>>> GOOD AFTERNOON.
I'M THE DEPUTY DIRECTOR OF THE
NHLBI AND I'M DELIGHTED TO
WELCOME DR. KATHY HIGH AS OUR
WALS LECTURER TODAY.
SHE IS THE DIRECTOR OF THE
CENTER FOR CELLULAR AND
MOLECULAR THERAPEUTICS AT THE
CHILDREN'S HOSPITAL IN
PHILADELPHIA.
AN ATTENDING HEMETOLOGIST AND
THE WILLIAM H BENNETT PROFESSOR
OF PEDIATRICS AT THE UNIVERSITY
PENNSYLVANIA SCHOOL OF MEDICINE
AND A HOWARD HUGHES INSTITUTE
FELLOW.
LAST MONTH, KATHI AND FREDRICO
PUBLISHED A REVIEW ARTICLE IN
THE JOURNAL, BLOOD, ENTITLED,
"IMMUNE RESPONSES TO AAV VECTOR,
OVERCOMING BARRIERS TO
SUCCESSFUL GENE THERAPY."
THIS IS AN ELEGANT SURVEY OF THE
STATE-OF-THE-ART GENE SURVEY,
USING AAV VECTORS.
GENE THERAPY PRODUCTS FOR THE
TREATMENT OF GENETIC DISEASES
ARE NOW CURRENTLY IN CLINICAL
TRIALS AND AN AAV PRODUCT HAS
BEEN LICENSED IN EUROPE.
THE AAV VECTORS HAVE ACHIEVED
POSITIVE RESULTS IN A NUMBER OF
CLINICAL AND PRECLINICAL
SETTINGS, INCLUDING HOMOLOGIC
DISEASES, SUCH AS HEME PHILIAS
AND HEMOCHROMETOSIS AND OTHERS.
THIS IS AN EXTREMELY HOT TOPIC
AND WE ARE DELIGHTED TO HAVE
CATHERINE HERE.
AAV VECTORS ARE ADMINISTERED TO
THE PATIENT AND THEREFORE LISTED
AS A HOST IMMUNE RESPONSE AND
THIS IS ACTUALLY A MAJOR TOPIC
OF DR. HIGH'S CURRENT RESEARCH.
SHE IS TRYING TO ACHIEVE A
COMPREHENSIVE UNDERSTANDING OF
THE DETERMINANTS IMMUNOGENICITY
OF THESE AAV VECTORS AND THE
POTENTIAL ASSOCIATED TOXICITIES.
IN COLLABORATION WITH GLOBAL
LEADERS IN THE FIELD, SHE IS
ENGAGED IN IMMUNOSURVEILLANCE OF
ONGOING CLINICAL STUDIES TO
PROVIDE THE BASIS FOR
UNDERSTANDING OF THE I WANT KEYS
OF THE IMMUNE RESPONSE IN AAV
MEDIATED GENE TRANSFER
FACILITATING SAFE AND EFFECTIVE
THERAPIES FOR GENETIC DISEASES.
KATHY HAS AN INTERESTING CAREER
TRAJECTORY.
SHE MAJORED IN CHEMISTRY AS AN
UNDERGRADUATE AND SPENT A YEAR
WORKING IN THE BIOLOGY OF
ARTHROSCLEROSIS IN COLLEGE.
THIS LED HER TO PURSUE AN MD
INSTEAD OF A PH.D. IN CHEMISTRY
AS SHE HAD ORIGINALLY PLANNED.
BUT WE ALMOST LOST HER TO
MEDICINE BECAUSE THE EMPHASIS ON
MEMORIZING FACTS AS A FIRST YEAR
MEDICAL STUDENT DISCOURAGED HER.
SHE TOOK A YEAR OFF TO DO
CHEMISTRY RESEARCH DURING WHICH
HER LANMENTOR IN CHEMISTRY
ENCOURAGED HER TO FINISH MEDICAL
SCHOOL.
FORTUNATELY FOR US, WHEN SHE
RETURNED TO MEDICAL SCHOOL,
THINGS CLICKED AND SHE DECIDED
TO STAY IN MEDICINE, BUT HER
SOLID GROUNDING IN SCIENCE AND
CHEMISTRY SERVED HER WELL.
KATHY'S WORK REGULARLY JOINS THE
BOUNDARIES BETWEEN THE BENCH AND
THE BEDSIDE.
SHE WAS DRAWN TO HEMATOLOGY
BECAUSE OF THE EXTREMELY
SOPHISTICATED UNDERSTANDING OF
THE BIOLOGY OF MANY HOMOLOGIC
DISEASES ON A MOLECULAR LEVEL.
THIS MATCHES HER INTEREST IN
BIOCHEMISTRY, CHEMISTRY AND
MOLECULES.
AS SHE WAS COMPLETING HER
HEMATOLOGY FELLOWSHIP AT YALE,
SCIENTISTS CLONED THE GENES FOR
FACTOR 8 AND FACTOR 9.
THIS FT. PRESENTED HER WITH AN
OPPORTUNITY TO SORT OUT THE
MOLECULAR BASIS OF DISEASE
UTILIZING HER CHEMISTRY TRAINING
AND HER TREMENDOUS INTEREST IN
THE BIOCHEMICAL ORIGINS OF
DISEASE.
HER INITIAL WORK FOCUSED ON
DEFINING MUTATIONS AND CLOTTING
ACTOR GENES THAT RESULT THE IN
HEMOPHILIA LEADING TO ABILITY TO
IDENTIFY CARRIERS AND PERMIT
PRENATAL COUNCILLING TO WOMEN
WHO CARRY THE MUTATION AND
ALLOWED HER TO ANALYZE THE
RELATIONSHIP OF THE STRUCTURE OF
THE MOLECULES AND THEIR FUNG.
KATHY AND HER COLLEAGUES
DEVELOPED MODELS OF HEMOPHILIA
IN MICE AND DOGS THAT WORK WITH
DOGS IN WHICH HEME PHILIA OCCURS
NATURALLY.
THEY DEMONSTRATED THEY CAN
SUCCESSFULLY TREAT THE DISEASE
IN MICE AND DOGS BY INSERTING A
NORMAL CLOTTING FACTOR GENE INTO
THE ANIMALS.
SHE IS NOW FOCUSED ON ADAPTING
THERAPIES FOR HUMANS.
HER ATTEMPTS AT GENE THERAPY
HAVE FOCUSED PRIMARILY ON HEME
PHILIA B, THE LESS COMMON FORM
OF THE DISEASE, EFFECTS 3000 MEN
AND BOYS IN THE UNITED STATES.
SHE DEMONSTRATEDS IN DOGS WITH
HEME PHILIA B THE PRINCIPLE THAT
GENE THERAPY CAN BE EFFECTIVE IN
TREATING THE DISORDER.
DOGS THAT RECEIVE A SINGLE
INJECTION FOR THE GENE FOR
FACTOR 8 PRODUCE THE CLOTTING
FACTOR FOR MORE THAN FIVE YEARS.
HUMAN TRIALS PROVEN TO BE A
LITTLE BIT MORE CHALLENGING.
IN SMALL CLINICAL TRIALS USING
AN AAV VECTOR, A FEW PATIENTS
PRODUCE THERAPEUTIC LEVELS OF
CLOTTING PROTEIN BUT ONLY FOR A
FEW WEEKS.
SHE IS NOW EXPLORING WAYS TO
MODULATE THE PATIENTS IMMUNE
RESPONSE LONG ENOUGH FOR THE
VIRUS TO DELIVER THE GENE SAFELY
TO THE CELLS.
HER MAJOR AREAS OF INVESTIGATION
INCLUDE THE STRUCTURE FUNDS
ANALYSIS AND FACTORS 7, 9 AND
10, RECOMBINANT MUTATIONS AND
THE REGULATION OF EXPRESSION OF
GENES.
PLEASE WELCOME DR. KATHY HIGH
WHO WILL TALK TO US ABOUT
GENETIC THERAPIES FOR GENETIC
DISEASE, RESULTS AND LESSONS
FROM RECENT SUCCESSES.
I'D ALSO LIKE TO REMIND YOU WE
HAVE A RECEPTION WHICH IS
SPONSORED BY THE FAES IN THE
LIBRARY DIRECTLY AFTER THE
LECTURE AND TO INVITE ALL OF YOU
TO ATTEND.
KATHY?
[ APPLAUSE ]
>> I CERTAINLY LIKE TO THANK
DR. SHER IN FOR THAT WONDERFUL
INTRODUCTION.
I HAVE TO SAY THAT MY CAREER
SOUNDED MUCH MORE INTERESTING AS
SHE TOLD IT THAN IT SEEMS TO
HAVE BEEN DURING THE LIVING OF
IT.
AT LEAST TO A CERTAIN EXTENT.
I THOUGHT THAT WHAT I WOULD DO
TODAY, BECAUSE THIS IS AN
NIH-WIDE LECTURE, IS TALK TO YOU
NOT OLE' ONLY ABOUT SOME OF THE
HURDLINGsS THAT WE ENCOUNTERED
IN ATTEMPTING TO WORK OUT THE
DETAILS OF GENE THERAPY FOR HEME
PHILIA, BUT ALSO TALK A LITTLE
BIT ABOUT THE WORK THAT WE HAVE
DONE IN COLLABORATION WITH THE
ALPHA MANAGEY GROUP AT THE
UNIVERSITY OF PENNSYLVANIA OF
CONDUCTING A TRIAL FOR A RARE
INHERITED FORM OF BLINDNESS.
AND THEN IF THERE IS A LITTLE
TIME AT THE END, I'D LIKE TO SAY
A LITTLE BIT ABOUT THE WORK WE
HAVE BEEN DOING MOST RECENTLY
USING GENOME EDITING TO APPROACH
TREATMENT OF GENETIC DISEASE.
SO I'LL JUST REMIND YOU AT THE
OUTSET THAT I WORK AT A
CHILDREN'S HOSPITAL AND THAT
GENETIC DISEASES ACCOUNTED FOR
ANYWHERE FROM 30-50% OF ALL
ADMISSIONS TO CHILDREN'S
HOSPITALS AND EVEN FOR A VERY
SUBSTANTIAL PROPORTION OF
ADMISSION TO ADULT HOSPITALS AND
FOR MANY OF THESE DISORDERS OUR
THERAPEUTIC ONES ARE FAIRLY
LIMITED AND ONE OF THE HOPES OF
THE HUMAN GENOME PROJECT WAS
THAT IT WOULD PROVIDE A PLATFORM
FROM WHICH WE COULD DEVELOP GENE
THERAPIES TO EXPAND THERAPEUTIC
OPTIONS FOR PEOPLE BORN WITH
GENETIC DISEASE.
THE GOAL OF ALL GENE THERAPY
STUDIES IS ESSENTIALLY TWO-FOLD
IN THE SETTING OF GENETIC
DISEASE.
ONE IS TO ACHIEVE LONG-TERM
EXPRESSION OF THE DONATED GENE
AND THE OTHER IS TO EXPRESS IT
AT LEVELS HIGH ENOUGH TO HAVE A
THERAPEUTIC EFFECT.
AND ALL GENE THERAPY STRATEGIES
CAN BE DESCRIBED ESSENTIAL
NETERMS OF THREE COMPONENTS,
FIRST THE GENE TO BE TRANSFERRED
IN, THE TRANSGENE, WHICH IN THE
CASE OF GENETIC DISEASE IS NOT
USUALLY UNDER DISPUTE IN
CONTRAST TO THE SITUATION IN
MANY ACQUIRED DISORDERS.
OF COURSE, IN ADDITION TO
SUPPLYING THE MISSING GENE, ONE
CAN DO VARIATIONS OF THIS
STRATEGY WHERE YOU SUPPLY
INSTEAD A DOWNSTREAM GENE
PRODUCT OR SOMETHING THAT
ENABLES AN ALTERNATE PATHWAY,
FOR EXAMPLE, 7A FOR SOMEBODY WHO
HEME PHILIA.
BUT IN GENERAL, THIS IS SPELLED
OUT CLEARLY IN GENETIC DISEASE
AND THEN A TARGET TISSUE INTO
WHICH IT IS INTRODUCED.
SO FOR EXAMPLE, HEMATOPOIETIC
CELLS FOR SICKLE CELL ANEMIA.
TO ACHIEVE LONG TERM EXPRESSION,
ONE CAN CHOOSE FROM TWO
DIFFERENT STRATEGIES.
SON TO USE INTEGRATING VECTOR
AND INTRODUCE IT INTO A STEM
CELL AND FROM THERE, IT IS
PASTED ON TO EVERY DAUGHTER CELL
AND ONE CAN ACHIEVE LONG-TERM
EXPRESSION ON THAT BASIS.
THE OTHER STRATEGY, WHICH IS THE
ONE THAT WE HAVE BEEN PURSUING,
IS TO TRANSDUCE LONG-LIVED POST
MITOTIC CELL TYPES.
THESE ARE CELL TYPES IN WHICH,
AS LONG AS THE DONATE THE DNA IS
STABILIZED, IT DOES NOT
NECESSARILY NEED TO BE
INTEGRATED.
ONE CAN ACHIEVE LONG TERM
EXPRESSION SIMPLY BECAUSE THE
CELL ITSELF IS LONG LIVED AND IS
NOT GOING TO DIVIDE.
SO, AS YOU MAY GUESS FROM THE
PICTURE HERE, MANY OF THESE
STRATEGIES USING INTEGRATING
VECTORS USED AS THEIR TARGET,
HEMATOPOIETIC STEM CELLS LEND
THEMSELVES WELL TO BEING EXTRACT
FRIDAY A PATIENT MANIPULATED IN
THE LABORATORY AND RETURNED TO
THE PATIENT.
WHEREAS MOST OF THESE LONG-LIVED
POST MITOTIC CELL TYPES,
INCLUDING CARDIAC MUSCLE SKELTAL
MUSCLE, CELLS IN THE BRAIN AND
THE EYE DO NOT LEND THEMSELVES
WELL TO BEING EXTRACTED FROM THE
PATIENT AND RETURNED BUT RATHER
REQUIRE THE VECTOR BE DELIVERED
IN VIVO.
SO, THAT OF COURSE, SPELLS OR
PREDICTS ONE OF THE MAJOR
PROBLEMS, WHICH IS THAT IF YOU
ARE GOING TO INTRODUCE A VIRAL
VECTOR DIRECTLY INTO THE
PATIENT, YOU'RE GOING TO BE
GRAPPLING AT CLOSE QUARTERS WITH
THE HUMAN IMMUNE RESPONSE.
SO, ALL OF OUR WORK HAS BEEN
WITH AN AAV VECTOR AND THESE
WERE OR HAVE BEEN ENGINEERED
FROM A MEMBER OF THE PARVO VIRUS
FAMILY.
THESE ARE NONENVELOPED VIRUSES
WITH LINEAR SSDNA GENOME OF 4.7
KILOBASES.
THERE ARE A NUMBER OF FEATURES
THAT MAKE IT ATTRACTIVE AS A
GENE DELIVERY VEHICLE.
IT WOULD BE FAIR TO SAY OF ALL
VIRAL VECTORS, IT'S ONE OF THE
SIMPLEST, WHICH IS GENERALLY A
POSITIVE FEATURE.
AS A VECTOR ENGINEERED FOR GENE
DELIVERY, IT IS NONPATHOGENIC
BECAUSE WILDTYPE AAV IS
NONPATHOGENIC.
IT IS NATURALLY REPLICATION
DEFECTIVE, AGAIN BECAUSE
WILDTYPE AAV IS WILDTYPE
REPLICATION DEFECTIVE.
AND THE DNA INTRODUCED AAV
VECTORS IS STABILIZED
REDOMESTICALLY IN A
NONINTEGRATING FORM.
SO PROBLEMS ARE MINIMIZED --
PREDOMINANTLY.
IN THE MID 90S TWO DIFFERENT
GROUPS DESCRIBED THE RESULT,
RECOMBINANT AAV VECT ERROR
INTRODUCED INTO SKELETAL IN MICE
AND HAS BECOME CLEAR THAT AAV
VECTORS IN THEIR NATURALLY
OCCURRING SEROTYPES AND IN
GENETICALLY ENGINEERED FORMS AS
WELL, HAVE TOPISM FOR A WIDE
VARIETY OF CELL TYPES, INCLUDING
A NUMBER OF POST MITOTIC CELL
TYPES THAT ARE LONG LIVED.
SO THE WILDTYPE VIRUS IN
CODES -INE CODES TWO TYPES OF
GENES REQUIRED FOR REPLICATION
AND THOSE THAT ENCODE THE CAPS
ID.
THE VEER YON IS COMPOSED OF
ESSENTIALLY A WILDTYPE CAPS ID
WHICH HAS 48 MOLECULES OF VIRAL
PROTEIN 3 AND 6 EACH OF VP1 AND
2 ALL ENCODED IN THIS CAPS ARE
ID SEQUENCE.
EEEE CAPS ID SEQUENCE.
THE VIRAL GENOME IS REPLACED
WITH THE THERAPEUTIC GENE OF
INTEREST UNDER THE CONTROL OF A
APPROPRIATE PROMOTOR.
SO IT IS COMPOSED OF 74% OF
PROTEIN AND 26% OF DNA.
AND SO WHAT YOU CENTRALLY HAVE
IN THIS RECOMBINANT VERYON IS A
HIGHLY-ORDERED SET OF PROTEINS,
THE CAPSID THAT ENCLOSE THE DNA,
THE THERAPEUTIC AGENT.
THIS MATERIAL IS DOSED IN VECTOR
GENOMES PER KILOGRAM OF BODY
WEIGHT OF THE RECIPIENT AND
SINCE IT IS PRETTY DIFFICULT TO
FIGHT PHYSICS, YOU CAN'T PACKAGE
MORE THAN ABOUT 5 KILL BASES OF
DNA INTO THE AAV CAPSID.
AND THERE ARE A LOT OF DIFFERENT
WAYS OF PRODUCING AAV.
THIS HIGHLIGHTS THE METHOD THAT
WE USE AT CHILDREN'S HOSPITAL
AND ESSENTIALLY, THREE DIFFERENT
PLASMIDS, ONE ENCODING THE GENE
OF INTEREST CLONED BETWEEN
THE -- AND THE THIRD PROVIDING
THE ADENOVIRAL HELPER GENES
NEEDED ARE INTRODUCED INTO 293
CELLS AND SUBSEQUENTLY THE
RECOMBINANT VERYONS CAN BE
HARVESTED FROM THE SUPERNATIN OR
FROM THE CELL LICE AID, AND ONE
CAN EITHER USE ALL OF THE
PRODUCT PURIFIED FROM THE CELL
LICE AID OR SEPARATED INTO THE
FULLS AND EMPTIES, WHICH STILL
CONSTITUTE A SUBSTANTIAL
PROPORTION OF WHAT IS GENERATED
IN MOST PRODUCTION
METHODS.
I WANT DOOB SURE TO SAY THAT THE
FIRST GENE THERAPY PRODUCT IN
ICH COUNTRY SIMULATOR, IF YOU
DON'T KNOW WHAT THAT MEANS, IT
MEANS THE UNITED STATES, EUROPE
OR JAPAN, THE FIRST AAV PRODUCT
WAS LICENSED LAST YEAR IN
EUROPE.
THIS IS A PRODUCT FOR LIKEO
PROTEIN LIPASE DEFICIENCY AND I
THINK IT IS IMPORTANT TO POINT
OUT THAT THIS PRODUCT IS BASED
VERY HEAVILY ON WORK DONE HERE
AT THE NIH BY ROB COTTON AND HIS
COLLEAGUES.
AS DR. SHURIN MENTIONED, I HAVE
A LONGSTANDING INTEREST IN
HEMOPHILIA.
AND I HAVE BEEN WORKING FOR A
NUMBER OF YEARS TO TRY TO
DEVELOP AN AAV GENE THERAPY FOR
HEMOPHILIA AND SINCE COLONIC
FACTORS ARE NORMALLY MADE IN THE
LIVER, LIVER HAS BEEN AN
IMPORTANT TARGET FOR US.
A NUMBER OF OBSTACLES HAD TO BE
OVERCOME TO MAKE AAV GENE IN THE
LIVER A REALITY.
WE COULD MAKE ENOUGH VECTOR TO
DO MICE IN THE ORLANDOY DAYS BUT
NOT TO DO HEMEPHILIC DOGS.
IN THE INITIAL TRIALS WHEN IT
WAS INTRODUCED INTO LIVER, WE
ENCOUNTERED THE PROBLEM, THAT
HAD NOT BEEN FORETOLD BY ANIMAL
MODELS AND THAT WAS THAT THE
VECTOR WAS DETECTEDDED IN THE
SEAMEN OF THE FIRST SUBJECTS AND
HAD TO WORK OUT STRATEGIES TO
MITIGATE THE RISK TO TRACK THAT
AND REDUCE RISK OF A PIECE OF
VECTOR DNA BEING INCORPORATED
INTO AN OFFSPRING.
I'M GOING TO TALK ABOUT THE
ISSUE THAT IS RESULTED FROM THE
HUMAN IMMUNE RESPONSE AND FOR
ANY NEW CLASS OF THERAPEUTICS,
ONE ISSUE THAT MUST BE ADDRESSED
IS WHAT IS THE LONG-TERM SAFETY
RELATED TO THE USE OF THE
PRODUCT.
AND FOR AAV, ISSUES ARE LIKELY
AT OR ABOUT RELATED TO
INTEGRATION.
BUT ANY TIME YOU INTRODUCE DNA
INTO A CELL, THERE IS A
POSSIBILITY THAT SOME OF IT WILL
INTEGRATE INTO THE HOST CELL
GENOME.
THIS IS TRUE FOR AAV AS WELL AND
THE QUESTION IS, WILL THIS HAVE
LONG TERM EFFECTS?
-- HAD TO BE SOLVED TO MAKE GENE
THERAPY FOR HEMOPHILIA THEY ARE
A HIGHLY ORDERED SET OF PROTEINS
THAT EXPRESS RECEPTORS ON THE
CELL SURFACE AND DELIVER DNA TO
THE NUCLEUS.
THE DELIVERY PROTEINS ARE
DEGRADED AND GET LONG TERM
EXPRESSION AND THAT WAS OBSERVED
OVER AND OVER AGAIN IN MANY
LARGE ANIMAL MODELS BEFORE HUMAN
TRIALS WERE CONDUCTED.
HOWEVER, AAV VECTORS VIEWED FROM
AN IMMUNOLOGIC STANDPOINT HAVE
BEEN ENGINEERED TO UNDERSTAND
EXACTLY WHAT ARE THE HUMAN
IMMUNE DOSE RESPONSES AND GET A
THERAPEUTIC OUTCOME.
SO I WOULD SAY THAT OUR LAB,
OVER THE LAST SEVERAL YEARS, HAS
BEEN FOCUSED ON THE FOLLOWING
QUESTIONS FROM A BIOLOGICAL
STANDPOINT.
WHAT HOST IMMUNE RESPONSES
ENCOUNTERED BY RECOMBINANT
VERYON THAT FAILS TO SYNTHESIZE
VIRAL ANTIGENS AND FAILS TO
SYNTHESIS THE VIRAL GENES THAT
NORMALLY DOWN REGULATE A HOST
IMMUNE RESPONSE AND FROM A
MEDICAL AND THERAPEUTIC
STANDPOINT, CAN THE HOST IMMUNE
RESPONSE THAT IS GENERATED BY
RECOMBINANT VERYON BE CONTROLLED
SO THE GENE TRANSFER OCCURS IN
THE HOST IS UNHARMED.
AND THIS IS MADE MORE COMPLEX
FOR THE FACT THAT EVERY TARGET
ISSUE, THE HUMAN IMMUNE RESPONSE
IS TISSUE-SPECIFIC.
SO THE ANSWER TO THESE QUESTIONS
IS ESSENTIALLY TISSUE-SPECIFIC
ANSWERS.
YOU HAVE TO WORK IT OUT ONE
TISSUE AT A TIME.
SO, LET ME JUST SAY A WORD OR
TWO ABOUT HEMOPHILIA.
IT'S THE EXCELLENT LEADING --
BLEEDING DISORDER THAT ARISES
BECAUSE OF MUTATIONS EITHER IN
THE GENE FOR FACTOR 9, AN
ENZYME, OR FOR FACTOR 8, THE
CO-ENZYME FOR THE SAME STEP IN
COAGULATION.
AND THAT CLINICALLY, THESE ARE
INDISTINGUISHABLE AND ARE
CHARACTERIZED BY FREQUENT BLEEDS
INTO THE JOINTS SOFT TISSUES BUT
PATIENTS, ESPECIALLY THOSE WHO
ARE SEVERELY EFFECTED CAN HAVE
BLEEDS INTO OTHER CLOSED SPACES
SUCH AS INTRACRANIAL SPACE WHERE
THE BLEEDS CAN BE RAPIDLY FATAL.
HEMOPHILIA A EFFECTS ABOUT
1-5000 MALE BIRTHS AND
HEMOPHILIA B1-30,000 MALE
BIRTHS.
THE OTHER PART THAT IS IMPORTANT
IS THIS, THE LARGEST CATEGORY OF
PATIENTS WITH BOTH DISEASES ARE
SEVERELY EFFECTED WITH LESS THAN
1% FACTOR 8 OR FACTOR 9.
BUT WE KNOW FROM THE NATURAL
HISTORY OF THE DISEASE THAT IF
THE CIRCULATING LEVELS ARE EVEN
SLIGHTLY BETTER, THE PATIENT IS
SPARED MOST OF THE LIFE THREAT
KNOWING BLEEDS AND MANY OF THE
JOINT BLEEDS AS WELL.
AND IF A PERSON'S LEVEL IS
RAISED TO AROUND 5%, THIS PERSON
MAY GO THROUGH CHILDHOOD WITHOUT
EVER BEING DIAGNOSED.
SO A RELATIVELY MILD PHENOTYPE.
WE KNOW FOR WHAT WE ARE
ATTEMPTING TO DO, THAT IF WE CAN
ELEVATE THE LEVELS BY JUST A FEW
PERCENT, WE ARE LIKELY TO
IMPROVE THE PHENOTYPE OF THE
DISEASE.
AND RIGHT NOW, OF COURSE,
HEMOPHILIA IS TREATED BY
INTERVENOUS INFUSION OF CLOTTING
FACTOR CONCENTRATES WITH PLASMA
DERIVED OR RECOMBINANT AND
TYPICALLY CHILDREN ARE TREATED
IN A CLINIC UNTIL THEY REACH THE
AGE OF ABOUT 4.
YOU GET THESE INFUSIONS TWO OR
THREE TIMES A WEEK AND THEN
STARTING AT AROUND 4, THE
PARENTS CAN BE TAUGHT TO DO THE
INTRAVENUS INFUSION AND
SUBSEQUENTLY CHILDREN LEARN TO
DO IT THEMSELVES.
THESE PRODUCTS COST AROUND
200,000 DOLLARS A YEAR FOR AN
ADULT MALE WHO IS INFUSING
HIMSELF 2-3 TIMES A WEEK.
AND BECAUSE OF THESE COSTS, IT'S
ESTIMATED THEY ARE AVAILABLE FOR
ABOUT 20% OF THE WORLD'S
HEMOPHILIA POPULATION.
IF YOU LOOK AT THE REST OF THE
WORLD'S HEMOPHILIA POPULATION
BASED ON A PER CAPITA GNP, THIS
GRAPH GUESS PLAYS CHILDREN WITH
HEMOPHILIA COMPARED TO ADULTS
WITH HEMOPHILIA AS A FUNCTION OF
THE GNP.
AND IN THE WESTERN WORLD, THERE
ARE MANY MORE ADULTS THAN
CHILDREN.
BUT THEN AS THE GNP FALLS, IT
REALLY BECOMES PROGRESSIVELY A
LETHAL DISORDER THAT KILLS MOST
PEOPLE BEFORE THEY REACH
ADULTHOOD IN RESOURCE-POOR
NATIONS.
SO, I'LL SKIP OVER A LOT OF
PRECLINICAL WORK THAT WE HAVE
OTHER PEOPLE HAVE DONE IN MICE
AND DOGS AND SAY THAT BY THE
LATE 1990S, WE HAVE THE
FOLLOWING RESULT IN THE
NATURALLY-OCCURRING HEMOPHILIA B
DOG MODEL THAT A SINGLE
INJECTION OF AN AAV VECTOR
INTRODUCED EITHER INTO SKELETAL
MUSCLE OR INTO THE LIVER IN A
DOG, WOULD RESULT IN LONG-TERM
EXPRESSION.
WE GOT BETTER LEVELS BOY GOING
INTO LIVER BECAUSE LIVER IS
BETTER AT SECRETING A GENE
PRODUCT DIRECTLY INTO THE
CIRCULATION.
BUT YOU CAN SEE THAT THESE
ANIMALS THAT WERE DOSED AT TIMES
ZERO WERE STILL EXPRESSING
THERAPEUTIC LEVELS, 140 WEEKS
LATER.
WE FOLLOWED SOME OF THESE
ANIMALS FOR AS LONG AS A DECADE
AND IT'S A VERY LONG LASTING
EFFECT FROM A SINGLE INFUSION OF
VECTOR INTO THE PORTAL VAIN OR
THE HEPATIC ARTERY.
AND SIMILARLY, WHEN WE DID
STUDIES IN HUMANS WHERE WE
INTRODUCED AN AAV TWO VECTOR
INTO SKELETAL MUSCLE IN A MAN
WITH SEVERE HEMOPHILIA, WE
RECENTLY HAD THE OPPORTUNITY TO
GO BACK AND SAMPLE MUSCLE IN A
PATIENT WHO HAD BEEN INJECTED
WITH AAV2 FACTOR 9 TEN YEARS
EARLIER AND THERE WAS STILL
FACTOR 9 BEING EXPRESSED IN THE
INJECTED MUSCLE.
THIS IS HISTOCHEMICAL STAINING
FOR FACTOR 9.
HOWEVER, IN THE FIRST SUBJECT IN
A LIVER TRIAL TO RECEIVE A
THERAPEUTIC DOSE, INSTEAD OF
SEEING LONG TERM EXPRESSION AS
WE HAD IN THE HEMEPHILIC DOG
LEVER AND IN HUMANS SKELETAL
MUSCLE, INSTEAD, WE SAW DIAGRAMS
HERE IN RED EXPRESSION AT A
THERAPEUTIC LEVEL IN THIS
PATIENT FOR ABOUT FOUR WEEKS AND
THEN HE SLOWLIY LOST FACTOR 9
EXPRESSION UNTIL HE RETURNED TO
HIS BASELINE OF LESS THAN 1%
THIS IS A SERIES OF DIFFERENT
ANIMALS MODELS TIME COURSE WE
HASN'T SEEN.
IT WAS A TRANSAMINANT ELEVATION
THAT OCCURRED IN TANDEM WITH THE
LOSS OF FACTOR 9 EXPRESSION.
SO, THE TIME COURSE OF THISSING
ISED IMMUNE MEIATED
RESPONSE - AGOD.
AND SUBSEQUENTLY WE WERE ABLE TO
DEVELOP REAGENTS TO LOOK AT THIS
SPECIFICALLY.
THIS IS USING A PANT MERTO
EXAMINE THE KAS ID SPECIFIC CD8
T-CELLS IN A SUBSEQUENT PATIENT
WHO WAS INFUSED WITH AAV FACTOR
9.
YOU CAN SEE WHAT HAPPENED IN
THIS PATIENT, HE DIDN'T GET
DETECTABLE FACTOR 9 EXPRESSION
AT ALL BUT HE DID EXPERIENCE
THIS TRANSAMNAS ELEVATION AND
WHEN WE USED THIS PANT MERTO
LOOK AT THE POPULATION OF
CAPSID-SPECIFIC CD8 T-CELLS, WE
COULD SEE THAT POPULATION OF
CELLS EXPANDS AFTER VECTOR
INJECTION, AND THEN IT CONTRACTS
N A TIME COURSE THAT MATCHES THE
RISE AND FALL AND IN LIVER
ENZYMES AND OTHER EXPERIMENTS WE
WERE ABLE TO SHOW THAT THE
SPECIFIC EPITOPE THAT WAS
RECOGNIZED BY THE PATIENT'S CD8
T-CELLS WAS HIGHLY CONSERVED IN
A NUMBER OF DIFFERENT AAV
CAPSIDS AND THAT THEY EVOKED THE
SAME RESPONSE WHEN THE PATIENT'S
LYMPHOCYTES WERE EXPOSED TO THE
APPROPRIATE EPITOPE.
SO, THERE WAS CROSS REACTIVITY
WITH A NUMBER OF STEREOTYPES FOR
THIS PARTICULAR EPITOPE.
SO, OUR WORKING HYPOTHESES SHOWN
HERE THAT THE CAPS SID
ESSENTIALLY A PREFORMED ANTIGEN,
THAT AFTER THE VECTOR ENTERS THE
CELL IN THE END ZOME AND ESCAPES
FROM THE END ZOME, IT EVENTUALLY
UNCODES AND THE DNA GOES TO THE
NUCLEUS AND BEGINS MAKING FACTOR
9 AND SOME OF THE CAPSID IS LEFT
BEHIND IN THE CYTOSOL AND WE
KNOW BASED ON WORK THAT WAS DONE
BY JOHN ENGEL HEART AND OTHERS,
THAT THAT CAPSID LEFT BEHIND
UNDER THOSE PROTOSTOMEAL
PROCESSING, AND CAN EVENTUALLY
BE TRANSPORTED INTO THE ER WHERE
IT GETS LOADED ON TO CLASS ONE,
DISPLAYED ON THE SURFACE OF THE
TRANSDUCED HE PAT SIGHT AND
MAKES THAT CELL A TARGET FOR
CIRCULATING CD8 T-CELLS, WHICH,
BECAUSE HUMANS HAVE MEMORY TO
AAV WHERE OTHERS SPECIES DO NOT,
RESULTS IN THE DESTRUCTION OF
THE TRANSDUCED CELL.
AND BASED ON OTHER EXPERIMENTS,
WE HAVE BEEN ABLE TO DEMONSTRATE
THE LEVEL OF ANTIGEN
PRESENTATION IS DOSE-DEPENDENT
SO THE HIGHER THE DOSE THAT IS
DELIVERED INTO THE CELL, THE
GREATER THE LIKELIHOOD THAT
THESE TRANSDUCED HEPATOCYTE WILL
BE DETECTED AND DESTROYED.
SO A NUMBER OF OTHER HYPOTHESIS
WERE PROPOSED TO EXPLAIN THAT
SERIES OF FINDINGS.
AND ONE WAS THAT THE VECTOR HAD
DURING MANUFACTURE, PACKAGED THE
REP/CAP PLASMID SO THE PATIENT
WAS CONTINUOUSLY EXPRESSING
CAPSID.
AND PREDICTED THAT SHORT-TERM
IMMUNOSUPPRESSION WOULDN'T WORK.
ANOTHER PIE POT SIS THERE WERE
ALTERNATE OPEN READING FRAMES IN
OUR -- HYPOTHESES -- AND THAT
THE IMMUNE RESPONSE WAS DIRECTED
TOWARDS THOSE.
AND THERE WERE PREDICTIONS THAT
IF WE SWITCHED SEROTYPES WE
WOULD AVOID THE RESPONSE BECAUSE
OF AAV2 SEROTYPE TRANSDUCED
ANTIGEN PRESENTING CELLS MORE
EFFICIENTLY THAN SOME OTHER
SEROTYPES.
OUR HYPOTHESES ABOUT PRE-FORMED
CAPSID LED TO THE PREDICTION
THAT SHORT-TERM
IMMUNOSUPPRESSION WOULD WORK AS
AN APPROACH IF ONE COULD SIMPLY
BLOCK THE IMMUNE RESPONSE UNTIL
THE PRE-FORMED CAPSID OF THE
DEGRADED IN THE CELL.
ONE COULD PERHAPS SEE LONG-TERM
EXPRESSION.
WE DID A SERIES OF EXPERIMENTS
TO TRY TO SUPPORT THAT
HYPOTHESES AND I'LL JUST POINT
OUT MAYBE A COUPLE OF THOSE.
WE EVENTUALLY CLONED THE T-CELL
RESPENTOR FROM A CAPSID-SPECIFIC
CD8 T-CELL AND THEN YOU'RE ABLE
TO USE THIS AS A DETECTING
REAGENTED TO DETECT PEPTIDE MHC
COMPLEXES CARRYING PEPTIDES
DERIVED FROM THE AAV CAPSID.
THIS IS SOME OF THE DATE A TOLD
YOU WE HAD THAT SUGGESTED THIS
IMMUNE RESPONSE WAS DOSE
DEPENDENT.
IF YOU TAKE THAT T-CELL RECEPTOR
AND CLONE IT INTO A T-CELL LINE
THAT EXPRESSES LUCIFERASE IN
PROPORTION TO THE DEGREE THAT IT
RECOGNIZES ITS COGNATE PEPTIDE
MHC COMPLEX, THEN, IF YOU TAKE
HUMAN HEPATOCYTE TRANSDUCE THEM
WITH AAV CAPSID, YOU CAN SHOW
THAT AT INCREASING LEVELS OF
CAPSID MULTIPLICITY OF INFECTION
OF THE VECTOR, YOU GET
PROGRESSIVELY INCREASING
EXPRESSION OF LUCIFERASE.
SO IT DEMONSTRATES THAT THE
AMOUNT OF ANTIGEN DISPLAYED ON
THE SURFACE OF THE TRANSDUCED
CELL IS DOSE DEPENDENT.
SO THE OTHER IMPORTANT THING
THAT WE LEARNED IN THAT INITIAL
TRIAL AND THE WORK THEY TOLD YOU
ABOUT WAS DONE OVER YEARS AFTER
THAT FIRST TRIAL, BUT THE OTHER
IMPORTANT THING WE LEARNED IN
THAT FIRST TRIAL, THESE TWO
PATIENTS BOTH GOT THE SAME
HIGH-DOSE OF THAT FIRST VECTOR.
AND ONE OF THEM HAD DETECTABLE
EXPRESSION FOR A PERIOD OF WEEKS
UNTIL THE CD8 T-CELL RESPONSE
SUPER VENED, WHEREAS THE OTHER
ONE, GOT VERY LITTLE DEDUCTIBLE
EXPRESSION AND FOUR WEEKS WAS
DOWN TO ZERO.
THIS PERSON HAD PRE-TREATMENT
NEUTRALIZING ANTIBODIES TO AAV,
FAIRLY HIGH TIGHTER, 1-17.
WHEREAS THIS PERSON HAD A LOW
TIGHTER OF NEUTRALIZING
ANTIBODIES.
SO, THE OTHER IMPORTANT THING WE
LEARNED THEN WAS THAT THIS
PRE-EXISTING HUMORAL IMMUNITY TO
AAV WOULD BE BLOCKING IF WE
CHOSE TO IN FUSE THE VECTOR
THROUGH THE CIRCULATION.
AND WE HAD CONSIDERABLE DATA IN
THE ORIGINAL MUSCLE TRIAL THAT
SAID THAT IF YOU'RE GOING
THROUGH SKELTAL MUSCLE, THESE
ANTIBODIES AREN'T A PROBLEM.
CLEARLY IF YOU'RE GOING THROUGH
THE CIRCULATION, THEY WOULD HAVE
AMPLE OPPORTUNITY TO NEUTRALIZE
THE INFUSED VECTOR.
THIS IS FROM A STUDY DONE BY JIM
WILSON.
THESE ARE PERCENT OF THE
POPULATION WITH NEUTRALIZING
ANTIBODY TITERS OF GREATER THAN
1-20 AND THE PERCENTAGE OF THE
POPULATION FAIRLY SUBSTANTIAL
ACROSS FOUR CONTINENTS.
AND THAT IS 1-20 FULLY BLOCKING.
SO, REALLY WE ARE INTERESTED IN
THE POPULATION WITH EVEN LOWER
NEUTRALIZING ANTIBODY TITERS.
SO THE SECOND AAV FACTOR 9 TRIAL
WAS CONDUCTED AT UNIVERSITY
COLLEGE LONDON AND ST. JUDE
CHILDREN'S RESEARCH HOSPITAL.
SO THE SOLUTION THEY PROPOSED TO
THOSE TWO PROBLEMS WITH THE
IMMUNE RESPONSE THAT HAD BEEN
SEEN IN THAT FIRST TRIAL WERE
SHOWN HERE.
FIRST JUST EXCLUDE EVERYBODY
WITH NEUTRALIZING ANTIBODIES.
SO THAT'S THE LONG TERM
SOLUTION.
BECAUSE IT INVOLVES TOO MANY
ADULTS AND THERE ARE A LOT OF
ADULTS WITH HEMOPHILIA.
BUT IT'S A GOOD SHORT-TERM
SOLUTION.
AND THEN, THEY ADDED A PROVISION
THAT A SHORT COURSE OF STEROIDS
WOULD BE ADMINISTERED IF THE
LIVER ENZYMES ROSE OR THE FACTOR
9 LEVEL BEGAN TO DECLINE.
SO, IN ADDITION TO THAT, THEY
DID A NUMBER OF THINGS TO MAKE
THEIR VECTOR MORE EFFICIENT.
THEY USED A SEROTYPE THAT HAS A
STRONG TROPISM FOR LIVER.
THEY USED A SOFT COMPLEMENTARY
VECTOR DESIGN SO NORMALLY THEY
ARE SINGLE STRANDED.
THEIRS WAS DOUBLE-STRANDED SO IT
DIDN'T HAVE TO RELY ON THE HOST
CELL MACHINERY FOR SECOND STRAND
SYNTHESIS AND IT WAS CODON
OPTIMIZED.
SO THE GOAL AND THE EXPECTATION
WAS THAT IT WOULD BE POSSIBLE TO
IN FUSE A LOWER DOSE AND STILL
GET THERAPEUTIC LEVELS OF
EXPRESSION AND PERHAPS AT THE
LOWER DOSE, THE T-CELL RESPONSE
WOULDN'T BE ENCOUNTERED AT ALL.
AND IN FACT IN THE FIRST SUBJECT
IT WAS INFUSED IN THAT STUDY,
THIS APPEARED TO BE THE CASE.
THIS PATIENT WAS INFUSED NOW
OVER THREE YEARS AGO AND HE
AFTER INFUSION OF TWO TIMES 10
TO THE 11 VECTOR GENOMES PER
KILOGRAM, HIS CIRCUMSTANCE
LATTING FACTOR 9 LEVEL WENT FROM
LESS THAN 1% UP TO 2%.
THAT'S A LOW-LEVEL IT'S MODEST.
BUT IT IS DEFINITELY DIFFERENT
FROM LESS THAN 1%.
THIS PATIENT HAS BEEN ABLE TO
REDUCE VERY SUBSTANTIALLY THE
AMOUNT OF CLOTTING FABBER HE
USES TO MANAGE HIS DISEASE.
SO FOR HIM, 2% REALLY MOSTLY
REMOVED THE NEED FOR
PROPHYLACTIC FACTOR 9 INFUSIONS.
HOWEVER, AS DOSE ESCALATION
CONTINUED, WHAT WAS SEEN AT THIS
DOSE, TWO TIMES 10 TO THE 12,
INITIALLY THE PATIENT HAD A
CIRCULATING LEVEL OF 6-8%, WHICH
IS GREAT, THAT REALLY CONVERTS
SEVERE HEMOPHILIA TO MILD.
BUT STARTING AT ABOUT SEVEN
WEEKS AFTER VECTOR INFUSION, THE
LIVER FUNCTION TESTS WHICH HAD
BEEN COMPLETELY NORMAL, BEGAN TO
RISE AND THEN THEY ROSE VERY
QUICKLY OVER THE PERIOD OF A FEW
DAYS.
AND THAT WAS OF COURSE THE
PRE-DETERMINED TRIGGER FOR
INITIATING AN EIGHT-WEEK COURSE
OF PREDNISOLONE.
AND THIS RAPIDLY RENORMALIZED
THE LIVER FUNCTION TEST AND
STABILIZED THE FACTOR 9 LEVEL
AND EVENTUAL AT THE STABILIZED
BETWEEN 1% AND 2%.
SO, WE HAVE THE OPPORTUNITY TO
STUDY THE LYMPHOCYTES FROM THIS
INDIVIDUAL USING INTERFERON
GAMMA ELISPOT AND YOU CAN SEE
THE TIME COURSE TRACKS CLOSELY
TO WHAT HAPPENED TO THE LIVER
FUNCTION TEST.
YOU BEGIN TO SEE SOME MODEST
LEVEL OF RESPONSE ABOVE THE
BASELINE AT 5-6 WEEKS.
AND THEN, AT EIGHT WEEKS IT
SHOOTS UP TO A MARKEDLY POSITIVE
RESPONSE IN PARALLEL WITH THIS
RISE IN THE LIVER ENZYMES.
AND THEN AFTER THE INITIATION OF
PREDNISOLONE, THESE CELLS
RAPIDLY DISAPPEAR FROM THE
CIRCULATION.
SO, THE CLINICAL INVESTIGATOR IN
LONDON, TED TUD NUMB ELEARNED A
GREAT DEAL FROM THAT.
IF YOU LOOK HERE AT THE
FREQUENCY WITH WHICH HE CHECKED
THE LIVER FUNCTION TEST, AFTER
THAT PREVIOUS PATIENT, YOU CAN
SEE HE WAS EXTREMELY VIGILANT.
AND AS SOON AS HE STARTED TO GO
UP AT ALL, HE STARTED THE
PATIENT ON PREDNISOLONE AND THAT
PATIENT HAS STABILIZED NOW TWO
YEARS LATER WITH A CIRCULATING
LEVEL OF AROUND 5%.
AGAIN, WHAT WE SAW HERE WAS A
VERY MODEST RESPONSE INITIALLY
AND THEN IT SHOOTS UP AT 9 WEEKS
JUST AT THE POINT WHERE IT
APPEARS THAT THE LFTs ARE
STARTING TO RISE EACH VERY
MODESTLY.
SO, SINCE THEN, THEY TREATED
SEVERAL MORE PATIENTS.
AND THIS WAS PRESENTED AT ASH
AND YOU CAN SEE MOST OF THESE
PEOPLE ARE STABILIZED IN A RANGE
OF 4-6% NOW AND SO, THAT'S VERY
EXCITING RESULTS.
FOUR OF THE SIX PEOPLE NEEDED
THAT COURSE OF STEROIDS.
TWO OF THEM WENT THROUGH WITH
NEVER RAISING THE LFTs AT ALL
SO ONE OF THE IMPORTANT GOALS
FOR THE FIELD IS TO TRY TO
DETERMINE WHAT PERCENTAGE OF
ADULTS ARE ACTUALLY GOING TO
NEED THIS COURSE OF STEROIDS.
HOW MANY PEOPLE WILL HAVE THIS
RESPONSE?
AND OF COURSE ANOTHER WAY TO
LOOK AT IT IS, THE EASY WAY TO
DO IT IS JUST IF YOU KNOW
EXACTLY WHEN THIS IS GOING TO
HAPPEN, CAN YOU JUST GIVE FOUR
WEEKS EVER STEROIDS AND NOT
WORRY ABOUT TRACKING IT?
AND SO I THINK WHAT IS IMPORTANT
ABOUT THAT HIGH DOSE COHORT IS
IMPOSSIBLE TO IDENTIFY DOSE THAT
LEADS TO LONG TERM EXPRESSION AT
THERAPEUTIC LEVELS.
THE TRANSAMNAS ELEVATION IS
ASYMPTOMATIC BUT SERVES AS A
MARKER OF THE CELLULAR IMMUNE
RESPONSE AND TRIGGERS THE
INVESTIGATOR TO START THE COURSE
OF STEROIDS WHICH SEEMS TO
STABLE ICE THE TRANSDUCED
HEPATOCYTES EVEN THOUGH
ADMINISTERED FOR FOUR-8 WEEKS.
FOR MOST HEMOPHILIA PATIENTS,
THE RISK AT LEAST SHORT-TERM, OF
THIS SERIES OF EVENTS WOULD FAR
OUTWAY OR BE FAR OUTWEIGHED BY
THE BENEFIT OF LONG TERM
EXPRESSION OF A LEVEL OF FACTOR
9 AND THE RANGE OF A FEW
PERCENTS.
SO WHAT I WOULD SAY ABOUT WHERE
THE FIELD IS NOW IN DEVELOPING
AV MEDIATED GENE TRANSFER TO
LIVER, WE UNDERSTAND MUCH BETTER
THE PROBLEMS POSED BY THE
HUMORAL IMMUNE RESPONSE AND
CELLULAR IMMUNE RESPONSE AND WE
BELIEVE THE DOSES SHOWN HERE TWO
TIMES 10 TO THE 12 PER KILOGRAM
IS POSSIBLE TO CONTROL THE
CELLULAR IMMUNE RESPONSE WAY
SHORT COURSE OF STEROIDS.
AND SO I THINK IT WILL ONLY BE
ANSWERED AND CONTINUING TO
FOLLOW LARGE ANIMALS AND HUMAN
SUBJECTS WHO HAVE BEEN INFUSED
WITH VECTOR AND SO, OBVIOUSLY
ALL OF THESE PATIENTS ARE
FOLLOWED FOR LIFE.
SO LET ME JUST SAY A WORD ABOUT
WHAT WE HAVE BEEN TRYING TO LOOK
AT AS A STRATEGY FOR THE 40% OF
THE ADULT POPULATION WITH
PRE-EXISTINGENTS TO AAV.
SO, WE GOT OUR IDEA FROM THIS BY
LOOKING AT COMPARING THE RESULTS
TO THE FIRST TWO TRIALS.
THE AAV2 FACTOR 9 TRIAL, WHICH
HAD A SINGLE STRANDED DNA
VECTOR, AND THE AV8 TRIAL WHICH
WAS SELF COMPLEMENTARY, BUT IN
THE END THE DOSES IN THESE TWO
TRIALS ENDED UP BEING ABOUT THE
SAME.
AND AT THE HIGH DOSE COHORT, AT
LEAST BEFORE THE T-CELL RESPONSE
SUPER VENED, YOU CAN SEE THAT
BOTH OF THEM LED TO A SIMILAR
CIRCULATING LEVEL OF FACTOR 9
WHICH SUGGESTS THAT THE POTENCY
WAS SIMILAR.
HOWEVER, IN THE AV2 TRIAL, THERE
WAS NO EXPRESSION AT THE
LOW-DOSE COHORTS WHEREAS IN THE
AV8 TRIAL, THERE WAS EXPRESSION,
AT LEAST A MODEST LEVEL AT THE
LOWER TO MEDIUM DOSE COHORT.
WHAT WAS THE DIFFERENCE?
WHAT ACCOUNTEDDED FOR THIS?
WAS IT THE STEREOTYPE?
THE DIFFERENCE IN THE DNA
CONFIRMATION?
WE WONDERED ABOUT ANOTHER
DIFFERENCE BETWEEN THESE TWO
PREPS.
THE PREP ADMINISTERED IN THE UCL
ST. JUDE TRIAL HAD A RATIO OF
ABOUT 10-1 EMPTIES TO FULLS.
WHEREAS THE PRODUCTION PROCESS
THAT WAS USED TO MAKE THAT
VECTOR, WHICH WAS MADE AT AFA
JEN, SEPARATED THE FULLS FROM
THE EMPTIES.
AND SO, WHAT WAS INFUSED INTO
THE TWO SUBJECTS DIFFERED IN
THAT WAY.
AND SO, WE WONDERED WHETHER THE
EMPTY CAPSIDS MIGHT IN FACT
SERVE AS DECOYS TO ABSORB
CIRCULATING ANTIBODIES TO AAV
RESULTING IN BETTER TRANSDUCTION
EFFICIENCY PARTICULARLY AT THE
LOWER DOSES.
SO, WE CARRIED OUT A SERIES OF
EXPERIMENTS DONE BY ANOTHER LAB,
AND THEY USED A MOUSE MODEL.
THE MOUSE WAS INJECTED WITH
HUMAN IVIG AT A RANGE OF DOSES
TO MIMIC NEUTRALIZING ANTIBODY
TITERS ENCOUNTERED IN THE HUMAN
POPULATION AND THEN THE MOUSE
WAS INJECTED WITH AAV FACTOR 9
VECTOR WITH NO EMPTY CAPSID OR
WITH A 10 FOLD EXCESS OF
EMPTIES.
HERE IS WHAT YOU SEE.
IF THE MOUSE HAS NO NEUTRALIZING
ANTIBODY TIGHTER, IT DOESN'T
MATTER WHETHER YOU ADD EMPTIES
AT 10 FOLD EXCESS OR YOU DON'T,
YOU GET THE SAME LEVEL OF FACTOR
9 EXPRESSION.
IF YOU GIVE THE MOUSE A LOW
TIGHTER NEUTRALIZING ANTIBODY TO
AAV, THEN IF YOU DON'T ADD ANY
EMPTIES, YOU DON'T GET MUCH
EXPRESSION AND IF YOU ADD A 10
FOLD ACCESS OF EMPTIES, YOU GET
VERY NICE LEVELS OF EXPRESSION.
AND SO, FREDRICO WENT ON TO DO A
SERIES OF EXPERIMENTS WHERE HE
JUST GAVE MICE PROGRESSIVELY
HIGHER NEUTRALIZING ANTIBODY
TITERS USING IVIG AND THEN
SHOWED THAT BY ADDING
PROGRESSIVELY HIGHER LEVELS OF
EMPTIES TO THE FINAL FORMULATION
THAT YOU COULD RESTORE LEVELS UP
TO THAT SEEN IN A NAIVE MOUSE
BUT THE HIGHER THE ANTIBODY
TIGHTER, THE HIGHER EXCESS OF
EMPTIES IS REQUIRED TO ACHIEVE
THAT EFFECT.
AND IF THE MOUSE HAS A VERY HIGH
NEUTRALIZING ANTIBODY, YOU CAN
NOT OVERCOME IT BECAUSE YOU
START TO INHIBITRY CEPTOR
MEDIATED UPTAKE WITH THE VAST
EXCESS OF EMPTIES.
SO, THE WAY TO MAKE THIS EVEN
SAFER OF COURSE IS TO ALTER THE
RECEPTOR BINDING SITES SO THE
EMPTIES, WHEN THEY ARE USED, CAN
CIRCULATE BUT THEY CAN NEVER
GAIN ACCESS TO THE TARGET CELL
AND CONTRIBUTE TO THE BURDEN OF
CAPSID THAT WILL BE PROCESSED
AND PRESENTED ON THE SURFACE OF
THE TRANSDUCED HEPATOCYTES.
SO THAT IS ANOTHER STRATEGY THAT
WE ARE INTERESTED IN.
ANDY WOO THINK THAT EVENTUALLY
FOR USE OF THIS KIND OF STRATEGY
IN PEOPLE WITH HEMOPHILIA, WE
WILL HAVE A SENSITIVE ASSAY WITH
NEUTRALIZING ANTIBODIES TO A
PERSONNELIDES FINAL FORMULATION
OF THE PRODUCT.
SO, ONE OF THE -- SO WE ARE
TESTING THIS AND IN OUR ONGOING
TRIAL, AT CHILDREN'S HOSPITAL.
AND SO, IN THAT TRIAL, WE WILL
BE USING THIS SAME EXPRESSION
THAT WE HAD USED IN THE FIRST
AAV2 TRIAL BUT IN AN AAV8
CAPSID.
AND THEN THE FINAL FORMULATION
WILL BE LINKED TO THE PATIENTS
NEUTRALIZING ANTIBODY TIGHTER.
SO THIS TRIAL IS OPEN AND
ONGOING AT CHILDREN'S HOSPITAL
AND UNIVERSITY OF PITTSBURGH
NOW.
SO, I THINK FOR THE FIELD AS A
WHOLE AROUND HEMOPHILIA,
OBVIOUSLY THERE ARE A NUMBER OF
VERY IMPORTANT QUESTIONS.
WHAT WILL BE THE DURATION OF
EXPRESSION IN THE UCL ACCEPT
JUDE TRIAL?
THERE IS NOW OVER THREE YEARS OF
EXPRESSION IN THE FIRST SUBJECTS
WITH OBSERVATION ONGOING.
WILL IT BE POSSIBLE TO
READMINISTER AND WHAT WILL BE
THE ROLE OF NEUTRALIZING
ANTIBODIES WHICH ALWAYS RISE
AFTER VECTOR INFUSION?
WILL IT BE POSSIBLE TO PURSUE
THE SAME APPROACH FOR HEMOPHILIA
A AND CERTAINLY FOR PROTEIN
THERAPEUTICS, THERE IS A MUCH
GREATER RISK OF IMMUNOGEISITY IN
FACTOR A DEFICIENCY COMPARED TO
FACTOR 9 DEFICIENCY.
AND THEN OF COURSE, THE LONG
TERM RELATED SIDE EFFECTS OF
THIS TYPE OF THERAPY?
WE HAVE FOLLOWED OVER 77 DOGS
WITH SEVERE HEMOPHILIA, A AND B,
FOR PERIODS OF OVER 10 YEARS,
AND HAVE NOT SEEN LONG TERM SIDE
EFFECTS RELATED TO THIS THERAPY.
AND THEN THE SUBJECTS ARE ON THE
FIRST TRIAL WERE ALL IN FUSED
BETWEEN 2001 AND 4, THEY ARE
PART OF A LONG TERM FOLLOW-UP
STUDY WITH NO EVIDENCE OF
PROBLEMS.
AND WILL IT BE POSSIBLE TO
EXTEND THIS TO THE PEDIATRIC
POPULATION?
SO, WHAT I'M GOING TO DO, I SEE
THAT AS USUAL, I HAVE BEEN
OVERLY AMBITIOUS ABOUT WHAT I
CAN COVER.
WHAT I WOULD LIKE TO DO, THOUGH,
IN THE REMAINING MINUTES, IS
TURN TO A SITUATION.
THIS IS AN APPLICATION THAT WE
HAVE BEEN WORKING WITH OVER THE
LAST 7 YEARS WITH JEAN BENNETT
AND ALBERT McGUIRE AT THE
DEPARTMENT OF OPTHALMOLOGY AT
PENN.
IF YOU SPEND A LOT OF TIME
WORRYING ABOUT IMMUNELY
RESPONSES TO YOUR VECTOR AND
TRANSGENE PRODUCT, IT'S ALWAYS
TEMPT TO THINK ABOUT A TARGET
TISSUE THAT IS RELATIVELY
IMMUNOPRIVILEGED.
AND OF COURSE, THAT IS WHAT THE
SUBRETINAL SPACE REPRESENTS.
AND IN A GENE THAT -- AND JEAN
WORKED FOR A NUMBER OF YEARS ON
A PARTICULAR GENETIC FORM OF
INHERITED BLINDNESS CONGENITAL
AMAUROSIS.
BUT IT IS AN AUTOSOMAL RECESSIVE
DEFECT AND ALL LEBER'S IS
CHARACTERIZED BY THE EARLY ONSET
OF RETINAL DEGENERATION AND
THESE CHILDREN ARE DIAGNOSED IN
IN FANCY OR EARLY CHILDHOOD WHEN
THE PARENTS NOTICE THE BABY DOES
NOT TRACK VISUALLY.
10-20% OF THE CASES ARE DUE TO
MUTATIONS IN RPE65 GENE.
AND THERE IS A NATURALLY
OCCURRING DOING MODEL.
-- DOG MODEL.
THIS IS A LIST OF GENES THAT IF
THEY CONTAIN MUTATIONS, CAN LEAD
TO LCA AND AS SOME MODEST
PROPORTION OF ALL OF THESE CASES
ARE DUE TO MUTATIONS IN RPE65.
SO FROM AN ANATOMIC STANDPOINT,
THE MUTATION EFFECTS THE RETINAL
PIGMENT EPITHELIAL CELLS WHERE
RPE ENCODES ISOMBERAISE.
SO WHEN LIKE STRIKES THE RETINA,
YOU GET THIS ISOMBERRIZATION
REACTION THAT THEN STARTS AN
ACTION POTENTIAL THAT GOES BACK
TO THE OPTIC NERVE.
EVENTUALLY THIS TRANSRETINAL IS
TRANSPORTED BACK OUT OF THE
PHOTORECEPTOR INTO THE RPE CELLS
WHERE IT MUST UNDERGO THIS
ISOMBERRIZATION TO GENERATE THIS
AGAIN.
IF YOU DO NOT HAVE THE ACTIVITY
OF THIS ENZYME THEN THE VISUAL
CYCLE IS BROKEN AND BLINDNESS
RESULTS -- COULD RESTORE VISION
IN THESE ANIMALS AS LONG AS THE
ANIMAL IS TREATED RELATIVELY
EARLY IN LIFE AND THE REASON FOR
THAT IS THAT AS THE RPE CELLS --
THAT FULL FUNCTION OF THE RPE
CELLS IS REEL -- REALLY REQUIRED
FOR THE PHOTORECEPTORS TO
SURVIVE.
AND THAT EVENTUALLY THESE WILL
DEGENERATE AND THEN IT'S NOT
POSSIBLE TO RESTORE VISION EVEN
IF THE ENZYME IS RE-INTRODUCED.
AND SHE AND HER COLLEAGUES WERE
ABLE TO FLUSH THAT OUT A LITTLE
MORE CLEARLY IN A MOUSE MODEL.
SO LET ME JUST SAY AMONG THE
MANY THINGS WE DID HERE, READY
FOR THIS TRIAL WERE DEVICE
STUDIES AND THIS IS ONE OF THE
DEVICES APPROVED FOR SUBRETINAL
INJECTION IN THE UNITED STATES.
AND ONE OF THE POINTS WE LEARN
IS ILLUSTRATEED HERE IF YOU
DON'T PUT A SMALL AMOUNT OF SER
FACT IN INTO YOUR FINAL
FORMATION, THE LOW
CONCENTRATIONS USED IN THIS
SETTING RESULT IN MOST OF THE
MATERIALS STICKING TO THE
DELIVERY DEVICE AND THE DELIVERY
IS LESS THAN WAS INITIALLY
LOADED INTO THE SYRINGE.
SO WE DO PUT A SMALL AMOUNT OF
SURFACT IN INTO OUR FINAL
PRODUCT.
SO AFTER COMPLETION OF A SERIES
OF STUDIES TO OPTIMIZE THE
VECTOR AND TO DECIDE ON THE
FINAL FORMULATION, THIS TRIAL
WAS INITIATED AT CHILDREN'S
HOSPITAL.
WE INITIALLY LIMITED -- LIMITED
IT TO CHILDREN AT LEAST 8 YEARS
OLD BUT YOUNG ADULTS NOT MORE
THAN 27, BECAUSE OF THAT DOG
DATA.
AND THE DOSE ESCALATION DESIGN
INCLUDED THREE SUBJECTS IN EACH
OF THREE DOSE COHORTS.
BECAUSE CHILDREN WERE GOING TO
BE INCLUDED IN THE TRIAL, THE
IRB INSISTED - EACH THE FIRST
DOSE WE USE WAS ONE THAT SHOWED
EFFICACY IN MORE THAN 90% OF THE
AFFECTED DOGS AND THE REASON FOR
THAT IS THAT IF CHILDREN ARE
INVOLVED IN CLINICAL TRIALS, IF
IT INVOLVES MORE THAN MINIMAL
RISK, WHICH THIS SORT OF
INJECTION DOES, THERE MUST BE
THE PROSPECT OF DIRECT BENEFIT
FOR THAT CHILD.
SO WE HAD TO START A NEW DOSE
THAT SEEMED TO WORK IN DOGS.
SO, AFTER EXTENSIVE BASELINE
TESTING, SUBJECTS ARE ENROLLED
IN THE STUDY AND IS INJECTED.
THE FIRST SUBJECTS WERE ENROLLED
IN THE FALL OF 2007 AND THIS
SHOWS THE DATA ON THE FIRST 12
PEOPLE WHO WERE IN THE PHASE
I-TWO STUDY.
THEY NOW HAVE BEEN FOLLOWED FOR
A PERIOD OF OVER THE LONGEST
PART, OVER 5 YEARS WE COLLECTED
DATA ON VISUAL ACUITY, VISUAL
FIELDS AND LIGHT RESPONSE AND
MOBILITY AS A THAT WAS SET UP IN
THE OPTHALMOLOGY CLINIC THAT HAS
A SERIES OF OBSTACLES THE
PATIENTS HAS TO STEP OVER AND
AROUND TO GET TO A DOOR AT THE
END AND THE RESULTS FOR THE
INJECTION OF THE FIRST EYE
SHOWED THAT FOR 9-10 SUBJECTS
THE INJECTED EYE DEVELOPS A
STRONGER POOP LARRY LIGHT REFLEX
AND NO UNINJECTEDDIZE DEVELOPED
A STRONGER PLR.
INCREASED LIGHT SENSITIVITY INTO
THE INJECTED EYE AND THAT DIDN'T
HAPPEN FOR ANY OF THE SUBJECTS
IN THE UNINJECTED EYE.
4 OF 8 SUBJECTS DEMONSTRATE THE
IMPROVED NAVIGATIONAL ABILITY
USING THE INJECTED EYE BUT
NOBODY DID THAT FOR THE
UNINJECTED EYE AND SIMILARLY,
50% OF THE PATIENTS SHOWED
IMPROVEMENT IN VISUAL ACUITY,
MORE THAN 3 LINES ON THE EYE
CHART IN THE INJECTED EYE
WHEREAS NOBODY DID THAT IN THE
UNINJECTED EYE.
SO, IF YOU NEVER SEEN THESE
VIDEOS OF CHILDREN GOING THROUGH
THE MOBILITY ASSAY, THEY ARE
VERY IMPRESSIVE.
I'LL SHOW JUST A MINUTE OR TWO.
WHAT YOU WILL SEE IN THIS FIRST
VIDEO IS THREE MONTHS AFTER
VECTOR INJECTION INTO THE WORSE
EYE.
THE SUBJECT FIRST HAS HIS
INJECTED EYE PATCHED AND HE IS
TRYING TO GO THROUGH THE
MOBILITY COURSE USING HIS
UNINJECTED EYE, WHICH WAS HE
WASLY HIS BETTER EYE.
AND WHAT YOU SEE, HE IS SUPPOSED
TO BE FOLLOWING A BLACK ARROW ON
THE FLOOR AND EVEN AT THE VERY
BEGINNING, HE WANDERS OFF THE
COURSE.
AND THAT IS EMBEDDED IN THE
BACKGROUND REDIRECTING HIM.
AND I THINK YOU DON'T HAVE TO
WATCH IT FOR VERY LONG TO
UNDERSTAND THAT THIS IS REALLY
BLIND BEHAVIOR.
AND THEN WHEN THE PATCH IS
APPLIED TO THE UNINJECTED EYE
AND THE PATIENT IS ASKED TO GO
THROUGH THE MOBILITY COURSE,
USING HIS INJECTED EYE, YOU CAN
SEE A VERY MARKED DIFFERENCE IN
HIS PERFORMANCE IN THESE TWO
SITUATIONS.
SO, TO MOVE THIS FORWARD, THE
FDA I WAS INTERESTED IN KNOWING
THE ANSWER TO WHETHER IT WAS
SAFE TO INJECT THE OTHER EYE.
AND SO, WE INITIATED THAT STUDY
IN NOVEMBER OF 2010.
WE HAD TO WRITE IN THE CONSENT
FORM FOR ALL OF THESE PEOPLE
THAT IT WAS POSSIBLE THAT THEY
WOULD DEVELOP IMMUNE RESPONSE
THAT WOULD WIPE OUT THE GAINS IN
THE FIRST EYE.
AND SO, FORTUNATELY FOR US,
THREE OF THE ADULTS WERE WILLING
TO SIGN THAT CONSENT FORM AND TO
UNDERGO THE INJECTION.
THERE WAS AN 8 WEEK STAGGER
BETWEEN SUBJECTS TO TRY TO
MONITOR FOR ANY EVIDENCE OF
IMMUNE RESPONSE, WHICH DID NOT
OCCUR.
AND BECAUSE IT APPEARED SAFE IN
THE FIRST THREE SUBJECTS IT WAS
POSSIBLE TO DO THE REPAINER OF
THE INDIVIDUALS EXCEPT FOR ONE
INDIVIDUAL WHO HAD GLAUCOMA IN
THE CONT LATERAL EYE.
SO HE COULDN'T UNDERGO INJECTION
OF THE SECOND EYE.
CURRENTLY, WE ARE ENGAGED IN A
PHASE III STUDY OF THIS AND I
THINK THAT YOU CAN UNDERSTAND
THAT ONE OF THE CONTROVERSIAL
ISSUES IS WHAT TO USE AS THE
CONTROL.
THE FDA WAS CONCERNED THAT IF WE
USED ONE EYE INJECTED AND THE
OTHER EYE IS CONTROL, THIS
CONTROLS FOR ANY AFFECTS DUE TO
THE SPECIFIC MUTATION THAT THE
PATIENT HAS, ALL OF THESE PEOPLE
HAVE DIFFERENT MUTATIONS IN
THEIR RP65 GENE BUT OF COURSE
THE PRODUCT WILL NOT, IN THE
END, BE ADMINISTERED TO ONE EYE.
IT WILL BE ADMINISTERED
BILATERALLY.
IN THE END, WHAT WE DECIDED TO
USE WAS SUBJECTS THAT THEY ARE
DEEMED ELIGIBLE, IF THEY MEET
ALL THE ELIGIBILITY CRITERIA,
THEY ARE RANDOMIZED 2-1 EITHER
TO BE INJECTED BILATERAL,
NONSIMULTANEOUS INJECTION WITHIN
ONE WEEK, OR THEY GO INTO A
CONTROL GROUP AND THE CONTROL
GROUP UNDERGOES ALL THE SAME
TESTING, BUT ONE YEAR LATER IS
PERMITTED TO CROSSOVER TO THE
TREATMENT GROUP.
THIS STUDY IS BEING CONDUCTED
BOTH AT CHILDREN'S HOSPITAL AND
AT THE UNIVERSITY OF IOWA AND
ENROLLMENT IS PROCEEDING VERY
NICELY AND WE HOPE TO HAVE
FINISHED ENROLLING THAT STUDY BY
EARLY 2014.
SO, I THINK WHAT I'M GOING TO DO
NOW IS JUST UNFORTUNATELY, I'M
NOT GO TO HAVE TIME TO TALK
ABOUT THE WORK WE HAVE BEEN
DOING WITH GENOME EDITING.
VERY INTERESTING APPROACH BUT
LET ME JUST SUMMARIZE AND SAY
THAT IN VIVO GENE TRANSFORWITH
THE AV VECT SOURCE A CLINICAL
REALITY.
IT'S A LICENSED CLINICAL PRODUCT
IN EUROPE.
RECOMBINANT AAV VECTORS ARE NOT
VIRUSES.
BUT THESE ENCOUNTER OR EVOKE
IMMUNE RESPONSES IN HUMANS AND
IT'S IMPORTANT TO TRY TO
UNDERSTAND THOSE TO CHARACTERIZE
THEM SO THAT THEY CAN BE
APPROPRIATELY MANAGED.
BUT FOR THOSE OF YOU WHO DO
ALLOGENEIC BONE MARROW
TRANSPLANTATION, UNDERSTANDING
AND MANAGING IMMUNE RESPONSES
WITH AAV VECTORS IS WAY EASIER
THAN TRANSPLANTING BONE MARROW.
AND EACH TARGET TISSUE OR ROUTE
OF ADMINISTRATION REPRESENTS A
UNIQUE PROBLEM IN TERMS OF
IMMUNE RESPONSES.
WE HAVE A VERY NICE PLATFORM FOR
DELIVERY TO THE SUBRETINAL SPACE
AND TO THE LIVER AT THE DOSES
THAT HAVE BEEN USED SO FAR AND
THAT THAT PROVIDES MUCH SCOPE
FOR FURTHER DEVELOPMENT IN THE
FIELD.
SO I WANT TO JUST SAY THAT IN
OUR GROUP AT THE CHILDREN'S
HOSPITAL, THE WORK ON IMMUNE
RESPONSES HAS BEEN LED BY
FREDRICO MINGOZZI WHO HAS TAKEN
A FACULTY POSITION AT GENATHON
AND ALSO BY THESE OTHERS.
READ READ.
[ READING ]
OUR COLLABORATOR FOR ALL OF THE
STUDIES IN HEMOPHILIC DOGS HAS
BEEN TIM NICHOLLS AT U IN.
C-CHAPEL HILL.
THAT JEAN BENNETT,AL McGUIRE
AND OTHERS HAVE BEEN OUR
PARTNERS IN THE LCA2 TRIAL AND A
NUMBER OF OTHER INVESTIGATORS AT
CHILDREN'S HOSPITAL INVOLVED
EITHER WITH CLINICAL GRADE
VECTOR PRODUCTION OR WITH
PRECLINICAL AND CLINICAL STUDIES
IN HEMOPHILIA AND GREG WITH
LCA2.
OUR COLLABORATOR ON MANY OF OUR
IMMUNOLOGY STUDIES GUNNEDY ERTL.
MAN COLLABORATORS FROM STAN
FORMED, AF I JEN, SIDNEY AND
BRAZIL.
AND THE AAV8 FACTOR TRIAL
CONDUCTED BY.
[ INDISCERNIBLE ]
THANK YOU VERY MUCH FOR YOUR
ATTENTION.
AND I'M HAPPY TO TAKE ANY
QUESTIONS IF THERE IS TIME.
00:57:55.633,00:00:00.000
[ APPLAUSE ]
