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Demystifying Medicine 2016: Cholesterol: Too Much and Too Little Are Bad for Your Health

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>> GOOD AFTERNOON. WE HAVE A SMALL BUT DISCRIMINATING AUDIENCE TODAY, SOMEWHERE ALWAYS BETWEEN TWO AND FOUR HUNDRED NIH COLLEAGUES WHO ARE WATCHING LIVE, SO WE ENCOURAGE YOU TO ASK QUESTIONS DURING TALKS, AFTERWARDS, THE ONLY THING I SUGGEST IS WAIT TILL WE CAN GET A MICROPHONE TO YOU BECAUSE I KEEP GETTING E-MAILS SAYING THE ANSWERS WERE WONDERFUL BUT I WONDER WHAT THE QUESTIONS WERE. YOU HAVEN'T BEEN HERE BEFORE, HAVE YOU? WHO HASN'T BEEN HERE BEFORE? OKAY, WHAT'S THAT? >> (INAUDIBLE). >> RIGHT. SO WHY DO WE SHOW THE BROOKLYN BRIDGE? THREE REASONS. ONE, BECAUSE MY GRANDFATHER TOOK THAT PICTURE BACK IN THE 1880s.

THE SECOND IS THE WHOLE PURPOSE OF THIS COURSE IS A BRIDGE-BUILDING EXERCISE, THE WHOLE IDEA IS TO GET PEOPLE WHO KNOW A GREAT DEAL ABOUT REDUCTION IN SCIENCE, PARTICULARLY AT THE STUDENT AND POSTDOC LEVEL, TO BE EXPOSED TO SOME OF THE MAJOR CLINICAL PROBLEMS BUT PRESENTED NOT IN A FORMAL CLINICAL WAY BUT MORE LIKE A SCIENTIFIC AMERICAN LEVEL TO ACQUAINT YOU WITH WHAT ARE DISEASES LIKE IN YOUR WORDS, AND MUCH OF THE PUBLIC INFORMATION ABOUT THESE DISEASES, MUCH OF IT IS BADLY (INDISCERNIBLE) SO WE BRING TOGETHER MOST OF THE TIME A LIVE PATIENT AND THE TRANSLATIONAL CLINICAL INVESTIGATOR AND MORE BASIC SCIENCE.

WE GET MIXED UP FROM TIME TO TIME. SO THE WHOLE IDEA IS WE'RE ON THE CATWALK CROSSING OVER TO BROOKLYN, AND ONCE YOU BUILD A BRIDGE LIFE IS NEVER THE SAME ON EITHER SIDE. THAT'S THE WHOLE IDEA. HOPEFULLY EVERYBODY WILL LEARN SOMETHING AND BE EXCITED BY WHAT THEY LEARN. THE INTENT IS NOT TO HAVE YOU WRITE DOWN THE OLD-FASHIONED MEDICAL STUDENT OR GRADUATE STUDENT EVERYTHING SOMEBODY SAYS. OKAY, HAVING SAID THAT, THERE'S A FINAL EXAM FOR THIS COURSE FOR THOSE LISTENING AND HERE. IF YOU ATTENDED 50% OF THE SESSIONS, LOGGED IN, AND STARTING NEXT WEEK YOU GO TO THE WEBSITE AND THERE'S A MULTIPLE CHOICE SET OF QUESTIONS. NOT GOING TO KEEP YOU UP LATE AT NIGHT BUT HOPEFULLY DEAL WITH IMPORTANT HIGHLIGHTS OF THE VARIOUS TOPICS AND THINGS THAT WERE DISCUSSED. YOU CAN TAKE IT AS MANY TIMES AS YOU WANT, ONCE YOU HIT THE PASSING GRADE YOU'LL GET A CERTIFICATE FROM THE NIH THAT HAS SOME INTRINSIC VALUE BUT I WOULDN'T WANT TO USE IT TO RIDE THE METRO WITH OR ANYTHING LIKE THAT.

OKAY. NOW, I WOULD ALSO LIKE TO MENTION TO FOLKS LISTENING THAT NEXT WEEK WE REALLY HAVE AN EXTRAORDINARY EVENT. TOM KROMEKIS, ANYBODY KNOW WHO HE IS? YOU RECALL THE EXPLORATION OF PLUTO ABOUT A YEAR AGO, THE INCREDIBLE PHOTOGRAPH? OKAY. SO THE PROFESSOR IS THE MAN WHO DIRECTS THE ENTIRE ROBOTIC SPACE PROGRAM FOR NASA, FOR HOPKINS, AND HE'S THE DIRECTOR OF THE APPLIED PHYSICS LAB AT JOHNS HOPKINS. EXTRAORDINARY MAN. AND HE HAS BEEN RESPONSIBLE FOR THE EXPLORATION OF EVERY PLANET IN OUR SOLAR SYSTEM. AND SO TOM IS GOING TO SPEAK ABOUT ROBOTICS, ENGINEERING, ASTROEXPLORATION HE'S THE CHAIRMAN OF THE GROUP DISCUSSING YOU AND ANALYZING PROBLEMS OF HUMAN FLIGHT. THAT'S WHERE THE LINK IS TO THE NIH BECAUSE SOMEWHERE ALONG THE LINE, THIS INTEREST EXPANDS FURTHER, THERE'S GREAT CONCERN ABOUT THE MAJOR BIOLOGICAL AND EVEN PHYSICAL PROBLEMS ASSOCIATED WITH PROLONGED SPACE FLIGHT. THAT'S WHERE SCIENCE COMES.

I DON'T KNOW THAT NASA WILL BECOME ANOTHER AREA LIKE NIH BUT SPACE TRAVELS THAT GREAT IMPLICATION FOR PEOPLE IN BIOLOGICAL SCIENCE ASIDE FROM THE WHOLE "STAR WARS" SIDE OF IT. THE LAST SESSION OF THE COURSE, THE WEEK AFTER THAT, ONE THAT WAS POSTPONED BECAUSE WE HAD A SNOWSTORM. HARD TO BELIEVE ON A DAY LIKE THIS. AND THAT'S WHEN — IT'S ON AGING, AND THE TWO SPEAKERS, ONE IS THE DIRECTOR OF THE AGING INSTITUTE, THE CLINICAL ASPECT OF IT, SO THE OTHER IS FROM ALBERT EINSTEIN AND STUDIES CENTINURIONS, IT'S AN EXTRAORDINARY ANALYSIS OF THE AGING PROBLEM, SO I JUST MENTION THOSE THINGS TO YOU. SO I THINK THAT THE TOPIC OF TODAY'S — OH, YEAH.

DOES ANYBODY KNOW WHAT THESE PEOPLE HAVE IN COMMON? SECOND PART OF THE QUIZ. OKAY. WELL, OF COURSE, THE TOP FOUR WON THE NOBEL PRIZE FOR STUDIES OF CHOLESTEROL. AND THE BOTTOM FOUR ALTHOUGH NOT SPECIFICALLY STUDYING CHOLESTEROL, THEIR WORK VERY MUCH WAS RELATED TO, SUCH AS KENDEL AND HENSCH AND OTHER STUDIES OF STEROID HORMONES AND RELATIONSHIP TO CHOLESTEROL. EVEN PAVLOV, INTERESTINGLY ENOUGH, I WAS SURPRISED TO FIND A PAPER BY PAVLOV WHERE HE ANALYZED GALLSTONES IN A PATIENT AND SAID THESE ARE CRYSTALS, AND HE DIDN'T NAME IT CHOLESTEROL, A FRENCHMAN DID, A SHORT TIME AROUND THE SAME TIME BUT CHOLESTEROL ATTENTION INTEREST CAME OUT OF THE FINDING OF THIS CRYSTAL MATERIAL THAT ACCUMULATED IN THE GALLBLADDER. AND THEN IT LED THROUGH A BIOCHEMICAL ERA, BLOCH AND LYNEN HAD 37 STOPS IN ACETATE, MAYBE MORE. STEROL CIRCULATES AROUND, PROTEINS, ESSENTIAL PART OF CELL MEMBRANES. IF YOU HAVE TOO MUCH OF IT, IT'S ASSOCIATED WITH ATHEROSCLEROSIS, STROKE AND SO FORTH. IF YOU HAVE TOO LITTLE OF IT, PARTICULARLY NOT IN THE RIGHT PLACE, THAT'S NOT GOOD EITHER BECAUSE IT'S REALLY ESSENTIAL FOR LIFE. CELL MEMBRANE STRUCTURE, IT'S A PRECURSOR FOR BILE ACID.

THAT'S HOW BILE ACIDS ARE FORMED, INTENSELY APOLAR SUBSTANCES CONVERTED INTO A DETERGENT, WHICH IS ESSENTIAL NOT ONLY FOR DIGESTION BUT ALSO FOR NOW WE'RE LEARNING MANY SIGNAL THINGS, VITAMIN D PRODUCTION AND STEROID HORMONE. ONE OF THE INTRIGUING QUESTIONS THAT WILL BE DISCUSSED TODAY ALSO IS THAT STEROLS LIKE THIS ARE NOT UNIQUE TO ANIMAL FOOD WHICH WE ALL ATTRIBUTE, EGGS, CHEESE, MEAT AND SO FORTH, THAT HAVE A GOOD BIT OF CHOLESTEROL BUT THE BIOLOGICAL WORLD HAS A LOT OF STEROL THAT ALSO HAS SIMILAR PROPERTIES, NOTABLY SITOSTEROL IN PLANTS, VEGETABLES, WE CONSUME HUGE AMOUNTS, THAT'S WHAT WE'RE SUPPOSED TO DO. EAT YOUR LETTUCE AND EXERCISE.

SO WHY DO WE GET INTO TROUBLE? BECAUSE SITOSTEROL CHEMICALLY IS VERY CLOSELY RELATED TO CHOLESTEROL. WE DON'T BECAUSE THE LIVER HAS A CAPACITY, A SPECIAL TRANSPORTER PAIR, THAT PUMP IT OUT, VERY EFFECTIVELY. NOT SO EFFECTIVELY FOR CHOLESTEROL. SO YOU MIGHT CONSIDER WHY DON'T PATIENTS GET CARDIOVASCULAR DISEASE FROM EATING TOO MUCH VEGETABLES? IT'S BECAUSE OF THESE TRANSPORTERS, ABC, G, 5 AND 8. WHEN THEY ARE MUTATED THINGS HAPPEN, WHICH WE'LL LEARN ABOUT TODAY. OUR SPEAKERS TODAY ARE EXPERTS IN THIS AREA, IN THIS FIELD, AND I WILL BRIEFLY INTRODUCE THEM TO YOU. THE FIRST SPEAKER IS BOB SHAMBURG WHO RECEIVED HIS MEDICAL DEGREE AT THE UNIVERSITY OF VIRGINIA WHERE HE TRAINED IN GASTROENTEROLOGY AND GERONTOLOGY, SINCE COMING TO THE NIH HE'S BEEN THE HEAD OF THE LIPID SERVICE, AND IS VERY MUCH INVOLVED, AND THE QUESTION PRIMARILY OF THE LIPID PROTEINS RELATED BY WHICH CHOLESTEROL GETS MOVED AROUND, BUT OTHER ASPECTS AS WELL.

AND OUR SECOND SPEAKER IS DENNY PORTER, M.D. AND Ph.D. FROM ST. LOUIS, TRAINED IN GENETICS AND PEDIATRICS CAME TO THE NIH IN 1993, HAS BEEN HERE SINCE. HE'S PARTICULARLY INTERESTED AND WELL KNOWN FOR STUDIES OF INHERITITTABLE DISORDERS PREDOMINANTLY RELATED TO CHOLESTEROL, HE WILL TELL US ABOUT IT AND PROGRESS MADE UNDERSTANDING MECHANISMS AND MANAGEMENT OF THESE DISEASES. SO WE'RE VERY GRATEFUL TO BOTH OF YOU. >> THANK YOU. I'D LIKE TO THANK BEN FOR INVITING ME HERE AND I THINK ONE OF THE IMPORTANT THINGS HE SAYS IS TO BE I THINK SUCCESSFUL IN TRANSLATIONAL MEDICINE YOU DO HAVE TO BE A BRIDGE BUILDER, AND WITH OUR ROUNDS WE INCLUDE — WE ACTIVELY INCLUDE THE Ph.D.s, I COULDN'T SURVIVE WITHOUT CELL BIOLOGISTS, TO UNDERSTAND THE LIPID DIS ORDERS.

I WOULD LIKE TO GIVE AN OVERVIEW WITHOUT A REAL PATIENT OF EIGHT DIFFERENT DISORDERS WHICH WE FOLLOW HERE BUT I THINK WE UNDERSTAND CHOLESTEROL AND WE WILL BE ABLE TO ANSWER THE QUESTION SHOWN NEAR THE END. SO I HAVE NO DISCLOSURES. TYPICALLY WHEN WE THINK OF PATIENTS WITH LIPID DIS ORDERS, ON THE LEFT WE SEE A HEART ATTACK WHERE CHOLESTEROL IS GREATER THAN 250. FREQUENTLY THAT'S A GENETIC DISORDER, ON THE RIGHT THE OTHER DISORDER WE SOMETIMES CONFUSE TRIGLYCERIDES IS NOT CHOLESTEROL, IT'S ANOTHER LIPID, AND IT CAN INFLAME THE PANCREAS AND GIVE PANCREATITIS. THE HEALTHY PERSON, WE KNOW ABOUT THE INCIDENCE OF OBESITY AND INCREASING, SO WHAT HAS REALLY HAPPENED? WE GO BACK TO 1504 WITH DAVID, GOOD LIFE, ACTIVE, YOU KNOW, HE'S QUIT EXERCISING, NO MORE MARATHONS, GAVE UP HIS MEDITERRANEAN DIET, HIGH FAT – CARBOHYDRATE DIET, ALCOHOL, HE DEVELOPED METABOLIC SYNDROME.

DIET, THIS IS HIS COPYCAT, TABITHA PICKED UP BAD HABITS. DIET AND LIFESTYLE PLAY AN IMPORTANT ROLE IN OBESITY BUT WHAT I WOULD LIKE DO DO IS FOCUS ON THE GENETIC FORMS. WE KNOW ATHEROSCLEROSIS, HARDENING OF THE ARTERIES, IS A TRANSITIONAL THING WHERE YOU GO FROM FATTY STREAKS TO LIPID TO THROMBUS, YOU CAN STUDY THOSE, BUT I'M INVOLVED MORE WITH THE LIPIDS THEMSELVES. SO ATHEROSCLEROSIS DOESN'T HAPPEN OVERNIGHT. ONE PERSON SAID THERE'S NO SUCH THING AS SUDDEN DEATH. IT TAKES DECADES BEFORE YOU END UP WITH FATAL PLAQUE. ONCE YOU DO A TREATMENT, YOU'RE LOOKING AT PATIENTS HERE, TRYING TO REVERSE, ARE YOU TRYING TO PREVENT PROGRESSION OR MAINTAIN IT HERE? SO OUR TREATMENTS I'M GOING TO JUST VERY BARELY TALK ON BUT EVERY FIELD IN HERE PROBABLY SOMEONE COULD HAVE AN EFFECT ON THE LIPID FIELD AS FAR AS TREATMENTS NOWADAYS. SO GENETIC CAUSES, WE KIND OF STARTED OUT HIGH CHOLESTEROL, LOW CHOLESTEROL. THIS IS A YOUNG PERSON, WE HAVE THEM AS YOUNG AS 3, 4, 5, 6 WITH HOMOZYGOUS FH, ONE IN A MILLION THAT GET HEART ATTACKS. ON THE RIGHT IS THE 17-YEAR-OLD WITH THIS METABOLIC DISEASE NOT TOO MUCH CHOLESTEROL, IT'S WHAT THE CHOLESTEROL TRANSPORTS, WHICH IS VITAMIN A AND VITAMIN E.

SO WHAT IS THE ROLE OF LDL, HDL AND LIPID METABOLISM? I THINK THE REASON WE HAVE THEM IS TO MOVE TRIGLYCERIDE. IT'S ENERGY. WE EAT THE FOOD, WE HAVE TO GET IT TO THE LIVER AND MUSCLE AND GET IT OUT. AND THE WAY WE DO THAT IS TRIGLYCERIDE IS ENERGY. SO CHOLESTEROL, IF YOU TRY TO MAKE A VESICLE OR SOMETHING MADE OUT OF JUST — YOU KNOW, PHOSPHOLIPID AND TRIGLYCERIDE IT'S HARD.

WHAT REALLY MAKES IT IN A HUMAN BODY WHEN YOU CAN PUT A PROTEIN AROUND IT AND CHOLESTEROL IS MORE OF A MEMBRANE STABILIZER, BUT WE DO NEED CHOLESTEROL AS PRECURSORS FOR OTHER THINGS. I THINK THIS IS PROBABLY A KEY SLIDE IN UNDERSTANDING A LIPOPROTEIN, NOT A BILAYER, IT'S A SINGLE LAYER OF PHOSPHOLIPIDS, THE MAJOR ONE IS LECITHIN, IN CORE IS TRIGLYCERIDE, ON THE OUTSIDE WATER SOLUBLE FREE CHOLESTEROL BECAUSE IT DOESN'T HAVE A FAT GROUP. ANOTHER NAME FOR A FAT GROUP IS AN ASILL GROUP. ONCE THE PLAQUE IS PUT DOWN, REMOVING IT, TO CONVERT FREE CHOLESTEROL INTO A CHOLESTEROL ESTER, BY ADDING AN ASILL GROUP, NOW IT'S HYDROPHOBIC. WHAT IS THAT GOING TO DO? CHANGE FREE CHOLESTEROL TO A CHOLESTEROL ESTER, WATER AND FAT SOLUBLE. LECITHIN, ACYL, WHAT THIS DOES IS TURNS IT INTO A TAXI. PLASMA HALF-LIFE IS 25 OR 30 MINUTES. IT IS I THINK ONE OF THE REALLY IMPORTANT PLAYERS.

IF YOU LOOK AT IT FROM A CARTOON, THIS DISC WITH THE FREE CHOLESTEROL CONVERTS OVER HERE, AND THAT'S GOING TO BE VERY IMPORTANT. I THINK AT THE END MAYBE WE CAN TALK ABOUT WHY HDL HAS BEEN GIVEN A BAD NAME BECAUSE I THINK THEY ARE GOING AFTER THE WRONG THINGS. THIS IS THE OVERVIEW OF LIPID METABOLISM. I'M GOING TO GIVE YOU INITIALLY A CARTOON ONE AND GO MORE ONE THROUGH A BIOCHEMICAL ONE. SO WE EAT FOOD, INTESTINE, WE GET TRIGLYCERIDE IN A TRUCK AND BASICALLY THESE TRIGLYCERIDE PARTICLES DUMP CARGO IN MUSCLE, THE REST GETS INTO THE LIVER. WE'VE HEARD LECTURES ON FATTY LIVER AND WHERE THIS COULD BECOME A PROBLEM. THIS OCCURS WHEN WE EAT A MEAL. IN THE FASTING STATE THE LIVER STARTS GETTING RID OF ITS TRIGLYCERIDE THROUGH THE ENDOGENOUS. THIS IS THE EXOGENOUS. THIS IS GOING TO GO THROUGH A CASCADE AS IT REMOVES TRIGLYCERIDE, AS IT GOES DOWN THE CASCADE A LOT OF IT WILL BE REMOVED BY THE LIVER BUT IF IT ISN'T COMPLETELY REMOVED, IT'S GOING TO GET TO A PERIPHERAL CELL AND START BEING DEPOSITED IN ARTERIES.

SO THAT'S THE SYNTHESIS PART, IN DEPOSITION. THEN WE HAVE TO HAVE THE CATABOLISM, THE CLEARANCE. THAT'S WHAT WE'LL CALL REVERSE CHOLESTEROL TRANSPORT. THAT'S WHERE WE'RE GOING TO CONVERT FREE CHOLESTEROL INTO CHOLESTEROL ESTER, WHICH THEN DEAFENS THE CHOLESTEROL. HERE CHOLESTEROL WAS JUST PASSIBLY ALONG, IT WAS GETTING RID OF TRIGLYCERIDES. HERE WE WANT TO GET THE CHOLESTEROL BACK TO THE LIVER AND OUT THROUGH THE BILE. SO THIS IS FOR A CLINICIAN KIND OF OUR FASTING LIPID PROFILE. WE GET A TOTAL CHOLESTEROL, WHICH IS ALL THE CHOLESTEROL. AND IN FACT THE TOTAL CHOLESTEROL IS WHAT'S IN THE LDL IN THE FASTING STATE, HDL, AND LDL. SO WE GET TWO MEASUREMENTS ON A LIPID PROFILE AND ALSO HAVE TRIGLYCERIDES, AND A RESEARCHER BACK 40 YEARS AGO HERE AT THE NIH FIGURED OUT THAT WE COULDN'T GET A GOOD MEASUREMENT OF THE LDL, BUT IN A FASTING SAMPLE UNDER 400 HE FOUND OUT IF YOU TAKE THE TRIGLYCERIDES AND DIVIDE BY 5, YOU CAN GET A GOOD ESTIMATE OF WHAT THE CHOLESTEROL IS.

IF YOU HAVE A TRIGLYCERIDE OF 100, THE CHOLESTEROL IS ABOUT 20. SO WITH THIS EQUATION, WE ALSO CAN'T USUALLY DIRECTLY MEASURE LDL, AND I CAN TALK ABOUT THAT LATER IF THERE'S INTEREST IN THAT. SO WE CAN MEASURE THIS, WE CAN MEASURE THIS, AND WE CAN ESTIMATE THIS AND HAVE A GOOD IDEA. SO THIS IS USUALLY HOW WE THINK LDL IS BAD, HDL IS GOOD. I'LL SHOW YOU WHERE NOW STUFF WE'RE DOING HERE KIND OF SHOWS THERE'S GOOD AND BAD OF BOTH OF THOSE. SO IF YOU JUST LOOK AT A SAMPLE, IF YOU ARE UP HERE AND I TOLD YOU WHO DO YOU THINK — WHICH SAMPLE HAS THE HIGHEST TOTAL CHOLESTEROL, LET'S SAY WHO HAS A THOUSAND, WELL, THE ANSWER IS NOT THIS ONE.

IT COULD BE THIS ONE. CHOLESTEROL DOESN'T DO ANYTHING TO THE SAMPLE. IT'S ONLY TRIGLYCERIDES. PUT THIS IN THE REFRIGERATOR OVERNIGHT, SPIN IT FOR TEN MINUTES, THE LABORATORY IS GOING TO SAY THIS IS LYTINA. THIS PART IS COMING FROM THE INTESTINE. IT'S CREAM JUST LIKE WHAT YOU TAKE OUT OF THE REFRIGERATOR. THIS IS TURBID, COMING FROM THE INTESTINE, THIS IS COMING FROM THE LIVER. I CAN UNDERSTAND A LITTLE BIT ABOUT THE PHYSIOLOGY. THEN WHAT WE ALSO NOTICED WAS THIS VLDL, IDL, GETTING RID OF TRIGLYCERIDE LEFT WITH THE CHOLESTEROL PARTICLE, IN THE NON-FASTING STATE WE HAVE TO DEAL WITH THE CHYLO MICRON, WHY I LIKE A FASTING SAMPLE, BUT THERE ARE DIFFERENCES IN THE PROTEINS, AND DIFFERENCES IN THE FAT CONTENT, ONE OF THE WAYS WE INITIALLY SEPARATED OUR PARTICLES. SO DR. FREDERICKSON HERE AT THE NIH WAS FAMOUS ABOUT TAKING THE PLASMA AND PUTTING IT ON PAPER, OR WE STILL DO THIS IN THE LAB, HE WOULD GET AN ALPHA, BETA AND PRE-BETA, AND PEOPLE WITH HEART ATTACKS HAD INCREASED BETA.

PEOPLE WITH NO HEART ATTACKS HAD HIGH ALPHA. ONE PATIENT WE SAW WITH A WHEELCHAIR HAD A-BETA. SOME ORIGINAL NAMES CAME FROM THIS F WE DON'T HAVE CHARGE, WE CAN LOOK AT DENSITY BECAUSE THERE'S DIFFERENT AMOUNTS OF FATS. SO THAT'S CLASSICALLY WHERE WE GET HIGH DENSITY, INTERMEDIATE AND LOW DENSITY, PUT IT IN POTASSIUM BROMIDE AND SPIN IT VERY HIGH, 40,000 REVOLUTIONS PER MINUTE, 24 HOURS, CUT IT, ADJUST, AND IT'S VERY TEDIOUS, GOES OVER A WEEK, BUT THAT'S REALLY HOW WE FOUND OUT THAT THIS LDL WAS THE BETA, BUT IN A LAB COMMERCIALLY NOWADAYS WE HAVE TO DO PRECIPITATION METHODS. SO WHAT WE'RE TRYING TO DO HERE AT THE NIH IS GO THROUGH ANOTHER THING. THIS IS USING NMR, AND WITH NMR YOU USE IT JUST LIKE AN MRI MACHINE BUT INSTEAD OF LOOKING AT DENSITY CAN YOU DO DIFFERENT SIZE AND PARTICLES BASED ON METHYL GROUPS SO WE CAN CHARACTERIZE CHYLO MICRON VIA LDL AND WE DO KNOW IT IS ATHEROGENIC AND SMALL AGAINST HDL IS BUT WE CAN'T PRACTICALLY DO ULTRACENTRIFIGATION.

SUPPOSE WE HAVE A PATIENT COME IN, TWO PATIENTS, THEY HAVE AN LDL OF 130. IDEALLY IT MIGHT BE 100. WHO NEEDS MORE TREATMENT? DO WE NEED TREATMENT? FROM DOZENS OF STUDIES, WITH MULTIPLE METHODS, WE FOUND OUT THE PATIENT WITH THE SMALLER PARTICLES WHICH ARE PROBABLY MORE ATHEROGENIC, OXIDIZABLE, HAVE A HIGHER RISK THAN THE ONES WITH LARGER ONES. IN FACT, DRUGS, STATINS, CAN CONVERT THIS OVER OR DECREASE THIS.

PEOPLE WITH GENETIC DISORDERS OFTEN HAVE THESE. SO WHAT WE CAN DO WITH NMR IS COUNT THE PARTICLE NUMBERS, THE SIZE AND PARTICLE NUMBERS. SO THE NUMBER ITSELF, IF IT'S GREATER THAN A THOUSAND, YOU'LL HAVE A BIG RISK, AND THIS PATIENT OVER HERE WOULD HAVE A BIG RISK, WE COULD ALSO LOOK AND SEE DOES HE HAVE SMALL OR LARGE PARTICLE, THAT WILL HELP US MAKE HIM EVEN A HIGHER RISK. THERE ARE MUST BE METHODS BUT THIS IS A PRACTICAL WAY. AND THIS IS KIND OF WHAT THE NUMBERS LOOK LIKE. THEY ARE VERY HARD FROM A CLINICAL STANDPOINT. HOW CAN I USE THAT WHEN SOMEONE SAYS DOES THIS PATIENT NEED TREATMENT? AND ONE OF THE THINGS WE'RE DOING HERE IN DEVELOPING HERE AT NIH IS A PROFILE WHERE WE TRY TO GET KIND OF WHAT THE AVERAGE ONE IS, AND WHEN I LOOK OVER HERE WITH HDL, THIS IS SMALL, MEDIUM AND LARGE, THIS IS INCREASED, WITH SMALL, MEDIUM, OR SMALL AND LARGE, THIS IS INCREASE. I'M NOT GOING TO GO THROUGH TRIGLYCERIDE, JUST AN EXAMPLE. WHAT IF I COULD FURTHER SUBCLASS THIS? THAT'S ONE OF THE THINGS IN THE LAST JUST MONTH OR TWO WE'RE STARTING TO DO.

AND IF YOU LOOK DOWN HERE, FOR HDL, NOW I CAN FURTHER DIVIDE THIS SMALL INTO VERY SMALL AND LARGE BUT THEY ARE BOTH SMALL. IN FACT, THIS ONE IS OFF THE CHART. THIS IS GENETICALLY VERY SPECIFIC ATHEROGENIC PARTICLE. EVEN OVER HERE WHERE THE LDL WAS UP HERE WHEN WE SUBGROUP THE SMALLEST IS THE WORST MOST ATHEROGENIC, IT'S OFF THE WALL. SO THIS PATIENT IS NOT GOING TO DO — I KNOW REMNANTS ARE BAD, THIS GUY HAS THREE STRIKES AGAINST HIM AND A GENETIC DISORDER AND WE ALSO NEED TREATMENT. WE CAN USE IT FOR TWO METHODS. THIS IS ONE OF THE THINGS THAT WE'RE ACTIVELY DOING HERE WITH OUR RESEARCH. SO NOW I'M GOING TO GO TO — IN THE NEXT 30 MINUTES, GO OVER ABOUT EIGHT OF THE GENETIC DISORDERS.

SO VERY EASY THING IS WHERE THE TRUCK IS SENDING OUT ITS PARTICLES, IT'S GOING TO THE MUSCLE, WHATEVER IS LEFT IS IN THE ARTERY, AND THEN THIS IS REVERSE CHOLESTEROL TRANSPORT, AND FOR ALL YOU ANIMAL PEOPLE, IN HUMANS WE HAVE CETP, TRANSFEATHER CHOLESTEROL ESTER FROM HERE TO HERE, ANIMALS DON'T. THERE ARE ARE DIFFERENCE BETWEEN ANIMAL MODELS AND HUMANS. THIS IS THE MORE ONE THAT EFFECT ENZYMES, STIMULATE THEM, TELL THE RECEPTORS WHERE TO GO. DEPENDING WHERE THE DEFECT IS, I CAN LOOK AT THEIR LIPID PROFILE AND HAVE A GOOD IDEA WHERE THE DEFECT IS.

ALL THREE OF THESE GENETICALLY WILL HAVE A TOTAL CHOLESTEROL OF 400. THIS ONE, TRIGLYCERIDE WILL BE 8000, THIS ONE WILL BE 400, THIS WILL BE ONE 1000, THAT GIVES ME A ROUGH IDEA. SO IF I HAVE A DEFECT RIGHT HERE, LIPO PROTEIN LIPASE, THAT CLEARS TRIGLYCERIDE PARTS FROM THE MUSCLES, IF I MISS ONE OF THESE I'LL HAVE A TRIGLYCERIDE OF 8000, BE UNABLE TO HYDROLYZE THE TRIGLYCERIDE IN THE LIVER LDL OR INTESTINAL TRIGLYCERIDE, AND IN FACT IF I DRAW THE SAMPLE, SET IT DOWN FOR FIVE MINUTES, IT WILL LOOK LIKE THIS.

AND I KNOW MY COLLEAGUES WORKING WITH ANIMAL MODELS THAT DONE IT BEFORE, THE SAME THINGS. IF YOU SEE THIS, THIS IS LIPEMIC SERUM. YOU DON'T HAVE TO SPIN IT. THAT MEANS IT'S VERY HIGH. YOU CAN IMAGINE AS A CLINICIAN IF I WOULD LOOK IN THE BACK OF THE EYE, IF I COULD SHINE A LIGHT IN AND SEE A LITTLE ARTERIAL OR SOMETHING, AND FLASH THE LIGHT, I MAY SEE THE SAME THING. AND IN FACT I SHOWED YOU LIPEMIC PLASMA, I'LL SHOW YOU A PICTURE, IT FLASHES, YOU CAN IMAGINE IN THIS TRIGLYCERIDE IS AROUND, IT MAY DEPOSIT ON THE SKIN. THAT'S WHAT WE CALL ERUPTIVE XANTHOMA, THE OUTCOME IS PANCREATITIS, IT WILL OCCUR IN A 2-YEAR-OLD AND 6-YEAR-OLD, A 12-YEAR-OLD.

SOMETHING THAT'S LIFE-THREATENING AND CERTAINLY CAN KILL THEM. THESE ARE THE ERUPTIVE XANTHOMA FULL OF TRIGLYCERIDES. I SAID I WOULD SHOW YOU THE BACK OF THE EYE. THIS IS THE OPTIC DISC WHERE WE SEE MOST OF OUR VISION, AND THIS IS THE NORMAL ARTERY. I SHINE THE LIGHT AND SURE ENOUGH I'M SEEING THE LIPID. I KNOW THE LIPID IS GREATER THAN 3000, UNTIL I'M ABLE TO GET THOSE LEVELS DOWN. THAT'S LIPEMIA RETINALIS. DO ANY OF YOU WORK WITH APO E KNOCKOUT MICE? THERE ARE SOME, YOU MAY NOT REALIZE, THE MOST COMMON MOUSE ON CAMPUS. USED IN RESEARCH. AND THIS IS THE HUMAN MODEL OF THE APO E KNOCKOUT MICE. IF YOU HAVE A DEFECT HERE, CHOLESTEROL AND TRIGLYCERIDE WILL BE EQUAL. IT'S AN IMPORTANT CLEARER, USED TO CLEAR IN THE RECEPTORS IN THE LIVER, WITHOUT THAT YOUR IDL PARTICLE BUILDS UP AND THIS IS SOMETHING BETWEEN A HIGH TRIGLYCERIDE AND A LOW TRIGLYCERIDE PARTICLE AND THIS IS HIGHLY OXIDIZABLE.

WE KNOW THAT. WE CAN PICK IT UP IN OUR NMR AS REMNANT PARTICLE. IT'S VERY INTERESTING BECAUSE THE HUMAN HAS WHAT WE CALL ISOFORMS, LIKE WE HAVE DIFFERENCES IN EYE COLOR, HAIR COLOR, APO E WITH ONE CYSTEINE IS THE MOST COMMON. IF YOU HAVE AN ISOFORM, YOU'RE UNABLE TO CLEAR THE LDL PARTICLE. THIS ONE IS ASSOCIATED WITH ALZHEIMER'S. WE HAD A TALK ON THAT. SO WHAT DO THEY GET? THEY GET XANTHOMAS ON THE PALM. WHY ON THE PALM? WE STILL DON'T KNOW. SO THEY CAN GET PALMAR AND ERUPTIVE XANTHOMA. BETWEEN AGE 30 AND 40 THEY WILL HAVE A HEART ATTACK. IF YOU RECOGNIZE AHEAD OF TIME I CAN PROBABLY TOTALLY PREVENT IT. SO WHEN YOU SEE CHEST PAIN, AS YOU'RE DIALING 911 LOOK AT THEIR PALMS, SEE IF YOU CAN DIAGNOSE WHERE THE DISORDER IS. SO PALMAR, TUBEROUS. THE LAST IS DEFECT OF LDL, THE THERE ARE MANY DEFECTS. APO B BINDS, ADAPTOR PROTEIN, THERE'S MORE THAN 2000 MUTATIONS AND THIS OCCURS IN ABOUT 1 IN 250 PEOPLE IN THE U.S SO THIS IS VERY IMPORTANT. WHAT THEY GET IS WHAT APPEARS TO BE A MORPHOUS AND CHOLESTEROL DEPOSITION AROUND THE EYE, CALLED ARCUS, BEFORE 50 IT'S SIGNIFICANT THAT IT'S CHOLESTEROL.

WE DON'T KNOW HOW THE CORNEA CLEARS CHOLESTEROL, AND THEY CAN GET THESE OTHER THINGS CALLED XANTHALASMA, IT APPEARS TO BE CHOLESTEROL IN THE INNER EYE AND TUBEROUS XANTHOMA. YOU CAN PICK THESE UP AND CUT THEM OFF. NOW WE HAVE SUCH GOOD TREATMENT WE'RE PRACTICALLY NEVER SEEING THESE IN PEOPLE ANYMORE UNLESS THEY COME IN COMPLETELY UNTREATED. SO THESE ARE TUBEROUS, AND WE HAVE TENDONNOUS ON THE KNUCKLES, FOR SOME REASON THEY OCCUR ON THE ACHILLES TEND. WE DON'T KNOW IF IT'S AN AREA OF HIGH FRICTION. WE HAVE NO IDEA WHY THEY OCCUR. SO THE OTHER REALLY WORRISOME THING IS ONE IN 250 ARE GOING TO BE UNTREATED, HAVE A HEART ATTACK IN THEIR 30s AND 40s. SO THIS IS SOMETHING THAT YOU'LL HAVE A STRONG FAMILY HISTORY OF IT, AND WITH DRUGS IS ALSO GENERALLY PREVENTIBLE.

SO WE REVIEWED DISORDERS THAT INCREASE DEPOSITION TO ARTERIES, LDL, IDL, AND WHAT WE CALL SOME REMNANT PARTICLES. BUT WHAT REMOVES THE CHOLESTEROL? WELL, THIS IS ANOTHER THERAPY AND THIS IS KIND OF WHERE A LOT OF OUR RESEARCH IS GOING, TRYING TO REMOVE IT. SO IT DOESN'T MATTER HOW IT GETS THERE., IF YOU CAN REMOVE IT. WE HEARD HOW THIS IS THE TRUCK GETTING RID OF ITS TRIGLYCERIDE, FOR HDL IT'S MORE TRYING TO GET A TAXI. I SAID WE HAVE TO CONVERT THIS NASCENT TO CHOLESTEROL ESTER, AND BY DOING THAT THIS HDL NOW IS DESTINED TO GO BACK AND POTENTIALLY BE SECRETED BY THE LIVER. SO THERE'S THE FIRST TWO STEPS WHAT WE'LL CALL REVERSE CHOLESTEROL TRANSPORT ARE VERY INTERESTING. THE FREE CHOLESTEROLS PUMP OUT BY AN ABCA 1 TRANSPORTER, THERE'S MORE THAN 50 OF THEM. MULTI-INSTITUTES ARE DEALING WITH DIFFERENT ABC TRANSPORTERS. THEY TEND TO PUMP OUT BAD THINGS, WHETHER IT'S CHLORIDE AND CYSTIC FIBROSIS, TO ANTIBIOTICS, TO CHEMOTHERAPY, ALL SORTS OF THINGS.

SO THEY ARE VERY IMPORTANT. IN THE BODY, IT PUMPS OUT CHOLESTEROL. AND THIS IS AN ENERGY DEPENDENT. THE SECOND STEP, WHICH IS WHERE MY RESEARCH IS INVOLVING WITH IS CONVERTING THIS FREE CHOLESTEROL TO CHOLESTEROL ESTER WHICH I SAID IS TRANSFERSING ASYL GROUP FROM LECITHIN. THERE HAVE BEEN NO REAL TREATMENTS DESIGNED FOR THIS. MOST TREATMENTS TO RAISE HDL ARE HERE OR ANOTHER THING OF BLOCKING IT. THAT'S WHY I THINK THIS IS THE RIGHT THERAPY, ANIMAL MODELS, ALL THAT SEEM TO POINT THAT THIS WILL BE THE RIGHT THERAPY IN THE FUTURE. HERE YOU'RE CONVERTING FREE CHOLESTEROL TO CHOLESTEROL ESTER, AND CORE CHOLESTEROL ESTER IS GOING TO BE TRANSFERRED BACK OVER. THIS DOESN'T OCCUR IN ANIMALS BY CHOLESTEROL ESTER TRANSFER PROTEIN. 75% GOES HERE, THE OTHER 25% GOES BACK AS FREE CHOLESTEROL THROUGH PROBABLY SRB 1. REVERSE CHOLESTEROL TRANSPORT AND DOING IT THE RIGHT WAY WHERE I THINK THIS IS THE RATE LIMITING IS VERY IMPORTANT.

THE DEFECT IN THE FIRST STEP IS REVERSE CHOLESTEROL TRANSPORT IS ABCA 1, TANGIER DECEMBER.
DISEASE, IF I ASK PEOPLE WHAT IT'S NAMED OVER MOST PEOPLE SAY TANGIER IN AFRICA. THIS IS THE ABCA1. THERE'S AN ISLAND IN THE CHESAPEAKE BAY CALLED TANGIER ISLAND. HERE'S THE BEST WAY TO GET IN. THAT'S THE RUNWAY FOR THE AIRPORT. YOU DON'T WANT TO OVERRUN IT. THERE ARE NO CARS. AND IT WAS SETTLED IN ABOUT THE EARLY 1600s BY ENGLISH. AND THE NEXT TIME ANYONE CAME BACK TO THE ISLAND, THEY BASICALLY WERE EITHER GOING TO STAY THERE OR GO OVER TO JAMESTOWN AND WINTER IT OUT AND WORRY ABOUT INDIANS, SO THERE ARE FIVE OR SIX FAMILIES THAT STAYED THERE, AND 1812 WAS THE FIRST TIME REALLY ANYONE MADE CONTACT. THE BRITISH CAME UP. THEY WINTERED THERE WITH A THOUSAND TROOPS, WENT UP AND BOMBED FORT McHENRY, LOST, CAME BACK AND SAID YOU WANT TO COME BACK TO ENGLAND WITH US? THEY SAID NO. REALLY IN 1960 WAS THE NEXT TIME THAT REALLY ANYONE WAS THERE. IF YOU VISIT THE ISLAND, THEY HAVE THE SHAKESPEREAN ENGLISH ACCENT. SO THEY HAVE IT FROM THE 1600s, IF YOU DON'T UNDERSTAND IT THE SECRETARIES SAY YOU GOT SOMEONE FROM A FOREIGN COUNTRY CALLING YOU.

YOU KNOW, IT'S A VERY INTERESTING DIALECT. WELL, DON FREDERICKSON, ONE OF OR FORMER DIRECTORS OF NIH, WAS THE ONE WHO DISCOVERED TANGIER AND FOUND THEM IN THE TONSIL, THE NERVES, YOU CAN SEE, BUT THE CLASSIC WAS ORANGE TONSILS. THIS IS YOUR MOUTH, UVULA AND TONGUE, THEY HAD ORANGE TONSILS. THIS IS ONE OF THE TWO CROW BANDS. THE ABNORMAL PARTICLES GET INTO THE SPLEEN, HAVE NO HDL, IT'S A DEFECT IN ABCA 1. ONCE AGAIN IT'S A DIMER, ABC TRANSPORT, AND WHY DON'T THEY DIE AT BIRTH? WHY DON'T THEY DIE IN UTERO? WE HAVE A COUPLE OTHER TRANSPORTERS OR EXCHANGERS THAT CAN EXCHANGE WITH LIPO SCENES, SRB 1, ABCG1 AND OTHERS. YOU DON'T HAVE HDL, YOU HAVE THOSE PROBLEMS, WHICH CAN BE NEUROLOGIC AND LOW HDL AND THINGS, BUT YOU'RE NOT — YOU'LL DIE OF EARLIER HEART DISEASE, BUT NOT IN UTERO OR EARLY AT BIRTH BECAUSE OF THAT.

SO THE SECOND STEP IN REVERSE CHOLESTEROL IS LCAT, THE ONE WHERE YOU FREE CHOLESTEROL TO CHOLESTEROL ESTER, FORMING THE TAXI, THE ONE THAT'S PRE-DISPOSED THAT I THINK IS VERY IMPORTANT. IF YOU'RE MISSING THIS IN LCAT DEFICIENCY THIS DISC PARTICLE CLUMPS WITH OTHERS AND FORMS A MULTI-LALELLAR, IT'S AQUEOUS AND CLOUDS UP THE KIDNEY, WHEN YOU INITIALLY CLOG UP KIDNEY YOU SPILL PROTEIN, PROTEINURIA AND WE HAVE ANIMAL MODELS WHERE YOU CAN FOLLOW THIS. IF YOU DAMAGE IT ENOUGH, THE PROTEINS AND THINGS THAT ARE EXCRETED WE CALL BLOODURIA AND NITROGEN AND CREATININE, THEY BUILD UP AND END UP NEEDING DIALYSIS AND ULTIMATELY KIDNEY TRANSPLANT WHICH RECURS UNFORTUNATELY.

THEY ALSO HAVE THE CORNEA AND RED CELLS ARE VERY SENSITIVE. SO THE RED CELLS ARE THE FOURTH BIGGEST POOL SIZE OF CHOLESTEROL IN THE BODY. SO THE CORNEA ALSO HAS AN INTRICATE INTERACTION WITH CLEARING, AND HDL IS VERY IMPORTANT. THEY HAVE THE APPEARANCE OF A FISH EYE. THEY HAVE THIS LPX PARTICLE. SO THIS IS WHAT A FISH EYE LOOKS LIKE. IT LOOKS LIKE IF YOU'RE AT THE MARKET, IT'S CLOUDY, THIS PERSON HAS 20/20 VISION BUT IF YOU SHINE A LIGHT AT THEM OR AT NIGHT IT GLOWS AND HAS PROBLEMS, THEY EVENTUALLY HAVE PROBLEMS WITH THAT. RED CELLS ARE IMPORTANT, THEY GET AN ANEMIA.

WHEN THE ANEMIAS MESS UP THE MEMBRANE, MEMBRANES GET BIG AND WE CALL THESE TARGET CELLS. SO YOU HAVE AN ANIMAL MODEL, YOU THINK THE MEMBRANE IS MESSED UP, I'M GOING TO SHOW YOU A COUPLE EXAMPLES, LOOK AT A BLOOD SMEAR AND I KNOW IT'S ABNORMAL, TO PROTEINURIA, TO DIALYSIS. IN FOUR SLIDES WE JUST PUBLISHED A MONTH AGO OUR FIRST-IN-HUMAN TREATMENT WITH RECOMBINANT HUMAN LCAT AND GAVE IT INITIALLY TO GET IT ON BOARD TO FOUR DIFFERENT KINDREDS WITH LOW HDL AND HEART DISEASE AND IT RAISED IT IN ALL THOSE AND THEN WE DID IN ONE PATIENT BEFORE WE'RE DOING THE LARGE ONE ENZYME REPLACEMENT OVER 8 MONTHS. SO WE DID A DOSES ESCALATION
AND TWO MAINTENANCE PHASES.

THIS PATIENT STARTED WITH NO HDL, NEVER SEEN ONE. AND WE DID THREE DIFFERENT DOSES, SO THIS IS OVER TIME, AND EVENTUALLY WITH THE HIGHEST DOSE WE COULD MAINTAIN AN HDL LEVEL, BUT ALSO HDL TRANSFERS WITH CHOLESTEROL ESTER OVER TO LDL, WE WERE SEEING IT BEING TRANSFERRED DOING ITS NORMAL FUNCTION IN VIVO. IF WE WANT TO SEE ACTION OF ENZYME LOOK AT PRECURSOR, THE PRODUCT BEING CHOLESTEROL ESTER. FREE CHOLESTEROL TO CHOLESTEROL ESTER. AND TO OUR HAPPINESS, EVEN AT THE LOW DOSE THE BASELINE CHOLESTEROL WAS 18%, EVEN AT THE LOWER ONE WHICH WASN'T REALLY MAKING THE PARTICLE, IT WAS MAKING CHOLESTEROL ESTER AND A NORMAL LEVEL IS ABOUT RIGHT UP HERE.

AND WHEN YOU HAVE CHOLESTEROL ESTER, YOU DON'T HAVE LPX PARTICLES. AND YOU DON'T GET KIDNEY DISEASE. SO ONE OF OUR INDIRECT METHODS WAS LOOKING AT PHOSPHOLIPID, DOWN THREE FOLD WITH THE HIGHEST DOSE, DISAPPEARANCE OF THE PARTICLE. L CAT DEFICIENT, THIS IS NORMAL, LDL, DIFFERENT LIPIDS ON THE LEFT, THIS IS LDL, THIS IS CLASSIC HDL, AND THE PRECURSOR. IF YOU LOOK UP HERE THEY HAVE NO HDL. THEY FORM NORMAL HDL. THIS IS PART OF THE MULTI-LAMELLAR PARTICLE, IT DISAPPEARED WITHIN AN HOUR OF THE INFUSION SO WE HAD VERY GOOD EVIDENCE, WE NOW HAD HDL AND HAD NO LPX. AND WE ALSO PROGRESSIVELY OVER 8 MONTHS, THIS IS THE INFUSION, IT WOULD GO UP, WE COULD STAIR STEP UP, IT WOULD GO DOWN A WEEK WHEN WE WENT TWO WEEKS BUT WE MAINTAINED THE INCREASE CHOLESTEROL ESTERS, AND THIS IS A LITTLE COMPLEX BUT THIS IS THE ONE WHERE WE STABILIZED OR IMPROVED THE RENAL.

THE ORANGE IS CREATININE, ONE OF THE FUNCTIONS, WE WENT DOWN HERE. UNFORTUNATELY THE PATIENT HAD ANOTHER GENETIC DISORDER FOR RAPID HEART RATE AFIB AND HAD TO BE PUT ON SOMETHING KNOWN TO INCREASE CREATININE, BUN STABILIZED OR REDUCED, ANOTHER STATIN MAINTAINED. THIS PATIENT WAS A WEEK AWAY FROM NEEDING DIALYSIS, HAD HIS FISTULE AND EVERYTHING. WE KNEW WE COULD MAINTAIN IT BUT WE KNEW ONCE THE KIDNEY IS DAMAGED WE'RE NOT GOING TO REVERSE IT SO WE NEED TO GET PEOPLE EARLY ON, TREAT BEFOREHAND, OR WHEN THEY ARE GOING FOR KIDNEY TRANSPLANT TO PREVENT THE REJECTION. WE SHOWED THAT IN SIX WEEKS A 25% INCREASE IN THE HEMOGLOBIN. WE ALSO FOUND OUT THAT WHEN YOU GO TO THE ICU THEY DRAW A LOT OF BLOOD. SO THAT WAS JUST MULTIPLE BLOOD STICKS. SO IT WAS WELL TOLERATED. WE THOUGHT, IMPROVEMENT TO RENAL AND HEMATOLOGIC, WE HOPE IN A YEAR OR TWO WE CAN — IT'S BEEN BOUGHT BY MAJOR PHARMACEUTICAL COMPANY AND THEY ARE GOING TO DEVICE IT FOR RAISING HDL AND HEART DISEASE, A ROTO-ROOTER, BUT WE'RE GOING TO BE ABLE TO PARALLEL ON WITH THAT AND HOPEFULLY DO THE RARE DISEASE PATIENTS AND CURE THEM OF THEIR RENAL DISEASE OR PREVENT IT.

SO IN THE NEXT COUPLE MINUTES I'M GOING TO DO TWO OF THE CLASSIC INTESTINAL LIVER DISORDERS, THAT LEAD TO LIPID DISORDERS. THE FIRST IS ABETALIPOPROTEINEMIA, IF YOU EAT FAT IT GETS IN THE ENTEROCYTE BUT YOU CANNOT ABSORB THE TRIGLYCERIDE AND CAN'T ABSORB FAT SOLUBLE VITAMINS. IT'S NOT GETTING IN VITAMIN E AND VITAMIN A AND I'LL SHOW YOU CLINICAL FINDINGS WHICH IN ANIMAL MODELS, OTHER THINGS, POTENTIALLY COULD BE USED. THE LIVER ITSELF CANNOT
SECRETE. SO THEY GET FAT MAL ABSORPTION, THEY WOULDN'T KNOW WHAT A POTATO CHIP WAS, THEY WOULD GET BLOATING, DIARRHEA. AND I'LL SHOW YOU THE NEUROLOGIC. THEY GET A SPECIFIC SPINAL CEREBELLAR. THAT'S VITAMIN E DEFICIENCY. WHAT'S SPINAL CEREBELLAR? PROPRIOCEPTION, VIBRATION, YOU WONDER WHY DOCTORS HAVE TUNING FORKS, VIBRATION IS ONE, DEEP TEND REFLEXES. THEY ARE CLUMSY WHEN THEY START EARLY VITAMIN E DEFICIENCY, NOT A MUSCLE ACHE OR WEAKNESS, PURELY DUE TO VITAMIN E.

WHEN YOU'RE NOT HAVING VITAMIN A, TAKING YOUR MULTI-VITAMIN OR WHATEVER, THEY GET ABNORMAL DEPOSITION IN THE BACK OF THE CENTRAL VISION, FIRST THING THAT GOES IN NIGHT VISION AND THEY LOSE VISION ENTIRELY AND THEN ONCE AGAIN WE SAID THE RED CELLS ARE VERY IMPORTANT, IF WE DON'T HAVE A POOL EXCHANGING WITH IT THEY ARE GOING TO HAVE WHAT'S CALLED ACANTHOCYTE, GREEK FOR SPINE, MESSED UP LIPID. YOU CAN SEE THAT IN ANIMAL MODELS TOO. SO THEY ARE MISSING MICROSOME AL TRIGLYCERIDE TRANSFER PROTEIN, BACK TO BASIC SCIENCE, RIBOSOME IN THE LIVER OR INTESTINE, MAKING APOB, THE DIMER, INTO APOB. YOU GET WHAT'S IN THIS CASE DLDL, AND THIS IS WHERE VITAMIN E AND A SLIP ALONG FOR THE RIDE. SO THE BIGGEST PROBLEM THERE IS NO OTHER TRANSPORTER REALLY FOR THESE AND THAT'S WHERE THEY GET THE NEUROLOGIC.

IF YOU LOOK HERE THE ONLY NORMAL CELL IS HERE. SO ONCE AGAIN THE MEMBRANES ARE MESSED UP. WITHOUT THE LIPOPROTEIN, RED CELLS MEMBRANE GETS MESSED UP. THIS IS AN INTESTINAL CELL. THIS IS ALL THIS PATIENT HAD WAS ONE OREO COOKIE THE DAY BEFORE. YOU CAN HAVE YOUR MICRO VILI FOLDED UP. ALL THIS, ALL THIS STUFF HERE, ALL THIS IS THEIR OREO COOKIE. THESE ARE NORMAL GOBLET CELLS BUT OTHERWISE IT LOOKS PERFECTLY NICE. IT CAN GET IN AND GOES BACK OUT, SLOUGHED OFF. AND IT CAN LOOK ORANGE IF YOU DO ENDOSCOPY, NOT PHYSIOLOGICALLY AFFECTS THE ENTEROCYTE ITSELF, AND THE NEUROLOGIC DEGENERATION, I SAID SPINAL CEREBELLAR
ATAXIA, VITAMIN E DEFICIENCY SO WE GIVING INCREDIBLE DOSES, RECOMMENDED DAILY IS ONE TABLET, THEY ARE GETTING 12 TABLETS 3 TIMES A DAY, WE'RE BARELY GETTING ENOUGH IN.

VITAMIN A, THIS IS THE OPTIC NERVE, VESSELS AND NERVES COME OUT, CENTRAL VISION, THEY GET DEPOSITION OF PROBABLY WE'RE NOT REALLY SURE WHAT IT IS BUT BASICALLY YOU CAN'T SEE CENTRAL VISION AND THEY BECOME FUNCTIONALLY BLIND. SO THAT'S THE FIRST ONE. THIS IS THE SECOND ONEMENT . THIS IS INTERESTING FROM A GENETIC ASPECT, ALSO WHEN YOU GO TO THE STORE TONIGHT OR OVER THE WEEKEND, YOU'RE GOING TO FIND OUT YOU ARE PART OF THE SITOSTEROL EXPERIMENT. THERE'S A DEFECT IN THE INTESTINE, AND BASICALLY A DEFECT IN THE LIVER. FOR SOME REASON THE HUMAN BODY DECIDED THE ONLY STEROL THEY WANT IS ANIMAL CHOLESTEROL. THERE ARE NUMEROUS OTHER STEROLS BUT FOR SOME REASON THESE EITHER ARE VERY OXIDIZABLE, THEY CAUSE INFLAMMATION, OR THINGS LIKE THAT.

SO THE BODY HAS DECIDED IT WANTS AN ABC TRANSPORTER, AND IT HAS DESIGNED AN ABCG 5 AND G8. WHERE DO WE HAVE OUR CHOLESTEROL? IN THE YOLK OF AN EGG. WHERE DO PLANTS FROM SITOSTEROL? IN THE SEEDS, HIGH IN SITOSTEROL. IF PATIENTS WITH THE DEFECT IN THE SPRING THEY GARDEN, FRESH VEGETABLES, THEY THINK THEY ARE DOING GREAT, XANTHOMAS GET BIGGER. THEY KNOW IT. THE ONLY DEFECT BETWEEN PLANT AND ANIMAL IS IN THE 24 POSITION. IF YOU HAVE ONE CARBON, IF YOU HAVE TWO CARBONS IT'S SITOSTEROL. ALGAE PUTS TWO, THERE ARE NUMEROUS ONES BUT THE BODY DOES NOT WANT ANYTHING OTHER THAN CHOLESTEROL. SO ONE CARBON, TWO CARBONS. SO WHY CAN'T WE JUST MEASURE THESE? WELL, WHEN YOU GO TO CLINICAL CHEMISTRY, THEY MEASURE CHOLESTEROL BY PUTTING A COLOR GROUP ON THE HYDROXYL GROUP. SO WE CAN'T REALLY DIFFERENTIATE. YOU GOT TO USE HPLC OR GC. THEY LOOK LIKE FAMILIAL, BUT THE ONE THING IN MY MIND IS THEY HAVE ARTHRITIS.

AND THERE'S A DEFECT HERE, SO IT'S A DIMER, AND WE KNOW CHOLESTEROL, THIS IS THE ENTEROCYTE. WE HAVE A STEROL PUMP, WHICH IS NPC 101, WHICH IS NOT NIEMANN-PICK BUT ALLOWS ANY STERILE IN, G 5 AND G 8 PUMP OUT ANY STEROL OTHER THAN CHOLESTEROL. IN THE CASE OF SITOSTEROLEMIA, THEY PUMP IT IN AND DON'T HAVE THIS. THIS GETS INTO THE BODY, IT'S VERY OXIDIZABLE, CAUSES ARTHRITIS, THINGS LIKE THAT. SO ONE OF THE INTERESTING THINGS WAS WE SAID WHAT IF WE COULD TARGET THIS? AND THERE IS A DRUG THAT WILL TARGET THIS, AND SURE ENOUGH YOU CAN REALLY VERY EFFECTIVELY DECREASE THE AMOUNT GETTING INTO THE BODY OF THE PATIENT.

WHAT DOES IT HAVE TO DO WITH YOU ALL? WHEN YOU'RE ABSORBING FOOD YOU HAVE PANCREATIC ENZYME, CHOLESTEROL, TRIGLYCERIDE, IN MICELLES. WE HAVE CHOLESTEROL FROM BILE, NOT THAT MUCH SITOSTEROL. ANY SITOSTEROL GETS PUMPED OUT. WHAT DID SOMEONE THINK? WHAT IF WE ENHANCED THE DIET WITH SITOSTEROL? SUCH AS BENOCOL, IT'S A MARGARINE, 100 PER CENT SITOSTEROL. AND IN THAT CASE ONE FOR ONE ONLY THE ORDER OF 2%, ENOUGH TO MAKE YOU INCREASED COST OF YOUR FOODS PROBABLY 25%.

IF YOU LOOK IN THE BUTTER, IT'S ON THE RIGHT SIDE, 100% SITOSTEROL. PROMISE ACTIVE, WHY CAN THEY SAY ACTIVELY REMOVES CHOLESTEROL? IT'S THE SITOSTEROL. HOW ABOUT MINUTE-MAID? REDUCES CHOLESTEROL. EVEN IN CHEESE, BREAD. IN HEALTHY HEART, KEEPS YOUR CHOLESTEROL LOW. ALL THEY HAVE TO DO IS SAY IT'S A SUPPLEMENT AND IT LOWERS IT 2 TO 8%. YOU'RE GOING TO PAY 25% MORE PROBABLY. BUT IT'S ALL OVER THE MARKETS. SO EVEN IN THE PHARMACY YOU'LL SEE CHOLEST OFF, CARDIO-CHOL, YOU BECOME A VEGETARIAN, YOU'RE GETTING A PLANT. I'M GOING TO PROBABLY END ON ONE LAST GENETIC ONE WHICH IS VERY IMPORTANT, NOT TALK LIKE ANYONE, BUT ANYONE IN THE LIPID FIELD KNOW WHAT THE CURRENT $15,000 QUESTION IS? THAT'S THE PCS K-9 INHIBITOR. WE'VE ARGUED AT STATINS AT $1200, $1500 A YEAR, NOW THERE'S TWO, PCSK9 INHIBITORS, A BARGAIN AT $14,000, DOUBLE OR TRIPLING OUR HEALTH INSURANCE PHARMACY IF EVERYONE WHO WOULD LIKE TO BE ON THEM IS ON THEM. SO BASICALLY, WHAT IT DOES IS WHEN LDL WAS CLEARED, WE KNOW THE BASIC PHYSIOLOGY, THE THING THAT REGULATES LDL RECEPTOR IS THIS PCSK9, CO-SECRETED, INSULIN, TRIGLYCERIDE, DIET, FREDP, ALL THOSE AFFECTING THE SRB 1 THAT FEEDS UPON THE LDL RECEPTOR.

IT ENDS UP THIS WAS FOUND IN A GENETIC STUDY WHICH I DON'T HAVE TIME TO TELL ABOUT, BUT IT ENDS UP IF YOU'RE MUTATED UP HERE, YOU GET A GAIN IN FUNCTION MEANING IT WORKS TOO WELL AND DESTROYS LDL RECEPTOR. IN THIS CASE YOU HAVE A LOSS OF FUNCTION, IT DESTROYS IT AND YOU HAVE LOW LDL. AND ACTUALLY GENETICALLY YOU DO BETTER THAN ANY DRUG THAT IS POSSIBLY DONE, BUT AS A RESULT THE PHARMACEUTICAL COMPANY NOTICED YOU GET LDL RECEPTORS, LDL COMES, YOU RECYCLE IT, THIS KEEPS GOING, 100, 150 TIMES. AT THE SAME TIME, PCSK9, WHICH IS REGULATED BY ANYTHING YOU COULD THINK OF, EVERYONE IN THIS FIELD HAS SOMETHING THAT REGULATES IT, AND WHAT IT DOES IS IT DETERMINES WHAT YOUR LDL VALUE ULTIMATELY WILL BE BECAUSE IT DESTROYS LDL AFTER 75 OR 110 TIMES. THE COMPANY SAID WHAT IF WE CAN DO SOMETHING TO THIS AND DEVELOPED MONOCLONAL ANTIBODIES, THEY DESTROY THIS AND IT ALLOWS YOUR LDL NOW TO HAVE MORE LDL RECEPTORS AND YOUR CHOLESTEROL GOES DOWN ANOTHER 60 TO 70%.

SO WE CAN GET LDL VALUES DOWN TO 40, 30, 20, PRETTY EASILY WITH THESE PCSK9s. WE DON'T KNOW THE LONG-TERM EFFECT OF A MONOCLONAL ANTIBODY INJECTED EVERY TWO WEEKS. PROBABLY KNOW THE LONG TERM OF A $15,000 A YEAR DRUG, VERY EXPENSIVE. BUT I CAN TELL YOU THERE ARE PEOPLE ON CAMPUS WORKING WITH PEOPLE I COLLABORATE DEVELOPING A VACCINE, WHICH YOU MAY BE ABLE TO TAKE ONCE A YEAR, SMALL MOLECULES PEOPLE ARE TRYING TO DO, ANYONE WHO CAN MAKE IT CHEAPER WILL BE THE NEXT REPLACEMENT FOR STATINS. SO THESE ARE THE TWO, ITS PRICE IS THE BIGGEST THING. I'M GOING TO FINISH IN TWO MINUTES. WELL, FIVE MORE MINUTES, THREE SLIDES ON THERAPIES TRANSITIONING TO DENNY TALKING ABOUT A COUPLE DISORDERS WHERE THEY ARE DOING LARGE CLINICAL TRIALS, I'M NOT GOING TO TALK ABOUT THOSE HERE. BUT WE'VE TALKED ABOUT WHERE THE TRIGLYCERIDE GOES DOWN TO LDL AND HAVE STATINS, WHICH ARE ON THE MARKET, THIS IS CHOLESTEROL ABSORPTION INHIBITORS, BILE ACID RESIN, THEY ALL INCREASE THE AMOUNT OF LDL RECEPTORS.

WE ALSO TALKED ABOUT PCSK9s THAT EITHER INHIBIT OR PREVENT THE DEGRADATION OF LDL RECEPTORS SO WE HAVE PCSK9s. WE HAVE APPROVED FOR THE HIGHEST OF THE HIGHEST CHOLESTEROL APOB, WHAT'S ON THE TRIGLYCERIDE PARTICLE. THAT WILL DEGRADE. THEY HAVE AN INHIBITOR OF NTP. WHAT DO THEY HAVE TO WORRY ABOUT? FATTY LIVER COULD BE A COMPLICATION SO SAFETY IS POTENTIALLY A FACTOR. I'M ALSO SHOWING WE NOW WENT BACK TO THE TRIGLYCERIDE PARTICLE, THIS IS THE TRIGLYCERIDE PARTICLE, THE ONLY THING I REALLY MENTIONED WAS LIPOPROTEIN LIPASE, IF YOU MISS THESE TRIGLYCERIDE IS 8000. OVER THE LAST 2-3 YEARS THEY HAVE DISCOVERED THAT APO A-5 STIMULATES THAT, THEY FOUND IF IT'S MISSING IN THE AMISH, IT LOWERED TRIGLYCERIDE, AND APOC2 HAS BEEN TRIED AND PUBLISHED, NEW ENGLAND JOURNAL, IT IS AN INHIBITOR, OR IT'S AN INHIBITOR SO THERE'S ALSO NEW ENGLAND JOURNAL, THE LAST TWO MONTHS, THIS ANGIOPOTIN-LIKE 3, 4 AND I HAVE TO ADD 8, HAVING EFFECT ON LIPOPROTEIN LIPASE, ALL AFFECTED BY INSULIN.

FATTY LIVER. ADIPOCYTES, YOU NAME IT. SO THIS HAS SUDDENLY BECOME A VERY COMPLEX THING BECAUSE IF THESE PARTICLES ARE NOT COMPLETELY METABOLIZED, WE CALL THEM REMNANT PARTICLES, THEY PROBABLY ACCOUNT FOR A LOT OF THE THE ATHEROSCLEROSIS WE CAN'T ACCOUNT FOR. THIS GOES TO TRANSCRIPTION, TRANSLATION, PROTEIN TARGETING, THEY ARE APPROVED, ADENO-ASSOCIATED VIRUSES. ABOUT $750,000 BUT IT'S APPROVED IN EUROPE. WE'VE HEARD ABOUT CRISPR AND HOW THAT MAY COME IN TO EVOLVE. THIS IS THE ONE I MENTIONED INVOLVED WITH APOB, ACOC 3, STUDIES IN THE CLINICAL CENTER RIGHT NOW WITH THAT. AND THERE'S ALSO OTHER OF THESE ATTACKS SINGLE-STRANDED DNA WITH APO-A, AND OTHER THINGS. THE PCSK9, AN ANTI-SENSE, IT MAY BE TOO EXPENSIVE, BUT THERE ARE ENZYME REPLACEMENTS, THIS ONE FOR CHOLESTEROL ESTER STORAGE DISEASE, RECOMBINANT PROTEINS THAT'S THE ONE THAT WE'RE DEVELOPING HERE AT THE NIH. RATHER THAN SPEND THE WHOLE PROTEIN USE A-1, THEY DON'T HAVE A-1.

APO IS USEFUL, APOC 2 IS USEFUL, DEVELOPED HERE AT THE NIH. THEN THIS MENTIONS THE PCSK9 INHIBITORS, P-PAR, ON AND ON. I'LL WRAP IT UP WITH THAT BUT I THINK I WOULD LIKE TO SAY WHERE I COULD NOT WORK WITH TRANSLATIONAL MEDICINE WITHOUT INTERACTING VERY CLOSELY WITH THE BASIC SCIENCE PEOPLE AND ALL THAT. AND THE THEME FOR WHEN I'M GOING TO END WITH MY BRIDGE TALK, HOWEVER I DON'T THINK HE SHOWED YOU SOME OF THE DETAILS. IF YOU LOOK REALLY CAREFUL, THERE'S A CLINICAL RESEARCHER RIGHT OVER HERE AND THERE'S A IS BASIC SCIENCIC SCIENCE.

THEY ARE WORKING VERY CAREFUL. BUT AS I THINK MOST OF THE PEOPLE IN THE AUDIENCE KNOW, IT'S REALLY THE POSTDOCS AND THE FELLOWS OUT HERE THAT ARE PROBABLY GETTING A LOT OF THAT DONE. IF YOU LOOK CAREFULLY THERE'S A FEW SUMMER STUDENTS, WAY OUT THERE. [LAUGHTER] BUT I THINK, YOU KNOW, AS I WOULD END MY ACKNOWLEDGMENTS ARE FOR THE BRIDGE MAKERS. WE CAN'T REALLY SUCCESSFULLY DO THAT AND I'M ONLY GOING TO SHOW THE FEW PEOPLE WHO ARE INVOLVED IN JUST OUR RECOMBINANT ENZYME. THERE'S MANY PEOPLE LEFT OUT, AND I HAVE NOT PUT IN MY NIH COLLABORATORS, AND I'LL TURN IT OVER TO DENNY AND WE'RE GOING TO SWITCH TO INTRACELLULAR CHOLESTEROL DISORDERS.

>> THAT WAS NICE. >> LET ME ASK YOU A QUICK QUESTION HERE. GIVEN THIS EXPANDED PLAYING FIELD, WITH MANY OPPORTUNITIES TO INTERACT PHARMACOLOGICALLY, ANTIBODIES AND SO FORTH, DIFFERENT POINTS, SO WHAT THEN BECOMES THE ROLE OF DIET AND EXERCISE? >> YOU KNOW, I THINK THAT'S A VERY TOUGH QUESTION, BUT THE WAY I KIND OF GIVE IT TO MY PATIENTS IS I TELL THEM BECAUSE I'M A METABOLISM PERSON TO THINK OF IT AS LIKE YOUR SINK. IF THE SINK IS FILLING UP, THERE'S SEVERAL WAYS THAT IT'S FILLING UP. ONE IS THE WATER IS COMING IN. THAT'S SYNTHESIS, MANY TIMES GENETIC DISORDERS, WE HAVE TO INHIBIT THAT. THE OTHER THING IS IT'S CLOGGED. THINGS LIKE THAT. BUT I SAY IF IT'S FILLED UP WHAT HAPPENS IF YOU COME AND POURED A BUCKET OF WATER IN? YOU'RE GOING TO INCREASE IT. SO I KIND OF TELL THEM THERE'S PROBABLY 25% OF THE POPULATION WHO GENETICALLY HAVE IT PERFECT, AND THEY CAN EAT A DOZEN EGGS A DAY. THEY WILL NOT HAVE A CHOLESTEROL — THERE'S FAMOUS PAPERS THAT HAVE SHOWN THAT.

25% HAVE DELAYED CATABOLISM, A MAJOR EEFFECT. THE OTHER 50% ARE IN BETWEEN DEPENDING ON EXERCISE. WE PROBABLY MAKE PUBLIC GUIDELINES FOR THAT 25% BUT IF WE HAD A WAY MAYBE THERE ARE SOME PEOPLE WHO DON'T, AND THERE HAVE BEEN GOOD QUESTIONS OF WHAT EVIDENCE DO WE HAVE FOR CHOLESTEROL AND DIET? WELL, YOU KNOW, SOME OF THE OLD ONES EVEN THAT WE QUESTION WE SHOULD EVEN QUESTION WHETHER OR NOT WE SHOULD USE THEM AT ALL. BUT, YOU KNOW, IT'S ONE OF THOSE TOUGH QUESTIONS WHERE DIET AFFECTS A SMALLER GROUP, IT'S DIFFICULT TO TELL WHICH GROUP THAT'S GOING TO HAVE AN EFFECT ON. >> LET ME ASK ONE MORE. SO THIS IS THE NIH. AND YOU'RE IN A POSITION TO LOOK AT ALL OF THESE PARAMETERS. >> UH-HUH. >> IN COLLABORATION WITH PEOPLE. YOU'RE THE PATIENT SEEING A COMPETENT INTERNIST SOMEPLACE WHO DOESN'T HAVE ACCESS TO THIS, AND YET THE PATIENT HAS A CHOLESTEROL CLOSE TO 200 OR 200-SOMETHING, AND, YOU KNOW, WHAT'S THE CURRENT STATUS OF MEDICINE WITH REGARD TO DEALING WITH THAT PATIENT? WE'RE ALL TOLD TO EXERCISE AND DON'T EAT SO MUCH BUT IT DOESN'T DO VERY MUCH.

>> YEAH, IT'S CHAOTIC, SCHIZOPHRENIC AND VERY MIXED UP BECAUSE, YOU KNOW, I THINK THE THING IS THAT WE HAVE GUIDELINES THAT ARE CONFUSING AND THINGS LIKE THAT. PEOPLE WOULD LIKE TO MAKE IT EASY. IT'S GETTING SO COMPLEX, YOU KNOW, WHEN YOU SAY THAT THERE'S LIKE 70 MILLION PEOPLE WHO WOULD LIKE TO GET IN THE NIH BUT DENNY AND I CAN'T SEE THAT MANY. I THINK ONE OF THE THINGS IS WE TRY TO MAKE GUIDELINES WHICH ARE EASY FOR A PRACTITIONERS, AND THE CURRENT ONES HAVE SLID TO ONE SIDE WHERE YOU SAY YOU JUST GIVE THIS DOSE AND YOU REALLY DON'T DO THAT MUCH FOLLOW-UP. BUT FOR THAT OTHER 25%, THEY PROBABLY NEED TO SEE WHAT THAT EFFECT IS, RATHER THAN SAYING GIVE A STATIN, 50%, PEOPLE NEED ENCOURAGEMENT AND LOOK AND SAY YOU'RE ONLY AT 30%.

THEY WILL SAY, WELL, I'VE NOT BEEN TAKING MY MEDICINES, HAVEN'T BEEN EXERCISING, WHEN YOU SHOULD GET THAT 50%. SO I THINK WE NEED SOMETHING WHERE WE KIND OF CHECK UP ON THEM. IF WE DON'T GET 50%, THEY'VE HAD A HEART ATTACK, WE PROBABLY NEED GUIDELINES THAT SAY WE NEED SOME ADDITIONAL MEDICINES. THE CURRENT GUIDELINES ALMOST ARBITRARILY SAID ANY STUDY THAT'S MORE THAN 10 YEARS OLD WE'RE GOING TO THROW OUT BECAUSE IT COULDN'T HAVE BEEN GOOD. SO THAT LEAVES OFF NIACIN, BUT WHEN YOU LOOK BACK AT BIOACID RESINS THEY SHOWED ANGIOGRAPHIC CHANGES IN 500 PEOPLE. WE'RE NOT SEEING CHANGES IN 20,000 PATIENTS BECAUSE THEY ARE SO QUICKLY RUSHING THROUGH THE STUDIES WITH, YOU KNOW, QUESTION NO ENDPOINTS. SO I THINK WE'RE KIND OF FORGETTING OUR PAST WHERE PEOPLE NEED ENCOURAGEMENT, NEED TO KNOW SCIENCE IS RELIABLE, AND THEY DO NEED SOME CHECKING UP ON I THINK FOR MOTIVATION.

>> RIGHT NOW THE STATINS ARE CHEAP AND AVAILABLE. >> CHEAP AND AVAILABLE, CRESTOR IS GOING GENERIC, THEY WILL ALL BE GENERIC. NOT QUITE PUTTING THEM IN THE WATER BUT, YOU KNOW, IT'S FOR THE APPROPRIATE PATIENT, YOU KNOW, I THINK IT'S — FROM A PUBLIC HEALTH STANDPOINT IT'S BEST MEDICINE. >> FOR THE APPROPRIATE PATIENT, THE ONE IN WHOM IT WORKS. >> RIGHT, YEAH, BUT MANY STUDIES ARE TRYING TO LOOK AT HEALTHY PEOPLE AND SAY CAN WE GET BETTER THAN HEALTHY. WELL, I DON'T THINK YOU CAN. >> OKAY. THANK YOU VERY, VERY MUCH. THAT WAS MARVELOUS. SO DENNY, WOULD YOU CONTINUE AND WE'LL HAVE QUESTIONS AFTERWARDS.

[APPLAUSE] >> OKAY. WHAT I'D LIKE TO TALK ABOUT THIS EVENING IS DISORDERS OF CHOLESTEROL HOMEOSTASIS, I PROBABLY SHOULD HAVE RENAMED THIS AND BEEN MORE SPECIFIC. I'M GOING TO TALK ABOUT DISORDERS OF INTRACELLULAR CHOLESTEROL HOMEOSTASIS IN CONTRAST TO THE SORT OF EXTRACELLULAR AND PLASMID HOMEOSTASIS. THE TWO DISORDERS THAT I'M GOING TO TALK ABOUT TODAY ARE SMITH-LENLI-OPITZ AND NEIMANN-PICK. SMITH-LENLI-OPITZ IS A MAL FORMATION COGNITIVE IMPAIRMENT, NEIMANN-PICK IS LETHAL. THERE'S AN INCIDENCE , AND NPC
IS LYSOSOMAL STORAGE DISEASE, ACCUMULATION OF FREE CHOLESTEROL, IT'S RARE, WITH INCIDENCE ON THE ORDER OF 1 IN 100,000, AND AGAIN THERE'S NO APPROVED THERAPIES. IN MY RESEARCH GROUP, OUR MAIN GOAL IS TRY TO COMBINE BASIC AND CLINICAL RESEARCH TO DEVELOP AND TEST VARIOUS THERAPEUTIC INTERVENTIONS.

SO I'M GOING TO GO THROUGH THESE TWO DISORDERS AND I'LL START WITH SMITH-LENLI-OPITZ SYNDROME, THIS IS AN INBORN CHOLESTEROL SYNTHESIS, IT HAS A VERY BROAD PHENOTYPIC SPECTRUM, CHARACTERISTIC OF A LOT OF THESE LYSOSOMAL — AUTO AUTOSOMAL DISORDERS. CLASSICAL DISEASE, PEOPLE FIGURE OUT TO DIAGNOSE THESE DISORDERS YOU END UP GETTING THE VARIATION, THE MORE SEVERE CASES AND MILDER CASES. SO IT RANGES FROM A SEVERE DISORDER AND THOSE INFANTS OFTEN DIE PRIOR TO BIRTH OR SOON AFTER DUE TO COMPLICATIONS OF MAJOR MALFORMATION, TO A MILD DISORDER THAT COMBINES MINOR PHYSICAL FINDINGS WITH NEURO COGNITIVE PROBLEMS. LEARNING AND BEHAVIORAL PROBLEMS. ON THIS SLIDE I'M SHOWING SOME OF THE TYPICAL CRANIOFACIAL FEATURES, THEY FREQUENTLY HAVE MICROCEPHALY, A SMALL HEAD, THEY WILL HAVE DROOPING OF THE EYELIDS WHICH IS KNOWN AS PTOSIS, THEY FREQUENTLY HAVE THIS SMALL UPTURNED NOSE SO YOU'RE LOOKING INTO THE NOSTRILS IN THESE KIDS, AND THEY HAVE FREQUENTLY HAVE SMALL JAW, THEY DON'T HAVE CLEFT LIFT BUT CLEFT PALATE IS FREQUENT. MANY OF THE KIDS MORE SEVERELY AFFECTED WILL HAVE POST AXIAL, SO ON THE SIDE OF THE HAND WITH THE SMALL FINGER OR SMALL TOES, POST AXIAL, POLY DACTYL, EXTRA FINGER OR SO.

SEVERELY AFFECTED CHILDREN CAN HAVE AMBIGUOUS GENITALIA, THIS IS A MALE CHILD, AS YOU CAN SEE HAS AMBIGUOUS GENITALIA. THE MOST COMMON PHYSICAL FINDING IN SMITH-LENLI-OPITZ. DON'T PANIC IF YOU HAVE THIS, THIS IS A NORMAL VARIANT, IT'S JUST IT'S VERY COMMON IN SMITH-LENLI-OPITZ SYNDROME. IN ADDITION TO THE PHYSICAL MANIFESTATIONS, SMITH-LENLI-OPITZ SYNDROME HAS A BEHAVIORAL PHENOTYPE. THESE BABIES ARE REALLY HARD TO CARE FOR. THEY ARE IRRITABLE. THEY HAVE A POOR SUCK, LACK OF INTEREST IN FEEDING. THEY COULD CARE LESS. THEY HAVE TACTILE DEFENSIVENESS, CRYING, WON'T EAT AND DON'T WANT TO BE PICKED UP. IF YOU CAN IMAGINE TRYING TO TAKE CARE OF A BABY AS A MOTHER OR FATHER THAT DOESN'T WANT TO FEED, DOESN'T WANT TO BE PICKED UP AND SITS THERE AND CRIES ALL THE TIME, IT'S REALLY ROUGH ON THE PARENTS.

OLDER CHILDREN HAVE AUTISTIC AND OBSESSIVE COMPULSIVE STRAITS, HYPERACTIVITY, SENSORY HYPER REACTIVITY. TRY CLIPPING THEIR NAILS AND THEY GO THROUGH THE ROOF. COMBING THEIR HAIR, THEY COULD GO THROUGH THE ROOF. SO IT'S A VERY INTERESTING BEHAVIOR PHENOTYPE. A LOT OF IT IS RELATED TO FOOD INTAKE. ONE CHILD WOULD EAT APPLES, THAT'S WHAT SHE WOULD EAT. HAD TO BE IN A SPECIFIC BOWL, HAD TO BE PEELED, AND HAD TO BE GALA APPLES. THAT'S HOW SPECIFIC IT WAS. OKAY. AS AN INBORN SYNTHESIS, DEFICIENCY OF 7 DEHYDRO CHOLESTEROL REDUCTASE, REDUCING THE DELTA 7 BOND, CHOLESTEROL IN THE LAST STEP OF THE SYNTHETIC PATHWAY, SO IN SMITH-LENLI-OPITZ SYNDROME WE HAVE A BLOCK HERE, CHOLESTEROL IN GENERAL IS DEFICIENT BUT YOU HAVE AN ACCUMULATION OF 7 DEHYDRO CHOLESTEROL, ACCUMULATES, CHOLESTEROL IS DEFICIENT. TO PUT IT INTO THE CONTEXT OF THE CHOLESTEROL SYNTHETIC PATHWAY THIS IS SMITH-LENLI-OPITZ SYNDROME, THE LAST STEP HERE.

THIS IS JUST THE LAST HALF OF THE CHOLESTEROL SYNTHETIC PATHWAY. WHAT I REFER TO AS AFTER YOU FORM THE CHOLESTEROL RING. SMITH-LENLI-OPITZ SYNDROME WAS THE FIRST DEFECT TO BE DEMONSTRATED IN THIS PATHWAY. SINCE THAT TIME A NUMBER OF OTHER GENETIC SYNDROMES HAVE BEEN SHOWN TO BE DUE TO DIFFERENT STEPS IN THE PATHWAY. I WON'T GO INTO THOSE TODAY BUT THEY TEND TO BE MORE RARE THAN SLOS. ONE OF THE QUESTIONS WE HAVE, YOU HAVE THIS DEFECT IN CHOLESTEROL SYNTHESIS, WHY DO YOU END UP WITH THIS DRAMATIC DEVELOPMENTAL SYNDROME? AND THIS RELATES TO THE FACT CHOLESTEROL ACTUALLY IS IMPORTANT, YOU HAVE TOO MUCH IT OBVIOUSLY CAUSES PROBLEMS BUT IF YOU DON'T HAVE ENOUGH THERE'S NORMAL CELLULAR PROCESSES THAT REQUIRE CHOLESTEROL, AND IT'S AN ESSENTIAL COMPONENT OF CELLULAR MEMBRANES, MAJOR COMPONENT MUCH MYELIN, IT'S A MAJOR COMPONENT IN WHAT ARE KNOWN AS LIPID RAPS, CONCENTRATED IN THE PLASMA MEMBRANE, HEDGE HOG SIGNALING, STEROID HORMONES, OXY STEROIDS AND BILE ACIDS.

SO IF YOU HAVE A CHOLESTEROL DEFICIENCY YOU COULD SAY WELL, YOU HAVE A DEFICIENCY THIS OF THESE NORMAL CELLULAR PROCESSES, AND THAT WOULD EXPLAIN WHY YOU HAVE A DEFECT IN SLOS. THIS ENTERS THE BIOLOGICAL PATHWAYS, TO CERTAIN DEGREE. IN SLOS YOU HAVE A QUESTION WHETHER IT'S DUE TO DEFICIENCY OF CHOLESTEROL OR WHETHER IT IS THE TOXIC EFFECT OF THE 7 DEHYDROCHOLESTEROL IT SELF. THAT'S A MAJOR QUESTION THAT WE'RE OFTEN TRYING TO REQUEST WHAT'S THE BIOCHEMICAL BASIS OF SMITH-LENLI-OPITZ, THE CHOLESTEROL DEFICIENCY OR 7 DEHYDRO CHOLESTEROL DEFICIENCY. IT DEPENDS. THERE ARE BIOLOGICAL PROCESSES, PROBABLY THE CHOLESTEROL DEFICIENCY, MY BIAS THERE'S A LOT OF BIOLOGICAL PROCESSES WITH DEHYDRO CHOLESTEROL. WE LOOKED AT WHETHER IT'S A DEFECT OF CHOLESTEROL OR DUE TO TOXICITY OF 7 DEHYDROCHOLESTEROL. THESE ARE INDUCED PLEURO POTENT THEM CELLS, CONTROL LINE, SLOS LINE.

AND WE CAN INDUCE FORMATION, THEY LOOK NORMAL, IF YOU MAINTAIN THEM IN MEDIA WITH CHOLESTEROL. THE CELLS TAKE IT UP FROM THEIR ENVIRONMENT. THEY HAVE NO NEED TO MAKE CHOLESTEROL THEREFORE THEY DON'T MAKE 7 DEHYDROCHOLESTEROL. IF YOU SWITCH TO LOW CHOLESTEROL MEDIA THE CONTROL CELLS ARE JUST FINE, THEY CONTINUE TO EXPRESS THE MARKERS OF UNDIFFERENTIATED MARKERS SUCH AS NANOG AND DON'T EXPRESS DIFFERENTIATED MARKERS SUCH AS PAX6. THE SLOL UNDERGOES DIFFERENTIATE TOWARDS NEURONAL LINEAGES, THEY LOSE THE PLURIPOTENT MARKER NANOG AND EXPRESS PAX6.

THIS SUGGESTS WHEN YOU INDUCE BIOCHEMICAL DEFECT YOU GET THIS PRECOCIOUS NEURONAL DIFFERENTIATION. AND WE WANT TO SEPARATE, WELL,S IS THAT BECAUSE WE MADE THE CELLS CHOLESTEROL DEFICIENT, BECAUSE THEY CAN'T MAKE THE CHOLESTEROL, OR DUE TO THE ACCUMULATION OF 7 DEHYDROCHOLESTEROL? WE MADE AN ITF CELL FROM ANOTHER PATIENT WHO HAS ANOTHER DEFECT IN CHOLESTEROL SYNTHESIS, THEY HAVE A DEFECT IN THE CONVERSION TO 7 DEHYDROCHOLESTEROL. SLOS IS RARE, AND THIS IS VERY RARE, THERE'S ABOUT 5 PATIENTS IN THE WORLD DESCRIBED WITH IT. WE'VE SEEN A COUPLE OF THESE, WE DIAGNOSED A COUPLE OF THESE PATIENTS, AND WE HAVE IPS, WE'RE ABLE TO MAKE IPS CELL LINES IN THESE PATIENTS AND USING THE SAME PARADIGM THIS IS THE CELL LINE IN A CHOLESTEROL REPLETE CULTURE, MAINTAINS PLUROPOTENCY, SUGGESTING IT WAS THE 7 DEHYDROCHOLESTEROL THAT WAS THE BAD ACTOR IN THIS PARTICULAR SITUATION.

THIS IS SORT OF A SUMMARY SLIDE FROM A LOT OF WORK WE DID. TRYING TO SAY WHAT IS HAPPENING, WHAT'S THE 7 DEHYDROCHOLESTEROL DOING? YOU WANT TO MAINTAIN BETA-CATENIN, YOU LOSE NORMAL WNT SIGNALING, IT TURNS OUT CHOLESTEROL IS ESSENTIAL FOR STABILIZATION OF WNT RECEPTOR COMPLEX, WHEN YOU PHUT IN 7 DEHYDROCHOLESTEROL YOU DESTABILIZE THAT RECEPTOR COMPLEX SO 7 DEHYDROCHOLESTEROL IS ACTING IN A TOXIC MANNER TO DISRUPT THE WYT SIGNALING COMPLEX AND YOU GET PRECOCIOUS DIFFERENTIATION OF NEURONS. WE ALSO THINK THAT THIS IS PROBABLY IS APPLICABLE CLINICALLY IN THE DEHYDROCHOLESTEROL MAY BE THE BAD ACTOR, AND WHAT THIS IS IS CORRELATION OF CEREBROSPINAL FLUID STEROLS, WITH NEURO COGNITIVE MEASURES, AND IN THIS CASE WE'RE LOOKING AT MEASURES RELATED TO THE ADIR OR TYPICAL AUTISM DIAGNOSTIC INTERVIEW SCORE. AND WHAT YOU CAN SEE IS THAT THE DEHYDRO — THERE'S BETTER CORRELATION WITH DEHYDROCHOLESTEROL AND ASPECTS, ONLY CORRELATIVE BUT SUGGESTING THIS MIGHT BE THE BAD ACTOR. THIS REALLY IS IN MY MIND THE FUNDAMENTAL QUESTION THAT WE HAVE TO ASK WHEN THINKING ABOUT TREATING SLOS. AND THAT'S REALLY TO WHAT EXTENT ARE THE MENTAL AND BEHAVIORAL PROBLEMS IN SLOS DUE TO FIXED DEVELOPMENTAL ABNORMALITIES VERSUS TO WHAT EXTEND DUE TO FUNCTIONAL PROBLEMS SECONDARY TO ABNORMAL STEROL COMPOSITION? TO USE ANOTHER ANALOGY, IF THIS IS AN ELECTRICAL SYSTEM MISWIRED WE'RE NOT GOING TO BE ABLE TO FIX IT.

IF IT'S AN ELECTRICAL SYSTEM AND THERE'S CORROSION AT THE CONTACT POINT, YOU CAN CORRECT THAT CORROSION. AND I THINK IN THE CLASSICAL TO MILD KIDS WE PROBABLY HAVE A COMPONENT OF THE BEHAVIORAL PROBLEMS THAT CAN BE ADDRESSED BY CORRECTING THE BIOCHEMISTRY AND CENTRAL NERVOUS SYSTEM. AND THIS IS AN OLD SLIDE, BUT IT GIVES A SENSE THAT PERHAPS WE CAN REVERSE THE DEFECT IN NEURONS, AND WE HAD SHOWN IN A NUMBER OF STUDIES THAT IN SLOS NEURONS THERE WAS A DEFECT IN NEURONAL TRANSMISSION, GLUTAMATE RECEPTORS.

AND WE STARTED UP ANOTHER COLLABORATION AND THIS WAS AN IN VITRO STUDY AND THE COLLABORATOR CALLED ME UP AND SAID DENNY, I THINK YOUR INITIAL STUDIES WERE WRONG, WE DID THE EXPERIMENT AND THERE'S NO DIFFERENCE BETWEEN THE CONTROL CELLS AND THE SLOS NEURON. WHAT'S GOING ON? YOU NEVER LIKE TO HEAR YOUR INITIAL STUDY MIGHT BE WRONG.

WE WENT OVER WHAT THEY DID. IT TURNED OUT WHILE THEY WERE PROCESSING THE NEURON, THERE WAS A SINGLE STEP IN WHICH THEY USED A MEDIA THAT CONTAINED CHOLESTEROL. WE CORRECTED THAT AND THEN WE COULD RESTORE THE DEFECT. SO IT GAVE YOU THE SENSE THAT A SINGLE EXPOSURE OF THE CHOLESTEROL WOULD ACTUALLY CORRECT — AT LEAST IN VITRO IN THIS STUDY CORRECT THE NEUROLOGICAL DEFECT. SO WE'VE BEEN WORKING IN THE CLINIC TO TRY TO SEE IF WE CAN DEVELOP A THERAPY FOR SLOS, AND IF YOU HAVE A DEFICIENCY OF CHOLESTEROL WE ALL KNOW HOW TO FIX THAT.

WE CAN FIGURE OUT WHERE TO GET CHOLESTEROL PRETTY EASILY. AND WHAT WE TRIED WAS DIETARY CHOLESTEROL SUPPLEMENTATION. AND THERE WERE MULTIPLE CASE REPORTS OUT THERE OF EFFICACY, BEHAVIOR GOT BETTER, YOU KNOW, KIDS ARE DOING BETTER. I AGREE THE KIDS ARE HEALTHIER. BUT IT'S HARD TO EXPLAIN BEHAVIORAL IMPROVEMENT BECAUSE CHOLESTEROL DOESN'T CROSS THE BLOOD-BRAIN BARRIER. WITH REGARDS TO CHOLESTEROL METABOLISM THE BRAIN IS ON ITS OWN, HAS TO MAKE ITS OWN CHOLESTEROL. SO WE SAID, WELL, OKAY, LET'S LOOK AT THIS. LET'S SET UP A RANDOMIZED PLACEBO CONTROL DOUBLE BLIND CROSSOVER TRIAL AND ANSWER THE QUESTION DOES DIETARY CHOLESTEROL DO ANYTHING, THIS WOULD BE IMPORTANT TO KNOW. I CAN THINK OF OTHER MECHANISMS. IF YOU KNEW THE MECHANISM YOU COULD TARGET THE THERAPY. WE USE PASTEURIZED EGG YOLK VERSUS SUBSTITUTE. DIETARY SUPPLEMENT WAS ENTRENCHED IN THE FIELD, IT WAS GOING TO BE IMPOSSIBLE TO GET PARENTS TO TAKE THE KIDS OFF OF CHOLESTEROL FOR ANY PROLONGED PERIOD OF TIME SO WE SAID FINE, WE'LL DO TWO WEEKS TREATED VERSUS UNTREATED BECAUSE WHAT WAS DESCRIBED IN THE LITERATURE WAS REALLY RAPID.

PUT THEM ON CHOLESTEROL, IT CORRECTS. IF YOU RUN OUT OF CHOLESTEROL BEHAVIOR GOES OFF. WE'LL TRY TWO WEEKS. WE WERE USING HYPERACTIVITY SUBSCALE, ABI CHECK LIST. REALLY UNDERPOWERED STUDY, HARD WHEN EVERYBODY IS CONVINCED IT WORKS. DR. PORTER, WE ALREADY KNOW THIS WORKS, WHY ARE YOU DOING THIS? HOW? WELL, WE KNOW IT WORKS. AFTER THREE YEARS GOT A LITTLE BIT FRUSTRATED, ENROLL 10 OF 40 SUBJECTS. ENOUGH IS ENOUGH. I'M NOT GOING TO GET THIS COMPLETED IN MY LIFESPAN. WE BROKE THE BLIND, STOPPED THE STUDY.

HAVE NEVER HAD THIS HAPPEN. ON THE PRIMARY OUTCOME MEASURE P-VALUE OF 1.0, WE NEED TO DO PLACEBO CONTROLLED STUDIES IF WE WANT TO UNDERSTAND THINGS WORK AND NOT JUST ACCEPT THE FACT THAT THEY WORK. JUST TRYING TO DO THINGS IS COMFORTING, AND PARENTS WILL THINK THAT THEY WORK. OKAY. A LITTLE BETTER, WE TRIED SIMVASTATIN, A STATIN DRUG WE HEARD ABOUT EARLIER, AND THE RATIONALE WAS MAYBE 70 HYDROCHOLESTEROL IS TOXICS SO YOU COULD DECREASE PRODUCTION OF 7 DEHYDROCHOLESTEROL BY INHIBITING AT AN EARLIER STEP. WE ALSO ADDED TO THAT HYPOTHESIS IN THAT WE PROPOSED THAT YOU COULD ALSO — YOU ALSO MIGHT GET A BENEFIT BECAUSE YOU INCREASED EXPRESSION, THE KIDS WITH MILD DON'T HAVE AN ABSENCE OF THE ENZYME.

THEY DON'T HAVE ENOUGH ENZYME. AND WHEN YOU GIVE SOMEBODY A STATIN DRUG, IT WAS WELL KNOWN IN THE LITERATURE THAT THEY UPREGULATE TO TRY TO GET AROUND THE BLOCK AND THESE ENZYMES ARE COORDINATELY REGULATED SO IF YOU TRY TO UPREGULATE YOU UPREGULATE VHCR 7, THE IDEA WAS OKAY, IF YOU HAVE AN ENZYME AT 5%, MAYBE DOUBLING TO 10% MIGHT BE BETTER. IN VITRO WE COULD SHOW THAT STATINS, SIMVASTATIN WOULD UPREGULATE, IN CELL CULTURE COULD INCREASE AND IMPROVE CHOLESTEROL TOTAL STEROL RATIO BY TREATING CELLS WITH A STATIN.

TWO-EDGED WORD, ONCE YOU GET TO HIGH ENOUGH DOSE YOU START SHUTTING DOWN CHOLESTEROL SYNTHESIS AND 70 HYDRO CHOLESTEROL IS BETTER THAN NOTHING. SO WE MOVED FORWARD WITH A PLACEBO-CONTROL TRIAL, CROSSOVER DISEASE, A SINGLE CHILD STARTS ON EITHER PLACEBO OR SIMVASTATIN AND HALFWAY THROUGH DO A CROSSOVER. THE ADVANTAGE IN A RARE DISEASE THAT HAS A HETEROGENEOUS PHENOTYPE, YOU CAN USE THE CHILD AS THEIR OWN CONTROL. IT'S NOT A TWO-ARMED STUDY WHERE YOU HAVE TO GET NUMBERS HIGH ENOUGH AND TRY TO MATCH THE TWO GROUPS. IN THIS CASE, BOTH GROUPS ARE MATCHED BECAUSE IT'S A CROSSOVER STUDY. OKAY? WE ENROLLED 23 PATIENTS AND HAD 18 PATIENTS COMPLETE THE STUDY WHICH WAS OUR GOAL. AND WE WERE ABLE TO SHOW THAT WE WERE ABLE TO ACTUALLY DECREASE HYDRO CHOLESTEROL TOTAL STEROL RATIO, DECREASE LEVELS CONSISTENT WITH THE HYPOTHESIS OF WHAT WOULD HAPPEN BIOCHEMICALLY, BUT ALSO WHEN WE LOOKED AT OUR PRIMARY OUTCOME MEASURE BEHAVIORAL OUTCOME MEASURE, IN THIS CASE IRRITABILITY SUBSCALE, WE COULD ACTUALLY SEE WE HAD A DECREASE IN THE SIMVASTATIN-TREATED GROUP VERSUS PLACEBO TREATED GROUP, FIRST DEMONSTRATING POTENTIAL OF DRUG THERAPY TO ALTER STEROL COMPOSITION AND IMPROVE SLOS BEHAVIOR.

SO SWITCHING GEARS TO THE SECOND DISORDER, THIS IS NPC 1, AS I MENTIONED EARLIER AUTOSOMAL RECESSIVE PROGRESSIVE LETHAL NEURODEGENERATIVE DISORDER DUE TO MUTATION OF NPC1 OR NPC2. WHEN CELLS TAKE UP CHOLESTEROL AT THE TAKE IT UP AS LDL WHICH WE HEARD EARLIER, IT MOVES INTO THE ENDOLYSOSOMAL SYSTEM. IN LDL CHOLESTEROL IS PRESENT AS LDL, AS CHOLESTEROL ESTERASE. REMOVE THE FATTY ACID, FREER CHOLESTEROL, NPC2 AND NPC1 COORDINATELY WORK TOGETHER THEN TO MOVE THE CHOLESTEROL ACROSS THE GLYCOCALYC OF THE SYSTEM AND TO MAKE IT BIOAVAILABLE TO THE REST OF THE CELL. SO IF YOU'RE MINUS NPC 2 OR NPC 1 YOU HAVE A BLOCK HERE AND GET AN ACCUMULATION OF THE FREE CHOLESTEROL AND THIS IS SHOWN IN THIS SLIDE, THIS IS A FIBROBLAST STAINED WITH A DRUG, A FLUORESCENT ANTIBIOTIC THAT BINDS TO CHOLESTEROL, YOU CAN SEE THESE NPC1 CELLS ACCUMULATE, AND IF YOU LOOK AT THE ORGANELLES, ENDOLYSOSOMAL SYSTEM. WE DID A SCREEN THROUGH SOME LARGE WHOLE EXOME SEQUENCING DATABASES, THERE MAY BE LATE ONSET VARIANT, SOME ALLELES LOOK TO BE DETRIMENTAL FREQUENT AT HIGH FREQUENCY, THE CLASSICAL DISEASE IS ON THE ORDER OF 1 IN 100,000.

OKAY. SO WHAT ARE THE THERAPEUTIC TRIAL ISSUES THAT WE'RE DEALING WITH? I PUT THIS IN THE NPC PART BUT IT'S ALSO TRUE OF SLOS. IT'S A RARE DISEASE. WE HAVE A HETEROGENEOUS PHENOTYPE. SO NPC WE'RE DEALING WITH A VARIABLE AGE OF ONSET, AND ALSO VARIABLE SYMPTOM COMPLEX. AND THEN CLINICAL PROGRESSION OCCURS OVER YEARS. IF YOU THINK ABOUT THIS, FROM THE STATISTICAL STANDPOINT WE'RE DEALING WITH A SMALL N, LARGE STANDARD DEVIATION, SMALL DELTA. IT DOESN'T GET ANY WORSE. IT'S WHAT MAKES CLINICAL TRIALS HARD IN RARE DISEASES. OUR GOAL IS ALSO TO STABILIZE OR DELAY NEUROLOGICAL DISEASE.

IF WE'VE LOST THE NEURONS, WE'RE NOT TALKING ABOUT METHODS CURRENTLY TO REPLACE THE NEURONS, CELLULAR THERAPIES WHERE WE GOING TO REPLACE NEURONS. SO WE'RE ACTUALLY LOOKING JUST TO STABILIZE IT AND STOP IT WHERE IT IS PRESENTLY OR SLOW IT DOWN. AND ONE OF THE ISSUES THAT WE HAVE IS THAT THERE WAS A LACK OF DEFINED AND ACCEPTED OUTCOME MEASURES, AND OUTCOME MEASURES ARE ESSENTIAL, IF YOU DO A CLINICAL TRIAL HOW DO YOU KNOW IT WORKS? SOME OF THE OUTCOME MEASURES I SHOWED YOU EARLIER WITH SLOS WERE USING THE ABERRANT BEHAVIOR CHECK LIST, NEURO COGNITIVE MEASURES. WE DIDN'T NECESSARILY HAVE THE EQUIVALENT IN NPC WHEN WE STARTED. SO HOW DO YOU TRY TO ADDRESS THAT PROBLEM? AND WITH ALL OF THESE RARE DISORDERS, IT'S ESSENTIAL THAT YOU DO NATURAL HISTORY TRIALS. YOU REALLY NEED TO UNDERSTAND THE DISORDER IN ORDER TO UNDERSTAND WHETHER IF YOU HAVE A THERAPY, IT WORKS OR NOT. AND IT'S REALLY TOO LATE TO START TRYING TO UNDERSTAND THE DISORDER WHEN YOU HAVE A POTENTIAL THERAPY AT HAND. IT'S BEST TO START IT AS SOON AS POSSIBLE.

WE STARTED SEEING PATIENTS HERE IN 2006 AND SINCE THAT TIME WE'VE SEEN 93 PATIENTS WITH NPC. AGE RANGE IS 3 MONTHS TO 54 YEARS OF AGE, BIASED TO THE PEDIATRIC AGE GROUP, MORE CLASSICAL DISEASE. I'LL TOUCH BASE ON THE SEVERITY SCORE WE DEVELOPED AND COME BACK TO IT LATER. OUR GOALS WERE TO IDENTIFY CLINICAL OR BIOCHEMICAL MARKERS USED AT OUTCOME MEASURES IN A THERAPEUTIC TRIAL. AND THEN ALSO TO IDENTIFY A BIOCHEMICAL MARKER THAT COULD BE USED FOR DIAGNOSTIC TESTING AND SCREENING.

AND THE REASON FOR THAT IS IN OUR COHORT OF PATIENTS OUR DIAGNOSTIC DELAYS WAS 4 TO 5 YEARS. FROM A POINT OF TIME A PARENTS IS SAYING SOMETHING IS WRONG WITH MY CHILD TO GETTING A DIAGNOSIS WAS 4 TO 5 YEARS. THAT'S BECAUSE IT AN INSIDIOUS DISEASE, SLOW ONSET, SOMETIMES IT'S HARD TO PICK OUT. OH, YOUR CHILD IS FINE. THEY ARE JUST A LITTLE BIT DELAYED. YOU KNOW, SO IT TOOK 4 TO 5 YEARS. AND THEY RANGED UP TO 19 YEARS FOR ONE FAMILY TO GET A DIAGNOSIS.

AND CONTRIBUTING TO THAT, SORRY, I GOT AHEAD OF MYSELF. I'LL COME BACK TO THAT. LET ME DISCUSS THIS NPC NEUROLOGICAL SEVERITY SCORE THAT I TALKED ABOUT. WE NEEDED A METHOD TO TRY TO DESCRIBE THE PATIENTS AND DESCRIBE THE PHENOTYPIC HETEROGENEITY, ALL THE PATIENTS WERE DIFFERENT SO HOW DO YOU DESCRIBE ONE PATIENT AND TRY TO DESCRIBE, YOU KNOW, ONE PATIENT HAS THIS SYMPTOM COMPLEX AND PROGRESSING AND THIS PATIENT HAS THIS SYMPTOM COMPLEX AND IS PROGRESSING.

HOW DO YOU TRY TO PUT IT TOGETHER TO GET AN OVERALL OF HOW DIFFERENT PATIENTS OF PROGRESSING? THIS SCALE INCLUDED NINE MAJOR DOMAINS, EIGHT MINOR, IT INCREASED FROM NORMAL TO HERE WITH AMBULATION, WHEELCHAIR DEPENDENT, INCREMENTAL AMOUNTS, A HIGHER SCORE RELATED TO INCREASED PHYSICAL IMPAIRMENT. WE CAN USE THIS RETROSPECTIVE AND PROSPECTIVE. THIS IS RETROSPECTIVE HERE, IT TURNED OUT THAT NPC WAS STUDIED AT THE NIH IN NINDS IN THE 1990s, IN FACT PETER WAS HERE AND WAS RESPONSIBLE FOR IDENTIFYING THE BIOCHEMICAL DEFECT OF THE INTRACELLULAR ACCUMULATION OF CHOLESTEROL, SO WE WERE ABLE TO GO BACK TO THOSE RECORDS AND FROM THOSE RECORDS RECONSTRUCT THE DISEASE PROGRESSION OVER 5, 10, 15 YEARS, A HUGE ADVANTAGE BECAUSE IF YOU'RE START DE NOVO YOU TAKE 15 YEARS BUT HERE WE COULD USE MEDICAL RECORDS.

A PLUG FOR ANY CLINICAL FELLOWS, GOOD RECORDS ARE VALUABLE. ESPECIALLY IN THESE RARE DISEASES. YOU REALLY CAN'T DO MUCH WITH, YOU KNOW, LUNGS CLEAR TO AUSCULTATION, SAME AS BEFORE. BUT WITH GOOD RECORDS WE WERE ABLE TO CONSTRUCT AND WE COULD SEE HOW PATIENTS PROGRESSED OVER YEARS, AND USING PROGRESSION MODELING WE CAN PREDICT PATIENTS PROGRESSED AT 2 POINTS PER YEAR ON THEY PARTICULAR SCALE. OKAY. COMING BACK TO THE DIAGNOSTIC DELAY, ONE OF THE THINGS CONTRIBUTING TO THE DIAGNOSTIC DELAY WAS THE DIFFICULTY IN MAKING THIS DIAGNOSIS. THERE WAS NOT A BLOOD TEST. THE CLASSICAL WAY OF MAKING THE DIAGNOSIS WAS THIS FILIPIN STAINING OF FIBROBLASTS, I SHOWED YOU THE PICTURE EARLY ON. TO GET FIBROBLASTS YOU HAVE TO DO A SKIN BIOPSY AND THERE'S A HIGH ACTIVATION ENERGY TO PEOPLE DOING A SKIN BIOPSY. OKAY. IT'S MUCH EASIER TO DRAW BLOOD, SEND IT TO THE LAB AND YOU DON'T HAVE TO WORRY ABOUT IT.

YOU DO SKIN BIOPSY, SEND IT TO THE LAB, GROW OUT THE CELLS, SEND TO A REFERENCE LABORATORY TO RUN TESTS, THERE'S REALLY ONLY 2 LABORATORIES IN THIS COUNTRY THAT RUN THE LIPID STAINING TEST AND DO IT WELL. THERE'S SOME TESTS IN EUROPE THAT DO IT VERY WELL TOO. SO IT WAS VERY SPECIALIZED TESTING REQUIRED TO SKIN BIOPSY, PEOPLE, OH, WE'LL TEST FOR ALL THESE OTHER THINGS SO LOTS OF KIDS CAME IN AND, YEAH, YOU LOOK AT THEIR RECORDS AND THEY EXCLUDED NEIMANN-PICK AB, A VARIATE BUT THERE'S BLOOD-BASED TESTS TO DO THAT BUT THEY DIDN'T GET TO NPC BECAUSE THAT WOULD HAVE TAKEN THIS EXTRA MAJOR STEP. THE OTHER APPROACH IS MOLECULAR TESTING, AND THAT REQUIRES A HIGH DEGREE OF SUFFICIENT, RELATIVELY EXPENSIVE, YOU HAVE TO BE THINKING ABOUT NPC 1 TO SEND OFF FOR SEQUENCING AT THE NPC GENE. FULL EXOME SEQUENCING ASK CHANGING THE DYNAMIC, WE'RE SEEING INDIVIDUALS DIAGNOSED BY WHOLE EXOME SEQUENCING. I HATE IT AS A GENETICIST BUT DIAGNOSIS GETS MADE. THE TESTS THAT WE DEVELOPED WAS BASED ON PLASMA OXY STEROL, NON-ENZYMATICALLY PRODUCED, IN NPC WE THINK YOU HAVE INTERSECTION OF STORAGE IN THE LIVER, FREE CHOLESTEROL AND YOU HAVE OXIDATIVE STRESS AND YOU END UP WITH THIS PRODUCTION OF THESE NON-ENZYMEATIC OXY STERILES.

THE BEST DIAGNOSTIC MARKER IS 3 BETA 5ALPHA 6 BETA, MARKEDLY ELEVATED IN NPC COMPARED TO CONTROLS, HETEROZYGOUS PATIENTS, WE EXTENDED THE STUDIES, LOOKED AT A SERIES OF OTHER LYSOSOMAL STORAGE DISEASES, NIH EXPERIMENT, I WALKED AROUND AND SAID WHAT DO YOU HAVE IN YOUR FREEZER. AND CAME UP WITH A WHOLE SERIES OF OTHER LYSOSOMAL STORAGE DISEASE WHERE I COULD GET A SMALL AMOUNT OF BLOOD FROM IT. AND THEN IN A LARGER STUDY WE CAN SHOW THAT IT HAS GOOD SENSITIVITY AND SPECIFICITY AND THIS IS REALLY MOVED INTO ABOUT 30 LABORATORIES NOW AND IS SORT OF BECOMING THE PRIMARY DIAGNOSTIC TEST FOR NPC. IT'S NOT PERFECT. IT DOES PICK UP NPA AND B BUT YOU CAN SORT THAT OUT QUICKLY IF YOU HAVE A POSITIVE THERE. SO WHAT ABOUT THERAPY FOR NPC? THE DRUG I'M GOING TO TALK ABOUT IS THIS DRUG CALLED 2 HYDROXYPROPYL BETA CYCLODEXTRIN. IT WAS DEVELOPED AS A PHARMACEUTICAL EXCIPIENT. IT WAS USED TO DISSOLVE DRUGS. IT'S A CYCLIC AL
OLIGOSACCHARIDE WITH HYDROPHOBIC CORE, AND THE OLIGOSACCHARIDE WILL ALLOW IT TO SOLUBLIZE IN WATER BUT THE HYDROPHOBIC CORE WOULD ALLOW IT TO BORN HYDROPHOBIC DRUGS, IT'S A GOOD CARRIER FOR HYDROPHOBIC SUBSTANCES, AND IT TURNED OUT THAT CINDY MELON'S GROUP WAS LOOKING AT THE NPC NOW AND IN NPC YOUR CHOLESTEROL IS BLOCKED IN THE ENDOLYSOSOMAL SYSTEM, SO THE REST OF THE CELL WAS ACTUALLY CHOLESTEROL STARVED, THEY HYPOTHESIZED MAYBE THERE'S A DEFECT IN NEUROSTEROID FORMATION AND FOUND THERE WAS A DEFICIENCY OF NEUROSTEROIDS IN THE NPC 1 MOUSE.

AND SO THEY SAID, WELL, THERE'S A DEFICIENCY, WHAT HAPPENS IF WE TREAT? THIS IS ALLOPREGNALONE, NOT WATER SOLUBLE, HOW ARE WE GOING TO SOLUBLIZE IT? DID IT IN CYCLODECKS CRITICAL CONDITION, WITH THE ALLOPREGNALONE AND GOT THIS REMARKABLE INCREASED SURVIVAL IN ANIMALS ESPECIALLY IF THEY STARTED TREATMENT YOUNG. IS ALLOPREGNALONE A THERAPY? A NUMBER OF LABS LIKE AT THIS, INCLUDING KRISTIN IN THE BACK HERE, THEY REALIZED IT'S NOT THE ALLOPREGNALONE BUT CYCLODEXTRIN IS THE ACTIVE SUBSTANCE HERE. THE TWO LABS WERE KRISTIN AND STEVE WACKILY'S AND BENNY LU IN JOHN'S LABORATORY, LOOKED AT THIS. BOTH LABS SHOWED INCREASED SURVIVAL OF THE NPC MICE TREATED WITH CYCLODEXTRIN AND COULD SHOW PATHOLOGICALLY THERE WAS IMPROVEMENT IN TH CEREBELLUM. IN NPC1 YOU LOSE THE CEREBELLAR CELLS STAINED, YOU CAN SEE THE CELL BODIES HERE, AND THEN THE CELL PROCESSES, AND NPC1 YOU HAVE LOTS OF THE POSITIVE CELLS, WHEN YOU TREAT WITH CYCLODEXTRIN YOU HAVE PRESERVATION OF THE CEREBELLAR CELLS, REVERSED PATHOLOGICAL CHANGES AND IMPROVED SURVIVAL. CHARLES VEET AT UNIVERSITY OF PENNSYLVANIA HAS A CAT MODEL OF NPC1, AND THIS IS A CLIP OF A KITTEN TREATED WITH INTRAFECAL CYCLODEXTRAN, AND AN UNTREATED KITTEN, NPC1 KITTEN HERE, JUMPING AROUND, BUT THIS ANIMAL SOON HAS TO BE EUTHANIZED.

AND THESE CYCLODEXTRIN TREATED ANIMALS CAN LIVE TO 2+ YEARS NOW, PRETTY REMARKABLE THE DEGREE OF SURVIVAL, PRESERVATION AND LACK OF PROKINGI CELLS AND LACK OF SYMPTOMS IN THE ANIMALS. THE QUESTION IS HOW DO WE MAKE THAT WORK IN THE KIDS? CAN WE TRANSLATE THAT FROM THE LABORATORY TO THE CLINIC? IN TELLING THIS STORY, I WANT TO TALK ABOUT ONE BIOMARKER, A PHARMACODYNAMIC MARKER. THE BIOMARKER IS 24S HYDROXYCHOLESTEROL. THIS IS AN OXY STEROL, IN CONTRAST TO THE OTHER OXYSTEROLS, ENZYMATICICALLY PRODUCED OXYSTEROL. CHOLESTEROL IS TAKEN UP, IT'S IN THE ENDOLYSOSOMAL SYSTEM, IT IS BY THE ACTION OF NPC2 AND 1 MOVED OUT OF ENDOLYSOSOMAL AND BECOMES BIOAVAILABLE.

WHEN CHOLESTEROL IS IN EXCESS IT MOVES TO THE ENDOPLASMIC RETICULUM, IT CAN MOVE INTO THE BLOOD, DEGRADED BY THE LIVER. IT TURNS OUT THAT 24 S HYDROXYCHOLESTEROL IS SPECIFIC FOR NEURONS WITH THE CENTRAL NERVOUS SYSTEM. THE REST OF THE CELLS IN THE BODY MAKE ANOTHER OXY STEROL KNOWN AS 27 HYDROXYCHOLESTEROL. SO 24S HYDROXYCHOLESTEROL IS A MARKER OF NEURONAL CHOLESTEROL HOMEOSTASIS. IN NPC1 YOU HAVE BLOCK IN THE EFFLUX OF CHOLESTEROL, OUT OF THE ENDOLYSOSOMAL SYSTEM, SO THE BIOAVAILABILITY TO THE REST OF THE CELL GOES DOWN, AND WHAT WE WERE ABLE TO SHOW IN NPC1 KIDS WHEN YOU AGE MATCH THEM FOR CONTROLS, THERE'S ACTUALLY DECREASE SERUM LEVELS OF 24 HYDROXYCHOLESTEROL. WHEN YOU GIVE CYCLODEXTRAN IT MOVES IT OUT OF THE LYSOSOMAL SYSTEM, CIRCUMVENTS THIS BLOCK, MAKES IT BIOAVAILABLE TO THE REST OF THE CELL AND WE PROPOSE THAT IF IT BECAME BIOAVAILABLE TO SIP 46 WE COULD SEE PRODUCTION OF 24 HYDROXYCHOLESTEROL, DONE IN THE MOUSE MODEL, SINGLE INJECTION, AND YOU CAN SEE INCREASED PRODUCTION OF THE 24 HYDROXYCHOLESTEROL COMPARED TO SALINE INJECTION. THIS IS A PHARMACODYNAMIC MARKER WE CAN USE TO ASK THE QUESTION IF WE GIVE THE DRUG TO THE KID ARE WE HAVING THE BIOCHEMICAL RESPONSE THAT WE EXPECT? SO HOW DO YOU GET THIS DRUG TO THE KID? YOU WANT TO GET IT TO THE BRAIN.

ANOTHER PROBLEM, LIKE CHOLESTEROL, IT DOESN'T CROSS THE BLOOD-BRAIN BARRIER. SO THE INITIAL APPROACH THAT WE USED WAS WHAT'S CALLED AMYORESERVOIR, BASICALLY A SMALL RESERVOIR OR BUBBLE THAT SITS UNDER THE SKIN AND HAS A SMALL CATHETER THAT ENTERS THE VENTRICULAR SYSTEM, MYORESERVOIRS WERE INVENTED AT THE NIH IN 1960s, USED TO STREET CENTRAL NERVOUS SYSTEM FUNGAL INFECTION BECAUSE FUNGAL DRUGS CAN'T GET IN, AND THEN CENTRAL NERVOUS SYSTEM TUMORS. SO THAT WAS OUR FIRST APPROACH. WE STARTED GIVING THE KID 50 MILLIGRAMS OF CYCLODEXTRIN, NO DRUG-RELATED ADVERSE EVENTS OR REACTIONS, AND TWO OUT OF THREE PATIENTS ALL HAD A POSITIVE RESPONSE WHEN WE LOOKED AT THE 24 HYDROXY CHOLESTEROL, BUT TWO OUT OF THE THREE ACTUALLY HAD WHAT WE HAD DEFINED AS SIGNIFICANT RESPONSE WHICH YOU COULD SEE THREE AND FOUR TIMES GREATER THAN THE STANDARD DEVIATION, AND YOU COULD SEE HERE THAT THE AREA UNDER THE CURVE OF THE TREATED IS HIGHER THAN THE CONTROL.

WE RAN INTO COLONIZATION, INFECTION, P ACNES INFECTION/COLONIZATION, IT MAY HAVE BEEN BAD LUCK OR PREDISPOSITION, WE DON'T KNOW. IT STOPPED US IN OUR TRACKS. WE SWITCHED THE APPROACH AND WE WENT TO A MUCH SIMPLER APPROACH, IT PUT US FURTHER AWAY FROM THE BRAIN, THAT WAS TO DO WHAT'S KNOWN AS SPINAL TAP OR A LUMBAR PUNCTURE IN WHICH CASE WE INTRODUCED AT THE BASE OF THE SPINE INTO THE CSF, RELYING ON IT TO MOVE UP TO THE BRAIN. OKAY. SO 14 SUBJECTS WERE ENROLLED, THEY GET MONTHLY LUMBAR INTRATHECAL ADMINISTRATION, OPEN LABEL DOSE ESCALATION, WE'RE LOOKING AT CLINICAL EFFICACY, BUT THAT'S NOT THE PRIMARY GOAL IN A PHASE 1 STUDY. IN A PHASE 1 STUDY YOU'RE FOCUSED ON SAFETY. TO DATE DOSE RANGE IS 50 TO 1200 MILLIGRAMS, CYCLODEXTRIN LUMBAR IT, MEAN DOSES AT 12 AND 18 MONTHS. SAFETY IS A PRIMARY CONSIDERATION. THE ISSUE IS OTOTOXICITY, IT'S WHAT WE CALL AN EXPECTED ADVERSE EVENT. WE KNEW FROM THE MOUSE, THE CAT, AND ALSO A PRE-CLINICAL DOG MODEL THAT CYCLODEXTRIN WAS OTOTOXIC.

IT'S VARIABLE. IT IMPACTS MID TO HIGH FREQUENCY, HERE YOU CAN SEE THE VARIABILITY IN THE VARIOUS PATIENTS. BUT TO DATE IT'S REALLY HAD MINIMAL IMPACT ON QUALITY OF LIFE. AND YOU CAN CORRECT THE ISSUES WITH THE HEARING AIDS, SO YOU HAVE TO PUT THAT IN CONTEXT OF A LETHAL DISEASE. HIGH FREQUENCY HEARING LOSS IS PART OF NPC, ABOUT HALF OF OUR PATIENTS STARTED THE TRIAL WITH SIGNIFICANT HIGH FREQUENCY HEARING LOSS, BUT DEAF FITLY DEFINITELY WE'VE MOVED THE PATIENTS DOWN TO WHERE THEY WOULD BE CANDIDATES FOR HEARING AIDS.

WE HAVE TARGET ENGAGEMENT, WHEN WE LOOKED AT THE 24 HYDROXYCHOLESTEROL, WORKED MUCH BETTER IN IT THE MICE AND CATS, HUMANS TEND TO BE MORE VARIABLE. WE TENDED TO HAVE POSITIVE RESPONSES IN THE SERUM, BUT WE HAD A LOT OF VARIABLE RESPONSES AND THIS IS JUST TWO PATIENTS AT 900 MILLIGRAMS. HERE IS THE SAME TWO PATIENTS, THE SAME TIME. WE'RE LOOKING AT THE CSF, 72 HOURS AFTER THE DOSE, WE CAN SEE BOTH OF THEM RESPOND WITH ABDUCTION OF 24 HYDROXYCHOLESTEROL. POTENTIAL CLINICAL EFFICACY, WE USE NEUROLOGICAL SCORE, WOULD PREDICT PROGRESSION OF 2 POINTS A YEAR, HERE IS 6, 12, 18 MONTHS, MOVED THE KIDS AWAY FROM THE PREDICTED PROGRESSION RATE. SMALL STUDY, SO YOU ALWAYS HAVE TO DEAL WITH ISSUES OF ALL OF A SUDDEN WE HAVE THIS PATIENT WHO DEFINITELY PROGRESSED, PROGRESSED AT WHAT LOOKS LIKE AN ACCELERATED RATE, IT TURNS OUT THAT THAT PATIENT IS THE OLDEST SUBJECT IN THE STUDY, MOST SEVERELY AFFECTED AND WE HAD MULTIPLE DOSE INTERRUPTIONS BECAUSE OF MULTIPLE ISSUES.

WE FLATTEN THIS CURVE OUT MORE. THIS DATA, IT'S AN OPEN STUDY BUT IT'S VERY PROMISING, REALIZING IT WAS A DOSE ESCALATION, IT FORMED THE BASIS FOR BREAKTHROUGH DESIGNATION FOR THIS DRUG BY THE FDA, AND IT FORMED BASIS OF A PHASE II/III CONTROL TRIAL NOW ENROLL AND ONGOING. THIS WAS A MAJOR COLLABORATION BETWEEN NCATS AND INTRAMURAL INVESTIGATORS FROM MULTIPLE INSTITUTES BUT ALSO INVOLVED EXTRAMURAL SITES. I'M JUST SHOWING THE PRINCIPALS HERE ON THIS. IT ALSO INVOLVED INDUSTRY, JOHNSON AND JOHNSON INITIALLY AND I DIDN'T UPDATE THE SLIDE BUT THE TEST IS A COMPANY FORMED SPECIFICALLY TO TAKE THIS ROUTE, PHASE II-III, A THANK YOU TO MY LAB AND THE PATIENTS, THAT'S THE WHOLE REASON WE'RE HERE.

THANK YOU. >> OKAY. [APPLAUSE] WELL, THANK YOU VERY MUCH. THAT WAS — WELL, DO YOU HAVE SOME QUESTIONS OR COMMENTS? DO YOU ALL WORK IN THIS FIELD? >> I'LL EMBARRASS HER, KRISTIN DOES. SHE PERFORMS THE MOUSE SURVIVAL EXPERIMENTS, ONE OF THE LABS, CHRIS LOCKLEY'S LABORATORY, AND YOU'RE ALSO RESPONSIBLE FOR SALVAGING THE CAT MODEL AND GETTING IT TO THE UNIVERSITY OF PENNSYLVANIA. SHE DROVE I ACROSS COUNTRY WITH THE KITTENS. >> HOW IS THE CAT MODEL GENERATED? >> (INAUDIBLE) >> DO YOU SEE A FUTURE FOR CRISPR APPROACHES TO THIS 5, 10, YEARS FROM NOW? >> YOU KNOW, IN TERMS OF ACTUALLY TRYING TO CORRECT THE PARTICULAR DEFECT, I THINK YOU WOULD HAVE TO COMBINE IT WITH A CELLULAR-BASED THERAPY.

IT'S GOING TO BE DIFFICULT BECAUSE OF THE NUMBER OF DIFFERENT MUTATIONS, THE MOST COMMON MUTATION ONLY ACCOUNTS FOR ABOUT 15% OF THE ALLELES. SO A LOT OF THE MUTATIONS ARE PRIVATE. YOU WOULD HAVE TO DO A LOT OF DEVELOPMENT IN TERMS OF INDIVIDUALLY TRYING TO PRODUCE THE REAGENT THAT WOULD WORK IN SMALL PERCENTAGE OF KIDS. >> PRENATAL SCREENING, SHOULD THIS BE PART OF SOME LEGAL MECHANISM WHERE EVERY CHILD THAT'S BORN GETS SCREENED? >> NEWBORN SCREENING IS AS MUCH POLITICAL AS IT IS RATIONAL. SO WE HAVE DEVELOPED A TRIAL THAT WORKS OKAY BUT THERE'S NEW VARIANT NOW THAT'S BILE ACID DERIVATIVE, IT WILL BE COMING OUT SOON. DEVELOPED IN COLLABORATION WITH DAN ORRY AT WASHINGTON UNIVERSITY. AND THAT WORKS REALLY WELL ON DRIED BLOOD SPOTS. SO IT'S ABSOLUTELY FEASIBLE TO HAVE A NEWBORN SCREEN. BUT THEN YOU'RE INTO POLITICS OF NEWBORN SCREENING IN TERMS OF GETTING THEM ON THE LIST AND THEN GETTING STATES TO ACCEPT IT. AND THEN GETTING ENOUGH PEOPLE TO ACTUALLY DEAL WITH — IT'S NOT ALL CUT AND TRIED. DRIED.

NOW YOU HAVE A SCREEN AND BRING IN EVERYBODY. NOW YOU MAY BE BRINGING IN KIDS THAT WEREN'T GOING TO GET THE DISEASE UNTIL 50, AND SO WHAT DO YOU DO WITH THEM AND TRYING TO SORT OUT HOW YOU HANDLE THE ACTUAL POSITIVES THERE. SO THAT'S REALLY A GENETIC COUNSELING AND POLITICAL ISSUE. >> ARE THERE ANY QUESTIONS? YES, PLEASE. USE THE MICROPHONE SO PEOPLE CAN HEAR YOU ELSEWHERE. >> SO FOR THE PATIENTS THAT ARE IN THE PHASE I TRIAL, I ASSUME ARE CONTINUING ON AS LIKE ON THE SIDE ARM OR WHATEVER, ARE YOU GOING TO CONTINUE TO FOLLOW THEIR — ARE THEY STILL BEING EVALUATED, THAT YOU'LL BE ABLE TO SEE THEIR SEVERITY SCORES? >> SO I MEAN WE'RE STARTING TO GET TWO-YEAR DATA ON A NUMBER OF THE KIDS.

SO FROM A PRACTICAL MANNER, THE PHASE I PATIENTS WILL MOVE TO WHAT WE CALL PART C OF THE PHASE II-III TRIAL, REALIZE COMING TO THE NIH AND LIVING A QUARTER OF YOUR LIFE HERE IS A SACRIFICE ON THESE FAMILIES, SO WE'RE TRYING TO IDENTIFY SITES, THE PHASE II SITES THAT ARE CLOSER TO THEM. SO THAT'S A LITTLE MORE PRACTICAL. BUT WE WILL CONTINUE TO BRING THEM BACK AT THE YEARLY HALF-YEAR INTERVALS. AND WE ALSO — THEY ARE A VERY GOOD SOURCE FOR LONG-TERM SAFETY INFORMATION STILL. >> ALL RIGHT. LISTEN, I WANT TO THANK YOU BOTH VERY MUCH. THIS IS REALLY VERY EXCITING AND INFORMATIVE. VERY GOOD. [APPLAUSE] CAN WE PUT YOUR — WE TRY TO PUT THE POWER POINTS ON THE WEB. >> CAN I SEND YOU A COPY?

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