| Project/Area Number |
06807058
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Neurology
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| Research Institution | Kumamoto University |
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Principal Investigator |
SUGINO Shigeto Kumamoto University, School of Medicine Child development TITLE OF POSITION : Assis.Prof., 医学部・附属病院, 助手 (60226446)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Duchenne muscular dystrophy / Gene therapy / Splicing / Antisense / Virus vector / デュシャンヌ型筋ジストロフィー / ジストロフィン |
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| Research Abstract |
Muscular Dystrophies based on mutations of dystrophin gene has been known as severe form DMD (Duchenne Muscular Dystrophy) caused by "out of frame" mutation of dystrophin gene and milder form BMD (Becker Muscular Dystrophy) caused by "in frame" mutation of dystrophin gene. It seems to rather difficult to repair the mutation or substitute entire dystrophin gene because of its huge size (14kb cDNA).
Here I report an experiment of new approach for gene therapy on DMD.This approach is to make a truncated but functionally almost normal dystrophin (Becker type dystrophin) by splicing control on dystrophin gene whicht has "out of frame" mutation.
Experiments have been done as following.
1.An "out of frame" mutation which is deletion from exon 48 to exon 50 was detected by PCR from a DMD patient. If, in this case, the splicing of the exon 51 can be skipped, transcripted exons of mRNA will connect from exon 47 to exon 52 which lead "in frame" strand and yield truncated dystrophin consequently.
2.The mini dystrophin gene was constructed as promoter-exon47-exon51-exon52 with each flanking intron sequences to analyze cell free splicing system as a substrait.
3.An 24mer antisense oligonucleotide complement to the upstream sequence of the 51 exon-intron border (splicing acceptor site) was designed and synthesized.
4.The exon skipping was not observed on in-vitro cell free splicing system adding this antisense oligonucleotide with above metioned mini dystrophin gene.
Future developmets : The another antisense oligonucleotide (cf.for splicing doner site and/or intron branch point) can be used for this system.
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