How the discovery of ISS-N1 led to the first medical therapy for spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA), a prominent genetic disease of infant mortality, is caused by low levels of survival motor neuron (SMN) protein owing to deletions or mutations of the SMN1 gene. SMN2, a nearly identical copy of SMN1 present in humans, cannot compensate for the loss of SMN1 because of predominant skipping of exon 7 during pre-mRNA splicing. With the recent US Food and Drug Administration approval of nusinersen (Spinraza), the potential for correction of SMN2 exon 7 splicing as an SMA therapy has been affirmed. Nusinersen is an antisense oligonucleotide that targets intronic splicing silencer N1 (ISS-N1) discovered in 2004 at the University of Massachusetts Medical School. ISS-N1 has emerged as the model target for testing the therapeutic efficacy of antisense oligonucleotides using different chemistries as well as different mouse models of SMA. Here, we provide a historical account of events that led to the discovery of ISS-N1 and describe the impact of independent validations that raised the profile of ISS-N1 as one of the most potent antisense targets for the treatment of a genetic disease. Recent approval of nusinersen provides a much-needed boost for antisense technology that is just beginning to realize its potential. Beyond treating SMA, the ISS-N1 target offers myriad potentials for perfecting various aspects of the nucleic-acid-based technology for the amelioration of the countless number of pathological conditions.

Figure 1

Diagrammatic representation of SMN2 gene and Spinraza mode of action. SMN2 exons are represented by colored boxes, while introns are shown as broken lines. Intronic sequence immediately down stream of exon 7 is given. ISS-N1 region within this sequence is highlighted in pink box. Positions to which Spinraza anneals are indicated. SMN2 pre-mRNA splicing results in exon 7-included (SMN2FL) and exon 7-skipped (SMN2Δ7) transcripts, translation of which leads to production of the full length functional SMN protein and a truncated less stable isoform, respectively. Targeting of ISS-N1 by Spriranza prevents exon 7 skipping and as a consequence increases levels of the full length SMN.

Figure 2

Mechanism of exon 7 splicing correction by an ISS-N1 targeting ASO. Only relevant portions of exon 7 and intron 7 are shown. Exonic and intronic sequences are presented in the context of experimentally derived structures.^, Neutral and positive numbering start from the first position of exon 7 and the first position of intron 7, respectively. Splicing regulatory cis-elements and structures, such as the 5′ ss of exon 7, ISS-N1, ISS-N2, TSL2 and ISTL1 are highlighted. Binding sites for hnRNPA1/A2 and TIA1 are indicated. Annealing positions of the ASO and U1 snRNA are shown. Targeting of ISS-N1 by ASO causes structural rearrangements, such as disruption of TSL3 and ISTL1 and blocks the binding sites of hnRNP A1/A2. As the result TIA1 binding sites become accessible and the recruitment of U1 snRNP to the 5′ ss of exon 7 is increased. Abbreviations: ASO, antisense oligonucleotide; ISS, intronic splicing silencer; ISTL, internal stem-loop structure; ss, splice site; TSL, terminal stem-loop structure.