COME IN WITH AN AAV ENCODING AQUAPORIN 1, 1 ENCODING LAGZ AND BY TWO WEEKS, WE SEE VERY LITTLE DIFFERENCE, MAYBE IT'S LIKE DECREASE FROM OUR BASELINE LEVELS, BUT NOW 4, 6, 8, NOW THROUGH 12 WEEKS, WE SEE SLOW BUT STEADY IMPROVEMENT IN GLAND ACTIVITY, AT THIS DOSE ABOUT 50% OF WHAT OUR STARTING LEVELS WERE. SO VERY SIMILAR IN THAT SENSE TO AT NOVI RUSS, BEING ABLE TO ENCODE AQUAPORIN AND TO SEE AQUAPORIN TRIGGER RECOVERY AND FUNCTION. SO WE'VE USED THIS AS EFFICACY DATA STARTING IN 2016 TO GO BACK AND START A DURABLE MORE LONG TERM STUDY USING AAV ENCODING AQUAPORIN. THE STUDY DESIGN AGAIN IS VERY SIMILAR, IT'S BEING DONE HERE IN THE CLINICAL CENTER, AND IT'S A SINGLE DOSE, DOSE ESCALATION-TYPE STUDY WITH THE PRIMARY MEASURE AGAIN BEING SAFETY WITH SECONDARY MEASURES OF EFFICACY LOOKING AT SUBJECTIVE AND OBJECTIVE IMPROVEMENTS IN GLAND ACTIVITY. IN KEEPING WITH THE FIRST STUDY, WE'VE EXTENDED IT AND ARE GOING OUT TO A TOTAL OF THREE YEARS WITH THIS STUDY. THE STUDY IS ONGOING SO I'M NOT GOING TO TALK A LOT ABOUT THE DATA FROM IT OARN OTHER THAN TO SAY WE'RE ENROLL, WE'VE ENROLLED 12 PATIENTS SO FAR AND DOSED OUR FIRST FIVE. WE'RE OUT 18 MONTHS FARTHEST AND OUR MOST RECENT PATIENT WAS DOSED EARLIER THIS SUMMER. THE VECTOR AS WE WERE SEEING IN OUR ANIMAL MODELS IS STAYING WHERE WE PUT IT. WE SEE IT LOCALIZED, THOUGH, WITHIN THE SALIVARY GLAND, WE'RE NOT DETECTING ANY IN THE SERUM AND NONE OF THE SAFETY STOPPING RULES HAVE BEEN MET SO WE'RE CONTINUING TO INCREASE OUR DOSE TO FIND A MAXIMUM TOLERATED DOSE WITHIN THESE STUDIES. AND WE'RE HOPING TO WRAP THE STUDY UP WITHIN THE NEXT YEAR OR TWO. THIS STUDY HAS REQUIRED A LOT OF RESOURCES FROM AROUND NIH. SPECIFICALLY THE CLINICAL TEAM THAT I'VE BEEN WORKING WITH IN NIDCR, A LOT OF THE PIONEERING WORK FOR THE BASIC PHYSIOLOGY WAS DONE BY BRUCE BAUM, WE'VE HAD A LOT OF RESPONSE FROM PEOPLE IN MY LAB, SANDRA, SANDRA,GIOVANNI, AND THE NICE THING ABOUT WORKING WITH THE CLINICAL CENTER IS WE HAVE& PHYSIOLOGY EXPERTS WITHIN NIDCR, BUT ALSO CLINICAL AND ANIMAL SUPPORT. WEE RELIED A LOT ON ROB DONAHUE, CINDY DUNBAR IN HEART, LUNG AND BLOOD FOR SOME OF THE MONKEY STUDIES THAT I DIDN'T HAVE TIME TO TALK ABOUT, ROB KOTIN FOR EARLY WORK WHEN HE WAS HERE AND OUR COLLEAGUES AT NIEHS DOWN IN NORTH CAROLINA, RON IRWIN AND MOLLY VALLIANT FOR TOXICOLOGY AND BIODISTRIBUTION STUDIES. THANK YOU. [APPLAUSE] >> I'M PLEASED TO INTRODUCE OUR SECOND SPEAKER, DR. LEONELA AM OWE SILL FROM THE UNIVERSITY OF TEXAS SOUTHWESTERN. LEONELA IS AN INSTRUCTOR THERE AND ALSO THE RECENTLY NAMED DIRECTOR OF GENE EDITING AT A BOSTON BASED GENE THERAPY STARTUP. SHE'S GOING TO BE TALKING ABOUT EXCITING RECENT WORK ON -- EDITING FOR DUCHENNE MUSCULAR DYSTROPHY USING CRISPR CAS9. LEONELA. >> GOOD MORNING. I WOULD LIKE TO THANK YOU FOR THE INVITATION AND OPPORTUNITY TO PRESENT OUR WORK ON THE CORRECTION OF MUSCULAR DYSTROPHY BY GENOME EDITING. ONE OF THE MOST SEVERE AND COMMON GENETIC DISORDER THAT IS DIAGNOSED IN CHILDHOOD, AND CURRENTLY THERE IS NO CURE FOR THIS DEVASTATING DISEASE. THE SYMPTOMS APPEAR IN THE PATIENTS QUITE EARLY BY THE AGE OF 2, 3 YEARS, AND ALREADY BY THE AGE OF 15 OR EVEN EARLIER, THEY LOSE THEIR MOBILITY AND SOME OF THE PATIENTS REQUIRE A WHEELCHAIR. CARDIAC PROBLEMS ARE QUITE OFTEN AND MOST COMMON -- OF THE DISEASE IN THOSE PATIENTS. IT'S CAUSED BY A GENE CALLED DISTROAPHIN, CODING FOR THIS PROTEIN THAT IS KNOWN TO BE THE MOLECULAR SHOCK ABSORBER, THE MUSCLE, SINCE IT'S LINKING THE CONTRACTILE UNITS OF THE MUSCLE. IT'S CONNECTING TO THE MEMBRANE FROM FROM THE DYSTROGLYCAN COMPLEX. SO YOU CAN SEE THE LOCALIZATION OF THIS PROTEIN IS AROUND THE MUSCLE FIBER AND IT'S SURROUNDING THE MUSCLE FIBER HERE. AND THE LOSS OF DYSTROPHIN CAUSES THE LOSS OF INTEGRITY OF THE MUSCLE SO IT BECOMES MORE SENSITIVE TO ANY MOVEMENT AND CONTRACTION. THE PROTEIN IS COMPOSED OF THESE DIFFERENT DOMAINS, AS ILLUSTRATED HERE, THE ACTIN, THE ROD AND THE CYSTINE DOMAIN THAT CONNECTS TO THE COMPLEX. THE ACTIN DOMAIN CONNECTS THE DYS -- THE MIDDLE PART DOMAIN, YOU CAN ASSOCIATE THAT TO THE SPRING OF THE SHOCK ABSORBER HERE COMPOSED OF DIFFERENT -- REPEATS THAT CAN BE ACTUALLY HAS BEEN SHOWN THAT A SHORTER VERSION OF THE PROTEIN WITH NOT ALL OF THE ROLE ROD DOMAINS IN THE CAN STILL BE FUNCTIONAL. THOSE EXAMPLES CAME FROM A LESS SEVERE FORM OF THE MUSCULAR DYSTROPHY CALLED BECKER DYSTROPHY. IT SHOWS IT CAN FUNCTION, IT'S NOT PERFECT BUT IT HAS THE EXTREMITY OF THE PROTEIN, IT CAN MAINTAIN THE INTEGRITY TO THE MUSCLE. THE SHAPE OF THE EXONS ILLUSTRATE THE MAINTENANCE OF THE READING FRAME AND THIS MAINTENANCE AND THIS SHAPE IS HIGHLY CONSERVED BETWEEN HUMANS AND OTHER SPECIES. SO THERE ARE MORE THAN 3,000 MUTATIONS THAT HAVE BEEN DESCRIBED FOR DUCHENNE MUSCULAR DYSTROPHY THAT CAN BE CATEGORIZED IN THOSE THREE GROUPS: EXON DELETIONS, POINT MUTATION AND DUPLICATIONS. THE EXON DELETIONS CLUSTER IN A PARTICULAR REGION OF THE GENE LISTED HERE, YOU CAN SEE BELOW, THESE ARE LISTING THE EXON OF THE GENE AND HEERT PERCENTAGE THAT EXONS ARE INVOLVED IN DELETIONS AND YOU CAN SEE ON THIS GRAPH THAT THERE'S A PARTICULAR REGION CALLED HOT SPOT WHERE THOSE DELETIONS OCCUR MORE OFTEN THAN IN OTHER REGIONS. ONE OF THESE REGIONS LISTED HERE IS FROM THE 43 TO 55, WHERE A LOT OF DELETIONS HAPPEN, AND SOME OF THE EXON IN THIS REGION CAN BE SKIPPED AND CORRECT THE READING FRAME OF THE PROTEIN. I'VE LISTED HERE THE TOP FOUR, THE EXON 51, FOR EXAMPLE, HAS BEEN SHOWN THAT BY SKIPPING THE EXON 51, THAT WOULD CORRECT 30% OF THE MUTATION THAT ARE IN THE POPULATION. SO TO BETTER UNDERSTAND HOW WE CAN CORRECT AND RESTORE THE READING FRAME USING THE SKIPPING OF EXON IF ONE, WE 51, WE WANTED TO GENERATE THE MODELS THAT WOULD MIMIC THIS REASONING FOR HOT SPOT 1 AND WE MADE AN EXON 50 DELETION IN THE MOUSE MODELS USING CRISPR CAS9, WHICH -- YOU CAN SEE HERE BY THE SHAPE OF THE EXON 9 COMPATIBLE AND THE MICE THAT ARE -- WERE LACKING THE EXON 50 WERE LACKING THE DYSTROPHIN -- THIS MOUSE MODEL PRO DUES I'DICALLY -- CREATING A CELL FIBROTIC TISSUE IN THE MUSCLE. SO THERE ARE MULTIPLE EFFORTS THAT HAVE BEEN DONE FOR -- OVER THE PAST AND THAT I LISTED HERE JUST A FEW EXAMPLES OF THE EFFORTS THAT HAD BEEN DONE. CURRENTLY THERE'S NO CURE AND WHAT WE'RE PROPOSING IS TO CORRECT -- TO DO -- TO HAVE A CORRECTION OF THE CAUSE OF THE DISEASE THAT WILL GO AT THE BASE AT THE DNA LEVEL LISTED HERE. HOW WE WANT TO DO THAT IS USING THE CRISPR CAS9, A KNEW CLEE NUCLEASE THAT SPECIFICALLY BINDS TO GENOMIC DNA USING A GUIDE RNA WHICH YOU CAN CONSIDER LIKE A GPS FOR THE CAS9 THAT WILL DIRECT THE CAS9 TO GO FOR A SPECIFIC LOCUS, WHERE IT WILL MAKE A CLEAVAGE CLOSE TO THIS SEQUENCE, DOUBLE STRAND BREAK, WHICH CAN BE REPAIRED BY THE DNA REPAIR MACHINERY BY TWO METHODS. ONE OF THE METHODS IS CALLED HOMOLOGY DIRECTED RECOMBINATION WHICH REQUIRED A DNA TEMPLATE AND WHICH WILL LEAD TO PRECISE MODIFICATION, PRECISE CORRECTION OF THE LESION. THIS PATHWAY OR OF THE OF THE DNA REPAIR APPEARS ONLY ON -- THAT ARE STILL DIVIDING. THE SECOND METHOD OF THE REPAIR CALLED NHEJ OR NON-HOMOLOGOUS END JOINING, CLEAVED ENDS THAT WERE GENERATED BY THE CLEAVAGE, GENERATES BY THIS WAY INSERTION DELETIONS AND THIS PATHWAY OCCURS IN MOST OF THE TISSUE POST MITOTIC AND PROLIFERATIVE TISSUE. SO IN OUR CASE, THE MUSCLE AND THE HEART WHICH ARE THE MAIN TISSUE THAT ARE AFFECTED IN DUCHENNE MUSCULAR DYSTROPHY -- POST MITOTIC TISSUE, WE HAD TO RELY ON THIS METHOD FOR THE CORRECTION OF THE MUTATIONS. SO THE WAY WE THOUGHT ABOUT CRISPR/CAS 9, WE'RE USING SP CASS 9 THAT HAS A PAM THAT HAS AN AG AND AGG. INTERESTINGLY. EVERY GENE IN THE GENOME HAS -- AND EVERY EXON OF THE GENE CONTAINS SPECIFIC MESSAGES THAT ACTUALLY TELL THE SPLICING MACHINERY TO INCLUDE OR NOT THE EXON IN THE GENE. THOSE SEQUENCES ARE HIGHLY CONSERVED SO BASICALLY IN EVERY EXOME THE GENE WE HAVE POTENTIALLY A PAM SEQUENCE FOR THE SP CASS 9 SO WE HAVE DIFFERENT POSSIBILITY TO DESIGN THOSE GUIDE RNAs FOR THE SPECIFIC EXONS. SO THE WAY WE DESIGN OUR STRATEGY TO SPECIFICALLY DESIGN A GUIDE RNA THAT IS LOCATED AT THE JUNCTION, IT'S MORE ILLUSTRATED HERE, AND STILL IN THE CODING SEQUENCE OF THE EXON 51 IN THIS WAY WILL GENERATE THE CUT OF THE EXON 51 AT THE VICINITY OF THAT SUPPLIES ACCEPTOR REGION. AND TO INCREASE OUR CHANCES TO RESTORE THE PROTEIN IN THE TWO WAYS, THAT WOULD LEAD TO SKIPPING OF EXON 51, ADDITIONALLY, POSSIBLY RESTORE THE READING FRAME OF THE PROTEIN BY THOSE EVENTS AND THE FACT THAT WE'RE STILL CUTTING IN THE THE CODING REGION. AND THIS, OF COURSE, IS ALSO HIGHLY CONSERVED REGION AND CAN BE EASILY TRANSLATED FROM SPECIES TO SHE'S, THE SPECIES, SO JUST TO ILLUSTRATE A BIT DIFFERENTLY, THE SKIPPING OF THE GENE, HERE'S A NORMAL GENE WHERE THE SHAPE OF THE EXON COMPATIBLE GENERATED RNA IS COMPATIBLE WITH THE PROTEIN, IT'S FUNCTIONAL AT THE END. IN OUR CASE FOR THE MICE, WE HAD DELETED, THERE'S NO PROTEIN THAT THIS PRODUCED. SO WE'RE HOPEING BY GENERATING A SINGLE CUT HERE, WE WOULD GENERATE -- THAT COULD SKIP EITHER THE EXON 51 AND POSSIBLY AS WELL REFRAME IT BY GENERATING INSERTION AND WE HAVE ONE IN THREE CHANCES TO REFRAME THE PROJECT SO YOU'LL SEE THAT IT'S ACTUALLY QUITE RESEISE, THE REFRAINING EVENTS. IN THIS CASE, WE'LL GENERATE A MINIMUM MODIFICATION, WE'RE NOT TALKING ABOUT BIG DELETION BUT JUST ONE EXON WHICH IS 71 AND TO GENERATE THE A REALLY SHORT VERSION OF DYSTROPHIN. THE WAY THAT WE'RE DELIVERING THE AV -- THE GENE HE EDITING MACHINERY USING AAV9 WHICH HAS GOOD -- FOR THE SCEL AT THAT TIME MUSCLE AND THE HEART AND HAS BEEN AS WELL HIGHLY USED IN THE PAST IN CLINICAL TRIALS, IT HAS SHOWN TO BE SAFE FOR HUMANS. FOR THE DELIVERY, WE USE TWO DIFFERENT VECTORS, ONE FORECAST 9, ANOTHER -- AND WE DELIVERED SYSTEMICALLY IN THE MOUSE AND ANALYZE DIFFERENT TIME POINTS. ADDITIONALLY, TO MAKE IT MORE SAFE AND REALLY SPECIFIC FOR THE TISSUE THAT WE'RE TARGETING, WE INTRODUCE THE MUSCLE SPECIFIC PROMOTER AS WELL IN THE VECTOR TO SPECIFICALLY EXPRESS THE CAS NINE ONLY ON THE TISSUE THAT WE WANT TO DO THE CORRECTION. THE GUIDE RNA IS CARRIED BY A DIFFERENT VECTOR, AND THE SAME GUIDE RNA WAS PUT IN THREE COPIES, USING THREE DIFFERENT SINGLE PROMOTOR TO INCREASE THE EFFICIENCY WITH OUR MACHINERY. YOU CAN SEE HERE OUR VECTOR IS HIGHLY SPECIFIC ONLY FOR THE MUSCLE IN THE HEART. HERE'S THE HEART EXPRESSION OF CAS NINE, THESE ARE THE -- INJECTED, HERE WE SEE THE EXPRESSION OF THE CAS NINE OF THE DIAPHRAGM, AND IN THE LIVER WE DIDN'T DETECT ANY EXPRESSION SO THERE'S NO EXPRESSION IN ANY OTHER TISSUE BESIDES SKELETAL MUSCLE AND THE HEART. SO AT THE GENOMIC LEVEL, THIS IS MORE AMONG -- I'M SHOWING THE RESULTS PLAYLY THE TRANSCRIPT AT THE -- LEVEL, I MENTIONED TO YOU THAT CRISPR/CAS 9 -- HERE'S A PAM SEQUENCE AT THE EXON 51. THIS IS A NON-EDITED VERSION OF THE LOCUS AND YOU CAN SEE THAT GENERATES A STOP -- SO WHAT -- IN OUR CASE USING THIS GUIDE RNA THAT THREE NUCLEOTIDES -- WE'VE SEEN REALLY ONE OF THE MOST COMMON MUTATION THAT WE SEE IN HIS CORRECTION -- CORRECTED MICE IS THIS ONE INSERTION, THE SAME POSITION AT THE CLEAVAGE SITE WE ALWAYS HAVE ONE INSERTION AND WAS THE MOST DOMINANT. IT'S REPRESENTED 60% OF OUR RNA TRANSCRIPTS. ADDITIONALLY AS WE EXPECTED THE DELETION WOULD LEAD TO THE SKIPPING OF THE EXON SO -- SKIPPING EVENTS WHICH WERE 20% OF THE RTPCR PRODUCTS. THE AT THE CORRECTION LEVEL AS YOU CAN SEE HERE, WE LOOKED AT DIFFERENT TISSUE HERE, WE HAVE ANTERIOR MUSCLE, DIAPHRAGM AND THE HEART. THIS IS A HEALTHY MUSCLE WITH THE DYSTROPHY SURROUNDING THE MUSCLE FIBERS AND THE DELTA 50 MICE THAT EXPRESS NO DYSTROPHIN HERE AS YOU CAN SEE, AND ONLY AFTER FOUR WEEKS AFTER INJECTION WE SEE A WIDESPREAD EXPRESSION IN DIFERENT TISSUES IN THE SCEL HEART AND WE SEE TSH AT EIGHT WEEKS AS WELL, HERE IS A WESTERN BLOT. BY FOUR WEEKS AFTER INJEK, INJECTION, THIS IS HEART TISSUE, THEY DON'T HAVE ANY DYSTROPHIN AND THE ONE THAT WE INJECTED WE OBSERVED UP TO 80% CORRECTION OF THE DYSTROPHIN IN ONLY FOUR WEEKS AFTER INJECTION. OF COURSE WE LOOKED AT THE -- IF THE CORRECTION OF THE DYSTROPHIN CORRELATES WITH A FUNCTIONAL CORRECTION OF THE MUSCLE, AND HERE'S THE RESULT OF THE GRIP STRENGTH ANALYSIS ON THOSE MICE, WHERE WE MEASURED THE FORCE THE MICE APPLIED TO THE SENSOR, YOU CAN SEE HERE THE HEALTHY MICE ARE QUITE STRONG AND THE DYSTROPHIC MICE HAVE 50% REDUCTION OF THEIR STRENGTH. AND THE MICE THAT WE INJECTED WITH THE AAV GENE EDITING MACHINERY, WE SEE -- FOUR WEEKS AFTER INJECTION. SO IN ORDER TO -- WE WERE QUITE IMPRESSED BY THE LEVEL OF CORRECTION IN THE MICE, AND WE WERE WONDERING HOW WELL IT WOULD WORK IN LARGER ANIMALS, IF THAT'S SOMETHING WE COULD TRANSLATE TO HUMANS, AND LUCKILY FOR US, THERE WAS A MOUSE MODEL ALMOST IDENTICAL GENETICALLY TO OUR MICE MODEL IN THE ROYAL VETERINARY COLLEGE THAT INSTEAD OF SPONTANEOUS MUTATION THAT IN THE CHARLES SPANIEL BEAGLE MODEL THAT PRESENTED FIVE DONOR SITE MUTATION THAT CAUSED THE EXON 50 DELETION. SO AS YOU CAN SEE HERE, THE EXON 50 DELETION, SAME RESULT, NO DYSTROPHIN AND A DOG THAT PRESENTS WITH A DYSTROPHIC PHENOTYPE -- IN THE SKELETAL MUSCLE AND THE HEART. SO WE USED A SIMILAR APPROACH, EXACT SIMILAR APPROACH TO LOOK AT THE CORRECTION AND THIS DOG MODEL, AND SINCE I MENTIONED, THE GUIDE RNA WAS DESIGNED IN AN EXON PART WHICH HIGHLY -- BASICALLY WE'RE CUTTING EXACTLY THE SAME LOCUS AS WELL IN THE DOG MODEL. AND FIRST WE WANTED TO TEST IF IT WORKS AND WE DID FIRST INTRAMUSCULAR INJECTION. HERE WE HAVE THE DYSTROPHIN STAINING IN THE HEALTHY DOG THAT'S ILLUSTRATED HERE. THIS IS AN UNTREATED DOG THAT WERE INJECTED -- IT'S AN UNTREATED -- THAT WERE NOT INJECTED AND HERE ARE TWO DOGS THAT WERE INJECTED AND ANALYZED SIX WEEKS AFTER INJECTION AND YOU CAN SEE HERE THE DYSTROPHIN EXPRESSION IS WIDESPREAD ALONG THE MUSCLE. WE CONFIRMED THIS CORRECTION BY WESTERN BLOT. YOU CAN SEE THE DYSTROPHIN THE DYSTROIN DY STROPHIN IN THE HEALTHY, UNTREATED AND TREATED ANIMALS, WE CAN CORRECT UP TO 60% OF THE DYSTROPHIN IN THOSE ONLY SIX WEEKS AFTER INJECTION. THEN WE WONDERED HOW WELL IT CORRECTS SYSTEMICALLY AND WE CORRECTED SEVERAL DOSES. THE HERE'S A WILD TYPE UNTREATED DIFFERENT MUSCLE, THIS IS TRICEPS AND BICEPS, CRANE YELL, HERE WE INJECTED THE DOSE OF -- WE COULD SEE SOME CORRECTION IN THOSE DIFFERENT GROUP OF MUSCLE. WHEN WE INCREASED THE DOSE, WE CAN SEE AN INCREASE OF THE CORRECTION IN THE CORRESPONDING MUSCLE TO REALLY STRIKING ALMOST ON THE BICEPS WAS ALMOST AS CLOSE AS TO THE WILD TYPE. OF COURSE WE QUANTIFY THAT BY WESTERN BLOT AND YOU CAN SEE HERE THE LEVELS OF CORRECTION THAT WE'VE SEEN IN THE CRANIAL TIBIALIS MUSCLE, TRY SPEPS AND TRICEPS AND BICEPS, EIGHT WEEKS POST INJECTION. ONE OF THE MAIN CAUSE OF DEATH IN THE PATIENTS ARE HEART FAILURE AND DIAPHRAGM INSUFFICIENCY, SO THOSE ARE TISSUE THAT ARE HIGHLY IMPORTANT FOR OUR CORRECTION AS WELL AND WE LOOKED AT THE EXPRESSION AND THE CORRECTION IN THOSE TISSUES. YOU CAN SEE HERE IS A DIAPHRAGM AND THE WILD TYPE UNTREATED AND THE ONE WE TREATED WITH GENE EDITING MACHINERY, AND SIMILAR THE HEART, WE SAW REALLY GOOD CORRECTION IN THE HEART TISSUES AS WELL. HERE'S ILLUSTRATED THE WESTERN BLOT ANALYSIS AND DIET FRAM DIAPHRAGM AND THE HEART, REACHED 90 TU% 992% OF& CORRECTION. THE ABSENCE OF DYSTROPHIN NOT ONLY LEADS TO LOSS OF INTEGRITY BUT ALSO LEADS TO THE -- DECREASE OF INTEGRITY OF THE MUSCLE. HERE YOU CAN SEE THE WILD TYPE LOCALIZATION FOR DYSTROGLYCAN COMPLEX PROTEIN, IT'S LOCALIZED SIMILAR TO THE DYSTROPHIN AT THE MEMBRANE. WE LOSE THE LOCALIZATION OF THE BETTER DYSTROGLYCAN, IT'S MISLOCALLIZED AND WHEN WE INJECT WITH AV9 CONTAINING THE JEAN GENE EDITING MACHINERY, WE COULDN'T CORRECT THE -- AND LOCALIZATION OF DYSTROGLYCAN PROTEIN IN THE AFFECTED MUSCLE. AT HISTOLOGICAL LEVEL, WE SEE AS WELL A CLEAR IMPROVEMENT, HAOERS A DIAPHRAGM. THIS IS A WILD TYPE UNTREATED ANIMAL. THIS IS THE DYSTROPICK, YOU CAN SEE THE FIBROTIC, THIS IS -- THE DIAPHRAGM IS HIGHLY AFFECTED IN THOSE DOGS, AND HERE WE HAVE THE DOG THAT WAS INJECTED WITH AAV9 WITH 1 -- EACH VIRUS DOSE AND YOU CAN SEE THE HISTOLOGICAL FEATURES OF THE DIAPHRAGM IS MUCH MORE IMPROVED THAN THE ONE THAT WERE NOT TREATED. SO WE HAVE DONE A LOT OF WORK IN OUR LAB IN VALIDATING FIRST THE GUIDE RNA, I HAVEN'T SHOWN ALL THE INFORMATION AND ALL THE RESULTS THAT WE HAVE DONE OVER THE PAST, BUT WE'VE HAD THIS -- WHERE WE CAN VALIDAE THE GUIDE RNA IPS DERIVED FROM PATIENTS AND WE'VE DONE SIMILAR FOR THIS GUIDE RNA, WE HAVE SHOWN THAT IT WORKS IN A SIMILAR WAY THAT -- LOCUS CORRECTION AS WE'VE SEEN IN MOUSE AND DOG AND HUMANS, AND GENERATES THE SAME CORRECTIONS AS I MENTIONED ONE INSERTION ACROSS THE SPECIES IN THE MOUSE, DOG AND HUMAN. WE CURRENTLY EVALUATE AND WE HAVE VALIDATED GUIDE RNAs USING THIS PLATFORM FOR ALL THE OTHER EXONS THAT WE'VE -- THE TOP 10 EXONS THAT ARE MOST COMMON. AND THAT CAN BE APPLICABLE FOR 80% OF THE POPULATION. THEN WE HAVE DEVELOPED AS WELL ADDITIONAL FIVE MODELS THAT WOULD LOOK INTO THE CORRECTION OF THE DISEASE USING THIS NEW GUIDE RNA SO WE'RE HOPING TO VALIDATE ALL OF THE GUIDE RNA THAT WE HAVE SELECTED IN VIVO, AND ULTIMATELY TRANSLATE OUR TECHNOLOGY IN HUMANS. I WANT TO POINT OUT AS I MENTIONED WE'RE EDITING THE DISEASE CAUSING MUTATIONS, WE'RE TRYING TO DO AS MINIMAL BUT USING A SINGLE CUT APPROACH, WE'RE DOING THE MINIMAL MODIFICATION TO THE GENE THAT WE CAN. IT'S IN PRIN PRINS PEL A ONE-TIME TREATMENT THAT AS YOU CAN SEE ALREADY BY FOUR WEEKS IN MICE AND EIGHT WEEKS IN DOGS, IT'S REACHED REALLY WELL THE SCEL AT THAT TIME MUSCLE IN THE SKELETAL MUSCLE IN THE HEART. ADDITIONALLY THE WILD TYPE LOCUS, WE'RE REALLY GETTING NORTH MALL EXPRESSION OF THE NORMAL -- OF COURSE THERE'S A LOT OF QUESTIONS TO BE ADDRESSED IN THE FUTURE. WHAT'S THE DURABILITY OF THE CORRECTION AND WE'RE HIGHLY LOOKING INTO ALL THE POTENTIAL OF TARGETS AND WHAT THE LONG TERM EXPRESSION AND ANALYZING OF TARGETS LONG TERM EXPRESSION AND AS WELL THE IMMUNE RESPONSE TO THE A ACHT V AND TO CAS NINE. SO THOSE ARE THE QUESTIONS WE'RE CURRENTLY LOOKING INTO IN THE LARGE EARLY ANIMALS WHICH IS CLOSER TO HUMANS, AND HOPEFULLY WE CAN ADDRESS THOSE QUESTIONS IN THE FUTURE. SO FINISH, I WOULD LIKE TO THANK ALL THE PEOPLE THAT CONTRIBUTED TO THIS WORK, AND AS WELL ALL OUR COLLABORATORS, AND FROM SOUTHWESTERN FROM LONDON, WHERE THE COLONY IS CURRENTLY HOUSED AND FROM GERMANY AND SEATTLE. I'D LIKE TO THANK ERIC OL SON, OLSON, HOPEFULLY WE CAN BRING THIS TECHNOLOGY FORWARD WITH THE RIGHT APPROPRIATE STEPS, OF COURSE, A LOT OF THINGS TO DO. AND I WOULD LIKE TO THANK YOU ALL FOR YOUR ATTENTION AND I'LL BE HAPPY LATER TO ADDRESS ANY QUESTIONS. THANK YOU. [APPLAUSE] >> I'D LIKE TO INTRODUCE OUR THIRD SPEAKER TODAY, DR. HARRY MALECH FROM THE INTRAMURAL RESEARCH PROGRAM HERE AT NIGHED A. DR. MALECH IS THE CHIEF OF THE GENETIC IMMUNOTHERAPY SECTION AND HE IS A FORMER PAST PRESIDENT OF THE ASGCT. TODAY HE'S GOING TO TALK ABOUT THE EFFECT OF MARROW CONDITIONING, LESSONS LEARNED FROM CLINICAL TRIALS AND NOVEL CONDITIONING APPROACHES IN PRE-CLINICAL AND EARLY CLINICAL DEVELOPMENT. DR. MALECH. >> SO I'M GRATEFUL TO THE ORGANIZERS FOR INVITE MEG TODAY. I WAS ORIGINALLY ASKED TO TALK ABOUT AN INTERESTING PAPER THAT WE PUBLISHED RECENTLY ABOUT USING CAR T-CELLS FOR CONDITIONING REGIMEN, AND WHEN ASKED TO TALK, I SAID, ACTUALLY, I'M GOING TO SPEND VERY LITTLE TIME ABOUT THAT, AND WHAT I'D LIKE TO DO TODAY IS TO TALK ABOUT THE CRITICAL ROLE OF CONDITIONING IN GENERAL AS AN ELEMENT OF HEMATOPOIETIC STEM CELL THERAPY. AS WITH ANYTHING LIKE THAT, SPECIFICS MATTER, SO I'M GOING TO REFER TO SOME OLD AND CURRENT TRIALS TO ILLUSTRATE THE POINTS AND THEN WRAP UP AT THE END TALKING ABOUT SOME NEW THINGS OUT THERE THAT ARE COMING ALONG FOR CONDITIONING. SO I'D LIKE TO START WITH SOME GENERAL PRINCIPLES RELATING TO SOME PRIOR CLINICAL TRIAL OUTCOMES DEMONSTRATING KEY PRINCIPLES RELEVANT TO TODAY'S DISCUSSION. ALMOST ALL OF YOU ARE FAMILIAR WITH THESE LANDMARK STUDIES FOR X LINK SEVERE -- WITH MIRRORING GAMMA RETROVIRUS. IT WAS SHOWN THAT THEY CAN RESTORE T-CELL IMMUNITY TO INFANTS ABOUT WITHOUT CONDITIONING -- B CELLS AND K CELLS OR MYELOID CELLS. A LANDMARK STUDY BY ALESSANDRO ALUTI AND FOLLOWED BY WORK BY BOBBY GASPAR SHOWED THAT IN THE CASE OF ADENOSINE DEAM NAIZ DEFICIENT SCID, THAT IF YOU ADDED LOW DOSE BUSULFAN BUT ONLY IF YOU ADDED IT, THEY ENDED UP WITH MARKED BROAD RESTORATION OF IMMUNITY TO INFANTS WITH THIS DISORDER, IN THIS CASE, THEY USED A VERY MODEST DOSING OF ABOUT 3 TO 4 MILLIGRAM PER KILOGRAM OF BUSULFAN. BIFFI -- IN TRYING TO DEVELOP TREATMENTS FOR LEUKODYSTROPHIES BY ALSO TARGETING HEMATOPOIETIC STEM CELLS FOUND THAT THEY GOT QUITE REMARKABLE RESULTS BUT ONLY IF THEY USED QUITE SIGNIFICANT LEVELS OF BEU SUL FAN CONDITIONING. SO IT WAS CLEAR THAT IT MATTERED, THAT YOU TAILORED WHAT YOU NEEDED TO THE DISEASE AND THE OUTCOMES THAT YOU NEEDED. WHAT I'D LIKE TO DO IS USE A MORE DETAILED DISCUSSION OF ANOTHER SPECIFIC EXAMPLE THAT WE'VE BEEN STUDYING IN OUR LABORATORY FOR A LONG TIME NOW, WITH CHRONIC GRANULOMATOUS DISEASE WHICH IS AN INHERITED DEFECT IN MICRO BUY SIGH DELL OXIDANT PRODUCTION BY MATURE MYELOID CELLS. THERE'S NO CELLULAR GROWTH OR SURVIVAL ADVANTAGE AT EVEN THE STEM CELL LEVEL OR IN MATURE MYELOID CELLS FROM GENETICALLY CORRECTING THIS. SO FOR THIS REASON, BONE MARROW CONDITIONING IS ABSOLUTELY NECESSARY AND I'LL SHOW YOU SOME HISTORICAL OUTCOMES TO PROVE THIS POINT. SO JUST A LITTLE MORE DETAIL, CHRONIC GRANULOMA TUS DISEASE IS A DEFECT IN OXIDANT PRODUCTION, PATIENTS FAIL TO PRODUCE A PHAGOCYTE OX DAYSIDDASE, I'M JUST GOING TO FOCUS ON THE X-LINKED TODAY, BUT TO SORT OF ILLUSTRATE A POINT HARKING BACK TO ONE OF THE EARLIER SPEAKERS, EVEN IN THIS ONE DISEASE, YOU'VE FIVE DIFFERENT GENES THAT YOU HAVE TO FIX FOR EACH TYPE OF GENE THERAPY FOR THIS DISORDER. BUT I'M GOING TO JUST TALK ABOUT X-LINKED RIGHT NOW. SO PRIOR EXPERIENCE, IN THE MID 90s, WE PUBLISHED SOME WORK SHOWING THAT WE COULD ACHIEVE TRANSIENT APPEARANCE OF VERY LOW LEVELS, VERY LOW LEVELS OF OX DAYS NORMAL NEUTROPHILS WITH GAMMA RETROVIRAL GENE THERAPY WITHOUT ADVERSE EVENTS AND THIS IS WITHOUT CONDITIONING. THIS WAS INTERESTING SCIENTIFICALLY. WE DID NOT BENEFIT THE PATIENTS. IT DIDN'T LAST VERY LONG. LATER STUDIES, AND HERE WE'RE TALKING ABOUT 2005 TO 2011, ALSO WITH MIRRORING RETROVIRAL VECTOR BUT NOW BUSULFATE CONDITIONING, THEY REPORTED IN USE OF THE SFV MURINE GAMMA RETROVIRAL VECTOR THAT THEY GOT INCREASINGLY HIGH MARKING WITH OXIDASE POSITIVE NEUTROPHILS BUT THIS DIDN'T LAST. WE HAD A STUDY USING THE MFG VECTOR, WE ACHIEVED WITH CONDITIONING, NOW THINK BACK TO THE PREVIOUS STUDY, WE BASICALLY USED THE SAME VECTOR BUT NOW WITH A CONDITIONING REGIMEN. EARLY ON WE ACHIEVE -- POSITIVE NEUTROPHILS EARLY BUT THIS CAME DOWN OVER SEVERAL MONTHS BUT WE DID ACHIEVE SOME MARKING IN TWO OF THREE PATIENTS FOR A VERY LONG TERM MEANING OUT TO SIX, SEVEN YEARS AT LAST LOOK. THERE WAS NO GENOTOXTOXICITY. THIS WAS ENOUGH TO PROVIDE A LITTLE BIT OF CLINICAL BENEFIT BUT NOT ENOUGH CERTAINLY, NOT ENOUGH FOR WHAT'S REALLY NEEDED FOR THESE PATIENTS. A SIMILAR STUDY BY THE KOREAN GROUP USED A VECTOR VERY SIMILAR TO OURS WITH SIMILAR CONDITIONING AND VERY SIMILAR RESULTS. SO THE CONCLUSION FROM EARLY STUDIES IS THAT SUBMYELOABLATIVE CONDITIONING IS REQUIRED TO ACHIEVE OX DAYS POSITIVE NEUTROPHILS WITH GENE THERAPY. EVEN WITH CONDITIONING -- IN THE LONG TERM. TROUBLING, OF COURSE, IS THAT THE USE OF GAMMA RETROVIRAL VECTORS FOR GENE THERAP HAS SIGNIFICANT RISK OF GENOTOXICITY INCLUDING MYELODYSPLASIAS AND LEUKEMIAS. SO OUR CURRENT TRIAL USES SULFAN-ACTIVATED LENTIVECTOR. THE KEY ISSUE IS THAT THIS IS ONE LENTIVECTOR. I'M NOT GOING TO GET INTO THE FINE DETAILS OF THE VECTOR ITSELF. IT'S A SELF-ACTIVATEING WITH A SPECIFIC PROMOTER DEVELOPED BY ADRIAN AND MANUEL. THERE WERE TWO TRIALS BUT HIGHLY COORDINATED. ONE IN THE U.S. WITH DON COHEN ABC AS THE SPONSOR AND THREE CENTERS TREATING PATIENTS AND ANOTHER TRIAL USING A VERY SIMILAR PROTOCOL AND EXACT SAME VECTOR AT UNIVERSITY COLLEGE LONDON. THE TRIAL WAS COORDINATED BY GENETHON, AND IT'S IMPORTAT FOR ME TO NOTE, WHICH IS TOO LOW ON THIS SLIDE SO MANY OF YOU CAN'T SEE IT BUT THE PROGRAM HAS NOW BEEN ACQUIRED FOR COMMERCIAL DEVELOPMENT BY ORCHARD THERAPEUTICS. THE PRIMARY OBJECTIVES WERE SAFETY, DETERMINATION OF OXIDASE ACTIVITY OUT AT 12 MONTHS AND STABILITY THEREAFTER TO 24 MONTHS, AS I MENTIONED, THIS IS A SELF ACTIVATING VECTOR WITH AN INTERESTING HYBRID CHIMERIC MYELOID PROMOTER EXPRESSING GP91 FROM CODO N OPTIMIZED CDNA, AND THIS USED A BEU SUL FAP REGIMEN OF 1.6 MILLIGRAM PER KILOGRAM TWICE A DAY FOR THREE DAYS THIS IS ALMOST MYELOABLATIVE, IMPORTANT TO POINT THAT OUT. SO A BRIEF SUMMARY, FIVE PATIENTS HAVE BEEN TREATED IN THE U.K., FOUR IN THE U.S. TWO PATIENTS DIED EARLY AFTER GENE THERAPY FROM PRE-EXISTING PROBLEMS, LUNG INFLAMMATION IN ONE, ANTI-PLATELET ABT BODIES WITH AN UNFORTUNATE BLEED THAT OCCURRED ABOUT ONE MONTH AFTERWARD IN THE OTHER. BUT THE OTHER SURVIVING PATIENTS OUT AT THIS POINT IN TIME BETWEEN EIGHT MONTHS AND 2 1/2 YEARS ARE AT 16, 30, 27, FOUR, 46, 50 AND 33% OF CIRCULATING NEUTROPHILS ARE OXIDASE-NORMAL. SO IN THREE OF THE SEVEN, WE'VE REACHED OUR GOAL. ALL SURVIVING PATIENTS WHO ENTERED THE STUDY WITH SEVERE INFECTION, RYAN SPON SIEVE CONVENTIONAL THERAPY ACHIEVED RESOLUTION OF THESE INFECTIONS BY SIX MONTHS POST TREATMENT. A PRELIMINARY REPORT OF THIS WAS PRESENTED BY DON COHEN AT AMERICAN SOCIETY OF GENE AND CELL THERAPY THIS PAST MAY. IT'S IMPORTANT TO NOTE THAT THE PATIENTS WHO CAME INTO THIS TRIAL WERE MOSTLY QUITE ILL. AND MANY OF THEM HAD ONGOING INFLAMMATORY AND INFECTIOUS PROBLEMS THAT WEREN'T RESPONDING TO THERAPY. AND I'D LIKE TO SHOW A REPRESENTATIVE PATIENT, SUBJECT N104, A 24-YEAR-OLD MAN FOLLOWED AT NIH, CLINICAL HISTORY OF MANY, MANY INFECTIONS, PAST LUNG LOBE RESECTION, GRANULOMA TUS INFLAMMATION OF LIVER, INFLAM BO TRI BOWEL DISEASE AND WHAT BORE OWED HIM MOST WAS HE HAD THIS HORRIBLE INFECTION UNDER HIS ARMS. HE THEN DEVELOPED AN ONGOING FUNGAL INFECTION NOT RESPONSIVE TO ANTIBIOTIC THERAPY, IN OCTOBER 2016, ALL THE WAY UNTIL THE TIME WHEN WE TREATED HIM IN MID JUNE OF 2017. SO JUST FOCUSING -- THIS IS A FLOW CYTOMETRY ASSAY. WHEN YOU EXACT VAIT CELLS, IF YOU HAVE THE OXIDASE, THE NEUTROPHILS APPEAR HERE. HERE'S AFTER HIS GENE THERAPY AT TWO MONTHS, HERE'S HIS INFECTION, WHICH KEPT GETTING WORSE OVER TIME. HERE WAS A BIOPSY THAT STILL GREW THE FUNGUS IN MAY. IN JUNE, HE HAD HIS GENE THERAPY. BY AUGUST THIS WAS RESOLVING AND AT NINE MONTHS AFTER GENE THERAPY, HE STILL HAD ALMOST 49% OXIDASE-POSITIVE CELLS, AND NOW SIX MONTHS, THIS IS FULLY RESOLVED, AND AT 12 MONTHS, HE HAS 46% DHR, NO NEW INFECTIONS SINCE TREATMENT, NO PNEUMONIA, AND HIS HIDRADINITIS, THAT IS WHAT HE'S HAPPIEST ABOUT, THAT IS COMPLETELY RESOLVED. THE SO SUBMYELOABLATIVE CONDITIONING IS BOTH NECESSARY AND EFFICIENT TO ACHIEVE STABLE LONG TERM CLINICALLY BENEFICIAL EVENTS OF GENE -- NEUTROPHILS AND LENTIVECTOR. I ACTUALLY MENTIONED MOST OF THE PEOPLE INVOLVED. I JUST WANT TO MOVE ON TO ANOTHER EXAMPLE OF THE NEED FOR MYELOID CONDITIONING. IN THE CASE OF CBG, WE NEED CONDITIONING TO GET THE NEUTROPHIL AND MONOCYTE CORRECTION, AND THERE WAS NO SELECTIVE ADVANTAGE HELPING US IN THE CASE OF X-LINKED SEVERE COMBINED IMMUNE DEFICIENCY, IT TURNS OUT YOU DO NOT NEED CONDITIONING TO GET CORRECTION OF KEY LYMPHOCYTES, BUT YOU DO NOT GET CORRECTION OF THE OTHER CELL TYPES THAT YOU NEED TO FINISH THE FULL CORRECTION OF IMMUNITY. THIS IS A PROFOUND IMMUNE DISORDER, MUTATIONS IN THE COMMON GAMMA CHAIN BY ALL OF THESE TO INTERLEUKIN RECEPTORS, THEY HAVE ABSENT T AND NK CELLS, LOW NUMBERS OF NON-FUNCTIONAL B CELLS, LIFE-THREATENING, RECURRENT AND OPPORTUNISTIC INFECTIONS AND A TRANSPLANT OR GENE THERAPY IS REQUIRED IN INFANCY. HISTORICALLY NO CENTERS HAVE USED ANY MYELOID CONDITIONING FOR EITHER OF THESE HAPLOTRAN PLANTS IN INTIMACY OR FOR GENE THERAPY. BUT WE FIND THAT MYELOID CONDITIONING IS NOT REQUIRED FOR THE T-CELL IMMUNE RECONSTITUTION BUT AT LEAST IN THE CASE OF -- IN INFANCY WITHOUT CONDITIONING, THERE'S NO -- THEY DON'T MAKE ANTIBODY AND REQUIRE LIFELONG IGG. THEY HAVE POOR TO ABSENT NK CELL PRO KUX, THEY HAVE PROGRESSIVE DESCRIPTION OF DONOR T-CELL REP TORE AND INCREASE FUNCTION OF NUMBERS AND MEDICAL PROBLEMS WHICH WE'VE PUBLISHED ABOUT IN THIS PAPER THAT MOST OF YOU PROBABLY CAN'T SEE AT THE BOTTOM OF THE SLIDE. SO I RETURN TO THE STUDIES OF USING GAMMA RETROVIRAL VECTOR. NONE OF THESE STUDIES USED CONDITIONING. 26 OF 29 OF ALL THE INFANTS TREATED WITH GAMMA RETROVIRAL GENE THERAPY WITHOUT MARROW CONDITIONING DID BENEFIT LONG TERM FROM GENE TRANSFER THAT WAS ROBUST RESTORATION OF T-CELL NUMBER AND FUNCTION, AND I UNDERLINE THIS, THERE WAS NO ROBUST RESTORATION OF T-CELL NUMBER AND FUNCTION, NOR -- NK CELLS. AND OF OLDER POST HAPLO-TRANSPLANT PATIENTS DO NOT EVEN ACHIEVE T-CELL RECONSTITUTION SO TO GET THAT, YOU NEED THE REALLY ACTIVE MARROW OF INFAN SEAL,FANCY AND THIS DOESN'T REALLY HAPPEN. IN A PAPER FROM ADRIAN THRASHER AND HIS GROUP IN LONDON AND OUR GROUP AT NIH, TREATING FIVE OLDER POST HAPLO TRANSPLANT PATIENTS, ONLY ONE ACHIEVED EVEN LOW LEVELS OF SUSTAINED GENE MARKING AND THEN ONLY OF T-CELLS. SO WITH THIS IN MIND WITH THE ST. JUDE GROUP, WE DEVELOPED THE SELF ACTIVATING LENTIVIRUS FOR XSCID -- WE CAN'T TREAT INFANTS HERE, AND THE ST. JUDE GROUP TOGETHER WITH THEIR COLLEAGUES IN SAN FRANCISCO AND IN SEATTLE HAVE BEEN TREATING INFANTS. WE'VE TREATED EIGHT OLDER CHILDREN AND YOUNG ADULTS, WE'VE REPORTED THE FIRST FIVE PATIENTS, THEY'VE TREATED NOW 10 INFANTS, THEY'VE REPORTED THE FIRST SEVEN IN AN ABSTRACT AT ASH THIS PAST SEPTEMBER. AND I'LL SORT OF SUMMARIZE THIS BY SAYING THAT LENTIVECTOR GENE THERAPY FOR X SCID WITH VERY LOW TO MODERATE DOSE BUSULFAN HAS RESULTED IN SUBSTANTIAL GENE MARKING OF B CELLS AND NK CELLS IN ADDITION TO THE EXPECTED MARKING OF T-CELLS, THERE WAS RESTORATION BY GM PRODUCTION, IGG PRODUCTION AND SUSTAINED INCREASES IN NK CELLS. MANY RESPONDED WITH PROTECTIVE TITERS OF ANTIBODIES AND THESE PATIENTS NO LONGER REQUIRE SUPPLEMENTAL IGG. SO I'M GOING TO GIVE YOU, AS WITH THE CGD, A REPRESENTATIVE CASE. THIS IS OUR SUBJECT ONE, BECAUSE HE'S THE LONGEST FOLLOWED OUT. HE WAS 23 AT THE TIME OF THE GENE THERAPY. WE WERE TOLD BY MANY OF OUR COLLEAGUES ARE, DON'T EVEN TRY THIS, THEY DON'T HAVE ANY MORE THYMIC FUNCTION, HOW ARE THEY GOING TO GET GOOD T-CELLS, HOW ARE THEY GOING TO DO ALL THE THINGS THEY NEED DO? HE RECEIVED A HAPLOIDENTICAL BONE MARROW TRANSPLANT WITHOUT CONDITIONING AS A PATIENT, NEEDED LIFELONG SUPPLEMENTAL IGG BUT DEVELOPED PROTEIN LOSING ENTEROPA THEE SO HIS IG GWAS NEEDED EVERY PAST TWO WEEKS FIVE YEARS BEFORE HIS GENE THERAPY. I DON'T KNOW IF ANY OF YOU KNOW THE NUMBERS, BUT WE'RE TALKING ABOUT ALMOST $100,000 A YEAR IN IVIG AND THE INCONVENIENCE OF EVERY TWO WEEKS GETTING THERAPY. HE HAD CHRONIC NOROVIRUS, THAT'S NOT GOOD TO HAVE, NOT VERY COMFORTABLE, WANING T-CELL NUMBERS AND RECURRENT PNEUMONIAS AND HIS BMI WAS VERY, VERY LOW. HE'S A VERY SKINNY GUY. IN OCTOBER 2012, WE TREATED HIM. AND I'M JUST GOING TO GIVE YOU A DETAILED LOOK AT THE LONG VIEW. HERE HIS T-CELLS, I DON'T SHOW FUNCTION BUT HE'S ACQUIRED NORMAL FUNCTION OF HIS T-CELLS. HE HAD VERY FEW B CELLS. HE NOW HAS NORMAL NUMBERS OF B CELLS. HIS NK CELLS WERE ALMOST UNDETECTABLE. HE'S ALMOST GETTING TOWARD THE NORMAL LEVEL. HE HAD NO MEMORY OF B CELLS, HE NOW HAS MEMORY B CELLS, HE STARTED MAKING IGM, NOW IN THE MORE MALL LEVELS AND WAS ABLE TO STOP IGG AT ABOUT ONE YEAR AND NOW MAINTAINS HIS IGG LEVELS. HIS PROTEIN LOSING ENTEROPATHY IS FULLY RESOLVED, HIS BMI IS WELL INTO THE NORMAL RANGE, HE'S OFF IGG, HE HAS PROTECTIVE TIGHTERS INCLUDING A Z. STER LIVE IMMUNIZATION AND HE'S HAD NO OPPORTUNISTIC INFECTIONS IN FOUR YEARS. I DON'T USUALLY TRY TO USE THE CURE WORD BECAUSE HE'S NOT -- NOT EVERYTHING IS FULLY FIXED, BUT TO HIM, HE FEELS LIKE HE'S BEEN CURED. HE NO LONGER NEEDS IGG, HE HEELDZ FULL TIME JOB AND HE HASN'T BEEN SICK EXCEPT LIKE THE REST OF US OCCASIONALLY GETTING THE FLU OR SOMETHING ELSE OVER THE LAST COUPLE YEARS. THIS IS A LARGE LIST OF PEOPLE, I PARTICULARLY WANT TO MENTION SUK SEE DERAVIN AND ELIZABETH KING, BRIAN SORRENTINO -- THE MAIN PRINCIPLE OF THIS WAS TO SAY THAT SUBMYELOABLATIVE CONDITIONING IS BOTH NECESSARY AND SUFFICIENT TO ACHIEVE LONG TERM CLINICALLY BENEFICIAL LEVELS OF GENE MARKED NK CELLS AND B CELLS PLUS RESTORATION OF HUMORAL IMMUNITY WITH LENTIVECTOR. SO WE NOT ONLY NEED CONDITIONING TO GET ENOUGH CELLS IN, BUT IN SOME CASES LIKE THIS, YOU NEED IT TO GET THE RIGHT REPERTOIRE OF THINGS TO BE RECONSTITUTED. SO WITH GENE THERAPY USING EX VIVO MODIFIED STEM CELLS, IT'S REQUIRED TO ACHIEVE EFFICIENT INGRAFTMENT AND THE BROADEST RANGE OF LINEAGE RECONSTITUTION BUT CURRENT AGENTS HAVE SIGNIFICANT SHORT AND LONG TERM TOXICITIES. IF YOU WANT IT TO FIX -- DISEASES --, RADIATION SENSITIVE DISEASES, WITH ITS POTENTIAL LONG TERM TOXICITIES. BUT IT'S ALL WE'VE GOT RIGHT NOW. FOR ALLOGENEIC TRANSPLANT, IMMUNOTHERAPIES ARE BECOMING ALL THAT IS USED. IMMUNOTHERAPY TARGETING HEMATOPOIETIC STEM CELLS MAY BE A FEASIBLE OPTION AND I'M GOING TO QUICKLY GO THROUGH SOME THINGS TO SHOW YOU HOW THIS MAY BE THE CASE. SO THESE ARE A BUNCH OF PAPERS. I PUT THEM UP THERE, I REALIZE IT'S HARD UNLESS YOU'VE GOT A CAMERA TO TAKE A QUICK PICTURE OF ALL OF THEM BUT I'M GOING TO GO THROUGH SOME OF THE CRITICAL ISSUES. I'LL START WITH SOME EARLY STUDIES WHICH USED ANTIC-KIT IN MOUSE MODELS. THIS IS -- C-KIT IS CD117. IT'S A TRANSMEMBRANE TYROSINE KINASE, ESSENTIAL FOR STEM CELL FUNCTION, THOUGH IT IS FOUND IN COMMON PROGENITORS OF MANY BLOOD LINEAGES, PROSTATE STEM CELLS AND SOME OTHER PLACES THAT WE SHOULD BE THINKING ABOUT WHEN USING THESE. THERE'S A RAT MONOCLONAL ANTIMOUSE K-KIT CALLED ACK2 THAT WAS USED IN BOTH OF THESE STUDIES. THE CZECHOWICZ TRIAL ESSENTIALLY USED THIS IN A RAG-DEFICIENT, GAMMA C DEFICIENT MOUSE AND IT WORKED QUITE WELL THERE, BUT FOR REASONS THAT WERE UNCLEAR, DIDN'T ACHIEVE MYELO CONDITIONING IN WILD TYPE MICE, BUT AT LEAST IN THESE RAG MICE, ACHIEVED 60 TO 90% DONOR CHIMERISM. WITH A LITTLE BIT OF RADIATION BUT NEEDED RADIATION, 300CGY IN WILD TYPE MICE ACHIEVED EFFICIENT MYELOCONDITIONING WITH 60 TO 80% DONOR CHIMERISM. THE CHHABRA GROUP FROM STANFORD SHOWED THAT IF YOU COMBINED ANTI-K KIT WITH ANTICD47 BEING A DON'T EAT ME SIGNAL ENDED UP BEING ABLE TO GET HIGH LEVELS OF CONDITIONING AND 40 TO 80% DONOR CHIMERISM IN WILD TYPE MICE. THOUGH HERE, THEY'RE USEING SYNGENEIC MODELS, SO YOU'RE NOT CROSSING MOUSE TISSUE-TYPE BARRIERS. I'LL JUST SORT OF SHOW JUST ONE SLIDE FROM THE CHHABRA STUDY SHOWING THAT NOT ONLY HEMATOPOIETIC STEM CELLS BUT DONOR MYELOID CELL, DOOR KNOW B CELLS, DOOR KNOW NK CELLS AND T-CELLS ALL INGRAFTED IN THESE ANIMALS USING THIS COMBINATION OF AND CKIT AND ANTICD47. IN LAND MARKMARK STUDY, PAICHAUDHURI SHOWED IF THEY USED ANTICD45 WITH A TOXIN CONGREGATED TO SEPORIN, RIBOSOME ACTIVATING PROTEIN, THEY COULD ACHIEVE QUITE SIGNIFICANT INGRAFTMENT AND IN ADDITION TO GETTING INGRAFTMENT IN NORMAL MICE, THEY ALSO USED A SICKLE CELL ANEMIA MOUSE MODEL AND AGAIN, THIS IS THE INGRAFTMENT IN THE -- SHOWING GFP MOUSE INTO THIS -- IN THIS MODEL AND GETTING QUITE HIGH LEVELS OF INGRAFTMENT. AND IN THE SICKLE MODEL, ACHIEVING CORRECTION OF SICKLING AND ALSO WITH FAIRLY HIGH LEVELS OF INGRAFTMENT. AND IF YOU'RE INTERESTED IN THIS AREA, I'D REALLY RECOMMEND LOOKING AT THE BOTH OF THE PAPERS I JUST TALKED ABOUT LAST. SO THE PAPER OUT THERE THAT LED TO AN INVITATION FOR ME ACTUALLY DERIVED FROM MY HAVING SOME DISCUSSIONS WITH PEOPLE ABOUT CONDITIONING, AND I SAID, YOU KNOW, MAYBE WE OUGHT TO SEE WHETHER CAR T-CELLS COULD DO THIS AS WELL, AND YOU CAN'T BELIEVE THE NUMBER OF PEOPLE WHO SAID YOU CAN'T POSSIBLY DO THAT. SO WE SAID, WELL, MAYBE WE COULD. I CONNECTED UP WITH STEVE FELDMAN, WHO I SERVE ON THE BIOINSTITUTIONAL SAFETY BOARD WITH AND WE HAD A BUNCH OF DISCUSSIONS ABOUT THIS. I SAID TO LOOK -- I DON'T KNOW ANYTHING ABOUT CAR T-CELLS. COULD YOU WORK WITH US ON THIS. WHICH WE DID, AND GENERATED THIS CAR T-CELL CONSTRUCT AND USE -- I'M NOT GOING TO GO THROUGH ALL THE DETAILS BECAUSE I DON'T HAVE A LOT OF TIME, THE PATIENT WAS RECENTLY PUBLISHED IN MOLECULAR THERAPY. ONE OF THE KEY THINGS WE HAD TO DO WAS THAT WE DID NEED TO USE A LITTLE BIT OF CYTOXAN TO MAKE ROOM AS IS DONE IN CLINICAL TRIALS WITH CAR T-CELLS WHERE YOU NEED TO MAKE A LITTLE ROOM FOR THE T-CELLS TO GET IN, AND THEN WE USED A THY1, THY2 SO WE COULD GET RID OF THE CAR T-CELLS WHEN IT WAS TIME TO TRANSPLANT. THOSE T-CELLS DIDN'T WANT TO FIND THEIR WAY AT VERY LARGE NUMBERS IN THE MARROW, MARROW, SO WE PUT A CX4 MOLECULE WHICH IS A TRAFFICKING ELEMENT INTO THOSE T-CELLS AND WELL AND THEN IT ALL WORKED. I ACTUALLY THINK THE PAPER IS MOST INTERESTING FOR ITS USE OF A TARGETING AND TETHERING ELEMENT IN THE CAR T-CELLS AND I THINK THAT'S A DIRECTION THAT PEOPLE SHOULD BE LOOKING AT TO TRY TO GET SOME OF THESE CARTEL CELLS INTO SOLID TUMORS AND SO ON. WE WENT AHEAD AND WERE ABLE TO SHOW THAT WE FIXED -- THAT WE GOT THIS INTO THE NORMAL MICE BUT MORE IMPORTANT, WHEN WE USED THE CGD MODEL, WE ENDED UP WITH 35.9% OF THIS PARTICULAR MOUSE I'M SHOWING HERE, OXIDASE-POSITIVE CELLS, AFTER TRANSPLANT USING THIS WHOLE SETUP. SO AGAIN I'M GOING VERY FAST BECAUSE I WANT TO FINISH UP WITH MY SUMMARY, AND I HAVE TO ALSO SAY THAT AMG191 CONDITIONING, WHICH IS A HUMANIZED ANTICKIT, IS IN CLINICAL TRIAL PUT FORWARD BY JUDY SHIZURU AT STANFORD UNIVERSITY, AND THEY'VE BEEN USING THIS AS A SORT OF RESCUE BOOST OF PATIENTS WITH SCID WHO HAVE THE PROBLEMS THAT I TALKED ABOUT EARLIER, WITH XSCID OLDER CHILDREN AND ADULTS. SO IT PILOTS THIS HAPLO DONOR STEM CELLS, THEY ACTUALLY PURIFY CD34N9 TOGETHER WITH A SINGLE DOSE CONDITIONING. AS A RECENT PUBLIC PRESENTATION OF DATA BY DR. SHI SEU. URU, AT LEAST PATIENTS WITH THREE VARIOUS FORMS OF SCID HAVE BEEN TREATED AND SHE HAS SEEN SOME LEVEL OF MYELOID INGRAFTMENT. AND MY LAST SLIDE, SO WHAT'S THE FUTURE OF IMMUNOTHERAPY MYELOCONDITIONING? WELL, AND THIS IS THE TIP OF THE ICEBERG. I'M AWARE OF, I'M NOT INVOLVED IN THIS, BUT I'M AWARE OF A MANUSCRIPT NEAR PUBLICATION FROM A COLLABORATIVE GROUP SHOWING THAT AND MOUSE C IT IS KIT CONGREGATE WITH MYELOCONDITIONING TOGETHER WITH LYMPHODEPLETION CONDITIONING CAN ACHIEVE VERY HIGH LEVELS OF INGRAFTMENT ACROSS SIGNIFICANT MOUSE MHC BARRIER. THERE'S ALSO A MANUSCRIPT NEAR PUBLICATION FROM ANOTHER COLLABORATIVE GROUP THAT I'M NOT INVOLVED WITH, BUT I KNOW ABOUT, USING ANTICD45SAP CONDITIONING, WHAT'S MOST IMPORTANT IS THAT COMPARED TO CONVENTIONAL REGIMENS, IT REALLY PROTECTS THE THYMUS. SO THERE MAY BE ANOTHER BENEFIT OF USING IMMUNOTHERAPIES RATHER THAN USING THINGS LIKE BUSULFAN, AND THAT IS THAT YOU PROTECT THE THYMUS. FINALLY I'M AN NIHER SO I OWN NOTHING AND I CAN'T BE PAID BY ANYBODY. I'M NOT ON THEIR BOARD AND SO ON, BUT I'M REALLY EXCITED ABOUT MAGENTA THERAPEUTIC, WHICH IS A COMPANY SORT OF DERIVING FROM WORK BY ROSSI AND SC ADDEN, FOCUSED ON DEVELOPING IMMUNOTHERAPIES FOR TRANSPLANT CONDITIONING TA BUILD ON A NUMBER OF THE CONCEPTS THAT I DISCUSSED IN THIS PRESENTATION, AND I'LL END AT THIS POINT. THANK YOU VERY MUCH. [APPLAUSE] >> OUR LAST PANELIST HAS JOINED, DR. IWEN WU, WHO IS THE BRANCH CHIEF AND THE PHARMACOLOGY AND TOXICOLOGY BRANCH IN THE DIVISION OF CLINICAL EVALUATION AND PHARMTOX AT THE FDA AND SHE'S JOINING THE PANEL TO HELP THE DISCUSSION, WHICH I BELIEVE IS NOW IN SESSION. SO WE WANTED TO KEEP THIS SORT OF INFORMAL SO WE WANTED TO SOLICIT QUESTIONS FROM THE AUDIENCE TO BEGIN. SORRY, SHE'S GOING TO HAVE REMARKS THEN WE'RE GOING TO OPEN IT UP. >> THANK YOU. I WANT TO THANK THE OTHER SPEAKER, I THINK THEY GAVE REALLY EXCELLENT PRESENTATIONS AND THEY REALLY HIGHLIGHT HOW DIFFERENT THESE GENE THERAPY PRODUCTS CAN BE AND ALSO HOW DIFFERENT THE PRE-CLINICAL TESTING PROGRAMS OFTEN ARE TO ADDRESS SOME OF THE SPECIFIC SAFETY AND ACTIVITY ISSUES THAT ARE SPECIFIC TO EACH PRODUCT. I JUST HAD A COUPLE OF ADDITIONAL REMARKS I WANTED TO MAKE TODAY. THE FIRST ONE IS FOR THOSE WHO ARE DEVELOPING THESE PRODUCTS, WE DO RECOMMEND THAT YOU TRY TO GET AS MUCH INFORMATION FROM YOUR PRE-CLINICAL STUDIES AS POSSIBLE. SO EVEN SOME OF THESE VERY EARLY PROOF OF CONCEPT STUDIES AND DIFFERENT ANIMAL MODELS OF DISEASE, I THINK THAT THEY CAN GIVE YOU GREAT OPPORTUNITY TO START TO UNDERSTAND WHAT YOUR PRODUCT IS DOING IN VIVO, SO NOT ONLY UNDERSTANDING, YOU KNOW, THE DIFFERENT ACTIVITY END POINTS BUT ALSO STARTING TO LOOK AT WHERE YOUR PRODUCT IS DISTRIBUTING TO, HOW LONG YOU MAY BE GETTING ASSISTANCE FOR AND HOW LONG YOU MAY BE GETTING EXPRESSION OF YOUR TARGET GENE. I THICK ALL OF THIS CAN BE REALLY CRUCIAL ESPECIALLY WHEN IT COMES TO -- THIS IS ALSO THE DATA THAT YOU CAN USE TO REALLY JUSTIFY SPECIFIC STUDY DESIGN PARAMETERS FOR THOSE DEFINITIVE TOXICOLOGY STUDIES. THE NEXT REMARK THAT I WANTED TO MAKE IS THAT WE ARE RECOMMENDING THAT PEOPLE -- THEIR PRE-CLINICAL VECTOR LOTS. WE NOTED THERE CAN BE VARIABILITY IN SOME OF THE DOSE DETERMINING ASSAYS USED ESPECIALLY ACROSS DIFFERENT MANUFACTURING FACILITIES AND ACROSS DIFFERENT LABS SO AT THIS STAGE, WE DO RECOMMEND THAT YOU KEEP RETAINS OF THOSE PRE-CLINICAL LOTS SO DOWN THE ROAD IF YOU ARE MAKING CHANGES TO YOUR VECTOR MANUFACTURING FACILITY OR TO THOSE ASSAYS, YOU STILL HAVE SAMPLES REMAINING THAT YOU CAN RE-TEST AND RE-CALCULATE THE DOSE LEVELS THAT WERE EVALUATE IN YOUR PLEA CLINICAL STUDIES. THE LAST POINT I WANT TO MAKE IS THAT WE DO UNDERSTAND THAT THE PRE-CLINICAL TESTING FOR A LOT OF THESE PRODUCTS CAN BE EXTREMELY CHALLENGING, SO WE DO ENCOURAGE SPONSORS TO COME TALK TO US EARLY. WE HAVE THE INTERACT PROGRAM, ESSENTIALLY A MECHANIC BY WHICH YOU CAN GET FEEDBACK ON YOUR PRE-CLINICAL DEVELOPMENT PROGRAM OR FEEDBACK AT AN EARLY STAGE OF DEVELOPMENT. I THINK FOLKS ARE FAMILIAR WITH -- ESSENTIALLY REPLACING -- IT'S GETTING REPLACED BY THE INTERACTIVE PROGRAM BUT IT WILL FUNCTION IN VERY MUCH THE SAME WAY. SO HOPEFULLY IF YOU'RE HAVING ISSUES WITH A PARTICULAR ANIMAL MODEL, OR IF YOU HAVE A NOVEL METHOT OF PRE-CLINICAL ASSESSMENT THAT YOU WANT TO LOOK AT, THOSE WOULD ALL BE GREAT QUESTIONS TO ASK AT AN INTERACTIVE MEETING. WITH THAT, I'LL TURN IT BACK OVER TO OUR SESSION CHAIRS. >> THANKS, DR. WU, AND ADD MY THANKS TO ALL THE SPEAKERS. TERRIFIC TALKS. THIS STAGE -- WE'D LIKE TO OPEN THE FLOOR TO DISCUSSION AND QUESTIONS BEGINNING RIGHT HERE. PLEASE LET US KNOW WHO YOU ARE. >> YES, LYN MCGRATH. I HAD A BROAD QUESTION FOR EVERYONE. IF YOU WERE GOING TO START FROM SCRATCH NOW, KNOWING WHAT YOU KNOW NOW, HOW COULD YOU EXPEDITE THE PRE-CLINICAL DEVELOPMENT RATHER THAN, YOU KNOW, DEVELOPING NEW ANIMAL MODELS AND STARTING FROM THE VERY BEGINNING LIKE YOU DID SEVERAL YEARS AGO, IS THERE SOME INFORMATION THAT YOU'VE LEARNED THAT CAN HELP MOVE FORWARD PROGRAMS FASTER SO THAT YOU WOULDN'T HAVE TO DO ALL THE WORK? I KNOW FDA LETS YOU BUILD ON SOME OF THE RESEARCH YOU'VE DONE, BUT WHAT SPECIFICALLY CAN YOU DO THAT WOULD MOVE THINGS ALONG FASTER BASED ON THE REACH RESEARCH YOU'VE DONE? >> I THINK WE COULD ASK JAY TO ANSWER FIRST IN TERMS OF HIS PRE-CLINICAL EXPERIENCE. >> SO IN LOOKING BACK OVER THE DEVELOPMENT PROCESS, WE GOT A LOT OF INSIGHT FROM LOCAL FDA PEOPLE, AND I WOULD ENCOURAGE PEOPLE TO CONTACT THEM JUST AS DR. WU POINTED OUT, THEY'RE VERY HELPFUL IN PROPOSING THE SCENARIO THAT WE COULD DISCUSS WITH THEM. THANK YOU. >> JAY, I WAS PARTICULARLY INTERESTED IN YOUR STORY THAT THE PIG'S SALIVARY GLAND IS LITERALLY DOUBLE THE SIZE OF THE HUMAN SALIVARY GLAND? THAT'S A NICE ADVANTAGE. >> YEAH. IT'S ALSO A DISADVANTAGE BECAUSE IT'S MORE TISSUE THAT WE HAD TO WORK WITH, SO PLUSES AND MINUSES. IT WAS A CHALLENGING MODEL, BUT FROM A PHYSIOLOGIC STANDPOINT, IT WAS THE BEST ONE THAT WE HAD AT THE TIME. PIGS VERY NICELY REPLICATE THE HUMAN RESPONSE TO RADIATION. >> I THINK THEY WERE OVER THERE BEFORE ME, BUT -- >> THANK YOU. I HAVE A COUPLE QUESTIONS ABOUT THIS. FIRST, DR. -- I -- WITH REGARD TO SAFETY -- THE TARGET YOU WERE LOOKING AT. >> SO WE'VE ANALYZED THE OFF TARGET IN VIVO, WE LOOKED AT THE MOST -- LIKE THE PREDICTABLE, WE WENT LOOKING WHERE IT CAN BIND, WE HAVEN'T FOUND ANY TARGETS SO FAR WITH THE GUIDE RNA, SO IT RELIES ON THE SPES SPECIFICITY OF THE GUIDE, WE HAVE TO KEEP IN MIND THOSE STUDIES ARE DONE BASICALLY WITH THE -- THEY'RE ALWAYS THE SAME GENES OR OTHER GENES THAT ARE KNOWN TO BE REPETITIVE AND NOT AS SPECIFIC SO THEY KIND OF LOOK LIKE THE WORST CASES, HOW THEY CAN IMPROVE THAT. THAT DOESN'T MEAN THAT FOR EVERY SINGLE LOCUS, WE'RE GOING TO SEE A SIMILAR PERCENTAGE SO IT'S REALLY TARGET-SPECIFIC AND GUIDE RNA SPECIFIC. SO FAR WE HAVEN'T IDENTIFIED BUT WE'RE LOOKING LONGER TERM, IT'S SOMETHING WE'RE ALWAYS ASSESSING ALONG THE WAY, PARTICULARLY IN VIVO, WHERE WE'RE LOOKING REALLY AT THE CONDITION THAT WE ACTUALLY ARE GOING TO HAVE BECAUSE IN CELLS, WE HAVE THIS LARGE AMOUNT OF -- CAS NINE GUIDE RNA THAT WILL NEVER ACHIEVE IN VIVO, SO WE'RE REDEVELOPING CURRENT METHODS USING NEXT GENERATION SEQUENCING WE'RE CURRENTLY USING. LOOKING AT THOSE TARGETS, AS WELL TRYING TO -- IN A MORE UNBIASED WAY, BUT THAT'S AN EXCELLENT POINT AS WE'RE REALLY KEEPING THAT AS ONE OF OUR MAJOR FOCUS FOR THE SAFETY. >> THANKS FOR THAT DETAILED RESPONSE. I FORGOT TO MENTION, I'M SCOTT KIVER FROM THE UNIVERSITY OF MINNESOTA. DR. MALECH, I WAS A LITTLE SURPRISED TO SEE THAT THE T-CELL MARKING WASN'T HIGHER IN THAT ONE PATIENT THAT YOU SHOWED? BECAUSE I DON'T KNOW HOW YOU GET T-CELLS DIFFERENTIATED WITHOUT THE GENETIC CORRECTION. WHAT DO YOU THINK ABOUT THAT? >> IT'S IMPORTANT TO REMEMBER THAT IN THE OLDER PATIENT/YOUNG ADULTS, THEY HAVE T-CELLS FROM THEIR PARENTAL DONOR. THEY JUST DON'T WORK TERRIBLY WELL. AND SO BY THE TIME THEY COME TO US, ALL OF THEIR T-CELL FUNCTIONS ARE PRETTY CRAPPY. THEY HAVE A REDUCED REPERTOIRE INSPECTOR TYPING SHOWS ABSENCES OF CERTAIN DOCTORS OF THE V BETA SUBTYPES, BUT AFTER THIS, EVEN THOUGH ONLY THAT PERCENT OF CELLS IS NOW REPRESENTED BY THE NEW CELLS COMING OUT FROM AUTOLOGOUS GENE-CORRECTED, THEY'RE CARRYING THE ENTIRE WEIGHT, THE SPECTROTYPING LOOKS GREAT, THE T-CELL FUNCTION IS NORMALIZED AND SO ON, SO THAT'S ALL CARRIED BY THAT 28% OR SO. >> THANKS. I FORGOT THAT THE PATIENT HAD ALREADY BEEN TRANSPLANTED. >> IT'S DIFFERENT IN THE INFANTS. I DIDN'T PRESENT ANYTHING ABOUT THE INFANTS. BUT IT'S ONLY THE CORRECTED T-CELLS THAT COME OUT. >> HI. JENNIFER LEVY FROM COALITION TO COMPARE, WE HEARD A LOT THIS MORNING ABOUT LARGE ANIMAL MODELS LIKE THE MINI PIG AND THE DOG. I WAS WONDERING IF YOU COULD SPEAK TO HOW ESSENTIAL THAT IS AND IF YOU'RE INTERESTED IN A DECEMBER THAT ONLY HAS A RODENT MODEL, WILL THAT EVER BE SUFFICIENT FOR STUDIES. >> MAYBE WE CAN ASK YOU IN TO REMARK ON THIS. >> SURE. SO FROM AN FDA PERSPECTIVE, IT'S NOT ABSOLUTELY NECESSARY THAT YOU ALWAYS HAVE A LARGE ANIMAL MODEL. I THINK IN SOME CASES, DEPENDING ON THE FEASIBILITY AND THE RELEVANT ANIMAL MODELS AVAILABLE, LARGE ANIMAL MODEL MAY BE THE MOST APPROPRIATE, BUT AGAIN, I THINK WE ALWAYS ASK SPONSORS TO START THINKING ABOUT WHAT THE MOST RELEVANT ANIMAL MODEL IS, AND IN SOME CASES IT MAY ONLY BE THAT THERE IS A ROLE MODEL THAT'S THE MOST TO EVALUATE IMROWRM DUCT. SO PRODUCT, WHAT IS GOING TO BE CONDUCIVE TO EVALUATING THE SAFETY OF YOUR PRODUCT AND WHAT MODEL IS GOING TO ALLOW YOUR PRODUCT TO BE ACTIVE. THANK YOU. >> NEXT QUESTION. >> HI. MY NAME IS CAROL VAN RYE 16, I'M A NURSE PRACTITIONER AT THE CLINICAL 16 TE I'VE BEEN HERE FOR ABOUT 20 YEARS. SO MY QUESTION ACTUALLY HAS TO DO WITH CLINICAL AND I FIND IT QUITE INTERESTING THAT TWO OF THE DISEASES THAT WE'RE TALKING ABOUT ARE X-LINKED, AND I'M WONDERING ABOUT GENE THERAPY IN FEMALE VERSUS MALE DISEASES, AND IF YOU HAVE ANY COMMENT ON THAT, WHETHER THAT'S JUST SORT OF A COINCIDENCE THAT WE HAVE THAT HAPPENING, AND THE PANEL OR IF IT'S BECAUSE IT'S A LITTLE BIT EASIER TO STUDY IN MALES WHO ARE NON-REPRODUCTIVE -- OR THEY'RE REPRODUCTIVE MAYBE BUT THEY'RE NOT GOING TO GIVE BIRTH. ANY COMMENT ON THAT? >> SO I COULD HAVE JUST AS EASILY TALKED ABOUT ADENOSINE DEAM NAIZ SCID, AUTOSOMAL RECESSIVE DISEASE. THERE'S A TREMENDOUS AMOUNT OF WORK BEING DONE IN THAT AREA. THERE'S ALREADY IN THE FORM OF STRAMVELLIS USING GAMMA RETROVIRAL VECTOR AND I'M NOT REVEALING ANYTHING TO SAY THAT GROUPS USING LENTIVECTOR ARE IN THE LATE PHASES OF COMMERCIAL DEVELOPMENT OF A LENTIVECTOR FOR ADA SCID, SO IT JUST HAPPENED TO BE MY CHOICE, IT WAS THE TWO DISEASES THAT I HAD A LOT OF DATA ABOUT AND PRESENTED, BUT IN NO WAY DID I MEAN TO SPEAK ABOUT X LINK DISEASES WHICH FOR THE MOST PART AFFECT ONLY MALES. >> I WAS JUST CURIOUS IF YOU COULD COMMENT ON WHETHER THERE WOULD BE DIFFERENT IMPACTS ON REPRODUCTIVE AGED FEMALES VERSUS MALES. THAT'S KIND OF WHERE MY QUESTION IS GOING. >> SO YOU ACTUALLY TOUCH ON THE ISSUE OF BUSULFAN. WHAT I CAN TELL YOU IS THAT BUSULFAN IN THE DOSES USED IN THE INFANTS WHICH IS ABOUT 2 MILLIGRAM PER KILOGRAM ALTHOUGH IT'S HIGHLY TARGETED AND EVEN IN THE OLDER CHILDREN/YOUNG ADULTS WHERE WE USE ALSO TARGETED BUT NOMINALLY AT 6 MILLIGRAM PER KILOGRAM, THOSE DOSES MAY HAVE ISSUES IN TOXICITIES BUT ACTUALLY FERTILITY IS FOR THE MOST PART LIKELY TO BE PRESERVED IN THOSE INFANTS AND ALSO SOMEWHAT FOR THE MOST PART IN THE OLDER CHILDREN/YOUNG ADULTS AT THOSE DOSES. THE DOSES WE USED FOR CGD ARE HIGH DOSES AND THOSE LIKELY ADVERSELY AFFECT FERTILITY. BUT THAT'S ONE OF THE OTHER VERY STRONG REASONS WHY WE NEED TO GET BETTER AGENTS FOR CONDITIONING. IT'S A SORT OF MUCH TALKED ABOUT BUT NOT MUCH SPOKEN ABOUT ISSUE FOR -- IN THE GENE THERAPY SETTING AND IS A CRITICAL ISSUE TO SOLVE. >> GREAT. THANK YOU FOR YOUR COMMENT. >> DR. WU, MAY I ASK YOU TO GIVE ET FDA PERSPECTIVE ON PRE-CLINICAL WORK FOR X-LINKED DISORDERS, FOR INSTANCE, DOES THE AGENCY PREFER THAT MALE ANIMALS BE STUDIED FOR PRE-CLINICAL TOX OR WHAT OTHER GENDER ISSUES DID YOU ALL CONSIDER? >> IDEALLY IT SHOULD REFLECT THE PATIENT POPULATION THAT YOU'RE GOING INTO, SO IF IT IS A DISORDER THAT WOULD BE ANTICIPATED TO AFFECT PRIMARILY MALES, TYPICALLY MALE ANIMALS ARE USED IN THOSE STUDIES. >> ALEX BAILEY. THIS IS A QUESTION PROBABLY FOR DR. WU. FOR THOSE THERAPEUTIC APPROACHES THAT MAY INCORPORATE MULTIPLE GENE THERAPY PRODUCTS OR SAY THE SAME PRODUCT BEING ADMINISTERED VIA MULTIPLE DIFFERENT ROUTES OF ADMINISTRATION, SAY TO TARGET DIFFERENT TISSUE COMPARTMENTS. I WAS WONDERING IF YOU COULD COMMENT ON HOW THE COMMUNITY SHOULD BE THINKING ABOUT PRE-CLINICAL TESTING STRATEGIES FOR THESE TYPES OF APPROACHES, PARTICULARLY SAFETY TESTING. >> SO I THINK FROM OUR PERSPECTIVE, WE'RE OBVIOUSLY GOING TO BE THE MOST CONCERNED ABOUT LOOKING AT THE SAFETY OF THE COMBINED ROUTES ADMINISTRATION OR THE COMBINED THERAPEUTIC INVESTIGATIONAL AGENTS THAT YOU'RE LOOKING TO ADMINISTER. I THINK THERE'S DEFINITELY GOING TO BE VALUE IN POTENTIALLY CHARACTERIZING THE SAFETY AND ACTIVITY OF EACH OF THOSE INDIVIDUALLY. BUT I THINK THAT WOULD BE UP TO THE INDIVIDUAL SPONSOR. WE'LL ULTIMATELY BE LOOKING AT THE SAFETY OF COMBINATION THAT YOU'RE PROPOSING TO USE. >> CATHY -- ONE QUESTION FOR DR. MALECH AN D ONE FOR DR. CHIORINI. DR. MALECH, CAN YOU ASSESS THE BENEFIT/RISK IN THE X-LINKED CGD TRIAL YOU TALKED ABOUT? YOU SAID THAT OUT OF NON-SUBJECTS, TWO INDIVIDUALS DIED. YOU AND MENTIONED, I THINK, THAT THE PATIENTS WHO PRESENT FOR INCLUSION IN THE TRIAL WERE MORE ADVANCED OR THEY WERE HAVING TROUBLE ON CONVENTIONAL MANAGEMENT OR SOMETHING LIKE THAT. BUT THAT SOUNDS LIKE A SOBERING NUMBER. AND MY OTHER QUESTION IS, DO YOU BELIEVE THAT THE CONDITIONING REGIMENTS THAT YOU TALKED ABOUT USING ANTICKIT AND THE OTHER ANTICD47, WOULD THOSE CONDITIONING REGIMENS HAVE MADE A DIFFERENCE IN THE OUTCOME FOR THOSE INDIVIDUALS? >> IT'S IMPORTANT TO NOTE YES TO THE FIRST COMMENT, FOR THE MOST PART THESE ARE PATIENTS WHO PRESENTED FOR A VERY NOVEL THERAPY, AND FOR THE MOST PART, IN TERMS OF RECRUITMENT, THEY WERE PATIENTS WHO IN FACT IN MANY CASES WANTED TRANSPLANT, BUT THERE WAS NO MATCH. IN FACT, THE PROTOCOL REQUIRES THAT YOU NOT HAVE EVEN A MATCHED UNRELATED DONOR OUT THERE. THE TWO PATIENTS WHO DIED IN THE STUDY, ONE WAS IN THE U.K., I'M MOST FAMILIAR WITH THE ONE WHO WAS HERE AT NIH WHO WAS A YOUNG CHILD WHO WAS REFERRED TO US AFTER ALMOST A YEAR OF A FUNGAL INFECTION THAT INVOLVED LUNG, BONES AND BRAIN. AND HE ACTUALLY HAD A BRAIN BLEED AT A SITE OF UP WITH OF ONE OF THESE INFECTIONS. SO WE COULD HAVE PROBABLY DONE BETTER BY JUST NOT CHOOSING SUCH PATIENTS, BUT HERE AT NIH, WE -- AT LEAST I TEND TO DEAL WITH LOST CAUSES, AND THIS WAS A LOST CAUSE WITHOUT THIS. IT WAS UNFORTUNATE, I CAN GO INTO ALL THE DETAILS, HE HAD HAD GRANULAR SITE TRANSFUSIONS WHICH HAD -- SO HE ALREADY CAME TO THE TABLE WITH ANTI-PLATELET ANTIBODIES THAT WE DIDN'T REALIZE THAT GOT WORSE WHEN HE GOT POST TRANSPLANT PLATELETS AND SO ON, ONE THING LED TO ANOTHER. IT WAS VERY DIFFICULT FOR ALL OF US, BUT IF YOU SAY WHAT I WOULD I HAVE ACCEPTED HIM AGAIN? YEAH, HERE AT NIH, WE HAVE DO THAT, THAT'S WHAT WE DO. BUT IF I WERE RUNNING A COMPANY ON THIS, I'D PROBABLY SAY LET'S NOT THE DO SUCH SICK PATIENTS, LET'S DO THEM WHILE THEY'RE DOING BETTER, AND WE WOULD AVOID THESE DEATHS. BUT WE RUN INTO THE SAME TROUBLE WITH OUR TRANSPLANT PROGRAM. WE HERE GET REFERRALS FROM ALL OVER THE COUNTRY OF PEOPLE WHOSE ROOF IS LEAKING, WHEREAS EVERYBODY ELSE TRANSPLANTS THEM WHEN THE ROOF -- WHEN IT'S NOT RAINING OUT. AND SO OUR OUTCOMES ARE A LITTLE LESS GOOD, EVEN IN OUR TRANSPLANT PROGRAM THAN THEY ARE IN OTHER PEOPLE'S TRANSPLANT PROGRAM WHO WOULD NEVER PLANS PLANT SOME OF TRANSPLANT SOME OF THE PATIENTS WE AGREE TO TRANSPLANT HERE. SO THAT'S MY WAY OF EXPLAINING THAT. YOU HAD ANOTHER QUESTION. >> WHICH WAS DO YOU THINK THAT THE MILDER CONDITIONING REGIMENS WOULD HAVE MADE A DIFFERENCE FOR EITHER OF THOSE PEOPLE? >> THE ANSWER IS ONLY MAYBE. THE IMMUNOAGENTS ARE LESS INFLAMMATORY. WE KNOW BUSULFAN ISINFLAMMATORY TO THE MARROW AND TO OTHER PLACES AS WELL, AND FOR LUNGS, TALKING ABOUT THE OTHER PATIENT IN THE U.K. WHO DIED OF HIS PRE-EXISTING LUNG INFLAMMATION THAT GOT WORSE THAN THAT POST CONDITIONING PERIOD. BUT IT'S ONLY I THINK THE IMMUNOTHERAPIES MAY BE BETTER, BUT YOU'RE STILL CONDITIONING.& YOU HAVE A NEUTROPENIC THROMBOCYTOPENIC PERIOD AND YOU HAVE TO DEAL WITH THAT. MAYBE THERE'S SOME MAGIC, MAYBE WE CAN DO IN VIVO GENE THERAPY FOR HEMATOPOIETIC DISORDERS, ALTHOUGH THAT PRESENTS ITS OWN PROBLEMS WHICH I WON'T GO INTO HERE. >> SO MY OTHER QUESTION ABOUT THE XEROSTOMIA TRIAL IS THAT WHEN YOU MOVE TO PHASE 3, YOU MENTIONED TWO END POINTS, ONE WAS PA RO TID GLAND FLOW AND THE OTHER WAS A SUBJECTIVE QUESTIONNAIRE. ARE YOU GOING TO USE ANY END POINTS THAT WOULD BE A MORE OBJECTIVE CLINICAL MEASURE, INFECTIONS. HAVE YOU THOUGHT ABOUT HOW YOU WOULD ADDRESS THAT? >> THAT'S GREAT QUESTION. I DIDN'T GO THROUGH ALL OF THE OTHER RESEARCH PARTS OF THE PHASE 1 THAT WE'RE DOING, LOOKING AT CHANGES IN MICROBIOME, CHANGES IN CARRY FORMATION, AS YOU POINT OUT THERE'S A LOT GOING ON IN THE ORAL CAVITY AND WE COULD USE THOSE AS END POINT MEASURES FOR IMPROVEMENT OF ORAL HEALTH OVERALL. FOR OTHER DRUGS THAT HAVE LOOKED AT CHANGING OR TREATING OTHER FORMS OF XEROSTOMIA FOR EXAMPLE IN AUTOIMMUNE FORM, THE OUT COME MEASURE USED IN PHASE 3 WAS AN OBJECTIVE RESPONSE, HOW DID YOU FEEL, DID YOU HAVE LESS ORAL PAIN, TO YOU HAVE MORE ORAL WETNESS. >> PARKINSON'S INSTITUTE AND CLINICAL CENTER. I HAVE A QUESTION, CAN THE PANEL PLEASE COMMENT ON THE USE OF PATIENT DERIVED IPS MODELING, ESPECIALLY FOR GENE THERAPY COMBINED WITH GENE EDITING? WE HAVE VERY SPECIFIC QUEENS SEQUENCES. >> GENERALLY IT'S NOT THE BEST MODEL TO LOOK AT LIKE OFF TARGET BECAUSE IPS IS NOT THE MOST STABLE LINE GENERALLY. SO THAT'S MOSTLY JUST TO VALIDATE THE GUIDE, WHICH WAS THE BEST GUIDE SCREEN, WE'RE USING MORE FOR THIS APPROACH AS STILL THE IN VIVO, WE FOUND IT MORE -- THE ENVIRONMENT AND AS WELL THE THERAPY THAT WE'RE GOING TO -- IT'S NOT WE'RE GOING TO USE IPS TO GIVE IT TO THE PATIENT SO THAT'S MOSTLY THE ENVIRONMENT THAT WE'RE LOOKING MORE CLOSELY INTO OFF TARGETS, ON TARGS, ANALYSIS. TARGETS, THE ANALYSIS, HOW WELL IT WORKS FOR THE IPS. >> I MIGHT ALSO JUST IN DEFENDING SOME OF THE IPS USE, WE'VE EXTENSIVELY USED IPS CELLS FOR SOME OF OUR EARLY MODELING OF EDITING. AND THEY CAN BE VERY IMPORTANT FOR LEARNING SOME KEY GENETIC AND PHYSIOLOGIC THINGS THAT OTHER MODELS ARE NOT, SO FOR EXAMPLE, WE'VE BEEN LOOKING AT GENE EDITING AS WELL FOR CHRONIC GRANULOMATOUS DISEASE. TO OUR SURPRISE, WE FIND THAT YOU SAY OKAY, IF YOU'RE GOING TO PUT A CDNA, WHY NOT PUT IT AT EXON 1, YOU GET THE WHOLE GENE. IT TURNS OUT YOU NEED THE FIRST INTRON TO MAKE THAT ALL WORK. YOU CAN'T PUT A CDNA THERE, YOU HAVE TO PUT IT AT EXON 2 OR FIGURE OUT WHAT'S GOING ON IN THAT INTRON. WE FOUND THAT WITH A NUMBER OF OTHER GENES AS WELL. SO THE IPS -- YOU YOU'RE REALLY ASKING TWO QUESTIONS, IS IT A GOOD MODEL IN THE EARLY DEVELOP. THINGS? ABSOLUTELY. IS IT GOING TO BE THE WAY TO GO TO TREAT? MAYBE FOR SOME KINDS OF DISEASES BUT FOR OTHERS, IT'S JUST A GREAT EARLY GENETIC MODEL FOR THESE THINGS. >> OVER HERE. >> THANK YOU. RALPH FROM -- BIOPHARMA. THE PRESENTATIONS WE SAW TODAY WERE REALLY ENGAGING BUT I NOTICED THAT THEY ALL INVOLVED VIRAL VECTORS. I WAS JUST WONDERING FOR THE PANEL IF THERE COULD BE SOME COMMENT ON THE STATUS OF THE POTENTIAL OF USING NON-VIRAL VECTORS, MOVING INTO THE CLINIC IN PATIENTS. >> FOR US, WE HAVE QUITE A CHALLENGING IT TISSUE, SKELETAL MUSCLE AND HEART, AND FOR THE NON-VIRAL DELIVERY METHODS SO FAR HAVE BEEN -- THERE IS A A LOT OF TECHNOLOGY THAT HAS BEEN DEVELOPED HOWEVER FOR SYSTEMIC DELIVERY, IT'S NOT THE BEST. LOCALLY IT WORKS, THE AAV SEEMS TO BE THE BEST CANDIDATE SO FAR CURRENTLY TO MOVE OUR TECHNOLOGY TO THE CLINIC AND TO MOVE TO THE 40% OF THE BODY MASS BECAUSE IT'S A LARGE TISSUE AND A LOT OF CELLS TO ACCESS AND TO DELIVER. SO FROM THE MUSCLE -- AAV IS STILL THE BEST CANDIDATE FOR US FROM THAT PERSPECTIVE. >> THANKS FOR THAT QUESTION. YOU KNOW, AS THE ORGANIZERS OF THE SESSION, WE DID SEEK THE PROSPECT OF SPEAKERS ON NON-VIRAL DELIVERY SYSTEMS, BUT THERE'S A LIMIT TO THE NUMBER OF SPEAKERS AND THE TIME OF COURSE, BUT I WOULD LIKE TO INVITE YOU TO TELL US A BIT ABOUT WHAT YOUR COMPANY IS ENGAGED IN. I ASSUME IT'S IN A NON-VIRAL ARENA. HONESTLY WE'D LIKE TO HEAR A BIT ABOUT IT. >> FIRST SLIDE, PLEASE? [LAUGHTER] THANK YOU. THANK YOU VERY MUCH FOR ASKING. I'D LOVE TO TELL YOU. WE ARE WORKING ON A LIPID NANOCRYSTAL WHICH IS VERY DIFFERENT FROM THE LIPID NANOPARTICLES THAT ARE CURRENTLY BEING USED. THE MECHANISM OF ENTRY ACROSS THE CELL MEMBRANE, WITH THE CURRENT PARTICLES, INVOLVES SOME SORT OF ARTIFICIAL CHEMIST SOLUTION TO TRY TO BASICALLY MAKE A HOLE IN THE MEMBRANE AND DELIVER THE MATERIAL INTO THE CYTOPLASM, THE LARGE NUCLEIC ACID POLYMER OF SOME SORT. THE OUTCOME OF THAT IS OFTENTIMES DESTRUCTION OF THE CELL WHICH INDUCES TOXICITY AND INFLAMMATION. THE UNIQUENESS OF OUR PARTICLE INVOLVES THE USE OF CALCIUM -- WHICH IS A NATURAL MEMBRANE FEWTION MECHANISM USED IN SIN APPS, WHAT WE'VE SEEN OVER THE YEARS IS NO INFLAMMATION, NO DESTRUCTION OF CELLS, YOU CAN GIVE IT TO 100% OF THE CELLS AND IN THE TISSUE CULTURE DISH U GET NO DESTRUCTION, AND HERE AT THE CLINICAL CENTER, THIS CAN DELIVER NUCLEIC POLYMERS AS WELL AS SMALL MOLECULE DRUGS. SO WE HAVE AM FI TEAR SEEN FOR EXAMPLE IN PHASE 2 TRIALS HERE IN THE CLIN CLINICAL CENTER, WE HAVE AMACASIN WE'VE BEEN ABLE TO USE TO GLIFER -- TO TREAT MICRO BACTERIUM IN CYSTIC FIBROSIS MODELS OR INFECTIONS WHERE YOU HAVE TO DELIVER THE DRUG INTO THE INTERIOR OF THE CELL. WE'VE DELIVERED DNA PLASMIDS IN ANIMALS, RNAI IN THE FLUID MODEL OF LUNG SO IT IS A VERY, VERY DIFFERENT TYPE OF PARTICLE IN THAT WE CAN SCALE IT UP, WE'RE MAKING 100-LITER BATCHES, WE'VE USED IT WITH BOTH SMALL MOLECULES AND NUCLEIC ACIDS AND IT HAS NEVER SHOWN THE FUNDAMENTAL MATRIX HAS SHOWN NO TOXICITY, NO INFLAMMATORY RESPONSES IN ANIMALS. SO THANK YOU VERY MUCH. >> NO, THAT'S GREAT. I MADE FOR IT TO BE AN ADVERTISEMENT, IT WASN'T, IT'S INFORMATIVE, AND ARE YOU IN A DEVELOPMENT PROGRAM WITH THE FDA FOR ANY OF THE PRODUCTS? >> WELL, WE HAVE THE AMPHITERASINE PRODUCT IN PHASE 2 AND THE O THER IN PHASE 1 AND WE'RE MOVING THOSE FORWARD, BUT WE ALSO HAVE DONE A NUMBER OF PRE-CLINICAL STUDIES WITH NUCLEIC ACID POLYMERS, BOTH RNA I AND LARGE 11 KILL OBEYS PLASMIDS. WE'VE ALSO DONE DNA PROTEIN COLLECTIONS EX VIVO IN STEM CELLS AND SHOWN VERY GOOD TRANSVEX. >> IN TERMS OF THE ANIMAL MODEL WORK, ANYTHING YOU'D LIKE TO SHARE IN TERMS OF YOUR PRE-CLINICAL STUDIES WITH RODENT OR LARGE ANIMAL MODELS WITH THIS DELIVERY METHOD? >> WE'VE NOT GN ON TO LARGE ANIMAL MODELS. THE CHOICE WE HAD TO MAKE AS A SMALL COMPANY IS TO EITHER PURSUE THE ANTIMICROBIALS VERSUS THE NUCLEIC ACID POLYMERS, WE FOCUSED ON THE ANTIMICROBIALS BUT NOW THAT WE HAVE ONE IN PHASE 2 AND 1 IN PHASE 1 AND WE CAN SEE THE UNIQUE DIFFERENCE BETWEEN OUR PARTICLE AND OUR NUCLEIC -- IT'S AN ANHYDROUS CRYSTAL, WHICH IS AN INTERMEDIATE MEMBRANE FUSION. SO THE SLIDE YOU SEE WHERE IT ATTACHES TO A CELL MEMBRANE, INFUSES AND DELIVERS RIGHT INTO THE CYTOPLASM IS VERY IMPRESSIVE. AGAIN, WE'VE SEEN NO TOXICITY OR INFLAMMATION. SO WE'RE SLITTING COLLABORATORS. >> OKAY. >> THANK YOU. >> STEVEN GRAY, UT SOUTHWESTERN. JUST A QUESTION COMING BACK TO DIFFERENT DISEASE MODELS. I'M ACTUALLY IN SOME SORT OF REAL LIFE SITUATIONS WHERE WE HAVE GENE THERAPY APPROACHES THAT WE FEEL MAYBE THERE'S A DISEASE THAT IT COULD APPLY TO, BUT THERE BASICALLY IS NO ANIMAL MODEL. AND IN ONE CASE, WE'VE BEEN TRYING FOR TWO YEARS TO DEVELOP AN APPROPRIATE MOUSE MODEL AND IT'S SIMPLY -- WE'VE BEEN BANGING OUR HEAD AGAINST THE WALL AND IT'S JUST NOT AMENABLE SO FAR TO CREATING AN ANIMAL MODEL, PERIOD, LARGE OR SMALL. SO YOU KNOW, I GUESS MY QUESTION, AND MAYBE THIS IS TO DR. WU, BUT ANYONE ELSE CAN CHIME IN, IF THERE'S A SITUATION WHERE WE FEEL LIKE, YOU KNOW, GENE THERAPY COULD ADDRESS THE TARGET, A KEY TARGET FOR A DISEASE, BUT THERE ARE NO APPROPRIATE ANIMAL MODELS, CAN YOU DO IB IN VITRO MODELING WITH SAFETY STUDIES AND IS THERE A PATH FOR THAT TO MOVE FORWARD ESPECIALLY FOR THESE DEVASTATING DISEASES? >> SO I CAN ONLY SPEAK VERY GENERALLY TO THAT, BUT I THINK THAT THERE IS A PATH FORWARD AND WE UNDERSTAND THAT THERE ARE LIMITATIONS IN THE ANIMAL MODELS THAT ARE AVAILABLE, AND AS SOME OF THESE PRODUCTS ARE GETTING MORE AND MORE SPECIFIC FOR HUMAN CONDITIONS DOWN TO SPECIFIC THANK MUTATIONS IN THE HUMAN GENOME, I THINK WE ARE GOING TO BE LIMITED BY THE ANIMAL MODELS AVAILABLE SO YES I THINK THERE'S A POSSIBILITY FOR MOVING FORWARD AND IT'S SOMETHING I THINK YOU SHOULD TAKE ALL THE DATA YOU DO HAVE AND ALL THE AVAILABLE TYPES OF MODALITIES IN WHICH YOU CAN ASSESS YOUR PRODUCT AND PRESENT THOSE AND WE CAN HOPEFULLY GIVE YOU SPECIFIC FEEDBACK ON YOUR PARTICULAR PROGRAM. >> THANK YOU VERY MUCH. >> ON THIS SAME THEME OF CREATING MOUSE MODELS OF DISEASE, I'D LIKE TO ASK A QUESTION OF LEONELA YOUR GROUP HAS CREATED MICE, RIGHT, TO MIMIC THE DUCHENNE MOLECULAR PATHOLOGIES, AND I ALSO -- I WONDERED SPECIFICALLY RELATED TO YOUR TALK, IN THE 60% OF THE ANIMALS THAT HAD THE INSERTION OF THE ADENINE NUCLEOTIDE TO RESTORE THE READING FRAME, WAS THAT A FUNCTIONAL MOLECULE OR DID IT -- IN OTHER WORDS, DID THE INSERTION OF THE A -- IT RESTORED THE READING FRAME BUT DID IT DISRUPT THE PROPER AMINO ACID SEQUENCE DOWNSTREAM? WOULD THOSE -- WERE THOSE ALLELES IN PART RESPONSIBLE FOSH THE RESCUE OR WA WR THOSE ALLELES IN WHICH THE EXON WAS DELETED? >> THAT'S A REALLY GREAT QUESTION. I'LL GO BACK TO THE FIRST ONE AND THE ANIMAL MODELS. >> RIGHT. COULD ANYONE WHO HAS AN ILLNESS, YOU KNOW, WORKING ON AN ILLNESS FOR WHICH THERE ISN'T A RODENT MODEL, WOULD CRISPR/CAS 9 BE SUITABLE FOR CREATING ONE? >> CRISPR/CAS 9, THE TECHNOLOGY IN THAT ONLY HAS AXILLARY TO THE GENERATION FOR THE MOUSE MODEL, IT OF COURSE DEPENDS ON THE CASE SPECIFIC BUT IN OUR LAB, WE HAVE BEEN GENERATING WITH OUR PREVIOUS METHOD REGULAR -- OR KNOCKOUT STRATEGY BEFORE AND THE -- MUCH EASIER AS LONG AS IT'S TARGETED AND WE CAN GENERATE IN A -- COLONY MOUSE MODEL FOR DIFFERENT MUTATIONS. SO THAT'S A REALLY GREAT ADVANTAGE SO WE HAVE GENERATED ADDITIONAL FIVE FOR ALL THE OTHER EXONS. RELATED TO THE -- INSERTION, INTERESTINGLY IT'S THE ALWAYS AT THE CLEAVAGE SITE, IT HAS BEEN SHOWN THAT IT'S -- THERE IS A RECENT -- THAT HAVE SHOWED THAT CRISPR GENERATED -- WERE -- CLEAVAGE WHERE THE NUCLEOTIDE THAT FOLLOWS THE AID AN A AS WELL SO IT'S THE SEQUENCE THAT'S THE NUCLEOTIDE ON THE SEQUENCE THAT FOLLOWS THE CLEAVAGE DIRECTS THE INSERTION OF THE NUCLEOTIDE. I WOULD SAY IT'S -- THE -- IF IT WILL BE A C THAT FOLLOWS, IT WILL BE A C -- IF IT WILL BE A G THAT FOLLOWS IT WILL BE A G THAT'S INSERTED. MOSTLY IT'S INCODED THAT IT'S MORE PRECISE THAT GIVES YOU THE LEVEL OF PREDICTABILITY TO THAT ONE INSERTION THAT CAN BE GENERATED BY ONE SINGLE CUT. WE HAVE LOOKED INTO SO THE IPS MODEL HAS ALLOWED US TO USE SINGLE CLONES SO WE CAN EASILY PRECISELY EVERY SINGLE WOULD SAY ONE INSERTION, IT'S A SIMILAR LEVEL, ON THE WESTERN BLOOD IT'S A SIMILAR -- SO IT'S FUNCTIONAL ALLELE AND THERE'S ONLY THREE NUCLEOTIDES DIFFERENCE FROM THE WILD TYPE ON THE EXON 51 SO IT DOESN'T DISTURB THE PROTEIN FUNCTION AT ALL, IT'S FUNCTIONAL AND IT'S REALLY -- THE IMPORTANT PART IS ONE OF THE MOST ABUNDANT SO IT CAN PREDICT IT'S NOT SO RANDOM, LET'S SAY, IT'S REALLY LIKE PRECISE, IT'S CONSERVED AL THE WAY SO THE SAME ONE INSERTION THAT WE SEEN AS I MENTIONED DURING MY TALK IN MOUSE AND DOGS AND IN HUMAN CELL LINES AND WITH A SIMILAR PATTERN, IT'S ALWAYS THE MOST COMMON THAT IS DETECTED THE DNA LEVEL THAN THE RNA LEVEL WE HAVE A 60% TO INCREASE THE PERCENT OF TRANSCRIPT -- ALONG THE SPECIES. >> OKAY. THANKS. ARE THERE OTHER QUESTIONS FROM THE AUDIENCE? IT'S BEEN A VERY INFORMATIVE SESSION, I THINK FROM OUR PERSPECTIVES. THINK WE SHOULD GIVE YOU ALL AN EXTRA HALF-HOUR FOR LUNCH, GET AN EARLY START ON THE CAFETERIA LINES. US A MAY KNOW, THERE ARE TWO OPTIONS, AT LEAST HERE IN THIS BUILDING. ONE IS DOWNSTAIRS IN THE B1 LEVEL, ANOTHER IS UPSTAIRS IN THE SECOND FLOOR. WE'RE DUE TO RESUME AT 12:30. LOOKS LIKE WILSON MAY COME AND MAKE SOME REMARKS BUT BEFORE I STOP SPEAKING, I WANT TO THANK CHUCK AND ALSO JAY, LEONELA, HARRY AND IWEN AND ALSO ANNE AND PETER AND CHRIS FOR THEIR OPENING REMARKS THAT LAUNCHED THE BEGINNING OF THIS MEETING. >> THANK YOU VERY MUCH. [APPLAUSE] >> MY UNDERSTANDING IS THERE'S A CAFETERIA ON B1 DOWNSTAIRS, AND WE'LL RECONVENE AT 12:30. >> GOOD AFTERNOON. LET'S GET STARTED. THIS AFTERNOON WE HAVE TWO SESSIONS, THE FIRST ON CLINICAL DEVELOPMENT FROM BENCH TO BEDSIDE AND THE SECOND ON QUALITY AND MANUFACTURING. FIRST SESSION ON CLINICAL DEVELOPMENT IS CHAIRED BY DR.S MENDELL AND CLARK. DR. JERRY MENDELL, I HAVE KNOWN DR. MENDELL, WORKED WITH HIM FOR OVER 25 YEARS NOW. HE'S A NEUROLOGIST AT NATIONWIDE CHILDREN'S AND PRINCIPLE INVESTIGATOR IN THE CENTER FOR GENE THERAPY AT THE RESEARCH INSTITUTE OF NATIONWIDE CHILDREN'S. HE'S ALSO PROFESSOR OF PEDIATRICS, NEUROLOGY, PATHOLOGY, AND PHYSIOLOGY AND CELL BIOLOGY AT OHIO STATE. DR. MENDELL MADE FUNDAMENTAL CIBCS IN CLINICAL -- CONTRIBUTIONS IN CLINICAL RESEARCH AND GENETICS AND NEUROMUSCULAR DISEASE. LIKE I SAID, 25 YEARS AGO DR. MENDELL AND I WERE WORKING ON TRYING TO FIND SOME TREATMENTS FOR PATIENTS WITH NEUROMUSCULAR DISEASE AND THEN -- AND I KNEW HIM THEN AS A DEDICATED CLINICIAN. BUT OVER THE LAST COUPLE OF DECADES HE DECIDED HE WANTED NOT JUST TO TREAT THESE DISEASES BUT CURE THEM. AND HE'S GONE AFTER THEM QUITE AGGRESSIVELY AND I THINK TO EVERYONE'S BENEFIT PARTICULARLY ATTACKING DUCHENNE AND SPINAL MUSCULAR ATROPHY AND OTHER DISEASES AS WELL. IN CONTRAST, I HAVE KNOWN DR. REEDE CLARK FOR ABOUT TWO MINUTES. AND DR. CLARK GOT HIS Ph.D. IN MOLECULAR GENETICS FROM OHIO STATE UNIVERSITY. AND IS CURRENTLY SENIOR VICE PRESIDENT WITH ULTRA GENICS GENE THERAPY. HE HAS HAD EXTENSIVE EXPERIENCE IN AAV GENETIC VECTOR ENGINEERING. ALSO PRE-CLINICAL AND CLINICAL GENE TRANSFER STUDIES. HE SERVED AS DIRECTOR OF NATIONWIDE CHILDREN'S HOSPITAL PRE-CLINICAL AND CLINICAL MANUFACTURING CORE FACILITIES. SO I WILL TURN IT OVER TO DR. MENDELL AND DR. CLARK. >> MY HONOR OR SHOULD I PUT IT ON HERE. >> THANK YOU VERY MUCH. WILSON, THANK YOU FOR YOUR GENEROUS INTRODUCTION. IT IS A PRIVILEGE BEING HERE. I STARTED MY CAREER AT NIH MANY YEARS AGO, WON'T SAY HOW MANY, BUT IT WAS A LONG TIME AGO. IT'S NICE TO COMPLETE THE CIRCLE. (OFF MIC) >> ALL RIGHT. SO I WILL MOVE -- CAN I MOVE MY COMPUTER OVER THERE? THANK YOU. >> SO THIS SESSION IS ON REALLY BASICALLY CLINICAL TRANSLATION BENCH TO BEDSIDE. AND I THINK THERE'S NO BETTER ILLUSTRATION OF THAT THAN OUR SPINAL MUSCULAR ATROPHY TRIAL AND IT WAS GENERALLY -- GENEROUSLY TALKED ABOUT THIS MORNING. THANK YOU. THATCH ABOUT DISCLOSURES, I DON'T HAVE ANY INVESTMENT DISCLOSURES TO MAKE, THESE ARE THE OTHERS THAT I DO HAVE. THE GENETICS OF SMA IS A LITTLE TRICKY AND WORTH JUST A MENTION ABOUT NORMALLY WE HAVE A DUPLICATED SMM 1 GENE, SMN, THE DUPLICATION IS CALLED SMN 2, IT HAS A MUTATION AND EXON 7. AND ONLY CAN PARTIALLY EXPRESS. SO IT CAN COMPENSATE IF THIS HAPPEN, IF SMN 1 IS LOST. THE TRIAL THAT WE PUT TOGETHER WE REQUIRE THAT THE PATIENTS HAVE NO MORE THAN TWO COPIES OF SMN 2. THIS MADE IT A HOMOGENOUS GROUP AND OBVIOUSLY MUCH EASIER TO DO OUTCOME MEASURES. YOU CAN SEE ON YOUR TOP RIGHT THAT THAT SMN 2 COPIES WILL MODIFY THE DISEASE ALL THE WAY UP TO ADULT LIFE. THIS ALSO SHOWS THAT COULD YOU FOCUS THAT A LITTLE BIT? I THINK IT'S A LITTLE OUT OF FOCUS. IS THERE A ARROW HERE? I DON'T SEE IT. I GOT IT. SO ONE OF THE ADVANTAGES OF DOING GENE THERAPY IN THIS PARTICULAR DISEASE IS WE HAVE HOMOGENEITY HERE AND TWO COPIES OF SMN 2 AND WE WERE ABLE TO THEN REPLACE THE FULL CDNA FOR SMN. THAT MAKES A AWFUL LOT OF DIFFERENCE AND FIT NICELY INTO AAV. A COUPLE OF COMMENTS ABOUT THE DISEASE ITSELF. IT'S A VERY SEVERE DISEASE. CHILDREN, INFANTS BORN WITH THIS ARE HYPOTONIC, NEVER LEARN TO TALK, HAVE DIFFICULTY SWALLOWING, ARE NUTRITIONAL FAILURES AND ULTIMATELY RESPIRATORY FAILURES. THE NATURAL HISTORY OF THE DISEASE HAS BEEN DEFINED WELL IN THIS SMA TYPE 1 GROUP. IF I CAN GET THIS TO BEHAVE I CAN SHOUGH E YOU WHAT IT WILL BE. THE FIRST THING IS THAT THE NATURAL HISTORY OF THE DISEASE, THIS SHOWS SURVIVAL CURVE, FOR ALL PRACTICAL PURPOSES MOST INFANTS ARE -- DIE BY AGE 2. BY 20 MONTHS THERE IS ONLY AN 8% SURVIVAL BY 13.6 MONTHS, 25% SURVIVAL. BY TEN MONTHS, TEN AND A HALF MONTHS, THERE'S 50% SURVIVAL. YOU CAN SEE THE CHALLENGES HERE FOR FOR TREATING THIS DISEASE. THAT PUT US INTO THE CHALLENGES FOR THE GENE THERAPY TRIAL. I SHOW THIS SLIDE BECAUSE WHAT THE OBJECTIVE OF THIS SESSION WAS TO ILLUSTRATE BENCH TO BEDSIDE. WE ARE FACED WITH THE FACT THAT MOUSE STUDIES DON'T REALLY TRANSLATE VERY WELL, THEY DON'T TELL US WHAT'S GOING TO HAPPEN. THESE STUDIES WERE DONE BY BRIAN KASPART AT NATIONWIDE CHILDREN'S HOSPITAL AND WHAT THEY SHOWED WERE THERAPEUTIC BENEFIT WAS DOSE TIME AND DEPENDENT IN A DELTA 7 MOUSE MODEL THE DELTA 7 MOUSE MODEL IS A GOOD REPRODUCTION OF CLINICAL DISEASE BECAUSE THESE MICE DIE BY 15 DAYS. IF FULL DOSE OF VIRUS IS GIVEN, FULL DOSE MEANING 2E TO THE 14th VECTOR GENOMES PER KILO, IT WILL ESSENTIALLY COMPLETELY NORMALIZE THE MOUSE WHERE SURVIVAL FOR LONG PERIODS OF TIME. WHAT'S IMPORTANT HERE IS THAT IF THERE'S A FIVE FOLD REDUCTION IN DOSE, THE 6.7, EITHER THE 13th VECTOR GENOMES WILL WE WERE FACED WITH HOW WE SHOULD DO THIS TRIAL AND HERE ARE OTHER MODIFICATIONS YOU SEE IF THE TREATMENT IS LAYERED AT FIVE DAYS, THESE ARE TREATMENT DAY ONE, AT FIVE DAYS THE MICE LIVE 28 DAYS AND IF TREATMENT IS DONE AT TEN DAYS IN THESE DELTA 7 MICE, THERE'S NO RECOVERY. WITH WRESTLE WITH ADVICE FROM THE FDA WHETHER THIS SHOULD BE A DOSE ASCENDING SCHEDULE. AND THAT WAS WHERE WE -- WHAT WE DECIDED ON, YOU SEE HOW PROFOUNDLY THAT AFFECTED THE PATIENTS. I SHOW THIS BECAUSE THIS IS THE FIRST DOSE OF VIRUS WE GAVE. AND THERE HAVE BEEN COMMENTS. IN THIS TRIAL AT THE TIME WE DID IT TO THE TIME WE DISCUSS WITH THE FDA IT WAS AT LEAST ONE HIGHEST DOSE OF VIRUS THAT'S EVER BEEN DELIVERED. THE DELIVERY WENT VERY WELL. NO REACTION AT THE TIME OF DELIVERY. WE HAD TWO IVs IN ONE EXTREMITY, WHERE THE IV WAS DELIVERED, WHERE THE VIRUS WAS DELIVERED AND WE KEPT THE OTHER ONE AS A BACK UP IN CASE THE VIRAL DELIVERY WAS COMPROMISED SO WE HAVE A CONTINUOUS DELIVERY. THIS VIRUS DELIVERY WAS OVER AN HOUR, SOMETIMES EXTENDED TO HOUR 15 MINUTES. THAT'S IMPORTANT. AND WE CAN TALK ABOUT THAT. HERE IS A SURVIVAL DATA WE HAD IN THIS TRIAL. SURVIVAL WAS THE PRIMARY OUTCOME MEASURE FOR THIS TRIAL BUT WE DID HAVE BACK UP FUNCTIONAL MEASURES AS I WILL SHOW YOU. THIS SHOWS THAT IN THE TOP THREE BRACKET, THE TOP THREE LINES HERE, HORIZONTAL LINES, WHAT YOU SEE IS THIS IS THE LOW DOSE, 6.7E TO THE 12th. 6.7E TO THE 13th VECTOR GENOMES PER KILO. WHEN THE NEXT COHORT WAS THE NEXT 12 PATIENTS WHO WERE ENROLLED IN THIS TRIAL AT 2E TO THE 14th VECTOR GENOMES PER KILO. IN TERMS OF OUTCOME MEASURES, ALL PATIENTS IN THIS TRIAL SURVIVED FOR THE 8% SURVIVAL PREDICTED AT 20 MONTHS. SO THAT WAS THE FIRST MILESTONE. THEY ALSO IF YOU LOOK HERE, THAT THIS IS CURRENT DATA THAT COHORT 1 NOW HAS SURVIVED FOR 48 MONTHS. COHORT 2 HAS SURVIVED 36 MONTHS. WE HAVE VERY LONG SURVIVAL BUT MARKED DIFFERENCES IN FUNCTION BETWEEN HIGH AND LOW DOSE. THIS IS THE CHOP AND FEN SCORE WHICH IS AN ACCURATE MEASURE OF FUNCTION IN INFANTS. WHAT YOU WANTED TO NOTICE ON THIS SLIDE, ALL THESE PATIENTS ARE SURVIVING FOR FOUR YEARS NOW BUT LEVEL OF FUNCTION NEVER REACHES 40 ON THE TOP AND YOU'LL SEE HOW THAT DIFFERS ON THE NEXT SLIDE WHEN WE LOOK AT THE HIGH DOSE COHORT. THERE WAS MILD IMPROVEMENT FROM BASELINE, PRETTY MILD SO ESSENTIALLY A PLATEAU. HERE IS THE NATURAL HISTORY LINE. ALL PATIENTS SURVIVE BEYOND WHAT WAS EXPECT IN NATURAL HISTORY. LOOK AT THE HIGH DOSE COHORT WE HAD DRAMATIC DIFFERENCES. THE CHOP AND PEN SCORES HERE WERE ALL OVER 40. THAT'S NEVER REACHED. THAT NUMBER IS NEVER REACHED IN THE NATURAL HISTORY OF THE DISEASE. MOST OF THEM ARE CLOSE TO THE HIGHEST CHOP AND PEN ACHIEVABLE. TWO HAVE EXCEEDED WHAT CAN BE MEASURE ON THE CHOP AND TEN AND HAVE TO BE MEASURED ANOTHER WAY USING THE BAILEY OUTCOME. THE OTHER THING ABOUT HERE IS NATURAL HISTORY, EXTENDED LINE I SHOWED ON THE LAST SLIDE. BUT ONE VERY IMPORTANT THING WHICH GOES BACK TO THIS PRINCIPLE OF TIME AND DOSE DEPENDENT, HERE IS THE PATIENT WHO WAS ENROLLED IN THE TRIAL AT LATEST TIME POINT, THIS PATIENT WAS ENROLLED 7.9 MONTHS. HAD ONSET THE SAME ADS THE OTHER PATIENTS WITH ONSET BEFORE SIX MONTHS OF AGE BUT ENROLLED AT 7.9 MONTHS AND MADE NO IMPROVEMENT DURING THE CLINICAL TRIAL. THESE TWO PATIENTS WERE ENROLLED AT THE EARLIEST TIME POINT. I'LL SHOW YOU PICTURES. THESE TWO PATIENTS WERE CONTACT WHEN THE PATIENTS PATIENTS WERE IN UTERO. WE FOLLOWED THEM ONE TREATED AT FOUR WEEKS, THE OTHER TREATED AT EIGHT WEEKS. THEY ARE ESSENTIALLY NORMAL NOW. THIS IS A FUNCTIONAL MEASURE AND MOTOR MILESTONES WRITING HAND TO MOUTH HEAD CONTROL, ROLLING OVER AND SITTING WITH AND WITHOUT ASSISTANCE. THIS IS THE PATIENT WHO WAS TREATED AT 7.9 MONTHS, NEVER ACHIEVED ANY OF THOSE MILESTONES. BUT VIRTUALLY EVERY PATIENT IN THIS CLINICAL TRIAL NOW HAS ACHIEVED THIS, 11 OF 12 CAN SIT MORE THAN FIVE SECONDS, 10 OF 12 SIT UNASSISTED FOR 10 SECONDS AND 9 OF 12 SIT UNASSISTED FOR 30 SECONDS. THE ADVERSE HE WANT IN THIS TRIAL WERE QUITE REMARKABLE. WE DID HAVE LIVER ENZYME ELEVATIONS AND JUST AS THE EARLY HEMOPHILIA STUDIES WERE REPORTING LIVER ENZYME ELEVATIONS, WE FOUND THE SAME HERE. THIS WAS NOT TARGETED TO LIVER, TARGETED TO CENTRAL NERVOUS SYSTEM BUT WE HAD TWO PATIENTS EXCEEDED TEN TIME IT IS NORMAL LIVER ENZYME ELEVATIONS AND THESE WERE LABELED SERIOUS ADVERSE EVENTS, WE HAD TWO UNDER TEN TIMES ELEVATED AND THEY WERE SIMPLY LABELED ADVERSE EVENTS RELATED TO VIRAL ADMINISTRATION. THE OTHER POTENTIALLY SERIOUS ADVERSE EVENTS WERE ALL NOT TREATMENT RELATED. THIS IS A SERIOUS DISEASE. IT HAS PATIENTS HAD TO BE CARED FOR VERY CAREFULLY. WE HAD A VERY GOOD TEAM OF INVESTIGATORS WHO WERE INVOLVE WITH THIS TRIAL. WE HAD PEOPLE IN INFECTIOUS DISEASE, PEOPLE IN PULMONARY CARE, THAT WAS ABSOLUTELY CRITICAL FOR THE SUCCESS OF THIS TRIAL. THESE ARE NATURAL EVENTS THAT OCCUR IN THE SMA GROUP, ALL SURVIVE, ANY PATIENT WHO IS ADMITTED TO THE HOSPITAL AS PART OF THIS TRIAL SURVIVED COMPLETELY. THIS -- I WILL SHOW YOU EXAMPLES OF PATIENTS AT VARIOUS TIMES, THIS IS EARLY ONE, DOSED 27 DAYS FOUR MONTHS POST GENE THERAPY, HAD VERY GOOD HEAD CONTROL AND FOR ALL PRACTICAL PURPOSES THIS IS A NORMAL MILESTONE. WE HAD ANOTHER ONE DOSED AT FOUR MONTHS. THIS IS SIX WEEKS POST GENE THERAPY, ABLE TO HOLD A BOTTLE INDEPENDENTLY AND DRINK WITHOUT HAVING ANY NUTRITIONAL SUPPORT THIS IS ONE I LIKE, PARTICULARLY YOU CAN SEE THIS IS A CHILD WITH GOOD HEAD CONTROL, SITS INDEPENDENTLY AND HAS A CERTAIN AMOUNT OF AFFECTION FOR HER DOCTOR WHICH GOES A LONG WAY. FROM THIS IS ANOTHER PATIENT, WHO HAS GOOD BODY CONTROL AND YOU CAN SEE, THIS BABY -- THIS IS ONE THAT WAS DOSED AT TWO MONTHS, AND THIS IS SIX MONTHS POST DELIVERY. SHE ALSO HAS GOOD TRUNKING CONTROL, SHE IS EXTENDED HER LEGS, I'M HOLDING HER RIGHT BELOW HER BELLY HERE. THIS IS END PENNSYLVANIA DENT SHE CAN HOLD HER LEGS OUT IN THIS FASHION. THIS IS A PATIENT WHO I TURNED OFF THE SOUND BECAUSE HIS MOM IS MAKING COMMENTS HERE. HE'S SITTING UNSUPPORTED, SPEAKS AND'S WITHOUT HELP, THIS IS A MILESTONE WE WOULD NEVER SEE IN THE NATURAL HISTORY OF SMA. THIS BABY GAINED HIS REFLEXES BACK. S IN THE EARLIEST TREATMENT IN THE TRIAL AND I LIKE TO SHOW THIS ONE BECAUSE IT'S DEAR TO THE HEART OF A NEUROLOGIST WHEN REFLEXES COME BACK. FINALLY ON THE LAST SLIDE THIS IS OUR AMERICAN NINJA WARRIOR, HE IS PERFORMING WELL. HERE HE IS CARRYING HIS BRIEFCASE GOING UP TO HIS OFFICE ON THE SECOND FLOOR PUSHING THE ELEVATOR. BASICALLY THIS TRIAL WAS VERY GRATIFYING AND VIRTUALLY EVERY WAY. VERY HIGH DOSE VIRUS, GOOD TRANSLATION AND PREDICTION FROM THE PRE-CLINICAL. MAYBE THE PEOPLE FROM THE FDA MAY WANT TO COMMENT ABOUT THAT. HOW OFTEN CAN WE REALLY ACHIEVE THAT. THAT IS REALLY A CRITICAL MILESTONE IN TERMS OF TRANSLATING FROM LAB TO PATIENT CARE. THERE ARE MANY PEOPLE WHO WERE CRITICAL IN THE PARTICIPATION, BRIAN KASPAR WAS CRITICAL IN LAB DEVELOPMENT, SAMUEL WAS A CRITICAL PERSON IN THE CLINICAL TRIAL PER SE. LOUISE RODINO CLAYPACK WAS VERY IMPORTANT IN TERMS OF MEASURING ANTIBODY LEVELS FOR US. WE HAD SEVERAL OTHER PEOPLE WHO WERE IMPORTANT IN THIS TRIAL. SO I'LL STOP THERE AND WE'LL GO ON IN THIS AND ANSWER ANY QUESTIONS. REED DO YOU WANT -- MOVE ON? I THINK WE HAVE TIME IN THIS SESSION. TURN THIS ONE OFF. AND INTRODUCE OUR NEXT SPEAKER. >> THE NEXT SPEAKER IS DR. CHET WHITLEY, PROFESSOR OF PEDIATRICS IN EXPERIMENTAL AND PHARMACOLOGY AND DIRECTOR OF ADVANCED THERAPIES UNIVERSITY OF MINNESOTA. HE HAS BEEN WORKING ON GENE THERAPY FOR LYSOSOMAL DISEASE AND HE'S GOING TO REVIEW THAT DATA FOR US TODAY. THANK YOU. >> THANK YOU VERY MUCH. THANKS FOR YOUR ATTENTION. THIS IS A FANTASTIC SYMPOSIUM THUS FAR. BY WAY OF DISCLOSURE. I'M INVOLVED IN ONE WAY OR ANOTHER WITH SEVERAL PHARMACEUTICAL ENTITIES AND DOING RESEARCH AND EDUCATION. NOTABLY IS THE LYSOSOMAL DISEASE NETWORK SUPPORTED BY NCATS SO SOME OF THE RESULTS I'LL SHOW YOU HERE WERE DEVELOPED BY VIRTUE OF THAT CLINICAL TRIAL READINESS PROGRAM GATHERING DATA WHICH WILL SUPPORT OR ENCOURAGE DEVELOPMENT OF PHARMACEUTICAL PROJECTS. I ALSO WANT TO THANK NICHD ON FRIDAY, SCOTT MCIVER AND I SENT IN FINAL REPORT FOR 20 YEAR PROGRAM PROJECT GRANT. VERY SAD PROGRAM PROJECT GRANTS ARE SUPPORTED THAT LONG BUT WE HAD TREMENDOUS PROGRESS IN GENE THERAPY AS A CONSEQUENCE OF THAT. I'LL ALLUDE TO SOME OF THOSE AND GIVE HISTORY ON THOSE. >> WHEN YOU LOOK AT LYSOSOMAL DISEASE, IT'S MANY DISEASE, SOMEBODY WILL SAY ABOUT 70 DISEASES, IF YOU REFLECT BACK ON DR. AUSTIN'S COMMENT EACH ONE OF THESE HAS OWN SEPARATE GROUP OF MUTATIONS. THERE MAYBE DIFFERENT FORMS WITHIN EACH GROUP SO IF YOU HAVE PALM DISEASE THERE'S A SEVERE INFANTILE FORM INTERMEDIATE AND ADULT SEVERE FORM. WE'LL SHOW OTHER EXAMPLES SO IN DESIGNING CLINICAL TRIALS COMING UP WITH THERAPIES AND THINKING ABOUT RAPID ADMINISTRATION AND THINKING ABOUT DOSE AND SO FORTH, ALL THOSE THINGS DO COME INTO CONSIDERATION. THE RARE DISEASE CLINICAL RESEARCH NETWORK WAS ESTABLISHED BY THE OFFICE OF RARE DISEASES AND IT'S NOW BEEN THROUGH ONE, TWO, THREE CYCLES OF FUNDING,LY GO ON MORE BUT IT INVOLVES MANY INSTITUTIONS LOOKING AT MORE THAN 200 DISEASE, CLOSER TO 10,000 SUBJECTS NOW INVOLVED IN THIS KIND OF WORK. IN FACT RIGHT NOW WE'RE AT THE END OF OUR NINTH YEAR OF PLANNING FOR FOURTH CYCLE OF FUNDING. ONE SURPRISING THING TO ME IS NIH ADMINISTRATORS HAVE COME UP WITH COUPLE OF PARTICULARLY CHALLENGING TASKS FOR US, ONE OF WHICH IS TO MAKE OURSELVES SELF-SUPPORTING AFTER THE NEXT FIVE YEARS. THIS THAT'S BEEN A MAJOR CHALLENGE TO THINK ABOUT. WE ARE LOOKING TOWARD DOING TRUE MULTI-SITE STUDIES FROM A SINGLE CENTRAL IRB. WE LEARNED WHAT USED TO BE FREE BY ACADEMIC IRB COSTS $50,000 PER YEAR TO DO FIVE STUDIES, SO MULTI-CENTER IRB MAY HAVE THEIR BENEFIT BUS THEY ARE NOT WITHOUT THEIR COSTS. THIS IS THE CURRENT FORMULATION. OF THE RARE DISEASE CLINICAL DISEASE NETWORK. HERE WE ARE LYSOSOMAL DISEASE NETWORK, SEVERAL DISORDER GROUPS, ALL TYPICALLY DISEASE BASED ORPHAN DISEASE BASED IN THEIR THEME. REPORT OUR DATA TO THE DNC, DATA MANAGEMENT AND COORDINATING CENTER, THIS DATA DOESN'T LINE THE FILE CABINETS BUT GOES TO A DATABASE PRESERVED IN PERPETUITY. WE ALSO WORK CLOSELY WITH COALITION OF PATIENT ADVOCATE GROUPS. WE WORK WITH 40 SMALL AND LARGE PATIENT ADVOCATE GROUPS TO DO THE WORK THAT WE DO. WE WORK IN A COLLABORATIVE WAY, WE HAVE CENTRALIZED DATA SUBMISSION AND ARCHIVING. WE USE OTHER PUB LOOK RESOURCES FOR EDUCATION AND TRAINING FELLOWS, SUCH AS PHARMAD ATTENDING THIS CONFERENCE IN FIRST MONTH OF TRAINING UNDER THE RDCRN PROGRAM. WE LOOK AT THE OVERALL STRUCTURE, YOU CAN SEE THAT DIFFERENT ICs NIH SUPPORTING UNDER NCATS LEADERSHIP, THE LYSOSOMAL DISEASE NETWORK ILLUSTRATING A SCHEMATIC HOW WE RELATE WITH DATA TO A CENTRAL MANAGEMENT COORDINATING CENTER IN A GROUP OF PATIENT ADVOCATE GROUPS THAT COLLABORATE IN THE RESEARCH. THIS IS ONE OF OUR PATIENTS. SOME OF YOU MAYBE FAMILIAR WITH INFANTILE G1, THIS IS ONE OF DR. VAIR SRIS'S PATIENTS AT TWO YEARS OF AGO. A RAPID LETHAL NEUROLOGIC DISEASE. HERE SHE IS A YEAR LATER, NOT ONLY MUCH MORE QUIET, SHE'S ON A SUPPORTED RESPIRATION AND IF YOU LOOK AT HER SKULL CAREFULLY THERE'S SIGNIFICANTLY LARGER. THAT'S NOT HYDROCEPHALUS, THAT'S MICROCEPHALY, IT'S THE BRAIN ITSELF GROWING OUT OF PROPORTION, GROWING BECAUSE OF STORAGE MATERIAL THAT'S THE DISORDER WHICH THERE IS NO TREATMENT CURRENTLY. LOOK AT THIS CEREBRAL CORTEX ON MRI AND UNDERSTAND WHAT WE ARE TALKING ABOUT. RAPIDLY PROGRESSIVE, CENTRAL NERVOUS SYSTEM DISEASE. I WANT SHOUGH TOE YOU DATA FROM DR. JARVIS STUDY, I'M NOT GOING TO UNDERSTAND ALL THE DETAIL BUT FOR SAKE OF UNDERSTANDING THAT HETEROGENEITY WITHIN ONE NAME OR WITHIN ONE GENE, HERE IN GREEN ARE THE PATIENTS THAT ARE GOING TO BE GRAFT OR STUDIED. JUVENILE AND INFANTILE DISEASE IN PATIENTS IN BLUE AND IN RED WE HAVE INFANTILE GM 1 GANGLIOCYTEOSES A PANOPLY OF DIFFERENT CLINICAL PHENOTYPES. FOR EXAMPLE WITHOUT GOING INTO DETAIL, THIS IS THE CEREBELLAR BRAIN VOLUME. NORMAL INDIVIDUALS INFANTS PLOT UP HERE, THIS IS THE NATURE OF THE CEREBELLUM IN GROUP OF PATIENTS STUDIED AGE 2, 4, 6, 8, 10. LIKEWISE WE CAN LOOK AT OTHER PARTS OF THE BRAIN SUCH ADS QUANTITATIVE MORPHOMETRIC ANALYSIS OF THE -- BY MRI. DIFFERENT GROUPS HAVING DIFFERENT PATTERNS. THESE ARE THE HETEROGENEOUS CHANGES THAT DEFINE NATURAL HISTORY OF THIS DISORDER. IF WE LOOK AT TUMOR NEE DROWSIEST FACTOR RECEPTOR 2, WE SEE IN SERUM AND CEREBRAL SPINAL FLUID, BETWEEN THESE LINES ARE NORMAL RANGE WITH CERTAIN GROUPS FALLING OUTSIDE THE NORMAL RANGE WHILE OTHERS STAY. AND SERUM IS DIFFERENT THAN CSF. IF YOU DO OTHER PATHOLOGIC LYSOSOMAL DISEASE THESE BY AND LARGE STAY IN THAT RANGE. WHEREAS OTHERS IN THE GANGLION SIDOSIS SO WE'RE IDENTIFYING THE FIRST TIME VERY IMPORTANT BIOMARKERS IN CSF. THIS IS UNTREATED PATIENTS, OTHER FACTORS LIKE EPIDERMAL NEUTRAPHIL ACTIVATING PROTEIN, SOME IN THE NORMAL RANGE OTHERS, OTHER SERUM BEING ABNORMAL OR VICE VERSA. FROM CHEMO TACTIC PROTEIN, AND MACROPHAGE INFLAMMATORY PROTEIN 1 ALPHA, SO THAT'S THE KIND OF DATA REQUIRING THESE DISORDERS PART OF CLINICAL TRIAL READINESS, HAVING OTHER INVESTIGATORS COME UP WITH THERAPIES, SUCH AS GENE THERAPY. SO THIS IS GOING FROM BENCH TO BEDSIDE. SOME OF THE WORK HAS TO BE DONE AT BEDSIDE AS YOU HAVE SEEN. THE LYSOSOMAL DISORDERS HAVE SOMEWHAT UNIQUE PLACE IN MEDICINE AND THERAPY AND EVEN IN GENE THERAPY, A LOT OF IT HINGES UPON DISCOVERY OF CROSS CORRECTION RIGHT HERE AT THE NIH. WHEN ELIZABETH AND COLLEAGUES MIXED CELLS FROM HUNDREDS OF PATIENTS AND FOUND OUT ONE CELL COULD CROSS CORRECT. BASICALLY ENZYME, WE DIDN'T KNOW AT THAT TIME, IT WAS A FACTOR LEAVING ONE CELL GOING THROUGH THE MEDIA, AND CORRECTING THE OTHER METABOLIC DEFECT FROM THE OTHER GENOTYPE, THAT LED TO A SERIES OF ADDITIONAL DISCOVERY. THE NEXT ONE WAS THE CONCEPT OF ENZYME REPLACEMENT THERAPY WHICH I NOTE BEGINNING ABOUT IN 1980. LIZ NEWFELD IDENTIFICATION OF CROSS CORRECTION, WHICH IS SUBSEQUENTLY FOUND IN MOST CASES TO BE MANOS OR MNO RECEPTOR MEDIATED UPTAKE. ROSCOE BRADY ATTENDED IV ADMINISTRATION FROM URINE, FLU OUT TO MINNESOTA BACK IN THE '70s TO ADMINISTER THAT TO A CHILD WITH SAN PHILIPPE SYNDROME, THAT'S HISTORICALLY THE FIRST ATTEMPT AT A HUMAN ENZYME GOING INTO A HUMAN BEING AS PART OF THERAPY. OVER THE YEARS MY COLLEAGUES AT MINNESOTA AND CHIEF LEADER -- ELSA SHAPIRO AND I WERE LOOKING AT MANY PATIENTS WITH HERLER SYNDROME, THOSE ARE THE EXAMPLES WE TALK ABOUT HERE, QUANTIFIED IS THE IQ OR DEVELOPMENTAL QUOTIENT ACROSS AT VARIOUS AGES HALF YEAR, A YEAR, TWO YEARS, THREE YEARS. UNTREATED WE FIND CHILDREN WITH HERLER SYNDROME LOST ON AVERAGE 20 IQ POINTS PER YEAR FOR WHICH THAT PERSON IS UNTREATED. IQ POINTS PER MONTH. THUS THE IMPORTANCE OF EARLY TREATMENT, THUS THE IMPORTANCE OF NEUROLOGIC TREATMENT WHICH IN CONTRAST MAY NOT BE NEEDED IN CHAI SYNDROME SO FINDING THE NATURAL HISTORY IS TREMENDOUSLY IMPORTANT. HERE IS THE BEGINNING OF ENZYME REPLACEMENT THERAPY IN SOME WAYS. IN LONDON, JACK HOBBS PUBLISHED IN LANSETT HURST FIRST ATTEMPTED TRAN PLANT FOR HERLER SYNDROME, BEING PUBLISHED IN THE LANSET IS A SIGN OF CREDIBLE WORK. I WAS BROUGHT IN BY MY DEPARTMENT CHAIR MAN BILL AT THAT POINT, HE THOUGHT FROM THE -- ONE OF THE WORLD'S -- THE WORLD'S LARGEST PEDIATRICS BONE MARROW TRANSPLANT CENTERS WE OUGHT TO BE INVESTIGATED SO BILL DID THE INITIAL BONE MARROW TRANSPLANT IN THE U.S. LYSOSOMAL PATIENTS WITH MARTOLA MAY SYNDROME, SHE HAD AIRWAY DISEASE AT AGE 60, THOUGHT SHE WOULDN'T LIVE BEYOND A YEAR, IT IT SEEMED A LEGITIMATE RISK BENEFIT RATIO. THAT LADY SURVIVED. SHE WAS ENGRAFTED AN DIED AD FEW YEARS AGO, TWO YEARS AGO IN SURGERY FROM UNRELATED BUT ONGOING CONSEQUENCE OF MART OLA MAY SYNDROME, I BECAME INVOLVED, BILL KNEW I KNEW THE CHEMISTRY AND BIOLOGY, IT WOULD BE NEEDED TO CONFIRM JACK HOBBS WAS RIGHT OR WRONG. SO ON SEPTEMBER 13th, 1983, HE AND I DID THE FIRST BONE MARROW TRANSPLANT IN THE i, THIS IS KELLY A LITTLE GIRL WHO UNDERWENT TRANSPLANT. NOW SOME 35 YEARS LATER, THIS WAS ACTUALLY THE FIRST AFFECTIVE SYSTEMIC TREATMENT FOR LYSOSOMAL DISEASE. WE NOW LOOK AT HEMATOPOIETIC STEM CELL TRANSPLANT FROM BONE MARROW OR UMBILICAL CORD BLOOD OR GENETIC MANIPULATED STEM CELLS AS STANDARD OF CARE FOR TREATMENT OF HERLER SYNDROME, NOT AS SUCCESSFUL FOR HUNTERS SYNDROME OR SAN PHILIPPE SYNDROME OR SOME OF THE OTHER LYSOSOMAL DISEASES SO THE CONCEPT WAS PROVEN WITH HERLER SYNDROME BUT THE DIFFERENT DISEASES OFTEN HAD DIFFERENT CLINICAL OUTCOMES WITH THE SAME TREATMENT. SO THUS BEGAN REPLACEMENT THERAPY AND GENZYME AND ROSCOE BRADY FROM NIH DID THEIR FIRST TREATMENT WITH PLACENTA DERIVED ENZYME WHICH RECEIVED FDA APPROVAL IN 1991. RECOMBINANT DNA TECHNOLOGY BECAME POSSIBLE THEY CONVERTED TO RECOMBINANT DNA PRODUCED GLOOM RACE, AND ENZYME REPLACEMENT THERAPY ADVANCE. IT WAS QUITE A GAP. BETWEEN 1994 AND 2001. SO PEOPLE ARE STARTING TO UNDERSTAND THE PRINCIPLE BUT THE BUSINESS MODEL WAS UNCLEAR. BUT EVENTUALLY GENZYME TOOK UP THE IDEA OF DOING DISEASE THERAPY TKT AND SHIRE TOOK OVER COMPETING DRUG AND THERE'S THE HISTORY OF THE RECENT ENZYME REPLACEMENT THERAPY FOR LYSESOMAL DISEASE PROVED HERE FOR SLICE THIS PAST YEAR. WE TALK ABOUT ORPHAN DISEASES, CONDITIONS THAT AFFECT 200 PEOPLE OR FEWER IN THE U.S.. THAT'S THE DEFINITION. BUT IN FACT MANY ARE BETTER DEFINED AS ULTRA ORPHAN DISEASE. WHETHER EPIYOU U THINK, -- WHEN YOU THINK ABOUT IT, ROSCOE BRADY HAD 12 PATIENTS IN FIRST DRUG APPROVAL. 12 PATIENTS. MORE WERE REQUIRED BY THE FDA AS TIME WENT ON BUT WE GOT DOWN TO SLICE SYNDROME AND THAT DISEASE WAS APPROVED ON A 12 PATIENT CLINICAL TRIAL. REMARKABLE. SO WE HAVE TO THINK ABOUT OUR STUDIES WITH THE EXISTING POPULATIONS AND WHAT IS RELEVANT IN TERMS OF PRODUCING -- PROVING OR DETERMINING SOMETHING IS SAFE AND EFFICACIOUS. SO THAT WAS THE ERA OF ENZYME REPLACEMENT THERAPY, SINCE THAT TIME SMALL MOLECULES ARE BROUGHT INTO THE ARMAMENTARIUM AS WELL AS COMBINING THERAPIES AND SYNTHETIC ENZYME REPLACEMENT, THINGS FOR EXAMPLE THAT ARE REDESIGNED ENZYMES AIMED AT CROSSING THE BLOOD BRAIN BARRIER. LUSHING ALL THESE YEARS HAS BEEN GENE THERAPY. THE TERMS POINTED OUT, COINED BY TED FREEMAN BACK IN THE 1972 ARTICLE AND THINGS HAVE NOT CHANGED MUCH SINCE THAT TIME. HE TALKED GENE THERAPY, THIS IS THE LANGUAGE FROM THAT ARTICLE, DNA COULD BE USED TOE TREAT DISEASE IN VIRAL VECTORS THOUGH WE COME UP WITH OTHER WAYS. THE FIRST CONCEPT OF VIRAL VECTORS AND AS I WAS IN ETHICS COMMITTEE IN COLLEGE I REMEMBER THE MEMORABLE EXPERIMENT WAS TAKING THE BETA GALACTASE ENZYME FROM E. COLI, PUT INTO A PLANT DERIVED VIRUS AND USING IT TO TRANSDUCE HUMAN BETA GALACTO DEFICIENT CELLS IN CULTURE. THAT WOULD BE TREATING MORFEUS SYNDROME OR GANGLIOSIDOSIS BUT IT'S NOT BEEN DONE. HERE IS IDEA OF GENE THERAPY BEGINNING HERE AGAIN AT THE NIH, MICHAEL BLAZE FRENCH ANDERSON, IN 1990 WERE TREATING WITH MLV VECTORS, AIMING AT CD34 CELLS BUT PROBABLY NOT GETTING REAL CD34 CELLS FOR ADA EFFICIENCY. WITH THAT, MILESTONE, THREE GROUPS IN THE LYSOSOMAL DISEASE BEGAN TREATING PATIENTS WITH GO CHAI DISEASE, THE SAME PROOF OF PRINCIPAL IF YOU WILL. THERE'S ALWAYS CONCERN THIS WILL NOT BE TREMENDOUSLY EFFICACIOUS BUT HOPES ARE HIGH. THIS TREATING THREE PATIENTS, PRODUCING NINE THERAPIES, ONE HAD LONG TERM E ENGRAFTMENT OF TRANSDUCED CELL FROM THAT PROCEDURE. MEANWHILE WE WERE INTERESTED IN PURSUING THAT, ONLY GET INTO CULTURED BETA DERIVED CELLS. SO SCOTT MCOVER AND OUR TEAM UNIVERSITY OF MINNESOTA BEGAN WORKING ON HUNTERS SYNDROME. IT'S LAUGHABLE NOW BUT SOME LESSONS FOR EVERYBODY GOING FROM BENCH TO BEDSIDE. WE WERE INTEREST IN THAT PROJECT WORKING IN THE LABORATORY, AND WE FOUND THERE WAS AN FDA ORPHAN DISEASE GRANT AVAILABLE SO WE QUICKLY PUT TOGETHER OUR GRANT APPLICATION FOR AN FDA GRANT AND NOTICED ON THE FORM YOU HAD TO HAVE AN IND NUMBER. SO WE FILLED OUT THE FORM, FIVE PAGES, THERE WAS A LOT LEFT EMPTY ON THAT FORM. WE HAD A ROUGH IDEA THE STRUCTURE, NO SAFETY DATA, WE HAVEN'T TREATED ANIMALS, THERE WAS NO ANIMAL MODEL BUT WE SUBMITTED AND LEARNED THE 38 TURN OVER WE GOT A LETTER YOU'RE ON CLINICAL HOLD. BUT ANY CASE WE DIDN'T GET THE GRANT BUT WELL ON OUR WAY AND IT TOOK ANOTHER FIVE YEARS WE DEVELOPED A GMP FACILITIES, GONE THROUGH THE FDA REQUIREMENTS, ANSWERED THE QUESTIONS, AND WE WERE TREATING FIRST ADULT PHASE 1 STUDY E VIVO GENE THERAPY. THAT PATIENT AFTER 12 INFUSIONS SO NO EFFICACY WE CAN DETERMINE. I WANT TO COMMENT THOUGH ONE LESSON WE LEARNED WAS FROM THE FDA, WHY NOT GIVE THE WHOLE DOSE TO THE PATIENT THE FIRST TIME. WHY DON'T YOU DO A DOSE ESCALATION STUDY. AND BACK THEN, WE WERE CONCERNED ABOUT MAKING ENOUGH CELLS TO PUT IN AN IV BAG AND GET TO PATIENT TO MEASURE ANYTHING. BUT THE FDA IN ITS WISDOM SAID WHY GIVE THE FIRST PATIENT 1 PERCENT OF MAXIMUM FEASIBLE DOSE, 10% NEXT MONTH AND GO UP TO FULL IN THE THIRD MONTH. REMEMBER GOING IN AFTER THE THIRD DOSE, THE CLINICAL RESEARCH CENTER, AND THE NURSE IS SAYING YOU KNOW, I DON'T THINK OUR SUBJECT IS -- HE HASN'T WAKED UP THIS MORNING. I I WALKED INTO THE ROOM, THIS IS EXPERIMENTAL, FACING THE WINDOW LYING IN BED, COULDN'T SEE HIS EYES BUT I YELLED AT HIM, DEAR MR. -- AND I SAID HIS NAME. AND HE DIDN'T RESPOND. SO I YELLED LOUDER. MR. SO AND SO, HOW ARE YOU DOING? HE STILL DID NOT MOVE, I WAS CONCERNED SO FINALLY I GRABBED HIM BY THE SHOULDERS AND HE STARTLED. THAT PATIENT WITH HUNTER SYNDROME DIDN'T HAVE HIS HEARING AIDS IN. SO HE COULDN'T HEAR ME. BUT GOING THROUGH THOSE EARLY DAYS, PROBABLY FOR ANYBODY DOING A PHASE 1 TRIAL IT GOES BEYOND LABORATORY NOTEBOOKS AND COMPUTERS AND ALL THE DESIGNS CALCULATIONS, TREATING A PATIENT AND TRYING TO UNDERSTAND WHAT IT'S LIKE TO DO SOMETHING THE FIRST TIME IN A HUMAN BEING. SO FOR ALL OF YOU GETTING INTO IT, THINK ABOUT THAT. WHAT'S REMARKABLE IS THAT IN THE WORLD OF GENE THERAPY AND PARTICULARLY LYSOSOMAL DISEASE THERAPY, THERE'S A BIG GAP. RON CRYSTAL OF ALL PEOPLE PERSISTED, WORKING ON LATE INFANTILE ACIDOSIS OR BAT DISEASE FOR A LONG TIME. MAKING THE PLASTIC CATHETERS, DRILLING BURR HOLES IN THE SKULL AND INSERTING THEM INTO THE BRAIN AND TRYING TO DELIVER VECTOR. THAT IS QUITE REMARKABLE. OTHERS HAVE PURSUED GENE THERAPY AND NOW HERE WE ARE, IN THE ERA OF MAYBE 2010, 2015, THINGS HAVE CHANGED QUITE A BIT. ALL THE FAILURES AND GETTING AROUND THOSE FAILURES AN LEARNING MORE IS STARTING TO GET TRACTION. RECENTLY PUBLISHED, 2011, HIS, AAV RH 10 BRAIN INFUSION, THE SAME TECHNOLOGY THAT RON CRYSTAL INVENTED FOR SAN FILIPPE A, WE HAVE PATIENT STUDIES FOR -- SAN FILIPPE A AND A AND B, THERE'S SOME DISORDERS WHICH ARE VERY RESISTANT TO THERAPY, THIS MAY NOT BE ALL THE CLINICAL TRIALS GONE ON BUT THESE ARE ONES FOR LYSOSOMAL DISEASE TAKEN OFF THE NIH RAC WEB SITED SO IT'S RELATIVELY COMPREHENSIVE. AS I LOOK BACK WITH HUNTERS SYNDROME SOME OF THE PEOPLE LINE LINDA MULE THANK YOU VERY MUCH FOR HELPING FIGURE WHY THAT PATIENT DID DEVELOP A TYPE 4 ARTHRUS REACTION, 99% BOVINE SERUM OUT OF CULTURE METHOD AND GIVE FOURTH AND FIFTH INFUSION. HARVEY KLINE WHO TAUGHT US TO DO CELL CELL CULTURE. WHERE ARE WE TODAY? THAT APPROACH BECAME REAL IN THE PHASE 1 CLINICAL TRIAL FOR SAN FILIPPE SYNDROME DIED IN PARIS, I'LL TALK ABOUT THAT MORE IN A MINUTE. I WILL PASS THIS UP HERE. THERE ARE OTHER STUDY GOING ON HERE WHICH ARE NOT USING INVASIVE PROCEDURES TO GET AROUND THE BLOOD BRAIN BARRIER. KEVIN FLANAGAN AND OTHERS FROM NATIONWIDE CHILDREN'S HOSPITAL ARE HERE. GREAT WORK. THIS SHOWS CURRENT STATUS AND SLIDES, WEEK OR SO ABOUT THE SAN PHILIPPE TRIAL, THIS IS STORAGE MATERIAL TO GET RID OF IN CELL, A NORMAL CELL. THIS TALKS ABOUT INTRAVENOUS ADMINISTRATION. WE DID SOME STUDIES LOOKING AT THE VARIOUS ARCAV VECTORS AVAILABLE, WHAT MIGHT GET ACROSS THE BRAIN BARRIER, THEY SHOW THAT AT LEAST IN ANIMAL STUDY MS. THE BRAIN >> DESIGN A STUDY THAT DOES THIS, AND WELL ON THEIR WAY TO SHOWING EFFECT ON NOT ONLY CSF BUT ALSO -- THOUGH AIMED AT THE BRAIN. THEY LOOK AT THE BINDING DECREASING SO GOOD SIGNS IMPACT THERE. BRAIN STRUCTURE, BRAIN ARCHITECTURE, CHANGES IN TREATMENT COMPARED TO NATURAL HISTORY GROUP AS WELL AS CONTAINMENT VOLUMES. WHAT'S COMING MORE IMPORTANT OR EQUAL IMPORTANT IS CAREGIVER REPORT OBSERVATIONS, GLAD THEY'RE IMPROVING THIS, IMPROVED SLEEP AND EVENTUALLY IMPROVED BEHAVIOR FOR THAT HORRIBLE DISEASE SO IN SUMMARY, THIS IS WHERE THEY ARE. AND THEIR PROGRESS AND THEIR STUDIES. THEY HAVE ALSO DONE THIS FOR PHASE B STUFF I -- STUDIES. MAKING PROGRESS THERE. ROGENICS IS A PLETHORA OF DIFFERENT AAV PSEUDOTYPES AND EXPLORING A NUMBER OF DISORDERS. PARTICULARLY RIGHT NOW DOING INTRACSF INJECTION OF MPS 1 AND 2 PATIENTS AND RECRUITING PATIENTS NOW SO EARLY ON BUT THIS IS YET A DIFFERENT ROUTE OF ADMINISTRATION, PUTTING A NEEDLE BETWEEN THE SKULL AND THE VERTEBRAL COLUMN TO HIT THE SPACE, IT WON'T GO THROUGH BRAIN TISSUE ITSELF. AND THEY ARE SHOWING ANIMAL STUDIES GOOD RESULTS STARTING TO ROLL TO FIVE PATIENTS FOR 104, ALMOST A TWO YEAR STUDY. LYSE GENE NOW JUST ANNOUNCED LAST WEEK TAKING ITS FIRST RESULTS, PHASE 1 STUDIES IN PARIS AND GOING AGAIN THROUGH THE CATHETERS INTO THE BRAIN, WITH NEW APPROVED VECTOR, SURGICAL PROCEDURE WHICH TAKES THREE HOURS, SEVEN, EIGHT DAYS IN HOSPITAL BUT CIRCUMVENTS THE BLOOD BRAIN BARRIER PHYSICALLY, NOT WITH MODIFIED VECTOR OR VECTOR THAT'S TROPIC FOR TRANSGUESSING THE BLOOD BRAIN BARRIER BUT GETTING TO NERVE CELLS, THAT'S THE NEEDLE THAT ILLUSTRATES HOW THE PROCEDURE IS DONE. WITH REDESIGN VECTOR, REMOVING BULK IF YOU WILL ADVANCING ON. FINALLY SANGAMO CAME TO US AT THE UNIVERSITY OF MINNESOTA, DR. MCOVER AND MYSELF, TEST GENE EDITING THE ZINC FINGER NUCLEASES. SO WE KNOW GENOME IS 20 CHROMOSOMES OR 20,500 GENES. WE WANT ONE GENE IN PARTICULAR AND SEE IF WE CAN EDIT, DO A TARGETED WITHOUT OFF TARGET EFFECTS. WE USE -- THEY HAVE DESIGNED OVER 20 YEARS MOLECULE DERIVED FROM NORMAL ZINC FINGER PEPTIDES. SERIES OF THEM, THAT BECOME A ZENC FINGER CONJUGATE WITH NUCLEASE TO BECOME ZINC FINGER NUCLEASE. YOU LINK UP 18 HERE, IN A DIFFERENT NUCLEOTIDE BINDING ZINC FINGER NUCLEASE HERE AN ANOTHER ONE HERE. WE CAN IDENTIFY VERY SPECIFIC PLACE IN THE HUMAN GENOME. THEY HAVE CHOSEN INTRON 1 OF THE ALBUMIN GENE. DOING THAT WE GET A CUT WHEN BOTH ZINC FINGER NUCLEASES WIND UP IN THE SAME PLACE AND ATTEMPT TO PUT INTEGRATION SITE, INTEGRATING GENE AT THAT SITE, IN FACT WE DO THIS IN THE PATIENT GIVING THREE VECTORS. ONE ON LEFT AND RIGHT CDNA WITHOUT PROMOTER INJECTED INTRAVENOUSLY, MOST GOES TO LIVER, WE INTEND TO EDIT BY INSERTION GENE INTO A FEW LIVER CELLS, THAT TRIAL IS ONGOING RIGHT NOW. THIS IS THE WORK WE HAVE DONE IN OUR LABORATORY IN REVIEW RIGHT HERE. AND COMPARABLE METHOD OF MCIVER AND HIS LABORATORY RIGHT HERE, PUBLISHED AND THOSE STUDIES ARE ONGOING. THIS IS A BACKGROUND PUBLISHED BUT THE IMPORTANT POINT IS IF YOU LOOK AT MOUSE TISSUES, LOOK AT ENZYME ACTIVITY, GRAY IS NORMAL. SO WE DO THIS PROCEDURE, LOOK AT FOUR MONTHS LATER WE SEE HIGHER LEVELS OF LOW MEDIUM AND HIGH DOSE THAN WHAT YOU FIND IN NORMAL MICE. LOOK AT PLASMA, ANALOGOUS FINDINGS, THERE'S EVIDENCE OF METABOLIC CORRECTION AND YOU SEE MORPHOLOGY SUCH ADS IN COMPARISON TO WILD TYPE MOUSE, THE PATHOLOGIC ACCUMULATION OF GLUCOSE GLYCANS IN A FEW LARGE CELLS, MACROPHAGES WHICH IS REVERSE PREVENTED IN TREATED MOUSE. SO THOSE TWO STUDYIES ARE ONGOING NOW. I THINK I SHOULD STOP EXCEPT TO INVITE YOU YOU TO WHERE WE PRESENT THIS EVERY YEAR, SYMPOSIUM, OCTOBER 4TH IS THE ABOUT RACKET DEADLINE. ALSO GO TO LYSOSOMAL DISEASE NETWORK AND JOAN THE LYSOSOMAL DISEASE NETWORK TO PARTICIPATE IN ONGOING NATURAL HISTORY STUDIES OF LYSOSOMAL DISEASES. I WILL STOP THERE. [APPLAUSE] THANK YOU. ON THAT SIMILAR THEME, I'M PLEASED TO INTRODUCE DR. DOUG MARTIN, PROFESSOR OF ANATOMY, PHYSIOLOGY, PHARMACOLOGY AND SCOTT RICHEY RESEARCH CENTER AT AUBURN. AT THE COLLEGE OF VETERINARY MEDICINE. HE'S 25 YEARS EXPERIENCE IN FELINE MODELS OF GANGLIOSIDOSIS AND NEUROPATHIC LYSESOMAL STORAGE DISORDERS INCLUDING TIE SACKS AND GM 1 GRANGE GLIOSIDOSIS. DR. MARTIN WILL BE TALKING ABOUT THAT. THANK YOU. >> THANK YOU. DO WE HAVE A POINTER? OKAY. GREAT. GREAT TO BE HERE TODAY AND PRESENT WITH COLLEAGUES THAT I HAVE EITHER KNOWN OR KNOWN OF FOR MANY YEARS AND INTERACT WITH THE FDA AND NIH EXPERTS WHO HOPEFULLY WILL HELP TAKE PROMISING GENE THERAPIES INTO HUMAN CLINICAL APPLICATION SOON. CHET MENTIONED GM 1 GANGLIOSIDOSIS. I'LL STEP BACK AND GO INTO THE BIOCHEMISTRY OF GM 1 GANGLIOSIDOSIS A LITTLE BIT WHICH WILL HELP UNDERSTAND WHAT WE'RE TRYING TO DO IN TERMS OF GENE THERAPY, WHAT IS GANGLIOSIGHOSIS, IT'S A DISEASE OF GAIN GEE OWE SIDE, IT'S A GLYCO LIPID MOLECULE WITH A MOI THE THEY LIVES IN THE PLASMA MEMBRANE. AND HANGING OFF INTO THE EXTRA CELLULAR SPACE IS A SUGAR SIDE CHAIN. LET'S REPLACE THAT ORGANIC DIAGRAM WITH SCHEMATIC DIAGRAM SO I CAN TALK A LITTLE BIT ABOUT THE LYSOSOMAL ENZYMES THAT BREAK DOWN GANGLIOSIDES. THE MOIETY HERE HAS TO BE REMOVED BY ENZYME CALLED GALACTO SIDASE, THE MAMMALIAN COUNTER PART OF BACTERIA LACK Z SO IT DOES THE SAME THING AT THE ACID PH OF A LYSOSOME. IF IF BETA GALACTO SIDASE DOES ITS JOB, GM 1 GAPE GLIOSIDE AND THE MOLECULE BECOMES GM 2 GANGLIOSIDE. GM 2 IS BROKEN DOWN BY HEXOSMINIDASE A, IT DOES ITS JOB AND GETS RID OF THE GALACTOSE AMINE, AND SO ON FOR A SERIES OF LYSOSOMAL ENZYME HYDROLYTIC EVENTS THAT BREAK DOWN THE ENTIRE MOLECULE. IT'S A STEP WISE DEGRADATED PROCESS SO THAT IF THE FIRST STEP DOESN'T OCCUR THE REST IN THE DEGRADATIVE PATHWAY CAN'T OCCUR EITHER. SO SAY FOR SOME REASON BETA GALACTO SIDASE IS NOT PRESENT OR FUNCTIONAL WE END UP WITH GM 1 GANGLIOSIDOSIS, STORAGE OF GM 1 GANGLIOSIDE, TYPICAL PATHOLOGICAL -- TYPICAL PATHOLOGY, THIS SWELLING OF THE INITIAL SEGMENT OF THE AXON CALLED A MEGA NEURORIOT AND OFF THAT ARE SEVERAL ECTOPIC NEURORIOTS CAUSED SHORT CIRCUIT THINK OF IT AS SHORT CIRCUITING OF THE NERVOUS SYSTEM. AT THE ULTRA STRUCTURAL LEVEL THESE LYSOSOMES UNDER ELECTRON MICROGRAPH ARE COMPLETELY FULL OF GANGLIOSIDE THAT FORMS THE MULTI-LAMELLAR OR MEMBRANE LIKE STRUCTURE. IF HEXOSEMINIDASE IS DEFICIENT, SUCH AS THERE, WE END UP WITH DISEASE CALLED GM 2 GANGLIOSIGHOSIS. THERE'S TWO,TY SACKS DISEASE AND GENE 2 ACTIVATOR DEFICIENCY. I WANT TO TAKE THE OPPORTUNITY TO PUT THE DISEASES IN CONTEXT AND CLINICALLY THEY ARE. THEY HAVE SIMILAR CLINICAL PRESENTATIONS AS WELL. FLORIAN EICHLER WHO IS UP NEXT HAS GOT A NICE HISTORY OFTY SACKS DISEASE ESPECIALLY. THIS FELLOW IS PORTER HEATHERLY, HE HAS GM 1 GANGLIOSIDOSIS. THAT'S WHAT I'LL SPEND MOST OF MY TIME TALKING ABOUT TODAY. PORTER LIVED ABOUT FIVE MILES FROM MY HOUSE. THIS IS PORTER WHEN HE WAS A HAPPY RELATIVELY HEALTHY YOUNG FELLOW. THEN PORTER AT ABOUT THREE YEARS OF AGE WITH -- AND RECLINING STROLLER WITH OXYGEN 24 HOURS A DAY ON MULTIPLE SEIZURE MEDS TO PREVENT AS MANY SEIZURES AS POSSIBLE. KIDS DEVELOP NORMALLY UNTIL SIX OR EIGHT MONTHS WHICH POINT THEY MISS DEVELOPMENTAL MILESTONES OR THEY LOSE PREVIOUSLY ACQUIRED SKILLS SUCH AS THE ABILITY TO ROLL OVER, ABILITY TO SIT. FROM THAT POINT IT'S JUST A CONTINUOUS SLOW PROGRESSION TO THE SEMIVEGETATIVE STATE, LIVE TO ABOUT FOUR YEARS OF AGE. THIS TIME LINE IS HERE, NOT TO GO OVER THE ENTIRE TIME LINE OF DISEASES BUT TO SAY THATTY SACKS DISEASE WAS FIRST REPORTED IN 1881 BY BRITISH OPHTHALMOLOGIST. SO WE HAVE KNOWN ABOUT THESE DISEASES SINCE THE CIVIL WAR. TODAY THE BEST THING THAT WE CAN YET DO FOR THE KIDS IS TO PUT A STOMACH TO THEM SO THEY DON'T ASPIRATE -- STOMACH TUBE. THEY HAVE SWALLOWING DIFFICULTIES. THAT IS TOTALLY UP ACCEPTABLE AND ALL YOU HERE AGREE WITH THAT. SO WHAT ARE WE GOING TO DO ABOUT IT? WE HAVE TWO PROGRAMS WE ARE WORKING ON, ONE ON GM 1, ONE ON GM 2. WE ARE TALKING GM 1 TODAY. WE DEVELOPED AN INTRACRANIAL APPROACH TO TREATING IT WITH AAV AND THEN INTRAVASCULAR APPROACH THAT I WILL FINISH UP MY TALK WITH TODAY. THIS IS THE VECTOR WE DECIDED TO USE IN OUR INITIAL PROOF OF CONCEPT STUDIES FOR THE INTRACRANIAL INJECTIONS FOR GM 1. WE USE CAG PROMOTER DEPENDING WHO YOU TALK TO. AND SPECIES SPECIFIC BETA GALACTO SIDASE WE USE FELINE CDNA IN THE CATS, MOUSE CDNA AND THE MICE SO FORTH. WE INCLUDED A WPR ELEMENT IN THIS INITIAL VECTOR, I DON'T THINK IT'S NECESSARY IN EVERY CONTEXT BASED ON WORK WE HAVE DONE BUT IT CERTAINLY INCREASES EXPRESSION LEVELS IN SOME CASES SO WE INCLUDED IN OUR PROOF OF CONCEPT VECTOR. WE DID BILATERAL INJECTIONS OF THE THALAMUS AND THE DEEP CEREBELLAR NUCLEI, BOTH STRUCTURES ARE WELL CONNECTED TO OTHER PARTS OF THE BRAIN AND SPINAL CORD SO WE THOUGHT WE MIGHT BE ABLE TO SET UP A CENTRALIZED DISTRIBUTION NODE FOR ENZYME DELIVERY TO OTHER PARTS OF THE BRAIN BY ADJUSTING THOSE STRUCTURES BILATERALLY. 16 WEEKS AFTER TREATMENT WE LOOK AT RESTORATION BETA GALACTASE ACTIVITY THROUGH THE BRAIN. WE CUT THE BRAIN IN SIX MILLIMETER BLOCKS FROM FRONTAL CORTEX TO THE CEREBELLUM, LAID OUT CORONALLY AND TOOK HALF THE SECTIONS AND STAINED FOR ENZYMATIC ACTIVITY. EVERYWHERE YOU SEE BLUE IS A FUNCTIONAL EVIDENCE OF BETA GALACTASE ACTIVITY, NOT EVIDENCE OF PROTEIN BUT BETA BLACK CO-SIDASE ACTIVITY. THIS IS THE NORMAL CAT BRAIN, NICE DISTRIBUTION OF ACTIVITY. UNTREATED CAT BRAIN WITH A SLIGHT LITTLE BIT OF RESIDUAL ACTIVITY IN THE WHITE MATTER THAT EQUATES TO ABOUT 3% NORMAL. THROUGHOUT THE BRAIN FROM THE FRONTAL CORTEX THROUGH THE CAUDAL CEREBELLUM WE SAW RESTORATION OF BETA GAL ACTIVITY RANGING FROM TWO TIMES NORMAL TO AROUND SEVEN TIMES NORMAL. THIS IS AFTER A SINGLE INTRACRANIAL SURGERY IN THE CATS TAKES AN HOUR 15 MINUTES. THE CATS WAKE UP FROM ANESTHESIA, GO BACK IN THE ROOMS WITH THE MOTHERS AND NEVER LOOK BACK. WE HAVE A LOW RATE OF INTRASURGICAL COMPLICATIONS FROM THIS APPROACH. WE DID NOT SPECIFICALLY TARGET THE SPINAL CORD WE LOOKED AT THAT TIME SPINAL CORD, FOUND ENZYMATIC ACTIVITY FROM THE LOSTAL SEARCHCAL REGIONS OF THE SPINAL CORD, AT LEVELS RANGING FROM TWO TIMES NORMAL TO SEVEN OR EIGHT TIMES NORMAL. WE THEN WANT TO LOOK AT STORAGE LEVELS IN THE TREATED CAT BRAIN. AND THAT'S WHAT THE TOP GRAPH IS SHOWING. LOOKING AT THE FOLD OF NORMAL LEVELS OF SIALIC ACID IN THESE PUNCH BIOPSIES OF BRAIN. SO ONE FOLD NORMAL WOULD BE NORMAL LEVELS OF SIALIC ACID IN DIFFERENT BRAIN REGIONS. YOU CAN SEE THE UNTREATED CATS RANGE FROM TWO AND A HALF TO FOUR TIMES NORMAL LEVELS OF SCIALIC ACID, IN ALMOST EVERY TREATED BLOCK THE LEVELS OF ACID COME BACK DOWN TO ROUGHLY NORMAL. THERE'S ONE CONSISTENT EXCEPTION TO THAT RULE AND THAT IS THE TEMPORAL LOBE WHICH IS D-3. AND IT'S SHOWN NICELY HERE IN OUR PAS SO THESE ARE PAS STAINS OF THE BRAIN. THIS IS UNTREATED GM 1 CAT BRAIN, ALL THAT DARK STAINING IN THE GRAY MATTER IS PAS STAIN, IT'S A RED STAIN BUT WE HAVE DONE BLACK AND WHITE BECAUSE IT'S EASIER TO SEE. IN THE TREATED CAT BRAIN MOST OF THAT STORAGE IS GONE EXCEPT THIS LITTLE POCKET OF THE TEMPORAL LOBE. AND THE TEMPORAL LOBE IS NOT VERY WELL CONNECTED TO THE THALAMUS WHICH IS OUR PRIMARY INJECTION SITE SO MAKES SENSE WE MIGHT NOT CLEAR ALL THE STORAGE IN TEMPORAL LOBE. OTHERWISE WE DID A GOOD JOB OF GETTING RID OF STORAGE LEVELS. SO WHAT DOES THAT MEAN IN TERMS OF SURVIVAL AND THERAPEUTIC BENEFIT TO THESE CATS? THIS IS THE SURVIVAL CURVE, IT'S A VERY NICE TIGHT SURVIVAL CURVE FOR UNTREATED GM 1 CATS WHICH SURVIVED TO EIGHT MONTHS OF AGE IN ANIMALS THAT WERE TREATED WITH EITHER AAV RH 8 OR AAV ONE VECTOR, WE TESTED BOTH CAPSIDS, THE CURVE LOOKS LIKE WITH SEVEN FOLD INCREASE IN LIFE SPAN. U THREE ARE STILL ALIVE AT SEVEN, EIGHT, ALMOST NINE YEARS OF AGE. SO THIS IS AN UNTREATED GM 1 CAT AT SIX AND A HALF MONTHS, ABOUT SIX WEEKS BEFORE END POINT. NOTICE ATAXIA, HIND LIMB WEAKNESS, TREMORS THAT DON'T SHOW UP TOO WELL ON VIDEO. BALANCE DIFFICULTIES, IT'S QUITE A CEREBELLAR PHENOTYPE. WE'RE ALL CAT LOVERS IN MY LAB, THESE GUYS ARE HELD AT LEAST TWICE A DAY AND PETTED, AND THEY'RE AFFECTIONATE, THEY'RE NOT IN ANY PAIN, THEY'RE FRUSTRATED NOT TO PLAY WITH THEIR COME PAD RAYS IN THE CAT COLONY. SO LOOK AT THE OLDEST TREATED GM 1 CAT, 8.7 YEARS OF AGE. THIS IS AMARI, HE WILL TURN AROUND AND REAR UP ON HIS HIND LIMBS. THE REASON THAT'S IMPORTANT IS HIND LIMB WEAKNESS IS FIRST SIGN OF CLINICAL DISEASE SO THOUGH HE HAS SOME HIND LIMB WEAKNESS AND LIGHTLY WIDE BASED HE HAS NO FUNCTIONAL DEFICIT -- NO REAL FUNCTIONAL DEFICITS IN HIS HIND LIMBS. THE NEXT SEGMENT WILL SHOW YOU HE'S PERFECTLY CAPABLE OF WALKING WITH NO BALANCE OR ATAXIA ISSUES. HIS MAIN ISSUE IS WEIGH LOSS. HE HOVERS AROUND 15% WEIGHT LOSS ALL THE TIME SO WITH TOE MAKE SURE HE'S GETTING ENOUGH NUTRITION AND HIS CAGE MATE IS NOT EATING ALL HIS FOOD. I WILL SHOW YOU ANOTHER CAT THOUGH THAT WAS TREATED. SHE'S ABOUT 8.2, 8.3 YEARS OF AGE. HER ISSUE IS DEFINITELY NOT WEIGH LOSS. THIS IS MANAGERS CINNAMON, WE HAD TO PUT -- MRS. CINNAMON. WE HAD TO PUT HER ON A DIET, OTHER THAN THAT SHE'S DOING QUITE WELL. THESE ARE CATS THAT WAVEGUIDED BY EIGHT MONTH OF AGE -- WOULD HAVE DIED BY 8 MONTHS BOUGHT TREATMENT. WE HAD A NICE OPPORTUNITY IN AUBURN WE HAVE A 7 TESLA MRI UNIT SO WE CAN FOLLOW THESE GUYS OVER TIME THROUGH THEIR DISEASE PROGRESSION. SHOWN ON THE FAR -- MY YOUR FAR LEFT IS A NORMAL CAT BRAIN, YOU CAN SEE THE NICE DARK WHITE MATTER, THE HYPOINTENSE WHITE MATTER ON THESE IMAGES SURROUNDED BY LIGHT OR GRAY MATTER. IN UNTREATED GM CAT THAT GRAY OR WHITE MATTER BOUNDARY IS BLURRED BECAUSE WE HAVE MYELIN PATHOLOGY, PRETTY SEVERE MYELIN PATHOLOGY IN LATER STAGES OF THE DISEASE. THESE TWO TREATED CATS AT FIVE FIVE YEARS, THAT GRAY WHITE MATTER BOUNDARY IS WELL PRESERVED, MAYBE JUST A BUILT BIT OF CORTICAL ATROPHY IN THE SIGNAL BUT OTHERWISE THEY LOOK GOOD. WITH DAN AURY'S LAB WE LOOK AT GANGLIOSIDE LEVELS IN CSF OF LONG TERM TREATED CATS BY LIVER CHROMATOGRAPHY MASS SPECTROMETRY. AND FOUND DRAMATIC ELEVATIONS IN UNTREATED CATS, THIS IS NORMAL LEVEL, NORMAL CAT AND LONG TERM TREATED CATS THAT GM LEVEL BROUGHT BACK DOWN TO NORMAL. FINALLY IN THE 40 YEAR HISTORY OF THE BREEDING COLONY WE HAVE NEVER BEEN ABLE TO REPRODUCE AFFECTED GM 1 CATS, NEVER BREED AFFECTED MALES AND FEMALES BECAUSE THEY WEREN'T CAPABLE OF REPRODUCING. SINCE WE STARTED TREATING THE ANIMALS WE HAVE HAD SEVERAL LITTERS PRODUCED BY AFFECTED GM 1 MALE AND FEMALE CATS TREATED WITH GENE THERAPY AND NO EVIDENCE OF GERM LINE GENE TRANSMISSION THAT WE HAVE SEEN LITTERS BORN FROM THESE MATINGS PROGRESS IN TERMS OF NEUROLOGICAL DISEASE, JUST LIKE THE OTHER ANIMALS DO. THEY DIE BY AGO MONTHS OF AGE. THIS IS GREAT. THIS BASICALLY PROVES OUR -- FULFILLS OUR DESIRE THE CONCEPT WE CAN TREAT DISEASE BUT IT'S AN INTRACRANIAL INJECTION SO WE WANT TO TRY TO DO SOMETHING LESS INVASIVE AND DURING THE COURSE OF THE INTRACRANIAL STUDIES WE AND EVERYBODY ELSE IN THE WORLD LEARNED THAT AAV 9 CAN TRANSDUCE NEURAL CELLS AFTER IV INJECTION. SO WE BEGAN THOSE STUDIES IN MICE. I LAB RA -- MY COLLABORATOR MIGUEL ESTEVEZ PUBLISHED THAT WORK AND THIS WORK IS NOT PUBLISHED YET. WE USE AN AAV 9 SAME BASIC BACKBONE WE USED FOR THE INTRACRANIAL VECTOR AND DOSE OF 1.5 E-13 VECTOR GENOMES PER KILOGRAM WHICH IS A MODERATE DOSE COMPARED TO SOME BEING TESTED TODAY. WE LOOK AT STAINING THROUGHOUT THE BRAIN OF SHORT TERM TREATED ANIMALS, THIS IS A 16 WEEK COHORT AND LONG TERM ALLOWED TO GO TO MAIN END POINT WHEN ANIMALS CAN NO LONGER STAND. WE FOUND ACTIVITY AGAIN FROM THE FRONTAL LOBE THROUGH CAUDAL CEREBELLUM AND SHORE TERM PERIOD AND LONG TERM TREATED ANIMALS IN THE SPINAL CORD, SHORT TERM AND SPINAL CORD LONG TERM, WE QUANTITATED THOSE LEVELS OF ENZYME ACTIVITY WE FOUND BETA GAL ACTIVITY RESTORED IN THE CEREBRUM TO SOMEWHERE AROUND 25% NORMAL ALL THE WAY UP TO ALMOST NORMAL LEVELS AND THAT'S THE SHORT TERM GROUP IN ORANGE. LONG TERM GROUP IS RESTORED UP TO TWO AND A HALF TIMES NORMAL. SAME SPINAL CORD LOWER LEVELS IN SHORT TERM GROUP BUT ROUGHLY NORMAL LEVELS, THEN WITH TIME OVER THE LIFE SPAN THAT ROSE TO ABOVE NORMAL LEVELS. THIS IS THE SURVIVAL CURVE FOR IV TREATED ANIMALS. WE DIDN'T HAVE A LOT OF FUNDING FOR THIS, SO ONLY ABLE TO TREAT A COUPLE OF ANIMALS LONG TERM BUT THEY LIVED THREE AND A HALF YEARS COMPARED TO UNTREATED LIFE SPAN OF EIGHT MONTHS FOR OVER FIVE FOLD INCREASE IN LIFE SPAN. FROM AGAIN WITH 7T MRI WE DID SPECTROSCOPY AS WELL. THIS STUDY WHAT WE DID IS LOOK AT SIX VOXELS OF THE CAT BRAIN, SHOWK CEREBELLUM HERE AND FIVE METABOLITES. IN THE UNTREATED COHORTS, WE LOOKED AT TWO MONTHS OLD ANIMALS FOUR MONTH OLD, SIXTY MONTHS AND EIGHT MONTHS, SAW THAT -- INCREASES OVER THAT TIME IN THE UNTREATED ANIMALS, THIS IS EVIDENCE OF GLIOSIS OR INFLAMMATION. NAA DECREASES OVER THAT TIME SPAN, THAT'S AN INDICATION THAT NEURONAL METABOLIC HEALTH IS NOT WHAT IT SHOULD BE. GPC PLUS PCH, RELIABLE MARKER FOR US INCREASES OVER TIME DEMONSTRATING THAT MYELIN IS BEING LOST OR MYELIN INTEGRITY IS BEING LOST. IN THE TREATED ANIMALS THE MYELIN IS RESTORED TO ROUGHLY NORMAL LEVELS, NAA IS NORMALIZED OR FULLY NORMALIZED DEPENDING TO GROUP WE LOOK AT. GPC IS REDUCED TO ROUGHLY NORMAL LEVELS AS WELL SO WHAT DO THESE WHAT DOES THIS MEAN IN TERMS OF THERAPEUTIC AFFECT? WE DECIDED TO LOOK AT BETA FLAK TOE SIDASE IN THESE TREATED ANIMALS, IN THE UNTREATED THERE'S NO BETA GAL ACTIVITY, SHORT TERM PERIOD ANIMALS, THERE'S 50% ACTIVITY AND LONG TERM TREATED ANIMALS TO EIGHTY FOLD NORMAL. ONE MARKER WE LIKE A LOT IS RELIABLE FOR US IS AST BUT NOT IN BLOOD. A, IS,T AND CSF. THERE'S GOOD MARKER OF NEURAL DISEASE PROGRESSION IN THE GM 2 CAT MODEL THAN GM 1 CAT MODEL. UNTREATED GM 1 CATS HAVE DRAMATIC ELEVATION OF AST IN CSF AND SHORT TERM TREATED ANIMAL NOT SO MUCH DECREASE. THEN THE LONG TERM TREATED ANIMALS THAT AST LEVELS COME BACK DOWN TO NORMAL. SO WHAT TREATED GM 1 CATS LOOK LIKE? THIS LADY IS CHRISTINE. SHE'S 27 MONTHS OF AGE HERE, BASICALLY A NORMAL CAT. SHE WILL TURN TO THE RIGHT AND GO INTO HER HIDING PLACE. THEN SHE WILL ALSO STAND UP ON HER BACK LEGS WHICH IS IMPORTANT TO SHOW US HOW STRONG THEIR LEGS ARE. SHE HAS NOT SHOWN ANY SIGNS OF CLINICAL REGRESSION YET. JUST FOR GRINS, I TOLD PEOPLE I SHOW THIS SO YOU CAN SEE ANIMALS ARE VISUAL BUT I LIKE TO SHOW THE LASER POINTER IMAGES. SOMETIMES WE HAVE TO MOTIVATE THEM BY GETTING OUT THE LASER POINTER AND THEY DON'T LIKE IT. HERE SHE GOES. NEED TO REAWE SURE MY FRIENDS AT THE FDA GENE THERAPY DOESN'T CAUSE TOXIC HATRED OF LASER POINTERS. COMPLETELY NORMAL FOR A CAT. SO WHERE ARE WE? WE ARE SCHEDULED FOR THE IV PROGRAM TO BEGIN VECTOR MANUFACTURING AT NATIONWIDE CHILDREN'S HOSPITAL. ON MONDAY. THAT MANUFACTURING IS SUPPOSED TO TAKE FOUR MONTHS. DR. CINDY TIFT AT NIH WILL BE PI OF CLINICAL TRIAL WE HOPE TO BEGIN EARLY 2019. SO WITH THAT, LET ME ACKNOWLEDGE THE ENORMOUS NUMBER OF PEOPLE AND THE HUGE AMOUNT OF EFFORT THAT HAS GONE INTO THIS, A LOT OF PEOPLE AT AUBURN ARE INVOLVED IN GENEROUSLY DONATEDDED THEIR TIME WHEN THEY DIDN'T HAVE TO. MY EXTERNAL COLLABORATORS ARE SO FANTASTIC, MIGUEL ESTAVEZ FROM UMASS IS THE EXPERT AND I HAVE LEARNED A LOT FROM HIM H. FLORIAN EICHLER IS PART OF THE TEAM WHO WILL TALK NEXT AND CINDY TIFT AT NIH AGAIN WILL RUN THE CLINICAL TRIAL SO WITH THAT, I WILL STOP, AND THANK YOU FOR YOUR TIME AND ATTENTION P [APPLAUSE] >> GREAT PLEASURE FOR ME TO INTRODUCE FLORIAN EICHLER. WE MET A WHILE BACK AT CLINICAL RESEARCH FORUM I HAVE GREAT RESPECT FOR WHAT HE'S ACCOMPLISHED FOR CEREBRAL ADRENAL LEUKODYSTROPHY. CURRENTLY ASSOCIATE PROFESSOR NEUROLOGY AT MASS GENERAL AND HARVARD MEDICAL SCHOOL. HIS CAREER DEDICATED TO ADVANCING CARE AND TREATMENT FOR DEVASTATING NEUROGENIC DISEASE. FOLLOWING NEUROGENETICS TRAINING AT JOHNS HOPKINS WITH THE LATE DR. HUGO MOSER AND RESIDENCY IN PEDIATRIC NEUROLOGY AT MGH. GREAT PLEASURE TO HAVE FLORIAN HERE WITH US. >> THANKS SO MUCH, JERRY, IT'S A PLEASURE TO BE HERE AND IN THIS FORUM THAT I FEEL BRINGS A LOT OF MY PASSIONS ALSO TOGETHER AND INJURY YOUR SMA WORK IS CERTAINLY AN INSPIRATION FOR US FOR SEVERAL YEARS. I THOUGHT I WOULD START WITH A LITTLE BIT OF A THOUGHT PROVOKING TITLE, PROGRESS IN THE ABSENCE OF ANIMAL MODEL, HEMATOPOIETIC STEM CELL, THIS IS REALLY THE SITUATION WE WERE IN TEN YEARS AGO TRYING TO EMBARK ON GENE THERAPY FOR CEREBRAL ALD WITHOUT AN ANIMAL MODEL OF THE DISEASE. LET ME ILLUSTRATE WHERE WE ARRIVED. SO DISCLOSURES. A WARD ABOUT CEREBRAL ALD. THIS IS A SINGLE GENE DISORDER, IN ITS MOST DEVASTATING FORM CAUSES INFLAMMATORY DEMONTYIZATION OF THE BRAIN. THOUGH YOU HEARD BEFORE THIS IS LYSOSOMAL DISORDER, IT WAS ON ONE OF THE SLIDES IT'S A PEROXISOMAL DISORDER, SO A DIFFERENT ORGANELLE. THIS GENE ENCODES A PROTEIN THAT SITS ON THE PEROXISOMAL MEMBRANE IMPORTS, VERY LONG CHAIN FATTY ACIDS INTO THE PEROXISOME. IF YOU HAVE MUTATIONS IN ABCD 1 THIS GENE YOU HAVE ELEVATIONS IN VERY LONG CHAIN FATTY ACIDS. HERE YOU SEE THE FIRST ENCOUNTERED DURING RESIDENCY NEUROLOGY AT MGH, PREVIOUSLY A HEALTHY 60-YEAR-OLD BOY PRESENTEDD WITH HYPERACTIVITY OVER SIX TO 12 MONTH PERIOD PUT ON RITALIN DUE TO THAT BUT QUICKLY PROGRESSED, GATE DIFFICULTIES, BUMPED INTO WALLS, LOST AMBULATION, SPEECH, TEN MONTHS AFTER ONSET HE DIED OF ASPIRATION PNEUMONIA. ON MRI YOU SAW THIS DEVASTATING PICTURE OF INFLAMMATORY DEMYELINATION. MORE RECENTLY, A BOY WHO CAME TO ME ALREADY WITH ADVANCED DISEASE, THIS WAS SIX MONTHS BEFORE HE ARRIVED IN MY CLINIC, IN KARATE CLASS AND QUITE PRECOCIOUS. YOU CAN SEE HE WAS UP TO PAR IN ALL HIS TASKS AND FEW MONTHS AFTER HE SAW ME HE WAS NON-AMBULATORY AND I WAS IN A VEGETATIVE STATE. PROGRESSION IS RAPID BUT DISEASE IS NOT A DISORDER OF DEVELOPMENTAL DELAY. THESE BOYS DEVELOP NORMALLY UNTIL THAT INCITING EVENT OF DEMONTYIZATION ENSUES IN THE BRAIN. HOW CAN WE CAPTURE THAT RAPID PROGRESSION? WE CAM UP WITH A NEUROLOGICAL FUNCTION SCALE ENCOMPASSING 25 POINTS ACROSS VARIOUS NEUROLOGICAL DOMAINS. OF THOSE DOMAINS WE CHOSE SIX MOST SERIOUS SYMPTOMS AS THE MAJOR FUNCTIONAL DISABILITIES WE FOCUS ON THAT ARE SHOWN HERE IN BOLD. ONE ADVANTAGE WORKING WITH THIS DISEASE IS MRI IS A VERY GOOD INDICATOR DISEASE PROGRESSION, SIMILAR TO SYMPTOMS PROGRESSES IN UNIFORM FASHION, IT STARTS MIDLINE WITHIN THE SPLEEN OF THE CORPUS CLOSE SUM, SPREADS OUT TO THE ADJACENT PERIVENTRICULAR WHITE MATTER AND YOU CAN ASSIGN A SCORING SYSTEM THE LARGER EXTENT LESIONS THE HIGHER THE UP TO POINT OF 34. WE CALL THIS THE LESS MRI SEVERITY SCORE. HALLMARK OF THE DISEASE SHOWED BEFORE WHICH IS CONTRAST ENHANCEMENT YOU CAN SEE BOYS IN ACTIVE STATE OF THE DISEASE, IF YOU SEE THIS ON MRI, IF THERE'S NO MASS INDICATING THIS IS GLIOBLASTOMA THIS IS X LINKED DYSTROPHY AND YOU HAVE TO SEND OFF LONG CHAIN FATTY ACIDS. WHAT LEADS TO THIS BLOOD BRAIN BAYIER DISRUPTION? THIS IS A UNIQUE FEATURE IN THIS PARTICULAR LEUKODYSTROPHY. SO THIS IS WORK BY TRADITIONAL -- EXAMINE IN MORE DETAIL. SHE FOUND WHEN SHE LOOKED AT AUTOPSY SAMPLES THE AREA DEMYELINATION ENSUED BEYOND THAT AREA, THERE WAS ACTUALLY LEAKAGE BLOOD BRAIN BARRIER WITH FIBRINOGEN AND DECIDED TO INVESTIGATE THIS FURTHER BY TAKING HUMAN BRAIN MICROVASCULAR END THEEIAL CELLS, SILENCING ABCD 1 AND TO HER SURPRISE WHAT SHE FOUND IS DECREASE IN CERTAIN TIGHT JUNCTION PROTEINS SUCH AS UPREGULATION OF ADHESION MOLECULES YOU CAN SEE NICELY THE TIGHT JUNCTION PROTEINS DON'T EXPRESS OF THE CELL SURFACE. I WILL CUT A LONG STORY SHORT. SO YOU ARE ABLE TO INTERPRET LATER STUDIES I'M SHOWING. DYSFUNCTIONAL ABCD 1 NOW ARE NOT EXPRESSING THE TIGHT JUNCTION PROTEINS ON THE CELL SURFACE, YOU HAVE UPREGULATION OF ADHESION MOLECULES SO THEY'RE BINDING TO ENDOTHELIAL CELLS AND CONFERS MIGRATION, AND WE CAN SHOWN IN IN CELL ADHESION ASSAYS. SO WHAT CAN WE DO TO PREVENT THIS RAPID PROGRESSION OF DISEASE? TURNS OUT THE ONLY MODALITY NOW USED FOR 20 YEARS IS BEING ALLOGENEIC BONE MARROW TRANSPLANTATION. THIS WORKS VERY WELL IN THE EARLY STAGES OF THE DISEASE AS ILLUSTRATED HERE ON THE LEFT WHERE YOU SEE THE UNTREATED COURSE IN RED, FIVE YEAR SURVIVAL COMPARED TO BOYS TREATED WITH EARLY HEMATOPOIETIC STEM CELL THERAPY, THAT HAVE BETTER OUTCOME. WHY DOES THIS WORK? WE THINK IT HAS TO DO WITH THE FACT THIS THERE'S A DISTINCT ZONE OF MICROGLIAL APOPTOSIS THAT IS OCCURRING AROUND THE DEMYELINATING ZONE. I EXAMINED THIS A FEW YEARS AGO AND FOUND THAT IN AREAS WHERE MYELIN WAS STILL RETAINED, THERE WERE MICROGLIAL CELLS THAT WERE DYING IN THE ZONAL DISTRIBUTION QUITE DIFFERENT FROM WHAT YOU SAW IN OTHER DISEASES SUCH AS MS. SO WHAT WE THINK BONE MARROW TRANSPLANTATION IS DOING, IT'S BRINGING IN NOW BONE MARROW DERIVED MONOCYTES INTO THE CNS DIFFERENTIATING INTO MICROGLIAL LIKE CELLS EXPRESSING NORMAL ALD PROTEIN, NORMAL ABCD 1. WHAT ARE THE RISKS OF BONE MARROW TRANSPLANTATION? IT WORKS WELL IN EARLY STAGES HOWEVER, THERE IS ABOUT 10% TREATMENT RELATED MORTALITY, EVEN THIS DAY AND AGE. WE HAVE 40 TO 50% OF GRAFT VERSUS HOST DISEASE SEEN AND SADLY, HALF THE BOYS DO NOT RECEIVE WELL MATCHED RELATED DONOR AND HIGH RISK FOR POOR OUTCOMES. SO SEMINOL PROOF OF CONCEPT STUDY CARRIED OUT BY PATRICK B ORGIN IN 2009 IN PARIS. WHEN PATRICK HAD TWO BOYS THAT CAME TO HIM FROM SPAIN, WHOM DIDN'T HAVE WELL MATCHED HLA DONOR, INSTEAD OF TRANSPLANTING THEM WITH A POORLY MATCHED DONOR, HE WOULD TAKE THEIR OWN BONE MARROW CELLS AND TRAN IF HE CAN WITH LENTIVIRAL VECTOR DELIVERING HEALTHY GENE. HE SAW AT 14 TO 16 MONTHS AFTER THAT PROCEDURE STABILIZATION ENSUED. SO AFTER THOSE RESULTS, WE DECIDED TO PURSUE A SIMILAR APPROACH WITH SELF-INACTIVATING LENTIVIRAL VECTOR DELIVERING A FUNCTIONAL COPY OF ABCD 1 TO AUTOLOGOUS BONE MARROW CELLS. I'LL PRESENT HERE THE RESULTS OF THE STAR BEAM STUDY, PUBLISHED LAST YEAR, OF CEREBRAL ALD. ENROLLMENT CRITERIA IDENTICAL TO THAT FOR CONVENTIONAL BONE MARROW TRANSPLANTATION, BOYS NEEDED TO HAVE EVIDENCE OF ACTIVE CEREBRAL ALD, EVIDENCE OF CONTRAST ENHANCEMENT BUT THEY HAVE TO HAVE EARLY DISEASE. WE'RE NOT ALLOWED TO HAVE A MATCH SIBLING DONOR. WE THOUGHT THOSE BOYS WOULD DO BETTER WITH CONVENTIONAL BONE MARROW TRANSPLANTATION. WE DECIDED TO LOOK AT OUTCOME MAJOR FUNCTIONAL DISABILITIES AND AT IMAGING SCORES AND GADOLINIUM ENHANCEMENT OVER TIME, CLEARLY BECAUSE THIS WAS EXPERIMENTAL TREATMENT, WE WERE INTERESTED IN WHETHER EVIDENCE OF INSERTIONAL MUTAGENESIS OCCURRED. HOW IS THIS PERFORMED? WE MOBILIZE BONE MARROW CELLS WITH GCSF, HARVEST THE CELLS, SELECT THE CD34 CELLS, TRANSDUCE THEM OVER LENTIVIRAL VECTOR DELIVERING HEALTHY COPY OF ABCD 1. BOYS GET A BRIEF AMOUNT OF CYCLOPHOSPHAMIDE, THEN CELLS REINFUSE. BOYS ARE FOLLOWED THEN FOR TWO YEAR PERIOD. IMPORTANT THING TO REMEMBER ABOUT THIS, EVERYTHING HERE IS IDENTICAL TO WHAT YOU WOULD SEE IN A CONVENTIONAL BONE MARROW TRANSPLANTATION WITH EXCEPTION THESE ARE AUTOLOGOUS CELLS SO CELLS FROM THE BOYS THEMSELVES. SO WE ENROLLED IN THE FIRST PHASE OF THE STUDY AS PUBLISHED 18 BOYS ONE WAS INELIGIBLE BECAUSE TOO ADVANCED UNTREATED 17, 11 OF THEM IN BOSTON. AS OF LAST AUGUST WE HAD DONE TOLD 21 NOW, WE HAVE 31 BOYS TREATED TO THE STATE. TO TO THIS DATE. ALL BOYS ARE IN EARLY STAGE OF DISEASE, YOU CAN SEE WITH BASELINE LESS SCORE OF TWO. AND BASELINE NFS SCORE OF ZERO. I'M FOCUSING ON THE FIRST 17 THAT WERE REPORTED LAST YEAR. SO YOU CAN SEE HERE IN THE NEUROLOGIC FUNCTION SCALES THAT THE VAST MAJORITY OF BOYS STABILIZE AFTER TREATMENT WE HAD ONE BOY WHO WAS ALREADY RAPIDLY PROGRESSING IN DETERIORATION AT TIME OF TREATMENT AND SUCCUMB TO DISEASE PROGRESSION. ONE BOY HAD REOCCURRENCE OF GADOLINIUM, DROPPED OUT OF THE STUDY UNDERWENT BONE MARROW TRANSPLANTATION AND DIED AFTER THE SECOND TRANSPLANT. HOWEVER YOU WILL APPRECIATE THIS IS STILL DRAMATICALLY DIFFERENT FROM WHAT YOU SEE IN THE NATURAL HISTORY, THE UNTREATED COURSE WHERE USUALLY THE NEUROLOGIC FUNCTION SCORES ARE RAPIDLY INCREASE AFTER ONSET OF DISEASE. IT IS SIMILAR TO CONVENTIONAL BONE MARROW TRANSPLANTATION IS NOW SHOWN ON THE RIGHT. WHAT HAPPENS TO THE IMAGING? I SHOWED YOU BEFORE THAT USUALLY THE LESION STARTS IN THE SPLEEN OF THE CORPUS CALLOSUM AND SPREADS OUT AFTER ONE AND TWO YEARS THE LESIONS STABILIZED, VERY DIFFERENT FROM WHAT IS SEEN IN THE UNTREATED PATIENT WHERE LESION SPREADS TO CENTRAL WHITE MATTER AND SUBCORTICAL YOUTH FIBERS CONTRAST ENHANCEMENT SEEN RIGHT BEFORE TREATMENT, USUALLY WENT AWAY AT ONE YEAR AND TWO YEAR, AGAIN VERY DIFFERENT FROM WHAT YOU SEE IN THE UNTREATED COURSE WHERE THERE'S USUALLY A GARLAND OF CONTRAST ENHANCEMENT. IS THIS TRUE FOR THE LARGER COHORT? YOU CAN SEE HERE THE LESS SCORES OF THE PATIENTS FROM BASELINE UP TO FOLLOW-UP. YOU CAN SEE HALF OF THE PATIENTS STABILIZE IN LESION SCORES IMMEDIATELY WHILE THE OTHER HALF AFTER ONE YEAR START TO STABILIZE. AGAIN SIMILAR WHAT YOU SEE UNDER CONVENTIONAL BONE MARROW TRANSPLANTATION. WHAT'S PUZZLING FOR US IS THE OCCASIONAL REOCCURRENCE OF GADOLINIUM ENHANCEMENT THAT WE SEE, THE VAST MAJORITY OF PATIENTS DON'T SHOW CONTRAST ENHANCEMENT SIX MONTHS AFTER TREATMENT BUT IN SOME PATIENTS WE DO SEE SOME FAINT REOCCURRENCE OF GADOLINIUM AND YOU SEE HERE SOME ILLUSTRATION OF THAT, IT'S QUITE DIFFERENT FROM WHAT WE SEE AT SCREENING WHERE THERE'S ALWAYS DISCRETE CONTRAST ENHANCEMENT AND QUITE STRONG HERE AT 12 MONTHS, IF IT RECURS, IT'S VERY FAINT AND DIFFUSE. SO DIFFERENT IN NATURE, WE HAVE TO FIGURE WHAT THIS MEANS AND WHAT IS HAPPENING TO THAT ENDOTHELIAL CELLS AND BLOOD BRAIN BARRIER. DID WE GET VECTOR IN AND DID IT EXPRESS NORMAL PROTEIN? YOU CAN SEE HERE VECTOR COPY NUMBER IN PERIPHERAL BLOOD UP TO LAST FOLLOW-UP THEN PERIPHERAL MONOCYTES EXPRESSING IN 20% ABCD 1. WHAT WE FOUND INTERESTING AS FOLLOWING PATIENTS OVER TIME IS THAT PATIENTS TENDED TO DO BETTER IF VECTOR COPY NUMBER IN DRUG PRODUCT WAS HIGHER THAN THOSE THAT HAD LOWER VECTOR COPY NUMBER AND YOU CAN SEE HERE IF PATIENTS THAT SUCCUMBED AFTER TREATMENT COMPARED TO THOSE THEY WERE LIVE AND DOING WELL. NO CHANGE IN NEUROLOGIC FUNCTION COMPARED TO THOSE THAT HAD SOME NEUROLOGICAL SIGNS OR SYMPTOMS. WE THINK IN GENERAL DETERMINANTS ARE LOWER SCORES AT ENTRY INTO THIS TRIAL IN TERMS OF IMAGING AND HIGH VECTOR COPY NUMBER, THOSE ARE THE ONES THAT DO BEST. WHAT KIND OF SIDE EFFECTS OCCURRED? WE DID NOT SEE ANY GRAPH FAILURE GRAPH VERSUS HOST DISEASE, WE DID NOT SEE ANY INSERTIONAL MUTAGENESIS, POLYCLONALTY WAS RETAINED UP TO LAST FOLLOW-UP. WE DID SEE ADVERSE EVENTS CONSISTENT WITH MYELOABLATION AND WHAT WE EXPECT ALSO UNDER CONVENTIONAL BONE MARROW TRANSPLANTATION. SO 17 PATIENT WHOSE COMPLETED 24 MONTHS FOLLOW-UP, 15 REMAIN ALIVE AND FREE OF FUNCTIONAL DISABILITIES. THIS IS ALL IN PUBLICATION LAST YEAR, WE ARE COMING TO THE FIVE YEAR MARK ON THE FIRST PATIENT TREATED SO THAT'S LAND MARK FOR US. NO GRAPH VERSUS HOST AND ADVERSE EVENT PROFILE AS ILLUSTRATED. BUT WE STILL NEED TO FOLLOW THIS AND MAKE SURE THERE'S DURABILITY OF EFFECT. I WILL SAY SIMILAR TO WHAT JERRY WAS SAYING, THIS IS VERY GRATIFYING TO SEE PATIENT POPULATION INITIALLY WHEN I STARTED MY CLINIC WAS COMING IN IN WHEELCHAIRS AND AT DEATH'S DOOR, NOW REPORTING BACK FROM SOCCER CAMP AND DOING WONDERFUL THINGS, HUNDRED YEARS AFTER THE FIRST DESCRIPTION OF CEREBRAL ALD WE HAVE BEEN ABLE TO PUT THE GENE BACK INTO THESE BOYS. I WANT TO STEP BACK A MOMENT AND REFLECT ON THE PURPOSE OF THIS PANEL. WHICH IS TO THINK ABOUT SOME OF THE STRATEGIC CONSIDERATIONS AROUND CLINICAL DEVELOPMENT THE BASIC BIOLOGICAL PREMISE FOR THE STUDY WAS TURN OVER OF BRAIN MICROGLIA FROM BONE MARROW AND FROM THE MONOCYTIC CELLS. AND JUST TO REFLECT ON THAT FOR A MOMENT, MICROGLIA ORIGINALLY EARLY DEVELOPMENT COME FROM THE YOLK SACK. NOT BONE MARROW DERIVED BUT THEN DURING LIFE AND PARTICULARLY UNDER DISEASE, WE DO SEE BONE MARROW CELLS TRAVELING THROUGH BLOOD COMING FROM MONOCYTES MONOCYTES AND CAN DEVELOP INTO MICROGLIA. THAT WAS SHOWN NICELY BY RANSAHOF AND PRINCE AL PRILLER AND OTHERS. THE BIGGEST OPPORTUNITY WAS FOR US IN RECOGNIZING THE CLINICAL RISK IN TRANSPLANTATION. BEYOND -- RECOGNITION OF THE CLINICAL SCENARIO PUTTING BOYS AT HIGHER RISK THAN THEY COULD BE WITH ALLOGENEIC COMPARED TO AUTOLOGOUS DONOR, REALLY OPENED THE DOOR FOR US TO PERFORM GENE THERAPY. I WANT TO JUST HERE SHOW YOU DATA IN THE PRE-CLINICAL REALM THAT LED TO GENE THERAPY TRIAL, IT'S HIDDEN IN THE SUPPLEMENTAL FILES OF THE SCIENCE PAPER FROM (INDISCERNIBLE) FROM 2009 AND IT SHOWS HERE CELLS WHICH ARE EQUIVALENT OF CD34 POSITIVE CELLS FROM MOUSE TRANSPLANTED FROM THE WILD TYPE INTO ABCD 1 KNOCK OUT MOUSE SURVIVE AND ARE -- GIVE RISE TO ABCD 1 POSITIVE MICROGLIA. THIS MOUSE MODEL DOES NOT DEVELOP CEREBRAL DISEASE, NEUROPATHOLOGICAL AND CLINICAL EFFECTS OF LENTIVIRAL GENE THERAPY COULDN'T BE ASSESSED IN THIS MODEL. I THINK IT'S INTERESTING TO SEE THAT IN THE CONTEXT OF THE RISK O BONE MARROW TRANSPLANTATION, THIS WAS REALLY THE ONLY PROOF OF CONCEPT NEEDED TO MOVE FORWARD. WHAT ARE STRATEGIC CONSIDERATIONS FOR CLINICAL DEVELOPMENT? IN OUR CASE SHOWING THE COLLABORATIONS ACROSS ORGAN SYSTEMS ARE USEFUL AND THERE'S INTERPLAY BETWEEN HEMATOPOIETIC SYSTEM AND NERVOUS SYSTEM WAS HUGE. I HAD TO SPEND A DECADE CONVINCING MY NEUROLOGY COLLEAGUE THERE WAS VALUE FROM BLOOD TO BRAIN AND THERE WAS CROSS TALK THERE. STILL SOMETHING THAT IS NOT IMMEDIATELY APPARENT TO MANY. AND WORTHY OF BEING EXPLORED FURTHER. I THINK UNDERSTANDING SELECTIVE VULNERABILITY OF THE ORGAN IS KEY TO UNDERSTANDING THE DISTRIBUTION OF GENE DISTRIBUTION NEEDED. NEEDS A LOT OF UNDERSTANDING OF TARGET CELLS AND STRUCTURE. THAT'S NOT NECESSARILY SOMETHING THAT NEEDS TO BE DONE IN ANIMAL MODELS. AS ONE GOES INTO HUMAN. AND THEN BRINGING TOGETHER ALIGNING PRE-CLINICAL AND CLINICAL EXPERTISE, THAT'S MENTIONED TODAY SEVERAL TIMES. WITHIN MGH AND THE HARVARD ENVIRONMENT WE TRIED TO DO THIS IN CENTER FOR RARE NEUROLOGICAL DISEASES WHERE WE TRY TO BRING TOGETHER VARIOUS INSTITUTES AND PEOPLE WHO ARE WORKING ON CLINICAL TRIALS WITHIN THE NEUROLOGICAL CLINICAL RESEARCH INSTITUTE OR DISCOVERING ARE GENES AT THE BROAD INSTITUTE AND BECOMING A HAVEN FOR THOSE RARE DISEASES THAT HAVE HUGE BIOLOGICAL AND CLINICAL UNMET NEED. SO RARE THAT MAYBE FEW PEOPLE WILL WANT TO ENGAGE WITH THEM. LEVERAGING THE BIOLOGY HERE WORKING WITH PATIENT ADVOCATES WORKING WITH INDUSTRY, BRINGING ALL STAKEHOLDERS TOGETHER YOU CAN MAKE A BIG DIFFERENCE. ONE KEY INCITE WE FOUND IS THAT AS WE LOOK AT THE ENTIRE LAND CAPE FROM BENCH TO BEDSIDE, WHICH IS SUBJECT OF THIS PANEL, WE CAN IDENTIFY NOT ONLY WHAT EXISTS BUT WHAT GAPS MIGHT BE APPARENT. DISORDERS MIGHT HAVE GOOD CELL BASED MODEL SYSTEM MISSING BIOMARKER MIGHT BE A COMPOUND BUT THERE'S NO NATURAL HISTORY. ANOTHER CASE IS EXISTING ANIMAL MODEL BUT THERE'S NO INDUSTRY PARTNER MISSING OUTCOME MEASURES. UNDERSTANDING FROM A PATIENT PERSPECTIVE WHAT IS NEEDED, WHAT THE CLINICAL UNMET NEED IS CAN REALLY HELP DRIVE THIS PROCESS FROM BENCH TO BEDSIDE, FOCUSING ON THE GAPS AND TRYING TO FILL THEM WITHOUT GOING OVERBOARD. IN CONCLUSION I HOPE I HAVE SHOWN YOU SAFETY DATA AND ENCOURAGING EFFICACY DATA FOR THE FIRST GENE THERAPY TRIALS. WE HOPE THIS PARTICULARLY HELPFUL FOR THOSE BOYS LACKING A MATCHED SIBLING DONOR. THERE WAS SUPPORTIVE& PRE-CLINICAL DATA WITHOUT ANIMAL MODEL OF DISEASE AND THAT WAS SUFFICIENT FOR US TO MOVE FORWARD AND WHY WAS THAT SO? BECAUSE RISKS OF CURRENT PRACTICE INFORMED OUR WINDOW OF INTERVENTION AND ROUTE OF ADMINISTRATION. WHILE EACH DISEASE IS DIFFERENT SOME THINGS AROUND THAT ARE GENERALIZABLE. CERTAINLY IN TERMS OF TIMING AND IN TERMS OF EXPERIENCE AND PROCEDURES AND THE RISKS THAT PATIENTS FACE. SO THANK YOU. [APPLAUSE] >> CAN WE INVITE THE SPEAKERS UP AS WELL AS CBER COLLEAGUES. >> WE'LL FOLLOW THE FORMAT THIS MORNING. JUST OPEN UP FOR QUESTIONS TO THE SPEAKERS AND PANEL. THERAPIES AT FDA, SHE RECEIVED HER Ph.D. IN BIOCHEMISTRY FROM THE UNIVERSITY OF NORTH TEXAS. WE ALSO HAVE CBER TEJASHRI PUROHIT SHETH. SHE'S DIRECTOR OF CLINICAL EVALUATION IN PHARMA TALKS OFFICE OF TISSUES AND ADVANCE THERAPIES AT CBER FDA. SHE COMPLETED AN INTERNAL MEDICINE RESIDENCY IN THE NAVY MEDICAL CENTER FOLLOWED BY ALLERGY IMMUNOLOGY AT WALTER REED. FOLLOWING THAT SHE BECAME SERVICE CHIEF OF ALLERGY IMMUNOLOGY CLINIC AT THE NATIONAL NAVAL MEDICAL CENTER IN BETHESDA. WE'LL OPEN THE SESSION FOR QUESTIONS. >>TY HAD A CURIOUS PHI QUESTION FOR JERRY WITH THE EXPERIMENTAL DESIGN OF THE PROTOCOL. OBVIOUSLY THE DATA IS TRANSFORMATIVE AND IMPRESSIVE. IT LOOKS LIKE IN YOUR FIRST COHORT THAT YOU WERE USING THE LOWER DOSE IN OLDER KIDS. THEN YOU SHIFTED TO YOUNGER KIDS IN HIGHER DOSE BUT YOU NOW SHARED WITH US DATA FROM ONE PATIENT, SEEMS TO BE A LITTLE BIT OLDER WITH HIGHER DOSE AND MAYBE DIDN'T RESPOND AS WELL. SO I GUESS CURIOSITY IS WHEN YOU CHANGED IT TO VARIABLES DO YOU THINK YOU'RE AT A DOSE THAT'S REQUIRED OR DO YOU THINK YOU'RE -- YOU MAY BE TOO HIGH, YOU NEED TO GO DOWN IN AGE IN ORDER TO SEE THE MOST SIGNIFICANT BENEFIT? >> COUPLE OF COMMENTS ABOUT THE DESIGN OF THE PROTOCOL WAS FIXED. THAT NEVER CHANGED. ALL S HAD BEENA ENROLLEES HAD TO HAVE DISEASE IDENTIFIED BY SIX MONTHS OF AGE. BUT IN THE BEGINNING BECAUSE OF TIMING AND SO FORTH AND ANXIETY ABOUT AAV GENE THERAPY, WE DIDN'T HAVE A RUSH TO TREATMENT. SO IT TURNED OUT THE OLDER KIDS MORE IN A LESS WELL-DEFINED POSITION TO RECOVER WAS ALREADY OBVIOUS TO THEIR PARENTS THAT THEY WERE ON A DOWNHILL SLOPE AND RAPIDLY PROGRESSING THAT WAY. THEY CAME FORWARD FIRST. THAT'S HOW THEY ENDED UP. ALL THE KIDS IN THE FIRST COHORT WERE SIX MONTHS OF AGE. WHEN WORD SPREAD THAT THERE WAS POTENTIAL HOPE HERE IN THE TRIAL THROUGH SOCIAL MEDIA, NOT THROUGH US, THEN WE STARTED HAVING ENROLLEES AT EARLY AGE, GOT DOWN WE HAD THE TWO BEST PATIENTS IN THIS TRIAL WERE PATIENTS THAT CAME TO US IN UTERO. SO WE FOLLOWED THEM, THAT WAS THE FOUR AND EIGHT WEEK OLD. BOTH OF THEM HAD KNOWN RELATIVES THAT HAD SMA. ONE WAS AN IMMEDIATE FAMILY MEMBER, THE EIGHT WEEK OLE WAS A SIBLING THAT DIED AT EIGHT MONTHS OF AGE, THE OTHER WAS PATIENT WHOSE HAD LITTLE COMPLEX SISTER WHO IDENTIFIED TO BE A CARRIER. SO THAT'S HOW OVER TIME WE HAD BETTER WORD THROUGH SOCIAL MEDIA THAT THIS MIGHT BE WORKING AND WE HAD MORE RECRUITS AT YOUNGER AGE, THAT'S BASICALLY HOW IT HAPPENED. THE PROTOCOL WAS FIXED REGARD TO THAT OLDER PATIENT, THE OLDER PATIENT I BELIEVE, I HAVE TO LOOK AT THE NUMBERS BUT I BELIEVE WAS PATIENT NUMBER 6. IN THE OLDER COHORT. WHAT THAT INDICATES IS WE WERE NOT BEING SELECTIVE. WE TOOK PATIENTS ONE AFTER ANOTHER. THE ONLY THING THAT NEGATED ENROLLMENT WAS ANTIBODY LEVEL TO AAB 9. THEY CAME IN ONE AFTER ANOTHER. THAT PATIENT WHO IS -- WHO DID POORLY INDICATES THAT TIME OF DELIVERY I THINK WE'RE FINDING THAT OUT IN VIRTUALLY EVERY GENE THERAPY STUDY, IT'S A THEME GOING THROUGH THIS ROOM AND THROUGH MY COLLEAGUES HERE, YOU NEED GENE THERAPY AS EARLY AS POSSIBLE IF YOU WANT TO SAVE TARGET ORGAN. >> SO GETS THE QUESTION SOUNDS LIKE YOU SUGGEST TIMING IS MORE IMPORTANT THAN DOSE, DO YOU THINK YOU HIT THE DOSE OR MAYBE HIGHER THAN NECESSARY? I THINK IT'S NOT BEEN ASKED IF AT LOWER DOSE SAME THING WITH A YOUNGER PATIENT. >> TRUTH IS, I DON'T KNOW. BUT THE FACT IS IT'S SAFE, HAS LIVER ENZYME ELEVATION BUT NO OTHER SAE AND WE ADOPTED IT NOW FOR ANOTHER TRIAL IN DUCHENNE. IT'S LOOKING FAVORABLE THAT'S WHAT I CAN SAY AT THIS POINT. >> THANK YOU FOR YOUR NICE TALK. THIS IS JOHN GRAY. IN YOUR SMA TRIAL, YOU HAVE OTHERS GOING ON MORE RELEVANT BUT IN THE SMA TRIAL THERE'S IMMUNOLOGICAL TOLERANCE OR EXPOSURE PRE-EXPOSURE PRIOR TO THERAPY. I WONDER IF YOU CAN COMMENT AND PERHAPS CLINICIAN FDA WITH REGARD TO THIS QUESTION FOR US, RAPIDLY TRANSLATING TRIALS IN THE CONTEXT OF IMMUNOLOGICALLY NULL PATIENT COHORT IS IT NECESSARY TO HIT IMMUNOSUPPRESSION REGIMEN RIGHT OFF THE BAT? HOW DIFFICULT IS THAT GOING TO BE? YOU THINK MIGHT BE POSSIBLE -- COULD YOU PLEASE COMMENT ABOUT YOUR STRATEGY MOVING THERAPIES RA PLI IN CONTEXT OF NOT HAVING EXPLORED THE IMMUNOSUPPRESSIVE REGIMENS WE CAN BE COMBINING WITH THESE PATIENTS, NULL PATIENTS PARTICULARLY. >> I WOULD SAY THAT THE WAY IT CAME TO MY ATTENTION WAS THE VERY FIRST PATIENT WE DOSED WITH AAV IN THE SMA TRIAL ALMOST FRIGHTENING TO ME BECAUSE WE HAD NEVER SEEN LIVER ENZYME ELEVATIONS TO THAT EXTENT. WE HAD AST AND ALT. THIS IS A PATIENT WHO WAS NOT PRE-TREATED WITH STEROIDS, WE HAD LIVER ENZYME ELEVATIONS TO ASD AND ALT IN THE 13 TO 1400 RANGE. I DIDN'T SLEEP WELL THAT NIGHT, SCARED THE HECK OUT OF ME. AND WHAT WERE OUR OPTIONS AT THAT POINT. I DIDN'T WANT TO WAIT FOR ANY FURTHER -- THERE WERE NO CLINICAL MANIFESTATIONS AT THAT TIME. MY GENERAL EXPERIENCE THROUGHOUT I GUESS ALONG DURATION OF CLINICAL TRIALS WAS THAT WE NEED TO SUPPRESS LIVER ENZYME ELEVATION. I CAN IMAGINE THERE WERE FOCAL AREAS OF INFLAMMATORY RESPONSE IN THE LIVER AT THAT TIME. I DID LEARN ONE THING COME BACK TO IT ABOUT ASD FROM THE BRAIN. BUT COME BACK AND TALK ABOUT THAT. IN ANY CASE, I ELECTED TO TREATED THAT PATIENT. I TREATED THAT PATIENT WITH 2-MILLIGRAMS PER KILOGRAM OF PREDNISONE AT THE TIME AND LOOKING AT EVERYTHING, WE DECIDED THAT IN FAVOR OF MOVING FORWARD WE WOULD PRETREAT WITH ONE PER KILO STARTING ONE DAY BEFORE DOSING. HAVING SAID THAT WE DIDN'T HAVE MUCH PROBLEM, WE HAD LIVER ENZYME ELEVATION THROUGHOUT THIS. CAN I INVARIABLY HONESTLY UNEQUIVOCALLY SAY PREDNISOLONE MANAGED ANY INFLAMMATORY RESPONSE IN THE LIVER? I DON'T KNOW. I WILL HAVE REED COMMENT BECAUSE WE HAVE DISCUSSED THIS QUITE EXTENSIVELY TODAY. THE ONE THING THAT DID -- WHEN WE MOVED TO THE DUCHENNE TRIAL WHAT WE SAW THERE WAS SAME PROTOCOL, THESE PATIENTS DUCHENNE PATIENTS ARE ON MAINTENANCE STANDARD OF CARE IN ONE PATIENT IT WAS VERY STRIKING, HAD MILD LIVER ENZYME ELEVATIONS THAT -- AND PROTOCOL CALLED FOR 30 DAYS OF PREED NISALONE AND WHEN THE LIVER ENZYMES DROPPED BELOW 120 ASD ALT, WE WOULD STOP THE STEROIDS, WHICH WE DID, THAT CHILD SPIKE -- HIS AST AND ALT AGAIN, AND THAT WAS THE MOST CONVINCING EVIDENCE I HAVE. CAN A SAMPLE OF ONE SOLVE MANY CLINICAL PROBLEM? ABSOLUTELY NOT. NOW, I WILL TURN THIS OVER TO REED >> I THINK IT'S A QUESTION OF IS THAT CAUSE AND EFFECT, WHAT'S THE DRIVER? I KNOW THIS HAS BEEN ACTIVELY DISCUSSED THROUGHOUT THE FIELD. OBVIOUSLY THERE'S AN IMPLICATION OF ADAPTIVE RESPONSE. IT APPEARS A DOSE RESPONSE OVERALL BUT EVEN LOW DOSE INDIVIDUAL PATIENTS WILL SEEMINGLY HAVE MAYBE UNCHARACTERISTIC AST ALT ELEVATION. SO I KNOW THE OTHER THING AT THE FIELD IS LOOKING AT POTENTIAL INNATE SIGNALING AND WE KNOW THAT THE LIVER PRIMARY TARGET FOR AT LEAST INITIAL UPTAKE,, INTERESTING POINT IS FOR TARGET THERAPIES EXTERNAL TO THE LIVER, WHERE HITTING EITHER MUSCLE WE HEARD THIS MORNING, HIGH TARGETING WITH A MUSCLE APPROACH. DOSE IN THE RANGE OF 14 DC PER KG IDENTICAL TO THE S HAD BEENA TRIAL SO THE QUESTION BECOMES IS THERE LEVELS WITHIN THE LIVER THAT THEN ALLOW OTHER TARGET ORGANS ESSENTIALLY LIVER DESATURATED AND ALLOWING MUSCLE AND CNS MOTOR NEURONS TO BE HIT EFFICIENTLY. MAYBE THE LIVER IS PRIMARY SITE FOR UPTAKE FOR SEROTYPE AND THEREFORE PRO-INFLAMMATORY MILIEU THAT CAN BE GENERATED, MAYBE NOT JUST SIMPLY ADAPTIVE T-CELL RESPONSE THOUGH CLEARLY MULTIPLE GROUPS HAVE SHOWN RECALL RESPONSES TO AD CAPSID BUT THE VECTOR GENOME ITSELF THROUGH INNATE TOLL 9 SIGNALING SHOULD HAVE AN EFFECT TO CREATE A MILIEU THAT IS PRO-INFLAMMATORY. SO THAT'S ESSENTIALLY WHAT AGAIN, I THINK WE WERE DISCUSSING. >> HI. MY NAME IS ERIC HARTMAN, I'M DIRECTOR OF ADVOCACY FOR THE RESEARCH FOUNDATION. WE ARE VERY -- WE ARE A NEGATIVE X LINK CHAIN THAT LEADS TO PROGRESSIVE LOSS OF PERIPHERAL VISION NIGHT BLINDNESS AND ULTIMATELY BLINDNESS. WE ARE FORTUNATE TO HAVE AMAZING RESEARCHERS THAT OVER THE LAST 18 YEARS OF FOUNDATION HAVE BEEN ABLE TO FUND AND WE HAVE HAD NUMEROUS NATURAL HISTORY STUDIES UNDERWAY. WE HAVE TWO GENE THERAPY TRIALS UNDERWAY. A THIRD ABOUT TO START. MY QUESTION MAYBE TO DR. WHITLEY AND TO ALL OF YOU, WHEN WE SPEAK ABOUT TRYING TO ADVANCE RARE DISEASE THERAPIES, GENE THERAPIES OR SITE TRANSPLANTATION THERAPY, ALL THESE THERAPIES THAT ARE OUT THERE, THEY'RE REQUIRING NATURAL HISTORY STUDIES. I HAVE BEEN IN NATURAL HISTORY STUDIES FOR 12 YEARS, I HAVE NO ACCESS TO THAT DATA. THE DUPLICITY AND THE COST OF EVERY TIME NATURAL HISTORY STUDY STARTS, I HAVE GOT TO GO BACK TO FULL BASELINE THOUGH I COULD GO TO FIVE OR SIX DIFFERENT ACADEMIC INSTITUTIONS I HAVE BEEN TO ACROSS THE UNITED STATES AND PRESENT THEM WITH A MODEL THAT IS THAT OLD, I NEED TO START AGAIN WITH ZERO. THESE NATURAL HISTORY STUDIES TAKE YEARS. AND WE LOSE OUR VISION EVERY DAY. SO MY QUESTION IS, HOW CAN PATIENTS GAIN RIGHTS BACK TO THEIR NATURAL HISTORY DATA? I'M TALKING OBJECTIVE DATA IN OUR CASE OCT, AUTOFLUORESCENCE, MICROPERIPHERY, MEASURED OBJECTIVELY, THAT OUR PATIENTS CAN MAINTAIN OR AT LEAST GET SO THEY CAN PRESENT WITHOUT NEEDING TO WAY TWO YEARS OR TO THE EXTENT IN OUR CASE, PATIENT POPULATION IS ONE IN 50,000. 6,000 IN THE UNITED STATES. WE ONLY KNOW 10% IN THE VAST MAJORITY OF EYE DISEASE UNDERDIAGNOSIS OF RETINITIS PIGMENTOSA. UNTIL EVERYBODY IS GENOTYPED THEY PROBABLY DON'T EVEN KNOW THEY HAVE IT SO WE'RE DEALING WITH A MINUTE PATIENT POPULATION THAT CAN BEGIN TO GET TO BURN OUT ON HOW MUCH TIME AND EFFORT AND EXPENSE THEY CAN PUT IN TO BEING PARTICIPANT IN NATURAL HISTORY STUDIES AND RECREATING IT EACH TIME. >> GREAT QUESTION. WHAT I HAVE SEEN NCATS NIH MECHANISM, TRY TO GET AROUND THAT PROBLEM BEST WE CAN. THE DATA WE HAVE GOES INTO A DATABASE, AS WE FINISH OUR STUDIES ACTUALLY MOVES TO NIH FUNDED DATABASE THAT DOES BECOME PUBLIC DOMAIN, SO THAT AS I UNDERSTAND, NEVER TRIED TO REQUEST IT, NEVER NEEDED IT CAN BE REQUESTED AND PROBABLY GIVEN FREELY. I THINK IT'S GREAT MODEL FOR THE PROBLEM YOU'RE TALKING ABOUT. I WILL SAY FOR THOSE PEOPLE WHO ARE NOT IN THOSE STUDIES OR PEOPLE IN OTHER STUFFS, THERE'S WAYS AROUND THINGS. ONE OTHER THING INVESTIGATORS USUALLY DO IS PUBLISH DATA. WHAT I HAVE SEEN OVER THE YEARS IS PUBLISHED DATA, SOON AS YOU ASK A QUESTION YOU HAVE TO DEAL WITH TEN NEW QUESTIONS SO THOUGH THE NATURAL HISTORY DATA MAY SEEM TO BE LOST OR DONE OR PUBLISHED, NOT GOING ANYWHERE EVEN INACCESSIBLE THERE'S A GOOD PROBABILITY LED TO THE NEXT QUESTIONS. BUT A THIRD THING TO CONSIDER, YOU CAN GO BACK AND RECOVER DATA. I WOULD BE HAPPY TO SIT DOWN AND TALK ABOUT IDEAS HOW TO DO THAT BUT EACH PATIENT TO SOME EXTENT OWNS THEIR DATA. THE REASON ONE CAN USUALLY NOT GET IT IS INVESTIGATOR IS GONE OR FILE CABINET OR DESTROYED. THE OTHER PROBLEMS ARE THAT IT MAY HAVE BEEN OBTAINED IN A WAY THAT LARGE GROUP NOT GO GET IT. SOMEONE DOES NOT GET MEDICAL RECORDS. IDENTIFY MEDICAL RECORDS, >> WE HAVE DONE THAT. MY POINT IS WE HAVE HAD INDIVIDUALS THAT HAVE TRIED. AND TURNED AWAY BECAUSE QUOTE THAT DATA HASN'T BEEN PUBLISHED YET. PATIENT DATA. OBSERVATIONAL STUDY. NOT PART OF THE CLINICAL TREATMENT. IT'S A NATURAL HISTORY STUDY. UNTIL OUR PATIENTS GET ACCESS TO THAT, THEY NEED TO WAIT FOR -- ACCORDING TO THEM, THE ACADEMIC END IS YOU HAVE TO WAIT FOR PUBLISHING AND IT CAN TAKE YEARS DEPENDING ON THE INSTITUTION. THE STRANGE THING IS, IN ALL THE AGREEMENTS WE HAVE SIGNED DISCLOSURES FOR OUR DATA, THAT SAY, IT GIVES ALL RESTRICTIONS WHEW WE CAN'T GET IT. THE ODD THING IS WE CAN REQUEST OUR DATA TO BE DESTROYED. >> WELL WHEN YOU TALKED ABOUT CONSOLIDATION OF YOUR STUDIES NATURAL HISTORY INTO ONE DATABASE, I'M JUST TRYING TO SEE IF THERE'S SOME WAY PATIENTS AND OTHERS TO BE COGNIZANT WHEN YOU HAVE RARE DISEASE PATIENTS, THERE'S A FINITE AMOUNT OF THEM. AND NOT EVERYBODY IS GOING TO BE ABLE TO PARTICIPATES. MAYBE SOMEWHAT FROM NINDS, NARCS CATS, IF YOU WANT TO COMMENT TOO. >> WE SHOULD BE MOVING TOWARDS PATIENT OPENING THEIR OWN DATA. THIS IS A QUESTION OF CONTENT AND MECHANISM BUT ULTIMATELY PATIENTS SHOULD OWN THEIR OWN DATA. THE SECOND THING YOU MIGHT BE ALLUDING TO WHICH WE SHOULD ALSO TAKE HEAD ON IS HOW MUCH DO WE NEED TO WAIT FOR NATURAL HISTORY DATA OR SHOULD WE HAVE ADAPTIVE TRIAL DESIGNS AND ACTUALLY ALLOWING US TO GATHER NATURAL HISTORY WHILE WE'RE TREATING RATHER THAN PRETENDING LIKE WE HAVE TO WAIT UNTIL CERTAIN AMOUNT OF NATURAL HISTORY DATA ARE COLLECTED. I DON'T THINK THAT'S REALLY SERVING THE COMMUNITY OR PATIENTS AND THERE ARE BETTER WAYS TO DO THAT. IT IS A CHALLENGE IN GENE THERAPY BECAUSE THERE'S SINGLE POST TREATMENT BUT CERTAINLY THIS IS BEING DONE IN OUTSIDE OF GENE THERAPY VERY SUCCESSFULLY NEURONEXT ADAPTIVE CLINICAL& TRIAL DESIGNS. >> I APPRECIATE YOUR INPUT. MY ONE CAVEAT ON THIS, IT DEPENDS ON THE DISEASE, IN OUR CASE WE'RE VERY FORTUNATE, IT'S A VERY SLOW PROGRESSING DISEASE. WHICH IN ORDER TO GAIN MEASUREMENT TAKES A LONG PERIOD OF TIME. WHICH IS PART OF THE PROBLEM. IN ESSENCE ALSO WITHIN THE TRIALS OF SHOWING EFFICACY. AS I APPRECIATE THE INPUT AND IF ANYBODY WANTS TO TALK TO ME AFTERWARDS ABOUT THIS WE CAN ESCALATE THAT FROM MY PATIENT POPULATION. I APPRECIATE IT. THANK YOU. >> THANK YOU ALL SO MUCH. JULIE KERNER, APRO BIO. WE HEARD ABOUT THE IMPORTANCE OF MYELOABLATION BUT THERE'S A LOOT WE NEED TO LEARN. WONDERING IF THE PANEL CAN TALK ABOUT HOW THEY ACHIEVE MYELOABLATION IN THEIR STUDIES AND LESSONS LEARNED AND MAYBE LESSONS LEARNED AT THE NIH AND FDA AS WELL. >> SO THE WADE WE APPROACH MYELOABLATION WAS SIMPLY TO SAY WHAT WAS CURRENT CLINICAL PRACTICE WITH CONVENTIONAL BONE MARROW TRANSPLANTATION AND WE WANTED TO CHANGE ONE VARIABLE AT A TIME. SO WE ADOPTED FULL MYELOABLATION WITH CYCLOPHOSPHAMIDE FOR AUTOLOGOUS GENE THERAPY TRIAL. NOW THAT WE HAVE SHOWN SAFETY OF THAT ITSELF AND NO ENGRAFTMENT PROBLEMS OR GRAFT VERSUS HOST WE ARE NOW GOING TO PROCEED WITH PEELING BACK SOME OF THE MYELOABLATION AND TAKING AWAY CYCLOPHOSPHAMIDE IN SECOND PHASE STUDY. WE SEE IT AS A SLOW ITERATIVE PROCESS, ALWAYS SAFETY FIRST AND TAKING ONE STEP AT A TIME. >> THERESA, CAN YOU MAKE A COMMENT ABOUT THAT? >> I THINK IN GENERAL IF -- IT'S KIND OF CLINICAL PRACTICE FOR MYELOABLATION. I THINK THE CONDITION, DOSE LEVEL AND REGIMEN THAT MEET LIKE YOU HAVE SUPPORTING SAFETY INFORMATION. I THINK THAT'S THE FIRST TO START. I'LL DEFER TO TEJASHRI FOR THE CLINICAL PART. >> I ECHO WHAT YOU HEARD FROM DR. EICHLER FROM MYELOABLATION AND CONDITION THERAPIES. WE ARE WILLING TO ENTERTAIN AND REVIEW NOVEL OR OTHER STRATEGIC IDEAINGS THAT SPONSORS AND STAKEHOLDERS MAY HAVE IN THIS REGARD. >> THANK YOU FOR WAITING FOR MY QUESTION. SAM JACKSON. JUST WANT TO FOLLOW-UP ON THE QUESTION OF THE ELEVATION AND IMMUNOTRANSFERASES THAT WE AND OTHERS AND YOU HAVE SEEN IN THESE TRIALS ASK WONDERING IF YOU HAVE INSIGHTS INTO INFLUENCE OF PATIENT AGE ON POTENTIAL FOR TOLERATION IN THE TRIALS THAT YOU HAVE SEEN. AND MAYBE COMMENT ON THE KINETICS OF THE IMMUNOTRANSFERASE ELEVATIONS. PRESUMABLY RESPONSES TO VIRAL CAPSID, THERE HAS TO BE TIME FOR MHC 1 PRESENTATION OF CAPSID ANTIGEN PRESUMING THIS IS A CYTOTOXIC T-CELL MEDIATED RESPONSE. A LOT IS PUBLISHED ON THIS, WE COULD SPEND AN ENTIRE DAY TALKING ABOUT IT. MAYBE THAT'S A GOOD IDEA. JUST WANT YOUR THOUGHTS ON KINETICS. >> WE'RE SLIGHTLY LIMITED IN THE ANSWER, THE DIRECT ANSWER TO THAT BECAUSE THE ENROLLMENT IS LIMITED NOW TO TWO TRIALS WITH CURRENT DOSE. IT'S THE DOSE THAT HAS VASTLY CHANGED. THE FIRST TRIAL I EVER DID IN GENE THERAPY IS BACK IN 1999 AND WE DID ONE TO THE 10TH VECTOR GENOMES PER KILO. WE WERE WAY OFF THE MARK. OVER TIME WE HAVE GOTTEN MORE AGGRESSIVE, I GUESS AS WE BEGAN TO SEE TRIALS THAT DIDN'T WORK OVER AND OVER AND OVER AGAIN. I THINK DR. AUSTIN AND OTHERS ADDRESS THIS THIS MORNING. AND NOW WE'RE BEGINNING TO SEE EFFICACY SINCE THE 2010s BUT THAT EFFICACY IS DEFINITELY RELATED TO DOSING. IN OUR CURRENT EXPERIENCE WE HAVE PATIENTS WHO ARE BASICALLY FOUR WEEKS TO SIX YEARS OF AGE. AND WE HAVE SEEN ELEVATIONS IN ALL OF THOSE. SO WE DON'T HAVE ANY AGE RELATED PATIENT DOSING AT THIS LEVEL BEYOND THAT. I DON'T KNOW WHETHER THE FDA MAY HAVE DATA INSIGHT THAT WE SIMPLY DON'T HAVE IN ONE CENTER. THERESA. >> I DON'T THINK WE CAN TALK INDIVIDUAL CASES. >> >> EACH CASE MAYBE DIFFERENT DEPENDING ON YOUR PRODUCT, INDICATION. SO I THINK FDA IS WILLING TO HELP TAKE HOLDERS SO IF YOU CAN PREPARE YOUR PACKAGE, COMING ENTERACT, THIS IS PRE-IND, INTERACT COMMUNICATION PRE-IND COMMUNICATION, WE'RE WILLING TO DISCUSS THE DETAIL WITH YOU TO WORK IT OUT. >> BEFORE WE LEAVE THAT, I'M CURIOUS, YOU ASKED THE QUESTION, SO CAN WE REVERSE THE QUESTION TO YOU, MAYBE YOU HAVE MORE DATA. IS THE IF YOU DO IT WOULD BE INFORMATIVE. >> TURN ABOUT IS FAIR PLAY. IN THIS SETTING THE DATA SETS ARE SO LIMITED. WHEN YOU ARE DEALING WITH CLINICAL TRIALS WITH NCIS OF FEWER THAN TEN PATIENTS IT'S CHALLENGING TO MAKE COMPARISONS BY AGE. WITHIN DEVELOPMENT OF IMMUNE SYSTEM THE YOUNGER PATIENTS SHOULD IN FACT HAVE BETTER ABILITY TO TOLLERRIZE FROM ANTIGEN STIMULI. THAT'S HOW THE IMMUNE SYSTEM EVOLVED. ONE POINT THERE MAYBE SOME METADATA EMERGE TO AT LEAST ALLOW US TO ANALYZE WHETHER THAT IS IN FACT TRUE. WHAT IS THE RAMIFICATIONS OF THAT TO PROSPECT OF GENE THERAPY. >> DEPENDS HOW YOU CONCEPTUALIZE WHAT'S GOING ON. I HAVE A SIMPLIFICATION HOW I VIEWED IT SINCE WE ORIGINALLY SAW. WE DELIVER MOST VIRUS TO LIVER. WE GET DELIVERY OF THE GENE TO HE HAT SITES AND CAPSID IS SITTING THERE WAITING TO BE DEGRADED. FOCAL AREAS OF INFLAMMATION RELATING TO DEGRADATION. I DON'T KNOW WHETHER SOMEBODY LIKE CATHY WOULD LIKE TO MAKE A COMMENT ABOUT THAT, THE LIVER WAS THE FIRST PLACE TO IDENTIFY IN -- FOR HEMOPHILIA, THE LIVER ENZYME ELEVATIONS THEY WERE FIRST TO REPORT IT. I WONDER IF SHE HAS AN IDEA ABOUT WHAT MECHANISM IS. >> I THINK YOU SUMMARIZED THE MECHANISM WELL AND MOST STUDIES WHEN WE'RE THE AAV VECTOR IS DELIVERED INTRAVENOUSLY, YOU WILL SEE DOSE DEPENDENT EFFECT AS YOU DESCRIBED, OF A TRANSIENT ELEVATION IN TRANSAMINASES. AND IT TYPICALLY OCCURS SOMEWHERE BETWEEN FOUR AND 12 WEEKS AFTER THE VECTOR GOES IN. AND IF IT'S ALT ELEVATION AFTER THAT, TO ME, IT MAYBE SOMETHING DIFFERENT. MAYBE SOMETHING UNRELATED TO THAT. >> SO UNFORTUNATELY WE JUST HAVE TO CLOSE THIS SESSION RIGHT NOW. IT'S 2:30. I THINK WE'LL RECONVENE AT 2:45. MANUFACTURING QUALITY. THANK YOU FOR THIS SESSION. >> THANK YOU VERY MUCH. WE HIT THE MARK HERE WITH A BROAD GROUP OF TOPICS AND TRANSLATION ISSUES. THANK YOU. [APPLAUSE] WE ARE NOW MOVING TO SESSION 3, QUALITY AND MANUFACTURING. CHAIRS DR. JUDE SAMULSKI ANDR- DR. ANDREW BYRNES. DR. SAMULSKI IS PROFESSOR PHARMACOLOGY AND GENE THERAPY AT THE UNIVERSITY OF NORTH CAROLINA SCHOOL OF MEDICINE. HE RECEIVED HIS Ph.D. IN MOLECULAR BIOLOGY FROM FLORIDA GAINESVILLE. HE HAS MANY FIRSTS BY HIS NAME, THE FIRST TO CLONE AAV, THE FIRST TO DO AAV GENE THERAPY IN THE BRAIN IN HUMANS. THE FIRST TO WIN SOCIETY ACHIEVEMENT AWARD, FIRST TO DEVELOP SUBCOMPLIMENTARY VECTORS AND MANY OTHERS THAT I'M SURE THAT I DIDN'T MENTION. ALSO I WOULD LIKE TO INTRODUCE DR. ANDREW BYRNES, CHIEF OF GENE TRANSFER IMMUNOGENICITY BRANCH, DIVISION OF CELLULAR AND GENE THERAPIES AT FDA CBER. HE HAS 18 YEARS EXPERIENCE IN FDA IN REVIEW AND MANUFACTURING OF GENE CELL THERAPIES, AND OTHER PRODUCTS FOR CLINICAL USE. HE HAS BACKGROUND IN VIROLOGY AND GENE THERAPY. AND HIS LAB RESEARCH FOCUS ON ADENOGENE THERAPY AND RODENT MODELS. SO THANK YOU VERY MUCH. >> OKAY. TODAY YOU CAN SEE THE LIST OF SPEAKERS, WE GOT KEN CORNETTA, JAYSSON, JOSH AND RICHARD. WHAT WE WANT TO ACHIEVE IN THIS SESSION IS WE DID A SAMPLING ACROSS ACADEMIC INTO BIOTECH TO THE CMO SO THAT WE CAN FLESH OUT THE CONCERNS THAT EVERYBODY IS EXPERIENCED IN ORDER TO BRING THINGS FORWARD. I WANT TO SHOW THE NEXT SLIDE BECAUSE I THINK WHAT'S REALLY GOING TO HAPPEN IN THIS SESSION IS WHAT WE REFER TO IS REVENGE OF THE NERDS, WE'RE ALL BACK. PEOPLE WHO DON'T APPRECIATE THE PRODUCTION GUYS, THEY'RE THE ONES THAT HAVE ESSENTIALLY CARRIED THE FIELD FOR THE LAST 20 SOMETHING YEARS. IT'S CYCLING BACK THAT ONCE WE GET THESE RESULTS IT TURNS TO THE QUESTION HOW WE MAKE IT TO TREAT PATIENTS. WE ARE QUITE AWARE WE'RE DEALING WITH WHAT WE REFER TO AS ROOT CAUSE THERAPY. THIS IS SOMEWHAT PERPLEXING BECAUSE WE NOTED THE DEFECT, WE KNOW THE GENE, ALL WE HAVE TO DO IS GET IT BACK. LIKE LOCATION, LOCATION, LOCATION. AND DELIVERY, DELIVERY, DELIVERY, WHICH TRANSLATES TO PRODUCTION. I THINK WHAT I WANT TO FRAME FOR YOU IS MOST OF THE EXPERIENCES THAT ARE COMING INTO THIS ARENA, HAS 20 YEARS OF VECTOR DELIVERY INTO ANIMALS. WHAT THIS SHOULD APPROPRIATELY BE PHRASED IS WHEN YOU STARTED USING VECTORS AND AS YOU WENT FROM SOMEONE WHO MADE SUPER BADGE THAT WORKED REALLY WELL BUT COULDN'T REPRODUCE, EVENTUALLY STARTED FINDING OUT THAT YOU HAD TO SETTLE FOR SOMETHING IN BETWEEN. WE WENT THROUGH THE BLUE FADES OF PICA SEW, THE CHLORIDE DAYS WHERE SESIUM WORKED BETTER THAN MANNITOL GETTING ACROSS THE BLOOD BRAIN BARRIER UNTIL WE STARTED TO FIND OUT WE HAD TO PURIFY THE AGENTS. AS THE FIELD REGRESSED WE GOT TO THE POINT WHERE DOING ALL THIS DEVELOPMENT IN RODENTS AS FORCED FIELD TO TRANSITION TO REGULATORY COLLEAGUES AND WHAT DO WE DO NEXT. TOX BIODISTRIBUTION. YOU SEE COLLECTION OF STUDIES, SPAN FROM RODENTS TO PRIMATES. BUT THIS ALSO REQUIRE THAT THE PRODUCTION TEAMS HAD TO STEP UP AND DECIDE HOW TO GENERATE ENOUGH VECTOR INSTEAD OF TREATING THE BRAIN OF MOUSE WE'RE TREATING BRAIN AND DOSES AS YOU HEARD IN SOME OF THE OTHER PRESENTATIONS THAT HAVE GONE ON EXPONENTIALLY. AT THIS POINT WE TRANSITION TO WHAT'S CALLED GOOD LABORATORY PRACTICES OR GLP. AND SOME SETTINGS, THIS IS NOTHING MORE THAN DOING LARGE SCALE OF THE SAME RESEARCH GRADE WE WERE DOING BEFORE. ULTIMATELY WE'RE ASKED TO BRING THINGS INTO CLINICAL TRIALS WHERE GMP IS BEING REQUESTED BUT I THINK THE VALUE AND OF THE GENE THERAPY IS WE MAY NOT GO THROUGH AS MANY PHASES AND MAYBE AS SIMPLE AS PHASE 1 OR PIVOTAL TRIAL THAT THEN MAKES SO CALLED VECTOR READY FOR GOING INTO PATIENTS AND TREATING. I THINK TODAY IF YOU LOOK AT THE ACADEMIC FACILITIES, THEY'RE NOT GNP FACILITIES, THEY'RE GNP LIKE AND THEY FOLLOW PROCESSES AND WE HOPE TO HEAR FROM THE SPEAKERS, THE DIFFERENCES BETWEEN WHEN YOU TRANSITION TO THESE VARIOUS PHASES. THIS GIVES YOU IMPRESSION OVER TIME HOW LONG THE COMMUNITY TRANSFORMATIVE THERAPIES TO COME ONLINE. WHEN YOU SEE THESE PROOF OF CONCEPT TYPE OF STUDIES NOT ONLY ARE THEY OVERWHELMING AND IMPRESSIVE BUT THEY ALSO BEG THE QUESTION NOW WHAT QUESTION GOES BACK TO THE PRODUCTION TEAM AS TO WHERE PRODUCTION WITH MEET EXPECTATIONS AND WHAT'S REQUIRED TO BRING THIS TO THE PATIENT. AND THE QUESTIONS WE HOPE TO ANSWER TODAY OR AT LEAST GET DISCUSSED HOW DOES ONE SCALE, WHAT'S -- HOW DO YOU REPRODUCE PRODUCTION PROCESSES? WHAT'S RELIABLE IN RELEASE ASSAYS, HOW AVAILABLE ARE SOURCE MATERIALS. AFTER A WHILE YOU CAN SEE WHEN YOU START ANSWERING THESE HOW YOU DON'T WANT TO TAKE THIS GUY OFF YOUR CHRISTMAS LIST BECAUSE HE'LL END UP BEING YOUR BEST FRIEND WHEN IT COMES TO LARGE PRODUCTION. I'M GOING TO FINAL WILL U CLOSE BY SHOWING YOU OUR PERSONAL EXPERIENCE, IN THE EARLY 2000s WE WERE ASKED TO HELP MAKE VECTOR FOR THE FIRST TRIAL OF AAV IN THE BRAIN FOR CANAVAN'S DISEASE, DONE WITH PAUL LEONE. WHAT U YOU'RE LOOKING AT IN THE RIGHT-HAND LANE IS PURIFIED VIRUS WE MADE BACK IN 2000. IT'S A LITTLE BIT SCARY TO THINK ABOUT PUTTING THAT INTO SOMEONE'S BRAIN BUT BEST COLLABORATORS, THE FDA WE'RE VERY JUDICIOUS SAYING IT'S ALL HUMAN PROTEIN SO WE FEEL SAFE THAT WE SHOULD BE ABLE TO DO THIS ONE. REMARKABLE THING IS THESE PATIENTS SOME ARE OUT OVER 18 YEARS AND THERE'S ZERO ADVERSE EVENTS RELATED TO THE VECTOR. I THINK WITHIN THAT PERIOD OF TIME WE LIKE EVERYONE ELSE LEARNED HOW TO PURIFY VECTORS TO HOMOGENEITY. THIS IS THE STANDARD WHICH EVERYBODY IS GOING FORWARD. SO I THINK YOU CAN SEE THAT JUST LIKE THE YEARS IN THE MICE, THE DEVELOPMENT OF THE PRODUCTION AND CAPABILITIES THAT GO ON IN THE HUMANS HAS EVOLVED LIKE THE VECTORS OVER TIME. THE QUESTIONS WE NOW ARE FACED WITH IS MAKING CELL LINES VERSUS USING PLASMIDS USING BACTERIA SYSTEMS, VERSUS HERPES VERSUS DIFFERENT APPROACHESES THAT ARE NOW BEING DEVELOPED. I PUT THIS SLIDE UP THERE BECAUSE WE HAD A MEETING NOT TOO LONG AGO ABOUT WHAT IT WOULD TAKE TO TREAT SUCH DISORDERS SUCH AS DND. WE HAVE ALWAYS FELT THIS WAS THE HERCULES OF ALL GENE THERAPY PROGRAMS WITH RESPECT TO THE AMOUNT OF VECTOR THAT HAD TO BE MADE. AND USING THE SMA DOSES THAT ARE NOW EFFECTIVELY BEING DONE IN THREE TRIALS FOR DND BY BOTH SEREPTA AND PFIZER YOU CAN SEE IF YOU'RE DOING A DOSE OF TEN TO 14 PER KILO IN YOUR PHASE 1, YOU ARE ALMOST APPROACHING 10 TO 17 PARTICLES THAT CARRY OUT PHASE 1. THAT QUESTION AFTER YOU GET PROOF OF CONCEPT WHAT DO YOU DO NEXT? IT HITS HOME WHEN YOU THINK A NUMBER OF PATIENTS WAITING ON THESE DRUGS THAT MAYBE ON ACCELERATED PATH. AND AT THE MOMENT IF ONE TOOK ON THIS PROBLEM AND WE'RE GOING TO GENERATE VECTOR TODAY, IT'S OVER 10 TO THE 20th PARTICLES. WE THINK OUR COLLABORATORS AT ADVERTISER ARE ATTEMPTING TO CHEAT THIS OBJECTIVE, IT WOULD TAKE 102,000 LITTERS TO MAKE ENOUGH MATERIAL TO TREAT EVERYONE. SO YOU CAN APPRECIATE THAT WHILE PRODUCTION USED TO BE FRONT AND CENTER FELL OFF THE TABLE. WE'RE NOW BACK, BACK WITH A VENGEANCE, WE REALIZE THESE GUYS CARRY ACROSS THE GOAL LINE AS FAR AS MAKING REAL DRUGS. I STOP THERE AND INTRODUCE OUR FIRST SPEAKER, KEN CORNETTA WHO HAS HELD DOWN THE FORT OVER THE LAST 20 YEARS PRODUCING THE LENTIVIRAL VECTORS RETROVIRAL VECTORS FROM THE NATIONAL VECTOR CORE FACILITY IN INDIANA. >> AS JUDE WAS ALLUDING TO I'M VERY OLD. HELLO, EVERYBODY. OVER THE TIME THEY GIVE US QUESTIONS WE'RE SUPPOSED TO TRY TO ANSWER. THEY'RE NEW QUESTIONS SO I'M EXCITED TO DO THAT. FIRST WHEN WE FIRST THOUGHT IT WAS LIKE IT COST HOW MANY? THEN IT WAS IT TAKES HOW LONG? NOW IT'S CAN YOU MOVE ME TO THE FRONT OF LINE? AND THE ANSWER IS NO. I HAVE BEEN INVOLVED WITH RETROVIRAL AND LENTIVIRAL VECTORS SO A TALE OF TWO VECTORS. I WORK AT INDIANA UNIVERSITY. VECTOR PRODUCTION FACILITY STARTED QUITE A WHILE AGO. WE STILL MAINTAIN THE SAME MISSION. THAT IS TO ASSIST INVESTIGATORS IN THEIR INITIAL GENE THERAPY TRIAL. WE FOCUS ON THE RETROVIRAL AND LENTIVIRAL VECTORS WE ARE AIMED AT PHASE 1 AND PHASE 1, 2 TRIALS. THAT'S BECAUSE WE AGAIN, WE WANT TO BE ABLE TO TAKE THE PREDOMINANTLY ACADEMIC INVESTIGATORS, MOVE THEM FORWARD. BUT ALSO OVER THE YEARS WORK FAIR AMOUNT ON SAFETY TESTING RELATED TO THIS AND TRY TO TAKE NOVEL IDEAS THAT FOLKS HAD AND TRY TO THINK HOW WE NEED TO DO THAT TO HELP THEM GET IT THROUGH THE FDA. OUR FOCUS IS ON E VIVO PRODUCTS, NOT THE IND AGENT, IT'S TRANSDUCTION OF VARIOUS CELL TYPES. OUR FUNDING WAS INITIALLY PROGRAM PROJECT GRANT WITH MARY DENOUER, BACK AT IEU IN 1994 TO MAKE VECTOR FOR FOLKS THAT WERE THINKING ABOUT DOING CLINICAL TRIALS IN INDIANA UNIVERSITY. THEN WE WERE SELECTED IN '95 AS ONE OF THE NATIONAL GENE VECTOR LABORATORY PROGRAM. THAT RAN THROUGH 2009. THAT WERE THE RETROVIRAL SITES, WE HAD OTHER SITES FOR PLASMID AND ADENOVIRUS VECTORS AT THE TIME. AAV WAS NOT EVEN BORN AT THIS TIME IN THE SAME WAY. AFTER THAT PROGRAM WAS DISCONTINUED NHLBI HAD A PROGRAM TO MAKE LENTIVIRAL VECTORS THE SCENE THERAPY RESOURCE PROGRAM WHICH RAN THROUGH 2017. WE WERE THE SITE FOR LENTIVIRAL FOR THAT. PRAY. WE WERE ASKED TO TALK ABOUT PROCESS. I USED TO SAY WE WOULD STOP GETTING REQUESTS FOR VECTORS, THAT'S NOT BEEN THE CASE CLEARLY. BUT WE HAVE BEEN DOING IT THE WAY WE HAVE DONE IT MANY YEARS. WE WILL TAKE A PLASMID THE INVESTIGATOR MAY PROVIDE TO US. AND TRANSFECT THAT AND MAKE A SMALL POOL OF VECTOR MATERIAL THAT WE THEN USE TO TRANSDUCE PG 13 CELLS WHICH WILL PACKAGE PARTICLES USING LEUKEMIA VIRUS ENVELOPE. WE'LL ACTUALLY BEFORE WE GO TO A MASTER CELL BANK DO CLONE SELECTION TO WORK WITH THE INVESTIGATORS TO FIND A HIGH TITER CLONE. WE'LL DO THIS ALL THIS WITHIN OUR CLEAN ROOM, WE THEN WILL GO AHEAD FIND OPTIMAL CLONE AND MAKE A MASTER CELL FOR 10 TO 12 VIALS. AFTER WE IDENTIFY THAT, WE DO KNOCK PRODUCTIONS LOOKING FOR OPTIMAL TEMPERATURE AND OPTIMAL TIME TO HARVEST, USUALLY 12 OR 24 HOURS BETWEEN HARVESTS. WE'LL THEN TAKE THAT TO A LARGE SCALE VECTOR PRODUCTION. WE CAN DO FOUR TO SIX DAYS COLLECTING MATERIAL. WE WILL CLARIFY AND FREEZE IT DOWN FOR USE BY INVESTIGATORS. SO ALL MATERIAL WE MADE TO DATE HAS BEEN IN VARIOUS TYPES OF RETROVIRAL VECTOR PRODUCER CELL LINES. EXCEPT ONE WE HAVE DONE TRANSIENT PRODUCTION. WE NOTICED EARLY ON VERY EARLY ON, THAT SOME OF THE EARLY -- CELL LINES WILL HAVE POTENTIAL TO MAKE REPLICATION COMPETENT VIRUS THROUGH RECOMBINATION. BACK IN THE DAY PA 317 WAS USED ON EARLY STUFF BACK IN THE '90s AND ACTUALLY TWO OF TWO WE HAD MADE NOT IN ALL BATCHES BUT SOME BATCHES WE CAN DETECT RCR. SIMILARLY WITH THE GPE 12 SO WE HAVE NOT USED THOSE LINES. NOW WE HAVE MADE OVER 40 PRODUCER CELL LINES WITH THE PG 13 CELL LINE WITHOUT ANY RCR. I THINK MOST INVESTIGATORS NOW HAVE CHOSEN THAT LINE, DIDN'T SPECIFY TO INVESTIGATOR WHICH TO USE BUT THE FIELD IS GENERALLY MOVED TO THAT PACKAGING CELL LINE. IN TERMS OF SCALE, WHAT WE'RE DOING IS IN ROLLER BOTTLES. WE HAVE THE ABILITY TO DO CLOSE CONNECTIONS BETWEEN THE ROLLER MODELS, WHAT'S NICE ABOUT THEM IS YOU CAN USE START WITH MINIMUM VOLUME AND GET HIGHEST TITER AND WE MONITOR GLUCOSE AND THEN INCREASE THE MEDIA OVER THIS FOUR TO SIX DAY HARVEST. WE'RE HARVESTING BETWEEN 3 AND 6-LITERS PER HARVEST, 12 TO 24 HOURS. SO WE GUARANTEE THAT WE'LL GIVE THE INVESTIGATOR A MINIMUM BUT GENERALLY IN 15 TO 20-LITER RANGE AFTER DONE WITH PURIFICATION. EXCUSE ME WITH THE PROCESS. WE DO THIS STEP FILTRATION WHICH IS JUST GETTING CELL MATERIAL, WE DON'T DO ANY SUBSEQUENT PURIFICATIONS AT THIS POINT FOR THE RETRO VIRUS. WE DON'T DO PARTICULAR CONCENTRATION. THAT'S BECAUSE APPLICATIONS THAT ARE ASKING FOR RETROVIRAL VECTORS ARE USUALLY CAR T OR TCR TYPE CANCER RELATED STUDIES AND WE HAVE MADE MATERIAL USED FOR KERATINCYTE GENE TRANSDUCTION. SO LOOKING AT OUR PRODUCT HISTORY, PRIOR TO 2008 WE HAD CERTIFIED 30 VIRAL VECTOR PRODUCTS AND TWO LENTIVIRAL VECTOR PRODUCTS. A LOT OF THOSE EARLY ONES WERE GETTING READY TO BE USED OR NOT USED BECAUSE OF THE DEVELOPMENT OF LEUKEMIA NOTED WITH RETROVIRAL USE IN HEMATOPOIETIC CELLS. A LOT OF OTHERS USED IN CANCER APPLICATIONS SPECIFICALLY TARGETING T-CELLS. SINCE 2008, I LOOKED AND WE CERTIFIED 52 PRODUCTS AND SHOW THIS UP HERE, BETWEEN 2008 AND 2012 WE WERE SPLIT BETWEEN NUMBER OF RETROVIRAL AND LENTIVIRAL PRODUCTS WE MADE. BUT SINCE 2013, WE SEE MORE REQUEST FOR LENTIVIRAL VECTORS TO BE MADE. LOOKING WHO WE MADE THEM FOR, 75% WERE FOR US ACADEMIC INVESTIGATORS. USUALLY FEE FOR SERVICE BUT THROUGH PROGRAMS LIKE GTRP. THAT COME TO US, WE DO COMMERCIAL WORK, 20% FOR COMMERCIAL FOLKS SINCE 2008. THE SECOND VECTORTOR IS LENTIVIRAL PRODUCTION. THIS IS SIGNIFICANTLY MORE COMPLICATED PROCESS WE DO, RATHER THAN USE STABLE CELL LINES THAT MAKE VECTOR WE ARE USING A TRANSIENT TRANSFECTION PROCESS TO MAKE THIS WE HAVE USED FOUR PLASMID SYSTEM USING HEK 293T CELLS, WE'RE USING TRANSFECTION INTO CELL FACTORIES. WE WILL HARVEST MATERIAL, SEND THROUGH PROCESS OF CLARIFICATION AND DEPENDING WHAT THE SPONSOR'S NEEDS ARE, TAKE THROUGH ION EXCHANGE AS ANOTHER PURIFICATION STEP BEFORE GOING TO DIFILTRATION AND CONCENTRATION ON TANGENTIAL FLOW FILTERS, PREDOMINANTLY FOR HEMATOPOIETIC APPLICATIONS, ADDITIONAL PURIFICATION STEP SEEMS TO GIVE HIGHER GENE TRANSFER. FOR OTHER APPLICATIONS SUCH AS CAR T-CELLS IT'S NOT CLEAR YOU NEED TO DO THAT SECOND PURIFICATION STEP. IT DOES MAKE SIGNIFICANT DIFFERENCE? YIELD SO YOU WILL CERTAINLY GET HIGHER YIELD IN MATERIAL WE ARE GENERATING. SO WE'LL COLLECT ONE TO TWO HARVESTS. DEPENDS ON THE NEED. PARTICULARLY ON THE TITER OF THE PLASMID WE ARE STARTING WITH. ACTUALLY THE HIGHER TITERS ARE BETTER WITH TWO HARVESTS IF YOU'RE WORRIED ABOUT YOUR TITER YOU WANT TO DO PROBABLY ONE COLLECTION. IN GENERAL THE SECOND HARVEST YIELD IS 35% OF WHAT THE FIRST IS FROM THE KINETICS OF THE TRANSFECTION PROCESS. YOU ARE GETTING -- YOU WANT THE MAKE SURE WHAT YOU ARE ADDING TO YOUR PROCESS BENEFITS FROM IT. A RANGE FOR PRODUCTIONS OF UNCONCENTRATED MATERIAL STARTS AT 10 TO 60-LITERS. DEPENDING ON INVESTIGATOR NEEDS AND AVERAGE CONCENTRATION REQUEST IS ABOUT 100 FOLD CONCENTRATED. IF YOU GO UP HIGHER AT LEAST FOR US WE FIND THAT YIELD IS PROBABLY WORSE AND IF YOU HAVE A VERY LOW TITER VECTOR YOUR YIELD MAYBE WORSE IF YOU PARTICULARLY TRY TO CONCENTRATE. WE ASK RELEASE TESTING, WON'T GO INTO GREAT DETAIL. THEY'RE OFTEN FOLLOWING THE GUIDANCE THAT WAS PUBLISHED QUITE A WHILE AGO AND GOING THROUGH A VARIETY OF TESTS ON THE MASTER CELL BANK. I THINK THESE ARE GENERALLY PRETTY STANDARD. ONE THINGS THAT ARE PARTICULARLY OF INTEREST IN THE SETTING OF RETROVIRAL LENTIVIRAL IS THESE REPLICATION COMPETENT VIRUS ASSAYS TO MAKE SURE THERE ISN'T ANY VIRUS THAT'S RECOMBINED AND NOW REGENERATEDDED IN THE PROCESS OF YOUR VECTOR PRODUCTION. SAME GOES FOR SUPERNATE HERE THESE RELEASE TESTS DO VARY WHICH TYPE OF METHOD YOU USE, WHICH VECTOR YOU WORK WITH. FOR EXAMPLE RESIDUAL BENZIDASE TO TRY TO CLEAR OUT PLASMIDS WITHIN THE PRODUCTION FOR LENTIVIRAL VECTORS TO TRY TO GET RID OF THEM AS MUCH AS WE CAN. AGAIN, DNA CONCENTRATION IS CURRENTLY BEEN REPORT RESULTS OBVIOUSLY MOVE PAST PHASE 1, MORE AND MORE AN ISSUE. TALK ABOUT VECTOR HURDLES, AT LEAST ON PRODUCTION. UNLIKE AAV WHICH I KNOW THERE ARE LOTS OF PRODUCTION ISSUES BUT WORKING WITH THE MEMBRANE BOUND VIRUS HAS CHALLENGES TOO. THAT IS -- LEADS TO RISKS ASSOCIATED WITH CELLS YOU ARE WORKING ON, HUMAN CELLS, MOUSE CELLS, WHAT MIGHT BE THE CONSEQUENCES OF MAKING MATERIAL THAT IS ACTUALLY BOUND BY THESE MEMBRANE PROTEINS FROM THESE DIFFERENT CELLS. ALSO WE NEED TO TRY TO PRESERVE THE INTEGRITY OF THE MEMBRANES AS MUCH AS POSSIBLE. GENERALLY THESE PARTICLES DON'T TOLERATE MANIPULATION AS WELL AS SOME THINGS LIKE AAV IN TERMS OF PROCESSING. THEY TEND NOT TO BE STABLE SO YOU GO THROUGH PROCESS AS QUICK AS POSSIBLE AND GET THE MATERIAL FROZEN DOWN. WITHOUT LOSING TOO MUCH VIABILITY. THE RETROVIRAL IS MORE PARTICULAR THAN LENTI. BECAUSE OF THAT YOU HAVE CARRY OVER OF SOME COMPONENTS USED IN MANUFACTURING. SO CELLULAR DNA THAT'S GOING TO BE ASSOCIATED WITH THOSE PARTICLES, VECTOR DNA, TRANSIENTS, PLASMID DNAs ARE BOUND WITHIN THE PARTICLE SO YOU CAN USE BENEFIT SHY DAYS BUT EVEN IF IF YOU LOOK WITHIN THE PARTICLES YOU WILL SEE THOSE COMPONENTS MAYBE THERE. AND RAISING PARTICULARLY AS FOLKS MOVE FROM THE EX-VIVO APPLICATIONS TO THE IN VIVO, ONE OF THE POTENTIAL IMMUNOGENIC POTENTIAL OR PROBLEMS ASSOCIATED WITH THE MEMBRANE AND VIRAL COMPONENTS. I THINK FOR THE FIELD, CONTINUE TO HAVE DEBATED TIME FOR STANDARDS, HAVING MORE REFERENCE MATERIALS, RIGHT NOW WE DON'T HAVE DEFINED METHODS FOR EXAMPLE OF TITERRING THIS MATERIAL. AND WHAT IS THE RELEVANCE TO TITER IN APPLICATIONS EX-VIVO? AND WE HAVE INSISTED TITERRING BE DONE BY INVESTIGATOR DOING THE TRIAL BECAUSE WE CAN GET A TITER THAT'S GENERATED IN OUR LAB BUT WE WANT TO KNOW HOW EFFICIENT IS THIS IN TRANSDUCING THE YES, SIR OF INTEREST THAT YOU ARE BECAUSE THAT'S THE CLINICALLY RELEVANT MATERIAL TO KNOW THIS BATCH MADE GIVEN LEVEL OF GENE TRANSFER AT THE APPROPRIATE LEVEL AND IT'S NOT TOO HIGH OR NOT TOO LOW. SO CONTRAST RETRO VERSUS LENTI. YOU SAW TWO MANUFACTURING SCHEMES, RETRO IS CERTAINLY LOWERED COST FOR MANUFACTURING. THERE'S WELL ESTABLISHED METHODS FOR USING THE MATERIAL TO TRANSDUCE T-CELLS AND OTHER ADHERENCE CELLS. PARTICULARLY PG 13 IS WELL ESTABLISHED HOW TO WORK WITH IT AND FOR YOUR BUSINESS, THE TECHNOLOGY AND PATENTS ARE GETTING PRETTY OLD. THAT MAY BE A POSITIVE FOR SOME FOLKS. THE NEGATIVES ABOUT THIS, IS RETROVIRAL -- THERE'S REQUIRES CELLS TO BE CYCLING SO NOT EFFICIENT TO CELLS NOT LIKE HEMATOPOIETIC STEM CELLS AND CLEARLY IN HEMATOPOIETIC CELLS IS A SAFETY PROFILE BECAUSE OF INSERTIONAL MUTAGENESIS AND THE NUMBER WE HAVE SEEN IN A NUMBER OF CLINICAL TRIALS. IN TERMS OF LENTI, THE POSITIVE, THERE APPEARS A LOWER RISK OF INSERTIONAL MUTAGENESIS. IT CEMENTS TO INTEGRATE MORE EFFICIENTLY INTO CYCLING CELLS SO HEMATOPOIETIC FOLKS TURN TO THIS AS MAIN STAY. LOWER RISK OF REPLICATION COMPETENT LENTIVIRUS OCCURRING COMPARED TO WHAT THE RISK IS IN THE REAVOW TRIAL SYSTEM AS I MENTION WELL SUITED TO CONCENTRATION PURIFICATION. THOSE ARE ALL THE POSITIVES. I THINK IF YOU'RE INDUSTRY OR OTHER FOLKS WHAT ARE THE IP ISSUES AROUND LENTIVIRUS VECTORS THAT CAN BE SOMEWHAT CHALLENGING TO TRY TO STORE OUT, SCALE UP IS IN DEVELOPMENT AND IS NEEDED. PACKAGING SYSTEMS THAT WE USE FOR RETRO ARE COMING ONLINE NOW FOR LENTIVIRAL VECTORS. HOW THEY WILL FIT IN IN THE FUTURE WITH SOMETHING WE ARE WAITING TO SEE. I TALKED ABOUT RETROVIRAL MAY IMPACT AND LENTIVIRUS TRANSIENT AND THE FIELD IS CHANGING. SO THERE'S FOLKS ON EITHER SIDE THAT ARE MIXING THOSE THINGS UP. IN TERMS OF METHODOLOGY, IF YOU ARE A NEW ACADEMIC INVESTIGATOR AND THINKING WHICH YOU MIGHT WANT TO USE, THE PACKAGING CELL LINES SEEM TO HAVE IMPROVED CONSISTENCY AND SOME HAVE ARGUED THOUGH NOT SURE I BUY IT COMPLETELY, MORE APPROPRIATE FOR LICENSE PRODUCTS, IN TERMS OF THE MANUFACTURING. THE CHALLENGE WITH THIS, AS YOU MENTION THERE'S CLONE SELECTION, A LONG LEAD TIME TO GET TO CLINICAL TRIAL, THERE'S HIGHER UP FRONT COST IN MAKING A PACKAGING CELL LINE AND PRODUCER CELL LINE. I THINK THERE'S INCREASE RISK FOR RCR, WE DON'T KNOW YET ABOUT RCL BECAUSE YOU JUST CARRYING THESE LINES FOR MANY GENERATIONS, BEFORE MAKING A PRODUCT. FOR TRANSIENT TRANSFECTION IS QUICKEST FOR PHASE 1 TRIAL. FOR EXAMPLE WE HAVE PACKAGING PLASMIDS IN OUR FACILITY WE SEQUENCE THEM SO WHEN YOU SEND PLASMID WE CAN GO AHEAD AND MOVE FORWARD QUICKLY. EXCEPT BIG BACKLOG FOR EVERYBODY. CAN MOVE QUICKLY THROUGH THE PROCESS. THERE'S FLEXIBILITY IMPLANTING CHANGE TO VECTOR DESIGN AND HAVING WORKED WITH ACADEMIC INVESTIGATORS, WHO DO ALL THIS WORK AND SELECT CLONES AND SAY I WANT TO USE DIFFERENT PRONOTTER, I CHANGE THIS. YOU HAVE DONE ALL THIS WORK AND NOW YOU WANT TO CHANGE IT WHERE IF YOU HAVEN'T MADE YOUR BATCH OF LENTIVIRAL VECTOR YET, YOU CAN SWAP OUT THE PLASMID AND BRING FORWARD AND MAKE CHANGES IN YOUR PROCESS. IT IS TECHNICALLY CHALLENGING TO PERFORM THESE TRANSIENT TRANSFECTIONS TO SCALE THEM UP. BECAUSE OF THAT PROCESS THE WAY CLLS ARE AT THE END OF PRODUCTION YOU NEED CLEAN UP TO GET A PRODUCT YOU WANT TO USE TO TREAT CELLS. I THINK GOING FORWARD I SEE FOLKS STRUGGLING, BALANCING THE -- TODAY AND TOMORROW. FOR A LOT OF PHASE 1 TRIALS, AGAIN A LOT OF ONES WE ARE MAKING ARE FOR CANCER, NOT NECESSARILY RARE DISEASES, CURRENT METHODOLOGY GET FOLKS THROUGH PHASE 1 TRIAL. LOWER COST AND I USE TO WORK WITH ACADEMIC FOLKS WHO MAY HAVE LIMITED BUDGET WE ALSO REALIZE THERE'S ACCUMULATING CLINICAL DATA NOW FOR CURRENT PRODUCTION METHODS. SO THERE'S A TRACK RECORD NOW IN THESE METHODS BEING USED. THOSE ARE SOME OF THE POSITIVE BUT WE HAVE TO REALIZE CLEARLY BETTER WAYS TO DO THESE THINGS. THERE'S A LOT OF WORK WITH BIOREACTORS AND SCALE UP METHODS, HOW MUCH YOU WANT TO INVEST IN THAT IN YOUR PHASE 1. PRODUCER CELLS LINES ARE THERE FOR LENTIVIRUSES, HOW MUCH TO INVEST IN THAT. A BALANCE MORE IMPORTANT TO GET PHASE 1 DONE QUICK WITH MINIMAL COST BUT THAT'S MAY HAVE IMPLICATIONS DOWN THE ROAD OF YOU GOING TO PHASE 3 AND TO LICENSE PRODUCT GO BACK AND REDO PART OF THAT WITH BRIDGING STUDY BECAUSE MANUFACTURING IS NOT RELEVANT AND SAFETY CONCERNS THAT MAYBE RAISED BY AGENCY OF YOUR MATERIAL. SO MAJOR HURDLES FOR ACADEMIC PRODUCTION CENTER IS ONE OF THE QUESTIONS WE WERE ASKED TO ADDRESS. FOR US WE HAVE BEEN TALKING WITH FOLKS IN INDUSTRY TO SEE ABOUT COLLABORATION. SO THAT CAN WE TRY TO HAVE A SYSTEM WHERE WE ARE NOW ABLE TO STREAMLINE FOLKS IF WE ARE MAKING SOMEBODY FOR THEIR PHASE 1 TRIAL AND IT'S BOUGHT UP BY A COMPANY AT SOME POINT, THERE'S MORE STREAMLINE MANUFACTURING, SO THERE AREN'T QUESTIONS THE MANUFACTURING HAS CHANGED SIGNIFICANTLY, THAT LEADS TO SAFETY QUESTIONS ABOUT THE AGENCY THAT MAY SET FOLKS BACK. I THINK THERE'S AN OPPORTUNITY FOR EXCHANGE OF EXPERIENCE AND INNOVATION BETWEEN INDUSTRY AND ACADEMIC CENTERS THAT HAVE BEEN WORKING AS PRODUCTION SITES OVER THE PAST 10, 20 YEARS. I THINK FOR US TOO, GONE AWAY. I DID TALK TO NCATS ORGANIZATION THAT'S SUPPOSED TO HELP INVESTIGATORS, DAY WILLIAMS AND DON CONE HAVE THIS GRANT, WHERE ARE YOU SENDING FOLKS TO? YOU SAY THAT'S A REALLY HARD QUESTION BECAUSE BECAUSE THE FACILITIES AT NIH HAVE BEEN FUNDING ARE REALLY NOT BEING FUNDED ANY MORE. SO WHERE ARE FOLKS GOING TO GO, AS FOLKS MOVE FORWARD THAT'S AS WE ARE LOOK AT PRODUCTION CENTER HOW WE CHANGE OUR MODEL, ARE WE GOING TO HAVE TO DO MORE INDUSTRY WORK, WHAT DOES THAT MEAN FOR THE INVESTIGATORS THAT WE WORKED WITH ALL THE TIME. FINALLY MEETING THE EXPECTATIONS OF INDUSTRY AFTER THE FACT, I PUT THAT IN THAT -- WE HAVE HAD NOW FOLKS WE MADE VECTOR FOR TEN YEARS AGO. AND NOW SOME COMPANY BOUGHT THE VECTOR AND QA WANTS TO RUN AND WE'RE SPENDING TIME, WHICH IS FINE. WE LEARN SOMETHING EVERY TIME QAs COME. IT'S NOT A GREAT SYSTEM TO WORK THAT WAY, GREAT THAT FOLKS, MATERIALS ARE BEING LICENSED AND TAKEN FORWARD BUT THAT PUTS MORE WORK ON FOLKS, THERE ARE IN TERMS OF JUST QA WORK WE'RE BUSY TRYING TO MAKE THAT FOR FOLKS. AND I THINK THERE'S CHALLENGE TO THE ACADEMIC INVESTIGATOR. ADDING COST CAN COMPLICATE THE GRANT REVIEW PUTTING IT MILDLY. THE CURRENTLY THE PRODUCTION CENTERS ARE BUSY, SO ACADEMICALLY LOSE PREFERRED STATUS, IS THAT GOING TO COME UP WITH AS WE THINK ABOUT HOW WE KEEP OUR PLACES OPEN. CHALLENGE OF ACADEMIC INVESTIGATORS AND THEY CAN BE CHALLENGING AT TIMES. ACADEMIC INVESTIGATOR AFFORD CMO PRICING WHAT I THINK THERE'S CONCERN IS THERE'S A CENTRALIZATION, A COUPLE OF UNIVERSITIES WHO MAY HAVE THEIR OWN PRODUCTION SITE THAT WILL BE THE ONE TO DO IN GENE THERAPY, IF YOU DON'T HAVE THAT THEN YOU ARE NOT GOING TO BE ABLE TO FIND SOMEBODY TO MAKE YOUR MATERIAL FOR YOU AND THAT'S GOING TO BE A CHALLENGE FOR FOLKS THAT ARE DOING REALLY GREAT WORK BUT THEY MAYBE IN A SMALL UNIVERSITY THAT DOESN'T HAVE THE CAPACITY FOR THAT. I MR. WRAP UP WITH QUICKLY MENTION, THERE'S COUPLE OF GUIDANCES OUT THERE, THAT FOLKS ARE -- FDA PUT FORWARD THAT ARE GOING TO HAVE US RETHINK SOME OF THE THINGS WE'RE DOING, PARTICULARLY AROUND SAFETY TESTING. AND JUST TO LET YOU KNOW WE HAVE BEEN INVOLVED WITH A LOT OF TESTING TO REPLICATION COMPETENT RETRO VIRUSES AND LENTIVIRUSES. IT'S THOUGHT OF IN THREE METHODS, VECTOR PRODUCT NEEDS TO BE TESTED, THE TRANSDUCED CELL PRODUCTS AND THE NEW GUIDANCE WOULD INCREASE THOSE PRODUCTS THAT NEED TO BE TESTED, BECAUSE CURRENTLY IT SAYS GREATER THAN FOUR DAYS BUT EPINOW LESS THAN FOUR DAYS IS POTENTIALLY NEEDING TO BE TESTED WHEN CELLS TRANSDUCE AND INFUSE INTO THE PATIENT. PATIENT MONITORING DOWN THE ROAD. I THINK THERE'S POTENTIAL THAT WE NEED TO THINK THIS AND TALK THIS MORE NOW IN DRAFT, GUIDANCE, PARTICULARLY AROUND TRANSDUCED CELL PRODUCTS, A PAPER THAT WAS ACCEPTED A WEEK OR SO AGO. WE HAD 14 CLINICAL TRIALS LOOKING AT TRANSDUCED PRODUCTS INFUSED FOR REPLICATION COMPETENT RETRO VIRUS, THESE WERE 282 TRIALS, THIS WAS A FOUR WEEK ASSAY, A VERY SENSITIVE ASSAY AND QUITE EXPENSIVE SO IT IS A BURDEN AND MOVING THIS FORWARD IN TERMS OF DEVELOPMENT AND ALL THESE 282 PRODUCTS WERE NEGATIVE FOR RCR SUGGESTING THE SCREEN WE DO ON THE VECTOR PRODUCT MAYBE SUFFICIENT FOR TESTING FOR RCR, AND THIS WAS 240 OF THE PATIENTS TREATED HAD FOLLOW-UP SHOWING NEGATIVE. REPLICATION COMPETENT RETRO VIRUS, STUDY THIS YEAR LOOKING AT LENTIVIRAL PRODUCTS 460 CELL PRODUCTS, 379 PATIENT, T-CELL PRODUCTS TCR STUDIES ALL NEGATIVE ALL REPLICATION COMPETENT VIRUS AND NATIONAL GENE VECTOR REPOSITORY TESTED, WE HAVE A COUPLE OF THOUSANDS PATIENTS MONITORED POST INFUSIO OF GENE THERAPY PRODUCTS BOTH RE RETROVIRAL AND AND ALL NEGATIVE TO DATE SO THANKS TO THE LAB, THANKS TO THE NGBB AND THANK YOU VERY MUCH. [APPLAUSE] >> THANKS, KEN, THAT WAS EXCELLENT. WHILE JAYSSON IS GETTING UP TO THE PODIUM, I WOULD LIKE TO ECHO SOME OF THE POINTS KEN MADE. SO WHEN WE HAVE AN OPPORTUNITY TO DISCUSS THAT MOST IF NOT ALL SETTINGS, IT IS TIME TO HAND THE BATON OFF TO THE COMMERCIAL ENTITIES. BUT I THINK AT THE SAME TIME IF WE END UP WITH A STRUCTURE WHERE IT'S THOSE WHO HAVE VERSUS THOSE WHO HAVE NOT, WE WILL SEE A LOT OF THESE ORPHAN DISEASES NOT GET TREATED OR TESTED BECAUSE OF THE COMMERCIAL VALUE MAY NOT REACH SOME OF THE THRESHOLDS THAT ARE REQUIRED BY THE RULES MOST COMPANY VERSUS TO PLAY BY SO I HOPE CONVERSATION EARLIER WILL BRING THIS BACK IN FOCUS TO DISCUSS. JAYSSON IS HEAD OF THE OHIO STATE GMP FACILITY, RESPONSIBLE FOR MAKING MOST IF NOT ALL VECTOR THAT WAS UTILIZED IN THE STUDIES. BEING DONE OUT THERE AS WELL AS OTHERS AND WILL GIVE A SLIGHTLY DIFFERENT PERSPECTIVE FROM HIS INSTITUTION WHICH IS NOT FUNDED BY THE NIH AS NATIONAL VECTOR LAB THAT RELIES ON GRANTS AND/OR CORPORATE STRUCTURES TO SUPPORT THE FACILITY GOING FORWARD. >> MY NAME IS JAYSSON EICHOLTZ, DIRECTOR OF OPERATIONS FOR NATIONWIDE GMP FACILITY, ALSO MEMBER OF THE FDA MEDICAL DEVICES MANUFACTURING PROCESS ADVISORY COMMITTEE. I HAVE 19 YEARS EXPERIENCE IN INDUSTRY. FOOD, VACCINES, CELL BASED THERAPIES, OVER THE COUNTER LIQUIDS, CAPSULES, GLOBAL SEAL TECHNOLOGY AND GENE THERAPY. I'M A BABY TO GENE THERAPY. I CAME ON BOARD IN 2015 AT NATIONWIDE CHILDRENS. SO I HAD LUXURY OF WALKING INTO A FULLY FUNCTIONING FACILITY MAKING AAV VECTORS. WITH GUIDANCE OF DR. MENDELL, DR. CLARK AND TEAM IN NATIONWIDE CHILDREN'S, THEY HAVE A FACILITY RUNNING, MY ROLE IS TO COME ON BOARD AND OPERATIONALIZE, GROW, MAKE THEM BIGGER FASTER STRONGER. SO OUR CORE WAS DEVELOPED IN 1999, TO SUPPORT NON-HUMAN PRIMATE CLINICAL STUDIES. AND FROM 2000 TO 2009 WE ARE LOOKING AT SEVERAL PROGRAMS TO SUPPORT PRE-CLINICAL STUDIES WE STARTED IN STABLE PRODUCER LINES USING CELL CUBES. YOU CAN SEE TARGET THERE IS AND WE HAVE VARIABLE VECTOR YIELDS AND LONG LEAD TIMES ASSOCIATED WITH THE PROCESS. IN 2007 DR. MENDELL AS WELL AS TEAM AT CENTER FOR GENE THERAPY WENT FORWARD TO ADMINISTRATION TO PUSH FOR CREATION OF A GMP FACILITY TO SUPPORT LAUNCH INTO PHASE 1 THE AVENUES OUT THERE TO MANUFACTURE VECTOR AT THAT TIME WERE LIMITED AND EXTREMELY EXPENSIVE. SO WE ARE LOOKING FOR A WAY TO GET TO PHASE 1 TRIALS AND DO IT IN AFFORDABLE MANNER. IN 2008 DESIGN WORK STARTED AND THE GMP FACILITY WENT ONLINE IN 2009. PART OF THAT COMING ONLINE 2009 WE DID A PROCESS CHANGE WE STEPPED AWAY FROM THE STABLE PRODUCER LINES AND WENT TO TRAININGS CENTS TRANSFECTION PROCESS. THIS IS BACK IN 2009. SIMPLE CONCEPT. 1200 SQUARE FEET, SINGLE PRODUCT FLOW. AND ISO 7 CORE AREA WITH ISO 5 PLACE INSIDE. FROM DETAILS, WEAPON A CELL CARRY SPACE, MAKE SURE SEGREGATION OF CELL LINE. WE HAD MANUFACTURING SPACE, PURIFICATION, THEN WE HAD AREA DESIGNATED FOR FINAL FILL. VERY SIMPLE SINGLE PRODUCT FACILITY. BUT DID THE JOB WELL. THE FIRST PROCESS WE USE, TRANSIENT TRANSFECTION AND WE'RE USING ADHERENT TECHNOLOGY, WE ARE IN TENT STACKS, YOU CAN SEE SCALE THERE, SO WE ARE RUNNING 12, TEN STACKS PER PRODUCTION RUN. AVERAGE YIELD WE ARE GETTING 1.5 E 15 CREW. FROM CELL LYSATES, WE'RE USING MICROFLUIDIZATION FOLLOWED BY CLARIFICATION STEPS, CONCENTRATION STEPS. GRADIANT, CHROMATOGRAPHY POLISHING AND FINAL CONCENTRATION TANGENTIAL FLOW FILTRATION TO GO TO FINAL FILL. THE FIRST LOT WE PRODUCED WAS FOR EU CLIENT. IT WAS IN 2011. WE ARE STILL WORKING WITH THAT CLIENT TODAY. MID 2012 WE MOVED AID WAY FROM THE -- MOVED AWAY FROM THE CELL LYSATE BUSINESS AND GOT TO MEDIA RELEASE. THE COUPLE OF REASONS THAT DROVE THAT DECISION, IT ALLOWED US TO GO FROM MICROPOLARRIZATION WHICH MEANT PROCESS COULD BE 100% DISPOSABLE SINGLE USE. THIS WAS KEY TO US BECAUSE WE DID NOT WANT TO HAVE TO PUSH FOR CLEANING VALIDATION BETWEEN DIFFERENT PRODUCTIONS WE ARE TRYING TO GO THROUGH. IT ALSO REDUCED CELL LOAD AND DEBRIS SEEING IN DOWNSTREAM PROCESSING FOR PURIFICATION. IT'SED UP SOME OF THE BUILD AND LOSS BECAUSE OF THAT DEBRIS. 2013 WE DID ANOTHER TRANSITION. AS I SPEAK, LOTS OF TRANSITIONS, LOTS OF EVOLUTION. WE HAVEN'T WITHIN BEEN ABLE TO KEEP OUR FEET STILL LONG. WE MOVED AWAY FROM TENT STACKS AND STARTED PROCESSING IN THE CORNING 36 LAYER HYPERSTACK. AND THEN WE USE THAT HYPERSTACK TECHNOLOGY TO HELP MANUFACTURE THE SMA PRODUCT WHICH WAS RELEASED OUT OF OUR FACILITY IN 2014 FOR TRIAL. IN 2015, RECOGNITION FROM THE ORGANIZATION FROM THE HOSPITAL AS WELL ADS CENTER FOR GENE THERAPY, THAT TO MEET THE VOLUME DEMANDS WE ARE SEEING, FOR SYSTEMIC DELIVERY WE NEED A LARGER FACILITY, WE NEED MORE CAPACITY, PROCESS MORE AND GET MORE VECTOR OUT. WE DIDN'T WANT TO BE THE BOTTLENECK. SO PUT TOGETHER A PLAN TO SHIFT AND MEET THOSE LARGER COHORT SIDESES LARGER DOSING SIZES WE ARE SEEING. 2016 WE WENT INTO CONSTRUCTION FOR DESIGNING THE FACILITY AND THE IDEA WAS TO CREATE A MULTI-PRODUCT FACILITY SUPPORTING VECTOR PRODUCTION BUT ALSO GET INTO CELL BASED THERAPY MANUFACTURING AS WELL. IT WAS UNTAPPED AREA, OF RESEARCH AND PHASE 1 TRIALS WE HADN'T HAD IN NATIONWIDE CHILDREN'S. IN 2017 WE OCCUPIED NEW SPACE. 9,000 SQUARE FEET, 7800 DEDICATED TO GNP USE FOR PRODUCTION AS WELL AS VIRAL VECTOR CORE FOR MAKING RESEARCH PRODUCTIONS. AND OUR QC ANALYTICAL LABS. WE HAD 1200 SQUARE FEET DEDICATED FOR THE CELL BASED THERAPY AREA AS WELL. AND THE IDEA WAS TO BE ABLE TO ACCOMMODATE FOUR PRODUCTION LOTS SIMULTANEOUSLY ON THE FLOOR. IN ESSENCE WE WERE MINIMALLY QUADRUPLING OUR CAPACITY FROM A VECTOR STANDPOINT. WE BUILD THE ENTIRE FACILITY TO BE FLEXIBLE WITHOUT PROCESSES, WE PLUMBED IN IN GAS LINES, WE MADE A LITTLE LARGER THAN WE NEEDED. MADE SURE WE DIDN'T HAVE EQUIPMENT NAILED DOWN. THAT WAY AS INDUSTRY CHANGED, AS WE EVOLVED AGAIN LATER ON, WE WOULD BE ABLE TO MOVE, TRANSITION INTO THAT SPACE WITHOUT HAVING TO DO FURTHER BUILDS. SO THIS IS OUR CURRENT FACILITY. SOME OF THE SAME CONCEPTS IN THE ORIGINAL ARE HERE AS WELL. SO WE HAVE DEDICATED CELL CARRIER SPACE. NOW WE HAVE THREE MANUFACTURING ROOMS. WE HAVE A PURIFICATION AND FILL SUITE DEDICATES TO THE FULL PRODUCT LINE. RESEARCH IN QC LABS LOCATED WITH THE GMP FACILITY, WE HAVE PASS THROUGH FROM GMP AREA INTO THE QC LABS SO WE HAVE ALL SAMPLES AND ALL PRODUCT DIRECTLY INTO THE QC LAB. AND CELL BASED THERAPY SPACE TO EXPLORE OPTIONS, ACADEMICALLY AS WELL AS CLINICALLY FOR NATIONWIDE CHILDREN'S. IN THE VECTOR SIDE A AV EXCLUSIVE PRODUCTION SYSTEM. WE COULD GET INTO LENTI. WE COULD GET INTO BACK LOW VIRUS BUT WOULDN'T BE GOOD AT IT. WE ARE TRYING TO STICK WHAT WE KNOW, WHAT WE HAVE EXPERTISE AT AND LEVERAGE CENTER FOR GENE THERAPY AT NIGHTS WIDE CHILDREN. WE HAVE GREAT MIND FOCUSED ON AAV, WE WANT TO KEEP THAT FOCUS UP. I MENTION BEFORE PROCESS, SINGLE USE FULLY DISPOSABLE SO WHEN WE TALK ABOUT CLEANING THE ROOM AND THE SPACE FACILITY ITSELF THAT WE HAVE TO CLEAN IN BETWEEN PRODUCTIONS. WE ARE IN HEK 293 ADHERENT CELL LINE, BASED OFF OUR MASTER CELL BANK CRETEIATED IN TWINE. WE ARE -- 2009. WE ARE A CONTINUOUS CARRY PROCESS. I WILL TALK ABOUT THAT IN A MINUTE. SINGLE LOT TRANSFECTION SCALE, 30 HYPERSTACKS. ABOUT 540,000 SQUARE CENTIMETERS. WE ARE IN MEDIA RELEASE PROCESS. AND OUR TIME LINE IS TWO WEEKS PER SUBLOT. I TALK ABOUT SUBLOT AS IN WE MANUFACTURE AND HARVEST, THEN WE CONCENTRATE DOWN AND PULL THOSE CONCENTRATES LATER FOR PURIFICATION. THIS ALLOWS TO REASON THE THREE MANUFACTURING ROOMS CONTINUOUSLY ALL YEAR LONG. THE POOLING WE DO IS BASED ON VOLUME AND DA REAR YIELDS ALONG THE WAY, WE ARE MAKING EDUCATED DECISIONS WHAT WE HANDLE FROM A PURIFICATION STAGE LOOKING AT SUBLOT TITERS. AND WE DO CHARACTERIZATIONS OF THOSE PULLED LOTS AS WE GO THROUGH. OUR CONTINUOUS CARRY PROCESS, OPERATIONALLY IS SLIGHTLY INEFFICIENT BECAUSE YOU'RE CARRYING CELLS BUT THE LITTLE INEFFICIENCY ALLOWS US TO RUN CONTINUOUSLY ALL YEAR LONG. WE DON'T FALL A BATCH OF CELLS AND RAMP UP INTO EACH PRODUCTION. WE MAINTAIN A LARGE VOLUME OF TEN STACKS CARRYING CELLS CONTINUOUSLY. THIS ALLOWS US TO MAINTAIN THE PROCESS ROOMS AND BE ABLE TO SUPPORT PRODUCTION EVERY WEEK. WE TAKE CELLS AND GIVE TO RESEARCH PRODUCTION AREA, WE GIVE TO PRODUCT DEVELOPMENT LABS, THEY DON'T NEED TO MAINTAIN OWN CELL GROWTH AND CELL PRODUCTION. THIS SIMPLIFIES THINGS. THE SMALL ADDED COST AND LABOR TO MAINTAIN CELL LINE AT THIS VOLUME IS ABSOLUTELY CANCELED OUT BY INABILITY AND FLEXIBILITY THAT WE HAVE. OUR PROCESS TALK ABOUT CONTINUOUS CARRY. WE HAVE CONTINUOUS CARRY SUCH A LARGE AMOUNT TO SEE THOSE 30 HYPERSTACKS ON A WEEKLY BASIS. TRIPLE TRANSFECTION WITH BENZINASE. IN WEEK TWO, WE DO A HARVEST, TO CLARIFY TANGENTIAL FLOW FILTRATION CONCENTRATION NEXT TO THE DIFILTERATION. THAT'S WHERE WE GO DOWN TO CONSENT TREATED STATE. 500 TO 750 MLs OF CONCENTRATED MATERIAL FROM THOSE 30 HYPERSTACKS. THAT MATERIAL IS WHERE WE START POLL STEP. ALL BASED ON CLIENT DEMAND HOW MANY LOTS TO PRODUCE. A THREE LOT PRODUCTION WE WILL CONCENTRATE DOWN EACH INDIVIDUAL LOT AND PULL THREE TOGETHER FOR PURIFICATION. OUR PURIFICATION PROCESS WE USE ULTRA CENTER PHICATION. WE INCREASE THROUGH PUT, USING SOME OPERATIONAL ADVANTAGES. WE HAVE INCREASED THE AMOUNT OF CENTER FUSION, USING MAN POWER AND DID POISON PHILOSOPHY HOW WE MOVE FROM THE SPIN CYCLES INTO DHAL COVERAGE CYCLES. CHROMATOGRAPHY POLISHING AND WE GO INTO FINAL FORMULATION TFF THEN STERILE FILL PRODUCT. SO WE CAN GO FROM START TO FILLED PRODUCT IN FOUR WEEKS. PRODUCTION WISE. 707 LOTS PER YEAR. WE YIELD ROUGHLY 1.5 EACH OF 15 PURIFIED. SO IF YOU DO THE NUMBERS WE END UP ABILITY 2017 IS MAXIMUM CAPACITY FOR THE YEAR. IT VARIES DEPENDING ON SEROTYPE YOU ENCOUNTER. OUR HISTORY OF SEROTYPE. THIS IS BASE -- DATES BACK TO 2009 WHEN WE OPENED THE DOORS FOR THE GMP AND GOES UP TO CURRENT. YOU CAN SEE VARIATION VARIABILITY SEROTYPES WE ENCOUNTERED. AND THIS IS JUST GMP PRODUCTION AT RESEARCH LEVEL, WE ENCOUNTER PROBABLY DOUBLE THIS AMOUNT MUTANT SEROTYPES, MODIFIED SEROTYPES ALONG THE WAY AS WELL. HER ASSAY INCLUDE WHAT WE DO FOR RELEASE. THIS IS AS LISTING FOR WHAT WE DO FOR RELEASE TESTING. THESE ARE ASSAYS WE HAVE IN PLACE, ABOUT 60, 60% WE DO IN HOUSE. THE REST WE TRY TO FARM OUT. WE DON'T HAVE A VERY LARGE STERILITY LAB. SO WE TRY TO KEEP IT SIMPLE, DO WHAT WE ARE GOOD AT AND FARM EVERYTHING ELSE OUT WHERE WE CAN. SOME SYSTEMS WE NEED TO EXIST AS GMP WE NEED VIRAL VECTOR CORE. WE NEED THE ACADEMIC CORE SMALL SCALE PRESENCE, SUPPORTING ALL THE NEW SCIENCE COMING IN, NEW TECHNOLOGY COMING FROM NATIONWIDE CHILDREN, AS WELL AS COMING FROM OUTSIDE CLIENTS. IT IS OUR FASTEST GROWING AREA THAT WE HAVE. WE HAVE MORE DEMAND AND MORE OF A QUEUE FOR RESEARCH AREA THAN WE DO IN GNP. I DESCRIBE IT AS A FUNNEL. TO GET TO ONE GNP LOT WE ARE SEEING MOST COMPANIES AND INVESTIGATORS ARE LOOKING TO AT LEAST ONE TOX, MOST LIKELY TWO. TO LEAD INTO THOSE TALKS WE'RE DOING MULTIPLE LARGE SCALE RESEARCH PRODUCTIONS FOR THEM. TO GO INTO THOSE MULTIPLE LARGE SCALE RESEARCH PRODUCTIONS WE HAVE A TON OF SMALLER PREPS ASKED TO MAKE. THE DEMAND IS HERE, THE PURPLE AND BLUE BOX, PART OF THE FUNNEL, WHERE WE SEE THE LARGEST PUSH FROM OUR CLIENTS WHO ARE COMING IN AND THE LACK OF SPACE. THERE SEEMS TO BE LACK OF WET LAB OR ACADEMIC LAB SPACE ACROSS THE COUNTRY. OTHER SUPPORT SYSTEMS WE HAVE TO HAVE IN PLACE TO FUNCTION. WE HAVE PRODUCT DEVELOPMENT GROUP, PRODUCT DEVELOPMENT GROUP IS FUNCTIONING SEPARATELY FROM GNP, SEPARATELY FROM RESEARCH PRODUCTION AREAS. NEW TECHNOLOGIES,NEW MANUFACTURING SYSTEMS WHAT IS OUR EVOLUTION. THEY'RE LOOKING TO OPTIMIZE WHAT WE CURRENTLY HAVE, LOOKING THE DEVELOP NEW TECHNIQUES AND SEE TO POSSIBLE FUTURE APPLICATIONS THAT WE NEED ALONG THE WAY. A TEAM THAT WE HAVE TO BUILD THAT WE NEVER THOUGHT WE WOULD NEED WAS OUR BUSINESS TEAM. WE HAVE GOTTEN TO THE POINT WHERE WE HAVE SO MANY ACADEMIC PRODUCTIONS, SO MANY OUTSIDE FOUNDATIONS AND SO MANY PRIVATE COMPANIES COMING INTO WORK WITH US WE HAVE TO ENGAGE THEM WITH A BUSINESS TEAM WHO HAS A PUBLIC FACE, RAN UP CONTRACTS OR AGREEMENTS, ALL THAT CUSTOMER SERVICE PIECE. THE THINGS THAT YOU DON'T THINK ABOUT SMALL ACADEMIC CORE LEGAL ENTANGLEMENTS AND CONTRACTS AND AGREEMENTS THAT ARE REQUIRED. THEY MANAGE THE PROJECTS ON THE FLOOR. WE TEAL WITH 50 PLUS CLIENTS ANY GIVEN TIME. SO WE HAVE TO HAVE A TEAM IN PLACE, JUST SPECIFICALLY ADDRESSING THEIR CONCERNS. THEY'RE SUPPORTING OUR FACILITY, KEEPING THE AIR HAMMER RUNNING AND KEEPING EVERYTHING MOVING ALONG VERY QUICKLY. OUR QUALITY CONTROL GROUP, WE HAVE A QUALITY CONTROL GROUP, THEY ARE TESTING OUR PRODUCTS WHETHER PRODUCT DEVELOPMENT, RESEARCH, OR GNP. BUT THEY'RE ALSO DOING ASSAY DEVELOPMENT. WE HAVE A MULTI-SHIFT GROUP. NEVER DREAM WE WOULD HAVE A SECOND SHIFT QUALITY CONTROL GROUP WORKING ALL NIGHT LONG TO MAKE SURE OPERATIONS TEAM COME IN THE MORNING TO TURN AND MAKE DECISIONS AND GET BACK ON THE FLOOR. WE HAVE SEPARATE QUALITY ASSURANCE TEAM, THEY DON'T REPORT TO OUR GROUP. THEY ARE SEPARATE UNIT REPORTING TO THE HOSPITAL. AND THAT GROUP RELEASES EVERY PRODUCT WE HAVE COMING OFF THE FLOOR FOR USE IN PATIENTS. TO KEEP THIS MOMENTUM GOING WE HAD TO BRING UP STAFFING TREMENDOUSLY. IN 2010 WE HAD SIX PEOPLE. WE HAVE 80 TODAY. IT'S HAD TO RAMP UP TO MEET VOLUME DEMAND, TO MEET CAPACITY THAT WE HAVE AS WELL AS ALL THE ANCILLARY PIECES REQUIRED TO MAINTAIN THE OPERATION. IT DOESN'T LOOK LIKE AN ACADEMIC CORE, IT LOOKS LIKE A SMALL BIOTECH. GO TO BIOTECH WEBSITE THAT'S WHAT THE ORG CHART LOOKS LIKE. INDIVIDUALIZED GROUPS, MANUFACTURING QUALITY CONTROL DEVELOPMENT AND QC QA. CURRENT BARRIERS. WHAT WE ARE FACING, WHAT WE ARE SEEING TODAY THAT IS -- IMPEDING PROGRESS DIRECTLY OR COULD BE IMPEDING SOON, FLAT WARE. SO IT'S ALWAYS SUPPLY AND DEMAND. YOU CAN'T REUSE, YOU HAVE TO BRING THEM IN. SO IT'S -- YOU'RE SUBJECT TO YOUR VENDOR. FILTERS, TUBING SETS, PLASMIDS, MEDIA, ALL THOSE THINGS. IT'S NOT A VERY MATURE ORGANIZATION OF VENDORS. A LOT OF THESE GROUPS ARE JUST MERELY BUILDING CAPACITY. SO YOU WILL SEE A HICCUP. A TREMENDOUS AMOUNT OF RAIN ON THE EAST COAST PUT US IN THE A BAD SITUATION ON SOME PRODUCTS COMING IN. THOSE LITTLE THINGS IMPACT US. IT'S NOT LIKE MY PREVIOUS HISTORY WORKING VACCINES, WE HAD MULTIPLE VENDORS, AND IF ONE WENT DOWN YOU HAD ANOTHER ONE STEP IN ITS PLACE. WE DON'T HAVE THAT YET FOR OUR INDUSTRY. CLIENT VOLATILITY. WE DEAL WITH FOUNDATIONS, INSTITUTIONS, PRIVATE COMPANIES. THEY ALL HAVE PLUSES AND MINUSES. THEY ALL HAVE DEEP SCHEDULING IMPLICATIONS. FOUNDATIONS, GENERALLY VERY EASY TO WORK WITH, YOU CAN GET CONTRACTS DONE QUICKLY. THE MONEY IS THE ISSUE. ARE THEY ABLE TO FINANCE, ARE THEY GOING TO PAY FOR PRODUCTION, ARE THEY GOING TO GET EVERYTHING TAKEN CARE OF. INSTITUTIONS. GENERALLY THE MONEY HAS ALREADY BEEN SPOKEN FOR, THEY HAVE GRANT, THEY HAVE SOMETHING ELSE IN PLACE. BUT WRITING THE CONTRACTS BETWEEN TWO INSTITUTIONS IS TREMENDOUSLY DIFFICULT. ESPECIALLY IF YOU ARE TWO PUBLIC ENTITIES IN TWO STATES WHO DON'T AGREE FROM LEGAL STANDPOINT OR INDEMNITY WISE. WE MAY GO SIX MONTHS IN CONTRACT NEGOTIATIONS TO TRY TO FIND COMMON GROUND. PRIVATE COMPANIES. PRIVATE COMPANIES ARE GREAT. VERY WELL FUNDED AND THEY WILL GENERALLY JUMP ON BOARD, GET AGREEMENTS PUT IN PLACE. THAT'S FANTASTIC. THEY WANT THEIR STUFF NOW. ABSOLUTELY NOW, GET OUT OF THE OF WAY, THEY WANT IT NOW. IF SOMEBODY FALSE OUT THEY WANT IT NOW NOW. THEY'RE BEING DRIVEN BY SOMETHING ELSE, SHAREHOLDER, TRYING TO GET TO MARKET. THEY HAVE A DIFFERENT AMOUNT OF DRIVE TO THEM THAT YOU DON'T SEE IN YOUR FOUNDATIONS OR YOUR INSTITUTIONS. CAPACITY WACK A MOLE. I SAY CAPACITY WACK A MOLL BECAUSE IF YOU FILL ONE CAPACITY, IF YOU THINK YOU'RE INCREASING CAPACITY IN ONE SPACE, SOMETHING ELSE POPS UP. WHAT WE HAVE SEEN, WE BUILT MORE GNP CAPACITY, AND MORE RESEARCH SPACE. WE NEED MORE PRODUCT DEVELOPMENT SPACE. IT'S JUST -- AS YOU TRY TO BUILD, IT KEEPS BRINGING A PROBLEM, YOU NEED MORE O TO SUPPORT, TO SUPPORT EACH PIECE ALONG THE WAY. MOVING PROGRAMS FORWARD. TODAY A SIX TO NINE PATIENT TRIAL, WE CAN MAKE THAT VECTOR. WITH SOME OF THE NEW CHANGES COMING ALONG, NEW IDEAS, CONCEPTS OF CREATING A PHASE 1 THAT COULD LAUNCH INTO COMMERCIAL, WHAT DOES THAT STUDY LOOK LIKE, WHAT DOES THE DESIGN LOOK LIKE? HOW MUCH PATIENTS ARE YOU TALKING ABOUT? WE CAN'T MAKE 30 PATIENTS WORTH OF MATERIAL, CAN'T MAKE 50, 100 OR A THOUSAND. THERE'S BIG IMPLICATIONS THERE. STUDY, TRIAL DESIGN DOES WHETHER OR NOT YOU CAN MANUFACTURE CERTAIN AREAS. WHAT WE ARE DOING TO TRY TO GET OVER SOME OF THESE BARRIERS AND TRY TO MOVE FORWARD AND EVOLVE. VECTOR DESIGN. WHERE YOU KNOW BIG THING I CAN TELL ANYONE BRINGING A PRODUCT TO US, HOW MUCH WORK PRE-CLINICALLY, HOW DID YOU DO DESIGN WORK. CAN WE GET VECTOR IN OUR HAND AND QUOTE UNQUOTE PLAY WITH IT? CAN WE MAKE PRESENCE? CAN WE SEE HOW IT PRODUCES IN OUR SYSTEM? WHAT WE ARE TRYING TO DO NOW IS WORK WITH THOSE CLIENTS BEFORE THEY GET TO THAT STAGE. BEARE REACHING OUT TO DO VECTOR DESIGN WITH THEM. USE BACKBONE AND SKILLS WE HAVE FROM CENTER FOR GENE THERAPY, AND THE ORGANIZATIONAL STRUCTURE WE HAVE BUILT, TRY TO SUPPORT THEM. TO BUILD BETTER VECTORS. VECTORS THAT PRODUCE WELL AND MORE. WE HAVE TO WORRY ABOUT HOW WELL DOES IT PERFORM IN ADHERENT SYSTEMS, HOW WELL DOES IT PERFORM IN SUSPENSION SYSTEMS. WE HAVE TO THINK ABOUT THAT IN DESIGN PHASE, IT'S EXTREMELY EXPENSIVE TO GO BACKWARD ONCE YOU KNOW YOU HAVE VECTOR THAT WON'T PRODUCE COMMERCIAL SCALE. PLASMA MANUFACTURING. RENOVATING SPACE TO BE ABLE TO SUPPORT THAT. THE IDEA IS BE ABLE TO TAKE CARE OF OUR OWN CLIENTS AS WELL AS TO BE ABLE TO SUPPORT MORE PHASE 1. WE ARE TRYING TO MAKE SURE THERE'S NO BOTTLENECKS FOR PEOPLE COMING TO WORK WITH US. RESEARCH PRODUCTION. I TALK ABOUT FUNNEL BEFORE, WE'RE EXPANDING SPACE, TRYING TO PUT A LAB WHEREVER WE CAN PUT A LAB. SOMEBODY HAS A BARN AT HOME, I'M JOKING BUT WE'RE TRYING TO FIGURE OUT A WAY TO INCREASE THAT CAPACITY TO BE ABLE TO WORK WITH CLIENTS EARLIER, TO GET THOSE CONSTRUCTS DESIGNED AND BUILT AND PUT IN PLACE. WE HAVE CLIENTS WHO HAVE 50 TO 606 CONSTRUCTS, THEY WANT TO START AND FUNNEL THROUGH US AND GET TO ONES THAT ARE PRODUCING HIGHEST AND GO BACK AND WORK ON THE ONES THAT HAVE LOWER LEVELS OF PRODUCTION. FROM QUALITY TESTING. WE ARE TRYING TO EVOLVE, MOVE AWAY FROM MANUAL QPCR AND DIGITAL DROP LOAD. WE ARE EXPANDING ASSAYS WE QUALIFY AND QUALIFICATIONS THAT WE DO. WE ARE ALSO LOOK AD SCREENING PROCESS OF INCOMING RAW MATERIALS, TRYING TO MAKE SURE OUR STARTING -- ALL RAW MATERIALS ARE HIGH ADS POSSIBLE. BIOREACTORS, TRYING TO CREATE A LINE WE CAN OFFER TO THE CLIENTS THAT HAS A PORTABLE FUNCTION TO IT. WE ARE LOOKING AT AAV MANUFACTURING HEK 293 LINE, 50 AND 200-LITER CAPACITIES AND TRY TO ROLE THEM OUT IN JANUARY OF NEXT YEAR. THE IDEA IS TO BE ABLE TO HAVE A PORTABLE PLATFORM. SOMETHING THAT CAN GO LARGE SYSTEMIC DOSING, LARGE TRIALS. CONCERNS, REGULATORY READINESS. WE SEE PHASE 1 BUSINESS COMING THROUGH WITH US. WE SEE A LOT OF CLIENTS WHO ARE LIKE I SAID, DOING 50, 60 CONSTRUCTS. ARE REGULATORS READY FOR THAT. IS THE FDA, NIH, ARE WE READY FOR THAT VOLUME OF NEW PHASE 1 TRIALS GOING. THERE'S A WAY. I WILL SKIP ONE, STANDARD STANDARDIZATION. AS KEN MENTION BEFORE HOW DO WE TITER? FINDING OUR PATHWAY. WHAT ARE OUR RULES, SPECIFICATIONS, REQUIREMENTS AND DIRECTIONS WE HAVE FOR GENE THERAPY. BACK UP COST OF ENTRY. COST OF ENTRY, WE SEE FOUNDATIONAL DOLLARS USED TO SEE NIH DOLLARS AND NOW PRICE IS GETTING MORE EXORBITANT, STEPPING UP RAW MATERIALS GETTING MORE EXPENSIVE, COST OF MANUFACTURING GETTING MORE EXPENSIVE. HOW DO WE MAKE SURE WE HAVE A PIPELINE FOR THOSE PRODUCTS THAT HAVE A COMMERCIAL FUTURE TO THEM AS WELL AS THE ONES THAT DON'T. RARE DISEASES OF THE TEN, 12 PATIENTS ACROSS THE COUNTRY, HOW DO WE GET THERE TO THE POINT WE CAN ACTUALLY SUPPORT BOTH TYPES. I WANT TO TAKE A SECOND TO THANK DR. CLARK AND MAN DELL WHO WERE THERE TO SUPPORT AND BUILD THIS PROGRAM AND KEN FLANAGAN FOR CONTINUED SUPPORT TO HELP MAINTAIN THE PROGRAM. THANK YOU. [APPLAUSE] >> >> THANK YOU JAYSSON. JOSH WILL STEP UP AND GIVE PERSPECTIVE FROM BIOTECH BUT HOPE WE DON'T LOSE SITE OF THE CONVERSATION THIS MORNING WHERE IT WAS DISCUSSED THAT THERE WAS 100 TO 200 RARE DISEASES DISCOVERED EACH YEAR AND IT'S CLEAR THAT THERE'S GOING TO BE A HANDFUL OF FACILITIES THAT ARE ACADEMICS THAT CAN HOUSE 80 OR SO INDIVIDUALS PRODUCING A VECTOR FACILITY. THERE IS SOME AREA FOR US TO THINK TANK WHAT'S GOING TO MAKE THIS WORK. GOING FORWARD. THANKS. >> MY NAME IS JOSH GRIEGER WORKING IN GENE THERAPY 17 YEARS AND GRADUATE SCHOOL AND WORKING WITH THEM EVER SINCE. I WILL TALK TO YOU LITTLE BIT ABOUT CAR MANUFACTURING PROCESS AND SOME OF THE HURDLES WE ARE TRYING TO GET THROUGH OURSELVES BUT I WANT TO START WITH SOME OF THE CHALLENGES AND ALSO SOME OF THE QUESTIONS THAT WE WERE GOING TO ADDRESS FIRST AND THEN TRANSITION TO MANUFACTURING PROCESS AND HISTORY AND HOW IT'S EVOLVED OVER TIME. MANUFACTURING SCALEUP IS A CRITICAL STEP IN ADVANCEMENT OF THERAPEUTIC DRUG BROUGHTS. WHY WE ARE HERE TODAY. BECAUSE -- PRODUCTS. BECAUSE THEY HAVE POTENTIAL TO DELIVER LIFE SAFING THERAPIES TO PATIENTS, CLINICAL PROGRAM DEVELOPMENT TIME LINES MAYBE ACCELERATED NOT ONLY BY COMPANY BUT ALSO BY LEGLATORY AGENCIES. IF THIS OCCURS, ALONG WITH COMPETITIVE PROGRAMS, THERE ARE OTHER COMPANIES OUT THERE THAT MAYBE INTEREST IN THE SAME PROGRAM YOU HAVE, YOUR TIME LINES BECOME COMPRESSED, IT FORCES YOU TO DEVELOP -- YOUR DEVELOPMENT TEAMS TO LOCK IN PROCESS STEPS DURING EARLY STAGES OF THE PROGRAM. WHICH MAKES IT DIFFICULT TO IMPLEMENT PROCESS IMPROVEMENTS ATZ WELL AS GET READY FOR COMMERCIAL MANUFACTURING, PRODUCE VECTORS AT THE RIGHT QUALITY, QUANTITY AND COST. FOR MANY GENE THERAPY PROGRAMS THE ACCELERATED TIME LINES LIMITED PROCESS CHARACTERIZATIONS AND LACK OF SCALABILITY DRIVE MANUFACTURERS TO SCALE OUT. ONE OF THESE IS TRANSIENT TRANSFECTION OF ADHERENCE CELLS OFTEN SUCCUMB TO THIS T. OUR EVOLUTION GOES THROUGH VECTOR CORE SO WE WORK THROUGH THESE ISSUES AS WELL AS JAYSSON IS TALKING TO PREVIOUSLY. GOING FORWARD YOU MUST START WITH SCALABLE MANUFACTURING PROCESS WITH ESTABLISHED PURIFICATION PLATFORM THAT IS ROBUST FOR MANY IF NOT ALL 80 SEROTYPES, THAT'S EASIER SAID THAN DONE. ONCE YOU HAVE THIS IN PLACE, IT LEADS TO MINOR TWEAKS BETWEEN EARLY AND LATE PHASE MANUFACTURING. THESE SMALL IMPROVEMENTS PROVIDE LOW RISK QUALITY TO THE SITUATION. IT'S ALSO AN ADVANTAGE IF YOU OWN THE PROCESS. BECAUSE IT'S YOUR -- YOU HAVE THE OPPORTUNITY TO DEVELOP IT ALONG THE WAY. AND NOT REQUIRE OTHERS TO OUTSOURCE THIS TYPE OF WORK. WHICH IS ADVANTAGE IF YOU'RE ABLE TO DO THAT SO COMPARABILITY IS ONE OF THE TOPICS THAT WE'RE HOPING TO DISCUSS TODAY AND WHEN CHANGES OR IMPROVEMENTS ARE MADE TO THE MANUFACTURING PROCESS HOW DO THESE CHANGES IMPACT QUALITY, PRODUCT QUALITY IN TERMS OF IMPURITY PROFILE SAFETY AND POTENCY SO WE HAVE DONE COMPARABILITY STUDIES THROUGH THE VECTOR CORE AND ALSO THREW OUR COMPANIES AND OBVIOUSLY COMPARABILITY STUDIES WHAT THEY ARE, THEY ASSESS COMPARE CRITICAL QUALITY ATTRIBUTES OF PREVIOUS DRUG PRODUCT TO NEW DRUG PRODUCT. THEY CAN BE STRAIGHT FORWARD AS ANALYTICAL COMPARISON, I HAVEN'T PUT IT IN PATIENTS YET. WE HAVE IT IN ANIMALS. HERE IS THE ANALYTICAL PROFILE PREVIOUS DRUG PRODUCT, THIS IS THE NEW DESTRUCT PRODUCT SIDE BY FORWARD. YOU MAY HAVE TO GO TO MORE COMPLEX ROUTE WHICH IS IN VIVO STUDY, REPEAT BIOTOX DEPENDING ON -- SWITCHING BETWEEN PLATFORMS. IN ADDITION TO THAT, YOU CAN ALSO GET EXTENSION OF PHASE 1 CLINICAL TRIAL BY ADDITION OF PATIENTS. ALLOWS YOU TO TEST YOUR VECTOR IN ACTIVE PHASE 1 CLINICAL TRIALS GETTING READY TO TRANSITION TO PHASE 3 PIVOTAL. AGAIN ALL OF THIS -- ALL THESE PATH FORWARDS, SURE THERE'S OTHERS, CAN BE IMPACTED BY TYPE OF MANUFACTURING CHANGES MOVING FORWARD WITH AND THE ANALYTICAL DATA YOU GENERATE. SO PROCESS CONTROLS MANUFACTURING AND TESTING, ADHERENCE TO GMP FOR QUALITY ASSURANCE, YOU HAVE TO TAKE INTO CONSIDERATION QUALITY OF RAW MATERIALS, SERUM, ANTIBODY, PLASMIDS OR CELL LINE. WHAT YOU KNOW ABOUT CMO WHO YOU ARE INTERACTING WITH WHO ARE MAKING THE MATERIALS WITH YOUR CLINICAL TRIAL, WHETHER IT'S YOUR ACTUAL DRUG PRODUCT OR STARTING MATERIALS. HOW DO THEY ASSURE QUALITY AGENTS OR MATERIAL? MOST QUALITY IS BUILT INTO GMP MANUFACTURING PROCESS, YOUR PROTOCOLS, QUALITY SYSTEMS, YOU CAN'T RETROACTIVELY TEST THIS BACK IN SO YOU HAVE TO QUALIFY YOUR CRITICAL SUPPLY AND RAW MATERIAL VENDORS AUDIT OF FACILITY, THEIR SUPPLY CHAIN, SOUND LICK THEY'RE DOING THEIR AUDITS, QUALIFY THEIR SUPPLY CHAIN VENDORS, QUALITY SYSTEMS, WHAT HAPPENS IF -- WHAT DO THEY DO, CAN THEY HANDLE DEVIATIONS IN MAKING GOOD DECISIONS EARLY ON PREVENT YOU HAVING TO REDO THINGS AT A LATER DATE. THESE QUALITY MEASURES GENE MIND SET IS WHAT YOU NEED FROM THE BEGINNING SO INSTEAD OF REACTIVE TO SITUATIONS, YOU ARE PROACTIVE. CONTROLLING CHANGES YOU WANT IN YOUR MANUFACTURING PROCESS, QUALITY CONTROL AND NOT HAVING TO MAKE CHANGES BECAUSE OF BAD DECISIONS YOU MADE EARLY ON. SO WE DO HAVE ONE CONTRACT MANUFACTURING ORGANIZATION, WE HAVE HAD A LONG STANDING RELATIONSHIP WITH UNC AND BAMBOO THERE ARE PLASMID SUPPLIER, WE HAVE LEARNED FROM ONE ANOTHER WHEN YOU REPLICATE IN BACTERIA. ONE THING THAT'S VERY IMPORTANT TO UNDERSTAND IS WHAT IS YOUR CLINICAL DESIGN. CLINICAL DESIGN IMPACTS HOW MUCH VECTOR YOU NEED. FOR MANY MANUFACTURING PROCESS WHETHER IT'S BACK LOW VIRUS OR HERPES OR TRANSFECTION, HOW LONG DOES IT TAKE TO GET STARTING MATERIALS AVAILABLE? WHEN YOU RECOGNIZE THIS AND PROACTIVE, ONCE YOU IDENTIFY YOUR PLASMID YOU MOVE FORWARD WITH, WE UNDERSTAND THEIR TIME LINES, THEY IMPROVED UPON BY BRINGING ON MORE PEOPLE, MORE SHIFTS. NEW GMP FACILITY. HERE IS OUR PLASMIDS, SEQUENCE ONCE SEQUENCE VERIFIED, GENERATE RESEARCH CELL BANK, IN ADDITION TO GENERATING MASTER CELL BANK, MASTER CELL BANK IS CRITICAL TO HAVE ESTABLISHED BEFORE GMP SOURCE PLASMID MANUFACTURING OCCUR. OF ALL THOSE ACTIVITIES GOING ON BANDWIDTH TO START MAKING HUNDREDS OF MILLIGRAMS OR GRAM QUANTITIES OF RESEARCH GRADE MATERIAL, PLASMID THAT WE CAN USE FOR PROCESS DEVELOPMENT WHICH ALSO SUPPORTS PROCESS IMPROVEMENTS ASSAY DEVELOPMENT, ASSAY QUALIFICATION. SO ALL THIS IS GOING ONLINE WHY WOULD BUILDING UP YOUR GMP TIME LINES. ONCE THE GMP SOURCE PLASMID IS AVAILABLE YOU CAN MOVE FORWARD SO GENERALLY SIX WEEKS, GET MASTER CELL BANK ESTABLISHED ONCE AGREE UPON SEQUENCE. ONCE THE GMP SOURCE PLASMID MASTER CELL BANK IS AVAILABLE YOU NEED TO TAKE ANYWHERE FROM FOUR, EIGHT, TEN WEEKS TO GET YOUR MATERIAL MADE SO THE TIME LINES AS LONG AS YOU'RE PROACTIVE WORKING WITH CONTRACT MANUFACTURING ORGANIZATION, YOU CAN GET HIGH AMOUNTS OF PLASMA DNA TURNED OVER QUICKLY. WITH NO PROBLEMS. THERE'S A PROCESS COMMERCIALLY VIABLE. I WILL WALK YOU THROUGH THAT. LATER SLIDES. BUT YES, OUR VECTOR PRODUCTS ARE MANUFACTURED FOR RESEARCH GRADE GLP, GMP AND HAS ABILITY TO SCALE UP THROUGH COMMERCIAL MANUFACTURING SCALES. WE HAVE ANY INHERENT DIFFICULTIES WITH REAGENTS THAT ARE NOT AVAILABLE TO COMMERCIAL MANUFACTURER? WE HAVE OUR CELLS I WILL DISCUSS TODAY. LIBRARY OF PATENTED PLASMIDS PRIMARY CAPSIDS THAT WE UTILIZE FOR SOME INDICATIONS. AGAIN, JUST EXPERIENCE HERE WE HAVE EVOLVED AS A FIELD HAS EVOLVED, UNC VECTOR CORE WAS MANUFACTURING BOTH RESEARCH GRADE THROUGH GMP USING 15-CENTIMETER CULTURE PLACE TO THE POINT WHERE IF YOU ARE GENERATING 10 TO 13th PURIFIED VECTOR GENOMES THAT WAS CONSIDERED GOOD, THIS WAS BACK IN THE EARLY 2000s. WE NEVER TRANSITION TO THE HYPERSTACKS, ROLLER BOTTLES AND NOW -- WE DECIDED TO DIVERT FROM THAT AND LOOK AT SUSPENSION BASED SYSTEM MOVING AWAY FROM ANIMAL DIRECT COMPONENTS EARLY ON IN 2008. EVERYBODY IS AWARE, I'M NOT TRYING TO BE NEGATIVE ABOUT ADHERENCE SYSTEM. IT DOES UTILIZE ANIMAL DERIVED COMPONENTS, NOT EASILY SCALABLE. THEY'RE ALL LIMITED BY SURFACE AREA. YOU HAVE TO SCALE OUT VERSUS SCALE UP. SO WE RECOGNIZE THAT EARLY ON. BACK IN 2008 IN OUR GMP FACILITY, WE TOOK OUR QUALIFIED ADHERENT MASTER CELL BANK WHICH HAVE GONE THROUGH SEVERAL ROUNDS OF SELECTION FOR TRANSFECTION. VECTOR MANUFACTURING. AND WE BEGAN TO WEAN FROM FES KNOWING ALL OF OUR GMP DOCUMENTATION, CELL CULTURE DOCUMENTATION AND FORMS UTILIZING ALL QUALITY ASSURANCE RELEASES AND SUPPLIES AND RECORDING ALL THIS INFORMATION SO WE HAVE A HISTORICAL OVERVIEW OF HOW WE TRANSITION FROM ADHERENT BANK TO NEW MASTER CELL BANK WE QUALIFIED IN 2010 WHICH IS WHY IT'S PRO10 PRODUCTION CELL 10. ONCE WE WEAN FROM FPS, THE CELLS TRANSFER INTO COMMERCIALLY AVAILABLE CHEMICALLY SYNTHETIC DEFINED OUR PROCESS DEVELOPMENT TEAM WAS AT THE TIME JUST ME AND SOMEBODY -- SOMEBODY ELSE AT THE TIME. WE ESTABLISHED A RESEARCH GRADE BANK AND FROM THERE WE STARTED LOOKING AT CHEMICALLY DEFINED MEDIAS TRANSFECTION AND GROWTH, NOT HAVE ONE THAT SUPPORTS GROWTH, YOU CAN'T TRANSFECT BECAUSE OF INHIBITORY COMPONENTS IN THE MEDIA, FOR FUSING THAT INTO A TRANSFECTION MEDIA AND TRANSFECTION MEDIA AND GROWTH MEDIA. SUPPORTED BOTH GROWTH AND TRANSFECTION AND PRODUCTION OF AAV HIGH LEVEL. T THEN ONCE WE HAD THAT IDENTIFIED, WE THEN GENERATED A GMP MASTER CELL BANK THAT WAS THEN QUALIFIED AND RELEASED FOLLOWING REQUIREMENTS THAT WAS DISCUSSED IN THE PREVIOUS SLIDE DECK. NOT GOING THROUGH THESE PARAMETERS, AT THE TIME WE WERE TRYING TO GENERATE CELL, PRO10 CELLS THAT GENERATE THE SAME VECTOR GENOME PER CELL AS ADHERENT SYSTEMS, WE DIDN'T WANT A HIT IN PRODUCTION. ALSO MOVE FORWARD WHEN SCALING. SO WE WORK OUT SEVERAL PARAMETERS AND TRANSITION TO -- DURING THIS PHASE WE ARE LOOKING AT FROM 2009 ONCE WE TRANSITION TO THE WAY BIOREACTOR WE ARE PURIFYING 10 TO THE 14th VECTOR GENOMES, FROM THE CULTURE LOW CELL CULTURE DENSITIES, ROUTINELY MAKE 10 TO THE 14th. WE SKIPPED OVER 10 TO THE 14th. THERE'S FIRST GENERATION PRO10 MANUFACTURING PROCESS, YOU CAN SEE HERE WE WERE IN SHAKER FLASKS AND WE TRANSITIONED TO BIOREACTOR AND ESTABLISHED DOWNSTREAM PROCESS THAT WAS ROBUST WE CAN PURIFY SEROTYPE AND CAPSID USING THE SAME METHODOLOGIES SAME ACADEMIC CEO PERSPECTIVE BECAUSE BEFORE IF IT'S 82 YOU CAN PURIFY BY HEPARIN, IF IT'S ANY OTHER SEROTYPE YOU CAN DO SESIUM OR ANOTHER METHODOLOGY. YOU ARE GETTING DIFFERENT VECTOR SEROTYPES, APPLES AND ORANGES THAT PURIFY DIFFERENTLY. SO WE CAN INPUT ONE MANUFACTURING PROCESS ONCE WE STARTED SCALING, WE WERE IN A WAY BIOREACTOR BETWEEN 10 AND 25 PUMP THE CELLS OUT, CENTRIFUGE, THAT WAS VOLUME REDUCTION STEP AND THEN SONCATION AND INCUBATION. FOLLOWED BY ANOTHER LOW SPEED CENTRIFICATION THAT CLARIFIED LYSATE INTO GRADIANT THAT WAS THE WORKHORSE OF ENRICHING FULL PARTICLES ALSO REMOVING ADDITIONAL CELL PROTEIN AND DNA THAT WAS STILL IN THERE WITH VECTOR TO ION EXCHANGE AND DIALYSIS, USING DIALYSIS STILL WORKING WITH SMALL VOLUMES AND THAT WOULD FORMULATE VIRUS AND THEN DO .2-MICRON FILTRATION. THIS WORKED GREAT, VERY HIGH THROUGH PUT, VERY FAST, THEN DEMANDS ON FACILITY WERE INCREASING AND WE WEREN'T GOING INTO LOW DOSE LOW PATIENT NUMBER TRIALS, WE TRANSITION SO HOW DO WE TRANSITION FROM THIS PROCESS. I THINK THAT'S BEFORE WE TELL YOU HOW WE TRANSITION, I WILL TELL YOU ABOUT THE ROBUSTNESS OF PREACHIFICATION. ON THIS SLIDE IS A SILVER STAIN LOOK AT SEROTYPES 1 THROUGH 9 PURIFIED THE SAME WAY BACK TO BACK. THESE ARE THE SINGLE STRANDED VECTORS AND THESE ARE THE COMPLIMENTARY VECTORS LOOKING AT INTEGRITY OF THE COMPLIMENTARY GENOME. SO THIS IS INAUGURAL SHELL SOUTHERN BLOT LOOKING AT INTEGRITY OF GREATER THAN 95% OF THE GENOMES PACKAGED ARE COMPLIMENTARY IN THAT MONOMERIC AND WE TRANSITIONED AWAY FROM USING NEGATIVE STAIN TEM, WE USED A COMPANY CALLED VERANOVA, CHARACTERIZING MP PARTICLES, USING CRYO-EM MORE STRAIGHT FORWARD METHOD DOLL AND ALSO COMPARED THESE NUMBERS AGAINST ANALYTICAL ULTRA CENTRIFICATION AND FOUND THEY CORRELATE WELL. AND THE NEGATIVE STAIN TEM CAN BIAS YOU TO 95% VERSUS WHAT WE'RE SEEING CONSISTENTLY BETWEEN 70 AND 80% PARTICLES FROM CENTRIFICATION. WEPT BACK AND SAID WE CAN'T PRODUCE VECTOR AND SHAKE FLASK WITH BIOREACTOR. FOR MOST CLINICAL MANUFACTURING WE NEED TO MOVE FORWARD, GO LARGER SCALES. WE DECIDED THESE IN C TRAIN, NO LONGER DO LOW SPEED CENTRIFICATION SONCATION, MECHANICAL METHODOLOGY TO LYSE THE CELLS, TRY TO REMOVE BENZINASE FROM THE MANUFACTURING PROCESS. WE ARE UTILIZING FOR SOME SEROTYPES, THE INGREDIENT MODIFIED SO WE CAN MAXIMIZE THE AMOUNT OF VIRUS PER GRADIANT. SO WE'RE NOT DOING AS MANY GRADE -- GRADIANTS FROM OUR EXPERIENCE WHAT WE HEARD FROM OTHERS IS PEOPLE ARE TRYING TO TRANSITION FROM THE GRADIANT CENTRIFICATION WHICH IS UNIVERSAL, DOESN'T MATTER THE SEROTYPE YOU'RE USING, CHROMATOGRAPHY AND SOMETIMES CHROMATOGRAPHY CAN BE PRODUCT SPECIFIC, SEROTYPE SPECIFIC, LENGTH OF GENOME IS IMPORTANT, SMALL GENOME VERSUS LARGE GENOME, IMPACT HOW VIRUSES ARE ALLUDED FROM THE COLUMN, SEPARATION YOU CAN GET FROM -- SO THERE'S MORE PROCESS DEVELOPMENT THAT HAS TO HAPPEN BEFORE WE FEEL CONFIDENT WE CAN REMOVE THIS BUT IT'S IN DEVELOPMENT. AND THEN OBVIOUSLY MOVING DIALYSIS CASSETTES OF OUR PROCESS AND REPLACE THAT WITH TFF UFDF, THEN AGAIN WE STILL DO THE .2-MICRON FILTRATION. IN ORDER TO MAKE THESE TRANSITIONS, WE ARE THE VECTOR CORE, WE -- DESIGN OF EXPERIMENT APPROACH, SO WHEN I WAS DOING MY INITIAL WORK IT WAS CHANGE ONE VARIABLE, DOES IMPACT VECTOR PRODUCTION YES, NO. WE LOOK AT CHANGING MULTIPLE FACTORS AND WHAT WE CALL THIS DESIGN SPACE AND AT THIS POINT NOW SMALL SCALE BIOREACTORS SO WE HAVE BETTER CONTROL OVER DISSOLVED OXYGEN, PH, HOW TO MAINTAIN THOSE THINGS. METABOLITES, WHAT ARE THE CELLS USING, CELLS ARE GROWING, WHAT ARE THEY USING PRODUCING VIRUS, WE HAVE A BETTER UNDERSTANDING OF WHAT'S HAPPENING IN THE BIOREACTOR FROM THE OTHER SITUATIONS WE WERE IN WITH FLASKS AND WEIGHTED BAGS. SO WE DID THIS SYSTEMIC METHODOLOGY TO LOOK AT FACTORS AND IF THEY CORRELATE CHANGING PH, CHANGING DNA CONCENTRATIONS, RATIOS OF THE PLASMAS LOOKING AT RESOLVED OXYGEN YOU CAN CHANGE THOSE ALL IN A SINGLE EXPERIMENT IF YOU HAVE THE RIGHT NUMBER OF REPLICATES AND THEN YOU CAN USE STATISTICAL ANALYSIS TO SEE WHICH ONES ARE CRITICAL. WHICH ARE CRITICAL AND LINKED AND THEN YOU CAN DEFINE YOUR CRITICAL PROCESS PARAMETERS VERY QUICKLY THROUGH THIS AND ALSO ROBUSTNESS AND CERTAIN PROCESS STEPS YOU ARE LOOKING AT, SO YOU'RE SOLVING PROBLEMS AT THE SAME TIME CAN ANYONE MOVE QUICKLY AND SUPPORT THE DECISIONS YOU ARE MAKING. SO THIS IS THE PROCESS, THE SECOND GENERATION PROCESS THAT WE HAVE DEVELOPED THE WAY BIOREACTOR IS IN OUR C TRAIN, WE'RE NOW 250-LITER SCALE HIGHER CELL DENSITIES, TRYING TO MAXIMIZE THE OUTPUT OF OUR PRODUCTION BIOREACTOR WITHOUT HAVING SCALE TO LARGER VOLUME. FOR SAME CELL DENSITY, HOW WE CAN GET MORE VIRUS, MORE CELLS FROM OUR BIOREKTOR. WE'RE NOW DOINGLYSIS, ALL THESE STEPS LOW SPEED CENTRIFICATION STEP, WE HAVE A CHROMATOGRAPHY FIGHT, IN AND OUT O OUR PROCESS DEPENDING HOW MUCH PROCESS DEVELOPMENT WE CAN DO AS WE TRANSITION THE PIPELINE. POLISHED CHROMATOGRAPHY STEP, .2-MICRON FILTRATION AND FINAL FILL. DEPENDING ON VECTOR YOU ARE MANUFACTURING, YOU MAY NEED TO MANUFACTURE SEVERAL BATCHES AS JAYSSON WAS ALLUDING TO WITH HIS PROCESS. COMBINING THEM INTO -- AND RELEASING A DRUG PRODUCT. A LOT IS TYPICAL WHAT YOU SEE WITH PEOPLE USING FOR DOWNSTREAM PURIFICATION. AND THEN SIMILAR TO WHAT JAYSSON WAS ECHOING WE GET BETWEEN A 30 TO 40% YIELD, THIS IS BASED ON IN THE BULK MATERIAL OF BIOREACTOR. 10 TO THE 14th PURIFIED VECTOR GENOMES FOR LITER. TWO AND FOUR TIMES TO THE 16th PURIFIED BIOPARTICLES FOR 250-LITER BIOREACTOR AT CURRENT CELL DENSITY. WON'T SPEND MUCH TIME ON RELEASE TESTING. JAYSSON SPENT TIME ON THAT. BUT AGAIN, COMPARE THESE, THEY ARE SIMILAR TO WHAT JAYSSON AND WE HAVE DONE IN THE PAST WITH THE UNC VECTOR CORE AND OHIO STATE AND OTHER GROUPS. ONE THING I WANT TO MENTION, COMPANIES LIKE PFIZER, NOVARTIS, DR. SNYDER, THEY ARE RAISING THE BAR ON QC RELEASE TESTING SO WE DO STANDARD WESTERN BLOT FOR IDENTIFICATION. WE KNOW THAT THE B-1 ANTIBODY RECOGNIZES MORE THAN ONE SEROTYPE AND I WOULD SAY AAV. NOW YOU CAN DO PEPTIDE MAPPING, IS TO SAY IT IS ARCVA AV 6. SO ONCE INDUSTRY STARTS GETTING MORE INVOLVED IN ANALYTICAL TESTING OF VIRUS WE WILL SEE THAT BARGAINING SET HIGHER AND LABS HAVE TO KEEP UP WITH THAT STANDARD. PROGRAM IS SIMILAR. COUPLE OF MINUTES THEN WRAP UP HERE. SECOND GENERATION UPSTREAM TECHNOLOGY, LOWER CELL DENSITY, WE'RE TRANSITIONING TO HIGHER CELL DENSITIES, THREE FOLD HIGHER CELL DENSITY, WE HAVE SEEN HOW MANY INCREASE IN CELL DENSITY, LINEAR INCREASE IN VECTOR PRODUCTION, THE SAME FOOTPRINT AND JUST AS REFERENCE, THERE'S ONE CAVEAT TO MENTION HERE, THE VECTOR PRODUCTION YIELDS FOR THESE ESTIMATES ENOUGH FACTOR IN PERCENTAGES FOR QC RELEASE TESTING STABILITY AND REGULATORY, FOR INDICATION USING SECOND GENERATION BATCH AND THIRD GENERATION BATCH, BETWEEN 350,000 PEOPLE, 10 TO THE 11th OR 700,000 Is AND OVER A MILLION PEOPLE, THIRD GENERATION FROM SINGLE BATCH. LOOKING AT SMA AND DMD, LOOKING ONE TIMES TEN TO THE FOURTH FOR BOTH DIFFERENT PATIENT WEIGHTS LOOKING AT 115 PATIENTS FROM OUR 423 FOR DND AND THEN 40 OR 80 WITH SECOND GENERATION, THIRD GENERATION BATCH. WE ARE PARTNERING WITH A GROUP IN SPAIN THEY HAVE THE CAPACITY TO DO 500 LITTER COMING ONLINE LATER NEXT YEAR. THEY HAVE DOWNSTREAM PROCESS UP AND RUNNING AS WELL. 250 LITTER SCALE COMING ON. OLEYLS WE HAVE BEEN ABLE THE ACHIEVE, PURITY AND PURITY PROFILES. BEING PROACTIVE, A LOT OF QC TESTING IS TRANSFERRED THEY HAVE IMPROVED UPON THAT AS WELL AS IN THE PROCESS OF QUALIFYING MAJORITY OF THEM THOSE THAT ARE QUALIFIED ARE NOT BEING TRANSFERRED INTO VALIDATION PHASE. SO AGAIN WE ARE SETTING OURSELVES UP FOR PHASE 1, 2 AND BEYOND SO WE CAN TRANSITION TO PIVOTAL PHASE 3 AND NOT BE REACTIVE TO STAGE NOW WE NEED TO VALIDATE THIS. WE'RE MOVING FORWARD AT A PACE THAT'S KEEPING UP WITH OUR PROGRAMS. MANYFING IS CRITICAL. WE ARE SIX MONTHS AGO NEW YORK TIMES HAD ARTICLE GENE THERAPY HITS A PECULIAR ROADBLOCK AND VIRUS SHORTAGE. US AS A FIELD TO FIGURE HOW TO MAKE VECTORS AND GET THEM OUT TO PATIENTS. SAFE EFFICIENT ROBUST WAY. THE BIOGEN UTILIZING A PROCESS AND PFIZER MADE AN INVESTMENT IN NORTH CAROLINA AS WELL AS NOVARTIS FOR GENE MANUFACTURING AND PFIZER AIMS TO BECOME INDUSTRY LEADER. THEY HAVE A HISTORY, A SUCCESSFUL HISTORY GENERATING PRODUCING COMMERCIAL DRUG PRODUCTS AND GENE THERAPY IS THEIR NEXT FIELD GETTING INTO FOR COMMERCIALIZATION. JUST LIKE TO THANK JUDE, MY BIO TEAM AND UNC VECTOR, WITHOUT THEM I'LL JUST PROCESS DEVELOPMENT AND MANUFACTURING BE CAPABLE OF DOING. [APPLAUSE] >> THANKS, JOSH. RICHARD IS GOING TO WRAP THIS UP. I HAVE GIVEN US THE EXPERIENCE OF A CMO AND WHAT THEY TAKE ON TODAY WITH RESPECT TO CUSTOMERS COMING INTO THE DOOR. I THOUGHT IT WAS INTERESTING THAT JAYSSON AND OTHERS IN ADDRESSING THE SHORTAGE OF RESOURCES BRINGING PLASMIDS IN HOUSE MAKING THEMSELVES DISCUSSION SECTION IF OUR COLLEAGUES ARE HERE, THEY MIGHT BE ABLE TO PROVIDE INSIGHT ON THIS SOURCING OF PLASMA MATERIALS FOR THE FIELDER THAT OKAYS EXPANDING FAIRLY QUICKLY. RICHARD. >> IN TODAY'S TALK I WANT TO QUICKLY LOOK AT THE DEFINITION OF A PRODUCT. AND REALLY GIVE YOU A PERSPECTIVE FROM OUR CLIENTS WHO ARE EXCLUSIVELY COMPANIES DEVELOPING PRODUCTS, SOME HAVE COME FROM KEN AND JAYSSON BUT THEY HAVE A BROAD SPECTRUM OF STRATEGIES AS WELL AS RISK TOLERANCES THAT THAT HAVE TO SYNCHRONIZE WITH OUR RISK TOLERANCE THRESHOLD AS WELL. APPROACHES IN TERMS OF SWITCHING PLATFORMS OR MAKING SMALL CHANGES TO BRUTE FORCING IT AND JUST GETTING TO MARKET AS FAST AS THEY CAN. OBVIOUSLY EARLY DECISIONS IMPACT LATER STAGES. AND THERE'S AN EVOLVING REGULATORY LANDSCAPE AS WELL ADS EVOLVING INDUSTRY BEST PRACTICES SOME IN THE PREVIOUS TALKS. PROCESS ANALYTICAL DEVELOPMENT IS ABSOLUTELY CRUCIAL AND WANT TO GIVE AN APPRECIATION OF THE TIME LINES THAT WE USE TO WORKING IT SO THE PRODUCT PROFILE VECTOR CONFIGURATION DETERMINES POTENCY AND AT SOME POINT YOU HAVE TO LOCK THAT IN WHATEVER THE TRANSGENE IS CODON OPTIMIZED ALL REGULATORY ELEMENTS, PARTICULAR CAPSID OR ENVELOPE. THEN IN TERMS OF TARGET PRODUCT PROFILE THE FORMULATION OF THAT VECTOR OBVIOUSLY IS SINGLE USE CONTAINERS, THAT'S GOING TO DRIVE LOT SIZE AND CONSUMPTION THE FILL VOLUME, DELIVERED VOLUME AS WELL AS THE LABEL AND PACKAGING CONFIGURATION AND STORAGE AND SHIPPING. CONDITIONS. SO SPECIFICATIONS OF THESE PRODUCTS ARE SET BASED ON DATA. WHAT ARE CRITICAL QUALITY ATTRIBUTES, JOSH WENT THROUGH SOME OF THAT AND DEFINING THOSE QUALITY ATTRIBUTES, BASED ON DATA THAT DRIVES THOSE LIMITS. THE OTHER QUESTION WE GET A LOT IS BY PRODUCTS EX-VIVO USE HAVE TO BE MANUFACTURED TO DRUG STANDARDS, OR CAN THEY REMAIN UNPURIFIED, THAT AN EVOLVING LANDSCAPE. SPECIFICATIONS OF THE PRODUCT WERE SET BEFORE MANUFACTURING BEGINS BUT INVOLVE AND GET NARROWED OVER THE COURSE OF CLINICAL DEVELOPMENT IN TERMS OF MANUFACTURING STRATEGIES. OBVIOUSLY DRIVEN BY INDICATION AND THE EXPECTED PATIENT POPULATION OR MARKET AND THE CHOICE OF THAT MANUFACTURING PLATFORM IS REALLY HAND IN HAND WITH THE SCALE THAT'S NEEDED. I WILL GET INTO THAT IN A MINUTE. FROM MOST CLIENTS LIKE YOU HEARD AND KEN AND JAYSSON'S UNITS WANT RAPID PROOF OF CONCEPT, THEY WANT TO GET INTO THE PATIENTS, DEMONSTRATE PROOF OF CONCEPT OR TREAT RARE DISEASE AS QUICKLY AS POSSIBLE. YOU HAVE OTHER CLIENTS WHO WANT TO INVEST MULTI-MONTHS, MORE THAN A YEAR INTO PROCESS THAT IS DESTINED FOR A SEAMLESS TRANSITION TO COMMERCIAL MANUFACTURING THEN THERE'S A WHOLE ELEMENT THAT I WON'T SPEND TOO MUCH TIME ON IN TERMS OF WHETHER CLIENTS WANT TO OWN THEIR OWN MANUFACTURING REAGENTS, WHETHER THEY LICENSED THOSE, WHAT THE STATE OF THOSE ARE. OBVIOUSLY THOSE DRIVE WHAT MANUFACTURING PLATFORM IS. SO IN TERMS OF PROCESS DESIGN AND DEVELOPMENT THAT IS ADS LONG AS YOU WANT TO MAKE IT REALLY. USUALLY MULTI-MONTH, NINE TO 12 MONTHS, IN TERMS OF CLONING CELL LINES, STARTING AT SINGLE CELL CLONES, OPTIMIZING GROWTH CONDITIONS LIKE JOSH WAS TALKING ABOUT. OPTIMIZING THE PRODUCTION WHETHER THOSE HAVE -- THOSE CELLS HAVE TO BE ADAPTED TO SERUM FREE SUGGESTION INTENTION CAN TAKE A LOT OF MONTHS, THE REASON THAT IS BECAUSE THE CYCLE TIME TO EVALUATE THOSE CHANGES IS LONG. WE ARE LOOKING FOR AN END POINT WHICH IS VIRAL VECTOR PRODUCTION AND REALLY INFECTIOUS VIRAL VECTOR PRODUCTION. TO GO THROUGH A CHANGE OR SERIES OF VARIABLES AND THEN GENERATE THAT VECTOR TITER THAT VECTOR AND COME BACK AND SAY THAT UPSTREAM CHANGE WAS BENEFICIAL OR NOT IS THE ANALYSIS. ASSAY DEVELOPMENT, OBVIOUSLY CANNOT PERFORM ROBUST PROCESS DEVELOPMENT UNLESS YOU HAVE THE ANALYTICS IN PLACE. TO MONITOR THOSE CHANGES. THE DRUG SUBSTANCE MAXIMIZING YIELD AS I SAID ARE REALLY BASED ON INFECTIOUS VECTOR, OPTIMIZING PURITY AND POTENCY ADS WELL AS STABILITY IDENTIFYING RAW MATERIALS, SOURCING THEM, MAKING SURE THEY'RE HIGH ENOUGH QUALITY AND AVAILABILITY IMPLEMENTING RAW MATERIALS TESTING AT LATER STAGES. AND IN SOME CASES HAVING TO MANUFACTURE RAW MATERIALS IN HOUSE THAT FEED INTO THE MANUFACTURING OF THE VECTOR. I THINK YOU HEARD FROM JAYSSON, THEY'RE INVESTING PLASMID MANUFACTURING AND THOSE THINGS TO SECURE THAT RAW MATERIAL SU PLY. WORKING IN CLOSED SYSTEMS, GETTING INTO AUTOMATION, AND SCALE UP, ADS WELL AS DEFINED SAMPLING POINTS WHERE TEST SAMPLES ARE PULLED FROM DIFFERENT MATRICES AND THEY HAVE THEIR OWN STABILITY PROFILES AS WELL. MONITORING THOSE, UNDERSTANDING THE DEGREES OF FREEDOM YOU HAVE WITH CERTAIN SAMPLES, IS CRITICAL WHEN YOU ARE THEN DETERMINING THE EFFICIENCIES OF PURIFICATION OR ABILITY TO HOLD THE PROCESS OF CERTAIN STEPS SUBSTRATES, THIS IS ONE DATA CLIP HERE, SHOWING SF 9 CELL LINE WHERE EITHER FOR ARCV PRODUCTION ON THE LEFT OR AV 5 AND AV 6 ON THE RIGHT. CLONING ONE IS A PRODUCER THAT'S ROUGHLY TWO TO FOUR TIMES BETTER THAN SF 9 POPULATION ITSELF. IN ADDITION WHEN YOU USE A DEVICE LIKE THE CELL SHOWN HERE YOU CAN PHOTOGRAPHICALLY DOCUMENT YOUR CELL SUBSTRATES COME FROM SINGLE CELL AND THEREFORE HAVE GENETIC PURITY. MANUFACTURING PLATFORMS ARE PRETTY VARIED. WE'RE MAKING AAV FIVE DIFFERENT WAYS, PRODUCE CELL LINES WITH ADENO, ADHERENCE TRANSIENT TRANSFECTION. SUSPENSION WITH HERPES VIRUS AS WELL AS SUSPENSION WITH BACULA VIRUS. SUSPENSION MANUFACTURING, LIPTY VIRAL VECTORS BY TRANSIENT TRANSFECTION AND PRODUCER CELL LINES, SOME COME FROM -- AS WELL AS TRANSIENT TRANSFECTION. THOSE ON A VARIETY OF SCALES AT OUR THREE LOCATIONS. 200 TO 500 LITEDDER SCALE AT TANK IN FLORIDA WITH FLAT STOCK PROCESSES UP TO 16 HYPE STACK 36 FOR -- 48 HYPER STACK 36s FOR PHASE 1, 2 MATERIAL. 48 BY -- HYPERSTACK 36 SCALE CARRIES THROUGH TO OUR COMMERCIAL FACILITIES AND MASSACHUSETTS. AND WE ARE ON A CELL LIST AS WELL, 500, THIS IS THE EQUIVALENT OF 300 HYPERSTACKS. HYPERSTACK IS EQUIVALENT TO FOUR CELL FACTORIES SO 1200 CELL FACTORIES WORTH ON ISL IS 500. THOSE UPSTREAM PRODUCTION PROCESSES ARE THEN OVERLAID ON TOP OF VARIETY OF PURIFYCATION TECHNOLOGIES, CHROMATOGRAPHIC SENTRY FEW GAL, FILTRATION SECONDNOLOGIES, ALSO DOING VIRAL ACTIVATION VIRAL CLEARANCE STUDIES FOR OUR CLIENTS, INSERTING STEPS INTO THE PROCESS AND ELIMINATE OR GREATLY REDUCE AGENTS. THIS IS A TIME LINE PROCESS REQUIRING FULL DEVELOPMENT, CLONING OF CELLS, CLONING OF BACCULA VIRUSES, SCREENING, UP NO 20 CLONES EACH OF CELLS AND TWO DIFFERENT BACCULAS, ESTABLISHING ANALYTICS. AND THEN GENERATING THE MASTER CELL AND MASTER VIRAL BANKS. THAT'S A TIME LINE THAT SPANS IN THIS EXAMPLE 16 MONTHS. WE ARE FEEDING IN PROCESS DEVELOPMENT THE PRE-CURSORS TO THOSE MASTER CELL BANKS AND INTO PRODUCTIONS THAT THEN GO INTO DOWNSTREAM PURIFICATION DEVELOPMENT AND THEN ULTIMATELY WE CAN GET TO A PRE-CLINICAL TOX LOT ABOUT MONTH 14 USING BACCULA. THAT THEN IN PARALLEL STARTING TO PREP AND THEN MANUFACTURE A CLINICAL LOT. SO THE POINT HERE IS THAT TIME LINE OF APPROXIMATELY 20 MONTHS GETS YOU A LOT THAT IS 10 TO 17 AAV VECTOR GENOMES, IF YOU DID THE SAME THING COMPARATIVELY IN TRIPLE TRANSFECTION, CLIENT DOING THIS IN 200 LITTER FORMAT, YOU CAN GET YOUR 10 TO 16th FOR YOUR TOX STUDY QUICKER, THERE IN MONTH AT START OF MONTH 11 AND THEN YOUR PRODUCTION OF CLINIC, FIRST LOT ROLLING OFF IN MONTH 16, BUT THEN YOU HAVE TO DO FIVE LOTS TO GET YOUR 10 TO THE 17th BATCH. SO YOU GET ROUGHLY SIMILAR TIME LINES THERE. REAL GAIN IS WHEN YOU GO TO MAKE SUBSEQUENT LOTS, YOU HAVE YOUR BANKS AND EVERYTHING READY TO GO. WITH BACK LA YOU GET 10 TO 17th IN FIVE MONTHS FROM PRODUCTION WHICH IS ROUGHLY 6 TO 8 WEEKS. THREE TO FOUR MONTHS FOR RELEASE TESTING. WITH TRIPLE TRANSFECTION YOU HAVE TO CONTINUE TO MAKE YOUR FIVE LOTS 200 LITTER TANK SCALE TO GET 10 TO THE 17th EACH TIME. THE DRUG PRODUCT ITSELF, THE FORMULATION REALLY WE WORK WITH CLIENTS A LOT ON THE FORMULATION WHERE IT HAS TO BE COMPATIBLE WITH THE TARGET ITSELF. COMPATIBLE WITH THE DELIVERY DEVICE, AS WELL AS STABILITY PROFILES THAT SUPPORT LONG TERM STORAGE AS WELL AS MANIPULATION AT THE BEDSIDE. SO IN SOME OF THESE CASES, INVESTIGATORS, PHARMACIST IS BUTTING OUT MULTIPLE VIALS AND BAGS, COMBINE THEM TO A SINGLE DRIP BAG INLINE FILTER ON THAT IV LINE, THAT TAKES TIME. DEMONSTRATING STABILITY ALL THE WAY TO THE MAIN IS IMPORTANT. ANOTHER AREA WE ARE WORKING IN IS THE PACKAGING. CZ CRYSTALIZE KNIT IS A CYCLICAL POLYMER TYPE VILE. IT'S A NEW ENTRANT ON THE MARKET. WE ARE PUTTING MORE AND MORE PRODUCT INTO THESE VIALS. THEY ARE REALLY EXCELLENT IN TERMS OF SHIPPING. THEY DON'T BREAK, TRADITIONAL GLASS VIALS YOU GET CHIPPING AND BREAKING IN TRANSIT. THEN THERE'S VERY GOOD COMPATIBILITY WITH VECTOR IN FORMULATION. STORE AND PACK, GETTING TO THE CLINICAL SITE. IN ITS MOST POW TERM FORM IS CRITICAL. WE DO A LOT OF WORK ON SHIPPING STUDIES AND VALIDATING SHIPPING TO THE CLIENT OR TO THE CLINICAL SITE. ONE BIGGEST HURDLE FOR US IS I GUESS WITH ANYONE IS TECH TRANSFER FROM THE CLIENT. WHAT IS THE STATUS OF MATERIALS, THE PROCESS, AND THEY'RE HANDING US? AND INVARIABLY ACCORDING TO THE CLIENT IT'S IN A MUCH MORE MATURE STATE THAN IT REALLY IS. IT WAS GREAT TO HEAR JOSH TALK ABOUT E. COLI MASTER CELL BANKS AND HAVING PLASMIDS THAT ARE DERIVED FROM QUALIFIED BANKS. SOMETIMES WE ARE GETTING A MATURE PROCESS, WE ARE GETTING A COHORT OF CLIENTS WHO ARE SWITCHING OVER TO US IN LATE PHASE 2. EVEN IN SOME CASES CLIENTS STARTED PHASE 3. THEY ARE BRINGING US A VERY MATURE PROCESS AT THAT POINT. WHETHER THEY'RE BRINGING RESEARCH CELL LINE OR FULLY QUALIFIED BANK WITH HISTORY MATTERS, AND FOR THAT TO COME IN TO OUR FACILITY AND NOT JEOPARDIZE OTHER PROGRAMS, WE HAVE A CERTAIN MINIMUM SET OF QUALITY STANDARDS THAT RAW MATERIALS, VIRAL BANKS CELL BANKS COMING INTO OUR FACILITY MUST MEET. WE ARE EXPLICIT WITH OUR CLIENTS, WHAT THEY ARE. MATURITY OF ANALYTICS, I WILL TALK ABOUT THAT IN A MINUTE. REFERENCE STANDARDS, WE HAVE CLIENTS PICKING UP PROGRAMS THAT HAVE LONG HISTORIES, AN DON'T HAVE ACCESS TO MATERIALS IN HUMAN BEINGS. SO TO ESTABLISH ANALYTICS OR OPTIMIZE ANALYTICS WITH REFERENCE OF WHAT HAS BEEN SAID IN THE PAST IN TERMS OF DOSE, IS PROTY CHALLENGING. ANOTHER ASPECT IS WHETHER THE PROCESS IN MOST CASES WE CHANGE DIFFERENT EQUIPMENT, WE ARE USING SINGLE USE AND CLOSED SYSTEMS. ONE EXAMPLE OF A FACILITY FIT IS PROCESSES WHERE GRAVITY TRANSFERS USED SMALL SCALE. WHEN YOU START USING PUMPS AND PARASTALL TICK PUMPS, WE TRIED TO SEED 2000 LATER REACTOR ARE YOU GRINDING THE CELLS WITH THE PARASTALTIC PUMP, YOU HAVE TO SWITCH PUMPS OR SHOW CELLS ARE VIABLE AT CERTAIN CONDITIONS. YOU ALSO NEED TO ESTABLISH HOLD STEPS. FOR THE EXPECTED. SO BETWEEN DRUG SUBSTANCE AFTER POLISHING COLUMN HERE AND DRUG PRODUCT IS USUALLY A HOLD STEP. THAT'S USUALLY A FROZEN HOLD STEP SO THAT Q SEQ CAN DO VECTOR GENOME TITER AND THEN THAT WILL DRIVE FORMULATIN DILUTED OR CONCENTRATED TO MEET THE TARGET CONCENTRATION IN THE VILE. YOU ALSO NEED TO DO HOLD STEPS FOR UNEXPECTED. HOUR OUTAGES, EQUIPMENT FAILURES, AND THE LIKE. IN TERMS OF QUALITY DESIGNING AS IS DE NOVO, WE DO A LOT OF THAT BUT WE'RE BRINGING IN ASSAYS FROM CLIENTS WHO HAVE BEEN RUNNING THEM FOR YEARS. THOSE SPAN NUCLEIC ACID BASED PROTEIN BASED ANALYTICAL CHEMCAL BASED ASSAYS. YOU NEED TO GENERATE AND QUALIFY CELL SUBSTRATES IN QUALITY CONTROL, FOR INFECTIOUS TITER AND OTHER CELL BASED ASSAYS. GENERATE ENOUGH STANDARDS AND CONTROLS TO SUSTAIN MULTI-PLOT RELEASE. THEN OBVIOUSLY SOURCE PROPER REAGENTS AND DEVELOP THOSE TEST RECORDS, SET THOSE LIMITS AND UNDERSTAND THE ASSAYS, QUALIFY AND VALIDATE THE ASSAY AND CERTAINLY IS A SUBSET OF ASSAYS THAT ARE OUTSOURCED, THINGS MOSTLY SAFETY TESTING AREA. WHERE THOSE HAVE TO BE TRACKED AND REPORTS REVIEWED AND FINALIZED. I WON'T GO INTO EACH OF THESE ANALYTICS FOR EACH VECTOR CLASS BUT SUFFICE IT TO SAY THERE ARE LOTS OF ANALYTICS WE PERFORM, WE HAVE OVER 90 QUALIFIED ASSAYS. AS NEW PRODUCTS COME IN, WE DO SUITABILITY STUDIES AND OTHER CHARACTERIZATIONS TO ESTABLISH THOSE ASSAYS. ASSAYS SPAN NOT ONLY PRODUCT REGENERATION THEMSELVES BUT ALSO PROCESS REAGENT RESIDUALS SUCH AS FINTY CHROMATOGRAPHY RESIN, MOLECULES AND BENZINASE AND THE LIKE. ADENO, SAME THING. WIDE VARIETY OF ASSAYS, WIDE VARIETY OF TECHNOLOGIES THAT WE HAVE ESTABLISHED AND EXECUTE FOR EACH OF THESE VECTOR CLASSES. QUALIFYING VALIDATE AND VERIFY ASSAYS PER ICH GUIDELINES, PREQUALIFIED ASSAYS SYSTEM STABILITY WE DO PROCESS RELEASE TESTING. GENERATE REFERENCE STANDARDS AND THEN CONDUCT THOSE STABILITY STUDIES THAT ARE MULTI-YEAR. THOSE ARE BACKED BY QMS WHERE WE ESTABLISH QUALITY AGREEMENTS WITH OUR CLIENTS, AND ENSURE EMPLOYEES ARE TRAINED, RAW MATERIALS ARE TESTED AND RELEASED CORRECTLY, BATCHES ARE REVIEWED AND RELEASED. DEVIATIONS IN CAPA PROGRAM, AUDITS, EM TRENDING AND EVERYTHING ON THE LIST. LAST PIECE IS PROCESS VALIDATION. MANY OF YOU MAY BE AWAYS AWAY FROM THIS BUT WE HAVE A LIFE CYCLE APPROACH THAT ADDRESSES THREE STAGES OF PROCESS VALIDATION. BEGINNING WITH PROCESS CHARACTERIZATIONS GENERATING SCALE DOWN MODEL OF THE PROCESS ITSELF. THEN EXECUTING MULTI-VARIANT ANALYSES AND ONE FACTORIAL ANALYSES, TO DEFINE WHAT EDGES OF FAILURE ON A PROCESS ARE. THAT LEADS TO QPP QUALITY PROCESS -- IF A BLA IS APPROVED, THOSE LOTS COULD BE SOLD AS COMMERCIAL PRODUCT. AFTER LICENSURE CONTINUE PROCESS VERIFICATION IS SOMETHING WE PERFORM. HERE IS AN HE WILL STRAYTIVE TIME LINE. DON'T GASP TOO MUCH, THIS IS THREE YEARS OF WORK TO DO THOSE THREE STAGES, PROCESS VALIDATION. BEGINNING WITH ASSAY TRANSFER. HAVE PIVOTAL RUNS, ENGINEERING RUNS, PROCESS CHARACTERIZATIONS AND PPQs WITH FINAL REPORT. THIS IS A TIME LINE IF SOMEONE CAME TO US WITH MATURE PROCESS ALREADY PAST PHASE 2, GETTING THIS ESTABLISHED, YOU HAVE TO ESTABLISH AND CONDUCT QPQ RUNS AT SITE OF COMMERCIAL MANUFACTURING. LASTLY SPEAKING WITH DEKNOW, I WANT TO BRING UP SOME COMMON QUESTIONS WE ENCOUNTER IS LATE STAGE COMMERCIAL SUPPLY AND GNP EQUAL TO SCALE? THE ANSWER IS NO. WE HAVE PLENTY OF PRODUCTS THAT ARE VERY SMALL SCALE ACTUALLY THAT STILL DEMAND THE RIGOR OF DATA ANALYTICS AND DEEP UNDERSTANDING OF THE PROCESS AND MATURE PROCESS CONTROLS. IN TERMS OF VILE FILL SIZE YOU HEARD OVER THE COURSE OF TODAY PATIENTS ARE RECEIVING VERY LARGE DOSES. COMBINE HUNDREDS OF VIALS IN BEDSIDE IS CUMBERSOME ADS WELL AS IMPOSES CERTAIN RISK. SO CAN LARGER FILLS BE CONDUCTED WHILE AT THE SAME TIME GENERATING SIDE SAMPLES FOR RELEASE AND STABILITY TESTING THAT ARE REPRESENTATIVE OF THE LARGE FILL TO CONSERVE VECTOR AND REFLECT PRODUCT IN ITS ULTIMATE CONTAINERS. THERE'S CREATIVE THINKING OUT THERE ON THIS. THAT WILL BECOME PRACTICE. WHAT ABOUT TERMINAL FILTRATION OF ENVELOPE VIRUS, WE HAVE CLIENTS WHO COME HISTORICALLY WITH .45-MICRON FILTERED LENTIAND WE HAVE OTHER CLIENTS DOING .22. OBVIOUSLY FOR RISK PROFILE WE WANT TO STICK TO .22 AND HAVING TO DO A WHOLE STUDIES AROUND FILTRATION IS SOMETHING PRETTY COMMON. DEFINITION OF DOSE. TWO MORE SLIDES. DEFINITION OF DOSE. IF A DOSE HUMAN BEING RECEIVED A TEN ML DOSE, THERAPEUTICALLY EFFECTIVE, TITERRED AT ORIGINAL MANUFACTURE OF 58 PER ML. IN OUR HAND WE GET THAT ASSAY, WE REALIZE THERE'S A MISMATCH AND PCR PRIMER ADS LONG AS THE PRODUCT HAS BEEN MADE. WE CORRECT THAT, WE GET HIGHER EFFICIENCY, ALL OF A SUDDEN WHAT'S IN THAT VILE 15 AND 12. SO GOING FORWARD, YOU HAVE TO REPORT THE NEW TITER ON THAT PRODUCT. THE PATIENT IS STILL GOING TO GET TEN MILLILITER OF THE PRODUCT BUT IT HAS A NEW UNIT DEFINITION. SWITCHING MANUFACTURING PLATFORMS WHEN SHOULD I DO THIS. DO I ADD AN ARM TO A CLINICAL STUDY, CAN I DO IT ANALYTICALLY, WHAT IS DEEPER BROADER ARRAY OF ANALYTICS TO DO IN ADDITION TO THE RELEASE, SWITCHING FROM 293T TO 293 CELLS FOR AV MANUFACTURING. SWITCHING FROM CENTRIFUGAL TO CHROMATOGRAPHIC SEPARATIONS INCLUDING POLISHING COLUMN, CELL LINE CLONALITY IS MULTI-MONTH ALONE AND ADVANTAGEOUS AGENT VIRAL AND CLINIC STUDIES ARE LIKELY AS WELL. ALL THESE IMPACT TIME LINE BUT I THINK THE INDUSTRY IS MOVING TOWARDS INCORPORATING MANY OF THESE CHANGES. SO LITTLE BIT ABOUT GRAMMAR. T ALL WE DO IS VIRAL VECTOR MANUFACTURING. AAV, ADENO, RETRO, LENTI, AND HERPES. WE ARE A TEAM OF 450 PEOPLE. OVER 12 YEAR TRACK RECORD, THOSE 600 COMMERCIAL LOTS CAME FROM OUR FACILITIES IN CAMBRIDGE WHEN WE ACQUIRED BIOGEN AS A BEIJING COMMERCIAL LOTS. THOSE ARE NOT GENE THERAPY COMMERCIAL LOTS BUT ON BOARDED A TEAM OF OVER 100 PEOPLE THAT MADE THOSE LOTS AND THAT EXPERTISE NOW. WE HAVE OVER 2000 SQUARE FEET OF FACILITIES WITH 16 CLEAN ROOMS, WE'RE BRINGING ANOTHER FOUR ONLINE BY Q1 AND ANOTHER TEN ONLINE AFTER THAT IN OUR LEXINGTON FACILITY IN THE SECOND QUARTER OF 2019. SO THANK YOU VERY MUCH. [APPLAUSE] >> THANK YOU TO OUR SPEAKERS. WE ARE GOING TO TAKE SOME QUESTIONS. WE'RE GOING OVER MAYBE FIVE MINUTES OR SO. WE WANT TO THANK SPEAKERS FOR THOUGHTFUL TALKS AND INTRODUCE DENISE GAVIN; SHE TRAINED IN DR. SAMULSKI'S LAB ON AAV VECTORS AND GENE THERAPY BRANCH CHIEF AND SHE'S YOUR POINT PERSON FOR QUESTIONS ABOUT MANUFACTURING. AND I ASK IF YOU ASK A QUESTION, PLEASE IDENTIFY YOURSELF FIRST. >> JAMES BROWN. THANKS, JUDE. FIRST TO ANSWER YOUR QUESTION, THEN I ASK MY QUESTION. I THINK THIS IS A GREAT PANEL AND THE WORD I KEPT HEAR HAG WAS EVOLVING SO I THINK ALL THESE THINGS ARE EVOLVING AND WE ADS CMO ARE TRYING TO DO WHAT Y'ALL ARE DOING AS WELL AS EVOLVE IN OUR CAPACITY AND THINGS LIKE THAT. AND SCALE UP AND SCALE OUT SO THERE'S A CLASS OF THERAPIES THAT ARE PATIENT SPECIFIC. MAKE SMALL AMOUNTS OF A LOT OF DIFFERENT MATERIALS THAT WERE EAGER TO SUPPORT AS WELL. SO ONE OF THE THINGS THAT I WANTED TO ASK IS WHEN YOU LOOK AT THE ASSAYS AND GREAT CRITERIA, YOU DID WESTERNS AND PIPETTING TO GET BETTER ANALYTICS. DO YOU SEE THAT EVOLVING AND IF SO IN THE DRUG PRODUCT OR RAW MATERIALS, WHAT ARE THE BIG CHANGES YOU SEE IN CHARACTERIZATIONSES OF PRODUCTS AND RAW MATERIALS? SO WHAT'S THE OTHER ASSAY LIKE THAT THAT YOU SEE, HEY, THIS IS GOING TO CHANGE DOWN THE LINE? SOME OF THOSE ARE STANDARD AND PROBABLY AREN'T GOING TO CHANGE BUT WHERE DO YOU SEE THE EVOLUTION WHEN IT COMES TO THE ANALYTICS FOR RAW MATERIALS AND FINAL PRODUCT? >> I KNOW SPECIFICALLY ONE WAY THAT WE'RE GOING TO HAVE TO START LOOKING AT THINGS ON RAW MATERIAL SIDE IS GOING DEEPER AND DIVING INTO THE CONTAMINANT PROFILE THAT COULD BE WITHIN ANY RAW MATERIALS THAT WE HAVE AND WHAT CAN BE PASSED ON INTO THE VECTOR. IT'S NOT THE SAME PURITY PROFILE YOU WOULD HAVE IN SAY A VACCINE BECAUSE YOU HAVE ACTIVE COMPONENTS WITH THE PRODUCT THAT WE ARE MAKING. I FOR SEE MORE STRINGENT VIEW WHAT CONTAMINANTS ARE AND WHAT THE KID MEANS THE ACTUAL PRODUCT ITSELF. >> JULIE (INAUDIBLE) THANK YOU FOR YOUR EXPERIENCE, INSIGHTS. MULTIPLE PEOPLE MENTIONED LOT TO LOT COMPARABILITY AND SHOWING THE ROBUSTNESS OF YOUR PROCESS. WITH SMALL ORPHAN DISEASE OFTEN YOU DOING VERY FEW LOTS DURING YOUR DEVELOPMENT PROCESS. ANY THOUGHT HOW YOU CAN CONTROL FOR THAT AND SHOW ROBUSTNESS AND -- FOR THE FDA ALSO THIS IS GOING TO BE A PARADIGM SHIFT COMPARED TO SMALL MOLECULE OLIGOS HOW WE SHOW COMPARABILITY ACROSS THE DIFFERENT LOTS. >> I CAN ANSWER FROM OUR PERSPECTIVE. DURING DEVELOPMENT YOU MAKE MULTIPLE LOTS AND YOU KEEP THE SAME PROCESS GOING YOU CAN CHARACTERIZE THOSE LOTS EARLY, THE MORE CHARACTERIZATIONS YOU& DO ON YOUR PRODUCT IN THE BEGINNING THE MORE DATA YOU HAVE WHEN IT COMES TO MAKING BIGGER SCALING UP OR MAKING CHANGES SO YOU CAN DO COMPARABILITY THAT WAY. IF YOU ONLY MAKE A FEW LOTS IT'S HARDER TO DO THAT. SO THERE'S -- YOU CAN ALSO MAKE -- THERE'S A CHANCE YOU CAN MAKE MULTIPLE SMALLER LOTS AND USE THOSE. THAT'S JUST ONE OPTION. I DON'T KNOW THAT'S THE WAY YOU WANT TO GO, WHAT YOUR PROCESS IS, CHARACTERIZING EACH TIME GETS MORE EXPENSIVE. WE UNDERSTAND THAT. SOMETIMES YOU CAN WORK SOME OF THESE PROCESS DEVELOPMENT INTO ENGINEERING RUNS AND THEN DO LIKE THOSE KIND OF THINGS, WORK THAT INTO SETTING PPQ SPECIFICATION AND GOING FORWARD THAT WAY. FROM KNOWING WHAT DEVELOPERS DO. >> COMMUNITIES LEARN FROM EXPERIENCE, I THINK ANYONE TODAY ANYONE MAKING A BATCH WOULD SET A VILE ASIDE REALIZENING THE FUTURE THAT MAYBE ALLOWS THEM TO DO COMPARABILITY STUDIES TO GO TO SOMETHING ELSE. WHEREAS IN THE EARLY DAYS YOU JUST USED EVERYTHING YOU HAD AND THEN MADE THE NEXT PREP. WE ARE SLOWLY BECOMING FAMILIAR WITH THOSE VARIABILITIES AND INCORPORATING THEM. >> THE FIRST LOTS ARE EXTREMELY USEFUL AS REFERENCE MATERIALS. >> NATHAN JONES, -- THERAPEUTICS. I HAVE TWO QUESTIONS, FIRST FOR JAYSSON. YOU MADE THE COMMENT YOU USE MEDIA RELEASE INSTEAD OF CELL DISRUPTION METHOD, CAN YOU GO INTO THAT IN MORE DETAIL. >> SLIGHTLY LITTLE MORE DETAIL. HOME COOKING BUT WE ARE USING THE SALT SPIKE TO REALS VECTOR INTO THE CELL -- RELEASE THE VECTOR AND WE GET SOLID RECOVERIES AND LESS DOWNSTREAM DEBRIS TO TAKE OUT WHEN WE GO INTO CLARIFICATION. >> MAKES SENSE. MY SECOND QUESTION, DR. SNYDER WAS THE ONLY ONE THAT MADE MENTION OF ADVENTITIOUS VIRUS SAFETY. A LOT OF PROCESSES WE SAW TODAY WERE FOR HEK 293 TRIPLE TRANSFECTION PROCESS. VERY LITTLE CAPABILITY OF VIRUS REMOVAL SO I'M CURIOUS WHAT IS THE EXPECTATION FOR ADVENTITIOUS VIRUS SAFETY? ARE VALIDATION STUDIES REQUIRED FOR A PROCESS THAT USES VECTOR 392 CELLS? >> DEPENDS ON THE VECTOR, CERTAINLY FOR AAV AND ADENO, YOU CAN DO CLEARANCE STEPS, FOR INSTANCE DETERGENT TREATMENT WILL GET RID OF ENVELOPE VIRUSES SO YOU DO VIRAL ACTIVATION VIRAL CLEARANCE STUDIES WHERE YOU'RE SPIKING IN MODEL VIRUSES. LARGE ENVELOPE, SMALL NON-ENVELOPE, U SEE WHAT THE REMOVAL IS THROUGHOUT THE PROCESS IS, WHAT THE STEP REDUCTIONS ARE ACROSS EACH STEP. >> YOU CAN DO IT. WHAT IS THE EXPECTATION? >> I THINK THIS IS GOING TOWARDS THE EVOLVING INDUSTRY BEST PRACTICES WHICH IS THE AGENCY IS SEEING SUBMISSIONS THAT INCORPORATE THESE STEPS SO IT WILL BECOME A STANDARD. >> A LOT OF VECTORS ARE LIVE VIRUSES SO IT DEPENDS, THERE ARE SO MANY THINGS YOU CAN DO TO REMOVE ADVENTITIOUS AGENTS IN A LIVE VIRUS SETTING. IT DEPENDS ON THE VECTOR YOU ARE MAKING, DEPENDS ON THE PROCESS YOU HAVE, WHETHER VALIDATION STUDIES WOULD BE NECESSARY, WHEN YOU HAVE KNOWN HELPER VIRUS LIKE SAY MAKING AAV AND YOU HAVE KNOWN HELPER VIRUS, YOUR PROCESS HAS TO BE QUALIFIED AND VALIDATED AT ONE POINT TO SHOW THOSE VIRUSES ARE REMOVED AND REMOVING THOSE VIRUSES YOU ALSO IN YOUR PROCESS LOOK -- CAN LOOK AT MODEL VIRUSES. WE FOUND CELL LINES ARE CONTAMINATED WITH VIRUSES, AND SO THE PROCESS SHOULD BE ABLE TO REMOVE ANY CONTAMINATING VIRUSES. IF YOUR PROCESS -- IF YOU ARE MAKING A VECTOR NOT SUPPOSED TO HAVE OTHER VIRUSES IN IT, THEN YOU SHOULDN'T HAVE OTHER VIRUSES IN IT. >> THANK YOU. >> KEITH (INAUDIBLE) PFIZER. SO MY QUESTION IS PETER MARK SAID THIS MORNING, WE HAVE SEEN THE NEW FDA GUIDANCE THAT THERE'S THE PUSH TO ESTABLISH COMMERCIAL READY MANUFACTURING FOR PHASE 1, MORE OR LESS. I HAVE CQAs IN PLACE, CQPs IN PLACE, AND HOST OF OTHER THINGS IN PLACE BEFORE YOU START YOUR PHASE 1. I'M JUST WONDERING FROM THE PERSPECTIVE OF OF THE PANELISTS WHERE ARE THE CHALLENGE AND OPPORTUNITIES IN PUSHING PROCESS AND PRODUCT DEVELOPMENT EARLIER AND EARLIER AND HOW CAN WE ALIGN WITH THE NEW RECOMMENDATIONS. >> REALLY GOOD QUESTION, GOES BACK TO OWNING YOUR PROCESS, HAVING THAT PROCESS THAT YOU'RE WORKING ON. THERE'S OBVIOUSLY THERE'S COMPANIES OUT THERE THAT DON'T HAVE PROCESS REQUIRED GOING TO SOMEBODY THAT'S GOT A PROCESS. THE RESPONSIBILITY IS ON THEM TO HAVE A QUALIFIED OR VALIDATED PROCESS. I THINK IT'S COMPLICATED. EASIER IF YOU HAVE A PROCESS YOU HAVE A CONTROL OF THAT PROCESS AND DEVELOP IT BUT THE COMPLICATED PART IS THE COMPETITION, NUMBER OF PEOPLE, NUMBER OF GROUPS ARE GOING TO PUT OFF GIVING VECTOR INTO CLINIC TO SEE IF IT WORKS. IT BEAT BY THOSE YOU GET IN, VALIDATED PROD SYCES, YOU MAYBE ABLE TO MAKE UP FOR IN THE END, GENE THERAPY SEEMS LIKE FIRST PERSON WHO GETS IN IS THE WINWINNER. IT'S A DIFFICULT QUESTION. >> KEITH, CAN I ADD CONTEXT TO THAT? ONCE YOU LOOK AT IT A COUPLE OF WAYS IF YOU'RE A STRUCTURER AND USING THE SAME SEROTYPE AND YOU HAVE TAKEN ALL THE WAY TO COMMERCIAL AND SWAPPING OUT AND PUTTING IN A DIFFERENT GENE, CHANCES ARE YOU WILL HAVE A REGULATORY PATH THAT WILL BE PRETTY MUCH IN PLACE THAT YOU MIGHT BE ABLE TO BORROW OFF OF. I THINK WHERE THE FIELD NEEDS TO COME TOGETHER AND GO THROUGH SOME GROWING PAINS IS WHEN YOU HAVE FOUNDATIONS VERSUS COMMERCIAL AND ACADEMIC AND DIFFERENCE IN EXPENDITURE CAPABILITY, SAME PASSION TO ADDRESS THESE DISEASES, HOW DO WE GO FORWARD WHERE WE KNOW THE INFORMATION IS AVAILABLE BUT IS NOT ACCESSIBLE. THIS IS GOING TO BE A TASK FOR THE COMMUNITY TO COME UP WITH WAYS OF FACILITATING THAT TYPE OF INFORMATION TRANSFER. ONE OF THE THINGS THAT I SEE GOING FORWARD FOR THIS COMMUNITY AS THINGS EVOLVE THOSE WHO HAVE AND THOSE WHO HAVE NOT, AND INSTITUTIONS THAT CAN AFFORD INDIVIDUALS STACKED AROUND CORE AND THOSE THAT DON'T HAVE ACCESS TO IT, WILL HAVE TO CONTINUE TO GO TO PLACES THAT ARE GOING TO BE LESS EXPENSIVE, HAVE LESS ASSAYS, LESS QUALITY CONTROL, SO FORTH, SO THIS IS KIND OF A PLUG TO GET THE NIH TO PUT MORE MONEY INTO OPERATION AND PROVIDING THESE REAGENTS BECAUSE THE FIELD IS COMING UP AGE AND THIS IS A BOTTLENECK. I THINK IT'S SOMETHING KEN CAN SPEAK TO DIRECTLY AND JAYSSON. >> I THINK THAT PIPELINE, VERY COMPLICATED FROM EVERY ASPECT YOU CAN IMAGINE. I SEE IN SOME WAYS NIH PULLING BACK ON RESOURCES TO LET -- TO EVEN GET FOLKS TO UNDERSTAND THAT PROCESS AND THAT'S WHERE I THINK THE NIH NEEDS TO THINK ABOUT IT. SO PRODUCTION CAPACITY COMES ON DIFFERENT PLACE BUT EARLY STEPS, BEING ABLE TO UNDERSTAND AT THE START WHEN THEY GOT SOMETHING THAT LOOKS GOOD, WHAT IS YOUR VECTOR START WITH, BASIC THINGS. PEOPLE ARE THROWN IN RIGHT NOW AND THERE'S BEEN A LOT OF THINGS THAT NOT GONE FORWARD BECAUSE PEOPLE DIDN'T SPEND THE TIME TO UNDERSTAND THE BASIC THINGS ABOUT WHAT IS THIS GOING TO MEAN TO YOUR CAPACITY TO MAKE VECTOR AND BE ABLE TO GET YOUR TRIAL FORWARD. THAT'S WHERE I THIS I THINGS ARE PULLING AWAY (INAUDIBLE). FROM GOING FORWARD I THINK WORKING WITH THE AGENCY TO HAVE A VISION ON EACH PROGRAM THAT'S COMING UP WHAT IS YOUR PATIENT POPULATION, WHAT IS THE NEED AND MAKING SURE WE ARE MAKING DECISION AS A COMMUNITY, AS REGULATORY BODY, AS MANUFACTURERS, TO MAKE SURE WE HAVE DUAL PATHWAY, WE HAVE THE ABILITY TO SERVICE THE FIVE PATIENT ULTRA RARE DISEASE AS WELL AS 10,000 PATIENT DISEASE. IT'S A COLLECTIVE GROUP THINK THAT WE HAVE TO GET INTO THERE IS NO ONE PATHWAY, THERE ARE SNOWFLAKES WE HAVE TO WORK TOGETHER TO MAKE SURE THAT WE HAVE AN ACADEMIC PATHWAY, A FOUNDATIONAL PATHWAY, AND A BIG PHARMA PATHWAY AS WELL. ALL THE PLAYERS HAVE TO BE ABLE TO COMPETE. >> I'M GOING TO ASK ONE LAST QUESTION AT THE COLLEAGUES DENISE DOWN AT THE END AND GOING FORWARD WITH THE EXPECTATION OF SHOWING PRODUCTION COMMERCIALIZATION, WE CAN SEE FROM RICHARD'S COMMENTS THE TIME THAT'S INVOLVED IN GOING THROUGH TRADITIONAL PROCESS. IS THE FDA STARTING TO HOOK AT THESE THINGS FROM A PERSPECTIVE HOW WE OFFICIALLY MOVE THESE DRUGS OUT VERSUS WHAT'S REQUIRED TO GET TO COMMERCIALIZATION. >> WE GREW EACH PACKAGE AS IT COMES IN, WE DON'T KNOW WHETHER THINKING DEVELOPING A PROCESS, MAYBE YOU THINK IS THIS SOMETHING I CAN USE IS THIS SOMETHING THAT CAN BE SCALABLE, ARE REAGENTS SOMETHING THAT ARE HIGH ENOUGH QUALITY TO GO FORWARD, INFORMATION I GET FROM THIS PHASE 1 TRIAL IS IT GOING TO BE ABLE TO BE CARRIED FORWARD TO THE NEXT TRIAL TO COMMERCIAL PROCESS. CAN YOU TRANSFER THAT INFORMATION, IS THERE SO MUCH OF THE INFORMATION THAT YOU HAVE FOR YOUR PHASE 1 TRIAL THAT YOU JUST HOPE TO GET INTO THE CLINIC AND SEE IF IT WORKS. SOMETIMES THAT MAY OR MAY NOT TRANSFER TO NEXT LEVEL BUT SOMETIMES IT MAY. DEPENDS HOW -- CAREFULLY YOU THINK ABOUT THINGS FROM THE VERY BEGINNING. SO YOU KNOW YOU HAD A GENE THAT YOU WANT TO TREAT, AND MAYBE YOU CAN CONSULT WITH PEOPLE TO HELP DEVELOP A PROCESS THAT WILL BE ABLE TO CARRY FORWARD. AND CONSULT WITH DIFFERENT CMOs OR DIFFERENT ACADEMIC CORES AND FIGURE OUT THE BEST WAY FORWARD FOR YOUR PRODUCT. I THINK THAT'S KIND OF WHAT HE WAS SAYING. I DON'T THINK THAT HE MEANT WHEN YOU STARTED PHASE 1 TRIAL YOU HAVE TO BE READY FOR COMMERCIALIZATION. >> I THINK AGAIN ONE OF THE TAKE HOME MESSAGES HERE ASK A LOT OF US SPENT THE LAST 20 YEARS TRYING TO GET PROOF OF CONCEPT AND THAT'S THE FORMULA GOING FORWARD IS PROOF OF CONCEPT IS THE NORM FOR THESE TRIALS COMING IN, NOW NEEDS TO BE THOUGHT THROUGH WITH THE MIND SET OF WHAT NEXT. WHAT ARE WE GOING TO DO NEXT. THAT'S GETTING PRODUCTION AND EVERYTHING ALIGNED SO IF IT DOES WORK HOW DO YOU TAKE IT THROUGH. I THINK TODAY IS TRYING TO ECHO IS THE PROCESS THAT ARE INVOLVED TO MAKE THIS HAPPEN EFFICIENTLY AND THE COST INVOLVED IN IT AND ALSO EVER CHANGING LANDSCAPE WHEN TECHNOLOGY KEEPS ADVANCING. SO I WILL LET ANDREW GIVE A FINAL COMMENT BEFORE WE CLOSE BUT APPRECIATE EVERYBODY'S STAYING THROUGH THIS SESSION AND LISTENING TO THE NERD TEAM ONE MORE TIME. >> THANK YOU. AND THANK YOU TO OUR SPEAKERS. AND DO YOU HAVE SOMETHING TO SAY ABOUT TOMORROW? WE'LL SEE EVERYBODY BRIGHT AND EARLY TOMORROW MORNING.