Cell-mediated cytotoxicity within CSF and brain parenchyma in spinal muscular atrophy unaltered by nusinersen treatment.

5q-associated spinal muscular atrophy (SMA) is a motoneuron disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Adaptive immunity may contribute to SMA as described in other motoneuron diseases, yet mechanisms remain elusive. Nusinersen, an antisense treatment, enhances SMN2 expression, benefiting SMA patients. Here we have longitudinally investigated SMA and nusinersen effects on local immune responses in the cerebrospinal fluid (CSF) - a surrogate of central nervous system parenchyma. Single-cell transcriptomics (SMA: N = 9 versus Control: N = 9) reveal NK cell and CD8+ T cell expansions in untreated SMA CSF, exhibiting activation and degranulation markers. Spatial transcriptomics coupled with multiplex immunohistochemistry elucidate cytotoxicity near chromatolytic motoneurons (N = 4). Post-nusinersen treatment, CSF shows unaltered protein/transcriptional profiles. These findings underscore cytotoxicity's role in SMA pathogenesis and propose it as a therapeutic target. Our study illuminates cell-mediated cytotoxicity as shared features across motoneuron diseases, suggesting broader implications.

CRISPR-dCas13d-based deep screening of proximal and distal splicing-regulatory elements.

Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs). However, determining promising targets is hampered by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically inactive CRISPR-RfxCas13d RNA-targeting system (dCas13d/gRNA) as a programmable platform to bind SREs and modulate splicing by competing against endogenous splicing factors. SpliceRUSH, a high-throughput screening method, was developed to map SREs in any gene of interest using a lentivirus gRNA library that tiles the genetic region, including distal intronic sequences. When applied to SMN2, a therapeutic target for spinal muscular atrophy, SpliceRUSH robustly identifies not only known SREs but also a previously unknown distal intronic SRE, which can be targeted to alter exon 7 splicing using either dCas13d/gRNA or ASOs. This technology enables a deeper understanding of splicing regulation with applications for RNA-based drug discovery.

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.

Efficient systemic CNS delivery of a therapeutic antisense oligonucleotide with a blood-brain barrier-penetrating ApoE-derived peptide.

Antisense oligonucleotide (ASO) has emerged as a promising therapeutic approach for treating central nervous system (CNS) disorders by modulating gene expression with high selectivity and specificity. However, the poor permeability of ASO across the blood-brain barrier (BBB) diminishes its therapeutic success. Here, we designed and synthesized a series of BBB-penetrating peptides (BPP) derived from either the receptor-binding domain of apolipoprotein E (ApoE) or a transferrin receptor-binding peptide (THR). The BPPs were conjugated to phosphorodiamidate morpholino oligomers (PMO) that are chemically analogous to the 2'-O-(2-methoxyethyl) (MOE)-modified ASO approved by the FDA for treating spinal muscular atrophy (SMA). The BPP-PMO conjugates significantly increased the level of full-length SMN2 in the patient-derived SMA fibroblasts in a concentration-dependent manner with minimal to no toxicity. Furthermore, the systemic administration of the most potent BPP-PMO conjugates significantly increased the expression of full-length SMN2 in the brain and spinal cord of SMN2 transgenic adult mice. Notably, BPP8-PMO conjugate showed a 1.25-fold increase in the expression of full-length functional SMN2 in the brain. Fluorescence imaging studies confirmed that 78% of the fluorescently (Cy7)-labelled BPP8-PMO reached brain parenchyma, with 11% uptake in neuronal cells. Additionally, the BPP-PMO conjugates containing retro-inverso (RI) D-BPPs were found to possess extended half-lives compared to their L-counterparts, indicating increased stability against protease degradation while preserving the bioactivity. This delivery platform based on BPP enhances the CNS bioavailability of PMO targeting the SMN2 gene, paving the way for the development of systemically administered neurotherapeutics for CNS disorders.

[Experience of the treatment of spinal muscular atrophy type 3 Kugelberg-Welander with Nusinersen]. / Opyt lecheniya nusinersenom spinal'noi myshechnoi atrofii 3-go tipa Kugel'berga­Velander.

Before the advent of pathogenetic therapy, the diagnosis of spinal muscular atrophy (SMA) meant the loss of all hopes for recovery and the patient's setting on the path of a steady decline in motor functions, a deterioration in the quality of life and, ultimately, inevitable early death. Currently, new methods of pathogenetic therapy with nusinersen and risdiplam, as well as etiological therapy with onasemnogene abeparvovec, are available in the Russia. Nusinersen is an antisense oligonucleotide that modifies splicing of the SMN2 gene to increase production of normal full-length motor neuron survival protein, which is deficient in SMA. The mechanism of action of Nusinersen is based on the activation of the disabled exon 7 of the SMN2 gene. The article describes an example of long-term effective treatment using pathogenetic therapy of a patient diagnosed with SMA type 3.

Early spinal muscular atrophy treatment following newborn screening: A 20-month review of the first Italian regional experience.

OBJECTIVES: Mandatory newborn screening (NBS) for spinal muscular atrophy (SMA) was implemented for the first time in Italy at the end of 2021, allowing the identification and treatment of patients at an asymptomatic stage. METHODS: DNA samples extracted from dried blood spot (DBS) from newborns in Apulia region were analysed for SMA screening by using a real-time PCR-based assay. Infants harbouring homozygous deletion of SMN1 exon 7 confirmed by diagnostic molecular tests underwent clinical and neurophysiological assessment and received a timely treatment. RESULTS: Over the first 20 months since regional NBS introduction, four out of 42,492 (0.009%) screened children were found to carry a homozygous deletion in the exon 7 of SMN1 gene, with an annual incidence of 1:10,623. No false negatives were present. Median age at diagnosis was 7 days and median age at treatment was 20.5 days. Three of them had two copies of SMN2 and received gene therapy, while the one with three SMN2 copies was treated with nusinersen. All but one were asymptomatic at birth, showed no clinical signs of disease after a maximum follow-up of 16 months and reached motor milestones appropriate with their age. The minimum interval between diagnosis and the treatment initiation was 9 days. INTERPRETATION: The timely administration of disease-modifying therapies prevented presymptomatic subjects to develop disease symptoms. Mandatory NBS for SMA should be implemented on a national scale.

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.

Carrier Screening and Diagnosis for Spinal Muscular Atrophy Using Droplet Digital PCR Versus MLPA: Analytical Validation and Early Test Outcome.

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular life-threatening disorder. Owing to high carrier frequency, population-wide SMA screening to quantify the copy number of SMN gene is recommended by American College of Medical Genetics and Genomics. An accurate, reliable, short runaround time and cost-effective method may be helpful in mass population screening for SMA. Methods: Multiplex ligation-dependent probe amplification (MLPA) is a gold standard to estimate the copy number variation (CNV) for SMN1 and SMN2 genes. In this study, we validated droplet digital polymerase chain reaction (ddPCR) for the determination of CNV for both SMN1 and SMN2 exon 7 for a diagnostic purpose. In total, 66 clinical samples were tested using ddPCR, and results were compared with the MLPA as a reference test. Results: For all samples, CNV for SMN1 and SMN2 exon 7 was consentaneous between ddPCR and MLPA test results (κ = 1.000, p < 0.0001). In addition, ddPCR also showed a significant acceptable degree of test repeatability, coefficient of variation < 4%. Conclusion: ddPCR is expected to be utilitarian for CNV detection for carrier screening and diagnosis of SMA. ddPCR test results for CNV detection for SMN1/SMN2 exon 7 are concordant with the gold standard. ddPCR is a more cost-effective and time-saving diagnostic test for SMA than MLPA. Furthermore, it can be used for population-wide carrier screening for SMA.

Repeated AAV9 Titer Determination in a Presymptomatic SMA Patient with Three SMN2 Gene Copies - A Case Report.

Adeno-associated viruses (AAV) are well-suited to serve as gene transfer vectors. Onasemnogene abeparvovec uses AAV9 as virus vector. Previous exposure to wild-type AAVs or placental transfer of maternal AAV antibodies, however, can trigger an immune response to the vector virus which may limit the therapeutic effectiveness of gene transfer and impact safety. We present the case of a female patient with spinal muscular atrophy (SMA) and three survival motor neuron 2 (SMN2) gene copies. The infant had elevated titers of AAV9 antibodies at diagnosis at 9 days of age. Being presymptomatic at diagnosis, it was decided to retest the patient's AAV9 antibody titer at two-weekly intervals. Six weeks after initial diagnosis, a titer of 1:12.5 allowed treatment with onasemnogene abeparvovec. The presented case demonstrates that, provided the number of SMN2 gene copies and the absence of symptoms allow, onasemnogene abeparvovec therapy is feasible in patients with initially exclusionary AAV9 antibody titers of >1:50.

5qSMA: standardised retrospective natural history assessment in 268 patients with four copies of SMN2.

Newborn screening for 5qSMA offers the potential for early, ideally pre-symptomatic, therapeutic intervention. However, limited data exist on the outcomes of individuals with 4 copies of SMN2, and there is no consensus within the SMA treatment community regarding early treatment initiation in this subgroup. To provide evidence-based insights into disease progression, we performed a retrospective analysis of 268 patients with 4 copies of SMN2 from the SMArtCARE registry in Germany, Austria and Switzerland. Inclusion criteria required comprehensive baseline data and diagnosis outside of newborn screening. Only data prior to initiation of disease-modifying treatment were included. The median age at disease onset was 3.0 years, with a mean of 6.4 years. Significantly, 55% of patients experienced symptoms before the age of 36 months. 3% never learned to sit unaided, a further 13% never gained the ability to walk independently and 33% of ambulatory patients lost this ability during the course of the disease. 43% developed scoliosis, 6.3% required non-invasive ventilation and 1.1% required tube feeding. In conclusion, our study, in line with previous observations, highlights the substantial phenotypic heterogeneity in SMA. Importantly, this study provides novel insights: the median age of disease onset in patients with 4 SMN2 copies typically occurs before school age, and in half of the patients even before the age of three years. These findings support a proactive approach, particularly early treatment initiation, in this subset of SMA patients diagnosed pre-symptomatically. However, it is important to recognize that the register will not include asymptomatic individuals.

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.

270th ENMC International Workshop: Consensus for SMN2 genetic analysis in SMA patients 10-12 March, 2023, Hoofddorp, the Netherlands.

The 270th ENMC workshop aimed to develop a common procedure to optimize the reliability of SMN2 gene copy number determination and to reinforce collaborative networks between molecular scientists and clinicians. The workshop involved neuromuscular and clinical experts and representatives of patient advocacy groups and industry. SMN2 copy number is currently one of the main determinants for therapeutic decision in SMA patients: participants discussed the issues that laboratories may encounter in this molecular test and the cruciality of the accurate determination, due the implications as prognostic factor in symptomatic patients and in individuals identified through newborn screening programmes. At the end of the workshop, the attendees defined a set of recommendations divided into four topics: SMA molecular prognosis assessment, newborn screening for SMA, SMN2 copies and treatments, and modifiers and biomarkers. Moreover, the group draw up a series of recommendations for the companies manufacturing laboratory kits, that will help to minimize the risk of errors, regardless of the laboratories' expertise.

Optimization of base editors for the functional correction of SMN2 as a treatment for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by mutations in SMN1. SMN2 is a paralogous gene with a C•G-to-T•A transition in exon 7, which causes this exon to be skipped in most SMN2 transcripts, and results in low levels of the protein survival motor neuron (SMN). Here we show, in fibroblasts derived from patients with SMA and in a mouse model of SMA that, irrespective of the mutations in SMN1, adenosine base editors can be optimized to target the SMN2 exon-7 mutation or nearby regulatory elements to restore the normal expression of SMN. After optimizing and testing more than 100 guide RNAs and base editors, and leveraging Cas9 variants with high editing fidelity that are tolerant of different protospacer-adjacent motifs, we achieved the reversion of the exon-7 mutation via an A•T-to-G•C edit in up to 99% of fibroblasts, with concomitant increases in the levels of the SMN2 exon-7 transcript and of SMN. Targeting the SMN2 exon-7 mutation via base editing or other CRISPR-based methods may provide long-lasting outcomes to patients with SMA.

Effect of nusinersen after 3 years of treatment in 57 young children with SMA in terms of SMN2 copy number or type.

BACKGROUND: Spinal muscular atrophy (SMA) is a rare genetic neuromuscular disorder due to an autosomal recessive mutation in the survival motor neuron 1 gene (SMN1), causing degeneration of the anterior horn cells of the spinal cord and resulting in muscle atrophy. This study aimed to report on the 36-month follow-up of children with SMA treated with nusinersen before the age of 3 years. Changes in motor function, nutritional and ventilatory support, and orthopedic outcomes were evaluated at baseline and 36 months after intrathecal administration of nusinersen and correlated with SMA type and SMN2 copy number. RESULTS: We found that 93% of the patients gained new motor skills during the 3 years-standing without help for 12 of 37 and walking with help for 11 of 37 patients harboring three SMN2 copies. No patients with two copies of SMN2 can stand alone or walk. Patients bearing three copies of SMN2 are more likely to be spared from respiratory, nutritional, and orthopedic complications than patients with two SMN2 copies. CONCLUSION: Children with SMA treated with nusinersen continue to make motor acquisitions at 3 years after initiation of treatment. Children with two SMN2 copies had worse motor, respiratory, and orthopedic outcomes after 3 years of treatment than children with three copies.

Real-Time PCR-Based Screening for Homozygous SMN2 Deletion Using Residual Dried Blood Spots.

The survival motor neuron 2 (SMN2) gene is a recognized modifier gene of spinal muscular atrophy (SMA). However, our knowledge about the role of SMN2-other than its modification of SMA phenotypes-is very limited. Discussions regarding the relationship between homozygous SMN2 deletion and motor neuron diseases, including amyotrophic lateral sclerosis, have been mainly based on retrospective epidemiological studies of the diseases, and the precise relationship remains inconclusive. In the present study, we first estimated that the frequency of homozygous SMN2 deletion was ~1 in 20 in Japan. We then established a real-time polymerase chain reaction (PCR)-based screening method using residual dried blood spots to identify infants with homozygous SMN2 deletion. This method can be applied to a future prospective cohort study to clarify the relationship between homozygous SMN2 deletion and motor neuron diseases. In our real-time PCR experiment, both PCR (low annealing temperatures) and blood (high hematocrit values and low white blood cell counts) conditions were associated with incorrect results (i.e., false negatives and positives). Together, our findings not only help to elucidate the role of SMN2, but also aid in our understanding of the pitfalls of current SMA newborn screening programs for detecting homozygous SMN1 deletions.

Daily Quantity and Kinematic Characteristics of Leg Movement in a Child With SMA (2 Copies SMN2).

PURPOSE: This case report describes daily leg movement quantity and kinematic characteristics of a child with spinal muscular atrophy (SMA) with 2 copies of SMN2, who was symptomatic at the time of treatment with disease-modifying therapies. KEY POINTS: Compared with infants with typical development, this child had differing values for leg movement quantity, duration, average acceleration, and peak acceleration measured across full days in the natural environment by wearable sensors. In addition, movement quantity and clinician-rated outcomes increased with age. CONCLUSIONS: Wearable sensors recorded movement quantity and kinematic characteristics in a treated infant with SMA (2 copies SMN2). These movement parameters were consistently different compared anecdotally with published data from infants with typical development, demonstrating their potential to add unique and complementary information to the assessment of motor function in SMA. RECOMMENDATIONS: Larger longitudinal studies are needed to determine the utility of wearable sensors as an assessment tool and an early predictor of motor outcomes in children with 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.

PRMT inhibitor promotes SMN2 exon 7 inclusion and synergizes with nusinersen to rescue SMA mice.

Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality. The advent of approved treatments for this devastating condition has significantly changed SMA patients' life expectancy and quality of life. Nevertheless, these are not without limitations, and research efforts are underway to develop new approaches for improved and long-lasting benefits for patients. Protein arginine methyltransferases (PRMTs) are emerging as druggable epigenetic targets, with several small-molecule PRMT inhibitors already in clinical trials. From a screen of epigenetic molecules, we have identified MS023, a potent and selective type I PRMT inhibitor able to promote SMN2 exon 7 inclusion in preclinical SMA models. Treatment of SMA mice with MS023 results in amelioration of the disease phenotype, with strong synergistic amplification of the positive effect when delivered in combination with the antisense oligonucleotide nusinersen. Moreover, transcriptomic analysis revealed that MS023 treatment has minimal off-target effects, and the added benefit is mainly due to targeting neuroinflammation. Our study warrants further clinical investigation of PRMT inhibition both as a stand-alone and add-on therapy for SMA.

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.