Generation of Humanized Zebrafish Models for the In Vivo Assessment of Antisense Oligonucleotide-Based Splice Modulation Therapies

Renske Schellens; Erik de Vrieze; Ralph Slijkerman; Hannie Kremer; Erwin van Wijk

doi:10.1007/978-1-0716-2010-6_19

Generation of Humanized Zebrafish Models for the In Vivo Assessment of Antisense Oligonucleotide-Based Splice Modulation Therapies

Methods Mol Biol. 2022:2434:281-299. doi: 10.1007/978-1-0716-2010-6_19.

Authors

Renske Schellens^{1

2}, Erik de Vrieze^{1

2}, Ralph Slijkerman^{1

2}, Hannie Kremer^{1

2

3}, Erwin van Wijk^{4

5}

Affiliations

¹ Department of Otorhinolaryngology, Radboud University Medical Center, Nijmegen, The Netherlands.
² Donders Institute for Brain Cognition and Behaviour, Nijmegen, The Netherlands.
³ Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.
⁴ Department of Otorhinolaryngology, Radboud University Medical Center, Nijmegen, The Netherlands. Erwin.vanWyk@radboudumc.nl.
⁵ Donders Institute for Brain Cognition and Behaviour, Nijmegen, The Netherlands. Erwin.vanWyk@radboudumc.nl.

Abstract

Antisense oligonucleotide (AON)-based splice modulation is the most widely used therapeutic approach to redirect precursor messenger RNA (pre-mRNA) splicing. To study the functional effect of human mutations affecting pre-mRNA splicing for which AON-based splice redirection would be a potential therapeutic option, humanized knock-in animal models are pivotal. A major limitation of using humanized animal models for this purpose is the reported poor recognition of human splice sites by the splicing machineries of other species. To overcome this problem, we provide a detailed guideline for the generation of functional humanized knock-in zebrafish models to assess the effect of mutation-induced aberrant splicing and subsequent AON-based splice modulation therapy .

Keywords: Antisense oligonucleotides; Inherited retinal dystrophies; Pre-mRNA splicing; Species-specific minigene splice assay; Usher syndrome; Zebrafish.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Humans
Mutation
Oligonucleotides, Antisense / pharmacology
RNA Precursors / genetics
RNA Splicing*
Zebrafish* / genetics
Zebrafish* / metabolism

Substances

Oligonucleotides, Antisense
RNA Precursors