CRISPR/Cas9-Mediated Treatment Ameliorates the Phenotype of the Epidermolytic Palmoplantar Keratoderma-like Mouse.

CRISPR/Cas9 has been confirmed as a distinctly efficient, simple-to-configure, highly specific genome-editing tool that has been used to treat monogenetic disorders. Epidermolytic palmoplantar keratoderma (EPPK) is a common autosomal dominant keratin disease resulting from dominant-negative mutation of the KRT9 gene, and it has no effective therapy. We performed CRISPR/Cas9-mediated treatment on a knockin (KI) transgenic mouse model that carried a small indel heterozygous mutation of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), which caused a humanized EPPK-like phenotype. The mutation within exon 1 of Krt9 generated a novel protospacer adjacent motif site, TGG, for Cas9 recognition and cutting. By delivering lentivirus vectors (LVs) encoding single-guide RNAs (sgRNAs) and Cas9 that targeted Krt9 sequence into HeLa cells engineered to constitutively express wild-type and mutant keratin 9 (K9), we found the sgRNA was highly effective in reducing expression of the mutant K9 protein in vitro. We injected the LV into the fore-paws of adult KI-Krt9 mice three times every 8 days and found that the expression of K9 decreased ∼14.6%. The phenotypic mitigation was revealed by restoration of the abnormal differentiation and aberrant proliferation of the epidermis. Our data are the first to show that CRISPR/Cas9 is a potentially powerful therapeutic option for EPPK and other PPK subtypes.

Eukaryotic translation initiation factor 5A2 regulates the migration and invasion of hepatocellular carcinoma cells via pathways involving reactive oxygen species.

Eukaryotic translation initiation factor 5A2 (eIF5A2) has been identified as a critical gene in tumor metastasis. Research has suggested that reactive oxygen species (ROS) serve as signaling molecules in cancer cell proliferation and migration. However, the mechanisms linking eIF5A2 and ROS are not fully understood. Here, we investigated the effects of ROS on the eIF5A2-induced epithelial-mesenchymal transition (EMT) and migration in six hepatocellular carcinoma (HCC) cell lines. Western hybridization, siRNA transfection, transwell migration assays, wound-healing assays, and immunofluorescence analysis were used. The protein levels of eIF5A2 in tumor and adjacent tissue samples from 90 HCC patients with detailed clinical, pathological, and clinical follow-up data were evaluated. Overexpression of eIF5A2 was found in cancerous tissues compared with adjacent tissues. We found that eIF5A2 overexpression in HCC was associated with reduced overall survival. Knockdown of eIF5A2 and intracellular reduction of ROS significantly suppressed the invasion and metastasis of HCC cells. Interestingly, N1-guanyl-1, 7-diaminoheptane (GC7) suppressed the intracellular ROS levels. After blocking the EMT, administration of GC7 or N-acetyl-L-cysteine did not reduce cell migration further. Based on the experimental data, we concluded that inhibition of eIF5A2 alters progression of the EMT to decrease the invasion and metastasis of HCC cells via ROS-related pathways.

A Small Indel Mutant Mouse Model of Epidermolytic Palmoplantar Keratoderma and Its Application to Mutant-specific shRNA Therapy.

Epidermolytic palmoplantar keratoderma (EPPK) is a relatively common autosomal-dominant skin disorder caused by mutations in the keratin 9 gene (KRT9), with few therapeutic options for the affected so far. Here, we report a knock-in transgenic mouse model that carried a small insertion-deletion (indel) mutant of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), corresponding to the human mutation KRT9/c.500delAinsGGCT (p.Tyr167delinsTrpLeu), which resulted in a human EPPK-like phenotype in the weight-stress areas of the fore- and hind-paws of both Krt9(+/mut) and Krt9(mut/mut) mice. The phenotype confirmed that EPPK is a dominant-negative condition, such that mice heterozygotic for the K9-mutant allele (Krt9(+/mut)) showed a clear EPPK-like phenotype. Then, we developed a mutant-specific short hairpin RNA (shRNA) therapy for EPPK mice. Mutant-specific shRNAs were systematically identified in vitro using a luciferase reporter gene assay and delivered into Krt9(+/mut) mice. shRNA-mediated knockdown of mutant protein resulted in almost normal morphology and functions of the skin, whereas the same shRNA had a negligible effect in wild-type K9 mice. Our results suggest that EPPK can be treated by gene therapy, and this has significant implications for future clinical application.