Splicing-Correcting Therapy for SMA.

Spinal muscular atrophy (SMA) is caused by deficiency of SMN protein, which is crucial for spliceosome subunits biogenesis. Most SMA patients have SMN1 deletions, leaving SMN2 as sole SMN source; however, a C→T substitution converts an exonic-splicing enhancer (ESE) to a silencer (ESS), causing frequent exon7 skipping in SMN2 pre-mRNA and yielding a truncated protein. Antisense treatment to SMN2 intron7-splicing silencer (ISS) improves SMN expression and motor function. To view this Bench to Bedside, open or download the PDF.

Regulated control of gene therapies by drug-induced splicing.

So far, gene therapies have relied on complex constructs that cannot be finely controlled1,2. Here we report a universal switch element that enables precise control of gene replacement or gene editing after exposure to a small molecule. The small-molecule inducers are currently in human use, are orally bioavailable when given to animals or humans and can reach both peripheral tissues and the brain. Moreover, the switch system, which we denote Xon, does not require the co-expression of any regulatory proteins. Using Xon, the translation of the desired elements for controlled gene replacement or gene editing machinery occurs after a single oral dose of the inducer, and the robustness of expression can be controlled by the drug dose, protein stability and redosing. The ability of Xon to provide temporal control of protein expression can be adapted for cell-biology applications and animal studies. Additionally, owing to the oral bioavailability and safety of the drugs used, the Xon switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans.

Real-world evidence: Risdiplam in a patient with spinal muscular atrophy type I with a novel splicing mutation and one SMN2 copy.

Spinal muscular atrophy (SMA), which results from the deletion or/and mutation in the SMN1 gene, is an autosomal recessive neuromuscular disorder that leads to weakness and muscle atrophy. SMN2 is a paralogous gene of SMN1. SMN2 copy number affects the severity of SMA, but its role in patients treated with disease modifying therapies is unclear. The most appropriate individualized treatment for SMA has not yet been determined. Here, we reported a case of SMA type I with normal breathing and swallowing function. We genetically confirmed that this patient had a compound heterozygous variant: one deleted SMN1 allele and a novel splice mutation c.628-3T>G in the retained allele, with one SMN2 copy. Patient-derived sequencing of 4 SMN1 cDNA clones showed that this intronic single transversion mutation results in an alternative exon (e)5 3' splice site, which leads to an additional 2 nucleotides (AG) at the 5' end of e5, thereby explaining why the patient with only one copy of SMN2 had a mild clinical phenotype. Additionally, a minigene assay of wild type and mutant SMN1 in HEK293T cells also demonstrated that this transversion mutation induced e5 skipping. Considering treatment cost and goals of avoiding pain caused by injections and starting treatment as early as possible, risdiplam was prescribed for this patient. However, the patient showed remarkable clinical improvements after treatment with risdiplam for 7 months despite carrying only one copy of SMN2. This study is the first report on the treatment of risdiplam in a patient with one SMN2 copy in a real-world setting. These findings expand the mutation spectrum of SMA and provide accurate genetic counseling information, as well as clarify the molecular mechanism of careful genotype-phenotype correlation of the patient.

Comprehensive SMN1 and SMN2 profiling for spinal muscular atrophy analysis using long-read PacBio HiFi sequencing.

Spinal muscular atrophy, a leading cause of early infant death, is caused by bi-allelic mutations of SMN1. Sequence analysis of SMN1 is challenging due to high sequence similarity with its paralog SMN2. Both genes have variable copy numbers across populations. Furthermore, without pedigree information, it is currently not possible to identify silent carriers (2+0) with two copies of SMN1 on one chromosome and zero copies on the other. We developed Paraphase, an informatics method that identifies full-length SMN1 and SMN2 haplotypes, determines the gene copy numbers, and calls phased variants using long-read PacBio HiFi data. The SMN1 and SMN2 copy-number calls by Paraphase are highly concordant with orthogonal methods (99.2% for SMN1 and 100% for SMN2). We applied Paraphase to 438 samples across 5 ethnic populations to conduct a population-wide haplotype analysis of these highly homologous genes. We identified major SMN1 and SMN2 haplogroups and characterized their co-segregation through pedigree-based analyses. We identified two SMN1 haplotypes that form a common two-copy SMN1 allele in African populations. Testing positive for these two haplotypes in an individual with two copies of SMN1 gives a silent carrier risk of 88.5%, which is significantly higher than the currently used marker (1.7%-3.0%). Extending beyond simple copy-number testing, Paraphase can detect pathogenic variants and enable potential haplotype-based screening of silent carriers through statistical phasing of haplotypes into alleles. Future analysis of larger population data will allow identification of more diverse haplotypes and genetic markers for silent carriers.

The diversity of splicing modifiers acting on A-1 bulged 5'-splice sites reveals rules for rational drug design.

Pharmacological modulation of RNA splicing by small molecules is an emerging facet of drug discovery. In this context, the SMN2 splicing modifier SMN-C5 was used as a prototype to understand the mode of action of small molecule splicing modifiers and propose the concept of 5'-splice site bulge repair. In this study, we combined in vitro binding assays and structure determination by NMR spectroscopy to identify the binding modes of four other small molecule splicing modifiers that switch the splicing of either the SMN2 or the HTT gene. Here, we determined the solution structures of risdiplam, branaplam, SMN-CX and SMN-CY bound to the intermolecular RNA helix epitope containing an unpaired adenine within the G-2A-1G+1U+2 motif of the 5'-splice site. Despite notable differences in their scaffolds, risdiplam, SMN-CX, SMN-CY and branaplam contact the RNA epitope similarly to SMN-C5, suggesting that the 5'-splice site bulge repair mechanism can be generalised. These findings not only deepen our understanding of the chemical diversity of splicing modifiers that target A-1 bulged 5'-splice sites, but also identify common pharmacophores required for modulating 5'-splice site selection with small molecules.

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.

Antisense oligonucleotides targeting the SMN2 promoter region enhance SMN2 expression in spinal muscular atrophy cell lines and mouse model.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease caused by homozygous deletions or mutations in survival motor neuron gene 1 (SMN1). Currently, the primary therapeutic strategy for SMA is to increase the level of SMN via correcting SMN2 splicing (nusinersen and risdiplam). However, some patients with SMA do not respond to such treatments, thereby warranting a need to develop new therapeutic strategies. We have previously reported that SMN2 expression is epigenetically regulated by DNA methylation levels of the SMN2 promoter region. In the present study, we determined that methyl-CpG-binding protein 2 (MeCP2) may bind to this critical promoter region (nt-167 to 43). Antisense oligonucleotides (ASO-P1 and ASO-P2) were designed to target the key methylation sites in the SMN2 promoter region, which enhanced the overall transcription and functional protein expression levels in the SMA cell lines. These results were similar to those observed in nusinersen-treated SMA cells. Moreover, a combined treatment of ASO-P1 and ASO-NUS in SMA cell lines further increases fl-SMN2 transcript and SMN protein levels. The delivery of ASO-P1 to the central nervous system of severe SMA mice corrected the molecular, pathological, and functional phenotypes of this disease and increased survival rates. Our findings suggest that the key methylation regions in the SMN2 promoter region may be a novel therapeutic target for SMA.

Clinical Phenotype of Pediatric and Adult Patients With Spinal Muscular Atrophy With Four SMN2 Copies: Are They Really All Stable?

OBJECTIVE: The aim of this study was to provide an overview of the clinical phenotypes associated with 4 SMN2 copies. METHODS: Clinical phenotypes were analyzed in all the patients with 4 SMN2 copies as part of a nationwide effort including all the Italian pediatric and adult reference centers for spinal muscular atrophy (SMA). RESULTS: The cohort includes 169 patients (102 men and 67 women) with confirmed 4 SMN2 copies (mean age at last follow-up = 36.9 ± 19 years). Six of the 169 patients were presymptomatic, 8 were classified as type II, 145 as type III (38 type IIIA and 107 type IIIB), and 8 as type IV. The remaining 2 patients were asymptomatic adults identified because of a familial case. The cross-sectional functional data showed a reduction of scores with increasing age. Over 35% of the type III and 25% of the type IV lost ambulation (mean age = 26.8 years ± 16.3 SD). The risk of loss of ambulation was significantly associated with SMA type (p < 0.0001), with patients with IIIB and IV less likely to lose ambulation compared to type IIIA. There was an overall gender effect with a smaller number of women and a lower risk for women to lose ambulation. This was significant in the adult (p = 0.009) but not in the pediatric cohort (p = 0.43). INTERPRETATION: Our results expand the existing literature on natural history of 4 SMN2 copies confirming the variability of phenotypes in untreated patients, ranging from type II to type IV and an overall reduction of functional scores with increasing age. ANN NEUROL 2023;94:1126-1135.

Improvement of TaC9-ABE mediated correction of human SMN2 gene.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Gene editing technology repairs the conversion of the 6th base T to C in exon 7 of the paralogous SMN2 gene, compensating for the SMN protein expression and promoting the survival and function of motor neurons. However, low editing efficiency and unintended off-target effects limit the application of this technology. Here, we optimized a TaC9-adenine base editor (ABE) system by combining Cas9 nickase with the transcription activator-like effector (TALE)-adenosine deaminase fusion protein to effectively and precisely edit SMN2 without detectable Cas9 dependent off-target effects in human cell lines. We also generated human SMA-induced pluripotent stem cells (SMA-iPSCs) through the mutation of the splice acceptor or deletion of the exon 7 of SMN1. TaC9-R10 induced 45% SMN2 T6 > C conversion in the SMA-iPSCs. The SMN2 T6 > C splice-corrected SMA-iPSCs were directionally differentiated into motor neurons, exhibiting SMN protein recovery and antiapoptosis ability. Therefore, the TaC9-ABE system with dual guides from the combination of Cas9 with TALE could be a potential therapeutic strategy for SMA with high efficacy and safety.

Analytical validation of the amplification refractory mutation system polymerase chain reaction-capillary electrophoresis assay to diagnose spinal muscular atrophy.

OBJECTIVES: Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by homozygous deletion and compound heterozygous mutations in survival motor neuron 1 (SMN1), with severity tied to the copy number of survival motor neuron 2 (SMN2). This study aimed to develop a rapid and comprehensive method for the diagnosis of SMA. METHODS: A total of 292 children with clinically suspected SMA and 394 family members were detected by the amplification refractory mutation system polymerase chain reaction-capillary electrophoresis (ARMS-PCR-CE) method, which targeted 19 reported mutations, and the results were compared with those in multiplex ligation-dependent probe amplification (MLPA). Individuals with identified point mutations were further confirmed by SMN1 long-range PCR and Sanger sequencing. RESULTS: A total of 202 children with SMA, 272 carriers, and 212 normal individuals were identified in this study. No difference was found in the R-value distribution of exons 7 and 8 in SMN1 and SMN2 among these cohorts, with coefficients of variation consistently below 0.08. To detect exon 7 and 8 copy numbers in SMN1 and SMN2, the ARMS-PCR-CE results were concordant with those of MLPA. Approximately 4.95 % (10/202) of the study patients had compound heterozygous mutations. CONCLUSIONS: The ARMS-PCR-CE assay is a comprehensive, rapid, and accurate diagnostic method for SMA that simultaneously detects copy numbers of exons 7 and 8 in SMN1/SMN2, as well as 19 point mutations in SMN1 and 2 enhancers in SMN2. This approach can effectively reduce the time frame for diagnosis, facilitating early intervention and preventing birth defects.

Prognostic factors for tube feeding in type I SMA patients treated with disease-modifying therapies: a cohort study.

The aim of this study was to assess the need for tube feeding in a cohort of treated infants with type I SMA and to identify predictive factors. All patients were classified at baseline, when treatment started, and at follow-up according to their functional level and the need for tube feeding. Fisher's exact test was used to examine the associations between the outcome at the last follow-up and SMA type, SMN2 copy number, and baseline nutritional status. ANOVA was performed to compare CHOP INTEND scores and age at treatment initiation with outcomes. The cohort includes 75 type I SMA infants treated between 0.1 and 5 years of age. At the last follow-up, 34 had no need for tube feeding, 9 had tube feeding but were also able to be fed by mouth, and 32 had tube feeding and were unable to be fed by mouth. Thirty of the 41 infants with tube feeding at follow-up already had feeding difficulties when treatment was started. The need for tube feeding at follow-up was associated with the level of feeding involvement at baseline and with CHOP INTEND scores [p < 0.001] but not with SMN2 copy number, SMA type 1 subtypes or age at treatment. The results of this study suggest that the need for tube feeding is not frequent in treated infants with type I SMA and, when occurring, can be predicted by the level of feeding involvement and low CHOP INTEND scores at baseline. What is Known: • The advent of disease-modifying therapies is increasingly changing the approach to swallowing and nutritional management in type I SMA. • Clinical trials and real-world data using all three disease-modifying therapies report a rather wide variability of feeding outcome and need for tube feeding that is often related to different cohorts that makes comparison between studies very difficult. What is New: • The real-world findings of this study, including all the children treated since treatments became available, confirmed that the need for tube feeding is not an invariable finding. • The level of feeding involvement at baseline appears to be a reliable prognostic indicator of bulbar outcome. • The results highlight the need for interventional studies with structured Speech and Language Therapist protocols that will help to better understand the extent to which bulbar function can be maintained or regained even in children requiring tube feeding.

Systematic characterization of short intronic splicing-regulatory elements in SMN2 pre-mRNA.

Intronic splicing enhancers and silencers (ISEs and ISSs) are two groups of splicing-regulatory elements (SREs) that play critical roles in determining splice-site selection, particularly for alternatively spliced introns or exons. SREs are often short motifs; their mutation or dysregulation of their cognate proteins frequently causes aberrant splicing and results in disease. To date, however, knowledge about SRE sequences and how they regulate splicing remains limited. Here, using an SMN2 minigene, we generated a complete pentamer-sequence library that comprises all possible combinations of 5 nucleotides in intron 7, at a fixed site downstream of the 5' splice site. We systematically analyzed the effects of all 1023 mutant pentamers on exon 7 splicing, in comparison to the wild-type minigene, in HEK293 cells. Our data show that the majority of pentamers significantly affect exon 7 splicing: 584 of them are stimulatory and 230 are inhibitory. To identify actual SREs, we utilized a motif set enrichment analysis (MSEA), from which we identified groups of stimulatory and inhibitory SRE motifs. We experimentally validated several strong SREs in SMN1/2 and other minigene settings. Our results provide a valuable resource for understanding how short RNA sequences regulate splicing. Many novel SREs can be explored further to elucidate their mechanism of action.

Ubiquitination Insight from Spinal Muscular Atrophy-From Pathogenesis to Therapy: A Muscle Perspective.

Spinal muscular atrophy (SMA) is one of the most frequent causes of death in childhood. The disease's molecular basis is deletion or mutations in the SMN1 gene, which produces reduced survival motor neuron protein (SMN) levels. As a result, there is spinal motor neuron degeneration and a large increase in muscle atrophy, in which the ubiquitin-proteasome system (UPS) plays a significant role. In humans, a paralogue of SMN1, SMN2 encodes the truncated protein SMNΔ7. Structural differences between SMN and SMNΔ7 affect the interaction of the proteins with UPS and decrease the stability of the truncated protein. SMN loss affects the general ubiquitination process by lowering the levels of UBA1, one of the main enzymes in the ubiquitination process. We discuss how SMN loss affects both SMN stability and the general ubiquitination process, and how the proteins involved in ubiquitination could be used as future targets for SMA treatment.

Potential of Cell-Penetrating Peptide-Conjugated Antisense Oligonucleotides for the Treatment of SMA.

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder that is caused by mutations in the survival motor neuron 1 (SMN1) gene, hindering the production of functional survival motor neuron (SMN) proteins. Antisense oligonucleotides (ASOs), a versatile DNA-like drug, are adept at binding to target RNA to prevent translation or promote alternative splicing. Nusinersen is an FDA-approved ASO for the treatment of SMA. It effectively promotes alternative splicing in pre-mRNA transcribed from the SMN2 gene, an analog of the SMN1 gene, to produce a greater amount of full-length SMN protein, to compensate for the loss of functional protein translated from SMN1. Despite its efficacy in ameliorating SMA symptoms, the cellular uptake of these ASOs is suboptimal, and their inability to penetrate the CNS necessitates invasive lumbar punctures. Cell-penetrating peptides (CPPs), which can be conjugated to ASOs, represent a promising approach to improve the efficiency of these treatments for SMA and have the potential to transverse the blood-brain barrier to circumvent the need for intrusive intrathecal injections and their associated adverse effects. This review provides a comprehensive analysis of ASO therapies, their application for the treatment of SMA, and the encouraging potential of CPPs as delivery systems to improve ASO uptake and overall efficiency.

Recent Advance in Disease Modifying Therapies for Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease characterized by progressive weakness and atrophy of skeletal muscles. With homozygous survival motor neuron 1 (SMN1) gene mutation, all SMA patients have at least one copy of the SMN2 gene, which provides an opportunity for drug targeting to enhance SMN expression. Current three disease modifying drugs, including nusinersen, onasemnogene abeparvovec, and risdiplam, have demonstrated impressive effectiveness in SMA treatment. Nusinersen is an antisense oligonucleotide targeting SMN2 pre-messenger RNA (mRNA) to modify alternative splicing and is effective in SMA children and adults, administrating via intermittent intrathecal injection. Onasemnogene abeparvovec is an adeno-associated viral vector carrying human SMN1 gene, featuring intravenous injection once in a lifetime for SMA patients less than 2 years of the age. Risdiplam is a small molecule also targeting SMN2 pre-mRNA and is effective in SMA children and adults with administration via oral intake once per day. Patients with SMA should receive these disease modifying therapies as soon as possible to not only stabilize disease progression, but potentially obtain neurological improvement. The development in these therapies has benefited patients with SMA and will potentially provide insight in future drug discovery for other neurodegenerative diseases. Keywords: Adeno-associated viral vector, antisense oligonucleotide, disease modifying therapy, gene therapy, motor neuron disease, spinal muscular atrophy.

[Application of CNVPLUS-array custom microarray in genetic analysis of spinal muscular atrophy].

OBJECTIVE: To assess the application value of CNVPLUS-array for the genetic analysis of Spinal muscular atrophy (SMA). METHODS: From June 2021 to December 2022, CNVPLUS-array technique was employed to test the SMN1 and SMN2 genes among peripheral blood samples from 17 suspected SMA patients, 18 core families with suspected SMA, and 25 healthy individuals. The results were compared with those of multiple ligation-dependent probe amplification (MLPA) assay. Samples with inconsistent results were subjected to nested PCR or comprehensive analysis of SMA. RESULTS: CNVPLUS-array has identified 35 SMA patients, 36 carriers, and 25 healthy individuals. In comparison, MLPA has identified 34 SMA patients, 36 carriers, and 26 healthy individuals. The two methods demonstrated a high consistency (Kappa = 0.968, P < 0.001). Additionally, CNVPLUS-array has identified one patient with compound heterozygous variants of SMN1 and one carrier with a [2+0] genotype. CONCLUSION: CNVPLUS-array not only can accurately determine the copy numbers of SMN1 and SMN2 genes, but also identify point mutations in SMN1 and [2+0] carriers, which has offered a new method for the genetic testing of SMA.

SMN protein is required throughout life to prevent spinal muscular atrophy disease progression.

Spinal muscular atrophy (SMA) is caused by the loss of the survival motor neuron 1 (SMN1) gene function. The related SMN2 gene partially compensates but produces insufficient levels of SMN protein due to alternative splicing of exon 7. Evrysdi™ (risdiplam), recently approved for the treatment of SMA, and related compounds promote exon 7 inclusion to generate full-length SMN2 mRNA and increase SMN protein levels. SMNΔ7 type I SMA mice survive without treatment for ~17 days. SMN2 mRNA splicing modulators increase survival of SMN∆7 mice with treatment initiated at postnatal day 3 (PND3). To define SMN requirements for adult mice, SMNΔ7 mice were dosed with an SMN2 mRNA splicing modifier from PND3 to PND40, then dosing was stopped. Mice not treated after PND40 showed progressive weight loss, necrosis, and muscle atrophy after ~20 days. Male mice presented a more severe phenotype than female mice. Mice dosed continuously did not show disease symptoms. The estimated half-life of SMN protein is 2 days indicating that the SMA phenotype reappeared after SMN protein levels returned to baseline. Although SMN protein levels decreased with age in mice and SMN protein levels were higher in brain than in muscle, our studies suggest that SMN protein is required throughout the life of the mouse and is especially essential in adult peripheral tissues including muscle. These studies indicate that drugs such as risdiplam will be optimally therapeutic when given as early as possible after diagnosis and potentially will be required for the life of an SMA patient.

LCM-seq reveals unique transcriptional adaptation mechanisms of resistant neurons and identifies protective pathways in spinal muscular atrophy.

Somatic motor neurons are selectively vulnerable in spinal muscular atrophy (SMA), which is caused by a deficiency of the ubiquitously expressed survival of motor neuron protein. However, some motor neuron groups, including oculomotor and trochlear (ocular), which innervate eye muscles, are for unknown reasons spared. To reveal mechanisms of vulnerability and resistance in SMA, we investigate the transcriptional dynamics in discrete neuronal populations using laser capture microdissection coupled with RNA sequencing (LCM-seq). Using gene correlation network analysis, we reveal a TRP53-mediated stress response that is intrinsic to all somatic motor neurons independent of their vulnerability, but absent in relatively resistant red nucleus and visceral motor neurons. However, the temporal and spatial expression analysis across neuron types shows that the majority of SMA-induced modulations are cell type-specific. Using Gene Ontology and protein network analyses, we show that ocular motor neurons present unique disease-adaptation mechanisms that could explain their resilience. Specifically, ocular motor neurons up-regulate (1) Syt1, Syt5, and Cplx2, which modulate neurotransmitter release; (2) the neuronal survival factors Gdf15, Chl1, and Lif; (3) Aldh4, that protects cells from oxidative stress; and (4) the caspase inhibitor Pak4. Finally, we show that GDF15 can rescue vulnerable human spinal motor neurons from degeneration. This confirms that adaptation mechanisms identified in resilient neurons can be used to reduce susceptibility of vulnerable neurons. In conclusion, this in-depth longitudinal transcriptomics analysis in SMA reveals novel cell type-specific changes that, alone and combined, present compelling targets, including Gdf15, for future gene therapy studies aimed toward preserving vulnerable motor neurons.

Recognition of single-stranded nucleic acids by small-molecule splicing modulators.

Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5'-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode.