SMN Protein Research Articles

Baseline Characteristics and Interim Safety in RESPOND: A Phase 4 Study in Children with Spinal Muscular Atrophy (SMA) Treated With Nusinersen After Onasemnogene Abeparvovec (P7-9.003)

Baseline Characteristics and Interim Safety in RESPOND: A Phase 4 Study in Children with Spinal Muscular Atrophy (SMA) Treated With Nusinersen After Onasemnogene Abeparvovec (P7-9.003)

The Use of Onasemnogene-abeparvovec at 33 weeks’ gestation in Spinal Muscular Atrophy with One Copy of SMN2 (P2-8.013)

<h3>Objective:</h3> To describe twin girls with spinal muscular atrophy (SMA) with1 copy of <i>SMN2</i> who received Onsasemnogene-abeparvove at 33 weeks’ gestation. <h3>Background:</h3> SMA is a neurodegenerative disorder caused by absence of <i>SMN1</i> resulting in loss of motor neurons and thus progressive muscle weakness and atrophy. <i>SMN2</i> copy number modifies phenotype. Patients with 1 <i>SMN2</i> copy have a severe form of the disease resulting in limited survival after birth. Pre-clinical and clinical data has shown that early treatment of SMA is critical for optimal outcomes. Onsasemnogene-abeparvove, an adeno-associated virus vector mediated gene therapy, is approved for treatment of SMA in children less than 2 years of age, but few cases have reported its impact in premature infants. <h3>Design/Methods:</h3> Exempt from consent by IRB review. <h3>Results:</h3> Twin monochorionic, monoamniotic girls were born at 30 weeks and 2 days gestation. SMA was suggested on newborn screen and confirmatory testing showed 0 copies of <i>SMN1</i> and 1 copy of <i>SMN2.</i> They received Onsasemnogene-abeparvove at 33 weeks’ gestation. Neither child had adverse events associated with the medication. Both girls sat without support and rolled over at 8 months (6 months corrected age), crawled, pulled to stand, and said one-word phrases at 12 months, and walked independently at 15 months of age. Neurological exam at 19 months of age showed normal muscle bulk, tone and strength and normal reflexes. <h3>Conclusions:</h3> This case report is important because it demonstrates the potential for excellent outcomes, even in children predicted to have extremely severe disease. Normally, SMN protein level falls during the last trimester and first few months after birth (Ramos, et al, 2019). These twins were treated at the beginning of the last trimester of pregnancy, very early in this proposed critical window for treatment, likely contributing to normal development. <b>Disclosure:</b> Ms. Ajjarapu has nothing to disclose. Ms. Ramachandra has nothing to disclose. Dr. Mathews has received personal compensation for serving as an employee of Avidity Bioscience. The institution of Dr. Mathews has received research support from NIH. The institution of Dr. Mathews has received research support from Centers for Disease Control and Prevention. The institution of Dr. Mathews has received research support from Muscular Dystrophy Association . The institution of Dr. Mathews has received research support from Friedreich’s Ataxia Research Alliance . The institution of Dr. Mathews has received research support from Sarepta . The institution of Dr. Mathews has received research support from Pfizer. The institution of Dr. Mathews has received research support from Reata . The institution of Dr. Mathews has received research support from PTC Therapeutics, Inc. The institution of Dr. Mathews has received research support from Italfarmaco . The institution of Dr. Mathews has received research support from AMO. The institution of Dr. Mathews has received research support from FibroGen. The institution of Dr. Mathews has received research support from Capricor. The institution of Dr. Mathews has received research support from edgewise. The institution of Dr. Mathews has received research support from biogen.

SMA Therapy in Poland: New Hopes and Challenges

Abstract: Spinal muscular atrophy (SMA) is a genetically inherited disease whose symptoms appear in children at a fairly early age. The main cause of the disease is a mutation of the SMN1 gene, which causes the lack of production of SMN This results in the disappearance of motoneurons, which consequently leads to the disappearance of the patient's ability to move and his death. An important element of coping with the disease is its early detection in newborn screening, because the earlier we start the drug, the greater the chance that the patient will maintain motor efficiency. The article describes treatment options for children in Poland, i. e. the use of such drugs as Nusinersen, which increases the amount of SMN protein, gene therapy Onasemnogen aeparvovec, which increases the amount of SMN gene, Ridisplam, which increases the amount of SMN protein, and salbutamol, which helps patients to breathe. The article also provides information about new drug therapies for the treatment of SMA and the stage of clinical trials at which they are currently being developed. It is important to look for new solutions in the treatment of SMA.&#x0D; Objective: To familiarize the readers of the article with the problems of SMA treatment in Poland, as well as to broaden their knowledge of new available drugs for the treatment of this disease.

Nifedipine Ameliorates Cellular Differentiation Defects of Smn-Deficient Motor Neurons and Enhances Neuromuscular Transmission in SMA Mice.

In spinal muscular atrophy (SMA), mutations in or loss of the Survival Motor Neuron 1 (SMN1) gene reduce full-length SMN protein levels, which leads to the degeneration of a percentage of motor neurons. In mouse models of SMA, the development and maintenance of spinal motor neurons and the neuromuscular junction (NMJ) function are altered. Since nifedipine is known to be neuroprotective and increases neurotransmission in nerve terminals, we investigated its effects on cultured spinal cord motor neurons and motor nerve terminals of control and SMA mice. We found that application of nifedipine increased the frequency of spontaneous Ca2+ transients, growth cone size, cluster-like formations of Cav2.2 channels, and it normalized axon extension in SMA neurons in culture. At the NMJ, nifedipine significantly increased evoked and spontaneous release at low-frequency stimulation in both genotypes. High-strength stimulation revealed that nifedipine increased the size of the readily releasable pool (RRP) of vesicles in control but not SMA mice. These findings provide experimental evidence about the ability of nifedipine to prevent the appearance of developmental defects in SMA embryonic motor neurons in culture and reveal to which extent nifedipine could still increase neurotransmission at the NMJ in SMA mice under different functional demands.

MiR34 contributes to spinal muscular atrophy and AAV9-mediated delivery of MiR34a ameliorates the motor deficits in SMA mice

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by the selective loss of spinal motor neurons (MNs) and concomitant muscle weakness. Mutation of SMN1 is known to cause SMA, and restoring SMN protein levels via antisense oligonucleotide treatment is effective for ameliorating symptoms. However, this approach is hindered by exorbitant costs, invasive procedures, and poor treatment responses of some patients. Here, we seek to circumvent these hurdles by identifying reliable biomarkers that could predict treatment efficacy. We uncovered that MiR34 exhibits consistent downregulation during SMA progression in both human and rodent contexts. Importantly, Mir34 family-knockout mice display axon swelling and reduced neuromuscular junction (NMJ) endplates, recapitulating SMA pathology. Introducing MiR34a via scAAV9 improved the motor ability of SMNΔ7 mice, possibly by restoring NMJ endplate size. Finally, we observed a consistent decreasing trend in MiR34 family expression in the cerebrospinal fluid (CSF) of type I SMA patients during the loading phase of nusinersen treatment. Baseline CSF MiR34 levels before nusinersen injection proved predictive of patient motor skills 1 year later. Thus, we propose that MiR34 may serve as a biomarker of SMA since it is associated with the pathology and can help evaluate the therapeutic effects of nusinersen.

Novel Alu-mediated deletions of the SMN1 gene were identified by ultra-long read sequencing technology in patients with spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by biallelic variants of the survival motor neuron 1 (SMN1) gene. In this study, our aim was to make a molecular diagnosis in two patients with SMA carrying only one SMN1 copy number. Using ultra-long read sequencing (Ultra-LRS), 1415 bp deletion and 3348 bp deletion of the SMN1 gene were identified in patient 1 and the father of patient 2, respectively. Ultra-LRS revealed two novel deletions, starting from the SMN1 promoter to intron 1. It also accurately provided the location of the deletion breakpoints in the SMN1 gene: chr5 g.70,924,798–70,926,212 for a 1415 bp deletion; chr5 g.70,922,695–70,926,042 for a 3348 bp deletion. By analyzing the breakpoint junctions, we identified that these genomic sequences were composed of Alu sequences, including AluJb, AluYm1, AluSq, and AluYm1, indicating that Alu-mediated rearrangements are a mechanism of SMN1 deletion events. In addition, full-length SMN1 transcripts and SMN protein in patient 1 were significantly decreased (p < 0.01), suggesting that a 1415 bp deletion that included the transcription and translation initiation sites of the SMN1 gene had severe consequences for SMN expression. Ultra-LRS can easily distinguish highly homozygous genes compared to other detection technologies, which is useful for detecting SMN1 intragenic mutations, to quickly discover structural rearrangements and to precisely present the breakpoint positions.

Risdiplam in Patients Previously Treated with Other Therapies for Spinal Muscular Atrophy: An Interim Analysis from the JEWELFISH Study.

Risdiplam is a survival of motor neuron 2 (SMN2) splicing modifier for the treatment of patients with spinal muscular atrophy (SMA). The JEWELFISH study (NCT03032172) was designed to assess the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of risdiplam in previously treated pediatric and adult patients with types1-3 SMA. Here, an analysis was performed after all patients had received at least 1year of treatment with risdiplam. Patients with a confirmed diagnosis of 5q-autosomal recessive SMA between the ages of 6months and 60years were eligible for enrollment. Patients were previously enrolled in the MOONFISH study (NCT02240355) with splicing modifier RG7800 or treated with olesoxime, nusinersen, or onasemnogene abeparvovec. The primary objectives of the JEWELFISH study were to evaluate the safety and tolerability of risdiplam and investigate the PK after 2years of treatment. A total of 174 patients enrolled: MOONFISH study (n = 13), olesoxime (n = 71 patients), nusinersen (n = 76), onasemnogene abeparvovec (n = 14). Most patients (78%) had three SMN2 copies. The median age and weight of patients at enrollment was 14.0years (1-60years) and 39.1kg (9.2-108.9kg), respectively. About 63% of patients aged 2-60years had a baseline total score of less than 10 on the Hammersmith Functional Motor Scale-Expanded and 83% had scoliosis. The most common adverse event (AE) was upper respiratory tract infection and pyrexia (30 patients each; 17%). Pneumonia (four patients; 2%) was the most frequently reported serious AE (SAE). The rates of AEs and SAEs per 100 patient-years were lower in the second 6-month period compared with the first. An increase in SMN protein was observed in blood after risdiplam treatment and was comparable across all ages and body weight quartiles. The safety and PD of risdiplam in patients who were previously treated were consistent with those of treatment-naïve patients.

An Overview of the Therapeutic Strategies for the Treatment of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a recessive, neurodegenerative disorder. It is one of the most common genetic causes of infant mortality and is characterized by muscle weakness, loss of ambulation, and respiratory failure. SMA is primarily caused by a homozygous deletion or mutation of the survival motor neuron 1 (SMN1) gene. Humans possess a second, nearly identical copy of SMN, known as the SMN2 gene. Although the disease severity correlates inversely with the number of SMN2 copies present, it can never completely compensate for the loss of SMN1 in patients with SMA; SMN2 expresses only a fraction of the functional SMN transcript. The SMN protein is ubiquitous in human cells and plays several roles, ranging from assembling the spliceosome machinery to autophagy, RNA metabolism, signal transduction, cellular homeostasis, DNA repair, and recombination. Although the underlying mechanism remains unclear, anterior horn cells of the spinal cord gray matter are highly vulnerable to decreased SMN protein levels. To harness SMN2's ability to provide SMN function, two treatment strategies have been approved by the Food and Drug Administration (FDA), including an antisense oligonucleotide, nusinersen (Spinraza), and a small molecule, risdiplam (Evrysdi). Onasemnogene abeparvovec (Zolgensma) is an FDA-approved adeno-associated virus 9-mediated gene replacement therapy that creates a copy of the human SMN1 gene. In this review, we summarize the SMA etiology and FDA-approved therapies, and discuss the development of SMA therapeutic strategies and the challenges we faced.

Comorbid pathology in children with type II–III spinal muscular atrophy on the background of acquired deformities of the skeleton bones

Spinal muscular atrophy (SMA) is a severe genetic disease associated with impaired SMN protein synthesis and degeneration of alpha motor neurons in the spinal cord. Developing neurogenic kyphoscoliosis and deformity of the chest against the background of symmetrical muscular hypotension sharply limit the activity of patients, worsening the concomitant diseases.Purpose. The study aims at determining the comorbid background of children with type II–III spinal muscular atrophy who underwent inpatient treatment for acquired skeletal bone deformities.Material and methods. A retrospective analysis of the data was carried out for the period from 2017 to 2021 based on the medical records of 31 children. The study group included 10 girls and 21 boys; 16 children were with type II and 15 — with type III spinal muscular atrophy. The following were assessed: comorbidity, neurological status, hemodynamic parameters, echocardiography, spirometry, laboratory research data.Results. In our study, comorbidity was associated with nutritional status (19% of patients overweight, 29% underweight), mental retardation (3%), gastroesophageal reflux disease (19%), diseases of the ENT organs (16%), eyes (19%), heart and lungs (93%). For health reasons, 61% of children required the use of non-invasive ventilation, and 71% of insufflator-aspirators. Limited motor abilities were registered based on the HFMSE and GMFCS scales, dysphagia based on the EDACS scale. A biochemical blood test revealed a low level of creatinine.Conclusion. Patients with spinal muscular atrophy require multidisciplinary care in diagnosis, treatment and rehabilitation. The use of objective rating scales, instrumental and laboratory methods of examination allow for a comprehensive analysis of the potential of children with spinal muscular atrophy, to select effective, family-oriented treatment regimens. Serum creatinine as a biomarker for the severity of muscle denervation makes it possible to monitor the progression of spinal muscular atrophy and predict response to treatment.

Adherence and Persistence to Nusinersen for Spinal Muscular Atrophy: A US Claims-Based Analysis.

Spinal muscular atrophy(SMA)is agenetic,neuromuscular diseasecaused bydeletionsand/or mutations in the survival of motor neuron 1(SMN1)gene leading toreduced SMNprotein levels.Nusinersen, an intrathecally administered antisense oligonucleotide therapy that increases SMN protein levels, is approved for use in adult and pediatric patients with SMA. Data to inform real-world patient adherence and persistence to nusinersen are limited, with disparities in the population with SMA, study design, and results. The objective of this study is to characterize real-world nusinersen adherence and persistence in patients with SMA. This retrospective study examined nusinersenadherence and persistence over a 2-year period in patients with SMA in the USA from the IQVIA PharMetrics Plus claims database.Patients were followed from the date of first evidence of nusinersen treatment (occurring after 1 July 2017) until the end of the study period (31 December 2019) or end of continuous pharmacy and medical benefit enrollment, whichever came first. Subgroup analyses for nusinersen adherence and persistence were performed on the basis of age and presence or absence of spinal complications. The final cohort consisted of 179 patients with SMA treated with nusinersen. Adherence to nusinersen treatment was 41% at 56weeks and 39% at 104weeks. In the base-case persistence analysis, there was a decrease in persistence before 6months (67%) and further decline at 1 (57%) and 2years (55%). Patients with spinal complication versus without had numerically higher persistence with nusinersen. The findings suggest that adherence and persistence to nusinersen treatment appear low. Demographic (age ≥ 18years) and clinical factors (no spinal complications) may contribute to nusinersen treatment discontinuation. Future research should explore possible reasons for low adherence and persistence to nusinersen treatment, such as clinical or logistical factors, patient preferences, and payer restrictions.

Review on Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is the second leading genetic, autosomal recessive disorder with progressive weakness of skeletal and respiratory muscles, leading to progressive paralysis with muscular atrophy, significant disability. SMA predominantly affects on children and represents the most common cause of hereditary infant mortality. Spinal muscular atrophy caused by mutations in the survival motor neuron 1 (SMN1) gene and a consequentdecrease in the SMN protein leading to lower motor neuron degeneration. The clinical features of Spinal muscular atrophy are caused by specific degeneration of a-motor neurons in the spinal cord, leading to muscle weakness, atrophy and, in the majority of cases, premature death. Encouraging results from phase II and III clinical trials have raised hope that other therapeutic options will enter soon in clinical practice. The common genetic etiology and recent progress in pre-clinical models suggest that SMA is well-suited for the development of therapeutic regimens. This review covers the available data and the new challenges of SMA therapeutic strategies.

A link between agrin signalling and Cav3.2 at the neuromuscular junction in spinal muscular atrophy

SMN protein deficiency causes motoneuron disease spinal muscular atrophy (SMA). SMN-based therapies improve patient motor symptoms to variable degrees. An early hallmark of SMA is the perturbation of the neuromuscular junction (NMJ), a synapse between a motoneuron and muscle cell. NMJ formation depends on acetylcholine receptor (AChR) clustering triggered by agrin and its co-receptors lipoprotein receptor-related protein 4 (LRP4) and transmembrane muscle-specific kinase (MuSK) signalling pathway. We have previously shown that flunarizine improves NMJs in SMA model mice, but the mechanisms remain elusive. We show here that flunarizine promotes AChR clustering in cell-autonomous, dose- and agrin-dependent manners in C2C12 myotubes. This is associated with an increase in protein levels of LRP4, integrin-beta-1 and alpha-dystroglycan, three agrin co-receptors. Furthermore, flunarizine enhances MuSK interaction with integrin-beta-1 and phosphotyrosines. Moreover, the drug acts on the expression and splicing of Agrn and Cacna1h genes in a muscle-specific manner. We reveal that the Cacna1h encoded protein Cav3.2 closely associates in vitro with the agrin co-receptor LRP4. In vivo, it is enriched nearby NMJs during neonatal development and the drug increases this immunolabelling in SMA muscles. Thus, flunarizine modulates key players of the NMJ and identifies Cav3.2 as a new protein involved in the NMJ biology.

Microvasculopathy in spinal muscular atrophy is driven by a reversible autonomous endothelial cell defect.

Spinal muscular atrophy (SMA) is a neuromuscular disorder due to degeneration of spinal cord motor neurons caused by deficiency of the ubiquitously expressed SMN protein. Here, we present a retinal vascular defect in patients, recapitulated in SMA transgenic mice, driven by failure of angiogenesis and maturation of blood vessels. Importantly, the retinal vascular phenotype was rescued by early, systemic SMN restoration therapy in SMA mice. We also demonstrate in patients an unfavorable imbalance between endothelial injury and repair, as indicated by increased circulating endothelial cell counts and decreased endothelial progenitor cell counts in blood circulation. The cellular markers of endothelial injury were associated with disease severity and improved following SMN restoration treatment in cultured endothelial cells from patients. Finally, we demonstrated autonomous defects in angiogenesis and blood vessel formation, secondary to SMN deficiency in cultured human and mouse endothelial cells, as the underlying cellular mechanism of microvascular pathology. Our cellular and vascular biomarker findings indicate microvasculopathy as a fundamental feature of SMA. Our findings provide mechanistic insights into previously described SMA microvascular complications, and highlight the functional role of SMN in the periphery, including the vascular system, where deficiency of SMN can be addressed by systemic SMN-restoring treatment.

Evaluation of Mean Percentage of Full-Length SMN Transcripts as a Molecular Biomarker of Spinal Muscular Atrophy.

The elevation of SMN transcript and protein level remains the principal aim of SMA therapy. Still, there is no standard molecular biomarker for the assessment of its efficacy. In the current study, we tested three methods of SMN transcript level measurement using real-time RT-PCR, quantitative fluorescent RT-PCR, and a semiquantitative RT-PCR gel densitometric assay. We examined several potential mRNA-based biomarkers and examined their sensitivity and reliability by comparing the obtained values in peripheral blood mononuclear cells of SMA patients, SMA carriers, and healthy individuals. We found that the mean percentage of full-length (FL-SMN) transcripts relative to the total sum of FL-SMN and exon 7-deleted (Δ7 SMN) transcripts detected by semiquantitative and quantitative fluorescence RT-PCR differed significantly between the three analyzed groups. The relevance of this biomarker was proven in an SMN2-targeting therapeutic experiment. We showed that the values of the biomarker changed significantly in SMA fibroblast cell cultures after treatment with therapeutic antisense oligonucleotides targeting the ISS-N1 site in intron 7 of the SMN2 gene. The obtained results indicate the convenience of using the mean percentage of FL-SMN transcripts determined by semiquantitative and quantitative fluorescence RT-PCR as a putative biomarker for the assessment of SMA therapy efficacy in vitro.

P.38 A novel splice site variant in a patient with spinal muscular atrophy and hypoplastic left heart syndrome

Spinal muscular atrophy (SMA) is an autosomal recessive disorder causing degeneration of motor neurones in the anterior horn of the spinal cord. SMA is caused by homozygous in activation of the SMN1 gene but a highly homologous copy (SMN2), which also produces a small amount of functional SMN protein, can mitigate disease severity. We present a patient with a severe SMA phenotype with hypoplastic left heart syndrome (HLHS), characterize a novel spice-site variant in the SMN1 gene and describe the unusual pathological features in muscle biopsy. The patient was born to consanguineous parents in a national heart center due to antenatal diagnosis of HLHS. Postnatal examination (day 4) revealed decreased tone, a lack of anti-gravity movements, absent deep tendon reflexes but no respiratory distress or feeding problems. Due to the combination of suspected muscle disease and HLHS, together with the results below, treatment was discontinued and the patient died on day 10 of life. Whole genome sequencing (WGS) revealed an apparently homozygous variant in SMN1 located in the donor splice-site between exon 7 and intron 7, c.*3+1G>T (NM_000344.3). MLPA identified one copy of SMN1 and SMN2. Analysis of RNA showed a transcript lacking exon 7 and western blotting of SMN protein from muscle showed reduced levels comparable to other SMA1 patients. Muscle biopsy and subsequent immunohistochemical and immunofluorescence showed unusual expression of embryonic myosin heavy chain in the patient compared to controls and other SMA patients. This case highlights the importance of rapid diagnosis of newborns to allow further treatment decisions to be made. Up to 30% of HLHS patients have associated conditions and the combination with SMA has been previously described, whether this is more than the coincidence of two relatively common conditions is unclear. A severe SMA phenotype has been previously seen in other patients with a splice site variant in intron 7.

P.102 Apitegromab in SMA: An analysis of multiple efficacy endpoints in the TOPAZ extension study

SMA manifests progressive denervation weakness caused by genetic constraint of the SMN protein expression. Apitegromab is an investigational, fully human, monoclonal antibody that specifically binds proforms of myostatin thereby inhibiting myostatin activation. Apitegromab is intended to improve motor function by targeting the residual innervated muscle fibers in patients with SMA. The TOPAZ phase 2 trial (NCT03921528) was designed to identify clinical and protocol features that will best enable further study of its potential therapeutic effect, in patients with SMA. TOPAZ was conducted in subjects with Type 2 or Type 3 SMA (age 2-21 years). Patients received IV apitegromab Q4W, either as monotherapy or as an adjunct to nusinersen. 58 patients were dosed in 3 cohorts. Primary and secondary efficacy endpoints (assessment of motor function improvements via the Hammersmith scale scores) and safety results from TOPAZ were previously reported. Following the initial treatment period in TOPAZ, participants were offered the opportunity to enter an open label 12 month extension Period A, followed by a 12 month extension Period B. The TOPAZ study showed positive results in motor function scores at 12 months. Incidence and severity of AEs were consistent with the underlying patient population and background therapy. Of 58 subjects enrolled in TOPAZ, 55 continued in extension Period A. We will present here multiple efficacy endpoints and safety analyses of the extension Period A, after 2 years of apitegromab treatment. TOPAZ provided proof-of-concept that apitegromab has the potential to be the first muscle-directed therapy, complementary to SMN-augmenting therapy, in patients with SMA. TOPAZ extension B is ongoing and will provide further long-term efficacy and safety data of apitegromab in SMA patients. This information is helpful to the understanding of latent myostatin dynamics, apitegromab's therapeutic role for SMA, and the planning of future studies.

P.53 Muscle microRNAs in the cerebrospinal fluid predict clinical response to nusinersen therapy in type II and type III spinal muscular atrophy patients

The antisense oligonucleotide nusinersen (spinraza) regulates splicing of the survival motor neuron 2 (SMN2) messenger RNA to increase SMN protein expression and has improved ventilator free survival and motor function outcomes in infantile onset forms of SMA, treated early in the course of the disease. However, the response in later onset forms of SMA is highly variable and dependent on symptom severity and disease duration at treatment initiation. Therefore, we aimed to identify novel noninvasive biomarkers that could predict the response to nusinersen in type II and III SMA patients. 34 SMA patients were included. We applied next-generation sequencing to identify microRNAs in the cerebrospinal fluid (CSF) as candidate biomarkers predicting response to nusinersen. Hammersmith Functional Motor Scale Expanded (HFMSE), was conducted at baseline and 6 months post initiation of nusinersen therapy to assess motor function. Patients changing by ≥ 3 or ≤0 points in the HFMSE total score were considered as responders or non-responders, respectively. Lower baseline levels of two muscle microRNAs (miR-206 and miR-133), alone or in combination, predicted the pre-determined clinical response to nusinersen after 6 months therapy. Moreover, miR-206 levels were inversely correlated with the HFMSE score. Lower miR-206 and miR-133 in the CSF predict more robust clinical response to nusinersen treatment in later onset SMA patients. These novel findings have high clinical relevance for identifying early treatment response to nusinsersen in later onset SMA patients and call to test the ability of miRNAs to predict more sustained long-term benefit.

VP.54 Amifampridine safety and efficacy in spinal muscular atrophy ambulatory patients: a randomized placebo-controlled, crossover, phase 2 trial

Spinal muscular atrophy (SMA) is an autosomal recessive disease where a deficient amount of SMN protein leads to progressive degeneration of bulbar and spinal motor neurons. Therapies for the restoration of SMN production are now available. Yet, fatigue and signs of impaired neuromuscular junction (NMJ) transmission have been documented as possible contributors to SMA phenotype. Amifampridine (3,4-diaminopyridine), a voltage-dependent K+ channel blocker, prolongs depolarization of the presynaptic NMJ terminal, enhancing neuromuscular transmission. Here, we evaluated the safety and efficacy of amifampridine in ambulatory patients with SMA Type 3. SMA-001 was a phase 2, 1:1 randomized, double-blind, placebo-controlled, 2-period, 2-treatment, crossover study. Genetically confirmed SMA Type 3, able to walk unaided for 30 m, not treated with nusinersen in the previous 6 months, entered the run-in phase for at least 21 days during which amifampridine was titrated up to 80 mg/day to reach an optimized stable dose. Then, patients with at least 3-points improvement in Hammersmith Functional Motor Score Extended (HFMSE) were randomized to receive either amifampridine or placebo for 2 weeks, alternatively, for a total of 28 days of double-blind treatment. Safety was evaluated from the first day of Run-In to the end of the study (primary safety outcome). Efficacy was evaluated by changes from randomization of HFMSE (primary efficacy outcome), quality of life, 6-minute walk test, and timed test (secondary outcomes). Descriptive analyses and a mixed effects linear model were used for statistic. Thirteen adult patients were included in the study, no serious adverse events (SAEs) attributed to Amifampridine were reported. Transient paresthesias (33,3%) were the only amifampridine-related AEs reported. Six patients for each sequence of treatment were randomized. Amifampridine treatment led to a statistically significant improvement in HFMSE (LS Mean Difference 0.792; 95% CI from 0.22 to 1.37; p=0.0083), compared to placebo, but not in the secondary endpoints. SMA-001 study provided Class I evidence that AP was safe and effective in treating ambulatory patients affected by SMA type 3.

P.110 JEWELFISH: 24-month safety and pharmacodynamic data in non-treatment-naïve patients with spinal muscular atrophy (SMA)

Risdiplam (EVRYSDI®) is a centrally and peripherally distributed, oral survival of motor neuron 2 (SMN2) pre-mRNA splicing modifier that has been approved in more than 70 countries worldwide. JEWELFISH (NCT03032172) is a multicenter, open-label study that assesses the safety, tolerability and pharmacokinetic/pharmacodynamic (PD) relationship of daily risdiplam in non-treatment-naïve patients with SMA (aged 6 months–60 years at enrollment) who previously received RG7800 (RO6885247), nusinersen (SPINRAZA®), olesoxime or onasemnogene abeparvovec (ZOLGENSMA®). The enrolled population (N=174) included a broad range of ages (1–60 years), SMA types (1–3), SMN2 copy numbers (1–4) and motor functions (non-sitters, sitters and walkers). We previously presented safety data from 173 patients (data cut-off: 29 January 2021) who received risdiplam for up to 47 months. One patient withdrew from the study at baseline; of the remaining patients, 13 previously received RG7800, 76 received nusinersen, 70 received olesoxime and 14 received onasemnogene abeparvovec. Risdiplam treatment led to a rapid and sustained >2-fold increase in SMN protein levels in blood compared with baseline levels (data cut-off: 29 January 2021), consistent with PD data from the SUNFISH (NCT02908685) study of treatment-naïve patients with Types 2/3 SMA. No drug-related safety findings leading to withdrawal were reported for any patients exposed to risdiplam in JEWELFISH at month 12. The risdiplam safety profile was consistent with the safety profile observed in risdiplam treatment-naïve trials. Here we will present the month 24 results from JEWELFISH for the first time. The JEWELFISH study is ongoing at sites across Europe and the USA and is providing important data on the safety and PD of risdiplam in a broad population of non-treatment-naïve patients with SMA.

Prevalence of Anti-Adeno-Associated Virus Serotype 9 Antibodies in Adult Patients with Spinal Muscular Atrophy.

5q-associated spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder that leads to progressive muscle atrophy and weakness. The disease is caused by a homozygous deletion or mutation in the survival of motor neuron 1 (<i>SMN1</i>) gene, resulting in insufficient levels of SMN protein. Onasemnogene abeparvovec-xioi (OA) is a nonreplicating vector based on adeno-associated virus serotype 9 (AAV9) that contains the full-length human <i>SMN1</i> gene. Recently, OA was approved for the treatment of SMA by the U.S. Food and Drug Administration and the European Medicines Agency. Because the presence of neutralizing antibodies caused by previous natural exposure to wild-type adeno-associated viruses (AAVs) may impair the efficiency of AAV-mediated gene transfer and thus reduce the therapeutic benefit of the gene therapy, an AAV9-binding antibody titer of &gt;1:50 was defined as a surrogate exclusion criterion in pivotal OA clinical trials. However, these studies were exclusively conducted in infants and children. Because data on anti-AAV9 antibody titers in adults are generally sparse and not available for adult patients with SMA, we determined the prevalence of anti-AAV9 antibodies in sera of adult individuals with SMA to evaluate the feasibility of AAV9-mediated gene therapy in this cohort. In our study population of 69 adult patients with SMA type 2 and type 3 from four German academic sites, only 3 patients (4.3%) had an elevated anti-AAV9 antibody titer of &gt;1:50. The prevalence of anti-AAV9 antibodies did not increase with age. The low and age-independent prevalence of anti-AAV9 antibodies in our cohort provides evidence that gene therapy with intravenous administered recombinant AAV9 vectors (rAAV9) might be feasible in adult patients with SMA, regardless of the patients' sex, SMA type, walking ability, or ventilatory status. This could also apply to the treatment of other inherited neurological diseases with rAAV9.