In-frame dystrophin following exon 51-skipping improves muscle pathology and function in the exon 52-deficient mdx mouse.
A promising therapeutic approach for Duchenne muscular dystrophy (DMD) is exon skipping using antisense oligonucleotides (AOs). In-frame deletions of the hinge 3 region of the dystrophin protein, which is encoded by exons 50 and 51, are predicted to cause a variety of phenotypes. Here, we performed functional analyses of muscle in the exon 52-deleted mdx (mdx52) mouse, to predict the function of in-frame dystrophin following exon 51-skipping, which leads to a protein lacking most of hinge 3. A series of AOs based on phosphorodiamidate morpholino oligomers was screened by intramuscular injection into mdx52 mice. The highest splicing efficiency was generated by a two-oligonucleotide cocktail targeting both the 5' and 3' splice sites of exon 51. After a dose-escalation study, we systemically delivered this cocktail into mdx52 mice seven times at weekly intervals. This induced 20-30% of wild-type (WT) dystrophin expression levels in all muscles, and was accompanied by amelioration of the dystrophic pathology and improvement of skeletal muscle function. Because the structure of the restored in-frame dystrophin resembles human dystrophin following exon 51-skipping, our results are encouraging for the ongoing clinical trials for DMD. Moreover, the therapeutic dose required can provide a suggestion of the theoretical equivalent dose for humans.
Aoki
Yoshitsugu
Y
Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.
Nakamura
Akinori
A
Yokota
Toshifumi
T
Saito
Takashi
T
Okazawa
Hitoshi
H
Nagata
Tetsuya
T
Takeda
Shin'ichi
S
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D003402:Creatine Kinase / Q000097:blood; D016189:Dystrophin / Q000502:physiology*; D005091:Exons / Q000502:physiology*; D020869:Gene Expression Profiling; D015316:Genetic Therapy; D007124:Immunoenzyme Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000473:pathology*; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D020411:Oligonucleotide Array Sequence Analysis; D016376:Oligonucleotides, Antisense / Q000627:therapeutic use*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015415:Biomarkers; D016189:Dystrophin; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D003402:Creatine Kinase
Biochem Biophys Res Commun. 1997 Sep 18;238(2):492-7
9299538
Genomics. 1988 Jan;2(1):90-5
3384440
Mol Ther. 2006 Sep;14(3):401-7
16753346
Neurology. 1989 Aug;39(8):1011-7
2668783
Neuromuscul Disord. 2010 Apr;20(4):251-4
20153965
Hum Mol Genet. 1998 Jul;7(7):1083-90
9618164
Gene Ther. 2010 Jan;17(1):132-40
19759562
Ann Hum Genet. 2005 May;69(Pt 3):253-9
15845029
Neuromuscul Disord. 1999 Jul;9(5):330-8
10407856
Proc Natl Acad Sci U S A. 2005 Jan 4;102(1):198-203
15608067
PLoS Genet. 2010 May 20;6(5):e1000958
20502633
Am J Hum Genet. 1995 Jul;57(1):22-33
7611292
Neuromuscul Disord. 2002 Oct;12 Suppl 1:S71-7
12206800
N Engl J Med. 2007 Dec 27;357(26):2677-86
18160687
Am J Phys Med. 1979 Feb;58(1):26-36
434131
Am J Respir Cell Mol Biol. 2005 Aug;33(2):178-85
15860797
Mol Ther. 2004 Nov;10(5):821-8
15509500
BMC Mol Biol. 2007 Jul 02;8:57
17601349
Arch Med Res. 1993 Spring;24(1):1-6
8292871
Am J Hum Genet. 1989 Oct;45(4):498-506
2491009
J Biol Chem. 1990 Mar 15;265(8):4560-6
2407739
Hum Mol Genet. 2003 Aug 1;12(15):1801-11
12874101
J Gene Med. 2006 Feb;8(2):207-16
16285002
Hum Mol Genet. 2001 Jul 15;10(15):1547-54
11468272
Am J Pathol. 1984 Sep;116(3):482-96
6476081
Nat Med. 2003 Aug;9(8):1009-14
12847521
Am J Hum Genet. 1989 Oct;45(4):507-20
2491010
Mol Ther. 2010 Jun;18(6):1210-7
20407428
Mol Ther. 2004 Aug;10(2):232-40
15294170
Muscle Nerve. 2006 Aug;34(2):135-44
16770791
Lab Invest. 2010 Sep;90(9):1396-402
20458276
Hum Mutat. 2009 Mar;30(3):293-9
19156838
Cell. 1987 Dec 24;51(6):919-28
3319190
Hum Gene Ther. 2007 Sep;18(9):798-810
17767400
FASEB J. 2008 Mar;22(3):659-61
17942826
Am J Hum Genet. 1991 Jul;49(1):54-67
2063877
Mol Ther. 2009 Aug;17(8):1418-26
19293776
Neuromuscul Disord. 2003 Mar;13(3):223-35
12609504
Nat Med. 2006 Feb;12(2):175-7
16444267
Lancet Neurol. 2009 Oct;8(10):918-28
19713152
Proc Natl Acad Sci U S A. 2002 Jul 9;99(14):9456-61
12077324
Oligonucleotides. 2005 Dec;15(4):284-97
16396622
J Clin Invest. 1991 Jun;87(6):2127-31
2040695
Lancet Neurol. 2003 Dec;2(12):731-40
14636778
Hum Mol Genet. 1995 Aug;4(8):1245-50
7581360
Mol Ther. 2009 Mar;17(3):548-53
18813282
Mol Ther. 2007 Jul;15(7):1288-96
17285139
Ann Neurol. 2009 Jun;65(6):667-76
19288467
Hum Mol Genet. 2003 Apr 15;12(8):907-14
12668614
J Gene Med. 2010 Apr;12(4):354-64
20235089
Neurology. 1989 Apr;39(4):465-74
2927671