A super minigene with a short promoter and truncated introns recapitulates essential features of transcription and splicing regulation of the SMN1 and SMN2 genes.

Here we report a Survival Motor Neuron 2 (SMN2) super minigene, SMN2Sup, encompassing its own promoter, all exons, their flanking intronic sequences and the entire 3'-untranslated region. We confirm that the pre-mRNA generated from SMN2Sup undergoes splicing to produce a translation-competent mRNA. We demonstrate that mRNA generated from SMN2Sup produces more SMN than an identical mRNA generated from a cDNA clone. We uncover that overexpression of SMN triggers skipping of exon 3 of SMN1/SMN2. We define the minimal promoter and regulatory elements associated with the initiation and elongation of transcription of SMN2. The shortened introns within SMN2Sup preserved the ability of camptothecin, a transcription elongation inhibitor, to induce skipping of exons 3 and 7 of SMN2. We show that intron 1-retained transcripts undergo nonsense-mediated decay. We demonstrate that splicing factor SRSF3 and DNA/RNA helicase DHX9 regulate splicing of multiple exons in the context of both SMN2Sup and endogenous SMN1/SMN2. Prevention of SMN2 exon 7 skipping has implications for the treatment of spinal muscular atrophy (SMA). We validate the utility of the super minigene in monitoring SMN levels upon splicing correction. Finally, we demonstrate how the super minigene could be employed to capture the cell type-specific effects of a pathogenic SMN1 mutation.

Diverse targets of SMN2-directed splicing-modulating small molecule therapeutics for spinal muscular atrophy.

Designing an RNA-interacting molecule that displays high therapeutic efficacy while retaining specificity within a broad concentration range remains a challenging task. Risdiplam is an FDA-approved small molecule for the treatment of spinal muscular atrophy (SMA), the leading genetic cause of infant mortality. Branaplam is another small molecule which has undergone clinical trials. The therapeutic merit of both compounds is based on their ability to restore body-wide inclusion of Survival Motor Neuron 2 (SMN2) exon 7 upon oral administration. Here we compare the transcriptome-wide off-target effects of these compounds in SMA patient cells. We captured concentration-dependent compound-specific changes, including aberrant expression of genes associated with DNA replication, cell cycle, RNA metabolism, cell signaling and metabolic pathways. Both compounds triggered massive perturbations of splicing events, inducing off-target exon inclusion, exon skipping, intron retention, intron removal and alternative splice site usage. Our results of minigenes expressed in HeLa cells provide mechanistic insights into how these molecules targeted towards a single gene produce different off-target effects. We show the advantages of combined treatments with low doses of risdiplam and branaplam. Our findings are instructive for devising better dosing regimens as well as for developing the next generation of small molecule therapeutics aimed at splicing modulation.

Human Survival Motor Neuron genes generate a vast repertoire of circular RNAs.

Circular RNAs (circRNAs) perform diverse functions, including the regulation of transcription, translation, peptide synthesis, macromolecular sequestration and trafficking. Inverted Alu repeats capable of forming RNA:RNA duplexes that bring splice sites together for backsplicing are known to facilitate circRNA generation. However, higher limits of circRNAs produced by a single Alu-rich gene are currently not predictable due to limitations of amplification and analyses. Here, using a tailored approach, we report a surprising diversity of exon-containing circRNAs generated by the Alu-rich Survival Motor Neuron (SMN) genes that code for SMN, an essential multifunctional protein in humans. We show that expression of the vast repertoire of SMN circRNAs is universal. Several of the identified circRNAs harbor novel exons derived from both intronic and intergenic sequences. A comparison with mouse Smn circRNAs underscored a clear impact of primate-specific Alu elements on shaping the overall repertoire of human SMN circRNAs. We show the role of DHX9, an RNA helicase, in splicing regulation of several SMN exons that are preferentially incorporated into circRNAs. Our results suggest self- and cross-regulation of biogenesis of various SMN circRNAs. These findings bring a novel perspective towards a better understanding of SMN gene function.

High-affinity RNA targets of the Survival Motor Neuron protein reveal diverse preferences for sequence and structural motifs.

The Survival Motor Neuron (SMN) protein is essential for survival of all animal cells. SMN harbors a nucleic acid-binding domain and plays an important role in RNA metabolism. However, the RNA-binding property of SMN is poorly understood. Here we employ iterative in vitro selection and chemical structure probing to identify sequence and structural motif(s) critical for RNA-SMN interactions. Our results reveal that motifs that drive RNA-SMN interactions are diverse and suggest that tight RNA-SMN interaction requires presence of multiple contact sites on the RNA molecule. We performed UV crosslinking and immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to identify cellular RNA targets of SMN in neuronal SH-SY5Y cells. Results of HITS-CLIP identified a wide variety of targets, including mRNAs coding for ribosome biogenesis and cytoskeleton dynamics. We show critical determinants of ANXA2 mRNA for a direct SMN interaction in vitro. Our data confirms the ability of SMN to discriminate among close RNA sequences, and represent the first validation of a direct interaction of SMN with a cellular RNA target. Our findings suggest direct RNA-SMN interaction as a novel mechanism to initiate the cascade of events leading to the execution of SMN-specific functions.

Activation of a cryptic 5' splice site reverses the impact of pathogenic splice site mutations in the spinal muscular atrophy gene.

Spinal muscular atrophy (SMA) is caused by deletions or mutations of the Survival Motor Neuron 1 (SMN1) gene coupled with predominant skipping of SMN2 exon 7. The only approved SMA treatment is an antisense oligonucleotide that targets the intronic splicing silencer N1 (ISS-N1), located downstream of the 5' splice site (5'ss) of exon 7. Here, we describe a novel approach to exon 7 splicing modulation through activation of a cryptic 5'ss (Cr1). We discovered the activation of Cr1 in transcripts derived from SMN1 that carries a pathogenic G-to-C mutation at the first position (G1C) of intron 7. We show that Cr1-activating engineered U1 snRNAs (eU1s) have the unique ability to reprogram pre-mRNA splicing and restore exon 7 inclusion in SMN1 carrying a broad spectrum of pathogenic mutations at both the 3'ss and 5'ss of the exon 7. Employing a splicing-coupled translation reporter, we demonstrate that mRNAs generated by an eU1-induced activation of Cr1 produce full-length SMN. Our findings underscore a wider role for U1 snRNP in splicing regulation and reveal a novel approach for the restoration of SMN exon 7 inclusion for a potential therapy of SMA.

Gender-Specific Amelioration of SMA Phenotype upon Disruption of a Deep Intronic Structure by an Oligonucleotide.

Spinal muscular atrophy (SMA), the leading genetic disease of children, is caused by low levels of survival motor neuron (SMN) protein. Here, we employ A15/283, an antisense oligonucleotide targeting a deep intronic sequence/structure, to examine the impact of restoration of SMN in a mild SMA mouse model. We show gender-specific amelioration of tail necrosis upon subcutaneous administrations of A15/283 into SMA mice at postnatal days 1 and 3. We also demonstrate that a modest increase in SMN due to early administrations of A15/283 dramatically improves testicular development and spermatogenesis. Our results reveal near total correction of expression of several genes in adult testis upon temporary increase in SMN during early postnatal development. This is the first demonstration of in vivo efficacy of an antisense oligonucleotide targeting a deep intronic sequence/structure. This is also the first report of gender-specific amelioration of SMA pathology upon a modest peripheral increase of SMN.

An intronic structure enabled by a long-distance interaction serves as a novel target for splicing correction in spinal muscular atrophy.

Here, we report a long-distance interaction (LDI) as a critical regulator of alternative splicing of Survival Motor Neuron 2 (SMN2) exon 7, skipping of which is linked to spinal muscular atrophy (SMA), a leading genetic disease of children and infants. We show that this LDI is linked to a unique intra-intronic structure that we term internal stem through LDI-1 (ISTL1). We used site-specific mutations and Selective 2'-Hydroxyl Acylation analyzed by Primer Extension to confirm the formation and functional significance of ISTL1. We demonstrate that the inhibitory effect of ISTL1 is independent of hnRNP A1/A2B1 and PTB1 previously implicated in SMN2 exon 7 splicing. We show that an antisense oligonucleotide-mediated sequestration of the 3' strand of ISTL1 fully corrects SMN2 exon 7 splicing and restores high levels of SMN and Gemin2, a SMN-interacting protein, in SMA patient cells. Our results also reveal that the 3' strand of ISTL1 and upstream sequences constitute an inhibitory region that we term intronic splicing silencer N2 (ISS-N2). This is the first report to demonstrate a critical role of a structure-associated LDI in splicing regulation of an essential gene linked to a genetic disease. Our findings expand the repertoire of potential targets for an antisense oligonucleotide-mediated therapy of SMA.

Spinal muscular atrophy: an update on therapeutic progress.

Humans have two nearly identical copies of survival motor neuron gene: SMN1 and SMN2. Deletion or mutation of SMN1 combined with the inability of SMN2 to compensate for the loss of SMN1 results in spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. SMA affects 1 in ~6000 live births, a frequency much higher than in several genetic diseases. The major known defect of SMN2 is the predominant exon 7 skipping that leads to production of a truncated protein (SMNΔ7), which is unstable. Therefore, SMA has emerged as a model genetic disorder in which almost the entire disease population could be linked to the aberrant splicing of a single exon (i.e. SMN2 exon 7). Diverse treatment strategies aimed at improving the function of SMN2 have been envisioned. These strategies include, but are not limited to, manipulation of transcription, correction of aberrant splicing and stabilization of mRNA, SMN and SMNΔ7. This review summarizes up to date progress and promise of various in vivo studies reported for the treatment of SMA.

Synergistic Effect of an Antisense Oligonucleotide and Small Molecule on Splicing Correction of the Spinal Muscular Atrophy Gene.

Spinal muscular atrophy (SMA) is treated by increasing the level of Survival Motor Neuron (SMN) protein through correction of SMN2 exon 7 skipping or exogenous expression of SMN through gene therapy. Currently available therapies have multiple shortcomings, including poor body-wide distribution, invasive delivery, and potential negative consequences due to high doses needed for clinical efficacy. Here we test the effects of a combination treatment of a splice-correcting antisense oligonucleotide (ASO) Anti-N1 with the small compounds risdiplam and branaplam. We show that a low-dose treatment of Anti-N1 with either compound produces a synergistic effect on the inclusion of SMN2 exon 7 in SMA patient fibroblasts. Using RNA-Seq, we characterize the transcriptomes of cells treated with each compound as well as in combination. Although high doses of each individual treatment trigger widespread perturbations of the transcriptome, combination treatment of Anti-N1 with risdiplam and branaplam results in minimal disruption of gene expression. For individual genes targeted by the 3 compounds, we observe little to no additive effects of combination treatment. Overall, we conclude that the combination treatment of a splice-correcting ASO with small compounds represents a promising strategy for achieving a high level of SMN expression while minimizing the risk of off-target effects.

Transcriptome- and proteome-wide effects of a circular RNA encompassing four early exons of the spinal muscular atrophy genes.

Spinal muscular atrophy (SMA) genes, SMN1 and SMN2 (hereinafter referred to as SMN1/2), produce multiple circular RNAs (circRNAs), including C2A-2B-3-4 that encompasses early exons 2A, 2B, 3 and 4. C2A-2B-3-4 is a universally and abundantly expressed circRNA of SMN1/2. Here we report the transcriptome- and proteome-wide effects of overexpression of C2A-2B-3-4 in inducible HEK293 cells. Our RNA-Seq analysis revealed altered expression of ~ 15% genes (4172 genes) by C2A-2B-3-4. About half of the affected genes by C2A-2B-3-4 remained unaffected by L2A-2B-3-4, a linear transcript encompassing exons 2A, 2B, 3 and 4 of SMN1/2. These findings underscore the unique role of the structural context of C2A-2B-3-4 in gene regulation. A surprisingly high number of upregulated genes by C2A-2B-3-4 were located on chromosomes 4 and 7, whereas many of the downregulated genes were located on chromosomes 10 and X. Supporting a cross-regulation of SMN1/2 transcripts, C2A-2B-3-4 and L2A-2B-3-4 upregulated and downregulated SMN1/2 mRNAs, respectively. Proteome analysis revealed 61 upregulated and 57 downregulated proteins by C2A-2B-3-4 with very limited overlap with those affected by L2A-2B-3-4. Independent validations confirmed the effect of C2A-2B-3-4 on expression of genes associated with chromatin remodeling, transcription, spliceosome function, ribosome biogenesis, lipid metabolism, cytoskeletal formation, cell proliferation and neuromuscular junction formation. Our findings reveal a broad role of C2A-2B-3-4, and expands our understanding of functions of SMN1/2 genes.

An antisense microwalk reveals critical role of an intronic position linked to a unique long-distance interaction in pre-mRNA splicing.

Here we report a novel finding of an antisense oligonucleotide (ASO) microwalk in which we examined the position-specific role of intronic residues downstream from the 5' splice site (5' ss) of SMN2 exon 7, skipping of which is associated with spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Our results revealed the inhibitory role of a cytosine residue at the 10th intronic position ((10)C), which is neither conserved nor associated with any known splicing motif. Significance of (10)C emerged from the splicing pattern of SMN2 exon 7 in presence of a 14-mer ASO (L14) that sequestered two adjacent hnRNP A1 motifs downstream from (10)C and yet promoted SMN2 exon 7 skipping. Another 14-mer ASO (F14) that sequestered both, (10)C and adjacent hnRNP A1 motifs, led to a strong stimulation of SMN2 exon 7 inclusion. The inhibitory role of (10)C was found to be tightly linked to its unpaired status and specific positioning immediately upstream of a RNA:RNA helix formed between the targeting ASO and its intronic target. Employing a heterologous context as well as changed contexts of SMN2 intron 7, we show that the inhibitory effect of unpaired (10)C is dependent upon a long-distance interaction involving downstream intronic sequences. Our report furnishes one of the rare examples in which an ASO-based approach could be applied to unravel the critical role of an intronic position that may not belong to a linear motif and yet play significant role through long-distance interactions.

Structural Context of a Critical Exon of Spinal Muscular Atrophy Gene.

Humans contain two nearly identical copies of Survival Motor Neuron genes, SMN1 and SMN2. Deletion or mutation of SMN1 causes spinal muscular atrophy (SMA), one of the leading genetic diseases associated with infant mortality. SMN2 is unable to compensate for the loss of SMN1 due to predominant exon 7 skipping, leading to the production of a truncated protein. Antisense oligonucleotide and small molecule-based strategies aimed at the restoration of SMN2 exon 7 inclusion are approved therapies of SMA. Many cis-elements and transacting factors have been implicated in regulation of SMN exon 7 splicing. Also, several structural elements, including those formed by a long-distance interaction, have been implicated in the modulation of SMN exon 7 splicing. Several of these structures have been confirmed by enzymatic and chemical structure-probing methods. Additional structures formed by inter-intronic interactions have been predicted by computational algorithms. SMN genes generate a vast repertoire of circular RNAs through inter-intronic secondary structures formed by inverted Alu repeats present in large number in SMN genes. Here, we review the structural context of the exonic and intronic cis-elements that promote or prevent exon 7 recognition. We discuss how structural rearrangements triggered by single nucleotide substitutions could bring drastic changes in SMN2 exon 7 splicing. We also propose potential mechanisms by which inter-intronic structures might impact the splicing outcomes.

Alternative splicing in spinal muscular atrophy underscores the role of an intron definition model.

Humans have two nearly identical copies of the Survival Motor Neuron (SMN) gene: SMN1 and SMN2. The two SMN genes code for identical proteins; however, SMN2 predominantly generates a shorter transcript due to skipping of exon 7, the last coding exon. Skipping of SMN2 exon 7 leads to production of a truncated SMN protein that is highly unstable. The inability of SMN2 to compensate for the loss of SMN1 results in spinal muscular atrophy (SMA), the second most prevalent genetic cause of infant mortality. Since SMN2 is almost universally present in SMA patients, correction of SMN2 exon 7 splicing holds the promise for cure. Consistently, SMN2 exon 7 splicing has emerged as one of the best studied splicing systems in humans. The vast amount of recent literature provides a clue that SMN2 exon 7 splicing is regulated by an intron definition mechanism, which does not require cross-exon communication as prerequisite for exon inclusion. Our conclusion is based on the prominent role of intronic cis-elements, some of them have emerged as the frontrunners among potential therapeutic targets of SMA. Further, the widely expressed T-cell-restricted intracellular antigen-1 (TIA1), a member of the Q-rich domain containing RNA-binding proteins, has recently been found to regulate SMN exon 7 splicing by binding to intron 7 sequences away from the 5' ss. These findings make a strong argument for an "intron definition model", according to which regulatory sequences within a downstream intron are capable of enforcing exon inclusion even in the absence of a defined upstream 3' ss of an alternatively spliced exon.

Desulfurization of dibenzothiophene (DBT) by a novel strain Lysinibacillus sphaericus DMT-7 isolated from diesel contaminated soil.

A new bacterial strain DMT-7 capable of selectively desulfurizing dibenzothiophene (DBT) was isolated from diesel contaminated soil. The DMT-7 was characterized and identified as Lysinibacillus sphaericus DMT-7 (NCBI GenBank Accession No. GQ496620) using 16S rDNA gene sequence analysis. The desulfurized product of DBT, 2-hydroxybiphenyl (2HBP), was identified and confirmed by high performance liquid chromatography analysis and gas chromatography-mass spectroscopy analysis respectively. The desulfurization kinetics revealed that DMT-7 started desulfurization of DBT into 2HBP after the lag phase of 24 hr, exponentially increasing the accumulation of 2HBP up to 15 days leading to approximately 60% desulfurization of the DBT. However, further growth resulted into DBT degradation. The induced culture of DMT-7 showed shorter lag phase of 6 hr and early onset of stationary phase within 10 days for desulfurization as compared to that of non-induced culture clearly indicating the inducibility of the desulfurization pathway of DMT-7. In addition, Lysinibacillus sphaericus DMT-7 also possess the ability to utilize broad range of substrates as sole source of sulfur such as benzothiophene, 3,4-benzo DBT, 4,6-dimethyl DBT, and 4,6-dibutyl DBT. Therefore, Lysinibacillus sphaericus DMT-7 could serve as model system for efficient biodesulfurization of diesel and petrol.

Spinal muscular atrophy: Broad disease spectrum and sex-specific phenotypes.

Spinal muscular atrophy (SMA) is one of the major genetic disorders associated with infant mortality. More than 90% of cases of SMA result from deletions of or mutations in the Survival Motor Neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, does not compensate for the loss of SMN1 due to predominant skipping of exon 7. The spectrum of SMA is broad, ranging from prenatal death to infant mortality to survival into adulthood. All tissues, including brain, spinal cord, bone, skeletal muscle, heart, lung, liver, pancreas, gastrointestinal tract, kidney, spleen, ovary and testis, are directly and/or indirectly affected in SMA. Accumulating evidence on impaired mitochondrial biogenesis and defects in X chromosome-linked modifying factors, coupled with the sexual dimorphic nature of many tissues, point to sex-specific vulnerabilities in SMA. Here we review the role of sex in the pathogenesis of SMA.

Lipid residues in pottery from the Indus Civilisation in northwest India.

This paper presents novel insights into the archaeology of food in ancient South Asia by using lipid residue analysis to investigate what kinds of foodstuffs were used in ceramic vessels by populations of the Indus Civilisation in northwest India. It examines how vessels were used in urban and rural Indus settlements during the Mature Harappan period (c.2600/2500-1900 BC), the relationship between vessels and the products within them, and identifies whether changes in vessel use occurred from the Mature Harappan to Late Harappan periods, particularly during climatic instability after 4.2 ka BP (c.2100 BC). Despite low lipid concentrations, which highlight challenges with conducting residue analysis in arid, seasonally-wet and alkaline environments, 71% of the vessels yielded appreciable quantities of lipid. Lipid profiles revealed the use of animal fats in vessels, and contradictory to faunal evidence, a dominance of non-ruminant fats, with limited evidence of dairy processing. The absence of local modern reference fats makes this dataset challenging to interpret, and it is possible that plant products or mixtures of plant and animal products have led to ambiguous fatty acid-specific isotopic values. At the same time, it appears that urban and rural populations processed similar types of products in vessels, with limited evidence for change in vessel use from the urban to the post-urban period. This study is a systematic investigation into pot lipid residues from multiple sites, demonstrating the potential of the method for examining ancient Indus foodways and the need for the development of further research in ancient organic residues in South Asia.

Functional characterization of alternatively spliced human SECISBP2 transcript variants.

Synthesis of selenoproteins depends on decoding of the UGA stop codon as the amino acid selenocysteine (Sec). This process requires the presence of a Sec insertion sequence element (SECIS) in the 3'-untranslated region of selenoprotein mRNAs and its interaction with the SECIS binding protein 2 (SBP2). In humans, mutations in the SBP2-encoding gene Sec insertion sequence binding protein 2 (SECISBP2) that alter the amino acid sequence or cause splicing defects lead to abnormal thyroid hormone metabolism. Herein, we present the first in silico and in vivo functional characterization of alternative splicing of SECISBP2. We report a complex splicing pattern in the 5'-region of human SECISBP2, wherein at least eight splice variants encode five isoforms with varying N-terminal sequence. One of the isoforms, mtSBP2, contains a mitochondrial targeting sequence and localizes to mitochondria. Using a minigene-based in vivo splicing assay we characterized the splicing efficiency of several alternative transcripts, and show that the splicing event that creates mtSBP2 can be modulated by antisense oligonucleotides. Moreover, we show that full-length SBP2 and some alternatively spliced variants are subject to a coordinated transcriptional and translational regulation in response to ultraviolet type A irradiation-induced stress. Overall, our data broadens the functional scope of a housekeeping protein essential to selenium metabolism.

RNA in spinal muscular atrophy: therapeutic implications of targeting.

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.

Characteristics of circular RNAs generated by human Survival Motor Neuron genes.

Circular RNAs (circRNAs) belong to a diverse class of stable RNAs expressed in all cell types. Their proposed functions include sponging of microRNAs (miRNAs), sequestration and trafficking of proteins, assembly of multimeric complexes, production of peptides, and regulation of transcription. Backsplicing due to RNA structures formed by an exceptionally high number of Alu repeats lead to the production of a vast repertoire of circRNAs by human Survival Motor Neuron genes, SMN1 and SMN2, that code for SMN, an essential multifunctional protein. Low levels of SMN due to deletion or mutation of SMN1 result in spinal muscular atrophy (SMA), a major genetic disease of infants and children. Mild SMA is also recorded in adult population, expanding the spectrum of the disease. Here we review SMN circRNAs with respect to their biogenesis, sequence features, and potential functions. We also discuss how SMN circRNAs could be exploited for diagnostic and therapeutic purposes.

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

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.