IV Injection

Treatment in mdx mice with FORCE-M23D:

• Promotes exon skipping

• Significantly restores dystrophin protein expression in muscles

including quadriceps, diaphragm and heart

• Significantly improves muscle function and health

• Significant reduction in secreted creatine kinase

• Improvement in multiple functional assessments

• Restoration of dystrophin expression in muscle membrane

• Delivers sustained therapeutic effect for at least 4 weeks

Targeted Delivery of ASOs Demonstrates Potential to Treat Duchenne Muscular Dystrophy

Monica Yao, Nelson Hsia, Kim Tang, Christina Shen, Leticia Fridman, Tim Weeden,

Mo Qatanani, Oxana Beskrovnaya, Romesh SubramanianDyne Therapeutics Inc., Massachusetts, USA

INTRODUCTION

Single-dose response study in the mdx mouse

model

Single-dose functional study in mdx mouse

model

Mdx mouse contains nonsense mutation in exon 23 of dystrophin gene, dystrophin not expressed

Key:

IV: intravenous dose

WT: Wild-type

RT-PCR: reverse transcription polymerase chain reaction

IHC: immunohistochemistry

ASO: antisense oligonucleotide

FORCE: Mouse Tfr1 targeting antibody ASO therapeutic

M23D: ASO targeting exon 23 mutation in mdx mouse. Utilized as “naked”

Figure 4. Dose dependent increase of dystrophin protein expression in mdx

mice.

(A) Western blot results showing the dystrophin levels in quadriceps two weeks

after a single IV dose. Mice were dosed with FORCE-M23D at 10, 20, 30 mg/kg.

Dystrophin production was detected in the heart as well (western blot not depicted

above).

(B) Bar graph representation of quadriceps western blot results. Each bar

represents an average n=3 mice except at 10mg/kg where n=2.

METHODS

CONCLUSIONS

Single dose of FORCE-M23D significantly decreases secreted creatine kinase in mdxmodel

FORCE achieves effective exon skipping in mdx

model

Figure 3. Exon-23 skipping in mdx mice. RT-PCR results showing the

exon skipping levels in different tissues 14 days after a single IV dose. Mice

were injected with 10, 20, 30 mg/kg of FORCE-M23D. Each bar represents

an average of 4 mice. Wild-type and mdx mice treated with saline show no

exon skipping in the tissues tested (not depicted in the graph).

RESULTS

www.dyne-tx.com

Figure 2. Dyne: Pioneering targeted therapies for muscle disease.

Rendering of Dyne therapeutic; not exact molecule

Figure 1. Duchenne Muscular Dystrophy (DMD) Overview.

1 4 64 32 16

20

mg/kg

Std. Curve (% WT) 10

mg/kg

30

mg/kg

Standard curve - Used pooled WT protein and pooled mdx protein, % indicates amt. of WT spiked into sample

Quadriceps

Dystrophin

427 kDa

Alpha-Actinin

103 kDa

0

Figure 8. Immunofluorescent staining of dystrophin expression in mdx mice

at 4 weeks post-dose.

Results showing dystrophin expression in quadriceps at four weeks after single IV

dose at 30mg/kg. Control wild-type (WT) and mdx mice were dosed with saline.

Figure 7. Home Wheel Running in mdx mice.

Functional results showing the distance travelled (meters) over a 24-hour

period, light and dark cycles, at four weeks post-treatment. Each data point

represents an average of 5 mice. Mice were dosed with M23D or FORCE-M23D

therapeutic at 30mg/kg. Control wild-type (WT) and mdx mice were treated with

saline.

Figure 5. Decrease in creatine kinase levels in mdx mice.

Results showing creatine kinase levels after 2 and 4-weeks treatment after a

single 30mg/kg IV dose of M23D or FORCE-M23D. Control wild-type (WT) and

mdx mice were dosed with saline. ( ****<0.0001).

A.

B.

Mouse activity wheel

with dual lickometer

FORCE-M23D results in dose-dependent

dystrophin production

Hindlimb FatigueChallenge

Home Cage

Running Wheel

2wd0 1wC57BL/10ScSn-Dmdmdx/J

IV injection

d0 3w1w 2w 4wC57BL/10ScSn-

Dmdmdx/J

Single dose of FORCE-M23D restores dystrophin expression to muscle membrane based on IHC

Quadriceps

Single dose of FORCE-M23D demonstrates

functional benefit in mdx model

Figure 6. Hind Limb Fatigue Challenge in mdx mice.

Functional results showing the percent change in total distance travelled in an

open-field after hind limb fatigue challenge at two weeks post-treatment. Each

bar represents an average of 5 mice. Mice were dosed with M23D or FORCE-

M23D therapeutic at 30mg/kg. Control wild-type (WT) and mdx mice were treated

with saline. ( **<0.01, ***<0.001, NS not significant).

0 10 20 30

0

5

10

15

%W

T

Dy

str

op

hin

(Mean +

/- S

D)

Dose (mg/kg)

• Muscle-specific TfR1 affinity

• Selectivity for TfR1 vs. TfR2

• Optimized for internalization

Linker

• Precise conjugation

• Circulating stability

• Endosomal release

Antibody• Matched to target biology

• Target specificity

• Chemistry & design

Oligo

Studies were conducted blinded at third-party CRO.

WT-s

alin

e

mdx-

salin

e

mdx-

M23

D

mdx-

FORCE-M

23D

-100

-80

-60

-40

-20

0

Distance Traveled In Open FieldFollowing Hind Limb Fatigue Challenge

(assessed 2 weeks after treatment)

% C

han

ge

in

To

tal

Dis

tan

ce T

ravele

d

**

***

NS

13:0

0

14:0

0

15:0

0

16:0

0

17:0

0

18:0

0

19:0

0

20:0

0

21:0

0

22:0

0

23:0

0

24:0

01:

002:

003:

004:

005:

006:

007:

008:

009:

00

10:0

0

11:0

0

12:0

0

0

250

500

750

1000

1250

Distance Traveled in Home Cage Running Wheel

(assessed 4 weeks after treatment)

Dis

tan

ce T

ravele

d (

m)

Du

rin

g R

un

nin

g W

heel S

essio

n

LightPeriod

Light PeriodDark Active Period

WT-saline

mdx-saline

mdx-M23D

mdx-FORCE-M23D

2-weeks post dose 4-weeks post dose

0

2

4

6

8

10

12

Creatine Kinase Levels

Rela

tive C

K A

cti

vit

y C

han

ge

No

rma

lized

to

W

T s

alin

e

WT-saline

mdx-saline

mdx-M23D

mdx-FORCE-M23D

✱✱✱✱ ✱✱✱✱

WT-Saline mdx-Saline

mdx-FORCE-M23Dmdx-M23D

IHC

Group Genotype N Treatment Dose

(mg/kg)

Endpoints

1 WT 4 Saline 0 • Exon skipping (RT-

PCR)

• Dystrophin Expression

(Western blot)

2 mdx 4 Saline 0

3 mdx 4 FORCE-M23D 10

4 mdx 4 FORCE-M23D 20

5 mdx 4 FORCE-M23D 30

Pe

rce

nt E

xo

n S

kip

pin

g

Quadriceps

Pe

rce

nt E

xo

n S

kip

pin

g

Diaphragm

Pe

rce

nt E

xo

n S

kip

pin

g

Heart

10 20 30

0

20

40

60

80

100

10 20 30

0

20

40

60

80

100

10 20 30

0

20

40

60

80

100

Total FORCE-M23D Dose (mg/kg)

Duchenne muscular dystrophy (DMD) is an X-linked

neuromuscular disease caused by mutations in the dystrophin

gene that lead to disruption in the mRNA reading frame,

resulting in lack of dystrophin protein production. It is the most

common pediatric-onset form of muscular

dystrophy, characterized by progressive muscle degeneration

and weakness that leads to early mortality as a result of

respiratory and cardiac failure.

Antisense oligonucleotides (ASOs) are promising therapeutics

that can alter the mRNA reading frame through exon skipping

to restore a more complete, functional dystrophin protein.

However, the efficacy of current exon skipping ASO therapies

has been limited by poor delivery and uptake to muscle cells.

The FORCE platform, an antibody-mediated targeting

technology developed by Dyne Therapeutics, improves

delivery of therapeutic oligonucleotides to skeletal, cardiac

and smooth muscles. We utilized the mdx mouse, a well-

established animal model that mimics key aspects of human

DMD pathophysiology, to evaluate the therapeutic potential of

a proprietary antibody-ASO therapeutic. A single dose of the

FORCE-M23D therapeutic increases exon skipping and

dystrophin protein production in the mdx model. Increased

dystrophin levels improved muscle function as measured by

multiple functional assessments. These data support the

therapeutic potential of Dyne's muscle-targeted

oligonucleotides for the treatment of key muscle groupsimportant in the pathobiology of DMD.

Open field test (10 min)

Open field test (10 min)

Distance recorded 1

Distance recorded 2

Fatigue

challenge

(10 min)