SummaryDuchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs) by TALENs or CRISPR-Cas9 holds promise for DMD gene therapy; however, the safety of such nuclease treatment must be determined. Using a unique k-mer database, we systematically identified a unique target region that reduces off-target sites. To restore the dystrophin protein, we performed three correction methods (exon skipping, frameshifting, and exon knockin) in DMD-patient-derived iPSCs, and found that exon knockin was the most effective approach. We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load. Finally, we differentiated the corrected iPSCs toward skeletal muscle cells and successfully detected the expression of full-length dystrophin protein. These results provide an important framework for developing iPSC-based gene therapy for genetic disorders using programmable nucleases.

No abstract

Mutations of the methyl CpG binding protein 2 (MeCP2) gene are a major cause of Rett syndrome. To investigate whether the expression of this gene was related to JC virus (JCV) infection, we examined brains of four progressive multifocal leukoencephalopathy (PML) patients. JCV infection was confirmed by immunohistochemical labeling with antibodies against JCV VP1, Agnoprotein and large T antigen. MeCP2 expression was examined by immunohistochemistry using a specific polyclonal antibody against MeCP2. In normal brains and uninfected cortices of PML brains, MeCP2 expression was observed in the nuclei of neurons, but not observed in glial and endothelial cell nuclei. In PML brains, however, intense immunolabeling was observed in abnormally enlarged glial nuclei of JCV-infected cells. The JCV infection was verified by immunolabeling against JCV VP1, Agnoprotein and large T antigen. Double immunolabeling using antibodies against large T antigen (visualized as blue) and MeCP2 (visualised as red) revealed purple JCV infected nuclei, which confirmed that the JCV infected nuclei expressed MeCP2. When we examined four MeCP2 related proteins, named as methyl CpG binding domains (MBD) 1, 2, 3, and 4, only MBD1 exhibited similar results to MeCP2. We conclude that MeCP2 is highly expressed in the JCV infected nuclei of PML brain and these results may provide a new insight into the mechanism which regulates the MeCP2 expression in glial cells by the infection of JCV.

No abstract

No abstract