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 .
Highlights
Precursor messenger RNA splicing is a tightly regulated and complicated process
For inherited retinal dystrophies (IRDs), it has been estimated that ~20% of all identified mutations affect pre-mRNA splicing [1]
In which a specific part of the species’ genomic DNA is replaced by the orthologous human sequence, are pivotal to study the functional effect of aberrant splicing and Antisense oligonucleotide (AON)-based splice correction therapy
Summary
Precursor messenger RNA (pre-mRNA) splicing is a tightly regulated and complicated process. For inherited retinal dystrophies (IRDs), it has been estimated that ~20% of all identified mutations affect pre-mRNA splicing [1]. Numerous of those mutations have already been described in literature [2–4], of which the recurrent deep-intronic. In which a specific part of the species’ genomic DNA is replaced by the orthologous human sequence, are pivotal to study the functional effect of aberrant splicing and AON-based splice correction therapy. We will discuss the step-by-step procedure to generate functional humanized zebrafish models, including optimization in cross-species splice-site recognition, to assess the effect of mutation-induced aberrant splicing and subsequent AON-based splice redirection therapies
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