Abstract:Antisense-mediated modulation of pre-mRNA splicing is an attractive therapeutic strategy for genetic diseases. Currently, there are few examples of modulation of pre-mRNA splicing using locked nucleic acid (LNA) antisense oligonucleotides, and, in particular, no systematic study has addressed the optimal design of LNA-based splice-switching oligonucleotides (LNA SSOs). Here, we designed a series of LNA SSOs complementary to the human dystrophin exon 58 sequence and evaluated their ability to induce exon skippi… Show more
“…Administration of this short LNA oligo by subcutaneous injection in patients at the highest dose (7 mg/kg) for 4 weeks was found to be safe and exhibited significant decrease in hepatitis C virus RNA, indicating the target specificity of short LNA-modified oligos 40 . Later, Shimo et al 41 . also reported the design of a series of LNA-modified AOs targeting DMD transcripts and evaluated their efficacy in inducing efficient exon 58 skipping in vitro.…”
Locked nucleic acid is a prominent nucleic acid analog with unprecedented target binding affinity to cDNA and RNA oligonucleotides and shows remarkable stability against nuclease degradation. Incorporation of locked nucleic acid nucleotides into an antisense oligonucleotide (AO) sequence can reduce the length required without compromising the efficacy. In this study, we synthesized a series of systematically truncated locked nucleic acid-modified 2′-O-methyl AOs on a phosphorothioate (PS) backbone that were designed to induce skipping exon 23 from the dystrophin transcript in H-2Kb-tsA58 mdx mouse myotubes in vitro. The results clearly demonstrated that shorter AOs (16- to 14-mer) containing locked nucleic acid nucleotides efficiently induced dystrophin exon 23 skipping compared with the corresponding 2′-O-methyl AOs. Our remarkable findings contribute significantly to the existing knowledge about the designing of short LNA-modified oligonucleotides for exon-skipping applications, which will help reduce the cost of exon-skipping AOs and potential toxicities, particularly the 2′-OMe-based oligos, by further reducing the length of AOs.
“…Administration of this short LNA oligo by subcutaneous injection in patients at the highest dose (7 mg/kg) for 4 weeks was found to be safe and exhibited significant decrease in hepatitis C virus RNA, indicating the target specificity of short LNA-modified oligos 40 . Later, Shimo et al 41 . also reported the design of a series of LNA-modified AOs targeting DMD transcripts and evaluated their efficacy in inducing efficient exon 58 skipping in vitro.…”
Locked nucleic acid is a prominent nucleic acid analog with unprecedented target binding affinity to cDNA and RNA oligonucleotides and shows remarkable stability against nuclease degradation. Incorporation of locked nucleic acid nucleotides into an antisense oligonucleotide (AO) sequence can reduce the length required without compromising the efficacy. In this study, we synthesized a series of systematically truncated locked nucleic acid-modified 2′-O-methyl AOs on a phosphorothioate (PS) backbone that were designed to induce skipping exon 23 from the dystrophin transcript in H-2Kb-tsA58 mdx mouse myotubes in vitro. The results clearly demonstrated that shorter AOs (16- to 14-mer) containing locked nucleic acid nucleotides efficiently induced dystrophin exon 23 skipping compared with the corresponding 2′-O-methyl AOs. Our remarkable findings contribute significantly to the existing knowledge about the designing of short LNA-modified oligonucleotides for exon-skipping applications, which will help reduce the cost of exon-skipping AOs and potential toxicities, particularly the 2′-OMe-based oligos, by further reducing the length of AOs.
“…compared the efficacies of LNA/DNA mixmers whose lengths are between 6 mer to 23 mer, and 13 mer showed the best efficacy for exon skipping 23 . The 8 mer was the shortest LNA/DNA mixmer which induced efficient exon skipping 23 . As such, we employed 13 mer and 8 mer oligos in this study.Figure 2Newly designed AONs induce SMN2 exon 7 inclusion.…”
Spinal muscular atrophy (SMA) is an autosomal recessive disorder affecting motor neurons, and is currently the most frequent genetic cause of infant mortality. SMA is caused by a loss-of-function mutation in the survival motor neuron 1 (SMN1) gene. SMN2 is an SMN1 paralogue, but cannot compensate for the loss of SMN1 since exon 7 in SMN2 mRNA is excluded (spliced out) due to a single C-to-T nucleotide transition in the exon 7. One of the most promising strategies to treat SMA is antisense oligonucleotide (AON)-mediated therapy. AONs are utilized to block intronic splicing silencer number 1 (ISS-N1) on intron 7 of SMN2, which causes exon 7 inclusion of the mRNA and the recovery of the expression of functional SMN protein from the endogenous SMN2 gene. We developed novel locked nucleic acid (LNA)-based antisense oligonucleotides (LNA/DNA mixmers), which efficiently induce exon 7 inclusion in SMN2 and restore the SMN protein production in SMA patient fibroblasts. The mixmers are highly specific to the targeted sequence, and showed significantly higher efficacy than an all-LNA oligonucleotide with the equivalent sequence. These data suggest that use of LNA/DNA mixmer-based AONs may be an attractive therapeutic strategy to treat SMA.
“…Furthermore, Shimo et al. () and Touznik, Maruyama, Hosoki, Echigoya, and Yokota () showed that 13‐mer SSOs with LNA modifications efficiently induced exon skipping and exon inclusion at the cellular level, respectively. These 13‐ to 16‐mer ASOs would theoretically have several tens to hundreds of complementary regions if one or more mismatches are tolerated (Table ).…”
Antisense oligonucleotide (ASO) therapeutics are single-stranded oligonucleotides which bind to RNA through sequence-specific Watson-Crick base pairings. A unique mechanism of toxicity for ASOs is hybridization-dependent off-target effects that can potentially occur due to the binding of ASOs to complementary regions of unintended RNAs. To reduce the off-target effects of ASOs, it would be useful to know the approximate number of complementary regions of ASOs, or off-target candidate sites of ASOs, of a given oligonucleotide length and complementarity with their target RNAs. However, the theoretical number of complementary regions with mismatches has not been reported to date. In this study, we estimated the general number of complementary regions of ASOs with mismatches in human mRNA sequences by mathematical calculation and in silico analysis using several thousand hypothetical ASOs. By comparing the theoretical number of complementary regions estimated by mathematical calculation to the actual number obtained by in silico analysis, we found that the number of complementary regions of ASOs could be broadly estimated by the theoretical number calculated mathematically. Our analysis showed that the number of complementary regions increases dramatically as the number of tolerated mismatches increases, highlighting the need for expression analysis of such genes to assess the safety of ASOs.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
