PMID- 20393509
OWN - NLM
STAT- MEDLINE
DCOM- 20100927
LR  - 20220408
IS  - 1476-5462 (Electronic)
IS  - 0969-7128 (Linking)
VI  - 17
IP  - 7
DP  - 2010 Jul
TI  - Meganucleases can restore the reading frame of a mutated dystrophin.
PG  - 846-58
LID - 10.1038/gt.2010.26 [doi]
AB  - Mutations in Duchenne muscular dystrophy (DMD) are either inducing a nonsense 
      codon or a frameshift. Meganucleases (MGNs) can be engineered to induce 
      double-strand breaks (DSBs) at specific DNA sequences. These breaks are repaired 
      by homologous recombination or by non-homologous end joining (NHEJ), which 
      results in insertions or deletions (indels) of a few base pairs. To verify 
      whether MGNs could be used to restore the normal reading frame of a dystrophin 
      gene with a frameshift mutation, we inserted in a plasmid coding for the dog 
      micro-dystrophin sequences containing a MGN target. The number of base pairs in 
      these inserted sequences changed the reading frame. One of these modified target 
      micro-dystrophin plasmids and an appropriate MGN were then transfected in 293FT 
      cells. The MGN induced micro-deletion or micro-insertion in the micro-dystrophin 
      that restored dystrophin expression. MGNs also restored micro-dystrophin 
      expression in myoblasts in vitro and in muscle fibers in vivo. The mutation of 
      the targeted micro-dystrophin was confirmed by PCR amplification followed by 
      digestion with the Surveyor enzyme and by cloning and sequencing of the 
      amplicons. These experiments are thus a proof of principle that MGNs that are 
      adequately engineered to target appropriate sequences in the human dystrophin 
      gene should be able to restore the normal reading frame of that gene in DMD 
      patients with an out-of-frame deletion. New MGNs engineered to target a sequence 
      including or near nonsense mutation could also be used to delete it.
FAU - Chapdelaine, P
AU  - Chapdelaine P
AD  - CHUL, Centre de Recherche du CHUQ, Quebec, Canada.
FAU - Pichavant, C
AU  - Pichavant C
FAU - Rousseau, J
AU  - Rousseau J
FAU - Paques, F
AU  - Paques F
FAU - Tremblay, J P
AU  - Tremblay JP
LA  - eng
GR  - Canadian Institutes of Health Research/Canada
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20100415
PL  - England
TA  - Gene Ther
JT  - Gene therapy
JID - 9421525
RN  - 0 (Dystrophin)
RN  - 0 (Homeodomain Proteins)
RN  - 128559-51-3 (RAG-1 protein)
RN  - EC 3.1.- (Endonucleases)
SB  - IM
MH  - Cell Line
MH  - Dystrophin/*genetics
MH  - *Endonucleases
MH  - *Frameshift Mutation
MH  - Gene Targeting/*methods
MH  - Homeodomain Proteins
MH  - Humans
MH  - Muscular Dystrophy, Duchenne/*genetics
MH  - *Protein Engineering
MH  - *Reading Frames
EDAT- 2010/04/16 06:00
MHDA- 2010/09/29 06:00
CRDT- 2010/04/16 06:00
PHST- 2010/04/16 06:00 [entrez]
PHST- 2010/04/16 06:00 [pubmed]
PHST- 2010/09/29 06:00 [medline]
AID - gt201026 [pii]
AID - 10.1038/gt.2010.26 [doi]
PST - ppublish
SO  - Gene Ther. 2010 Jul;17(7):846-58. doi: 10.1038/gt.2010.26. Epub 2010 Apr 15.