>> ALL RIGHT.
WELL, GREETINGS AND SALUTATIONS.
I'M DAN KASTNER.
I'M THE SCIENTIFIC DIRECTOR
EMERITUS AND STILL ACTIVE
PHYSICIAN SCIENTIST IN THE
INTRAMURAL RESEARCH PROGRAM OF
THE NATIONAL HUMAN GENOME
INSTITUTE AND IT IS MY ENORMOUS
PLEASURE TO WELCOME YOU TO THIS
THE 13th INSTALLMENT OF
DEMYSTIFYING MEDICINE, 2025.
A SERIES OF PRESENTATIONS THAT
BRIDGE THE EXCITING DEVELOPMENTS
IN BIOLOGY, ENGINEERING AND
COMPUTER SCIENCE WITH MEDICINE.
AND OF COURSE YOU SEE THAT
ICONIC PHOTO UP IN THE UPPER
RIGHT HAND CORNER OF THE
CONSTRUCTION OF THE BROOKLYN
BRIDGE BACK IN THE LATE 1800s.
AND YOU SEE THOSE TWO GUYS IS UP
THERE ON THE CATWALK.
ONE PROBABLY A PHYSICIAN FROM
MANHATTAN OR MAYBE A BASIC
SCIENTIST FROM BROOKLYN OR
OPPOSITE, I DON'T KNOW.
MAYBE THEY COULD HAVE BEEN
DISCUSSING COAST POSULATES AND
WE'LL HAVE A BONANZA DISCUSSION
OF MUSCULOSKELETAL DISEASE AND
WE'LL HAVE A GREAT DEAL OF FUN
WITH TODAY'S SPEAKERS.
THE SERIES, DEMYSTIFYING
MEDICINE OCCURS TUESDAY
AFTERNOONS FROM 3:00 TO 5:00
EASTERN TIME FROM JANUARY
THROUGH MAY OF 2025.
YOU CAN WATCH IT ON THE LINK
SHOWN HERE ON THE SCREEN, CME
CREDIT IS AVAILABLE IF YOU
SIGNED UP AND THE CME CODE IS
SHOWN HERE, 58792.
FOR THOSE WHO HAVE QUESTIONS
DURING THE TALKS, THERE ARE
GOING TO BE TWO TALKS, WHILE THE
TALKS ARE GOING ON, YOU CAN
SUBMIT YOUR QUESTIONS SO THAT WE
CAN DISCUSS THEM AT THE END OF
THE SESSION USING THE SEND LIVE
FEEDBACK BUTTON ON THE VIDEOCAST
DISPLAY.
ALL THE PREVIOUS SESSIONS ARE
ARCHIVED SHOWN AT THE LINK THERE
ON THE SCREEN.
AND FOR ADDITIONAL INFORMATION
YOU CAN GO TO DEMYSTIFYING
MED@NIH.gov.
THIS IS A SERIES BRIDGING SILOS
AT THE NIH SINCE 2003.
LET'S GET RIGHT ON TO IT.
SO AS I MENTIONED, WE HAVE TWO
WONDERFUL SPEAKERS TODAY.
THE FIRST IS GOING TO BE TALKING
TO US ABOUT A VERY DIABOLICAL
THEME INFILTRATERS HOW THEY
DISRUPT MUSCLE CELLS FROM
WITHIN.
LIKE A TROJAN HORSE, I GUESS.
HE'S A SENIOR INVESTIGATOR AND
CHIEF OF THE MUSCLE DISEASE
SECTION OF THE LABORATORY OF
MUSCLE STEM CELLS AND GENE
REGULATION IN THE NATIONAL
INSTITUTE OF ARTHRITIS AND
MUSCULOSKELETAL AND SKIN
DISEASES.
AND THE ADJUNCT PROFESSOR AT
JOHNS HOPKINS AND GOT HIS
MEDICAL DEGREE AT JOHNS HOPKINS
AND Ph.D. IN NEUROSCIENCE ALSO
FROM JOHNS HOPKINS AND DID HIS
RESIDENCY NEURO MUMUSCULAR
FELLOWSHIP AT JOHN HOPKINS AND
THE CO-FOUNDER MYOCYTIS AND THE
RECIPIENT OF MANY AWARDS AND ON
AND ON.
HIS RESEARCH LABORATORY FOCUSES
ON DEFINING SUB TYPES OF
AUTOIMMUNE DISEASE AND
ELUCIDATING THE ROLE OF MIOSITIS
AND UNDERSTANDING HOW
ENVIRONMENTAL EXPOSURES TRIGGER
AUTOIMMUNE MUSCLE DISEASE AND
DEVELOPING TARGETED THERAPIES.
AND IT THE SECOND SPEAKER WILL
TALK TO US ABOUT ADENO MEDIATED
GENE THERAPY AND OPPORTUNITIES
AND CHALLENGES.
DR. BONNEMAN IS CHIEF OF THE
NEUROMUSCULAR SECTION AT THE
NEUROGENETICS BRANCH OF THE
NATIONAL INSTITUTE FOR
NEUROLOGIC DISEASES AND STROKE.
HE'S ALSO THE ADJUNCT FULL
PROFESSOR OF NEUROLOGY AT THE
UNIVERSITY OF PENNSYLVANIA.
HE GOT HIS MEDICAL DEGREE SUMMA
CUM LAUDE AND DID CLINICAL AND
RESEARCH FELLOWSHIPS AT THE MASS
GENERAL AND BOSTON CHILDREN'S
HOSPITAL AT HARVARD.
HE'S AN ELECTED MEMBER OF THE
ASSOCIATION OF AMERICAN
PHYSICIANS, RECIPIENT OF THE
GEORGE CA JOBY AWARD OF THE NERN
NEUROLOGICAL ASSOCIATION AND
RECIPIENT OF THE HONORARY AWARD
OF THE GERMAN NEUROPEDIATRIC
SOCIETY.
HIS RESEARCH GROUP STUDIES THE
GENETICS AND PATHOGENESIS OF
EARLY ONSET NEUROMUSCULAR
DISORDERS OF CHILDHOOD.
HIS GROUP WORKS ON GENE EDITING
AND RNA DIRECTED THERAPEUTICS
FOR ALLELE SPECIFIC KNOCK DOWN
OF SOME KIND OF ACTING MUTATIONS
THERE, DOMINANTLY ACTING
MUTATIONS AND STUDIES OUTCOME
MEASURES IN AAV9 MEDIATED GENE
TRANS FOREGIANT AXONAL
NEUROPATHY.
WITH THAT HAVING BEEN SAID I
THINK THAT'S IT FOR MEF FOREGIA
NEUROPATHY.
WITH THAT HAVING BEEN SAID I
THINK THAT'S IT FOR MEER FOREGI
NEUROPATHY.
WITH THAT HAVING BEEN SAID I
THINK THAT'S IT FOR ME FOREGIAN
NEUROPATHY.
WITH THAT HAVING BEEN SAID I
THINK THAT'S ITOF FOREGIANT AXO
NEUROPATHY.
WITH THAT HAVING BEEN SAID I
THINK THAT'S IT FOREGIANT AXONA
NEUROPATHY.
WITH THAT HAVING BEEN SAID I
THINK THAT'S ITA FOREGIANT AXON
NEUROPATHY.
WITH THAT HAVING BEEN SAID I
THINK THAT'S IT FOR MEOREGIANT
NEUROPATHY.
WITH THAT HAVING BEEN SAID I
THINK THAT'S ITREGIANT AXONAL
NEUROPATHY.
WITH THAT HAVING BEEN SAID I
THINK THAT'S IT FOR MEGIANT AXO
NEUROPATHY.
WITH THAT HAVING BEEN SAID I
THINK THAT'S IT FOR MEIANT AXON
NEUROPATHY.
WITH THAT HAVING BEEN SAID I
THINK THAT'S ITNT AXONAL
NEUROPATHY.
WITH THAT HAVING BEEN SAID I
THINK THAT'S ITT AXONAL
NEUROPATHY.
WITH THAT HAVING BEEN SAID I
THINK THAT'S IT AXONAL NEUROPAT.
WITH THAT HAVING BEEN SAID I
THINK THAT'S ITAXONAL NEUROPATH.
WITH THAT HAVING BEEN SAID I
THINK THAT'S IT FOR MEXONAL NEU.
WITH THAT HAVING BEEN SAID I
THINK THAT'S IT FOR ME
>> THE TOPIC OF MY TALK IS GOING
TO BE INTRACELLULAR INFILTRATERS
HOW ANTIBODIES DISRUPT FROM
WITHIN.
DESIGN TO BE A PROVOCATIVE
TITLE.
AND I HAVE A COUPLE DISCLOSURES.
THE FIRST IS I AM ON A PATENT
FOR AN AUTOANTIBODY TO DEFECT
ANTIBODIES BUT HAVE RECEIVED
COMPENSATION FOR THAT.
AND HAVE TREATMENTS FOR
MYOSITIS.
AN OVERVIEW OF MY TALK IS TO
TELL YOU ABOUT MYOSITIS A
HETEROGENEOUS FAMILY OF RARE
AUTOIMMUNE DISEASES.
I WANT TO TELL YOU ABOUT THE
FACT THERE'S MYOCYSITIS SPECIFI
DISTINCT CLINICAL PHENOTYPE.
EACH MUCH THESE ANTIBODIES
RECOGNIZES A PROTEIN TARGET THAT
IS INTRACELLULAR, NOT ON THE
SURFACE OF CELLS.
THEN I'M GOING TO MOVE ON TO
PRESENT EVIDENCE WE NOW HAVE THE
MYOSITIS SPECIFIC ANTIBODIES AND
ASSOCIATED ANTIBODIES ARE
INTERNALIZED INTO MUSCLE CELLS
AND DISRUPT THE FUNCTION OF
THEIR TARGET PROTEINS AND WE
THINK IT'S A POTENTIALLY
IMPORTANT PART OF THE DISEASE
PATHOGENESIS.
I'LL TOUCH ON THE FOUR.
ONE IS DEFINED BY AUTOANTIBODIES
AND THERE'S IMMUNE-MEDIATED
NECROTIZING MYOSITIS AND OVERLAP
WHERE PATIENTS HAVE ANOTHER
DEFINED CONNECTIVE TISSUE
DISEASE WITH AN INFLAMMATORY
DISEASE.
DERMATOMYOSITIS IS THEY HAVE
BOTH DERMO LOGIC FEATURES AND
GET RASHES ONE YOU CAN SEE.
THE EMETHRITIS LESIONS AND THE
KNUCKLES HAVE A RED SCALY
LESION, THAT'S THE GOTRIN SIGN.
HERE'S MY PICTORIAL
REPRESENTATION OF PROXIMAL
MUSCLE WEAKNESS THAT OCCURS IN
MOST IF NOT ALL PATIENTS WITH
DERMATOMYOSITIS AND ABOUT 70% TO
80% OF THESE PATIENTS WILL HAVE
ONE OF SEVERAL KNOWN DERMATO
MYOCYSITIS ANTIBODIES AND TAY
TARGET INTRACELLULAR PROTEINS.
THERE ARE FOUR I'M GOING TO
MENTION.
THEY ARE RECOGNIZE TIP 1 GAMMA
AND KNOWN WITH CHROMATIN
REMODELLING AND BOTH HAVE
PREDOMINANTLY NUCLEAR
LOCALIZATION.
AND THERE'S MI2 WITH A NUCLEAR
LOCALIZATION.
THERE ARE OTHER PATIENTS WHO
HAVE AUTOANTIBODIES THAT
RECOGNIZE CYTOPLASMIC AND
USUALLY MDA5 THAT SITS IN THE
CYTOPLASM TO RECOGNIZE DOUBLE
STRANDED RNA AND TRIGGERS THE
INNATE IMMUNE RESPONSE AND AGAIN
MDA5 IS A CYTOPLASMIC ROW TEEN.
I WANT TO SPEND A FEW MINUTES
SPENDING A FEW MINUTES THAT
THOUGH THEY'RE ALL FOUND IN
PATIENTS THEY DEFINE UNIQUE
CLINICAL PHENOTYPES AND
IMPORTANT TO RECOGNIZE THAT
ALMOST ALL JUST HAVE ONE OF EACH
OF THESE FOUR DIFFERENT MYOSITIS
SPECIFIC ANTIBODIES.
THE FIRST ARE FOUR WITH
ANTIBODIES AGAINST THE PROTEIN
CALLED TIP 1 GAMMA.
THIS IS THE MOST COMMON ANTIBODY
AND THE CLASSIC -- THEY HAVE THE
CLASSIC FEATURES AND THE
ADDITIONAL THING IS THEY HAVE AN
INCREASED RISK OF CO-EXISTING
MALIGNANCY COMPARED TO OTHER
DERMATOCYTIS PATIENTS AND
HEALTHY CONTROLS.
THAT'S ASSOCIATED WITH THE TIF1
GAMMA ANTIBODIES.
AND THE SECOND MOST COMMON IS
THE RECOGNITION OF THE PROTEIN
NXP2.
IN CONTRAST TO OTHER PATIENTS,
THEY'RE MORE LIKELY TO HAVE SUB
CUTANEOUS EDEMA.
THE FOREARM OF THE PATIENT WITH
PITTING EDEMA.
NOTHING IN THE LEGS.
THIS IS A FEATURE OF THE
DERMATOMYOSITIS.
IT'S NOT BECAUSE OF HEART
FAILURE OR ANYTHING.
MOST DERMATOMYOSITIS PATIENTS IS
TRYING TO FULLY EXTEND THEIR
FINGERS AND THEY'RE WEAK AND
THAT'S AS FAR AS THEY CAN GO.
THEY CAN DEVELOP A DREADED
COMPLICATION WHICH IS SUB
CUTANE
CUTANEOUS KAL -- CALCINOSIS AND
YOU CAN SEE THE RADIO DENSE
CALCIUM DEPOSITION THAT OCCURS
IN THE PATIENT.
AND THEY'LL HAVE DIFFICULTY
SWALLOWING WHERE WE ONLY SEE
THIS IN A QUARTER OF THE
PATIENTS AND THE ONLY
DERMATOMYOSITIS PATIENTS I'VE
HAD TO HAVE A FEEDING TUBE HAVE
THE ANTRI-NXP2 DERMATOMYOSITIS.
SOME PATIENTS WITH
DERMATOMYOSITIS HAVE ANTIBODIES
AGAINST THE PROTEIN MI2 PART OF
THE NUCLEOSOME ACETYLATION AND
HAVE THE MOST SEVERE WEAKNESS
AND HIGHEST MUSCLE ENZYME
VALUES.
THE CK200 OR LOWER IN THE
ANTI-MI2 DERMATOMYOSITIS THE
MEAN REACHES 4,000 WHEREAS IN
OUR OTHER DERMATOMYOSITIS
PATIENTS WITH OTHER AUTOIMMUNE
PATIENTS THE MEAN IS IN THE 200
THOUGH IT CAN GET HIGHER IN SOME
PATIENTS.
AGAIN, THESE PATIENTS HAVE NOT
JUST MORE SEVERE WEAKNESS AND
HIGHER MUSCLE ENZYMES, WHEN YOU
LOOK HISTOLOGICALLY AT THEIR
MUSCLE TISSUE AND THE MOST
NECROSIS AND INFLAMMATION IN THE
MUSCLE BIOPSY.
THE LAST ANTIBODY I WANT TO TELL
YOU ABOUT RECOGNIZES MDA5 WHICH
AGAIN IS A CYTOPLASMIC
DOUBLE-STRANDED RNA SENSOR.
THESE PATIENTS HAVE UNIQUE
CUTANEOUS FEATURES.
THEY GET THE CLASSIC GOTRIN SIGN
OVER THEIR FINGERS AND
ULTERATING LESIONS ON THE
SURFACE OF THEIR PALMS AND --
ULCERATING.
AND THESE OFTEN DEVELOP
INTERSTITIAL LUNG DISEASE AND A
RAPID FORM THAT UNFORTUNATELY
HAS A VERY HIGH MORTALITY RATE.
AND THE MUSCLE BIOPSIES CAN BE
RELATIVELY BLAND COMPARED TO
PATIENTS WITH OTHER TIMES OF
DERMATOMYOSITIS.
HOPEFULLY I CONVINCED YOU THOUGH
ALL THESE PATIENTS ARE SAID TO
HAVE DRAMATIC DERMATOMYOSITIS,
IN SOME WAYS EACH IS A UNIQUE
DISEASE DEFINED BY THE TYPE OF
RECOGNIZED.
THE SECOND IS THE ANTISNTHETASE.
IT'S DEFINED BY THE PRESENCE OF
AUTOBEYONDSS -- ANTIBODIES AND
THEY CORRECT TO THE APPROPRIATE
TRNA.
YOU HAVE A BUNCH OF DIFFERENT
TRN As AND SYNTHETASE AND
THEY'RE REFERRED TO AS ANTI-JO-1
AND OTHERS ARE RECOGNIZED AND
THESE PATIENTS HAVE A UNIQUE
SYNDROME THAT INCLUDES SOME OF
FEATURES I ALREADY TOLD YOU
ABOUT LIKE MYOSITIS AND
INTERSTITIAL LUNG DISEASE AND A
CLASSIC DERMATOMYOSITIS LIKE
RASH AND THESE PATIENTS WILL
OFTEN DEVELOP ARTHRITIS.
THEY HAVE UNIQUE MANIFESTATIONS.
THE HYPERCARE -- FEATURES AND
THEY'LL HAVE SOME COMBENGS.
AND THE LAST TYPE BEFORE WE MOVE
ON IS THE TYPE OF MYOSITIS
CALLED NECROTIZING MYOSITIS.
I HAVEN'T TOLD YOU TOO MUCH
ABOUT MUSCLE BIOPSIES SO FAR BUT
WHETHER THEY'RE DERMATOMYOSITIS
OR ANTISNTHETASE HAVE A
SIGNIFICANT AMOUNT OF
INFLAMMATORY CELLS IN THEIR
TISSUE.
IN CONTRAST THESE PATIENTS WITH
NECROTIZING HAVE MINIMAL
INFILTRATION BUT HAVE MYOFIBER
NECROSIS.
THIS IS THE EARLY STAGE OF
NECROSIS AND THESE PATIENTS HAVE
ONE OF TWO AUTOANTIBODIES.
THEY CAN RECOGNIZE THE SIGNAL
RECOGNITION PART OR THEY CAN
RECOGNIZE THE PHARMACOLOGIC
STATINS.
ONE OF THE FASCINATING THINGS
ABOUT ANTI-HMGCR IS THEY'RE AN
IMPORTANT PART OF MYOSITIS.
IT OCCURS IN THE CONTEXT OF
ANOTHER RHEUMATOLOGIC DISEASE
LIKE LUPUS OR RHEUMATOID
ARTHRITIS.
THESE PATIENTS DON'T HAVE THE
MYOSITIS SPECIFIC ANTIBODIES BUT
HAVE ASSOCIATED ANTIBODIES.
THEY'RE FOUND IN PATIENTS WITH
MYOSITIS AND OTHER RHEUMATOLOGIC
CONDITIONS.
THE ONE I'M GOING TO TALK ABOUT
IS ANTIBODIES RECOGNIZING A
PROTEIN COMPLEX CALLED PMSCL
OFTEN FOUND IN PATIENTS WITH A
SCLERO DERMAPHENOTYPE.
ALL THESE ANTIBODIES I'VE TOLD
YOU ABOUT RECOGNIZE
INTRACELLULAR PROTEINS AND HAVE
BEEN THOUGHT OF FOR THE MOST
PART AS BIO MARKERS OF THESE
PHENOTYPES RATHER THAN PLAYING A
PATHOGENIC ROLE.
THAT'S MOSTLY BECAUSE THE
ANTIBODIES HAVE THOUGHT -- IT'S
BEEN THOUGHT THEY CAN'T REACH
THEIR INTRACELLULAR TARGETS.
ONE THING WE'LL TRY TO CONVINCE
YOU OF TODAY IS THEY DO AND ARE
LIKELY TO HAVE A PATHOGENIC
ROLE.
IT WAS REALLY A DIFFERENT
RESEARCH QUESTION THAT LED US TO
CONSIDER WHETHER AUTOANTIBODIES
CAN ENTER MUSCLE CELLS.
WE CAME AT IT FROM A DIFFERENT
DIRECTION WHICH IS TRYING TO
UNDERSTAND WHETHER SOME GENES
ARE UNIQUELY EXPRESSED IN THE
MUSCLE TISSUE OF PATIENTS WHO
HAVE A PARTICULAR TYPE OF
AUTOANTIBODY MYOSITIS.
WE HAD 49 PATIENTS WITH
NECROTIZING MYOSITIS AND SOME
HAD SRP AND SOME HAD
DERMATOMYOSITIS AND EACH WITH A
DEFINED ANTIBODY AND 18 MUSCLE
BIOPSIES AND ONE FROM A DISEASE
CALLED INCLUSION BODY MYOSITIS
AND 20 MUSCLE BODY SPECIMENS
FROM HEALTHY NORMAL SUBJECTS.
WE DID BULK RNA SEQ.
THE QUESTION WE ASKED ARE THERE
GENES UNIQUELY EXPRESSED IN ONE
TYPE OF ANTIBODY DEFINED
MYOSITIS.
THE REASON WE ASKED THAT TO WHAT
IS UP OR DOWN REGULATED COMPARED
TO NORMAL, WE RECOGNIZED MANY
GENES DYSREGULATED IN MYOSITIS
ARE GOING TO BE DYSREGULATED
BECAUSE THERE'S MUSCLE
RESTRICTION OR REGENERATION
GOING ON AND TELL US ABOUT
PATHOGENESIS.
WE FIGURED IF WE FOCUSSED ON
THOSE UNIQUE TO A PARTICULAR
TYPE OF MYOSITIS WE MIGHT GET
SOME CLUES.
FOR THIS STUDY ONE OF THE KEY
THINGS THAT STOOD OUT TO US FOR
THOSE WITH POSITIVE MYOSITIS
THEY HAD A UNIQUE SET OF 135
GENES NOT UPREGULATED IN OTHER
TYPES OF MYOSITIS OR NORMAL
TISSUE.
SO 135 GENES THAT DEFINED
SOMETHING UNIQUE ABOUT THE
MUSCLE TISSUE MUCH PATIENTS WITH
THE MYOSITIS.
AND HERE'S AN UPREGULATED GENE
AND IT'S A TRANSCRIPTOMIC
REPRESSOR AND HERE YOU GOT THE
VALUES OF EXPRESSION OF SER21 IN
NORMAL TISSUE AND THE MUSCLE
BIOPSIES WITH EACH DOT
REPRESENTING THE EXPRESSION AND
MUSCLE BIOPSY AND PATIENTS WITH
OTHER AUTOANTIBODIES AT THE GENE
EXPRESSION LEVEL IT'S ONLY
EXPRESSED IN THE ANTI-MI2
SPECIFIC MUSCLE BIOPSIES.
WE WANTED TO SEE IF IT WAS
TRANSLATED TO PROTEINS AND IT
IS.
THE STAINING BY
IMMUNOFLUORESCENCE SHOWED IT'S
ONLY FOUND IN ANTI-MI2 POSITIVE
MUSCLE TISSUE AND HAS A NUCLEAR
LOCALIZATION PREDOMINANTLY AS WE
WOULD EXPECT.
SO WE ALSO LOOKED TO SEE IF
THERE WAS CORRELATION BETWEEN
THE EXPRESSION OF SRT1 AND
MUSCLE BIOPSIES.
YOU SEE A RELATIVELY NORMAL
MUSCLE BIOPSY MORE NORMAL THAN
THE REST WITH VERY LITTLE SER21
EXPRESSION AND 0.0.
MOVING RIGHT TO LEVEL AND HERE
YOU SEE INCREASING EXPRESSION
LEVELS OF SER21.
AS THE EXPRESSION LEVELS OF SEV
ERT1 INCREASED YOU GET MORE AND
MORE MUSCLE DAMAGE.
THERE'S A CORRELATION BETWEEN
THE AMOUNT OF DAMAGE AND THE
EXPRESSION OF THIS GENE.
THE OTHER THING WE FOUND IS THAT
THE SER T1 EXPRESSION LEVELS IN
AN INDIVIDUAL MUSCLE BIOPSIES
ARE CORRELATED WITH THE ANTI-MI2
ANTIBODY LEVELS IN THE SERUM OF
THE PATIENTS THAT THE MUSCLE
BIOPSIES CAME FROM.
THERE'S A LINK THERE AS WELL.
SO THE NEXT THING WE DID WAS
THINK WELL, WHAT IS IT SRT1 AND
THE OTHER GENES INCLUDED IN THE
MIT2 POSITIVE IS SPECIFIC GENE
SET WHAT DO THEY HAVE IN COMMON?
AND WE STARTED THINKING ABOUT,
WELL, WHAT IS THE FUNCTION OF
MIT AND WHAT DO THE ANTI-MI2
ANTIBODIES RECOGNIZE?
THEY RECOGNIZE MI2 ALPHA AND
BETA BOTH IN THE NUCLEUS AND
BOTH ARE COMPONENTS OF THE
NUCLEOSOME REMODELLING AND
DECEYTLATION AND WE LOOKED HOW
IT'S EXPRESSED IN MUSCLE TISSUE.
WE FOUND THE AND IT'S ENRICHED
FOR GENES KNOWN TO BE REPRESSED
BY THE COMPLEX.
WE GOT THE IDEA MAYBE THE
ANTIBODIES ARE SOMEHOW
DISRUPTING THE FUNCTION OF MI2
COMPLEX AND ALLOWING THE GENES
TO BE EXPRESSED WHICH ARE NORM
HI REPRESSED BY THE COMPLEX.
THIS ONLY WORKS IF THEY GET IN
THE MUSCLE CELLS AND WE ALL KNEW
THAT DOESN'T HAPPEN.
IMMUNOGLOBULIN IS A LARGE CELL
AND CAN'T GET IN MUSCLE CELLS.
WE DECIDED TO LOOK ANYWAY TO SEE
WHAT WE CAN SEE.
WE WOUND UP DOING DIRECT
IMMUNOFLUORESCENCE AND USED AN
ANTI-IMMUNOGLOBULIN AGAINST IGG
LABELLED GREEN AND LOOKED TO SEE
WHAT THE DISTRIBUTION OF
IMMUNOGLOBULIN WAS IN PATIENTS
WITH THE MI-2 POSITIVE
DERMATOMYOSITIS AND HERE YOU SEE
THE IMMUNOGLOBULIN AND THIS IS
THE NUCLEUS.
THE IMMUNOGLOBULIN IS
ACCUMULATING IN THE MUSCLES AND
THIS IS REPRODUCIBLE AND ROBUST.
YOU SEE IT JUST IN THE NUCLEI IN
THE PATIENTS BUT NOT HEALTHY
CONTROLS.
THAT WAS INTRIGUING.
WE WANTED TO PROVE THE
ANTIBODIES CAN CAUSE THIS
DYSREGULATED EXPRESSION OF THE
GENE SET WE SEE IN THE PATIENTS
WITH ANTI-MI2 POSITIVE
DERMATOMYOSITIS.
HOW CAN WE DO THAT?
WE FOUND UP GROWING HUMAN
MYOBLASTS IN CULTURE AND BATHING
THEM IN THE IMMUNOGLOBULIN FROM
AN INDIVIDUAL PATIENT AND THEN
MAKE SURE IT GOT IN THE CELLS.
TO DO THAT WE ELECTROPOREATED
THE CELLS AND GOT IN THE FIBERS
AND THE CELLS SEAL UP AND THEN
DO BULK RNA SEQ ON THE CELLS
GROWING IN CULTURE WITH
INTERNALIZED ANTIBODIES AND SEE
THE FULL SET AND SEE IF THERE'S
DYSREGULATED GENE EXPRESSION AS
A CONSEQUENCE OF INTERNALIZING
THE ANTIBODY.
WE DID THIS USING THE
IMMUNOGLOBULIN AND EACH COLUMN
IS THE EXPRESSION OF A GENE SET
FROM AN INDIVIDUAL PATIENT.
THE GENE SET WE'RE LOOKING AT IS
ALL THE GENES OVER EXPRESSED IN
THE PATIENTS AND SER21 AND THE
NEXT TOP 19 OR 20 OF THE 135
GENE SET.
HERE WE HAVE INDIVIDUAL CONTROLS
INTERNALIZED AND THEN FIVE
DIFFERENT IMMUNOGLOBULIN FROM
PATIENTS AND IMMUNOGLOBULIN FROM
OTHER ANTIBODIES AND
ANTISNTHETASE AND THOSE
OVEREXPRESSED THE SAME GENE SET
THAT IS FOUND IN THE MUSCLE
TISSUE FROM THOSE PATIENTS.
WE WANTED TO SEE WHAT PART IS
RECOGNIZED BY THE ANTIBODIES.
THIS IS WORK WE DID WITH BEN
LARMON AT JOHNS HOPKINS AND DOES
PHAGE IMMUNOPRECIPITATION
SEQUENCING AND SUFFICE TO SAY IS
YOU CREATE A LIBRARY WHERE EACH
IS DISPLAYED IN THE HUMAN
SEGMENT OF ALL THE PROTEOMES AND
THEY'RE STAGGERED WITH
OVERLAPPING PEP SIDES.
YOU ADD YOUR MIXTURE AND SINCE
YOU DON'T HAVE AUTOANTIBODIES
AND THEY'LL RECOGNIZE A
PARTICULAR FAITH YOU CAN
PRECIPITATE IT AND DO DNA
SEQUENCING AND IDENTIFY THE
REGION OF THE FULL LENGTH
PROTEIN RECOGNIZED BY THE
AUTOANTIBODIES.
WHAT WE FOUND WITH PATIENTS WITH
ANTI-MI2 IS THERE'S AN
IMMUNODOMINANT EPITOPE THAT
RECOGNIZE BOTH MIT ALMA --
ALPHA AND BETA A.
AND WHAT IS INTERESTING ABOUT
THIS IS AND THIS LINE TO THE PH1
ONE DOMAIN AND THE REGION IS
CRITICAL FOR ENABLING IT TO
RECOGNIZE HISTOMODIFICATIONS AND
REGULATION THE TARGET GENE
EXPRESSION.
Ph.D -- PHD1 ALLOWS IT TO
REGULATE THE GENE SET.
BASED ON THIS WE HAVE A MODEL
FOR HOW WE THINK AUTOANTIBODIES
AGAINST MIT MAY BE PATHOGENIC.
THIS IS MY RUDE UMENT RYUMENTRY
RUDIMENTARY AND THE GENOME
INCLUDES GENES INCLUDING SERT1
AND THE TRANSCRIPTION IS
REPRESSED BY THE NURD COMPLEX.
AND FOR REASONS NOT WELL KNOWN,
SOME PATIENTS DEVELOP
AUTOANTIBODIES AND WE'VE SHOWN
THEY CAN GET INTO THE NUCLEUS
AND OUR MODEL IS THEY BIND TO
THE MIT COMPLEX PROBABLY AT THE
PHD DOMAIN AND THAT ALLOWS FOR
THE TRANSCRIPTION OF THE GENE
AND THE EXPRESSION OF SERT T1
PROTEIN AND OUR HYPOTHESIS IS
THAT IS CAUSING DAMAGE TO THE
MUSCLE FIBERS AND CONTRIBUTING
TO THE PATHOGENESIS OF THE
DISEASE.
THOUGH WE STILL HAVE TO DO A
BETTER JOB DEMONSTRATING THE
CASE.
ARE THERE OTHER MYOSITIS
ANTIBODIES THAN CAN GET IN CELLS
AND DISRUPT THEIR FUNCTION
PROTEINS.
WHEN WE DID IMMUNOFLUORESCENCE
FROM THE PATIENTS WITH THE
DIFFERENT TYPES OF MYOSITIS
ANTIBODIES WE FOUND
IMMUNOGLOBULIN ALWAYS GETS
CO-LOCALIZED WITH THE TARGET
PROTEINS.
I SHOWED YOU THIS THE NUCLEUS IS
WHERE IMMUNOGLOBULIN FOR THOSE
WHO HAVE IT LOCALIZE AND
PATIENTS WITH ANTIBODIES AGAINST
INTRACELLULAR TARGETS FROM
IMMUNOGLOBULIN -- IF THEY'RE
CYTOPLASMIC NOT NUCLEAR, THE
IMMUNOGLOBULIN GOES TO WHERE THE
TARGET ANTIGEN IS.
I WANT TO SHOW YOU ONE MORE
EXAMPLE OF HOW THE
AUTOANTIBODIES CAN DISRUPT THE
FUNCTION.
THESE ARE ANTIANTIBODIES FOR
PEOPLE WITH SCLERO DERMA-AND
MIXED CONNECTED TISSUE DISEASE
AND THEY'RE UNIQUE IN THAT THEY
HAVE MORE SEVERE WEAKNESS IN
THEIR ARMS THAN LEGS.
THAT'S THE OPPOSITE OF OTHER
PATIENTS.
THEY HAVE MANY MORE EXTRA MUSS
LAR MANIFESTATIONS COMPARED TO
OTHER PATIENTS I TOLD YOU ABOUT
INCLUDING THEY OFTEN HAVE SKIN
MANIFESTATION.
WHAT DO ANTIBIOTICEDS RECOGNIZE?
THEY RECOGNIZE TWO SUB UNITS.
EXO C9 AND 10 OF THE NUCLEAR RNA
EXPOSOME COMPLEX AND WHAT'S IT
DO?
THE NORMAL DPUNG AL FUNCTION I
DEGRADE LONG CODING RNAs.
THAT'S A MAJOR FUNCTION.
WE SAW THE IMMUNOGLOBULIN
ACCUMULATES IN THE NUCLEI.
EACH HAVE IMMUNOGLOBULIN
DEPOSITION AND APPEARS TO HAVE A
NUCLEAR POWER.
WE DID A SIMILAR STUDY AS
PRESENTED ALREADY FOR MI2 AND
INCLUDE 669 MUSCLE ANTIBODIES
AND WE'RE NOW ASKING THE
QUESTION ARE THERE GENES THAT
ARE ONLY DYSREGULATED IN MUSCLE
BIOPSIES IN PATIENTS WITH THIS
AND THERE ARE 236 GENES.
THERE ARE ONLY UPREGULATED IN
ANTI-PMSCL NOT THE OTHER TYPES
OF MYOSITIS OR HEALTHY CONTROLS.
I'LL SHOW A LIST OF THE TOP 20
OR SO AND YOU PROBABLY DON'T
RECOGNIZE THE NAMES BECAUSE
THEY'RE ALL NUMBERS BECAUSE THE
TOP 20 AND GOING DOWN THE LIST
INTO THE 200s IS ALMOST
EXCLUSIVELY NOT LONG ENCODING
RNAs AND NON-DIVERGENT
TRANSCRIPTS AND THESE ARE THE
GENES NORMALLY DEGRADED BY
PMSCL.
OUR HYPOTHESIS WAS BINDING TO
THE NUCLEAR EXPOSOME AND LEAD TO
THE SURVIVAL OF LONG CODING RNAs
AND DIVERGENT TRANSCRIPTS.
AGAIN WE WANTED TO PROVE THE
ANTIBODIES CAN HAVE THE EFFECT
IN CULTURED MYOBLASTS.
THIS IS BASICALLY THE SAME
EXPERIMENT.
WE TAKE THE HUMAN MYOBLAST AND
BATHE THEM AND CREATE PORES AND
LET THE ANTIBODIES GET IN AND DO
BULK RNA SEQ.
THIS BASICALLY THE SAME
EXPERIMENT AS BEFORE ONLY NOW
THE GENES LISTED ON THE RIGHT
ARE THE TOP 20 GENES ONLY FOUND
IN MUSCLE BIOPSIES FROM PATIENTS
WITH ANTI-PMSCL AND NOT LONG
ENCODING RNAs AND DIVERGENT
TRANSCRIPTS.
WHEN WE INTERNALIZE THE
ANTIBODIES INTO THE MYOBLASTS WE
GET HIGH LEVELS OF EXPRESSION OF
THAT PMSCL SPECIFIC GENE SET AND
DON'T SOO THE SAME WHEN WE
INTERNALIZE FROM OTHER MYOSITIS
OR HEALTHY CONTROLS.
IT'S A VERY SPECIFIC EFFECT %F
THE ANTIBODIES AND THE
EXPRESSION OF THESE GENES IN
CULTURED CELL AND TISSUES.
WE HAVE A MYOSITIS WHERE
PATIENTS DEVELOP THE
AUTOANTIBODIES.
NORMALLY THE EXOSOME DEGRADES
THE LONG-ENCODING RNAs.
SOMEHOW THEY GET IN AND CAN SEE
THEM IN THE MUSCLE TISSUE.
OUR HYPOTHESIS IS THEY DISRUPT
THE ABILITY OF THE NUCLEAR
COMPLEX TO DEGRADE THESE LONG
ENCODING RNAs AND THAT RESULTS
IN THEIR OVER EXPRESSION.
AGAIN, WE HYPOTHESIZE THAT IS AN
IMPORTANT PART OF THE
PATHOGENESIS AND ACCOUNTS FOR
WHY PATIENTS HAVE A UNIQUE
PHENOTYPE.
THE QUESTION CAN THEY GET TO
OTHER CELL TYPES.
WE STARTED EXPLORING THIS AND
HAPPEN TO HAVE SKIN BIOPSY
TISSUE FROM AN PATIENT.
IMMUNOGLOBULIN IS STAINED AND
YOU SEE IT'S ACCUMULATING IN THE
NUCLEI.
WE THINK IT'S THE SAME AS IN
MUSCLE TISSUE WHERE THE
IMMUNOGLOBULIN IS ACCUMULATING
IN THE NUCLEI.
NOT JUST IN THE MUSCLE BUT IN
THE SKIN AND WE'RE DOING FURTHER
WORK ON THAT.
HOPEFULLY I'VE CONVINCED YOU AT
LEAST IN MYOSITIS FOR THE
SAMPLES I'VE SHOWN YOU AND CAN
SHOW YOU SAMPLES FROM OTHER
ANTIBODIES AS WELL, HOPEFULLY
I'VE CONVINCED YOU THE
ANTIBODIES GET INTO CELLS, REACH
THEIR INTRACELLULAR TARGETS AND
DISRUPT THEIR FUNCTION.
WHAT I ALSO WANT TO MENTION IS
THAT WHEN WE LOOKED AT THE
HISTORY OF THIS TURNS OUT
THEY'RE INTERNALIZED HAS BEEN
AROUND FOR THE LUPUS FIELD AND
THERE'S NUMEROUS EXAMPLES AND
LUPUS ANTIBODIES SUCH AS THOSE
AGAINST DOUBLE STRANDED DNA ALL
CAN PENETRATE CELLS AND LOCALIZE
INTRACELLULARLY.
THOUGH IT'S BEEN LESS CLEAR WHAT
THE RESULT OF THAT IS.
HOUR ANTIRIBOSOMAL ANTIBODIES
COULD DISRUPT PROTEIN SYNTHESIS
AND THEY CAN ACTIVATE IMMUNE
CELLS AND ACTUALLY INDUCE A P
POPTOSIS AND IT'S BEEN KNOWN HOW
IT OCCURS FOR SOME LUPUS
ANTIBODIES.
THERE'S A MOUSE MODEL OF LUPUS
AND IT THEY'RE A DOUBLE STRANDED
DNA MONOCLONAL ANTIBODY HAS BEEN
STUDIED CAREFULLY AND THIS MOUSE
DOUBLE-STRANDED DNA ANTIBODY
BINDS EXTRA CELLULAR DNA AND
THEN IT IS TRANSPORTED ACROSS
THE CELL MEMBRANE BY A PROTEIN
AND ACTIVELY TRANSPORTED IN AND
ONCE INSIDE THE CELL DOUBLE
STRANDED DNA ANTIBODIES CAN BIND
AND GET IN THE NUCLEUS, BIND THE
CHROMATIN WHERE THEY INHIBIT DNA
REPAIR.
SO THIS IDEA IN SOME OF THE
MECHANISMS FOR HOW ANTIBODIES
ENTER CELLS HAS BEEN WORKED OUT
FOR SOME OF THESE LUPUS
ANTIBODIES.
SO WHAT ARE THE THERAPEUTIC
IMPLICATIONS FOR THIS?
THE OBVIOUS ONE IS IF
AUTOANTIBODIES ARE PLAYING AN
IMPORTANT ROLE IN THE
PATHOGENESIS OF THESE AND
GETTING RID OF B CELLS SHOULD BE
HELPFUL FOR THERAPY.
CORTICOSTEROIDS REDUCE THE
LEVELS AND AND MYCOPHENOLATE AND
RET
RETUXOMAB CAN REDUCE IT IN THE
LYMPH NODES BUT THEY'RE WELL
ESTABLISHED TREATMENTS FOR
MYOSITIS.
WHAT'S REALLY EXCITING IS WHAT
WE HEARD ABOUT RECENTLY WITH
REGARD TO USING CAR THERAPY FOR
OTHER AUTOIMMUNE CELLS AND
THEY'RE THE PATIENT'S OWN CELLS
TO REPLICATE A CHIMERIC RECEPTOR
AND WHEN THESE ARE INFUSED AND
INFUSED BACK IN THE PATIENT THE
CAR-T CELLS BIND TO THE TARGET
AND THAT TRIGGERS THE T CELLS TO
KILL THE TARGET CELL.
SO THESE ACTIVATED CAR-T CELLS
ORIGINALLY DESIGN TO GET RID OF
B CELL LYMPHOMA CAN GET RID OF
NORMAL B CELLS THAT EXPRESS THE
TARGET.
THE CD-19 CAR-T CELLS HAVE BEEN
USED TO TREAT MALICE --
MALIGNANCIES INCLUDING MYOSITIS.
THIS IS ONE OF THE FIRST CASE
REPORTS OF A PATIENT AND ON THE
Y AXIS WE HAVE THE CK LEVEL AND
THEY GET TREATED AND THEN BOUNCE
BACK UP AND EVENTUALLY THE
PATIENT BECOMES REFRACTORY TO
MULTIPLE TREATMENTS AND TREATED
WITH CAR-T THERAPY AND CAUSES
THE PATIENT TO GO TO LONG-TERM
DRUG-FREE REMISSION.
THIS IS KIND OF THE PROMISE OR
THE HOPE FOR CD 19 AND OTHER
CELLULAR B CELL THERAPIES BEEN
NUMEROUS CASE REPORTS OF THOSE
CAR-T CELLS TO PUT PATIENTS WITH
DIFFERENT TYPES OF MYOSITIS INTO
DRUG-FREE WHAT APPEARS TO BE
DURABLE REMISSION.
I SHOWED YOU ONE WAY
AUTOANTIBODIES CAN ENTER LIVE
CELLS.
WE'RE INTERESTED IN FEATURES OF
ANTIBODIES THAT ALLOW THEM TO
ENTER LIVE CELLS.
IN MUSCLE AND SKIN TISSUE,
ANTIBODIES CAN GAIN ACCESS TO
THE NUCLEI FOR WE'D LIKE TO SHOW
WHICH WE HAVEN'T DONE AS WELL AS
WE'D LIKE THE RESULTS IN THE
DYSFUNCTION OF THOSE CELLS AND
CONTRIBUTE TO THE SIGNIFICANT
WAY TO THE PATHOGENESIS DISEASE
AND IN ADDITION TO LUPUS AND
MYOSITIS WE'RE INTERESTED IN
WHETHER AUTOANTIBODY
INTERNALIZATION PLAYS A ROLE OR
OCCURS IN PATIENTS WITH OTHER
TYPES OF AUTOIMMUNE DISEASES
CHARACTERIZED BY SCLERODERMA AS
ANOTHER RHEUMATIC DISEASE.
THE WORK WAS DONE BY A TEAM OF
PEOPLE.
I WANT TO ESPECIALLY HIGHLIGHT
THE ROLE AND WILL IAGO FERNANDEZ
SPEARHEAD THE WORK AND WE HAVE A
STAFF SCIENTIST.
AND WE HAVE A BIOLOGIST AND HAS
DONE A LOT OF WORK.
AND TALENT ED POST-BAC DOING WOK
ON AUTOANTIBODY INTERNALIZATION
AND THERE WERE PROGRAMS
INCLUDING STANFORD AND DUKE AND
UNIVERSITY OF MICHIGAN AND JOSÉ
MILISENDA WORKED IN OUR LAB AND
BACK IN BARCELONA AND CENTER IS
UM SAMPLES FROM THE PATIENTS
THAT'S MADE THE WORK POSSIBLE.
I'LL END MY PRESENTATION THERE
AND IF I LEFT MYSELF TIME I'D BE
HAPPY TO TAKE QUESTIONS.
IF NOW'S THE APPROPRIATE TIME TO
DO THAT.
>> NO, IT'S NOT.
>> OKAY.
>> WRONG AGAIN.
>> THANK YOU VERY MUCH.
FOR GIVING ME THE OPPORTUNITY TO
PRESENT TODAY.
EQUAL SWITCH TO GENETIC MUSCLE
DISEASE.
AND HOPEFULLY YOU'LL SEE AT THE
END OF THE PRESENTATION THERE IS
AN IMPORTANT INTERFACE AND THE
IMMUNOLOGY OF THE MUSCLES.
DISCLOSURES AND I'M THE P.I. ON
THE TRIALS I'LL DESCRIBE BRIEFLY
IN THIS PRESENTATION AND WANT TO
TALK TO A LOT OF GENE THERAPIES
AND PROVIDE SCIENTIFIC ADVICE
AND I HAVEN'T RECEIVED
COMPENSATION FOR ANYTHING IN
THIS SPACE.
I'LL START AT A HIGH LEVEL BY
INTRODUCING A SIM MYSTIC CONCEPT
WHICH IS THE PROMISE OF GENETIC
THERAPIES FOR GENETIC DISEASE.
THIS IS A SUPER SIMPLE DIAGRAM
OF THE FLOW OF GENETIC
INFORMATION FROM DNA TO
TRANSCRIPT TO PATIENT WHEN YOU
HAVE A MUTATION IT'S ENCODED IN
THE DNA AND TRANSCRIBED AND
CAUSES IT TO FORM THE PROTEIN IN
THE AFFECTED PATIENT.
THE PROMISE OF GENE THERAPY IS
SIMPLE.
THE CLOSER YOU GO TO THE GENETIC
ORIGIN OF THE DISEASE, THE
HIGHER YOU EXPECT THE
THERAPEUTIC IMPACT TO BE BECAUSE
YOU GO TO THE SOURCE OF IT.
THAT'S BEEN THE BASIS OF
DEVELOPING TECHNOLOGIES TO
DIRECT THERAPIES TO THE
GENE-DIRECTED THERAPIES
INCLUDING GENE REPLACEMENT I'LL
TALK ABOUT TODAY AND ALSO
UPCOMING GENOMIC EDITING AND
TRANSCRIPT DIRECTED THERAPIES
THAT'S MAINLY THE SPACE OF THE
NUCLEOTIDES TO MODULATE SPLICING
AND REGULATE GENES AND
TRANSCRIPTS.
AND THE BEAUTY OF THIS IF YOU
WILL IS THESE TOOLS ARE DISEASE
OG -- AGNOSTIC AT WHAT THE
MECHANISM MIGHT BE AND PERFECT
TO PROVIDE A GENETIC TOOLBOX TO
ADDRESS GENETIC DISORDERS AND
PART OF THE TOPIC OF TODAY'S
TALK AND ANOTHER INPET --
IMPETUS OF DEVELOPING THE
THERAPIES.
THE AV MEDIATED GENE REPLACEMENT
IS THE TOPIC OF WHAT I'LL TODAY
AND YOU PROVIDE A PIECE OF DNA
THAT ENCODES A GENE THAT MAY BE
MISSING AND THE DNA WILL BE
UPLOADED INTO THE NUCLEUS AND
DOES NOT INTEGRATE INTO THE
CHROMOSOMES AND HOPEFUL HAVE A
THERAPEUTIC EFFECT ON THE
PATIENT.
THE AND I'LL BE SPENDING A FEW
WORDS ON WHAT THAT IS AND WHAT'S
IMPORTANT TO SUCCEED IS YOU
TARGET WHERE YOU WANT TO GO AND
HAVE SUFFICIENT EXPRESSION OF
WHY YOU ARE DESIRED PRODUCT AND
THE EXPRESSION IS DURABLE AND
THERE'S NO TOXICITY TO THIS
APPROACH.
THIS LITTLE GUY HERE STARTS WITH
A SINGLE-STRANDED DNA VIRUS THAT
CANNOT EXIST ON ITS OWN.
IT NEEDS BIG GUYS LIKE TO
COMPLEMENT THE GENOME.
AND IT HAS HELPER GENES FROM THE
CO-EFFECTING VIRES TO BE ABLE TO
CAUSE THE INFECTION.
IT'S NON-PATHOGENIC.
IT COMES ALONG WITH THE RIDE FOR
BIGGER VIRUS ARE PATHOGENIC.
THAT'S A GREAT FEATURE TO THINK
ABOUT GENE THERAPY.
AND THE CONCEPT OF GENE THERAPY
IS TO USE THIS VIRUS TO DELIVER
THINGS TO CELLS.
IT'S ABLE TO USE DIVIDING AS
WELL AS NON-DIVIDING CELLS AND
GENE EXPRESSION AND WE SEE
LIMITATIONS TO THAT.
BECAUSE IT'S SO SMALL AND GENOME
IS SO SMALL, IF YOU USE IT TO
DELIVER OTHER THINGS, THE
PACKAGING CAPACITY IS LIMITED
COMPARED TO THE NATURAL CAPACITY
OF THE VIRUS AND THAT'S 4.5KG
RELEVANT IN THERAPEUTIC
APPLICATIONS.
IT'S USED IN CLINICAL
APPLICATIONS IN NATIONALLY
OCCURRING STEREOTYPES AND OTHERS
THAT WE'LL TOUCH ON.
THIS IS WHAT IT LOOKS LIKE.
AND IN GENE THERAPY YOU REPLACE
THE GENOME WITH WHAT YOU WANT TO
DELIVER AND THE ONLY THING LEFT
OVER FROM THE GENOME ARE THE
ITRs HERE AND THERE'S SOMETHING
YOU WANT TO DELIVER IN BETWEEN
AND PROMOTER AND TRANS GENE YOU
WANT TO EXPRESS AND GENETIC
ELEMEN
ELEMENTS.
THE WAY YOU DO THAT IS IN
TRANSFECTION AND THAT'S ONE
PLASMID AND THEY CONTAIN THE
ORIGINAL AAV GENES AND THE THIRD
PLASMID THAT IS PACKAGED AND NOW
BECAUSE OF THE FEATURE, THIS IS
WHAT ENDS UP IN THIS CAPSID.
SO NOW YOU HAVE A CAPSID THAT
LOOKS FROM THE OUTSIDE LIKE A
VIRUS AND WE HAVE REPLICATION
AND IT'S A ONE-TIME INFECTION
THAT DELIVERS TO THE CELLS.
AND THAT LOOKS LIKE THIS BINDS
TO A SURFACE AND TAKEN UP BY THE
CELL AND BROUGHT TO THE NUCLEUS
AND ENCODES IN THE NUCLEUS AND
THEN YOU HAVE A SINGLE STRANDED
GENE AND IT'S DOUBLE-STRANDED.
YOU MAKE IT AS A DOUBLE-STRANDED
CONSTRUCT AND WE CALL IT
COMPLEMENTARY.
BUT THEN YOU ONLY HAVE HALF THE
LOADING CAPACITY OF THE ORIGINAL
SIN
SINGLE-STRANDED ONE.
IT LIVES IN THE NUCLEUS THAT DO
NOT FOR THE MOST PART INTEGRATE
IN THE HOST GENOME.
A LITTLE BIT OF THAT HAPPENS BUT
MOST DOES NOT.
WHEN THE CELLS DIVIDES THEY
DON'T GET REPLICATED.
THE HEAVILY DIVIDING CELL GETS
LOST.
LET'S SWITCH GEARS AND TALK
ABOUT MUSCLE DISEASE.
LET'S START WITH MUSCULAR
DYSTROPHY AND MYOPATHIES.
IT'S DEGENERATIVE DISORDER OF
MUSCLE.
IT'S A GROUP OF DISORDERS WHERE
THE MUSCLE UNDER GOES
DEGENERATION FOR VARIOUS REASONS
THAT WE'LL TOUCH UPON AND
ATTEMP
ATTEMPTS AS LONG AS IT CAN AND
AT THE END REGENERATION IS NO
LONGER SFRNT.
THERE'S A NORMAL MUSCLE BYPASS
AND SEE THE MYOFIBERS AND YOU
SEE THE PERIPHERY OF THE FIBER.
YOU GET DEGENERATIVE FEATURES
AND MACROPHAGES MOVING AND
INFLAMMATORY RESPONSE AND IT
TRIES TO REGENERATE AND GETS
REPLACED BY CONNECTIVE TISSUE
AND LOOKS SOMETHING LIKE THIS
WHICH IS REALLY TOUGH TO
ENVISION TO REPAIR.
YOU CAN SEE THAT IN THE MRI THE
DARK MUSCLE IS THIS IS ADVANCED
MUSCULAR DYSTROPHY AND IT'S
TRANSFORMED AND JUST ANOTHER
VIEW OF THIS AND NORMAL LOOKING
MUSCLE AND TWO BIOPSIES YOU SEE
THE PROCESS AND THE MUSCLE
TIGERS DEGENERATE BEING
FIBROC
FIBROCYTOSED.
AND THAT IS THE CYCLE AND
INCREASING FIBROSIS IN THE
MUSCLE.
THE MUSCULAR DYSTROPHY IS
DIVERSE.
MANY THINGS CAN CAUSE THE
DEGENERATIVE MUSCLE DISEASE AND
THERE'S DISTRIBUTION OF WEAKNESS
OF A SYSTEM INVOLVED.
WE'LL TOUCH ON TWO AND FOCUS ON
THE MOST COMMON, DUCHENNE
MUSCULAR DYSTROPHY.
THIS IS THE FOCAL POINT WHICH IS
THE INTERFACE BETWEEN THE MUSCLE
FIBER AND THE EXTRA CELLULAR
PLAY TRIX AND IT'S DISASSOCIATED
AND I'LL HIGHLIGHT THIS HERE AND
THIS IS THE PLASMA MEMBRANE.
THIS IS THE EXTRA CELLULAR
MATRIX AND THIS IS WITHIN THE
MUSCLE FIBER AND THERE'S A
PROTEIN CALLED DYSTROPHY THAT
LINKS TO A COMPLEX OF PROTEINS
ACROSS THE PLASMA MEMBRANE THAT
FORM A RECEPTOR FOR THE EXTRA
CELLULAR MATRIX AND THE
CHROMATIN TYPE 6 AND THIS IS
LINKED AND MUSCLE BEING A
CONTRACTILE TISSUE THE BOLD
BETWEEN THE INTRACELLULAR AND
EXTRA CELLULAR WERE IMPORTANT
FOR THE TISSUE.
IT'S INTERESTING IF YOU HAVE
MUTATIONS UP HERE IN THE EXTRA
CELLULAR MATRIX YOU GET
CONGENITAL MUSCULAR DYSTROPHY
AND AT BIRTH AFFECTING THE
COLLAGEN AND GLYCAN HERE.
YOU CAN SEE HOW STRIKING IT CAN
BE.
THIS IS THE RECEPTOR AND NOT
ONLY MUSCLE BUT BRAIN
DEVELOPMENT INVOLVEMENT.
IT'S A DEFICIENCY WITH STRIKING
WEAKNESS AT BIRTH AND
DESTRUCTIVE LOOKING MUSCLE.
THESE ARE CHILDREN WITH COLLAGEN
TYPE 6 DISORDERS WHERE THERE'S A
PROMINENT CONNECTIVE TISSUE
COMPONENT AS WELL.
ALL AT PRESENT HAVE EARLY ONSET
MUSCULAR DYSTROPHIES.
THE COMPLEX CAUSES DYSTROPHIES
AND THAT'S WHERE MUTATIONS CAUSE
DUCHENNE MUSCULAR DYSTROPHY AND
THE MOST COMMON AFBT BOYS AND
THERE'S STRIKING IMAGES HERE AND
YOU CAN SEE A BOY WHO LOOKS LIKE
HE HAS BIG MUSCLES BUT IT'S WHAT
WE CALL MUSCLE ALREADY REPLACED
BY CONNECTIVE TISSUE GIVEN THE
APPEARANCE OF HYPERTROPHY OF THE
MUSCLE A CHARACTERISTIC FEATURE
OF THIS DISEASE.
IT'S QUITE COMMON AS YOU CAN
SEE.
1 IN 3,500 TO 5,000 MALE BIRTHS.
IT LEARNINGS TO THE COMPLEX OF
GLYCOPROTEINS AND THIS IS ONE
THAT WAS IDENTIFIED AT THE TIME.
THIS IS A YOUNG BOY WITH
DUCHENNE MUSCULAR DYSTROPHY AND
LATER IN LIFE HERE HE'S 10 OR
11.
HE'S WALKING BUT NOT ABLE TO GET
UP FROM THE FLOOR AND THREE OR
FOUR MONTHS LATER HE LOSES
AMBULATION AND THERE'S A PHASE
OF WHEELCHAIR USE AND LOSS OF
AMBULATION AND THE MYOPATHY AND
SCOLIOSIS.
THIS HAS TO BE MANAGED TO AVOID
THE SITUATION WHERE YOU HAVE
UNTREATED SCOLIOSIS IN THIS BOY
AND WHAT CAUSES THE DISEASE IS
MOSTLY DELETIONS IN THE LARGE
GENOMIC LOCUS.
IT'S PRONE TO DELETIONS IN THE
GENE AND THEY'RE TO THE MISTAKE
COMMON THESE ARE 75 EXONS AND
HOW THEY BOUNDARY IN THE
TRANSCRIPT.
THEY DEFINE WHERE IN THE READING
FRAME AN EXON WOULD START AND
THE NEXT WOULD STOP.
THERE'S A DELETION AND THE
FRAMEWORK IS PRESERVED BECAUSE
BUT IF YOU HAVE IT HERE THEN
IT'S INTERRUPTED.
AND THEY CLONED THE GENE A BOY
HAD A DELETION LARGE ENOUGH TO
BE SEEN AND THEY PUT THIS NEXT
TO THE OTHER IN PAINSTAKING WORK
AND THIS IS ORIGINAL SLIDE AND
SAW THE DELETIONS HAVE THE
PROTEIN TRUNCATED AND ABSENT.
AND THEY HAD MODULATION LONGER.
THE OXIDATION SLIDE MOVED THE
THERAPEUTICS FIELD YOU HAVE THE
PROTEIN IN THE SCHEMATIC.
THIS WAS A FAMILY OF PATIENTS
OBSERVED AND 45% OF THE PROTEIN
WAS
WASS
WASSING AND THEY -- AND THEY
ALLOWED FOR A LONGER FUNCTION
THAN YOU WOULD IN THE DUCHENNE
MUSCULAR DYSTROPHY.
S AND THERE'S WORK THAT ARE
INTERNALLY DELETED AND MUCH OF
THE REPEAT DOMAIN STRUCTURE IS
DELETED BUT THE BUSINESS ENDS
ARE PRESERVED.
THESE ARE IN CLINICAL TRIAL OR
APPROVED AND THEY WERE DESIGN
THIS WAY.
S AND THERE'S VARIOUS GENOTYPES.
S AND THIS WAS A CLINICAL TRIAL
AND GO THE WORK AND GO RIGHT TO
THE PHASE III TRIALS AND A MIXED
PICTURE.
FOR THE PFIZER OUTCOME SHOWN
HERE, THIS WAS UNAFFECTED.
IN A PHASE III TRIAL AT THE END
USING AN ASSESSMENT CALLED THE
NORTH STAR AMBULATORY ASSESSMENT
WAS NO DIFFERENCE BETWEEN THE
PLACEBO GROUP AND TREATMENT
GROUP.
ANOTHER CONSTRUCT USED THINGS
LOOKED A LITTLE BIT MORE
PROMISING AND THE PRIMARY END
POINT WAS NOT MET HERE BUT
SECONDARY END POINT IN THE
COMPOSITE ANALYSIS DID REACH A
SIGNIFICANT WHEN THEY SAW YOUNG
PATIENTS TREATED WITH THIS HAD
BETTER SCORES AND THE EXPRESSION
HAD BETTER OUTCOMES AND HERE IN
THE BLOT AS WELL BEING
EXPRESSED.
THAT LED TO APPROVAL OF THIS IN
THE UNITED STATES AS GENE
THERAPY FOR DUCHENNE MUSCULAR
DYSTROPHY.
THE TWO OBSERVATIONS WERE SHOWN
AND YOU CAN SEE HERE AFTER TWO
YEARS THE SHIFT OF BENEFICIAL
CHANGES IS MAINTAINED IN THE
MEASURES IT'S NO LONGER PLACEBO
CONTROLLED.
IT'S OPEN LABEL BUT STILL
SHOWING SOME BENEFIT SHIFTING
DISEASE.
ONE REASON IS THIS IS NOT IN THE
GENOME OF THE PATIENT GIVEN
LATER IN THE PROGRESSION OF THE
DISEASE.
THESE BOYS WERE TREATED BETWEEN
5 AND 7 YEARS OF AGE.
IT'S INCOMPLETE RESTORATION
COMPARED TO A GENOTYPE YOU'RE
BORN WITH AND THERE'S A
DIFFERENCE AND HAVING IT IS
BETTER AND THAT'S UNDER
DEVELOPMENT KNOW AS WELL.
WORK BY JEFF CHAMBERLAIN WHERE
HE GIVES FRAGMENTS AND ABLE TO
TRANS SPLICE ON TO THE PROTEIN
LEVEL TO FORM FULL LENGTH
DYSTROPHY AND IN THE PRECLINICAL
DEVELOPMENT AT THE MOMENT.
LET'S SWITCH GEARS TO CONGENITAL
MYOPATHIES.
THEY'RE LARGELY NON-DYSTROPIC.
HERE'S THE PATHOPHYSIOLOGY IS
PLAYING OUT MOSTLY IN THE
INTRACELLULAR COMPARTMENT BUT
ALSO WHERE EXCITATION
CONTRACTION HAPPENS DEEP IN THE
MUSCLE FIBERS.
THEY'RE CHARACTERIZED BY THE
PATHOLOGY AND YOU CAN SEE EARLY
ONSET WEAKNESS AND STABILITY
OVER THE YEARS HOWEVER, ARE MUCH
MORE SEVERE AT BIRTH BUT STILL
WITH STABILITY.
THE DISEASE NAME ARRIVES FROM
THE HISTOLOGICAL APPEARANCE AND
THAT'S CHARACTERISTIC.
THIS I
THIS IS A PROTO TYPICAL
APPEARANCE AND THIS IS MORE
STABLE HISTOLOGICALLY OVER THE
YEARS.
THESE ARE THE CORE MYOPATHY.
AND THIS IS THE ONE I'M GOING TO
FOCUS ON NOW BECAUSE OF GENE
THERAPY DEVELOPED.
THE MYOPATHY IS THE MOST SEVERE
IN THAT IT'S AN EXCELLENT
DISEASE.
BOYS ARE SEVERELY WEAK FROM
BIRTH AND THEY NEVER ---IBLE AND
THEY ARE DO NOT WALK BUT THEY'RE
DEPENDENT ON SUPPORT FOR THE
LIFE TIME.
A LOT OF THEM, 50% DO NOT
SURVIVE PAST 18 MONTHS BECAUSE
OF COMPLICATIONS OF THE
RESPIRATORY INSUFFICIENCY.
AND HERE THE GENE PRODUCT IS A
PHOSPHOTASE AN ENZYMATIC
ACTIVITY INVOLVED IN MARKERS.
THEY'RE MARKERS OF MEMBRANES
THAT ALLOW MEMBRANES TO BE
SORTED AND HAVE STRUCTURES SUCH
AS THE CYTOPLASTIC RETICULUM AND
GIVE THE NAME TO THE DISEASE.
HERE GENE THERAPY WAS DEVELOPED
AND THE PROMOTER THAT IS CALLED
AAV8 AND THIS WAS TRIED AND THE
NATURAL HISTORY OF THE DISEASE.
YOU CAN SEE A FEW IN THE NATURAL
HISTORY AND THOSE WICK THE LOWER
DOZE ARE VERY DIFFERENT.
THEY GOT TO STANDING OR WALKING
IN THE YOUNG GROUP AND THE
SUPPORT DROPPED OFF AND GOT OFF
THE RESPIRATORY SUPPORT IN THAT
TRIAL.
THE TRIAL IS ON HOLD BECAUSE OF
LIVER TOXICITY THAT DEVELOPED
AND I'LL COME BACK TO THAT IN A
SECOND.
THERE'S A CLEAR EFFECT ON THE
HISTOLOGY OF THE BIOPSIES.
THESE ARE BIOPSIES TAKEN AT
BASELINE AND THEN 48 WEEKS
LATER.
AND YOU CAN SEE THAT A NUMBER OF
THE FEATURES NORMALIZED WITH THE
THERAPY.
IN PARTICULAR THE APPEARANCE AND
THE MUSCLE FIBERS GREW.
THE NUCLEAR PLACEMENT DID NOT
CHANGE.
IT REMAINS IN THE MIDDLE OF THE
FIBER.
THERE WAS CLEARLY A HISTOLOGICAL
RESPONSE AS WELL.
THE MUSCLE IS A LARGE ORGAN.
40% OF BODY MASS IS DEPENDENT ON
THE BODY MASS INDEX AND FITNESS
AND SO ON BUT IT IS A LARGE
ORGAN.
CRUCIAL MUSCLES AND THE AXON
MUSCLES FOR PREVENTING SCOLIOSIS
AND SOME MUSCULAR DYSTROPHIES
AND ORGANS MAY BE AFFECTED AND
NEED TO BE CONSIDERED.
AND THE OTHER IS OF COURSE THE
MUSCLE IS NOT JUST ONE BUT
CONTAINED DIFFERENT TYPES THAT
MAY BE OF IMPORTANCE.
THERE'S THE MYONUCLEAR AND
SATELLITE CELLS MAY BE IMPORTANT
IN THE THERAPY AS IMMUNE CELL
PRESENT THERE'S IMPORTANT CELLS
FOR THE COLLAGEN 6 DYSTROPHY AND
TARGET THE CELLS THAT DRIVE THE
DISEASE EFFECTIVELY.
AND SOME CELLS ARE DIVIDING OR
TURNING OVER AND YOU HAVE AN AAV
CORRECTION YOU WOULD LOSE IT ALL
THE TIME.
IN ADDITION, ONE MUSCLE FIBER IS
A NUCLEATED STRUCTURE.
WHEN WE THINK OF GENE THERAPY
CORRECTING THESE, IF YOU THINK
OF MUSCULAR DYSTROPHY AND YOU
HAVE THIS HERE BUT THEN YOU HAVE
GAPS, THIS IS A POINT OF OUR
ABILITY TO CONSIDER.
ONE IS THE BRIGHT WINDOW.
IS IT EARLY STAGES OF THE
DISEASE.
WHAT CAN YOU ESTABLISH AND WHAT
IT IS THE OPPORTUNITY WINDOW OF
TREATMENT.
THAT MUST DEPEND ON THE NATURE
OF THE DISEASE.
AND IT DECLINED OVER TIME AND
YOU CAN ADDRESS THE GENE THERAPY
AND DEGENERATIVE DISEASE
PROGRESSES IF YOU HAVE A MORE
STABLE DISEASE WITH LESS
DEGENERATION YOUR OPPORTUNITY IS
LONGER.
IN DUCHENNE DISEASE THE MUSCLE
BIOPSY SHOWS THE DEGENERATIVE
NATURE OF THE DISEASE.
YOU'RE DOSING INTO SOMETHING
THAT IS ALREADY ACTIVE AND
DEGENERATIVE.
IT'S DIVIDING AND ACTIVE COULD
BE LOST OVER TIME IS DIFFERENT
FROM GENE THERAPY IN A TISSUE
THAT IS POST-MYTOTIC AND UNIQUE
TO MUSCLE DISEASE.
THERE WILL BE MUSCLE COMBROEJ
DRIVEN BY SATELLITE CELLS TO
CREATE ADDITIONAL NUCLEI TO
CREATE THIS.
YOU CAN IMAGINE IF A GENE
THERAPY IS GIVEN EARLY ADD TO
THAT THE MUSCULAR DYSTROPHY AND
YOU COULD ENVISION THERE'S A
DURABILITY PROBLEM WITH THIS
APPROACH.
AND WE'VE HAD A MYOTROPIC AAD
AND BETTER AT THE SATURATION OF
THE TARGET TISSUE IS EXPECTED TO
BE BETTER.
THEY ARE BIND TO THE MOTIVE
HERE.
THESE ARE THE COMMON
DETERMINATES AND THE TISSUE OF
THE FIBERS TIMES AND DURABILITY
OF EXPRESSION.
ONE IS SUBSTRATE YOU'RE TRYING
TO RESCUE HAS TO BE IN A STATE
THAT STILL ALLOWS FOR
REVERSIBILITY.
I WANT TO RAISE THE IMMUNOLOGY
IN THE CONTEXT OF MUSCLE DISEASE
AND IT'S SOMETHING WE HAVE TO
PHASE.
HERE ARE THE IMMUNOLOGICAL
EVENTS THAT HAPPEN BEFORE AND
AFTER GENE THERAPY.
AAV IS A NATURAL OCCURRING VIRUS
AND AS THE A PERCENTAGE OF
PEOPLE ALREADY HAVE ANTIBODIES
AND AFTER TRANSFER THERE'S
INMATE IMMUNITY.
IT REACT TO THE HIGH VIRUS LOAD
AND THERE'S IMMUNITY WHERE IT
HAS AN ADAPTIVE IMMUNE RESPONSE
AGAINST THE CAPSID BUT THEN ALSO
AS A TRANS GENE THAT'S EXPRESSED
AGAINST THE TRANS GENE ITSELF.
THERE'S DISEASE FACTORS AND
STAGES BECAUSE OF THE INDIVIDUAL
ENVIRONMENT OF THE DISEASE AND
THE HOST.
THESE SHOW THE DIFFERENT PHASES
OF IMMUNE REACTION.
I'M GOING TO HIGHLIGHT A FEW.
ONE IS INNATE IMMUNE ACTIVATION.
THERE'S AN IMMEDIATE REACTION TO
THE HIGH LOWS OF VIRUSES AND
THAT'S SOMETHING THAT CAN HELP
EVEN WITH INFECTION.
IT'S AN ACTIVATION OF THE
COMPLEMENT SYSTEM THAT THEN GOES
OUT OF CONTROL AND CAUSES THE
DAMAGES AND CYTO PENA AND
HEMOLYSIS AND ORGAN DAMAGE.
THERE'S HIGH DOZE TRIALS
PARTICULARLY THOSE USING AAV9
SEEMS TO BE A PARTICULAR
VIABILITY OF THAT CAPSID.
THIS SEEN IN THE EARLY TRIALS
AND CYTOTOXIC TRIALS WERE
ATTACKING THE LIVER TRYING TO
MAKE THE PROTEIN FACTOR AND THAT
LED TO DECREASING EFFICACY AND
IN THE TRIAL WHERE THE LIVER IS
A BYSTANDER IF YOU WILL.
SOMETIMES DRAMATICALLY SO AND
CONTROLLED BY IMMUNE MODULATION
SUCH AS STEROIDS.
THERE'S ONE SPECIFIC LIVER
TOXICITY THAT BRINGS THE TRIAL
AND M1 TRIAL AND FOUR PATIENTS
IN THAT TRIAL DIED OF SEVERE
LIVER FAILURE.
DIFFERENT FROM THE TOXIC EVENT
IN THE OTHER HEMOPHILIA AND
DUCHENNE TRIALS AND A DIFFERENT
TYPE THAT HAPPENED IN THE BOYS.
THAT LED TO THE RECOGNITION THIS
NOT A PURE DISEASE BUT A CHO
CHOLESTATIC LIVER DISEASE AND
AND HAS PROPENSITY TO
CHOLESTASIS AND THEY'RE
SOMETIMES PART OF VIRAL
INFECTIONS.
THEY WERE ABLE TO SHOW TUBULAR
MYOPATHY HAS A CHOLESTATIC
DELIVER PHENOTYPE DUE TO DEFECT
IN MEMBRANE TRAFFIC IKING WITH E
TRANSPORTER NOT IN PLACE AND IT
GETS OVERLOADED AND THE SYSTEM
GETS STRESSED TO A POINT IT GOES
INTO CHOLESTATIC FAILURE.
THE GENE PRODUCT IS UNDER A
PROMOT
PROMOTER THAT LEAVES IT TO THIS
PARTICULAR COMBINATION OF THE
VIRAL LOAD AND THE DISEASE
SPECIFIC SUSCEPTIBILITY.
WE CAN DISCUSS POSSIBLE
SOLUTIONS TO THIS PROBLEM IN THE
DISCUSSION PHASE.
LASTLY I WANT TO MENTION GENE
THERAPY IS A TRANSPLANTATION OF
A PROTEIN INTO A HOST WHO MAY
NOT HAVE SEEN THIS PEPTIDE EVER
AND REACTING WITH ANTI-TRANS
IMMUNITY.
IT COMES BACK TO ANDY'S TALK AND
WHEN YOU TRANSFECT THE MUSCLE
GIVING IT A NEW PROTEIN TO
EXPRESS IT WILL BE PRESENTED TO
THE IMMUNE SYSTEM.
WHEN THE IMMUNE SYSTEM IT WILL
TAKE NOTICE.
IN THE DUCHENNE MUSCULAR
DYSTROPHY TRIALS IT WAS CLEAR
SOME PATIENTS DEVELOPED ALL OF A
SUDDEN A VERY SEVERE MYOSITIS
AND EVEN MYOCARDITIS.
THERE WAS A GROUP OF AND COME
TOGETHER VERY RAPIDLY AND TRY TO
FIGURE OUT WHAT WAS GOING ON.
ALL THAT'S GOING ON IS THE
PATIENTS HAD GENOMIC DELETIONS
AS PART OF THEIR GENOTYPE FOR
THE MUSCULAR DYSTROPHY AND
OVERLAPPED THE PEPTIDE SEQUENCE
DELIVERED BY THE TRANS GENES.
IN THESE PARTS ARE PATIENTS
WHERE THEY'RE IMMUNE NAIVE.
THEY HAVEN'T SEEN THE EPITOPES
AND THE IMMUNE SYSTEM WAS TRYING
TO REJECT IT.
AND THIS WAS HIGHLY EXPOSED AND
MORE RECENT CASE WITH A DELETION
WAS ENOUGH TO CAUSE SEVERE
MYOSITIS AND THEY WERE ABLE TO
SHOW THIS WAS RECOGNIZED BY CYTO
TOXIC T CELLS AND THAT ALLOWS US
TO UNDERSTAND DEVELOP RISK
PROFILES AND I CAN'T EMPHASIZE
THE SHARING AROUND THIS IS KEY
TO RECOGNIZE THIS AND DEVELOP
MITIGATING STRATEGIES.
HERE THE LIVER COMES TO THE
RESCUE AS A BY-STANDER AND
INDUCING ORGAN NOW INVOLVED IN
THE RESEARCH AND WE NEED TO
UNDERSTAND THE RISKS BETTER.
I'LL LEAVE YOU WITH THESE
THOUGHTS AND THANK THE
COLLABORATORS AND MY TEAM AND
THOSE INSTRUCTIONAL IN THE
MUSCULAR DYSTROPHY TRIAL AND
MOVE ON TO QUESTIONS.
THANK YOU VERY MUCH.
>> ALL RIGHT.
THANK YOU VERY MUCH FOR THAT
REALLY ANFANTASTIC SET OF
PRESENTATIONS.
IT'S AMAZING.
IT'S A TEST -- TESTIMONY OF THE
HARD WORK INTO THIS AND HOW
IMPORTANT MUSCLE IS AT ALL
STAGES OF LIFE AND DOES MAKE UP
A MAJOR PART OF OUR BODIES AND
PHYSIOLOGY.
WE HAVE A GREAT GROUP OF
QUESTIONERS IN THE AUDIENCE WITH
INTERESTING THOUGHTS AND
PROBABLY ONE OF THE MOST
IMPORTANT THINGS WE CAN TRY TO
DO IS TIE TOGETHER THE THOUGHTS
OF THESE TWO PRESENTATIONS.
ONE LINE OF QUESTIONS HAS TO DO
WITH WHERE THE AUTOBEYOANTIBOY
COMING FROM AND WHAT'S
STIMULATING THEM AND THEN
CONSIDER THE ISSUES WITH REGARD
TO THE DYSTROPHIES AND
MYOPATHIES.
I'LL PUT THE GOOD DR. MAMMEN IN
THE HOT SEAT.
ONE POSSIBLE SOURCE, AND WHAT IS
STIMULATING THEM IS THE
QUESTION?
>> THAT'S A HOLY GRAIL TO TRY TO
UNDERSTAND THAT.
WE KNEW A COUPLE THINGS.
ONE IS NOT EVERYBODY HAS THE
SAME RISK FOR DEVELOPING A
PARTICULAR TYPE OF AUTOIMMUNITY.
THERE'S IMMUNOGENETIC RISK
FACTORSES THAT PREDISPOSE
PATIENT TO DEVELOPING DISEASE.
MANY SHARE THE RISK FACTORS BUT
ONLY A RELATIVELY SMALL SUBSET
WILL DEVELOP AUTOIMMUNITY.
WE THINK THERE'S ENVIRONMENTAL
TRIGGERS.
AN ENVIRONMENT TRIGGER OR
TRIGGERS THAT OCCUR TO A PATIENT
WHO HAS THE 2346R9 -- DEVELOP
THE RISK FACTORS AND I'LL GIVE
AN EXAMPLE OF WHAT IS PROBABLY
THE BEST UNDERSTOOD ONE.
SOME WHO DEVELOP NECROTIZING
MYOSITIS AND HAVE THE
PHARMACOLOGIC TARGET OF STATINS.
THERE'S A POTENT IMMUNOGENETIC
RISK FACTOR FOR DEVELOPING THIS
FORM AND ABOUT 70% OF PATIENTS
WITH THESE ANTIBODIES HAVE THE
CLASS 2HLA ALLELE WHERE WE ONLY
FOUND IT IN 15% OF THE HEALTHY
POPULATIONS.
IT'S ONE OF THE STRONGEST
IMMUNOGENETIC RISK FACTORS FOR
AN AUTOIMMUNE DISEASE.
AND WE ALSO KNOW WHAT THE
ENVIRONMENTAL RISK FACTOR IS AND
IT'S EXPOSURE TO STATIN
MEDICATIONS.
AS I'M SURE THE AUDIENCE KNOW,
THEY LOWER CHOLESTEROL LEVELS BY
INHIBITING THE FUNCTION SO
THERE'S LESS BILE SYNTHESIS AND
THERE'S A DIRECT LINK BETWEEN
THE TARGET OF THE IMMUNE
RESPONSE AND THE THING
PREDISPOSING IT.
WHAT WE HYPOTHESIZE BUT DON'T
KNOW FOR SURE IS THAT BINDING OF
THE STATIN TO HMGCR MAY GENERATE
NOVEL EPITOPES AND YOU
SUBSEQUENTLY DEVELOP
AUTOIMMUNITY AGAINST THESE
SLIGHTLY DIFFERENT FRAGMENTS OF
HMGCR THE SYSTEM HASN'T SEEN
BEFORE AND THAT ANTIBODY
RESPONSE SPREAD TO ALSO
RECOGNIZE THE NATIVE WILD TYPE
PROTEINS.
SO THAT'S AN EXAMPLE WHERE WHERE
TWO THINGS HAVE TO COME
TOGETHER.
THE IMMUNOGENETIC RISK FACTOR
AND THE STATIN EXPOSURE.
BUT IT'S NOT THAT SIMPLE BECAUSE
THE MAJORITY OF PATIENTS WHO
HAVE BOTH THE IMMUNOGENETIC RISK
FACTOR AND EXPOSURE TO STATINS
NEVER DEVELOP THAT AUTOIMMUNITY
SO THERE HAVE BEEN ADDITIONAL
FACTORS OR ENVIRONMENTAL FACTORS
WE DON'T KNOW.
TO THROW IN A TIDBIT, THE RISK
FOR DEVELOPING THE DISEASE
VARIES WIDELY ACROSS ETHNIC
GROUPS.
IN THE UNITED STATES AMONG BLACK
AND WHITE AMERICANS 1 IN 30,000
PATIENTS DEVELOP WITH A STATIN
WILL DEVELOP THIS AND AMONG THE
NAVAJO IN NEW MEXICO IS 1 IN
300.
THEY HAVE ANOTHER FACTOR I
PRESUME IT'S GENETIC AND THAT'S
MY LONG WINDED EXAMPLE.
IT'S NOT JUST ONE THING.
MULTIPLE THINGS HAVE TO BE WITH
THE RIGHT IMMUNOGENETICS AND THE
RIGHT ENVIRONMENTAL EXPOSURES TO
DEVELOP IT AND ONE FURTHER THING
ABOUT THE CLASS 2HLA ALLELE WE
THINK IT'S PRE DISPOSING BECAUSE
THOSE ARE VERY INVOLVED IN
PRESENT
PRESENTING ANTIGENS LEADING TO
THE DEVELOPMENT OF ANTIBODIES.
FOR MOST FORMS OF AUTOIMMUNITIES
AND ANTIBODIES WE DON'T
UNDERSTAND IT NEARLY AS WELL.
>> THAT'S VERY HELPFUL AND
THAT'S A GREAT EXAMPLE IN TERMS
OF AN EXOGENOUS ANTIGEN IN A
MEDICATION ORDINARILY AN
INDIVIDUAL WOULDN'T ENCOUNTER IN
NATURAL LIFE THAT MIGHT THEN
CREATE THIS KIND OF SITUATION IN
WHICH THE IMMUNE SYSTEM GETS
SORT OF HOOKWINKED --
HOOKWINKED AND BEFORE I BRING IT
OVER, WHAT ABOUT RATHER THAN IT
BEING A MEDICATION IT BE AN
INFECTIOUS AGENT.
COULD YOU LOOK AT THE
SPECIFICITY OF THE ANTIBODIES.
THERE'S ANTIBODIES AGAINST THE
INTRACELLULAR MOLECULES.
USING SEQ COULD YOU DEDUCE THE
SMALL PIECE OF ANTIGEN AND THEN
TRY TO LOOK AT THE DATABASE OF
PATHOGENIC SEQUENCES TO DEDUCE
WHAT MIGHT IT BE THAT MAY BE
STIMULATING THAT IF YOU WANT TO
BUY INTO THAT MODEL?
>> THAT INSTEAD OF EXPRESSING
THE WHOLE HUMAN PROTEOME YOU
EXPRESS THE VIRUSES AND THE
IMMUNE RESPONSE AND LOOK TO SEE
IF PATIENTS WITH THE AUTOIMMUNE
DISEASES HAVE BEEN EXPOSED TO A
VIRUS IN GREATER FLRMZ.
SOME VIRUSES MAY HAVE SOME ROLE
BUT IT'S NOT A SLAM DUNK.
MAYBE THE BEST MODEL IS GILLAN
BU
BARRE -- GUILLAIN-BARRE.
I'M SPACING ON THE NAME.
THERE'S EXAMPLES OF THAT AND
IT'S A MOLECULAR MIMICRY.
WE SO FAR HAVEN'T NAILED THAT
DOWN FOR MOST THE RHEUMATIC
DISEASES.
>> TO TRY TO TAKE IT OVER THEN,
YOU MIGHT THINK PATIENTS WITH
SOME OF THE DYSTROPHIES OR MY N
-- MYOPATHIES AND LIKE THE ONE
ANDY TALKS ABOUT HIS PATIENTS
GET ANTIBODIES AGAINST, IS THERE
EVIDENCE THOSE PATIENTS OR HAS
ANYBODY LOOKED TO SEE WHETHER
THOSE PATIENTS GET
AUTOANTIBODIES AGAINST PROTEINS
THAT ARE INSIDE MUSCLE CELLS?
>> WE DID LOOK AND IT'S NOT SO
STRAIGHTFORWARD.
WE LOOKED AT GENETIC DISEASE OF
HMGCR.
THE GENETIC MUTATION OCCURRED IN
THE PROTEIN AND WE LOOKED TO SEE
IF THEY'D BE PART OF THE DISEASE
DRIVER.
THAT'S NOT THE CASE.
THERE'S BIG INCLUSIONS OF THE
PROTEIN AND THERE'S ANTIBODIES
KNOWN AGAINST THE PROTEIN TO SEE
IF IT WOULD TRIGGER AN
ADDITIONAL ANTIBODY RESPONSE.
IT'S KNOWN ON THE NERVE SIDE
IT'S KNOWN SOME OF THE FORMS ITS
LOOKS LIKE THEY HAVE A HIGHER
PROPENSITY OF DEVELOPING
AUTOINFLAMMATORY RESPONSE CALLED
CIDP.
THE QUESTION IS THAT AN EXAMPLE
WHERE WHAT YOU DESCRIBED IS
HAPPENING.
AND IT'S PRESENTED TO THE
AUTOIMMUNE SYSTEM AND THE
PROBLEM WITH GENE THERAPY IN
DYSTROPHY IS THE DEGENERATIVE
DISEASE CAUSES ITS OWN
ENVIRONMENT.
THERE'S CELLULAR INFILTRATION
AND INFLAMMATION HAPPENING AND
THAT CAUSES THE HYPER
INFLAMMATORY ENVIRONMENT AND IS
THE POTENTIAL EXAMPLE OF THAT
ONE A PATIENT MORE ADVANCED IN
THE MUSCULAR DYSTROPHY TREATMENT
AND IT WAS PUBLISHED IN THE NEW
ENGLAND JOURNAL OF MEDICINE.
THAT PATIENT HAD UNFORTUNATELY A
FATAL RESPONSE TO THE GENE
THERAPY WHERE HE GOT INTO A
HYPER INFLAMMATORY STATE AND
CALLED MULTI-ORGAN DAMAGE THAT
PROBABLY WAS A COMBINATION OF
THE DISEASE STAGE AND THE
ADDITIONAL STRESS FROM THE HIGH
DOSE OF THE AAV GOING INTO HIS
BODY.
SO THERE IS THAT INTERPLAY
BETWEEN HEIGHTENED IMMUNITY FROM
THE UNDERLYING DISEASE AND THE
ADDITIONAL CHALLENGE,
THERAPEUTIC CHALLENGE OF THE
GENE THERAPY THAT CAN OCCUR.
>> TO EXPAND ON THAT A LITTLE
BIT, WE HAVE SEEN SEVERAL
PATIENTS WHO HAVE BOTH PRETTY
WELL DEFINED AUTOIMMUNE MYOSITIS
AND ALSO A KNOWN GENETIC
DISEASE.
I HAVE A PATIENT WHO HAS BOTH
ANTI-MI2 POSITIVE
DERMATOMYOSITIS AND MUSCULAR
DYSTROPHY AND ONE HAS A
DYSTROPHY WHO HAS THE
ANTISYNTHETASE SYNDROME AND
THERE'S OTHER EXAMPLES IN THE
LITERATURE OF PEOPLE HAVING
BOTH.
I DON'T THINK WE'VE DONE A GOOD
JOB OF PINNING DOWN WHETHER
THEY'RE AT RISK OR NOT.
CERTAINLY WHEN YOU SEE THE
PATIENTS -- I ALSO HAVE A
PATIENT WHO'S A CARRIER FOR
DUCHENNE MUSCULAR DYSTROPHY AND
HAVE MYOSITIS.
MAYBE PATIENTS WHO HAVE A
GENETIC MUSCLE DISEASE MAY ALSO
DEVELOP AUTOIMMUNITY TARGETING
THE MUSCLE BUT IT'S NOT SUPER
WELL STUDIED BUT SUPER
INTERESTING.
>> THE IDEA THE MOLECULES INSIDE
THE CELL WHETHER IT'S MUSCLE
CELLS OR OTHER KIND OF CELL.
THOSE MOLECULES COULD UNDER SOME
CIRCUMSTANCES STIMULATE THE
IMMUNE SYSTEM IS REALLY I THINK
EYE OPENING AND WE THOUGHT THAT
WERE A PHENOMENON AGAINST THE
PROTEINS AND MAY HAVE AN
IMPORTANT BIOLOGICAL ROLE.
THAT IS QUITE CONSIDERING.
WHAT'S GOING ON AND MAYBE YOU
SAID IT BUT MAYBE I MISSED IT,
SHOULD THE STANDARD OF TREATMENT
BE AN IMMUNE EXPRESSIVE THERAPY
TO TAMP DOWN THAT INFLAMMATORY
MILIEU YOU'RE MENTIONING?
>> BEYOND STEROID ADMINISTRATION
IS NOW DOWN IN I THINK ALL THE
GENE THERAPIES THAT I WAS
INVOLVED IN.
COULD YOU REFINE IT AND I THINK
THE ANSWER IS INTEREST.
WE HAVE TO UNDERSTAND MORE TO DO
RESPONSES INNATE AND ADAPTIVE TO
UNDERSTAND THIS BETTER.
AND YOU CAN COMBINE THE STEROID
REGIMENT WITH ANTI-T CELL DRUGS
TO PREVENT AGAINST THE CAPSID
AND THE GENE TO AVOID REJECTIONS
LIKE I DESCRIBED IN THE DUCHENNE
TRIAL PUTTING THE GENE THERAPY
AT RISK.
I THINK WE HAVE DONE SO MANY
PROTOCOLS OUT THERE AND THERE'S
NO COMPARATIVE ANALYSIS IN THE
GENE THERAPY CONTEXT.
WE NEED TO COME TOGETHER AS A
FIELD.
WE'RE DOING THAT TO LOOK AT THE
LENS AND COMPARE THAT WITH THE
REGIMENTS AND THERE'S A GENOTYPE
MENTIONED AND IT'S PROBABLY
SOMETHING WE NEED TO TAKE NOTE
OF IN GENE THERAPY TO TAILOR
THERAPIES FOR PATIENTS AND
INDIVIDUAL MAKE UP.
THAT HOPEFULLY IS THE PRECISION
MEDICINE ASPECT OF GENE THERAPY
COMING IN THE FUTURE.
>> YOU KNOW, THAT'S WHAT YOU'RE
SAYING IS REALLY IMPORTANT WITH
REGARD TO GENE THERAPY BUT YOU
CAN EVEN IMAGINE FOR A PATIENT
THAT ISN'T UNDERGOING GENE
THERAPY, IF THEY'RE HAVING THIS
INFLAMMATORY PROCESS GOING ON
THAT MAYBE IS EXACERBATING
THINGS THAT MAYBE THEY SHOULD BE
ON SOME SORT OF
IMMUNOSUPPRESSIVE AGENT AS WELL.
>> THAT'S COMMON WITH
DEGENERATIVE DISORDERS.
>> MAYBE COMMENT ON THE LONG
STANDING TRADITION OF TREATING
BOYS WITH DYSTROEPENOPATHY WITH
STEROIDS.
>> THERE'S PROBABLY A MEMBRANE
HEALING ASPECT AND IMMUNOLOGICAL
AND BIOLOGICAL EFFECT OF THE
STEROID.
>> ANOTHER ASPECT YOU RAISED IN
YOUR TALK LIKE THE $64 MILLION
QUESTION NOW, I GUESS, THE WHOLE
QUESTION OF WHAT IS THE RIGHT
TIME TO DO GENE THERAPY ON ANY
OF THESE PARTICULAR THERAPIES
AND COMES DOWN TO THE
COMBINATION OR MORE.
>> THAT'S THE QUESTION.
AS SOON AS YOU IDENTITY THIS YOU
CAN TREAT THIS BUT PARTICULARLY
IN MUSCLE DISEASE IS WHAT
HAPPENS AND THE DISEASE HAD
DURABILITY IF IT'S TREATED EARLY
ON IN THE IMPACT OF THE DISEASE
AND IF ANYTHING WERE TO STAY THE
SAME BUT IN A POST-MYCROTIC
TISSUE HAS NEURONS AND IF YOU
CAN TREAT THEM ALL EFFECTIVELY,
THAT IS PROBABLY FOR YEARS NOT
DECADES BECAUSE THERE'S NO
TURNOVER AND HERE YOU HAVE
NUCLEI AND THEY'RE DIVIDING AND
LOSING THE GENE THERAPY IN THE
CASE OF AAD.
THE DURABILITY IS STILL
PROMISING EVEN IN MUSCULAR
DYSTROPHY.
THE EFFECT IS QUITE DURABLE
HOPEFULLY BUT WE CANNOT REDOSE.
YOU CAN'T REDOSE CURRENTLY
UNLESS YOU DO INTERVENTIONS THAT
ALLOW YOU TO CREATE AN IMMUNE
WINDOW TO OVERCOME THE
ANTIBODIES AND REDOSE.
IT'S NOT TRIVIAL.
IT'S NOT GOING TO BE EASY.
IF WE LOOKED TO THE FUTURE THAT
WOULD BE A PUSH TO GENOME
EDITING.
IF WERE ABLE TO CORRECT THE
GENETIC MUTATION IN THE GAIN OME
ITSELF THEN IT COMES AS A
BENEFIT AND YOU'RE PROLIFERATING
THE CORRECTION AND AMPLIFYING
THE CORRECTION OF THE CELLS THAT
ARE CORRECT AND PROLIFERATING
AND HEALING THE TISSUE.
THEN OF COURSE TREATING EARLY IS
NO QUESTION THAT THAT SHOULD BE
DONE THEN YOU HAVE THE
INTERACTION.
>> YEAH.
IT DOES SOUND LIKE THE EDITING
OPTION IS SOMETHING THAT MAKES A
LOT OF SENSE FROM THE CONCEPTUAL
POINT OF VIEW YOU OUTLINED.
THE EARLY TREATMENT IS DESTINED
IF YOU'RE USING AN AAV APPROACH
IT'S DESTINED TO EVENTUALLY
WEARING OFF BECAUSE MUCH THE
FACT YOU'RE DEALING WITH A
MITOTIC TISSUE.
WHAT IF YOU GAVE THE AAV AS AN
INFUSION TO TOLERATING THEM.
THEY NEED IT ANYWAY BECAUSE
THEY'RE.
YOU HAVE IT SO THEY'RE
CONSTANTLY BEING DOSED?
HAS ANYONE TRIED THAT?
>> WHAT YOU INDUCED IS HIGHER
ORDERS OF MAGNITUDE.
SO YOU'RE CREATING A
NEUTRALIZING ANTIBODY AND THE
FIRST EXPOSURE OF THE EDITED
GENE THERAPY THAT IS STILL THERE
DECADES OUT.
AND YOU CAN CREATE ANTIBODIES
THAT PREVENT YOU FROM
READMINISTER.
THE QUESTION IS CAN YOU BUILD
TOLERATION PERMANENTLY IS TO BE
CONSIDERED.
>> HERE'S OTHER QUESTIONS COMING
IN FROM THE AUDIENCE.
DO THEY HAVE TO BE
AUTOANTIBODIES OR DO THEY IN
GENERAL ENTER CELLS?
IS THIS SOMETHING HAPPENING ALL
THE TIME AND WE HAPPEN TO LOOK
AT THE ONES THAT ARE
AUTOANTIBODIES.
THERE'S A LOW LEVEL OF
IMMUNOGLOBULIN IN THE CYTOPLASM.
WHAT WE FIND THAT'S UNIQUE ABOUT
MYOSITIS IS THE IMMUNOGLOBULIN
CONCENTRATING WHERE THE TARGET
ANTIGEN IS.
THE OTHER THING I'D MENTION IS
THERE'S A WHOLE INTRACELLULAR
SYSTEM THAT SEEMS TO EXIST FOR
THE PURPOSE OF DEGRADING
INTRACELLULAR IMMUNOGLOBULIN AND
THAT'S ROW 52 OR TRIM 21 WITH AN
FC BINDING DOMAIN THAT'S
CYTOPLASMIC AND BIND TO
INTRACELLULAR IMMUNOGLOBULIN AND
THAT'S TO A VIRUS OR SOMETHING
AND WILL DEGRADE THE
IMMUNOGLOBULIN AND WHATEVER
ANTIGEN IS BOUND TO THE
IMMUNOGLOBULIN.
THERE'S EVIDENCE WE'VE EVOLVED
TO COPE WITH INTRACELLULAR
IMMUNOGLOBULIN.
>> NOW THAT YOU BROUGHT UP ROW
52, WE HAVE TO MENTION THE FACT
THAT IN SOME AUTOIMMUNE
DISEASES, PATIENTS HAVE
ANTIBODIES AGAINST R52 AND MAYBE
THAT'S SUBVERTING THE PROCESS
AND ALLOWING INTRACELLULAR
ANTIBODIES TO DO THE SAME THING.
>> THAT'S OUR HYPOTHESIS.
WE HAVE PRELIMINARY DATA THAT IF
YOU TAKE AN ANTIBODY IN THE CASE
WHERE WE DID IT AGAINST MDA5 AND
YOU CAN GET A SIGNIFICANT AMOUNT
OF INTERFERON EXPRESSION IF YOU
JUST INTRODUCE MDA5 ANTIBODIES
IF YOU INTRODUCE THE ROW 5
ANTIBODIES YOU DON'T SEE
ANYTHING BUT IF YOU PUT THEM
TOGETHER THERE'S A MASSIVE
INCREASE OF INTERFERON THAT GETS
PRODUCED AND THE ANTIBODIES
POTENTIATE THE AFFECT AND
PATIENTS WHO HAVE THIS WITH THE
AUTOSPECIFIC ANTIBODIES HAVE
MORE SEVERE DISEASE
MANIFESTATIONS.
WE THINK THAT'S PROBABLY THE
MECHANISM FOR WHY THEY HAVE MORE
SEVERE DISEASE MANIFESTATION.
THEY'RE BLOCKING THE DEGRADATION
OF THE AUTOANTIBODIES.
>> THAT'S REALLY INTERESTING.
SO ANOTHER MEMBER OF OUR
AUDIENCE IS A LITTLE BIT FAR OUT
IN TERMS OF THEIR THINKING BUT
WHAT THE HECK.
I'LL ASK THE QUESTION ANYWAY.
DO PETS GETS MYOSITIS AND IF SO,
CAN THEIR OWNERS CATCH IT FROM
THEIR PETS?
>> WELL, THE ANSWER TO THE FIRST
QUESTION IS YES.
THERE'S A BREED OF DOGS THAT GET
DERMATOMYOSITIS.
I SHOULD KNOW WHICH ONE IT IS.
I HAVEN'T THOUGHT ABOUT IT FOR A
WHILE BUT THERE'S A BREED OF
DOGS SUSCEPTIBLE TO IT.
AS THERE ARE DOGS THAT GET THESE
AUTOIMMUNE DISEASES.
NO, TO THE BEST OF MY KNOWLEDGE
THERE'S NO EVIDENCE OR REASON TO
BELIEVE THE AUTOIMMUNITY WOULD
BE TRANSFERRED TO THEIR OWNERS.
LIKE WE DON'T SEE IT TRANSFERRED
FROM ONE PERSON TO ANOTHER BY
PROXIMITY.
WE WOULDN'T EXPECT IT TO OCCUR
BUT YES, THERE'S A DOG MODEL OF
DERMATOMYOSITIS SOME PEOPLE
STUDY.
>> WOW.
WELL, THERE YOU GO.
I NEVER WOULD HAVE THOUGHT THE
ANSWER IS YES.
ANYWAY, WE'RE UP AT 4:58 P.M.
AND LESS THAN TWO MINUTES AWAY.
AT THIS POINT I THINK I NEED TO
THANK BOTH OF YOU FOR YOUR
PRESENTATIONS.
BREATHTAKING THE PROGRESS
MEDICAL RESEARCH HAS TAKEN IN
THE LAST FEW YEARS AND OUR LIFE
TIMES.
AND IT'S REALLY GREAT TO SEE.
CONGRATULATIONS TO BOTH OF YOU
AND YOUR RESPECTIVE FIELDS.
IT'S CLEAR THERE'S A LOT OF
REALLY EXCITING THINGS YET TO
COME.
ANYWAY, I THINK WITH THAT, WE'LL
SIGN OFF MAYBE A MINUTE EARLY
AND LOOK FORWARD TO SEEING YOU
AGAIN NEXT YEAR OR THE YEAR
AFTER, WHATEVER, TO GIVE US A
PROGRESS REPORT ON HOW THINGS
ARE DEVELOPING THEN.
>> THANKS, DAN.
THANKS FOR THE STIMULATING
CONVERSATION AND THE INVITE.
>> YES, THANKS FOR COMING.
BYE-BYE, EVERYONE.