A Prospective Study in the Rational Design of Efficient Antisense Oligonucleotides for Exon Skipping in the DMD Gene

Abstract

Antisense oligonucleotide (AON)-mediated exon skipping to restore dystrophin expression in Duchenne muscular dystrophy (DMD) therapy shown promise in a number of human clinical trials. Current AON design methods are semi-empirical, involving either trial-and-error and/or preliminary experimentations. Therefore, a rational approach to design efficient AONs to address the wide spectrum of patients' mutations is desirable. Retrospective studies have extracted many AON design variables, but they were not tested prospectively to design AONs for skipping DMD exons. Not only did the variables differ among the various studies, no numerical cutoff for each variable was inferred, which makes their use in AON design difficult. The challenge is to thus select a minimal set of key independent variables that can consistently design efficient AONs. In this prospective study, a novel set of design variables with respective cutoff values was used to design 23 novel AONs, each to skip one of nine DMD exons. Nineteen AONs were found to be efficacious in inducing specific exon skipping (83% of total), of which 14 were considered efficient (61% of total), i.e., they induced exon skipping in > 25% of total transcripts. Notably, the satisfactory success rates were achieved by using only three design variables; namely, co-transcriptional binding accessibility of target site, presence of exonic splicing enhancers, and target length. Retrospective analyses revealed that the most efficient AON in every exon targeted has the lowest average cumulative position (ACP) score. Taking the prospective and retrospective studies together, we propose that design guidelines recommend using the ACP score to select the most efficient AON for each exon.

Quantitative Antisense Screening and Optimization for Exon 51 Skipping in Duchenne Muscular Dystrophy

“…[4][5][6][7][8][9][10][11]13 Accordingly, quantitative screening of exon 51 skipping AOs based on their ability to rescue dystrophin protein has never been reported in primary DMD cells. 4,7,10,13,31 In this study, we demonstrated the feasibility of AO screening via quantification of dystrophin protein induced by exon 51 skipping using immortalized DMD patient cell lines. Through a combination of in silico and in vitro screening, morpholino-based AOs showed significantly greater effectiveness at facilitating exon 51 skipping and rescuing dystrophin protein expression than the eteplirsen sequence.…”

“…2 Currently one of the most promising therapeutic avenues is exon skipping using antisense oligonucleotides (AOs). 3 Exon skipping can restore the reading frame by removing the mutant exon and/or its flanking exon(s) from the DMD pre-mRNA, [4][5][6][7][8][9][10][11][12][13] enabling the production of truncated but partly functional dystrophin protein as seen in the milder counterpart disorder, Becker muscular dystrophy (BMD). [14][15][16] A majority of DMD patients harbor deletion mutations, and 20% of these are amenable to exon 51 skipping.…”

“…However, the exon skipping effectiveness of designed AOs has not been evaluated both quantitatively and statistically. 4,7,10,13,31 Although restoring dystrophin protein expression is necessary to improve dystrophic muscle function, the ability of AOs to rescue dystrophin protein expression has not been reported with sufficient methods of quantification in previous AO screening studies. They highly relied on RT-PCR from primary DMD muscle cells.…”

Introduction to Cotranscriptional RNA Splicing

“…Some of the genes that undergo dysregulated cotranscriptional alternative splicing are currently being investigated as targets for antisense oligonucleotide (AON) therapy. For example, antisense oligonucleotides, which induce exon skipping in the DMD gene, have shown promise as therapeutic intervention in Duchenne muscular dystrophy [76]. Further analysis to better understand which genes are cotranscriptionally spliced and the mechanisms of cotranscriptional splicing will likely uncover many genes whose dysregulation leads to disease or defects in development.…”

Mutational spectrum of dystrophinopathies in Singapore: Insights for genetic diagnosis and precision therapy

“…Among some of the most promising approaches are those targeted toward specific mutations. In the exon skipping strategy, antisense oligonucleotides (AONs) targeted to specific sequences mediate the exclusion of out‐of‐frame exon(s) in the transcript to convert out‐of‐frame mutations to in‐frame mutations leading to a milder phenotype for DMD patients (Aartsma‐Rus et al, ; Aartsma‐Rus et al, ; Kinali et al, ; Nakamura, ; Pramono et al, ; Shimizu‐Motohashi, Miyatake, Komaki, Takeda, & Aoki, ; Takeshima et al, ; Wee et al, ; Wilton et al, ). The recent breakthrough in exon skipping therapeutics came with the release of the first and the only FDA approved drug, Eteplirsen (Exondys 51) which is applicable for DMD patients amenable for exon 51 skipping (https://www.fda.gov/newsevents/newsroom/pressannouncements/ucm521263.htm).…”