Development of an AAV5 gene therapy for Fabry disease Ying Poi - - PowerPoint PPT Presentation

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

• 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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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 symptoms in red: late symptoms

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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

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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 and Kytidou et al. 2017 (PMID: 28680430) Licenced from Prof. Sakuraba, Tokyo

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Two approaches: liver specific or constitutive promoter

AAV5 ModNAGA

C1 NAGA coNAGA spNAGA SV40pA GLA signal peptide L1

• r

ModNAGA L1 Liver produces and secretes protein can be taken up into target organs C1 Constitutive protein expressoin from target

• rgans

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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

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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.

250 500 750 1000 1250 1500 1750 2000

GLA activity (nmol/h/mL) M6P no M6P

N A G A u n t r . N A G A u n t r .

25 50 75 100 125

GLA activity (nmol/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

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Glycosylation pattern of in vitro expressed modNAGA indicates presence of high mannose oligosacharides

modNAGA

Cells Sup

PNGase endoH

• 50

37

M KDa

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

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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 - vehicle vehicle AAV5-C7-NAGA AAV5-C7-coNAGA AAV5-EF1a-NAGA AAV5-EF1a-coNAGA 4 8 12 16 20 25 50 75 100

GLA-activity (nmol/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

w t

• v

e h i c l e v e h i c l e A A V 5

• C

7

• N

A G A A A V 5

• C

7

• c
• N

A G A A A V 5

• E

F 1 a

• N

A G A A A V 5

• E

F 1 a

• c
• N

A G A 60 120 180 240 300 360 420

lysoGb3 (pmol/ml)

AAV AAV wt - v vehic AAV AAV

Wt mice vehicle L1- NAGA L1- coNAGA C1- NAGA C1- coNAGA

Fabry mice

2 to 8wks

2 4 12 wks

/sac.

6 8 10

2 to 12wks

Collaboration with Hans Aerts, Leiden and Carlie de Vries, Amsterdam

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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 10 20 30 40 50 60

liver

12 wks post IV lysoGb3 (pmol/mg protein) Wt Vehicle Fabry Vehicle AAV5-C7-NAGA AAV5-C7-coNAGA AAV5-EF1a-NAGA AAV5-EF1a-coNAGA

5 10 15

kidney

12 wks post IV lysoGb3 (pmol/mg protein) Wt Vehicle Fabry Vehicle AAV5-C7-NAGA AAV5-C7-coNAGA AAV5-EF1a-NAGA AAV5-EF1a-coNAGA

2 4 6

heart

12 wks post IV lysoGb3 (pmol/mg protein) 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

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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

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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
• rgans of AAV-injected Fabry mice (> 6 mo)
• Determine AAV vector distribution and expression in target
• rgans (>6 mo)
• Glycosylation characterization of in vivo expressed

modNAGA

• Detect Zebrabodies in kidney cells and localization of active

modNAGA in lysosomes

Conclusions Future plans

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Acknowledgements

• Chi-Lin Kuo
• Maria J. Ferraz
• Johannes M.F.G. Aerts

Research

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

Immunology

Valerie Ferreira

Vector and process development

Erich Ehlert Tamar Grevelink Mustafa Kyamil Richard van Logtenstein Maroeska Oudshoorn Mark van Veen Jacek Lubelski

• Emily Mallet
• H. Fogg
• Linda Tan
• Aurelia Gondrand
• Dhivina Gagoomal

Analytical development

Eddy Berthier Monika Golinska Jaap Twisk

• Carlie J.M. de Vries
• Roelof Ottenhoff
• Jan Aten
• Histoshi Sakuraba
• Ryoko Tsukahara
• Juan Manuel Iglesias
• Michael Roberts

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GLA and NAGA protein expression

NAGA GLA

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liver

7 wks post IV Genome copies /g DNA 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

5 10 15 30 60 90 120

plasma

2, 4 and 7 wks post IV GLA-activity (nmol/h/mL) Vehicle AAV5-L1-NAGA AAV5-L1-coNAGA AAV5-L1-spNAGA Vehicle AAV5-C1-NAGA

GLA-activity Vector DNA

AAV-vector Wild type mice IV injected with 5e13 gc/kg AAV5-modNAGA

2 4 7 wks

/sac.

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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

50 100 500 1000 1500

liver

7 wks post IV GLA-activity (nmol/h/mg protein) Vehicle AAV5-C7-NAGA AAV5-C7-coNAGA AAV5-C7-spNAGA Vehicle AAV5-EF1a-NAGA

50 60 80 100 120

kidney

7 wks post IV GLA-activity (nmol/h/mg protein) Vehicle AAV5-C7-NAGA AAV5-C7-coNAGA AAV5-C7-spNAGA Vehicle AAV5-EF1a-NAGA

2 4 6 8 10

heart

7 wks post IV GLA-activity (nmol/h/mg protein) Vehicle AAV5-L1-NAGA AAV5-L1-coNAGA AAV5-L1-spNAGA Vehicle AAV5-C1-NAGA

GLA-activity in organs

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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 30 60 90 500 1000 1500 2000 2500

liver

12 wks post IV GLA-activity (nmol/h/mL) Wt Vehicle Fabry Vehicle AAV5-L1-NAGA AAV5-L1-coNAGA AAV5-C1-NAGA AAV5-C1-coNAGA

GLA-activity

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Vector DNA and NAGA mRNA expression in target organs

• f Fabry (GLA-KO) mice upon AAV5-modNAGA injection

101 102 103 104 105 106 107

liver

12 wks post IV Genome copies /g DNA 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 Genome copies /g DNA 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 Genome copies /g DNA 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 NAGA copies /g RNA Wt Vehicle Fabry Vehicle AAV5-C7-NAGA AAV5-C7-coNAGA AAV5-EF1a-NAGA AAV5-EF1a-coNAGA LLOQ

* *

* below LLOQ

103 104 105 106 107 108 109

kidney

12 wks post IV NAGA copies /g RNA Wt Vehicle Fabry Vehicle AAV5-C7-NAGA AAV5-C7-coNAGA AAV5-EF1a-NAGA AAV5-EF1a-coNAGA LLOQ

* *

* below LLOQ

103 104 105 106 107 108 109

heart

12 wks post IV Genome copies /g DNA AAV5-L1-NAGA AAV5-L1-coNAGA AAV5-C1-NAGA AAV5-C1-coNAGA

* *

* below LLOQ Wt Vehicle Fabry Vehicle LLOQ