Challenges of Assessing Exon 53 Skipping of the Human Transcript with Locked Nucleic Acid-Modified Antisense Oligonucleotides in a Mouse Model for Duchenne Muscular Dystrophy.

Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy (DMD) patients to restore dystrophin expression by reframing the disrupted open reading frame of the transcript. However, the treatment efficacy of the already conditionally approved AONs remains low. Aiming to optimize AON efficiency, we assessed exon 53 skipping of the transcript with different chemically modified AONs, all with a phosphorothioate backbone: 2'-O-methyl (2'OMe), locked nucleic acid (LNA)-2'OMe, 2'-fluoro (FRNA), LNA-FRNA, αLNA-FRNA, and FANA-LNA-FRNA. Efficient exon 53 skipping was observed with the FRNA, LNA-FRNA, and LNA-2'OMe AONs in human control myoblast cultures. Weekly subcutaneous injections (50 mg/kg AON) for a duration of 6 weeks were well tolerated by hDMDdel52/ males. Treatment with the LNA-FRNA and LNA-2'OMe AONs resulted in pronounced exon 53 skip levels in skeletal muscles and heart up to 90%, but no dystrophin restoration was observed. This discrepancy was mainly ascribed to the strong binding nature of LNA modifications to RNA, thereby interfering with the amplification of the unskipped product resulting in artificial overamplification of the exon 53 skip product. Our study highlights that treatment effect on RNA and protein level should both be considered when assessing AON efficiency.