Alan Babuji

Introduction. Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease and motor neuron disease (MND), is a progressive condition caused by the deterioration of the motor neurons in the spinal cord and brain, resulting in paralysis.¹ To date, more than 100 mutations in the SOD1 gene have been implicated in ALS, and although the role of mutant SOD1 in the pathogenesis of the disorder remains incompletely understood. It is thought that mutant SOD1 expression in both motor neurons.² Altered RNA processing leading to prion-like self-aggregation, superoxide dismutase type 1 SOD1 mutations leading to free radical toxicity, cascading inflammatory responses, and excessive concentrations of glutamate, among others.³ Riluzole prolongs survival by 3–6 months.⁴ Although ALS is currently not curable, CRISPR base editors provide a promising future by disabling the expression of mutant SOD1 gene.⁵ Methods. SOD1G93A transgenic mice (B6SJL-TgN [SOD1-G93A] 1Gur) were obtained from Jackson Laboratories.⁶ Guide sequences for a luciferase reporter assay for Staphylococcus aureus Cas9 had 5’-CACC and 5’-AAAC overhangs; if the guide sequence did not begin with a “G,” a G was added to the 5’ end to enhance U6 promoter-driven transcription.⁶ The SaCas9-sgRNA plasmid and the luciferase reporter plasmid were then cotransfected into HEK293T cells. ⁶  Neonatal mice were anesthetized first, then administered a 10-μL injection of the vector. ⁶ H&E stains were analyzed for pathological differences, and fluorescence microscopy visualized the protective effects of motor neurons provided by AAV9 SOD1 sgRNAs.⁶ Western blot quantified SOD1 protein. Behavioral analysis including weight, motor function, rotarod performance, and locomotion were noted in a blinded manner. ⁶ Results. The motor neurons showed NeuN positivity, a marker for mature motor neurons and a cell body diameter greater than 20 µm at the anterior horn of the spinal cord. Higher IBA1 protein activity in the mutant control displays the larger macrophages/microglial activation. ⁶ In the Cas9 treated group, there was lower inflammation as seen with lower expression of IBA1. ⁶ Treatment group lived 11% longer than the mutant control. ⁶ Higher rotarod activity for the treatment group displays more muscle activity and less musical atrophy. ⁶ This translated to a higher muscle mass in the treatment group. Conclusion. Decreased muscle atrophy improved neuromuscular function and an overall slowing of the progression of the disease in a mouse model with the ALS disorder were consistent with the lower amounts of mRNA production. ^6,7 Therefore, utilization of CRISPR opens potential therapy for ALS.

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