Interaction of modified oligonucleotides with nuclear proteins, formation of novel nuclear structures and sequence-independent effects on RNA processing

Ll, Flynn; R, Li; Mt, Aung-Htut; Il, Pitout; Jal, Cooper; Hubbard, Alysia; L, Griffiths; Bond, Charles S.; Wilton, Steve D.; Fox, Archa H.; Fletcher, Sue

doi:10.1101/446773

Cited by 5 publications

(8 citation statements)

References 45 publications

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“…Immunofluorescent staining of NONO showed that all 2′-Me PS AOs sequestrated NONO, at various subcellular locations (Figure 4A), while the identical sequences synthesised as PMOs had no such effects (Figure 4B). Consistent with the report by Flynn et al (2018) [18], the Gene Tools control sequence seemed to induce the highest amount of NONO-containing nuclear inclusions when applied as a 2′-Me PS AO. All 2′-Me PS sequences tested in this study did induce NONO inclusions in most cells, some inclusions were located within the nucleus while others appear to be peri-nuclear or distributed in the cytoplasm (Figure 4A).…”

Section: Resultssupporting

confidence: 90%

“…Phosphorothioate backbone AOs have resulted in severe injection site reactions, thrombocytopenia and renal toxicity in clinical studies [16,17]. An in vitro study by Flynn et al (2018), demonstrated severe, sequence-independent backbone-specific effects of 2′- O -methyl modified bases on a phosphorothioate backbone (2′-Me PS) AOs, including altered distribution of nuclear proteins, the appearance of abnormal but highly structured nuclear inclusions and aggregates and global disturbance of the transcriptome [18].…”

Section: Introductionmentioning

confidence: 99%

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

McIntosh¹,

Aung-Htut²,

Fletcher³

et al. 2019

IJMS

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Spinocerebellar ataxia type 3 (SCA3) is a devastating neurodegenerative disease for which there is currently no cure, nor effective treatment strategy. One of nine polyglutamine disorders known to date, SCA3 is clinically heterogeneous and the main feature is progressive ataxia, which in turn affects speech, balance and gait of the affected individual. SCA3 is caused by an expanded polyglutamine tract in the ataxin-3 protein, resulting in conformational changes that lead to toxic gain of function. The expanded glutamine tract is located at the 5′ end of the penultimate exon (exon 10) of ATXN3 gene transcript. Other studies reported removal of the expanded glutamine tract using splice switching antisense oligonucleotides. Here, we describe improved efficiency in the removal of the toxic polyglutamine tract of ataxin-3 in vitro using phosphorodiamidate morpholino oligomers, when compared to antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone. Significant downregulation of both the expanded and non-expanded protein was induced by the morpholino antisense oligomer, with a greater proportion of ataxin-3 protein missing the polyglutamine tract. With growing concerns over toxicity associated with long-term administration of phosphorothioate oligonucleotides, the use of a phosphorodiamidate morpholino oligomer may be preferable for clinical application. These results suggest that morpholino oligomers may provide greater therapeutic benefit for the treatment of spinocerebellar ataxia type 3, without toxic effects.

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Section: Resultssupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

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

McIntosh¹,

Aung-Htut²,

Fletcher³

et al. 2019

IJMS

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“…However, the cells did not survive. Possibly due to their toxicity [ 29 ], 2′-OMe AOs targeting COL7A1 exons 10 and 73 tested at concentrations higher than 50 nM (approximately one femtomoles per cell) using Lipofectamine 3000 showed reduced transfection efficiency and a notable increase in cell death. The 150 µM concentration of PMO used in this study was nucleofected into a larger number of cells and hence the calculated number of molecules per cell (approximately 0.1 femtomoles) was roughly tenfold less compared to the highest 2′-OMe AO treatments.…”

Section: Discussionmentioning

confidence: 99%

Nonsequential Splicing Events Alter Antisense-Mediated Exon Skipping Outcome in COL7A1

Ham

Aung-Htut

Fletcher

et al. 2020

IJMS

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The COL7A1 gene encodes homotrimer fibrils essential for anchoring dermal and epidermal layers, and pathogenic mutations in COL7A1 can cause recessive or dominant dystrophic epidermolysis bullosa. As a monogenic disease gene, COL7A1 constitutes a potential target for antisense oligomer-mediated exon skipping, a therapy applicable to a growing number of other genetic disorders. However, certain characteristics of COL7A1: many exons, low average intron size, and repetitive and guanine-cytosine rich coding sequence, present challenges to the design of specific and effective antisense oligomers. While targeting COL7A1 exons 10 and 73 for excision from the mature mRNA, we discovered that antisense oligomers comprised of 2′-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers produced similar, but distinctive, splicing patterns including excision of adjacent nontargeted exons and/or retention of nearby introns in some transcripts. We found that the nonsequential splicing of certain introns may alter pre-mRNA processing during antisense oligomer-mediated exon skipping and, therefore, additional studies are required to determine if the order of intron removal influences multiexon skipping and/or intron retention in processing of the COL7A1 pre-mRNA.

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“…In addition, gapmers modified with LNAs show acute hepatotoxicity in mice (Kasuya et al, 2016). We and others have previously reported that AOs on a fully modified phosphorothioate backbone recruited nuclear proteins involved in RNA processing and induced global disturbance of cellular processes (Shen et al, 2014;Flynn et al, 2018).…”

Section: Discussionmentioning

confidence: 98%

Consequences of Making the Inactive Active Through Changes in Antisense Oligonucleotide Chemistries

et al. 2019

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Antisense oligonucleotides are short, single-stranded nucleic acid analogues that can interfere with pre-messenger RNA (pre-mRNA) processing and induce excision of a targeted exon from the mature transcript. When developing a panel of antisense oligonucleotides to skip every dystrophin exon, we found great variation in splice switching efficiencies, with some antisense oligonucleotides ineffective, even when directed to canonical splice sites and transfected into cells at high concentrations. In this study, we reevaluated some of these ineffective antisense oligonucleotide sequences after incorporation of locked nucleic acid residues to increase annealing potential. Antisense oligonucleotides targeting exons 16, 23, and 51 of human DMD transcripts were synthesized as two different chemistries, 2′-O-methyl modified bases on a phosphorothioate backbone or mixmers containing several locked nucleic acid residues, which were then transfected into primary human myotubes, and DMD transcripts were analyzed for exon skipping. The ineffective 2′-O-methyl modified antisense oligonucleotides induced no detectable exon skipping, while all corresponding mixmers did induce excision of the targeted exons. Interestingly, the mixmer targeting exon 51 induced two unexpected transcripts arising from partial skipping of exon 51 with retention of 95 or 188 bases from the 5′ region of exon 51. These results indicated that locked nucleic acid/2′-O-methyl mixmers are more effective at inducing exon skipping, however, this improvement may come at the cost of activating alternative cryptic splice sites and off-target effects on gene expression.

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Interaction of modified oligonucleotides with nuclear proteins, formation of novel nuclear structures and sequence-independent effects on RNA processing

Cited by 5 publications

References 45 publications

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

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

Nonsequential Splicing Events Alter Antisense-Mediated Exon Skipping Outcome in COL7A1

Consequences of Making the Inactive Active Through Changes in Antisense Oligonucleotide Chemistries

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