Exon exchange approach to repair Duchenne dystrophin transcripts.
BACKGROUND
Trans-splicing strategies for mRNA repair involve engineered transcripts designed to anneal target mRNAs in order to interfere with their natural splicing, giving rise to mRNA chimeras where endogenous mutated exons have been replaced by exogenous replacement sequences. A number of trans-splicing molecules have already been proposed for replacing either the 5' or the 3' part of transcripts to be repaired. Here, we show the feasibility of RNA surgery by using a double trans-splicing approach allowing the specific substitution of a given mutated exon.


RESULTS
As a target we used a minigene encoding a fragment of the mdx dystrophin gene enclosing the mutated exon (exon 23). This minigene was cotransfected with a variety of exon exchange constructions, differing in their annealing domains. We obtained accurate and efficient replacement of exon 23 in the mRNA target. Adding up a downstream intronic splice enhancer DISE in the exon exchange molecule enhanced drastically its efficiency up to 25-45% of repair depending on the construction in use.


CONCLUSIONS
These results demonstrate the possibility to fix up mutated exons, refurbish deleted exons and introduce protein motifs, while keeping natural untranslated sequences, which are essential for mRNA stability and translation regulation. Conversely to the well-known exon skipping, exon exchange has the advantage to be compatible with almost any type of mutations and more generally to a wide range of genetic conditions. In particular, it allows addressing disorders caused by dominant mutations.
Université Pierre et Marie Curie (UMR S 974)-Institut National de la Santé et de la Recherche Médicale (U974)-Centre National de la Recherche Scientifique (UMR 7215), Paris, France. stephanie.lorain@upmc.fr
Stéphanie
S
Lorain
Cécile
C
Peccate
Maëva
M
Le Hir
Luis
L
Garcia
D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis; D016189:Dystrophin / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005091:Exons / Q000235:genetics*; D007438:Introns / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009137:Muscular Dystrophy, Animal / Q000235:genetics*; D041681:NIH 3T3 Cells; D012333:RNA, Messenger / Q000235:genetics; D020040:Trans-Splicing / Q000235:genetics
D016189:Dystrophin; D012333:RNA, Messenger