CRISPR CAS9 human neuromuscular

Tandem repeats are a common, highly polymorphic class of variation in human genomes. Their expansion beyond a pathogenic threshold is a process that contributes to a wide range of neurological and neuromuscular genetic disorders, of which over 60 have been identified to date. The last few years have seen a resurgence in repeat expansion discovery propelled by technological advancements, enabling the identification of over 20 novel repeat expansion disorders. These expansions can occur in coding or non-coding regions of genes, resulting in a range of pathogenic mechanisms...

We report the first correction from prime editing a mutation in the RYR1 gene, paving the way to gene therapies for RYR1-related myopathies. The RYR1 gene codes for a calcium channel named Ryanodine receptor 1, which is expressed in skeletal muscle fibers. The failure of this channel causes muscle weakness in patients, which leads to motor disabilities. Currently, there are no effective treatments for these diseases, which are mainly caused by point mutations. Prime editing allows for the modification of precise nucleotides in the DNA...

De novo heterozygous missense mutations in EEF1A2, encoding neuromuscular translation-elongation factor eEF1A2, are associated with developmental and epileptic encephalopathies. We used CRISPR/ Cas9 to recapitulate the most common mutation, E122K, in mice. Although E122K heterozygotes were not observed to have convulsive seizures, they exhibit frequent electrographic seizures and EEG abnormalities, transient early motor deficits and growth defects. Both E122K homozygotes and Eef1a2-null mice develop progressive motor abnormalities, with E122K homozygotes reaching humane endpoints by P31...

BACKGROUND: A key mechanism in the neuromuscular disease GNE myopathy (GNEM) is believed to be that point mutations in the GNE gene impair sialic acid synthesis - maybe due to UDP- N -acetylglucosamine 2-epimerase/ N -acetylmannosamine kinase (GNE) activity restrictions - and resulting in muscle tissue loss. N -acetylmannosamine (ManNAc) is the first product of the bifunctional GNE enzyme and can therefore be regarded as a precursor of sialic acids. This study investigates whether this is also a suitable substance for restoring the sialic acid content in GNE -deficient cells...

Chemical synaptic transmission involves neurotransmitter release from presynaptic active zones (AZs). The AZ protein Rab-3-interacting molecule (RIM) is important for normal Ca2+ -triggered release. However, its precise localization within AZs of the glutamatergic neuromuscular junctions of Drosophila melanogaster remains elusive. We used CRISPR/Cas9-assisted genome engineering of the rim locus to incorporate small epitope tags for targeted super-resolution imaging. A V5-tag, derived from simian virus 5, and an HA-tag, derived from human influenza virus, were N-terminally fused to the RIM Zinc finger...

Many devastating neuromuscular diseases currently lack effective treatments. This is in part due to a lack of drug discovery platforms capable of assessing complex human neuromuscular disease phenotypes in a scalable manner. A major obstacle has been generating scaffolds to stabilise mature contractile myofibers in a multi-well assay format amenable to high content image (HCI) analysis. This study describes the development of a scalable human induced pluripotent stem cell (iPSC)-neuromuscular disease model, whereby suspended elastomer nanofibers support long-term stability, alignment, maturation, and repeated contractions of iPSC-myofibers, innervated by iPSC-motor neurons in 96-well assay plates...

BACKGROUND: Charcot-Marie-Tooth disease type 1X is caused by mutations in GJB1, which is the second most common gene associated with inherited peripheral neuropathy. The GJB1 gene encodes connexin 32 (CX32), a gap junction protein expressed in myelinating glial cells. The gene is X-linked, and the mutations cause a loss of function. AIMS: A large number of disease-associated variants have been identified, and many result in mistrafficking and mislocalization of the protein...

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder brought on by mutations in the DMD gene, which prevent muscle cells from expressing the dystrophin protein. CRISPR/Cas9 technology has evolved as potential option to treat DMD due to its ability to permanently skip exons, restoring the disrupted DMD reading frame and leading to dystrophin restoration. Even though, having potential to treat DMD, the delivery, safety and efficacy of this technology is still challenging. Several delivery methods, including viral vectors, nanoparticles, and electroporation, have been explored to deliver CRISPR/Cas9 to the targeted cells...

There is still no effective drug treatment available for Charcot-Marie-Tooth disease (CMT). Current management relies on rehabilitation therapy, surgery for skeletal deformities, and symptomatic treatment. The challenge is to find disease-modifying therapies. Several approaches, including gene silencing (by means of ASO, siRNA, shRNA, miRNA, CRISPR-Cas9 editing), to counteract the PMP22 gene overexpression in the most frequent CMT1A type are under investigation. PXT3003 is the compound in the most advanced phase for CMT1A, as a second phase-III trial is ongoing...

Duchene muscular dystrophy (DMD) is an X-linked neuromuscular disorder that affects about one in every 5000 live male births. DMD is caused by mutations in the gene that codes for dystrophin, which is required for muscle membrane stabilization. The loss of functional dystrophin causes muscle degradation that leads to weakness, loss of ambulation, cardiac and respiratory complications, and eventually, premature death. Therapies to treat DMD have advanced in the past decade, with treatments in clinical trials and four exon-skipping drugs receiving conditional Food and Drug Administration approval...

SURF1 deficiency (OMIM # 220110) causes Leigh syndrome (LS, OMIM # 256000), a mitochondrial disorder typified by stress-induced metabolic strokes, neurodevelopmental regression, and progressive multisystem dysfunction. Here, we describe two novel surf1-/- zebrafish knockout models generated by CRISPR/Cas9 technology. While gross larval morphology, fertility, and survival into adulthood appeared unaffected, surf1-/- mutants manifested adult-onset ocular anomalies and decreased swimming activity, and classical biochemical hallmarks of human SURF1 disease, including reduced complex IV expression and enzymatic activity and increased tissue lactate...

BACKGROUND: Astrocytes play a crucial, yet not fully elucidated role in the selective motor neuron pathology in amyotrophic lateral sclerosis (ALS). Among other responsibilities, astrocytes provide important neuronal homeostatic support, however this function is highly compromised in ALS. The establishment of fully human coculture systems can be used to further study the underlying mechanisms of the dysfunctional intercellular interplay, and has the potential to provide a platform for revealing novel therapeutic entry points...

BACKGROUND: Becker muscular dystrophy (BMD) is a genetic neuromuscular disease of growing importance caused by in-frame, partial loss-of-function mutations in the dystrophin (DMD) gene. BMD presents with reduced severity compared with Duchenne muscular dystrophy (DMD), the allelic disorder of complete dystrophin deficiency. Significant therapeutic advancements have been made in DMD, including four FDA-approved drugs. BMD, however, is understudied and underserved-there are no drugs and few clinical trials...

At the neuromuscular junction (NMJ), DOK7 enhances the phosphorylation of muscle-specific kinase (MuSK) and induces clustering of acetylcholine receptors (AChRs). We identified a patient with congenital myasthenic syndrome (CMS) with two heteroallelic mutations in DOK7, c.653-1G>C in intron 5 and c.190G>A predicting p.G64R in the pleckstrin homology domain. iPS cells established from the patient (CMS-iPSCs) showed that c.653-1G>C caused in-frame skipping of exon 6 (120 bp) and frame-shifting activation of a cryptic splice site deleting seven nucleotides in exon 6...

CRISPR-based gene editing technology represents a promising approach to deliver therapies for inherited disorders, including amyotrophic lateral sclerosis (ALS). Toxic gain-of-function superoxide dismutase 1 (SOD1) mutations are responsible for ~20% of familial ALS cases. Thus, current clinical strategies to treat SOD1-ALS are designed to lower SOD1 levels. Here, we utilized AAV-PHP.B variants to deliver CRISPR-Cas9 guide RNAs designed to disrupt the human SOD1 (huSOD1) transgene in SOD1G93A mice. A one-time intracerebroventricular injection of AAV...

Charcot-Marie-Tooth disease is an inherited peripheral neuropathy that is clinically and genetically heterogenous. Mutations in IGHMBP2, a ubiquitously expressed DNA/RNA helicase, have been shown to cause the infantile motor neuron disease spinal muscular atrophy with respiratory distress type 1 (SMARD1), and, more recently, juvenile-onset Charcot-Marie-Tooth disease Type 2S (CMT2S). Using CRISPR-cas9 mutagenesis we developed the first mouse models of CMT2S (p.Glu365del (E365del) and p.Tyr918Cys (Y918C)). E365del is the first CMT2S mouse model to be discovered and Y918C is the first human CMT2S allele knock-in model...

Triosephosphate isomerase deficiency (TPI Df) is a rare, aggressive genetic disease that typically affects young children and currently has no established treatment. TPI Df is characterized by hemolytic anemia, progressive neuromuscular degeneration, and a markedly reduced lifespan. The disease has predominately been studied using invertebrate and in vitro models, which lack key aspects of the human disease. While other groups have generated mammalian Tpi1 mutant strains, specifically with the mouse mus musculus, these do not recapitulate key characteristic phenotypes of the human disease...

The immune response is a primary hurdle in the development of gene therapy for neuromuscular diseases. Both innate and adaptive immune responses have been observed in human trials. The canine model is an excellent platform to understand immunological consequences of gene therapy. Over the last several decades, we have conducted gene replacement and gene repair therapies in the canine model of Duchenne muscular dystrophy (DMD) using adeno-associated virus (AAV)-mediated expression of the highly abbreviated micro-dystrophin gene, the larger mini-dystrophin gene, and the Cas9-based CRISPR genome editing machinery...

GNE Myopathy is a rare, recessively inherited neuromuscular worldwide disorder, caused by a spectrum of bi-allelic mutations in the human GNE gene. GNE encodes a bi-functional enzyme responsible for the rate-limiting step of sialic acid biosynthesis pathway. However, the process in which GNE mutations lead to the development of a muscle pathology is not clear yet. Cellular and mouse models for GNE Myopathy established to date have not been informative. Further, additional GNE functions in muscle have been hypothesized...

GNE myopathy is an adult onset neuromuscular disorder characterized by slowly progressive distal and proximal muscle weakness, caused by missense recessive mutations in the GNE gene. Although the encoded bifunctional enzyme is well known as the limiting factor in the biosynthesis of sialic acid, no clear mechanisms have been recognized to account for the muscle atrophic pathology, and novel functions for GNE have been hypothesized. Two major issues impair studies on this protein. First, the expression of the GNE protein is minimal in human and mice muscles and there is no reliable antibody to follow up endogenous expression...