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.

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 The Necessity of a Systematic Approach for the Use of MSCs in the Clinical Setting

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Christophe Michel Raynaud ◽  
Arash Rafii
Keyword(s):  
Clinical Trials ◽  
Stromal Cells ◽  
Therapeutic Strategy ◽  
Systematic Approach ◽  
Cell Types ◽  
Clinical Applications ◽  
Physiological Properties ◽  
Different Cell Types

Cell therapy has emerged as a potential therapeutic strategy in regenerative disease. Among different cell types, mesenchymal stem/stromal cells have been wildly studiedin vitro,in vivoin animal models and even used in clinical trials. However, while clinical applications continue to increase markedly, the understanding of their physiological properties and interactions raises many questions and drives the necessity of more caution and supervised strategy in their use.

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 HNRNPM controls circRNA biogenesis and splicing fidelity to sustain cancer cell fitness

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jessica SY Ho ◽  
Federico Di Tullio ◽  
Megan Schwarz ◽  
Diana Low ◽  
Danny Incarnato ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Growth ◽  
Solid Tumors ◽  
Cancer Cell ◽  
Antisense Oligonucleotides ◽  
Therapeutic Strategy ◽  
Splicing Factors ◽  
Normal Cells

High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified HNRNPM as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and back-splicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM dependent linear splicing events using splice-switching-antisense-oligonucleotides (SSOs) was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors.

scholarly journals Myoblasts and macrophages are required for therapeutic morpholino antisense oligonucleotide delivery to dystrophic muscle

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
James S. Novak ◽  
Marshall W. Hogarth ◽  
Jessica F. Boehler ◽  
Marie Nearing ◽  
Maria C. Vila ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Duchenne Muscular Dystrophy ◽  
Antisense Oligonucleotide ◽  
Antisense Oligonucleotides ◽  
Therapeutic Strategy ◽  
Exon Skipping ◽  
Efficient Production ◽  
Dystrophic Muscle ◽  
Oligonucleotide Delivery ◽  
Dystrophin Protein

Abstract Exon skipping is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), employing morpholino antisense oligonucleotides (PMO-AO) to exclude disruptive exons from the mutant DMD transcript and elicit production of truncated dystrophin protein. Clinical trials for PMO show variable and sporadic dystrophin rescue. Here, we show that robust PMO uptake and efficient production of dystrophin following PMO administration coincide with areas of myofiber regeneration and inflammation. PMO localization is sustained in inflammatory foci where it enters macrophages, actively differentiating myoblasts and newly forming myotubes. We conclude that efficient PMO delivery into muscle requires two concomitant events: first, accumulation and retention of PMO within inflammatory foci associated with dystrophic lesions, and second, fusion of PMO-loaded myoblasts into repairing myofibers. Identification of these factors accounts for the variability in clinical trials and suggests strategies to improve this therapeutic approach to DMD.

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 Sam68 binds Alu-rich introns in SMN and promotes pre-mRNA circularization

2019 ◽  
Vol 48 (2) ◽  
pp. 633-645 ◽  
Author(s):  
Vittoria Pagliarini ◽  
Ariane Jolly ◽  
Pamela Bielli ◽  
Valentina Di Rosa ◽  
Pierre De la Grange ◽  
...  
Keyword(s):  
Binding Sites ◽  
Muscular Atrophy ◽  
Circular Rna ◽  
Cross Link ◽  
Bioinformatics Analyses ◽  
Protein Coding ◽  
Inverted Orientation ◽  
Human Genes

Abstract The Spinal Muscular Atrophy (SMA) gene SMN was recently duplicated (SMN1 and SMN2) in higher primates. Furthermore, invasion of the locus by repetitive elements almost doubled its size with respect to mouse Smn, in spite of an almost identical protein-coding sequence. Herein, we found that SMN ranks among the human genes with highest density of Alus, which are evolutionary conserved in primates and often occur in inverted orientation. Inverted repeat Alus (IRAlus) negatively regulate splicing of long introns within SMN, while promoting widespread alternative circular RNA (circRNA) biogenesis. Bioinformatics analyses revealed the presence of ultra-conserved Sam68 binding sites in SMN IRAlus. Cross-link-immunoprecipitation (CLIP), mutagenesis and silencing experiments showed that Sam68 binds in proximity of intronic Alus in the SMN pre-mRNA, thus favouring circRNA biogenesis in vitro and in vivo. These findings highlight a novel layer of regulation in SMN expression, uncover the crucial impact exerted by IRAlus and reveal a role for Sam68 in SMN circRNA biogenesis.

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.

Sign in / Sign up

Export Citation Format

Share Document