The Study Of Stem Cell-based Platelet Targeted Gene Therapy For Hemophilia A

Hemophilia A(HA),a severe congenital bleeding disorder caused by the loss of clotting factor VIII(FVIII),is an X-linked recessive hereditary disease.Protein replacement therapy in clinical is limited by the cost and its easy to produce inhibitors in patient’s plasma,however gene therapy may be an effective means of solving these problems and curing the disease.Targeting FVIII expression in platelets is an ideal way to treat hemophilia A with inhibitors,but the use of viral vectors may have potential insertional mutagenesis,and lower gene transduction and expression efficiency also constrain the clinical application.To this end,we intend to overcome the bottlenecks by targeting integration of induction of pluripotent stem cells(iPSCs)via gene editing tool clustered regμlarly interspaced short palindromic repeats(CRISPR/Cas9).IPSCs have infinite proliferative potential and can differentiate into mμlti-lineage cells,including hematopoietic stem progenitor cells(HSPCs),which play a significant role in gene therapy for a variety of hereditary diseases.CRISPR/Cas9-mediated site-specific integration coμld efficiently guide the gene into the target site to eliminate the potential mutagenesis,and the iPS gene editing combined with platelet-targeted gene therapy for HA has a wide range of application prospects.For this purpose,we obtained HA-miPSC by reprogramming the newborn HA mouse tail tip fibroblasts,then targeted the codon-optimized BDD-FVIII under the control of the GPαIIb promoter into the mROSA26 safety locus of miPS and screened for targeting integration monoclone.Furthermore,because of high-efficiency in inducing pluripotent stem cells hematopoietic differentiation,we established a hHoxB4 stable expression clone 2bopF8-HAiPSC,and induced the iPSCs differentiate into HSPCs by in vivo teratoma formation and transplanted into lethal irradiated HA mice.We analyzed the hematopoietic reconstitution and platelet function,the expression levels and activity of FVIII in platelets afer bone marrow transplantation,meanwhile,we evaluated the improvement of bleeding phenotype via tail clip assay.In addition,we obtained chimeric mice through blastocyst injection of targeted-integration iPS cells,which can participate in the development of hematopoietic system,and be able to produce FVIII expression platelets.After secondary bone marrow transplantation,the hematopoietic reconstruction and therapeutic efficiency coμld also be achieved,indicating that the differentiated hematopoietic stem cells have complete biological function.Based on these data,this study suggest that the combination of iPS-induced hematopoietic differentiation and target platelet gene therapy can effectively correct the hemorrhagic phenotype of HA mice and doesn’t induce the inhibitor formation,which is worth further investigation.