| Over 3000 human diseases are monogenic diseases that result from some single-gene mutations. Due to the specialty of their origination, most monogenic diseases cannot be eradicated by common drugs and the only cure left is the gene therapy. Traditional gene therapies use viruses as vectors to deliver normal genes into the human beings while leaving the mutations uncorrected. However, the newly emerging CRISPR-Cas9 system that holds unlimited potential in genome reprogramming gives high hope for curing the monogenic diseases.The core principle of the CRISPR-Cas9 system is that it can efficiently create double strand breaks on the targeted DNA site with the guidance of a single guide RNA (sgRNA), which can be readily designed. Because of this simplicity and efficiency, the CRISPR-Cas9 system has overtaken ZFN and TALEN, becoming the most widely used gene editing tool in the study of genetic disease. Researchers are currently trying to remove or repair different disease-related gene mutations in situ by using the CRISPR-Cas9 system. Hemophilia B, which is one of those genetic diseases is an ideal model for validating the CRISPR-Cas9 system in gene therapy. So in this study, we manipulated the CRISPR-Cas9 system to treat Hemophilia B by correcting the point mutation on the F9 gene in mouse models.First, we identified a family with hemophilia B carrying a novel point mutation, Y371D. In order to develop the corresponding, we compare the protein sequence of human F9 (hF9) with the counterparts in some other species. We found that the Y371 in hF9 is highly homologous through all species chosen, and the site in the mouse is Y381. After microinjecting of Cas9 mRNA, sgRNA and ssODN into one-cell stage embryos, we succeeded in constructing the novel point mutation F9Y38ID mouse to mimic human hemophilia B. Besides, we also obtained the reported point mutation F9Y381S mouse and the knock-out mouse F93835STOP. By measuring the coagulation activity and the tail-clip challenge among these mice, we found that the novel mutation Y371D has a stronger pathogenicity than the reported Y371S.Next, we attempted to treat our hemophilia B mouse model by the CRISPR-Cas9 system. We used the hydrodynamic delivery method to inject the plasmid expressing Cas9 and sgRNA, and the double or single strand DNA as homologous recombination donor template to repair the point mutation of the F9 gene in liver. After the treatment, we found that 0.56% and 1.55% of F9 alleles in hepatocytes had been corrected in the group injected with single and double strand template respectively. Besides, there was no immune response in liver cells after plasmids injection. We also found that the activated partial thromboplastin time is shortened and survival rate of the tail-clip challenge is improved, which suggested that the correction rate of over 0.56% of F9 alleles was sufficient to restore hemostasis.To improve the efficiency of infection and correction, we used the adenovirus to substitute the naked DNA as the delivery vector. After intravenous injection of adenovirus vectors which contained Cas9, sgRNA and donor template,6.2% of F9 alleles were corrected in the liver cells. However, the coagulation activity was not improved in adenovirus treated F9Y38ID mice. Through the analysis of aminotransferase, inflammatory cytokines and morphology of liver cells, we found that adenovirus system caused severe hepatic toxicity, which may be the reason why adenovirus treatment had no therapeutic effects.In conclusion, our studies suggest that CRISPR/Cas9-mediated in situ genome editing is beneficial for treating hemophilia B, which could be a feasible therapeutic strategy for human hereditary diseases. |
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