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Development of an
AAV5 gene therapy
for Fabry disease
Ying Poi Liu, PhD
uniQure Biopharma B.V.
Amsterdam, The Netherlands
This presentation contains forward-looking statements. All statements other than statements of historical fact are
forward-looking statements, which are often indicated by terms such as “anticipate,” “believe,” “could,”
“estimate,” “expect,” “goal,” “intend,” “look forward to,” “may,” “plan,” “potential,” “predict,” “project,” “should,”
"will,” “would” and similar expressions. Forward-looking statements are based on management's beliefs and
assumptions and on information available to management only as of the date of this press release. These
forward-looking statements include, but are not limited to, statements regarding the development of our gene
therapies, the success of our collaborations, and the risk of cessation, delay or lack of success of any of our
ongoing or planned clinical studies and/or development of our product candidates. Our actual results could differ
materially from those anticipated in these forward-looking statements for many reasons, including, without
limitation, risks associated with collaboration arrangements, our and our collaborators’ clinical development
activities, regulatory oversight, product commercialization and intellectual property claims, as well as the risks,
uncertainties and other factors described under the heading "Risk Factors" in uniQure’s Quarterly Report on
Form 10-Q filed on November 1, 2017. Given these risks, uncertainties and other factors, you should not place
undue reliance on these forward-looking statements, and we assume no obligation to update these forward-
looking statements, even if new information becomes available in the future.
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 3
Fabry disease: a lysosomal storage disease
- X-linked genetic disorder
- Deficiency of α-galactosidase A (α-Gal A or GLA)
- Females also suffer from Fabry and severity depends on X-inactivation despite having GLA activity in the plasma
- Systemic accumulation of substrate; Gb3 and LysoGb3 in plasma, tissues and organs
- Bi-weekly ERT infusions may not be effective due to poor organ incorporation due to lack of cross correction (uptake into lysosomes)
- Furthermore, a significant number of patients develop antibodies to GLA
* Spada M et al. Am J Hum Genet 2006, Inoue T et al J Hum Genet 2013
in black: early symptomsin red: late symptoms
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 4
What is cross correction and why is it important?
Disadvantages of ERT:
• Poor cross correction of GLA
• Heterozygous females are also symptomatic
• Thus, unaffected cells produce GLA but uptake into lysosomes via the Mannose 6-phosphate receptor is poor
• In MPS II, there are asymptomatic carriers due to sufficient cross correction
• Poor cross correction hamper clearance of all substrate in target organs
• Long-term ERT does not prevent disease progression
Adapted from Parenti et al. Int J Mol Med 2013
Mannose-6-
phosphate receptor
ERT
GLA protein
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 5
uniQure’s approach: modified NAGA
Expression of
endogenous
NAGA in classic
Fabry patients
More stable in blood and low pH
More efficient uptake
Better distribution
Novel Approach
• Expression of modified NAGA (modNAGA) using AAV5 vector (constant supply)
• ModNAGA has GLA activity and is able to reduce LysoGb3 accumulation
More effective (cross-correction) than ERT
Patients with and without inhibitors
Non-immunogenic
modNAGA is
active in the
presence of
GLA inhibitors
Tajima et al. 2009 (PMID: 19853240) Tajima et al. 2009 andKytidou et al. 2017 (PMID: 28680430)
Licenced from Prof. Sakuraba, Tokyo
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 6
Two approaches: liver specific or constitutive promoter
AAV5 ModNAGA
C1
NAGA
coNAGA
spNAGA
SV40pA
GLA signal peptide
L1
or
ModNAGA
L1
Liver produces and
secretes protein can
be taken up into
target organs
C1
Constitutive protein
expressoin from target
organs
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 7
Studies to show proof of concept of modNAGA in vitro and in vivo
Wt mice Fabry mice
GLA activity
(plasma and target organs)
GLA activity
LysoGb3
(plasma and target organs)
In vitro, cells
GLA activity
M6P-receptor
mediated uptake
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 8
Expression of modNAGA results in GLA activity in cells and cellular uptake is mediated via M6P-receptor
Conclusions:
- Expression and secretion of modNAGA, that exhibits GLA-activity
- M6P-receptor blockage results in increased GLA activity in the supernatant,
suggesting M6P-receptor mediated uptake of NAGA.
NAGA
untr.
NAGA
untr.
0
250
500
750
1000
1250
1500
1750
2000
GL
A a
cti
vit
y (
nm
ol/h
/mL
)
M6P
no M6P
NAGA
untr.
NAGA
untr.
0
25
50
75
100
125
GL
A a
cti
vit
y (
nm
ol/h
/mL
)
M6P
no M6P
GLA activity in
cells
GLA activity in
supernatant
More NAGA in cell
supernatant due
to M6P-receptor
blockage
50
37
M
50
37
KDa
modNAGA
expression
Plasmid transfection
+/- M6P
supernatant
cells
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 9
Glycosylation pattern of in vitro expressed modNAGA indicates presence of high mannose oligosacharides
modNAGA
Cells
Sup
PNGase endoH-
50
37
MKDa
50
37
PNGaseF• Cleaves high mannose, hybrid and complex oligosacharides
EndoH:• Is unable to cleave N-linked complex type glycans
In vitro expressed modNAGA:
Contains both high mannose
and complex N-linked glycans
EndoH PNGaseF
PNGaseF
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 10
GLA activity and reduction of LysoGb3 in Fabry (GLA-KO) mice plasma following AAV5-modNAGA injection
AAV-vector
GLA-activity
GLA KO mice (n=4) IV injected with
5e13 gc/kg AAV5-modNAGA
wt -
veh
icle
vehic
le
AAV5-
C7-
NAG
A
AAV5-
C7-
coNAG
A
AAV5-
EF1a-N
AG
A
AAV5-
EF1a
-coN
AG
A
0
4
8
12
16
20
25
50
75
100
GL
A-a
cti
vit
y (
nm
ol/h
/mL
)
wt - vehicle
vehicle
AAV5-C7-NAGA
AAV5-C7-coNAGA
AAV5-EF1a-NAGA
AAV5-EF1a-coNAGA
Wt mice vehicle L1-
NAGA
L1-
coNAGA
C1-
NAGA
C1-
coNAGA
Fabry mice
LysoGb3 levels
wt -
veh
icle
vehic
le
AAV5-
C7-
NAG
A
AAV5-
C7-
coNAG
A
AAV5-
EF1a-N
AG
A
AAV5-
EF1a
-coN
AG
A
0
60
120
180
240
300
360
420
lysoG
b3 (
pm
ol/m
l)
AAV5-EF1a-NAGA
AAV5-EF1a-coNAGA
wt - vehicle
vehicle
AAV5-C7-NAGA
AAV5-C7-coNAGA
Wt mice vehicle L1-
NAGA
L1-
coNAGA
C1-
NAGA
C1-
coNAGA
Fabry mice
2 to 8wks
20 4 12 wks
/sac.
6 8 10
2 to 12wks
Collaboration with Hans Aerts, Leidenand Carlie de Vries, Amsterdam
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 11
LysoGb3 reduction in target organs in GLA-KO mice upon AAV-injection
AAV-vector
Conclusion: LysoGb3 reduction in all target organs upon AAV5-modNAGA injection in GLA-KO mice
GLA-KO mice IV injected with
5e13 gc/kg AAV5-modNAGA
0
10
20
30
40
50
60liver
12 wks post IV
lyso
Gb
3 (
pm
ol/m
g p
rote
in)
Wt Vehicle
Fabry Vehicle
AAV5-C7-NAGA
AAV5-C7-coNAGA
AAV5-EF1a-NAGA
AAV5-EF1a-coNAGA
0
5
10
15kidney
12 wks post IV
lyso
Gb
3 (
pm
ol/m
g p
rote
in)
Wt Vehicle
Fabry Vehicle
AAV5-C7-NAGA
AAV5-C7-coNAGA
AAV5-EF1a-NAGA
AAV5-EF1a-coNAGA
0
2
4
6heart
12 wks post IV
lyso
Gb
3 (
pm
ol/m
g p
rote
in)
Wt Vehicle
Fabry Vehicle
AAV5-L1-NAGA
AAV5-L1-coNAGA
AAV5-C1-NAGA
AAV5-C1-coNAGA
lysoGb3 levels in organs
Collaboration with Hans Aerts, Leiden
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 12
ModNAGA poses a low predicted immunogenicity risk versus endogenous NAGA
• Step 1 – In silico
• Algorithms to screen potential T-cell epitopes
• Identify linear motifs (9-10 aa) that bind to
HLA MHC class I or II molecules A*01:01, A*02:01, A*03:01, A*11:01,
A*24:02, A*29:02, B*07:02, B*08:01,
B*14:02, B*15:01, B*27:05, B*35:01,
B*40:01
Collaboration with Abzena and Pro-immune
ModNAGA Moderate
affinity
High affinity
MHC I peptides 1 1
MHC II peptides - -
• Step 2 – In vitro
• 2 MHC class I peptides tested to common
MHC class I (HLA) alleles:
• Quantitative and qualitative analysis:
• Peptide binding properties demonstrate that
the two peptides do not pose an increased
immunogenicity risk compared to
endogenous NAGA
Immunogenicity evaluation of modNAGA
uniQure Research Immunology
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 13
Conclusions and future plans
• AAV5-NAGA results in therapeutic GLA activity and Lyso-Gb3
reduction in plasma and target organs at already 12wks
• Plasma GLA activity is not indicative for efficacy of therapy
• ModNAGA may be a more effective therapy (Tajima et al. 2009)
▪ More stable
▪ Can be used in GLA inhibitor patients
▪ Better organ distribution
• Expressed modNAGA contains high mannose glycans and is
taken up via M6P-receptor
• Determine GLA activity and Lyso-Gb3 reduction in target
organs of AAV-injected Fabry mice (> 6 mo)
• Determine AAV vector distribution and expression in target
organs (>6 mo)
• Glycosylation characterization of in vivo expressed
modNAGA
• Detect Zebrabodies in kidney cells and localization of active
modNAGA in lysosomes
Conclusions Future plans
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 14
Acknowledgements
• Chi-Lin Kuo• Maria J. Ferraz• Johannes M.F.G. Aerts
ResearchBetty Au
Sander van DeventerMelvin Evers
Pavlina KonstantinovaJolanda Liefhebber
Ying Poi LiuLieke Paerels
Vanessa ZancanellaTom van der Zon
ImmunologyValerie Ferreira
Vector and process developmentErich Ehlert
Tamar GrevelinkMustafa Kyamil
Richard van LogtensteinMaroeska Oudshoorn
Mark van VeenJacek Lubelski
• Emily Mallet• H. Fogg
• Linda Tan• Aurelia Gondrand• Dhivina Gagoomal
Analytical developmentEddy Berthier
Monika GolinskaJaap Twisk
• Carlie J.M. de Vries• Roelof Ottenhoff• Jan Aten
• Histoshi Sakuraba• Ryoko Tsukahara
• Juan Manuel Iglesias• Michael Roberts
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 16
GLA and NAGA protein expression
NAGAGLA
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 17
101
102
103
104
105
106
107
liver
7 wks post IV
Gen
om
e c
op
ies /
g D
NA Vehicle
AAV5-C7-NAGA
AAV5-C7-coNAGA
AAV5-C7-spNAGA
Vehicle
AAV5-EF1a-NAGA
GLA-activity in plasma of wild type mice injected with AAV5-mod-NAGA
05
1015
30
60
90
120plasma
2, 4 and 7 wks post IV
GL
A-a
cti
vit
y (
nm
ol/h
/mL
)
Vehicle
AAV5-L1-NAGA
AAV5-L1-coNAGA
AAV5-L1-spNAGA
Vehicle
AAV5-C1-NAGA
GLA-activityVector DNA
AAV-vector
Wild type mice IV injected with
5e13 gc/kg AAV5-modNAGA
20 4 7 wks
/sac.
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 18
GLA-activity in the liver and target organs of wild type animals injected with AAV5-modNAGA
AAV-vector
Conclusions: All AAV5-modNAGA vectors increase GLA-activity in the liver by at least 10 times above wild type
AAV5-EF1a-modNAGA might increase GLA-activity in target organs
Wild type mice IV injected with
5e13 gc/kg AAV5-modNAGA
0
50
100
500
1000
1500liver
7 wks post IV
GL
A-a
cti
vit
y
(nm
ol/
h/m
g p
rote
in)
Vehicle
AAV5-C7-NAGA
AAV5-C7-coNAGA
AAV5-C7-spNAGA
Vehicle
AAV5-EF1a-NAGA
0
50
60
80
100
120kidney
7 wks post IV
GL
A-a
cti
vit
y
(nm
ol/
h/m
g p
rote
in)
Vehicle
AAV5-C7-NAGA
AAV5-C7-coNAGA
AAV5-C7-spNAGA
Vehicle
AAV5-EF1a-NAGA
0
2
4
6
8
10heart
7 wks post IV
GL
A-a
cti
vit
y
(nm
ol/
h/m
g p
rote
in)
Vehicle
AAV5-L1-NAGA
AAV5-L1-coNAGA
AAV5-L1-spNAGA
Vehicle
AAV5-C1-NAGA
GLA-activity in organs
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 19
GLA activity in liver of Fabry (GLA-KO) mice upon AAV5-modNAGA injection
AAV-vector
Conclusion: Increased GLA activity in the liver of AAV-modNAGA injected GLA-KO mice
GLA-KO mice IV injected with
5e13 gc/kg AAV5-modNAGA
0
30
60
90
500
1000
1500
2000
2500liver
12 wks post IV
GL
A-a
cti
vit
y (
nm
ol/h
/mL
)Wt Vehicle
Fabry Vehicle
AAV5-L1-NAGA
AAV5-L1-coNAGA
AAV5-C1-NAGA
AAV5-C1-coNAGA
GLA-activity
D E L I V E R I N G G E N E T H E R A P Y T O P A T I E N T S F a b r y p r o g r a m | 20
Vector DNA and NAGA mRNA expression in target organs of Fabry (GLA-KO) mice upon AAV5-modNAGA injection
101
102
103
104
105
106
107
liver
12 wks post IV
Gen
om
e c
op
ies /
g D
NA Wt Vehicle
Fabry Vehicle
AAV5-C7-NAGA
AAV5-C7-coNAGA
AAV5-EF1a-NAGA
AAV5-EF1a-coNAGA
101
102
103
104
105
106
107
kidney
12 wks post IV
Gen
om
e c
op
ies /
g D
NA Wt Vehicle
Fabry Vehicle
AAV5-C7-NAGA
AAV5-C7-coNAGA
AAV5-EF1a-NAGA
AAV5-EF1a-coNAGA
101
102
103
104
105
106
107
heart
12 wks post IV
Gen
om
e c
op
ies /
g D
NA Wt Vehicle
Fabry Vehicle
AAV5-L1-NAGA
AAV5-L1-coNAGA
AAV5-C1-NAGA
AAV5-C1-coNAGA
Vector DNA
NAGA mRNA
103
104
105
106
107
108
109
liver
12 wks post IV
NA
GA
co
pie
s /
g R
NA Wt Vehicle
Fabry Vehicle
AAV5-C7-NAGA
AAV5-C7-coNAGA
AAV5-EF1a-NAGA
AAV5-EF1a-coNAGALLOQ
* * * below LLOQ103
104
105
106
107
108
109
kidney
12 wks post IV
NA
GA
co
pie
s /
g R
NA Wt Vehicle
Fabry Vehicle
AAV5-C7-NAGA
AAV5-C7-coNAGA
AAV5-EF1a-NAGA
AAV5-EF1a-coNAGALLOQ
* * * below LLOQ103
104
105
106
107
108
109
heart
12 wks post IV
Gen
om
e c
op
ies /
g D
NA
AAV5-L1-NAGA
AAV5-L1-coNAGA
AAV5-C1-NAGA
AAV5-C1-coNAGA
* *
* below LLOQ
Wt Vehicle
Fabry Vehicle
LLOQ