Hemophilia A gene therapy: AAV-mediated delivery of an ...
Transcript of Hemophilia A gene therapy: AAV-mediated delivery of an ...
Hemophilia A gene therapy:
AAV-mediated delivery of an enhanced F8 cassette
to the liver produces supraphysiological levels of
human FVIII in vivo
Brigit E. RILEY
Director Discovery and Translational Research
Sangamo BioSciences
USA
1
Disclosures for: Brigit E. RILEYIn compliance with the PIM* policy, WFH requires the following disclosures be made at each presentation
CONFLICT DISCLOSURE — IF CONFLICT OF INTEREST EXISTS
RESEARCH SUPPORT Sangamo BioSciences
DIRECTOR, OFFICER, EMPLOYEE DIRECTOR
SHAREHOLDER
HONORARIA
ADVISORY COMMITTEE
CONSULTANT
* Postgraduate Institute for Medicine
2
C L I C K TO E D I T M A S T E R T I T L E S T Y L E
3
• Background – Hemophilia A and AAV Gene Therapy
• Overview of AAV Factor 8 cDNA
• Approach to Optimizing AAV Factor 8 cDNA
• Evaluating the Optimized AAV Factor 8 cDNA
– in vitro: HepG2 Cells
– in vivo: Wild Type and Hemophilia A mouse models
– in vivo: Non-Human Primates (NHPs)
• Summary and Next Steps
I N T R O S L I D E / A G E N D A
C L I C K TO E D I T M A S T E R T I T L E S T Y L E
4
• Background – Hemophilia A and AAV Gene Therapy
• Overview of AAV Factor 8 cDNA
• Approach to Optimizing AAV Factor 8 cDNA
• Evaluating the Optimized AAV Factor 8 cDNA
– in vitro: HepG2 Cells
– in vivo: Wild Type and Hemophilia A mouse models
– in vivo: Non-Human Primates (NHPs)
• Summary and Next Steps
I N T R O S L I D E / A G E N D A
5
• Modest increases in Factor VIII levels (>1% of normal) have a positive impact
on patient lives
• Adeno-associated virus (AAV) vectors have shown efficacy in preclinical and
clinical studies; stable Factor IX (FIX) expression ~ five years in the clinic
• Lag in the clinic of AAV human Factor 8 (hF8) gene therapy – poor vector
yields at clinical scale and dose required to achieve therapeutic FVIII levels
• We optimized an AAV hF8 cDNA vector cassette to improve both vector yields
and liver-specific hFVIII expression
• Administration of the enhanced AAV hF8 cDNA vector in vivo resulted in 2-8X
normal circulating hFVIII levels
* Higher circulating hFVIII levels will enable lower dose in the clinic
H E M O P H I L I A A
A N I D E A L D I S E A S E F O R L I V E R - D I R E C T E D G E N E T H E R A P Y
P liver- specific promoterTG therapeutic gene (F8)
H E M O P H I L I A A
L I V E R - D I R E C T E D A AV F 8 C D N A G E N E T H E R A P Y
7
Transgene packaged into
AAV vectorsTherapeutic delivered by a
single infusion
}
}
AAV vectors
P TGtransgene
Transgene packaged into
AAV vectors
P liver- specific promoterTG therapeutic gene (F8)
H E M O P H I L I A A
L I V E R - D I R E C T E D A AV F 8 C D N A G E N E T H E R A P Y
8
Transgene packaged into
AAV vectorsTherapeutic delivered by a
single infusion
}
}
AAV vectors
P TGtransgene
Transgene packaged into
AAV vectors
AAV is delivered by a
single infusion
P liver- specific promoterTG therapeutic gene (F8)
H E M O P H I L I A A
L I V E R - D I R E C T E D A AV F 8 C D N A G E N E T H E R A P Y
9
Transgene packaged into
AAV vectorsTherapeutic delivered by a
single infusion
Liver produces and secretes
therapeutic hFVIII protein
}
}
AAV vectors
Liver Cell DNA
Promoter
TherapeuticGene (hF8)
Nucleus
Liver Cell
P TGtransgene
Transgene packaged into
AAV vectors
AAV is delivered by a
single infusion
AAV traffics to liver to deliver
transgene into nucleus of liver cells
P liver- specific promoterTG therapeutic gene (F8)
H E M O P H I L I A A
L I V E R - D I R E C T E D A AV F 8 C D N A G E N E T H E R A P Y
10
Transgene packaged into
AAV vectorsTherapeutic delivered by a
single infusion
Liver produces and secretes
therapeutic hFVIII protein
}
}
AAV vectors
Liver Cell DNA
Promoter
TherapeuticGene (hF8)
Nucleus
Liver Cell
Transgene is expressed from the liver,
but remains separate from the cell’s DNA
P TGtransgene
Transgene packaged into
AAV vectors
AAV is delivered by a
single infusion
AAV traffics to liver to deliver
transgene into nucleus of liver cells
P liver- specific promoterTG therapeutic gene (F8)
H E M O P H I L I A A
L I V E R - D I R E C T E D A AV F 8 C D N A G E N E T H E R A P Y
11
Transgene packaged into
AAV vectorsTherapeutic delivered by a
single infusion
Liver produces and secretes
therapeutic hFVIII protein
}
}
AAV vectors
Liver Cell DNA
Promoter
TherapeuticGene (hF8)
Nucleus
Liver Cell
Transgene is expressed from the liver,
but remains separate from the cell’s DNA
P TGtransgene
Transgene packaged into
AAV vectors
AAV is delivered by a
single infusion
AAV traffics to liver to deliver
transgene into nucleus of liver cells
W I L D T Y P E A AV
12
• Single-stranded DNA virus which requires a helper virus for replication
• No pathology associated with AAV infection
• Tissue selectivity is determined by capsid composition
Vance et al, DOI: 10.5772/61988
Liver,
Heart,
Skeletal
muscle
Liver,
Heart, and
muscle
Heart, Liver Liver, Heart,
Brain, Lung,
Skeletal
muscle
Heart,
Lung, Liver
Liver Liver,
Heart,
Skeletal
muscle
Liver,
Skeletal
muscle
Liver,
Heart,
Brain,
Muscle
Tissue Selectivity of AAV Serotypes
AAV1 AAV2 AAV3 AAV4 AAV5 AAV6 AAV7 AAV8 AAV9
R E C O M B I N A N T A AV
13
• Recombinant AAV (rAAV) has been used extensively for nearly 20 years as a
gene therapy vector in preclinical and clinical studies
• Efficient transduction and long term, stable transgene expression in
non-dividing tissues:
- Liver, brain and muscle
• Replication deficient
• High degree of stability which allows for rigorous methods of vector
purification
• AAV vectors carrying capacity is small (~4.7 kb of DNA)
G E N E R AT I O N O F R E C O M B I N A N T A AV
14
ITR ITR
Transgene of Interest
Recombinant AAV
• Contains the transgene in place of
wild type genes
• Harvest crude rAAV extract
• Purify rAAV
- Density gradient
- Column purification
Highly purified rAAV
Within Cell
• Capsid Assembly
• Virus Packaging
Manufacturing rAAV
ITR ITR
Wild-Type AAV
Replace wild type
genes
wild type genes
ITR ITR
Transgene
of interest
+cell line
• Helper plasmid is supplied in trans, together with the
transgene to a packaging cell line
Helper plasmid
C L I C K TO E D I T M A S T E R T I T L E S T Y L E
15
• Background – Hemophilia A and AAV Gene Therapy
• Overview of AAV Factor 8 cDNA
• Approach to Optimizing AAV Factor 8 cDNA
• Evaluating the Optimized AAV Factor 8 cDNA
– in vitro: HepG2 Cells
– in vivo: Wild Type and Hemophilia A mouse models
– in vivo: Non-Human Primates (NHPs)
• Summary and Next Steps
I N T R O S L I D E / A G E N D A
W H AT I S O P T I M A L F O R A AV H F 8 C D N A ?
16
hF8 is not an ideal gene for AAV:
• Constrained by hF8 gene size
– Optimal AAV transgene size is ~4.7 kb; full length hF8 is ~7 kb
• AAV dose required to achieve therapeutic hFVIII levels
– Low efficiency of transcription/translation
• Low manufacturing yields of AAV hF8
– Clinical scale manufacturing feasibility is limiting
Optimal AAV hF8 cDNA requires:
• Shorter coding sequence for hF8
– Potential solution is the use of an optimized B-domain deleted sequence (BDD)
• An optimized robust liver-specific promoter module to drive hF8 expression
• Improved virus yields
C L I C K TO E D I T M A S T E R T I T L E S T Y L E
17
• Background – Hemophilia A and AAV Gene Therapy
• Overview of AAV Factor 8 cDNA
• Approach to Optimizing AAV Factor 8 cDNA
• Evaluating the Optimized AAV Factor 8 cDNA
– in vitro: HepG2 Cells
– in vivo: Wild Type and Hemophilia A mouse models
– in vivo: Non-Human Primates (NHPs)
• Summary and Next Steps
I N T R O S L I D E / A G E N D A
O P T I M I Z AT I O N O F A AV H F 8 C D N A R E Q U I R E D
M U LT I - FA C TO R I A L M O D I F I C AT I O N S
18
ITR
Liver Promoter Human Factor 8 B-Domain Deleted (BDD) polyA
ITR
Promoter module
modifications
• Assembled different permutations of
liver-specific promoter elements
• Identified regions of the promoter
module that could be improved upon
• For other elements a systematic
mutational design approach was used
Transgene
modifications
• Optimized the F8 cassette
Other modifications
• Identified minimal
synthetic polyA
• Removed un-necessary
nucleic acids
• Reduced size
• Optimized sequences
outside transgene hFVIII protein has the same amino acid sequence
as biologics currently in clinic
I T E R AT I V E P R O C E S S TO I D E N T I F Y A N I M P R O V E D
A AV H F 8 C D N A C A S S E T T E
19
• Virus Yields
• In Vitro secreted
hFVIII levels
(mRNA/protein) and
activity
• In Vivo circulating
hFVIII levels
(mRNA/protein)
Transgene
modifications
Promoter module
modifications
Other
modifications
3 .01 0 12
6 .01 0 12
9 .01 0 12
1 .21 0 13
1 .51 0 13
1 .81 0 13
2 .11 0 13
2 .41 0 13
2 .71 0 13
3 .01 0 13
Yie
ld (
vg
)
M O D I F I C AT I O N S I M P R O V E V I R U S Y I E L D
20
Stage 4Stage 3Stage 2Stage 1
Iterative Process to Improve Cassette
3 .01 0 12
6 .01 0 12
9 .01 0 12
1 .21 0 13
1 .51 0 13
1 .81 0 13
2 .11 0 13
2 .41 0 13
2 .71 0 13
3 .01 0 13
Yie
ld (
vg
)
M O D I F I C AT I O N S I M P R O V E V I R U S Y I E L D
21
Stage 4Stage 3Stage 2Stage 1
Iterative Process to Improve Cassette
3 .01 0 12
6 .01 0 12
9 .01 0 12
1 .21 0 13
1 .51 0 13
1 .81 0 13
2 .11 0 13
2 .41 0 13
2 .71 0 13
3 .01 0 13
Yie
ld (
vg
)
M O D I F I C AT I O N S I M P R O V E V I R U S Y I E L D
22
Stage 4Stage 3Stage 2Stage 1
Iterative Process to Improve Cassette
3 .01 0 12
6 .01 0 12
9 .01 0 12
1 .21 0 13
1 .51 0 13
1 .81 0 13
2 .11 0 13
2 .41 0 13
2 .71 0 13
3 .01 0 13
Yie
ld (
vg
)
M O D I F I C AT I O N S I M P R O V E V I R U S Y I E L D
23
Stage 4Stage 3Stage 2Stage 1
Iterative Process to Improve Cassette
8-10 fold
improvement
at the cell
factory scale
At clinical scale; greater than 5-fold improvement in vector yields
C L I C K TO E D I T M A S T E R T I T L E S T Y L E
24
• Background – Hemophilia A and AAV Gene Therapy
• Overview of AAV Factor 8 cDNA
• Approach to Optimizing AAV Factor 8 cDNA
• Evaluating the Optimized AAV Factor 8 cDNA
– in vitro: HepG2 Cells
– in vivo: Wild Type and Hemophilia A mouse models
– in vivo: Non-Human Primates (NHPs)
• Summary and Next Steps
I N T R O S L I D E / A G E N D A
S E V E R A L M E T H O D S U S E D TO A S S E S S H F V I I I
L E V E L S A N D A C T I V I T Y
25
hFVIII
Enzyme-Linked
Immunosorbent Assay (ELISA)
LEVELSSpecific for human FVIII
magnet magnet
Clotting times
Clotting Assay
FVIII
deficient
plasma
hFVIII
Activator +
phospholipids
Add Ca2+
Generation of Factor Xa and
thrombin yielding clot formation
ACTIVITYNot specific for human FVIII
Factor X
Factor Xa
Factor Xa
Factor IXa,
Ca2+, Phospholipid
hFVIII
Chromophore
405 nmChromogenic
substrate
Chromogenic Assay
ACTIVITYNot specific for human FVIII
I N V I T R O : H E P G 2 C E L L S
E X P E R I M E N TA L D E S I G N
26
50
Test article
addition
7 days
Supernatant collection schedule
Optimized rAAV hF8 cDNA cassette packaged into AAV2/6
Endpoints• ELISA for hFVIII levels
• Clotting assay for hFVIII activity
• Chromogenic assay for
hFVIII activity
AAV2
ITR
Liver-specific
promoter module
PolyAhFVIII BDDAAV2
ITR
3
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
0
2 0
4 0
6 0
8 0
1 0 0
hF
VII
I (P
erc
en
t N
orm
al)
hF
VII
I A
cti
vit
y (
U/m
L)
C lo tt in g A s s a y
C h ro m o g e n ic A s s a y
E L IS A
D o s e
I N V I T R O : H E P G 2 C E L L S
G O O D C O R R E L AT I O N B E T W E E N H F V I I I A C T I V I T Y / L E V E L S
27
Values will be reported as hFVIII (Percent Normal) for
ELISA, Chromogenic or Clotting where 1 U/mL = 100% Normal
4.8E6 / 2.4E6 / 1.2E6 / 0.6E6 MOI
C L I C K TO E D I T M A S T E R T I T L E S T Y L E
28
• Background – Hemophilia A and AAV Gene Therapy
• Overview of AAV Factor 8 cDNA
• Approach to Optimizing AAV Factor 8 cDNA
• Evaluating the Optimized AAV Factor 8 cDNA
– in vitro: HepG2 Cells
– in vivo: Wild Type and Hemophilia A mouse models
– in vivo: Non-Human Primates (NHPs)
• Summary and Next Steps
I N T R O S L I D E / A G E N D A
I N V I V O : W I L D T Y P E M O U S E D ATA
E X P E R I M E N TA L D E S I G N
29
Optimized rAAV hF8 cDNA cassette packaged into AAV2/6
AAV2
ITR
Liver-specific
promoter module
PolyAhFVIII BDDAAV2
ITR
Endpoints• ELISA for hFVIII levels
• hF8 mRNA tissue
biodistribution
Dose
• 7.2E+12 vg/kg
70 14 21 28 days
Plasma collection schedule
Immunosuppression regimen of cyclophosphamide (50 mg/kg)
IV injection of
test article
I N V I V O : W I L D T Y P E M O U S E D ATA
S U P R A P H Y S I O L O G I C A L L E V E L S
30Levels were determined by ELISA
hFVIII Levels
F o rm u la tio n A A V h F 8 c D N A
0
1 0 0
2 0 0
3 0 0
4 0 0
hF
VII
I (P
erc
en
t N
orm
al)
2 4 1 .6 %
I N V I V O : W I L D T Y P E M O U S E D ATA
H F 8 E X P R E S S I O N I S R E S T R I C T E D TO L I V E R
31
hF8 mRNA
Represents two independent
mouse studies
Liv
er
Bra
in
Kid
ney
Teste
s
Hear t
Sp
leen
Lu
ng
0
2
4
6
8
No
rm
ali
ze
d h
FV
III
mR
NA
Va
lue
s
I N V I V O : H E M O P H I L I A A M O U S E D ATA
E X P E R I M E N TA L D E S I G N
32
Optimized rAAV hF8 cDNA cassette packaged into AAV2/6
AAV2
ITR
Liver-specific
promoter module
PolyAhFVIII BDDAAV2
ITR
Dose
• 7.2E+12 vg/kg
70 14 21 28 days 3 months
Plasma collection schedule
Endpoint• Chromogenic assay for
hFVIII activity
• Tail vein transection
(TVT) for hemostasis
IV injection of
test article
I N V I V O : H E M O P H I L I A A M O U S E D ATA
O V E RV I E W O F H E M O P H I L I A A R 5 9 3 C M I C E
33
• Hemophilia A R593C mice are tolerized to hFVIII because they contain a
hF8-R593C transgene under control of a murine albumin promoter
• hFVIII-R593C is frequently observed in Hemophilia A patients, and in mice
produces no detectable hFVIII protein
- Thought to be rapidly degraded in mice, with peptide fragments
presented to the immune system
• Mice also contain a knockout of the mouse F8 gene and are deficient for
endogenous mouse FVIII protein
• Studies were conducted in collaboration with Dr. David Lillicrap at
Queen’s University
I N V I V O : H E M O P H I L I A A M O U S E D ATA
S U P R A P H Y S I O L O G I C A L L E V E L S
34
Day 14 Day 42
Activity determined by Chromogenic Activity Assay
hFVIII Activity
F o rm u la tio n A A V h F 8 c D N A
0
2 0 0
4 0 0
6 0 0
hF
VII
I (P
erc
en
t N
orm
al)
3 3 4 .1 %
F o rm u la tio n A A V h F 8 c D N A
0
2 0 0
4 0 0
6 0 0
hF
VII
I (P
erc
en
t N
orm
al)
3 3 0 .9 %
I N V I V O : H E M O P H I L I A A M O U S E D ATA
R E D U C E D B L E E D T I M E I N T R E AT E D M I C E
35
TVT method based on
Johansen et al., Haemophilia, 1-7, 2016.
Tail Vein Transection (TVT)
F o rm u la tio n A A V h F 8 c D N A
0
1 0
2 0
3 0
4 0
5 0
p < 0 .0 0 0 1
To
tal
Ble
ed
ing
Tim
e (
min
)
Normal bleeding time
C L I C K TO E D I T M A S T E R T I T L E S T Y L E
36
• Background – Hemophilia A and AAV Gene Therapy
• Overview of AAV Factor 8 cDNA
• Approach to Optimizing AAV Factor 8 cDNA
• Evaluating the Optimized AAV Factor 8 cDNA
– in vitro: HepG2 Cells
– in vivo: Wild Type and Hemophilia A mouse models
– in vivo: Non-Human Primates (NHPs)
• Summary and Next Steps
I N T R O S L I D E / A G E N D A
I N V I V O : N O N - H U M A N P R I M AT E D ATA
E X P E R I M E N TA L D E S I G N
37
Optimized rAAV hF8 cDNA cassette
packaged into AAV2/6
AAV2
ITR
Liver-specific
promoter module
PolyAhFVIII BDDAAV2
ITR
Doses
• 2.0E+12 vg/kg
• 6.0E+12 vg/kg
70 14 21 28 days 247 days
Plasma collection schedule
Immunosuppression regimen of Rituxan and Solu-Medrol (10 mg/kg for both)
IV injection of
test article
Endpoints• ELISA for hFVIII levels
• Clotting for hFVIII activity
• Bethesda Units (BU) for
inhibitory hFVIII antibodies
• Liver enzymes
I N V I V O : N O N - H U M A N P R I M AT E D ATA
S U P R A P H Y S I O L O G I C A L L E V E L S
38Levels were determined by ELISA
hFVIII Levels
2 E + 1 2 6 E + 1 2
1 0
1 0 0
1 0 0 0
T o ta l D o s e (v g /k g )
hF
VII
I (P
erc
en
t N
orm
al)
4 8 1 .6 %6 5 4 .1 %
Follow up dose-finding
studies are aimed at
determining the minimal
effective dose for the clinic
• Dose selection was based on
published studies
I N V I V O : N O N - H U M A N P R I M AT E D ATA
G O O D C O R R E L AT I O N B E T W E E N A C T I V I T Y / L E V E L S
39
Shown in the Formulation Group:
• Detection of ~100 % Normal NHP
FVIII activity
• No detection of NHP FVIII levels given
the ELISA is specific for human FVIII
1101
1101
1102
1102
3101
3101
3102
3102
3103
3103
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
A n im a l ID
hF
VII
I (P
erc
en
t N
orm
al)
A c tiv ity E L IS A L e v e ls
F o rm u la tio n 6 E + 1 2 v g /k g G ro u p
0 5 0 1 0 0 1 5 0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
0
1
2
3
D a y s P o s t D o s in g
hF
VII
I (P
erc
en
t N
orm
al)
BU
/mL
S o lu -M e d ro l
R e m o v e d
3 1 0 2 A n t ig e n L e v e ls
3 1 0 2 B e th e s d a U n its (B U )
I N V I V O : N O N - H U M A N P R I M AT E D ATA
D U R A B I L I T Y
40
Stable hFVIII levels for over 8-weeks in the
absence of all immunosuppression
Levels were determined by ELISA
0 5 0 1 0 0 1 5 0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
0
1
2
3
D a y s P o s t D o s in g
hF
VII
I (P
erc
en
t N
orm
al)
BU
/mL
3 1 0 1 B e th e s d a U n its (B U )
3 1 0 1 A n t ig e n L e v e ls
S o lu -M e d ro l
R e m o v e d
I N V I V O : N H P I M M U N E TO L E R A N C E C H A L L E N G E
D U R A B I L I T Y
41
• A simplified view of tolerance is induction of B-cell/T-cell anergy
and/or apoptosis in the presence of sustained levels of agent (hFVIII)
• Do the sustained hFVIII levels produced from the rAAV-hF8 prevent
re-induction of neutralizing antibodies?
• rAAV-hF8 treated NHP were challenged with hFVIII biologic
• hFVIII biologic challenge consisted of 4 weekly infusions of 25 U/kg
of Xyntha®
I N V I V O : N H P I M M U N E TO L E R A N C E C H A L L E N G E
N O A P P E A R A N C E O F I N H I B I TO RY A N T I B O D I E S
42
+ Regions shaded gray are
above the BU cutpoint thus
positive for inhibitory antibodies
Control Group
1 5 0 2 0 0 2 5 0
0
5 0
1 0 0
1 5 0
2 0 0
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
D a y s P o s t D o s in g
hF
VII
I (P
erc
en
t N
orm
al)
BU
/mL
1 1 0 1 B e th e s d a U n its (B U )
1 1 0 1 A n t ig e n L e v e ls
X y n th a
C h a l le n g e
P o s it iv e fo r
In h ib ito ry A n t ib o d ie s
AAV hF8 cDNA Dose Groups
hFVIII levels ~150% hFVIII levels ~10%
1 5 0 2 0 0 2 5 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
D a y s P o s t D o s in g
hF
VII
I (P
erc
en
t N
orm
al)
BU
/mL
3 1 0 1 B e th e s d a U n its (B U )
3 1 0 1 A n t ig e n L e v e ls
X y n th a
C h a l le n g e
N o E v id e n c e o f
In h ib ito ry A n t ib o d ie s
1 5 0 2 0 0 2 5 0
0
1 0
2 0
3 0
4 0
5 0
6 0
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
D a y s P o s t D o s in g
hF
VII
I (P
erc
en
t N
orm
al)
BU
/mL
4 1 0 3 B e th e s d a U n its (B U )
4 1 0 3 A n t ig e n L e v e ls
X y n th a
C h a l le n g e
N o E v id e n c e o f
In h ib ito ry A n t ib o d ie s
I N V I V O : N O N - H U M A N P R I M AT E D ATA
W E L L TO L E R AT E D
43
ALT = Alanine Aminotransferase, upper limit of normal,126 U/L
AST = Aspartate Aminotransferase, upper limit of normal,120 U/L
*Elevated levels observed post-liver biopsies (day 41)
Control Group High Dose Group
0 5 0 1 0 0 1 5 0
0
1 0 0
2 0 0
3 0 0
4 0 0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
D a y s P o s t D o s in g
Liv
er E
nz
ym
e (
U/L
)
hF
VII
I (P
erc
en
t N
orm
al)
A S T
A L T
*
0 5 0 1 0 0 1 5 0
0
1 0 0
2 0 0
3 0 0
4 0 0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
D a y s P o s t D o s in g
Liv
er E
nz
ym
es
(U
/L)
hF
VII
I (P
erc
en
t N
orm
al)
A S T
A L T
*upper limit of normal upper limit of normal
0 5 0 1 0 0 1 5 0
0
1 0 0
2 0 0
3 0 0
4 0 0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
D a y s P o s t D o s in g
Liv
er E
nz
ym
es
(U
/L)
hF
VII
I (P
erc
en
t N
orm
al)
A S T
A L T
*
h F V III L e v e ls
0 5 0 1 0 0 1 5 0
0
1 0 0
2 0 0
3 0 0
4 0 0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
D a y s P o s t D o s in g
Liv
er E
nz
ym
e (
U/L
)
hF
VII
I (P
erc
en
t N
orm
al)
A S T
A L T
*
h F V III L e v e ls
I N V I V O : N O N - H U M A N P R I M AT E D ATA
W E L L TO L E R AT E D
44
ALT = Alanine Aminotransferase, upper limit of normal,126 U/L
AST = Aspartate Aminotransferase, upper limit of normal,120 U/L
*Elevated levels observed post-liver biopsies (day 41)
Control Group High Dose Group
upper limit of normal upper limit of normal
I N V I V O : N O N - H U M A N P R I M AT E D ATA
P R E L I M I N A RY R E S U LT S D O S E - F I N D I N G S T U D Y
45Levels were determined by ELISA
hFVIII Levels
Preliminary results.
Dose-finding study
demonstrates high hFVIII
production from GMP-clinical
scale manufacturing process
6 E + 1 1 9 E + 1 1 2 E + 1 2 6 E + 1 2
1
1 0
1 0 0
1 0 0 0
T o ta l D o s e (v g /k g )
hF
VII
I (P
erc
en
t N
orm
al)
5 .7
5 6 .4
2 2 9 .0
1 2 .0
S U M M A RY A N D F U T U R E P L A N S
46
• Administration of AAV hF8 cDNA, engineered to improve vector yields and
liver-specific hFVIII expression, resulted in supraphysiological levels in vivo- Wild type mice
- Hemophilia A R593C mice
- NHPs
• Good correlation between assays used to measure circulating hFVIII protein- Levels by ELISA and activity by Chromogenic or Clotting assays
• Sustained hFVIII levels from the rAAV-hF8 prevented re-induction of
neutralizing antibodies with biologic challenge suggestive of induced tolerance
(even in the context of a xenogeneic setting)
• Ongoing studies are aimed at determining the minimal effective dose
• Goal of filling the IND, second half of 2016
A C K N O W L E D G E M E N T S
47
Richard Surosky
Alicia Goodwin
Andrea Kang
Tim Gabriele
Hung Tran
Jennifer Huang
Judy Greengard
Lisa King
Eudean Garces
Stephen Ballaron
Daniel Richards
Melanie Butler
Carolyn Gasper
Kathy Meyer
Dale Ando
Mike Holmes
Jeff Boonsripisal
Derek Liu
Rainier Amora
Lei Zhang
David Lillicrap
Christine Hough
Dominique Cartier
Kate Nesbitt
Courtney Dwyer
Kassandra Herbert