1. Duchenne muscular dystrophy (DMD)
(H. Lodish et al., in Molecular Cell Biology, p999)
(I. Nonaka, in Muscle Pathology)
Normal muscle
DMD muscle
H&E
Dystrophin
IHC
・X-linked
・1/3500 among newborn boys
・Mutations in the DMD gene and complete loss of dystrophin from the sarcolemma
・Progressive muscle weakness
・Early death due to respiratory or
heart failure
・Corticosteroid treatment is
effective

2. Generation of micro-dystrophin cDNA
actin-binding
domain
cysteine-rich
domain
C-terminal
domain
rod domain
full-length dystrophin
14 kb
mini-dystrophin
6.4 kb
micro-dystrophin 1 2 3 24
4.9 kb; this truncated micro-dystrophin has an
enough function as a molecule.
Yoshimur M et al. Mol Ther, 2004.
DDys CS1 1 24
DDys AX11
4.4 kb
hinge
2/23
rod repeat
DDys M3
3.7 kb
1/24

3. Introduction of micro-dystrophin by using AAV9 vector
Improvement in fibrosis
24 weeks after the injection
mdx, AAV9-Dys(+)
Sirius Red staining (24 weeks after the injection
AAV9-microdystrophin(+)
mdx
BL10
AAV(-), mdx
Ant.
Rt.
mdx, AAV(-)
Rt.
Ant.
Improvement in LV function
We also reported that AAV-uDys treatment improves cardiac dysfunction in the mdx mice.
AAV-uDys was injected via tail vein, then 24 weeks after the transduction, extensive uDys expression was observed.
This transgene expression sustained for 74 weeks after the transduction.
Cardiac fibrosis observed in the mdx mice was significantly improved with microdystrophin transduction.
Then we also certified that left ventricular dysfunction was considerably improved by some cardiac assessments, such as fractional shortening and tissue doppler.
500 μm
P<0.01
74 weeks after the injection
AAV9-CMV-microdystrophin
3x1012 v.g./body via tail vein
Anti-Dystrophin antibody
Fractional shortening (FS) and Doppler tissular velocities of left ventricular posterior wall in systole (DTVS) and diastole (DTVD)
Shin JH et al., Gene Ther.,18(9):910-9, 2011

4. Systemic delivery of Morpholinos to skip exon 6 and 8, into dystrophic dogs, CXMDJ
Ex6A
Ex6B
Ex8A
(119 bp)
(173 bp)
(182 bp)
(93 bp)
(129 bp)
Out-of-frame mutation
(188 bp)
Alternative splice
Ex 9
Ex 10
Ex 79
STOP
Ex 6
Ex 7
Ex 8
Actin binding domain
Hinge 1 X
Rod
Skipping 474 bp or
603 bp (in frame)
Day 0
observation
Weekly morpholino injection
sacrifice
2wk
MdxマウスについてのX-gal活性での発現効率の相違では、AAV5 はどの時期でも効率が良くなっており、とくに10日齢以下の時期で著しくなっています。ただし、3日齢ではバラツキがあり有意差はみられませんでした。
１. Exon 6 and 8 skipping induced dystrophin expression
and amelioration of the phenotype.
２. No obvious adverse effects
Yokota T, et al., Ann Neurol, (2009) 65:

5. exon51 skipping by Morpholino treatment is effective in mdx52 mice
Non treated　
mdx52
Weekly intravenous injection of Morpholino （1000 nmoles）
Treated　mdx52
Analysis
2 wk
Day 0
Observation GC
Quad GC
EDL
前脛骨筋
Positive fibers (%)
50 mm
0.3 80 63 42
8 wk mdx52
Serum CK level
Tetanic force
U/L
p = 0.003
mN/mm2
p = 0.045
We have challenged exon 51 skipping by morpholino treatment in exon 52 deficient dystrophic mice which our research group developed long time ago.
This is a summary slide of exon51 skipping. We injected morpholino into tail vein 7times and analyzed dystrophin expression in whole body. We found bodywide expression of dystrophin by Immunohistochemistry Especially in Quadriceps muscle and gastrocnemius muscle. Ｓｅｒｕｍ　CK level was significantly decreased and specific tetanic force of EDL muscle was significantly improved. Exon 51 skipping looks favorable. .
But we have a little concerned whether exon 51 skipping is really favorable for dystrophy patients or not.
120
4000
3000 80
2000 40
1000
C57
Non treated
mdx52
Treated
C57
Non-treated
mdx52
Treated
mdx52
n= 5-9
n= 4
Y Aoki et al., Mol Ther, 2010

6. Intravenous injections of vPMOs to skip exon 45 to 55 in mdx52
restore dystrophin expression throughout the body
5xIV vPMOs
BL6TA
Quad TA GC
Bi-weekly i.v. injection
of vPMO (1.2 mg/kg each)
Analysis
2 wks
Day 0
No-treat TA TB
Diaph
Heart
Observation
100 mm
Western blotting
5x IV vPMOs
BL6TA
(10%)
No-treat TA
kDa
Quad TA GC TB
Abd
Para
Diaph
Heart
dystrophin
427
del ex45-55 dys
380
a-tubulin 55
Aoki Y et al., PNAS., 21;109(34): , 2012

7. Molecular Mechanism of PMO Uptake in Skeletal Muscle:
Results of Exon Skipping and BrdU-labeling
Triple staining for dystrophin,
BrdU and DAPI (TA)
Histogram of fibers size
BrdU-labeled
BrdU-unlabeled
Dystrophin positive fibers mm
Dystrophin
BrdU
DAPI
Diameter of muscle fibers
Aoki Y et al., Hum Mol Genet, 2013