Towards AAV5-mediated Gene

Therapy for Hemophilia A with a

Factor IX Variant that functions

independently of FVIII

Ying Poi Liu, PhDuniQure 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.

2 0 1 9 | 3CONFIDENTIAL

Hemophilia A; a bleeding disorder due to lack of FVIII

- X-linked bleeding disorder

- Deficiency in coagulation factor FVIII that serves as a cofactor for factor IX for activation of the coagulation cascade

- Spontaneous bleeds

- Classified into severe, moderate and mild (<1%, 1-5% and >5-40% of FVIII activity)

- ~66,000 patients with severe HemA in US and Europe

- Treatment: FVIII concentrates

- 30% of patients develop inhibitors

2 0 1 9 | 4CONFIDENTIAL

Why not express FVIII in the liver?

• Endogenous FVIII synthesis in endothelial cells and not hepatocytes

• Production site and protein load may activate the unfolded protein response in vitro and in vivo (Dorner et al. 1989, Malhotra et al 2008, Brown et al 2011, Zolothukhin et al 2016, uniQure unpublished data)

• Expression in the liver may not be sustainable

PBS Tunicamycin Vector AAV-FVIII AAV-FIX

2 0 1 9 | 5CONFIDENTIAL

uniQure’s approach: FIX variant

Non

thrombogenic

Normal

activation

Low predicted

immunogenicity

risk

Hepatocyte friendly

Non immunogenic

Novel Approach

• Expression of a FIX variant with FVIII-independent FX activity using AAV5 vector

Long-term expressionEfficacious in patients

with and without inhibitorsSafety

Correction of

hemophilia

phenotype

2 0 1 9 | 6CONFIDENTIAL

FIX-FIAV activates FX in the absence of FVIII

Adapted from Kristensen L. H. et al Biochem J. 2016

FIX-FIAV:

L6F, V181I, K265A and I383V

FIX-FIAV wt FIX

FIX-FIAV

L6F

V181I

K265A

I383V

Not hyperactive

and normally

activated

FIX FX

FVIII

FVIII-independent

activation of FX

2 0 1 9 | 7CONFIDENTIAL

AMT-180: AAV5-Q1-FIX-FIAV is physiologically activated

FIX

Q1

Promoter

AAV5-FIX-FIAV

AAV5

ss AAV vector genome

hFIXco-FIAV gene

L6F, K265A, V181I, I383V

- The inactive FIX-FIAV zymogen is expressed

- Activation is required

FIX-FIAV

FX activation in the

absence of FVIII

2 0 1 9 | 8CONFIDENTIAL

Studies to show proof of concept of FIX-FIAV in vitro and in vivo

Wt mice HemA mice Cynomolgus Macaques

FIX protein FIX protein

FVIII-independent

activity

FIX protein

Safety / tolerability of

the AAV product

In vitro, cells

FIX protein

FVIII-independent

activity

2 0 1 9 | 9CONFIDENTIAL

FIX-FIAV shows 32% of FVIII-independent activity in APTT and thrombin generation assay

L6F

V181I

K265A

I383V

Catalytic

Residues Stable FIX

expression in

human cell line

Protein

Purification

Functional

Evaluation

FIX

variant

FVIII

independent

activity (%)

WT < 6

FIAV 32 ± 6NPP = Normal

Pooled Plasma0 1 0 2 0 3 0 4 0

0

5 0

1 0 0

1 5 0

2 0 0

T im e (m in )

Th

ro

mb

in (

nM

)

F V III-d e p le te d p la s m a

0 .5 p M T is s u e F a c to r

F V IIId + F IX -F IA V

F V IIId

F V IIId + N o v o E ig h t

29%

- FIX-FIAV (5 µg/ml) shows 32% and 29% of FVIII-independent activity by

APTT and thrombin generation relative to a FVIII standard

- FIX-FIAV thrombin generation curve overlaps with the normal curve

Thrombin generation assayOne stage clotting assay (APTT)

2 0 1 9 | 10CONFIDENTIAL

FIX-FIAV is not hyperactive and requires physiologicalactivation (same as FIX)

55kDa = FIX

45kDa = FIXa

FIXa FIX-FIAV

t=60

FIX-WT

188

98

62

49

38

28

t=0 t=60t=0

Western blot

2 0 1 9 | 11CONFIDENTIAL

0 4 0 0 8 0 0 1 2 0 0 1 6 0 0 2 0 0 0

0

2 0

4 0

6 0

8 0

1 0 0

W e e k 5 F V I I I i n d e p e n d e n t a c t i v i t y v s F I X a n t i g e n

F I X p r o t e i n ( % )

FV

III

ind

ep

en

de

nt a

ctiv

ity

(%

)

F I X - w t

F I X - F I A V

v e h i c l e

FIX protein level by ELISA; FVIII activity by APTT

• FIX-FIAV shows FVIII independent activity in hemophilic

mice

• Measured in APTT assay

Week 5

FVIII-independent activity vs FIX protein

FVIII-independent activity upon AAV injection in hemophilic mice

n=10, male

FVIII KO mice

IV dose 5e13 gc/kg

Clotting (APTT) assay

Activator +

phospholipids

FVIII deficient

plasma

magnet

FIX-FIAV in

sample

+ Calcium

generation FXa

clotting time

FVIII-independent activity

Relative to a serial dilution

of a FVIII standard

2 0 1 9 | 12CONFIDENTIAL

• Normalisation of the FVIII-independent activity to 100% of FIX protein

• ~24% of FVIII-independent activity in hemophilic mice

Summary efficacy AMT-180

• Recombinant FIX-FIAV

✓ 29% FVIII-like activity in thrombin generation assay

✓ 32% FVIII like-activity in clotting assay

• AMT-180 in hemophilic mice

✓ 24% FVIII-like activity in clotting assay

• AMT-180 expected to show clinical meaningful efficacy (per 100% protein)

FIX-FIAV shows a therapeutic meaningfulFVIII-independent activity in hemophilic plasma

FI X

- wt

FI X

- FI A

V

0

1 0

2 0

3 0

4 0

5 0

S p e c i f i c F V I I I i n d e p e n d e n t a c t i v i t y

w e e k 5

Sp

ec

ific

FV

III

ind

ep

en

de

nt

ac

tiv

ity

(%

)

2 0 1 9 | 13CONFIDENTIAL

FIX-FIAV expression in NHPs expected to translate to therapeutically relevant FVIII independent activity in humans

male Cynomolgus macaque

n=2

IV, 9e13 gc/kg

adapted delivery

1 vehicle treated NHP

1) AAV5-LP1-FIAV

2) AAV5-Q1-FIAV

Q1= a proprietary liver specific

promoter- 2 0 2 4 6 8 1 0 1 2 1 4

5 0

1 0 0

1 5 0

2 0 0

2 5 0

h F I X p r o t e i n ( % ) i n N H P s

w e e k s p o s t - i n j e c t i o nh

FIX

pr

ote

in (

%)

v e h ic le

A A V 5 - L P 1 - F I A V

A A V 5 - Q 1 - F I A V

8-folds increased protein expression using Q1

2 0 1 9 | 14CONFIDENTIAL

Safety assessments: non thrombogenic & low predicted immunogenicity risk

Thrombogenicity

• No elevation of coagulation activation markers: TAT + D-dimer levels in AAV-injected mice and

NHPs

• Histopathological examination of the NHP organs did not show signs of thrombus formation

Immunogenicity

• In silico assessment of potential T-cell epitopes

• 9-10 aa peptides that bind to HLA MHC Class II or I molecules

• 4 moderate affinity peptides found for MHC Class I and no peptides for MHC Class II

• Quantitative and Qualitative analysis of MHC Class I peptide binding properties predict a non

significant risk compared to FIX-wt

2 0 1 9 | 15

Conclusions and future plans

• AMT-180 is expected to prevent bleeds; sufficient thrombin generation & clot formation

• Hepatocyte friendly

• Safe; non thrombogenic (normal activation & regulation) & low predicted immunogenicity risk

• Effective for HemA patients with and without inhibitors

• Full biochemical characterization of recombinant FIX-FIAV protein

• Thrombin generation & clotting activity of AAV-injected NHP plasma samples (with or without addition of FVIII antibodies)

• GLP tox study in NHPs ongoing

• IND enabling

Conclusions Ongoing & Future plans

2 0 1 9 | 16CONFIDENTIAL

Acknowledgements

• Mettine H.A. Bos

• Viola J.F. Strijbis

• Pieter Reitsma

• Joachim Schwäble

• Karin Huber

• Erhard Seifried

ResearchBetty Au

Sander van Deventer

Pavlina Konstantinova

Jolanda Liefhebber

Ying Poi Liu

Vanessa Zancanella

Tom van der Zon

• Keiran Sinclair

• Caroline Brennan

ImmunologyNikki Timmer

Valerie Ferreira

Non ClinicalMartin de Haan

Paula Miranda

Srijana Tripathi

Corina van der Kruijssen

Vector and process developmentErich Ehlert

Tamar Grevelink

Mustafa Kyamil

Richard van Logtenstein

Maroeska Oudshoorn

Lisanne Schulte

Mark van Veen

Jacek Lubelski

• Emily Mallet

• H. Fogg

• T. Jones

• Linda Tan

Analytical developmentEddy Berthier

Monika Golinska

Elina Hessels

Kamille Pekcan

Jaap Twisk

• Juan Manuel Iglesias

• Michael Roberts

2 0 1 9 | 18CONFIDENTIAL

FVIII-independent activity in AAV-FIX-FIAV transduced cells

• AAV transduction of Huh-7 cells

• Dose-dependent FIX-FIAV protein expression (ELISA) and FVIII-independent activity as

measured by APTT clotting assay relative to a FVIII standardd

FI X

- wt

FI X

- FI A

V

0

2

4

6

8

F I X p r o t e i n l e v e l s i n H u h - 7

FIX

pr

ot

ein

le

ve

l (

%)

M O I = 1 x 1 05

M O I = 1 x 1 07

M O I = 1 x 1 06

FI X

- wt

FI X

- FI A

V

0 . 0

0 . 5

1 . 0

1 . 5

2 . 0

F V I I I i n d e p e n d e n t a c t i v i t y i n H u h - 7

FV

III

mim

et

ic a

ct

ivit

y (

% o

f n

or

ma

l)

M O I = 1 x 1 05

M O I = 1 x 1 07

M O I = 1 x 1 06

2 0 1 9 | 19CONFIDENTIAL

• Some mice were excluded for analyses based on bioanalyses data

• Dose = 5e13 gc/kg

0 2 4 6 8 1 0

0

4 0 0

8 0 0

1 2 0 0

1 6 0 0

2 0 0 0

2 4 0 0

F I X - w t e x p r e s s i o n i n h e m o p h i l i c m i c e

w e e k s p o s t - in j e c t io n

FIX

pr

ot

ein

(%

)

1 2 L

1 2 R

1 L

1 R L

1 X

3 X

1 1 X

A

0 2 4 6 8 1 0

0

4 0 0

8 0 0

1 2 0 0

1 6 0 0

2 0 0 0

2 4 0 0

F I X - F I A V e x p r e s s i o n i n h e m o p h i l i c m i c e

w e e k s p o s t - i n j e c t io n

FIX

pr

ote

in (

%)

1 4 L

1 5 L

1 5 R

7 R

8 L

8 R

B

n=10, male

FVIII KO mice

IV dose 5e13 gc/kg

FIX-FIAV protein expression in hemophilic mice upon AAV injection

2 0 1 9 | 20CONFIDENTIAL

Dose-dependent increase in FVIII independent activity upon increased FIX-FIAV expression in hemophilic mice

Day 0

IV tail vein injection

Weeks 1, 2, 5 and 8

Collect blood

Week 8

Sacrifice

FVIII-/- mice

n=11

Dosis= high:1,44e14, mid:

5e13, low: 1e13 gc/kg

1) Vehicle (n=6)

2) AAV-FIX-wt (high)

3) AAV-FIX-FIAV (high)

4) AAV-FIX-FIAV (mid)

5) AAV-FIX-FIAV (low)

6) AAV-FVIII (high)

Phenotypic correction

assay

Week 8 FVIII mimetic activity