Spinal muscular atrophy (SMA) is an intractable neuromuscular disorder primarily caused by homozygous deletions in exon 7 of the SMN1 gene. Early diagnosis and prompt treatment of patients with SMA have a significant impact on prognosis, and several therapies have recently been developed. Current SMA screening tests require a significant turnaround time to identify patients with suspected SMA, due both to the interval between the birth of a newborn and the collection of blood for newborn mass screening and the difficulty in distinguishing between SMN1 and SMN2, a paralog gene that requires testing in specialized laboratories. The aim of this study was therefore to develop a novel SMA screening assay that can be rapidly performed in ordinary hospitals and clinics to overcome these issues. We designed over 100 combinations of forward and reverse primers with 3\' ends targeting SMN1-specific sites around exon 7, and evaluated their specificity and amplification efficiency by quantitative PCR to identify the best primer pair. Furthermore, we performed a single-stranded tag hybridization assay after PCR. To evaluate the accuracy and practicality of the newly developed assay, we analyzed saliva specimens from five patients with SMA and two SMA carriers collected in an outpatient clinic and DNA specimens from three patients with SMA and four SMA carriers from a biobank, together with those from healthy individuals. DNA and raw saliva specimens from all patients with SMA demonstrated a biallelic loss of SMN1, whereas those from carriers and healthy individuals did not. The results of 50 independent experiments were consistent for all samples. The assay could be completed within one hour. This simple and convenient new screening tool has the potential to allow patients with SMA to receive disease-modifying therapies within a shorter timeframe.

4例患儿均因新生儿筛查发现运动神经元存活（SMN）1基因7号外显子纯合缺失就诊，均无脊髓性肌萎缩症（SMA）临床症状，神经系统查体均无阳性体征，电生理检查尺神经、腓总神经复合肌肉动作电位（CMAP）波幅均处于同年龄段正常范围。4例患儿经基因检测提示SMN2基因拷贝数均为3，均诊断为症状前SMA。患儿在出现症状前接受诺西那生钠疾病修正治疗，随访14~18个月，均可实现正常运动里程碑，监测CMAP波幅均处于同年龄段正常范围，无患儿发病。.

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.

Advances in the treatment of spinal muscular atrophy (SMA) have revolutionized the field. SMA is a rare autosomal recessive neurodegenerative motor neuron disease in which wide phenotypic variability has been described. The rate of increase in neurological deficit and the severity of the disease is mainly determined by the amount of functional SMN (Survival of Motor Neuron) protein. However, the clinical picture may differ significantly in patients carrying homozygous deletions of the SMN1 gene (Survival of Motor Neuron 1) and an identical number of copies of the SMN2 gene (Survival of Motor Neuron 2). A family clinical case of adult patients with spinal muscular atrophy 5q with a homozygous deletion of the SMN1 gene and the same number of copies of the SMN2 gene, having a different clinical picture of the disease, is presented, and the dynamics of the condition against the background of oral pathogenetic therapy is presented.
В настоящее время достигнуты прорывные успехи в терапии спинальной мышечной атрофии (СМА). СМА — редкое аутосомно-рецессивное нейродегенеративное заболевание двигательных нейронов, описана его широкая фенотипическая вариабельность. Скорость нарастания неврологического дефицита и тяжесть заболевания в основном определяются количеством функционального белка SMN (англ.: Survival of Motor Neuron). Вместе с тем клиническая картина может значимо отличаться у пациентов, несущих гомозиготные делеции гена SMN1 и идентичное количество копий гена SMN2. Представлен семейный клинический случай взрослых пациентов со СМА 5q с гомозиготной делецией гена SMN1 и одинаковым количеством копий гена SMN2, имеющих различную клиническую картину заболевания, описана динамика состояния на фоне пероральной патогенетической терапии.

OBJECTIVE: Compare efficacy of gene therapy alone (monotherapy) or in combination with an SMN2 augmentation agent (dual therapy) for treatment of children at risk for spinal muscular atrophy type 1.
METHODS: Eighteen newborns with biallelic SMN1 deletions and two SMN2 copies were treated preemptively with monotherapy (n = 11) or dual therapy (n = 7) and followed for a median of 3 years. Primary outcomes were independent sitting and walking. Biomarkers were serial muscle ultrasonography (efficacy) and sensory action potentials (safety).
RESULTS: Gene therapy was administered by 7-43 postnatal days; dual therapy with risdiplam (n = 6) or nusinersen (n = 1) was started by 15-39 days. Among 18 children enrolled, 17 sat, 15 walked, and 44% had motor delay (i.e., delay or failure to achieve prespecified milestones). Those on dual therapy sat but did not walk at an earlier age. 91% of muscle ultrasounds conducted within 60 postnatal days were normal but by 3-61 months, 94% showed echogenicity and/or fasciculation of at least one muscle group; these changes were indistinguishable between monotherapy and dual therapy cohorts. Five children with three SMN2 copies were treated with monotherapy in parallel: all sat and walked on time and had normal muscle sonograms at all time points. No child on dual therapy experienced treatment-associated adverse events. All 11 participants who completed sensory testing (including six on dual therapy) had intact sural sensory responses.
CONCLUSIONS: Preemptive dual therapy is well tolerated and may provide modest benefit for children at risk for severe spinal muscular atrophy but does not prevent widespread degenerative changes.

BACKGROUND: This study presents the findings of a newborn screening (NBS) pilot project for 5q-spinal muscular atrophy (5q-SMA) in multiple regions across Russia for during the year 2022. The aim was to assess the feasibility and reproducibility of NBS for SMA5q in diverse populations and estimate the real prevalence of 5q-SMA in Russia as well as the distribution of patients with different number of SMN2 copies.
METHODS: The pilot project of NBS here was based on data, involving the analysis of 202,908 newborns. SMA screening assay was performed using a commercially available real-time polymerase chain reaction kit, the Eonis SCID-SMA.
RESULTS: In one year, 202,908 newborns were screened, identifying 26 infants with homozygous deletion of SMN1 exon 7, yielding an estimated 5q-SMA incidence of 1:7804 newborns. It was found that 38.46% had two SMN2 copies, 42.31% had three copies, 15.38% had four copies, and 3.85% had five copies of SMN2. Immediate treatment was proposed for patients with two or three SMN2 copies. Infants with four or more SMN2 copies warranted further investigation on management and treatment. Short-term monitoring after gene therapy showed motor function improvements. Delays in treatment initiation were observed, including the testing for adeno-associated virus 9 antibodies and nonmedical factors.
CONCLUSIONS: The study emphasizes the need for a standardized algorithm for early diagnosis and management through NBS to benefit affected families. Overall, the NBS program for 5q-SMA in Russia demonstrated the potential to improve outcomes and transform SMA from a devastating disease to a chronic condition with evolving medical requirements.

BACKGROUND: Spinal muscular atrophy (SMA) is a neuromuscular disease, causing progressive muscle weakness due to loss of lower motoneurons. Since 2017, three therapies, two modifying gene transcription and one adding the defective gene, have been approved with comparable efficacy on motor outcome. Data on cognitive outcomes of treated SMA type 1 patients is limited. The aim of this study was to evaluate cognitive function in symptomatic and presymptomatic SMA type 1 patients with two or three SMN2 copies who received SMN-modifying or gene-addition therapy in the first year of life.
METHODS: Cognitive testing was performed in 20 patients, including 19 symptomatic SMA type 1 patients with up to three SMN2 copies and 1 pre-symptomatically treated patient. Children were tested using Bayley Scales of Infant Development (BSID-III) at the age of 2 or 3 years or the Wechsler Preschool and Primary Scale of Intelligence (WPSII-IV) at the of age of 5 years.
RESULTS: 11/20 patients showed subnormal cognitive development. Boys had significantly lower cognitive scores. Patients requiring assisted ventilation or feeding support were more likely to have cognitive deficits. Achieving more motor milestones was associated with a better cognitive outcome.
CONCLUSIONS: Treated patients with SMA type 1 have heterogeneous cognitive function with 55 % of patients showing deficits. Risk factors for cognitive impairment in our cohort were male gender and need for assisted ventilation or feeding support. Therefore, cognitive assessment should be included in the standard of care to allow early identification of deficits and potential therapeutic interventions.

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.

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.

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.