scholarly journals Nucleobase-modified antisense oligonucleotides containing 5-(phenyltriazol)-2′-deoxyuridine nucleotides induce exon-skipping in vitro

RSC Advances ◽  
2017 ◽  
Vol 7 (86) ◽  
pp. 54542-54545 ◽  
Author(s):  
Bao T. Le ◽  
Mick Hornum ◽  
Pawan K. Sharma ◽  
Poul Nielsen ◽  
Rakesh N. Veedu
Keyword(s):  
Antisense Oligonucleotides ◽  
Exon Skipping

We investigated the potential of nucleobase-modified antisense oligonucleotides to induce exon-skipping, and found that 5-(phenyltriazol)-2′-deoxyuridine-modified antisense oligonucleotides induced efficient exon-skipping in vitro.

Investigation of twisted intercalating nucleic acid (TINA)-modified antisense oligonucleotides for splice modulation by induced exon-skipping in vitro

RSC Advances ◽  
2016 ◽  
Vol 6 (97) ◽  
pp. 95169-95172 ◽  
Author(s):  
Bao T. Le ◽  
Vyacheslav V. Filichev ◽  
Rakesh N. Veedu
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Antisense Oligonucleotides ◽  
Exon Skipping

We have investigated the applicability of twisted intercalating nucleic acids (TINA)-modified antisense oligonucleotides (AOs) in exon skipping. We found that TINA-modified AOs induced exon skipping.

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

2021 ◽  
Vol 22 (7) ◽  
pp. 3479
Author(s):  
Jessica M. Cale ◽  
Kane Greer ◽  
Sue Fletcher ◽  
Steve D. Wilton
Keyword(s):  
Antisense Oligonucleotide ◽  
Antisense Oligonucleotides ◽  
Exon Skipping ◽  
Mrna Splicing ◽  
Immunofluorescent Staining ◽  
Proof Of Concept ◽  
Connective Tissue Disorders ◽  
Fibrillin 1 ◽  
Healthy Control

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.

scholarly journals Alpha-l-Locked Nucleic Acid-Modified Antisense Oligonucleotides Induce Efficient Splice Modulation In Vitro

2020 ◽  
Vol 21 (7) ◽  
pp. 2434
Author(s):  
Prithi Raguraman ◽  
Tao Wang ◽  
Lixia Ma ◽  
Per Trolle Jørgensen ◽  
Jesper Wengel ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Antisense Oligonucleotides ◽  
Exon Skipping ◽  
Initial Study ◽  
Locked Nucleic Acid ◽  
Mdx Mouse ◽  
Complementary Oligonucleotide ◽  
Low Cytotoxicity ◽  
Target Binding

Alpha-l-Locked nucleic acid (α-l-LNA) is a stereoisomeric analogue of locked nucleic acid (LNA), which possesses excellent biophysical properties and also exhibits high target binding affinity to complementary oligonucleotide sequences and resistance to nuclease degradations. Therefore, α-l-LNA nucleotides could be utilised to develop stable antisense oligonucleotides (AO), which can be truncated without compromising the integrity and efficacy of the AO. In this study, we explored the potential of α-l-LNA nucleotides-modified antisense oligonucleotides to modulate splicing by inducing Dmd exon-23 skipping in mdx mouse myoblasts in vitro. For this purpose, we have synthesised and systematically evaluated the efficacy of α-l-LNA-modified 2′-O-methyl phosphorothioate (2′-OMePS) AOs of three different sizes including 20mer, 18mer and 16mer AOs in parallel to fully-modified 2′-OMePS control AOs. Our results demonstrated that the 18mer and 16mer truncated AO variants showed slightly better exon-skipping efficacy when compared with the fully-23 modified 2′-OMePS control AOs, in addition to showing low cytotoxicity. As there was no previous report on using α-l-LNA-modified AOs in splice modulation, we firmly believe that this initial study could be beneficial to further explore and expand the scope of α-l-LNA-modified AO therapeutic molecules.

scholarly journals Systematic Approach to Developing Splice Modulating Antisense Oligonucleotides

2019 ◽  
Vol 20 (20) ◽  
pp. 5030 ◽  
Author(s):  
May Aung-Htut ◽  
Craig McIntosh ◽  
Kristin Ham ◽  
Ianthe Pitout ◽  
Loren Flynn ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Therapeutic Strategy ◽  
Systematic Approach ◽  
Muscular Atrophy ◽  
Exon Skipping ◽  
Human Genes ◽  
Gene Transcripts ◽  
Primary Gene ◽  
Selection Of

The process of pre-mRNA splicing is a common and fundamental step in the expression of most human genes. Alternative splicing, whereby different splice motifs and sites are recognised in a developmental and/or tissue-specific manner, contributes to genetic plasticity and diversity of gene expression. Redirecting pre-mRNA processing of various genes has now been validated as a viable clinical therapeutic strategy, providing treatments for Duchenne muscular dystrophy (inducing specific exon skipping) and spinal muscular atrophy (promoting exon retention). We have designed and evaluated over 5000 different antisense oligonucleotides to alter splicing of a variety of pre-mRNAs, from the longest known human pre-mRNA to shorter, exon-dense primary gene transcripts. Here, we present our guidelines for designing, evaluating and optimising splice switching antisense oligomers in vitro. These systematic approaches assess several critical factors such as the selection of target splicing motifs, choice of cells, various delivery reagents and crucial aspects of validating assays for the screening of antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone.

Evaluation of anhydrohexitol nucleic acid, cyclohexenyl nucleic acid andd-altritol nucleic acid-modified 2′-O-methyl RNA mixmer antisense oligonucleotides for exon skipping in vitro

2016 ◽  
Vol 52 (92) ◽  
pp. 13467-13470 ◽  
Author(s):  
Bao T. Le ◽  
Suxiang Chen ◽  
Mikhail Abramov ◽  
Piet Herdewijn ◽  
Rakesh N. Veedu
Keyword(s):  
Nucleic Acid ◽  
Antisense Oligonucleotides ◽  
Exon Skipping ◽  
Antisense Oligos

We have investigated the potential of anhydrohexitol, cyclohexenyl and altritol nucleic acid-modified antisense oligos in exon skipping, and found that they efficiently inducedDmdexon 23 skipping.

scholarly journals Targeted SMN Exon Skipping: A Useful Control to Assess In Vitro and In Vivo Splice-Switching Studies

Biomedicines ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 552
Author(s):  
Loren L. Flynn ◽  
Chalermchai Mitrpant ◽  
Abbie Adams ◽  
Ianthe L. Pitout ◽  
Anja Stirnweiss ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Exon Skipping ◽  
Positive Control ◽  
In Vivo Models ◽  
Specific Effects ◽  
Smn Gene ◽  
Human And Mouse ◽  
Antisense Oligomer

The literature surrounding the use of antisense oligonucleotides continues to grow, with new disease and mechanistic applications constantly evolving. Furthermore, the discovery and advancement of novel chemistries continues to improve antisense delivery, stability and effectiveness. For each new application, a rational sequence design is recommended for each oligomer, as is chemistry and delivery optimization. To confirm oligomer delivery and antisense activity, a positive control AO sequence with well characterized target-specific effects is recommended. Here, we describe splice-switching antisense oligomer sequences targeting the ubiquitously expressed human and mouse SMN and Smn genes for use as control AOs for this purpose. We report two AO sequences that induce targeted skipping of SMN1/SMN2 exon 7 and two sequences targeting the Smn gene, that induce skipping of exon 5 and exon 7. These antisense sequences proved effective in inducing alternative splicing in both in vitro and in vivo models and are therefore broadly applicable as controls. Not surprisingly, we discovered a number of differences in efficiency of exon removal between the two species, further highlighting the differences in splice regulation between species.

In vitro evaluation of hexitol nucleic acid antisense oligonucleotides

1999 ◽  
Author(s):  
Arthur Van Aerschot ◽  
Mark Vandermeeren ◽  
Johan Geysen ◽  
Walter Luyten ◽  
Marc Miller ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Antisense Oligonucleotides ◽  
In Vitro Evaluation

scholarly journals Personalized Development of Antisense Oligonucleotides for Exon Skipping Restores Type XVII Collagen Expression in Junctional Epidermolysis Bullosa

2021 ◽  
Vol 22 (7) ◽  
pp. 3326
Author(s):  
Michael Ablinger ◽  
Thomas Lettner ◽  
Nicole Friedl ◽  
Hannah Potocki ◽  
Theresa Palmetzhofer ◽  
...  
Keyword(s):  
Epidermolysis Bullosa ◽  
Antisense Oligonucleotides ◽  
Exon Skipping ◽  
Junctional Epidermolysis Bullosa ◽  
Reading Frame ◽  
Collagen Expression ◽  
Promising Tool ◽  
Mucous Membranes ◽  
Personalized Approach ◽  
Xvii Collagen

Intermediate junctional epidermolysis bullosa caused by mutations in the COL17A1 gene is characterized by the frequent development of blisters and erosions on the skin and mucous membranes. The rarity of the disease and the heterogeneity of the underlying mutations renders therapy developments challenging. However, the high number of short in-frame exons facilitates the use of antisense oligonucleotides (AON) to restore collagen 17 (C17) expression by inducing exon skipping. In a personalized approach, we designed and tested three AONs in combination with a cationic liposomal carrier for their ability to induce skipping of COL17A1 exon 7 in 2D culture and in 3D skin equivalents. We show that AON-induced exon skipping excludes the targeted exon from pre-mRNA processing, which restores the reading frame, leading to the expression of a slightly truncated protein. Furthermore, the expression and correct deposition of C17 at the dermal–epidermal junction indicates its functionality. Thus, we assume AON-mediated exon skipping to be a promising tool for the treatment of junctional epidermolysis bullosa, particularly applicable in a personalized manner for rare genotypes.

scholarly journals Inhibition of Kirsten-Ras reduces fibrosis and protects against renal dysfunction in a mouse model of chronic folic acid nephropathy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Lucy J. Newbury ◽  
Jui-Hui Wang ◽  
Gene Hung ◽  
Bruce M. Hendry ◽  
Claire C. Sharpe
Keyword(s):  
Folic Acid ◽  
Kidney Disease ◽  
Mouse Model ◽  
Renal Dysfunction ◽  
Antisense Oligonucleotides ◽  
Underlying Mechanism ◽  
Therapeutic Benefit ◽  
End Stage

Abstract Chronic Kidney Disease is a growing problem across the world and can lead to end-stage kidney disease and cardiovascular disease. Fibrosis is the underlying mechanism that leads to organ dysfunction, but as yet we have no therapeutics that can influence this process. Ras monomeric GTPases are master regulators that direct many of the cytokines known to drive fibrosis to downstream effector cascades. We have previously shown that K-Ras is a key isoform that drives fibrosis in the kidney. Here we demonstrate that K-Ras expression and activation are increased in rodent models of CKD. By knocking down expression of K-Ras using antisense oligonucleotides in a mouse model of chronic folic acid nephropathy we can reduce fibrosis by 50% and prevent the loss of renal function over 3 months. In addition, we have demonstrated in vitro and in vivo that reduction of K-Ras expression is associated with a reduction in Jag1 expression; we hypothesise this is the mechanism by which targeting K-Ras has therapeutic benefit. In conclusion, targeting K-Ras expression with antisense oligonucleotides in a mouse model of CKD prevents fibrosis and protects against renal dysfunction.

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