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