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Reducing DYRK1A expression in human down syndrome cells using Exon-Skipping antisense oligonucleotides
Thesis

Reducing DYRK1A expression in human down syndrome cells using Exon-Skipping antisense oligonucleotides

Aidan Murphy
Honours, Murdoch University
2022
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Whole Thesis4.37 MB
Embargoed Access, Embargo ends: 01/03/2027

Abstract

Down syndrome (DS) occurs in approximately 1 in 500 live births worldwide, making it the most common genetic form of severe childhood intellectual disability (ID). Interestingly, all individuals with DS present with some form of ID. Research suggests that the overexpression of the DYRK1A gene, due to the triplication of chromosome 21, plays a critical role in the development of this phenotype and that its normalisation can recover ID. With little to no treatment options available, we aim to validate an in vitro proof-of-concept therapeutic strategy for DS, by inducing exon skipping with splice-switching antisense oligonucleotides (AOs) that downregulates DYRK1A gene and protein expression. Initially, we screened 18 AOs targeted to skip exons 2, 5 and 6, as this would induce a reading frame shift, and the resulting DYRK1A mRNA may be subjected to degradation via non-sense mediated decay. The four most efficient were then assessed for their gene knockdown efficiency in patient fibroblast cells. Then, the three most efficient were chosen to be synthesised in a clinically relevant AO chemistry. Finally, these were assessed for their exon-skipping ability and protein knockdown in patient cells. At a concentration of 5 μM, a significant 88% ±5% and 84% ±5% knockdown of protein expression was recorded from two of our candidate AOs. This supports that inducing a frameshift in the DYRK1A transcript using AOs can downregulate gene and protein expression in vitro. This finding is highly encouraging, as it provides a promising basis for developing a therapeutic intervention for individuals with DS.

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