Generation of Human iPSC-Derived Myotubes to Investigate RNA-Based Therapies In Vitro

Pablo Herrero-Hernandez; Atze J. Bergsma; W. W.M.Pim Pijnappel

doi:10.1007/978-1-0716-2010-6_15

Generation of Human iPSC-Derived Myotubes to Investigate RNA-Based Therapies In Vitro

Pablo Herrero-Hernandez, Atze J. Bergsma, W. W.M.Pim Pijnappel^*

^*Corresponding author for this work

Research output: Chapter/Conference proceeding › Chapter › Academic

2 Citations (Scopus)

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Abstract

Alternative pre-mRNA splicing can be cell-type specific and results in the generation of different protein isoforms from a single gene. Deregulation of canonical pre-mRNA splicing by disease-associated variants can result in genetic disorders. Antisense oligonucleotides (AONs) offer an attractive solution to modulate endogenous gene expression through alteration of pre-mRNA splicing events. Relevant in vitro models are crucial for appropriate evaluation of splicing modifying drugs. In this chapter, we describe how to investigate the splicing modulating activity of AONs in an in vitro skeletal muscle model, applied to Pompe disease. We also provide a detailed description of methods to visualize and analyze gene expression in differentiated skeletal muscle cells for the analysis of muscle differentiation and splicing outcome. The methodology described here is relevant to develop treatment options using AONs for other genetic muscle diseases as well, including Duchenne muscular dystrophy, myotonic dystrophy, and facioscapulohumeral muscular dystrophy.

Original language	English
Title of host publication	Methods in Molecular Biology
Pages	235-243
Number of pages	9
DOIs	https://doi.org/10.1007/978-1-0716-2010-6_15
Publication status	Published - 2022

Publication series

Series	Methods in Molecular Biology
Volume	2434
ISSN	1064-3745

Bibliographical note

Funding Information:
We thank Erik van der Wal for the critical reading and the revisions of this manuscript. This work was funded by Tex net, the Prinses Beatrix Spierfonds/Stichting Spieren voor Spieren (grant W. OR13-21), the Sophia Children’s Hospital Foundation (SSWO) (grant S-687 and S17-32), and Metakids (grant 2016-063).

Publisher Copyright:
© 2022, The Author(s).

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978-1-0716-2010-6Final published version, 11 MBLicence: CC BY

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abstract = "Alternative pre-mRNA splicing can be cell-type specific and results in the generation of different protein isoforms from a single gene. Deregulation of canonical pre-mRNA splicing by disease-associated variants can result in genetic disorders. Antisense oligonucleotides (AONs) offer an attractive solution to modulate endogenous gene expression through alteration of pre-mRNA splicing events. Relevant in vitro models are crucial for appropriate evaluation of splicing modifying drugs. In this chapter, we describe how to investigate the splicing modulating activity of AONs in an in vitro skeletal muscle model, applied to Pompe disease. We also provide a detailed description of methods to visualize and analyze gene expression in differentiated skeletal muscle cells for the analysis of muscle differentiation and splicing outcome. The methodology described here is relevant to develop treatment options using AONs for other genetic muscle diseases as well, including Duchenne muscular dystrophy, myotonic dystrophy, and facioscapulohumeral muscular dystrophy.",

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Generation of Human iPSC-Derived Myotubes to Investigate RNA-Based Therapies In Vitro

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