RNA in spinal muscular atrophy: therapeutic implications of targeting

Abstract

Introduction: Spinal muscular atrophy (SMA) is caused by low levels of the Survival Motor Neuron (SMN) protein due to deletions of or mutations in the SMN1 gene. Humans carry another nearly identical gene, SMN2, which mostly produces a truncated and less stable protein SMNΔ7 due to predominant skipping of exon 7. Elevation of SMN upon correction of SMN2 exon 7 splicing and gene therapy have been proven to be the effective treatment strategies for SMA.

Areas covered: This review summarizes existing and potential SMA therapies that are based on RNA targeting.We also discuss the mechanistic basis of RNA-targeting molecules.

Expert opinion: The discovery of intronic splicing silencer N1 (ISS-N1) was the first major step towards developing the currently approved antisense-oligonucleotide (ASO)-directed therapy (SpinrazaTM) based on the correction of exon 7 splicing of the endogenous SMN2pre-mRNA. Recently, gene therapy (Zolgensma) has become the second approved treatment for SMA. Small compounds (currently in clinical trials) capable of restoring SMN2 exon 7 inclusion further expand the class of the RNA targeting molecules for SMA therapy. Endogenous RNA targets, such as long non-coding RNAs, circular RNAs, microRNAs and ribonucleoproteins, could be potentially exploited for developing additional SMA therapies.

Keywords: ISS-N1; RNP; SMA; SMN; Spinal muscular atrophy; SpinrazaTM; Survival Motor Neuron; antisense; circular RNA; nusinersen; pre-mRNA splicing.

Figure 1.

Diagrammatic representation of relevant splicing regulatory cis-elements within SMN exon 7 and the flanking intronic sequences. Positive and negative regulatory cis-elements/transfactors are indicated by (+) and (−), respectively. In addition, cis-elements are highlighted in different colors. 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. Exinct, the Conserved tract and the 3′-Cluster were identified by the in vivo selection of the entire exon 7. ISS-N1 spans from the 10^th to the 24^th position of intron 7. For details of other splicing regulators depicted here, please refer to the main text and the recent reviews [13,14]. The 3′ and 5′ss of exon 7 are shown. SMN2-specific nucleotides are marked. Exonic and intronic binding sites of hnRNP A1/A2 are highlighted in pink. The binding site of Sam68 within exon 7 is indicated as well. ISS-N1 harbors two hnRNP A1 motifs. Additional two hnRNP A1/A2 motifs are located at/near the 3′ss of exon 7. One of these hnRNP A1/A2 motifs is specific to SMN2 due to a C-to-T substitution (C6U) at the 6^th position of exon 7. Another SMN2-specific hnRNP A1/A2 motif is created due to A-to-G substitution (A100G) at the 100^th position of intron 7. The C residue at the 10^th intronic position, ¹⁰C, associated with an inhibitory RNA structure (ISTL1) formed by long-distance interactions is indicated by an arrow. GC-rich sequence (GCRS) that overlaps ISS-N1 is boxed. For other relevant terminologies and abbreviations see Table 2.

Figure 2.

Diagrammatic representation of RNA secondary structures, either local or formed by long-distance interactions, identified within SMN exon 7 and intron 7. The structures were experimentally validated by enzymatic and chemical probing. Exonic sequences are shown in uppercase letters, intronic, in lowercase letters. Negative and neutral numbering of nucleotides starts from the end of exon 7 and the beginning of intron 7, respectively. Secondary structures TSL2 and ISTL1 fully sequester the 5′ss of exon 7. Regulatory cis-elements that affect exon 7 splicing, including ISS-N1 with its hnRNP A1/A2 binding sites, GC-rich sequence and TIA1 binding sites, are highlighted in different colors (see the insert). The native and the cryptic (Cr1 and Cr2) 5′ss are indicated by arrows. For terminologies and abbreviations see Table 2.

Figure 3.

Structure-specific interactions of small molecules that are known to promote SMN2 exon 7 inclusion. (A) Interactions of SMN-C5 with the 54A (highlighted with a white circle) in the context of U1:5′ss duplex formed between the U1 snRNA and the 5′ss of exon 7. 54A is located at the last position of exon 7 and is boxed. Exon 7 sequence is shown in uppercase letters; intron 7, in lowercase letters. The U1 snRNA sequence (red uppercase letters in bold) and its base-pairing with the 5′ss of exon 7 are shown. The 5′ss is indicated by an arrow. For comparison reasons the chemical structure of SMN-C5 (C5) is given. In the bottom panel, C5 is represented as a hexagon. 54A forms a bulge in the context of the U1:5′ss duplex. C5 stabilizes the bulged 54A by specifically interacting with it. This promotes SMN2 exon 7 inclusion. For terminologies and abbreviations see Table 2. (B) Interactions of SMN-C3 with the internal stem (IS), formed by the central region of exon 7 and intron 6. The probed structure formed by the entire exon 7 and adjacent intronic sequences is presented. Exon 7 sequence is shown in uppercase letters, intron 6 and intron 7, in lowercase letters. Neutral, negative and positive nucleotide numbering starts from the beginning of exon 7, the end of intron 6 and the start of intron 7, respectively. The 5′ss and the 3′ss are indicated by arrows. The binding site of SMN-C3 in the context of IS, which is boxed, is highlighted in red. Residues, whose sensitivity to a structure probing agent, NAI, is increased and decreased upon SMN-C3 binding are highlighted using a pink and a blue circle, respectively. For comparison reasons the chemical structure of SMN-C3 is given as well. For terminologies and abbreviations see Table 2. (C) Interactions of PK4C9 with TSL2. Exonic and intronic sequences are shown in upper- and lowercase letters, respectively. The 5′ss of exon 7 is indicated by an arrow. Chemical structure of PK4C9 is given. In the bottom panel, PK4C9 is represented by a star. Upon PK4C9 binding to TSL2, several residues at the base of the stem highlighted with pink circles form hydrogen bonds, hydrophobic interactions or stack with the compound. These interactions destabilize TSL2 and increase the accessibility of the 5′ss. In addition, binding of PK4C9 to TSL2 stabilizes the triloop of this RNA structure. For terminologies and abbreviations see Table 2.