P.6.6 Splice switching Antisense Oligonucleotides: A “kink” in the design?

Abstract

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by the lack of a functional SMN1 gene product, resulting in loss of motor neurons in the anterior horn. This leads to muscle wastage and early death. However, SMA phenotype and disease progression are modified by the presence of one or more copies of SMN2 a near identical gene that potentially encodes the same protein. While multiple copies of SMN2 can reduce disease severity, a base change in exon 7 alters splicing fidelity leading to exon 7 exclusion from the majority of transcripts and a non-functional protein. Antisense Oligonucleotides (AOs) to enhance recognition and retention of exon 7 in the SMN2 transcript have been explored as a therapy for SMA. Others have shown that a 20 base AO sequence targeted to ISS-N1 , a splice silencing domain within intron 7, can promote exon 7 recognition and retention in the mature SMN2 transcript. Five bases added to the 5′ end of this sequence further improves levels of exon 7 retention in SMA cells in vitro. However, contrary to expectation, we found that some mismatched AOs are as effective as the previously reported sequences. We tested a series of mismatch AO sequences for both Anti-ISS-N1 and Anti-ISS-N1+5. Different mismatches were evaluated, including the deletion of either a Uracil (U) or Adenine (A) base, and an U → A base change at various positions along the oligomer sequence. We show that AOs carrying the deletion of an Adenine base or the transversion from U → A to be more efficient, inducing 100% exon inclusion at an AO concentration of 10 nM. We speculate that the AOs are acting in a similar way to microRNAs, where selected mismatches between the mRNA target and the oligomer are biologically active. Perhaps the mismatch creates a “kink” or change in the mRNA secondary structure, ultimately affecting mRNA and potentially protein binding. Further studies aim to investigate this mechanism and its therapeutic potential for SMA and other disorders.