Utrophin and therapy of DMD

PPMD Annual Conference

Kay E. Davies
MRC Functional Genetics Unit,
Henry Wellcome Building of Gene Function
Department of Physiology, Anatomy and Genetics,
University of Oxford

Acknowledgements
Rebecca Fairclough
Dave Powell
Sarah Squire
Ally Potter
Laura Giles

Jon Tinsley
Richard Storer
Graham Wynne

Jon Tinsley
Ed Burton
Qing Bai
Kelly Perkins
Andrew Weir
Rosie Fisher
Thank you to all the families that inspire us to find the cure

Aim: to reconstruct this link by

replacement with an alternative,
but similar protein, utrophin

Evidence for utrophin as a replacement

for dystrophin
Utrophin expression at the sarcolemma in early
development
Structural studies of the actin binding domain of utrophin
and dystrophin show extensive similarities (KendrickJones)
Binds similar complex of proteins at carboxyterminal end
Differences in localisation are transcriptionally controlled
Occupy similar sites at costamere (Ervasti)
Utrophin transgene expression prevents muscular
dystrophy in mdx mice

Questions to be addressed
Can we replace dystrophin with increased utrophin? yes
How much utrophin do we need? 3-4 fold above normal
How early in life and how often would we have to increase
utrophin? as early as possible like dystrophin
Does increased utrophin throughout the body have any
side effects? no
What features of the utrophin promoter enable us to
increase the gene expression?
What drug would work to increase utrophin?

5`Region of the Utrophin Gene

Promoter A

Promoter B

1A 2ADUE

1B

ATG

ATG

120 Kb

Burton et al (1999) PNAS 96:14025-14030.

Utrophin in DMD patients

Nguyen thi Man et al (1991)

J Cell Biol, 115: 1695-1700.

Kleopa et al Hum Mol Genet. 15: 1623-8 (2006)

Naturally occurring utrophin correlates with disease severity in DMD

Important questions
Are there features of the utrophin promoter that
enable us to increase the gene expression?

Proximal promoter region of utrophin B

Perkins et al FEBS Lett (2003) 538:168-72

Utrophin-luciferase
The new PAC-utrophin luciferase construct contains about 35kb upstream region of
promoter 1A and the luciferase gene is in-frame fused within exon 3.
Currently utrophin-luciferase construct used for screening contains ~10kb upstream region of
promoter 1A and the luciferase gene is in-frame fused within exon 1A.
~35kb

LUCIFERASE

10kb

LUCIFERASE

SX

X C C

N
F
A
T

NFATc

1B

1A 2A mDUE

GABP

X C

S
p
1

A
P
2

Utrophin

Myogenic Transcription Activating

regulatory
factor
enhancer-binding
factor
protein-2

~ 5kb

Perkins et al Nucl Acids Res (2001) 29:4873-50 C=ClaI

S=SalI X=XhoI

456
S

Utrophin regulation

Chakkalakal et al FASEB J. 19:880-891(2005)

Regulation of utrophin expression

Taken from Khurana and Davies

Nature Drug Discovery 2003 2: 379

Taken from Davies and Khurana

Nature Med 2007 13: 538

Utrophin in non-muscle tissues

Utrophin is expressed at relatively high levels in
the kidney
A-utrophin is the predominant isoform
Important areas of the promoter for kidney
expression have been identified, but the
mechanism of promoter activation is not clear

Pharmacological approach to increase utrophin

levels
Develop rapid cell based assay
Set up high throughput screen in
collaboration with VASTox plc
Advantage of not being mutation specific,
avoiding the immune response and
potentially targeting all muscles via
systemic delivery

Pharmacological approach to increase utrophin

levels
Elisa assay
Problems because utrophin takes ~16 hours to
transcribe

Transcriptional assay
Need rapid readout

Tissue Culture screen

H2K/mdx cell line stably transfected with a
construct containing promoter A of
utrophin plus a luciferase reporter.
Luciferase levels were measured, any hits
were repeated for reproducibility

In vitro Screening: Identification of Small Molecule Inducers of

Utrophin

Mdx myoblasts cloned from H-2K-tsA58 x mdx

IFN / ts SV40 T Ag controlled proliferation
IFN medium / 34oC -no fusion
Stable integration Hu UTRN promoter luciferase
Significant mu/hu promoter conservation

Unique, patented screen - FTO

ID UTRN inducing compounds
Robust, homogeneous assay
Reproducible
Relevance: human UTRN promoter
HT (>2000 compounds/week)
Simple endpoint

10kb
LUCIFERASE

1A

SX

2A

1B

X CC
N
F
A
T

P
P
R
E

NFATc PPAR GABP

response
element
~ 5kb

XC
S
p
1

Myogenic Transcription
regulatory
factor
factor

A
P
2

C
Utrophin

Activating
enhancer-binding
protein-2

456

DMD- disease modifying profile of an

ideal drug
The primary cause
Reduce membrane damage
The catastrophic secondary effects
Reduce muscle degeneration and necrosis
Reduce fibrosis and fatty deposition within the
gaps
Reduce chronic inflammation
Drug like
Systemic
Synthesis,scale up etc.
Safe

Screening Cascade
mdx mouse is Gold
Standard model for DMD
Constant progression of
compounds through 5
week mdx study

Iterative Cycle

7 week cycle

Chemistry

H2K hit

ZF Tox

ADME

Decision

In vivo PK

Decision

5 week cycle

5 week Mdx

Decision

Nomination

EC50 of active compounds

Cell based screen
H-2K immortal myoblasts
UTRN promoter A -luciferase
Blasts assayed
Luciferase output

Dose dependent luciferase induction

VOX A
VOX B

9000
8000

RFU

7000
6000
5000
4000
3000

VOX A
VOX B

2000
1000
-7.5

EC50
0.8
M
0.9
M

-7.0

-6.5

-6.0

log [cmpd]

-5.5

-5.0

VOX A
Control

Mouse 1

TA

C19 1:150 (20X) 2 sec exposure

Functional rescue with VOX A

25

20

15

% force
drop
10
vehicle mdx
5
treated mdx

0
n=2

% Force drop measured in mdx soleus muscle after 5 stretches.

12 weeks of treatment with compound 185.

Analysis of earlier compound from development series

muscle sizes
0.009

p<0.05

0.008
0.007

CSA (cm2)

0.006
0.005
0.004
0.003
0.002

n=4

n=5

Vehicle

VoxB

0.001
0.000

VOX B reduced the hypertrophy of the mdx EDL

Analysis of earlier compound from development series

80.000

p<0.01
70.000
60.000

% FD

50.000
40.000
30.000
20.000
10.000
0 000

n=5

Vehicle

n=5

VoxB

VOXB improved the integrity of the sarcolemma enough to

significantly prevent damage in eccentric contractions

DMD- disease modifying profile of the

VASTox candidate

The primary cause

Reduce membrane damage
Normalises Ca2+ balance

Protects against forced contraction damage

Normalises relaxation rate
The catastrophic secondary effects
Reduce muscle degeneration and necrosis

Reduction %CNFs and fibre atrophy

Reduce fibrosis and fatty deposition

Reduce chronic inflammation
Drug like
Systemic

ip

Synthesis, scale up etc.
Safe

Daily ip for 28 days

Conclusion

Utrophin upregulation has been achieved in
mdx mice using compounds from a number
of chemical series.

Candidate for pre-clinical development
identified

Aim to do first in man trials in 2008

The ZF Partnership

Medicines for Duchenne Muscular Dystrophy

A drug development programme with

VASTox plc and Parent Project UK
Parent Project MD Annual Meeting
13th July 2007

The Concept single targets vs. black box

Utrophin RNA

Whole
muscle
Bundle of fibres

Fibre membrane
lacking dystrophin

Utrophin Reporter Cell Based Screen

The drug target is a fragment of the
region which controls expression of the
utrophin gene. A positive outcome is an
increase the amount of Utrophin RNA

Dystrophin Deficient Zebrafish Screen

The drug target is any modulator of muscle
growth or development which results in a
reduction in skeletal muscle dystrophy

Direct whole animal screening

Dystrophin deficient zebrafish mutant screen

Screen for compounds stabilising or promoting muscle growth
Opportunity to develop additional chemotypes targeting novel mechanisms

Assay development
Quantify differences in maximal velocity between mutants and sibs (EthoVision
Tracking system)
6

Max. Velocity

5dpf

6dpf

5
6

4
3

2
2

1
0

0
Sibs-Max Velocity

Sibs-Max Velocity

Muts-Max Velocity

Muts-Max Velocity

Quantitative method for measuring mutant

muscle birefringence phenotype

% Area occupied by
high-intensity pixels
(160-225)

80

70

60

50

40

30

20

10

Sibs
n=47
Muts
n=11

Time and cost estimates

Phase 1

VOX
Staff

Cost

Q3 07

Q4 07

41K

30K Library acquisition

Q1 08

Q2 08

30K

Q4 08

ZF Screen

1
3FTE 254K
2FTE

Q3 08

Current Reporter Screen

Total 325K

Phase 2

4FTE

Chemistry

1FTE 480K
5 cmpds/month

Preclinical
Candidates

Outsourcing ADMET, efficacy

Immediate integration into VASTox DMD pre-clinical and clinical infrastructure

Current screening hits

8611 cmpds screened to date

Estimated Completion 30K- end Sept 07

Backup Follow up Programme

VASTox

ZF Partnership

Chemical approaches

Candidate nominated for preclinical

development
Alternative series optimisation

ongoing

Screening Approaches

Transcriptional upregulation of
utrophin

New candidate Q3 08

Chemical approaches

Alternative scaffolds

New libraries

Screening Approaches

Zebrafish dystrophin model

Transcriptional upregulation of
utrophin

New candidates Q3 08

Partnerships

ZF Partnership

Patient Registries

www.dmdregistry.org

TREAT-NMD