Towards AAV5-mediated Gene Therapy for Hemophilia A with a Factor - - PowerPoint PPT Presentation

Towards AAV5-mediated Gene Therapy for Hemophilia A with a Factor IX Variant that functions independently of FVIII

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

• ngoing 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.

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CONFIDENTIAL

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

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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

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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 expression Efficacious in patients with and without inhibitors Safety

Correction of hemophilia phenotype

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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

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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

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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

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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 ± 6

NPP = Normal Pooled Plasma

1 0 2 0 3 0 4 0 5 0 1 0 0 1 5 0 2 0 0

T im e (m in ) T h ro m b in (n M )

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 assay One stage clotting assay (APTT)

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

55kDa = FIX 45kDa = FIXa

FIXa FIX-FIAV

t=60

FIX-WT

188 98 62 49 38 28

t=0 t=60 t=0

Western blot

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4 0 0 8 0 0 1 2 0 0 1 6 0 0 2 0 0 0 2 0 4 0 6 0 8 0 1 0 0 F I X p r o t e i n ( % ) F V I I I i n d e p e n d e n t a c t i v i t y ( % ) F I X - w t F I X - F I A V v e h ic le

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

• f a FVIII standard

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• 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 meaningful FVIII-independent activity in hemophilic plasma

F I X - w t F I X - F I A V 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

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 ( % )

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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

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 - in je c t io n h F I X p r o t e i n ( % )

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

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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

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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

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Acknowledgements

• Mettine H.A. Bos
• Viola J.F. Strijbis
• Pieter Reitsma
• Joachim Schwäble
• Karin Huber
• Erhard Seifried

Research

Betty Au Sander van Deventer Pavlina Konstantinova Jolanda Liefhebber Ying Poi Liu Vanessa Zancanella Tom van der Zon

• Keiran Sinclair
• Caroline Brennan

Immunology

Nikki Timmer Valerie Ferreira

Non Clinical

Martin de Haan Paula Miranda Srijana Tripathi Corina van der Kruijssen

Vector and process development

Erich 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 development

Eddy Berthier Monika Golinska Elina Hessels Kamille Pekcan Jaap Twisk

• Juan Manuel Iglesias
• Michael Roberts

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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

F I X - w t F I X - F I A V 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

F I X p r o t e i n l e v e l ( % ) F I X - w t F I X - F I 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

F V I I I m i m e t i c a c t i v i t y ( % o f n o r m a l ) M O I = 1 x 1 0

5

M O I = 1 x 1 0

7

M O I = 1 x 1 0

6

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• Some mice were excluded for analyses based on bioanalyses data
• Dose = 5e13 gc/kg

2 4 6 8 1 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 je c t io n F I X p r o t e i n ( % ) 1 2 L 1 2 R 1 L 1 R L 1 X 3 X 1 1 X

A

2 4 6 8 1 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 - in je c t io n F I X p r o t e i n ( % ) 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

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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