Format

Send to

Choose Destination
Hum Mol Genet. 2015 Jul 15;24(14):4049-60. doi: 10.1093/hmg/ddv141. Epub 2015 Apr 22.

Dysferlin rescue by spliceosome-mediated pre-mRNA trans-splicing targeting introns harbouring weakly defined 3' splice sites.

Author information

1
Université de Versailles St-Quentin, INSERM U1179, LIA BAHN Centre Scientifique de Monaco, 2 Avenue de la Source de la Bievre, Montigny-le-Bretonneux 78180, France, Muscle Research Unit, Experimental and Clinical Research Center, a Joint Cooperation Between Max-Delbrück-Center for Molecular Medicine and Charité Medical Faculty, Berlin, Germany and Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS974, CNRS FRE3617, Myology Research Center, Paris, France.
2
Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS974, CNRS FRE3617, Myology Research Center, Paris, France.
3
Université de Versailles St-Quentin, INSERM U1179, LIA BAHN Centre Scientifique de Monaco, 2 Avenue de la Source de la Bievre, Montigny-le-Bretonneux 78180, France.
4
Muscle Research Unit, Experimental and Clinical Research Center, a Joint Cooperation Between Max-Delbrück-Center for Molecular Medicine and Charité Medical Faculty, Berlin, Germany and.
5
Université de Versailles St-Quentin, INSERM U1179, LIA BAHN Centre Scientifique de Monaco, 2 Avenue de la Source de la Bievre, Montigny-le-Bretonneux 78180, France, luis.garcia@uvsq.fr.

Abstract

The modification of the pre-mRNA cis-splicing process employing a pre-mRNA trans-splicing molecule (PTM) is an attractive strategy for the in situ correction of genes whose careful transcription regulation and full-length expression is determinative for protein function, as it is the case for the dysferlin (DYSF, Dysf) gene. Loss-of-function mutations of DYSF result in different types of muscular dystrophy mainly manifesting as limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi muscular dystrophy 1 (MMD1). We established a 3' replacement strategy for mutated DYSF pre-mRNAs induced by spliceosome-mediated pre-mRNA trans-splicing (SmaRT) by the use of a PTM. In contrast to previously established SmaRT strategies, we particularly focused on the identification of a suitable pre-mRNA target intron other than the optimization of the PTM design. By targeting DYSF pre-mRNA introns harbouring differentially defined 3' splice sites (3' SS), we found that target introns encoding weakly defined 3' SSs were trans-spliced successfully in vitro in human LGMD2B myoblasts as well as in vivo in skeletal muscle of wild-type and Dysf(-/-) mice. For the first time, we demonstrate rescue of Dysf protein by SmaRT in vivo. Moreover, we identified concordant qualities among the successfully targeted Dysf introns and targeted endogenous introns in previously reported SmaRT approaches that might facilitate a selective choice of target introns in future SmaRT strategies.

PMID:
25904108
DOI:
10.1093/hmg/ddv141
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Silverchair Information Systems
Loading ...
Support Center