The First Orally Deliverable Small Molecule for the Treatment of Spinal Muscular Atrophy

Abstract

Spinal muscular atrophy (SMA) is 1 of the leading causes of infant mortality. SMA is mostly caused by low levels of Survival Motor Neuron (SMN) protein due to deletion of or mutation in the SMN1 gene. Its nearly identical copy, SMN2, fails to compensate for the loss of SMN1 due to predominant skipping of exon 7. Correction of SMN2 exon 7 splicing by an antisense oligonucleotide (ASO), nusinersen (Spinraza™), that targets the intronic splicing silencer N1 (ISS-N1) became the first approved therapy for SMA. Restoration of SMN levels using gene therapy was the next. Very recently, an orally deliverable small molecule, risdiplam (Evrysdi™), became the third approved therapy for SMA. Here we discuss how these therapies are positioned to meet the needs of the broad phenotypic spectrum of SMA patients.

Keywords: Branaplam; Evrysdi; ISS-N1; SMA; SMN; Spinraza; Zolgensma; antisense oligonucleotide; nusinersen; risdiplam; splicing.

Figure 1.

Diagrammatic representation of cis-elements and transacting factors that regulate SMN2 exon 7 splicing. (a) Relative positioning of cis-elements within exon 7 and downstream intron 7 of SMN. Cis-elements and transacting factors that bind them are highlighted in different colors. Note, the diagram presented here is not inclusive of all reported exon 7 splicing regulators. Please refer to recent reviews for more comprehensive information.^, Positive and negative regulators of exon 7 splicing are indicated by (+) and (−), respectively. Neutral numbering of nucleotides starts from the first position of exon 7. Positive numbering of nucleotides starts from the first position of intron 7. Exonic and intronic sequences are shown in upper- and lower-case letters, respectively. SMN2-specific C6U substitution is marked. Exinct, the Conserved tract and the 3′-Cluster were identified by in vivo selection of the entire exon 7. In vivo selection of the entire exon also revealed the strong negative effect of an “A” residue at the 54th position (54A) of exon 7 and (b) structural context of the 5′ss of SMN exon 7. Numbering is the same as described in panel A. Only a portion of exon 7 and intron 7 is shown. Cis-elements that promote exon 7 skipping are highlighted in colors. Abbreviations: 3′ss, 3′ splice site; 5′ss, 3′ splice site; Exinct, extended inhibitory context; GCRS, GC-rich sequence; ISS-N1, intronic splicing silencer; ISTL1, an internal stem (inhibitory RNA structure) formed by long-distance interaction; TSL2, terminal stem-loop structure 2; TSL3, terminal stem-loop structure 3.

Figure 2.

Structure of orally available small molecules used in pre-clinical and clinical studies for the treatment of SMA. SMN-C1, SMNC-2, and SMN-C3 were the first set of compounds reported by PTC-Roche (PTC Therapeutics, South Plainfield, New Jersey and Hoffmann-La Roche, Basel, Switzerland) to correct SMN2 exon 7 splicing with high specificity. Most mechanistic studies have been done using SMN-C3 and SMN-C5. Clinical trial of RG-7800 by PTC-Roche was terminated due to its toxicity in cynomolgus monkeys. Risdiplam has gone through multiple clinical trials by PTC-Roche and has recently been approved by FDA. Branaplam is in clinical trial by Novartis Pharmaceuticals. PK4C9 and TEC-1 are the newly reported compounds to show specific splicing correction of SMN2 exon 7.

Figure 3.

Potential mechanism of action of risdiplam. Risdiplam analogs SMN-C3 and -C5 are depicted as red stars. SMN-C3 has been shown to interact with an AG-rich motif (shown in green letters) located in the middle of exon 7. SMN-C3 has been proposed to recruit splicing factors FUBP1 and KHSRP. SMN-C5 has been proposed to promote recruitment of U1 snRNP by directly binding to 54A at the 5′ss of exon 7. Interaction of U1 snRNP with the 5′ss of exon 7 has been depicted. Drawing is not to scale.

Figure 4.

Off-target effects of risdiplam or its analog SMN-C3. (a) Splicing pattern of SMN2 exon 7 and ten other splicing events affected by SMN-C3 treatment as reported by Naryshkin and coworkers. Y axis indicates the proportion of total spliced transcript that has the exon in question included. X axis labels indicate the host gene and exon number of the target exons. NE: novel (unannotated) exon, (b) top enriched sequence motifs near the 3′ and 5′ss of the exons, splicing of which was changed by SMN-C3. Letter height in each motif corresponds to nucleotide enrichment at that position, (c) the sequences of 5“off-target” exons, splicing of which was affected by risdiplam, as reported by Ratni and coworkers. Numbering is given relative to the first position of each exon. Uppercase letters represent exonic sequences, lowercase letters represent intronic sequences. The longest AG-rich motif in each exon is boxed. The last two exonic nucleotides and the first six intronic nucleotides of the 5′ss are shown in bold. Each shaded/clear area “cover” ten consecutive nucleotides. An additional 5′ss within exon 9 of FOXM1 is indicated with an asterisk, and (d) genomic overview of two examples of splicing events induced by SMN-C3 treatment. POMT2 (top panel) contains a novel, unannotated exon located in the region between exons 11 and 12. Inclusion of this unannotated exon is caused by SMN-C3 treatment, as shown by the increased read depth. SNAP23 (bottom panel) has a novel exon (between exons 3 and 4) that undergoes inclusion. This is coupled with intron retention, as indicated by increased read depth in the flanking introns.