Removal of the Polyglutamine Repeat of Ataxin-3 by Redirecting pre-mRNA Processing

McIntosh, Craig S.; Aung-Htut, May T.; Fletcher, Sue; Wilton, Steve D.

doi:10.3390/ijms20215434

Cited by 12 publications

(13 citation statements)

References 59 publications

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“…While we demonstrated efficient and consistent exon skipping using PMO52, the concentrations used are relatively high, when compared to similar studies targeting other genes [59,68]. One of the possible explanations for the high concentration required is the abundance of fibrillin-1 transcripts.…”

Section: Discussionmentioning

confidence: 64%

Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52

Cale

Greer

Fletcher

et al. 2021

IJMS

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Marfan syndrome is one of the most common dominantly inherited connective tissue disorders, affecting 2–3 in 10,000 individuals, and is caused by one of over 2800 unique FBN1 mutations. Mutations in FBN1 result in reduced fibrillin-1 expression, or the production of two different fibrillin-1 monomers unable to interact to form functional microfibrils. Here, we describe in vitro evaluation of antisense oligonucleotides designed to mediate exclusion of FBN1 exon 52 during pre-mRNA splicing to restore monomer homology. Antisense oligonucleotide sequences were screened in healthy control fibroblasts. The most effective sequence was synthesised as a phosphorodiamidate morpholino oligomer, a chemistry shown to be safe and effective clinically. We show that exon 52 can be excluded in up to 100% of FBN1 transcripts in healthy control fibroblasts transfected with PMO52. Immunofluorescent staining revealed the loss of fibrillin 1 fibres with ~50% skipping and the subsequent re-appearance of fibres with >80% skipping. However, the effect of exon skipping on the function of the induced fibrillin-1 isoform remains to be explored. Therefore, these findings demonstrate proof-of-concept that exclusion of an exon from FBN1 pre-mRNA can result in internally truncated but identical monomers capable of forming fibres and lay a foundation for further investigation to determine the effect of exon skipping on fibrillin-1 function.

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Section: Discussionmentioning

confidence: 64%

Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52

Cale

Greer

Fletcher

et al. 2021

IJMS

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“…Partial exon 9 skipping from the 21Q transcript produces a product 16 nt smaller than the canonical 8Q transcript and could not be confirmed by the methods used in this study. Cryptic donor activation in the transcript with fewer CAG repeats dominates in some AO treatments but not others 32,33 . The CAG expansion occurs in the following exon 10, separated by a 10 kb intron from the AO target.…”

Section: Srsf2 Exonmentioning

confidence: 99%

Induction of cryptic pre-mRNA splice-switching by antisense oligonucleotides

Ham

Keegan

McIntosh

et al. 2021

Sci Rep

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Antisense oligomers (AOs) are increasingly being used to modulate RNA splicing in live cells, both for research and for the development of therapeutics. While the most common intended effect of these AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven AO-induced cryptic splice sites in six genes. Five of these cryptic splice sites were discovered through our own experiments, and two originated from other published reports. We modelled the predicted effects of AO binding on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was disruption of the exon definition signal within the exon’s excluded segment.

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“…Complete and partial exon 9 skipping was observed only from the 8Q and not the 21Q transcript. Cryptic donor activation in the transcript with fewer CAG repeats dominates in some AO treatments but not others 28,29 . The CAG expansion occurs in the following exon 10 that is separated by a 10 kb intron from the AO target.…”

Section: Atxn3 Exonmentioning

confidence: 99%

Cryptic U2-dependent Pre-mRNASplice Site Usage Induced by Splice Switching Antisense Oligonucleotides

Ham¹,

Keegan²,

McIntosh³

et al. 2021

Preprint

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Antisense oligomers (AOs) are increasingly being used for modulating RNA splicing in live cells, both for research and for therapeutic purposes. While the most common intended effect of these AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven such examples of AO-induced cryptic splice site activation – five new examples from our own experiments and three from reports published by others. We modelled the predicted effects that AO binding would have on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was a disruption to the exon definition signal within the exon’s excluded segment.

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Removal of the Polyglutamine Repeat of Ataxin-3 by Redirecting pre-mRNA Processing

Cited by 12 publications

References 59 publications

Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52

Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52

Induction of cryptic pre-mRNA splice-switching by antisense oligonucleotides

Cryptic U2-dependent Pre-mRNASplice Site Usage Induced by Splice Switching Antisense Oligonucleotides

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