hemophilia a gene therapy: aav-mediated delivery of an

Hemophilia A gene therapy:

AAV-mediated delivery of an enhanced F8 cassette

to the liver produces supraphysiological levels of

human FVIII in vivo

Brigit E. RILEY

Director Discovery and Translational Research

Sangamo BioSciences

USA

1

Disclosures for: Brigit E. RILEYIn compliance with the PIM* policy, WFH requires the following disclosures be made at each presentation

CONFLICT DISCLOSURE — IF CONFLICT OF INTEREST EXISTS

RESEARCH SUPPORT Sangamo BioSciences

DIRECTOR, OFFICER, EMPLOYEE DIRECTOR

SHAREHOLDER

HONORARIA

ADVISORY COMMITTEE

CONSULTANT

* Postgraduate Institute for Medicine

2

C L I C K TO E D I T M A S T E R T I T L E S T Y L E

3

• Background – Hemophilia A and AAV Gene Therapy

• Overview of AAV Factor 8 cDNA

• Approach to Optimizing AAV Factor 8 cDNA

• Evaluating the Optimized AAV Factor 8 cDNA

– in vitro: HepG2 Cells

– in vivo: Wild Type and Hemophilia A mouse models

– in vivo: Non-Human Primates (NHPs)

• Summary and Next Steps

I N T R O S L I D E / A G E N D A


4










5

• Modest increases in Factor VIII levels (>1% of normal) have a positive impact

on patient lives

• Adeno-associated virus (AAV) vectors have shown efficacy in preclinical and

clinical studies; stable Factor IX (FIX) expression ~ five years in the clinic

• Lag in the clinic of AAV human Factor 8 (hF8) gene therapy – poor vector

yields at clinical scale and dose required to achieve therapeutic FVIII levels

• We optimized an AAV hF8 cDNA vector cassette to improve both vector yields

and liver-specific hFVIII expression

• Administration of the enhanced AAV hF8 cDNA vector in vivo resulted in 2-8X

normal circulating hFVIII levels

* Higher circulating hFVIII levels will enable lower dose in the clinic

H E M O P H I L I A A

A N I D E A L D I S E A S E F O R L I V E R - D I R E C T E D G E N E T H E R A P Y


L I V E R - D I R E C T E D A AV F 8 C D N A G E N E T H E R A P Y

6

P liver- specific promoterTG therapeutic gene (F8)



7

Transgene packaged into

AAV vectorsTherapeutic delivered by a

single infusion

}

}

AAV vectors

P TGtransgene


AAV vectors




8



single infusion

}

}

AAV vectors

P TGtransgene


AAV vectors

AAV is delivered by a

single infusion




9



single infusion

Liver produces and secretes

therapeutic hFVIII protein

}

}

AAV vectors

Liver Cell DNA

Promoter

TherapeuticGene (hF8)

Nucleus

Liver Cell

P TGtransgene


AAV vectors


single infusion

AAV traffics to liver to deliver

transgene into nucleus of liver cells




10



single infusion



}

}

AAV vectors

Liver Cell DNA

Promoter


Nucleus

Liver Cell

Transgene is expressed from the liver,

but remains separate from the cell’s DNA

P TGtransgene


AAV vectors


single infusion






11



single infusion



}

}

AAV vectors

Liver Cell DNA

Promoter


Nucleus

Liver Cell

Transgene is expressed from the liver,

but remains separate from the cell’s DNA

P TGtransgene


AAV vectors


single infusion



W I L D T Y P E A AV

12

• Single-stranded DNA virus which requires a helper virus for replication

• No pathology associated with AAV infection

• Tissue selectivity is determined by capsid composition

Vance et al, DOI: 10.5772/61988

Liver,

Heart,

Skeletal

muscle

Liver,

Heart, and

muscle

Heart, Liver Liver, Heart,

Brain, Lung,

Skeletal

muscle

Heart,

Lung, Liver

Liver Liver,

Heart,

Skeletal

muscle

Liver,

Skeletal

muscle

Liver,

Heart,

Brain,

Muscle

Tissue Selectivity of AAV Serotypes

AAV1 AAV2 AAV3 AAV4 AAV5 AAV6 AAV7 AAV8 AAV9

R E C O M B I N A N T A AV

13

• Recombinant AAV (rAAV) has been used extensively for nearly 20 years as a

gene therapy vector in preclinical and clinical studies

• Efficient transduction and long term, stable transgene expression in

non-dividing tissues:

- Liver, brain and muscle

• Replication deficient

• High degree of stability which allows for rigorous methods of vector

purification

• AAV vectors carrying capacity is small (~4.7 kb of DNA)

G E N E R AT I O N O F R E C O M B I N A N T A AV

14

ITR ITR

Transgene of Interest

Recombinant AAV

• Contains the transgene in place of

wild type genes

• Harvest crude rAAV extract

• Purify rAAV

- Density gradient

- Column purification

Highly purified rAAV

Within Cell

• Capsid Assembly

• Virus Packaging

Manufacturing rAAV

ITR ITR

Wild-Type AAV

Replace wild type

genes

wild type genes

ITR ITR

Transgene

of interest

+cell line

• Helper plasmid is supplied in trans, together with the

transgene to a packaging cell line

Helper plasmid


15










W H AT I S O P T I M A L F O R A AV H F 8 C D N A ?

16

hF8 is not an ideal gene for AAV:

• Constrained by hF8 gene size

– Optimal AAV transgene size is ~4.7 kb; full length hF8 is ~7 kb

• AAV dose required to achieve therapeutic hFVIII levels

– Low efficiency of transcription/translation

• Low manufacturing yields of AAV hF8

– Clinical scale manufacturing feasibility is limiting

Optimal AAV hF8 cDNA requires:

• Shorter coding sequence for hF8

– Potential solution is the use of an optimized B-domain deleted sequence (BDD)

• An optimized robust liver-specific promoter module to drive hF8 expression

• Improved virus yields


17










O P T I M I Z AT I O N O F A AV H F 8 C D N A R E Q U I R E D

M U LT I - FA C TO R I A L M O D I F I C AT I O N S

18

ITR

Liver Promoter Human Factor 8 B-Domain Deleted (BDD) polyA

ITR

Promoter module

modifications

• Assembled different permutations of

liver-specific promoter elements

• Identified regions of the promoter

module that could be improved upon

• For other elements a systematic

mutational design approach was used

Transgene

modifications

• Optimized the F8 cassette

Other modifications

• Identified minimal

synthetic polyA

• Removed un-necessary

nucleic acids

• Reduced size

• Optimized sequences

outside transgene hFVIII protein has the same amino acid sequence

as biologics currently in clinic

I T E R AT I V E P R O C E S S TO I D E N T I F Y A N I M P R O V E D

A AV H F 8 C D N A C A S S E T T E

19

• Virus Yields

• In Vitro secreted

hFVIII levels

(mRNA/protein) and

activity

• In Vivo circulating

hFVIII levels

(mRNA/protein)

Transgene

modifications

Promoter module

modifications

Other

modifications

3 .01 0 12

6 .01 0 12

9 .01 0 12

1 .21 0 13

1 .51 0 13

1 .81 0 13

2 .11 0 13

2 .41 0 13

2 .71 0 13

3 .01 0 13

Yie

ld (

vg

)

M O D I F I C AT I O N S I M P R O V E V I R U S Y I E L D

20

Stage 4Stage 3Stage 2Stage 1

Iterative Process to Improve Cassette

3 .01 0 12

6 .01 0 12

9 .01 0 12

1 .21 0 13

1 .51 0 13

1 .81 0 13

2 .11 0 13

2 .41 0 13

2 .71 0 13

3 .01 0 13

Yie

ld (

vg

)


21



3 .01 0 12

6 .01 0 12

9 .01 0 12

1 .21 0 13

1 .51 0 13

1 .81 0 13

2 .11 0 13

2 .41 0 13

2 .71 0 13

3 .01 0 13

Yie

ld (

vg

)


22



3 .01 0 12

6 .01 0 12

9 .01 0 12

1 .21 0 13

1 .51 0 13

1 .81 0 13

2 .11 0 13

2 .41 0 13

2 .71 0 13

3 .01 0 13

Yie

ld (

vg

)


23



8-10 fold

improvement

at the cell

factory scale

At clinical scale; greater than 5-fold improvement in vector yields


24










S E V E R A L M E T H O D S U S E D TO A S S E S S H F V I I I

L E V E L S A N D A C T I V I T Y

25

hFVIII

Enzyme-Linked

Immunosorbent Assay (ELISA)

LEVELSSpecific for human FVIII

magnet magnet

Clotting times

Clotting Assay

FVIII

deficient

plasma

hFVIII

Activator +

phospholipids

Add Ca2+

Generation of Factor Xa and

thrombin yielding clot formation

ACTIVITYNot specific for human FVIII

Factor X

Factor Xa

Factor Xa

Factor IXa,

Ca2+, Phospholipid

hFVIII

Chromophore

405 nmChromogenic

substrate

Chromogenic Assay

ACTIVITYNot specific for human FVIII

I N V I T R O : H E P G 2 C E L L S

E X P E R I M E N TA L D E S I G N

26

50

Test article

addition

7 days

Supernatant collection schedule

Optimized rAAV hF8 cDNA cassette packaged into AAV2/6

Endpoints• ELISA for hFVIII levels

• Clotting assay for hFVIII activity

• Chromogenic assay for

hFVIII activity

AAV2

ITR

Liver-specific

promoter module

PolyAhFVIII BDDAAV2

ITR

3

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

0

2 0

4 0

6 0

8 0

1 0 0

hF

VII

I (P

erc

en

t N

orm

al)

hF

VII

I A

cti

vit

y (

U/m

L)

C lo tt in g A s s a y

C h ro m o g e n ic A s s a y

E L IS A

D o s e

I N V I T R O : H E P G 2 C E L L S

G O O D C O R R E L AT I O N B E T W E E N H F V I I I A C T I V I T Y / L E V E L S

27

Values will be reported as hFVIII (Percent Normal) for

ELISA, Chromogenic or Clotting where 1 U/mL = 100% Normal

4.8E6 / 2.4E6 / 1.2E6 / 0.6E6 MOI


28










I N V I V O : W I L D T Y P E M O U S E D ATA


29


AAV2

ITR

Liver-specific

promoter module

PolyAhFVIII BDDAAV2

ITR


• hF8 mRNA tissue

biodistribution

Dose

• 7.2E+12 vg/kg

70 14 21 28 days

Plasma collection schedule

Immunosuppression regimen of cyclophosphamide (50 mg/kg)

IV injection of

test article


S U P R A P H Y S I O L O G I C A L L E V E L S

30Levels were determined by ELISA

hFVIII Levels

F o rm u la tio n A A V h F 8 c D N A

0

1 0 0

2 0 0

3 0 0

4 0 0

hF

VII

I (P

erc

en

t N

orm

al)

2 4 1 .6 %


H F 8 E X P R E S S I O N I S R E S T R I C T E D TO L I V E R

31

hF8 mRNA

Represents two independent

mouse studies

Liv

er

Bra

in

Kid

ney

Teste

s

Hear t

Sp

leen

Lu

ng

0

2

4

6

8

No

rm

ali

ze

d h

FV

III

mR

NA

Va

lue

s

I N V I V O : H E M O P H I L I A A M O U S E D ATA


32


AAV2

ITR

Liver-specific

promoter module

PolyAhFVIII BDDAAV2

ITR

Dose

• 7.2E+12 vg/kg

70 14 21 28 days 3 months


Endpoint• Chromogenic assay for

hFVIII activity

• Tail vein transection

(TVT) for hemostasis

IV injection of

test article


O V E RV I E W O F H E M O P H I L I A A R 5 9 3 C M I C E

33

• Hemophilia A R593C mice are tolerized to hFVIII because they contain a

hF8-R593C transgene under control of a murine albumin promoter

• hFVIII-R593C is frequently observed in Hemophilia A patients, and in mice

produces no detectable hFVIII protein

- Thought to be rapidly degraded in mice, with peptide fragments

presented to the immune system

• Mice also contain a knockout of the mouse F8 gene and are deficient for

endogenous mouse FVIII protein

• Studies were conducted in collaboration with Dr. David Lillicrap at

Queen’s University



34

Day 14 Day 42

Activity determined by Chromogenic Activity Assay

hFVIII Activity


0

2 0 0

4 0 0

6 0 0

hF

VII

I (P

erc

en

t N

orm

al)

3 3 4 .1 %


0

2 0 0

4 0 0

6 0 0

hF

VII

I (P

erc

en

t N

orm

al)

3 3 0 .9 %


R E D U C E D B L E E D T I M E I N T R E AT E D M I C E

35

TVT method based on

Johansen et al., Haemophilia, 1-7, 2016.

Tail Vein Transection (TVT)


0

1 0

2 0

3 0

4 0

5 0

p < 0 .0 0 0 1

To

tal

Ble

ed

ing

Tim

e (

min

)

Normal bleeding time


36










I N V I V O : N O N - H U M A N P R I M AT E D ATA


37

Optimized rAAV hF8 cDNA cassette

packaged into AAV2/6

AAV2

ITR

Liver-specific

promoter module

PolyAhFVIII BDDAAV2

ITR

Doses

• 2.0E+12 vg/kg

• 6.0E+12 vg/kg

70 14 21 28 days 247 days


Immunosuppression regimen of Rituxan and Solu-Medrol (10 mg/kg for both)

IV injection of

test article


• Clotting for hFVIII activity

• Bethesda Units (BU) for

inhibitory hFVIII antibodies

• Liver enzymes




hFVIII Levels

2 E + 1 2 6 E + 1 2

1 0

1 0 0

1 0 0 0

T o ta l D o s e (v g /k g )

hF

VII

I (P

erc

en

t N

orm

al)

4 8 1 .6 %6 5 4 .1 %

Follow up dose-finding

studies are aimed at

determining the minimal

effective dose for the clinic

• Dose selection was based on

published studies


G O O D C O R R E L AT I O N B E T W E E N A C T I V I T Y / L E V E L S

39

Shown in the Formulation Group:

• Detection of ~100 % Normal NHP

FVIII activity

• No detection of NHP FVIII levels given

the ELISA is specific for human FVIII

1101

1101

1102

1102

3101

3101

3102

3102

3103

3103

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

A n im a l ID

hF

VII

I (P

erc

en

t N

orm

al)

A c tiv ity E L IS A L e v e ls

F o rm u la tio n 6 E + 1 2 v g /k g G ro u p

0 5 0 1 0 0 1 5 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

0

1

2

3

D a y s P o s t D o s in g

hF

VII

I (P

erc

en

t N

orm

al)

BU

/mL

S o lu -M e d ro l

R e m o v e d

3 1 0 2 A n t ig e n L e v e ls

3 1 0 2 B e th e s d a U n its (B U )


D U R A B I L I T Y

40

Stable hFVIII levels for over 8-weeks in the

absence of all immunosuppression

Levels were determined by ELISA

0 5 0 1 0 0 1 5 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

0

1

2

3


hF

VII

I (P

erc

en

t N

orm

al)

BU

/mL



S o lu -M e d ro l

R e m o v e d

I N V I V O : N H P I M M U N E TO L E R A N C E C H A L L E N G E

D U R A B I L I T Y

41

• A simplified view of tolerance is induction of B-cell/T-cell anergy

and/or apoptosis in the presence of sustained levels of agent (hFVIII)

• Do the sustained hFVIII levels produced from the rAAV-hF8 prevent

re-induction of neutralizing antibodies?

• rAAV-hF8 treated NHP were challenged with hFVIII biologic

• hFVIII biologic challenge consisted of 4 weekly infusions of 25 U/kg

of Xyntha®

I N V I V O : N H P I M M U N E TO L E R A N C E C H A L L E N G E

N O A P P E A R A N C E O F I N H I B I TO RY A N T I B O D I E S

42

+ Regions shaded gray are

above the BU cutpoint thus

positive for inhibitory antibodies

Control Group

1 5 0 2 0 0 2 5 0

0

5 0

1 0 0

1 5 0

2 0 0

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0


hF

VII

I (P

erc

en

t N

orm

al)

BU

/mL



X y n th a

C h a l le n g e

P o s it iv e fo r

In h ib ito ry A n t ib o d ie s

AAV hF8 cDNA Dose Groups

hFVIII levels ~150% hFVIII levels ~10%

1 5 0 2 0 0 2 5 0

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

6 0 0

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0


hF

VII

I (P

erc

en

t N

orm

al)

BU

/mL



X y n th a

C h a l le n g e

N o E v id e n c e o f


1 5 0 2 0 0 2 5 0

0

1 0

2 0

3 0

4 0

5 0

6 0

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0


hF

VII

I (P

erc

en

t N

orm

al)

BU

/mL



X y n th a

C h a l le n g e

N o E v id e n c e o f



W E L L TO L E R AT E D

43

ALT = Alanine Aminotransferase, upper limit of normal,126 U/L

AST = Aspartate Aminotransferase, upper limit of normal,120 U/L

*Elevated levels observed post-liver biopsies (day 41)

Control Group High Dose Group

0 5 0 1 0 0 1 5 0

0

1 0 0

2 0 0

3 0 0

4 0 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0


Liv

er E

nz

ym

e (

U/L

)

hF

VII

I (P

erc

en

t N

orm

al)

A S T

A L T

*

0 5 0 1 0 0 1 5 0

0

1 0 0

2 0 0

3 0 0

4 0 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0


Liv

er E

nz

ym

es

(U

/L)

hF

VII

I (P

erc

en

t N

orm

al)

A S T

A L T

*upper limit of normal upper limit of normal

0 5 0 1 0 0 1 5 0

0

1 0 0

2 0 0

3 0 0

4 0 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0


Liv

er E

nz

ym

es

(U

/L)

hF

VII

I (P

erc

en

t N

orm

al)

A S T

A L T

*

h F V III L e v e ls

0 5 0 1 0 0 1 5 0

0

1 0 0

2 0 0

3 0 0

4 0 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0


Liv

er E

nz

ym

e (

U/L

)

hF

VII

I (P

erc

en

t N

orm

al)

A S T

A L T

*

h F V III L e v e ls


W E L L TO L E R AT E D

44

ALT = Alanine Aminotransferase, upper limit of normal,126 U/L

AST = Aspartate Aminotransferase, upper limit of normal,120 U/L

*Elevated levels observed post-liver biopsies (day 41)

Control Group High Dose Group

upper limit of normal upper limit of normal


P R E L I M I N A RY R E S U LT S D O S E - F I N D I N G S T U D Y


hFVIII Levels

Preliminary results.

Dose-finding study

demonstrates high hFVIII

production from GMP-clinical

scale manufacturing process

6 E + 1 1 9 E + 1 1 2 E + 1 2 6 E + 1 2

1

1 0

1 0 0

1 0 0 0

T o ta l D o s e (v g /k g )

hF

VII

I (P

erc

en

t N

orm

al)

5 .7

5 6 .4

2 2 9 .0

1 2 .0

S U M M A RY A N D F U T U R E P L A N S

46

• Administration of AAV hF8 cDNA, engineered to improve vector yields and

liver-specific hFVIII expression, resulted in supraphysiological levels in vivo- Wild type mice

- Hemophilia A R593C mice

- NHPs

• Good correlation between assays used to measure circulating hFVIII protein- Levels by ELISA and activity by Chromogenic or Clotting assays

• Sustained hFVIII levels from the rAAV-hF8 prevented re-induction of

neutralizing antibodies with biologic challenge suggestive of induced tolerance

(even in the context of a xenogeneic setting)

• Ongoing studies are aimed at determining the minimal effective dose

• Goal of filling the IND, second half of 2016

A C K N O W L E D G E M E N T S

47

Richard Surosky

Alicia Goodwin

Andrea Kang

Tim Gabriele

Hung Tran

Jennifer Huang

Judy Greengard

Lisa King

Eudean Garces

Stephen Ballaron

Daniel Richards

Melanie Butler

Carolyn Gasper

Kathy Meyer

Dale Ando

Mike Holmes

Jeff Boonsripisal

Derek Liu

Rainier Amora

Lei Zhang

David Lillicrap

Christine Hough

Dominique Cartier

Kate Nesbitt

Courtney Dwyer

Kassandra Herbert

hemophilia a gene therapy: aav-mediated delivery of an

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