RESUMEN
Spinal muscular atrophy is a progressive motor neuron disorder caused by deletions or point mutations in the SMN1 gene. It is not known why motor neurons are particularly sensitive to a decrease in SMN protein levels and what factors besides SMN2 underlie the high clinical heterogeneity of the disease. Here we studied the methylation patterns of genes on sequential stages of motor neuron differentiation from induced pluripotent stem cells derived from the patients with SMA type I and II. The genes involved in the regulation of pluripotency, neural differentiation as well as those associated with spinal muscular atrophy development were included. The results show that the PAX6, HB9, CHAT, ARHGAP22, and SMN2 genes are differently methylated in cells derived from SMA patients compared to the cells of healthy individuals. This study clarifies the specificities of the disease pathogenesis and extends the knowledge of pathways involved in the SMA progression.
Asunto(s)
Células Madre Pluripotentes Inducidas/fisiología , Neuronas Motoras/fisiología , Atrofia Muscular Espinal/genética , Diferenciación Celular , Células Cultivadas , Metilación de ADN , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Neurogénesis , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/metabolismoRESUMEN
Spinal muscular atrophy (SMA) is a genetic disease, which characterized by the degeneration of motor neurons in the spinal cord and further striated muscle atrophy. The research of the processes in diseased neurons is complicated due to the impossibility of obtaining them safely from patients. Thus, we generated SMA type III induced pluripotent stem cell lines via using non-integrated episomal plasmid vectors. The resulting cell line expresses the major pluripotency markers and can differentiate in vitro into derivatives of three germ layers. The iPSC line can be used for further studies by providing in vitro the relevant cell types.
Asunto(s)
Células Madre Pluripotentes Inducidas , Atrofia Muscular Espinal , Atrofias Musculares Espinales de la Infancia , Línea Celular , Humanos , Neuronas Motoras , Atrofia Muscular Espinal/genéticaRESUMEN
The CRISPR/Cas technology has a great potential in the treatment of many hereditary diseases. One of the prospective models for the CRISPR/Cas-mediated therapy is spinal muscular atrophy (SMA), a disease caused by deletion of the SMN1 gene that encodes the SMN protein required for the survival of motor neurons. SMA patients' genomes contain either single or several copies of SMN2 gene, which is a paralog of SMN1. Exon 7 of SMN2 has the single-nucleotide substitution c.840C>T leading to the defective splicing and decrease in the amounts of the full-length SMN. The objective of this study was to create and test gene-editing systems for correction of the single-nucleotide substitution c.840C>T in exon 7 of the SMN2 gene in fibroblasts, induced pluripotent stem cells, and motor neuron progenitors derived from a SMA patient. For this purpose, we used plasmid vectors expressing CRISPR/Cas9 and CRISPR/Cpf1, plasmid donor, and 90-nt single-stranded oligonucleotide templates that were delivered to the target cells by electroporation. Although sgRNA_T2 and sgRNA_T3 guiding RNAs were more efficient than sgRNA_T1 in fibroblasts (p < 0.05), no significant differences in the editing efficiency of sgRNA_T1, sgRNA_T2, and sgRNA_T3 was observed in patient-specific induced pluripotent stem cells and motor neuron progenitors. The highest editing efficiency in induced pluripotent stem cells and motor neuron progenitors was demonstrated by the sgRNA_T1 and 90-nt single-stranded oligonucleotide donors.