Arbab Ahmed

Introduction. Amyotrophic Lateral Sclerosis (ALS) is a heterogeneous neurodegenerative disease that is characterized by the degeneration of both the upper and lower motor neurons.¹ ALS is subdivided into Familial ALS, 5-15% of cases, and the majority as Sporadic ALS.²  There are 30 genes associated with familial ALS.² SOD1 gain-of-function mutations is one of the first genetic cause identified for this disease.³ Sporadic ALS is thought to result from both environmental and genetic risk factors such as smoking, exposure to pesticides, organic toxins, and electromagnetic radiation.^1,2 The median onset for the disease is 65 years old.² SOD1 is an antioxidant enzyme that breaks down superoxide radicals, preventing cell damage. Mutations in SOD1 (G93A) lead to neuronal excitability, endoplasmic reticulum stress, mitochondrial dysfunction, oxidative stress, impaired protein transport, accumulation of misfolded SOD1 protein, and inflammation, all of which may increase neuronal cell death.⁴  Patients can present with predominantly upper motor neuron dysfunction such as spasticity and weakness or lower motor neuron dysfunction such as fasciculation, cramps, and muscle wasting.¹ Currently, pharmacological treatment Riluzole exists, but only prolongs survivability by 3 months. This brings up the need for alternative treatment. Studies have shown that treatment with CRISPR genome editing to reduce misfolded SOD1 proteins could be a potential treatment for ALS.  Methods.  The Luciferase reporter plasmid contains a stop codon and the SOD1 DNA flanked by sequences homologous to the Gaussia luciferase (Gluc) gene. The SaCas9-sgRNA plasmid and the luciferase reporter plasmid were cotransfected into mice. SOD1G93A transgenic mice were used to study the effects of the CRISPR treatment. Mutant SOD1 protein was measured via Quantitative RT- PCR, immunohistochemistry, Western blot, and using deep sequencing. Rotarod was used for muscle strength tests, and weight loss was used to measure disease progression.  Results. First we determined that CRISPR was effective in its intended effect to reduce mutant SOD1 in motor proteins. Fluorescence was used to determine that treatment of CRISPR to reduce mutant SOD1 reduced neuron degeneration and inflammation. Improvement in ALS disease progression resulted due to a reduction in mutant SOD1 and neurodegeneration. Conclusion. From the experiments, we concluded that G93A-SOD1 mice had improved motor function and improved hindlimb grip strength, lost weight 43% slower and lost weight ∼75% slower than did control animals.⁵ The treatment of mutant SOD1 improved the lifespan of SOD1G93A mice by 54.6%.⁶

1. Hardiman O, Al-Chalabi A, Chio A, et al. Amyotrophic lateral sclerosis [published correction appears in Nat Rev Dis Primers. 2017 Oct 20;3:17085]. Nat Rev Dis Primers. 2017;3:17071. Published 2017 Oct 5. doi:10.1038/nrdp.2017.71
2. van Es MA, Hardiman O, Chio A, et al. Amyotrophic lateral sclerosis. Lancet. 2017;390(10107):2084-2098. doi:10.1016/S0140-6736(17)31287-4
3. Miccio A, Antoniou P, Ciura S, Kabashi E. Novel genome-editing-based approaches to treat motor neuron diseases: Promises and challenges. Mol Ther. 2022;30(1):47-53. doi:10.1016/j.ymthe.2021.04.003
4. Yun Y, Ha Y. CRISPR/Cas9-Mediated Gene Correction to Understand ALS. Int J Mol Sci. 2020;21(11):3801. Published 2020 May 27. doi:10.3390/ijms21113801
5. Lim CKW, Gapinske M, Brooks AK, et al. Treatment of a Mouse Model of ALS by In Vivo Base Editing. Mol Ther. 2020;28(4):1177-1189. doi:10.1016/j.ymthe.2020.01.005
6. Duan W, Guo M, Yi L, et al. The deletion of mutant SOD1 via CRISPR/Cas9/sgRNA prolongs survival in an amyotrophic lateral sclerosis mouse model. Gene Ther. 2020;27(3-4):157-169. doi:10.1038/s41434-019-0116-1