Abstract Background Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with limited therapeutic options. The circulating metabolome comprises small molecules present in plasma/serum which are the intermediates and end-products of cellular metabolism, and is linked to ALS pathogenesis. Methods We conducted hypothesis-free two-sample Mendelian randomisation (MR) analysis of the concentration of 575 plasma/serum metabolites, to determine which are causally linked to risk of ALS. Significant metabolites were validated in an independent GWAS of plasma/serum metabolite concentrations and evaluated for sex-specific effects. Correlations between directly measured patient biofluid metabolite concentrations and ALS risk/severity were examined in 94 ALS patients and 40 controls. We experimentally assessed metabolic function in a murine neurons and human astrocytes carrying an ALS-associated G4C2-repeat expansion within C9orf72. Results MR causally associated five metabolites with ALS risk after multiple-testing correction. Higher serum concentration of glycoprotein acetyls (P = 9.7e − 9, β = 0.21) and the peptide DSGEGDFXAEGGGVR (P = 8.0e − 6, β = 0.22) was associated with increased ALS risk, whereas higher plasma concentration of phenylalanylserine, isobutyrylcarnitine, and acetylcarnitine was protective (P < 5e − 5, β = − 0.29 to − 0.72). DSGEGDFXAEGGGVR has been linked to glucose metabolism but we have used genetic fine-mapping to link DSGEGDFXAEGGGVR, neuronal glucose uptake through GLUT3, and ALS risk. Direct measurement of metabolite concentrations in patient biofluids revealed elevated acetylcarnitine levels in patients with ALS, which were associated with delayed symptom onset (Cox regression, P = 0.02, HR = 0.4). Similarly, lactate is elevated in ALS patient CSF (ANOVA, P = 1.3e − 3) and in patients with longer survival time (Cox regression, P = 0.03, HR = 0.3). Plasma fructose is elevated in ALS patients with shorter survival time (Cox regression, P = 0.02, HR = 1.1). In vitro, neurons and astrocytes carrying an ALS-associated G4C2-repeat expansion within C9orf72 demonstrated reduced metabolic flexibility. Conclusions We provide evidence that impaired energy substrate availability contributes to ALS risk and severity. CNS cell types differ in their use of energy substrates and therefore we postulate the relative importance of different cell types for different stages of disease. Our findings support further investigation of metabolic interventions to treat or prevent ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by the death of motor neurons leading to paralysis and death, generally 3-5 years post-symptom onset. The most frequent genetic cause of ALS is a hexanucleotide repeat expansion (HRE) in the chromosome 9 open reading frame 72 (C9orf72) gene, that has three major hypothesised pathological mechanisms including the production of dipeptide repeat proteins (DPRs). Our laboratory has previously identified purine metabolism dysfunction in induced neural progenitor cell-derived astrocytes (iAstrocytes) from C9orf72 ALS (C9-ALS) cases (C9-iAstrocytes), driven by loss of the enzyme adenosine deaminase (ADA). Here, we have demonstrated that loss of ADA along with changes to ecto-5 '-nucleotidase and hypoxanthine-guanine phosphoribosyl transferase led to disruption in purine metabolite levels including purine dNTP output. These changes were recapitulated in patient CSF, whilst loss of ADA was recapitulated in patient white matter. Immunofluorescence also demonstrated purinosome formation dysfunction in C9-iAstrocytes. These changes are likely driven by DPRs as ADA loss was recapitulated in in vitro and in vivo DPR models. Finally, ADA levels could be recovered by reducing DPR levels either by inhibiting serine/arginine-rich splicing factor 1 or overexpressing RuvB-like 2. Our data demonstrate that DPR production negatively affects purine function in C9-ALS suggesting a potentially pivotal role for purine metabolism dysfunction in C9-ALS pathology.

Abstract COL4A1-related disorders cause early-onset stroke, intracerebral haemorrhage, visual impairment and kidney disease, often affecting children and young adults, yet no disease-modifying therapies exist. These disorders arise from pathogenic COL4A1 variants that disrupt type IV collagen and impair small-vessel integrity, leading to cerebral small-vessel disease and endothelial dysfunction. We performed a mechanism-guided screen in human brain endothelial cells using a CRISPR-engineered COL4A1 p.G755R line and patient-specific COL4A1 p.G773R iPSC-derived endothelial cells. Simvastatin, L-carnosine, and XPD-101 restored impaired endothelial proliferation, migration, and other markers of endothelial function, including transendothelial electrical resistance (TEER). In a Col4a1 Svc/+ mouse model, simvastatin increased pre-weaning survival, improved functional behaviour and reduced cerebral microhaemorrhage burden. These findings identify mechanism-informed candidates that rescue COL4A1-mutant endothelial dysfunction in vitro, with simvastatin demonstrating in vivo efficacy, supporting prioritisation for further preclinical development.

Abstract Gene therapies are promising for diseases previously considered incurable. Adeno-associated virus serotype 9 (AAV9) demonstrates remarkable tropism for motor neurons (MNs) and represents an exciting candidate to target genetic causes of motor neuron diseases like amyotrophic lateral sclerosis (ALS). However, systemic delivery risks immunogenicity and off-target effects, therefore localised delivery to the CNS is advantageous. We assessed MN transduction in wild-type post-natal mice using AAV9-controlled, cytomegalovirus-promoter driven, enhanced GFP expression. Intra-cisterna magna (ICM) and intra-cerebroventricular (ICV) methods were compared. Four weeks post-delivery, GFP positivity in MN and astrocytes were quantified via immunohistochemical approaches and viral genome copy number determined by qPCR. All delivery methods achieved high MN transduction in lumbar spinal cord (> 68%). Unilateral ICV delivery provided the highest and most consistent levels (89 ± 3%), and minimal peripheral viral copies. ICV delivery resulted in higher astrocytic transduction, most notably in the cortex. Brainstem MN transduction was high with all methods (> 55%). We failed to find evidence of neuronal transduction in motor cortex. Viral genome copies trended higher in spinal cord and brainstem with ICV approaches, however further work is required to understand how bilateral repeated dose delivery leads to more profound increases. Whilst several routes of administration into cerebrospinal fluid exist, direct comparisons for targeting MNs in vivo remain limited. Overall, all methods of CNS-directed delivery result in high levels of motor neuron transduction in the lumbar spinal cord and brainstem, but not in motor cortex. Unilateral ICV appears to provide the best balance between consistent, high levels of transduction and low off-target effects. However, ICM might be the better option if seeking to avoid astrocytic transduction.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenetic hereditary small-vessel disorder characterised by recurrent subcortical ischemic strokes, cognitive deterioration, and other neurological symptoms. Single nucleotide mutations within the NOTCH3 gene can impair NOTCH3 processing and/or signalling, resulting in the accumulation of granular osmiophilic material (GOM) in blood vessel walls, and consequently CADASIL. Despite its significant clinical impact, there is currently no definitive treatment for CADASIL. This review provides a comprehensive analysis of the pathophysiological mechanisms underlying CADASIL, focusing on NOTCH3 mutations and their effects on protein processing and signalling. The review proposes a hypothesis that explains how NOTCH3 mutations may alter the signalling process and result in GOMs. Additionally, the review explores published therapy strategies aimed at restoring normal NOTCH3 function.

Collagen disorders encompass a wide range of genetic conditions caused by pathogenic variants in collagen genes for which there is an unmet need for treatments. They present various clinical features, ranging from localised tissue abnormalities to severe systemic complications. Symptoms differ significantly and depend on the pathogenic variant, which can affect various systems, including the musculoskeletal, cardiovascular, and respiratory systems, highlighting the complex implications of collagen gene pathogenic variants and the wide range of expression patterns among different collagen types. Gene-editing technologies, particularly Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas systems, have emerged as promising therapeutic options for these disorders, representing a putative one-for-all treatment strategy. This review provides an overview of current gene-editing strategies aimed at collagen-related diseases, including osteogenesis imperfecta, Alport syndrome, and dystrophic epidermolysis bullosa. We explore the application of CRISPR-Cas9, which facilitates targeted DNA modifications, base editing (BE), and prime editing (PE), enabling precise single-nucleotide alterations without double-strand breaks (DSB). Preclinical and clinical studies have shown the potential of gene therapy to enhance collagen production, restore tissue integrity, and alleviate symptoms. However, challenges persist, including the lack of recurring mutations, the need for improved delivery methods, the reduction of off-target effects, and the development of novel therapies. Despite these challenges, advancements in gene editing techniques appear promising in enhancing editing efficiency while minimising unintended mutations, paving the way for more precise and safer genetic interventions for collagen disorders. Gene editing is fundamentally transforming medicine and biotechnology. Its applications encompass advanced diagnostics, tailored therapeutic strategies, and solutions for rare genetic disorders. By enabling precise genetic modifications, gene editing is paving the way for treatments of previously untreatable diseases, including those linked to collagen pathogenic variants. This review discusses the latest advancements in gene therapy techniques targeting collagen-related disorders. It explores innovative approaches like CRISPR-Cas9-mediated gene editing and highlights emerging strategies, such as allele-specific inactivation and base editing (BE). By examining these cutting-edge therapies and their potential clinical applications, this review highlights the transformative impact of gene editing in treating collagen-related conditions, while also identifying critical challenges and future directions for research.

Gene therapy medicinal products (GTMPs) are currently undergoing intense industrial expansion and technological advancement. However, one issue facing development of most GTMPs is the generation of unwanted immune responses. Immunomodulatory strategies are also often applied in conjunction with GTMP administration to suppress or enhance these responses. This review focusses on the global regulatory requirements for immunogenicity assessments and immunomodulation in relation to GTMPs. The specific aims are to (1) identify the principal international guidelines; (2) identify areas of concordance and discrepancy between guidelines; (3) propose areas where guidelines could be harmonized; and (4) predict areas, which future guidance may address. Methodologies used included surveillance of literature, international guidelines, advocacy initiatives, and compilation of previous regulatory advice received. Overall, there is a clear absence of and need for GTMP-specific guidance on immunogenicity and immunomodulation. Several specific measures and areas for future regulatory harmonization and coverage are proposed.

Translational medicine for neurodegenerative diseases can advance through the use of in vitro models incorporating human neural cells derived from patient-specific induced pluripotent stem cells (iPSCs). Previously, we investigated whether motor neuron (MN) progenitors derived from human iPSCs could differentiate to MNs within 3D-printed scaffolds. While extensive neurite arborization was observed on the scaffold surface, no neurite outgrowth occurred within the scaffold interior. Here we show the extensive growth of the neurites from iPSC-derived neural progenitors, imbedded into the gelatin scaffolds during 30 days of experimental time. We present a bioink formulation that softens the scaffold while preserving its 3D structure, thereby facilitating neurite outgrowth throughout the scaffold. MN differentiation, evidenced by extensive neurite arborization and the expression of choline acetyltransferase (ChAT) was verified in 3D images deep within the scaffold structure. Notably, the degree of MN differentiation appeared to depend on two factors: the delivery of MN differentiation factors via mesoporous silica particles (MSPs) embedded in the bioink and the method used to generate MN progenitors prior to 3D printing. We provide a detailed protocol for 3D printing human iPSC-derived MN progenitors, enabling their differentiation and survival within gelatin scaffolds. This protocol could be expanded to incorporate additional cell types, allowing the creation of more complex and standardized 3D neural tissues. Such advancements could facilitate investigations into the pathophysiology of motor neuron diseases and the development of new therapeutic strategies.

Abstract Spastic paraplegia 47 (SPG47) is a neurological disorder caused by mutations in the adaptor protein complex 4 β1 subunit ( AP4B1 ) gene leading to AP-4 complex deficiency. SPG47 is characterised by progressive spastic paraplegia, global developmental delay, intellectual disability and epilepsy. Gene therapy aimed at restoring functional AP4B1 protein levels is a rational therapeutic strategy to ameliorate the disease phenotype. Here we report that a single delivery of adeno-associated virus serotype 9 expressing hAP4B1 (AAV9/hAP4B1) into the cisterna magna leads to widespread gene transfer and restoration of various hallmarks of disease, including AP-4 cargo (ATG9A) mislocalisation, calbindin-positive spheroids in the deep cerebellar nuclei, anatomical brain defects and motor dysfunction, in an SPG47 mouse model. Furthermore, AAV9/hAP4B1-based gene therapy demonstrated a restoration of plasma neurofilament light (NfL) levels of treated mice. Encouraged by these preclinical proof-of-concept data, we conducted IND-enabling studies, including immunogenicity and GLP non-human primate (NHP) toxicology studies. Importantly, NHP safety and biodistribution study revealed no significant adverse events associated with the therapeutic intervention. These findings provide evidence of both therapeutic efficacy and safety, establishing a robust basis for the pursuit of an IND application for clinical trials targeting SPG47 patients.

A G4C2 hexanucleotide repeat expansion inC9orf72is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Bidirectional transcription and subsequent repeat-associated non-AUG (RAN) translation of sense and antisense transcripts leads to the formation of five dipeptide repeat (DPR) proteins. These DPRs are toxic in a wide range of cell and animal models. Therefore, decreasing RAN-DPRs may be of therapeutic benefit in the context of C9ALS/FTD. In this study, we found that C9ALS/FTD patients have reduced expression of the AAA+ family members RuvBL1 and RuvBL2, which have both been implicated in aggregate clearance. We report that overexpression of RuvBL1, but to a greater extent RuvBL2, reduced C9orf72-associated DPRs in a range of in vitro systems including cell lines, primary neurons from the C9-500 transgenic mouse model, and patient-derived iPSC motor neurons. In vivo, we further demonstrated that RuvBL2 overexpression and consequent DPR reduction in ourDrosophilamodel was sufficient to rescue a number of DPR-related motor phenotypes. Thus, modulating RuvBL levels to reduce DPRs may be of therapeutic potential in C9ALS/FTD.

The emergence of adeno-associated virus (AAV)-based gene therapy has brought hope to patients with severe monogenic disorders. However, immune responses to AAV vectors and transgene products present challenges that require effective immunosuppressive strategies. This systematic review focuses on the immunosuppressive protocols used in 38 clinical trials and 35 real-world studies, considering a range of monogenic diseases, AAV serotypes, and administration routes. The review underscores the need for a deeper understanding of immunosuppressive regimens to enhance the safety and effectiveness of AAV-based gene therapy. Characterizing the immunological responses associated with various gene therapy treatments is crucial for optimizing treatment protocols and ensuring the safety and efficacy of forthcoming gene therapy interventions. Further research and understanding of the impact of immunosuppression on disease, therapy, and route of administration will contribute to the development of more effective and safer gene therapy approaches in the future.

Autoimmune polyglandular syndrome type 1 (APS-1) is an inherited autosomal disorder. The most common clinical features of the disease include adrenocortical failure, hypoparathyroidism (HP), and chronic mucocutaneous candidiasis (CMC). APS-1 is caused by mutations in the autoimmune regulator (AIRE) gene. AIRE is a transcriptional factor involved in the regulation of thousands of genes in the thymus. It facilitates central tolerance by promoting the ectopic expression of tissue-specific antigens (TSAs) in medullary thymic epithelial cells (mTECs), leading to the deletion of self-reactive thymocytes. Several Aire-deficient mice were developed separately, on different backgrounds; seven published Aire knockout mice show a variety of phenotypes depending on the strain used to generate the experimental model. The first Aire-deficient mice were generated on a "black 6" background almost 20 years ago. The model showed mild phenotype with relatively modest penetrance compared to models generated on BALBc or NOD backgrounds. The generation of all these experimental models is crucial for development and testing new therapeutics as well as reading the response to treatments.

Hexanucleotide repeat expansions in C9ORF72 are the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies have shown that the hexanucleotide expansions cause the noncanonical translation of C9ORF72 transcripts into neurotoxic dipeptide repeat proteins (DPRs) that contribute to neurodegeneration. We show that a cell-penetrant peptide blocked the nuclear export of C9ORF72 -repeat transcripts in HEK293T cells by competing with the interaction between SR-rich splicing factor 1 (SRSF1) and nuclear export factor 1 (NXF1). The cell-penetrant peptide also blocked the translation of toxic DPRs in neurons differentiated from induced neural progenitor cells (iNPCs), which were derived from individuals carrying C9ORF72 -linked ALS mutations. This peptide also increased survival of iNPC-differentiated C9ORF72-ALS motor neurons cocultured with astrocytes. Oral administration of the cell-penetrant peptide reduced DPR translation and rescued locomotor deficits in a Drosophila model of mutant C9ORF72-mediated ALS/FTD. Intrathecal injection of this peptide into the brains of ALS/FTD mice carrying a C9ORF72 mutation resulted in reduced expression of DPRs in mouse brains. These findings demonstrate that disrupting the production of DPRs in cellular and animal models of ALS/FTD might be a strategy to ameliorate neurodegeneration in these diseases.

Due to their low pathogenicity, immunogenicity, and long-term gene expression, adeno-associated virus (AAV) vectors emerged as safe and efficient gene delivery tools, over-coming setbacks experienced with other viral gene delivery systems in early gene therapy trials. Among AAVs, AAV9 can translocate through the blood-brain barrier (BBB), making it a promising gene delivery tool for transducing the central nervous system (CNS) via systemic administration. Recent reports on the shortcomings of AAV9-mediated gene delivery into the CNS require reviewing the molecular base of AAV9 cellular biology. A more detailed understanding of AAV9's cellular entry would eradicate current hurdles and enable more efficient AAV9-based gene therapy approaches. Syndecans, the transmembrane family of heparan-sulfate proteoglycans, facilitate the cellular uptake of various viruses and drug delivery systems. Utilizing human cell lines and syndecan-specific cellular assays, we assessed the involvement of syndecans in AAV9's cellular entry. The ubiquitously expressed isoform, syndecan-4 proved its superiority in facilitating AAV9 internalization among syndecans. Introducing syndecan-4 into poorly transducible cell lines enabled robust AAV9-dependent gene transduction, while its knockdown reduced AAV9's cellular entry. Attachment of AAV9 to syndecan-4 is mediated not just by the polyanionic heparan-sulfate chains but also by the cell-binding domain of the extracellular syndecan-4 core protein. Co-immunoprecipitation assays and affinity proteomics also confirmed the role of syndecan-4 in the cellular entry of AAV9. Overall, our findings highlight the universally expressed syndecan-4 as a significant contributor to the cellular internalization of AAV9 and provide a molecular-based, rational explanation for the low gene delivery potential of AAV9 into the CNS.

Introduction: Gene therapy provides the exciting opportunity of a curative single treatment for devastating diseases, eradicating the need for chronic medication. Adeno-associated viruses (AAVs) are among the most attractive vector carriers for gene replacement in vivo. Yet, despite the success of recent AAV-based clinical trials, the clinical use of these vectors has been limited. For instance, the AAV packaging capacity is restricted to similar to 4.7 kb, making it a substantial challenge to deliver large gene products. Areas covered: In this review, we explore established and emerging strategies that circumvent the packaging limit of AAVs to make them effective vehicles for gene replacement therapy of monogenic disorders, with a particular focus on diseases affecting the nervous system. We report historical references, design remarks, as well as strengths and weaknesses of these approaches. We additionally discuss examples of neurological disorders for which such strategies have been attempted. Expert opinion: The field of AAV-gene therapy has experienced enormous advancements in the last decade. However, there is still ample space for improvement aimed at overcoming existing challenges that are slowing down the progressive trajectory of this field.

Disruption to protein homeostasis caused by lysosomal dysfunction and associated impairment of autophagy is a prominent pathology in amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). The most common genetic cause of ALS/FTD is a G4C2 hexanucleotide repeat expansion in C9orf72 (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 repeat transcripts gives rise to dipeptide repeat (DPR) proteins that have been shown to be toxic and may contribute to disease etiology. Genetic variants in TMEM106B have been associated with frontotemporal lobar degeneration with TDP-43 pathology and disease progression in C9ALS/FTD. TMEM106B encodes a lysosomal transmembrane protein of unknown function that is involved in various aspects of lysosomal biology. How TMEM106B variants affect C9ALS/FTD is not well understood but has been linked to changes in TMEM106B protein levels. Here, we investigated TMEM106B function in the context of C9ALS/FTD DPR pathology. We report that knockdown of TMEM106B expression exacerbates the accumulation of C9ALS/FTD-associated cytotoxic DPR proteins in cell models expressing RAN-translated or AUG-driven DPRs as well as in C9ALS/FTD-derived iAstrocytes with an endogenous G4C2 expansion by impairing autophagy. Loss of TMEM106B caused a block late in autophagy by disrupting autophagosome to autolysosome maturation which coincided with impaired lysosomal acidification, reduced cathepsin activity, and juxtanuclear clustering of lysosomes. Lysosomal clustering required Rab7A and coincided with reduced Arl8b-mediated anterograde transport of lysosomes to the cell periphery. Increasing Arl8b activity in TMEM106B-deficient cells not only restored the distribution of lysosomes, but also fully rescued autophagy and DPR protein accumulation. Thus, we identified a novel function of TMEM106B in autophagosome maturation via Arl8b. Our findings indicate that TMEM106B variants may modify C9ALS/FTD by regulating autophagic clearance of DPR proteins. Caution should therefore be taken when considering modifying TMEM106B expression levels as a therapeutic approach in ALS/FTD.

New therapeutic targets are a valuable resource in the struggle to reduce the morbidity and mortality associated with the COVID-19 pandemic, caused by the SARS-CoV-2 virus. Genome-wide association studies (GWAS) have identified risk loci, but some loci are associated with co-morbidities and are not specific to host-virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins; EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. Lung-specific eQTLs were identified from GTEx (v7) for each of the 332 host proteins. Aggregating COVID-19 GWAS statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19 which survived stringent multiple testing correction. EXOSC2 is a component of the RNA exosome and indeed, LC-MS/MS analysis of protein pulldowns demonstrated an interaction between the SARS-CoV-2 RNA polymerase and the majority of human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression, impeded SARS-CoV-2 replication and upregulated oligoadenylate synthase ( OAS) genes, which have been linked to a successful immune response against SARS-CoV-2. Reduced EXOSC2 expression did not reduce cellular viability. OAS gene expression changes occurred independent of infection and in the absence of significant upregulation of other interferon-stimulated genes (ISGs). Targeted depletion or functional inhibition of EXOSC2 may be a safe and effective strategy to protect at-risk individuals against clinical COVID-19.

An understanding of the biodistribution and shedding profile of a gene therapy product following in vivo administration is an important element of the development program.Recommendations for biodistribution studies have been issued by various regulatory authorities with the most recent draft International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guideline S12 'Nonclinical biodistribution considerations for gene therapy products' released for public comment on 3 June 2021.In this paper the Innovative Medicines Initiative, Accelerating Research & Development for Advanced Therapies consortium provides an overview of the current regulatory landscape for conducting shedding and biodistribution studies and makes a call for harmonization across regions.In addition, over the last three decades, a significant body of literature on biodistribution and shedding of AAV-based gene therapies has amassed, and we describe herein the initial stages of construction of a formal database of published biodistribution

Spinal muscular atrophy, the leading genetic cause of infant mortality, is a motor neuron disease caused by low levels of survival motor neuron (SMN) protein. SMN is a multifunctional protein that is implicated in numerous cytoplasmic and nuclear processes. Recently, increasing attention is being paid to the role of SMN in the maintenance of DNA integrity. DNA damage and genome instability have been linked to a range of neurodegenerative diseases. The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. Instability in rDNA has been associated with cancer, premature ageing syndromes, and a number of neurodegenerative disorders. Here, we report that SMN-deficient cells exhibit increased rDNA damage leading to impaired ribosomal RNA synthesis and translation. We also unravel an interaction between SMN and RNA polymerase I. Moreover, we uncover an spinal muscular atrophy motor neuron-specific deficiency of DDX21 protein, which is required for resolving R-loops in the nucleolus. Taken together, our findings suggest a new role of SMN in rDNA integrity.