Construction Of Cell Model Of Hemophilia A With Nonsense Mutation Based On IPS And Base Editing Technology

Objective: Hemophilia is an X chromosome recessive hemorrhagic disease caused by the loss of coagulation factor.At present,the treatment of hemophilia is still based on infusion of coagulation factor replacement therapy,but this traditional treatment requires long-term infusions and high costs.As a single gene genetic disease,gene therapy is the only cure for hemophilia.In this study,we generated human induced pluripotent stem cells(i PSCs)from urine cells of hemophilia A patients with nonsense mutation(F8 C.2440C>T)previously discovered in the laboratory.Meanwhile,Cytosine Base Editor(CBE)was used to construct hemophilia A cell model with nonsense mutation(F8 C.6682C>T).In order to lay a foundation for the research on the pathogenesis,gene therapy and personalized therapy of hemophilia caused by nonsense mutation.Methods: 1.Establishment and identification of hemophilia-specific i PSCs with nonsense mutation(F8 C.2440C>T)First,the mid-stream urine of hemophiliac patients was collected,and human urine cells(UCs)were isolated.When UCs reached a good state,we introduced reprogrammed fourfactor OSKM into urine cells,and then selected and cultured monoclonal cells after the appearance of stem-like formation.At this time,UCs had been reprogrammed into i PSCs.Finally,AP staining and karyotype analysis were used to verify that i PSCs had no chromosomal aberrations,and they were preliminarily determined to be pluripotent.High expression of pluripotent markers could be observed by immunofluorescence staining.2.Construction of hemophilia A cell model with nonsense mutation(F8 C.6682C>T)by using CBE.sgRNA targeting specific mutation sites was designed and integrated with single base editing system YE1-BE3 as the target vector.In HEK-293 T cells,sgRNA identified c.6682,and resulting in C>T nonsense mutation at this site by using three transfection kits.Results: 1.Human induced pluripotent stem cells were successfully reprogrammed from urine cells of hemophilia A patients with nonsense mutation(F8 C.2440C>T).2.The cell model of hemophilia A with nonsense mutation(F8 C.6682C>T)was successfully constructed.Conclusion: 1.In this study,ori P/EBNA1 system was used to transfect PEP4-EO2S-ET2 K and p CEP4-M2 L,which contained reprogramming factors OCT4,SOX2,KLF4 and C-MYC,into urine cells.Urine cells were successfully reprogrammed into human induced pluripotent stem cells(i PSCs).2.Using CBE single-base editing system,the target vector YE1-BE3-sgRNA was transfected into HEK-293 T cells,and then the specific cell model of hemophilia A with nonsense mutation(F8 C.6682C>T)was successfully constructed,thus proving that base editing system is feasible and effective in HEK-293 T cells.