The X‐linked genetic bleeding disorder caused by deficiency of coagulator factor IX, hemophilia B, is a disease ideally suited for gene therapy with genome editing technology. Here, we identify a family with hemophilia B carrying a novel mutation, Y371D, in the human F9 gene. The CRISPR/Cas9 system was used to generate distinct genetically modified mouse models and confirmed that the novel Y371D mutation resulted in a more severe hemophilia B phenotype than the previously identified Y371S mutation. To develop therapeutic strategies targeting this mutation, we subsequently compared naked DNA constructs versus adenoviral vectors to deliver Cas9 components targeting the F9 Y371D mutation in adult mice. After treatment, hemophilia B mice receiving naked DNA constructs exhibited correction of over 0.56% of F9 alleles in hepatocytes, which was sufficient to restore hemostasis. In contrast, the adenoviral delivery system resulted in a higher corrective efficiency but no therapeutic effects due to severe hepatic toxicity. Our studies suggest that CRISPR/Cas‐mediated in situ genome editing could be a feasible therapeutic strategy for human hereditary diseases, although an efficient and clinically relevant delivery system is required for further clinical studies.
No effective targeted therapies exist for cancers with somatic KRAS mutations. Here we develop a synthetic lethal chemical screen in isogenic KRAS-mutant and wild-type cells to identify clinical drug pairs. Our results show that dual inhibition of polo-like kinase 1 and RhoA/Rho kinase (ROCK) leads to the synergistic effects in KRAS-mutant cancers. Microarray analysis reveals that this combinatory inhibition significantly increases transcription and activity of cyclin-dependent kinase inhibitor p21WAF1/CIP1, leading to specific G2/M phase blockade in KRAS-mutant cells. Overexpression of p21WAF1/CIP1, either by cDNA transfection or clinical drugs, preferentially impairs the growth of KRAS-mutant cells, suggesting a druggable synthetic lethal interaction between KRAS and p21WAF1/CIP1. Co-administration of BI-2536 and fasudil either in the LSL-KRASG12D mouse model or in a patient tumour explant mouse model of KRAS-mutant lung cancer suppresses tumour growth and significantly prolongs mouse survival, suggesting a strong synergy in vivo and a potential avenue for therapeutic treatment of KRAS-mutant cancers.
Forkhead box D3 (FOXD3), as a transcriptional repressor, is well known to be involved in the regulation of development. Although FoxD3 is associated with several cancers, its role in colon cancer and the underlying mechanism are still unclear. Here, we first showed that FOXD3 knockdown dramatically increased the proliferation of human colon cancer cells, enhanced cell invasive ability and inhibited cell apoptosis. In vivo xenograft studies confirmed that the FOXD3-knockdown cells were more tumorigenic than the controls. Silencing FOXD3 markedly activated EGFR/Ras/Raf/MEK/ERK pathway in human colon cancer cells. In addition, blocking EGFR effectively decreased the activity of MAPK induced by FOXD3 knockdown. In human cancer tissue, the expression of FOXD3 was reduced, however, the EGFR/Ras/Raf/MEK/ERK pathway was activated. Our study indicates that FOXD3 may play a protective role in human colon formation by regulating EGFR/Ras/Raf/MEK/ERK signal pathway. It is proposed that FOXD3 may have potential as a new therapeutic target in human colon cancer treatment.
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