DMD antisense oligonucleotide mediated exon skipping efficiency is affected by flanking intron retention time and target position within the exon

Mutations in the DMD gene are causative for Duchenne muscular dystrophy (DMD). Antisense oligonucleotide (AON) mediated exon skipping to restore disrupted dystrophin reading frame is a therapeutic approach that allows production of a shorter but functional protein. As DMD causing mutations can affect most of the 78 exons encoding dystrophin, a wide variety of AONs are needed to treat the patient population. Design of AONs is largely guided by trial-and-error, and it is yet unclear what defines the skippability of an exon. Here, we use a library of phosphorodiamidate morpholino oligomer (PMOs) AONs of similar physical properties to test the skippability of a large number of DMD exons. The DMD transcript is non-sequentially spliced, meaning that certain introns are retained longer in the transcript than downstream introns. We tested whether the relative intron retention time has a significant effect on AON efficiency, and found that targeting an exon flanked at its 5′ by an intron that is retained in the transcript longer ('slow' intron) leads to overall higher exon skipping efficiency than when the 5′ intron is 'fast'. Regardless of splicing speed of flanking introns, we find that positioning an AON closer to the 5′ of the target exon leads to higher exon skipping efficiency opposed to targeting an exons 3′-end. The data enclosed herein can be of use to guide future target selection and preferential AON binding sites for both Duchenne and other disease amenable by exon skipping therapies. ### Competing Interest Statement Salary of RG is paid by an unrestricted grant from Sarepta Therapeutics and the Ammodo Organisation. FS is an employee of Sarepta Therapeutics AAR discloses being employed by LUMC which has patents on exon skipping technology, some of which has been licensed to BioMarin and subsequently sublicensed to Sarepta. As co-inventor of some of these patents AAR is entitled to a share of royalties. AAR further discloses being ad hoc consultant for PTC Therapeutics, Sarepta Therapeutics, Regenxbio, Alpha Anomeric, BioMarin Pharmaceuticals Inc., Eisai, Entrada, Takeda, Splicesense, Galapagos and Astra Zeneca. Past ad hoc consulting has occurred for: CRISPR Therapeutics, Summit PLC, Audentes Santhera, Bridge Bio, Global Guidepoint and GLG consultancy, Grunenthal, Wave and BioClinica. AAR also reports having been a member of the Duchenne Network Steering Committee (BioMarin) and being a member of the scientific advisory boards of Eisai, hybridize therapeutics, silence therapeutics, Sarepta therapeutics. Past SAB memberships: ProQR, Philae Pharmaceuticals. Remuneration for these activities is paid to LUMC. LUMC also received speaker honoraria from PTC Therapeutics and BioMarin Pharmaceuticals and funding for contract research from Italfarmaco, Sapreme, Eisai, Galapagos, Synnaffix and Alpha Anomeric. Project funding is received from Sarepta Therapeutics.