Disruption of splicing-regulatory elements using CRISPR/Cas9 to rescue spinal muscular atrophy in human iPSCs and mice
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ABSTRACT:
SUBMITTER: Li J
PROVIDER: S-EPMC8446915 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Spinal muscular atrophy (SMA) is one of the major genetic disorders associated with infant mortality. More than 90% cases of SMA result from deletions or mutations of Survival Motor Neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, does not compensate for the loss of SMN1 due to predominant skipping of exon 7. However, correction of SMN2 exon 7 splicing has proven to confer therapeutic benefits in SMA patients. The only approved drug for SMA is an antisense oligonucleotide (Spinraza™/Nusinersen), which corrects SMN2 exon 7 splicing by blocking intronic splicing silencer N1 (ISS-N1) located immediately downstream of exon 7. ISS-N1 is a complex regulatory element encompassing overlapping negative motifs and sequestering a cryptic splice site. More than 40 protein factors have been implicated in the regulation of SMN exon 7 splicing. There is evidence to support that multiple exons of SMN are alternatively spliced during oxidative stress, which is associated with a growing number of pathological conditions. Here, we provide the most up to date account of the mechanism of splicing regulation of the SMN genes.
| S-EPMC6026014 | biostudies-literature
Project description: Not available
| S-EPMC7264321 | biostudies-literature
Project description:Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, arises from survival motor neuron (SMN) protein insufficiency resulting from SMN1 loss. Approved therapies circumvent endogenous SMN regulation and require repeated dosing or may wane. We describe genome editing of SMN2, an insufficient copy of SMN1 harboring a C6>T mutation, to permanently restore SMN protein levels and rescue SMA phenotypes. We used nucleases or base editors to modify five SMN2 regulatory regions. Base editing converted SMN2 T6>C, restoring SMN protein levels to wild type. Adeno-associated virus serotype 9-mediated base editor delivery in Δ7SMA mice yielded 87% average T6>C conversion, improved motor function, and extended average life span, which was enhanced by one-time base editor and nusinersen coadministration (111 versus 17 days untreated). These findings demonstrate the potential of a one-time base editing treatment for SMA.
| S-EPMC10270003 | biostudies-literature
Project description:Spinal muscular atrophy (SMA) is a potentially devastating and lethal neuromuscular disease frequently manifesting in infancy and childhood. The discovery of the underlying mutation in the survival of motor neurons 1 (SMN1) gene has accelerated preclinical research, leading to treatment targets and transgenic mouse models, but there is still no effective treatment. The clinical severity is inversely related to the copy number of SMN2, a modifying gene producing some full-length SMN transcript. Drugs shown to increase SMN2 function in vitro, therefore, have the potential to benefit patients with SMA. Because several drugs are now on the horizon of clinical investigation, we review recent clinical trials for SMA and discuss the challenges and opportunities associated with SMA drug development. Although an orphan disease, SMA is well-positioned for successful trials given that it has a common genetic etiology in most cases, that it can be readily diagnosed, that preclinical research in vitro and in transgenic animals has identified candidate compounds, and that trial networks have been established.
| S-EPMC4514700 | biostudies-other
Project description:Spinal Muscular Atrophy results from loss-of-function mutations in SMN1 but correcting aberrant splicing of SMN2 offers hope of a cure. However, current splice therapy requires repeated infusions and is expensive. We previously rescued SMA mice by promoting the inclusion of a defective exon in SMN2 with germline expression of Exon-Specific U1 snRNAs (ExspeU1). Here we tested viral delivery of SMN2 ExspeU1s encoded by adeno-associated virus AAV9. Strikingly the virus increased SMN2 exon 7 inclusion and SMN protein levels and rescued the phenotype of mild and severe SMA mice. In the severe mouse, the treatment improved the neuromuscular function and increased the life span from 10 to 219 days. ExspeU1 expression persisted for 1 month and was effective at around one five-hundredth of the concentration of the endogenous U1snRNA. RNA-seq analysis revealed our potential drug rescues aberrant SMA expression and splicing profiles, which are mostly related to DNA damage, cell-cycle control and acute phase response. Vastly overexpressing ExspeU1 more than 100-fold above the therapeutic level in human cells did not significantly alter global gene expression or splicing. These results indicate that AAV-mediated delivery of a modified U1snRNP particle may be a novel therapeutic option against SMA.
| S-EPMC6698663 | biostudies-literature
Project description:Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. The almost identical SMN2 gene is unable to compensate for this deficiency because of the skipping of exon 7 during pre-messenger RNA (mRNA) processing. Although several splicing factors can modulate SMN2 splicing in vitro, the physiological regulators of this disease-causing event are unknown. We found that knockout of the splicing factor SAM68 partially rescued body weight and viability of SMA?7 mice. Ablation of SAM68 function promoted SMN2 splicing and expression in SMA?7 mice, correlating with amelioration of SMA-related defects in motor neurons and skeletal muscles. Mechanistically, SAM68 binds to SMN2 pre-mRNA, favoring recruitment of the splicing repressor hnRNP A1 and interfering with that of U2AF65 at the 3' splice site of exon 7. These findings identify SAM68 as the first physiological regulator of SMN2 splicing in an SMA mouse model.
| S-EPMC4602033 | biostudies-literature
Project description:Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by mutations of the survival of motor neuron 1 (SMN1) gene. In the pathogenesis of SMA, pathological changes of the neuromuscular junction (NMJ) precede the motor neuronal loss. Therefore, it is critical to evaluate the NMJ formed by SMA patients' motor neurons (MNs), and to identify drugs that can restore the normal condition. We generated NMJ-like structures using MNs derived from SMA patient-specific induced pluripotent stem cells (iPSCs), and found that the clustering of the acetylcholine receptor (AChR) is significantly impaired. Valproic acid and antisense oligonucleotide treatment ameliorated the AChR clustering defects, leading to an increase in the level of full-length SMN transcripts. Thus, the current in vitro model of AChR clustering using SMA patient-derived iPSCs is useful to dissect the pathophysiological mechanisms underlying the development of SMA, and to evaluate the efficacy of new therapeutic approaches.
| S-EPMC4400613 | biostudies-literature
Project description:Spinal muscular atrophy (SMA) is a neurological disorder characterized by motor neuron degeneration and progressive muscle paralysis. The disease is caused by a reduction in survival of motor neuron (SMN) protein resulting from homozygous deletion of the SMN1 gene. SMN protein is also encoded by SMN2. However, splicing of SMN2 exon 7 is defective, and consequently, the majority of the transcripts produce a truncated, unstable protein. SMN protein itself has a role in splicing. The protein is required for the biogenesis of spliceosomal snRNPs, which are essential components of the splicing reaction. We now show that SMN protein abundance affects the splicing of SMN2 exon 7, revealing a feedback loop inSMN expression. The reduced SMN protein concentration observed in SMA samples and in cells depleted of SMN correlates with a decrease in cellular snRNA levels and a decrease in SMN2 exon 7 splicing. Furthermore, altering the relative abundance or activity of individual snRNPs has distinct effects on exon 7 splicing, demonstrating that core spliceosomal snRNPs influence SMN2 alternative splicing. Our results identify a feedback loop in SMN expression by which low SMN protein levels exacerbate SMN exon 7 skipping, leading to a further reduction in SMN protein. These results imply that a modest increase in SMN protein abundance may cause a disproportionately large increase in SMN expression, a finding that is important for assessing the therapeutic potential of SMA treatments and understanding disease pathogenesis.
| S-EPMC2989896 | biostudies-literature
Project description:Spinal muscular atrophy is a leading genetic cause of infantile death and occurs in approximately 1/6000 live births. SMA is caused by the loss of Survival Motor Neuron-1 (SMN1), however, all patients retain at least one copy of a nearly identical gene called SMN2. While SMN2 and SMN1 are comprised of identical coding sequences, the majority of SMN2 transcripts are alternatively spliced, encoding a truncated protein that is unstable and nonfunctional. Considerable effort has focused upon modulating the SMN2 alternative splicing event since this would produce more wild-type protein. Recently we reported the development of an optimized trans-splicing system that involved the coexpression of a SMN2 trans-splicing RNA and an antisense RNA that blocks a downstream splice site in SMN2 pre-mRNA. Here, we demonstrate that in vivo delivery of the optimized trans-splicing vector increases an important SMN-dependent activity, snRNP assembly, in disease-relevant tissue in the SMA mouse model. A single injection of the vector into the intracerebral-ventricular space in SMA neonates also lessens the severity of the SMA phenotype in a severe SMA mouse model, extending survival approximately 70%. Collectively, these results provide the first in vivo demonstration that SMN2 trans-splicing leads to a lessening of the severity of the SMA phenotype and provide evidence for the power of this strategy for reprogramming genetic diseases at the pre-mRNA level.
| S-EPMC2836862 | biostudies-other
Project description: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.
| S-EPMC10325915 | biostudies-literature