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

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

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

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) 65:667-76.

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

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):13763-8, 2012

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