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Amyotrophic lateral sclerosis (ALS) is a fatal motoneuronal disorder that leads to progressive degeneration of upper and lower motor neurons in the primary motor cortex, spinal cord and brainstem. The degeneration and loss of motor neurons lead to progressive weakness and atrophy of skeletal muscles, which usually progress to paralysis and death within 3 to 5 years. At present, no effective therapy exists for this disease.
Most cases of ALS are sporadic (SALS), but a few have been classified as 'familial ALS' (FALS) due to the identification of some causative genes in the past 30 years. Sporadic ALS accounts for 85-90% of all cases and has a median age of onset ranging from 58-63 years old. Familial ALS represents for the remaining 10-15% of cases and has a slightly younger age of onset (47-53 years old); potential causative mutations have been identified in more than 50 genes. The most commonly mutated genes, accounting for about 75% of FALS cases, are C9orf72, FUS, TARDBP and SOD1. Dominantly inherited mutations in the SOD1 gene were described for the first time and are linked to 15% of FALS cases. More than 180 genetic variants of SOD1 have been identified in patients with ALS. Most of these genes confer dominant inheritance, whereas some of them are recessive (FUS, SOD1, and Optineurin) and others can confer only autosomal recessive (Alsin, Spastacin, and SIGMAR1) or are even X-linked (Ubiquilin-2) inheritance.
Figure 1. Localization and role of the targets (proteins) of the main causative genes of FALS. (Mathis S, et al., 2019)
The CRISPR/Cas9 system is a novel genome modification method in which gRNA direct the nuclease Cas9 to selected sequences of genomic DNA, and Cas9 cuts both strands at a precise location. The genomic DNA is then repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR), leading to mutations that can interrupt the open reading frame and cause gene inactivation. Genome editing by CRISPR/Cas9 can be used in disease research to study genetic modifications in various cell types. With the development of reprogramming technology, somatic cells have been used for induced pluripotent stem cell (iPSC) generation. Patient-derived iPSCs provide an unlimited source of cells for study and treat disease. Especially for genetic disorders, pathologic effects of mutations can be investigated in iPSCs using CRISPR/Cas9 to correct mutations. Therefore, the correction of specific mutations related to ALS can provide important insights into verifying the pathologic effects of these mutations and identifying targets for therapeutic interventions.
Currently, the application of the CRISPR/Cas9 system in ALS treatment has a dual purpose: gene therapy for inherited ALS and the expression of genes encoding neurotrophic factors (NTFs). Gaj et al. demonstrated that the CRISPR/Cas9 can be used to modify mutant expression in the G93A-SOD1 mouse model of ALS, which was delivered in vivo by an adeno-associated virus vector (AAV). Gene disruption contributed to the reduced expression of mutant SOD1 protein in the lumbar and thoracic spinal cord resulting in diminished muscle atrophy and enhanced motor function. Importantly, compared with the control group, ALS mice treated by CRISPR/Cas9 had increased survivability of motor neurons, delayed disease onset, and a prolonged lifespan. This study has significantly confirmed the potential of CRISPR/Cas9 for the treatment of SOD1-linked forms of ALS and other neurodegenerative diseases. The second method of applying CRISPR/Cas9 technology in ALS therapy is based on the meaningful potential of neurotrophic factors to exert neuroprotective effects on damaged motor neurons in familial and sporadic ALS cases. Genetically modified mesenchymal stem cells (MSCs) expressing neurotrophic factors might be a less invasive method of administration, which can enhance the neuroprotective effects of MSCs on impaired motor neurons. Van den Akker et al. found that MSCs are able to steadily express CRISPR/Cas9 components along with the standard differentiation features of these cells. Once the expression of a gene is evaluated and optimized, the cells can be implanted into the central nervous system regions to play a local role in providing neurotrophic factors. As the combination of several growth factors might be more successful in the context of ALS treatment, stem cells equipped with NTFs genes would be another possibility.
CRISPR/Cas9 PlatformCB is committed to providing the most professional and comprehensive genetic editing technology solutions for our clients. To support your projects, we offer a comprehensive custom CRISPR/Cas9 gene editing service from strategy design to final ALS model generation.
➢ Human Cell Models Generation of ALS by CRISPR/Cas9 System
➢ Animal Models Generation of ALS by CRISPR/Cas9 System
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