Abstract
Splice-switching oligonucleotides (SSOs) have been used to modulate gene expression by interfering with pre-mRNA splicing with the intent to treat disease. For Duchenne muscular dystrophy, splicing modulation has been used to induce the skipping of exon 51 of the dystrophin transcript, allowing the production of a truncated but functional protein. Although oligonucleotide-based therapies are promising, the rapid degradation of oligonucleotides (ONs) by intracellular nucleases has been a major obstacle. Locked nucleic acid (LNA) substitution in SSOs protects oligonucleotides from nuclease degradation and enhances the hybridization properties of the oligo. However, the best optimum size of the oligo depends on the LNA substitution rate. Here we show that 16-mer DNA SSOs with 60% LNA substitution and full phosphorothioate (PS) linkage backbone efficiently induce exon 51 skipping in myogenic cells derived from a DMD patient, allowing expression of the dystrophin protein.
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Acknowledgments
We acknowledge the Association Française Contre les Myopathies and the Platform for the Immortalization of Human Cells and Collaboration, Institut de Myologie, Paris, for the immortalized human cell lines, and Capucine Trollet (Centre de Recherche en Myologie, Sorbonne Universités, Paris) for oligonucleotide AO51. We thank Ricardo Simões, Vanessa Borges Pires, and Kamel Mamchaoui for collaboration and Ana de Jesus and Ana Margarida Nascimento for technical support. We would like to thank Marcia Triunfol for assistance in preparing this manuscript and Noélia Custódio for critical review.
We further acknowledge funding from Fundação para a Ciência e Tecnologia and FEDER/POR Lisboa 2020—Programa Operacional Regional de Lisboa, PORTUGAL 2020 (LISBOA-01-0145-FEDER-016394; 007391).
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Carvalho, C., Carmo-Fonseca, M. (2020). Design and Application of a Short (16-mer) Locked Nucleic Acid Splice-Switching Oligonucleotide for Dystrophin Production in Duchenne Muscular Dystrophy Myotubes. In: Ørom, U. (eds) RNA-Chromatin Interactions. Methods in Molecular Biology, vol 2161. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0680-3_4
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DOI: https://doi.org/10.1007/978-1-0716-0680-3_4
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