Intramuscular Evaluation of Chimeric Locked Nucleic Acid/2′OMethyl-Modified Antisense Oligonucleotides for Targeted Exon 23 Skipping in Mdx Mice

Abstract

Duchenne muscular dystrophy (DMD) is a fatal disorder characterised by progressive muscle wasting. It is caused by mutations in the dystrophin gene, which disrupt the open reading frame leading to the loss of functional dystrophin protein in muscle fibres. Antisense oligonucleotide (AON)-mediated skipping of the mutated exon, which allows production of a truncated but partially functional dystrophin protein, has been at the forefront of DMD therapeutic research for over two decades. Nonetheless, novel nucleic acid modifications and AON designs are continuously being developed to improve the clinical benefit profile of current drugs in the DMD pipeline. We herein designed a series of 15mer and 20mer AONs, consisting of 2′O-Methyl (2′OMe)- and locked nucleic acid (LNA)-modified nucleotides in different percentage compositions, and assessed their efficiency in inducing exon 23 skipping and dystrophin restoration in locally injected muscles of mdx mice. We demonstrate that LNA/2′OMe AONs with a 30% LNA composition were significantly more potent in inducing exon skipping and dystrophin restoration in treated mdx muscles, compared to a previously tested 2′OMe AON and LNA/2′OMe chimeras with lower or higher LNA compositions. These results underscore the therapeutic potential of LNA/2′OMe AONs, paving the way for further experimentation to evaluate their benefit-toxicity profile following systemic delivery.

Efficiency of genome editing using modified single-stranded oligodeoxyribonucleotides in human cells

“…2, Group 2). Indeed, the binding a nity and stability of ssODNs vary depending on the number and position of LNAs [14,21,22,25]. However, no data have been reported on the optimal position of 60-nt or longer ssODNs for genome editing in human cells.…”

“…The introduction of LNA into oligonucleotides increases their melting point and induces a C3'-terminal (N-type) sugar structure in the molecular backbone, resulting in a higher binding a nity to DNA and RNA than natural deoxyribose of the same size [18][19][20]. Although several examples of genome editing using ssODNs of LNA: DNA chimeras have been reported [12,17,[21][22][23][24], there are no reports of genome editing of sense strands longer than 60 nt using such chimeric ssODNs. Therefore, in this study, we produced various ssODNs with different numbers and positions of LNA modi cations of ssODNs longer than 60 nt, which can now be synthesised with improved technology, and performed genome editing using them to investigate the optimal number and positions of LNAs that signi cantly contribute to improving the e ciency of genome editing.…”