(assessed 4 weeks after treatment) · group genotype n treatment dose (mg/kg) endpoints 1 wt 4...
TRANSCRIPT
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)