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

Item Type:Article
Title:Dysferlin rescue by spliceosome-mediated pre-mRNA trans-splicing targeting introns harbouring weakly defined 3' splice sites
Creators Name:Philippi, S. and Lorain, S. and Beley, C. and Peccate, C. and Precigout, G. and Spuler, S. and Garcia, L.
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.
Keywords:Computational Biology, Cultured Cells, Inbred C57BL Mice, Introns, Knockout Mice, Limb-Girdle Muscular Dystrophies, Membrane Proteins, Muscle Proteins, Muscular Dystrophies, Myoblasts, RNA Precursors, RNA Splice Sites, Skeletal Muscle, Spliceosomes, Trans-Splicing, Animals, Mice
Source:Human Molecular Genetics
Publisher:Oxford University Press
Page Range:4049-4060
Date:15 July 2015
Official Publication:https://doi.org/10.1093/hmg/ddv141
PubMed:View item in PubMed

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