Combined Method of Neuronal Cell-Inducible Vector and Valproic Acid for Enhanced Gene Expression under Hypoxic Conditions.

BACKGROUND: Gene therapy shows the ability to restore neuronal dysfunction via therapeutic gene expression. The efficiency of gene expression and delivery to hypoxic injury sites is important for successful gene therapy. Therefore, we established a gene/stem cell therapy system using neuron-specific enolase promoter and induced neural stem cells in combination with valproic acid to increase therapeutic gene expression in hypoxic spinal cord injury. METHODS: To examine the effect of combined method on enhancing gene expression, we compared neuronal cell-inducible luciferase levels under normoxia or hypoxia conditions in induced neural stem cells with valproic acid. Therapeutic gene, vascular endothelial growth factor, expression with combined method was investigated in hypoxic spinal cord injury model. We verified gene expression levels and the effect of different methods of valproic acid administration in vivo. RESULTS: The results showed that neuron-specific enolase promoter enhanced gene expression levels in induced neural stem cells compared to Simian Virus 40 promoter under hypoxic conditions. Valproic acid treatment showed higher gene expression of neuron-specific enolase promoter than without treatment. In addition, gene expression levels and cell viability were different depending on the various concentration of valproic acid. The gene expression levels were increased significantly when valproic acid was directly injected with induced neural stem cells in vivo. CONCLUSION: In this study, we demonstrated that the combination of neuron-specific enolase promoter and valproic acid induced gene overexpression in induced neural stem cells under hypoxic conditions and also in spinal cord injury depending on valproic acid administration in vivo. Combination of valproic acid and neuron-specific enolase promoter in induced neural stem cells could be an effective gene therapy system for hypoxic spinal cord injury.

CRISPR-mediated gene correction links the ATP7A M1311V mutations with amyotrophic lateral sclerosis pathogenesis in one individual.

Amyotrophic lateral sclerosis (ALS) is a severe disease causing motor neuron death, but a complete cure has not been developed and related genes have not been defined in more than 80% of cases. Here we compared whole genome sequencing results from a male ALS patient and his healthy parents to identify relevant variants, and chose one variant in the X-linked ATP7A gene, M1311V, as a strong disease-linked candidate after profound examination. Although this variant is not rare in the Ashkenazi Jewish population according to results in the genome aggregation database (gnomAD), CRISPR-mediated gene correction of this mutation in patient-derived and re-differentiated motor neurons drastically rescued neuronal activities and functions. These results suggest that the ATP7A M1311V mutation has a potential responsibility for ALS in this patient and might be a potential therapeutic target, revealed here by a personalized medicine strategy.

Malignant gliomas can be converted to non­proliferating glial cells by treatment with a combination of small molecules.

Gliomas, the most highly malignant central nervous system tumors, are associated with an extremely poor patient survival rate. Given that gliomas are derived from mutations in glial precursor cells, a considerable number of them strongly react with glial precursor cell­specific markers. Thus, we investigated whether malignant gliomas can be converted to glial cells through the regulation of endogenous gene expression implicated in glial precursor cells. In the present study, we used three small­molecule compounds, [cyclic adenosine monophosphate (cAMP) enhancer, a mammalian target of rapamycin (mTOR) inhibitor, and a bromodomain and extra­terminal motif (BET) inhibitor] for glial reprogramming. Small­molecule­induced gliomas (SMiGs) were not only transformed into exhibiting a glial­specific morphology, but also showed positive reactions with glial­specific markers such as glial fibrillary acidic protein (GFAP), 2',3'­cyclic nucleotide 3'­phosphohydrolase (CNP) and anti­oligodendrocyte (RIP). A microarray analysis indicated that SMiGs exhibited a marked increase in specific gene levels, whereas that of a malignant cancer­specific gene was greatly decreased. Moreover, proliferation of the cells was markedly suppressed after the conversion of malignant glioma cells into glial cells. Our findings confirmed that malignant gliomas can be reprogrammed to non­proliferating glial cells, using a combination of small molecules, and their proliferation can be regulated by their differentiation. We suggest that our small­molecule combination (with forskolin, rapamycin and I­BET151) may be the next generation of anticancer agents that act by reprogramming malignant gliomas to differentiate into glial cells.