title: Dysferlin rescue by spliceosome-mediated pre-mRNA trans-splicing targeting introns harbouring weakly defined 3' splice sites.
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.
authors.affiliation: 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.
authors.forename: Susanne
authors.initials: S
authors.lastname: Philippi
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS974, CNRS FRE3617, Myology Research Center, Paris, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Lorain
authors.affiliation: 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.
authors.forename: Cyriaque
authors.initials: C
authors.lastname: Beley
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS974, CNRS FRE3617, Myology Research Center, Paris, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Peccate
authors.affiliation: 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.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Précigout
authors.affiliation: 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.
authors.forename: Simone
authors.initials: S
authors.lastname: Spuler
authors.affiliation: 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.
authors.forename: Luis
authors.initials: L
authors.lastname: Garcia
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D019295:Computational Biology; D000073939:Dysferlin; D006801:Humans; D007438:Introns; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009124:Muscle Proteins / Q000235:genetics* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D009136:Muscular Dystrophies / Q000235:genetics; D049288:Muscular Dystrophies, Limb-Girdle / Q000235:genetics; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D022821:RNA Splice Sites; D017381:Spliceosomes / Q000235:genetics* / Q000378:metabolism; D020040:Trans-Splicing
chemical_list: C114839:DYSF protein, human; C497505:Dysf protein, mouse; D000073939:Dysferlin; D008565:Membrane Proteins; D009124:Muscle Proteins; D012322:RNA Precursors; D022821:RNA Splice Sites