Construction And Hematopoietic Differentiation Of Hemophilia A Patients IPSCs With F8 Site-Specific Integration

Background Hemophilia A(HA)is an X-linked recessive hereditary disease caused by mutation of factor VIII(FVIII)gene(F8).Current protein-replacement therapies are limited by their high cost,the need for repeated infusions,and the presence of FVIII inhibitors in nearly 30%of patients.Emerging gene therapies are promising to cure HA,but there are still problems such as insufficient FVIII expression and inhibitor production.It has been proposed that the coagulation efficiency of FVIII can be effectively improved and the production of FVIII inhibitors can be avoided by ectopic expression of FVIII in platelets,the expression of v WF in platelets and the aggregation at bleeding sites and the release ofαparticles to promote coagulation.In addition,although viral vector has been approved for clinical application,its limited load,immunogenicity,as well as the risk of insertional mutagenesis still existing.Therefore,this study intends to target the B domain deletion F8 gene(BDDF8)driven by the megakyocyte(MK)specific ITGA2B gene promoter at the ribosomal DNA(rDNA)locus of HA patient induced pluripotent stem cells(iPSCs)by combining our self-developed rDNA targeting vector with the single-strand cleaving nuclease TALENickases.The gene-integrated iPSCs(2b F8-iPSCs)will be differentiated into hematopoietic progenitor cells(HPCs),MKs and mesenchymal stem cells(MSCs),of which the expression of functional FVIII be detected to provide experimental basis for application study of the platelet-based HA targeted gene therapy.Objective This study aims to establish the therapeutic cells of HSCs,MKs and MSCs with highly efficient and specific expression of functional FVIII differentiated from the F8-modified iPSCs by jointly targeting a MK-specific promoter and BDDF8 at HA-iPSCs,laying a foundation for application of the platelet-targeted HA gene therapy.Methods(1)The truncated ITGA2B gene promoter fragment was amplified by PCR,and the target vector minip Hrn-2b F8 was designed and constructed which expressing BDDF8 gene in rDNA region;(2)Combined with TALENickases and minip Hrn-2b F8,BDDF8 driven by ITGA2B gene promoter was site-integrated into HA-iPSCs by nuclear transferring.After screening,the single-cell clone was selected as site-specific integration,2b F8-iPSCs;(3)2b F8-iPSCs were differentiated into induced HPCs(i HPCs)and induced MKs(i MKs)in vitro,and signature identification analysis was performed on the differentiated cells,and the expression of FVIII was detected;(4)2b F8-iPSCs were differentiated in the form of teratoma in the immune deficiency HA model mice,and the proportion of HPCs in the teratoma and human cells in the mouse peripheral blood were detected,and the changes of coagulation activity of the mice were analyzed;(5)2b F8-iPSCs were differentiated into induced MSCs(iMSCs)in vitro,and the signature identification analysis was performed on the differentiated cells,and the expression of FVIII was detected.Results(1)The rDNA targeting vector minip Hrn-2b F8 was successfully constructed;(2)2b F8-iPSCs with rDNA site-specific integration of ITGA2B promoter driven BDDF8 were obtained;(3)2b F8-iPSCs can be efficiently differentiated into 2b F8-i HPCs and 2b F8-i MKs in vitro,and the CD34 positive rate of i HPCs purified by beads can reach more than 90%.The expression level of FVIII in the lysates of 2b F8-i HPCs was 10.31 ng/10~6,which was much higher than that of HA-i HPCs was 1.56 ng/10~6;(4)HPCs were successfully differentiated from 2b F8-iPSCs in the form of teratoma in the immune deficiency HA model mice.Human CD45 cells accounted for 5.57%of the total peripheral blood cells,and FVIII coagulation activity of tumor-forming mice transplanted with 2b F8-iPSCs was significantly increased;(5)2b F8-iPSCs can be efficiently differentiated into 2b F8-iMSCs in vitro,and the differentiated iMSCs conform to the characteristics of MSCs.The expression level of FVIII in 2b F8-iMSCs lysate was 3.64 ng/10~6 cells.However,the expression level of FVIII in the supernatant of cell culture was only 0.05ng/10~6 cells,while the expression level of iMSCs which integrated BDDF8 driven by universal promoter(T-7-iMSCs)was 1.31 ng/10~6 cells,while the expression level of FVIII in supernatant was 0.23 ng/10~6cells.Conclusion This study obtains the therapeutic cells of HSCs and MSCs,which derived from F8-modified HA-iPSCs,with highly efficient and specific expression of functional FVIII,laying a foundation for the further study on the treatment of HA.The results also indicate that this innovative approach of autologous platelet-targeted gene therapy holds promise for clinical application.